Phylogeny and Polyploidy: Resolving the Classification of Cyprinine Fishes (Teleostei: Cypriniformes).

Abstract

Cyprininae is the largest subfamily (>1300 species) of the family Cyprinidae and contains more polyploid species (∼400) than any other group of fishes. We examined the phylogenetic relationships of the Cyprininae based on extensive taxon, geographical, and genomic sampling of the taxa, using both mitochondrial and nuclear genes to address the phylogenetic challenges posed by polyploidy. Four datasets were analyzed in this study: two mitochondrial gene datasets (465 and 791 taxa, 5604bp), a mitogenome dataset (85 taxa, 14,771bp), and a cloned nuclear RAG1 dataset (97 taxa, 1497 bp). Based on resulting trees, the subfamily Cyprininae was subdivided into 11 tribes: Probarbini (new; Probarbus + Catlocarpio), Labeonini Bleeker, 1859 (Labeo & allies), Torini Karaman, 1971 (Tor, Labeobarbus & allies), Smiliogastrini Bleeker, 1863 (Puntius, Enteromius & allies), Poropuntiini (Poropuntius & allies), Cyprinini Rafinesque, 1815 (Cyprinus & allies), Acrossocheilini (new; Acrossocheilus & allies), Spinibarbini (new; Spinibarbus), Schizothoracini McClelland, 1842 (Schizothorax & allies), Schizopygopsini Mirza, 1991 (Schizopygopsis & allies), and Barbini Bleeker, 1859 (Barbus & allies). Phylogenetic relationships within each tribe were discussed. Two or three distinct RAG1 lineages were identified for each of the following tribes Torini, Cyprinini, Spinibarbini, and Barbini, indicating their hybrid origin. The hexaploid African Labeobarbus & allies and Western Asian Capoeta are likely derived from two independent hybridization events between their respective maternal tetraploid ancestors and Cyprinion.
Copyright © 2015 Elsevier Inc. All rights reserved.

Full text

This article appeared in a journal published by Elsevier. The attached copy is
furnished to the author for internal non-commercial research and educational
use, including for instruction at the author’s institution and sharing with
colleagues.
Other uses, including reproduction and distribution, or selling or licensing
copies, or posting to personal, institutional or third party websites are
prohibited.
In most cases authors are permitted to post their version of the article (e.g. in
Word or Tex form) to their personal website or institutional repository. Authors
requiring further information regarding Elsevier’s archiving and manuscript
policies are encouraged to visit:
http://www.elsevier.com/copyright

Phylogeny and polyploidy: Resolving the classification of cyprinine
fishes (Teleostei: Cypriniformes)
Lei Yang
a,
⇑
,1
, Tetsuya Sado
b
, M. Vincent Hirt
c
, Emmanuel Pasco-Viel
d
, M. Arunachalam
e
, Junbing Li
f
,
Xuzhen Wang
f
, Jörg Freyhof
g
, Kenji Saitoh
h
, Andrew M. Simons
i
, Masaki Miya
b
, Shunping He
f
,
Richard L. Mayden
a,
⇑
a
Department of Biology, Saint Louis University, St. Louis, MO 63103, USA
b
Department of Zoology, Natural History Museum and Institute, Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan
c
Graduate Program in Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108, USA
d
Evo-devo of Vertebrate Dentition, and Molecular Zoology, Institut de Ge
´nomique Fonctionnelle de Lyon (IGFL), UMR CNRS 5242, Universite
´de Lyon, Université Claude Bernard
Lyon 1, Ecole Normale Supe
´rieure de Lyon, Lyon 69007, France
e
Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India
f
Laboratory of Fish Phylogenetics and Biogeography, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
g
German Centre for Integrative Biodiversity Research (iDiv), Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
h
National Research Institute of Fisheries Science, Aquatic Genomics Research Center, Fukuura, Kanazawa, Yokohama 236-8648, Japan
i
Department of Fisheries, Wildlife, and Conservation Biology and Bell Museum of Natural History, University of Minnesota, St. Paul, MN 55108, USA
article info
Article history:
Received 12 April 2014
Revised 29 January 2015
Accepted 30 January 2015
Available online 16 February 2015
Keywords:
Biogeography
Cyprinidae
Evolution
Hexaploids
Taxonomy
Tetraploids
abstract
Cyprininae is the largest subfamily (>1300 species) of the family Cyprinidae and contains more polyploid
species (400) than any other group of fishes. We examined the phylogenetic relationships of the Cypri-
ninae based on extensive taxon, geographical, and genomic sampling of the taxa, using both mitochon-
drial and nuclear genes to address the phylogenetic challenges posed by polyploidy. Four datasets
were analyzed in this study: two mitochondrial gene datasets (465 and 791 taxa, 5604 bp), a mitogenome
dataset (85 taxa, 14,771 bp), and a cloned nuclear RAG1 dataset (97 taxa, 1497 bp). Based on resulting
trees, the subfamily Cyprininae was subdivided into 11 tribes: Probarbini (new; Probarbus +Catlocarpio),
Labeonini Bleeker, 1859 (Labeo & allies), Torini Karaman, 1971 (Tor,Labeobarbus & allies), Smiliogastrini
Bleeker, 1863 (Puntius,Enteromius & allies), Poropuntiini (Poropuntius & allies), Cyprinini Rafinesque,
1815 (Cyprinus & allies), Acrossocheilini (new; Acrossocheilus & allies), Spinibarbini (new; Spinibarbus),
Schizothoracini McClelland, 1842 (Schizothorax & allies), Schizopygopsini Mirza, 1991 (Schizopygopsis &
allies), and Barbini Bleeker, 1859 (Barbus & allies). Phylogenetic relationships within each tribe were dis-
cussed. Two or three distinct RAG1 lineages were identified for each of the following tribes Torini, Cypri-
nini, Spinibarbini, and Barbini, indicating their hybrid origin. The hexaploid African Labeobarbus & allies
and Western Asian Capoeta are likely derived from two independent hybridization events between their
respective maternal tetraploid ancestors and Cyprinion.
Ó2015 Elsevier Inc. All rights reserved.
1. Introduction
Within vertebrates, phylogenetic resolution of the largest
group, Actinopterygii or ray-finned fishes, lags behind other taxa,
due to the large number of species and the difficulties obtaining
and identifying specimens. Another challenge in actinopterygian
phylogeny is the evolution of genome duplications producing poly-
ploids. Genome duplications have occurred multiple times in
actinopterygian evolution but are particularly prevalent in Cyprini-
formes, the largest clade of freshwater fishes (Amores et al., 1998;
Leggatt and Iwama, 2003; Taylor et al., 2003). Within Cyprini-
formes, the Cyprininae contains more than 1300 freshwater spe-
cies in over 120 genera, accounting for nearly 4% of bony fish
diversity (Eschmeyer, 2015). Most species of this subfamily inhabit
waters of southern Eurasia and Africa. Some are well known, such
as the common carp (Cyprinus carpio) and Goldfish (Carassius aura-
tus). The Cyprininae contains around 400 closely related polyploid
http://dx.doi.org/10.1016/j.ympev.2015.01.014
1055-7903/Ó2015 Elsevier Inc. All rights reserved.
⇑
Corresponding authors at: Department of Biology, 3507 Laclede Ave, Saint Louis
University, St. Louis, MO 63103, USA. Fax: +1 314 977 3658.
E-mail addresses: leiyangslu@gmail.com (L. Yang), cypriniformes@gmail.com
(R.L. Mayden).
1
Present address: Hollings Marine Laboratory, College of Charleston, Charleston, SC
29401, USA.
Molecular Phylogenetics and Evolution 85 (2015) 97–116
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Author's Personal Copy

species (Arai, 2011; Froese and Pauly, 2015), more than other poly-
ploid fish lineages, such as Acipenseriformes, Salmoniformes, and
Catostomidae (all <250 species; Eschmeyer, 2015). Over 30 genera
in this subfamily consist only of polyploid species (Leggatt and
Iwama, 2003; Arai, 2011). Most polyploids in this subfamily are
either tetraploids (2n= ca. 100) or hexaploids (2n= ca. 150). The
species Ptychobarbus dipogon has an amazingly large number of
chromosomes (2n= 446, Cui et al., 1991;2n= 424–432, Wu et al.,
1999).
The classification of the subfamily Cyprininae is still under dis-
cussion as various numbers of formal or informal groups have been
recognized within it (Tables 1 and S1). Many molecular studies
have been conducted on the phylogenetic relationships of the
Cyprininae; however, most were limited by restricted taxon or
character sampling or limited geographical sampling. Most impor-
tantly, these studies either did not use nuclear genes or used, but
ignored the issue of paralogy associated with polyploid taxa. If a
nuclear gene has only one copy in diploids, it is expected to have
two copies in tetraploids, three copies in hexaploids, and four
copies in octoploids. In polyploids, especially allopolyploids, the
different nuclear gene copies could be quite divergent and belong
to distinct clades in a gene tree (e.g. Evans et al., 2005; Saitoh
et al., 2010). Direct amplification of these nuclear genes with PCR
(polymerase chain reaction) without using specially designed par-
alog-specific primers will likely result in a mixture of gene copies.
If nuclear copies are not sorted appropriately, homology cannot be
confidently established, potentially misleading phylogenetic stud-
ies. In the present study, extensive DNA cloning was performed
and multiple alleles were used in phylogeny reconstruction of all
major diploid and polyploid lineages of the subfamily Cyprininae.
These data and molecular phylogenies inferred from mitochondrial
genes made it possible for us to explore the phylogenetic relation-
ships and subdivisions of this subfamily as well as the evolution of
polyploidy.
The major objectives of this study are: (1) to investigate the
phylogenetic relationships and subdivisions of the subfamily
Cyprininae based on the largest taxon, geographical, and genomic
sampling to date; and (2) to propose a classification for this group.
The evolution of polyploid lineages; the distribution of some mor-
phological characters important in classification; and biogeogra-
phy of some taxa will be discussed.
2. Materials and methods
2.1. Taxon sampling and datasets
Four datasets were analyzed in this study: (1) a mitochondrial
gene dataset with 465 taxa (465-taxon mt dataset); (2) an expand-
ed mitochondrial gene dataset with 791 taxa (791-taxon mt data-
set); (3) a mitochondrial genome dataset with 85 taxa
(mitogenome dataset); and (4) a cloned RAG1 dataset with 97 taxa
(RAG1 dataset).
In the 465-taxon mt dataset, most taxa (97.4%) were represent-
ed by sequences from five mitochondrial genes: cytochrome oxi-
dase subunit I (COI), Cytochrome b(Cyt b), 16S ribosomal RNA
(16S rRNA), NADH dehydrogenase subunit 4 (ND4), and NADH
dehydrogenase subunit 5 (ND5); remaining taxa (2.6%) were repre-
sented by sequences from at least three genes. This dataset has
good representation for major lineages of cyprinines with only
9.2% missing data and thus should provide a good estimate of
the mitochondrial phylogeny (Wiens, 2006; Roure et al., 2013).
The 791-taxon mt dataset was constructed by adding to the
465-taxon mt dataset, 326 taxa represented by sequences for Cyt
bor Cyt bplus another gene. All taxa in the 791-taxon mt dataset
were represented by Cyt bsequences, providing comparable data
for all included taxa. This dataset was assembled to expand taxon
sampling to include most of the generic diversity, nearly half of the
species diversity, and important biogeographic diversity for the
subfamily Cyprininae. Analysis of this dataset was constrained
using results from analysis of the 456-taxon mt dataset.
The mitogenome dataset was assembled and used to create a
strongly supported estimate of mitochondrial phylogeny that
could be used to place five enigmatic species whose phylogenetic
position could not be established using the other mitochondrial
datasets. Of the five enigmatic species, Semiplotus semiplotum,
Luciocyprinus striolatus,Eirmotus octozona, and Oreichthys cosuatis
were represented by five mitochondrial genes, while Aaptosyax
grypus was represented only by Cyt b.
Table 1
Brief summary of some previous hypotheses regarding the classifications of the
subfamily Cyprininae.
Author Classification
Rainboth (1981) Subfamily Cyprininae
1. Cyprinini
2. Barbini
Chen et al. (1984) Series Barbini
Tribe Barbines
1. Barbinae (includes
Schizothoracinae)
2. Cyprininae
3. Labeoninae
Tribe Tincanes
4. Tincinae
Howes (1987, 1991) Subfamily Cyprininae
1. Squaliobarbin
2. Barbin
3. Labein
4. Schizothoracin
5. Cyprinion-Onychostoma (Howes,
1991)
6. Other taxa
Rainboth (1991, 1996a) and
Rainboth et al. (2012)
Subfamily Cyprininae
1. Cyprinini
2. Labeonini
3. Systomini
4. Oreinini (Rainboth, 1991;
Rainboth et al., 2012)
5. Catlini (Rainboth, 1996a;
Rainboth et al., 2012)
6. Semiplotini (Rainboth, 1991)
7. Squaliobarbini (Rainboth, 1991)
Cavender and Coburn (1992) Subfamily Cyprininae
1. Barbins
2. Cyprinins
3. Labeonins
Nelson (1994) Subfamily Cyprininae
1. Barbus,Carassius, etc.
2. Bangana,Cirrhinus, etc.
3. Ctenopharyngodon etc.
4. Balantiocheilos,Cyclocheilichthys,
etc.
5. Cyprinion,Semiplotus
6. Puntius
7. Acrossocheilus,Hampala,
Poropuntius
8. Pseudobarbus,Varicorhinus
Yue et al. (2000) Series Barbini
1. Barbinae
2. Labeoninae
3. Schizothoracinae
4. Cyprininae
Wang et al. (2007) Subfamily Cyprininae
1. Cyprinini
2. Labeonini
Note: In Nelson (2006), members of the Cyprininae scatter within the Cyprinidae.
98 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

The RAG1 dataset was assembled to create a phylogeny based
on a nuclear locus and used to assess ploidy and the pattern and
impact of gene duplication on phylogeny. Nuclear RAG1 gene
(recombination activating gene 1, exon 3) is widely used in phylo-
genetic studies. This gene is single copy in diploids (Evans et al.,
2005; Saitoh et al., 2010) and our results corroborated this obser-
vation (see below).
See Table 2for the composition of each dataset and Table S2 for
detailed sample information. Nomenclature for genera and species
generally follows Eschmeyer (2015). To avoid confusion, the classi-
fication system proposed by us is adopted throughout the paper
(Table 3).
2.2. DNA extraction, PCR, cloning, and sequencing
Genomic DNA was isolated from ethanol-preserved muscle or
fin clips using DNeasy tissue extraction kits (Qiagen, USA). The five
mitochondrial genes were amplified and sequenced using primers
and protocols from Yang et al. (2010). Complete mitochondrial
genomic sequences used in this study were downloaded from Gen-
Bank except for three sequences provided by M.M. (Table S2).
The RAG1 sequences of 34 diploid ingroup and 26 outgroup taxa
were either downloaded from GenBank or obtained following Yang
and Mayden (2010). We cloned RAG1 for all 30 polyploids sampled,
including tetraploids and hexaploids, as well as six possible
diploids, to represent all major ingroup clades recovered in ana-
lyses of mitochondrial data (Arai, 2011;Table S2). Cloning was also
performed for Catostomus commersoni, a tetraploid outgroup taxon.
Conditions for PCR followed Yang and Mayden (2010) using pri-
mers R1_2533F, R1_4090R, and R1_4078R and 30 amplification
cycles rather than 35. The PCR products were ligated into pGEM-
T vector (Promega, USA) and cloned into JM109 competent Escher-
ichia coli (Promega, USA). Standard blue–white colony screening
was performed following cloning and incubation. We picked 12
or more positive clones for diploids and tetraploids, and 24 positive
clones for hexaploids. Those clones were then used as templates
for second-round PCR amplifications, also using 30 cycles.
The PCR products of mitochondrial and nuclear genes were
purified using one of three methods: AMPure (Agencourt Bio-
science), QIAquick Gel Extraction Kits (Qiagen), or outsourced com-
mercial purification with ExoSAP-IT (USB/Affymetrix). Sequencing
Table 2
Statistics of the four datasets used in the present study. Numbers separated by two
slashes are (left to right) number of genera, species, and individuals, respectively.
Taxon/Character 465-
taxon
mt
791-
taxon
mt
Mitogenome RAG1
a
Cyprininae 102/
348/395
117/
612/721
27/56/58 53/68/
68
Probarbini 2/2/2 2/3/3 1/1/1 2/2/2
Labeonini 32/135/
139
34/141/
158
5/9/9 8/10/
10
Torini 7/30/38 13/83/
103
1/2/2 7/7/7
Smiliogastrini 19/72/
77
20/132/
153
5/5/5 8/12/
12
Poropuntiini 17/34/
37
17/37/
42
2/3/3 9/11/
11
Cyprinini 6/17/17 7/48/50 4/9/9 6/9/9
Acrossocheilini 3/18/23 3/19/26 2/9/9 2/3/3
Spinibarbini 1/3/6 1/4/7 1/2/3 1/2/2
Schizothoracini 3/15/26 4/42/54 2/6/7 2/3/3
Schizopygopsini 6/11/15 9/26/35 2/7/7 3/3/3
Barbini 6/11/15 7/77/90 3/3/3 7/7/8
Outgroup 63/70/
70
63/70/
70
26/27/27 26/27/
27
Total 165/
418/465
180/
682/791
53/83/85 80/97/
97
Nucleotides (bp) 5604 5604 14,771 1497
Missing data (%) 9.2 37.3 0.0 0.6
Variable characters (bp) 3775 3869 8132 728
Parsimony-informative
characters (bp)
3392 3429 6787 599
A% 30.5 30.3 28.8 26.0
C% 27.1 27.2 26.4 23.5
G% 15.3 15.3 17.9 26.1
T% 27.1 27.2 26.9 24.4
a
A total of 31 polyploids (includes tetraploids and hexaploids) and six diploids
were cloned for RAG1 (see Table S2). Cloned species usually have multiple RAG1
sequences and the RAG1 dataset contains a total of 162 sequences (see Fig. 4).
Table 3
Revised classification of the subfamily Cyprininae. Type genus of each tribe is underlined.
Classification Taxon Author/year Genus/group
Family Cyprinidae Rafinesque, 1815
Subfamily Cyprininae Rafinesque, 1815
Tribe Probarbini –This study– Catlocarpio,Probarbus
Tribe Labeonini Bleeker, 1859 40 genera (see Yang et al., 2012b)
Tribe Torini Karaman, 1971 Hypselobarbus Clade (Hypselobarbus,Osteochilichthys,Lepidopygopsis), Naziritor,Neolissochilus,Tor,Labeobarbus
Clade (Labeobarbus,Varicorhinus,Carasobarbus,Mesopotamichthys,Arabibarbus,Pterocapoeta,‘Labeobarbus’)
Tribe Smiliogastrini Bleeker, 1863 Chagunius,Oreichthys,Osteobrama,Sahyadria,Dawkinsia,Striuntius,Oliotius,Barbodes,Hampala,Puntigrus,
Desmopuntius,Rohtee,Puntius,Haludaria,Pethia,Systomus,Enteromius,Barboides,‘Pseudobarbus’, Pseudobarbus,
Clypeobarbus
Tribe Poropuntiini
a
Eirmotus,‘Puntius’snyderi,‘P.’ semifasciolatus,Discherodontus,Cyclocheilos,Balantiocheilos,Cosmochilus,
Amblyrhynchichthys,Puntioplites,Albulichthys,Cyclocheilichthys,Barbonymus,Scaphognathops,Sawbwa,
Poropuntius,‘Barbonymus’gonionotus,Hypsibarbus,Sikukia,Mystacoleucus
Tribe Cyprinini Rafinesque, 1815 Procypris,Luciocyprinus,Cyprinus,Carassioides,Carassius,Sinocyclocheilus,Aaptosyax
Tribe Acrossocheilini –This study– Onychostoma,Acrossocheilus,Folifer
Tribe Spinibarbini –This study– Spinibarbus
Tribe Schizothoracini McClelland, 1842 Percocypris,Schizothorax,Schizopyge,Aspiorhynchus
Tribe Schizopygopsini Mirza, 1991 Diptychus,Gymnodiptychus,Ptychobarbus,Oxygymnocypris,Platypharodon,Gymnocypris,Schizopygopsis,
Herzensteinia,Chuanchia
Tribe Barbini Bleeker, 1859 Scaphiodonichthys,Cyprinion,Semiplotus,Aulopyge,Barbus,Luciobarbus,Capoeta
Incertae sedis Schizocypris,Barbopsis,Caecobarbus,Coptostomabarbus,Kalimantania,Laocypris,Neobarynotus,Parasikukia,
Paraspinibarbus,Parator,Rohteichthys,Typhlobarbus
Note: Sanagia velifera (CO1: HM418112) belongs to the Labeobarbus lineage of the tribe Torini. The following genera probably belong to the subfamily Cyprininae: Troglo-
cyclocheilus Kottelat & Bréhier 1999, Xenobarbus Norman, 1923.
a
We think that the name Poropuntii Rainboth, 1991 is not available (see Section 4). Here we use Poropuntiini only for convenience.
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 99
Author's Personal Copy

was conducted at two facilities: htSEQ High-Throughput Genomics
Unit (University of Washington, USA) and Macrogen (South Korea).
Primers used for PCR amplifications were also used for sequencing.
All novel mitochondrial sequences and RAG1 sequences were
deposited in GenBank, see Table S2 for accession numbers for all
sequences included in this study. Vouchered specimens were
deposited at Saint Louis University or other museums associated
with the Cypriniformes Tree of Life (CToL) Project.
2.3. Sequence alignment and phylogenetic analyses
MT DATASETS: Multiple alignment of the mitochondrial gene
datasets followed Yang et al. (2012b,c) and the final alignment
was 5604 bp in length. Partitioned Maximum Likelihood (ML) ana-
lysis of the 465-taxon mt dataset was conducted using RAxML
v.8.0.26 (Stamatakis, 2006, 2014). The optimal partitioning scheme
and models of nucleotide substitution for this dataset as well as
other datasets in this study were analyzed using PartitionFinder
v1.1.1 (Lanfear et al., 2012, 2014). See Table S3 for parameter set-
tings and results. A total of 200 distinct runs were performed based
on 200 random starting trees using the default algorithm of the
program. The tree with the best likelihood score was chosen as
the final tree. In analysis of the 791-taxon mt dataset, the optimal
ML tree was constructed through constraint searching (200 runs)
using as the constraint the best ML tree from analysis of the 465-
taxon mt dataset. Maximum Likelihood searches were not con-
ducted directly on the 791-taxon mt dataset due to the large
amount of missing data. Our strategy was designed to minimize
the impact of missing data on tree topology. During constraint
searches, the relationships of nearly 60% of taxa in the 791-taxon
dataset were stabilized and other taxa were placed with respect
to the constraint tree. The inclusion of Cyt bfor all taxa was expect-
ed to facilitate this process. Maximum likelihood bootstrap ana-
lyses were conducted for both mitochondrial datasets using
RAxML (Felsenstein, 1985; Stamatakis et al., 2008) using the same
partitioning strategy and nucleotide substitution model as in the
initial maximum likelihood search. The number of nonparametric
bootstrap replications was set as 1000 and other parameters were
set as default. The resulting trees were imported into PAUP⁄4.0.b10
(Swofford, 2002) to obtain the 50% majority rule consensus tree
and bootstrap values (BP).
MITOGENOME DATASET: Mitochondrial genomic sequences
were aligned following Saitoh et al. (2006, 2011). The complemen-
tary strand sequences were used for L-strand-encoded genes (ND6
and eight tRNA genes). The final alignment had 14,771 nucleotide
sites in total, including 11,337 sites of 13 protein-coding genes,
2089 sites of two rRNA genes and 1345 sites of 22 tRNA genes.
All gaps and ambiguous sites were excluded during alignment
and the final dataset contained no missing data. Gaps were exclud-
ed because they are frequently adjacent to or within ambiguously
aligned regions. Some parsimony-informative sites may have been
pruned in this process, but these represent a small percentage of
the entire alignment. The partitioning scheme and model of
nucleotide substitution used is included in Table S3. Partitioned
Maximum Likelihood analysis and bootstrap analysis were con-
ducted using RAxML with the same settings as other mitochondrial
analyses. Five independent ML analyses and bootstrap analyses
were then performed to determine the placement of each of the
five enigmatic species described above. For each of these five
species, the mitogenome tree built previously was used as a con-
straint and 100 independent ML and bootstrap analyses were per-
formed using RAxML. Other settings for the ML analyses and
bootstrap analyses were the same as previous analyses.
For the 465-taxon mt dataset, 791-taxon mt dataset, and mito-
genome dataset, corresponding RY-coding datasets were built by
coding ‘‘A’’ and ‘‘G’’ as ‘‘R’’, and ‘‘C’’ and ‘‘T’’ as ‘‘Y’’ for the third
codon positions of all protein-coding mitochondrial genes, thus
taking only transversions into account. According to Phillips et al.
(2001, 2004), RY-coding can remove noise from third codon posi-
tions effectively while retaining all available positions in the data-
set. Phylogenetic analyses were conducted on each of these
datasets with the same settings as used for their corresponding
normally coded datasets. See Table S3 for partitioning schemes
and nucleotide substitution models used.
RAG1 DATASET: For each species, RAG1 sequences of all
sequenced clones were aligned. Consensus sequences of each puta-
tive allele of each species were phased following Saitoh and Chen
(2008). Chimeric sequences, sequences with private sites (unique
variant nucleotide appeared in a single clone), and sequences with
private combinations (unique nucleotide combinations of variable
sites appeared in a single clone), were discarded during the process
to reduce cloning artifacts for recovery of real allelic sequences of
RAG1. Alleles of cloned species and sequences from other species
were then aligned, producing the RAG1 dataset (1497 bp in
length). The dataset was partitioned by codon positions and
GTR + I + G model was used according to results of PartitionFinder
(Table S3). A total of 1000 distinct runs were performed based on
1000 random starting trees using the default algorithm of RAxML.
The tree with the best likelihood score was chosen as the final tree.
3. Results
MT DATASETS: The subfamily Cyprininae was resolved as
monophyletic in both ML trees derived from the 465-taxon mt
dataset (BP = 100%; full tree embedded in Fig. 2) and the 791-taxon
mt dataset (BP = 99%) (Figs. 1 and 2). Eleven major clades were
resolved: Probarbini, Acrossocheilini, Spinibarbini, Poropuntiini,
Labeonini, Torini, Smiliogastrini, Cyprinini, Schizothoracini,
Schizopygopsini, and Barbini. The first three clades represent tribes
described herein (see Section 4). Support for these clades was
strong with the exception of Smiliogastrini (BP 664%), Poropunti-
ini (BP 672%), and Cyprinini (BP < 50%). The position of some gen-
era such as Sinocyclocheilus,Procypris,Chagunius, and Aaptosyax
were weakly supported (BP < 50%). To facilitate discussion
of relationships and paralogy of RAG1, we identified two
unnamed clades: Clade B (BP P98%) ((Barbini, Schizopygopsini),
((Schizothoracini, Spinibarbini), Acrossocheilini)) and Clade A
(BP P94%) (Smiliogastrini, (Poropuntiini, (Cyprinini, Clade B))).
Clade A and three other clades constituted the subfamily Cyprini-
nae with the following relationships: (((Clade A, Torini), Labeo-
nini), Probarbini). Relationships within each tribe were illustrated
in Fig. 2b–h. For Labeonini, we refer reader to Yang et al. (2012b)
for a full description of relationships.
MITOGENOME DATASET: The subfamily Cyprininae was robust-
ly supported (BP = 100%; Fig. 3). The eleven major clades recovered
by the analyses of the two mt datasets were also recovered here
and nearly all clades were strongly supported, including Clades A
and B (BP = 100%). Relationships of the major clades are largely
consistent with the analyses of the mt dataset, except in Clade B
where the following relationships were supported: (((Acrossochei-
lini, Spinibarbini), Schizothoracini), (Schizopygopsini, Barbini)),
and the position of the genera Chagunius and Oreichthys which fell
within the Smiliogastrini (BP = 80%). The phylogenetic position of
the other four enigmatic genera were identified as follows:
Aaptosyax and Luciocyprinus in Cyprinini (BP = 92%); Eirmotus in
Poropuntiini (BP = 98%), and Semiplotus confirmed as part of the
Barbini clade (BP = 75%). Phylogenetic trees resulting from
RY-coded datasets were consistent with other analyses (see
Figs. S1 and S2).
RAG1 DATASET: The number of copies recovered by cloning was
less than or equal to the number predicted by ploidy level of each
100 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

species (Fig. 4 and Table S2). The two unnamed clades, A and B,
were recognized, corresponding to Clade A and Clade B from the
mt analyses (Figs. 1–3). Most major clades recovered in analyses
of mitochondrial data were also recovered by the RAG1 phylogeny
with the polyploid taxa Cyprinini, Spinibarbini, Barbini, and Torini
represented by two or three clades in the tree (Fig. 4).
4. Discussion
4.1. Polyploid taxa
Polyploid taxa pose significant challenges for phylogenetic sys-
tematists. This is particularly true in taxa such as the Cyprininae,
which contains large numbers of diploid, tetraploid, and hexaploid
species. Our analyses, comparing trees based on mitochondrial
data with trees based on cloned nuclear data, allows the construc-
tion and comparison of complex gene trees including paralogous
allele sequences. Our data provide the best estimate of phylogeny
of the Cyprininae and allow development of a classification system
that reflects the phylogenetic history of these organisms.
The RAG1 analysis demonstrates that polyploid lineages are
embedded in branches of paralogs (Fig. 4). Some polyploid tribes
have two distant (inter-tribal) RAG1 lineages. For example, Cypri-
nini has two RAG1 paralogs, one close to the mostly diploid Porop-
untiini + Smiliogastrini lineage and the other close to the tetraploid
Probarbini. Polyploids are in many cases of hybrid origin (Wu et al.,
2001; David et al., 2003; Saitoh et al., 2010). As a probable expla-
nation for our results, the cyprinin common ancestor established
itself via hybridization between the diploid common ancestor of
Poropuntiini and/or Smiliogastrini and a diploid common ances-
tor of Probarbini (paternal source; Figs. 3 and 4). Likewise, some
other tribes (Spinibarbini, Torini, and Barbini) have distant gene
lineages indicating their hybrid origin. Alleles of Probarbini,
Schizothoracini, and Schizopygopsini fall in a single tribal lineage
respectively. Both probarbin species show 2 + 2 allelic configura-
tion, and we postulated hybridization of closely related ancient
diploids within the tribe. On the other hand, alleles of Schizotho-
racini and Schizopygopsini do not show clear 2 + 2 relationships,
and therefore alternative mechanisms such as autopolyploidy,
gene conversion between paralogs, or counter-diploidization in
which tetrasomy evolves from amphidiploidy through incidental
onset of paralogous (homeologous) synapsis and crossing-over
(Saitoh, 2003) may better explain polyploidy in these taxa.
The hexaploid members of Torini (Labeobarbus,Varicorhinus,
and Carasobarbus) and Barbini (Capoeta) formed a clade with the
Fig. 1. Cladogram showing topologies of Maximum Likelihood trees resulting from 465-taxon mt dataset (full tree embedded in Fig. 2) and 791-taxon mt dataset (full tree
shown as Fig. 2). Outgroups are not shown. Numbers above and below branches are the bootstrap support values for each node in trees resulting from the 465-taxon dataset
and the 791-taxon dataset, respectively. Values lower than 50% are indicated by ‘‘’’. Values that are not applicable, because some taxa in the 791-taxon dataset are either not
included or have only one individual in the 465-taxon dataset, are indicated by ‘‘n.a.’’. Two strongly supported large clades are labeled ‘‘A’’ and ‘‘B’’ to facilitate discussion of
the phylogeny. See Section 4for nomenclature of each major clade (tribe).
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 101
Author's Personal Copy

Fig. 2. The optimal Maximum Likelihood tree (ln L= 546800.981288) resulting from the 791-taxon mt dataset. Bootstrap support values are shown beside each node. Values
lower than 50% are indicated by ‘‘’’. Chromosomes numbers (2n), if known, were shown in parentheses following each species (Sahoo et al., 2009; Arai, 2011; Ganai et al.,
2011; Donsakul et al., 2012). Some species of Sinocyclocheilus were denoted as ‘‘tetraploids’’ following Xiao et al. (2002). The type species of each genus was also indicated. (a)
Outgroups; (b) Probarbini, Labeonini (see Yang et al. 2012b), and Torini; (c) Smiliogastrini (Part I); (d) Smiliogastrini (Part II); (e) Poropuntiini and Cyprinini; (f)
Acrossocheilini, Spinibarbini, and Schizothoracini; (g) Schizopygopsini; (h) Barbini. The name Xenocypridinae rather than Oxygastrinae is used, following Kottelat (2013: 69).
102 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

Fig. 2 (continued)
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 103
Author's Personal Copy

Fig. 2 (continued)
104 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

diploid Cyprinion (Fig. 4). The genus Capoeta did not group with any
other torin lineage (i.e. Torini 1&3; Fig. 4). A probable explanation
for these results is that these hexaploid lineages were derived from
two independent hybridization events between their respective
tetraploid ancestors and Cyprinion. For the tribe Torini, this
hexaplodization event could be ascribed to hybridization between
tetraploid torins (maternal source; Fig. 2b) and Cyprinion (paternal
source; Fig. 4). Because all hexaploid members of Torini are found
in Africa and West Asia but all tetraploid members (Tor etc.) are
found in southern Asia and Cyprinion are mainly distributed in
West Asia, we hypothesized that this hexaplodization event
happened during their dispersal toward Africa but predated their
arrival in Africa (see Section 4below). For the tribe Barbini,
because Capoeta is placed within the tetraploid Luciobarbus in the
mitochondrial tree (Fig. 2h), the hexaplodization event of Capoeta
might be due to ancient hybridization between a Luciobarbus spe-
cies (maternal source; see also Levin et al., 2012) and a Cyprinion
species (paternal source). All discussion above was based only on
mitochondrial trees and the RAG1 tree. Further analyses based
on more nuclear loci and inheritance test may clarify the state
Fig. 2 (continued)
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 105
Author's Personal Copy

Fig. 2 (continued)
106 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

Fig. 2 (continued)
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 107
Author's Personal Copy

and origin of Schizothoracini and Schizopygopsini and test our
observations on other polyploid tribes as well.
4.2. Phylogenetic relationships among tribes and the revised
classification
Multiple lines of evidence provided in this study supported the
subdivision of Cyprininae into eleven major clades/tribes. Relation-
ships among these clades were shown in our trees (Figs. 1–4). The
following seven clades consistently received strong support in all
analyses: Probarbini, Labeonini, Torini, Acrossocheilini, Spinibarbi-
ni, Schizothoracini, and Schizopygopsini, except for Torini 2 & 3,
and Schizothoracini in the RAG1 tree (Figs. 1–4). The following
three clades Smiliogastrini (less Oreichthys and Chagunius), Porop-
untiini (less Eirmotus,‘Puntius’semifasciolatus, and ‘P.’ snyderi), and
Barbini (less Cyprinion,Semiplotus, and Scaphiodonichthys) received
strong support in most analyses (Figs. 1–4). Oreichthys and Chagu-
nius are likely members of Smiliogastrini, while Eirmotus and the
two ‘Puntius’ species are likely members of Poropuntiini. Analyses
based on mitochondrial data indicate that Cyprinion,Semiplotus,
and Scaphiodonichthys are members of Barbini (Figs. 1–3); howev-
er, it was difficult to sort RAG1 paralogs of barbins with confidence.
We chose to be conservative and included these taxa in the tribe
Barbini. The tribe Cyprinini was supported in the mitogenome tree
(BP = 92%; Fig. 3), and the RAG1 tree (Cyprinini 1, BP = 99%; Cypri-
nini 2, BP = 53%; Fig. 4).
See Table 3for the new classification we proposed for the
subfamily Cyprininae. Species-level relationships depicted in our
phylogenetic trees should be interpreted with caution as they may
change when more species are added to the analyses. Sequences
from GenBank are also more likely to be problematic at the spe-
cies-level, although we have tried our best to ensure the sequences
we used were from trusted institutions and are authentic.
4.3. Nomenclature and phylogenetic relationships within tribes
4.3.1. Tribe Probarbini (new tribe)
This clade includes two genera native to Southeast Asia, Catlo-
carpio and Probarbus (Figs. 2b, 3 and 4). These genera were previ-
ously placed in two distinct tribes or subtribes (Rainboth, 1991,
1996a; Rainboth et al., 2012) but were resolved as sister taxa in
our mitochondrial trees (Fig. 2b and 3); their RAG1 alleles mixed
with each other in the nuclear tree (Fig. 4). A formal description
of this new tribe is provided at the end of the discussion.
4.3.2. Tribe Labeonini Bleeker, 1859
This clade contains all species of Labeo,Garra,and their allies
widely distributed in tropical Asia and Africa (Figs. 3 and 4). The
oldest available family-group name for this clade is Labeonini
Bleeker, 1859. Our tribe Labeonini is essentially equivalent to the
Labeines of Reid (1982, 1985), the subfamily Labeoninae of Chen
et al. (1984) and Yue et al. (2000), and the tribe Labeonini of
Rainboth (1981, 1991, 1996a) and Rainboth et al. (2012). See
Yang et al. (2012b) for details of phylogenetic relationship within
the Labeonini.
4.3.3. Tribe Torini Karaman, 1971
Large-sized barbs of Asia (e.g. Tor)and Africa (e.g. Labeobarbus)
are included in this clade (Figs. 2b, 3 and 4). Torinae Karaman, 1971
is the oldest family-group name available for this clade. Our tribe
Fig. 2 (continued)
108 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

Fig. 2 (continued)
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 109
Author's Personal Copy

Torini is generally equivalent to the subtribe Tores of Rainboth
(1981).
Large-sized barbs of southern Asia constituted the basal clades.
They may all be tetraploids and share the same evolutionary histo-
ry despite difference in external morphology (Fig. 4). Type species
of both Osteochilichthys and Lepidopygopsis are found within
Hypselobarbus (Fig. 2b). Further taxonomic revision on this clade
is warranted.
The genus Barbus formerly included approximately 800 species
distributed in Eurasia and Africa. Many of these species and species
groups were later reclassified into other, presumably monophylet-
ic, genera based on diagnostic morphological characters (e.g. Tor,
Fig. 3. The optimal Maximum Likelihood tree (ln L= 313101.148476) resulting from the mitogenome dataset. Bootstrap support values are shown beside each node. Values
lower than 50% are indicated by ‘‘’’. Two strongly supported large clades are labeled ‘‘A’’ and ‘‘B’’ to facilitate discussion of the phylogeny. The phylogenetic position of the
five enigmatic species listed on the left are indicated using dashed lines and relevant bootstrap values are shown.
110 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

Fig. 4. The optimal Maximum Likelihood tree (ln L= 20257.574486) resulting from the RAG1 dataset. Bootstrap support values are shown beside each node. Values lower
than 50% are indicated by ‘‘’’. Different putative RAG1 alleles of each species are denoted by A1-n, B1-n, or C1-nfollowing species names. The letters A, B, or C were randomly
assigned to alleles. Numbers following family-group names denote different clades (e.g. Cyprinini 1 and Cyprinini 2) of each polyploid lineage. From bottom to top of this
figure, smaller numbers were assigned to clades of the same lineage that appeared earlier. Ploidy levels (2n,4n,or6n) of tribes and some genera are shown in brackets.
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 111
Author's Personal Copy

Puntioplites,Pseudobarbus). Currently, there remain around 400
species in Barbus s.l.(Barbus s.s. +‘Barbus’). In our trees, members
of ‘Barbus’ were located in Torini (hexploids) and Smiliogastrini
(diploids and tetraploids). Our phylogeny on hexaploid barbs of
the Torini is similar to that of Tsigenopoulos et al. (2010) except
for the position of several weakly supported small clades. Here
we suggest that all hexaploid ‘Barbus’ be allocated to the genus
Labeobarbus. The genus name Pterocapoeta Günther, 1902 should
be revalidated to accommodate its type species Varicorhinus
maroccana. We tentatively recognize three informal groups within
the Labeobarbus clade: ‘‘Labeobarbus lineage’’, ‘‘Carasobarbus lin-
eage’’, and ‘‘Pterocapoeta lineage’’. According to Borkenhagen and
Krupp (2013),Mesopotamichthys,‘Barbus’grypus, and ‘B.’ reinii of
the Carasobarbus lineage should not be simply moved to Carasobar-
bus, because they seem more similar in some morphological char-
acters to Labeobarbus or Varicorhinus than to Carasobarbus.
Borkenhagen (2014) moved ‘Barbus’grypus to the new genus Ara-
bibarbus. In our present study, we put ‘Barbus’reinii under the
genus-group name ‘Labeobarbus’ to clearly separate them from Bar-
bus s.s. We tested the position of the monotypic African genus
Sanagia based on a 633 bp CO1 fragment of S. velifera from Gen-
Bank (HM418112) and found that it is a member of the ‘‘Labeobar-
bus lineage’’ sister to Labeobarbus sp. Kongou (results not shown).
4.3.4. Tribe Smiliogastrini Bleeker, 1863
This clade includes small-sized barbs of Asia (Puntius and allies)
and Africa (‘Barbus’ and allies) (Fig. 2c and d, 3&4). At least five
family-group names are available for this clade: Barbini Bleeker,
1859, Systomi Bleeker, 1863, Osteobramae Bleeker, 1863, Smilio-
gastrini Bleeker, 1863 (Smiliogaster Bleeker, 1860 is a junior syn-
onym of Osteobrama Heckel, 1843), and Puntii Karaman, 1971.
The name Barbini Bleeker, 1859 was used to designate another
major clade resolved in this study, because the type species of Bar-
bus,B. barbus, fell within that clade. The name Puntii appeared later
than other names, and thus could not be considered. The names
Systomi, Osteobramae, and Smiliogastrini were established simul-
taneously, but the last name was proposed at a higher rank and
takes precedence (Art. 24.1, ICZN, 1999). Therefore, we name this
clade as the tribe Smiliogastrini Bleeker, 1863. No equivalent
family-group has been proposed before.
Sixteeen lineages are recognized from what was Puntius and its
Asian allies (Puntius s.l.). Besides the genera redefined or recently
described, i.e. Puntius s.s. Systomus,Dawkinsia,Haludaria,Pethia,
Barbodes,Oliotius,Puntigrus,Striuntius,Desmopuntius, and Sahya-
dria (Pethiyagoda et al., 2012; Pethiyagoda, 2013; Kottelat, 2013;
Raghavan et al., 2013), there are five more lineages: Rohtee (includ-
ing Osteobrama belangeri and O. sp.), Hampala,Osteobrama,Ore-
ichthys, and Chagunius. Most of these lineages received strong
bootstrap support (Oliotius is monotypic), except for several large
lineages, Systomus,Pethia,Puntius, and Barbodes. Relationships
among these lineages are, in general, unresolved. More studies
are warranted for Puntius s.l. and nuclear genes with appropriate
rate variation should be exploited to further explore relationships
among these diploid lineages.
Our results show that Asian Puntius and allies are closely related
to African small ‘Barbus’, confirming previous hypotheses based on
scale morphology (Rainboth, 1981) and karyology (Golubtsov and
Krysanov, 1993; Ráb et al., 1995). We propose a revalidation of
the generic name Enteromius Cope, 1867 (type species: Barbus
potamogalis) to accommodate all African diploid ‘Barbus’, as Enter-
omius is the oldest available genus-group name for these fishes.
Further studies are required to more fully resolve the phylogenetic
relationships among species of the non-monophyletic Enteromius
(as resolved herein). We also suggest moving all southern African
tetraploid barbs into ‘Pseudobarbus’, if they cannot be placed in
the genus Pseudobarbus.
4.3.5. Tribe Poropuntiini
This clade is comprised of genera primarily distributed in
Southeast Asia (Figs. 2e, 3 and 4). Poropuntii Rainboth, 1991 and
Puntioplitini Nguyen & Ho, 2003 are the only family-group names
proposed for various members of this clade. However, neither is
available because no diagnoses were provided (art. 13.1.1, ICZN,
1999) and it is impossible to make any of them available (art.
13.2, ICZN, 1999; see also Van der Laan et al., 2014). For conve-
nience, here we refer this clade as the tribe Poropuntiini. Most
members of this tribe are diagnosed by the combination of the fol-
lowing characters (Rainboth, 1981, 1996a,b; Shan et al., 2000;
Kottelat et al., 1993; Kottelat, 2001a,b; Arai, 2011): (1) diploid,
with a chromosome number of 2n= ca. 50; (2) serrated dorsal
fin-ray; (3) scales with a moderate number of parallel to converg-
ing posterior radii and no or few lateral radii; and (4) body color
plain, without bars, blotches or spots, may have a stripe along
the body, sometimes with a reticulate pattern.
Species in this tribe exhibit great diversity in pharyngeal teeth
morphology, which has recently been addressed by Pasco-Viel
et al. (2014). The genus Barbonymus is not monophyletic. A taxo-
nomic revision on B. gonionotus is warranted, as this species did
not group with the type species of the genus, B. schwanenfeldii.
The former species should be a member of Hypsibarbus as they
are quite similar in many morphological characters (Chaiwut
Grudpan pers. comm.) and form a clade (despite BP = 51%;
Fig. 2e). Rainboth (1996b) argued that the genus Hypsibarbus is
most closely related to Poropuntius and Barbodes (=Barbonymus).
Our results suggest that Hypsibarbus is most closely related to ‘Bar-
bonymus’gonionotus,Sikukia, and Mystacoleucus.
4.3.6. Tribe Cyprinini Rafinesque, 1815
This clade contains Cyprinus and allies found in Eurasia (Figs. 2e,
3 and 4). This tribe was named based on the oldest available
family-group name of this clade, Cyprinia Rafinesque, 1815. Our
tribe Cyprinini is most similar to the Cyprinini of Rainboth
(1981) but contains more genera.
Sinocyclocheilus has usually been considered a barbin genus (e.g.
Shan et al., 2000). Yang et al. (2010) was the first to hypothesize
that Sinocyclocheilus was a member of Cyprinini and we are now
highly confident that Sinocyclocheilus is a member of that tribe.
Shan et al. (2000) treated Luciocyprinus as a member of their Barbi-
nae while Rainboth et al. (2012) placed this genus in their sub-
family Cyprininae, tribe Oreinini. Multiple lines of evidence
provided in this study strongly support Luciocyprinus as a member
of the tribe Cyprinini. The monotypic genus Aaptosyax is very rare
(Tomoda, 2011) and our efforts to obtain specimens and tissue of
Aaptosyax failed. The Cytochrome bgene sequence used in He
et al. (2004) is the only known available molecular data for this
taxon (Tomoda, 2011). Using these data the genus was grouped
with Cyprinus and Carassius in the molecular trees of He et al.
(2004) and Rüber et al. (2007).Rainboth et al. (2012) allocated this
species to their subfamily Danioninae, tribe Chedrini, without jus-
tifiable evidence. Our analyses suggest that Aaptosyax is likely a
member of the tribe Cyprinini but further studies are certainly
needed (Fig. 3). Even if Aaptosyax is not considered, it will be chal-
lenging to find morphological synapomorphies for our Cyprinini,
because both Sinocyclocheilus and Luciocyprinus are quite different
from other members of the Cyprinini (Cyprinus etc.) in external
morphology (see Yang et al., 2010).
The possession of both a serrated anal and dorsal spine (spinous
fin-ray) has been the most important, and sometimes only, charac-
ters used to determine membership of a fossil species in the tribe
Cyprinini. Our study, however, illustrates that some taxa (e.g.
Puntioplites falcifer) have these characters but do not belong to
Cyprinini, whereas other members of Cyprinini lack these charac-
ters (e.g. Sinocyclocheilus,Luciocyprinus). Therefore caution is
112 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

advised when identifying cyprinin fossils. The lack of explicit mor-
phological synapomorphies suggests that diversity of cyprinin fos-
sils and the age of the clade are likely underestimated.
4.3.7. Tribe Acrossocheilini (new tribe)
This clade includes three genera: Acrossocheilus,Onychostoma,
and Folifer (Figs. 2f, 3 and 4), distributed in Southeast Asia and Chi-
na. Members of this tribe have been previously placed in one or
more tribes/subtribes together with many other cyprinines
(Rainboth, 1981, 1991; Chen et al., 1984; Yue et al., 2000). In our
analyses, neither Acrossocheilus nor Onychostoma are monophyletic
(see also Wang et al., 2007; Li et al., 2008). A thorough taxonomic
revision of Acrossocheilus and Onychostoma is highly warranted.
The Asian genus Onychostoma was once considered a synonym
(but a valid subgenus) of the African genus Varicorhinus (e.g.
Ba
˘na
˘rescu, 1971; Wu, 1977; Chu and Chen, 1989) but our results
demonstrate that Onychostoma and Varicorhinus fall in two distinct
major clades. These two genera are also very distinct in chromo-
some numbers (Arai, 2011), species of Onychostoma are usually
diploid (2n= ca. 50), whereas species of Varicorhinus are hexaploid
(2n= ca. 150). The genera Acrossocheilus and Folifer have the same
chromosome number (2n= ca. 50) as Onychostoma. The genus Folif-
er was treated as a junior synonym of Tor by some researchers (e.g.
Shan et al., 2000) because this genus possesses a median lobe on its
lower lip, a character shared by species of Tor. However, Tor is tet-
raploid (2n= ca. 100), while Folifer is diploid (Arai, 2011). They also
exhibit differences in scale size, Folifer has relatively small scales,
whereas Tor usually has large scales. Zhou and Cui (1996) thought
that these genera belonged to two different lineages. Our results
indicate that they belong to distinct clades (Acrossocheilini vs.
Torini).
4.3.8. Tribe Spinibarbini (new tribe)
This clade currently contains only the genus Spinibarbus,which
is distributed in Laos, northern Vietnam and southern China
(Figs. 2f, 3 and 4). No available family-group name was found for
this group. The tribe is formally described later in Section 4. The
genus Spinibarbus was usually included in Barbini or Barbinae
(Rainboth, 1981; Chen et al., 1984; Yue et al., 2000). Rainboth
(1991) put it in his tribe Cyprinini subtribe Tores. Wang et al.
(2007) also placed it in his tribe Cyprinini.
Species of Spinibarbusare tetraploids and have a chromosome
number of 2n= 100 (Arai, 2011). It remains to be seen whether
other genera should be assigned to this tribe. The genus Spinibarbus
and several other cyprinine genera, including Mystacoleucus,
Paraspinibarbus,Rohtee, and Parator (P. zonatus), share one mor-
phological character: the presence of a procumbent predorsal
spine. The genera Spinibarbus,Mystacoleucus, and Rohtee were
included in our analyses and the latter two were found in Poropun-
tiini and Smiliogastrini, respectively.
4.3.9. Tribe Schizothoracini McClelland, 1842
This clade includes Schizothorax and allies, as well as Perco-
cypris, and included species are mainly found on Asian plateaus
and adjacent regions (Figs. 2f, 3 and 4). At least three available
family-group names could be applied to this clade of fishes,
Schizothoracinae McClelland, 1842, Oreini Bleeker, 1863, and Opis-
tocheili Bleeker, 1863. There is some debate as to whether
Schizothoracinae in McClelland (1842) is a family-group name
(e.g. Kullander et al., 1999; Kottelat, 2013). Here we follow
Kottelat (2013) and name this clade as the tribe Schizothoracini
McClelland, 1842. It is equivalent to Mirza’s (1991) tribe
Schizothoracini plus the genus Percocypris.
Our results illustrate that Percocypris is sister to a clade mainly
formed by Schizothorax, consistent with previous studies (Wang
et al., 2007, 2013; Kong et al., 2007; Li et al., 2008). Two species
of Schizopyge including the type species (S. curvifrons) formed a
small distinct lineage within Schizothorax. The monotypic Aspi-
orhynchus (A. laticeps) constituted a sister group with Schizothorax
biddulphi. As discussed below, it is too early to make taxonomic
revisions for these taxa. We analyzed the Cytochrome bsequences
of Schizocypris altidorsalis from GenBank (JN790240). It fell with
Cyprinion and Semiplotus in our tree; however the branch to this
taxon was quite long and we doubt the result. Hence, it is not
shown (but see Rainboth, 1991: 200). More discussion on our tribe
Schizothoracini can be found below.
4.3.10. Tribe Schizopygopsini Mirza, 1991
This clade contains a group of genera native to Asian Plateaus
and adjacent regions (Figs. 2g, 3 and 4). There are two available
family-group names Diptychini and Schizopygopsini both pro-
posed by Mirza (1991). Here, we name this clade as the tribe
Schizopygopsini Mirza, 1991, because Diptychini (now Diptychi-
nae) proposed by Janse (1933) is currently used for a group of
insects (Lepidoptera: Geometridae). This tribe contains all mem-
bers of Mirza’s (1991) tribes Schizopygopsini and Diptychini.
Fishes of our Schizothoracini (less Percocypris), Schizopygopsini,
and sometimes the genus Lepidopygopsis (Torini) are referred to as
‘‘snowtrouts.’’ They share a character called the anal shield, ‘‘a
peculiar cleft in the ventral side of the body in front of the anal
fin, which is bounded laterally with scales of a peculiar form placed
vertically like eave-tiles’’ (Mirza, 1991) which may be correlated
with spawning behavior (Cao et al., 1981). In our analyses, these
fishes were found in three different tribes suggesting multiple,
independent evolution of anal shield. Cao et al. (1981) divided
snowtrouts (except Lepidopygopsis) into three groups. Members
of the ‘‘primitive group’’ (Schizothorax,Schizocypris, and
Aspiorhynchus) usually possess 1 or 2 pairs of barbels, 3 or 4 rows
of pharyngeal teeth, and scales usually covering the whole body
except for the belly area; members of the ‘‘specialized group’’ (Dip-
tychus,Ptychobarbus, and Gymnodiptychus) usually possess 1 pair of
barbels, 2 rows of pharyngeal teeth, and moderately degenerated
scales (degenerated entirely in Gymnodiptychus); and members of
the ‘‘highly specialized group’’ (Gymnocypris,Oxygymnocypris,
Schizopygopsis,Platypharodon,Chuanchia, and Herzensteinia) usual-
ly do not have barbels, possess 1 or 2 rows of pharyngeal teeth, and
entirely degenerate scales. Mirza’s (1991) tribes Schizothoracini,
Diptychini, and Schizopygopsini are equivalent to the three groups
of Cao et al. (1981), respectively. In the present study, the primary
group (Schizothorax) is found in the tribe Schizothoracini, whereas
the specialized and highly specialized groups are located in the
tribe Schizopygopsini, with the former being more basal in the
trees than the latter.
Recent studies demonstrate that the alpha taxonomy and
nomenclature of snowtrouts, especially the former, are problemat-
ic (e.g. Kullander et al., 1999; He and Chen, 2006; Yang et al.,
2012a; Kottelat, 2013). Similarity in morphological characters
widely used in their taxonomy, e.g. mouth and lip structure, may
be the result of convergence or parallelism, a situation similar to
that in the tribe Labeonini (Yang and Mayden, 2010). This may
explain the non-monophyly of several taxa of Schizothoracini
and Schizopygopsini in our analyses. Until issues associated with
the taxonomy and nomenclature of snowtrouts are resolved, there
can be no reasonable phylogenetic discussion on the intra-
relationships of either Schizothoracini (less Percocypris)or
Schizopygopsini.
4.3.11. Tribe Barbini Bleeker, 1859
All Barbus s.s. and allies, Cyprinion,Semiplotus, and Scaphiodo-
nichthys are contained in this clade (Figs. 2h, 3 and 4). These taxa
are distributed in Eurasia and northwestern Africa. The name
Barbini Bleeker, 1859 is the oldest available family-group name
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 113
Author's Personal Copy

for this clade. Our tribe Barbini contains fewer genera than many
previously proposed Barbini or Barbinae (Tables 1 and S1).
In our phylogenetic trees, the relationships between Luciobar-
bus,Barbus,Capoeta,and Aulopyge are largely the same as depicted
in previous studies (Machordom and Doadrio, 2001; Tsigenopoulos
and Berrebi, 2000; Tsigenopoulos et al., 2003) likely due to the fact
that many of our sequences were retrieved from these studies. The
genus Capoeta was monophyletic and fell within Luciobarbus. The
same result was hypothesized in previous studies (Machordom
and Doadrio, 2001; Tsigenopoulos et al., 2003; Levin et al., 2012).
Taki (1974: 135) found that the Southeast Asian genus Scaphiodo-
nichthys shows a close affinity to Scaphognathops in many respects.
However, in our trees, the former genus was found in the Barbini,
whereas the latter in the Poropuntiini. The ‘‘Cyprinion-Onychos-
toma lineage’’ considered by Chen (1989) to be a monophyletic
group, contains Onychostoma,Cyprinion,Semiplotus, and Scaphiodo-
nichthys. However, the present results show that this lineage is not
monophyletic, as Onychostoma was located in the Acrossocheilini
but the other three genera are in the Barbini.
4.4. Implications for biogeography
Biogeography is not the focus of this study, but we briefly
discuss several interesting points. The current distribution of
cyprinines in Eurasia and Africa may not have been shaped by
the breakup of Gondwana, because their diversification began
55–80 Mya (million years ago; Saitoh et al., 2011; Chen et al.,
2013; Pasco-Viel et al., 2014), after the separation of the Indian
landmass from Africa (130 Mya; Smith et al., 1994).
We hypothesize that there are at least five independent
dispersal events from Eurasia to Africa. Besides the two (one in
Labeonina, the other in Garraina) in the Labeonini (Yang and
Mayden, 2010; Yang et al., 2012b), three more such events are
identified in Torini, Smiliogastrini, and Barbini, respectively. In Tor-
ini, all large-sized African and Western Asian barbs (Labeobarbus,
Carasobarbus, etc.) are located at the apical clade of the tree,
whereas large-sized southern Asian barbs (Hypselobarbus,Tor,
etc.) are located in the basal clades (Fig. 2b). In Smiliogastrini,
the small-sized African barbs and allies and small-sized Asian Pun-
tius and allies are located at the apical clade and basal clade of the
tree, respectively (Fig. 2c and d). In Barbini, all northwestern Afri-
can species constituted a monophyletic group in the apical clade of
the tree (from Luciobarbus moulouyensis to L. massaensis,Fig. 2h)
whereas the rest species of this tribe occur in Eurasia.
Because all African cyprinines can be allocated to the four tribes
mentioned above, we hypothesize that ancestors of all these spe-
cies invaded Africa via these dispersal events. The oldest Labeo-like
and Barbus-like fossils found in Africa are around 17 and 12–
13 million years old, respectively (Van Couvering, 1977). These fos-
sils may represent some of the earliest cyprinines in Africa,
although we have no evidence to which tribe the Barbus-like fossil
belongs because it was described based on pharyngeal teeth only.
4.5. Description of three new tribes
4.5.1. Probarbini, new tribe
Type genus
Probarbus Sauvage, 1880
4.5.1.1. Diagnosis. Members of this tribe are diagnosed by combina-
tion of the following characters (Smith, 1945; Arai, 2011; Roberts,
1992; Rainboth, 1996a): (1) pharyngeal teeth arranged in a single
row with 4 teeth on each side; (2) tetraploid, with a chromosome
number of 2n= 98; (3) scales covering most of the body; and (4)
last simple anal fin-ray segmented and flexible.
4.5.1.2. Composition. Probarbus Sauvage, 1880 and Catlocarpio Bou-
lenger, 1898. Currently, Probarbus includes three valid species,
while Catlocarpio has one valid species.
4.5.1.3. Distribution. Mekong, Chao Phraya, and Maeklong basins of
Indo-China, and the Pahang and Perak basins of Malaysia.
4.5.2. Acrossocheilini, new tribe
Type genus
Acrossocheilus Oshima, 1919
4.5.2.1. Diagnosis. Members of this tribe are diagnosed by combina-
tion of the following characters (Chen, 1989; Shan et al., 2000;
Kottelat, 2001a,b; Arai, 2011): (1) diploid, with a chromosome
number of 2n= ca. 50; (2) last simple ray of dorsal fin strong, with
a serrated or smooth posterior margin; (3) 8 branched dorsal-fin
rays; (4) pharyngeal teeth arranged in three rows; and (5) horny
sheath on lower jaw.
4.5.2.2. Composition. Includes over 40 valid species in three genera
Acrossocheilus Oshima, 1919, Onychostoma Günther, 1896, and
Folifer Wu, 1977.
4.5.2.3. Distribution. Mainland China, Taiwan, Viet Nam, Laos, Thai-
land, and Cambodia.
4.5.3. Spinibarbini, new tribe
Type genus
Spinibarbus Oshima, 1919
4.5.3.1. Diagnosis. Members of this tribe are diagnosed by combina-
tion of the following characters (Yang and Chen, 1994; Shan et al.,
2000; Kottelat, 2001a,b; Arai, 2011): (1) the presence of a procum-
bent predorsal spine (i.e. an external anterior pointed process of
the first proximal radial); (2) tetraploid, with a chromosome num-
ber of 2n= 100; and (3) lower lip not developed, confined to side of
lower jaw.
4.5.3.2. Composition. Spinibarbus Oshima, 1919 and around eight
valid species. Further studies are needed to determine if there
are other genera to be included into this tribe.
4.5.3.3. Distribution. Laos, northern Viet Nam, and southern China.
Acknowledgments
This study was developed from the Ph.D. Dissertation of LY
(Saint Louis University, 2010; Advisor: RLM). LY would like to
express his gratitude to Larry Page, Jonathan Armbruster, Milton
Tan, Jarungjit Grudpan, and Chaiwut Grudpan and his students
for either organizing or participating in the fieldwork in Thailand.
Preserved specimens in Saint Louis University, University of Kan-
sas, and Auburn University and some type materials stored at Insti-
tute of Hydrobiology (Wuhan), Kunming Institute of Zoology,
Institute of Zoology (Beijing) were examined for this study. We
thank Wei-Jen Chen, Mary Agnew and Qiu Ren for help with data
collection. Kevin Tang, Lanping Zheng, and Leyang Yuan are
thanked for helpful discussion. David Neely is thanked for provid-
ing samples. We are grateful to Hsin-Hui Wu and Joe Besser for
help using the Cluster computing system. Maurice Kottelat and
Carl Ferraris are thanked for discussion on the availability of sever-
al family-group names. Maurice Kottelat kindly commented on the
discussion part of this paper. LY sincerely appreciates the enor-
mous support from Gavin Naylor. We are very grateful to the asso-
ciate editor and two anonymous reviewers for their valuable
comments and suggestions that helped improve this paper. This
114 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy

research was supported in part by personal funds of RLM, by the
USA National Science Foundation grants including The Cyprini-
formes Tree of Life initiative (CToL) (EF 0431326 to RLM; EF
0431132 to AMS) and the Collaborative PBI All Cypriniformes Spe-
cies – Phase II of An Inventory of the Otophysi (ACSI-II)
(DEB1021840 to RLM), by the Japan Society for the Promotion of
Science (17207007 and 22370035) and by Saint Louis University,
St. Louis, Missouri.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2015.01.
014.
References
Amores, A., Force, A., Yan, Y.L., Joly, L., Amemiya, C., Fritz, A., Ho, R.K., Langeland, J.,
Prince, V., Wang, Y.L., Westerfield, M., Ekker, M., Postlethwait, J.H., 1998.
Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711–
1714.
Arai, R., 2011. Fish Karyotypes: A Check List. Springer, Japan.
Ba
˘na
˘rescu, P.M., 1971. A review of the species of the subgenus Onychostoma s. str.
with description of a new species (Pisces, Cyprinidae). Rev. Roum. Biol. Ser. Zool.
16, 241–248.
Borkenhagen, K., 2014. A new genus and species of cyprinid fish (Actinopterygii,
Cyprinidae) from the Arabian Peninsula, and its phylogenetic and
zoogeographic affinities. Environ. Biol. Fish. 97, 1179–1195.
Borkenhagen, K., Krupp, F., 2013. Taxonomic revision of the genus Carasobarbus
Karaman, 1971 (Actinopterygii, Cyprinidae). ZooKeys 339, 1–53.
Cao, W.X., Chen, Y.Y., Wu, Y.F., Zhu, S.Q., 1981. Origin and evolution of
schizothoracine fishes in relation to the upheaval of the Xizang Plateau. In:
The Team of the Comprehensive Scientific Expedition to the Qinghai-Xizang
Plateau, Chinese Academy of Sciences (Eds.), Collection in Studies on the Period,
Amplitude and Type of the Uplift of the Qinghai-Xizang Plateau. Science Press,
Beijing, pp. 118–130 (in Chinese).
Cavender, T.M., Coburn, M.M., 1992. Phylogenetic relationships of North American
Cyprinidae. In: Mayden, R.L. (Ed.), Systematics, Historical ecology, and North
American Freshwater Fishes. Stanford University Press, Stanford, CA, pp. 293–
327.
Chen, Y.Y., 1989. Anatomy and phylogeny of the cyprinid fish genus Onychostoma
Günther, 1896. Brit. Mus. Nat. Hist. (Zool.) 55, 109–121.
Chen, X.L., Yue, P.Q., Lin, R.D., 1984. Major groups within the family Cyprinidae and
their phylogenetic relationships. Acta Zootaxon. Sin. 9, 424–440 (in Chinese
with English summary).
Chen, W.-J., Lavoué, S., Mayden, R.L., 2013. Evolutionary origin and early
biogeography of otophysan fishes (Ostariophysi: Teleostei). Evolution 67,
2218–2239.
Chu, X.L., Chen, Y.R. (Eds.), 1989. The Fishes of Yunnan, China. Part I. Cyprinidae.
Science Press, Beijing, China.
Cui, J.X., Ren, X.H., Yu, Q.X., 1991. Nuclear DNA content variation in fishes. Cytologia
56, 425–429.
David, L., Blum, S., Feldman, M.W., Lavi, U., Millel, J., 2003. Recent duplication of the
common carp (Cyprinus carpio L.) genome as revealed by analyses of
microsatellite loci. Mol. Biol. Evol. 20, 1425–1434.
Donsakul, T., Magtoon, W., Rangsiruji, A., 2012. Karyotypes of Five Cyprinid Fishes
(Family Cyprinidae): Macrochirichthys macrochirus,Scaphiodonichthys
acanthopterus,Epalzeorhynchos munensis,Opsarius koratensis and Parachela sp.
from Thailand. In: Proceedings of the 50th Kasetsart University Annual
Conference, Kasetsart University, Thailand, 31 January – 2 February 2012, vol.
1. Subject: Animals, Veterinary Medicine, Fisheries, pp. 439–446.
Eschmeyer, W.N. (Ed.), 2015. Catalog of Fishes. <http://research.calacademy.org/
ichthyology/catalog/fishcatmain.asp>.
Evans, B.J., Kelley, D.B., Melnick, D.J., Cannatella, D.C., 2005. Evolution of RAG-1 in
polyploid clawed frogs. Mol. Biol. Evol. 22, 1193–1207.
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the
bootstrap. Evolution 39, 783–791.
Froese, R., Pauly, D. (Eds.), 2015. FishBase. <www.fishbase.org> version (1/2015).
Ganai, F.A., Yousuf, A.R., Dar, S.A., Tripathi, N.K., Wani, S.U., 2011. Cytotaxonomic
status of Schizothoracine fishes of Kashmir Himalaya (Teleostei: Cyprinidae).
Caryologia 64, 435–445.
Golubtsov, A.S., Krysanov, E.Y., 1993. Karyological study of some cyprinid species
from Ethiopia. The ploidy differences between large and small Barbus of Africa.
J. Fish Biol. 42, 445–455.
He, D.K., Chen, Y.F., 2006. Biogeography and molecular phylogeny of the genus
Schizothorax (Teleostei: Cyprinidae) in China inferred from cytochrome b
sequences. J. Biogeogr. 33, 1448–1460.
He, S.P., Liu, H.Z., Chen, Y.Y., Kuwahara, M., Nakajima, T., Zhong, Y., 2004.
Molecular phylogenetic relationships of Eastern Asian Cyprinidae (Pisces:
Cypriniformes) inferred from cytochrome bsequences. Sci. China Ser. C – Life
Sci. 47, 130–138.
Howes, G.J., 1987. The phylogenetic position of the Yugoslavian cyprinid fish genus
Aulopyge Heckel, 1841, with an appraisal of the genus Barbus Cuvier and
Cloquet, 1816 and the subfamily Cyprininae. Bull. Brit. Mus. Nat. Hist. (Zool.) 52,
165–196.
Howes, G.J., 1991. Systematics and biogeography: an overview. In: Winfield, I.J.,
Nelson, J.S. (Eds.), Cyprinid Fishes: Systematics, Biology and Exploitation.
Chapman and Hall, London, pp. 1–33.
ICZN (International Commission on Zoological Nomenclature), 1999. International
Code of Zoological Nomenclature, fourth ed. The International Trust for
Zoological Nomenclature, London.
Janse, A.J.T., 1933. The Moths of South Africa, vol. 2. Geometridae. E. P. &
Commercial Printing, Durban, 448 p.
Kong, X.H., Wang, X.Z., Gan, X.N., Li, J.B., He, S.P., 2007. Phylogenetic relationships of
Cyprinidae (Teleostei: Cypriniformes) inferred from the partial S6K1 gene
sequences and implication of indel sites in intron 1. Sci. China Ser. C – Life Sci.
50, 780–788.
Kottelat, M., 2001a. Freshwater Fishes of Northern Vietnam. A Preliminary Check-
list of the Fishes known or expected to occur in Northern Vietnam with
Comments on Systematics and Nomenclature. Environment and Social
Development Unit, East Asia and Pacific Region. The World Bank, Washing,
DC, i–iii + 1–123 + 1–18 (June).
Kottelat, M., 2001b. Fishes of Laos. Wildlife Heritage Trust Publications, Colombo.
Kottelat, M., 2013. The fishes of the inland waters of Southeast Asia: a catalogue and
core bibliography of the fishes known to occur in freshwaters, mangroves and
estuaries. Raff. Bull. Zool. 27 (Suppl.), 1–663.
Kottelat, M., Whitten, A.J., Kartikasari, S.N., Wirjoatmodjo, S., 1993. Freshwater
Fishes of Western Indonesia and Sulawesi. Periplus Editions, Hong Kong, 259
pp, 84 pls.
Kullander, S.O., Fang, F., Delling, B., Åhlander, E., 1999. The fishes of the Kashmir
Valley. In: Nyman, L. (Ed.), River Jhelum, Kashmir Valley, Impacts on the Aquatic
Environment. Swedmar, Göteborg, pp. 99–167.
Lanfear, R., Calcott, B., Ho, S.Y.M., Guindon, S., 2012. PartitionFinder: combined
selection of partitioning schemes and substitution models for phylogenetic
analyses. Mol. Biol. Evol. 29, 1695–1701.
Lanfear, R., Calcott, B., Kainer, D., Mayer, C., Stamatakis, A., 2014. Selecting optimal
partitioning schemes for phylogenomic datasets. BMC Evol. Biol. 14, 82.
Leggatt, R.A., Iwama, G.K., 2003. Occurrence of polyploidy in the fishes. Rev. Fish
Biol. Fish. 13, 237–246.
Levin, B.A., Freyhof, J., Lajbner, Z., Perea, S., Abdoli, A., Gaffaro lug, M., Özuluh, M.,
Rubenyan, H.R., Salnikov, V.B., Doadrio, I., 2012. Phylogenetic relationships of
the algae scraping cyprinid genus Capoeta (Teleostei: Cyprinidae). Mol.
Phylogenet. Evol. 62, 542–549.
Li, J.B., Wang, X.Z., Kong, X.H., Zhao, K., He, S.P., Mayden, R.L., 2008. Variation
patterns of the mitochondrial 16S rRNA gene with secondary structure
constraints and their application to phylogeny of cyprinine fishes (Teleostei:
Cypriniformes). Mol. Phylogenet. Evol. 47, 472–487.
Machordom, A., Doadrio, I., 2001. Evolutionary history and speciation modes in the
cyprinid genus Barbus. Proc. Roy. Soc. Lond. B Biol. Sci. 268, 1297–1306.
McClelland, J., 1842. On the fresh-water fishes collected by William Griffith, Esq., F.
L. S. Madras Medical Service, during his travels under the orders of the Supreme
Government of India, from 1835 to 1842. Calcutta J. Nat. Hist. 2, 560–589.
Mirza, M.R., 1991. A contribution to the systematics of the Schizothoracine fishes
(Pisces: Cyprinidae) with the description of three new tribes. Pakistan J. Zool.
23, 339–341.
Nelson, J.S., 1994. Fishes of the World, third ed. Wiley, New York.
Nelson, J.S., 2006. Fishes of the World, fourth ed. Wiley, New York.
Pasco-Viel, E., Yang, L., Veran, M., Balter, V., Mayden, R.L., Laudet, V., Viriot, L., 2014.
Stability versus diversity of the dentition during evolutionary radiation in
cyprinine fish. Proc. Roy. Soc. B 281, 20132688. http://dx.doi.org/10.1098/
rspb.2013.2688.
Pethiyagoda, R., 2013. Haludaria,a replacement generic name for Dravidia
(Teleostei: Cyprinidae). Zootaxa 3646, 199–199.
Pethiyagoda, R., Meegaskumbura, M., Maduwage, K., 2012. A synopsis of the South
Asian fishes referred to Puntius (Pisces: Cyprinidae). Ichthyol. Explor.
Freshwaters 23, 69–95.
Phillips, M.J., Lin, Y.-H., Harrison, G.L., Penny, D., 2001. Mitochondrial genomes of a
bandicoot and a brushtail possum confirm the monophyly of australidelphian
marsupials. Proc. Roy. Soc. Lond. B Biol. Sci. 268, 1533–1538.
Phillips, M.J., Delsuc, F., Penny, D., 2004. Genome-scale phylogeny and the detection
of systematic biases. Mol. Biol. Evol. 21, 1455–1458.
Ráb, P., Machordom, A., Perdices, A., Guegan, J.-F., 1995. Karyotypes of three « small
»Barbus species (Cyprinidae) from Republic of Guinea (Western Africa) with a
review on karyology of African small Barbus. Caryologia 48, 299–307.
Raghavan, R., Philip, S., Ali, A., Dahanukar, N., 2013. Sahyadria,a new genus of barbs
(Teleostei: Cyprinidae) from western Ghats of India. J. Threatened Taxa 5, 4932–
4938.
Rainboth, W.J., 1981. Systematics of the Asiatic Barbins (Pisces, Cyprinidae). Unpubl.
Ph.D. Dissertation. The University of Michigan, Ann Arbor.
Rainboth, W.J., 1991. Cyprinid fishes of Southeast Asia. In: Winfield, I.J., Nelson, J.S.
(Eds.), Cyprinid Fishes: Systematics, Biology and Exploitation. Chapman and
Hall, London, pp. 156–210.
Rainboth, W.J., 1996a. Fishes of the Cambodian Mekong. FAO Species Identification
Field Guide for Fishery Purposes. FAO, Rome.
Rainboth, W.J., 1996b. The Taxonomy, Systematics, and Zoogeography of
Hypsibarbus. A New Genus of Large Barbs (Pisces, Cyprinidae) from the Rivers
of Southeastern Asia, vol. 129. Univ. Calif. Publ. Zool., i–xiii, 1–199.
L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116 115
Author's Personal Copy

Rainboth, W.J., Vidthayanon, C., Mai, D.Y., 2012. Fishes of the Greater Mekong
Ecosystem with Species List and Photographic Atlas, vol. 201. Miscellaneous
Publications, Museum of Zoology, University of Michigan, Berlin (i–vi, 1–173,
121 pls).
Reid, G.Mc.G., 1982. The form, function and phylogenetic significance of the
vomeropalatine organ in cyprinid fishes. J. Nat. Hist. 16, 497–510.
Reid, G.Mc.G., 1985. A Revision of African Species of Labeo (Pisces: Cyprinidae) and
A Re-definition of the Genus, vol. 6. Theses Zool. Cramer, Braunschweig, pp. 1–
322.
Roberts, T.R., 1992. Revision of the Southeast Asian cyprinid fish genus Probarbus,
with two new species threatened by proposed construction dams on the
Mekong River. Ichthyol. Explor. Freshwaters 3, 37–48.
Roure, B., Baurain, D., Philippe, H., 2013. Impact of missing data on phylogenies
inferred from empirical phylogenomic data sets. Mol. Biol. Evol. 30, 197–214.
Rüber, L., Kottelat, M., Tan, H.H., Ng, P.K.L., Britz, R., 2007. Evolution of
miniaturization and the phylogenetic position of Paedocypris, comprising the
world’s smallest vertebrate. BMC Evol. Biol. 7, 38.
Sahoo, P.K., Nanda, P., Barat, A., 2009. Chromosomal studies on a threatened fish
Cyprinion semiplotus (Teleostei: Cyprinidae) from Arunachal Pradesh. Asian Fish.
Sci. 22, 501–504.
Saitoh, K., 2003. Mitotic and meiotic analyses of the ‘large race’ of Cobitis striata,a
polyploid spined loach of hybrid origin. Folia Biol. (Krakow) 51 (Suppl.), 101–
105.
Saitoh, K., Chen, W.-J., 2008. Reducing cloning artifacts for recovery of allelic
sequences by T7 endonuclease I cleavage and single re-extension of PCR
products – a benchmark. Gene 423, 92–95.
Saitoh, K., Sado, T., Mayden, R.L., Hanzawa, N., Nakamura, K., Nishida, M., Miya, M.,
2006. Mitogenomic evolution and interrelationships of the Cypriniformes
(Actinopterygii: Ostariophysi): The first evidence toward resolution of higher-
level relationships of the World’s largest freshwater fish clade based on 59
whole mitogenome sequences. J. Mol. Evol. 63, 826–841.
Saitoh, K., Chen, W.-J., Mayden, R.L., 2010. Extensive hybridization and
tetrapolyploidy in spined loach fish. Mol. Phylogenet. Evol. 56, 1001–1010.
Saitoh, K., Sado, T., Doosey, M.H., Bart Jr., H.L., Inoue, J.G., Nishida, M., Mayden, R.L.,
Miya, M., 2011. Evidence from mitochondrial genomics supports the lower
Mesozoic of South Asia as the time and place of basal divergence of cypriniform
fishes (Actinopterygii: Ostariophysi). Zool. J. Linn. Soc. 161, 633–662.
Shan, X.H., Lin, R.D., Yue, P.Q., Chu, X.L., 2000. Barbinae. In: Yue, P.Q. et al. (Eds.),
Fauna Sinica. Osteichthyes: Cypriniformes III. Science Press, Beijing, pp. 3–170.
Smith, H. M., 1945. The freshwater fishes of Siam, or Thailand. Bull. Am. Mus. Nat.
Hist. 188, ix + 622pp.
Smith, A.G., Smith, D.G., Funnel, B.M., 1994. Atlas of Mesozoic and Cenozoic
coastlines. Cambridge University Press, Cambridge, pp. 778–826.
Stamatakis, A., 2006. RAxML–VI–HPC: Maximum Likelihood-based Phylogenetic
Analyses with Thousands of Taxa and Mixed Models. Bioinformatics 22, 2688–
2690.
Stamatakis, A., 2014. RAxML version 8: A tool for phylogenetic analysis and post-
analysis of large phylogenies. Bioinformatics. http://dx.doi.org/10.1093/
bioinformatics/btu033.
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the
RAxML web-servers. Syst. Biol. 57, 758–771.
Swofford, D.L., 2002. PAUP⁄: Phylogenetic Analysis Using Parsimony (⁄and other
methods), Version 4.0b10. Sinauer Associates, Sunderland, MA.
Taki, Y., 1974. New species of the genus Scaphognathops, Cyprinidae, from the Lao
Mekong River system. Jpn. J. Ichthyol. 21, 129–136.
Taylor, J.S., Braasch, I., Frickey, T., Meyer, A., Van de Peer, Y., 2003. Genome
duplication, a trait shared by 22000 species of ray-finned fish. Genome Res. 13,
382–390.
Tomoda, Y., 2011. A note on Aaptosyax grypus, a very rare, unique cyprinid fish.
Ichthyol. Res. 58, 288–290.
Tsigenopoulos, C.S., Berrebi, P., 2000. Molecular phylogeny of North Mediterranean
Barbs (Genus Barbus: Cyprinidae) inferred from Cytochrome bsequences:
biogeographic and systematic implications. Mol. Phylogenet. Evol. 14, 165–179.
Tsigenopoulos, C.S., Durand, J.D., ünlü, E., Berrebi, P., 2003. Rapid radiation of the
Mediterranean Luciobarbus species (Cyprinidae) after the Messinian salinity
crisis of the Mediterranean Sea, inferred from mitochondrial phylogenetic
analysis. Biol. J. Linn. Soc. 80, 207–222.
Tsigenopoulos, C.S., Kasapidis, P., Berrebi, P., 2010. Phylogenetic relationships of
hexaploid large-sized barbs (genus Labeobarbus, Cyprinidae) based on mtDNA
data. Mol. Phylogenet. Evol. 56, 851–856.
Van Couvering, J.A.H., 1977. Early records of freshwater fishes in Africa. Copeia
1977, 163–166.
Van der Laan, R., Eschmeyer, W.N., Fricke, R., 2014. Family-group names of recent
fishes. Zootaxa Monogr. 3882, 1–230.
Wang, X.Z., Li, J.B., He, S.P., 2007. Molecular evidence for the monophyly of East
Asian groups of Cyprinidae (Teleostei: Cypriniformes) derived from the nuclear
recombination activating gene 2 sequences. Mol. Phylogenet. Evol. 42, 157–170.
Wang, M., Yang, J.X., Chen, X.Y., 2013. Molecular phylogeny and biogeography of
Percocypris (Cyprinidae, Teleostei). PLoS ONE 8, e61827. http://dx.doi.org/
10.1371/journal.pone.0061827.
Wiens, J.J., 2006. Missing data and the design of phylogenetic analyses. J. Biomed.
Inform. 39, 34–42.
Wu, H.-W., 1977. The Cyprinid Fishes of China. Part 2. Technical Printing House,
Shanghai, pp. 229–598 (in Chinese).
Wu, Y.F., Kang, B., Men, Q., 1999. Chromosome diversity of Tibetan fishes. Zool. Res.
20, 258–264.
Wu, R., Gallo-Meagher, M., Littell, R.C., Zeng, Z.B., 2001. A general polyploid model
for analyzing gene segregation in outcrossing tetraploid species. Genetics 159,
869–882.
Xiao, H., Zhang, R.D., Feng, J.G., Ouyang, M., Li, W.X., Chen, S.Y., Zan, R.G., 2002.
Nuclear DNA content and ploidy of seventeen species of fishes in
Sinocyclocheilus. Zool. Res. 23, 195–199.
Yang, J.X., Chen, Y.R., 1994. Systematic revision of Spinibarbus fishes (Cyprinifromes:
Cyprinidae). Zool. Res. 15, 1–10 (In Chinese with English abstract).
Yang, L., Mayden, R.L., 2010. Phylogenetic relationships, subdivision, and
biogeography of the Cyprinid tribe Labeonini (sensu Rainboth, 1991) (Teleostei:
Cypriniformes), with comments on the implications of lips and associated
structures in the labeonin classification. Mol. Phylogenet. Evol. 54, 254–265.
Yang, L., Mayden, R.L., Sado, T., He, S.P., Saitoh, K., Miya, M., 2010. Molecular
phylogeny of the fishes traditionally referred to Cyprinini sensu stricto
(Teleostei: Cypriniformes). Zool. Scr. 39, 527–550.
Yang, J., Yang, J.X., Chen, X.Y., 2012a. A re-examination of the molecular phylogeny
and biogeography of the genus Schizothorax (Teleostei: Cyprinidae) through
enhanced sampling, with emphasis on the species in the Yunnan-Guizhou
Plateau, China. J. Zool. Syst. Evol. Res. 50, 184–191.
Yang, L., Arunachalam, M., Sado, T., Levin, B.A., Golubtsov, A.S., Freyhof, J., Friel, J.P.,
Chen, W.-J., Hirt, M.V., Manickam, R., Agnew, M.K., Simons, A.M., Saitoh, K.,
Miya, M., Mayden, R.L., He, S.P., 2012b. Molecular phylogeny of the cyprinid
tribe Labeonini (Teleostei: Cypriniformes). Mol. Phylogenet. Evol. 65, 362–379.
Yang, L., Hirt, M.V., Sado, T., Arunachalam, M., Manickam, R., Tang, K.L., Simons,
A.M., Wu, H.-H., Mayden, R.R., Miya, M., 2012c. Phylogenetic placements of the
barbin genera Discherodontus,Chagunius, and Hypselobarbus in the subfamily
Cyprininae (Teleostei: Cypriniformes) and their relationships with other
barbins. Zootaxa 3586, 26–40.
Yue, P.Q. et al. (Eds.), 2000. Fauna Sinica. Osteichthyes: Cypriniformes III. Science
Press, Beijing, pp. 1–654.
Zhou, W., Cui, G.H., 1996. A review of Tor species from the Lancangjiang River
(Upper Mekong River), China (Teleostei: Cyprinidae). Ichthyol. Explor.
Freshwater 7, 131–142.
116 L. Yang et al. / Molecular Phylogenetics and Evolution 85 (2015) 97–116
Author's Personal Copy