A remarkable finding of Centroptilum Eaton, 1869 (Ephemeroptera: Baetidae) in Georgia, Turkey and Iran: one new species evidenced by morphology and DNA

Abstract The genus Centroptilum Eaton, 1869 is widely distributed in the West Palaearctic region, but poorly diversified as it previously only encompassed two valid species, C. luteolum (Müller, 1776) and C. pirinense Ikonomov, 1962. The discovery of a new congener, C. volodymyri Martynov, Godunko and Palatov, sp. nov., is therefore remarkable; the new species is described based on larvae collected in Georgia, Turkey and Iran in 2012–2019. This new species can be distinguished by the structure of the cuticle with flattened, stretched shallow, triangular and semilunar-shaped corrugations; scales and their bases densely scattered on body surface; features of the mouth parts, including the presence of an expanded laterally labrum, in combination with segment III of maxillary palp and superlinguae of hypopharynx rounded apically, without any pointed projections at the tip; numerous small teeth scattered proximally in two rows along inner margin of the pretarsal claw; the shape of denticulation along the posterior margin of the abdominal terga, and the number of prominent posterolateral spines on tergum VII; and the shape of gills II–VII, with a distinctly concave posterior margin. Together with a morphological description, data on the distribution and mitochondrial cytochrome c oxidase subunit I gene (COI) barcode sequences of the species are provided. The species delimitation of C. volodymyri Martynov, Godunko and Palatov, sp. nov. and C. luteolum is tested using COI barcode sequences. The data obtained show that the new species can be distinctly recognised as a separate lineage. The habitat preferences associated with the different parts of the area of C. volodymyri Martynov, Godunko and Palatov, sp. nov. are described and compared. Distribution of C. volodymyri Martynov, Godunko and Palatov, sp. nov. confirms the connections between Caucasian and Asian fauna of Ephemeroptera, as already demonstrated in various families and genera. Short notes on the distribution of mayfly species common to the Caucasus, north-western Iran and Central Asia are presented. http://www.zoobank.org/urn:lsid:zoobank.org:act:1D2B7557-0DEC-4ACD-8CA3-A6B50FCC4A51.


Introduction
The mayfly fauna of the Caucasus is still largely unexplored, although considerable progress has been achieved recently. Important taxonomic studies, checklists and critical reviews of literature data were published in the last decade (e.g. Kluge et al. 2013;Godunko et al. 2015;Martynov et al. 2015Martynov et al. , 2016Salur et al. 2016;Martynov & Godunko 2017;Bojková et al. 2018;Gabelashvili et al. 2018;Hrivniak et al. 2018Hrivniak et al. , 2020. The family Baetidae Leach, 1815 represents an important component of mayfly diversity; however, it remains incompletely known in the Caucasus and adjacent regions, especially when considering taxa from lentic habitats and low altitudes. They include mainly species belonging to the subfamily Cloeoninae Kazlauskas, 1972. A new species from this group, Procloeon (Pseudocentroptilum) caspicum Sroka, 2018, was recently described from Iran based on larvae (Bojková et al. 2018). Other genera of this subfamily, mainly Cloeon Leach, 1815 and Centroptilum Eaton, 1869, remain unexplored or their published records are doubtful. Importantly, the taxonomy of these genera is not fully resolved and a critical review of Cloeoninae is necessary. This study is hence focused on the genus Centroptilum, the concept of which has greatly evolved since its original description by Eaton (1871).
During more than a century, it conventionally encompassed all species of Cloeoninae with hind wings from all over the world (including Nearctic, Afrotropical, Oriental and Australasian regions). The concept was circumscribed step by step, for instance by excluding all African species (Gillies 1990). Kazlauskas (1972) and Sowa (1975, 1984) listed the distinguishing characters for the C. luteolum species group, including the structure of larval mouthparts and cerci, the shape of the gills and the surface of abdominal terga, and the shape of the hind wing and forceps-base projection in male imago. Jacob (1973Jacob ( , 1991 recognised the polyphyletic origin of the species grouped into Centroptilum sensu Eaton (1871) and divided them into C. luteolum and the C. stenopteryx complex. McCafferty and Waltz (1990) discussed diagnostic characters for Centroptilum, including Nearctic representatives originally described within this genus, Cloeon and Neocloeon Traver, 1932. Neocloeon was considered a junior synonym of Centroptilum. Novikova (1992a, 1992b) summarised previous data and proposed additional diagnostic characters for genera and subgenera within Cloeoninae. Because these authors applied a broader generic concept, several taxa traditionally recognised as genera of Cloeoninae were subordinated as subgenera to Cloeon. The presence/absence of hind wings was no longer considered a reliable character to separate genera; Centroptilum was distinguished from Procloeon Bengtsson, 1915 by the absence of spines on the lateral margin of tergites VII to IX and the absence of greatly enlarged spines on the outer margin of cerci, and from Anafroptilum Kluge, 2012 by the presence of a patella-tibial suture on fore legs of larvae (Kluge 2012(Kluge , 2016. Finally, Jacobus and Wiersema (2014) reinstated the genus Neocloeon and transferred seven Nearctic species originally described in Centroptilum or Cloeon to the genus Anafroptilum.
Currently, the distribution of Centroptilum s.s. is limited to the Palearctic region only, encompassing two species, the widely distributed C. luteolum (Müller, 1776) and the poorly known C. pirinense Ikonomov, 1962. The latter was originally described from the Balkans and only its larval stage is known. Keffermüller and Sowa (1984) considered C. pirinense to be closely related to C. luteolum. Novikova (1992a, 1992b) did not cite this species in the revision of the systematics of the subfamily Cloeoninae. Bauernfeind and Soldán (2012) reviewed the distinguishing characters, distribution and ecology of its larvae. They pointed out its possible synonymy with C. luteolum and considered it a species inquirenda. Jacobus and Wiersema (2014) considered it a valid Eurasian species in the discussion of species belonging to Centroptilum s.s. They noted the need for additional studies of C. pirinense, and also mentioned a single larva identified as C. pirinense by R. D. Waltz, that was partly mounted on the slide and deposited in the collection of the Purdue University Entomological Research Collection (USA). The specimen was labelled as collected in Turkey, without an exact locality -not even an indication of whether it was collected in the European or Asian part of the country.
This study aims to describe a new species of Centroptilum, C. volodymyri Martynov, Godunko and Palatov, sp. nov., based on material collected during field trips to Turkey, Georgia and Iran in 2012-2019; to discuss in detail morphological differences of the new species from C. luteolum (Müller, 1776) and C. pirinense Ikonomov, 1962; and to briefly discuss affinities of Baetidae faunas of the Caucasus and adjacent regions.

Sampling of material, preservation and morphological study
The present contribution is based on the larval material collected by A. V. Martynov, D. Palatov andJ. Bojková in 2012-2019, in several regions of Georgia, Turkey and Iran. Administrative divisions and geographic coordinates of localities are taken from Google Earth (http:// earth.google.com). All specimens were collected by kick sampling in different freshwater habitats and preserved in the field in 96% ethanol.

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A. V. Martynov et al. The holotype (from Georgia) is preserved in ethanol; paratypes from Georgia are preserved in ethanol (one larva), mounted on slides in Canada balsam (three larvae) (see below for the inventory numbers (IN)), preserved on scanning electron microscopy (SEM) stubs for taking SEM photos (one larva), and preserved as the external skeleton only in alcohol (two larvae -used for molecular study); paratypes from Turkey are preserved on a slide (one larva) and in ethanol (48 larvae Also, some non-type material of Centroptilum volodymyri Martynov, Godunko and Palatov, sp. nov. from Iran was used in the current study: 19 larvae deposited in IEE; one larva in NMNH NASU; four larvae in MZL; and nine larvae in the Department of Botany and Zoology of Masaryk University, Brno [MU]. The inventory numbers of samples from the type locality (Georgia) deposited in the NMNH NASU are: Grg24Censp/1 and Grg24Censp/2 for vials with larvae in alcohol; and 648, 661 and 662 for larvae mounted on slides.
For morphological analysis using SEM, larval mouthparts, legs and parts of the cuticle, and eggs dissected from a female larva were obtained from C. volodymyri sp. nov. (from type locality) and C. luteolum (Russian Federation (RF)

Terminology
The terminology and abbreviations proposed by Godunko et al. (2015) for the subgenus Rhodobaetis Jacob, 2003 are used to describe body setation of larvae. Their applicability was confirmed also for the genus Nigrobaetis Novikova and Kluge, 1987. The larval characters used to distinguish the the genus Centroptilum from its congeners were taken from and adapted based on references cited in the Remarks to Table II. DNA extraction, polymerase chain reaction amplification and sequencing, and DNA sequence analysis Total genomic DNA from XY specimens was extracted using the BioSprint 96 extraction robot (Qiagen Inc., Hilden, Germany), following the supplier's instructions. The non-destructive protocol described in Vuataz et al. (2011), which enables post-extraction morphological study of specimens, was implemented. We then amplified a 658-bp fragment at the 5' end of the mitochondrial cytochrome c oxidase subunit I gene (COI), corresponding to the standard animal barcode region, using the HCO2198 and LCO1490 primers (Folmer et al. 1994). Polymerase chain reaction (PCR) was conducted in a volume of 30 μL, consisting of 9 μL of template DNA, 1. 5 μL (10 μM) of each primer, 0.24 μL (25 mM) of dNTP solution (Promega), 6 μL of 10X buffer (Promega) containing 7.5 mM of MgCl 2 , 3 μL (25 mM) of MgCl 2 , 1.5 U of Taq polymerase (Promega), and 8.46 μL of sterile ddH 2 O. Optimised PCR conditions included initial denaturation at 95°C for 5 min, 38 cycles of denaturation at 95°C for 40s, annealing at 50°C for 40s, and extension at 72°C for 40s, with final extension at 72°C for 7 min. Purification and automated sequencing were carried out in Microsynth (Balgach, Switzerland).
In order to provide a comprehensive taxon sampling for the species delimitation, we analysed COI sequences from 62 specimens. We included all obtained sequences (three for C. volodymyri and three for C. luteolum; Table I) and added sequences from related taxa published in the GenBank database (https://www.ncbi.nlm.nih.gov/) as well as unpublished sequences from the platform FREDIE (Freshwater Diversity in Europe: https://www.fredie. eu/). In total, 43 sequences of C. luteolum, one sequence of C. victoriae McDunnough, 1938 and the outgroup sequences of Cloeon dipterum (Linnaeus, 1761), Cloeon simile Eaton, 1870, Procloeon pennulatum (Eaton, 1870) and P. bifidum (Bengtsson, 1912) were used for the analysis. Sequences were edited and aligned using Geneious Prime v. 2019.0.4 (Biomatters Ltd.). Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018;Stecher et al. 2020; http://www.megasoftware.net). We applied the GTR+G + I model of molecular evolution as it produces the lowest Bayesian information criterion (BIC) scores and is therefore considered to describe the substitution pattern the best.
Tree topology was reconstructed with MEGA X using the maximum likelihood method based on the Tamura-Nei model (Tamura & Nei 1993). The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap with 1000 replicates). For grouping sequences, the Automatic Barcode Gap Discovery (ABGD) method was performed on the webserver (http://wwwabi.snv. jussieu.fr/public/abgd/abgdweb.html; 09.09.2021) using as input the multiple sequence alignment obtained below, default values for the prior intraspecific divergences, relative gap width and distance distribution, and a Jukes-Cantor (JC69) distance matrix. The genetic variability between groups of sequences obtained above was calculated using Kimura twoparameter distances (K2P, Kimura 1980), with MEGA X (http://www.megasoftware.net).
( Tables I-III  . Larvae of C. volodymyri sp. nov. can be distinguished from the two other Western Palearctic representatives of the genus Centroptilum, namely C. luteolum and C. pirinense, by a combination of the following diagnostic characters: (1) the cuticle with flattened, stretched shallow triangular and semilunar-shaped corrugations, and relatively numerous scales and their bases covering the body surface; (2) labrum expanded laterally, with the U-shaped incurvation on the anterior margin; (3) apically rounded shape of the segment III of maxillary palps; (4) apically rounded shape of superlinguae of hypopharynx without a projection at the tip; (5) pretarsal claw with more than 60 small teeth in basal half of the claw, these teeth arranged into two rows; (6) the posterior margin of terga I-IV(V) with robust equilateral triangle-shaped spines scattered along the segment (equilateral spines sparse on terga (V)VI-X); (7) tergum VII near gill base with 3-4 relatively prominent posterolateral spines and sometimes several small spines; (8) gills II-VII with distinctly concave posterior margin.
Description. Based on specimens from the type locality only.
General body colouration intensively brown/dark brown and blackish; base of fore wing pads, lateral and ventral side of meso-and metathorax darker,

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A. V. Martynov et al. yellow to brownish black and black. Abdominal segments paler than thorax; legs and cerci lightest ( Figure 1). Head. Colour yellowish brown to light brown, with markedly darker area between ocelli, diffused light brown maculation on frons and vertex; clypeus and genae slightly darker, brown. Larval compound eyes brown. Antennae unicolor, light brown, distinctly longer than head and thorax. Frontal suture V-shaped. Head cuticle flattened, without pronounced corrugation. Surface of two first antennal segments and frons covered with sparse B and Hr setae (Figure 3(a,c,e)); clypeus additionally with two groups of long setae grouped on both sides of body axis.
Labrum expanded laterally, approximately 1.8-2.0× broader than long; median notch flattened; anterior margin concave, with U-shaped median incurvation relatively deep; anterior margin laterally from medial notch relatively symmetrically rounded. Dorsal surface of labrum covered with dense long and short Hr not grouped in rows and sparse B (Figures 4(a) and 5(b)); ventral surface with row of submarginal short setae ( Figure 5(a)).
Mandible incisors clearly divided into two groups, deeply separated throughout their length. Left mandibular incisor groups terminated by 4 + 2 stout denticles; left prostheca terminated by a group of slender setae (2-3 setae larger than other ones). Right mandibular incisor groups terminated by 3 + 2 stout denticles; right prostheca is stick-like ( Figure 4(c,d)). Maxillae with long slender canines and dentisetae; 1st dentiseta is simple, 2nd and 3rd dentisetae are bifid; dentisetae not pressed, and well separated from canines. Maxillary palps 3-segmented: segment I distinctly wider than segment II; segment III distinctly longer than segment II, rounded apically, without stout setae apically of surface; surface of maxillary palps covered with sparse Hr and FT (Figures 4(b,e,f) and 5(f)).
Labial palps 3-segmented. Segment III distinctly expanding apically, nearly trapezoidal with rounded outer angles; inner margin distinctly concave apically; posterolateral and posteromedian corners of different shape; stout setae along of apical margin and numerous Hr scattered on surface. Segment II distinctly wider than base of segment III; ventral side of segments I and II with scattered Hr; dorsal side of segment II with 6-10 elongated setae near to apicointernal margin ( Figure 5(g,f)).
Glossae nearly as broad as paraglossae ( Figure 5  (d,e)). Paraglossae slightly longer; dorsal surface with single row of elongated stout setae along inner

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A. V. Martynov et al. margin; ventral surface with fine long setae scattered over entire surface (apical setae shorter). Glossae with irregular row of submarginal short stout setae along outer and inner margins; denser setation shaped by fine long setae on dorsal side. Basal part of glossae and paraglossae with numerous fine long setae.
Thorax. Pronotum narrow, approximately 3.0-3.5× longer than broad. Pronotum with two yellowish to light brown maculae centrally; two spreading maculae of same colour on darker background laterally [yellow to yellowish brown, with occasional dirty yellow diffuse spots laterally in specimens from Iran]. Mesonotum with elongated brown strips on light brown background; coxae intensively brown to black brown [slightly paler in specimens from Iran]. Metanotum with spreading yellowish spot centrally, yellowish brown to dark brown laterally. Thoracic pleura slightly darker than terga (Figure 1(a,b)).

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A. V. Martynov et al. whitish yellow to intensively brown; coxae of middle and hind legs darker, intensively brown to dark brown. Trochanters with diffused brownish spot. Brownish elongated diffused macula along outer margin of all femora; tibiae yellowish to light brown; tarsi of the same colour as tibiae, occasionally slightly darker distally; claws yellowish brown. Cuticle of the legs formed by flattened equilateral  )). Numerous short, stout, pointed setae along outer margin of femora; numerous longer pointed setae along inner margin of tibiae and tarsi; outer margin of tibiae and tarsi covered with Hr and occasional B setae. Pretarsal claw length less than or equal to 1/2 of tarsus length (Figure 8(a-c)); more than 60 small teeth arranged into two rows distributed in basal half to 2/3 of claw length (Figure 7(a,c)).
Abdomen. Terga dark, yellowish brown to intensively brown (Figure 1(a,b)); terga I with two broad spots centrally, and two smaller spots anterolaterally. Terga II-V with uniform colour pattern, viz. a small pale spot centrally near anterior margin of segment; large brownish diffused spot centrally, with paler maculae near posterior margin of segment; additionally, yellowish-brown to light brown elongated strips surrounding pale maculae. Tergum VI

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A. V. Martynov et al. darkest, with colour pattern similar to those on previous segments, except of more diffused light spot anteriorly, and presence of two pale dots and two oblique strokes centrally. Terga VII-VIII with colour pattern similar to those on terga II-V (occasionally without small pale spot anteriorly), or uniformly light brown. Tergum IX uniformly light brown to brown without conspicuous pattern. Tergum X pale, yellow to light brown, with light transversal macula anteriorly, two unclear maculae centrally near anterior margin of segment and two brown small spots laterally. Anterolateral area of terga I-IX pale, yellowish brown. Abdominal sterna predominantly uniformly coloured, whitish yellow to light brown, without distinct pattern. Occasionally sterna with contrasting pattern: sternum I whitish yellow, with dark brown spots anterolaterally; sterna II-V (VI) with diffused triangular brownish spot on yellowish background; sterna VII-VIII darker than previous segments, light brown to brown, with longitudinal pale band and two yellowish spots centrally; sterna VIII-IX darkest, intensively brown, with diffused paler maculation centrally and laterally; sternum X slightly paler than two previous segments, light brown.
Surface of terga with cuticle formed by flattened equilateral triangle-and semilunar-shaped corrugations; terga I-IV covered with micropores, Hr and B, dominant SC-et and more sparse SCS-et; terga V-X with the same type of micropores, Hr and B, sparse SC-et and dominant SCS-et (Figure 9(a,c,e,g)). Posterior margin of terga I-IV (V) with robust equilateral triangle-shaped   A. V. Martynov et al. spines, alternating with sparse shorter ones and B setae; posterior margin of terga VI-X (occasionally tergum V) with thin and robust isosceles and equilateral triangle-shaped spines, alternating with shorter ones and sparse B setae. Tergum VII near gill bases with 3-4 relatively prominent posterolateral spines, and sometimes with several additional small spines; terga VIII-IX with 2-4 relatively prominent posterolateral spines, and sometimes with several additional small spines (Figure 10(h)).  (Figure 7(e,g)). Posterior margin of sterna with robust equilateral triangle-shaped spines alternating with occasional short B setae.
Gills whitish yellow, with well visible tracheisation, light brown to intensively brown coloured. All gills asymmetrical, rounded apically; gill I distinctly sinuous, narrow, lanceolate; gills II-VII with posterior margin distinctly concave (Figure 10(a-g)). Paraproct plate with 13-15 pointed strong teeth along inner margin; ventral surface of paraproct plate covered with leaf-shaped SC and their SCS grouped mainly centrally, elongated Hr and B setae (Figures 10(i) and 11(a,c)).
Cerci and paracercus slightly paler than body, light brown, without coloured rings. Posterior margin of caudal filament segments with combination of robust marginal spines, occasional Hr and B setae; submarginal area with solitary SC-et (Figure 11(e)). Cerci (inner margin) and paracercus (both margins) up to the apex bear well-developed primary swimming setae, except last one or two segments. Outer margin of cerci with relatively short Hr and B setae.
[4] For C. pirinense, figures by Ikonomov (1962: 115, fig. 25.1), in the original description the shape the of labrum and setation are described as follows: the labrum is almost twice wider than long; lateral side of the labrum with sharp setae that look like a feather; two long sharp setae apically, and 3-4 smaller pointed setae more medially [ventral side]; setae evenly scattered over the entire surface [dorsal side].
[13] Keffermüller and Sowa (1984: 310) reported a lack of posterolateral spines on abdominal terga of C. luteolum. McCafferty and Waltz (1990: 784) listed for several Nearctic larvae the presence of posterolateral spines on abdominal terga VIII-IX, as an exception in comparison with all other species of the genus. However, later, Kluge and Novikova 1992: 69, fig. 5.1) clarified this information, and reported small posterolateral spines in C. luteolum, typical also for the species of the genus Cloeon s.l. According to Bauernfeind and Soldán (2012), posterolateral part of tergum IX in C. luteolum occasionally with a group of 3-6 small and rather insignificant spines. [14] For C. pirinense, based on fig. 26.13 in Ikonomov (1962). [15] Using scanning microscopy, the chorion surface of the egg was depicted for the first time by Jacob (1991: 284, fig. 9b). Ubero-Pascal and Puig (2007: 332, fig. 4a) and Ubero-Pascal et al. (2005: 266, 268, figs 2b, 4c) depicted a general view of eggs and their chorionic surface.
Distinguishing characters of C. volodymyri sp. nov. discussed in the Table II have been analysed based on type material only. Some differences of body colouration between Caucasian and Iranian specimens of C. volodymyri sp. nov. are discussed in the text.

Etymology
The species is named in honour of Volodymyr Martynov, senior son of the first author, for his field assistance in mayfly sampling during a series of field trips.

Distribution and habitat
In Georgia, the larvae of C. volodymyri sp. nov. were collected in small pools, where small brooks flowed in, in the valley of the Kintrishi River at 325 m a.s.l. (Figure 13(a,b)). They were shaded by the ancient colchic forests (chestnut, hornbeam and box trees). Pools were about 0.3 m deep, with bed substrate composed of silt and detritus, without macrophytes. No other mayflies were found there.
In Iran, the larvae were collected in two small brooks (1-3 m wide) flowing in steep terrain below 300 m a.s.l. on the northern slopes of the Alborz Mts., close to the Caspian Sea coastal area in Guilan Province (Figures 13(c,d) and 14). They were shaded by humid deciduous forest and shallow, with an average depth of 0.05-0.15 m (max. 0.3 m in pools). They were characterised by alternating riffles and pools, where Centroptilum larvae were collected. Coarse bed substratum in riffles alternated with finer substrates formed by coarse and fine gravel in pools. Bedrock and boulders were sparsely covered with mosses. Water was relatively cold (16°C) and clear, although one brook was slightly turbid. The mayfly taxocene consisted of the following species: C. luteolum (dominant), Baetis (Rhodobaetis) cf. gadeai Thomas, 1999, Nigrobaetis (Alainites) muticus (Linnaeus, 1758) (all Baetidae), Electrogena pseudaffinis (Braasch, 1980)
In Turkey, larvae were collected in a small stream (1.5-2.5 m width; 0.1-0.2 m depth) flowing in the forest at 337 m a.s.l. (Figure 13(e,f)). Its bed substrate was composed of pebbles, detritus and silted stones and the flow velocity was 0.

Affinities
We attribute C. volodymyri sp. nov. to the genus Centroptilum based on larval and egg characters depicted by Jacob (1991), Novikova (1992a, 1992b), and Bauernfeind and Soldán (2012). The attribution is confirmed by the presence of (1) epicranial suture met at an obtuse angle; (2) (3) segment III of maxillary palp not shortened, longer than segment II; (4) narrow pronotum, approximately 3.0-3.5 × longer than its width [4.0 times according to Novikova (1992a, 1992b)]; (5) pretarsal claws with two rows of small teeth; (6) simple gills with pinnate tracheisation [according to Bauernfeind and Soldán (2012), in Centroptilum simple gills acutely pointed apically and almost symmetric, except for the very narrow gill I]; (7) abdominal segments almost without strong posterolateral setation, except for tergum VII covered by 1-3 relatively prominent spines [no strong posterolateral setation in Centroptilum according to Bauernfeind and Soldán (2012)]; (8) surface of abdominal terga with flattened, nonpointed corrugation and micropores [pointed chagrin structure in the form of minute spines, and circular sensillae according to Bauernfeind and Soldán (2012)]; (9) posterior margin of terga with robust spines alternating with shorter ones [the same for some terga of C. luteolum (see Figure 9(h)) and C. pirinense (according to Ikonomov 1962); the posterior margin of abdominal terga in Centroptilum with spines of subequal length according to Bauernfeind and Soldán (2012)]; (10) no long setation on last two caudal segments [basal and apical 1/ 5 part of caudal filaments are without setation according to Bauernfeind and Soldán (2012)]; and (11) chorionic surface of eggs with dense uniformly reticulated ridges, forming irregular polygonal cells ["meshes" according to Bauernfeind and Soldán (2012)]. Centroptilum volodymyri sp. nov. occupies a relatively independent position within the genus due to characteristics of the cuticle, setation of the posterolateral margin of tergum VII and the gill shape. At the larval stage, it clearly differs from the Western Palearctic representatives C. luteolum and C. pirinense by numerous scales and scale bases densely scattered on the body surface, some features of the mouthparts (particularly the shape of the labrum and superlinguae of the hypopharynx, and the proportions and setation of maxillary and labial palps; see also Table II), and dense denticulation of claws. Minor differences are found in the size of eggs and their cells within the chorionic mesh. The structure of the chorionic surface is thus very similar to that of C. luteolum (it was not described for C. pirinense). A detailed comparative overview of the distinguishing characters for the delimitation of the three species discussed above is given in Table II.
Specimens of C. volodymyri sp. nov. collected in the Caucasus and Iran show some variability in colouration and morphology. Caucasian specimens are distinctly darker: their pleural sclerites and particularly anterior part of mesonotum and fore wing pads are markedly brown to dark brown. In contrast, Iranian specimens are distinctly paler, with body whitish yellow to brown. While Caucasian specimens had brown abdominal tergum and sternum of segment IX, Iranian specimens show a whitish-yellow central part of the tergum IX lined by two brownish oblique strips (Figure 2(a)) and small light brown smudges on the sternum IX. Some minor differences between Caucasian and Iranian populations can be recognised in the structure of mouthparts and arrangement of scales and setae along the posterior margin of flagellar segments. Iranian specimens had a more elongated labrum width/length ratio (1.6-1.7, compared to 1.8-2.0 in Caucasian specimens). Some Iranian specimens have fewer setae (fewer than six) on the dorsal side of segment II of the labial palp than the Caucasian specimens (6-8 setae). The setae under the maxillary crown are arranged in a nearly distinct row in specimens from the Caucasus, while they are Figure 14. Map of Centroptilum volodymyri sp. nov. distribution. Red star -type locality; green pentagrams -non-type localities.

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A. V. Martynov et al. mostly scattered in some specimens from Iran. The specimens from Georgia and Turkey have bluntly pointed or apically rounded scales on posterior margins of flagellar segments, while two or three pointed apices are found on each marginal scale in specimens examined from Iran. Nevertheless, these differences are not consistent in all specimens and characters, and we consider the specimens from Caucasus, Turkey and Iran to be conspecific, belonging to C. volodymyri sp. nov.

Molecular results
The molecular reconstruction highly supported C. volodymyri sp. nov. as a monophyletic clade, with a bootstrap support of 99% (Figure 15). Georgian and Iranian sequences are separated by genetic distances (13%, Table III) that are generally considered higher than intraspecific. Centroptilum volodymyri sp. nov. is recovered as the sister species of C. luteolum; the genetic distances between C. volodymyri sp. nov. and the two clades of C. luteolum are higher than 25%. Centroptilum luteolum is composed of two monophyletic lineages (clade 1 and clade 2 in Table III and Figure 15); both are highly supported as monophyletic and may represent distinct species (K2P distances higher than 20%).

Discussion
Integrative approaches clearly confirm the validity of the new species C. volodymyri sp. nov. This species is closely related to the type species of Centroptilum, C. luteolum, both morphologically and genetically. The two species can be morphologically distinguished by the shape and length of the maxillary palp, the length and denticulation of the claws and the shape of the gills. High molecular distances strengthen the validity of the new species. At present, three populations of C. volodymyri are known, from Georgia, Turkey and Iran. As these three countries represent the limit of distribution of C. luteolum, these two species of Centroptilum are partially sympatric. The molecular distances between the Georgian and Iranian population of C. volodymyri sp. nov. are rather high and may suggest the presence of two cryptic species. We refrain from considering the two as separate species as no morphological characters support this division. The high molecular distance could also be due the geographical gap between the populations and the absence of gene flow between them. The discovery of additional populations between them might challenge the present interpretation and reduce the average intraspecific distances.
In addition to C. luteolum and C. pirinense known from the West Palaearctic region, Bauernfeind and Soldán (2012) considered three extralimital Palaearctic species of Centroptilum. Two of them are poorly known species described based on adults, namely C. chinense You and Gui, 1995 based on a male imago from China and C. rotundum Takahashi, 1929 based on a female imago from Japan. Comparing the species described here with these taxa is impossible because the imago is unknown in C. volodymyri sp. nov. The third species, C. kazlauskasi Kluge, 1983, distributed in Mongolia, and the Far East and the Republic of Buryatia in the Russian Federation, was designated as the type species of the genus Anafroptilum Kluge, 2011. The genus is established for non-African taxa, initially attributed to Centroptilum (Kluge 2012). In contrast to Centroptilum (incl. C. volodymyri sp. nov. as well as the closely related Nearctic genus Neocloeon), the representatives of Anafroptilum are characterised by the absence of the patella-tibial suture in larvae and adults of both sexes on the fore legs, and therefore are considered to belong to a completely separate lineage from Centroptilum.
There are some other taxa nominally placed within Centroptilum and distributed in the Palearctic Region. Centroptilum hungaricum Pongrácz, 1913 was described as the female imago from the Trenčianska župa [Trencsén County in the former Kingdom of Hungary; now Western Slovakia] (Pongrácz 1913). Centroptilum obtusum Navás, 1915 from Spain is known from the old material only (Navás 1915), but Alba-Tercedor and Peters (1985) reported the absence of the types of this species in the collection of Longinos Navás at the Museo de Zoologia del Ayuntamiento (Barcelona, Spain). These two taxa are mentioned as dubious species (for additional information and respective references see Jacobus & Wiersema 2014). Additionally, C. striatum Bengtsson, 1904 is considered a nomen nudum (see Bauernfeind & Soldán 2012) and the generic placement of C. lituratum (Pictet, 1843) is considered unclear, because the taxon was described from adult material from Switzerland and the type material is lost (Keffermüller & Sowa 1984). The comparison of the presently discovered species with the taxa referred to above is impossible, again because the imaginal stage of C. volodymyri sp. nov. is unknown.
Summarised data on the diversity of the subfamily Cloeoninae of Iran were published by Bojková et al. (2018): five species of this subfamily were reported, belonging to Cloeon, Centroptilum and Procloeon. Regarding biogeographic patterns of Iranian Cloeoninae, the dominant part of the species exhibits a broad Palearctic and Holarctic distribution. This is in clear contrast to the distribution of C. volodymyri sp. nov., which is limited to Northern Middle East, Southern Caucasus and Eastern Anatolia. A single species of Procloeon (P. caspicus) was recorded from Northwest Iran, close to the border with Azerbaijan (Bojková et al. 2018).
The fauna of Caucasian Cloeoninae is more diverse. Altogether, 11 species belonging to three genera were recorded. While Centroptilum luteolum is distributed throughout the region (see Cherchesova 2004;Palatov 2013;Martynov et al. 2016;Hrivniak et al. 2018), the taxon listed as Centroptilum sp. from the Kintrishi River (AR Adzharia, Georgia) corresponds to the new species described here (see the list of synonymies and Martynov et al. 2016). The species of Cloeon are