Group Of Orchids Research Articles

Phylogenetic climatic niche conservatism and evolution of climatic suitability in Neotropical Angraecinae (Vandeae, Orchidaceae) and their closest African relatives

In the present study we investigate the concept of phylogenetic niche conservatism (PNC) within the American species of angraecoid orchids (Campylocentrum and Dendrophylax) and their closest relatives in the Old World (Angraecum) using ecological niche modelling (ENM). The predicted niche occupancy profiles were matched with the outcomes of previous phylogenetic studies to reconstruct the evolution of climatic suitability within the orchid group studied and evaluate the role of niche differentiation in the speciation of Angraecinae. No correlation between preferred niches and taxonomic relationships within the orchid group studied was revealed. The climatic suitability of the majority of the species overlapped each other, either fully or partially. This pattern is also present in the species of other orchid genera. Our research confirms a significant level of PNC in Orchidaceae, even within taxa exhibiting a transatlantic disjunction. The analysis of the evolution of climatic suitability indicated that the adaptation to various climatic conditions is not a factor that has driven speciation within orchids studied.

Intrachromosomal karyotype asymmetry in Orchidaceae.

The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimitation efforts. However, there is no consensus about the best method to calculate the intrachromosomal asymmetry. The present study aimed to compare different intrachromosomal asymmetry indexes in order to indicate which are more efficient for the estimation of asymmetry in different groups of orchids. Besides, we aimed to compare our results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971). Through a literature review, karyotypes were selected and analyzed comparatively with ideal karyotypes in a cluster analysis. All karyotypes showed some level of interchromosomal asymmetry, ranging from slightly asymmetric to moderately asymmetric. The five tested intrachromosomal asymmetry indexes indicated Sarcoglottis grandiflora as the species with the most symmetrical karyotype and Christensonella pachyphylla with the most asymmetrical karyotype. In the cluster analysis, the largest number of species were grouped with the intermediary ideal karyotypes B or C. Considering our results, we recommend the combined use of at least two indexes, especially Ask% or A1 with Syi, for cytotaxonomic analysis in groups of orchids. In an evolutionary perspective, our results support Stebbins’ hypothesis that asymmetric karyotypes derive from a symmetric karyotypes.

Evidence of reward production and pollination by Centris in Encyclia (Orchidaceae: Laeliinae): the reproductive biology of Encyclia mapuerae

Encyclia is a neotropical orchid genus distributed from Florida to South Brazil and comprises ~120 taxa, including Encyclia mapuerae (Huber) Brade & Pabst. Besides its diversity and wide distribution, conclusive data on pollinators and reproductive biology of this orchid genus is scarce. Furthermore, nothing is known about the production of floral reward in Encyclia but the pollinators of their species are assumed to be attracted through food deception. Based on data on phenology, floral morpho-anatomy, histochemistry, pollinators, pollination mechanisms, and breeding system, the reproductive biology of E. mapuerae was studied in a nature reserve at Central Amazonia, Brazil. The flowers of E. mapuerae show longitudinal lines on the labellum that act as a nectar guide. The secretory epidermis has papillae whose cells are covered by an ornamented cuticle. The subtended tissue is composed of a tree-layered collenchyma. The flowers attract several species of Hymenoptera. However, a single species of Centris was recorded as pollinator. The bees collect the nectar produced inside the cuniculus. When the bees leave the flower the pollinarium is attached to their heads. Encyclia mapuerae is self-compatible and pollinator-dependent. The reproductive success in natural conditions is low because of deficient pollen transference due to the scarcity of pollinators. As far we know this is the first study that reports and production of floral reward in Encyclia. This discovery provides new insights on the function of the cuniculus in Laeliinae, and sheds light on the evolution of floral rewards and pollination mechanisms within this diverse group of Neotropical orchids.

Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes

Genetic diversity was assessed in the four orchid species using NGS based ddRAD sequencing data. The assembled nucleotide sequences (fastq) were deposited in the SRA archive of NCBI Database with accession number (SRP063543 for Dendrobium, SRP065790 for Geodorum, SRP072201 for Cymbidium and SRP072378 for Rhynchostylis). Total base pair read was 1.1 Mbp in case of Dendrobium sp., 553.3Kbp for Geodorum sp., 1.6 Gbp for Cymbidium, and 1.4 Gbp for Rhynchostylis. Average GC% was 43.9 in Geodorum, 43.7% in Dendrobium, 41.2% in Cymbidium and 42.3% in Rhynchostylis. Four partial gene sequences were used in DnaSP5 program for nucleotide diversity and phylogenetic relationship determination (Ycf2 gene of Dendrobium, matK gene of Geodorum, psbD gene of Cymbidium and Ycf2 gene of Ryhnchostylis). Nucleotide diversity (per site) Pi (π) was 0.10560 in Dendrobium, 0.03586 in Geodorum, 0.01364 in Cymbidium and 0.011344 in Rhynchostylis. Neutrality test statistics showed the negative value in all the four orchid species (Tajima's D value -2.17959 in Dendrobium, -2.01655 in Geodorum, -2.12362 in Rhynchostylis and -1.54222 in Cymbidium) indicating the purifying selection. Result for these gene sequences (matK and Ycf2 and psbD) indicate that they were not evolved neutrally, but signifying that selection might have played a role in evolution ofthese genes in these four groups of orchids. Phylogenetic relationship was analyzed by reconstructing dendrogram based on the matK, psbD and Ycf2 gene sequences using maximum likelihood method in MEGA6 program.

Integrative taxonomy of the fly orchid group: insights from chemical ecology.

Several authors have recently stressed the need to develop an integrative approach in taxonomy, but studies applying such an approach to Mediterranean orchids are scarce. In sexually deceptive orchids from the taxonomically difficult genus Ophrys, pollination is specific and performed by male insects attracted to the flowers by sex pheromone-mimicking floral scents. Floral compounds are therefore of primary importance for reproductive isolation and species delimitations in this genus. In the fly orchid group, molecular, morphological, and ecological characters have been extensively studied, but a comprehensive survey of floral scents is still lacking. In the present study, the blends of floral compounds of its three members, Ophrys insectifera, Ophrys aymoninii, and Ophrys subinsectifera, were extracted and analyzed by gas chromatography-mass spectrometry. A total of 107 compounds were found, with a majority of saturated and unsaturated hydrocarbons. Significant differentiation, both qualitative and quantitative, was found among the three taxa. This result, pooled with those from the literature, forms a comprehensive and congruent dataset that allows us to elucidate the taxonomic rank of the three members of the fly orchid group.

Evolutionary diversification and historical biogeography of the Orchidaceae in Central America with emphasis on Costa Rica and Panama

Históricamente, el Istmo de Costa Rica y Panamá ha sido fuente de fascinación por su posición estratégica, uniendo América del Norte con América del Sur. En términos de biodiversidad, el Istmo es considerado una de las regiones más ricas del mundo. Orchidaceae es la familia de plantas más diversa en la zona, y el número de especies triplica a otras familias de angiospermas bien representadas como Rubiaceae, Fabaceae y Poaceae. Aunque todavía estamos lejos de conocer el número exacto de especies de orquídeas existentes en ambos países, en la actualidad la flora de orquídeas registradas para Costa Rica (1574 spp.) y Panamá (1372 spp.) suman en conjunto alrededor de 2010 especies; lo que representa 6,5-8,0% de todas las especies de orquídeas en apenas alrededor de 1% de la superficie terrestre del planeta. Pleurothallidinae y Laeliinae son los grupos más ricos en especies y contienen la mayor géneros: Lepanthes, Pleurothallis, Stelis y Epidendrum. Estos grupos superan significativamente los otros géneros registrados en términos de riqueza de especies. Algunos de los factores que explican esta diversidad taxonómica regional de orquídeas son el puente natural que une tres de los 25 puntos reconocidos en todo el mundo (Mesoamérica, Chocó / Darién / Ecuador Occidental y Andes tropicales), la influencia climática de los océanos Pacífico y Atlántico, y la reciente elevación de la Cordillera de Talamanca y la formación de estribaciones de Majé, Darién y San Blas, en Panamá y el oeste de Colombia. Aunque estos factores pueden explicar la alta diversidad de orquídeas en términos generales, se necesita información detallada para comprender la diversificación de especies, así como la evolución de la composición florística. Los inventarios florísticos actualizados (con una tasa de 25 nuevas especies / año) y el estudio de los mecanismos biológicos que han llevado a la diversificación evolutiva de Lepanthes (como uno de los géneros más grandes de orquídeas) son los principales proyectos de investigación en curso para dilucidar la evolución de Orchidaceae en Costa Rica y Panamá. Con este fin, presentamos algunos resultados preliminares de la investigación llevada a cabo en esta dirección que incluye la integración de estudios filogenéticos, ecología de la polinización, taxonomía y biogeografía.

Crane flies and microlepidoptera also function as pollinators in Epidendrum (Orchidaceae: Laeliinae): the reproductive biology of E. avicula

Crane flies and microlepidoptera have been recorded as pollinators in unrelated orchid groups, but these insects have never been recorded in Epidendroideae, the most species‐rich orchid subfamily, which includes one of the most diverse genera among Orchidaceae, Epidendrum. Based on data on phenology, floral morpho‐anatomy, pollinators, pollination mechanisms and breeding system, the reproductive biology of E. avicula was studied in south‐eastern Brazil. Epidendrum avicula possess osmophores that produce a citric fragrance at night. The flowers attract Tipulidae flies and several families of microlepidoptera that drink the nectar produced in a tube formed by the adnation of the labellum and column. As is common in Epidendrum, after removing the pollinarium, both crane flies and micro‐moths get trapped by the proboscis, which frightens the insects and inhibits any possible intent to immediately visit another flower. The behavior of the pollinators on flowers, plus the retention of the anther cap by the pollinarium, results in a reduction in the occurrence of geitonogamy. Because E. avicula is self‐incompatible, the consequence of pollinator behavior and the floral mechanisms tend to reduce the pollen loss. As far as we know, this is the first study to report the reproductive biology of a species of Epidendroideae pollinated by crane flies and microlepidoptera. Based on more recent concepts of plant–pollinator interactions, although E. avicula is pollinated by several species belonging to two distinct orders, suggesting an unspecialized pollination system is involved, nectar‐seeking microlepidoptera and Tipulidae flies can be recognized as a single functional group.

Factors affecting the distribution and abundance of orchids in grasslands and herbaceous wetlands

Grasslands, wet meadows, fens, bogs, and marshes represent important ecosystems that host many orchid species. However, research on the joint effect of the multi-scalar abiotic drivers on orchid distribution and abundance in these habitats is limited. In this study, we investigated factors associated with the occurrence and abundance of 42 orchid taxa in grasslands and herbaceous wetlands of 29 mountains and the surrounding low altitude areas in western Serbia (eastern Dinaric Alps, southeastern Europe). Niche analysis of orchids was performed using outlying mean index analysis (OMI). In addition, we used similarity profile analysis (SIMPROF) to classify orchid taxa into ecological groups, multi response permutation procedure (MRPP) to analyse the differences in orchid composition between vegetation types, and indicator species analysis (ISA) to detect orchids with an affinity to a certain type of vegetation and geological bedrock. The most important gradients that separate orchids within the sampled environmental conditions were moisture, temperature, altitude and soil pH. Moreover, this study underlines the importance of certain vegetation types and geological bedrocks in determining the distribution and abundance of orchids. In total, eight ecological groups of orchids were distinguished. The results have shown that 11 orchid taxa are indicators of specific vegetation types, whereas 13 orchid taxa are indicators of specific bedrock types. The study area provides suitable conditions for survival of some rare and endemic orchid species that have southern limits of their distribution in this part of southeastern Europe. The study suggests that the joint effect of ecological factors, especially vegetation communities and bedrock types, is important to consider in the planning and management of orchid species conservation.

Toward a phylogeny of Maxillariinae orchids: multidisciplinary studies with emphasis in Brazilian species

<div class="page" title="Page 1"><div class="layoutArea"><div class="column"><p><span>It has been demonstrated that the neotropical sub- tribe Maxillariinae comprises a well-supported mono- phyletic orchid group, according to cladistic analyses of nuclear and chloroplast DNA regions </span></p></div></div></div>

Seven New Complete Plastome Sequences Reveal Rampant Independent Loss of the ndh Gene Family across Orchids and Associated Instability of the Inverted Repeat/Small Single-Copy Region Boundaries.

Earlier research has revealed that the ndh loci have been pseudogenized, truncated, or deleted from most orchid plastomes sequenced to date, including in all available plastomes of the two most species-rich subfamilies, Orchidoideae and Epidendroideae. This study sought to resolve deeper-level phylogenetic relationships among major orchid groups and to refine the history of gene loss in the ndh loci across orchids. The complete plastomes of seven orchids, Oncidium sphacelatum (Epidendroideae), Masdevallia coccinea (Epidendroideae), Sobralia callosa (Epidendroideae), Sobralia aff. bouchei (Epidendroideae), Elleanthus sodiroi (Epidendroideae), Paphiopedilum armeniacum (Cypripedioideae), and Phragmipedium longifolium (Cypripedioideae) were sequenced and analyzed in conjunction with all other available orchid and monocot plastomes. Most ndh loci were found to be pseudogenized or lost in Oncidium, Paphiopedilum and Phragmipedium, but surprisingly, all ndh loci were found to retain full, intact reading frames in Sobralia, Elleanthus and Masdevallia. Character mapping suggests that the ndh genes were present in the common ancestor of orchids but have experienced independent, significant losses at least eight times across four subfamilies. In addition, ndhF gene loss was correlated with shifts in the position of the junction of the inverted repeat (IR) and small single-copy (SSC) regions. The Orchidaceae have unprecedented levels of homoplasy in ndh gene presence/absence, which may be correlated in part with the unusual life history of orchids. These results also suggest that ndhF plays a role in IR/SSC junction stability.

Molecular identification of endangered Korean lady’s slipper orchids (Cypripedium, Orchidaceae) and related taxa

Reliable and accurate species identification is essential to establish a strategy for monitoring and manipulation of plant populations for conservation biology. However, identification of nonflowering plants is difficult. Based on recent advances in molecular biological techniques, various molecular identification methodologies have been proposed and applied in plant science, with a focus on medicinally valuable species. Lady’s slipper orchids (Cypripedium) are a representative terrestrial orchid group that includes many endangered species. Of 46 species, 3 (Cypripedium japonicum Thunb., Cypripedium macranthos Sw., and Cypripedium guttatum var. koreanum Nakai) were reported from Korea and categorised as endangered species or at risk of becoming endangered because of the rapidly decreasing population. We generated an accurate molecular identification system for these species using the sequence variation and species-specific SNPs of two plastid loci, rpoC2 and the IGS region between atpF and atpH. We selected the atpF-H region for molecular identification of the Korean Cypripedium and related Taiwan-endemic taxon Cypripedium formosanum Hayata. Cypripedium guttatum var. koreanum contained large deletions of over 300 bp, and could be distinguished by electrophoresis. For the other three species, we designed new primers based on a specific insertion (C. macranthos) and SNPs (C. japonicum and C. formosanum). We confirmed that molecular identification enables detection of each species using species-specific primers.

Nectary structure and nectar in Sobralia and Elleanthus (Sobralieae: Orchidaceae)

With approximately 200 species, the tribe Sobralieae is a dominant and common Neotropical group of orchids, yet little is known of variation in floral morphology as it relates to their pollination. As currently circumscribed, the tribe includes four genera that differ considerably in flower size and morphology: Elleanthus, Epilyna, Sertifera, and Sobralia. Although nectar-foraging pollinators are known for some species, the relationships of pollination to deceit and to nectar production are all poorly understood. We examined pollination-related of nectaries and nectar characteristics (presence/absence, volume, and concentration) for major clades of Sobralieae. Some species produce abundant nectar, but many species offer no reward. When present, nectar is secreted by thickened calli at the lip base. The cells of the nectariferous calli contain starch, which is rapidly converted to sugar during a brief anthesis (often lasting only one day). Most Sobralia flowers are relatively large, bee-pollinated, with a gullet-shaped lip, false nectary, large pollinia, and offer no reward. Elleanthus flowers are relatively small with a legitimate nectar reward, and most species are hummingbird- pollinated. Hummingbird-pollinated Sobralieae flowers are relatively small, brightly colored in the perianth and/or the subtending bracts, somewhat tubular, with a lip that forms a cup around the callus for storing nectar, and pollinia that are dark and relatively small.

A macro-ecological perspective on crassulacean acid metabolism (CAM) photosynthesis evolution in Afro-Madagascan drylands: Eulophiinae orchids as a case study.

Crassulacean acid metabolism (CAM) photosynthesis is an adaptation to water and atmospheric CO2 deficits that has been linked to diversification in dry-adapted plants. We investigated whether CAM evolution can be associated with the availability of new or alternative niches, using Eulophiinae orchids as a case study. Carbon isotope ratios, geographical and climate data, fossil records and DNA sequences were used to: assess the prevalence of CAM in Eulophiinae orchids; characterize the ecological niche of extant taxa; infer divergence times; and estimate whether CAM is associated with niche shifts. CAM evolved in four terrestrial lineages during the late Miocene/Pliocene, which have uneven diversification patterns. These lineages originated in humid habitats and colonized dry/seasonally dry environments in Africa and Madagascar. Additional key features (variegation, heterophylly) evolved in the most species-rich CAM lineages. Dry habitats were also colonized by a lineage that includes putative mycoheterotrophic taxa. These findings indicate that the switch to CAM is associated with environmental change. With its suite of adaptive traits, this group of orchids represents a unique opportunity to study the adaptations to dry environments, especially in the face of projected global aridification.

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

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. 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. 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. 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.

Caladenia revisited: Results of molecular phylogenetic analyses of Caladeniinae plastid and nuclear loci.

The classification of the primarily Australasian group of orchids Caladenia and allied genera (Caladeniinae: Diurideae) containing 71 federally listed threatened species has proven controversial. Analyzing these species using genetic material will provide a sound basis for their classification and the capacity to ensure accurate conservation measures can be implemented. We present a multigene analysis based on nuclear ribosomal ITS and five plastid regions from 54 species representing all major taxonomic groups within Caladeniinae. In our combined analysis, apart from Leptoceras and Praecoxanthus, all Caladenia ingroup taxa form a strongly supported clade that is also supported by morphological synapomorphies (parallel leaf venation; leaf solitary, lanceolate, covered with glandular or eglandular trichomes). Characters and character states historically used to delimit taxa were revealed to be homoplasious and therefore do not support recognition of Arachnorchis, Cyanicula, Drakonorchis, Ericksonella, Jonesiopsis, Petalochilus, Pheladenia, and Stegostyla as previously proposed. Glossodia and Elythranthera are shown to be a specialist group embedded within Caladenia. Based on our results, none of the current systems of classification of the subtribe is satisfactory. Instead our results point to Lindley's 1840 interpretation of Caladenia, but including Glossodia and Elythranthera, as being the most accurate reflection of the group. Accordingly, a renewed reclassification of Caladeniinae is proposed as well as several new combinations.

Root and leaf anatomy of some terrestrial representatives of the Cranichideae tribe (Orchidaceae)

The Orquidaceae family has more than 25,000 species, of which 20 % are terrestrial Cranichideae mainly belonging to the tribe, which is inserted in Orchidoideae, one of the five subfamilies of the Orchidaceae. This group of terrestrial orchids presents a confusing taxonomy and is a poorly known group in terms of their vegetative anatomy in opposition to the epiphytic representatives that have been extensively investigated. This paper describes the root and leaf anatomy of ten species of Cranichideae tribe found in Brazil, belonging to subtribes Cranichidinae, Goodyerinae, and Spiranthinae, comparing their organization to those of the epiphytic plants. We also point out some features that can be useful for the characterization of each subtribe. The roots have studied characteristics that may assist in the absorption and retention of water as the occurrence of velamen and exodermis system, and in the presence of tilossomos Cranichidinae and Spiranthinae. The leaves are narrow with a homogeneous mesophyll, covered by uniseriate epidermis and covered by thin cuticle. The xylem cells are arranged in pairs or inverted V-shape, as in the other orchids Cranichideae tribe.

First flowering hybrid between autotrophic and mycoheterotrophic plant species: breakthrough in molecular biology of mycoheterotrophy

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed fiveyears after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.

Molecular phylogenetics of <i>mormolyca</i> (orchidaceae: maxillariinae) based on combined molecular data sets

The Neotropical orchid genus Mormolyca Fenzl, as currently circumscribed, encompasses a diverse group of ca. 27species. Many of these were included traditionally in Maxillaria sect. Rufescens, when similarity of floral morphology was considered foremost in their classification rather than the evolutionary history of the taxa. In order to begin revising species delimitation and clarifying the evolution and biology of the genus, we present a phylogenetic hypothesis using sequence data from five plastid loci (rpoC1, matK gene and flanking trnK intron, atpB-rbcL intergenic spacer, and the 3’ portion of ycf1) and the nuclear ribosomal internal and external transcribed spacers (ITS, ETS). Resulting trees using both Bayesian and parsimony inference are congruent with each other, and generally well resolved. Based on current level of sampling across Maxillariinae, these molecular data support the monophyly of Mormolyca and shed light on the interspecific phylogenetic patterns within the genus. These include an early divergent paraphyletic grade of Mormolyca species successively sister to a clade with at least two definable subclades within. The latter are characterized by two different flower morphologies that are likely related to their pollination systems. Although not all relationships within the genus are fully resolved or supported, these results offer a first glimpse into the phylogeny of a small group of epiphytic orchids characterized by an unusually high level of variable vegetative characters, floral fragrance profiles, and pollination systems.

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen-stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.

Morphological, ecological and genetic aspects associated with endemism in the Fly Orchid group

The European genus Ophrys (Orchidaceae) is famous for its insect-like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives.