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

Figures & data

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.

Species	Specimen catalogue number	Country	Locality	Latitude	Longitude	Date	GenBank ID	GenSeq nomenclature
Centroptilum volodymyri sp. nov.	GBIFCH00895431	Georgia	Adjara, Kobulety District, vicinity of Chakhati village, valley of the Kintrishi River, small lentic water bodies [Grg24Censp]	41.762222	41.978111	18 April 2016	OL960099	genseq-2 COI
Centroptilum volodymyri sp. nov.	GBIFCH00895432	Iran	Guilan Province, SW from Amlash town, unnamed small brook	37.036944	50.082500	21 May 2016	OL960097	genseq-3 COI
Centroptilum volodymyri sp. nov.	GBIFCH00895437	Iran	Golestan Province, Kalaleh County, vicinity of Zav-e-Bala village, unnamed stream [Iran3Censp/1]	37.529433	55.791550	23 June 2019	OL960098	genseq-3 COI
Centroptilum luteolum	GBIFCH00895434	Georgia	Adzharia, territory of Kobulety town, Kintrishi River [Grg6Cenlut]	41.803889	41.775833	4 June 2013	OL960101	genseq-4 COI
Centroptilum luteolum	GBIFCH00895435	Ukraine	Luhansk Region, Antratsyt District, vicinity of Khrustal’nyi village, Khryshtaleva River [Lug 94]	48.182321	38.877363	30 April 2012	OL960102	genseq-4 COI
Centroptilum luteolum	GBIFCH00895441	Switzerland	Burtignière, Vallée de Joux	46.561667	6.170000	23 August 2001	OL960100	genseq-4 COI

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.

No.	Characters [remarks; references; figures]	Centroptilum volodymyri sp. nov. [type material]	Centroptilum luteolum (Müller, 1776) [Europe, Middle Asia, Caucasus]	Centroptilum pirinense (Ikonomov, 1962) ^[1] [Macedonia, Bulgaria]
Larva
I. Head (colour, shape and setation)
1	Colour pattern Figures 1(a,b) and 2(a–c)	Yellowish-brown to light brown; diffused light brown maculation on frons and vertex; clypeus and genae brown	Light brown; clypeus and genae brown	Light brown; diffused pale maculation
2	Antenna length^[2] Figures 1(a,b) and 2(a,b)	Distinctly longer than head and thorax	Slightly longer than head and thorax	Reaching approximately the bases of fore wings or longer than head and thorax
3	Surface of scape and pedicel: setation Figure 3(a,b)	Sparse Hr and B	Sparse Hr and B	−
4	Posterior margin of flagellar segment (central part of flagellum): shape Figure 3(c,d)	Sparse row of 10–14 well separated, robust, bluntly pointed or rounded apically spines 1 or grouped by 2 B setae and sparse Hr	Dense row of 16–20 robust, bluntly pointed or rounded apically spines, grouped by 2–3 spines with common basis; 1 or grouped by 2–3 B setae	−
5	Surface of clypeus and frons: setation Figure 3(e,f)	Sparse B and long Hr	Sparse B and long Hr	−
6	Frontal suture: shape ^[3] Figure 3(f)	V-shaped	V-shaped to U-shaped	−
II. Mouthparts
1	Labrum: width/length ratio^[4]	1.8–2.0	1.4–1.6	approx. 2.0
2	Labrum: dorsal surface Figures 4(a) and 5(b)	Dense short and long Hr; sparse B	Dense short and long Hr; sparse B	Dense short and long Hr; sparse B
3	Labrum: anterior margin Figures 4(a) and 5(a,b)	Slightly concave; median incurvation relatively deep, angular, U-shaped	Nearly straight; median incurvation shallow, angular	Nearly straight; small medial semicircular incurvation
4	Ratio of width of maxillary palp segments I/II^[5] Figures 4(f) and 5(f)	1.3–1.6	1.2–1.8	1.2
5	Segment III of maxillary palp; ratio length segments III/II Figures 4(e,f) and 5(f)	Rounded apically; distinctly longer than segment II – 1.6–1.7	Pointed or bluntly pointed apically; subequal to distinctly longer than segment II – 1.1–1.7	Pointed apically; subequal to segment II – 1.1
6	Right mandible: number of incisors of mandibular incisor groups^[6] Figure 4(d)	3 + 2	3 + 2	3 + 2
7	Left mandible: number of incisors of mandibular incisor groups^[6] Figure 4(c)	4 + 2	4 + 2 (occasionally 4 + 3)	4 + 3
8	Mandibles: surface Figure 4(c,d)	Occasionally sparse B and Hr	No setae; occasionally sparse B and long Hr, small prominent isosceles and equilateral triangle-shaped corrugations, and micropores	Occasionally sparse B and Hr
9	Hypopharynx: shape of superlinguae^[7] Figure 5(c)	Rounded; no triangular apicomedial projection	Rounded; small triangular bluntly pointed apicomedial projection	Rounded; no triangular projection
10	Segment II of labial palp [dorsal view]: setation Figure 5(h)	6–10	2–9	6–7
11	Segment II of labial palp: distal end Figures 5(g,h)	Distinctly wider than segment III base	Equal width or slightly wider than segment III base	Equal length or slightly wider than segment III base
12	Segment III of labial palp: shape^[8] Figures 5(g,h)	Distinctly expanding apically; nearly trapezoidal; inner margin concave; posterolateral and posteromedian corners of different shape	Moderately expanding apically; roughly quadrangular; inner apical margin slightly concave; posterolateral and posteromedian corners of different shape	Slightly expanding apically; roughly quadrangular; inner margin slightly concave; posterolateral and posteromedian corners of very similar shape
III. Thorax and legs (colour, shape and setation)
1	Colour pattern: pronotum^[9] Figures 1(a,b) and 2(a,b)	Dark brown to blackish brown maculation centrally; pale diffused spots laterally	Light yellowish brown; brownish maculation centrally and laterally	Light brown; two pairs of large dissolved spots centrally and laterally
2	Surface of pronotum: cuticle structure Figure 6(a,b)	Flattened, stretched shallow triangular and semilunar-shaped corrugations	Prominent rounded and broadly triangular corrugations	−
3	Surface of pronotum: setation Figure 6(a,b)	Hr, B, micropores, sparse SC-et and SCS-et	Sparse Hr and B	−
4	Colour pattern: legs Figures 1(a,b) and 2(b)	Yellowish to dirty brown; all leg segments slightly darker distally	Femora with brown transversal band distally; tibiae darkened proximally	Unicolour light brown
5	Surface of legs: cuticle structure Figure 6(c–f)	Flattened equilateral triangle- and semilunar-shaped corrugations	Prominent isosceles and equilateral triangle-shaped corrugations	−
6	Outer margin of femora: stout setae Figure 8(a–c)	Numerous short, pointed	Occasional short, pointed	Short, pointed setae
7	Surface of femora [dorsal view]: setation Figure 6(c,d)	SC-et, SCS-et, Hr, B	Hr, B, pointed stout setae with feathered margins mainly near inner margin [sparse]	−
8	Surface of tibiae [dorsal view]: setation Figure 6(e,f)	SC-et, SCS-et [dominant], Hr, B	Long Hr, B	−
9	Surface of tarsi [dorsal view]: setation	SC-et, SCS-et [dominant], Hr, B	Hr, B, pointed stout setae with feathered margins mainly near inner margin [sparse]	−
10	Pretarsal claw length relative to tarsus length^[10] Figures 7(a,b) and 8(a–c)	≤ 1/2	≈ 1/2	1/2–2/3
11	Pretarsal claw: number of small teeth per row Figure 7(a–d)	More than 30 in basal half to 2/3 of claw length	About 18–27 in basal third	About 15–20 in basal half [13–16 in original description]
IV. Abdomen (colour, shape and setation)
1	Colour pattern: terga II–VIII (IX) Figures 1(a,b) and 2(a,b)	Terga dark, yellowish brown to intensively brown; terga II–VIII often with more or less distinct colour pattern consisting of pale spots, maculae and strips centrally, and darker spots laterally	Terga pale, ivory-white, usually with a pair of small, dark brown spots in the middle; tergum VI usually darkest; terga II and VI often with dark spot centrally on paler background	Terga unicoloured, brownish, with indistinct pale diffused area and two unclear spots centrally; clearer colour pattern on terga VII–IX (occasionally VI)
2	Surface of terga: cuticle structure^[11] Figure 9(a–h)	Flattened equilateral triangle- and semilunar-shaped corrugations	Prominent isosceles and equilateral triangle-shaped corrugations	Prominent isosceles and equilateral triangle-shaped corrugations
3	Surface of terga I–IV: setation^[12] Figure 9(a–d)	SC-et [dominant], SCS-et [sparse], micropores, Hr and B	Micropores, Hr and B	SC-et
4	Surface of terga V–X: setation^[12] Figure 9(e–h)	SC-et [sparse], SCS-et [dominant], micropores, Hr and B	Micropores, Hr and B	SC-et
5	Posterior margin of terga I–IV (V)^[12] Figure 9(a–d)	Robust equilateral triangle-shaped spines, B [occasionally]	Robust isosceles triangle-shaped spines, B [occasionally]	Long and pointed spines alternating with shorter ones
6	Posterior margin of terga (V) VI–X^[12] Figures 9(e–h) and 11(d)	Thin and robust isosceles and equilateral triangle-shaped spines of different length, B [occasionally]	Slim isosceles triangle-shaped spines of different length, B [occasionally]	Long and pointed spines of different length
7	Posterolateral part of terga VII/VIII–IX ^[13] Figure 10(h)	3–4 relatively prominent spines and several small spines sometimes/2–4 relatively prominent spines and several small spines sometimes	0–6 small spines/3–13 small spines	0/−
8	Surface of sterna: cuticle structure Figure 7(e–g)	Flattened undulating corrugations	Flattened equilateral triangle-shaped corrugations	−
9	Surface of sterna: setation Figure 7(e–g)	SC-et [sparse], SCS-et [dominant], micropores, elongated Hr and B	Micropores, elongated Hr and B	−
10	Posterior margin of sterna: setation Figure 7(f,g)	Robust equilateral triangle-shaped spines, B [sparse]	Robust equilateral triangle-shaped spines, B [sparse]	−
11	Gill I: shape Figure 10(a)	Asymmetrical, distinctly sinuous, narrow, lanceolate	Asymmetrical, distinctly sinuous, narrow, lanceolate	Slightly asymmetrical, nearly straight, narrow, lanceolate
12	Gills II–VI: shape Figure 10(b–f)	Asymmetrical, rounded apically, posterior margin distinctly concave	Nearly symmetrical, leaf-shaped, acutely pointed apically	Nearly symmetrical, leaf-shaped, pointed or bluntly pointed apically
13	Gill VII: shape Figure 10(g)	Asymmetrical, rounded apically, posterior margin distinctly concave	Symmetrical, leaf-shaped, acutely pointed apically	Nearly symmetrical, leaf-shaped, rounded apically
14	Paraproct [inner margin]: stout spines^[14] Figures 10(i) and 11(a,b)	13–15 pointed strong teeth	More than 17–23 pointed, strong teeth, alternating with smaller ones	Approximately 17 pointed teeth of different lengths
15	Surface of paraproct: setation Figures 10(i) and 11(a–c)	Leaf-shaped SC and their SCS, elongated Hr, B	Elongated Hr, B	−
16	Colour pattern: cerci and paracercus Figures 1(a) and 2(b)	Slightly paler than body, unicoloured light brown; no rings	Slightly paler than body, darker distally; ringed by narrow dark reddish-brown rings	Paler than body, unicoloured brown to dark brown; no rings
17	Posterior margin of caudal filament segments: shape Figure 11(e,f)	Robust marginal spines; occasional Hr and B, solitary submarginal SC-et	Elongated marginal spines; occasional Hr and B	Long and pointed spines alternating with shorter ones
18	Cerci length: relative to body length	approx. 0.5	approx. 0.5–0.6	approx. 0.5 [slightly longer than body]
19	Terminal filament length: relative to cerci length	Distinctly shorter	Subequal or slightly shorter	Subequal or slightly shorter
Egg
1	Length/width [µm] Figure 12(a–d)	130–140/65–70	180–215/120–135	−
2	Chorionic surface: shape of polygonal ridges^[15] Figure 12(e–h)	Thin and flat	Relatively extended	−
3	Cells of chorionic mesh: diameter [µm] Figure 12(e–h)	2.5–4.2	3.0–6.4	−

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.

Table III. Genetic distances (COI) between sequenced species of Centroptilum, Cloeon and Procloeon and within them, using Kimura two-parameter.

	C. volodymyri	C. luteolum clade 1	C. luteolum clade 2	C. victoriae	P. bifidum	P. pennulatum	C. dipterum	C. simile
C. volodymyri sp. nov.	0.131
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	-

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.

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.

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.

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.

Figure 8. Larvae of Centroptilum volodymyri sp. nov., paratypes. (a) Fore leg; (b) middle leg; (c) hind leg.

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.

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.

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.

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.

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).

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