High level population differentiation of finless porpoises(Neophocaena phocaenoides) in Chinese waters revealedby sequence variability of four nuclear introns

Jianfeg Ju • Mei Yang • Shixia Xu •

Kaiya Zhou • Guang Yang

Received: 2 November 2011 / Accepted: 15 February 2012 / Published online: 15 March 2012

� Springer Science+Business Media B.V. 2012

Abstract In the present study, sequence variations at four

nuclear introns which were respectively from the parathy-

roid hormone-like (PTH) gene, isolate Pdalz1692 inter-

feron (IFN) gene, peripherin-like (RDS) gene, and tyrosine

kinase receptor-like (KIT) gene, were examined to analyze

genetic diversity and population structure of the finless

porpoise (Neophocaena phocaenoides) in Chinese waters.

High among-population differentiation was revealed, with

a significant genetic structure between populations (PTH:

FST = 0.29, P \ 0.001; IFN1@: FST = 0.23, P \ 0.001;

RDS: FST = 0.12, P \ 0.001; KIT: FST = 0.16, P \0.001) shown by the analysis of molecular variance.

Although common haplotypes accounted for more than one

half of all samples examined, many haplotypes were found

to be population-specific. The Tajima’s D, Fu’s tests and

mismatch distributions all suggested a recent colonization

and population expansion of finless porpoises in Chinese

waters. In view of special reference to the conservation

priority of the Yangtze finless porpoises, special protection

measures must be taken urgently for this population.

Keywords Finless porpoise � Nuclear intron � Genetic

diversity � Population structure � Conservation

Introduction

The finless porpoise (Neophocaena phocaenoides) inhabits

shallow and often partially enclosed marine waters along

coastlines of tropical and temperate Asia [1–3]. It has long

been accepted that the genus Neophocaena is monospecific

[4]. Based on obvious external differentiation [5] and

stepwise discriminant analysis of skeletal morphology [6,

7], three different geographical populations (subspecies)

were identified for finless porpoises in Chinese waters: the

south China Sea population (N. p. phocaenoides) distrib-

uted in the south China Sea and southern east China Sea,

the Yellow sea population (N. p. sunameri) distributed in

the Yellow-Bohai sea and the northern east China sea, and

the unique Yangtze river population (N. p. asiaeorientalis,

with a common name of ‘Yangtze finless porpoise’)

endemic to the middle and lower reaches of the Yangtze

river [8]. Jefferson [9] based on cranial morphology sug-

gested that the ‘‘wide’’ (phocaenoides) and ‘‘narrow’’

(asiaeorientalis and sunameri) forms were differentiated

enough to warrant separate species status, which is con-

gruent with a recent study using morphological characters

and molecular markers [10]. However, no data is available

to support further sub-division of the narrow form into the

asiaeorientalis and sunameri subspecies. Besides the tax-

onomic inconsistency, this coastal species has been under

threats from various anthropogenic factors such as gillnets

and habitat destruction, over the past years [1, 2].

Jianfeg Ju and Mei Yang equally contributed to this study.

J. Ju � M. Yang � S. Xu � K. Zhou � G. Yang (&)

Jiangsu Key Laboratory for Biodiversity and Biotechnology,

College of Life Sciences, Nanjing Normal University, Nanjing

210046, China

e-mail: gyang@njnu.edu.cn

J. Ju

e-mail: jujianfeng@njnu.edu.cn

M. Yang

e-mail: yangmei200801@163.com

S. Xu

e-mail: xushixia78@163.com

K. Zhou

e-mail: kyzhounj@126.com

123

Mol Biol Rep (2012) 39:7755–7762

DOI 10.1007/s11033-012-1614-z

Especially, the Yangtze finless porpoise has been catego-

rized as ‘endangered’ in the IUCN Red List [11].

In order to complement conservation measures for this

species, it is prerequisite to develop a comprehensive

population genetic analysis. Previous studies were mainly

used mitochondrial and microsatellite data, indicated that

the finless porpoise with a high level of among-population

genetic structure and to follow relatively low level of

within-population genetic diversity [12–16].

The intervening introns are presumed to be under low or

no selection pressure and therefore potentially more vari-

able than the exonic regions. The application of nuclear

intron sequences, therefore, would be helpful to provide a

better understanding of ecology and population biology

relevant to conservation issues of the finless porpoise. In

this study, we surveyed four nuclear DNA (nDNA) introns,

which were respectively from parathyroid hormone-like

gene (PTH), isolate Pdalz1692 interferon gene (IFN),

peripherin-like gene (RDS) and tyrosine kinase receptor-

like gene (KIT), to examine the genetic diversity and

population structure of finless porpoises in Chinese waters.

We aimed to: (i) determine the level of population differ-

entiation and genetic structure of finless porpoises, (ii)

address their phylogeographic pattern and population his-

tory, and (iii) recommend conservation and management

measures for the Yangtze finless porpoise based upon

genetic information obtained in this study in combination

with those revealed previously.

Materials and methods

Sampling

A total of 144 samples of muscle or skeleton were collected

from incidentally killed individuals in China coastal waters

and Yangtze river (Fig. 1; Table 1). These samples were

a priori assigned to the aforementioned three populations

Fig. 1 Schematic map showing

the sampling localities of finless

porpoises examined in this

study, with sampling size for

each sampling locality in the

parenthesis. Sampling localities

abbreviations are as follows: the

south China sea population

(inverted filled triangle): PT, DS

and BH; the Yellow sea

population units (filled triangle)

LS, ZS and NB; the Yangtze

river population (filled circle):

HK, CZ, WH, CMD, YX, YZ,

ZJ and NJ. See Table 1 for

abbreviations of sampling

localities

7756 Mol Biol Rep (2012) 39:7755–7762

123

primarily according to their sampling localities as shown in

Fig. 1. Voucher specimens are preserved in the Jiangsu

Key Laboratory for Biodiversity and Biotechnology, Col-

lege of Life Sciences, Nanjing Normal University, China.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from 0.1 to 0.2 g of 124

muscle samples using standard overnight proteinase K

digestion followed by phenol–chloroform extraction and

ethanol precipitation [17]. In order to avoid crosslink

contaminated external DNA, the additional 20 skeletal

samples from the Yangtze river were washed using 5%

H2O2 first, then used double distilled water to rinsed, and

dried at 67�C, finally irradiated under ultraviolet for

30 min. Following ground the samples into powder and

transferred into an Eppendorf tube of 2 ml to extract DNA

using CTAB method [18]. The extracted DNA was detec-

ted by gel electrophoresis before being used as template

DNA for amplification.

Four introns were amplified using the primers identified in

Lyons et al. [19] (Table 2). The polymerase chain reactions

(PCRs) were carried out in a total volume of 30 ll containing

2.0 mM MgCl2, 10 mM Tris–HCl (pH 8.4), 50 mM KCl,

0.2 mM each dNTP, 0.3 lM each primer, 2 unit Ex-Taq

DNA polymerase (Takara, Japan), and 100 ng DNA tem-

plate. For PTH, IFN1@ and KIT loci, the touchdown PCR

cycling scheme included an initial denaturation of 2 min at

94�C; first cycle (94�C, 1 min; decreasing annealing tem-

perature from 64 to 48�C of -1�C per cycle, 30 s; 72�C,

30 s) 16 times; followed by a second cycle (94�C, 1 min;

48�C, 30 s; 72�C, 30 s) 20 times; and a final extension at

72�C for 4 min. For the RDS locus, the touchdown PCR

cycling scheme included an initial denaturation of 10 min at

95�C; first cycle (95�C, 30 s; decreasing annealing temper-

ature from 60 to 50�C of -0.5�C per cycle, 1 min; 72�C,

1 min) 20 times; followed by a second cycle (95�C, 30 s;

50�C, 1 min; 72�C, 1.5 min) 20 times; and a final extension

at 72�C for 10 min. The PCR-products were purified using

Wizard PCR Preps DNA purification kit (Promega, USA)

according to the manufacturer’s instruction.

For the skeletal samples from the Yangtze river popu-

lation, the purified PCR-products were cloned into the

pMD-18T vectors (Takara, Japan). PCR-products or clones

were chosen from each individual for direct sequencing, in

the forward and/or reverse directions using the BigDye

terminator cycle sequencing ready reaction kit (ABI) on an

ABI 3730 automated genetic analyzer.

Data analysis

Sequences were aligned with Clustal X version 1.83 using

the multiple alignment default parameters, and compared

by eye to establish the optimal alignment [20]. The number

of polymorphic sites (S), nucleotide diversity (p), haplo-

type diversity (h), and the average number of nucleotide

differences between sequences (K) were estimated for each

population and overall populations using the DnaSP ver-

sion 5.0 [21]. An analysis of molecular variance (AMOVA)

[22, 23] was performed in Arlequin 3.11 to determine the

Table 1 Summary list of samples used in this study

Population Sampling site

South China sea Pingtan (PT, 40), Dongshan (DS, 18), Fujian

Province

(SS, 60) Beihai (BH, 2), Guangxi Zhuang autonomous

region

Yellow sea (YS,

50)

Lvsi (LS, 23), Jiangsu Province

Ningbo (NB, 26) and Zhoushan (ZS, 1),

Zhejiang Province

Yangtze river

(YR, 34)

Hukou (HK, 1), Jiangxi Province

Chizhou (CZ, 1) and Wuhu (WH, 3), Anhui

Province

Chongmingdao (CMD, 1), Shanghai city

Yixing (YX, 1), Yizheng (YZ, 2), Zhenjiang

(ZJ, 1), and

Nanjing (NJ, 24), Jiangsu Province

Abbreviation and sample size for each sampling locality are shown in

parenthesis

Table 2 PCR primers used to

amplify the target fragment in

the present study

F forward, R reverse

Locus Length in bp Sequences Reference

PTH 283 F: ACCAGGAAGAGATCTGTGAGTG [18]

R: TGCCCTATGCTGTCTAGAGC

IFN1@ 351 F: TTCTCCTGCCTGAAGGACAG

R: GGATCTCATGATTTCTGCTCTGAC

RDS 498 F: TTTGACCAGAAGAAGCGGGT

R: TTGCTGATCCACTGAATCTC

KIT 679 F: CCTGTGAAGTGGATGGCACC

R: GCATCCCAGCAAGTCTTCAT

Mol Biol Rep (2012) 39:7755–7762 7757

123

partitioning of variation between and within populations

[24]. For each locus, phylogenies of all haplotypes were

reconstructed using Neighbor-joining (NJ) in MEGA ver-

sion 3.0, with both bootstrap and interior branch support

tests [25]. Phylogenetic trees were also estimated under the

maximum likelihood (ML) criterion in PAUP* software

package version 4.0b10 [26]. In addition, a median-joining

(MJ) was generated for all haplotypes using the software

network 4.5.1.6 [27], to represent phylogeographic struc-

ture. Two tests of selective neutrality, Tajima’s D [28] and

Fu’s F [29] values were used to determine whether or these

introns are evolving in a neutral manner. Arlequin 3.11 was

used to calculate and show in graphic form the distributions

of observed and expected pairwise nucleotide site differ-

ences, also called mismatch distributions, to address recent

demographic changes in populations. Finally, we carried

out the Mantel test to research correlations between geo-

graphical distance and genetic differentiation, the log of

minimum geographical distance in km was compared to

FST with the isolation by distance (IBD) software [30].

Results

Genetic diversity

Among the 144 individuals, at PTH locus(GenBank num-

ber: FJ 176375), Hap4 and Hap15 were found to be shared

by all three populations, whereas Hap5 and Hap13 were

only found in the south China sea population and the

Yellow sea population.

For IFN1@ sequences (GenBank number: HQ 585525),

86% haplotypes were present in only one population,

whereas three (Hap2, Hap3 and Hap5) were shared by all

three populations and three were found in two populations

(Hap8 and Hap12 found in the south China sea population

and the Yellow sea population, and Hap11 found in the

south China sea population and the Yangtze river

population).

At RDS locus (GenBank number: FJ 176376), only

Hap1 was shared by all three populations and Hap6 and

Hap11 were found in the south China sea population and

the Yangtze river population. In the all KIT sequences

(GenBank number: FJ 176363), Hap1 and Hap3 were

shared by all three populations in Chinese waters, whereas

Hap10, Hap14, and Hap15 were found in the south China

sea population and the Yellow sea population, and Hap2

and Hap19 were found in the south China sea population

and the Yangtze river population.

When the four nuclear introns were combined as a

whole, the overall nucleotide diversity was comparatively

low, while the overall haplotype diversity was relatively

high. Comparatively, the Yellow sea population had the

highest nucleotide diversity and haplotype diversity

(Table 3).

Phylogeographic structure

The AMOVA analysis showed significant genetic structure

in finless porpoises in Chinese waters (PTH: FST = 0.29,

P \ 0.001; IFN1@: FST = 0.23, P \ 0.001; RDS:

FST = 0.12, P \ 0.001; KIT: FST = 0.16, P \ 0.001).

Pairwise population comparisons showed the highest

structure between the south China sea population and the

Yangtze river population, meanwhile between the Yellow

sea population and either the south China sea population or

the Yangtze river population has a relatively low level of

population genetic structure (Table 4).

The median-joining network proved that the haplotypes

could not be clearly divided into three geographical groups

(Fig. 2), and the result was also supported by the phylo-

genetic reconstructions (data not presented). The network

was characterized by a star-like phylogeny with closely

related haplotypes derived from the most common

haplotype.

Furthermore, in Chinese waters as a whole, IBD ana-

lyzes revealed no obvious correlation between geographi-

cal distances and genetic (Fig. 3).

Demographic analysis

Tajima’s D statistic showed negative values (PTH: D = -

1.99, P = 0.002; IFN1@: D = -2.29, P \ 0.001; RDS:

D = -2.67, P \ 0.001; KIT: D = -2.13, P \ 0.001) and

Fu’s test of neutrality based on 1,000 simulating samplings

was clearly negative (PTH: FS = -25.50, P \ 0.001;

IFN1@: FS = -24.32, P \ 0.001; RDS: FS = -6.05,

P \ 0.001; KIT: FS = -25.98, P \ 0.001). In addition,

the mismatch distributions showed a distinct unimodal

curve for finless porpoise populations in Chinese waters.

All these results indicated a recent population expansion in

Chinese waters. Implemented the Tajima’s D statistic, Fu’s

test of neutrality and mismatch analyzes for the three

populations separately, expansion was also found for each

population (Fig. 4).

Discussion

Previous mtDNA, microsatellite, and morphological ana-

lyzes suggested three population units of finless porpoises

in Chinese waters [5–8, 12–15], and with an area of

sympatry in the Taiwan Strait, species-level differentiation

between marine finless porpoises in the east China sea and

south China sea [10]. Nuclear intron sequences, which are

presumed to be under low selection pressure, may be

7758 Mol Biol Rep (2012) 39:7755–7762

123

helpful to provide novel information of ecology and pop-

ulation biology relevant to conservation issues of the fin-

less porpoise. Our analyzes showed that the overall

nucleotide and haplotype diversities of the south China sea

population, the Yellow sea population and the Yangtze

river population were 0.5% and 0.98, 0.6% and 0.99, 0.5%

and 0.95 respectively, all of which were much higher than

those reported using mitochondrial control region sequen-

ces. Meanwhile the overall FST (PTH: 0.29, P \ 0.001;

IFN1@: 0.23, P \ 0.001; RDS: 0.12, P \ 0.001; KIT:

0.16, P \ 0.001) suggested that 10–30% of the intron

variation is distributed among populations. AMOVA

showed that the highest divergence occurred between the

south China sea population and the Yangtze river popula-

tion, followed by that between the south China sea popu-

lation and the Yellow sea population, and the lowest

divergence occurred between the Yellow sea population

and the Yangtze river population. These findings are

basically concordant with those revealed previously by

mitochondrial [12–14] and nuclear microsatellite [15]

markers, which strongly confirmed the significant genetic

differentiation between finless porpoise populations in

Chinese waters.

The present study showed low levels of nucleotide

diversity, in contrast with relatively high haplotype diver-

sity. In combination with the negative Tajima’s D and Fu’s

F values, the star-like phylogeny and closely related hap-

lotypes derived from the common haplotype (Fig. 2), and

the unimodal curve in mismatch analyzes (Fig. 4), it is

reasonable to infer a rapid demographic expansion from a

small effective population size occurring in the finless

porpoises. As previously suggested by mitochondrial con-

trol region sequences [14], in the quaternary glaciations,

finless porpoises that colonized refugial region in the

Yellow sea dispersed into different parts of Chinese waters

including the freshwater Yangtze river due to great fluc-

tuations in climatic and environmental conditions [31, 32].

The dispersed populations usually show small population

sizes with consequently limited genetic diversities [33, 34].

The refugial status of the Yellow sea population was fur-

ther corroborated with its highest nucleotide and haplotype

diversities among the populations examined in this study.

Although there is a significant genetic structure in finless

porpoises in Chinese waters, no obvious phylogeographic

pattern was revealed. Neither network analysis (Fig. 2) nor

phylogenetic reconstruction (data not shown) divided the

intron haplotypes into independent groups coinciding with

one of the three populations in Chinese waters. As shown

in Fig. 2, all shared haplotypes of the four loci were at the

relatively central position in the median-joining network,

whereas other haplotypes are derived directly or indirectly

from them. It is possible that these common haplotypes are

ancestral in the evolutionary history of finless porpoises,Ta

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Mol Biol Rep (2012) 39:7755–7762 7759

123

although their frequency could equally have been aug-

mented due to past episodes of genetic drift.

Up to date, multiple lines of evidence have been accu-

mulated to suggest significant genetic differentiation among

finless porpoise populations in Chinese waters, including

evidences not only came from morphometrics but also from

molecular markers such mtDNA, microsatellite, and the

present nuclear introns, which supported their subspecies or

species status as suggested by Gao and Zhou [5–7], Wang

et al. [10], and Chen et al. [15]. For this reason, in designing

conservation programs, each of these population units in

Chinese waters should be treated as different management

units (MUs) [35]. Although the Yangtze finless porpoise has

a relatively small population differentiation from the

adjacent Yellow sea population, special attention should be

given to its conservation. This distinct freshwater popula-

tion, having evolved some adaptive characteristics in order

to survive in the freshwater environment, represents special

and important genetic composition of the finless porpoise. Its

genetic uniqueness and distinct group status were recently

evidenced with Structure analysis using microsatellite data

[15]. Those serious anthropogenic pressures (e.g., agricul-

tural and industrial pollution, riverine development, etc.) that

made the Baiji go extinction are still in effect on the finless

porpoise [36]. Protection measures must be taken urgently to

alleviate the human impacts and protect the Yangtze finless

porpoise from gradual degradation and catastrophic

collapse.

Table 4 Pairwise FST values for three porpoises populations in Chinese waters

PTH IFN1@ RDS KIT

SCS YS SCS YS SCS YS SCS YS

YS 0.080* 0.092* 0.069* 0.073*

YR 0.155* 0.075* 0.174* 0.087* 0.162* 0.079* 0.169* 0.068*

SCS the south China sea population, YS the Yellow sea population, YR the Yangtze river population

* P \ 0.01

Fig. 2 Neighbor-joining (NJ) tree reconstructed by using PAUP based on HKY distance among the mitochondrial control region haplotypes of

the Chinese long snout catfish. Bootstrap percentage values are indicated next to nodes of 1,000 bootstrap simulations

7760 Mol Biol Rep (2012) 39:7755–7762

123

Acknowledgments This research was financially supported by the

National Natural Science Foundation of China (NSFC) (No.

30830016), the Program for New Century Excellent Talents in Uni-

versity, the Ministry of Education of China (No. NCET-07-0445), and

the Priority Academic Program Development of Jiangsu Higher

Education Institutions (PAPD) of Jiangsu Province, China to GY.

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Fs=-24.61, P=0.00

0102030405060708090

100

1 3 5 7 9 11 13 15 17 19 21 23 25

Yangtze River PopulationTajima's D=-2.21, P=0.001

Fs=-19.24, P= 0.00

0

10

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50

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1 3 5 7 9 11 13 15 17 19 21 23 25

Exp

Obs

South China Sea PopulationTajima's D=-1.70, P=0.02

Fs=-24.66, P=0.00

0

20

40

60

80

100

120

140

160

180

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Fig. 4 Distribution of the number of pairwise differences between haplotypes of finless porpoise

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