Molecular Phylogenetics and Evolution 99 (2016) 44–52

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Species phylogeny and diversification process of Northeast AsianPungitius revealed by AFLP and mtDNA markers

http://dx.doi.org/10.1016/j.ympev.2016.03.0221055-7903/� 2016 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: hiroshi@fish-u.ac.jp (H. Takahashi).

1 Present address: University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa903-0213, Japan.

Hiroshi Takahashi a,⇑, Peter R. Møller b, Sergei V. Shedko c, Temirbekov Ramatulla d, Sang-Rin Joen e,Chun-Guang Zhang f, Valentina G. Sideleva g, Keisuke Takata h, Harumi Sakai a, Akira Goto i,Mutsumi Nishida j,1

aNational Fisheries University, 2-7-1 Nagata-honmachi, Shimonoseki, Yamaguchi 759-6595, JapanbNatural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, Copenhagen DK-2100, Denmarkc Institute of Biology and Soil Science, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690022, RussiadKarakalpak Research Institute of Natural Science, Karakalpak Branch of Uzbekistan Academy of Sciences, Nukus, Uzbekistane Sang Myung University, Chongno-gu, Seoul 110-743, Republic of Koreaf Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuan Xilu, Haidian, Beijing 100080, PR Chinag Zoological Institute, Russian Academy of Science, Universitetskaya Emb. no. 1., St. Petersburg 199034, RussiahDepartment of Biology, Faculty of Science, Shinshu University, Matsumoto 390-8621, Japani Field Science Center for Northern Biosphere, Hokkaido University, Hakodate 041-8611, JapanjAtmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan

a r t i c l e i n f o

Article history:Received 8 January 2016Revised 16 March 2016Accepted 17 March 2016Available online 17 March 2016

Keywords:Ninespine sticklebackAFLPSpeciationSea of JapanIntrogression

a b s t r a c t

Pungitius is a highly diversified genus of sticklebacks (Gasterosteidae) occurring widely in northern partsof the Northern Hemisphere. Several ecologically and genetically divergent types that are largely isolatedreproductively but occasionally hybridize in sympatry have been discovered in Northeast Asia, althoughthe taxonomy and evolutionary relationships among them remain unclear. We used amplified fragmentlength polymorphism (AFLP) and mitochondrial DNA (mtDNA) markers to infer phylogenies among indi-viduals collected from sympatric and allopatric populations, including the type localities of the describedspecies. Phylogenetic analyses based on 2683 polymorphic AFLP loci confirmed seven species, each ofwhich (except for one entirely allopatric species P. platygaster) was clearly differentiated from one ortwo other sympatric species and constituted a highly supported monophyletic clade with conspecificallopatric populations. The phylogeny showed that two lineages arose early; one gave rise to two species(circumpolar species P. pungitius and Paratethys species P. platygaster) and the other to five species ende-mic to Northeast Asia (P. sinensis, P. tymensis, P. polyakovi, P. kaibarae, and P. bussei). The brackish-water,freshwater, and Omono types previously discovered in Japan were reidentified as P. pungitius, P. sinensis,and P. kaibarae, respectively. A marked incongruence was noted between the phylogenies of AFLP andmtDNA markers, suggesting the occasional occurrence of hybridization and mtDNA introgression amongdistinct species. Our results highlight that the marginal seas of Northeast Asia played a key role as barriersto or facilitators of gene flow in the evolution of species diversity of Pungitius concentrated in this region.

� 2016 Elsevier Inc. All rights reserved.

1. Introduction

The genus Pungitius comprises a highly variable group ofsticklebacks (Gasterosteidae) found widely in northern parts of theNorthernHemisphere (Münzing, 1969). Of the five genera of stickle-backs, only two, Pungitius and Gasterosteus, are morphologically

variable and geographically widespread, while the remaining three,Apeltes, Culaea, and Spinachia, are monotypic and geographicallyrestricted (Keivany and Nelson, 2000). The former two generacontain several reproductively isolated sympatric populations atvarious levels of evolutionary divergence, providing opportunitiesfor investigating the process of speciation (e.g., Boughman, 2007;Ishikawa et al., 2013). This, however, has caused taxonomic confu-sion, as denoted by the fact that the actual number of species withinboth genera remains unknown (Taylor, 1999; Nelson, 2006;Mattern, 2007).

H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52 45

Northeast Asia is one of the richest reservoirs of phylogeneticallydivergent lineages in both Pungitius and Gasterosteus (Takahashiand Goto, 2001; Watanabe et al., 2003). This fact may indicate thatthis regionwas not glaciated extensively during the Pleistocene andtherefore fostered diversification of endemic lineages, as with othercircumboreal, euryhaline fish genera, such as Acipenser and Cottus(Ludwig et al., 2001; Yokoyama and Goto, 2005).

In contrast to the widespread distribution of Pungitius, sym-patric, reproductively isolated lineages within the genus have beenfound only in Northeast Asia. Recent molecular studies based onallozyme, mitochondrial DNA (mtDNA), and microsatellite DNAmarkers revealed four lineages in Japan: P. tymensis and threegenetically divergent types of the P. pungitius–sinensis complex(brackish-water, freshwater, and Omono types; Takata et al.,1987a,b; Takahashi and Goto, 2001; Table 1). The correspondenceof the latter three types with the previously recognized morpho-logical species, namely P. pungitius and P. sinensis, remains to beverified, because the latter, which differs from the former only inhaving a complete row of lateral plates, is clearly paraphyletic(Haglund et al., 1993; Takahashi et al., 2001; Wang et al., 2015).

Under the biological species concept (BSC; Dobzhansky, 1937;Mayr, 1942), these four lineages are considered to be independentspecies, because they are largely isolated reproductively in sympa-try, with very few hybrids produced (Takahashi et al., 2001;Tsuruta and Goto, 2006; Tsuruta et al., 2008; Ishikawa et al.,2013). Furthermore, both reciprocal crosses between thebrackish-water and freshwater types show hybrid male sterility,while those between P. tymensis and the freshwater type producefertile hybrids of both sexes (Kobayashi, 1959; Takahashi et al.,2005), which indicates a long divergence time between the formertwo types, as intrinsic postzygotic isolation generally increaseswith the time of divergence (Coyne and Orr, 2004). Comparativeanalyses of the various levels of postzygotic reproductive isolationamong these lineages may deepen our understanding of the speci-ation process (Russell, 2003; Rogers and Bernatchez, 2006).

Compared with Pungitius, the taxonomy and phylogenetic rela-tionships in Gasterosteus are relatively well understood in North-east Asia, serving as a basis for testing evolutionary hypotheses.Two reproductively isolated species exist, the threespine stickle-back Gasterosteus aculeatus with a nearly circumpolar distributionand G. nipponicus endemic to Northeast Asia (Higuchi and Goto,1996; Higuchi et al., 2014). In addition, several ancient freshwaterpopulations with a long history of isolation have derived from theformer, which is in marked contrast to the postglacially establishedfreshwater populations of the European and North American three-spine stickleback (Watanabe et al., 2003; Cassidy et al., 2013).These divergent lineages have been studied intensively as an excel-lent model system for evolutionary biology and have providedenormous insights into speciation and phenotypic evolution usingcomparative genetic approaches (e.g., Colosimo et al., 2005; Kitanoet al., 2009; Yoshida et al., 2014). Considering the higher levels ofdiversification, the Northeast Asian Pungitius will likely emergeas a model system for evolutionary biology like Gasterosteus, oncea comprehensive species phylogeny is obtained.

Inferring phylogenetic relationships among closely relatedhybridizing species is often challenging, as incomplete lineagesorting and introgression are involved (Maddison, 1997). RecentmtDNA and allozymic studies on the Northeast Asian Pungitiusrevealed repeated instances of mitochondrial introgression amongthe different lineages in areas of sympatry (Takahashi and Takata,2000, 2003; Takahashi et al., 2003). Given the complex evolution-ary history caused by introgression among species, phylogeneticinformation from multiple nuclear loci is necessary to estimate aspecies phylogeny (Maddison, 1997). In recent years, amplifiedfragment length polymorphism (AFLP) has proven to be an effec-tive method for resolving relationships and detecting mtDNA

introgression among closely related species (Sullivan et al., 2004;Bossu and Near, 2009; Sturmbauer et al., 2010).

Here, we applied AFLP andmtDNAmarkers to unravel the taxon-omy and phylogenetic relationships of the Northeast AsianPungitius through comprehensive sampling of sympatric andallopatric populations including those collected from their typelocalities. We examined whether each reproductively isolated pop-ulation in areas of sympatry represents a single lineage togetherwith other geographically isolated populations based on phyloge-netic analysis of AFLP data. This can be achieved by relatively highintraspecific gene flow after lineage splitting, resulting in genotypiccohesion of the assemblage of populations (i.e., biological species;Mayr, 2000). We also estimated a time-calibrated mtDNA gene treeand overlaid it onto the AFLP-based species tree to examine the spa-tiotemporal patterns ofmitochondrial introgression among species.

2. Materials and methods

2.1. Sampling

Altogether, 86 specimens of Pungitius were collected from 30sites chosen as representative sympatric and allopatric areas(Fig. 1; Table 1). Importantly, our sampling included specimensfrom the type localities for five of seven previously recognized spe-cies, such as P. pungitius, P. sinensis, P. tymensis, P. polyakovi, andP. bussei (Table 1). One of the remaining two species, P. kaibarae,is extinct at its type locality (Kyoto, Japan). Although specimensof P. platygaster were collected far from its type locality, this islikely less of an issue in our study, because it is an entirely allopa-tric species. The specimens collected from the type locality corre-sponded well with the original descriptions in their morphology(see references in Table 1). Except for a geographically isolatedpopulation (J5) called ‘‘Musashi-tomiyo” (Nakamura, 1963;Igarashi, 1968), samples collected from Japan (H1–H4, J1–J4) wereidentified as one of the three types (i.e., brackish-water, freshwa-ter, and Omono types) in our previous allozymic and mtDNA-based phylogeographic studies (Takahashi et al., 2001, 2003;Tsuruta et al., 2002). In addition, 2 and 10 specimens of Culaeainconstans and Gasterosteus spp., respectively, were collected asoutgroups (Fig. 1; Table 1). The latter included three species (i.e.,G. aculeatus, G. wheatlandi, and G. nipponicus) among whichintrinsic postzygotic isolation occurred (Hendry et al., 2009). Allspecimens were preserved in 99% ethanol for DNA extraction.

2.2. DNA extraction, AFLP genotyping, and mtDNA sequencing

Total genomic DNA was extracted from the fin or muscle tissueusing the AquaPure Genomic DNA Isolation Kit (Bio-Rad, Hercules,CA, USA) following the manufacturer’s protocol. The quality andquantity of DNA were estimated spectrophotometrically and visu-ally via ethidium bromide staining of agarose gels.

The AFLP procedure followed a standard method (Vos et al.,1995), with only minor modifications, as described by Takahashiet al. (2015) using 23 EcoRI/MseI selective primer combinations(Table S1). AFLP loci ranging from 50 to 500 bp were scored auto-matically using the GeneMapper software version 3.7 (AppliedBiosystems, Foster City, CA, USA) and then proofed manually onthe basis of fragment size reproducibility, proximity to other loci,and signal intensity. Genotyping errors were quantified by com-paring the AFLP profiles of 12 individuals replicated in the twoAFLP analyses (Table S1; Pompanon et al., 2005). AFLP loci withhigh genotyping error rates (5%/locus) measured for two replicatedgenotypes were also excluded. The mean error rate per locus was0.38%, which is within the range reported in many other studies(Pompanon et al., 2005).

Table 1List of samples used in this study. Locality codes correspond to those in Fig. 1. Asterisks of sample ID indicate specimens from the type locality.

Locality code and sampling locality (habitat) Species or type Sample ID Coordinates GenBank # Sympatric? (Ref.)

North AmericaN1 Moncton, NB, Canada (brackish) Pungitius pungitius N1-Ppu 46�160N, 64�350W LC108069-70 No

Gasterosteus wheatlandi N1-Gwh LC108095-96N2 Rozon Lake, ON, Canada (fresh) Pungitius pungitius N2-Ppu 48�150N, 79�520W LC108071 NoN3 Guelph, ON, Canada (fresh) Culaea inconstans N3-Cin 43�320N, 80�130W -N4 Mud Lake, AK, USA (fresh) Pungitius pungitius N4-Ppu 61�330N, 149�30W LC108075 No

Gasterosteus aculeatus N4-Gac LC108076

EuropeE1 Isefjord, Denmark (marine) Pungitius pungitius E1-Ppu⁄ 55�470N, 11�370E LC108089-90 No

Gasterosteus aculeatus E1-Gac LC108074E2 Copenhagen, Denmark (fresh) Pungitius pungitius E2-Ppu⁄ 55�410N, 12�350E LC108072-73 NoE3 St. Petersberg, Russia (fresh) Pungitius pungitius E3-Ppu 59�580N, 30�290E LC108055 NoE4 Azov Sea, Russia (brackish) Gasterosteus aculeatus E4-Gac 46�400N, 38�350E LC108093-94

Central Asia (Paratethys)UZ Sudochie Lake, Uzbekistan (brackish) Pungitius platygaster UZ-Ppl 43�330N, 58�280E LC108092 No

Magadan and AnadyrMA Magadan, Russia (fresh, brackish) Pungitius pungitius MA-Ppu 59�430N, 150�20E LC108097 Yes (Takahashi and Goto, 2001)

Pungitius sinensis MA-Psi LC108098-99AN Anadyr, Russia (fresh) Pungitius pungitius AN-Ppu 64�440N, 177�310E LC108075, 86 No

Northeast Asia (China)CH Xisiduhe, Hai R., China (fresh) Pungitius sinensis CH-Psi⁄ 40�200N, 116�290E LC108083 No

Northeast Asia (Primorskii)P1 Kiya River, Amur, Russia (fresh) Pungitius bussei P1-Pbu 47�570N, 135�50E LC108077-8 NoP2 Ilistaya River, Khanka Lake, Russia (fresh) Pungitius bussei P2-Pbu⁄ 44�230N, 132�260E LC108061-2 Yes (Bogutskaya et al., 2008)

Pungitius kaibarae P2-Pka LC108058P3 Vladivostok, Russia (fresh) Pungitius kaibarae P3-Pka 43�200N, 132�180E LC108056-7 Yes (Bogutskaya et al., 2008)

Pungitius sinensis P3-Psi LC108059-60

Northeast Asia (Korean Peninsula)K1 Buk River, Korea (fresh) Pungitius kaibarae K1-Pka 38�240N, 128�280E LC108068 NoK2 Yeon-gok River, Korea (fresh, brackish) Pungitius kaibarae K2-Pka 37�510N, 128�500E LC108067 Yes (Yang and Min, 1990)

Pungitius sinensis K2-Psi LC108065-6K3 Hwangbo River, Korea (fresh) Pungitius sinensis K3-Psi 36�450N, 129�280E LC108066 NoK4 Nakdong River, Korea (fresh) Pungitius kaibarae K4-Pka 36�10N, 128�590E LC108063 NoK5 Hyeongsan River, Korea (fresh) Pungitius kaibarae K5-Pka 35�470N, 129�120E LC108064 No

Northeast Asia (Sakhalin)S1 Tym River, Sakhalin, Russia (fresh) Pungitius sinensis S1-Psi 50�380N, 142�460E LC108084 Yes (Takahashi and Goto, 2001)

Pungitius tymensis S1-Pty⁄ LC108048-9S2 Ayrup River, Sakhalin, Russia (fresh) Pungitius polyakovi S2-Ppo 46�470N, 143�250E LC108050, 87 NoS3 Ozerskiy, Sakhalin, Russia (fresh, brackish) Pungitius polyakovi S3-Ppo⁄ 46�370N, 143�90E LC108050 Yes (Shedko et al., 2005; Takahashi and Goto, 2001)

Pungitius pungitius S3-Ppu LC108045, 47Pungitius sinensis S3-Psi LC108079-80Pungitius tymensis S3-Pty LC108081-2

Northeast Asia (Hokkaido)H1 Teshio River, Hokkaido, Japan (fresh) Freshwater type H1-FWT 45�50N, 141,450E LC108105-6 Yes (Takahashi and Takata, 2000)

Pungitius tymensis H1-Pty LC108107-8H2 Bekanbeushi River, Hokkaido, Japan (fresh, brackish) Brackish-water type H2-BWT 43�40N, 144�510E LC108053, 105 Yes (Takata et al., 1987a; Takahashi and Goto, 2001)

Freshwater type H2-FWT LC108046, 54Pungitius tymensis H2-Pty LC108043Gasterosteus aculeatus H2-Gac LC108042, 85Gasterosteus nipponicus H2-Gni LC108040-1

H3 Asahikawa, Hokkaido, Japan (fresh) Freshwater type H3-FWT 43�530N, 142�190E LC108088 No

46H.Takahashi

etal./M

olecularPhylogenetics

andEvolution

99(2016)

44–52

Table1(con

tinu

ed)

Locality

code

andsamplinglocality

(hab

itat)

Speciesor

type

SampleID

Coo

rdinates

Gen

Ban

k#

Sympa

tric?(Ref.)

H4

IshikariRiver,H

okka

ido,

Japa

n(fresh

)Freshwater

type

H4-FW

T42

�530N,1

41�390E

LC10

8102

-3Yes

(Tak

ahashian

dTa

kata,2

000)

Pung

itiustymen

sis

H4-Pty

LC10

8088

,104

Northea

stAsia(H

onshu)

J1Ta

zawak

o,Omon

oRiver,Jap

an(fresh

)Omon

otype

J1-O

MT

39�460N,1

40�410E

LC10

8091

No

J2Hirak

a,Omon

oRiver,Jap

an(fresh

)Freshwater

type

J2-FW

T39

�160N,1

40�300E

LC10

8051

Yes

(Tak

ataet

al.,19

87b;

Tsuru

taan

dGoto,

2006

)Omon

otype

LC10

8052

J3Gak

koRiver,Y

amag

ata,

Japa

n(fresh

)Freshwater

type

J3-FW

T39

�10 N

,139

�540E

LC10

8044

No

J4Mog

amiRiver,Y

amag

ata,

Japa

n(fresh

)Omon

otype

J4-O

MT

38�240N,1

40�210E

LC10

8100

No

J5Motoa

raka

wa,

Saitam

a,Japa

n(fresh

)Pu

ngitiussp

.J5-Psp

36�8

0 N,1

39�240E

LC10

8101

No

Theoriginal

reference

andtype

locality

ofea

chsp

ecieswas

summarized

inSa

kaian

dYab

e(200

3)an

dMattern

(200

7).

H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52 47

The entire region of themitochondrial cytochrome b gene (cyt b)was amplified using the primer pair L-Glu (50-CTAACCAGGACTAATGGCTTGAA-30) and H-Thr (50-CGGCTTACAAGACCGGCGCTCTGA-30), which were designed based on conserved regions inthe flanking tRNA genes of sticklebacks (Kawahara et al., 2009).Two Culaea specimens were omitted from analysis due toamplification difficulties. Amplified products were sequenceddirectly using the BigDyeTM Terminator version 3.1 Cycle SequencingKit (PerkinElmer, San Jose, CA, USA) and ABI 3130 Genetic Analyzer(Applied Biosystems). The DNA sequences were compared withpublished sequences for sticklebacks (Kawahara et al., 2009) toverify the boundaries and alignment of mitochondrial genes. Allunique mtDNA sequences (haplotypes) used in the analyses weredeposited in GenBank/EMBL/DDBJ (accession nos. LC108040–LC108108).

2.3. Phylogenetic analyses

For the AFLP data, neighbor-joining (NJ) trees based on Nei–Lidistances (Nei and Li, 1979) and unweighted maximum parsimony(MP) trees were estimated using the NEIGHBOR andMIX programs,respectively, of the PHYLIP package (ver. 3.67; Felsenstein, 2007).To assess the robustness of each node in the trees, 1000 bootstrapreplications were conducted using the SEQBOOT and CONSENSEprograms in the same package. In addition, we inferred phyloge-netic relationships using the Metropolis-coupled Markov chainMonte Carlo algorithm implemented in MrBayes (ver. 3.1.2;Ronquist and Huelsenbeck, 2003). The Bayesian analysis consistedof two runs of 24,000,000 generations to ensure that the standarddeviation (SD) of split frequencies was <0.01. Trees were sampledevery 100 generations, with the first 25% discarded as burn-in.

To infer evolutionary relationships among mtDNA haplotypes,NJ, MP, and maximum likelihood (ML) trees were estimated usingthe MEGA software (ver. 6.0; Tamura et al., 2013). We used theBayesian information criterion to select the best-fitting nucleotidesubstitution models for the NJ and ML analyses. A Tamura–Neimodel, with gamma-distributed rate heterogeneity and a propor-tion of invariant sites (T93 + G + I), was used in these analyses. AMP tree was inferred using tree–bisection–reconnection branchswapping. A bootstrapping analysis with 1000 replicates was per-formed to estimate support for nodes within each phylogenetictree. We used the Shimodaira–Hasegawa (SH) test (Shimodairaand Hasegawa, 1999), as implemented in PAUP 4.0b10 (Swofford,2003), to assess incongruence between trees obtained from themtDNA and AFLP data sets.

A time-calibrated mtDNA gene tree was estimated using theBEAST software (ver. 1.8.2; Heled and Drummond, 2010). We usedbasically the same setting parameters used by Wang et al. (2015),except for the following three modifications: (1) individualsbelonging to Pungitius and Gasterosteus were treated as mono-phyletic, considering the occurrence of mitochondrial introgres-sion between congeneric species. (2) The T93 + G + I model wasused according to the above selection. (3) We previously set a log-normal distribution for the ucld.mean parameter (mean of thebranch rates) as 0.00095 ± 0.03 (mean ± SD) based on the molecu-lar evolutionary rate of 1.9%/million year for the cyt b sequence ofPungitius (Wang et al., 2015).

3. Results and discussion

3.1. The AFLP tree revealed seven species

Phylogenetic analyses based on 2683 polymorphic AFLP locirevealed seven highly supported monophyletic clades [bootstrapprobabilities (BPs) of the NJ and MP methods were >98%; Bayesian

Fig. 1. (a) The circumboreal distribution of Pungitius and (b) its southern stretches in Northeast Asia. Solid and open circles indicate the sampling sites for sympatric andallopatric populations, respectively, of Pungitius. Collection sites for the outgroups (Culaea inconstans and Gasterosteus spp.) are also indicated by open squares. The locationcodes correspond to those in Table 1.

48 H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52

posterior probabilities (PPs) were >0.75] corresponding to sevenpreviously recognized species of Pungitius (Fig. 2a). Except forone entirely allopatric species, P. platygaster, each species wasclearly differentiated from one or two other sympatric speciesand characterized by a distinct assemblage of sympatric and allo-patric populations. Our data, therefore, demonstrate the validityof applying the BSC to the six species to facilitate the study of spe-ciation in this genus (Mayr, 2000; Coyne and Orr, 2004).

Although the BSC cannot be applied directly to P. platygaster,this species will likely be placed as an equally-ranking taxon withthe other six ‘biological’ species in terms of genetic distance. TheNei–Li distance between P. platygaster and its sister speciesP. pungitius (0.0539 ± 0.0029; mean ± SD) was comparable to thosebetween P. pungitius and P. sinensis (0.0615 ± 0.0030) and betweenG. aculeatus and G. nipponicus (0.0356 ± 0.0012), both involving theevolution of intrinsic postzygotic isolation, and was significantlyhigher than those among the remaining five species (Table S2).By adding this species, our data confirm the genetic distinctivenessof the seven Pungitius species.

The AFLP-based phylogeny showed that two lineages aroseearly in the evolution of Pungitius with 100% BPs: one of them gaverise to two species (circumpolar species P. pungitius and Paratethysspecies P. platygaster) and the other to five species endemic toNortheast Asia (P. sinensis, P. tymensis, P. polyakovi, P. kaibarae,and P. bussei; Fig. 2a). In the latter lineage, a sister relationshipbetween P. kaibarae and P. bussei, sympatric in the Khanka Lakebasin, was strongly supported (100% BPs), while the relationshipsamong the remaining three species and the common ancestor ofthe former two species were not resolved fully.

Based on clustering patterns, the brackish-water, freshwater,and Omono types in Japan were reidentified as P. pungitius,P. sinensis, and P. kaibarae, respectively, without regard to theirlateral plate morphologies. Only the latter two species exhibit thelateral plate polymorphism. In addition, the geographically isolatedpopulation ‘‘Musashi-tomiyo” was reidentified as P. sinensis. Note,however, that specimens from the type locality of P. kaibarae were

not included in our analysis, because this species is extinct from itstype locality as described above. Nonetheless, the populations con-stituting the P. kaibarae cluster, including the three populations ofthe Omono types, have synapomorphic characters consistent withthe type specimens, such as a short body, black dorsal and anal spi-nous membranes, and mostly seven or eight dorsal spines (Tanaka,1915; Tanaka et al., 1982; Tsuruta and Goto, 2006). Furthermore,although these populations are geographically isolated from oneanother, their distributions are clearly associated with the south-western half of the Sea of Japan coast, where the type locality iscentrally located. Therefore, one can reasonably assume that theOmono type is conspecific with P. kaibarae, together with the pop-ulations distributed in the Korean Peninsula and the southern partof Primorsky Krai, Russia (see Bogutskaya et al., 2008; Bae and Suk,2015).

3.2. Extensive introgression of mtDNA among species

All of the NJ, MP, and ML trees based on the cyt b sequencesrevealed three major mitochondrial lineages corresponding to lin-eages A, B, and C in Takahashi and Goto (2001), which were wellsupported by high bootstrap values using all methods (>93%BPs). In addition to these lineages, P. platygaster was representedby a highly divergent haplotype sister to lineage C, although thenode was not supported by a high bootstrap value for the MPmethod (Fig. 2b). Apart from this exception, all haplotypes of theremaining six Pungitius species belonged to one of the three majormitochondrial lineages.

The mitochondrial gene tree exhibited three significant topo-logical incongruences with the AFLP-based tree (Fig. 2, Table S3).(i) Perhaps the most remarkable incongruence is that all haplo-types of P. pungitius and P. sinensis originating from different ances-tral lineages in Pungitius were included in lineage C, together witha few haplotypes of P. tymensis and P. kaibarae. (ii) The remainingP. tymensis haplotypes were paraphyletic with respect to those ofP. polyakovi within lineage A. (iii) Haplotypes of P. bussei formed

Fig. 2. A comparison of phylogenies based on nuclear and mitochondrial DNA (mtDNA) markers. (a) A neighbor-joining (NJ) tree based on 2683 polymorphic amplified fragment lengthpolymorphism (AFLP) loci. Bootstrap probabilities (BPs, >50%) for NJ and maximum parsimony (MP) methods and Bayesian posterior probabilities (PPs, >0.5) of major lineages are shownabove the nodes (NJ/MP/PPs). (b) A NJ tree based on the entire mtDNA cytochrome b gene sequences. BPs for NJ, MP, maximum likelihood (ML) methods of major lineages are shown abovethe nodes (NJ/MP/ML). Seven species of Pungitius are indicated by different colors. The location codes and the species abbreviations correspond to those in Table 1.

H.Takahashi

etal./M

olecularPhylogenetics

andEvolution

99(2016)

44–52

49

50 H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52

a strongly supported clade (100% BPs) with those of P. kaibaraefrom Primorsky Krai, resulting in paraphyly of the P. kaibarae hap-lotypes. The results of the SH tests also revealed incongruencesbetween the mtDNA and AFLP-based topologies, namely the mono-phyly of haplotypes within each of the three species (i.e., P. sinensis,P. tymensis, and P. kaibarae) was significantly rejected as well as themonophyly of haplotypes of the five species endemic to NortheastAsia (Table S3).

Fig. 3. (a) A time-calibrated phylogeny based on the entire mtDNA cytochrome b genelineages are shown above the nodes. The 95% highest posterior density of node age estimaNortheast Asia. Six species are indicated by different colors (as in Fig. 2).

The sister relationship between the P. platygaster haplotype andlineage C would be key to understanding the directions of mtDNAintrogression among species. Comparing the results of crossexperiments may imply a relatively close relationship betweenP. pungitius and P. platygaster compared with that betweenP. pungitius and P. sinensis (formerly the brackish-water andfreshwater types, respectively), because no intrinsic postzygoticisolation occurred between the former two species (Ziuganov and

sequences inferred by BEAST. Bayesian posterior probabilities (PPs, >0.5) of majortes are shown by gray bars. (b) Geographic distributions of six species of Pungitius in

H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52 51

Gomeluk, 1985; cf. Takahashi et al., 2005). In addition, several phy-logenetic studies on circumboreal, euryhaline fish genera, such asAcipenser, Cottus, and Esox, have shown a close biogeographic con-nection between the Paratethys and northern circumpolar regionscompared with that between Northeast Asia and the latter regions(e.g., Ludwig et al., 2001; Kinziger et al., 2005; Skog et al., 2014).Considering these findings together with our AFLP-based phy-logeny, lineage C probably represents the original mtDNA lineageof P. pungitius. Given this argument, the major incongruencebetween mtDNA- and AFLP-based phylogenies (i) would beexplained by mtDNA introgression from P. pungitius to P. sinensis,resulting in complete replacement of the original lineages withthe lineage C mtDNA.

Except for a few divergent haplotypes in lineage C, the topolog-ical incongruences may be attributed to local introgression ofmtDNA, as will be obvious from the regional assemblages ofmtDNA haplotypes (Fig. 2b, see also Fig. 3). The likelihood existsthat several cases of the lineage C mtDNA introgression occurredfrom P. pungitius to P. tymensis or to P. kaibarae via P. sinensis,because these took place in regions where P. pungitius is absent.For example, Takahashi and Takata (2000) reported that mtDNAintrogression occurred independently in the northern and centralHokkaido regions (H1 and H4) from P. pungitius to P. tymensis,but the former should be reidentified as P. sinensis based on thepresent study. In addition, clear regional assemblages of mtDNAhaplotypes were also found in the remaining two cases of topolog-ical incongruences (ii and iii), suggesting that local mtDNA intro-gression occurred from P. tymensis to P. polyakovi and fromP. kaibarae to P. bussei in the southeastern Sakhalin and KhankaLake basin, respectively (Fig. 3b). Thus, mtDNA lineages A and Bwould represent the original lineages of P. tymensis and P. kaibarae,respectively. In both cases, the original mtDNA lineages of theintrogressed species were not observed in either the sympatric orallopatric populations sampled, implying that introgressed mtDNAhas extensively displaced their original lineages. However, thedetailed processes of the ancient mtDNA introgression in lineageC involving isolated inland populations of P. kaibarae (J2 and J4)remain unclear.

3.3. Diversification of the Northeast Asian Pungitius

The tree topology produced by the uncorrelated relaxed-clockmethod based on the cyt b sequences was basically congruent withthe above-mentioned mtDNA-based trees (Fig. 3). The monophylyof the three major mitochondrial lineages and the sister relation-ship between the P. platygaster haplotype and lineage C werestrongly supported (>0.98 PPs). Our divergence estimates revealedthat the initial diversification between the two Pungitius lineagespredated that between G. aculeatus and G. wheatlandi: the age ofthe former was estimated to be 5.97 million years ago (Mya)[95% highest posterior density (HPD), 3.97–8.76 Mya], and that ofthe latter was estimated to be 4.92 Mya (HPD, 2.40–8.40 Mya)—consistent with the levels of postzygotic reproductive isolation,since intrinsic postzygotic isolation was observed between eachof them (Takahashi et al., 2005; Hendry et al., 2009).

Our time-calibrated phylogeny revealed that diversification ofthe five Pungitius species endemic to Northeast Asia occurred dur-ing the middle Pliocene and early Pleistocene. The age of the diver-gence between lineages A and B, which corresponds to the time tothe most recent common ancestor of P. tymensis and P. kaibarae,was estimated to be 4.42 Mya (HPD, 2.66–6.55 Mya). The diver-gence time among the five Pungitius species endemic to NortheastAsia may approximate this estimate, considering the similargenetic distances between these species in the AFLP data: specifi-cally, the Nei–Li distance between P. tymensis and P. kaibarae was0.0459, and those between the other species were 0.0367–0.0594

(Table S2). Note that our estimates are somewhat older than thoseof the oldest node in Pungitius (4.44 Mya) and the node betweenP. tymensis and P. kaibarae (2.67 Mya) reported in previous studieson the European P. pungitius and P. laevis and on the P. kaibaraepopulations in the Korean Peninsula (Bae and Suk, 2015; Wanget al., 2015). The disparity is probably due to differences in themitochondrial regions assessed, taxon sampling, and calibrationpoints, which suggests that further evaluations are required.Nonetheless, one can safely conclude that the diversification of thefive species occurred from the middle Pliocene to early Pleistocene,followed by regional differentiation within the species.

Our results demonstrated that P. sinensis has a wide and contin-uous distribution with relatively low genetic subdivision across thespecies range, in contrast to the remaining four species withrestricted and discontinuous distributions (Fig. 3b). In the middlePliocene, the ocean environment of the Sea of Japan had changeddramatically, because the warm Tsushima Current began to flowinto this semi-enclosed marginal sea (Kitamura and Kimoto,2006). This possibly promoted diversification of cold-water-adapted euryhaline fish, such as Pungitius and Cottus, whose distri-bution surrounds the Sea of Japan, through the isolation of lineagesin discontinuous freshwater systems. However, the developmentof low-salinity surface water during Pleistocene glacial periods(Tada et al., 1999) might have allowed Pungitius, especially thehighly euryhaline species P. sinensis (Tanaka and Shinbo, 1985;Ishikawa et al., 2013), to disperse through coastal waters in theSea of Japan and the adjacent Sea of Okhotsk (Takahashi andGoto, 2001; Takahashi et al., 2001). Thus, the contrasting phylogeo-graphic patterns among the five species could be attributable tothe evolution of the marginal seas, which played a key role asbarriers to or facilitators of gene flow in the evolution of speciesdiversity in Pungitius concentrated in Northeast Asia.

Acknowledgments

We are grateful to D.L.G. Noaks, B. Robinson, R.A. Curry, M.A.Bell, K.N. Toderich, B. Karimov, S.B. Tuniev, K. Tanaka, Y. Touge,S.V. Frolov, H. Ida, K. Iguchi, K. Watanabe, S. Mori, H. Sugiyama,Y. Uematsu, H. Kanazawa, R. Kawahara-Miki, R. Yokoyama,Y. Yamazaki, T. Tsuruta, T. Kuwahara, T. Kitamura, M. Kume,Y. Meguro, and the late S.F. Safronov for providing samples andassistance with the collection. We also thank H. Motomura,S.J. Raredon, and M.V. Nazarkin for sending the digital images ofthe Pungitius bussei syntypes. This work was supported in part byJSPS KAKENHI (Nos. 15405008, 19405011, 19770074, 21370035,and 22687006).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2016.03.022.

References

Bae, H.G., Suk, H.Y., 2015. Population genetic structure and colonization history ofshort ninespine sticklebacks (Pungitius kaibarae). Ecol. Evol. 5, 3075–3089.

Bogutskaya, N.G., Naseka, A.M., Shedko, S.V., Vasileva, E.D., Chereschnev, I.A., 2008.The fishes of the Amur River: updated check-list and zoogeography. Ichtyol.Explor. Freshwaters 19, 301–366.

Bossu, C.M., Near, T.J., 2009. Gene trees reveal repeated instances of mitochondrialDNA introgression in orangethroat darters (Percidae: Etheostoma). Syst. Biol. 58,114–129.

Boughman, J.W., 2007. Speciation in sticklebacks. In: Östlund-Nilsson, S., Mayer, I.,Huntingford, F.A. (Eds.), Biology of the Three-Spined Stickleback. CRC Press,Boca Raton, pp. 83–126.

Cassidy, L.M., Ravinet, M., Mori, S., Kitano, J., 2013. Are Japanese freshwaterpopulations of threespine stickleback derived from the Pacific Ocean lineage?Evol. Ecol. Res. 15, 295–311.

Colosimo, P.F., Hosemann, K.E., Balabhadra, S., Villarreal, G., Dickson, M., Grimwood,J., Schmutz, J., Myers, R.M., Schluter, D., Kingsley, D.M., 2005. Widespread

52 H. Takahashi et al. /Molecular Phylogenetics and Evolution 99 (2016) 44–52

parallel evolution in sticklebacks by repeated fixation of ectodysplasin alleles.Science 307, 1928–1933.

Coyne, J.A., Orr, H.A., 2004. Speciation. Sinauer Associates, Sunderland.Dobzhansky, T., 1937. Genetics and the Origin of Species. Columbia Univ. Press, New

York.Felsenstein, J., 2007. PHYLIP (phylogeny inference package) Version 3.67. University

of Washington, Seattle. <http://evolution.genetics.washington.edu/phylip.html> (accessed 12.04.15).

Haglund, T.R., Buth, D.G., Lawson, R., 1993. Allozyme variation and phylogeneticrelationships of Asian, North American, and European populations of theninespine stickleback, Pungitius pungitius. In: Mayden, R.L. (Ed.), Systematics,Historical Ecology, and North American Freshwater Fishes. Stanford Univ. Press,Stanford, pp. 438–452.

Heled, J., Drummond, A.J., 2010. Bayesian inference of species trees from multilocusdata. Mol. Biol. Evol. 27, 570–580.

Hendry, A.P., Bolnick, D.I., Berner, D., Peichel, C.L., 2009. Along the speciationcontinuum in sticklebacks. J. Fish Biol. 75, 2000–2036.

Higuchi, M., Goto, A., 1996. Genetic evidence supporting the existence of two distinctspecies in the genus Gasterosteus around Japan. Environ. Biol. Fish. 47, 1–16.

Higuchi, M., Sakai, H., Goto, A., 2014. A new threespine stickleback, Gasterosteusnipponicus sp. nov. (Teleostei: Gasterosteidae), from the Japan Sea region.Ichthyol. Res. 61, 341–351.

Igarashi, K., 1968. Observation on the development of the scutes in a ten-spinedstickleback.Musashi Tomiyo, Pungitius sp. Bull. Jpn. Soc. Sci. Fish. 34, 1083–1087.

Ishikawa, A., Takeuchi, N., Kusakabe, M., Kume, M., Mori, S., Takahashi, H., Kitano, J.,2013. Speciation in ninespine stickleback: reproductive isolation andphenotypic divergence among cryptic species of Japanese ninespinestickleback. J. Evol. Biol. 26, 1417–1430.

Kawahara, R., Miya, M., Mabuchi, K., Near, T.J., Nishida, M., 2009. Sticklebackphylogenies resolved: evidence from mitochondrial genomes and 11 nucleargenes. Mol. Phylogenet. Evol. 50, 401–404.

Keivany, Y., Nelson, J.S., 2000. Taxonomic review of the genus Pungitius, ninespinesticklebacks (Gasterosteidae). Cybium 24, 107–122.

Kinziger, A.P., Wood, R.M., Neely, D.A., 2005. Molecular systematics of the genusCottus (Scorpaeniformes: Cottidae). Copeia 2005, 303–311.

Kitamura, A., Kimoto, K., 2006. History of the inflow of the warm Tsushima Currentinto the Sea of Japan between 3.5 and 0.8 Ma. Palaeogeogr. Palaeoclimatol.Palaeoecol. 236, 355–366.

Kitano, J., Ross, J.A., Mori, S., Kume, M., Jones, F.C., Chan, Y.F., Absher, D.M.,Grimwood, J., Schmutz, J., Myers, R.M., Kingsley, D.M., Peichel, C.L., 2009. A rolefor a neo-sex chromosome in stickleback speciation. Nature 461, 1079–1083.

Kobayashi, H., 1959. Cross-experiments with three species of stickleback, Pungitiuspungitius (L.), Pungitius tymensis (Nikolsky), and Pungitius sinensis (Guichenot),with special reference to their systematic relationship. J. Hokkaido GakugeiUniv., Sect. B 10, 363–384.

Ludwig, A., Belfiore, N.M., Pitra, C., Svirsky, V., Jenneckens, I., 2001. Genomeduplication events and functional reduction of ploidy levels in sturgeon(Acipenser, Huso and Scaphirhynchus). Genetics 158, 1203–1215.

Maddison, W.P., 1997. Gene trees in species trees. Syst. Biol. 46, 523–536.Mattern, M.Y., 2007. Phylogeny, systematics, and taxonomy of sticklebacks. In:

Östlund-Nilsson, S., Mayer, I., Huntingford, F.A. (Eds.), Biology of the Three-Spined Stickleback. CRC Press, Boca Raton, pp. 1–40.

Mayr, E., 1942. Systematics and the Origin of Species from the Viewpoint of aZoologist. Columbia Univ. Press, New York.

Mayr, E., 2000. The biological species concept. In: Wheeler, Q.D., Meier, R. (Eds.),Species Concepts and Phylogenetic Theory: A Debate. Columbia Univ. Press,New York, pp. 17–29.

Münzing, V.J., 1969. Variabilität, Verbreitung and Systematik der Arten undUnterarten in der Gatting Pungitius Coste, 1848 (Pisces, Gasterosteidae). Z.Zool. Syst. Evol. Forsch. 7, 208–233.

Nakamura, M., 1963. Keys to the Freshwater Fishes of Japan Fully Illustrated inColors. Hokuryukan, Tokyo.

Nei, M., Li, W.-H., 1979. Mathematical model for studying genetic variation in termsof restriction endonucleases. Proc. Natl. Acad. Sci. USA 76, 5269–5273.

Nelson, J.S., 2006. Fishes of the World, fourth ed. John Wiley & Sons Inc., Hoboken.Pompanon, F., Bonin, A., Bellemain, E., Taberlet, P., 2005. Genotyping errors: causes,

consequences and solutions. Nat. Rev. Genet. 6, 847–859.Rogers, S.M., Bernatchez, L., 2006. The genetic basis of intrinsic and extrinsic post-

zygotic reproductive isolation jointly promoting speciation in the lake whitefishspecies complex (Coregonus clupeaformis). J. Evol. Biol. 19, 1979–1994.

Ronquist, R.R., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inferenceunder mixed models. Bioinformatics 19, 1572–1574.

Russell, S.T., 2003. Evolution of intrinsic post-zygotic reproductive isolation in fish.Ann. Zool. Fennici. 40, 321–329.

Sakai, H., Yabe, M., 2003. Problems in classification and taxonomy of Gasterosteidae.In: Goto, A., Mori, S. (Eds.), The Natural History of Sticklebacks. HokkaidoUniversity Press, Sapporo, pp. 23–45.

Shedko, S.V., Shedko, M.B., Pietsch, T.W., 2005. Pungitius polyakovi sp n., a newspecies of ninespine sticklebask (Gasterosteiformes, Gasterosteidae) fromsoutheastern Sakhalin Island. In: Storozhenko, Yu (Ed.), Flora and Fauna ofSakhalin Island. Part 2. Dalnauka, Vladivostok, pp. 223–233.

Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods withapplications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.

Skog, A., Vøllestad, L.A., Stenseth, N.C., Kasumyan, A., Jakobsen, K.S., 2014.Circumpolar phylogeography of the northern pike (Esox lucius) and itsrelationship to the Amur pike (E. reichertii). Front. Zool. 11, 67.

Sturmbauer, C., Salzburger, W., Duftner, N., Schelly, R., Koblmüller, S., 2010.Evolutionary history of the Lake Tanganyika cichlid tribe Lamprologini(Teleostei: Perciformes) derived from mitochondrial and nuclear DNA data.Mol. Phylogenet. Evol. 57, 266–284.

Sullivan, J.P., Lavoué, S., Arnegard, M.E., Hopkins, C.D., 2004. AFLPs resolvephylogeny and reveal mitochondrial introgression within a species flock ofAfrican electric fish (Mormyroidea: Teleostei). Evolution 58, 825–841.

Swofford, D.L., 2003. PAUP⁄. Phylogenetic Analysis Using Parsimony (⁄and OtherMethods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts.

Tada, R., Irino, T., Koizumi, I., 1999. Land-ocean linkages over orbital and millennialtimescales recorded in Late Quaternary sediments of the Japan Sea.Paleoceanography 14, 236–247.

Takahashi, H., Goto, A., 2001. Evolution of East Asian ninespine sticklebacks asshown by mitochondrial DNA control region sequences. Mol. Phylogenet. Evol.21, 135–155.

Takahashi, H., Nagai, T., Goto, A., 2005. Hybrid male sterility between the fresh- andbrackish-water types of ninespine stickleback Pungitius pungitius (Pisces,Gasterosteidae). Zool. Sci. 22, 35–40.

Takahashi, H., Takata, K., 2000. Multiple lineages of the mitochondrial DNAintrogression from Pungitius pungitius (L.) to Pungitius tymensis (Nikolsky).Can. J. Fish. Aquat. Sci. 57, 1814–1833.

Takahashi, H., Takata, K., Goto, A., 2001. Phylogeography of the lateral platedimorphism in the freshwater type of ninespine sticklebacks, genus Pungitius.Ichthyol. Res. 48, 143–154.

Takahashi, H., Tsuruta, T., Goto, A., 2003. Population structure of two ecologicallydistinct forms of ninespine stickleback, Pungitius pungitius: gene flow regimesand genetic diversity based on mtDNA sequence variations. Can. J. Fish. Aquat.Sci. 60, 421–432.

Takahashi, H., Takeshita, N., Tanoue, H., Ueda, S., Takeshima, H., Komatsu, T.,Kinoshita, I., Nishida, M., 2015. Severely depleted genetic diversity andpopulation structure of a large predatory marine fish (Lates japonicus)endemic to Japan. Conserv. Genet. 16, 1155–1165.

Takahashi, K., Takata, K., 2003. Hybridization and mtDNA introgression amongspecies of ninespine sticklebacks, genus Pungitius. In: Goto, A., Mori, S. (Eds.),The Natural History of Sticklebacks. Hokkaido Univ. Press, Sapporo, pp. 102–113.

Takata, K., Goto, A., Yamazaki, F., 1987a. Biochemical identification of a brackishwater type of Pungitius pungitius, and its morphological and ecological featuresin Hokkaido, Japan. Jpn. J. Ichthyol. 34, 176–183.

Takata, K., Goto, A., Yamazaki, F., 1987b. Genetic differences of Pungitius pungitiusand P. sinensis in a small pond of the Omono River System, Japan. Jpn. J. Ichthyol.34, 384–386.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecularevolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

Tanaka, S., 1915. Ten new species of Japanese fishes. Doubutsugaku Zasshi 27, 565–568.

Tanaka, S., Hirai, K., Joen, S.-R., 1982. Morphological comparison of ‘‘minami-tomiyo” Pungitius kaibarae between the Korean forms and those from Kyoto.Tansuigyo 8, 70–72.

Tanaka, S., Shinbo, C., 1985. Salinity tolerance of three species of ninespinestickleback (genus Pungitius) in Japan. Bull. Fac. Edu. Toyama Univ. 33, 2–22.

Taylor, E.B., 1999. Species pairs of north temperate freshwater fishes: Evolution,taxonomy, and conservation. Rev. Fish Biol. Fish. 9, 299–324.

Tsuruta, T., Goto, A., 2006. Fine scale genetic population structure of the freshwaterand Omono types of nine-spined stickleback Pungitius pungitius (L.) within theOmono River system, Japan. J. Fish Biol. 69 (Suppl. B), 155–176.

Tsuruta, T., Machida, Y., Goto, A., 2008. Nesting habitat use and partitioning of threesympatric ninespine sticklebacks (genus Pungitius): implications forreproductive isolation. Environ. Biol. Fish. 82, 143–150.

Tsuruta, T., Takahashi, H., Goto, A., 2002. Evidence for type assortative matingbetween the freshwater and Omono types of nine-spined stickleback in naturalfresh water. J. Fish Biol. 61, 230–241.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot,J., Peleman, J., Kuiper, M., 1995. AFLP: new technique for DNA fingerprinting.Nucl. Acids Res. 23, 4407–4414.

Wang, C., Shikano, T., Persat, H., Merilä, J., 2015. Mitochondrial phylogeography andcryptic divergence in the stickleback genus Pungitius. J. Biogeogr. http://dx.doi.org/10.1111/jbi.12591.

Watanabe, K., Mori, S., Nishida, M., 2003. Genetic relationships and origin of twogeographic groups of the freshwater threespine stickleback, ‘hariyo’. Zool. Sci.20, 265–274.

Yang, S.Y., Min, M.S., 1990. Genetic variation and systematics of the sticklebacks(Pisces, Gasterosteidae) in Korea. Korean J. Zool. 33, 499–508.

Yokoyama, R., Goto, A., 2005. Evolutionary history of freshwater sculpins, genusCottus (Teleostei; Cottidae) and related taxa, as inferred from mitochondrialDNA phylogeny. Mol. Phylogenet. Evol. 36, 654–668.

Yoshida, K., Makino, T., Yamaguchi, K., Shigenobu, S., Hasebe, M., Kawata, M., Kume,M., Mori, S., Peichel, C.L., Toyoda, A., Fujiyama, A., Kitano, J., 2014. Sexchromosome turnover contributes to genomic divergence between incipientstickleback species. PLoS Genet. 10, e1004223. http://dx.doi.org/10.1371/journal.pgen.1004223.

Ziuganov, V.V., Gomeluk, V.Y., 1985. Hybridization of two forms of ninespinestickleback, Pungitius pungitius and P. platygaster, under experimentalconditions and an attempt to predict the consequences of their contact innature. Environ. Biol. Fish. 13, 241–251.