A remarkable finding of Centroptilum Eaton, 1869 (Ephemeroptera: Baetidae) in Georgia, Turkey and Iran: one new species evidenced by morphology and DNA Author Martynov, A. V. 0000-0002-6506-5134 National Museum of Natural History, National Academy of Sciences of Ukraine, Kyiv, Ukraine Author Palatov, D. M. Independent Researcher, Lviv, Ukraine Author Gattolliat, J. - L. 0000-0001-5873-5083 Author Bojková, J. 0000-0003-0996-3308 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic Author Godunko, R. J. 0000-0003-2196-3327 Institute of Entomology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, České Budějovice 37005, Czech Republic roman.hodunko@biol.uni.lodz.pl text The European Zoological Journal 2022 2022-07-22 89 1 826 855 journal article 10.1080/24750263.2022.2090625 b19fe223-b5ce-44d5-b783-ef05b7d3f034 7741484 1D2B7557-0DEC-4ACD-8CA3-A6B50FCC4A51 Centroptilum volodymyri Martynov, Godunko and Palatov , sp. nov. ( Tables I –III; Figures 1 , 2 , 3 (a,c,e), 4 (a–e), 5, 6 (a,c,e), 7 (a,c,e,g), 8, 9 (a,c,e,g), 10, 11 (a,c,e), 12(a,c,e,g)) http://www.zoobank.org/urn: lsid:zoobank.org: a c t: 1 D 2 B 7 5 5 7 - 0 D E C - 4 A C D - 8 C A 3 - A6B50FCC4A51 Centroptilum sp. : Martynov et al. 2016: 170 [distribution in Georgia (Kintrishi River valley), with remark on belonging to undescribed new species] Centroptilum sp. : Bojková et al. 2018: 91 , 96, tables 1, 2 [distribution in Iran within Zayanderud (based on Mahboobi Soofiani et al. 2012 ) and Gilan (two localities listed also in present contribution) provinces] Table I. Codes and origin of new sequences used in molecular study. For each specimen, the specimen catalogue number (GBIF code), the sample information (country, locality, coordinates and date of sampling), the GenBank accession number of the COI sequence, and nomenclature of genetic sequences (GenSeq; follows Chakrabarty et al. 2013 ) are provided.

Specimen	GenBank	GenSeq
Species	catalogue number	Country	Locality	Latitude	Longitude	Date	ID	nomenclature
Centroptilum	GBIFCH00895431	Georgia	Adjara, Kobulety District, vicinity of Chakhati village, valley of	41.762222	41.978111	18 April	OL960099	genseq-2 COI
volodymyri sp.	the Kintrishi River, small lentic water bodies [Grg24Censp]	2016
nov.
Centroptilum	GBIFCH00895432	Iran	Guilan Province, SW from Amlash town, unnamed small brook	37.036944	50.082500	21 May 2016	OL960097	genseq-3 COI
volodymyri sp.
nov.
Centroptilum	GBIFCH00895437	Iran	Golestan Province, Kalaleh County, vicinity of Zav-e-Bala	37.529433	55.791550	23 June 2019	OL960098	genseq-3 COI
volodymyri sp.	village, unnamed stream [Iran3
nov.	Censp/1]
Centroptilum	GBIFCH00895434	Georgia	Adzharia, territory of Kobulety town, Kintrishi River	41.803889	41.775833	4 June 2013	OL960101	genseq-4 COI
luteolum	[Grg6Cenlut]
Centroptilum	GBIFCH00895435	Ukraine	Luhansk Region, Antratsyt District, vicinity of Khrustal’nyi	48.182321	38.877363	30 April	OL960102	genseq-4 COI
luteolum	village, Khryshtaleva River [Lug 94]	2012
Centroptilum	GBIFCH00895441	Switzerland	Burtignière, Vallée de Joux	46.561667	6.170000	23 August	OL960100	genseq-4 COI
luteolum	2001

? Centroptilum sp. : Mahboobi Soofiani et al. 2012: 137 [Zayandeh-Roud River in the Zayanderud Province; this record should be verified] Diagnosis [ based on larvae ]. 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. Mature larva. Body length 9.5–10.2 mm (male larvae slightly smaller than female larvae), length of cerci 5.5–5.8 mm (approx. 0.5× body length); length of paracercus 3.0– 3.3 mm . General body colouration intensively brown/dark brown and blackish; base of fore wing pads, lateral and ventral side of meso- and metathorax darker, yellow to brownish black and black. Abdominal segments paler than thorax; legs and cerci lightest ( Figure 1 ). Figure 1. Larva of Centroptilum volodymyri sp. nov. , Georgia, holotype. (a, b), Body in lateral view. Scale bars: 1 mm. Figure 2. Larva of Centroptilum volodymyri sp. nov. , Iran. (a) Body in dorsal view; (b) body in lateral view; (c) head and part of thorax in lateral view. Scale bars: 1 mm. 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)). Superlinguae of hypopharynx rounded apically, without any projections ( Figure 5 (c)). 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)). Figure 3. Larvae of Centroptilum volodymyri sp. nov. , paratype (a, c, e) and C. luteolum (Müller, 1776) (b, d, f). (a, b) Scapus and pedicellus; (c) flagellum, basal part; (d) flagellum, central part; (e, f) surface of frons. Scale bars: a, c, d = 20 μm; b = 30 μm; e = 10 μm; f = 100 μm. 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 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. Figure 4. Larvae of Centroptilum volodymyri sp. nov. , paratype (a–e) and C. luteolum (Müller, 1776) (f). (a) Labrum; (b) galea-lacinia; (c, d) mandibles, left (c) and right (d); (e, f) maxillary palp. Scale bars: a, b, e, f = 30 μm; c, d = 100 μm. 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)). Hind wing pads well developed. Thoraxal cuticle formed by flattened, stretched shallow triangular and semilunar-shaped corrugations. Surface of pronotum covered with Hr, B , micropores [ chloride cells ], sparse SC-et [6–8 μm in length] and SCS-et ( Figure 6 (a)). Legs relatively pale, whitish yellow to dirty brown [paler in specimens from Iran]; all leg segments slightly darker distally ( Figure 1 (a,b)). Legs slender; femora parallel-sided; patella-tibial suture developed on all legs ( Figure 8 (a–c)). Fore leg coxae pale, 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 triangle- and semilunar-shaped corrugations. Surface of femora with SC-et, SCS-et, Hr, B setae; the same types of scales and setae, with dominance of SCS-et distributed of surface of tibiae and tarsi ( Figure 6 (c,e)). 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)). Figure 5. Larvae of Centroptilum volodymyri sp. nov. , paratypes. (a, b) Labrum, ventral (a) and dorsal (b) view; (c) hypopharynx (left side – dorsal view; right side – ventral view); (d, e) glossa and paraglossa, dorsal (d) and ventral (e) view; (f) maxilla; (g, h) labial palp, ventral (g) and dorsal (h) view. Figure 6. Larvae of Centroptilum volodymyri sp. nov. , paratype (a, c, e) and C. luteolum (Müller, 1776) (b, d, f). (a, b) Pronotum, dorsal surface; (c, d) hind femur, dorsal surface; (e, f) hind tibia, dorsal surface. Scale bars: a–c, e, f = 30 μm; d = 10 μm. Figure 7. Larvae of Centroptilum volodymyri sp. nov. , paratype (a, c, e, g) and C. luteolum (Müller, 1776) (b, d, f). a, b = hind pretarsal claw (apex of claw not visible in (a)); (c, d) denticles of hind pretarsal claw; (e) surface of sternum IV; (f) surface and posterior margin of sternum VI; (g) surface and posterior margin of sternum VII. Scale bars: a = 50 μm; b = 100 μm; c–e, g = 30 μm; f = 10 μm. 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 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. Figure 8. Larvae of Centroptilum volodymyri sp. nov. , paratypes. (a) Fore leg; (b) middle leg; (c) hind leg. 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 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 9. Larvae of Centroptilum volodymyri sp. nov. , paratype (a, c, e, g) and C. luteolum (Müller, 1776) (b, d, f, h). Surfaces and posterior margins of terga: (a, b) tergum II; (c, d) tergum IV; (e, f) tergum VI; (g, h) tergum VIII. Scale bars: a, b = 20 μm; c–h = 30 μm. Figure 10. Larvae of Centroptilum volodymyri sp. nov. , paratypes. (a) Gill I; (b) gill II; (c) gill III; (d) gill IV; (e) gill V; (f) gill VI; (g) gill VII; (h) posterolateral part of tergum VII; (i) paraproct plate. Cuticle of sterna shaped by flattened undulating corrugations covered with micropores, elongated Hr and B setae, sparse SC-et and dominant SCS-et ( 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)). Figure 11. Larvae of Centroptilum volodymyri sp. nov. , paratype (a, c, e) and C. luteolum (Müller, 1776) (b, d, f). (a, b) Apical part of paraproct; (c) surface of paraproct; (d) tergum X and caudal filaments; (e) caudal filaments, basal part; (f) caudal filament, medial part. Scale bars: a, b, e, f = 30 μm; c = 20 μm; d = 100 μm. 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. Egg (taken from larva). Oval; 130–140 µm long, 65–70 µm wide. Chorionic surface shaped by thin, flat reticulated ridges forming irregular polygonal mesh; cells (2.5–4.2 µm in diameter) of this mesh mostly with rounded margins; no knob-terminated coiled thread inside of chorionic cells ( Figure 12 (a,c,e,g)). One, occasionally two oval micropyles in equatorial area, 5.5–5.8 µm long and 4.8–5.0 µm wide; no micropylar rim surrounding micropyle. Imago and subimago. Unknown. Figure 12. Eggs of Centroptilum volodymyri sp. nov. , from paratype (a, c, e, g) and C. luteolum (Müller, 1776) (b, d, f, h); provided by Nicolás Ubero-Pascal). (a–d) General view of egg; (e, f) chorionic surface; (g, h) micropyle. Scale bars: a, c = 30 μm; b = 80 μm; d = 20 μm; e, g = 5 μm; f = 10 μm; h = 5 μm. Table II. Morphological characters of larvae that distinguish Centroptilum volodymyri sp. nov. from C. luteolum (Müller, 1776) and C. pirinense Ikonomov, 1962 . Distinct differential characters are in bold.

Centroptilum luteolum (Müller,
Characters [remarks;	Centroptilum volodymyri sp. nov.	1776) [Europe, Middle Asia,	Centroptilum pirinense (Ikonomov,
No.	references; figures]	[type material]	Caucasus]	1962) [1] [Macedonia, Bulgaria]
Larva
I. Head (colour, shape and setation)
1	Colour pattern	Yellowish-brown to light brown;	Light brown; clypeus and genae	Light brown; diffused pale
Figures 1(a,b) and 2	diffused light brown	brown	maculation
(a–c)	maculation on frons and vertex;
clypeus and genae brown
2	Antenna length [2]	Distinctly longer than head and	Slightly longer than head and	Reaching approximately the bases
Figures 1(a,b) and 2	thorax	thorax	of fore wings or longer than
(a,b)	head and thorax
3	Surface of scape and	Sparse Hr and B	Sparse Hr and B	−
pedicel: setation
Figure 3(a,b)
4	Posterior margin of	Sparse row of 10–14 well	Dense row of 16–20 robust,	−
flagellar segment	separated, robust, bluntly	bluntly pointed or rounded
(central part of	pointed or rounded apically	apically spines, grouped by 2–3
flagellum): shape	spines	spines with common basis;
Figure 3(c,d)	1 or grouped by 2 B setae and	1 or grouped by 2–3 B setae
sparse Hr
5	Surface of clypeus and	Sparse B and long Hr	Sparse B and long Hr	−
frons: setation
6	Figure 3(e,f) Frontal suture: shape [3]	V-shaped	V-shaped to U-shaped	−
Figure 3(f)
II. Mouthparts
1	Labrum: width/ length ratio [4]	1.8–2.0	1.4–1.6	approx. 2.0
2	Labrum: dorsal surface	Dense short and long Hr ; sparse B	Dense short and long Hr ; sparse B	Dense short and long Hr ; sparse B
Figures 4(a) and 5(b)
3	Labrum: anterior	Slightly concave; median	Nearly straight; median	Nearly straight; small medial
margin	incurvation relatively deep,	incurvation shallow, angular	semicircular incurvation
Figures 4(a) and 5(a,	angular, U-shaped
b)
4	Ratio of width of	1.3–1.6	1.2–1.8	1.2
maxillary palp segments I/II[5]
Figures 4(f) and 5(f)
5	Segment III of	Rounded apically;	Pointed or bluntly pointed	Pointed apically;
maxillary palp;	distinctly longer than segment	apically;	subequal to segment II – 1.1
ratio length	II – 1.6–1.7	subequal to distinctly longer
segments III/II	than segment II – 1.1–1.7
Figures 4(e,f) and
5(f)
6	Right mandible:	3 + 2	3 + 2	3 + 2
number of incisors of
mandibular incisor groups [6]
Figure 4(d)
7	Left mandible:	4 + 2	4 + 2 (occasionally 4 + 3)	4 + 3
number of incisors
of mandibular incisor groups [6]
Figure 4(c)
8	Mandibles: surface	Occasionally sparse B and Hr	No setae; occasionally sparse	Occasionally sparse B and Hr
Figure 4(c,d)	B and long Hr , small prominent
isosceles and equilateral
triangle-shaped corrugations,
and micropores

( Continued ) Table II. (Continued).

Centroptilum luteolum (Müller,
Characters [remarks;	Centroptilum volodymyri sp. nov.	1776) [Europe, Middle Asia,	Centroptilum pirinense (Ikonomov,
No.	references; figures]	[type material]	Caucasus]	1962) [1] [Macedonia, Bulgaria]
9	Hypopharynx: shape	Rounded; no triangular	Rounded; small triangular bluntly	Rounded; no triangular projection
of superlinguae [7]	apicomedial projection	pointed apicomedial projection
Figure 5(c)
10	Segment II of labial	6–10	2–9	6–7
palp [dorsal view]:
setation
Figure 5(h)
11	Segment II of labial	Distinctly wider than segment III	Equal width or slightly wider than	Equal length or slightly wider than
palp: distal end	base	segment III base	segment III base
Figures 5(g,h)
12	Segment III of labial	Distinctly expanding apically;	Moderately expanding apically;	Slightly expanding apically;
palp: shape [8]	nearly trapezoidal; inner	roughly quadrangular;	roughly quadrangular; inner
Figures 5(g,h)	margin concave; posterolateral	inner apical margin slightly	margin slightly concave;
and posteromedian corners of	concave;	posterolateral and
different shape	posterolateral and	posteromedian corners of very
posteromedian corners of	similar shape
different shape
III. Thorax and legs (colour, shape and setation)
1	Colour pattern:	Dark brown to blackish brown	Light yellowish brown; brownish	Light brown; two pairs of large
pronotum [9]	maculation centrally; pale	maculation centrally and	dissolved spots centrally and
Figures 1(a,b) and 2	diffused spots laterally	laterally	laterally
(a,b)
2	Surface of pronotum:	Flattened, stretched shallow	Prominent rounded and broadly	−
cuticle structure	triangular and semilunar-	triangular corrugations
Figure 6(a,b)	shaped corrugations
3	Surface of pronotum:	Hr, B , micropores, sparse SC-et	Sparse Hr and B	−
setation	and SCS-et
Figure 6(a,b)
4	Colour pattern: legs	Yellowish to dirty brown; all leg	Femora with brown transversal	Unicolour light brown
Figures 1(a,b) and	segments slightly darker distally	band distally; tibiae darkened
2(b)	proximally
5	Surface of legs:	Flattened equilateral triangle- and	Prominent isosceles and	−
cuticle structure	semilunar-shaped corrugations	equilateral triangle-shaped
Figure 6(c–f)	corrugations
6	Outer margin of	Numerous short, pointed	Occasional short, pointed	Short, pointed setae
femora: stout setae
Figure 8(a–c)
7	Surface of femora	SC-et, SCS-et, Hr, B	Hr, B , pointed stout setae with	−
[dorsal view]:	feathered margins mainly near
setation	inner margin [sparse]
Figure 6(c,d)
8	Surface of tibiae	SC-et, SCS-et [dominant], Hr, B	Long Hr, B	−
[dorsal view]:
setation
Figure 6(e,f)
9	Surface of tarsi	SC-et, SCS-et [dominant], Hr, B	Hr, B , pointed stout setae with	−
[dorsal view]:	feathered margins mainly near
setation	inner margin [sparse]
10	Pretarsal claw length	≤ 1/2	≈ 1/2	1/2–2/3
relative to tarsus length[10]
Figures 7(a,b) and 8
(a–c)
11	Pretarsal claw:	More than 30 in basal half to 2/3	About 18–27 in basal third	About 15–20 in basal half [13–16
number of small	of claw length	in original description]
teeth per row
Figure 7(a–d)
IV. Abdomen (colour, shape and setation)

( Continued ) Table II. (Continued).

Centroptilum luteolum (Müller,
Characters [remarks;	Centroptilum volodymyri sp. nov.	1776) [Europe, Middle Asia,	Centroptilum pirinense (Ikonomov,
No.	references; figures]	[type material]	Caucasus]	1962) [1] [Macedonia, Bulgaria]
1	Colour pattern: terga	Terga dark, yellowish brown to	Terga pale, ivory-white, usually	Terga unicoloured, brownish, with
II–VIII (IX)	intensively brown; terga II–VIII	with a pair of small, dark brown	indistinct pale diffused area and
Figures 1(a,b) and 2	often with more or less distinct	spots in the middle; tergum VI	two unclear spots centrally;
(a,b)	colour pattern consisting of	usually darkest; terga II and VI	clearer colour pattern on terga
pale spots, maculae and strips	often with dark spot centrally	VII–IX (occasionally VI)
centrally, and darker spots	on paler background
laterally
2	Surface of terga:	Flattened equilateral triangle- and	Prominent isosceles and	Prominent isosceles and
cuticle structure [11]	semilunar-shaped corrugations	equilateral triangle-shaped	equilateral triangle-shaped
Figure 9(a–h)	corrugations	corrugations
3	Surface of terga I–IV:	SC-et [dominant], SCS-et	Micropores, Hr and B	SC-et
setation [12]	[sparse], micropores, Hr and B
Figure 9(a–d)
4	Surface of terga V–X:	SC-et [sparse], SCS-et	Micropores, Hr and B	SC-et
setation [12]	[dominant], micropores, Hr
Figure 9(e–h)	and B
5	Posterior margin of	Robust equilateral triangle-shaped	Robust isosceles triangle-shaped	Long and pointed spines
terga I–IV (V) [12]	spines, B [occasionally]	spines, B [occasionally]	alternating with shorter ones
Figure 9(a–d)
6	Posterior margin of	Thin and robust isosceles and	Slim isosceles triangle-shaped	Long and pointed spines of
terga (V) VI–X [12]	equilateral triangle-shaped	spines of different length,	different length
Figures 9(e–h) and	spines of different length,	B [occasionally]
11(d)	B [occasionally]
7	Posterolateral part of	3–4 relatively prominent spines	0–6 small spines/3–13 small spines	0/−
terga VII/VIII–IX	and several small spines
[13]	sometimes/2–4 relatively
Figure 10(h)	prominent spines and several
small spines sometimes
8	Surface of sterna:	Flattened undulating corrugations	Flattened equilateral triangle-	−
cuticle structure	shaped corrugations
Figure 7(e–g)
9	Surface of sterna:	SC-et [sparse], SCS-et	Micropores, elongated Hr and B	−
setation	[dominant], micropores,
Figure 7(e–g)	elongated Hr and B
10	Posterior margin of	Robust equilateral triangle-shaped	Robust equilateral triangle-shaped	−
sterna: setation	spines, B [sparse]	spines, B [sparse]
Figure 7(f,g)
11	Gill I: shape	Asymmetrical, distinctly sinuous,	Asymmetrical, distinctly sinuous,	Slightly asymmetrical, nearly
Figure 10(a)	narrow, lanceolate	narrow, lanceolate	straight, narrow, lanceolate
12	Gills II–VI: shape	Asymmetrical, rounded apically,	Nearly symmetrical, leaf-shaped,	Nearly symmetrical, leaf-shaped,
Figure 10(b–f)	posterior margin distinctly	acutely pointed apically	pointed or bluntly pointed
concave	apically
13	Gill VII: shape	Asymmetrical, rounded apically,	Symmetrical, leaf-shaped, acutely	Nearly symmetrical, leaf-shaped,
Figure 10(g)	posterior margin distinctly	pointed apically	rounded apically
concave
14	Paraproct [inner	13–15 pointed strong teeth	More than 17–23 pointed, strong	Approximately 17 pointed teeth of
margin]: stout	teeth, alternating with smaller	different lengths
spines [14]	ones
Figures 10(i) and 11
(a,b)
15	Surface of paraproct:	Leaf-shaped SC and their SCS ,	Elongated Hr, B	−
setation	elongated Hr, B
Figures 10(i) and 11
(a–c)
16	Colour pattern: cerci	Slightly paler than body,	Slightly paler than body, darker	Paler than body, unicoloured
and paracercus	unicoloured light brown; no	distally; ringed by narrow dark	brown to dark brown; no rings
Figures 1(a) and 2(b)	rings	reddish-brown rings

( Continued ) Table II. (Continued).

Centroptilum luteolum (Müller,
Characters [remarks;	Centroptilum volodymyri sp. nov.	1776) [Europe, Middle Asia,	Centroptilum pirinense (Ikonomov,
No.	references; figures]	[type material]	Caucasus]	1962) [1] [Macedonia, Bulgaria]
17	Posterior margin of	Robust marginal spines;	Elongated marginal spines;	Long and pointed spines
caudal filament	occasional Hr and B , solitary	occasional Hr and B	alternating with shorter ones
segments: shape	submarginal SC-et
Figure 11(e,f)
18	Cerci length: relative to	approx. 0.5	approx. 0.5–0.6	approx. 0.5 [slightly longer than
body length	body]
19	Terminal filament	Distinctly shorter	Subequal or slightly shorter	Subequal or slightly shorter
length: relative to
cerci length
Egg
1	Length/width [µm]	130–140/65–70	180–215/120–135	−
Figure 12(a–d)
2	Chorionic surface:	Thin and flat	Relatively extended	−
shape of polygonal ridges [15]
Figure 12(e–h)
3	Cells of chorionic mesh:	2.5–4.2	3.0–6.4	−
diameter [µm]
Figure 12(e–h)

Notes: [1] Based on Ikonomov (1962) and Bauernfeind and Soldán (2012) . [2] Character tested by Bauernfeind and Soldán (2012: 180) . [3] Character tested by Kluge and Novikova 1992a: 63 ). [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]. [5] For C. luteolum , figured by Kluge and Novikova 1992: 63 , fig. 2.1); for C. pirinense , figured by Ikonomov (1962: 117 , fig. 27.1). [6] For C. pirinense , described and figured in detail by Ikonomov (1962: 114 , 115, figs 25.3–7). [7] Apicomedial projection in the middle of anterior margin was considered a generic character of Centroptilum by Bauernfeind and Soldán (2012: 178) ; character also figured for some Indocloeon ( Kaltenbach and Gattolliat 2017: 52 , fig. 4d). [8] For C. luteolum , figured by Kluge and Novikova 1992: 63 , fig. 2.4); for C. pirinense , figured by Ikonomov (1962: 115 , figs 25.8, 25.9). [9] For C. pirinense , described and figured in detail by Ikonomov (1962: 114 , fig. 24). [10] According to Bauernfeind and Soldán (2012: 181) , claws as long as 2/3 of tarsus length; Ikonomov (1962: 116 , figs 26.9–12) figured the legs with claws as long as about 1/2–2/3 of tarsus length. [11] For C. pirinense , the fragmentary cuticle structure figured by Ikonomov (1962: 116 , fig. 26.14) and most likely close to that in C. luteolum ; C. volodymyri sp. nov. similar to Anafroptilum kazlauskasi ( Kluge, 1983 ) in the presence of transparent scale-shaped bristles on abdominal terga of larvae ( Kluge 2012: 373 , fig. 43), in contrast to C. luteolum which lacks stout setae or scales on surface of terga. [12] For C. pirinense , briefly described and fragmentary figured by Ikonomov (1962: 116 , 117, fig. 26.14); the structure most likely close to that in C. luteolum . [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. Type material Holotype . Female mature larva , GEORGIA, Autonomous Republic of Adjara , Kobuleti District , vicinity of Chakhati village, valley of the Kintrishi River , small lentic water bodies, 41.762°N , 41.978° E , ′ 325 m a.s.l., 18 April 2016 , Martynov A. V. leg. (inventory number in NMNH NASU collection: Grg24Censp/1 ) . Table III. Genetic distances (COI) between sequenced species of Centroptilum , Cloeon and Procloeon and within them, using Kimura twoparameter.

C. luteolum	C. luteolum
C. volodymyri	clade 1	clade 2	C. victoriae	P. bifidum	P. pennulatum	C. dipterum	C. simile
C. volodymyri sp.	0.131
nov.
C. luteolum clade 1	0.254	0.041
C. luteolum clade 2	0.251	0.206	0.021
C. victoriae	0.262	0.271	0.257	-
P. bifidum	0.279	0.271	0.255	0.224	-
P. pennulatum	0.253	0.263	0.239	0.234	0.181	-
C. dipterum	0.264	0.282	0.26	0.242	0.184	0.192	-
C. simile	0.259	0.281	0.262	0.218	0.205	0.188	0.168	-

Paratypes . Six larvae , GEORGIA, same locality and date as holotype (three larvae in slides 648 , 661 , 662 ; one larva in ethanol deposited in NMNH NASU with inventory number Grg24Censp/2 ) ; 49 larvae , TURKEY , Rize il [Province], Fındıklı Region , KaÇkar Mountains , Büyük [ Çağlayan ] stream, 41.236°N , 41.270°E , 337 m a.s.l., 18 August 2012 , Palatov D. M . leg .; Non-type material. 10 larvae , IRAN Guilan Province , unnamed small brook (left tributary of Shalmanrud River ), SW of Amlash town, 37.046°N , 50.095°E , 185 m a.s.l., 21 May 2016 , Bojková J. , Soldán T. , Imanpour Namin J. leg .; One larva , IRAN , ibid., SW of Amlash town, 37.037°N , 50.083°E , 287 m a.s.l., 21 May 2016 , Bojková J. , Soldán T. , Imanpour Namin J. , leg .; 22 larvae , IRAN , Golestan Province , unnamed small brook (right tributary of Rude-Tengen River ), S of Qarangi-ye Jangal town, 37.529°N , 55.792°E , 604 m a.s.l., 23 June 2019 , Palatov D. M. leg . 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) ( Heptageniidae ), and Caenis macrura Stephens, 1835 ( Caenidae ). 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 0.3–0.6 m .s −1 . The mayfly taxocene consisted of the following taxa: Electrogena sp. ( Heptageniidae ) (dominant), Habroleptoides cf. confusa Sartori and Jacob, 1986 ( Leptophlebiidae ), B . ( R .) cf. gadeai , and N. ( A. ) muticus ( Baetidae ). Affinities We attribute C. volodymyri sp. nov. to the genus Centroptilum based on larval and egg characters depicted by Jacob (1991) , Kluge and 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) mandibular incisors deeply separated into two groups from their base; (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 Kluge and 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) ]. Figure 13. Habitats of Centroptilum volodymyri sp. nov. in Georgia, Iran and Turkey. (a, b) Small lentic waterbody, Georgia, Adjara, type locality (April 2016; photos by A. Martynov); (c, d) small brook, left tributary of Shalmanrud River, Guilan Province, Iran (May 2016; photos by J. Bojková); (e, f) small stream, Rize Province, Turkey (August 2012; photos by D. Palatov). Figure 14. Map of Centroptilum volodymyri sp. nov. distribution. Red star – type locality; green pentagrams - non-type localities. 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 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. Figure 15. Maximum likelihood tree including several representatives of the genus Centroptilum . Bootstrap supports (BS) are indicated on branches for Centroptilum volodymyri sp. nov. and C. luteolum (Müller, 1776) . 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 ( K 2 P distances higher than 20%).