NATURA SLOVENIAE 26(2): 35-39 Prejeto / Received: 5. 11. 2024 SHORT COMMUNICATION Sprejeto / Accepted: 18. 11. 2024 DOI: 10.14720/ns.26.2.35-39 Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2024 Three new species of Niphargidae family added to the list of subterranean amphipods (Crustacea: Amphipoda) in Slovenia Cene FIŠER, Teo DELIĆ, Maja ZAGMAJSTER University of Ljubljana, Biotechnical Faculty, Department of Biology, SubBio Lab, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia; E-mails: cene.fiser@bf.uni-lj.si; teo.delic@bf.uni-lj.si; maja.zagmajster@bf.uni-lj.si Abstract. Since the last comprehensive checklist of amphipods in 2021, three new species of subterranean amphipods have been confirmed for Slovenia: Niphargus danielopoli, N. lurensis and N. julius. Identification of all three species was based on molecular comparative analyses of the mitochondrial CO1 gene fragment, usually used in DNA barcoding identification. With these additions, we reached a total of 201 amphipod species confirmed for Slovenia, 60 of which are groundwater inhabitants, with 57 species belonging to the genus Niphargus. Key words: groundwater, checklist, additional species, Niphargus, hotspot, barcoding, species richness Izvleček. Tri nove vrste iz družine Niphargidae dodane na seznam podzemnih postranic (Crustacea: Amphipoda) Slovenije – Od zadnjega izčrpnega seznama postranic leta 2021 so bile za Slovenijo potrjene tri nove vrste podzemeljskih postranic: Niphargus danielopoli, N. lurensis in N. julius. Identifikacija treh vrst je temeljila na molekularnih primerjalnih analizah fragmenta mitohondrijskega gena CO1, ki se običajno uporablja pri identifikaciji s črtnim kodiranjem DNA. S temi dodatki je za Slovenijo potrjenih skupno 201 vrst postranic, od katerih je 60 podzemnih, s 57 vrstami iz rodu Niphargus. Ključne besede: podzemne vode, seznam, dodatne vrste, Niphargus, vroča točka, barkodiranje, vrstno bogastvo Introduction In the recently published checklist of amphipods of Slovenia, 198 different species inhabiting marine, brackish, freshwater, and terrestrial habitats have been listed (Fišer et al. 2021). More than one-fourth (57 species) of them inhabit groundwater and can be treated as obligatory subterranean species. All but three belong to the family Niphargidae. The genus Niphargus is the most diverse genus of freshwater amphipods in the world and is distributed throughout the western Palearctic region (Borko et al. 2021). Due to its wide distributional range and high species richness, Niphargus is the focal group for various taxonomical, ecological, evolutionary and biodiversity studies (e.g. Stoch et al. 2020; Borko et al. 2021, 2022). 36 Cene FIŠER et al.: Three new species of Niphargidae family added to the list of ... / SHORT COMMUNICATION NATURA SLOVENIAE 26(2): 35-39 Subsequently, Borko et al. (2022) published a study with an extensive list of Niphargus species with corresponding CO1 barcoding sequences. Although some sequences could have been ascribed to morphologically described species, many of them present potential new species based on molecular differences. Further sequencing of the Niphargus CO1 marker in Ljubljana (SubBioLab, at Biotechnical Faculty, University of Ljubljana) and elsewhere allowed us to link the identity of two other MOTUs (molecular operational taxonomic units) in Borko et al. (2022) with valid species names. To keep the amphipod checklist for Slovenia updated, we report herewith on all three additional species for the country. Material and methods Information on the new species for Slovenia was registered in the SubBioDB database, a spatial relational database managed by SubBioLab. Info on the new species and MOTUs were extracted from Borko et al. (2022), where the sequences were originally used in the analyses of biodiversity patterns of subterranean amphipods in the Western Balkans. DNA extraction from amphipods stored in the SubBioLab collection was based on a single appendage; hence the specimens remained in the collection for potential subsequent morphological identification. The protocol of DNA extraction and amplification of the Folmer’s CO1 barcoding fragment (Folmer et al. 1994) for two species followed the one described in Borko et al. (2021), with post-sequencing species identification using the BLAST tool (Altschul et al. 1990). Species identification was made based on widely used 97% match (Ratnasingham & Hebert 2007) with the barcoding fragments of CO1 stored in GenBank. All sequences were deposited in GenBank, with access numbers given in the Results section. Results and discussion Three new groundwater amphipod species were recorded at four different localities in Slovenia (Tab. 1). One of them, N. danielopoli, was identified to the species level by Borko et al. (2022), while the other two were referred to as different MOTUs (Tab. 1). A comparison of acquired sequences with those stored in GenBank or originating in neighbouring countries (Stoch et al. 2020) allowed us to unambiguously identify two additional Niphargus species. Niphargus danielopoli Karaman G., 1994 was described from Austria. It is a small species that has been reported from several localities geographically scattered in central Europe and northern Italy (Karaman 1994), and confirmed in Northern Slovenia (Borko et al. 2022). Cene FIŠER et al.: Three new species of Niphargidae family added to the list of ... / SHORT COMMUNICATION 37 NATURA SLOVENIAE 26(2): 35-39 Niphargus lurensis Schellenberg, 1935 was first described as a form of N. tatrensis Wrześniowski, 1888. In their molecular study, Stoch et al. (2020) provided evidence that this form should be treated as a valid species distributed in Styrian karst in Austria. Based on the barcoding fragment published by Stoch et al. (2020), the identification of material from Northeastern Slovenia revealed that this species is also present at two sites in Slovenia (Tab. 1). The third species, N. julius Stoch, 1997, has been reported from several caves and springs in the Nadiža/Natisone and neighbouring river valleys in northeastern Italy, close to the Slovenian border (Stoch 1997; Karaman 2020). Its presence in Slovenia was confirmed after comparing sequences of individuals collected in a cave close to Kobarid with sequences from Italy (Stoch et al. 2023; Tab.1). Table 1. The list of new groundwater amphipod species confirmed as occurring in Slovenia, with reported details on findings (taken from the SubBioDB database). Coordinates (Lat: N latitude; Long: E longitude) are given in WGS84 decimal degrees. The Acc. No. refers to the sequence’s GenBank access number and a code in brackets to the specimen’s voucher number in the SubBioDB. The name, under which it was reported in Borko et al. (2022), is given in the last column. Tabela 1. Seznam novih vrst podzemnih postranic, ki se potrjeno pojavljajo v Sloveniji, s podrobnejšimi podatki o najdbah (povzeto po podatkovni bazi SubBioDB). Koordinate (Lat: severna geografska širina; Long: vzhodna geografska dolžina) so podane v WGS84 decimalnih stopinjah. Okrajšava Acc. No. se označuje GenBank identifikacijsko številko, medtem ko se oznaka v oklepaju nanaša na številko osebka (voucher) v SubBioDB. V zadnjem stolpcu je navedeno ime, pod katerim je bila vrsta navedena v Borko et al. (2022). Species Locality Lat, Long Date Collectors Acc. No. (voucher) Name in Borko et al. (2022) Niphargus danielopoli G. Karaman, 1994 Izvir Veliki Javornik, Javorniški rovt, Jesenice (spring) 46.458228, 14.108519 27.5.2017 Simona Prevorčnik OK156541 (NC216), OK157026 (ND518), OK157027 (ND519) N. danielopoli Niphargus lurensis Schellenberg, 1935 Vodnjak pri hiši Cven 1c (well) 46.551063, 16.208525 14.8.2011 Jasmina Kotnik OK156762 (ND186) N. spn ND186 Vodnjak ob hiši Ribiška 24 in Veržej (well) 46.586558, 16.165139 9.6.2017 Maja Zagmajster, Simona Prevorčnik, Nataša Mori OK157130 (ND642) N. spn ND186 Niphargus julius Stoch, 1997 Turjeva jama (Slovene cave Registry No. 821), Kred, Kobarid (cave) 46.241212, 13.500463 15.1.2018 Teo Delić, Špela Borko, Klemen Koselj, Maja Zagmajster OK157116 (ND624) N. spn ND624 Even though Slovenia has already been recognised as a hotspot of groundwater amphipod diversity in the Western Balkans (Bregović et al. 2019), the ongoing analyses, run by the SubBioLab members, revealed new species for the country. The new observations reported herein increase the number of amphipod species for Slovenia to 201, with 60 of them from groundwater, and 57 belonging to the genus Niphargus. 38 Cene FIŠER et al.: Three new species of Niphargidae family added to the list of ... / SHORT COMMUNICATION NATURA SLOVENIAE 26(2): 35-39 Povzetek V zadnjem objavljenem pregledu postranic Slovenije je bilo naštetih 198 vrst iz različnih habitatov, od teh 57 iz podzemnih voda (Fišer et al. 2021). Od slednjih vse razen treh vrst pripadajo družini Niphargidae. Rod Niphargus je vrstno najpestrejši rod postranic, ki je razširjen po območju cele zahodne Palearktike (Borko et al. 2021). V študiji Borko et al. (2022) je bil uporabljen in objavljen seznam številnih vrst rodu Niphargus s pripadajočimi CO1 sekvencami. V tem seznamu je navedena nova vrsta za Slovenijo, še dve pa sta bili v tej študiji navedeni kot molekulski operativni taksonomski enoti, ki sta potencialno svoji vrsti. Za identifikacijo smo uporabili postopek BLAST (Altschul et al. 1990) in na podlagi 97-% ujemanja s sekvencami na GenBanku potrdili identiteto teh osebkov. Skupno so bile identificirane tri nove vrste postranic za Slovenijo, najdene na štirih različnih lokalitetah (Tab. 1). Niphargus danielopoli Karaman G., 1994 je bil znan iz srednje Evrope. Niphargus lurensis Schellenberg, 1935 je razširjen po Štajerskem delu Avstrije, potrjena je bila tudi v severovzhodni Sloveniji. Tretja vrsta, N. julius Stoch, 1997, je bila znana iz doline reke Nadiže/Nattisone v Italiji, zdaj smo jo potrdili tudi v jami blizu Kobarida. Nova opažanja so povečala skupno število potrjenih postranic v Sloveniji na 201, od katerih jih 60 živi v podzemnih vodah. Acknowledgements We are grateful to the collectors of the material. The species identity of reference sequences from GenBank was confirmed by Dr. Fabio Stoch (Italy). The preparation of updates to the checklist has been encouraged by the need of NarcIS – Nature Conservation Information System (http://narcis.gov.si), established via the EU-funded project LIFE NarcIS (LIFE19 GIE/SI/000161). The research of Slovenian amphipod fauna was cofounded by the Slovenian Research Agency through core program P1-0184. The study was additionally funded by Biodiversa+, the European Biodiversity Partnership under the 2021-2022 BiodivProtect joint call for research proposals, co-funded by the European Commission (GA N°101052342), and with the funding organisations Ministry of Universities and Research (Italy), Agencia Estatal de Investigación – Fundación Biodiversidad (Spain), Fundo Regional para a Ciência e Tecnologia (Portugal), Suomen Akatemia – Ministry of the Environment (Finland), Belgian Science Policy Office (Belgium), Agence Nationale de la Recherche (France), Deutsche Forschungsgemeinschaft e.V. – BMBF-VDI/VDE INNOVATION + TECHNIK GMBH (Germany), Schweizerischer Nationalfonds zur Forderung der Wissenschaftlichen Forschung (Switzerland), Fonds zur Förderung der Wissenschaftlichen Forschung (Austria), Ministry of Higher Education, Science and Innovation (Slovenia), and the Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania); and Biodiversa+, the European Biodiversity Partnership, in the context of the Sub-BioMon – Developing and testing approaches to monitor subterranean biodiversity in karst project under the 2022-2023 BiodivMon joint call. It was co-funded by the European Commission (GA N°101052342) and the following funding organisations: Ministry of Higher Education, Science and Innovation (Slovenia), The Belgian Science Policy (Belgium), Ministry of Universities and Research (Italy), National Research, Development and Innovation Office (Hungary), Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania) and self-financing partner National Museum of Natural History Luxembourg (Luxembourg). References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology. 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2 Cene FIŠER et al.: Three new species of Niphargidae family added to the list of ... / SHORT COMMUNICATION 39 NATURA SLOVENIAE 26(2): 35-39 Borko Š, Trontelj P, Seehausen O, Moškrič A, Fišer C. 2021. A subterranean adaptive radiation of amphipods in Europe. Nature Communications. 12: 3688. https://doi.org/10.1038/s41467-021- 24023-w Borko Š, Altermatt F, Zagmajster M, Fišer C. 2022. A hotspot of groundwater amphipod diversity on a crossroad of evolutionary radiations. Diversity and Distributions. 28(12): 2765-2777. https://doi.org/https://doi.org/10.1111/ddi.13500 Bregović P, Fišer C, Zagmajster M. 2019. Contribution of rare and common species to subterranean species richness patterns. Ecology and Evolution 9(20): 11606-11618. https://doi.org/10.1002/ece3.5604 Fišer C, Mavrič B, Govedič M, Pekolj A, Zagmajster M. 2021. Checklist of amphipod crustaceans (Crustacea: Amphipoda) in Slovenia. Natura Sloveniae. 23(2): 5-24. https://doi.org/10.14720/ns.23.2.5-24 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 3: 294-299. Karaman GS. 1994. Contribution to the knowledge of the Amphipoda. Further studies on genus Niphargus Schioedte 1849 from Europe, N. ivokaramani, n. sp and N. danielopoli, n. sp. The Montenegrin Academy of Sciences and Arts: Glasnik of the Section of Natural Sciences. 10: 113-133. Karaman GS. 2020. Biodiversity of subterranean waters: redescription of poorly known Niphargus julius Stoch, 1997 (Crustacea: Amphipoda: Niphargidae) from Italy (Contribution to the knowledge of the Amphipoda 314). Agriculture & Forestry. 66(4): 239-258. https://doi.org/10.17707/AgricultForest.66.4.19 Ratnasingham S, Hebert PDN. 2007. BOLD: The Barcode of Life Data System (www.barcodinglife.org). Molecular Ecology Notes. 7: 355-364. https://doi.org/10.1111/j.1471-8286.2006.01678.x Stoch F. 1997. La fauna delle acque carsiche sotterranee delle Valli del Natisone. In: Il fenomeno carsico delle Valli del Natisone. Memorie dell'Istituto Italiano di Speleologia. s. II, vol. IX. p. 89-100. Stoch F. Christian E, Flot JF. 2020. Molecular taxonomy, phylogeny and biogeography of the Niphargus tatrensis species complex (Amphipoda, Niphargidae) in Austria. Organisms Diversity & Evolution. 20: 701-722. https://doi.org/https://doi.org/10.1007/s13127-020-00462-z Stoch F, Salussolia A, Flot JF. 2023. Polyphyly of the Niphargus stygius species group (Crustacea, Amphipoda, Niphargidae) in the Southern Limestone Alps. bioRxiv. https://www.biorxiv.org/content/10.1101/2022.04.28.489871v2.full © 2024 Cene Fišer, Teo Delić, Maja Zagmajster To je prostodostopen članek, objavljen pod določili licence Creative Commons Priznanje avtorstva 4.0 Mednarodna, ki dovoljuje neomejeno rabo, razširjanje in kopiranje v kakršnemkoli mediju ter obliki, pod pogojem, da sta navedena avtor in vir. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.