A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

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.

An Overview of Orthoptera Mass Occurrences in Croatia from 1900 to 2023.

During the last century, well-known locust species, such as Calliptamus italicus and Dociostaurus maroccanus, have produced outbreaks of varying degrees in the Balkans. The literature data on outbreaks in the region are scarce, and Croatia is not an exception. This study summarized the data on 23 Orthoptera mass occurrences in Croatia from 1900 to 2023 from 28 localities, representing 12 species. This is a low level of outbreak activity compared with other locust and pest grasshopper species in other parts of the world. The species with the most reporting is C. italicus with altogether six mass occurrences, while second is Barbitistes ocskayi and Miramella irena with three records, and in the third, place D. maroccanus and Gryllotalpa sp., each with two mass occurrences having been reported. One of the most notable swarms is that of Anacridium aegyptium which occurred around Sibenik in 1998, and this paper provides the first account of it, 25 years after it took place. The most recent outbreaks took place in 2022, and the most notable one was that of D. maroccanus swarm in Stikovo. The 2022 and 2023 reports were brief and muted, despite the affected agriculturists claiming significant damages.

Molecular analyses of pseudoscorpions in a subterranean biodiversity hotspot reveal cryptic diversity and microendemism.

Nested within the Mediterranean biodiversity hotspot, the Dinaric Karst of the western Balkans is one of the world's most heterogeneous subterranean ecosystems and renowned for its highly diverse and mostly endemic fauna. The evolutionary processes leading to both endemism and diversity remain insufficiently understood, and large-scale analyses on taxa that are abundant in both subterranean and surface habitats remain infrequent. Here, we provide the first comprehensive molecular study on Croatian pseudoscorpions, a lineage of arachnids that is common and diverse in both habitats. Phylogenetic reconstructions using 499 COI sequences derived from 128 morphospecies collected across the Dinaric Karst show that: (i) occurrence in karstic microhabitats boosters speciation and endemism in the most diverse genera Chthonius C.L. Koch, 1843 (37 morphospecies) and Neobisium Chamberlin, 1930 (34 morphospecies), (ii) evidence for ongoing diversification is found in many species and species complexes through low optimal thresholds (OTs) and species delineation analyses, and (iii) landscape features, such as mountain ranges, correlate with patterns of genetic diversity in the diverse genus Neobisium. We present two synonymies: Protoneobisium Curcic, 1988 = Neobisium, syn. nov., and Archaeoroncus Curcic and Rada, 2012 = Roncus L. Koch, 1873, syn. nov. Overall, our study suggests that karstic microhabitats promote diversification in soil- and cave-dwelling arthropods at all taxonomic levels, but also provide important refugia for invertebrates in past and present periods of environmental change.

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.

DNA barcoding of the horsefly fauna (Diptera, Tabanidae) of Croatia with notes on the morphology and taxonomy of selected species from Chrysopsinae and Tabaninae.

In the Croatian fauna, horseflies (Tabanidae) are represented by 78 species belonging to two subfamilies, five tribes, and 10 genera. Identification of these species is based on morphological characteristics. In this study, 43 species of horseflies were analyzed. The highest number of species (19) belongs to the genus Tabanus, followed by the genera Hybomitra with seven species, Haematopota with six species, Chrysops with four species, Atylotus and Philipomyia with two species each, and the genera Silvius, Dasyrhamphis, and Heptatoma with one species each. The standard DNA barcoding region of the mitochondrial cytochrome c oxidase gene, subunit I (COI), was sequenced and compared to the Barcode of Life Database (BOLD). Our analyses confirmed our morphological identifications and added 16 new Barcode Index Numbers (BINs) for Tabanidae to BOLD. Potential problems in the systematics and taxonomy of this family are highlighted.

DNA barcoding of hard ticks (Ixodidae), notes on distribution of vector species and new faunal record for Croatia.

Molecular methods are increasingly being utilized for accurate identification of ticks (Acari: Ixodidae), especially in cases of morphologically highly similar species. In this study, we performed molecular research of the tick fauna in Croatia using DNA barcoding method. Ticks were sampled in three biogeographical regions and thirteen species were recorded: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma marginatum, Ixodes frontalis, Ixodes hexagonus, Ixodes kaiseri, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus. Ixodes kaiseri is for the first time recorded in the fauna of Croatia. Of the thirteen hard tick species analyzed in this study, pathogens from different groups (bacteria, protozoa and viruses) have been detected in eight species in Croatia so far. For the important vector species R. sanguineus s.s., new distributional data for Croatia are given. The standard COI barcoding region was amplified, and the sequences were analyzed by species delimitation methods together with the sequences of conspecific and congeneric species from the public BOLD database. Our specimens of H. punctata represent a new, genetically distinct MOTU. A brief overview of the available public DNA barcoding data for Ixodidae is presented, highlighting the need for an integrative approach for the clarification of the taxonomic status of problematic Ixodid taxa. The results provide a basis for the establishment of a molecular data platform for the Ixodidae of the Croatian fauna.

The Rhyacophila fasciata Group in Europe: Rhyacophila fasciata Hagen 1859 and formerly synonymized species (Trichoptera: Rhyacophilidae), with new description of Rhyacophila fasciata and Rhyacophila septentrionis McLachlan 1865 (stat. prom.).

The presence and distribution of Rhyacophila fasciata Hagen 1859 in Europe were revised, based on bibliographic study, collection specimens, and new material collected in different countries. The status of formerly synonymized species, Rhyacophila ferruginea (Scopoli 1763) and Rhyacophila septentrionis McLachlan 1865 was also assessed. The type of R. ferruginea is missing, the taxon is still unidentified, and thus we propose Rhyacophila ferruginea as a nomen dubium. Morphological features and genetic evidence revealed that R. septentrionis differs from R. fasciata, so we propose to change its status to status resurrectus. We therefore include new descriptions of the different stages (larva, pupa, male, and female) of R. fasciata and of R. septentrionis, together with a molecular analysis based on mitochondrial cytochrome oxidase I (mtCOI) and ecological notes. The species R. coppai Oláh 2020 NEW SYNONYM and R. soreda Coppa Oláh 2020 NEW SYNONYM are synonyms of R. sociata Navás 1916; the species R. kopasa Oláh Coppa 2020 NEW SYNONYM and R. rova Oláh Coppa 2020 NEW SYNONYM are synonyms of R. denticulata McLachlan 1879; the species R. matrensis Oláh Szczesny 2020 is probably a synonym of R. fasciata, so more study of this species is needed in order to confirm or deny that it is a valid species.

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.

Diversity of mosquito fauna (Diptera, Culicidae) in higher-altitude regions of Croatia.

Global climate change and the accompanying rise in temperature could affect the biology and ecology of a number of vectors, including mosquitoes. High altitude areas that were previously unsuitable for the spread of mosquito vector populations could become suitable. The aim of this research was to study the distribution of mosquito species in higher altitude regions of Croatia. Samples were collected in three areas: Slavonian Mountains, Gorski Kotar, and Middle Velebit. Specimens were morphologically determined and confirmed by DNA barcoding and other genetic markers and showed the presence of 16 species belonging to six genera. The most abundant species were the Culex pipiens complex with 50% of the collected specimens. Both pipiens (Linnaeus, 1758) and molestus (Forskal, 1775) biotypes and their hybrids were identified within the complex, followed by Culex torrentium (Martini, 1925) (20.2%), Culiseta longiareolata (Macquart, 1838) (8.5%), and the invasive species Aedes japonicus (Theobald, 1901) (7.8% of the total number of collected specimens). The remaining 12 species made up 14.7% of the collected specimens. Intraspecific COI p-distances were within the standard barcoding threshold for OTUs, while interspecific genetic distances were much higher, confirming the existence of barcoding gaps. Mosquito fauna of Croatian mountains showed a moderate variety and made 30.8% of the total number of recorded mosquito species in Croatia thus far.

Data on pyraloid moth specimens (Lepidoptera: Pyraloidea) held in the collections of CNHM in Zagreb, Croatia.

Natural history museum collections can be a helpful tool in documenting changes in biodiversity throughout decades or even centuries. This article uncovers information on the content of three different museum collections, collected over 100 years. It deepens the knowledge on the distribution of pyraloid moth species occurring predominantly in the Continental parts of Croatia, but also in other areas of this country.                The article presents the first published listing of pyraloid moth species held in three museum collections of the Croatian Natural History Museum in Zagreb. The list contains 148 taxa in total, 61 from the Igalffy collection, 96 from the Koca collection, and 96 from the Kucinic collection. Altogether, 96 species from the family Crambidae and 52 species from the family Pyralidae are listed.

The Rhyacophila fasciata Group in Croatia and Bosnia and Herzegovina: Rhyacophila f. fasciata Hagen 1859 and the description of two new subspecies, Rhyacophila fasciata delici Kucinic Valladolid (ssp. nov.) from Croatia and Bosnia and Herzegovina and Rhyacophila fasciata viteceki Valladolid Kucinic (ssp. nov.) from Bosnia and Herzegovina (Trichoptera: Rhyacophilidae).

We present the description of two new subspecies of the Rhyacophila fasciata Group: Rhyacophila fasciata delici Kucinic Valladolid (ssp. nov.), broadly distributed in Croatia and present also in Bosnia and Herzegovina, and R. fasciata viteceki Valladolid Kucinic (ssp. nov.), found in Bosnia and Herzegovina. Our study of the morphology of adults, as well as our analysis of the barcode region of the mitochondrial cytochrome oxidase I (mtCOI) gene and geographical distribution confirm the differences of the two new subspecies with the nominal species R. f. fasciata, also found in both countries.

Integrative taxonomy by molecular species delimitation: multi-locus data corroborate a new species of Balkan Drusinae micro-endemics.

BACKGROUND: Taxonomy 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. RESULTS: Here 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. CONCLUSIONS: Our 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.

Morphological features of larvae of Drusus plicatus Radovanovic (Insecta, Trichoptera) from the Republic of Macedonia with molecular, ecological, ethological, and distributional notes.

A description of the larva of Drusus plicatus Radovanovic is given for the first time. The most important diagnostic characters enabling separation from larvae of the other Drusinae from the southeast Europe are listed. Molecular, ecological, and ethological features and distribution patterns of the species are given. Additionally, information on the sympatric caddisfly species of the three springs where larvae and adults of Drusus plicatus were found and presented.

Drusus sharrensis sp. n. (Trichoptera, Limnephilidae), a new species from Sharr National Park in Kosovo, with molecular and ecological notes.

In this paper we describe Drusus sharrensis sp. n., from the Sharr Mountains in Kosovo. Males of the new species are morphologically most similar to Drusus krusniki Malicky, 1981, Drusus kerek Oláh, 2011 and Drusus juliae Oláh, 2011 but differ mainly in exhibiting (1) a differently shaped spinose area on tergite VIII; (2) intermediate appendages anteriorly curved in lateral view with broad tips in dorsal view; (3) inferior appendages with a distinct dorsal protrusion in the proximal half. Females of the new species are morphologically most similar to Drusus krusniki, Drusus kerek, Drusus juliae, and Drusus plicatus Radovanovic, 1942 but mainly differ in (1) segment X that is longer than the supragenital plate with distinctly pointed tips; (2) supragenital plate quadrangular with a distinct round dorsal protrusion; (3) a vulvar scale with a small median lobe. Results of phylogenetic species delimitation support monophyly of Drusus sharrensis sp. n. and recover it as sister to a clade comprising (Drusus pelasgus Oláh, 2010 + Drusus juliae + Drusus arbanios Oláh, 2010 + Drusus plicatus + (Drusus dacothracus Oláh, 2010 + Drusus illyricus Oláh, 2010)). The new species is a micro-endemic of the Sharr Mountains, a main biodiversity hotspot in the Balkan Peninsula. Main threats to the aquatic ecosystems of this part of the Balkan Peninsula are discussed.

Larval morphology of the Western Balkans endemic caddisflies Drusus krusniki Malicky 1981, D. vernonensis Malicky 1989, and D. vespertinus Marinkovic 1976 (Trichoptera, Limnephilidae, Drusinae).

Drusinae (Trichoptera, Limnephilidae) are highland caddisflies inhabiting high-gradient, turbulent running water and spring habitats. They are disjunctly distributed over the Eurasian mountain ranges, and the majority of species is endemic to particular mountain areas. The most diverse of three main groups of the Drusinae, the grazer clade, consists of species in which larvae feed on epiltihic biofilm and algae. In this paper we describe three previously unknown grazer-clade Drusinae larvae: Drusus krusniki Malicky 1981 (endemic to the Dinaric western Balkans), D. vernonensis Malicky 1989 (endemic to the Hellenic western Balkans), and D. vespertinus Marinkovic 1976 (endemic to the Dinaric western Balkans). The larvae of these species have toothless mandibles typical of the Drusinae grazer clade. Larvae and adults were unambiguously associated using molecular genetic data, i.e., the mitochondrial cytochrome oxidase I gene fragment (mtCOI3-P). Morphological characteristics of the larvae are described and the diagnostic features enabling species-level identification are illustrated. We further discuss the ecology and distribution of three Western Balkan endemic species.

The larva of Rhyacophila balcanica Radovanovic 1953 (Trichoptera: Rhyacophilidae) with notes on ecology.

The previously unknown larva of Rhyacophila balcanica Radovanovic 1953 is described. The diagnostic features of the species are listed and illustrated and some information on its ecology and distribution is included. In addition, diagnostic characters for larvae of the known Greek Rhyacophila species are provided.

New records for the Kosovo caddisfly fauna with the description of a new species, Drusus dardanicus sp. nov. (Trichoptera: Limnephilidae).

The Balkan Peninsula is one of the most important European hotspots of freshwater biodiversity. The region is, however, to a large extent insufficiently investigated. Here we present data on distribution of caddisflies in one particularly understudied area, the Republic of Kosovo. Our data include the first records of Adicella altandroconia Botosaneanu & Novak and Halesus tessellatus (Rambur) for the Kosovo caddisfly fauna, and a new locality for the recently described Ecclisopteryx keroveci Previsic, Graf, & Vitecek. Further, we describe the new caddisfly species Drusus dardanicus sp. nov. from the Kopaonik Mountains. The new species belongs to the D. discophorus Species Group and differs morphologically from its most similar congeners (D. discophorus Radovanovic, D. balcanicus Kumanski, and D. bureschi Kumanski) mainly in exhibiting (1) subtrianglar superior appendages; (2) a narrow, dorsal spinate area of tergite VIII; and (3) evenly rounded tips of intermediate appendages in caudal view. In phylogenetic analysis, D. dardanicus sp. nov. is well delineated and recovered as a sister taxon to D. osogovicus Kumanski, a species recorded from Bulgaria. The recent discovery of a new species and other rare or microendemic species presents important contributions to the knowledge on the rich freshwater biodiversity in Kosovo. These species face increasing anthropogenic pressure and threats to their conservation.

A hairy case: The evolution of filtering carnivorous Drusinae (Limnephilidae, Trichoptera).

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.

Larval description of Drusus bosnicus Klapálek 1899 (Trichoptera: Limnephilidae), with distributional, molecular and ecological features.

In this study we present morphological, molecular and ecological features of the last instar larvae of Drusus bosnicus with data about distribution of this species in Bosnia and Herzegovina. We also included  the most important diagnostic features enabling separation of larvae of D. bosnicus from larvae of the other European Drusinae and Trichoptera species.