The material studied comprises 7 larvae of Drusus plicatus collected on 23 August 2009 from the spring Vevčani (Fig. 1B), 12 larvae collected on 25 August 2009 (4th and 5th instar larvae), 24 larvae collected on 2 July 2010 (4th and 5th instar larvae), 4 larvae collected on 29 May 2013 from the spring of the River Galička reka (Fig. 1C), Mavrovo National Park, and 5 larvae of the same species collected on 2 July 2010 from the spring of the River Strežimirska reka, Mavrovo National Park (Table 1). Larvae were collected by handpicking and adults with an entomological net during the day. Collected specimens were stored in containers with 80% and 96% EtOH for morphological and molecular analysis, respectively.

Additionally, adult caddisfly communities in three springs in Macedonia (Vevčani spring, spring of the River Strežimirska reka, and the spring of the River Galička reka) were sampled using light traps. Identification of the adults was conducted using the works of Malicky (2004) and Kumanki (1988). The larval morphological terminology follows Wiggins (1996) and the systematics follow Morse (2015). Most of the collected specimens of larvae and adults are deposited in the collections of the first (Croatian Natural History Museum in Zagreb) and second authors (Faculty of Science, University of Zagreb). Some adults are deposited in the Macedonian Museum of Natural History in Skopje (collection Trichoptera Kučinić, Mihoci & Krpač).

We have included literature data for caddisfly species collected in Vevčani spring (Rhyacophila trescavicensis Botosaneanu, Wormaldia occipitalis Pictet, Tinodes rostocki McLachlan, Ecclisopteryx keroveci Previšić, Graf & Vitecek, Potamophylax luctuosus Piller & Mitterpacher) (Oláh and Kovács 2014) which were not found during our investigation of this spring.

DNA was extracted from two adult males and two larvae of D. plicatus from the spring of the River Galička reka and one adult male and two larvae from Vevčani spring to confirm the association of the larvae with the adults. DNA extraction, amplification of the 541–bp–long fragment of the mitochondrial cytochrome oxidase I (mtCOI) using primers S20 and Jerry (Simon et al. 1994, Pauls 2004) were accomplished as outlined by Previšić et al. (2009). Sequences were edited manually using the program BioEdit v7.0.9 (Hall 1999) and aligned using ClustalX (Thompson et al. 1997). Sequences were deposited in GenBank under accession numbers listed in Table 2. Intraspecific p-distances were calculated using the software Mega 4.0.1 (Tamura et al. 2007).

Electron microscopy of larvae of D. plicatus (specimens from Vevčani spring) was carried out using a Tescan TS 5136 variable pressure scanning electron microscope (SEM). Samples were mounted with graphitic adhesive tape on the SEM stub and coated with carbon. The samples were examined by SEM operating in secondary electron (SE), or back-scattered (BSE) mode, at an accelerating voltage of 20 kV, running current of 110 pA, and variable pressure of 30 Pa to 5”10-1 Pa; sometimes the pressure was increased to 10 Pa to eliminate sample charging. Macrophotography and assessment of morphometric characteristics of pupae, larvae and larval cases were carried out using a Leica Wild MZ8 stereomicroscope and Olympus SP-500 UZ digital camera; photographs were processed with the software Olympus Quick Photo Camera 2.2. In the larvae of D. plicatus the following features were measured (in mm): head width, total body length, length of the anterior sclerites, their width at the widest median part and the distance between them, and also the length of the posterior sclerites. The following characters of cases were measured: total length, width of the anterior part, and width of the posterior part.

Larval case constructed of mineral particles (Figs 3, 4), slightly curving, total length 9.97–19.19 mm, width of anterior part 2.30–2.70 mm, width of posterior part 1.64–2.01 mm. Overall body shape eruciform (Fig. 5).

Head capsule hypognathous (width 1.40–1.46 mm, n = 5) (Figs 5, 6, 7), in lateral view rounded in posterior dorsal part. Head (dark) brown to black, dorsally darker and laterally lighter (Fig. 6), with granular surface sculpturing and dark muscle attachment spots posteriorly. Genae reddish-brown to yellow with lighter (yellow) ring around each eye (Fig. 6). Frontoclypeal suture bell-shaped with narrow central region (Fig. 8). Antennae short, brown to dark brown (black), each positioned on small prominences (Fig. 6). Other primary setae positioned as shown in Fig. 8. Spinules (Figs 9, 10) present in small numbers, positioned around and between primary setae 15 and 16 (Fig. 8). Labrum symmetrical, brown to yellowish, with setal brush at anterolateral margins. Anterior part of labrum usually lighter. Mandibles black (Fig. 11), mesal part reddish. Typical for grazers, mesal margin with yellowish setal brush. Two setae present laterobasally on each mandible (Fig. 11). Labium and maxillae light-brown (yellowish). Each maxillary palp 5-segmented.

Thorax. Pronotum brown to black with granular surface sculpturing (Figs 6, 7, 12). Posterior margin rounded, both posterior and lateral margins thick and darkly sclerotized. In lateral view, anterior half of pronotum slightly concave, almost flat, posterior half slightly rounded (Figs 6, 7, 12). Pronotum bearing dark setae, especially laterally and on anterior margin, some of them long and conspicuous. Dorsal and lateral regions of pronotum bearing short, white, recumbent setae (Fig. 9).

Mesonotum sclerites brown, lighter than pronotum, with dark muscle attachment spots, dark setae and irregular surface (Fig. 13). Posterior and lateral margins thick and darkly sclerotized (Fig. 13).

Metanotum with 3 pairs of dorsal sclerites: anterior sclerites, posterior sclerites and lateral sclerites (Figs 5, 13). Anterior sclerites (sa1) elongated, triangular with rounded apices (Fig. 13), covered by setae, mainly in anterior part, color similar to mesonotum. Length of anterior sclerites 0.45–0.52 mm; width of anterior sclerites 0.25–0.31 mm; distance between anterior sclerites 0.07–0.11 mm. Posterior sclerites (sa2) smaller and lighter than sa1 sclerites (Fig. 13), triangularly or irregularly ellipsoid and with many setae. Length of posterior sclerites 0.26–0.31 mm. Lateral sclerites (sa3) (Fig. 5) longitudinally prolonged, sickle-shaped, lighter brown with dark median region, and group of setae anteriorly.

Legs (Figs 14, 15, 16) yellow-brown to brown or black, with dark ventral and dorsal margins. Foreleg coxae with dark setae on ventral and dorsal edges. Foreleg trochanters without dorsal setae, each with few light yellow setae on ventral margin, trochanteral brush present (Fig. 14). Mid- and hind leg coxae and femora (Figs 15, 16) with dark setae on both ventral and dorsal edges. Additional setae present on anterior and posterior faces of all femora. Setae on dorsal edges of tibiae present only distally on all legs. Foreleg coxae and femora wide compared to those of mid- and hind legs (Figs 14, 15, 16). Mid- and hind legs similar in shape and size (Figs 15, 16), with slender coxae, trochanters, femora and tibiae.

Abdomen. Abdominal segment I with well-developed dorsal and lateral humps (protuberances) with numerous ventral setae, some of them with small sclerites at bases. Lateral protuberances with few setae. Some of them (1-2) with small sclerites at bases. Single-filament gills (Fig. 5) present on segments II–VII. Lateral gills present on segments II-V (on segment V only pre-segmental gills are present). Lateral fringe extending from second half of segment III to first half of segment VIII (Fig. 18).

Segment IX bearing irregular, semicircular, light brown dorsal sclerite, with few long dark setae on posterior margin (Fig. 17). The anal prolegs typical of limnephilids (Fig. 18). Each with lateral sclerite longitudinally prolonged, sickle-shaped, yellowish, with small setae and 2 large, dark setae posteriorly (Fig. 18). Anal claws brown to dark brown.

Mandible morphology of the larvae and observations during fieldwork suggest Drusus plicatus is a member of the Drusinae grazer clade (Previšić et al. 2014b). Species of this clade feed on epilithic algae and biofilms and can be found on stream bottoms, generally on cobbles, small pebbles and moss.

Based on the number of adults observed during the day, the most abundant population of D. plicatus was present in the spring of the River Galička reka (Fig. 1C). In this spring we observed two emergence peaks in spring and in late summer/autumn periods.

We collected D. plicatus larvae, adults or both in eight localities in the Republic of Macedonia (Table 1). Altitudes of locations where D. plicatus were collected range between approx. 950 m and 1410 m a.s.l. (Table 1).

We collected adult caddisflies at the three springs inhabited by Drusus plicatus. In the Vevčani spring the following species were recorded: Rhyacophila balcanica Radovanović, R. trescavicensis (literature data), Wormaldia occipitalis (literature data), Tinodes rostocki (literature data), Tinodes sp. (female), Ecclisopteryx keroveci (literature data), D. tenellus Klapálek, D. plicatus, Potamophylax latipennis Curtis, P. luctuosus (literature data), in the spring of River Strežimirska reka: R. balcanica, R. laevis Pictet, Synagapetus iridipennis McLachlan, Tinodes sp. (female), Hydropsyche sp. (females), Philopotamus montanus Donovan, Annitella cf. triloba Marinković-Gospodnetić, D. plicatus, Potamophylax pallidus Klapálek, Allogamus sp. (male), Thremma anomalum Mclachlan and in the spring of the River Galička reka the following species: R. balcanica, D. plicatus, Philopotamus montanus, Thremma anomalum, D. plicatus and Potamophylax lemezes Oláh & Graf.

Association of larvae and adults of D. plicatus is supported by the similarity of partial COI haplotypes. Since the association of larvae and adults is not completely reliable based solely on comparisons of sequences of a single gene from one specimen each (e.g., Zhou et al. 2007), we analysed specimens from two different populations. At each locality some adult males of D. plicatus and unassigned larvae shared identical COI haplotypes (Table 2). Observed variability in COI haplotypes within populations (Table 2) is in line with the variability of the same COI fragment in populations of some other Drusus species (e.g., Pauls et al. 2009, Previšić et al. 2009). Variability between populations in D. plicatus (Table 2), however, seems to be lower than observed in some other Dinaric Drusus endemics (e.g., D. croaticus, Previšić et al. 2009, D. krusniki Malicky, Previšić et al. 2014b).

Moreover, additional data, such as larvae and adults of D. plicatus recorded in 3 springs in Republic of Macedonia (Vevčani spring, spring of the River Galička reka, and the spring of the River Strežimirska reka), confirm our association of larvae and adults of D. plicatus. In these springs D. plicatus is sympatric with the following Drusinae species: Drusus tenellus, D. botosaneanui Kumanski and D. biguttatus, and larvae of these species exhibit different morphological characteristics from those observed in larvae of D. plicatus (Waringer and Graf 1997, Waringer et al. 2015).

Morphological features of the known larvae from the subfamily Drusinae are usually species specific and stable, enabling separation and identification of the species (e.g., Hickin 1967, Waringer and Graf 1997, Waringer et al. 2010, 2015). This is not the case for some other groups of Trichoptera in which larvae of many species are still not described or for which the separation of known larvae of some genera (e.g., Hydroptila Dalman, Chaetopteryx Stephens, Rhyacophila Pictet) is either very difficult or generally not possible (Waringer and Graf 1997).

Larvae from the subfamily Drusinae can be separated from other European Trichoptera larvae by the following morphological features (e.g., Waringer and Graf 1997, Graf et al. 2005, Kučinić et al. 2015): 1. A fully sclerotized pronotum and mesonotum; 2. Metanotum with six sclerites; 3. Gills with one filament; 4. Additional setae present on anterior and posterior faces of mid- and hind leg femora.

From the total of 49 Drusinae species recorded in southeast Europe, larval descriptions and taxonomic tools exist for the following 25 species: Drusus balcanicus Kumanski, D. biguttatus, D. botosaneanui, D. bosnicus Klapálek, D. chrysotus Rambur, D. crenophylax Graf & Vitecek, D. croaticus, D. discolor, D. klapaleki Marinković-Gospodnetić, D. krpachi Kučinić, Graf & Vitecek, D. krusniki, D. macedonicusSchmid, D. medianus Marinković-Gospodnetić, D. meridionalis Kumanski, D. radovanovici Marinković-Gospodnetić, D. ramae Marinković-Gospodnetić, D. septentrionis Marinković-Gospodnetić, D. serbicus Marinković-Gospodnetić, D. siveci Malicky, D. tenellus, D. vernonensis Malicky, D. vespertinus Marinković-Gospodnetić, Ecclisopteryx dalecarlica Kolenati, E. ivkae Previšić, Graf & Vitecek and E. keroveci (Kučinić et al. 2008, 2010, 2011a, 2011b, 2015, Previšić et al. 2014a, Vitecek et al. 2015a, 2015c, Waringer et al. 2010, 2015, 2016).

Drusus plicatus larvae can be easily distinguished from larvae of these species by the following morphological features:

– D. chrysotus, D. discolor, D. krpachiD. meridionalis and D. siveci have mandibles with terminal teeth and filtering bristles on legs and the first abdominal sternite, D. plicatus does not have any of the listed morphological features;

– D. chrysotus, D. discolor, D. krpachi, D. meridionalis and D. siveci have a head capsule concavity, a typical characteristic for larvae of these species, which is absent in D. plicatus larvae;

– Larvae of D. plicatus, D. bosnicus and D. ramae differ in head capsule shapes in lateral view. In D. bosnicus and D. ramae the head vertex is flat, while in D. plicatus the vertex is slightly rounded;

– D. ramae has a specific shape of the pronotum with two prominent acute humps on the posterior part, while the posterior part of the pronotum in D. plicatus is rounded; D. plicatus has areas of spinules on the head capsule that are absent in D. ramae;

– Larvae of D. bosnicus, D. klapaleki, D. krusniki, D. medianus, D. septentrionis and D. vespertinus have a pronounced hump in the central part of the pronotum in lateral view which is absent in D. plicatus, in which the pronotum is flat in the anterior part and slightly rounded in the posterior part;

– Larvae of D. serbicus have a recognizable shape of the pronotum in lateral view with an annular crest highest at dorsal center and gradually declining laterally, while the pronotum of D. plicatus larvae has a different shape (flat in the anterior part and slightly rounded in the posterior part);

– Larvae of D. serbicus lack lateral gills on the abdomen, D. plicatus has lateral gills on abdominal segments II throughout V;

– Larvae of D. croaticus lack prominent, long median setae dorsally on the anterior border of the pronotum and spinule areas on the head, which can be found in D. plicatus;

– Larvae of D. radovanovici and D. vernonensis have the dorsal part of the pronotum covered with numerous thin long, yellow (yellowish) setae, which are lacking in D. plicatus;

– Larvae of D. botosaneanui, D. tenellus, Ecclisopteryx dalecarlica, E. ivkae and E. keroveci have distinct parietal spines on the head, which are absent in D. plicatus;

– The whole pronotum of D. plicatus larvae is covered in white recumbent setae, D. crenophylax lacks these setae in a semicircular area anterior to the pronotal ridge, D. biguttatus generally lacks these recumbent setae on the whole pronotum;

– Larvae of D. balcanicus and D. biguttatus lack spinule areas on the head, which can be found in D. plicatus.

Interestingly, the last larval instar of D. plicatus differs from the earlier larval stages not only in head capsule width, but also in the larger extent of spinule fields (Fig. 19, fourth instar larva). So far, this feature was noticed only for the earlier larval stages of D. bosnicus (M. Kučinić, unpublished data) and for last instars of D. vernonensis (Waringer et al. 2016).

Faunistic research conducted in western Macedonia, for the last eight years recoverd besides D. plicatus, eight more species from the genus Drusus: D. biguttatus, D. vernonensis, D. botosaneanui, D. discolor, D. discophorus Radovanovic, D. macedonicus, D. krpachi and D. tenellus (Radovanović 1942, Botosaneanu 1960, Vitecek et al. 2015a, 2015b, Waringer et al. 2015, 2016). From all the above listed species only larva of D. discophorus was not described yet. Of these species only D. biguttatus and D. plicatus larvae cannot be easily distinguished (Figs 20, 21). Differentiation of D. biguttatus larvae from D. plicatus larvae can be done by careful examination of morphological features on the pronotum (Figs 20, 21) and on the head.

Drusus discophorus larvae have not been described yet, but this species seem to be limited to the type locality consists of a spring and little mountain stream at Labuniško Lake (Jablanica Mt.). In this locality we never found larvae or adults of D. plicatus during several years of repeated collections. Radovanović described both species from the Jablanica Mt. and stated that D. discophorus inhabits higher elevations (1900 m a.s.l.), while D. plicatus inhabits lower altitudes (approx. up to 900 m a.s.l., Labunište village) (Radovanović 1942). In this investigation we recorded D. plicatus in localities at higher elevation (approx. 1410 m a.s.l., spring of the River Galička reka), and Oláh and Kovács (2013) found this species in one location in Albania at an elevation of approx. 1600 m a.s.l. (Table 1). However, the morphology of male genitalia of D. plicatus and D. discophorus is very similar (Radovanović 1942, Malicky 2004), and a comprehensive study using morphology and molecular genetic data is necessary to enable clear separation of all stages of these two species.

Based on shared morphological (dark coloring of the imago, morphology of genitalia), and behavioral features (diurnal activity), Drusus plicatus could be closely related to the Drusus bosnicus group that is represented by a great number of species in southeast Europe (Marinković-Gospodnetić 1976, 1978, Kučinić et al. 2014, Vitecek et al. 2015c). Most Drusus bosnicus group species exhibit highly similar male genital morphology (Mariković-Gospodnetić 1978, Malicky 2004, Kučinić et al. 2011a, 2011b, Vitecek et al. 2015c). Analysis of the molecular data of D. plicatus, as well as of the other Drusus species (Malicky 2004, 2005, Oláh 2010, 2011, Oláh and Kovács 2013, Kučinić et al. 2014, Vitecek et al. 2015c), could show which species belong to the Drusus bosnicus group and clarify their phylogenetic and evolutionary relationships.

The subfamily Drusinae has been shown to comprise 3 groups differing in larval feeding ecology and morphology (Pauls et al. 2008). Also, these groups represent distinct evolutionary lineages (Pauls et al. 2008; Vitecek et al. 2015a). Based on the morphology of the larvae mandibles of Drusus plicatus are grazers. In addition to species with grazing larvae (e.g., species from Drusus bosnicus group, D. plicatus) (Kučinić et al. 2014, Viteck et al. 2015c), southeast Europe, along with western Alps, is a center of diversity for species with different larval feeding behaviors, for example, carnivorous filters (D. meridonalis, D. macedonicus, D. krpachi, D. siveci) (Vitecek et al. 2015a, 2015b). The mandibles of grazers are morphologically different from larvae that have carnivorous filtering feeding behavior (Pauls et al. 2008, Kučinić et al. 2011a, 2011b, 2015, Vitecek et al. 2015a). Molecular data from grazers and carnivorous filterers indicate a closer phylogenetic relationship among species in each group and also suggest certain evolutionary processes of speciation that probably happened in the ancestors of each feeding group (Marinković-Gospodnetić 1978, Kučinić et al. 2011a, Pauls et al. 2008, Vitecek et al. 2015a). Data suggest greater similarity for species that are geographically closer and have a similar feeding behaviour (Previšić et al. 2014b, Vitecek et al. 2015a) with Drusus plicatus grouping with grazers from Albania, for example D. arbanios Oláh, D. dacothracus Oláh, D. illyricus Oláh and D. pelasgus Oláh (Previšić et al. 2014b). Speciation of these and other Drusus is driven not only by the allopatric distribution caused by distinct geological and hydrological processes (e.g., karstification) in the past (Previšić et al. 2014b), but also by specific biologies that also condition this type of distribution, such as limited dispersal ability of adults (Kučinić et al. 2014, Geismar et al. 2015).

According to Schmid (1956), species of the Drusus bosnicus group are distributed in southeast Europe and the Alps. Generally, all are endemics or micro-endemics with small distribution areas and known only one or a few populations per species (Marinković-Gospodnetić 1979, Kučinić et al. 2008, Oláh 2010, 2011, Oláh and Kovács 2013, Vitecek et al. 2015c). Drusus krusniki is an exception, as more populations of this species are known (Previšić et al. 2014b). We collected D. plicatus at 8 localities in the Republic of Macedonia and the species is further reported from two localities in Albania (Oláh and Kovács 2013) (Table 1), rendering this also one of the more widely distributed endemic Drusus bosnicus group species in the southeast of Europe. We did not find D. plicatus at the type locality in Labunište village (Radovanović 1942), but we collected larvae and adults of this species in Vevčani spring (Table 1, Fig. 1B), several kilometres from Labunište village. Type locality in Labunište village was destroyed by anthropogenic influence: high level of urbanisation, pollution, stream canalisation.

The distance between the southern-most (Vevčani spring) (Fig. 1B) and the northern-most sampling location (spring of the River Strežimirska reka) of D. plicatus is about 100 km (Fig. 1A). Compared to the other species of the Drusus bosnicus group in the southeast Europe, this is a relatively large distance (Marinković-Gospodnetić 1978, 1979, Kučinić et al. 2014).

Drusus plicatus inhabits the creanal zone of streams and rivers with adults day-active at or near the spring. Diurnal activity is reported for several Drusus species in southeast Europe, e.g., D. krusniki, D. vespertinus, D. medianus, D. klapaleki, D. radovanovici (Kučinić et al. 2014, M. Kučinić, A. Previšić, unpublished data). However, a small number of D. plicatus specimens were collected also during the night using UV light traps at the spring of the River Galička reka, which is an exception for dark colored species of caddisflies that generally are active during day (Kučinić et al. 2014). At this locality, the highest abundance of D. plicatus has been recorded, with several hundreds of adults, during the day.

A similar mass emergence of adults has been previously recorded in D. septentrionis at two localities in Bosnia and Herzegovina (springs of the rivers Bistrica and Sturba, Kučinić et al. 2008, M. Kučinić, unpublished data) and in D. krusniki at Alipaša’s springs in Montenegro (A. Previšić unpublished data). We observed two peaks in the emergence of D. plicatus at the spring of the River Galička reka, the first one in spring (May - June) and the second one in autumn (September). The same emergence pattern was recorded for some other Drusus species in the Balkan Peninsula, e.g. D. croaticus and D. septentrionis (Kučinić 2002, Kučinić et al. 2008).

Among the three springs encompassed in this study the highest biodiversity (species richness) of caddisflies was recorded in the spring of the River Strežimirska reka, and the lowest in the spring of the River Galička reka. Only two species, Rhyacophila balcanica and Drusus plicatus, were recorded in all three springs. Also during this study, Synagapetus iridipennis was recorded for the first time for the Trichoptera fauna of the Republic of Macedonia.

Potamophylax lemezes was described based on specimens collected in the spring of the River Galička reka (Oláh et al. 2013). The exact taxonomic status of this population would ideally be assessed using molecular methods for a comparison of this population with some other populations of Potamophylax nigricornis Pictet, from which P. lemezes was delineated (Oláh et al. 2013).

According to the literature Wormaldia occipitalis was recorded from Vevčani spring (Oláh and Kovács 2014). During our investigation we did not collect specimens of any Wormaldia from this locality. The taxonomic status of this species will be evaluated in future studies following Neu (2015), because this species is not present in the Republic of Macedonia.