/

' J

^/

A3 9?

Volume SO

Number 1

January-March 2000

QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY

Variation in Tree Fern Stipe Length with Canopy Height: Tracking Preferred Habitat

Through Morphological Change AT. C. Arens and R Sanchez Baracaldo

Cryopreservalion of In Vitro Grown Fern Gametophytes

V C Pence

Vessels in Roots and RJiizomes of Dryopteris crassirhizoma (Dryopteridaceae) from Hei-

longjiang Province, China
SEM Studies on Vessels in Ferns. 19. Marsilea

R. Li, X. Yan and D. Zhang

E. L. Schneider and S. Carlquist

1

16

24

32

Shorter Notes:

6-C-P-CellohiosylisoscutelIarein-8-methyl ether, a new flavonoid from Pteris vittata

F. Imperato and A. Telesca

42

Ophloglossum pendulum L. Naturalized in Miami, Dade County, Florida

A. Tejedor and B. W. McAIpin

46

Reviews:

Flora Malesiana, Series H-Ferns and Fern Allies, Volume 3

G. Yatskiexych

48

Bibliografia sobre Gametofitos de Helechos y Plantas Afines, 1699-1996

G. Yatskievych

49

I

The American Fern Society

Council for 1999

90004

CHRISTOPHER H. HAUFLER

President

JAMES

Yice-Pres ide i x t
Public Museum, Milwaukee, WI 53233-1478.

Secretary

Treasurer
DAVID B. LELLINGER, 326 West St. NW., Vienna, VA 22180-4151. Membership Secretary

JAMES D. MONTGOMERY, Ecology HI, R.D. 1, Box 1795, Berwick, PA 18603-9801.

Back Issues Curator
GEORGE YATSKIEVYCH, Missouri Botanical Garden, FO. Box 299, St. Louis, MO 63166-0299.

Journal Editor
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Smithsonian Institution, Washington, DC 20560-0166. Memoir Editor

CINDY JOHNSON-GROH. Depl. of Biology, Gustavus Adolphus College,

800 W. College Ave., St. Peter, MN 56082 1498. Bulletin Editor

American Fern Journal

EDrroR

R. JAMES HICKEY

Botany Department,

Miami University, Oxford, OH 45056
ph. (513) 529-6000, e-mail: hickeyrj@muohio.edu

ASSOCIATE EDITORS

GERALD J. GASTONY Dept. of Biology, Indiana University, Bloomington, IN 47405-6801

CHRISTOPHER H. HAUFLER .... Dept. of Botany, University of Kansas, Lawrence. KS 66045-2106

ROBBIN C. MORAN New York Botanical Garden, Bronx, NY 10458-5126

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2801 S. University Ave., Little Rock, AR 72204

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American Fern Journal 90(1]:1-15 (2000)

MISSOURI BOTANICAL

MM 8 2000

^A^DEN LIBRARY

Variation in Tree Fern Stipe Length with Canopy
Height; Tracking Preferred Habitat Through

Morphological Change

Nan Crystal Arens and Patricia Sanchez Baracaldo

Department of Integrative Biology, University of California, Berkeley, CA 94720-3140

Abstract. — Cyathea caracasana is a common open-habitat tree fern in the Andes of Colombia.
In full sun, stem growth rates are high (up to 2 cm/month) and individuals regularly produce
spores. However, even the fastest growing ferns are overtopped by woody angiosperms after 10 to
15 years of natural forest regeneration. As individuals are overtopped, C. caracasana produces
nearly vertical fronds with long stipes (commonly over 3 m] apparently to place the photosynthetic
surface into the canopy. We compared stipe length and blade length and width among individuals
growing in open sites and in the understories of two regenerating forests: one with a canopy of
20-25 m, and one with a canopy of 5-8 m. Stipes and blades were shortest in open habitat and
longest in the low-canopy forest. Ferns in the high-canopy forest had intermediate measurements.
Despite the change in frond length, the number of primary pinnae per-frond did not differ among
the habitats sampled. This suggests that elongation cues are received late in the development of
the frond. This conclusion is supported by a positive relationship between stipe length and the
distance of the fern meristem below the canopy. Because both understory populations show stipe
elongation relative to open-hapitat ferns, the cue to elongate is likely a low red/far-red wavelength
ratio of the light received by the apical meristem. Extraordinary elongation is probably made
possible by extra carbon resources available to low-canopy plants, which still have leaves in full
sun. This sense and response mechanism allows individual plants to produce elongated fronds as
their apical meristems are overtopped. Functionally, the long-stiped plants remain in full sun even
after they are overtopped, thus they "track" their preferred, open habitat.

Plant populations respond to environmental variation in several ways. When
the environment fluctuates infrequently relative to the life span of individuals,
adaptive segregation may produce differential response to environmental cues
among separate populations. Consider variation in light environment. Sun-
adapted populations of Impatiens capensis L. showed enhanced growth in
response to changes in the ratio of red (600-700 nm) to far-red (700-850 nm)
light relative to shade-adapted populations (Dudley and Schmitt, 1995). Sim-
ilar sun/shade segregation in growth response to red/far-red (R/FR) has heen
observed in many angiosperm species in open [as compared to understory)
communities (Morgan and Smith, 1979). In contrast, when the environment
varies frequently relative to the life span of individuals, adaptive segregation
cannot occur and individuals must rely on morphological and physiological
flexibility (plasticity) (Thompson, 1991; Sultan, 1993; Ackerly and Bazzaz,
1995; Arens, 1997). In such cases, plants may change form or physiology to
suit new conditions, for example switching from sun-leaf to shade-leaf anat-
omy (Arens, 1997). Alternatively, individuals *'track" or follow their preferred
habitat as it moves. Conventionally, habitat-tracking in plants has been applied
to intergenerationa! migration (Davis et al., 1986; Webb, 1987; Davis and Sa-

2

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

80

5°N

W

100 km

Reserva Natural
La Planada

Fig. 1.

America

Within

(Salzman

form of habitat-tracking may also be applied to differential organ growth in
non-clonal plants.

When growing in low-canopy secondary forest, some individuals of the tree
fem Cyathea caracasana (Klotzsch) Domin produce unusually long, vertically
oriented stipes that place the photosynthetic portion of the frond into the can-

full sun. This naoer documents oiu'

an example oi habitat-tracking bv means

wth

dividual plant organs.

Study Site and Methods

Planada

Andean

"N, 77°59'13"W (Fig. 1). La Planada receives about 4,500 mm

ARENS & SANCHEZ BARACALDO: VARIATION IN TREE

3

of rainfall annually (cumulative data from 1982 to 1997) with a '*dry" season
from June to August. Cloud-harvesting approximately doubles the moisture
available to plants (C. Rios, Instituto von Humboldt, Villa de Leyva, Colombia,
unpublished data). Average daily temperature is ig^'C, with no seasonal vari-
ation. Vegetation within the reserve is typical of Pacific slope cloud forest at
this elevation, with a natural canopy approximately 25 m in height composed
of relatively few tree species. The most common canopy trees include ^7-
chornea (Euphorbiaceae), Clusia (Guttiferae), Inga (Fabaceae], Miconia (Melas-
tomataceae), Myrica and Psidium (Myrtaceae), and Otoba (Myristicaceae).
Eleven described tree fern species have been reported from the reserve (Arens
and Sanchez Baracaldo, 1998]. The majority of the reserve's 3,200 hectares are
covered with primary forest that has been little-disturbed by human activity.
Within primary forest, canopy gaps are an important part of the habitat dy-
namic. Primary forest turnover due to gap formation and canopy closure was
estimated at 3% annually (Samper K., 1992). This suggests that at a given point
in the forest, a canopy gap will open every 30 years, on average — well within
the life span of most tree ferns. The reserve also contains areas of recently
abandoned pasture and secondary forest, which represent less than 50 years
of natural regeneration. Secondary forest was identified by relatively homo-
geneous size class of canopy trees; land-use records establish the time of aban-
donment. Regeneration of abandoned pasture is rapid, with canopies of 5 to 6
meters closing within 10 to 20 years after abandonment. Land use history in-
formation available at the reserve permits precise estimation of regeneration-
time. This makes La Planada a valuable site for studies of successional dynam-
ics in the cloud forest ecosystem.

Field work was initiated in 1992 and completed in 1994. We identified co-
occurring individuals of Cyathea caracasana whose apical meristems experi-
enced three distinct habitats: (1) open habitat, [2) the understory of 35-year-
old secondary forest, and (3) the understory of 10-year-old secondary forest.
Open habitat plants grew in recently abandoned pasture and cleared areas,
commonly within 1,000 m of reserve buildings. Abandoned pastures were in
the earliest stages of forest regeneration. Vegetation was characterized by grass-
es, herbaceous angiosperms, moss, lycophytes, ferns, and Miconia saplings. In
open habitat, tree ferns grew in full sun. Understory individuals grew in the
shade below the closed canopy of secondary forest [approximately 35 years of
regeneration); this sample is referred to as "high canopy" forest in subsequent
discussion. The canopy was approximately 25 m in height with no emergent
trees. Canopy gaps were uncommon and none were observed greater than 2 m
in diameter. The younger secondary forest represented approximately 10 years
of regeneration based on land-use history records, and had a multi-layered
canopy ranging from 5 m to 8 m (uncommonly up to 10 m) in height; this
sample is referred to as "low canopy" forest in subsequent discussion. Most
ferns in low-canopy forest placed their blades in the canopy; however, the
apical meristems of these individuals resided below canopy. For this study,
we chose only those individuals in the low-canopy forest that had placed their
photosynthetic surfaces in the canopy, because they maintain open-habitat

4

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

c

Fig. 2. Frond of Cyothea caracasana showing the position of measurements made in this study.
Length of stipe was measured from A-B; length of frond blade was measured along the rachis
from B-C; distance between first-order pinnae at three standardized positions (D) along the rachis.
Blade width was measured at the widest part of the blade E-F. Original drawing by Caroline A.E.
Stromberg.

Sanchez

as habitat-trackers. Co-occurring individuals (commonly with trunk heights

their blades in the canopy and hmctioned

and

measured

length, blade length, blade width, number of primary pinnae [Cyathea cara-
casana is twice-pinnate-pinnatifid), and distance between first-order pinnae at

standardized

range

sam

(p

0.001). Since trunk height is commonly used as a rough proxy for age in
erns (Conant, 1976; Seller. 1981: Bittner and Breckle. 1995: Sfiilpr iqqrI

this result suggests that each habitat sam

ARENS & SANCPIEZ BARACALDO: VARIATION IN TREE

5

Table 1. Pearson product-moment correlation coefficients (R) for log-transformed frond measurements.
Stem height, stipe length, blade length, and blade width at widest point measured in centimeters. Log
(distance) is the logarithmic transformation of average distance along the rachis between primary pinnae.
NS ^ not statistically significant; * significant at p < 0.01; ** significant at p < 0.001.

Log Log Log Log Log

(stem height) (stipe length) (blade length) (blade width) (distance)

Log (stem height) 1

Log (stipe length) -0.03 NS 1

Log (blade length) 0.34* 0.44** 1

Log (blade width) 0.29 NS 0.30 NS 0.19 NS 1

Log (distance) -0.02 NS 0.90** 0.53** 0.30 NS 1

mensurate range of age-size classes. Furthermore, there was no statistically-
significant correlation between trunk height and stipe length (R = -0.03, Table
1). Thus, differences in frond measurements among habitats were not due to
different age-size populations sampled, or age- or size-related allometric var-
iation.

Light environment experienced by individual ferns was estimated using
photo-paper (Azon non-erasable diazo sepia paper, Preprint) light sensors
placed at the apical meristem of each tree fern (Friend, 1961; Kitajima and
Augspurger, 1989). Paper sensors were calibrated for a 24 hour interval with
a LI-COR 1000 light meter (Lambda Instruments Corp., Lincoln, NE). Photo-
synthetically active radiation (PAR) was calculated for each paper photo sensor
increment using the regression derived from LI-COR calibration data. Although
this method is less precise than instantaneous light meter measurements, it
does give integrated measures of light environment that can be compared
among individuals. From the plant's perspective, integrated measurements are
more ecologically meaningful than instantaneous light meter readings, which
are subject to the vagaries of cloud cover and time of day. While this method
does provide useful information on light quantity, it is insensitive to light
quality, which is also ecologically important (Perez-Garcia et al., 1994; Ritchie,
1997; van Hinsberg and van Tienderen, 1997).

Manipulations and analyses were performed in SYSTAT 5.2 (Wilkinson,
1989) and MS Excel 5.0a for Macintosh.

Results

We compared frond measurements in pair-wise fashion between habitats
using a t-test assuming unequal variances. Mean stipe length differed among
all three habitats (Fig. 3A, p < 0.001). This result held (p < 0.001) when stipe
lengths were logarithmetically transformed to compensate for unequal vari-
ances. Open habitat ferns possessed the shortest stipes (66.4 cm ± 15.22 cm.
Appendix 1); ferns living in the high-canopy forest had intermediate stipe
lengths (102.9 cm ± 21.7 cm); ferns growing in low-canopy forest produced
significantly longer and more variable stipes (207.9 cm ± 49.1 cm). Blade
length differed between low- and high-canopy forest samples, and low-canopy

6

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

B

o

o

300

250 -

200 -

L^O -

100

50 -

Open

High canopy Low canopy

E

o

o

c

300

250

B

200

150

100

50

Open

High canopy Low canopy

B

o

5

o

^ ^^H^^^

^

200

150

100 -

50

Open

High canopy Low canopy

Q TJ

> PL,
<

25

20 -

15

10 -

5

Open

High canopy Low canopy

13

o

35

Z 30 1-
25 I-

C

o

I

o
E

20

15 -

? 10 -

5

Open

High canopy Low canopy

Fig. 3. Average frond measurements compared among habits. Data (Appendix 1] are averages of
20 individuals sampled per habitat; error bars are one standard deviation. A) Length of stipe from
trunk to the first primary pinna; B) length of the frond blade measured along the rachis from the
first primary pinna to the frond tip; C) width of the frond blade at its widest point; D) average (of
three measurements taken at standard positions] distance along the rachis between primary pinnae
on the same side of the frond; E) average number of first-order pinnae per frond.

ARENS & SANCHEZ BARACALDO: VARIATION IN TREE FERN STIPE LENGTH

7

^

Log (blade length) R = 0.45 p < 0.001

Log (average distance between first-order pinnae) R = 0.90 p < 0.001

2.5

2

1.5

1

0.5

1.6

1,8

2

2.2

2.4

Log (stipe length)

Fig. 4. Logarithmically-transformed data for stipe length, blade length, and the average distance
between first-order pinnae. Linear relationships between these parameters suggest an allometric
growth relationship between stipe length and both blade length and average distance between
first-order pinnae.

forest and open habitat (Fig. 3B, p < 0.001). However, there was no significant
difference in blade length between open habitat and high-canopy forest. Sim-
ilarly, blade width differed between low- and high-canopy forest, and low-

canopy forests and open habitat (Fig. 3C, p < 0.001); blade width did not differ

and

tance between first-order pinnae also differed among the three habitats (Fig.
3D, p < 0.001). However, the number of first-order pinnae per frond did not
differ statistically (p < 0.001) among any of the three habitats: open habitat

mean =
= 26.9

27.5

mean

27.7

mean

4.0 (Appendix 1).

Variation in stipe length might be allometrically related to size or age of the
plant. To evaluate this hypothesis, we performed a logarithmic transformation
on measurement data and calculated Pearson product-moment correlation co-
efficients fR) for all parameter combinations (Table 1). If an allometric rela-

tionship was present, such transformed data would show high correlations.
Non-significant correlations between trunk height and all parameters except
blade length (p < 0.01, Table 1) indicate that differences in frond size were
not due primarily to an allometric relationship with size-age class. Moderate
correlation between stipe length and blade length (Fig. 4), and between blade

8

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

B

'S

X

30

25

20

15

10

5

T

T

T

Canopy height (m)
Fern trunk height (m)

5

10

15

20
Age (yrs)

25

30

35

40

Fig. 5. Canopy height in regenerating secondary forest at La Planada and average stem height of
Cyatbea caracasana plotted through time. Average canopy heights were estimated based on plots
of regenerating forest of known age within the reserve. Tree fern stem heights based on average
growth rates of plants within open and understory habitats.

parameters

growth

blade length, and the distance betv/een first-order pinnae (Table 1, Fig. 4) show
a clear, allometric growth relationship between these parameters.

In contrast to stipe and blade lengths, the number of first-order pinnae per
frond does not vary with environment (Fig. 3E). The invariance of this param-
eter, compared with differences in stipe, blade, and distance between first-
order pinnae, suggests that variation in frond dimension arise by elongation
of support structures (stipe and rachis) during the elongation phase of fi-ond

expansion, rather than early in development when the number of pinnae are
set.

Discussion

among the

(Arens

cies was also very common along roadsides and other open habitats. Within
mature forest, C. caracasana produced spores only when growing in forest

many years, hi full

an

ARENS & SANCHEZ BARACALDO: VARIATION IN TREE FERN STIPE LENGTH

9

6

C/D

200

400

600

800

1000

Distance of meristem below canopy (cm)

Fig. 6. Untransformed measurements of stipe length plotted against the distance of the plant's
meristem below the canopy. The linearity of this relationship suggests a direct gradient response
of stipe length to the light gradient below the low canopy.

Sanchez Baracaldo, 1998) and reached a height of up to two meters before
woody species formed a canopy. Individuals of C. caracasana in sunny habitat
produced abundant spores and recruited new sporoph3^es. In contrast, C. car-
acasana grew slowly in the understory of the closed canopy forest and seldom
produced spores (Arens, 1996; Arens and Sanchez Baracaldo, 1998). These
data suggest that C. caracasana prefers open, sunny environments such as
human-disturbed landscapes or forest gaps. Since mature forest canopy turn-
over is high in this montane forest, long-lived ferns like C. caracasana might
be expected to show habitat-tracking or plastic responses to such changes in

light environment.

Growth rate data for forest ferns showed that individuals placing their pho-
tosynthetic blades in the canopy by means of long, vertical stipes have statis-
tically indistinguishable growth rates compared to those in open habitats (Ar-
ens and Sanchez Baracaldo, 1998). Similarly, ferns in a low-canopy forest that
do not produce stipes sufficiently long to reach the canopy had low growth
rates, similar to individuals growing below the closed canopy (Arens and San-
chez Baracaldo, 1998). These observations support the conclusion that stipe
elongation in Cyathea caracasana allows individual plants to continue growth
and spore production as open-habitat plants — to track their preferred habitat
by means of a morphological change.

10

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

a

o

N

O

6

o

c

00

1000

Distance of meristem below canopy (cm)

Fig. 7. Untransformed measurements of stipe angle plotted against the distance of the plant's
meristem below the canopy. The Unearity of this relationship suggests a direct gradient response
of stipe length to the light gradient below the low canopy.

For this strategy to work, individual plants must sense and respond to their
environment in appropriate ways. How does Cyathea caracasana detect a low
canopy into which it might extend its frond? '
observed only in the low-canopy forest?

In angiosperms, changes in light quality (R/FR) can induce internode and
petiole elongation. Low R/FR induced significant internode elongation in Gly-
cine max (LJ Merr. (Thomas and Raper, 1985), Phaseolus vulgaris L. (Beall et

Why is extreme

Meerb

(Morgan and Smith, 1979), Sinapis alba L., and Datura fi

stim

ulated by low R/FR (van Hinsberg and van Tienderen, 1997). Low R/FR pro-
duced petiole elongation in Citrullus lanatus (Thunb.) Matsum. & Nakai
(Graham and Decoteau, 1997), and Tblaspi arvense L. (Metzger, 1988). Com-
pared with plants grown in high R/FR light, elongated structures result pri-
marily from lengthening individual cells with a minor component of addition-
al cell division in support structures (Beall et al.. 1996). These data are con-
sistent with our observation that support structure (stipe and rachis) length in
Cyathea caracasana increases to produce fronds that can reach the canopy of
young secondary forest; however, the architecture of the frond (number of first-
order piimae) does not chai

ARENS & SANCHEZ BARACALDO: VARIATION IN TREE FERN STIPE LENGTH 11

Light in the forest understory generally has low R/FR (Chazdon and Fetcher,
1984; Lee, 1987; Lee, 1989; Endler, 1991; Turnbull and Yates, 1993). Because
R/FR reliably indicates overtopping, plants have evolved phytochrome-medi-
ated sensory systems that trigger morphological or physiological responses,
such as petiole elongation. Although no research has systematically evaluated
a R/FR trigger for petiole elongation in non-angiosperms, ferns do possess phy-
tochrome systems similar to those of angiosperms and these systems function
as light-environment sensors (Haupt, 1985; Sugai and Furuya, 1990). Light
quality signals for spore germination have been explored in some detail
(Haupt, 1985; Sugai and Furuya, 1985; Psaras and Haupt, 1989; Sugai and
Furuya, 1990; Esteves and Felippe, 1991; Perez-Garcia et al., 1994). It seems
reasonable, therefore, that low R/FR experienced by meristems in the under-
story cues stipe and blade elongation in Cyathea caracasana. This is consistent
with our data that show both stipe and blade are elongated in all forest plants,
relative to open habitat individuals.

As they differentiate from the apical meristem, Cyathea caracasana crosiers
may use a phytochrome mechanism to detect that they reside in the low R/FR
environment of the understory. This cue triggers the stipe and blade elongation
observed in all understory plants. In low canopy ferns, having fronds already
in the full sun of the canopy provides carbon and energy resources needed for
dramatic stipe elongation. In contrast, ferns under the high canopy may simply
lack sufficient photosynthetic resources to produce extremely elongated stipes.
This conclusion is supported by the lower average trunk growth rates (0.35
cm/month) observed in understory ferns at La Planada (Arens, 1996). Figure
5 shows that overtopping of tree ferns begins at about 10 years of forest regen-
eration, as the growth rate of angiosperm trees exceeds that of the ferns. It is
in the 10-year-old forest that we observe dramatically elongated stipes as ferns
attempt to forestall overtopping of their photosynthetic surfaces.

To explore the effect of meristem position in the multi-layered canopy, we
selected a second sample of 20 individuals in the low-canopy forest and re-
corded the distance of each apical meristem below the canopy. Position below
the canopy was positively correlated with both stipe length (R = 0.58, p =
0.007, Fig. 6) and with the angle of the stipe measured from the horizontal (R
= 0.61, p ^ 0.004, Fig. 7). These data show that meristems well below the
canopy produce fronds with longer and more erect stipes, capable of projecting
the frond's photosynthetic surface into the sunny canopy. This supports the
conclusion that stipe elongation in Cyathea caracasana is triggered by low R/
FR conditions present at the meristem and terminated when the frond reaches
light of high R/FR in the canopy.

From these results, we conclude that at La Planada, Cyathea caracasana
responds to overtopping of its apical meristem by producing elongated, erect
stipes that place blades into the full sun of the canopy. In this way, the plant
fine-tunes its morphology to specific conditions in its environment. This al-
lows the plant to continue growth and maintain spore production rates similar
to those of individuals growing alone in open habitat, even in the early stages
of overtopping by fast-growing angiosperm trees. These morphological chang-

12

AMERICAN

es, likely stimulated by low R/FR, allow the plant to continue growth and
reproduction even as forest regeneration relegates it to the understory.

Acknowledgments

We thant La Reserva Natural La Planada and La Fundacidn para la Educacion Superior [FESJ,
Call, Colombia for permitting our research within the reserve. D. Ackerly assisted in the field.
This manuscript was improved as the result of comments from D.C. Kendrick, Y.-J. Liu, C.A.E.
Stromberg, and A. Thompson. D. Ackerly gave this manuscript thoughtful review and offered
creative ideas on the mechanisms underlying stipe elongation. We are grateful to an anonymous
reviewer for detailed and constructive comments.

Literature Cited

Ackerly, D.D,. and F.A. Bazzaz. 1995. Seedling crown orientation and interception of diffuse
radiation in tropical forest gaps. Ecology 76:1134-1146.

Arens. N.C. 1996. Demography of the tree fern Cyothea caracasana across the successional mosaic
of an Andean cloud forest. Amer. J. Bot. 83:123 [Abstract].

Arens, N.C. 1997. Responses of leaf anatomy to light environment in the tree fern Cyatbea cara-
casana (Cyatheaceae) and its application to some ancient seed ferns. Palaios 12:84-94.

Arens, N.C. and P. Sanchez Baracaldo. 1998. Distribution of tree ferns (CyatheaceaeJ across the
successional mosaic in an Andean cloud forest, Nariiio, Colombia. Amer. Fern. J. 88:60-71.

Ballare, C.L., A.L. Scopel, and R.A. Sanchez. 1991. Photocontrol of stem elongation in plant
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ARENS & sAnCHEZ BARACALDO

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14

AMERICAN

APPENDIX 1. Frond measurement data analyzed in this study. Open habitat individuals are indicated
by *'open''; individuals in the understory of high-canopy forest are indicated by "high**; young secondary
forest individuals that place iheir fronds in the low canopy are indicated by **low*\

Indi-
vidual

Open 1
Open 2
Open 3
Open 4
Open 5
Open 6
Open 7
Open 8
Open 9
Open 10
Open 11
Open 12
Open 13
Open 14
Open 15
Open 16
Open 17
Open 18
Open 19
Open 20
A\erage
High 1
High 2
High 3
High 4
High 5
High 6
High 7
High 8

Trunk
height (cm)

59
57
68
76
73
70
54
51
99
55
46
42
71
121

86
107

78
102

57

85
72.7 ± 21,7

90

91
150

21
107
103

33

29

Stipe length
(cm)

73
90
75
72
49
68
67
65
44

77
73
86
86
83
74
57
52

51
43
43

66.4

15,2

High 9

88

High 10

77

High 11

23

High 12

63

High 13

57

High 14

65

High 15

82

High 16

77

High 17

59

High 18

34

High 19

55

High 20

70

Average

68.7 ^ 32.3

126
131

89
102
101

90.5

83

78

101.5
130

76
120
140
104
113

83-5
124
120

66

80

Frond
leneth (cm)

Distance
between
Frond width pinnae (cm) Number of

(cm)

(average)

pinnae

176
120
173
177
171
155
153
151
200
88
131
129
175
150
148
183
181
174
145
152

135
125

137
154
121
111
111

99
103
109

91

no

126
136
107

133

127.5

113

103

121

12.7
13.0
10.7
13.7
13.7
12.0
13.7

9.7
10.0
12.7
10.0
10.3
11.3
15.0
11.0
10.0
10.3
10.3
10.0

8.0

204

226

179

122

224

190

126

163.5

195.5

193

153

205
257
58
175
180
174

175
170
165

141
143
123

99
128
135
109

11.5
165
147

97
131
137
119

135
11

105

79

81
110

14.7
17.0
13.3
12.7
14.7
15.3
12.3
12.3
14.0
15.0
11.7
17.3
21.0
12.0
17.3
12.7
12.3
18.7
10.0
11.0

30
26

32

28
30
22
24
27
32
26
22
21
30
30
22
28
32
22
33
32

156.6 ± 26.0 118.6 ± 15.6 11.4 ± 1.8 27.5 ± 4.1

33
32
30
23
34
30
21
24
28
28
28
26
34
29
25
31
28
22
20
28

102.9 ± 21.7 176.8 ± 42.3 110.3 ± 40.4 14.3 ± 2.8 27.7 ± 4.2

ARENS & SANCHEZ BARACALDO: VARIATION IN TREE FERN

15

APPENDIX I. Continued

Indi-
vidual

Low 1
Low 2
Low 3
Low 4
Low 5
Low 6
Low 7
Low 8
Low 9
Low 10
Low II
Low 12
Low 13
Low 14
Low 15
Low 16
Low 17
Low 18
Low 19
Low 20
Average

Trunk
height (cmj

Stipe length
(cm)

Frond

length (cm)

Distance
between
Frond width pinnae fcm) Number of

(cm)

(average)

pmnae

44
37
77
96

110
39

101

118
60
44

148
68
76
42
46
69
73
43
45
61

200

229

181

314

226

180

201

151

181

180.5

168

249

290

163

207

303

204

220

172

138

177
223
161
228
282
182
279
266
222
173
222
204
176
181
190
205
248
240
157
220
69.9 ± 30.8 207.9 ±49.1 211.8 ± 37.8 144.4 ± 19.2 21.0 ± 2.9 26.9 ± 4.0

132
141
169
149
115
139
171

189
119
139

161
147
167
129
129
149
141
147
125
129

20.7
23.0
22.0
21.0
22.3
20.7
16.7
19.0
20.3
17.0
17.7
21.3
23.0
18.0
24.3
22.7
26.3
26.7
17.0
20.7

26
28
30
28
32
24
32
34
26
20
30
24
30
22
24
28
26
26
19
28

American Fern Journal 90(l):16-23 (2000)

Cryopreservation of In Vitro Grown

Fern Gametophytes

Valerie C. Pence

Center For Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden,

3400 Vine Street, Cincinnati, Ohio 45220

Abstract.— Two methods of protecting fern gametophyte tissues through exposure to liquid ni-
trogen (LN) were examined. In vitro grown gametophytic tissues from six fern species were ex-
posed to LN after open drying or after encapsulation dehydration, with and without preculture
on ahscisic acid (ABAJ. Open drying itself decreased survival with little further effect from LN
exposure, although survival was somewhat improved by preculture on ABA. In contrast, encap-
sulated tissues survived drying and LN exposure at rates comparable to controls (86-100%) irre-
spective of ABA preculture. Sucrose pretreatment of the encapsulated tissues w^as important for
their subsequent survival through these procedures. Tissues prepared by encapsulation dehydra-
tion were successfully regrown after 3.5 years in LN storage. Thus, cryopreservation appears to
be a technique which could be used for the stable preservation of in vitro cultures of fern game-
tophytes and for the long-term storage of rare or endangered germplasm of ferns.

and

focus of study, the small, fragile thallus of the fern gametophyte has also been

tophytes are easily grown in culture, m

Mill

physiological and developmental studies, but maintaining stock lines or lines
from a number of species requires a consistent input of time and labor.
Cryopreservation, or storage in liquid nitrogen (LN) at -196°C, has been used

to preserve a variety ot hvmg tissues of both vascular and nonvascular plants.
Seeds, shoot tips, cell cultures, callus, protoplasts, pollen, and embryos of seed
plants (Kartha and Engelmann, 1994; Stanwood, 1985; Pence, 1991), as well as
fern spores and the gametophytes of bryophytes (Christians on, 1998; Pence,
1998; and Pence, submitted) have all been successfully maintained in LN.

In this study, the possibility of using cryopreservation to preserve fern ga-
metophytic tissue was explored. Two protocols, which have been used suc-
cessfully with a variety of other tissues, were tested for preparing gametophyte
tissue for cryopreservation: open drying of the tissues before LN exposure and
the encapsulation dehydration procedure of Fabre and Dereuddre (1990).

Materials and Methods

Drynaria quercifolia (L.) John
Adiantum trapeziforme L., Ao

Davallia fejeensis

Krohn Conservatory

Mr

wrapped in small packages made

PENCE: CRYOPRESERVATION OF IN VITRO GROWN FERN GAMETOPHYTES 17

folding pieces of Whatman No. 1 filter paper. The spores and packages were
immersed in a 1:20 dilution of commercial sodium hypochlorite for 5 min,
followed by two rinses in sterile distilled water. The packages were then
opened, and the spores were blotted onto sterile germination medium, con-
sisting of half-strength Linsmaier and Skoog (LS) (1965] salts and organics,
with 1.5% sucrose and 0.22% Phytagel (Sigma Chemical Co.), in 60 X 15 mm
disposable plastic petri dishes, approximately 15 ml/dish. The spores were
incubated at 26*'C under CoolWhite fluorescent lights in a 16/8 hr light/dark
cycle. Once germination occurred and gametophytes were formed, the cultures
were maintained by subculturing the tissue every 2-3 months onto fresh me-
dium. In some experiments, gametophytes were precultured for one week on
this same medium, with and without 10 |xM abscisic acid (ABA), which was
added to the medium after autoclaving.

For open drying, tissues were cut into pieces, approximately 2-5 mm long,
blotted onto sterile filter paper to remove excess moisture and placed in a
sterile petri dish under the air flow of the laminar flow hood for 3 hrs.

For encapsulation dehydration, the method of Fabre and Dereuddre (1990]
was followed. Tissues were cut into small pieces, approximately 2-3 mm long,
and transferred to a solution of 3% alginic acid in calcium-free MS medium
plus 0.75 M sucrose. This solution, containing one or more pieces of game-
tophyte tissue, was then pipeted dropwise into a solution of 100 mM CaCl2,
which caused the alginic acid to gel, encasing the tissue in an alginate bead.
After 20 min, the beads were removed fi:'om the calcium solution and trans-
ferred to liquid MS medium containing 0.75 M sucrose, 25 ml in 125 ml flasks,
and placed onto a gyratory shaker, 125 rpm, for 18 hr as a pretreatment. In
one experiment, different concentrations of sucrose, ranging from 0-30% were
tested in the pretreatment step. The pretreated beads were then blotted on
sterile filter paper to remove excess moisture and placed on dry filter paper in
sterile petri dishes under the air flow of the laminar flow hood to dry for 3-4

hours.

Open dried tissues and dry encapsulated tissues were then placed into ster-
ile 2 ml polypropylene cryovials and immersed directly into LN where they
were left either for 1 hr or overnight (no difference was observed between these
two LN exposure times). Tissues were thawed by placing the cryovials on the
benchtop at ambient temperature for 20 min, after which the tissues or beads
were removed and placed onto growth medium for rehydration and recovery.
Survival was measured as the recovery of growth from each tissue piece for
open dried tissue or the number of beads containing tissues resuming growth.
As controls, some tissues were transferred to recovery medium after drying
but without LN exposure. With the encapsulation dehydration procedure, tis-
sues which had been pretreated for 18 hours in .75 M sucrose but which had
not been dried were cultured as an additional control.

Fern gametophyte tissue from each species was also prepared by the encap-
sulation dehydration method for long-term cryostorage and banked in LN. Af-
ter 3.5 years, samples of each were removed from storage and placed onto
medium for rehydration and recovery growth.

18

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

100

0)

80

I ^^^A

o

60

40

^

20

1

2

3

4

Drying Time (hrs)

Fig. 1. Moisture loss from open dried gametophytic tissue of D. fejeensis during a 4 hour drying
period (n = 3).

Moisture determinations were made on tissues of D. fejeense

the

groups of small tissue pieces weighing between 0.15 and 0.25 g before drying

moisture

the weights of the tissues before and after drying overnight in an oven at 95°C.
Samples of tissues encapsulated in alginate beads were also analyzed for mois-
ture after drying in the laminar flow hood.

Results

fejeense

reduced to approximately 10% within 3 hours (Figure 1). The gametophytes
of the other five species appeared to dry similarly from visual and tactile ex-
amination. When tissues and their surrounding alginate beads were dried,
moisture levels were somewhat higher, decreasing to only 19-27% during the

3-4

tophyt

furth

trapezifi

glaucum were particularly sensitive to drying, while the other species showed

some

gametophytes through both drying and LN exposure. However, in only one
species, A. tenerum, was survival of ABA treated tissues equivalent to that of
the undried controls.

In contrast, there was excellent survival when gametophytes were encap-
sulated, pretreated with sucrose and dehydrated in alginate beads prior to LN
exposure (Table 2). Tissues showed 100% survival through the 18 hr sucrose
pretreatment, with no decrease in viability when the encapsulated tissues were

PENCE: CRYOPRESERVATION OF IN VITRO GROWN FERN GAMETOPHYTES

19

Table 1. Percentage survival and growth of gametophyle tissue pieces of six fern species through 3
hours of open drying followed by LN exposure, with and without preculture on medium containing 10
|xM ABA- (n = 8 for controls; n = 10-32 for dried and LN exposed).

Species

Preculture
on ABA

% Sur\i\al^

Control

Dried

LN exposed

C glaucum

+

A. tenenim

+

D. quercifoUa

+

D. fejeensis

+

-i-

P. aureum

■f

+

.4. trapezifonne

+

+

100

100

100

100

100

100

100

100

nd

nd

100

100

nd

nd

100

100

nd

nd

45

20

100

23

88

58

92

18

35

5

47

6

67

33

10
88
60
100
62
71
50
83
30
69

33

16
69

39

^ nd = not determined

dried. In a few cases, there was a slight decline in survival after LN exposure,
but this effect was small (<15%). Because of the high survival rate without
ABA, there was no apparent effect of the ABA preculture on survival when
encapsulation was used.

Although survival of encapsulated material was good, some damage of the
tissues was still evident. Whereas pretreated controls remained consistently
green when placed on recovery medium, tissues which were dried or dried
and exposed to LN often had some areas which were brownish green in color.
Survival came from areas which remained bright green and which eventually
grew out and reestablished the culture.

When encapsulated tissues of D. fejeense were exposed to different concen-
trations of sucrose during the 18 hr pretreatment, there was little or no survival
through drying and LN exposure when sucrose was omitted completely from
the pretreatment medium (Figure 2). However, good survival was observed at
all but the highest sucrose concentrations.

Samples of encapsulated gametophyte tissues from these six species showed
good survival after 3.5 years in LN storage (Table 3; Figure 3). Survival rates
ranged from 50-100%, depending on the species.

Discussion

These results indicate that the encapsulation dehydration procedure can be
ted successfully to cryopreserve gametophytes of at least six fern species and

20 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Table 2. Percentage survi\ al and growth of tissue pieces of six fern gametophytes through encapsulation

pretreatment, drying and LN exposure, (n = 5-17). Survival through encapsulation without pretreatment
was I007o.

% Survival^

Species Preculture on ABA Sucrose pretrmt Dried LN exposed

C glaiicum

A, Xenerum

D. quercifolia

D. fejccnsis

P. aiireum

A. trapezlfonne

100 100 100

+ 100 100 100

100 100 94

100 100 100

+ 100 100 93

100 KX) 100

- . 100 100 100

+ 100 100 100

100 100 100

+ 100 100 100

100 100 100

100 100 100

+ 100 100 100

100 100 86

+ nd 100 100

80 82 82

+ 100 100 100

100 100 100

+ nd 100 100

100 100 86

+ 100 100 100

' nd = not determined.

suggest that this technique might be broadly applicable to the gametophytes

ameto

phytic tissues through LN exposure was generally equivalent to that of con-

trols.

The ability of fem gametophytes to survive drying without encapsulation

sur

moist

implicated in increasing stress tolerance in a number

gametophyt

impro

with

phytes, the effects of ABA on improving tolerance to open drying were also
variable, depending on the species [Pence, 1998). However, with these clonal
cultures, even a low percentage of survival will regenerate the culture, and
open drying with ABA preculture could provide a straightforward method for
freezing gametophytes of a number of species.

Survival through the encapsulation dehydration procedure, however, pro-

the fern same

an important

fejeense through

GAMETOPHYTES

21

CO

100

re

(D

00

■M

y_

$

o

o

a;

o

O)

TO

o:

c

c

rce

o

<D

^

CL

CO

Pretr
Dried
LN Exposed

5

10 15 20 25 30

Sucrose Concentration (%)

Fig. 2. Growth after drying and LN exposure of gametophytic tissue of D. fejeensis pretreated in
several concentrations of sucrose using the encapsulation dehydration method (n = 8-10).

procedure. Research in this laboratory with byrophytes has shown that a su-
crose pretreatment can improve the survival of open dried tissues (Geiger et
al., unpublished). In addition, disaccharides and oligosaccharides have been
implicated in the natural desiccation tolerance of several types of plant tissues,

Fig. 3. Gametophyte tissue of C. glaucum resuming growth after 3.5 years of storage in LN,
prepared for storage with the encapsulation dehydration method. 3.5X

22 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Table 3. Regrowth from fern gametophyte pieces which were prepared by the encapsulation dehydration
method and stored in LN for 3.5 years.

Number of Percent of tissue

Species pieces pieces growing

C. glaucum
A. tenerum

D. quercifolia
D. fejeensis
P. aiireum

20 75

8 88

12 67

10 100

10 90

A. trapezifonne 6

50

including seeds (Koster and Leopold, 1988], bryophytes (Smirnoff, 1992), and
pteridoph5^es (Adams et al., 1990).

Encapsulation appears to be a technique that should be useful for long-term
germplasm storage of fern gametophytes. Tissues resumed growth and ap-
peared normal after 3.5 years of storage in LN, and it is likely that much longer
storage times will be achieved. Less than 100% survival was observed with
these tissues, in contrast to the experiments done with short-term LN exposure.
While the possibility that the longer storage time was detrimental cannot be
discounted, it is also possible that other factors, such as the state of the cul-
tures (time since last subculture, etc.), may account for this difference. More
tissues will be removed from storage over the next few years in order to de-
termine whether viability does decline with time in storage.

Although survival was less than 100%, each culture was easily regenerated

useful

tophyt

Gametophyt

maintain
occur in

known onlv from the game

some

more

Sheffield

ame

which may or may not be easily grown into sporophytes (Dyer and Lindsay,
1992; Dyer, 1994). In such situations, cryopreservation could be used for main-
taining in vitro cultures of such gametophytes for future study.
Nonseed plants have not received as much attention as seed plants with

are

tions that preserving the biodiversity of these organisms could be potentially

Cryopreservation

useful

the

genetic diversity, providing a stable resource of valuable genetic lines as well
as an ex situ back-up for species which are rare or endangered in the wild.

CRYOPRESERVATION

23

Literature Cited

Adams, R. R, Kendall, E. and K. K. Kartha. 1990. Comparison of free sugars in growing and
desiccated plants of Selaginella lepidopbylla. Biochem. Systemat. EcoL 18:107-110.

Christianson, M.L. 1998. A simple protocol for cryopreservation of moss. The Bryol. 101:32-35.

Dyer, A. F. 1979. The culture of fern gametophytes for experimental investigation. Pp. 253-305
in A.F. Dyer. The experimental biology of ferns. Academic Press, New York.

Dyer, A. F. 1994. Natural soil spore hanks-can they be used to retrieve lost ferns? Biodivers.
Conserv. 3:160-175.

Dyer, A. F. and S. Lindsay. 1992. Soil spore banks of temperate ferns. Amer. Fern. J. 82:89-122.
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Hartung, W. and W. J. Davies. 1991. Drought-induced changes in physiology and ABA. Pp. 63-

79, in W. J. Davies and H. G. Jones. Abscisic acid. Bios Sci. Pub., Oxford.
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Koster, K. L. and A. C. Leopold. 1988. Sugars and desiccation tolerance of seeds. Plant Physiol.
88:829-832.

Linsmaier, E. M. and F. Skoog. 1965. Organic growth factor requirements of tobacco tissue cul-
tures. Physiol. Plant. 18: 100-127.

Miller, J. H. 1968. Fern gametophytes as experimental material. Bot. Rev. 34:361-367.

Penge, V. C. 1991. Cryopreservation of seeds of Ohio native plants and related species. Seed Sci.
Technol. 19:235-251.

Penge, V. C. 1998. Cryopreservation of bryophytes: The effects of ABA and encapsulation dehy-
dration. The Bryol. 101:278-281.

Raine, C. a. and E. Sheffield. 1997. Establishment and maintenance of aseptic culture of Tricho-

manes speciosum gametoph3rtes from gemmae. Amer. Fern J. 87:87-92.
Smirnoff, N. 1992. The carbohydrates of bryophytes in relation to desiccation tolerance. J. Bryol.

17:185-191.
Soeder, R. W, 1985. Fern constituents: Including occurrence, chemotaxonomy and physiological

activity. Bot. Rev. 51:442-536.
Stanwood, p. C. 1985. Cryopreservation of seed germplasm for genetic conservation. Pp. 199-226.

in K. K. Kartha. Cryopreservation of plant cells and organs. CRC Press, Inc., Boca Raton,

Florida.
Stokey, a. G. 1940. Spore germination and vegetative stages of the gametophytes of Hynieno-

pbyllum and Trichomanes. Bot. Gaz. 101:759-790.

American Fern Journal 90(l):24-31 [2000]

Vessels in Roots and Rhizomes of
Dryopteris crassirhizoma (Dryopteridaceae)

from Heilongjiang Province, China

RuijUN U\ XiUFENG Yan^ and Dawei Zhang^

^Department of Biology, Harbin Normal University, Harbin 150080, China
Open Research Laboratory of Forest Plant Ecology, Northeast Forestry University,

Harbin 150040, China

Abstract.— In the present study, tracheary elements in roots and rhizomes of Dryopteris crassi-
rhizoma were observed with scanning electron microscopy (SEM). SEM observation revealed that
all tracheary elements in both organs were vessels. These vessel elements have end-wall perfo-
ration plates and lateral-wall perforation plates. End-wall perforation plates in roots are more
specialized than in rhizomes; they are all scalariform and obliquely positioned in end walls. Most
end-wall perforations, especially in the center portion of end-wall perforation plates, lack pit
membrane remnants, but pit membrane remnants are relatively abundant in some end-wall per-
forations of roots and rhizomes. It is noteworthy that several larger perforations on lateral walls
usually are grouped together and form local lateral-wall perforation plates. Wide perforations
alternating with narrow perforations characterize vessels of roots and rhizomes. In addition, the
majority of perforations in lateral-walls have porose pit membranes or pit membrane remnants,
range from intact pit membrane to nearly devoid of pit membrane remnants. Some vessels in
rhizomes have several facets in which long scalariform pits have various degrees of porose mem-
branes. These vessels contact several other tracheary elements for transferring materials.

In his classic study on fern tracheary elements, White (1961,1963) not only
reported that vessels occur in Pteridum and Marsilea, but also used such terms
''presumptive vessel" or "tracheary elements with end plates" to describe ves-

Woodsia

Dry opt

parison with the lateral walls; scalariform pits on the end walls are wider than

White

(1963) was unable to determine the degree of pit membranes on end plates of
those vessel-like tracheary elements (i.e. whether entire pit membranes on end
plate were present or absent). The nature and presence of vessels are not re-

with

means of SEM

scribed by White (1963) in Astrolepis and Woodsia are vessels. SEM obser-
vations on those ferns showed that so-called pits on end walls are actually
perforations with various degrees of pit membrane remnants from minutely to
clearly absent, and revealed that there are lateral-wall perforations. It was not-
ed that those genera, which grow in seasonally dry and cold places, have
varying differentiated vessel elements in their roots and rhizomes in these
authors' studies (Carlquist and Schneider 1997a,b, 1998a,b, 1999; Carlquist.
Schneider and Yatskievych, 1997; Schneider and Carlquist 1997, 1998a,b, c,
1999). These studies seem to further sunnort the hvnnthpsi« that flnrtnatinn

RHIZOMES

25

in water availability appears basic to evolution of vessels in vascular plants
(Carlquist 1975). Ferns distributed in Heilongjiang province can be formally
divided into four different ecological groups according to plant-water relation-
ships as follows (Ao and Li, 1987):

1. Xerophytes: Selaginella sibihca, S.tamariscina, Aleuritopteris argen-
tea, Dryopteris fragrans, Lepisorus ussriensis and Pyrrosia petiolosa etc.

2. Mesophytes: Adiantum pedutum, Athyrium multidentatum,
A.yokoscense, Dryopteris crassirhizoma, Equisetum pratense and
E.silvaticum etc.

3. Helophytes: Matteuccia struthipteris, Osmunda cinnamomeo
vai.asiatica, Onocleo sensibilis and Thelypteris polustris etc.

4. Hydrophyte: Equisetum fluviatilo, Marsilea quadrifolia, Azolla filico-
loides and Salvinia natans etc.

We have observed features of vessel elements in roots and rhizomes from Mat-
teuccia struthipteris and Osmunda cinnamomea vai.asiatica and found that
even if they belong to the same ecological grouping, their vessel elements show
different patterns of specializtion, i.e. primitive O. cinnamomea var. asiatica
had little differentiation between perforation plate on the end wall and lateral
wall pitting whilst M. struthipteris was markedly differentiated (Li et al., 1999).
Thus we choose those different taxa, which are in different systematic posi-
tions, in the same ecological group as research materials to observe the micro-
structures of trachery elements by means of SEM for understanding evolution-
ary trends of treachery elements of ferns in similar and/or the same environ-
ment.

Dryopteris Adans. (Dryopteridaceae) has about 400 species distributed main-
ly in the North Temperate Zone (Wu & Ching, 1991). Russow (1873) considered
that roots of Nephrodium (= Dryopteris) had true vessels because of the com-
bination of lateral pitting which was most often alternate or opposite with
short scalariformly pitted overlapping areas between the tracheary elements.
Schneider and Carlquist (1997) have documented presence of vessels in Po-
lystichum of the same family by means of SEM.

Dryopteris crassirliizoma Nakai is common in Heilongjiang province, which
belongs to the second ecological species group but it also intrudes into other
habitats including dry places. We attempt to demonstrate with SEM whether
tracheary elements in root and rhizomes of this species are vessels or tracheids
and make a comparison of microstructures of trachery elements to these of
Adiantum pedatum in the same ecological group and Polysticlium acrosti-
cboides in the same family.

Materials and Methods

Fresh roots and rhizomes of Dryopteris crassirhizoma were collected from
3-5 wild plants of the Maoershan population, Shangzhi County, Heilongjiang
province, and fixed in 3:1 absolute ethanol-glacial acid for 2-24 hours. Fixed
materials were transferred into 70% aqueous alcohol and stored. We used mac-

26

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

t

Figs. 1-9. SEM photographs from tracheary elements in root oi Dryopteris crossirhizoma. 1. Lat-
eral view of relative short end wall. 2. Face view of elongate end wall, scale bar - 50|xm. 3. The
lower part of the end wall, to show wider scalariform perforations in end wall that contrast with

LI ET AL.: VESSELS IN ROOTS AND RHIZOMES OF DRYOPTERIS CRASSIRHIZOMA 27

erated and sectioned materials for SEM observation. We use two maceration
methods [Flanklin's method: solution consisted of glacial acetic acid and 3%
hydrogen peroxygen (1:1) and Jeffrey*s method used by Carlquist et al. 1997).
Roots and rhizomes were macerated in Franklin solution at 60°C for 4-5 days,
while in Jeffery solution at 23°C for 2-3 days. These macerated materials were
transferred into 70% alcohol, then spread onto the surface of SEM aluminum
stubs. After drying, they were sputtercoated and examined with a Hitachi S-
520 SEM. The sectioned materials were made as follows: the materials fixed
in FAA were cut longitudinally, and dehydrated by an ethanol series. Then
the ethanol was removed by isoamylacetate. The materials were dried in a
critical point dryer with carbon dioxide and coated with gold.

Results

In Dryopteris crassirhizoma roots, all end walls of tracheary elements ex-
amined bear perforations. These perforation plates range widely in morphol-
ogy. Some perforation plates are relatively short with numerous bars [Fig.l),
whereas other perforation plates are very long with numerous bars (Fig. 2).
Perforation plates are markedly differentiated from lateral walls; the perfora-
tions on end walls are larger and more elongate than lateral-wall pits or per-
forations arranged in alternate or opposite pattern (Figs. 3—5). In figure 5, the
end wall is at the left, but some lateral-wall perforations are similar to the end-
wall perforations in absence of pit membrane remnants. In some plates, pit
membrane remnants are very few (Fig. 6), but sometimes relatively abundant
(Fig. 7). In the end wall shown in figure 8, monomorphic perforations mostly
lack pit membranes. Some end walls show dimorphism between wide and
narrow perforations in figure 6. In addition to perforation plates on end walls,
lateral-wall perforations are also found (Figs. 5,8,9). It is interesting that lateral-
wall perforations range from very small to large; several larger perforations
(usually two to four perforations) usually group together (Fig. 9).

Most tracheary elements in rhizomes possess end walls with perforations,
which are less specialized than those in roots (Fig. 10). Some wider perfora-
tions on end walls clearly lack pit membranes in this figure and those narrow
perforations that alternate with wide perforations in the same perforation plate
usually have various degrees of pit membrane remnants (i.e. from intact pit

the alternate or opposite pits or narrower perforations with pit membrane remnants in lateral wall.
4. The lower part of end walL to show those perforations with absence of pit membrane. 5. The
lower part of end wall, to show those perforations with absence of pit membranes and lateral-wall
perforations in the right of end-wall perforation plate. 6. End wall, to show perforations with few
pit membrane remnants and dimorphism in wideness of perforations; grain of unknown materials
lodged on perforation plate. 7. End wall, to show perforations with many pit membrane remnants.
8. Central portion of an end-wall perforation plate, to show monomorphic perforations that com-
pletely lack pit membrane. 9. Lateral-wall perforation plate, to show larger perforations grouping
in lateral wall. Scale bars = 30^.m.

28

VOLUME

Figs. 10-13. SEM photographs from tracheary elements in rhizomes of Dry^opteris crassirbizoma.
10. Perforations with absence of pit membrane and the lower perforations with some pit membrane
remnants on end wall; 11. Central section of this vessel, to show four perforation plates. 12.
Lateral-wall perforations with porose pit membranes of vessel element shown in figure 10. 13.
Lateral wall, to show various sizes of pores in pit membrane, scale bar = lO^im. Scale bars in the
other figures = 30^m.

membrane

walls consist of scalariform perforations in which there are porose pit mem-
branes or pit membrane remnants (Figs. 12, 13), but in some lateral walls,
perforations markedly lack pit membranes as is the case for end-wall perfo-
rations. One can find many tracheary elements that have several facets in con-
tact with other tracheary elements. The tracheary elements possess as many

membrane remnants

membranes

LI ET AL.: VESSELS IN ROOTS AND RHIZOMES OF DRYOPTERIS CRASSIRHIZOMA 29

Discussion

In the present study, Dryopteris crassirhizoma has been shown to possess
vessels in both roots and rhizomes, and the same characters of these vessel
elements in materials made by three methods are observed by mean of SEM.
Our SEM observations not only reveal that pits on oblique end walls as seen
in the light microscopy are perforations which lack pit membranes, we also
find perforations with degrees of pit membrane remnants on lateral walls.
Dryopteris crassirhizoma therefore has end-wall perforation plates and lateral-
perforation plates. End-wall perforation plates in roots are more specialized
than in rhizomes; these perforation plates are obliquely positioned in end
walls. End walls in roots are relatively short with wider scalariform perfora-
tions that contrasts with the alternate or opposite pits or narrow perforations
with pit membrane remnants on lateral walls. Thus end walls are markedly
differentiated from the lateral walls. Moreover their perforation plates are more
specialized than those of Adiantum pedatum belonged into the same ecolog-
ical group [Li et al., 1999) and Polystichum acrostichoides in the same family
(Schneider and Carlquist, 1997) based on morphology of perforation plate on
the end wall. Dryopteris crassirhizoma not only occurs in mesic habitat, but
also ranges into other habitats such as dry place and grow normally. In the
genus Woodsia, W. scopuhna and W, ilvensis, which occurs in places where
winter freezing and summer drought abbreviate the growing season, all had
high differentiation in morphology between perforation plate and lateral wall
pitting, in contrast, W, obtusa growing in a mesic habitat had little differenti-
ation (Carlqusit, Schneider and Yatskievych, 1997; Schneider and Carlquist,
1998a; Carlquist and Schneider, 1998a]. This phenomenon was explicated to
markedly differentiated vessels as a benefit to increase water supply when
ferns occupied area where there was water availability stress in some seasons.

Carlquist and his colleagues observed transverse perforation plates with sev-
eral bars in vessels of Pteridium roots (Carlquist and Schneider, 1997a) and no
lateral-wall perforation plates in Woodsia obtusa (Carlqusit, Schneider and
Yatskievych, 1997). It is evident that vessels of Dryopteris crassirhizoma are
moderately specialized. In rhizome vessels, narrow scalariform perforations of
end walls are in contrast to the scalariform pits and perforations with pit mem-
brane remnants on the lateral walls; there is less differentiation between end
walls and lateral walls. This result is observed in several other ferns (Carlquist
& Schneider, 1997a, b; Carlquist et al., 1997; Schneider & Carlquist, 1997),
which supplies evidence that vessels may have first occurred in fern roots.
Dimorphic perforations are common in vessels of roots and rhizomes; Figure
9 shows the end walls of some root vessels which have perforations differ-
entiated into two forms. This dimorphism in width of perforations also occurs
in end walls of rhizome vessels, but it is less pronounced.

Lateral-wall perforations are common in Dryopteris crassirhizoma. We noted
some particularly larger perforations which differ markedly from the other
perforations in the same lateral wall shown in figs. 5, 8 and 9, which (two to
four) group together to form a local perforation district or plate. In Astropis,

30 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Wood si a

formation

further

needed on the development of this type of perforations in lateral walls of
Dryopteris crassirhizoma.

The lateral walls of rhizome vessels have porose pit membranes. Perforations
of the terminal end wall of vessels in roots and rhizomes may also have this
type of membrane. Carlquist and Schneider (1997a) considered that presence
of porose pit membrane in Pteridium vessels could enhance lateral transport
of water without markedly increasing the vulnerability of vessels to transfer
of air embolism from one vessel element to another laterally. End walls are
less specialized, because numerous pits with porose pit membranes and/or pit
membrane remnants occur in lateral wall of rhizomes. We note that some ves-
sels in rhizomes have several facets, in which long scalariform pits have var-
ious degrees of porose pit membrane, ranging from smaller pores in pit mem-
branes to absolute absence of pit membranes. These vessel elements contact
with several other cells for transferring materials.

Acknowledgements

We greatly thank Dr. E. L. Schneider, Santa Barbara Botanic Garden. USA, for critical reading
of the manuscript and for giving fruitful suggestions. The authors are grateful to anonymous re-
viewers for their helpful comments.

Literature Cited

Zhiwen

Forestry University, Harbin.

Schneider

Bot, 84:581-587.

-and 1997b. SEM studies on vessels in ferns. 4. Astrolepis. Amer. Fern J. 87:43-50.

-and 1998a. SEM studies on vessels in ferns. 6. Woodsia ilvensis. Flora 193:179-185.

-and — — 1998b. SEM studies on vessels in ferns. 10. Selected Osmundaceae and Schi-
zaeaceae. Int. J. Plant Sci. 159:788-797.

-and 1999. SEM studies on vessels in ferns. 12. Marattiaceae, with comments on

vessel patterns in Eusporangiate ferns. Amer. J. Bot. 86:457-464.

, and Yatskievych, G. 1997. SEM studies on vessels in ferns. 1. Woodsia obtusa.

Amer. Fern. J. 87:1-8.

Gwynne-Vaughan, D. T. 1908. On the real nature of the tracheae in the ferns, Ann Bot 22- 517-
523.

Li Ruijun, Zhang Davvei and Zhang Hengqing. 1999. Scanning electron microscope observations
on the vesels of ferns: Adiantum, Matteuccia and Osmunda from Heilongjiang Province. Int.
J. Plant Sci. 160:595-602.

Russow, E. 1873. Vergleichende Untersuchungen beterffend die Histologie(Histographic und His-
togenie) der vegetativen und sporebildenen Organe und die Entwickelung der Sporen der
Leibundel Kryptogamen, mit Beriicksichtigung der Histologie der Phanerogamen, ausgehend
von der Betrachtung der Marsiliaceen. St. Petersburg Acad Sci Mem, ser. 7. 19(l):l-207.

Schneider, E. L. and Carlquist, S. 1997. SEM studies on vessels in ferns. 3. Phlebodiuni and
Polystichum. Int. J. Plant Sci. 158:343-349.

and 1998a. SEM studies on vessels in ferns. 5. Woodsia scopuUna, Amer Fern J

88:17-23.

LI ET AL.: VESSELS IN ROOTS AND RHIZOMES OF DRYOPTERIS CRASSIRHIZOMA 31

-and 1998b. SEM studies on vessels in ferns. 7. Microgramina nitida. Anales de

Biologia, ser. Botanica 69:1-7.

-and 1998c. SEM studies on vessels in ferns. 9. Dicranopteris (Gleicheniaceae) and

vessel patterns in leptosporangiate ferns. Amer. J. Hot. 85:1028-1032.
and 1999. SEM studies on vessels in ferns. 11. Ophicoglossum. Hot. J. Linn. Soce.

129:105-114.

Wu Zhaohong and Ching Renchang. 1991. Fern families and genera of China. Science Press,
Beijing.

White, R. A. 1961. Vessels in roots of Marsilea. Science 133:1073-1074.

-. 1963. Tracheary elements of the ferns. 2. Morphology of tracheary elements:conclusions.
Amer. J. Bot. 50:514-522.

American Fern Journal 90(l):32-41 (2000)

SEM Studies on Vessels in Ferns. 19. Marsilea

Edward L. Schneider and Sherwin Carlquist

Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, CA 93105

Abstract.— Tracheary elements from roots and rhizomes of M. drummondii, M. quadrifolia, and
M. vestita supplement the work of other authors by showing features of Marsilea vessels not
previously reported or not commonly reported. Pit dimorphism [wide pits lacking pit membranes
alternating with narrow pits covered with pit membranes] form perforation plates in some vessel
elements. Scalariform perforation plates with perforations like lateral wall pitting in size are com-
mon, and have not been previously reported in Marsilea by workers using light microscopy; fur-
ther SEM studies may reveal such perforation plates to be more common in the family than pres-
ently thought. Circular to oval pits arranged in alternate, opposite, and transition fashion are
characteristic of some vessels in the genus, as are tracheary element facets with few or no pits.
The occurrence of perforation plates with few bars (found also in xeric and boreal ferns) and
simple perforation plates in Marsilea may represent a capability for rapid uptake of water in
habitats with relatively short periods of water availability.

Marsileaceae have been exnlored more

for presence and morphology of vessels. Following the initial reports of vessels

Marsilea by White (1961. 1962), Mehra

Marsilea

genus. Tewari (1975) reported vessels in roots of Regnellidium. Bhardv^aja and

zomes of only two Marsilea

Marsilea

Marsilea

rhizome

Takker

Marsilea were added by J

sileaceae, but does not add original observations.

Johnson's on vessels in Mar

that

light microscopy. Although vessels with well-defined perforation plates con-
sisting of few perforations may be observed with reasonable accuracy with
light microscopy, observation of fern vessels with SEM (scanning electron mi-
croscopy) seems a procedure that can add more information and is therefore
of value. In most of the fern families studied in our papers, we have described
vessel elements in which perforation plates are very similar to, or identical to,
lateral walls of tracheary elements, and differ in absence of primary walls in
the perforations, which are like lateral wall pits in size and shape; the distri-
bution of perforations as seen by SEM is also distinctive and diagnostic, and
not at all random, We believe SEM is valuable in identifying such probable
perforations. In some carefully observed preparations utilizing particular
stains, light microscopy has been used to establish presence or absence of
primary walls in possible perforation plates in vessel elements. SEM. however.

SCHNEIDER & CARLQUIST: VESSELS IN MARSILEA

33

clearly reveals presence of cell walls, which reflect the electron beam. Presence
of primary walls and of perforations can be established accurately by means
of transmission electron microscopy (TEM). Ultimately, one would wish for
observations by all kinds of microscopy, as well as the utilization of particles
that can be transmitted through perforations but not through intact pit mem-
branes.

We believe that our results with SEM are accurate indications of vessel pres-
ence in ferns because the macerations (and in some instances, sections) in our
studies reveal pit membranes with few artifacts. Some pit membranes are rup-
tured by handling, but removal of all pit membranes on an end wall while pit
membranes are intact on adjacent lateral walls cannot, in our opinion, be at-
tributed to the maceration process. Moreover, we frequently find at ends of
perforation plates pit membranes that are transitional to perforations by virtue
of thinness and presence of porosities or even minimal weblike or strandlike
remnants of primary walls. Such incipient perforations can also be found on
end walls that have no perforations devoid of pit membranes. By eliminating
from consideration all instances we could ascribe to pit membrane absence
that might represent artifacts induced by preparation methods, we believe that
our reports of perforation plate presence are reliable.

The studies on xylem of Marsileaceae cited above report vessel presence in
rhizomes much less frequently than in roots. However, our studies on vessels
in ferns have found that in any species in which vessels could be demonstrated
in roots, they could also be demonstrated in rhizomes. On account of this
finding, we were motivated to search for vessels in rhizomes of Marsilea. In
fact, the sheaths of fibers around the steles of Marsilea roots provided severe
difficulties in isolating tracheary elements for observation by SEM, whereas
rhizome steles yielded somewhat more easily to the maceration process. These
maceration difficulties, greater than those we have encountered in any other
fern families in our series of papers, have limited the number of vessel ele-
ments we could observe clearly. Consequently, the observations we report here
should not be taken as representative of kinds of vessels in these species. Our
observations do show the presence of tracheidlike vessel elements in which
scalariform perforation plates resemble scalariform lateral wall pitting. These
have not been reported by earlier authors and thus we add to the picture of
perforation plates in Marsilea.

Materials and Methods

Species studied and the sources of collections are as follows: M. drummori'
da A. Braun, cultivated at the University of California Botanic Garden, Berke-
ley (74.0212); M. quadrifolia L., cultivated by San Marcos Growers, Santa Bar-
bara, California; M. vestita Hook & Grev, cultivated in the Biological Sciences
greenhouses, University of California, Santa Barbara. Voucher specimens pre-
pared from these plants have been deposited in the herbarium of Santa Barbara
Botanic Garden. Portions of roots and rhizomes were fixed and stored in 50%
aqueous ethanol. Macerations were prepared by means of Jeffrey's Fluid (Jo-

34

AMERICAN

Figs. 1-6.

SEM photographs of tracheary elements from macerations of roots (Fig. 1) and rhi-
zomes (Figs. 2-6] oi Marsilea drumniondiL 1) Element with scalariform facet (right), facet with
small circular pits (center), and facets with few, sparse pits (left). 2) Slender tracheary element
with facets hearing perforations. 3) Portion of perforation plate in which perforations are large,
the result of pit dimorphism. 4) Lateral wall with pits that have thin, incipiently porose pit mem-
branes, 5) Two adjacent elements with apparent groups of perforations separated from each other

form

SCHNEIDER & CARLQUIST: VESSELS IN MARSILEA

35

hansen, 1940), stored in 50% ethanol, spread onto aluminum stubs, air-dried,

15 KV.

SEM

Results

Marsilea drummondii root xylem proved unusually difficult to isolate by
means of maceration. The root tracheary element shown in Fig. 1 illustrates
scalariform lateral wall pitting, at right. The facet at the center of the photo-
graph is so narrow that the pits are circular in outline, whereas the facet at
left bears pits that are few and small.

Rhizome tracheary elements of M. drummondii (Figs. 2-6) were more readily
recovered by maceration and proved to bear abundant and diverse perfora-
tions. In Fig. 2, pit membranes are absent from all of the facets of a slender
tracheary element, and perforations are of various sizes. The phenomenon of
dimorphic pits is illustrated in the facet shown in Fig. 3; wide perforations
alternate with several pits compressed parallel to the long axis of the tracheary
element. The compressed pits are so narrow that one cannot readily observe
pit membranes on them. Pit membrane remnants certainly are absent on the
wide perforations. Pit membranes are present on the scalariform pits of the
lateral wall illustrated in Fig. 4. We cannot certify that pores are present in
these pit membranes, but thin areas are discernible in the pit membranes. In
a tracheary element shown in Fig, 5 (facet at right), on the other hand, pits
that apparently lack pit membranes (and would therefore be perforations) are
present in groups, and these groups alternate with groups of several pits that
bear intact pit membranes (some membranes torn by handling). If this inter-
pretation is valid and does not represent a condition caused by artifact for-
mation, various facets of the tracheary elements illustrated have what one can
regard as intermittent perforation plates. This situation is also present in the
pair of adjacent tracheary elements in Fig. 6, although perforations are some-
what more abundant in the tracheary element at right. In the tracheary element
at left in Fig. 6, the left facet consists of pits with intact membranes, and the
right facet bears, above, narrowed pits and slightly widened perforations al-
ternating with each other, an example of pit dimorphism.

Tracheary elements from roots of M. quadrifolia are illustrated in Figs. 7-
10. The lateral wall in Fig. 7 bears pits with intact pit membranes, and is
noteworthy only in that the pits are irregularly spaced: a few pits appear in
close pairs. In Fig. 8, pit membrane remnants of an incipient perforation plate
bear striking pores or weblike strands of primary wall material can be seen.
Tears due to handling may be seen in the pit membrane remnants, but these
can easily be detected and the porose nature of the pit membranes is clear. In

among the perforations; facet left of center shows, above, alternation of perforations and narrow
pits (pit dimorphism); facet at left is composed of scalariform pitting. Scales in all figures = 5
jjim.

36

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 [2000)

3__ /

^fWift^r^f

Figs. 7-10. SEM photographs of tracheary elements from roots ol MarsUea. 7) Facet bearing nar-
row pits, some of which are in close pairs. 8) Facet in which pit membrane remnants are markedly
porose; some tearing present but most areas are intact. 9) Scalariform perforation plate portion;
bars are narrow, perforations wide, and thus the perforations differ in width from lateral wall pits
(compared with Fig. 7]. 10] Perforation plate bearing a single bar. Scales in all figures = 5 ^m.

SCHNEIDER & CARLQUIST: VESSELS IN MARSILEA

37

Fig. 9 a scalariform perforation plate with perforations wider (with respect to

axis

We

certainty observe any tracheary elements with simple perforation plates.

Rhizome tracheary elements of M. quadrifolia are relatively wide in diam-
eter, and at least some have well-defined perforation plates (Figs. 11-14). Sca-
lariform perforation plates are illustrated in Fig. 11, left, and Fig. 12, right; the
perforations contain pit membrane remnants that do not appear to have re-
sulted from handling. In both Fig. 11 and Fig. 12, alternately arranged circular
to oval pits cover other facets. Pitting of this type was reported in drawings of
Loyal and Singh (1978, Figs. 35 and 36). However in the tracheary elements
in our figures, there is wide diversity in pit size on a single facet not reported
in previous studies: a kind of pit dimorphism. The larger pits clearly lack pit
membranes, and we have no reason to believe that this membrane absence is
an artifact. The scalariform perforation plate in Fig. 13 is noteworthy in that
the bars are very slender, the perforations are correspondingly large, and some
bars fork or anastomose. The facet at left in Fig. 13 bears intact pit membranes
with the exception of two pits, which seem to lack pit membranes, probably
a result of handling. The scalariform perforation plate of Fig. 14 has both wide
bars and wide perforations and is the kind of perforation plate that could be
seen with light microscopy. The other facets of the wide tracheary element in
Fig. 14 lack pitting, a condition also shown for Marsilea vessels by Loyal and
Singh (1978, Figs. 23 and 25). The slender tracheary element at right in Fig.
14 may be a late protoxylem or early metaxylem element; in any case, the facet
at left bears intact pit membranes, whereas the facet at right has porose pit

m

element

15) was found in the rhizome; no perforations were observed on this element.
Scalariform perforations were observed on other rhizome tracheary elements
(Fig. 16, left), and root tracheary elements (Fig. 17, right; porose pit membranes
at bottom of perforation plate and intact pit membranes above perforation
plate). The facet at right in Fig. 16 would be termed transitional in dicotyle-
dons; this pitting type has apparently not been previously reported in Marsi-
lea.

Discussion and Conclusions

Our investigation revealed features that have not previously been reported
in tracheary elements of MarsiJea. Among the features newly reported are pit
dimorphism; transitional pits on lateral walls; and scalariform perforation
plates with perforations like lateral wall pits in size and shape.

The pit dimorphism we report consists of wide pits, which lack pit mem-
branes and are therefore perforations, that alternate with one or more extreme-
ly narrow pits that have pit membranes. This feature has not been reported to
any appreciable extent before SEM studies, although Bierhorst (1960) figures
pit dimorphism in Asplenium, We have found pit dimorphism (resulting in

38

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Figs. 11-14. SEM photographs of tracheary elements from rhizomes oi Marsilea quadrifolia. 11)
Portions of two tracheary elements; element at left bears scalariform perforation plates with min-
imal pit membrane remnants; element at right bears alternate pitting (some larger perforations
appear to be perforations]. 12j Portions of two tracheary elements; element at left has pit dimor-
phism, the larger openings are perforations; element at right has a scalariform perforation plate
with pit membrane remnants in some perforations. 13) A well demarcated perforation plate (cen-
ter) with slender bars, some of which anastomose. 14) Portions of two tracheary elements; perfo-
ration plates with wide perforations, left; slender element at right has porosities in pit membranes
of right facet, but left facet has intact pit membranes. Scales in all figures = 5 ^im.

SCHNEIDER & CARLQUIST: VESSELS IN MARSILEA

39

Figs. 15-17. SEM photographs of tracheary elements from rhizome (Figs. 15, 16) and root (Fig.
17) of Marsilea vestita. 15) Wide tracheary elements with linear scalariform pitting on each of
three facets. IB] Scalariform pitting with porose pit membranes in facet at left; facet at right bears
transitional pitting. 17) Scalariform perforation plate; several pits with intact membranes at top,
several porose pit membranes at bottom of plate. Scales in all figures = 5 [im.

perforation plates) in a number of fern families and genera, such as Phlebod-
ium (Schneider and Carlquist, 1997), Anemia (Carlquist and Schneider, 1998),
Angiopteris and Danaea (Carlquist and Schneider, 1999).

Loyal and Singh (1978) have figured alternately arranged circular to oval

Marsilea vessels, such as we have illustrated in M.

ifolia

M.

Marsilea

Marsilea

cular strands in the genus, but macerations cannot offer a clear interpretation.
This matter should be studied, particularly because unpitted facets on trache-

Marsilea

an

reported for Marsilea by several authors (White 1961, 1962; Mehra and Soni,
1971; Bhardwaja and Baijal, 1977; Loyal and Singh, 1978). Our material did
not yield vessel elements with simple perforation plates, but we did observe
a perforation plate traversed by a single bar. The maceration techniques used
for our SEM studies did. however, reveal scalariform perforation plates similar

an

Mar

40 AMERICAN FERN JOURNAL; VOLUME 90 NUMBER 1 (2000]

silea, and those perforation plates are rather unlike lateral wall pitting in mor-
phology, and are therefore likely to be seen with light microscopy. Our finding
of perforation plates similar to scalariform lateral wall pitting is not unex-
pected in view of the Loyal and Singh (1978) findings, but in addition, per-
foration plates that are similar in secondary wall framework to lateral wall

pits, may h
microscopy

many

Marsilea

material did not vield vessel elements

When one views intact stele portions with light microscopy, tracheary ele-
ments can be seen even when they are sheathed by fibers. In such preparations,
simple perforation plates can be located more readily [e.g., White, 1962). Mar-
silea is unique among ferns in having simple perforation plates. Perforation

Marsilea

(Carlquist

Schneider, 1997b). The ecological nature of ferns with such vessel elements is
illuminating: all grow in areas with marked extremes of moisture availability

an

Marsilea

grow in temporary ponds in which water is available for only a relatively brief
portion of the year. Simple perforation plates and perforation plates with few
bars potentially offer the least resistance to rapid flow of a given volume of
water per unit time, and the occurrence of such perforation plates in vessels
(especially root vessels) of plants (especially monocotyledons) that live in hab-
itats with brief wet seasons (Carlquist, 1975) is understandable.

Literature Cited

Bhardwaja, T. N., and J. Baijal. 1977. Vessels in rhizome oi Marsilea. Phytomorphology 27:206-
208.

Bhardwaja, T. N., and T Takker. 1979. Tracheary elements oi PiMoria, Phytomorphology 29:
388-389.

Bierhorst, D. W. 1960. Observations on tracheary elements. Phytomorphology 10:249-305.
Carlquist, S. 1975. Ecological strategies of xylem evolution. University of California Press, Berke-
ley and Los Angeles.

and E. L. Schneider. 1997a. SEM studies on vessels in ferns. 2. Pteridium. Am. J. Bot. 84:

581-587.

and E. L. Schneider. 1997b. SEM studies on vessels in ferns. 4. Astrolepis. Am. Fern }. 87:

43-50.

- and . 1998. SEM studies on vessels in ferns. 10. Selected Osmundaceae and Schi-

zaeaceae. Int. J. Plant Sci. 159:788-797.

™d . 1999, SEM studies on vessels in ferns. 12. Marattiaceae, with comments on

vessel patterns in eusporangiate ferns. Am. J. Bot. 86:457-464.
Johansen, D. A. 1940. Plant microtechnique. McGraw Hill, New York.

Monogr. 11:1-87.

species

Loyal, D. S., and H. Singh. 1978. A further investigation of the morphology of vessels in Marsilea.
Proc. Indian Acad. Sci. (Plant Sciences-4) 876:335-346.

SCHNEIDER & CARLQUIST: VESSELS IN MARSILEA

41

Mehra, p. N., and S. L. Soni. 1971. Morphology of tracheary elements in Marsilea emd Pteridium.
Phytomorphology 21:68-71.

Sharma, B. D. 1988. Tracheary elements in pteridophytes. Bionature 8:113-134.
Schneider, E. L., and S. Carlquist. 1997. SEM studies on vessels in ferns. 3. Phlebodium and
Polystichum. Int. J. Plant Sci. 158:343-349.

Tewari, R. B. 1975. Structure of vessels and tracheids oi Regnellidium dipbyllum Lindm. (Mar-

sileaceae). Ann. Bot., n.s., 39:229-231.
White, R. A. 1961. Vessels in roots of Marsileo. Science 133:1073-1074.

. 1962. A comparative study of the tracheary elements of the ferns. Ph.D. dissertation, Uni-

versity of Michigan, Ann Arbor.

American Fern Journal 90(l]:42-47 (2000)

Shorter Notes

6-C-P-Cellobiosylisoscutellarein-8-inethyl ether, a new flavonoid from Pteris
vittata. — In spite of the fact that analyses of fern flavonoids are of chemotax-
onomic interest, data relating to flavonoids of some fern families (e.g. Pteri-
daceae) are limited. Previous v\^ork on the flavonoids of Pteris vittata L. has
led to the identification of luteolinidin 5-O-glucoside by Harborne (Phyto-
chemistry 5: 589-600, 1966] and acid hydrolysis of extracts of this fern led to
the identification of kaempferol, quercetin, leucocyanidin and leucodelphini-
din by Voirin (Ph. D. thesis, University of Lyon, p. 151, 1970); very recently
3-C-(6'"-0-acetyl-(3-cellobiosyl) apigenin has been isolated from Pteris vittata
L. by Imperato and Telesca (Amer. Fern J., 89:217-220,1999).

The present paper deals with the isolation of three flavonoid glycosides (I—
III) from Pteris vittata growling in the Botanic Garden of the University of Na-
ples (Italy). This fern w^as collected and identified by Dr. R. Nazzaro (Univer-
sity of Naples); a voucher specimen (149.001.001.01) has been deposited in
the Herbariun Neapolitanum (NAP) of the University of Naples.

Flavonoids I-III were isolated by preparative paper chromatography in BAW
(/i-butanol-acetic acid-water, 4:1:5, upper phase), 15% HOAc (acetic acid) and
BEW (n-butanol-ethanol-water, 4:1:2.2) from an ethanolic extract of aerial parts
of Pteris vittata L.. Further purification was carried out by Sephadex LH-20
column chromatography, eluting with methanol.

Rf values on Whatman No 1 paper (0.33 in BAW; 0.56 in 15% HOAc), color
reactions (brown to yellow in UV + NH3) and ultraviolet spectral analysis in
the presence of the customary shift reagents (X^^^ (nm) (MeOH) 273, 301 (sh),
332; +NaOMe 283, 334, 400 (increase in intensity); +NaOAc 282, 306 (sh),
383; +AICI3 279, 304, 348, 385, +AICI3/HCI 280, 304, 344, 383 were consistent
with flavonoid (I) being a flavone glycoside with free hydroxyl groups at po-
sition 5 (shifts with AICI3 and AlCl^/HCl), 7 (shift with NaOAc) and 4' (shift
with NaOMe). Acid hydrolysis (2N HCl/MeOH (1:1); 1 hr at 100°C) gave D-
glucose and two flavonoids (IV and V) which showed free hydroxyl groups
(detected by UV spectral analysis) at position 5, 7 and 4' and behaved as
flavonoid glycosides in paper chromatography; FeCl, oxidation of both flavo-
noids IV and V gave D-glucose. These results suggest that the isolated flavo-
noid (I) is a C-glycosylflavone in which the hydrolyzable D-glucose is not
linked to phenolic hydroxyl groups whereas flavonoids IV and V may be a

Wessely-Moser

thylformamide

(PM)

trum (MS) with [M]^ at m/z (% base peak) 764 (4) and fragment ions at 749

(8), 733 (10), 718 (12), 705 (6), 691 (7), 675 (8), 663 (12), 648 (15), 633 (8), 623
(14), 603 (21), 529 (30), 515 (21), 410 (18), 399 (37), 397 (51), 383 (100), 368

(33), 341 (36), 314 (33), 287 (63). The molecular ion of PM flavonoid fll fm/z

SHORTER NOTES

43

HOCH

H

HOCH

Fig. 1. Structural formula of flavonoid (I). 6-C-p-Cellobiosylisoscutel!arein-8-methyl ether.

764] and the above UV spectral data suggested that flavonoid (I) may be a
flavone C-diglucoside based on a flavone moiety with three hydroxyl groups
and one methoxyl group. Acid hydrolysis (2N HCl/MeOH (1:1); 1 hr at lOOX)
gave 2, 3, 4, 6-tetra-O-methyl-D-glucose and a partially methylated C-glyco-
sylflavone which gave 2,3, 6-tri-O-methyl-D-glucose by FeCl3 oxidation. Hence
a disaccharide (O-D-glucosyl- (1 — > 4)-D-glucose) is attached to flavone moiety
of I. 'H NMR spectrum (DMSO-dJ of flavonoid (I) showed signals at 5 3.12-

3.91 (diglucosyl 12 protons, m), 8 3.84 (3H, s, methoxyl group), 8 4.13 (IH, d,
J = 9 Hz, glucosyl anomer), 8 4.79 (IH, d, J = 9 Hz, glucosyl anomer), 8 6.81
(IH, s, H-3), 8 6.96 (2H, d, J = 8.8 Hz, H-3' and H-5') and 8 7.91 (2H, d, J =
8,8 Hz, H-2' and H-6'). Since anomeric protons appeared as doublets with
coupling constants J = 9 Hz, the disaccharide of flavonoid (I) is cellobiose (O-
3-D-glucosyl-(l — > 4)-D-glucose) and this sugar is attached to the flavone moi-
ety by a p-linkage. In addition cellobiose and methoxyl group must be on A-
ring of flavone moiety since signals of H-6 and H-8 were absent in ^H NMR
spectrum of flavonoid (I) and B-ring protons appeared as an ortho coupled
system [A,BJ. The bathochromic shift with AICI3/HCI (51nm) of band I in UV
spectrum of flavonoid (I) showed the absence of 6-oxygenation according to
Markham [pp. 197-235 in J.B. Harborne ed., Methods in Plant Biochemistry,
Academic Press, London, 1989); hence methoxyl group is at C-8 and cellobiose
is attached to C-6 of flavonoid (I) which must be 6-C-p-cellobiosylisoscutellar-
ein-8-methyl ether (Fig. 1), a new natural product. The following characteris-
tics of ^^C NMR spectrum of flavonoid (I) supported (Table 1) this structure
according to a review of Markham and Chari (pp. 19-134 in J.B. Harborne and
T.J. Mabry eds., The Flavonoids, Advances in Research, Chapman and Hall,
London, 1982). A shift (due to C-glucosylation) of C-6 to lower field (+9.3
ppm) in comparison with the corresponding carbon of apigenin and a shift of
C-8 (due to methoxyl group) to lower field (+30.8 ppm) in comparison with
the corresponding carbon of isovitexin were observed. In addition C-4" showed

44

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Table 1. "C NMR spectral data (DMSO-drt) of flavonoid I. ^''Assignments with the same superscripts
may be interchanged.

C-2

C-3
C-4

C-5
C-6

C-7
C-8
C-9
C-10

c-r

c-2'
C-3'
C-4'
C-5'
C-6'
OCH

Isoscutellarein-8-methyl

ether

163.9
101.9
182.2
155.6
108.1
153.9
124.8
144.1

103.1
121.1

128.8
115.8
161.4

115.8

128.8

59.9

C-1"
C-2"
C-3"
C-4"
C-5"
C-6"

C-Glucosyl

_U _ ^_

74.2
70.8
77.1
80.8
79.3
61.5

O-Glucosyl

C-l"'

104.4

c-2" '

74. P

C-3" '

75. 9^'

C-4" '

69.6

C-5" '

76.1*>

c-e"'

60.8

a shift to lower field ( + 10.1 ppm) whereas C-3" and C-5" showed small upheld
shifts [-1.9 ppm and -2.0 pppm respectively) in comparison with the cor-
responding carhons of isovitexin; these shifts are due to O-glucosylation at C-
4". The signals of O-glucosyl moiety of flavonoid (I) were similar to those of
the hydrolyzable D-glucose of 6-C-sophorosylapigenin-7-methyl ether. The fol-
lowing characteristic features of EI-MS of PM flavonoid (I) corroborated this
structure as shown by Chopin et al. (pp. 487-490 in J.B. Harborne and T.J.
Mabry eds., The Flavonoids, Advances in Research, Chapman an Hall, Lon-
don, 1982) as well as by Bouillant et al (Phytochemistry 17: 527-533, 1978).
The presence of fragment ions at m/z 749 (M-CH3) and m/z 733 (M-^OCHa)

showed the absence of 1 -^ 2 interglucosidic linkage because these fragment
ions are generally absent in EI-MS of 2"-0-glycosyl-6-C-glycosylflavones. The
presence of a fragment ion at m/z 529 ([S]% derived from the loss of PM O-
glucosyl moiety whitout oxygen of glucosidic bond) higher than the fragment
ion at m/z 515 ([S-14l^, due to elimination of C-5" PM glucosylloxymethyl side
chain) showed the absence of 1 — > 6 interglucosidic link since EI-MS of PM
6"-0-glycosyl-6-C-glycosylflavone show [S-14]^ > [Sl^, The base peak at m/z
383 (PM aglycone-CH = ""OCHy) showed the absence of 1 — > 3 integlucosidic
link since EI-MS of 3"-0-glycosyl-6-C-glycosylflavones show PM aglycone-CH
= ^OH as base peak. The absence of fragment ion at m/z 545 ([50]^, derived
from the loss of PM O-glucosyl moiety with the oxygen of glucosidic bond) as
well as the absence of fragment ions at m/z 589, 575 and 559 (in which frag-

ments of PM O-glucosyl moiety (-CH

-t-

OMe, -CH

OH, -^CHJ are bound

to [SO]^) confirmed the proposed structure since these fragment ions are very
weak or absent in EI-MS of PM 4"-0-glycosyl-6-C-glycosylflavones.

Flavonoid cellobiosides are rare plant constituents; in addition cellobiose

SHORTER NOTES

45

has been reported for the first time in association with fern flavonoids only
recently as shown by Imperato and Telesca (above reference). According to
Swain (pp. 1097-1129 in J.B. Harborne, TJ. Mabry and H. Mabry eds., The
Flavonoids, Chapman and Hall, London, 1975) flavonoid (I) may be considered
an ''advanced" biochemical character from the phylogenetic point of view
since a methoxyl group is present at C-8. A large number of flavonoid agly-
cones has been found on the outside of fronds of gymnogrammoid ferns as
shown in a review of Markham (pp. 427-468 in J.B. Harborne ed., The Fla-
vonoids, Advances in Research since 1980, Chapmann and Hall, 1988); some
of these "external" flavonoids have an hydroxyl group (often acylated) or a
methoxyl group at C-8. However hydroxyl and methoxyl groups at C-8 are near
absent from ''internal" vacuolar flavonoids of ferns since there is only the
report of 3-O-glucosides of herbacetin 8-O-methyl ether and gossypetin 8-0-
methyl ether from one fern species, Humata pectinata (Sm) Desv. (Davalli-
aceae) by Wu and Furukawa (Phytochemistry 22: 1061-1065, 1983). The iso-
lation of flavonoid (I) from Pteris vittata represents the first occurrence in ferns
of a C-glycosylflavone with hydroxyl or methoxyl group at C-8.

Flavonoid (11) has been identified as quercetin 3-0-|3-glucuronide by UV
spectral analysis with the customary shift reagents, total acid hydrolysis
(which gave quercetin, glucuronic acid and glucuronolactone), ^H NMR spec-
trum, ^^C NMR spectrum and DEPT experiments. Quercetin 3-O-glucuronide
has been found previously in Adiantum capillus-veneris L. (Pteridaceae) by
Akabori and Hasegawa (Bot. Mag., Tokyo 82: 294-299. 1969); glucuronic acid
has previously been found in association with fern flavonoids only in the ge-
nus Adiantum as shown in the above review of Markham (1988).

Flavonoid (III) has been identified as rutin by UV spectral analysis in the
presence of usual shift reagents, total acid hydrolysis (which gave quercetin,
D-glucose and L-rhamnose), controlled acid hydrolysis (which gave rutinose
in addition to the products of total acid hydrolysis) and co-chromatography
with an authentic sample (four solvent systems); this identification was con-
firmed by Kuhn methylation followed by acid hydrolysis which gave 2, 3, 4-
tri-O-methyl-L-rhamnose, 2, 3, 4, 6 tetra-O-methyl-D-glucose and quercetin 5,
7, 3', 4'-tetra-0-methyl ether. Rutin is here reported for the first time in the
genus Pteris; as shown in the above review of Markham (1988), rutin has pre-
viously been identified in ten species oi Adiantum (Adiantaceae), five species
of Gymnopteris (Sinopteridaceae), all four species of Bommeria (Sinopterida-
ceae), four species of Hemionitis (Sinopteridaceae), the genus Trachypteris
(Sinopteridaceae), Paesia anfractuosa (Dennstaedtiaceae), Pteridium aquilin-
um (Dennstaedtiaceae) and Loxsoma cunninghamii (Loxsomaceae); recently
rutin has been identified in Polypodium decumanum Wild (Polypodiaceae) by
Vasange et al. (Planta Medica 63: 511-517, 1997).

The authors thank MURST (Rome) for financial support. Mass spectral data
were provided by SESMA (Naples). — Filippo Imperato, Dipartimento di Chim-
ica, Universita della Basilicata, 1-85100 Potenza, Italy, and Antonella Teles-
ca, Istituto di Orticoltura e Colture Industriale- CNR, Via S. Loja-Zona Indus-
triale, 85050-Tito Scalo (PZ), Italy.

46 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

Ophioglossum pendulum L. Naturalized in Miami, Dade County, Florida.

Ophioglossum pendulum L. has been discovered separately by Adrian Tejedor
and Craig Allen, growing on cultivated palm trees in Miami, Florida. Ophio-
glossum pendulum is an Old World epiphyte, which grows from Madagascar
through tropical Asia and into Polynesia. It has been infrequently cultivated
in tropical fern collections in Miami since the mid-1970s. This is the first
report of established plants growing outside of strictly man-made, horticultural
conditions in the New World. This is quite a surprising discovery due to the
relative difficulty of maintaining this exotic species in cultivation.

Two populations are known in two separate sites in Coral Gables, a com-
munity located in southeast Miami. One population (discovered by Adrian
Tejedor, in April, 1998) is groAAring in the persistent leaf bases of Canary Island
date palms {Phoenix canariensis) along a public street. In this location, three
colonies are on adjacent palms and a fourth is some distance away, in the
same row of planted palms. A second, small population, is growing on a sugar
palm [Arenga pinnata), inside of Fairchild Tropical Gardens. It was discovered
during the summer of 1995, by Craig Allen. In both locations, mature, sporu-
lating plants are growing among the old persistent leaf bases on the palm
trunks. Two of the date palm colonies were relatively large and vigorous in
1998, the other two were smaller. The largest colony covered 1.5 square meters
of the palm trunk, about ten feet from the ground, with an estimated number
of 60 fronds. Considering the slow growth typical of O. pendulum, this largest
colony is estimated to be in excess of 15 years old, and may be much older.
In March 1999, only 12 fronds were observed. The majority of the fronds ob-
served the previous year had died and remained in place, completely dried
and shriveled. Only the largest fronds bore sporangia. One fertile appendage
is borne on the undersurface of the large fronds, which are from 45 to 90 cm.
long. Most of the fronds in all the colonies are small, infertile and average 45
cm. in length. Specimens have been taken from this population to document
its occurrence and are on deposit at the Fairchild Tropical Garden Herbarium
(A. Tejedor, Fairchild Herbarium #81775).

In Phoenix and other palm genera, a compact and spongy mass of old leaf
bases remains attached to the palm's upper trunk for many years after the
leaves are shed. This is where O. pendulum and other epiphytes become es-
tablished. In the case of Ophioglossum, the dangling fronds are the only visible
part of the plant. The rhizome and root system are hidden under the substrate
of old leaf bases of the palm. Adventitious shoot buds that develop on the root
system eventually produce a colony of plants on the south-east side of the
trunk, sheltered by the leaf crown. The dead leaf bases have an ability to
remain remarkably wet for days after a rain. The palm leaf-base habitat seems
favorable for these ferns, which otherwise may not survive to South Florida's
long, late winter and spring, dry season. During the dry winter of 1999 the
colonies seemed to have suffered and appeared decidedly smaller. Other epi-
phytes that coexist with O. pendulum in this habitat are the Boston fern {Ne-
phrolepis cordifolia) and young individuals of Ficus aurea (strangler fig) and

SHORTER NOTES

47

Brassaia actinophylla (schefflera]. A young staghorn fern [Platycerium sp.) was
observed among the other epiphytes.

The Ophioglossum population growing on a sugar palm inside Fairchild
Tropical Garden was known to Craig Allen, the gardener in the Rare Plant
House, since 1995. He told B. McAlpin, in June, 1998, about the location of
this plant, and said that it has grown approximately five times larger, since he
first discovered it. However, Fairchild Tropical Garden has never accessioned
this plant into its collection. Nonetheless, plants of O. pendulum from private
collections have been exhibited many times over the last fifteen years at the
annual Fern Show sponsored by the South Florida Fern Society, on the pre-
mises of the Fairchild gardens, Innoculation by wind blown spores from ma-
ture, sporulating plants could have occurred during movement of plants into
the fern shows, or during the fern show itself, which occurs for a full weekend.
Established horticultural plants, growing in open-air, screened shade houses,
could also release spores into the general environment of South Florida. It is
still a mystery when these exotic colonies first became established, and, if in
fact, the spores, and hence the adventative plants are from cultivated sources.

O. pendulum is grown in very few South Florida fern collections. Snails and
poor watering practices usually are responsible for the demise of cultivated
plants of this taxon. Successful growers use long-fiber Sphagnum moss,
mounted on plaques, tied with wire or mono-filament fishing line, in which
to grow this fern. Most successful growers also employ automated irrigation
systems in shade houses to provide protection fi:*om drying winds and to main-
tain high humidity. In cultivation plants may achieve impressive size, having
up to 100 fronds that may reach two meters in length. Plants in cultivation
are relatively slow growing. They are seldom divided because sections could
easily decay, leading to the death of the division and/or the parent plant. Less
than six growers in the Miami area are presently known to have cultivated
plants in their possession. — Adrian Tejedor, Biology Department, University
of Miami, Miami, Florida, 33124 and Bruce W. McAlpin, Biology Department,
Miami-Dade Community College, 11011 SW 104 Street, Miami, Fl. 33176.

American Fern Journal 90(l):48-50 (2000)

Reviews

Flora Malesiana, Series II-Ferns and Fern Allies, Volume 3, edited by the
Flora Malesiana Editorial Committee. 1998. Rijksherbarium/Hortus Botanicus.
Publications Department, P.O. Box 9514, 2300 RA Leiden, The Netherlands,
iv, 334 pp. Softcover (ISBN 90-71236-39-0). 100 Dfl. [available in the U.S. from
Balogh Scientific Books, www.balogh.com, for $60 + shipping].

The present volume is the seventh installment of the Malesian pteridoflora
and includes treatments of the Polypodiaceae (by P. H. Hovencamp and five
collaborators), Davalliaceae (by H. P. Nooteboom), Azollaceae (by R. M. K,
Saunders), Cheiropleuriaceae (by J. E. Laferriere), Equisetaceae (by J. E. Laf-
erriere), Matoniaceae (by M. Kato), and Plagiogyriaceae (by X. C. Zhang and
H. P. Nooteboom). Those familiar with this long-running series (the first pte-
ridophyte fascicle was published in 1959) will find the format quite similar to
that of previous parts, with the exception that the species entries are now in
a single rather than double column and have been set in a slightly larger type-
face, making the work easier to read. As with previous installments, the text
is "dense" with discussions and listing of synonyms, typification, and taxo-
nomic interpretations, as well as literature citations, all quite valuable as few
other detailed sources of information such as this presently exist for paleo-
tropical regions. There are also lengthy discussions of economic uses, phyto-
chemistry, cytology, spore morphology, and other topics as pertinent. Genera
and species are treated alphabetically within families. The descriptions are
relatively complete, although (as in previous parts), the distributional and eco-
logical data are relatively brief There are a number of excellent drawings and
photographs, which are numbered as figures independently within each family
treatment.

Most of the volume is devoted to the Polypodiaceae, with 18 genera and 183
species in the region. The remaining 6 families account for only 9 total genera
and 45 species. Interestingly, the treatments of the two small paleotropical
relict families Cheiropleuriaceae and Matoniaceae cover all of the known spe-
cies and amount to small monographs of these groups. The treatment of Azol-
laceae (including only a single Malesian species, Azolla pinnata] also has five
pages of thorough and interesting summary of the symbiotic relationship with
cyanobacteria and the concommitant economic importance of the plant in the
region.

The family Davalliaceae is of particular horticultural interest. Fern growers
interested in reading about Humata species will find these submerged in a
broadly circumscribed Davallia, in keeping with recent systematic studies. The
SEM photos of enlarged segments with sori are of particular help in determin-
ing the 23 species treated in this genus, and there are two keys to species, so
if a given specimen doesn't seem to key out well the first time an alternative
set of characters is available.

REVIEWS

49

In the Polypodiaceae, another family of considerable horticultural interest
to North American growers, the generic classification generally follows that of
Hennipman et al. in the 'Tamilies and Genera of Vascular Plants" volume. An
exception is the inclusion of Pbymatosorus in Microsorum. For this genus and
Selliguea, there are secondary keys to species in different geographic subsets

Malesian

Malesiana

addition to the inclusion of some groups of relatively great horticultural and

ance

seeking to understand the modern generic classification of the taxonomically
complex Polypodiaceae, which, except for the aforementioned very expensive
''Families and Genera'* volume, previously has not been summarized in detail

World

Mis

MO

Bibliografia sobre Gametofitos de Helechos y Plantas Afines, 1699-1996, by

Blanca Perez-Garcia and Ramon Riba. Monographs in Systematic Botany from
the Missouri Botanical Garden, volume 70. 1998. Missouri Botanical Garden
Press, 4344 Shaw Blvd., St. Louis, MO 63110. 98 pp. Softcover [ISBN 0-
915279-61-4, ISSN 0161-1542). $20.00 plus $4.00 shipping/handling.

This useful bibliography covers nearly 300 years of publications on various
aspects of pteridophyte gametophytes. The brief introduction is in Spanish
and might have been printed in English as well. However, most non-Spanish
speaking pteridologists will be able to understand the gist, if not the details,
of the half page that this covers, and the introduction is not necessary to the
use of the remaining matter. The main 75 pages of the volume contain the
lengthy bibliography itself, arranged in a single alphabetical sequence of 2195
entries. Each entry is followed by one or more numerical codes in parentheses,
referring to numbered headings in a subject index that follows. Similarly, a
taxonomic index containing a single alphabetical sequence of genera and fam-
ilies has these numerical codes following each author/date citation.

A key to the contents of the subject index appears on p. 2, between the
introduction and the main text, again in Spanish. The technical terms are
sufficiently similar to their English equivalents as to be usable without trans-
lation. The subject index has two main subject headings, spores and gameto-
phytes. The Spores heading is further broken into five subheadings ranging
from factors affecting germination to ultrastructure. The factors affecting ger-
mination are further subdivided into eight subject areas, ranging from meth-
odological concerns to environmental stimuli like temperature, light, and
chemicals. The Gametophjrtes heading is similarly broken into a number of
subject headings. As with any attempt to organize a large body of diverse
literature into discrete subject headings, there are inevitable problems of se-
lection of headings and overlapping subject areas in a given paper. The authors
have done a creditable job of balancing the tendency to divide the subject

50 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)

headings ever more finely at one extreme with the loss of utility in headings
that are too hroad at the other. Nevertheless, individual readers probably will
have minor quibbles here. For example, I would have preferred a discrete
section on studies dealing with antheridiogens, but these are immersed in a
more general subject heading entitled Metabolism, Biochemistry, Molecular
Biology, and Chemical Components.

Anyone who develops an interest in fern reproduction will find this bibli-
ography a convenient starting point for delving into the surprisingly large body
of literature on various aspects of spore and gametophyte structure, physiol-
ogy, ecology, biochemistry, and genetics. Even more seasoned prothalliists will
gain a more complete historical perspective on their field. It is hoped that the
authors will periodically issue updates to this work. — George Yatskievych,
Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166.

Missouri Botanical Garden Libra

3 1753 00299 6749

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2A. Lellinger, David B. 1989. The Ferns and Fern-allies of Costa Rica,

Panama, and the Choco (Part 1: Psilotaceae through Dicksoniaceae). 364 pp.
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Volume 90

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QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY

The Chloroplast Genome Structure of the Vascular Plant Isoetes is Similar to that of the

Liverwort Marchantia H. Joel Duff and Edward E. Schilling

i of Growth am
acrostich oides

Gary K, Greer and Brian C. McCarthy

The Effects of Rhizome Severing and Nutrient Addition on Growth and Binmass Allocation

in Diphasiastrum digitatum

Carrie A. Raihng and Brian C. McCarthy

Obituary

Joseph A. Ewan (1909-1999]

51

60

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The American Fern Society

Council for 1999

BARBARA JOE HOSHIZAKI, 557 N. Westmoreland Ave., Los Angeles, CA 90004-2210.

President
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American Fern Journal 90(2):51-59 (2000)

The Chloroplast Genome Structure of the VascuK^

X\\C''^'^

Plant Isoetes is Similar to That o^\ffi9 ^a^^

Liverwort Marchantia ^^^^ a ^ ^

R. Joel Duff ar)0^^^^

Department of Biology, University of Akron, Akron, OH 44315-39fil0k

Edward E. Schilling

Department of Botany, University of Tennessee, Knoxville, TN 37996-1100

Abstract. — Restriction site mapping was used to characterize the chloroplast genome of the ly-
cophyte, Isoetes melonopodo. The Isoetes chloroplast genome is approximately 139-145 kb in size
with an inverted repeat of 12-13 kb. The gene content and consensus gene order are similar to
that of Marchantia. A distinctive feature of the Isoetes genome is an increased size of the small
single copy (SSC) region possibly due to the insertion of a piece of DNA (3-8 kb) of unknown
composition. The inferred insertion, along with a slightly larger inverted repeat, are responsible
for the apparent size difference in the total genome relative to Marchantia. Patterns of restriction
fragments were also consistent with the presence of a small inversion (2-3 kb) in the large single
copy (LSC) region.

Structural differences in chloroplast genomes have proven to be povirerful
characters in understanding relationships among land plants because of their
rarity and corresponding apparent low levels of homoplasy (Palmer, 1985a, b,
1987, 1991; Palmer and Stein, 1986; Palmer et al., 1988; Manhart and Palmer,
1990; Raubeson and Jansen, 1992; Downie and Palmer, 1992; Doyle, 1992; Lew
and Manhart, 1993; Raubeson and Stein, 1995). The chloroplast genome ex-
hibits a remarkably consistent structure and gene order and content among a
wide range of members of the plant kingdom (reviewed in: Stein et al., 1986;
Palmer et al., 1988; Olmstead and Palmer, 1994). Among photosynthetic plants
this circular molecule is usually 120—160 kilobases (kb) in length and has a
capacity to code for approximately 120 genes.

Our understanding of the chloroplast genome comes primarily from angio-
sperms, but little has been reported about the size, overall structure, or vari-
ation of the chloroplast genomes of lower vascular plants (Lycophyta, Psilo-
toph)^a, and Sphenopsida) or most of the bryophytes. Among the lower vas-
cular plants only the presence or absence of a 30 kb inversion (Raubeson and
Jansen, 1992) has been demonstrated. Among the pteridophytes Stein et al.
(1992) and Conant, et al. (1994) have shown that multiple structural rearrange-
ments exist among the diverse groups of ferns and that these structural features
may be useful phylogenetic markers at the familial and higher levels. More
information about the genomes of the primitive land plants will be needed to
attain a comprehensive understanding of the chloroplast genome.

In this study we provide the first complete restriction site maps of the chlo-
roplast genome of a lycophyte, the quillwort Isoetes melanopoda. We find that
the Isoetes chloroplast genome shares significant features with those of two

52 AMERICAN FERN JOURNAL: VOLUME

non-vascular plants, the bryophytes Marchantia (liverwort) and Physcomitrel-
la (moss). The Isoetes chloroplast genome has the following features: (1) its
inverted repeat (IR) is several kb larger than the IR of either of the bryophytes
but is significantly smaller than the IR of most ferns and seed plants, (2) the
small single copy region (SSC) has a large (3-8 kb) region of extra genetic

unknown origin, and

(2-4

Materials and Methods

Leaves oi Isoetes melanopoda Gay & Durieu (Isoetaceae) were collected from
a single population in Alabama and a voucher specimen {Duff 9201) v^as de-
posited at the University of Tennessee Herbarium. Total DNA was isolated
using the procedure of Doyle and Doyle (1987). Single digests of sixteen re-
striction endonucleases [BamHI, Banl, Banll, Dral, EcoRl, EcoRV, Haell,
Hindlll, Neil, Ncol, Pstl, Pvull, Sad, SaR, Stul, and Xhol) and selected double
digests [Banl/Banll, EcoRl/EcoRW, Pstl/SaR, SaclfPvull, and StuVXhol] were
made and the fragments separated on 0.9% agarose gels run out 15 cms and
transferred by dry blotting to Amersham (Hybond N+) nylon membranes.
Cloned cpDNA fragments from lettuce (Jansen and Palmer, 1987) and tobacco
(Olmstead and Palmer, 1992; Shinozaki et al., 1986) were used as probes for
physical mapping and gene localization. Membrane-bound DNAs were hy-
bridized to 32P-dCTP labeled probes using random primer oligolabeling (Fein-
berg and Vogelstein, 1983, 1984; Gibco BRL labeling kit) for 24 hr at 55° C.
Hybridization buffers and conditions were used as described by the manufac-
turer (Gibco BRL) except that hybridizations were done at 55° C. Tobacco DNA

fy

from Hin dill

Mappin

Palmer (1982, 1986) and Jansen and Palmer (1987).

Results

Hybridization to both lettuce and tobacco probes generally gave good results

mapping

limited

probes. As a result it was possible to generate complete maps only for restric-

enzymes

The problematic regions are those covered by tobacco probes 3, 20a, 21, 29,

and

March

Adiantum also reported a lack of hybridization with tobacco probe 3, which

March

March

(ORFs) that

map

STRUCTURE

* 4

53

double digests of Isoetes melanopoda were successfully generated (Fig. 1). Par-
tial maps for seven additional enzymes were obtained for a great majority of
the genome including the inverted repeat and small single copy region, and
were utilized in determining portions of the genome structure. Completed re-
striction site maps gave total chloroplast genome size estimates for Isoetes of
139-145 kb with an average of 141 kb. The estimates for the minimum size of
the inverted repeat varied from 11.8 [EcoRl + EcoRV] to 13.2 kb [Nsn). The
size of the large single copy region (LSC) was approximately 85 kb and for the
small single copy region (SSC) was 24-29 kb. Consistent with the results of
Raubeson and Jansen [1992), the Isoetes genome exhibits the presumed ances-
tral gene order exhibited in bryophytes and lycophtes. Evidence of this genome
architecture came from the fact that two non-adjacent pairs of tobacco probes,
31-11 and 2-12, respectively, consistently hybridized to overlapping frag-
ments (Fig. 1). A large insertion in the SSC, relative to both Marchantia and
tobacco, was inferred from mapping studies and appears to be a feature unique
to Isoetes. An additional feature of the Isoetes genome was the apparent pres-
ence of a small inversion (1.5-3.0 kb) found in the large single copy region
(LSC) as well as several other smaller inversions which may be postulated from
individual restriction site maps but cannot be characterized more completely
due to the resolution of the current data set.

Inverted Repeat. — The size of the inverted repeat in Isoetes (11.8-13.2 kb) is

Marchantia

IR

of regions homologous to those of tobacco fragment 32 (rps' 12, Tps7) and a
very small portion of probe 31, which are found in the IR of tobacco but are
restricted to the large single copy region adjacent to IR^ in Marchantia (Ohya-
ma et al., 1986). Just as in Marchantia, regions of cpDNA homologous to to-
bacco probes 28, 29, 30, 36 and 1, all of which are part of the IR of tobacco,
were mapped to the large single copy region of the Isoetes chloroplast genome.

Small Single Copy Region. — The single copy region was estimated to be 24-
29 kb in size. The variation in this estimate was due to lack of hybridization
and presence of genetic material in Isoetes not represented in the tobacco
probes resulting in difficulties in resolving the boundaries of the region. The
lack of hybridization to several probes combined with the size of spanning
fragments made precise estimations of the size of the SSC and the entire ge-
nome difficult. Figure 1 shows that the amount of DNA in the area adjacent
to the edge of IR^ was more than could be accounted for from the sizes of the

tobacco probes used and than its expected content compared with the Mar-
chantia genome (Ohyama et al., 1986). For example, a 17 kb chloroplast DNA
fragment, the result of digestion by PvuII, only hybridized to probes 36, 37,
and very weakly to 35 which account for a maximum of 10 kb of DNA in
tobacco. The best estimate of total size of the SSC came from the map of SacL
This enzyme yielded only two fragments that span the SSC; a 23-27 kb frag-
ment and a 1.8 kb fragment each of which hybridized to probe 35. For the
same enzyme two 5.4 kb fragments hybridized strongly to probe 35 and very

54

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

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DUFF & SCHILLING: CHLOROPLAST GENOME STRUCTURE OF ISOETES 55

weakly to probe 34 indicating that the 5.4 kb fragment accounts for the ma-
jority of the 4.7 kb tobacco probe 35. The approximately 27 kb and 1.8 kb
fragments that weakly hybridize to probe 35 must lie primarily within the SSC,
This gives an upper estimate of the SSC of approximately 29 kb. Considering
maps of more than 10 enzymes completed for the SSC, an estimate of 24-29
kb can be made. Compared with Marchantia (20 kb SSC), PhyscomitreJJa (21
kb SSC, Calie and Hughes, 1986], and Adiantum (20-22 kb SSC, Hasebe and
Iwatsuki, 1990), this would place the amount of extra DNA in the Isoetes SSC
at 3-8 kb.

Absence of 30 KB Inversion, — Based on the presence of fragments from
BamHl and Hindlll digests that show overlap to tobacco probes 31 and 10,
and 12 and 2, Raubeson and Jansen (1992) report the absence of a 30 kb in-
version found in ferns and seed plants. Our data supported the absence of this
inversion in these higher plants. Figure 1 shows that genetic material homol-
ogous to tobacco probes 29, 30 and a portion of probe 1 were detected between
fragments homologous to probes 2 and 12. Tobacco probe 1 hybridized to frag-
ments in two areas: between probes 2 and 29 and between probes 28 and 32
at the edge of IRg. This is what would be expected were the genome oi Isoetes
to have the same arrangement as Marchantia. In Marchantia the transfer RNA,
H(GUG), found in probe 1 of tobacco, is present next to probe 2 but the re-
maining portion of probe 1; frnl(CAU), rp723, rp/2, can be found in the LSC at
the edge of IR^^ in Marchantia, The presence of very weak hybridization to
probe 1 adjacent to probe 2 suggested that the arrangement of genes in Isoetes,
in the region of this 30 kb inversion in ferns and seed plants, is identical to
that in Marchantia,

Additional Inversions. — A small (1-3 kb) inversion was detected in the LSC
but its precise size was difficult to determine. The restriction enzymes used
produced DNA fragments that were too large to allow localization of the end-
points of the inversion. The presence of the inversion was supported by the
detection of multiple fragments that hybridized identically to both tobacco
fragments 14 and 15. Additional, smaller inversions maybe hypothesized from
several individual restriction maps. These inversions could not be accurately
characterized because they are located in a portion of the genome for which
there was poor hybridization signal. This region corresponds to the position
of a large open reading frame (ORF2136 = tobacco fragments 29,30). Only

Fig. 1. Linearized restriction site maps of the chloroplast genome of Isoetes melanopodo. En-
zymes used in single or double digestions to map the genome are indicated at each end of the
map. The top line represents the tobacco probe set (Olmstead and Palmer, 1992) in positions
relative to their hybridization with Isoetes cpDNA. Large solid bars indicate relative position and
boundaries of the inverted repeat in Isoetes. Asterisks denote tobacco probes for which very week
hybridization was observed to Isoetes fragments. Fragments less than 0.5 kb were not consistently
observed and therefore are postulated based on data from double digests. Fragment sizes larger
than 18 kb could not be accurately calculated and so reflect estimations based on additive sizes
of fragments detected by double digests.

56 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

fragments spanning the entire region were utilized in the mapping data pre-
sented here. Raubeson and Jansen (1992) also report the probable occurrence
of up to two small inversions in this region as well, but did not characterize

further

Discussion

Although Isoetes is a highly distinctive genus, whose evolutionary lineage
is separated from the majority of vascular plants by up to 300 million years
and from the non-vascular plants for as long or longer, it exhibits a chloroplast
genome remarkably similar to the liverwort Morchantia. Most vascular plants
appear to have significantly larger chloroplast genomes than the bryophytes
because the inverted repeat is larger. The Isoetes cpDNA genome is also larger
than the bryophyte genome in size but the difference is the result of an in-
creased size of the SSC relative to other characterized cpDNAs and to a lesser
extent an increase in the size of the IR. The overall size, including the IR, and
structure of the chloroplast genome of Selaginella (Duff, unpublished data)

March

Sim

Marchantia

of the 30 kb inversion confers some structural integrity to these molecules.
The 30 kb inversion that took place in the ancestor of the ferns and seed plants
appears to be correlated with a relaxation of physical constraints on the size
of the inverted repeat region. Hence the IR subsequently underwent rapidly
expand to incorporate genetic material from the LSC in the vast majority of
ferns and seed plants (Palmer and Thompson 1982; Jansen and Palmer 1987;
Howe et al. 1988]. Stein et al. (1992] has proposed that the region around the

the

Marchantia
mlike those

integrity of the genome not to affect the IR. These taxa are by all accounts
highly divergent and yet the sizes of their respective inverted repeats are not
significantly different nor have they been demonstrated to have undergone
inversion events to the extent observed in plants which contain the 30 kb

March

Physcomitrella, and Isoetes is directly related to their gene order.

March

the

fficulty

29 kb) in Isoetes was unex-

homol

example, it has been shown that the chIL gene has been lost from angiosperm
cpDNAs although it is present in Isoetes as well as all bryoph5^es. lycophytes,
ferns except Psilotum, and gymnosperms except Welwitschia (Burke et al.,
1993]. This gene is typically located in the SSC adjacent to the IR^ though the
restriction map generated for Isoetes does not clearly define this region due to
the present of several large fragments spanning this region. Furthermore, the

DUFF & SCHILLING: CHLOROPLAST GENOME STRUCTURE OF ISOETES 57

tobacco probes used in constructing the map lack this gene region and thus it
is possible that several small fragments generated by Nsil, Sad, and Pvull
could have escaped detection in this region between probes 40 and 35. Even
accounting for these sources of uncertainty the total size of the SSC appears
to be increased over that of all other previously characterized land plant chlo-
roplast genomes. Furthermore this increase in DNA content is most apparent
in the fragments that hybridized to tobacco probe 35, found in the IR of both
tobacco and Isoetes, and tobacco probe 36. The latter represents genetic ma-
terial found in the IR of tobacco but whose homologous content can be found
in the SSC of Marchantia adjacent to IRg. This extra DNA had insufficient
sequence similarity with any portion of the tobacco or lettuce genomes to
produce hybridization signals and its presence was only determined by the
presence of fragments that spanned the entire region (see Stul, SaR, Pstl in
Fig. 1). This DNA found in the Isoetes SSC may be the result of either an
increased size of spacer regions between the genes or may be due to an inser-
tion of foreign DNA. Very few definitive cases of extra, non-homologous DNA
found in the chloroplast genome are known. Conant et al. (1994) report the
presence of about 0.9 kb of DNA in the IR of Cyathea furfuracea (Cyatheaceae)
for which there is no counterpart in the Adiantum genome and thus cannot
be explained by duplication or inversion events. A more dramatic example
can be seen in the unusual choloroplast genome of Trachelium (Campanula-
ceae) in which as many as seven insertions of foreign DNA have been postu-
lated (Cosner et al. 1997). In any case, more detailed analyses will need to be
undertaken to confirm the existence and to determine the nature of any ad-
ditional sequences in the Isoetes chloroplast genome.

In summary, we have established that the genome of Isoetes has the overall
structure of the bryophytes, Marchantia and Physcomitrella. The only signif-
icant difference between the bryophyte genome and that of Isoetes is the pos-
sible increase in DNA content of the SSC region of the Isoetes genome and
increased IR content. Further work to characterize the insertion in Isoetes and
to map the genomes of Lycopodium sens, lat., Equisetum, and Psilotum will
most likely result in a better understanding of the evolution of the chloroplast
genome and possibly provide clues regarding the relationships of the lower
vascular plants.

Acknowledgments

This paper represents a portion of a Ph.D. dissertation submitted to the University of Tennessee.
We thank Diana Stein and Linda Raubeson for their advice and help on the mapping of the genome
and the comments of one anonymous reviewer. This research was supported by National Science
Foundation Doctoral Dissertation Improvement Grant DEB-9321767, and grants from the A. J.
Sharp Fund, Department of Botany, University of Tennessee.

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STRUCTURE

59

Raubeson, L. a., and R. K. Jansen. 1992. Chloroplast DNA evidence on the ancient evolutionary

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American Fern Journal 90(2):60-76 (2000)

Patterns of Growth and Reproduction in a Natural
Population of the Fern Polystichum acrostichoides

Gary K. Greer

Department of Biology. West Virginia State College, Institute. WV 25112-1000

Brian C. McCarthy

Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701

Abstract. — Patterns of growth and reproduction were documented in a natural population of
Polystichum acrostichoides in southeastern Ohio during the 1994 and 1995 growing seasons. The
proportion of biomass allocated to fronds increased with plant biomass, indicating fronds are an
increasingly dominant component of the body of P. acrostichoides. Regression analysis indicated
a minimum size threshold exists at which this species first becomes reproductive. Both repro-
ductive status and frequency of reproduction were positively associated with greater plant biomass
and above-ground growth rates. A cost of reproduction to growth was apparent; above-ground
growth rates increased during non-reproductive years among individuals that reproduced in only
1994. Minor increases in reproductive effort were associated with increasing plant biomass; rang-
ing from approximately 0.01% to 2.11%. Nevertheless, reproductive effort may be plastic in R
acrostichoides; the frequency of reproduction correlated negatively with cation concentrations and
positively with phosphorous concentrations, and reproductive effort increased with decreasing
canopy cover. Together, these observations suggest reproduction in P. acrostichoides only occurs
when resources are sufficient to offset it*s cost to future growth; a life history that may optimize
the advantages of early reproduction and life-time fecundity in a species whose colonizing phases
(i.e., gametophyte and juvenile sporophyte) have high risks of mortality.

Patterns of vegetative growth and reproduction are major components of
plant life history evolution. They are the basis for investigating the relation-
ships betvi^een age, size, growth, and reproduction, as well as the roles of ge-
notypic and allometric constraints versus plasticity in allocation patterns
(Harper, 1977; Stearns, 1992). According to resource allocation theory, repro-
duction and growth compete for the same pool of resources and, therefore,
occur at the expense of the other (Schaffer and Rosenzweig, 1977; Harper,
1977; Watson, 1984). The size of an individual's resource pool is a function of
its capacities for storage and acquisition balanced against its current and past
expenditures. Life history theory predicts that, within the boundaries set by
genetic and allometric constraints, a species allocation pattern will evolve to
maximize it's contribution to the gene pool (Harper, 1977; Stearns, 1992). In
populations where juvenile mortality rates are lower than adult mortality rates,
selection will favor the demographic advantage conferred by early reproduc-
tion. Conversely, in populations where juvenile mortality rates are higher than
adult mortality rates, life time fecundity will be favored.

Among angiosperms and gymnosperms, the challenge of studying patterns
of resource allocation is compounded by a number of factors, including the
existence of structures that serve both reproductive and vegetative functions
(e.g., petals, pedicels, bracts, and ovary walls), loss of meristems to reproduc-

GREER & McCarthy: growth, reproduction in p. ACROSTICHOIDES 61

tion, production of nectar, and other floral and fruit characters that effect, or
are affected by, pollinator visitation and behavior, pollination success, fertil-
ization, and seed set (Harper, 1977; Willson, 1983; Riska, 1986). Because pte-
ridophytes do not produce seeds or flowers, they represent a major group of
vascular plants which lack most of the complications listed above. Despite
their advantageous simplicity, very little attention has been given to patterns
of allocation to growth versus reproduction among pteridophytes, at devel-

[but

Moreover

perennial life histories occur among pteridophytes, permitting study of a va-
riety of life history categories.

This study describes patterns of vegetative and reproductive performance in
a natural population of the homosporous fern, Polystichum acrostichoides
from which hypotheses regarding the life history of this species were devel-
oped.

Methods

Polystichum acrostichoides is an iteroparous perennial that possesses a
short-creeping rhizome (i.e., rarely longer than 40 cm) and semi-evergreen
fronds that fully senesce as new fronds emerge in the spring. It is a species
common to forests of eastern North America and a major component of the
understory at the study site, Watterloo Wildlife Research Station (WWRS),
Ohio. WWRS is located in Athens County, Southeastern Ohio. The forest at
the study site is a mixed hardwood assemblage dominated by species of Acer,
Quercus, and Carya,

To survey the population of R acrostichoides at WWRS, we established two
200 m transects [streambank and upper-slope) in each of three ravines. Upper-
slope transects were initiated at points perpendicular in elevation to the be-
ginning of each streambank transect, and were defined as 70% of the eleva-
tional distance from streambank to ridgetop. Elevations were determined using
a 7.5 minute USGS topographic map (Athens, OH 1985) and an American
Paulin Systems model Ml -6 altimeter.

An individual plant was identified every 20 m along each transect using the
nearest individual method, for a total of 120 plants. To facilitate unobtrusive
observation of growth and reproduction during consecutive growing seasons,
above-ground morphological measurements [number of fronds, number of fer-
tile fronds, frond area, and fertile area) of each plant were made between mid-
June and mid-July, the approximate month of spore release, in 1994 and 1995.
Because sporangial maturation is mixed in P. acrostichoides, and the inherent
difficulty in efficiently collecting spores in the wild, an indirect measure of
spore output (i.e., area of the fertile tip of a frond) was used. Fertile pinnae of
P. acrostichoides occur at frond tips and are densely covered with sori on their
lower surface. Separation between fertile and non-fertile pinnae is typically
abruptly dimorphic, accompanied by a reduction in size (Fig. 1). Occasionally,
the lower-most fertile pinnae are not reduced in size and possess only a few

62 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

Fertile Vegetative 4 cm

Fig. 1. Silhouette of a fertile frond of P. acrostichoides showing the abrupt change in pinnae size
between vegetative and fertile pinnae.

scattered sori. To minimize error, these pinnae were not included in the mea-
surement of fertile area. These methods assume the metabolic cost of produc-
ing vegetative tissues, sporangia, and spores is relatively constant from year to
year (e.g., 1.0 g of spores requires the same absolute metabolic expenditure in
1995 as it did in 1994).

At the end of the 1995 growing season, each plant was collected, stored in
a paper bag, dried at 70°C for 72 h, and total frond and rhizome-root mass dry
weight determined. Dead rhizomatous tissues and persistent frond bases were
removed from the rhizome prior to drying. Eight environmental variables were
measured for each plant; canopy cover, moisture status, soil nitrate-nitrogen,

potassium

measure

Moisture
Moisture

mo

— -. ,.-__ ^ — — ^ — ^--* wr ^ ^^A If _i t_^ *_* # X V^A XX J. 1^ \-XXXX

cation details). Soil was collected in summer 1995 from the rhizome-root mass
mantle of each plant, stored in a paper bag, and shipDed to the Ohio Aericul-

Wooster

Data Analyses

Mann-Whitney

environmental

an

reproducers; i.e., those that reproduced in only 1994 or 1995 versus those that
reproduced in both years. Comparisons of plant size and morphology used
estimates of 1994 + 1995 plant biomass ftotal frond + rhiznme + rnnt hinmacQl

minimize

ance between years. Relative growth rate (reffered to hereon as "growth") was
estimated using the equation "(1995 total frond area - 1994 total frond area)/
1994 frond area." Environmental variables were represented by loading scores

GREER & McCarthy: growth, reproduction in p. ACROSTICHOIDES 63

from Principle Components Analysis (PCA), PCA axes representing more than
10% of total environmental variance, and environmental variables with factor
scores greater than 0.50, were interpreted. PCA is sensitive to highly correlated
variables within each matrix. Among the environmental variables surveyed,
soil pH, available calcium, magnesium, and potassium, were closely correlat-
ed, however, removal of all cations except pH did not substantially effect the
outcome of the PCA and were therefore retained in both analyses.

Simple linear regressions were used to: 1] explore the relationship between
frond and rhizome-root mass biomass throughout development, 2) describe the
relationship between plant size and allocation to reproduction versus vegeta-
tive growth (i.e., reproductive effort, RE 1), and 3) investigate the cost of re-
production to growth in the following year. RE 1 as well as estimates of above-
ground growth, used total fertile area and total frond area to minimize error
that would result from conversion to estimates of biomass.

A second measure of reproductive effort (RE 2), based on Willson's (1983)
suggestion that a more appropriate measure of reproductive effort for peren-
nials may be yearly allocation to reproductive versus vegetative structures, w^as
used for comparison. RE 2 was estimated for each plant by dividing its 1995
fertile biomass by its change (1995-1994) in plant biomass. Spearman's rank
correlations were used to investigate the relationship between reproductive
effort and PCA axes representing environmental gradients; mean reproductive
effort values were used for each individual (e.g., mean RE 1 = (1994 + 1995
RE 1/ 2)).

The relationship between plant biomass and above-ground growth rate was
investigated using linear regression model fitting (SPSS PC+, v. 6.1). Interpre-
tation of regression used to investigate the relationship between reproductive
effort and plant size followed Samson and Werk (1986). Their method, which
eliminates the autocorrelation that may occur between reproductive biomass
and total plant biomass, but also requires untransformed data, permits simple
and direct interpretation of the slope and y-intercept. Here, we regressed 1994
+ 1995 total fertile biomass against 1994 + 1995 vegetative biomass. To in-
vestigate the relationship between plant size and reproductive effort, as de-
fined by Willson (1983) for perennials, we regressed 1995 RE 2 against 1994
plant biomass. Although the potential for autocorrelation exists, total fertile
biomass was included within plant biomass for this analysis. Fertile tips typ-
ically remain photosynthetic after spore-release (i.e., mid June through early
July) and are not reproductive organs. Therefore, deletion of total fertile bio-
mass from estimates of plant biomass would result in under-representation of
vegetative biomass.

The D'Agostino-Pearson omnibus test statistic (D'Agostino et al., 1990) was
used to determine whether data met requirements of normality. Homoscedasc-
ity was tested using the F-max test (Dowdy and Wearden, 1985). All statistics
were analyzed using SPSS PC+ v.6.1 (Norusis, 1994). A number of data trans-
formations were used to meet statistical assumptions, including natural log,
and power transformations. A critical value of P = 0.05 was used in all anal-
yses. All reported r^ values were adjusted for degrees of freedom.

64

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

O

4 -

3

2 -

p 1

4.5

5.5

6.5

7.5

8.5

9.5

1995 Laminar area {cm^f^ )

Fig. 2. Regression analysis of 1995 total laminar area (LA) prediction of 1995 total frond biomass
(FrB).

Results

Linear regressions indicated frond area and the area of the fertile tip are
robust predictors of frond biomass (Fig. 2) and reproductive biomass (Fig. 3),
respectively. Consequently, the linear equation "frond biomass = (0.007902 *

frond area) - 0.071802

JJ

from

1994 total frond area. Likewise, the linear equation "fertile biomass = 1.85X

- 7.66" was used to estimate fertile biomass from fertile area for both 1994
and 1995.

axes

15.3%, 12.7%, and 12.0%

cumulative

axes

potassium

whereas, PCA 2 represented moisture and nitrate concentrations [Fig. 4a). PCA

3 and PCA 4 represented canopy cover and phosphorous concentrations, re-
spectively (Fig. 4b).

1994 + 1995 plant biomass correlated negatively with PCA 1 and positively
with PCA 4, indicating plant biomass was adversely affected by soil cation

Mean

any

ij. ineretore, ditterences m plant biomass associated with environmental gra-
dients were not associated with differences in biomass allocation to above-
ground versus below-ground organs.

ant

Gowth

numb

total frond area, 32 (91.4%) exhibited a decrease in frond number. Likewise,

GREER & McCarthy: growth, reproduction in p. acrostichoides

65

2.0

2.2

2.4

2.6

2.8

3.0

3.2

Fertile tip area (cm^)

Fig. 3. Regression analysis of total fertile area prediction of sporangial + spore biomass

54 of 55 individuals (98.2%) exhibited an increase in total frond number.
Growth rate correlated significantly and positively with PCA 2 (Table 1), in-
dicating growth increased with moisture and soil nitrate concentrations. How-
ever, the low r^-value indicates considerable variability exists over the range
of observations.

The proportion of biomass allocated to fronds increased significantly with

plant biomass (r^ - 0.31, P < 0.001, Y = l.OlX - 0.69). Mean frond: rhizome-
root biomass ratio for the entire population was 1.3 ± 1.2, indicating that
fronds constituted 56.5% ± 51.7% and rhizome-root mass biomass 43.5% ±
40.2% of plant biomass, respectively. Thus, as plant size increased, allocation
shifted from favoring the rhizome-root mass to fronds. As a result, rhizome-

root biomass was a poor predictor of frond biomass (r^ = 0.19, P < 0.001, Y

= 0.09 + 1.12).

Of the 120 plants surveyed, 41 (34.2%) were non-reproductive and 79
(65.8%) were reproductive in one or both years. Mann-Whitney U Rank Sum
Tests indicate that reproductive individuals were larger (1994 + 1995 plant
biomass) and fronds composed a greater proportion of their biomass (mean
frond: rhizome-root biomass ratio) than non-reproductives (Table 2). Never-
theless, substantial overlap in plant biomass between reproducers and non-
reproducers (Fig. 6) indicates reproduction was not entirely size dependent.
Reproducers also exhibited significantly greater growth rates than non-repro-
ducers (Table 2). No significant differences were observed between reproducers
and non-reproducers for PCA axes representing environmental gradients (Ta-
ble 2), indicating their distributions were not influenced by current environ-
mental conditions.

Regression of 1994 + 1995 total fertile biomass versus 1994 + 1995 plant
biomass for the entire reproductive population was significant; both slope and
y-intercept differed significantly from zero (Fig. 7), The negative y-intercept
(—0.29) indicates a minimum size at first reproduction exists in P. acrosti-
choides. Allocation of biomass to reproduction ranged from approximately

66

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

0.8
0.6
0.4

(a)

CN 0.2

<
O

Q. -0.2

0.0

-0,4
-0.6

0.75

0.5
"^ 0.25

(b)0 0.0

-0.25

-0.5

1

1

1

I ' I

1

1

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

PCA1

-0.75 -0.5 -0.25 0.0 0.25 0.5 0.75

PCA3

Fig. 4.

Ordination from principle components analysis (PCA) of edited data matrix. Arrows in-
dicate strength and direction of environmental gradients based on Spearman's rank correlations
between ordination scores and observed environmental values; correlation coefficients can be de-
termined from values on each axis. Symbols represent reproductive status of individual plants as
follows: open triangles represent non-reproductives; open circles represent non-sequential repro-
ductives, and closed circles represent sequential reproductives.

0.01% among the smallest individuals to 2.11% among the largest individuals
(Fig. 7). The absolute value of the slope v^as small (0.03) compared to the y-
intercept (-0.29), therefore, only a minor increase in the proportion of carbon
allocated to reproduction vi^as associated with increasing plant size.

Mean RE 1 and RE

mean RE

and PCA 1. These observations suggest allocation to reproduction versus

TOWth

trations may have had a small negative effect on reproductive effort.

Of the 79 reproductives, 31 (39.2%) reproduced in only one growing season
(i.e., non-sequential reproducers), whereas, 48 (60.8%) reproduced in both
growing seasons (i.e.. sequential reproducers). Mann-Whitney U Rank Sum
Tests indicate sequential reproducers were larger and allocated a greater pro-
portion of their biomass to fronds than non-sequential reproducers; however.

GREER & McCarthy: growth, reproduction in p. acrostichoides

67

Table I. Spearman's rank correlation coefficients and associated probabilities for morphological traits,
measures of above-ground growth, reproductive effort, and PCA axes representing environmental gradi-
ents.

PCA 1

PCA 2

PCA 3

PCA 4

IRAIT

1995)

(Ca,

rs2

K, Mg)

- -0.23

(H,0
r s^ -

, NO3)
-0.04

(Canopy)
r s2 - -0.08

(P)

Plant Biomass (1994 +

r s^ - 0.18

P

= 0.01

P

0.34

P =

= 0.24

P 0.04

Mean Frond

.* Rhizome-

■root

rs2

- -0.04

rs^ -

0.08

rs2 =

= 0.03

r s^ - 0.09

Biomass

P

= 0.36

P

0.22

P =

= 0.39

P - 0.21

Growth rate

rs2

= -0.02

rs^ -

0.19

rs^ -~

= O-Il

r s^ - -0.08

P

= 0.49

P -

0.04

P =

= 0.16

P 0.23

Reproductive

Effort

rs2

= -0.17

rs2

0.16

rs2 --

= -0.28

r s2 - 0.09

Mean RE 1

P

= 0.09

P

0.10

P =

= 0.0 1

P - 0.24

1995 RE 2

rs2

= 0.01

r s^s —

0.08

rs^ --

= -0.24

r s2 = -0.04

P

= 0.46

P -

0.29

P =

= 0.04

P - 0.39

a substantial overlap in 1994 + 1995 plant biomass indicates that frequency
of reproduction was not entirely a function of plant size (Figure 6). Sequential
reproducers also grew more rapidly than non-sequential reproducers (Table 3).
Moreover, the mean size of sequential reproducers (Figure 6) corresponded
with the beginning of an asymptotic increase in growth rate (Figure 5), indi-
cating the ability to reproduce in consecutive years was associated with a high
rate of growth.

Sequential reproducers exhibited greater 1994 + 1995 RE 1 and greater RE
2 than non-sequential reproducers (Table 3), indicating, non-sequential repro-
ducers did not compensate for non-reproductive years by increasing allocation
to reproduction in reproductive years. Sequential reproducers also exhibited
greater growth / 1994 RE 1 than non-sequential reproducers (Table 3), indi-
cating sequential reproducers suffered a smaller cost of reproduction than non-
sequential reproducers. Among non-sequential reproducers, individuals that
reproduced during only 1994 exhibited greater growth rates than those that
reproduced during only 1995 (Table 4). Thus, reproduction was associated
with a cost to growth and, subsequently, the probability of reproducing in the
following year.

Sequential reproducers possessed lower PCA 1 and greater PCA 4 scores
(Table 3) than non-sequential reproducers, indicating environments lower in
soil cation concentrations and higher in phosphorus were conducive to repro-
duction (Figure 4a-b].

Discussion

Vegetative Allometry. — As sporophytes of P. acrostichoides develop be-
md an establishment nhase, biomass allocation annears to switch from fa-

68

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

E
o

4.5

4.3

P 4.1

D)

O
I

(D

3.9

o

3.7

T

T

T

T

T

T

2 ^ ^^3

Y = 3.85 + 1.17X - 3.16X + 2.95

r

0.37, P < 0.001

1

1

1

1

X

1

1

1

1

<

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1994 Plant biomass (In g)

Fig. 5. Cubic model of 1994 plant biomass prediction of above-ground growth rate (i.e., A Laminar
area). The arrow labeled "R" marks the size at first reproduction determined in Figure 3, whereas
the arrow labeled "S" marks the mean size of sequential reproductives (see Figure 6). Growth
rates initially increase with plant size during establishment. Reproduction among smaller indi-
viduals and/or those in less favorable habitats, may drain resources sufficiently to affect growth,
resulting in sporadic (i.e., non-sequential) reproduction and a plateau in growth rates. At some
point determined by genotype X environment interactions, resource acquisition and storage may
become sufficient to consistently overcome the cost of reproduction to growth.

voring the rhizome-root mass to fronds. The proportion of plant biomass com-

among

among

age of plant biomass composed of rhizome-root mass reported here [43.5%
40.2%) is considerably lower than the 19.0% ± 5.4% root and 60.8% ± 5.8%
rhizome reported for this species by Minoletti and Boerner (1993). These dif-
ferences may have resulted from differences in plant sizes and/or because we
removed necrotic tissues from the distal end of the rhizome as v^ell as the
relatively large, persistent, frond bases. Early investment to the rhizome-root
mass is very likely a trait that has evolved in response to factors that limit
survival during establishment. These might include limitation of below-
ground resources due to competition, herbivory, environmental composition,

and

ophyt

most

primary storage organs. The fronds of P. acrosticboides are evergreen, photo-

3aith

throughout the autumn, winter, and spring (Minoletti

important in reducine rates of nutrient loss and

maintainin

semi-evergreen, may

environmental

GREER & MCCARTHY: GROWTH, REPRODUCTION IN P. ACROSTICHOIDES

69

Table 2. Mann-Whitney U rank sum tests of morphological traits and PGA axes representing environ-
mental gradients (see text) between reproducti\es (R = 79) and non-reproductives (NR = 41). Ranks are
ascending.

TRAIT/PCA AXIS

Plant Biomass (1994 + 1995)

STATUS

NR
R

MEAN
RANK

34.3
71.3

SUM OF

RANKS

1339
5564

U, P- VALUE

U = 559
P < 0.001

Mean Frond : Rhizome-root
Biomass

NR
R

39.4
68.8

1536
5367

U = 756
P < 0.001

Growth Rate

NR
R

52.6

62.2

2053
4850

U = 1273
P = 0.08

Reproductive Effort
PGA 1 (Ga, K, Mg)

NR

R

47.2

45.5

1274
2912

U = 832
P = 0.39

PGA 2 (H,0, NO3)

NR
R

51
43.9

1377
2809

U = 842
P = 0.12

PGA 3 (Ganopy)

NR
R

46.8
45.6

1264
2922

U = 842
P = 0.43

PGA 4 (P>

NR
R

44.0
46.8

1189
2997

U = 811
P = 0.65

tions» and may explain why rhizome-root biomass was a poor predictor of
frond biomass. If fronds are the primary storage component in larger plants,
then the rhizome may function primarily as a genitor of new fronds and roots.
This architectural strategy may be common among fern species with evergreen
fronds, particularly those with short-creeping or erect rhizomes.

Relationships Between Plant Size, Growth Rate, and Reproduction. — Re-
production in P. acrosticboides appears to be size dependent. A minimum size
at reproduction was apparent; reproducers and sequential reproducers were
significantly larger than their respective counterparts, and reproductive effort
increased with size. A general relationship between plant size and reproduc-
tion has been well established for a variety of species (Sohn, 1977; Pitelka et
al., 1980; Young, 1981; Lee and Hamrick, 1983; Willson, 1983; Lacey, 1986;
Samson and Werk, 1986; Lotz, 1990; Primack and Hall, 1990; Thompson et al.,
1990; Hanzawa and Kalisz, 1993; Linvelle, 1995), and evidence for a minimum
size at first reproduction and a relationship between size and frequency of
reproduction have been reported for a few iteroparous perennials (Young,
1981; Samson and Werk, 1986; Hanzawa and Kalisz, 1990; Primack and Hall,
1990). Nevertheless, the overlap in plant biomass observed between non-re-
producers and reproducers, and between non-sequential and sequential repro-
ducers, indicate reproduction in R acrosticboides is not determined entirely
by size.

70

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

100

o

50

-

Q_
in

CD

+

CT>

-50

hJorvreprcxIuctives

Reprocfcjctives

Sequentials

Non-sequenfials

Fig. 6. Box plots of 1994 + 1995 plant biomass (PB) for non-reproductives, reproductives, non-
sequential reproductives, and sequential reproductives. Horizontal lines mark the following in
increasing order, mean-2* standard deviation, mean-2* standard error, mean, mean -(-2* standard er-
ror, mean -I- 2* standard deviation.

Three observations indicate that reproduction in P. acrostichoides occurs
only when stored resources exceed that necessary to maintain a threshold rate
of growth. First, reproducers possessed higher growth rates than non-repro-
ducers. Second, sequential reproducers possessed higher growth rates and suf-
fered lower costs of reproduction to growth than non-sequential reproducers.

amon

reproducers; i.e., those that reproduced in 1994 exhibited significantly greater
growth rates than those that reproduced in 1995. Similar models of growth-
limited reproduction have been suggested for iteroparous perennials by Reekie
and Bazzaz (1987c), Saulnier and Reekie (1995), Kozlowski (1992), and Galen
and Stanton (1993). According to these models, the curvilinear relationship
between growth and plant size may be a function of the threshold of resources
necessary for both growth and reproduction.

The value of reproduction is expected to increase with age in iteroparous
plants (Kozlowski, 1992; Stearns, 1992). Consequently, iteroparous plants are
expected to gradually increase the proportion of resources that are diverted to
reproduction with age, or to reach a maximum size, beyond which all resourc-
es are diverted to reproduction (Kozlowski and Uchmanski, 1987; Pugilese and
Kozlowski, 1990; Kozlowski, 1992; Stearns, 1992; Worley and Harder, 1996).
Assuming that size corresponds with age, our observations do not support to
these models. We observed no evidence of a maximum size and growth rates
increased with size, particularly among the largest individuals. Furthermore,

1 . ■ -• «

amon

metabol

minor increases in reproductive allocation were associated with increases in
plant size (i.e., 0.01% to 2.11% of total biomass). Similar patterns of sporadic
reproduction and low and/or constant reproductive allocation were reported

GREER & McCarthy: growth, reproduction in p. acrostichoides

71

<D

in

+

10

30

50

70

90

1994 + 1995 Vegetative biomass (g)

Fig. 7. Regression analysis of 1994 + 1995 vegetative biomass prediction of 1994 + 1995 fertile
segment biomass.

for Chamaenerion angustifolium (Van Andel and Vera, 1977) and Cypripedium
acaule (Primack et al., 1994).

Relative construction costs between vegetative and reproductive tissues
probably remain constant in most ferns, since they lack the ancillary structures
that complicate this relationship [e.g., peduncles, petals, or ovary walls);
whether they selectively abort or provision spores is less clear. This is partic-
ularly true for species with monomorphic fronds. Nevertheless, life history and
demographic traits such as growi:h, reproduction, and survival, may be more
useful measures of the cost of reproduction than limiting metabolic currencies,
which are difficult to identify and vary among organs, individuals, popula-
tions, and environments (Ricklifs and Miles, 1994; Muir, 1995; Bazzaz, 1997).
Assessing the life history and demographic costs of reproduction is also more
straightforward for most ferns than seed plants, because, reproduction does
not result in the loss of a meristem that could otherwise assist vegetative ac-
tivity (with the exception of ferns with indeterminate and clone-forming
fronds). However, phenological patterns of development may complicate the
analysis of resource allocation. Species that reproduce early in the growing
season often initiate new vegetative reproductive structures a year or more in
advance of emergence (Gerber et al., 1997).

Costs of reproduction were evident among both sequential and non-sequen-
tial reproducers as reductions in growth rates and the likelihood of reproduc-
ing in the following year. A number of studies have also reported costs of
reproduction to survival, growth, the amount of resources allocated to repro-
duction, and the likelihood of reproducing in the following year (Sohn, 1977;
Reekie and Bazzaz, 1987c; Pugilese and Kozlowski, 1990; Fox and Stevens,
1991; Muir, 1995; Worley and Harder, 1996; Gerber et al., 1997). Sporadic

72

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000J

Table 3. Mann- Whitney U rank sum tests of morphological traits and PCA axes representing environ-
mental gradients between non-sequential (NS = 31) and sequential (S = 48) reproducers. Ranks are

ascending.

TRAIT/PCA AXIS

Plant Biomass (1994 + 1995)

Mean Frond : Rhizome-root
Biomass

Growth Rate

Reproductive Effort
1994 + 1995 RE 1

1995 RE 2

Cost of Reproduction
(Growth rate/RE 1 )

Environment
PCA 1

PCA 2

PCA 3

PCA 4

STATUS

* 1994 non-sequentials only; N

15

NS
S

NS
S

NS
S

NS
S

NS
S

NS
S

NS
S

NS
S

NS
S

NS
S

MEAN
RANK

30.5

45.1

34.1
42.8

31.4
44.6

31.9
44.3

27,4
36.2

17.5
35.0

37.2
29.1

30.3
34.1

33.0

32.2

27.6
36.1

SUM OF
RANKS

915
2166

1023
2058

942
2139

956
2125

822
1194

245
1646

1005
1075

819
1261

889
1191

746
1334

U, P- VALUE

U = 450
P < 0.002

U - 558
P = 0.05

U - 477
P = 0.01

U = 491
P = 0.01

U = 357
P = 0.03

U

140

P < 0.001

U = 372
P = 0.04

U = 441
P = 0.22

U = 488
P = 0.44

U = 368
P = 0.04

common amon

maintenance

^ECTS ON Allocation Patterns.— No patterns in mean
biomass ratio were observed relative to environmental gra-

observations
and ]

ground

In contrast, allocation to reproduction may be plastic in P. acrostichoides;

with

reproducers were more frequent in habitats low in cations and high in phos-

observations

and Monk

GREER & MCCARTHY: GROWTH, REPRODUCTION IN P. ACROSTICHOIDES

73

Table 4. Mann- Whitney U rank sum tests of morphological traits between 1994 and 1995 non-sequcn-
tial reproducers (NS). Ranks are ascending

TRAIT

STATUS

Plant Biomass (1994 + 1995)

1994 NS

1995 NS

MEAN
RANK

15.3

15.7

SUM OF
RANKS

230
235

U, P- VALUE

U

no, P = 0.92

Mean Frond : Rhizome -root Bio-

mass

1994 NS

1995 NS

16.7
14.3

251

214

U = 94, P = 0.44

Growth Rate

1994 NS

1995 NS

19.1
11,9

286
179

U = 59, P = 0.01

Greer, et al., 1997) and the hypothesis that reproduction in this species occurs
only when a threshold growth rate can be maintained. Given the above, growth
rate is predicted to be greatest in habitats characterized by sparse canopy, low
cation concentrations, and high phosphorous concentrations. However, no re-
lationship between growth rate and any of these environmental gradients was
evident. Increased allocation to reproduction in favorable habitats may have
maintained growth rates near a minimum threshold. Plasticity of reproductive
effort, resulting in increased sexual and asexual reproduction in favorable hab-
itats, was also reported for Dennstaedtia punctilobula (Hammen, 1993).

In contrast to the positive correlation between growth and reprodutive effort
among sporophytes, the gametophytes of P. acrostichoides increase reproduc-
tive effort in habitats that are unfavorable to rapid growth (Greer and McCar-
thy, 1999). This opposing pattern in plasticity of reproductive effort may be
common among species of ferns where the gametophyte is ephemeral and the
sporophyte long-lived (Greer and McCarthy, 1999).

A Model of Life History Evolution in Polystichum acrostichoides. — Like

many iteroparous plants, the colonizing stages of ferns (i.e., gametophyte and
juvenile sporophyte) have much greater risks of mortality than mature sporo-
phytes (Peck et al., 1990). Under these circumstances, fitness is highly corre-
lated with life time fecundity, and the value of reproduction is expected to
increase with age (Harper, 1977; Willson, 1983; Steams, 1992). Nevertheless,
rates of reproductive success may be sufficiently low in P, acrostichoides and
many other pteridophytes, that life time fecundity, and therefore fitness, is
maximized through essentially "immortal" life histories that maintain growth
across all age classes. Indeed, low reproductive output is favored when envi-
ronmental heterogeneity affects reproductive success more than adult survival
(Murphy, 1968; Pianka, 1972; Schaffer, 1974; Goodman, 1979)— a likely con-
dition among most pteridophytes.

Environmentally induced heterogeneity in recruitment favors plasticity in
reproductive allocation (Hirshfield and Tinkle, 1975). Even when conditions
that confer a fitness advantage to early or delayed reproduction are infrequent,
the onset of reproduction may be optimized through phenotypic plasticity

74 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

(Travis, 1994). Thus, plasticity rather than simple allometric relationships may
account for much of the variation in age and size at first reproduction (Stearns,
1992), and variation in size at first reproduction may account for much of the
suhsequent variation in allocation (Samson and Werk, 1986; Herndon, 1987).
Physiological and reproductive plasticity that facilitate growth and longevity
may explain the wide distribution and abundance of P acrostichoides in East-
ern North America.

Although observations from unmanipulated studies of natural populations
are ambiguous, this study provides essential baseline data for experimental
studies of allometry, resource allocation, and phenotypic plasticity in P. ac-
rostichoides.

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American Fern Journal 90(2]:77-86 (2000)

The Effects of Rhizome Severing and Nutrient
Addition on Growth and Biomass Allocation in

Diphasiastrum digitatum

Carrie A. Railing and Brian C. McCarthy*

Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, U.S. A

Abstract. — The effects of severing and fertilization on ramets of Diphasiastrum digitatum in an
Ohio hardwood forest were examined to determine the extent of young ramet integration. The
extent of integration was measured by overall vigor, and by architectural biomass, and growth
variables. Vigor decreased with any plant manipulation. Likewise, all measured architectural and
biomass variables were smaller in manipulated treatments. Similar negative effects in growth and
biomass were seen with severing or fertilization, while a combination of both showed a further
decrease in growth variables compared to either of the individual treatments. Severing reduced
growth and biomass fifty percent compared to the control, indicating that integration is important
in young multi-module ramet development. The results are consistent with other studies sug-
gesting clonal lycopods are highly integrated and emphasizes the importance of integration in
young ramets of D. digitatum for survival and growth.

Clonal plants are constructed of basic units, called modules, that are iterated
throughout the development of a clone and can potentially function as inde-
pendent members (ramets] of a genetic individual (genet) (Harper, 1977). The
existence of connections between ramets or modules of a clone is inherent to
its architecture. These underlying vascular connections facilitate the move-
ment and sharing of plant substances (Pitelka and Ashmun, 1985), and thereby
permit the clone to function as a physiologically integrated system (Watson
and Casper, 1984). As long as connections persist among modules, there is a
potential for resource movement (Marshall, 1990; Pitelka and Ashmun, 1985).
However, a great deal of variation in the degree of integration within and
among clonal plant species has been demonstrated (Jonsdottir and Watson,
1997). Specifically, clonal plants may vary in their longevity and/or function-
ality of connections (months to decades), the substances that are moved (min-
erals, nutrients, carbohydrates, photoassimilates, hormones), and the direction
of flow, whether acropetal, basipetal, or bidirectional (see references in Hutch-
ings and Bradbury, 1986; Jonsdottir and Watson, 1997; Klimes et al., 1997;
Marshall, 1990; Pitelka and Ashmun, 1985).

Such physiological integration can be a major factor influencing survival of
clonal plants that retain ramet connections for any period of time (Eriksson
and Jerling, 1990; Jonsdottir and Watson, 1997; Pitelka and Ashmun, 1985).
Adaptive advantages incurred by ramets participating in such integration in-
clude successful establishment and survival, accelerated maturity, and local-
ized stress recovery (Caraco and Kelly, 1991; Hutchings and Bradbury, 1986;
Jonsdottir and Watson, 1997; Pitelka and Ashmun, 1985). The significance of
these adaptive features are seen in cases where disintegration, or fragmenta-

78

J ME

occur,

mental

through

Ashmun, 1985). However this fragmentation or disintegration occurs, it has
consequences on the growth, biomass and overall survival of the plant in ques-
tion, most of which remain relatively unexplored in a number of clonal plant
species (Charpentier et al., 1998).

Although physiological integration in clonal plants has attracted a great deal
of attention in recent years (see references in Marshall and Price, 1997; Jons-
dottir and Watson, 1997; Pitelka and Ashmun, 1985), most studies have fo-
cused on angiosperms rather than lower plants such as pteridophytes. Exam-
ining the clonal patterns and processes of integration in pteridophytes offers
not only a complete view of the plant kingdom, but also offers an evolutionary
view of integration in lineages of clonal plants that have existed in many hab-
itats and geologic time periods (Dyer, 1979). In particular, few studies have
investigated the extent of physiological integration in lower plants such as
lycopods (Callaghan, 1980; Carlsson et al., 1990; Headley et al., 1985; ig88a;
1988b) and those that do concern lycopods involve relatively few, common
species that do not reside in North America. Diphasiastrum digitatum (Dillen-

that

American

Watson

1997; Lau and Young, 1988). Previous examination of this species' physiolog-
ical integration has focused on the survival of older modules (Lau and Young,
1988), whereas generalized studies of clonal plants indicate that younger ra-

mets

Bradbury

In this study, we examine the effects of severing and nutrient addition on
newly developing ramet growth in Diphasiastrum digitatum in order to deter-
mine the general extent of its physiological integration and to determine how
it directly responds to disintegration from the parent clone and to a localized
increase in nutrients. We hypothesize that during early development, artifi-
cially severing rhizome connections to the parent will significantly decrease
the vigor, growth, and biomass of young ramets. Furthermore, we hypothesize
that adding nutrients to the sterile edaphic environment characteristic of D.

ramets

Methods and Materials

Organism.— Diphasiastrum

form

(Wagner and Beitel, 1996) that forms
e growth. This evergreen oerennial dis

fan-like, dimorphic (vegetative and reproductive) vertical shoots and adven-

■ • ■ ' «-^fc ■ 1 # _ « _

(Wagner and

common

RAILING & MCCARTHY: RHIZOME

79

habitats, including hardwood and conifer forests as well as open shrubby areas
(Cody and Brltton, 1989; Wagner and Beitel, 1996). Many members of this and
other closely related genera in the temperate region are characterized as weedy
and are believed to grow in relatively infertile soil conditions (Page, 1979;
Tryon and Tryon, 1982).

The life span of most vertical shoots is approximately four to six years (Pri-
mack, 1973; Railing and McCarthy, unpublished data). Rhizome connections
may persist for some time before decaying and thereby separating previously
connected ramets. This type of vegetative reproduction has been noted as more
common than sexual reproduction (Primack, 1973) in Diphasiastrum digita-
tum, most likely reasoned so because of the unsuccessful or poor spore ger-
mination witnessed in gametophyte cultures, rather than in nature (Roberts
and Herty, 1934; Whittier, 1981; 1998).

Study Area. — Strouds Run State Park (SRSP) is located in Athens County,
Ohio at 39^20'N (latitude) and 82^5'W (longitude), in the southeastern foothills
of the state. In the early twentieth century, the area was disturbed by grazing
and timbering activities. As such, most of the forests are considered to be even-
aged second-growth. What is now known as SRSP was acquired for conser-
vation purposes in the late 1940's and early 1950's by the State of Ohio and
eventually became a state park in 1959. The park spans over 1100 ha on Ap-
palachian Ohio's unglaciated Allegheny plateau, and is centered around a man
made watershed, Dow Lake. The topography consists of a number of steep
hills and ravines believed to be remnants of glacial meltways. Soils in this
region of Ohio are classified as Inceptisols (Brady, 1984) and soil conditions
throughout the park are characterized as being moderately deep to deep, well-
drained, and mostly loamy (typic hapludalfs), originating from sandstone, silt-
stone, shale, and limestone parent material (National Cooperative Soil Survey,
1985). Precipitation is relatively evenly distributed throughout the year and
ranges from 5—10 cm per month, with July being the wettest month and Oc-
tober the driest. Mean temperatures (high/low) are 29/18 C for July and 2/— 8
C for January (Midwestern Climate Center, 1999).

The vegetation of SRSP is primarily deciduous forest cover and can be clas-
sified as mixed mesophytic (^cer spp., Aesculus octandra, Fagus grandifolia,
and Liriodendron tulipifera) in the lowlands and mixed oak or oak-hickory in
the uplands [Quercus alba, Q. rubra, Q. velutina and Caryo glabra, C. ovata),
A number of white pine plantations [Pinus strobus) are also present in the
park (<10% of vegetation), having been planted by conservation corps for
watershed protection. There are also sporadic old-fields, grassy meadows, and
maintained turf grass habitats. The herbaceous layer of the woodlands is large-
ly dependent upon the overstory vegetation and there is considerable diversity
of both microhabitats and species (Payne, 1957; Greer et ah, 1997).

Field Methods. — Ten Diphasiastrum digitatum patches were located within
SRSP. In June 1998, 4 apical rhizome tips, approximately 1-2 m apart, were
haphazardly located around the perimeter of each patch. Rectangular quadrats
(0.25 X 2 m) were established around each of the four rhizome tips such that

80 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 2 (2000)

the rhizome was situated 0.5 m into the quadrat along the longest dimension.
Quadrats within each patch were randomly assigned to one of four treatment
groups. The first treatment group (Cj served as the control, and remained un-
manipulated throughout the duration of the experiment. Rhizomes in the sec-
ond treatment group (S) were severed at their entrance into the rectangular
quadrat. Quadrats assigned to the third treatment group (F] were treated with
a standard 10-10-10 inorganic fertilizer (Dragon All Purpose Plant Food) at a

prescribed monthly rate (ca. 100 g fertilizer per m^) for perennials. Rhizomes
in the fourth treatment group (SF] were subject to both severing and fertiliza-
tion treatments. Other above ground vegetation within quadrats was clipped
and treatments were applied after quadrat establishment in June 1998.

In August 1998 vigor observations were made in situ before excavating all
rhizomes. Rhizomes were assigned to one of three vigor categories that were
based on health and color. High vigor (HI) indicated a rhizome or ramet with
high health and a green to bright green color, similar to the rest of its original
patch. Moderate vigor (MD) and low vigor (LO) referred to a ramet that dis-
played moderate health and green to yellow color and poor health with yellow
to brown color, respectively. Excavated ramets were subsequently measured,
oven dried at 80 **C for ca. 48 hours and weighed. Data were collected to reflect
growth and biomass patterns since the initial treatment application. Variables
examined upon ramet collection include total growth length (cm), number of
new shoots and roots initiated, mean internode and shoot length (cm), and
aboveground (internode and shoot) biomass (g) for each ramet.

Analytical Methods. — A G-test (Chi-Square Goodness-of-Fit Test) was used

to analyze the homogeneity of vigor categories among treatment groups (Sokal
and Rohlf, 1995) with the null hypothesis that there are no differences in ramet
frequency among the twelve possible classes (i.e., treatment/vigor combina-
tions). In order for G to better anDroximate the chi-sauare statistic, the Wil-

and

(1995).

Multivariate Analysis of Variance (MANOVA)

significant treatment effects across all growth and biomass parameters mea-
sured using the Wilk's Lambda test statistic (Hintze, 1997). A randomized com-
plete block design MANOVA was employed using treatment group as a fixed
effect and patch as a random effect. Data sets were checked for normality, equal
variance, and equality of covariance using the D'Agostino Omnibus test

Modified-Levene Equal- Variance test, and
he assumptions of MANOVA. Those failini

transfer

mations.

Planned (a priori) comparisons (Dowdy and Wearden, 1991) were used to
test for significant differences among treatment groups according to the three
hypotheses. We used the first hypothesis (HJ to test for significant differences
between the control and all other treatments (C vs. S,F,SF), the second (H^) to
test for differences between treatment types (S vs. F) and the third (HJ to test

RHIZOME

81

Table 1. Observed frequency of ramets within each treatment group according to vigor category, in-
cluding G-lest (Chi-Square Goodness-of-Fit) results. Note that when the cell element marked with an
asterisk is removed, G = 14.8 and P > 0.1.

Vigor Category

Treatment Group HI MD LO

Control (C)

Sever (S)

9* I

5 5

Fertilize (F) 3 6 1

Sever and Fertilize (SF) 4 6

G = 24.9, P < 0.01

for an additive effect of treatment types (SF vs. S,F). The above planned com-
parisons were only used on those variables significantly contributing to treat-
ment effect within the MANOVA. All statistical analyses mentioned above
were conducted using NCSS statistical software (Hintze, 1997) with an alpha
of 0.05 used to test hypotheses.

Results

The results of the G-test used for the vigor study indicated that significant

differences (P < 0.01) among vigor-treatment combinations existed (Table 1),
thereby rejecting the overall null hypothesis and indicating that Diphasiastrum
digitatum ramets within vigor categories did reflect a treatment effect. How-
ever, upon closer examination, one class (Treatment C — HI vigor) may be driv-
ing the significance in this analysis (Table 1), In an exploratory procedure, the
aforementioned class was removed from the analysis to test for significant dif-
ferences among the remaining other classes. The resultant G-Test indicated
that the classes were then homogeneous (Table 1). Hence, the control group
displayed the highest vigor, and any perturbation (treatments S, F, SF) of the
ramets affected their health and coloring, reducing vigor to either the moderate

(MD) or poor (LO) categories.

Although there are no significant (P = 0.347) differences among patches for
the measured variables (Table 2), there are significant differences among treat-
ment groups (P = 0.044). Individual one-way ANOVAs for each of the growth
and biomass variables in the MANOVA analysis indicate that shoot biomass,
total growth, new shoots initiated and new roots initiated are the specific var-
iables measured that reflect significant differences among treatment groups
with high power (Table 3). In addition, relatively little to no significant differ-
ences in mean internode and shoot height among treatment groups were wit-
nessed (Table 3). However, confidence in this finding is low due to the minimal
statistical power in the respective F-tests. In order to meet the multicollinearity
assumption of MANOVA, both above ground biomass and internode biomass
variables were dropped from the analysis and examined seperately using one-
way ANOVAs. Above ground biomass (P = 0.004) and internode biomass (P
= 0.002) did differ significantly among treatment groups (Table 3).

82

FERN JOURNAL: VOLUME 90 NUMBER

Table 2. Multivariate Analysis of Variance (MANOVA) test for differences among treatments and
patches across all variables measured. Above-ground biomass and intcmode biomass variables were re-
moved to meet multicollinearity assumption.

Wilk's

Source Lambda dfl df2 F-ratio P-value

Treatment 0.306 18 63 L81

Patch 0.125 54 117 L09

0.044
0.347

Overall, the plants in any manipulated group (S, F, or SF] displayed a 50%
or more decrease in growth and biomass (Fig. 1). The first planned comparison
[H,: C vs. S,F,SF) was significant across all variables examined (P < 0.01] with
any manipulation of the rhizomes leading to a decrease in the measured var-
iables (Fig. 1). There was no significant difference between treatment types for
all variables as indicated by the second planned comparison (H2: S vs. F).
Results of the third planned comparison {H3: SF vs. S,F) indicate that only the
new shoots initiated (P - 0.03) and new roots initiated (P = 0.08) showed an

treatment

Discussion

The fact that severing showed a decrease in growth and biomass compared

ramets

show dependency upon parental connections and are therefore highly inte-

number

losperms

biomass in severed versus non-severed ramets (Ashmun et al., 1982; Charpen-

Watson

ever, the severity of such disintegration often depends upon the size and age
of the ramet severed from the parent clone

ramet

Jonsdottir and Watson

(Hartnett

when comparing the current study using younger, interconnected ramets to

ramets

1988) the effects of severing are essentially the same. This suggests that the
increased hardship likely incurred by younger ramets is not seen when those

ramets

Jonsdottir and Watson (1997} classify Diphasiastrum digitatum as displaying
full integration in large clonal fragments, and in turn place other pteridophytes
and angiosperms within this category. Bracken {Pteridium aquilinum (L.)

digitatum

number of growth

more

that grow by means of a perennating, indeterminate rhizome

Werth

1993). Experiments originally aimed at finding a method of controlling bracken

RAILING & McCarthy: rhizome severing

83

Table 3. Results of one-way ANOVA tests for variables significantly contributing to treatment effect
in previous MANOVA. Asterisks denote one-way ANOVAs for two variables originally omitted from
MANOVA.

Variable Measured

Above-Ground Biomass (g)*
Intemode Biomass (g)*
Shoot Biomass (g)
Total Growth (cm)
Mean Internode Length (cm)
Mean Shoot Height (cm)
Number New Shoots
Number New Roots

df

3
3
3
3
3
3
3
3

MS

0.28
0,57
1.69
0.37
0.51
0.50
9.90
0.34

F-ratio

5.71

6.33
6.92
4.69
0.10
2.69
8.36
5.67

P

0.004
0.002
0.001
0.009
0.959
0.066
0.001
0.004

Power

0.82
0.86
0.89
0.73
0.06
0.47
0.95
0.82

have revealed that severing bracken rhizomes does not ehcit the same severe

and

concerning D. digitatum (Lau and Young, 1988). In fact, seasonally-timed, re-
petitive cutting of above and below ground rhizomes, as well as above ground
fronds of bracken is needed to significantly affect respiration and assimilate
translocation and ultimately reducing the plant's spread within an area (Low-

reaffirms the

anoth

dophyte.

Comparisons between the physiological integration of the mayapple [Podo-
phyllum peltatum, L.) and D. digitatum are also useful as these two herbs often
occupy similar habitats in the eastern deciduous forest and are frequently
found growing near each other. Although the P. peltatum tends to have a great-
er degree of lateral spread than D, digitatum, both are extensive integrators
(Jonsdottir and Watson, 1997) that reproduce mainly through vegetative pro-
cesses, and thereby are severely affected by severing treatments that interfere
with acropetal transport to younger ramet fragments in particular. However,

ramet

ment severed (Tonsdottir and Watson

ramets

same reduction in growth

■^ ^^ s^

comparison suggests that Dipbasiastrum digitatum is dependent upon integra-
tion at any scale within a ramet, indicating that integration is an adaptive trait,
possibly because of the resource-poor environments it inhabits (Jonsdottir and
Watson. 19971.

treatment

TOWth

treatment group (S) compared to the control group (C). This resuh does not

and

biomass of Diphasiastrum digitatum. Upon closer examination of the experi-
mental methods, the nitrogen source within the fertilizer may be a possible
explanation. The fertilizer used within the current experiment delivered ni-
trogen in two forms: ammonium nitrogen f3.91%l and urea nitropen ffi.n9%V

84

AMERICAN FERN JOURNAL: VOLUME

o ^

a

-H

V)

o

CiO

o

60

40

20

12

8

4

4
3
2
1

Hi**

C S F SF
Treatment Group

■o

1.25

v:

2 a

o o

o CQ

-H

"So

0.75

<

0.25

o

B

o

-H

CO

0.4
0.3
0.2
0.1

a>

o

E

C

is

0.8
0.6
0.4

0.2

C S F SF
Treatment Group

Fig. 1. Mean values (± 1 standard error) of growth and biomass variables under each of four
treatment conditions (A = control, B = severed, C = fertilized, D = severed and fertilized). Sig-
nificance levels for each of the three planned comparisons, (H,, H^, HJ, are denoted as such: ***

= P < 0.001,

= P < 0.01,

P < 0.05,+ = P < 0.1.

A number of studies suggest that nitrate is the ideal deliverable form of nitro-
gen in fertilizers, whereas ammonium and urea forms can negatively affect
health of some plants, particularly in combination with lower edaphic pH

Ma

further

ammonium

accumulation of ammonium

plants

ammonium

accompanied by plant wilting, chlorosis, and necrosis of above-ground parts
(Cao and Tibbitts, 1998; Kpodar et al., 1992). Similar symptoms of ammonium
toxicity were observed in this study for the two treatment groups receiving
fertilizer (F and SF).

For the most part, studies concerning physiological integration in lycopod
species have focused strictly on documenting water and/or nutrient movement
and storage throughout the organism (Callaghan, 1980; Headley et al., 1985;

I ■ I V ■ I ■ _^^ ^^H fl « H 1 F S ■ ■ ^r 1 ■ ■ ■ ^ ^m ^H ^ ^H ^M "^h

further

ramet

lycopod or clonal plant survival in addition to determining short and long-

plant architectural

examinations would lend greatlv to the

RAILING & MCCARTHY: RHIZOME SEVERING IN DIPHASIASTRUM DIGIT ATUM 85

understanding of the presence of clonal plants throughout evolutionary and
ecological history.

Acknowledgments

This study was funded in part by the Department of Environmental and Plant Biology at Ohio
University, Athens, Ohio. We would like to thank Robert Madden and Teresa Dennis for their help
in field work and data collection.

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88:106-113.

American Fern Journal 90(2):87-89 (2000)

Obituary: Joseph A. Ewan

(1909-1999)

The American Fern Society lost a longtime member and supporter on the
morning of Sunday, December 5, 1999, when Joseph Ewan passed away peace-
fully at the age of 90. Joe was a fine botanist and plant taxonomist, but also
was reknowned as one of the leading botanical historians of our time and a
skilled commentator on the literature of natural history and exploration.

Born on October 24, 1909, in Philadelphia, Joseph Andorfer Ewan, lived
briefly in Pennsylvania and New Jersey, before moving to California in 1912.
In high school, he was influenced strongly by a librarian and teachers in bi-
ology and Latin, which in 1928 led him to enroll as an undergraduate at the
University of California, Los Angeles and later the University of California,
Berkeley, from which he graduated in 1934. He worked at the UCLA library
and Los Angeles County Public Library during these years to help fund his
studies. Continuing graduate studies at Berkeley allowed him to work as a
research assistant under Willis Linn Jepson, but a variety of difficulties, in-
cluding the birth of the first of three daughters and other financial hardships,
forced him to seek more lucrative emplojinent elsewhere. In 1937, Joe ac-
cepted his first professional appointment as a lecturer in the biology depart-
ment at the University of Colorado, in Boulder, where he remained until 1944.
There followed a series of appointments, including a year in Colombia with

88

FERN JOURNAL: VOLUME

overnment's World War

sources for the fight against malaria, as well as subsequent appointments at
the Smithsonian Institution and the U.S. Department of Agriculture. In 1947,
Ewan accepted a faculty appointment at Tulane University, in New Orleans!
where he remained until his retirement in 1977. In 1972, Tulane awarded him
the Ida Richardson professorship in biology, in which he continued in an

emeritus

During his more than three decades at Tulane, Ewan spent several terms as
visiting professor at other schools, such as the University of Hawaii, University
of Oregon, and Ohio State University. He also was the recipient of a Guggen-
heim Fellowship in 1954 to allow him to complete research at the British
Museum, as well as receiving a Regent's Fellowship at the Smithsonian Insti-
tution in 1984. Although he was unable to realize his earlier dream of obtain-
ing a graduate degree in botany, Ewan was awarded two honorary doctorates,
one from The College of William and Mary (1972) and the other from Tulane
University (1980). He was a fellow of the Linnaean Society of London and

Medal

Missouri

Medal

While taking a botany course as an undergraduate at UCLA, Joe Ewan met
a student of Canadian descent who would change his life immeasurably, Nesta

Dunn (1908-)

him

J

J

their ever-growing library over the years. Their final project, a bibliographic

commentary

Miss

future

Joe and Nesta Ewan's interest in the history of natural history led them to
assemble huge quantities of correspondence and biographical files. Their fas-
cination with history and naturalists also led to a keen interest in the books
themselves, and they acquired a large personal library. These became well
known and were frequently consulted by scholars from around the world,
much to their delight. The book collection is impressive not only for its size!
but also the "special" nature of some of its contents. For example, the copy of
William Jackson Hooker's Genera Filicum (an 1858 printing, handsomely
leatherbound) is one that was presented by the author to his father-in-law.
Dawson Turner, and a letter from Hooker and a
tipped in by Turner.

After Joe retired from Tulane, there came a time when the future of these
collections was in doubt, particularly as there appeared no ideological suc-
cessor at the university. After investigating various options, the entire collec-
tion was sold to the Missouri Botanical Garden in 1986. Arrangements were
made for the Ewans to join their treasures in St. Louis and continue research
projects with them. At that time the library contained some 4,600 volumes and
had to be removed from the facility New Orleans through an unner stnrv win-

youthful

OBITUARY

89

dow using a large fork lift. Since arriving in St. Louis, another 600 titles have
been added to the library, vi^hich continues to be stored as a separate special
collection. The Ewans' papers, correspondence, and other biographical files

M. Rilev. and M

Missouri

stated by the compilers to occupy some 88 linear feet of space. In 1997, fol-
lowing Joe's stroke, the Ewans decided to lay dovirn their books and moved to

J

Mandeville, Louisiana, near their daughter's family

Keith

of Joe and Nesta Ewan, The American Botanist, Chillicothe, IL, 1989) His re-
views and commentary of other publications are treasured as much for their
eloquent language as for the inciteful opinions, and are treasure troves of ref-
erence information pertinent to the covered topics. Ewan's historical works
include several books, including the much-cited "Rocky Mountain Natural-
ists" (University of Denver Press, 1950) and his masterful biography of John
Banister (University of Illinois Press, Urbana, 1970). He also edited **A Short
History of Botany in the United States" (Hafner Publishing Company, New
York, 1969; reprinted 1981), which was published in conjunction with the
Eleventh International Botanical Congress, in Seattle. His taxonomic studies
focused primarily on the neotropical Gentianaceae and Delphinium (Ranun-
culaceae), but he published on other groups as well. He was also active in
floristic work in Arizona, California, and Colorado.

Joe's love of ferns began rather early. He joined the American Fern Society
in 1930 and published his first paper in the American Fern Journal in 1931
(21:106-109), entitled "Recent Fern Notes from "Southern California." By our
count, Ewan's publications relating directly to ferns total to some 15 titles, but
he also included fern species in some of his more general floristic writings.
These contributions spanned a breadth of subjects equal to his other writings,

taxonomic

masterful writin

Morton (Taxon 22:271-274, 1973), another

W,

The Californian species of Pellaea and Polystichum were among his favorite
subjects for taxonomic study, although he made contributions to our knowl-

Joe was an officer in the American

more

an

president 1947-1951.

In his long and fruitful career, Joe Ewan witnessed the growth and evolution
of the American Fern Society for nearly seven decades, even as he observed
the growth and changes in botany and other natural history as a whole. He

an

writin

future

missed. — George Yatskievych and Doug Holland

len, P.O. Box 299. St. Louis. MO 63166-0299.

Missouri Botanical Garden Ubra

1753 00299 6871

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Volume 90

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JOURNAL

July-September 2000

QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY

The effect of spore density on germination and development in Pteridium, monitored using

a novel culture technique Carl f. Ashcroft &■ Elizabeth Sheffield 91

On the Lectotypification of Danaea elliptica David B. Leilinger 100

Shorter Notes

New Records for the Pteridoflora of the State of Chiapas, Mexico

Ramon Riba and Miguel Angel P^rez Farrera 104

Production of Adventitious Buds on the Leaves in Dicksonia sellowiana

Eduardo Calderdn-Sdenz 105

On the itineraries of Alfred and Alexander Curt Brade in Costa Rica

Paulo G. Windisch 108

Asplenium Xalte mi folium in the Black Hills of South Dakota

Mollis Marriott, fan Conn, and Herb Conn 109

Dryopteris goldiana and its Hybrid with D. celsa New to Arkansas

James K Peck, C. Theo Witsell and Earl Hendrix 110

Obituary

Ramon Riba y Nava Esparza: (1934-1999) Leticia Pacbeco and Blanco Perez-Garcia 112

•.

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The American Fern Society

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BARBARA JOE HOSHIZAKI, 557 N. Westmoreland Ave., Los Angeles, CA 90004-2210.

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Secretary
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American Fern Journal 90[3):91-g9 [2000)

The Effect of Spore Density on Germination and
Development in Pteridium, Monitored using a Novel

Culture Technique

Carl J. Ashcroft^ and Elizabeth Sheffield^

School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road,

Manchester M13 9PT.

Abstract. — Percentage germination and percentage transition to two-dimensional growth in Pter-
idium (bracken fern) were recorded for spores sown on a solid Phytagel^-based growth medium
at known densities of between 3 and 9883 spores mm 2. The maximum germination recorded
occurred at sowing densities higher than those at which maximum transition occurred. Percentage
germination was greatest (43 to 52% of spores sown) at intermediate densities (187 to 2114 spores
mm-^j, being highest (52% of spores sown] at 360 spores mm"2. Percentage transition was highest
at the lowest densities used. It was concluded that germination and transition have different
density optima and that investigations of these two phenomena in ferns should take account of
this.

Fern gametophytes are accepted as excellent models for the study of many
biological systems. Their utility in the studies of both pure and applied de-
velopmental biology and genetics was described by Hickok et aL (1987), see
also Miller (1968). Their usefulness for the study of developmental selection
was illustrated by Klekowski (e.g. 1982). Fern gametophytes have also been
used in studies of herbicide tolerance and herbicide mode of action, (Hickok
et ah, 1987; Keary et al, 2000). However, the density of fern spores on their
substrate affects both percentage germination and gametophyte development
(see Dyer, 1979 for review). The findings reviewed by Dyer which related to
the sowing of spores at known densities on artificial media indicated that per-
centage germination is inhibited at both high and low densities, yet few fern
researchers before or since have specified the spore densities used in their
experiments. It follows that fern gametoph3^e studies would benefit from being
conducted at optimal densities for the phenomenon under study; there is a
need for quantification.

Two problems arise in attempting to quantify the effects of spore sowing
density on germination and transition. Transition is a term used to describe
the progression from (ID) filamentous growth to (2D) growth involving divi-
sions in more than one plane, which generates the thallus in most fern ga-
metophytes. The first problem is obtaining an even distribution of spores
across the culture medium. Carboxy-methyl cellulose (CMC, manufactured by
BDH) has been used to help spread spores evenly over media surfaces (e.g.

^ Current address: Life and Environmental Sciences, University of Nottingham, University Park,

Nottingham, NG7 2RD.
^ Corresponding author.

MJSSOURt BOTANICAL

FEB 2 Z001

GARDEN LISaARY

92

NUMBER

Sheffield et ah, 1997). CMC does aid spore dispersal in liquid but does not
ensure even distribution across the entire surface of solidified growth media.
Even spore distribution can be achieved by spraying using a diffuser (see Dyer,
1979). However, this method only works well on a large scale and is wasteful,
an important consideration in work with rare or irregularly fertile species.
Given the toxicity of fern spores (Povey et al, 1995; Siman et ah 1999; Siman
et ah 2000), the generation of aerosols containing spores raises health and
safety considerations. A second problem concerns counting spores and scoring
germination and transition in cultures sown at high densities. At high densi-
ties, spores and gametophytes cover one another and make germination diffi-
cult to detect.

The aim of this work was to quantify the effect of spore sowing density on
germination and transition to two-dimensional growth in Pteridium. A new
culture technique was developed to allow this.

Methods

Two experiments were conducted under identical conditions, unless other-

Experiment

Mai

(Spain). These spores are E.S. Collection Number 196. At the time of collec-
tion, rhizome material was also taken. This was planted at the University of
Manchester Botanical Grounds where the specimen now forms part of the liv-
ing collection. Spores were stored in sealed plastic vials at 4 ""C after collection
and until surface sterilisation in 1999.

Moore's medium (after Moore, 1903), pH 6.5, was used as the growth me-
dium, with the addition of Phytagel® (manufactured by Sigma) gelling agent
(0.2 % weight/volume). Phytagel® is a gelling agent that forms a solid similar
in form to that of conventional agar media. Phytagel® is distinct from tradi-
tional gelling agents in that when agitated, it changes phase from a solid to a
liquid. The Phytagel®-based growth medium was autoclaved then allowed to
cool to approximately 40°C. In the first experiment (Experiment 1). 100 ^1 of
Phytagel®-based medium was dispensed into each 2.35 mm diameter well
microtitre plate (approximate well volume 175 \il]. A microtitre plate is a
transparent plastic tray, with 96 individual wells). In the second experiment
(Experiment 2), 200 \x\ of Phytagel®-based medium was dispensed into each
6.65 mm diameter well microtitre plate (approximate well volume 370 \jA).
Microtitre plates were left open until the surface of the medium was dry. Lids
were then sealed in place with Parafilm (manufactured by NESCO). All equip-
ment and solutions were autoclaved or purchased sterile. Procedures were

Envar laminar

temperature

dH^O, to which had been added 2 drops of Tween 80 detergent (BDH). Tween

then

clumps

What

ASHCROFT & SHEFFIELD: Spore Density in Pteridium

93

Table 1. The spore sowing densities used (spores mm^^ of medium surface).

Treatment dl d2 d3 d4 d5 d6 d7 d8 d9 dlO dll dl2 dI3

Experiment 1122122222 122

Density 3 35 187 360 538 618 1137 2414 4445 5671 7813 7859 9883

man). The Tween 80 solution was then fihered off from the spores. The spores
were then sm-face steriUsed by re-suspending in 50 ml of 5% (vol/vol) aq.
sodium hypochlorite (approximately 10-14% available chlorine, BDH) for
three minutes. The sodium hypochlorite solution was filtered off and the
spores rinsed three times each with 50 ml of sterile dH.O.

In order to facilitate the inoculation of the growth medium, surface sterilised
spores were suspended in a solution of 0.5% (w/vol) CMC (BDH) in sterile
dH^O. This was done by removing the nitro-cellulose membrane from the filter
unit and eluting the spores from the membrane using a 1 ml Gilson pipette.
In the case of the highest density treatments, inoculation was conducted using
a fixed volume of undiluted stock spore suspension. In the case of other den-
sity treatments, aliquots were taken from this stock spore suspension and made
up to the same fixed volume of inoculum that was used to inoculate at the
highest densities. The 2.35 mm diameter microtitre plate wells used in Exper-
iment 1 were inoculated with 10 [xl of inoculum. The 6.65 mm diameter mi-
crotitre plate wells used in Experiment 2 were inoculated with 20 |jlI of in-
oculum. The spore suspension flowed across the surface of the growth medi-
um, distributing the spores evenly.

In Experiment 1, spores were sown at four densities (Table 1). In Experiment
2, spores were sown at nine densities (Table 1). A total of thirteen densities
were sown, dl to dl3. For each experiment, four replicate blocks were sown
on the same microtitre plate. The experiment was designed so that there was
an overlap in the ranges of spores sown in the two experiments. To illustrate
the repeatability of the cultm^e technique, one spore density was repeated in
both experiments; dll from Experiment 1 was sown at approximately the same
density as dl2 from Experiment 2 (see Table 2),

Spores were grown in sealed microtitre plates maintained at 20 ± 2 ""C with
12 h light, 12 h dark cycles of illumination for 14 days. Approximate photon

-2o-l

flux density was 120 |xmol.m"^s

At the end of the growth period spores were re-suspended using gradual
additions of known volumes of 0.5% (w/vol) CMC (BDH) in sterile dH^O to
each treatment until all spores were re-suspended. Gentle fluxing back and
forth using a 1 ml Gilson pipette caused the Phytagel® to change phase firom
a solid to a liquid, thereby re-suspending the spores and gametophytes that
had previously been evenly spread across surface of the solid medium. Each
final spore suspension was rendered sufficiently dilute to enable convenient
scoring using a Sedgwick Rafter counting chamber and viewed under a Leitz
Dialux 20EB binocular compound microscope at X400 magnification. Final
volumes of spore suspension (including the volume of Phytagel®-based

94 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 (2000)

Table 2. Mann-Whitney U test of dl I versus dl2 for density, germination and transition.

U-Value Tied Z- Value Tied P- Value

Density 51222 0.079 0.9369

Germination 5 1 1 20 1.27 1 0.2037

Transition 4886 1 1 .086 0.2775

growth medium)
on spore density.

ml

were taken from each treatment final spore suspension, with the exception of
treatments with final suspension volumes of less than 2 ml. In such cases, the
whole final spore suspension was sampled. Between 266 and 431 one-micro-
litre counting chamber cells were scored for each treatment sowing density. A
total of 4386 one-microlitre counting chamber cells were examined and 7228
spores or gametophytes were recorded. Germination was considered to have
occurred with the emergence of a rhizoid.

Results

Preliminary analysis

Mann-Whitney

showed there to be no difference between replicate treatments (p < 0.01] and

that th

taken from a given treatment spore suspension [p < O.OlJ. Consequently, val-
ues recorded from each counting chamber cell were treated as individual es-
timates of spore density, germination and transition. Means and confidence
intervals were calculated using these estimates.

The data were not normally distributed. Accordingly, spore density esti-
mates were log-transformed and percentage germination and percentage tran-
sition data were arc-sin transformed. Ninety five percent confidence intervals
were calculated using transformed data according to Wheater & Cook [1999].
All statistics were conducted using StatView and graphs produced using Excel.

The spore densities tested are shown in Figure 1 and listed in Table 1. Per-
centage germination was greatest (43 to 52% of spores sown] at intermediate
densities, 187 to 2114 spores mm ^ [d3 to d8], being highest (52% of spores

^ (d4). Percentage transition was greatest [between
0.5 and 1.4% of spores sown) at lower densities (3 to 538 spores mm-^]. At
densities of 4445 spores mm"^ and above, percentage transition did not exceed
0.00005%. Percentage transition was zero at densities of 7813 and 9883 spores
nmi"^. Far greater variance was observed for transition than for germination.

The two treatments sown at the same density in different experiments, dll
and dl2, showed the same proportions of germination and transition, see
Table 2.

sown at 360 snores mm

ASHCROFT & SHEFFIELD: Spore Density in Pteridium

95

(a)

<N

Q

R

fO

f*^

00

»c

fS

^ I

*n

2 {

^

5 f

00 90

dl d2 d3 d4 d5 d6 d7 d8 d9 dlO dll dl2 dl3

%

(b)

60 -^

50

40

30

20 -

10 -

I

I

^ t , . '

dl d2

1 I I — ^ ^ , J 1 1 1

d3 d4 d5 d6 d7 d8 d9 dlO dll dl2 dl3

(C)

2.0

1.5

_

%

1.0

0.5-

t

t

t

dl dl d3 d4 d5 d6 d7 d8 d9 dlO dll dl2 dl3

Fig. 1. Sowing density (a), percentage germination [b) and percentage transition (c) for Pteridium
spores at 13 densities (dl-dl3). Open symbols + Experiment 1, closed symbols = Experiment 2.
Error bars = 95% confidence interv-als.

96 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 [2000)

Discussion

The period of time for which Pteridium spores remain viable varies. Pteri-
dium spores do not remain viable for long in soil under field conditions (Con-
way, 1953; Lindsay et ah, 1994). In contrast, it seems that spores stored in
sealed containers at 4*'C may remain viable for many years (Simpson & Dyer,
1999). Our findings show that germination percentage as high as 52% may
occur in spores stored in this way for 9 years, this is higher than the percentage
germination obtained from 9 year old spores in the study of Lindsay et ah
(1994). It should be noted that subsequently, transition was low, less than 1.5%
in all cases, but the reasons for this were not sought or identified. Sheffield
(1996) reported that Pteridium spores collected from the same location in dif-
ferent years can differ considerably in percentage germination. It may be that
the environmental conditions to which a frond is exposed have an effect on

that

known

for example, presented evidence temperature influences germination in three
species of Asplenium.

Percentage germination of Pteridium spores collected in the same year from
different localities also varies, as does germination in spores stored for differ-
ent lengths of time, but there is no direct relationship between Pteridium spore
age and viability (Lindsay et al„ 1994; Sheffield, 1996). Given effects of hy-
dration (Lindsay, Williams and Dyer, 1992), growing temperature and storage
time on percentage germination, it is clear that storage conditions have an
important influence on fern spore percentage germination. It would be valu-
able if standardised spore conditions could be agreed, or at least that future-
authors were encouraged to include a statement describing spore storage con-
ditions. Although germination of many species might be higher following fully
hydrated storage (Lindsay et al, 1992) this method is space and time consum-
ing and inappropriate for species, such as Pteridium, capable of dark germi-
nation. We therefore suggest that pteridophytes spores should routinely be
stored dry, in sealed containers at 4°C, in the dark.

Not only are spores inherently variable in terms of germination, but our
experiments show that they are also sensitive to growing conditions. The den-
sity at which Pteridium spores are sown on their substrate affects germination
and development. Percentage germination was highest at densities interme-
diate in the range tested herein. This is in keeping with earlier findings, such
as those reviewed by Dyer (1979). Smith & Rogan (1970) found an inhibition
of development by gametophytes of Polypodium vulgare to increase with spore
sowing density. Smith & Robinson (1971) identified three growth factors re-
sponsible for the inhibition of cell division in high densities of Polypodium
vulgare. They concluded that other factors, such as competition for nutrients

an

mportant at later stages of development and

sities. Light is not required for germination of Pteridium spores (Lindsay et al.,
1994) but is required for photosynthesis to drive gametophyte development.

ASHCROFT & SHEFFIELD: Spore Density in Pteridium

97

Smith & Robinson's (1971) investigations into the effect of high spore density
were done in liquid growth media. We have expressed our densities on the
basis of numbers on the surface of the medium, and undoubtedly depth of
medium and speed of diffusion of nutrients into, and exudates from, game-
tophytes influences results in different vessels or types of solidified medium.
We attribute impeded development of Pteridium at extremely high sowing
densities to both resource limitations and inhibition due to gametophyte ex-
udates. As for storage conditions, discussed above, it is clear that density does
have an important influence on germination and growth, and we recommend
that future authors standardise, or at least state, their spore sowing densities.

There are ecological implications to density-dependent germination rates.
The majority of fern spores probably fall within a few metres of their sporangia
but a few travel great distances, see Sheffield (1996) and Siman et qL (1999).
Our results imply that spores landing close to the parental sporophyte could
accumulate to such a high density that germination and gametophyte devel-
opment are inhibited; the reverse would apply to spores dispersed over great
distances. Ecological implications are that lone or sparse spores are unlikely
to germinate but if they do, they are likely to undergo transition and therefore
go on to produce female gametophytes. This will mean that these female ga-
metophytes will secrete antheridiogens and promote germination in any other
spores in the area, and cause those neighbouring spores germinate develop
into male gametophytes (Naf, 1958).

The new culture technique employed in this work provides an economical
and straightforward means of determining spore density and quantifying ger-
mination and transition. The repeatability of the technique was illustrated by
the identical germination and transition percentages observed in dll from Ex-
periment 1 and dl2 from Experiment 2, two treatments sown at identical den-
sities in separate experiments.

Laboratory conditions were optimised during the course of preliminary
work. In particular, the volume of inoculum used is critical. The volumes pre-
sented here were optimised in a laminar flow cabinet at room temperature and
are intended only for guidance. Other laboratories may have slightly different
environmental conditions, e.g. room temperature, and it may be necessary to
fine-tune procedures for local conditions. Preliminary trial and error optimi-
sation is recommended. It was observed during our preliminary experiments
that sub-optimal conditions lead to shrinkage of inoculum upon drying, re-
sulting in a distribution of spores which did not extend to the full circumfer-
ence of the microtitre plate well. In such situations it may still be possible to
quantify spore density by estimating the diameter of dried spore inoculum and
re-calculating accordingly, using this diameter, rather than that of the micro-
titre plate well.

In conclusion, Pteridium spores stored for nine years at 4°C and sown at 360
spores mm"^ had a mean maximum germination rate of 52%. Lower mean
maximum germination rates were observed above and below this density. In-
hibition of germination at high densities may be due to resource limitation,
physical impediment or biochemical inhibition caused by the exudation of

98 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 (2000)

growth inhibitors. Inhibition of germination at low densities may have a bio-
chemical basis; germination may be stimulated by the exudation of growth
promoters from other spores. At higher spore densities, the effects of growth
inhibitors, physical impediment or resource limitation overcome the effects of
growth promoters. The low percentage transition observed could be a conse-
quence of the age of the spores used. Inhibition of transition at high densities

last two hypotheses.

furth

Acknowledgments

Thanks to Dr Rod Cullen for his help with the statistics and Dr Caroline Bowsher for the Phy-
tagel®. Carl Ashcroft was in receipt of a Natural Environmental Research Council Studentship.

Literature Cited

Conway. E. 1953. Spore and sporeling survival in bracken [Pteridium aquilinum L. Kuhn.). J. Ecol.
80:289-294.

Dyer, A. F. 1979. The culture of fern gametophytes for experimental investigation. Pp. 253-305

in A. F. Dyer, Ed. The experimental biology of ferns. Academic Press. London.

Dyer, A. F. and S. Lindsay. 1996. Soil spore banks — a new resource for conservation. Pp. 153-

160 in ]. M. Camus, M. Gibby and R. J. Johns. Eds. Pteridology in perspective. Royal Botanic
Gardens, Kew.

HiCKOK, L. G., T. R.Warne, and M. C. Slocum. 1987. Cerotopteris richardii: applications for ex-
perimental plant biology. Amer. J. Bot. 74:1304-1316.

Keary, L R, C. Thomas, and E. Sheffield. 2000. The effects of the herbicide Asulox on the ga-
metophytes oi Pteridium aquilinum, Cryptogomma crispa and Dryopteris filix~mas. Ann. Bot.
85, Suppl. B:47-51.

Klekowski. E. J. 1982. Genetic load and soft selection in ferns. Hered. 49:191-197.

Lindsay, S., E. Sheffield, and A. F. Dyer. 1994. Dark germination as a factor limiting the formation

of soil spore banks by bracken. Pp 47-51 in R. T. Smith and J. A. Taylor, Eds. Bracken: an

environmental issue. International Bracken Group. Aberystwyth. UK.
Lindsay, S., N. Williams, and A. ¥. Dyer. 1992. Wet storage of fern spores: unconventional but

far more effective! Pp 285-294 in J. M. Ide, A. C. Jermy and A. M. Paul Eds. Fern horticulture:

past present and future perspectives. Intercept. Andover.
Miller, J. H. 1968. Fern gametophytes as experimental material. Bot. Rev. (Lancaster) 34:361-440.
Moore, G. T. 1903. Methods for growing pure cultures of algae. J. App. Microscop. Lab. Meth. 6:

2309-3214.

Naf, U. 1958. On the physiology of antheridium formation in the bracken fern [Pteridium aquil-
inum). Physiol. Plant. 11:728-246.

Pangua, E., S. Lindsay and A. F. Dyer. 1994. Spore germination and gametophyte development

in three species of Asplenium. Ann. BoL 73:587-593.
PovEY, A. C, I. A. Evans, J. A. Taylor, and P. J. O'conner. 1995. Detection of DNA adducts by

32P-postlabeIling in mice treated whh bracken extract and bracken spores. Pp 95-98 in R.T.

Smith and J. A. Taylor. Eds. Bracken: an environmental issue. International Bracken Group.

Aberystwyth.

Sheffield, E. 1996. From spore to sporophyte in the natural environment. Pp. 541-549 in J. M.

Camus, M. Gibby and R. J. Johns. Eds. Pteridology in perspective. Royal Botanic Gardens,
Kew,

Sheffield, E., G. E. Douglas and D. J. Cove. 1997. Growth and development of fern gametophytes

in an airlift fermenter. Plant Cell Rep. 16: 561-564.
Siman,S. E., a. C. Povey, and E. Sheffield. 1999. Human health risks from fern spores— a review.

Fern Gaz. 15:275-287.

ASHCROFT & SHEFFIELD: Spore Density in Pteridium

99

SiMAN, S. E., A. C. PovEY, T. H. Ward, G. Margison and E. Sheffield. 2000. Fern spore extracts
can damage DNA. Brit. J. Cancer 83:69-73.

Simpson, K, and A. Dyer. 1999. The survival of dormant fern spores. PteridoJ. 3:98-103.

Smith, D. L., and P. M. RoBrNSON. 1971. Growth factors produced by germinating spores oi Poly-
podium vulgareL. New Phytol. 70:1043-1052.

Smith, D. L., and P. G. Rogan. 1970. The effects of population density on gametophyte morpho-
genesis in Polypodium vulgare L. New Phytol. 69:1039-1051.

Wheater, C. p. and P. A. Cook. 1999. Statistics in environmental investigations. Routledge. Lon-
don. UK.

WiLKiE, D. 1963. Genetic analysis of variation in the bracken prothallus. Bot. J. Linn. Soc. 58:333-
336, 373.

American Fern Journal 90(3):100-103 (2000)

On the Lectotypification of Danaea elliptica

David B. Lellinger

U. S. National Herbarium, Smithsonian Institution, Washington, DC 20560-0166

Abstract.— Prior lectotypifications oi Danaea elliptica are rejected because they did not take into
account the one adequate specimen cited by the author of the species. This specimen is chosen
here as lectotype, which enables the name D. elliptica to continue to be used in its usual sense.

J. E. Smith in Rees (1808), in his treatment of Danaea, included D. alata J.

Smith, D. nodosa (L.) J. Smith, D. simplicifolia Rudge, and one new species,

D. elliptica J. E. Smith in Rees. The latter three species are closely related,

although D. simplicifolia is obviously different in having simple, rather than

pinnate laminae. J. E. Smith was careful to distinguish his new species from

the related species D. nodosa, as his descriptions and excerpts from his notes
illustrate:

1. D. nodosa: "Stalk scarcely winged; leaflets linear-oblong, sessile, pointed,
nearly entire, covered with capsules to the edge. Radical scales acute.

"Each frond is about four feet high . . . Leaflets ... six or eight inches long,
oblong, almost linear, entire, wavy, with a taper point . . . Capsules . . . Ihaving]
each row extending from the main rib very nearly to the margin ..."

2. D. elliptica: "Stalk scarcely winged; leaflets elliptic-oblong, stalked, point-

near

"The fronds are but half as tall as in the former [D. nodosa], and their leaflets
half as long, though somewhat broader and elliptical. The latter, moreover,
stand on short partial footstalks. The rows of capsules scarcely extend to near
the edge of the leaflet on which they grow, but are more remarkably separated
from each other, at least in a half-ripe state, by a double prominent undulated

membrane

from the foregoing that J

between these two species, which have been maintained as separate taxa by
virtually all authors since the time of his publication. Danaea elliptica is a
smaller plant with nodose stipes and fewer, shorter, more elliptical pinnae
(Lellinger, 1989, p. 85). Roller! (pars, comm.) has found additional differences

What is less clear is J

urface

the pre-Linnaean phrase name

Sloane (1707, p. 85) and the reference "Sloane J

the

commented, '^Observed by Sloane in J

cite any specimens as if he had seen them
standards, but was not unusual in his day.
p. 201), J. E. Smith was very familiar with

LELLINGER: Lectotypification of Danaea

101

baria. As it turns out, two specimens are involved, both collected by Sloane
in Jamaica,

The specimen from Mt. Diablo, Jamaica (cat. no. 1183, BM-SL) consists of
three sterile fronds of D. nodosa. One is large and mature, the other two small-
er and juvenile. The juvenile fronds, which were drawn for Sloane's illustra-
tion t. 41, f. 1, bear some resemblance to D. elliptica in size and outline. How-
ever, the laminae have more lateral pinna pairs for their size, the pinnae are
sessile or nearly so, and the pinna bases are distinctly acute and almost sym-
metrical, all characters of D. nodosa. This specimen may be viewed at http://
www.nhm.ac.uk/botany/, in the Sloane database.

Although it seems unlikely, the Mt. Diablo specimen could be a mixed col-
lection, for both D. nodosa and D. elliptica occur in Jamaica (Proctor, 1985,
pp. 61-62). The veins of the juvenile fronds are ca. 1 mm distant with slightly
diverging veins in D. elliptica, but ca. 0.75 mm distant with strictly parallel
veins in D. nodosa. These differences can be seen in all but the most juvenile
fronds and can be used to make a positive identification of the juvenile fronds.

The specimen from an unknown locality in Jamaica (**1245 Acrostichum
nodosum,'" LINN) was added to the Linnean Herbarium after 1767 Qackson,
1912, pp. 26, 28), which is consistent with J. E. Smith's statement about it in
his description. By 1945, this specimen was not present in LINN (Savage, 1945
p. 186), nor was it on the IDC microfiche of the Linnean Herbarium made a
few years later. Fortunately, the specimen is in the J. E. Smith collection (cat.
no. 1645.7, BM). Although the specimen lacks a rhizome, it does have one
sterile frond with a nodose stipe and three pairs of lateral, ascending pinnae.
The pinnae are elliptic-oblanceolate, relatively wide, and taper rather abruptly
to an acuminate, entire to subcrenate apex. No centimeter scale is present on
the microfiche; 1 estimate that the pinnae are ca. 13-15 cm long and 3.4 cm
wide at their widest point distal to the middle of the pinna. A much smaller,
fertile lamina has a partial stipe broken off, presumably above the most distal
node, and also three pinna pairs; the middle pair is broken off. The pinnae
are oblong-oblanceolate and are estimated to be ca. 3.7-4.8 cm long and 1.1-
1.4 cm wide at their widest point distal to the middle of the pinna. According
to Savage's handwritten and unpublished catalogue accompanying the IDC
microfiche of the J. E. Smith collection, it is labelled "Ind. Occ. H[erb] L. fil.,"
which can be seen on the microfiche itself. 1 suppose the specimen was moved
from the Linnean Herbarium to the J. E. Smith collection between 1912 and
1945 because it was type material of one of J. E. Smith's own species.

It is clear that J. E. Smith saw both Sloane specimens, as he was familiar
with both collections (Steam, 1988, p. 201). Because his description men-
tioned a difference in the position of the synangia and because the Sloane
specimen then in the Linnean Herbarium was mature and fertile, his descrip-
tion of D. elliptica must have been based principally or entirely on that spec-
imen, and tJie information about the fertile frond exclusively so. The role that
the Mt. Diablo specimen played is problematical. On it, an annotation ''As-
plenium nodosum p" was written between the two juvenile fronds and the
larger frond by Solander, Linnaeus' amanuensis. The position of the annotation

102 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 (2000)

might signal Solander's differentiating between the two, or J. E. Smith might
have thought that it did. He may have included the reference to t. 41, f. 1
because the drawing somewhat resembles D. elliptica or because he thought
the two fronds on which the drawing was based were D. elliptica. In either

dentify

ecimen

Underwood (1902, p. 672), who was the first to choose a lectotype of D.
elliptica, stated 'Type from Jamaica, Sloane, pi. 41, f. 1/' This is clearly in-
tended as the illustration, rather than the Sloane specimen from which it was
drawn, which he did not cite. According to Curtis [1908, p. 10), Underwood
**made repeated visits to the herbaria of Europe for comparison and study of
material." Because his monogranh flJndRrwnnd. IQfl?. n fi7l1 ritprl matprial

and P, but none from BM, presum

J. E. Smith

oane

protologue that he himself had seen. His choice may have even been mechan-
ical, the illustration being the first element in the protologue mentioned by J.
E. Smith. Because the specimen underlying the illustration is D, nodosa, be-
cause the specimen and/or illustration were misidentified, and because the
selection may have been mechanical, I believe that Underwood's lectotype
should be set aside.

Proctor (1985, p. 62) concluded that the lectotype was Sloane no. 1183,
without comment or mentioning Underwood's prior choice. Baksh-Comeau
(2000, p. 25) misstated Underwood and also cited the same lectotype. Because
Proctor's later lectotype is D. nodosa and because he did not show cause to
reject Underwood's prior lectotype, I believe this lectotype also should be set
aside.

more

unknown, Jamaica, Sloane (BM-Hb. J. E. Smith

no. 1645.7 ex LINN] This choice of lectotype has the advantage of fixing the
name D. elliptica J. E. Smith in Rees on a specimen inferentially cited by and
undoubtedly seen by J. E. Smith, and the additional advantage of allowing the
name to be used in its usual sense, rather than becoming a synonym of D.
nodosa.

Acknowledgments

I thank Dr. Robbin Moran, Dr. Jefferson Prado, and Dr. Alan Smith for their reading of the
manuscript and helpful comments.

Literature Cited

Baksh-Comeau, Y. S. 2000. Checklist of the pteridophytes of Trinidad & Tobago. Fern Gaz 16:11-
122.

Curtis, C. C. 1908. A biographical sketch of Lucien Marcus Underwood. Bull. Torrey Bot. Club
35:1-12.

Jackson, B. D. 1912. Index to the Linnean Herbarium. . . Linnean Society, London.

LELLINGER: Lectotypification of Danaea

103

Lellinger, D. B. 1989. The Ferns and Fern-allies of Costa Rica. Panama, and the Chocd (Part 1:

Psilotaceae through Dicksoniaceae). Pteridologia 2A:l-364.
Proctor, G. R. 1985. Ferns of Jamaica. British Museum (Natural History), London.
Rees, a. 1808. The Cyclopaedia, vol. 11, DANAEA. Longman. . ., London.
Savage, S. 1945. A Catalogue of the Linnean Herbarium. Linnean Society of London, London.
Stearn, W. T. 1988. J. E. Smith (1759-1828]: First President of the Linnean Society and his her-

barium. Bot. J. Linn. Soc. 96:199-216.
Underwood, L. M. 1902. American Ferns-V. A review of the genus Danaea. Bull. Torrey Bot. Club

29:669-679.

American Fern Journal 90(3):104-111 (2000)

SHORTER NOTE(S)

New Records For The Pteridoflora Of The State Of Chiapas, Mexico, — ^The

State of Chiapas, Mexico, is the second richest State of the country in regard
to the diversity of pteridophytes, only surpassed by Oaxaca. Smith [Fh Chiapas
2:1-370, 1981) treated 609 taxa, Breedlove [Listados floristicos de Mexico, W,
flora de Chiapas, Institute de Biologia, UNAM, 1986) cited 675, and Riba et
al. (Amer. Fern J. 77:69-71, 1987) added 16 new records for the State, totaling
691 taxa. As a result of intensive field work in the western part of the State,
two more species are added, whose presence in Chiapas was already antici-
pated by Smith (Fl. Chiapas 2:1-370, 1981). They are Hemionitis levyi E.
Fourn., previously collected in Mexico (Oaxaca) and Central America, and
Doryopteris concolor (Langsd. et Fisch) Kuhn var. concolor, otherwise known
from southern Mexico (Veracruz, Oaxaca) and widely distributed in Central
and South America, Antilles, Asia, Africa, Australia, and Pacific Islands.

Hemionitis levyi [Perez Farrera 670, CHIP, UAMIZ) was collected in the Mu-
nicipality of Jiquipilas, El Campanario, 2.5 km N from Ejido Andres Quintana
Roo, north of the Biosphere Reserve La Sepultura, in tropical deciduous forest
with species of Bursera, Mimosa, Plumeria, together with Clieilanthes , Selagi-
nella, Lygodium, and several cacti, at 650 m altitude (16°37'N; 93°34'W) in the
Central Depression de Chiapas. The specimens were found in small caves in
the sandstone, with shallow soil. This species differs from H. pinnatifida Baker
and H. palmata L. in its smaller size (leaves 2.5-16 cm long, 1.7-5 cm wide;

Fig. 1- Pressed voucher specimens of Hemionitis levyi (left) and Doryopteris concolor var. con-
color (right), showing general frond morphology. Scale bars = 1 cm.

SHORTER NOTE(S)

1U5

Fig. 1), simple blade, orbicular to shallowly 3-5 lobed, without proliferous buds,
and with short laminar hairs.

Doryopteris concolor var. concolor [Perez Farrera 442, CHIP, UAMIZ) was
collected in the Municipality of Jiquipilas, Cerro Hojas Moradas, 6 km W Ran-
cho Alpes, in the Biosphere Reserve La Sepultura, in tropical deciduous forest,
1300 m altitude [16^20'30"N; 93°42'30"W), in the Sierra Madre of Chiapas. This
taxon (Fig. 1) differs from the other species of Doryopteris growing in Mexico,
D. palmata (Willd.) J. Sm.. in its free venation, lack of proliferous buds at the
base of the blade, and the basically glabrous petiole.

A complete list of the plant species found in the Reserve is in preparation
by the junior author. — Ramon Riba (deceased), Universidad Autonoma Metro-
poUtana-Iztapalapa, Ap. Postal 55-535. Mexico, D. F. 09340, and Miguel Angel
Perez Farrera, Instituto de Historia Natural, Depto. de Botanica, Calzada de
los Hombres Ilustres s/n, Tuxtla Gutierrez, Chiapas, Mexico 29000.

Production of Adventitious Buds on the Leaves in Dicksonia sellowiana. —

The genus Dicksonia L'Her. has been successfully propagated through the cul-
ture of spores and gametophytes [Constantino et al., Memorias del I Congreso
Nacional sohre Biodiversidad, Cali, Colombia, Die, 4-7 1994, p. 303-308,
1995}. No secondary or adventitious budding is know^n for this genus to date.
Evidence is presented here for the first time on the production of foliar (peti-
olar) adventitious buds in Dicksonia sellowiana Hook., and on the feasibility
of small scale propagation of the species using these buds.

I have been able to propagate Dicksonia sellowiana through the culture of
adventitious buds that are naturally produced on the basal parts of the petioles,
and which become especially noticeable on the old leaves [Figs. 1-2). Such
buds are spontaneously produced in some populations inhabiting the West
Cordillera of Colombia, at altitudes between 1900 and 2200 m, between Fara-
llones de Call and La Cumbre (in Depto. Valle). The species has been consid-
ered locally threatened for Colombia, according to Constantino et al. [1995)
and Instituto Humboldt-Colombia [Informe Nacional sohre el Estado de la Bio-
diversidad, 1998), mainly due to over-exploitation of stems (for construction)
and roots [as a substrate for orchid cultivation), but also as a consequence of
land clearing.

Two subpopulations have been carefully observed in Depto. Valle: One at
Reserva El Refugio-Torremolinos (Mpio. Dagua, 2000 m alt.), the other one at
Reserva Himalaya [Mpio. Bitaco, 2050 m alt.). Both localities are currently
being protected by private landowners and are located on the continental di-
vide of the Colombian West Cordillera, slightly towards the Pacific side.
Voucher material fi'om El Refugio-Torremolinos has been deposited at FMB
[Calderon-Sdenz 103).

*

The buds are produced on the adaxial side of the leaf bases, e.g. on the
proximal and widest parts of the petioles. Although barely noticeable on young
or even on mature leaves (they are covered by a dense layer of hairs), buds

106

AMERICAN FERN JOURNAL

Figs. 1-5. Adventitios buds in Dicksonia selloiviana and Alsophih erinacea. 1] Mature D. sel-
lowiana at Reserva El Refugio-Torremolinos showing incipient, petlolar buds. 2) Base of an old,
detached leaf of D. sellovviana depicting thickened, adventitious buds, prior to germination. 3)
Mature D. sellovviana at Reserva Himalaya, with sprouting petiolar buds; leaves on the right belong
to (petiolarj buds that have adhered to the stem. 4) Incipient adventitious stem buds oi Alsophila
ennacea, m a garden specimen at Reserva El Refugio-Torremolinos. 5) Two year-old D. sellowiana
obtained through bud propagation and planted at Reserva El Refugio-Torremolinos. Arrows point
to petiolar buds in Dicksonia sellowiana. Scale bars = 5 cm (Figs. 1-3 and 5) or 1 cm (Fio 4).

SHORTER NOTE(S)

107

become larger in overmature ones and are able to keep growing, indepen-
dently, on the old, decaying leaves. In some cases, when decaying leaves re-
main hanging from the stem, adventitious buds tend to adhere to the latter,
giving the impression of stem buds, but actually being petiolar buds (Fig. 3).
Cultivation of the buds is relatively simple under nursery conditions if hu-
midity is kept high and constant.

Foliar adventitious buds can be produced at the edges of the lamina in a
number of unrelated fern genera (see Tryon & Tryon, Ferns and allied plants
with special reference to tropical America, 1982]. Adventitious buds on the
latero-adaxial surface of the leaf bases are already known for a tree fern spe-
cies, namely Alsophila polystichoides H. Christ [also known as Nephelea po-
lystichoides (Christ) Tryon], as cited by Gastony [A revision of the fern genus

Nephelea, Contr. Gray Herb., No. 203, p. 86, 1973).

The position of such buds on the petiole suggests that they represent the
first one or two pairs of pinnae, which remain undeveloped or dormant while
the leaf is still young. Ontogenically, petiolar buds also resemble the aphlebia
of some fern species, like those of Alsophila capensis (L.f.) J. Sm. (see Tryon
and Tryon, 1982).

Initial observations on the spatial distribution of individuals of Dicksonia
sellowiana in the cloud forest at Reserva El Refugio-Torremolinos suggested
that these buds can play a role in the natural propagation of the species, at
least in some local subpopulations living near the continental divide, on top
of the West Cordillera of Colombia, between 1900 and 2200 m alt. Other pop-
ulations of this species inhabiting the cooler, montane belt at 2500-3200 m in
Colombia (for example at Laguna de la Cocha, Depto. Narino, and at the East
Cordillera in Depto. Cundinamarca) apparently do not produce any adventi-
tious buds at all.

By comparison, adventitious buds in Alsophila are usually formed on the
stem, near its base or on the upper parts (Fig. 4). Such stem buds can grow to
'*diageotropic rhizomes", and might also play a role in the natural propagation
of some populations of Alsophila. This is suggested by the ease with which
they detach from the mother plant and by the retained capacity to develop
into new plants, as exemplified by cultivated specimens of Alsophila erinacea
(H. Karst.) D.S. Conant at Reserva El Refugio-Torremolinos (unpublished ob-
servation).

Is the capability of producing petiolar buds in Dicksonia sellowiana genet-
ically determined or is it triggered by envionmental conditions? More studies
are needed for an adequate answer and for a better understanding of the phys-
iological, reproductive-ecological and phylogenetical implications. In the
meantime, several two-year old specimens of Dicksonia sellowiana obtained
from petiolar buds are growing well at Reserva El Refugio-Torremolinos (Fig.
5). Clearly, bud cultivation is another option for the propagation and conser-
vation of this locally threatened and horticulturally desirable species.

The author thanks the Colombian Network of Private Nature Preserves (Red
Nacional de Reservas Naturales de la Sociedad Civil) for logistic support dur-
ing the field work. — Eduardo Calderon-SAenz, Instituto de Investigacion de

108

JOURNAL: VOLUME

Recursos Biologicos "Alexander von Humboldt", Calle 37 No. 8-40, Mezza-
nine, Santafe de Bogota, D.C., Colombia. Correspondence to the author: Av.
3CN No. 62N-77, Villas de San Martin. Casa 43. Call, rnlnmbia

Rica. — Al-

exander Curt Brade stayed in Costa Rica with his brother Alfred, between 1908

herbarium

imens

taricense" of A. C. Brade. Duplicate series were initially distributed by Gold-
schmidt and later (but still in 1908) by Rosenstock as "Filices Costaricenses
Exsiccate", indicating "A. & A. C. Brade" as collectors. Alexander kept inten-
sive correspondence with Rosenstock at least up to 1932. His costarican her-
barium had 912 pteridophyte specimens representing 502 species of which 60
were described as new, as well as 27 new varieties and 5 forms. The collection
is now part of the Herbarium Bradeanum— HB (Rio de Janeiro). Markgraf (Bot.
Jahrb. Syst. 93(1): 1-8. 1973) in his biosranhv of A. C. Rradp inrlnr^prl

some

here presented in more detail.

and a general itinerary map. Complementin

durine that nerind and snmp mainr PTrpntc ar

1908: February 22, A. C. Brade arrives at Puerto Limdn.

February 23, train trip to San Jose.

March 4, Tablazo; 17, La Palma; 26, idem.

April 10, Carpintera; 25, idem.

June 10-14, Farm of Mr. Zent fll, farm in Chirind: 12.

July

lantic coastal region).

At-

August 1, La Palma; 4, Candelaria; 11, Granadilla; 28, Tablazo.
September 8, Irazii; 17, Tablazo; s.d., Carpintera; s.d., Candelaria.
October s.d., La Palma.

November s.d., Tablazo; 25, Carpintera.

December 2, La Palma; 9, San Domingo; 14, La Verbena; 21-23, Tablazo (Finca
Haberl.).

1909: January 14, Rio Grande; 21-

February 1-27, trip to Guanacaste

23, Volcan Barba.

Buigdorf

Pitahaja, Isla de Chira, rio Tempisque. Bebedero. Mo

jica, Miravalles, Aguas Calientes, Mocole).
March 5, La Palma; 12, Candelaria; 20, La Palma,
April 7, Candelaria; s.d., Barba-Poas; May s.d., (

Burgdorf (rio Surubres); s.d., Cartago.
June 16-22, Carrillo (La Palma, Hondura).
July 20, Tablazo.

August 5-8, Turrialba; 18, La Palma; 28, Tablazo.

SHORTER NOTES

109

September 2-22, Finca of the brothers Hundriesser in the Atlantic coastal re-
gion, between the rivers Reventazon and Parasmina].
October and November: sick.
December 4, Carpintera; 17, Candelaria.
1910: January s.d., Turrialba; 20, Finca Schild-Burgdorf (rio Surubres).

February 9, Finca Schild-Burgdorf [rio Surubres); 10-13, Cerro Turubales; 26,
Granadilla.

March 5, La Verbena; 11, Candelaria; 17, La Palma; 28-31, Juan Vinas, region
of the rio Chis; April s.d., Turrialba [14? eruption of Volcan Poas]; 16,
Cartago; 22, San Jeronimo de Grecia (region of Zisma, Poas); 25-May 7,
trip to Llanuras de San Carlos (Naranjo, Zarcero, Buena Vista, Finca Kos-
chny) .

April (see above).

May 1-7, conclusion of trip to Llanuras de San Carlos [7, catastrophic earth-
quake destroys Cartago]; 10, Cartago, Aguacaliente.
June 2, Tablazo; 12, Turrialba; 22, La Palma.
July s.d., Quassimo; s.d., Finca de los Padres.
August 21, departure from Puerto Limon.

Paulo G. Windisch, Universidade do Vale do Rio dos Sinos— UNISINOS,
CCS— Botanica, C. Postal 275, 93022-000 S. Leopoldo— RS, Brazil.

Asplenium x alternifolium in the Black Hills of South Dakota. — On Septem-
ber 19, 1998, we discovered one individual of Asplenium Xalternifolium Wul-
fen on the north side of the Iron Creek drainage ca. 14 air km northeast of the
town of Custer, Custer County, South Dakota. This area has numerous large
outcrops of the Precambrian Harney Peak granite. The plant was found on the
south face of a large rock massif ca. 100 meters in height, on a shaded microsite
in a deep crack 30 meters above the ground. The elevation of the site is 1023
m (5400 ft.]. Many fronds with sori were present. No other individuals were
found in a survey of the massif and other nearby outcrops. The individual was
growing vigorously when revisited on July 5, 1999. That year, two additional
individuals were found in similar habitat at a second site ca. 6.5 km southwest
of the original discovery. Material was sent to Dr. Robbin Moran (NY) for iden-
tification; vouchers (Marriott 11782, 11786} with photographs have been de-
posited at NY and RM.

Asplenium Xalternifolium is a sterile hybrid between Asplenium septen-
trionale and Asplenium trichomanes, both of which occur at the sites. As-
plenium septentrionale is common throughout the granitic Central Core region
of the Black Hills. A. trichomanes is uncommon in the area, and is sufficiently
rare to be tracked by the South Dakota Natural Heritage Program. The Black
Hills populations most likely are the diploid cytotype, which occurs primarily
on acidic rocks, including granites (Moran, Amer. Fern J. 72: 5-11. 1982). The
hybrid was first collected in North America in West Virginia (Wagner et ah.

110 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 (2000)

Castanea 56: 128-134. 1991). It is considered common in Europe and rare in
the eastern United States. The South Dakota locations are disjunct by more
than 2000 km from the sites reported by Wagner et al — Mollis Marriott, 655
N. Cedar St., Laramie, WY 82072, and Jan and Herb Conn, HCR 83 Box 93,
Custer, SD 57730.

■Wood

Dryopteris goldiana and its Hybrid with D. celsa New to Arkansas -

Ferns {Dryopteris) were studied intensively in Arkansas for twenty years (Peck
et aL, Amer. Fern J. 75:71, 1985; Peck and Peck, Proc. Arkansas Acad. Sci. 42:
74-78, 1988; Peck and Taylor, Proc. Arkansas Acad. Sci. 49:130-137,1995), yet
surprises remain to be found in a state with no less than 16 previous lists of
its pteridophyte flora. Dryopteris celsa [W. Palmer) Knowlton, Palmer & Pollard

amore

J

unsuccessful

men's label coordinates were incorrect, being off by six miles to the west. Earl

Hendrix

Drvi

d Peck on 20 July 199 conf
mmunity for Arkansas. We

to Arkansas. Additional specimens, locality data, and habitat observations

posited at LRU.

Witsell on 27 Novemb

The locality (Merrill Ridge Road Blowing Cave, Baxter Co., AR, T17N, R12W,
S30, Norfolk SE Quad.), is located in a remote portion of north-central Arkan-
sas within the Sylamore District, Ozark National Forest. The locality has three
special microhabitats for Arkansas, including limestone bed-rock, a breakdown
strewn and partially blocked entrance of a blowing-cave, and a hillside seep
and stream located on a north-facing toe-slope. These microhabitats occur at
the bottom of a forested ravine with 100 m of vertical relief. The associated
vegetation is composed of canopy trees such as Carya spp., Nyssa sylvatica,
Quercus alba, and Quercus rubra, with understory plants of Cornus florida,

stream

rotundifolia

environment

This is a very protective habitat for ferns of a more northerly habitat and
range. The humus-rich soils on the toe-slope supports three species of Dryop-
teris, including five plants of D. goldiana (Hooker ex Goldie] A. Gray [Peck
994^7 (LRU)]. Adjacent to the seepage area and stream are several dozen plants
of D. celsa (Palmer) Knowlton, Palmer, & Pollard. Dryopteris marginalis (L.)
A. Gray occurs throughout the valley at various elevations on or near rock
outcrops. This population of D. goldiana occurs at the extreme southwestern
edge of its range, modestly disjunct about 200 km from outliers in Missouri.

SHORTER NOTES

111

Missouri

Minnesota (see Fig. 2, Werth. Svst. Bot. 16:446-461

moist

Small

Wlierry

Dry op t

celsa X goldiana was discovered for the first time in Arkansas. D xleedsii
Wherry was noted at its second Arkansas location. The Arkansas range D.
Xaustralis Small, was expanded from its previously known occurrence in the
Ouachita Mountain Region to include the Ozark Mountain Region as well.
Although not found at this locality, another hybrid might be present in the

Jersey

jowherryi W. H. Wagner [D. goldiana X
. Xneowherryi in Arkansas Q. D. Mont
Forum 8:25-31,1981; T. D. Montgomej

Xleedsii.

With this report, five Dryopteris species [D. carthusiana (three localities in
three counties; D. celsa (23 localities in five counties), D. goldiana (one locality
in one county), D. ludoviciana (one locality in one county), and D. marginalis
(numerous localities in 38 counties)] plus three Dryopteris hybrids [D. Xaus-
trails (nine localities in four counties), D. xleedsii (two localities in two coun-
ties), and D. celsa X goldiana (one locality in one county)] are known from
Arkansas. — James H. Peck, Department of Biology, University of Arkansas at
Little Rock, 2801 S. University Ave., Little Rock, AR 72204; C. Theo Witsell,
Arkansas Dept. Natural Heritage, Little Rock, AR 72201; Earl Hendrix, Syla-
more District, Ozark National Forest. Mt. View. AR 72560.

American Fern Journal 90(3):112-118 (2000]

Obituary: Ramon Riba y Nava Esparza

(1934-1999)

Mexico

Mexico

and

otany

noma Metropolitana
the taxonomy, floris!
genesis of ferns.
He earned a John

1999). His interests in botany included

wrote

(Cyatheaceae]" under the direction of Rolla M. Try

Mexico

in 1967.

man of the Department

Metrop

and the Herbario MetropoUtano (UAMIZ)

Mexico

an

of Pteridology (1998

Volume

Mesoamericana and author of several monoeraDhs. He held mem

American

Mexican Academy

OBITUARY

113

ences. Botanical Society of Mexico and National Council for the Teaching of
the Biology.

He was very active in research and earned an international reputation as a
distinguished pteridologist, having been one of the first people to study this
group of plants in Mexico. For these efforts he was named by the Botanical
Society of Mexico and by the Universidad Autonoma Metropolitana, as one of
their distinguished professors and by the Sistema Nacional de Investigadores
in Mexico as a member. His field notebooks and the principal set of his fern
collections, are deposited in the herbarium of the Universidad Aut6noma Me-
tropolitana Iztapalapa (UAMIZ) and the National Herbarium of Mexico
(MEXU).

Ramon Riba also excelled as a teacher and was awarded by the Universidad
Nacional Autonoma de Mexico and Universidad Autonoma Metropolitana Iz-
tapalapa for teaching excellence. He has co-authored five books on teaching.
Ramon's enthusiasm for teaching in the field was remarkable and he was fun
to be in the field with. He could drive several hours nonstop to Oaxaca or
Veracruz, and when he arrived, he always knew the best place to eat. He spent
a lot of time in the field teaching his students about ferns, and he enjoyed it
tremendously when someone discovered the beauty of ferns for the first time.

He was patient with all his students and his office was open to them all
day-no appointment necessary. Whether the topic was academic or personal,
he always had an encouraging word for everybody, and he would listen care-
fully to what everyone had to say, usually while drinking a warm black coffee
and enjoying a cigarette.

Ramon was highly disciplined, demanding in his work, in the presentation
of his papers and correspondence. He wrote and spoke well and frequently
liked to explain the meaning of a term or to improve the accuracy of the man-
uscripts that his students presented to him. He hated the lack of commitment
and determination in other people, and he was persistent and tenacious in
what he wanted to get accomplished. He motivated co-workers to do the same
thing. He would frequently come to our offices unexpectedly to "check our
discipline," to make sure we were working. Actually, he just wanted to see
how things were going, whether our research and classes were going well.

Ramon had a well-developed sense of friendship and solidarity with his
collaborators and pupils, and he did not tolerate any kind of dishonesty on
the part of those associated with him. As a man, he was intelligent, talented,
creative, imaginative, humble, generous, and a gentile person with all those
that he worked with. He was a religious man, faithful to his beliefs, marriage,
and family, always giving love to his wife, children, and grandsons. He never
forgot to call his wife every afternoon before leaving work. He was an inter-
national scientist but he always remembered his roots, the country necessities;
he encouraged his students and colleagues to follow up on and conclude their
work. He was a man with a lot of character, his professional charisma and
leadership converted him into a successful man. He loved Latin American and
romantic music and he transmitted this love to his children. He also had a

114

AMERICAN FERN JOURNAL

beautiful

make iokes and laugh at them himself

Ramon

that we accompany him to eat, and once
and amusing anecdotes. He commented

We

refused

appointment with his doctor for the following day. Later during the meal we
insisted that he go to the doctor, but he insisted that we were worrying about
nothing. At about 7:30 that evening, he died in his house of a heart attack. He
is survived by his wife Maria Estela Ramirez de Riba, their sons, Ramon Mi-
guel, Arturo and Roberto Carlos, their daughters, Laura Estela and Alejandra,
three grandchildren, and a sister Maria Eugenia. We were privileged to count
ourselves among his doctoral students (at Universidad Nacional Autonoma de
Mexico) and we have been **in touch with the divine's ray" as he said to all
who loved and worked with ferns.

Ramon is commemorated by the species Callipteris ribae Pacheco & R. C.
Moran and Bursera ribana Rzed. & Calderon.

Ramon has not left; people that knew him and had the privilege to know
him and to live next to him for more than eight hours of work a day all knew
what he told us:

Everything is all right . . .

The death is not anything, it does not matter

I have only slipped to the next room . . .

There is an absolute and intact continuity

What is death but an accident without importance?

Why must I remain outside of your mind

Only for not being visible?

I am not but waiting for you, for an interval,

In some place, very close, to the turn of the corner,

Everything is all right . . .

Publications of Ramon Riba

Riba, R. 1958. Contribucidn al estudio de algunas especies de AspergilU aisladas en la Repiiblica

Mexicana. Bol. Soc. Bot. Mexico. 23:10-25.
Riba, R. 1963. Notas sobre la familia Loranthaceae y el parasitismo secundario. Bol. Soc. Bot.

Mexico. 28:1-11.
Riba, R. 1963. Notas sobre los helechos arbdreos en Mexico. Anales Inst. Biol. Univ. Nac. Mexico.

34:51-52.

Riba, R. 1964. Los generos DIcksonIa y Cibotium en America. Revista Soc. Mex. Hist. Nat. 25:163-
171.

RiBA, R. 1965. Helechos arboreos en Guerrero. Anales Inst. Biol. Univ. Nac. Mexico. 36:81-82.

RjBA, R. 1965. Mougeotiopsis calospora Palla en Mexico. Anales Inst. Biol. Univ. Nac. Mexico. 36:
79-80.

Riba, R. 1967. New taxa in the genus Alsophila. Rhodora 69:65-68.

Riba, R. 1967. Revision monografica del complejo Alsophila swartziana Martins (Cyatheaceae).
Anales Inst. Biol. Univ. Nac. Mexico. Bot. 38 (1]:61-100.

OBITUARY

115

RiBA, R. 1969. The Alsopbjla swartziana complex (Cyatheaceae). Rhodora 71:7-17.

GOMEz-PoMPA, A., A. Barrera, G. Halffter, C. Beyer, M. Russek, J. Comas, C. Savin, M. T.
Garcia, J, M. Gutierrez-Vazquez, B. G6mez-Lepe, R. Riba, J. kohashi, M. Servin, L. Bo-
jorquez, R. Villalobos, J. Valdes, G. Yankelevich and A. Le6n de Gar^-w. 1970. Biologia:

Unidad, Diversidad y Continuidad de los Seres Vivos. Consejo Nacional para la Ensenanza

de la Biologia. Mexico, D. R 960 pp.
Riba, R. 1970. Investigaciones de laboratorio y de campo. Consejo Nacional para la Ensenanza de

la Biologia. Mexico, D. R 439 pp.
Riba, R. 1971. El Herbario Nacional. Pasado, presente y futuro. Revista Soc. Mex. Hist. Nat. 30:

25-37.
Tryon, R. M., B. Voeller, A. Tryon and R. Riba. 1973. Fern Biology in Mexico. BioScience 23(1):

28-33.
Riba, R. and T. Herrera. 1973. Ferns, lichens and hummingbirds'nests. Amer. Fern J. 63(3):128.
Riba, R. 1974. Biologia: Modelos y Procesos. Ed. Trillas. Mexico, D. F. 336 pp. [Adaptation and

translation of the original version in English].
Riba, R. B. Perez-Garcia and M. Perez-Garcia. 1976. A new locality for Lycopodiuin serratum in

Mexico. Amer. Fern J. 66:21.
Riba, R. 1976. Comentarios sobre la vida y obra de Jose N. Rovirosa. In: Rovirosa, J. N. Pteridograffa

del sur de Mexico. 1909. Edicidn Facsiniilar de la Sociedad Mexicana de Historia Natural.
Riba, R. 1976. Actualizaci6n de los nombres cientificos de las pteridofitas. In: Rovirosa, J. N.

Pteridograffa del sur de Mexico. 1909. Edicidn Facsimilar de la Sociedad Mexicana de His-
toria Natural.
Riba, R. 1978. Los helechos arborescentes y el "maquique". INIREB Informa 25:1-4.
Gregory, D. and R. Riba. 1979. Selaginellaceae. In Flora de Veracruz. 6:1-35. Institute Nacional

de Investigaciones sobre Recursos Bioticos. Xalapa, Ver. Mexico.
Riba, R. and B. Perez-Garcia 1979. Estudios botanicos y ecoldgicos de la region del n'o Uxpanapa.

No. 9. Las pteridofitas de Uxpanapa, Veracruz. Bidtica 4(3):135-139.
Riba, R. 1979. Cyatheaceae. In Flora de Veracruz. 17:1-35. Tnstituto Nacional de Investigaciones

sobre Recursos Bioticos. Xalapa, Ver. Mexico.
Fraile, Ma. E. and R. Riba. 1981. Distribucion esporangial en estrobilos de especies de Selaginella.

Bol. Bot. Soc. Bot. Mexico. 41:33-40.
P6rez-Garcia, B. and R. Riba. 1982. Germinacion de esporas de helechos arborescentes (Cyathea-
ceae) bajo diferentes condiciones de temperatura. Biotropica. 14(4):281-287.
Perez-Garcia, B., A. Orozco S. and R. Riba. 1982. El banco de esporas de helechos en suelos de

Los Tuxtlas. Bol. Soc. Bot. Mexico. 43:89-92.
Palacios RiOS, M. and R. Riba. 1983. Helechos de Veracruz: Adiantum (Pteridaceae). Bol. Soc.

Bot. Mexico. 44:43-62.
Lira, R. and R. Riba. 1984. Aspectos fitogeograficos y ecoldgicos de la flora pteridofita de la sierra

de Santa Marta, Veracruz, Mexico, Bidtica 9{4):45 1-467.
Riba, R. and A. Butanda. 1987. Bibliografia comentada sobre pteridofitas de Mexico. Consejo

Nacional de la Flora de Mexico, A. C. 87 pp.
Riba, R., L. Pacheco and E. Martinez S. 1987. New records of pteridophytes from the State of

Chiapas. Mexico. Amer. Fern J. 77(2):69-70.
Riba, R. 1989. A new species of Thelypteris (subg. Goniopteris] from the State of Veracruz, Mexico.

Amer. Fern J. 79:122-124.
Lorea, F. and R. Riba. 1989. Guia para la recoleccidn y preparacidn de ejemplares para herbaria

de pteridofitas. Consejo Nacional de la Flora de Mexico, A. C. 18 pp.
Riba, R. and I. Reyes J. 1990. Pityrogromma colomelanos (L.) Link in layers of volcanic ash in Los

Tuxtlas, Veracruz, Mexico. Ann. Missouri Bot. Card. 77(2):287-289.
Perez-Garcia, B. and R. Riba. 1990. Glosario para Pteridophyta (espanol-ingles). Consejo Nacional

de la Flora de Mexico, A. C. 82 pp.
Pacheco, L. and R. Riba. 1990. Hymenophyllaceae. In Flora de Veracruz. 63:1-54. Instituto de

Ecologfa, A. C. & University of California, Riverside.
Riba, R., B. Perez-Garcia, and M. Perez-Garcia. 1991. Schaffneria nigripes Fee (Aspleniaceae):

116

AMERICAN FERN

morfogenesis del gametofito, anatomia y morfologia del esporofito. Bol. Soc. Bot. Mexico. 52:
105-113.

Perez-Garcia, B., I. Reyes, L. Pacheco and R. Riba. 1992. Manual de practicas de laboratorio de

briofitas y pteridofitas. Universidad Autdnoma Metropolitana Iztapalapa. Mexico, D. F. 119 pp.
Rtba, R. 1992. Lophosoriaceae. Flora de Mexico 6 (l):13-16. Consejo Nacional de la Flora de

Mexico, A. C. Mexico.
Riba, R. 1992. Reflexiones pteridoldgicas. Ciencias. 6:41-46.
Riba, R. 1993. Mexican pteridophytes: distribution and endemism. in T. P. Ramamoorthy, R. Bye,

A. Lot. & J. Fa, eds. Biological Diversity of Mexico: Origins and Distribution, pp. 379-396.

Oxford University Press. New York.

Riba, R., B. Perez-Gargia and A. Orozco S. 1993. Las pteridofitas en la Historia de las Plantas
de la Nueva Espafia. Acta Bot. Mex. 25:27-48.

RiBA, R. and S. Torres-Pecm. 1993. First Continental record of Thelypteris guadolupensis in Amer-
ica. Anier. Fern J. 83 (1):39.

Perez-Garcia, B. and R. Riba. 1993. Observaciones sobre los gametofitos de Woodwardia martinezii
Maxon ex Weatherby y W. spinulosa Mart, et Gal. (Blechnaceae). Acta Bot. Mex. 21:7-14.

Lira, R. and R. Riba. 1994. Las pteridofitas (helechos y plantas afines) de Mexico. Revista Soc.
Mex. Hist. Nat. 44:99-108.

Perez-Garcia, B., a. Orozco S. and R. Riba. 1994. The effects of white fluorescent light, far-red

light, darkness and moisture on spore germination oi Lygodium heterodoxum (Schizaeaceae).

Amer. J. Bot 81(11]:1367-1369.
Perez-Garcia, B., A. Mendoza and R. Riba. 1994. Observaciones sobre el gametofito de Metaxya

rostrata (H. B. K.j C. Presl (Metaxyaceae]. Revista Biol. Trop. 42(3):455-460.
Perez-Garcia, B., R. Riba and A. Mendoza. 1994. Observaciones sobre el gametofito de Thelypteris

rhachiflexuosa Riba (Thelypteridaceae). Acta Bot. Mex. 28:63-69.
Riba, R. 1994. Desarrollo de los estudios sobre pteridofitas de Mexico. In Llorente B., J. & L Luna

v., (comps.). La taxonomia en Mexico. Fondo de Cultura Econdmica. Mexico, pp. 333-339.
Perez-Garcia, B. and R. Riba. 1994. Dicksoniaceae. Flora de Mexico. 6(3):1-13. Consejo Nacional

de la Flora de Mexico, A. C. Mexico.
Perez-Garcia, B., R. Riba and I. Reyes Jaramillo. 1995. Helechos mexicanos: formas de creci-

miento, habitat y variantes edaficas. Contactos 11:22-27.
Riba, R. and B. Perez-Garcia. 1995. Perspectivas en el estudio de las pteridofitas. Bol. Soc. Bot.

Mexico 55:127-133.

Riba, R. and B. Perez-Garcia. 1995. Pteridophyta. in G. Ibarra Manriquez, & S. Sinaca Colin. Lista
floristica comentada de la Estacion de Biologia Tropical "Los Tuxtlas". Veracruz, Mexico.
Rev. Biol. Trop. 43(l-3):75-115.

Riba, R. & L. Pacheco. 1995. Actinostochys. in R. C. Moran & R. Riba, pteridophyte eds. Flora
Mesoamericana. Vol. 1. Psilotaceae a Salviniaceae. pp. 52-53. Universidad Nacional Autdn-
oma de Mexico, Mexico City.

Riba. R. and L. Pacheco. 1995. Schizaea. in R. C. Moran, & R. Riba, pteridophyte eds. Flora
Mesoamericana. Vol. 1. Psilotaceae a Salviniaceae. pp. 57. Universidad Nacional Autdnoma
de Mexico, Mexico City.

Riba. R. 1995. Lophosoriaceae. in R. C. Moran & R. Riba, pteridophyte eds. Flora Mesoamericana.

Vol. 1. Psilotaceae a Salviniaceae. pp. 85. Universidad Nacional Autdnoma de Mexico, Mex-
ico City.

Riba, R. 1995. Metaxyaceae. in R. C. Moran & R. Riba, pteridophyte eds. Flora Mesoamericana.
Vol. 1. Psilotaceae a Salviniaceae. pp. 85-86. Universidad Nacional Autdnoma de Mexico,
Mexico City.

Riba, R. 1995. Ahophila. in R. C. Moran & R. Riba, pteridophyte eds. Flora Mesoamericana. Vol.
1. Psilotaceae a Salviniaceae. pp. 88-90. Universidad Nacional Autdnoma de Mexico Mexico
City.

Riba, R. 1995. Onocleopsis. in R. C. Moran & R. Riba. pteridophyte eds. Flora Mesoamericana. Vol.
1. Psilotaceae a Salviniaceae. pp. 246-247. Universidad Nacional Autdnoma de Mexico, Mex-
ico City.

Riba, R. 1995. Schoffneria. in R. C. Moran & R. Riba, pteridophyte eds. Flora Mesoamericana. Vol.

OBITUARY

117

1. Psilotaceae a Salviniaceae. pp. 324-325. Universidad Nacional Autdnoma de Mexico, Mex-
ico City.

SouSA, M., R. RiBA, F. Chiang, B. Perez-Garcia, S. Zarate and L. Pacheco. 1995. Glosario. in R.

C. Moran & R. Riba, pteridophyte eds. Flora Mesoamericana. Vol. 1. Psilotaceae a Salvini-
aceae. pp. 411-432. Universidad Nacional Autonoma de Mexico, Mexico City.

Perez-Garcia, B. and R. Riba. 1995. Dicksoniaceae. Flora de Mexico. 6(3):1-18. Consejo Nacional

de la Flora de Mexico, A. C. Mexico.
Riba, R. 1995. A manera de conclusion, in E. Linares, et al., eds. Conservacion de plantas en

peligro de extincion: diferentes enfoques. Universidad Nacional Autdnoma de Mexico, pp.

171-175.

Riba, R. and R. Lira. 1996. Flora del Valle de Tehuacan-Cuicatlan. Fasciculo 10. Pteridophyta

sensu R. Sadebeck: Familias Equisetaceae DC, Lycopodiaceae Mirb. y Selaginellaceae Milde.

Institute de Biologia, U.N. A.M. Mexico. 23 pp.
Perez-Garcia, B., A. Mendoza, R. Riba and M. Ricci. 1996-1997. Estudio del gametofito de Tb}T-

sopteris elegons (Filicales: Thyrsopteridaceae, Dryopteridaceae.). Rev. Biol. Trop. 44(3)/45(l):

59-65.

Riba, R., L. Pacheco, A. Valdes and Y. Sandoval. 1996. Pteridoflora del estado de Morelos,
Mexico. Lista de familias, generos y especies. Acta Bot. Mex. 37:45-65.

Riba, R. and B. Perez-Garcia. 1996. El pico de los helechos. Contactos. 14:20-21. Translation of
the paper: Moran, R. C. The fern spike. Fiddlehead Forum. 21(4&5):32-33.

Riba, R. and B. Perez-Garcia. 1996. La historia de la molesta Salvinia, Contactos. 15:42-46. Trans-
lation of the paper: Moran, R. C. 1992. The story of the molesting Salvinia. Fiddlehead Forum

19(4&5}:26-28.

Riba, R. and B. Perez-Garcia. 1996. Bracken, el ponzonoso. Contactos 16:30-32. Translation of
the paper: Moran, R. C. 1993. Bracken, the poisoner. Fiddlehead Forum. 20(3):18-19, 22.

Riba. R. and B. PEREZ-GARCfA. 1997. Pteridofitas. In Gonzalez Soriano, E., R. Dirzo & R. C. Vogt.
Historia Natural de los Tuxtlas. Universidad Nacional Autonoma de Mexico. Mexico. Mexico,

D. F. pp.175-181.

RiBA, R., B. Perez-Garcia and A. Orozco Segovia. 1997. Las pteridofitas en la Historia de las
Plantas de la Nueva Espana de Francisco Hernandez, Protomedico espanol. Actas Etnobo-

tanicas. 92:75-80.

Riba, R. and B. Perez-Garcia. 1997. En busca de la semilla de los helechos. Contactos. 23:35-38.
Translation of the paper: Moran, R. C. 1995. In search of the fern seed. Fiddlehead Forum
22(6]:37-40.

Riba, R. and B. Perez-Garcia. 1997. Pueden las pteridofitas ser arboles? Contactos. 24:5-9. Trans-
lation of the paper: Moran, R. C. 1994. Pteridophytes as trees. Fiddlehead Forum. 21[2):10-13.

RiBA, R. and B. Perez-Garcia. 1998. Helechos, linternas y bosques del Terciario. Contactos. 30:
30-34. Translation of the paper: Moran, R. C. 1998. Ferns, flashlights and Tertiary forests.
Fiddlehead Forum. 25(1):1, 4-7.

Perez-Garcia, B. and R. Riba. 1998. Bibliografia sobre gametofitos de helechos y plantas afines
(1609-1992). Monogr. Syst. Bot. Missouri Bot. Card. 70:1-78.

Perez-Garcia, B., R. Riba, A. Mendoza & I. Reyes J. 1998. Compared gametophytic development
of three species of Phlebodium (Polypodiaceae s. str.J. Rev. Biol. Trop. 46:1059-1067.

Ramirez, M. R., R, Riba and B. Perez-Garcia. 1998. Marsilea. . .trebol de cuatro hojas?. ContactoS
(3' Epoca). 28:44--46.

Riba, R. and B. Perez-Garcia. 1999. Dryopteridaceae. Flora de Mexico. 6(4]:l-48. Consejo Na-
cional de la Flora de Mexico, A. C. Mexico.

Ramirez, M. R., B. Perez-Garcia and R. Riba 1999. Psilotum, la planta que otro poco y no existe,

ContactoS (3=^ Epoca). 31:68-70.

Ramirez, M. R., B. Perez-Garcia and R. Riba. 1999. Solanopteris. . . hogar, dulce hogar. ContactoS

(3" Epoca) 33:11-13.

Perez-Garcia, B., R. Riba and D. M. Johnson. 1999. Marsileaceae. Flora de Mexico. 6(5):1-17.

Consejo Nacional de la Flora de Mexico, A. C. Mexico.
Mendoza, A., B. Perez-Garcia and R. Riba. 1999. Morfologia y anatomia del gametofito de Di-

dymochlaena truncatula (Dryopteridaceae). Rev. Biol. Trop. 47(l):87-93.

118 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 3 (2000]

Perez-Garci'a, B., R. Riba and A. R. Smith. 1999. Thelypteridaceae. Flora del Bajfo y Regiones
Adyacentes. 79:1-35. Instituto de Ecologia, A. C. Centro Regional del Bajio. Patzcuaro, Mich.,
Mexico.

Mendoza, a.. B. Perez-Garcia and R. Riba. 1999. Desarrollo protalico de Lygodium heterodoxum
y Lygodium venustum (Schizaeaceae). Rev. Biol. Trop. 47(l-2):83-92.

Perez-Garcia, B., A. Mendoza, I. Reyes J. and R. Riba. 1999. Morfogenesis de la fase sexual de
seis especies mexicanas del genero Dry^opieris (Dryopteridaceae). Rev. Biol. Trop. 47(1-2):
69-81.

Mendoza, A., B. Perez-Garcia and R. Riba. 1999. Morfogenesis de la fase sexual del helecho

Aracbniodes denticulota (Dryopteridaceae). Rev. Biol. Trop. 47(4):00-00.

Riba, R., B. Perez-Garcia, A. Mendoza and 1. Reyes Jaramillo. 2000. Temas selectos de Botanica:

Morfologia de gametofitos de helechos. Universidad Autonoma Metropolitana-Iztapalapa.
132 pp

Riba, R. and M. A. Perez Farri^a. In press. New records for the pteridoflora of Chiapas, Mexico.
Amer. Fern J.

We are grateful to Mr. Jorge Lodigiani for preparing the photograph of Ramon Riba. Leticia
Pacheco & Blanca Perez-Garcfa, Universidad Autonoma Metropolitana-Iztapalapa, Depto. de Biol-
ogia, Apdo. Postal 55-535, 09340 Mexico, D. F.

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QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY

Survival of Chlorophyllous and Nonchlorophyllous Fern Spores Through Exposure to

Liquid Nitrogen Valerie C. Pence

119

Morphology of Gametophytes and Young Sporophytes of Sphaeropteris lepifera

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American Fern Journal 90(43:119-126 (2000) ^ . ^__

GARDEN LIBRARY

Survival of Chlorophyllous and Nonchlorophyllous
Fern Spores Through Exposure to Liquid Nitrogen

Valerie C. Pence

Center for Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden,

3400 Vine Street, Cincinnati, Ohio 45220

Abstract. — Thirty-three species of ferns with nonchlorophyllous spores and five species with
chlorophyllous spores were studied in regard to their ability to survive exposure to liquid nitrogen
(LN). Air dried spores showed no inhibition of germination after LN exposure when planted on
soil or growth medium. Spores of three species that were stored for 75 months either at 4°C, -20°C»
or in LN showed no decrease in viability over that time, and spores of four other species were
maintained successfully for 52 months in LN, Fresh chlorophyllous spores that were air dried,
dried over silica gel, or prepared with the encapsulation dehydration procedure also showed good
survival through desiccation and LN exposure. Spores of Osmunda regalis germinated well after
18 months of LN storage. These results indicate that both nonchlorophyllous and chlorophyllous
spores are candidates for long-term germplasm storage at low temperatiires, including storage in
LN.

Ex situ preservation of spores, particularly of rare species from threatened
habitats, can be an important supplement to the maintenance of pteridophyte
species in the wild. Many species of homosporous ferns produce nonchlor-
phyllous spores that can be stored for relatively long periods of time, although
they can eventually lose viability in storage (Beri and Bir, 1993; Camloh, 1999J.
It has also been shown that hydrated spores of some of these species can
remain viable in the soil spore bank for a number of years, and moist storage
of spores ex situ has also been effective (Lindsay et ah, 1992; Dyer, 1994).
Other species of ferns and fern allies produce chlorophyllous spores, which
generally have limited viability, even though they may survive desiccation
(Lloyd and Klekowski, 1970].

Low temperature storage has been shown to improve the longevity of dry
seeds (Dickie et ah, 1990), and lower temperatures improved the survival of
spores of the tree fern, Cyathea delgadii during two years of storage (Sima-
bukuro et ah, 1998). Cryopreservation, or storage at -196°C in liquid nitrogen
(LN), has the potential for maintaining viability in living tissues over long
periods of time. Many seeds can survive direct exposure to LN (Stanwood,
1985; Pence, 1991), and spores of the endangered tree fern, Cyathea spinulosa
have been successfully germinated after cryopreservation (Agrawal et qL,
1993).

In order to examine the possibility of extending cryopreservation to other
species of ferns, spores of several nonendangered chlorophyllous and non-
chlorophyllous species were tested for their ability to withstand LN exposure,
and storage in LN was compared with other low temperature storage methods.
Results from tests with these common species should provide direction for

120 AMERICAN FERN JOURNAL: VOLUME

future work with rare or endangered germplasm, which is often in limited
supply.

Materials and Methods

Spores were obtained from a variety of sources, including from the American
Fern Society Spore Exchange and from fronds collected in northwest Trinidad,
at the Krohn Conservatory (Cincinnati] and at the Cincinnati Zoo and Botan-
ical Garden.

When fronds were collected, they were air dried for several days under am-
bient conditions in the laboratory in paper envelopes, with spores collected

am

transferred

LN. After 1 hr, the vials were removed and placed on the benchtop to warm
at ambient temperature for 20 min.

LN exposed and control nonchlorophyllous spores of 33 species were tested
for germination using one of the two following methods.

1) Spores of some species were sown on moist sterile soil (Metro Mix 250)
in Magenta boxes or baby food jars with Magenta caps, and incubated at 26°C
under CoolWhite fluorescent lights in a 16/8 hr light/dark cycle.

2) Spores of other species were germinated in vitro. Spores were wrapped
in a small package made from Whatman No. 1 filter paper, and surface ster-
ilized in a 1:20 dilution of commercial sodium hypochlorite for 5 minutes,
followed by two rinses in sterile, ultra-pure water. The spores were then blot-
ted onto medium consisting of half-strength Linsmaier and Skoog (1965) (LS)
medium with 1.5% sucrose and 0.22% Phytagel (Sigma Chemical Co.), in 60
X 15 mm disposable petri dishes, approximately 15 ml/dish.

For longer storage, nonchlorophyllous spores of three species were placed
in multiple cryovials and stored at three temperatures: LN (-196°C), -20°C,
and 4°C. The cryovials stored at -20°C and 4°C were placed inside 20 ml
borosilicate scintillation vials with plastic screw caps, containing 2-2.3 g of
silica gel. Spores from the three storage temperatures were sampled at 7, 13,
34, and 75 months by germinating on soil, as described above. Spores from
four species collected in Trinidad were also cryostored in LN.

Chlorophyllous spores of five species were also tested. These were obtained
either from the Spore Exchange or from plants growing at the Cincinnati Zoo
and Botanical Garden. In most cases these were an dried, as for the nonchlo-
rophyllous spores.

However,^ Osmunda regalis spores which were collected from plants grown
at the Cincinnati Zoo and Botanical Garden were tested using two other pro-
cedures. One portion of the spores was dried in a desiccator with silica gel
overnight, placed in a cryovial, and frozen and thawed as described above.
The spores were then placed in a 100% humidity chamber for 3 hours, and
surface sterilized and germinated in vitro as described above. A second portion
of the spores was first surface sterilized and then encapsulated in alginate
accordmg to the procedure of Fabre and Dereuddre (1990). The spores were

PENCE: SURVIVAL OF FERN SPORES

121

blotted into a 3% solution of low viscosity alginic acid (Sigma Chemical Co.)
and the spore suspension was added dropwise to a 100 mM solution of cal-
cium chloride, where the drops gelled and formed alginate beads containing
the spores. After 20 min the beads were transferred to a solution of LS medium
containing .75M sucrose and incubated on a gyratoray shaker, 125 rpm, for 18
hours. They were then dried on filter paper under the air flow of a laminar
flow hood for 4 hours, transferred to 2 ml polypropylene cryovials, immersed
directly into LN, and maintained there for 1 hour. The beads with spores were
warmed at room temperature for 20 minutes and then transferred to half-
strength LS medium for rehydration and growth. Some dried beads were also
put into long-term LN storage, and a sample was removed after 18 months for
growth and evaluation.

Only a qualitative evaluation of spore germination was made, both on soil
and in vitro, although an attempt was made to maintain approximately equal
amounts of control and LN exposed spores. Positive spore germination was
recorded if any germination was observed. With the nonchlorophyllous spores,
no great differences in the rate of germination were observed between control
and frozen spores of any species, as determined by gross observation. With
chlorophyllous spores, a distinction was made between the germination of
many spores and the germination of one or only a few spores, as indicated in
Table 3.

Results

The germination of air-dried nonchlorophylous spores exposed to LN was
equivalent to that of nonexposed spores for the species tested (Table 1). Ger-
mination rates varied between species, as determined by visual examination
of the germination of similar amounts of spores, but there were no obvious
differences between LN exposed and control spores of the same species. In the
case of three species, there was no germination from either control or LN ex-
posed spores.

There was also no apparent difference between the germination of spores of
three species stored at 4°C, -20'^C, or in LN, after more than six years of storage
(Table 2). Four species from Trinidad [Adiantum tenerum, Cyclopeltis semi-
cordata, Macrothelyptris torresiana, and Tectaria incisa) were also placed into
long-term LN storage and germinated well after 52 months of storage.

Chlorophyllous spores of five species were also tested (Table 3). There was
no germination observed for Blechnum nudum, Matteuccia struthiopteris, or
Osmunda cinnamomea spores, that were obtained after storage at the Spore
Exchange for several months. With Onoclea sensibilis spores obtained from
the Spore Exchange, only one spore germinated in the nonexposed samples
and none in the LN exposed sample, suggesting a low viability of the spores
overall. However, when fresh spores of O. sensibilis were collected locally and
tested, there was germination of the dried spores, both with and without LN
exposure. Similarly, when spores of Osmunda regalis were obtained from a
nonlocal source after about a year of dry storage, there was very little germi-

122

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

Table L Survival of 33 species with nonchlorophyllous spores through LN exposure after air drying;
germination = +; no germination observed = -; contaminated, germination could not be observed = C.
Spore sources: K - Krohn Conservatory; SB = Spore Exchange; T = Trinidad collection; Z = collection
at the Cincinnati Zoo and Botanical Garden.

Species

Germinated in vitro

Adiantum caiidatum L.

Adiantum tenerum Sw.

Adiantum trapeziforme L.

Cibotium ghiucum (Sm.) Hook & Am.

Cyrtomium falcatum (L.f.) C. Presl

Daxallia fejeensis Hook.

Dtynaria quercifolia (L.) J. Sm.

Phlebodium aureiim (L.) J. Sm.

Germinated on soil

Adiantum tenerum Sw.

Adiantum tetraphyllum Humb. & Bonpl. ex Willd.

Asplenium ruta-muraria L.

Asplenium platyneuron (L.) Britton, Stems, & Poggenb
Athyrhun filix-femina (L.) Roth

Athyrhim thelypteroides (Michx.) Desv.
Bolbitis sp.

Cibotium schiedei Schltdl. & Cham.
Cyathea arborea (L.) Sm.
Cyclopehis semicordata (Sw.) J. Sm.
Cyrtomium falcatum (L.f.) C. Presl.
Cyrtomium fortunei John Sm.
Dryopteris carthusiana (Vill.) H. R Fuchs
Dryopteris celsa (W. Palmer) Small
D/yopteris clintoniana (D.C. Eaton) Dowell
Dryopteris goldiana (Hook.) A. Gray
Dryopteris marginalis (L.) A. Gray

Macrothelyptris torresiana (Gaud.) Ching
Nephrolepis sp.

Phegopteris connectilis (Michaux) Watt
Polystichum acrostichoides (Michx.) Schott
Polystichum aculeatum (L.) Roth
Polystichum hraunii (Spenn.) Fee

Polystichum tsus-simense (Hook.) J. Sm.
Pteris sp.

Rumohra adiantifonnis (G. Forst.) Ching
Tectaria incisa Cav.

Source

K
K
K
K
K
K
K
K

T
T

SE
Z

z

z

K
K
SE

T

Z, K
Z

z

SE
SE
Z

z

K, T
Z

SE

Z

z
z

K
K
K
T

Germination

Control

C

+
+

+
+
+
+
+

+

+
+
+

+

+

+
+
+
+
+

LN expose

+
+

+
+

+

+
+

+
+
+
+

+

+
+
+
+

+
+

+
+
+

+

+

nation with or without LN exposure. However, when locally collected spores
were processed immediately after collection, there was good survival through
drying and LN exposure, using either drying over silica gel or the encapsula-
tion dehydration procedure. In addition, fresh spores of O. regalis that were
cryostored using the encapsulation dehydration procedure retained good via-
bility after 18 months of LN storage [Table 3).

PENCE: SURVIVAL OF FERN SPORES

123

Table 2. Survival of nonchlorophyllous spores of three species for up to 75 months at three storage

temperatures.

Species

Pteris sp.

Cyrtomium falcatum

Pohstichiwn tsus-sinense

Storage time
(months)

7
13

34
75

7
13
34
75

7
13
34
75

4°C

+
+
+
+

Storage temperatures

20°C

+

+

+
+
+
+

+

196°C

+

+
+

+
C

Discussion

These results demonstrate that dried spores from a number of species of
ferns can survive exposure to liquid nitrogen. In addition, dry, nonchloro-
phyllous spores of four species survived LN storage for 52 months, and three
other species survived well for over 75 months when kept at either 4''C, -20°C,
or — 196°C (in LN). Chlorophyllous spores of Osniundo regalis maintained vi-
ability in LN for at least 18 months.

It is known that nonchlorophyllous spores of many species can be main-
tained in the dry state at room temperature for a number of years, or even
decades (e.g., Windham et aL, 1986). Viability does decrease over time, and
this loss has been correlated with a loss of protein, sugar, and amino acids in
Pteris vittata (Beri and Bir, 1993). Hydrated storage has also been explored and
has been shown to maintain viability longer than dry storage with spores of
several species (Lindsay et aL, 1992). Spores of Cry^pto gramma crispa showed
a decreased rate of germination when subjected to freezing at -18''C, although
the degree of moisture in the spores was not determined (Pangua et aL, 1999).

Nonchlorophyllous spores are similar to orthodox seeds, which can be dried
and stored for a number of years. The longevity of orthodox seeds is extended
proportionately with a decrease in the storage temperature, and such seeds
have traditionally been stored at 4°C or -20X. Dried orthodox seeds are also
generally tolerant of LN storage, which may extend longevity significantly
(Stanwood, 1985; Pence, 1991). Nonchlorophyllous spores appear to have a
similar tolerance for cryostorage, and, in the case of three species, there was
no apparent difference in the germination of spores stored at 4°C, -20°C, or
in LN for up to six years. More detailed studies are needed to determine if
there are differences in the rate or the quality of germination after longer stor-
age times, but all three temperatures should extend the storage life of non-
chlorophyllous spores well beyond the mean of 3 years at room temperature,
determined by Lloyd and Klekowski (1970).

124

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 [2000)

Table 3. Survival of spores of five chlorophyllous species through LN exposure, prepared either with
air drying, drj^ng under the air flow of the laminar flow hood or with the encapsulation/pretreatment/
dehydration procedure (Fabre and Dereuddre, 1990). Spore sources: SE = Spore Exchange; Z = collection
at the Cincinnati Zoo and Botanical Garden. Germination of many spores = +; germination of one or
only a few spores = ( + ); no germination observed = — .

Species

Blechnum nudum (Labill.) Matt ex Lerss

Matteiiccia struthiopteris (L.) Tod

Onoclea sensibilis L.

Osmunda cinnamomea L.

Osmunda regalis L.

Source

Z

Z

z

Treatment

SE Air drying

SE Air drying

SE Air drying

Air drying

SE Air drying

SE Air drying

SE Air drying

SE Air drying

Drying over silica gel

Encapsulation/dehydration

Time

in LN Survival

Ohr

18 mos

(+)

(+)
(+)
(+)

+

+
+
+

In contrast with nonchlorophyllous spores, chlorophyllous spores germinate
rapidly, within an average of two days, and have a mean storage life of only
48 days (Lloyd and Klekowski, 1970), although there is evidence that in the
moist soil spore bank they can survive at least 10 months (Dyer and Lindsay,
1992). No more than 5% viability was reported for Osmunda regalis and O.
claytoniana spores maintained for 3.5 years at 4°C (Stokey, 1951], The limited
viability of dry spores of Equisetum hyemale has been shown to result from
damage to photosynthetic function that occurs within two weeks of storage
(Lebkuecher, 1997), although their longevity has been increased by storage at
4°C and at -70T [Lloyd and Klekowski, 1970; Whittier, 1996). Of the chlo-
rophyllous species studied here, spores that had been stored for a year had
lost most or all viability by the time of testing, whereas fresh spores were
viable and maintained viability through desiccation and LN storage. In the case
of Osmunda regalis spores, viability was maintained for at least 18 months in
LN storage, and it is likely that this may extend for even longer periods of

time. Freeze storage of dried chlorophyllous spores might be used by spore
banks to maintain viabilitv in tV»Pc^ cnor-Toc r.^T^r^ ir^^r^r.^ ^^^^^^^ ^i* ^^^^ ^i

PENCE: SURVIVAL OF FERN SPORES

125

though further research is needed to determine whether conventional freezing
temperatures are equally as effective as storage in LN.

Chlorophyllous spores that can tolerate desiccation but have limited viabil-
ity under natural conditions resemble seeds classified as sub-orthodox (Bon-
ner, 1986]. Seeds of species of Populus and Salix, for example, can tolerate
drying, but are generally short-lived either as dried or hydrated seeds. How-
ever, when they are dry, they can also tolerate exposure to freezing tempera-
tures (^20*^C and LN], and when stored under these conditions their longevity
is increased compared with storage at 4°C (Pence, 1996, and unpublished]. LN
storage should offer a similar advantage for the long-term storage of the gen-
erally short-lived chlorophyllous spores.

The results of these studies suggest that low temperature storage, including
cryopreservation, is an option for the storage of both nonchlorophyllous and
chlorophyllous fern spores. Although each species is unique, these results
with nonendangered species provide an indication that there is a high prob-
ability of success with spores from a variety of species, including rare or en-
dangered species, for which spores may be in limited supply. Low temperature
spore banking could be used to supplement traditional spore storage, as well
as sporophyte collections, soil spore banks (Dyer, 1994), and LN storage of fern
gametophytes (Pence, 2000] for the maintenance of rare or endangered species.
For the long-term ex situ preservation of rare or endangered germplasm, low
temperature storage, including cryopreservation, can be used as one tool in an
integrated approach to fern species preservation.

Acknowledgments

The author gratehilly acknowledges Jeff Kapella (Krohn Conservatory) and Jocelyn Horder and
Wa}Tie Baxter [American Fern Society Spore Exchange) for providing several of the species used
in this study; Alvin Jose for making the collections at the Cincinnati Zoo and Botanical Garden;
William Stiver, Alvin Jose, Mary Ann Feist, and Natasha Cavanaugh for excellent technical assis-
tance; and Yasmin Comeau (National Herbarium of Trinidad and Tobago] and her staff for iden-
tifying species collected in Trinidad.

Literature Cited

Agrawal, D. C, S. S. Pa war, and A. F. Mascarenhas. 1993. Cryopreservation of spores oiCyotbea

spinulosa Wall. ex. Hook. f. An endangered tree fern. J. Plant Physiol. 142:124-126.
Beri, a., and S. S. BiR. 1993. Germination of stored spores of Pteris vittata L. Amer. Fern J. 83:

73-78.

Bonner, F. 1986. Technologies to maintain tree germplasm diversity. Pp. 630-672 in Technologies
to maintain biological diversity, vol 2, part D. Office of Technology Assessment, Washington,
DC.

Camloh, M. 1999. Spore age and sterilization affects germination and early gametophyte devel-
opment of Platycerium bifurcatum. Amer. Fern J. 89:124-132.

Dickie, J. B., R. H. Ellis, H. L. Kraak, K. R"iXiER, and P. B. Tompsett. 1990. Temperature and seed
storage longevity. Ami, Bot. 65:197-204.

Dyer A. F. 1994. Natural soil spore banks: Can they be used to retrieve lost ferns? Biodiversity &
Conservation 3:160-175.

Dyer, A. F., and S. Lindsay. 1992. Soil spore banks of temperate ferns. Amer. Fern J. 82: 89-122.

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Fabre, J., and J. Dereuddre. 1990. Encapsulation-dehydration: A new approach to cryopreservation

of Solanum shoot-tips. Cryoletters 11:413-426.

Jermy

and R S. Green. Springer Verlag, Berlin.

Kramer

Lebkuecher, J. G. 1997. DesIccatlon-time limits of photosynthetic recovery in Equisetum hyemale
{Ec[uisetaceae) spores. Amer. J. Bot. 84:792-797.

Lindsay, S., N. Williams, and A. F. Dyer. 1992. Wet storage of fern spores: unconventional but
far more effective. Pp. 285-294 in J. M. Ide, A. C. Jermy, and A. M. Paul, eds. Fern horticul-
ture: Past, present, and future perspectives. Intercept, Andover.

Linsmaier, E. M., and R Skoog. 1965. Organic growth factor requirements of tobacco tissue cul-
tures. Physiol. PI. 18:100-127.

Lloyd, R. M., and E. J. Klekowski, Jr. 1970. Spore germination and viability in Pteridophyta:
Evolutionary significance of chlorophyllous spores. Biotropica 2:129-137.

Pangua, E., L. Garcia-Alvarez, and S. Pajaron. 1999. Studies on Cryptogramma crispa spore
germination. Amer. Fern J. 89:159-170.

Pence. V. C. 1991. Grvonreservatinn nf cppric nf OViin natiiro nlor»tc ^,^A ^^}r^^^A ..^^r.;«« c ^ c^:

Technol. 19:235-251.

Pence

Bartr. Seed Sci. Technol. 24:151-157.

Pence, V. C. 2000. Cryopreservation of in vitro grown fern gametophytes. Amer. Fern J. 90:16-23.
Simabukuro E. a., a. R Dyer, and G. M. Felippe. 1998. The effect of sterilization and storage

conditions on the viability of the spores of Cyotbea delgadii. Amer. Fern J. 88:72-80.
Soeder, R. W, 1985. Fern constituents: Including occurrence, chemotaxonomy and physiological

activity. Bot. Rev. 51:442-536.

Stanwood, R C. 1985. Cryopreservation of seed germplasni for genetic conservation. Pp. 199-226.

in K, K. Kartha. Cryopresen^ation of plant cells and organs. CRC Press, Boca Raton, Florida.

Stokey, a. G. 1951. Duration of viability of spores of the Osmundaceae. Amer. Fern J, 41-111-
115.

Walter, K. S.. and H. J. Gillett. 1998. 1997 lUCN Red List of Threatened Plants. lUCN-The World
Conservation Union, Gland, Switzerland.

Whittier, D. R 1996. Extending the viability of Equisetum hyemale spores. Amer. Fern J. 86-114-
118.

Windham, M. D., P G. Wolf, and T. A. Ranker. 1986. Factors affecting prolonged spore viability
in herbaceous collection of three species of Pellaea. Amer. Fern J. 76:141-148.

American Fern Journal 90(4j:127-137 (2000J

Morphology of Gametophytes and Young Sporophytes

of Sphaeropteris lepifera

Yao-Moan Huang

Department of Forestry, National Taiwan University, 1, sec. 4, Roosevelt Rd„ Taipei 106, Taiwan

Shao-Shun Ying

Department of Forestry, National Taiwan University, 1, sec. 4, Roosevelt Rd., Taipei 106, Taiwan

Wen-Liang Chiou^

Division of Forest Biology, Taiwan Forestry Research Institute, 53 Nan-Hai Rd.,

Taipei 100, Taiwan

AhsiracL— Sphaeropteris lepifera is one of the largest tree ferns in Taiwan. On average, it produces
50.7 sporangia per sorus, and 64 spores per sporangium. Spore germination, after 2 years of storage
at 4°C was over 95%. The pattern of spore germination was "Cyathea-type", and the gametophytes
exhibited mainly Drynaria-type development with occasional Adiantum-type development. Typ-
ical gametophytes were heart-shaped but had the potential to elongate and become elliptical.
Multicellular hairs on the dorsal and ventral surfaces of the midrib cushion increased in size and
changed shape with age. They w^ere usually uniseriate when young, and became muhiseriatc with
age. Gametophytes initiated antheridia about 1 month after spores were sown, and did not become
hermaphrodites until 7 weeks later. During ontogeny, the gametangial sequence was from the male
to hermaphroditic. Antheridia formed on the wings of the ventral and dorsal surfaces of gameto-
phj^es. The wall of each antheridium was composed of 5 cells. Archegonia appeared on the
cushion of the ventral surface of gametophytes. Some gametophytes initiated clone-formation
through vegetative regeneration. When sufficient water was provided, young sporophytes began
to appear 12 weeks after spores were sown. The first fronds were midribless. The uniseriate,
multicellular hairs on young sporophytes were similar to those on gametophytes.

:emiination

types of early development, trichomes, and gametangia, have been used to

taxa

and Kaur, 1971; Atkinson, 1973]. These characteristics provide information
relevant to fern phytogeny (e.g., Nayar and Kaur, 1971] and the ecology and
reproductive biology of different species [Chiou and Farrar, 1997a, b; Chiou et
aL, 1998; Masuyama, 1975a, b, 1979].

In the Cyatheaceae, spore germination is Cyathea-type and gametophyte de-
velopment is Adiantum-type or nearly Drynaria-type (Nayar and Kaur, 1971].
Gametophytes are long-lived (2-6 years] and tend to elongate slightly with age.
The multicellular, bristle-like hairs that appear on gametophytes in this family
indicate the Cyathaceae is phylogenetically close to the Loxsomaceae (Atkin-

same

gametophytes, with some vigorous gametophytes being strictly archegonial
(Stokey, 1930]. Apogamy was reported in some species in the Cyatheaceae

' Corresponding author.

128

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

Table L Spore sources, usage, culture medium and storage period.

Spores

Location

Observation

Stored
Medium (years)

Huang 25
Huang 26
Huang 27
Huang 25
Chiou 14495
Chkni 14787
Huang 25
Huang 26
Huang 27

Wulai, 370 m
Wulai, 370 m
Yamingshan, 250 m
Wulai, 370 m
Wulai, 150 m
Hsinihsien, 200 m
Wulai, 370 m
Wulai, 370 m
Yamingshan, 250 m

sporangium and spore number
sporangium and spore number
sporangium and spore number
germination rate
germination rate
germination rate
gametophyte morphology
gametophyte morphology
gametophyte morphology

agar
agar
agar
peat

peat
peat

2
1

(Slokey, 1918), but Walker (1966) reinvestigated two of these species and found
them to be sexual diploids.

Spbaeropteris lepifera (Hook.) Tryon (Cyatheaceae) is the largest, and one of
the most abundant, species in this family in Taiwan. Wang et al. (1977) de-
scribed its sporophyte morphology, scales, stomata, epidermal cells, and the
anatomy of the stipes and trunks. However, except for the gametangial se-
quence (Masuyama, 1975b)» characteristics of the gametophyte of S. lepifera
have not been described.

The number of spores per sporangium has also been used in the systematic
classification of ferns (Sen, 1964; Gastony, 1974). Spore viability, gametangial
sequence, and gametophyte growth habits are important components of fern
reproductive biology (Chiou and Farrar, 1997a; Chiou et al, 1998). Complex
interactions among gametophytes in multispore cultures may affect the se-
quence of gametangial development in individual plants. Detailed analysis of
sexual status, in relation to the age and size of gametophytes, is essential for
determining the most probable breeding system employed by S. lepifera
(Chiou and Farrar, 1997b; Chiou et al, 1998). The present study explores spore

m

ogy and growth habits of the gametophytes and the morphology of young spo-
rophytes of S. lepifera.

Materials and Methods

Spores were collected from sporophytes growing on mountain slopes in
northern Taiwan (Table 1). Voucher specimens were deposited in the Herbar-
ium of Taiwan Forestry Research Institute (TAIF). Spores were stored in the

refrigerator at 4°C.

The number of sporangia per sorus (from 50 randomly sampled sori per
plant) and the number of spores per sporangium (from 50 randomly sporangia

estimated

medium

spores stored at 4°C for one or two years was assessed (Table 1). Spores were
sterilized with 2.5% Clorox for 5 min, rinsed with sterilized water for 10 min.

HUANG ET AL.: GAMETOPHYTES AND SPOROPHYTES OF SPHAEROPTERIS

129

Tari f 2.

Mean geiniination

(%) of fresh and stored (4°C) S. lepifera spores

sown on agar medium.

Storage _

5

0.0 (0)
35.8 (26.0)
1.3 (1.9)

Days after sowing spores
7 9

(years)

11

1
2

81.2 (7.2) 96.7 (4.0)
97.9 (2.0) 98.7 (1.5)
29.7 (16.8) 84.0 (7.4)

99.0(1.2)
98.9 (1.5)
95.4 (4.0)

Standard error in parentheses.

and then sown on 1.1% agar-solidified medium containing Gantt and Arnotts'
(1965) mineral nutrients and one drop of 1% FeCl3 per 400 ml of medium.
The pH of the media ranged from 5,0-5.2. Germination rates were estimated
from six agar-medium petri dish cultures with 50 spores per dish. Rupture of
the spore wall was used as an indicator of germination. Gametophyte mor-
phology was observed for 8 months. The sexual expression, age and size of 50
gametophytes from each of 3 multispore peat cultures were recorded every 2
weeks. Gametophyte width was measured at the widest point.

Because the peat medium provided conditions more similar to those in the
field than the agar medium, gametophyte morphology and sexual expression
were assessed only for gametophytes in peat-grown cultures. However, game-
tophyte cultures on agar medium were used to estimate germination because
spores and their germination status could be seen more easily on agar medium.
All cultures were maintained at 20-25''C under white fluorescent illumination
of 1000-1500 lux for 12 hrs per day.

Microscopes (Lecica, Wild M8; Leitz, Dialux 20) were used to make mor-
phological observations of gametophytes and young sporophytes. Pictures
were made with the aid of a drawing tube or photomicrography. Gametangia
were also observed with a scanning electron microscope. Gametophytes were
dry mounted on double-stick mending tape, coated with gold, and observed
with a Hitachi S2400 Scanning Electron Microscope.

Results

The number of sporangia per sorus averaged 50.7 ± 13.1 s.d. and ranged
from 19 to 78. Each sporangium contained 64 spores.

mm

began about 5 days after sowing, whereas germination of fresh spores did not
begin until seven days after sowing. The viability of spores maintained for one
or two years in cold storage was not significantly less than that of fresh spores.
Two weeks after sowing, over 95% of fresh and stored spores had germinated

(Table 2).

lepifera are tetrahedral. Wh

rupt

formed

and did not contain chloroplasts. The second division was perpendicular to

130

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

Figs. 1-9. Morphology of different stages of S. leplfera gametophytes. Figs. 1-3. Spore germi-
nation. Figs. 4-6. Filamentous stages. Fig. 7. A spatulate gametophyte. Fig. 8. A filament with a

leart-shaped gametophyte. Bar = 50(jLm for Figs. 1-8; 1 mm
for Fig. 9.

wedge-shaped apical cell. Fig. 9. A heart-shaped g;

the first division (Fig. 3) and, by a series of transverse divisions, initiated a
uniserlate filament that was usually 4-8 cells long (Figs. 4-8). About 4 days
after spore germination, the apical, and sometimes subapical, cells divided
parallel to the long axis of the filament (Fig. 5). However, in a few gameto-
phytes the apical cell of the filament became sluggish and did not divide (Fig.
6). This apical cell was pushed to the subapical margin by intercalary divi-
sions. Next, a spatulate plate formed. When gametophytes were about 5 cells
wide, a wedge-shaped meristematic cell formed in the apical region (Fig. 7).

some

(Fig. 8] that underwent repeated oblique divisions until it was replaced by a
pluricellular meristem, the cells of which divided actively, producing a

mi

me

phyte formed (Fig. 9). The wings were one cell thick and were usually flat,
but some curved upward at the margin. Rhizoids were restricted mostly to the
ventral surface of the midrib. They were transparent but became light brown
to brown with age.

Gametophytes initiated antheridium production in the spatulate stages (<0.5
mm wade). In heart shaped gametophytes, antheridia usually appeared on the
ventral surface of the wings (Fig. 9), with a few forming on the dorsal surface.
If the apical notch had not formed, antheridia sometimes formed near the api-
cal region (Fig. 17), or on the margin, especially on filamentous proliferations
(Fig. 20). Mature antheridia were about 50 jjim wide and 40 jxm high (Fig. 10).
The antheridium wall was composed of five cells: a basal cell, a lower ring
cell, an upper ring cell, a crescent-shaped cell, and an elliptical opercular cell.
Upon dehiscence, the opercular cell was thrown off, and sperm were released.
Gametophytes did not produce archegonia until 7 weeks after spore sowing

mi

observed

HUANG ET AL.: GAMETOPHYTES AND SPOROPHYTES OF SPHAEROPTERIS

131

•

X 1 .

15K'-.' 50u»

Figs. 10-11. SEM of S. lepifera gametangia. Fig. 10. An antheridium. Fig. 11. An archegonium.

Mature archegonia were about 40 jjim wide and 50 jxm high (Fig. 11], and the
neck was 4 to 5 cells long. Antheridia continued to form after archegonia were
initiated. Male gametophytes were usually less than 3 mm wide, whereas her-
maphroditic gametophytes were usually greater than 3 mm wide. By 11 weeks
after spore sowing, all gametophytes bore antheridia, that is they were male
or hermaphroditic (Table 3].

Trichomes occurred on both the dorsal and ventral surfaces of the gameto-
phyte midrib. Usually, they were mixed with the archegonia on the cushion,
especially near the apical notch of the gametophyte. Trichomes did not appear
until the cushion and archegonia formed. The size of the pluricellular hairs
varied greatly. Those on young gametophytes were uniseriate or, occasionally,
bi- or triseriate (Figs. 12, 13], and usually were composed of less than 20 cells.
Hairs began as uniseriate structures that grew by intercalary division into larg-
er, multiseriate structures with age. On older gametophytes (e.g., 8 months
old), the hairs could grow to 8 cells wide and be comprised of more than 50
cells in total, which were with a uniseriate, needle-like tip (Fig. 14].

Some gametophytes of S. lepifera elongated and became elliptical as they
aged (e.g., 15 weeks old) (Figs. 15-17). Older gametophytes often became pale,
or even necrotic, at their posterior end but kept growing at their anterior end.
Some were slender, with many antheridia, but without archegonia or an ap-
parent apical meristem. Other elongate gametophytes formed discrete cushions
(Figs. 16, 17).

Some gametophytes began to produce vegetative proliferations when they
were 13 weeks old. Daughter gametophytes formed from one or a few cells on
the dorsal or ventral surfaces or on the margin (Fig. 18). Each of the daughter
gametophytes developed a pluricellular meristem and gametangia and even-
tually produced large clones. Some gametophytes formed many filamentous
proliferations, one to several cells wide (Fig. 20], that produced only anther-
idia. Occasionally, the anterior wings of gametophytes elongated and prolif-
erated (Fig. 19). As proliferations grew, their posterior died, separating them
from the parent gametophyte.

132

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

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HUANG ET AL.: GAMETOPHYTES AND SPOROPHYTES OF SPHAEROPTERIS

133

Figs. 12-29. Morphology of gametophytes and juvenile sporophytes of S. lepifera. Figs. 12-14.
Multicellular hairs of ganietophytes. Figs. 15-17. Elongate gametophytes. Figs. 18, 19. Gameto-
phytes with proliferations. Fig. 20. Filamentous proliferation with antheridia. Figs. 21-24. Mid-
rihless first few fronds. Figs. 25-27. Midribbed fronds. Fig. 28. Unicellular hairs of a young spo-
rophyte. Fig. 29. Multicellular hair of a young sporophyte. Bar = 100p.ni for Figs. 12-14, 28, 29;
0.2 mm for Fig. 20; 1 mm for Figs. 15-17, 21-27; 2 mm for Figs. 18, 19.

134

FERN JOURNAL: VOLUME

sufficient

peared in peat cultures about 12 weeks after spore sowing. The gametophyte
usually senesced and disappeared after the sporophyte formed. However, some
gametophytes with a young sporophyte formed proliferations that produced
additional sporophytes. Laminae of the first few fronds of young sporophytes
branched dichotomously (Figs. 21-24). Subsequent juvenile fronds were pin-
nately divided (Figs. 25-27). Except for the first few fronds, unicellular hairs
and scales were evenly distributed on the frond margin (Figs. 25-29).

Discussion

Sen (1964) noted that the diagnostic characters for the sporangia of most

simil

spores in each sporangium is 64 or 16. The sporangia of Lophosoria, Sphaer-

Metaxya

Nephelea

have 16 spores (Gastony, 1974). In this study, 64 spores were found in S. lep-

ifera sporangia, the same number found in the sporangia of the majority of

more highly evolved leptosporangiate ferns (Holttum and Sen, 1961; Gastony,
1974).

Page (1979) reported that the spores of some Cyatheaceae lost their viability
after a few weeks, but did not describe the conditions under which they were
stored. Germination of Cyathea delgadi spores was less than 20% after 2 years
of dry storage at -12°C (Simabukuro et al, 1998). However, the high germi-

that

lepifi

be dependent on the storage conditions.

After S. lepifera spores germinated, the filament grew along the polar axis,
while the first rhizoid elongated along the equatorial plane. This pattern of
spore germination has been classified as "Cyathea-type" and is considered
typical of ferns in the Cyatheaceae (Nayar and Kaur, 1971).

Using the definitions of Nayar and Kaur (1971), S. lepifera gametophyte de-
velopment was mostly of the Drynaria-type, but with some Adiantum-type
features. Our results are somewhat different from those of Nayar and Kaur
(1971), who described S ' "
or nearly Drynaria-type.

lepifi

if'

became more elongated. Elongate gametophytes were paler and more slender
than heart-shaped gametophytes, and some remained in the antheridial stage
for 8 months. Stokey (1930) reported that elongate gametophytes of Cyathea
arborea were very much pronounced and produced only antheridia when they

gametophyt

lepifi

developed after being overgrown by other gametophytes. This simulates partial
burial, which may occur in nature. Although elongate gametophytes are in-

HUANG ET AL.: GAMETOPHYTES AND SPORGPHYTES OF SPHAEROPTERIS 135

capable of supporting sporophytes, they may play a significant role in provid-
ing sperm and thus promoting cross-fertilization.

In the Cyatheaceae, the antheridial wall is usually composed of 5 cells, but
some variation occurs (Stokey, 1930; Atkinson and Stokey, 1964]. The wall of
each antheridum of S. lepifera in this study was always comprised of 5 cells,
including a basal cell, 2 ring cells, a crescent-shaped cell and an elliptical
opercular cell that was usually shed at antheridial dehiscence. The archego-
nium neck is straight, more or less, as has been reported for other Cyatheaceae
and some less advanced ferns (Stokey, 1930; Atkinson and Stokey, 1964).

The peculiar, multicellular hairs on the archegonial cushion of the game-
tophyte are characteristic of Cyatheaoid gametophytes (Stokey, 1930). Outside
the Cyatheaceae, similar multicellular hairs are found only on gametophytes
in the Loxsomaceae (Stokey, 1960; Atkinson and Stokey, 1964). Homology of
the multicellular hairs on gametophytes in the Cyatheaceae and Loxsomaceae
is supported by molecule-based cladistic analysis (Pryer et aL, personal com-
munication).

Gametophytes of S. lepifera can produce large clones and enhance their
longevity by vegetative growth, as has been described for some other species
in the Cyatheaceae and many other fern taxa (Stokey, 1930; Atkinson and Sto-
key, 1964; Chiou and Farrar, 1997a). The type of vegetative regeneration pre-

lepifi

gametophyt

Atkinson and Stokey (1964). They suggested that this type of vegetative regen-
eration usually occurred on old or injured gametophytes. However, many
clones of S. lepifera formed from gametophytes that were green and very
healthy. Although each new gametophyte could produce male and female gam-
etangia, few produced sporophytes while still attached to the parent gameto-
phyte if the parent plant had produced a sporophyte. Perhaps, the developing
embryo of the parent gametophyte inhibited the fertilization of eggs of the
daughter gametophytes, or inhibited zygote development in fertilized eggs.

lepifi

further investig

Most

mal

i only gametophytes were 0.5-3.0 mm wide, and hermaphroditic
gametophytes were usually more than 3.0 mm in width. Initial antheridium
formation was followed by the attainment of a prolonged hermaphroditic
phase during which antheridia continued to form. However, in a previous
study of the gametangial sequence of S. lepifera [= Cyathea lepifera), anther-
idium formation ceased once plants became hermaphroditic (Masuyama,
1975b). Gametophyte sexual expression may be affected by the culture medi-
um (Masuyama, 1975a). Masuyama s (1975b) observations were on gameto-
phytes grown on agar medium, whereas gametophytes in this study were
grow^n on peat medium.

A gametangial sequence from male to hermaphroditic may favor intraga-

metophytic selfing (Klekowski, 1969; Masuy

lepifi

136 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

Based on isozyme data, Chen (1995] suggested that the mating system of S.
lepifera tended toward intergametophytic crossing. Similarly, three other tree
ferns, Alsophila firma (Cyatheaceae), Cyathea stipularis (Cyatheaceae) and Lo-
phosoria quadripinnata (Lophosoriaceae) are outcrossing (Soltis et ah, 1991).
It has been suggested that inbreeding of bisexual gametophytes may be limited
by high genetic load (Klekowski, 1969, 1973; Masuyama, 1979; Peck et ah,
1990; Hooper and Haufler, 1997). Thus, if bisexual gametophytes are common
in natural populations of S. lepifera, genetic load may be the most important
factor favoring outcrossing in S. lepifera.

Young sporophytes were produced when sufficient water was supplied to
sexually mature plants. Gametophytes would not bear sporophytes when water
was not added to cultures, although they had mature gametangia. In addition,
young sporophytes were always born on the archegonia, suggesting that S.
lepifera sporophytes likely resulted from fertilization. The 64-spore sporangi-
um also indicates that it is not an apogamous species.

In S. lepifera, the blades of juvenile leaves are dichotomously branched,

whereas those of succeeding juvenile fronds are pinnately dissected. This is

the most common type of frond ontogeny in leptosporangiate ferns (Wagner,
1952).

Acknowledgments

The authors thank two anonymous reviews for their useful comments, Dr. Alan Warneke for
editing the manuscript, and Miss Chien-Rong Huang for SEM assistance. This research was sup-

Research Institute (contribution No. 137).

Forestry

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biology in Elapho gloss urn Schott. Canad. J. Bot. 76:1967-1977.
Gantt. E., and I. T. Arnott. 1965. Spore germination and development of the young gametophytes

of the ostrich ferns (Matteucia stnithiopteris). Amer. J. Bot. 52:82-94.
Gastony, G. J. 1974. Spore morphology in the Cyatheaceae. I. The perine and sporangial capacity:

general considerations. Amer. J. Bot. 61:672-680.

UM

phology 11:406-420.

Haufler

neotropical epiphytic ferns [Pleopeltis; Polypodiaceae). Amer. J. Bot. 84:1664-1674.

Volume 90

2000

QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY

Editor

R. James Hickey

Botany Department,

Miami University,

Oxford, OH 45056

Associate Editors

Gerald J. Gastony, Department of Biology, Indiana University,

Bloomington, IN 47405-6801

er, Department of Botany,
Lawrence, KS 66045-2106

Robbin C. Moran, New York Botanical Garden,

Bronx, NY 10458-5126

James H. Peck, Department of Biology,
University of Arkansas-Little Rock, Little Rock, AR 72204

The American Fern Society

Council for 2000

BARBARA

President

HAUFLER

Vice-President

CARL

CAPONE

Secretary

Treasurer
DAVID B. LELLINGER, 326 West St. NW., Vienna, VA 22180-4151. Membership Secretary

JAMES D. MONTGOMERY, Ecology TIT, R.D. 1, Box 1795, Berwick, PA 18603-9801.

Back Issues Curator

GEORGE YATSKIEVYCH, Missouri Botanical Garden, RO. Box 299, St. Louis, MO 63166-0299.

4

Journal Editor
DAVID B. LELLINGER, U.S. National Herbarium MRC-166,

Smithsonian Institution, Washington, DC 20560-0166. Memoir Editor

CINDY JOHNSON-GROH, Dept. of Biology, Gustavus Adolphus College,

800 W. College Ave., St. Peter, MN 56082-1498. Bulletin Editor

American Fern Journal

EDITOR

^

R. JAMES HICKEY

Botany Department,

Miami University, Oxford, OH 45056
ph. (513) 529-6000, e-mail: hickeyrj@muohio.edu

ASSOCIATE EDITORS

GERALD

HAUFLER

ROBBIN C. MORAN New York Botanical Garden, Bronx, NY 10458 5126

JAMES H. PECK Dept. of Biology, University of Arkansas — Little Rock,

2801 S. University Ave., Little Rock, AR 72204

The "American Fern Journal" (ISSN 0002-8444) is an illustrated quarterly de\oted to the general
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11

Table of Contents for Volume 90

(A list of articles ananged alphabetically by author)

Arens, N. C. & P. Sanchez Baracaldo. Variation in Tree Fern Stipe Length with

Canopy Height: Tracking Preferred Habitat through Morphological Change .. 1

AsHCROFT, Carl J, & Elizabeth Sheffield. The Effect of Spore Density on Ger-
mination and Development in Pteridium, Monitored using a Novel Culture
Technique 91

Baracaldo, P. Sanchez (see N. C. Arens) 1

Calderon-Saenz, Eduardo. Production of Adventitious Buds on Leaves in Dick-

sonia sellomana

Carlquist, Sherwin (see E. L. Schneider)

Chiou, Wen-Liang (see Y. Huang)

Conn, Herb (see H. Marriott)

Conn, Jan (see H. Marriott)

Duff, R. Joel, & Edward E. Schilling. The Chloroplast Genome Structure of the

105
32
127
109
109

Vascular Plant Isoetes is Similar to that of the Liverwort Marchantia 51

Farrera, Miguel Angel Perez (see R. Riba)

104

Gottlieb, Joan Eiger. Dryopteris fiUx-mas New in Pennsylvania 144

Greer, Gary K. & Brl\n C, McCarthy. Patterns of Growth and Reproduction in

a Natural Population of the Fern Polystichum acrosdchoides 60

Hendrix, Earl (see J. H. Peck)

Holland, Doug (see G. Yatskievych)

110

87

42

Huang, Yao-Moan, Shao-Shun Ying, & Wen-Liang Chiou. Morphology of Ga-

metophytes and Young Sporophytes of Sphaeropteris lepifera 127

Imperato, Filippo, & Antonella Telesca. 6-C-{i-CellobiosyHsoscutellarein-8-meth-

yl ether, a New Flavonoid from Pteris vittaTa

Imperato, Filippo. Kaempferol and Quercetin 3-0-(X", X"-di-protocatechuoyl)-glu-

curonides from Pteris vittata

Labiak, Paulo H. Notes on LeUmgena oreophila (Grammitidaceae), a poorly known

species from Colombia

141

24

138

Lellinger, David B. On the Lectotypification of Danaea elliptica 100

Li, Ruijun, Xiueeng Yan, & Dawei Zhang. Vessels in Roots and Rhizomes of

Dryopteris crassirhizoma (Dryopteridaceae) from Heilongjiang Province, Chi-
na

Marriott, Hollis, Jan Conn, & Herb Conn. Asplenium Xaltemifolium in the Black

Hills of South Dakota

McAlpin, Bruce W. (see A. Tejedor)

McCarthy, Brian C. (see C. A. Railing)

McCarthy, Brian C. (see G. K. Greer)

MoRAN, RoBBTN C. Review: Helechos de Mbaracayu

Pacheco, Leticia, & Blanca Perez-Garcia. Obituary: Ramon Riba y Nava Esparza

(1934-1999)

109

46

77

60

145

112

ui

Peck, James H., C. Theo Witsell, & Earl Hendrix. Dryopteris goldiana and its Hybrid

with D. celsa New to Arkansas 110

119

Pence, Valerie C. Survival of Chlorophyllous and Nonchlorophyllous Fern Spores

Through Exposure to Liquid Nitrogen

Pence, Valerie. Cryopreservation of In Vitro Grown Fern Gemetophytes 16

Perez-Garcia, Blanca & Ramon Riba. Bibliografia sobre Gametofitos de Helechos

y Plantas Afines, 1699-1996 [Reviewed]

PEREZ-GARCfA, Blanca (see L. Pacheco)

Railing, Carrie A.. & Brlvn C. McCarthy. The Effects of Rhizome Severing and

Nutrient Addition on Growth and Biomass Allocation in Diphasiastrum digi-
tatum

Riba, Ramon, & Miguel Angel Perez Farrera. New Records for the Pteridoflora

of the State of Chiapas, Mexico

Schilling, Edward E. (see R. J. Duff)

Schneider, Edward L., and Sherwin Carlquist. SEM Studies on Vessels in Ferns.

19. Marsilea

Sheffield, Elizabeth (see C. J. Ashcroft)

Tejedor, Adrian, & Bruce W. McAlpin. Ophioglossum pendulum L. Naturalized in

Miami, Dade County, Florida

Telesca, Antonella (see F. Imperato)

Windisch, Paulo G. On the Itineraries of Alfred and Alexander Curt Brade in Costa

Rica

Witsell, C. Theo (see J. H. Peck)

Yan, Xiufeng (see R. Li)

Yatskievych, George, & Doug Holland. Obituary: Joseph A. Ewan (1909-

1999)

Yatskievych, George. Review: Flora Malesiana, Series II-Fems and Fern Allies,

Volume 3

Yatskievych, George. Review: Bibliografia sobre Gametofitos de Helechos y Plan-
tas Afines, 1699-1996

YiNG, Shao-Shun (see Y Huang)

Zhang, Dawei (see R. Li)

49
112

77

104

51

32
91

46
42

108
110

24

87

48

49

127

24

Volume 90, Number 1, January-March, pages 1-50, issued 25 April 2000
Volume 90, Number 2, April-June, pages 51-90. issued 9 August 2000
Volume 90. Number 3, July-September, pages 91-118, issued 1 February 2001
Volume 90, Number 4, October-December, pages 119-151, issued 5 June 2001

IV

HUANG ET AL.: GAMETOPHYTES AND SPOROPHYTES OF SPHAEROPTERIS 137

Klekowski, E. J. 1969. Reproductive biology of the Pteridophyta. II.Theoretical Considerations.
Bot. J. Linn. Soc. 62:347-359.

Klekowski, E. J. 1973. Genetic load in Osmunda regolis populations. Amer. Fern J. 60:146-154.

Masuyama S. 1975a. The sequence of the gametangium formation in homosporous fern gameto-
phytes. I. Patterns and their possible effect on the fertilization, with special reference to the
gametophytes oi Athyrium. Sci. Rep. Tokyo Kyoiku Daigaku., B 16(240]:l-22.

Masuyama S. 1975b. The sequence of the gametangium formation in homosporous fern gameto-
phytes. II. Types and their taxonomic distribution. Sci. Rep. Tokyo Kyoiku Daigaku., B
16(241]:71-86.

Masuyama S. 1979. Reproductive biology of the fern Phegopteris decursive-pinnata. The dissim-
ilar mating systems of diploids and tetraploids. Bot. Mag. (Tokyo] 92:275-289.

Nayar, B. K. and S. Kaur. 1971. Gametophytes of homosporous ferns. Bot. Rev. (Lancaster) 37:
295-396.

Page, C. N. 1979. Experimental aspects of fern biology. Pp. 552-585 in A. F. Dyer, ed. The exper-
imental biology of ferns. Academic Press, London.

Peck, J. H., C. J. Peck, and D. R. Farrar. 1990. Influences of life history attributes on formation
of local and distant fern populations. Amer. Fern J. 80:126-142.

Sen, U. 1964. Importance of anatomy in the phylogeny of tree ferns and their allies. Bull. Bot.
Soc. Bengal 18:26-34.

SiMABUKURO, E. A., A. F. Dyer, and G. M. Felippe. 1998. The effect of sterilization and storage
conditions on the viability of the spores of Cyathea delgadii. Amer, Fern J. 88:72-80.

SoLTis, D. E., P. S. SoLTis, and A. R. Smith. 1991. Breeding systems of three tree ferns: Alsophila
firma (Cyatheaceae), Cyathea stipularis (Cyatheaceae), and Lophosoria quadripinnata (Lo-
phosoriaceae). PI. Sp. Biol. 6:19-25.

Stokey, a. G. 1918. Apogamy in the Cyatheaceae. Bot. Gaz. 65:97-102.

Stokey, a. G. 1930. Prothallia of the Cyatheaceae. Bot. Gaz. 90:1-45.

Stokey, A. G. 1960, Multicellular and branched hairs on the fern gametophyte. Amer. Fern J. 50:
78-87.

Wagner, W. H. Jr. 1952. Types of foliar dichotomy in living ferns. Amer. J. Bot. 39: 578-592.

Walker, T. G. 1966. A cytotaxonomic survey of the pteridophytes of Jamaica. Trans. R. Soc. Ed-
inburgh 66:169-237.

Wang, H-C., T-S. Liu, and F-S. Liew. 1977. On the species of Cyatheaceae. Quart. J. Exp. Forest,
Natl. Taiwan Univ. 119:239-267.

American Fern Journal 90(4]:138-140 (2000J

Notes on Lellingeria oreophila (Grammitidaceae)

Poorly Known Species from Colombia

Paulo H. Labiak^

University of Sao Paulo, Institute of Systematic Botany,

Sao Paulo— SP, Brazil

Abstract.

known

lombia, is described, illustrated, and discussed on the basis of a new find of this collection from
Northern Andes in Colombia.

herbarium

kn

(Maxon) A.R.Sm. & R.C.Moran

Maxon

Moran
future

known

Maxon

Lellingeria oreophila (Maxon) A. R. Sm. & R. C. Moran, Amer. Fern J. 81: 85.
1991. Polypodium oreophilum Maxon, Contr. Gray Herb. 165: 72. 1947.
Type: COLOMBIA. Santander: Cerro Armas, 6° 21' N, 73° 50' W, 1200-1500
m, 26 July 1936, Haught 1959 (HOLQ-nTE US; isotype K). Fig. 1. A-F

Stems short-creeping, c. 0.5 cm thick, scaly, the scales 0.2-0.3 cm long, cas-
taneous, linear-lanceolate, with hyaline, ciliate margins. Fronds 10-23 X 2-3
cm, erect; stipes 2-4 X 0.04 cm, dark brown, densely covered with simple,
castaneous hairs ca. 0.1 mm long; laminae pinnatisect, lanceolate to slightly
elliptic, chartaceous, gradually tapering to the base and to the apex; rachises
sclerenchymatous, black; segments 1-2 X 0.15-0.2 cm, linear-deltate, slightly
asymmetrical at the base, oblique to the costa, the margins flat, entire, the apex
obtuse to slightly acute; indument of small, appressed, castaneous, un-
branched hairs (up to 1 mm) on the rachises and abaxial laminar tissue; si-
nuses narrower than the width of the segments; veins free, 1-furcate, ending
submarginally, inconspicuous. Sori round, arising at the vein apex, superficial
or slightly sunken, glabrous. Sporangia non setose.

ADDITIONAL SPECIMEN EXAMINED.— Colombia. Antioquia: Guatape, Vereda Santa Rita, finca Mon-
tepinar, 06° 15'N, 75° lO'W, 1850 m, 06 Feb 1990, Contreras & Echeveni 159 (NY).

Lellingeria oreophila is apparently restricted to the northern region of the
Andes, between 1200 and 1850 meters in elevation, where it occurs as an

» Current Address: Caixa Postal 1644, 80011-970, Curitiba, PR, Brasil.

LABIAK: NOTES ON LELLINGERIA OREOPHILA

139

Fig. 1. A-F. Lellingeria oreophila [Contreras Er Echeverri 159]. A. Habit. B. Blade detail. C. Lam-
inar hair detail. D. Stem scale. E. Stem scale detail. F. Geographic distribution.

140 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

epiphyte in cloud forests. This is the only specimen I found of this species
after a search for it at BM, BR, GH, K, NY and US. This suggests the species
is rare, but field worl; is needed to substantiate this.

Leilingeria oreophila is characterized by having glabrescent rachises or hav-
ing only scattered hairs abaxially, stem scales that are linear-lanceolate, cas-
taneous centrally with hyaline, ciliate margins, and by having appressed, un-
branched, castaneous hairs on the abaxial surface of the laminae and rachises.

It is related to Leilingeria apiculata (Kunze ex Klotzsch) A. R. Sm. & R. C.
Moran, which can be distinguished by its rachises densely covered by hairs
on both sides, and its abaxial laminar surfaces glabrous or with scattered, hy-
aline, branched hairs, these acicular and spreading. Leilingeria oreophila al-
though similar to L. hirsuta A. R. Sm. & R. C. Moran, can be recognized by
having castaneous, unbranched hairs on the abaxial surface of the laminae;
while L. hirsuta is glabrous.

Leilingeria stuebelii (Hieron.) A. R. Sm, & R. C. Moran is also similar but,
according to Maxon (1947), can be distinguished by having conspicuous hairs

on the rachises, margins, and abaxial laminar tissue, stems only 1 mm thick,
and by the small and few ciliate scales on the stems.

Acknowledgments

I thank the Andrew W. Mellon Foundation for support of a six-month studies at the New York
Botanical Garden (including the GH and US herbaria in the United States), and the Margaret Mee
Foundation, which provided financial support for a visit to the BM, BR and K herbaria in Europe.
I also thank Dr. Robbin C. Moran and Dr. Jefferson Prado by their helpful comments on the man-
uscript. The drawings were prepared by the author.

Literature Cited

Maxon, W. R. 1947. New Ferns from the Northern Andes. Contr. Gray Herb. 165: 69-75.
Smith, A. R., R. C. Moran, and L. E. Bishop. 1991. Leilingeria^ a new genus of Grammitidaceae

Amer. Fern J. 81: 76-88.

American Fern Journal 90(4):141-144 [2000]

Shorter Notes

Kaempferol and quercetin 3-0-(X",X''-di-protocatecliuoyl)-glucuronides from
Pteris vittata. — Previous work on the flavonoids oiPteris vittata L. has shown
the presence of an anthocyanin (luteohnidin 5-0-glucoside) which was isolated
by Harborne (Phytochemistry 5:589-600, 1966); in addition acid hydrolysis of
extracts of this fern has led to the identification of kaempferol. quercetin, leu-
cocyanidin and leucodelphinidin by Voirin (Ph.D. thesis, University of Lyon,
p. 151, 1970). Recently 3-C-(6"'-acetyl-cellobiosyl]-apigenin (Am. Fern J. 89:
217-220, 1999) and 6-C-cellobiosylisoscutellarein 8-methyl ether together with
quercetin 3-0-glucuronide and rutin (Am. Fern J. 90:42-47, 2000) have been
reported from P. vittata by Imperato and Telesca.

In the present paper four flavonoids (I-IV) have been isolated from Pteris
vittata L. This fern was collected in the Botanic Garden of the University of
Naples and has been identified by Dr. R. Nazzaro (University of Naples); a
voucher specimen (149.001.001.01) has been deposited in the Herbarium Nea-
politanum (NAP) of the University of Naples.

Flavonoids (I-IV) have been isolated by preparative paper chromatography
in BAW (n-butanol-acetic acid-water, 4:1:5, upper phase), 15% HOAc (acetic
acid) and BEW (22-butanol-ethanol-water, 4:1:2.2) from an ethanolic extract of
aerial parts of Pteris vittata. Further purification was carried out by Sephadex
LH-20 column chromatography eluting whith methanol.

Ultraviolet spectral analysis with the customary shift reagents X^^ (nm)
(MeOH) 257, 296 (sh), 348; +AICI3 273, 303, 347, 398; +AICI3/HCI 273, 303,
346, 393; +NaOAc 271, 302, 367; +NaOMe 272, 324, 392 (increase in inten-
sity); +NaOAc/H3B03 258,351 suggested that flavonoid (I) may be a flavonol
glycoside with free hydroxyl groups at positions 5, 7 and 4'.

Chromatographic behaviour on Whatman No 1 paper (Rf values: 0.65 in
BAW; 0.40 in 15% HOAc; 0.82 in H^O) suggested that flavonoid (I) may be a
flavonoid 0-glucuronide since it has a high mobility in H2O according to an
observation of Harborne (pp. 376-441 in J.B. Harborne, T.J. Mabry and H. Ma-
bry, eds., Tiie Falvonoids, Chapman and Hall, London, 1975).

Both total acid hydrolysis (2N HCl/MeOH 1:1; 1 hr at 100°C) and controlled
acid hydrolysis (10% HOAc; 3.5 hr under reflux) gave kaempferol, glucuronic
acid, glucuronolactone and 3,4-dihydroxybenzoic acid (protocatechuic acid).
Alkaline hydrolysis (2N NaOH; 2 hr at room temperature in a sealed tube)
gave kaempferol 3-0-glucuronide and protocatechuic acid. Since electrospray
mass spectrum (ESMS) showed a pseudomolecular ion at m/z 779
[(M+H) + 2Na]^, two 3,4-dihydroxybenzoyl groups are present as acyl substit-
uents. The above results show that flavonoid (I) is kaempferol 3-0-(X", X"-di-
protocatechuoyl)-glucuronide (Fig. 1), a new natural product.

Protocatechuic acid is reported for the first time as acyl substituent in fla-
vonoid glycosides.

142

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000)

H

H

Fig. 1. The structure of flavonoid I (R=H] and flavonoid II (R=OH), kaempferol and quercetin 3-
0-(X",X"-di-protocatechuoyl]-gIucuronides.

Ultraviolet spectral analysis with the usual shift reagents [X ^^^ [nm) (MeOH)

256, 265 [sh), 306 (sh), 355; +AICI3 271, 301 (sh), 370 (sh), 427; +AICI3/HCI
269, 300 (sh), 399; +NaOAc 270, 327 (sh), 367; +NaOMe 271, 330 (sh), 400
(increase in intensity); +NaOAc/H3B03 260, 374) suggested that flavonoid (II)
may he a flavonol glycoside with free hydroxyl groups at positions 5, 7, 3' and
4'. Chromatographic behaviour (Rf values on Whatman No 1 paper: 0.48 in
BAW; 0.42 in 15% HOAc; 0.81 in H,0) suggested that flavonoid (II) may be a
flavonoid 0-glucuronide since it has high R, value in Hp according to Har-
borne and Williams (pp. 376-441 in J.B. Harborne, TJ. Mabry and H. Mabry,
eds.. The Flavonoids, Chapman and Hall, London, 1975).

Both total acid hydrolysis (2N HCl/MeOH 1:1; 1 hr at 100°C) and controlled
acid hydrolysis (10% HOAc; 3.5 hr under reflux) gave quercetin, glucuronic
acid, glucuronolactone and protocatechuic acid. Alkaline hydrolysis (2N
NaOH; 2 hr at room temperature in a sealed tube) gave quercetin 3-0-glucu-
ronide and protocatechuic acid. Electrospray mass spectrum showed a pseu-
domolecular ion at m/z 795 [(M + H) + 2Na]^ and an ion at m/z 1523

[(MX2)+H+Na]" which corresponds to a dimer; hence two 3,4-dihydroxyben-
zoyl groups are present in flavonoid (II) which is identified as quercetin 3-0-
(X",X"-di-protocatechuoyl)-glucuronide (Fig. 1), a new natural product.
Flavonoid (III) was identified as kaempferol 3-0-glucuronide by ultraviolet

;is with the customary shift reagents, acid ]
NMR spectrum, DEPT experiments, ESMS

NMR

raphy with an authentic sample. The electrospray mass spectrum showed a
pseudomolecular ion at m/z 507 [(M + H) + 2

[(M

Na]+ and ions m/z 992

m/z 1475 [(M

[(M

+

which showed that kaempferol 3-0-glucuronide forms a dimer, a trimer and a
tetramer. Kaempferol 3-0-glucuronide has previously been found in several
species of Adiantum as shown in a review of Markham (pp. 427-468 in I.E.

SHORTER NOTES

143

Harborne, ed., The Flavonoids, Advances in Research since 1980, Chapman
and Hall, London and New York, 1988).

Flavonoid (IV) was identified as kaempferol 3-0-glucoside (astragalin) by ul-
traviolet spectral analysis with the usual shift reagents, acid hydrolysis, co-
chromatography with an authentic sample and ESMS. The electrospray mass
spectrum showed a quasimolecular ion at m/z 471 [(M+H)-hNa]+ and an ion
at m/z 919 [(]V[X2)+H+Na]+ which showed that kaempferol 3-0-glucoside
forms a dimer. Astragalin has been found previously in a large number of fern
species as shown in a review of Markham (above reference) but is here re-
ported for the first time in the genus Pteris.

Until 1985, flavonoid glucuronides have been reported in only one fern ge-
nus [Adiantum] as shown in the review by Markham. More recently, quercetin
3-0-glucuronide has been found in Pteris vittata (above reference), luteolin 7-
O-glucuronide has been isolated from Pyrrosia serpens by Markham and An-
dersen (Phytochemistry 29: 3919-3920, 1990), and eriodyctiol 7-0-glucuronide
has been found in Davallia mariesiihy Cui et al. (Chem. Pharm. Bull. 38: 3218-
3220, 1990).

The presence of flavonoid glucuronides (I-III) in Pteris vittata as well as
isolation of quercetin 3-0-glucuronide from this fern by Imperato and Telesca
(above reference) show advancement from the phylogenetic point of view
since glycosylation with sugars other than glucose and rhamnose may be con-
sidered phylogeneticly advanced. Acylated flavonoids (I, II) from P vittata and
the previous report of two acetylated flavonol glycosides from P. grandifolia
by Tanaka et al. (Chem. Pharm. Bull. 26: 3580-3582, 1978) also indicate phy-
logenetic advancement. In addition, recent isolation of flavone 0-glycosides
from P. cretica by Imperato (Phytochemistry 37: 589-590, 1994; Experientia
50: 1115-1116, 1994) and by Imperato and Nazzaro (Phytochemistry 41: 337-
338, 1996) and isolation of 0-glucosides of C-glycosylflavones from P vittata
by Imperato and Telesca (above references) confirm the phylogenetic advance-
ment of the genus Pteris since the presence of flavone 0-glycosides and 0-
glycosides of C-glycosylflavones may be considered phylogeneticly derived
according to the review by Markham.

The electrospray mass spectra showed that flavonoids (II and IV) form di-
mers whereas flavonoid (III) forms dimers, trimers and tetramers. These data
are of interest since dimers and polimers based on hydrogen bonding have
been observed in crystal structures of flavonoid aglycones as reported by Can-
trell (pp. 391-394 in V. Cody, E. Middleton and J.B. Harborne, eds., Plant
Flavonoids in Biology and Medicine, Alan R. Liss, New York, 1986) whereas
data relating to crystal structures of flavonoid glycosides are scanty.

The author thanks University of Basilicata (Potenza, Italy) for finacial sup-
port (ex 60%), Mass spectral data were provided by SESMA (Naples). — Filippo
Imperato, Dipartimento di Chimica, Universita della Basilicata, 1-85100 Po-
tenza, Italy.

144 AMERICAN FERN JOURNAL: VOLUME 90 NUMBER

Dryopteris fil

Dryopteris fil

mas (L.) Schott has not been recorded previously in Pennsylvania. The species
is generally rare in the eastern part of North America, but does occur in cal-
careous areas and moist woods around the Great Lakes. Clnsest to Pennsvl-

km distant, are sites in Mich

Bruce Peninsula).

Recently, the author discovered a natural population of male fern near Pitts-

Man

there

plants

7.6 m of the 4.5 m high, east-facing slope, and two more are disjunct about 29
m south of the main cluster. Most of the plants are fully fertile with frond
lengths averaging 66 cm. The colony appears to be between 15-20 years old,
possibly older, estimated from growth rates for cultivated specimens in Pitts-
burgh. The origin of Dryopteris filix-mas here is unknown. Fern spores can
travel on prevailing winds over considerable distances, and it is possible that
a "lake effect" windstorm carried this species south. It may be a relict popu-
lation from a colder era or it may have started from spores locally produced
from cultivated specimens in our general area.

Dead Man's Hollow is located along the Youghiogheny River about 1.6 km
north of Boston Bridge (Route 48) in Lincoln and Liberty Boroughs of Alle-
gheny County. The preserve was acquired in sections starting in 1996 and
consists of about 160 hectares [400 acres) that have been largely untouched
since the mid-20*^ century. Prior to that, the hollow was quarried, logged,
farmed, and most recently housed a clay sewer pipe factory, the remains and
shards from which are still quite evident. However, no roads or utility lines
penetrated the hollow, and today it is a quiet place of small streams in bottom
land that is surrounded by relatively steep slopes. The secondary forest has
mixed eastern deciduous trees, introduced white pine and eastern hemlock,
and native as well as exotic undergrowth species. Residential neighborhoods
and two community parks border the preserve on three sides. An abandoned
rail line (a rails to trails site) and the Youghiogheny River form the remaining
border. The area where the male fern grows is near a footpath, but appears
little disturbed. The author found the colony during an exploratory hike in

November

Museum

CM)

filix-mas are dozens of robust plants of Asplen.
of the fern flora in the hollow consists of common western Pennsylvania spe-
cies, e.g., D. intermedia, D. carthusiana, D. marginalis, and Polystichum ac-
rosticboides.~]OAN Eiger Gottlieb, 2310 Marbury Road, Pittsburgh, PA 15221.

American Fern Journal 90[4]:145-146 (2000)

Review

Helechos de Mbaracayii, by Maria Pefia-Chocarro, Griselda Marin, Belen
Jimenez, and Sandra Knapp. 1999. The Natural History Museum, London. 142
pages. Softcover [ISBN 565 09137 9) £3.00, Available from The Natural His-
tory Museum, Cromwell Road, London SW7 5BD, United Kingdom, and from
Fundacion Moises Bertoni, Casilla 714, Asuncion, Paraguay.

This book, written in Spanish, is a popular identification guide to the 115
species of pteridophytes growing in Mbaracayu, a 650 km^ reserve in eastern
Paraguay. Nearly 80% of the reserve consists of relatively undisturbed wet
forest, and the rest is mostly savanna, or cerrado, that occurs on well-drained
soils. Both habitats harbor pteridophytes.

What distinguishes this guide is its highly visual approach. There are a lot
of illustrations. Each species treatment is allotted a full page, and that page is
filled with illustrations, including silhouettes of leaves and drawings of ve-
nation and sorus shape. The rhizomes are usually shown in the silhouettes if
the plant is of small size. Toward the bottom of the page is a short description
with the main characters in boldface, followed by a statement of geographic
distribution and notes. Below this, at the bottom of the page, are five boxes
containing standardized pictures that give the plant's habit and size (first box),
sorus shape, habitat, growth habit, and (last box) any characteristic helpful in

identifying the plant.

The main key at the beginning of the book repeats these five boxes so that,

again, identification is primarily visual. The key is divided into ten groups
based primarily on leaf cutting, from entire to more highly divided. The spe-
cies treatments are grouped in the text by this characteristic, and in the middle
of the fore-edge of each page a shaded side-box encloses a leaf symbol showing
the degree of cutting. This allows you to flip through the guide and quickly
find the main leaf-division sections.

At the end of the book is a list of the pteridophytes ordered by family (22
families and 46 genera are recognized). This is followed by a bibliography and
index to scientific names. There is no glossary because technical terms have
been kept to a minimum. Unfamiliar terms can be sought in the introduction

that explains the parts of the fern plant.

My only complaint is that several of the drawings are inaccurate or of poor
quality. For instance, the drawing of Salvinia minima does not accurately show
the arrangement of the sori on the submerged leaf, nor does it depict the nu-
merous and conspicuous hairs on the submerged segments. Also, one of the
sori appears to be fused with another sorus— something that does not occur.
In the illustration oi Psilotum nudum, the synangia are poorly drawn, the bifid
enation beneath it is missing, and a long stalk is shown attaching to the side,
not the base, of one synangium (were the synangia really that long-stalked?).
The indusia on Adiantum raddianum are shovra separated from (i.e., within)

146

JOURNAL

the leaf margin, not formed from an enrolling of it. I find it hard to believe
that Cyathea atrovirens, or any extant tree fern, has thick prop roots as is
illustrated for that species.

useful

botanists working in Paraguay and nearby regions in adjacent countries. At a
price of £3.00, it is easily affordable. The authors should be congratulated on
the care they have taken in making this guide easy-to-use and for their highly
visual approach.— ROBBIN C. Moran, The New York Botanical Garden, Bronx,

NY 10458-5126, U.S.A.

American Fern Journal 90(4]:147 (2000)

Referees for 2000

All papers submitted to the journal are peer reviewed. Members of the editorial board and the
Society, as well as additional scientists in cognate areas, do these reviews on a voluntary basis. It
is their work that contributes to the high quality of articles in the American Fern Journal and to
its continued success. The American Fern Society and I extend our thanks to the following re-
viewers for their assistance, diligence, and patience in the year 2000 — R. James Hickey.

David Barrington David Johnson Lar.'K Strittmatter

James Caponetti Carolynn Keiffer W. Carl Taylor

Davud Conant James Montgomery Michael Vincent

Richard Edelmann Robbin Moran Charles Werth

Gerald Gastony Benjamin 0llgaard Dean Whittier

Daniel Gladish James Peck Niklas Wikstrom

Christopher Haufler Alan Smith George Yatskievych

Index to Volume 90

6-C-3-Cellobiosylisoscutellarein-8-methyl Blechnum nudum, 121, 124
ether, a new flavonoid from Pteris vittato, Bolbitis sp., 122

42

Acer spp., 79

Acrostichum nodosum, 101

Adiantunh 45, 52, 57, 142-143
capillus-veneris, 45

cau datum, 122

pedatum, 25, 29

tenerum, 16, 18-20, 22, 121-122

tetrophyllum, 122

trapeziforme, 16, 18-20, 22, 122
i4escu/us octandra, 79
A'.'-hornea, 3

y^7e. ntopteris argentea, 25
Alsophila, 107, 134

capensis, 107

erinacea, 106, 107

excelsQ, 135

^rma, 136

polystichoides, 107
^fiejiiia, 39
Angiopteris, 39
^re77ga pinnata, 46

Bommeria, 45
Brade, itineraries, 108
Brassaia actinophyllo, 47
Bursera, 104

Calderon-Saenz, Eduardo. Production of Ad-
ventitious Buds on Leaves in Dicksonia
sellowiana, 105

Carlquist, Sherwin (see E. L. Schneider). 32
Gary a glabra, 79

ovafa 79; sp. 110
Chamaenerion angustifolium, 71
Cheilanthes, 104
Chloroplast genome, Isoetes, 51
Chiou, Wen-Liang (see Y. Huang], 127
Cibotium glaucum, 16, 18-20, 22, 122

schiedei, 122
Citrullus lanatus, 10
Clusia, 3
Cnemidaria, 134
Colombia, Lellingeria in, 138
CkDNN, Herb (see H. Marriott), 109

Arens, N. C. & P. SANCHEZ Baracaldo. Varia- Conn. Jan (see H. Marriott), 109
tion in Tree Fern Stipe Length with Can- Cornus flodda, 110
opy Height: Tracking Preferred Habitat Cryopreservation of In Vitro Grown Fern Ge-

through Morphological Change, 1
Arkansas, Dryopteris, 110
Ashcroft, Carl J, & Elizabeth Sheffield. The Cyotbea, 134

metophytes, 16
Cryptogramma oris pa, 123

Effect of Spore Density on Germination
and Development in Pteiidium, Moni-
tored using a Novel Culture Technique, 91
Asplenium, 37

Xalternifolium, 109
septentrionale, 109
platyneuron, 122, 144
ruta-muraria, 122
trichomanes, 109
Asplenium Xalternifolium in the Black Hills of

South Dakota, 109
Astrolepfs, 24, 29, 40

Athyrium filix-femina, 122

multidentatum, 25

thelypteroides, 122

yokoscense, 25
AzoIIa filicuhides, 25

arborea, 122,134

caracasano, 1-4, 8-11

delgadi, 134

furfuracea, 57

(Alsophila) lepifera, 135

sp/niiiosa, 119

stipu]aris, 136
Cyclopeltis semicordata, 121-122
Cyrtomium falcatum, 122-123

fortune!, 122

SANCHEZ (see N. C. Arens]

Danaea, 39
a/afa, 100
elUptica, 100-102

nodosa, 100-102
simplicifolia, 100
Datura ferox, 10

Davallia fejeensis, 16, 18-20, 22, 122
mariesii, 143

Bibliografia sobre Gametofitos de Helechos y Dennstedtia punctilobula, 73
Plantas Afines, 1699-1996. [Reviewed], Dicicsonia sei/oiv/ana, 105-107

49

Diphasiastrum digitatum, 77-80, 82-83

INDEX TO VOLUME 90

149

Doryopteris concolor vai. concolor, 104-105

palmata, 105
Drynaria quercifolia, 16, 19-20, 22, 122
Dryopterls, 25

Xaustralis, 110, 111

carthusiana, 111, 122, 144

celsa X goldiana, 110, 111

celsQ, 110, 111, 122

celsa X morginalis. 111

clintoniana, 122

crassirhizoma, 24-30

fragrans, 25

filix-mas, 144

goldlana, 110, 111

goldiana X marginalis, 111

intermedia, 144

Xleedsii, 111

ludoviciana. 111

marginalis, 110, 111, 122, 144

Xneowherryi, 111

thelypteris, 24;

and biomass allocation in Diphasiastrum, 77
Gymnopteris, 45

Helechos de Mbaracayu [Reviewed], 145
Hemionitis, 45

/evyi, 104

palmofa, 104

pinnotifida, 104

Hendrix, Earl [see J, H, Peck), 110
Holland, Doug (see G. Yatskievych), 87
Huang, Yao-Moan, Shag-Shun Ying, & Wen-
Liang Chiou. Morphology of Gameto-
phytes and Young Sporophytes oi Sphaer-
opteris lepifera, 127
Humata pectinata, 45

Impatiens capensis, 1, 10

Imperato, Filippo, & Antonella Telesca. 6-C-
P-Cellobiosylisoscutellarein-8-methyl
ether, a new flavonoid from Pteris vittata,
42

Dryopteris filix-mas New in Pennsylvania, 144 Imperato, Filippo. Kaempferol and quercetin 3-

Dryopteris goldiana and its Hybrid with D. cel-
sa New to Arkansas, 110

Duff, R. Joel, & Edward E. Schilling, The inga, 3

Chloroplast Genome Structure of the Vas- Isoetes, 51, 53, 56-5

0-(X", X"-di-protocatechuoyl)-glucuro-
nides from Pteris vittata, 141

cular Plant Isoetes is Similar to that of the
Liverwort Marchantia, 51

melanopoda, 51-53, 55

Equisetum, 57
fluviatile, 25
hyemale, 124
pratense, 25
sylvaticum, 25

Kaempferol and quercetin 3-0-(X", X"-di-pro-
tocatechuoylj-glucuronides from Pteris
vittata, 141

Labiak, Paulo H. Notes on Lellingeria oreophi-
la (Grammitidaceae), a poorly known spe-
cies from Colombia, 138

Lectotypification oi Danaea elliptica, 100
Florida, Ophioglossum pendulum naturalized Lelunger, David B. On the Lectotypification of

Flavonoids, in Pteris, 42; 141

in, 46
Fagus grandifolia, 79

Farrera, Miguel Angel Perez (see R. Riba),

104
Ficus aurea, 46

Danaea elliptica, 100
Lellingeria apiculata, 140

oreophila, 138-140

stuebelii, 140
Lepisorus ussriensis, 25

Flora Malesiana, Series II-Ferns and Fern Al- Li, RuijUN, Xiufeng Yan, & Dawei Zhang. Ves-

lies, Volume 3. [Reviewed], 48

Gametophytes, cryopreservation, 16

in Sphaeropteris, 127
Glycine max, 10

Gottlieb, Joan Eiger. Dryopteris filix-mas New
in Pennsylvania, 144

sels in Roots and Rhizomes of Dryopteris
crassirhizoma (Dryopteridaceae) from
Heilongjiang Province, China, 24

Liriodendron tulipifera, 79

Lopbosoria, 134

quadripinnata, 136

Loxsoma cunninghamii, 45

Greer, Gary K. & Brian C. McCarthy. Patterns Lycopodium, 57

of Growth and Reproduction in a Natural Lygodium, 104

Population of the Fern Polystichum acros-
tichoides, 60
Growth, and reproduction in Polystichum, 60

Macrothely pteris torresiana, 121, 122
Marchantia, 51-53, 55-57

150

AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000J

Marriott, Hollis, Jan Conn, & Herb Conn. As-

plenium Xalterni folium in the Black Hills
of South Dakota, 109.
Marsilea, 24, 32, 36-37, 39-40

drummondii, 33-35

quadrifolia, 25, 33, 35, 37-39

vestita, 33, 37, 39
Matteucia struthiopteris, 25, 121,124
Mexico, new records, 104
McAlpin, Bruce W. (see A. Tejedor), 46
McCarthy, Brian C. (see.C. A. Railing), 77
McCarthy, Brian C. (see G. K. Greer), 60
Metaxya, 134
Micon'm, 3
Mimosa, 104
Moran, Robbin C, Review: Helechos de Mbar- Perez-Garcia, Blanca (see L. Pacheco), 112

ural Population of the Fern Polystichum
Qcrostichoides, 60

Peck, James H., C. Theo Witsell, & Earl Hen-

DRix. Dryopteris goldiana and its Hybrid
with D. celsa New to Arkansas, 110

Pence, Valerie C. Survival of Chlorophyllous
and Nonchlorophyllous Fern Spores
Through Exposure to Liquid Nitrogen, 119

Pence, Valerie. Cryopreservation of In Vitro
Grown Fern Gemetophytes, 16

Pennsylvania, Dryopteris filix~mas, 144

Perez-Garcia, Blanca & Ramon Riba. Biblio-
graffa sobre Gametofitos de Helechos y
Plantas Afines, 1699-1996. [Reviewed],
49

acayii, 145

PiiQseolus vulgaris, 10

Morphology of Gametophytes and Young Spo- Phegopteris connectilis, 122

rophytes of Sphaeropteris lepifera, 127

Myrica, 3

Nephelea, 134

polystichoides, 107
Nephrodium, 25

Nephrolepis cordifolia, 46; sp., 122

Phlehodium, 30, 39

aureum, 122
Phoenix canariensis, 46
Physcomitrella, 52-53, 55-57
Pilularia, 32
Pinus strobus, 79
Plantago lanceolata, 10

New Records for the Pteridoflora of the State of ^^"^^"^' ^^^

Chiapas, Mexico, 104

Podophyllum peltatam, 83

Notes on LeIIingeria oreophih (Grammitida- P^^YP^^i""' aureum. 16, 18-20, 22

ceae), a poorly known species from Co-
lombia, 138

Nyssa sylvatico, 110

Obituary: Joseph A. Ewan (1909 — 1999), 87
Obituary: Ramon Riba y Nava Esparza (1934
1999], 112

On the Itineraries of Alfred and Alexander Curt Populus, 125

decumanum, 45
oreophilum, 138
vulgare, 96
Polystichum, 30

acrostichoides, 25, 29, 60-62, 65, 67-70, 72

74, 122, 144
braunii, 122
tsu-sinense, 122, 123

Brade in Costa Rica, 108

On the Lectotypification of Danaea elliptica,
100

Onoclea sensibilis, 25, 121
Ophioglossum pendulum, 46, 47
Ophioglossum pendulum L. Naturalized in Mi-
ami, Dade County, Florida, 46
Osmunda cinnamomea, 124

cinnamomea var. asiafica, 25

claytoniana, 124

regalis, 119-124
Otoba, 3

Production of Adventitious Buds on Leaves in

Dicksonia sellowiana, 105
Psidium, 3

Psilotum, 56, 57

Pteridium, 24. 29-30, 40, 91, 96-97

aquilinum, 45, 82, 92
Pteris grandi folia, 143

vittata, 42, 45, 123, 141, 143

sp., 122, 123
Pyrrosia petiolosa, 25

serpens, 143

Pacheco, Leticia, & Blanca Perez-Garcia.

Obituary: Ramon Riba y Nava Esparza

(1934—1999), 112

Paesia onfracfuosa, 45

Patterns of Growth and Reproduction in a Nat-

Quercus alba, 79, 110
rubra, 79, 110
velutina, 79

Railing, Carrie A,, & Brian C. McCarthy, The

Effects of Rhizome Severing and Nutrient

INDEX TO VOLUME 90

151

Addition on Growth and Biomass Alio- The Effect of Spore Density on Germination and

cation in Diphasiastrum digitatum, 77
Regnellidium, 32

Rhamnus caroliniana, 110

RiBA, Ramon [see B. Perez-Garcia), 49

RiBA, Ramon, & Miguel Angel Perez Farrera

New Records for the Pteridoflora of the Thelypterls palustris, 25

Development in Pteridium, Monitored us-
ing a Novel Culture Technique, 91
Effects of Rhizome Severing and Nutrient
Addition on Growth and Biomass Allo-
cation in Diphasiastrum digitatuin, 77

State of Chiapas, Mexico, 104
Rumohra adiantiformis, 122

Salix, 125
Salvinia natans, 25

Schilling, Edward E. (see R. J. Duff], 51
Schneider, Edward L., and Sherwin Carlqu-

IST. SEM Studies on Vessels in Ferns. 19.
Marsilea, 32
Selaginella, 56, 104
sibirica, 25
tamariscina, 25
SEM Studies on Vessels in Ferns. 19. Marsilea, ^^^^^ rotundifolia, 110

Thlaspi arvense, 10
Trachelium, 45, 57
Tricbipteris, 134

Vaccinium pallidum, 110

Variation in Tree Fern Stipe Length with Can-
opy Height: Tracking Preferred Habitat
through Morphological Change, 1

Vessels in Roots and Rhizomes of Dryopteris
crassirbizoma (Dryopteridaceae] from
Heilongjiang Province, China, 24

Vessels, in Marsilea, 32

32

Sheffield, Elizabeth (see C. J. Ashcroft), 91

Sinapsis alba, 10

South Dakota, Asplenium, 109
Sphaeropteris, 134

lepifera, 127-136
Spbagnum, 47

Survival of Chlorophyllous and Nonchloro-
phyllous Fern Spores Through Exposure
to Liquid Nitrogen, 119.

Spores, germination and development, 91
survival after freezing, 119

Tectaria incisa, 121-122

Tejedor, Adrian, & Bruce W. McAlpin.
Opbioglossum pendulum L. Naturalized
in Miami, Dade County, Florida, 46

Telesca, Antonella (see R Imperato], 42

Teucrium scorodonia, 10

Wehvitscbia, 56

Windisch, Paulo G. On the Itineraries of Alfred

and Alexander Curt Brade in Costa Rica,

108

Witsell, C. Theo [see J. H. Peck), 110

Woodsia, 24, 29-30
ilvensis, 29
obtusa, 24, 29
scopuUna, 29

Yan, Xiufeng [see R. Li), 24

Yatskievych, George, & Doug Holland. Ohit-
uary: Joseph A. Ewan (1909 — 1999), 87

Yatskievych, George. Review: Flora Malesi-
ana, Series Il-Ferns and Fern Allies, Vol-
ume 3, 48

Yatskievych, George. Review: Bibliografia so-
bre Gametofitos de Helechos y Plantas Af-
ines, 1699-1996, 49

The Chloroplast Genome Structure of the Vas- YiNG, Shao-Shun [see Y. Huang), 127

cular Plant Isoetes is Similar to that of the
Liverwort Marchantia, 51

Zhang, Dawei (see R. Li), 24

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