Phylogenetic studies of Chiropteran Fauna in FATA region of Pakistan through classical and molecular approaches

Abstract

The present study conducted on morphogenetic evaluation of chiropteran fauna from Federally Administered Tribal Areas (FATA), region of Pakistan. A total of 217 specimens were collected during May 2014 to October 2017. Investigative visits were made to locate Chiropteran nest in study areas as possible. For capturing Chiropteran species Ultra Violet unchangeable, deep black and heavy duty nets were used. During the field work various parameters i.e. sex, age, locality, habitat, date of collection, time of collection and field number were recorded in the notebook. The specimen were identified up to species level through morphological characterization, yielding in to 2 sub orders i.e. Mega and Micro chiropterans spreading into 3 families i.e. Pteropodiade, Vespertilionidae and Minopteridae with 6 genera and consisting into 9 species. The sub order Mega chiroptera was represented by only one family i.e. Pteropodiade with single species i.e. Cynopterus sphinx. Sub order Micro chiroptera was included into two families i.e. Vespertilionidae and Minopteridae. Family Vespertilionidae was consisted into 7 species i.e. Myotis mystacinus, Pipisterulus khulli, P. javinicus, P. coromandra, P. tenius, Scotophilus heathi, Nuctalus leisleri. While family Minopteridae was resulted into single genus with one species i.e. Minopterus fuliginosis. All the species were recorded first time from FATA region. Details of collected material i.e. names, synonyms and measurement of body (Body length, Fore arm thumb, metacarpals, Hind foot and Cranial), habitat description, distribution range in Pakistan as well as international distribution, IUCN Red list status, colored images of all nine species were presented. Moreover, taxonomic xi keys for families, genera and species were also presented. Morphometry of the various body parts were done and this morphometric data was analyzed statistically based on Cluster, Principal coordinate analysis (PCoA), Principal Component analysis (PCA) and Detrended correspondence analysis (DCA) to understand the species relationship. Further we used diagnostic characters and multivariate analysis to determine morphological differences between the genera and species. Our data clearly revealed wide variation in the cranial and body parameters of the species, supported by statistical differences. These results serve as the basis for the future assessment and taxonomic position of Chiropteran species. Second part of the present study was molecular characterization and phylogenetic analysis. Relationships among members of the Chiroptera were examined using 658 bp of mitochondrial Cytochrome Oxidase I (COI) gene sequence. Species with COI gene sequences were obtained for molecular characterization and their phylogenetic relationships. The successfully obtained sequences were studied with their genetic distance with (K2P) parameter. Composition of nucleotide, genetic distance and transitional ratio were revealed among the genera and species of Chiroptera. Neighbor Joining, Maximum likelihood and Maximum Parsimony methods were used to construct the phylogenetic tree and also found the divergence and genetic relations among the recorded species of Chiroptera. Cytochrome Oxidase one (COI) gene sequences of Chiropteran species were compared to other gene sequences BLAST in NCBI GenBank data base. The similarity indices were resulted 96-100% with other sequences of the same species xii already reported in GenBank. Intraspecific divergence was founded 0.06 – 1.6% and interspecific divergence was 6.8 – 22.2 % respectively. The mean A+T contents were observed 56.6% (±2.35) and C+G were resulted 45.1% (±5.1). Genetic similarities with bootstrap values of Neighbor Joining method were resulted = 70 -100 %, while the boot strip of Maximum likelihood method were observed= 65 -100 %, and the bootstrap value of Maximum Parsimony method were sorted out= 50 -100%. All findings of the present study revealed the taxonomical position of the Chiropteran species, with morphological characters as well as molecular and phylogenetic characterizations. Present research provides pioneer report for phylogenetic elaboration and cataloguing of Chiropteran species in the FATA regions of Pakistan. In short this research based on both method i.e. classical taxonomic approach and modern molecular techniques, which provide baseline study of Chiropteran species found in FATA.