Reproductive Biology of Cyrtopodium Punctatum in Situ: Implications for Conservation of an Endangered Florida Orchid

Plant Species Biology (2009) 24, 92–103 doi: 10.1111/j.1442-1984.2009.00242.x

Reproductive biology of Cyrtopodium punctatum in situ: implications for conservation of an endangered Florida orchid

DANIELA DUTRA,*1 MICHAEL E. KANE,* CARRIE REINHARDT ADAMS* and LARRY RICHARDSON† *Department of Environmental Horticulture, University of Florida, PO Box 110675, Gainesville, Florida 32611, USA and †Florida Panther National Wildlife Refuge, US Fish and Wildlife Service, 3860 Tollgate Boulevard, Suite 300, Naples, Florida 34114, USA

Abstract

Cyrtopodium punctatum (Linnaeus) Lindley is an endangered epiphytic orchid restricted in the USA to southern Florida. This species has been extensively collected from the wild since the early 1900s, and today only a few plants remain in protected areas. As part of a conservation plan, a reproductive biology study was conducted to better understand the ecology of this species in Florida. Cyrtopodium punctatum relies on a deceit pollination system using aromatic compounds to attract pollinators. Nine aromatic compounds were identified as components of the fragrance of C. punctatum inflorescences, including two compounds that are known to be Euglossine bee attractants. However, this group of bees is not native to Florida. Of the four bee species observed to visit C. punctatum flowers in the present study, carpenter bees (Xylocopa spp.) are likely to be the main pollinators. Pollination experiments demonstrated that C. puntatum is self-compatible, but requires a pollinator and thus does not exhibit spontaneous autogamy. In addition, the rates of fruit set were significantly higher for flowers that were outcrossed (xenogamy) than for those that were self-crossed. Thus, the species has evolved a degree of incompatibility. Examples of natural pollination and fruit set were observed during the present study (2007–2008), but the rates of reproduction were modest as a consequence of the low plant numbers and possible changes in insect densities as a result of anthropogenic influences. Keywords: breeding system, Cyrtopodium punctatum, Orchidaceae, pollination. Received 11 March 2009; accepted 21 May 2009

Introduction often linked to plant rarity given the dependence on insect species for cross pollination as this relationship is Several types of plant rarity exist and different natural required for seed production and fruit set to take place. processes may affect species density and distribution Decreased abundance or loss of pollinators often results in (Rabinowitz 1981; Fiedler & Ahouse 1992). Species may be increased rarity of the plant. The Orchidaceae is a very considered rare based on habitat specificity, local popula- diverse family, and pollination systems may range from tion size and/or the size of their geographical range. being very specific with a plant being pollinated by only However, plants that were once common in their range one insect, to very generalized with a plant being polli- may now be rare as a result of anthropogenic influences nated by many species (van der Pijl & Dodson 1966; (Partel et al. 2005). The factors influencing rarity need to Dressler 1981; van der Cingel 1995). A comprehensive be considered for an effective conservation plan to be investigation of the pollination system is a prerequisite to designed for any target species. Pollination systems are developing a conservation plan for any rare species. Cyrtopodium punctatum (L.) Lindl. is an epiphytic orchid Correspondence: Daniela Dutra Email: [email protected] found in southern Florida, Cuba, Hispaniola, Puerto 1Present address: Botany Department, University of Hawai’i at Rico and the north-western Caribbean coast of South Manoa, 3190 Maile Way, Room 101, Honolulu, HI 96822, USA. America (Romero-González & Fernández-Concha 1999).

In Florida, the species is listed as endangered (Coile & Fragrances produced by plants attract pollinators and Garland 2003); populations have been depleted because of may function to select the type and number of visitors cypress logging and over-collecting during the past (Hills et al. 1972). Floral fragrances have been associated century. Early accounts in the literature to C. punctatum as with deception pollination systems based on generalized abundant throughout south and south-west Florida, par- food deception (Little 1983) and can be an extra tool in the ticularly in the cypress swamps in the Big Cypress region determination of pollination systems. Different fragrance (Ames 1904; Luer 1972). Photographs from the early 1900s compounds or groups of compounds may attract different show wagons loaded with plants being extracted from the pollinators. A larger array of compounds may be benefi- wild. Consequently, only a few plants still remain in very cial in deceit pollination systems because a higher number small populations within inaccessible protected areas, of visitors will be attracted to the plant. such as Everglades National Park, Big Cypress National In many orchid pollination strategy studies, capsule Preserve and the Florida Panther National Wildlife Refuge formation is commonly observed as the final result of the (FPNWR). Over the past 10 years, observations of plant breeding system study without taking into consideration populations in the FPNWR have indicated very low rates seed viability (Wong & Sun 1999; Lehnebach & Robertson of capsule formation and seed production in the remain- 2004). Other researchers have used seed number and ing plants (L. Richardson, pers. comm. 2005). Additional weight as a further measurement for breeding system information regarding the breeding strategies and polli- determination (Robertson & Wyatt 1990b; Sipes & Tepe- nation mechanisms of natural populations of C. punctatum dino 1995). However, this type of measurement produces in Florida is critical to develop strategies for the recovery uncertain results because of differences in capsule matu- of the species. rity. Sipes and Tepedino (1995) reported that fruit weight The study of pollination systems, including breeding and fruit size were not correlated with seed number, and systems, is critical in rare plant conservation because it instead used seed number as another measurement may influence the genetic diversity of populations for breeding system determination. As capsules of C. (Hamrick & Godt 1996). Pollination aids the long-term punctatum require approximately 11 months to mature, survival of most species because population persistence repeated capsule measurement over the course of capsule depends on the recruitment of new plants from geneti- development may minimize this limitation. cally diverse seeds (Dixon et al. 2003). Most of the perti- Another tool used to evaluate breeding systems is tet- nent literature on the pollination of C. punctatum refers to razolium seed viability testing (TZ) and it has been used observations conducted in only one area of the species’ to assess the viability of European and North American range (e.g. Puerto Rico; Ackerman 1995). In different parts temperate orchid species (Van Waes & Debergh 1986; of its geographical range, a species may possess different Lauzer et al. 1994; Vujanovic et al. 2000; Bowles et al. 2002) breeding strategies, which is not uncommon for tropical and tropical epiphytic species (Singh 1981). Assessing orchid species in Florida. For example, both Epidendrum seed viability and germinability is crucial for in vitro nocturnum and Secoila lanceolata produce seeds by agamo- propagation of orchid species and their conservation and spermy in Florida, but use animal cross-pollination in the may be a helpful tool to more precisely determine effec- more southern part of their range (Catling 1987; Brown tive pollination strategies for threatened and endangered 2002). taxa. Pollinator species may also vary from one location in the As a prerequisite to developing a comprehensive con- geographical range to another. Pollination ecotypes have servation plan for C. punctatum, integrated field and labo- been reported by Robertson and Wyatt (1990a) in Plantan- ratory studies were conducted with the objectives of: (i) thera ciliaris. In the present study, the pollination ecology determining the breeding system of C. punctatum through of two disjunct populations was compared and the controlled pollination experiments and the effects on both primary pollinators were found to differ between the capsule formation and seed viability; (ii) determining the sites. Cyrtopodium punctatum is reported to be pollinated pollinator(s) of C. punctatum in Florida; and (iii) determin- by Euglossa bees (van der Pijl & Dodson 1966; Jeffrey et al. ing the volatile potential attractant compounds present in 1970; Luer 1972; Dressler 1993). However, as Florida lies C. punctatum flowers. outside the range of euglossine bees, C. punctatum’s pollinator(s) in Florida may vary (Chase & Hills 1992). In Puerto Rico, C. punctatum is reported to be pollinated by Materials and methods Centris or Xylocopa bees (Ackerman 1995). Bees of the Study species genus Centris were reported by Dodson and Adams (in Luer 1972) to visit flowers of C. punctatum in Florida. In the FPNWR (Collier County, FL, USA), C. punctatum However, these observations have not been fully plants are found growing epiphytically on cypress (Taxo- described or verified. dium distichum) and less often on cabbage palm (Sabal

Plant Species Biology 24, 92–103 © 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology 94 D. DUTRA ET AL. palmetto) or old stumps of massive cypress trees that were linear procedures and Waller–Duncan mean separation logged in the 1940s. The vegetation classification for the (a=0.05) with SAS v. 9.1 (SAS Institute 2003). habitat is cypress forest moderately to densely distributed cypress with an open canopy. The understory is com- posed of Fraxinus caroliniana, Cladium jamaicense and Tetrazolium seed viability test Annona glabra, among others. In other areas of Florida Seed capsules produced from the breeding system study (e.g. Everglades National Park), C. punctatum can also be treatments (2006–2007) were collected on 23 February found growing on Buttonwood trees (Conocarpus erecta)in 2007 and dried over silica desiccant for 70 days at full sun. The inflorescences of C. punctatum are up to 1 m 23 Ϯ 2°C. The seeds harvested from each capsule col- long and can have 30–40 flowers. lected were transferred to 20 mL scintillation vials. The vials were then maintained in cold storage (-10 Ϯ 2°C). Three 5 mg seed subsamples (75–100 seeds) from each Breeding system determination capsule of each treatment were dispensed into a 1.5 mL One population with 15 plants located in the FPNWR microcentrifuge tube and homogenized with the aid of a served as the source of plants for the determination of the vortex shaker. The methods of tetrazolium viability breeding system. Six plants located approximately 30 m testing follow Lakon (1949). A pretreatment test con- apart were used for the breeding system experiment ducted prior to experimentation indicated that a 15 min during two consecutive years (2006–2007 and 2007–2008). treatment in a seed scarification solution of 5% CaOCl2 Seven breeding system treatments (Table 1) were applied was ideal for C. punctatum seeds. Percentage seed viability to five flowers of each plant. Flowers were randomly data were collected for each of the three subsamples per selected on the inflorescences. A total of 245 flowers were capsule by placing seeds that were suspended in water used in this study. A plant located approximately 1.61 km droplets into Petri dishes and the seeds were then scored from the breeding system study population served as the with the aid of a dissecting microscope. Approximately pollen donor. Pollen was applied to flowers within 1 h. 100 seeds were counted per subsample. Embryos that con- The breeding system determination methods were tained any degree of red or pink coloration were scored as adapted from Wong and Sun (1999). An extra treatment viable and white embryos were scored as non-viable (induced xenogamy) was added based on Stewart (2007). (Fig. 2). Percentage viability was calculated for each rep- The breeding system treatments are described in Table 1. licate by dividing the number of stained embryos by the Pollen used in the artificial xenogamy treatment was total number of embryos. Data were analyzed using obtained from a plant originating from a different popu- general linear procedures and Waller–Duncan mean sepa- lation in the FPNWR. Inflorescences were produced in ration (a=0.05) using SAS v. 9.1. February and were bagged before the flowers opened with 95 mm nylon mesh to prevent pollination events Asymbiotic seed germination test prior to initiating the experiment (Fig. 1). Plants remained bagged until signs of capsule formation were observed. Seeds from the capsules produced from each specific The percentage of flowers forming capsules was recorded breeding system treatment were sampled (5 mg sub- and capsule development was recorded bimonthly for samples of 75–100 seeds). Seeds from the same treatment 1 year by recording capsule length, width and abortion. were then pooled and dispensed into 1.5 mL microcentri- The breeding system study was conducted during 2006– fuge tubes. The seeds were surface sterilized in a solution 2007 and 2007–2008. Data were analyzed using general containing 5 mL ethanol (100%), 5 mL 6.0% (v/v) sodium

Table 1 Pollination treatments used to determine orchid breeding systems

Breeding system test Flowers bagged Treatment Pollen source

Control No None Open pollination Agamospermy Yes Emasculate No pollination Spontaneous autogamy Yes None Same flower Induced autogamy Yes Emasculate Same flower Artificial geitonogamy Yes Emasculate Different flower, same plant Artificial xenogamy Yes Emasculate Different population Induced xenogamy Yes Emasculate Same population, different plant

Adapted from Wong and Sun (1999) and Stewart (2007).

Fig. 1 Breeding system experiment. (a) Flower, (b) artiﬁcial removal of pollinia, (c) capsule measurement, (d) Cyrtopodium. punctatum plant with pollination bag in natural habitat at the Florida Panther National Wildlife Refuge (Collier County, FL, USA). hypochlorite and 90 mL sterile distilled water for 3 min, KOH prior to the addition of 0.8% TC agar (Phyto- followed by three repetitive 30 s rinses in sterile distilled Technology Laboratories, LLC) and autoclaving at water. 117.7 kPa for 40 min at 121°C. The autoclaved medium P723 Orchid Seed Sowing Medium was prepared from (approximately 50 mL medium/plate) was dispersed into concentrated stock solutions using the formulation devel- square 100 mm ¥ 15 mm Petri plates (Falcon ‘Integrid’ oped by PhytoTechnology Laboratories, LLC (Shawnee Petri Dishes; Becton Dickinson Woburn, MA, USA). The Mission, KS, USA) and adjusted to pH 5.8 with 0.1 mol/L bottom of each dish was divided into 36, 13 mm ¥ 13 mm

Fig. 2 Seed viability and asymbiotic germination. (a) Germinating seeds (stage 2). (b) Protocorm at stage 4 of development showing two leaves. (c) Stained and unstained seeds from tetrazolium. (d) Falcon ‘Integrid’ Petri dishes showing cells. cells (Fig. 2). Only the 16 cells in the middle of the plate Table 2 Seed developmental stages of Cyrtopodium punctatum were considered for inoculation because the cells at the (modified from Stewart et al. 2003) outer edges of the plate were more susceptible to drying. Stage Description Five of the 16 interior cells were selected randomly for sowing using a computerized random number generator. 1 Intact testa Surface-disinfected seeds were sown onto the surface of 2 Embryo enlarged, testa ruptured (= germination) the sterile germination medium using a sterile bacterial 3 Appearance of protomeristem inoculating loop. Plates were sealed with NescoFilm 4 Emergence of two first leaf primordia 5 Elongation of shoot and further development (Karlan Research Products, Santa Rosa, CA, USA) and incubated under 12/12 h light/dark (60 mmol/m2/s) pho- toperiod at 25 Ϯ 3°C. Approximately 82 seeds were sown onto each plate (mean seeds/plate = 82, mean seeds per cell = 16.4). Five replicate plates were used for each breed- tion percentages were calculated by dividing the number ing system treatment. Seed germination and protocorm of seeds in each germination and development stage by development stage percentages were recorded weekly for the total number of viable seeds in the subsample. Data 10 weeks. Seedling development was scored on a scale of were analyzed using general linear model procedures and 1–5 (Table 2; modified from Stewart et al. 2003). Germina- Waller–Duncan at a=0.05 with SAS v. 9.1. Percentage

© 2009 The Authors Plant Species Biology 24, 92–103 Journal compilation © 2009 The Society for the Study of Species Biology REPRODUCTIVE BIOLOGY OF CYRTOPODIUM PUNCTATUM 97 germination counts were arcsine transformed to normal- ize variation.

Pollinator identification Pollinator observations were conducted during the 2007 and 2008 flowering seasons on a population consisting of 15 plants. Observations took place on plants in the population that had the largest number of inflorescences during both flowering seasons (12 and 14 inflorescences in 2007 and 2008, respectively). Observations took place from 07.00 to 18.00 hours for the first 2 days (13 and 14 March 2007). When peak visitation time was identified, observations took place from 10:00 am to 4:00 pm for the remaining days (15, 23–25 March 2007). In 2008, observations took place on 18–19 March (10.00–15.00 hours) and 26 March (11.00–15.00 hours). Flower visitors were pho- tographed, collected and identified, and their behavior in Fig. 3 Percentage capsule set among seven breeding system the flowers was documented. Flower visitors capable of treatments. Data from 2006 and 2007. Six plants and 30 flowers pollinating the flowers were determined either by direct per treatment were used per year. Percentages with the same letter are not significantly different. Control (no treatment, open observation or by the detection of pollinaria on their pollination); agamospermy (emasculated flower, no pollination); bodies. The insects collected were identified at the Florida spontaneous autogamy (no treatment, pollen from the same State Collection of Arthropods (Gainesville, FL, USA). flower); induced autogamy (selfing with pollen from the same flower); artificial geitonogamy (selfing with pollen from the same plant, different flower); artificial xenogamy (pollen from Fragrance analysis different population); induced xenogamy (pollen from a plant in the same population). Two inflorescences with 30 open flowers each from two C. punctatum plants were collected from the FPNWR during the 2007 flowering season. The inflorescences were collected while still attached to the pseudobulbs to preserve spermy, spontaneous autogamy or open pollination the flowers and taken to the US Department of Agricul- (control) treatments resulted in capsule formation (Fig. 3). ture Chemistry Research Unit (Gainesville, FL, USA). The induced geitonogamy (pollen from the same flower) The inflorescences were placed into a 15 cm diameter ¥ and artificial geitonogamy (pollen from the same plant, but 40 cm tall glass volatile collection chamber. Volatiles from different flowers) treatments resulted in the produc- were collected over three time periods: 9:00–12:00 am, tion of significantly fewer capsules (14.6 and 27.1% capsule 12:00 am–5:00 pm and 5:00 pm–7:00 am. The volatile traps formation, respectively) than the artificial xenogamy were extracted with 150 mL methylene chloride. Gas (pollen from a different population) and induced chromatography-mass spectroscopy (GC/MS) analyses of xenogamy (pollen from a different plant in the same popu- the collected volatiles were carried out on an HP-6890 gas lation) treatments (48.9 and 73.9%, respectively) (Fig. 3). chromatograph coupled to an HP5973 mass spectrometer Capsule widths and lengths were significantly different (Agilent, Santa Clara, CA, USA). The GC/MS peaks of among the pollination treatments (Figs 4,5). During the interest were identified by comparing their mass spectral 2006–2007 growing season, the length of the capsules data with data from three mass spectral libraries. Only formed following induced autogamy was significantly two inflorescences could be used because very limited less than the length of the capsules produced following plant material from wild populations is available owing to the other treatments 240 and 300 days after pollination the endangered status of the species. (Fig. 5). In the 2007–2008 season, the capsule length of capsules formed following both the induced autogamy (pollen from the same flower) and artificial geitonogamy Results treatments (pollen from the same plant, different flower) were significantly less than the length of capsules formed Breeding system determination in the other treatments (Fig. 5). In addition, in the 2007– Capsule formation occurred in four out of the seven treat- 2008 season, the width of the capsules formed from the ments tested during both the 2006–2007 and 2007–2008 induced autogamy treatment was significantly less than seasons (Figs 1c,3). There was no evidence that the agamo- that in all other treatments throughout the year (Fig. 4).

Fig. 4 Effect of breeding system treatment on mean capsule Fig. 5 Effect of breeding system treatment on mean capsule width (Ϯ standard error). The interaction between the 2006–2007 length (standard error). The interaction between the 2006–2007 and 2007–2008 flowering seasons was significant. and 2007–2008 flowering seasons was significant.

Viability and asymbiotic seed germination tests and Megachile xylocopoides were only observed during the We found no differences among treatments for either seed 2007 flowering season (Table 4). Both Xylocopa micans and viability or asymbiotic seed germination. In general, seed Xylocopa virginica were frequent visitors to the flowers germination was very low (induced autogamy, 2.9%; arti- during both flowering seasons (Table 4). Flowers were ficial geitonomy, 1.9%; artificial xenogamy, 4.3%; induced observed without pollinia; however, no visitors were xenogamy, 4.7%). This was unexpected considering the observed carrying pollen to or from flowers. high seed viability observed across treatments that resulted in seed production, particularly induced autogamy (79.9%), artificial geitonogamy (67.6%), artificial Fragrance analysis xenogamy (87.2%) and induced xenogamy (79.1%; Nine fragrance compounds were identified as being Table 3). However, capsules that originated from selfing produced by C. punctatum flowers: benzaldehyde, (induced autogamy and artificial geitonogamy) had sig- myrcene, benzyl alcohol, Z-b-ocimene, E-b-ocimene, nificantly lower seed germination (2.9 and 1.9%, respec- 1,3,8-para-menthatriene, methyl salicylate, indole and tively) than seeds that originated from outcrossing E,E-a-farnesene. The relative abundance of these com- (induced xenogamy, 4.7%). pounds was recorded (Fig. 7).

Pollinator identification Discussion During the two flowering seasons (2007 and 2008), four Breeding system bee species were observed visiting the flowers of C. punctatum (Fig. 6). Apis mellifera was the most frequent visitor Our results show that C. punctatum is not capable of repro- in 2007 (53 visits in 46.5 h; Table 4). However, A. mellifera ducing autogamously and requires a pollinator for

Table 3 Seed viability, germination and development stage percentages generated from Cyrtopodium punctatum pollination treatments in the 2006–2007 ﬂowering season

Seed Total Seedling development stages (%)† Pollination treatment viability (%) germination 1234

Control‡ 0.0 0.0 0.0 0.0 0.0 0.0 Agamospermy‡ 0.0 0.0 0.0 0.0 0.0 0.0 Spontaneous autogamy‡ 0.0 0.0 0.0 0.0 0.0 0.0 Induced autogamy 79.9 ab 2.9 bc 17.9 ab 1.2 b 1.7 a 0.35 a Artiﬁcial geitonogamy 67.6 b 1.9 c 18.1 a 1.2 b 0.49 b 0.44 a Artiﬁcial xenogamy 87.2 a 4.3 ab 17.6 b 2.7 a 1.9 a 0.55 a Induced xenogamy 79.1 ab 4.7 a 17.5 b 4.0 a 1.1 ab 0.29 a

† See Table 2 for descriptions of the seedling developmental stages. ‡ Treatments produced no capsules. Six plants were used and 30 ﬂowers were used per treatment. Values with the same letter are not signiﬁcantly different (a=0.05).

Fig. 6 Bees observed visiting ﬂowers. (a) Apis mellifera (honey bee). (b) Xylocopa virginica. (c) Xylocopa micans (female). (d) Megachile xylocopoides. (e) X. micans (male).

capsule set. Given that no capsules were formed from ferent flower, 27.2%), significantly more capsules were the agamospermy or spontaneous autogamy treatments produced following artificial and induced xenogamy implemented in the field, we demonstrated that no spon- (48.9 and 73.9%, respectively; Fig. 3). Species within the taneous self-fertilization occurs. Although a few capsules Orchidaceae are predominantly self-compatible; however, were formed from induced autogamy (selfing with pollen species normally require an insect vector to facilitate from the same flower; 17.2%) and artificial geitonogamy pollen movement (Dressler 1981). This is supported in C. (selfing with pollen from the same plant, but from a dif- punctatum; spontaneous autogamy treatments resulted in

Fig. 7 Fragrance analysis. (a) Relative amounts of fragrance compounds extracted from Cyrtopodium punctatum inﬂorescences.

Table 4 Insect visitors to Cyrtopodium punctatum flowers provide no food reward to floral visitors. There are implications of having a deceit pollination strategy. Insect visitors 2007 2008 Neiland and Wilcock (1998) found that orchids that offer Xylocopa micans 28 17 food rewards (e.g. nectar) had double the probability of Xylocopa virginica 15 2 setting capsule than non-rewarding species across all geo- Apis melifera 53 0 graphical areas. In North America, the rate of capsule Megachile xylocopoides 20set was 19.5% for species that offer no nectar reward compared with 49.3% for rewarding orchids (Neiland & Total number of observation hours for 2007 and 2008 were Cyrtopo- 46.5 h and 14.2 h, respectively. Wilcock 1998). A closely related species in Brazil, dium polyphyllum, also uses a deceit pollination system. This plant mimics the yellow flowers of a reward- producing unrelated species that occurs in the same no capsule formation, but both induced autogamy and habitat. This species also has low capsule set as a result of artificial geitonogamy treatments, where pollen was low pollinator visitation (Pansarin et al. 2008). Pollinator manually moved to simulate the action of the pollinator, limitation has also been observed in other deceptive resulted in capsule formation. orchids, such as Serapias vomeracea and Pogonia japonica Other studies with epiphytic orchid species show a (Matsui et al. 2001; Pellegrino et al. 2005). degree of self-incompatibility and the need for pollinators Historically, populations of C. punctatum in Florida (Ackerman 1989; Jaimes & Ramírez 1999; Borba et al. 2001; were numerous and plants were abundant. In this situa- Lehnebach & Robertson 2004). Lehnebach and Robertson tion, a deceit pollination system with low capsule produc- (2004) and Borba et al. (2001) reported that capsules were tion was viable because the probability of capsule not produced following agamospermy and spontaneous formation was higher owing to plant abundance. How- autogamy treatments on the epiphytic orchid species that ever, populations are now small and fragmented; thus, they examined, showing dependence on a pollinator. the deceit pollination system may minimize future repro- Some degree of self-incompatibility was also found for ductive success for the species as the likelihood of cap- these species (Borba et al. 2001; Lehnebach & Robertson sules being produced is so low. The open pollination 2004). Significantly fewer capsules were formed from arti- treatment resulted in no capsule formation during both ficial selfing than from induced outcrossing. Similarly, the 2006–2007 and 2007–2008 growing seasons in the some degree of self-incompatibility exists in C. punctatum population studied. However, two capsules were formed because fewer capsules were formed from selfing than on two different plants during the 2006–2007 season in outcrossing treatments. inflorescences not used in the breeding system study, sug- Cyrtopodium punctatum uses deceit via aromatic com- gesting that natural pollination does occur, albeit at very pounds and visual signals to attract insect visitors. Our low levels. The deceit pollination system alone may not be fragrance analysis and compound identification showed a the cause of the low capsule formation at the FPNWR. A floral bouquet strategy in which a diverse array of attrac- decrease in pollinator populations may also be affecting tant compounds are produced. However, C. punctatum natural capsule set. A more detailed study on the insect

© 2009 The Authors Plant Species Biology 24, 92–103 Journal compilation © 2009 The Society for the Study of Species Biology REPRODUCTIVE BIOLOGY OF CYRTOPODIUM PUNCTATUM 101 populations of the area should be conducted, particularly Fragrance analysis because of the closeness of the populations to agricultural The compounds identified in the fragrance analysis of C. fields that continually use pesticide applications. punctatum are considered relatively common pollinator The capsule size measurements (length and width) attractants produced by many other orchid species (Kaiser taken during the 11-month maturation period in both 1993). However, two compounds identified, indole and years indicated that the induced autogamy and artificial methyl salicylate, can be associated with the Euglossini geitogamy treatments resulted in capsules that were pollination system (Williams & Whitten 1983). Although relatively smaller than capsules produced from other Florida is located outside the range of Euglossini bees, C. treatments (except for capsule length in 2006–2007). punctatum is reported to be pollinated by Euglossa bees in However, capsule size may vary from year to year owing part of its range (van der Pijl & Dodson 1966; Jeffrey et al. to significant variation in environmental factors, such as 1970; Luer 1972; Dressler 1993). An introduced species of rainfall and temperature, which can influence capsule for- Euglossa (E. viridissima) has recently become naturalized in mation and maturation over time. Our research suggests southern Florida (Skov & Wiley 2005). Pemberton and that breeding system studies should be conducted across Wheeler (2006) extracted and identified compounds col- multiple growing seasons. lected by E. viridissima in southern Florida. Although methyl salicylate and indole have been detected in many Seed viability and asymbiotic germination other Euglossini species in the tropics (Ramírez et al. 2002), these compounds were not found in the collection Tetrazolium seed viability testing showed high seed storage organs of E. viridissima in southern Florida (see viability in all breeding system treatments that formed Pemberton & Wheeler 2006). It is possible that even if E. capsules in C. punctatum. However, germination was very viridissima was found in the same location as C. punctatum, low across all treatments. This may be because of subop- it would not be attracted by the types of compounds that timal in vitro cultural conditions for asymbiotic germina- the plant produces and would not affect pollination. tion. Light may inhibit germination of C. punctatum seeds (Dutra et al. 2009). By sowing seeds and initially incubat- Conservation implications ing them in the dark, germination could be significantly improved in this species. Understanding a plant’s breeding system and pollinator diversity can help conservation and management strategies because plant populations may be greatly impacted Pollinator identification by a reduction in pollinator visitations (Kearns et al. 1998; During the 2007 flowering season, honey bees (Apis Havens 1999). For example, Sahli and Conner (2006) melifera) were the most common visitors to C. punctatum found that pollinator visitation and not pollinator effec- flowers. However, these bees were too small to remove tiveness is the main driving factor in determining pollina- pollinaria and efficiently act as pollinators. In the 2008 tion importance. In a generalized pollination system, such flowering season, honey bees were not seen visiting C. as that in C. punctatum, the pollinators that visit the plants punctatum flowers. Xylocopa bees (X. micans and X. vir- the most are most likely to be the ones responsible for ginica) were observed visiting the flowers, but removal capsule formation and not simply infrequent visitors. or deposition of pollinia was not observed during the Xylocopa bees visited the plants during both seasons and field observation period, despite being large enough to were responsible for most of the visits during 2008. fit inside the flowers. In Puerto Rico, C. punctatum is Although A. melifera was the most frequent visitor in 2007, reported to be pollinated by Xylocopa bees (Ackerman it did not visit the plants during the 2008 season. 1995). Pemberton and Liu (2008) observed two species of Observations made during the present study and by Centris bees, visiting the flowers of C. punctatum in south- biologists over the past 10 years at the FPNWR indicate east Florida. However, these pollinator observations were that very few capsules were formed by C. punctatum. Pes- conducted at Fairchild Tropical Botanical Garden and in ticide use in nearby agricultural areas is another possible Fort Lauderdale, Florida, in an artificial setting with direct cause of low capsule formation as pesticide use may plants of unknown origins. Observations conducted in a decrease pollinator populations at the study site. Similarly, garden setting do not accurately reflect the real ecological habitat degradation may affect insect populations. The links because a mixture of exotic and native plants are area has been impacted from hydrological changes that planted in unnatural arrangements causing insect pollina- have shortened the hydroperiod. Local insect population tor densities to change. Pollination observations con- dynamics should be studied for conservation purposes ducted in situ are more representative because it is where and land management in areas were C. punctatum occurs. natural conditions prevail and where future plant reintro- Our study indicates that sexual reproduction in C. punc- ductions will take place. tatum is severely depressed, suggesting that recruitment is

Plant Species Biology 24, 92–103 © 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology 102 D. DUTRA ET AL. highly unlikely. Consequently, the long-term viability/ Brown P. M. (2002) Wild Orchids of Florida. University Press of persistence of the existing population is at risk. In Florida, Gainesville. response to a likely need for a reintroduction program, Catling P. M. (1987) Notes on the breeding systems of Sacoila lanceolata (Aublet) Garay (Orchidaceae). Annals of the Missouri additional studies aimed at determining the population Botanical Garden 74: 58–68. genetic diversity and structure of the existing populations Chase M. W. & Hills H. G. (1992) Orchid phylogeny, and developing efficient asymbiotic germination proce- flower sexuality, and fragrance seeking. BioScience 42:43– dures for reintroduction are underway. Although genetic 49. diversity studies are being conducted, manual pollination Coile N. & Garland M. A. (2003) Notes on Florida’s Endangered and should be conducted within populations to ensure that Rare Plants Page, 4th edn. Botany Section No. 38. Bureau of seeds are being produced for in situ recruitment and ex Entomology, Nematology and Plant Pathology, Florida Department of Agriculture and Consumer Services, Division situ propagation for future reintroductions. of Plant Industry, Gainesville. Pemberton and Liu (2008) suggested that restoration of Dixon K. W., Kell S. P., Barrett R. L. & Cribb P. J. (2003) Orchid C. punctatum could be aided by planting Brysonima lucida, Conservation. Natural History Publications, Kota Kinabalu. a rare species in the Malpighiaceae that is restricted to Dressler R. L. (1981) Orchids Natural History and Classification. southern Florida, which attracts Centris bees. However, B. Harvard University Press, Cambridge. lucida is not naturally found growing in the big cypress Dressler R. L. (1993) Phylogeny and Classification of the Orchid region where natural populations of C. punctatum are Family. Cambridge University Press, Cambridge. Dutra D., Kane M. E. & Richardson L. (2009) Asymbiotic seed found (Wunderlin & Hansen 2004). We suggest that local germination and in vitro seedling development of Cyrtopo- recovery plans for the species be developed given the dium punctatum: a propagation protocol in an endangered high habitat variation (e.g. between Everglades National Florida native orchid. Plant Cell Tissue and Organ Culture 96: Park vs FPNWR) and fragmentation found in Florida. Rec- 235–243. ommendations should not be made solely from the rela- Fiedler P. L. & Ahouse J. J. (1992) Hierarchies of cause: toward an tively few plants in cultivation and should be based on understanding of rarity in vascular plant species. In: Fiedler P. in-depth studies that will likely affect the survival of the L. & Jain S. K. (eds). Conservation Biology: the Theory and Prac- tice of Nature Conservation, Preservation, and Management. species in the wild. Chapman and Hall, New York, pp. 23–47. Hamrick J. L. & Godt M. J. W. (1996) Effects of life history traits on Acknowledgments genetic diversity in plant species. Philosophical Transactions of the Royal Society in London 351: 1291–1298. We thank Hans Alborn (USDA) for his help with the Havens K. (1999) Pollination biology: implications for restoring fragrance analysis, Jim Wiley (Florida Department of Agri- rare plants. Ecological Restoration 17: 216–218. culture and Consumer Services) for his help with insect Hills H. G., Williams N. H. & Dodson C. H. (1972) Floral fragrances and isolating mechanisms in the genus Catasetum identification and the Florida Panther National Wildlife (Orchidaceae). Biotropica 4: 61–76. Refuge staff for logistical support in the field. Special Jaimes I. & Ramírez N. (1999) Breeding systems in a secondary thanks to Nancy Philman, Timothy Johnson, Scott Stewart deciduous forest in Venezuela: The importance of life form, and Philip Kauth. This project was supported by both the habitat, and pollination specificity. Plant Systematics and Evo- US Fish and Wildlife Service and the American Orchid lution 215: 23–36. Society. Jeffrey D. C., Arditti J. & Koopowitz H. (1970) Sugar content in floral and extrafloral exudates of orchids: pollination, myrme- cology and chemotaxonomy implication. New Phytologist 69: References 187–195. Kaiser R. (1993) The Scent of Orchids: Olfactory and Chemical Inves- Ackerman J. D. (1989) Limitations to sexual reproduction in tigations. Elsevier, Amsterdam. Encyclia krugii (Orchidaceae). Systematic Botany 14: 101–109. Kearns C., Inouye D. & Waser N. (1998) Endangered mutualisms: Ackerman J. D. (1995) An Orchid Flora of Puerto Rico and the Virgin the conservation of plant–pollinator interactions. Annual Islands. The New York Botanical Garden, Bronx. Review of Ecology, Evolution and Systematics 29: 83–112. Ames O. (1904) A Contribution to Our Knowledge of the Orchid Flora Lakon G. (1949) The topographical tetrazolium method for deter- of Southern Florida. Contributions from the Ames Botanical mining the germination capacity of seeds. Plant Physiology 24: Laboratory I. E. W. Wheeler, Cambridge. 389–394. Borba E. L., Semir J. & Shepherd G. J. (2001) Self-incompatibility, Lauzer D., St-Arnaud M. & Barabe D. (1994) Tetrazolium staining inbreeding depression and crossing potential in five Brazilian and in vitro germination of mature seeds of Cypripedium acaule Pleurothallis (Orchidaceae) species. Annals of Botany 88: 89– (Orchidaceae). Lindleyana 9: 197–204. 99. Lehnebach C. A. & Robertson A. W. (2004) Pollination ecology of Bowles M. L., Jacobs K. A., Zettler L. W. & Wilson Delaney T. four epiphytic orchids of New Zealand. Annals of Botany 93: (2002) Crossing effects on seed viability and experimental 773–781. germination of the Federal threatened Platanthera leucophaea Little R. J. (1983) A review of floral food deception mimicries (Orchidaceae). Rhodora 104: 14–30. with comments on floral mutualism. In: Jones C. E. & Little R.

J. (eds). Handbook of Experimental Pollination Biology. Van Nos- SAS Institute (2003) SAS for Windows, version 9.1. SAS Institute, trand Reinhold, New York, pp. 294–309. Cary. Luer C. A. (1972) The Native Orchids of Florida. New York Botanical Sahli H. F. & Conner J. K. (2006) Characterizing ecological Garden, New York. generalization in plant–pollination systems. Oecologia 148: Matsui K., Ushimaru T. & Fujita N. (2001) Pollinator limitation in 365–372. a deceptive orchid, Pogonia japonica,onaﬂoatingpeatmat. Singh F. (1981) Differential staining of orchid seeds for viability Plant Species Biology 16: 231–235. testing. American Orchid Society Bulletin 50: 416–418. Neiland M. R. M. & Wilcock C. C. (1998) Fruit set, nectar reward, Sipes S. D. & Tepedino V. J. (1995) Reproductive biology of the and rarity in the Orchidaceae. American Journal of Botany 85: rare orchid, Spiranthes diluvialis: breeding system, pollination, 1657–1671. and implications for conservation. Conservation Biology 9: 929– Pansarin L. M., Pansarin E. R. & Sazima M. (2008) Reproductive 938. biology of Cyrtopodium polyphyllum (Orchidaceae): a Cyrtopo- Skov C. & Wiley J. (2005) Establishment of the Neotropical orchid diinae pollinated by deceit. Plant Biology 10: 650–659. bee Euglossa viridissima (Hymenoptera: Apidae) in Florida. Partel M., Kalamees R., Reier U., Tuvi E. L., Roosaluste E., Vellak Florida Entomologist 88: 225–227. A. & Zobel M. (2005) Grouping and prioritization of vascular Stewart S. L. (2007) Integrated conservation of Florida Orchi- plant species for conservation: combining natural rarity and daceae in the genera Habenaria and Spiranthes model orchid management need. Biological Conservation 123: 271–278. conservation systems for the Americas (PhD dissertation). Pellegrino G., Gargano D., Noce M. E. & Musacchio A. (2005) University of Florida, Gainesville, FL. Reproductive biology and pollinator limitation in a deceptive Stewart S. L., Zettler L. W., Minso J. & Brown P. M. (2003) Sym- orchid, Serapias vomeracea (Orchidaceae). Plant Species Biology biotic germination and reintroduction of Spiranthes brevilabris 20: 33–39. Lindley, an endangered orchid native to Florida. Selbyana 24: Pemberton R. W. & Liu H. (2008) Potential of invasive and native 64–70. solitary specialist bee pollinators to help restore the rare van der Cingel N. A. (1995) An Atlas of Orchid Pollination: Euro- cowhorn orchid (Cyrtopodium punctatum) in Florida. Biological pean orchids. Balkema Publishers, Rotterdam. Conservation 141: 1758–1764. van der Pijl L. & Dodson C. H. (1966) Orchid Flowers: Their Pemberton R. W. & Wheeler G. S. (2006) Orchid bees don’t need Pollination and Evolution. University of Miami Press, Coral orchids: Evidence from the naturalization of an orchid bee in Gables. Florida. Ecology 87: 1995–2001. Van Waes J. M. & Debergh P. C. (1986) In vitro germination of Rabinowitz D. (1981) Seven forms of rarity. In: Synge H. (ed.). The some Western European orchids. Physiologia Plantarum 67: Biological Aspects of Rare Plant Conservation. Wiley, New York, 253–261. pp. 205–217. Vujanovic V. St, Arnaud M., Barabe D. & Thibeault G. Ramírez S., Dressler R. & Ospina M. (2002) Abejas euglosinas (2000) Viability testing of orchid seed and the promotion (Hymenoptera: Apidae) de la Región Neotropical: listado de of colouration and germination. Annals of Botany 86:79– especies con notas sobre su biología. Biota Colombiana 1: 7–118. 86. Robertson J. L. & Wyatt R. (1990a) Evidence for pollination Williams N. H. & Whitten W. M. (1983) Orchid ﬂoral fragrances ecotypes in the yellow-fringed orchid, Platanthera ciliaris. Evo- and male Euglossine bees: methods and advances in the last lution 44: 121–133. sesquidecade. Biological Bulletin 164: 355–395. Robertson J. L. & Wyatt R. (1990b) Reproductive biology of the Wong K. C. & Sun M. (1999) Reproductive biology and conser- yellow-fringed orchid, Platanthera ciliaris. American Journal of vation genetics of Goodyera procera (Orchidaceae). American Botany 77: 388–398. Journal of Botany 86: 1406–1413. Romero-González G. A. & Fernández-Concha G. C. (1999) Notes Wunderlin R. P. & Hansen B. F. (2004) Atlas of Florida Vascular on the species of Cyrtopodium (Cyrtopodinae, Orchidaceae) Plants. Institute for Systematic Botany, University of South from Florida, Greater Antilles, Mexico, Central and northern Florida, Tampa. [Cited 15 June 2009.] Available from URL: South America. Harvard Papers in Botany 4: 327–341. http://www.plantatlas.usf.edu/