Morphological features of larvae of Drusus plicatus Radovanović (Insecta, Trichoptera) from the Republic of Macedonia with molecular, ecological, ethological, and distributional notes.

A description of the larva of Drusus plicatus Radovanović is given for the first time. The most important diagnostic characters enabling separation from larvae of the other Drusinae from the southeast Europe are listed. Molecular, ecological, and ethological features and distribution patterns of the species are given. Additionally, information on the sympatric caddisfly species of the three springs where larvae and adults of Drusus plicatus were found and presented.

Introduction

Radovanović (, subfamiliy ), was described by Radovanović based on specimens collected in Labunište village situated in the southwest part of the Republic of Macedonia (Radovanović 1942) in southeast Europe (Fig. 1A). This region (southeast Europe) is delimited by the Croatia on the west and north, by the Serbia on the north, by the Bulgaria on the east and by the Greece on the south (Ecoregions: 5, 6, 7, 11, 12; Graf et al. 2008). In the area delimited in this way, 46 species have been recorded (e.g., Malicky 2004, 2005, Oláh 2010, 2011, Oláh and Kovács 2013, Kučinić et al. 2014, Ibrahimi et al. 2015, 2016, Vitecek et al. 2015a, 2015b, 2015c), from which six species are widely distributed (e.g., Pictet, Rambur, Marinković-Gospodnetić, Rambur). The remaining 40 species are endemics of southeast Europe. Most species of from southeast Europe are reported from Bulgaria, Albania, Macedonia, and Bosnia and Herzegovina (e.g., Marinković-Gospodnetić 1979, Kumanski 1988, Malicky 2004, Oláh 2010, 2011, Oláh and Kovács 2013, Vitecek et al. 2015a, 2015b, 2015c), while the lowest number of species is recorded in Croatia (Kučinić et al. 2014). In recent years intensive research focussing on caddisfly diversity in southeast Europe has resulted in the description of 16 new species from the subfamily (Oláh 2010, 2011, Oláh and Kovács 2013, Previšić et al. 2014a, Ibrahimi et al. 2015, 2016, Vitecek et al. 2015b, 2015c).

Figure 1.

A Southeast Europe with distribution of (grey) B Spring Vevčani C Spring of the River Galička reka.

The subfamily consists of eight genera with some 110 described species (Hickin 1967, Oláh 2010, 2011, Malicky 2004, Oláh and Kovács 2013, Oláh et al. 2015, Previšić et al. 2014a, Ibrahimi et al. 2015, 2016, Vitecek et al. 2015a, 2015b, 2015c), from which 95 belong to the genus , which is one of the largest genera in the European fauna (Malicky 2004).

Faunistic, phylogenetic, and phylogeographic characteristics of have been studied extensively (e.g., Pauls et al. 2006, 2008, 2009; Previšić et al. 2009, 2012, 2014a, 2014b, Previšić and Popijač 2010, Ibrahimi et al. 2012, 2014, Stanić-Koštroman et al. 2012, 2015, Vitecek et al. 2015a). Additionally, taxonomic interest in the group was demonstrated by a number of studies focussing on the delineation of new species (Sipahiler 1992, Urbanič et al. 2002, Oláh 2010, 2011, Oláh and Kovács 2013, Previšić et al. 2014a, Ibrahimi et al. 2015, 2016, Vitecek et al. 2015b, 2015c) and larval taxonomy (e.g., Waringer et al. 2007, 2011, 2015, 2016, Kučinić et al. 2008, 2015, Vitecek et al. 2015a, 2015c). Larval morphology of all widely distributed species (e.g., , . ) of this genus recorded in southeast Europe is well known (Lepneva 1966, Waringer and Graf 1997, Previšić et al. 2012, Vitecek et al. 2015a); this is also valid for 16 of the southeast Europe endemic species (Kučinić et al. 2008, 2010, 2011a, 2011b, 2015, Vitecek et al. 2015a, 2015c, Waringer et al. 2015, 2016).

The present study has three main objectives: 1. present the morphological features of the final larval instar of ; 2. present molecular and ecological features and new data on the distribution of ; 3. provide information on the caddisfly fauna in three springs in which larvae and adults of (Fig. 2) were found. Two of the springs are located in Mavrovo National Park, highlighting the importance of these data for the continued conservation of the protected areas of the Republic of Macedonia.

Figure 2–4.

Radovanović. 2 Adult (spring of the River Galička reka) 3 Larva in case, 5th instar, right lateral view 4 Case, right lateral view.

Material and methods

Fieldwork and sampling

The material studied comprises 7 larvae of 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 PageBreakcollected 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.

Table 1.

Sampling sites of in Republic of Macedonia and literature data of in Albania (Oláh and Kovács 2013).

Locality	Country	Altitude	Longitude (E)	Latitude (N)
Vevčani spring	Macedonia	ca 950 m	20.5844	41.2396
Modrič	Macedonia	ca 960 m	20.3425	41.2156
Tresonče	Macedonia	ca 1030 m	20.7223	41.5606
Mavrovska reka	Macedonia	ca 1290 m	20.4465	41.3843
spring of the river Galička reka	Macedonia	ca 1410 m	20.6646	41.5934
spring Sveta voda, Ničpur, river Radika	Macedonia	ca 980 m	20.4034	41.4435
spring - Rosočka Reka, Rosoki village	Macedonia	ca 1200 m	20.6933	41.5694
spring of River Reč	Macedonia	ca 1280 m	20.6348	41.7902
Mt Kaptinë, brooks	Albania	ca 1600 m	20.2889	41.3866
Cermenikë Mts, Zalli and Steblevës streams	Albania	ca 1270 m	20.4425	41.3083

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 Kučinić, Mihoci & Krpač).

We have included literature data for caddisfly species collected in Vevčani spring ( Botosaneanu, Pictet, McLachlan, Previšić, Graf & Vitecek, Piller & Mitterpacher) (Oláh and Kovács 2014) which were not found during our investigation of this spring.

DNA extraction and PCR amplification

DNA was extracted from two adult males and two larvae of 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 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).

Table 2.

Intraspecific uncorrected pairwise distances (p) of partial sequences observed in (shown as percent). Abbreviations are used to denote life stages; IM (M) = adult male, L = larva.

mitochondrial cytochrome oxidase I

Locality	Specimen codes	Stage	DpMAIM1	DpMAIM2	DpMAL1	DpMAL2	DpVEIM1	DpVEL1	DpVEL2	GenBank accession nos
Spring of Galičnka reka, Mavrovo National Park	DpMAIM1	IM (M)								KT598014
	DpMAIM2	IM (M)	0.7							KT598015
	DpMAL1	L	0.7	0.0						KT598016
	DpMAL2	L	0.7	0.0	0.0					KT598017
Vevčani	DpVEIM1	IM (M)	1.1	1.1	1.1	1.1				KC881523
	DpVEL1	L	1.1	1.1	1.1	1.1	0.0			KT598018
	DpVEL2	L	0.6	0.9	0.9	0.9	0.9	0.9		KT598019

Electron microscopy, macrophotography and biometry

Electron microscopy of larvae of (specimens from Vevčani spring) was carried out using a Tescan TS 5136 variable pressure . Samples were mounted with graphitic adhesive tape on the SEM stub and coated with carbon. The samples were examined by SEM operating in , or 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; PageBreakphotographs were processed with the software Olympus Quick Photo Camera 2.2. In the larvae of 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.

scanning electron microscope

secondary electron

back-scattered

Results

Description of the fifth instar larva of

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).

Figure 5–7.

Radovanović, 5th instar larva. 5 Larva, right lateral view 6 Larva, head and pronotum, right lateral view 7 Head, pronotum and mesonotum, right lateral view.

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.

Figure 8–10.

Radovanović, 5th instar larva. Head, fronotclypeus, and setae (with number), frontal view 9 Head, spinules (sp) and anterior part of pronotum with recumbent setae (rs), right lateral view 10 Spinules (sp) on the head, right lateral view.

Figure 11–13.

Radovanović, 5th instar larva. 11 Right mandible 12 Pronotum, right lateral view 13 Mesonotum, metanotum with anterior (an) and posterior sclerites (po), dorsal view.

Thorax. Pronotum brown to black with granular surface sculpturing (Figs 6, 7, 12). Posterior margin rounded, both posterior and lateral margins thick and darkly PageBreaksclerotized. 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; PageBreakPageBreakdistance 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) PageBreakwith 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.

Figure 14–18.

Radovanović, 5th instar larva. 14 Left foreleg 15 Left midleg 16 Left hindleg 17 Sclerites (sc) on segment IX, dorsal view 18 Anal proleg (ap), anal claws (ac), and lateral fringe (lf), right lateral view.

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.

Ecology, ethology and distribution of

Mandible morphology of the larvae and observations during fieldwork suggest is a member of the 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 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 larvae, adults or both in eight localities in the Republic of Macedonia (Table 1). Altitudes of locations where were collected range between approx. 950 m and 1410 m a.s.l. (Table 1).

Sympatric caddisfly communities in three springs

We collected adult caddisflies at the three springs inhabited by . In the Vevčani spring the following species were recorded: Radovanović, (literature data), (literature data), (literature data), sp. (female), (literature data), Klapálek, , Curtis, (literature data), in the spring of River Strežimirska reka: , Pictet, McLachlan, sp. (female), sp. (females), Donovan, Marinković-Gospodnetić, , Klapálek, sp. (male), Mclachlan and in the spring of the River Galička reka the following species: , , , , and Oláh & Graf.

Discussion

Association of larvae and adults of

Association of larvae and adults of 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 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 species (e.g., Pauls et al. 2009, Previšić et al. 2009). Variability between populations in (Table 2), however, seems to be lower than observed in some other Dinaric endemics (e.g., , Previšić et al. 2009, Malicky, Previšić et al. 2014b).

Moreover, additional data, such as larvae and adults of 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 . In these springs is sympatric with the following species: , Kumanski and , and larvae of these species exhibit different morphological characteristics from those observed in larvae of (Waringer and Graf 1997, Waringer et al. 2015).

Separation of larvae of from other European larvae

Morphological features of the known larvae from the subfamily 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 in which larvae of many species are still not described or for which the separation of known larvae of some genera (e.g., Dalman, Stephens, Pictet) is either very difficult or generally not possible (Waringer and Graf 1997).

Larvae from the subfamily can be separated from other European 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 species recorded in southeast Europe, larval descriptions and taxonomic tools exist for the following 25 species: Kumanski, , , Klapálek, Rambur, Graf & Vitecek, , , Marinković-Gospodnetić, Kučinić, Graf & Vitecek, , PageBreakPageBreakSchmid, Marinković-Gospodnetić, Kumanski, Marinković-Gospodnetić, Marinković-Gospodnetić, Marinković-Gospodnetić, Marinković-Gospodnetić, Malicky, , Malicky, Marinković-Gospodnetić, Kolenati, Previšić, Graf & Vitecek and (Kučinić et al. 2008, 2010, 2011a, 2011b, 2015, Previšić et al. 2014a, Vitecek et al. 2015a, 2015c, Waringer et al. 2010, 2015, 2016).

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

– , , and have mandibles with terminal teeth and filtering bristles on legs and the first abdominal sternite, does not have any of the listed morphological features;

– , , , and have a head capsule concavity, a typical characteristic for larvae of these species, which is absent in larvae;

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

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

– Larvae of , , , , and have a pronounced hump in the central part of the pronotum in lateral view which is absent in , in which the pronotum is flat in the anterior part and slightly rounded in the posterior part;

– Larvae of 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 larvae has a different shape (flat in the anterior part and slightly rounded in the posterior part);

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

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

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

– Larvae of , , , and have distinct parietal spines on the head, which are absent in ;

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

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

Interestingly, the last larval instar of 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 (M. Kučinić, unpublished data) and for last instars of (Waringer et al. 2016).

Figure 19.

Radovanović, 4th instar larva. Head, and area with , frontal view.

frontoclypeus

spinules

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

Figures 20–21.

20 Pictet. Pronotum, right lateral view 21 Radovanović. Pronotum, showing , right lateral view.

recumbent setae

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 during several years of repeated collections. Radovanović described both species from the Jablanica Mt. and stated that inhabits higher elevations (1900 m a.s.l.), while inhabits lower altitudes (approx. up to 900 m a.s.l., Labunište village) (Radovanović 1942). In this investigation we recorded 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 and 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.

Ecological and ethological aspects and distribution of

Based on shared morphological (dark coloring of the imago, morphology of genitalia), and behavioral features (diurnal activity), could be closely related to the 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 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 , as well as of the other species (Malicky 2004, 2005, Oláh 2010, 2011, Oláh and Kovács 2013, Kučinić et , Vitecek et al. 2015c), could show which species belong to the group and clarify their phylogenetic and evolutionary relationships.

The subfamily 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 are grazers. In addition to species with grazing larvae (e.g., species from group, ) (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 (, , , ) (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 grouping with grazers from Albania, for example Oláh, Oláh, Oláh and Oláh (Previšić et al. 2014b). Speciation of these and other 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 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). is an exception, as more populations of this species are known (Previšić et al. 2014b). We collected 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 group species in the southeast of Europe. We did not find 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 is about 100 km (Fig. 1A). Compared to the other species of the group in the southeast Europe, this is a relatively large distance (Marinković-Gospodnetić 1978, 1979, Kučinić et al. 2014).

inhabits the creanal zone of streams and rivers with adults day-active at or near the spring. Diurnal activity is reported for several species in southeast Europe, e.g., , , , , (Kučinić et al. 2014, M. Kučinić, A. Previšić, unpublished data). However, a small number of 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 has been recorded, with several hundreds of adults, during the day.

A similar mass emergence of adults has been previously recorded in at two localities in Bosnia and Herzegovina (springs of the rivers Bistrica and Sturba, Kučinić et al. 2008, M. Kučinić, unpublished data) and in at Alipaša’s springs in Montenegro (A. Previšić unpublished data). We observed two peaks in the emergence of 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 species in the Balkan Peninsula, e.g. and (Kučinić 2002, Kučinić et al. 2008).

Caddisfly species richness

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, and , were recorded in all three springs. Also during this study, was recorded for the first time for the fauna of the Republic of Macedonia.

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 Pictet, from which was delineated (Oláh et al. 2013).

According to the literature was recorded from Vevčani spring (Oláh and Kovács 2014). During our investigation we did not collect specimens of any 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.