Skip Navigation


AOBPreview originally published online on September 28, 2006
Annals of Botany 2006 98(6):1207-1213; doi:10.1093/aob/mcl202
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
98/6/1207    most recent
mcl202v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Agricola
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Low Genetic Structure in an Epiphytic Orchidaceae (Oncidium hookeri) in the Atlantic Rainforest of South-eastern Brazil

SUZANA ALCANTARA1,*, JOÃO SEMIR2 and VERA NISAKA SOLFERINI1

1 Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas Caixa Postal 6109, Campinas, SP, 13083-970, Brazil
2 Departamento de Botânica, Instituto de Biologia, Universidade Estadual de Campinas Caixa Postal 6109, Campinas, SP, 13083-970, Brazil

*For correspondence. E-mail suzanaalcantara{at}yahoo.com.br

Received: 10 May 2006    Returned for revision: 5 July 2006    Accepted: 7 August 2006    Published electronically: 28 September 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

• Background and Aims Oncidium hookeri is a neotropical species of epiphytic Orchidaceae found in the Brazilian Atlantic rainforest at the top of the Mantiqueira Range of mountains. The genetic variation of O. hookeri was studied to assess the distribution of genetic variability within and among six populations localized in Atlantic rainforest remnants. Gene flow among populations and the occurrence of recent bottlenecks were investigated in order to infer the degree of isolation of these populations.

• Methods Thirteen polymorphic loci were used for allozyme electrophoresis. The data were analysed by means of standard statistical approaches, to estimate gene diversity and the genetic structure of the populations.

• Key Results The mean gene diversity and allelic richness were He = 0·099 and A = 1·75, respectively. F-statistics revealed high heterozygote deficiencies in all populations (FIS = 0·43–0·82). Several rare alleles were found in all the populations, and three populations presented private alleles. Low genetic differentiation among O. hookeri populations was detected (FST = 0·029); natural selection may be involved in PGM locus differentiation among populations. The genetic differentiation between paired populations was low, bearing no correlation with geographic distance (Mantel test: r = –0·34, P = 0·72). Only two populations showed signs of recent bottlenecks.

• Conclusions The heterozygote deficiency found seems to be caused by pollinator behaviour; the low frequencies of several alleles of different loci can be maintained due to clonal propagation. Despite the stochastic nature of the wind-dispersal of seeds to long distances, this process may promote an effective gene flow among populations, thus avoiding genetic differentiation.

Key words: Orchidaceae, Oncidium hookeri, allozyme, genetic variability, genetic structure, tropical Atlantic rainforest, wind-dispersed seeds, vegetative propagation, insect-pollination


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
During the last two decades, several studies have concentrated on the distribution of genetic variability in orchid populations (see Hanrick and Godt, 1996; Forrest et al., 2004). This family represents an excellent example for adaptive radiation and rapid diversification, facilitating research on a myriad of ecological and evolutionary aspects (Nilsson, 1992; Dressler, 1993; Tremblay et al., 2005). In this context, the evolution of floral morphology, the dynamics of plant–pollinator interactions, the effects of habitat fragmentation on the genetic diversity of species, and the effectiveness of gene flow in natural conditions are some of the relevant points highlighted (Sun, 1996; Ackerman and Ward, 1999; Wong and Sun, 1999; Tremblay and Ackerman, 2001; Trapnell and Hamrick, 2005; Tremblay et al., 2005).

In general, breeding systems are regarded to be the main factor in determining the organization of genetic variability in plant populations (Loveless and Hamrick, 1984; Lande and Shemske, 1985). In orchids, the population level of genetic structure is greatly influenced by pollinator behaviour and, under natural conditions, the fruit set is predominantly pollinator-dependent (Neiland and Wilcock, 1998; Cozzolino and Widmer, 2005; Tremblay et al., 2005). In some cases, seeds may be even more important than pollen for interpopulational gene flow (Peakall and Beattie, 1991; Rasmussen, 1995; Cozzolino et al., 2003; Brzosko et al., 2004). In general, it is predicted that Orchidaceae seeds, which are very small and light, can be wind-dispersed over long distances (Dressler, 1981, 1993; Ackerman and Ward, 1999; Chung et al., 2004), promoting genetic homogeneity among populations. However, recent studies have shown that most orchid seeds settle down near to the maternal plant (Murren and Ellison, 1998; Machon et al., 2003), causing a spatial genetic structure on a small scale (among individuals within populations) (Peakall and Beattie, 1996; Chung et al., 2004; Trapnell et al., 2004).

Even though most species (approx. 70 %) of Orchidaceae are epiphytic (Dressler, 1993), data on genetic diversity are biased towards terrestrial species. Some studies have shown high genetic diversity and low differentiation in populations of Brazilian rupicolous orchids; these findings are thought to be the consequence of self-incompatibility and seed dispersal by wind in an open environment (Borba et al., 2001). It has been proposed that the gene flow of epiphytes could be more susceptible to environmental changes than other species due to the habitat, patchy distribution and specific pollination strategies of these species (Ackerman and Ward, 1999; Tremblay and Ackerman, 2001; González-Astorga et al., 2004; Trapnell et al., 2004). Furthermore, changes in pollinator dynamics affect small populations restricted to remnants of vegetation more intensely than they affect large ones (Ellstrand and Ellam, 1993; Young et al., 1996; Lande, 1999; Oostermeijer et al., 2003).

In this study, the genetic variability of Oncidium hookeri populations in an Atlantic rainforest area of south-eastern Brazil is analysed. Oncidium is a neotropical orchid genus, including more than 600 epiphytic, terrestrial and rupicolous species (Chase, 1986; Dressler, 1993). The pollinators reported are several genera of native bees (Apidae, which gather oils produced by specialized glands); however, fruit setting in the field is rare, despite the reward offered by the flowers (Singer and Cocucci, 1999; Reis et al., 2000; Singer, 2003). Although uncommon in orchids, there is a suggestion that the Subtribe Oncidiinae is self-incompatible, due to frequencies of abortion in geitonogamic breeding (i.e. between flowers of the same individual) (Singer and Koehler, 2003). A large number of Oncidium populations are currently restricted to remnants of the tropical Atlantic rainforest and the aim of the present study was to determine whether the populations of O. hookeri in these vegetation fragments are genetically structured. To do this, inbreeding and intrapopulational genetic variability of six populations of Oncidium hookeri were estimated, and the genetic differentiation and gene flow among these populations evaluated. The genetic variability of the species was assessed by means of allozyme electrophoresis. The results may highlight the processes that influence the distribution of genetic variability in a tropical epiphytic orchid.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Species studied
Oncidium hookeri Rolfe is a perennial, long-lived, small, epiphytic orchid, occurring in the mountains of the south-eastern and southern regions of Brazil. Species identification followed the criteria of Pabst and Dungs (1977). The leaves are inserted into pseudobulbs, and the inflorescence is lateral, ramified, with a high number of flowers. The pendant inflorescence possesses 15–30 small flowers (approx. 1 cm) with yellow petals and elaiophores (glands which produce floral lipids). Floral morphology, lipid-oil production (gathered by several genera of Apidae) and field observations have shown that Oncidium species are pollinated mainly by native bees (Singer and Coccuci, 1999; Reis et al., 2000; Singer, 2003). However, the fruits are rare because the behaviour of the pollinators promotes some degree of geitonogamy, and the self-incompatibility (abortion) may be responsible for the low pollination success in this group (Singer, 2003; Singer and Koehler, 2003). Oncidium species can also grow clonally through pseudobulbs, which are capable of maintaining clones for many generations.

Sampling sites
Specimens were collected in six rainforest fragments located in the Southern Mantiqueira Range in Minas Gerais state (S22°28'16''/W46°08'42'') (Fig. 1). The area is situated above 1400 m a.s.l., and was originally characterized as evergreen vegetation (Rizzini, 1979). The region's climate is classified as Cwb (Köppen, 1948), where the mean temperature of the hottest month is below 22 °C. The area contains a high number of forest fragments of various sizes in an agricultural matrix. In general, the fragments are isolated and occur at the top of the mountains.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1 Geographical locations of Oncidium hookeri populations studied: Pop. 1, ‘Mata dos Damázios'; Pop. 2, ‘Mata Campo Alegre’; Pop. 3, ‘Mata dos Garcias’; Pop. 4, ‘Mata Alto Caxambu’; Pop. 5, ‘Morro Alto do Serro’; Pop. 6, ‘Pedreira dos Garcias’.

 


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2 Allele frequencies of PGM loci in six populations of Oncidium hookeri. A, B, C and D represent the four different alleles. Pop. 1, ‘Mata dos Damázios’; Pop. 2, ‘Mata Campo Alegre’; Pop. 3, ‘Mata dos Garcias’; Pop. 4, ‘Mata Alto Caxambu’; Pop. 5, ‘Morro Alto do Serro’; Pop. 6, ‘Pedreira dos Garcias’.

 
A ramet from each individual was collected and cultivated in the greenhouse of the Genetics and Evolution Department of the State University of Campinas, from September 2002 to February 2003. Special care was taken in order to collect individuals isolated in different trees to avoid sampling clones. At least 17 individuals from each fragment were collected and considered as one sample (Pop. 1, 24 individuals sampled; Pop. 2, 18; Pop. 3, 21; Pop. 4, 26; Pop. 5, 28; Pop. 6, 17).

Allozyme electrophoresis
A small portion of fresh leaf tissue (approx. 5 mg) was crushed in 0·5 mL of grinding buffer [0·1 mol L–1 Tris, 0·2 mol L–1 sucrose, 0·6 % PVP (w/v), 1 mmol L–1 EDTA, 0·15 % BSA, 0·03 mol L–1 DIECA, 0·03 mol L–1 sodium tetraborate and 0·2 % mercaptoethanol; pH 7·0], modified from Sun and Ganders (1990). The extracts were absorbed in pieces of Whatman paper no. 3 and loaded onto an 8·5 % starch gel (Sigma). Different buffer systems and running conditions were employed: System 1, 0·026 mol L–1 LiOH, 0·095 mol L–1 boric acid and 0·0033 mol L–1 EDTA, with electrode diluted 1 : 10, 15 mA; System 2, 0·04 mol L–1 citric acid, pH 6·1 adjusted with N-(3-aminopropyl)-morpholine and electrode diluted 1 : 20 (Clayton and Tretiak, 1972), 20 mA; System 3, 0·047 mol L–1 LiOH, 1·91 mol L–1 boric acid, pH 8·4, and electrode of 0·008 mol L–1 citric acid, Tris 0·015 mol L–1, pH 8·1, diluted 1 : 20, 24 mA.

Standard electrophoreses were performed until the inner marker (bromophenol blue) reached 8 cm from the application site. The gels were stained for 11 enzymatic systems, according to Alfenas et al. (1991): System 1 was used to analyse alcohol dehydrogenase (ADH1, ADH2 and ADH3; EC 1.1.1.1 [EC] ), diaphorase (DIA; EC 1.8.1.4 [EC] ), malic enzyme (ME; EC 1.1.1.40 [EC] ), phosphoglucoisomerase (PGI; EC 5.3.1.9 [EC] ) and phosphoglucomutase (PGM1; EC 5.4.2.2 [EC] ). System 2 was used to analyse glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC 1.2.1.12 [EC] ), isocitrate dehydrogenase (IDH; EC 1.1.1.42 [EC] ), malate dehydrogenase (MDH; EC 1.1.1.37 [EC] ) and shikimate dehydrogenase (SKDH; EC 1.1.1.25 [EC] .). System 3 was used to analyse fumarase (FUM; EC 4.2.1.2 [EC] ) and glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49 [EC] ). Alleles were identified by their mobility in relation to the alleles of the same Oncidium hookeri reference individual applied in all gels.

Data analysis
Allele frequencies were determined by visual interpretation of gels. The genetic variability for each population was estimated according to the mean number of alleles per locus (A), the proportion of polymorphic loci (P: 0·95 criterion), and the observed (Ho) and expected (He) mean heterozygosity per locus. Analyses were performed using the BIOSYS 1.0 software package (Swofford and Selander, 1981).

Departures from frequencies expected under Hardy–Weinberg equilibrium were tested by randomization using the software Genetix (Belkhir et al., 1996–2001). The FIS values obtained were compared using a {chi}2-test. F-statistics (Wright, 1978) were calculated in order to determine the levels of allelic variation within and among populations of O. hookeri. The confidence interval of F-coefficients was estimated through bootstrap procedures (Manly, 1997). Pairwise FST were estimated according to Weir and Cockerham (1984) for six populations of O. hookeri and the confidence interval calculated with 5000 permutations using the Fstat software (Goudet, 1995). A Mantel test (Mantel, 1967) was used to test the correlation between pairwise FST and geographical distance matrices with Genetics software.

A bottleneck test (Cornuet and Luikart, 1996) was carried out to determine signs of excess expected heterozygosity (He) in relation to heterozygosity expected under mutation-drift equilibrium (Heq).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Genetic variability within populations
The genetic variation observed in O. hookeri was high. The mean number of alleles per locus was 1·75 [s.d. = 0·1], whereas the percentage of polymorphic loci was between 15·4 and 61·5 (Table 1). The allelic frequencies did not fit those expected by the Hardy–Weinberg equilibrium (P < 0·05), with a much lower observed heterozygosity (Ho = 0·034, s.d. = 0·01) than expected (He = 0·099, s.d. = 0·02); FIS values ranged from 0·43 to 0·824.


View this table:
[in this window]
[in a new window]

  		TABLE 1 Genetic variability in six populations of Oncidium hookeri

 
Four loci were monomorphic in all populations, and most showed one allele with a frequency of near to 1·0 (Table 2). The number of alleles in polymorphic loci ranged from two to four. Among the six populations, Pop. 2 and Pop. 6 (the populations with the lowest number of individuals found in the field) showed signs of recent bottlenecks (P-values: 0·048 and 0·029, respectively).


View this table:
[in this window]
[in a new window]

  		TABLE 2 Allelic frequencies of 13 loci for six populations of the epiphyte orchid Oncidium hookeri

 
Genetic structure and gene flow among populations
Estimated FIS values were high for most loci (Table 3), and a great heterogeneity of values per locus among populations was observed. A low, but significant, genetic structure was observed among populations (FST = 0·029). According to Wright (1978), the FST of PGM locus indicates a moderate structure (0·073; Table 3).


View this table:
[in this window]
[in a new window]

  		TABLE 3 F-statistics of nine polymorphic loci of six populations of Oncidium hookeri

 
Populations 1, 3 and 4 showed private alleles. However, only one in a total of six alleles had a frequency >0·05 (on SKDH locus in Pop. 3; Table 2). There was no correlation between FST values and the geographical distances matrices (Mantel test: r = –0·34, P = 0·72; Table 4). The lowest genetic differentiation (0·001) was estimated between populations 2 and 5 (5·6 km apart) and the highest differentiation (0·096) occurred between populations 3 and 6 (2·12 km apart).


View this table:
[in this window]
[in a new window]

  		TABLE 4 Genetic differentiation and geographical distance for pairwise Oncidium hookeri populations

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Genetic variability within Oncidium hookeri
There was a higher genetic variability within Oncidium hookeri compared with other plant species, be they herbaceous or perennial (Hamrick and Godt, 1990). However, the A and P values reported here are similar to those observed in other insect-pollinated orchids (Scacchi et al., 1990, 1991; Sharma et al., 2000; Borba et al., 2001; Sun and Wong, 2001; Trapnell et al., 2004). It is generally agreed that wind-dispersed seeds can reach long distances (Dressler, 1981; Ackerman and Ward, 1999), which could maintain enough gene flow to counterbalance losses due to genetic drift (Sharma et al., 2000, 2003). However, the few available studies of seed dispersal and genetic variability in orchids show a significant fine-scale structure (Peakall and Beattie, 1996; Chung et al., 2004). According to Trapnell et al. (2004), this pattern is caused mainly by highly localized (on a scale of centimetres) seed dispersal within the population. Other studies also show that, in the great majority of orchids, seeds are dispersed short distances, near to the maternal plants (Murren and Ellison, 1998; Machon et al., 2003).

In contrast with the high variability found, there were four monomorphic loci (ADH2, FUM, G6PDH and PGI), and most of the polymorphic loci showed one allele at a frequency of close to 1 and some rare alleles. The absence of allozyme diversity has already been reported in highly inbred orchids, such as the terrestrials Cephalanthera damasonium (Scacchi et al., 1991), Eulophia sinensis and Zeuxine gracilis (Sun and Wong, 2001) and the epiphyte Spiranthes hongkongensis (Sun, 1997). However, assuming the self-incompatibility observed in Oncidium species (Singer, 2003), including O. hookeri (see next paragraph), the absence of variability in four loci is surprising. Chung et al. (2004) found a similar pattern in one population of Cephalanthera longifolia, a predominantly outcrossing orchid species, and suggested that it was genetically isolated and had drifted to fixation, or had undergone a recent bottleneck. In O. hookeri, there are signs of recent bottlenecks in two out of six populations, suggesting that other processes may also be influencing genetic variability. A long generation time due to vegetative propagation, as well as the overlapping generations, can also mitigate the loss of genetic variation by drift and maintain alleles in low frequencies (Ellstrand and Rose, 1987; Gustafsson, 2000; Borba et al., 2001). During the sampling of plant material, more individuals were observed at the limits of the forest than inside, suggesting that the environmental conditions at the boundaries of the fragments (Murcia, 1995) are suitable for O. hookeri. Furthermore, tests for genetic bottlenecks, revealing a putative effect of habitat fragmentation, were significant for only two of the populations studied.

There are no previous reports on the mating system of O. hookeri. Although orchids are usually self-compatible (van der Pijl and Dodson, 1966; Dressler, 1993), hand self-pollination carried out on several individuals maintained in the greenhouse (approx. 40 flowers of different individuals) was not successful (S. Alcantara et al., unpubl. data). Additionally, fruits were rarely observed in the field, suggesting the rarity of natural matings (both cross- and self-), as was also observed in other Oncidium species (Singer and Cocucci, 1999; Singer, 2003; Singer and Koehler, 2003). The rarity of self-pollination contrasts with the high fixation indices and the heterozygote deficiencies observed. Studies on the genetic variability of orchids showed different patterns of heterozygosity, probably reflecting various life histories, pollination systems, geographical distributions and bottlenecks, as well as fragmentation events (Scacchi et al., 1990; Sun, 1996; Wong and Sun, 1999; Ehlers and Pedersen, 2000; Gustafsson, 2000; Borba et al., 2001; Sharma et al., 2003). In some cases, pollinator behaviour promotes inbreeding in plant populations (Levin and Kerster, 1969; Schmitt, 1980; Fritz and Nilsson, 1994; Kearns et al., 1998). It is well established that the foraging strategies of food-seeking pollinators can lead to high levels of pollen transfer within the same plant and mean pollen dispersal distances are typically less than several metres (Richards, 1997). The pollinators reported for Oncidium species are, in general, solitary bees (Trigona, Tetrapedia, Centris), with short flight ranges, that exhaust the feeding sources in an area before moving on to another (Dodson, 1962; Singer and Cocucci, 1999; Parra-Tabla et al., 2000; Singer and Koehler, 2003), promoting inbreeding and a low numbers of heterozygotes. Thus, the high inbreeding coefficients found could be attributed to pollinator behaviour in these rewarding orchids (Dressler, 1981; Peakall and Schiestl, 2004).

The high genetic variability and high FIS values observed can be explained by the life history of the species, with most seed dispersal occurring within populations, self-incompatibility mechanisms and pollinators with restricted foraging areas. In this scenario, a high genetic variability and a high number of alleles at low frequencies can be maintained by clonal propagation, since each genotype can remain for many generations (Ellstrand and Rose, 1987; Borba et al., 2001).

Genetic structure and gene flow
Despite the fact that populations were sampled in small, isolated fragments, they presented a modest genetic structure (FST = 0·029). This differentiation is low, as reported by Hamrick and Godt (1996), who noted a very low differentiation in orchids, compared with other perennial herbaceous, due to species-specific pollinators and wind-dispersed seeds. Other studies on recently fragmented orchid populations found contrasting patterns in perennials and allogamous species (Sun, 1996; Wong and Sun, 1999; Ehlers and Pedersen, 2000; Gustafsson, 2000; Tremblay and Ackerman, 2001; Forrest et al., 2004; Trapnell and Hamrick, 2004). Although wind-dispersal of seeds may be highly localized, generating a fine scale genetic structure, Trapnell and Hamrick (2004) pointed out that some seeds can enter the air column and be dispersed over long distances (kilometres), as predicted for small and light seeds. The long-distance tail of the seed dispersal distribution (Murren and Ellison, 1998) is also important because even small amounts of gene flow have significant consequences for the homogenization of genetic variation among populations (Chung et al., 2004; Trapnell and Hamrick, 2004), minimizing the effects of isolation and population differentiation. Assuming the scenario that the majority of the seeds settle near the maternal plant, the high FIS values reported here indicate low pollinator mobility. On the other hand, the open matrix may promote efficient seed exchange, hindering the differentiation of populations. The moderate value of FST in the PGM locus and its different allele frequencies among populations suggest the action of selection on this locus or nearby loci (hitchhiking effect; Maynard Smith and Haigh, 1974). For the remaining loci, there are no differences in population frequencies or significant structure to suggest non-neutral dynamics.

In Oncidium hookeri, seeds seem to play a more important role in gene flow between populations than pollen. A comparative analysis of nuclear and organellar DNA markers in natural populations would help in testing this hypothesis. Despite the small number of fruits, the large number of seeds per fruit and the proximity of the populations seem to promote sufficient gene flow to avoid the structuring of populations, independently of the spatial distance between them. The differentiation observed between Pop. 3 and Pop. 6 and the other populations could be due to their location in isolated, inclined fragments, surrounded by stone formations. The private alleles observed in Pops 1, 3 and 4 could either have appeared recently or represent a transitory polymorphism, which could be maintained due to clonal propagation.

Conclusions
The low differentiation among Oncidium hookeri populations, the absence of correlation between genetic structure and spatial distance among populations, the high genetic variability, the weak evidence for bottlenecks, and the high number of individuals at the boundaries of the fragments indicate that the isolation of these populations within remnants of the Atlantic rainforest apparently does not directly threaten this species. The heterozygote deficiency found seems to be caused by the pollinator behaviour coupled with mechanisms of self-incompatibility, in a situation where the majority of seed dispersal occurs near to the maternal plants, and the low frequencies of several alleles at different loci may be maintained by vegetative propagation. Despite the stochastic nature of the wind-dispersion of seeds over long distances, this process may promote effective gene flow among populations, as in other orchid species, thus, preventing the build-up of genetic differentiation.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank J. M. Alcantara, G. H. Aguirre and M. C. Crevellaro for their help with field work, and J. José, F. N. Ramos, C. F. Verola, the handling editor Dr Christian Lexer, and the anonymous referees for valuable comments and suggestions on the manuscript. This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico 5879597362603248).


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

    Ackerman JD and Ward S. (1999) Genetic variation in a widespread, epiphytic orchid: where is the evolutionary potential? Systematic Botany 24:282–291.[CrossRef]

    Alfenas AC, Peters I, Brune W, Passador GC. (1991) Eletroforese de proteínas e isoenzimas de fungos e essências florestais(Imprensa da Universidade Federal de Viçosa, Viçosa).

    GENETIX, logiciel sous Windows TM pour la génétique des populations Belkhir K, Borsa P, Chikhi L, Raufuste N, Bonhomme F. (1996–2004) Laboratoire Génome, Populations, Interactions, CNRS UMR 5000, Université de Montpellier II, Montpellier, France.

    Borba EL, Felix JM, Solferini VN, Semir J. (2001) Fly pollinate Pleurothallis (Orchidaceae) have high genetic variability: evidence from isozyme markers. American Journal of Botany 88:419–428.[Abstract/Free Full Text]

    Brzosko E, Wróblewska A, Talalaj I. (2004) Genetic variation and genotypic diversity in Epipactis helleborine populations from NE Poland. Plant Systematics and Evolution 248:57–69.

    Chase MW. (1986) A reappraisal of the oncidioid orchids. Systematic Botany 11:477–491.[CrossRef]

    Chung MY, Nason JD, Chung MG. (2004) Spatial genetic structure in populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae). American Journal of Botany 91:52–57.[Abstract/Free Full Text]

    Clayton J and Tetriak D. (1972) Amine-citrate buffers for pH control in starch gel electrophoresis. Journal of the Fisheries Research Board of Canada 29:1169–1172.

    Cornuet JM and Luikart G. (1996) Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144:2001–2014.[Abstract]

    Cozzolino S and Widmer A. (2005) Orchid diversity: an evolutionary consequence of deception? Trends in Ecology and Evolution 20:487–494.[CrossRef]

    Cozzolino S, Cafasso D, Pellegrino G, Musacchio A, Widmer A. (2003) Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation. Molecular Ecology 12:2783–2792.[CrossRef][Medline]

    Dodson CH. (1962) The importance of pollination in the evolution of the orchids of tropical America. American Orchid Society Bulletin 31:525–534.

    Dressler RL. (1981) The orchids: natural history and classification(Harvard University Press, Cambridge, MA).

    Dressler RL. (1993) Phylogeny and classification of the orchid family(Dioscorides Press, Portland, OH).

    Ehlers BK and Pedersen HAE. (2000) Genetic variation in three species of Epipactis (Orchidaceae): geographic scale and evolutionary inferences. Biological Journal of the Linnean Society 69:411–430.[CrossRef]

    Ellstrand NC and Elam DR. (1993) Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24:217–242.[CrossRef][ISI]

    Ellstrand NC and Rose ML. (1987) Patterns of genotypic diversity in clonal plant species. American Journal of Botany 74:123–131.[CrossRef][ISI]

    Forrest AD, Hollingsworth ML, Hollingsworth PM, Sydes C, Bateman RM. (2004) Population genetic structure in European populations of Spiranthes romanzoffiana set in the context of other genetic studies on orchids. Heredity 92:218–227.[CrossRef][ISI][Medline]

    Fritz AL and Nilsson A. (1994) How pollinator-mediated mating varies with population size in plants. Oecologia 100:451–462.[CrossRef]

    González-Astorga J, Cruz-Angón A, Flores-Palacios A, Vovides AP. (2004) Diversity and genetic structure of the Mexican endemic epiphyte Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys (Bromeliaceae). Annals of Botany 94:545–551.[Abstract/Free Full Text]

    Goudet J. (1995) FSTAT(version 1.2): a computer program to calculate F-statistics. Journal of Heredity 86:485–486.[Free Full Text]

    Gustafsson S. (2000) Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid. Molecular Ecology 9:1863–1872.[CrossRef][Medline]

    Hamrick JL and Godt MJW. (1990) Allozyme diversity in plant species. In Brown AHD, Clegg MT, Kahler AL, Weir BS (Eds.). Plant population genetics, breeding, and genetics resources(Sinauer, Sunderland, MA).

    Hamrick JL and Godt MJW. (1996) Effects of life history traits on genetic diversity in plant species. Philosophical Transactions of the Royal Society of London, B 351:1291–1298.

    Kearns C, Inouye D, Waser N. (1998) Endangered mutualism: the conservation of plant–pollinator interactions. Annual Review of Ecology and Systematics 29:83–112.[CrossRef][ISI]

    Köppen W. (1948) Climatologia com un estudio de los climas de la Tierra(Fondo de Cultura Economica, Mexico).

    Lande R. (1999) Extinction risks from anthropogenic, ecological, and genetic factors. In Landweber LA and Dobson AP (Eds.). Genetic and extinction of species(Princeton University Press, Princeton, NJ).

    Lande R and Schemske D.W. (1985) The evolution of self-fertilization and inbreeding depression in plants. I. Genetic models. Evolution 39:24–40.[CrossRef][ISI]

    Levin DA and Kerster HW. (1969) The dependence of bee-mediated pollen and gene dispersal upon plant density. Evolution 23:560–571.[CrossRef]

    Loveless MD and Hamrick JL. (1984) Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15:65–95.[CrossRef][ISI]

    Machon N, Bardin P, Mazer SJ, Moret J, Godelle B, Austerlitz F. (2003) Relationship between genetic structure and seed and pollen dispersal in the endangered orchid Spiranthes spiralis. New Phytologist 157:677–687.[CrossRef]

    Manly BFJ. (1997) Randomization, bootstrap and Monte Carlo methods in biology(Chapman & Hall, London).

    Mantel N. (1967) The detection of disease clustering and generalized regression approach. Cancer Research 27:209–220.[ISI][Medline]

    Maynard Smith J and Haigh J. (1974) The hitch-hiking effect of a favourable gene. Genetic Research, Cambridge 23:23–35.

    Murcia. (1995) Edge effects in fragmented forests: implications for conservation. Trends of Ecology and Evolution 10:58–62.

    Murren CJ and Ellison AM. (1998) Seed dispersal characteristics of Brassavola nodosa (Orchidaceae). American Journal of Botany 85:675–680.[Abstract]

    Neiland MR and Wilcock CC. (1998) Fruit set, nectar reward and rarity in the Orchidaceae. American Journal of Botany 85:1657–1671.[Abstract/Free Full Text]

    Nilsson LA. (1992) Orchid pollination biology. Trends in Ecology and Evolution 7:255–259.[CrossRef]

    Oostermeijer JGB, Luijten SH, den Nijs JCM. (2003) Integrating demographic and genetic approaches in plant conservation. Biological Conservation 113:389–398.[CrossRef]

    Pabst GFJ and Dungs F. (1977) Orchidaceae Brasiliensis(Brücke-Verlag, Hildesheim) Vol. II:.

    Parra-Tabla V, Vargas CF, Magana-Rueda S, Navarro J. (2000) Female and male pollination success of Oncidium ascendens Lindey (Orchidaceae) in two contrasting habitat patches: forest vs agricultural field. Biological Conservation 94:335–340.[CrossRef]

    Peakall R and Beattie AJ. (1991) The genetic consequences of worker ant pollination in a self-compatible, clonal orchid. Evolution 45:1837–1848.[CrossRef]

    Peakall R and Beattie AJ. (1996) Ecological and genetic consequences of pollination by sexual deception in the orchid Caladenia tentaculata. Evolution 50:2207–2220.[CrossRef]

    Peakall R and Schiestl FP. (2004) A mark–recapture study of male Colletes cunicularius bees: implications for pollination by sexual deception. Behavioral Ecology and Sociobiology 56:579–584.[CrossRef]

    van der Pijl L and Dodson CH. (1966) Orchid flowers—their pollination and evolution(University of Miami Press, Coral Gables, FL).

    Rasmussen HN. (1995) Terrestrial orchids from seed to mycotrophic plant(Cambridge University Press, Cambridge).

    Reis MG, Faria AD, Bittrich V, Amaral MCE, Marsaioli A. (2000) The chemistry of flower rewards—Oncidium (Orchidaceae). Journal of Brazilian Chemistry Society 11:600–608.

    Richards AJ. (1997) Plant breeding systems(Chapman and Hall, London).

    Rizzini CT. (1979) Tratado de fitogeografia do Brasil. Aspectos sociológicos e florísticos(Hucitec, São Paulo) v. 2:.

    Scacchi R, De Angelis G, Corbo RM. (1991) Effect of breeding system on the genetic structure in three Cephalanthera sp. (Orchidaceae). Plant Systematics and Evolution 176:53–61.[CrossRef]

    Scacchi R, Lanzara P, De Angelis G. (1990) Allozyme variation among and within eleven Orchis species (fam. Orchidaceae) with special reference to hybridizing aptitude. Genetica 81:143–150.

    Schmitt J. (1980) Pollination foraging behaviour and gene dispersal in Senecio (Compositae). Evolution 34:934–943.[CrossRef]

    Sharma IK, Clements MA, Jones DL. (2000) Observation of high genetic variability in the endangered Australian terrestrial orchid Pterostylis gibbosa R. Br. (Orchidaceae). Biology Systematics and Ecology 28:651–663.[CrossRef]

    Sharma IK, Jones DL, French CJ. (2003) Unusually high genetic variability revealed through allozymic polymorphism of an endemic and endangered Australian orchid, Pterostylis aff. picta (Orchidaceae). Biochemical Systematics and Ecology 31:513–526.[CrossRef]

    Singer RB. (2003) Orchid pollination: recent developments from Brazil. Lankesteriana 7:111–114.

    Singer RB and Cocucci AA. (1999) Pollination mechanism in four sympatric southern Brazilian Epidendroideae orchids. Lindleyana 14:47–56.

    Singer RB and Koehler S. (2003) Notes on the pollination biology of Notylia nemorosa (Orchidaceae: Oncidiinae): do pollinators necessarily promote cross-pollination? Journal of Plant Research—Japan 116:19–25.

    Sun M. (1996) Effects of population size, mating system and evolutionary origin on genetic diversity in Spirantes sinensis and S. hongkongensis. Conservation Biology 10:785–795.[CrossRef]

    Sun M. (1997) Genetic diversity in three colonizing orchids with contrasting mating systems. American Journal of Botany 84:224–232.[Abstract]

    Sun M and Ganders FR. (1990) Outcrossing rates and allozime variation in rayed and rayless morphs of Bidens pilosa. Heredity 62:139–143.

    Sun M and Wong KC. (2001) Genetic structure of three orchid species with contrasting breeding systems using rapid and allozyme markers. American Journal of Botany 88:2180–2188.[Abstract/Free Full Text]

    Swofford DL and Selander RB. (1981) BIOSYS-1: a FORTAN program for the comprehensive analysis of eletrophoretic data in populations genetics and systematics. Journal of Heredity 72:282–283.

    Trapnell DW and Hamrick JL. (2004) Partitioning nuclear and chloroplast variation at multiple spatial scales in the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 13:2655–2666.[CrossRef][Medline]

    Trapnell DW and Hamrick JL. (2005) Mating patterns and gene flow in the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 14:75–84.[CrossRef][Medline]

    Trapnell DW, Hamrick JL, Nason JD. (2004) Three-dimensional fine-scale genetic structure of the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 13:1111–1118.[CrossRef][Medline]

    Tremblay RL and Ackerman JD. (2001) Gene flow and effective population size in Lepanthes (Orchidaceae): a case for genetic drift. Biological Journal of the Linnean Society 72:47–62.[CrossRef]

    Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN. (2005) Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification. Biological Journal of the Linnean Society 84:1–54.[CrossRef]

    Weir BS and Cockerham CC. (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370.[CrossRef][ISI]

    Wong KC and Sun M. (1999) Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae). American Journal of Botany 86:1406–1413.[Abstract/Free Full Text]

    Wright S. (1978) Evolution and the genetics of populations(University of Chicago Press, Chicago, IL) Vol. 4:.

    Young AG, Boyle T, Brown T. (1996) The population genetic consequences of habitat fragmentation for plants. Trends in Ecology and Evolution 11:413–419.[CrossRef]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
98/6/1207    most recent
mcl202v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Agricola
Right arrow Articles by ALCANTARA, S.
Right arrow Articles by SOLFERINI, V. N.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?