A new cyprinid fish, Nipponocypris takayamai sp. nov. is described from the Middle Pleistocene Nogami Formation in Oita Prefecture, Northern Kyushu, Japan. Nipponocypris takayamai differs from its congeners in the following combination of characters: the sensory canal of the parietal not reach to the medial edge of the parietal, and its length longer than half the width of the parietal; the notch of the orbital margin of the frontal weak; the dorsal margin of opercle concave; the posterodorsal margin of the fourth infraorbital L-shaped; the sensory canal of the dentary running slightly ventrally; 42–44 vertebrae; the flanges of the neural spines of the second and third preuralcentra elongated to the dorsal ends of the neural spines; eight supraneurals between the dorsal fin and the supraneural 3 bone; the dorsal fin base located slightly more posterior than the pelvic fin base; first three dorsal fin rays unbranched and unsegmented; eight dorsal fin rays, ten dorsal fin pterygiophores; and eleven anal fin pterygiophores with thirteen anal fin rays. A cladistic analysis suggests that this new species is related to N. sieboldii, N. koreanus and N. temminckii, and is probably the sister taxon to N. temminckii. This new species shows that an extinct species closely related to Recent Nipponocypris existed until the Middle Pleistocene in East Asia.
Introduction
The Middle Pleistocene Nogami Formation of Oita Prefecture, Northern Kyushu, Japan is a lacustrine deposit, and yields many complete fossils of freshwater fishes, which belong to Salmonidae (Oncorhyncus cf. masou, O. rhodums or Oncorhyncus sp.), Gobiidae (Rhinogobius similis and R. brumous), and Cyprinidae (Zacco cf. temminckii, Hemibarbus barbus or Hemibarbus sp., and Acheilognathus lanceolata) (e.g. Takahashi and Okumura, 1996; Uyeno et al., 1975, 2000; Yabumoto, 1987). All of these fossils provide significant information about the freshwater fish fauna of East Asia in the Quaternary, and further understanding of the origin of the Recent freshwater fish fauna in East Asia.
Among these, the genus Zacco is a member of the opsariichthine group (Chen, 1982) which is one of the common Asian endemic cyprinid fishes. The opsariichthine group dwells in Vietnam, Taiwan, China, Japan, Korea, and eastern Russia, and consists of the following five genera: Opsariichthys Bleeker, 1863, Zacco Jordan and Evermann, 1902, Candidia Jordan and Richardson, 1909, Parazacco Chen, 1982, and Nipponocypris Chen et al., 2008, which is treated as Zacco by Chen (1982) and other ichthyologists (see Chen et al., 2008).
Recently, the studies of the taxonomy and molecular phylogeny of this group have been rapidly increasing (e.g. Chen et al., 2008, 2009; Hosoya et al., 2003; Kim et al., 2005; Tang et al., 2010, 2013a, b; Wang et al., 2007). Hosoya et al. (2003) redescribed Japanese “Zacco” temminckii and demonstrated that it should be divided into two species, “Z.” temminckii and “Z.” sieboldii, based on diagnostic characters such as the number of anal fin rays, the coloration of the anterior margin of the pectoral and pelvic fins, and the number of lateral line scales. In the same way, Kim et al. (2005) divided “Z.” temminckii of southern Korea into two species, “Z.” temminckii and “Z.” koreanus based on the color of the upper margin of the eyes and the anterolateral part of the body, and the number of scales between the lateral line and the dorsal fin origin. Moreover, Chen et al. (2008) suggested that Z. platypus was closely related to the genus Opsariichthys, while “Z.” sieboldii, “Z.” temminckii and “Z.” koreanus form a monophyletic group that is closer to the genera Candidia and Parazacco than Z. platypus. In other words, the genus Zaceo is a paraphyletic group. Thus, Chen et al. (2008) advocated the new genus Nipponocypris for “Z.” sieboldii, “Z.” temminckii and “Z.” koreanus. As the result of molecular phylogenetic study by Tang et al. (2010), Candidia, Parazacco and Nipponocypris are polyphyletic, but with low bootstrap support. In Tang et al. (2013 a, b), Opsariichthys + Zacco and Nipponocypris + Candidia form a monophyletic group, and the genus Parazacco is the basal taxon for opsariichthins.
However, there is no detailed phylogenetic study of the Recent opsariichthins and fossil opsariichthins from the Nogami Formation. With the increase of the taxonomic and molecular phylogenetic studies mentioned above, the study of morphology of fossil taxa becomes important. In the present study, a new fossil opsariichthin is described from the Nogami Formation of the Middle Pleistocene, Oita Prefecture, Japan and its phylogenetic position within the Recent opsariichthine group is discussed.
Geological setting
The southern part of the Kusu Basin, Oita Prefecture of Northern Kyushu, Japan belongs to the Hohi volcanic zone, and consists of volcanic rocks and lacustrine deposits (Figure 1). Stratigraphical, sedimentological, volcanic, paleontological, and paleoecological investigations have been conducted since the 1950s (e.g. Hayashi, 1959; Ishihara et al., 2010; Iwauchi, 1998; Iwauchi and Hase, 1987; Julius et al., 2006; Kamata and Muraoka, 1982; Shuto, 1953a, b).
The lower part of the Nogami Formation that yielded the fossils is mainly composed of lacustrine diatomaceous beds with the Nakamura Lava that inflowed into the paleo-lake (Iwauchi and Hase, 1987). The Nogami Formation yields well preserved fossils of plants (e.g. Iwauchi and Hase, 1987), diatoms (e.g. Julius et al., 2006) and freshwater fishes (e.g. Uyeno et al., 1975, 2000).
The age of the Nogami Formation is the Middle Pleistocene, which is supported as follows: the K-Ar age of the Haneyama Lava that is covered by the Nogami Formation (e.g. 0.8±0.3 Ma, 0.7±0.3 Ma: Kamata and Muraoka, 1982; 0.67±0.01 Ma, 0.69±0.01 Ma, 0.71±0.01 Ma: Yamada et al., 2006); the K-Ar age of the Kabushidake Lava that covers the Nogami Formation (0.27±0.11 Ma, 0.32±0.05 Ma, 0.33±0.02 Ma: Kamata, 1985); and the K-Ar age of the Hiwaku (Hwk) tephra (Shiromaru band of Iwauchi and Hase, 1987), which is within the Nogami Formation (0.58±0.03 Ma, 0.57±0.03 Ma; Machida and Arai, 2003; Figure 1).
Material and methods
Comparative material.—The repository of the specimens of the Recent opsariichthins examined is the Kitakyushu Museum of Natural History and Human History, Kitakyushu, Fukuoka Prefecture, Japan, with the prefix KMNH. These specimens were cleared and stained by a method modified from Kawamura and Hosoya (1991). Zacco platypus (Temminck and Schlegel, 1846), from Onogawa River, Takeda City, Oita Prefecture, Japan: KMNH VR 100,128, Standard length (SL) = 104.0 mm; KMNH VR 100,129, SL = 101.0 mm; KMNH VR 100.130, SL = 91.0 mm. Nipponocypris temminckii (Temminck and Schlegel, 1846), from Onogawa River, Takeda City, Oita Prefecture, Japan: KMNH VR 100, 134, SL = 103.2 min; KMNH VR 100,135, SL = 101.4 mm; KMNH VR 100,136, SL = 80.7 mm. Nipponocypris sieboldii (Temminck and Schlegel, 1846), from Ashimorigawa River, Okayama City, Okayama, Japan: KMNH VR 100,138, SL = 92.7 mm; KMNH VR 100,139, SL 90.8 mm; KMNH VR 100,140, SL = 100.9 mm. Nipponocypris koreanus (Kim et al., 2005), from Korea: KMNH VR 100,141, SL = 84.1 mm ; KMNH VR 100,142, SL = 87.4 mm; KMNH VR 100,143, SL = 101.0 mm, KMNH VR 100,144, SL = 95.0 mm. Parazacco spilurus (Günther, 1868), from Guangdong, China: KMNH VR 100,145, SL = 79.5 mm ; KMNH VR 100,146, SL = 87.7 mm; KMNH VR 100,147, SL = 107.0 mm. Candidia barbata (Regan, 1908), from Kaohsiung, Taiwan: KMNH VR 100.131, SL = 84.9 mm; KMNH VR 100,132, SL = 91.6 mm; KMNH VR 100,133, SL = 88.3 mm. Opsariichthys uncirostris (Temminck and Schlegel, 1846), from Lake Biwa, Shiga, Japan: KMNH VR 100,148, SL = 204.7 mm; KMNH VR 100,149, SL = 196.0 mm; KMNH VR 100,150, SL = 191.8 mm. Opsariichthys bidens Günther, 1873 from China: KMNH VR 100,151, SL = 102.0 mm; KMNH VR 100,152, SL = 79.5 mm. Opsariichthys evolans (Jordan and Evermann, 1902, from Taiwan: KMNH VR 100,155, SL = 73.7 mm; KMNH VR 100,156, SL = 86.5 mm; KMNH VR 100,157, SL = 70.1 mm.
Counts and measurements.—Standard length measurements were done for the estimated tip of the snout to the posterior end of the hypural. Fin ray counts were made according to Chen and Chu (1998) and vertebral counts were made according to Uyeno (1984).
Osteological terminology.—Names of the skull bones and the anterior part of vertebrae follow Britz and Conway (2009), Chen et al. (1984), Dahdul et al. (2010), and Harrington (1955), and of the caudal bones follow Fujita (1990).
Figure 1.
Locality maps of the fossil fish. A, geological map of the Nogami area, southern part of the Kusu Basin (modified from Iwauchi and Hase, 1987). Fish silhouette indicates the locality of Nipponocypris takayamai sp. nov. B, stratigraphic sequence of Late Cenozoic formations in the southern part of the Kusu basin (modified from Iwauchi and Hase, 1987). Radiometric age from Kamada and Muraoka (1982), Iwauchi and Hase (1987), Machida and Arai (2003), and Yamada et al. (2006).
Cladistic analysis.—A cladistic analysis of the data matrix of Appendixes 1 and 2 in this study with the data set of the present fossil species, Nipponocypris takayamai sp. nov. was carried out using PAUP* (v. 4.0 Beta, Windows ver., Swofford, 2003), PaupUPv.1.0.3.1 (Calendini and Martin, 2005), and Mesquite (v. 2.75 Windows, Maddison and Maddison, 2011) and MS-Excel. Aphyocypris chinensis Günther, 1868 (breed, KMNH VR 100,164, SL = 51.1 mm; KMNH VR 100,165, SL = 56.6 mm) A. normalis (Nichols and Pope, 1927) (China, KMNH VR 100,166, SL = 52.7 mm; KMNH VR 100,167, SL = 54.4 mm; KMNH VR 100,168, SL = 77.6 mm), Hemiculter leucisculus (Basilewsky, 1855) from Yabumoto et al. (2008), and Xenocypris macrolepis Bleeker, 1871 from Yabumoto et al. (2010) (= Xenocypris argentea Günther, 1868) were selected as the outgroup, because Tang et al. (2010, 2013a, b) found that Aphyocypris, Hemiculter, and Xenocypris are among the most closely related taxa to the opsariichthine group among the cyprinid fishes.
The characters of numbers 5–8, 20, 27, 28, 30, 32, and 35 are from Dai and Yang (2003), characters of number 31 is from Dai et al. (2005). The osteological characters of Opsariichthys pachycephalus Günther, 1868 are from Ashiwa and Hosoya (1998) and the examination of additional specimens (Taiwan, KMNH VR 100,162, SL = 102.0 mm; KMNH VR 100,163, SL = 110.0 mm). The character of cephalic lateral line system of Candidia barbata is from Ito and Hosoya (2017) besides our specimens. The external morphological data are from Chen et al. (2008, 2009), Chen et al. (2011), Chen and Chang (2005), Chen and Chu (1998), Hosoya et al. (2003), Kim et al. (2005), Yang and Huang (1964). The characters were optimized to the tree using the ACCTRAN option. Analysis of PAUP using the branch-and-bound search option with apomorphic states of each clade and bootstrap values (%) for 1,000 replicates.
Systematic description
Order Cypriniformes Bleeker, 1859
Family Cyprinidae Rafinesque, 1815
Genus Nipponocypris Chen et al., 2008
Type species.—Nipponocypris temminckii (Temminck and Schlegel, 1846).
Nipponocypris takayamai sp. nov.
Figures 2–10
Zaceo temminckii (Temminck and Schlegel, 1846); Uyeno et al., 1975, pl. 7. fig. b. pl. 8. fig. c.
Zaceo cf. temminckii (Temminck and Schlegel); Uyeno et al., 2000, p. 72, pl. 9.
Etymology.—The species is named after Akinori Takayama, a former factory manager of Hakusan Kogyo Campany, who collected the holotype and donated it to the Kitakyushu Museum of Natural History and Human History.
Material.—Holotype, KMNH VP 102,036 (SL = 114.0 mm); paratypes, KMNH VP 102,028 (SL = 107.0 mm), KMNH VP 102,034 (SL = 111.4 mm), KMNH VP 102,062 (SL = 136.5 mm), NSM (National Museum of Nature and Science, Tsukuba) PV22663 (SL = 158.0 mm), and NSM PV22664 (head length = 24.1 + mm).
Diagnosis.—A member of the Cyprinidae distinguished by the following combination of characters: sensory canal of the parietal not reaching to the medial edge of the parietal, and its length longer than half the width of the parietal (1 [1], see Appendixes 1, 2); a weak notch present at the posterolateral margin of the frontal (2 [1]); dorsal margin of the operele concave (20 [1]); posterodorsal margin of the fourth infraorbital L-shaped (11 [1]); sensory canal of the dentary running slightly ventrally; 42–44 vertebrae; flanges of the neural spines of the second and third preuralcentra extended to the top of the neural spines (33 [3], 34 [3]); eight supraneurals between the dorsal fin and the supraneural 3 bone (31 [0]); dorsal fin base located in the middle of the body, with its origin approximately posterior to the pelvic insertion; unbranched and unsegmented first three dorsal fin rays; eight dorsal fin rays, ten dorsal fin proximal pterygiophores; and 11 anal fin proximal pterygiophores with thirteen anal fin rays (32 [2]).
Description
Skull.—The skull is preserved in all specimens, the skulls of the holotype, KMNH VP 102,036 and the paratypes, KMNH VP 102,042 and NSM PV22664 are especially almost completely preserved (Figures 2–6). The kinethmoid, ethmoid, preethmoid and prevomer are fragmentary and not clearly visible.
The lateral ethmoid, which is well observed in KMNH VP 102,036 and KMNH VP 102,042, contacts the anterolateral margin of the frontal and expands laterally. The frontal is a large, elongate bone that forms the dorsal roof of the orbit, and there is a notch at the posterolateral margin (orbital margin) of the frontal in the holotype, KMNH VP 102,036 (Figures 2, 3).
The parietal is almost square in shape and there is a sensory canal on the bone. The sensory canal does not reach to the medial edge of the parietal, and its length is longer than half the width of the parietal (KMNH VP 102,036, NSM PV22664). The supraoccipital connects with the posterior margin of the frontal, and has a crest on its dorsal medial surface (NSM PV22664).
Other bones of the otic region (e.g. prootic, autosphenotic) and the occipital region (e.g. exoccipital, basioccipital) are not clearly visible, because these are fragmentary or covered by the opercular bone and the shoulder girdle.
Infraorbital bones.—The infraorbital bones are partially preserved in KMNH VP 102,028, NSM PV22663 and NSM PV22664. The first infraorbital (= lachrymal by Harrington, 1955) is preserved in KMNH VP 102,034, NSM PV22663 and NSM PV22664. It is a large, flat, almost pentagonal bone. The sensory canal runs along the middle of the bone and is slightly curved in KMNH VP 102,028. The second infraorbital is observable in KMNH 102,042 and KMNH 102,034. The width of the second infraorbital is almost the same as the third one, and the sensory canal runs down the middle. The third and fourth infraorbitals are preserved in KMNH 102,042 and KMNH 102,028, the width of both is the same, and the third one is slightly longer than the fourth one. The posterodorsal corner of the fourth infraorbital has a small projection which is L-shaped. The sensory canal runs along the dorsal margin of the third and fourth infraorbitals. The fifth infraorbital is not preserved.
Figure 2.
Nipponocypris takayamai sp. nov. A, photo of KMNH VP 102,036 (holotype); B, line drawing of A.
Jaws.—The jaw bones are well preserved in the holoty pe, KMNH VP 102,036 and paratypes, KMNH VP 102,042 and NSM PV22664 (Figures 3, 5, 7). The anteroventral margin of the maxilla is rounded in KMNH VP 102,042. The posterior margin of the posterodorsal process on the maxilla is triangular, slightly convex such as the genus Zacco or Nipponocypris in KMNH VP 102,042 and NSM PV22664. The premaxilla is located anterior to the maxilla. The anterior end of the premaxilla forms a process ascending to the dorsal in KMNH VP 102,042. The dentary is a large, deep bone, and the mandibular sensory canal runs in the ventral part. The flat coronoid process is present and inclines about 45 degrees dorsally. The oral margin of the dentary is slightly convex. The anguloarticular is located behind the dentary, and its dorsal margin is almost triangular. The suspensorial articulation facet is present at the posterior part of the anguloarticular. The sensory canal runs along the ventral part of the anguloarticular. The small retroarticular contacts with the ventral surface of the anguloarticular in KMNH VP 102,042 and NSM PV22664.
Figure 3.
Nipponocypris takayamai sp. nov. skull in lateral view. A, photo of KMNH VP 102,036 (holotype); B, line drawing of A. Abbreviations: ACH = anterior ceratohyal; ANG = anguloarticular; BRA = branchiostegal rays = CLE, cleithrum; CRA = coracoid; DEN = dentary; ETH LAT = lateral ethmoid; EPO = epiotic; FRO = frontal; IOP = interopercle; 104 = 4th infraorbital; MET = metapterygoid; OP = operele; PAR = parietal; PCL = postcleithrum; PCH = posterior ceratohyal; PMX = premaxilla; PTO = pterotic; SOC = supraoccipital; SOP = subopercle; SRPE = supraethmoid.
Figure 4.
Nipponocypris takayamai sp. nov. A, photo of KMNH VP 102,042 (paratype); B, line drawing of A.
Opercular series.—The operele is large and trapezoidal, and the posterior margin is concave. Its anterodorsal corner is slightly projected anteriorly and the opercular canal is present. In KMNH VP 102,042 (Figure 5), the preopercle is marginally observable. The preoperculomandibular sensory canal is preserved, and it is enclosed in a bony tube and reaches to the opercular canal.
The interopercle is almost the same size as the subopercle. The bone is narrow at its anterior end and gradually widens posteriorly. Its dorsal margin is concave and its ventral margin is slightly convex on KMNH VP 102,042 (Figure 5) and NSM PV22664 (Figure 6). The subopercle is a knife-like shape and gradually narrows posteriorly.
Figure 5.
Nipponocypris takayamai sp. nov. skull in lateral view. A, photo of KMNH VP 102,042 (paratype); B, line drawing of A. Abbreviations: ANG, anguloarticular; BRA = branchiostegal rays; CHY = ceratohyal; CLE = cleithrum; DEN = dentary; ECT = ectopterygoid; ETH = ethmoid; ETH LAT = lateral ethmoid; EPO = epiotic; FRO = frontal; HYO = hyomandibular; IOP = interopercle; 102 = 2nd infraorbital; IO3 = 3rd infraorbital; IO4 = 4th infraorbital; MAX = maxilla; MET = metapterygoid; OP = opercle; P = autopalatine; PAR = parietal; PARA = parasphenoid; PCL = postcleithrum; PMX = premaxilla; POP = preopercle; PTO = pterotic; Q = quadrate; RET = retroarticular; R4 + OS = rib4+os suspensorium; RD = pectral radial; SC = scapula; SCL = supracleithrum; SN3 = supraneural 3 bone; SOP = subopercle.
Figure 6.
Nipponocypris takayamai sp. nov. skull in lateral view. A, photo of NSM PV22664 (paratype); B, line drawing of A. Abbreviations: ACH = anterior ceratohyal; AN G = anguloarticular; BRA = branchiostegal rays; CLE = cleithrum; DEN = dentary; EB5 = 5th ceratobranchial; ETH LAT = lateral ethmoid; FRO = frontal; IOP = interopercle; 101 = 1st infraorbital; MAX = maxilla; MET = metapterygoid; OP = opercle; PAR = parietal; PCL = postcleithrum; PCH = posterior ceratohyal; pt = pharyngeal teeth; Q = quadrate; RD = pectral radial; RET = retroarticular; SC = scapula; SCL = supracleithrum; SN3 = supraneural 3 bone; SN4 = supraneural 4 bone; SOC = supraoccipital.
Figure 7.
Caudal fin skeleton of Nipponocypris takayamai sp. nov. A, photo of KMNH VP 102,036 (holotype); B, line drawing of A. Abbreviations: EPU = epural; HPU2 = haemal spine of the second preural centrum; HPU3 = haemal spine of the third preural centrum; HYP = hypural; NPU1 = neural spine of first preural centrum; NPU2 = neural spine of the second preural centrum; NPU3 = neural spine of the third preural centrum; PAR = parhypural; PLE = pleurostyle; PP = hypurapophysis; PU1+U1 = first preuralcentrum+first ural vertebra; PU2 = second preuralcentrum; PU3 = third ural vertebra; PU4 = fourth ural vertebra.
Figure 8.
Supraneural bones of Nipponocypris takayamai sp. nov. A, photo of KMNH VP 102,036 (holotype); B, line drawing of A.
Suspensorium.—The suspensorium series bones are preserved in KMNH VP 102,042 (Figure 5), and in the other specimens, the bones are fragmental or covered by other bones. The posterior margin of the autopalatine articulates with the endopterygoid in KMNH VP 102,042. The endopterygoid is a large and flat bone. Its anterior margin is saddle-shaped and thick, and articulates with the autopalatine in KMNH VP 102,042. The ectopterygoid is a thin and oval-shaped bone in KMNH VP 102,042. The metapterygoid is a broad and flat plate with a strut along its dorsal margin. The quadrate is shallow, and consists of a fan-like part and a thick ventral projection (articulation facet for the mandibular). The projection articulates with the suspensorial articulation facet of the anguloarticular in NSM PV22664. The symplectic cannot be observed because it is fragmentary or covered with the opercular bones. The hyomandibular is barely observable in KMNH VP 102,042, and the most part of the bone is covered by the opercular and infraorbital bones. The elongated opening of the hyomandibular branch of the facial nerve is present in the lower portion of the strut.
Hyoid arch.—The upper and lower hypohyals are fragmentarily preserved in NSM PV22664 (Figure 6).
The anterior and posterior ceratohyals are partially preserved in KMNH VP 102,036 (Figure 3) and NSM PV22664. The anterior ceratohyal is a thick bone that is narrow at the anterior part, expanded at the posterior part, and convex at the ventral margin. The posterior ceratohyal is narrow toward the posterior. There are three branchiostegals, which are curved flat bones and relatively deep.
Pharyngeal bone and teeth.—The pharyngeal bone and teeth are observed only in NSM PV22664. In the other specimens, these are covered by the opercular bones. In NSM PV22664, only one pharyngeal tooth is preserved, which is conical in shape. There is a cycloid vestige of the pharyngeal tooth on the pharyngeal bone. This shows that the dental formula is 4 or 5, 4, and 2 or 3.
Vertebrae.—In the present study, the estimated number of vertebrae is based on the number of neural and haemal spines and ribs, although some abdominal and caudal vertebrae are missing. There are 23 or 24 abdominal vertebrae, 19 or 20 caudal vertebrae, and the total number of vertebrae are 42 to 44 (Figures 2, 4). The first to fourth abdominal vertebrae relate to the Weberian ossicles (Figures 3, 5). The first vertebra is not visible. The Weberian apparatus is not well preserved. In KMNH VP 102,036, the upper part of supraneural 3 bone is comb-like in shape and connects with the dorsal margin of the neural arch of the third and fourth vertebrae. The neural spine of the fourth vertebra is shorter than the fifth. The supraneural 4 bone is located between the supraneural 3 bone and the neural spine of the fifth vertebra, and they do not contact each other. There are 15 to 16 pairs of ribs in total in KMNH VP 102,036 and NSM PV22663. The ribs except for the first one are elongate, arch-shaped, and reaching to the abdominal edge. The dorsal margin of the neural spine of the third vertebra turns downward in KMNH VP 102,042.
Figure 9.
Anal fin skeleton of Nipponocypris takayamai sp. nov. A, photo of NSM PV22663 (paratype); B, line drawing of A. Abbreviations: PTR dis = distal ptery giophore; PTR med = median pterygiophore; PTR pro = proximal pterygiophore.
Caudal fin skeleton.—The first uroneural is fused with the preural centrum 1 and the ural centrum 1 to form the pleurostyle (Figure 7). The pleurostyle is stout and straight. The hypural 1 is the largest in hypural series, and its posterior margin is about 1.3 times that of the parhypural. The hypural 2 is fused with the preural centrum 1 and ural centrum 1, and is the same size as the hypural 3. A large gap exists between hypurals 2 and 3. The hypural 3 is articulated with the preural centrum 1 and ural centrum 1. The hypural 4 spreads out in the posterior area, is fan-shaped, and its width at the posterior margin is almost the same as the hypural 1. The hypural 5 is almost the same width as the hypural 3, and is almost the same length as half of the hypural 3. The hypural 6 is the smallest in the hypural series, which is well observed in KMNH VP 102,028 and KMNH VP 102,036. A single long, narrow epural is present and does not reach to the neural spine of the first preuralcentrum that is especially observed in NSM PV22663 and KMNH VP102,028. The neural spine of the first preuralcentra is sharp and is shorter than one-third the length of the neural spine of the second preuralcentrum. The neural spines of the second and third preuralcentra are almost the same size, and the flanges of these extend to the top of the neural spines. The haemal spines of the second and third preuralcentra are almost the same length. The posterior margin of the haemal spine of the second preuralcentrum is about twice as large as that of the third.
Figure 10.
Pelvic girdle of Nipponocypris takayamai sp. nov. A, photo of KMNH VP 102,062 (paratype); B, line drawing of A. Abbreviations: BASI = basiptery gium.
Figure 11.
The single most parsimonious tree (length 113, CI = 0.56, RI = 0.62, RC = 0.34) resulting from the character matrix of Appendix 2. Numbers to the branches are the apomorphic states of each clade (reversed characters are in bold). Numbers to the box of branches are bootstrap values (%) based on 1,000 replicates.
Figure 12.
Phylogenetic relationship of the genus Nipponocypris. The divergence times of each species are unknown.
Intermuscular bones.—Eight supuraneural bones are located between the dorsal fin skeleton and the supuranerural 3 bones (Figure 8). Supraneural 4–11 bones are not in contact with each neural spine. The anteriormost supraneural (supraneural 4 bone) is the largest, a thin, approximately rectangular sheet. The fifth to eleventh are small and thin in the series. There are numerous epipleurals and epineurals. These are hair-thin and almost all of these divide dichotomously.
Dorsal fin and skeleton.—The dorsal fin is located in the middle of the body, with its origin approximately posterior to the pelvic insertion (Figures 2, 4). The length of the dorsal fin base is shorter than that of the anal fin. In KMNH VP 102,036, there are three unbranched and unsegmented dorsal fin rays, and seven branched dorsal fin rays. The first dorsal fin ray is short. In KMNH VP 102,036, KMNH VP 102,028, KMNH VP 102,042 and NSM PV22663, the proximal pterygiophores are preserved. The first one forms a large bony plate with struts along the anterior and posterior margins, and divides dichotomously. The other proximal pterygiophores are shorter than the first one, and have median struts with developed anterior and posterior wings. The posterior and median pterygiophores are drum-shaped.
Anal fin and skeleton.—The anal fin is well preserved in NSM PV22663 (Figure 9) It is suspended under the haemal spines of the anterior caudal vertebrae. Its origin is well behind the posterior end of the dorsal fin base. The proximal pterygiophores are bony plates, and have median struts with anterior and posterior wings. The posterior and median pterygiophores are drum-shaped. Anal fin rays are fragmented and not clearly visible. However, it is estimated that the anal fin rays number 13, because the number of anal proximal pterygiophores is 11 based on comparison with Recent species, which have 13 anal fin rays (e.g. Nipponocypris temminckii and N. koreanus).
Shoulder girdle.—The postcleithrum is fragmentary and not clearly visible (Figure 5). The supracleithrum is well observable in KMNH VP 102,042 and KMNH VP 102,028, which is slender and slightly thick along the posterior margin. The sensory canal runs along the posterior upper edge of the supracleithrum. The cleithrum is L-shaped, and the anterior margin of its upper arm is thick. The posteroventral margin of the cleithrum is concave. The coracoid is thin and the ventral margin is convex, and the posterior margin is slightly thick and connects with the scapula. The mesocoracoid is observable only in KMNH VP 102,062; it is a reedy bone gradually becoming thinner dorsally, and is attached to the cleithrum at the dorsal margin. The postcleithrum is long, thin and slightly sigmoidal. The scapula is a short and stout bone and attached to the cleithrum at the dorsal part. Four pectoral radiais are preserved between the coracoid and pectoral fin rays in KMNH VP 102,042. There are 14 or 15 pectoral fin rays in KMNH VP 102,028, KMNH VP 102,042, and NSM PV22663.
Pelvic girdle.—The pelvic fin is located in the middle of the ventral margin of the body and its insertion is approximately anterior to the origin of the dorsal fin base (Figures 2, 4). There are ten ventral fin rays in NSM PV22663 (Uyeno et al., 1975, pl. 7, fig. b) and KMNH VP 102,028, although almost all of these are fragmented. The basipterygium is bifurcated and forms the external deep wing and the internal shallow wing with a trough-like depression at the ventral view of KMNH VP 102,062 (Figure 9). The anterior ends of both wings are apart and the bifurcation reaches beyond the two-thirds point of the bone. The posterior part is thick, and has projections reaching internally.
Result and discussion
Phylogenetic relationship of Nipponocypris takayamai sp. nov.
In the present study, the fossil species is assigned to the family Cyprinidae based on toothless jaws, short anteriorly curved ribs of the fourth vertebra, the upper jaw bordered only by the premaxilla, and presence of pharyngeal teeth (see Nelson, 2006; Nelson et al., 2016). Chen et al. (2008) proposed the new genus Nipponocypris for “Zacco temminckii”, “Z. sieboldii” and “Z. koreanus”, because the molecular phylogenetic study supported that Zacco forms a paraphyletic group. The diagnostic characters of Nipponocypris are the following combination of characters: there are three unbranched, unsegmented dorsal fin rays with seven to nine branched, segmented dorsal fin ray; there are three unbranched, unsegmented anal fin rays with eight to ten branched, segmented dorsal fin rays; pharyngeal tooth in three rows (dental formula = 1 – 2, 3 – 4, 5 – 5, 2- 4, 1 – 2); and some external characters (Chen et al., 2008). In addition, there are 42–45 vertebrae in the genus Nipponocypris (see Hosoya et al., 2003; Kim et al., 2005). The present fossil specimens are included in Nipponocypris because they have the diagnostic characters mention above.
Analysis of PAUP using the branch-and-bound search option resulted in one parsimonious tree (length 113, CI = 0.56, RI = 0.62, RC = 0.34) shown in Figure 11. About 44% of the 39 data for Nipponocypris takayamai sp. nov. are designated as question marks in the matrix. The genus Nipponocypris forms a monophyletic group, which is supported by four synapomorphies (the dorsal margin of the fourth infraorbital bone is U shaped (11 [2]); the fifth infraorbital bone has a projection (14 [1]); the posterodorsal process on the maxilla is triangular (18 [1]); and the flange of the neural spine of preuralcentrum 2 is extended over the middle of the neural spine of preuralcentrum 2 (33 [1])). The character 14 is unknown in N. takayamai sp. nov.
The clade of Nipponocypris koreanus + N. temminckii + N. takayamai sp. nov. has one synapomorphy (the number of proximal pterygiophores of the anal fin is 11 (33 [2])).
Nipponocypris temminckii + N. takayamai has four synapomorphies (the posterolateral margin of the frontal is notched (2 [1]); the dorsal margin of the fourth infraorbital bone is L-shaped (11 [1]) (= reversed character); the pores of the sensory canal of the dentary open posteroventral (15 [2]); and the pectoral fin's color is yellow (39 [2])), although the character states in the characters 15 and 39 are unknown in N. takayamai sp. nov.
In addition to the characters mentioned above, this fossil species is distinguished from Nipponocypris temminckii by these characters: the wings of neural spines of preural centrura 2 and 3 are developed (33 [3]; 34 [3]); and the notch at the frontal margin is weaker than that of N. temminckii (2 [1]).
On the other hand, the fossil species, Zacco honggangensis Li and Wang, 1981 (Li and Wang in Wang et al., 1981) from the Buxin Formation, Sanshui Basin, China is related to Z. platypus with almost the same numbers of vertebrae and fin rays. Zaceo sp. from the Eozen Formation in Ishikawa Prefecture reported by Nakajima (1975) and Tomoda and Nakajima (1975) is similar to Z. platypus in the shape of the dentary and the number of vertebrae. Therefore, it is possible that these fossils are closer to or belong to Zacco rather than Nipponocypris.
As a result, the cladistic analysis and comparisons with the Recent species in the present study demonstrate that this fossil species is the first record of the genus Nipponocypris from the Cenozoic of East Asia and a sister species of N. temminckii (i.e., it is the stem taxon of N. temminckii).
Divergence time of the genus Nipponocypris
The divergence time of the genus Nipponocypris is uncertain, although it has been estimated by several previous molecular phylogenetic studies. Lee et al. (1989) analyzed 9 restriction endonucleases and estimated that the divergence time of N. temminckii (Zacco temminckii MM Type in Lee et al., 1989) from N. koreanus (Zacco temminckii MS Type in Lee et al., 1989) was 2.1 Ma. Its estimation of divergence times was based on the general rate for mitochondrial genes (2% nucleotide substitution per million years) by Brown et al. (1979). Min and Yang (1991) estimated that the divergence of lineages Nipponocypris (Zacco temminckii group in Min and Yang, 1991) from Candidia took place 2.6 Ma, and N. temminckii (Zaceo temminckii type A in Min and Yang, 1991) from N. koreanus (Zacco temminckii type B in Min and Yang, 1991) took place 0.8 Ma. This estimation was based on Nei's (1972) genetic distance coefficients (D values) and Nei's protein calibrations (Nei, 1975). In Okazaki et al. (1991), the divergence time of N. sieboldii (Zacco temminckii Type A in Okazaki et al., 1991) from N. temminckii (Zacco temminckii Type B in Okazaki et al., 1991) was 3 Ma, based on Nei's formula (Nei, 1975). Wu et al. (2007) hypothesized that the estimation for divergence times of mitochondrial D-loop sequences was 6% per million years, and estimated that separation between Candidia and Nipponocypris (Zacco temminckii complex in Wu et al., 2007) was 0.75 Ma. Chen et al. (2011) hypothesized that the earliest possible divergence time of Candidia lineages was between 5 and 3 Ma. This estimation was based on the time when Taiwan Island was lifted above sea level (Chemenda et al., 2001).
However, these molecular clocks should not be applied to the estimation of divergence times, because evolutionary rates are never constant (Thorne and Kishino, 2005). Okazaki et al. (1991) remarked that the fossil record is important to set up a standard molecular clock for cyprinid fishes. Recently, molecular geneticists have suggested that the fossil record is important for molecular phylogenetic studies to calibrate divergence time (e.g. Thorne and Kishino, 2005; Inoue et al., 2010).
The fossil-bearing strata, the Nogami Formation, is considered to be deposited in the Middle Pleistocene based on the radiometric age of the basement igneous rock, and the Hiwaku Tephra in the uppermost part of the Nogami Formation as mentioned above.
The present study suggests that the divergence of Nipponocypris temminckii from N. takayamai sp.nov. took place in the Middle Pleistocene, while the time of divergence of N. koreanus is earlier than that. At the least these results are incongruent with those of Wu et al. (2007), because the divergence time of Candidia and Nipponocypris by Wu et al. (2007) was younger than the result of this study. On the other hand, other calibrated divergence times of previous molecular studies (Chen et al., 2011; Lee et al., 1989; Min and Yang, 1991; Okazaki et al., 1991) basically support the present study although evolutionary rates are never constant as mentioned above. Because the divergence time of Candidia or N. koreanus is earlier than the Middle Pleistocene, Nipponocypris takayamai sp. nov. provides an age constraint for fossil calibrations of the lineage of the genus Nipponocypris and indicates that the genus Nipponocypris appeared in East Asia by Middle Pleistocene time at the latest (Figure 12).
Acknowledgments
The authors are greatly indebted to Teruya Uyeno of the National Museum of Nature and Science for his encouragement and suggestions throughout this study. The authors would like to express their sincere gratitude to Akinori Takayama of the Hakusan Kogyo Corporation for the donation of the holotype and paratypes to the Kitakyushu Museum of Natural History and Human History and his help in collecting other specimens and fieldwork. Naoki Eto of Kokonoe town is thanked for donating the paratypes to the National Museum of Nature and Science. The authors are grateful to Hideo Takagi, Yoshihide Ogasawara, Ren Hirayama and Tohru Ohta of Waseda University for their valuable suggestions and comments to the first author. The authors also deeply thank Akinori Takahashi of Waseda University for his comments on an early version of this manuscript. The authors also wish to thank the following people for the loan of specimens and their help in the fieldwork: Makoto Manabe (National Museum of Nature and Science) for helping in examining NSM specimens; and Tokuji Mitsugi (The Factory of Education and Welfare Science of Oita University) and the staff of Hakusan Kogyo Corporation for their help and support to the fieldwork in Oita Prefecture. The authors also deeply thank Mizuki Murakami (Shumei University) and Seike Kazuma (Saitama Museum of Natural History), graduate and undergraduate students of Hirano Laboratory, and all of the staff of the Department of Earth Sciences, School of Education, Waseda University for their valuable discussions, support, and warm encouragement. The authors are grateful to Brito M. Paulo (Universidade do Estado do Rio de Janeiro), and anonymous referees who reviewed this paper and provided many valuable comments and suggestions that helped improve the final version. This study was partly supported by JSPS KAKENHI Grant Number JP26400506 to Y. Yabumoto.
References
Appendices
Appendix 1.
List of the characters. The consistency index (CI), retention index (RI), for the most parsimonious tree (Figure 11) is shown for each character.
Character 1.—Sensory canals of parietals. Character states: in contact with each other (0); not in contact with each other, and lengths are longer than half width of parietal (1); not in contact with each other, and length shorter than half width (2). ci = 0.67. ri = 0.00.
Character 2.—Posterolateral margin of frontal. Character states: smooth (0); convex (1); notched (2). ci = 0.67. ri = 0.67.
Character 3.—Projection of intercalar. Character states: present (0); absent (1). ci = 0.25. ri = 0.25.
Character 4.—Width of supraorbital. Character states: almost the same as half its length (0); narrower than half its length (1). ci = 0.33. ri = 0.33.
Character 5.—Sensory canal on first infraorbital. Character states: curved (0); straight (1) (Dai and Yang, 2003; character no. 10). ci = 1.00. ri = 1.00.
Character 6.—Number of pores of sensory canal on first infraorbital. Character states: 2 (0); 3 (1) (modified from Dai and Yang, 2003; character no. 11), ci = 0.33. ri = 0.00.
Character 7.—Width of anterior half of second infraorbital. Character states: same as its posterior half (0); wider than its posterior half (gradually narrow from its anterior end to its posterior end) (1) (Dai and Yang, 2003; character no. 12). ci = 0.33. ri = 0.00.
Character 8.—Width of third infraorbital. Character states: significantly wider than fourth (0); almost as wide as fourth (1) (modified from Dai and Yang, 2003; character no. 13). ci = 1.00. ri = 1.00.
Character 9.—Width of fourth infraorbital. Character states: enrtirely same (0); abruptly widened below at its middle point (1); gradually widened from its anterior end to its posterior end (2). ci = 0.67. ri = 0.75.
Character 10.—Nub at posterodorsal margin of fourth infraorbital bone. Character states: absent (0); present (1). ci = 0.33. ri = 0.67.
Character 11.—Posterior margin of fourth infraorbital bone. Character states: (0) straight; bend to L-shaped (1); dent into U-shaped (2). ci = 0.40. ri = 0.40.
Character 12.—Fifth infraorbital bone. Character states: approximately quadrangle (0); approximately triangle (1); tube shaped (2); absent (3). ci = 1.00. ri = 1.00.
Character 13.—Width of the fifth infraorbital bone. Character states: atrophied or narrower than its length (0); approximately the same as its length of these (1); wider than its length (2). ci = 0.50. ri = 0.50.
Character 14.—Projection of fifth infraorbital bone at posterodorsal margin. Character states: absent (0); present (1). ci = 0.50. ri = 0.67.
Character 15.—Direction of openings of dentary sensory canals. Character states: laterally (0); ventrally (1); posteroventrally (2). ci = 0.33. ri = 0.00.
Character 16.—Anterior end of dentary. Character states: not constricted (0); constricted (1); hooked (2). ci = 0.50. ri = 0.67.
Character 17.—Dorsal margin of dentary. Character states: straight (0); downward (1). ci = 1.00. ri = 1.00.
Character 18.—Posterodorsal process of maxilla. Character states: trapezoidal shaped (0); triangular shaped (1); dorsally convex or straight, and that the dorsal margin of these elongated to the posterior part (2). ci = 0.67. ri = 0.83.
Character 19.—Ventral margin of premaxilla. Character states: straight (0); convex (1). ci = 1.00. ri = 1.00.
Character 20.—Posterior margin of opercule. Character states: convex or straight (0); concave (1) (modified from Dai and Yang, 2003; character no. 21). ci = 0.33. ri = 0.67.
Character 21.—Quadrate-pterygoid fenestra (Greenwood et al., 1966). Character states: absent (0); present (1). ci = 1.00. ri = 1.00.
Character 22.—Shape of metapterygoid. Character states: not constricted (0); constricted to H-shaped (1). ci = 1.00. ri = 1.00.
Character 23.—Shape of quadrate. Character states: snicked to a V-shaped (0); snicked to an L-shaped or weakly U-shaped, and the upper arm of the quadrate is shorter than the lower arm (1); snicked a strong U-shaped, the upper arm of the quadrate is elongate, and the length of the upper arm is the same size in length of the lower arm (2). ci = 0.67. ri = 0.67.
Character 24.—Symplectic. Character states: in contact with anteroventral margin of metapterygoid (0); not in contact with anteroventral margin of metapterygoid (1). ci = 1.00. ri = 1.00.
Character 25.—Posterior margin of urohyal. Character states: concave (0); smooth (1); snicked (2). ci = 0.50. ri = 0.33.
Character 26.—Shape of urohyal at ventral view. Character states: slender (0); posterior region wider than anterior one (1). ci = 0.33. ri = 0.60.
Character 27.—Dentition of pharyngeal tooth. Character states: 3 rows (0); 2 rows (1). (Dai and Yang, 2003; character no. 24) ci = 0.50. ri = 0.50.
Character 28.—Anterior branch of pharyngeal bone. Character states: as long as its posterior branch (0); longer than its posterior branch (1) (Dai and Yang, 2003; character no. 26). ci = 1.00. ri = 1.00.
Character 29.—End of dorsal branch of parapophysis of 4th vertebra. Character states: blunt or shovel-shaped (0); tipped (1) (modified from Dai and Yang, 2003; character no. 42). ci = 0.33. ri = 0.33.
Character 30.—Fourth supraneural bone. Character states: convex ventrally or sigmoidal (0); snicked at the anterior margin (1); approximately quadrangle (2) (modified from Dai et al., 2005; character no. 56). ci = 0.67. ri = 0.50.
Character 31.—Number of supraneural bones. Character states: more than 8 (0); 6–7 (1) (modified from Dai et al., 2005; character no. 57) ci = 0.40. ri = 0.40.
Character 32.—Number of proximal pterygiophores of anal fin. Character states: more than 13 (0); 12 (1); 11 (2); 10 (3); 9 (4). ci = 1.00. ri = 1.00.
Character 33.—Flange of neural spine of preuralcentrum 2. Character states: not extended over middle of neural spine of preuralcentrum 2, or to middle (0); extended over middle of neural spine of preuralcentrum 2(1); projected at base (2); extended to top of neural spine of preuralcentrum 2 (3). ci = 0.75. ri = 0.80.
Character 34.—Flange of neural spine of preuralcentrum 3. Character states: not extended over middle of neural spine of preuralcentrum 3, or to middle (0); extended over middle of neural spine of preuralcentrum 3(1); projected at base (2); extended to top at neural spine of preuralcentrum 3 (3). ci = 0.60. ri = 0.50.
Character 35.—Anterodorsal margin of cleithrum. Character states: straight (0); convex (1) (Dai and Yang, 2003; character no. 30). ci = 0.50. ri = 0.50.
Character 36.—Ventral keel. Character states: from pelvic fin base to anus (0); absent on anterior half region from pelvic fin base to anus (1); absent or indistinct (2). ci = 0.50. ri = 0.50.
Character 37.—Vertical stripes. Character states: absent or blurred (0); irregular pattern and black (1); bandlike and black (2). ci = 0.67. ri = 0.67.
Character 38.—Crossband of body. Character states: absent (0); bandlike (1); irregular pattern and blue (2) ci = 0.67. ri = 0.67.
Character 39.—Color of pectoral fins. Character states: transparent or light yellow (0); yellow, and orange or red at margin (1); yellow (2). ci = 0.67. ri = 0.75.
