Jurnal Eng Acara II Var Intra

ISSN 1022�7954, Russian Journal of Genetics, 2011, Vol. 47, No. 3, pp. 322–331. © Pleiades Publishing, Inc., 2011.Original Russian Text © O. V. Apalikova, A. V. Podlesnykh, A. D. Kukhlevsky, S. Guohua, V. A. Brykov, 2011, published in Genetika, 2011, Vol. 47, No.3, pp. 368–378.

322

INTRODUCTION

Polyploidy plays an important role in evolution [1].Multiple polyploidization events occurred indepen�dently in various fish groups [2]. Polyploidy is directlyrelated with hybridization and unisexual reproduction[3–9]. There is a hypothesis that the genera Carassiusand Cyprinus (family Cyprinidae) originate from acommon tetraploid ancestor and that the genome havestabilized in the diploid state in the majority of species[10]. The genera Carassius and Cyprinus include bisex�ual species whose chromosome numbers correspond�ing to tetraploid (2n = 100), while the species are actu�ally diploid. The species include crucian carp Caras�sius carassius, common carp Cyprinus carpio, andcertain others.

The genus Carassius includes the crucian carp Car�assius carassius and silver crucian carp Carassius aura�tus, which are well differentiated morphologically. Theformer inhabits plant�grown water bodies and bottom�land lakes of North and East Europe and Siberia. TheC. auratus species area is broader and includes EastEurope, Siberia, the Russian Far East, Sakhalin, thebasins of the largest Central Asian rivers Syr Darya andAmu Darya, Korea, China, and Japan [11].

Seven subspecies were recognized until recently inthe genus Carassius auratus on the basis of severalmorphological characters [11, 12]: C. a. auratus L.,which is widespread in China and is an ancestor of allornamental goldfish; C. a. gibelio, which is widespreadin Russia and northern China; and five Japanese sub�species (C. a. grandoculis Temm. et. Schl., C. auratussubsp., C. a. langsdorfi Temm. et Schl., C. a. cuvieriTemm. et Schl., and C. a. buergeri Temm. et Schl.)[13].

There is another substantial difference between C.auratus and C. carassius in addition to their differencesin morphology and species area. While C. carassius (2n= 100) is a typical diploid species like the vast majorityof vertebrates, C. auratus occurs in two forms, whichhave different types of reproduction (gonochory andgynogenesis) and differ in ploidy [14]. The diploidchromosome set of the bisexual form consists of 100chromosomes, while both triploid and tetraploidchromosome sets are found in the gynogenetic form(2n ≈ 150 and 4n ≈ 200) [15]. According to the currentclassification, the above C. auratus subspecies are con�sidered to be separate species: Carassius langsdorfi,C. grandoculis, C. buergeri, C. cuvieri, C. auratussubsp., and C. gibelio, although the taxonomic status of

Phylogenetic Relationships of Silver Crusian Carp Carassius auratus gibelio, C. auratus cuvieri, Crucian Carp Carassius carassius, and

Common Carp Cyprinus carpio As Inferred from Mitochondrial DNA Variation

O. V. Apalikovaa, A. V. Podlesnykha, A. D. Kukhlevskya, b, S. Guohuac, V. A. Brykova, b

aZhirmunsky Institute of Marine Biology, Russian Academy of Sciences, Vladivostok, 690041 Russia;e�mail: [email protected], [email protected]

bDepartment of Genetics, Far East State University, Vladivostok, 690600 RussiacMarine Fisheries Research Institute of Shandong, 264006 China

Received July 29, 2009

Abstract—PCR–RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotidesequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the fam�ily Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic sta�tus. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after itsseparation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japa�nese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggestingdivergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possiblyrelated to the formation of two silver crucian carp groups with different types of reproduction, triploid gyno�genetic and diploid gonochoric. At the same time, the results supported the high probability of current geneticexchange between the forms. In view of these findings and high morphological similarity of the two forms,they were not considered to be separate species.

DOI: 10.1134/S1022795411020025

ANIMAL GENETICS

Fajar

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RUSSIAN JOURNAL OF GENETICS Vol. 47 No. 3 2011

PHYLOGENETIC RELATIONSHIPS OF SILVER CRUSIAN CARP 323

C. auratus is still open for discussion [13, 16]. This isrelated primarily to the fact that both diploid and poly�ploid forms were found for the majority of these spe�cies. The exceptions are Carassius auratus subsp. andC. grandoculis, which lack gynogenetic forms. Theavailable data on a triploid form of Carassius cuvieriare discrepant [17].

Karyotype [14, 18–21] and biochemical (alloz�yme) [22–24] studies demonstrate an intricate geneticnature of Carassius auratus. The origin of the polyp�loid gynogenetic form is still poorly understood [13,16, 23]. Since such forms are known for several C.auratus subspecies, their origin via a common mecha�nism seems questionable. Hybridogenesis is the mostlikely mechanism yielding a triploid form in C. aura�tus. However, if this is true, the parental species thathave given origin to a gynogenetic form are stillunknown. It is unclear if one or two species might beinvolved in the formation of the polyploid gynogeneticform of C. auratus [25–27]. The objective of this workwas to study the phylogenetic relationships of the carpspecies on the basis of their interspecific and intraspe�cific mtDNA variations. Owing to the specifics ofmtDNA evolution, species and genetically isolateddivergent intraspecific units form separate, well distin�guishable mtDNA phylogroups as a result of randomprocesses or selection.

MATERIALS AND METHODS

Sample collection and DNA isolation. Biologicalmaterial for a cytological examination for ploidy andanalysis of mtDNA variation included samples of thesilver crucian carps Carassius auratus gibelio (Bloch)and C. auratus cuvieri, crucian carp Carassius caras�sius, and common carp Cyprinus carpio and was col�lected from 2001 to 2009 in water bodies of Primorye,Kamchatka, Khabarovsk krai, Uzbekistan, EuropeanRussia, China, and Japan (Table 1, Fig. 1). Total DNAwas isolated according to a standard protocol [28].

DNA amplification. Two mtDNA regions were ampli�fied using two oligonucleotide primer pairs. TheND3/ND4L/ND4 region was amplified with primers5’�CACGGCCCCCTTATGACA�3’ and 5’�TGGGA�CAAAAATTAGGGAGTAGTG�3’, and the 12S/16SrRNA region was amplified with primers 5’�CTAC�CCGGGGACGAGGAG�3’ and 5’�ATAGCGGCTG�CACCATTAGG�3’.

The PCR mixture (50 μl) contained 2 units of TaqDNA polymerase, 5 μl of a 10 × Taq buffer (SibEn�zyme), 2 mM each dNTP, 0.25 μM each primer, andapproximately 150 ng of total DNA.

Amplification included initial denaturation at94°C for 5 min; 35 cycles of denaturation at 94°C for0.5 min, primer annealing at 54°C (in the case of theND3/ND4L/ND4 mtDNA fragment) or 56°C (in thecase of the 12S/16S rRNA fragment) for 1 min, andelongation at 72°C for 2.4 min; and last synthesis at68°C for 4 min. The amplified fragments were electro�phoretically separated in 1% agarose gel in 50 mM

Table 1. Collection sites and samples used for RFLP analysis of mtDNA fragments

No. Locality, year Species representedin the sample Sample size Haplotypes

Ploidy Carassius

auratus gibelio

1 Kamchatka River, Kamchatka, Far East, Russia, 2004

Carassius auratus gibelio

29 H 2n

2 Bezymyannoe Lake, Sakhalin, Far East, Russia, 2006

C. auratus gibelio 50 A, H 2n, 3n

3 Lake near the town of Dal’negorsk, Primorye, Far East, Russia, 2006

C. auratus gibelio 2 A 3n

4 Razdol’naya River, Primorye, Far East, Russia, 2003 and 2006

C. auratus gibelio 52 A, H 2n, 3n

5 Amur River, Far East, Russia, 2005 C. auratus gibelio(27), Cyprinus carpio(3)

30 A, K***, L***, M***

2n, 3n

6 Khanka Lake, Primorye, Far East, Russia, 2006

Carassius auratus gibelio

20 A, B, F 2n, 3n

7 Iijima, Hasama, Tome Miyagi, Moguri Pum�ping Site, Japan, 2008

C. auratus cuvieri 4 I* 2n

8 Yantai, China, 2008 C. auratus gibelio 9 A 3n9 Syr Darya River, Uzbekistan, 2006 C. auratus gibelio 20 A, B, C, D, E 2n, 3n

10 Rybinsk Water Reservoir, Russia, 2007 C. auratus gibelio(34), Carassius carassius(2)

36 A, G, J** 2n, 3n

11 Tumangan Riber, 2004 and 2007 C. auratus gibelio 64 A, B, H 2n, 3n

Note: Haplotypes characteristic of (*) C. auratus cuvieri, (**) Carassius carassius, and (***) Cyprinus carpio are indicated.

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APALIKOVA et al.

Tris–borate. Gels were stained with ethidium bro�mide, and DNA was visualized in transmitted UV light[28].

Electrophoresis of the restriction fragments of theamplified mtDNA regions. Each of the amplified frag�ments was digested with several restriction enzymes.We used AvaI, BsuRI, Cfr13I, FnuDII, Hin6I, HinfI,MspI, MvaI in the case of the ND3/ND4L/ND4region and AvaI, BsuRI, Cfr13I, HinfI, MspI, MvaI,and RsaI in the case of the 12S/16S rRNA region; theenzymes were from Fermentas (Lithuania) andSibEnzyme (Novosibirsk).

After digestion, the samples were electrophoreti�cally separated in 1.8% agarose gel in 50 mM Tris–borate [28]. The DNA fragments were stained withethidium bromide and photographed in transmittedUV light. The molecular weight of the fragments wasestimated against a 100�bp DNA ladder (Gibco,Grand NY) and phage λ DNA digested with PstI.

Restriction fragment length polymorphism (RFLP)analysis. Based on the linear arrangement of therestriction fragments, a restriction map was con�structed for each of the restriction enzymes and eachof the amplified mtDNA regions. The maps were usedto establish the presence or absence of particular sites.Variants differing by the presence or absence of arestriction site as a result of mutations were designatedwith letters for each region and each restrictionenzyme. A binary code was used to designate the seg�regation variants for each fragment and each restric�tion enzyme, 1 suggesting the presence of a restrictionsite, and 0 suggesting its absence. The results obtainedfor the two mtDNA regions were pooled over all indi�vidual fish, all sites, and all restriction enzymes toyield combined haplotypes, which each were desig�

nated with one letter (Table 2). In total, we observed104 restriction sites, of which 64 were polymorphic.

Haplotype diversity h, nucleotide diversity π, andhaplotype divergence p were calculated according toNei [29], using the REAP software package [30]. Theresulting REAP matrix of the haplotype divergencevalues was employed in clustering and constructing aphenogram by UPGMA (NTSYS package [31])(Table 3).

Sequencing of the cytochrome b gene fragment. A frag�ment of the cytb gene was amplified in a reaction mix�ture (12.5 μl), which contained 1.25 μl of a 10 × PCRbuffer (750 mM Tris–HCl, pH 8.8, 200 mM(NH4)2SO4), 1.25 μl of 10 mM dNTPs (2.5 mM eachdNTP), 1.25 μl of a solution containing 2.5 μM eachof the primers, 1.25 μl of 25 mM MgCl2, 10 ng ofDNA, and 1 unit of Taq DNA polymerase. The prim�ers were FishCytB�F (5'�ACC ACC GTT GTT ATTCAA CTA CAA GAA C�3'') and THR�Fish�R (5'�ACC TCC GAT CTT CGG ATT ACA AGA CC�3').Amplification of the cytb gene included initial dena�turation at 94°C for 3 min; 35 cycles of 94°C for 30 s,57°C for 30 s, and 72°C for 1 min 30 s; and last synthe�sis at 68°C for 5 min. The purity and length of the reac�tion products were checked in 1% agarose gel.

The purified amplification product was sequencedusing a Big Dye Terminator v. 3.1 Cycle Sequencing kit(Applied Biosystems). The DNA sequence was estab�lished using an ABI Prism 3130 sequencer. For eachsample, the forward and reverse sequencing productswere aligned using the SeqScape v. 2.5 program(Applied Biosystems). The resulting fragments of thecytb gene sequence were deposited in NCBI/Gen�Bank (Table 4). Sequence alignment utilized theClustalW algorithm. The final length of the cytb gene

10

9 11

6 4

35

8

7

2

1

Russia

China

Ja

pa

n

Fig. 1. Localities where the samples were collected.



sequence was 765 bp, including 76 (9.9%) variable and92 (12.0%) parsimony�informative sites. The optimalnucleotide substitution model was computed usingModeltest 3.7 [32]. Phylogenetic trees were con�structed by the maximum parsimony (MP) and Baye�sian maximum likelihood (BI) methods, using thePAUP 4.0 b 10 [33] and MrBayes v. 3.1.2 [34] pro�grams, respectively. In either case, we used the nucle�otide substitution model HKY + G (k = 4) andMSMC with 1100000 generations and discretizationby every 200 generations. The stability of the resulting

phylogenetic trees was checked by bootstrap analysis[35] with 1000 bootstrap replications (in the case ofthe MP tree).

Ploidy Analysis in Carassius auratus Individuals

Determination of the erythrocyte nucleus area. Tomeasure the erythrocyte nucleus area, preparationswere obtained using the blood taken from the tailartery [36]. The preparations were examined under aLeica DM2500 microscope (magnification ×100).

Table 2. Combined haplotypes of the species under study

Haplo�type Species

ND3/ND4 12S/16S rRNA

Ava

I

Bsu

RI

Cfr

13I

Fnu

DII

Hin

6I

Hin

fI

Msp

I

Mva

I

Ava

I

Bsu

RI

Cfr

13I

Fnu

DII

Hin

fI

Msp

I

Mva

I

Rsa

I

A C. a. gibelio A A B A A A B A A A A A A A A A

B '' A A B A A A A A A A A A A A B A

C '' A A B A A B A C A C A A A A A A

D '' A C C A A B A C A A A A A A A A

E '' A D B A A B A C A A A A A A A A

F '' A A B A A A B E A A A A A A B A

G '' A A B A A A B D A A A A A A A A

H '' A B B A A B C B A A B A A B A B

I C. a. cuvieri A E B B B C D G A A C A A B B A

J C. carassius B F A C C D E C A B C A A B C A

K Cyprinus carpio A G A D D E F F A A D A A C C C

L '' A G A D D E F F A A E A A C C C

M '' A H A D D E F F A A D A A C C C

Table 3. Matrix of the distances between haplotypes as inferred from the RFLP data (see Table 1 for haplotype designations)

A B C D E F G H I J K L M

A 0.000

B 0.004 0.000

C 0.009 0.009 0.000

D 0.013 0.013 0.007 0.000

E 0.015 0.015 0.009 0.013 0.000

F 0.008 0.007 0.017 0.020 0.022 0.000

G 0.002 0.005 0.010 0.015 0.016 0.010 0.000

H 0.028 0.029 0.023 0.026 0.025 0.036 0.030 0.000

I 0.037 0.033 0.035 0.039 0.037 0.040 0.034 0.026 0.000

J 0.065 0.067 0.069 0.074 0.067 0.069 0.067 0.064 0.059 0.000

K 0.068 0.068 0.075 0.080 0.073 0.071 0.069 0.070 0.066 0.050 0.000

L 0.069 0.070 0.077 0.082 0.075 0.073 0.071 0.072 0.067 0.052 0.002 0.000

M 0.069 0.070 0.077 0.082 0.075 0.073 0.071 0.072 0.068 0.052 0.002 0.003 0.000

326


APALIKOVA et al.

To identify the ploidy, a frequency distribution wasconstructed for erythrocyte nucleus area values, whichwere expressed in conventional units (pixels) [37].

Number of nucleoli in erythrocyte nuclei. As an addi�tional test for ploidy of Carassius auratus individuals,we counted the nucleoli in erythrocyte nuclei, usingblood preparations stained with 50% AgNO3 [38]. Thenumber of nucleoli in cell nuclei, which is suggestiveof the ploidy [39], was analyzed statistically. The pro�portion of nuclei with undetectable nucleoli, nucleihaving one nucleolus each, and nuclei having twonucleoli each was approximately 10% : 60% : 30%,respectively, in diploid fish. Some diploid fish in bothfirst and second mtDNA phylogroups had a certainportion of erythrocytes (up to 5%) whose nuclei con�tained three detectable nucleoli. In triploids, the pro�portion of cells with one, two, and three nucleoli wasapproximately 30% : 40% : 40%, respectively. In tetra�ploids, the proportion was biased towards a larger por�tion of cells with three or more nucleoli and wasapproximately 10% : 30% : 40% : 20%, respectively.

RESULTS

To study the phylogenetic relationships of two silvercrucian carp Carassius auratus gibelio forms, one sub�species of the Japanese silver crucian carp C. auratuscuvieri, crucian carp Carassius carassius, and common

carp Cyprinus carpio, we compared their mtDNAs.Assuming that the gynogeneic Carassius auratus formresults from hybridization, mtDNA analysis allows areliable identification of at least one of the speciesinvolved in the process.

PCR�RFLP analysis of two mtDNA regions,ND3/ND4 and 12S/16S rRNA, in Cyprinus carpio,Carassius carassius, the Japanese subspecies of C. aura�tus cuvieri, and C. auratus gibelio showed that theC. auratus forms belong to a separate cluster, whichincludes two groups of C. auratus gibelio haplotypes(Fig. 2). One phylogroup included haplotypes A, B, C,D, E, F, and G, which were found in both diploid andpolyploid silver crucian carps from all regions exam�ined, including Primorye, China, Middle Asia, andEuropean Russia. The other phylogroup included onehaplotype, H, which was found only bisexual (diploid)fish from the Russian Far East (Table 1, Fig. 2). The sec�ond C. auratus gibelio mtDNA phylogroup clusteredtogether with C. auratus cuvieri haplotype I (Fig. 2).

The cluster of the first C. auratus gibelio phylogroupincludes two haplotype subgroups. One includes hap�lotype A, which was the most common throughout thespecies area; haplotype G, which was found only infish from the Rybinsk Water Reservoir; and haplotypeB, which was detected in fish from the Khanka Lake,the basin of the Tumangan River in southern Pri�morye, and in the basin of Syr Darya (Table 1). The

Table 4. Fish of the genus Carassius used to analyze the cytochrome b gene sequence

Sample Species and subspecies C. auratus gibelio ploidy NCBI/GenBank accession no. Ordinal number (see Table 5)

1 C. auratus gibelio 2n FJ822044 4


FJ478016 12



2n FJ478017 13

FJ822045 14

5 Cyprinus carpio FJ478020 16


4n FJ822041 17

7 C. auratus cuvieri FJ822042 2

FJ822043 3


3n FJ822047 7

3n FJ822048 8

10 C. auratus gibelio 2–3n FJ478012 9

10 Carassius carassius FJ478014 10



haplotype clustered with haplotype F, which wasfound in one triploid male from the Khanka Lake. Aseparate group within the first phylogroup consisted ofthree unique haplotypes (C, D, and E), which werefound in fish from the basin of Syr Darya (Fig. 2).

The genetic distance between the two most com�mon haplotypes A and H, which belonged to two dif�ferent phylogroup, was 2.8% nucleotide substitutions.The separation of the two phylogroups had a bootstrapsupport of 99%. The distance between haplotype Hand haplotype I, which was found in Japanese C. aura�tus cuvieri, was 2.6% nucleotide substitutions, whilethe distance between haplotype I and haplotypes of thefirst C. auratus gibelio group varied from 3.3% to 4%nucleotide substitutions. Carassius carassius mtDNAdiffered from the haplotypes of the two C. auratus gibe�lio phylogroups by approximately 7% (from 6.5%between haplotypes A and J or H and J to 7.4%between haplotypes J and G). The distance betweenthe Carassius carassius haplotype and C. auratuscuvieri haplotype I was 6.9% nucleotide substitutions(Table 3).

The distance between the Cyprinus carpio haplo�types (K, L, and M) and the Carassius auratus gibeliohaplotypes ranged from 6.8% to 8% nucleotide substi�tutions. The Cyprinus carpio haplotypes differed fromC. auratus cuvieri haplotype I by 6.6–6.8% nucleotidesubstitutions. The distance between Cyprinus carpioand Carassius carassius haplotypes ranged from 5% to5.2% nucleotide substitutions (Table 3).

To study the nucleotide sequence variation of thecytochrome b gene, we sequenced a 765�bp region.Cyprinus carpio from the Amur River was used as anoutgroup. According to the topology of a NJ tree, Car�assius carassius and the two C. auratus subspeciesformed two separate clusters (Fig. 3). The C. auratusbranch diverged into a branch of Japanese C. auratuscuvieri and a branch of C. auratus gibelio with a highbootstrap support (80%). In turn, the latter divergedinto two clusters. One of these included diploidC. auratus gibelio fish, which represented the secondhaplotype group, while the other combined diploid andtriploid C. auratus gibelio fish, which belonged to thefirst phylogroup. The bootstrap support of the separa�tion of the two clusters was higher than 90% (Fig. 3).Note that the position of Carassius carassius slightlydiffered between the two phylogenetic tress. The spe�cies clustered with Cyprinus carpio on the tree based onthe PCR–RFLP data (Fig. 2) and with various Caras�sius auratus forms on the tree based on the cytochromeb gene sequence (Fig. 3).

DISCUSSION

The wo methods, RFLP analysis of theND3/ND4L/ND4 and 12S/16S rRNA regions andanalysis of the nucleotide sequence variation of thecytochrome b gene, yielded similar divergence esti�mates for the two Carassius auratus subspecies, Caras�

sius carassius, and Cyprinus carpio and produced simi�lar phylogenetic trees (Tables 3, 5; Figs 2, 3). ThemtDNA variants of the Carassius auratus mtDNAhaplogroups formed a separate cluster on both of thephylogenetic trees. One branch of the cluster includeda group of diploid and triploid Carassius auratus gibeliofish. The haplotypes of this group were found in thesamples from the Razdol’naya Riber, Khanka Lake,the lake close to the town of Dal’negorsk, the basin ofSyr Darya, the Rybinsk Water Reservoir, and a waterbody from Shangdong Province (China). The othercluster included the second�phylogroup haplotype,which was observed in diploid C. auratus fish from theRazdol’naya Riber, Bezymyannoe Lake (Sakhalin),and Kamchatka River (Kamchatka) (Figs. 2, 3). Thus,our results support a certain relationship between thefish ploidy and particular mtDNA phylogroup [37].Takada et al. [40] studied the mtDNA variation in theCarassius auratus complex, which seems to be identi�cal to Carassius auratus gibelio. Analysis of a large fishsample from many water bodies of the JapaneseIslands, Taiwan, and continental Eurasia similarlyrevealed two mtDNA phylogroups (which weretermed “superclades”). At the same time, the results[40] did not associate the ploidy with a particularmtDNA superclade.

A comparison showed that Carassius carassiusmtDNA differs from Carassius auratus gibelio mtD�NAs of the two phylogroups by approximately 6%nucleotide substitutions. The major haplotypes (A andH) of the two C. auratus mtDNA phylogroups areequidistant from C. carassius mtDNA. The mitochon�drial lineages of the two Carassius species were approx�imately equidistant from Cyprinus carpio mtDNA; i.e.,the distance between Carassius auratus and Cyprinuscarpio mtDNA haplotypes was 6–8%, while the dis�tance between Carassius carassius and Cyprinus carpio

AGBFCDEHIJKML

7655

64

92

514699

55

88 48100

0.07 0.05 0.02 0

Fig. 2. UPGMA phenogram of genetic distances betweenC. auratus gibelio, C. auratus curviari, C. carassius, andCyprinus carpio haplotypes. Bootstrap supports (1000 rep�lications) of the branches on a topologically similar tree isindicated at the nodes.

328


APALIKOVA et al.

haplotypes was 7% nucleotide substitutions (Table 3).The phylogenetic position of Carassius carassius isquestionable because of this similarity of the distances,since Carassius carassius clustered with Cyprinus car�pio in one tree (Fig. 2) and with Carassius auratus inthe other tree (Fig. 3). Hence, it is possible to assumethat the ancestral forms of the three species divergedapproximately at the same time. If molecular clock isused to estimate the divergence time, early divergencecorresponds to the early Pliocene 4–6 Myr ago). Thisperiod is characterized by dramatic paleoclimaticchanges, which affected species evolution [41, 42].

Our results support the idea that the two Carassiusauratus forms are genetically close to Carassius caras�sius and Cyprinus carpio. The divergence between thesecond mtDNA phylogroup, which represents thebisexual C. auratus gibelio form, and Japanese C. aura�tus cuvieri was 2.6% (Table 3). It is noteworthy thatmuch the same divergence, approximately 2.5%, wasobserved between the two mtDNA phylogroups of C.auratus gibelio. The divergence between the first C.auratus gibelio phylogroup and C. auratus cuvieri washigher, up to 4% nucleotide substitutions. A distance

of 3.7% separated the Japanese C. auratus cuvieri hap�lotype and the most common haplotype (A) of thegynogenetic C. auratus gibelio form. This findingmakes it possible to assume that the C. auratus cuvierihaplotype, which represents the second mtDNA phy�logroup and occurs exclusively in the Russian FarEast, is younger than haplotype A, characteristic of thegynogenetic form. It is still unclear why the differencein mtDNA between the two C. auratus gibelio forms isso great, comparable with the difference between thesubspecies C. auratus cuvieri and C. auratus gibelio.

Our findings and published data [40] suggest onemore step of divergent evolution for the group. TheJapanese species C. auratus cuvieri separated from theancestral form approximately at the same time whenthe two mtDNA phylogroups formed within C. auratusgibelio. The two phylogroups were independent for along time, as evident from the absence of transitionalhaplotypes, which were eliminated via sorting [37].However, the two mtDNA forms mixed during a morerecent secondary contact, and both of them are foundnow in the same populations. This step of divergencewas possibly related to the origin of the gynogenetic

C. auratus gibelio FJ822048













C. auratus cuvieri FJ822042

C. auratus cuvieri FJ822043

Carassius carassius FJ478014

Cyprinus carpio FJ478020

10

85/97

100/94

100/97

84/98100/94

86/77

62/93

60/74

100/100

I

II100/100

Fig. 3. MP tree (50% consensus) constructed to illustrate the phylogenetic relationships of the genus Carassius and Cyprinus car�pio on the basis of the cytb gene sequence. Bootstrap support of the branches of the MP tree (n = 1000) and support of the brancheson the BI tree (%) are indicated at the nodes. The MP and BI trees were constructed using the nucleotide substitution modelHKY+G. Phylogroups I and II are indicated on the right.



form. The computed divergence time, 1 Myr ago, cor�responds to the early Pleistocene, which was charac�terized with changes in the paleoclimate of the North�ern hemisphere with periodical climatic fluctuationand extension or reduction of glaciers [41, 42]. Evi�dence against the assumption that mtDNA divergencewas due to the origin of the gynogenetic form is pro�vided by the fact that triploid gynogenetic forms arefound in different Carassius auratus subspecies [13,16], suggesting their independent origin [40]. Our datasupport the idea that genetic exchange between theforms is highly probable now. Thus, based on our find�ings and the high morphological similarity of the twoforms, the forms cannot be considered to be separatespecies.

Our results fail to provide a certain answer as towhether the other species, Carassius auratus and Cyp�rinus carpio, were involved in the formation of the C.auratus polyploid gynogenetic form. The cause is thatmtDNA is inherited exclusively maternally. It is clearthat only Carassius auratus females could be involvedin the formation of the gynogenetic form via hybrid�ization. Hybridization with unidirectional mtDNAtransfer from only one parental species to a hybrid

form is known for fish [43]. To identify the otherancestral species, it is necessary to compare nucleargenes, which are inherited from both of the parents bythe hybrid form.

ACKNOWLEDGMENTS

We are grateful to Yu.M. Kovalev for help in mate�rial collection and to Kanji Saitoh (Tohoku NationalFisheries Research Institute).

This work was supported by the Far East Divisionof the Russian Academy of Sciences (project no. 09�III�A�192) and the Russian Foundation for BasicResearch (project no. 10�04�91164 GFEN_a).

REFERENCES

1. Soltis, D.E. and Soltis, P.S., Polyploidy: RecurrentFormation and Genome Evolution, Trends Ecol. Evol.,1999, vol. 14, pp. 348–352.

2. Leggatt, R.A. and Iwama, G., Occurrence of Polyp�loidy in Fishes, Rev. Fish Biol. Fisher., 2003, vol. 13,pp. 237–246.

Table 5. Genetic distances between the cytochrome b gene regions sequenced in samples 1–17 (ordinal numbers of the samplesare shown in Table 4)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 92.7 92.7 94.9 99.3 99.3 99.3 99.3 99.9 91.5 95.0 95.2 95.0 99.3 99.5 87.1 99.6

2 7.9 99.7 92.0 92.3 92.3 92.3 92.3 92.5 90.5 92.2 92.3 91.9 92.3 92.4 84.8 92.5

3 7.9 0.3 92.0 92.3 92.3 92.3 92.3 92.5 90.5 92.2 92.3 91.9 92.3 92.4 85.0 92.5

4 5.4 8.6 8.6 95.0 95.0 95.0 95.0 94.8 90.6 99.9 99.7 99.9 95.0 95.2 86.3 95.3

5 0.7 8.3 8.3 5.2 100.0 100.0 100.0 99.2 91.4 95.2 95.3 95.2 100.0 99.6 87.1 99.7

6 0.7 8.3 8.3 5.2 0.0 100.0 100.0 99.2 91.4 95.2 95.3 95.2 100.0 99.6 87.1 99.7

7 0.7 8.3 8.3 5.2 0.0 0.0 100.0 99.2 91.4 95.2 95.3 95.2 100.0 99.6 87.1 99.7

8 0.7 8.3 8.3 5.2 0.0 0.0 0.0 99.2 91.4 95.2 95.3 95.2 100.0 99.6 87.1 99.7

9 0.1 8.0 8.0 5.5 0.8 0.8 0.8 0.8 91.4 94.9 95.0 94.9 99.2 99.3 86.9 99.5

10 9.2 10.4 10.4 10.3 9.4 9.4 9.4 9.4 9.4 90.7 90.7 90.5 91.4 91.4 86.1 91.5

11 5.2 8.5 8.5 0.1 5.1 5.1 5.1 5.1 5.1 10.1 99.9 99.7 95.2 95.3 86.4 95.4

12 5.1 8.3 8.3 0.3 4.9 4.9 4.9 4.9 5.2 10.1 0.1 99.6 95.3 95.4 86.5 95.6

13 5.2 8.8 8.8 0.1 5.1 5.1 5.1 5.1 5.4 10.5 0.3 0.4 95.2 95.3 86.4 95.4

14 0.7 8.3 8.3 5.2 0.0 0.0 0.0 0.0 0.8 9.4 5.1 4.9 5.1 99.6 87.1 99.7

15 0.5 8.2 8.2 5.1 0.4 0.4 0.4 0.4 0.7 9.4 4.9 4.8 4.9 0.4 87.2 99.9

16 14.5 17.4 17.3 15.5 14.5 14.5 14.5 14.5 14.7 15.7 15.4 15.2 15.4 14.5 14.4 87.3

17 0.4 8.0 8.0 4.9 0.3 0.3 0.3 0.3 0.5 9.2 4.8 4.7 4.8 0.3 0.1 14.2

Note: Percent similarity and percent difference are shown above and below the diagonal, respectively.

330


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