Philippine Journal of Science 148 (S1): 133-140, Special Issue on Genomics ISSN 0031 - 7683 Date Received: 18 Mar 2019 DNA Barcodes Reveal High Genetic Diversity in Philippine Fruit Bats Adrian U. Luczon1*, Sofia Anne Marie M. Ampo1,2, John Gregor A. Roño1, Mariano Roy M. Duya1, Perry S. Ong1, and Ian Kendrich C. Fontanilla1,3 1Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101 Philippines 2Rizal Medical Center, Pasig Blvd., Barangay Bagong Ilog Pasig City 1600 Philippines 3Philippine Genome Center, University of the Philippines Diliman, Quezon City 1101 Philippines Fruit bats of the family Pteropodidae is the third largest family in the order Chiroptera. There are 26 recorded species in the Philippines, 17 of which are endemic to the country. However, the number of species in the archipelago may still be underestimated. With the growing threats to biodiversity and dwindling number of taxonomists, DNA barcodes can assist with the problem by providing an accurate, rapid, and effective method of species recognition. To contribute to the barcoding endeavor and determine the diversity of Philippine fruit bats, a 542-base-pair portion of the cytochrome c oxidase subunit 1 (COI) gene was sequenced from 111 individuals belonging to 19 pteropodid species. A neighbor-joining (NJ) and maximum likelihood (ML) tree was generated using the sequences in this study and other available sequences in Genbank and Barcode of Life Data Systems (BOLD). DNA barcodes were effective in delineating Philippine species. Closer inspection of the NJ tree revealed distinct [> 6% mean Kimura-2-parameter (K2P) distance] Philippine lineages for Macroglossus minimus, Rousettus amplexicaudatus, Megaerops wetmorei, and Cynopterus brachyotis relative to conspecifics from Southeast Asia. Between-island differentiation was also observed for the Philippine endemic Haplonycteris fischeri (> 7% mean between-island K2P distance). From this study, these species may be flagged for taxonomic reevaluation. Keywords: COI gene, cryptic species, DNA barcoding, Pteropodidae INTRODUCTION (Campbell et al. 2004, Galimberti et al. 2010). Looking at Vespertilionidae, Phyllostomidae, and Pteropodidae – the The catalog of mammalian species found in the Philippine three largest families of bats – the number of recognized archipelago is far from complete. Just recently, 28 new species in 2005 were 407, 160, and 186, respectively species of non-volant mammals were discovered by (Simmons 2005). However, based on the latest record Heaney et al. (2016a). Certainly, more species can still in 2017 (ASM 2019), the number of species increased be discovered, especially in remote and unexplored parts considerably (Vespertiolionidae, 24.07%; Phyllostomidae, of the archipelago (Heaney et al. 2010). Species with 35%; and Pteropodidae 5.4%). cryptic morphology and habits may also contribute to the number of overlooked taxa, a common occurrence in bats Nevertheless, species are still endangered throughout the world and in the Philippines through activities such as *Corresponding Author: [email protected] habitat disturbance and overharvesting of animals (Francis 133 Special Issue on Genomics Luczon et al.: DNA barcoding of Philippine fruit bats et al. 2010). Globally, a quarter of the total mammalian robust record gathered by morphological taxonomists species is threatened with extinction, and half have (Hebert et al. 2003). declining populations (Myers et al. 2000). Conservation measures must therefore be put in place to maintain the The family of fruit bats, Pteropodidae, is one of the largest local biodiversity, and proper steps cannot be taken unless bat families within the order Chiroptera having around 45 species are correctly identified. genera and about 196 recognized species (ASM 2019). In the Philippines, there are about 26 recognized species As the number of classically trained taxonomists is (Tanalgo and Hughes 2018) – 17 of which (about 65%) dwindling (Hebert et al. 2003), a molecular method for are endemic to the country. identifying species known as DNA barcoding is gaining increasing importance as source of data for taxonomy. COI barcodes for native and endemic Philippine fruit bat DNA barcoding uses a standard region of the genome as species are lacking. To contribute to current published a genetic marker for identifying species. In animals, the 5’ barcoding data for pteropodids, the study aims to end of the COI is the DNA barcode of choice. Although the obtain sequences of the COI gene of the fruit bats in the task of identifying and describing new species is ultimately Philippines. The study also aims to test the viability of the achieved through comprehensive taxonomic work, DNA marker in delineating the new COI records from existing barcoding and the data derived from it can significantly pteropodids in the database. A gene tree for Philippine bats facilitate the process by serving as a supplement to the and those in existing databases were constructed to assess identification provided by morphological taxonomists the fruit bat species diversity in the archipelago based on (Hajibabaei et al. 2007). It can also facilitate the COI. The study will not only be able to document DNA “democratization” of taxonomic knowledge by making diversity of bats but also have practical applications such expert taxonomic information available to non-experts as in wildlife forensics. in an applied context (Holloway 2006). As DNA sequences have become the major source of new information for advancing understanding of evolutionary MATERIALS AND METHODS and genetic relationships (Hajibabaei et al. 2007), the Sampling data obtained in this study may contribute substantially to current knowledge on chiropteran classification. In the Fruit bats were collected from 17 sites in the Philippines same way, DNA barcode libraries can serve as effective (see Figure 1). Samples used in this study came from identification systems for any regional bat fauna (Clare et various collections by the authors, as well as collections al. 2007). DNA barcode analysis and the methodology it by other researchers mentioned in the acknowledgments. employs can be applied in a standardized manner across large domains of life – overcoming the difficulty presented by conventional taxonomy, where multiple data types may be required, depending on the taxa being studied (Hajibabaei et al. 2007). Use of DNA barcodes translates to more species being examined in a shorter span of time with less cost. The profiles obtained from barcodes will be cost-effective in many taxonomic contexts. It is important to acknowledge that, at present, access to this new technology is not always readily available to everyone. Nevertheless, innovations in sequencing technology promise future reductions in the cost of DNA- based identifications (Hebert et al. 2003). DNA barcoding may also serve as the basis for a global bioidentification system for animals that will overcome the deficiencies of morphological approaches to species discrimination and allow single laboratories to execute taxon diagnoses across the full spectrum of animal life (Hebert et al. 2003). However, genetic differentiation has its limitations as it cannot incorporate the fossil record or older museum specimens with degraded material. A database of COI profiles coupled with other data (images, geographic Figure 1. Map showing the collection sites of specimens in this study. location, and other specimen data) can also serve as a 134 Special Issue on Genomics Luczon et al.: DNA barcoding of Philippine fruit bats Species identification was based on Ingle and Heaney Almeida et al. (2011) – was included as an outgroup in the (1992). Tissue samples collected came in the form of final data set. The list of specimens in this study can be found 2-mm wing punches from individuals that were captured in Appendix I, while the list of sequences from Genbank and released, or muscle from vouchered specimens. and BOLD used in the dataset can be found in Appendix II. Tissues collected were kept frozen or placed in a microcentrifuge tube with 95–100% ethanol. At least three The dataset was then aligned using MAFFT version 7 individuals per species were used for the study. (Katoh et al. 2017), followed by manual alignment as needed. The lengths of sequences were made uniform to 542 base pairs to accommodate sequences that were DNA Extraction, PCR Amplification, and shorter in length. The complete dataset included 817 Sequencing sequences, of which 111 are pteropodid sequences Genomic DNA was obtained from the tissues using the generated from this study. Promega™ Wizard® SV Genomic DNA Purification Kit (USA) following the manufacturer’s protocols. MEGA 7 (Kumar et al. 2016) was used to calculate The COI gene was amplified using the primers VF1 pairwise K2P distances needed to construct a COI gene (TTCTCAACCAACCACAARGAYATYGG) and VR1 tree. An NJ tree (Nei and Saitou 1987) with 1000 bootstrap (TAGACTTCTGGGTGGCCRAARAAYCA) (Ivanova replicates was constructed to view the groupings of the et al. 2006). Alternatively, the mammal primer cocktail sequences. An ML tree using RaxML (Stamatakis 2014) (Ivanova et al. 2012) was used for other specimens. A 30- with 1000 bootstraps (rapid bootstrap algorithm) and a μL reaction mix was prepared using the genomic DNA, best-fit model of substitution acquired from jModelTest 2 primers, and Taq dNTPack (Roche, USA). The PCR reaction (Darriba et al. 2012) was also constructed to see if there is consisted of 5 μL 10x polymerase