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 Ćurčić, 1988 = Neobisium, syn. nov., and Archaeoroncus Ćurčić and Rađa, 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.
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.
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.
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.
Abstract
Despite the recent advent of molecular data to assess the phylogeny of many invertebrate groups, the systematics of the pseudoscorpion family Chernetidae is unresolved, even though it ...comprises a quarter of the world’s generic pseudoscorpion diversity. We derive a preliminary molecular phylogeny of chernetids to assess subfamilial and generic monophyly using mitochondrial (COI) and nuclear (28S rRNA, 18S rRNA, and H3) markers. Three chernetid subfamilies have previously been recognized and Lamprochernetinae, originally defined based on T-shaped spermathecae, is recovered here but expanded to include the Old-World tropical genus Verrucachernes. In contrast, the genera Conicochernes and Calymmachernes of the subfamily Goniochernetinae are nested within the largest subfamily, Chernetinae. Three new subterranean species of the Palearctic genus Lasiochernes are also described from the Dinaric Karst: L. marinaeHlebec & Harvey, sp. nov., L. jalziciHlebec & Harvey, sp. nov., and L. pavlekaeHlebec & Harvey, sp. nov.. The former two species are single-cave endemics but L. pavlekae sp. nov. is more widespread. The relative lack of genetic structuring in this species, including haplotype sharing between sampling localities, together with a patchy distribution, suggests that its biogeography is probably shaped by multiple vector-mediated dispersal events, rather than geomorphological history. Due to their rarity, we discourage further collecting of Lasiochernes in this subterranean biodiversity hotspot.
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.
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.
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
Zwick, 1984. A total of 84% morphological species could be unambiguously identified using
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
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.
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 Koča collection, and 96 from the Kučinić collection. Altogether, 96 species from the family Crambidae and 52 species from the family Pyralidae are listed.
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
, followed by the genera
with seven species,
with six species,
with four species,
and
with two species each, and the genera
,
, and
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.
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. Popijač’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.
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.
,
and
), except for
. Additionally, our study has identified isolated populations that merit further in-depth investigation concerning morphology, genetics and ecology.
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 dispersal—diversification processes or allopatric diversification following karstrelated 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 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.
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.
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.
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.
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.
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