Evolutionary history and systematics of European blind mole rats (Rodentia: Spalacidae: Nannospalax): Multilocus phylogeny and species delimitation in a puzzling group.

Species delimitation is a powerful approach to assist taxonomic decisions in challenging taxa where species boundaries are hard to establish. European taxa of the blind mole rats (genus Nannospalax) display small morphological differences and complex chromosomal evolution at a shallow evolutionary divergence level. Previous analyses led to the recognition of 25 'forms' in their distribution area. We provide a comprehensive framework to improve knowledge on the evolutionary history and revise the taxonomy of European blind mole rats based on samples from all but three of the 25 forms. We sequenced two nuclear-encoded genetic regions and the whole mitochondrial cytochrome b gene for phylogenetic tree reconstructions using concatenation and coalescence-based species-tree estimations. The phylogenetic analyses confirmed that Aegean N. insularis belongs to N. superspecies xanthodon, and that it represents the second known species of this superspecies in Europe. Mainland taxa reached Europe from Asia Minor in two colonisation events corresponding to two superspecies-level taxa: N. superspecies monticola (taxon established herewith) reached Europe c. 2.1 million years ago (Mya) and was followed by N. superspecies leucodon (re-defined herewith) c. 1.5 Mya. Species delimitation allowed the clarification of the taxonomic contents of the above superspecies. N. superspecies monticola contains three species geographically confined to the western periphery of the distribution of blind mole rats, whereas N. superspecies leucodon is more speciose with six species and several additional subspecies. The observed geographic pattern hints at a robust peripatric speciation process and rapid chromosomal evolution. The present treatment is thus regarded as the minimum taxonomic content of each lineage, which can be further refined based on other sources of information such as karyological traits, crossbreeding experiments, etc. The species delimitation models also allowed the recognition of a hitherto unnamed blind mole rat taxon from Albania, described here as a new subspecies.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex-ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex-reversing stimuli more frequently, which may lead to higher sex-reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR-based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L-1 17α-ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L-1 EE2 and a glyphosate-based herbicide with 3 µg L-1 or 3 mg L-1  glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

Phenotypic plasticity rather than genotype drives reproductive choices in Hydra populations.

Facultative clonality is associated with complex life cycles where sexual and asexual forms can be exposed to contrasting selection pressures. Facultatively clonal animals often have distinct developmental capabilities that depend on reproductive mode (e.g., negligible senescence and exceptional regeneration ability in asexual individuals, which are lacking in sexual individuals). Understanding how these differences in life history strategies evolved is hampered by limited knowledge of the population structure underlying sexual and asexual forms in nature. Here we studied genetic differentiation of coexisting sexual and asexual Hydra oligactis polyps, a freshwater cnidarian where reproductive mode-dependent life history patterns are observed. We collected asexual and sexual polyps from 13 Central European water bodies and used restriction-site associated DNA sequencing to infer population structure. We detected high relatedness among populations and signs that hydras might spread with resting eggs through zoochory. We found no genetic structure with respect to mode of reproduction (asexual vs. sexual). On the other hand, clear evidence was found for phenotypic plasticity in mode of reproduction, as polyps inferred to be clones differed in reproductive mode. Moreover, we detected two cases of apparent sex change (males and females found within the same clonal lineages) in this species with supposedly stable sexes. Our study describes population genetic structure in Hydra for the first time, highlights the role of phenotypic plasticity in generating patterns of life history variation, and contributes to understanding the evolution of reproductive mode-dependent life history variation in coexisting asexual and sexual forms.

Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution.

Pulsatilla (Anemoneae, Ranunculaceae) is sister to Anemone s.s. and contains ca 40 perennial species of considerable horticultural and medical importance. We sequenced 31 of those species, plus nine subspecies, two cultivars and six outgroups, for two nuclear regions (high-copy nrITS and low-copy MLH1) and three plastid regions (rbcL, accD-psaI, trnL intron) in order to generate the first comprehensive species-level phylogeny of the genus. Phylogenetic trees were constructed using both concatenation-based (maximum likelihood and Bayesian inference) and coalescence methods. The better supported among the internal nodes were subjected to molecular clock dating and ancestral area reconstruction, and karyotypic characters identified by us using Fluorescence In Situ Hybridization were mapped across the tree. The preferred species tree from the coalescence analysis formed the basis of a new infrageneric classification based on monophyly plus degree of divergence. The earliest divergent of the three subgenera, Kostyczewianae, is represented by only a single species that is morphologically intermediate between Anemone s.s. and 'core' Pulsatilla. Subgenus Pulsatilla is considerably richer in species than its sister subgenus Preonanthus and contains three monophyletic sections. Species possessing nodding flowers and pectinately dissected leaves are phylogenetically derived compared with groups possessing erect flowers and palmately lobed leaves. Pulsatilla separated from Anemone s.s. at ca 25 Ma. Our results indicate a central Asian mountain origin of the genus and an initial diversification correlated with late Tertiary global cooling plus regional mountain uplift, aridification and consequent expansion of grasslands. The more rapid and extensive diversification within subgenus Pulsatilla began at ca 3 Ma and continued throughout the Quaternary, driven not only by major perturbations in global climate but also by well-documented polyploidy.

Iterative allogamy-autogamy transitions drive actual and incipient speciation during the ongoing evolutionary radiation within the orchid genus Epipactis (Orchidaceae).

BACKGROUND AND AIMS: The terrestrial orchid genus Epipactis has become a model system for the study of speciation via transitions from allogamy to autogamy, but close phylogenetic relationships have proven difficult to resolve through Sanger sequencing. METHODS: We analysed with restriction site-associated sequencing (RAD-seq) 108 plants representing 29 named taxa that together span the genus, focusing on section Epipactis. Our filtered matrix of 12 543 single nucleotide polymorphisms was used to generate an unrooted network and a rooted, well-supported likelihood tree. We further inferred genetic structure through a co-ancestry heat map and admixture analysis, and estimated inbreeding coefficients per sample. KEY RESULTS: The 27 named taxa of the ingroup were resolved as 11 genuine, geographically widespread species: four dominantly allogamous and seven dominantly autogamous. A single comparatively allogamous species, E. helleborine, is the direct ancestor of most of the remaining species, though one of the derived autogams has generated one further autogamous species. An assessment of shared ancestry suggested only sporadic hybridization between the re-circumscribed species. Taxa with the greatest inclination towards autogamy show less, if any, admixture, whereas the gene pools of more allogamous species contain a mixture alleles found in the autogams. CONCLUSIONS: This clade is presently undergoing an evolutionary radiation driven by a wide spectrum of genotypic, phenotypic and environmental factors. Epipactis helleborine has also frequently generated many local variants showing inclinations toward autogamy (and occasionally cleistogamy), best viewed as incipient speciation from within the genetic background provided by E. helleborine, which thus becomes an example of a convincingly paraphyletic species. Autogams are often as widespread and ecologically successful as allogams.

Integrating restriction site-associated DNA sequencing (RAD-seq) with morphological cladistic analysis clarifies evolutionary relationships among major species groups of bee orchids.

Background and Aims: Bee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus. Methods: At least two accessions of each of the ten species groups previously circumscribed from large-scale cloned nuclear ribosomal internal transcibed spacer (nrITS) sequencing were subjected to restriction site-associated sequencing (RAD-seq). The resulting matrix of 4159 single nucleotide polymorphisms (SNPs) for 34 accessions was used to construct an unrooted network and a rooted maximum likelihood phylogeny. A parallel morphological cladistic matrix of 43 characters generated both polymorphic and non-polymorphic sets of parsimony trees before being mapped across the RAD-seq topology. Key Results: RAD-seq data strongly support the monophyly of nine out of ten groups previously circumscribed using nrITS and resolve three major clades; in contrast, supposed microspecies are barely distinguishable. Strong incongruence separated the RAD-seq trees from both the morphological trees and traditional classifications; mapping of the morphological characters across the RAD-seq topology rendered them far more homoplastic. Conclusions: The comparatively high level of morphological homoplasy reflects extensive convergence, whereas the derived placement of the fusca group is attributed to paedomorphic simplification. The phenotype of the most recent common ancestor of the extant lineages is inferred, but it post-dates the majority of the character-state changes that typify the genus. RAD-seq may represent the high-water mark of the contribution of molecular phylogenetics to understanding evolution within Ophrys; further progress will require large-scale population-level studies that integrate phenotypic and genotypic data in a cogent conceptual framework.

Reconstructed historical distribution and phylogeography unravels non-steppic origin of Caucasotachea vindobonensis (Gastropoda: Helicidae).

Existing data on the phylogeography of European taxa of steppic provenance suggests that species were widely distributed during glacial periods but underwent range contraction and fragmentation during interglacials into "warm-stage refugia." Among the steppe-related invertebrates that have been examined, the majority has been insects, but data on the phylogeography of snails is wholly missing. To begin to fill this gap, phylogeographic and niche modeling studies on the presumed steppic snail Caucasotachea vindobonensis were conducted. Surprisingly, reconstruction of ancestral areas suggests that extant C. vindobonensis probably originated in the Balkans and survived there during the Late Pleistocene glaciations, with a more recent colonization of the Carpatho-Pannonian and the Ponto-Caspian regions. In the Holocene, C. vindobonensis colonized between the Sudetes and the Carpathians to the north, where its recent and current distribution may have been facilitated by anthropogenic translocations. Together, these data suggest a possible non-steppic origin of C. vindobonensis. Further investigation may reveal the extent to which the steppic snail assemblages consist partly of Holocene newcomers.

Molecular phylogeny and evolutionary history of the Eurasiatic orchid genus Himantoglossum s.l. (Orchidaceae).

BACKGROUND AND AIMS: Lizard orchids of the genus Himantoglossum include many of Eurasia's most spectacular orchids, producing substantial spikes of showy flowers. However, until recently the genus had received only limited, and entirely traditional, systematic study. The aim of the current work was to provide a more robust molecular phylogeny in order to better understand the evolutionary relationships among species of particular conservation concern. METHODS: All putative species of Himantoglossum s.l. were sampled across its geographical range. A large subsample of the 153 populations studied contributed to an initial survey of nuclear ribosomal internal transcribed spacer (nrITS) ribotypes. Smaller subsets were then sequenced for four plastid regions and the first intron of the low-copy-number nuclear gene LEAFY. Rooted using Steveniella as outgroup, phylogenetic trees were generated using parsimony and Bayesian methods from each of the three datasets, supplemented with a ribotype network. KEY RESULTS: The resulting trees collectively determined the order of branching of the early divergent taxa as Himantoglossum comperianum > H. robertianum group > H. formosum, events that also involved significant morphological divergence. Relaxed molecular clock dating suggested that these divergences preceded the Pleistocene glaciations (the origin of the H. robertianum group may have coincided with the Messinian salinity crisis) and occurred in Asia Minor and/or the Caucasus. Among more controversial taxa of the H. hircinum-jankae clade, which are only subtly morphologically divergent, topological resolution was poorer and topological incongruence between datasets was consequently greater. CONCLUSIONS: Plastid sequence divergence is broadly consistent with prior, morphologically circumscribed taxa and indicates a division between H. hircinum-adriaticum to the west of the Carpathians and H. jankae-caprinum (plus local endemics) to the east, a distinction also suggested by nrITS ribotypes. LEAFY phylogenies are less congruent with prior taxonomic arrangements and include one likely example of paralogy. Himantoglossum metlesicsianum fully merits its IUCN Endangered status. Potentially significant genetic variation was detected within Steveniella satyrioides, H. robertianum and H. hircinum. However, confident circumscription of the more derived species of Himantoglossum s.s., including local endemics of hybrid origin, must await future morphometric and population-genetic analyses.

The draft genome of Spiraea crenata L. (Rosaceae) - the first complete genome in tribe Spiraeeae.

Spiraea crenata L. is a deciduous shrub distributed across the Eurasian steppe zone. The species is of cultural and horticultural importance and occurs in scattered populations throughout its westernmost range. Currently, there is no genomic information on the tribe of Spiraeeae. Therefore we sequenced and assembled the whole genome of S. crenata using second- and third-generation sequencing and a hybrid assembly approach to expand genomic resources for conservation and support research on this horticulturally important lineage. In addition to the organellar genomes (the plastome and the mitochondrion), we present the first draft genome of the species with an estimated size of 220 Mbp, an N50 value of 7.7 Mbp, and a BUSCO score of 96.0%. Being the first complete genome in tribe Spiraeeae, this may not only be the first step in the genomic study of a rare plant but also a contribution to genomic resources supporting the study of biodiversity and evolutionary history of Rosaceae.

Breeding Polyommatus icarus Serves as a Large-Scale and Environmentally Friendly Source of Precisely Tuned Photonic Nanoarchitectures.

The colour of the butterfly wing serves as an important sexual and species-specific signal. Some species produce structural colouration by developing wing scales with photonic nanoarchitectures. These nanostructures are highly conservative, allowing only a ±10 nm peak wavelength deviation in the reflectance spectra of the blue structural colour in natural Common Blue (Polyommatus icarus) populations. They are promising templates of future artificial photonic materials and can be used in potential applications, too. In this work, we present methodology and infrastructure for breeding laboratory populations of Common Blue as a cost-effective and environmentally friendly source of nanostructures. Our technology enables the production of approximately 7500 wing samples, equivalent to 0.5-1 m2 of photonic nanoarchitecture surface within a year in a single custom-made insectarium. To ascertain the reliability of this method, we compared reflectance properties between different populations from distant geographic locations. We also provide genetic background of these populations using microsatellite genotyping. The laboratory population showed genetic erosion, but even after four generations of inbreeding, only minimal shifts in the structural colouration were observed, indicating that wild Common Blue populations may be a reliable source of raw material for photonic surfaces.

Phylogenomic insights into the polyphyletic nature of Altai falcons within eastern sakers (Falco cherrug) and the origins of gyrfalcons (Falco rusticolus).

The Altai falcon from Central Asia always attracted the attention of humans. Long considered a totemic bird in its native area, modern falconers still much appreciated this large-bodied and mighty bird of prey due to its rarity and unique look. The peculiar body characteristics halfway between the saker falcon (Falco cherrug) and the gyrfalcon (F. rusticolus) triggered debates about its contentious taxonomy. The weak phylogenetic signal associated with traditional genetic methods could not resolve this uncertainty. Here, we address the controversial evolutionary origin of Altai falcons by means of a genome-wide approach, Restriction-site Associated DNA sequencing, using sympatric eastern sakers falcons, allopatric western saker falcons and gyrfalcons as outgroup. This approach provided an unprecedented insight into the phylogenetic relationships of the studied populations by delivering 17,095 unlinked SNPs shedding light on the polyphyletic nature of Altai falcons within eastern sakers. Thus we concluded that the former must correspond to a low taxonomic rank, probably an ecotype or form of the latter. Also, we found that eastern sakers are paraphyletic without gyrfalcons, thus, these latter birds are best regarded as the direct sister lineage of the eastern sakers. This evolutionary relationship, corroborated also by re-analyzing the dataset with the inclusion of outgroup samples (F. biarmicus and F. peregrinus), put eastern sakers into a new light as the potential ancestral genetic source of high latitude and altitude adaptation in descendent populations. Finally, conservation genomic values hint at the stable genetic background of the studied saker populations.

Seasonal variation of genotypes and reproductive plasticity in a facultative clonal freshwater invertebrate animal (Hydra oligactis) living in a temperate lake.

Facultative sexual organisms combine sexual and asexual reproduction within a single life cycle, often switching between reproductive modes depending on environmental conditions. These organisms frequently inhabit variable seasonal environments, where favorable periods alternate with unfavorable periods, generating temporally varying selection pressures that strongly influence life history decisions and hence population dynamics. Due to the rapidly accelerating changes in our global environment today, understanding the population dynamics and genetic changes in facultative sexual populations inhabiting seasonal environments is critical to assess and prepare for additional challenges that will affect such ecosystems. In this study, we aimed at obtaining insights into the seasonal population dynamics of the facultative sexual freshwater cnidarian Hydra oligactis through a combination of restriction site-associated sequencing (RAD-Seq) genotyping and the collection of phenotypic data on the reproductive strategy of field-collected hydra strains in a standard laboratory environment. We reliably detected 42 MlGs from the 121 collected hydra strains. Most of MLGs (N = 35, 83.3%) were detected in only one season. Five MLGs (11.9%) were detected in two seasons, one (2.4%) in three seasons and one (2.4%) in all four seasons. We found no significant genetic change during the 2 years in the study population. Clone lines were detected between seasons and even years, suggesting that clonal lineages can persist for a long time in a natural population. We also found that distinct genotypes differ in sexual reproduction frequency, but these differences did not affect whether genotypes reappeared across samplings. Our study provides key insights into the biology of natural hydra populations, while also contributing to understanding the population biology of facultative sexual species inhabiting freshwater ecosystems.

Range-wide phylogeography of the flightless steppe beetle Lethrus apterus (Geotrupidae) reveals recent arrival to the Pontic Steppes from the west.

The Eurasian Steppe belt is one of the largest biomes in the Northern Hemisphere. We provide here a range-wide phylogeography of the flightless steppe beetle Lethrus apterus that inhabits the western part of the Steppe belt through the study of population-level variance of mitochondrial cytochrome oxidase I sequences and nuclear microsatellites. We detected a concordant geographic structure of genetic data with a significant isolation-by-distance pattern. We found more genetic variation in the western part of the area and identified Northern Bulgaria and the Pannonian Basin as possible refugia. Genetic clusters were separated by main rivers in the eastern part of the area. This implies west-to-east colonisation and argues for an evolutionarily recent arrival of this species to its current main distribution area, the Pontic Steppes. This contradicts the classical biogeographical wisdom that assumed an east-to-west colonisation pattern.

Concordance of the spectral properties of dorsal wing scales with the phylogeographic structure of European male Polyommatus icarus butterflies.

The males of more than 80% of the Lycaenidae species belonging to the tribe Polyommatini exhibit structural coloration on their dorsal wing surfaces. These colors have a role in reinforcement in prezygotic reproductive isolation. The species-specific colors are produced by the cellular self-assembly of chitin/air nanocomposites. The spectral position of the reflectance maximum of such photonic nanoarchitectures depends on the nanoscale geometric dimensions of the elements building up the nanostructure. Previous work showed that the coloration of male Polyommatus icarus butterflies in the Western and Eastern Palearctic exhibits a characteristic spectral difference (20 nm). We investigated the coloration and the de novo developed DNA microsatellites of 80 P. icarus specimens from Europe from four sampling locations, spanning a distance of 1621 km. Remarkably good concordance was found between the spectral properties of the blue sexual signaling color (coincident within 5 nm) and the population genetic structure as revealed by 10 microsatellites for the P. icarus species.

A comparison of microsatellites and genome-wide SNPs for the detection of admixture brings the first molecular evidence for hybridization between Mustela eversmanii and M. putorius (Mustelidae, Carnivora).

Introgressive hybridization can pose a serious threat to endangered species which have an overlapping distribution such as in the case of two polecat species, Mustela eversmanii and M. putorius, in Europe. The population size of steppe polecat is known to continuously shrink, whereas its sister species, the European polecat, is still somehow widespread. In this study, we perform an analysis using microsatellite (SSR) and genomic (SNP) data sets to identify natural hybrids between polecats. Four populations were genotyped for eight polymorphic SSR loci, and thousands of unlinked SNPs were generated using a reduced-representation sequencing approach, RADseq, to characterize the genetic make-up of allopatric populations and to identify hybrids in the sympatric area. We applied standard population genetic analyses to characterize the populations based on their SSR allelic frequency. Only a single sample out of 48 sympatric samples showed exact intermediacy that we identified as an F1 hybrid. Additionally, one specimen was indicated in the genomic data sets as backcrossed. Other backcrosses, indicated by SSRs, were not validated by SNPs, which highlights the higher efficacy of the genomic method to identify backcrossed individuals. The low frequency of hybridization suggests that the difference in habitat preference of the two species may act as a barrier to admixture. Therefore, it is apparently unlikely that polecat populations are threatened by significant introgression. The two species showed a clear genetic differentiation using both techniques. We found higher genetic diversity values in the sympatric steppe polecat population than in the other studies on polecat populations. Although M. putorius is a hunted species in most countries, genetic diversity values indicate worse conditions in Europe than in the protected sibling species M. eversmanii. Suspending hunting and providing protected status of the former seems to be reasonable and timely.

Development and characterization of novel SSR markers in the endangered endemic species Ferula sadleriana.

PREMISE: Ferula sadleriana (Apiaceae) is a polycarpic, perennial herb with a very limited range and small populations. It is listed as "endangered" on the IUCN Red List of Threatened Species. Microsatellite markers can contribute to conservation efforts by allowing the study of the genetic structure of its shrinking populations. METHODS AND RESULTS: We used a microsatellite-enriched library combined with an Illumina sequencing approach to develop simple sequence repeat markers in our target species. Out of 44 tested primer pairs, 22 provided specific products, and 13 showed heterologous amplification in the target species. Cross-species amplification was achieved at 20 and 19 loci in two congeneric species, F. soongarica and F. tatarica, respectively. CONCLUSIONS: The primers described here are the first tools that enable the population genetic characterization of F. sadleriana. Our results suggest a wider applicability in the genus Ferula.

Seed mass, hardness, and phylogeny explain the potential for endozoochory by granivorous waterbirds.

Field studies have shown that waterbirds, especially members of the Anatidae family, are major vectors of dispersal by endozoochory for a broad range of plants lacking a fleshy fruit, yet whose propagules can survive gut passage. Widely adopted dispersal syndromes ignore this dispersal mechanism, and we currently have little understanding of what traits determine the potential of angiosperms for endozoochory by waterbirds. Results from previous experimental studies have been inconsistent as to how seed traits affect seed survival and retention time in the gut and have failed to control for the influence of plant phylogeny. Using 13 angiosperm species from aquatic and terrestrial habitats representing nine families, we examined the effects of seed size, shape, and hardness on the proportion of seeds surviving gut passage through mallards (Anas platyrhynchos) and their retention time within the gut. We compiled a molecular phylogeny for these species and controlled for the nonindependence of taxa due to common descent in our analyses. Intact seeds from all 13 species were egested, but seed survival was strongly determined by phylogeny and by partial effects of seed mass and hardness (wet load): species with seeds harder than expected from their size, and smaller than expected from their loading, had greater survival. Once phylogeny was controlled for, a positive partial effect of seed roundness on seed survival was also revealed. Species with seeds harder than expected from their size had a longer mean retention time, a result retained after controlling for phylogeny. Our study is the first to demonstrate that seed shape and phylogeny are important predictors of seed survival in the avian gut. Our results demonstrate that the importance of controlling simultaneously for multiple traits and relating single traits (e.g., seed size) alone to seed survival or retention time is not a reliable way to detect important patterns, especially when phylogenetic effects are ignored.

Isolation and characterization of 15 SSR loci for the endangered European tetraploid species Gladiolus palustris (Iridaceae).

PREMISE: Gladiolus palustris (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species. METHODS AND RESULTS: We utilized a 454 pyrosequencing approach to identify simple sequence repeat (SSR) regions in a microsatellite-enriched library. Of all SSR regions, 46 were screened for specific PCR amplification, and 15 were found to be applicable in the target species. We found 1.62-3.08 alleles per population (effective alleles: 1.58-2.08) that indicated moderate to high genetic diversity values (0.28-0.44) in three pilot populations. Cross-species amplification was less effective in G. imbricatus and G. tenuis. CONCLUSIONS: The primers reported here can be used for the population genetic characterization of G. palustris. They will help us to better understand the conservation genetics of this highly endangered species.

In situ morphometric survey elucidates the evolutionary systematics of the Eurasian Himantoglossum clade (Orchidaceae: Orchidinae).

BACKGROUND AND AIMS: The charismatic Himantoglossum s.l. clade of Eurasian orchids contains an unusually large proportion of taxa that are of controversial circumscriptions and considerable conservation concern. Whereas our previously published study addressed the molecular phylogenetics and phylogeography of every named taxon within the clade, here we use detailed morphometric data obtained from the same populations to compare genotypes with associated phenotypes, in order to better explore taxonomic circumscription and character evolution within the clade. METHODS: Between one and 12 plants found in 25 populations that encompassed the entire distribution of the Himantoglossum s.l. clade were measured in situ for 51 morphological characters. Results for 45 of those characters were subjected to detailed multivariate and univariate analyses. KEY RESULTS: Multivariate analyses readily separate subgenus Barlia and subgenus Comperia from subgenus Himantoglossum, and also the early-divergent H. formosum from the less divergent remainder of subgenus Himantoglossum. The sequence of divergence of these four lineages is confidently resolved. Our experimental approach to morphometric character analysis demonstrates clearly that phenotypic evolution within Himantoglossum is unusually multi-dimensional. CONCLUSIONS: Degrees of divergence between taxa shown by morphological analyses approximate those previously shown using molecular analyses. Himantoglossum s.l. is readily divisible into three subgenera. The three sections of subgenus Himantoglossum-hircinum, caprinum and formosum-are arrayed from west to east with only limited geographical overlap. At this taxonomic level, their juxtaposition combines with conflict between contrasting datasets to complicate attempts to distinguish between clinal variation and the discontinuities that by definition separate bona fide species. All taxa achieve allogamy via food deceit and have only weak pollinator specificity. Artificial crossing demonstrates that intrinsic sterility barriers are weak. Although we have found evidence of gene flow among and within the three sections of subgenus Himantoglossum, reports of natural hybrids are surprisingly rare, probably because putative parents are sufficiently similar to questionably warrant the status of species. Phenological separation and increased xeromorphy characterise the origin of subgenus Barlia. Several individual morphological characters show evidence of parallel acquisition, and loss of features is especially frequent in floral markings among members of section caprinum. Detailed patterns of gain and loss demonstrate that several different categories of flower markings are inherited independently. Along with the dimensions of labellar lobes, these pigmentation characters have been over-emphasised in previous taxonomic treatments. Increased plant vigour was a crucial element of the origin of the genus, but vegetative characters underwent remarkably little subsequent evolution. Attempts to reconstruct hypothetical ancestors at internal nodes of the phylogeny are weakened by (a) uncertain placement of Steveniella as sister to Himantoglossum s.l. and (b) uncertain relationships among subtly different putative species within section caprinum. Nonetheless, heterochronic/allometric trends, ultimately limited by functional constraints, clearly dictate transitions between contrasting flower sizes and complex labellum shapes.