Onychostoma minnanensis, a new cyprinid species (Teleostei: Cyprinidae) from Fujian, southern mainland China, with comments on the mitogenetic differentiation among related species

Abstract

Onychostoma minnanensis sp. nov. is described from tributaries of the Julong River in Fujian Province, China, based on morphological and mitogenetic evidence. It is characterized by the following unique combination of features: (1) a flexible and nonserrated last simple ray, (2) lateral-line scales always 41–44 modally 42 and predorsal scales 12 or 13 modally 12, (3) longer maxillary barbels in head length, (4) smaller cornified rostral cap with thick upper lip and lower part of upper lip exposed, and (5) coloration of young individuals with random grayish-black spots near or above lateral-line scales. Morphological surveys and mitochondrial DNA analyses reveal that O. minnanensis and Onychostoma barbatulum in the neighboring Min River Basin are the most closely related species.

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Onychostoma minnanensis, a new cyprinid species (Teleostei:
Cyprinidae) from Fujian, southern mainland China,
with comments on the mitogenetic differentiation
among related species
Nian-Hong Jang-Liaw •I-Shiung Chen
Received: 2 May 2011 / Revised: 6 August 2012 / Accepted: 19 August 2012
ÓThe Ichthyological Society of Japan 2012
Abstract Onychostoma minnanensis sp. nov. is described
from tributaries of the Julong River in Fujian Province,
China, based on morphological and mitogenetic evidence.
It is characterized by the following unique combination of
features: (1) a flexible and nonserrated last simple ray, (2)
lateral-line scales always 41–44 modally 42 and predorsal
scales 12 or 13 modally 12, (3) longer maxillary barbels in
head length, (4) smaller cornified rostral cap with thick
upper lip and lower part of upper lip exposed, and (5)
coloration of young individuals with random grayish-black
spots near or above lateral-line scales. Morphological
surveys and mitochondrial DNA analyses reveal that
O. minnanensis and Onychostoma barbatulum in the
neighboring Min River Basin are the most closely related
species.
Keywords Onychostoma minnanensis sp. nov. 
Morphology CO1 cyt bmtDNA phylogeny
Introduction
Onychostoma Gu
¨nther 1896 is a cyprinid genus comprising
19 or 20 valid species distributed in hill-stream habitats in
East and South East Asia (Wu et al. 1977; Shan et al. 2000;
Kottelat 2001a,b; Nguyen and Ngo 2001; Xin et al. 2009).
Onychostoma is characterized by a lower lip restricted only
to the lower jaw sides, which bears a sharp cornified sheath
on the cutting edge (Chen 1989; Shan et al. 2000). Xin
et al. (2009) concluded that 19 nominal species were
documented from South East Asia including Laos, Cam-
bodia, Vietnam, as well as mainland China and Taiwan.
Among them, 15 valid species of Onychostoma are rec-
ognized in mainland China and Taiwan (Shan et al. 2000;
Chen and Chang 2005; Xin et al. 2009). Kottelat (2001b)
considered Onychostoma uniforme (Mai 1978) as a valid
species, and also suggested that further confirmation was
required. Xin et al. (2009) also modified the catalog of
species-groups, which was proposed by Chen (1989), by
reconsideration of the criteria of the width of the mouth,
mouth-opening shape, and postlabial groove length as
rather important diagnostic features, and they suggest that
members of Onychostoma could be subdivided into three
major groups.
Among them, Onychostoma barbatulum (Pellegrin
1908) is considered to be a widely distributed species in
Taiwan and southeastern mainland China, including the
tributaries of the Changjiang (Yangtze), Lin, Min, and
Pearl River systems, as well as the Julong River Basin in
Fujian Province (Shan et al. 2000). During a series of field
expeditions to the Julong R. Basin in 2008 and 2009, we
collected unusual specimens of Onychostoma rather
resembling O. barbatulum.
To provide more evidence regarding speciation histo-
ries, we employed molecular biological approaches to
reveal its relationship with related congeners from the same
geographical region. Molecular phylogenetic analysis from
several mitochondrial (mt)DNA sequences has been suc-
cessfully used to reconstruct intra- and interspecific
Electronic supplementary material The online version of this
article (doi:10.1007/s10228-012-0312-0) contains supplementary
material, which is available to authorized users.
N.-H. Jang-Liaw I-S. Chen (&)
Institute of Marine Biology, National Taiwan Ocean University,
Keelung 202, Taiwan, Republic of China
e-mail: iscfish@yahoo.com.tw; isc@ntou.edu.tw
N.-H. Jang-Liaw
Department of Zoology, National Museum of Natural Science,
Taichung 404, Taiwan, Republic of China
123
Ichthyol Res (2013) 60:62–74
DOI 10.1007/s10228-012-0312-0

relationships among many fish groups (e.g., Wang et al.
2004; Watanabe et al. 2007; Wu et al. 2007; Chen and
Chang 2007; Chiang and Chen 2008; Chen et al. 2009).
The aims of this study are not only to describe this species
of Onychostoma as new to science, but also to provide a
phylogenetic assessment of all the available related con-
generic species in the same region inferred from the
mitochondrial cytochrome (cyt) band cytochrome coxi-
dase subunit 1 (CO1) genes.
Materials and methods
Both morphological and molecular biological surveys were
employed in this research. According to the specimens
used for morphology purpose were fixed by formalin, some
specimens used for DNA analysis (including holotype and
some of paratypes) were taken fin clips before fixing in
10 % formalin, or fixed in 95 % ethanol directly before
storage in laboratory. The fin clips samples were preserved
in 99.5 % ethanol before the DNA extraction procedure.
Morphological study. The holotype and most of the
paratypes were fixed in 10 % formalin and kept in 70 %
ethanol for permanent storage. Other paratypes were first
directly fixed in 80–95 % ethanol for later use for mole-
cular biological analyses. Methods of measurements,
counts, and terminology of the mouth structure followed
Kottelat and Freyhof (2007). All measurements were made
point to point, never by projections. The number of spec-
imens with a given count is indicated in parenthesis after
the count. All fish lengths are indicated as standard length
(SL). Among the specimens of Onychostoma collected in
the Julong R. Basin, only adult specimens (SL [74 mm)
were used for morphological study. Observations of
cephalic lateral-line systems were mainly conducted fol-
lowing the method of Kurawaka (1977). Terminology for
each cephalic lateral-line system (canals) generally fol-
lowed Takeuchi et al. (2011).
The type specimens and comparative material are
deposited in the National Museum of Natural Science,
Taichung (NMNS) and the Pisces collection of National
Taiwan Ocean University, Keelung (NTOUP). Additional
comparative materials from Institute of Zoology, Chinese
Academy of Sciences, Beijing (IOZCAS) were checked
and are listed below.
DNA extraction, amplification, and sequencing. Crude
DNA was isolated from a piece of pectoral or pelvic fin
using a Tissue and Cell Genomic DNA Purification Kit
(Hopegen Biotechnology Development Enterprises,
Taiwan). Two fragments of mtDNA protein-coding genes
were selected as genetic markers. A 1080-bp cyt bgene
sequence was amplified by polymerase chain reaction
(PCR) using the primer set of Glu-L (50-GAAGAACCA
CCGTTGTTATTCAA-30) and Ptacek 2-H Cyt b (50-TC
TTCTACTGGTTGTCCTCCGATTCA-30) designed by
Palumbi et al. (1991) and Ptacek et al. (1994), respectively.
Another fragment as 1,480 bp of the CO1 gene sequence
was amplified by PCR using the primer set of JLCO1
(50-TCTGTCTTCGGGGCTACAAC-30) and JLCO1R (50-AT
GTGRCTGGCTTGAAACCAG-30). PCR consisted of 35
cycles of denaturation (95 °C, 50 s), annealing (52 °C for
cyt b,56°C for CO1, 1 min), and extension (72 °C, 80 s)
on a RoboCycler Gradient 96 temperature cycler (Strata-
gene, USA) with a PCR Master Mix Kit (Hopegen
Biotechnology Development Enterprises). PCR products
were purified with a HiYield Gel/PCR DNA Fragments
Extraction Kit (RBC Bioscience, Taiwan) and used for
sequencing. Sequences were obtained by automated
sequencing following a multiple fluorescent dye method
using an ABI PRISM 3130xl genetic analyzer with Big-
Dye
Ò
Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems, USA), and were aligned with the aid of Meg-
Align version 4.0 (DNA Star, USA) and then adjusted
manually by eye, using the complementary strand for ver-
ification. The primers used for DNA sequencing for both
cyt band CO1 markers were the same as used in PCRs.
Phylogenetic analysis of mtDNA. DNA sequence anal-
yses employed MEGA version 4.02 (Tamura et al. 2007)
for pairwise distance analysis, and DNA SP ver-
sion 5.00.07 (Librado and Rozas 2009) for haplotype
determination, polymorphic sites, parsimony informative
sites, haplotype diversity (Hd), and nucleotide diversity (p)
analyses. Preliminary phylogenetic and molecular evolu-
tionary analyses of these species of Onychostoma and
outgroup taxa were constructed by neighbor-joining (NJ),
maximum-likelihood (ML) analyses using unique haplo-
types of the two datasets from mtDNA fragments with
PAUP* version 4.0 beta8 (Swofford 2001), and Bayesian
analyses with MrBayes version 3.1.2 (Ronquist and Huel-
senbeck 2003). NJ analyses were based on the two-
parameter method of Kimura (K2P). For the cyt bdataset,
likelihood settings were used from the best-fit model
TVM ?G (gamma shape 0.1879) with base frequencies of
A of 0.2866, C of 0.3165, G of 0.1344, and T of 0.2625.
For the CO1 dataset, likelihood settings were used from the
best-fit model HKY ?G (gamma shape 0.1455) with base
frequencies of A of 0.2744, C of 0.2742, G of 0.1689, and
T of 0.2825, and a transition/transversion ratio of 10.5997.
Both were selected by hierarchical likelihood ratio tests
(hLRTs) in MODELTEST 3.7 (Posada and Crandall 1998).
ML analysis was conducted using random-addition heu-
ristic search with subtree pruning–regrafting (SPR) branch
swapping. Bootstrapping {1,000 replicates for NJ [NJ
option] and ML [full heuristic search (TBR) branch
swapping]} was performed to obtain a relative measure of
nodal support for the resulting tree (Felsenstein 1985).
A new cyprinid species 63
123

Besides, Bayesian analyses were performed with four
chains run simultaneously for 3,000,000 generations for
both cyt band CO1 datasets; trees were sampled every 100
cycles. The first 20 % of sampled trees was discarded as
burn-in. The models of nucleotide substitution best fitting
the data were selected using the Bayesian information
criterion (BIC; Schwarz 1978) as implemented in
jModeltest 0.1.1 (Posada 2008). For the cyt bdataset, the
HKY ?I model was selected as the best-fit model with a
fixed transition/transversion rate ratio (tratio) of 7.469; for
the CO1 dataset, the HKY ?G model was selected as the
best-fit model with a gamma distribution with shape
parameter 0.1450. Bayesian inference (BI) using Mr. Bayes
3.1.2 was also used to yield trees and to assess support on
both genes.
The estimation of the posterior probability distribution
of divergence times among haplotypes of Onychostoma
minnanensis and O. barbatulum employed the computer
program Bayesian Evolutionary Analysis Sampling Trees
(BEAST) 1.6.1 (Drummond and Rambaut 2007). Estima-
tion of divergence time in this study was based on the
cyt bdataset only. The posterior distributions were
obtained by the Markov chain Monte Carlo method run for
twenty million generations, sampling and saving every
1,000th tree under the HKY model with a fixed clock rate
of 0.76 %/Myr, following Zardoya and Doadrio (1999),
with a strict clock model. The results were viewed using
Tracer version 1.5 (Rambaut and Drummond 2007) with
effective sample sizes (ESS) exceeded 200.
Onychostoma minnanensis sp. nov.
(Figs. 1,2,4a, Table 1)
Holotype. NMNSF 01970, 124.0 mm SL, a small dammed
pool of Yuanfeng R. near Guongya Mt., in the Julong R.
Basin, 24°55046.9900N, 117°25015.6000E (site 2 in Fig. 3),
980 m in elevation, Hua-an County, Julong R. Basin,
Fujian Province, China, Coll. N.-H. Jang-Liaw, S.-C.
Cheng, and C.-C. Liu, 29 March 2009.
Paratypes. NMNSF 01970-1 (2 specimens),
111.1–121.4 mm SL; NTOUP-2009-10-077 (1), 121.6 mm
SL; NMNSF 01969 (3), 77.9–97.1 mm SL; NTOUP-2009-
10-078 (1), 95.7 mm SL, data same as for holotype; NTOUP-
2012-07-731 (3), 67.98–107.44 mm SL, a small tributary of
Chuanchang R., 24°33017.0800N, 117°12058.5300E (site 4 in
Fig. 3), 442 m in elevation, Nanjing County, Julong R.
Basin, Fujian Province, China, Coll. I-S. Chen, 15 June 2012.
Nontypes. NMNSF 01965 (12 specimens), 60.2–74.0 mm
SL; NTOUP-2009-10-078 (8), 60.5–70.5 mm SL, a small
tributary of Yuanfeng R. in the Julong R. Basin nearGuongya
Mt., 24°54038.3000N, 117°24059.4000E (site 1 in Fig. 3),
1,015 m in elevation, Hua-an County, Julong R. Basin, Fujian
Province, China, Coll. N.-H. Jang-Liaw and W.-H. Chou, 28
Oct. 2008. NMNSF 01971 (3), 49.9–72.6 mm SL; NTOUP-
2009-10-079 (2), 49.7 and 62.1 mm SL; NMNSF01972 (2),
44.5 and 50.8 mm SL; NMNSF 01973 (9), 22.5–45.4 mm SL;
data same as for holotype. NMNSF 02000 (1), 37.9 mm SL,
Longchuang R., Julong R. Basin, 24°59.403400N117°0036.
0400E(site3inFig.3), 440 m in elevation, Longyen City,
Fujian Province, China, Coll. N.-H. Jang-Liaw, S.-C. Cheng,
and C.-C. Liu, 31 March 2009.
Diagnosis. Onychostoma minnanensis can be well dis-
tinguished from other congeners by the following unique
combination of features: (1) a flexible and nonserrated last
simple ray of dorsal fin, (2) squamation: lateral-line scales
always 41–44 modally 42 and predorsal scales 12–14
modally 12, (3) longer maxillary barbels: 9.7–12.4 %
(mean 11.2 %) in head length, (4) smaller rostral cap with
thick upper lip and lower part of upper lip exposed, and (5)
coloration of young individuals (\75 mm SL) with random
grayish-black spots near lateral-line scales.
Description. Meristic and morphometric data of eight
adult type specimens are listed in Table 1. Body elongate,
moderately compressed. Dorsal body profile convex with a
slight hump posterior to nape, ventral profile somewhat
rounded. Head longer than deep, dorsal profile strongly
convex. Snout stout and rounded, longer than eye diameter
in adults. Interorbital area slightly convex. Mouth subter-
minal. Maxillary reaching vertical of anterior margin of
orbit. Upper lips thick, upper region covered by rostral fold
(rostral cap) and lower 1/2 mostly exposed. Barbels in two
pairs (Figs. 1,2); one smaller pair on maxillary, another
larger mandibular pair at corner of mouth measuring
9.7–12.4 % (mean 11.2 %, 12.4 % in holotype) of head
length (HL) and 34.8–53.9 % (mean 45.4 %, 53.9 % in
holotype) of eye diameter.
Dorsal fin with 2 simple and 8 branched rays; last one
split to base; last simple ray nonserrated; dorsal fin origin
inserted slightly in front of vertical with pelvic fin origin;
distal margin slightly concave. Pectoral fin with 1 simple
ray and 12 (3) or 13 (6, including holotype) branched rays,
its length shorter than HL. Pelvic fin with 1 simple ray and
8 (7, including holotype) or 9 (2) branched rays, shorter
than HL, not reaching anal-fin origin when adpressed. Anal
fin with 3 simple and 5 branched rays, last one split to base,
origin behind vertical through posterior end of dorsal fin
base, distal margin concave. Caudal fin forked. Lateral-line
complete, with 41–44 (42 in holotype, frequencies in order:
2, 3, 2, 2) perforated scales, bent ventrally before pelvic-fin
origin and running parallel with ventral margin to anal-fin
origin, rear extended mid-laterally along caudal peduncle.
Scale rows between lateral line and origin of dorsal fin 5 (2,
including holotype) or 6 (7), as is count between lateral line
and middle of belly. Predorsal scales 12–14 (14 in holo-
type, frequencies in order: 6, 2, 1); circumpeduncular
scales 14–16 (15 in holotype, frequencies in order: 2, 4, 3).
64 N.-H. Jang-Liaw, I-S. Chen
123

Pelvic axillary scales long, reaching beyond base of pelvic
fin. Pharyngeal teeth triserial as 1, 3, 4-4, 3, 1 (examined in
1 paratype and 1 nontype).
Cephalic lateral-line canal system (Fig. 4a). The
cephalic lateral-line canal system consists of the infraor-
bital canal (IOC, with 11 pores in each side), preopercu-
lomandibular canal (POM, 10 pores), the supraorbital canal
(SOC, 10 pores), and the supratemporal canal (STC, 7
pores, including the middle one of both sides)
(observations based on a paratype, NTOUP-2009-10-077,
121.6 mm SL). A connection of IOC and STC, and the
unification of STC (connection of both sides of STC) were
observed. The SOC was not connected with the other
canals. The IOC was not connected with POM either
(observed from one).
Coloration when fresh. In holotype, dorsum grayish,
belly and lower half of body silver. Head grayish-brown on
dorsal 1/3 region, and lower remaining area pale to light
Table 1 Morphometric and meristic data of adult Onychostoma minnanensis sp. nov. [including the holotype (H) and paratypes; ranges and
means include the holotype] and O. barbatulum
O. minnanensis (n=9) O. barbatulum (n=13)
H Range Mean Range Mean
Standard length (SL, mm) 124 77.9–124 105.7 90.9–197 136.8
Morphometrics
%SL
Head length (HL) 22.5 22–25 22.9 20.7–24.1 22.5
Body depth at dorsal-fin origin 23.4 23.3–26.3 24.8 24.2–26.9 26
Body width at dorsal-fin origin 15.1 11.4–17.2 14.1 10.8–17.1 14.3
Predorsal length 46.3 44.7–47.7 46.5 44.5–47.7 45.9
Postdorsal length 42.4 40.6–42.4 41.6 39.4–45.4 42.8
Prepelvic length 50.2 49.4–52.1 50.4 46.3–52.3 49.8
Preanal length 73.4 69.9–73.5 71.9 68.2–73.6 70.9
Distance between pectoral- and pelvic-fin origins 29.1 24.7–31.5 29.2 27.4–31.5 29
Distance between pelvic- and anal-fin origins 27.2 23.2–27.2 24.7 21.7–24.9 23
Depth of caudal peduncle 9.6 9.4–10 9.7 9.7–11.9 10.7
Length of caudal peduncle 21.2 18.2–21.8 19.9 17.9–24.7 21.3
Depth of dorsal fin 21.2 20.3–22.2 21.4 18.4–22.8 20.8
Length of dorsal fin base 14.7 14.1–15.9 14.9 11.8–15.8 14.4
Length of anal fin base 8.5 7.6–9.5 8.4 7.8–9.8 9
Length of pectoral fin 17.5 17.5–20.9 18.9 18.3–21.6 19.7
Length of pelvic fin 17.5 15.7–18.3 17 15.5–18.7 17.1
Length of anal fin 20.6 17.9–22.7 20.5 13.2–22.2 17.4
%HL
Head depth at nape 83.8 83.8–93.6 90 78.8–91.5 85.2
Head depth at eye 63.8 58.6–64.9 62 47.5–63.3 57.2
Snout length 34.2 29–37.7 33.2 32.0–39.1 36.3
Eye diameter 23 21.5–32.2 25 19.9–27.3 22.8
Postorbital distance 47.9 42.8–51.7 46.8 43.0–52.8 49.1
Maximum head width 70 56.3–70 63.9 58.5–73.1 67.1
Interorbital width 43.3 37.8–45.9 41.9 40.3–50.1 46.5
Mouth width 46.3 37.9–46.3 41.3 37.2–49.2 43.1
Mandibular barbel length 12.4 9.7–12.4 11.2 2.9–4.3 3.5
Counts
Lateral line scales 42 41–44 42.3 45–47 46.2
Scales between lateral line and origin of dorsal fin 5 5–6 5.8 6 6
Scales between lateral line and middle of belly 6 5–6 5.7 6–7 6.2
Scales between lateral line and origin of anal fin 5 5–6 5.4 4–6 4.7
Circumpeduncular scales 15 14–16 15.1 14–17 15.9
Predorsal scales 14 12–14 12.7 13–15 13.3
A new cyprinid species 65
123

Table 2 Sampling localities, sample sizes (N), haplotypes, and specimen numbers of the mtDNA samples analyzed in this study
Species/sampling locality River system NHaplotypes (nof individuals) Specimen voucher and cyt b/CO1 GenBank no.
cyt bCO1
O. minnanensis 21
1. Guongyashan Mt. (GYS1) Julongjiang R. (FJ) 10 JCyb-1 (10) JCO1-1 (9), JCO1-2 NMNSF01965 (10); HM142529-538/HM142478-487
2. Guongyashan Mt. (GYS2) Julongjiang R. (FJ) 10 JCyb-2 (10) JCO1-3 (10) NMNSF01969 (3), 70, 70-1, 71 (2), 72 (2), NTOUP-2009-10-077; HM142539-548/
HM142488-497
3. Longyen (LY) Julongjiang R. (FJ) 1 JCyb-1 JCO1-4 NMNSF02000; HM142549/HM142498
O. barbatulum 27
5. Fuchiu (FC) Minjiang R. (FJ) 1 BCyb-1 BCO1-1 NMNSF01815; HM142550/HM142499
6. Siyangjian Town (SYJ) Minjiang R. (FJ) 1 BCyb-2 BCO1-2 NMNSF01987; HM142551/HM142500
7. Shanping (SP) Minjiang R. (FJ) 1 BCyb-3 BCO1-3 NTOUP2009-09-074; HM142552/HM142501
8. Taipei (TP) Tamsui R. (TW) 1 BCyb-4 BCO1-4 NTOUP2009-09-068; HM142553/HM142502
9. Miaoli (ML) Houlong R. (TW) 1 BCyb-5 BCO1-5 NMNSF02011; HM142554/HM142503
10. Taichung (TC) Dajia R. (TW) 1 BCyb-6 BCO1-6 NMNSF01520; HM142555/HM142504
11. Puli (PL) Wu R. (TW) 2 BCyb-7, BCyb-8 BCO1-7 (2) NMNSF02020 (2); HM142556-557/HM142505-506
12. Jhushan (Jh) Jhuoshuei R. (TW) 2 BCyb-7 (2) BCO1-8 (2) NMNSF01409 (2); HM142558-559/HM142507-508
13. Shueili (SL) Jhuoshuei R. (TW) 1 BCyb-8 BCO1-9 NMNSF02018; HM142560/HM142509
14. Chiayi (CY) Bajhang R. (TW) 1 BCyb-9 BCO1-10 NTOUP2009-09-069; HM142561/HM142510
15. Alishan Mt. (ALS) Zengwun R. (TW) 7 BCyb-9 (7) BCO1-11 (7) NTOUP2009-09-071 (7); HM142562-568/HM142511-517
16. Meishan (MS) Gaoping R. (TW) 5 BCyb-9 (5) BCO1-11 (4), BCO1-12 NTOUP2009-09-073 (5); HM142569-573/HM142518-522
17. Hualien (HL) Hualien R. (TW) 1 BCyb-4 BCO1-13 NTOUP2009-09-070; HM142574/HM142523
18. Jiaosi (JS) Yilan R. (TW) 2 BCyb-10 (2) BCO1-14 (2) NMNSF02014 (2); HM142575-576/HM142524-525
O. lepturum
Nankai 1 – – NTOUP2009-09-075; HM142578/HM142528
O. alticorpus
Ligang Gaoping R. (TW) 1 – – NMNSF01433; HM142577/HM142527
Acrossocheilus paradoxus
Miaoli Houlong R. (TW) 1 – – NMNSF02008; HM142579/HM142526
66 N.-H. Jang-Liaw, I-S. Chen
123

greenish-silver. Basal region of scales grayish-black except
scales on belly. Lateral body with reticulate pattern on
scales on dorsal 2/3 region. All fins pale to grayish and
translucent. Somewhat pinkish-orange on pectoral, pelvic,
and caudal fins. Dorsal fin membrane with a series of
vertical black bars distally (Fig. 1; see also Fig. 2showing
paratype condition). Most young specimens smaller than
75 mm SL characterized as having random arranged
grayish-black spots on scales near or above lateral-line
scale series (Fig. 1b, c).
Distribution. Onychostoma minnanensis is so far known
from three tributaries of the Julong R. Basin in Fujian
Province, southern China (solid triangle sites in Fig. 3).
These tributaries are the Yuanfeng R. in Hua-an County,
Longchuang R. in Longyen City, and Chuanchang R. in
Nanjing County, located at 440–1,015 m elevation.
Etymology. The specific name, minnanensis, is derived
from ‘‘Min-Nan,’’ which meaning the southern region of
Fujian Province, China.
Habitats. Onychostoma minnanensis was found in small
tributaries in mountain areas. Three of these habitats can be
classified as running brooks with large rocks (sites 1, 3, 4
in Fig. 3), similar to the habitat preference of O. barbatu-
lum. The other collection site (site 2), at which we caught
several adult individuals, was a slow-flowing pool in a
Fig. 1 a Onychostoma minnanensis sp. nov., holotype, NMNSF
01970, 124.0 mm SL, Guongya Mt., Hua-an County, Julong R.,
Fujian Province, China. Photo by N.-H. Jang-Liaw; and spotted
individuals of Onychostoma minnanensis sp. nov., bNMNSF
01965-4, 73.2 mm SL, ca juvenile of about 30 mm TL hiding under
a rock in a brook at Guongya Mt. (site 1 in Fig. 3). Photo by N.-H.
Jang-Liaw, 29 March 2009 (specimen not collected)
Fig. 2 a Front view and blateral view of head of Onychostoma
minnanensis sp. nov., paratype, NTOUP-2009-10-077, 121.6 mm SL.
cFront view and dlateral view of head of O. barbatulum, NMNSF
00724-1, 197 mm SL
A new cyprinid species 67
123

mountain stream; the pool bed consisted of mud and
aquatic plants.
Sequence analyses and molecular phylogenetics.
Twenty-one sequences from sampled individuals of
O. minnanensis, 27 from O. barbatulum, one from Ony-
chostoma lepturum (Boulenger 1900), one from Onychos-
toma alticorpus (Oshima 1920), and one assigned outgroup,
Acrossocheilus paradoxus (Gu
¨nther 1868), were deposited
in the GenBank database (accession nos. HM142478–
HM142579). From 21 samples of O. minnanensis, the
amplification of partial cyt bgene sequences of 1,080 bp
yielded two distinct haplotypes, containing two polymor-
phic/parsimony informative sites. On the other hand, the
CO1 fragments of 1,480 bp represented four haplotypes,
containing four polymorphic sites and two parsimony
informative sites. Comparison of both cyt band CO1 gene
sequences from 27 samples of O. barbatulum yielded 10
and 14 haplotypes, 39 and 43 polymorphic sites, and 31 and
27 parsimony informative sites from the cyt band CO1
fragments, respectively. Generally, the haplotype diversity
(Hd) of O. minnanensis samples was lower than that of
O. barbatulum samples used in this study in both cyt band
CO1 segments. Furthermore, the nucleotide diversity (p)
values of O. minnanensis were very low, only about a tenth
of that revealed by O. barbatulum samples used in this
study (Table 3). Relationships between sampled sequences
and haplotypes are listed in Table 2.
Based on the mtDNA dataset (cyt band CO1 genes), the
estimated pairwise distances using the Kimura 2-parameter
model (K2P; Kimura 1980) between O. minnanensis and
O. barbatulum were 7.52 ±0.76 % and 7.22 ±0.70 %
(mean ±se), which were more distant than those within O.
minnanensis (0.19 ±0.13 % and 0.14 ±0.07 %) and local
(Taiwanese and Chinese) populations of O. barbatulum
(1.28 ±0.21 % and 0.92 ±0.14 %) [Electric Supple-
mentary Material (ESM) Table S1]. The lineage of
O. barbatulum from the Min R., Fujian Province showed
the closest relationships with O. minnanensis (7.52 ±
0.81 % and 6.95 ±0.74 %).
Totally 5 species of Onychostoma including O. min-
nanensis and both populations of O. barbatulum have been
applied to construct the molecular phylogenetic analysis.
Fig. 3 Map of Fujian Province,
China and Taiwan showing
sampling locations for
Onychostoma minnanensis sp.
nov. and O. barbatulum
samples used in this study. See
Table 2for locality information
except site 4. The specimens of
O. minnanensis collected at
site 4 (paratype NTOUP-2012-
07-731) were used for the
morphological study only.
Sites 1 and 2 are in tributaries of
the Julong R. in Guongya Mt,
and site 2 is the holotype
locality
68 N.-H. Jang-Liaw, I-S. Chen
123

The Bayesian trees inferred from partial cyt band CO1
sequences are shown in Fig. 5. Both NJ and ML trees (not
shown) exhibited minor discrepancies with the Bayesian
tree in intraspecific relationships. Thus, the phylogenetic
results for these two genes were not congruent in part of
interspecific relationships. In the cyt bdata, O. alticorpus
was placed at a basal position with respect to all of the
remaining ingroup taxa in the phylogenetic tree, whereas in
the CO1 data, O. lepturum was placed at a basal position.
Furthermore, in the cyt bdata, O. alticorpus,O. lepturum,
and A. paradoxus were separated from the remaining
O. minnanensis and two main populations of O. barbatu-
lum in that order. In the CO1 data, the order was
O. lepturum,O. alticorpus, and A. paradoxus. Among
these comparable taxa, only the grouping [(O. minnanen-
sis ?O. barbatulum)O. lepturum] was well supported by
high bootstrap values (97//95/1.00) from the CO1 data.
However, the tree topologies constructed by the two genes
were very similar among populations of O. minnanensis
and O. barbatulum, and consistently indicated the close
relationship of these two species by composing a mono-
phyletic group [with supporting values for the NJ analysis
of 100/100 (cyt b/CO1), ML of 67/97, and BI of 1.0/1.0]
rather than the two other Onychostoma species. The analyses
revealed only one lineage of O. minnanensis from the three
localities, which was also supported by the presence of two
Fig. 4 Diagram of the cephalic
lateral-line system in
Onychostoma minnanensis sp.
nov. and O. barbatulum.
aO. minnanensis, paratype,
NTOUP-2009-10-077,
121.6 mm SL, bO. barbatulum,
NTOUP-2012-03-278,
105.2 mm SL. Grey
dashed lines indicate the
presumed canal system: IOC
infraorbital canal, POM
preoperculomandibular canal,
SOC supraorbital canal, STC
supratemporal canal. Scales
10 mm
Table 3 Intragroup variation based on cytochrome (cyt)b(left) and cytochrome csubunit 1 (CO1) (right) sequences in species of Onychostoma.
Standard deviations are not shown
Group Sample size No. of haplotypes No. of polymorphic
sites
Haplotype
diversity
Nucleotide diversity
in percent
1. O. minnanensis 21 2/4 2/4 0.524/0.614 0.097/0.084
2. O. barbatulum (all) 27 10/14 39/43 0.761/0.835 0.927/0.637
3. O. barbatulum (Taiwan) 24 7/11 27/34 0.696/0.790 0.690/0.508
4. O. barbatulum (Fujian) 3 3/3 4/4 1.000/1.000 0.247/0.180
A new cyprinid species 69
123

major distinct lineages of O. barbatulum as Taiwanese (as
the type locality) and Chinese (Fujian) groups (Fig. 5).
Monophyly of each species (and local population) was
confirmed based on molecular analyses.
According to the estimated divergence times,
O. minnanensis diverged from O. barbatulum during the
Pliocene, and the mean age was estimated to about 3.09
[2.20–4.06, 95 % highest posterior density (HPD)] million
Fig. 5 Bayesian tree of haplotypes inferred from partial cytochrome
(cyt) b(left) and cytochrome csubunit 1 (CO1) (right) sequences.
Gray boxes indicate lineages of Onychostoma barbatulum from
Fujian (above) and Taiwan (below). Dark-gray box indicates the
O. minnanensis sp. nov. group. Branch lengths are proportional to the
scale, given in nucleotide substitutions per site. Numbers at major
internal nodes are bootstrap probabilities ([50 %) for the values only
in order for NJ/ML (above) and Bayesian posterior probability values
(BI; below) based on 1,000 replications for each tree
Table 4 Mean estimated time of the most recent common ancestor within/between Onychostoma barbatulum and O. minnanensis sp. nov.,
based on cytochrome bsequences by Bayesian analysis using BEAST
O. minnanensis ?O. barbatulum O. minnanensis O. barbatulum O. barbatulum
(Taiwan)
O. barbatulum
(Fujian)
Mean (MYA) 3.091 0.087 0.740 0.529 0.147
95 % HPD lower (MYA) 2.220 0.080 0.491 0.333 0.038
95 % HPD upper (MYA) 4.056 0.185 1.000 0.738 0.264
The upper and lower values of 95% highest posterior density (HPD) are listed
MYA million years ago
70 N.-H. Jang-Liaw, I-S. Chen
123

years ago (MYA). Within the O. barbatulum, the most
recent common ancestor (tMRCA) was estimated as 0.74
(0.49–1.00, 95 % HPD) MYA (Table 4).
Discussion
Morphological comparison. This new species, Onycho-
stoma minnanensis, can be easily distinguished from 11
congeners of mainland China and Taiwan by no serration
of last simple dorsal ray in O. minnanensis versus presence
of posteriorly serrated last simple dorsal ray in Onycho-
stoma simum (Sauvage and Dabry de Thiersant 1874),
Onychostoma fusiforme Kottelat 1998,Onychostoma
gerlachi (Peters 1881), Onychostoma rarum (Lin 1933),
Onychostoma ovale Pellegrin and Chevery 1936, Ony-
chostoma laticeps Gu
¨nther 1896,Onychostoma lini (Wu
1939), Onychostoma angustistomata (Fang 1940), Ony-
chostoma breve (Wu and Chen in Wu et al. 1977), Ony-
chostoma daduense (Ding 1994), and Onychostoma
virgulatum (Xin, Zhang and Cao 2009) (Kottelat 1998;
Shan et al. 2000; Xin et al. 2009). Therefore, O. minnan-
ensis indeed shares the common feature of a smooth last
simple dorsal fin ray with the following seven nominal
species of mainland China and Taiwan: Onychostoma
macrolepis (Bleeker 1871), O. lepturum,O. barbatulum,
O. alticorpus,Onychostoma barbatum (Lin 1931), Ony-
chostoma elongatum (Pellegrin and Chevey 1934), and
Onychostoma meridionale Kottelat 1998. Furthermore, this
new Onychostoma can be well distinguished by lower
counts of lateral-line scales of 41–44 in O. minnanensis
versus 50–53 and 45–47 in O. macrolepis and O. barba-
tum, respectively; by higher depth of the caudal peduncle
with caudal peduncle length/depth of about 2 in O. min-
nanensis versus 2.5–3.1 in O. lepturum; by the different
body shape of adults as body rather elongate, moderate
compressed with body depth 23.3–26.3 % SL in O. min-
nanensis versus oval body shape with higher depth of
29.8–36.8 % SL in O. alticorpus; and by the absence of a
longitudinal black stripe in O. minnanensis versus presence
of the stripe extending along the lateral-line scales in
O. elongatum and O. meridionale (Fang et al. 1996;
Kottelat 1998,2001a; Shan et al. 2000).
In general, O. minnanensis most resembles O. barbatu-
lum morphologically when compared with congeners in
mainland China and Taiwan. Among the collected speci-
mens in this study, however, most young individuals of
O. minnanensis were very easy to differentiate from
O. barbatulum by having random grayish-black spots on
the scales near or above the lateral-line scale series
(Fig. 1b, c). Adult specimens, including the holotype,
which were over about 80 mm in SL showed no such
spotted pattern. Due to the great similarity of adult
coloration patterns, these two species had previously been
overlooked as the same species under O. barbatulum in
Fujian Province, China. After detailed measurements and
comparisons of specimens of both species, O. minnanensis
can be well distinguished from O. barbatulum by the
following combination of features: (1) a lower count of
lateral-line scales usually 41–44 modally 42 versus 45–47
modally 46, (2) a smaller rostral fold with a thick, promi-
nent upper lip versus a larger rostral fold fully covering the
upper lip anteriorly, merely being present the rather thin
edge of the lip in ventral view, and (3) longer maxillary
barbels as 34.8–53.9 % (average 45.4 %) of eye diameter
versus 10.9–13.5 % (average 11.9 %) of eye diameter.
In addition, the major differentiations of cephalic lateral-
line canal systems of these two closely related species of
Onychostoma (Fig. 4) are also observed as follows: (1)
larger general size of canal pores observed in O. minnan-
ensis versus O. barbatulum. POM, SOC, and STC canals
are well developed with more pores in O. minnanensis
compared with those in O. barbatulum, (10 vs. 8, 10 vs. 8,
7 vs. 6, respectively); for IOC, O. barbatulum shows more
pores than O. minnanensis (13 vs. 11); and (2) IOC con-
nected with POM in O. barbatulum versus well separated
in O. minnanensis.
mtDNA analyses. Nevertheless, both the taxonomic
status and phylogenetic relationships of these two species
of Onychostoma were difficult to properly clarify simply
based on morphological examination. Molecular approa-
ches are useful for discussing phylogeography and a
species’ or population’s history. Wang et al. (2004) docu-
mented the population structure of O. barbatulum from
Taiwanese waters using both a mitogenetic marker (D-loop
sequences) and allozyme data. However, they mainly
focused on genetic variations among Taiwanese popula-
tions; a few specimens collected from the Min R. in
mainland China were sequenced and compared with
Taiwanese O. barbatulum sequences, but there was no
further discussion of specimens of Onychostoma popula-
tions from other river systems in mainland China.
Our molecular analyses, both cyt band CO1 gene
results, revealed obvious phylogenetic structures of
haplotypes of O. barbatulum and O. minnanensis. Tree
topologies indicated that they are sibling species, and the
pairwise distances between O. barbatulum populations
from Taiwan and Fujian (i.e., across the Taiwan Strait)
were smaller than those between the two sibling species
(0.0175/0.0120 vs. 0.0752/0.0722 for cyt b/CO1; ESM
Table S1). Generally, the geographical distance between
the Julong R. and Min R. is less than that between Taiwan
and the Min R. The Taiwan Strait, as the geographic bar-
rier between Taiwan and the Min R., is very efficient as an
isolation mechanism for inland aquatic animals such as
frogs (Jang-Liaw et al. 2008; Jang-Liaw and Chou 2011)
A new cyprinid species 71
123

and freshwater fishes (Chen and Fang 1999; Chen et al.
2009). Obviously the separation leading to speciation event
(O. barbatulum vs. O. minnanensis) occurred before
Taiwan was last isolated from mainland China (2.22–4.06
vs. 0.49–1.0 MYA). We suppose that the speciation event
of the new species and O. barbatulum was much earlier
than the population isolation of O. barbatulum between
Fujian and Taiwan.
The sequence results within our O. barbatulum speci-
mens showed different trends for populations from Taiwan
and the Min R., Fujian. In the Taiwanese population,
nucleotide diversity was 0.69 %/0.51 % (cyt b/CO1); for
the Min R. population it was 0.24 %/0.18 % (Table 3). On
the other hand, O. minnanensis showed much lower genetic
diversity according to both nucleotide diversity (0.10 %/
0.08 %; Table 3) and within-group distance values inferred
from the haplotypes (0.0019/0.0014 for cyt b/CO1; ESM
Table S1). The issue of conservation of this new species of
Onychostoma is worthy of further attention, due to its
limited distribution and low mitogenetic diversity.
Species diversity of Onychostoma in southeastern
mainland China. The validity of O. minnanensis as a
species is well supported by both overall morphological
and molecular evidence. This new cyprinid is very close to
its sibling species, O. barbatulum, and was probably mis-
identified previously because of the high similarities in
their adult overall morphology and geographic distribution.
Shan et al. (2000) described O. barbatulum as a wide-
spread species from the Yangtze R. south to the Pearl R.
Basin, but the results of this study disagree with this
statement for all basins in Fujian Province. Actually, our
fish collections in the Pearl R. Basin also suggest similar
result that another cryptic and undescribed congener has
also been recorded recently (Chen and Jang-Liaw, unpub-
lished data). Even between geographically close basins
such as the Min R. and Julong R. in Fujian Province,
southern China, distinct species differentiations of sibling
species of Onychostoma are represented. Based on these
findings, it is highly possible that Onychostoma is a highly
divergent group in different river systems separated by
topographic barriers, at least for the current O. barbatulum
species complex. It therefore could be predicted that the
higher species diversity and complicated sympatric rela-
tionships to those species of Onychostoma that occur in
other large river basins such as the Yangtze and even Pearl
R. Basins in southern mainland China. Detailed surveys of
high-mountain and hill streams must be carried out to
better understand the real species diversity of the genus in
Chinese waters. Detailed and comprehensive surveys such
as taxonomic revision, geographical distributions, and
molecular phylogenetic reconstructions for different sub-
groups of species of Onychostoma in southeastern main-
land China are essential.
Comparative materials
Onychostoma alticorpus (Oshima 1920): NTOUP-2010-05-
214 (3 specimens), 93.5–105 mm SL, Shulukuan R., Hu-
alien, Taiwan, 20 May 2004. Onychostoma angustistomata
(Fang 1940): IOZCAS-72043 (1), 173 mm SL, Ebian,
Sichuan, China, 1978. Onychostoma barbatulum (Pellegrin
1908): NMNSF00010 (1), 132.6 mm SL, Dajia R.,
Taichung, Taiwan, 2 July 1990; NMNSF00724 (4),
116.9–197 mm SL, Dajia R., Taichung, Taiwan, 1 Sep.
2004; NMNSF00755 (1), 90.9 mm SL, Danshuei R., New
Taipei City, Taiwan, 4 Sep. 2004; NMNSF01409 (3),
100–145.1 mm SL, Jhuoshuei R., Nantou, Taiwan, 20 Nov.
2005; NMNSF01520 (2), 98.5 and 153.6 mm SL, Dajia R.,
Taichung, Taiwan, 4 Apr. 2006; NMNSF02020 (2), 129 and
159.5 mm SL, Wu R., Nantou, Taiwan, 23 May 2009;
NTOUP-2010-05-213 (8), 73.1–121.2 mm SL, Keelung R.,
Keelung, Taiwan, 18 Mar. 2010; NTOUP- 2012-03-278 (1),
105.2 mm SL, Keelung R., Keelung, Taiwan, 17 Oct. 2011.
Onychostoma elongatum (Pellegrin and Chevey 1934):
IOZCAS-65934 (1), 152.8 mm SL, Dawantan Reservoir,
Guangxi, China, 27 Sep. 1975. Onychostoma gerlachi
(Peters 1881): IOZCAS-39785 (1), 179 mm SL, Minjiang
River, Sichuan, China, 1957; IOZCAS-69584 (1),
157.7 mm SL, Fangcheng, Guangxi, China, 3 May 1999;
IOZCAS-78036 (1), 118 mm SL, Pingle, Guangxi, China,
28, Oct. 1975. IOZCAS-78037–38 (2), 127.7 and 131.8 mm
SL, Guangxi, China, unknown; NMNSF00070 (2), 143 and
164 mm SL, Tian’e, Guangxi, China, 1 Oct. 2002. Ony-
chostoma lepturum (Boulenger 1900): NTOUP-2010-05-
212 (2), 31.3 and 39.8 mm SL, Nandu R. Basin, Hainan,
China, 29 Apr. 2010; NTOUP-2012-02-378 (1), 103.2 mm
SL, Wuzhishan, Hainan, China, 26 Oct. 2010. Onychostoma
lini (Wu 1939): IOZCAS-185095–96 (2), 135.5 and
138.8 mm SL, Sanjiang, Guangxi, China, 7 Mar. 2003.
Onychostoma macrolepis (Bleeker 1871): IOZCAS-
156807–09, 15–17 (6), 124.1–173 mm SL, Zhouzhi, Sha-
anxi, China, 29 May 2004; IOZCAS-168184 (1), 254 mm
SL, Qinyang, Henan, China, 4 Sep. 2007; IOZCAS-
194201–05 (5), 97.9–118.3 mm SL, Yun, Hubei, 1 Apr.
2012. Onychostoma ovale Pellegrin and Chevey (1936):
IOZCAS-61596 (1), 260.7 mm SL, unknown. Onychos-
toma rarum (Lin 1933): IOZCAS-63709 (1), 229 mm SL,
Sanjiang, Guangxi, China, 13 Oct. 1975; IOZCAS-
77980–81 (2), 115.7 and 131.9 mm SL, Guangxi, China,
unknown. Onychostoma simum (Sauvage and Dabry de
Thiersant 1874): IOZCAS-56831–34 (4), 181–216 mm SL,
Xian’xi, Hubei, China, unknown; IOZCAS-63419 (1),
204 mm SL, Guangxi, China, Oct. 1975.
Acknowledgments We are very grateful to Wen-Hao Chou
(NMNS), Jia-Lin Zhu (Xiamen Daily, Xiamen), Ruei-Yun Lin (Hwa-
An Science Association, Hwa-An), Shun-Chi Cheng and Shih-Pin
Huang (NTOU), and Chien-Chin Liu (National Taiwan Normal
72 N.-H. Jang-Liaw, I-S. Chen
123

University, Taipei) for their assistance on the many field trips to
mainland China and Taiwan. Special thanks are due to Chun-Guang
Zhang, En-Qi Ye, Ya-Hui Zhao (IOZCAS), and Ho-Tien Hon
(NMNS) for making partial material available for comparative stud-
ies. The research was partly supported by a grant from the National
Museum of Natural Science, Taichung, Taiwan. The research of the
second author was financed by a grant from the National Science
Council, Taiwan, 2009–2010.
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