In Vitro Cell.Dev.Biol.—Plant (2007) 43:178-186
DOI 10.1007/s11627-006-9023-4
SIVB SYMPOSIUM PROCEEDINGS-MICROPROPAGATION
Symbiotic seed germination and evidence for in vitro
mycobiont specificity in Spiranthes brevilabris (Orchidaceae)
and its implications for species-level conservation
Scott L. Stewart & Michael E. Kane
Received: 26 July 2006 / Accepted: 27 December 2006 / Published online: 13 March 2007 / Editor: B. R. Reed
# The Society for In Vitro Biology 2007
Abstract Orchid–mycobiont specificity in the Orchidaceae
was considered controversial and not well understood for
many years. Differences in mycobiont specificity during
germination in vitro vs in situ have lead some to consider
orchid–mycobiont specificity as being generally low;
however, others have suggested that specificity, especially
in vitro, is surprisingly high. Mycobiont specificity may be
genus or species specific. An in vitro symbiotic seed
germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid
Spiranthes brevilabris using mycobionts isolated from both
the study species and the endemic congener Spiranthes
floridana. In a screen of mycobionts, isolates Sflo-305
(99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1)
seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only
isolate Sbrev-266 supported advanced germination and
protocorm development to stage 5 (53.1%) after 12 wk
culture. These findings suggest that S. brevilabris maintains
a high degree of mycobiont specificity under in vitro
symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the
rare status of this orchid in Florida.
Keywords Ceratorhiza . Epulorhiza . Fungal co-culture .
Mycorrhizae . Native . Protocorm
Introduction
In nature, orchids utilize naturally occurring endophytic
mycorrhizal fungi as sources of carbohydrates, nutrients,
S. L. Stewart (*) : M. E. Kane
Department of Environmental Horticulture, University of Florida,
P.O. Box 110675, Gainesville, FL 32611, USA
e-mail: slstewar@ufl.edu
and water through the action of mycotrophy. The digestion
of these mycobionts and subsequent uptake of nutrients by
the immature orchid embryo stimulates seed germination,
protocorm development, and seedling growth (Arditti,
1966; Clements, 1988; Rasmussen, 1995). For this reason,
the survival of orchids in managed or restored habitats may
require the presence of appropriate mycobionts to support
plant development and subsequent seedling recruitment
(Zettler, 1997a). Symbiotic seed germination techniques
represent an efficient way to promote the orchid–fungus
association under in vitro conditions and to study in vitro
orchid–mycobiont specificity (Dixon, 1987; Zettler, 1997a,
b; Stewart and Kane, 2006). While a number of symbiotic
seed germination techniques exist for terrestrial orchid
species, their germination efficiency is often lower than
expected (Anderson, 1991, 1996; Zettler and McInnis,
1992; Zettler and Currah, 1997; Zettler and Hofer, 1998;
Zettler et al., 2001; Stewart and Zettler, 2002; Sharma et al.,
2003; Stewart et al., 2003; Zettler et al., 2005; Stewart and
Kane, 2006), especially when compared to asymbiotic
germination studies of the same taxa. This low seed germination efficiency is likely because of a degree of specificity
many terrestrial orchids appear to have for certain mycobionts at the time of germination vs later life stages. However, this specificity has apparently been overlooked by
previous symbiotic culture practitioners. Mycobiont specificity was shown to play an important role in symbiotic
orchid propagation and is thought to play a critical role in
the establishment of orchids into field sites (Zettler, 1997a, b;
Stewart et al., 2003; Batty et al., 2006a, b).
Orchid–mycobiont specificity was considered controversial for many years. Many researchers have considered the
orchid–fungus relationship to be happenchance and nonspecific both in vitro and in situ (Knudson, 1922; Curtis,
1939; Hadley, 1970; Masuhara and Katsuya, 1989; Masuhara
et al., 1993). Differences in orchid–fungal specificity were
identified under in vitro vs in situ conditions (Masuhara and
MYCOBIONT SPECIFICITY IN S. BREVILABRIS
Katsuya, 1994; Taylor and Bruns, 1999; Taylor et al., 2003;
Bidartondo and Bruns, 2005), and these differences have led
some to consider orchid–mycobiont specificity as generally
low (Hadley, 1970; Stewart and Zettler, 2002). However,
others have suggested that specificity, especially under in
vitro conditions, is surprisingly high (Clements, 1988;
Smreciu and Currah, 1989; Taylor and Bruns, 1997;
McKendrick et al., 2002; Selosse et al., 2002; McCormick
et al., 2006; Stewart and Kane, 2006). In the most general
sense, it appears that orchid–mycobiont specificity may be
genus or even species specific.
Spiranthes brevilabris Lindley, the short-lipped ladies’
tresses, is an endangered terrestrial orchid historically found
throughout the southeastern United States coastal plain, but is
now restricted to one roadside population in Levy County in
west central Florida (Brown, 2002; Fig. 1a). The orchid’s
current habitat is a grassy roadside, although little is known
about the species’ existence in its more natural sunny pine
flatwood habitat (S. L. Stewart, personal observation).
Information exists on the symbiotic seed germination of S.
brevilabris. Stewart et al., (2003) provided a symbiotic seed
germination protocol using mycobionts originating from S.
brevilabris and the Florida epiphytic orchid Epidendrum
magnoliae Mühlenberg var. magnoliae (syn.=Epidendrum
conopseum R. Brown ex Aiton). However, the mycobiont
specificity in S. brevilabris using mycobionts originating
from within the genus was not explored. No further research
exists concerning the symbiotic germination or mycobiont
specificity in this species.
Spiranthes floridana (Wherry) Cory emend. P.M. Brown,
the Florida ladies’ tresses, is an endemic terrestrial orchid
historically ranging throughout north–central Florida, but is
currently restricted to two populations in Bradford and
Duval Counties in northeastern Florida (Brown, 2002;
Figure 1. (a) Spiranthes brevilabris inflorescence in situ. (b) S.
floridana inflorescence in situ. Scale bars=5 mm.
179
Fig. 1b). The orchid’s typical habitats are grassy roadsides,
cemeteries, and pine flatwoods—all of which are becoming
restricted and degraded in Florida because of habitat loss
through urban development and habitat mismanagement
(Main et al., 1999; Marshall and Pielke, 2004). No information exists concerning the symbiotic seed germination,
mycobiont specificity, or mycobiont diversity of this
species. S. brevilabris and S. floridana were shown to be
two independent species with a high degree of relatedness
based on genetic evidence (L. Dueck, unpublished data),
and are considered congeners for this reason.
The present study investigates the in vitro mycobiont specificity of the rare Florida terrestrial orchid S. brevilabris
using mycobionts originating from both study species and the
endemic congener S. floridana through the use of symbiotic
seed germination techniques. A concise description and
tentative identification of all mycobionts are provided. The
role of fungal specificity in the distribution, current status,
and conservation of S. brevilabris is also given consideration.
Materials and Methods
Fungal isolation and identification. Four mycobionts were
chosen for in vitro symbiotic seed germination and mycobiont specificity trials of S. brevilabris (Table 1). Mycobiont
Sbrev-266 was previously isolated by Stewart et al. (2003).
S. floridana mycobionts were isolated on 28 April 2004
following the procedure outlined by Stewart et al. (2003),
modified by taking only root sections and not entire
flowering plants because of the rarity of the species. Root
systems of five adult flowering plants at the Bradford
County (Florida) population of S. floridana were carefully
excavated and root sections (<10 cm) were removed. Only
five plants, representing 20% of the total population, were
sampled because of the small size of this population. The
Duval County population was not sampled because, at the
time of sampling, only two plants were known from this site.
The root sections were wrapped in paper towels moistened
with sterile deionized water, placed in plastic bags, stored in
darkness at ca 10°C, and transported to the laboratory (<4 h).
Root sections were rinsed with cold tap water to remove
surface debris and were surface cleansed 1 min in a solution
containing absolute ethanol:6.00% NaOCl:sterile deionized
distilled (dd) water (5:5:90 v/v/v). Clumps of cortical cells
containing fungal pelotons were removed, placed on corn
meal agar (CMA; Sigma-Aldrich, St. Louis, MO) supplemented with 50 mg l−1 novobiocin sodium salt, and
incubated at 25°C for 4 d. Hyphal tips were excised from
actively growing pelotons and subcultured onto one-fifthstrength potato dextrose agar (1/5-PDA): 6.8 g PDA (BD
Company, Sparks, MD), 6.0 g granulated agar (BD
Company), 1 l dd water. The pH of all previously mentioned
180
Table 1. Sources of fungal
mycobionts used in the
inoculation of Spiranthes
brevilabris seed
All mycobionts hosted within
the roots of adult flowering
plants.
STEWART AND KANE
Isolate
Host
Collection
information
Sflo-305
Sflo-306
Sflo-308
Sbrev-266
S.
S.
S.
S.
Collected
Collected
Collected
Collected
floridana
floridana
floridana
brevilabris
media was adjusted to 6.0 with 0.1 N KOH before autoclaving
at 117.7 kPa and 121°C for 20 min.
Mycobionts showing cultural characteristics similar to
those orchid endophytic fungi previously described in the
literature (Currah et al., 1987; Currah et al., 1997; Zettler,
1997b; Richardson and Currah, 1993; Zelmer et al., 1996;
Stewart et al., 2003) were assigned a reference number and
stored at 10°C on oat meal agar (OMA): 3.0 g pulverized
rolled oats (Quaker Oats, Chicago, IL), 7.0 g granulated
agar, 100 mg yeast extract (BD Company, Sparks, MD), and
1 l dd water (Dixon, 1987). Isolates were also stored on 1/5
PDA in continual darkness at 25±2°C until use in seed
germination experiments.
Mycobiont characterization and identification followed
methods described by Davidson (1938), Smith (1977), Zelmer
and Currah (1995), Currah et al. (1987, 1990, 1997), and
Zelmer et al. (1996) for cultural morphology, polyphenol
oxidase production, and cellulase activity. Hyphal and
monilioid cell characteristics were assessed using a Nikon
Labophat-2 light microscope (Nikon USA, Melville, NY)
fitted with a Nikon Coolpix 990 digital camera (Nikon USA).
Fungal staining procedures followed those outlined by
Phillips and Hayman (1970) modified by the use of acid
fuchsin as the stain (Stevens, 1974).
Seed collection. Seeds of S. brevilabris were collected before
capsule dehiscence from mature fruits on 17 April 2005.
Seeds were collected from a roadside population on statemanaged land in Levy County (FL). Immediately after
collection, capsules were dried over silica gel desiccant for
2 wk at 25°C, followed by storage at −10°C in darkness for
192 d. Before the initiation of symbiotic seed germination
cultures, seed viability was visually assessed (Stewart and
Zettler, 2002). Viable seeds were considered those seeds
containing a distinct, rounded, and hyaline embryo. Seeds of
S. floridana could not be obtained because the species
apparently aborts seed capsules soon after pollination and
fertilization (S.L. Stewart, personal observation).
Symbiotic seed germination and fungal specificity. Four
mycobionts (Table 1) were evaluated for their ability
to support the in vitro symbiotic seed germination of
S. brevilabris and for the demonstration of any in vitro
mycobiont specificity. Seeds were sown according to the
procedures described by Stewart et al. (2003). Seeds were
28
28
25
30
Identification
April
April
April
April
2003;
2003;
2004;
1999;
Bradford Co., FL
Bradford Co., FL
Bradford Co., FL
Levy Co., FL
Ceratorhiza sp.
Ceratorhiza sp.
Ceratorhiza sp.
Epulorhiza repens
removed from cold–dark storage, allowed to warm to room
temperature (ca 25°C), surface disinfected 1 min in the
same solution used during root surface cleansing, and
placed on the surface of a 1 cm×4 cm filter paper strip
(Whatman No. 4, Whatman International, Maidstone, UK)
within a 9-cm diameter Petri plate containing ca 25 ml
OMA. Germination medium pH was adjusted to 5.8 with
0.1 N HCl before autoclaving at 117.7 kPa and 121°C for
40 min. Seeds were transferred to the filter paper strips
using a sterile bacterial inoculating loop. Between 60 and
100 seeds were sown per plate. Each plate was inoculated
with a 1-cm3 block of fungal inoculum, one mycobiont per
plate, and 10 replicate plates per mycobiont. Plates without
fungal mycobiont served as controls. Plates were sealed
with Nescofilm (Karlan Research Products, Santa Rosa,
CA) and maintained in darkness (0/24 h L/D) for 86 d at
25±2°C. Plates were examined weekly during dark incubation for signs of germination or contamination, exposing the
seeds to brief (<10 min) periods of illumination. Plates were
returned to experimental conditions after visual inspection.
Every 3 wk after the start of dark incubation, seed
germination and protocorm development were assessed
using a dissecting stereomicroscope.
Germination and seedling growth and development were
scored on a scale of 0–5 (Table 2; Stewart et al., 2003).
Seed germination percentages were based on viable seeds
determined by visual inspection with the aid of a dissecting
stereomicroscope. Germination percentages for each developmental stage were calculated by dividing the number of
seeds in that particular germination and development stage
by the total number of viable seeds in the sample. Data
were analyzed using general linear model procedures and
Table 2. Seed germination and protocorm development in Spiranthes
brevilabris, adapted from Stewart et al. (2003)
Stage
Description
0
1
2
3
4
5
No germination, viable embryo
Enlarged embryo, production of rhizoid(s) (=germination)
Continued embryo enlargement, rupture of testa
Appearance of protomeristem
Emergence of first leaf
Elongation of first leaf
MYCOBIONT SPECIFICITY IN S. BREVILABRIS
181
Figure 2. Fungal mycobionts isolated from Spiranthes floridana (Sflo)
and S. brevilabris (Sbrev). (a) Sflo-305 whole culture morphology at
20 d, scale bar=1 cm. (b) Sflo-305 monilioid cells stained with acid
fuchsin at 20 d (×100), scale bar=30 μm. (c) Sflo-306 whole culture
morphology at 20 d, scale bar=1 cm. (d) Sflo-306 monilioid cells
stained as above (×100), scale bar=30 μm. (e) Sflo-308 whole culture
morphology at 20 d, scale bar=1 cm. (f) Sflo-308 monilioid cells
stained as above (×100), scale bar=30 μm. (g) Sbrev-266 whole
culture morphology at 20 d, scale bar=1 cm. (h) Sbrev-266 monilioid
cells stained as above (×400), scale bar=10 μm.
Waller–Duncan mean separation at α=0.05 by SAS v 8.02
(SAS, 1999). Germination counts were arcsine transformed
to normalize variation.
negative, which typically is indicative of orchid mycobionts
from the form genus Epulorhiza Moore (Moore, 1987).
However, a rapid average daily growth rate (Sflo-305=
11.3 mm d−1, Sflo-306=10.6 mm d−1, Sflo-308=12.1 mm
d−1) and the production of large, broadly connected barrelshaped monilioid cells (Sflo-305=36×24.8 μm, Sflo-306=
39.6×21.6 μm, Sflo-308=39.2×29.2 μm) support the
tentative identification of these isolates as Ceratorhiza
species (Fig. 2a–f ). Only superficial differences in cultural
morphology were identified among the group of three
mycobionts. Isolate Sflo-305 formed smaller and more
numerous loose aerial sclerotia than either isolate Sflo-306
or Sflo-308, whereas isolate Sflo-306 formed more irregularly shaped aerial sclerotia than the other isolates. Isolate
Sbrev-266 was previously recovered from the roots of S.
Results
Fungal mycobionts. Three fungal mycobionts were recovered from root sections of flowering plants of S. floridana
(Table 1; Fig. 2a–f ). All three mycobionts were identified
as members of the anamorphic genus Ceratorhiza Moore
(Moore, 1987). Isolates Sflo-305 and Sflo-306 tested
cellulase-negative, a typical Ceratorhiza-like response
(Zelmer and Currah, 1997). Isolate Sflo-308 tested cellulase-positive. All three isolates tested polyphenol oxidase-
182
STEWART AND KANE
brevilabris and identified as a strain of Epulorhiza repens
(Bernard) Moore (Table 1; Fig. 2g–h; Stewart et al., 2003).
Symbiotic seed germination and fungal specificity. Seeds in
all fungal treatments began to swell within 2 wk after
sowing, and germination commenced within 3 wk. Visual
contamination rate of cultures was 1%. Visual inspection
revealed S. brevilabris seeds to be 94.6% viable.
An effect of fungal mycobiont was found on the in vitro
symbiotic germination of S. brevilabris. Germination after
3 wk was highest when seeds were inoculated with
mycobionts Sflo-305, Sflo-306, and Sflo-308 (99.5, 99.5,
89.9%, respectively; Fig. 3a). However, these fungal isolates
only promoted seed germination to stage 1, whereas isolate
Sbrev-266 supported stage 2 germination (32.4%) after 3 wk
dark incubation (Fig. 3a). Mycobionts Sflo-305 and Sflo-306
did support stage 2 germination after 10 wk dark incubation,
but only to a minimal percentage (10.6, 0.6%, respectively;
Fig. 3b). In contrast, mycobiont Sbrev-266 supported a
maximum of stage 5 germination (46.2%) after 10 wk dark
incubation (Fig. 3b). After a total of 12 wk dark incubation,
only mycobiont Sbrev-266 supported germination to an
advanced developmental stage (i.e., stage 3 or greater;
Figs. 3c and 4). Control treatments supported only stage 1
germination after a total of 12 wk dark incubation (Fig. 3c).
Discussion
The conservation of rare, endangered, and endemic Spiranthes species in Florida depends on an understanding of
not only the requirements for in vitro symbiotic seed propagation, but also the degree of mycobiont specificity exhibited by each species within the genus. Most studies on
orchid–mycobiont specificity examine the topic at either the
generic level within the Orchidaceae or among species representing extremes in the family (i.e., terrestrial vs epiphytic;
nonphotosynthetic), without examining mycobiont specificity effects on in vitro seed germination. This study represents
the first report of in vitro mycobiont specificity in S.
brevilabris using mycobionts originating from the study
species and from the endemic congener S. floridana. This
study also represents the first report of successful mycobiont
isolation from the roots of the S. floridana.
Successful in vitro symbiotic seed germination of S.
brevilabris was previously reported (Stewart et al., 2003).
Using mycobionts isolated from S. brevilabris (Sbrev-266)
and the Florida epiphytic species Epidendrum magnoliae
var. magnoliae (syn.=E. conopseum; Econ-242), Stewart et
al. (2003) reported a maximum percent germination of 40.8
and 49.8%, respectively, on modified OMA after 55 d in
vitro culture. This was a similar final percent germination as
found in the present study for those seeds inoculated with
isolate Sbrev-266 on OMA after 12 wk (53.1% stage 5;
Fig. 3c; Table 2). Stewart et al. (2003) concluded that
S. brevilabris may be nonspecific for mycobionts as
comparable germination percentages were supported by
mycobionts originating from the study species and an
epiphytic Florida species. Unfortunately, Stewart et al.
(2003) only tested mycobiont specificity using two mycobionts, none of which originated from closely related
Spiranthes species in Florida, such as S. floridana. To
better demonstrate any orchid–mycobiont specificity within
S. brevilabris, mycobionts from both S. brevilabris and its
endemic congener, S. floridana, should have been tested.
This was the aim of our study.
Of further interest is that the same mycobiont Stewart
et al. (2003) utilized in the symbiotic seed germination of S.
brevilabris continued to support in vitro symbiotic germination in the present study. Some authorities have suggested that
the effectiveness of orchid mycobionts at supporting symbiotic germination may lessen if the mycobionts are stored over
long periods of time or subjected to multiple subcultures (L.
W. Zettler, personal communication). Our present data
suggest that mycobiont Sbrev-266 does not demonstrate any
reduced symbiotic germination capacity despite being routinely subcultured and stored at 25±2°C for 7 yr.
Similarly to both the current study and Stewart et al.
(2003), Zettler et al. (1999) used the previously mentioned
mycobiont Econ-242, originating from the epiphytic species Epidendrum magnoliae var. magnoliae, to germinate
seeds of the Florida epiphytic orchid Encyclia tampensis
(Lindley) Small. While mycobiont Econ-242 did support
the in vitro symbiotic seed germination of E. tampensis to a
final percent germination of 0.3% (stage 5; Table 2) after
13 wk, it is unlikely that this represented a true test of
mycobiont specificity in E. tampensis because only one
mycobiont was tested (Econ-242) and no mycobionts from
E. tampensis were incorporated. While of some basic interest,
mycobiont specificity across widely diverse genera does not
elucidate orchid–mycobiont specificity at the generic level,
thus, circumventing questions of orchid–mycobiont specificity
and possible mycobiont sharing within closely related species
pairs, such as S. brevilabris and S. floridana in Florida.
Moreover, the use of widely diverse mycobionts in the in vitro
symbiotic seed germination of widely diverse genera yields
little practical information on the symbiotic propagation or
conservation of orchid species. As previously stated, the
conservation of orchid species by symbiotic seed germination
relies on an understanding of not only mycobiont diversity
and specificity, but also the physiological role specific
mycobionts may provide to their orchid hosts (i.e., seed
germination). This can only be investigated once a thorough
understanding of generic or species level mycobiont specificity was achieved.
MYCOBIONT SPECIFICITY IN S. BREVILABRIS
100
80
(c)
b
Sbrev-266
Sflo-305
Sflo-306
Sflo-308
Control
b
b
b
a
(%)
60
40
a a
a
a
a
a
20
a
a
a
aa
0
(b)
100
b
b
(%)
80
b
b
60
a
40
a a
a
a
a
20
a
a
a
a
aa
0
b b
100
(a)
b
b
80
60
(%)
Figure 3. Effects of four fungal
mycobionts on percent seed
germination and protocorm development (Table 2) of Spiranthes brevilabris cultured on
oat meal agar after (a) 3 wk
symbiotic in vitro culture, (b)
10 wk symbiotic in vitro culture,
and (c) 12 wk symbiotic in vitro
culture. Histobars with the same
letter are not significantly different within germination and
development stage (α=0.05).
183
a
40
a
a
20
bb b
0
Stage 0
Stage 1
Stage 2
Stage 3
Developmental Stage
Stage 4
Stage 5
184
STEWART AND KANE
Figure 4. Effect of fungal
mycobiont Sbrev-266 on percent seed germination and protocorm development (Table 2)
of Spiranthes brevilabris cultured on oat meal agar at 3, 10,
and 12 wk symbiotic in vitro
culture. Histobars with the
same letter are not significantly
different within development
time (α=0.05).
70
d
60
a
d
50
Stage 0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
(%)
40
c
30
20
a
a
b
b
b
c c
10
c
c c
c
0
3 wks
10 wks
12 wks
Development Time
Previous reports demonstrating the in vitro symbiotic
seed germination of other Spiranthes species using mycobionts isolated from divergent genera may have questionable application to the conservation of Spiranthes species,
in general. Zettler and McInnis (1993) reported the
successful symbiotic seed germination of two spiranthoid
orchids—Spiranthes cernua (Linnaeus) L.C. Richard and
Goodyera pubescens (Willdenow) R. Brown—using mycobionts isolated from S. cernua, Platanthera integrilabia
(Correll) Luer, and Platanthera ciliaris (Linnaeus) Lindley.
In their study, only S. cernua seeds inoculated with the
mycobiont originating from P. ciliaris germinated and
developed to a leaf-bearing stage, and only those resulting
seedlings were acclimatized in the greenhouse. Zettler and
McInnis (1993) suggest that based on these data, mycobiont
specificity in S. cernua is rarely species specific therefore
partially explaining the wide distribution of S. cernua in
North America. They go on to suggest that the orchid–
mycobiont association is rarely species specific, in general.
Given that the aforementioned study did not utilize multiple
mycobionts from S. cernua or closely related species (i.e.,
S. odorata) from a wide geographic range, the conclusion
of low mycobiont specificity in S. cernua seems premature,
especially in light of more recent data.
Moreover, the use of a mycobiont originating from P.
ciliaris in the symbiotic germination of S. cernua seeds
presents an ethical dilemma for those interested in S. cernua
conservation—what are the potential ecological impacts of
releasing a mycobiont not originating from S. cernua into S.
cernua habitats? As was the finding in our current study, a
closer examination of mycobiont diversity in S. cernua and
closely related species may have revealed a higher-thanexpected degree of mycobiont specificity during in vitro
symbiotic seed germination. Our current study demonstrated
this trend, with mycobionts isolated from S. floridana supporting no advanced stage symbiotic seed germination when
cocultured with seed of S. brevilabris. These two closely
related species appear to not share mycobionts based on
mycobiont isolations and in vitro symbiotic seed germination
trails. Unfortunately, symbiotic seed germination trials were
not possible using seed of S. floridana because it appears the
species aborts seed capsules soon after pollination and
fertilization (S.L. Stewart, personal observation).
Otero et al. (2005) reported varied performance of
mycobionts during in vitro symbiotic seed germination of
the tropical epiphytic species Tolumnia variegata (Swartz)
Braem. They go on to hypothesize that, given the presumed
geographic heterogeneous distribution of orchid mycobionts, these mycobionts may affect orchid distribution
and population size. This conclusion appears valid based on
our current findings and may help to explain the rarity of S.
brevilabris in Florida.
The isolation of the mycobiont E. repens from the roots
of S. brevilabris was previously reported (Stewart et al.,
2003). Subsequent mycobiont isolations from other plants
within the only known S. brevilabris population have
consistently yielded isolates identifiable as strains of the
species E. repens (S.L. Stewart, unpublished data). This
Epulorhiza species is considered ubiquitous throughout
many orchid habitats worldwide (Anderson, 1991; Zelmer,
MYCOBIONT SPECIFICITY IN S. BREVILABRIS
2001). Thus, its repeated isolation from S. brevilabris was
not surprising, and its isolation from S. floridana was
expected. Moreover, given the relatedness between S.
brevilabris and S. floridana, one would suspect that the
two species may share related mycobionts. However,
repeated mycobiont isolations from the roots of S. floridana
at the Bradford County site have consistently yielded
isolates identifiable as strains of the anamorphic fungal
genus Ceratorhiza. This apparent species level orchid–
mycobiont specificity was surprising given the previously
mentioned taxonomic relatedness of the two orchid species.
High mycobiont specificity on the generic and species
level, as is hypothesized between S. brevilabris and S.
floridana, was previously reported. Shefferson et al. (2005)
reported high mycobiont specificity at the generic level in a
study of the terrestrial orchid genus Cypripedium. Of the
seven Cypripedium species surveyed, five of the species
shared mycobionts in the Tulansnellaceae. Two species surveyed, Cypripedium californicum Gray and Cypripedium
parviflorum Salisbury, demonstrated a higher degree of mycobiont diversity than all other surveyed species. Interestingly,
C. californicum and C. parviflorum demonstrated high
mycobiont specificity at the generic level in comparison to
other five Cypripedium species studied, especially for
mycobionts not commonly associated with the other five
species surveyed. This trend is similar to the mycobiont
specificity apparently demonstrated between S. brevilabris
and S. floridana.
Likewise, Taylor et al. (2003) reported a high degree of
specificity between two closely related nonphotosynthetic
orchids in the genus Hexalectris. Four distinct types of fungi
were identified from samples of H. spicata (Walter)
Barnhardt var. spicata, whereas only one type was identified
from samples of H. spicata var. arizonica (S. Watson)
Catling and Engel. Taylor et al. (2003) hypothesized that the
divergence seen in mycobiont specificity between these two
varieties of the same orchid species represents evidence for
the contribution of mycobiont specificity to the evolutionary
diversification of the Orchidaceae. High mycobiont specificity between closely related varieties may require special
consideration for the in vitro symbiotic propagation and
conservation of those species, but these factors were not
examined by Taylor et al. (2003). Moreover, Taylor and
Bruns (1999) reported that two species of the nonphotosynthetic orchid genus Corallorhiza each associated with
several species of Russula, but never shared fungi despite
the plants growing sympatrically. Again, these data can
prove to be crucial for those interested in the symbiotic
propagation or conservation of these orchid species. A
similar specificity trend may be occurring between S.
brevilabris and S. floridana; however, further experimentation is necessary to hypothesize on the habitat preference or
selectivity pressures acting between these two species.
185
The current study presents a first look at orchid–
mycobiont specificity in one endangered terrestrial orchid
from Florida—S. brevilabris. Specificity, in this case, was
demonstrated through not only the isolation and identification of different mycobionts from S. brevilabris and its
Florida endemic congener S. floridana, but also the use of
those mycobionts in the in vitro symbiotic seed germination
of S. brevilabris. Data such as reported here may prove
invaluable in the conservation of both S. brevilabris and S.
floridana in Florida, especially given their rare status within
the state. While this study represents the first report of a
high degree of mycobiont specificity in S. brevilabris,
further investigation into mycobiont diversity, and in vitro
and in situ specificity between S. brevilabris and S.
floridana should be conducted.
Acknowledgements The authors thank Tim Johnson (Environmental Horticulture Department, University of Florida) and James
Kimbrough, Ph.D. (Plant Pathology Department, University of
Florida) for their helpful reviews of this manuscript. Carrie Reinhardt
Adams, Ph.D. (Environmental Horticulture Department, University of
Florida) provided access to microscopic equipment. Appreciation is
also extended to the San Diego County Orchid Society, the U.S. Fish
and Wildlife Service—Florida Panther National Wildlife Refuge, and
the Florida Division of Forestry for providing financial support of this
research. Brand names are provided for references; the authors do not
solely endorse these particular products.
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