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Heredity (2014) 112, 415–427 & 2014 Macmillan Publishers Limited All rights reserved 0018-067X/14 www.nature.com/hdy

ORIGINAL ARTICLE Tempo and mode of recurrent polyploidization in the auratus complex (, )

JLuo1,6, Y Gao2,6,WMa1, X-y Bi1, S-y Wang2,3, J Wang1, Y-q Wang1, J Chai1,RDu1, S-f Wu2,AMeyer4, R-g Zan1, H Xiao1, RW Murphy2,5 and Y-p Zhang1,2

Polyploidization is an evolutionarily rare but important mechanism in both and because it increases genetic diversity. Goldfish of the Carassius auratus species complex can be tetraploids, hexaploids and octaploids. Polyploidization events have occurred repeatedly in goldfish, yet the extent of this phenomenon and its phyletic history are poorly understood. We explore the origin, tempo and frequency of polyploidization in Chinese and Japanese goldfish using both mitochondrial (mtDNA) and nuclear DNA sequences from up to 1202 individuals including the outgroup taxon, carpio. Analyses of de novo nuclear gene data resolve two clusters of alleles and the pattern supports the prior hypothesis of an ancient allotetraploidization for Carassius. Alleles shared by tetraploid and hexaploid individuals indicate recent autoploidizations within the C. auratus complex. Sympatric tetraploids and hexaploids share mtDNA haplotypes and these frequently occur independently within six well-supported lineages and sublineages on a small spatial scale. Gene flow estimates (Fst values) indicate that hexaploids differ only slightly from sympatric tetraploids, if at all. In contrast, allopatric populations of tetraploids and hexaploids differ from one another to a far greater extent. Gene flow between sampled localities appears to be limited. Coalescence-based time estimations for hexaploids reveal that the oldest within any sampled locality is around one million years old, which is very young. Sympatric, recurrent autoploidization occurs in all sampled populations of the C. auratus complex. Goldfish experience polyploidization events more frequently than any other . Heredity (2014) 112, 415–427; doi:10.1038/hdy.2013.121; published online 8 January 2014

Keywords: goldfish; autopolyploidization; tetraploid; hexaploid

INTRODUCTION in some lineages of invertebrates such as ostracods and . Some The frequency and hence the evolutionary importance of polyploidi- species of fishes, amphibians and reptiles are polyploids and in some zation for diversification differs greatly between plants and animals. evolutionary lineages of fishes polyploidization is not uncommon More than 70% of angiosperms experienced one or more episodes of (Dawley, 1989; Otto and Whitton, 2000; Leggatt and Iwama, 2003). polyploidization and about 95% of species are polyploids (Soltis Cyprinid fishes are of special interest in this regard because they are and Soltis, 1999; Van de Peer et al., 2009). Notwithstanding, whole- unusually rich in lineages, several of which are ancient genome duplications have contributed to the evolutionary success of (Otto and Whitton, 2000), including the Barbinae . Extensive comparative genomic work reveals three rounds (Tsigenopoulos et al., 2002), and Schizothoracinae of whole-genome duplication early in teleost diversification (Ohno, (Yu et al., 1989; Buth et al., 1991; Le Comber and Smith, 2004). Full 1970; Vandepoele et al., 2004; Van de Peer et al., 2009). The genomes genomic duplications have occurred in at least 40 cyprinid species of all 25 000 or so species in this lineage of vertebrates reflect this (Yu et al., 1989; Buth et al.,1991). whole-genome duplication event. In fact, whole-genome duplication Recurrent polyploidization events occur either by interspecific appears to be causally related to their evolutionary success (Wittbrodt hybridization and the fusion of two or more genomes (allopoly- et al., 1998). Generally speaking, polyploidization occurs only rarely ploidy) or by genome doubling within a species or a population in animals, especially in vertebrates, in part because sex determination (autopolyploidy). In plants, most cases of polyploidization occur via and development are disrupted by polyploidization (Mable, 2004; allopolyploidization, and this process is thought be an important Mable et al., 2011). Among animals, polyploidization is common only mechanism for the rapid formation of species (Soltis and Soltis, 1999;

1Laboratory of Conservation and Utilization of Bio-resources and Key Laboratory for Genetic Diversity and Evolution of High Education in Yunnan Province, School of Sciences, Yunnan University, Kunming, Yunnan, ; 2State Key Laboratory of Genetic Resources and Evolution and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China; 3School of life sciences, University of Science and Technology of China, Hefei, Anhui, China; 4Lehrstuhl fu¨ r Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, Konstanz, Baden-Wu¨ rttemberg, and 5Centre for and , Royal Ontario Museum, Toronto, ON, Canada 6These authors contributed equally to this work. Correspondence: Professor Y-p Zhang, State Key Laboratory of Genetic Resources and Evolution and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China. E-mail: [email protected] Received 13 February 2013; revised 29 September 2013; accepted 1 October 2013; published online 8 January 2014 Repeated polyploidization in Carassius JLuoet al 416

Hegarty and Hiscock, 2008; Van de Peer et al.,2009;Huet al., 2011). Japanese (silver crucian ), nagobuna, and Polyploidization may be involved in about 2–4% of the gengorobuna; the latter is sometimes considered to be the species events in angiosperms and , respectively (Otto and Whitton, Carassius cuvieri (Takada et al., 2010 and therein). C. carassius occurs 2000), but only a handful of these cases are autopolyploidization from the Ertis River in China westwards to Central and Eastern events (Segraves et al., 1999; Parisod and Besnard, 2007). In contrast, and the British Isles (Luo and Yue, 2000; Wheeler, 2000); only in a single case of multiple autopolyploid origins among C. carassius may no longer occur in the Ertis River in China, based on invertebrates is known: the lepidopteron Solenbia triquetrella (Psy- our attempts to locate the species. C. auratus (Cypriniformes, chidae) (Lokki and Saura, 1980). Little is known about the extent of Cyprinidae) is a complex of tetraploid and hexaploid fishes. Indivi- multiple autopolyploid origins in vertebrates, and some possible cases duals within each lineage are morphologically similar yet their are now believed to represent allopolyploidization (for example, numbers and reproductive strategies vary. Except for Holloway et al., 2006). exclusively tetraploid C. cuvieri, bisexual tetraploid and gynogenetic The genus Carassius has a recent, allotetraploid origin (Buth, 1983; hexaploid and occasional octaploid forms occur sympatrically in Risinger and Larhammar, 1993; Yang and Gui, 2004; Luo et al., 2006). China, Russia and (Golovinskaya et al., 1965; Chen et al., Within Carassius, at least two species are recognized: Carassius 1996; Murakami et al., 2001; Brykov et al., 2002). Multiple levels of carassius (the ) and C. auratus, a complex of several polyploidy occur in Fangzheng, Puan and Lake Dianchi, China (Zan, lineages (Table 1) that includes the silver crucian (gibel) carp and 1982; Zan et al., 1986 and therein; Chen et al., 1996 and therein).

Table 1 Breed, distribution, chromosome number and of the genus Carassius

Species, Distribution Reproductive Male/female Chromosome Ploidy Karyotype formula Source or local types strategy no.

Carassius carassius Central &Eastern Sexual Male, 100 4n ? Vinogradov, 1998 (Crucian carp) Europe, female British Isles C. auratus auratus Euro-Asia Sexual Male, 100 4n 22 M þ 30SM þ 48 T. Zan et al., 1986; Wang et al.,1988 (Goldfish) Continent female ST 24 M þ 30SM þ 46ST-T C. auratus gibelio Euro-Asia Gynogenesis Female 156± 6n 42 M þ 74SM þ 40ST-T Shen et al.,1983 (Gibel carp) Continent C. auratus gibelio Euro-Asia \ Male, 162± ?48Mþ 56SM þ 18ST þ 40 T Shan et al.,1988 (Gibel carp) Continent female C. auratus (back Yuannan Gynogenesis Female 162± 6n 33 M þ 53SM þ 76ST-T Zan, 1982 high type) mainly C. auratus Hunan Sexual Female 156 4n 36 M þ 36SM þ 84ST Yu et al.,1987 (Suogu breed) C. auratus (Qihe Henan Gynogenesis ? 156± 6n ? Lou et al.,1989 breed) C. auratus (Puan Guizhou Gynogenesis Female 156± 4n 38 M þ 28SM þ 18ST þ 72 T Yu et al.,1992 breed, A) C. auratus Jiangxi Gynogenesis Male, 166± 4n 32 M þ 40SM þ 18ST þ 76 T Chen et al.,1996 (Pengze breed) female C. a. langsdorfi Japanese Islands Gynogenesis Female 152–158 6n 34 M þ 62SM þ 60 T Kobayashi, 1978; Ueda and Ojima, (Ginbuna) 1978; Ojma and Yamano, 1980 C.s a. langsdorfi Japanese Islands Sexual Male, 100 4n 12 M þ 36SM þ 52 T Kobayashi, 1978; Ueda and Ojima, (Ginbuna) female 1978; Ojma and Yamano, 1980 C. cuvieri Japanese Islands Sexual Male, 100 4n 12 M þ 36SM þ 52 T Kobayashi, 1978; Ueda and Ojima, (Gengorobuna) female 1978; Ojma and Yamano, 1980 C. auratus. buergeri Japanese Islands Sexual Male, 100 4n 12 M þ 36SM þ 52 T Kobayashi, 1978; Ueda and Ojima, (Nagabuna) female 1978; Ojma and Yamano, 1980 C. a.buergeri Japanese Islands Gynogenesis Female 150± 6n ? Kobayashi, 1978; Ueda and Ojima, (Nagabuna) 1978; Ojma and Yamano, 1980 C. a. grandoculis Japanese Islands Sexual Male, 100 4n 12 M þ 36SM þ 52 T Kobayashi, 1978; Ueda and Ojima, (Nigorobuna) female 1978; Ojma and Yamano, 1980

Abbreviations: M, metacentric; SM, submetacentric; ST, subtelcentric; T, telcentric.

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The occurrence of allopatric polyploids questions may owe to two de novo sequences (total 346) that were accompanied by genome sizes and/or phenomena: independent origins or a rather ancient, single origin number of (Supplementary Table S1, part 1 and 2); (2) 18 gibel followed by dispersal to different drainage systems within Eurasia carp studied by Li and Gui (2008) (Supplementary Table S1, part 1); (3) six (Table 1). For Carassius,preferenceforonealternativeovertheother Japanese sequences from the study by Gao et al. (2012) along with 725 involves biogeography as well as the tempo and mode of polyploidiza- sequences they used from GenBank that had ploidy information (Supplementary Table S2); (Murakami et al., 2001; Iguchi et al.,2003; tion. (Takada et al., 2010) and (Gao et al., 2012) detailed the Takada et al., 2010); (4) three samples of C. cuvieri (Supplementary biogeography of the complex. RisingerandLarhammar(1993)and Table S3); (5) three samples of C. carassius from the study by Gao et al. (Buth et al., 1991 and therein) documented the mode of polyploidiza- (2012); (Supplementary Table S3); (6) 94 haplotypes downloaded from tion; allozymic and paralog information identified allo- and autopo- GenBank without ploidy information; and (7) seven sequences from carp lyploidization events. The tempo of polyploidization remains unknown. (C. carpio) for the outgroup (Supplementary Table S3). The second data set Analyses involving both maternally inherited mitochondrial DNA contained complete sequences of the gene encoding cytochrome b (cytb)plus (mtDNA) and nuclear genes can identify the maternal progenitor CR for 180 individuals taken from the study by Gao et al. (2012) including all (Evans et al., 2004) of polyploids. If two paralogs cluster in different taxa of C. carassius and the C. auratus complex (Supplementary Table S3). and with different species, then allopolyploidization provides Among these sequences, 1095 had ploidy information and these were used for the best explanation. When this pattern does not occur, we cannot population analyses. The remaining 107 sequences were used for constructing matrilineal trees only. reject the null hypothesis of autopolyploidization. The mtDNA gene- We investigated whether polyploidization in the goldfish occurred by either tree can identify the maternal progenitor within one of the nuclear auto-or allopolyploidization at the nuclear genomic level (Woods and Buth, gene clusters (Evans et al.,2004). 1984). We sequenced three nuclear genes (see below) from 15 individuals as The origin hexaploids, their population genetic features and their follows: four individuals of C. carassius, one silver crucian carp, one tetraploid relationship to tetraploids remains unclear. Although initial reports and one hexaploid goldfish each from Lake Dianchi, Zhejiang and Heilong- suggested that all hexaploid C. auratus are females, males have been jiang (six total), three gengorobuna (C. cuvieri) and one recently discovered in several populations (Lu et al., 2006; our (C. carpio). unpublished data). Hexaploids might have originated from tetraploid C. auratus auratus (Allendorf and Thorgaard, 1984). Normally, Ploidy determination bisexual tetraploid fish do not exclude the second polar cell until We used two methods to newly determine ploidy levels for 340 individuals from fertilization (Gui et al., 1995), but hexaploids possibly originate when the study by Gao et al. (2012) plus six new individuals: karyotyping, following the second diploid polar cell is not excluded from a tetraploid egg as a the study by Yu et al. (1989), and flow cytometry, as in the study by Luo et al. consequence of either hydrostatic pressure shock (Gui et al., 1995) or (2006). The chromosomal numbers of 167 individuals were visually determined heat/cold stock (Pandian and Koteeswaran, 1998). Most hexaploid from either using photographs or under a microscope (Olympus, individuals are females and they reproduce gynogenetically as sexual Tokyo, Japan). Flow cytometry used a fluorescence-activated cell sorter (FACS Vantage SE System, BD Biosciences, San Jose, CA, USA) to estimate ploidy parasites; females require exogenic sperm to stimulate egg develop- levels of 179 individuals. We modified the method of Luo et al. (2006) by ment (Gui, 2007). Because hexaploids are closely related to tetra- staining blood cells for 10 min and using 1:10 concentration of domesticated ploids, they can serve as a model system to explore the origin, history goldfish blood cells: target specimen cells to standardize genome size estimates. and mechanisms of autopolyploidization within species. We use our published sequence data (Gao et al.,2012)andthose DNA extraction, PCR amplification and nucleotide sequencing for the Japanese goldfish from GenBank along with de novo ploidy MtDNA fragment amplification and sequencing for six individuals with information for Eurasian Carassius to investigate the history and known ploidy levels were performed as in the study by Gao et al. (2012). possible multiple origins of polyploidy within the C. auratus complex. We targeted the CR because of its high level of variability. If hexaploidization is rare and originated only once or very few times We used the following primers for amplifying and sequencing three nuclear in independent drainages, we would expect hexaploids to be restricted genes: a partial fragment of the gene encoding macrophage migration to one or a few matrilines (Supplementary Figure S1). Alternatively, if inhibitory factor (MIF), 4MIF-50-RCGCCCAAAATAARCAATACT-30,4MIF- hexaploidization is not rare but rather independently reoccurs often, 50-FAAGATGTTGTCTGCTGTAAG-30; a partial fragment of the gene encoding then hexaploids should be widespread on the tree (Supplementary steroidogenic acuteregulatory (StAR), 2StAR-50-RGTGATGCTGGAA 0 0 0 Figure S1). We track the maternal history and population structure of CAGAAGAC-3 ,2StAR-5-FGCACAACGGACACTTACAAA-3 ;andapartial 0 tetraploid and hexaploid populations within the C. auratus complex fragment of the gene for growth hormone (GH)5-GH-U2TGCTGGTTAGTT TGTTGGTG-30, GH-50-L2GCTCYTCTGYGYTTCATCTTT-30. Amplifications using mtDNA sequence data, and then map polyploidization events were performed using the Gene Amp PCR system 9700 (ABI, Foster City, CA, onto the tree. Subsequently, we investigate the origin of the hexaploids USA). The total PCR volume was 25 ml with final concentrations of 1x buffer by calculating population coalescence times for each lineage. We containing 0.15 mM MgCl2 (TaKaRa, Dalian, China), 0.25 mM dNTPs sequence three nuclear genes to investigate the characteristics of (TaKaRa), 1U Taq DNA polymerase (TaKaRa) and 25–50 ng total DNA. duplicated genomes and autopolyploidy within each species complex. Following an initial 5 min denaturing step at 94 1C, PCR comprised 35 cycles at 94 1C for 30 s, 58 1Cfor30sand721C 30 s, followed by a final extension at 72 1C for 7 min. The corresponding PCR products were purified on agarose MATERIALS AND METHODS gels and extracted using a kit (Watson BioMedical Inc., Shanghai, China). Samples Subsequently, the purified products were ligated and cloned with the pUC18 Sampling included all taxonomically recognized forms of C. carassius and the DNA (TaKaRa). Each clone was picked individually from a Luria broth (LB) C. auratus complex, the latter from its native distribution in mainland China. plate and cultured in LB liquid medium. Plasmid DNA was manually isolated We downloaded all available mtDNA sequences in GenBank (Supplementary via the standard alkaline lysis miniprep method. The plasmid DNA was Tables S1–S3) and combined them with six de novo sequences to form two sequenced using an ABI 3730 with an ABI PRISM BigDye Terminator mtDNA data sets for constructing a matrilineal genealogy: control region (CR) Cycle Sequencing Ready Reaction Kit according to the manufacturer’s and CR þ cytb. The first data set consisted of 1202 mitochondrial CR sequences instructions. The following universal primers were used for sequencing the that comprised 237 unique haplotypes, including the outgroup taxa. This data plasmids: BOO12 50-CGCCAGGGTTTTCCCAGTCACGAC-30 and BOO13 set consisted of seven parts: (1) 340 sequences from Gao et al. (2012) plus six 50-AACAGCTATGACCATG-30.

Heredity Repeated polyploidization in genus Carassius JLuoet al 418

Data analysis among ingroup representatives the transition/transversion ratio We added 21 CR haplotypes from downloaded sequences to the 216 averaged 6.57. Inclusion of the outgroup dropped the transition/ haplotypes of Gao et al. (2012) and constructed genealogies. Ploidy levels transversion ratio to 5.01. Pairwise p-distances between Carassius and were then mapped onto the trees, and the proportions of ploidy were also Cyprinus ranged from 10.53 to 17.63%, averaging 13.72%. Both shown. genera were distinct genetically. MtDNA variation and divergence within and between populations of Gao et al. (2012) sequenced cytb for 104 selected samples (accession different ploidy levels were explored using several indices as implemented in no. GU135503–GU135605; NC_010768) to resolve deeper level ARLEQUIN 3.5 (Excoffier et al., 2005). Between population Fst (Reynolds et al., 1983; Slatkin, 1995) and within population haplotype diversity (H) and relationships than those revealed from CR data. We mapped our nucleotide diversity (p) (Nei, 1987) were assessed. Neutrality tests estimated ploidy information onto their matrilineal genealogy. population stability, expansion and bottlenecking (Tajima, 1989; Fu, 1997). Sequencing of the three nuclear genes used a total of 15 individuals Coalescence time (Tco) and the maternal effective population size (Nef) including one common carp as the outgroup. Among the 269 clones (Ruvolo, 1997) were calculated using a generation time (t) of 2 y because sequenced for GH, 43 alleles were identified (accession no. the sexual maturity of the wild goldfish was shown to be 1–2 y (Lorenzoni JX406582—406620, KC462749-KC462762), including 2–6 alleles et al., 2007). The frequency of polyploidy events was estimated by evaluating belonging to two copies in each individual. Thirty-two alleles were the number of shared haplotypes and the coalescence time (Tco) between the identified from 272 clones of StAR (accession no. JX406621—406650, most divergent haplotypes. KC462770-KC462782), including 2–5 alleles belonging to two copies The three nuclear gene sequences were aligned with DNASTAR 5.0 (DNASTAR Inc. Madison, WI, USA) and refined by . DAMBE 4.1.19 in each individual. Finally, for MIF, 32 alleles were identified from 269 (Xia and Xie, 2001) was used to identify shared haplotypes, and MEGA 4.1 sequenced clones (accession no. JX406651–JX406681, KC462763– (Tamura et al., 2007) was used to extract information on variable sites. These KC462769), including 1–8 alleles belonging to at least two copies in fragments were used to reconstruct polyploidization events in Carassius. each individual (Supplementary Table S4). There were 75/65, Maximum parsimony (MP) analyses were implemented in PAUP* 4.0b10 62/62 and 92/23 potentially parsimony-informative/uninformative (Swofford, 2002), maximum likelihood (ML) used RAxML (Stamatakis et al., nucleotide positions in GH, MIF and StAR, respectively. Phylogenetic 2008) and Bayesian inference (BI) was implemented in MrBayes 2.01 analyses recovered two major groups each for GH and (Huelsenbeck and Ronquist, 2001). Best-fitting models for the BI StAR (Figure 1). In GH, the maximum and minimum distances and ML analyses used likelihood ratio tests (Goldman, 1993; Huelsenbeck within group I were 4.5% and 0.3%, respectively, and within and Crandall, 1997) as implemented in the jMODELTEST 0.0.1 group II 4.1% and 0.3%, respectively. In StAR,themaximum (Posada, 2008). For BI, four independent MCMC chains were simultaneously run for 5 000 000 generations while sampling one tree per 500 and minimum distances within group I were 5.3% and 0.3%, replicates, Burnin ¼ 0, and Burninfrac ¼ 0.5 (0.1 or 0.2 and so on), with respectively, and within group II 4.6% and 0.3%, respectively. two runs conducted independently. Sampled trees were used to construct a Only one group was recognized for MIF, and the maximum 50% majority rule consensus tree. Frequency of nodal resolution was and minimum distances were 5.2% and 0.3%, respectively. Distances termed a Bayesian posterior probability (BPP) and these values were mapped for GH between ingroup sequences and carp sequences varied onto the consensus tree. Nodal support in the MP and ML analyses from 13.7 to 11.3% in group I, and 13.2 to 6.1% in group II. For was assessed using nonparametric bootstraps (BS) (Felsenstein, 1985) StAR, distances ranged from 12.2% to 5.0% in group I and from calculated for MP in PAUP* and for ML in RAxML with 1000 replicates 13.4 to 3.5% in the group II. Finally, within from MIF distances each. We assessed the robustness of our genealogy by comparing trees obtained ranged from 23.2 to 9.1%. from MP, ML and BI analyses. Autopolyploidization or allopolyploidization based on nuclear genes? RESULTS Analyses of GH and StAR obtained very similar sets of relationships. Ploidy level and sequence variation Both trees depicted two primary clusters and one or two outgroup We successfully determined the ploidy levels of 346 fishes alleles occurred in at least one of the ingroup clusters (Figure 1). from mainland China and to this we added 18 samples from Further, within one cluster (Figure 1), the topology was similar to that Li and Gui (2008) (total 364; Supplementary Table S1 part 1). derived from the mtDNA data: C. carassius diverged first, and the C. auratus complex had 57 tetraploids and 146 hexaploids from Japanese gengorobuna, the gibel carp and the sympatric Chinese Yunnan (Southwest China), 27 hexaploids from Guizhou (Southwest tetraploids and hexaploids followed this in sequence. Finally, for all China), 23 tetraploids and 21 hexaploids from Zhejiang (East China), sampling localities, alleles of hexaploid individuals did not cluster 5 tetraploids and 6 hexaploids from Hunan (East China), together, not even within the collecting sites including Yunnan (Lake 14 hexaploids from Guangdong (East China) and 29 tetraploids Dianchi), Zhejiang and Heilongjiang (Figures 1a and b). Unlike for and 36 hexaploids from Heilongjiang (Northeast China). In total, GH and StAR, the tree derived from sequences of MIF did not resolve these included 114 tetraploids and 250 hexaploids (Supplementary two distinct groups (Figure 1c) even after either including or deleting Table S1). Further, the ploidy levels of 725 Japanese fish were gaps in the phylogenetic analyses. Within each locality, hexaploids previously determined (Supplementary Table S2; Murakami et al., independently nested with sympatric tetraploids. 2001; Iguchi et al., 2003; Takada et al., 2010). For six Japanese individuals whose CR sequences were used by (Gao et al.,2012),we Maternal genealogy of the C. auratus complex determined their ploidy levels. Matrilineal relationships of Carassius reconstructed using BI from 237 For mitochondrial regions, the 320–426 nucleotide sites of partial haplotypes of CR were used to visualize the distribution of the CR sequences from 1202 fishes yielded 237 haplotypes tetraploid and hexaploid lineages (Figure 2). In comparison, MP and (Supplementary Table S3). In the CR sequences, 130 nucleotide ML analyses produced very similar topologies for the main lineages positions varied of which 106 were potentially parsimony-informative except for the relative positions of C. carassius and the Japanese and 24 were invariant. Uncorrected pairwise p-distances between gengorobuna. In the BI and ML trees, C. carassius formed the sister haplotypes ranged from 0.24 to 11.52%. Within Carassius, p-distances group of C. auratus, yet in the MP tree, the Japanese gengorobuna averaged 3.84%. The majority of substitutions were transitions and diverged first followed by C. carassius and then the other forms of

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Figure 1 BI 50% majority rule consensus trees for three nuclear genes, GH (a) StAR (b)andMIF (c) indicate autopolyploidization within the genus Carassius. Numbers represent nodal support inferred from BPPs, ML bootstrapping and MP bootstrapping, respectively. In all genes, most sequences from sympatric tetraploid and hexaploid fishes clustered together. GH and StAR had two major groups, which nested with carp alleles and indicated an earlier round of allotetraploidization in the ancestor of goldfish and carp. Within each cluster of alleles for GH and StAR, sympatric tetraploid and hexaploid individuals share the same alleles (in yellow), a result of autoploidization. A full color version of this figure is available at the Heredity journal online.

C. auratus. The conflicting nodes were not well supported. The and hexaploids, and even octaploids, shared the same haplotypes. In Japanese gengorobuna rooted at the base of the BI tree with strong sublineages C2, C5 and C6, 18 haplotypes (marked in yellow in support (Figure 2; BPP ¼ 100%; BS ¼ 100%). Next, C. carassius from Figure 2; Supplementary Tables S1 and S2) were shared among Central Europe and Russia branched off (Figure 2; BS ¼ 93–100%). tetraploid and hexaploids and these different haplotypes were The remaining samples of the C. auratus complex formed five highly substantially diverged from one another. The most divergent haplo- supported groups. types, h39 and h41, differed by 12 substitutions yet both contained Analysis of the concatenated (CR þ cytb) sequences resolved the tetraploid and hexaploid individuals. In total, 15.38% (18/117) main nodes of the CR tree and with stronger support (Figure 3). of the haplotypes were shared by tetraploid and hexaploid C. carassius split first and with high support (Figure 3; BPP ¼ 100%; samples in sublineages C2, C5 and C6 (Figure 2; Supplementary BS ¼ 100%). Sequentially, the next lineages included the Japanese Tables S1 and S2). In Yunnan, China, 12 of 20 (60%) haplotypes of gengorobuna and ginbuna (lineages A and B, Figure 3), both of which the hexaploids were shared with tetraploids, and 93.84% (137/146) of received high bootstrap support. Compared with the CR tree, six the hexaploid individuals shared one of the 12 haplotypes. In the sublineages were revealed in lineage C (sublineages C1–C6, Figure 3), Japanese samples, haplotype CR58 was shared between tetraploids excluding the Japanese ginbuna. and hexaploids in sublineages C5 and C6. Goldfish from Japan and the Kyusyu Islands exhibited a similar pattern; 22 of 72 haplotypes Hexaploid lineages in the C. auratus complex (30.56%) were shared by tetraploids and hexaploids, including one Hexaploids were scattered on the maternal genealogies based on both haplotype, ht2 occurred in tetraploid, hexaploid and octaploid CR (Figure 2) and the concatenated data (CR þ cytb) (Figure 3). In individuals (Figure 2; Supplementary Table S2). the Japanese goldfish, the hexaploids mapped onto two highly supported branches. Similarly, hexaploid C. auratus were scattered Genetic variation within/between populations of differing ploidy among the six sublineages. This distribution of hexaploids received Levels of variation in CR for tetraploid and hexaploid individuals high nodal support and various estimated dates of origin. Thus, were estimated after assigning populations to China, Kyusyu and hexaploids had multiple independent origins in both the C. auratus Japan (Table 2). Among Chinese C. auratus, hexaploid haplotypes complex and the Japanese ginbuna; at least six polyploidization events (40) were more common than tetraploid haplotypes (35) (Table 2). were identified in well-supported matrilines. Similarly, hexaploid haplotypes (5) outnumbered tetraploid haplo- The proportion of tetraploids/hexaploids was summarized on types (41) in Japan, including the Kyusyu Islands (Table 2; sublineages C2, C5, C6, Japanese ginbuna and goldfishes from Anhui, Supplementary Table S2). Similarly, hexaploid populations showed Fujian and south-central Ryukyus (Figure 2). The mapping depicted higher levels of than did the tetraploids within two clear features. First, tetraploids and hexaploids were intermixed China (H4n ¼ 0.9360±0.0109; H6n ¼ 0.9413±0.0048), Japan among lineages. Hexaploidy occurred repeatedly. Second, tetraploids (H4n ¼ 0.9252±0.0097; H6n ¼ 0.9417±0.0074) and the Kyusyu

Heredity Repeated polyploidization in genus Carassius JLuoet al 420

CR64 CR63 CR61 (0/13/0) CR60 (0/16/0) CR55 CR46 CR45 CR44 ht44 (0/1/0) h103 h81 h80 h78 h72h71 (0/1/0) h70 (0/1/0) h65 (3/0/0) h64 (0/5/0) h62 (1/0/0) h61 (0/1/0) h60 (0/1/0) h56 (9/22/0) h55 (4/17/0) h49 (0/1/0) h46 (0/1/0) h23 h39 (1/15/0) h22 h21 (1/0/0) h20 (9/8/0) h19 (0/1/0) 100/70/- h18 (5/2/0) h14 (1/0/0) h13 (8/21/0) Sublineage h12 h11 (0/4/0) C5&C6 h10 h9 (0/2/0) h8 h7 (3/25/0) h6 (2/5/0) h5 (0/1/0) h40 (0/12/0) h54 h24 h63 (2/0/0) h16 (0/4/0) h50 (0/1/0) 4n h38 h47 (0/1/0) h67 6n h15 (1/4/0) h87 Shared by 4n and 6n CR57 (8/1/0) h59 (0/1/0) Shared by 4n, 6n and 8n h68 (2/9/0)() ht43 (0/1/0) CR58 (8/14/0) h66 (0/2/0) h1h2 (16/23/0) h3 (3/0/0) BPP/ML/MP h4 (1/0/0) h48 (1/0/0) h74 (1/0/0) h75 (1/0/0) h76 (1/0/0) 0.1 h102 h100 h77 h37 h33 (3/0/0) h32 (2/0/0) h31h30 h29 94/-/- h28 h27 h26 (1/2/0) h25 h53 h73 (1/0/0) h36 h52 h34 h35 h79 h51 (0/4/0) ht20 (0/1/0) CR6 (0/1/0) ht40 (0/1/0) ht19 (0/1/0) CR7 (2/9/0) ht29 (18/0/0) CR5 (3/5/0) CR8 (0/10/0) CR9 (17/13/0) ht27 (1/0/0) CR12 (3/0/0) ht13 (0/3/0) CR11 (3/7/0) ht18 (0/2/0) ht32 ht9 (0/3/0) Sublineage B CR13 (14/15/0) CR14 (0/1/0) CR15 (0/8/0) CR10 ht14 (0/1/0) CR1 ht6 (0/10/0) ht7 (4/1/0) 100/91/81 CR3 (10/17/0) C. a. CR4 CR2 (3/29/0) CR20 langsdorfii ht12 (0/6/0) CR27 (3/2/0) CR28 (1/5/0) ht2 (9/6/1) ht24 (1/0/0) ht5 (2/5/0) ht41 (0/1/0) ht15 (0/8/0) CR24 (12/1/0) ht28 (1/0/0) Japan ht1 (2/14/0) CR18 (11/4/0) ht4 (14/2/0) ht8 (0/7/0) ht10 (0/2/0) ht11 (0/3/0) ht16 (0/1/0) ht17 (0/1/0) ht22 (0/3/0) ht23 (2/0/0) ht25 (3/0/0) ht26 (1/0/0) ht31 ht30 ht33 ht39 (0/1/0) CR16 (3/6/0) CR17 100/82/52 CR19 (3/0/0) CR21 CR22 CR23 CR25 (2/0/0) CR26 (4/17/0) CR29 (1/0/0) CR30 (19/0/0) CR31 (1/0/0)

Figure 2 BI 50% majority rule consensus tree depicting the matrilineal relationships within Carassius based on 320–426 nucleotides positions of mtDNA CR sequences. Numbers represent nodal support inferred from BPPs, MLBS and MPBS, respectively. Branch support values are given for major lineages only. Colors indicate ploidy levels. Haplotype name is followed by the number of tetraploid, hexaploid and octaploid individuals. C. carpio formed the outgroup. A full color version of this figure is available at the Heredity journal online.

Heredity Repeated polyploidization in genus Carassius JLuoet al 421

gh5 gh11 (1/3/0) gh20(0/1/0) gh3 gh24 Sublineage C2 gh4 (0/1/0) gh14 (0/2/0) gh15 (2/0/0) C. a. gibelio gh27 gh28 Northern mainland gh1 (18/28/0) China & Russia & gh2 (0/3/0) gh10 (1/0/0) gh18 gh19 (1/0/0) gh21 (1/0/0) gh23 gh25 gh26 gh29 gh30 CR56 CR62 100/84/85 gh6 (3/1/0) gh7 4n gh8 CR59 6n gh12 (3/3/0) gh13 (0/5/0) gh31 Shared by 4n and 6n CR48 Shared by 4n, 6n and 8n CR49 (5/0/0) CR50 Sublineage C3 CR51 CR53 (0/4/0) Taiwan & 100/97/95 CR54 (0/19/0) southern-central CR52 BPP/ML/MP CR47 Ryukyus CR33 (1/0/0) CR34 (6/0/0) 0.1 CR32 (0/7/0) CR35 (1/0/0) CR36 (16/11/0) CR37 (0/6/0) h41 (1/6/0) h42 h43 Anhui & Fujian & h44 h94 southern-central h99 Ryukyus h101 100/92/74 CR38 (0/1/0) CR39 CR40 CR41 (1/0/0) CR42 (132/51/0) CR43 (1/0/0) h95 Sublineage C1b h96 Fujian h82 h83 h84 h85 h86 100/83/65 h91 h92 h97 h93 h98 Sublineage C1a 100/99/97 h88 Vietnam h89 h90 100/93/93 CC1 C. carassius CC2 CC3

100/100/100 NC C. a. cuvieri ht37 cuvieri4 Carp2 Carp4 Carp5 Carp6 100/100/100 Carp7 // Carp3 Carp1 Figure 2 Continued.

Islands (H4n ¼ 0.6720±0.0325; H6n ¼ 0.8896±0.0134). The same (Table 2). Estimated coalescence times for tetraploids and patterns occurred when all data were combined. hexaploids from Japan were the same as were those for all Estimated coalescence times for Chinese hexaploids were three groups combined. Thus, female effective population slightly younger than their sympatric tetraploids and the size (Table 2) was concordant with the estimated coalescence inverse pattern occurred for populations from the Kyusyu Islands times.

Heredity Repeated polyploidization in genus Carassius JLuoet al 422

h3B22 h4B22 h1_2B22 h46B22 h6B22 h59B75 CR58B75 h11B8 h15B8 h14B11 CR57B11 h40B9 h54B9 h64B82 h64B84 h19B13 CR63B13 CR64B13 h7B33 h39B33 CR60B62 CR61B62 h10B7 h10B10 h81B89 h87B89 h6B38 h8B39 h18B14 h60B79 h16B12 h50B12 C6 h65B83 h70B85 Mainland h1_2B9 h5B14 China h6B9 h7B9 h9B9 h12B9 h13B10 4n h13B9 h18B15 6n h19B9 h20B9 Shared by 4n and 6n h21B9 h22B16 h23B9 h24B76 D-loop + cytb 98/-/- h38B32 BPP/ML/MP h47B9 0.18 h49B10 h55B10 h56B9 0.02 h56B37 h56B78 100/-/56 h61B81 h62B82 h63B80 h103B10 h41B9 CR34B13 CR56B37 CR62B9 CR59B9 h26B18 h29B21 h30B23 h25B17 h26B17 100/100/99 h27B19 h31B24 h102B24 h36B30 C5 h33B27 Yangtze h37B31 h51B40 River h53B27 h33B26 h32B25 h32B26 h28B20 0.15 h100B103 h34B28 h35B29 CR39B57 CR40B55 CR41B55 100/70/97 CR42B55 CR43B55 CR32B53 C4 CR33B53 CR35B53 South-central 95/73/- CR36B53 CR37B53 Ryukyus& CR38B56 h43B36 Anhui & Fujian h101B104 h44B77 0.11 h99B102 h42B35 0.25 h41B34 h94B34

Figure 3 BI 50% majority rule consensus tree depicting the matrilineal relationships within Carassius based on 426 bp from the mitochondrial CRplus 1141 bp from the complete gene encoding cytochrome b. Numbers represent nodal support inferred from BPPs, MLBS and MPBS, respectively. Branch support values are given for major lineages only. Colors indicate ploidy levels. Haplotype name is followed by the number of tetraploid, hexaploid and octaploid individuals. C. carpio formed the outgroup. A full color version of this figure is available at the Heredity journal online.

Heredity Repeated polyploidization in genus Carassius JLuoet al 423

-/-/96 C3 CR48B59 South-central CR49B60 CR50B61 Ryukyus & 100/97/94 CR51B59 100/97/99 CR53B59 Taiwan CR54B59 CR52B59 & Guizhou CR47B58 96/-/- 0.17 h10B6 GH3B3 GH4B1 0.68 GH1B1 GH14B1 GH21B88 GH1B2 100/95/62 GH2B2 C2 GH10B2 4n GH12B2 Mainland 6n GH15B2 GH11B87 Shared by 4n and 6n GH20B87 China GH6B4 GH8B5 & Europe 100/87/81 GH13B86 D-loop + cytb GH15B86 GH13B73 BPP/ML/MP 0.77 0.18 CR45B73 CR46B74 0.02 h93B97 h98B97 100/100/99 h95B98 h96B99 h82B92 C1 80/83/- h86B101 h97B100 h85B94 Fujian & h92B96 h83B92 Vietnam h84B93 h91B95 0.53 h88B90 h89B90 100/100/100 h90B91 CR27B71 99/87/- CR28B72 CR24B69 CR26B70 2.25 CR30B54 CR31B54 ht1B45 CR16B45 B CR18B67 CR19B68 ht13B41 100/90/81 ht13B42 ht4B43 C. a. CR14B66 CR15B66 2.64 100/-/78 CR11B41 langsdorfii CR12B65 CR13B52 100/94/98 CR2B63 CR5B64 1.02 CR7B49 100/100/100 CR8B50 100/-/99 CR9B51 4.33 CR6B48 100/100/100 ht37B44 A NC_010768 0.02 C. a. cuvieri 100/100/100 C. carassius 1 9.34 C. carassius 2 0.01 Carp2 Carp4 Carp3 100/100/- Carp5 // Carp6 0.32 Carp1

Figure 3 Continued.

We evaluated the possibility of population expansion by assuming years. Thus, population expansion was unlikely to explain the pattern. that the 39 (17 in China plus 22 in Japan) incidences of haplotypes Further, a recent population expansion was not identified by using being shared by tetraploids and hexaploids represented independent either the Tajima’s D or Fu’s neutrality test among the different ploidy polyploidization events. Subsequently, we estimated the frequency of levels of either the Chinese or the Japanese populations. Populations polyploidization by coalescence times. The suggested coalescence time in the species complex appeared to be established and stable. for the 17 Chinese populations with hexaploid individuals was When individuals were assigned to one of three populations 6 Tco ¼ 0.94 Â 10 years (Table 2), that is, a rate of at least 0.55 (China, Kyusyu and Japan), Fst values indicated that most sympatric polyploidization events per 105 years. For the 14 populations on the tetraploid and hexaploid populations were more similar to each other 6 main islands of Japan, the coalescence time was Tco ¼ 1.18 Â 10 years than to individuals in non-sympatric populations (Table 3). The Fst (Table 2), or a minimum rate of 0.84 polyploidization events per 105 values of sympatric tetraploid/hexaploid populations in China, years. Finally, the eight occurrences of hexaploids on the Ryukyu Ryukyu Islands and Japan were less than 0.140. Except for Chinese 6 5 Islands had Tco ¼ 1.13 Â 10 year (Table 2), or 1.41 events per 10 tetraploids and hexaploids from the Kyusyu Islands, for which

Heredity Repeated polyploidization in genus Carassius JLuoet al 424

Table 2 Genetic diversity of tetraploids and hexaploids in the Ca.auratuscomplex based on mitochondrial control region, defined by lineage (1) and sympatric and non- sympatric tetraploid and hexaploid populations (2)

N Haplotype (HN) Polymorphic sites (PN) Gene diversity (H) Nucleotide diversity (p)% Tco (years) Nef

(1) C5 and C6 4n 91 27 18 0.9316±0.0122 0.8846±0.5233 3.77 Â 105 9.43 Â 104 C5 and C6 6n 322 35 25 0.9044±0.0102 0.8110±0.4834 4.24 Â 105 1.06 Â 105 subB 4n 162 30 25 0.9357±0.0065 1.9364±1.0265 6.60 Â 105 1.65 Â 105 subB 6n 215 38 25 0.9506±0.0054 2.0829±1.0944 6.60 Â 105 1.65 Â 105 C2 4n 30 8 7 0.6322±0.0959 0.2731±0.2024 2.36 Â 105 5.89 Â 104 C2 6n 47 9 8 0.6318±0.0766 0.2819±0.2068 2.83 Â 105 7.08 Â 104

(2) C. a. auratus 4n (CH) 114 35 37 0.9360±0.0109 1.9912±1.0286 0.99 Â 106 2.48 Â 105 C. a. auratus 6n (CH) 250 40 39 0.9413±0.0048 1.7456±0.9069 0.94 Â 106 2.36 Â 105 C. a. auratus 4n (KY) 238 18 36 0.6720±0.0325 2.0887±1.0967 1.08 Â 106 2.70 Â 105 C. a. auratus 6n (KY) 193 18 34 0.8896±0.0134 3.2436±1.6543 1.13 Â 106 2.83 Â 105 C. a. auratus 4n (JAP) 114 23 24 0.9252±0.0097 2.1181±1.1159 1.18 Â 106 2.95 Â 105 C. a. auratus 6n (JAP) 185 38 39 0.9417±0.0074 2.2277±1.1642 1.18 Â 106 2.95 Â 105 C. a. auratus 4n (All) 466 76 53 0.9064±0.0110 3.1954±1.6195 1.13 Â 106 2.83 Â 105 C. a. auratus 6n (All) 628 93 57 0.9750±0.0017 3.7081±1.8612 1.13 Â 106 2.83 Â 105

Abbreviations: CH, China; JAP, Japan; KY, Kyusyu; Nef, estimated effective population size; Tco, estimated time to coalescence.

Table 3 Pairwise FST between (1) different lineages and (2) sympatric and non-sympatric tetraploid and hexaploid populations

(1) C5 and C6 4n C5 and C6 6n sub 4n sub 6n C2 4n C2 6n

C5 and C6 4n \ C5 and C6 6n 0.05951** \ subB 4n 0.72781** 0.77716** \ subB 6n 0.70736** 0.75823** 0.03692** \ C2 4n 0.83776** 0.82471** 0.66209** 0.66848** \ C2 6n 0.84308** 0.82348** 0.67655** 0.67913** À0.00281 \

(2) China 4n China 6n Kyusyu 4n Kyusyu 6n Japan 4n Japan 6n

China 4n \ China 6n 0.01413 \ Kyusyu 4n 0.39061** 0.41156** \ Kyusyu 6n 0.21249** 0.25333** 0.14008** \ Japan 4n 0.60135** 0.62542** 0.40157** 0.31834** \ Japan 6n 0.59791** 0.62181** 0.40037** 0.32612** 0.03397** \

Note: ** denotes P ¼ 0.000.

Fst ¼ 0.212, all other values of Fst of non-sympatric tetraploid/ simultaneously. Irrespective of the particular topologies of the trees, hexaploid exceeded 0.253, and most were greater than 0.318. Non- the Japanese gengorobuna has been treated as a species, C. cuvieri. sympatric tetraploid/hexaploid populations showed higher pairwise C. carassius has 100 chromosomes (Vinogradov, 1998) as does the divergence than those in . Japanese gengorobuna, along with its diagnostic C-banding pattern (Ueda and Ojima, 1978). Considering that both species are tetraploids DISCUSSION and sequentially root at the base of the tree (Figures 2 and 3), their Genealogical relationships and recurrent polyploidy common ancestor appears to be a tetraploid (Vinogradov, 1998). C. carassius (Linnaeus 1758) and C. auratus (Linnaeus 1758) are The taxonomic classification of the C. auratus complex is compli- clearly distinguished only in the total evidence mtDNA tree cated (Figures 2 and 3). Other than the Japanese gengorobuna, two (Figure 3); they are resolved as polyphyletic (polygenealogical main lineages exist: lineage B (Japanese ginbuna) and a lineage C Murphy and Me´ndez de la Cruz, 2010) in the CR trees because the (mainly from China and the Kyusyu Islands). Lineage C contains six Japanese gengorobuna splits earlier than C. carassius (Figure 2). sublineages (Gao et al., 2012). Recurrent polyploidizations occur Analyses of nuclear GH and StAR resolve the Japanese gengorobuna within both lineages and tetraploid and hexaploid individuals fre- as a unique lineage following after C. carassius (Figures 1a and b). In quently share identical haplotypes. Every case implies the independent contrast, analyses of MIF suggest that the Japanese gengorobuna origin of a hexaploid. Several hexaploid lineages of C. auratus occur in diverged before C. carassius (Figure 1c). These inconsistent results China, for example, Lake Dianchi (Luo et al.,1999),andinthesecases suggest that these two lineages might have diverged near they are most closely related to local tetraploids (Zan et al., 1986 and

Heredity Repeated polyploidization in genus Carassius JLuoet al 425 therein; Zhang et al., 1998 and therein). Curiously, hexaploids from Convergent can explain a single origin but two lines of Guizhou cluster with both local tetraploid C. auratus and the gibel evidence reject this possibility. First, the absence of saturated substitu- carp. This population contains mixed, divergent lineages. Regardless, at tions precludes convergence as an explanation. Second, because these this locality the hexaploids are not monogenealogical (sensu Murphy unisexual fishes are gynogenetic, divergent 4n and 6n individuals and Me´ndez de la Cruz, 2010); the mtDNA data show that they do not should occur within both sister lineages if the common ancestor was a share a single common maternal ancestor. hexaploid; this does not occur in any of the incidences of sympatry. Hexaploid C. a. auratus show greater variation than tetraploids. Shared haplotypes occur in divergent lineages that differ by up to 13 Hexaploid individuals appear to be more common than tetraploids in polymorphic sites. Independent polyploidization events best explain nature, as suggested by previous collections (Xiao et al., 2011). the numerous co-occurrences of tetraploids and hexaploids. Kleptogenesis—the temperature-dependent incorporation of sperm in Laboratory experiments identify some possible mechanisms for otherwise gynogenetic polyploids (Bogart et al., 2007)—may be a hexaploidization. Hydrostatic pressure, cold and heat shock following contributing factor. Alternatively, unidirectional gene flow and recent fertilization results in the retention of the second polar cell. When this expansion of the hexaploid populations of C. auratus might also occurs, three nuclei combine yielding an additional set of chromo- promote higher levels of polymorphism in the hexaploid goldfish. somes and a hexaploid individual (Gui et al., 1995; Pandian and These possibilities require further testing. Koteeswaran, 1998). Thus, possible environmental perturbations may In Japan, both tetraploid and hexaploid forms exist in the ginbuna promote the formation of hexaploids. Variation in hydrostatic (Murakami et al., 2001; Takada et al., 2010 and therein). Because pressure or temperature is a potential factor and environmental data gene flow can only occur between tetraploids, the mixture and future analyses may confirm this possibility. Regardless, after of tetraploids and hexaploids indicates additional recurrences of formation, hexaploids reproduce gynogenetically. polyploidization (Figures 2 and 3). The frequent dispersal of historically isolated populations might facilitate mixture of the tetraploids and hexaploids. This scenario does The tempo, mode and origin of tetraploid and hexaploid Carassius not appear to be true for Carassius. Geographic structure exists in both The ancestor of Carassius is most likely a tetraploid carp (Buth et al., Japan and mainland China (Takada et al.,2010;Gaoet al., 2012). 1991 and therein; our analyses). All Carassius have at least 100 Migration does not seem to have blurred the geographic structure and, chromosomes, twice as many than the standard cyprinid karyotype, again, the evidence suggests site-specific-independent polyploidization and their genome sizes are also about twice as large as diploid events. This mechanism may explain elevated levels of variation in cyprinids (Ohno and Atkin, 1966; Ohno, 1970; Yu et al., 1989; Risinger hexaploids; hexaploids would have two paternal contributions. and Larhammar, 1993). The initial tetraploidization might have been Gynogenesis is common in hexaploid Carassius, and this might an alloploidization event that occurred about 14.2–14.5 Ma, as significantly influence population structure. It occurs sporadically estimated from nuclear gene studies (Risinger and Larhammar, among invertebrates (Smith, 1978), and in vertebrates it is especially 1993; Yang and Gui, 2004; Luo et al., 2006). Our analyses from both widespread in polyploid teleosts (Gui, 1997; Le Comber and Smith, mitochondrial and nuclear data place tetraploid C. carassius and 2004). Smith (1978) argued that short-term selection can favor Japanese C. cuvieri at the base of the tree for Carassius. Analyses of GH unisexuality. Gynogenetic forms a barrier to gene flow and StAR also support a model of allopolyploidization of the genus from hexaploid to tetraploid individuals. Hexaploid females only because at least one allele of each gene clusters within the outgroup require sperm from tetraploid males to trigger egg development; egg carp, and two clusters are indicated (Figures 1a and b). Hence, our and sperm nuclei do not fuse (Gui, 1997). Where tetraploids, results confirm the assumption of allotetraploidization of the genus hexaploids and, rarely, octaploids coexist in natural waters, hexaploid Carassius. Analyses of the nuclear gene data cannot reject the male C. auratus occur but they do not contribute genetically to hypothesis of recurrent polyploidization via autoploidization because tetraploid reproduction. A combination of hexaploid and tetraploid we did not detect either C. carassius or C. cuvieri within the C. auratus nuclei would form an irregular number of chromosomes (about 125) complex. For example, tetraploid and hexaploid individuals share and no such individuals are known (Chen et al., 1996; Xiao et al., localized alleles within clusters of both GH and StAR (Figures 1a and 2011). Thus, hexaploids depend on tetraploids even though the b) only in the C. auratus complex. Thus, ancient alloploidization and former do not fertilize eggs of the later and their syntopic occurrence recent autopolyploidization are indicated for Carassius. is widespread. The tempo indicates that recurrent autopolyploidization occurred after the origin of the C. auratus complex. Gao et al. (2012) estimated Polyploid Carassius: a model system for investigations in divergence time of C. carassius and the C. auratus complex to be 3.6– vertebrates 4.0 Ma. Hexaploids in different geographic regions are younger than With its large genome, Carassius possesses many duplicated genes. the two species themselves (Table 2) and no recurrent event occurred Ancestral carp appear to have about 50 chromosomes (Yu et al.,1989) in their common ancestor. Analyses suggest a rate of about 0.55–1.41 and a genome size of about 2.0 pg per cell. In contrast, tetraploid events per 105 years. Carassius have from 3.52 pg to 3.84 pg per cell, almost twice that of The most intriguing discovery lies in the ubiquitous, recurrent diploid barbs (http://www.genomesize.com/; Luo et al.,2006).Hex- hexaploidization. The mapping of polyploids on the matrilineal aploid Carassius have about 5.25 pg per cell (http://www.genomesi- genealogy precludes the possibility of one polyploidization event ze.com/). Our results indicate that tetraploids have 2–4 copies for (Supplementary Figure S1). The nuDNA allele tree, matrilineal each nuclear gene, and hexaploids normally have 3–6 copies genealogy, coalescence times and scale of divergence in sympatric/ (Supplementary Table S4). Thus, Carassius is likely to be a polyploid non-sympatric tetraploid/hexaploid individuals indicate that the genus, and not a diploid/triploid system as sometimes claimed (for hexaploidization has several independent origins (Figures 2 and 3; example, Takada et al.,2010). Tables 2 and 3). Shared mtDNA haplotypes reject the hypothesis of a The existence of both allo- and autopolyploidization events single origin for the hexaploids and in doing so they support the facilitates investigations into post-polyploidization events at both alternative hypothesis of multiple formations of polyploids. genetic and epigenetic levels. The origin of the allopolyploidization in

Heredity Repeated polyploidization in genus Carassius JLuoet al 426

Carassius is known (Yang and Gui, 2004; Luo et al.,2006).The Dawley RM (1989). An introduction to unisexual vertebrates. In: Dawley RM, Bogart JP importance of allopolyploidization and how its evolutionary effects (eds) Evolution and Ecology of Unisexual Vertebrates. New York State Museum: Albany, NY, USA, pp 1–18. differ from those of autopolyploidization (Parisod and Besnard, 2007) Evans BJ, Kelley DB, Tinsley RC, Melnick DJ, Cannatella DC (2004). A mitochondrial DNA is a topic of considerable interest (Soltis and Soltis, 1999; Vandepoele phylogeny of African clawed frogs: and implications for polyploid et al., 2004; Van de Peer et al., 2009). This level of interest differs from evolution. Mol Phylogenet Evol 33: 197–213. Excoffier L, Laval G, Schneider S (2005). Arlequin (version 3.0): an integrated software that for autopolyploidization (Parisod and Besnard, 2007). Autopo- package for population genetics data analysis. Evol Bioinform 1:47–50. lyploidization might be advantageous under conditions of environ- Felsenstein J (1985). Confidence limits on phylogenies: an approach using the bootstrap. mental instability because of genic redundancy and polysomic Evolution 39: 783–791. Fu YX (1997). Statistical tests of neutrality of mutations against population growth, inheritance (Parisod and Besnard, 2007). Thus, this genus is an ideal hitchhiking and background selection. Genetics 147: 915–925. model system for investigating genetic and epigenetic changes soon Gao Y, Wang SY, Luo J, Murphy RW, Du R, Wu SF et al. (2012). Quaternary after polyploidizations, either allo- and autopolyploidy. palaeoenvnvironmental oscillations drove the evolution of East Asian Carassius auratus complex (Carassius, Cypriniformes, Cyprinidae). JBiogeogr39: 2264–2278. In summary, intraspecific polyploidization occurs multiple times in Goldman N (1993). Statistical tests of models of DNA substitution. J Mol Evol 36: angiosperms, yet this phenomenon is less common and not yet fully 182–198. Golovinskaya KA, Romashov DD, Cherfas NB (1965). Unisexual and bisexual forms of understood in vertebrates. We document recurrent polyploidization silver crusian carp (Carassius auratus gibelio Bl.). Vopr Ikhtiol 5: 614–629 (in Russian). events in the genus Carassius based on matrilineal history. The Gui JF (1997). Retrospectus and prospects of studies on the mechanism of natural C. auratus complex has recurrent formations of hexaploids; tetra- gynogenesis in silver crucian carp (Carassius auratus gibelio). Bull Nat Sci Found China 1:11–16(inChinese). ploids and hexaploids share identical haplotypes in three main Gui JF (2007). Genetic Basis and Artificial Control of Sexuality and Reproduction in . lineages and within localities. The origin of the gibel carp likely owes Science Press: Beijing, China. to a single hexaploidization event in China within the C. auratus Gui JF, Xiao W, Liang S, Jiang Y (1995). Preliminary study on the cytological mechanism of triploidy and tetraploidy induced by hydrostatic pressure shock in transparent colored complex. The other two main lineages, the Chinese C. auratus crucian carp. Acta Hydrob Sinica 19:49–55. complex and the ginbuna of Japan, have at least 22 independent Hegarty MJ, Hiscock SJ (2008). Genomic clues to the evolutionary success of polyploid hexaploidization events, plus one octaploidization. These events make plants. Curr Biol 18:R435–R444. Holloway AK, Cannatella DC, Gerhardt HC, Hillis DM (2006). Polyploids with different the species of the genus Carassius an ideal model system for the study origins and ancestors form a single sexual polyploid species. Am Nat 167:E88–101. of recurrent polyploidization and the evolution of genomic complex- Hu T, Pattyn P, Bakker E, Cao J, Cheng J, Clark R et al. (2011). The Arabidopsis lyrata ity. How polyploidy contributes to and evolution, as well genome sequence and the basis of rapid genome size change. Nat Genet 43:476–481. Huelsenbeck JP, Crandall KA (1997). Phylogeny estimation and hypothesis testing using as speciation in vertebrates, is a very promising area of future research. maximum likelihood. Annu Rev Ecol Syst 28: 437–466. Huelsenbeck JP, Ronquist F (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. DATA ARCHIVING Iguchi K, Yamamoto G, Matsubara N, Nishida M (2003). Morphological and genetic Sequences were deposited in GenBank: Accession numbers: analysis of fish of a Carassius complex (Cyprinidae) in Lake Kasumigaura with reference JX406582-JX406681 and KC462749-KC462782. to the taxonomic status of two all-female triploid morphs. Biol J Linn Soc 79: 351–357. Kobayashi H (1978). Classification of the genus Carassius and cellular genetics of CONFLICT OF INTEREST gynogenesis of ginbuna (C. auratus langsdorfi). Genetics 32: 28–38 (in Japanese). Le Comber SC, Smith C (2004). Polyploidy in fishes: patterns and processes. Biol J Linn The authors declare no conflict of interest. Soc 82: 431–442. Leggatt RA, Iwama GK (2003). Occurrence of polyploidy in the fishes. Rev Fish Biol ACKNOWLEDGEMENTS Fisheries 13: 237–246. Li FB, Gui JF (2008). Clonal diversity and genealogical relationships of gibel carp in four This work was supported by grants from National Science Foundation of hatcheries. Anim Genet 39:28–33. China (31360514 and 30870291 to J Luo; 30600065 to Y Gao; and Lokki J, Saura A (1980). Polyploidy in evolution. In: Lewis W (ed) Polyploidy: 31061160189 to Y-p Zhang; 30930071 to SJ Liu), Bureau of Science and Biological Relevance. Plenum Press: New York, NY, USA, pp 277–312. Technology of Yunnan Province and the State Key Laboratory of Genetic Lorenzoni M, Corboli M, Ghetti L, Pedicillo G, Carosi A (2007). Growth and reproduction of the goldfish Carassius auratus: a case study from . In: Gheradi F (ed) Biological Resources and Evolution, Kunming Institute of Zoology, CAS. Manuscript Invaders in Inland Waters: Profiles, Distribution, and Threats.Springer-Verlag:New preparation was supported by a Discovery Grant from the Natural Sciences York, NY, USA, pp 259–273. and Engineering Research Council of Canada (3148) and by a Visiting Lou YD, Zhang YP, Weng ZH, Zhao L (1989). Preliminary studies on cytogenetics and Professorship for Senior International Scientists from the Chinese Academy of isoenzymes of crucian carp (Carassius auratus) in Qihe River of Henan province. JFishChina13: 254–258 (in Chinese). Sciences to RW Murphy. We thank CL Zhu, L Zhang, SG Fang, YF Ge, JH Liu Lu CY, Yang YH, Tong GX, Hao J, Sun XW, Liang LQ et al. (2006). Comparative studies of and X Chen et al. for sample collection and technical help. We also thank the genetic diversity between C. auratus gibelio (Bloch) and C. auratus auratus (L.) in the two anonymous reviewers for constructive suggestions. same reservoir. Chinese J Fisheries 19:42–50. Luo J, Zhang YP, Zhu CL, Xiao WH, Huang SY (1999). Genetic diversity in crucian carp (Carassius auratus). Biochem Genet 37: 267–279. Luo J, Lang M, Salzburger W, Siegel N, Stolting KN, Meyer A (2006). A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes). J Exp Zool Part B Mol Allendorf FW, Thorgaard GH (1984). Tetraploidy and the evolution of salmonid fishes. Dev Evol 306:567–574. In: Turner BJ (ed) Evolutionary Genetics of . Plenum Press: New York, NY, USA, Luo YL, Yue PQ (2000). Cyprinidae. In: Yue PQ (ed) Fauna Sinica. Osteichthyes pp 1–53. Cypriniformes III. Science Press: Beijing, China, pp 399–433 (in Chinese). Bogart JP, Bi K, Fu JZ, Noble DWA, Niedzwieki J (2007). Unisexual salamanders (genus Mable BK (2004). Why polyploidy is rarer in animals than in plants: myths and Ambystoma) present a new reproductive mode for eukaryotes. Genome 50:119–136. mechanisms. Biol J Linn Soc 82: 453–466. Brykov VA, Poliakova NE, Skurikhina LA, Dolganov SM, Eliseikina MG, Mlu Kovalev Mable BK, Alexandrou MA, Taylor MI (2011). Genome duplication in amphibians and fish: (2002). Mitochondrial DNA variation in goldfish Carassius auratus gibelio from Far an extended synthesis. JZool284:151–182. Eastern water reservoirs. Genetika 38: 1387–1392 (in Russian). Murakami M, Matsuba C, Fujitani H (2001). The maternal origins of the triploid ginbuna Buth DG (1983). Duplicate isozyme loci in fishes: origins, distribution, phyletic (Carassius auratus langsdorfi): phylogenetic relationships within the C. auratus taxa by consequences, and locus nomenclature. Isozymes: Curr Top Biol Med Res 10: partial mitochondrial D-loop sequencing. Gene Genet Syst 76:25–32. 381–400. Murphy RW, Me´ndez de la Cruz FR (2010). The herpetofauna of Baja California and its Buth DG, Dowling TE, JR (1991). Molecular and cytological investigations. In associated islands: a conservation assessment and priorities. In: Wilson LD, Townsend Winfield IJ, Nelson JS (eds) Cyprinid Fishes: Systematics, Biology, and Exploitation. JH, Johnson JD (eds) Conservation of Mesoamerican Amphibians and Reptiles.Eagle Chapman & Hall: London, UK, pp 83–126. Mountain Publishing, LC: Eagle Mountain, UT, USA, pp 238–273. Chen MR, Yang XQ, Yu XM, Chen HX (1996). Karyotype studies on the bisexual natural Nei M (1987). Molecular Evolutionary Genetics. Columbia University Press: New York, gynogenetic crucian carp (Carassius auratus)ofPengze.Acta Hydrob Sinica 20: NY, USA. 25–31. Ohno S (1970). Evolution by Gene Duplication. Springer-Verlag: New York, NY, USA.

Heredity Repeated polyploidization in genus Carassius JLuoet al 427

Ohno S, Atkin NB (1966). Comparative DNA values and chromosome complements of Tsigenopoulos CS, Rab P, Naran D, Berrebi P (2002). Multiple origins of polyploidy in the eight species of fishes. Chromosoma 18:455–466. phylogeny of southern African barbs (Cyprinidae) as inferred from mtDNA markers. Ojma Y, Yamano T (1980). The assignment of the nuclear organizer in the chromosome of Heredity 88: 466–473. the fauna (Carassius,Cyprindae,Pisces).Proc Jpn Acad 56B: 551–556. Ueda T, Ojima Y (1978). Differential chromosomal characteristics in the funa subspecies Otto SP, Whitton J (2000). Polyploid incidence and evolution. Annu Rev Genet 34: (Carassius). Proc Jpn Acad 54B: 283–288. 401–437. Van de Peer Y, Maere S, Meyer A (2009). The evolutionary significance of ancient genome Pandian TJ, Koteeswaran R (1998). Ploidy induction and sex control in fish. Hydrobiologia duplications. Nat Rev Genet 10: 725–732. 384: 167–243. Vandepoele K, De Vos W, Taylor JS, Meyer A, Van de Peer Y (2004). Major events in the Parisod C, Besnard G (2007). Glacial in situ survival in the Western Alps and polytopic genome evolution of vertebrates: paranome age and size differ considerably between ray-finned fishes and land vertebrates. Proc Natl Acad Sci USA 101: 1638–1643. autopolyploidy in Biscutella laevigata L. (Brassicaceae). Mol Ecol 16: 2755–2767. Vinogradov AE (1998). Genome size and GC-percent in vertebrates as determined by flow Posada D (2008). jModelTest: phylogenetic model averaging. Mol Biol Evol 25: cytometry: the triangular relationship. Cytometry 31:100–109. 1253–1256. Wang RF, Shi LM, He WS (1988). A comparative study of the Ag-NORs of Carassius Reynolds J, Weir BS, Cockerham CC (1983). Estimation for the coancestry coefficient: auratus from different geographic districts. Zool Res 9: 165–169 (in Chinese). basis for a short-term genetic distance. Genetics 105:767–779. Wheeler A (2000). Status of the crucian carp, Carassius carassius (L.), in the UK. Risinger C, Larhammar D (1993). Multiple loci for synapse protein SNAP-25 in the Fisheries Manage Ecol 7: 315–322. tetraploid goldfish. Proc Natl Acad Sci USA 90: 10598–10602. Wittbrodt J, Meyer A, Schartl M (1998). More genes in fish? Bioessays 20: 511–515. Ruvolo M (1997). Molecular phylogeny of the hominoids: inferences from multiple Woods TD, Buth DG (1984). High level of gene silencing in the tetraploid goldfish. independent DNA sequence data sets. MolBiolEvol14:248–265. Biochem Syst Ecol 12: 415–421. Segraves KA, Thompson JN, Soltis PS, Soltis DE (1999). Multiple origins of polyploidy Xia X, Xie Z (2001). DAMBE: software package for data analysis in molecular biology and and the geographic structure of Heuchera grossulariifolia. Mol Ecol 8:253–262. evolution. JHered92: 371–373. Shan SX, Jiang YG (1988). Karyotype studies of crucian carp Carassius auratus gibelio. Xiao J, Zou TM, Chen YB, Chen L, Liu SJ, Tao M et al. (2011). Coexistence of diploid, Acta Hydrobiol Sin 12:381–384(inChinese). triploid and tetraploid crucian carp (Carassius auratus)innaturalwater.BMC Shen JB, Wang GR, Fan ZT (1983). The ploidy of crucian carp and its geographical Genet 12:20. distribution in Heilongjiang main river. JFishChina7: 79–94 (in Chinese). Yang L, Gui JF (2004). Positive selection on multiple antique allelic lineages of transferrin Slatkin M (1995). A measure of population subdivision based on microsatellite allele in the polyploid Carassius auratus. Mol Biol Evol 21: 1264–1277. frequencies. Genetics 139: 457–462. Yu HX, Xu H, Guan HW (1992). Preliminary study on the karyotypes of the natural Smith M (1978). The Evolution of Sex. Cambridge University Press: Cambridge, Great gynogenetic crucian carp Carassius auratus (A form) of Puan, Guizhou province. Acta Britain. Hydrobiol Sin 16: 87–89 (in Chinese with English abstract). Soltis DE, Soltis PS (1999). Polyploidy: recurrent formation and genome evolution. Trends Yu HX, Zhang HM, Lin LY (1987). A preliminary study on the biology and culture experiment of the gynogenetic crucian carp Carassius auratus of Guangdong. Acta Ecol Evol 14:348–352. Hydrobiol Sin 11: 278–288 (in Chinese with English abstract). Stamatakis A, Hoover P, Rougemont J (2008). A rapid bootstrap algorithm for the RAxML Yu XJ, Zhou D, Li YC, Li K, Zhou M (1989). Chromosomes of Chinese Fresh Water Fishes. web servers. Syst Biol 57: 758–771. Science Press: Beijing, China, pp 135–143 (in Chinese with English Abstract). Swofford DL (2002). PAUP*. Phylogenetic Analysis Using Parsimony (* and other Zan RG (1982). Studies on sex chromosomes and C-banding karyotypes of two forms of Methods). Version 4.0b10*. Sinauer Associates: Sunderland, MA, USA. Carassius auratus in Kunming Lake. Acta Genet Sinica 9: 32–39 (in Chinese). Tajima F (1989). Statistical method for testing the neutral mutation hypothesis by DNA Zan RG, Song Z, Liu WG (1986). Studies on karyotypes and nuclear DNA contents of some polymorphism. Genetics 123: 585–595. cyprinoid fishes, with notes on fish polyploids in China. In: Uyenno T et al. (eds) Takada M, Tachihara K, Kon T, Yamamoto G, Iguchi K, Miya M et al. (2010). Biogeography Indo-Pacific Fish Biology: Proceedings of the Second International Conference on and evolution of the Carassius auratus-complex in . BMC Evol Biol 10:7. Indo-Pacific Fish. Ichthyological Society of Japan: Tokyo, Japan, pp 877–885. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: molecular evolutionary genetics Zhang H, Dong XH, Ye Y, Wu QJ (1998). Comparative studies of the mtDNA from three analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599. strains of hexaploid and C. auratus auratus. Acta Genet Sinica 25: 330–336.

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