Larval morphology and phylogenetic position of Drusus balcanicus, Drusus botosaneanui, Drusus serbicus and Drusus tenellus (Trichoptera: Limnephilidae: Drusinae)

Abstract

In a recent 3–gene phylogeny of the Trichoptera subfamily Drusinae Banks, 1916 molecular data clearly correlated with the morphology and feeding ecology of larvae. The largest of three main groups, the Drusinae grazer clade, exhibits an unusual larval feeding ecology for Limnephilidae, and is the most diverse group. In this paper we describe four previously unknown Drusinae larvae from this clade: Drusus balcanicus Kumanski, 1973 (micro–endemic to Eastern Balkans); Drusus botosaneanui Kumanski, 1968 (Dinaric Western Balkans, Hellenic and Eastern Balkan, Asia Minor), Drusus serbicus Marinković-Gospodnetić, 1971a (micro–endemic to Dinaric Western Balkans); and Drusus tenellus (Klapálek, 1898) (Carpathians, Dinaric Eastern Balkans). Characteristically, the larvae of these species develop toothless mandibles typical for the Drusinae grazer clade. Larvae and adults were unambiguously associated by a phylogenetic approach based on two mitochondrial (mtCOI, mtLSU= 16S rDNA) and two nuclear genes (nuWG, nuCAD). In addition, information on the morphology of the larvae is given and the diagnostic features necessary for identification are illustrated.

INTRODUCTION

Geographically the Drusinae Banks, 1916 are restricted to Eurasian mountain ranges from Iran and the Caucasus in the East to the Iberian Peninsula in the south-west. Three quarters of the known species are endemic to a single or very few mountain ranges, making the group an ideal model for studying evolutionary processes like speciation and diversification (Schmid, 1956; Kumanski, 1973; Marinković-Gospodnetić, 1976; Sipahiler, 2002; Malicky, 2005). As cold-water adapted aquatic insects that occupy fragmented montane sky-island populations, Drusinae are also very sensitive to global change and are among the most threatened species under a warming climate. The taxon currently comprises eight genera (Drusus Stephens, 1837; Monocentra Rambur, 1842; Ecclisopteryx Kolenati, 1848; Cryptothrix McLachlan, 1867; Metanoea McLachlan, 1880; Leptodrusus Schmid, 1955; Anomalopterygella Fischer, 1966 and Hadimina Sipahiler, 2002) and more than hundred species (Malicky, 2004, 2005; Graf et al., 2008; Kučinić et al., 2011a; Olah, 2010, 2011, 2013, Previšić et al., 2014). Unfortunately, larvae from only 45 species have been described so far (references in Waringer, 2013a, b). In this paper we augment our knowledge in Drusinae larval taxonomy by presenting descriptions of the hitherto unknown larvae of Drusus balcanicus Kumanski, 1973, D. botosaneanui Kumanski, 1968, D. serbicus Marinković-Gospodnetić, 1971a, and Drusus tenellus (Klapálek, 1898). In the present study, the putative larvae of those four species were associated with co-occurring adults by using molecular data from four gene regions following the methods outlined by Pauls et al. (2006, 2008).

As caddisfly larvae are considered primary indicator taxa for monitoring water quality (Barbour et al., 1999; Barbour & Yoder, 2000; AQEM consortium, 2002; Graf et al., 2002; Hering et al., 2006) and are very frequently used as bioindicators (sensitive species) (Moog et al., 2002; Graf et al., 2002), the inclusion of the newly-described larvae will improve resolution of ecological assessment procedures. Further, larval morphology is also seen as an important tool in phylogenetics and taxonomy (van Emden, 1957; Meier & Lim, 2009; Minoshima et al., 2013). The descriptions of the four new Drusinae larvae are, therefore, also expected to increase our present knowledge of the phylogenetic structure of the Drusinae grazer clade sensu Pauls et al., 2008.

MATERIAL AND METHODS

Species collection

Adults and larvae were collected at the Balkan Peninsula using hand picking at the following locations: Drusus serbicus: Serbia, Golija Mt, spring Ilinac (Serbia), 43°20′00″ N, 20°16′55″ E, 1500 m a.s.l., 22 June 2013, leg. Kučinić, Bjelanović, Živić. Drusus botosaneanui: Gornje Lukovo polje (Kosovo), 41°52′3″ N, 20°42′0″ E, 1642 m a.s.l., 31 May 2012, leg. Kučinić, Krpač. Drusus balcanicus: Troyan Pass, South Side, brook (Bulgaria), 42°47′21″N, 24°37′05″E, 1450 m a.s.l., 12 June 2013, leg. Keresztes, Torok, Kolocsar. Drusus tenellus: Mavrovo, Lukovo Pole (Makedonija), 41°42′03″N, 20°39′52″E, 1665 m a.s.l., 3 July 2010, leg. Previšić); Murinska Rijeka (Montenegro), 42° 39′ 17.6″N, 19° 52′ 47.64″E, 878 m a.s.l., 5 July 2012, leg. Previšić.

The material intended for sequencing was transferred to 100 % alcohol, the material for morphological analyses in pure 70% ethanol in order to keep the specimens more flexible.

Morphological study

Morphological terminology follows Wiggins (1998). The larvae were described following the set of morphological characters for Drusinae defined by Waringer & Graf 2011. Larvae were studied and photographed using a Nikon SMZ 1500 binocular microscope with DS-Fi1 camera and NIS-elements D 3.1 image stacking software to combine 8 to 42 frames in one focused image. The two 5th instar larvae of D. balcanicus and the three larvae of D. botosaneanui, D. serbicus and D. tenellus are deposited in the collection of J. Waringer (Vienna, Austria).

For SEM microscopy, two fifth instar larvae of Drusus serbicus were gold coated with a BAL-TEC SCD 005 sputter coater and examined using a JEOL JSM-6390lv scannning electron microscope.

Comparative material of other Drusinae species included the following (all larvae preserved in pure 70% ethanol): Drusus franzressli Malicky, 1974 (two 5th instar larvae), Drusus spelaeus (Ulmer, 1920) (five 5th instar larvae), Drusus schmidi Botosaneanu, 1960 (six 5th instar larvae), Drusus improvisus (McLachlan, 1884) (eight 5th instar larvae), Drusus nigrescens Meyer-Dür, 1875 (five 5th instar larvae), Drusus rectus McLachlan, 1868 (six 5th instar larvae), Drusus brunneus Klapálek, 1898 (one 5th instar larva), Drusus bosnicus Klapálek, 1899 (one 5th instar larva), Drusus radovanovici Marinković-Gospodnetić, 1976 (one 5th instar larva), Drusus septentrionis Marinković-Gospodnetić, 1976 (two 5th instar larvae), D. trifidus McLachlan, 1868) (three 5th instar larvae), Ecclisopteryx dalecarlica Kolenati, 1848 (one 5th instar larva), E. guttulata Pictet, 1834 (three 5th instar larvae), E. madida (McLachlan, 1867) (one 5th instar larva), Hadimina torosensis Sipahiler, 2002 (one 5th instar larva), Metanoea rhaetica Schmid, 1956 (seven 5th instar larvae) and Metanoea flavipennis (Pictet, 1834) (ten 5th instar larvae). The material is deposited in the collection of J. Waringer (Vienna, Austria).

Molecular study

We used a phylogenetic approach to associate the larvae. To this end we inferred phylogenetic trees based on molecular sequence data from two nuclear and two mitochondrial genes with the four target species and 43 previously published Drusinae species (Appendix Table A1). We extracted DNA of larval and adult specimens using the DNEasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol. PCR reactions were set up in 10μl reactions. PCR procedures and primers are listed in Table 1. PCR products were sequenced on an ABI 3177XL capillary sequencer at the Biodiversity and Climate Research Laboratory Centre. Sequences were edited in Geneious vR7 (biomatters). Sequences were aligned using the Muscle-plugin in Geneious vR7.

Table 1.

PCR primers and PCR cycling conditions.

Fragment	Primers	Primer concentration	PCR cycling conditions	Taq Kit	Additional reagents
COI-5P (barcode region)	HCOI, LCOI (Folmer et al. 1994)	0.25 μM	5′ 95°C, 5 × (30″ 95°C, 1′ 44°C, 1′ 72°C), 15× (30″ 95°C, 30″ 48°C, 1′ 72°C), 20 × (30″ 95°C, 30″ 50°C, 1′ + (10″ * n) 72°C), 5′ 72°C	peqGOLD HotTaq	-
COI-3P	Jerry, S20 (Pauls et al. 2006)	0.25 μM	5′ 95°C; 35 × (45″ 95°C, 30″ 45°C, 45″ 72°C); 5′ 72°C	peqGOLD HotTaq	-
16SrDNA	Lepto-F, Lepto-R	0.75 μM	3′ 95°C, 35 × (30″ 95°C, 30″ 52°C, 40″ 72°C), 5′ 72°C	peqGOLD HotTaq	4 ug BSA
WG	WGbDrrev 5′-ACCCTCTCCCGCARCACTTGAG-3’	0.5 μM	5′ 95°C, 35 × (45″ 95°C, 45″ 60°C, 90″ 72°C), 7′ 72°C	Qiagen Hotstar Taq Plus	-
	WGbDrfwd 5 ′-CTTGCTGGATGCGTCTGCC-3′			Master mix
CAD	1028r-ino, 743nF-ino (Johanson & Malm 2010)	0.25 μM	5′ 95°C, 35 × (45″ 95°C, 30″ 50°C, 45″ 72°C), 5′ 72°C	peqGOLD HotTaq	-

We inferred phylogenetic trees for each locus separately using a Bayesian/MCMC approach implemented in MrBayes v3.2.1 (Ronquist et al., 2012). Nucleotide substitution models were selected using the Bayesian Information Criterion in the model test module of MEGA v5.2 (Tamura et al. 2011). In the protein coding genes, nucleotide substation models were identified separately for each codon position (see Table 2 for codon-specific selected models). We did not partition the 16SrDNA fragment. All model estimations were performed on all sites, i.e. including the gaps in LSU. The B/MCMC analysis was based on 2 parallel runs with six chains each that explored tree space for 10 million generations. Phylogenetic trees were based on 15,000 trees (2×7,500) following a 25% burn-in phase. We assessed the parameter files in Tracer Version 1.4.6 (Drummond & Rambaut 2007) to assess if each run had reached stationarity. We used the average standard deviation of split frequencies between runs after 2500000 generations if both runs reached the same optimality space.

Table 2.

Characteristics of the molecular data sets used for phylogenetic analysis and larval-adult associations.

Locus	N sequences	Length (bp)	Variable sites (N / %)	Substitution model by codon position (1st / 2nd / 3rd)	Average standard deviation of split frequencies after 25mio & 100mio generations
COI	187	1210*	533 / 44	TN93+G / HKY+G / GTR+I+G	0.014 - 0.007
LSU	197	362	162 / 44	T92+G	0.011 - 0.005
CAD	163	848	644 / 76	HKY+G / HKY / K2+G	0.008 - 0.004
WG	181	352	147 / 42	JC+G / JC / T92	0.008 - 0.005

^*11 Ns were added between the two fragments of mtCOI

RESULTS

Identification of the larvae

The putative conspecific larvae always clustered in monospecific clades with adults (Table 3, Appendix Figures A2a-A2d). There are, however, some weaknesses in the resolution of the larval association clades. In the WG phylogeny, clades of D. botosaneanui and D. balcanicus were not significantly supported (pp < 0.95). In COI and CAD all sequences of D. balcanicus are basal to a highly supported clade of D. discophoroides (pp = 1.0), but are not grouped in supported clades. In LSU there is a similar situation regarding D. tenellus. However, these topological inconsistencies only insignificantly weaken the overall associations of adults to larvae, which are further supported by identical haplotypes in all species except D. tenellus.

Table 3.

Results of larval associations based on the phylogenetic reconstruction for each of the four loci. The 5th instar larvae from the target species are deposited in the collection of J. Waringer (Vienna, Austria).

Fragment Taxon	pp	CAD Association criterion	N (m/f/l)	pp	COI Association criterion	N (m/f/l)	pp	WG Association criterion	N (m/f/l)	pp	LSU Association criterion	N (m/f/l)
D. serbicus	1.0	monophyly; identical haplotypes	4/1/2	1.0	monophyly; identical haplotypes	4/1/2	0.98	monophyly; identical haplotypes	4/1/2	0.99	identical haplotypes	4/1/2
D. tenellus	1.0	monophyly	1/1/1	1.0	monophyly	1/1/2	1.0	sister clades	1/1/2	0.98	monphyly (by exclusion to D. botosaneanui)	1/1/2
D. botosaneanui	1.0	monophyly; larvae nested within males	3/0/7	1.0	monophyly	3/1/3	0.89	monophyly; identical haplotype to ♀	0/1/4	1.0	identical haplotype	3/1/4
D. balcanicus	1.0	monophyly; identical haplotypes	5/0/2	0.9	monophyly; identical haplotypes	6/1/2	0.82	identical haplotypes	6/1/2	0.97	identical haplotype	6/1/2

Description of the larva of Drusus serbicus Marinković-Gospodnetić, 1971a

Material examined

3 ex. of fifth instar, Serbia, Golija Mt, spring Ilinac (Serbia), 43°20′00″ N, 20°16′55″ E, 1500 m a.s.l., 22 June 2013, leg. Kučinić, Bjelanović, Živić.

General morphology

Larva erucoid, head and sclerotized parts chestnut to blackish brown, nonsclerotised parts whitish. Body length ranging from 9.8 to 10.8 mm, head width from 1.33 to 1.43 mm.

Head

Head capsule coarsely granulated, almost circular in shape and hypognathous (Figs 1A–C), dorsally with blackish muscle attachment spots. Ventral parietalia sections, submentum, maxillolabial sclerites and premandibular areas medium to orange brown (Figs 1C, D). Yellowish-white ring around each eye (Fig. 1C). In lateral view, head capsule with carina (0.40–0.45 mm long and approximately 0.04 mm wide) starting a short distance from anterior eye margin and extending to frontomedian corner of frontoclypeus (Fig. 1C, arrow).

Fig. 1.

Drusus serbicus (5th instar larva). A – head and pronotum, frontal view (arrow: median notch); B – head, frontal view; C – head, pro- and mesothorax, right lateral view (arrow: lateral carina); D – head, ventral view; E–F – head, dorsolateral view, details of spinule area (white ovals) (arrow= antenna). Scale bars: A – E = 1 mm; F = 0.02 mm.

Head capsule with complete set of 18 pairs of primary setae (nomenclature by Wiggins, 1998) and lacking any additional spines or bristles known from other Drusinae larvae (e.g., Ecclisopteryx spp., Drusus trifidus McLachlan, 1868). However, posterior of each eye, a spinule area surrounding the bases of setae 15 and 16 (nomenclature by Wiggins, 1998) is present (diameter 0.13-0.18 mm; Figs 1E, F; white ovals). Such spinule areas are well known from members of the Drusus bosnicus Group sensu Marinković-Gospodnetić (1971a), e.g., Drusus klapaleki Marinković-Gospodnetić, 1971. Frontoclypeus bell-shaped, with narrow central constriction (Figs 1A, B).

Antennae based at dorsal rim of lateral carina and halfway between eye and anterior head margin (Fig. 1E, arrow), each consisting of 1 short cylindrical base and 1 short flagellum. At each parietale, 10 dorsal and 2 ventral primary setae present, with primary setae 5, 9 and 14 long and conspicuous (Figs 1B, C, E). Each side of frontoclypeus with 6 primary setae, 3 of them along anterior border. Labrum medium to light brown, with setal brush and primary setae 1–3 at anterolateral margins; on dorsal area, setation consisting of primary setae 4–6 (Figs 1A, E).

Ventral apotome elongated triangular, medium to light brown, postgenal suture approximately 55% of apotome length (Fig. 1D). Blackish brown to dark brown mandibles lacking terminal teeth along edges as well as lacking ridges in central concavity (Figs. 1D, E).

Thorax

Pronotum black brown to chestnut brown, very coarsely granulated (Figs 1C, 2A, B); its posterior margin thickened and darkly striped (Fig. 2C). Pronotal transverse groove at end of anterior 3rd lacking. Dorsal profile in lateral view with annular crest highest at dorsal center and gradually fading laterally (Figs 2B, C). With semicircular step directly posterior of crest center and in front of posterior pronotal rim (Fig. 2A, arrows). In anterior view, pronotal crest with dorsal center notch (Fig. 1A, black arrow). Two setal rows along anterior border of pronotum: (1) Dense fringe of short, curved, fine, yellow setae; (2) widely-spaced, continuous row of long, straight, dark setae meeting at anterior pronotal midline (Figs 2A, B); in total, 80–90 dark setae of varying lengths distributed over each pronotal half (Figs 1C, 2A,C). In addition, pronotal surface covered by high number of tiny, pale, recumbent setae (Fig. 2D); spines present in other Drusinae (e.g., Ecclisopteryx dalecarlica Kolenati, 1848) lacking. Pentangular prosternite light brown with medium brown posterior rim; prosternal horn present.

Fig. 2.

Drusus serbicus (5th instar larva). A–C – pronotum, dorsal and right lateral view (arrows: semicircular step posterior of pronotal annular crest); D – pronotum, central posterior section, showing white recumbent setae; E – mesonotum, metanotum and 1st abdominal segment, dorsal view; F – left fore leg, posterior view. Scale bars: A, C, E–F = 1 mm; B = 0.2 mm; D = 0.5 mm.

Mesonotum completely covered by 2 medium to yellowish brown sclerites with fine granulation except along posterior border and at lateral half of anterior border; their lateral and posterior margins with black sclerotization (Fig. 2E). Counts for mesonotal setae are as follows (nomenclature sensu Wiggins, 1998): anterior setal group sa1: 15–18, posterior group sa2: 15–20, lateral group sa3: 28–30.

Metanotum partially covered by 3 pairs of medium to dark brown sclerites. Anterior metanotal sclerites (sa1, sensu Wiggins, 1998) very large, broadly ovoid, strongly tapering laterally, each with black anterior margin; their intermediate separation shorter as length of each of them (Fig. 2E). Approximately 15 setae per sclerite (Fig. 2E). Row of setae present between small posteromedian sclerites (sa2, sensu Wiggins, 1998); 14–17 setae per sclerite. Small setal group present between each lateral (sa3, sensu Wiggins, 1998) and posteromedian sclerite (sa2); sa3 sclerites with approximately 25–30 setae per sclerite, concentrated anteriorly (Fig. 2E). Legs orange brown with numerous setae on coxae, trochanters and femora; tibiae and tarsi with only small number of setae (Figs. 2F, 3A, B). On all femora several proximodorsal setae present. Coxa, femur and tibia of each foreleg wider than those of mid- and hind legs. Setae present at proximal sections of trochanters of all three pairs of legs. Additional setae present at both anterior and posterior faces of all femora; ventral trochanteral brush present at distal sections of fore- and mid-trochanters. Fore femora each with 5 yellow ventral-edge setae, mid- and hind femora each with 4 dark ventral edge setae. Dorsal setae only at distal third of mid and hind tibiae (Figs 2F, 3A, B).

Fig. 3.

Drusus serbicus (5th instar larva). A – left mid leg, posterior view; B – left hind leg, posterior view; C – metathorax and 1st abdominal segment, right lateral view; D – 1st abdominal sternum; E – abdominal segments VIII-IX, dorsal view (arrows: posterolateral setae; dotted oval: posterodorsal setae); F – apex of abdomen, right lateral view; G – case, right lateral view. Scale bars: A–G = 1 mm.

Abdomen

First abdominal segment with 1 dorsal and 2 lateral fleshy protuberances (Figs 2E, 3C). Setal areas sa1, sa2 and sa3 (sensu Wiggins, 1998) fused, thereby creating continuous transverse row of setae anterior to dorsal protuberance until dorsal section of each lateral protuberance. Sharply delimited basal sclerites present for about 30% of these setae; without setal group posterior to dorsal protuberance (Fig. 2E). Posterior sclerites lacking at lateral protuberances (Fig. 3C). In front of each lateral protuberance, continuous band of anterolateral setae linking with each dorsal and ventral sa3 setal group (Fig. 3C). On 1st abdominal sternum, setal areas sa1, sa2 and sa3 fused, creating continuous field of setae; setal bases at the central section of the first abdominal sternum mostly small and inconspicuous except four larger bases near midline and immediately ventral of the lateral protuberances. Setal bases never fuse with neighbouring ones (Fig. 3D). On 8th abdominal dorsum, two to four long posterodorsal setae (pds) present (Fig. 3E, dotted oval). Only 1 posterolateral seta present on each half of 9th abdominal dorsum (Fig. 3E, arrows).

All gills single filaments. Dorsal gills present at most from 2nd segment (presegmental position) to 7th segment (presegmental position). Ventral gills ranging from 2nd (postsegmental) to 8th segment (presegmental). Lateral gills lacking. Lateral fringe extending from posterior third of 2nd to middle of 8th abdominal segments; in addition, a prominent seta surrounded by a small number of isolated lateral fringe setae at anterior border of 2nd segment. Light brown sclerite on 9th abdominal tergum semicircular (Fig. 3E); along its posterior border, 7–8 long and several shorter setae present, 1–2 of these long setae having position of central intermediate c setae (Fig. 3E). Anal prolegs of limnephilid type, light to medium brown, with light muscle attachment spots. Anal claws medium brown, each with 1 small accessory hook (Fig. 3F).

Case

Larval case 10.2–10.5 mm long (n= 3), curved, conical (width at anterior opening 2.45–2.7 mm and at posterior opening 1.7–2.3 mm), consisting of mineral particles (sand grains of mixed size; Fig. 3G).

Habitat

This species inhabits the epirhithral section of oxygen-rich streams with high to moderate current, but is also encountered near the source (hypocrenal region) down to the metarhithral zone. Drusus serbicus is a grazer feeding on epilithic biofilms and associated algae.

Larval key to Drusus species of the grazer clade with spinule areas at the head capsule

As in the other species of the Drusinae grazer clade, the mandibles are spoon-shaped (terminal teeth and central cavity ridges lacking; Fig. 1E). The larva of Drusus serbicus is similar to six Drusinae species from the Balkan Peninsula where a small field of spinules (= small spines approximately 0.03 mm long) is present posterior of the eyes (Figs 1E, F). Due to the recent detailed descriptions given by Kučinić et al. (2008, 2010, 2011a, b, 2014) and Previšić et al. (2009), D. serbicus can be integrated in the following dichotomous key:

1	Head with flat vertex (Fig. 4A)	Drusus bosnicus Klapálek, 1899
-	Vertex evenly rounded (e.g., Fig. 1C)	2
2	Pronotum with thin long, yellow setae (Fig. 4B)	Drusus radovanovici Marinković-Gospodnetić, 1971
-	Pronotum without thin long, yellow setae	3
3	Pronotum with numerous short, white, recumbent setae (Fig. 2D)	4
-	Pronotum without numerous short, white, recumbent setae	Drusus medianus Marinković-Gospodnetić, 1976
4	Dorsal pronotal hump smoothly rounded	Drusus medianus Marinković-Gospodnetić, 1976
-	Dorsal pronotal hump with distinct ridge (e.g., Fig 2 A–C)	5
5	Anterior metanotal sclerites elongated triangular (width / length ratio ≥ 2.0)	Drusus vespertinus Marinković-Gospodnetić, 1976
-	Anterior metanotal sclerites broadly triangular (width / length ratio < 2.0; e.g., Fig. 2E)	6
6	In lateral view, posterior section of dorsal ridge gently descending	Drusus klapaleki Marinković-Gospodnetić, 1971b
-	In lateral view, dorsal ridge annular, posterior section sharply descending (Figs 2A–C)	Drusus serbicus Marinković-Gospodnetić, 1971a

Fig. 4.

A – Drusus bosnicus (5th instar larva), head and pronotum, right lateral view (arrow: vertex flattened); B – Drusus radovanovici (5th instar larva), pronotum, right lateral view (arrows: thin long yellowish setae); C–F – Drusus balcanicus (5th instar larva). C – head, frontal view; D – head and pronotum, right anterolateral view; E – pronotum, dorsal view; F – pronotum, right lateral view. Scale bars: A–F = 1 mm.

Description of the larva of Drusus balcanicus Kumanski, 1973

Material examined

2 ex. of fifth instar, Troyan Pass brook, south Side (Bulgaria), 42°47′21″N, 24°37′05″E, 1450 m a.s.l., 12 June 2013, leg. Keresztes, Torok, Kolocsar.

General morphology

Larva erucoid, head and sclerotized parts dark brown, nonsclerotised parts whitish. Body length ranging from 11.0 to 11.2 mm, head width from 1.27 to 1.30 mm.

Head

Head capsule granulated, roundish (Figs 4C, D). Labrum dark brown, with setal brush (Fig. 4D). Ventral apotome yellowish to light brown and with postgenal suture approximately 70–75% of apotome length. Head capsule lacking any additional spines, bristles or spinule areas.

Thorax

Pronotum with adjacent series of granuli creating ribbed structures (Fig. 4F). Dorsal profile in lateral view with posterior half of pronotum rounded, this curvature creating distinct step leading down to anterior, lower section of pronotum (Fig. 4 F). Lateral ridge lacking. In total, 35–40 dark setae of varying lengths distributed over each pronotal half. Prosternite very light and indistinct.

Mesonotum completely covered by 2 medium brown to yellowish sclerites with dark brown muscle attachment spots (Fig. 4E). Their anterolateral corners, lateral and posterior margins darkly sclerotized. Numbers of setae in anterior setal group sa1 are 30–40, in posterior group sa2 25–30 and in lateral group sa3 25–30.

Anterior sa1 metanotal sclerites triangular, with approximately 25–30 setae per sclerite (Fig. 5A). Setal counts of posteromedian sa2 sclerites are 15–20 setae per sclerite and of lateral sa3 sclerites 15–20, respectively. Legs light brown. All other details as in D. serbicus.

Fig. 5.

A–E – Drusus balcanicus (5th instar larva). A– mesonotum, metanotum and 1st abdominal dorsum, dorsal view; B – 1st abdominal sternum, ventral view; C–D – details of central sections of 1st abdominal sternum, ventral view; E – case, right lateral view. F–G – Drusus franzressli (5th instar larva). F – pronotum, right lateral view (arrows: lateral ridge); G – detail of central section of 1st abdominal sternum, ventral view. H–I – Drusus botosaneanui (5th instar larva). H – head and pronotum, dorsolateral view; I – head and pronotum, frontal view. Scale bars: A–B, E–F, H–I = 1 mm; C–D, G = 0.3 mm.

Abdomen

Center of 1st abdominal sternum with large or medium concentrations of fused basal sclerites of setae, creating multilobed patterns of sclerotized areas arranged mostly posteriorly of the two largest basal sclerites (Figs 5B–D).

Dorsal gills present at most from 2nd segment (presegmental position) to 5th segment (presegmental position). Ventral gills ranging from 2nd (presegmental) to 7th segment (presegmental). Lateral gills lacking. Lateral fringe, details of 9th abdominal sclerite and of anal prolegs as in D. serbicus.

Case

Larval case 10.6–10.7 mm long (n= 2), curved, slightly conical. Width at anterior opening 2.5–2.6 mm and at posterior opening 1.8–1.9 mm, consisting of mineral particles (sand grains of mixed size; Fig. 5E).

Habitat

Drusus balcanicus is confined to oxygen-rich headwater streams and spring regions. Data logger records over a full year revealed arithmetric means of water temperatures for typical habitats of D. balcanicus (e.g., springs under the Troyan pass, Bulgaria) of 6.65°C (range 1.08–15.44°C). The species grazes epilithic algae.

Larval key to Drusus species of the grazer clade without spinule areas or additional spines at the head capsule

The species belongs to the Drusinae grazer clade where the mandibles are spoon-shaped (terminal teeth and central cavity ridges lacking; Fig. 1E). The larva of Drusus balcanicus lacks spinule areas and additional bristles and spines at the head capsule (Fig. 4C; Table 4). Dorsal gills present; basal sclerites of setae at the first abdominal sternum fused to sclerotized plates or multilobed patterns (Fig. 5B); anterior-row setae present near the dorsal pronotal midline (Fig. 4C); dorsal edge setae restricted to distal third of mid- and hind tibiae. These features are shared with Drusus franzressli Malicky, 1974, D. improvisus (McLachlan, 1884), D. nigrescens Meyer-Dür, 1875, D. rectus McLachlan, 1868, D. spelaeus (Ulmer, 1920), Ecclisopteryx malickyi Moretti, 1991, Metanoea flavipennis (Pictet, 1834) and M. rhaetica Schmid, 1956. Due to the presence of a low central ridge at the pronotum, the larva of D. balcanicus is similar to that of Drusus franzressli (Table 5); the two species can be easily separated by the fact that a distinct lateral ridge (Fig. 5F, arrows) is present in D. franzressli which is lacking in D. balcanicus (Fig. 4F). In addition, the basal sclerites of the center setae at the first abdominal sternum are fused, thereby creating a large, uniform central plate (Fig. 5G) in D. franzressli whereas a multilobed sclerotized pattern is present in D. balcanicus (Figs 5B–D).

Table 4.

Synopsis of characters separating the currently known Drusinae larvae (5th instars) where mandibles are spoon-shaped and spinule areas, additional bristles and spines at the head capsule are lacking (i.e., only the standard set of 18 pairs of primary setae is present).

Species/character	Dorsal gills present?	Basal sclerites of setae at first abdominal sternum fused to sclerotized plates or multilobed patterns?	Anterior-row setae present near dorsal pronotal midline?	Dorsal edge setae at entire length of mid- and hind tibia?	Pronotum evenly rounded?	References
Drusus carpathicus	no	no	yes	no	yes	Szczesny (1978)
Drusus improvisus
Drusus rectus
Drusus spelaeus	yes	yes	yes	no	yes	Table 5 (present paper)
Metanoea flavipennis
Metanoea rhaetica
Drusus balcanicus
Drusus franzressli	yes	yes	yes	no	no	Table 5 (present paper)
Drusus nigrescens
Ecclisopteryx malickyi
Drusus camerinus	yes	yes	no	no	yes	Waringer et al. 2008a
Drusus melanchaetes	yes	no	yes	yes	yes	Graf (1993)
Drusus adustus						Waringer et al. (2008b)
Anomalopterygella chauviniana						Urbanič et al. (2003)
						Vieria-Lanero et al. (2005)
Drusus bolivari	yes	no	yes	no	no	Kučinić et al. (2010)
Drusus monticola						Waringer et al. (2010)
Drusus ramae						Waringer & Graf (2011)
Drusus annulatus
Drusus biguttatus
Drusus ingridae
Drusus rectus						Moretti & Pirisinu (1981)
Drusus vinconi	yes	no	yes	no	yes	Sipahiler (2002)
Ecclisopteryx asterix						Urbanič et al. (2003)
Hadimina torosensis						Waringer et al. (2013b)
Leptodrusus budtzi
Drusus aprutiensis
Drusus camerinus						Kučinić et al. (2008)
Drusus croaticus	yes	no	no	no	yes	Waringer et al. (2008a, 2010, 2011)
Drusus mixtus
Drusus trifidus

Table 5.

Synopsis of characters separating the currently known Drusinae larvae (5th instars) which share the following group morphomatrix: spoon-shaped mandibles; lack of additional head bristles, spines or spinule areas; anterior-row setae present near dorsal pronotal midline; dorsal gills present; dorsal edge setae restricted to distal third of mid- and hind tibiae; basal sclerites of setae at first abdominal sternum fusing to sclerotized plates or multilobed patterns.

Species/character	Dorsal outline of pronotum / median incision present?	Pronotal sculpturing	Sclerotization at 1st abdominal sternum	Posterolateral gills present at 2nd and 3rd abdominal segment?	Start of lateral fringe	Distribution	References
Drusus nigrescens	high ridge / yes	coarsely granulated, ribbed	multilobed sclerotized pattern	yes	last third III	western alpine	Waringer et al. (2007)
Ecclisopteryx malickyi	high ridge / no	coarsely granulated	multilobed sclerotized pattern	yes	last third III	southern alpine	Graf et al. (2011)
Drusus franzressli	low central ridge / no	coarsely granulated	central plate	no	first third II	Hellenic Western Balkans	Waringer et al. (2013a)
Drusus balcanicus	low central ridge / no	coarsely granulated, ribbed	multilobed sclerotized pattern	no	first third II	Eastern Balkans	present paper
Drusus improvisus	evenly rounded, high profile / no	coarsely granulated, ribbed	multi-lobed sclerotized pattern	yes	last third II	Apennines	Waringer et al. (2008a)
Drusus rectus	evenly rounded, low profile/no	coarsely granulated, ribbed	multilobed sclerotized pattern	yes	last third III	Pyrenees, Massif central	unpublished data
Drusus spelaeus	evenly rounded, low profile / no	coarsely granulated, ribbed	central plate or multi-lobed sclerotized pattern	yes	last third II	western alpine	Waringer et al. (2013a)
Metanoea flavipennis	evenly rounded, low profile / no	finely granulated	central plate	yes	last third II	western alpine	Waringer et al. (2000)
Metanoea rhaetica	evenly rounded, low profile / no	finely granulated	central plate	no	last third II	eastern alpine	Waringer (1985)

Description of the larvae of Drusus botosaneanui Marinković-Gospodnetić, 1971a and Drusus tenellus (Klapálek, 1898)

Material examined

Drusus botosaneanui: 2 ex. of fifth instar, Gornje Lukovo polje (Kosovo), 41°52′3″ N, 20°42′0″ E, 1642 m a.s.l., 31 May 2012, leg. Kučinić, Krpač. Drusus tenellus: 3 ex. of fifth instar, Mavrovo, Lukovo Pole (Makedonija), 41°42′03″N, 20°39′52″E, 1665 m a.s.l., 3 July 2010, leg. Previšić; 2 ex. of fifth instar, Murinska Rijeka (Montenegro),42° 39′ 17.6″N, 19° 52′ 47.64″E, 878 m a.s.l., 5 July 2012, leg. Previšić.

General morphology

Larva erucoid, head and sclerotized parts dark brown, nonsclerotised parts whitish. Body length of final instar larva of D. botosaneanui ranging from 10.3 to 10.5 mm, head width from 1.26 to 1.30 mm. Body length of final instar larva of D. tenellus ranging from 10.7 to 12.9 mm, head width from 1.24 to 1.34 mm.

Head

Head capsule roundish, dark brown with lighter orange areas around foramen occipitale and with smooth surface sculptured by shallow wrinkles (Figs 5H, I, 7A, B). At each parietale, 20–25 long bristles and short, strongly tapering spines present in addition to standard set of 12 primary setae, mostly anterior and dorsally of the eye. Also on frontoclypeus, 12–18 long bristles and short, strongly tapering spines present in addition to standard set of 6 pairs of primary setae, mostly at anterolateral corners (Figs 5H, I, 7A).

Fig. 7.

A–C, Drusus tenellus (5th instar larva). A – head and pronotum, frontal view; B – head and pronotum, right lateral view; C – detail of central notch, right dorsolateral view. D–E – Drusus schmidi (5th instar larva). D – detail of central notch, right dorsolateral view; E– head, pro- and mesonotum, right lateral view; F– Ecclisopteryx madida (5th instar larva), detail of central notch, right dorsolateral view; G–I – schematic cross sections (right lateral view) of central pronotal ridges (5th instar larvae). G – Ecclisopteryx madida; H – Drusus schmidi; I – Drusus tenellus. Scale bars: A, F–I = 1 mm; B–E = 0.5 mm.

Ventral apotome orange, in D. botosaneanui broadly bell-shaped, in D. tenellus more narrow and parallel-sided; postgenal suture approximately 60–66% of apotome length.

Thorax

Pronotum dark brown to blackish brown. Pronotal surface relatively smooth, sculptured by shallow wrinkles in D. botosaneanui (Figs 6C, D) but coarsely granulated in D. tenellus with adjacent series of granuli creating ribbed structures (Fig. 7B). Dorsal profile in lateral view with low ridge not elongated laterally. In D. botosaneanui, ridge gently ascending from posterior pronotal border and creating a distinct step leading down to anterior, lower 2/3 of pronotum (Figs 6B, D). In anterior view with deep central notch flanked by two anteriorly directed hooks (Figs 6B-D). In D. tenellus, posterior and anterior section of ridge joining closely without creating distinct step; anterior section gently sloping down to anterior part of pronotum (Figs 7B, C, 7I). Central notch very shallow, flanking anteriorly directed hooks totally missing (Figs 7A, C). In total, 60–75 long dark bristles and short, strongly tapering spines distributed over each pronotal half. Prosternite light brown, with medium brown anterolateral corners, trapezoidal in shape and tapering posteriorly.

Fig. 6.

Drusus botosaneanui(5th instar larva). A– head, detail of spines and bristles at the right parietale; B – head, pro- and mesonotum, right lateral view; C – detail of central ridge at pronotum showing median notch flanked by anteriorly directed hooks; D – pronotum, mesonotum and anterior part of metanotum, dorsal view; E – mesonotum, metanotum and 1st abdominal segment, dorsal view; F – 1st abdominal sternum, ventral view; G – case, right lateral view; Scale bars: B, D–G = 1 mm; A, C = 0.5 mm.

Mesonotal sclerites dark brown to blackish brown with lateral and posterior margins darkly sclerotized. Numbers of setae in anterior setal group sa1 are 15–25, in posterior group sa2 17–35 and in lateral group sa3 18–25 (Fig. 6D).

Dark brown anterior sa1 metanotal sclerites ovoid, with approximately 25–30 setae per sclerite in D. botosaneanui (Fig. 6D) and with 15–20 setae in D. tenellus. Setal counts of medium brown posteromedian sa2 sclerites are 15–20 setae per sclerite and lateral sa3 sclerites 17–25, respectively, for both species; the latter sclerites medium brown with black brown markings (Fig. 6E). Legs medium to dark brown. All other details as in D. serbicus.

Abdomen

Posterior sclerite present at lateral protuberances in D. botosananui but lacking in D. tenellus. On 1st abdominal sternum, setal areas sa1, sa2 and sa3 fused, creating continuous field of 70–100 setae; setal bases at the central section of the first abdominal sternum mostly small and inconspicuous except four larger bases near midline and immediately ventral of the lateral protuberances (Fig. 6F). On 8th abdominal dorsum, 2 to 4 long and 4 short posterodorsal setae (pds) present. Only 1 posterolateral seta present on each half of 9th abdominal dorsum. Light to medium brown sclerite on 9th abdominal tergum semicircular; along its posterior border, 10 long and several shorter setae present, 2 of these long setae having position of central intermediate c setae.

Dorsal gills present at most from 2nd segment (presegmental position) to 7th segment (postsegmental position). Ventral gills ranging from 2nd (presegmental) to 7th segment (postsegmental). Dorsolateral gills ranging from 2nd (presegmental) to 4th (presegmental) and ventrolateral gills ranging from 2nd (postsegmental) to 4th segment (postsegmental). Lateral fringe extending from onset of 3rd to first third of 8th abdominal segments.

Case

Drusus botosaneanui: larval case 10.2–10.4 mm long (n= 2), curved, conical. Width at anterior opening 3.1–3.3 mm and at posterior opening 1.9–2.2 mm. Drusus tenellus:11.0–11.5 mm long (n= 3), curved, conical. Width at anterior opening 2.7–3.0 mm and at posterior opening 1.9–2.0 mm. In both species case consisting of mineral particles (sand grains of mixed size; Fig. 6G).

Habitat

Drusus botosaneanui inhabits springs and the upper sections of headwater streams as well as midstream sections of rivers at 655 to 1450 m above sea level (Ibrahimi et al., 2012) whereas D. tenellus prefers the epi- and metarhithral section of oxygen-rich streams with high to moderate current at altitudes > 1450 m a.s.l. Mean annual water temperatures for stream sections inhabited by D. botosaneanui (e.g., tributary of Beli Iskar, Bulgaria) were found to be 5.66°C (annual range 0.08–14.38°C) and for D. tenellus sampling sites (e.g., Strežimirska reka, Mavrovo, Makedonija) 6.87°C (annual range 5.11–8.54°C). Both species are grazes of biofilms and epilithic algae.

Larval key to Drusus species of the grazer clade with additional spines at the head capsule

As in the other species of the Drusinae grazer clade, the mandibles are spoon-shaped (terminal teeth and central cavity ridges lacking; Fig. 1E). Drusus botosaneanui and D. tenellus belong to the group of Drusinae species which, in addition to their standard set of 18 pairs of primary setae (nomenclature by Wiggins, 1998), have short, thick spines or long, tapering bristles at each parietale (and frontoclypeus in some species): Drusus brunneus Klapálek, 1898, D. schmidi Botosaneanu, 1960, D. trifidus McLachlan, 1868, Ecclisopteryx dalecarlica Kolenati, 1848, E. guttulata Pictet, 1834, E. ivkae Previšić, Graf & Vitecek, 2014, E. keroveci Previšić, Graf & Vitecek, 2014 and E. madida (McLachlan, 1867) (Table 6). Because the number of frontoclypeal setae is > 20 (Figs 5I, 7A) and the dorsal ridge (Figs 6B, 7B) does not extend to the anterolateral corners of the pronotum as in D. brunneus (Fig. 8A), D. botosaneanui and D. tenellus key with Ecclisopteryx madida and Drusus schmidi. D. botosaneanui can be easily separated from the other three species by its deep center notch in the pronotal ridge, flanked by two anteriorly directed hooks (Figs 6B-D). In Drusus schmidi, D. tenellus and Ecclisopteryx madida the central notch is very shallow (Figs 7A, C-F, 8B), and anteriorly directed hooks are completely missing. These species can be separated by the profile of the central pronotal ridge: in E. madida, the ridge is rather sharp and almost concave in its anterior section (Figs 7F, G, 8B), in D. schmidi there is a distinct step (Figs 7D, E, H) and in D. tenellus the anterior section of the ridge gently slopes down to the anterior part of the pronotum (Figs 7B, C, I).

Table 6.

Synopsis of characters separating the currently known Drusinae larvae (5th instars) where mandibles are spoon-shaped and additional bristles and spines at the head capsule are present (in addition to the standard set of 18 pairs of primary setae).

Species/character	Pronotum with ridge extending laterally to the anterior pronotal margin?	Dorsal outline of pronotum / Deep median incision present?	Number of setae, spines and bristles at the fronto-clypeus?	Colour of pronotal setae, spines and bristles?	References
Drusus brunneus	yes	high ridge / no	> 20	black brown	Szczesny (1978)
Drusus botosaneanui	no	low central ridge / yes	> 20	black	present paper
Ecclisopteryx guttulata	no	low central ridge / yes	12-16	black brown	Szczesny (1978); Pitsch (1993); Waringer & Graf (2011)
Ecclisopteryx madida 1	no	low central ridge / no	> 20	black brown	Szczesny (1978); Pitsch (1993); Waringer & Graf (2011)
Drusus schmidi 1	no	low central ridge / no	> 20	black	Kučinić et al. (unpublished data)
Drusus tenellus 1	no	low central ridge / no	> 20	black	present paper
Ecclisopteryx keroveci 2	no	low central ridge / no	12	black brown	Previšić et al. (2014)
Ecclisopteryx ivkae 2
Drusus trifidus	no	evenly rounded / no	12	pale yellow	Szczesny (1978)
Ecclisopteryx dalecarlica	no	evenly rounded / no	12-16	black brown	Szczesny (1978); Pitsch (1993); Waringer & Graf (2011)

¹In E. madida, the pronotal ridge is rather sharp and almost concave in its anterior section (Figs 7F, G; 8B), in D. schmidi there is a distinct step (Figs 7D, E, H) and in D. tenellus the anterior section of the ridge gently slopes down to the anterior part of the pronotum (Figs 7B, C, I).

²In E. keroveci, the number of additional spines on each parietale is 12-20, in E.ivkae 1-7.

Fig. 8.

5th instar larvae. A – Drusus brunneus, head and pronotum, right lateral view; B– Ecclisopteryx madida, head and pronotum, frontal view; C– Ecclisopteryx dalecarlica, head and pronotum, right lateral view; D – Ecclisopteryx guttulata, head and pronotum, frontal view; E– Drusus trifidus, head and pronotum, right lateral view. Scale bars: A–E = 1 mm.

DISCUSSION

Previous studies that associated adults and larvae in caddisflies have generally used COI sequences only (e.g. Graf et al., 2009; Waringer et al., 2008b), COI & WG (Waringer et al., 2013a), COI & 28S rDNA sequences (Zhou et al., 2007) and COI, WG & LSU sequences (Previšić et al., 2014). The additional use of WG, LSU and CAD did not bring additional information to our 1200 bp long COI sequences. However, the use of unlinked nuclear markers provides independent support for the sorting of mitochondrial lineages, that could also result from historical isolation of presently admixed populations (e.g. Elbrecht et al., 2014). It is thus advisable to use both nuclear and mitochondrial markers for life stage associations. The nuclear genes we used, WG and CAD proved sufficiently variable to discern species in this study. The level of variation is similar in WG and even higher in CAD than in COI. Among the two genes we used, CAD performed somewhat better in our study, but both seem suitable for associating life stages in caddisflies.

All four species described in the present paper belong to the largest group of epiltihic grazers where terminal teeth are lacking at the mandible (Figs 1D, E, 4D). Based on the presence or absence of bristles and setae in addition to the standard set of 18 pairs of primary setae at the larval head capsule, the grazer clade is separated in three subgroups: subgroup 1 where, posterior of each eye, a spinule area is present (Figs 1E, F; white ovals). Such spinule areas are well known from members of the Drusus bosnicus Group. In her important paper on the species of genus Drusus in former Yugoslavia, Marinković-Gospodnetić (1971a) assigned D. bosnicus Klapálek, 1899, D. klapaleki Marinković-Gospodnetić, 1971b, D. plicatus Radovanović, 1942, D. radovanovici Marinković-Gospodnetić, 1971b and D. ramae Marinković-Gospodnetić, 1971b to the D. bosnicus Group because main structures of the male genitalia showed a high degree of structural similarity. Later, D. krusniki Malicky, 1981, D. medianus Marinković-Gospodnetić, 1976, D. septentrionis (Marinković-Gospodnetić, 1976) and D. vespertinus Marinković-Gospodnetić, 1976 were added to this group (see discussion in Kučinić et al., 2011a). Interestingly, the small field of spinules at the larval head capsule situated posterior of the eyes lacks in D. ramae of the D. bosnicus Group but could be also detected in the hitherto unknown larva of D. serbicus. In subgroup 2 of the grazer clade, additional spines and bristles are present at the parietalia and/or the frontoclypeus; this is the case in Ecclisopteryx dalecarlica, E. guttulata, E. ivkae, E. keroveci, E. madida, Drusus brunneus, D. schmidi, D. trifidus and the hitherto unknown larvae of D. botosaneanui and D. tenellus (Figs 5H, I, 6A, B, 7A). In this group D. trifidus is a special case because only a very small number of 1–2 additional spines per parietale are present in this species and it seems that primary setae simply took the shape of such spines, thereby maintaining the set of 18 pairs of primary setae at the parietalia. Finally, D. balcanicus belongs to the largest subgroup of the grazer clade where only the standard set of primary setae is present at the head capsule (Figs 4C, D).

The spines which split subgroup 2 of the grazer clade from the other two subgroups (e.g., Figs 5H, I, 7A) have a length of 0.4 mm or more in Ecclisopteryx guttulata and are one magnitude longer than the spinules observed in Drusus serbicus and associated species where a length of up to 0.03 mm has been measured. These morphological traits are in line with distinct differences in longitudinal distribution patterns: the center of longitudinal distribution of species bearing additional spines at the head capsule (morphological features summarized in Table 6) is in the epi- and metarhitral section, whereas Drusinae species without spines or with spinules are only found in spring or springbrook sections (eucrenal-hypocrenal). Statzner & Higler (1985) have shown that the eucrenal and hypocrenal sections of streams (source and springbrooks) are frequently characterized by relatively low hydraulic stress. The hypocrenal-epirhithral transition zone is followed by a reach of high hydraulic stress, which, after the next zone of transition at the break-point of the slope curve, is then followed by a zone of lower hydraulic stress (Statzner & Higler, 1985). As Drusinae larvae arrange their heads most frequently towards the current, the presence of spines at the head capsule of the species group summarized in Table 6 may be associated with their presence in such hydrological high-stress sections within the stream continuum. Videler (1995) has shown that small irregularities at the scales of fish can reduce shear stress in the boundary by a maximum of 10% compared with the shear stress of a smooth surface, a mechanism based on the impedance of cross flow under well-defined conditions. The function of roughness probably reduces total drag by generating premature turbulence and by boundary layer thinning, despite an increased friction over the surface (Videler, 1995).

The adults and larvae of Drusus serbicus, D. botosaneanui and D. balcanicus were sampled in the months of May and June in 2012 and 2013, of D. tenellus in July 2010. This is in accordance with the reported spring flight period of D. serbicus as the type and paratype specimens were collected on 30 May, 1970 (Marinković-Gospodnetić, 1971a). Drusus balcanicus is also a spring and early summer species with a rather short flight period, whereas it is elongated in D. botosaneanui being on the wing from spring to autumn (Graf et al., 2008). An elongated flight period has also been observed in D. tenellus where adults were still collected in the first week of October (Olah, 2013).

With respect to distribution, D. balcanicus is a species (micro-) endemic to the eastern Balkan Peninsula where it is restricted to the Stara Planina and Witoscha, whereas D. serbicus is (micro-) endemic to the Dinaric Western Balkans and restricted to the Dinaric Alps. Drusus tenellus has a wider range, with records from the Carpathians and the Dinaric Eastern Balkans. The distribution of D. botosaneanui is even wider, covering the Dinaric Western Balkans, the Hellenic and Eastern Balkans as well as Asia Minor.

Appendix

Table A1.

Specimen and sequence information for the data used in the present study.

BOLD Process ID	Sample ID	Institution Storing	Species	Life Stage / Sex	Collection Information	Lat	Lon	Elevation	WG	16S	COI-5p	COI-3p	CAD	Source of Sequences
SPDRU147-14	fAns0101L	University of Natural Resources and Life Sciences	Anisogamus nov sp.	L	France, 09-Jun-2013, leg. Graf				n.a.	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU117-14	AC003	Senckenberg Forschungsinstitut und Naturmuseum	Anomalopterygella chauviniana	L	Germany, Rossbach, Bieber, 12-Jul-2004, leg. Pauls	50,156	9,306		346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU145-14	fAch0101L	University of Natural Resources and Life Sciences	Anomalopterygella chauviniana	L	Germany, Rossbach, Grosser Rossbach, 17-Jun-2013, leg. Vitecek	50,156	9,296		346[0n]	362[0n]	658[0n]	541[0n]	844[4n]
SPDRU146-14	fAch0102L	University of Natural Resources and Life Sciences	Anomalopterygella chauviniana	L	Germany, Rossbach, Grosser Rossbach, 17-Jun-2013, leg. Vitecek	50,156	9,296		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU152-14	fCsp0101M	University of Natural Resources and Life Sciences	Chaetopteryx sp.	M	Montenegro, 02-Nov-2013, leg. Pesic				n.a.	361[0n]	658[0n]	541[0n]	816[32n]
SPDRU150-14	fCne0203M	University of Natural Resources and Life Sciences	Cryptothrix nebulicola	M	Italy, Piedmont, Torino, Traversella, Fondo, Burdeiver brook, 12-Jul-2012, leg. Graf	45,5204	7,66128	1500	352[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU151-14	fCne0204M	University of Natural Resources and Life Sciences	Cryptothrix nebulicola	M	Italy, Piedmont, Torino, Traversella, Fondo, Burdeiver brook, 12-Jul-2012, leg. Graf	45,5204	7,66128	1500	352[0n]	362[0n]	634[0n]	541[0n]	841[0n]
SPDRU309-14	fDmu0102F	University of Natural Resources and Life Sciences	Cryptothrix nebulicola	F	Italy, Piedmont, Torino, Traversella, Fondo, Alpe Gias del Prete, 12-Jul-2012, leg. Vincon	45,519	7,65074		352[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU153-14	fDad0101M	University of Natural Resources and Life Sciences	Drusus adustus	M	Austria, Kaernten, Sankt Oswald, 08-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	329[0n]	658[0n]	541[0n]	819[29n]
SPDRU412-14	fDsp2301F	University of Natural Resources and Life Sciences	Drusus adustus	F	Austria, Kaernten: Sankt Oswald, 08-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU413-14	fDsp2302F	University of Natural Resources and Life Sciences	Drusus adustus	F	Austria, Kaernten: Sankt Oswald, 08-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	841[7n]
SPDRU154-14	fDal0102M	University of Natural Resources and Life Sciences	Drusus alpinus	M	Italy, Piedmont, Torino, Traversella, Fondo, Alpe Gias del Prete, 12-Jul-2012, leg. Graf	45,5317	7,68669	1063	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU155-14	fDal0201M	University of Natural Resources and Life Sciences	Drusus alpinus	M	Italy, Piedmont, Torino, Traversella, Fondo, Alpe Gias del Prete, 12-Jul-2012, leg. Vincon	45,519	7,65074		346[0n]	363[0n]	658[0n]	541[0n]	845[3n]
SPDRU156-14	fDal0202F	University of Natural Resources and Life Sciences	Drusus alpinus	F	Italy, Piedmont, Torino, Traversella, Fondo, Alpe Gias del Prete, 12-Jul-2012, leg. Vincon	45,519	7,65074		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU072-14	DAn001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus annulatus	L	Slovakia, Muranska Planina, SK, Muranska Planina, Havranik tributary, 22.05.2003, 22-May-2003, leg. Blanar & Pauls				466[0n]	497[0n]	658[0n]	658[0n]	n.a.	Pauls et al. 2008
SPDRU118-14	Dan002	Senckenberg Forschungsinstitut und Naturmuseum	Drusus annulatus	M	Slovakia, Muranska Planina, Havranik tributary, 22-May-2003, leg. Blanar, Pauls				346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU157-14	fDan0101M	University of Natural Resources and Life Sciences	Drusus annulatus	M	France, Rhone-Alpes, Chamrousse, 06-Jul-2012, leg. Graf	45,1231	5,86548		346[0n]	362[0n]	658[0n]	541[0n]	841[7n]
SPDRU158-14	fDan0102M	University of Natural Resources and Life Sciences	Drusus annulatus	M	France, Rhone-Alpes, Chamrousse, 06-Jul-2012, leg. Graf	45,1231	5,86548		346[0n]	362[0n]	658[0n]	541[0n]	840[8n]
SPDRU163-14	fDar0106M	University of Natural Resources and Life Sciences	Drusus arbanios	M	Albania, South Albania: Skraper district, Ostrovice Mt, spring, 02-Jun-2013, leg. Kucinic, Cukusic			1950	346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU164-14	fDar0107M	University of Natural Resources and Life Sciences	Drusus arbanios	M	Albania, South Albania: Skraper district, Ostrovice Mt, spring, 02-Jun-2013, leg. Kucinic, Cukusic			1950	346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU124-14	DBal003	Senckenberg Forschungsinstitut und Naturmuseum	Drusus balcanicus	M	Bulgaria, Plovdiv, Karnare, Hristo Danovo, 12-Jun-2008, leg. Balint, Neu	42,7734	24,6186		346[0n]	362[0n]	658[0n]	541[0n]	846[1n]
SPDRU125-14	DBal004	Senckenberg Forschungsinstitut und Naturmuseum	Drusus balcanicus	M	Bulgaria, Plovdiv, Karnare, Hristo Danovo, 12-Jun-2008, leg. Balint, Neu	42,7734	24,6186		346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU183-14	fDbl0203F	University of Natural Resources and Life Sciences	Drusus balcanicus	F	Bulgaria, Loveci region: Central Balkan Range, Karnare, Troyanski Prohod, springs, 12-Jun-2012, leg. Ujvarosi, Kolcsar & Torok	42,7724	24,6176	1386	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU184-14	fDbl0203M	University of Natural Resources and Life Sciences	Drusus balcanicus	M	Bulgaria, Loveci region: Central Balkan Range, Karnare, Troyanski Prohod, springs, 12-Jun-2012, leg. Ujvarosi, Kolcsar & Torok	42,7724	24,6176	1386	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU185-14	fDbl0206M	University of Natural Resources and Life Sciences	Drusus balcanicus	M	Bulgaria, Loveci region: Central Balkan Range, Karnare, Troyanski Prohod, springs, 12-Jun-2012, leg. Ujvarosi, Kolcsar & Torok	42,7724	24,6176	1386	346[0n]	362[0n]	658[0n]	541[0n]	738[0n]
SPDRU186-14	fDbl0207M	University of Natural Resources and Life Sciences	Drusus balcanicus	M	Bulgaria, Loveci region: Central Balkan Range, Karnare, Troyanski Prohod, springs, 12-Jun-2012, leg. Ujvarosi, Kolcsar & Torok	42,7724	24,6176	1386	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU187-14	fDbl0302M	University of Natural Resources and Life Sciences	Drusus balcanicus	M	Bulgaria, Loveci region: Central Balkan Range, Karnare, Troyanski Prohod, springs, 12-Jun-2012, leg. Ujvarosi, Kolcsar & Torok	42,7724	24,6176	1386	346[0n]	362[0n]	658[0n]	541[0n]	500[3n]
SPDRU414-14	fDsp2401L	University of Natural Resources and Life Sciences	Drusus balcanicus	L	Bulgaria, Central Balkan, Troian Pass, South side, brook, 12-Jun-2013, leg. Keresztes, Torok, Kolocsar	42,7725	24,6181	1450	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU415-14	fDsp2402L	University of Natural Resources and Life Sciences	Drusus balcanicus	L	Bulgaria, Central Balkan, Troian Pass, South side, brook, 12-Jun-2013, leg. Keresztes, Torok, Kolocsar	42,7725	24,6181	1450	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU174-14	fDbg0301L	University of Natural Resources and Life Sciences	Drusus biguttatus	L	Bosnia and Herzegovina, Sutjeska NP, Jabucnica brook, 02-Jun-2009, leg. Graf	43,3472	18,5999		346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU176-14	fDbg0303L	University of Natural Resources and Life Sciences	Drusus biguttatus	L	Bosnia and Herzegovina, Sutjeska NP, Jabucnica brook, 02-Jun-2009, leg. Graf	43,3472	18,5999		346[0n]	362[0n]	653[5n]	541[0n]	848[0n]
SPDRU178-14	fDbg0401L	University of Natural Resources and Life Sciences	Drusus biguttatus	L	Macedonia, Alilica cave, 04-Jul-2010, leg. Graf	41,552	20,7181		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU179-14	fDbg0402L	University of Natural Resources and Life Sciences	Drusus biguttatus	L	Macedonia, Alilica cave, 04-Jul-2010, leg. Graf	41,552	20,7181		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU180-14	fDbg0501M	University of Natural Resources and Life Sciences	Drusus biguttatus	M	France, 30-Jun-2012, leg. Graf	45,2103	6,96985		346[0n]	325[0n]	658[0n]	541[0n]	848[0n]
SPDRU181-14	fDbg0601M	University of Natural Resources and Life Sciences	Drusus biguttatus	M	Montenegro, Murinska rijeka, 30-May-2009, leg. Graf	42,6438	19,8692		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU333-14	fDpe0103L	University of Natural Resources and Life Sciences	Drusus biguttatus	L	Albania, Korab, populacija 1, 28-Jul-2012, leg. Kucinic, Ibrahimi, Surina, Mihoci				346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU515-14	MN0787	Senckenberg Forschungsinstitut und Naturmuseum	Drusus biguttatus		Austria, Vorarlberg, Klostertal, 29-May-2008, leg. Aistleitner			1370	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU188-14	fDbo0101M	University of Natural Resources and Life Sciences	Drusus bolivari	M	Spain, Ezcaray, 15-Jun-2013, leg. Graf	43,2816	−2,96075	1371	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU189-14	fDbo0102M	University of Natural Resources and Life Sciences	Drusus bolivari	M	Spain, Ezcaray, 15-Jun-2013, leg. Graf	43,2816	−2,96075	1371	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU126-14	Dbot001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus botosaneanui	M	Bulgaria, Bankso, Pirin Mountains, Demyanica river, leg. Kumanski, Pauls	41,75	23,46		n.a.	361[0n]	658[0n]	541[0n]	841[7n]
SPDRU127-14	Dbot002	Senckenberg Forschungsinstitut und Naturmuseum	Drusus botosaneanui	M	Bulgaria, Bankso, Pirin Mountains, Demyanica river, leg. Kumanski, Pauls	41,75	23,46		n.a.	361[0n]	658[0n]	541[0n]	848[0n]
SPDRU128-14	Dbot003	Senckenberg Forschungsinstitut und Naturmuseum	Drusus botosaneanui	M	Bulgaria, Bankso, Pirin Mountains, Demyanica river, leg. Kumanski, Pauls	41,75	23,46		n.a.	361[0n]	658[0n]	541[0n]	846[2n]
SPDRU197-14	fDbt0503L	University of Natural Resources and Life Sciences	Drusus botosaneanui	L	Kosovo, 31-May-2012, leg. Kucinic, Krpac	41,875	20,7	1642	346[0n]	361[0n]	658[0n]	541[0n]	848[0n]
SPDRU198-14	fDbt0504L	University of Natural Resources and Life Sciences	Drusus botosaneanui	L	Kosovo, 31-May-2012, leg. Kucinic, Krpac	41,875	20,7	1642	346[0n]	361[0n]	658[0n]	541[0n]	848[0n]
SPDRU206-14	fDbu0201L	University of Natural Resources and Life Sciences	Drusus botosaneanui	L	Macedonia, CrnKamen, 03-Jul-2010, leg. Graf	41,84	20,6251	1905	346[0n]	361[0n]	658[0n]	541[0n]	844[4n]
SPDRU207-14	fDbu0202L	University of Natural Resources and Life Sciences	Drusus botosaneanui	L	Macedonia, CrnKamen, 03-Jul-2010, leg. Graf	41,84	20,6251	1905	346[0n]	361[0n]	658[0n]	541[0n]	830[18n]
SPDRU436-14	fDsp4001F	University of Natural Resources and Life Sciences	Drusus botosaneanui	F	Bulgaria, Beli Iskar region, Borovets, waterfall, 25-Aug-2013, leg. Keresztes, Kolcsar, Torok	42,269	23,568	1437	346[0n]	361[0n]	658[0n]	541[0n]	n.a.
SPDRU077-14	DBr001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus brunneus	L	Romania, Carpathians, RO, Caliman Mts, Toplita, Lomas river, 29.07.2003, 29-Jul-2003, leg. Pauls & Ujvarosi				465[1n]	498[0n]	658[0n]	658[0n]	n.a.	Pauls et al. 2008
SPDRU079-14	DBr002	Senckenberg Forschungsinstitut und Naturmuseum	Drusus brunneus	L	Romania, Carpathians, RO, Apuseni Mts, Buscat springs, 03.08.2003, 03-Aug-2003, leg. Pauls & Ujvarosi				n.a.	497[1n]	658[0n]	658[0n]	n.a.	Pauls et al. 2008
SPDRU330-14	fDpc0106M	University of Natural Resources and Life Sciences	Drusus cf. plicatus	M	Macedonia, populacija Strezimirska reka spring, 31-May-2012, leg. Kucinic, Krpac	41,7902	20,6348	1279	346[0n]	360[0n]	658[0n]	n.a.	847[1n]
SPDRU080-14	DCh001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus chrysotus	L	Austria, Alps, AT, Soboth, Krumbach tributary, 18.05.2002, 18-May-2002, leg. Graf & Pauls				466[0n]	497[0n]	658[0n]	658[0n]	845[3n]	Pauls et al. 2008
SPDRU081-14	DCh002	Senckenberg Forschungsinstitut und Naturmuseum	Drusus chrysotus	L	Austria, Alps, AT, Soboth, Krumbach tributary, 18.05.2002, 18-May-2002, leg. Graf & Pauls				466[0n]	n.a.	658[0n]	541[0n]	838[10n]	Pauls et al. 2008
SPDRU220-14	fDcy0101M	University of Natural Resources and Life Sciences	Drusus chrysotus	M	Austria, Kaernten, Sankt Oswald, 08-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU221-14	fDcy0102F	University of Natural Resources and Life Sciences	Drusus chrysotus	F	Austria, Kaernten, Sankt Oswald, 08-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU222-14	fDcy0201M	University of Natural Resources and Life Sciences	Drusus chrysotus	M	Austria, Kaertnen, Glitzalm, Bodenbach, 07-Jul-2013, leg. Graf	46,7625	15,0221		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU227-14	fDda0204M	University of Natural Resources and Life Sciences	Drusus dacothracus	M	Albania, Diber district, Deje Mt., stream above the cave spring, 07-Jun-2013, leg. Kucinic, Ibrahimi, Kihoci, Cukusic			1045	346[0n]	360[0n]	658[0n]	541[0n]	n.a.
SPDRU230-14	fDda0208M	University of Natural Resources and Life Sciences	Drusus dacothracus	M	Albania, Diber district, Deje Mt., stream above the cave spring, 07-Jun-2013, leg. Kucinic, Ibrahimi, Mihoci, Cukusic			980	346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU231-14	fDdd0801M	University of Natural Resources and Life Sciences	Drusus discolor	M	Montenegro, Brodavac, right tributary of Perucica, 10-Jul-2013, leg. A. Previsic	42,6859	19,7364	960	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU232-14	fDdd0802F	University of Natural Resources and Life Sciences	Drusus discolor	F	Montenegro, Brodavac, right tributary of Perucica, 10-Jul-2013, leg. A. Previsic	42,6859	19,7364	960	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU364-14	fDrm0102L	University of Natural Resources and Life Sciences	Drusus discolor	L	Montenegro, Murinska, 01-Oct-2009, leg. Graf	42,6438	19,8692		346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU407-14	fDsp2001M	University of Natural Resources and Life Sciences	Drusus discolor	M	Kosovo, Lumbardhi i Pejes, 10-Jun-2009, leg. H. Ibrahimi				346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU235-14	fDdi0101M	University of Natural Resources and Life Sciences	Drusus discophoroides	M	Greece, Central Macedonia, Serres, Vyroneia, Beabies, 02-May-2008, leg. Gordon Ramel	41,3167	23,2333	1150	346[0n]	362[0n]	658[0n]	541[0n]	840[8n]
SPDRU236-14	fDdi0102M	University of Natural Resources and Life Sciences	Drusus discophoroides	M	Greece, Central Macedonia, Serres, Vyroneia, Beabies, 02-May-2008, leg. Gordon Ramel	41,3167	23,2333	1150	346[0n]	362[0n]	658[0n]	490[0n]	836[12n]
SPDRU243-14	fDds0110M	University of Natural Resources and Life Sciences	Drusus discophorus	M	Macedonia, Jablanica Mt, spring of the stream near Labunisko Lake, 29-May-2013, leg. Kucinic, Krpac, Cukusic				346[0n]	360[0n]	658[0n]	495[0n]	848[0n]
SPDRU244-14	fDds0111M	University of Natural Resources and Life Sciences	Drusus discophorus	M	Macedonia, Jablanica Mt, spring of the stream near Labunisko Lake, 29-May-2013, leg. Kucinic, Krpac, Cukusic				346[0n]	360[0n]	658[0n]	n.a.	848[0n]
SPDRU131-14	DDisDis01	Senckenberg Forschungsinstitut und Naturmuseum	Drusus discophorus discophorus	M	Greece, Central Macedonia, Serres, Vyroneia, Beabies, leg. Ramel	41,3167	23,2333	1150	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU132-14	DDisDis02	Senckenberg Forschungsinstitut und Naturmuseum	Drusus discophorus discophorus	M	Greece, Central Macedonia, Serres, Vyroneia, Beabies, leg. Ramel	41,3167	23,2333	1150	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU092-14	DFrll	Senckenberg Forschungsinstitut und Naturmuseum	Drusus franzi	M	Austria, Alps, AT, Koralpe, Weinebene, 27.5.2006, 27-May-2006, leg. Graf				466[0n]	497[0n]	n.a.	n.a.	n.a.	Pauls et al. 2008
SPDRU251-14	fDfl0101F	University of Natural Resources and Life Sciences	Drusus franzressli	F	Greece, Central Greece, Phocis, Vargiani, 07-May-2012, leg. Balint	38,6417	22,4274	908	466[0n]	570[0n]	n.a.	n.a.	n.a.
SPDRU257-14	fDfl0107F	University of Natural Resources and Life Sciences	Drusus franzressli	F	Greece, Vargiani, 07-May-2012, leg. Balint	38,6417	22,4274	908	n.a.	329[0n]	658[0n]	541[0n]	848[0n]
SPDRU268-14	fDil0109M	University of Natural Resources and Life Sciences	Drusus illyricus	M	Albania, Mat district: Kreshtes Mt, Vajkal, 06-Jun-2013, leg. Kucinic, Cukusic	41,5358	20,2279	1830	346[0n]	360[0n]	658[0n]	541[0n]	847[1n]
SPDRU114-14	DImp001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus improvisus	M	Italy, Equi Terme at the Alpi Apuane, 17-Jul-2007, leg. Graf				n.a.	489[0n]	n.a.	n.a.	n.a.	Waringer et al. 2008
SPDRU115-14	DImp003	Senckenberg Forschungsinstitut und Naturmuseum	Drusus improvisus	M	Italy, Abetone Val V. luce, 18-Jul-2007, leg. Graf	44,1432	10,6329	1417	n.a.	460[6n]	n.a.	n.a.	n.a.	Waringer et al. 2008
SPDRU116-14	DImp004	Senckenberg Forschungsinstitut und Naturmuseum	Drusus improvisus	L	Italy, Abetone Val V. luce, 18-Jul-2007, leg. Graf	44,1432	10,6329	1417	432[5n]	467[1n]	n.a.	n.a.	n.a.	Waringer et al. 2008
SPDRU285-14	fDkl0101L	University of Natural Resources and Life Sciences	Drusus klapaleki	L	Bosnia and Herzegovina, stream and spring Toplica, 17-May-2008, leg. Kucinic	43,5943	18,4949	995	n.a.	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU286-14	fDkl0101M	University of Natural Resources and Life Sciences	Drusus klapaleki	M	Bosnia and Herzegovina, Toplica, 06-Jul-2009, leg. Graf	43,8338	18,1046		346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU287-14	fDkl0102F	University of Natural Resources and Life Sciences	Drusus klapaleki	F	Bosnia and Herzegovina, stream Toplica, 03-Jun-2009, leg. Kucinic	43,5943	18,4949	995	346[0n]	362[0n]	658[0n]	541[0n]	774[3n]
SPDRU288-14	fDkl0102M	University of Natural Resources and Life Sciences	Drusus klapaleki	M	Bosnia and Herzegovina, Toplica, 06-Jul-2009, leg. Graf	43,8338	18,1046		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU289-14	fDkl0103F	University of Natural Resources and Life Sciences	Drusus klapaleki	F	Bosnia and Herzegovina, Toplica, 06-Jul-2009, leg. Graf	43,8338	18,1046		346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU290-14	fDkl0104F	University of Natural Resources and Life Sciences	Drusus klapaleki	F	Bosnia and Herzegovina, Toplica, 06-Jul-2009, leg. Graf	43,8338	18,1046		346[0n]	362[0n]	656[2n]	541[0n]	847[1n]
SPDRU291-14	fDkl0104M	University of Natural Resources and Life Sciences	Drusus klapaleki	M	Bosnia and Herzegovina, stream Toplica, 03-Jun-2009, leg. Kucinic	43,5943	18,4949	995	346[0n]	362[0n]	658[0n]	541[0n]	843[5n]
SPDRU294-14	fDkr0101M	University of Natural Resources and Life Sciences	Drusus krusniki	M	Montenegro, Plav, Murino, Murinska rijeka, 30-May-2009, leg. Graf	42,6438	19,8692		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU295-14	fDkr0102M	University of Natural Resources and Life Sciences	Drusus krusniki	M	Montenegro, Plav, Murino, Murinska rijeka, 30-May-2009, leg. Graf	42,6438	19,8692		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU302-14	fDme0101M	University of Natural Resources and Life Sciences	Drusus melanchaetes	M	Italy, Piedmont, Torino, Traversella, Fondo, Alpe Gias del Prete, 12-Jul-2012, leg. Vincon	45,519	7,65074	2150	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU313-14	fDmx0101M	University of Natural Resources and Life Sciences	Drusus mixtus	M	Switzerland, Le Brassus, 24-Aug-2012, leg. Graf	46,5856	6,20848		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU314-14	fDmx0102F	University of Natural Resources and Life Sciences	Drusus mixtus	F	Switzerland, Le Brassus, 24-Aug-2012, leg. Graf	46,5856	6,20848		346[0n]	329[0n]	658[0n]	541[0n]	848[0n]
SPDRU095-14	DMon001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus monticola	L	Austria, Alps, AT, Soboth, Krumbach tributary, 18.05.2002, 18-May-2002, leg. Graf & Pauls				466[0n]	497[0n]	658[0n]	658[0n]	805[5n]	Pauls et al. 2008
SPDRU306-14	fDmt0101M	University of Natural Resources and Life Sciences	Drusus monticola	M	Romania, Maramures, Eastern Carpathians, Baile Borsa, Maramures, Tasla stream, 25-May-2007, leg. Csuzdi & Kovacs	47,6995	24,8297	1011	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU307-14	fDmt0201f	University of Natural Resources and Life Sciences	Drusus monticola	F	Austria, Kaernten, Sankt Oswald, 07-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	826[22n]
SPDRU097-14	DMue003	Senckenberg Forschungsinstitut und Naturmuseum	Drusus muelleri	M	Switzerland, Alps, CH, Alps, Meienreuss tributary, Sustenpass, 18.07.2004, 18-Jul-2004, leg. Lubini, Pauls & Sundermann				466[0n]	496[1n]	658[0n]	658[0n]	848[0n]	Pauls et al. 2008
SPDRU098-14	DMue004	Senckenberg Forschungsinstitut und Naturmuseum	Drusus muelleri	M	Switzerland, Alps, CH, Alps, Furkapass, Springs of Mutt tributary, 17.07.2004, 17-Jul-2004, leg. Lubini, Pauls & Sundermann				466[0n]	481[0n]	658[0n]	658[0n]	848[0n]	Pauls et al. 2008
SPDRU099-14	DMue005	Senckenberg Forschungsinstitut und Naturmuseum	Drusus muelleri	M	Switzerland, Alps, CH, Alps, Grimselsee Zulauf, 18.07.2004, 18-Jul-2004, leg. Lubini, Pauls & Sundermann				466[0n]	497[0n]	658[0n]	658[0n]	792[12n]	Pauls et al. 2008
SPDRU311-14	fDmu0201F	University of Natural Resources and Life Sciences	Drusus muelleri	F	Switzerland, Furkapass, 21-Jul-2006, leg. Graf	46,5885	8,43023	2348	346[0n]	362[0n]	658[0n]	541[0n]	425[11n]
SPDRU312-14	fDmu0202M	University of Natural Resources and Life Sciences	Drusus muelleri	M	Switzerland, Furkapass, 21-Jul-2006, leg. Graf	46,5885	8,43023	2348	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU100-14	DNig001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus nigrescens	M	Switzerland, Alps, CH, Alps, Furkapass, Springs of Mutt tributary, 17.07.2004, 17-Jul-2004, leg. Lubini, Pauls & Sundermann				466[0n]	483[0n]	n.a.	n.a.	n.a.	Pauls et al. 2008
SPDRU101-14	DNig005	Senckenberg Forschungsinstitut und Naturmuseum	Drusus nigrescens	M	Switzerland, Alps, CH, Alps, Furkapass 21.7.2006, 21-Jul-2006, leg. Graf				464[2n]	497[0n]	n.a.	n.a.	n.a.	Pauls et al. 2008
SPDRU319-14	fDni0101m	University of Natural Resources and Life Sciences	Drusus nigrescens	M	Switzerland, Furkapass, 21-Jul-2006, leg. Graf	46,6706	8,64278	2348	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU320-14	fDni0102m	University of Natural Resources and Life Sciences	Drusus nigrescens	M	Switzerland, Furkapass, 21-Jul-2006, leg. Graf	46,6706	8,64278	2348	346[0n]	362[0n]	657[1n]	541[0n]	n.a.
SPDRU321-14	fDni0103m	University of Natural Resources and Life Sciences	Drusus nigrescens	M	Switzerland, Furkapass, 21-Jul-2006, leg. Graf	46,6706	8,64278	2348	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU334-14	fDpe0105M	University of Natural Resources and Life Sciences	Drusus pelasgus	M	Albania, Korab, populacija 1, 28-Jul-2012, leg. Kucinic, Ibrahimi, Surina, Mihoci				346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU335-14	fDpe0106F	University of Natural Resources and Life Sciences	Drusus pelasgus	F	Albania, Korab, populacija 1, 28-Jul-2012, leg. Kucinic, Ibrahimi, Surina, Mihoci				346[0n]	327[0n]	658[0n]	541[0n]	846[2n]
SPDRU336-14	fDpe0108F	University of Natural Resources and Life Sciences	Drusus pelasgus	F	Albania, Korab Mountains, 15-Sep-2013, leg. Kucinic				346[0n]	360[0n]	658[0n]	541[0n]	844[4n]
SPDRU337-14	fDpe0109F	University of Natural Resources and Life Sciences	Drusus pelasgus	F	Albania, Korab Mountains, 15-Sep-2013, leg. Kucinic				346[0n]	360[0n]	658[0n]	n.a.	846[2n]
SPDRU338-14	fDpe0205M	University of Natural Resources and Life Sciences	Drusus pelasgus	M	Albania, Korab, populacija 2, 30-Jul-2012, leg. Ibrahimi			1756	346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU331-14	fDpc0401L	University of Natural Resources and Life Sciences	Drusus plicatus	L	Macedonia, populacija Vevcani spring, 23-Sep-2009, leg. Kucinic	41,2396	20,5844	951	346[0n]	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU332-14	fDpc0405M	University of Natural Resources and Life Sciences	Drusus plicatus	M	Macedonia, populacija Vevcani spring, 23-Sep-2009, leg. Kucinic	41,2396	20,5844	951	n.a.	360[0n]	658[0n]	541[0n]	848[0n]
SPDRU358-14	fDrd0103M	University of Natural Resources and Life Sciences	Drusus radovanovici	M	Bosnia and Herzegovina, Sutjeska, spring Cemerno, 16-May-2008, leg. Kucinic	43,265	18,5928	1109	346[0n]	362[0n]	658[0n]	541[0n]	687[2n]
SPDRU359-14	fDrd0104M	University of Natural Resources and Life Sciences	Drusus radovanovici	M	Bosnia and Herzegovina, Sutjeska, spring Cemerno, 16-May-2008, leg. Kucinic	43,265	18,5928	1109	346[0n]	362[0n]	658[0n]	n.a.	700[2n]
SPDRU431-14	fDsp3501L	University of Natural Resources and Life Sciences	Drusus radovanovici	L	Kosovo, 16-May-2008, leg. Graf, Ibrahimi				346[0n]	362[0n]	658[0n]	541[0n]	844[4n]
SPDRU361-14	fDre0102M	University of Natural Resources and Life Sciences	Drusus rectus	M	France, Languedoc-Roussillon, Pyrenees-Orientales, Vallee d’Err, 11-Jul-2012, leg. Graf	42,3859	2,09584	2225	346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU362-14	fDre0202M	University of Natural Resources and Life Sciences	Drusus rectus	M	France, Languedoc-Roussillon, Pyrenees-Orientales, Canigou, 12-Jul-2012, leg. Graf	42,4864	2,41395	1858	346[0n]	362[0n]	658[0n]	541[0n]	844[4n]
SPDRU104-14	DR001	Senckenberg Forschungsinstitut und Naturmuseum	Drusus romanicus	M	Romania, Carpathians, RO, Apuseni Mts, Buscat springs, 03.08.2003, 03-Aug-2003, leg. Pauls & Ujvarosi				450[4n]	497[0n]	658[0n]	658[0n]	n.a.	Pauls et al. 2008
SPDRU105-14	DR003	Senckenberg Forschungsinstitut und Naturmuseum	Drusus romanicus	M	Romania, Carpathians, RO, Retezat Mts, Rausor Valley, 08.08.2003, 08-Aug-2003, leg. Pauls & Ujvarosi				466[0n]	497[0n]	658[0n]	658[0n]	847[1n]	Pauls et al. 2008
SPDRU106-14	DR007	Senckenberg Forschungsinstitut und Naturmuseum	Drusus romanicus	M	Bulgaria, BG, Pirin Mts, Banderishka River, 18.08.2003, 18-Aug-2003, leg. Beskov, Kumanski & Pauls				n.a.	497[0n]	658[0n]	658[0n]	847[1n]	Pauls et al. 2008
SPDRU134-14	DR002	Senckenberg Forschungsinstitut und Naturmuseum	Drusus romanicus	M	Romania, Cluj, Apuseni, Muntele Baisorii, Buscat springs, 03-Aug-2003, leg. Pauls, Keresztes	46,5375	23,2913		346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU355-14	fDrb0201M	University of Natural Resources and Life Sciences	Drusus romanicus	M	Romania, Capatenii Ungurenii, Southern Carpathiasn, Fagaras Mountains, Poarta Zmailor, 27-Jul-2013, leg. Keresztes, Peter	45,6046	24,6848	2000	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU356-14	fDrb0202M	University of Natural Resources and Life Sciences	Drusus romanicus	M	Romania, Capatenii Ungurenii, Southern Carpathiasn, Fagaras Mountains, Poarta Zmailor, 27-Jul-2013, leg. Keresztes, Peter	45,6046	24,6848	2000	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU357-14	fDrb0203F	University of Natural Resources and Life Sciences	Drusus romanicus	F	Romania, Capatenii Ungurenii, Southern Carpathiasn, Fagaras Mountains, Poarta Zmailor, 27-Jul-2013, leg. Keresztes, Peter	45,6046	24,6848	2000	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU379-14	fDro0201M	University of Natural Resources and Life Sciences	Drusus romanicus	M	Romania, Retezat Mts, Bucara Stream, 150 m below Bucara lake, 10-Aug-2013, leg. Bajka Balogh Borics Borics	45,3571	22,875	2015	346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU380-14	fDro0202F	University of Natural Resources and Life Sciences	Drusus romanicus	F	Romania, Retezat Mts, Bucara Stream, 150 m below Bucara lake, 10-Aug-2013, leg. Bajka Balogh Borics Borics	45,3571	22,875	2015	346[0n]	362[0n]	658[0n]	540[0n]	847[1n]
SPDRU367-14	fDrm0203M	University of Natural Resources and Life Sciences	Drusus romanicus meridionalis	M	Bulgaria, Vihren, Pirin Mountains, Okotovo-Banserishka, marshy spring, 23-Aug-2013, leg. Keresztes, Torok, Kolcsar	41,7389	23,4462	2200	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU368-14	fDrm0204F	University of Natural Resources and Life Sciences	Drusus romanicus meridionalis	F	Bulgaria, Vihren, Pirin Mountains, Okotovo-Banserishka, marshy spring, 23-Aug-2013, leg. Keresztes, Torok, Kolcsar	41,7389	23,4462	2200	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU396-14	fDsm0107M	University of Natural Resources and Life Sciences	Drusus schmidi	M	Croatia, Dubocanka, Papuk, 18-Sep-2012, leg. A. Previsic, M. Milisa, M. Ivkovic	45,4867	17,6787	451	346[0n]	362[0n]	658[0n]	n.a.	848[0n]
SPDRU397-14	fDsm0108F	University of Natural Resources and Life Sciences	Drusus schmidi	F	Croatia, Dubocanka, Papuk, 18-Sep-2012, leg. A. Previsic, M. Milisa, M. Ivkovic	45,4867	17,6787	451	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU398-14	fDsm0401F	University of Natural Resources and Life Sciences	Drusus schmidi	F	Bosnia and Herzegovina, Sturba: bridge, 05-Jul-2008, leg. Kucinic, Delic, Mihoci	43,7779	17,0189	734	346[0n]	362[0n]	n.a.	n.a.	n.a.
SPDRU399-14	fDsm0403M	University of Natural Resources and Life Sciences	Drusus schmidi	M	Bosnia and Herzegovina, Sturba: bridge, 05-Jul-2008, leg. Kucinic, Delic, Mihoci	43,7779	17,0189	734	345[1n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU400-14	fDsm0409L	University of Natural Resources and Life Sciences	Drusus schmidi	L	Bosnia and Herzegovina, Sturba: bridge, 24-Apr-2009, leg. Kucinic, Delic, Mihoci	43,7779	17,0189	734	n.a.	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU401-14	fDsm0410L	University of Natural Resources and Life Sciences	Drusus schmidi	L	Bosnia and Herzegovina, Sturba: bridge, 24-Apr-2009, leg. Kucinic, Delic, Mihoci	43,7779	17,0189	734	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU387-14	fDse0103M	University of Natural Resources and Life Sciences	Drusus septentrionis	M	Bosnia and Herzegovina, spring of the Sturba river, 23-Jun-2012, leg. Kucinic	43,7744	17,0234	750	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU388-14	fDse0104M	University of Natural Resources and Life Sciences	Drusus septentrionis	M	Bosnia and Herzegovina, spring of the Sturba river, 23-Jun-2012, leg. Kucinic	43,7744	17,0234	750	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU389-14	fDse0105M	University of Natural Resources and Life Sciences	Drusus septentrionis	M	Bosnia and Herzegovina, spring of the Sturba river, 23-Jun-2012, leg. Kucinic	43,7744	17,0234	750	345[1n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU390-14	fDse0106F	University of Natural Resources and Life Sciences	Drusus septentrionis	F	Bosnia and Herzegovina, spring of the Sturba river, 23-Jun-2012, leg. Kucinic	43,7744	17,0234	750	346[0n]	362[0n]	658[0n]	n.a.	847[1n]
SPDRU440-14	fDsr0101F	University of Natural Resources and Life Sciences	Drusus serbicus	F	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU441-14	fDsr0102M	University of Natural Resources and Life Sciences	Drusus serbicus	M	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU442-14	fDsr0103M	University of Natural Resources and Life Sciences	Drusus serbicus	M	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU443-14	fDsr0104M	University of Natural Resources and Life Sciences	Drusus serbicus	M	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU444-14	fDsr0105M	University of Natural Resources and Life Sciences	Drusus serbicus	M	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	326[0n]	658[0n]	541[0n]	848[0n]
SPDRU445-14	fDsr0106L	University of Natural Resources and Life Sciences	Drusus serbicus	L	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU446-14	fDsr0107L	University of Natural Resources and Life Sciences	Drusus serbicus	L	Serbia, Golija Mt, spring Ilinac, 22-Jun-2013, leg. Kucinic, Bjelanovic, Zivic			1500	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU447-14	fDsu0101M	University of Natural Resources and Life Sciences	Drusus spelaeus	M	France, Bruyant Engins, 07-Jul-2012, leg. Graf	45,1466	5,17086	1012	464[2n]	522[0n]	n.a.	n.a.	n.a.
SPDRU456-14	fDsu0107F	University of Natural Resources and Life Sciences	Drusus spelaeus	F	France, Bruyant Engins, 07-Jul-2012, leg. Graf	45,1466	5,17086	1012	346[0n]	n.a.	657[1n]	541[0n]	n.a.
SPDRU461-14	fDte0202L	University of Natural Resources and Life Sciences	Drusus tenellus	L	Montenegro, Small stream, tributary of Perucica, 08-Jul-2012, leg. Previsic	42,6944	19,7616	885	346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU462-14	fDte0302L	University of Natural Resources and Life Sciences	Drusus tenellus	L	Montenegro, Murinska rijeka, 05-Jul-2012, leg. Previsic	42,6549	19,8799	878	346[0n]	362[0n]	658[0n]	541[0n]	n.a.
SPDRU463-14	fDtl0101M	University of Natural Resources and Life Sciences	Drusus tenellus	M	Romania, Arges, Southern Carpathians, Fagaras Mts., Capra hut surroundings, small springs around ponds, 30-Aug-2012, leg. Olah & Muranyi	45,58	24,6236	1486	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU464-14	fDtl0102F	University of Natural Resources and Life Sciences	Drusus tenellus	F	Romania, Arges, Southern Carpathians, Fagaras Mts., Capra hut surroundings, small springs around ponds, 30-Aug-2012, leg. Olah & Muranyi	45,58	24,6236	1486	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU108-14	DTri006	Senckenberg Forschungsinstitut und Naturmuseum	Drusus trifidus	M	Austria, Alps, AT, Ennstaler Alps, Gesaeuse, 02.07.2006, 02-Jul-2006, leg. Graf				433[2n]	486[12n]	n.a.	n.a.	n.a.	Pauls et al. 2008
SPDRU109-14	DTri007	Senckenberg Forschungsinstitut und Naturmuseum	Drusus trifidus	M	Austria, Alps, AT, Ennstaler Alps, Gesaeuse, 02.07.2006, 02-Jul-2006, leg. Graf				434[1n]	498[0n]	n.a.	n.a.	n.a.	Pauls et al. 2008
SPDRU479-14	fDvs0202M	University of Natural Resources and Life Sciences	Drusus vespertinus	M	Croatia, Una spring, 18-May-2012, leg. Kucinic, Delic, Mihoci, Vuckovic	44,3994	16,1043	386	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU470-14	fDvi0103L	University of Natural Resources and Life Sciences	Drusus vinconi	L	France, Pyrenees-Atlantiques, St. Pierre, 23-Jul-2012, leg. Graf	42,9555	−0,826958		346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU471-14	fDvi0103M	University of Natural Resources and Life Sciences	Drusus vinconi	M	France, Pyrenees-Atlantiques, St. Pierre, 22-Jul-2012, leg. Graf	42,9555	−0,826958		346[0n]	362[0n]	658[0n]	541[0n]	846[2n]
SPDRU044-14	East003	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx asterix	L	Austria, Karawanken, AT, Karawanken, Babniakgraben, leg. Graf	46,5201	14,2345	683	444[0n]	362[0n]	658[0n]	658[0n]	n.a.	Pauls et al. 2008
SPDRU130-14	DDes002	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx asterix	L	Austria, Styria, Soboth, Krumbach, Krumbach, 20-May-2002, leg. Graf	46,7053	15,0971	841	n.a.	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU003-14	fEda0801M	University of Natural Resources and Life Sciences	Ecclisopteryx dalecarlica	M	Norway, Hedmark, E 9.96412, N, Hedmark (HEN), Folldal, Streitlie, leg. Andersen	62,095	9,964	804	346[0n]	362[0n]	658[0n]	541[0n]	841[7n]	Previsic et al. 2014 Ecclisopteryx
SPDRU007-14	fEda0701F	University of Natural Resources and Life Sciences	Ecclisopteryx dalecarlica	F	Romania, Carpathians, RO, Carlibaba, Tibau Valley, leg. Neu	47,4642	24,8425	980	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]	Previsic et al. 2014 Ecclisopteryx
SPDRU008-14	fEda0901F	University of Natural Resources and Life Sciences	Ecclisopteryx dalecarlica	F	Bulgaria, Rhodope Mts, BG, Mursalica, Tesel, stream close to Tesel S, Devin, leg. Neu	41,666	24,366	870	346[0n]	362[0n]	658[0n]	541[0n]	843[5n]	Previsic et al. 2014 Ecclisopteryx
SPDRU140-14	ED003	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx dalecarlica	M	Austria, Styria, Bruck an der Lafnitz, 20-May-2002, leg. Graf	47,4354	15,9243		346[0n]	362[0n]	658[0n]	n.a.	842[6n]
SPDRU408-14	fDsp2101L	University of Natural Resources and Life Sciences	Ecclisopteryx dalecarlica	L	Serbia, Radovanska River, spring, 18-Sep-2011, leg. Zivic, Bielanovic	43,8967	21,7844	415	346[0n]	362[0n]	658[0n]	541[0n]	841[7n]
SPDRU048-14	Egut009	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx guttulata	M	Austria, AT, Jogland, Lafnitz tributary, leg. Graf & Pauls	47,43	15,48	1170	466[0n]	362[0n]	658[0n]	658[0n]	n.a	Pauls et al. 2008
SPDRU049-14	fEgu0101M	University of Natural Resources and Life Sciences	Ecclisopteryx guttulata	M	Spain, Catalonia, Pyrenees, ES, Pyrenees, Val d’Aran, Salardu, leg. Graf	42,706	0,897	1220	342[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU050-14	fEgu0102F	University of Natural Resources and Life Sciences	Ecclisopteryx guttulata	F	Spain, Catalonia, Pyrenees, ES, Pyrenees, Val d’Aran, Salardu, leg. Graf	42,706	0,897	1220	346[0n]	362[0n]	658[0n]	541[0n]	843[5n]	Previsic et al. 2014 Ecclisopteryx
SPDRU051-14	fEgu0103M	University of Natural Resources and Life Sciences	Ecclisopteryx guttulata	M	Spain, Catalonia, Pyrenees, ES, Pyrenees, Val d’Aran, Salardu, leg. Graf	42,706	0,897	1220	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU052-14	fEgu0104M	University of Natural Resources and Life Sciences	Ecclisopteryx guttulata	M	Spain, Catalonia, Pyrenees, ES, Pyrenees, Val d’Aran, Salardu, leg. Graf	42,706	0,897	1220	328[0n]	362[0n]	658[0n]	541[0n]	839[9n]	Previsic et al. 2014 Ecclisopteryx
SPDRU141-14	EGut01	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx guttulata	M	Germany, Bavaria, Unterfranken, Hassberge, Riedbach, 26-Jun-2004, leg. Graf	50,1	10,4333		346[0n]	362[0n]	n.a.	n.a.	848[0n]
SPDRU036-14	fEda0101M	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	M	Croatia, HR, Cetina River, Crveni most, leg. Previsic	43,9603	16,4295	370	346[0n]	362[0n]	658[0n]	541[0n]	847[1n]	Previsic et al. 2014 Ecclisopteryx
SPDRU037-14	fEda0102M	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	M	Croatia, HR, Cetina River, Crveni most, leg. Previsic	43,9603	16,4295	370	346[0n]	362[0n]	658[0n]	541[0n]	847[1n]	Previsic et al. 2014 Ecclisopteryx
SPDRU038-14	fEda0101F	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	F	Croatia, HR, Cetina River, Crveni most, leg. Previsic	43,9603	16,4295	370	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]	Previsic et al. 2014 Ecclisopteryx
SPDRU039-14	fEda0102F	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	F	Croatia, HR, Cetina River, Glavas spring, leg. Previsic	43,9767	16,4301	386	346[0n]	362[0n]	658[0n]	541[0n]	844[4n]	Previsic et al. 2014 Ecclisopteryx
SPDRU040-14	fDsp3301L	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	L	Croatia, HR, Cetina River, Glavas spring, leg. Kucinic & Previsic	43,9767	16,4301	386	346[0n]	362[0n]	658[0n]	541[0n]	844[4n]	Previsic et al. 2014 Ecclisopteryx
SPDRU042-14	fEda1202L	University of Natural Resources and Life Sciences	Ecclisopteryx ivkae	L	Croatia, HR, Cetina River, Glavas spring, leg. Kucinic & Previsic	43,9767	16,4301	386	346[0n]	362[0n]	658[0n]	541[0n]	834[14n]	Previsic et al. 2014 Ecclisopteryx
SPDRU015-14	fDs30101M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Bosnia and Herzegovina, BH, Sutjeska NP, mouth of the Jabucica River, leg. Ivkovic, Mihaljevic, Milisa & Previsic	43,2902	18,6173	765	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU017-14	fDs30103M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Bosnia and Herzegovina, BH, Sutjeska NP, mouth of the Jabucica River, leg. Ivkovic, Mihaljevic, Milisa & Previsic	43,2902	18,6173	765	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU018-14	fDs30105M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Bosnia and Herzegovina, BH, Sutjeska NP, mouth of the Jabucica River, leg. Ivkovic, Mihaljevic, Milisa & Previsic	43,2902	18,6173	765	346[0n]	329[0n]	658[0n]	541[0n]	842[6n]	Previsic et al. 2014 Ecclisopteryx
SPDRU019-14	fDs30106F	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	F	Bosnia and Herzegovina, BH, Sutjeska NP, mouth of the Jabucica River, leg. Ivkovic, Mihaljevic, Milisa & Previsic	43,2902	18,6173	765	346[0n]	362[0n]	658[0n]	541[0n]	843[5n]	Previsic et al. 2014 Ecclisopteryx
SPDRU020-14	fDs30109F	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	F	Bosnia and Herzegovina, BH, Sutjeska NP, mouth of the Jabucica River, leg. Ivkovic, Mihaljevic, Milisa & Previsic	43,2902	18,6173	765	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU030-14	fEda1001M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Croatia, HR, Velika Belica, bridge at Kuzelj, leg. Popijac	45,4755	14,8051	242	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]	Previsic & Popijac 2010
SPDRU031-14	fEda0201M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Montenegro, MN, Brodavac, right tributary of Perucica, leg. Milisa	42,6859	19,7364	960	346[0n]	362[0n]	658[0n]	541[0n]	842[6n]	Previsic et al. 2014 Ecclisopteryx
SPDRU032-14	fEda0202F	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	F	Montenegro, MN, Brodavac, right tributary of Perucica, leg. Milisa	42,6859	19,7364	960	346[0n]	362[0n]	658[0n]	541[0n]	846[2n]	Previsic et al. 2014 Ecclisopteryx
SPDRU034-14	fEns0101M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Montenegro, MN, Grncar, Gusinje, leg. Graf	42,5659	19,8334	922	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU035-14	fEda0301M	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	M	Montenegro, MN, Perucica, leg. Previsic	42,6947	19,7566	884	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU296-14	fDle0101L	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	L	Albania, Mirdite district, Kurbnesh, Urake River, 08-Jun-2013, leg. Kucinic			800	346[0n]	362[0n]	658[0n]	541[0n]	845[3n]
SPDRU297-14	fDle0102L	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	L	Albania, Mirdite district, Kurbnesh, Urake River, 08-Jun-2013, leg. Kucinic			800	346[0n]	362[0n]	658[0n]	541[0n]	843[5n]
SPDRU298-14	fDle0103L	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	L	Albania, Mirdite district, Kurbnesh, Urake River, 08-Jun-2013, leg. Kucinic			800	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU402-14	fDsp1401F	University of Natural Resources and Life Sciences	Ecclisopteryx keroveci	F	Montenegro, tributary of Perucica, 08-Jul-2012, leg. Previsic	42,6944	19,7616	885	346[0n]	362[0n]	658[0n]	541[0n]	844[4n]
SPDRU056-14	spEM004	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx madida	M	Austria, Alps, AT, Nockberge, St. Oswald stream, leg. Graf	46,8644	13,7877	1570	n.a.	362[0n]	658[0n]	658[0n]	846[2n]	Pauls et al. 2008
SPDRU058-14	spEM006	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx madida	M	Austria, Alps, AT, Nockberge, St. Oswald stream, leg. Graf	46,8644	13,7877	1570	445[0n]	362[0n]	658[0n]	658[0n]	848[0n]	Pauls et al. 2009
SPDRU059-14	fEma0101M	University of Natural Resources and Life Sciences	Ecclisopteryx madida	M	Austria, AT, Bruck/Lafnitz, leg. Graf	47,4396	15,9138	560	329[0n]	362[0n]	658[0n]	541[0n]	845[3n]	Previsic et al. 2014 Ecclisopteryx
SPDRU060-14	fEma0102M	University of Natural Resources and Life Sciences	Ecclisopteryx madida	M	Austria, AT, Bruck/Lafnitz, leg. Graf	47,4396	15,9138	560	346[0n]	362[0n]	658[0n]	541[0n]	848[0n]	Previsic et al. 2014 Ecclisopteryx
SPDRU143-14	EMal001	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx malickyi	M	Italy, Friuli-Venezia Giulia, Udine, Camporosso, 15-Oct-2006, leg. Graf				346[0n]	363[0n]	658[0n]	541[0n]	848[0n]
SPDRU144-14	EMal002	Senckenberg Forschungsinstitut und Naturmuseum	Ecclisopteryx malickyi	M	Italy, Veneto, Verona, Velo Veronese, Campeggio Camposilvano, 17-Jul-2007, leg. Graf				n.a.	363[0n]	658[0n]	541[0n]	839[9n]
SPDRU496-14	fMelaus0101M	University of Natural Resources and Life Sciences	Melampophylax austriacus	M	Austria, spring Schwarze Sulm, 20-Oct-2013, leg. Graf	46,8106	14,9931		n.a.	361[0n]	658[0n]	541[0n]	842[6n]
SPDRU497-14	fMelaus0102F	University of Natural Resources and Life Sciences	Melampophylax austriacus	F	Austria, spring Schwarze Sulm, 20-Oct-2013, leg. Graf	46,8106	14,9931		n.a.	361[0n]	658[0n]	n.a.	843[5n]
SPDRU110-14	MF001	Senckenberg Forschungsinstitut und Naturmuseum	Metanoea flavipennis	M	Switzerland, Alps, CH, Val Muenstair, 20.07.2006, 20-Jul-2006, leg. Graf				445[0n]	497[0n]	658[0n]	658[0n]	848[0n]	Pauls et al. 2008
SPDRU513-14	MF002	Senckenberg Forschungsinstitut und Naturmuseum	Metanoea flavipennis		Switzerland, Graubunden, Inn, Val Muestair, 20-Jul-2006, leg. Graf				346[0n]	362[0n]	658[0n]	541[0n]	847[1n]
SPDRU514-14	MF003	Senckenberg Forschungsinstitut und Naturmuseum	Metanoea flavipennis		Switzerland, Graubunden, Inn, Val Muestair, 20-Jul-2006, leg. Graf				346[0n]	362[0n]	658[0n]	541[0n]	848[0n]
SPDRU111-14	MR001	Senckenberg Forschungsinstitut und Naturmuseum	Metanoea rhaetica	M	Austria, Alps, AT, Nockberge, St.Oswald Stream, 01.07.2003, 01-Jul-2003, leg. Graf				n.a.	470[0n]	658[0n]	658[0n]	842[6n]	Pauls et al. 2008
SPDRU113-14	MR005	Senckenberg Forschungsinstitut und Naturmuseum	Metanoea rhaetica	M	Austria, Alps, AT, Nockberge, St.Oswald Stream, 01.07.2006, 01-Jul-2006, leg. Graf, Pauls & Schmidt-Kloiber				465[1n]	497[0n]	658[0n]	658[0n]	843[5n]	Pauls et al. 2008
SPDRU506-14	fMrh0101M	University of Natural Resources and Life Sciences	Metanoea rhaetica	M	Austria, Kaernten, Sankt Oswald, 07-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	541[0n]	843[5n]
SPDRU507-14	fMrh0102M	University of Natural Resources and Life Sciences	Metanoea rhaetica	M	Austria, Kaernten, Sankt Oswald, 07-Jul-2013, leg. Graf	46,8652	13,7874		330[0n]	362[0n]	658[0n]	541[0n]	843[5n]
SPDRU508-14	fMrh0103F	University of Natural Resources and Life Sciences	Metanoea rhaetica	F	Austria, Kaernten, Sankt Oswald, 07-Jul-2013, leg. Graf	46,8652	13,7874		346[0n]	362[0n]	658[0n]	n.a.	847[1n]

Fig. A2.

Phylogenetic trees used for larval associations of D. balcanicus, D. botosaneanui, D. serbicus, and D. tenellus. Shown are the 50% majority rule consensus tree from the B/MCMC phylogenetic inferences for the a) mitochondrial COI; b) mitochondrial LSU; c) nuclear WG; and d) nuclear CAD datasets. Posterior probabilities above 0.94 are shown at the supported nodes. The specimen level topology is shown for those taxa that were subject to life stage associations (highlighted with grey boxes; specimen codes as in Appendix Table A1). Species-clades were collapsed for the other taxa for clarity.