Geographic Distribution of Pelteobagrus fulvidraco and Pelteobagrus vachelli in the Yangtze River Based on Mitochondrial DNA Markers

Three populations of Pelteobagrus vachelli and Pelteobagrus fulvidraco of the Yangtze River were examined by PCR-RFLP analysis of mitochondrial DNA fragments. ND5/6 and D-loop fragments were digested by 10 restriction endonucleases. Significant geographic variations between upstream and mid-downstream populations in the haplotype frequencies and restriction patterns were revealed. This suggested that the diversity of P. vachelli was high; 11 haplotypes were obtained from all the samples. The upstream population shared seven haplotypes and the middle and downstream populations shared another four haplotypes. Among all of the haplotypes, one haplotype was shared in 30 samples of the populations from middle and downstream, but it was not found in the upstream population. Any haplotype found in the upstream population was not detected in the middle and downstream populations. Genetic diversity of P. fulvidraco was low and only five haplotyes were detected from all 60 samples. Phylogenic relationships also indicated that the fishes from upstream and mid-downstream were apparently divided into two populations.     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Length–weight relationships of two fish species from the Yangtze River,China

Fish samples for the study were collected from the Yangtze River in China. Length–weight relationships were determined for two endemic fish species (Leiocassis longirostris Günther, 1864 and Schistura fasciolata Nichols & Pope, 1927) for the first time.     

Population genetic structure and evolutionary history of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> in the Yangtze River,China

Ample studies have been conducted to investigate the population genetic structure of grass carp Ctenopharyngodon idella in the Yangtze River, China. However, samples from the upper reaches were not included. In this study, we collected samples from the entire river, including three locations in the upper reaches: Yibin, Banan and Yunyang, two locations in the middle reaches: Shishou and Ruichang and one location in the lower reaches: Hanjiang, and sequenced three mitochondrial coding genes (ND5, ND6 and Cytb) and one control region (i.e., the D-loop). Nineteen haplotypes were observed in grass carp of the Yangtze River through the analysis of combined sequence data sets (around 4428 bp). Haplotype diversity indices (0.6000 ∼ 0.9333) and nucleotide diversity indices (0.0002 ∼ 0.0020) demonstrated low genetic diversity in the Yangtze grass carp. The analysis of molecular variance and the fixation index (F _ST = 0.0202) revealed insignificant genetic difference between samples from different reaches. Two monophyletic lineages of haplotypes were identified, with the lineage A experiencing potential expansion events. Along with previous findings, this study provides a better understanding of genetic diversity and variation of grass carp in the Yangtze River and will be served as an important baseline to evaluate the long-term impact of the Three Gorges Dam and other hydroelectric facilities on fish biodiversity.     

Genetic diversity and population structure of Schizopygopsis younghusbandi Regan in the Yarlung Tsangpo River inferred from mitochondrial DNA sequence analysis

The genetic diversity and population structure of Schizopygopsis younghusbandi from six sites in the middle reach of the Yarlung Tsangpo River were examined based on mitochondrial DNA cytochrome b (Cyt b) gene and control region (D-loop) sequences. 1141 bp of complete Cyt b sequences and 737 bp of partial D-loop sequences for 153 individuals of S. younghusbandi were obtained by using PCR amplification and sequencing. The results showed that S. younghusbandi populations had high haplotype diversity and low nucleotide diversity, and the population genetic diversity of this species was at a low level. Analysis of molecular variance revealed that most genetic variation occurred within populations, and that genetic differentiation was at low or moderate levels. The network of haplotypes based on Cyt b gene showed that there were two groups within the examined populations. Neutrality tests and mismatch distribution analysis suggested that this species might have undergone a population expansion and the expansion time was estimated to be 0.25–0.46 Ma BP. All these results would be crucial to establish scientific strategies for effective conservation and sustainable exploitation of wild resources of S. younghusbandi.     

Genetic divergence of sympatric charrs of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> from Nachikinskoe Lake (Kamchatka)

We studied genetic differentiation of two charr species, Dolly Varden Salvelinus malma malma Walbaum and resident lacustrine charr Salvelinus sp., which sympatrically inhabit Nachikinskoe Lake (the Bol’shaya River basin) in southwestern Kamchatka Peninsula. Using restriction analysis (RFLP), three mitochondrial DNA fragments (ND1/ND2, ND5/ND6, and Cyt b/D-loop) amplified in polymerase chain reaction (PCR) were compared. The divergence of the mtDNA sequences between Salvelinus sp. and S. malma malma was 2.8%; Salvelinus sp. and S. taranetzi, 0.36%; Salvelinus sp. and S. krogiusae, 0.21%; Salvelinus sp. and S. alpinus, 3.0%. These results point to reproductive isolation of charrs in Nachikinskoe Lake and support the earlier suggestion on a close relationship between Salvelinus sp., S. taranetzi, and S. krogiusae.     

Population genetics and sympatric divergence of the freshwater gudgeon,Gobiobotia filifer,in the Yangtze River inferred from mitochondrial DNA

The ecosystem and Pleistocene glaciations play important roles in population demography. The freshwater gudgeon, Gobiobotia filifer, is an endemic benthic fish in the Yangtze River and is a good model for ecological and evolutionary studies. This study aimed to decode the population structure of G. filifer in the Yangtze River and reveal whether divergence occurred before or after population radiation. A total of 292 specimens from eight locations in the upper and middle reaches of the Yangtze River were collected from 2014 to 2016 and analyzed via mitochondrial DNA Cyt b gene sequencing. A moderately high level of genetic diversity was found without structures among the population. However, phylogenetic and network topology showed two distinct haplotype groups, and each group contained a similar proportion of individuals from all sampled sites. This suggested the existence of two genetically divergent source populations in G. filifer. We deduced that a secondary contact of distinct glacial refugia was the main factor creating sympatric populations of G. filifer, and climate improvement promoted population expansion and colonization.     

Genetic variation at mtDNA and microsatellite loci in Chinese longsnout catfish (<Emphasis Type="Italic">Leiocassis longirostris</Emphasis>)

Genetic variability and population structure of the Chinese longsnout catfish Leiocassis longirostris Günther in the Yangtze River was examined with mitochondrial control region sequences and nuclear microsatellite markers. A 705-bp segment of the mitochondrial DNA control region was sequenced from 132 samples, which identified a total of 61 haplotypes. The Chinese longsnout catfish in the Yangtze River was characterized with high haplotype diversity (h = 0.9770 ± 0.0041) but low nucleotide diversity (π = 0.0081 ± 0.0043). Median-joining network analysis revealed a star-shaped pattern and mismatch distribution analysis found a smooth unimodal distribution, which suggested that this species in the Yangtze River underwent a population expansion following bottlenecks and/or they originated from a small size of founding population. It was estimated that the possible time of population expansion was 139,000–435,000 years before present, a time period in the middle Pleistocene. The analysis of molecular variance and phylogenetic reconstructions did not detect significant geographic structure between different river sections. This pattern of genetic variation was further evidenced with nuclear microsatellite markers. The genetic differentiation between above and below the Gezhouba Dam and Three Gorges Dam is very small at mitochondrial and nuclear levels, which suggested that these recently developed dams might have not significantly resulted in population genetic fragmentation in the Chinese longsnout catfish. However, the potential exacerbation of genetic structuring by the dams should not be overlooked in the future.     

Nuclear DNA PCR-RFLPs that distinguish African and European honey bee groups of subspecies. II: Conversion of long PCR markers to standard PCR

Nuclear DNA PCR-RFLPs previously found in amplifications of three long (>5 kbp) anonymous regions of DNA were made analyzable using standard PCR procedures. RFLP analyses were simplified by restricting the amplifications to sections, within each locus, that contained most of the informative polymorphic sites. AluI digests of locus L-1 section 2 (L-1S2) revealed three suballeles of which one was African-specific (Apis mellifera scutellata Lepeletier) and one was east European-predominant (A. m. ligustica Spinola, A. m. carnica Pollman, and A. m. caucasica Gorbachev). Alleles found originally at locus L-2 with Avawere determined in RFLP analysis of two sections, L-2S1int and L-2S2, resulting in two African-specific and two east European-predominant suballeles. Suballele identity was determined by the combination of banding patterns from both fragments. revealed by HaeIII in locus L-2 were analyzed in amplifications and digests of L-2S1int, an 830 bp fragment within L-2S1. Seven suballeles were found of which two were African-specific and three were east European-specific or predominant, including one suballele specific to the east European subspecies A. m. caucasica. In locus L-5, RFLPs were detected with HaeIII, DdeI, and SpeI. HaeIII polymorphisms were analyzed by amplification and digestion of fragments L-5S1xt and L-5S1ter. Five suballeles were found of which three were African-specific and one east European-predominant. For DdeI, all five alleles originally found with long PCR could be identified in RFLP analyses of three sections. Two African-specific, one east European-specific, and one west European-predominant (A. m. mellifera L. and A. m. iberica Goetze) suballeles were found. A west European-predominant suballele was also found in RFLP analysis of L-5S3 with SpeI. Allele frequency data from Old World and U.S. populations are presented.     

Genetic divergence and phylogeography in the genus <Emphasis Type="Italic">Nyctalus</Emphasis> (Mammalia,Chiroptera): implications for population history of the insular bat <Emphasis Type="Italic">Nyctalus azoreum</Emphasis>

We used three mitochondrial DNA fragments with different substitution rates (ND1, Cyt b and the CR) to infer phylogenetic relationships among six species of the genus Nyctalus, and compare levels of genetic divergence between the insular, vulnerable Nyctalus azoreum and its continental counterpart to assess the origins of the Azorean bat. The larger species found throughout the Palaearctic region (N. lasiopterus, N. aviator and N. noctula) share a unique chromosome formula (2n = 42) and form a monophyletic clade in our reconstructions. Nyctalus plancyi (= velutinus), a Chinese taxon with 2n = 36 chromosomes, is sometimes included in N. noctula, but is genetically very divergent from the latter and deserves full species status. All Cyt b and CR haplotypes of N. azoreum are closely related and only found in the Azores archipelago, but when compared to continental sequences of N. leisleri, levels of mtDNA divergence are unusually low for mammalian species. This contrasts with the high level of differentiation that N. azoreum has attained in its morphology, ecology, and echolocation calls, suggesting a recent split followed by fast evolutionary change. The molecular data suggest that N. azoreum originated from a European population of N. leisleri, and that the colonisation of the Azores occurred at the end of the Pleistocene. The Madeiran populations of N. leisleri also appear to have a European origin, whereas those of the Canary Islands probably came from North Africa. In spite of its recent origin and low genetic divergence, the Azorean bat is well differentiated and consequently represents a unique evolutionary unit with great conservation value.     

Mitochondrial DNA restriction map for the Caribbean fruit fly,Anastrepha suspensa,and occurrence of mitochondrial DNA diversity within highly inbred colonies

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIII site. After cloning of the 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruitflies.     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Phylogeny of Nannizzia incurvata,N. gypsea,N. fulva and N. otae by restriction enzyme analysis of mitochondrial DNA

Sequence divergence among Nannizzia incurvata, N. gypsea, N. fulva and N. otae was studied genetically using digestion profiles of mitochondrial DNA with restriction enzymes, Hae III, Hha I, Hind III and Xba I. Only N. fulva was subdivided into two types on restriction profiles. Phylogenetic relationships suggest that 1) N. gypsea is more closely related to N. fulva than N. incurvata, 2) the phylogenetic distance between N. otae and the other three species is larger than the distances between the other three species.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

Fine structure of polyoma virus DNA.

A fine structure map of polyoma DNA has been made based on cleavage with a number of restriction endonucleases (including HaeII and III, BamI, HindII and III, BumI, HpaII, and in part, HphI) and depurination of wild-type DNA, the eight HpaII restriction fragments and some HaeIII fragments. This analysis has made possible some correlation with simian virus 40 DNA, and has facilitated detailed examination of various polyoma strains and variants. Sequences from the region of the origin of DNA replication have been examined.     

Unique repetitive sequences of 170 bp inChlorella

Summary Cot analysis ofChlorella DNA revealed that the genome of the unicellular green alga contained a small amount of repetitive sequences (at most 15% of the total DNA). Short repetitive sequences (SRS) of 170 bp produced by enzymatic digestion of algal DNA with eitherHaeIII,HinfI, orPstI, were found by polyacrylamide gel electrophoresis, and their copy number was estimated to be a few hundred (about 2% of the total repetitive sequences). All three members showed high sequence homology and could be be unified into one family, HaeIII family. The family was divided further into two subfamilies,HinfI- (HaeIII-andHinfI-SRS) andPstI-(PstI-SRS) subfamilies, based on small sequence differences among the members. TheHaeIII family had characteristic structural features, including a considerable number of small unique sequence units (purine-CC) and both direct and inverted repeats, and were organized in tandem arrays in the genome.     

Mitochondrial DNA variation, effective female population size and population history of the endangered Chinese sturgeon, Acipenser sinensis

The anadromous Chinese sturgeon (Acipenser sinensis), mainly endemic to the Yangtze River in China, is an endangered fish species. The natural population has declined since the Gezhouba Dam blocked its migratory route to the spawning grounds in 1981. In the near future, the completion of the Three Gorges Dam, the world's largest hydroelectric project, may further impact this species by altering the water flow of the Yangtze River. Little is currently known about the population genetic structure of the Chinese sturgeon. In this study, DNA sequence data were determined from the control region (D-loop) of the mitochondrial genome of adult sturgeons (n = 106) that were collected between 1995–2000. The molecular data were used to investigate genetic variation, effective female population size and population history of the Chinese sturgeon in the Yangtze River. Our results indicate that the reduction in abundance did not change genetic variation of the Chinese sturgeon, and that the population underwent an expansion in the past. AMOVA analysis indicated that 98.7% of the genetic variability occurred within each year's spawning populations, the year of collection had little influence on the diversity of annual temporary samples. The relative large effective female population size (N _ef) indicates that good potential exists for the recovery of this species in the future. Strikingly, the ratio of N _ef to the census female population size (N _f) is unusually high (0.77–0.93). This may be the result of a current bottleneck in the population of the Chinese sturgeon that is likely caused by human intervention. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River

The Yangtze River is the longest river in China and is divided into upstream and mid‐downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.     

Variation in blood proteins and mitochondrial DNA within and between local populations of longtail macaques,Macaca fascicularis on the Island of Java, Indonesia

We examined 31 blood protein loci, and restriction fragment length profiles of a PCR product of mitochondrial DNA containing the D-loop region using five kinds of restriction endonucleases (HaeIII,HinfI,MboII,MspI, andSau3 AI) in order to quantify the level of genetic variation of longtail macaques,Macaca fascicularis. Samples were collected from nine social groups in five localities of West Java, Indonesia. The average heterozygosity per individual was 0.060 and 15.7% of the loci were polymorphic (P_poly) over all populations in the protein analysis. There was no mtDNA haplotype variation within either social groups or local populations. To the contrary, great diversity was observed among local populations. Both nuclear diversity (measured byNei's standard genetic distance) and mitochondrial diversity (measured by sequence divergence) showed a significantly positive correlation with geographic distance. There was no significant correlation between these two genetic markers, however. The genetic structure of the population was evaluated in terms of local inbreeding and temporal changes in allele frequency. Differences between nuclear and mitochondrial data are discussed in relation to gender specific migration and lineage sorting.     

PM2 bacteriophage plaque morphology mutants exhibit differences in DNA length and restriction endonuclease patterns

A large plaque (LP) and a small plaque (SP) variant of PM2 bacteriophage were isolated from a mixture of the two plaque variants and were grown separately in the appropriate host bacterium,Alteromonas espejiana. They have remained pure for approximately one year from the original isolation. Restriction endonuclease analyses revealed differences in theHaeIII restriction profile between the two variants.HaeIII fragment 1 of the SP DNA was found to be smaller than the corresponding fragment from the LP variant DNA, whereas fragment 7 from the SP DNA was slightly larger than the same fragment from LP DNA. Electron microscopy of heteroduplexes formed between the DNAs from the two variants revealed that the deletion in fragment 1 mapped very close to the junction betweenHaeIII fragments 1 and 13 on the physical map of PM2 DNA. The difference in DNA length between the two variants results from addition or deletion mutations.Deceased.