Thin film structural color is widespread in slime molds (Myxomycetes, Amoebozoa).

Brilliant colors in nature arise from the interference of light with periodic nanostructures resulting in structural color. While such biological photonic structures have long attracted interest in insects and plants, they are little known in other groups of organisms. Unexpected in the kingdom of Amoebozoa, which assembles unicellular organisms, structural colors were observed in myxomycetes, an evolutionary group of amoebae forming macroscopic, fungal-like structures. Previous work related the sparkling appearance of Diachea leucopodia to thin film interference. Using optical and ultrastructural characterization, we here investigated the occurrence of structural color across 22 species representing two major evolutionary clades of myxomycetes including 14 genera. All investigated species showed thin film interference at the peridium, producing colors with hues distributed throughout the visible range that were altered by pigmentary absorption. A white reflective layer of densely packed calcium-rich shells is observed in a compound peridium in Metatrichia vesparium, whose formation and function are still unknown. These results raise interesting questions on the biological relevance of thin film structural colors in myxomycetes, suggesting they may be a by-product of their reproductive cycle.

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Contrasting evolutionary origins of two mountain endemics: Saxifraga wahlenbergii (Western Carpathians) and S. styriaca (Eastern Alps).

BACKGROUND: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock. RESULTS: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage. CONCLUSIONS: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

Pollen metabarcoding as a tool for tracking long-distance insect migrations.

Insects account for a large portion of Earth's biodiversity and are key players for ecosystems, notably as pollinators. While insect migration is suspected to represent a natural phenomenon of major importance, remarkably little is known about it, except for a few flagship species. The reason for this situation is mainly due to technical limitations in the study of insect movement. Here, we propose using metabarcoding of pollen carried by insects as a method for tracking their migrations. We developed a flexible and simple protocol allowing efficient multiplexing and not requiring DNA extraction, one of the most time-consuming part of metabarcoding protocols, and apply this method to the study of the long-distance migration of the butterfly Vanessa cardui, an emerging model for insect migration. We collected 47 butterfly samples along the Mediterranean coast of Spain in spring and performed metabarcoding of pollen collected from their bodies to test for potential arrivals from the African continent. In total, we detected 157 plant species from 23 orders, most of which (82.8%) were insect-pollinated. Taxa present in Africa-Arabia represented 73.2% of our data set, and 19.1% were endemic to this region, strongly supporting the hypothesis that migratory butterflies colonize southern Europe from Africa in spring. Moreover, our data suggest that a northwards trans-Saharan migration in spring is plausible for early arrivals (February) into Europe, as shown by the presence of Saharan floristic elements. Our results demonstrate the possibility of regular insect-mediated transcontinental pollination, with potential implications for ecosystem functioning, agriculture and plant phylogeography.

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.

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Rhizomarasmius epidryas (Physalacriaceae): phylogenetic placement of an arctic-alpine fungus with obligate saprobic affinity to Dryas spp.

As a part of a large-scale biogeographical study we examined the evolutionary relationships and taxonomic position of Marasmius epidryas, one of the most typical circumpolar arctic-alpine fungi, characterized by a specific, saprobic affinity to dead tissues of Dryas spp. A phylogenetic analysis based on nLSU and RPB2 DNA regions unequivocally indicated the phylogenetic placement of this species within the Physalacriaceae. The Bayesian MCMCMC analysis as well as other inference methods tested (ML, NJ) revealed a well supported affinity of M. epidryas to Rhizomarasmius pyrrhocephalus, type species of a recently circumscribed genus, Rhizomarasmius. As a consequence, based on these results, we introduce a new combination, Rhizomarasmius epidryas (Kühner ex A. Ronikier) A. Ronikier and M. Ronikier. Thus our results demonstrate that neither the traditional taxonomic placement of the fungus in genus Marasmius nor the recent transfer into genus Mycetinis are phylogenetically correct. In contrast they support the importance of the third lineage of the polyphyletic Marasmius s. l., having evolutionary links with taxa forming the Physalacriaceae clade of agaricoid fungi. In addition the lineage of Rhizomarasmius was confirmed to be closely related to the representatives of Gloiocephala, comprising small, often narrowly specialized saprobic species previously also classified within Marasmius s. l.

Rediscovery of Alnicola cholea (Cortinariaceae): taxonomic revision and description of its mycorrhiza with Polygonum viviparum (Polygonaceae).

Alnicola cholea, a little-known species so far reported only from the two original localities in the French Alps, is redefined here based on revision of herbarium materials and studies of recent field collections. A detailed morphological and anatomical description of fruit bodies of Alnicola cholea, including a discussion on its taxonomic status and distribution data is provided. Due to the unique combination of characters of Alnicola cholea within the genus, a new monospecific section is introduced for this species: Alnicola sect. Cholea, sect. nov. Mycorrhizal symbiosis of A. cholea with an arcticalpine plant Polygonum viviparum was observed in the Tatra Mountains (Poland). A description of these mycorrhizae is given, providing first detailed data on an identified herbaceous plant mycorrhiza.

The use of AFLP markers in conservation genetics--a case study on Pulsatilla vernalis in the Polish lowlands.

Pulsatilla vernalis is a rare species in the Polish lowlands, strongly threatened by anthropogenic disturbance of its habitats. A grave decrease in its populations has been observed during the past 60-80 years (analogous populations in Eastern Austria and the Czech Republic are almost or completely extinct). An analysis of the genetic diversity of populations in the Polish lowlands was performed to estimate its level and distribution. The AFLP method was used for the study of seven populations. An analysis using five pairs of selective primers revealed 446 scorable fragments; 62.1% of them were polymorphic. The average gene diversity indices was 0.17 (the mean value for all the populations), ranging from 0.139 to 0.204. A weak relationship between diversity and population size was revealed. Most of the genetic diversity was contributed to by the within-population level (AMOVA) and only a weak geographical structure was shown by UPGMA clustering. Four populations formed population-specific clusters while three others (from one region) were intermixed. These preliminary results show a moderate genetic diversity of the studied populations, which was still rather high when compared with their size. This result, together with the low between-population differentiation in the region, suggests that these populations are the remnants of larger populations that, only a few decades ago, were much less isolated.