Epeorus (Caucasiron) Hrivniak, Sroka, Türkmen, Godunko, and Kazanci 2019

(Figs 1 – 18)

Description. Larva (females: n = 12; males: n = 6). Body length of mature larvae: 14 mm (female), 12 – 13 mm (male). Length of cerci approximately 1.2 × body length. Head. Shape trapezoidal; anterior and lateral margin rounded, posterior margin rounded in female, slightly rounded or nearly straight in male (Figs 4, 5). Anterior margin with shallow concavity medially (female). Head dimensions of mature larvae: length 2.9 mm, width 4.3 – 4.4 mm (female); length 2.6 mm, width 3.8 mm (male). Head width / length ratio: 1.41 – 1.51 (female); 1.42 – 1.47 (male). Colouration yellowish-brown with brown markings. Dorso-medial part with brown rectangular or oval pattern with edges often extending to antennae. Dark triangular maculae located near inner edges of compound eyes; pair of rounded maculae located anteriorly to triangular maculae. Pale stripes extending from lateral ocelli to lateral edges of head (Figs 4, 5). Pair of maculae between compound eyes. Compound eyes dark grey to black in female; grey to brown and basally darkened in male. Ocelli dark grey to black, basally pale. Antennae yellowish-brown. Anterior margin densely covered with hair-like setae extending to lateral margins and directed medio-dorsally. Dorsal surface of head covered with fine hair-like setae and sparsely distributed stick-like setae. Sparse longer fine hair-like setae located posteriorly to eyes. Mouthparts. Labrum (Fig. 6) widened anteriorly, with anterior margin slightly rounded or nearly straight (in dorsal view), medial emargination present. Lateral angles rounded (shape of labrum may vary among individual specimens). Dorsal surface (Fig. 6 b, lower half) sparsely covered with setae of different size; 4 – 8 longer bristlelike setae located antero-medially and two antero-laterally (n = 6). Ventral surface (Fig. 6 a, upper half) with longer, shortly plumose bristles situated along lateral to anterior margin (only bases of setae figured), and brush of fine hair-like setae medially (not figured). Group of 6 – 12 setae located near brush of fine hair-like setae. Posterior margin of labrum irregularly concave. Outer incisors of both mandibles (Figs 7, 8) with three apical teeth; outer tooth blunt in both mandibles. Inner incisor of left mandible with three apical teeth, right inner incisor bifurcated; lateral margins of both inner incisors covered with variously situated setae and outgrowths. Prostheca represented by tuft of plumose setae. Maxillae and maxillary palpi without diagnostic characters. Lingua of hypopharynx oblong-shaped, with two latero-apical lobes, covered with dense hair-like setae. Each superlingua with inner apical lobe, covered with hair-like setae that extend laterally; outer margin of superlinguae slightly extended, rounded and pigmented. Dorsal surface of glossae with longitudinal brush of fine setae; ventral surface with scattered short setae; proximal half of outer margins covered by longer setae. Inner margins with long bristles extending medially near apex. Paraglossae densely covered with fine setae on its anterior half. Shape of glossae, paraglossae, shape and setation of labial palpi without diagnostic characters (for shape of glossae and paraglossae in Caucasiron see e. g Braasch 1979: 286, fig. 1 i). Thorax. Yellowish-brown with pale markings dorsally. Pronotum anteriorly narrowed, lateral edges slightly rounded or nearly straight. Posterior margin of metanotum medially bluntly pointed. Dorsal surface covered with fine hair-like setae (as on abdominal terga and head); pro, meso- and metanotal suture with sparse longer hair-like setae. Legs. Yellowish with dark brown markings; all legs with same colouration pattern. Femora with contrasting dark brown markings and hypodermal medial spot (Fig. 9). Shape of medial spot and colour intensity vary between specimens. Tibio-patellar suture and anterior margin of tibiae darkened; tarsi proximally and distally darkened. Coxal projections of forelegs pointed, of middle legs blunt and of hind legs pointed or bluntly pointed. Trochanters with spatulate setae. Tibiae of forelegs longer than femora (1.07 – 1.18 × femur length), tibiae of middle legs nearly equal length as femora (0.97 – 1.01 × femur length), tibiae of hind legs slightly shorter than femora (0.75 – 0.9 × femur length). Tarsi of all legs 0.29 – 0.35 × tibia length. Posterior margin of femora with row of long blade-like setae and sparse row of short, bluntly pointed spines. Dorsal surface of femora covered by flat, short (Fig. 10 a – c), and sporadically elongated (Fig. 10 d) spatulate setae, hair-like setae and sparsely distributed stick-like setae. Anterior margin of femora with short, pointed and / or bluntly pointed spines. Tibiae and tarsi with row of long, shortly plumose setae dorsally and irregularly situated spines of various size ventrally. Tarsal claws with 3 – 4 teeth. Abdomen. Abdominal terga. Yellowish-brown, with contrasting dark brown to blackish pattern, that consists of: i) narrow macula along anterior margin on terga II – IX (contiguous or medio-laterally interrupted by pair of blanks); ii) medial macula on terga II – IV (variable in shape, usually in form of blurred stripe, triangle or anteriorly and posteriorly widened macula); iii) medial longitudinal anteriorly widened stripe stretching between anterior and posterior margins on terga V – VII (VIII); iv) elongated medial macula on terga (VIII) IX (sometimes longitudinal stripe as on terga V – VII); v) lateral maculae on terga I – IX extending postero-medially, sometimes forming contiguous stripe along posterior margin (usually on terga II – V). Tergum X without distinct colouration. Pair of sigilla located antero-laterally to medial maculae usually not expressed (sometimes present in form of short dark stripes). Denticles on posterior margin on terga irregular and pointed; its density vary between terga and instars. Each abdominal tergum with medial longitudinal band of relatively sparse hair-like setae. Surface of terga covered with hair-like setae and sparsely with stick-like setae (as on head). Tergum X without postero-lateral projections. Supra-tergal projections short and blunt. Abdominal sterna. Yellowish, without distinct colouration pattern. Nerve ganglia usually darkened; sigilla not expressed. Sternum IX with V-shaped medial emargination (shape may vary between individuals) and covered by scattered hair-like setae of different size (Figs 17 – 18). Larval protopenis. Penis lobes with deep medio-apical emargination (n = 2). Gills. Dorsally grey to brownish; ventrally yellowish. Gill plates I overlapping beneath thorax. Projection on costal margin of gill plates II – VII well developed (its size vary among specimens and segments) (Fig. 13). Gill plates VII relatively narrow (in natural position from ventral view) (Figs 14 – 16). Each gill plate with filaments, without distinct colouration. Filaments of gills II – VI reaching to 0.37 – 0.58 × length of respective plate, filaments of gill VII to 0.23 – 0.26 × (in late-instar larvae). Cerci. Yellowish-brown, basally darkened. Dorsal surface with hair-like setae. Posterior margin of basal segments with pointed and bluntly pointed denticles. Imago, subimago and egg. unknown. Molecular analyses. Final alignment of COI gene fragment consisted of sequences with 631 base pairs. Of the 193 variable sites, 179 were parsimony informative positions. No insertions and deletions were identified. GMYC analysis. The likelihood of GMYC model was significantly higher than the likelihood of null model expecting uniform coalescent braching rates across entire tree (Table 1). The GMYC estimated 11 species with a confidence interval ranging between 10 – 14. The newly described species E. (C.) turcicus sp. nov. was confirmed and the number of delimited GMYC species well correspond to morphologically defined species (Fig. 19), with monophyly of all species clusters highly supported (PP = 1). Pairwise genetic distances. The ABGD analysis of the COI distance matrix recognized 10 groups. Except for the two species, E. (C.) sinitshenkovae and E. (C.) longimaculatus that were merged into one group, all groups well correspond to morphologically defined species. The newly proposed species E. (C.) turcicus sp. nov. was recognised as a distinct group (Fig. 19). delimitation methods. Abbreviations: SGMYC – Single threshold General Mixed Yule Coalescent model, ABGD – Automatic Barcode Gap Discovery. TABLE 1. Threshold T N GMYC CI L 0 L GMYC LR single 0.02795126 11 10 – 14 335.4751 343.4486 0.0003444511 *** T — threshold genetic distance from branch tips where transitions occured (shown only for single threshold analysis). N GMYC — number of delimited species (sum of clusters and singletons). CI — confidence intervals of putative species. L 0 — likelihood for null model. L GMYC — likelihood for GMYC model. LR — results of likelihood ratio test (*** indicates the test statistically significant). The mean pairwise genetic distances between the newly proposed species E. (C.) turcicus sp. nov. and all other Caucasiron species included in the analysis ranged between 10.45 % (species nigripilosus) and 16.95 % (species alpestris).

Morphological diagnosis and affinities. E. (C.) turcicus sp. nov. belongs to the subgenus Caucasiron based on the presence of projections on the costal rib of gill plates II – VII and the medio-dorsal direction of hair-like setae located on the anterior margin of the head. At the larval stage, the main diagnostic characters of E. (C.) turcicus sp. nov. are: i) presence of rounded hypodermal medial femur spot (Fig. 9, colouration intensity varies among specimens); ii) colouration of abdominal terga (Fig. 1); iii) absence of colouration pattern of abdominal sterna (Fig. 2); vi) shape of gill plate VII, relatively narrow in natural position from ventral view (Figs 14 – 16); v) fine hair-like setae on surface of abdominal terga (Fig. 11); vi) absence of postero-lateral projections on tergum X. The presence of a rounded hypodermal medial femur spot, narrowed gill plates VII and the colouration of the abdominal terga and sterna clearly distinguish E. (C.) turcicus sp. nov. from the species E. (C.) znojkoi (see Braasch 1980: 172, fig. 4 c) and E. (C.) alpestris (see Braasch 1979: 284, figs 1 b, c). However, E. (C.) turcicus sp. nov. is similar to several other Caucasiron species occurring in the Caucasus and adjacent areas. The rounded hypodermal medial femur spot is characteristic also for the species E. (C.) nigripilosus and E. (C.) caucasicus (this may be less visible in the latter species and not emphasized in the description of holotype, see Braasch 1979: 294). The larvae of both species differ from E. (C.) turcicus sp. nov. in the presence of a contrasting hypodermal colouration pattern on the abdominal sterna (see Braasch 1979: 284, figs 3 b, 4 b; Sinitshenkova 1976: 89, fig. 28). Additionally, E. (C.) nigripilosus differs by wider and apically rounded gill plate VII (in natural position from ventral view). The gill plate VII in E. (C.) turcicus sp. nov. exhibits similar shape as in E. (C.) longimaculatus, E. (C.) soldani, E. (C.) bicolliculatus and E. (C.) sinitshenkovae. The first three species named above differ from E. (C.) turcicus sp. nov. by their basally, distinctly enlarged flattened setae on the surface of the abdominal terga (see Hrivniak et al. 2017: 359, figs 23 – 25) in contrast to the new species which bears only fine setae (Fig. 11). E. (C.) longimaculatus additionally differs in: i) displaying characteristic elongated medial femur spot (see Braasch 1980: 172, fig. 11); ii) absence of distinct projection on costal rib of gill plates (see Braasch 1980: 172, fig. 6 b), and iii) absence of medial longitudinal stripe of hair-like setae along abdominal terga. The species E. (C.) sinitshenkovae can be distinguished from E. (C.) turcicus sp. nov. by: i) absence of hypodermal medial femur spot and general colouration of femora (see Braasch & Zimmerman 1979: 106, fig. 10); ii) less developed projection on costal rib of gill plates (see Braasch & Zimmerman 1979: 106, fig. 8 b); iii) only slightly enlarged setae on surface of abdominal terga. Moreover, all four above mentioned species differ from E. (C.) turcicus sp. nov. in the colouration pattern of their abdominal terga (see Braasch 1979: 284, fig. 2 b; Braasch & Zimmerman 1979: 105, fig. 1; Braasch 1980: 172, fig. 2 B; Hrivniak et al. 2017: 356, fig. 10). The species E. (C.) magnus, which lacks the colouration pattern of abdominal sterna as well as E. (C.) turcicus sp. nov., differs in the presence of a distinct posterolateral projection of abdominal segment X.

Distribution, habitat and biology. E. (C.) turcicus sp. nov. occurs in streams and brooks in the Eastern Black Sea Mountains of the Lesser Caucasus (NE Turkey — Eastern part of Black Sea Region of Turkey). Its presence on the northern slopes of the Lesser Caucasus (SW Georgia) and in other parts of the Eastern Black Sea Mountains (especially in Kaçkar Mountain Range) is possible (the materials from these areas were not available for the present study). However, the collection sites of E. (C.) turcicus sp. nov. are in a very restricted area in the Camili (Machakheli) District (near the border between Turkey and Georgia) (Fig. 20). We found no specimens of E. (C.) turcicus sp. nov. in the samples collected throughout the Caucasus region (Russian Federation, Georgia, Armenia, Azerbaijan, Iran) during our previous investigations. These data indicate that the distribution of E. (C.) turcicus sp. nov. might be restricted to the area of the Eastern Black Sea Mountains or distributed westwards, rather than in other parts of the Caucasian region. The larvae of E. (C.) turcicus sp. nov. inhabit the hypocrenal and epirhithral zones of streams and brooks at relatively high altitudes (recorded in the alpine zone, between 2050 and 2388 m a. s. l.). The species was found in small-sized streams and brooks (approx. 1 – 2 m wide in the dry season and 1.5 – 3.5 m wide in the rainy season, at maximum depths of 0.1 – 0.5 m) (Figs 21, 22). Like many other Epeorus species, E. (C.) turcicus sp. nov. is a rheophilic species and prefers a moderate to fast current (current velocity recorded from 0.33 m / s to 2.3 m / s) with rocky and stony substrate. The environment around sampling sites varied, from riverbanks fully covered by riparian vegetation (small or large leaved-plants) with 100 % shade, to exposed with 0 % shade. The larvae of E. (C.) turcicus sp. nov. were found in waters with a temperature range of 7 – 13 ° C. Therefore, the species was classified as a psychrophilic, preferring highly oxygenated water (concentrations of dissolved oxygen ranging from 9.8 mg / l to 12.2 mg / l, and the saturation of dissolved oxygen ranging from 87 % to 102.2 %). Electrical conductivity (spEC) values were 29 – 45 µS / cm and TDS (total dissolved solid) values were 0.026 – 0.042 mg / l. All the values of physico-chemical variables mentioned above correspond to Class I water quality, according to the Legislation of Surface Water Quality Management of Turkey (Ministry of Forestry and Water Affairs, 2015). The pH values of the collecting sites ranged from 2.8 to 7.4. The lowest pH values were observed in localities CAM 8 and CAM 10 (sites 2 and 1, see section Type material) 2.8 and 3.7, respectively. These pH values correspond to Class IV water quality. We assume these values to be the result of the episodic acidification caused by melting snow mass. The melting period of the snow mass in the upper parts of the mountains was still in progress when the specimens were collected. Episodic acidification is a natural and a temporary situation observed in freshwater ecosystems, resulting in low pH values and high PO 4 - P concentrations during the melting period (Davies et al. 1993; Jacks et al. 1986). Episodic acidification is a common phenomenon for the Eastern Part of the Black Sea Region (Kazancı 2009 b). The other aquatic invertebrates frequently found with the larvae of E. (C.) turcicus sp. nov. were Heptageniidae, Baetidae, Leptophlebiidae (Ephemeroptera), Athericidae, Chironomidae, Tipulidae, Simuliidae, Blephariceridae (Diptera), Hydropsychidae, Rhyacophilidae, Brachycentridae, Sericostomatidae (Trichoptera), Perlidae, Perlodidae, Nemouridae, Chloroperlidae, Leuctridae (Plecoptera) and Oligochaeta. The taxa only occasionally found in the collecting sites included Empididae, Dixidae, Pediciidae, Limoniidae, Ceratopogonidae (Diptera), Elmidae, Curculionidae (Coleoptera), Dugesiidae (Plathelminthes), Asellidae (Isopoda), Gammaridae (Amphipoda), and Gordiidae (Nematomorpha). The TR-BMWP, a national-based biotic index (Kazancı et al. 2016), was also applied to the collecting sites. The biotic index scores of the collecting sites ranged from 119 to 160, which correspond to Class I water quality in all sites. This also reflects that all sites have high ecological status and hosts very diversified macroinvertebrate assemblages. Therefore, considering the results of physico-chemical variables and habitat quality evaluations, all collecting sites of E. (C.) turcicus sp. nov. are determined as reference sites.

Type material (bold numbers in parentheses correspond to the localities in Fig. 20). Holotype: female mature larva: TURKEY, Artvin Province, Camili Village, Merata Plateau, unnamed mountain stream, 2190 m a. s. l.; 41 ° 26 ' 30 " N / 42 ° 04 ' 41 " E; code: CAM 10; 26.7. 2016, G. Türkmen & N. Kazancı leg. (1). Paratypes: 3 female (1 mounted on slide) and 1 male larvae, same locality as holotype. 1 male larva: TURKEY, Artvin Province, Camili Village, Merata Plateau, unnamed mountain stream, 2092 m a. s. l.; 41 ° 26 ' 08 " N / 42 ° 04 ' 00 " E; code: CAM 8, 26.7. 2016, (2). 4 female and 1 male larvae: TURKEY, Artvin Province, Camili Village, Lekoban Plateau, unnamed mountain stream, 2386 m a. s. l.; 41 ° 24 ' 05 " N / 42 ° 02 ' 18 "; code: CAM 5, 25.7. 2016, (3). 2 female larvae (1 mounted on slide): TURKEY, Artvin Province, Camili Village, Merata Plateau, unnamed mountain stream, 2050 m a. s. l.; 41 ° 29 ' 18 " N / 42 ° 04 ' 31 " E; code: CAM 7, 26.7. 2016, (4). 2 female larvae (both mounted on slides) and 3 male larvae (2 mounted on slides): TURKEY, Artvin Province, Camili Village, Lekoban Plateau, unnamed mountain stream, 2388 m a. s. l.; 41 ° 24 ' 04 " N / 42 ° 24 ' 04 " E; code: CAM 6, 25.7. 2016, (5). All type material was collected by G. Türkmen and N. Kazancı. Holotype and 12 paratypes are deposited in the collection of the Biology Centre of the Czech Academy of Sciences, Institute of Entomology, České Budějovice, Czech Republic. Four paratypes (2 larvae in EtOH and 2 larvae mounted on slides) are housed in the collection of N. Kazancı and G. Türkmen at Hacettepe University, Department of Biology, Biomonitoring Laboratory, Turkey.