Studies On The Molecular Phylogeny And Molecular Population Genetics Of The Slow Lorises (Genus Nycticebus)

I Background information of the present study 1.IntroductionGenus Nycticebus belongs to Mammalia;Eutheria;Primates;Suborder Strepsirhini;Family Loridae. It is generally believed that the slow lorises (Nycticebus coucang and Nycticebus bengalensis) and pygmy lorises (Nycticebus pygmaeus) are species belong to Nycticebus. They are small nocturnal primates. N. coucang is widely distributed across the mainland and islands of Southeast Asia. In China, it is confined to west, southeast and south Yunnan and southwest Guangxi. The lesser or pygmy slow loris (N. pygmaeus) is mainly found in Vietnam, Laos, China, and possibly also in Cambodia. The number of N. coucang has severely decreased due to habitat loss and hunting. Moreover, a number of entered into China through the border trade, it is contributed to the population declining throughout its distribution range. N. coucang listed as one of the first protected animals in China. It is listed in Appendix II of CITES and IUCN (The World Conservation Union).Habitat loss from loggingalong with destruction from military activities and hunting, has severely decreased thenumber of the pygmy loris (N.pygmaeus). The species is poorly pretected in the wild.N.pygmaeus is one of the least studied species of all prosimians. N.pygmaeus is listedin Appendix II of CITES. It is listed as vulnerable by IUCN, it is also listed as one ofthe first protected animals in China.2.Origin of the study projectThe significance of the reality: The slow lorises and pygmy lorises (genusNycticebus) are the lower advanced primates. In the field of the phylogenetic studies of primates, they become one of the hotpot and focus, and to more attention paid, because of their specially phylogenetic positions. Both of the slow lorises and pygmy lorises are rare species. It is most urgent that we study and conserve the species of Nycticebus.The significance of the theory: the scarce of samples, because it is difficult to obtain available samples to study. The slow lorises and pygmy lorises are noctural, arboreal and cryptic prosimian primates, they are difficult to observe and study in the wild. Thus, it is great urgency to introduce new study methods.There is still controversy over taxonomy in genus Nycticebus. The views that there are one to three species in genus Nycticebus are current. Classic classifications of Nycticebus relied mainly on morphological differences, including body size, pelage color, head forks and other characters. Since the species and subspecies boundaries in Nycticebus ate still unfixed, more taxonomic characters other than morphological ones may be useful for further evaluation. Molecular studies attempting to reconstruct the whole phytogeny of all the taxa in Nycticebus have not so far been undertaken.II Purpose, Methods, contents and results of the studies1. Molecular Phylogeny of the Slow Lorises( Genus Nycticebus) Revealed by D— loop sequences and complete Cytochrom b Gene sequences of MitochondrialDNAPartial sequences of the D-loop and the complete sequences of cytochrome b gene (1140bp) of the slow lorises (genus Nycticebus) were undertaken to investigate evolutionary relationships among species of genus Nycticebus.Sequences were visually checked and aligned using DNASTAR program. Initial sequence comparisons, base composition and measures of variability were performed using MEGA version 2.1. The polymorphic sites and synonymous/non-synonymous substitutions were calculated using software DNASP3.0. Phylogenetic analyses were performed using PAUP, (version 4.0b8). Neighbor-joining trees based on P-distance (Nei & Kumar, 2000) were computed, and 1000 bootstrap replicates were conductedto assess the statistical confidence of each node. The maximum parsimony analysis was performed using heuristic search, and the majority-rule consensus tree was obtained from 1000 bootstrap replicates. The maximum likelihood analysis was also carried out using PAUP.There was an individual, namely, Npyg5, which was identified as Nycticebus intermedius, according to morphological information. However, the analysis of the D-loop sequences revealed that the sequence of Npyg5 was identical to that of three of four individuals of N. pygmaeus, (namely, Npygl, Npyg2, Npyg4). They were identified as being the same haplotype. This provides new taxonomic evidence with molecular genetic study for supporting N. intermedius is merely the adult of N. pygmaeus.The cytochrome b data set consisted of 1140 nucleotides. No insertions or deletions were observed. No terminal codon was found between the first and last codons, and all of these sequences start with initial codon ' TGA' and end in terminal codon ' AGA'. These 1140 bp of cytochrome b sequences code 379 amino acids in length. It was clear that all sequences obtained were for the genuine cytochrome b gene. The analyses of base composition suggested that the characteristics are consistent with base compositional patterns found in various mammalian species. The analysis also revealed that the sequence of Npyg5 was identical to that of three individuals of N. pygmaeus (namely, Npygl, Npyg3). They were identified as the same haplotype. These results derived from cytochrome b sequences were same to that of D-loop sequences.The result of the PHT test revealed that combined data analysis was appropriate. Whether analysis combining D-loop sequences data with complete sequences of mitochondnal cytochrome b gene data was performed or analysis which employed each of them separately, all of results derived revealed that genus Nycticebus is comprised of two monophyletic clades that were well supported by high bootstrap value. The first cluster comprised all individuals of N. pygmaeus and one individual of N. intermedius (i.e. Npyg5) characterized by morphological information;the second cluster was grouped with all individuals of N. coucang.Analysis results are consistent to provide new taxonomy evidence at the DNA level for supporting Ratajszczak and Groves' viewpoint, namely, N.intermedus was considered to be merely the adult of N. pygmaeus.2. Molecular Phytogeny of Slow Loris (Nycticebus) Inferred from Mitochondrial GenesPartial D-loop and cytochrome b genes were sequenced from 22 specimens. A molecular phylogeny were established, covering all recognized taxa in Nycticebus. The purpose of this study was to test whether or not the evolutionary relationship of all the taxa in Nycticebus would be concordant with those based on morphology and to provide molecular information for further evaluating the classification of Nycticebus.Summary statistics such as the number of variable sites were calculated by Mega2 version 2.1. Saturation of transversions was tested by DAMBE (Xia, Xie, 2001) using K80 distance (Kimura, 1980). Phylogenetic trees were constructed using both ML and MP methods with the help of PAUP4.0* with a 1000 step bootstrap. The DNA substitution model for the ML tree was selected by Model Test 3.5.About 390bp of partial D-loop fragment and 425 bp of partial Cytb fragments were obtained. No insertions or deletions were found in Cytb sequence. Most substitutions were transitions: 79.3% in D-loop and 80.5% in Cytb. No unexpected stop codon was found while translating Cytb sequences, which obviates one of the main suspicions of being numts.In both trees (Cytb tree and the second D-loop tree), taxa in Nycticebus are separated into two major lineages corresponding to N.pygmaeus and the others. The lineage containing taxa other than N.pygmaeus further divides into two subclades, one of which contains only N.c.menagensis and the other N.c.javanicus, Nccoucang and Nbengalensis. Unexpectedly, Nccoucang and N.bengalensis are mixed together;although the N.bengalensis sequences form a clade, the Nccoucang sequences do not. Together, they form a sister clade to N.cjavanicus. Most of the major groups wereseparated except for some mixing of N.coucang.coucang and N.bengalensis. N.pygmaeus diverged earlier from the ancestral stock than the other taxa. N.c.menagensis was well discriminated from N.c.coucang. The mixing of N.c.coucang and N.bengalensis could perhaps be explained by the hybridization between these two groups in the field. Although our data did not provide direct evidence for or against the new proposal that N.c.javanicus be raised to the rank of a distinct species (N.javanicus), we have good evidence for regarding N.c.menagensis as a species.3. Population Genetic Patterns of Bengal Slow Loris (Nycticebus bengalensis) and Pygmy Slow Loris (Nycticebus pygmaeus): a Study of the Mitochondrial Control RegionDue to the difficulties in field study and the scarcity of samples, the population patterns of these two species are still poorly understood, especially those from genetics. The main purpose of this paper is to study and report their population genetic patterns. We sequenced and analyzed the partial fragment of the first hyper variable region (HVRI) of mitochondrial D-loop of 21 N.bengalensis and 119 N.pygmaeus individuals from the boundary of China and Vietnam where they are sympatric.Median-joining network were constructed to show the relationship of haplotypes (Bandelt et al., 1995) . The nucleotide diversity (Nei, 1987) was obtained by DNASP version3. Population parameters such as mutation parameter 9, population growth parameter ￡ and TMRCA were estimated by Griffiths and Tavare's maximum likelihood method using the program GeneTree v9.0. One millions of simulations were used for each parameter set to ensure accurate results. Other 0 estimates such as Watterson's estimate (8W) (Watterson, 1975 ) and Tajima's estimate(Gp) (Tajima, 1983) as well as Tajima's neutrality test (Tajima, 1989) were also calculated by DNASP version3.There were 9 haplotypes among 21 bengal samples and 10 haplotypes among 119 pygmy samples. The nucleotide diversity (Nei, 1987) for N.bengalensis andN.pygmaeus were 1.378% and 0.192%,respectively. Although with a much larger sample size, the polymorphism level of N.pygmaeus was much lower than that of N.bengalensis, which may be caused by four factors: (1) the extern geneflow from other habitats of N.bengalensis;(2) the gene ingression from N.coucang.coucang to N.bengalensis;(3) a skewed birth sex ratio in N.pygmaeus;(4) a possible low survival rate of N.pygmaeus babies. Compared and referred by N.bengalensis, we speculated that the poor polymorphism of the pygmy might be the result of a founder effect. N.pygmaeus in southern China and northern Vietnam might have expanded from middle or southern Vietnam, possibly at 380-1500 years (p=95%) or 300-1800 years (p=99%) ago.In the network of the pygmy slow loris, there were two dominant haplotypes (P2 and P3). In the network of the bengal slow loris, we could not find any dominant haplotypes. The networks for these two species showed different topologies. The topologies of the networks show that the bengal slow loris population was more structured and the pygmy was more growth like.The Tajima's estimate of 8 (Tajima, 1983) for the bengal slow loris population (5.038) was bigger than its Watterson's counterpart (4.725) (Watterson, 1975). But for the pygmy slow loris, the Tajima's estimate (0.746) was smaller than Watterson's (1.495). Tajima's D was significant in neither Bengal (positive) nor pygmy population (negative), which also means that the bengal population was more prone to be structure like while the pygmy was more growth like.Griffiths and Tavare's ML method could estimate both 9 and 8 simultaneously, and for the pygmy population, 9 was 2.3 and B was 1.6 and for the bengal, 9 was 5.0 and 6 was 1.575. III. The significance of the study and originalityHair samples are critical part of the whole of genus Nycticebus. Hair-DNA sampling, Non-invasive molecular genetics approach has been established for genus Nycticebus in the present study. In the studies on the molecular phylogeny and genetic diversity in wild population of Nycticebus, the difficulties derived from sampling have been solved to a certain degree. It provides a valuable potential usefulness of thenon-invasive genetic approach to the study of the molecular phylogeny, biodiversity and conservation genetics foi Nycticebus.The results of molecular genetic analysis are consistent to provide new taxonomy evidence at the DNA level for supporting Ratajszczak and Groves' viewpoint, namely, N.intermedus was considered to be merely the adult of N. pygmaeus.Molecular studies attempting to reconstruct the whole phylogeny of all the taxa in Nycticebus have been undertaken firstly in the present study. It provides the new taxonomy evidence at the DNA level for supporting genus Nycticebus comprises three species: N.pygmaeus, N.coucang andN.bengalensis.The present study first reported the new results of population genetics of Nycticebus.