Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E. puniceoluteum (Orchidaceae)

The hypothesis of gene flow between species with large differences in chromosome numbers has rarely been tested in the wild, mainly because species of different ploidy are commonly assumed to be reproductively isolated from each other because of instantaneous and strong postzygotic barriers. In this study, a broad‐scale survey of molecular variation was carried out between two orchid species with different ploidy levels: Epidendrum fulgens (2n = 2x = 24 chromosomes) and Epidendrum puniceoluteum (2n = 4x = 52 chromosomes). To test the strength of their reproductive barriers, we investigated the distribution of genetic variation in sympatric and allopatric populations of these two species and conducted crossing experiments. Nuclear and plastid microsatellite loci were used to genotype 463 individuals from eight populations across the geographical range of both species along the Brazilian coastal plain. All six sympatric populations analysed presented hybrid zones, indicating that hybridization between E. fulgens and E. puniceoluteum is a common phenomenon. Bayesian assignment analysis detected the presence of F₁ and F₂ individuals and also signs of introgression, demonstrating a high potential for interspecific gene flow. Introgression occurs preferentially from E. fulgens to E. puniceoluteum. Pure parental individuals of both species display strong genotype–habitat associations, indicating that environment‐dependent selection could be acting in all hybrid zones. This study suggests that hybridization and introgression are evolutionary processes playing a role in the diversification of Epidendrum and indicates the importance of investigations of hybrid zones in understanding reproductive barriers and speciation processes in Neotropical orchid species.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.     

A narrow group of monophyletic Tulasnella (Tulasnellaceae) symbiont lineages are associated with multiple species of Chiloglottis (Orchidaceae): Implications for orchid diversity

? Premise of the study: The Orchidaceae is characterized by exceptional species diversity. Obligate orchid mycorrhizae are predicted to determine orchid distributions, and highly specific relationships between orchids and fungi may drive orchid diversification. In this study, mycorrhizal diversity was examined in the terrestrial, photosynthetic orchid genus Chiloglottis to test the hypothesis of mycorrhizal-mediated diversification in the genus Chiloglottis. This orchid genus secures pollination by sexual deception, an obligate and highly specific pollination strategy. Here we asked whether the obligate orchid-fungal interactions are also specific. ? Methods: Two sequenced loci, the internal transcribed spacer region (ITS) and mitochondrial large subunit (mtLSU), were used to identify fungal isolates and assess fungal species diversity. Symbiotic germination of two species Chiloglottis aff. jeanesii and C. valida were used to assess germination potential of isolates and confirm mycorrhizal association. ? Key results: Phylogenetic analyses revealed that six representative Chiloglottis species spanning a broad survey of the genus were all associated with a narrow group of monophyletic Tulasnella fungal lineages. ? Conclusions: The Chiloglottis-Tulasnella interaction appears to be the first known case of such a narrow symbiont association across a broadly surveyed orchid genus. It appears that the specific pollination system of Chiloglottis, rather than specific orchid-fungal interactions has been the key driving force in the diversification of the genus. These findings also indicate that plant groups with highly specific mycorrhizal partners can have a widespread distribution.     

Isolation, characterization and cross-amplification of polymorphic microsatellite loci in Laelia speciosa (Orchidaceae)

Laelia speciosa is an endangered epiphytic orchid endemic to central Mexico. Here, we report on the development of 14 perfect and imperfect microsatellite repeat loci for this species. Numbers of alleles ranged from two to 16 and levels of observed heterozygosities among the 14 loci ranged from 0.28 to 1.00 across two widely divergent populations. All loci were also tested for cross-amplification in four other Laelia species and other selected genera of the subtribe Laeliinae.     

Home-loving boreal hare mitochondria survived several invasions in Iberia: the relative roles of recurrent hybridisation and allele surfing

Genetic introgression from a resident species into an invading close relative can result from repeated hybridisation along the invasion front and/or allele surfing on the expansion wave. Cases where the phenomenon is massive and systematic, such as for hares (genus Lepus) in Iberia, would be best explained by recurrent hybridisation but this is difficult to prove because the donor populations are generally extinct. In the Pyrenean foothills, Lepus europaeus presumably replaced Lepus granatensis recently and the present species border is parallel to the direction of invasion, so that populations of L. granatensis in the contact zone represent proxies of existing variation before the invasion. Among three pairs of populations sampled across this border, we find less differentiation of mitochondrial DNA (mtDNA) across than along it, as predicted under recurrent hybridisation at the invasion front. Using autosomal microsatellite loci and X- and Y-linked diagnostic loci, we show that admixture across the border is quasi-absent, making it unlikely that lack of interspecific mtDNA differentiation results from ongoing gene flow. Furthermore, we find that the local species ranges are climatically contrasted, making it also unlikely that ongoing ecology-driven movement of the contact account for mtDNA introgression. The lack of mtDNA differentiation across the boundary is mostly due to sharing of mtDNA from a boreal species currently extinct in Iberia (Lepus timidus) whose mitochondria have thus remained in place since the last deglaciation despite successive invasions by two other species. Home-loving mitochondria thus witness past species distribution rather than ongoing exchanges across stabilised contact zones.     

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae)

Background and Aims The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus.Methods The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank.Key Results Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids.Conclusions Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.     

Characterization of microsatellite loci in the endangered grand spider orchid Caladenia huegelii (Orchidaceae)

The orchid genus Caladenia is species rich with many threatened and endangered taxa. We report on the isolation and characterization of microsatellite loci in the rare Caladenia huegelii for molecular evaluation of this and the closely related C. thinicola as part of the development of conservation initiatives. Eight di‐ and trinucleotide loci were screened using 30 samples from each species. All loci were highly variable, with similar levels of heterozygosity and number of alleles across both species. These markers will be highly informative for population studies in both species.     

Transferability and characterization of microsatellite markers in two Neotropical Ficus species

Microsatellite markers were transferred and characterized for two Neotropical fig tree species, Ficus citrifolia and Ficus eximia. Our study demonstrated that microsatellite markers developed from different subgenera of Ficus can be transferred to related species. In the present case, 12 of the 15 primer pairs tested (80%) were successfully transferred to both of the above species. Eleven loci were polymorphic when tested across 60 F. citrifolia and 60 F. eximia individuals. For F. citrifolia, there were 4 to 15 alleles per locus, whereas expected heterozygosities ranged from 0.31 to 0.91. In the case of F. eximia, this was 2 to 12 alleles per locus and expected heterozygosities from 0.42 to 0.87.     

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.     

Isolation and characterization of microsatellite loci in Epidendrum puniceoluteum, an endemic orchid from the Atlantic Rainforest

Epidendrum puniceoluteum is an endemic orchid of Atlantic Rainforest, restricted to few populations only due to the destruction and fragmentation of its native habitat. Here, we report on the development of 10 microsatellite markers isolated from this orchid species. Genetic variability was characterized in two distant populations from Brazil coast. The number of alleles observed for each locus ranged from two to 12 and with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying both fine-scale genetic structure of scattered E. puniceoluteum population and patterns will be useful genetic markers for other closely related taxa.     

Orthoptera, a new order of pollinator

Background and Aims

Pollinator-mediated selection and evolution of floral traits have long fascinated evolutionary ecologists. No other plant family shows as wide a range of pollinator-linked floral forms as Orchidaceae. In spite of the large size of this model family and a long history of orchid pollination biology, the identity and specificity of most orchid pollinators remains inadequately studied, especially in the tropics where the family has undergone extensive diversification. Angraecum (Vandeae, Epidendroideae), a large genus of tropical Old World orchids renowned for their floral morphology specialized for hawkmoth pollination, has been a model system since the time of Darwin.

Methods

The pollination biology of A. cadetii, an endemic species of the islands of Mauritius and Reunion (Mascarene Islands, Indian Ocean) displaying atypical flowers for the genus (white and medium-size, but short-spurred) was investigated. Natural pollinators were observed by means of hard-disk camcorders. Pollinator-linked floral traits, namely spur length, nectar volume and concentration and scent production were also investigated. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified in natural field conditions weekly during the 2003, 2004 and 2005 flowering seasons (January to March).

Key Results

Angraecum cadetii is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator species was observed, an undescribed species of raspy cricket (Gryllacrididae, Orthoptera). These crickets, which are nocturnal foragers, reached flowers by climbing up leaves of the orchid or jumping across from neighbouring plants and probed the most ‘fresh-looking’ flowers on each plant. Visits to flowers were relatively long (if compared with the behaviour of birds or hawkmoths), averaging 16·5 s with a maximum of 41·0 s. At the study site of La Plaine des Palmistes (Pandanus forest), 46·5 % of flowers had pollen removed and 27·5 % had pollinia deposited on stigmas. The proportion of flowers that set fruit ranged from 11·9 % to 43·4 %, depending of the sites sampled across the island.

Conclusions

Although orthopterans are well known for herbivory, this represents the first clearly supported case of orthopteran-mediated pollination in flowering plants.     

Evolution and diversity of floral scent chemistry in the euglossine bee-pollinated orchid genus Gongora

•Background and Aims Animal-pollinated angiosperms have evolved a variety of signalling mechanisms to attract pollinators. Floral scent is a key component of pollinator attraction, and its chemistry modulates both pollinator behaviour and the formation of plant–pollinator networks. The neotropical orchid genus Gongora exhibits specialized pollinator associations with male orchid bees (Euglossini). Male bees visit orchid flowers to collect volatile chemical compounds that they store in hind-leg pouches to use subsequently during courtship display. Hence, Gongora floral scent compounds simultaneously serve as signalling molecules and pollinator rewards. Furthermore, because floral scent acts as the predominant reproductive isolating barrier among lineages, it has been hypothesized that chemical traits are highly species specific. A comparative analysis of intra- and inter-specific variation of floral scent chemistry was conducted to investigate the evolutionary patterns across the genus.•Methods Gas chromatography–mass spectrometry (GC-MS) was used to analyse the floral scent of 78 individuals belonging to 28 different species of Gongora from two of the three major lineages sampled across the neotropical region. Multidimensional scaling and indicator value analyses were implemented to investigate the patterns of chemical diversity within and among taxonomic groups at various geographic scales. Additionally, pollinator observations were conducted on a sympatric community of Gongora orchids exhibiting distinct floral scent phenotypes.•Key Results A total of 83 floral volatiles, mainly terpenes and aromatic compounds, were detected. Many of the identified compounds are common across diverse angiosperm families (e.g. cineole, eugenol, β-ocimene, β-pinene and terpinen-4-ol), while others are relatively rare outside euglossine bee-pollinated orchid lineages. Additionally, 29 volatiles were identified that are known to attract and elicit collection behaviour in male bees. Floral scent traits were less variable within species than between species, and the analysis revealed exceptional levels of cryptic diversity. Gongora species were divided into 15 fragrance groups based on shared compounds. Fragrance groups indicate that floral scent variation is not predicted by taxonomic rank or biogeographic region.•Conclusions Gongora orchids emit a diverse array of scent molecules that are largely species specific, and closely related taxa exhibit qualitatively and quantitatively divergent chemical profiles. It is shown that within a community, Gongora scent chemotypes are correlated with near non-overlapping bee pollinator assemblies. The results lend support to the hypothesis that floral scent traits regulate the architecture of bee pollinator associations. Thus, Gongora provides unique opportunities to examine the interplay between floral traits and pollinator specialization in plant–pollinator mutualisms.     

Revision of the orchid bee subgenus Euglossella (Hymenoptera, Apidae), Part I, The decorata species group

Euglossella, one of the most distinctive subgenera of orchid bees of the genus Euglossa, is composed of two characteristic assemblages of species, one of them comprising bees bearing the strongly metallic integument trademark of the genus (viridis species group), and the other consisting of bees with a brown integument shaded with metallic iridescence (decorata species group). Here we provide the first of two parts of a revision of Euglossella, providing diagnostic definitions for the subgenus, the decorata species group, and all the species included therein. Six species are included in the decorata group, one new: Euglossa (Euglossella) aurantia, sp. n.; Euglossa (Euglossella) apiformis Schrottky, resurrected status; Euglossa (Euglossella) decorata Smith, revised status; Euglossa (Euglossella) singularis Mocsáry, revised status; Euglossa (Euglossella) cosmodora Hinojosa-Díaz and Engel; and Euglossa (Euglossella) perpulchra Moure and Schlindwein. Euglossa meliponoides Ducke and Euglossa urarina Hinojosa-Díaz and Engel are newly synonymized under Euglossa decorata, Euglossa decorata ruficauda Cockerell is synonymized under Euglossa singularis, and a neotype is designated for Euglossa apiformis.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

Background and Aims Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale.Methods Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia.Key Results With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent.Conclusions The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.     

Isolation of polymorphic microsatellite loci in the genus Clusia (Clusiaceae)

Microsatellite flanking region sequences may provide phylogenetically useful information. We isolated 13 polymorphic microsatellite loci from two species, Clusia minor (five loci) and Clusia nemorosa (eight loci), to aid in the determination of phylogenetic relationships within the genus Clusia. Eleven loci amplified across all 17 Clusia species tested, while two loci amplified in 10 out of 17 species. The extensive cross‐species amplification suggests that these loci may be useful for an examination of phylogenetic relationships in this genus.     

High degree of conservation of nuclear microsatellite loci in the genus Clusia.

In plants, microsatellites and their flanking DNA are rarely conserved across a whole genus, let alone other genera in the same family. Therefore, the possibility of using microsatellite primers developed for a species across a large number of plant species in the same genus is often limited. Remarkably, dinucleotide nuclear microsatellites developed for Clusia minor and for Clusia nemorosa amplified homologous microsatellites in species across the whole genus Clusia. In this present study, we report on the DNA sequence variation across the genus of 3 microsatellite loci with varying levels of variation. Compared over the species, there was a correlation between the lengths of the microsatellite loci. Interrupts occurred multiple times and did not seem to lead to the death of the microsatellite. These highly conserved markers will be useful for studying the variable reproductive systems in the genus Clusia.     

Spatial and demographic population genetic structure in Catasetum viridiflavum across a human-disturbed habitat

Spatial and temporal genetic structures were examined across sites on islands and mainland (continuous forest) populations of an epiphytic orchid, Catasetum viridiflavum, using 17 polymorphic allozyme loci. I tested whether patches on islands or at mainland sites comprised small local populations or a large population. Low among population differentiation was observed across the landscape suggesting that the species-specific pollinator and tiny wind-dispersed seeds maintain interconnections among distant patches. Temporal genetic structure among stage classes, and among breeding individuals are important components of the maintenance of genetic variation in this orchid. The natural history of this species including small breeding populations, probable high frequency of mating among relatives, and the high rates of seed movement among sites contribute to the high FIS. These data show that physically isolated patches in this epiphytic orchid comprise a single larger genetic population, which is independent of the physical distances among sites. Although quite different in ecological and life history characteristics, the genetic structure of this orchid demonstrates a pattern similar to temperate and tropical trees in fragmented landscapes.     

Orchid seed removal by ants in Neotropical ant‐gardens

• Most plants that inhabit ant‐gardens (AGs) are cultivated by the ants. Some orchids occur in AGs; however, it is not known whether their seeds are dispersed by AG ants because most orchid seeds are tiny and dispersed by wind.
• We performed in situ seed removal experiments, in which we simultaneously provided Azteca gnava ants with seeds of three AG orchid species and three other AG epiphyte species (Bromeliaceae, Cactaceae and Gesneriaceae), as well as the non‐AG orchid Catasetum integerrimum.
• The seeds most removed were those of the bromeliad Aechmea tillandsioides and the gesneriad Codonanthe uleana, while seeds of AG orchids Coryanthes picturata, Epidendrum flexuosum and Epidendrum pachyrachis were less removed. The non‐AG orchid was not removed. Removal values were positively correlated with the frequency of the AG epiphytes in the AGs, and seeds of AG orchids were larger than those of non‐AG orchids, which should favour myrmecochory.
• Our data show that Azt. gnava ants discriminate and preferentially remove seeds of the AG epiphytes. We report for the first time the removal of AG orchid seeds by AG ants in Neotropical AGs.