View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Institute of Hydrobiology, Chinese Academy Of Sciences Chinese Science Bulletin © 2008 SCIENCE IN CHINA PRESS ARTICLES Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) ZHANG YuBo1,2 & HE ShunPing1† 1 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; 2 Graduate University of Chinese Academy of Sciences, Beijing 100039, China The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodonti- dae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this ge- nus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsi- mony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the mo- lecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our re- sults also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data. phylogeny, cytochrome b gene, 12S rRNA gene, Takifugu Abe Pufferfishes of the genus Takifugu Abe, 1949 uted in marine regions of the Indian Ocean[2－4] (Figure (Tetraodontiformes, Tetraodontidae) occur in Southeast 1). Most of species can be found along coastal regions of Asia and the genus is composed of comparatively few the Yellow Sea, Bohai Sea, East China Sea and South species. There are 22 recognized species, and all of China Sea, with some species entering the estuaries of which have been recorded in China (including two pre- rivers in China[1,4,5]. viously unknown forms) according to the description in Due to patchy distribution, it is difficult to collect the Fauna Sinica[1] (cf. 23 Valid taxonomic names could be specimens of Takifugu. Few molecular phylogenetic found in records in fishbase: http://www.fishbase.org). It analyses have been conducted to date. The published consists of primarily brackish and freshwater species but phylogenetic studies were based on morphological[5], also includes species that are reef-dwelling, a few that cytogenetic[6] and isozyme variations[7]. Additionally, are entirely pelagic, and a small number of benthic some researchers have investigated the genus-level tax- slope-dwellers species. Members of this genus are onomy for the purpose of species identification[4,8―14]. mainly distributed along the coastal regions of the west- Received December 27, 2006; accepted September 25, 2007 GENETICS ern part of the Sea of Japan and the East China Sea from doi: 10.1007/s11434-008-0066-2 the Yellow Sea northward to Muroran, Hokkaido, Japan. †Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant No. Additionally, a few species of the genus are also distrib- 30225008) www.scichina.com | csb.scichina.com | Chinese Science Bulletin | January 2008 | vol. 53 | no. 2 | 233-244 Figure 1 The sampling localities of the pufferfishes used in this study (a), and the distribution of the Takifugu Abe (shaded, (b)). Before the 1950s, members of Takifugu Abe were as- the data set of RAPD analysis and sequences of mito- cribed to Sphoeroides. Based on their taxonomic work, chondrial 16S rRNA (572 bp). Up to now, little system- Fraser-Brunner[8], Abe[9,10] and Whitley[11] showed the atic effort has been put forth to generate a DNA-based morphological diversity of these fishes was distinct and complete phylogeny and species identification of the a new name Takifugu was given to discriminate them genus Takifugu. from Sphoeroides. Abe[9] also indicated that there were 6 Takifugu rubripes’s role has become more important subgenera comprised of 18 species, all of which oc- not only in commerce, but also in functional genome curred in marine habitats near China and Japan. Cheng studies as the model organism since the 1990s[15]. A draft et al.[5] examined the classification and interspecific re- sequence covering about 95% of the genome, using the lationships of 15 species (including 2 new species), “whole-genome shotgun” sequencing strategy, results of found in China using morphological characters. Wang et the assembly annotation, and a preliminary analysis of al.[7] examined interspecific differences among 13 spe- the genome was reported in Science[15], and later cies within Takifugu, using allozyme variation of myo- amended in July 2005 (http://www.fugu-sg.org/in- gen. Miyaki et al.[6] performed karyological analyses for dex.html). It has a uniquely compact genome with un- 6 species of Takifugu and revealed their remarkably usually small introns and lacks extensive repetitive se- similar chromosome complements. More recently, quences and pseudo-genes, making it a useful model for DNA-based analyses such as random amplified poly- gene discovery, annotating the human and other verte- morphic DNA (RAPD) have been used to analyze the brate genomes[4,16,17]. A reliable phylogeny of the group, phylogenetic relationships among: (1) T. pseudommus, T. which is crucial for a better interpretation of the evolu- obscurus, and two forms of T. rubripus (from Japan and tionary trends for speciation of the group, is still ab- China)[12]; (2) T. pseudommus, T. rubripus, T. xanthop- sent[18]. Although the phylogenetic relationships among terus, T. vermicularis and T. niphobles; and (3) the the genera of the family have been recently discussed by population structure of two species, T. pseudommus and Holcroft[19,20], in which the relationships of the order T. rubripus[4,13]. Song et al.[4] contributed topological Tetraodontiformes and the interrelationships of phylogenetic trees for T. pseudommus, T. rubripus, T. tetraodontiform fishes were laid out, it is essential to xanthopterus, T. vermicularis and T. niphobles, which determine the phylogenetic position of T. rubripes by were generated from neighbor-joining analysis based on identifying its sister species and to place Takifugu Abe 234 ZHANG YuBo et al. Chinese Science Bulletin | January 2008 | vol. 53 | no. 2 | 233-244 into a comparative context. T. guttulatus, T. plagiocellatus and T. reticularis were The purpose of this study is to infer the phylogenetic not included in this study. 17 spececies of Takifugu Abe, ARTICLES relationships of the species within the genus Takifugu, to about three fourths of all described species (according to evaluate the monophyly of the group, and to estimate the Su and Li[1]; Figure 1), were collected, including the validity of some of the currently recognized species Chinese coastal samples, in an effort to represent the within this genus. We used molecular data to infer the diversity of this genus as much as possible (Table 1). All phylogeny of Takifugu from mitochondrial cytochrome b samples were preserved in 95% ethanol, and deposited (Cyt b) and 12S rRNA genes. Cyt b gene has been con- in the Freshwater Fish Museum of the Institute of sidered one of the most useful genes for phylogenetic Hydrobiology, the Chinese Academy of Sciences. reconstruction, and is probably the best-known mito- 1.2 DNA extraction, PCR amplification and se- chondrial gene with respect to structure and function of quencing its protein product[21]. Cyt b gene contains both slowly and rapidly evolving codon positions, as well as more Total DNA was extracted from fins following phe- [26] conservative and more variable regions or domains nol/chloroform extraction procedure . Target regions overall[22―24]. Moreover, in order to add more informa- of the mitochondrial DNA were amplified from the total tive sites, complete sequences of the 12S rRNA gene DNA extracts using the polymerase chain reaction from 16 species were added to the Cyt b gene sequences (PCR). The primers for both Cyt b and 12S rRNA, Cyt b to generate a robust phylogeny of the genus Takifugu. F (5′-GGCGTGAAAAACCATCGTTG-3′) and Cyt b R Two species, Tetraodon nigroviridis and Lagocephalus (5′-CCCCGACATTCGGTTTACAAGAC-3′), and 12sF gloveri, were included as outgroups. They are the puta- (5′-GCAGAGTACTGAAGATGCTAAG-3′) and 12sR tive closest relatives to Takifugu according to recent (5′-CGTCAACTCGGTGTAAGG-3′) were adapted studies[19,20]. from Xiao et al.[27] and Liu[28], modified with the mito- chondrial sequence of T. rubripes[16] (GenBank Acces- 1 Materials and methods sion No. AJ421455). The PCR contained approximately 100 ng of template DNA, 1.5 µL of each primer, 6 µL of 1.1 Species examined and sample collection 10× reaction buffer, 1.5 µL dNTPs (each 2.5 mmol/L), Due to recent over-fishing, wild stocks of Takifugu have and 1.5 U Taq DNA polymerase in total 60 µL in a total been decreasing rapidly[25], which increased the difficul- volume. The reaction conditions were conducted as the ties of sampling; therefore, T. porphyreus, T. pardalis, following: an initial 94℃ denaturation for 4 min; fol- Table 1 Pairwise p-distance of the complete of Cyt b gene data matrix of 19 taxa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 T. rubripes T. pseudommus 0.003 T. stictonotus 0.005 0.003 T. flavidus 0.034 0.037 0.04 T.