Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Mitochondrial cytochrome b gene sequences of Old World monkeys: With special reference on evolution of Asian colobines

The classification and phylogenetic relationships of the Old World monkeys are still controversial. For Asian colobines, from three to nine genera were recognized by different primatologists. In the present study, we have sequenced a 424 bp mitochondrial tRNA(Thr) gene and cytochrome b gene fragment from Macaca mulatta, Mandrillus sphinx, Mandrillus leucophaeus, Semnopithecus entellus, Trachypithecus vetulus, T. johnii, T. phayrei, T. francoisi, Pygathrix nemaeus, Rhinopithecus roxellanae, R. bieti, R. avunculus, Nasalis larvatus, and Colobus polykomos in order to gain independent information on the classification and phylogenetic relationships of those species. Phylogenetic trees were constructed with parsimony analysis by weighting transversions 5 or 10 fold greater than transitions. Our results support the following conclusions: (1) the Old World monkeys are divided into two subfamilies; (2) that among the colobines, Colobus, the African group, diverged first, and Nasalis and Rhinopithecus form a sister clade to Pygathrix; (3) that there are two clades within leaf monkeys, i.e. 1) S. entellus, T. johnii, and T. vetulus, and 2) T, phayrei and T. francoisi; (4) that Rhinopithecus avunculus, R. roxellanae, and R. bieti are closely related to each other, and they should be placed into the same subgenus; (5) that Rhinopithecus is a distinct genus; and (6) that the ancestors of Asian colobines migrated from Africa to Asia during the late Pliocene or early Pleistocene.

Phylogeny and evolution of the Drosophila nasuta subgroup based on mitochondrial ND4 and ND4L gene sequences

The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genesis strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five, distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D, sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The Kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D.. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D, nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous. (C) 1999 Academic Press.

Mitochondrial cytochrome b sequences variation of Protura and molecular systematics of Apterygota

Mitochondrial cytochrome b genes of about 450 bp fragments from 3 proturan species, 5 collembolan species and 2 dipluran species have been sequenced. The number of nucleotide substitutions and Kimura 2-parameter distances have also been calculated, and a series of molecular phylogenetic trees reconstructed by using parsimony and distance methods. The proturan, collembolan and dipluran species have evolved monophyletic groups. The results suggest that Protura and Collembola are sister groups, while Diplura is more or less demonstrating a closer phylogenetic relationship to the pterygotan insects. The phylogeny and their systematic position of protura and other groups are also discussed.

Mitochondrial DNA variation in cattle of South China: origin and introgression

Ten restriction endonucleases were used to investigate the mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) of 11 native cattle breeds and one cultivated cattle breed in South China. Twenty-three restriction morphs were detected, which can be sorted into five haplotypes, A phylogenetic tree of the haplotypes was constructed by using the 'upgma' method. Our study showed that haplotype I and II are identical to the zebu (Bos indicus) and taurine (Bos taurus) haplotypes, respectively. Zebu and taurine were the two major origins of cattle populations in South China, and the zebu probably had more influence on the native cattle population than taurine did. Haplotype III is identical to haplotype I of yak (Bos grunniens), which was only detected in the Diqing cattle breed. Haplotype IV was detected for the first time. This haplotype, found only in Dehong cattle, might be from an independent domestication event, probably from another Bos indicus population. Divergence of haplotypes I and IV occurred about 268,000-535 000 years ago, much earlier than the 10,000-year history of cattle husbandry. Our results also suggest a secondary introgression of mtDNA from yak to Diqing cattle.

Evolutionary trends of the mitochondrial lineage differentiation in species of genera Martes and Mustela

We compared partial sequences (402 bp) of the mitochondrial cytochrome b gene in 68 individuals of martens (Martes), weasels (Mustela) and their relatives from the Northern Hemisphere to identify the modes of geographic differentiation in each species. We then compared complete sequences (1140 bp) of the gene in 17 species of the family Mustelidae to know the spatial and temporal modes of speciation, constructing linearized trees with transversional substitutions for deeper lineage divergences and with transversions and transitions for younger lineages. Our data suggested that these lineages of Martes and Mustela differentiated in a stepwise fashion with five radiation stages from the generic divergences (stage I) to the intraspecific divergences (stage V), during the last 10 or 20 million years as the fossil evidence suggests. In the lineage of Martes, the first offshoots are of Martes flavigula, M. pennanti, and Gulo gulo (stage II), the second is M. foina (stage III), and the third are M. americana, M. martes, M. melampus, and M. zibellina (stage IV). The divergence of the lineages of Mustela is likely to have taken place concurrently with the radiations of the Martes. These divergence processes are attributable in part to the geographic allocation along the two continents, North America and Eurasia, as well as among peripheral insular domains, such as Taiwan and the Japanese Islands. In addition, the Eurasian continent itself was shown to have been involved in the species diversification in the martens and weasels.

Sequence of mitochondrial DNA cytochrome oxidase II in Cryptopygus nanjiensis and phylogeny of apterygota

The mitochondrial cytochrome oxidase II (Co II) from four different apterygotens Cryptopygus nanjiensis (Collembola), Neanura latior (Collembola), Gracilentulus maijiawensis (Protura) and Lepidocampa weberi (Diplura) were sequenced. Their A+T content, number of nucleotide substitutions, TV/TV ratio; and Tamura-Nei's distance were calculated. A series of phylogenetic trees were constructed by parsimony and distance methods using a crustacean Artemia franciscana as outgroup, Finally the evolutionary trend A+T content of CO II genetic divergence and phylogenetic relationship of apterygotan groups were discussed.

Mitochondrial cytochrome b gene sequence diversity in the Korean hare, Lepus coreanus Thomas (Mammalia, Lagomorpha)

Partial sequences of the mitochondrial cytochrome b gene of the Korean hare (Lepus coreanus) were analyzed to determine the degree of genetic diversity. Nine haPlotyes were observed, and the maximum Tamura-Nei nucleotide distance among them was 2.8%, indicating that genetic diversity of L. coreanus is moderate. In order to clarify the Korean hare's taxonomic status and relationship with the Manchurian hare (L. mandshuricus) and the Chinese hare (L. sinensis), these nine haplotypes of the Korean hare were compared with 13 haplotypes from five other species of eastern Asian Lepus including L. mandshwicus and L. sinensis. The Korean hare was distinct in its cytochrome b gene, and it is confirmed that L. coreanus is a valid species, as noted by Jones and Johnson (1965, Univ. Kansas Publ. (Mus. Nat. Hist.) 16:357). Further analyses of mtDNA cytochrome b gene with additional specimens of L. coreanus from North Korea and other species of Lepus from eastern Asia are needed to clarify the taxonomic status of the divergent mtDNA clades of L. mandshuricus and L. sinensis.