Microsatellite Content in 397 Nuclear Exons and Their Flanking Regions in the Fern Family Ophioglossaceae.

Microsatellites or SSRs are small tandem repeats that are 1-6 bp long. They are usually highly polymorphic and form important portions of genomes. They have been extensively analyzed in humans, animals and model plants; however, information from non-flowering plants is generally lacking. Here, we examined 29 samples of Ophioglossaceae ferns, mainly from the genera Botrychium and Sceptridium. We analyzed the SSR distribution, density and composition in almost 400 nuclear exons and their flanking regions. We detected 45 SSRs in exons and 1475 SSRs in the flanking regions. In the exons, only di-, tri- and tetranucleotides were found, and all of them were 12 bp long. The annotation of the exons containing SSRs showed that they were related to various processes, such as metabolism, catalysis, transportation or plant growth. The flanking regions contained SSRs from all categories, with the most numerous being dinucleotides, followed by tetranucleotides. More than one-third of all the SSRs in the flanking regions were 12 bp long. The SSR densities in the exons were very low, ranging from 0 to 0.07 SSRs/kb, while those in the flanking regions ranged from 0.24 to 0.81 SSRs/kb; and those in the combined dataset ranged from 0.2 to 0.81 SSRs/kb. The majority of the detected SSRs in the flanking regions were polymorphic and present at the same loci across two or more samples but differing in the number of repeats. The SSRs detected here may serve as a basis for further population genetic, phylogenetic or evolutionary genetic studies, as well as for further studies focusing on SSRs in the genomes and their roles in adaptation, evolution and diseases.

Population Genetic Structure and Diversity of Cryptic Species of the Plant Genus Macrocarpaea (Gentianaceae) from the Tropical Andes.

The Pleistocene climatic oscillations (PCO) that provoked several cycles of glacial-interglacial periods are thought to have profoundly affected species distribution, richness and diversity around the world. While the effect of the PCO on population dynamics at temperate latitudes is well known, considerable questions remain about its impact on the biodiversity of neotropical mountains. Here, we use amplified fragment length polymorphism molecular markers (AFLPs) to investigate the phylogeography and genetic structure of 13 plant species belonging to the gentian genus Macrocarpaea (Gentianaceae) in the tropical Andes. These woody herbs, shrubs or small trees show complex and potentially reticulated relationships, including cryptic species. We show that populations of M. xerantifulva in the dry system of the Rio Marañón in northern Peru have lower levels of genetic diversity compared to other sampled species. We suggest that this is due to a recent demographic bottleneck resulting from the contraction of the montane wet forests into refugia because of the expansion of the dry system into the valley during the glacial cycles of the PCO. This may imply that the ecosystems of different valleys of the Andes might have responded differently to the PCO.

Organellar phylogenomics of Ophioglossaceae fern genera.

Previous phylogenies showed conflicting relationships among the subfamilies and genera within the fern family Ophioglossaceae. However, their classification remains unsettled where contrasting classifications recognize four to 15 genera. Since these treatments are mostly based on phylogenetic evidence using limited, plastid-only loci, a phylogenomic understanding is actually necessary to provide conclusive insight into the systematics of the genera. In this study, we have therefore compiled datasets with the broadest sampling of Ophioglossaceae genera to date, including all fifteen currently recognized genera, especially for the first time the South African endemic genus Rhizoglossum. Notably, our comprehensive phylogenomic matrix is based on both plastome and mitogenome genes. Inferred from the coding sequences of 83 plastid and 37 mitochondrial genes, a strongly supported topology for these subfamilies is presented, and is established by analyses using different partitioning approaches and substitution models. At the generic level, most relationships are well resolved except for few within the subfamily Ophioglossoideae. With this new phylogenomic scheme, key morphological and genomic changes were further identified along this backbone. In addition, we confirmed numerous horizontally transferred (HGT) genes in the genera Botrypus, Helminthostachys, Mankyua, Sahashia, and Sceptridium. These HGT genes are most likely located in mitogenomes and are predominately donated from angiosperm Santalales or non-Ophioglossaceae ferns. By our in-depth searches of the organellar genomes, we also provided phylogenetic overviews for the plastid and mitochondrial MORFFO genes found in these Ophioglossaceae ferns.

Hybridization fuelled diversification in Spialia butterflies.

The importance of hybridization and introgression is well documented in the evolution of plants but, in insects, their role is not fully understood. Given the fact that insects are the most diverse group of organisms, assessing the impact of reticulation events on their evolution may be key to comprehend the emergence of such remarkable diversity. Here, we used an insect model, the Spialia butterflies, to gather genomic evidence of hybridization as a promoter of novel diversity. By using double-digest RADseq (ddRADseq), we explored the phylogenetic relationships between Spialia orbifer, S. rosae and S. sertorius, and documented two independent events of interspecific gene flow. Our data support that the Iberian endemism S. rosae probably received genetic material from S. orbifer in both mitochondrial and nuclear DNA, which could have contributed to a shift in the ecological preferences of S. rosae. We also show that admixture between S. sertorius and S. orbifer probably occurred in Italy. As a result, the admixed Sicilian populations of S. orbifer are differentiated from the rest of populations both genetically and morphologically, and display signatures of reproductive character displacement in the male genitalia. Additionally, our analyses indicated that genetic material from S. orbifer is present in S. sertorius along the Italian Peninsula. Our findings add to the view that hybridization is a pervasive phenomenon in nature and in butterflies in particular, with important consequences for evolution due to the emergence of novel phenotypes.

The Evolution of Trait Disparity during the Radiation of the Plant Genus Macrocarpaea (Gentianaceae) in the Tropical Andes.

The evolutionary processes responsible for the extraordinary diversity in the middle elevation montane forests of the Tropical Andes (MMF; 1000-3500 m) remain poorly understood. It is not clear whether adaptive divergence, niche conservatism or geographical processes were the main contributors to the radiation of the respective lineages occurring there. We investigated the evolutionary history of plant lineages in the MMF. We used the vascular plant genus Macrocarpaea (Gentianaceae) as a model, as it consists of 118 morphologically diverse species, a majority of which are endemic to the MMF. We used a time-calibrated molecular phylogeny and morphological and climatic data to compare a set of evolutionary scenarios of various levels of complexity in a phylogenetic comparative framework. In this paper, we show that the hypothesis of adaptive radiation for Macrocarpaea in the MMF is unlikely. The genus remained confined to the upper montane forests (UMF > 1800 m) during more than a half of its evolutionary history, possibly due to evolutionary constraints. Later, coinciding with the beginning of the Pleistocene (around 2.58 Ma), a phylogenetically derived (recently branching) clade, here referred to as the M. micrantha clade (25 species), successfully colonized and radiated in the lower montane forests (LMF < 1800 m). This colonization was accompanied by the evolution of a new leaf phenotype that is unique to the species of the M. micrantha clade that likely represents an adaptation to life in this new environment (adaptive zone). Therefore, our results suggest that niche conservatism and geographical processes have dominated most of the diversification history of Macrocarpaea, but that a rare adaptive divergence event allowed a transition into a new adaptive zone and enabled progressive radiation in this zone through geographical processes.

New genetic markers for Sapotaceae phylogenomics: More than 600 nuclear genes applicable from family to population levels.

Some tropical plant families, such as the Sapotaceae, have a complex taxonomy, which can be resolved using Next Generation Sequencing (NGS). For most groups however, methodological protocols are still missing. Here we identified 531 monocopy genes and 227 Short Tandem Repeats (STR) markers and tested them on Sapotaceae using target capture and NGS. The probes were designed using two genome skimming samples from Capurodendron delphinense and Bemangidia lowryi, both from the Tseboneae tribe, as well as the published Manilkara zapota transcriptome from the Sapotoideae tribe. We combined our probes with 261 additional ones previously published and designed for the entire angiosperm group. On a total of 792 low-copy genes, 638 showed no signs of paralogy and were used to build a phylogeny of the family with 231 individuals from all main lineages. A highly supported topology was obtained at high taxonomic ranks but also at the species level. This phylogeny revealed the existence of more than 20 putative new species. Single nucleotide polymorphisms (SNPs) extracted from the 638 genes were able to distinguish lineages within a species complex and to highlight geographical structuration. STR were recovered efficiently for the species used as reference (C. delphinense) but the recovery rate decreased dramatically with the phylogenetic distance to the focal species. Altogether, the new loci will help reaching a sound taxonomic understanding of the family Sapotaceae for which many circumscriptions and relationships are still debated, at the species, genus and tribe levels.

Rapid colour shift by reproductive character displacement in Cupido butterflies.

Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction-site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies.

Targeted Capture of Hundreds of Nuclear Genes Unravels Phylogenetic Relationships of the Diverse Neotropical Palm Tribe Geonomateae.

The tribe Geonomateae is a widely distributed group of 103 species of Neotropical palms which contains six ecologically important understory or subcanopy genera. Although it has been the focus of many studies, our understanding of the evolutionary history of this group, and in particular of the taxonomically complex genus Geonoma, is far from complete due to a lack of molecular data. Specifically, the previous Sanger sequencing-based studies used a few informative characters and partial sampling. To overcome these limitations, we used a recently developed Arecaceae-specific target capture bait set to undertake a phylogenomic analysis of the tribe Geonomateae. We sequenced 3,988 genomic regions for 85% of the species of the tribe, including 84% of the species of the largest genus, Geonoma. Phylogenetic relationships were inferred using both concatenation and coalescent methods. Overall, our phylogenetic tree is highly supported and congruent with taxonomic delimitations although several morphological taxa were revealed to be non-monophyletic. It is the first time that such a large genomic dataset is provided for an entire tribe within the Arecaceae. Our study lays the groundwork not only for detailed macro- and micro-evolutionary studies within the group, but also sets a workflow for understanding other species complexes across the tree of life.

A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris.

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double-digest restriction-site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex-biased asymmetries, NUMTs, natural selection, introgression or Wolbachia-mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.

Genomics of extreme ecological specialists: multiple convergent evolution but no genetic divergence between ecotypes of Maculinea alcon butterflies.

Biotic interactions are often acknowledged as catalysers of genetic divergence and eventual explanation of processes driving species richness. We address the question, whether extreme ecological specialization is always associated with lineage sorting, by analysing polymorphisms in morphologically similar ecotypes of the myrmecophilous butterfly Maculinea alcon. The ecotypes occur in either hygric or xeric habitats, use different larval host plants and ant species, but no significant distinctive molecular traits have been revealed so far. We apply genome-wide RAD-sequencing to specimens originating from both habitats across Europe in order to get a view of the potential evolutionary processes at work. Our results confirm that genetic variation is mainly structured geographically but not ecologically - specimens from close localities are more related to each other than populations of each ecotype from distant localities. However, we found two loci for which the association with xeric versus hygric habitats is supported by segregating alleles, suggesting convergent evolution of habitat preference. Thus, ecological divergence between the forms probably does not represent an early stage of speciation, but may result from independent recurring adaptations involving few genes. We discuss the implications of these results for conservation and suggest preserving biotic interactions and main genetic clusters.

Haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) and potential insect vectors of their transmission.

Sedentary bird species are suitable model hosts for identifying potential vectors of avian blood parasites. We studied haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) in the Ore Mountains of the Czech Republic using molecular detection methods. Sex of owl nestlings was scored using molecular sexing based on fragment analysis of PCR-amplified CHD1 introns. Observed infection prevalences in nestlings and adult owls were 51 and 86 %, respectively. Five parasite lineages were detected. Most of the infections comprised the Leucocytozoon AEFUN02 and STOCC06 lineages that probably refer to distinct Leucocytozoon species. Other lineages were detected only sporadically. Mixed infections were found in 49 % of samples. The main factor affecting the probability of infection was host age. No effect of individual sex on infection probability was evidenced. The youngest infected nestling was 12 days old. High parasite prevalence in the Tengmalm's Owl nestlings suggests that insect vectors must enter nest boxes to transmit parasites before fledging. Hence, we placed sticky insect traps into modified nest boxes, collected potential insect vectors, and examined them for the presence of haemosporidian parasites using molecular detection. We trapped 201 insects which were determined as biting midges from the Culicoides genus and two black fly species, Simulium (Nevermannia) vernum and Simulium (Eusimulium) angustipes. Six haemosporidian lineages were detected in the potential insect vectors, among which the Leucocytozoon lineage BT2 was common to the Tengmalm's Owl and the trapped insects. However, we have not detected the most frequently encountered Tengmalm's Owl Leucocytozoon lineages AEFUN02 and STOCC06 in insects.

Hidden diversity in Senegalese bats and associated findings in the systematics of the family Vespertilionidae.

INTRODUCTION: The Vespertilionidae is the largest family of bats, characterized by high occurrence of morphologically convergent groups, which impedes the study of their evolutionary history. The situation is even more complicated in the tropics, where certain regions remain under-sampled. RESULTS: Two hundred and thirteen vespertilionid bats from Senegal (West Africa) were studied with the use of non-differentially stained karyotypes and multi-locus sequence data analysed with maximum likelihood and Bayesian methods. These bats were identified as 10 different taxa, five of which were distinctive from their nominate species (Pipistrellus hesperidus, Nycticeinops schlieffenii, Scotoecus hirundo, Neoromicia nana and N. somalica), based on both karyotypes and molecular data. These five cryptic taxa are unrelated, suggesting that these West African populations have long been isolated from other African regions. Additionally, we phylogenetically analysed 166 vespertilionid taxa from localities worldwide using GenBank data (some 80% of the genera of the family) and 14 representatives of closely related groups, together with our Senegalese specimens. The systematic position of several taxa differed from previous studies and the tribes Pipistrellini and Vespertilionini were redefined. The African Pipistrellus rueppellii was basal to the Pipistrellus/Nyctalus clade and the Oriental species Glischropus tylopus was basal to the East Asian pipistrelles within the tribe Pipistrellini. The African genus Neoromicia was confirmed to be diphyletic. Based on GenBank data, Eptesicus was polyphyletic, with the Asian E. nasutus and E. dimissus both supported as phylogenetically distinct from the Eptesicus clade. The subfamily Scotophilinae was confirmed as one of the basal branches of Vespertilionidae. CONCLUSIONS: New taxa and new systematic arrangements show that there is still much to resolve in the vespertilionids and that West Africa is a biogeographic hotspot with more diversity to be discovered.