Plant Ecol (2010) 211:7–17
DOI 10.1007/s11258-010-9765-2
Comparative water balance profiles of Orchidaceae seeds
for epiphytic and terrestrial taxa endemic to North America
Jay A. Yoder • Samantha M. Imfeld • Derrick J. Heydinger
Chloé E. Hart • Matthew H. Collier • Kevin M. Gribbins •
Lawrence W. Zettler
•
Received: 24 November 2009 / Accepted: 25 March 2010 / Published online: 9 April 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The Orchidaceae have dust-like seeds that
use wind currents for long-distance dispersal. Lacking
endosperm, orchid embryos consume free-living,
mycorrhizal fungi as a carbon source (mycotrophy)
after settling on a substrate. Few studies have investigated orchid seed morphology as it relates to ecology,
but conceivably variations in seed size and testa characteristics could be linked to water loss rates aimed at
maximizing germination in a particular habitat. Seeds
of 2 epiphytic, 1 aquatic, and 7 terrestrial orchids
native to North America were compared with respect to
water balance profiles: Cleistes bifaria, Encyclia
tampensis, Epidendrum nocturnum, Habenaria repens,
Isotria medeoloides, Liparis elata, L. hawaiensis,
Platanthera holochila, P. integrilabia, and P. leucophaea. Water content, water loss rate, activation energy,
and equilibrium humidity were assessed for each
species. Seeds of epiphytic orchids were smaller,
lighter, more porous, and had higher water loss rates
J. A. Yoder S. M. Imfeld D. J. Heydinger C. E. Hart M. H. Collier (&) K. M. Gribbins
Department of Biology, Wittenberg University,
Ward Street at North Wittenberg Avenue, Springfield,
OH 45501, USA
e-mail: mcollier@wittenberg.edu
L. W. Zettler
Department of Biology, Illinois College, Jacksonville,
IL 62650, USA
compared to terrestrials. No active mechanism for
water absorption exists in seeds of either group. Water
loss appears to be a species-specific phenomenon that
may be linked to the ecological niches these species
occupy.
Keywords Mycotrophy Orchidaceae Permeability Platanthera Seeds Water balance
Introduction
A unifying theme to all members of the Orchidaceae
worldwide is their diminutive, dust-like seeds (ca.
0.18–3.80 mm in length; Clifford and Smith 1969)
that generally lack nutrient reserves (endosperm;
Rasmussen and Whigham 1993). Each orchid seed
consists merely of a tiny, approximately 120-celled
embryo cloaked by a papery thin testa (Arditti 1966;
Clements 1988). The convoluted external surface of
the tests probably facilitates long-distance wind
dispersal (Healey et al. 1980). The lack of endosperm
has made this extreme reduction in seed size possible,
enabling these plants to colonize arboreal substrates
as well as new geographical areas where speciation
can occur. The trade-off occurs once the seed settles
upon a substrate. Lacking access to nutrient reserves,
the embryo’s fate is dependent on the acquisition
of an external source of carbon. For orchids, the
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exploitation of free-living fungi as a carbon source
(mycotrophy) serves this crucial role (Rasmussen
1995; Zettler et al. 2003).
The Orchidaceae has received considerable study
in recent years (e.g., taxonomy, pollination mechanisms, in vitro seed germination), but comparatively
few studies have investigated orchid seed physiology
and morphology with respect to ecology. Classic
studies by Burgeff (cited in Arditti 1966) estimated
that the weight of an orchid seed ranges between 0.3
and 14 lg. Orchid seeds vary in length from 0.18 to
3.85 mm (Clifford and Smith 1969), but are typically
1 mm. Yoder et al. (2000) concluded that orchid
seeds are incapable of absorbing water from subsaturated air, probably because of the impervious,
waterproofing nature afforded by the testa. They also
concluded that seeds of a terrestrial orchid, Platanthera integrilabia (Correll) Luer, had lower water
loss rates compared to seeds of an epiphyte, Epidendrum conopseum (=syn. E. magnoliae Muhlen.).
From an ecological standpoint, seeds that are incapable of absorbing water from air would likely
remain lightweight and airworthy. Exactly why seeds
of epiphytes would lose water faster than terrestrials,
however, remains unclear but could be an adaptation
for colonizing a specific habitat or substrate (e.g., tree
limbs). It is conceivable that variations in seed size,
testa characteristics, and presumably phylogeny
could be linked to water loss rates, at least in part.
We attempted to explore this possibility by comparing water balance profiles for a range of taxa inhabiting different habitat extremes.
In this study, seeds of two epiphytic and seven
terrestrial orchids native to North America were
compared with respect to water balance profiles (i.e.,
the ability to retain water at different relative humidities and temperatures). Of the 10 taxa, six are listed in
the United States as state or Federally endangered/
threatened [Epidendrum nocturnum Jacquin, Isotria
medeoloides (Pursh) Raf., Liparis elata Lindl., Platanthera holochila (Hillebr.) Kraenzlin, P. integrilabia (Correll) Luer, P. leucophaea (Nutt.) Lindl.]. The
objectives of our study were to: (1) determine the
intrinsic, water-related features critical for orchid seed
survival prior to germination; (2) correlate these
features with habitat (substrata); and (3) provide new
information that might be applied to conservation
programs for endangered taxa in North America and
abroad.
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Plant Ecol (2010) 211:7–17
Materials and methods
Seeds
Seeds from 10 orchid taxa native to North America
were utilized in this study (Table 1). These species
were selected because of their habitat preference,
horticultural appeal, and/or rarity. All seeds were
collected from mature, yellowing capsules just prior
to dehiscence and promptly dried over anhydrous
CaSO4 desiccant (Drierite, W. A. Hammond Drierite
Co., Xenia, Ohio, USA) immediately after collection
for 7–14 days at ambient temperature (Zettler 1997).
Seeds were subsequently stored at -7 ± 1°C until
use (Table 2). Seeds were dried and stored in this
manner according to standard protocols (e.g., Pritchard 1985; Seaton and Hailes 1989) to maintain
embryo viability in storage for eventual germination
studies.
Instrumentation, reagents, and test conditions
Basic temperature for all observations was 25°C
(±1°C) and 14 h:10 h light:dark (averaging 179
lmol m-2 s-1). All other temperatures used programmable environmental cabinets (±0.5°C). A
drying oven set at 90°C was used to dry the seeds
to complete dryness. Relative humidities (still air
conditions) were adjusted using saturated salt solutions (Winston and Bates 1960) or glycerol distilled
water mixtures (Johnson 1940) placed at the bottom
of a sealed 3000 cc glass desiccator; seeds were
placed on a porcelain plate so that they were suspended above the material at the base of the desiccator. Relative humidity (% RH) was measured using
a hygrometer (SD ± 0.5% RH; Thomas Scientific,
Philadelphia, Pennsylvania, USA) and varied less
than 2% RH. Drierite (CaSO4) provided 0% RH
(1.5 9 10-2 % RH; Toolson 1978) and doubledistilled deionized (DI) water provided 100% RH. A
microbalance was used to weigh the seeds (precision
of SD ± 0.2 lg, accuracy of ±lg at 1 mg; CAHN,
Ventron Co., Cerritos, California, USA). Each seed
was weighed and monitored individually and returned
to test conditions in less than 1 min. An aspirator was
used to handle the seeds. The tetrazolium reduction
method was used to confirm seed viability (i.e.,
embryonic respiration) by soaking the seeds in 1.0%
tetrazolium-DI water solution for 1 day (five seeds/
Plant Ecol (2010) 211:7–17
9
Table 1 The 10 North American orchid species utilized as a source of seed, with reference to abundance, ecological habitat
preference, and distribution for each
Taxon
Status
Notes
Cleistes bifaria (Fernald) Catling &
Gregg
Common
Terrestrial
E USA, S Florida, W to Texas
Upland Spreading Pogonia
Encyclia tampensis (Lindley) Small
Savannas, meadows, bogs, oak/pine forests
Common
Florida Butterfly Orchid
Epiphytic, especially on live oak (Quercus virginiana)
C and S Florida, Bahamas
Horticulture value; specimens often collected from wild
Epidendrum nocturnum Jacquin
State endangered (Florida) Epiphytic, especially in humid swamps (hammocks)
Night-fragrant Epidendrum
S Florida, W Indes, C and S America
Horticultural value; often poached from wild populations
Habenaria repens Nuttall
Common
Water Spider Orchid
Isotria medeoloides (Pursh)
Rafinesque
Aquatic (one of a few aquatic orchids in North America)
SE Coastal Plain, W to Texas, W Indes, C and S America
Federally listed
(threatened)
Terrestrial, occupies rich, shaded deciduous forests
Michigan to Maine, S to Missouri and Carolinas
Small Whorled Pogonia
Liparis elata Lindley
State endangered (Florida) Terrestrial to semi-epiphytic on rotting logs in swamps
Tall Twayblade
L. hawaiensis H. Mann
Florida, W Indes, C and S America
Uncommon
Aapu pa’i niu a hina
Platanthera holochila (Hillebr.)
Kraenzlin
Federally listed
(threatened)
Puahala a kane
P. integrilabia (Correll) Luer
Monkey-face Orchid
P. leucophaea (Nuttall) Lindley
Eastern Prairie Fringed Orchid
Terrestrial to semi-epiphytic in cloud forests
One of three orchids native and endemic to Hawaii
Terrestrial in cool, elevated cloud forests
One of three orchids native and endemic to Hawaii
Only ca. 36 individual plants remain
Federal candidate
(threatened)
Federally listed
(threatened)
Terrestrial, in shaded swamps and on seepage slopes
Restricted in range (Tennessee, Georgia, S Carolina, and
Kentucky)
Terrestrial, mostly in moist open prairies in the Midwestern
United States
Taxonomic treatment follows Brown (2003)
0.5-ml tube containing 40 ll tetrazolium, 24 h at
37°C), rinsing (vortex with 2–3 volumes of clean DI
water, 1 min each), sectioning the seed, and observing under a light microscope (809) for the presence
of pink coloration as a positive indicator of respiratory activity (Lakon 1949). The tetrazolium was purchased from Sigma (Sigma Chemical Co., St. Louis,
Missouri, USA).
Sample sizes and statistics
An analysis of variance (ANOVA; SPSS 14.0 for
Windows, Microsoft Excel and Minitab, Chicago,
Illinois, USA) was used to compare data. Percentages
were arcsine-transformed prior to analysis. Characteristics derived from regression lines were analyzed
using a test for the equality of slopes of several
regressions (Sokal and Rohlf 1995). Total sample
size (N) for each water balance characteristic was 45,
divided into three replicates of 15 seeds each, with
each replicate coming from a separate plant. The
same group of 45 seeds was used to determine fresh
mass, dry mass, water mass, and percentage water
content. For the water loss rate determination, a
different group of 45 seeds was used. The same group
of 45 seeds was tracked (10, 20, 30, 40, 50, and 60°C)
for the activation energy determination. A different
group of 45 seeds was used at each of the relative
humidities (75, 85, 93, and 97% RH) to find the
equilibrium humidity. Expression of units conforms
to standard water balance physiology, based on
activation energy as a relative measure or porosity
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Plant Ecol (2010) 211:7–17
Table 2 Seed sources and collection information for the 10 orchid taxa utilized
Taxon
Seed
source #
Collection information
Cleistes bifaria
S 118
West Virginia, Barbour Co.
Encyclia
tampensis
S 144
Epidendrum
nocturnum
S 78
6 September 2002 by Katherine B. Gregg
Florida, Collier Co., Florida Panther National Wildlife Refuge
13 November 2005 by William Kutosky and Larry Richardson
Florida, Collier Co., Florida Panther National Wildlife Refuge
1 August 2003 by Scott L. Stewart
Habenaria repens S 121
Seed from greenhouse-grown plant. Original population from Florida, Highlands Co., Avon Park, 16
September 2002 by Scott L. Stewart
Isotria
medeoloides
S 102
New Hampshire, Belknap Co., E Alton
Liparis elata
S 126
Florida, Collier Co., Fakahatchee Strand State Preserve
L. hawaiensis
S 128
8 December 2002 by Mike Owen
Hawaii, Kamakou Preserve, Molokai
Platanthera
holochila
S 145
P. integrilabia
S 10
P. leucophaea
S 111
12 October 1999 by William E. Brumback
9 August 2003 by Steve Perlman and Lawrence W. Zettler
Hawaii, Kamakou Preserve, Molokai
25 October 2005 by Steve Perlman
Tennessee, McMinn Co., Cherokee National Forest
16 October 1989 by Lawrence W. Zettler
Illinois, Henry Co., Munson Cemetery prairie
1 September 2002 by Lawrence W. Zettler
Seed source numbers are those cataloged and currently in storage at Illinois College, Jacksonville, Illinois
and water loss rate as the measure of permeability
(Yoder et al. 2000).
Determination of water balance characteristics
Pretreatment of the seeds consisted of exposing them
to 100% RH for 24 h and then to 33% RH, 30°C until
they lost 4–6% of their mass so that mass changes
reflect changes in water levels (Arlian and Ekstrand
1975; Yoder et al. 2000). Dry mass of the seed, d,
was determined at the end of each experiment by
placing the seed at 90°C and weighing until mass
remained constant for three consecutive days (Wharton 1985). The dry mass was subtracted from each
mass measurement to convert mass measurement into
a measure of water mass, m. Water mass (m) was
expressed as a percentage of initial mass (f, fresh
mass) to determine the percentage water content of
the seed according to Eq. 1 (Wharton 1985):
percentage m ¼ 100ðf dÞ=f :
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ð1Þ
The seed’s water loss rate was determined at 0%
RH, which is the only relative humidity where water
loss rate is exponential permitting the rate of loss to
be calculated from the slope of a regression line on a
plot of ln mt/m0 against time according to Eq. 2
(Wharton 1985):
mt ¼ m0 ekt
ð2Þ
where mt is the water mass at any time t and m0 is
the initial water mass. Each water loss rate was based
on a total of five consecutive mass measurements.
The first measurement was taken at m0 and five
subsequent measurements (time variable depending
on species) represent mt values, and was expressed as
%/h.
The permeability of the seed coat was assessed by
using activation energy for water loss (Ea) that
describes the seed coat as an energy barrier and the
amount of energy that is needed by a molecule of
water to cross (Yoder et al. 2000). Thus, activation
energy reflects a permeability constant and the
Plant Ecol (2010) 211:7–17
11
porosity of the barrier (Yoder et al. 2005). Activation
energy was calculated from the slope (= -Ea/Rgas) of
a regression line on an Arrhenius plot of water loss
rate (%/h, determined according to Eq. 2) versus
reciprocal absolute temperature (1/K) based on Eq. 3
(Wharton 1985; Yoder et al. 2000):
k ¼ AeEa =ðRgas TÞ
ð3Þ
where k is the water loss rate, A is the frequency
(steric) factor, Ea is the activation energy, R is the
universal gas constant, and T is the absolute temperature. Activation energy was expressed as kJ/mol. A
change in activation energy (change in slope on the
Arrhenius plot) denotes the temperature threshold of
a dramatic water loss known as the critical transition
temperature, CTT (Rourke and Gibbs 1999; Gibbs
2002).
The capacity of the seed to absorb water vapor was
determined based on a change in mass after exposure
to different relative humidities according to Eq. 4
(Wharton 1985):
percentage change in w ¼ 100ðwt w0 Þ=w0
ð4Þ
where wt is the mass after 24 h exposure to a certain
relative humidity and w0 is the initial mass. Data were
expressed as a %/h rate. The relative humidity that
corresponds to the point where change in mass is 0%
on a plot of percentage change in mass against
percentage relative humidity denotes where water
balance (mass change = 0; hence, water loss =
water gain) is estimated to be achieved under these
conditions and is an equilibrium humidity, EH, a
relative humidity that if exceeded results in water
gain (Wharton 1985; Yoder et al. 2000).
Results
Water content
Seeds of the 10 orchid taxa varied significantly with
respect to fresh mass, dry mass, and water mass
(Table 3). Platanthera holochila and P. integrilabia
seeds exhibited the largest mass values, whereas seeds
from the two Liparis species had the lowest values
(Table 3). Despite these differences in mass, no significant differences were detected in percentage water
content among the 10 species (Table 3) regardless of
their size, geographical origin, and ecological niche.
Similarities in water content are supported by the
closeness in water mass to dry mass (m/d) ratios:
C. bifaria, E. tampensis, and P. holochila = 0.85;
L. elata = 0.83; I. medeoloides = 0.80; E. nocturnum = 0.79; P. leucophaea = 0.77; H. repens =
0.76; P. holochila and P. integrilabia = 0.73. Water
mass (m) was also positively correlated with dry mass
(d) for the 10 taxa: R2 C 0.89 for H. repens; 0.91 for
E. tampensis, P. holochila; 0.92 for P. integrilabia,
P. leucophaea; 0.94 for C. bifaria, I. medeoloides, and
L. elata; 0.95 for L. hawaiensis; 0.96 for E. nocturnum
(ANOVA, P \ 0.001). Visually, seeds were generally
alike with respect to overall size and shape, suggesting that they deliver a standardized water flux. We
conclude that seeds of these 10 distinct taxa have the
same general water content, but seeds of the epiphytes
are smaller and lighter compared to those of terrestrial
orchids.
Water loss rate
Regardless of the species, all seeds displayed a
proportionate, exponential pattern of water loss over
time at 0% RH, with R2 C 0.99 (ANOVA, P \
0.001). This value, used to derive water loss rates
depicted in Fig. 1, revealed that seeds of the two
Liparis species had significantly higher (ANOVA,
P \ 0.05) water loss rates than the other eight taxa
(Table 4). Moreover, seeds of the epiphytes had significantly higher (ANOVA, P \ 0.05) water loss
rates than those of the non-Liparis terrestrials
(Table 4). Water loss rates for the 10 taxa varied
inversely with dry mass of the seed, y = -2.6x,
R2 = 0.90 (Fig. 2a; ANOVA, P \ 0.001), indicating
a positive size–rate relationship. However, not all
species fit this profile, i.e., smallest seeds did not
consistently lose water the fastest (Fig. 1; Tables 3
and 4). This phenomenon was best exemplified by the
prairie-inhabiting terrestrial, P. leucophaea. Seeds of
this species were small, comparable to those of the
epiphytes (Table 3), and yet it had the slowest water
loss rates of the 10 taxa (0.82%/h). By comparison, other species with small seeds (E. tampensis,
E. nocturnum, both Liparis spp.) had higher water
loss rates (Fig. 2a), suggesting that P. leucophaea
might be an unusual case. Seeds of the other
terrestrial orchids tested (excluding Liparis) had
larger seeds than P. leucophaea, but slower water
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Table 3 Seed water content for 10 North American orchid species
Species
Mass
Water content
f (mg)
C. bifaria
d (mg)
m (mg)
%
0.0048 ± 0.002a
0.0026 ± 0.001a
0.0022 ± 0.002a
45.83 ± 0.76a
b
b
b
E. tampensis
0.0037 ± 0.001
0.0020 ± 0.003
0.0017 ± 0.001
45.95 ± 0.81a
E. nocturnum
0.0034 ± 0.003b
0.0019 ± 0.001b
0.0015 ± 0.002b
44.12 ± 0.79a
a
a
a
43.14 ± 1.01a
a
46.67 ± 0.92a
c
45.45 ± 0.72a
c
H. repens
0.0051 ± 0.002
0.0029 ± 0.001
a
I. medeoloides
a
0.0045 ± 0.001
0.0025 ± 0.002
c
L. elata
c
0.0022 ± 0.001
0.0012 ± 0.002
c
0.0020 ± 0.001
0.0010 ± 0.001
L. hawaiensis
0.0026 ± 0.001
0.0015 ± 0.001
0.0011 ± 0.001
42.31 ± 0.85a
P. holochila
0.0063 ± 0.001d
0.0034 ± 0.002d
0.0029 ± 0.001d
46.03 ± 0.74a
d
d
d
42.11 ± 0.88a
b
43.59 ± 0.92a
P. integrilabia
c
0.0022 ± 0.003
0.0057 ± 0.003
0.0033 ± 0.002
b
P. leucophaea
b
0.0039 ± 0.002
0.0022 ± 0.001
0.0024 ± 0.001
0.0017 ± 0.001
f Fresh (initial) mass, d dry mass, m water mass, % water content
Data are mean ± SE. Values denoted by the same superscript letter within each column are not significantly different (ANOVA,
P [ 0.05). N = 3, with each replicate consisting of 15 seeds weighed individually
loss rates. Thus, it appears that seeds of the terrestrial
orchids retain water more effectively than those of
the epiphytes. Although seed size is an important
factor in water loss rates, it is not the sole factor.
Water loss appears to be a complex, species-specific
phenomenon linked to other factors (e.g., ecological
0.00
ln (mt /m0)
-0.04
P. leucophaea
-0.08
-0.12
E. tampensis
-0.16
0
1
2
3
4
5
Days
Fig. 1 Proportion of water mass lost at 0% RH and 25°C in
seeds of Encyclia tampensis (epiphyte) and Platanthera
leucophaea (terrestrial) used to determine the water loss rate
(seed coat plus respiratory water loss). The slope of the
regression line is the water loss rate. mt, water mass at any time
t; m0, initial water mass. Each point represents the mean of 45
seeds (±SE B 0.004)
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niche, phylogeny), and is not intrinsic to surface-tovolume differences in the seed.
Activation energy
When water loss rates were examined by Arrhenius
analysis, single component curves (continuous slope;
R2 C 0.96; ANOVA, P \ 0.001), depicted by Fig. 3
(e.g., E. tampensis and P. leucophaea), were exhibited by seeds of all 10 species, and were consistent
with a regular Boltzman temperature function. In no
case did the Arrhenius plot display evidence of
accelerated water loss attributed to a temperature
threshold. There is no CTT because the activation
energy did not change (reflected by a lack of change
in the slope given a rise in temperature). The steeper
slope on the Arrhenius plot (hence the higher
activation energy) for E. tampensis showed that seeds
of this epiphytic orchid lost twice as much water from
one temperature jump to the next than those of the
terrestrial, P. leucophaea. Water loss occurred for
seeds transferred from low to high temperatures
(ramp up) and high to low temperatures (ramp down),
as well as for seeds measured at each temperature
(data not shown). The lower activation energy
observed in P. leucophaea led to a lower frequency
(steric) factor A (y-intercept; Fig. 3), and this was
used to measure the resistance of the testa to water
loss, coinciding with water loss rate data (Fig. 1). For
Plant Ecol (2010) 211:7–17
13
Table 4 Water retention (water loss rate = WLR, activation energy = Ea) and water vapor adsorption (equilibrium humidity = EH) for seeds of 10 North American orchid species
Species
Water loss
Water gain
Ea (kJ/mol)
EH (% RH)
1.44 ± 0.03a
46.6 ± 0.7a
86 ± 1a
b
b
93 ± 1b (Fig. 3)
53.7 ± 0.9
b
92 ± 2b
29.4 ± 0.6
c
85 ± 1a
45.1 ± 0.8
a
84 ± 2a
WLR (%/h)
C. bifaria
E. tampensis
2.67 ± 0.07 (Fig. 1)
56.2 ± 1.1 (Fig. 2)
2.36 ± 0.06
b
1.02 ± 0.04
c
I. medeoloides
1.52 ± 0.05
a
L. elata
3.28 ± 0.09d
58.5 ± 0.6b
96 ± 1b
3.41 ± 0.04
d
55.4 ± 0.7
b
96 ± 2b
0.90 ± 0.03
c
31.9 ± 0.6
c
86 ± 1a
1.23 ± 0.08
c
44.0 ± 0.4
a
83 ± 2a
c
84 ± 1a (Fig. 3)
E. nocturnum
H. repens
L. hawaiensis
P. holochila
P. integrilabia
P. leucophaea
c
0.82 ± 0.06 (Fig. 1)
24.7 ± 1.0 (Fig. 2)
Data are mean ± SE. Values denoted by the same superscript letter within each column are not significantly different (ANOVA,
P [ 0.05). N = 3, with each replicate consisting of 15 seeds weighed individually
4.0
B
A
LE
LH
LH
LE
3.0
ET
ET
WLR (%/h)
Fig. 2 Correlation between
water loss rate (WLR) and
dry mass (R2 = 0.90) and
equilibrium humidity (EH;
R2 = 0.95) in 10 species of
orchid seeds. Complete
names are given in Table 1,
and data are from Tables 3
and 4
EN
EN
2.0
PL CB
IM
CB
PI
PI
1.0
PL
HR
HR
PH
PH
IM
0.0
-6.8
-6.4
ln dry mass
all 10 species examined, high water loss rates were
associated with high activation energies (Table 4).
Thus, seeds of epiphytic orchids are extremely porous,
and this physical feature appears to correspond to the
higher water loss rates observed.
Equilibrium humidity
Water vapor adsorption varied depending on the
species (Fig. 4). Seeds of E. tampensis, for example,
adsorbed water vapor at and above 93% RH, but for
seeds of P. leucophaea, rehydration occurred at or
above 84% RH (Fig. 4). Seed water retention was
positively correlated with amount of water vapor in
-6.0
-5.6
80
85
90
95
100
EH (% RH)
the air: y = 0.076x, R2 = 0.97 for P. leucophaea, and
y = 0.096x, R2 = 0.99 for E. tampensis (ANOVA,
P \ 0.001; Fig. 4). This strong positive correlation implies that a higher relative humidity prompts
water adsorption. Moreover, the seed equilibrium
humidities of the 10 species (Table 4) also correlate positively with the water loss rate for each
species (Fig. 2b), y = 0.18x, R2 = 0.95 (ANOVA,
P \ 0.001). Thus, seeds that lose water the fastest
have equilibrium humidities that are closer to saturation compared to seeds that have lower water loss
rates, and this is a function of seed permeability. That
is, reduction of water loss rate reveals evidence for
water vapor adsorption that is obscured when water
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Plant Ecol (2010) 211:7–17
loss rates are higher. This phenomenon is indicative
of water gain by passive processes alone (passive
chemisorption and physical adsorption of water
vapor) and not active processes.
100
WLR (%/h)
10
Discussion
1
E. tampensis
P. leucophaea
0.1
0.0028
0.0030
0.0032
0.0034
0.0036
1/K
Fig. 3 Arrhenius analysis of water loss rate (WLR) and
temperature in seeds of Encyclia tampensis (epiphyte) and
Platanthera leucophaea (terrestrial) used to determine the
activation energy, Ea. The slope of the regression line
corresponds to the activation energy (slope = -Ea/Rgas). No
critical transition temperature (CTT) is present as indicated by
the continuous slope and lack of a biphasic relationship. Each
point is the mean of 45 seeds (±SE B 0.002). Results were
similar for freezer killed seeds (-40°C for 1 week/thawed to
room temperature) that were used to rule out respiratory
artifacts
2.0
P. leucophaea
Change in mass (%/h)
1.0
E. tampensis
0.0
-1.0
-2.0
70
75
80
85
90
95
100
%RH
Fig. 4 Changes in water mass (m) at different relative
humidities at 25°C for seeds of Encyclia tampensis (epiphyte)
and Platanthera leucophaea (terrestrial) for determination of
the equilibrium humidity, EH. The equilibrium humidity was
identified on x-axis by dropping a perpendicular line from the
point corresponding to 0% change in mass (gain = loss). Each
point is the mean of 45 seeds (±SE B 0.12)
123
The Orchidaceae is widely regarded as the most
diverse plant family containing between 17,000 and
35,000 species worldwide (Dressler 1993). During
the course of their 100? million year-old evolutionary history (Chase 2001), orchids have successfully
exploited all vegetated continents (Dressler 1981).
This feat is particularly noteworthy because, as monocots, they lack the capacity to produce woody tissues
and must, therefore, often affix themselves to illuminated, arboreal surfaces. About 73% of the known
species exist as epiphytes within the tropics (Atwood
1986), and the remainder occupy soil (terrestrials),
rocks (lithophytes), and even semi-aquatic areas (e.g.,
Habenaria repens Nuttall). Part of the success of the
Orchidaceae is attributed to the coevolution of the
family with specific pollinators, especially insects
(Roberts 2003). Recently, mycorrhizal fungi have also
been implicated in the evolutionary diversification of
the family (Taylor et al. 2003; Otero and Flanagan
2006) because orchids exploit (consume) free-living
fungi as a food source (mycotrophy) that supplement
photosynthesis. Thus, for an orchid to complete its life
cycle in situ, the seed must encounter a specific
mycorrhizal fungus present in the substrate on or near
the impact site where the seed settles.
The first critical step in initiating the orchid life
cycle begins with the physical transfer of seed from
mature capsule to fungus-laden substrate. Seeds of
terrestrial and epiphytic orchids alike rely on wind
currents for dispersal to new, suitable habitats, and
remaining lightweight while aloft is of paramount
importance. Thus, it is no surprise that orchid seeds
would resist water vapor absorption from subsaturated
air. Upon contacting a substrate, however, differences
in water balance profiles between the two groups
would be expected to differ. Compared to their
terrestrial counterparts, epiphytic orchids do not have
the capacity to inhabit a spatially fixed, ubiquitous
surface such as soil. Therefore, their seeds must
contact and quickly affix themselves to a limited
number of arboreal substrates or otherwise the
Plant Ecol (2010) 211:7–17
resulting seedlings will perish from lack of photosynthesis. To do this effectively, epiphytic seeds are
porous and capable of absorbing liquid water rapidly.
Although the convoluted surface of the testa probably
aids in the attachment process, water absorption
facilitated by a leaky seed would enhance this process
further by the simple act of cohesion. Indeed, all
epiphytic taxa examined in our study displayed greater
porosity (higher activation energy), suggesting a leaky
nature. In fact, this phenomenon is frequently observed
in the laboratory when seeds of epiphytic species often
settle, not float, while undergoing surface sterilization.
Once in contact with liquid water on the substrate,
embryos quickly imbibe water, swell, and commence
the germination process, assisted by fungi. As arboreal
substrates are prone to desiccation during dry periods,
mycotrophy is thought to supply a critical source of
water to the epiphytes, not just carbon (Yoder et al.
2000).
The ecological strategy employed by terrestrial
orchids is somewhat different but not without
potential problems. As revealed by this study, seeds
of terrestrials are larger, lose water at a slower rate,
and are less porous than the epiphytes. This is
especially true of P. leucophaea and H. repens. Both
orchids, particularly the latter, occupy moist to wet
soils, and they are often accompanied by other plants
capable of growing vigorously during the growing
season. In the prairie ecosystem, seeds of P. leucophaea, released in the fall, would be expected to settle
onto the surface of dense mats of senescing organic
debris composed of grasses and forbes. Through the
action of natural weathering (precipitation), decomposition, and even invertebrate activity, it is conceivable that seeds of P. leucophaea settle into the
substrate at a slower rate because of the low porosity
of the seed coat. Without this feature, seeds might
otherwise trickle down into the sod too deep for the
emergent (basal) leaves to break above the surface,
making it impossible for seedlings to initiate photosynthesis. In nature, roots of mature P. leucophaea
are typically found at a depth of 6–10 cm below the
soil surface, about the same length of the basal leaves
of seedlings cultivated in vitro (Zettler et al. 2005).
Thus, it seems likely that the low porosity of
P. leucophaea seeds might serve a useful purpose
by keeping the seeds ‘‘afloat’’ in a sod layer for
extended periods, within a zone where microbial
activity (e.g., mycorrhizal fungi) might be most
15
active, yet deep enough to withstand dry periods
without compromising initial photosynthetic ability.
Similarly, seeds of H. repens displayed an even lower
porosity, probably because this species frequents
wetter habitats. In nature, mature H. repens is often
found rooted on floating mats on standing water, and
having buoyant seeds seems like the most logical
establishment and survival strategy. The exception to
this trend is seen in the two terrestrial species from
the genus Liparis that have very similar seed
morphologies to those of the epiphytes. These
terrestrial Liparis species also have high water loss
rates, activation energies, and equilibrium humidities
similar to the epiphytes. Even though the Liparis
species are terrestrial, these data suggest their
germination requirements are similar to those seen
in epiphytes.
Implications for orchid ecology and conservation
Mycoheterotrophy is not unique to the Orchidaceae.
Worldwide, at least 10 plant families containing 400
species and 87 genera are known to exploit fungi as a
primary source of carbon: Burmanniaceae, Corsiaceae, Gentianaceae, Geosiridaceae, Monotropaceae,
Orchidaceae, Petrosaviaceae, Polygalaceae, Pyrolaceae, and Triuridaceae (Baskin and Baskin 2001).
Although most members of the Orchidaceae are
autotrophic at maturity, all are unable to photosynthesize during the young seedling (protocorm) stage
(Harley and Smith 1983) and exist as obligate
mycoheterotrophs. Even as autotrophs, orchids continue to utilize fungi as a carbon source supplementing photosynthesis well into maturity (Rasmussen
1995). Leake (1994) proposed that mycoheterotrophy
evolved several times among different taxonomic
groups, but yet these plants share many similarities
including dust-like seeds dispersed by wind. Likewise, small seed size, facilitated by a reduction in the
embryo and endosperm, is also observed in parasitic
angiosperms (Baskin and Baskin 2001). Moreover,
these plants initiate contact with the host during
earlier stages of ontogenesis (Baskin and Baskin
2001). Among the parasitic plants, orchids and two
other families (Pyrolaceae, Monotropaceae) differ in
that the haustorium is never formed, instead being
replaced by fungi (Baskin and Baskin 2001; Teryhokhin and Nikiticheva 1982). Recently, Cameron
et al. (2006) proposed that the orchid–fungal
123
16
association is mutualistic, based on experiments
carried out under special laboratory conditions.
However, as Rasmussen and Rasmussen (2007) aptly
state, ‘‘such conditions would hardly ever occur
under field conditions where complex carbon sources
abound.’’ Given the complexity of the orchid–fungal
association, some degree of reciprocal carbon
exchange might be expected to occur in situ under
limited circumstances. As interesting as this possibility may be, however, it is both legitimate and
practical to regard orchids as the aggressor (parasite)
and the fungus the ‘‘host’’ (Baskin and Baskin 2001;
Rasmussen and Rasmussen 2007). Clearly, orchids
are highly specialized mycotrophs on a level that
remains unparalleled in the plant kingdom. Not
coincidentally, the Orchidaceae is also the most
diverse plant family.
Typical of other parasites (e.g., tapeworms, roundworms), orchids produce numerous offspring (seeds),
very few of which reach their target (the host) and
survive to maturity. Teryhokhin and Nikiticheva
(1982) suggested that parasitic plants evolved smaller
and more numerous seeds to ensure contact with their
host plants having scattered distributions. It is equally
conceivable that orchids did the same to ensure
contact with a suitable fungus required for germination and/or development. Although fungi are ubiquitous and may attain massive sizes within a substrate
(Raven et al. 2005), specific fungi required by orchids
may or may not be widespread. Curtis (1939)
predicted that orchid species restricted to a particular
habitat would harbor fewer fungal associates compared to those found in different habitats. Indeed,
fungal specificity has been documented in the Orchidaceae (e.g., Warcup 1973, 1985; Zettler and Hofer
1998; Otero et al. 2007), and this may explain, in
part, an orchid’s limited distribution. Orchids that are
closely tied to specific fungi are also at greater risk
for extinction (Swarts and Dixon 2009). When
orchids are viewed for what they really are—parasites
of fungi—their conservation takes on new meaning.
Like animals, young orchid seedlings are consumers,
not producers, and should be treated accordingly.
Thus, to promote a self-sustainable orchid population
in situ, there must be suitable prey (fungi) available at
the site (S.L. Stewart, pers. commun.).
It is probably no coincidence that seeds of both
epiphytic and terrestrial orchids have dust-like
seeds—seedlings of both groups must make physical
123
Plant Ecol (2010) 211:7–17
contact with suitable fungi on a particular substrate to
propel the orchid life cycle to completion. By
comparing water balance profiles for a range of
orchids inhabiting different habitat extremes in North
America, seeds of epiphytes fundamentally differ
from those of terrestrials. The high seed/embryo
volume ratio (=air space within the seed coat) appears
to a common characteristic of all orchids (Baskin and
Baskin 2001); however, our results clearly demonstrate that seeds of epiphytic orchids are more porous
(have higher activation energy) than those of terrestrials. As to why these differences are evident
remains unclear but could be linked to the ecological
niches these orchids occupy. The water balance
profiles revealed herein represent a start, but additional studies are needed to demonstrate the true
relationship of the seed’s physical traits and their
success in a given niche.
Acknowledgments We express sincere gratitude to the
following individuals for their assistance with seed
collections: William E. Brumback (New England Wildflower
Society), Katherine B. Gregg (West Virginia Wesleyan
University), William Kutosky (Illinois College), Mike Owen
(Fakahatchee Strand State Preserve, Florida), Steve Perlman
(National Tropical Botanical Garden, Hawaii), Larry
Richardson (Florida Panther National Wildlife Refuge, U.S.
Fish and Wildlife Service), and Scott L. Stewart (Kankakee
Community College, Illinois).
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