Philippine Journal of Science 150 (3): 837-846, June 2021 ISSN 0031 - 7683 Date Received: 05 Oct 2020

Genetic Diversity of Philippine Carabao (Bubalus bubalis) Using Mitochondrial DNA D-loop Variation: Implications to Conservation and Management

Lilian P. Villamor1,2*, Yukimizu Takahashi2, Koh Nomura2, and Takashi Amano3

1Department of Agriculture, Philippine Carabao Center National Headquarters and Gene Pool Science City of Muñoz, Nueva Ecija 3120 2Tokyo University of Agriculture, Atsugi, Kanagawa 243-0034 Japan 3Yamazaki University of Animal Health Technology, Hachioji, Tokyo 192-0364 Japan

Developing rational conservation programs for Philippine carabao (PC) (Bubalus bubalis) requires knowledge on the pattern of genetic variability within and between populations of swamp buffalo. Studying the genetic diversity and phylogeographic structure is essential to understand the distribution of the Philippine swamp buffalo maternal haplotypes towards the PC conservation. This study aimed to determine the genetic diversity and the phylogeographic structure of PC using molecular approaches. D-loop of the mitochondrial DNA (mtDNA) on the PC B. bubalis (n = 107) were randomly selected from 23 populations across the islands of , Visayas, and as major islands sub-groups. DNA was isolated from the whole blood, and the D-loop region of the swamp buffalo was amplified using the polymerase chain reaction (PCR). Purified PCR products were sequenced with the Applied Biosystems Automated 3730. Results showed that the phylogenetic analyses detected 16 mtDNA haplotypes observed with 12 variable sites and haplotype and nucleotide diversities of 0.695 ± 0.042 and 0.004 ± 0.001, respectively. This study also identified the delineation of swamp buffalo populations into major and minor groups. Among the major groups, 14 haplotypes were included, and sequences were incorporated in the maternal lineage A. Minor groups which fell in the maternal lineage B identified two new haplotypes. Thus, the current findings revealed the moderate mtDNA haplotype diversity and weak phylogeographic structure of the PC. In addition, the results of this study will serve as a vital starting point in planning effective strategies and prioritizing the genetic resources for PC conservation and management programs.

Keywords: genetic diversity, mtDNA D-loop, phylogenetic, Philippine carabao, conservation

INTRODUCTION have a worldwide distribution in the Indian subcontinent, China, Brazil, Italy, and mostly across Southeast Asia Water buffaloes (Bubalus bubalis) are widely distributed (Lau et al. 1998; Kierstein et al. 2004; Cruz 2015). In the and economically important livestock species in many Philippines, swamp-type buffalo is locally known as native developing countries regarding their significant contribution carabao or PC. The animal is considered the farmer's great to agriculture and rural economy (Zhang et al. 2020). They partner in providing draft power in his farm works and has the potential for meat, milk, and hide sources. *Corresponding Author: [email protected]

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The Philippine buffalo population has decreased by maternal origins: A and B (Lei et al. 2011). Previous 130,345 heads and at a rate of –0.216% annually between studies on mtDNA D-loop sequence variation have 1998–2018, and these animals are commonly reared in shown the genetic differentiation between the swamp the backyard or at commercial farms. However, they are and the riverine buffalo types in Southeast Asia (Lau et not distinguished between two types: riverine or swamp al. 1998; Lei et al. 2007b), Brazil, and Italy (Kierstein et (http://www.fao.org/faostat/en/#data/QA). Thus, this leads al. 2004). Similarly, the mtDNA D-loop was useful in the to a primary concern to conserve and manage buffalo genotypic analysis to characterize the Malaysian water breeds – particularly the swamp buffaloes – across the buffaloes and their crossbreds, which strongly indicated islands in the Philippines. The Philippine archipelago swamp maternal lineage (Shaari et al. 2019). In addition, is comprised of Luzon, Visayas, and Mindanao major Chinese swamp buffaloes shed light on the two distinct island groups. The Luzon island is in the northern part maternal lineages A and B. After the domestication in the of the country and considered the largest island group in China region, the domesticated Chinese swamp buffaloes terms of land area with dimensions of 740 by 225 km2 revealed two dispersal pathways: first, through Taiwan and covers approximately one-third of the land area of the and the Philippines to the eastern islands of Borneo and Philippines. The Visayas is situated in the central portion Sulawesi; and second, through the mainland Southeast of the country and the third major island group in terms of Asia and then to the western islands of Indonesia (Lau et land area. The Mindanao island group is in the southern al.1998; Lei et al. 2007a). part and the second-largest landmass group of the country (https://www.britannica.com/place/Philippines). The genetic diversity of Philippine swamp buffaloes has few published data and remained unresolved. The mtDNA The buffalo species are divided into swamp and riverine cytochrome c oxidase I (COI) gene was used to identify based on the differences of cytogenic and morphological the species of buffaloes in Calayan Island as swamp-type characterizations. There are no formally recognized breeds buffaloes and suitable for establishing the PC sanctuary and variation in breed characteristics among the swamp (Paraguas et al. 2018). Previous studies on mtDNA D-loop buffaloes (Qui 1986). For example, swamp buffaloes sequences of Philippine swamp buffaloes from the three in other countries such as China and the Philippines Philippine Carabao Center (PCC) institutional herds and are considered a single and homogeneous breed (Qui farms were all incorporated in the maternal lineage A (del 1986; Paraguas et al. 2018). However, swamp buffalo Barrio et al. 2009; Yue et al. 2013). It was reported that no is identified with 48 chromosomes, and riverine buffalo significant geographical structure was identified among with 50 chromosomes. The common physical features swamp-types in Luzon and Visayas islands using the of swamp buffaloes are the white or light gray markings mtDNA COI gene (Bondoc 2013). Lack of studies relating in the lower jaw and brisket (chevron) and the lighter to wide genetic diversity and phylogeographic analyses skin and coat color than the riverine buffaloes. The horn of Philippine swamp buffaloes with more individuals and is sickle-shaped, which extends backward compared to populations were reported. Assessment of the Philippine riverine buffaloes having heavily curled horns (Castillo swamp-type genetic variation with the rationale for 1998). However, the chromosome karyotyping and conservation and management of PC has been conducted morphological-based identification systems could be with the mtDNA control region (D-loop) of swamp buffalo limited to identify swamp buffaloes. The genetic diversity populations. Therefore, this study aimed to determine the assessment of PC could complement a more accurate genetic diversity and the phylogeographic structure using technique such as the DNA-based approach. This entails molecular approaches. using informative genetic markers that could be used to understand the genetic diversity and phylogenetic relationship of swamp buffalo in the country. MATERIALS AND METHODS In Asia, previous studies have been reported that genetic diversity and phylogenetic analyses of swamp buffaloes using the variable regions in the mtDNA markers (Lau Site Selection and Specimen Collection et al. 1998; Kierstein et al. 2004; Lei et al. 2011; Shaari The coordination of collection sites was identified and et al. 2019; Winaya et al. 2019). For instance, the assisted by various regional managers of the PCC and phylogenetic relationships of Indonesia's swamp buffalo local government units. Moreover, the selection of sites groups corresponded to their biogeographic origin and highly considered isolated areas such as mountainous their closer genetic affinity to the Philippines swamp and coastal parts of the country to ensure no introduction buffalo based on the cytochrome b (cyt b) gene partial of artificial insemination program of the river-type was sequences (Winaya et al. 2019). Genetic diversity of cyt done. In this study, 107 fresh blood samples of Philippine b gene in Chinese swamp buffaloes confirmed the two swamp buffalo were randomly selected from 23 different populations of Luzon, Visayas, and Mindanao islands

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(Table 1). Approximately 5 ml of blood was collected DNA Extraction, Amplification, and Sequencing into heparinized tubes from the swamp buffalo's jugular Genomic DNA was extracted from the whole blood vein and was stored on ice before transportation to the samples using the commercially available DNA extraction laboratory for molecular analysis. All animal handling kit, and the manufacturer's recommended procedure and techniques involved were approved by the PCC Ethics with little modifications was followed (Promega Committee, Nueva Ecija, Philippines. ReliaPrep™). After the third washing using column wash solution (CWS), additional centrifugation (14,000 Nine reference D-loop sequences were retrieved from rpm) for 1 min was performed to remove traces of CWS NCBI GenBank. These were represented by six water before the first elution. The incubation time of DNA in buffaloes from China (GenBank Accession Numbers nuclease-free water was extended to 5 min before the DQ658064.1, DQ658077.1, EF053542.1, GQ260390.1, elution step to obtain a high yield and good quality of KX758295.1, and KX758304.1) with 912-940 bp, one DNA. The purity and quantity of DNA were determined Indian river buffalo (AF547270.1) with 926 bp long, one using a spectrophotometer following the manufacturer's Philippine swamp buffalo (FJ873678) with 485 bp long, recommendation (Thermo Scientific™ NanoDrop 2000 and one Thai wild buffalo (Bubalus arnee) (KU687004) and 2000c). with 405 bp long (Appendix Figure I; Appendix Table I).

Table 1. Source and genetic diversity of 107 PC samples. Population Code Sample size aHap bｈ cπ Island Luzon 56 12 0.560 0.003 Visayas 27 7 0.761 0.004 Mindanao 24 6 0.761 0.003 Collection site Calayan Island CAL 7 5 0.857 0.005 BTS 2 1 – – Benguet BGT 7 2 0.286 0.001 Kalinga KA 5 1 – – Aurora AU 6 1 – – La Union LA 4 4 1.000 0.006 Pangasinan PA 4 3 0.833 0.004 Zambales ZAM 5 3 0.700 0.003 Camarines CAM 6 3 0.733 0.003 Sorsogon SOR 2 2 1.000 0.006 Carabao Island CAR 2 1 – – Occidental Mindoro OCM 6 2 0.533 0.006 Capiz CPZ 8 5 0.857 0.006 Gigantes Island GILL 5 4 0.900 0.005 Island GMR 4 1 – – Island BHL 3 2 0.667 0.002 Talibon TAL 4 2 0.667 0.004 Leyte LYT 3 2 0.667 0.002 Bukidnon BKD 6 5 0.933 0.006 North Cotabato NCOT 3 2 0.667 0.002 South Cotabato SCOT 5 3 0.800 0.003 Sultan Kudarat SK 7 3 0.762 0.003 Saranggani SRN 3 3 1.000 0.004 aNumber of haplotypes; b haplotype diversity; c nucleotide diversity

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The DNA was amplified via PCR in an ABI Thermal RESULTS Cycler (Biorad, Hercules, CA, USA) using the mtDNA D-loop, a primer designed from the Laboratory of Animal Genetic Diversity and Pairwise Difference (Fst) Genetics and Breeding, Tokyo University of Agriculture. The 107 sequences from PC were successfully submitted The total volume of 10-μL PCR reactions was as follows: to NCBI GenBank (MT642457-MT642563) and included 6.2 μL of sterile ultrapure water, 1 μL of 10X Buffer, 16 mtDNA haplotypes with 12 polymorphic nucleotide 0.8 μL of 2.5mM dNTPs, 0.70 μL (6.6pmol/μL) of each sites (Table 1). Luzon had 12 haplotypes, followed by forward (DL-1) and reverse (DH-1) primers, 0.1 μL of seven haplotypes for Visayas and six for Mindanao. 5U/μL, and 0.5 μL of at approximately at least 75 ng/μL Haplotype diversity was higher in Visayas and Mindanao DNA template. The optimized PCR amplification program at 0.761 and the lowest in Luzon at 0.560. Nucleotide included: initial denaturation of 2 min at 95 °C followed diversity was highest in the Visayas at 0.004, followed by 30 cycles of denaturation of 30 s at 95 °C, annealing by Mindanao at 0.003 and Luzon at 0.003. The average of 30 s at 53 °C and extension of 30 s at 72 °C, and final of haplotype and nucleotide diversities in 23 populations extension of 5 min at 72 °C. of PC was 0.695 ± 0.042 and 0.004 ± 0.001, respectively. Electrophoresis was performed for 30 min at 100 Significant pairwise differences were based on Fst among V. The bands were visualized with ultraviolet light the swamp buffalo populations from the three major and photographed using the Alphalmager™ 2200 islands, ranging from –0.014 to 0.058 (Table 2). The electrophoresis documentation and analysis system highest significant genetic distance was between Luzon (Cell Biosciences, CA, USA). The sizes of the and Mindanao is Fst = 0.102, followed by pairwise amplified PCR products were estimated using 72–1353 distances between Luzon and Visayas is Fst = 0.058. bp ladder Marker 4 (Ф× 174/HaeIII digest). Then, However, a lack of pairwise difference was observed the purified PCR products were sequenced with the between Visayas and Mindanao (P < 0.05; Fst = –0.014). Applied Biosystems Automated 3730 using the Big Dye Terminator chemistry and AmpliTaq-FS DNA Table 2. Pair-wise differences (Fst) of PC from major island groups. polymerase following the standard protocol in the Laboratory of Animal Genetics and Breeding, Tokyo Island Group Luzon Visayas Mindanao University of Agriculture, Tokyo, Japan. Luzon 0 Visayas 0.058* 0 Data Analysis Mindanao 0.102* –0.014* 0 The sequence editing, alignment, model test, and *P-value < 0.05 neighbor-joining (NJ) tree construction of 348-bp mtDNA D-loop PCR amplified fragments were done A closer look at the 23 PC populations revealed uneven in MEGA7.0.26 software (Saitou and Nei 1987; distribution of haplotypes across the major islands of the Thompson et al. 1994). Insertions and deletions in the Philippines. The highest frequency of five haplotypes aligned sequences were excluded from the analyses. was detected in three populations: Calayan (CAL), Capiz The sequences of mtDNA (coding region) D-loop of (CPZ), and Bukidnon (BKD). La Union (LA), Sorsogon PC were deposited in the NCBI GenBank. All retrieved (SOR), and Sarangani (SRN) had the highest haplotype NCBI sequences except GenBank Accession Numbers diversity (1.00), but the highest nucleotide diversity was FJ873678 and KU687004 were aligned with the PC detected in CPZ (0.006) (Table 1). Only one haplotype D-loop sequences. However, the percent sequence in the populations – including Batanes (BTS), Kalinga identity of GenBank Accession Nos. KU687004, (KA), Aurora (AU), Carabao Island (CAR), and Guimaras FJ873678, KX758304.1, and DQ658064.1 were (GMR) – exhibited a lack of haplotype and nucleotide analyzed using Nucleotide BLAST. Haplotype diversity diversities (Table 1; Appendix Tables II and III). (h), nucleotide diversity (π), and neutrality tests based on Tajima's D and Fu's Fs were computed using DnaSP v6 program (Rozas et al. 2017). Genetic distance NJ Tree Phylogenetic Relationship based on Fst was computed to determine significant The phylogenetic tree of PC showed two maternal lineages differences in mitochondrial diversity between indicating 16 mtDNA haplotypes. The results also showed Philippine swamp buffalo breeds using ARLEQUIN that the PC sequences are incorporated in lineage A 3.01 (Excoffier and Lischer 2010). The NETWORK (97%, 104/107), and the minority of the sequences fell 10.1 program was used to generate the median-joining in lineage B (3%, 3/107). Fourteen of the 16 haplotypes (MJ) network (Bandelt et al. 1999). were observed in lineage A – including H1-H9, H11-14, and H16 – which could further be separated into two sub- groups. The first sub-group had nine haplotypes and can

840 Philippine Journal of Science Villamor et al.: Genetic Diversity of Philippine Carabao Vol. 150 No. 3, June 2021 be divided into two clusters, indicating four haplotypes haplotypes, none of these are common to all the PC for the first cluster (H1, H3, H11, and H13) and five populations (Figure 2). However, nine haplotypes (H1– haplotypes for the second cluster (H2, H6, H7, H14, and H9) were distributed among various populations, with H16). The second sub-group showed closer affinity among haplotype H1 having the highest frequency. Seven of the four haplotypes incorporating H4, H8, H9, and H12. 16 haplotypes occurred only once (H10–H16) (Appendix Among the group of haplotypes, H5 clustered separately, Tables II and III). The MJ network delineated lineages with a very low statistical support (PB-11%). However, A and B, which were separated by four mutation steps. among the generally low bootstrap values precluded the Lineage A incorporated 14 haplotypes, while Lineage B interpretation of the groupings. In contrast, the minor detected two haplotypes. group – which belonged to lineage B – showed a closer relationship between haplotypes H10 and H15, with high The two predominant haplotypes (H1 and H2) can be statistical support (PB = 96; Figure 1). traced to lineage A, and they were separated by a mutation step at the 207th base position. The most prominent The phylogeny analysis of D-loop sequences obtained haplotype H1 represented 55 individuals (51% of 107 from this study and retrieved from the NCBI GenBank individuals). In lineage A, haplotype H1 was separated showed a clear delineation between the swamp and by one, two, three, and four mutation steps for five riverine clusters. The haplotype distribution of PC D-loop haplotypes (H2, H3, H4, H5, and H9), for five haplotypes sequences in maternal lineages A and B was confirmed by (H6, H8, H11, H12, and H16), for haplotype H7, and the reference sequences obtained from the NCBI GenBank haplotype H14, respectively. All populations exhibited (Appendix Figure I). The nucleotide BLAST analysis individuals belonging to haplotype H1 except for SOR of representative PC sequences revealed 97–100% and TAL, and the same haplotype showed a pattern of similarity to D-loop sequences of Chinese swamp buffalo decreasing distribution of 37 in Luzon, 10 in the Visayas, (KX758304.1). Moreover, Thai wild buffalo (KU687004) and eight in Mindanao. This pointed to haplotype H1 indicated 92.57–94.16% similarity to those sequences of as the ancestral haplotype of the PC samples included various breeds of Chinese and Philippine swamp buffaloes in the study. The second prominent haplotype (H2) (Appendix Table I). represented 21 individuals (17% of 107 individuals). The haplotype distribution in H1 was opposite to and had a smaller number of haplotypes in H2 – indicating four in MJ Network and Phylogeography Luzon, nine in the Visayas, and eight in Mindanao. Thus, The MJ network analysis revealed that out of 16

Figure 1. NJ tree of 16 PC swamp buffalo haplotypes.

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Figure 2. MJ network of 107 PC samples. the pattern of haplotype distribution demonstrated the of swamp buffaloes along the Yangtze valley throughout existence of an evolutionary relationship among them. history. In addition, the people in Southern China who preferred to take along buffaloes during their migration The maternal lineage B comprised of two haplotypes (H10 and were involved in the frequent trading of buffaloes for and H15) and was separated by a mutation step at the 267th their farm works contributed to the improved homogeneity base mutation. Haplotype H1 was connected to haplotype of swamp buffaloes in China (Yue et al. 2013). H10 with four mutation steps. The phylogeography showed similar haplotype distribution to that of the NJ The average genetic distances of swamp buffaloes from phylogenetic tree and the MJ network analyses (Figure 3). the three major island groups agreed with two factors: geographic distances and human migration. Proximity and accessibility of Luzon to Visayas and Visayas to Mindanao, as compared with Luzon to Mindanao and vice versa, DISCUSSION could affect geographical distances and human migration patterns. The closer geographical locations and easy Genetic Diversity and Pairwise Difference accessibilities to inter-island transportations could cause The average haplotype diversity indicated abundant a lack of genetic differences between the two closer major genetic diversity in 23 PC populations of this study. It was island groups. The lack of genetic differentiation between comparable to genetic diversity in Asian swamp buffalo Visayas and Mindanao swamp buffalo populations can populations that were previously reported (h = 0.786– also be traced to the historical event of the early migration 0.866) (Lau et al. 1998; Lei et al. 2007a, b; Yue et al. of farmers from the Visayas to Mindanao, who carried 2013). However, the average nucleotide diversity obtained with them their native carabaos to cultivate the agricultural from this study of PC was lesser (π = 0.004) than those land of Bukidnon province, in the Mindanao island reported by previous studies (π = 0.007–0.049) (Lau et subgroup (Pendleton 1942). Consequently, the pattern al. 1998; Lei et al. 2007a; Yue et al. 2013). This could on the genetic information revealed by mtDNA D-loop indicate that the PC has lower genetic variation than in sequences highly suggested that swamp buffaloes are Asian swamp buffaloes. However, an extensive gene flow not native species but are introduced farm animals in among Chinese swamp buffalo populations was reported Mindanao. Hence, the distribution of haplotypes across to be associated with frequent trading and transportation the three major islands in the Philippines supported the

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Figure 3. Map showing the frequency of distribution of the 16 haplotypes (retrieved from https://www.sporcle.com/games/FilipinoBreloom/ find-the-provinces).

pairwise distance (Fst values), which clarified the closer provincial accessibility of LA and PA to ZAM and vice relationship of swamp buffaloes in Visayas and Mindanao versa. A similar pattern showed closer relationships and the widespread differences in Luzon. The widespread between H3 and H11 and between H10 and H15, which haplotype distribution in Luzon's population indicated the could be due to the availability of inter-island and intra- high migration of swamp buffaloes within and between province transportations from CPZ to GILL and from Luzon and the islands of Visayas and Mindanao. OCM to CPZ and vice versa, respectively. These research findings showed biased by the number of samples from Luzon populations, which could contribute to the wide Phylogenetic Analyses distribution of the haplotypes in the country. However, In this study, the relationship of the 16 haplotypes the genetic patterns based on mtDNA D-loop sequences indicated a weak phylogeography structure. Although of PC demonstrated a closer affinity between few the bootstrap support on separating the two maternal haplotypes with geographically close populations and lineages using the NJ phylogenetic tree was low, the had improved accessibility to transportations. In fact, a MJ network confirmed the similar delineation of the related study on COI reported that a high gene flow of major and minor groups – which belonged to maternal PCs could be due to wide transportation among local lines A and B, respectively. For example, haplotype that carabao populations caused by improved accessibility to fell in H3 showed intermixing of CPZ, GILL, BKD, inter-provincial and inter-island (Bondoc 2013). SK, and SRN (Appendix Table III). Swamp buffaloes that belonged to haplotype H11 were predominantly The mtDNA D-loop sequences of Philippine swamp found in GILL, while individuals detected in H10 were buffaloes were all identified in maternal lineage A (del from OCM. The closer affinities between H4–H12 Barrio et al. 2009; Yue et al. 2013). The undetected swamp and between H8 and H9 indicated the high gene flow buffalo samples to the maternal lineage B in the previous among populations that could be attributed to the intra- studies could be attributed to the small sample size and

843 Philippine Journal of Science Villamor et al.: Genetic Diversity of Philippine Carabao Vol. 150 No. 3, June 2021 restricted sampling sites. This study first reported the unique haplotypes and a high number of shared among detection of the two haplotypes in lineage B, which were the different PC populations. However, the PC's unique traced from OCM and CPZ, implying a genetic exchange haplotype distribution was opposite to and had a smaller between the two populations. The combined genetic number of haplotypes in domestic goats. The study of information revealed in the NJ tree and MJ network Vacca et al. (2010) reported that domestic goats showed structure provided sufficient evidence on the moderate a higher number of unique haplotypes, wherein a single haplotype diversity of the PC populations. sequence was each represented in 35 haplotypes, than shared haplotypes among the animals. The haplotypes In lineage A, haplotype H1 was the most predominant represented by one individual or shared by only a few haplotype with the highest frequency and could be samples were common since mtDNA variation is more considered the ancestral haplotype. In fact, haplotype frequent within breeds, especially in domestic goats H1 was found in 21 PC populations and with five direct (Naderi et al. 2007). The detection of single individuals mutational links to haplotypes H2, H3, H4, H5, and in haplotype suggested that little gene flow existed H13. Moreover, the haplotype H1 was linked to H10, among some PC populations. Therefore, the set of 107 B. which belonged to lineage B. A related study described bubalis mtDNA D-loop sequences from this study that haplotype that displayed affinity by mutations steps to was deposited in the NCBI GenBank would serve as a more than one haplotype and generally demonstrating reference dataset for future research on differentiating a high frequency would be referred to as interior haplotypes within and among PC populations. or ancestral haplotype (Posada and Crandall 2001). However, the non-significant neutral test values suggested that the Philippine swamp buffaloes included Implication to Conservation and Management in the study did not experience population expansion. The presence of two maternal lineages, A and B, would On the other hand, the intermixing of the individuals strengthen the rationale to enhance the in situ and ex from various populations of the ancestral haplotype H1 situ conservation and management of the PC. In in lineage A could probably reflect polymorphism in situ conservation entailed an institutional herd at the recently diverged wild water buffalo. In fact, a Thai PCC regional satellite at State University in wild buffalo (B. arnee) was reported to have sequence Cagayan. The other in situ conservation sites of swamp similarity using mtDNA D-loop with domesticated buffaloes are maintained by the farmers organized swamp buffaloes from the maternal lineage A from group in four sites – namely, Tabuk in Kalinga, Batanes Thailand, China, and the Philippines (Sarataphan et Island and Calayan Island in Cagayan, and Pres. Carlos al. 2017). Due to lacking Asian wild buffalo complete P. Garcia Island in Bohol (Cruz 2012; Paraguas et mtDNA D-loop sequences available in NCBI GenBank, al. 2018). The ex situ cryo-conservation through the partial D-loop sequences of this study did not allow Cryobanking facility serves as a biological vault of the further analysis to determine the relationship of PC animal genetic resources (AnGR) of water buffaloes with the Thai wild buffalo. However, the Thai wild and other economically important livestock. In fact, the buffalo (KU687004) D-loop sequence indicated AnGR are preserved for future animal management and more similarities with a Chinese swamp buffalo utilization (Villamor 2015). As to date, swamp buffalo (KX758304.1), which fell in haplotype H1 from lineage genetic resources for in situ and ex situ conservation A than swamp buffalo haplotypes from lineage B of and management are still limited to haplotypes belonged this study; this agreed with the previous study, which to maternal lineage A. Hence, the useful insights was reported by Sarataphan et al. (2017). Thus, the obtained on newly detected individuals from maternal evolutionary relationship between swamp buffaloes and lineage B would be highly recommended for strategic wild water buffaloes could be elucidated by following conservation and management program. The program the recommendation of Sarataphan et al. (2017) that entails collecting, cryopreserving, and storing viable larger representative samples could be considered to germplasm in frozen semen and embryos from swamp identify the genetic diversity and integrity of Thai wild buffaloes that belonged to maternal lineage B. The PC buffalo and other Asian wild buffaloes. genetic resources in the Cryobanking will be used for breeding programs in institutional herd and distribution The NJ, MJ, and population demography analyses to smallholder farmers for increased productivity of confirmed the unique haplotypes, indicating one swamp buffalo. individual in the haplotype. These were five haplotypes, including H12 to H16 – found in CAL, LA, ZAM, CPZ, Future population genetic research on B. bubalis swamp and BKD, respectively. The high unique haplotype of PC type in the Philippines should encompass a larger area was observed in Luzon and belonged to maternal lineage and number of local sampled individuals, particularly in A. The Philippine swamp buffaloes displayed a few Visayas and Mindanao populations. The genetic diversity of PC obtained from this study could be compared with

844 Philippine Journal of Science Villamor et al.: Genetic Diversity of Philippine Carabao Vol. 150 No. 3, June 2021 other mitochondrial genes such as cyt b and nuclear genes NOTES ON APPENDICES that would provide more significant genetic structure insights. It is recommended that additional research The complete appendices section of the study is accessible findings are needed to determine the relationship and at http://philjournsci.dost.gov.ph position of PC with other Asian origins.

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