全 文 ：䛾⇆↌⇠䅤➦ᰶḺ➖㑯㇌㣟㟞⮱Ԋ៑䖄э႓᥁
Ⴥ㤹㤹ϕᕷԦᕷ 侙঒ ⦋ϕᕷ 哇঒ ≢ϕ᥁᥁
ཽϕ ͚ప⻾႓䮏ᬳᬻḺ➖ⵁ⾣᝭䉱⎽Ḻ➖̻⩌➖ឭᱜ䛺◦჋侹ბᖈ ξࢄ ᬳᬻ঒ ᣷጖ԗԦԗϕଟ
Ԧ ͚ప⻾႓䮏๔႓ᖈ ࡄϙ঒ ϕԗԗԗ࿋ࣷɥ
ᦅ㺮ᧅ ℈㡈⻾⮱㑯㇌㣟㟞 ཽዛႥᓣቂᅳዞᤀᓣቂᐹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂɥ ᭜͚ప㺬ࢄ䘕䛾⇆↌⇠䅤ౝፓ➦ᰶ⮱ࢂ⻺ᆋḺ➖ᤙ ᱙
ⵁ⾣ݖ⩕ܳၽ⩌➖႓᝸⃢ ཽଢ଼પ۠໵ɥᖈ ᄦ㑯㇌㣟㟞⮱䖄э็ᵤᕔহ䖄э㐀Ჱ䔈㵹γⵁ⾣ᤙ ̶͗ण㐬Ҁ❴⃢
ཽ ʗᡓ໧ϕ᣷ ڲक़ၽᖈ ᡓ໧ᤇዪ᤿ᡙʗႥ̶ ധ఍䬡䯁ࡧВࣷ ᡙʗႥऀ᤿ᡥዞஏ᣷ ധ఍䬡䯁ࡧɥ 㖁वܳᲽ⮱㐀᳉᭫⹧ѻ⮱䖄э็ᵤᕔহ
倅⮱䖄эܳࡃᤙ 䔆ज㘪᭜⩞λᅲ㓑䬡䪬᱌⮱ౝ⤳䯁⻨ᖈ ⠚ᄼ⮱ܳጰࡧВࣷ⩌ධ❴⃢ࡃ䕍᜽⮱ᰶ䭽⮱ധ఍≮
᝭ᑂ䊤⮱ᤙ ຯϷౕ䛾⇆↌̷ԛᐧⅡ⩢〆⮱Ѻ㒛̻㑯㇌㣟㟞⮱ܳጰࡧᰶ䘕ܳ䛺ऍᖈ 䔆ψⅡ⩢〆ч⌦⇎㑯㇌㣟
㟞⮱䘕ܳ͗Ҁহ⩌ධᖈ Ꭳ́ᩦअ⩌ᔮ⣜ධᖈ ༮㗮㑯㇌㣟㟞⮱⩌ႅᤙ ᱙ⵁ⾣ᄦ㑯㇌㣟㟞䖄э็ᵤᕔ⮱ⵁ⾣ͧ
ݣჇᰶ᩵⮱Ԋ៑ゃ⪒᣽ӈγԎᖜᤙ
ڠ䩛䃺ᧅ 㑯㇌㣟㟞ଟ ण㐬Ҁ ᧉપዪଟ ࢂᵥ㠤䚥็ᵤᕔଟ 䖄э็ᵤᕔଟ Ԋ៑䖄э႓
͚ఫܳㆨतᧅ ͆ ϕ᣷ᖈ ͆ Ꭲ጖঒ ঒ ঒ ঒ ঒ ᪴⡛ᴴ䃳⴮ᧅ ዪ঒ ঒ ঒ ঒ ঒ ঒ ᪴」㑃तᧅ Ԧԗࣷ጖ͧԗआ࿋጖ཽԦԗϕሉɥԗ጖ͧ጖጖጖ͧԗआ
ࣷᅳႥ໧ᓣʗצᐹᡙᔠᅳႥ ᤩᓣႥᓣᡙᔠዞ໧ ᅳஏ ᐹႥ ጛႥʐᓣቂᔠዞ ۗᤀᐹႥᡙᖈ ዛႥᓣቂᅳዞᤀᓣቂᐹ
੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂᖈ ᔠႥ ᡙ၊ᓣ ȣᔠႥ໧၊ᐹ ቴᔠצᓣʗ Ⴌᐹᤀᤀᓣᡥ
᤼Јዪપ ᥌ᓴႳ੡᤿᥌ᓴႳ੡ϕᖈԦᖈ ᥌ዪ ኃࣲᔱϕᖈ ᤼᧷પ᤼ ᣢࣲႳϕ᥁᥁
ཽϕ ᣞᓣᡥ ᒦᐹᤇᅳʗᐹᡙᅳʗᡥ ᅳஏ ጛዞᅳႥᅳቂᔠዞ ۗᤀᐹႥᡙ ᐹႥʐ ଧᔠᅳᡙᓣዞ၊Ⴅᅳᤀᅳ੖ᡥᖈ ᣞࣩႥቂᔠႥ੖ ᧇႥ໧ᡙᔠᡙࣩᡙᓣ ᅳஏ ଧᅳᡙᐹႥᡥᖈ ࣷ၊ᔠႥᓣ໧ᓣ ዛዞᐹʐᓣቂᡥ ᅳஏ ୒ዞᔠᓣႥዞᓣ໧ᖈ
ᣱࣲႳቑᔱႳ੡ ᣷጖ԗԦԗϕᖈ ऀၘᔱႳᑉଟ Ԧ ІႥᔠצᓣʗ໧ᔠᡙᡥ ᅳஏ ࣷ၊ᔠႥᓣ໧ᓣ ᅳஏ ୒ዞᔠᓣႥዞᓣ໧ᖈ ଲᓴᔱᢰᔱႳ੡ ϕԗԗԗ࿋ࣷᖈ ऀၘᔱႳᑉɥ
ዛᤇ໧ᡙʗᐹዞᡙ᧙ ዛႥᓣቂᅳዞᤀᓣቂᐹ ۲঄ ௿঄ ۲ᑉႳ੡ᖈ ᑉ ቑᆁႳᆁᡫᡷᡥᔱይ ੡ᓴႳࣲ໵ ᆁச ኃᑉႳࣲႳይࣲᤓᑉይᓴᑉᓴᖈ ᔱ໵ ᓴႳʑᓴቑᔱይ ᡫᆁ ᡫၘᓴ ȤᔱႳ໵ၘᑉ ኃᔱׯᓴʘ Ⴚᑉᤓᤓᓴᡷ ᔱႳ
໵ᆁࣲᡫၘऊᓴ໵ᡫ ऀၘᔱႳᑉ঄ ऀᆁቑᤚᔱႳᔱႳ੡ சᔱᓴᤓʑ ᔱႳׯᓴ໵ᡫᔱ੡ᑉᡫᔱᆁႳ ऊᔱᡫၘ ቑᆁᤓᓴይࣲᤓᑉʘ ቑᑉʘҪᓴʘ ᑉႳᑉᤓᡷ໵ᔱ໵ ཽଢ଼પ۠໵ɥᖈ ऊᓴ ᔱႳׯᓴ໵ᡫᔱ੡ᑉᡫᓴʑ ᡫၘᓴ ੡ᓴႳᓴᡫᔱይ
ʑᔱׯᓴʘ໵ᔱᡫᡷ ᑉႳʑ ੡ᓴႳᓴᡫᔱይ ໵ᡫʘࣲይᡫࣲʘᓴ ᆁச ዛॹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂ঄ ዪႳᑉᤓᡷ໵ᔱ໵ ᆁச ᡫၘʘᓴᓴ ይၘᤓᆁʘᆁᡥᤓᑉ໵ᡫ ᧉપዪ ཽይᡥᧉપዪɥ ʘᓴ੡ᔱᆁႳ໵ ཽ ʗᡓ໧ϕ᣷ ᔱႳ᤿
ᡫʘᆁႳᖈ ᡓ໧ᤇዪ᤿ᡙʗႥ̶ ᔱႳᡫᓴʘ੡ᓴႳᔱይ ໵ᡥᑉይᓴʘ ᑉႳʑ ᡙʗႥऀ᤿ᡥዞஏ᣷ ᔱႳᡫᓴʘ੡ᓴႳᔱይ ໵ᡥᑉይᓴʘɥ ʘᓴׯᓴᑉᤓᓴʑ ᑉ ᤓᆁऊ ᤓᓴׯᓴᤓ ᆁச ੡ᓴႳᓴᡫᔱይ ʑᔱׯᓴʘ໵ᔱᡫᡷ ऊᔱᡫၘᔱႳ
ᡫၘᓴ ໵ᡥᓴይᔱᓴ໵ᖈ ᤚࣲᡫ ၘᔱ੡ၘ ʑᔱׯᓴʘ੡ᓴႳይᓴ ᑉቑᆁႳ੡ ᡥᆁᡥࣲᤓᑉᡫᔱᆁႳ໵঄ ௿ၘᔱ໵ ੡ᓴႳᓴᡫᔱይ ໵ᡫʘࣲይᡫࣲʘᓴ ᔱ໵ ᡥᆁ໵໵ᔱᤚᤓᡷ ይᑉࣲ໵ᓴʑ ᤚᡷ ᑉ ᤓᆁႳ੡ ᡥᓴʘᔱᆁʑ ᆁச
ၘᔱ໵ᡫᆁʘᔱይᑉᤓ ੡ᓴᆁ੡ʘᑉᡥၘᔱይ ᔱ໵ᆁᤓᑉᡫᔱᆁႳᖈ ᑉ ʘᓴᤓᑉᡫᔱׯᓴᤓᡷ Ⴓᑉʘʘᆁऊ ʑᔱ໵ᡫʘᔱᤚࣲᡫᔱᆁႳ ʘᑉႳ੡ᓴ ᑉႳʑ ᤓᔱቑᔱᡫᓴʑ ੡ᓴႳᓴ சᤓᆁऊ ʑࣲᓴ ᡫᆁ ၘᑉᤚᔱᡫᑉᡫ சʘᑉ੡ቑᓴႳᡫᑉ᤿
ᡫᔱᆁႳ঄ ̶ᡷʑʘᆁᡥᆁऊᓴʘ ໵ᡫᑉᡫᔱᆁႳ໵ ᑉʘᓴ ໵ይၘᓴʑࣲᤓᓴʑ ᡫᆁ ᤚᓴ ᤚࣲᔱᤓᡫ ᔱႳ ᡫၘᓴ ȤᔱႳ໵ၘᑉ ኃᔱׯᓴʘ ʑʘᑉᔱႳᑉ੡ᓴ ໵ᡷ໵ᡫᓴቑ ᔱႳ ᑉʘᓴᑉ໵ ᡫၘᑉᡫ ᆁׯᓴʘᤓᑉᡥ ᡫၘᓴ
ʘᑉႳ੡ᓴ ᆁச ዛॹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂ ᑉႳʑ ᑉ໵ ᑉ ʘᓴ໵ࣲᤓᡫ ᆁச ᡫၘᓴᔱʘ ይᆁႳ໵ᡫʘࣲይᡫᔱᆁႳ ᡫၘᓴ ၘᑉᤚᔱᡫᑉᡫ໵ ᆁச ዛॹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂ ऊᔱᤓᤓ ᤚᓴ சᤓᆁᆁʑᓴʑ ᆁʘ ᑉʑׯᓴʘ໵ᓴ᤿
ᤓᡷ ᑉசசᓴይᡫᓴʑ ᔱႳ ᆁᡫၘᓴʘ ऊᑉᡷ໵ᖈ ᡫၘࣲ໵ ᡫၘʘᓴᑉᡫᓴႳᔱႳ੡ ᡫၘᓴ ໵ࣲʘׯᔱׯᑉᤓ ᆁச ᡫၘᓴ ໵ᡥᓴይᔱᓴ໵঄ ௿ၘᓴ ʘᓴ໵ࣲᤓᡫ໵ ᆁச ᆁࣲʘ ᑉႳᑉᤓᡷ໵ᔱ໵ ᆁச ੡ᓴႳᓴᡫᔱይ ʑᔱׯᓴʘ໵ᔱᡫᡷ
ᔱႳ ዛॹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂ ᑉʘᓴ ᆁச ׯᑉᤓࣲᓴ சᆁʘ ʑᓴׯᓴᤓᆁᡥᔱႳ੡ ᑉႳ ᑉᡥᡥʘᆁᡥʘᔱᑉᡫᓴ ይᆁႳ໵ᓴʘׯᑉᡫᔱᆁႳ ໵ᡫʘᑉᡫᓴ੡ᡷ சᆁʘ ᡫၘᔱ໵ ׯࣲᤓႳᓴʘᑉᤚᤓᓴ ໵ᡥᓴይᔱᓴ໵঄
ᣞᓣᡥ ँᅳʗʐ໧᧙ ዛႥᓣቂᅳዞᤀᓣቂᐹ ੖ᤀᐹࣩዞᔠஏᅳᤀᔠࣩቂଟ ይᡥᧉપዪଟ ଢ଼પ۠໵ଟ ᤼ᓴႳᓴᡫᔱይ ʑᔱׯᓴʘ໵ᔱᡫᡷଟ ऀᆁႳ໵ᓴʘׯᑉᡫᔱᆁႳ ੡ᓴႳᓴᡫᔱይ໵
঒ ȤᔱႳ໵ၘᑉ ኃᔱׯᓴʘ Ⴚᑉᤓᤓᓴᡷ ᤓᆁይᑉᡫᓴ໵ ᑉᡫ ᡫၘᓴ ᤚᑉ໵ᑉᤓ ओᆁႳᓴ ᆁச
ᡫၘᓴ ̶ᓴႳ੡ʑࣲᑉႳ ᥌ᆁࣲႳᡫᑉᔱႳ໵ ʘᓴ੡ᔱᆁႳᖈ ऊၘᔱይၘ ᔱ໵ ᆁႳᓴ ᆁச ᡫၘᓴ
Ԧ጖ ੡ᤓᆁᤚᑉᤓ ᤚᔱᆁʑᔱׯᓴʘ໵ᔱᡫᡷ ၘᆁᡫ໵ᡥᆁᡫ໵ ᑉ໵ சᓴᑉᡫࣲʘᔱႳ੡ ᓴନይᓴᡥ᤿
ᡫᔱᆁႳᑉᤓ ይᆁႳይᓴႳᡫʘᑉᡫᔱᆁႳ໵ ᆁச ᓴႳʑᓴቑᔱይ ໵ᡥᓴይᔱᓴ໵ ཽ᥌ᡷᓴʘ໵ ᓣᡙ
Ḻ ➖ ܳ ㆨ ̻ 䉱 ⎽ ႓ ្঒ Ԧԗϕሉᕷ ᇺጇ ཯጖ɤᧅ ጖጖጖ ጦ ጖᣷Ԧ
ۗᤀᐹႥᡙ ᦵᔠצᓣʗ໧ᔠᡙᡥ ᐹႥʐ ቴᓣ໧ᅳࣩʗዞᓣ໧঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ঒ ᧉ᧷᧛ᧅ ϕԗ঄ Ꭲ᣷ᎢᎢ ᣤ ᡷႳओऊᡷᢰԦԗϕሉϕԦϕሉԗ
᥁
᥁᥁
ÞࣲႳʑᔱႳ੡᧙ ௿ၘᔱ໵ ໵ᡫࣲʑᡷ ऊᑉ໵ ໵ࣲᡥᡥᆁʘᡫᓴʑ ᤚᡷ ੡ʘᑉႳᡫ໵ சʘᆁቑ ᡫၘᓴ પᑉᡫᔱᆁႳᑉᤓ પᑉᡫࣲʘᑉᤓ ଢ଼ይᔱᓴႳይᓴ ÞᆁࣲႳʑᑉᡫᔱᆁႳ ᆁச ऀၘᔱႳᑉ ཽሉԗआᎢԗԦ࿋Ԧɥ
ዪࣲᡫၘᆁʘ சᆁʘ ይᆁʘʘᓴ໵ᡥᆁႳʑᓴႳይᓴଟ ጪ᤿ቑᑉᔱᤓ᧙ ੡ᆁႳ੡ନࣲႳܶ ቑᑉᔱᤓ঄ Ҫᔱᤚ঄ ᑉይ঄ ይႳ
ኃᓴይᓴᔱׯᓴʑ ʑᑉᡫᓴ᧙ ԦԗϕԦͧϕԗͧԦ጖ᖈ ዪይይᓴᡥᡫᓴʑ ʑᑉᡫᓴ᧙ Ԧԗϕሉͧԗϕͧϕ጖
҉㔲キϸ᧙ Ⴥ㤹㤹 ཽϕࣷआआͧɥ ຠᖈ ⶂธᖈ ͨ㺮ϻθԊ៑䖄э႓ⵁ⾣ᤙ
al., 2000). It is a special and vulnerable ecosystem
characterized by aridity, high temperatures, semi鄄
savanna vegetation and relatively few plant species
(Jin et al., 1994). However, there are especially
rich for endemic genera (e. g. , Anemoclema, Noue鄄
lia, Musella, Ostryopsis, Trailliaedoxa) and species
(e. g. , Munronia delavayi, Aristolochia delavayi,
Vitex dulouxii, Cotinus nana, Mastixia microcarpa)
(Jin et al., 1994). Due to natural, historical and
anthropogenic factors, this ecosystem has become
seriously degraded, which has resulted in soil and
water losses and significant difficulties in vegetation
restoration.
Jinsha River, the upper part of the Changjiang
River, is one of the largest rivers in southwest Chi鄄
na, which originates from the Qinghai鄄Tibet plateau.
A total of 14 large鄄scale hydropower stations are
planning to be built along Jinsha River drainage, of
which, some have been building, such as, Xiangjia鄄
ba hydropower station, Xiluodu hydropower station
and so on. The construction of hydroelectric power
stations along Jinsha River will cause the rise of wa鄄
ter level, so that various types of ecosystem would
suffer serious destruction, and vertical climate would
disappear. Moreover, many species would extinct
owing to the submerged habitat in Jinsha River Val鄄
ley. Firstly, the habitat of some species would be
submerged, so that these species, especially endemic
species, might extinct. For example, the construc鄄
tion of the Three Gorges Dam caused the native area
of Myricaria laxiflora to be submerged. Fortunately,
ex situ conservation of M. laxiflora in advance pre鄄
served some individuals (Wu et al., 1998; Wang et
al., 2003 ). Secondly, various ecological types
would be destroyed after dams retained water. Verti鄄
cal climate would be changed, especially the tem鄄
perature and humidity of the valley. This change
would threaten the existence of species that had a鄄
dapted to dry鄄hot or dry鄄warm environment. Last,
there would be some direct influence on species in
construction area. The original vegetation covered
area would be occupied by construction and living
facilities, so that the area might lose ability to regu鄄
late the ecological environment.
If there is no effective measures to conduct con鄄
servation strategy and population reconstruction,
large鄄scale construction of hydropower stations along
Jinsha River would cause some species to extinct in
a short time, especially those populations close to
the river. Studies show that before implementing
conservation measures, genetic diversity and genetic
structure of the species must be studied and potential
benefits and risks of conservation strategy must be
analyzed. Results of Marshall and Brown爷 s study
show that in order to preserve species and maintain
normal evolution, more than 95% of the genetic di鄄
versity must be preserved ( Marshall and Brown,
1975).
Anemoclema W. T. Wang, a monotypic genus of
Ranunculaceae, is endemic to Jinsha River Valley in
southwest China (Zhang and Gong, 2002). Anemo鄄
clema glaucifolium (Franch. ) W. T. Wang is peren鄄
nial herb, 45-80 (-150) cm tall. The blossom is in
July to September (Liu, 1980) (Fig. 1). A. glaucifo鄄
lium grows on the rock slope of Jinsha River Valley,
where the climate is dry and hot or warm and the alti鄄
tude ranges from 1 500 m to 3 000 m. As the common
character species of this area, it is valuable in the
study of phylogeny and adaption to specific environ鄄
ment of plants (Jin, 2002). Due to high ornamental
value, A. glaucifolium may be cultivated as orna鄄
mental or act as breeding materials after being accli鄄
mated. Because A. glaucifolium is a monotypic ge鄄
nus species, there are rare relevant researches except
a few studies about molecular systematics and cytolo鄄
gy (Zhang and Gong, 2002; Wang et al., 2009).
Considering from the level of the state to deve鄄
lop Jinsha River爷s water resources, it is urgent to
protect threatened species. Detailed analysis of the
levels and spatial distribution of genetic diversity is
important for the development of effective conserva鄄
tion strategies and management practices for endan鄄
gered species (Hedrick and Miller, 1992). Therefore,
a conservation genetics research of A. glaucifolium
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before construction of hydropower stations on Jinsha
River is indispensable. The research explored the
distribution of A. glaucifolium by field investigation
and sampling, and studied the genetic diversity and
genetic structure through SNPs ( Single Nucleotide
Polymorphisms). Then we could elaborate genetic
background and the distribution of genetic variation
within and among populations, so that effective con鄄
servative strategy can be made. The research can not
only protect the endemic species A. glaucifolium,
but also provide experience and conservative mea鄄
sures to protect other biological group in this area.
Materials and methods
Population sampling
In our study, leaf tissue was collected from 133
individuals, representing 7 populations of A. glauci鄄
folium from its distribution range along Jinsha River
during July in 2008 to September in 2009. All popu鄄
lations were collected in Yunnan Province, except
for population QS, which was collected in Sichuan
province. Information about each sampling location
is presented in Fig. 2 and Table 1. Fresh and
healthy leaves were dried with silica gel and stored
at 4 益 .
Fig. 1摇 Anemoclema glaucifolium in wild. (A) The leaves of A. glaucifolium; (B) The flower of A. glaucifolium
Fig. 2摇 Map showing the distribution of cpDNA haplotypes in the species. The pie sizes of sampled
populations are proportional to their sample sizes
7555 期摇 摇 摇 摇 GUAN Meng鄄Meng et al. : Conservation Genetics of an Endemic Plant, Anemoclema glaucifolium …摇 摇 摇 摇
Table 1摇 Information of sampled populations of A. glaucifolium
Population
Code Locality
Sample
size
Latitude(N)
/ 毅
Longitude(E)
/ 毅
Altitude
/ m
摇 Haplotypes
摇 (no. of individuals)
1 QS Qiansuo, Yanyuan, Sichuan 29 28. 340 101. 258 2 650 摇 Hap2(2), Hap3(1)
2 YS Around Yongsheng, Yunnan 10 27. 374 100. 726 2 150 摇 Hap1(1), Hap2(2)
3 SNC Shuinichang, Ninglang, Yunnan 26 27. 724 101. 365 2 390 摇 Hap2(4)
4 LGH Luguhu, Ninglang, Yunnan 12 28. 083 101. 150 2 700 摇 Hap2(3)
5 HLT Xiangshan, Lijiang, Yunnan 24 27. 399 100. 517 2 530 摇 Hap4(3)
6 DJ Daju, Yulong, Yunnan 17 27. 799 100. 484 1 860 摇 Hap2(3)
7 ZD Hutiaoxia, Xianggelila, Yunnan 24 27. 699 100. 217 2 300 摇 Hap1(2), Hap2(1)
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted following the
CTAB protocol ( Doyle and Doyle, 1987 ). After
preliminary screening of a range of nucleic and or鄄
ganelle DNA, we chose three cpDNA fragments
( rps16、 psbA鄄trnH、 trnC鄄ycf6) for the full survey
because they contained the highest number of poly鄄
morphic sites.
PCR amplification was carried out in a 20 滋L
reaction volume, containing 30 ng template DNA,
2. 0 滋L 10 伊Taq Buffer (25 mM), 1. 5 - 1. 6 滋L
MaCl2 (25 mM), 1. 2-1. 5 滋L dNTPs (2. 5 Mm),
1. 0 滋L dimethyl sulfoxide (10 滋M), 0. 3-0. 35 滋L
each primer (10 滋M), 1. 5 unit of Taq polymerase
and double鄄distilled water. PCR was performed in a
T1 thermocycler ( Biometra, G觟ttingen, German),
with an initial denaturation at 94 益 for 3 min, fol鄄
lowed by 30-36 cycles of denaturation at 94 益 for
45 sec, annealing at 52-55 益 for 30 sec to 70 sec,
extension at 72 益 for 50 sec to 1. 5 min, and a final
extension cycle of 7 min at 72 益 . All PCR products
were purified directly using a PCR product purifica鄄
tion kit (Sangon, Shanghai). Purified PCR products
were sequenced in both directions with the same
primers for the amplification reactions, using a
3730xl DNA analyzer by Sangon Biotech ( Shang鄄
hai) Co., Ltd.
Sequences were aligned using Clustal X version
1. 83 (Thompson et al., 1997) and manually adjus鄄
ted using BioEdit 7. 0. 5 (Hall, 1999). Indels were
treated as the single mutation as substitution (Caice鄄
do and Schaal, 2004). DnaSP version 4. 10 (Rozas
et al., 2003) was used to calculate: (1) the num鄄
ber of haplotypes and variable sites; (2) nucleotide
diversity per site (仔) (Nei and Li, 1979); (3)
haplotype diversity (Hd) (Nei and Tajima, 1983).
Within鄄population diversity ( HS ), total diversity
(HT), and two measures of population differentia鄄
tion GST and NST were calculated using the HAP鄄
LONST. Program Arlequin version 3. 1 (Excoffier et
al., 2005) was used to conduct an analysis of mo鄄
lecular variance (AMOVA) (Excoffier et al., 1992)
and thus to estimate genetic variations within and a鄄
mong populations. Genealogical haplotype networks
(with 95% most parsimonious connection limits )
were constructed using TCS version 1. 21 (Temple鄄
ton and Sing, 1993; Clement et al., 2000). To in鄄
fer possible demographic expansion of A. glaucifoli鄄
um, mismatch distribution analysis, based on the
sudden population expansion model ( Rogers and
Harpending, 1992) using the observed number of
differences between pairs of haplotypes, was conduc鄄
ted with DnaSP.
Results
Nucleotide and haplotype diversity
The aligned cpDNA rps16 data matrix was 801
bp in length and contained two polymorphic sites
that resulted in two haplotypes. For the psbA鄄trnH
region, the aligned length was 354 bp, with three
haplotypes derived from two polymorphic sites. Re鄄
gion trnC鄄ycf6 was 429 bp in length, producing two
haplotypes with only one mutation.
The total length of the combined rps16、 psbA鄄
trnH、 trnC鄄ycf6 was 1 584 bp long with four substi鄄
tutions (84, C / A; 525, A / C; 972, T / G; 1453,
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C / A) and one indel (1138). A total of four haplo鄄
types (Hap1鄄Hap4) were identified when all the se鄄
quences were combined (Table 2). The nucleotide
diversity (仔) at the species level was 0. 00053 and
the total haplotype diversity (Hd) was 0. 325. In the
TCS network of cpDNA haplotypes, the most widely
distributed haplotype (Hap2) was in the interior po鄄
sition. The other three haplotypes were in the tip po鄄
sition, originating from Hap2 with one to three muta鄄
tional steps (Fig. 3).
Table 2摇 Variable sites and distribution of four
haplotypes of A. glaucifolium
Haplotype
Variable sites
rps16 psbA鄄trnH trnC鄄ycf6
84 525 972 1138 1453
Hap1 C A T - C
Hap2 C A T T C
Hap3 C A T T A
Hap4 A C G T C
-: indicates deletion
Fig. 3摇 Network of the four cpDNA haplotypes of A. glaucifolium.
Hap1 to Hap4 represent haplotypes. The size of each circle is
proportional to the haplotype frequency. Each solid line represents
one mutational step that interconnects two haplotypes. The small
bold circles indicate inferred intermediate haplotypes not
detected in this investigation
Haplotype frequencies in each population and
the distribution of the four haplotypes are presented
in Table 1 and Fig. 2. Hap2 was widely distributed
in every population except population HLT and fixed
in population SNC, LGH and DJ. Hap1 occurred in
population YS and ZD. Unique haploypes within
population were detected in population QS and HLT.
Population analysis
On the population level, total diversity (HT )
was estimated to be 0. 566 and within鄄population di鄄
versity (HS) was 0. 286 (Table 3). The comparison
between NST and GST did not show a significant
difference (NST = 0. 435, GST = 0. 495; P>0. 05).
For the total dataset, 90. 31% of variation existed
among populations, only 9. 69% within populations,
and a significant genetic differentiation was detected
(FST =0. 90) (Table 4).
Table 3摇 Genetic diversity and differentiation parameters
based on the cpDNA fragment of A. glaucifolium
HS HT GST NST
0. 286
(0. 1347)
0. 566
(0. 1527)
0. 495
(0. 2518)
0. 435
(0. 2866)
Standard errors are shown in parentheses
Table 4摇 AMOVA of the cpDNA fragment of A. glaucifolium
Source of
variation d. f. SS
Variance
components
Variation
/ %
Fixation
Index
(FST)
Among
populations 6 8. 06 0. 41 90. 31 0. 90
**
Within
populations 15 0. 67 0. 04 9. 69
Total 21 8. 73 0. 45
d. f. Degrees of freedom, SS sum of squares, **P<0. 001
Mismatch distribution analysis
The mismatch distribution based on the cpDNA
haplotype dataset for the total samples was multimo鄄
dal (Fig. 4) and inconsistent with the curve expec鄄
ted for an expanding population, indicating a demo鄄
graphic equilibrium (PSSD<0. 01).
Discussion
Genetic diversity
In this study, four haplotypes were found by
analyzing conjointly three chloroplast non鄄coding ar鄄
eas ( rps16 intron, psbA鄄trnH intergenic spacer and
trnC鄄ycf6 intergenic spacer). The nucleotide diver鄄
sity (仔) is 0. 00053 and the haplotype diversity
9555 期摇 摇 摇 摇 GUAN Meng鄄Meng et al. : Conservation Genetics of an Endemic Plant, Anemoclema glaucifolium …摇 摇 摇 摇
Fig. 4摇 Distribution of the number of pairwise nucleotide differences
for cpDNA haplotypes in A. glaucifolium. The dashed line shows
observed values, whereas the solid line represents expected values
under a model of sudden (stepwise) population expansion
(Hd) is 0. 325, both are lower compared with herb
plants in the similar distribution area (Chen et al.,
2008). Genetic diversity is the consequence of long鄄
term evolution. Its formation is the comprehensive
result of sorts of factors, including current range,
breeding system, seed dispersal mechanism and
some historical causes like Quaternary glacial fluctu鄄
ation, which can lead to repeated and drastic climate
changes. All of these changes may cause bottleneck
effect and founder effect, eventually, affecting dis鄄
tribution and genetic diversity of plants (Hamrick et
al., 1992). Unfortunately, information concerning
pollination mechanisms and breeding system for this
species is scarce. So we deduce the low genetic di鄄
versity of A. glaucifolium is the comprehensive result
of limited individuals and some historical cause. Low
average population genetic diversity (HS = 0. 286)
was also detected. The reason is that there is only
one haplotype in each four population out of the total
seven populations, which also leads to high genetic
differentiation among populations (GST =0. 495, NST
=0. 435).
Genetic structure
Concerning the geographic distribution of haplo鄄
types, the highest frequency Hap2 was distributed in
six populations except population HLT and was fixed
in three populations (SNC, LGH and DJ). Accord鄄
ing to coalescent theory, the haplotype in the interior
position likely represents the ancestral haplotype,
haplotype in tip position evolve from the ancestors
(Wakeley, 2008 ). Therefore, inferred from the
TCS network of cpDNA haplotypes (Fig. 4), we can
conclude that the widely distributed Hap2 is ances鄄
tral haplotype and the other haplotypes ( Hap1、
Hap3 and Hap4) all evolve from Hap2. Hap1 and
Hap3 originate from Hap2 with only one mutational
step. Moreover, Hap1 and Hap2 coexist in popula鄄
tion ZD and YS, while Hap2 and Hap3 coexist in
population QS. These further illustrate the evolution
relationship among Hap1, Hap3 and Hap2. In addi鄄
tion, the most southwestern population HLT fixes
Hap4, which may result from geographical isolation
or population expansion. But the mismatch distribu鄄
tion (Fig. 4) showed that a recent population expan鄄
sion was unlikely in this case, so the unique Hap4
fixed in population HLT may be the consequence of
long鄄term geographical isolation.
AMOVA showed that 90. 31% of genetic varia鄄
tion existed among population while variation within
population only accounted for 9. 69% of the whole
variation. In addition, we detected high level of ge鄄
netic differentiation (FST =0. 90), much higher than
the statistics studied by Petit (1999), which showing
average value of genetic markers of 97 plant (FST =
0. 70 ). Generally, species in fragmented habitat
tend to have lower population genetic diversity and
high genetic differentiation (Young et al., 1996),
which is in accordance with our results. Seven popu鄄
lations of A. glaucifolium in this study were distribu鄄
ted in separate habitat of fragmentation. The land鄄
scape characteristics of distribution area in Hengdu鄄
an Mountains, where high mountains and deep val鄄
leys are distributed alternately, block gene exchange
among populations ( Li and Li, 1993 ). Besides,
seeds of A. glaucifolium do not have special struc鄄
tures that facilitate long distance distribution or at鄄
tachment to the animals, so the seed dispersal ability
is limited. According to formula Nm = ( 1鄄GST ) /
4GST, we calculated the Nm of A. glaucifolium (Nm
= 0. 255), which is less than 1. Wright thought that
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if the value of gene flow among populations is less
than 1, limited gene flow is the main reason leading
to the genetic differentiation of the species (Wright,
1931).
Implications for conservation
A. glaucifolium is an endemic species in Jinsha
River Valley, which are in small numbers. The ge鄄
netic diversity of A. glaucifolium is relative low with
high genetic differentiation among populations.
Small populations of rare and endangered species
have higher risks of extinction than larger stable
ones, especially when gene flow between populations
is restricted ( Frankham et al., 2002 ). As small
populations are more susceptible to the loss of genet鄄
ic diversity caused by genetic drift and inbreeding,
which reduces heterozygosity and the performance of
various fitness related traits.
According to field investigation, in some popu鄄
lations (YS、 LGH), not only the population size is
small, but also the number of adult individuals is
rare, showing A. glaucifolium is in dangerous situa鄄
tion of extinction. Moreover, the narrow distribution
area and fragmented habitat result in limited gene
flow among populations, so that genetic drift of this
species occurred easily. Human activities also cause
significant damages to A. glaucifolium, especially
developing hydroelectric power in Jinshan River Val鄄
ley. 1) The construction of hydroelectric power sta鄄
tions in Jinsha River Valley will change the local cli鄄
mate with greatly increasing the water surface area.
On one hand, humidity will increase, which is
harmful to A. glaucifolium. Because A. glaucifolium
has adapted to drought environment. On the other
hand, the daily and annual temperature ranges will
narrow, which may alter the phenophase of A.
glaucifolium. 2) The wider river will interfere with
the exchange of genes between populations located
on opposite sides of the Jinsha River. 3) The hydro鄄
power station construction process produces a large
amount of waste, which will change the soil struc鄄
ture, influence the local ecological environment and
affect local plant growth (Mu et al., 2010). 4) The
current available farmland will be flooded, so the
wilderness, where A. glaucifolium may grow, will be
cultivated as farmland leading to the loss of the habi鄄
tat of A. glaucifolium. Therefore, it is urgent to con鄄
duct effective conservation strategy.
Modern conservation biology aims at protecting
not only species and habitats, but also genetic diver鄄
sity of extant species for its evolutionary potentiality
(Caughley and Gunn, 1996). Protection of genetic
diversity needs to carry out corresponding measures
according to genetic differences among populations.
As to the development of protection strategy, genetic
uniqueness is the main reference to identify prior
population to protect (Arthur et al., 2004). In our
study, A. glaucifolium showed relatively low genetic
diversity and high differentiation among populations.
Population QS, HLT and YS contribute most to the
total genetic diversity and allelic richness, and Hap
4 in HLT is unique. Therefore, these three popula鄄
tions should be given priority of protection.
Taking into account current distribution and
conservation status of A. glaucifolium, for population
QS, HLT and YS, in situ conservation strategy
would be of practical value. Haplotypes in popula鄄
tion ZD is the same as YS. But population ZD is
close to Jinsha River and will be affected by hydro鄄
power station construction. Therefore, it is necessary
to conduct ex situ conservation in population ZD,
and a new population could be reestablished in the
location with similar habitat afterwards. The seeds
from all populations can be collected and preserved
in Germplasm Bank of Wild Species in Southwest
China. For the rest populations, in situ conservation
is recommended.
Acknowledgments: We appreciate lab members for their
generous help.
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