| The subfamily Cultrinae sensu stricto, a group of East Asian cyprinids (Teleostei: Cypriniformes), comprises18genera with64species or subspecies, among which only genus Ischikauia is distributed in Japanese Archipelago and the remaining cultrins are mainly distributed in China. As Howes (1979) stated:"Of all the cyprinid sub familial assemblages recognized at present there is probably none so ill-defined as the Cultrinae", the monophyly, the systematic position, and the intergeneric relationships of this group of fishes remain controversial. Along with the wide use of molecular phylogenetics in cypriniform systematics, the monophyly and phylogenetic relationships of the cultrin fishes have attracted more attention. In addition, Megalobrama pellegrini is one of the endemic species in China belonging to the Cultrinae, which is only distributed in the main streams and tributaries along the upper reaches of the Yangtze River in the Sichuan basin. Recently, the population size of this species has decreased sharply because of the loss of habitats and overfishing. However, the population genetic studies on M. pellegrini are limited. Hence, the main themes of this thesis were two-fold:First, to explore the origin and evolution of the cultrins, we used mitochondrial genome sequences as a molecular marker to reconstruct the phylogenetic relationships of the Cultrinae sensu stricto and related groups, and estimated the divergence times of the Cultrinae sensu lato based on the method of relaxed molecular clock; Second, to better understand and manage the current M. pellegrini populations, we first developed polymorphic microsatellites for M. pellegrini and then using some of these microsatellite markers combined with mitochondrial DNA sequences (cytb&D-loop) to study the population genetic diversity of M. pellegrini. The main results were as follows: 1. We determined25complete mitochondrial genome sequences with16,610bp16,747bp in size, of which20mitogenomes representing6genera with12species in the Cultrinae sensu stricto, and5mitogenomes representing5genera with5species of other cyprinids. And we retrieved7mitogenomes from GenBank representing6genera with7species belonging to the Cultrinae sensu stricto, and21mitogenomes representing17genera with21species belonging to other cyprinids. Totally55mitogenome sequences were used to infer the polygenetic relationships of the Cultrinae sensu stricto and related groups, with Myxocyprinus asiaticus as outgroup. We reconstructed the phylogenetic trees by partitioned and unpartitioned Bayesian inference (BI) and maximum likelihood (ML) methods. Based on relaxed molecular clock and8fossil records plus one geological event as the carlibration points, we estimated the divergence times of the Cultrinae sensu stricto and related groups using mitogenomic sequences. Furthermore, we compared the phylogenetic performance of different mitochondrial segments by using the Partitioned Bremer Support (PBS). The results show that:(1) The Cultrinae sensu stricto was not a monophyletic group with the representatives of Macrochirichthys and Paralaubuca not grouping with the remaining9genera of the Cultrinae; the remaining9genera were clustered together and could be divided into3groups:the Cultrine group with two branches (the Parabramis branch and the Culter branch), the Ischikauia group, and the Hemiculter group; Megalobrama, Parabramis, Sinibrama, Culter, Cultrichthys, and Chanodichthys clustered within the Cultrine group, of which the former three genera formed the Parabramis branch and the later three genera formed the Culter branch; the Ischikauia group only contained genus Ischikauia; Hemiculter and Pseudolaubuca clustered as Hemiculter group;(2) Removing the genera Macrochirichthys, Paralaubuca, and Metzia out of the Cultrinae sensu stricto, the remaining15genera formed a monophyletic group and could be redefined as a new subfamily "Cultrinae sensu stricto’", which was sister to the subfamily Xenocyprinae;(3) The genera of Megalobrama, Sinibrama and Pseudolaubuca were not monophyletic groups, respectively;(4) The Cultrinae sensu lato and redefined "Cultrinae sensu stricto" in this study most probably originated in middle Eocene (～42.7million years ago, Mya) and early Oligocene (～31.7Mya), respectively;(5) The partitioned strategies on the mitogenomic dataset had no prominent effect not only on the resolving of the intergeneric relathionships of the Cultrinae sensu stricto and its related groups, but also on the improvement of the node supports; and (6) Of the different mitochondrial fragments, ND4and ND5genes showed the best phylogenetic performance, while ND4L, ND3,12S, and ATP8genes were the poorest. 2. In the present study, we developed polymorphic microsatellites on M. pellegrini using454sequencing. In addition, we tested the cross-amplification and polymorphism for all microsatellite loci that amplified successfully in M. pellegrini on four other related species (M. amblycephala, M. skolkovii, M. terminalis, and Sinibrama wui). The results showed that:(1) Of53microsatellite loci screened in this study,33were amplified successfully with26polymorphic loci and7monomorphic loci;(2) The mean number of alleles (NA), mean observed (Ho) and expected (HE) heterozygosity, mean polymorphic information content (PIC) were3.08,0.47,0.51, and0.42, respectively, showing a moderate level of polymorphism; of the26polymorphic microsatellite loci,5loci (MP15, MP16, MP17, MP32, and MP40) showed a high level of polymorphism (PIC>0.5) with no deviation from Hardy-Weinberg equilibrium (HWE), no null alleles and no evidence of linkage equilibrium (LD), which were suitable for the further population genetic studies of M. pellegrini;(3) High success rates of cross-amplification on four related species were obtained (78.8%-97.0%) as well as high proportions of polymorphism (64.3%-78.1%).3. Ten microsatellite loci and two mitochondrial DNA (cytb&D-loop) markers were used to study the genetic diversity of two wild M. pellegrini populations that collected from Longxi River (LXH) and Qiuxi River (QXH), respectively. The results showed that:(1) Both of the wild populations appeared to have a moderate level of genetic diversity based on10microsatellite loci (LXH: NA=3.8, Ho=0.63, HE=0.62; QXH:NA=3.7, Ho=0.64, HE=0.64);(2) The genetic diversity of LXH population and QXH population evaluated by two mitochondrial DNA markers (cytb&D-loop) were low, and the former was much lower than the later (LXH_cytb:H (haplotype)=1, Hd (haplotype diversity)=0.000, V (variation site)=0, P,(nucleotide diversity)=0.00000; QXH_cytb:H=2,Hd=0.272, V=4, Pi=0.00097; LXH_D-loop:H=2, Hd=0.239, V=3, P,=0.00077; QXL_D-loop:H=5,Hd=0.423, V=15, Pi=0.00243);(3) Analysis of molecular variation (AMOVA) inferred from both microsatellites and mitochondrial DNA (cytb&D-loop) revealed that the degree of genetic divergence was moderate (Fst=0.083～0.124, P<0.05), which were occurred mainly within the population (87.62%～91.66%) rather than between populations. Our studies indicate that the M. pellegrini population habitated in Qiuxi River could be a better candidate for conservation management than that from Longxi River. |
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