J. Agric. Sci., Tokyo Univ. Agric.

, 66 (3), 65-74 (2021)

東京農大農学集報，66（3），65-74（2021）

Genetic Divergence and Phylogenetic Relationship

of Philippine Carabao (Bubalus bubalis) and Other
Swamp Buffalo Populations in Neighboring
Countries Revealed by the Mitochondrial DNA
D-loop Region
By
Lilian P. Villamor*, Koh Nomura**, Takashi Amano*** and Yukimizu Takahashi** †
(Received May 20, 2021/Accepted September 14, 2021)

Summary：The swamp buffalo (Bubalus bubalis) provides a major source of draft power in cultivating
rice farms and has great potential for meat, milk, and hide sources, particularly to smallhold farmers. To
date, there is limited published information on mtDNA D-loop sequence variation, genetic divergence, and
genetic relationship between the Philippine carabao and other swamp buffalo populations in Asia. Thus,
the objective of the study is to determine the phylogenetic relationship among the swamp buffalo popula-
tions with Asiatic origins. Dataset construction and data analyses were performed from 307-bp of 367
mtDNA D-loop sequences of swamp buffaloes from the Philippines and Asian countries which were
retrieved from NCBI GenBank. The research findings highlighted the genetic relationship among the
modern Asian swamp buffalo populations and could be explained in two points : First, the Philippine
carabao (native swamp buffalo), particularly the Visayas and Mindanao populations, had the closest
affinity to Taiwan swamp buffalo population based on the low pair-wise distance (FST), and, second, the
Chinese swamp buffalo could be the ancestral population of the modern population of the swamp buffaloes
in Asia, as inferred by mtDNA haplotype phylogenetic tree. The results of this research agreed with the
hypothesis of Lau et al. 1998 that after the domestication in the China region, the domesticated swamp
buffalo spread with rice farming into Taiwan to the Philippines and the eastern islands of Borneo and
Sulawesi. This study revealed that native buffaloes in the Philippines have various genes of buffaloes of
which numbers are declining in Southeast Asian countries and showed that it is necessary to conserve
and maintain them as valuable genetic resources while utilizing them in the Philippines.

Key words：genetic differentiation, mtDNA D loop, native carabao, phylogenetic relationship

cultivating rice farms and has great potential for meat,

1. Introduction
milk, and hide sources, particularly to smallhold farmers3, 7）.
The domestic swamp buffalo (Bubalus bubalis) is wide- In the Philippines, the swamp buffalo is considered the
spread and contributes tremendous economic importance national animal and is locally recognized as the Philippine
to several Asian countries. This species is widely dis- carabao (PC) or native carabao. The Philippine buffalo
tributed in the Philippines, China, Laos, Thailand, Taiwan, population between 1998 and 2018 had decreased by 130,
Vietnam, Indonesia, Myanmar, and Malaysia 1-6）. The 345 heads and at a rate of －0.216% yearly (Retrieved from
swamp buffalo provides a major source of draft power in http://www.fao.org /faostat/en/#data/QA on 21 January

* Department of Agriculture, Philippine Carabao Center-National Headquarters and Gene Pool, Science City of Munoz, Nueva Ecija,
3120 Philippines
** Department of Animal Science, Faculty of Agriculture, Tokyo University of Agriculture
*** Professor Emeritus, Tokyo University of Agriculture
†
Corresponding author（E-mail : [email protected]）
 66 Villamor, Nomura, Amano and Takahashi

2021)8）. These animals are non-descript between river or

2. Materials and Methods
swamp buffalo and commonly raised in the backyard or
at commercial farms. Thus, this becomes part of the 2. 1Sample Collection
priority to conserve and manage the water buffaloes, A total of 376 D-loop sequences of swamp buffaloes from
particularly the swamp buffaloes, across the major island the Philippines and Asian countries were retrieved from
groups in the Philippines. The archipelagic features of NCBI GenBank. The 107 sequences from Philippine carabao
the Philippines consist of Luzon, Visayas, and Mindanao, previously submitted to NCBI GenBank (MT642457-
which are situated in the northern, central, and southern MT642563) were retrieved and included in the analysis.
parts of the country, respectively. Luzon is considered These samples were grouped within the original areas of
the largest island group, the Visayas is the third major the sub-populations in Luzon, Visayas, and Mindanao.
island group, and the Mindanao is the second-largest The detailed information of the samples and the down-
landmass group of the country in terms of land area stream molecular analysis were described in Villamor et
(https://www.britanicca.com/place/Philippines). al.14）. A further 269 reference D-loop sequences were
Previous studies reported the genetic diversity, phylo- also retrieved from NCBI GenBank (Table 1). These
genetic, and phylogeography analyses of swamp buffaloes were represented by swamp buffaloes from the Asia
in Asia using diverse mitochondrial DNA (mtDNA) region, including China, Myanmar, Laos, Thailand,
markers. For instance, genetic diversity of mitochondrial Vietnam, Taiwan, and Malaysia6, 9, 10, 15, 16）. Additional
cytochrome b (cyt b) and mtDNA D-loop genes in Chinese D-loop sequences from two river buffaloes were included
native buffalo reported the two maternal lineages A and in the analysis to be used as outgroups (AF197209.1 and
B1, 4, 9, 10）. The closer genetic affinity between the Indone- AF475218.1).
sian and Philippine swamp buffalo groups was affirmed
based on the cytochrome b (cyt b) gene partial sequences5）. 2. 2Dataset construction and Data Analyses
In another study, the Malaysian water buffaloes and Sequence editing, alignment, and model test of 307 bp
their crossbreds showed their close relationship to of 367 mtDNA D-loop PCR amplified fragments were con-
swamp maternal lineage based on the mtDNA D-loop6）. ducted using MEGA version X17）. Insertions/deletions in
The PC population genetic divergence and phylogenetic the aligned sequences were excluded in the analyses.
analyses within the major island sub-groups and between The software Arlequin version 3.118） was conducted to (1)
the neighboring countries have little published informa- perform an Analysis of Molecular Variance (AMOVA), (2)
tion and are still unidentified. The mtDNA cytochrome calculate the population pair-wise FST, and (3) calculate
c oxidase I (COI) gene was informative to confirm the the population-specific FST indices. The criterion for
species identification of buffaloes in Calayan Island in genetic differentiation (FST) by Wright (1978) was used
Cagayan as swamp buffaloes and appropriate for selecting to define the varying degrees of FST, as low for FST＜
this animal for conservation and management in the PC 0.05, moderate for 0.05＜FST＜0.15, high for 0.15＜FST＜
sanctuary12）. Previous studies showed that mtDNA D-loop 0.25, and very high for FST＞0.25. Inferred phylogenetic
sequences of Asian buffaloes, including the Philippine tree using Neighbour-joining (NJ) and UPGMA methods
swamp buffaloes, originated from China9）. The data ob- were constructed based on population differentiation
tained from other related studies showed the closer affinity (FST) using the using MEGA version X17）. Haplotype
of Chinese swamp buffaloes to Philippine swamp buffaloes, diversity (h) and nucleotide diversity (Pi) were computed
which were identified in maternal lineage A11, 13）. However, using DnaSP v6 program19）. All the analyses were per-
the recent findings on the Philippine swamp buffalo formed in two levels : the PC sub-populations and the
mtDNA D-loop sequence variation first reported the neighboring Asian swamp buffalo populations.
detection of the two haplotypes in lineage B, which were
3. Results
traced from the two populations in Luzon and Visayas14）.
All these studies were good baseline information which 3. 1 Philippine Carabao populations in Major Island
provided a glimpse of the genetic relationship of swamp Sub-groups
buffaloes in Asian countries. However, it is still unresolved AMOVA analysis at the PC sub-populations or the
to understand the spread of domesticated Chinese swamp island groups showed significant genetic differentiation
buffaloes to the major islands of the Philippines. Thus, of 3.10% among the populations and 96.90% within the
the objective of the study is to determine the genetic populations (P＜0.05) (Table 2). The significant population-
differentiation and phylogenetic history of the modern specific FST indices had an average of 0.031 and obtained
Philippines swamp buffaloes with selected swamp buffalo the highest in Luzon at 0.063, followed by Mindanao at
populations in Asia using the mtDNA D-loop sequences. 0.061, and the least in the Visayas at 0.055. The phyloge-
 Genetic Divergence and Phylogenetic Relationship of Philippine Carabao (Bubalus bubalis) and Other Swamp Buffalo Populations in Neighboring Countries Revealed by the Mitochondrial DNA D-loop Region 67

Table 1 Sample information of 269 Asian swamp buffaloes retrieved from NCBI Genbank included in the analysis

Table 2 AMOVA among Philippine swamp buffalo

sub-populations.

netic relationship using the NJ method based on the

genetic distance (FST) showed the separation of popula-
tions between the swamp buffaloes in Luzon and those in
Visayas and Mindanao (Fig. 1). The result of NJ tree
was concordant with the topology and relationship of Fig. 1 Unrooted Neighbour-joining tree using the genetic
distance (FST) for Philippine swamp buffaloes from
swamp buffaloes among the major islands obtained using
major island sub-groups
the UPGMA method (data not shown).

3. 2Asian Swamp Buffaloes specific FST indices among countries had an average of
AMOVA analysis among the swamp buffalo sub- 0.101, ranging from the highest 0.122 in Myanmar and
populations in the Asia region showed significant genetic Malaysia to the lowest 0.093 in Vietnam. FST index
variation of 10.45% among the populations and 89.55% 0.118 in Taiwan was second to the highest and followed
within the populations (P＜0.05) (Table 3). Population- by 0.113 in the Philippines. However, the Philippines
 68 Villamor, Nomura, Amano and Takahashi

Table 3 AMOVA among Asian swamp buffalo populations.

Table 4 Pair-wise distance among Asian swamp buffalo

populations.

Fig. 2 Neighbour-joining tree for eight Asian swamp

buffalo populations, based on average pair-wise
distance (FST) between populations.

indicated higher FST indices than those of 0.103 in

Thailand, 0.099 in China, 0.096 in Laos, and 0.092 in
Vietnam.
The values of population pair-wise FST ranged from
－0.005 to 0.352. Out of 28 iterations, 17 significant popu-
lations pair-wise differences (FST, P＜0.050) were detected
and ranged from relatively low to very high genetic
variations (Table 4). The highest significant pair-wise Fig. 3 Neighbour-joining tree for Philippine swamp
FST value (0.308) was indicated between the Philippines buffalo from major island sub-groups and Asia
and Vietnam, suggesting that swamp buffaloes living in region, based on average pair-wise distance
these countries had very high genetic differentiation. (FST) between populations.
Conversely, the pair-wise FST value (0.029) between the
Philippines and Taiwan suggested a relatively low genetic encompassing China, Malaysia, Laos, Thailand, and
differentiation. The non-significant pair-wise FST values Vietnam. The topology of the phylogenetic trees was
between Laos-China, Laos-Thailand, Vietnam-Thailand, congruent using the NJ (Fig. 3) and UPGMA (data not
and Vietnam-Laos populations indicated that swamp shown) methods.
buffaloes from these countries had the absence of genetic
differentiation. However, Myanmar was the only popula- 3. 3 Genetic Diversity in Asian countries
tion that revealed non-significant FST values, showing a The 376 sequences of swamp buffaloes from the Philip-
lack of genetic differentiation among swamp buffaloes pines and neighboring countries revealed 39 haplotypes
from other Asian countries included in the study. with 23 polymorphic nucleotide sites (Table 5). Haplotype
The phylogenetic relationship was analyzed in the NJ and nucleotide diversities were highest in Vietnam at
method and showed two main clusters. The inferred 0.810 and 0.010, with the lowest in Malaysia at 0.473 and
tree formed Cluster 1 that showed closer affinity among 0.002, respectively. The Philippines had higher haplotypes
Philippines, Taiwan, Myanmar, and China while Cluster 2 and nucleotide diversities than those in Taiwan, Myanmar,
entailed closer affinity among Vietnam, Thailand, Laos, and Malaysia. Focusing on the eight Asian swamp popu-
and Malaysia (Fig. 2). The evolutionary history of the lations revealed uneven distribution of haplotypes. China
Philippines’ three major islands, including Luzon, Visayas, had 24 haplotypes, followed by 17 haplotypes for the
and Mindanao, together with other Asian origins, Philippines and less than ten haplotypes for other Asian
confirmed further the formation of two clusters (Fig. 3). countries. The frequency of unique haplotypes detected
Cluster 1 consisted mostly of the three major islands in 17 in China, 11 in the Philippines, two in Laos, and one in
the Philippines, Myanmar, and Taiwan, while Cluster 2 Malaysia (Table 6 & Fig.4).
was found predominantly from the mainland populations
 Genetic Divergence and Phylogenetic Relationship of Philippine Carabao (Bubalus bubalis) and Other Swamp Buffalo Populations in Neighboring Countries Revealed by the Mitochondrial DNA D-loop Region 69

Table 5 Source and genetic diversity indices of Asian swamp buffaloes in mtDNA D-loop.

Table 6 Sources information on lineages and frequency of haplotype distribution of swamp buffaloes from Asia region.

Table 6 Continuation

3. 4 NJ Tree Phylogenetic Relationship ＝11%), which entailed SW1-SW8, SW12-SW13, SW18,

The phylogeny analysis of D-loop sequences obtained SW20, SW22-SW26, SW28-SW29, SW35-SW39. Among
from the Philippine swamp buffaloes and retrieved from the group of haplotypes, SW39 clustered separately, with
the NCBI GenBank confirmed the separation between the meager statistical support (PB＝33%). On the other hand,
swamp and riverine groups (Fig. 5). The phylogenetic 12 haplotypes belonged to lineage B (PB＝27%), which
tree of Asian swamp buffaloes confirmed four maternal showed a closer affinity among haplotypes SW9-SW11,
lineages, A, B, C, and E (Fig. 5). Moreover, the NJ tree SW14, SW16-17, SW19, SW21, SW27, and SW30-SW32.
showed delineation between two lineages, A and B, but However, the groupings of the haplotypes (SW) that be-
with low bootstrap support (PB＝14%). The majority of longed to maternal lineages A and B were affirmed by
sequences (80%, 300/376) were included in lineage A, and the pair-wise difference (FST＝0.532 ; (P＜0.000), implying
the minority of the sequences were observed in lineage a very high genetic differentiation between the two lin-
B (19%, 73/376). Few sequences were observed in lineage eages (data not shown). The groupings of swamp haplo-
C (0.5%, 2/376) and lineage E (0.3%, 1/376). Twenty-five types were also confirmed by the reference sequences
of the 39 haplotypes were incorporated in lineage A (PB obtained from the NCBI GenBank from the previous
 70 Villamor, Nomura, Amano and Takahashi

Fig. 4 Map showing the frequency of geographic distribution of the 39 haplotypes from Asian swamp buffaloes.
(Asian map retrieved from https://whatsanswer.com/world-map/blank-map-of-asia-printable-outline-
map-of-asia/)

Fig. 5 Neighbour-joining tree for 39 mtDNA D-loop region haplotypes of swamp buffaloes from selected Asia region,
with the riverine sequences as outgroups.

studies on Asian swamp buffaloes11, 14, 15, 16） (Table 1). The
4. Discussion
other rare haplotypes SW33 in lineage C and SW34 in
lineage E were clustered separately from lineages A and The molecular variance analysis for mtDNA D-loop
B and showed recent divergence from the other swamp sequences among the Philippine swamp buffaloes implied
haplotypes. that the genetic variation was attributed by the differences
between individuals rather than among populations. The
 Genetic Divergence and Phylogenetic Relationship of Philippine Carabao (Bubalus bubalis) and Other Swamp Buffalo Populations in Neighboring Countries Revealed by the Mitochondrial DNA D-loop Region 71

AMOVA from this study showed reasonable agreement and Mindanao and Taiwan than in Luzon and China. In
with previous reports on the Philippine carabao haplotype contrast, the moderate and significant genetic differentia-
network that little population differentiation existed. tion between PC in Luzon and Taiwan implied the closer
The unique haplotypes in the Philippines were also pre- affinity between the two populations compared with
viously reported but they were determined in small China. In this analysis, Taiwan swamp buffaloes were
numbers14）. It would be interesting to include additional obtained from an old population from the conservation
samples from other populations of the Philippines in sites16）. The relatively low genetic divergence (FST)
future studies. For instance, Palawan Island in Luzon obtained from this study did not agree with the results
and Tawi-Tawi Island in Mindanao, which have been the obtained by Barker et al.20）, which stated that one of the
central ports for settlers and serve as the hub of trans- dispersal routes of the domestication of swamp buffalo
porting goods and passengers, could be more likely be from mainland Asia followed from China through the
genetically differentiated. However, the genetic data to Philippines to the eastern islands of Borneo and Sulawesi.
support this hypothesis is lacking. In fact, in other study, the inferred phylogenetic tree
The average population-specific FST index of the based on mtDNA D-loop sequences of PC from the major
modern Philippine carabao populations obtained from island groups of the Philippines was confirmed to be
this present study indicated low genetic differentiation. closely related to the Chinese swamp buffaloes13）. The
This finding was concordant with the topology of inferred hypothesis that swamp buffalo of the Philippines origi-
phylogenetic relationship of PC’s sub-group of major nated from China was further supported by the mtDNA
islands obtained from this study (Fig. 1). The low genetic D-loop sequences and mitogenomic data from the previous
differentiation of PC obtained from this study could be studies21-23）. However, the results of the genetic divergence
attributed to lack of population pair-wise difference (FST and phylogenetic relationship of this research indicated
＝－0.014, P＜0.050) between Visayas and Mindanao, otherwise. Our results agreed with the hypothesis by
indicating the high genetic exchange of swamp buffaloes Lau et al.1） that after domestication in the China region,
living in these sub-groups of major islands14）. However, a the domesticated Chinese swamp buffaloes had the first
closer look at the population-specific FST indices at the dispersal pathway through Taiwan and the Philippines
PC sub-populations from this present study indicated to the eastern islands of Borneo and Sulawesi1, 9）.
moderate genetic variation within the three sub-groups In contrast, a lack of genetic differentiation of Myanmar
of major islands. Thus, the highest genetic differentiation swamp buffaloes among other Asian countries included
within Luzon indicated swamp species’ restricted gene in this study could indicate a high gene flow of swamp
flow due to geographical features of the island surrounded buffaloes from Myanmar to other neighboring countries.
by the mountainous and coastal areas. This genetic pattern can be partially elucidated by the
The AMOVA showed higher genetic variation obtained geographical positioning of Myanmar as a Southeast
in Asia region swamp buffalo populations than Philippine Asian nation bordered by various countries and linked to
carabao population, but most of the differences in the historical events of human migration together with their
former populations could be described by the variation swamp buffaloes. The additional research findings are
between individuals rather than among populations. pertinent to understand the relationship of swamp buffaloes
While the unique haplotypes were detected in China, from Myanmar and with other neighboring countries.
Philippines, Laos, and Malaysia, they were found in small The mtDNA haplotype analysis confirmed the previous
numbers and were obtained from the three most numer- reports on the two maternal lineages A and B in swamp
ous haplotypes. This implied that more individuals from buffalo haplotypes1, 9-11）. Yue et al.11） previously reported
Asian regions like Cambodia and Indonesia would be that no sequences in the Philippines and Thailand fell
needed to be certain that low population differentiation is into lineage B. In contrast, the present study highlighted
not an artefact of sampling from the Asia populations. that Philippines, Taiwan, Thailand, Vietnam, and Laos
The genetic divergence (FST) explained further the had haplotypes which were incorporated to maternal
genetic differentiation of Asian region. The population lineage B. The undetected haplotypes from the previous
pair-wise differentiation elucidated the closer affinity of studies could be attributed to the limited samples and
swamp buffaloes between the Philippines and Taiwan sampling sites from other Asiatic origins since the previ-
than in China. Moreover, among the Philippines’ major ous study focused mainly on the mtDNA D-loop sequences
islands, there was a very low genetic variation detected of Chinese swamp buffaloes. It would also be worth
between Visayas and Mindanao in the Philippines and mentioning that rare haplotypes from Chinese buffaloes
Taiwan. This shed light on the closer relationship between were detected and fell to the maternal lineages C and E,
the Philippine carabao’s modern population in Visayas which was previously identified by Wang et al.15）. This
 72 Villamor, Nomura, Amano and Takahashi

study also reported that the Philippine swamp buffaloes of farmers from the Visayas to Mindanao, who brought
predominantly fell in lineage A, very little in lineage B, with them their native swamp buffaloes for rice cultivation
and none in lineages C and E, which was consistent with and second, it would be possible that access within and
the previous study on PC mtDNA genetic diversity14）. between inter-island transportation could cause genetic
However, three sequences of swamp buffaloes from exchange of swamp buffaloes among sub-populations in
Northeast Thailand and South Thailand, which were pre- the Philippines. Furthermore, this study supported the
viously reported to belong to lineage D based on the genetic analysis that showed a lack of genetic differentiation
mitogenomes from the study of Wang et al. 201715）, all fell between Visayas and Mindanao, suggesting that swamp
to haplotype SW10 in Lineage B of this study. The low buffaloes are introduced farm animals in Mindanao14, 25）.
resolution between haplotypes in Lineage D and Lineage Thus, the spread of domesticated Chinese swamp buffaloes
B, which failed to delineate, could imply the need for from Taiwan to the Philippines would probably be intro-
additional representative samples to confirm the relation- duced to the Visayas rather than Mindanao Island.
ship of rare haplotypes to predominant haplotypes in The research findings highlighted the phylogenetic
maternal lineages A and B. history among the modern Asian swamp buffalo popula-
The predominant haplotype SW1 in lineage A could be tions and could be explained in two points : (1) Philippine
considered the ancestral haplotype. In fact, SW1 had the carabao had the closest affinity to Taiwan swamp buffalo
highest frequency of intermixing of the individuals from population based on the low pair-wise distance (FST) and
all Asian countries except Thailand. The present study (2) Chinese swamp buffalo could be the ancestral popula-
that indicated one individual or shared by only a small tion of the modern population of the swamp buffaloes in
number of samples could be attributed to restricted gene Asia, which was spread to Taiwan then introduced to
flow among some swamp buffalo sub-populations, including the Visayas island of the Philippines, as supported by
the Philippines, China, Laos, and Malaysia. The presence inferred mtDNA haplotypes.
of unique haplotypes was common in breeds within the Future studies will compare these Asian swamp buffalo
mtDNA variation, according to the previous study of mtDNA D-loop sequences to Asian wild buffalos and
Naderi et al.24）. would consider the utility of other polymorphic mtDNA
The other pertinent findings from the mtDNA haplotype markers such as cyt b to gain a better perspective on
analysis explained the pattern and frequency of the phylogenetic history of Philippine carabao.
swamp buffaloes’ haplotype distribution across the Asian
5. Conclusion
neighboring populations. The Philippines shared the
highest haplotypes with China, followed by Laos. These The mtDNA D-loop sequences of swamp buffaloes were
research findings could be attributed to the bias by num- informative to establish the genetic divergence and eluci-
ber of swamp buffaloes and sampling sites derived date the relationship of swamp buffaloes in eight Asian
particularly from China and Philippines, which could populations. Low genetic divergence between the PC
contribute to a higher number of haplotypes and haplotype sub-populations in Visayas and Mindanao and Taiwan
diversity. However, the inferred phylogenetic tree based shed light on the most likely route of the swamp buffalo’
on the mtDNA haplotypes showed that the two unique s introduction and spread in the Philippines. Also, the
haplotypes SW34 and SW33 from China had the earliest dataset supported China as the most likely ancestor of
divergence from the clusters of swamp buffaloes and the modern swamp buffalo populations in Asia. This will
were the closest to the riverine buffaloes. In lineage B, provide essential insights on phylogenetic relationship of
haplotype SW27 had a closer relationship to haplotype Asian swamp buffaloes which will have implications for
SW9, which had an individual source from Capiz in the the utilization, conservation, and management of the
Philippines’ western Visayas. An earlier study of Lau et swamp buffalo genetic resources of the swamp buffaloes
al.1） indicated a pattern of differentiation in the phyloge- with Asiatic origins.
netic tree, wherein three haplotypes were detected and
Acknowledgement
traced predominantly in the eastern island populations of
the Philippines. From this study, the Philippine swamp All the authors would like to extend gratitude to the
populations from the Visayas and Mindanao sub-group of Philippine Carabao Center National Headquarters and
major islands seemed to be closely related to Taiwan Gene Pool (PCC-NHQGP), the Tokyo University of
swamp buffaloes and suggested an extensive level of gene Agriculture-Southeast Asian Regional Center for Graduate
flow. However, the high genetic exchange of swamp Study and Research in Agriculture (Tokyo NODAI-
buffaloes between the two major islands could be explained SEARCA) Scholarship for Dissertation Doctorate Program,
by two perspectives: first, the history of early migration Laboratory of Animal Genetics and Breeding in Tokyo
 Genetic Divergence and Phylogenetic Relationship of Philippine Carabao (Bubalus bubalis) and Other Swamp Buffalo Populations in Neighboring Countries Revealed by the Mitochondrial DNA D-loop Region 73

University of Agriculture Atsugi Campus (TUA), and the C A S, Herrera J R V, Del Barrio A N. (2009) Molecular
Department of Agriculture Livestock Biotech Program characterization of the Philippine carabao (Bubalus bubalis
L.) from the major island groups of the Philippines using
and the Bureau of Agricultural Research through the
molecular cloning and sequence analysis of the D-loop of
DABIOTECHR1506 project for the research funds. mitochondrial DNA. [BS thesis]. Los Baños, Laguna : Uni-
versity of the Philippines-Los Baños. 75. (Available at the
References UPLB library).
1） Lau C H, Drinkwater R D, Yusoff K, Tan S G, Hetzel D J, 14） Villamor L P., TakahashI Y., Nomura K., Amano T. (2021)
Barker J S. (1998) Genetic diversity of Asian water buffalo Genetic Diversity of Philippine Carabao (Bubalus bubalis)
(Bubalus bubalis) : mitochondrial DNA D-loop and cyto- using Mitochondrial DNA D-loop Variation : Implications
chrome b sequence variation. Anim. Genet. 29 : 253-264. to Conservation and Management. Philipp. J. Sci. 150 :
2） Kierstein G, Vallinoto M, Silva A, Schneider M P, Iannuzzil, 839-848.
Brenig B. (2004) Analysis of mitochondrial D-loop region 15） Wang, S, Chen N, Capodiferro M R, Zhang T, Lancioni H,
casts new light on domestic water buffalo (Bubalus bubalis) Zhang H, Miao Y, Chanthakhoun V, Wanapat M, Yindee
phylogeny. Mol. Phylogenet. Evol. 30 : 308-324. M, Zhang Y, Lu H, Caporali L, Dang R, Huang Y, Lan X,
3） Cruz L C. (2015) Institutionalization of swamp buffalo de- Plath M, Chen H, Lenstra J A, Achilli A. Lei C. (2017)
velopment in the Philippines. Proceedings of International Whole Mitogenomes Reveal the History of Swamp Buffalo:
Seminar on Improving Tropical Animal Production for Initially Shaped by Glacial Periods and Eventually
Food Security ; 2015 November 3-5 Southeast Sulawesi, Modelled By Domestication. Sci. Rep. 7 : 4708.
Indonesia : 15-37. 16） Zhang Y, Lu Y, Yindee M, Li K-Y, Kuo H-Y, Ju Y-T, Barker
4） Lei C Z, Zhang C M, Weining S, Campana M G, Bower M A, J S F. (2016). Strong and Stable Geographic Differentiation
Zhang X M, Liu L, Lan X Y, Chen H. (2011) Genetic diversity of Swamp Buffalo Maternal and Paternal Lineages
of mitochondrial cytochrome b gene in Chinese native Indicates Domestication in the China/Indochina Border
buffalo. Anim. Genet. 42 : 432-436. Region. Mol. Ecol. 25 : 1530-1550.
5） Winaya A, Sukri A, Gofur A, Amin M. (2019) The genetic 17） Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. (2018)
divergence and phylogenetic relationship of Indonesia MEGA X : Molecular Evolutionary Genetics Analysis across
swamp buffalo (Bubalus bubalis) based on partial sequences computing platforms. Mol. Biol. Evol. 35: 1547-1549.
of cytochrome b gene of mitochondrial DNA. Int. J. Eng. 18） Excoffier L, Lischer H E. (2010) Arlequin suite ver 3.5 : a
Technol. 8 : 96-100. new series of programs to perform population genetics
6） Shaari N A L, Jaoi-Edward M, Loo S S, Salisi M S, Yusoff R, analyses under Linux and Windows. Mol. Ecol. Resour 10 :
Ab Ghani N I, Saad M Z, Ahmad H. (2019) Karyotypic and 564-567.
mtDNA based characterization of Malaysian water buffalo. 19） Rozas J, Ferrer-Mata A, Sánchez-Delbarrio J C, Guirao-
BMC Genet. 20 : 37. Rico S, Librado P, Ramos-Onsins S E, Sánchez-Gracia A.
7） Villamor L P. (2015) Conserving Animal Genetic Diversity (2017) DnaSP 6 : DNA sequence polymorphism analysis of
to adapt to Climate Change in the Philippines. In : Learning large datasets. Mol. Biol. Evol. 34 : 3299-3302.
and Coping with Change : Case Stories of Climate Change 20） Barker J. S .F., Moore S. S., Hetzel D. J. S., Evans D., Tan S. G.,
Adaptation in Southeast Asia. SEARCA Philippines. 235 p. Byrne K. (1997) Genetic diversity of Asian water buffalo
8） Food and Agriculture Organization of the United Nations (Bubalus bubalis) : microsatellite variation and a comparison
(FAO) FAOSTAT, http://www.fao.org/faostat/en/#data/ with protein-coding loci. Anim. Genet. 28 : 103-115.
QA (Last Access January 21 2021) 21） Zhang Y, Vankan D, Zhang Y, Barker J S. (2011) Genetic
9） Lei C Z, Zhang W, Chen H, Lu F, Ge Q L, Liu R Y, Dang R H, differentiation of water buffalo (Bubalus bubalis) popula-
Yao Y Y, Yao L B, Lu Z F, Zhao Z L. (2007a) Two maternal tions in China, Nepal and south-east Asia : inferences on
lineages revealed by mitochondrial DNA Dloop sequences the region of domestication of the swamp buffalo. Anim.
in Chinese native water buffaloes (Bubalus Bubalis). Asian- Genet. 42 : 366-77.
Aust. J. Anim. Sci. 20 : 471-476. 22） Licia C et al. (2018) New Insights on Water Buffalo Genomic
10） Lei C Z, Zhang W, Chen H, Lu F, Liu R Y, Yang X Y, Zhang Diversity and Post-Domestication Migration Routes From
H C, Liu Z G, Yao L B, Lu Z F, Zhao Z L. (2007b) Indepen- Medium Density SNP Chip Data. Front. Genet. 9 : Article
dent maternal origin of Chinese swamp buffalo (Bubalus 53.
bubalis). Anim. Genet. 38 : 97-102. 23） Zhang K, Lenstra JA, Zhang S, Liu W, Liu J. (2020) Evolu-
11） Yue X P, Li R, Xie W M, Xu P, Chang T C, Liu L, Cheng F, tion and domestication of the Bovini species. Anim. Genet.
Zhang R F, Lan X Y, Chen H, Lei C Z. (2013) Phylogeography 51 : 637-657.
and domestication of Chinese swamp buffalo. PLoS One 8 : 24） Naderi S, Rezaei H R, Taberlet P, Zundel S, Rafat S A,
e56552. Naghash H R, El-Barody M A, Ertugrul O, Pompanon F.
12） Paraguas A M, Cailipan T P C, Flores E B, Villamor L P. (2007) Large-scale mitochondrial DNA analysis of the
(2018) Morphology and phylogeny of swamp buffaloes domestic goat reveals six haplogroups with high diversity.
(Bubalus bubalis) in Calayan Island, Cagayan. Philipp. J. PLoS One 2 : e1012
Vet. Anim. Sci. 44 : 59-67. 25） Pendleton R L. (1942) Land Utilization and Agriculture of
13） Del Barrio L A M, Dela Vina C B, Yebron JR. M G N, Estrella Mindanao, Philippine Islands. Geogr. Rev. 32 : 180-210.
74 Villamor, Nomura, Amano and Takahashi

DNA D-loop

Lilian P. Villamor*・野村こう**・天野 卓***・髙橋幸水** †

（令和 3 年 5 月 20 日受付/令和 3 年 9 月 14 日受理）

：沼沢スイギュウ（Bubalus bubalis）は，小規模な稲作農家の役畜として大変重要であり，乳，肉およ
び皮も活用されている。これまでにフィリピンとその他周辺諸国スイギュウの遺伝的分化や系統遺伝学的関
係は明確にされていない。本研究は，フィリピンで飼養されているスイギュウ集団と中国を含むインドシナ
半島集団間の遺伝的分化と系統遺伝学的関係を明らかにすることを目的とした。367 頭のミトコンドリア
DNA D-loop 領域の塩基配列を用い解析を行った。その結果，遺伝的分化の程度を示す指数からフィリピン
の Visayas および Mindanao 集団は，台湾集団と近いことが推定された。また，系統樹からもフィリピンお
よび台湾集団は近い関係を示し，ミャンマーおよび中国集団と同一のクラスターを形成した。これまでに報
告された仮説と一致していることから，中国から台湾，フィリピンへスイギュウが伝わってきたことが推察
された。本研究は，頭数が減少しつつある東南アジアのスイギュウの多様な遺伝子をフィリピンスイギュウ
が保有していることから，国内で利活用しながら貴重な遺伝資源として保全・維持する必要があることを示
した。

：遺伝的分化，ミトコンドリア DNA D-loop，在来スイギュウ，系統遺伝関係

* Department of Agriculture, Philippine Carabao Center-National Headquarters and Gene Pool, Science City of Munoz, Nueva Ecija,
3120 Philippines
** 東京農業大学農学部動物科学科
*** 東京農業大学名誉教授
†
Corresponding author（E-mail : [email protected]）