Mayfly response to different stress types in small and mid-sized lowland rivers.

Freshwater ecosystems are endangered worldwide by various human pressures, resulting in dramatic habitat and species loss. Many aquatic invertebrates respond to disturbances in their habitat, and mayflies are among the most sensitive ones. Therefore, we investigated mayfly response to anthropogenic disturbances at 46 study sites encompassing slightly to heavily modified small and mid-sized lowland streams and rivers. Mayfly nymphs were sampled between April and September 2016 using a benthos hand net. A total of 21 species was recorded, with Cloeon dipterum (Linnaeus, 1761) being the most frequently recorded one. Nevertheless, the taxa richness was rather low per site, i.e., between zero and nine. Assemblage structure had a high share of lower reaches and lentic (potamic and littoral) elements, and detritivores (gatherers/collectors and active filter feeders). This indicates that hydromorphological alterations lead to assemblage "potamisation" in small and mid-sized rivers. More mayfly species were related to higher oxygen concentration and lower water temperature, abundance of aquatic vegetation and total organic carbon. Additionally, the assemblage diversity and abundance were negatively associated with increasing intensive agriculture area at the catchment scale. This study confirms mayfly bio-indicative properties, i.e., their sensitivity to alterations of their habitat and pollution, but also provides new data related to mayfly response to the impacted environment. Those data can be used for management and protection activities of lowland rivers and their biota according to the requirements of the European Water Framework Directive.

New species of aquatic oligochaetes (Annelida: Clitellata) from tufa barriers in Croatia.

Two new aquatic oligochaete species are described from tufa barriers in Croatia. The reproductive system in the phallodriline Aberrantidrius mihaljevici Vuckovic et al. n. sp. conforms to the general pattern in the genus but it is distinguished by a combination of characters, including the shape and length of penial chaetae (3 times the mean length of somatic chaetae), atrium shape and spermathecal pore position (anterior in the postatrial segment). The lumbriculid Stylodrilus tofaceus Rodriguez et al. n. sp. is mainly distinguished from congeners by the spindle-shaped spermathecal ducts with high lining epithelium, and position of the spermathecal pores. These two new taxa reveal that aquatic oligochaete communities in tufa barriers of karstic regions may be of particular interest with respect to diversity of endemic taxa, and also for study of colonization routes of this interesting habitat, with possible relations with groundwaters. The finding of one specimen attributable to the genus Eclipidrilus may open ainteresting research on the biogeography of this Nearctic genus.

Croatian freshwater oligochaetes: species diversity, distribution and relationship to surrounding countries.

This paper presents a checklist of the freshwater oligochaetes of Croatia, including data related to the ecoregions, river basins and habitat types in which species were recorded. The checklist is based on literature data and new records from unpublished research conducted since 1975. A total of 71 freshwater oligochaete species were recorded at 345 sampling sites. The family Naididae was most species rich (31 species), followed by the family Tubificidae (27 species), Lumbriculidae (6 species) and Pristinidae (4 species). The families Haplotaxidae, Propappidae and Lumbricidae were least diverse, with only one species recorded for each. Approximately two-thirds of the oligochaete species occurred in both the Dinaric western Balkan and the Pannonian lowland ecoregions. Similar patterns in species richness were detected for the Black Sea and the Adriatic Sea basins. Species richness varied between habitat types, with rivers as the most speciose habitats. In comparison to adjacent countries, the Croatian oligochaete fauna was most similar to that of Bosnia and Herzegovina, Hungary and Austria.

Spatial distribution and seasonal changes of mayflies (Insecta, Ephemeroptera) in a Western Balkan peat bog.

Peat bogs are unique wetland ecosystems of high conservation value all over the world, yet data on the macroinvertebrates (including mayfly assemblages) in these habitats are still scarce. Over the course of one growing season, mayfly assemblages were sampled each month, along with other macroinvertebrates, in the largest and oldest Croatian peat bog and an adjacent stream. In total, ten mayfly species were recorded: two species in low abundance in the peat bog, and nine species in significantly higher abundance in the stream. Low species richness and abundance in the peat bog were most likely related to the harsh environmental conditions and mayfly habitat preferences. In comparison, due to the more favourable habitat conditions, higher species richness and abundance were observed in the nearby stream. Three of the recorded species, Caenis luctuosa from the peat bog, and Eurylophella karelica and Leptophlebia marginata from the stream are new records for the Croatian mayfly fauna. Typical Central European life cycle patterns were confirmed for several species (e.g. Baetis vernus, Nigrobaetis niger, Electrogena ujhelyii), while for several others (e.g. Habrophlebia fusca, Paraleptophlebia submarginata) some discrepancies were observed. Therefore, these results provide new and valuable information on the ecology of mayflies in peat bog habitats.

Microscale vicariance and diversification of Western Balkan caddisflies linked to karstification.

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 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 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.

Estimation of DNA integrity in blood cells of eastern mosquitofish (Gambusia holbrooki) inhabiting an aluminium-polluted water environment: an alkaline comet assay study.

To estimate the impacts of an Al-contaminated aquatic environment on DNA integrity in the blood cells of eastern mosquitofish Gambusia holbrooki Girard 1859 inhabiting Lake Njivice (Island of Krk, Croatia), an evaluation using the alkaline comet assay was carried out. Genome integrity was studied in parallel with the same fish species inhabiting the nearby, unpolluted Lake Ponikve. The amount of DNA damage in cells was estimated from three different parameters: comet tail length as the extent of genetic material migration, tail intensity (% DNA in the comet tail) and tail moment. The results indicate the loss of genome integrity in blood cells of mosquitofish inhabiting Lake Njivice and the genotoxicity of this aquatic environment. Using the same assay, acute genotoxicity of contaminated water and sediment was evaluated and confirmed on fish, mouse and human blood cells treated ex vivo. Results of the present study indicate that the alkaline comet assay applied to fish blood cells is a valuable tool for determining the potential genotoxicity of water pollutants and confirm its usefulness in the evaluation of DNA damage in fish living in Al-polluted waters.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: a case study.

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.

Pleistocene divergence of Dinaric Drusus endemics (Trichoptera, Limnephilidae) in multiple microrefugia within the Balkan Peninsula.

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.