19

External Characters and Karyotypes of the Captive Tamaraws, Bubalus (B.) mindorensis, at the Gene Pool in the Island of Mindoro, Philippines

Takao Namikawa 1), Joseph S. Masangkay 2), Kei-ichiro Maeda 1), Roberto F. Escalada3), Kanjyun Hirunagi 1) and Vicente G. Momongan4)

1) School of Agricultural Sciences , Nagoya University,

Chikusa, Nagoya 464-01, Japan. 2) College of Veterinary Medicine and 4)Institute of

Animal Science, University of the Philippines at Los

Bað¬os, College, Laguna 3720, the Philippines. 3) Tamaraw Conservation Program , San Jose, Occidental

Mindoro, the Philippines.

(Received December 12, 1994, Accepted January 5, 1995)

Abstract Measurement values of sixteen external characters and karyotypes were described for one female and five males of the adult captive tamaraws, Bubalus (B.) mindorensis endemic to the island of Mindoro. Principal component analyses among bubaline species, using nine or ten of the characters as variables, derived more than 95% of total variance on the first component axis, in which the tamaraw was located almost at the midpoint of two distant bubaline groups : the dwarf water buffaloes of Sulawesi (Anoa depressicornis and A. quarlesi) and the Asiatic water buffaloes (the swamp and the river types of Bubalus bubalis). All the six tamaraws had 46 chromosomes in diploid form (2N) and 58 as the fundamental number (FN) including the sex chromosomes. In contrast, all the bubaline species of Syncerus, Bubalus and Anoa were reported to have FN=60 with exception of the swamp type of the Asiatic buffalo (FN=58 but 2N 48). The X chromosome of the tamaraws was the largest acrocentric, and the Y was also acrocentric, probably among the smallest acrocentrics. The karyotype of the tamaraws differs from that of the swamp type buffalo both by one additional pair of metacentrics and the two decreased pairs of acrocentrics in the autosomes.

Key words : Bubalus mindorensis, karyotype, external morphology, tamaraw, water buffalo

Introduction Fischer & Hohn, 1976). This may partly be due to

The tamaraw (synonymous with tamarao, the shortage of biological characterization of this tamarau or tamarou, or Mindoro Buffalo) animal. In this paper, we follow the systematics inhabits nowhere except in the island of Mindoro, of Groves (1969 ; 1976), in which the tamaraw was the Philippines. Different scientific names have given the position of Bubalus mindorensis in the been given for the tamaraw by different authors subgenus Bubalus, not in the subgenus Anoa of such as Bubalus mindorensis in the subgenus the genus Bubalus.

Bubalus (Groves,1969 ; Nowak & Paradiso,1983), The tamaraw is the rarest wild bubaline species

B. arnee mindorensis (Klð±s & wð¹nschmann, on earth and has some characteristics similar to

1972), or Anoa mindorensis (Hediger,l965 ; the two species of Anoa, which are the smallest 20 J. Anim. Genet., 23(1): 19-28, 1995

Table 1. The adult captive tamaraws (Bubalus mindorensis) at the Gene Pool on the island of Mindoro , a the Philippines, whose external characters and karyotypes were examined.

a) Estimated by wear on the permanent incisors.

b)X•}SE in males.

Table 2. Measurements(cm) of external characters on the live tamaraws (Bubalus mindorensis) at the Gene Pool on the island of Mindoro.

a)See in the text for detailed measurement points .

b) Coefficient of variation (ƒÐ2n-1 /X) in males. c) Not measured. d)Circumference length . Namikawa et al., : Morphology and karyotype of the Tamaraw 21 bubaline species endemic to the island of Withers height : at the highest point above /

Sulawesi, Indonesia (Klð±s & Wð¹nschmann, around the scapulas, Hip height : at a midpoint

1972). The 1990 IUCN-Red List of Threatened between proximal ends of Spina iliaca dorsalis,

Animals of the Red Data Book categorized the Body length : horizontal distance from the frontal tamaraw in •gE•h next to •gEx•h. In the early 1900's, edge of the scapula to the caudal end of Os ischii in there were still 10,000 head of tamaraws but its parallel to the sagittal plane, Chest width : population declined tremendously in 1949 to 1,000 maximum distal distance between a pair of the head, and presently, the number of tamaraws has fifth costae, Chest depth : vertical distance from been reported to be about 200 to 300 head only the bottom of the fifth costae to the top of the back,

(Momongan & Walde, 1993). Conservation Chest girth : crossing both the fifth and sixth schemes have been conducted to increase the costae in a right angle to ground, Rump width : population size and to conserve their natural between the distal ends of Spina iliaca ventralis, habitats on the island of Mindoro under the Thurl width : between the distal ends of

Tamaraw Conservation Program (TCP) Acetabulum, Rump length : distance from the supported by the University of the Philippines at distal end of Spina iliaca ventralis to the caudal

Los Bað¬os Foundation Incorporated (UPLBFI) end of Os ischii, Cannon bone circumference : the and related organizations. The Gene Pool of the minimum on the mid-part of the metacarpals

TCP occupies an area of 280 ha of fenced (Mc3+Mc4), Head length : distance from the grassland, bush and forests. With the supports of muzzle to the midpoint of the parietale, Head the TCP of UPLBFI in 1993, we examined the width : maximum width between the processes of external characters and karyotypes of the captive Os temporale behind the orbital, Ear length : tamaraws in order to increase phylogenetical from the base to top behind the lobe, Horn knowledge on this animal. circumference : around the basement, Horn length

: the base to top along its outer curve, Tail length

:•@ the base to end.

Materials and Methods Of these sixteen characters, nine or ten were

Handling of animals : Measurement of external used in the principal component analysis (PCA) characters and blood collection were made in one to compare the tamaraws with other bubaline female and five males of adult tamaraws in species. captivity (Table 1). Prior to the examination, we treated each animal with anesthetic injection of Preparation of chromosome specimens : Each xylazine (Ceractal, Bayer) at a controlled nucleated cell layer on the red blood cells of 10 ml dosage determined by Masangkay et al. (1991 ; whole blood in a heparinized tube was transfered

1993), so that the animal could keep its standing into the culture vial containing Chromosome position as normal as possible but never lay Medium 4 (cat.no.124-1674A,GIBCO Labs.). This down. This pretreatment seemed to be essential procedure was carried out in a transparent vinyl for making measurements on the ferocious bag, that was sterirized and large enough in size tamaraw and to facilitate collection of to operate the non-contaminated blood samples noncontaminated blood from the jugular vein. inside. The cell vials were immediately cooled in

However we lost several measurement values for a box containing ice, and transported to the the animals (Table 2). laboratory of UPLB. The cell vials were put into

an incubator about 50 h after the blood sample

Measurements of external characters : Measure- collection. 15 min before termination of the 70 h ment points of the sixteen external characters on incubation at 37•Ž, 50ƒÊ 1 of warmed Colcemid the live animals (Table 2) are detailed as follows (GIBCO Labs.) was added to each vial, and

reincubated for an additional 2h. Further 22 J. Anim. Genet., 23(1): 19-28, 1995

procedure for the chromosome preparation agreed with those of Groves (1969). The tamaraw followed the direction given by GIBCO Labs. is much smaller and robuster than the large Asiatic water buffalo, with not only short thick horns directed more backward but shortened and Results and Discussion thickened limbs. The general shape of the horns, Morphological characterization : We examined with a considerable outward initial direction and one female and five male tamaraws in captivity, the stockier limbs of the tamaraw, are completely whose origin, approximate age and body mass different from the closely approximated and are shown in Table 1. All males were obviously backwardly directed horns and the thin limbs of fully matured but the female was two or three the anoas. years, when they were captured. Body mass of the The raw data of the sixteen external characters female seemed to be over-estimated, as compared of the tamaraws are tabulated since there has been to wild-grown tamaraws, due to her increased very little description on the live animals body mass with concentrated managements for (Table 2). The relatively large coefficients of the six years since she was captured. This variation (CVs) in the thurl width, cannon bone possibility is confirmed with the large chest girth circumference and head length of the males might of the female, compared to the constant values be partly due to difficulties in making accurate among the five males (Table 2). We may therefore measurements. But the large CVs in the horn regard the average body mass of the adult length, horn base circumference and tail length tamaraw to be about 274 kg in male and less than were based on reliable measurements. No 260 kg in the female (Table 1). Our general significant correlation coefficient was found observation of external features of the tamaraw between the horn length and the individual age in

Table 3. Comparison of measurements (X•}SE cm) of external characters among the bubaline species ;

Anoa quarlesi, A, depressicornis, Bubalus mindorensis, and the swamp and the river types of

the Asiatic water buffalo (B. bubalis).

a)Amano et al ., b)Present data, and c)Amano et al., 1976 a)See Table 2 . e) Estimated value based on correlation between WH and HH in the female individuals of A.quarlesi. f) No data available. Namikawa et al.,: Morphology and karyotype of the Tamaraw 23 males, nor between the horn base circumference of the tamaraws, as expressed by (WH-CD) / and their age. The measurement values for horn WH, were not significantly smaller than those of length seemed to become widely between the Asiatic water buffaloes. individuals by the tamaraw's habit of sharpening Table 4 and Fig.l show the results of PCA in the the horns frequently, however the horns of each three categories of the bubaline species, that is, animal were observed to be remarkably uniform male-female pooled (MFP), Female group (FG) in length. and male group (MG). The PCI summarised Measurements on the ten external characters more than 95 % of total variance in every category, examined in the tamaraws are compared with and the PCI and PCII derived more than 98% of the those available in other bubaline species cumulative variance from total variance in every (Table 3). The tamaraws showed all midvalues in category. The large eigenvalues of the PCI these characters, that is, larger than the anoa obviously indicate that in size is the most striking species and smaller than the Asiatic water morphological differentiation between the anoa buffaloes. Of the ten characters, the values of CW species, B. mindorensis and the Asiatic water and CB of the tamaraws were, however, relatively buffaloes (Table 4). This is further confirmed close to those of the large Asiatic water buffaloes with remarkably uniform eigenvectors (EVEs) in (Table 3). This may be a reason for the fact that the PCs for the ten characters (Table 4). Although the tamaraw gives an impression of being the PCII contributes little to the total variance, it robuster and stockier than the Asiatic water indicates the relatively wide chest width, thick buffaloes, and to have shortened and thickened cannon bone and deep chest depth of the tamaraws limbs in contrast to the very thin cannon bones of with their large absolute eigenvector values ; EVE the anoa species (Table 3), as also mentioned by of CW = 0.523 in MFP, EVE of CB = 0.593 in FG, Groves (1969). But the proportion of limb length and EVE of CD = -0.543 in MG, respectively

Table 4. Result of principal component (PC) analysis based on the correlation matrix of the body- measurement variables in the bubaline species in Table 3. 24 J. Anim. Genet., 23(1): 19-28, 1995

Fig. 1. Morphological comparison of Bubalus and Anoa species on the axes for the first (I) and second (II) principal component scores, derived by the principal component analysis with the nine or ten external characters as variables (Table 3). Top : male-female pooled (MFP), Middle: Female group (FG), Bottom : male group (MG). Aq : A, quarlesi, Ad : A. depressicornis, Bm : B. mindorensis, BS : the swamp type of the Asiatic water buffaloes (B. bubalis), BR : the river type of B. bubalis.

(Table 4 and Fig.1) . The PCA indicated that the chromosomes were found in all the metaphase tamaraw has a medium body size and a relatively spreads of the female (Table 5). This large stocky shape in comparison with the anoa species acrocentric chromosome was then determined to and the Asiatic water buffaloes. be the X chromosome of the tamaraw. The karyotypes of the six tamaraws are shown in Karyotypes : We made ten or more preparatory Fig.2. The twelve uniarmed autosomes comprised photoprints of the metaphase spreads of the six of four pair of metacentrics and two pair of tamaraws to determine the diploid number and to submetacentrics. The fundamental number of the roughly classify the chromosomes according to chromosomes (FN) was therefore fifty-eight in shape (Table 5). The mode of chromosome both sexes, including the pair of acrocentric sex numbers was forty-six in diploid, twelve for the chromosomes (Table 6). biarmed chromosomes, and thirty-four for the Fischer & Hohn (1976) examined the karyotype uniarmed or acrocentric chromosomes in most of a female tamaraw and indicated the same metaphase spreads of each animal. This set was chromosome composition as in the present female therefore concluded to be a basic chromosome tamaraw. The present work confirmed the largest composition of the tamaraw. Among the acrocentric chromosome to be the X chromosome uniarmed chromosomes, a significantly large by comparing the chromosome sets of the female sized acrocentric chromosome was recognized in and the five males. The Y chromosome was all of the five males and, also, a pair of such apparently acrocentric, but we did not identify Namikawa et al., : Morphology and karyotype of the Tamaraw 25 directly by using banding methods because of the the great variation in 2Ns in thoes species. But, limited experimental condition. there are two exceptional cases ; one is the swamp The X chromosome was the largest of the type of the Asiatic water buffalo, B. bubalis acrocentrics and the Y was the smallest or a small (Fischer & Ulbrich, 1968; Di Berardino & acrocentrics in all the bubaline species according Iannuzzi, 1981) and the other is the tamaraw, B. to previous reports on the two types of Bubalus mindorensis (Fischer & Hohn, 1976; the present bubalis (Fischer & Ulbrich,1968 ; Ulbrich & data). Both of these species have fifty-eight in the Fischer, 1968; Di Berardino & Iannuzzi, 1981), FNs. But, the karyotype of the tamaraws the two species of Anoa (Amano et al., 1983 ; obviously differs from that of the swamp type of Schreiber et al., 1993), and the two subspecies of the Asiatic water buffalo because the tamaraw has Syncerus (Heck et al., 1968; Ulbrich & Fischer, six pairs of biarmed and sixteen pairs of 1967). acrocentric autosomes (Table 6), while the The above authors reported that the FNs, swamp type buffalo has five pairs of biarmed and including the pair of acrocentric sex chromoso- eighteen pairs of acrocentric autosomes. The mes, were sixty in those bubaline species inspite of difference may suggest a single event of

Table 5. Distribution of chromosome numbers counted on the preparatory photoprints of the metaphase spreads from the six tamaraws (Bubalus mindorensis).

a)Number of uniquely large acrocentric chromosomes found among the uniarmed chromosomes , which were considered to be the X-chromosome. 6 J. Anim. Genet., 23(1): 19-28, 1995

Fig. 2. Chromosome sets of the five male and one female tamaraws (Bubalus mindorensis) in captivity at the Gene Pool in the island of Mindoro, the Philippines. M, metacentric ; S, submetacentric; A, acrocentric autosomes. The largest acrocentric chromosome was determined to be the X, and the Y was considered to be one of the smallest acrocentric chromosomes as in all species of Syncerus, Bubalus and Anoa previously reported (Ulbrich & Fischer, 1967 ; Fischer & Ulbrich, 1968 ; Di Berardino & Iannuzzi, 1981 ; Amano & Martojo, 1983 ; Schreiber et al., 1993).

Table 6. The chromosome sets determined in the captive tamaraws, Bubalus mindorensis.

a) Fundamental number including the sex chromosomes. b)The largest acrocentric chromosome . e) Probably one of the smallest acrocentric chromosomes according to reports on other bubaline species (see in the text for detail). Namikawa et al., : Morphology and karyotype of the Tamaraw 27

chromosomal fusion or fission occurred between References the tamaraw and the swamp type buffalo, but it is Amano, T., & H. Martojo, 1983. Karyotypes of ambiguous because, unfortunately, the present water buffaloes and anoas. Rep. Soc. Res. karyotypic analysis did not confirm homologies Nat. Livest.,10 : 98-110. of biarmed chromosomes between the two Amano, T., T. Namikawa, T. I. Azmi, & H. animals. It is however noticed that all the Hilmi, 1976. Body measurements of water tamaraws have a pair of the large metacentric buffaloes in Malaysia. Rep. Soc. Res. Nat. autosomes that are much bigger than the X Livest., 7:93-95. chromosome (Fig.2). This very similar type of Di Berardino,D., & L.Iannuzzi,1981. Chromoso- autosomes was also found in the swamp type me banding homologies in Swamp and buffalo but not in the river type of the Asiatic Murrah buffalo. J. Hered., 72: 183-188. buffaloes (Fischer & Ulbrich, 1968; Ulbrich & Fischer, H., & H. Hohn, 1976. Der Karyo-

Fischer, 1968 ; Di Berardino & Iannuzzi, 1981). typeines weiblichen Tamarau (Anoa

Kakoi et al. (1994) showed two and five amino mindorensis). Giess. Beitr. Erbpathol. acid substitutions in the hemoglobin ƒÀ chains of Zð¹cht. hyg. , 6: 173-177. the two species of Anoa in comparison with those Fischer, H., & F. Ulbrich, 1968. Chromosomes of the two types of the Asiatic water buffaloes but of the Murrah buffalo and its crossbreds found no difference between the two types of water with the Asiatic swamp buffalo (Bubalus buffaloes. bubalis). Z.Tierzð¹cht.Zð¹cht.biol., 84:110-

The unique karyotype of the tamaraw may 114. support the view that it should be given a distinct Groves, C. P., 1969. Systematics of the Anoa taxonomic position at least at the species level in (Mammalia, Bovidae). Beauf ortia, 17: 1- the subgenus Bubalus. The present analyses of the 11. external characters and karyotypes of the six Groves,C.P., 1976. The origin of the mammalian tamaraws and the previous studies on bubaline fauna of Sulawesi(Cerebes). Z. Sð ugetierk., species elsewhere do not contradict the Groves' 41: 201-216. systematics based on morphology (Groves, 1969; Heck, H., D.Wulster, & K.Benirschke, 1968.

1976) that classifies the tamaraw into a separate Chromosome study of members of the

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Bubalus. Musk Ox, Ibex, Aoudad, Congo Buffalo

and Gaur. Z. Sð ugetierk., 33: 172-179.

Hediger,H., 1965. Der Mindoro-Buffel, das

Acknowledgments seltenste Wildrind. Z.Sð ugetierk., 30: 249-

The authors thank Drs. M. F. Manuels of CVM 253.

of UPLB, C.B.UIep of Protected Areas and Kakoi,H., T.Namikawa, O.Takenaka, A.

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SEARCA, the Philippines, and Dr. T. Tomita of Divergence between the anoas of Sulawesi

Nagoya University, for their invaluable and the Asiatic water buffaloes, inferred

suggestion to complete the present work. We also from their complete amino acid sequences of

thank H. Nagabukuro and K. Tanaka of Nagoya hemoglobin ƒÀ chains. Z. zool. Systemat.

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