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Genetic Diversity of Green Peafowl (Pavo Muticus) in Northern Thailand Based on Mitochondrial DNA Control Region Sequences

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Genetic Diversity of Green Peafowl (Pavo Muticus) in Northern Thailand Based on Mitochondrial DNA Control Region Sequences Tropical Natural History 18(1): 11–23, April 2018 2018 by Chulalongkorn University Genetic Diversity of Green Peafowl (Pavo muticus) in Northern Thailand Based on Mitochondrial DNA Control Region Sequences THANAPORN SAWANGTHAM AND AMPORN WIWEGWEAW* Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330 THAILAND * Corresponding author. Amporn Wiwegweaw ([email protected], [email protected]) Received: 21 September 2017; Accepted: 3 March 2018 ABSTRACT.– The population size of green peafowl (Pavo muticus L.) has declined dramatically in Thailand. It is currently found in two geographically isolated regions in the northern and western areas. Here, we assessed the level of genetic variation of this endangered bird species to examine the influence of habitat fragmentation. We collected moulted feather and egg specimens of wild individuals from three populations in the North: Wiang Lor Wildlife Sanctuary (WLO-1 and WLO-2) and Tappaya Lor Non-Hunting Area (TPL). Mitochondrial control region sequences were obtained and analyzed. The results revealed that P. muticus in all populations showed a high level of haplotype diversity (0.784, n = 72), implying high genetic diversity. The haplotype diversity in WLO-1 population was higher than in other two populations. On phylogenetic trees and a haplotype network, the haplotypes of P. muticus individuals did not form a separate cluster, regardless of their distribution ranges, and three haplotypes were found in the three populations. By combining the present results with the previous data, our genetic investigations revealed that P. muticus populations in Thailand exhibited a low genetic difference (4.31%) between the two regional populations with no statistical significance. This suggests that there is no subdivision of wild populations. KEY WORDS: D-loop, genetic variation, mitochondrial DNA, peafowl, phylogeny INTRODUCTION loss and fragmentation, environmental pollution and human persecution (McGowan Green peafowl (Pavo muticus Linnaeus, et al., 1999; Meckvichai et al., 2007; Pinthong, 1766) is a well-known galliform bird in the 2009), resulting in local extinction in some family Phasianidae which is highly areas; such as in the southern part of threatened in many countries in East and Thailand. Therefore, the endangered green South-east Asia (IUCN, 2014). It is peafowl is currently considered as one of the commonly found in tropical forests, nationally protected species by the particularly in mixed deciduous and dry Thailand’s Office of Natural Resources and dipterocarp forests, as well as in the Environmental Policy and Planning (ONEP) agricultural areas adjacent to forests for and is listed under Appendix II of the foraging (Johnsgard, 1999; BirdLife Convention on International Trade in International, 2015). In Thailand, this Endangered Species of Wild Fauna and species was historically distributed across Flora (CITES). almost all of Thailand except in the central Nowadays, within Thailand, green valley of the Chao Phraya River and the peafowl is found only in two geographically northeastern and eastern provinces isolated regions in the northern and western (Delacour, 1977). In the last few decades, areas of the country. Within each region, however, the green peafowl population has they are distributed in patchy areas undergone a serious decline owing to habitat particularly close to shallow streams or 12 TROPICAL NATURAL HISTORY. 18(1), APRIL 2018 rivers with exposed sand bars, for example, Hong Khrai Royal Development Study along the Ping, Ing, Yom and Nan river Center (HHK; northern Thailand) and HKK basins in the North and in the Huai Kha (western Thailand) was previously reported Khaeng, Huai Tab Salao, Huai Song Tang in a preliminary study based on mtDNA and Mae Klong basins in the West control region sequences. The results showed (Ponsena, 1988; Meckvichai et al., 2007). a high level of genetic diversity in the two The population sizes of the western populations (Sawangtham et al., 2015). populations are larger than the northern However, the sample sizes and number of populations, with the largest population locations analyzed were both small, and so being found in Huai Kha Khaeng Wildlife those results need confirmation and Sanctuary (HKK), with an estimate of 300 expanding upon by an analysis of a more birds (McGowan et al., 1999). extensive sample collection to understand the Most studies on green peafowl in local and overall population genetic diversity Thailand have principally focused on their within and between wild populations of this ecology, particularly distribution range and endangered bird species in Thailand. habitat use, and some behaviors (e.g., Therefore, the present study examined the Ponsena, 1988; Arrathrakorn, 2001; genetic diversity and structure of green Saridnirun et al., 2016; Sukumal et al., peafowl in the two localities in the North: 2017). However, research on genetic Wiang Lor Wildlife Sanctuary (WLO) in variability and structure is relatively rare, Phayao Province and Tappaya Lor Non- particularly in the wild populations, Hunting Area (TPL) in Chiang Rai Province. probably because it is not easy to find and These are recently-discovered populations catch them in nature. Since habitat near Ing River in which the information on fragmentation can increase the chance of population genetics of each location is genetic drift and inbreeding, and so unknown. The results gained from this study potentially result in a reduced genetic are discussed with the previous study in order diversity that could directly influence the to better understand the green peafowl’s survivability and adaptability of the animals genetic diversity in Thailand. (Frankham, 1995). Therefore, knowledge of the current genetic diversity and structure of MATERIALS AND METHODS wild populations is necessary for long-term conservation strategies and management of Study areas and sampling the species to maintain and increase the Geographically, WLO can be considered population size. as a fragmented area comprised of two The non-coding control region or geographically isolated fragments, namely displacement-loop (D-loop) region is WLO-1 and WLO-2, which are at least 15 considered to be the most variable portion of km apart and separated from each other by the mitochondrial DNA (mtDNA) genome roads and human settlements (Fig. 1). (Desjardins and Morais, 1990; Randi and However, WLO-1 is adjacent to TPL, only Lucchini, 1998). Therefore, it is suitable as a 1.5 km apart and separated from each other molecular marker for investigating genetic by the road and river, whereas WLO-2 is variation and phylogenetic relationships about 40 km from TPL. According to this within species. The genetic variability in two geography, we therefore considered WLO-1, natural populations of green peafowl in Huai SAWANGTHAM AND WIWEGWEAW — GENETIC DIVERSITY OF GREEN PEAFOWL 13 FIGURE 1. Map of Thailand showing the approximate locations of WLO in Phayao Province and TPL in Chiang Rai Province. The enlarged map of WLO-1, WLO-2 and TPL showing the locations of collection sites. Sites with and without feather collection are represented by black and white circles, respectively. WLO-2 and TPL as different populations extensively collected moulted feather isolated by geographic barriers. specimens of green peafowl from 22 Because green peafowl birds are collection sites, of which six, eight and eight relatively large and very wary birds, it is sites were from WLO-1 (47 feathers), hard to catch them in nature to take blood or WLO-2 (80 feathers) and TPL (104 tissue samples. Thus, noninvasive samples feathers), respectively (Fig. 1). We collected such as feathers are excellent specimens for these specimens in May 2015 and a study of mtDNA diversity in wild discriminated individuals in each location populations. Therefore, this study used based on two criteria: distance and sex. In feathers as a source for DNA extraction. We the former case, since it has been reported 14 TROPICAL NATURAL HISTORY. 18(1), APRIL 2018 that the home range size of green peafowl in template, 0.5 µM of each primer and breeding season was about 148 hectares or 2 1Xpremix of EmeraldAmp®GT PCR master km in distance (Ponsena, 1988), we mix (Takara, Japan). Negative controls were collected feather specimens from within included in all reactions. The PCR collection sites that were at least 2 km away proceeded on a BioRad thermal cycler with from each other, which means that we were a PCR reaction cycle of 95°C for 4 min initially restricted to one individual from followed by 40 cycles of 95°C for 40 sec, each of six sites from WLO-1, eight from 60.6°C for 1.15 min and 72°C for 3 min, WLO-2 and eight from TPL (Fig. 1). For the and then a final 72°C for 10 min. The latter criterion, we identified additional reaction was terminated at 4°C. The size of individuals within each site based on the amplification product was checked by morphological differences in the feathers electrophoresis through 0.8% (w/v) agarose between males and females as previously gel, stained with SYBR®Safe DNA gel stain reported (Meckvichai and Arsirapoj, 2009). (Invitrogen™) and visualized via blue light. In addition to these two criteria, we also The desired PCR products were subsequently performed DNA sequencing of some sent to Bioneer Inc., Daejeon, South Korea remaining specimens within a site for for commercial purification and sequencing screening of different mtDNA haplotypes to services. Each PCR product was sequenced increase the sample sizes for each site. in both directions using the forward and Additionally, egg samples (broken or un- reverse primers. hatched eggs) of green peafowl were also obtained. Eight and two eggs were collected Sequences and the phylogenetic and from WLO-1 and TPL, respectively, haplotype network analyses preserved in 95% (v/v) ethanol and kept in Seventy-two sequences were obtained the laboratory at room temperature for and subsequently aligned and edited subsequent DNA extraction. manually using Clustal W (Thompson et al., 1994) via the program included in version DNA extraction, amplification and 6.06 of MEGA (Tamura et al., 2013).
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