Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei: Cypriniformes)

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Abstract

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenetic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitorinae + (Cobitinae + Nemacheilinae)))), which indicated that Sawada’s Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae.

Introduction

Mitochondrial DNA (mtDNA) sequences, especially the cytochrome b (cyt b) gene and the control region (CR) are frequently utilized for population genetic and phylogenetic studies of fishes (Liu and Chen, 2003, Moum and Árnason, 2001, Peng et al., 2004, Perdices et al., 2004). The cytochrome b gene encodes a protein and evolves relatively slowly, whereas the non-coding CR in vertebrates, presumably because of the lack of coding constraints, evolves rapidly. Sequence variation in the CR consists not only of substitutions but also of indels of various lengths and of variation in number of copies of tandem repeats (Sbisà et al., 1997). Control region, especially the tRNApro end, has been suggested to have one of the highest substitution rates of all the mitochondrial genes (Brown, 1985, Meyer, 1993). Mutation rate of the CR can be two to five times higher than that of mitochondrial protein-coding genes (Meyer, 1993). However, several reports have challenged the generality of this observation, especially in different fish groups. In rat and mouse, Brown et al. (1986) found a slower rate of substitution in CR than that of protein-coding genes. A slower rate of substitution in CR was also found in salmonid fishes (Bernatchez and Danzmann, 1993, Shedlock et al., 1992), and butterflies of the genus Jalmenus (Taylor et al., 1993). Zhu et al. (1994) compared relative rates and patterns of sequence evolution in CR and cyt b sequences from different populations and species of freshwater rainbow fishes of the genus Melanotaenia, and discovered that the overall levels of divergence were similar for these two gene segments but patterns of sequence evolution varied. Crochet and Desmarais (2000) provided evidence for a lower-than-expected interspecific divergence among CRs of gulls and proposed that the slow rate of evolution of CR part III of the gulls could be partly explained by the existence of secondary structures. All these and other studies have been confined to species or genera. Comparison at different levels including species, genera, families, and for genealogical patterns of molecular evolution of these important genera is needed.

Fishes of the family Cobitidae are part of a major lineage of the order Cypriniformes, which is the largest group of freshwater fishes in the world. Presently, five families (Gyrinocheilidae, Catostomidae, Cobitidae, Balitoridae, and Cyprinidae) are recognized as valid in Cypriniformes (Nelson, 1994). However, their phylogenetic relationships remain controversial. Two main hypotheses had been proposed by Wu et al. (1981) and Siebert (1987) (Fig. 1). Wu et al. (1981) suggested that the Balitoridae (=Homalopteridae) was closest to the Cyprinidae and the other families form another monophyletic group. Siebert (1987) proposed that the Cyprinidae forms a single monophyletic group and the non-cyprinid cypriniforms form another monophyletic group, a conclusion supported by some recent investigations (He et al., 1997, Liu et al., 2002). The fact that Gyrinocheilidae and Catostomidae form their own monophyletic group has also been accepted widely. Therefore, the relationship between Cobitidae and Balitoridae and their relationship to other families is the key to resolve the phylogenetic relationship of the whole Cypriniformes.

Regan (1911) first defined the group Cobitidae and divided the family into the subfamilies Cobitinae and Nemacheilinae. Hora (1932) classified the family Homalopteridae, an apparent clade that has been replaced by name as Balitoridae (Kottelat, 1988), into two subfamilies Gastromyzoninae and Homalopterinae (=Balitorinae), and considered the former as a derivative of the Cobitidae with the latter as a descendant of the family Cyprinidae. Berg (1940) divided the Cobitidae into three subfamilies, Botiinae, Cobitinae, and Nemacheilinae, a change that was accepted by many authors at that time (Chen and Zhu, 1984, Nalbant, 1963, Ramaswami, 1953, Wu et al., 1981). After examining 52 characters of 48 species or subspecies, Sawada (1982) transferred the subfamily Nemacheilinae from the family Cobitidae to the family Balitoridae, these two clades form a monophyletic group, the superfamily Cobitoidea. The former group is differentiated into two monophyletic groups Botiinae and Cobitinae which are considered sister groups, and the latter consists of Nemacheilinae and Balitorinae. This classification has been widely accepted (Kottelat, 2001, Nelson, 1994, Siebert, 1987). However, based on molecular phylogenetic analysis of the Cypriniformes, Liu et al. (2002) proposed that the relationships within the Cobitoidei are: Catostomidae + (Gyrinocheilidae + (Botiinae + (Balitoridae + (Cobitinae + Nemacheilinae)))). Thus, the Botiinae forms the basal group to other loaches, a conclusion in general agreement with Nalbant (1963). Furthermore, Nalbant (2002) treated the Botiinae, Cobitinae, and Nemacheiliane as three valid families Botiidae, Cobitidae, and Nemacheilidae. The analyses by Liu et al. (2002) included only a few loach species, precluding an adequate test of the phylogenetic relationship of loaches.

In the present study, we sequenced mitochondrial cytochrome b gene and CR of the so-called loaches (including the families Cobitidae and Balitoridae) to compare the evolutionary rate of these two segments at different classification levels that has been examined previously, and study the phylogenetic relationship of the Cobitoidea.

Section snippets

Samples and DNA extraction

In present study, 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea were selected for analysis. Two sequences of Myxocyprinus asiaticus were obtained from GenBank [AF036176 (cyt b), AY017140 (CR)]. Detailed information of specimens is listed in Table 1. The cyt b and CR sequences of Cyprinus carpio and Danio rerio were used as outgroups (NC001606 and NC002333). Muscles from alcohol fixed museum specimens were used for DNA extraction. All specimens belong to

Base compositions

Following alignment of the 1140 bp of cyt b gene obtained for 95 individuals (including outgroups), no deletions or insertions were observed. Plots of the number of substitutions against HKY distances revealed no saturation for Ti or Tv for all positions (not shown). Base frequencies were heterogenous across all taxa for all three codon positions (χ2 = 383.168, df = 282, P = 0.000 < 0.001). Nucleotide composition at the third position exhibited significant heterogeneity: first position, χ2 = 43.789, df = 

Dynamics of the evolutionary rate of CR

Generally, the CR sequences evolve more rapidly than cyt b sequences in the Cobitoidea, however, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene. Roukonen and Kvist (2002) reported a similar finding in a survey of 68 avian species. They proposed that the trend of the ratio of CR versus cyt b divergences seems to be somewhat genus specific; many avian lineages were shown to have more rapidly evolving CR (e.g., among the Cyanoramphus species,

Acknowledgments

We are grateful to L. Zhang, J. Yang, H. Hu, Z. Peng, X. Guo, and L. Yang for help in collecting specimens. Thanks are also to Dr. M. Kottelat for advices on species identification and to the anonymous reviewers for their suggestions to this paper. Funding support was provided by Grants of NSFC 30170137, State Key project of NSFC 40432003, and Innovation Project of the Chinese Academy of Sciences (Grant No. KZCX-SW-126). Our work is also part of the project, the Cypriniformes Tree Of Life,

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