Diversity and distribution of Epeorus (Caucasiron) (Ephemeroptera, Heptageniidae) in Iran, with descriptions of three new species

Abstract Combining morphological and molecular data in an integrative approach, three new mayfly species of Epeorus (Caucasiron) are described. These include Epeorus (Caucasiron) alborzicus Hrivniak & Sroka, sp. nov. and Epeorus (Caucasiron) shargi Hrivniak & Sroka, sp. nov. from northern Iran, and Epeorus (Caucasiron) zagrosicus Hrivniak & Sroka, sp. nov. from central Iran. They are unambiguously delimited using both distance-based and likelihood-based approaches in the analyses of barcode COI sequences. Each new species is compared with other species of the subgenus and morphological diagnostic characters are provided. Based on extensive sampling of streams throughout the country, the distribution and habitat preferences of all Caucasiron species in Iran are assessed. Altogether, there are now six species recorded, among them also E. (C.) nigripilosus Sinitshenkova, 1976 is reported for the first time in Iran. Five species are distributed in the Alborz Mts. in northern Iran, one species was found in the Zagros Mts. in central Iran.


Introduction
The genus Epeorus Eaton, 1881, subgenus Caucasiron Kluge, 1997 represents a group of mountainous mayflies distributed in Palaearctic region. Kluge (1997) defined Caucasiron based on a unique larval apomorphy, a projection on the costal margin of gill plates II-VII. Other larval diagnostic characters include the presence of medio-dorsally directed hair-like setae along anterior margin of head and gill plates forming a so-called "adhesive disc", consisting of enlarged gill plate I and overlapping gill plates II-VII. Gill plate VII has a longitudinal fold allowing to bend the plate ventrally under the abdominal segments. The systematic position of Caucasiron within Epeorus-related taxa was unclear for a long time (e.g., Braasch 2006, Kluge 2015. The recent study by Hrivniak et al. (2020) confirmed its monophyly and subgeneric position within Epeorus s.l. Moreover, the study pointed out its close phylogenetic relationship with the subgenus Iron Eaton, 1883 distributed in Central Asia and Nearctic realm.
Caucasiron occurs in the Eastern Mediterranean (Samos and Cyprus Island), Anatolia, Caucasus, and central and western Asia (Hrivniak et al. 2019(Hrivniak et al. , 2020. Their larvae inhabit riffle sections of montane and submontane streams with coarse bed substrate (Nguyen et al. 2004;Bauernfeind and Soldán 2012). At present there are 17 species described (Hrivniak et al. 2020), but apparently several Central Asian taxa described in the genus Iron rather belong to Caucasiron (Chen et al. 2010;Hrivniak et al. 2017). In any case, a taxonomic revision of these species is needed to clarify their systematic position.
Individual Caucasiron species exhibit different distribution patterns within the Caucasus region varying from an endemic distribution in the Greater Caucasus to a wide distribution covering distant regions in the Pontic Mountains, Lesser Caucasus, Zagros, and Alborz Mountains (Hrivniak et al. 2020). The highest species richness and endemism of Caucasiron is concentrated in the western and central part of the Greater Caucasus, the most prominent mountain range in the Caucasus region. However, the individual mountain ranges of the Caucasus have been studied to a different extent until now. Especially the Alborz Mountains, a southeast part of the Caucasus biodiversity hotspot, and the Zagros Mountains, a dominant part of the Irano-Anatolian biodiversity hotspot, have been left unattended without detailed investigation (Bojková et al. 2018). The only Caucasiron species described and known exclusively from Iran, E. (C.) iranicus (Braasch & Soldán, 1979), is distributed in the Alborz and most likely represents an endemic species of this mountain range. However, given the size and diversity of the Iranian territory and stream habitats, the diversity and endemism within Caucasiron can be expected to be much higher in Iran. Summarizing recent knowledge on the diversity and distribution of Iranian mayflies, Bojková et al. (2018) reported two species of Caucasiron from Iran, namely E. (C.) iranicus and E. (C.) znojkoi.
Based on morphology and molecular analyses, we describe in this integrative study, two new species of Caucasiron from the Alborz Mountains and one new species from the Zagros Mountains. We provide morphological diagnostic characters of the three new species and differential diagnoses between all species known from the Caucasus and adjacent areas, plus an analysis of respective COI sequences. Following recent studies on Iranian mayflies by Bojková et al. (2018), Sroka et al. (2019), , we also sum up all records of Caucasiron species from our recent Iranian field trips to further contribute to a systematic research of mayflies in Iran.
The main objectives of this study are to (i) describe the morphology of three new Caucasiron species and provide their differential diagnoses, (ii) apply the molecular species delimitation methods using analytical tools for the single-locus COI dataset, (iii) provide basic information about habitat requirements of the new species, and (iv) summarize the distribution of all Caucasiron species recently known from Iran.

Materials and methods
The material used for this study was collected by J. Bojková, T. Soldán, J. Imanpour Namin, and S. Bagheri in April and May 2016, and A. Staniczek, M. Pallmann, R. J. Godunko, and F. Nejat in April and May 2017. All specimens were preserved in 75-96% EtOH and are deposited in the collections of the Biology Centre of the Czech Academy of Sciences, Institute of Entomology, České Budějovice, Czech Republic (IECA), State Museum of Natural History, Stuttgart, Germany (SMNS) and Natural History Museum and Genetic Resources, Department of Environment, Tehran, Iran (MMTT_DOE). Material of other Caucasiron species used for the morphological and molecular comparisons was obtained from the collection of IECA. This publica-tion and the nomenclatural acts therein are registered with ZooBank under the LSID urn:lsid:zoobank.org:pub:3297FBE4-111C-4849-9533-225A53F7DB3C.

Morphological examination
Parts of specimens were mounted on microscopic slides using HydroMatrix (Micro-Tech Lab, Graz, Austria) mounting medium. In order to remove the muscle tissue for an investigation of the cuticular structures, specimens were left overnight in a 10% solution of NaOH prior to slide mounting. Drawings were made using a stereomicroscope Olympus SZX7 and a microscope Olympus BX41, both equipped with a drawing tube. Photographs were obtained using Leica DFC450 camera fitted with macroscope Leica Z16 APO and folded in Helicon Focus version 5.3 X64. All photographs were subsequently enhanced with Adobe Photoshop CS5. Diagnostic characters for the description of larva were chosen according to Braasch and Soldán (1979) and Braasch (2006). The terminology was used mostly according to Kluge and Novikova (2011) and Kluge (2004Kluge ( , 2015.

DNA extraction, PCR, sequencing and alignment
Total genomic DNA of the species (4-8 specimens/species) was extracted from legs using the DEP-25 DNA Extraction Kit (TopBio s.r.o., Prague, Czech Republic) according to the manufacturer's protocol. Mitochondrial cytochrome oxidase subunit I (COI) was sequenced according to Hrivniak et al. (2017). COI sequences of other Caucasiron species used for comparisons were obtained from Hrivniak et al. (2017) (GenBank accession nos KY865691-KY865725) and Hrivniak et al. (2019) (GenBank accession nos KY865691-KY865725). Three specimens of E. (C.) iranicus were additionally sequenced. The PCR amplification of COI and reaction volumes was carried out as described in Hrivniak et al. (2017). Sequences were assembled in Geneious 7.0.6 (http:// www.geneious.com) and aligned in the same software using the Mafft 7.017 (Katoh et al. 2002) plugin with default settings. Newly obtained sequences are deposited in Gen-Bank with accession numbers (GB) MN856180-MN856198.

Molecular species delimitation
Species were delimited using the single locus (COI) coalescence based General Mixed Yule Coalescent model (GMYC, Pons et al. 2006;Fusijawa and Barraclough 2013). We used the single-threshold GMYC model as it has been found to outperform the multithreshold (Fusijawa and Barraclough 2013) and was found to be highly suitable for species delimitation within Caucasiron (Hrivniak et al. 2019). The GMYC model identifies independent evolutionary clusters by detecting a threshold value at the transition from interspecific to intraspecific branching patterns (Bryson et al. 2013). A maximum likelihood approach is used to optimize the shift in branching patterns. A likelihood ratio test assesses if the mixed model fits the data significantly better than a null model that assumes a single coalescent process for the entire tree (Pons et al. 2006;Monaghan et al. 2009). Analyses were performed using the SPLITS package for R (http://r-forge.rproject.org/projects/splits). An ultrametric COI gene tree was reconstructed under relaxed molecular clock (uncorrelated lognormal distribution) using BEAST 2 (Bouckaert et al., 2014) on CIPRES Science Gateway 3.3 (Miller et al. 2010). An input file was generated in BEAUti 2. The substitution model was selected by bModelTest (Bouckaert and Drummond 2017) implemented in BEAUti 2 using a model averaging approach. A coalescent constant population tree prior was preferred, because the GMYC null model constitutes a single coalescent cluster (Monaghan et al. 2009;Zaldívar-Riverón et al. 2010;Vuataz et al. 2011). Other settings were default. Two analyses of MCMC chains were run for 50 million generations sampled every 5000 generations. Convergence and effective sample size (ESS > 200) were verified using Tracer 1.6. The first 10% of trees (1000) from each run were discarded as burn-in. The files from both independent runs were combined using LogCombiner 1.8.4. The maximum clade credibility tree was constructed from 18000 trees using TreeAnnotator 1.8.4 with default settings.
Inter-and intra-specific K2P pairwise genetic distances were calculated in MEGA 7 (Kumar et al. 2016). The distance matrix was analysed using Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012) (online version: http://wwwabi.snv.jussieu. fr/public/abgd/) with default settings. The method identifies so-called barcode gap that corresponds to threshold between intra-and inter-specific genetic distances and splits sequences to groups corresponding to putative species accordingly.

Results and discussion
Taxonomy All of the species described below are attributed to the subgenus Caucasiron within the genus Epeorus based on the presence of projections on the costal rib of gill plates II-VII, and the presence of medio-dorsally directed hair-like setae located on the anterior margin of the head (see Kluge 2015 for a revision of the subgenus). The holotype and 50 paratypes are deposited in SMNS, 50 paratypes (including DNA extracted specimens) are deposited in IECA and 29 paratypes in MMTT_DOE.

Epeorus (Caucasiron) alborzicus
Other material examined. 8 larvae: same data as holotype, SMNS_EPH_010056; young instars or damaged specimens. Etymology. The species name refers to the type locality and distribution of the species in the Alborz mountain range.
Localities and habitat preferences of larvae. Larvae inhabit small streams (2-8 m width, 20-50 cm depth) at high altitudes (six of eleven localities at approx. 2000 m a.s.l.) in the central Alborz (Fig. 9). One larva was found in the eastern Alborz (Fig. 9). Larvae were found only in cold and clear streams where they dwelled on large stones in riffles with very fast flow. All localities were situated in deep valleys with rivers draining high mountains. They were mostly treeless, only sometimes with sparse solitary shrubs and trees at the banks (Fig. 10A, B). Streams had a very coarse bed substrate with prevailing boulders and stones and a low share of fine sediments, and turbulent to strongly turbulent flow. They were characteristic of high fluctuation of discharge, with sudden peaks of discharge after spates on the mountains (Fig. 10A).
Head. Shape trapezoidal; anterior and lateral margin rounded, posterior margin rounded in female, slightly rounded or nearly straight in male (Fig. 1D, E). Anterior margin with shallow concavity medially. Head dimensions of mature larvae: length 2.8-3.1 mm, width 4.0-4.6 mm (female); length 2.2-2.7 mm, width 3.2-3.7 mm (male). Head width/length ratio: 1.4-1.5 (both male and female). Dorso-medial part with pair of stripes. Pair of maculae located between ocelli (sometimes fused into single macula). Rounded maculae ventrolateral of lateral ocelli and blurred maculae near in- ner edges of compound eyes. Pale stripes extending horizontally from lateral ocelli to lateral edges of head. Pair of elongated, curved maculae located along coronal suture. Compound eyes grey to black in female, brownish or greyish and basally black in male mature larva. Ocelli blackish, basally paler. Antennae yellowish brown, scapus and pedicellus darkened. Anterior margin of head 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 and fine hair-like setae located posteriorly to eyes.
Mouthparts. Labrum ( Fig. 2A) widened anteriorly, with anterior margin slightly rounded or nearly straight (in dorsal view). Lateral angles rounded (shape of labrum may vary among individual specimens). Dorsal surface ( Fig. 2A, right half ) sparsely covered with setae of different size; 4-6 longer bristle-like setae located antero-medially and two bristles antero-laterally. Epipharynx with longer, slightly plumose bristles situated along lateral to anterior margin ( Fig. 2A, left half, range of setation figured as large black dots), and cluster of fine, hair-like setae medially (not figured). Posterior margin of labrum irregularly concave; group of 6-17 setae of various size located on ventral surface close to posterior margin. Outer incisors of both mandibles ( Fig. 2B, C) with three apical teeth; outer tooth blunt in both mandibles. Inner incisor of left mandible with three apical teeth, right inner incisor bifurcated.
Legs. Colour pattern of femora as in Fig. 1F. Femora without medial hypodermal spot. Patella-tibial suture darkened; tarsi proximally and distally darkened. Coxal projections of fore-and hind legs pointed or bluntly pointed; in middle legs blunt. Trochanteres with spatulate setae as on dorsal surface of femora (Fig. 2D). Tibiae of forelegs 1.20-1.37× femur length, tibiae of middle legs 1.0-1.2× femur length, and tibiae of hind legs 0.92-1.08× femur length. Tarsi of all legs 0.26-0.34× tibia length. Dorsal surface of femora covered by short and sporadically elongated spatulate setae ( Fig. 2D), hair-like setae, and sparsely distributed stick-like setae. Anterior margin of femora with short, pointed or bluntly pointed spine-like setae; posterior margin with row of long blade-like setae and sparse row of bluntly pointed, spine-like setae. Dorsal margin of tibiae and tarsi with row of long setae; ventral margin of both with irregular row of spine-like setae accumulated distally. Tarsal claws with 2-3 denticles.
Abdominal terga. Colour pattern of abdominal terga (Fig. 1A, H-J) consists of transversal stripe along anterior margin of terga I-IX (X), medially extending to single blurred macula or pair of rounded maculae on terga II-IV and short triangular or nearly rectangular macula on terga V-IX. Terga VIII and IX (X) medially darkened. Pattern of abdominal terga sometimes poorly expressed, only with medially thickened transversal stripe along anterior margin.
The presence of postero-lateral projections on the abdominal tergum X is characteristic for two species distributed in the Caucasus, E. The holotype (SMNS_EPH_010057) and 50 paratypes (SMNS_EPH_010057) are deposited in SMNS, 50 paratypes (including DNA extracted specimens) are deposited in IECA, and 17 paratypes in MMTT_DOE.
Other material examined (not paratypes): 3 larvae: same data as holotype; young instars or damaged specimens.
Etymology. The species name derives from shargi ‫,)شرقی(‬ which means eastern in Farsi. It refers to the distributional range of the species in the eastern part of the Alborz mountain range. Localities and habitat preferences of larvae. Larvae were found in three clear streams at middle altitude (740-1450 m a.s.l.) in the eastern Alborz (Fig. 9). Habitat conditions of these streams differed from each other. Larvae were abundant in a cold, alkaline brook (water conductivity 1320 μS/cm) with patches of precipitated calcium crusts on the bed and in the non-alkaline water (with water conductivity reaching the values of clear montane streams in the region, 433 μS/cm) of the type locality. Both localities were characterised by stony bed sediment with leaf litter debris and fine gravel along the banks, and by fast, turbulent flow (Fig. 10C, D). Lower abundance of larvae was found in a river with uniform coarse substrate flowing in a wide gravel river channel. All streams were surrounded by deciduous forests (Fig. 10C, D). The species was not found in urban and agricultural areas in this region where many localities were investigated.
Head. Shape trapezoidal; anterior and lateral margin rounded, posterior margin rounded in female, slightly rounded in male (Fig. 3D, E). Anterior margin with shal- low concavity medially. Head dimensions of mature larvae: length 3.0-3.2 mm, width 4.1-4.4 mm (female); length 2.70-2.95 mm, width 3.5-4.0 mm (male). Head width/ length ratio: 1.33-1.40 (both male and female). Dorso-medial part with brown, rectangular or oval smudge, sometimes reduced to pair of stripes. Pair of maculae located between ocelli (sometimes fused into single macula). Rounded maculae lateroventral of lateral ocelli and blurred maculae near inner edges of compound eyes. Pair of pale stripes extending from lateral ocelli to lateral edges of head. Pair of maculae located along coronal suture. Compound eyes dark grey to black in female, brownish and basally blackish in male mature larva. Ocelli dark grey to black, basally paler. Antennae yellowish-brown, scapus and pedicellus darkened. Anterior margin of head 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 sticklike setae. Sparse longer fine hair-like setae located posteriorly to eyes.
Mouthparts. Labrum (Fig. 4A) widened anteriorly, with anterior margin slightly rounded or nearly straight (in dorsal view). Lateral angles rounded (shape of labrum may vary among individual specimens). Dorsal surface (Fig. 4A, right half ) sparsely covered with setae of different size; 4-6 longer bristle-like setae located antero-medially and two antero-laterally. Epipharynx with longer, shortly plumose bristles situated along lateral to anterior margin (Fig. 4A, left half ), range of setation figured as large black dots), and brush of fine hair-like setae medially (not figured). Posterior margin of labrum irregularly concave; with group of 5-10 setae of various size located on ventral surface close to posterior margin. Outer incisors of both mandibles (Fig. 4B, C) with three apical teeth; outer tooth blunt in both mandibles. Inner incisor of left mandible with three apical teeth, right inner incisor bifurcated (inner side of right tooth usually with small denticle).
Legs. Colour pattern of femora as in Fig. 3F. Femora with rounded or slightly elongated medial hypodermal femur spot. Patella-tibial suture darkened; tarsi proximally and distally darkened. Coxal projections of fore-and hind legs pointed or bluntly pointed; of middle legs blunt. Trochanteres with spatulate setae as on dorsal surface of femora (Fig. 4D). Tibiae of forelegs 1.23-1.28× femur length, tibiae of middle legs 1.03-1.50× femur length, and tibiae of hind legs 0.87-1.06× femur length. Tarsi of all legs 0.28-0.32× tibia length. Dorsal surface of femora covered by short, sporadically elongated spatulate setae (Fig. 4D), hair-like setae, and sparsely distributed stick-like setae. Anterior margin of femora with short, pointed and/or bluntly pointed spine-like setae; posterior margin with row of long blade-like setae and sparse row of bluntly pointed spine-like setae. Dorsal margin of tibiae and tarsi with row of long setae; ventral margin of both with irregular row of spine-like setae accumulated distally. Tarsal claws with 2-3 denticles.
Abdominal terga. Colour pattern of abdominal terga (Fig. 3A, H-K) consists of transversal stripe along anterior margin of terga I-IX (X) medially extending to i) triangular or blurred macula on terga (II) III-IV; ii) triangular or T-shaped macula on terga V-IX, reaching to half or stretching to posterior margin of corresponding tergum (medial macula of terga VIII and IX often widened). Transversal stripe along anterior margin of terga laterally extends to pair of short maculae. Medial maculae often surrounded by pale background. Tergum X without distinct maculation. Pair of sigilla sometimes coloured and forming pair of short stripes adjacent laterally to medial macula. Lateral margins of abdomen with oblique maculae on terga I-IX. Denticles along posterior margin on terga of various size, irregular and pointed (Fig. 4E). Surface of terga covered with hair-like setae and sparsely with stick-like setae. Supra-tergalial projections short and blunt. Tergum X without distinct postero-lateral projections (Fig. 4H). Longitudinal row of hair-like setae along abdominal terga present medially.
Gills. Dorsal surface of gill plate I yellowish, of gill plates II-VII greyish on anterior half and brownish to reddish on posterior half. Ventral margin of all gill plates yellowish. Projection of gill plate III well developed (Fig. 4G). Gill plate VII relatively wide (in natural position of ventral view, Figs 3G, 4L). Gill filaments reaching to 0.41-0.50× length of respective plate, filaments of gill VII to 0.24-0.28× (in lateinstar larvae).
Epeorus (C.) magnus can be reliably distinguished by the presence of distinct postero-lateral projections on abdominal tergum X and characteristic setation of labrum (numerous thickened bristle-like setae, Hrivniak et al. in prep.).
The holotype and 100 paratypes are deposited in SMNS, 50 paratypes (including DNA extracted specimens) are deposited in IECA and 32 paratypes in MMTT_DOE.
Etymology. The species name refers to its known records in the Zagros mountain range.
Localities and habitat preferences of larvae. Larvae were found in five streams of different size at high altitude, above 1700 m a.s.l. Three streams were strongly turbulent rivers with very coarse bed substrate flowing in high-mountain valleys (Fig. 10E). Larvae were found also in a shallow, slow-flowing brook with finer, gravel substrate flowing in the forest (locality near Yasuj fall, Fig. 10F), and in a small stream with moderate, slightly turbulent flow and stony bed substrate with fine gravel, silt, and macrophytes (Chehme-Dimeh River). The species was not found in streams that were polluted or seasonally drying out.
Head. Shape trapezoidal; anterior and lateral margin rounded, posterior margin slightly rounded or nearly straight (Fig. 5D, E). Anterior margin with shallow concavity medially.
Dorso-medial part with indistinct brown rectangular or oval macula, sometimes reduced to pair of stripes. Rounded maculae under lateral ocelli and blurred or triangular maculae near inner edges of compound eyes. Pair of pale stripes extending from lateral ocelli to lateral edges of head. Pair of maculae located along coronal suture. Compound eyes dark grey to black in female, brownish and basally blackish in male mature larva. Ocelli dark grey to black, basally paler. Antennae yellowish-brown, scapus and pedicellus darkened. Anterior margin densely covered with hair-like setae extending to lateral margins and directed medio-dorsally. Dorsal surface 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. 6A) widened anteriorly, with anterior margin slightly rounded or nearly straight (in dorsal view). Lateral angles rounded (shape of labrum may vary among individual specimens). Dorsal surface (Fig. 6A, right half ) sparsely covered with setae of different size; four longer, bristle-like setae located antero-medially and two antero-laterally. Epipharynx with longer, shortly plumose bristles situated along lateral to anterior margin (Fig. 6A, left half; range of setation figured as large black dots), and brush of fine hair-like setae medially (not figured). Posterior margin of labrum irregularly concave; with group of 6-10 setae of various size located on ventral surface close to posterior margin. Outer incisors of both mandibles (Fig. 6B, C) with three apical teeth; outer tooth blunt in both mandibles. Inner incisor of left mandible with three apical teeth, right inner incisor bifurcated.
Thorax. Pronotum anteriorly narrowed, lateral edges nearly straight. Metanotum with slight postero-medial projection. Dorsal surface covered with fine hair-like setae (as on abdominal terga and head); sparse longer hair-like setae along pro, meso-and metanotal suture.
Legs. Colour pattern of femora as in Fig. 5F. Femora with rounded medial hypodermal femur spot. Patella-tibial suture darkened; tarsi proximally and distally darkened. Coxal projections of fore-and hind legs pointed or bluntly pointed; of middle legs blunt. Trochanteres with spatulate setae as on dorsal surface of femora (Fig. 6D). Tibiae of forelegs 1.20-1.31× femur length, tibiae of middle legs 1.06-1.14× femur length, and tibiae of hind legs 0.90-1.04× femur length. Tarsi of all legs 0.25-0.34× tibia length. Dorsal surface of femora covered by elongated and sporadically short rounded spatulate setae (Fig. 6D); hair-like setae and sparsely distributed stick-like setae. Anterior margin of femora with short, pointed and/or bluntly pointed spinelike setae; posterior margin with row of long blade-like setae and sparse row of bluntly pointed spine-like setae. Dorsal margin of tibiae and tarsi with row of long setae; ventral margin of both with irregular row of spine-like setae accumulated distally. Tarsal claws with two or three denticles.
Abdominal terga. Colour pattern of abdominal terga includes transversal stripe along anterior margin of terga I-IX (X) medially extending to triangular, short rectan- gular or stripe-like medial macula on terga (III) IV-IX (transversal stripe sometimes not distinctly extended, for variability see Fig. 5A, H-J). Pair of sigilla sometimes coloured, in form of short stripes or spots located antero-laterally to medial macula. Tergum X without distinct maculation. Lateral margins of abdomen with oblique maculae on terga I-IX extending to dorso-posterior margin. Denticles along posterior margin on terga of various size, irregular and pointed, sometimes curved (Fig. 6E). Surface of terga covered with hair-like setae and sparsely with stick-like setae. Supra-tergalial projections short and blunt. Tergum X with more or less developed postero-lateral projections (Fig. 6H, I, arrows). Longitudinal row of hair-like setae medially along abdominal terga present.
Abdominal sterna. Yellowish, with distinct colouration pattern consisting of anteriorly widened pair of stripes (medio-anterior sigilla) on terga II-VIII (Fig. 5B, G, K). Sometimes only oblique stripes are present, without anterior widening (especially on sterna VI-VIII). Nerve ganglia occasionally darkened. Intensity of colouration varies among individuals. Sternum IX with V-shaped medial emargination; surface covered by irregularly distributed short hair-like setae, and medially accumulated longer hairlike setae (Fig. 6J, K).
Gills. Dorsal surface of gill plate I yellowish; of gill plates II-VII greyish on anterior half and brownish to reddish on posterior half. Ventral margin of all gill plates yellowish. Projection of gill plate III well developed (Fig. 6G). Shape of gill plate VII (in natural position from ventral view) varies from narrow to relatively wide (Figs 5G, 6M-O). Gill filaments reaching to 0.4-0.5× length of respective plate, filaments of gill VII to 0.24-0.30× (in late-instar larvae).
Affinities. Based on the colour pattern of abdominal sterna, E. (C.) zagrosicus sp. nov. is most similar to E. (C.) caucasicus and E. (C.) iranicus. Both latter species possess pigmented medio-anterior sigilla forming a pair of oblique stripes on abdominal sterna II-VIII (e.g., Braasch 1979: 284, fig. 3b), and a medial hypodermal femur spot. However, E. (C.) zagrosicus sp. nov. differs by the distinct widening at the anterior margin of medio-anterior sigilla of abdominal sterna. If the sternal colour pattern is not fully developed (sporadically only stripes are present on all or several sterna), E. (C.) zagrosicus sp. nov. is distinguishable by the colour pattern of abdominal terga (Fig.  5A, H-J), which is different in E. (C.) caucasicus (Braasch 1979: 284, fig. 3a) and E. (C.) iranicus (Braasch and Soldán 1979: 264, fig . 12). In E. (C.) zagrosicus sp. nov., the postero-lateral projections on the tergum X are well-developed, whereas they are not significantly pronounced in either of the two species mentioned above (only small projections may be sporadically present).  (Braasch 1980: 172, fig. 4b, c).

Results from molecular species delimitation
The GMYC model provided significantly better fit to COI gene tree than the null model expecting uniform coalescent branching rates across entire tree (likelihood ratio test = 3.671927e-06***). The GMYC estimated 15 species (CI=13-19) consisting of 14 ML clusters and one singleton (CI = 12-16). All three newly described species were confirmed, and the overall number of delimited GMYC species corresponded well to morphologically defined species within Caucasiron (Fig. 7C). Monophyly of all species clusters were highly supported (PP = 1).
The ABGD analysis of the COI distance matrix recognized 15 stable groups within initial partition. All groups corresponded well to morphologically defined species and were congruent with GMYC analysis. All three newly proposed species were recognized as distinct groups (Fig. 7C). The mean pairwise genetic K2P distances between all Caucasiron species, including newly described, ranged between 6.71% (E.

Distribution of Caucasiron in Iran
Specimens of the genus Epeorus were found in 68 localities of all 254 localities investigated by us in 2016-2018 (Fig. 8) and in seven additional localities investigated by others (Braasch and Soldán 1979;Mousavi and Hakobyan 2017) (Table 1). Their occurrence was limited to streams with good water quality at altitudes between -4 and 2440 m a.s.l. (Table 1). They were neither found in polluted streams of agricultural and urban areas, nor in seasonally drying streams. Most of the species and records were found in the Alborz in northern Iran (Fig. 9). These mountains host five species of the subgenus Caucasiron and one species of the subgenus Epeorus (E. zaitzevi). Except for the newly described species, E. (C.) nigripilosus found in five localities in the Alborz is new for Iran (its genetic data from the Alborz were used in phylogeographical analyses in Hrivniak et al. 2020). It is a widely distributed species ranging from Cyprus and Turkey to Georgia, Russia and Iraq (Sinitshenkova, 1976;Braasch 1979;Al-Zubaidi et al. 1987;Salur et al. 2016;Gabelashvili et al. 2018;Hrivniak et al. 2020). The identification of E. (C.) nigripilosus was confirmed by both morphological characters and molecular delimitation. The specimen from the Alborz (coded as NI10I in Fig. 7C) clustered within the clade containing conspecific individuals from Russia and Turkey in the analysis of COI. It differed from these conspecifics in 2.8-3.6 % of K2P distance.
The most common Caucasiron species in the Alborz is E. (C.) cf. znojkoi distributed from the Talysh Mts. in the west to the Golestan NP in the east (Fig. 9). However, our study dealing with the molecular diversity of Caucasiron species in the Caucasus and adjacent regions (Hrivniak et al. 2020) indicated that E. (C.) znojkoi might represent a complex of cryptic species (only a subset of sequences included in the present study). The lineage Caucasiron sp. 4 (see Hrivniak et al. 2020) occurring in Iran (here called E. (C.) cf. znojkoi) differed from the Central Caucasian lineage. The delimitation of species within E. (C.) znojkoi s. l. requires further study. Nevertheless, the morphotype of E. (C.) cf. znojkoi has a wide geographical and ecological range, occurring at altitudes from -4 to 2290 m a.s.l. in northern Iran (Table 1). It was often found in shallow warm streams with good water quality flowing in humid broadleaved forests in the Caspian Sea lowland; approximately half of its localities was below 350 m a.s.l. At higher altitude, it may co-occur with E. (C.) alborzicus sp. nov., E. (C.) shargi sp. nov., E. (C.) nigripilosus, and E. (C.) iranicus (Fig. 9).
Three Caucasiron species, E. (C.) iranicus, E. (C.) alborzicus sp. nov., and E. (C.) shargi sp. nov., were described from the Alborz and are so far only known from there. E. (C.) iranicus is reliably reported from 12 localities, eight of them above 2000 m a.s.l. These include the Sabalan Mt. slopes in the western Alborz and the central Alborz, where it can co-occur with E. (C.) alborzicus sp. nov. (Fig. 9). It was found only in very cold streams fed by glaciers and melting snow from the highest mountains, with Alborz (coded as NI10I in Fig. 7C) clustered within the clade containing conspecific individuals from Russia and Turkey in the analysis of COI. It differed from these conspecifics in 2.8-3.6 % of K2P distance.
The most common Caucasiron species in the Alborz is E. (C.) cf. znojkoi distributed from the Talysh Mts. in the west to the Golestan NP in the east (Fig. 9). However, Alborz (coded as NI10I in Fig. 7C) clustered within the clade containing conspecific individuals from Russia and Turkey in the analysis of COI. It differed from these conspecifics in 2.8-3.6 % of K2P distance.
The most common Caucasiron species in the Alborz is E. (C.) cf. znojkoi distributed from the Talysh Mts. in the west to the Golestan NP in the east (Fig. 9). However, our study dealing with the molecular diversity of Caucasiron species in the Caucasus and adjacent regions (Hrivniak et al. 2020) indicated that E. (C.) znojkoi might represent a complex of cryptic species (only a subset of sequences included in the present study). The lineage Caucasiron sp. 4 (see Hrivniak et al. 2020) occurring in Iran (here called E. (C.) cf. znojkoi) differed from the Central Caucasian lineage. The delimitation of species within E. (C.) znojkoi s. l. requires further study. Nevertheless, the morphotype of E. (C.) cf. znojkoi has a wide geographical and ecological range, occurring at altitudes from -4 to 2290 m a.s.l. in northern Iran (Table 1). It was often found in shallow warm streams with good water quality flowing in humid broadleaved forests in the Caspian Sea lowland; approximately half of its localities was below 350 m a.s.l. At higher altitude, it can co-occur with E. (C.) alborzicus sp. nov., E. (C.) shargi sp. nov., E. (C.) nigripilosus, and E. (C.) iranicus (Fig. 9).
Three Caucasiron species, E. (C.) iranicus, E. (C.) alborzicus sp. nov., and E. (C.) shargi sp. nov., were described from the Alborz and are so far only known from there. E. (C.) iranicus is reliably reported from 12 localities, eight of them above 2000 m a.s.l. These include the Sabalan Mt. slopes in the western Alborz and the central Alborz, where it can co-occur with E. (C.) alborzicus sp. nov. (Fig. 9). It was found only in very cold streams fed by glaciers and melting snow from the highest mountains, with very rapid flow and strongly turbulent riffle sections. Four records of E. (C.) iranicus published by Mousavi and Hakobyan (2017) should be revised, because they included a wide range of altitude (20-2120 m a.s.l.) and were very close to our records of E. (C.) alborzicus sp. nov. and E. (C.) cf. znojkoi. The two new species from the Alborz seem to differ in habitat requirements. Epeorus (C.) alborzicus sp. nov. was only found in higher altitudes. These were all treeless localities in montane valleys with harsh climatic conditions, whereas E. (C.) shargi sp. nov. was found well below in submontane streams that were flowing in forests. The latter species was recorded only in the eastern Alborz near Gorgan (Fig. 9).
Other streams investigated in Iranian mountain ranges were dominated by Baetidae, and Heptageniidae were generally only scattered there. Larvae of E. (C.) zagrosicus sp. nov. and E. (E.) zaitzevi were found only in five and seven localities respectively, relatively distant to each other in the Zagros (Fig. 8). However, most of the streams explored in the Zagros were polluted or seasonally drying out due to the water storage in dams and water abstraction for irrigation of surrounding fields. Moreover, streams at higher altitude with presumably better water quality were almost inaccessible for us in April and May during our field trips. As E. (C.) zagrosicus sp. nov. was mostly found in natural streams in high-mountain valleys only with sparse villages, we expect that its distribution is limited to clear and cold mountain streams. However, a more detailed investigation of mayflies in high-mountain streams in Iran is needed.