The Balkan Peninsula is one of three major European refugial areas. It has high biodiversity and endemism, but data on the age and origin of its fauna, especially endemics, are limited. Mitochondrial sequence data (COI and 16S genes) were used to study the population structure and phylogeography of the caddisfly Drusus croaticus and the phylogeny and divergence of seven other Drusus species, mostly range-restricted endemics of the Dinaric region of the Balkan Peninsula. The divergence of D. croaticus populations in Croatia and allopatric Drusus species in Bosnia dated to the Pleistocene, showing the importance of this time period for the origin and diversification of Balkan endemic taxa. The divergence of more distantly related species dated to the Late Miocene/Early Pliocene. Population genetic and phylogeographic analysis of 115 individuals from 11 populations of D. croaticus revealed a high level of genetic differentiation and absence of gene flow between populations separated by more than 10 km. The existence of allopatrically fragmented lineages in D. croaticus and the endemic Bosnian species is most likely the result of long-term isolation in multiple microrefugia, probably due to the specific habitat requirements and life-history traits of Drusinae coupled with the topographic complexity and historical changes in geomorphology of the region. Overall, these findings shed light on the processes generating the high genetic complexity of this refugial region that parallels the 'refugia within refugia' pattern widely reported from the Iberian refugium.

The karst areas in the Dinaric region of the Western Balkan Peninsula are a hotspot of freshwater biodiversity. Many investigators have examined diversification of the subterranean freshwater fauna in these karst systems. However, diversification of surface-water fauna remains largely unexplored. We assessed local and regional diversification of surface-water species in karst systems and asked whether patterns of population differentiation could be explained by dispersaldiversification processes or allopatric diversification following karst-related microscale vicariance. We analyzed mitochondrial cytochrome c oxidase subunit I (mtCOI) sequence data of 4 caddisfly species (genus Drusus) in a phylogeographic framework to assess local and regional population genetic structure and Pliocene/Pleistocene history. We used BEAST software to assess the timing of intraspecific diversification of the target species. We compared climate envelopes of the study species and projected climatically suitable areas during the last glacial maximum (LGM) to assess differences in the species climatic niches and infer potential LGM refugia. The haplotype distribution of the 4 species (324 individuals from 32 populations) was characterized by strong genetic differentiation with few haplotypes shared among populations (16%) and deep divergence 6 ana.previsic@biol.pmf.hr 7 jan.schnitzler@senckenberg.de 8 mladen.kucinic@biol.pmf.hr 9 wolfram.graf@boku.ac.at 10 halilibrahimi@yahoo.com 11 mladen.kerovec@biol.pmf.hr 12 steffen.pauls@senckenberg.de AP and SUP designed the research; AP, MK, MK, WG, and HI contributed new reagents/analytic tools; AP, JS, and SUP performed the research, analyzed the data, and wrote the paper. All authors edited the drafts and approved the final version of the manuscript. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts among populations of the 3 endemic species, even at local scales. Divergence among local populations of endemics often exceeded divergence among regional and continental clades of the widespread D. discolor. Major divergences among regional populations dated to 2.0 to 0.5 Mya.Species distribution model projections and genetic structure suggest that the endemic species persisted in situ and diversified locally throughout multiple Pleistocene climate cycles. The pattern for D. discolor was different and consistent with multiple invasions into the region. Patterns of population genetic structure and diversification were similar for the 3 regional endemic Drusus species and consistent with microscale vicariance after the onset of intensified karstification in the Dinaric region. Karstification may induce microscale vicariance of running surface-water habitats and probably promotes allopatric fragmentation of stream insects at small spatial scales. et al. , Milá et al. 2007). Differentiation of species and populations on small scales also are often associated with limited dispersal (e.g., Brändle et al. 2007, Johannesen et al. 2010, and smal...

The caddisfly subfamily Drusinae BANKS comprises roughly 100 species inhabiting mountain ranges in Europe, Asia Minor and the Caucasus. A 3-gene phylogeny of the subfamily previously identified three major clades that were corroborated by larval morphology and feeding ecologies: scraping grazers, omnivorous shredders and filtering carnivores. Larvae of filtering carnivores exhibit unique head capsule complexities, unknown from other caddisfly larvae. Here we assess the species-level relationships within filtering carnivores, hypothesizing that head capsule complexity is derived from simple shapes observed in the other feeding groups. We summarize the current systematics and taxonomy of the group, clarify the systematic position of Cryptothrix nebulicola, and present a larval key to filtering carnivorous Drusinae. We infer relationships of all known filtering carnivorous Drusinae and 34 additional Drusinae species using Bayesian species tree analysis and concatenated Bayesian phylogenetic analysis of 3805bp of sequence data from six gene regions (mtCOI5-P, mtCOI3-P, 16S mrDNA, CADH, WG, 28S nrDNA), morphological cladistics from 308 characters, and a total evidence analysis. All analyses support monophyly of the three feeding ecology groups but fail to fully resolve internal relationships. Within filtering carnivores, variation in head setation and frontoclypeus structure may be associated with progressive niche adaptation, with less complex species recovered at a basal position. We propose that diversification of complex setation and frontoclypeus shape represents a recent evolutionary development, hypothetically enforcing speciation and niche specificity within filtering carnivorous Drusinae.

Adult Trichoptera were observed at the Plitvice Lakes, Croatia during two years (2000 and 2001) using pyramid-type emergence traps. A total of 1350 individuals and 37 species were collected. Throughout the study, the emergence patterns showed only minor variations regarding differences in particular habitats. During 2000 most species emerged in June, and in 2001 in May. The complete emergence period is shown for all collected species and studied in detail for abundant species. Excluding Allogamus uncatus, all abundant species emerged in summer, with most exhibiting a long emergence period. Rhyacophila fasciata had the longest emergence period in both years of the study and also showed winter activity. Trichopteran diversity and equitability were calculated and trophic relationships of the collected species were identified for each sampling site in order to obtain better insight into trichopteran community structure. Results indicated that the trichopteran community could be grouped according to travertine barriers and stream habitat types. IntroductionTo date, there have been various studies focusing on trichopteran communities in karst habitats, but mostly based on larval stages (e.g. BONADA et al., 2005;HABDIJA, 1988; HAB-DIJA et al., 2002). Besides, data of adult Trichoptera ecology and faunistics are commonly obtained by sampling with light and Malaise traps (e.g. SMITH et al., 2002;SVENSSON, 1974;WARINGER, 1991;. Thus emergence patterns and the length of the emergence periods are still insufficiently investigated for many species inhabiting karst areas.The emergence method has been used for various studies all over the world. At first, the method was used for quantitative production studies (ILLIES, 1971;MALICKY, 1976), but more recently it is mostly used for faunistic, phenological and biodiversity studies of running waters (FREITAG, 2004;FÜREDER et al., 2005;PETERSEN et al., 1999;WARINGER, 1996) due to its advantages over some other methods, e.g. benthos sampling (MALICKY, 2002). Hence, the emergence method was applied to investigate trichopteran community composition and emergence patterns in different karstic habitats.The Balkan Peninsula is known to be an important evolutionary centre for a number of trichopteran genera (e.g. Drusus, Rhyacophila, Tinodes, Potamophylax) resulting in a high species endemism (KUMANSKI and MALICKY, 1999). The Plitvice Lakes National Park was chosen for this study because of the presence of different habitat types typical of the karst systems (springs, streams, lakes and travertine barriers). Moreover, some endemic species from the mentioned genera inhabit the Plitvice Lakes area (KUČINIĆ and MALICKY, 2002;MARINKOVIĆ-GOSPODNETIĆ, 1971) and were thus encompassed in the study. The objectives of the study were (1) to define emergence patterns of species inhabiting karst but also to compare these with data from different habitats and regions (2) to investigate the composition, diversity and similarity of trichopteran fauna at different karst habitats, (3) to determine ...

BackgroundTaxonomy offers precise species identification and delimitation and thus provides basic information for biological research, e.g. through assessment of species richness. The importance of molecular taxonomy, i.e., the identification and delimitation of taxa based on molecular markers, has increased in the past decade. Recently developed exploratory tools now allow estimating species-level diversity in multi-locus molecular datasets.ResultsHere we use molecular species delimitation tools that either quantify differences in intra- and interspecific variability of loci, or divergence times within and between species, or perform coalescent species tree inference to estimate species-level entities in molecular genetic datasets. We benchmark results from these methods against 14 morphologically readily differentiable species of a well-defined subgroup of the diverse Drusinae subfamily (Trichoptera, Limnephilidae). Using a 3798 bp (6 loci) molecular data set we aim to corroborate a geographically isolated new species by integrating comparative morphological studies and molecular taxonomy.ConclusionsOur results indicate that only multi-locus species delimitation provides taxonomically relevant information. The data further corroborate the new species Drusus zivici sp. nov. We provide differential diagnostic characters and describe the male, female and larva of this new species and discuss diversity patterns of Drusinae in the Balkans. We further discuss potential and significance of molecular species delimitation. Finally we argue that enhancing collaborative integrative taxonomy will accelerate assessment of global diversity and completion of reference libraries for applied fields, e.g., conservation and biomonitoring.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0972-5) contains supplementary material, which is available to authorized users.

A description of the hitherto unknown larvae of Drusus radovanovici septentrionis Marinković-Gospodnetić, 1976 and Drusus croaticus Marinković-Gospodnetić, 1971 is given. The most important diagnostic features enabling separation from larvae of the other Drusinae are listed and illustrated. Larvae of D. croaticus are distinguished by the combination of specific morphological features (e.g. shape of head and pronotum, the absence of mandible teeth, the absence of prominent, black median setae on frontal margin of pronotum etc.). Besides the above mentioned, larvae of D. radovanovici septentrionis are primarily distinguished by distinct spinules on the head capsule. Some ecological notes are given and the life cycle of both species is discussed. Regarding the feeding behaviour, both analyzed species seem to be predominantly grazers. Both species were recorded in different spring types in the Dinarides. D. radovanovici septentrionis is known only from three springs in Bosnia and Herzegovina (springs of the Rivers Pliva, Bistrica and Sturba), while D. croaticus was recorded in 19 different springs in Croatia. Additionally, all Trichopteran species recorded from the sampled springs are listed.

Two new species of the genus Drusus (Trichoptera, Limnephilidae, Drusinae) from the Western Balkans are described. Additionally, observations on the biodiversity and threats to the region’s endemic aquatic fauna are discussed. Drusus krpachi sp. n. is a micro-endemic of the Korab Mountains, Macedonia, and Drusus malickyi sp. n. is a micro-endemic of the Prokletije Mountains, Albania. Both new species are most similar to Drusus macedonicus but differ from the latter in the shape of segment IX, the shape of the tips of the intermediate appendages in lateral view, the shape of the inferior appendages, and the form and shape of the parameres. In addition, males of the European species of filtering carnivore Drusinae are diagnosed and illustrated, including Cryptothrix nebulicola McLachlan, Drusus chrysotus Rambur, Drusus discolor Rambur, Drusus macedonicus Schmid, Drusus meridionalis Kumanski, Drusus muelleri McLachlan, Drusus romanicus Murgoci and Botosaneanu, and Drusus siveci Malicky. These additions to the Western Balkan fauna demonstrate the significance of this region for European biodiversity and further highlight the importance of faunistic studies in Europe.

Species of the genus Drusus usually inhabit cold springs and spring areas of mountain streams. Isolation of populations and a patchy distribution are reasons for high diversity and endemism in this genus. Nine taxa from the bosnicus group (genus Drusus) live in the Balkan Peninsula (Croatia, Bosnia and Herzegovina, Montenegro, Macedonia). This study has two main objectives. First, morphological, ecological, and genetic features (mitochondrial COI and 16S genes) of the final larval instar of Drusus radovanovici radovanovici from Bosnia and Herzegovina are analysed. Association of larvae and adults of D. radovanovici radovanovici is carried out according to genetic data (mitochondrial DNA) of adults and larvae, and supported by their distribution in Bosnia and Herzegovina. Second, phylogeny, taxonomy and distribution of taxa of the bosnicus group in the Balkan Peninsula are discussed. The analysis includes morphological and genetic data for the known larvae of the genus Drusus distributed in the Balkans. As a consequence, both subspecies of Drusus radovanovici are ranked as species (D. radovanovici and D. septentrionis stat. n.), and the status of other taxa from the bosnicus group is discussed. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)