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.

Summary1. Herbarium collections contain long-term data for a wide range of taxa and provide unique opportunities to evaluate the importance of life-history components in driving species-specific responses to climate change. In this paper, we analyse the relationships between change in flowering dates and life-history traits within a phylogenetic framework. The study is based on an extensive data set of herbarium specimens of orchids collected in Hungary between 1837 and 2009, supplemented by recent field observations . 2. Of the 39 taxa investigated, 31 (79%) showed apparent advancement in mean flowering time. Among these, advancement was statistically significant in nine taxa. The rest (eight taxa) showed non-significant delays in flowering. Averaging across all taxa, flowering time advanced by 3 days (3.8% of flowering period) during the last 50 years compared with the period before 1960. In taxa showing significant advancement, flowering times advanced by 7.7 days (8.6% of the flowering period). The most extreme advancement was 13.9 days. 3. Multivariate models were used to evaluate ways in which life history may affect phenological responses to climate change. Pollination mode (i.e. deceptive vs. rewarding vs. autogamous), life span (i.e. short-lived vs. long-lived), biogeographical distribution type (i.e. Mediterranean vs. nonMediterranean) and flowering time (i.e. mean date of blooming) emerged as important factors that influence changes in flowering through time. Phylogenetic relatedness did not predict phenological response. The strongest response was observed in orchids that flower relatively early in spring, exhibit an autogamous or deceptive pollination mechanism, have a long life span and possess a Mediterranean centre of distribution. 4. Synthesis. Our investigation demonstrates that the majority of Hungarian orchids have shifted their yearly mean flowering to earlier dates during the past 50 years. Certain life-history traits, but not phylogenetic relatedness, were found to be important in predicting climatic responsiveness in European terrestrial orchids.

The phylogeography of species associated with European steppes and extrazonal xeric grasslands is poorly understood. This paper summarizes the results of recent studies on the phylogeography and conservation genetics of animals (20 taxa of beetles, butterflies, reptiles and rodents) and flowering plants (18 taxa) of such, ''steppic'' habitats in Eastern Central Europe. Most species show a similar phylogeographic pattern: relatively high genetic similarity within regional groups of populations and moderate-to-high genetic distinctiveness of populations from currently isolated regions located in the studied area. This distinctiveness of populations suggests a survival here during glacial maxima, including areas north of the Bohemian Massif-Carpathians arc. Steppic species generally do not follow the paradigmatic patterns known for temperate biota (south-north ''contraction-expansion''), but to some extent are similar to those of arctic-alpine taxa. There are three main groups of taxa within Eastern Central Europe that differ in their contemporary distribution pattern, which may reflect historical origin and expansion routes. Present diversity patterns of the studied steppic species suggest that they share a unique genetic signature and distinct assemblages exist in each of the now isolated areas rich in steppic habitats. At least some of these areas probably act as present ''interglacial refugia'' for steppic species. This study strongly supports the need to protect steppic species throughout their entire ranges in the region, as the continuous destruction of steppic habitats in some areas may lead not only to the disappearance of local populations, but also to the extinction of unique evolutionary units.

Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest.

Background and AimsBee 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.MethodsAt 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 ResultsRAD-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.ConclusionsThe 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.

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.

for moisture (EIVM) was used to describe plant hygrophily. Phylogenetic analysis was carried out, 37 at a broader taxonomic scale on 128 species, and then refined on strong isoprene emitters (Salix and 38Populus species) based on isoprene synthase gene sequences (IspS). 39 40Results. Isoprene emitters were significantly more common, and isoprene emission was higher in 41 hygrophilous EIVM classes, whereas monoterpene emitters were more widespread, and 42 monoterpene emission was higher, in xeric classes. However, when controlling for phylogeny, 43 isoprene emission was not associated with EIVM, possibly due to the large presence of Salicaceae 44 among hygrophilous isoprene emitters. Moreover, the distribution of isoprene emitters among 45 EIVM classes was not related to IspS-based phylogenesis in Populus and Salix, suggesting that the 46 gene has not undergone evolution linked to ecological pressure. In contrast, monoterpene emission 47 pattern is independent of phylogeny, suggesting that the evolution of monoterpenes is associated 48 with transitions to more xeric habitats. 49 50 Main conclusions. Our results reveal an interesting ecological pattern linking isoprenoids and 51 water availability. The idea is surmised that isoprene is a trait that i) evolved in plants adapted to 52 high water availability; ii) is replaced by more effective protection mechanisms, e.g. more stable 53 isoprenoids, in plants adapting to more xeric environments; iii) being strongly constrained by 54 phylogeny, persists in Salicaceae adapted to more xeric environments. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Leaves of many woody and perennial plants constitutively emit volatile isoprenoids (isoprene and 66 monoterpenes) to the atmosphere, at rates that often exceed 1-2% of the photosynthetic carbon 67 fixation, especially in stressed leaves (Loreto & Schnitzler 2010). Isoprene and monoterpenes are 68 formed from photosynthetic metabolism in the chloroplasts (Loreto & Schnitzler 2010). Generally, 69 either isoprene or monoterpenes are emitted but not both (Harrison et al. 2013). However, some 70 species (e.g. Myrtales) show significant storage of monoterpenes in specialized structures and these 71 species can emit both isoprene and monoterpenes (Niinemets et al. 2004). 72Isoprene is believed to play a role against thermal and oxidative stresses, possibly because of the 73 capacity of this molecule to stabilize thylakoidal membranes (Singsaas et al. 1997, Velikova et al. The emission of isoprene and monoterpenes is widespread across plant families (Harley et al. 79 1999). A recent study has indicated a strong phylogeographic signal for monoterpenes; the emission 80 of monoterpenes is qualitatively different in cork oaks across their distribution range in Europe 81 (Loreto et al. 2009). Alien species of Hawaii emit more monoterpenes than native one...

Background and AimsThe 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.MethodsBetween 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 ResultsMultivariate 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.ConclusionsDegrees 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 t...