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

Abstract 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.

Faunistic, phylogenetic, and phylogeographic characteristics of Drusinae have been studied extensively (e.g., Pauls et al. 2006Pauls et al. , 2008Pauls et al. , 2009Previšić et al. 2009, 2014a, 2014b, Previšić and Popijač 2010, Ibrahimi et al. 2012, Stanić-Koštroman et al. 2012, 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, Vitecek et al. 2015b, 2015c and larval taxonomy (e.g., Waringer et al. 2007, 2011, Kučinić et al. 2008, Vitecek et al. 2015a, 2015c. Larval morphology of all widely distributed species (e.g., D. biguttatus, D. chrysotus. D. discolor) 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, Vitecek et al. 2015a, 2015c. The present study has three main objectives: 1. present the morphological features of the final larval instar of Drusus plicatus; 2. present molecular and ecological features and new data on the distribution of D. plicatus; 3. provide information on the caddisfly fauna in three springs in which larvae and adults of D. plicatus (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.

Fieldwork and sampling
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

DNA extraction and PCR amplification
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, macrophotography and biometry
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.
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).
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).

Ecology, ethology and distribution of Drusus plicatus
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).

Sympatric caddisfly communities in three springs
We collected adult caddisflies at the three springs inhabited by Drusus plicatus. In the Vevčani spring the following species were recorded: Rhyacophila balcanica

Association of larvae and adults of D. plicatus
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 Graf 1997, Waringer et al. 2015).

Separation of larvae of Drusus plicatus from other European Trichoptera larvae
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. 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., Graf 1997, Graf et al. 2005, Kučinić et (Kučinić et al. 2008, 2010, 2011a, 2011b, Previšić et al. 2014a, Vitecek et al. 2015a, 2015c, Waringer et al. 2010. Drusus plicatus larvae can be easily distinguished from larvae of these species by the following morphological features: 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 .
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. 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.

Ecological and ethological aspects and distribution of Drusus plicatus
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, 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, 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) , 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(Vitecek et al. , 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, 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 , 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. Drusus plicatus inhabits the creanal zone of streams and rivers with adults dayactive 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 . 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.

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, 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 . 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 .
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.
of Zagreb and Hrvoje Posilović, Croatian Geological Survey for assistance in photographing of the larvae, Ivana Maguire, Faculty of Science, University of Zagreb for her assistance with the English, Petar Kružić, Faculty of Science, University of Zagreb and Mladen Plantak, Elektroprojekt, for technical help. This paper is part of the project "The Drusinae (Insecta: Trichoptera) in a world of global change" (project number P23687-B17, PI: J. Waringer) funded by the Austrian Science Foundation (FWF). It was also funded by the Croatian Ministry of Science, Education and Sports (Project: 119-1193080-1206, PI: M. Kučinić and project: 119-1193080-3076, PI: M. Kerovec) and the University of Zagreb. We are also very grateful to two anonymous reviewers.