Identification of Tamaraw (Bubalus Mindorensis) from Natural

Animal Science Journal (2010) 81, 635–641 doi: 10.1111/j.1740-0929.2010.00794.x

ORIGINAL ARTICLE Identiﬁcation of tamaraw (Bubalus mindorensis) from natural habitat-derived fecal samples by PCR-RFLP

analysis of cytochrome b geneasj_794 635..641

Shinya ISHIHARA,1 Rommel J. HERRELA,2 Daichi IJIRI,1 Hisashi MATSUBAYASHI,3 Miho HIRABAYASHI,1 Arnel N. DEL BARRIO,2 Rodel M. BOYLES,4 Medardo M. EDUARTE,5 Ronilo L. SALAC,6 Libertado C. CRUZ7 and Yukio KANAI1 1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 2Philippine Carabao Center – University of the Philippines at Los Baños, Laguna, 3Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia, 4Tamaraw Conservation Program, Department of Environment and Natural Resources (DENR), 5Protected Areas and Wildlife Bureau (PAWB), DENR, Quezon City, 6Provincial Environment and Natural Resources Ofﬁce Region IV, DENR, Occidental Mindoro and 7Philippine Carabao Center, National Headquarters, Munoz, Nueva Ecija, Philippines

ABSTRACT

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit-Baco National Park and species-known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species-unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus- and tamaraw-specific primers for PCR of cytochrome b gene were newly designed. The Bubalus-specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw-specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR-RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR-RFLP analysis also showed that the species-unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.

Key words: cytochrome b, fecal DNA, species identiﬁcation, tamaraw.

INTRODUCTION tinuing decline of 25% over the next three generations Tamaraw (Bubalus mindorensis) is a wild dwarf buffalo or the next 30 years (Hedges et al. 2008). endemic to the Philippine island of Mindoro. It was The population of tamaraw in the early 1900s was formerly abundant and well-distributed across the estimated to be around 10 000 individuals across the island, but its current range is restricted to three small whole of Mindoro Island, but over-hunting and protected areas: Mount Iglit-Baco National Park exploitation of their habitat with an increasing human (MIBNP), Mount Aruyan and Mount Calavite population resulted in a drastic decline of the tamaraw Tamaraw Preserve in Occidental Mindoro (Cox & population to less than 1000 in 1949 (Harrison 1969). Woodford 1990). International Union for Conserva- To save the tamaraw from extinction, the Philippine tion of Nature and Natural Resources (IUCN) ﬁrst cat- Government prohibited by law the killing, wounding egorized tamaraw as an endangered species in 1986, and then as a critically endangered species in 2000. Correspondence: Yukio Kanai, University of Tsukuba, Recent estimation of tamaraw populations by IUCN Tsukuba 305-8572, Japan. (Email: kanaiy@sakura. indicates that the number of mature individuals is less cc.tsukuba.ac.jp) than 250 with more than 90% of individuals assigned Received 10 September 2009; accepted for publication 25 to the MIBNP subpopulation, giving an estimated con- January 2010.

© 2010 The Authors Journal compilation © 2010 Japanese Society of Animal Science 636 S. ISHIHARA et al. or taking away of tamaraw from their habitat as early designed genus Bubalus-specific or tamaraw-specific as 1936, and also initiated official conservation mea- primer pair can amplify the tamaraw cytochrome b sures called the Tamaraw Conservation Program (TCP) gene fragment, and the combination of PCR and in 1979, which consists of three major components, restriction fragment length polymorphism (RFLP) namely, conservation of habitat, captive breeding and analysis with restriction enzymes could successfully education of the populace in cooperation with various discriminate tamaraw from domestic buffalo. non-government organizations (NGOs) and Depart- ment of Environment and Natural Resources of the MATERIALS AND METHODS Philippines, or DENR (Caringal-Pangga et al. 1994). Despite such conservation efforts, the tamaraw popu- Fecal sampling lation still remains low and up to now only limited Fecal samples from putative tamaraw (Bubalus mindorensis), information has become available regarding ecological domestic buffalo (Bubalus bubalis) and cattle (Bos taurus) were behavior of tamaraw and tamaraw–human conflict used in the present study. Eighteen fecal samples presumed to be from the tamaraw by its size and shape were collected (Maala 2001). One reason for these constraints could at 11 different observation sites within the tamaraw conser- be the difficulty in accessing their habitat and in sight- vation area (approximately 16 000 ha) of MIBNP in April, ing tamaraw in the field. The tamaraw are known to 2007. Species-known fecal samples were collected from each be fierce, nocturnal and wary animals, and therefore it two individuals of swamp buffaloes, crossbred buffaloes is difficult to directly observe them in the daytime (swamp ¥ river) and cattle (crossbred Brahman) in the Phil- (Kuehn 1977, 1986). At present, confirmed sightings ippine Carabao Center, University of Phillipines at Los Banos are largely limited to MIBNP where the vegetation is (PCC-UPLB), immediately after sighting of defecation from each animal. Additionally, one species-unknown fecal dominated by grasses with small scattered forest areas sample obtained at the ridge area of Mount Aruyan Preserve (Ishihara et al. 2007). In contrast to MIBNP, little infor- in our previous study (Matsubayashi et al. 2009), was also mation is available for the other two protected areas of included to determine the host species of this fecal sample at Mount Aruyan and Mount Calavite where human– the DNA level. The collection site of this sample is the eastern tamaraw conflict or the dense secondary forest makes part of Aruyan near the Buayan and Kapihan creeks (altitude the sighting of tamaraw more difficult (Matsubayashi range 210–350 m), where the sighting of tamaraw has not been reported in recent years. et al. 2009). Intestinal epithelial cells on the surface of feces were col- As the technique of molecular analysis has devel- lected using a sterilized cotton swab, and then dipped into oped, DNA became a useful tool in conservation 1 mL of lysis buffer (White & Densmore 1992) in a 1.5 mL biology and wildlife management in terms of acquiring micro-test tube. The lysis buffer contained 0.5% sodium valuable information such as behavioral and ecological dodecyl sulfate (SDS), 100 mmol/L ethylenediaminetetra- features. Mitochondrial DNA (mt-DNA) is known to acetic acid (EDTA: pH 8.0), 100 mmol/L Tris-HCl (pH 8.0), evolve much faster than nuclear DNA and thus con- and 10 mmol/L NaCl. All tubes with samples were trans- ferred to PCC-UPLB and kept in a regulator at 4°C until tains more sequence diversity compared to nuclear analyzed. DNA, facilitating the identification of closely related species (Brown et al. 1979, 1982). Above all, the DNA preparation nucleotide sequence of the cytochrome b gene on DNA extraction was conducted using the standard method of mt-DNA which contains species-specific information proteinase K digestion, phenol/chloroform extraction, and has been used in forensic analysis in a number of precipitation with ethanol by Sambrook et al. (1989). Pro- studies (Irwin et al. 1991; Smith & Patton 1991; Ces- teinase K and NaCl were added into the fecal sample tube to pedes et al. 1998). Tanaka et al. (1996) successfully reach a final concentration of 250 mg/mL and 0.15 mol/L, sequenced the whole cytochrome b gene (1140 bp) respectively, and incubated for 1 h at 55°C and then for 16 h using blood samples from captured tamaraw and dem- at 37°C. After removing the residue of the feces and centrifu- gation (25 000 ¥ g, 5 min at 4°C), DNA was extracted and onstrated that tamaraw is phylogenetically closer to purified as described by Sambrook et al. (1989). The swamp buffaloes than riverine buffaloes. Furthermore extracted DNA was dissolved in 50 mL Tris-EDTA buffer (each it has been shown that non-invasive samples such as 1 mmol/L of Tris-HCl and EDTA, pH 8.0). feces are a valuable source for mt-DNA analysis (Höss et al. 1992; Constable et al. 1995). Paxinos et al. (1997) Oligonucleotide primers discriminated fecal DNA of sympatric canid species In order to establish a method for species identification using using three restriction enzymes on cytochrome b gene. fecal DNA, the genus Bubalus-specific primer pair and Meanwhile, Kurose et al. (2005) distinguished carni- tamaraw-specific primer pair for PCR were designed based vore species using fecal DNA amplified with a species- on the mtDNA cytochrome b gene. Sequences of whole cyto- specific primer on PCR. chrome b gene regions (1140 bp) from different species were referred to the GenBank database and Tanaka et al. (1996) The object of this study was to establish the method (GenBank accession; Bos taurus, D34635; Bubalus bubalis, for identifying tamaraw from its close relative species D82894; Bubalus mindorensis, D82895). Primers for amplify- by using cytochrome b gene extracted from outdoor- ing cytochrome b gene of the genus Bubalus and the species derived fecal samples. We present here that the newly tamaraw were designed on the mutated regions between the

© 2010 The Authors Animal Science Journal (2010) 81, 635–641 Journal compilation © 2010 Japanese Society of Animal Science IDENTIFICATION OF TAMARAW FROM FECAL SAMPLES 637 three species as indicated in Figure 1. The genus Bubalus- RESULTS specific primer, PBb-CbF (5′-CATTCATTGACCTCCCTGCT- 3′)/PBb-CbR (5′-GGCTGTCCTCCAATTCATGT-3′), and the Sequencing of cytochrome b gene tamaraw-specific primer, PBm-CbF (5′-GCACAAACCTAG The 976 bp fragment of cytochrome b was successfully TTGAGTGA-3′)/PBm-CbR (5′-CGATGGTAATATATGGGTGT amplified for all fecal samples, from both the putative ′ TCG-3 ), were newly designed. Expected amplified products tamaraw and domestic buffalo, by the genus Bubalus- for PBb-CbF/PBb-CbR and PBm-CbF/PBm-CbR were 976 bp and 582 bp in length, respectively. Additionally, a universal specific primer pair (PBb-CbF/PBb-CbR). However, primer pair for cytochrome b gene (Irwin et al. 1991), due to sequence ambiguity at the end of some ampli- L14724B (5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′)/ fied fragments, only 854 bp (position 113 to 966) of H15915R (5′-GGAATTCATCTCTCCCGGTTTACAAGAC-3′), 976 bp fragments of PCR products were used in the was also used as a positive control for PCR amplification. analysis. The nucleotide sequence of the species- unknown sample collected in Mount Aruyan Preserve PCR showed complete homology to the sequence of The final PCR mixture contained 0.4 mL EX Taq polymerase tamaraw cytochrome b reported by Tanaka et al. (Takara Bio Inc., Tokyo, Japan), 2 mLof10¥ EX taq buffer (1996), while all of the putative tamaraw samples (Takara), 1.6 mL of deoxynucleotide triphosphate (dNTP: from MIBNP showed single nucleotide transition 2.5 mmol/L of each dNTP; Takara), 1.2 mLof10mmol/L of → each primer and l.6 ng DNA, in a total volume of 20 mL. PCRs (T C) at position 255 bp (registered in DDBJ with were performed in an automated DNA thermal cycler accession no. AB526220). (TP600; Takara). The thermal cycling was performed by As for the sequence of cytochrome b in domestic initial denaturation at 98°C for 10 s, followed by 30 cycles of buffalo, one sequence from crossbred buffalo had a denaturation at 98°C for 30 s, primer annealing at 65°C for single nucleotide mutation (T→C) at position 471 bp. 30 s, and extension at 72°C for 2 min. The PCR products This mutation (registered in DDBJ with accession no. (9 mL) were examined by electrophoresis for 20 min in 1% AB529514) was not matched with any haplotype pre- agarose gel, stained with ethidium bromide, visualized on a UV trans-illuminator, and photographed with a Polaroid viously reported in the GenBank database. The other camera (FAS-III, Toyobo, Tokyo, Japan) three domestic buffaloes showed entirely the same sequence in the database (GenBank accession D34637 Direct sequencing for one buffalo, and FJ556563 for the other two PCR products from 18 putative tamaraw feces from MIBNP, a buffaloes). species-unknown feces from Mount Aruyan Preserve and four domestic buffalo feces were subjected to direct sequenc- PCR with the genus Bubalus or ing to confirm whether the products actually matched with tamaraw-specific primer cytochrome b gene for each species. A fragment of cytochrome b for sequencing was obtained by a PCR with PBb- The PCR amplification of a fragment of the cyto- CbF/PBb-CbR primer pair. Each PCR product was mixed chrome b gene by different primer pairs in represen- with 1 mL dye terminator (Big Dye Terminator, Applied Bio- tative samples are shown in Figure 2. The universal systems, Tokyo, Japan), 1 mL primer (3 mmol/L PBb-CbF/ primer pair (L14724B /H15915R) yielded an approxi- PBb-CbR), 6.5 mL distilled purified water and 1.5 mL mate 1200 bp fragment in all fecal samples irrespective sequencing buffer. PCR conditions were: initial denaturation of the species (Fig. 2a), whereas the genus Bubalus- at 96°C for 3 min followed by 25 cycles of denaturation at specific primer pair (PBb-CbF/PBb-CbR) allowed PCR 96°C for 10 s, and annealing at 50°C for 5 s and at 60°C for 4 min. The PCR products were subjected to capillary gel amplification of the 976 bp fragment of cytochrome electrophoresis in an ABI Prism 310 genetic analyzer b only for the samples from domestic buffaloes and (Applied Biosystems), according to the manufacturer’s putative tamaraw (Fig. 2b), On the other hand, the instructions. tamaraw-specific primer pair (PBm-CbF/PBm-CbR) yielded the 582 bp fragment for all 18 fecal samples PCR-RFLP analysis from putative tamaraw and one out of four fecal For PCR-RFLP, two restriction endonucleases, AvrII (Takara) samples from crossbred buffaloes (Fig. 2c). and BsaXI (New England Biolabs Inc., Tokyo, Japan) were used. AvrII recognizes the 5′-CCTAG-3′ and BsaXI recognizes RFLP of PCR product the 5′-13(N)AC(N)5CTCC(N)11-3′ (Fig. 1). The PCR products amplified with PBb-CbF/PBb-CbR were used for digestion. The restriction endonuclease digestion of the PCR Digestions were carried out in 50 mL volumes using reaction products with AvrII yielded two distinct fragments conditions recommended by the manufacturer. The restric- (589 and 387 bp) for domestic buffalo feces, but its tion products were separated on 2% agarose gel by electro- restriction site was not present for putative tamaraw phoresis, visualized with ethidium bromide staining, and feces (Fig. 3. upper panel). Another restriction endo- sized with reference to a DNA size marker. Expected digested nuclease BsaXI resulted in profiles that distinguished products by AvrII were two fragments of 589 and 387 bp for all of the putative tamaraw from domestic buffaloes, domestic buffaloes and one undigested fragment of 976 bp for tamaraw, while those by BsaXI were three fragments of visualizing two bands each with different sizes as 679, 267 and 30 bp for domestic buffalo and five fragments of shown in the lower panel of Figure 3 (679 and 267 bp 367, 282, 267, 30 and 30 bp for tamaraw. fragments for domestic buffaloes and 367 and 282/

Figure 1 Figure 1, Whole cytochrome b (1140 bp) sequence from three bovidae species referred to Genbank; tamaraw (Bubalus mindorensis), D82895; domestic buffalo (Bubalus bubalis), D82894; and cattle (Bos taurus), D34635. The sites of each primer are surrounded by a square. The genus Bubalus-speciﬁc primer, PBb-CbF/PBb-CbR, and the tamaraw-speciﬁc primer, PBm-CbF/PBm-CbR, were newly designed. Endonuclease restriction sites of AvrII and BsaXI are underlined and the cutting locus is shown by a vertical line. Arrows indicate a mutation of tamaraw at 255 bp and domestic buffalo at 471 bp.

chrome b gene fragment from outdoor-derived fecal samples, and that RFLP analysis of the PCR products with restriction enzymes, AvrII or BsaXI, could accu- rately discriminate tamaraw from domestic buffalo and cattle. To design tamaraw-specific PCR primers for the cytochrome b gene, two sites were selected where the nucleotides sequence of tamaraw differed from both domestic buffalo and cattle. The newly designed primer set successfully amplified the expected DNA fragments in all 18 fecal samples of putative tamaraw, but also reacted to one of the four domestic buffalo feces. Sequences with a nucleotide mutation at the 3′ end are often chosen for species-specific primer designing; however, in the present study there was a lack of nucleotide difference at the 3′ end between the two species, which could have reduced the specificity Figure 2 Electrophoretic analysis (1 % agarose) of PCR of the tamaraw-specific primers. This should be con- products of the cytochrome b amplified with L14724B/H15915R (a), PBb-CbF/PBb-CbR (b), and firmed by further analysis. Degeneration may have PBm-CbF/PBm-CbR (c). Samples 1 and 2 = cattle (Bos taurus), also affected the specificity of the fecal DNA PCR 3 and 4 = domestic buffalo (Bubalus bubalis), and 5 and amplification, as it has been shown that damaged DNA 6 = tamaraw (Bubalus mindorensis). M = molecular weight reduces the fidelity of Taq DNA polymerase (Sikorsky marker 100 bp ladder. et al. 2007). Kurose et al. (2005) successfully discriminated the four carnivore species of the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus) from fecal samples, using a species-specific primer on PCR to amplify cytochrome b fragments. They also found that in a small number of the samples, a PCR band appeared in a different species-specific primer pair in addition to a band in one species- specific primer pair, and they recommended using other PCR primers simultaneously to guarantee high accuracy. Nevertheless, PCR for cytochrome b fragment with a tamaraw-specific primer pair used in the present study can be a useful tool for the species identification of tamaraw, since it is rare for tamaraw to Figure 3 Electrophoretic analyisis (2% agarose) of PCR share their habitat with domestic buffaloes. This is products of the cytochrome b amplified with supported by a recent report that indigenous peoples PBb-CbF/PBb-CbR and digested with AvrII (upper) and BsaXI of Mangyan are living within the vicinity of tamaraw (lower). Samples 1 and 6 = undigested sole PCR product, 2 to 5 = domestic buffalo (Bubalus bubalis), 7 to 10 = tamaraw conservation areas and they still conduct slash-and- (Bubalus mindorensis), and 11 = species unknown fecal burn farming with no domestic animals (Matsubayashi sample collected in Aruyan area. M = molecular weight et al. 2009). marker 100 bp ladder. The method to identify a species from fecal samples requires high accuracy. Restriction enzyme digestion of PCR-amplified species-specific mtDNA has been 267 bp fragments for tamaraw). The PCR product from widely used for species identification in a variety of the species-unknown fecal sample from Mount animals such as canid species (Paxinos et al. 1997), Aruyan Preserve showed the same band patterns as flatfish species (Cespedes et al. 1998) and Chinese alli- the putative tamaraw feces from MIBNP after treat- gators (Yan et al. 2005). In the present study, the genus ment with AvrII or BsaXI. Bubalus-specific PCR primer pair for a 976 bp fragment of cytochrome b was able to discriminate domestic buffalo and tamaraw from cattle, and restriction DISCUSSION enzyme digestion of the PCR products with AvrII or Results of the present study demonstrate that both the BsaXI could identify tamaraw in all fecal samples genus Bubalus-specific and tamaraw-specific primer which were presumed to be from tamaraw. Thus, the pair could successfully amplify the tamaraw cyto- methods developed in the present study enabled the

Animal Science Journal (2010) 81, 635–641 © 2010 The Authors Journal compilation © 2010 Japanese Society of Animal Science 640 S. ISHIHARA et al. species identification of tamaraw from outdoor- Tamaraw may have already been affected by the derived fecal samples with high precision. The rate of bottleneck effect or by inbreeding because of their successful PCR amplification for outdoor-derived fecal small population size. If we can collect putative samples in the present study (100%, 18/18 samples) is tamaraw feces on a large scale, it may be possible to higher than that (80%, 24/30 samples) reported in investigate any inbreeding effects, after the species sympatric carnivore species by Kurose et al. (2005). identification of fecal samples and amplification of Since fecal DNA is often damaged by ultraviolet rays, target DNA markers such as 12sRNA, D-loop and mic- the time process and environmental conditions before rosatellites which can be used for genetic diversity fecal sampling might affect the success rate of PCR analysis. amplification. In the present study, relatively fresh Poaching by outsiders, not by indigenous people, is feces were sampled to minimize possible DNA damage. still a serious threat to tamaraw, and it has been pre- Further study is recommended to determine how time sumed that tamaraw meat can be traded on the black and environmental factors affect the success rate of market at a high price, two to three times the price of PCR amplification from fecal samples. buffalo meat (Matsubayashi et al. 2009). The method Fecal DNA analysis enables us to acquire valuable of species identification developed in this study can be information for the conservation of endangered theoretically applied to any tissues of tamaraw. There- animals, namely, phylogeny (Irwin et al. 1991; Smith fore, it may also be a useful tool to intensify govern- & Patton 1991; Hammond et al. 2001), population mental regulation against the poaching of tamaraw genetics (Constable et al. 1995; Nielsen et al. 2008), and smuggling of tamaraw meat, as has been applied distribution (Kurose et al. 2005) and feeding behavior in other endangered animal species (Fang & Won (Höss et al. 1992). Currently, the habitat range of 2002; Yan et al. 2005). tamaraw is isolated into three regions in the Occiden- tal Mindoro, and the largest population has been found in the tamaraw conservation area of MIBNP ACKNOWLEDGMENTS where annual population surveys by simultaneous We sincerely thank Ms. Josefina L. de Leon in DENR- multiple vantage-point counts has been undertaken as PAWB for her dedicated support to the conclusion of a part of TCP operation under the assignment of DENR the Memorandum of Agreements (MOA) between since 1999 (Ishihara et al. 2007). However, only PCC and DENR, and the technical staff of the PCC/ limited information is available in the other two DENR and the tamaraw rangers in MIBNP/Mount tamaraw habitats, due to the difficulty in accessing Aruyan Preserve for their technical support to our field these locations and in sighting tamaraw (Hedges et al. work. The study was reviewed and approved under 2008). Recently, Matsubayashi et al. (2009) succeeded the MOA, and partly supported by a Grant-In-Aid for in identifying one male tamaraw by camera trap in Scientific Research (No. 17405004 and no. 21274) Mount Aruyan Preserve. They also observed some from the Japan Society for the Promotion of Science signs of tamaraw presence such as footprints, foraging to YK. prints and fresh feces, but failed to obtain actual sightings. Fecal DNA analysis to identify tamaraw estab- REFERENCES lished in the present study makes it possible to screen large areas of habitat for the presence of tamaraw by Brown WM, George M, Wilson AC. 1979. Rapid evolution of collecting feces. In fact, the present study confirmed an animal mitochondrial DNA. Proceedings of the National Academy of Sciences 76, 1967–1971. unidentified fecal sample collected in Aruyan area as Brown WM, Prager EM, Wang A, Wilson AC. 1982. Mito- originating from tamaraw. 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