RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016 659 *For correspondence. (e-mail: archana.bahuguna65@gmail.com) Molecular phylogeny of rediscovered Travancore flying squirrel (Petinomys fuscocapillus) and its conservation implications Ashutosh Singh and Archana Bahuguna* Molecular Systematic Laboratory, Northern Regional Centre, Zoological Survey of India, 218, Kaulagarh Road, Dehradun 248 195, India Petinomys fuscocapillus (Travancore flying squirrel), Jerdon 1847, is a near threatened species, native to India and Sri Lanka. Deforestation, wood plantation, infrastructure development, poaching and natural predators are major threats to the species. This study reports for the first time the molecular phylogenetic position and level of genetic divergence of P. fusco- capillus among the flying squirrel species of South and Southeast Asia, based on two mitochondrial genes. The phylogenetic analysis confirms that the P. fusco- capillus and Petinomys setosus (Temminck’s flying squirrel) are sister taxa and share most recent com- mon ancestry. Phylogenetic position of other flying squirrels obtained in the present study was also sup- ported by the previous studies. We also emphasize on the extensive survey for population sampling, need for plantations to maintain a continuous canopy and enforcement of strict laws at the potential geographi- cal distribution of the species in two countries. Keywords: Conservation, molecular phylogeny, Peti- nomys fuscocapillus. THERE is global interest in the rediscovery of extinct spe- cies1,2. An estimated 351 species have been rediscovered, mostly from the tropics over the last 122 years3. Redis- covery of previously thought extinct species requires new conservation efforts to preserve such species and helps to understand the reason for their population decline as a consequence of human disturbance4,5. Genus Petinomys consists of eight species6, distributed in South and Southeast Asia. The genus is represented by only one species, Petinomys fuscocapillus Jerdon, 1847 from India and Sri Lanka. P. fuscocapillus was consid- ered as extinct in India, but after 100 years it was redis- covered in 1989 in a coconut grove in Kerala, India7 and in Sri Lanka, it was rediscovered after a gap of 78 years in Knuckles mountain range8. P. fuscocapillus is differen- tiated from other species of genus due to the peculiar honeycombed bones in their ear. Two subspecies are known, i.e. P. f. fuscocapillus Jerdon, 1847 found in Western Ghats of southern India and P. f. layardi Kelaart, 1850 from Sri Lanka. In India, the species is distributed in Brahmagiri Wildlife Sanctuary and Makutta, Coorg in Karnataka9 and in the states of Tamil Nadu10–12 and Kerala7,13 (Figure 1). In Sri Lanka, it has been reported from Central Provinces and Sabaragamuwa Provinces14,15. The species is arboreal (lives in tree canopy) and noctur- nal and it occurs in evergreen, deciduous and montane forest. Kumara and Suganthasakthivel16 predicted the potential distribution of P. fuscocapillus using Genetic Algorithm for Rule Set Prediction (GARP) and their result indicates that the potential distribution of P. fusco- capillus in India is restricted to the narrow band on the western slope of the Western Ghats; and in Sri Lanka, the distribution is predominant in the lowlands of wet and intermediate zones. Koprowski and Nandini17 mentioned that there is a lack of knowledge on distribution, population and conserva- tion status of flying squirrels in the tropical countries and tropical flying squirrels are at a high risk of extinction due to high deforestation rate (mainly through expansion of agriculture, small-scale logging, small wood planta- tion, infrastructure development and harvesting for local consumption), natural predators and poaching15. Accord- ing to IUCN18, the species is Near Threatened because its level of occurrence could be approximately 30,000 sq. km and its habitats are probably declining and it occurs as a severely fragmented population, thus making the species close to qualifying as vulnerable. Wildlife conservation programmes usually integrate molecular techniques for the ecological studies of species of concern. Not much is known about the ecology of P. fuscocapillus; therefore, it is very difficult to develop an efficient conservation programme. Mitochondrial genes were extensively used for the molecular phylogenetics of rediscovered and possibly extinct species19,20. This study is the first earnest attempt to construct the phylogenetic relationship and level of genetic divergence of P. fusco- capillus with other flying squirrels from South and Southeast Asia (Table 1) and to know the utility of mark- ers for identification of the species. We hope that identi- fication of P. fuscocapillus closest relative through phylogeny for which ecological data could be available would help in its conservation.
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016660 Materials and methods Ethical statement Sample used in the study was obtained from specimens deposited in the Mammals collection of Zoological Survey of India, Kolkata, India with the permission of the Director, Zoological Survey of India. Our sampling did not violate any law, rule or regulation thus required no ethical approval. Sampling In this study, we used one museum skin sample. The spe- cimen was collected by D. R. Sugathan from Idukki, Kerala, India on 22 March 2005 (Registration no. 25793). All tools were flame sterilized prior to sample collection. A small fragment of skin (approx. 0.5 cm 0.5 cm) was sliced in such a way that there was no significant loss of skin that could compromise further studies. Additionally, 12s rRNA and cytochrome b available sequences of South and Southeast Asian Sciuridae were obtained from Genebank (Table 1). Thus, in the present study, the phy- logenetic position of Petinomys fuscocapillus was searched among the species of flying squirrels (9 and 15 species of flying squirrel for 12s rRNA and cytochrome b analysis, respectively) of South and Southeast Asian countries. DNA preparation and sequencing Fur from the skin sample was removed using a sterile scalpel. Then, the sample was washed with sterile milliQ Figure 1. Distribution of Petinomys fuscocapillus (Jerdon, 1847) in India and Sri Lanka. water and ethanol 70% (v/v) respectively. The skin sample was hydrated before digestion by incubating the dried skin sample for 24 h in 1 ml TE solution (Tris 10 mM and EDTA 1 mM, pH 7.6)21. After 24 h of hydra- tion, the DNA was isolated from the skin sample using HiPur ATM Forensic Sample Genomic DNA Purification Table 1. Sciuridae species used for phylogeny reconstruction with their accession numbers of respective sequences obtained from Genbank Accession number Taxon 12s rRNA Cytochrome b Flying squirrels Petaurista alborufus AY227541 AB092614 Petaurista elegans – AB092610 Petaurista xanthotis – DQ072111 Petaurista philippensis – JQ928697 Petaurista petaurista D50282 AB092608 Petaurista leucogenys D50280 AB433269 Belomys pearsonii AY227537 AB126245 Eoglacomys fimbriatus AY227562 AB126248 Petinomys fuscocapillus KP973561* KP973562* Petinomys setosus AY227544 AB030260 Eupetaurus cinereus AY227538 AY331668 Hylopetes alboniger – DQ093187 Hylopetes spadiceus – DQ093189 Hylopetes phayrei AY227539 – Hylopetes nigripes – DQ093190 Hylopetes Lepidus – AB126251 Petaurillus kinlochii AY227542 – Non-flying squirrels Ratufa affinis AY227547 – Ratufa bicolour AY227548 – Callosciurus nigrovittatus – AB499917 Callosciurus inornatus – AB499907 Callosciurus finlaysonii – AB499911 Callosciurus caniceps – AB499919 Callosciurus erythraeus – AB499909 Callosciurus notatus AY227510 AB499913 Callosciurus prevostii – AB499915 Tamiops mcclellandii – EF539333 Tamiops maritimus – HQ698387 Tamiops rodolphii – HQ698400 Tamiops swinhoei AY227522 EF539334 Dremomys rufigenis AY227511 EF539341 Dremomys lokriah – EF539335 Dremomys pernyi – EF539336 Dremomys pyrrhomerus – EF539342 Dremomys gularis – EF539339 Funambulus layardi FJ861245 – Funambulus sublineatus FJ861259 – Funambulus palmarum FJ861251 – Funambulus pennantii FJ861254 – Marmota himalayana NC_018367 GQ329721 Menetes berdmorei AY227516 – Rhinosciurus laticaudatus AY227519 JF417972 Outgroup Rattus norvegicus AY012115 AB033713 *Novel DNA sequence data from this study. –, Sequence not available.
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016 661 Kit (HIMEDIA) following the manufacturer’s protocol. The quantity of isolated DNA was estimated using Invi- trogen, Qubit® 2.0 Fluorometer. 12s rRNA sequences were amplified using a set of primer pair, L1091 and H1478, and a primer set of L14841 and H15149 was used to amplify cytochrome b gene22. The PCR reaction was performed in Q-cycler, Quanta Biotech, in a total volume of 25 l of reaction mixture (10X PCR with MgCl2, 2.5 l; 10 mM dNTPs, 2.5 l; 5 pmol primer, 0.45 l each; 15 ng of DNA template; 1.5 U Taq enzyme). Poly- merase chain reaction consisted of initial denaturation of 94C for 4 min and each cycle of denaturation for 1 min at 94C, hybridization for 1 min at 55C (50C for cyto- chrome b) and extension for 1 min at 72C followed by final elongation for 10 min at 72C. The cycle was repeated 35 times. The PCR products were sequenced using ABI’s AmpliTaq FS dye terminator cycle sequenc- ing chemistry on an automated ABI 3100 Genetic Ana- lyser. All experiments were performed in a PCR workstation (Bangalore GeNeiTM). Negative controls were used in all DNA extractions and PCR amplifications to control for potential contamination. Mitochondrial DNA analysis Nucleotide sequences were proofread using MEGA5.0 (ref. 23) and aligned using ClustalW24. To cross-check the quality of sequence generated, query sequences were compared with NCBI/GenBank (http://www.ncbi.nlm. nih.gov/) database using BLAST tool. Quantitative pair- wise comparisons between the species in study were per- formed, and the uncorrected percentage differences (p- distance) between phylogenetic clades were calculated using Kimura’s25 2-parameter (K2P) method. To elucidate the phylogenetic position of P. fuscocapil- lus among the Southeast and South Asian species, phy- logenetic analyses was performed to assess maximum likelihood with RAxML 7.4.2 (ref. 26) and implemented in ra x mlGUI 1.3 (ref. 27) and Bayesian inference (BI) using MrBayesv3.2.2 (ref. 28). The best-fit evolutionary model was calculated using jModeltestv2.1.3 (ref. 29) and determined using Bayesian information criterion (BIC). The chosen models were GTR + I + G for 12s rRNA sequence and TPM3uf +I + G for cytochrome b data. The selected model, i.e. TPM3uf + I + G for cyto- chrome b data cannot be implemented in RaxmlGUI and MrBayes. So, the TPM3uf model was replaced by closed parameterized model, GTR model30,31. ML tree calculation was performed using the GTRGAMMA (general time-reversible model with gam- ma distribution) substitution model32, and a rapid boot- strap analysis and search for a best-scoring ML tree (ML + rapid bootstrap) was carried out26 with 1000 repe- titions. Bayesian analyses were executed using a random start- ing tree and program’s default distribution for model parameter. The analyses were repeated twice and each analysis included 3 million generation. The results were sampled every 1000th generation. Convergences were assessed by calculating the effective sample sizes (ESS) using Tracer v1.6 (ref. 33). Conservatively, the first 25% of the sampled trees were discarded as ‘burn in’ and the remaining 75% of the sampled trees were used to calcu- late the Bayesian posterior probabilities (BPP). The 12s rRNA and cytochrome b gene sequence of Rattus norve- gicus was used as an outgroup for rooting the trees34. Result and discussion Genes Partial sequences of the 12s rRNA gene (410 bp) and cytochrome b gene ( 370 bp) of Petinomys fuscocapillus were determined. Both generated sequences (12s rRNA and cytochrome b) showed 96% and 95% identity with their congeneric sequences of accession numbers AY227544 and AB030260 respectively. Thus, BLAST search confirms the specific identity of the studied spe- cimen of P. fuscocapillus. These generated sequences were analysed with previously published sequences (Table 1). The average nucleotide composition of the 12s rRNA gene was 35% A, 22.6% C, 17.7% G and 24.6% T. The aligned sequences included 471 variable sites along with 273 parsimony informative sites (25.41% of the entire sequence). Cytochrome b gene sequences shows a deficiency in guanine (12.8% G), while the remaining three nucleotide were more balanced, i.e. 27.8% A, 29.3% C and 30.1% T. The alignment of cytochrome b gene sequence gives 566 variable sites and 476 parsimony informative sites (41.21% of the entire sequence). These sites depicted the overall variability among the species examined leading to evolution of the species. Phylogenetic analyses of flying squirrels On the basis of fossil records, Mein35, Black36 and De Brujin37 mentioned that the diverse fauna of flying squirrels were dominant in Eurasian continents during the Miocene and Oligocene. Fossil records and molecular data38 indicate that the flying squirrels diverge from Europe and their distribution may have shifted to Southeast Asia and North America. In this study, which is based on the 12s rRNA and cytochrome b da
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016662 Figure 2. Maximum likelihood (ML) and Bayesian Inference (BI) phylogram based on 12s rRNA sequence (ML and BI analyses generated the same tree topology). Significance values are listed in the order ML (bootstrap support)/BI (Bayesian posterior probability). Terminal triangle represents the non-flying squirrels used in tree generation (detailed topology of non-flying squirrels is not shown as it is not necessary for this study). Number in parentheses is the number of non-flying squirrel species. Rattus norvegicus was used as an out-group. Table 2. Pairwise comparison of partial 12s rRNA sequence of Petinomys fuscocapillus with 10 flying squirrels. Data above the diagonal represents nucleotide substitution. Data below the diagonal are uncorrected percentage difference (p-distance) 1 2 3 4 5 6 7 8 9 10 Petaurista alborufus (AY227541) 9 19 43 35 37 31 48 46 43 Petaurista petaurista (D50282) 0.024 20 42 37 40 28 49 44 47 Petaurista leucogenys (D50280) 0.051 0.054 41 38 38 34 47 49 45 Belomys pearsonii (AY227537) 0.116 0.114 0.111 35 44 36 46 46 50 Eoglaucomys fimbriatus (AY227562) 0.095 0.100 0.103 0.095 39 30 35 42 43 Petinomys setosus (AY227544) 0.100 0.108 0.103 0.119 0.105 34 36 39 10 Eupetaurus cinereus (AY227538) 0.084 0.076 0.092 0.097 0.081 0.092 37 39 42 Hylopetes phayrei (AY227539) 0.130 0.132 0.127 0.124 0.095 0.097 0.100 36 39 Petaurillus kinlochii (AY227542) 0.124 0.119 0.132 0.124 0.114 0.105 0.105 0.097 49 Petinomys fuscocapillus 0.116 0.127 0.122 0.135 0.116 0.027 0.114 0.105 0.132 other ground squirrels (non-flying squirrels) (Figure 3). All the nodes were supported by moderate Bayesian pos- terior probability (BPP) while low Bootstrap support (BS) was observed at some nodes. The phylogram ob- tained from ML and BI analysis of 12s rRNA sequence (Figure 2), generated three main clades, i.e. Clade I is represented by the genus Petaurista (giant flying squir- rels), Clade II is represented by other flying squirrels and Clade III comprises non-flying squirrels (ground and tree squirrels). The genus Petaurista (giant flying squirrels) includes a group of diverse species that are adapted for arboreal life and are distributed from Western Himalayas to East Asia, North Indo-China and Southeast Asia39–42. In this study, this genus was noted to form a separate clade from other flying squirrels, i.e. genus Belomys Thomas, 1908; Eupetarus Thomas, 1888; Eoglaucomys Howell, 1915; Hylopetes Thomas, 1908; Petinomys Thomas, 1908 and Petaurillus Thomas, 1908 (Figure 2). The phylogenetic position of Genus Petaurista with other flying squirrels is noted to be in accordance with previous studies by Oshida et al.34,38; Li et al.43; Thorington et al.44 and Mercer and Roth45. Phylogeny obtained from the analysis of cytochrome b gene showed an early divergence of Marmota himalayana from rest of the squirrel species and is supported by high BPP value (100% BPP, Figure 3). Additionally, the genera Belomys and Eupetaurus also show an early divergence from rest of the flying squirrels (BS = 66%, BPP = 100%, Figure 3). This kind of early divergence of B. pearsonii and E. cinereus has also been reported in earlier studies
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016 663 Figure 3. Maximum likelihood (ML) and Bayesian Inference (BI) phylogram based on cytochrome b gene sequence (ML & BI analyses generated the same tree topology). Significance values are listed in the order ML (bootstrap support)/BI (Bayesian posterior probability). Terminal triangle represents the non-flying squirrels used in tree generation (detailed topology of non-flying squirrels is not shown as it is not necessary for this study). Number in parentheses is the number of non-flying squirrel species. Rattus norvegicus was used as an out-group. on flying squirrels by Oshida et al.38,46; Li et al.43 and Yu et al.47. On the other hand, our 12s RNA phylogenetic tree (Figure 2) and previous studies by Thorington et al.44 and Mercer and Roth45 do not show the early divergence of these two species from other flying squirrels. Phylogenetic position of P. fuscocapillus This is the first study ever conducted on the molecular phylogeny of P. fuscocapillus based of two mitochondrial sequences. Travancore flying squirrel is known to be an example of discontinuous distribution of mammals in India and adjacent countries48. Its distribution is restricted to Western Ghats and Sri Lanka. Based on 12s rRNA and cytochrome b data analysis, P. fuscocapillus and P. seto- sus (Temminck’s flying squirrel), which are distributed in Malaysia, Myanmar and Thailand44, are clustered to- gether and their phylogenetic relationship was strongly supported with high BS and BPP in 12s rRNA tree (BS and BPP = 100, Figure 2); while moderate BS and BPP was observed in cytochrome b tree (BS = 70% and BPP = 87%; Figure 3). The p-distance of P. fuscocapillus was found to be the highest in Belomys pearsonii (0.135, Ta- ble 2) and Eoglaucomys fimbriatus (0.210, Table 3) and lowest in P. setosus (0.027 and 0.095, Tables 2 and 3). Thus, the topology of the phylogenetic tree and low genetic divergence between P. fuscocapillus and P. seto- sus strongly supports that these two species are mono- phyletic and share recent common ancestry. In this study, P. fuscocapillus and P. setosus together form a sister clade with Hylopetes phayrei (from China, Myanmar, Thailand and Vietnam) (Figures 2 and 3). So, our result strongly supports the Corbet and Hills40 hy- pothesis that Petinomys is closely related to Hylopetes and the two genera are distinguished by number of septa in the auditory bullae. In addition to the Corbet and Hills hypothesis, our study also supports the phylogeny pro- vided Oshida et al.48 and Herron et al.49 based on Cyto- chrome b gene in which they reported that genus Petinomys is closely related to genus Hylopetes. Moreover, in a study by Mercer and Roth45, using three genes (IRBP, 12s and 16s rRNA sequence) found that genus Petinomys is phylogenetically closely associated with genus
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016664 Table 3. Pairwise comparison of partial cytochrome b gene sequence of Petinomys fuscocapillus with 15 flying squirrels. Data above the diagonal represents nucleotide substitution. Data below the diagonal are uncorrected percentage difference (p-distance) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Petaurista philippensis (JQ928697) 24 55 46 49 52 61 57 59 55 53 61 60 61 59 Petaurista alborufus (AB092614) 0.069 56 42 46 45 59 63 56 58 52 56 57 63 59 Petaurista elegans (AB092610) 0.172 0.176 47 61 51 67 64 63 63 60 56 61 68 60 Petaurista xanthotis (DQ072111) 0.139 0.125 0.142 40 37 56 62 61 59 51 59 59 56 53 Petaurista petaurista (AB092608) 0.149 0.138 0.192 0.118 50 61 68 67 63 59 69 62 71 65 Petaurista leucogenys (AB433269) 0.161 0.136 0.157 0.108 0.152 52 55 62 66 56 57 52 55 57 Belomys pearsonii (AB126245) 0.187 0.180 0.210 0.171 0.188 0.156 61 57 53 59 51 57 57 59 Eoglaucomys fimbriatus (AB126248) 0.173 0.194 0.199 0.191 0.213 0.166 0.187 68 55 60 62 59 63 67 Eupetaurus cinereus (AY331668) 0.180 0.170 0.196 0.187 0.208 0.191 0.173 0.212 60 58 48 58 61 66 Hylopetes alboniger (DQ093187) 0.166 0.176 0.195 0.180 0.196 0.206 0.160 0.166 0.185 47 47 49 43 43 Hylopetes spadiceus (DQ093189) 0.159 0.156 0.184 0.153 0.181 0.170 0.182 0.186 0.178 0.142 44 30 44 40 Hylopetes nigripes (DQ093190) 0.188 0.170 0.170 0.181 0.219 0.173 0.153 0.193 0.143 0.143 0.134 42 60 55 Hylopetes lepidus (AB126251) 0.184 0.173 0.188 0.181 0.192 0.156 0.174 0.182 0.177 0.149 0.087 0.126 47 52 Petinomys setosus (AB030260) 0.187 0.194 0.214 0.170 0.225 0.166 0.174 0.195 0.189 0.130 0.132 0.189 0.142 33 Petinomys fuscocapillus (KP973562) 0.180 0.180 0.183 0.159 0.203 0.173 0.183 0.210 0.206 0.128 0.118 0.169 0.158 0.095
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016 665 Petaurillus. Similar findings were obtained in our study, where genus Petinomys formed a sister clade with Petau- rillus kinlochii (Figure 2). Species of genus Petinomys is distributed in South and Southeast Asia. Seven among the eight species of the genus is distributed across the Maritime and Mainland Southeast Asian countries6,15. Petinomys fuscocapillus is the only species representing the genus in the South Asia and it appears that it has become endemic to two regions of Western Ghats, India and Sri Lanka because of geo- graphical barriers as well as adaptations to climatic con- ditions. This study has indicated that the species is forming a separate lineage from other species of flying squirrel and can be identified by using the two molecular markers, i.e. cytochrome b and 12S rRNA. Thus, the two markers used in this study are useful in providing scientific proof for wildlife forensic cases as well as for status survey and will thus strengthen the conservation efforts. Implications for conservation Ashraf et al.10; Umapathy50 and Karanth51 reported the presence of this squirrel, but unfortunately there were no sight records available. Few decades later, Kurup7, Kumara and Singh52 sighted this small flying squirrel from Makut Reserve Forest, Karnataka, India and Jayase- kara et al.8 sighted the squirrel in Sinharaja tropical rain forest, Sri Lanka. This study provides the species-specific gene sequence by using two markers, useful for generating scientific proof for wildlife forensic cases and status survey. Since half of the potential distribution of P. fuscocapillus lies in human inhabited areas16, strict laws should be enforced in these areas to control hunting in the forest. Kumara and Suganthasakthivel16 modelled the potential distribution of Travancore flying squirrel in the two countries thus by using this study for identification of the species as well as by applying conservation plans such as afforestation for continuous canopy with height of the forest profile around 25 m as suggested by Koprowski and Nandini17 and Nanayakkara et al.53, the population of the species can be restored. Moreover, an extensive survey is pivotal at these potential distributions for a thorough population sampling to determine the probable reason behind the presence of a genetic bottleneck that reduced the genetic richness. Conclusion This study was designed to determine the molecular phylogenetic position of rediscovered Travancore flying squirrel (P. fuscocapillus) using two mitochondrial genes, i.e. 12s rRNA and cytochrome b. With respect to the phylo- genetic position of the species, tree topology of both genes provided the same result. Thus, both markers can be used for identification of the species. The Travancore flying squirrel was found to be monophyletic with the Temminck’s flying squirrel (Petinomys setosus). Accord- ing to IUCN (2008), the population of P. fuscocapillus was continuously decreasing; so, there is a strong need for some conservation action plans for the species. Con- servation plans may include extensive sampling, captive breeding, maintenance of continuous forest canopy and enforcement of strict laws to control hunting. 1. Crowley, B., Extinction and rediscovery: where the wild things are. J. Biogeogr., 2011, 38, 1633–1634. 2. Ladle, R. J., Jepson, P., Malhado, A. C. M., Jennings, S. and Barua, M., The causes and biogeographical significance of species rediscovery. Front. Biogeogr., 2011, 3, 111–117. 3. Scheffers, B. R., Yong, D. L., Harris, J. B. C., Giam, X. and Sodhi, N. S., The world’s rediscovered species: back from the brink? PLoS ONE, 2011, 6(7), e22531. 4. Fisher, D. O., Cost, effort and outcome of mammal rediscovery: neglect of small species. Biol. Conserv., 2011, 144, 1712–1718. 5. Fisher, D. O. and Blomberg, S. P., Correlates of rediscovery and the detectability of extinction in mammals. Proc. R. Soc. Lond. Series B, Biol. Sci., 2011, 278, 1090–1097. 6. Jackson, S. M. and Thorington Jr, R. W., Gliding Mammals: Tax- onomy of living and extinct species. Smithsonian Institution Scho- larly Press, Smithsonian Contribution to Zoology, 2012, p. 638. 7. Kurup, G. U., Rediscovery of the small Travancore flying squirrel. Oryx, 1989, 23, 2–3. 8. Jayasekara, P., Weerasinghe, U. R., Wijesundara, S. and Takatsuki, S., Identifying diurnal and nocturnal frugivores in the terrestrial and arboreal layers of a tropical rain forest in Sri Lanka. Ecotropica, 2007, 13, 7–15. 9. Hutton, A. F., Notes on the snakes and mammals of the High Wavy Mountains, Madurai district, South India, Part II-Mammals. J. Bom. Nat. Hist. Soc., 1949, 48, 681–894. 10. Ashraf, N. V. K., Kumar, A. and Johnsingh, A. J. T., On the rela- tive abundance of two sympatric squirrels of Western Ghats, India. J. Bom. Nat. Hist. Soc., 1993, 90, 158–162. 11. Sridhar, H., Raman, T. R. S. and Mudappa, D., Mammal persis- tence and abundance in tropical rainforest rem
RESEARCH ARTICLES CURRENT SCIENCE, VOL. 110, NO. 4, 25 FEBRUARY 2016666 19. Gonzalez, J., Phylogenetic position of the most endangered Chil- ean bird: the Masafuera Rayadito (Aphrastura masafuerae; Fur- nariidae). Trop. Conserv. Sci., 2014, 7(4), 677–689. 20. Jowers, M. J., Caut, S., Garcia-Mudarra, J. L., Alasaad, S. and Ineich, I., Molecular phylogenetics of the possibly extinct Martin- ique ground snake. Herpetologica, 2013, 69, 227–236. 21. Barros, N. D. M. and Morgante, J. S., A simple protocol for the extraction and sequence analysis of DNA from study skin of museum collections. Genet. Mol. Biol., 2007, 30(4), 1181–1185. 22. Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X. and Wilson, A. C., Dynamics of mitochon- drial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci., 1989, 86, 6196– 6200. 23. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S., MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 2011, 28, 2731–2739. 24. Thompson, J. D., Higgins, D. G. and Gibson, T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence align- ment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res., 1994, 22, 4673–4680. 25. Kimura, M., A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 1980, 16, 111–120. 26. Stamatakis, A., RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 2006, 22, 2688–2690. 27. Silvestro, D. and Michalak, I., raxmlGUI: a graphical front-end for RaxML. Org. Div. Evol., 2011, 12(4), 335–337. 28. Ronquist, F. et al., MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol., 2012, 61, 539–542. 29. Darriba, D., Taboada, G. L., Doallo, R. and Posada, D., jModelTest 2: more models, new heuristics and parallel computing. Nat. Meth., 2012, 9, 772. 30. Huelsenbeck, J. P. and Rannala, B., Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Syst. Biol., 2004, 53, 904–913. 31. Lecocq, T. et al., Patterns of genetic and reproductive traits differ- entiation in mainland vs Corsican populations of bumblebees. PLoS ONE, 2013, 8(6), e65642. 32. Yang, Z., Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol., 1994, 39, 306–314. 33. Rambaut, A. and Drummond, A. J., Tracer v1.5; http:// beast.bio.ed.ac.uk/Tracer, 2007. 34. Oshida, T., Masuda, R. and Yoshida, M. C., Phylogenetic relation- ships among Japanese species of the family Sciuridae (Mammalia, Rodentia), inferred from nucleotide sequences of mitochondrial 12S ribosomal RNA genes. Zool. Sci., 1996, 13, 615–620. 35. Mein, P., Les Sciuropteres (Mammalia, Rodentia) Neogenes d’Europe Occidentale. Geobios (Lyon), 1970, 3, 7–77. 36. Black, C. C., Holarctic evolution and dispersal of squirrels (Rodentia: Sciuridae). Evol. Biol., 1972, 6, 305–322. 37. De Bruijn, H., Superfamily Sciuroidea. In The Miocene land mammals of Europe (eds Rösner, G. E. and Heissing, K.), Verlag Dr. Friedrich Pfeil, Müchen, 1999, pp. 271–280. 38. Oshida, T., Lin, L.-K., Yanagawa, H., Endo, H. and Masuda, R., Phylogenetic relationships among six flying squirrel genera, inferred from mitochondrial cytochrome b gene sequences. Zool. Sci., 2000, 17, 485–489. 39. Allen, G. M., The Mammals of China and Mongolia, American Museum of Natural History, New York, 1940. 40. Corbet, G. B. and Hill, J. E., The Mammals of the Indomalayan Region: A Systematic Review, Oxford University Press, Oxford, 1992. 41. Hoffmann, R. S., Anderson, C. G., Thorington Jr, R. W. and Hea- ney, L. R., Family Sciuridae, Mammal Species of the World: A Taxonomic and Geographic Reference (eds Wilson, D. E. and Reeder, D. M.), 2nd ed., Smithsonian Institution Press, Washing- ton DC, 1993. 42. Thorington Jr, R. W. and Hoffmann, R. S., Family Sciuridae, Mammal Species of the World: A Taxonomic and Geographic Ref- erence (eds Wilson, D. E. and Reeder, D. M.), 3nd edn, Washing- ton DC, The Johns Hopkins University Press, 2005. 43. Li, S., He, K., Yu, F.-H. and Yang, Q.-S., Molecular phylogeny and biogeography of Petaurista inferred from the cytochrome b gene, with implications for the taxonomic status of P. caniceps, P. marica and P. sybilla. PLoS ONE, 2013, 8(7), e70461. 44. Thorington Jr, R. W., Pitassy, D. and Jansa, S. A., Phylogenies of flying squirrels (Pteromyinae). J. Mammalian Evol., 2002, 9, 99– 135. 45. Mercer, J. M. and Roth, V. L., The effects of Cenozoic global change on squirrel phylogeny. Science, 2003, 299, 1568–1572. 46. Oshida, T., Shafique, C. M., Barkati, S., Fujita, Y., Lin, L.-K. and Ma