A new species of Epeorus (Caucasiron) (Ephemeroptera, Heptageniidae) from Azerbaijan and Iran

Abstract A new species, Epeorus (Caucasiron) hyrcanicussp. nov., is described based on larval morphology and molecular data (COI) containing sequences from all Caucasian Caucasiron species described to date. The species is distributed in the Hyrcanian forest of southeastern Azerbaijan and northwestern Iran. Based on our wide-range sampling, the new species is likely endemic to this area. The most pronounced larval morphological diagnostic characters are the coloration pattern of abdominal sterna (a pair of oblique stripes and stripe-like medio-lateral maculae) and terga (triangular medial maculae), poorly developed projection of the costal margin of gill plates III, presence of hair-like setae on the surface of abdominal terga, and relatively wide shape of gill plates VII (in natural position from ventral view). The diagnostic characters are compared to related species, and primary information to habitat is provided.


Introduction
Epeorus Eaton, 1881 s.l. is one of the most diverse mayfly genera in the Caucasus region. Except for a single representative of Epeorus s.str., Epeorus (Epeorus) zaitzevi Tshernova 1981, all Epeorus species distributed in the region belong to the subgenus Caucasiron Kluge, 1997 (hereinafter Caucasiron).
The global distribution of Caucasiron includes the eastern Mediterranean islands (Samos and Cyprus), Turkey, the Caucasus, Iraq, Iran, Central Asia (Kazakhstan, Tajikistan, Nepal, and India), and south-western China (Guizhou province) (e.g. Chen 2010; Bojková et al. 2018;Hrivniak et al. 2020a;Khudhur and Sroka 2021). The highest diversity is known in the Caucasus and adjacent areas (including Samos Island), where 15 species have been described to date (Hrivniak et al. 2020a). Central Asia and south-western China are each inhabited by a single species, altogether numbering 17 confirmed Caucasiron species worldwide.
In this study, we provide a detailed morphological investigation and description of the lineage labelled as Caucasiron sp. 3 by Hrivniak et al. (2020c).
This species is distributed in the Hyrcanian forest of southeastern Azerbaijan and northwestern Iran (Fig. 1). The forest stretches along the southern shores of the Caspian Sea in Azerbaijan and Iran (Soofi et al. 2018) and covers the lowlands and foothills of the northern slopes of the Alborz Mountains (Gholizadeh et al. 2019). It has a high conservation value and represents a tertiary relict temperate forest (Soofi et al. 2018).
Our extensive sampling in the Caucasus and surrounding areas ( Fig. 1) points to a relatively narrow distribution of the new species, which is likely restricted to the Hyrcanian forest. It may represent a species endemic to this area, similar to E. (C.) iranicus, E. (C.) alborzicus, and E. (C.) shargi (Hrivniak et al. 2020a(Hrivniak et al. , 2020b. The new species was found to be related to E. (C.) caucasicus, E. (C.) nigripilosus, and E. (C.) turcicus, and its origin is dated to the Pliocene (Hrivniak et al. 2020c). During this period, intensive orogenic activity and climate cooling took place in the Caucasus region, causing fragmentation of ancient forests to isolated patches (Popov 2004;Naderi et al. 2014;Tarkhnishvili 2014). The Hyrcanian forest, together with the Pontic Mountains and Colchis lowland, was an important forest refugium where forest-associated species found suitable and stable environmental conditions during this period (cf. Tarkhnishvili 2014). This allowed the survival of tertiary relicts (Erichsen et al. 2017), resulting in a high number of local endemics (Naderi et al. 2014). We assume that these historical events could affect the evolutionary diversification of lineages in Caucasiron distributed along the southern Caspian Sea coastal areas.
Although the discovery of distinct lineages represents an essential step for evolutionary studies, their morphological determination is required for practical purposes of nature conservation and biomonitoring surveys. Thus, we aim to complete our phylogenetic studies with morphological investigations and taxonomy of evolutionary lineages delimited by molecular data.
The main aims of this study are to: i) investigate larval morphology of the lineage Caucasiron sp. 3 delimited as a putative species by Hrivniak et al. (2020c) and provide morphological comparison with other Caucasian Caucasiron species, ii) apply molecular species delimitation methods to our COI dataset containing all currently described Caucasian Caucasiron species, and iii) provide diagnostic characters for identification of the new species together with basic information on its habitat requirements.

Material and methods
The material used for this study was collected by J. Bojková, T. Soldán, and J. Imanpour Namin in Iran (May 2016), and Ľ. Hrivniak, P. Manko, D. Murányi, and M. Žiak in Azerbaijan (September 2018). Larvae were collected by hand net and preserved in 75-96% EtOH. Other Caucasiron species, used for morphological comparisons and molecular analyses, were obtained from the collections of the Biology Centre of the Czech Academy of Sciences, Institute of Entomology, České Budějovice, Czech Republic (IECA).

Morphological examination
Parts of larval specimens were mounted on microscopic slides using HydroMatrix (Mi-croTech 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 an Olympus SZX7 stereo microscope and an Olympus BX41 microscope, both equipped with a drawing tube. Photographs were obtained using a Leica DFC450 camera on a Leica Z16 APO macroscope and stacked in Helicon Focus v. 5.3 X64. All photographs were subsequently enhanced with Adobe Photoshop CS5. Diagnostic characters for the description of larva were chosen according to Hrivniak et al. (2020a).

DNA extraction, PCR, sequencing, and alignment
Total genomic DNA of the 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 were obtained from seven specimens (three from Iran and four from Azerbaijan). COI sequences of other Caucasiron species were obtained from Hrivniak et al. (2017)  The PCR amplification of COI and reaction volumes was carried out as described in Hrivniak et al. (2017). Sequences were assembled in Geneious v. 7.0.6 (http://www.geneious.com) and aligned in the same software using the Mafft v. 7.017 (Katoh et al. 2002) plugin with default settings. Newly obtained sequences are deposited in GenBank with accession numbers MZ389776-MZ389782.

Molecular species delimitation
Species were delimited using the single locus (COI) coalescence based General Mixed Yule Coalescent model (GMYC, Pons et al. 2006;Fujisawa and Barraclough 2013). We applied the single-threshold GMYC model as it has been found to outperform the multi-threshold (Fujisawa and Barraclough 2013) and was found to be highly suitable for species delimitation within Caucasiron (Hrivniak et al. 2019(Hrivniak et al. , 2020b. Analyses were performed using the SPLITS package for R (http://r-forge.rproject.org/projects/splits). An ultrametric COI gene tree was reconstructed in BEAST 2 (Bouckaert et al. 2014) with the settings as described in Hrivniak et al. (2020b).

Results and discussion
Epeorus ( Etymology. The name refers to the distribution of the species in the Hyrcanian forest. Distribution and habitat preferences of larvae. The species is distributed in northwestern Iran and southeastern Azerbaijan (Fig. 1) at -4 to 1345 m a.s.l. Larvae were found in streams and rivers flowing to the Caspian Sea in the humid forested slopes of western Alborz. They likely inhabit only cold and clear streams and rivers with stony bed substrate and turbulent flow. The species was not found in urban and agricultural areas in this region where many localities were investigated. Larvae were not abundant in either locality and co-occurred with the more abundant E. (C.) znojkoi.
Description of larva. General coloration of larvae yellowish-brown, with dark brown to reddish maculation. Body length of male mature larva 8.25 mm (n = 1); cerci broken. Body length of female mature larvae unknown.
Head. Shape oval to trapezoidal (Fig. 4D, E). Anterior margin with shallow concavity medially. Head dimensions: length 2.20 mm, width 3.04 mm (male); dimensions of female mature larva unknown. Head width/length ratio 1.36-1.41 (male), 1.40-1.44 (female). Coloration pattern as in Figure 4D, E. Dorsal surface of head covered with fine hair-like setae and sparsely distributed stick-like setae. Sparse longer hair-like setae located posteriorly to eyes.
Mouthparts. Labrum (Fig. 5A) widened anteriorly, with anterior margin slightly rounded (in dorsal view). Lateral angles rounded (shape of labrum may vary among specimens). Dorsal surface (Fig. 5A, right half ) sparsely covered with setae of different size; four longer bristle-like setae located antero-medially and two antero-laterally. Epipharynx with longer, slightly plumose bristles situated along lateral to anterior margin (Fig. 5A, 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 11-13 setae of various sizes located on ventral surface close to posterior margin. Outer incisors of both mandibles (Fig. 5B, C) with three apical teeth; outer tooth blunt in both mandibles. Inner incisor of left mandible with three apical teeth (Fig. 5B), right inner incisor bifurcated (Fig. 5C).  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.
Abdominal terga. Colour pattern of abdominal terga (Fig. 4A, H-I) consisting of transversal stripe along anterior margin of terga I-IX (X), medially extending, forming rectangular or triangular macula on terga II-IV (sometimes blurred), triangular macula on terga V-VII, and triangular or rectangular macula on terga VIII-IX (X). Lateral margins with oblique maculae on terga I-VIII (IX). Denticles on posterior margin of terga relatively dense, of various sizes, pointed and sometimes curved (Fig. 5E). Surface of terga covered with hair-like setae and sparsely with stick-like setae. Tergum X with short or without postero-lateral projections (Fig. 5L, M; arrow). Medial longitudinal row of hair-like setae along abdominal terga present.
Gills. Dorsal surface of gill plates I yellowish; of gill plates II-VII yellowish on anterior half, brownish to reddish on posterior half. Ventral margin of all gill plates yellowish to greyish. Projection of gill plates III poorly developed ( Fig. 5G; arrow). Gill plates VII relatively wide (in natural position of ventral view; Fig Results from molecular species delimitation. The GMYC species delimitation model applied to COI gene tree provided significantly better fit for a speciation branching than null model suggesting uniform coalescent branching across the entire tree (likelihood ratio test = 6.258895e-07***). The GMYC estimated 16 species (CI = 11-18) consisting of 15 ML clusters and one singleton (E. (C.) insularis). Morphologically defined E. (C.) hyrcanicus sp. nov. was delimited as a distinct species based on both GMYC and ABGD species molecular delimitation analyses. All species clusters were highly supported (PP = 1; Fig. 3).
The oblique stripes on abdominal sterna II-VI in E. (C.) zagrosicus are anteriorly widened (Hrivniak et al. 2020a: fig. 46I). This feature separates this species from E. Well-defined triangular medial maculae on abdominal terga V-VII are characteristic also for E. (C.) soldani (distributed in the western and central Greater Caucasus Mountains). Epeorus (C.) hyrcanicus sp. nov. can be separated from E. (C.) soldani by hair-like setae on abdominal terga (Fig. 5E), in contrast to the wide setae in E. (C.) soldani (Hrivniak et al. 2020a: fig. 20E). Additionally, the gill plates VII (in natural position from ventral view) are wider in E. (C.) hyrcanicus sp. nov., in contrast to E. (C.) soldani with narrow shape (Hrivniak et al. 2020a: fig 19L), and the projection on gill plates III is poorly developed in E. (C.) hyrcanicus sp. nov., in contrast to E. (C.) soldani with well-developed projection (Hrivniak et al. 2020a: fig. 20G).