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Title: Analysis of Genetic Diversity in Grey Langur (Semnopithecus spp.) Populations of Pakistan
Authors: Minhas, Riaz Aziz
Keywords: Zoology
Issue Date: 2019
Publisher: University of Azad Jammu and Kashmir Muzaffarabad, Pakistan
Abstract: Grey langurs (Semnopithecus spp., Colobinae, Cercopithecidae, Primata, Mammalia) is a group of the old-world leaf eating monkeys widely distributed in the Indian subcontinent, with two species viz. Semnopithecus ajax and S. schistaceus reported from higher altitudes of the Himalayan hills extending into northern Pakistan. Species status of these populations is still debated, though S. ajax is regarded as Endangered globally. In Pakistan, small declining population of these grey langurs is distributed in pockets, but the level of isolation is still unknown. To resolve such uncertainties, the present study was undertaken to assess intrapopulation genetic diversity, and to settle taxonomic status of different populations, using modern molecular biology tools. We collected 86 noninvasive (feces 64, hair 13, blood 5, tissues 4) samples from 5 geographic langur populations of Pakistan and Azad Jammu and Kashmir (AJK) and succeeded in extraction of DNA from 23 samples, which were used for further genetic analysis. We used nuclear (Random Amplified Polymorphic DNA-RAPD and microsatellites) and mitochondrial DNA (Cytochrome oxidase-I, Cytochrome b and 16S rRNA) markers. RAPD makers (n=8) produced 245 bands (30.62±2.87 mean±SE / primer) of different molecular weights (126-3342 bp), of which, 96 were population specific. Polymorphism was (37.71±5.29%; mean ± SE), with the highest in Muzaffarabad population (54.29%), followed by Poonch (43.67%) and Neelum (36.73%). Values of Shannon’s (I: 0.129-0.200) and Nei's genetic diversity (He: 0.082-0.117) indices were low. Total heterozygosity (Ht: 0.144±0.007), genetic diversity within population (Hs: 0.096±0.005), between populations (Dst: 0.018±0.003), genetic differentiation constants among populations (Gst: 0.153±0.025) and within populations (Rst: 0.847±0.025) were calculated. Gene flow (Nm: 3.246 0.448) and genetic similarity (97-98%) between populations was high. UPGMA (unweighted pair group method with arithmetic mean) based dendrogram identified five distinct geographic groups, and Mantel tests (Rxy=-0.008, P0.05) suggested a non-significant relationship between genetic distance and geographic distance. Phist (PT) value suggested a significance difference within population and between populations (PT=0.042; p=0.006) variances, suggesting that within populations variation was higher (96%) than variation between populations (4%). Microsatellite analysis, using 16 primers, exhibited successful cross-species amplification suggesting high discriminatory powers (PIC = 0.94±0.01). A total of 256 polymorphic bands comprising on 97 different sized (88-383 bp) alleles (2-10 alleles/marker) were amplified in different genotypes sampled. Mean level of polymorphism in different populations was 45±6.06%. Tests for linkage disequilibrium between different loci exhibited no significant deviations from expected values (p>0.05). Mean values of Shannon’s (0.357±0.05), Nei’s genetic diversity (0.241±0.03), fixation indices (-0.894±0.03), genetic differentiation coefficient (Fst: range = 0.223 - 0.898, mean = 0.438±0.097) and mean gene flow (1.185±0.374) were calculated. The largest Nei’s genetic distance (0.752) was between Mansehra and Neelum populations, while the least (0.255) between Mansehra and Kohistan populations. UPGMA based dendrogram identified two main clusters, Cluster one subdivided into Poonch population (as outgroup) and a monophyletic clade of Muzaffarabad and Neelum populations. Second cluster included Mansehra and Kohistan populations. Principal Coordinates Analysis (PCoA) also indicated three clusters: (1) AJK (Muzaffarabad-Neelum-Poonch), (2) Mansehra, and (3) Kohistan. The value of Rxy=0.302 (P<0.01) revealed a significant association between genetic distance and geographic distance. Phylogenetic analysis, based on different mitochondrial genes partial sequences (COI, Cyt b and rDNA), using Maximum likelihood, Neighbor-Joining, and Minimum Evolution methods, suggested close relationship of grey langurs of Pakistan with S. schistaceus and S. entellus populations from different regions of the Indian subcontinent. Estimated evolutionary divergence values showed a low genetic distance (<0.01) indicating that different populations belong to a single species. Analysis for species delimitation using 4×-rule or K/ϴ (D/ϴ) method also indicated status of a single species. Present study suggested a low level of isolation and inbreeding between grey langur populations of Pakistan and Azad Jammu and Kashmir. This study confirmed that Semnopithecus ajax is the only species found in different areas of Pakistan and AJK. Further molecular, as well as morphological, studies using larger sample size and analysis of complete mitochondrial genome sequences are suggested.
Gov't Doc #: 20113
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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