Confronting Assumptions About Spontaneous Autogamy

Botanical Journal of the Linnean Society, 2009, 161, 78–88. With 4 ﬁgures

Confronting assumptions about spontaneous autogamy in populations of Eulophia alta (Orchidaceae) in south Florida: assessing the effect of pollination treatments on seed formation, seed germination and seedling

developmentboj_992 78..88

TIMOTHY R. JOHNSON1*, SCOTT L. STEWART2, PHILIP KAUTH1, MICHAEL E. KANE1 and NANCY PHILMAN1

1Plant Restoration, Conservation and Propagation Biotechnology Lab, Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611-0675, USA 2Horticulture and Agriculture Programs, Kankakee Community College, 100 College Drive, Kankakee, IL 60901, USA

Received 1 June 2009; accepted for publication 7 July 2009

The breeding system of the terrestrial orchid Eulophia alta was investigated in south Florida where it has previously been reported as an auto-pollinated species. The effect of breeding system on seed viability and germinability and seedling development was also investigated. Incidences of spontaneous autogamy in E. alta were rare at the study site, resulting in only 7.1% of observed flowers forming capsules. In addition, hand pollination resulted in significantly greater capsule formation when flowers were subjected to induced autogamy (46.4%), artificial geitonogamy (64.3%) and xenogamy at both short (pollen source 10–100 m away; 42.9%) and long (pollen source > 10 km away; 67.9%) distances. Pollen source had little effect on seed viability and germinability or seedling growth rates. However, seed resulting from spontaneous autogamy developed more slowly than seed originating from the other treatments. These data indicate that spontaneous autogamy is rare in E. alta and that naturally forming capsules may be the result of unobserved pollination events. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88.

ADDITIONAL KEYWORDS: asymbiotic germination – auto-pollination – breeding system – ﬁtness – optimal out-crossing distance – orchid – sexual reproduction.

INTRODUCTION plants is vital to conservation planning and imple- mentation. The importance of maintaining functional Pollination can be a limiting stage in the life cycles of breeding systems may be particularly relevant in flowering plants, especially for those for which fitness Orchidaceae as pollinators have played a critical role depends on pollination by insect vectors (Weekley & in the diversification and speciation of the family Brothers, 2006). Recent evidence of pollinator decline (Darwin, 1885; Dressler, 1981; Tremblay et al., 2005). attributed to anthropogenic habitat loss (Biesmeijer Orchids have also evolved many mechanisms to et al., 2006; Taki & Kevan, 2007) has drawn attention enhance out-crossing and prevent or reduce incidents from conservation and pollination biologists alike. of self-pollination, but genetically regulated self- Understanding threats to the reproductive success of incompatibility appears to be rare in the family, or at least rarely reported (see Tremblay et al., 2005). This may reflect the effectiveness with which orchid floral *Corresponding author. E-mail: timjohn@ufl.edu structures, such as pollinia and rostellae, prevent

78 © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88 BREEDING SYSTEM OF EULOPHIA ALTA 79 self-fertilization by effectively separating pollen from ment, leading to more informed decisions about the the stigma on the same flower during flower matura- status of orchid populations, conservation concerns tion and immediately after pollen removal (Dressler, and conservation management. 1981). Other structural, phenological and evolution- In contrast with the numerous examples of floral ary mechanisms that promote out-crossing without adaptations in orchids that promote out-crossing, the need for genetic incompatibility include delayed some orchid species have evolved various methods of caudicle taxis, shrinkage of pollinia after removal, ensuring pollination in the absence of pollinators. In incomplete flowers, dichogamy and attraction of pol- auto-pollination (= spontaneous autogamy) systems, linators with large foraging ranges (Dressler, 1981; pollinia are deposited on the stigmatic surface Borba & Semir, 1999; Johnson & Edwards, 2000; without a pollinator through a number of mecha- Tremblay et al., 2005; Tremblay, Pomales-Hernandez nisms, including development of powdery pollinia, & Mendez-Cintron, 2006). Evidence from nectar expansion of the pollinia, malformation of the rostel- addition studies with orchid species also indicates lum and bending of the caudicle (Gonzalez-Diaz & that the evolution of deceptive pollination strategies, Ackerman, 1988; Catling & Lefkovitch, 1989; Catling, strategies in which pollinators are attracted to 1990; Gale, 2007; Micheneau et al., 2008). Autogamy flowers with the promise of a reward without one is thought to facilitate (or to be a consequence of) being supplied, may have evolved to enhance out- range expansion into areas in which pollinators are crossing, as nectar rewards increase pollinator visita- rare or absent (Catling, 1990). Autogamy may be tion time and lead to increased self-pollination more widespread than previously thought; Fenster & (Johnson & Nilsson, 1999; Johnson, Peter & Agren, Martén-Rodriguez (2007) suggested that pollinator 2004; Cozzolino & Widmer, 2005; Jersáková & specificity and auto-pollination may have co-evolved Johnson, 2006; Jersáková et al., 2008). as adaptations to the same selection pressures: polli- Early studies of orchid pollination systems in nator limitation and the uncertainty of seed set. In Florida and elsewhere have emphasized the identifi- this scenario, auto-pollination could be a means of cation of visitors but, in many cases, do not report reproductive assurance given the ‘risky’ strategy of capsule formation data (see, for example, Dodson & specialized pollinator attraction exhibited by many Frymire, 1961; Dodson, 1962). Undoubtedly, identifi- orchid species. cation of visitors and pollinators is an important Reports of auto-pollination are not uncommon in aspect of integrated orchid conservation, as protecting Orchidaceae. Over 350 species have been reported to breeding systems is vital to the in situ persistence be autogamous in some part of their range, and of populations and species (Swarts & Dixon, 2009). estimates of the number of autogamous orchid species However, confirming successful breeding by quantify- of some regional floras, such as Ecuador and Puerto ing capsule formation and identifying potential post- Rico, are 20% or greater (Ackerman, 1985; Catling, pollination reproductive barriers, such as genetic 1990). Several species of orchids in peninsular Florida incompatibility, inbreeding depression and/or out- are reportedly autogamous (Leur, 1971; Leur, 1972; breeding depression, is equally as important. Given Goss, 1973; Dressler, 1981; McCartney, 1985; Catling, many recent reports of successful asymbiotic and 1987, 1990; Calvo, 1990). However, most reports of symbiotic culture of rare, threatened and endangered autogamy do not include investigations of capsule orchid taxa (see, for example, Stenberg & Kane, 1998; formation under experimental conditions. Such Huynh et al., 2004; Zettler et al., 2005, 2007; studies are needed to confirm that auto-pollination is Znaniecka et al., 2005; Stewart & Kane, 2006, 2007; a successful mechanism of fruit production. Dutra, 2008; Dutra et al., 2008; Kauth et al., 2008; To quantify the rates of spontaneous autogamy and Dutra, Kane & Richardson, 2009b), examining the post-pollination reproductive isolating mechanisms, effects of breeding system studies on effective capsule the pollination system of Eulophia alta (L.) Fawc. & formation, seed viability and germinability and Rendle (subfamily Epidendroideae, tribe Cymbidieae; seedling vigour could be particular illuminating in Cameron et al., 1999) was studied. This terrestrial investigations of orchid breeding systems. Although species is distributed in Africa, the neotropics and self-fertilization has been shown to decrease the seed Florida, USA. It has been reported to be auto- production and seed weight of some orchid species pollinated in Mexico, South Africa, Zambia and (Beardsell et al., 1986; Gonzalez-Diaz & Ackerman, Florida (Goss, 1973; Williamson, 1984; Catling, 1990), 1988; Peakall, 1989; Robertson & Wyatt, 1990), but its breeding system has not yet been fully studied. virtually nothing is known about how breeding Consequently, several questions remain. (1) How system affects germinability and early seedling devel- common is spontaneous autogamy in this species in a opment in orchids. Such integrated data would help single population? (2) How efficient is spontaneous to clarify possible effects of different pollination autogamy as a mode of fertilization and capsule systems on reproductive success and stand establish- formation? (3) Does out-breeding enhance capsule

flatwoods (Brown, 2005). Although not listed as state threatened, E. alta is state protected, listed by the Federal Government as a wetlands indicator species (USDA, 2009) and described as commercially exploit- able (Brown, 2005). At the study location, the Florida Panther National Wildlife Refuge (FPNWR), E. alta is locally abundant, but patchy in distribution (Stewart & Richardson, 2008). Concentrated populations are found in only five of 54 burn units on FPNWR, total- ling approximately 2000 plants (S.L. Stewart, pers. observ.). Plants are found in the understorey of pine flatwoods, especially along mowed access roads. Eulophia alta is among the largest North American terrestrial orchids. Indeterminate, unbranched racemes can be up to 2 m in height and contain over 50 flowers (although some carry as few as five flowers; Fig. 1A) that are 3.5–4.5 cm wide (Brown, 2005). Flowers open in succession and often have red hues (pink, red and maroon flowers are common). Green flowering phenotypes can also be found. The pollen of E. alta flowers is coalesced into two hard pollinia joined by a single motile stipe. The base of the pollinarium has a sticky viscidium that readily attaches to contacting objects. Flowers have no discernible scent.

DETERMINATION OF POLLINATION MECHANISM Pollination experiments following the methods of Wong & Sun (1999) were conducted at the FPNWR (Collier County, FL, USA). In order to assess the breeding system of E. alta, the effects of seven polli- Figure 1. Study of the breeding system of Eulophia alta. nation treatments (Table 1) on capsule formation, (A) Flower. Scale bar, 1 cm. (B) Inflorescence with pollina- capsule length and capsule width were examined. tor exclusion netting. (C) Infructescence. (D) Capsule. Pollinia were harvested by touching a toothpick to the Scale bar, 1.5 cm. (E) Seeds stained with tetrazolium viscidium of flower pollinaria (to which the viscidium chloride. Scale bar, 250 mm. (F) Germinated seeds at readily adheres), removing the anther cap and placing week 8. Scale bar, 250 mm. the harvested pollinia into a plastic bag for transport. Harvested pollinia were used within 8 h of collection. formation frequency? (4) Are there indications of To test for asexual fruit set (agamospermy), flowers genetic post-pollination reproductive barriers, such were emasculated and covered (bagged) with pollina- as self-incompatibility, in this species? In order to tor exclusion netting (2 mm2 mesh fabric; Fig. 1B). examine these questions, several experiments were The ability of flowers to transfer pollinia to the stigma designed with the objective of describing the pollina- without a pollination vector (spontaneous autogamy) tion mechanisms of E. alta in south Florida. In addi- was tested by bagging flowers with their pollinia left tion, pollination treatment effects on seed viability, in place and without hand pollination. Within-flower germination and early seedling development were self-incompatibility was tested for by emasculat- also examined. ing flowers and pollinating them with their own pollinia (induced autogamy) before bagging. Self- incompatibility among flowers of the same inflores- MATERIALS AND METHODS cence was tested for by pollinating flowers with STUDY SPECIES pollinia from another flower on the same inflorescence Eulophia alta (common name: wild coco; Fig. 1A) is a (induced geitonogamy) before bagging. The effective- terrestrial orchid found in Africa, the West Indies and ness of outbreeding at great distances on capsule the American tropics and subtropics. The occurrence formation (artificial xenogamy) was examined by of E. alta in the USA is restricted to south Florida, emasculating flowers and pollinating them with pol- where it is found in moist to saturated soils of pine linia harvested from plants > 10 km from the study

Table 1. Pollination treatments tested in the study of the pollination mechanism of Eulophia alta

Treatment Pollinators Pollinia number Pollination treatment excluded removed Pollen treatment Assessment

1 Control (open pollination) No No No pollination Capsule set under natural conditions 2 Agamospermy Yes Yes No pollination Frequency of non-sexual capsule set 3 Spontaneous autogamy Yes No No pollination Need for pollinators for reproduction 4 Induced autogamy Yes Yes Same flower Self-compatibility of flowers 5 Artificial geitonogamy Yes Yes Different flower on Self-compatibility of plants same plant 6 Artificial xenogamy Yes Yes Flower from a distant Potential for out-breeding population at long distances (pollen source > 10 km) 7 Induced xenogamy Yes Yes Distant plant in the Potential for out-breeding same population at short distances (pollen source 10–100 m)

site before bagging. The effect of out-crossing at short was analysed using general linear modelling and distances (induced xenogamy) was examined by pol- Waller–Duncan mean separation (a = 0.05). All data linating emasculated flowers with pollinia collected were analysed using SAS v.9.1 (SAS Institute). from plants at a distance of 10–100 m away from manipulated plants. Fourteen inflorescences from 14 individual plants SEED COLLECTION, STORAGE AND GERMINATION were selected with at least seven open, unpollinated In order to examine the effect of pollination treatment flowers. Each inflorescence received all seven previ- on seed viability, germinability and seedling develop- ously described pollination treatments. Treatments ment, capsules formed in 2006 as the result of the were randomly assigned to seven flowers on each pollination treatments were collected after 12 weeks inflorescence. Senesced lower flowers were removed and dried over silica gel desiccant at room tempera- as they were encountered and 0–5 flowers immedi- ture until the capsules dehisced. At least one capsule ately above manipulated flowers were removed as was collected from six of the pollination treatments. needed for the attachment of pollinator exclusion Seed from each capsule was stored separately over netting. Upper flowers that opened later in the season silica gel desiccant at room temperature for up to 2 were not removed. For each treatment, n = 14 for each months prior to experimentation. For each of the of two study years, the first beginning on 9 October treatments that produced more than one capsule, 2006 and then again on 11 November 2008. Potential 5.0 mg samples from all capsules were collected and pollinators were excluded by securing 2 mm2 mesh pooled to create a proportional representative sample. fabric around all flowers, except those in treatment 1 Approximately 5.0 mg of bulked seed from each treat- (Fig. 1B), the open pollination control. Capsules ment was surface sterilized for 45 s in a solution of (Fig. 1C, D) were examined 3, 6 and 12 weeks after 100% ethanol–6.0% NaOCl–sterile distilled deionized pollination treatments were initiated. water (1:1:20), and then rinsed three times in Inflorescences were used as block treatments. This sterile distilled water. design was chosen because of the limited number of PhytoTechnology Orchid Seed Sowing Medium flowering individuals and to avoid possible effects of (P723) was used for asymbiotic germination. This has individual plant genotypes on the results. Logistic been shown previously to support germination and regression was used to assess the effect of pollination early seedling development of E. alta (Johnson et al., treatment on capsule formation using proc glimmix in 2007). The medium was adjusted to pH 5.7 with 1.0 M SAS v.9.1 (SAS Institute, 2003). Only capsules that NaOH and autoclaved for 40 min at 121 °C and developed to at least week 11 were used in this 117.1 kPa. Sterilized medium was dispensed as analysis. Least-squares means were used for mean 30 mL aliquots into Petri plates (diameter, 9.0 cm). separation (a = 0.05). The effect of pollination treat- Approximately 30 surface-sterilized seeds from each ment on capsule dimensions (length and diameter) pollination treatment were sown separately onto

Table 2. Developmental stages used to score Eulophia alta seed germination and seedling development

Stage Description

0 Hyaline embryo, testa intact 1 Embryo swollen, rhizoids present (= germination) 2 Continued embryo enlargement, testa ruptured 3 Appearance of protomeristem 4 Emergence of ﬁrst leaf

eight replicate Petri plates. The plates were then sealed with a single layer of Nesco film (Karlan Research Products Corp.), wrapped in two layers of aluminium foil to exclude light and incubated at 22±3°Cfor12weeks. Seeds were exposed to short periods of light (less than 30 min) during data collection. Germination and seedling development was scored on a scale of 0–5 (Table 2) with a dissecting microscope after 4, 8 and 12 weeks of culture. The percentage seed and seedlings in each developmental stage and the percentage germination were calculated. Germination and developmental data were arcsine transformed and analysed using general Figure 2. Effect of pollination treatment on capsule for- linear modelling and Waller–Duncan mean separa- mation 10 weeks after pollination treatments. Data from tion (a = 0.05). two study years are combined. Error bars represent standard error of means for each treatment. Means with the same letters are not significantly different at P > 0.05. SEED VIABILITY TESTING Tetrazolium chloride staining was used to assess seed mentation began was confirmed by observing frost- viability by pretreating three replicates of 100–200 damaged capsules at week 12. Undamaged capsules seeds from each pollen treatment with 0.5% calcium were used in the analysis of the effect of pollination hypochlorite for 3 h. The seed was then rinsed three on capsule dimensions. times in sterile distilled deionized water and soaked Statistical analysis indicated that pollination treat- for 24 h in darkness at 22±2°C.Theseed was then ment had a significant effect on capsule formation soaked for 24 h in 1.0% tetrazolium chloride at (F6,6 = 6.29, P = 0.02). Data on the effect of pollination 30±2°Cin darkness. The percentage viability was treatment on capsule formation from both observation calculated for each replicate by dividing the number years were combined because significant differences of pink/red-stained embryos (viable; Fig. 1E) by the were not observed between years (F1,6 = 0.28, total number of embryos counted. Three replications P = 0.62). All pollen treatments resulted in capsule were performed for each treatment and this experi- formation in 2006, but capsules that formed from ment was repeated once (n = 6). Statistical analysis spontaneous agamospermy aborted before 6 weeks was conducted as described for the germination data. of development. Similarly, the spontaneous agamospermy treatment resulted in only a single capsule being formed in 2008. Overall, agamospermy only RESULTS produced a single mature capsule (Fig. 2; 3.6% ± POLLINATION SYSTEM AND CAPSULE FORMATION 3.6%; mean ± 3.6%). Open pollination resulted in In 2008, a late frost destroyed developing capsules on 14.3% ± 6.7% capsule formation and was not signifi- all but five plants 11 weeks after the initiation of cantly different from the observed capsule formation experimentation. All capsules resulting from agamo- from spontaneous autogamy (7.1% ± 5.0%) or agamospermy (1) and artificial xenogamy (4) were damaged, spermy. A greater proportion of capsules formed from as well as six of seven induced autogamy capsules, induced xenogamy (42.9% ± 9.5%) than spontaneous seven of 10 artificial geitonogamy capsules and seven autogamy or agamospermy. Rates of capsule forma- of nine artificial xenogamy capsules. The presence of tion resulting from induced xenogamy, induced auto- capsules at the time of frost 11 weeks after experi- gamy (46.4% ± 9.6%), geitonogamy (64.3% ± 9.2%)

Figure 3. Effect of pollination treatment on Eulophia alta capsule length (A) and diameter (B). Data from two study years are combined. The data for agamospermy (*) at week 6 are for the length and width of a single capsule (a single capsule formed in 2006, but aborted after week 3 and a single capsule that formed in 2008 was destroyed by frost). and artificial xenogamy (67.9% ± 9.0%) were not significantly different among treatments at 6 and 12 significantly different. weeks (Fig. 3A). At week 3, the capsule diameters of The study year did not have a significant effect on the agamospermous treatment were not statistically capsule length or capsule diameter at week 3 (length: siginificantly different from those of capsules result-

F1,72 = 0.00; P = 0.99; diameter: F1,72 = 1.62; P = 0.21) ing from spontaneous autogamy treatment, but were or week 12 (length: F1,72 = 1.13; P = 0.30; diameter: signiﬁcantly lower than those of all other treatments.

F1,72 = 1.33; P = 0.26). Signiﬁcant differences were The diameters of capsules resulting from different detected between study years during week 6 obser- pollination treatments were not signiﬁcantly different vations (length: F1,57 = 15.32; P = 0.00; diameter: at any time examined (Fig. 3B).

F1,57 = 26.31; P = 0.00). However, when the capsule length and capsule diameter data for each study year were analysed separately, the same result was EFFECT OF POLLINATION TREATMENT ON SEED obtained for both study years; no signiﬁcant differ- VIABILITY AND EARLY SEEDLING DEVELOPMENT ences were detected among treatments in 2006 In 2006, seed was generated from all pollination

(length: F6,30 = 0.60; P = 0.70; diameter: F6,30 = 0.46; treatments except agamospermy. Seed viability was

P = 0.80) or 2008 (length: F6,27 = 0.88; P = 0.52; not signiﬁcantly affected by pollination treatment diameter: F6,27 = 0.66; P = 0.68) at week six. Because (F5,30 = 0.57; P = 0.57) and ranged from 88.2% ± 2.4% of these results, capsule dimension data for both to 92.7% ± 1.8%. Pollination treatment had a signiﬁ- study years were combined. cant effect on germination after 12 weeks of culture

Pollination treatment did not have a signiﬁcant (F5,37 = 2.88; P = 0.03) and ranged from 94.7% ± 1.4% effect on capsule dimensions at weeks 3 (length: to 99.3% ± 0.5% (Fig. 4). At week 4, most seeds were

F6,72 = 0.92; P = 0.48; diameter: F6,72 = 0.54; P = 0.77), 6 observed at stage 1 (swollen embryo; 74.2% ± 3.4% to

(length: F6,72 = 1.33; P = 0.26; diameter: F6,30 = 0.65; 86.8% ± 2.4%) and were considered to have germi-

P = 0.69) or 12 (length: F6,33 = 0.82; P = 0.55; diameter: nated. Pollination treatment had a signiﬁcant affect

F6, 33 = 1.00; P = 0.43). Capsule lengths were not on the percentage of seedlings at stage 2 at this time

Figure 4. Effect of pollination treatment on Eulophia alta seed germination and seedling development after 4, 8 and 12 weeks culture. Seed and seedling stages are reported in Table 2. Capsules resulting from the 2006–2007 pollination experiment were used. Error bars represent the upper limit of the standard error. Treatments within each stage with different letters are signiﬁcantly different (a = 0.05).

(F5,37 = 3.08; P = 0.02); significantly fewer seeds from autogamy treatment had developed to stage 3 (7.3% ± the spontaneous autogamy treatment had developed 2.5%) compared with other treatments. Statistically to stage 2 (6.8% ± 2.5%) than all other treatments, significant differences were not found among treat- except for artificial geitonogamy (11.5% ± 2.5%). At ments at stages 1 (F5,37 = 0.86; P = 0.51), 2 (F5,37 = week 8 (Fig. 1F), pollination treatment had a signifi- 0.48; P = 0.79) or 4 (F5,37 = 0.86; P = 0.51) after 8 cant effect on the percentage of seeds/seedlings at weeks of culture. After 12 weeks of culture, pollina- stage 0 (F5,37 = 6.41; P = 0.00) and stage 3 (F5,37 = 7.85; tion treatment was found to have a significant effect

P = 0.00). Signiﬁcantly more nongerminated seeds on the percentage of seedlings at stages 0 (F5,37 = 3.10;

(stage 0) were observed from spontaneous autogamy P = 0.02), 2 (F5,37 = 7.10; P = 0.00), 3 (F5,37 = 5.96;

(6.7% ± 1.7%) and artiﬁcial autogamy (6.7% ± 1.3%) P = 0.00) and 4 (F5,37 = 38.20; P = 0.00), but not at treatments. Fewer seeds from the spontaneous stage 1 (F5,37 = 0.37; P = 0.86). At this time, signiﬁ-

© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88 BREEDING SYSTEM OF EULOPHIA ALTA 85 cantly fewer seedlings from the spontaneous auto- ing have been reported for other orchid genera. Seed gamy treatment were observed at stage 4 (7.2% ± germination of Dactylorhiza sambucina (L.) Soó and 1.2%) compared with other treatments. Seed gener- the subsequent survival of reintroduced plants pro- ated from the spontaneous autogamy treatment also duced from induced autogamy were lower than that of had signiﬁcantly lower seedling development than seeds and seedlings produced from out-crossing polli- other treatments, as evidenced by a signiﬁcantly nation treatments (Juillet, Dunand-Martin & Gigord, higher percentage of seedlings at stage 3 (56.2% ± 2007). Induced autogamy also decreased the seed

4.1%; F5,37 = 5.96; P = 0.00). viability of Barlia robertiana (Loisel.) Greuter and Anacamptis morio (L.) R.M.Bateman, Pridgeon & M.W.Chase and reduced seed weight and the number DISCUSSION of seeds with embryos of Platanthera ciliaris Lindl. Eulophia alta infructescences with abundant capsules (Robertson & Wyatt, 1990; Smithson, 2006). are often observed at FPNWR (T.R. Johnson pers. Most pollination treatments did not have a signifi- observ.; Fig. 1C, D). Goss (1973) reported that, soon cant effect on seed viability of E. alta. In all pollination after flowers open, the caudicle of the pollinarium of treatments, tetrazolium chloride staining under- south Florida E. alta plants bends forwards bringing estimated the observed germination by as much as the pollinia into contact with the stigma. However, 10.8% (artificial geitonogamy: estimated, 88.2% ± this mechanism was not observed during the course of 2.4%; observed, 99.0% ± 0.7%), but provided a good our study. Given previous reports of an autogamous relative estimate of germinability among treatments. breeding mechanism of E. alta in Florida (Goss, 1973) This difference in tetrazolium chloride-estimated via- and the lack of documented E. alta pollinators in bility and germinability highlights the need to perform North America, there has been little reason to inves- tests of both seed respiration and germinability. tigate the pollination biology of this species. However, One consequence of using a blocked design in the the low success rate of induced autogamy in forming current study was the inability to control for the capsules during this investigation indicates that field- possible selective abortion of low-quality fruits. It is observed capsules could be the result of unobserved possible that low capsule formation in spontaneous pollinator visitation. autogamous treatments is reduced when higher Further evidence of the scarcity or lack of auto- quality pollen is available for fertilization (Bookman, pollination in E. alta at the study site comes from the 1984). However, the observations that glasshouse- observation that glasshouse-grown plants do not grown plants do not form capsules without hand produce capsules unless pollinated by hand (T.R. pollination suggests that resource limitation does not Johnson, pers. observ.). Discrepancies between this regulate spontaneous autogamy. In addition, self- report and previous reports of autogamy in E. alta pollination resulted in a high degree of capsule for- could be a result of differences in the frequency of mation and vigorous seed on tested plants. These auto-pollination of E. alta throughout its range in data indicate that E. alta populations at FPNWR are Florida, although this requires further study. Capsule not autogamous, as has been reported previously for formation in other auto-pollinated orchid species is the species in Florida (Goss, 1973; Catling, 1990). typically much more efficient than observed for E. Whether autogamy of E. alta is common throughout alta (Tremblay et al., 2005). The fruit set of Nervilia its range or limited to certain populations also nipponica Makino under pollinator exclusion netting remains to be studied. was 93% and that of Jumellea stenophylla Schltr. was Capsule formation resulting from induced auto- 66.7%–83.9% (Gale, 2007; Micheneau et al., 2008). gamy in self-compatible orchids can be highly effec- This is to be expected given the strong selection tive: 85% in Listera ovata (L.) R.Br. and 71.9–90% in pressure that pollinator absence would impose. Platanthera ciliaris at two study sites (Ackerman & Seed that formed from the autogamous treatment Mesler, 1979; Robertson & Wyatt, 1990). Although in 2006 exhibited a slower rate of germination and induced autogamy in E. alta resulted in fewer cap- seedling development compared with seed resulting sules than did artificial geitonogamy and induced from other pollination treatments. This is an indica- xenogamy, the means were not statistically different. tion that rare spontaneous autogamy may produce These data indicate that there are no significant seed of lower fitness than other pollination treat- reproductive barriers to within-flower self- ments. It is possible that the difference in capsule fertilization for E. alta in Florida. However, the trans- formation and seedling development between sponta- fer of pollinia to stigma may still be vital to capsule neous autogamy and induced autogamy treatments is formation, given the significant difference in capsule a consequence of pollination with aged pollen (Rosell, formation between treatments that did not involve Galan Sauco & Herrero, 2006; Castro, Silveira & hand pollination (control, agamospermy and sponta- Navarro, 2008). More pronounced effects of inbreed- neous autogamy) and those that did (induced

© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161, 78–88 86 T. R. JOHNSON ET AL. autogamy, artificial geitonogamy, artificial xenogamy orchid conference. Miami, FL: International Press Co Ltd., and induced xenogamy). 98–101. Pollination by induced autogamy did not signifi- Ackerman JD, Mesler MR. 1979. Pollination biology of cantly reduce seed viability, germination or the devel- Listera cordata (Orchidaceae). American Journal of Botany opment of seeds and seedlings relative to other pollen 66: 820–824. supplementation treatments. Agamospermy appears Beardsell DV, Clements MA, Hutchinson JF, Williams to be a rare event in this species as no capsules EG. 1986. Pollination of Diuris maculata R Br (Orchi- formed from emasculated flowers in 2006 and a single daceae) by floral mimicry of the native legumes Daviesia spp and Pultenaea scabra RBr.Australian Journal of Botany capsule formed in this treatment in 2008. Although 34: 165–173. only visibly unpollinated flowers were used during Biesmeijer JC, Roberts SPM, Reemer M, Ohlemuller R, the experimental set-up, undetected pollination of Edwards M, Peeters T, Schaffers AP, Potts SG, some flowers could have occurred. Agamospermy Kleukers R, Thomas CD, Settele J, Kunin WE. 2006. appears to be rare in E. alta and is not expected to Parallel declines in pollinators and insect-pollinated plants contribute to viable seed production. in Britain and the Netherlands. Science 313: 351–354. The hypothesis of insect-mediated pollination of E. Bookman SS. 1984. Evidence for selective fruit production in alta in Florida may be speculative, but there is evi- Asclepias. Evolution 38: 72–86. dence that plants could be pollinated by an uniden- Borba EL, Semir J. 1999. Temporal variation in pollinarium tified vector. Comparison with other Eulophia spp. size after its removal in species of Bulbophyllum: a different suggests that E. alta could be pollinated by beetles (as mechanism preventing self-pollination in Orchidaceae. is Eulophia foliosa Bolus) or Xylocopa bees (as are E. Plant Systematics and Evolution 217: 197–204. cristata Lindl. and E. rosea (Lindl.) A.D.Hawkes [= E. Brown PM. 2005. Wild orchids of Florida. Gainesville, FL: horsfallii (Bateman) Summerh.]) (Kullenberg, 1961; University Press of Florida. Lock & Profita, 1975; Peter & Johnson, 2006). Scarce Calvo RN. 1990. Inflorescence size and fruit distribution pollinators have been linked to poor capsule forma- among individuals in three orchid species. American tion in Cyrtopodium punctatum (L.) Lindl., an orchid Journal of Botany 77: 1378–1381. found in the vicinity of E. alta at FPNWR. 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