Description of two new filtering carnivore Drusus species (Limnephilidae, Drusinae) from the Western Balkans

Abstract Two new species of the genus Drusus (Trichoptera, Limnephilidae, Drusinae) from the Western Balkans are described. Additionally, observations on the biodiversity and threats to the region’s endemic aquatic fauna are discussed. Drusus krpachi sp. n. is a micro-endemic of the Korab Mountains, Macedonia, and Drusus malickyi sp. n. is a micro-endemic of the Prokletije Mountains, Albania. Both new species are most similar to Drusus macedonicus but differ from the latter in the shape of segment IX, the shape of the tips of the intermediate appendages in lateral view, the shape of the inferior appendages, and the form and shape of the parameres. In addition, males of the European species of filtering carnivore Drusinae are diagnosed and illustrated, including Cryptothrix nebulicola McLachlan, Drusus chrysotus Rambur, Drusus discolor Rambur, Drusus macedonicus Schmid, Drusus meridionalis Kumanski, Drusus muelleri McLachlan, Drusus romanicus Murgoci and Botosaneanu, and Drusus siveci Malicky. These additions to the Western Balkan fauna demonstrate the significance of this region for European biodiversity and further highlight the importance of faunistic studies in Europe.

The subfamily Drusinae Banks comprises roughly 110 species in 8 genera (Malicky 2004(Malicky , 2005Oláh 2010Oláh , 2011Oláh and Kovács 2013;Previšić et al. 2014a;Vitecek et al. 2015;Ibrahimi et al. pers. comm.). Ecologically, most species are crenobiont , and as larvae fall into one of three different feeding groups: filtering carnivores, omnivorous shredders and scraping grazers (Pauls et al. 2008). The adults of each larval feeding group are also characterized by a set of synapomorphies (Vitecek et al. in press). Filtering carnivorous Drusinae males uniquely exhibit laterally positioned gland openings at the fifth abdominal sternite and parallel wing veins in the hind wing anal field (depicted in Vitecek et al. in press). The largest genus Drusus is paraphyletic (Pauls et al. 2008) and comprises 86 species of all feeding types (Malicky 2004(Malicky , 2005Graf et al. 2008;Kučinić et al. 2011a;Oláh 2010Oláh , 2011Oláh and Kovács 2013;Vitecek et al. 2015;Vitecek et al. in press;Ibrahimi et al. pers. comm.). The monotypic genus Cryptothrix (C. nebulicola McLachlan) is also a filtering carnivore (Bohle 1987, and thus represents another filtering carnivorous Drusinae sensu Pauls et al. 2008, the systematic position of which is discussed in Vitecek et al. (in press).
Here we describe two new filtering carnivore Drusus species. Additionally, we provide re-descriptions of filtering carnivorous Drusinae sensu Pauls et al. (2008) in order to facilitate identification of known filtering carnivorous Drusinae, and identification of new species.

Materials and methods
Adults were collected using sweep nets and by handpicking. Collected specimens were stored in 96% EthOH. Male and female genitalia were examined after being cleared in either KOH or lactic acid. Nomenclature of male genitalia of Drusus follows Nielsen (1957, for Limnephilus flavicornis Fabricius) using the simplifying terms "superior appendages" for the lateral processes of segment X (cerci sensu Snodgrass 1935), and "intermediate appendages" for the sclerite and the anterior process of segment X (paraproct sensu Snodgrass 1935). Nomenclature of larval morphological features follows Wiggins (1998) and Waringer and Graf (2011), nomenclature of primary setae and setal areas follows Wiggins (1998). Illustrations were prepared according to Thomson and Holzenthal (2010)  Type specimens will be deposited in museum collections upon completion of the taxonomic work. (2) a dorsally straight tip of the intermediate appendage distinctly separated by a proximal indentation and with small proximal and distal rough protrusions; (3) a conical inferior appendage with a proximal dorsal triangular protrusion; (4) parameres with three tines in the distal third in dorsal view. Drusus macedonicus males have a distally concave ventral half of segment IX, intermediate appendages with two rough rounded dorsad protrusions but lacking a distinct proximal indentation, distally tapering inferior appendages, and parameres with a single tine in the distal third in dorsal view.
Male genitalia (Fig. 1). Tergite VIII (tVIII) fawn, setae absent; spinose area in lateral view approximately flat with slight dorsocaudal protrusion in cranial half, in dorsal view suboval; flanked by membranous, less sclerotized areas. Segment IX (IX) in lateral view ventrally straight distally; in caudal view dorsally approximately as wide as ventrally; with rounded lateral protrusion in the dorsal half (best seen in ventral view). Superior appendages (sup) in lateral view suboval, curved obtusely caudad in proximal third, proximally with slight dorsal protrusion, longest in anterioposterior axis: approximately 2.5 times longer than high; in dorsal view proximally slightly concave medially; medial transverse section oval. Intermediate appendages (int) in lateral view with subtriangular tip, rough areas concentrated on dorsal proximal and dorsal distal aspect; in dorsal view tips separated, oval, distally converging; in caudal view approximately triangular. Inferior appendages (inf; gonopods sensu Snodgrass 1935) in lateral view conical, proximally wide, distally slender, with proximal triangular protrusion dorsally; in ventral and dorsal view with small medial projection and slight notch. Parameres simple, in dorsal view with 3 tines in distal third: 2 mediolateral, 1 dorsal.
Female and pupa unknown. Larval description and indentification key provided by Vitecek et al. (in press).
Etymology. Named after V. Krpač, Macedonian entomologist and collector of the species.

Drusus malickyi
Male genitalia (Fig. 2). Tergite VIII fawn, setae scarce; spinate area in lateral view approximately flat with slight dorsad protrusion in anterior half, in dorsal view suboval; flanked by membraneous, less sclerotized areas. Segment IX in lateral view with sharp medial caudal protrusion, ventrally concave distally; in caudal view wider dorsally than ventrally; with irregular triangular, rounded lateral protrusion in dorsal half (best seen in dorsal and ventral views). Superior appendages in lateral view suboval, curved obtusely caudad in proximal quarter, proximally with slight dorsal and distinct ventral protrusions, longest in anterioposterior axis: approximately 2.5 times longer than high; in dorsal view proximally concave medially; medial transverse section oval. Intermediate appendages in lateral view with subtriangular, dorsally rough tip; in dorsal view tips separated, wedge-shaped, approximately parallel; in caudal view approximately triangular. Inferior appendages in lateral view subtriangular, proximally somewhat bulbous, distally slender and distinctly constricted; in ventral and dorsal views with small medial protrusion and slight notch; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple, in lateral view with 1 tine in distal third.
Male genitalia (Fig. 3). Tergite VIII brown, with lighter areas around alveoli; setation abundant; spinose area approximately rectangular in dorsal view; flanked by membraneous, less sclerotized areas. Segment IX in lateral view ventrally slightly concave distally; in caudal view wider dorsally than ventrally; with long, round, wedgeshaped protrusion in dorsal half (best seen in ventral view). Superior appendages in lateral view suboval, curved obtusely caudad in proximal fifth, proximally dorsal somewhat protuberant, tips slightly curved dorsad, longest in anterioposterior axis: approximately 2 times longer than high; in dorsal view medially concave, tips converging; medial transverse section lateroventrally curviconvex suboval. Intermediate appendages in lateral, dorsal and caudal views dorsally with 2 distinct tips, the proximal tip rounded, rough, the distal tip pointed, smooth; in caudal view approximately an isoceles trapezium. Inferior appendages in lateral view roughly triangular, proximally constricted, ventrocaudally slightly concave; in dorsal and ventral views tips converging; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple, rodlike, medially and distally somewhat bulbous. Female depicted by Schmid (1956), Malicky (2004); larva in key presented by Waringer and Graf (2011), Vitecek et al. (in press); pupa unknown.

Drusus chrysotus
Male genitalia (Fig. 4). Tergite VIII light brown, with short, pale, translucent setae; spinose area in lateral view with distinct dorsal protrusion and dorsomedial caudal protrusion, in dorsal and caudal views tripartite; flanked by membraneous, less sclerotized areas. Segment IX in lateral view ventrally irregular concave distally; dorsally approximately as wide as ventrally in caudal view; with distinct approximately subtriangular, rounded protrusion in dorsal half (best seen in dorsal and ventral views). Superior appendages in lateral view curved obtusely ventrocaudad in proximal third, proximally with distinct dorsocranial protrusion, approximately as long as high, in dorsal view proximally concave medially; medial transverse section oval. Intermediate appendages in lateral view medially protruding caudad, dorsally with long, rough tip; in dorsal view fused into approximately heart-shaped, centrally indented structure; in caudal view ventrally broad with bulbous lateral protrusions, dorsally narrow, fused. Inferior appendages in lateral view conical, short; in ventral and dorsal views blunt, with blunt, short medial protrusion and slight notch; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple with several tines on common base in distal third.

Drusus discolor
Male genitalia (Fig. 5). Tergite VIII light brown, setation scarce, in lateral view with distinct cranial dorsal protuberance; spinose area in lateral view with distinct dorsal protrusion and dorsomedial caudal protrusion, in dorsal view suboval, caudally straight; flanked by membraneous, less sclerotized areas. Segment IX in lateral view ventrally distinctly concave distally; in caudal view dorsally approximately as wide as ventrally; with a distinct, caudally straight rounded protrusion indorsal half (best seen in ventral view). Superior appendages in lateral view suboval, curved obtusely caudad in proximal half, proximal half with distinct dorsal protrusion, approximately as long as high; in dorsal view medially concave; medial transverse section suboval. Intermediate appendages in lateral view medially approximately straight, dorsally with rounded, rough tip; in dorsal view tips separate, oval, distally converging; in caudal view approximately triangular with dorsally diverging tips. Inferior appendages in lateral view conical; in ventral and dorsal views with distinct medial protrusion and distinct notch; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple with single bulbously based tine in distal third.

Drusus macedonicus Schmid, 1956
Male genitalia (Fig. 6). Tergite VIII fawn, setation lateral, scarce; spinose area in lateral view approximately flat, in dorsal view suboval, tapering cranially; flanked by membraneous, less sclerotized areas. Segment IX in lateral view ventrally deeply concave distally, with distinct medial and ventral caudad protrusion; in caudal view slightly wider dorsally than ventrally; with sharp, caudally approximately straight protrusion in dorsal half (best seen in dorsal and ventral views). Superior appendages in lateral view irregularly suboval, curved obtusely caudad in proximal quarter, proximally with an irregular dorsal and irregular ventral protuberance, longest in anterioposterior axis: approximately 2.5 times longer than high; in dorsal view proximally slightly concave medially; medial transverse section suboval. Intermediate appendages in lateral view with two rough tips: 1 curved dorso-posteriorly, 1 central, rounded; in dorsal view posterior tips adjacent, parallel; in caudal view subtriangular with slender lateral projections. Inferior appendages in lateral view approximately conical, proximally wide, distally slender; in ventral and dorsal views with medial tip and notch, separated by slight notch; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple, with single dorsal tine in distal third.
Male genitalia (Fig. 7). Tergite VIII yellow to brown, setae absent; spinose area in lateral view approximately flat, in dorsal view suboval, somewhat rectangular cranially; flanked by membraneous, less sclerotized area bearing single seta. Segment IX in lateral view ventrally slightly concave distally; in caudal view wider ventrally than dorsally; with distinct, approximately triangular, rounded protrusion in dorsal half (best seen in dorsal view). Superior appendages in lateral view suboval, curved obtusely caudad in proximal third, proximally with distinct dorsal protrusion, longest in anterioposterior axis: approximately 3 times longer than high; in dorsal view proximally slightly concave medially; medial transverse section oval. Intermediate appendages in lateral view with rounded, rough tip; in dorsal view 2 separate parallel tips, each oval, rough; in caudal view subtriangular, dorsally with 2 separate tips. Inferior appendages in lateral view conical; in ventral and dorsal views slender with minute subtriangular medial protrusion and shallow notch; in ventral view with longitudinal groove delimiting medial lobe. Parameres simple, with single, bulbously based tine in distal third.

Drusus muelleri McLachlan, 1868
Male genitalia (Fig. 8). Tergite VIII brown, setae absent; spinose area in lateral view convex with caudal ventral lobe, in dorsal view suboval with small medial protrusion; flanked by membraneous, less sclerotized areas. Segment IX in lateral view ventrally slightly concave distally; in caudal view wider dorsally than ventrally; with sharp caudally straight subtriangular protrusion in the dorsal half (best seen in dorsal and ventral views). Superior appendages in lateral view irregular, curved obtusely caudad in proximal quarter, proximally distinctly dilated, distally clavate, longest in anterioposterior axis: approximately 5 times longer than high; in dorsal view the proximal third concave medially; medial transverse section circular. Intermediate appendages in lateral view with long, rounded rough tip; in dorsal view tips separate, approximately parallel, proximally bulbous; in caudal view subtriangular. Inferior appendages in lateral view subtriangular, ventrally irregular, proximally sightly concave dorsally; in ventral and dorsal views broad, with small subtriangular medial protrusion and distinct notch; in ventral view with longitudinal groove delimiting medial lobe; in caudal view broad. Parameres simple, with single dorsal tine in distal third.

Drusus romanicus Murgoci and Botosaneanu, 1953
Male genitalia (Fig. 9). Tergite VIII brown, setae present; spinose area in lateral view approximately flat with slight dorsal protrusion, in dorsal view suboval, distally straight; flanked by membraneous, less sclerotized areas. Segment IX in lateral view dorsally with distinct notch distally, ventrally irregularly concave distally; in caudal view ventrally wider than dorsally; with distinct subtriangular rounded protrusion in dorsal half (best seen in dorsal view). Superior appendages in lateral view elongate suboval, curved obtusely dorsocaudad in proximal quarter, proximally with round dorsal protrusion and irregular ventral protrusion, longest in anterioposterior axis: approximately 4.5 times longer than high; in dorsal view proximally distinctly concave medially; medial transverse section circular. Intermediate appendages in lateral view with rounded, rough tip; in dorsal view tips separate, laterally diverging; in caudal view subtriangular. Inferior appendages in lateral view conical, long, dorsally irregular, proximally slightly concave dorsally; in ventral and dorsal views proximal half robust, distal half slender with slight medial protrusion and shallow notch. Parameres simple, with medial hook-shaped tip bearing several smaller tines.
Male genitalia (Fig. 10). Tergite VIII fawn, setation concentrated dorsally and posterolaterally, with slight dorsal protrusion; spinose area in lateral view approximately flat, in dorsal view oval; flanked by membraneous, less sclerotized areas. Segment IX in lateral view with medial caudad protrusion, ventrally irregularly concave distally; in caudal view wider dorsally than ventrally; with distinct rounded triangular protrusion in dorsal half (best seen in dorsal and ventral views). Superior appendages in lateral view suboval, curved obtusely caudad in proximal half, proximal half with distinct rounded protrusion, in dorsal view slightly concave medially; medial transverse section subcircular. Intermediate appendages in lateral view with pointed, hook-like tip arching dorsad; in dorsal and caudal views the tips fused; in caudal view subtriangular. Inferior appendages in lateral view conical, short, blunt, posteroventrally somewhat concave; in ventral view with medial protrusion and distinct notch. Parameres simple, with single bulbously based tine in distal third.

Drusinae micro-endemics of the Western Balkans
Morphology of the new species as well as molecular phylogenetic analyses (Vitecek et al. in press)  appendages, and are discretely distributed. Also, they are highly supported in phylogenetic analysis and form a highly supported clade comprising (D. malickyi + (D. krpachi + D. macedonicus) in the phylogenetic analysis of Vitecek et al. (in press). To our knowledge, the new species are small-scale endemics restricted to single mountain ranges. Interestingly, the type localities of the new species are close to the known range of D. macedonicus (Fig. 11). Such small-scale distribution of distinct Drusinae species is well documented from the Western Balkans (Marinković-Gospodnetić 1976; Kučinić et al. 2011a;Oláh 2010Oláh , 2011Oláh and Kovács 2013;Previšić et al. 2014a, b;Vitecek et al. 2015). Similarly, other taxa exhibit comparable distribution patterns, in which single mountain ranges represent the range of a species, or haplogroups (Ursenbacher et al. 2008, Stevanović et al. 2009, Zogaris et al. 2009, Karaman et al. 2011). The intriguing distribution patterns exhibited by some groups of species potentially result from the geological history of the region and historic and present-day climate conditions. Small-scale speciation of Drusinae presumably is facilitated by intrinsic traits of the subfamily, such as their occurrence at higher elevations (Pauls et al. 2006), a putatively low long-distance dispersal potential (Müller-Peddinghaus 2011, Geismar et al. 2015), and might be further enhanced by habitat fragmentation, e.g., by regional karstification. Occurrence of Drusinae could therefore serve as proxy to occurrence of other aquatic invertebrate taxa, particularly to crenobiont taxa exhibiting the same or similar traits. Figure 11. Distribution of filtering carnivore Drusinae. Single records of endemic species are depicted as symbols, stroked or filled areas denote ranges of more widely distributed species with a higher number of occurrence records.

Western Balkan aquatic diversity
The Western Balkans represent a hot-spot of species richness and endemicity in Europe , Guéorguiev 2007, Kenyeres et al. 2009, Jaskuła 2011. In particular, the faunas of isolated habitats such as coldwater springs and streams, caves or the profundal zone of large lakes contribute to high species richness in the region (Petkovski et al. 2009, Wilke et al. 2010, Pešić and Glöer 2013. Such taxa probably are more susceptible to factors promoting speciation, such as climatic and geological processes (e.g., karstification, see Previšić et al. 2009Previšić et al. , 2014b, especially if their dispersal potential is low. The description of the two new micro-endemic Drusus species increases the number of Western Balkan Drusinae species. Drusinae richness in the Western Balkans currently comprises 40 species including 13 species (30 %) that were discovered since 2010, of which 32 are endemic to the Western Balkans Oláh 2010Oláh , 2011Schmidt-Kloiber and Hering 2012;Oláh and Kovács 2013;Previšić et al. 2014b;Vitecek et al. 2015;Ibrahimi et al. pers. comm.;this study).
Thus, endemism rates of Western Balkan Drusinae are high, and are further augmented by the description of the two new micro-endemic Drusus species. Global and anthropogenic habitat changes are among the greatest threats to micro-endemic and endemic freshwater species (Hering et al. 2009, Tierno de Figueroa et al. 2010, Bálint et al. 2011, Conti et al. 2014. Water extraction for human consumption intensified by tourism, agriculture, and hydroelectricity are the primary modes of global anthropogenic habitat modification of freshwaters (Foster 1991, Polhemus 1993, Dudgeon 2006. Hydropower plants were identified as the greatest threat to European freshwater biodiversity (Freyhof 2012, Schwarz 2012, Zarfl et al. 2014, http://riverwatch.eu).
Recent published taxonomic works treating the Western Balkans, including the present one describing two new micro-endemic Drusus species, have demonstrated the significance of the region for European biodiversity. However, progressing socioeconomic change and anthropogenic habitat modification threaten the freshwater biodiversity of the Western Balkans, and potentially will result in the loss of yet-to-be discovered species. thanked for their help with distribution data and permission to use their data on sampling localities. Ian Stocks and Ralph Holzenthal are thanked for their rigorous reviews on an earlier version of the manuscript, and very quick handling of the manuscript.