A surprising finding of Ecclisopteryx asterix Malicky, 1979 (Insecta, Trichoptera) in Croatia with notes to DNA barcoding and new distributional data of the subfamily Drusinae Author Kladarić, Lidija Hrvatske vode, Central Water Management Laboratory, Ulica Grada Vukovara 220, 10000 Zagreb, Croatia Author Popijač, Aleksandar Kopački rit Nature Park Public Institution, Mali Sakadaš 1, 31327 Kopačevo, Bilje, Croatia Author Hlebec, Dora Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia & Zoological Museum Hamburg, Leibniz Institute for the Analysis of Biodiversity Change (LIB), Martin-Luther-King- Platz 3, 20146 Hamburg, Germany Author Previšić, Ana Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia Author Ćuk, Renata Hrvatske vode, Central Water Management Laboratory, Ulica Grada Vukovara 220, 10000 Zagreb, Croatia Author Vučković, Ivan Elektroprojekt, Alexandera von Humboldta 4, 10000 Zagreb, Croatia * Corresponding author: mladen. kucinic @ biol. pmf. hr Author Kučinić, Mladen Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia mladen.kucinic@biol.pmf.hr text Ecologica Montenegrina 2021 2021-12-14 48 71 85 http://dx.doi.org/10.37828/em.2021.48.11 journal article 10.37828/em.2021.48.11 2336-9744 13250260 1. Ecclisopteryx asterix Malicky, 1979 : An Alpine endemic lives in Croatia too In the current study, we found larvae of Ecclisopteryx asterix from northern Croatia , at the Ivanščica Mt. (i.e. three larvae of species E. asterix were collected at the Šumi Spring during two visits, on March 10 th 2015 and on March 6 th 2021 ; Table 1 , Figure 1 ). Up till now, E. asterix was considered a stenoendemic species of the southeastern Alps , distributed in the limnoecoregion 4 only (the Alps; Figure 1A , Graf et al . 2021 ). Thus, by adding one more Drusinae species to the list of caddisfly species in Croatia ( Kučinić et al. 2012 ), our findings bring new insights into the distribution of this species. This suggests that E. asterix has a broader distribution, or at least that some additional disjunct populations exist in the area. Even though we only found larvae, these could be identified after morphological examination and comparison with Ecclisopteryx malickyi Moretti, 1991 , Drusus annulatus (Stephens, 1837) and Drusus biguttatus (Pictet, 1834) . These species differ in the contours of the pronotum in lateral view, the presence/absence of the pronotal transverse groove, the shape of the median notch of the pronotum (in anterior view), pronotal sculpturing, presence/absence of the lateral carina of the head capsule, the number of proximo-dorsal setae on the mid-and hind femora, where the lateral fringe starts on the abdomen, and also in distribution ( Graf et al . 2011 ). Larva of E. asterix look similar to D. annulatus and D. biguttatus . It is characterized by the shape of the case, which is smooth, slightly curved and consists completely of mineral particles ( Figure 2A ). The dorsal line of the pronotum is rounded in the last third, creating a small dorsal hump, and a step-like interruption of the dorsal silhouette ( Figure 2B ). The metanotum is partially covered by three pairs of sclerites of which the anterior pair is large, ovoid and has a very narrow median separation ( Figure 2C ). The lateral fringe starts on the last third of the third abdominal segment ( Figure 2D ). Figure 2 . A Larva of Ecclisiopteryx asterix in its case; B morphological character important for identification: shape of the pronotum (p); C pronotum (p), mesonotum (me), and sclerites at metanotum (mt) in dorsal view; D abdominal segments I-VI with gills (g) and lateral fringe (lf), right lateral view. Additionally, molecular analyses supported our morphological identification as obtained sequences were identified as the same species we identified using morphological characters. The new findings of E. asterix were confirmed with the use of BOLD Identification Engine (98.17 – 98.32 %, private sequence, sample from Austria ). Obtained COI gene sequences were 658 bp long and formed two COI haplotypes with low sequence divergence (0.00153 uncorrected p -distance) and with low sequence divergence between the single E. asterix sequence available on BOLD ( Table 2 ). The topology was congruent during two conducted analyses ( Figure 3 ). Sequences of E. asterix from Croatia group together with the single sequence of E. asterix (HMKKT864-11) available in the BOLD database. A separate taxonomic status of E. asterix is possible for populations in Croatia . For now, this is indicated by DNA barcoding data, as well as the separation of populations from the Ivanščica Mt. However, for any taxonomic study it is hard to make a conclusion with only three collected larvae. Future investigations are needed and planned as it is necessary to collect more larvae and adults of both sexes for detailed morphological analyses. Figure 3 . Maximum likelihood phylogram based on the COI sequences of Ecclisopteryx asterix from Croatia and haplotypes of Ecclisopteryx species available in BOLD database. Numbers at the nodes indicate maximum likelihood (ML) bootstrap support values (BS). Terminal codes represent BOLD Process IDs. Ecclisopteryx asterix is mainly montane species, distributed from 400 to 1100 m above sea level (Urbanič et al . 2003). It inhabits springs, spring reaches and upper stream reaches, and is considered as a lithal habitat specialist and a cold water stenotherm ( Graf et al. 2021 ). Because of these characteristics, it is also highly vulnerable to climate changes ( Graf et al. 2021 ). According to the morphology of the mouth parts, larvae of this species are scrapers, caddisflies that feed on periphyton on stone pebbles and aquatic plants. The location at the Šumi Spring is at an altitude of ca. 400 m a.s.l., in line with E. asterix being a submontane species ( Graf et al. 2021 ). Generally, the habitat where it was collected is similar to the ecology from the Alpine region ( Graf et al. 2021 ). We collected larvae in a small (ca. 1 m wide, average 15 cm deep), fast flowing brook, around 200 m downstream of the spring (spring is partially captured). This is typical of the habitat preference of this species, as it mainly inhabits hypocrenal, but also eucrenal and epirithral areas, and zones with higher current velocity ( Graf et al. 2021 ). The substrate consists of bare microlithal ( 2-6 cm ) (60%) and mesolithal ( 6-20 cm ) covered by moss (35%) with only 5% of akal (0,2- 2 cm ), what is also in accordance with species substrate preferences. The temperature in March 2015 at 3:00 PM was 9.3°C, which corresponds with cold stenotherm temperature preferences according to Graf et al. (2021) . Electric conductivity was 429 µS/cm. Hence, we found E. asterix in a typical habitat, but geographically dislocated. It is therefore highly likely that the species inhabits additional similar habitats in the vicinity of the Šumi Spring, but also in areas in between the known distribution in the southeastern Alps and the current finding.