全 文 ：东亚———北美东部间断透骨草属的扩增片段长度多态性
?
聂泽龙1 , 2 , 文 军1 ,3 ,4 , 孙 航1
??
(1 中国科学院昆明植物研究所生物多样性与生物地理重点实验室 , 云南 昆明 650204; 2 中国科学院
研究生院 , 北京 100049; 3 美国史密森研究院国家自然历史博物馆植物系 , 华盛顿 20013-7012;
4 中国科学院植物研究所系统与进化植物学重点实验室 , 北京 100093 )
摘要 : 透骨草属 ( Phryma) 是一个单种属 , 间断分布于东亚与北美东部。尽管东亚与北美东部居群形态差
异非常小 , 但分子变异却非常明显。本研究进一步运用 AFLP 两对引物来衡量透骨草属的遗传多样性并评
估其形态保守性。结果发现透骨草的遗传差异主要存在于两大洲的居群之间。聚类与 PCA 分析显示透骨
草分成两大支与其地理分布相吻合 , 一支全部来自东亚 , 另一支则是北美东部的居群。我们的结果强烈支
持透骨草东亚———北美东部居群存在明显的遗传分化和形态保守。
关键词 : AFLP; 洲际间断 ; 东亚 ; 北美东部 ; 遗传多样性 ; 透骨草 ; 透骨草科
中图分类号 : Q 16 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 04 - 289 - 07
AFLP Analysis of Phryma (Phrymaceae) Disjunct between
Eastern Asia and Eastern North America
NIE Ze-Long
1 , 2
, WEN Jun
1 , 3 , 4
, SUN Hang
1 *
(1 KeyLaboratory of Biodiversity and Biogeography, Kunming Instituteof Botany, ChineseAcademy of Sciences, Kunming 650204 , China;
2 GraduateUniversity of ChineseAcademy of Sciences, Beijing 100049 , China; 3 Department of Botany, National Museumof
Natural History, MRC 166 , Smithsonian Institution, Washington, DC 20013-7012 , USA ; 4 Key Laboratory of Systematic
and Evolutionary Botany, Instituteof Botany, Chinese Academy of Sciences, Beijing 100093 , China)
Abstract : Although littlemorphological differentiation is detected between intercontinental disjunct populations of Phryma,
this monotypic genus shows distinct molecular divergencecorresponding to its distribution in eastern Asia and eastern North
America . This study further employs amplified fragment length polymorphism (AFLP) analyses using two selective primer
pairs to quantify genetic diversity and evaluate the morphological stasis . Most of themolecular variance is accounted for by
variance among populations betweenregions .Cluster and PCA analyses revealedthat Phryma constitutes twomajor groupsin line
with their geographic distribution, with one genetically distinct group fromthe eastern Asia and the other with accessions from
eastern North America . The results robustly supportedthe distinct genetic divergenceand morphological stasis in Phryma .
Key words: AFLP; Intercontinental disjunction; Eastern Asia; EasternNorth America; Genetic diversity; Phryma; Phrymaceae
Phryma L . ( Phrymaceae) is well - known for its
unusual morphology with a derived pseudomonomerous
gynoecium ( bicarpellate withone carpel reduced devel-
opmentally) and the classical intercontinental disjunct
distribution between eastern Asia and eastern North
America (Li , 1952; Wen, 1999) . It is also oneof the
云 南 植 物 研 究 2009 , 31 (4) : 289～295
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.09075
?
?? ?Author for correspondence; E-mail : hsun@ mail . kib. ac. cn
Received date: 2009 - 04 - 16 , Accepted date: 2009 - 06 - 09
作者简介 : 聂泽龙 (1973 - ) 男 , 博士 , 副研究员 , 硕导 , 主要从事植物分子系统发育与生物地理以及细胞地理学研究。 ?
Foun ?dation item: Xibuzhiguang (Light in Western China) project of the ChineseAcademy of Science (to Z . -L . Nie) , theNatural Science Foun-
dation of China ( NSFC 30625004 and 40771073 to H . Sun) , the Yunnan Natural Science Foundation (2008CC013 to H . Sun) and the John
D . and Catherine T . MacArthur Foundation (to J . Wen) .
very few monospecific genera with such an interconti-
nental disjunction . Two varieties ( Hara, 1966;
Thieret, 1972 ) or subspecies (Kitamura and Miurata,
1957; Li , 2000) have been recognized within the sin-
gle intercontinental disjunct species Phryma lep-
tostachya L ., corresponding to its biogeographic distri-
bution in eastern Asia and eastern North America . A
high level of morphological similarity has been reported
for the two disjunct subspecies ( Hara, 1969; Ramana
et al. , 1983) , but companied with distinct molecular
divergence (Nie et al. , 2006) .
Hara (1966) pointed out that the intercontinental
disjunct populations of Phryma were identical in most
morphological characters, cytology, and ecological
habitats . For example, populations from both conti-
nents bear flowers that are erect in bud but later
spreading or becoming deflexed; they have the same
chromosome number of 2 n= 28; and they share similar
habitats of deciduous or mixed forests . Plants fromthe
two different regions only differ slightly in leaf size,
shape of upper lip of the corolla, and length of theup-
per spinulose calyx-lobes (Hara, 1962 , 1966 , 1969;
Li , 2000; Nie et al. , 2006) .
Although a high level of morphological similarity
has been found in Phryma, recent molecular studies
revealed substantial molecular divergence between the
two intercontinental varieties ( Beardsley and Olmstead,
2002; Lee et al. , 1996; Nie et al. , 2006; Xiang et
al. , 2000 ) . A low level of isozyme identity ( 0 . 291)
from allozyme loci and high ITS divergence (4 . 46% )
were detected between the disjunct pair in Phryma
(Lee et al. , 1996) .
The eastern Asian-eastern North American dis-
junction is a well-known and classical biogeographic
pattern in theNorthern Hemisphere that has received
considerable attention in thelast 15 years (Donoghue et
al. , 2001; Meng et al. , 2003; Milne, 2006; Milne and
Abbott, 2002; Ran et al. , 2006; Wen, 1998 , 1999 ,
2001; Wen et al. , 1996; Wen et al. , 1998; Wen and
Stuessy, 1993; Wen and Zimmer, 1996; Xiang et al. ,
1996 , 1998, 2000; Zhou et al. , 2006 ) . Most studies
on taxa with disjunct distribution in these two regions
(especially for the disjunct species) have focused on
phylogenetic relationship and biogeographic history at
the interspecific level ( Nie et al. , 2006 ) , yet few
have analyzed patterns of genetic variation among the
disjunct taxa at the population level . The objectives of
this preliminary study are to exploregenetic divergence
between intercontinental disjunct populations of Phry-
ma and examine thephylogeographic structure and bio-
geographic implications of Phryma in the Northern
Hemisphere .
1 Materials and Methods
Seven populations were analyzed with three from eastern
Asia and four from eastern North America (Table 1) . Genomic
DNA was extractedfrom15 mg silicadried leaf material usingthe
modified CTAB method (Doyle and Doyle, 1987) .
Procedures for AFLPswere as described by Vos et al. with
somemodifications (Vos et al. , 1995) . 400 - 500 ng DNA of
each sample was digested and ligated with5 U of EcoRI and1 U
of MseI in areactionmixturethat contained1X T4 restrictionand
ligase buffer with ATP, 0. 5 μmol L - 1 EcoRI adapter, 5μmol
L - 1 MseI adapter, 50 mmol L - 1 NaCl , 5 ngBSA , 1 U T4-DNA
ligase, and water to a final volume of 10μl . After digestion and
ligation, products were diluted 1∶20 with distilled water . In the
first round of PCR ( pre-selective amplification) , each reaction
contained2 .0μl Promega 10X reaction buffer, 2. 0μl dNTPs ( 2
mM) , 10.0 μl water, 2 . 0 μl Promega Taq Polymerase ( 5 U
μL - 1 ) , 0 . 5μl MseI + C primer ( 50 ngμL - 1 ) , 0. 5μl EcoRI
+ A primer (50 ngμL - 1 ) , and 2 .0μl of the diluted digestion?
Table 1 Voucher information of Phryma populations sampled in this study
Taxon Abbreviation Voucher Locality
EN1 ?Wen 7140 ( US) USA : Illinois
Phryma leptostachya L . var . leptostachya EN2 ?Wen 7161 ( US) USA : Illinois
(eastern North America) EN3 ?Wen 7188 ( US) USA : Alabama
EN4 ?Wen 7292 ( US) USA : Wisconsin
Phryma leptostachya var ?. asiatica Hara EA1 Nie 102 K( KUN) China: Yunnan
(eastern Asia) EA2 ?Yue 122 [(KUN) China: Sichuan
EA5 ?Wen 5757 ( US) China: Yunnan
092 云 南 植 物 研 究 31 卷
ligation product . The PCR conditions were: 94℃ for 20 sec,
56℃ for 30 sec, and 72℃ for 2 min, for 19 cycles . The PCR
products were diluted 1∶20 with water . Samples used in the
bulked DNA study were run individually through the preselective
amplification step . Subsequently, 10μl of the diluted preselec-
tiveamplification from each of three individuals was combined .
In the secondround of PCR (selectiveamplification) , each reac-
tion contained1 .5μl Promega10X reaction buffer, 1 . 0μl dNTPs
(2 mM) , 11 .275μl water, 1 . 5μl PromegaTaq Polymerase (5 U
μL - 1 ) , 0 .5 μl MseI + XXX primer ( 50 ngμL - 1 ) , 0 . 5 μl
EcoRI + XXX ( + dye) primer (50 ngμL - 1 ) , and 1.5μl of the
preselective product . The PCR conditions were: 94℃ for 20
sec, 66℃ for 30 sec, and 72℃ for 2 min, 9 cycles; 94℃ for
20 sec, 56℃ for 30 sec, and 72℃ for 2 min, 30 cycles . All
sampleswere processed in randomorder and subsampleswere re-
run for internal control . Selective amplification products were
separated usingLongRangerSingel packs ( BioWhittakerMolecu-
lar Applications, cat . # 50691 ) running for about 2 hours on
the ABI Prism377 genetic analyzer by combining0 .4μl of selec-
tive amplification product with 1 .2μl of loading buffer containing
0 .75μl formamide, 0. 30 μl GeneScan ROX-500 internal size
standard, and 0 .15μl loading dye .
We identified and sized the peaks between 50 and500 bp in
the ABI gel images using GeneScan 3 .1 (Applied Biosystems) to
create sample files . Sample trace fileswere imported into Genog-
rapher (ver 1.6 , Montana StateUniversity, 2001 ; http:??horde-
um.msu.montana. edu?genographer?) for visualization and scor-
ing . AFLP loci were analyzed using the“thumbnail”option of
Genographer, which allows for comparison of signal strength at
each locusfor all samples . Onlypolymorphic AFLP markerswere
scoredfor each sample and recorded as a binary character with
presence as“1”and absence as“ 0”. Monomorphic markers
were not scored .
The genetic differentiation between populations of Phryma
basedon all AFLP loci was calculated using Nei′s genetic dis-
tance (Nei, 1978 ) . A cluster analysis usingtheunweighted pair-
group method with arithmetic averaging (UPGMA ) (Sneath and
Sokal, 1973) was performed using the software POPGENE 1 .32
(Yeh et al. , 1997) . A genetic distances matrix was also used to
perform a hierarchical analysis of molecular variance (AMOVA)
(Excoffier et al. , 1992 ) , using ARLEQUIN version 3 .11 ( Ex-
coffier et al. , 2005 ) , to partition the total genetic variation
among individualswithin populations, between populationswithin
a region, and between regions of eastern Asia and eastern North
America . These are not directly comparable to the traditional F-
statistics (Wright, 1951) and are therefore referred to asΦ-sta-
tistics ( Excoffier et al. , 1992 ) . The significance of Φ values
was tested by 1000 permutations .
The AFLP data were also subjected to a principal compo-
nents analysis ( PCA) , which may help reveal unexpected rela-
tionships amonga large number of variables into twoor three new
uncorrelated variables so that they retain most of the original in-
formation . Similarity matrices usingthe Jaccard′s similarity coef-
ficient were generated . Eigenvalue and eigenvector matrices were
calculated fromthe similarity matrix . The standardized data were
projected onto theeigenvectors of thecorrelationmatrix and repr-
esented in a two-dimensional scatter plot . Plots of samples in re-
lation to the first three principal components were constructed
with populations designated as either eastern Asian or eastern
North American distribution . PCAs were performed using the
computer programNTSYSpc, version 2 .11h ( Rohlf , 2000) .
2 Results
The two AFLP primer pairs M-CTC?E-ACT and
M-CTC?E-AGG generated a total of 426 polymorphic
markers from80 individuals for seven populations . Ta-
ble 2 shows the genetic differentiation [ Nei′genetic
distance; (Nei , 1978 ) ] between Phryma populations .
The genetic distance matrix was used to establish the
level of genetic divergence between the populations
(Table 2 ) . Estimates of genetic distance using AFLP
data ranged from 0 .027 for the most closely related
populations within each region ( EN1 and EN2 ) , to
0 .811 in the most divergent populations between re-
gions ( EA1 and EN3) . These results indicate that ge-
netic distance is solely dependent on geographical dis-
tance, and clustering based on genetic distances re-
flects geographical relationships .
Table 2 Nei′s genetic distance of Phryma populations estimated
by AFLP analysis ( Population codes are as in Table 1)
Population EA1 {EA2 ?EA5 EN1 EN2 EN3 ?EN4
EA1 3-
EA2 30 ?. 1367 -
EA5 30 ?. 1510 0 ?. 1248 -
EN1 30 ?. 6233 0 ?. 5133 0 +. 4235 -
EN2 30 ?. 6214 0 ?. 5438 0 +. 4379 0 J. 0274 -
EN3 30 ?. 8114 0 ?. 6483 0 +. 5826 0 J. 0496 0 i. 1032 -
EN4 30 ?. 6526 0 ?. 5948 0 +. 5361 0 J. 0689 0 i. 0535 0 .1179 -
The results of the AMOVA based on AFLP varia-
tion are shown in Table 3 . Most of the total variance
was found between regions ( 60 .57% , P < 0 .0001 ) .
The correspondingΦCT value was 0.61 ( P < 0 .0001) .
The among-populations?within-regions percentage was
relatively low (18 .2% , P < 0 .0001) when compared
1924 期 NIE Ze-Long et al . : AFLP Analysis of Phryma (Phrymacea) Disjunct between Eastern Asia . . .
Table 3 Results of hierarchical analysis of molecular variance ( AMOVA ) based on AFLP markers between eastern North American and eastern
Asian populations of Phryma . P-values, calculated from a random permutation test (1000 replicates) , andΦ-statistics represent the
probability of obtaining by chance alone a more extreme variance than theobserved values ( Excoffier et al. , 1992)
Sourceof variation d ?. f . SS Variance component % of variance Φ-statistics P
Between regions 1 ?396 .084 9 . 38894 60 L. 57 ΦCT = 0 .60574 < 0 .0001
Among populations within regions 7 ?187 .236 2 . 82036 18 L. 20 ΦSC = 0 .46152 < 0 .0001
Within populations 73 &240 ?. 216 3 . 29064 21 L. 23 ΦST = 0 .78770 < 0 .0001
Total 81 &823 ?. 537 15 3. 49993
to the within-population variance component, close to
that of the among-individuals within populations
(21 .23% , P < 0 .001) . All threeΦ-values were sig-
nificant based on 1000 permutation tests (Table 3 ) .
The UPGMA dendrogram based on the genetic
distances between populations showed two main clusters
of populations that correlated to their continental distri-
bution (Fig . 1) . ThePCA of the AFLP-based distance
data was performed to examine relationships among
Phryma populations . The first and second principal co-
ordinates described approximately 44% and 11% of
the total variation, respectively ( Fig . 2 ) . Similar to
the UPGMA dendrogram, the PCA showed that eastern
Asian populations clustered apart fromthose of eastern
North America .
3 Discussion
The AMOVA analyses showed that thelargest por-
tion ( 61% ) of genetic variance is contributed by ge-
netic variation between intercontinental regions of east-
ern Asia and eastern North America and only a small
part is due to divergence among populations within re-
gions (18% ) and within populations ( 21% ) . The re-
sults are similar to the pairwise sequence divergenceof
ITS, rps16 , and trnL-F markers (Nie et al. , 2006 ) .
For example, the ITS divergence of the two varieties
between eastern Asia and eastern North America
(3 . 11% - 4 .41% ) was higher than that amongpopula-
tions within each continent ( 1 . 63% - 0 .65% ) . The
high level of genetic divergence between regions sug-
gests that variations between the two continents display
a large proportion of genetic loci of high allelic varia-
tions .
The long timeof geographic isolation (Lee et al. ,
1996; Nie et al. , 2006; Xiang et al. , 2000 ) may
contribute to thehighmolecular differentiationof Phry-
ma between regions observed . Several previous studies
estimated the divergence times for the disjunct Phryma
varieties using various dating approaches . Lee et al .
(1996) reported the divergence time to be over 20 mil-
lion years ago (mya) using allozymedata and 12 .35 mya
using ITS sequences . Xiang et al . (2000) estimated the
divergence times for thegenus with rbcL sequence data
and a molecular clock calibrated with Cornus fossils .
They estimated that the two varieties of Phryma di-
verged about 5 .85±2.66 mya . Recently we estimated
their divergencetimeas3.68±2.25 to 5.23±1 .37 mya
based on combined chloroplast data using both the
Bayesian dating and the penalized likelihood methods
with relaxed molecular clocks (Nie et al. , 2006) .
Genetic divergence within the eastern North
American populations is less than that within the east-
ern Asian populations ( Table 2) , although only a few
populations sampled in this study . A similar situation
is found with the DNA sequences with more extensive
sampling fromour previous study (Nie et al. , 2006 ) .
The eastern Asian populations are reported to be more
heterogeneous in ITS sequence variation than those in
the eastern North America ( maximum divergence of
1 .63% vs . 0 . 65% ) . As suggested by Nie et al .
( 2006 ) , the more genetic variation in the eastern
Asian Phryma may be due to higher level of geographic
isolation with the more heterogeneous and pronounced
topographies in eastern Asia ( Qian and Ricklefs,
2000; Wen, 1999; Xiang et al. , 2004) .
Morphological stasis in Phryma is also supported
by genetic analysis at the population level . Little mor-
phological variationhasbeenobserved fromthedisjunct
pairs of Phryma (Holm, 1913; Li , 2000) . Multivari-
ate analysis based on 23 quantitative morphological
characters also revealed that no significant variations
werefound between them (Nie et al. , 2006) . However,
292 云 南 植 物 研 究 31 卷
Fig . 1 UPGMA dendrogram based on pairwisegenetic distance showing relationships between Phryma
populations fromAFLP data . (● = EN samples; ○ = EA samples)
3924 期 NIE Ze-Long et al . : AFLP Analysis of Phryma (Phrymacea) Disjunct between Eastern Asia . . .
Fig . 2 Principal component analysis of AFLP data . Accessions are
plotted according to thevalues of the first ( x-axis) and the second
( y-axis) components and with different symbols according to
geographical origin (● = EN samples; ○ = EA samples)
AFLP analysis revealed a clear geographical pattern of
genetic variation, consistent with our findings fromthe
sequence data (Nie et al. , 2006 ) . The results showed
that Phryma is agenetically diverse entity, and AFLP
markers can be effectively employed to assess genetic
diversity and to measure genetic relationship among
populations .
Morphological stasis was suggested to explain the
discordanceof thegenetic andmorphological ratesof ev-
olution (Nie et al. , 2006) . Among various possible ex-
planations for stasis in morphology, a relatively constant
environment with the concomitant action of stabilizing
selection, might be the most plausible ( Nie et al. ,
2006; Wen, 1999) . The intercontinental populations of
Phryma occupy similar habitats in rich mesic to moist,
deciduous or mixed deciduous and evergreen forests in
both eastern Asia and eastern North America . These
types of forests covered most of the temperate regions of
the Northern Hemisphere in the Tertiary , but can be
found today only in southeast North America and in
eastern to central China and central to southern Japan
(Milne, 2006; Milne and Abbott, 2002; Nie et al. ,
2008; Wen, 1999) .
In present study, our sampling is limited with
only three eastern Asian populations from SW China
and four populations fromeastern North America . Nev-
ertheless, our AFLP results at thepopulation level cor-
roborateour findings in the sequence analyses (Nie et
al. , 2006) . Both the AFLP ( Table 2 ) and DNA se-
quence data ( Nie et al. , 2006 ) suggested that the
eastern Asian populations havemuch higher genetic and
molecular divergence than that of eastern North Ameri-
ca . Comparative phylogeographic analysis ( Avise,
2000) of the disjunct taxa is very important to our un-
derstandingof mechanisms responsible for their phylo-
genetic relationships and disjunct distribution . Future
studies are planned to extensively sample the popula-
tions on both continents to better understand the rela-
tionships amonggenetic divergence, morphological dif-
ferentiation, and the development of the intercontinen-
tal disjunct pattern . We intend to employ phylogeo-
graphic analysis, as implemented in Gao et al . (2007)
and Meng et al . ( 2007 ) , but analyze the Phryma
populations intercontinentally and intracontinentally .
Acknowledgements : Laboratory work was conducted in and par-
tially supported by the Pritzker Laboratory for Molecular System-
atics and Evolution of the Field Museum .
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