Kathrin Theissinger

Patterns of population structure in two closely related, partially sympatric caddisflies in Eastern Europe: historic introgression, limited dispersal, and cryptic diversity1

Abstract We used 2 caddisflies, Drusus discolor and Drusus romanicus, to test explicitly whether closely related species that occupy similar niches and occur in partial sympatry maintain comparable population structure and share a similar population history. We used mitochondrial sequence data to analyze and compare the population structure and the phylogeography of 105 specimens of D. discolor and 74 individuals of D. romanicus collected in southeastern Europe. We examined the relationship between both species with phylogenetic inference and coalescent modeling and used the results to assign larvae to species. We were able to unambiguously assign larvae to species level based on clearly defined association criteria within a phylogenetic analysis of all specimens. The species were closely related and not reciprocally monophyletic in our haplotype phylogeny. One D. romanicus haplotype from the Bucegi Mountains was nested within D. discolor, a result that suggests isolation with migration, introgression, or incomplete lineage sorting between the 2 species. For each species, we examined population genetic structure with median joining networks, analysis of molecular variance (AMOVA), exact tests of population differentiation, and Mantel tests of isolation by distance. We used tests for selective neutrality (Tajimas D, Fus F) to infer potential population growth and expansion. Species differed in their genetic population structure. Drusus discolor had haplotype overlap among several mountain ranges in the study region. No D. romanicus haplotypes were shared among any regions examined, and levels of divergence between haplotype clades exceeded those of D. discolor by a factor of up to 2.1. The different degree of population differentiation and divergence of both species probably reflects different Pliocene/Pleistocene population histories and might be related to differences in dispersal capabilities or competitive exclusion of D. romanicus by D. discolor in the mountain ranges north and west of the Western Carpathians. Based on our results, we discuss the importance of the Carpathian Mountains and Bulgarian highlands as Pliocene/Pleistocene refugia and centers of diversification.

Modelling range shifts and assessing genetic diversity distribution of the montane aquatic mayfly Ameletus inopinatus in Europe under climate change scenarios

Genetic diversity is one of the most important criteria to identify unique populations for conservation purposes. In this study we analyze the genetic population structure of the endangered montane mayfly Ameletus inopinatus in its European range. The species is restricted to unpolluted cold-water streams, and exhibits an insular distribution across highlands of Central Europe and a more continuous distribution across Fennoscandia and Northern Euro-Siberia. We genotyped 389 individuals from 31 populations for eight highly polymorphic microsatellite loci to investigate genetic diversity and population structure within and among European mountain ranges. Genetic diversity of A. inopinatus decreases along an east–west gradient in Central Europe and along a north–south gradient in Fennoscandia, respectively. Centres of exceptionally high genetic diversity are located in the Eastern Alps (Andertal Moor, Austria), the High Tatra, the Beskides, the Sudety Mountains and the Eastern German Highlands. Species distribution modelling for 2080 projects major regional habitat loss, particularly in Central Europe mountain ranges. By relating these range shifts to our population genetic results, we identify conservation units primarily in Eastern Europe, that if preserved would maintain high levels of the present-day genetic diversity and continue to provide long-term suitable habitat under future climate warming scenarios.

The Carpathians as a Major Diversity Hotspot in Europe

The Carpathians are one of the major mountain ranges of Europe, but still one of its least studied regions. It is increasingly recognized that they played a major role in the formation and Pleistocene survival of numerous continental, arctic, and arctic–alpine taxa. Many endemic taxa have been described from these mountains. The number of phylogeographic/phylogenetic studies covering at least partially the Carpathians is also increasing. These studies reveal unevenly distributed genetic and taxonomic diversity. In this work, we analyse population genetic structures in the Carpathians revealed by case studies on aquatic insects, comparing them to existing literature data on plants, butterflies, vertebrates, and the distribution of several microendemics. The distribution of molecular lineages and/or microendemics show strong biogeographic structures within the Carpathians. The overlap between the distribution barriers of microendemics and intraspecific molecular lineages suggests that isolation of populations among the major Carpathian ranges (Western Carpathians, Eastern Carpathians, Apuseni Mts, Southern Carpathians, Banat and Serbian Carpathians) played a major role in promoting Carpathian diversity.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.

Determination of the minimum number of microsatellite markers for individual genotyping in wild boar (Sus scrofa) using a test with close relatives

In the context of developing a noninvasive, practicable method for population size estimation in wild boar, we present a stepwise procedure to reduce the number of required microsatellite markers for individual genotyping. Step1: an initial marker set of 12 microsatellite loci was tested for species specificity with nontarget DNA and resulted in an exclusion of two markers. Step 2: a variability test regarding heterozygosity and deviations from Hardy–Weinberg equilibrium led to the rejection of two further markers. Step 3: the remaining eight markers were tested for transferability across populations with three separate wild boar sample sets. Step 4: on the basis of probability of identity values, a reduction from eight to five markers was possible. Step 5: a novel test using tissue samples from female wild boars and their embryos provided evidence that four variable microsatellite markers and one sex marker are sufficient for individual identification of close relatives. Step 6: feces samples were finally used to estimate PCR (PS) and genotyping success (GS). In conclusion, we recommend a specific four-marker combination with both PS and GS >50% for a reliable individual identification in noninvasive population size estimation of wild boar.

Pros and cons of external swabbing of amphibians for genetic analyses

Non-invasive DNA sampling is an important tool in amphibian conservation. Buccal swabs are nowadays replacing the wounding toe-clipping method. Skin and cloaca swabbing are even less invasive and easier to handle than buccal swabbing, but could result in contaminations of genetic material. Therefore, we test if external skin and cloaca swabs are as reliable as buccal swabs for genetic analysis of amphibians. We analysed eight microsatellite loci for the common frog (Rana temporaria, Linnaeus 1758) and compared genotyping results for buccal, skin and cloaca swabs regarding allelic dropouts and false alleles. Furthermore, we compared two DNA extraction methods regarding efficiency and cost. DNA quality and quantity (amplification success, genotyping error rate, in nanogram per microlitre) were comparable among DNA sources and extraction methods. However, skin and cloaca samples exhibited high degrees of contamination with foreign individuals, which was due to sample collection during mating season. Here, we established a simple low budget procedure to receive DNA of amphibians avoiding stressful buccal swabbing or harmful toe clipping. However, the possibility of contaminations of external swabs has to be considered.

Isolation and characterization of 11 polymorphic trinucleotide microsatellite markers in the stonefly Arcynopteryx compacta (Plecoptera: Perlodidae)

We describe the isolation of 11 polymorphic trinucleotide microsatellite loci from the stonefly Arcynopteryx compacta. Loci were highly variable with 3 to 14 alleles (mean = 6.45). Observed heterozygosity ranged from 0 to 0.867. Seven loci showed significant deviation from Hardy–Weinberg equilibrium across both populations. There was no evidence for null alleles, and thus, Hardy–Weinberg departures could have resulted from genetic structure between populations or subpopulations. No linkage between loci was found. The 11 loci should prove highly informative for population genetic studies.

Isolation and characterization of 10 highly polymorphic di- and trinucleotide microsatellite markers in the mayfly Ameletus inopinatus (Ephemeroptera: Siphlonuridae)

We describe the isolation of ten polymorphic microsatellite loci from the mayfly Ameletus inopinatus. Loci had di‐ or trinucleotide repeat motifs and were highly variable with three to 17 alleles (mean = 7.15). Observed heterozygosity ranged from 0.143 to 0.905. One locus (Ami_202) showed significant deviation from Hardy–Weinberg equilibrium in one population, but no evidence for null alleles. One locus (Ami_73) was significantly linked with three other loci. The remaining nine loci should prove highly informative for population genetic studies.

Genetic characterization of Western European noble crayfish populations ( Astacus astacus ) for advanced conservation management strategies

One central goal of conservation biology is to conserve the genetic diversity of species in order to protect their adaptive potential. The main objective of this study was to identify management units (MUs) for the threatened noble crayfish (Astacus astacus) in Western Europe by utilizing sequence and microsatellite analysis to determine populations in need of focused conservation programs. With the analysis of noble crayfish from 31 sampling sites from Belgium, France, The Netherlands and Germany, and further comparison of this data with a European-wide dataset, we propose four distinct MUs: the French Meuse (MU 1), the French Rhine (MU 2), the Belgian Scheldt and Meuse (MU 3) as well as populations from the French Seine (MU 4). This knowledge enables advanced A. astacus conservation management practises in these catchments by distinguishing between outbreeding and inbreeding populations and by preserving the maximum genetic diversity. When required, a high genetic diversity can be conserved by strengthen existing populations via stocking with populations that either bear the most common haplotype or population-specific private haplotypes in order to maintain recent and regional adaptions. Above all, stocking with populations that exhibit haplotypes from outside Western Europe should be avoided in these catchments. This study supports the preservation of the genetic diversity of noble crayfish in Western Europe and provides thus a proposition for advanced conservation management.

Evaluation of Fecal Storage and DNA Extraction Methods in Wild Boar (Sus scrofa

The wild boar (Sus scrofa) is one of the most common and widely distributed ungulates in Europe. Population sizes have been growing rapidly in recent years, leading to agricultural damage and farmer compensation costs (Toigo et al. 2008). Furthermore, wild boars play an important role in the transmission of diseases (Fickel and Hohmann 2005). Reliable information on absolute population sizes is of crucial importance for effective wildlife management; however, conventional methods based on hunting harvests, direct sightings, or fecal drop counts yield only relative estimates or predictions about population trends. A useful alternative, with great potential as a feasible census method, may be noninvasive genetic sampling without the need of individual capture (Sloane et al. 2000; Fickel and Hohmann 2005). Feces as a DNA source are attractive because of easy sampling and the possibility for an almost equal capture probability (Wehausen et al. 2004). Fecal genotyping, however, has some pitfalls, including low amplification and genotyping