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DA DANG DI TRUYEN VA MQT SO DAC DIEM SEVH THAI, SESH HOC CUA THO VAN TRUNG BQ {NESOLAGUS TIMMINST) 6 VIET NAM
PHAM TRQNG ANH, DANG TAT THE, PHAM DlTC TIEN, HO THI LOAN, HA VAN TUE, NGUYEN TRUONG SON
Vien Sinh thdi vd Tdi nguyen sinh vgt Hg thd (Leporidae) thi gidi gom cd 11 giong, 54_ loai, trong dd cd ba loai thudc hai gidng (Lepus vi Nesolagus) phan bd d Viet Nam. Rieng gidng thd vin Nesolagus chi cd hai loai: tho vin Sumatra (A^. netscheri) phan bo d Indonesia va thd van trung bd (A'', timminsi) phan bo d Vift Nam. Thd vin tmng bd dugc cdng bo loai thii mdi d Viet Nam nam 2000 (Averianov et al., 2000). Die dilm hinh thii va phan bd cua thd van da dugc cdng bo gan day (Pham Trgng Anh va cs, 2007). Trong nghien ciru nay, chiing tdi bd sung nhiing thdng tin mdi ve da dang di tmyIn vi mdt sd die dilm sinh hgc, sinh thii sinh hgc cua thd van trung bd.
I. PHUONG PHAP NGHIEN ClTU
Nghien cuu da dang di tmyen su dung mau vat gdm: Mau sd 1 (ma hieu A'', timminsi. I), lay tir miu sg ma hifu N02.VTS cua Vifn sinh thii v i Tii nguyen Sinh vat, thu dugc tai xa Lam thuy, Lf Thuy, Quang Binh. Mau so 2 (ma hifu N. timminsi.2), lay tu md co thu dugc tai xa San Tiy - Huomg Son - Ha TTnh.
ADN tdng sd cua cac mau tren dugc tich chiet bang bg kit DNeasy blood and tissue (QAGEN Inc.). Cip mdi dugc thiet ke de nhan ban doan ADN dich thudc gen cytb cua he gen ty thi cd ma hifu vi trinh ty TF: 5'-GAGGTGGACTGCTGCTAGG-3''va TR: 5'-ATCCAAC ATCTCAGCATGATGAA-3'. Sir dung bd hoa chat TaqPCR mastrmix de nhan bin doan gen dich.Chu truih nhift cua PCR tren miy gdm: Budc biln tinh 94° C - 3 phiit tilp theo 35 chu ki:
94° C - 1 phiit, 54° C - 45giay, 72° C - 1 phut chu ki cudi keo dii 3 phiit d 72° C. Sin phim PCR dugc tinh sach bing bg hoi chat Gel extraction mini Elute (QAGEN Inc). Giai trinh ty tryc tiep sin phim PCR, sir dung bd hda chit FS-DNA sequencing kit (Perkin Elmer, My) va may giai trinh frr ty dgng ABI 3100 - Avant Genetic Analyzer (PE Applied Biosystems, My).
Phan tich di tmyen vi tien hda phan tu bang chuong trinh MEGA4.0 (Molecular Evolutionary Genetics Analysis) ciia Kumar et al., (2007). Cay phit sinh chimg loai dugc xay dyng theo phuong phip NJ, vdi cic die diem bien ddi cd gii trj ngang nhau va khoing cich tien boa theo md hinh Kimura hai tham sd. Gia trj bootstrap cua ciy phit sinh chung loai dugc tinh vdi 1000 miu thir. Cic loai thd khic cd trinh ty tuong ddng dugc sir dyng de tham khao va phan tich gdm: Lepus hainanus, ed ma so trong ngan hing Genbank li AY745116, tuong ty, L sinensis -AJ279420, L. timidus - AY745I09, L. comus - AJ 279402, Romerolagus diazi - AY292734, Oryctolagus cuniculus - NC001913, Bunolagus monticulari - AY292718.
Poetalagus fumssi - AY292720, Sylvilagus aquaticus - AY292726, S. floridanus - AY292724, Pronolagus randesis-AY292737, P. saundersia - AY292736, P. mpestris-AY292735.
Nghien ciru sinh thai, sinh hgc cua thd van dya tren co sd khao sat, thu thap cic sd lifu ve kifn ty nhien, •viing hoat dgng, sinh cinh sdng, noi kilm an vi tap tinh boat dgng cua cic quin the thd van trong vung nghien ciiu. Quan sat ciu tnic dan, ghi nhan cay thiic an cua thd van de budc dau tim hieu ve sinh hgc vi phan tich thanh phan cay thiic in cua chung.
n . KET QUA NGHIEN CUtJ 1. Phan tich di truyen
Da iihan ban dugc doan ADN trong hai miu nghien cuu bing ky thuat PCR vdi cap moi fren.
San pham PCR dai gin 400 nucleotide da dugc giii trinh ty vi xic nhan trinh frr dich khi so sanh vdi cac trinh ty tijong dong bing chuong trinh tim kilm trinh ty tuong ddng trong
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Genbank.. Ddi chieu trinh ty nucleotide tir hai mau nghien cim dugc the hien trong hinh 1 va cho thiy giira chung khdng cd sy khac bift nucleotide nao.
N.tinroinsi. 2 N.timminsi. 1
N.timminsi.2 N.timminsi.1
N.timminsi.2 N.timminsi.1
N.timminsi.2 N.timminsi.1
N.timminsi.2 N.timminsi.1
N.timminsi.2 N.timminsi.1
N.timminsi.2 N.timminsi.1
CACCCTCTGCTAAAAATTATTAATCACTCACTAGTGGACCTTCCAGCTCCATCCAATATC CACCCTCTGCTAAAAATTATTAATCACTCACTAGTGGACCTTCCAGCTCCATCCAATATC + * * * + * * * * * • * * * * * * + * + * * * + * * + * + + + * * * * * * + * + * + * + + * * * + * + + + + + * * * * *
TCTACTTGATGAAACTTTGGTTCCCTTTTAGGACTCTGCCTAATCATCCAAATCTTAACT TCTACTTGATGAAACTTTGGTTCCCTTTTAGGACTCTGCCTAATCATCCAAATCTTAACT
* * + * * + * + + * + * * * * + * * * * * * * * * . + * * * * * * + + * * * • + * * * * * * * * + * + * * + + * * * * * +
GGTCTTTTCCTCTCTATACACTATACAGCTGATACAACAACAGCATTTTCATCTGTAACC GGTCTTTTCCTCTCTATACACTATACAGCTGATACAACAACAGCATTTTCATCTGTAACC
CATATTTGCCGTGATGTAAACTACGGCTGACTCCTCCGATATCTCCACGCCAACGGAGCA CATATTTGCCGTGATGTAAACTACGGCTGACTCCTCCGATATCTCCACGCCAACGGAGCA
* * * * * * * + + + + + * * + + * * * * * * * * * * * * • + + * * + + * + + * • * * * * * * * * * * * * * * * * * * * *
TCTATATTTTTTATCTGCCTATATATGCATATTGGTCGAGGAATTTATTACGGCTCATAC TCTATATTTTTTATCTGCCTATATATGCATATTGGTCGAGGAATTTATTACGGCTCATAC
* * + * * * * + * * * * * * * * + * * * * * * * + * * * * * + * * + * * * + + * * + * * * * + * * * * * • * * * + * * +
ACCTATTTAGAGACCTGAAATATTGGCATCATTCTACTATTCGCAGTTATAGCTACCGCA ACCTATTTAGAGACCTGAAATATTGGCATCATTCTACTATTCGCAGTTATAGCTACCGCA
TTCATAGGCTATGTCCTCCCATGAGGC TTCATAGGCTATGTCCTCCCATGAGGC
Hinh 1: Doi chieu trinh ty nucleotide di xac djnh dugc cua 2 mau nghien cihi
Trong Ngan hing trinh ty (Genbank) hifn khdng cd trinh ty tuong dong cua loai thd vin duy nhit (N netscheri) cung giong Nesolagus vdi thd van trung bg, nen chiing tdi da chgn dai dien cac loai thd trong Hg thd (Leporidae) dl phan tich mdi quan hf di tmyen vi xay dyng ciy phit sinh chiing loai ciia chiing.
Khoing cich di truyin giira cic trinh ty nghien ciru va tham khio da dugc tinh toan theo md hinh thich hgp vdi bg sd lieu trinh ty niy la md hinh 2 tham sd ciia Kimura vi dugc trinh bay trong bing 1.
Bdng I Khoang each di truyen giira cac trinh t y nghien ciru va trinh t y tham khao
I . L . h a i n a n u s 2 . L . s i n e n s i s 3 . L . t i m i d u s
< . l . c o m u a i . B . m o n t i c u l a r i s fi.P.futnessi
^ 0 . c u n i c u l u s 8 . S . a q u a t i c u s 9 . S . f l o r i d a n u s l O . P . s a u n d e r s i a a U . P . r u p e s t r l s 1 2 . P . l a n d e n s i s I J . R . d l a z i 1 * . N . t i m m i n s i . 2
• - . N . t i r w i i n s i . 2 0 . 0 6 3 -
0 . 0 7 B 0 . 0 9 6 0 . 1 6 5 0 . 1 6 1 0 . 144 0 . 1 7 6 o . i a o 0 . 2 2 1 0 . 2 0 8 0 . 2 2 3 0 . 1 6 4 0 . 2 1 B 0 . 2 1 9 0 0 0 0 0 0 0 0 0 . 0 . 0 . 0 . 0 .
. 0 7 5 -
. 0 9 9 . 1 7 5 . 1 6 8 . 1 4 7 . 1 8 3 . 1 9 0 , 2 1 4 . 2 1 3 . 2 0 9 1 7 1 2 0 6 2 0 6
0 0 0 0 0 0 . 0, 0 . 0 , 0 . 0 . 0 .
. 0 7 8 -
. 1 6 5 . 1 7 1 . 1 5 7 . 1 7 6 . 1 9 1 . 2 1 7 . 2 0 1 , 2 0 4 164 1 9 6 1 9 6 0 0 0 0 0 0, 0 . 0 , 0 , 0 , 0 .
. 1 6 1 -
. 1 8 1 . 1 6 1 . 1 9 7 . 1 8 3 . 2 4 6 , 2 3 8 , 2 2 6 , 1 8 0 2 0 3 2 0 3 0 0 0 0 0, 0 , 0 , 0 . 0 . 0 .
. 1 3 7 -
. 1 5 2 , 1 7 2 , 1 7 9 , 1 9 2 , 1 8 1 1 9 9 1 7 9 2 2 0 2 2 0 0 0 0 0 0 0 . 0, 0, 0 ,
. 1 2 9 -
. 1 5 1 . 1 6 0 . 2 1 7 , 1 9 8 , 1 9 4 , 1 8 1 , 2 3 3 2 3 3 0 0 0 0 0 0 0 0 ,
. 1 8 1 -
. 1 7 0 . 2 1 5 . 1 9 5 . 1 9 5 . 1 8 2 . 2 1 1 , 2 1 1 0 0 0 0 0 0 0
. 1 2 7 -
. 2 3 5 . 2 1 0 . 2 3 6 . 2 2 0 , 2 3 3 , 2 3 3 0 0 0 0 0 0,
-
, 2 6 1 . 2 5 2 . 2 3 6 . 2 2 0 , 2 4 7 , 2 4 7 0 0 . 0 0 0
. 1 0 0 -
. 1 5 8 , 2 3 1 , 2 5 7 , 2 5 7
0 . 0 . 0, 0,
, 1 4 9 -
, 2 0 5 , 2 2 3 , 2 2 3 0 0 0
. 2 1 3 -
. 2 4 1 . 2 4 1
0 . 2 2 9 -
0 . 2 2 9
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Bang 1 cho thiy khoang each di truyen giiia cac gidng trong Hg Leporidae tii 16,1% din 25,7%. Trong khi giira mgt so loai cimg giong cd khoang each di tmyIn nhd hon nhilu, nhu giira cic loai thugc gidng Lepus l i giira 6,3% din 9,9%) va cao nhit cung chi den 15,0% (ba loai thugc giong Pronolagus). Thd vin trung bg cd khic bift di truyin ldn nhit so vdi dai difn ciia cic gidng khac frong Hg Leporidae, tir 19,6% den 25,1%. Dilu nay thi hifn thd vin trung bg cd gii frj cao frong bao tdn. Giira cac gidng, thd van tmng bd cd quan he di tmyIn gin gui nhit vdi gibng Lepus, vdi khoang cich di tmyIn tir 19,6% din 21,8%) va cd quan hf di fruyin xa nhit vdi gidng Pronolagus phan bo d nam va tay nam chiu Phi, vdi khoang each di fruyin tdi 25,7%.
Cay phat sutii chung loai Neighbor Jioning (NJ) frong huih 2 cho thay cac loai thugc giong Pronolagus phin bd d nam vi tay nam chau Phi phan hda thinh mgt nhanh rieng va cac gidng cdn lai phin bd trong mgt nhinh ldn cdn khic va cung thi hifn thd vin tmng bg cd quan hf di tmyen gin giii nhit vdi dai difn mgt sd loai frong gidng Lepus.
Lepus hainanus 'Lepus sinensis ' Lepus timidus
Lepus comus
,001 Nesolagus timminsi 1
Nesolagus timminsi 2 Romerolagus diazi
Oryctolagus cuniculus
— Bunolagus monticulari
Poetalagus fumessi Sylvilagus aquaticus
Sylvilagus floridanus - Pronolagus randensis
Pronolagus saundersia - Pronolagus rupestris Ochotono huangensis
Hmh 2: Cay phit sinh chiing loai Neighbor Joining (NJ). So d goc cac nhanh la gia fri boostrap.
2. Dac diem sinh thai va tap tinh
Thd vin frmng bg la thii in dem, kilm an tir 7 gid toi den 3 gid sing, thdi gian thudng gip la tir 10 gid tdi den 2 gid sing. Vimg sinh tiiii chfrih cua thd vin tmng bg l i rimg im thudng xanh CO dg che phu nhg, rimg thii sinh hon giao tre mia, rimg tii sinh sau khai thac cd thim thyc vat cay bui thip vi tang cd quyet. Mgt sd tmdng hop thd ra kilm in d nuong bai bd hoang hoic dang canh tic c ^ rung. Vimg sdng cua thd vin thudng gin khe sudi, mac dii nude khdng phii la yeu td chi phdi, m i tham thyc v^t canh sudi la ylu to anh hudng quylt djnh. Thd vin tmng bg a Viet Nam phan bo d binh dg tu 500m trd xuong so vdi mat biln, trong ldc dd hg hang gin gui cua chiing thd vin Sumatra (A^. netscheri) a Indonesia phan bd d 600m trd len, d rimg tren niii (Sterling E. J. et a l , 2007).
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Thd Vin tmng bg d hang ndng, dya vio cic hoc cay, hoc da, bd hem sudn nui, hd dudi cic bui cay rim, khdng dao xudng Idng dit. Thd vin song dgc than, kilm in rieng le tru tmdng hgp dan cd td me con.
Ke thii cua thd vin tmng bg gom cac loai thu in thit nhd vi nhd nhu frilt (Mustela sp.), chin vang (Mertes flavigula), Idn tranh (Herpestes javanicus), ciy gim (Pronodon paradicolor), meo rimg (Prionailums bengalensis), bio lira (Catopumur temminekii) vi rin hd mang chiia (Ophiophagus hannah), rin hd mang (Naja naja), frin dit (Python monlutus). Ihu canh tranh ciia thd vin li thd^ niu (Lepus pyguensis), nhumg so lugng thd nau frong vimg phan bd cua thd vin khdng ding ke.
3. Dac diem sinh hgc
Chua cd nhieu thdng tin ve sinh hgc sinh sin ciia thd vin tmng bg, ngoai t\i lifu quan sit mgt tl tho vin 2 con non khoang 1 5 - 2 0 ngiy tudi vao thing 8/2003 tai khe Thi Ldi, xa San Tay, huyfn Huong Son, Ha TTnh.
KeJ qui thdng ke thanh phin loai thyc vat thirc an cua thd vin trung bg gdm 44 loai thugc 6 hg vi 16 chi (bing 2), hau het la cicloii ciy thio, day leo, ciy bui thip. Thd vin in li, cudng li va nggn ciia ciy cd, nhung ua thich la phin Ii vi chdi non. Tuy thuc in ciia thd vin trung bg da dang, gom nhieu loii va hg, nhumg cic khio sit thyc dja cho thiy cic ciy thiirc in ua thich li cac loii cay thao, ciy byi day leo thugc ldp hai Ii mim, ciy thugc Idp mgt Ii mim rit it.
Bdng 2 Mgt so loai thyc vat dugc ghi nhan la thirc in ciia thd vin trung bf
TT
1.
«2.
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10.
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13.
14,
Ten Vift Nam I. HQ Rau muong
/. Chi Bim bim Bim Ba thiiy Bim chan cgp Bim canh IL Hp C i phe 2. Chi An dien An dien lan Lii dong An dien hai hoa An dien ngin Coc min An dien dau An dien ao An dien lo An dien phun Rim nili An dien sat
Ten khoa hijc Convolvulaceae
Ipomoea
Ipomoea triloba L.
/. pestigridis L.
/. hederifolia L.
Rubiaceae Hedyotis
Hedyotis diffusa Willd.
H. heynii R. Br.
H biflora L. Lamk.
H. contracta (Pierre ex Pitard) Phamh.
H. corymbosa (L.) Lamk.
H. capitellata Wall.
H. verticillata (L.) Lamk.
H. grandis (Pitard) Phamh.
H. diffusa Will.
H. merguensis Hook. f.
H. pressa Pierre ex Pitard
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15.
16.
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29.
30.
31.
33.
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34.
35.
36.
3. Chi gang Chim chich Ging henr>' Gap ngheo III. Hg Hoa thao 4. Chi CO ddng San frirng San cap San la dai San fron
5. Chi CO chdn nh^n Tlic hinh
Tuc hinh dao simo Tlic hinh
Tlic hinh ria
6. Chi CO long sucmg Dang hoa sole DSng hoa ebe IV. HpGac 7. Chi Ld han Mau la dai 8. Chi Gai Id If eh Cao hiing Cao hiing da 9. Chi phu I?
Phy If mdng nhd Phu If re 10. Chi de gia Nai
Nai petelo V. H9 6 ro 11. Chi nhj thgp Nhi thap yeu Nhi thap Nees
Randia
Randia canthioides Champ ex Benth.
R. henryi E. Pritz.
Fagerlindia depauperata (Drake) tinveng Poaceae
Paspalum
Paspalum commersonii Lamk.
P. confugatum Berg.
P. longifolium Roxb.
P. serobiculatum L.
Digitaria
Digitaria henrifi Rendl.
D. radicosa (Pres) Miq.
D. bicomis (Lamk.) Roem. Schult.
D. ciliaris (Retz.) Koel.
Isachne
Isachne dispa Trin.
/. eberhardtii A. Camus Urticaceae
Laportea
Laportea disepala (Gagnep.) Chew Elastoma
Elastoma disseetum Wedd.
E. rupestre (Buch. - Ham.) Wedd.
Pellionia
Pellionia eberhardtii Gagnep.
P. rhizomatosa Gagnep.
Villebrunea
Villebmnea frutescens Bl.
V. petelotii Gagnep.
Acanthaceae Staurogyne
Staurogyne debilis (T. Anders.) C.B. Clarke S. neesii (Vidal) Merr.
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37.
38.
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41.
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43.
44.
12. Chi dinh lich
Dinh lich dimg Dinh lich Vo ich 13. Chi qua no No ong to Song dye VI. H9 Hoa tan 14. Chi rau md
Rau ma
15. Chi rau md nho Rau ma nhd 16. Chi hoa khdm Khom
Hygrophila
Hygrophila erecta (Burm. f.) Hochr.
H. salicifolia (Vahl.) Nees H. phlomoides Nees Ruellia
Ruellia macrosiphon Kurz R. repens L.
Apiaceae Celltella
Celltella asiatica (L.) Urb. in Mart.
Hydrocotyle
Hydrocotyle sibthorpioides Lamk.
Trachysperm urn
Trachyspermum roxburghianum (DC.) H.
Wolff.
HI. KET LUAN
1. Da xic djnh dugc mgt doan trinh ty gin 400 nucleotide thugc gen Cytochrome b ciia hf thugc hf gen ty the tir hai mau thd vin trung bg, nhung khdng cd sai khiC trinh ty nucleotide gitra hai mau niy, nen cd the sy da dang di truyen quan the ciia thd vin tmng bg la thap.
2. Thd vin tmng bg cd khic bift di truyin Idn nhit so vdi d^i difn ciia cic gidng khic trong Hg thd - Leporidae, vdi khoing cich di tmyIn tii 19,6% din 25,7%, thi hifn chung cd gii frj cao vl bio ton. Thd vin trung bg cd quan bf di truyen gin nhit vdi giong thd Lepus.
3. Thd vin tmng bg song d rimg im thudng xanh, cd dg che phii thip, d dg cao dudi 500m so vdi mit biln. Vimg boat dgng ua thich li viing thim thyc vit gin ngudn nude. Hang td ndng, dya vao cic hang hdc la chinh, khdng dio xudng Idng dit. Thd vin tmng bg in dem song don dfc, cd nhilu ke thii l i thu in thjt nhd vi trin rin.
4. Da xic djnh dugc 44 loii thyc vat thugc 6 hg, 16 chi la tiiirc in cua thd vin tmng bg.
TAI LI?U THAM KHAO
1. Pham Trgng Anh, Pham Due Tien, D3ng Tat The, Nguyen Tnrdng Som, Ngd Kim Thai, 2007: Die diem hinh thii vi phan bo cua thd vin Nesolagus timminsi a Vift Nam, Bao cio khoa hgc vl Sinh thai va Tii nguyen sinh vat lan thir 2, 26-31.
2. Averianov A. O., A. V. Abramov, A. N. Tikhonov, 2000: A new species of Nesolagus (Lagomorpha, Leporidae) from Vietaam with osteological description confribution from the zoological institute, S.T. Peteriburg, 6 -12
3. Sterling E. J., M. M. Hurley, Le Due Minh, 2007: Ljch sii ty nhien Vift Nam, Yale University Press, New Haven and London.
4. Tamura K., J. Dudley J, M. Nei, S. Kumar, 2007: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.
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5. Web http://animaldiversity.tmimz.umich.edue 6. Web http://www.ncbi.nlm.nih.gov
GENETIC DIVERSITY AND BIOECOLOGICAL FEATURES OF ANNAMITE STRIPED RABBIT {Nesolagus timminsi) IN VIET NAM
PHAM TRONG ANH, DANG TAT THE, PHAM DUC TIEN, HO THI LOAN, HA VAN TUE AND NGUYEN TRUONG SON
SUMMARY
In the world, family of the rabbit (Leporidae) includes 11 genera, 54 species, of which three species belong to two genera distributed in Vietnam (Lepus and Nesolagus). As for Striped Rabbit genera (Nesolagus), there are only two species: Sumatra Stripped Rabbit (A^. netscheri) distributed in Indonesia, Annamite Striped Rabbit (N. timminsi) distributed in Viet Nam and announced as a new species in Viet Nam (Averianov et al., 2000). The mophological features of Annamite Striped Rabbit and their distribution described before (Pham Trong Anh et al., 2007).
In this smdy, some information on genetic diversity as well as biological and ecological features and the habit of Armamite Striped Rabbit were described:
Two mtDNA sequences with 400bp of Cytochrome b gene were identified from two Annamite Striped Rabbit samples. Analysis of these sequenses with some homology sequences from other species belonging family Leporidae showed that genetic differentiation of Annamite Striped Rabbit is highest compared between the other species of Leporidae and indicated that Armamite Striped Rabbit has high conservation value.. Annamite Sfriped Rabbit often lives in evergreen moistiue forest at 500 high metiers above sea levels. The favourable area for activities is vegetation near water. Their habitat is mainly in woody cave. They are animal nightwalker and single life. Thefr enemies in the natural are small carnivores and snakes. 44 plant species belonguig to 6 families, 16 genera are identified as food of Annamite Striped Rabbit.
