Rhodeus caspius, a new bitterling from Iran (Teleostei: Cypriniformes Acheilognathidae) Author Esmaeili, Hamid Reza Ichthyology and Molecular Systematic Research laboratory, Zoology Section, Department of Biology, School of Science, Shiraz University, Shiraz, Iran. Author Sayyadzadeh, Golnaz 0000-0003-0758-6502 Ichthyology and Molecular Systematic Research laboratory, Zoology Section, Department of Biology, School of Science, Shiraz University, Shiraz, Iran. & g. sayyadzadeh 92 @ gmail. com; https: // orcid. org / 0000 - 0003 - 0758 - 6502 g.sayyadzadeh92@gmail.com Author Japoshvili, Bella 0000-0003-0966-0622 Department of Hydrobiology and Ichthyology, Institute of Zoology, School of Natural Sciences and Medicine, Ilia State University, Tbilisi, Georgia. bela _ japoshvili @ iliauni. edu. ge; https: // orcid. org / 0000 - 0003 - 0966 - 0622 bela_japoshvili@iliauni.edu.ge Author Eagderi, Soheil Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran. Author Abbasi, Keivan 0000-0001-5095-2905 Inland Waters Aquaculture Research Center, Iranian Fisheries Sciences Research Institute, Agricultural Research, Education and Extension Organization, Bandar Anzali, Iran. keyvan _ abbasi @ yahoo. com; https: // orcid. org / 0000 - 0001 - 5095 - 2905 Department of Fisheries, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran. keyvan_abbasi@yahoo.com Author Mousavi-Sabet, Hamed text Zootaxa 2020 2020-09-10 4851 2 319 337 journal article 9134 10.11646/zootaxa.4851.2.6 f856b95c-dd39-4a84-b152-11bce44818e2 1175-5326 4407661 589A7843-16F6-4829-A98D-933801B06024 Rhodeus caspius , new species ( Figs. 2–11 ) Holotype . ZM-CBSU H1005, 46 mm SL; Iran : Guilan prov. : Shakhraz (=Shakhazar or Siahdarvishan) River , the Anzali Lagoon drainage, the Caspian Sea basin, at Tolamshahr , 37°16’3.98”N 49°21’53.82”E . Paratypes . ZM-CBSU H1001, 4, 42–46 mm SL; VMFC RC4 , 4 , 35–42 mm SL; same as holotype . ZM- CBSU H1010 , 1 , 34 mm SL; Iran : Mazandaran prov. : Siah River , near Qaemshahr , 36°26’6.13”N 52°56’58.91”E ). ZM-CBSU H1011, 2, 36–44 mm SL; Iran : Mazandaran prov. : Tajan River at Goharbaran , the Caspian Sea basin, 36°42’44.3’’N 53°07’51.8’’E . ZM-CBSU H1013, 2, 31–34 mm SL; Iran : Mazandaran prov. : Babol River , the Caspian Sea basin, 36°30’50.2’’N 52°40’36.2’’E . ZM-CBSU D193, 1, 37 mm SL; ZM-CBSU D196, 1, 35 mm SL; –ZM-CBSU D199, 1, 28 mm SL; ZM-CBSU D203, 1, 35 mm SL; ZM-CBSU D205, 1, 35 mm SL; Iran : Kordestan prov. : Zarrineh River at Adinan village , Urmia Lake basin, 36°12’13.7’’N 46°25’44.9’’E . Material used in the molecular genetic analysis. Rhodeus caspius : ZM-CBSU M 526; Iran : Kermanshah prov. : Razavar River at Qazanchi village , Tigris basin, 34°26’40.5’’N 47°01’30.1’’E ( GenBank accession numbers: MT 878140 ). ZM-CBSU M527; Iran : Kermanshah prov. : Kamiaran River at Kamiaran , the Tigris basin, 34°48’00.34’’N 46°56’27.65’’E ( GenBank accession numbers: MT 878141 ) . ZM-CBSU M1303; Iran : Mazandaran prov. : Babolrud River , Caspian Sea basin, 36°30’50.2’’N 52°40’36.2’’E . ( GenBank accession numbers: MT 878142 ) . IMNRFI-UT 24A; Iran : Guilan prov. : Anzali Lagoon , the Caspian Sea basin, 37°26’21”N 49°27’21”E ( GenBank accession numbers: MT 878143 ) . IMNRFI-UT 22B; Iran : Mazandaran prov. : Babol River , Caspian Sea basin, 36°30’50.2’’N 52°40’36.2’’E ( GenBank accession numbers: MT 878144 ) . IMNRFI-UT 23 T ; Iran : Mazandaran prov. : Tajan River , Caspian Sea basin, 36°42’44.3’’N 53°07’51.8’’E ( GenBank accession numbers: MT 878145 ) . Rhodeus colchicus : ZM-CBSU M2487 , M2490, M2491, M2492; Georgia : Samegrelo : Khobi River , Black Sea basin, 42° 15’ 50.7708’’ N 41° 46’ 4.242’’ E (GenBank accession numbers: MT 878146 , MT 878147 , MT 878148 , MT 878149 ). Diagnosis. Rhodeus caspius sp. nov. is distinguished morphologically from its closest species, R. amarus by having short post-dorsal distance (28–30 vs. 31–34% SL); and from R. colchicus by having long caudal peduncle length (26–30 vs. 23–25% SL) and head length (27–30 vs. 25–26% SL). It is also characterized by one fixed diagnostic nucleotide substitutions and a K2P nearest neighbor distance of 1% to R. amarus and R . colchicus in the mtDNA COI barcode region ( Table 2 ). Based on Bogutskaya & Komlev (2001) , Rhodeus colchicus is distinguished from congeners by a suite of characters which includes large scales (34 to 37, commonly 35, in the lateral row), commonly 9 branched rays in both the dorsal and anal fins, a low number of vertebrae (33 to 36, commonly 35, total, and 16 to 18, commonly 17, in the abdominal region), a deep shortened second infraorbital, a broad fourth infraorbital and a well-developed fifth one with a widened lamellate portion. Rhodeus caspius sp. nov. is distinguished from R. meridionalis by having a subinferior mouth (vs. terminal), upper lip covered completely or more than half (vs. rostral cap covering upper part of upper lip (according to Kottelat & Freyhof 2007 )) and low number of pored scales in lateral line (3–8, usually 6 vs. 11 (according to Karaman (1924)) . FIGURE 1. Maximum Likelihood and Bayesian phylogeny reconstructed based on COI gene sequences. The values beside the branches before and after a slash are BI posterior and ML bootstrap probability values, respectively. Black bars to the right of specimen labels indicate species delimitation results from PTP followed by the results of the mPTP approach as dashed bars. FIGURE 2. Rhodeus caspius , holotype, a, ZM-CBSU H1005, 46 mm SL; paratypes, b, ZM-CBSU H1003, 44 mm SL; c, ZM- CBSU H1004, 43 mm SL; d, ZM-CBSU H1008, 38 mm SL; Iran: Shakhraz River. TABLE 2. Estimates of evolutionary divergence (%) over sequence pairs between species found in the COI barcode region of Rhodeus species studied.

R. amarus	R. atremius	R. caspius sp. nov.	R. colchicus	R. fangi	R. lighti	R. meridionalis	R. notatus	R. ocellatus	R. sericeus	R. shitaiensis	R. sinensis	R. uyekii
R. amarus
R. atremius	15
R. caspius sp. nov.	1	14
R. colchicus	1	16	1
R. fangi	15	3	15	16
R. lighti	17	19	17	17	19
R. meridionalis	2	16	2	1	16	16
R. notatus	15	3	15	16	0.4	19	16
R. ocellatus	17	19	17	17	19	1	16	19
R. sericeus	20	20	20	20	19	21	20	19	21
R. shitaiensis	13	13	13	14	13	18	14	12	18	16
R. sinensis	18	20	18	18	20	5	17	20	6	20	20
R. uyekii	16	17	15	15	16	17	15	15	17	20	13	18

In the R. sericeus group, R. caspius sp. nov. is distinguished from R. sericeus by having low number of scales in lateral series (34–37 vs. 36–40 according to Holcik & Jedlicka 1994 ). Rhodeus caspius sp. nov. is distinguished from Rhodeus sinensis which was formerly placed in R. sericeus as a subspecies, by having low number of branched dorsal fin rays 9–10, usually 9 (vs. 12–13, based on Bogutskaya & Komlev 2001 ), low number of branched anal fin rays 8–9, usually 9 (vs. 10, based on Bogutskaya & Komlev 2001 ), higher number of gill rakers 10-13 vs. 6–7, based on Arai et al. 2001 ), and low range of maximum body depth (35–39 vs. 32–55, based on Arai et al . 2001 ). Rhodeus caspius sp. nov. is distinguished from R. pseudosericeus , another species in the Rhodeus sericeus group, by low number of anal fin branched ray (8–9 usually 9 vs. 9–11, mode 10 based on Arai et al . 2001 ), iris color of males (bright red vs. blackish). Rhodeus caspius sp. nov. differs from the R. smithii complex including R. smithii (Regan, 1908) , R . notatus Nichols, 1929 , R . suigensis (Mori, 1935) , R . atremius ( Jordan & Thompson, 1914) and R . fangi (Miao, 1934) , distributed in China , Korea , and Japan , in the gill raker number on the outer side of the first gill arch (10-13, vs. 4–8, based on Arai et al . 2001 ). Rhodeus caspius sp. nov. is distinguished from R. ocellatus by having lower number of branched dorsal fin ray [(9–10, usually 9 vs. 10-12 (according to Kottelat & Fryhof (2007))], and females and juveniles with a black blotch in anterior part of dorsal (vs. no blotch). Rhodeus caspius sp. nov. is also distinguished from East Asian Rhodeus species. Rhodeus caspius sp. nov. is distinguished from R. albomarginatus Li & Arai, 2014 from the Lvjiang River, a tributary flowing into Poyang Lake of Yang-tze River basin, in Anhui Province , China by branched anal-fin rays 9–10, usually 9 (vs. modally10–11 based on Li & Arai 2014 ); and colour pattern of adult males (dorsal and anal fins of males edged with black margin vs. white margin, according to Li & Arai (2014)) . Rhodeus caspius sp. nov. is also distinguished from R. shitaiensis Li & Arai, 2011 by usually low number of pored scale (3–8 vs. 6–12) and transverse scale 10-12 (vs. 10 according to Li & Arai (2011)) . Rhodeus caspius sp. nov. is distinguished from R . haradai Arai, Suzuki & Shen, 1990 , R . spinalis Oshima, 1926 , from southern China ( Arai et al . 1990 ), and R. laoensis Kottelat, Doi & Musikasinthorn in Kottelat (1998) from Laos , in having fewer anal fin branched rays (8–9 usually 9, vs. more than 11, based on Arai et al . 2001 ). FIGURE 3. Rhodeus caspius , Uncatalogued ; a , and c , male, b , female, Anzali wetland; d , male, Razavar River; e , female, Tajan River and f , male, Babol River. Rhodeus caspius sp. nov. differs from R . rheinardti (Tirant, 1883) from Vietnam in having fewer dorsal (9–10, usually 9, vs. 12–13) and anal fin branched rays (8–9 usually 9, vs. 13–14, based on Arai et al . (2001)) . Description. For general appearance and morphometric data see Figures 2–4 and Table 3–4 . Small sized and deep and compressed body species with short head. Head length 27–30% SL, considerably less than the maximum body depth, 34–42% SL. The greatest body depth at dorsal-fin origin and shallowest near caudal-fin base. Dorsal body surface profile convex and ventral body surface profile rounded. Snout usually shorter than eye diameter (22–30 and 28–35% HL, respectively). Mouth sub-inferior. Lower lip well developed but not thick, not interrupted in the middle. Barbels absent. Dorsal fin with 3 simple and 9–10 (usually 9) branched rays. Anal fin with 2–4 (usually 3) simple and 8–9 (usually 9) branched rays. Pectoral fin with 10-12 (usually 12) rays. Pelvic fin with 7–8 (usually 8) rays. Caudal fin forked with 9 + 8 branched rays; its lobes rounded distally; lobes nearly equal in length. First simple rays of dorsal and anal fins very small, embedded under scales at base. Longest simple ray of dorsal fin strong and stiff, distally segmented; width of basal part about twice that of first branched dorsal-fin ray base. Body covered with large cycloid scales. Lateral line incomplete: pored scales 3–8, usually 6. Longitudinal scale series 34–37. Transverse scale rows 10-12. Predorsal scale rows 14–16 including one deeply notched in front of first dorsal fin ray. Scale rows around caudal peduncle 12–14. Cephalic sensory canals ( Fig. 4 ) categorized as Type II with incomplete infraorbital canal (IOC). Supraorbital canal pores (SOC) 6–8; infraorbital + temporal canal pores (IOC+TC) 10-15; preopercular + mandibular canal pores (PC+MC) 8–11 and supratemporal canal pores (STC) 0-4 ( Table 4 ). Dark spot on scales behind gill cleft. Total vertebrae 35–36. Gill rakers on external side of first gill arch 10-13. Pharyngeal teeth in one row, 0.0.5–5.0.0. A developed ovipositor in females during breeding season. TABLE 3. Morphometric data of Rhodeus caspius sp. nov. , holotype ZM-CBSU H1005, paratypes ZM-CBSU H1003– H1004, H1006–H1009, Shakhraz River; H1010, Siah River; H1011–H1012, Tajan River; H1013-H1014, Babol River; D193, D196, D199, D203, D205, Zarrineh River (n=17). The holotype is included in calculations of range, mean and SD.

Holotype	Range	Mean	SD
Standard length (mm)	45.8	28.2–45.8	37.4
In percent of standard length
Head length	28.1	26.8–29.8	28.0	0.8
Predorsal length	56.9	55.1–58.4	56.8	1.1
Postdorsal length	28.1	28.1–30.4	29.6	0.8
Dorsal fin length	21.5	16.9–22.2	19.5	1.4
Anal fin length	19.4	14.3–19.4	16.4	1.2
Preanal length	66.1	63.6–67.7	65.6	1.3
Pectoral fin length	20.5	16.4–21.3	18.7	1.2
Pelvic fin length	16.9	14.4–17.4	16.0	0.8
Prepelvic length	51.0	48.7–53.6	50.7	1.2
Maximum body depth	40.5	34.4–42.0	36.9	2.1
Minimum body depth	12.9	10.2–12.9	11.5	0.8
Distance between pectoral and pelvic-fin origins	25.2	20.6–26.0	23.3	1.4
Distance between pelvic and anal-fin origins	16.5	12.7–16.9	15.6	1.1
Distance between pectoral and anal-fin origins	40.8	35.6–42.0	39.3	1.7
Length of caudal peduncle	27.9	26.2–29.9	27.6	1.0
Maximum body width	14.1	11.9–14.2	13.0	0.7
In percent of head length
Maximum head depth	76	73–85	78.2	3.3
Snout length	30	22–30	28.2	1.7
Postorbital distance	47	39–48	42.4	2.8
Interorbital width	39	33–44	37.8	2.5
Eye diameter	31	28–35	32.1	1.4
Maximum head width	54	47–56	51.7	2.5

TABLE 4. Cephalic sensory canals, canal pores in the studied Rhodeus species.

R. amarus	R. caspius sp. nov.	R. colchicus
Supraorbital canal pores (SOC)	6-7	6-8	6-8
Infraorbital + temporal canal pores (IOC+TC)	11-12	10-15	11-12
Preopercular + mandibular canal pores (PC+MC)	8-10	8-11	8-10
Supratemporal canal pores (STC)	1-2	0-4	0-3

Osteological characteristics ( Figs. 5–11 ). In ethmoid region of neurocranium, supraethmoid with two horizontal and vertical portions; horizontal part wider possessing three posterior lobes; presence of two small processes in vertical part of supraethmoid-ethmoid with two small nasals positioned on its lateral sides; lateral ethmoid blade-shaped; vomer V-shaped anteriorly and pointed posteriorly; two cartilaginous preethmoid I in antero-lateral part of vomer; In in orbital region, frontal wide posteriorly with two lateral pointed process; two oval supraorbitals located on antero-lateral part of frontal; orbitosphenoid blade-shaped; ptersphenoid situated between orbitosphenoid and sphenotic; anterior and posterior half of parasphenoid elongated with two central wings bended dorsally; circumorbital series with four infraorbital, one supraorbital and lachrymal elements. In otic region, epiotic almost circular in shape; pterotic oval in shape the parietal tetragonal with its edges overlaping frontal, pterotic, epiotic and supraoccipital; sphenotic with an anteroventral pointed process fused to posterolateral process of frontal; ventral edge of prootics connected to each other. In occipital region, supraoccipital with a triangular crest. In ventral view, exoccipital almost L-shaped; lateral margin of basioccipital reclined dorsally having a pharyngeal process with a ventral process. FIGURE 4. Cephalic sensory canal system of Rhodeus caspius , holotype, ZM-CBSU H1005, 46 mm SL; Iran: Shakhraz River. Upper jaw with two paired maxillaries and premaxillaries; anterior part of maxillary broader having a developed mid-dorsal ascending process; anterior part of premaxillae possessing rostral ascending process with posterior narrower part. In suspensorium, dorsal part of hyomandibular wider; dorsal process of metapterygoid triangular and bended medially; endopterygoid with a pointed process just above its facet; ectopterygoid under anteroventral side of endopterygoid; quadrate with two dorsal and ventral portions; symplectic elongated and narrow and anterior part of palatine pointed bearing two dorsal and one ventral processes. In opercular series, anterior part of subopercle wider bearing an anterodorsal small process; interopercle with a knife-like shape overlapping with ventral part of crescent shaped interopecle. In hyoid arch, narrow basihyal with a ventral blade-shaped process; urohyal with two horizontal and vertical parts; posterior part of ceratohyal wider and epihyal triangular in shape and a small interhyal bone located on its posterior part; hypohyal with two paired dorsal and ventral elements. Three pairs of branchiostegal rays, branchial apparatus with four pairs of ceratobranchial, four pairs of epibranchial, three pairs of hypobranchial, two pairs of oval shaped pharyngobranchial and three unpaired basibranchial bones. Pharyngeal teeth in a row with a dental formula of 5-5. Pectoral girdle with an almost L-shaped cleithrum having a large and narrow postcleithrum in its rear; coracoid triangular in shape connecting to cleithrum with a spiral mesocoracoid; scapula broadened ventrally and pointed dorsally with a large foramen in its middle part; supracleithrum narrow, attached to a triangular-shaped posttemporal dorsally; four radials in pectoral fin. In pelvic girdle, paired anteriorly bifurcated basipterygium attached to each other; L-shape paired lateral-pterygium positioned in lateral side of basipterygium and three pairs of meta-pterygium located behind it. FIGURE 5. Skeleton of R. caspius (X-ray images), a, ZM-CBSU H1002, b, ZM-CBSU H1001; Iran: Shakhraz River. Dorsal fin with 3 unbranched and 9½ branched rays, 10 pterygiophores and one stay; first dorsal pterygiophore adjacent to 11 th or12 th vertebra; 7–8 supraneurals in front of dorsal fin; anal fin with 3 unbranched and 9½ branched rays (10½ found in one species), 10 pterygiophores and a small stay, first pterygoid is next to 18 th or 19 th vertebra. 35–36 vertebrae in axial skeleton. Caudal fin forked, and its skeletal structure including epural, parahypural, pleurostyle, uroneural and six hypurals bones. Caudal fin with 19 branched rays and various numbers of procurrent rays. Sexual dimorphism. The female has an ovipositor near the genital opening; males develop tubercle-bearing scales on the snout, mature males are colorful during the spawning season. Coloration. Adult males are especially colorful in the spawning season: Dorsal surface of head and back olive to bright green, reddish or dark violet, iris bright red, flanks iridescent with violet and steel-blue colors most evident, throat and belly orange to red, anal fin bright red and margined with black, a well-pronounced bright red spot on the first four rays of dorsal fin, caudal fin bright green at the base and yellow distally, pectoral and pelvic fins yellowish with dark pigments specially on pelvic fin membranes, females more yellowish and less iridescent than males in the spawning season, both sexes similar in color with a grey-green back, silvery flanks and yellowish belly outside the breeding season, a grey-green to greenish-blue stripe originates under the dorsal fin and extends back to the tail base, broadening posteriorly being broader in males, the dorsal fin blackish and other fins reddish to yellowish, the dorsal, and sometimes the anal fin, with a dark interrupted stripe, the iris silvery or yellowish. FIGURE 6. Dorsal (a), lateral (b) and ventral (c) views of the neurocranium in Rhodeus caspius . Abbreviations: Bo: basioccipital; Epo: epiotic; Exo: exoccipital; Fr: frontal; Le: lateral ethmoid; Nas: nasal; Orb: orbitosphenoid; Pa: parietal; Pe: preethmoid I; Ptt: posttemporal; Pr-Pp: posterior pharyngeal process; Pro: prootic; Ps: parasphenoid; Pts: pterosphenoid; Pto: pterotic; Stt: supratemporal; Scl: supracleithrum; So-cr; supraoccipital crest; So: supraorbital; Soc: supraoccipital; Sp: sphenotic; Se: supraethmoid; Vo: vomer. Scale bar is 2 mm. FIGURE 7. Internal view of circumorbital series (a) and the upper (b) and lower (c) jaws of Rhodeus caspius . Abbreviations: An: Angular; Crb: coronoid process; Cm: coronomeckelian; Dn: dentary; Io 2-5: infraorbitals 2-5; Keth: kinethmoid; Ra: retroarticular; Mx: maxillary; Pmx: premaxillary; Mdip: maxillary descending process; Mip: maxillary mid-lateral ascending process. FIGURE 8. The suspensorium and opercular series (c) in Rhodeus caspius . Abbreviations: Ect: ectopterygoid; End: endopterygoid; Hm: hyomandibulare; Iop: interopercle; Mtp: metapterygoid; Op: opercle; P: palatine; Pop: preopercle; Q: quadrate; Sop: subopercle; Sym: symplectic. FIGURE 9. Ventral view of branchial (a) and hyoid (b) arches in Rhodeus caspius . Bhy: basihyal; Brs;branchiostegal rays; Chy: ceratohyal; Epy: epihyal; Hhy: dorsal and ventral hypohyal; Ihy: interhyal; Uhy: urohyal; Bbr: basibranchial; Cbr: ceratobranchial; Ebr: epibranchial; Hbr: hypobranchial; Pbr: inphrapharyngobranchial. FIGURE 10. Medial view of pectoral girdle (left side) (a) and pelvic girdle (b) of Rhodeus caspius . Abbreviations: Cl: cleithrum; Co; coracoid; Mco: mesocoracoid; Mlp; mid-lateral process; Pb: pelvic bone; Pcl; postcleithrum; Ps: pelvic splint; R: radials; Sc: scapula. Distribution. Rhodeus caspius sp. nov. is originally known from the Caspian Sea basin but it has been translocated to two the Tigris River drainage and Urmia Lake basin ( Fig. 12 ). Habitat. Rhodeus caspius sp. nov. favors heavily vegetated areas of lakes, ponds and slow-running rivers, rarely in the faster waters. It is found in the lower reaches of rivers on the Caspian Sea coast, Urmia Lake and Tigris River drainage of Iran ( Fig. 13 ). Biology. Reproductive season in the Siah River from March to June, peaking in April, average absolute fecundity 329.74 eggs and average relative fecundity 187.4 eggs/g based on Patimar et al . (2010) . Average number of eggs 78.8 for examined fishes from the Sefid River ( Norouzi & Abbasi 2015 ). Ovipositor length up to 126.5% of standard length in the Anzali Lagoon in April (Holcik in Bănărescu 1999 ). Maximum life span 8–9 years ( Norouzi et al . 2014 , Norouzi & Abbasi 2015 ). FIGURE 11. Lateral view of the dorsal and anal fins (a) and caudal skeleton (b) in Rhodeus caspius . Abbreviations: Dfs: dorsal fin spine; Dpt: distal pterygiophore; Epu: epural; Hp 1-6: hypural plates 1-6; Hsp: hemal spine; Mtp: medial pterygiophore; Ns: neural spine; Ppt: proximal pterygiophore; Ph: parhypurale; Pls: pleurostyle; Rna: rudimentary neural arch; Sn: supraneural; Sty: stay; Un: uroneural. FIGURE 12. Records of Rhodeus caspius from the southern Caspian Sea basin, Urmia Lake basin and Tigris River drainage in Iran. Mirhashemi Nasab et al . (2017) reported a trematode worm Diplostomum spathaceum in fish from the Anzali Lagoon with a prevalence of 18.18% and a range of 1–4 worms per fish. Etymology. The species is named for the Caspian Sea basin where the type materials were collected.