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

Evaluating the impact of scoring parameters on the structure of intra-specific genetic variation using RawGeno, an R package for automating AFLP scoring

<p>Abstract</p> <p>Background</p> <p>Since the transfer and application of modern sequencing technologies to the analysis of amplified fragment-length polymorphisms (AFLP), evolutionary biologists have included an increasing number of samples and markers in their studies. Although justified in this context, the use of automated scoring procedures may result in technical biases that weaken the power and reliability of further analyses.</p> <p>Results</p> <p>Using a new scoring algorithm, RawGeno, we show that scoring errors – in particular "bin oversplitting" (i.e. when variant sizes of the same AFLP marker are not considered as homologous) and "technical homoplasy" (i.e. when two AFLP markers that differ slightly in size are mistakenly considered as being homologous) – induce a loss of discriminatory power, decrease the robustness of results and, in extreme cases, introduce erroneous information in genetic structure analyses. In the present study, we evaluate several descriptive statistics that can be used to optimize the scoring of the AFLP analysis, and we describe a new statistic, the information content per bin (I_bin) that represents a valuable estimator during the optimization process. This statistic can be computed at any stage of the AFLP analysis without requiring the inclusion of replicated samples. Finally, we show that downstream analyses are not equally sensitive to scoring errors. Indeed, although a reasonable amount of flexibility is allowed during the optimization of the scoring procedure without causing considerable changes in the detection of genetic structure patterns, notable discrepancies are observed when estimating genetic diversities from differently scored datasets.</p> <p>Conclusion</p> <p>Our algorithm appears to perform as well as a commercial program in automating AFLP scoring, at least in the context of population genetics or phylogeographic studies. To our knowledge, RawGeno is the only freely available public-domain software for fully automated AFLP scoring, from electropherogram files to user-defined working binary matrices. RawGeno was implemented in an R CRAN package (with an user-friendly GUI) and can be found at <url>http://sourceforge.net/projects/rawgeno</url>.</p

A Total Evidence Approach to Understanding Phylogenetic Relationships and Ecological Diversity in Selanginella subg. Tetragonostachys

• Premise of the Study: Several members of Selaginella are renowned for their ability to survive extreme drought and “resurrect” when conditions improve. Many of these belong to subgenus Tetragonostachys , a group of ~45 species primarily found in North and Central America, with substantial diversity in the Sonoran and Chihuahuan Deserts. We evaluated the monophyly and the age of subgenus Tetragonostachys and assess how drought tolerance contributed to the evolution of this clade. • Methods: Our study included most Tetragonostachys species, using plastid and nuclear sequences, fossil and herbarium records, and climate variables to describe the species diversity, phylogenetic relationships, divergence times, and climatic niche evolution in the subgenus. • Key Results: We found that subgenus Tetragonostachys forms a monophyletic group sister to Selaginella lepidophylla and may have diverged from other Selaginella because of a Gondwanan–Laurasian vicariance event ca. 240 mya. The North American radiation of Tetragonostachys appears to be much more recent and to have occurred during the Early Cretaceous–late Paleocene interval. We identifi ed two signifi cant and nested ecological niche shifts during the evolution of Tetragonostachys associated with extreme drought tolerance and a more recent shift to cold climates. Our analyses suggest that drought tolerance evolved in the warm deserts of southwest North America and may have been advantageous for colonization of cold and dry boreal climates. • Conclusions: Our investigation provides a foundation for future research addressing the genomics of ecological niche evolution and the potential role of reticulate evolution in Selaginella subgenus Tetragonostachys .The authors thank P. Korall for sharing data. S. Buerki, N. Alvarez, and B. Marazzi provided technical assistance and valuable comments on the manuscript. N.A. was funded by the Swiss National Science Foundation (grant no. PBNEP3-132747). C.L.A. was funded by the Swedish Research Council

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques—Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species’ monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence

Biogeography and ecological diversification of a mayfly clade in New Guinea

Understanding processes that have driven the extraordinary high level of biodiversity in the tropics is a long-standing question in biology. Here we try to assess whether the large lineage richness found in a New Guinean clade of mayflies (Ephemeroptera), namely the Thraulus group (Leptophlebiidae) could be associated with the recent orogenic processes, by applying a combination of phylogenetic, biogeographic and ecological shift analyses. New Guinean representatives of the Thraulus group appear monophyletic, with the possible exception of a weakly-supported early-diverging clade from the Sunda Islands. Dating analyses suggest an Eocene origin of the Thraulus group, predating by several million years current knowledge on the origin of other New Guinean aquatic organisms. Biogeographic inferences indicate that 27 of the 28 inferred dispersals (96.4%) occurred during the Eocene, Oligocene and Miocene, while only one dispersal (3.6%) took place during the Pliocene-Pleistocene. This result contrasts with the higher number of altitudinal shifts (15 of 22; 68.2%) inferred during the Pliocene-Pleistocene time slice. Our study illustrates the role played by – potentially ecological - diversification along the elevation gradient in a time period concomitant with the establishment of high-altitude ecological niches, i.e., during orogenesis of the central New Guinean mountain range. This process might have taken over the previous main mode of diversification at work, characterized by dispersal and vicariance, by driving lineage divergence of New Guinean Leptophlebiidae across a wide array of habitats along the elevation gradient. Additional studies on organisms spanning the same elevation range as Thraulus mayflies in the tropics are needed to evaluate the potential role of the ecological opportunity or taxon cycles hypotheses in partly explaining the latitudinal diversity gradient

DiscoSnp-RAD: de novo detection of small variants for RAD-Seq population genomics

International audienceRestriction site Associated DNA Sequencing (RAD-Seq) is a technique characterized by the sequencing of specific loci along the genome that is widely employed in the field of evolutionary biology since it allows to exploit variants (mainly Single Nucleotide Polymorphism—SNPs) information from entire populations at a reduced cost. Common RAD dedicated tools, such as STACKS or IPyRAD, are based on all-vs-all read alignments, which require consequent time and computing resources. We present an original method, DiscoSnp-RAD, that avoids this pitfall since variants are detected by exploiting specific parts of the assembly graph built from the reads, hence preventing all-vs-all read alignments. We tested the implementation on simulated datasets of increasing size, up to 1,000 samples, and on real RAD-Seq data from 259 specimens of Chiastocheta flies, morphologically assigned to seven species. All individuals were successfully assigned to their species using both STRUCTURE and Maximum Likelihood phylogenetic reconstruction. Moreover, identified variants succeeded to reveal a within-species genetic structure linked to the geographic distribution. Furthermore, our results show that DiscoSnp-RAD is significantly faster than state-of-the-art tools. The overall results show that DiscoSnp-RAD is suitable to identify variants from RAD-Seq data, it does not require time-consuming parameterization steps and it stands out from other tools due to its completely different principle, making it substantially faster, in particular on large datasets

Naturalised Vitis Rootstocks in Europe and Consequences to Native Wild Grapevine

The genus Vitis is represented by several coexisting species in Europe. Our study focuses on naturalised rootstocks that originate in viticulture. The consequences of their presence to the landscape and to native European species (Vitis vinifera ssp. silvestris) are evaluated. This study compares ecological traits (seven qualitative and quantitative descriptors) and the genetic diversity (10 SSR markers) of populations of naturalised rootstocks and native wild grapevines. 18 large naturalised rootstock populations were studied in the Rhône watershed. Wild European grapevines are present in four main habitats (screes, alluvial forests, hedges, and streamside hedges). In contrast, naturalised rootstock populations are mainly located in alluvial forests, but they clearly take advantage of alluvial system dynamics and connectivity at the landscape level. These latter populations appear to reproduce sexually, and show a higher genetic diversity than Vitis vinifera ssp. silvestris. The regrouping of naturalised rootstocks in interconnected populations tends to create active hybrid swarms of rootstocks. The rootstocks show characters of invasive plants. The spread of naturalised rootstocks in the environment, the acceleration of the decline of the European wild grapevine, and the propagation of genes of viticultural interest in natural populations are potential consequences that should be kept in mind when undertaking appropriate management measures

Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression

Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sopfixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds

Hybridization as a threat in climate relict Nuphar pumila (Nymphaeaceae)

Field studies and conceptual work on hybridization-mediated extinction risk in climate relicts are extremely rare. Nuphar pumila (Nymphaeaceae) is one of the most emblematic climate relicts in Europe with few isolated populations in the Alpine arc. The extent of introgression with related lowland and generalist species Nupharlutea has never been studied using molecular methods. All biogeographical regions where N.pumila naturally occurs in the neighbourhood of the Alpine arc were sampled and studied using nuclear microsatellite markers. Furthermore, we used forward-in-time simulations and Approximate Bayesian Computation to check whether an introgression scenario fits with the observed admixture patterns and estimated the demographic parameters associated with this process. Our study confirms ongoing hybridization between N.pumila and N.lutea and validates it by the use of population models. More than 40 % of investigated N.pumila individuals were admixed and hybrids were found in over 60 % of studied populations. The introgression is bidirectional and is most likely a result of very recent gene flow. Our work provides strong evidence for rapid extinction risk and demographic swamping between specialized climatic relicts and closely related generalists. The remaining pure populations of N.pumila are rare in the Alpine arc and deserve high conservation priority