| The Cobitoidea is an important group within Cypriniformes, where the relationships of "loaches", within the families of Cobitidae and Balitoridae are unclear. Similarly, the relationship between loaches and the famlily Cyprinidae remains unresolved. Many species of loaches are confined to China, where they show the highest diversity. The relationships among loaches are complex. So far, few reports have been published on the phytogeny of loaches. Without accurate knowledge of their phylogeny, it is impossible to examine the biogeographic events within the group. The investigation on classification and phylogeny of loaches is also important to resolve the relationships among Cypriniformes. Reports about the phylogeny of the whole Cobitoidea clade are scarce, especially at the molecular level. To help resolve this situation, in the present study, we sequenced mitochondrial cytochrome b (cyt b) gene and the control region (d-loop) of Cobitoidea to study the phylogenetic relationships of this group. Based on our result we focused on the major divergent events and evolutionary processes of morphological characters. The following are the main results:1. We analyzed cyt b gene and d-loop in 93 specimens representing 67 species of the Cobitoidea. The results showed that average base composition of cyt b gene (1140bp) was A=28.1%, T=28.7%, C=28.2% and G=15.0%. Transitions (Ti) outnumbered transversions (Tv), and the average rate of Ti/Tv was 2.093. The length of d-loop sequences of specimens ranged from 834 bp to 944 bp and many deletions or insertions were observed. The average base composition was A=34.5 %, T=31.9%, C=19.8% and G=13.8%. Compared to cyt b, d-loop showed a strong bias in A+ T content. And the average rate of Ti/Tv was 1.096. The comparison of evolution rate between cyt b and d-loop indicated that sequence divergences of d-loop were broadly higher than those of cyt b (about 1.83 times). However, considering for the closely related species only (cyt b <0.10), d-loop was evolving slower than cyt b gene (d-loop /cyt b - 0.78). A test for the partition homogeneity between the cyt b and d-loop revealed no significant differences(P=0.07>0.01), so we used combined data to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony (MP), neighbor-joining (NJ), and Bayesian methods (BI). The three analyses resulted in almost the same topology. The phylogenetic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitoridae + (Cobitinae + Nemacheilinae)))). Based on this relationship, we raised four groups of loaches to family level as Botiidae, Balitoridae, Cobitidae and Nemacheilidae, which were composed of the Cobitoidea together with Catostomidae and Gyrinocheilidae.2. Combining cyt b and some morphological characters, we discussed the phylogenetic relationship of the Botiinae in detail. Four methods, MP, NJ, maximum likelihood (ML) and BI, were adopted to construct the phylogenetic relationship of the botiine fishes. All these analyses consistently showed that the botiine fishes are monophyletic and distantly related to other loaches with a high support, which suggested that botiine fishes should be raised to form a separate family, the Botiidae. The analysis on morphological charaters indicated that the Botiidae are different from other loaches with two apomorphic characters. This again suggested that the Botiidae form a monophyletic group. Three monophyletic clades, Botia, Parabotia and Leptobotia, were found, with Botia at basal position. The Parabotia and Leptoutia are more derived and form a sister-group relationship.3. With the Botiidae as an example, we analyzed the structure of d-loop of the Botiidae and the phylogenetic implications on this group. Three blocks, Extended termination associated sequence (ETAS), Central conserved domain (CD) and Conserved sequence block (CSB) were included in the d-loop. Some conserved sequences were identified and their general sequences were given. In ETAS, ETAS1 was identified; in the CD, three conserved sequences, CSB-D, CSB-E and CSB-F were identified; in the CSB, we successfully identified CSB-1, CSB-2 and CSB-3. And all these conserved sequences were similar to those of the Vertebrate. The phylogenetic relationships resulted is almost the same situation as using cyt b.4. Combined with some European cobitids, we studied the phylogeny of the Cobitidae. The result suggested that Lepidocephalus octocirrhus was basal, and the genera Cobitis and Misgurnus were not monophyletic. But some subgroups can form monophyletic groups. Three analyses using NJ, MP and BI methods confirmed four monophyletic groups: Cobitis I, Misgurnus I, Cobitis II and Misgurnus II. However, the phylogenetic relationships of other cobitids cannot be resolved. Among Eurasia Cobitis, Cobitis II is basal, and Cobitis I is more derived with a place on the top of the phylogenetic tree. Our result also showed that European and Asian Cobitis formed sister-group relationship in many clades, which suggested that European Cobitis is polyphyletic origins. So, we cannot determine whether the current distribution pattern of Cobitis resulted from dispersal or vicariance.5. We discussed some important divergent events of the Cobitoidea and the evolution of some morphological characters. The analytic results with programs of PALM and Multidivtime suggested that the divergence of the Botiidae ancestry from other loach species was the oldest (about 150 million years ago, mya), which is similar to the divergence between Gyrinocheilus aymonieri and Myxocyprinus asiaticus (about 145 mya). The divergence of Balitoridae from the clade (Cobitidae + Nemacheilidae) was estimated to be 129 mya, while the divergent time between the Cobitidae and Nemacheilidae was 120 mya. However, the distribution pattern of the recent Cobitoidea cannot be interpreted using the geographic events happened within 100 mya. The analyses of morphogical characters of the Cobitoidea showed that the evolution of characters corresponds to the habitat shift from middle layer to the bottom of water bodies.
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