Rhinogobius yonezawai, Suzuki & Oseko & Kimura & Shibukawa, 2020

Suzuki, Toshiyuki, Oseko, Naoharu, Kimura, Seishi & Shibukawa, Koichi, 2020, Rhinogobius, Bulletin of the Kanagawa Prefectural Museum (Natural Science) 49, pp. 7-28 : 15-25

Rhinogobius yonezawai sp. nov.

(New Standard Japanese name: Kemmun-hirayoshinobori)

( Figs. 7–12; Tables 2 & 3)

Rhinogobius brunneus (not of Temminck & Schlegel): Nakayama, 1975: 114 (in part: Amami-oshima Island of Amami Group, the Ryukyu Islands, Japan).

Rhinogobius sp. DL: Kawanabe & Mizuno, 1989: 589 (in part: Amami-oshima Island of Amami Group, the Ryukyu Islands, Japan); Akihito et al., 1993: 1080 (in part: the Nansei Islands, Japan); Akihito et al., 2000: 1252 (in part: the Nansei Islands, Japan); Sakai et al., 2001: 117 (in part: Tanega-shima and Yaku-shima islands of Osumi Group, and Amami-oshima Island of Amami Group, the Ryukyu Islands, Japan); Akihito et al., 2002: 1252 (in part: the Nansei Islands, Japan); Yonezawa, 2002: 1 (Tanega-shima and Yaku-shima islands of Osumi Group, the Ryukyu Islands, Japan); Suzuki et al., 2004: 450 (in part: Tanega-shima and Yaku-shima islands of Osumi Group, Amami-oshima Island of Amami Group and Okinawa-jima Island of Okinawa Group, the Ryukyu Islands, Japan); Yonezawa et al., 2010: 256 (Yaku-shima Island of Osumi Group, the Ryukyu Islands, Japan); Akihito et al., 2013: 1454 (in part: Tanega-shima and Yaku-shima islands of Osumi Group, Amami-oshima Island of Amami Group and Okinawa-jima Island of Okinawa Group, the Ryukyu Islands, Japan); Motomura & Harazaki, 2017: 139 (Yaku-shima Island of Osumi Group, the Ryukyu Islands, Japan).

Holotype. OMNH-P 44049 , male, 75.2 mm SL, Issogawa River , Yaku-shima Island of Osumi Group, the Ryukyu Islands, Japan, 30°26'22.45"N 130°28'23.65"E, 16 November 2018, Fig. 7.

Paratypes. Total 16 specimens (six males and 10 females), 45.8–71.0 mm SL, collected from Osumi Group , Amami Group and Okinawa Group , the Ryukyu Islands , Japan. Tanega-shima Island (Osumi Group): KAUM-I. 5753, 5754 (stained with Alizarin Red S), 5755, 5761, 5763, two males and three females, 49.2–64.5 mm SL O’okouda-gawa River, 30°37'34.77"N 131°2'4.02"E, 11 August 2007 . Yaku-shima Island (Osumi Group): KPM-NI 53488 and 53489 (formerly OMNH-P 44058 and 44059), male and female, 67.9 and 68.9 mm SL, stained with Alizarin Red S, Jyono-gawa River, 30°23'58.19"N 130°35'22.49"E, 9 December 2018 ; OMNH-P 44050 , female, 60.2 mm SL, collected with holotype, Fig. 8. Amami-oshima Island (Amami Group): OMNH-P 43718 , 43720 (stained with Alizarin Red S), 43724, 43726 (stained with Alizarin Red S), two males and two females, 45.8–71.0 mm SL, Kawauchi-gawa River, 28°18'46.10"N 129°25'13.28"E, 31 May 2017 . Okinawa-jima Island (Okinawa Group): OMNH-P 43973 and 44082, male and female, 48.7 and 51.3 mm SL, stained with Alizarin Red S, Ufu-gawa River, Aha-gawa River System, 26°44′04.0″N 128°15′12.0″E, 3 October 2005 ; SPMN-PI 45463 and 45464 (formerly OMNH-P 43692 and 43693), male and female, 59.4 and 63.8 mm SL, Fun-gawa River of Aha-gawa River System , 26°43'29.27"N 128°17'12.65"E, 25 June 2015 .

Diagnosis. Rhinogobius yonezawai is distinguished from all congeneric species by the following unique combination of features: 35–39 longitudinal scales; 10+16=26 vertebrae; first dorsal fin in males high and falcate, non-filamentous, longest second and/or third spine(s) extending posteriorly to a space between bases of first and fourth second dorsal-fin branched rays when adpressed; fifth pelvic-fin segmented ray divided into four branches at its first (most proximal) segmenting point; pectoral-fin base and prepelvic areas naked; belly with small cycloid scales except for a narrow area around ventral midline or around anterior half of ventral midline; longitudinal rows of sensory-papillae on cheek with no transverse rows; a black oval spot on base of pectoral fin; two red stripes on temporal region, reaching posteriorly to, or around, dorsum of body below first dorsal fin; six to eight vertical deep-red or reddish-orange lines on caudal fin in males; a black bifurcated blotch posteriorly at base of caudal fin in females when alive or freshly collected.

Description. Dorsal-fin rays VI-I, 8* (17); anal-fin rays I, 8* (17); pectoral-fin rays 19 (3), 20* (13), or 21 (1); pelvic-fin rays I, 5* (17); segmented caudal-fin rays 8+8 (1), or 9+8* (16); branched caudal-fin rays 7+7* (11), 7+8 (1), or 8+7 (5); longitudinal scales 35 (1), 36 (1), 37* (9), 38* (5), or 39 (1); transverse scales 9 (1), 10 (5), 11* (10), or 12 (1); scales between origin of dorsal fin and dorsal insertion of pectoral fin 8 (7), 9* (4), or 10 (6); predorsal scales 2 (1), 4 (1), 6* (3), 7 (1), 8 (2), 9 (1), 11 (4), 12 (1), 13 (2), or 14(1); P-V 3/21210/9 (1), or 3/22110/9* (14); vertebrae 10+16=26* (15).

Proportional measurements based on holotype and seven paratypes ( KAUM-I 5753, 5763; OMNH-P 43718, 43724, 44050; SPMN-PI 45463, 45464,) are given in Table 2. Body slender, slightly compressed anteriorly, compressed posteriorly. Head moderately large, slightly depressed. Snout nearly pointed and long, longer than eye diameter; snout length of males greater than that of females. Eye large, dorsolateral on head, located slightly behind a vertical through midpoint between snout tip and posterior margin of preopercle. Cheek somewhat bulbous, fleshy. Lips thick and fleshy; upper lip slightly protruding anteriorly beyond, or equal to, lower lip; gape slightly oblique; posterior margin of lower jaw not extending posteriorly to a vertical through anterior margin of eye. Anterior naris a short tube without skin flap at its tip, located slightly before the midpoint between snout tip and anterior margin of eye; posterior naris a round pore with low rim, closer to anterior naris than to eye. Gill opening extending anteriorly to a vertical through posterior margin of preopercle. Gill membranes broadly attached to isthmus. No fleshy papillae- or finger-like projections on lateral margin of shoulder girdle. Tongue free from floor of mouth, with rounded anterior margin. Genital papillae cone-shaped in males and oval in females.

Origin of first dorsal fin slightly behind a vertical through dorsal insertion of pectoral fin; first dorsal fin falcate and much higher than second dorsal fin in males, whereas in females, trapezoid, triangle or semicircular, and subequal to second dorsal fin in height; second and/or third spine(s) longest; all dorsal-fin spines slender and flexible, not filamentous; tip of longest spine (= posterior tip of the fin) extending posteriorly to a space between bases of first and fourth second dorsal-fin branched rays in males, but not extending to origin of second dorsal fin in females when adpressed. First and second dorsal fins not continued by membrane; all segmented dorsal-fin rays usually branched; usually seventh and second or third branched rays longest in males and females, respectively; posterior tip of second dorsal fin usually extending to caudal fin in males, but not reaching there in females when adpressed; posterior end of base of second dorsal fin above posterior end of anal-fin base. Origin of anal fin below a space between bases of first and third second dorsal-fin branched rays; anal fin slightly lower than second dorsal fin in height; all segmented anal-fin rays usually branched; sixth or seventh and fifth, sixth or seventh branched rays longest in males and females, respectively; posterior tip of anal fin not extending to caudal fin when adpressed. Pectoral fin oval, extending posteriorly to a vertical through a space between base of fifth spine and end of first dorsal-fin base; pectoral-fin rays branched, except dorsalmost and/or ventralmost ray(s) unbranched. Pelvic fins fused medially by well-developed frenum (between spines) and connecting membrane (between innermost rays), forming a circular cup-like disc at least in large adults; pelvic fins usually extending posteriorly to a vertical through a space between bases of second and fifth first dorsal-fin spines, and not reaching to anus; pelvic-fin spine with a rounded membranous lobe at its tip; all segmented rays of pelvic fin branched; fifth pelvic-fin segmented ray divided into four branches at its most proximal point with segment of each branch ( Fig. 9A). Caudal fin elliptical or fan-shaped.

Scales on body largely ctenoid, becoming smaller anteriorly; a part of basal region of caudal fin, anterodorsal part of body before a diagonal line from base of sixth spine and fourth second dorsal-fin branched ray to dorsal insertion of pectoral fin, belly with small cycloid scales; scaled area of belly not extending anteriorly to pelvic-fin insertion. Predorsal squamation usually with trifurcate anterior edge; mid-anterior extension extending anteriorly to a space between transverse lines through a point slightly before origin of first dorsal fin and sensory-canal pore K'; anterior extensions of lateral sides extending anteriorly to a space between transverse lines through anterodorsal end of gill membrane and pore K' ( Fig. 9B). The other part of head, pectoral-fin base, pelvic-fin axil and following narrow triangular area around ventral midline or around anterior half of ventral midline of belly ( Fig. 9C), and prepelvic areas naked.

Cephalic sensory systems are illustrated in Fig. 10. Nasal extension of anterior oculoscapular canal with terminal pore B' located above anterior naris. Anterior interorbital sections of anterior oculoscapular canal separated bilaterally, with paired pore C and a single pore D. Pore E present just behind posterior edge of eye. Lateral section of anterior oculoscapular canal with anterior pore F and terminal pore H'. Posterior oculoscapular canal with two terminal pores K' and L'. Gap between anterior and posterior oculoscapular canals much shorter than length of posterior oculoscapular canal. Preopercular canal present, with three pores M', N, and O' (a paratype without pore N). Sensory-papillae row “a” oblique and uniserial, composed of loosely-arranged papillae, extending anteriorly to a vertical through anterior margin of eye. Row “b” longitudinal, composed of densely-arranged papillae, extending anteriorly to a vertical through pupil; its length slightly longer than eye diameter. Row “c” composed of loosely-arranged papillae, extending posteriorly to a vertical through posterior margin of eye. Row “d” composed of densely-arranged papillae, extending posteriorly to a vertical through posterior margin of pupil. Row “cp” comprising a single papilla. Row “f” comprising paired papillae. Anterior end of row oi slightly separated from a vertical row “ot”.

Coloration of males when alive [ Figs. 11A, C, E, G; Suzuki et al., 2004: 451 (middle figure)]. Ground color of head and body bluish green or yellow green. Ventral side of belly pale yellow. Cheek and lower half of operculum grayish, with many small deep-red dots, spots and/or short lines forming an irregular network pattern (indistinct in a population in Okinawa-jima Island). Snout with a broad, oblique deep-red stripe between eye and anterior one fourth of upper lip; lateral side of snout sometimes tinged with yellow. Several irregular-shaped, deep-red short stripes and botches on dorsal surface of snout, interorbital space, occipital region and nape; dorsal margin of cheek edged by a narrow deep red line and bluish green line; upper part of operculum with two oblique deep red or black stripes. Temporal region with two deep-red stripes reaching posteriorly to, or around dorsum of body below the first dorsal fin. Many of scale pockets on body with reddish orange spots. Dorsum of body with four saddle-like, large black blotches; anteriormost one at base of first dorsal fin, middle one at base of second dorsal fin, and the other two on caudal peduncle, sometimes indistinct. A longitudinal series of seven large black blotches on midlateral body; first and second ones ventral to first dorsal fin, third one ventral to between dorsal fins, fourth and fifth ones ventral to second dorsal fin, sixth one at middle of caudal peduncle, seventh one at end of caudal peduncle; all these blotches nearly rounded or rectangular, and sometimes indistinct. Membranes of vertical fins similar to body in coloration, with deep-red or reddish-orange spines/ rays; distal margins of vertical fins pale yellow. Caudal fin becoming blackened distally, with a narrow pale-yellow edge; central part of caudal fin with six vertical deep-red or reddish-orange lines. Pectoral fin greenish or yellowish, with a central part tinged with red; base of pectoral fin paler, with a distinct black oval spot. Pelvic fins bluish gray.

Coloration of female when alive ( Figs. 11B, D, F, H; Suzuki et al., 2004: 451, lower figure). Resembles that of male, except as follows. Ground color of head, body and fins bluish gray, pale sky or pale yellow-green. In spawning season, belly bright yellow ( Fig. 11F). Orange dots on cheek indistinct, fewer in number. Many scale pocket spots with pale spots. Stripes and blotches on head and body darker. Seventh midlateral black blotch, located at base of caudal fin, bold and bifurcated posteriorly. Caudal fin without vertical rows.

Coloration when freshly collected. Freshly-collected coloration of males ( Figs. 7, 12A) resembles that when alive in underwater photograph, except as follows. Ground color of head and body light or medium gray; head bluish; branchiostegal membrane pale yellow, with many small indistinct bright orange spots. Red and orange markings on head and body darker. Saddle-like black blotches on dorsum and a longitudinal series of black blotches on midlateral body hardly visible. Membranes of vertical fins grayish or reddish brown; distal margins of vertical fins white. Central part of caudal fin with usually six to eight vertical deep-red or reddish-orange lines. Pectoral and pelvic fins similar to, or slightly more darkened than, body in coloration. Freshly-collected coloration of females ( Figs. 8, 12B) resembles that of males, except as follows. Ground color of head and body dull yellow or grayish yellow; head and midlateral body bluish. Scale pockets with orange spots, forming a network pattern. Fin membranes light gray. Caudal fin without barred pattern. In some specimens, pectoral fin with two vertical rows of orange dots.

Coloration when preserved in alcohol. All blue, green, orange, red and yellow color faded; ground color of head and body turns to grayish brown and pale yellow in males and females respectively; blackish markings on body turn to brown.

Habitat and Distribution. Known from upper reaches of swift freshwater streams in montane areas of Tanega-shima and Yaku-shima islands of Osumi Group, Amami-oshima Island of Amami Group and Okinawa-jima Island of Okinawa Group, the Ryukyu Islands, Japan; it is an amphidromous species ( Takagi et al., 2015), although landlocked in few freshwater reservoirs of Okinawa-jima Island (present study).

Etymology. The specific name, yonezawai , refers to Mr. Toshihiko Yonezawa, who offered much information and specimens to us for our study.

Discussion

Nakayama (1975) named a species of Rhinogobius collected from Iriomote-jima Island of Yaeyama Group of the Ryukyu Islands as “ Iriomote kokusyoku oogata (Iriomote large dark morph type)” with a note that it was also distributed in the Amami group of the Ryukyu Islands. Hayashi (1984) applied a new name “ Minami kokusyoku oogata (South large dark morph type)” for it. Kawanabe & Mizuno (1989) assumed this as a distinct unnamed species, and tentatively named it “ Rhinogobius sp. DL” with a new standard Japanese name “Hira-yoshinobori”. Takagi et al. (2015) surveyed the genetic structure of Rhinogobius sp. DL in the Ryukyu Islands using three microsatellite loci and clarified that the populations from Yaku-shima/ Tanega-shima/Amami-oshima islands are placed in a single cluster, different from a population of Iriomote-jima Island. Our investigation revealed that these two groups are also clearly distinguished from each other in their morphological characters; we thus describe these two as distinct species ( R. yaima and R. yonezawai ) here.

Rhinogobius is currently known as the most specious freshwater gobiid genus, comprising 85 valid species ( Endruweit, 2018; Suzuki et al., 2019; present study). As indicated by Chen & Shao (1996) and Suzuki et al. (2015), the genus is divided into two distinct groups; one comprises only a single species R. similis , whereas the other includes all the remaining species. Rhinogobius similis differs from the other congeners by having large ctenoid scales on the nape (vs. nape naked or with cycloid scales in the others) and several short transverse rows of sensory papillae on the cheek (vs. no distinct transverse rows of sensory papillae on cheek). Suzuki et al. (2019) assigned all species of the genus but R. similis to the “ Rhinogobius brunneus complex”, following Chen & Shao (1996). Both of the two new species described here, Rhinogobius yaima and R. yonezawai , also belong to the R. brunneus complex.

Furthermore, Suzuki et al. (2019) attempted to divide the R. brunneus complex into two subgroups: one almost always has 27 or more vertebrae (they named it "Group I"), whereas the others have lower counts (25–27, almost always 26) ("Group II"). The groups I and II, both of which appear to be phylogenetic grades merely assembled by the vertebral counts ( Suzuki et al., 2019), hitherto comprise at least 46 and 30 described species, respectively. Unfortunately, Suzuki et al. (2019) failed to assign the remaining six species to these subgroups due to the lack of information of their vertebral counts (Endruweit, 2019; Suzuki et al., 2019; present study). Rhinogobius yaima and R. yonezawai , having 26 vertebrae, belong to Group II, making the total number of species in the group 32.

Species of Group II and the assemblage with no information about vertebral counts (total 38 species) are compared in Table 3. Within 38 species, R. yaima is most similar to Rhinogobius bucculentus ( Herre 1927) and Rhinogobius philippinus ( Herre 1927) , by having the following combination of characters: 40 or more longitudinal scales (40–43 in R. yaima ; 40–44 in R. bucculentus ; 36–40 in R. philippinus ); a low first dorsal fin in males, not extending posteriorly to the origin of the second dorsal fin when adpressed. Rhinogobius yaima is, however, distinguished from those two species by having a naked area around the ventral midline of the belly (vs. scaly in R. bucculentus and R. philippinus ); and six first dorsal spines (vs. seven spines in R. bucculentus ; six or seven spines in R. philippinus ). Data on R. bucculentus and R. philippinus follow Herre (1927).

Rhinogobius yonezawai can be distinguished from all other congeners of group II except Rhinogobius bedfordi ( Regan 1908) , Rhinogobius brunneus ( Temminck and Schlegel, 1845) , Rhinogobius fluviatilis Tanaka 1925 and Rhinogobius ogasawaraensis Suzuki, Chen & Senou 2012 by having the following combination of characters: 35–39 longitudinal scales (35–39 in R. yonezawai ; 36–38 in R. bedfordi ; 32–35 in R. brunneus ; 32–38 in R. fluviatilis ; 31–35 in R. ogasawaraensis ); a falcate first dorsal fin extending posterior to, or beyond, the origin of the second dorsal fin in males; no scales on the pectoral-fin base and prepelvic area (both naked in R. yaima and R. ogasawaraenisis ; naked or scaly on the base of pectoral fin, naked on prepelvic area in R. brunneus and R. fluviatilis ; both unknown in R. bedfordi ). However, R. yonezawai is distinguished from R. bedfordi by having the second and/ or third spine(s) of first dorsal fin longest, not filamentous and extending posteriorly to the space the bases of the first and fourth second dorsal-fin branched rays when adpressed (vs. second spine longest, its tip filamentous and extending to sixth ray base in R. bedfordi ); the head in males is longer, its length 32.1–35.6 % SL (vs. 30.0 and 30.8% SL in males of R. bedfordi ); a more slender body in males, its depth at the anal-fin origin 14.7–16.3 % SL (vs. 16.8 and 18.2 % SL in males). Rhinogobius . yonezawai is distinguished from R. brunneus by being naked around the ventral midline or around the anterior half of the ventral midline of the belly (belly entirely scaly in R. brunneus ); a black oval spot on upper half of base of the pectoral fin (vs. a dusky crescent mark extending ventrally to ventral half); the midlateral body with no longitudinal series of brown spots or short lined (vs. a longitudinal series of brown spots or short lines); the dorsolateral side of body with no black spots (vs. 3–4 longitudinal series of black spots). Rhinogobius yonezawai is distinguished from R. fluviatilis by having around ventral midline or around anterior half of the ventral midline of belly naked (belly entirely scaly in R. fluviatilis ); the temporal region with two red stripes reaching posteriorly to, or around the dorsum of the body below the first dorsal fin (only temporal region with two stripes); a longitudinal series of seven large black blotches on the midlateral body (vs. eight blotches); a black blotch at the base of the caudal fin bold and bifurcated posteriorly (vs. a broad black band); the caudal fin with six to eight vertical red lines in males (vs. no line). Rhinogobius yonezawai is distinguished from R. ogasawaraensis by having the fifth pelvic-fin segmented ray divided into four branches at its first (most proximal) segmenting point (vs. bifurcated in R. ogasawaraensis , Fig.13B). Data on R. bedfordi , R. brunneus R. fluviatilis , and R. ogasawaraensis were taken from Regan (1908), Oijen et al. (2011), Suzuki et al. (2004), Suzuki & Chen (2011), Akihito et al. (2013), Suzuki et al. (2012) and comparative materials.

Yamazaki et al. (2015) analyzed nuclear DNA of the Japanese species of Rhinogobius and concluded that Rhinogobius sp. DL collected from Iriomote-jima Island (= R. yaima ) has a sister relationship with R. fluviatilis and R. ogasawaraensis . Rhinogobius yaima is distinguished from R. fluviatilis and R. ogasawaraensis by having a low first dorsal fin in males, not extending posteriorly to the origin of the second dorsal fin when adpressed (vs. a high first dorsal fin in males, extending posteriorly to a space between bases of the second and sixth second dorsal-fin branched rays in R. fluviatilis and R. ogasawaraensis ); 40–43 longitudinal scales (vs. 34–38 in R. fluviatilis ; 31– 35 in R. ogasawaraensis ); the fifth pelvic-fin segmented ray usually divided into five branches at its first (most proximal) segmenting point (vs. four and two branches in R. fluviatilis and R. ogasawaraensis , respectively) ( Fig. 13); a naked area around the ventral midline of the belly and the pectoral-fin base (vs. scaly in R. fluviatilis ). Data on R. fluviatilis and R. ogasawaraensis were taken from Suzuki & Chen (2011) and Suzuki et al. (2012), respectively, and comparative materials.

Because their coloration and habitat are similar to each other, R. yaima and R. yonezawai have been regarded as a single species by previous researchers. Rhinogobius yaima is, however, distinguished from R. yonezawai by having a low first dorsal fin in males, not extending posteriorly to the origin of the second dorsal fin when adpressed (vs. a high first dorsal fin in males, extending posteriorly to a space between the bases of the second and sixth second dorsal-fin branched rays in R. yonezawai ); the fifth pelvic-fin segmented ray usually divided into five branches at its first (most proximal) segmenting point (vs. four in R. yonezawai ); a more depressed head, its depth at pelvic-fin origin 14.4–15.5 % SL in males, 14.6–15.8 % SL in females (vs. 15.5–16.7 % SL in males, 15.0–16.8 % SL in females); a more slender body, its depth at the anal-fin origin 13.7–14.2 % SL in males, 13.8–14.5 % SL in females (vs. 14.7–16.3 % SL in males, 15.0–19.6 % SL in females); a more slender caudal peduncle, its depth 11.4–11.6 % SL in males, 11.5–12.2 % of SL in females (vs. 12.7–13.8 % SL in males, 13.1–15.1 % SL in females); the caudal fin with four vertical rows of bright orange dots in males (vs. six to eight vertical deep-red or reddish-orange lines); the base of the caudal fin with a pair of vertically-arranged, rounded or rectangular black blotches in females (a black bifurcated blotch posteriorly).

Rhinogobius yaima is an endemic species of Ishigaki-jima and Iriomote-jima islands, Yaeyama Group of the Ryukyu Islands, and R. yonezawai is also endemic to Tanega-shima and Yaku-shima islands of Osumi Group, Amami-oshima Island of Amami Group and Okinawa-jima Island of Okinawa Group, the Ryukyu Islands. In the RDB of Okinawa Prefecture, Rhinogobius sp. DL (= Rhinogobius yaima and R. yonezawai ) is designated toas NT (Near Threatened) ( Maeda, 2017). Rhinogobius yaima is one of the species to have the smallest distribution area and population among the genus from Japan. The habitat of R. yonezawai is limited into the Okinawa Island, and the population decreases sharply because of the for influence of the dam construction and the flood caused by the heavy rain. The prompt protection and maintenance of both species are necessary. This account is very important as the first step for their protection and maintenance.

Comparative materials. Rhinogobius bedfordi : a radiograph of syntypes [ BMNH (British Museum of Natural History, London)1907.12.30.42-46 (https://data. nhm.ac.uk)] of Ctenogobius bedfordi ; Rhinogobius brunneus : 3 specimens: OMNH-P 32106, 32107 and 43683, 2 males and a female, 47.0– 54.2 mm SL, stained with Alizarin Red S, To-gawa River, Shimoda, Shizuoka Prefecture, Japan, 29 July 1985. Rhinogobius fluviatilis : 7 specimens: OMNH-P 18391 and 18393, 2 males, 70.7 and 86.1 mm SL, Komenotsu-gawa River, Izumi, Kagoshima Prefecture, Japan, 3 September 2003; OMNH-P 18429, female, 67.2 mm SL, Nabeno-gawa River, Izumi, Kagoshima Prefecture, Japan, 24 October 2003; OMNH-P 42882, female, 69.7 mm SL, Sendaigawa River, Satsumasendai, Kagoshima Prefecture, Japan, 30 September 2014; OMNH-P 32104, 32105 and 43684, 2 males and a female, 50.6–58.1 mm SL, stained with Alizarin Red S, Seto-gawa River, Okabe, Shizuoka Prefecture, Japan, 15 October 1984. Rhinogobius nagoyae : 6 specimens: OMNH-P 37977 and 37978, a male and a female, 70.7 and 63.1 mm SL, Inaosawa-gawa River, Shimoda, Shizuoka Prefecture, Japan, 28 August 2011; OMNH-P 43560 and 43565, a male and a female, 60.3 and 58.1 mm SL, Takeno-gawa River, Toyooka, Hyogo Prefecture, Japan, 8 August 2016; OMNH-P 32109 and 43685, a male and a female, 46.9 and 56.3 mm SL, stained with Alizarin Red S, Takizawa-gawa River, Fujieda, Shizuoka Prefecture, 05 March 1985. Rhinogobius ogasawaraensis : NTOU P 2007-02-882, male, 38.6 mm SL, stained with Alizarin Red S, a small stream of Oki village, Haha-jima Island, Ogasawara Islands, Japan, 24 November 2001.

Acknowledgments

We are very grateful to Masahiro Aizawa (formerly Biological Laboratory of Imperial Palace , Tokyo), Shigeru Harazaki ( Yakushima Diving Service Mori-to-umi), Masao Kasai (Mr.SAKANA Diving Service , Iriomote-jima Island ), Ken Maeda (Okinawa Institute of Science and Technology Graduate University ), Toshifumi Saeki ( Rivus , Okinawa), Masatomi Suzuki ( Kawanishi , Hyogo), Motohiro Takagi ( South Ehime Fisheries Research Center ), Takatoshi Tsunagawa (Tochigi Prefecture Fisheries Experimental Station ), Yo Yamasaki ( National Institute of Genetics ) and Toshihiko Yonezawa ( Foundation of Kagoshima Environmental Research and Service ) gave us valuable information for the present study; Kiyotaka Hatooka and Shoko Matsui ( OMNH), Hiroyuki Motomura ( KAUM) and Hiroshi Senou ( KPM) helped registration and/or loan of the specimens examined here; David Greenfield (California Academy of Science ) and Hiroshi Senou ( KPM), read the manuscript and gave helpful comments .