Utilising public sequence databases to investigate genetic diversity of stoneflies in Medvednica Nature Park.

In Medvednica Nature Park, near Croatia's capital Zagreb, urbanisation significantly impacts the fauna. Comprehensive field research has never been conducted in this area, despite the presence of diverse microhabitats and the discovery of several rare species previously unknown in the Croatian fauna. This study provides the Park with first insight into the genetic and morphological diversity of stoneflies, one of the most endangered groups of organisms. Phylogenetic reconstructions and species delineation methods revealed intraspecific haplotype variation in most species (e.g. Brachypteraseticornis, Isoperlagrammatica and Leuctrabraueri), except for Leuctraprima. Additionally, our study has identified isolated populations that merit further in-depth investigation concerning morphology, genetics and ecology.

DNA barcoding for biodiversity assessment: Croatian stoneflies (Insecta: Plecoptera).

Background: The hemi-metabolous aquatic order Plecoptera (stoneflies) constitutes an indispensable part of terrestrial and aquatic food webs due to their specific life cycle and habitat requirements. Stoneflies are considered one of the most sensitive groups to environmental changes in freshwater ecosystems and anthropogenic changes have caused range contraction of many species. Given the critical threat to stoneflies, the study of their distribution, morphological variability and genetic diversity should be one of the priorities in conservation biology. However, some aspects about stoneflies, especially a fully resolved phylogeny and their patterns of distribution are not well known. A study that includes comprehensive field research and combines morphological and molecular identification of stoneflies has not been conducted in Croatia so far. Thus, the major aim of this study was to regenerate a comprehensive and taxonomically well-curated DNA barcode database for Croatian stoneflies, to highlight the morphological variability obtained for several species and to elucidate results in light of recent taxonomy. Methods: A morphological examination of adult specimens was made using basic characteristics for distinguishing species: terminalia in males and females, head and pronotum patterns, penial morphology, and egg structures. DNA barcoding was applied to many specimens to help circumscribe known species, identify cryptic or yet undescribed species, and to construct a preliminary phylogeny for Croatian stoneflies. Results: Sequences (658 bp in length) of 74 morphospecies from all families present in Croatia were recovered from 87% of the analysed specimens (355 of 410), with one partial sequence of 605 bp in length for Capnopsis schilleri balcanica Zwick, 1984. A total of 84% morphological species could be unambiguously identified using COI sequences. Species delineation methods confirmed the existence of five deeply divergent genetic lineages, with monophyletic origin, which also differ morphologically from their congeners and represent distinct entities. BIN (Barcode Index Number) assignment and species delineation methods clustered COI sequences into different numbers of operational taxonomic units (OTUs). ASAP delimited 76 putative species and achieved a maximum match score with morphology (97%). ABGD resulted in 62 and mPTP in 61 OTUs, indicating a more conservative approach. Most BINs were congruent with traditionally recognized species. Deep intraspecific genetic divergences in some clades highlighted the need for taxonomic revision in several species-complexes and species-groups. Research has yielded the first molecular characterization of nine species, with most having restricted distributions and confirmed the existence of several species which had been declared extinct regionally.

Morphological and molecular characterisation of the Popijac's Yellow Sally, Isoperlapopijaci sp. nov., a new stenoendemic stonefly species from Croatia (Plecoptera, Perlodidae).

A new species of the Yellow Sally genus (Isoperla Banks, 1906) is described, based on morphological (males and females adults, larval and egg) and molecular (the barcode region of the cytochrome c oxidase subunit I gene (COI)) features. Popijac's Yellow Sally, I.popijaci Hlebec & Sivec, sp. nov. inhabits two karstic sources of the Krasulja rivulet in Croatia. Male and female of the new species are characterised by colouration patterns of the head and pronotum; the dimensions of the female subgenital plate; the medial penial armature and oval-shaped egg without collar and anchor. The larvae differ from their congeners by the uniquely coloured head and pronotum. Based on morphological characteristics I.popijaci sp. nov. belongs to the I.tripartita species group. Phylogenetic and taxonomic relationships were reconstructed using three methods of phylogenetic inference and three species delimitation methods. As I.popijaci sp. nov. occurs at a narrow area of the Krasulja rivulet in Krbava field, the study puts emphasis on the conservation and hotspot importance of the temporary rivers in the Dinaric karst. Furthermore, the study accentuates the necessity for further research on the genetic diversity of Plecoptera in Croatia.

Systematic notes on Sinacroneuria Yang amp; Yang, 1995 (Plecoptera: Perlidae), with descriptions of two new species from China.

Two previously unrecognized species of Sinacroneuria Yang Yang, 1995 are formally described from Chinese specimens collected in Fujian Province. In addition, S. orientalis Yang Yang, 1995 type of the genus, is a synonym of Kamimuria flavata Navás, 1933, however K. flavata is a secondary homonym of Perla (Kamimuria) flavata Navás, 1924 and is invalid. A checklist of valid Sinacroneuria species is provided.

A stonefly species extinct in Europe (Taeniopteryx araneoides Klapalek, 1902, Taeniopterygidae, Plecoptera) is thriving in the Irtysh River in West Siberia and North Kazakhstan.

Taeniopteryx araneoides Klápalek, 1902, a stonefly species with brachypterous males, known historically from the Danube, Elbe, and Dniester rivers, but considered to be extinct at least in Europe for more than a century. This species has also been doubtfully reported from Krasnoyarsk, Central Siberia. However, we report this species to be still thriving in the Irtysh River at the cities of Omsk (West Siberia, Russia) and Pavlodar (North Kazakhstan). The occurrence of this species reported least fifty years ago from the Yenisey River at Krasnoyarsk, Russia is considered possible. Unlike the widespread Palaearctic T. nebulosa (Linnaeus, 1758) that occurs in a broad range of stream types, T. araneoides is a potomon species, apparently confined to large rivers. In this habitat, it appears vulnerable to anthropogenic impacts and may be unable to compete with T. nebulosa. The construction of dams and reservoirs has apparently extirpated T. araneoides from most of its former geographical range.