In Vitro Cell.Dev.Biol.— (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 () and its implications for -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 and water through the action of mycotrophy. The digestion was considered controversial and not well understood for of these mycobionts and subsequent uptake of nutrients by many years. Differences in mycobiont specificity during the immature orchid embryo stimulates seed germination, germination in vitro vs in situ have lead some to consider protocorm development, and seedling growth (Arditti, orchid–mycobiont specificity as being generally low; 1966; Clements, 1988; Rasmussen, 1995). For this reason, however, others have suggested that specificity, especially the survival of orchids in managed or restored habitats may in vitro, is surprisingly high. Mycobiont specificity may be require the presence of appropriate mycobionts to support genus or species specific. An in vitro symbiotic seed plant development and subsequent seedling recruitment germination experiment was designed to examine myco- (Zettler, 1997a). Symbiotic seed germination techniques biont specificity of the endangered Florida terrestrial orchid represent an efficient way to promote the orchid–fungus Spiranthes brevilabris using mycobionts isolated from both association under in vitro conditions and to study in vitro the study species and the endemic congener Spiranthes orchid–mycobiont specificity (Dixon, 1987; Zettler, 1997a, floridana. In a screen of mycobionts, isolates Sflo-305 b; Stewart and Kane, 2006). While a number of symbiotic (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (origi- seed germination techniques exist for terrestrial orchid nating from S. floridana) supported higher initial (stage 1) species, their germination efficiency is often lower than seed germination than isolate Sbrev-266 (32.4%) (originat- expected (Anderson, 1991, 1996; Zettler and McInnis, ing from S. brevilabris) after 3 wk culture. However, only 1992; Zettler and Currah, 1997; Zettler and Hofer, 1998; isolate Sbrev-266 supported advanced germination and Zettler et al., 2001; Stewart and Zettler, 2002; Sharma et al., protocorm development to stage 5 (53.1%) after 12 wk 2003; Stewart et al., 2003; Zettler et al., 2005; Stewart and culture. These findings suggest that S. brevilabris maintains Kane, 2006), especially when compared to asymbiotic a high degree of mycobiont specificity under in vitro germination studies of the same taxa. This low seed germi- symbiotic seed germination conditions. High orchid–myco- nation efficiency is likely because of a degree of specificity biont specificity in S. brevilabris may be indicative of the many terrestrial orchids appear to have for certain myco- rare status of this orchid in Florida. bionts at the time of germination vs later life stages. How- ever, this specificity has apparently been overlooked by Keywords Ceratorhiza . Epulorhiza . Fungal co-culture . previous symbiotic culture practitioners. Mycobiont speci- Mycorrhizae . Native . Protocorm ficity 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; Introduction Stewart et al., 2003; Batty et al., 2006a, b). Orchid–mycobiont specificity was considered controver- In nature, orchids utilize naturally occurring endophytic sial for many years. Many researchers have considered the mycorrhizal fungi as sources of carbohydrates, nutrients, orchid–fungus relationship to be happenchance and non- specific both in vitro and in situ (Knudson, 1922;Curtis, * : S. L. Stewart ( ) M. E. Kane 1939;Hadley,1970; Masuhara and Katsuya, 1989; Masuhara Department of Environmental Horticulture, University of Florida, – P.O. Box 110675, Gainesville, FL 32611, USA et al., 1993). Differences in orchid fungal specificity were e-mail: [email protected] identified under in vitro vs in situ conditions (Masuhara and MYCOBIONT SPECIFICITY IN S. BREVILABRIS 179

Katsuya, 1994; Taylor and Bruns, 1999; Taylor et al., 2003; Fig. 1b). The orchid’s typical habitats are grassy roadsides, Bidartondo and Bruns, 2005), and these differences have led cemeteries, and pine flatwoods—all of which are becoming some to consider orchid–mycobiont specificity as generally restricted and degraded in Florida because of habitat loss low (Hadley, 1970; Stewart and Zettler, 2002). However, through urban development and habitat mismanagement others have suggested that specificity, especially under in (Main et al., 1999; Marshall and Pielke, 2004). No infor- vitro conditions, is surprisingly high (Clements, 1988; mation exists concerning the symbiotic seed germination, Smreciu and Currah, 1989;TaylorandBruns,1997; mycobiont specificity, or mycobiont diversity of this McKendrick et al., 2002; Selosse et al., 2002; McCormick species. S. brevilabris and S. floridana were shown to be et al., 2006; Stewart and Kane, 2006). In the most general two independent species with a high degree of relatedness sense, it appears that orchid–mycobiont specificity may be based on genetic evidence (L. Dueck, unpublished data), genus or even species specific. and are considered congeners for this reason. Spiranthes brevilabris Lindley, the short-lipped ladies’ The present study investigates the in vitro mycobiont spe- tresses, is an endangered terrestrial orchid historically found cificity of the rare Florida terrestrial orchid S. brevilabris throughout the southeastern United States coastal plain, but is using mycobionts originating from both study species and the now restricted to one roadside population in Levy County in endemic congener S. floridana through the use of symbiotic west central Florida (Brown, 2002; Fig. 1a). The orchid’s seed germination techniques. A concise description and current habitat is a grassy roadside, although little is known tentative identification of all mycobionts are provided. The about the species’ existence in its more natural sunny pine role of fungal specificity in the distribution, current status, flatwood habitat (S. L. Stewart, personal observation). and conservation of S. brevilabris is also given consideration. 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. Materials and Methods brevilabris and the Florida epiphytic orchid Epidendrum magnoliae Mühlenberg var. magnoliae (syn.=Epidendrum Fungal isolation and identification. Four mycobionts were conopseum R. Brown ex Aiton). However, the mycobiont chosen for in vitro symbiotic seed germination and myco- specificity in S. brevilabris using mycobionts originating biont specificity trials of S. brevilabris (Table 1). Mycobiont from within the genus was not explored. No further research Sbrev-266 was previously isolated by Stewart et al. (2003). exists concerning the symbiotic germination or mycobiont S. floridana mycobionts were isolated on 28 April 2004 specificity in this species. following the procedure outlined by Stewart et al. (2003), Spiranthes floridana (Wherry) Cory emend. P.M. Brown, modified by taking only root sections and not entire the Florida ladies’ tresses, is an endemic terrestrial orchid flowering because of the rarity of the species. Root historically ranging throughout north–central Florida, but is systems of five adult flowering plants at the Bradford currently restricted to two populations in Bradford and County (Florida) population of S. floridana were carefully Duval Counties in northeastern Florida (Brown, 2002; 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) supple- mented 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-fifth- strength potato dextrose agar (1/5-PDA): 6.8 g PDA (BD Figure 1. (a) Spiranthes brevilabris in situ.(b) S. Company, Sparks, MD), 6.0 g granulated agar (BD floridana inflorescence in situ. Scale bars=5 mm. Company), 1 l dd water. The pH of all previously mentioned 180 STEWART AND KANE

Table 1. Sources of fungal mycobionts used in the Isolate Host Collection Identification inoculation of Spiranthes information brevilabris seed Sflo-305 S. floridana Collected 28 April 2003; Bradford Co., FL Ceratorhiza sp. All mycobionts hosted within Sflo-306 S. floridana Collected 28 April 2003; Bradford Co., FL Ceratorhiza sp. the roots of adult flowering Sflo-308 S. floridana Collected 25 April 2004; Bradford Co., FL Ceratorhiza sp. plants. Sbrev-266 S. brevilabris Collected 30 April 1999; Levy Co., FL Epulorhiza repens media was adjusted to 6.0 with 0.1 N KOH before autoclaving removed from cold–dark storage, allowed to warm to room at 117.7 kPa and 121°C for 20 min. temperature (ca 25°C), surface disinfected 1 min in the Mycobionts showing cultural characteristics similar to same solution used during root surface cleansing, and those orchid endophytic fungi previously described in the placed on the surface of a 1 cm×4 cm filter paper strip literature (Currah et al., 1987;Currahetal.,1997;Zettler, (Whatman No. 4, Whatman International, Maidstone, UK) 1997b; Richardson and Currah, 1993;Zelmeretal.,1996; within a 9-cm diameter Petri plate containing ca 25 ml Stewart et al., 2003) were assigned a reference number and OMA. Germination medium pH was adjusted to 5.8 with stored at 10°C on oat meal agar (OMA): 3.0 g pulverized 0.1 N HCl before autoclaving at 117.7 kPa and 121°C for rolled oats (Quaker Oats, Chicago, IL), 7.0 g granulated 40 min. Seeds were transferred to the filter paper strips agar, 100 mg yeast extract (BD Company, Sparks, MD), and using a sterile bacterial inoculating loop. Between 60 and 1 l dd water (Dixon, 1987). Isolates were also stored on 1/5 100 seeds were sown per plate. Each plate was inoculated PDA in continual darkness at 25±2°C until use in seed with a 1-cm3 block of fungal inoculum, one mycobiont per germination experiments. plate, and 10 replicate plates per mycobiont. Plates without Mycobiont characterization and identification followed fungal mycobiont served as controls. Plates were sealed methods described by Davidson (1938), Smith (1977), Zelmer with Nescofilm (Karlan Research Products, Santa Rosa, and Currah (1995), Currah et al. (1987, 1990, 1997), and CA) and maintained in darkness (0/24 h L/D) for 86 d at Zelmer et al. (1996) for cultural morphology, polyphenol 25±2°C. Plates were examined weekly during dark incuba- oxidase production, and cellulase activity. Hyphal and tion for signs of germination or contamination, exposing the monilioid cell characteristics were assessed using a Nikon seeds to brief (<10 min) periods of illumination. Plates were Labophat-2 light microscope (Nikon USA, Melville, NY) returned to experimental conditions after visual inspection. fitted with a Nikon Coolpix 990 digital camera (Nikon USA). Every 3 wk after the start of dark incubation, seed Fungal staining procedures followed those outlined by germination and protocorm development were assessed Phillips and Hayman (1970) modified by the use of acid using a dissecting stereomicroscope. fuchsin as the stain (Stevens, 1974). Germination and seedling growth and development were scored on a scale of 0–5 (Table 2; Stewart et al., 2003). Seed collection. Seeds of S. brevilabris were collected before Seed germination percentages were based on viable seeds capsule dehiscence from mature fruits on 17 April 2005. determined by visual inspection with the aid of a dissecting Seeds were collected from a roadside population on state- stereomicroscope. Germination percentages for each devel- managed land in Levy County (FL). Immediately after opmental stage were calculated by dividing the number of collection, capsules were dried over silica gel desiccant for seeds in that particular germination and development stage 2 wk at 25°C, followed by storage at −10°C in darkness for by the total number of viable seeds in the sample. Data 192 d. Before the initiation of symbiotic seed germination were analyzed using general linear model procedures and 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 Table 2. Seed germination and protocorm development in Spiranthes apparently aborts seed capsules soon after pollination and brevilabris, adapted from Stewart et al. (2003) fertilization (S.L. Stewart, personal observation). Stage Description

Symbiotic seed germination and fungal specificity. Four 0 No germination, viable embryo mycobionts (Table 1) were evaluated for their ability 1 Enlarged embryo, production of rhizoid(s) (=germination) to support the in vitro symbiotic seed germination of 2 Continued embryo enlargement, rupture of testa S. brevilabris and for the demonstration of any in vitro 3 Appearance of protomeristem 4 Emergence of first leaf mycobiont specificity. Seeds were sown according to the 5 Elongation of first leaf procedures described by Stewart et al. (2003). Seeds were MYCOBIONT SPECIFICITY IN S. BREVILABRIS 181

Figure 2. Fungal mycobionts isolated from Spiranthes floridana (Sflo) stained as above (×100), scale bar=30 μm. (e) Sflo-308 whole culture and S. brevilabris (Sbrev). (a) Sflo-305 whole culture morphology at morphology at 20 d, scale bar=1 cm. (f) Sflo-308 monilioid cells 20 d, scale bar=1 cm. (b) Sflo-305 monilioid cells stained with acid stained as above (×100), scale bar=30 μm. (g) Sbrev-266 whole fuchsin at 20 d (×100), scale bar=30 μm. (c) Sflo-306 whole culture culture morphology at 20 d, scale bar=1 cm. (h) Sbrev-266 monilioid morphology at 20 d, scale bar=1 cm. (d) Sflo-306 monilioid cells cells stained as above (×400), scale bar=10 μm.

Waller–Duncan mean separation at α=0.05 by SAS v 8.02 negative, which typically is indicative of orchid mycobionts (SAS, 1999). Germination counts were arcsine transformed from the form genus Epulorhiza Moore (Moore, 1987). to normalize variation. 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 barrel- Results shaped monilioid cells (Sflo-305=36×24.8 μm, Sflo-306= 39.6×21.6 μm, Sflo-308=39.2×29.2 μm) support the Fungal mycobionts. Three fungal mycobionts were recov- tentative identification of these isolates as Ceratorhiza ered from root sections of flowering plants of S. floridana species (Fig. 2a–f ). Only superficial differences in cultural (Table 1; Fig. 2a–f ). All three mycobionts were identified morphology were identified among the group of three as members of the anamorphic genus Ceratorhiza Moore mycobionts. Isolate Sflo-305 formed smaller and more (Moore, 1987). Isolates Sflo-305 and Sflo-306 tested numerous loose aerial sclerotia than either isolate Sflo-306 cellulase-negative, a typical Ceratorhiza-like response or Sflo-308, whereas isolate Sflo-306 formed more irregu- (Zelmer and Currah, 1997). Isolate Sflo-308 tested cellu- larly shaped aerial sclerotia than the other isolates. Isolate lase-positive. All three isolates tested polyphenol oxidase- Sbrev-266 was previously recovered from the roots of S. 182 STEWART AND KANE brevilabris and identified as a strain of Epulorhiza repens found in the present study for those seeds inoculated with (Bernard) Moore (Table 1; Fig. 2g–h; Stewart et al., 2003). isolate Sbrev-266 on OMA after 12 wk (53.1% stage 5; Fig. 3c; Table 2). Stewart et al. (2003) concluded that Symbiotic seed germination and fungal specificity. Seeds in S. brevilabris may be nonspecific for mycobionts as all fungal treatments began to swell within 2 wk after comparable germination percentages were supported by sowing, and germination commenced within 3 wk. Visual mycobionts originating from the study species and an contamination rate of cultures was 1%. Visual inspection epiphytic Florida species. Unfortunately, Stewart et al. revealed S. brevilabris seeds to be 94.6% viable. (2003) only tested mycobiont specificity using two myco- An effect of fungal mycobiont was found on the in vitro bionts, none of which originated from closely related symbiotic germination of S. brevilabris. Germination after Spiranthes species in Florida, such as S. floridana.To 3 wk was highest when seeds were inoculated with better demonstrate any orchid–mycobiont specificity within mycobionts Sflo-305, Sflo-306, and Sflo-308 (99.5, 99.5, S. brevilabris, mycobionts from both S. brevilabris and its 89.9%, respectively; Fig. 3a). However, these fungal isolates endemic congener, S. floridana, should have been tested. only promoted seed germination to stage 1, whereas isolate This was the aim of our study. Sbrev-266 supported stage 2 germination (32.4%) after 3 wk Of further interest is that the same mycobiont Stewart dark incubation (Fig. 3a). Mycobionts Sflo-305 and Sflo-306 et al. (2003) utilized in the symbiotic seed germination of S. did support stage 2 germination after 10 wk dark incubation, brevilabris continued to support in vitro symbiotic germina- but only to a minimal percentage (10.6, 0.6%, respectively; tion in the present study. Some authorities have suggested that Fig. 3b). In contrast, mycobiont Sbrev-266 supported a the effectiveness of orchid mycobionts at supporting symbi- maximum of stage 5 germination (46.2%) after 10 wk dark otic germination may lessen if the mycobionts are stored over incubation (Fig. 3b). After a total of 12 wk dark incubation, long periods of time or subjected to multiple subcultures (L. only mycobiont Sbrev-266 supported germination to an W. Zettler, personal communication). Our present data advanced developmental stage (i.e., stage 3 or greater; suggest that mycobiont Sbrev-266 does not demonstrate any Figs. 3c and 4). Control treatments supported only stage 1 reduced symbiotic germination capacity despite being rou- germination after a total of 12 wk dark incubation (Fig. 3c). tinely 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 spe- Discussion cies Epidendrum magnoliae var. magnoliae, to germinate seeds of the Florida epiphytic orchid Encyclia tampensis The conservation of rare, endangered, and endemic Spi- (Lindley) Small. While mycobiont Econ-242 did support ranthes species in Florida depends on an understanding of the in vitro symbiotic seed germination of E. tampensis to a not only the requirements for in vitro symbiotic seed prop- final percent germination of 0.3% (stage 5; Table 2) after agation, but also the degree of mycobiont specificity ex- 13 wk, it is unlikely that this represented a true test of hibited by each species within the genus. Most studies on mycobiont specificity in E. tampensis because only one orchid–mycobiont specificity examine the topic at either the mycobiont was tested (Econ-242) and no mycobionts from generic level within the Orchidaceae or among species repre- E. tampensis were incorporated. While of some basic interest, senting extremes in the family (i.e., terrestrial vs epiphytic; mycobiont specificity across widely diverse genera does not nonphotosynthetic), without examining mycobiont specific- elucidate orchid–mycobiont specificity at the generic level, ity effects on in vitro seed germination. This study represents thus, circumventing questions of orchid–mycobiont specificity the first report of in vitro mycobiont specificity in S. and possible mycobiont sharing within closely related species brevilabris using mycobionts originating from the study pairs, such as S. brevilabris and S. floridana in Florida. species and from the endemic congener S. floridana.This Moreover, the use of widely diverse mycobionts in the in vitro study also represents the first report of successful mycobiont symbiotic seed germination of widely diverse genera yields isolation from the roots of the S. floridana. little practical information on the symbiotic propagation or Successful in vitro symbiotic seed germination of S. conservation of orchid species. As previously stated, the brevilabris was previously reported (Stewart et al., 2003). conservation of orchid species by symbiotic seed germination Using mycobionts isolated from S. brevilabris (Sbrev-266) relies on an understanding of not only mycobiont diversity and the Florida epiphytic species Epidendrum magnoliae and specificity, but also the physiological role specific var. magnoliae (syn.=E. conopseum; Econ-242), Stewart et mycobionts may provide to their orchid hosts (i.e., seed al. (2003) reported a maximum percent germination of 40.8 germination). This can only be investigated once a thorough and 49.8%, respectively, on modified OMA after 55 d in understanding of generic or species level mycobiont specific- vitro culture. This was a similar final percent germination as ity was achieved. MYCOBIONT SPECIFICITY IN S. BREVILABRIS 183

Figure 3. Effects of four fungal 100 mycobionts on percent seed b (c) Sbrev-266 germination and protocorm de- Sflo-305 b velopment (Table 2)ofSpi- Sflo-306 b ranthes brevilabris cultured on 80 Sflo-308 b oat meal agar after (a)3wk Control symbiotic in vitro culture, (b) 10 wk symbiotic in vitro culture, and (c) 12 wk symbiotic in vitro 60 a culture. Histobars with the same letter are not significantly dif-

ferent within germination and (%) development stage (α=0.05). 40 a a a a a a 20

a a a a a 0 (b) 100 b

b b b 80

60 a (%)

40 a a a a a a 20 a a a a a 0 bb b (a) 100 b

80

60 a (%)

40 a

a 20

b b b 0 Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Developmental Stage 184 STEWART AND KANE

Figure 4. Effect of fungal 70 mycobiont Sbrev-266 on per- cent seed germination and pro- Stage 0 tocorm development (Table 2) Stage 1 d of Spiranthes brevilabris cul- 60 Stage 2 tured on oat meal agar at 3, 10, a Stage 3 and12wksymbioticin vitro d Stage 4 culture. Histobars with the 50 Stage 5 same letter are not significantly different within development time (α=0.05). 40 c a a (%) 30 b b

b 20

c c 10 c c c c

0 3 wks 10 wks 12 wks Development Time

Previous reports demonstrating the in vitro symbiotic closer examination of mycobiont diversity in S. cernua and seed germination of other Spiranthes species using myco- closely related species may have revealed a higher-than- bionts isolated from divergent genera may have question- expected degree of mycobiont specificity during in vitro able application to the conservation of Spiranthes species, symbiotic seed germination. Our current study demonstrated in general. Zettler and McInnis (1993)reportedthe this trend, with mycobionts isolated from S. floridana sup- successful symbiotic seed germination of two spiranthoid porting no advanced stage symbiotic seed germination when orchids— (Linnaeus) L.C. Richard and cocultured with seed of S. brevilabris.Thesetwoclosely pubescens (Willdenow) R. Brown—using myco- related species appear to not share mycobionts based on bionts isolated from S. cernua, Platanthera integrilabia mycobiont isolations and in vitro symbiotic seed germination (Correll) Luer, and Platanthera ciliaris (Linnaeus) Lindley. trails. Unfortunately, symbiotic seed germination trials were In their study, only S. cernua seeds inoculated with the not possible using seed of S. floridana because it appears the mycobiont originating from P. ciliaris germinated and species aborts seed capsules soon after pollination and developed to a leaf-bearing stage, and only those resulting fertilization (S.L. Stewart, personal observation). seedlings were acclimatized in the greenhouse. Zettler and Otero et al. (2005) reported varied performance of McInnis (1993) suggest that based on these data, mycobiont mycobionts during in vitro symbiotic seed germination of specificity in S. cernua is rarely species specific therefore the tropical epiphytic species Tolumnia variegata (Swartz) partially explaining the wide distribution of S. cernua in Braem. They go on to hypothesize that, given the presumed North America. They go on to suggest that the orchid– geographic heterogeneous distribution of orchid myco- mycobiont association is rarely species specific, in general. bionts, these mycobionts may affect orchid distribution Given that the aforementioned study did not utilize multiple and population size. This conclusion appears valid based on mycobionts from S. cernua or closely related species (i.e., our current findings and may help to explain the rarity of S. S. odorata) from a wide geographic range, the conclusion brevilabris in Florida. of low mycobiont specificity in S. cernua seems premature, The isolation of the mycobiont E. repens from the roots especially in light of more recent data. of S. brevilabris was previously reported (Stewart et al., Moreover, the use of a mycobiont originating from P. 2003). Subsequent mycobiont isolations from other plants ciliaris in the symbiotic germination of S. cernua seeds within the only known S. brevilabris population have presents an ethical dilemma for those interested in S. cernua consistently yielded isolates identifiable as strains of the conservation—what are the potential ecological impacts of species E. repens (S.L. Stewart, unpublished data). This releasing a mycobiont not originating from S. cernua into S. Epulorhiza species is considered ubiquitous throughout cernua habitats? As was the finding in our current study, a many orchid habitats worldwide (Anderson, 1991; Zelmer, MYCOBIONT SPECIFICITY IN S. BREVILABRIS 185

2001). Thus, its repeated isolation from S. brevilabris was The current study presents a first look at orchid– not surprising, and its isolation from S. floridana was mycobiont specificity in one endangered terrestrial orchid expected. Moreover, given the relatedness between S. from Florida—S. brevilabris. Specificity, in this case, was brevilabris and S. floridana, one would suspect that the demonstrated through not only the isolation and identifica- two species may share related mycobionts. However, tion of different mycobionts from S. brevilabris and its repeated mycobiont isolations from the roots of S. floridana Florida endemic congener S. floridana, but also the use of at the Bradford County site have consistently yielded those mycobionts in the in vitro symbiotic seed germination isolates identifiable as strains of the anamorphic fungal of S. brevilabris. Data such as reported here may prove genus Ceratorhiza. This apparent species level orchid– invaluable in the conservation of both S. brevilabris and S. mycobiont specificity was surprising given the previously floridana in Florida, especially given their rare status within mentioned taxonomic relatedness of the two orchid species. the state. While this study represents the first report of a High mycobiont specificity on the generic and species high degree of mycobiont specificity in S. brevilabris, level, as is hypothesized between S. brevilabris and S. further investigation into mycobiont diversity, and in vitro floridana, was previously reported. Shefferson et al. (2005) and in situ specificity between S. brevilabris and S. reported high mycobiont specificity at the generic level in a floridana should be conducted. study of the terrestrial orchid genus Cypripedium.Ofthe seven Cypripedium species surveyed, five of the species Acknowledgements The authors thank Tim Johnson (Environmen- shared mycobionts in the Tulansnellaceae. Two species sur- tal Horticulture Department, University of Florida) and James veyed, Cypripedium californicum Gray and Cypripedium Kimbrough, Ph.D. (Plant Pathology Department, University of Florida) for their helpful reviews of this manuscript. Carrie Reinhardt parviflorum Salisbury, demonstrated a higher degree of myco- Adams, Ph.D. (Environmental Horticulture Department, University of biont diversity than all other surveyed species. Interestingly, Florida) provided access to microscopic equipment. Appreciation is C. californicum and C. parviflorum demonstrated high also extended to the San Diego County Orchid Society, the U.S. Fish — mycobiont specificity at the generic level in comparison to and Wildlife Service Florida Panther National Wildlife Refuge, and the Florida Division of Forestry for providing financial support of this other five Cypripedium species studied, especially for research. Brand names are provided for references; the authors do not mycobionts not commonly associated with the other five solely endorse these particular products. species surveyed. This trend is similar to the mycobiont specificity apparently demonstrated between S. brevilabris and S. floridana. References Likewise, Taylor et al. (2003) reported a high degree of specificity between two closely related nonphotosynthetic Anderson, A. B. Symbiotic and asymbiotic germination and growth of – orchids in the genus Hexalectris. Four distinct types of fungi (Orchidaceae). Lindleyana 6:183 186; 1991. were identified from samples of H. spicata (Walter) Anderson, A. B. The reintroduction of Platanthera ciliaris in Canada. Barnhardt var. spicata, whereas only one type was identified In: Allen, C., ed. North American native terrestrial orchids: from samples of H. spicata var. arizonica (S. Watson) propagation and production. Washington D.C.: North American – Catling and Engel. Taylor et al. 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