Biodiversity Data Journal 9: e64486 OO) doi: 10.3897/BDJ.9.e64486 open access Taxonomic Paper Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species from Bjeshket e Nemuna National Park in the Republic of Kosovo, with molecular and ecological notes Halil Ibrahimit, Astrit Bilalli8, Simon Vitecek!'l*, Steffen U. Pauls", Felicitas Erzinger”, Agim Gashi*, Linda Grapci Kotori+, Donard Gecit, Milaim Musliu8, Edison Kasumaj* + University of Prishtina "Hasan Prishtina", Faculty of Mathematics and Natural Sciences, Department of Biology, Eqrem Cabej nn., Prishtina, Kosovo § University of Peja "Haxhi Zeka", Faculty of Agribusiness, UCK str. nn., Peja, Kosovo | University of Vienna, Department of Functional and Evolutionary Ecology, AlthanstraRe 14, 1090, Vienna, Austria 4 University of Natural Resources and Life Sciences, Vienna Institute of Hydrobiology and Aquatic Ecosystem Management (IHG) Gregor-Mendel-Strake 33, 1180, Vienna, Austria # WasserCluster Lunz — Biologische Station, Dr. Carl Kupelwieserpromenade 5, 3293, Lunz am See, Austria = Senckenberg Research Institute and Natural History Museum Frankfurt, Senckenberganlage 25, 60388, Frankfurt a. M., Germany « Institute of Insect Biotechnology, Justus-Liebig-University GieRen, Heinrich-Buff-Ring 26, 35392, Gie&en, Germany » Senckenberg Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60388, Frankfurt a. M., Germany Corresponding author: Astrit Bilalli (astritbilalli@yahoo.com) Academic editor: Henrique Paprocki Received: 16 Feb 2021 | Accepted: 05 Apr 2021 | Published: 07 Apr 2021 Citation: Ibrahimi H, Bilalli A, Vitecek S, Pauls SU, Erzinger F, Gashi A, Grapci Kotori L, Geci D, Musliu M, Kasumaj E (2021) Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species from Bjeshkét e Nemuna National Park in the Republic of Kosovo, with molecular and ecological notes. Biodiversity Data Journal 9: e64486. https://doi.org/10.3897/BDJ.9.e64486 ZooBank: urn:lsid:zoobank.org:pub:ACD9E29A-CF 15-4373-9224-98499F 1 DFD01 Abstract Background The Western Balkans are an important hotspot of caddisfly diversity in Europe, with several microscale endemics, many of which were discovered during the recent years. The genus © Ibrahimi H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 lbrahimi H et al Potamophylax Wallengren, 1891 likely originated and diversified in Europe, with the Balkan Peninsula being one of the most important diversity hotspots. New information In this paper, we describe the new species Potamophylax coronavirus sp. n. from Bjeshkét e Nemuna National Park in the Republic of Kosovo. The new species belongs to the Potamophylax winneguthi species group and is morphologically most similar to Potamophylax juliani Kumanski, 1999, currently known only from Bulgaria and Potamophylax winneguthi Klapalek, 1902, known from Bosnia and Herzegovina and Serbia. The male of the new species differs from its most similar congeners mainly in exhibiting: (1) elongated subrectangular superior appendages in lateral view; (2) hardly acuminate, almost rounded apex of intermediate appendages; (3) differently shaped, irregular and higher inferior appendages; (4) narrow spinate area, roughly rectangular in dorsal view, slightly wider at the base and (5) different paramere shape and/or spine pattern. The new species also differs by its considerably smaller size and association with open, high altitude eucrenal zones. The uncorrected interspecific pairwise distance between P coronavirus and other species of the P winneguthi species group is on par with those amongst other recognised species in the group, as well as with the yet ambiguously identified taxa from the Sharr, Rila and Bajgoré Mountains. The new species is most probably a microendemic of Bjeshkét e Nemuna, thus highlighting further this area as an important hotspot of caddisfly biodiversity in Europe. Keywords rare species, caddisflies, Balkans, Potamophylax winneguthi species group, microscale endemism Introduction The genus Potamophylax likely originated and diversified in Europe, with only seven taxa distributed outside the continent, in nearby Asia (Mey 1979, Darilmaz and Salur 2015, Sipahiler 2014). Species of this genus are frequently found in different habitats of freshwater ecosystems, such as spring areas, small brooks, large rivers and lakes as well (Graf et al. 2008). Larvae of some species have also been found in man-made habitats and brackish waters (e.g. Williams and Williams 1998). Adults are found from early spring up to late autumn, with some species undergoing a summer diapause. While many species of this genus are widely distributed, several others have a narrow area of distribution and are mostly found in the Balkan Peninsula and the Iberian Peninsula, which are probably the main speciation areas (Martinez et al. 2016, Olah et al. 2013, Olah and Kovacs 2012). Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 3 The ongoing caddisfly investigations in the Balkan Peninsula during the past decade have discovered several new species, have increased the knowledge about the ecology of many other species and contributed to raising awareness about conservation of this order of aquatic insects and associated habitats (e.g. lbrahimi et al. 2015, lbrahimi et al. 2016, lbrahimi et al. 2019a, Olah et al. 2018, Vitecek et al. 2017). For example, this increased knowledge about Balkan caddisflies has culminated in the listing of 53 species into the Red Book of Fauna of the Republic of Kosovo (lbrahimi et al. 2019c). This is the first inclusion of Trichoptera in Red Lists in south-eastern Europe. However, there are still areas in the Balkan Peninsula which are under-investigated or not investigated at all. Several new records and species have been reported lately from the Bjeshkét e Nemuna Mountains, from Albania and Kosovo (e.g. lbrahimi et al. 2014, lbrahimi et al. 2019a, Ibrahimi et al. 2019b, Malicky 1981, Malicky 1986, Olah et al. 2017, Vitecek et al. 2015b); however, the number of caddisfly species from this mountainous massif is still low. Considering the great diversity of freshwater habitats in the Bjeshkét e Nemuna Mountains that expand into three countries (Albania, Montenegro and Kosovo), we posit that the number of caddisfly species is considerably higher than currently known. In this paper, we describe a new species of Potamophylax from Bjeshkét e Nemuna in the Republic of Kosovo, with molecular and ecological notes included and also discuss morphological and ecological features of other species of the Potamophylax winneguthi species group. Materials and methods Fieldwork, identification and taxonomic work We collected adult caddisflies with entomological nets and handpicking from the riparian vegetation near the streams, as well as with ultraviolet light trapping in the vicinity of the streams. Nocturnal light trapping followed Malicky (2004). Specimens were stored directly in 80% ethanol. Specimens of the new species were collected at two localities in the Llogan Mountain, belonging to the Bjeshkét e Nemuna massif in the Degan Municipality (Fig. 1). Specimens of three other, still unidentified, taxa of the Potamophylax winneguthi species group were collected in two areas in the Republic of Kosovo: Sharr Mountains and Bajgoré and Rila Mountain in Bulgaria. Details of the sampling stations and specimens are given in the examined material list further on and in Table 1. The collected material is deposited at the Department of Biology, Faculty of Mathematics and Natural Sciences, University of Prishtina “Hasan Prishtina’, Prishtiné, Republic of Kosovo. In addition, for comparative assessments of morphological features of Potamophylax coronavirus sp. Nn., we used specimens collected in Osogovo Mountain in Bulgaria (Potamophylax juliani) and Ziatibor Mountain in Serbia (Potamophylax winneguthi). For the lacking species, comparative assessment was done, based on literature (Malicky 2004, Kumanski and Malicky 1999, Olah and Kovacs 2012). Table 1. lbrahimi H et al Collection data, specimen IDs and BOLD IDs of specimens used in phylogenetic analysis. Abbreviations used: HI — Halil lbrahimi, AB — Astrit Bilalli, AG — Agm Gashi, LGK — Linda Grapci- Kotori, EK — Edison Kasumaj, DG — Donard Geci, MM — Milaim Musliu, WG — Wolfram Graf, JK - Jen6é Kontschan, DM - David Muranyi, TS - Timea Szederjesi, AE - A. Ecsedi, TK - T. Kovacs, GP - G. Puskas, GM - G. Magos. SpecimenID BOLDID Collectors Collection Latitude Longitude Altitude Taxon date (N) (E) m Pgo0101M SPLIM298-21 HI,AG,EK 12.08.2014 42.5518 20.1624 1666 Potamophylax seprus Psp40101M SPLIM299-21 AE, TK, 07.10.2011 42.139028 23.46452 1935 Potamophylax sp. GP (Rila, Bulgaria) Psp30101F SPLIM300-21 HI, AB 13.10.2014 42.549100 20.138330 2066 Potamophylax coronavirus sp. n. Psp30102M SPLIM301-21 HI 13.10.2014 42.549100 20.138330 2066 Potamophylax coronavirus sp. n. Pju0101M SPLIM302-21 JK, DM, 23.10.2013 42.174383 22.63443 1520 Potamophylax TS juliani Pju0102F SPLIM303-21 JK, DM, 23.10.2013 42.174383 22.63443 1520 Potamophylax TS juliani Psp20101M SPLIM304-21 HI, DG 29.10.2013 42.979 21.0509 1262 Potamophylax sp. (Bajgoré, Kosovo) Psp10101M SPLIM305-21_ HI 11.11.2012 42.17506 20.97593 1410 Potamophylax sp. (Sharr, Kosovo) Psp10201M SPLIM306-21_ HI 18.10.2012 42.17506 20.97593 1410 Potamophylax sp. (Sharr, Kosovo) Ppa0101M SPLIM307-21 HI, MM 21.08.2013 42.5185 20.9788 721 Potamophylax pallidus Pro0101M SPLIM308-21 HI, LGK 01.10.2013 42.5185 20.9788 721 Potamophylax rotundipennis Pwi0101F SPLIM309-21 TK, GM 03.11.2011 43.63127 19.77166 1150 Potamophylax winneguthi Pwi0102M SPLIM310-21 TK, GM 03.11.2011 43.63127 19.77166 1150 Potamophylax winneguthi fMelaus0101M SPDRU496-14 WG 20.10.2013 46.8106 14.9931 N.A. Melampophylax austriacus Morphological characteristics of male terminalia of the new species were examined in cleared specimens. Nomenclature of male terminalia follows Nielsen (1957), for Limnephilus flavicornis (Fabricius, 1787) and Kumanski and Malicky (1999). The rest of the specimens were identified using publications by Kumanski (1988), Malicky (2004) and Olah et al. (2017). Systematic nomenclature follows Morse (2020). Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 5 Morphological features of genitalia of Potamophylax coronavirus sp. n. were analysed from seven male specimens. Illustrations were prepared in Adobe Illustrator (version Creative Cloud 2018) by digitising pencil templates made with a camera lucida. Figure 1. EES] Pictures of type locality of Potamophylax coronavirus sp. n.: Krojet e Ali Pashé Gucisé. DNA extraction and PCR amplification Whole genomic DNA was extracted from the abdomen of adult or individual legs of larval specimens using the DNEasy Blood and Tissue Kit (Qiagen, Hilden), following the manufacturer’s protocol. Three mitochondiral gene regions (mtCOI5-P, mtCOI3-P and 16S rDNA) and three nuclear gene regions (CADH, WG and 28S nrDNA) were amplified using standard PCR procedures and primers as described in Vitecek et al. (2015a). All these genes have been shown to successfully discriminate closely-related species in various Trichoptera families (e.g. Vitecek et al. 2015a, Hjalmarsson et al. 2019). Reactions for PCR amplifications were set up in 10 ul reactions. PCR products were visualised on 1.5% agarose gels and purified in the case of nuclear 28S nrDNA (Qiagen Gel Extraction; Qiagen) and sequenced in both directions on an ABI 3730XL sequencer (Applied Biosystems, Waltham) at the Senckenberg Biodiversity and Climate Research Centre, Frankfurt. In order to obtain full length information of the 1627 bp long 28S gene (D1—D3 regions), two internal primers (D2-UP-4 / D2DN-B; Zhou et al. 2007) were used. Sequence alignment and phylogenetic analyses Raw ABI sequence reads were edited and merged in Geneious R6 (https:/Awww.ge neious.com). Sequences were aligned using MAFFT (Katoh and Standley 2013) and manually trimmed to exclude regions with > 80% missing data in the peripheral regions of 6 lbrahimi H et al the alignments. Phylogenetic relationships were inferred using Bayesian and Maximum- Likelihood methods, based on a concatenated alignment of 2918 bp length (Table 1). Nucleotide substitution models were selected for each method separately, using automatic substitution model selection via bModelTest (Bouckaert and Drummond 2017) and ModelFinder (Kalyaanamoorthy et al. 2017), as implemented in BEAST2 (Bouckaert et al. 2014) and IQ-TREE2 (Minh et al. 2020), respectively. For Bayesian Inference through MrBayes 3.2 (Ronquist et al. 2012), appropriate nucleotide substitution models for each partition were selected using MEGA (Stecher et al. 2020). Protein coding genes were partitioned by codon; 16S rDNA and 28S nrDNA were not. Bayesian Inference through BEAST2 to estimate phylogenetic relationships was done by running analyses of 10x10°9 generations and sampling every 10,000"" generation. Bayesian Inference through MrBayes was conducted in 2 parallel runs with 2 chains each, running for10x10° generations and sampling every 10,000" generation. BEAST log files and MrBayes parameter files were visualised in Tracer1.6 (Rambaut et al. 2014) and checked for convergence of model parameters. Maximum-clade-credibility trees were estimated with TreeAnnotator from Bayesian tree samples obtained through BEAST2 after discarding 80% of the samples as runs failed to become stationary before this threshold. A majority rule consensus tree was estimated from the trees sampled by MrBayes after discarding the first 50% of the sample as average standard deviation of split frequencies reached values < 0.01 past that point. Finally, a Maximum-Likelinood tree was inferred using IQ-TREE2 and support values estimated, based on 1000 ultrafast bootstrap samples (Hoang et al. 2018), as well as 1000 replicates of an SH-like approximate likelihood ration test (Guindon et al. 2010). Phylogenetic reconstruction was conducted twice with each method to assure stability of recovered tree topologies. Results of phylogenetic analyses are presented using the Maximum-Likelinood topology where node support values also include Bayesian posterior probabilities; topologies inferred through Bayesian methods are provided in the Supplementary files (Suppl. materials 1, 2). Taxon treatment Potamophylax coronavirus \brahimi, Bilalli & Vitecek, sp. n. ° ZooBank A0208ED4-C6EC-43FB-8FED-6E16AF16DABB Materials Holotype: a. class: Insecta; order: Trichoptera; family: Limnephilidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Potamophylax; specificEpithet: coronavirus; scientificNameAuthorship: lbrahimi, Bilalli & Vitecek; continent: Europe; waterBody: Adriatic Watershed; country: Kosovo; countryCode: XKS; municipality: Degan; locality: Bjeshkét e Nemuna, Llogqan Mountain.; verbatimLocality: A tributary of Lumbardhi i Deganit River, Krojet e Ali Pashé Gucisé springs.; verbatimElevation: 2066; decimalLatitude: 42.5491; decimalLongitude: 20.13833; samplingProtocol: entomological net; year: 2014; month: 10; day: 13; individualCount: 1; sex: male; lifeStage: adult; recordedBy: Halil lbrahimi; datasetName: Trichoptera Bjeshket e Nemuna Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 7 Other material: a. class: Insecta; order: Trichoptera; family: Limnephilidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Potamophylax; specificEpithet: coronavirus; scientificNameAuthorship: lbrahimi, Bilalli & Vitecek; continent: Europe; waterBody: Adriatic Watershed; country: Kosovo; countryCode: XKS; municipality: Degan; locality: Bjeshkét e Nemuna, Llogqan Mountain.; verbatimLocality: A tributary of Lumbardhi i Deganit River, Krojet e Ali Pashé Gucisé springs.; verbatimElevation: 2066; decimalLatitude: 42.5491; decimalLongitude: 20.13833; samplingProtocol: entomological net; year: 2014; month: 10; day: 13; individualCount: 1; sex: female; lifeStage: adult, with slightly damaged terminalia; recordedBy: Halil lbrahimi; datasetName: Trichoptera Bjeshket e Nemuna Paratypes: a. class: Insecta; order: Trichoptera; family: Limnephilidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Potamophylax; specificEpithet: coronavirus; scientificNameAuthorship: lbrahimi, Bilalli & Vitecek; continent: Europe; waterBody: Adriatic Watershed; country: Kosovo; countryCode: XKS; municipality: Degan; locality: Bjeshkét e Nemuna, Llogan Mountain.; verbatimLocality: A tributary of Lumbardhi i Deganit River, Gurrat e Hasan Agés springs.; verbatimElevation: 2218; decimalLatitude: 42.560696; decimalLongitude: 20.153876; samplingProtoco!l: entomological net; year: 2014; month: 10; day: 15; individualCount: 2; sex: male; lifeStage: adult; recordedBy: Halil Ilbrahimi, Agim Gashi; datasetName: Trichoptera Bjeshket e Nemuna class: Insecta; order: Trichoptera; family: Limnephilidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Potamophylax; specificEpithet: coronavirus; scientificNameAuthorship: lbrahimi, Bilalli & Vitecek; continent: Europe; waterBody: Adriatic Watershed; country: Kosovo; countryCode: XKS; municipality: Degan; locality: Bjeshkét e Nemuna, Llogan Mountain.; verbatimLocality: A tributary of Lumbardhi i Deganit River, Krojet e Ali Pashé Gucisé springs.; verbatimElevation: 2066; decimalLatitude: 42.5491; decimalLongitude: 20.13833; samplingProtocol: entomological net; year: 2014; month: 11; day: 21; individualCount: 4; sex: male; lifeStage: adult; recordedBy: Halil lbrahimi, Astrit Bilalli, Linda Grapci-Kotori, Donard Geci, Edison Kasumaj; datasetName: Trichoptera Bjeshket e Nemuna class: Insecta; order: Trichoptera; family: Limnephilidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Potamophylax; specificEpithet: coronavirus; scientificNameAuthorship: lbrahimi, Bilalli & Vitecek; continent: Europe; waterBody: Adriatic Watershed; country: Kosovo; countryCode: XKS; municipality: Degan; locality: Bjeshkét e Nemuna, Llogan Mountain.; verbatimLocality: A tributary of Lumbardhi i Deganit River, Krojet e Ali Pashé Gucisé springs.; verbatimElevation: 2066; decimalLatitude: 42.5491; decimalLongitude: 20.13833; samplingProtocol: entomological net; year: 2020; month: 12; day: 03; individualCount: 2; sex: male; lifeStage: adult; recordedBy: Halil Ibrahimi, Astrit Bilalli, Linda Grapci-Kotori, Donard Geci, Edison Kasumaj; datasetName: Trichoptera Bjeshket e Nemuna Description Male. General appearance (Figs 2, 3). Head and appendages brown, prothorax, sclerites of meso and metathorax and coxae dark brown to black; femora and tibiae brown, tarsi gradually darkening towards the apex. Wings dark brown with dark setae. Male maxillary palp 3 segmented. Forewing length 10 — 11.5 mm, spur formula 1-3-4. Ibrahimi H et al Figure 2. EESI Adults of Potamophylax coronavirus sp. n. in copulation. Figure 3. EE Male adult of Potamophylax coronavirus sp. n. Male genitalia (Figs 4, 5, 6). Tergite VIII dark brown to black, darker than the preceding tergites, in dorsal view roughly quadratic in shape, with dorsal portion slightly narrower, posterior margin with distinct median lobe; setation concentrated on proximal portion of segment VIII, which is well sclerotised, spinate area roughly rectangular in shape with a slightly wider base in dorsal view, located on the semi-membranous distal portion of segment VIII, covered by small black spines, which are more abundant at the apex. Segment IX laterally broad, with rounded proximal areas entering into segment VIII, with short and narrow dorsal and ventral portions. Superior appendages in lateral view long, subrectangular, with rounded tips, slightly narrowing at their basal part, covered Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 9 with thin setae of medium length. Intermediate appendages long, sickle-shaped with slightly rounded apex, turned upwards. Inferior appendages long, asymmetric, broadly connected with the segment IX and fused throughout most of their length, the separation line between them and segment IX visible only in basal third, bulging dorsally at the area between them and segment IX, their protruding upper portion truncated squarely, both dorsal and ventral corners of the upper portion forming rounded points, parallel to each other, directed mesally. Phallic apparatus consists of an aedeagus of medium height and a pair of parameres. Aedeagus bulbous, narrow in the middle, enlarged at the tip with bifid apex, apicomesal excision wide-U-shaped. Parameres robust, brown-black in colour, with a wider base, narrowing gradually towards the apex, with a bunch of short and very thick spines originating mainly at the distal third and a few of them ventraly. Figure 4. EES] Left lateral view of the male genitalia of Potamophylax coronavirus sp. n. VIII - tergite VIII, IX - segment IX, ia - inferior appendages, ima - intermediate appendages, sa - superior appendages. Female. A single female specimen collected during the field trip generally resembles the female of Potamophylax juliani, but smaller in size. Smaller and of lighter colour than the male. Head and appendages brown, prothorax, sclerites of meso and metathorax light brown to brown; femora and tibiae brown, tarsi gradually darkening towards the apex. Brachypterous. Forewings light brown in colour, shorter than abdomen, with very long and strong erect setae, mostly on the longitudinal veins. Forewing length 8.5 mm. Spur formula 1-3-4. Antennae slender. It has slightly damaged terminalia and thus we currently refrain from describing genitalia. 10 Ibrahimi H et al Figure 5. EE Caudal (A) and dorsal (B) view of the male genitalia of Potamophylax coronavirus sp. n. VIII - tergite VIII, ia - inferior appendages, ima - intermediate appendages, sa - superior appendages. Figure 6. EES Aedeagus (a) and parameres (p) of the male genitalia of Potamophylax coronavirus sp. n. in lateral (A) and ventral view (B). Diagnosis Males of the new species are most similar to Potamophylax juliani, currently known only from Bulgaria and Potamophylax winneguthi, known from Bosnia and Herzegovina and Serbia, but differ in exhibiting: (1) elongated subrectangular superior appendages in lateral view, slightly narrowing at their base, rounded at the apex; (2) hardly acuminate, almost rounded apex of intermediate appendages in lateral view; (3) differently shaped inferior appendages, bulging dorsally at the area between them and segment IX in lateral view, with high and broad upper protruding portion, with a narrow Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 11 distance between dorsal and ventral corners of the upper portion, which are set parallel to each other and directed mesially; (4) spinate area narrow, roughly rectangular in dorsal view, only slightly wider at the base and (5) short stout parameres with base wider than the apex and short, very thick spines originating below the apex, only slightly reaching above the apex and few other smaller ones proximally. Potamophylax juliani males have: (1) small, laterally rounded, ovoid superior appendages; (2) long, slender intermediate appendages with sharply acuminate apex in lateral view; (3) rather short inferior appendages, with a shortened protruding upper portion as high as half of the entire appendage’s height, with a wide distance between dorsal and ventral corners, which are set parallel to each other and directed mesially; (4) spinate area in dorsal view narrow at the apex and almost three times wider at the base and (5) short stout parameres with very wide basal third and narrow apex, with 15 — 20 thick spines of medium length originating mostly from the tip. Potamophylax winneguthi males have: (1) small, laterally semicircular superior appendages; (2) long slender intermediate appendages with acuminate apex; (3) inferior appendages parallel-edged, dorsally truncated in a rectangular manner, longer on their ventral edge, directed dorsad; (4) spinate area wide, covering almost the entire width of the distal portion of segment VIII in dorsal view and (5) short stout parallel-edged parameres with almost same width along the entire length, only slightly narrower at their middle part, with 5-7 very long spines originating from distal half. The new species also differs from both of its most similar congeners by its considerably smaller size and different type of habitat, inhabiting open high altitude eucrenal zones. Etymology The species epithet coronavirus relates to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which caused a global pandemia starting 2020. The current paper was written during the quarantine time due to the pandemics. The species epithet also emphasises figuratively another silent pandemic occurring on freshwater organisms in Kosovo rivers, due to the pollution and degradation of freshwater habitats, including particularly the increased activity of mismanaged hydropower plants. Distribution During the field survey in the Bjeshkét e Nemuna Mountains, we found Potamophylax coronavirus sp. n. at only two localities within a 3 km perimeter, although several other springs and brooks were sampled. Ecology Both sampling stations are open spring areas, located above 2000 m a.s.l. The substrate of streams close to the sampling sites was dominated by meso- to macrolithal, surrounded by riparian vegetation. The species was collected during the day by handpicking and entomological nets. The species was not observed flying, 12 lbrahimi H et al implying low flying activity. No specimen was caught with light traps. The species was collected during late September, October, November and early December, implying it has an autumn flying period. In both sites, the specimens of the new species were collected only within a one-kilometre perimeter from the spring area downwards along the stream, implying that it is a typical eucrenal species. In both sampling stations, Potamophylax coronavirus sp. n. was found in sympatry with the following species: Rhyacophila tristis Pictet, 1834, Allogamus uncatus (Brauer, 1857), Drusus botosaneanui Kumanski, 1968, Drusus krusniki Malicky, 1981 and Drusus fortos |lbrahimi and Olah, 2017. Psp10101M Potamophylax sp. 100/100/1 E hd e 95.9/86/1 Psp10201M Potamophylax sp. Psp40101M Potamophylax sp. 77.5/50/0.72 Pju0102F P. juliani 100/100/1 N Pju0101M P. juliani Psp20101M Potamophylax sp. 74.9/66/0.9 60.4/54/0.58 Pwi0101F P. winneguthi 100/100/1 100/100/1 Pwi0102M P. winneguthi Psp30101F P. coronavirus sp. nov. 100/100/1 Psp30102M P. coronavirus sp.nov. Pgo0101M P. seprus Pro0101M P. rotundipennis Ppa0101M P. pallidus fMelaus0101M Melampophylax austriacus 0.02 Figure 7. EES Maximum Likelihood tree of phylogenetic relationships within the Potamophylax winneguthi species group and closely related congeners. The P. winneguthi group comprises a highly supported clade with new species P coronavirus sp. n. supported as sister to other species of the group, including yet ambiguously identified taxa from the Sharr, Rila and Bajgoré Mountains. Support from SH-like approximate likelihood ratio tests/Ultrafast Bootstrap/ Bayesian posterior probabilities are presented above nodes; outgroup: Melampophylax austriacus. Results of phylogenetic analysis Both methods recovered the same supported topology and highly similar support values. The phylogenetic analyses reveal that the P winneguthi species group is monophyletic with regard to the other included Potamophylax species and Melampophylax (Fig. 7, Suppl. materials 1, 2). Within the P winneguthi species group, relationships are largely unresolved. Only the proposed conspecificity of males and females is confirmed in all four species (posterior probabilities of 1.0, high bootstrap support). In addition, the Potamophylax spp. from Kosovo and Bulgaria appear to have Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 13 a sister relationship in this group. The uncorrected interspecific pairwise distance between P coronavirus and other species of the winneguthi species group is on par with those amongst other recognised species in the group, as well as with the yet ambiguously identified taxa from the Sharr, Rila and Bajgoré Mountains (0.03-0.05;Suppl. materials 2, 3). Discussion Potamophylax coronavirus sp. n. is a member of the Potamophylax winneguthi species group. This group contains the following species: Potamophylax winneguthi, Potamophylax haidukorum Malicky, 1999, Potamophylax juliani, Potamophylax gurunaki Malicky, 1992, Potamophylax alsos Olah, 2014, Potamophylax hajlos Olah, 2012, Potamophylax kesken Olah, 2012, Potamophylax tagas Olah & Kovacs, 2012 and Potamophylax coronavirus sp. n. All of them are restricted to certain areas in the Balkan Peninsula: P winneguthi in Varosh and some nearby localities around Sarajevo (Bosnia and Herzegovina) and Zlatibor (Serbia), P. haidukorum nearby Hajducka Voda (Bosnia and Hercegovina), P juliani in Osogovo Mountain (Bulgaria), P gurunaki in Vernon (Greece), P. a/sos in Jabllanica (North Macedonia), P hajlos in Mali i Gropés (Albania) and P kesken and P. tagas in Korab (Albania) (Kumanski and Malicky 1999, Olah and Kovacs 2015). Morphologically, Potamophylax coronavirus sp. n. forms a cluster together with Potamophylax winneguthi, Potamophylax haidukorum and Potamophylax juliani. This cluster is characterised by a bilobed apical margin of inferior appendages in lateral and caudal views. In this cluster, Potamophylax coronavirus sp. n. is small (male wing length 10-11.5 mm), while the other species of the group have wing lengths from 11-18 mm. The other cluster of this species group, the Potamophylax tagas species cluster, comprises P alsos, P. hajlos, P. kesken and FP. tagas and is characterised by a rounded apical margin of the inferior appendages without any significant projection. The remaining species of this species group, Potamophylax gurunaki is characterised by its remarkable pointed long apicodorsal corner of inferior appendages and longer rounded apicoventral corner in lateral view. During this investigation in Kosovo, we sampled specimens of two other taxa closely resembling Potamophylax juliani: one from the Bajgoré area in northern Kosovo and the second one from the Sharr Mountains in western Kosovo. The comparison of these specimens with the specimens of Potamophylax juliani from the type locality showed that there are certain differences, mostly in the shape of inferior appendages and phallic apparatus. In Potamophylax juliani, the ventral margin of inferior appendages is sharply angled at its distal third, while, in the taxon from Bajgoré area, the ventral margin is curved, but not sharply angled and in the taxon from the Sharr Mountain, it is almost concave. The aedeagus, parameres and superior appendages of both populations have also certain differences from those of P juliani. The male specimen collected in Rila Mountian in Bulgaria differs from P juliani too by the shape of inferior appendages and width of parameres. However, the number of specimens examined in all these three taxa is too small to draw a conclusion related to the morphology of these populations. More 14 Ibrahimi H et al specimens from these areas are needed to show if these differences are stable and consequently to resolve their taxonomic position. The phenology of Potamophylax coronavirus sp. n. is similar to the other species of the Potamophylax winneguthi species group. All of them are typical autumn species. However, unlike other species of this group, specimens of P coronavirus sp. n. were also collected during early December in 2018, when the daily temperatures were above 13°C. Habitat requirements of the new species are different from all other species of the Potamophylax juliani species cluster collected throughout Balkans. All other species inhabit mostly spring areas or rivers inside the forested areas, while the new species was only found at open spring areas, well above the tree line. Similar to the other species of the group, the single female Potamophylax coronavirus sp. n. specimen collected during this investigation was brachypterous and unable to fly. Freshwater ecosystems in the area where the new species was found, have been extremely threatened during the past years by anthropogenic activities, such as deforestation, habitat destruction, hydropower plants and touristic activities. In addition, many springs in the area are mismanaged and endangered by individual water intake pipes from nearby houses and touristic facilities. The change in water regime may greatly threaten aquatic diversity in the near future. For example, the downstream segments of the Lumbardhi i Deganit River, whose tributaries are springs where the new species was found, were severely deteriorated during the past years due to the construction of a hydropower plant. The prescribed minimal flows are not implemented in practice and most of the time are too low to sustain aquatic biota. The description of a new micro-endemic Potamophylax species from this area documents that the species of Potamophylax winneguthi species group are restricted to isolated highland freshwater habitats, similar to some species of the genera Drusus Stephens, 1837 and Chaetopteroides Kumanski, 1987. The high number of micro-endemics in Potamophylax may also be the result of a combination of geological processes and low dispersal capacity, as hypothesised for the Balkan Drusus species (Previsic et al. 2014). Very often, species of Potamophylax winneguthi species group live in sympatry with species of these two genera, although unlike Drusinae, species of this species group were found also in warmer and smaller springs and streams of lower altitudes. This investigation highlights the Bjeshkét e Nemuna Mountains as an important area, which harbours rare species of caddisflies, which are, at the same time, critically endangered by ongoing activities causing the degradation of freshwater habitats. Acknowledgements We would like to express our gratitude postmortem to Mr. Arif Kasumaj who showed us the type locality of Potamophylax coronavirus sp. n. and drove us there several times during 2014 and 2015 and to many other localities in the Llogan Mountain. We would also like to thank Professor Janos Olah for loaning specimens of Potamophylax juliani, Potamophylax winneguthi and Potamophylax sp. from the Rila Mountain. We would like to thank Subject Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 15 Editor, Henrique Paprocki and two reviewers, Allan Paulo Santos and Roger Blahnik, whose comments greatly improved the earlier version of the manuscript. References ° Bouckaert R, Heled J, Khnert D, Vaughan T, Wu c, Xie D, Suchard MA, Rambaut A, Drummond AJ (2014) BEAST 2: a software platform for Bayesian evolutionary analysis. PLOS Computational Biology 10: e1003537. https://doi.org/10.1371/journal.pcbi. 1003537 ° Bouckaert RR, Drummond AJ (2017) bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evolutionary Biology 17: 42. https://doi.org/ 10.1186/s12862-017-0890-6 ° Darilmaz MC, Salur A (2015) Annotated Catalogue of the Turkish Caddisflies (Insecta: Trichoptera). Munis Entomology & Zoology 10 (Supp.): 521-734. ° Graf W, Murphy J, Dahl J, Zamora-Munoz C, Lopez-Rodriguez MJ (2008) Volume 1 - Trichoptera. In: Schmidt Kloiber A, Hering D (Eds.), Distribution and ecological preferences of European freshwater organisms. Pensoft Publishers, 338 pp. ° Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelinood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59 (3): 307-321. https://doi.org/10.1093/ sysbio/syq010 ° Hjalmarsson AE, Graf W, Vitecek S, Jhnig SC, Cai Q, Sharma S, Tong X, Li F, Shah DN, Tachamo Shah RD, Pauls SU (2019) Molecular phylogeny of Himalopsyche (Trichoptera, Rhyacophilidae). Systematic Entomology 44: 973-984. https://doi.org/ 10.1111/syen.12367 ° Hoang DT, Chernomor O, Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35: 518-522. https://doi.org/10.1093/molbev/msx281 ° Ibrahimi H, Kuini M, Gashi A, Grapci Kotori L (2014) Trichoptera biodiversity of the Aegean and Adriatic Sea basins in Kosovo. Journal of Insect Science 14: 1-8. ° Ibrahimi H, Kucinic M, Vitecek S, Waringer J, Graf W, Previsic A, Balint M, Keresztes L, Pauls S (2015) New records for the Kosovo caddisfly fauna with the description of a new species, Drusus dardanicus sp. nov. (Trichoptera: Limnephilidae). Zootaxa 4032 (5): 551-568. https://doi.org/10.11646/zootaxa.4032.5.5 ° Ibrahimi H, Vitecek S, Previsi¢ A, Kucini¢ M, Waringer J, Graf W, Balint M, Keresztes L, Pauls S (2016) Drusus sharrensis sp. n. (Trichoptera, Limnephilidae), a new species from Sharr National Park in Kosovo, with molecular and ecological notes. ZooKeys 559: 107-124. https://doi.org/10.3897/zookeys.559.6350 ° Ibrahimi H, Grapci-Kotori L, Bilalli A, Qamili A, Schabetsberger R (2019a) Contribution to the knowledge of the caddisfly fauna (Insecta: Trichoptera) of Leqinat lakes and adjacent streams in Bjeshkt e Nemuna (Kosovo). Natura Croatica 28 (1): 35-44. ° Ibrahimi H, Kugi R, Bilalli A, Musliu M, GashiA, Sinani N, Emrllahu B (2019b) Distribution of two rare taxa of caddisflies (Trichoptera: Rhyacophilidae, Polycentropodidae) from the Republic of Kosovo. Biodiversity Data Journal 7: e46466. https://doi.org/10.3897/BDJ.7.e46466 16 Ibrahimi H et al Ibrahimi H, Gashi A, Regjepaj D, Zhushi F, Grapci-Kotori L, Feher Z, Bino T, Jelaska LS, Theou P, Mesaros G (2019c) The Red Book of Fauna of the Republic of Kosovo. In: Ibrahimi H (Ed.) The Red Book of Fauna of the Republic of Kosovo. Ministry of Environment and Spatial Planning, 410 pp. Kalyaanamoorthy S, Minh BQ, Wong TK, Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587-589. https://doi.org/10.1038/nmeth.4285 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772-780. https://doi.org/10.1093/molbev/mst010 Kumanski K (1988) Trichoptera, Integripalpia. Fauna Bulgarica 19. Bulgarska Akademi na Naukite Sofia: 354. Kumanski K, Malicky H (1999) A survey of the genus Potamophylax Wallengren 1891 in the Balkan Peninsula, with description of two new species (Trichoptera: Limnephilidae). Braueria 26: 27-30. Malicky H (1981) Weiteres neues Uber Kécherfliegen aus dem Mittelmeegebiet (Trichoptera). Entomofauna Zeitschrift fur Entomologie 2 (27): 335-355. Malicky H (1986) Beschreibung von vier neuen Kcherfliegen-Arten aus der Trkei und aus Jugoslawien (Trichoptera. Opuscula Zoologica Fluminensia 4: 1-7. Malicky H (2004) Atlas of European Trichoptera. 2nd Edition. Springer, Netherlands, 359 pp. Martinez J, Martin L, Gonzalez MA (2016) A new species of Potamophylax from Spain with a key to the Iberian species (Trichoptera: Limnephilidae). Acta Entomologica Musei Nationalis Pragae 56 (2): 837-844. Mey W (1979) Die Potamophylax luctuosus PILLER Verwandtschaftsgruppe im Kaukasus. Deutsche Entomologische Zeitschrift 36 (I-III): 39-4. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Haeseler A, Lanfear R (2020) |Q-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution 37: 1530-1534. https://doi.org/10.1093/molbev/msaa015 Morse JC (2020) Trichoptera World Checklist. http://entweb.clemson.edu/database/ trichopt/ index.htm. Accessed on: 2020-10-19. Nielsen A (1957) A comparative study of the genital segments and their appendages in male Trichoptera. Biologiske Skrifter udgivet af Det Kongelige Dankse Vedenskabernes Sleskab 8: 1-159. Olah J, Koves T, H | (2018) Agaphylax, a new limnephilid genus (Trichoptera) from the Balkan: Lineage ranking by adaptive paramere. Opuscula Zoologica 49 (1): 77-89. https://doi.org/10.18348/opzool.2018.1.77 Olah J, Kovacs T (2012) New records of Chaetopterygini species (Trichoptera: Limnephilidae). Folia Historico Naturalia Musei Matraensis 36: 81-88. Olah J, Andersen T, Chvojka P, Coppa G, Graf W, lbrahimi H, Lodovici O, Previsic A, Valle M (2013) The Potamophylax nigricornis group (Trichoptera, Limnephilidae): resolution of phylogenetic species by fine structure analysis. Opuscula Zoologica 44 (2): 167-200. Olah J, Kovacs T (2015) New species and records of Balkan Trichoptera Ill. Folia Historico-Naturalia Musei Matraensis 38: 97-131. Potamophylax coronavirus sp. n. (Trichoptera: Limnephilidae), a new species ... 17 Olah J, Beshkov S, Chvojka P, Ciubuc C, Coppa G, lbrahimi H, Kovacs T, Mey W, Olah J (2017) Revision of Drusinae subfamily (Trichoptera, Limnephilidae): divergence by paraproct and paramere: speciation in isolation by integration. Opuscula Zoologica 48 (Supplementum 1): 3-228. https://doi.org/10.18348/opzool.2017.S1.3 Previsi¢ A, Schnitzler J, Kucini¢ M, Graf W, lbrahimi H, Kerovec M, Pauls SU (2014) Micro-scale vicariance and diversification of Western Balkan caddisflies linked to karstification. Freshwater Science 33: 250-262. httos://doi.org/10.1086/674430 Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) http://tree.bio.ed.ac.uk/software/ tracer. Tracer v.1.6. Ronquist F, Teslenko M, Mark P, Ayres DL, Darling A, Hhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MRBAYES 3.2: Efficient Bayesian phylogenetic inference and model selection across a large model space. Systematic Biology 61: 539-542. https://doi.org/10.1093/sysbio/sys029 Sipahiler F (2014) Three new species of Trichoptera (Odontoceridae, Leptoceridae) and the faunistic list for Zonguldak and KarabUk provinces in northwestern Turkey. Munis Entomology & Zoology 9 (1): 542-55. Stecher G, Tamura K, Kumar S (2020) Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Molecular Biology and Evolution 37 (4): 1237-1239. httos://doi.org/ 10.1093/molbev/msz312. Vitecek S, Graf G, Previsi¢ A, Kucini¢ M, Olah J, Balint M, Keresztes L, Pauls SU, Waringer J (2015a) A hairy case: the evolution of filtering carnivorous Drusinae (Limnephilidae. Trichoptera). Molecular Phylogenetics and Evolution 93 (24960): 249-260. https://doi.org/10.1016/j.ympev.2015.07.019 Vitecek S, Kucini¢ M, Olah J, Previsic A, Balint M, Keresztes L, Waringer J, Pauls SU, Graf W (2015b) Description of two new filtering carnivore Drusus species (Limnephilidae, Drusinae) from the Western Balkans. ZooKeys 513: 79-104. https://doi.org/10.3897/zookeys.513.9908 Vitecek S, Kuéinié M, Previ8ié A, Zivié |, Stojanovié K, Keresztes L, Balint M, Hoppeler F, Waringer J, Graf W, Pauls S (2017) Integrative taxonomy by molecular species delimitation: multi-locus data corroborate a new species of Balkan Drusinae micro- endemics. BMC Evolutionary Biology 17: 129. https://doi.org/10.1186/s12862- 017-0972-5 Williams DD, Williams EN (1998) Aquatic insects in an estuarine environment: densities, distribution and salinity tolerance. Freshwater Biology 39: 411-421. https://doi.org/ 10.1046/j.1365-2427.1998.00285.x Zhou X, Kjer KM, Morse JC (2007) Associating larvae and adults of Chinese Hydropsychidae caddisflies (Insecta: Trichoptera) using DNA sequences. Journal of the North American Benthological Society 26: 719-742. httos://doi.org/10.1899/06-089.1 18 lbrahimi H et al Supplementary materials Suppl. material 1: Maximum clade credibility tree of phylogenetic relationships within the Potamophylax winneguthi species group and closely related groups inferred from one of two Bayesian tree samples obtained via BEAST. The new species P. coronavirus sp. n. is supported as sister to other taxa of the highly supported P. winneguthi group. Bayesian posterior probabilites are presented next to nodes; outgroup: Melampophylax austriacus. FE] Authors: Halil Ibrahimi, Astrit Bilalli, Simon Vitecek, Steffen U. Pauls, Felicitas Erzinger, Agim Gashi, Linda Grapci-Kotori, Donard Geci, Milaim Musliu and Edison Kasumaj Data type: Genomic Download file (61.05 kb) Suppl. material 2: Uncorrected pairwise distances amongst study specimens on the basis of the concatenated alignment of all six gene fragments. Highlighted in grey are species and specimens from the P. winneguthi-complex. In the matrix, light grey fields indicate intraspecific distances; dark grey fields interspecific distances.*Values in specimen are out of range, because the CAD locus was completely missing and coded in Ns. EES] Authors: Halil Ibrahimi, Astrit Bilalli, Simon Vitecek, Steffen U. Pauls, Felicitas Erzinger, Agim Gashi, Linda Grapci-Kotori, Donard Geci, Milaim Musliu and Edison Kasumaj Data type: Genomic Download file (208.07 kb) Suppl. material 3: Majority rule consensus tree of phylogenetic relationships within the Potamophylax winneguthi species group and closely related groups inferred from two Bayesian tree samples obtained via MrBayes. The new species P. coronavirus sp. n. is supported as sister to other taxa of the highly supported P. winneguthi group. Bayesian posterior probabilites are presented next to nodes; outgroup: Melampophylax austriacus. F&5] Authors: Halil Ibrahimi, Astrit Bilalli, Simon Vitecek, Steffen U. Pauls, Felicitas Erzinger, Agim Gashi, Linda Grapci-Kotori, Donard Geci, Milaim Musliu and Edison Kasumaj Data type: Genomic Download file (26.36 kb)