Description of a new species of Wormaldia from Sardinia and a new Drusus species from the Western Balkans (Trichoptera, Philopotamidae, Limnephilidae)

Abstract New species are described in the genera Wormaldia (Trichoptera, Philopotamidae) and Drusus (Trichoptera, Limnephilidae, Drusinae). Additionally, the larva of the new species Drusus crenophylax sp. n. is described, and a key provided to larval Drusus species of the bosnicus-group, in which the new species belongs. Observations on the threats to regional freshwater biodiversity and caddisfly endemism are discussed. The new species Wormaldia sarda sp. n. is an endemic of the Tyrrhenian island of Sardinia and differs most conspicuously from its congeners in the shape of segment X, which is trilobate in lateral view. The new species Drusus crenophylax sp. n. is a micro-endemic of the Western Balkans, and increases the endemism rate of Balkan Drusinae to 79% of 39 species. Compared to other Western Balkan Drusus, males of the new species are morphologically most similar to Drusus discophorus Radovanovic and Drusus vernonensis Malicky, but differ in the shape of superior and intermediate appendages. The females of Drusus crenophylax sp. n. are most similar to those of Drusus vernonensis, but differ distinctly in the outline of segment X. Larvae of Drusus crenophylax sp. n. exhibit toothless mandibles, indicating a scraping grazing-feeding ecology.


Introduction
The Mediterranean area is a flora and fauna biodiversity hot-spot. The Tyrrhenian islands and the Balkans, in particular, are noteworthy for their high number of plant endemics Quezél 1997, 1999;Nikolić et al. 2008;Fenu et al. 2010;Bacchetta et al. 2012), and mammal and invertebrate endemics (Holdhaus 1924, Vigne 1992, Muccedda et al. 2002, Griffiths et al. 2004, Grill et al. 2007. Freshwater biodiversity has recently become a focus of attention throughout Europe, including the Mediterranean region with the Western Balkans and Sardinia (e.g., di Sabatino 2003, Zakšek et al. 2009, Tierno de Figueroa et al. 2013, Klobučar et al. 2013, Weiss et al. 2014. The genus Wormaldia currently comprises 204 species (Morse 2014) of which 36 species occur in Europe (Malicky 2005. Most species are widely distributed, but also several apparently highly endemic species have been described (Graf et al. 2008, Martínez-Menéndez andGonzález 2011). Aquatic stages of the genus, with few exceptions, prefer crenal and rhithral sections of alpine to lowland streams, are caseless and behave as passive filter feeders using characteristic nets ). Species in the genus exhibit characteristic male genitalia, but also comparatively high variability, particularly of the phallic structures (Malicky 2004, Martínez-Menéndez andGonzález 2011, Neu pers. comm.), resulting in the description of several subspecies.
The genus Drusus is in the subfamily Drusinae Banks, and comprises 84 species (Malicky 2004(Malicky , 2005Kučinić et al. 2011a;Oláh 2010Oláh , 2011Oláh and Kovács 2013). Larvae of the group prefer eucrenal to epirhithral sections of cold alpine or montane streams and brooks. Feeding ecology of Drusus larvae is complex, and three different feeding guilds can be distinguished based on the shape of larval mandibles and leg setation: filtering carnivores, omnivorous shredders, and scraping grazers (Pauls et al. 2008). Taxonomic richness of Drusinae is particularly high in the Western Balkans, including a high number of micro-endemics (Malicky 2004;Graf et al. 2008;Oláh 2010Oláh , 2011Kučinić et al. 2011a, b;Kovács 2013, Previšić et al. 2014a, b).
In this paper we describe a new species of Wormaldia and a new grazer Drusus species, including a key to the hitherto known larval stages of the bosnicus-group, in which Drusus crenophylax sp. n. belongs.

Materials and methods
Adults were collected using sweep nets and immature stages by handpicking. Collected specimens were stored in 70% and 96% EthOH, for morphological and molecular analyses, respectively.
Male and female genitalia were examined after being cleared in either KOH or lactic acid. Nomenclature of male genitalia of Wormaldia McLachlan follows Nielsen (1957, for Wormaldia occipitalis Pictet), 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) in which pencil drawings made with a camera lucida are digitized, edited and inked in Adobe Illustrator (v. 16.0.4,Adobe Systems Inc.).

Wormaldia sarda
Male genitalia (Fig. 1A-D). Segment IX in lateral view subrectangular, bulging anteriad; dorsal quarter reduced to a narrow transverse bridge, ventral 3/4ers broad (Fig.  1A). Segment X in lateral view trilobate: unpaired dorsal lobe strongly convex with a bicuspid apex, dorsoproximally membraneous; 1 lateromedian lobe, subovate, pointed on either side; 1 ventral lobe, posteriad, pointed on either side (Fig. 1A Female and larva unknown. Etymology. The species epithet refers to the island of Sardinia, the type locality.  Females of the new species show the reduced median lobe of the vaginal sclerite and high base of the lateral lobe of segment IX as typical for Balkan Drusinae, and are most similar to Drusus vernonensis, but exhibit (1) a sharp dorsal notch of segment X in lateral view, and (2) segment X with 2 round median lobes in dorsal view. Drusus vernonensis females have a rounded dorsal outline of segment X and lack the median lobes of segment X.
Male genitalia (Fig. 3A-E). Tergite VIII dark brown, in dorsal view cranially distinctly incised, with lighter areas around fused alveoli; setation concentrated at laterocranial borders of spinate areas; spinate area as two ± triangular laterocaudal lobes medially connected by a band of spines, embracing a medial, indent less sclerotized area (translucent in cleared specimens) with scarce spines. Ninth abdominal segment (IX) ventrally wider than dorsally in caudal view; in lateral view medially with a sharp caudad protrusion and a ventral protrusion, embracing the base of the inferior appendices. Superior appendages in lateral view subtriangular, somewhat Y-shaped with a shorter dorsal and a longer ventral protrusion separated by a slight indentation. Intermediate appendages in lateral view blocky with 2 tips, the proximal sharp, the distal high, rounded, rough; in dorsal view the tips parallel, extending laterally: a bar-shaped, laterally rounded distal tip and a sharp proximal tip, separated by a rounded excision with round edges; in caudal view approximately triangular, tips rounded. Inferior appendages (gonopods sensu Snodgrass 1935) in lateral view proximally wide, medially slightly constricted with a slight dorsal triangular protrusion, curved dorsadly in the slender posterior third; in dorsal, ventral and caudal view proximal part laterad, distal part approximately straight in dorsoventral plane, curved dorsad; in caudal view tips distinctly slender; setal alveoli fused, creating a rugged, less sclerotized ventral area. Parameres simple, with a distinct medial thorn-like spine and 2 proximal spines in the proximal half.
Female genitalia (Fig. 3F-I). Segment IX setation abundant, concentrated in the caudal half; lateral lobe of segment IX membraneous, in lateral view oblique triangular, the ventral edge about twice as long as the dorsal edge, with a dorsal sclerotized setose part protruding caudally; in dorsal and ventral view slender, projecting lateradly; in caudal view dorsal sclerotized setose part somewhat triangular. Segment X in lateral view with a proximal and a distal part, defined by a sharp dorsal notch; in dorsal view trapezoidal, with rounded shoulders, 2 small dorsal median lobes, and distally with 2 triangular, sharp-tipped lateral lobes, each with a lateral rounded setose and a small median rounded protrusion; ventrally unsclerotized, open. Supragenital plate in lateral view sinuously-edged quadrangular with a small, rounded dorsal protrusion, caudal line slightly indent; in ventral view quadrangular, in caudal view quadrangular, dorsally slightly wider than ventrally. Vulvar scale in lateral view triangular, rather straight, longer than the supragenital plate; in ventral view slender with 3 lobes: 2 lateral lobes, digitiform, roundly oval, straight; 1 median, short (reduced), of greater width than length: length approximately 1/6th of that of lateral lobes.
Molecular species delimitation and larval affiliation. Analysis of the genetic distance of mtCOI between Drusus crenophylax sp. n. and the in the adult stage morpho- logically most similar species, D. discophorus and D. vernonensis, clearly supports the recognition of the new species. Uncorrected p-distances recorded in a fragment of the mtCOI gene (ranging from 2-8%; Fig. 5), agree with the interspecific distances commonly recorded in Limnephilidae (e.g., Graf et al. 2005;Kučinić et al. 2011a;Previšić et al. 2014a, b) and other caddisfly families (e.g., Hydropsychidae; Pauls et al. 2010). Also, all haplotypes of Drusus crenophylax sp. n. adults were completely identical to another and those of undescribed Drusus-larvae collected at the locus typicus, enabling confident affiliation of larvae and adults of D. crenophylax sp. n.
Ecology and distribution. Drusinae species typically are members of crenal species communities, and mainly inhabit crenal sections of cold streams. Larval D. crenophylax were collected at eucrenal sections of the Cvrcka River (Fig. 6A, B) and behave as epilithic grazers, as indicated by mandible morphology (Pauls et al. 2008). Based on regional collection data, we assume that the species is a microendemic restricted to the watershed of the Cvrcka river.
Etymology. The species epithet is a compound name, combining κρηνον ('well, spring, fountain' in Ancient Greek) and φυλαξ ('guard, keeper, protector' in Ancient Greek), terms that reflect the high degree of niche specificity of Drusus species, the majority of which inhabit crenal sections of streams .
Larvae of the bosnicus-group also develop, with the exception of D. ramae (Kučinić et al. 2010), a field of microspinules close to each eye (Kučinić et al. 2011a, b;Waringer et al. 2015). Further, carinae of D. bosnicus, D. radovanovici, D. septentrionis and D. medianus are high and curved mediad. Larvae of D. crenophylax sp. n. share those characters and can be integrated in the following dichotomous key (Waringer et al. 2015

Discussion
Systematic significance of Wormaldia sarda sp. n.
The Tyrrhenian islands and Sardinia in particular have been renowned for their relictual fauna and flora for a long time (Holdhaus 1924) and represent one of the Mediterranean biodiversity hotspots (Grill et al. 2007). Wormaldia sarda sp. n. represents an addition to the distinct Sardinian biodiversity. As no species similar to W. sarda sp. n. are recorded from neither northern Africa nor mainland Europe, it is likely that this species is restricted to Sardinia, as are several other species such as Crunoecia irrorata sarda Curtis, Stactobia ericae Malicky or Hydropsyche sattleri Tobias ). However, the geological history and geographic proximity of the Tyrrhenian islands -Sardinia and Corsica in particular (Vigliotti et al. 1990)suggest that some species may occur on both islands. For instance, Leptodrusus budtzi Ulmer or Micrasema togatum Hagen occur also on Corsica, or other Mediterranean islands ). The distinct apomorphic characters, particularly the modified segment X and the very different pupal characters (mandibles, dorsal abdominal sclerites; Fig. 2D-F), might warrant establishing a new genus for this species. The pupal characteristics alone are strikingly different from those of either Wormaldia or Philopotamus (Lepneva 1964 ; Fig. 2). However, since pupae of only three species of Wormaldia are described (Nielsen 1942, Lepneva 1964) the range of genus-level pupal characters remains unknown. Further, modifications of segment X are common in southeast Asian species of Wormaldia (Malicky 2010). Tooth-like structures on segment X similar to the ones observed in W. sarda sp. n. are present in Wormaldia species from Thailand (e.g., W. acheloos Malicky & Chantaramongkol, W. congina Malicky & Chantaramongkol, W. lot Malicky & Chantaramongkol), or Sulawesi (W. otaros Neboiss). Nevertheless, Wormaldia species with a phallus shaped as in W. sarda sp. n. have not yet been described. Since the whole genus is in need of revision (Malicky 2005, Malicky unpubl. data), we refrain, in the interest of taxonomic stability, from creating a new genus.

Aquatic diversity of the Western Balkans under threat
Endemic freshwater species are particularly vulnerable to global change and (anthropogenic) habitat degradation (Hering et al. 2009, Tierno de Figueroa et al. 2010, Bálint et al. 2011, Conti et al. 2014). The Balkans is rich in apparently endemic freshwater species (Griffiths et al. 2004). Recent taxonomic efforts in the Western Balkans increased the number of endemic Drusinae taxa to 31 of 39 described Drusinae species (Previšić et al. 2014b, Vitecek et al. unpubl. data). Further, several endemic species of Chaetopteryx were recently described from the Western Balkans (Oláh et al. 2012, Kučinić et al. 2013 indicating the need for further systematic investigations on an underestimated diversity of southeastern Europe. The construction of hydropower dams in emerging economies is currently one of the greatest threats to freshwater biodiversity (Zarfl et al. 2014). Small hydropower plants fed by small cold-water mountain rivers such as the Cvrcka River are currently under construction throughout the Western Balkans (Freyhof 2012, Schwarz 2012, and gravely threaten the habitats that harbour endemic highland caddisflies such as Drusinae (Previšić et al. 2014a, Vitecek et al. unpubl. data, this study), or Chaetopteryx species (Kučinić et al. 2013). The description of Drusus crenophylax sp. n. highlights the importance of biodiversity research in southern Europe, and demonstrates that the currently prevailing energy policy will likely result in the loss of known and unknown biodiversity.