Molecular Phylogenetics and Evolution 51 (2009) 405–411

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Molecular Phylogenetics and Evolution

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Short Communication

Population genetic structure and geographical differentiation of the Chinese catfish

Hemibagrus macropterus (Siluriformes, Bagridae): Evidence for altered
drainage patterns
Lei Yang a,b, Richard L. Mayden a, Shunping He b,*
a
Department of Biology, Saint Louis University, Saint Louis, MO 63103-2010, USA
b
Laboratory of Fish Phylogenetics and Biogeography, Institute of Hydrobiology, Chinese Academy of Sciences, 7# Donghu South Road, Wuhan, Hubei 430072, China

a r t i c l e i n f o a b s t r a c t

Article history:
Received 8 September 2008 Ó 2009 Published by Elsevier Inc.
Revised 7 January 2009
Accepted 10 January 2009
Available online 19 January 2009

Keywords:
Analysis of molecular variance (AMOVA)
Basin connection
Changjiang
China
Nested clade analysis (NCA)
Range expansion

1. Introduction Three Gorges separate many fast-flowing rivers and streams of

Primary freshwater fishes provide excellent opportunities for
studying biogeographic patterns. Their distributions are restricted
to lake and river drainage systems, so their evolution and disper-
sion are closely related to paleogeography. Historical basin connec-
tion events resulting from the geological activities may shape the
genetic structure of fish populations in associated basins (e.g. Ber-
mingham and Martin, 1998; Durand et al., 1999; Pfrender et al.,
2004).
The geomorphology of China has been altered extensively and
the trajectories of main drainages have changed greatly since the
late Pliocene (Chinese Academy of Sciences, 1980; Clark et al.,
2004). Up until now, relatively few biogeographical studies have
been conducted over the vast area south of Changjiang (Yangtze)
River and east of the Red River (e.g. Perdices and Coelho, 2006;
Yang and He, 2008), although several important drainages of China
such as the Changjiang, Zhujiang (Pearl), Minjiang and Hanjiang
Rivers and numerous small rivers and streams flow through this
area.
The Changjiang River, the largest river in China and the third
longest river in the world, has more than 3000 tributaries and
4000 lakes (Zeng, 1990). It is usually divided into three sections:
the upper reaches, the middle reaches and the lower reaches. The
* Corresponding author. Fax: +86 27 68780123.
E-mail addresses: leiyangslu@gmail.com (L. Yang), clad@ihb.ac.cn (S. He).
the upper reaches from the rest of the Changjiang River, which
has a completely different character (Clark et al., 2004). The differ-
ences between the upper Changjiang River and the middle and
lower Changjiang River may be partially because they were once
two independent rivers. The upper Changjiang River once drained
into the South China Sea through the Paleo-Red River until it was
captured by the middle–lower Changjiang River and reversed its
route to flow into the East China Sea prior to or coeval with the ini-
tiation of Miocene (see Clark et al., 2004, notice different divisions).
In the middle and lower reaches, many tributaries flow into the
main river channel and numerous lakes interlace with them and
form a complete riverine–lacustrine network (Fu et al., 2003).
Therefore, the Changjiang River has a very complex and diverse
range of habitats and harbors an extremely high fish biodiversity
(Fu et al., 2003). Many tributaries of the upper and middle Chang-
jiang River are geographically very close to that of the Zhujiang
(Fig. 1). The lower reaches of the Changjiang River and the drain-
ages of southeast China are also very close geographically
(Fig. 1). The historical relationships of these drainages and possible
influence on fish distribution patterns are thus very interesting.
Ren and Han (1959) have proposed that the upper reaches (the
Jinshajiang River) of the Changjiang River were once connected
with the upper reaches (the Beipanjiang River) of the Zhujiang Riv-
er in the northwestern area of Yunnan Province based on geologi-
cal studies. Liu (1998) studied the biogeographical process of three
East Asian freshwater fish groups, the sinipercine fishes, the

1055-7903/$ - see front matter Ó 2009 Published by Elsevier Inc.

doi:10.1016/j.ympev.2009.01.004
406 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411

Fig. 1. Map showing the 12 sampling sites of Hemibagrus macropterus (see Table 1 for details). Dotted lines indicate the boundaries of the three river drainages studied, the
Changjiang (Yangtze) River, the Zhujiang (Pearl) River and the Hanjiang River. Unrelated drainages were not shown. Shaded areas show the most probably genetic structures
of H. macropterus populations inferred based on the analysis of molecular variance (AMOVA) (see Table 2).

gobiobotiine fishes and the pseudogobionine fishes, and revealed
that the Changjiang River and the Zhujiang River were closely re-
lated. Based on the biogeographic study of the cyprinid genus
Sinilabeo, Zhang (1999) suggested that the upper reaches of these
two rivers were once connected. Yap (2002) also supported this
view, based on the phenogram built from the distribution of 21
common fish genera. The possible connection of the upper Chang-
jiang River and the upper Zhujiang River (Ren and Han, 1959) may
have left imprints in the genetic structure of some freshwater
fishes distributed across both of these river basins. Li and Zheng
(1998) studied the fish fauna of the Hanjiang River System and pro-
posed that some fish taxa have migrated from the north (the lower
reaches of the Changjiang River System) before the early Pleisto-
cene. At the end of the early Pleistocene, the Wuyishan Mountains
rose up more than 200 m and thus prevented the migration of
fishes from the Changjiang River System. This possible connection
of the lower Changjiang River and the Hanjiang River as hypothe-
sized by Li and Zheng (1998) may also manifest at the population
(infraspecific) level for some of these fishes.
Hemibagrus macropterus (Bleeker, 1870), which is endemic to
Mainland China, is a moderate-sized, bottom-dwelling freshwater
catfish of the family Bagridae. It inhabits clear, swiftly flowing riv-
ers with gravel bottoms and mainly feeds on small fishes, mollusks
and other invertebrates (Chu et al., 1999). It is distributed in the
Changjiang, Zhujiang, Hanjiang and Jiulongjiang Rivers, with the
majority of diversity found in the Changjiang (Chu et al., 1999).
The objectives of this study are: (1) to investigate the genetic
structure and geographical differentiation of H. macropterus popu-
lations throughout its distribution range; (2) to infer historical pro-
cesses or events, especially basin connection events, that may have
shaped its phylogeographical pattern. The possible historical basin
connection events as stated in the hypotheses of Ren and Han
(1959) and Li and Zheng (1998) will be tested using the genetic
structure of H. macropterus.
 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411 407

Table 1
Sampling information, and haplotypes found in 12 populations of Hemibagrus macropterus and corresponding lineages/sub-lineages and nested clades.

Population Lineage A (Clade 3-1) Lineage B (Clade 1-7) Lineage C (Clade 1-8)
a1 (Clade 2-1 & Clade 1-4) a2 (Clade 1-5) a3 (Clade 1-6)
YS1 YS2 LP HC1 HC2 HC3 MD JK TY2 TY3 CX2 TY1 CX1 CX3 SR WY1 WY2 WY4 WY3 CT
Zhujiang River
Yangshuo (YS, 19/3/1) 16 1 2
Zhaoping (ZP, 7/2/0) 6 1
Lipu (LP, 8/2/1) 5 3
Upper Changjiang River
Nanchong (NC, 1/1/0) 1
Hechuan (HC, 15/3/0) 9 5 1
Mudong (MD, 7/3/1) 5 1 1
Middle Changjiang River
Jinkou (JK, 4/2/1) 3 1
Chenxi (CX, 4/3/1) 1 2 1
Taoyuan (TY, 12/6/3) 1 3 1 2 4 1
Lower Changjiang River
Shangrao (SR, 11/3/1) 1 4 6
Wuyuan (WY, 4/4/2) 1 1 1 1
Hanjiang River
Changting (CT, 7/2/1) 5 2
Total (99) 27 1 3 18 8 2 1 1 1 3 1 1 4 5 1 1 6 1 12 2

Numbers in parentheses following locality abbreviation show total numbers of individuals, different and unique haplotypes, respectively. Lineages A, B and C are main clades
recovered in the phylogenetic trees (Fig. 2A and B) and a1, a2 and a3 are sub-lineages found within lineage A. Clades denote the nested clades in Fig. 2C.

2. Materials and methods
2.1. Sampling
A total of 96 specimens of H. macropterus were collected from
12 localities covering most of its distribution range (Table 1,
Fig. 1). As a part of the sampling plan for H. macropterus and its sis-
ter species Hemibagrus guttatus (Ku et al., 2007; Yang and He,
2008), more sampling localities have actually been chosen across
the entire Zhujiang River, the Hanjiang River and the Jiulongjiang
River. But the sampling of H. macropterus in these areas resulted
in a failure, possibly partially due to environmental change and
the existence of another very abundant species, H. guttatus, whose
primary distribution area is in these southern rivers and has simi-
lar ecological requirements as H. macropterus (Chu et al., 1999). All
tissues for genomic DNA extraction were preserved in 75–95% eth-
anol and deposited in the collection of the Freshwater Fish Mu-
seum of The Institute of Hydrobiology, Chinese Academy of
Sciences. Three sequences were retrieved from GenBank under
the Accession Nos. AF416890, AF430373 and AF430374. These
three specimens were sampled from Mudong (Chongqing Munici-
pality), Wuyuan (Jiangxi Province) and Yangshuo (Guangxi Auton-
omous Region), respectively. Hemibagrus nemurus (Valenciennes,
1840) (AF499600) was selected as outgroup in our phylogenetic
analyses according to the bagrid phylogeny (Ku et al., 2007).
2.2. DNA extraction, amplification and sequencing
Total genomic DNA was extracted from a piece of muscle tissue
following the procedure of Ausubel et al. (1992). Polymerase chain
reaction (PCR) was used to amplify the sequence of the mitochon-
drial cytochrome b gene. The primers L14724 (50 -GAC TTG AAA
AAC CAC CGT TG-30 ) and H15915 (50 -CTC CGA TCT CCG GAT TAC
AAG AC-30 ) were adapted from Xiao et al. (2001). Amplified DNA
was fractionated by electrophoresis through 0.8% low-melting aga-
rose gels and purified using BioStar Glassmilk DNA purification Kit
according to manufacturer’s instructions. The purified PCR product
was sequenced using the same PCR primers with the Perkin-Elmer
BigDye DNA Sequencing Kit according to the manufacturer’s proto-
col. All sequences (each 1103 bp in length) have been deposited in
GenBank under the Accession Nos. AY907572–AY907667.
2.3. Phylogenetic analyses
Multiple alignment of sequences was performed using CLUSTAL
X (Thompson et al., 1997), SEAVIEW alignment editor (Galtier
et al., 1996) and verified by eye. All sequences were translated into
amino acids to check the occurrence of stop codons within the se-
quences, which might imply that pseudogenes had been amplified.
The complete data set was analyzed using neighbor joining (NJ),
maximum parsimony (MP) and maximum likelihood (ML) ap-
proaches in PAUP v. 4.0b10 (Swofford, 2002). ModelTest v.3.7 (Po-
sada and Crandall, 1998) was used to find the best model of
evolution of the complete dataset. The model selected by hLRT
was HKY + G (0.2443). In the NJ analysis, the corrected distance
HKY85 + G (0.2443) was adopted. MP analysis was conducted
using heuristic searches, TBR branch swapping and 100 replicates
of random addition of taxa. For ML analysis, heuristic searches,
TBR branch swapping was also employed and the number of ran-
dom addition replicates was set as 10. Bootstrap analysis (1000
replicates) was used to assess the relative robustness of branches
of MP and ML trees.
Bayesian analyses were also performed using MrBayes 3.1.2
(Huelsenbeck and Ronquist, 2001). Four simultaneous Markov
chains were run for 1,000,000 generations, with trees being sam-
pled every 100 generations for a total of 10,000 trees in the initial
sample. Two independent analyses were conducted.
2.4. Population genetic analyses
An analysis of molecular variance (AMOVA) (Excoffier et al.,
2000) was performed to assess the genetic structure and differen-
tiation of H. macropterus populations using ARLEQUIN 2.000
(Schneider et al., 2000). Due to small sampling size (n = 1), the pop-
ulation Nanchong (NC) of the upper Changjiang was analyzed to-
gether with its geographically closest population which is
Hechuan (HC). More than 20 hierarchical arrangements of popula-
tions were tested. The arrangement that maximized hCT, the among
408 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411

group variation, and was statistically significant, was assumed to
be the most plausible geographical subdivision. Genetic differenti-
ation between populations was assessed by comparing average
number of pairwise differences (uncorrected P distance) between
populations (PiXY), average number of pairwise differences within
populations (PiX and PiY) and corrected average pairwise differ-
ence (PiXY  (PiX + PiY)/2) using ARLEQUIN.
2.5. Nested clade analysis (NCA)
Nested clade analyses (Templeton et al., 1995) were also con-
ducted. The haplotype network was constructed using TCS
ver1.2.1 (Clement et al., 2000) and was used to define an evolution-
ary clade hierarchy. Then the Clade distance (Dc) which measures
the geographical range of a given clade, and nested clade distance
(Dn) which measures how this clade is geographically distributed
relative to its closest evolutionary sister clades, were calculated.
The statistical significance level of the association of phylogenetic
and geographical positions was assessed with a permutational con-
tingency test (10,000 resamples) using Geodis 2.5 (Posada et al.,
2000). When the null hypothesis (i.e. random geographical distri-
bution of all clades or haplotypes within a nested clade) is rejected,
the biological causes for the haplotype-geography associations
were inferred using the latest inference keys (December 2008
version).
Both River distances and Geographic (great circle) distances
were employed in the nested clade analyses. River distances were
determined by measuring along river courses to connect sampling
localities using tools implemented in Google Earth version 4.2
(http://earth.google.com). The river distances between localities
of the upper Changjiang River and the Zhujiang River were mea-
sured according to the hypothesis of Ren and Han (1959) and along
the most parsimonious course. The river distances between locali-
ties of the lower Changjiang River and the Hanjiang River were
determined by tracing the most parsimonious river course across
the Wuyishan Mountains to connect their tributaries following
the hypothesis of Li and Zheng (1998). In the measuring of river
distances from the Zhujiang River populations to the middle
Changjiang River populations, we generally followed the course
of the artificial Lingqu Canal, which was dug over 2000 years ago
to connect the Guijiang River (Zhujiang) and the Xiangjiang River
(Changjiang). Since so many hypotheses were involved in the mea-
suring of River distances, Geographic distances were also used to
conduct an independent NCA as was suggested by Fetzner and
Crandall (2003) that cross-drainage migration of individuals may
be highlighted by the comparison of results from both analyses.
Geographic distances were determined directly from latitudinal
and longitudinal coordinates.
2.6. Demographic analyses
Mismatch distributions (i.e. the frequency distribution of pair-
wise differences among all haplotypes in a sample) were per-
formed on some nested clades to infer their demographic history
using ARLEQUIN. Given the fact that mismatch distributions have
been found to be very conservative (Ramos-Onsins and Rozas,
2002), Fu’s (1997) Fs test was performed by comparing Fs against
a distribution generated from 10,000 random samples under the
hypothesis of selective neutrality and population equilibrium
using ARLEQUIN. Fs tend to be negative under an excess of recent
mutations and a significant negative value will be taken as evi-
dence of population growth. We also obtained maximum likeli-
hood estimates of ancestral population parameters using the
Metropolis-Hastings sampling procedure as implemented in the
program FLUCTUATE 1.4 (Kuhner et al., 1998). This coalescent-
based approach produces a likelihood surface and maximum like-
lihood estimates of effective population size (H = 2lNf) and expo-
nential growth rate (g). We set the transition–transversion ration
to 20, as there was no transversion in our datasets. The base fre-
quencies were obtained from likelihood ratio tests performed with
the ModelTest v.3.7 (Posada and Crandall, 1998). Stability of the
parameter estimation was ensured by conducting 100 short Mar-
kov Chain Monte Carlo (MCMC) chains of 2000 steps each and 10
long chains of length 500,000 steps with a sampling increment of
20, and one independent re-run. The relationship s = 2ut (Rogers
and Harpending, 1992) was then used to estimate an approximate
time of most recent range expansion in generations (t) for H. macr-
opterus populations, where s is the model of the mismatch distri-
butions measured in units of evolutionary time and u is the
mutation rate per sequence and per generation. The value u was
calculated from the formula u = 2lk, where l is the mutation rate
per nucleotide and k is the number of nucleotides of the analyzed
fragment. Ku et al. (unpublished data) estimated that the evolu-
tionary rate of mitochondrial cyt b of East Asian bagrids was
approximately 0.5–0.7%/Ma, based on the bagrid fossil record from
China (Cheng, 1962) and the phylogeny of the East Asian bagrids
(Ku et al., 2007). This rate was used in the dating of population
expansion events. A generation time of around 4 years for

Table 2
Hierarchical AMOVA (Analysis of molecular variance) of Hemibagrus macropterus populations. Groups represent some of the rearrangements tested in the hierarchical analysis.
The number of groups is shown in front of each grouping. Fixation indexes: total variance hST; among group variance hCT, and hSC intra-group variation among populations. The
maximum hCT is highlighted in bold.

Groups hST hSC hCT

*
1 [All populations] 0.40748
2 [YS, ZP, LP, HC&NC, MD, JK, CX, TY]; [SR, WY, CT] 0.88840* 0.57331* 0.73846
3 [YS, ZP, LP]; [NC&HC, MD, JK, CX, TY, SR, WY]; [CT] 0.81760* 0.75567* 0.25348
3 [YS, ZP, LP, NC&HC, MD]; [JK, CX, TY]; [SR, WY, CT] 0.85079* 0.36704* 0.76427*
3 [YS, ZP, LP, NC&HC, MD, JK]; [CX, TY]; [SR, WY, CT] 0.86140* 0.20695 0.82523*
4 [YS, ZP, LP, NC&HC, MD, JK]; [CX, TY]; [SR, WY]; [CT] 0.85797* 0.14511 0.83386*
4 [YS, ZP, LP, NC&HC, MD]; [JK], [CX, TY]; [SR, WY, CT] 0.85175* 0.20853* 0.81269*
4 [YS, ZP, LP, JK, CX, TY]; [HC&NC, MD]; [SR, WY]; [CT] 0.83298* 0.59084* 0.59179
4 [YS, ZP, LP]; [HC&NC, MD, JK, CX, TY]; [SR, WY]; [CT] 0.82833* 0.54743* 0.62067
4 [YS, ZP, LP]; [HC&NC, MD]; [JK, CX, TY, SR, WY]; [CT] 0.81445* 0.68876* 0.40383
4 [YS, ZP, LP]; [HC&NC, MD, JK]; [CX, TY]; [SR, WY, CT] 0.83498* 0.13388 0.79793*
4 [YS, ZP, LP, HC&NC, MD]; [JK, TY]; [CX]; [SR, WY, CT] 0.84816* 0.36958* 0.75914*
5 [YS, ZP, LP]; [HC&NC, MD, JK]; [CX, TY]; [SR, WY]; [CT] 0.82165* 0.02075 0.81787*
5 [YS, ZP, LP, NC&HC, MD]; [JK]; [CX]; [TY]; [SR, WY, CT] 0.84952* 0.22226* 0.80652*
5 [YS, ZP, LP]; [NC&HC, MD]; [JK, CX, TY]; [SR, WY]; [CT] 0.81798* 0.33367 0.72684
6 [YS, ZP, LP]; [NC&HC, MD]; [JK]; [CX, TY]; [SR, WY]; [CT] 0.81807* 0.01319* 0.81564*
*
Significant at P < 0.001 after 10,100 permutations.
 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411 409

HC1
HC3
HC2
MD
78 (84) LP
98 a1 YS2
a1
TY1

Lineage A YS1
88 (78) JK
89 TY2 Lineage A
a2 a2
CX2
CX3
93 (83)
TY3
98 a3 a3
CX1

Lineage B SR
87 (84) WY4
Lineage B
91 WY2
WY1
85 (66) WY3
87 CT
Lineage C Li
Lineage C
H. nemurus

0.005 substitutions/site

A B

HC2 JK

HC3 YS1 CX1

HC1

MD CX2
TX3
1-4 1-5
LP

YS2
S 1-1
1 1 CX3
1-6
TY2
1-2
TY1 1-3
2-1
31
3-1

WY1 WY3
WY4 CT

WY2
1-8
SR 1-7
25
2-5

C
Fig. 2. (A) Strict consensus tree of 18 equally parsimonious trees (L = 234). Numbers above branches indicate the bootstrap values (1000 replicates) from the maximum
parsimony (MP) analysis and maximum likelihood (ML) analysis (in parentheses). Only those higher than 50% are shown. The MP bootstrap consensus tree also supports the
clade (sub-lineages a1 + a2) at 54%, and the ML bootstrap consensus tree also supports lineages A and B to be sister groups at 54%. Numbers below branches show the
posterior possibility values of the Bayesian analysis that are greater than 80% (0.80). (B) Neighbor-joining tree. Haplotypes are named as in Table 1. Hemibagrus nemurus
(AF499600) was included as an outgroup. Groups a1, a2 and a3 denote sub-lineages revealed within lineage A. (C) The haplotype network and nested clade design for the
mitochondrial cyt b haplotypes of H. macropterus. Haplotypes are named as in Table 1. Haplotype labels: black, the upper Changjiang River populations; gray, the Zhujiang
River populations; white, the middle Changjiang River populations; dark upward diagonals, the lower Changjiang River populations; and gray stipple, the Hanjiang River
population. Circle size represents haplotype frequency. Each solid line represents a mutational change that interconnects two haplotypes that has a probability greater than
95% level. The dashed line represents more than 14 mutational steps between two main groups. Missing intermediate haplotypes are indicated by little black dots.

H. macropterus (Wang and Luo, 1992) was used to translate gener-
ations into years.
3. Results and discussion
As is revealed by AMOVA, Hemibagrus macropterus of the upper
Changjiang River populations and the Zhujiang River populations
are closely related to each other, while the genetic divergence
among populations of the three parts of the Changjiang River
(upper, middle and lower) is significant (Table 2). This result is fur-
ther supported by the corrected average number of differences be-
tween these populations. The number of differences between the
Zhujiang River populations and the upper Changjiang River popu-
lations is only 0.67, whereas this value ranges from 4.29 to 15.88
410 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411
among the three population groups within the Changjiang River.
The same conclusion can be drawn from the topology of phyloge-
netic trees and haplotype networks (Fig. 2). The haplotypes of the
Zhujiang River populations (YS1, YS2, LP and HC1) are only con-
fined to the sub-lineage a1 or clade 2-1, while the Changjiang River
haplotypes (HC1-3, MD, JK, TY1-3, CX1-3, SR and WY1-4) can be
found across all major lineages or clades.
The close relationship of H. macropterus populations of the
upper Changjiang River and the Zhujiang River is congruent with
the hypothesis of Ren and Han (1959). Intense tectonic activity
in this region from the late Pliocene might account for the connec-
tion of rivers of the upper Changjiang River and the upper Zhujiang
River (Chinese Academy of Sciences, 1980; Clark et al., 2004). The
characteristics of the Changjiang River and the migration ability
of H. macropterus may have contributed to the formation of the ge-
netic pattern of the Changjiang River populations. The Changjiang
River is an enormous river, which includes many tributaries and
lakes with complex and diverse habitats (Fu et al., 2003). As many
as 361 species have been recorded from this river, of which 117 are
endemic (Fu et al., 2003). The characteristics of the Changjiang Riv-
er that lead to its enormous diversity at the species level may also
manifest at the population level of many species. As is shown in
Table 1, most of the haplotypes of H. macropterus were found with-
in the Changjiang River. The two big nested clades (3-1 and 2-5)
that clearly show a west–east division stimulate us to relate it with
the hypothesis that the upper Changjiang River and the other two
parts of the Changjiang River were once two independent rivers
(Clark et al., 2004). But we certainly need more data to test it.
Hemibagrus macropterus usually inhabits river rapids with gravel
bottoms and the numerous lakes (e.g. the Dongtinghu Lake and
the Poyanghu Lake) of the middle and lower Changjiang River
might serve as an impediment to dispersal (Perdices et al., 2004).
For the upper and middle Changjiang River populations, geograph-
ical barriers, such as the Three Gorges, may restrict gene flow be-
tween them (Xiao et al., 2001; Wang et al., 2004). Xiao et al.
(2001) suggested that populations of Xenocypris argentea of the
upper and middle reaches of the Changjiang River were genetically
separated according to their geographical distributions; Perdices
et al. (2004) examined the genetic structure of Zacco platypus pop-
ulations from six main tributaries of the upper and middle Chang-
jiang River and suggested that these populations might correspond
to four different species. Berrebi et al. (2005) also agreed with this
view. However, the isolation among populations of H. macropterus
of the Changjiang River is not clear-cut. Gene flow may still exist
between the populations of the upper and middle reaches. This is
mirrored by the existence of a shared haplotype (HC2) between
them. There is also a haplotype (WY2) that is shared between pop-
ulations of the middle and lower Changjiang River. The historical
overflow of rivers in the eastern plain of China since the early Qua-
ternary may have facilitated gene flow between them (Chen et al.,
1986). Perdices et al. (2005) showed that all samples of Opsariich-
thys bidens in the tributaries of the upper and middle Changjiang
River were genetically homogeneous. The genetic structure of H.
macropterus appears to be intermediate between that of Z. platypus
and O. bidens. The distinct genetic structures of these three species
in this region might reflect their differential migratory abilities.
As can be seen from the haplotype networks, some H. macropte-
rus haplotypes of the middle Changjiang River are more closely re-
lated to that of the Zhujiang River populations than the other
populations. In Clade 1-2, two haplotypes JK (1 individual from Jin-
kou) and TY2 (1 individual from Taoyuan) from the middle Chang-
jiang River are connected with YS1 (shared by 27 individuals from
the Zhujiang River) by only one step, respectively (Fig. 2C). A his-
torical connection may also have existed between the tributaries
of the Zhujiang River and the middle Changjiang River. This con-
nection may also have been created by the intense tectonic activity
from the late Pliocene (Chinese Academy of Sciences, 1980). Based
on their study of the phylogeographic structure of Z. platypus and
O. bidens, Berrebi et al. (2005, 2006) have concluded that the
south-middle tributaries of the Changjiang were once connected
with the north-middle tributaries of the Zhujiang. This view was
also supported by the studies of Perdices et al. (2005) and Perdices
and Coelho (2006). It has been noted that the Lingqu Canal may
have promoted fish exchange between the middle Changjiang Riv-
er and the Zhujiang River (e.g. Berrebi et al., 2006; Perdices and
Coelho, 2006). The existence of this Canal might have also pro-
moted the exchange of H. macropterus between these two drain-
ages. It is possible that some haplotypes evolved in one drainage
but some of these individuals (e.g. the two middle Changjiang sam-
ples in clade 1-2) dispersed to the other drainage in a relatively re-
cent time.
Hemibagrus macropterus of the lower Changjiang populations
and the Hanjiang population are also closely related to each other.
The haplotype WY3 is shared by seven individuals from the lower
Changjiang River populations and five individuals from the Hanji-
ang River population. In the phylogenetic trees, the haplotype CT
(2 individuals), which is unique to the Hanjiang population, is clus-
tered with the haplotype WY3 and they form the sub-lineage a3
(Fig. 2A and B). In the Clade 1-8 of the haplotype networks, the
haplotype CT is connected with the haplotype WY3 by only one
step (Fig. 2C). The corrected average pairwise differences between
the lower Changjiang and Hanjiang populations are also signifi-
cantly low (1.65). It was probably caused by a very recent (proba-
bly late Pleistocene) connection between the lower Changjiang
River and the upper Hanjiang River, although we can not exclude
the possibility of human-mediated movement. Our results gener-
ally agree with the hypothesis of Li and Zheng (1998) on the prob-
able connection of river basins, but are not congruent with the
time of the connection, which they hypothesized was the early
Pleistocene.
Although NCA based on River distances failed to identify spe-
cific historical events for Clade 2-1, NCA based on Geographic dis-
tances, Mismatch distribution analysis (unimodal, s = 1.197), Fu’s
Fs test (Fs = 3.355, P = 0.037) and the coalescence-based analysis
(g = 3669.32 ± 420.86; ln L = 32.20) all revealed a range expansion
for H. macropterus populations nested in Clade 2-1. This clade in-
cludes all the haplotypes from the Zhujiang River (YS1, YS2 and
LP) and the upper Changjiang River (HC1-3 and MD) and several
haplotypes from the middle Changjiang River (JK, TY1 and TY2)
(Table 1). Range expansion in this vast area was made possible
by the connection of the upper Changjiang River and the Zhujiang
River and also the Zhujiang River and the middle Changjiang River
(as discussed above). The time of the range expansion event was
dated back to approximately 77,000–109,000 years, which falls
into the late Pleistocene.
Acknowledgments
We are grateful to Zuogang Peng, Xuzhen Wang, Xianguang
Guo, Junbing Li, Haipeng Zhao, Zhijian Wang, Qing Xue, Yanwei
Ding, Qiuling Li, Yawei Han, Jinquan Yang, Qiongying Tang and Hai-
tao Zhao for assistances in collecting specimens. Dr. Huanzhang Liu
generously provided some specimens. We thank Xiying Ku for pro-
viding her unpublished data on evolutionary rate. We would also
like to thank those people who have helped during the early stage
of this paper, Drs. Zuogang Peng, Xianguang Guo, Huanzhang Liu,
Hungdu Lin (National Cheng Kung University) and Carl Smith (Uni-
versity of Leicester, UK). Lijun He is thanked for her help in making
figures. We are also grateful to Kevin Conway, Dr. Kevin Tang, Dr.
Casey Dillman and Dr. Wei-Jen Chen of Saint Louis University for
their valuable comments on the paper and help with language. Fi-
nally, the authors thank the two anonymous reviewers for their
 L. Yang et al. / Molecular Phylogenetics and Evolution 51 (2009) 405–411
very insightful comments. This research was supported by the
Grants from National Natural Science Foundation of China (NSFC)
2007CB411600 and 30530120 to S.H.
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