Comparison research and phylogenetic implications of mitochondrial control regions in four soft-shelled turtles of Trionychia (Reptilia, Testudinata)

The mitochondrial control regions (CRs) and flanking sequences of Pelodiscus sinensis, Apalone ferox, Palea steindachneri and Carettochelys insculpta were obtained using Long-PCR with gene-specific primers. The CR lengths of the four species were 1843 bp, 1356 bp, 1725 bp, and 969 bp. The base composition percentages of A+T were 60.5%, 60.7%, 65.7%, 64.7%, respectively. Combined with CR sequences of other three soft-shelled turtles published in GenBank (Pelodiscus sinensis, Korea, AY962573; Dogania subplana, AF366350; Lissemys punctata, EF050073), we compared the CR structures and identified three functional domains (TAS, CD and CSB) in which conserved sequence blocks (TAS, CSB -F, CSB-1, CSB-2 and CSB-3) were also successfully identified according to their sequence similarities to those of other turtles. The variable numbers of tandem repeats (VNTRs 1) with 50–52 bp motif were identified at 5′-end of CR among the five soft-shelled turtles P. sinensis (China), P. sinensis (Korea), A. ferox, P. steindachneri, D. subplana. The copy number of the VNTRs varied from 5 to 15. VNTRs 2 with 2–11 bp motif were identified in the 3′- end of CR among all of the six soft-shelled turtles with variable number of motifs from 4 to 29. Moreover, VNTRs 3 with 6 bp motif were identified between CSB-1 and CSB-2 of CR both in P. sinensis (China) and P. sinensis (Korea), in which the number of motifs varied from 19 to 29. The types and distribution of VNTRs of the six soft-shelled turtles were also discussed. With Alligator mississippiensis as an outgroup, combined with the CR sequences (excluding VNTRs) of other five turtles which were published in GenBank, the molecular phylogenetic trees were constructed using PAUP 4.0b10 and MrBayes ver. 3.0. The results strongly supported the monophyly of Carretochelyidae and Carettochelyidae as sister group to an assemblage of Cryptodira. Our research suggested that the earliest phylogenetic tree splits into three separated basal branches; the Pelomedusidira (Pelomedusa subrufa), the Carettochelyidae (C. insculpta), and an assemblage of Cryptodira and the C. insculpta that might be a representation of distinctive suborder.     

The mitochondrial DNA control region comparison studies of four hinged turtles and its phylogentic significance of the genusCuora sensu lato (Testudinata: Geoemydidae)

The complete sequences of the mitochondrial DNA (mtDNA) control region (CR) ofCistoclemmys flavomarginata, Cistoclemmys galbinifrons, Cuora aurocapitata andCyclemys atripons were amplified by long-polymerase chain reaction (Long-PCR). The lengths were 1207 bp, 1722 bp, 1379 bp and 980 bp, respectively. Combining with the CR sequence ofPyxidea mouhotii (DQ659152), we compared the CR structure, and identified three functional domains (TAS, CD and CSB) in which the conservation sequences (TAS, CSB-F, CSB-1, CSB-2 and CSB-3) were also successfully identified according to their homology to those of other turtles. These 5 turtles have the identical CSB-2 and CSB-3 sequences, and 4 of them have the same CSB-1 sequence while there is one base transversion (T → A) inCy. atripons. We analyzed the variable number of tandem repeat (VNTR) sequences or microsatellites at the 3′ end of CR. The motifs of tandem repeats (7 types) are made up of 2–8 nucleotides, and the copy numbers are from 4 to 48. All of the 5 turtles exceptCy. atripons have the “TATTATAT” repeats and are ended by TA. The results of CR structure analysis displayed that theCuora, Cistoclemmys, andPyxidea have many similarities, but differ fromCyclemys. WithIndotestudo elongate (DQ080043) andIndotestudo forstenii (DQ080044) as outgroups, using the CR sequences (1123bp) excluded the microsatellites at the 3′ end of CR, we constructed the molecular phylogenetic trees using the MP, ML and BI methods. The results showed that there was a strong support to the monophyly of theCuora group consisting ofCuora,Cistoclemmys andPyxidea, which has a close relationship withMauremys andChinemys but far fromCyclemys, which are consistent with the analysis of the CR structure of the 5 turtles.     

Structure of mitochondrial DNA control region of Fenneropenaeus chinensis and phylogenetic relationship among different populations

This paper deals with the structure of mitochondrial DNA control region of Fenneropenaeus chinensis. The termination-associated sequence (TAS), cTAS, CSB-D-CSB-F, and CSB-1 are detected in the species. The results indicate that the structures of these parts are similar to those of most marine organisms. Two conserved regions and many stable conserved boxes are found in the extended TAS area, central sequences blocks, and conserved sequences blocks (CSBs). This is the special character of F. chinensis. All the mtDNA control region sequences do not have CSB2 and CSB3 blocks, which is quite different from most vertebrates. In addition, the complete mtDNA control region sequences are used to analyze the phylogenetic relationships of F. chinensis. The phylogenetic trees show a lack of genetic structure among populations, which is similar to many previous studies.     

Phylogeny of the Eurasian freshwater turtles of the genus Mauremys Gray 1869 (Testudines), with special reference to a close affinity of Mauremys japonica with Chinemys reevesii

Phylogenetic relationships of the freshwater turtles of the genus Mauremys and representatives of several other batagurid genera were inferred from variations in 863 base positions of mitochondrial 12S and 16S rRNA genes. Results strongly suggested the non-monophyly of Mauremys by indicating the closest affinity of Mauremys japonica with Chinemys reevesii , the type species of the genus Chinemys . Recent morphological analyses of the batagurid genera suggested that Mauremys is a basal stock of the subfamily Geoemydinae, whereas Chinemys is a member of the subfamily Batagurinae as supported by several putative synapomorphs with other batagurine genera. It is thus probable that the morphological character states used to define Mauremys actually represent symplesiomorphy, and that morphological character states shared between Chinemys and other batagurine genera have resulted from convergence. Also, our results did not support a sister-group relationship between Mauremys annamensis and Mauremys mutica , which has been implicitly or explicitly assumed by a number of previous authors on the basis of morphological data. Instead, M. annamensis was indicated to be closest to Mauremys i versoni , a species assumed to be most divergent among the East Asian Mauremys by previous authors.     

Mitochondrial genome of the eurasian otterLutra lutra (Mammalia, Carnivora, Mustelidae)

We determined the complete mitochondrial genome of the Eurasian otterLutra lutra, which is an endangered species in Korea. The circle genome (16,536 bp in size) consists of 13 protein-coding, 22 tRNA, and 2 rRNA genes, and a control region, as found in other metazoan animals. Out of the 37 genes, 28 are encoded on the H-strand, and the nine (ND6 and 8 tRNA genes) on the L-strand. Three overlaps among the 13 protein-coding genes were found: ATP8-ATP6, ND4L-ND4, and ND5-ND6. A control region (1090 bp) including the origin of H-strand replication (OH), TAS (a conserved motif TACAT-16bp-ATGTA) and CSB (CSB-1, CSB-2. and CSB-3) was observed between tRNA-Pro and tRNA-Phe genes, and O_L, with 36 highly conserved nucleotides between tRNA-Asn (N) and tRNA-Cys (C) within a cluster of five tRNA genes (WANCY), as typically found in vertebrates. The other important characteristics of theL. lutra mitochondrial genome were described in detail. In addition, a maximum likelihood and Bayesian trees of 9 mustelid species and 1 outgroup were reconstructed based on the nucleotide sequences of 11 protein-coding genes excluding ATP8 and ND6. It showed that Lutrinae formed a monophyletic group with Mustelinae that is not monophyletic. Within the subfamily Lutrinae,L. lutra andEnhydra lutris were grouped together and thenLontra canadentis placed as a sister of the clade. The present result is the first complete mitochondrial genome sequence reported from the genusLutra, and is applicable to molecular phylogenetic, phylogeographic, conservation biological studies for mustelid members. In particular, exploration of sequence variations of the control region may be helpful for analyzing inter-and intra-species variations in the genusLutra.     

Recombination and Evolution of Duplicate Control Regions in the Mitochondrial Genome of the Asian Big-Headed Turtle,Platysternon megacephalum

Complete mitochondrial (mt) genome sequences with duplicate control regions (CRs) have been detected in various animal species. In Testudines, duplicate mtCRs have been reported in the mtDNA of the Asian big-headed turtle, Platysternon megacephalum, which has three living subspecies. However, the evolutionary pattern of these CRs remains unclear. In this study, we report the completed sequences of duplicate CRs from 20 individuals belonging to three subspecies of this turtle and discuss the micro-evolutionary analysis of the evolution of duplicate CRs. Genetic distances calculated with MEGA 4.1 using the complete duplicate CR sequences revealed that within turtle subspecies, genetic distances between orthologous copies from different individuals were 0.63% for CR1 and 1.2% for CR2app:addword:respectively, and the average distance between paralogous copies of CR1 and CR2 was 4.8%. Phylogenetic relationships were reconstructed from the CR sequences, excluding the variable number of tandem repeats (VNTRs) at the 3′ end using three methods: neighbor-joining, maximum likelihood algorithm, and Bayesian inference. These data show that any two CRs within individuals were more genetically distant from orthologous genes in different individuals within the same subspecies. This suggests independent evolution of the two mtCRs within each P. megacephalum subspecies. Reconstruction of separate phylogenetic trees using different CR components (TAS, CD, CSB, and VNTRs) suggested the role of recombination in the evolution of duplicate CRs. Consequently, recombination events were detected using RDP software with break points at ≈290 bp and ≈1,080 bp. Based on these results, we hypothesize that duplicate CRs in P. megacephalum originated from heterological ancestral recombination of mtDNA. Subsequent recombination could have resulted in homogenization during independent evolutionary events, thus maintaining the functions of duplicate CRs in the mtDNA of P. megacephalum.     

Complete mitochondrial genome sequence of the Korean water deer Hydropotes inermis argyropus (Cervidae, Capreolinae)

The complete mitochondrial genome sequence of Hydropotes inermis argyropus consists of 13 protein-coding, 22 tRNA, and two rRNA genes, and 1 control region (CR). Three overlaps among the 13 protein-coding genes were found: ATP8/ATP6, ND4L/ND4, and ND5/ND6. The CR was located between the tRNA-Pro and tRNA-Phe genes and is 928 bp in length. The typical conserved domains, such as TAS and CSB, were identified in the CR.     

不同产地中华鳖的线粒体控制区序列分析及结构比较

采用PCR特异引物,扩增了两产地中华鳖（Pelodiscus sinensis）个体的mtDNA控制区（CR）及其邻近片段,测序获得了长度分别为1830bp和1630bp的序列。结合GenBank中已发表的韩国产中华鳖mtDNA的CR区序列,比较了3个产地中华鳖的CR区结构。分析显示：中华鳖不同产地mtDNA CR区DNA中的A＋T含量分别为60.5%、63.6%和64.8%,它们的5′、3′末端以及CSB1-CSB2之间均存在丰富的可变数目串联重复序列（variable numbers oftandem repeats,VNTR）。基于mtDNA CR区序列和结构分析,显示中华鳖不同产地的野生个体中存在丰富的遗传多样性。     

太湖新银鱼线粒体D-loop和Cytb片段序列结构与进化速率比较

共获得49个太湖新银鱼(Neosalanx taihuensis)个体的线粒体细胞色素b(Cyt b)全序列和控制区(D-loop)部分序列。所测线粒体D-loop部分序列长度变化范围为648～680bp,识别到位于前端的一个串联重复序列、一个终止相关序列(ETAS),3个中央保守区保守序列(CSB-F、CSB-E、CSB-D)及一个保守序列区保守序列(CSB-1),结构与其他鱼类的研究结果类似。太湖新银鱼线粒体Cyt b和D-loop片段的相对进化速率的比较研究结果表明,太湖新银鱼D-loop总的序列多态性位点的比例为0.83%,低于线粒体Cyt b部分总的序列多态性位点的比例(1.31%)。假设太湖新银鱼Cyt b基因平均进化速率相对值为1,贝叶斯(Bayes)MCMC模拟给出Cyt b基因的相对速率区间估计为1.000±0.131,而D-loop基因的相对速率为0.859±0.261,表明太湖新银鱼D-loop基因的进化速率低于Cyt b基因,同时,后验概率分布的变异方差也比较大。说明Cyt b基因比D-loop基因具有相对较高的进化速率,也相对更接近分子钟假设。因此,可以认为Cyt b基因比D-loop基因更适于太湖新银鱼种内及近缘种间相关分子生态及系统地理格局的研究。     

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.     

A genetic survey of heavily exploited, endangered turtles: caveats on the conservation value of trade animals

Asian turtles face an extinction crisis, and so it is imperative that systematists accurately determine species diversity in order to guide conservation strategies effectively. We surveyed mitochondrial and nuclear DNA (mtDNA and nuDNA) variation of the heavily exploited Mauremys mutica complex, a clade of Asian turtles that contains the endangered M. mutica from Japan, Taiwan, China and Vietnam, and the critically endangered Mauremys annamensis from central Vietnam. We discovered extensive mtDNA and nuDNA variation among samples that did not correspond to the currently recognized taxonomy. Both nuDNA and mtDNA data suggest that M. mutica is paraphyletic with respect to M. annamensis . Surprisingly, M. annamensis exhibits a previously unknown mtDNA structure in the form of two clades that are paraphyletic to M. mutica . These data reveal that the currently recognized taxonomy of the mutica complex does not reflect the genetic diversity of our samples. Unfortunately, many conservation-oriented captive-breeding efforts for turtles are also based on trade samples such as the ones studied here. These efforts include plans to breed trade-rescued individuals and release their progeny into the wild. Because our genetic survey reveals that the taxonomic identity of these samples does not reflect genetic diversity, we raise serious questions about the efficacy of these programs. In order to address conservation issues and provide more accurate estimates of evolutionary lineages within Mauremys , we recommend continued surveys for wild populations of the mutica complex to provide new genetic material and additional distributional data, attempts to extract DNA from historic museum specimens and a shift in conservation focus to in situ preservation of wild populations and associated habitat.     

Heteroplasmy, length, and sequence characterization of the mitochondrial control region in Tomistoma schlegelii

This study describes the organization of the repetitive pattern in the mtDNA control region of Tomistoma schlegelii. Using newly designed primers, we detected length variations of approximately 50-100?bp among individuals, and only one individual showed a heteroplasmic band. Sequencing the region after CSB III revealed two main patterns: a repeat motif and a variable number tandem repeat (VNTR) pattern. The VNTR region, with a core unit of 104?bp, consisting of four motifs and a short AT chain, is implicated in the length variation seen among individuals of Tomistoma. A conserved motif seen in a family unit indicated that the repeat pattern was stably inherited from the maternal parent to all offspring. A combination of VNTR patterns specific to different crocodilians was seen in Tomistoma, and the overall secondary structure was shown to be similar to that in Crocodylus and Gavialis.     

Analysis of the novel VNTR polymorphisms ofMUC8 gene

Analysis of mucin genes has identified the presence of several features that may represent important functional domains in mucin glycoproteins. In the central region of each mucin, there are a variable number of tandem repeats (VNTR; minisatellites). However, their genomic levels are unclear because of complex genomic properties. We report here the distribution of VNTR and polymorphic analysis ofMUC8. We searched for VNTR ofMUC8 using the Tandem Repeat Finder program and found nine VNTR motif. Six (MUC8 MS1～MS6) among the nine VNTRs were evaluated in this study. Each VNTR inMUC8 region was analyzed in genomic DNA obtained from 200 unrelated individuals and multi-generational families. All VNTRs (MUC8 MS1, -MS2, -MS3, -MS4,-MS5 and -MS6) were genotyped as polymorphic. The degree of polymorphism within theMUC8-MS5 showed the highest heterozygosity (h = 0.786) in theMUC8 region. In order to perform a segregation analysis of the VNTRs inMUC8, we analyzed genomic DNA obtained from two generations of five families and from three generations of two families. Six of the polymorphic VNTRs were transmitted through meiosis following a Mendelian inheritance, which suggests that polymorphic VNTRs could be useful markers for paternity mapping and DNA fingerprinting.     

Sequence evolution in and around the mitochondrial control region in birds

By cloning and sequencing 3.4 kilobases of snow goose mtDNA we found that the ND5 gene is followed by the genes for cytochrome b, tRNA^Thr, tRNA^Pro, ND6, tRNA^Glu, the control region, tRNA^Phe, and srRNA. This order is identical to that of chicken, quail, and duck mtDNA but differs from that of mammals and a frog (Xenopus). The mean extent of difference due to base substitution between goose and chicken is generally closer to the same comparison between rat and mouse but less than that between human and cow. For one of the nine regions compared (tRNA^Glu), the bird differences appear to be anomalous, possibly implicating altered functional constraints. Within the control region, several short sequences common to mammals are also conserved in the birds. Comparison of the goose control region with that of quail and chicken suggests that a sequence element with similarity to CSB-1 duplicated once prior to the divergence of goose and chicken and again on the lineage leading to chicken. Between goose (or duck) and chicken there are four times more transversions at the third positions of fourfold-degenerate codons in mitochondrial than in nuclear genes.Abbreviations CSB conserved sequence block - cytb cytochrome b - ND NADH dehydrogenase - srRNA small-subunit ribosomal RNA Deceased July 21, 1991 Correspondence to: T.W. Quinn at the University of Denver     

Structural conservation and variation in the mitochondrial control region of fringilline finches (Fringilla spp.) and the greenfinch (Carduelis chloris)

We sequenced the entire control region and portions of flanking genes (tRNA(Phe), tRNA(Glu), and ND6) in the common chaffinch (Fringilla coelebs), blue chaffinch (F. teydea), brambling (F. montifringilla), and greenfinch (Carduelis chloris). In these finches the control region is similar in length (1,223-1,237 bp) and has the same flanking gene order as in other birds, and contains a putative TAS element and the highly conserved CSB-1 and F, D, and C boxes recognizable in most vertebrates. Cloverleaf-like structures associated with the TAS element at the 5' end and CSB-1 at the 3' end of the control region may be involved with the stop and start of D-loop synthesis, respectively. The pattern of nucleotide and substitution bias is similar to that in other vertebrates, and consequently the finch control region can be subdivided into a central, conserved G-rich domain (domain II) flanked by hypervariable 5'-C-rich (domain I) and 3'-AT-rich (domain III) segments. In pairwise comparisons among finch species, the central domain has unusually low transition/transversion ratios, which suggests that increased G + T content is a functional constraint, possibly for DNA primase efficiency. In finches the relative rates of evolution vary among domains according to a ratio of 4.2 (domain III) to 2.2 (domain I) to 1 (domain II), and extensively among sites within domains I and II. Domain I and III sequences are extremely useful in recovering intraspecific phylogeographic splits between populations in Africa and Europe, Madeira, and a basal lineage in Nefza, Tunisia. Domain II sequences are highly conserved, and are therefore only useful in conjunction with sequences from domains I and III in phylogenetic studies of closely related species.      

The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae): genome characterization and phylogenetic considerations

The complete mitochondrial genome sequence of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) was determined and phylogenetic analysis was conducted to elucidate the evolutionary relationship of the marbled rockfish with other Sebastinae species. This mitochondrial genome, consisting of 17301 bp, is highly similar to that of most other vertebrates, containing the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one putative control region. Most of the genes are encoded on the H-strand, while the ND6 and seven tRNA genes (for Gln, Ala, Asn, Tyr, Ser (UCA), Glu, and Pro) are encoded on the L-strand. The reading frame of two pairs of genes overlapped on the same strand (the ATPase 8 and 6 genes overlapped by ten nucleotides; ND4L and ND4 genes overlapped by seven nucleotides). The possibly nonfunctional light-strand replication origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNA(Cys) gene. An extent termination-associated sequence (ETAS) and conserved sequence blocks (CSB) were identified in the control region, except for CSB-1; unusual long tandem repeats were found at the 3' end of the control region. Phylogenetic analyses supported the view that Sebastinae comprises four genera (Sebates, Hozukius, Helicolenus, and Sebasticus).     

Structure of mitochondrial DNA control region and molecular phylogenetic relationship among three flounders of genus Pleuronectes

Structure of mitochondrial DNA control region about three flounders – Pleuronectes yokohama, Pleuronectes schrenki and Pleuronectes herzensteini – were reported. The TAS, cTAS, CSB-A to CSB-F and CSB-1 to CSB-3 were detected in these three flounders. The results indicated that the structures of these parts were different from most fishes. All the mtDNA control region sequences of the three founders have tandem repeat sequences in the downstream of CSB-3, which is different from most vertebrates. According to the structure of the mtDNA control region, P. yokohama was more similar with P. schrenki and P. herzensteini was much different from the other two species. In addition, three segments such as control region, Cytb and COI are used to analyze the phylogenic relationships of the three species. The genetic distances and phylogenetic tree results support the classification by traditional morphology. It is not clear if P. yokohama and P. schrenki belong to the same species, and this should be accepted with caution.     

Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis

Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5^′-GCCGG-3^′) was found at the base of the stem within the tRNA ^Cys gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.     

Mitochondrial DNA structure and organization of the control region of South American camelids

This work presents the mitochondrial DNA molecular organization of the control region (CR) of South American camelids. Sequencing of five individuals each of guanaco, llama, alpaca and vicuna species showed that this region spans 1060 bp including three conserved sequence blocks (CSB I–III) adjacent to the tRNAPhe gene, a conserved central domain and one extended termination‐associated sequence in the 3′ domain of the CR close to the tRNAPro gene. A repeated array formed by three units of 26 bp was detected between CSB I and II. Alignment of the CR sequences from the four species shows a 337‐bp segment that includes most of the nucleotide variability with 10 polymorphic sites. We suggest the use of this segment as a molecular marker to infer data on camelid genetic relationships and population diversity studies.