Two new species from the Hygrobates nigromaculatus-complex (Acariformes, Hydrachnidia, Hygrobatidae), based on morphological and molecular evidence Vladimir Pešić, Milica Jovanović, Ana Manović, Andrej Zawal, Aleksandra Bańkowska, Łukasz Broda, Peter Martin, Miroslawa Dabert To cite this version: Vladimir Pešić, Milica Jovanović, Ana Manović, Andrej Zawal, Aleksandra Bańkowska, et al.. Two new species from the Hygrobates nigromaculatus-complex (Acariformes, Hydrachnidia, Hygrobatidae), based on morphological and molecular evidence. Acarologia, Acarologia, 2020, 60 (4), pp.753-768. 10.24349/acarologia/20204400. hal-02972682 HAL Id: hal-02972682 https://hal.archives-ouvertes.fr/hal-02972682 Submitted on 20 Oct 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 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Two new species from the Hygrobates nigromaculatus-complex (Acariformes, Hydrachnidia, Hygrobatidae), based on morphological and molecular evidence Vladimir Pešića , Milica Jovanovića , Ana, Manovića , Andrej Zawalb , Aleksandra Bańkowskac , Łukasz Brodad , Peter Martine , Miroslawa Dabertf a Department of Biology, University of Montenegro, Cetinjski put b.b., 81000 Podgorica, Montenegro. b Institute of Marine and Environmental Sciences, Center of Molecular Biology and Biotechnology, University of Szczecin, Waska 13, 71–415 Szczecin, Poland. c Institute of Biology, University of Szczecin, Waska 13, 71–415 Szczecin, Poland. d Department of Animal Morphology, Institute of Environmental Biology, Adam Mickiewicz University, Umultowska 89, 61–614 Poznan, Poland. e Zoological Institute: Limnology, Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany. f Molecular Biology Techniques Laboratory, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland. Original research ABSTRACT We analyse the taxonomic structure of the Hygrobates nigromaculatus-complex from the Balkan Peninsula. We describe two new species: Hygrobates lacrima Pešic sp. nov. (Montenegro) and H. limnocrenicus Pešic sp. nov. (Montenegro, North Macedonia). Although both species are morphologically similar, the average K2P-distance between DNA-barcode sequences from H. limnocrenicus sp. nov. and its closest relative H. setosus was 12.43% (SD = 1.47), and between H. lacrima sp. nov. and its closest relative H. nigromaculatus 15.87% (SD = 1.74). The new species exhibit distinct differences in terms of habitat preference: H. lacrima sp. nov. inhabits pools and shallow eddies along faster flowing waters, whereas H. limnocrenicus sp. nov. prefers deeper, fast flowing water, typically found in the outflow of a limnocrenic springs or lake outlets. The finding of these two new species suggests that efforts to investigate mites of the H. nigromaculatus-complex in the Balkans should be intensified. Keywords water mites; DNA-barcoding; species delimitation; new species; running waters; Montene- Received 18 August 2020 Accepted 16 October 2020 gro; Macedonia Published 20 October 2020 Zoobank http://zoobank.org/754BE1B0-A316-409B-8008-556CE54ED5E4 Corresponding author Vladimir Pešić: [email protected] Introduction Academic editor Water mites of the genus Hygrobates Koch, 1837 are often the most ubiquitous and usually the Mąkol, Joanna most abundant representatives of the group in different types of running and standing waters over the Palaearctic (Pešić et al. 2017). In terms of ecology, many species of this genus have been DOI reported in literature to be present in both standing (lakes) and running (streams) water habitats. 10.24349/acarologia/20204400 Such kind of bipolar habitat preference, for example has been reported for H. nigromaculatus ISSN 0044-586X (print) Lebert, 1879 and H. longipalpis (Hermann, 1804), both often reported as common species in ISSN 2107-7207 (electronic) Europe living both in lakes and streams (see Martin et al. 2010 for a discussion). Recently, Copyright the status of these species has been questioned by the integrative studies using DNA barcodes, Pešic V. et al. proving that lake and stream populations, indeed, represent morphologically and genetically Distributed under Creative Commons CC-BY 4.0 How to cite this article Pešic V. et al. (2020), Two new species from the Hygrobates nigromaculatus-complex (Acar- iformes, Hydrachnidia, Hygrobatidae), based on morphological and molecular evidence. Acarologia 60(4): 753-768; DOI 10.24349/acarologia/20204400 distinct lineages (see Martin et al. 2010 for H. nigromaculatus; Pešić et al. 2019a for H. longipalpis). Hygrobates nigromaculatus has been subject of controversial debate in taxonomy for a long time (see Martin et al. 2010 for an overview). The status of populations from Northern and Central Europe was resolved by a molecular study: using the DNA barcode region of the mitochondrial cytochrome c subunit I (COI) gene and the nuclear D2 region of 28S rRNA gene, the presence of two well-defined species could be revealed, H. setosus Besseling, 1942 living in streams, and H. nigromaculatus in lakes (Martin et al. 2010). In this study we used morphological data and results of DNA-barcoding to analyse specimens of the H. nigromaculatus s.l. from the Balkans, with the aim to evaluate potentially cryptic species and establish the nigromaculatus species complex in the genus Hygrobates. As a result, two species new to science are described. Materials and methods Water mites were collected by hand netting, sorted live in the field, and immediately preserved in 96% ethanol. Specimens for molecular analysis were examined without dissecting under a compound microscope in ethanol, using a cavity well slide with a central depression. After DNA extraction, some specimens were dissected and slide mounted in Faure’s medium. Morphological nomenclature follows Pešić et al. (2017; for explanations concerning morphology and measurements of Hygrobates species see there Figs. 1B-D). The holotypes of the new species are deposited in Naturalis Biodiversity Center in Leiden (RMNH). DNA sequences prepared in the course of this study are published in BOLD with accession numbers indicated in Table 1. All measurements are given in µm. The following abbreviations are used: Ac-1 = most anterior acetabulum; Cx-I = first coxae; dL = dorsal length; H = height; I-L-4-6 = fourth to sixth segments of first leg; L = length; mL = median length; n = number of specimens examined; P-1– P-5 = palp segments 1 to 5; W = width; χ = mean value. Molecular analysis Molecular analysis was conducted in the Department of Invertebrate Zoology and Hydrobiology, University of Łódź, Poland. For methods used for COI gene amplification and sequencing see Pešić et al. (2017). For this study, DNA was extracted from a total number of three specimens of genus Hygrobates from Montenegro and North Macedonia (Table 1). For DNA-barcoding and phylogenetic analysis we used previously published COI sequence data from Martin et al. (2010). In total, we used 51 sequences representing COI haplotypes of Hygrobates lacrima sp. nov. (1), H. limnocrenicus sp. nov. (2), H. setosus (n=33), H. nigromaculatus (n=11), with H. trigonicus (2), H. prosiliens (1) and Limnesia undulata (1) as outgroup taxa; the latter ones were chosen following Martin et al. (2010). Sequences were aligned by MUSCLE 3.8.425 algorithm as implemented in Geneious Prime 2020.1.1 (Biomatters Ltd.). Phylogenetic tree for species delimitation was constructed Table 1 List of newly sequenced specimens used in this study. For previously published sequences of the Hygrobates nigromaculatus-complex see Martin et al. (2010). Locality (country, name) Lat/Long Voucher code BOLD Acc. nos. Hygrobates limnocrenicus sp. nov. Montenegro, Mareza 42°28'44.01"N, 19°10'52.50"E 13. M19_20_5_E4 DNAEC051-20 North Macedonia, Struga 41°11'3.39"N, 20°40'40.88"E 19. MECD2019_3.2_C2 DNAEC024-20 Hygrobates lacrima