全 文 ：Assessing Biogeographic Relationships of Some
Species in the Lichen Family Umbilicariaceae
Based on Nuclear ITS rDNA Sequence

GUO Shou-yu1 , HUANG Man-rong2
(1.State Key Laboratory of Mycology , Institute of Microbiology , Chinese Academy of Sciences , Beijing
100101 , China ;2.Beijing Museum of Natural History , Beijing 100050 , China)
Abstract:A close biogeographic relationship between the macrolichens of eastern North America and
eastern Asia has rarely been tested within a phylogenetic context.Here we examine relationships of four
species within two genera , Lasallia and Umbilicaria of the lichen family Umbilcariaceae chosen as exam-
ples of disjunct and vicarious distribution patterns.Nuclear ribosomal DNA ITS sequences for 10 samples
belonging to 8 species were obtained and the data set was used to construct the phylogeny and estimate the
divergence dates for species examined.All of the three tested putative disjunct species pairs were sup-
ported.U.muehlenbergii and L .pennsylvanica are conspecific disjuncts , while U.esculenta from
eastern Asia and U.mammulata from eastern North America are allospecific disjunct.The divergence
time estimated show that the speciation of U.esculenta and U.mammulata occurred in the middle of
Miocene (23—5.33 Mya), during the period of the formation of Bering strait.
Key words:biogeography;disjunct distribution;Umbilicaria;Lasallia;Lichen
CLC number:Q949.34　　　Document code:A　　　Article ID:1672-3538(2012)03-0147-07
几种石耳科地衣生物地理学关系的分子评估
郭守玉1＊＊ , 黄满荣2
(1.中国科学院微生物研究所真菌学国家重点实验室 , 北京 100101;2.北京自然博物馆 ,北京
100050)
摘　要:对具有北美东部和亚洲东部近缘生物地理学关系的大型地衣种类进行系统发育检测较少。笔者获
得了石耳科中 8个种 10 个样本nrDNA的 ITS完整序列 ,并对本科中在分布上具有重要意义的 4 个种的生物地
理学关系进行了分析和评估。 通过对细胞核核糖体 DNA 变异性较强的 ITS 段碱基序列的比较分析 ,支持放
射盘石耳(Umbilicaria muehlenbergii)、宾州疱脐衣(Lasallia pennsylvanica)为同种型间断分布 , 美味石耳(U.escu-
lenta)和大叶石耳(U.mammulata)间已经达到了相当高的分化程度 , 为异种型间断分布。 地理学分析显示该
两种石耳的分化可能是由于太平洋的扩展和隔离所造成的结果。同种型间断分布种类的不同地区的样本之
间的差异较小。推测了不同类型间断分布种类的分化时间 , 美味石耳与大叶石耳的分化发生在中新世(23 ～
5.33 Mya)中期 , 白令海峡形成期间。

Corresponding author:GUO Shou-yu , E-mail:guosy@im.ac.cn
Foundation item:National Natural Science Foundation of China(30770012)
Biography:GUO Shou-yu , male , PhD , majoring in lichenology , biogeography and molecular systematics.
Received date:2012-06-14
　第 10卷第 3 期 菌　物　研　究 Vol.10 No.3　
　2012 年 9月 Journal　of　Fungal　Research Sep.2012　
关键词:生物地理学;间断分布;石耳属;疱脐衣属;地衣
中图分类号:Q949.34　　　文献标识码:A　　　文章编号:1672-3538(2012)03-0147-07
引文格式:郭守玉 , 黄满荣.几种石耳科地衣生物地理学关系的分子评估[ J] .菌物研究 , 2012 , 10(3):147-153.
　　Lichens , as the special fungi , usually have wider
distributions than vascular plants.However , some
species have well-defined disjunction patterns , where
populations of the same species or closely related taxa
present discontinuous distributions.One of the intrigu-
ing and understudied questions in the biogeography of
macrofungi and macrolichens is the relationships be-
tween putative eastern Asian and eastern North Ameri-
can disjunct species[ 1] .Results from the studies on the
north tropical and temperate disjunct distributions of
some macrofungi (including macrolichens) demon-
strate:1)the similarity between the mycota of eastern
Asia and eastern North America is relatively high com-
pared to that observed between other regions in the
north tropical and temperate zones;2)the disjunct dis-
tribution of macrofungi is usually only seen at species or
lower taxonomic levels.The results , however , are
greatly influenced by authors′species concepts and in-
terpretations of morphological characters.Thus , the
phylogeny analysis of putative disjunct fungal and/or
lichen species is necessary to rigorously test the eastern
Asian-eastern North American affinity hypothesis.
Studies on phylogeny and its implications for under-
standing the biogeography of macrolichens become pos-
sible in recent years due to the advancements in DNA
sequencing and phylogenetic analysis methods.
Similarities between the biota of eastern North
America and eastern Asia have been noted and dis-
cussed for nearly 300 years[ 2] .This distribution pattern
is widely documented for plants and fungi , and similar
biogeographic relationships have been noted for
lichens[ 3-6] .The objective of this study is to investigate
genetic difference and divergence time between geo-
graphic isolated populations of selected taxa in Umbil-
cariaceae from China and North America using se-
quencing data of ITS region in the nuclear ribosome
RNA gene.
1　Materials and methods
The materials for the research were based on the
collections preserved in the Herbarium of Mycology ,
Chinese Academy of Sciences , Lichenology Section
(HMAS-L).Some freshmaterials were collected from
China and USA in 1996 and 1997 , and stored at
-20℃(Table 1).
Table 1.The species studied with GenBank accession numbers and their geographical distribution
Genus Subgenus Species　　 GenBank accession Dist ribution
Lasallia L.pensylvanica (CN) AF297674 Multirange(Alpine)
L.pensylvanica (US) AF297675
Umbi licaria Actinogyra U.muehlenbergii(CN) AF297666 North American-Asian
U.muehlenbergii(US) AF297667
Agyrophora U.lyngei AF297669 Holarctic
Omphalodiscus U.virginis AF297673 Multirange(Alpine)
Umbilicaria U.esculenta AF284447 East Asian
U.flocculosa AF297670 Multirange(Alpine)
U.loboperipherica AF297671 East Asian
U.mammulata AF284448 North American
148 菌　物　研　究 2012 年
　　DNA preparation.The DNA was isolated following
the Puregene DNA Extraction Protocol (FMNH Bio-
chemistry Lab) and the procedure of Gardes and
Bruns
[ 7] .Fragments were amplified with the primers
ITS5 and ITS4 , ITS1 and ITS4[ 8] .
Both complementary strands were sequenced using
Dye Terminator Cycle Sequencing Ready Reaction Kit
(Perkin Elmer)according to the manufacturer′s in-
struction.ITS5 , ITS1 , and ITS4 were used as se-
quencing primers , Cycle sequencing was carried out for
25 cycles with denaturation at 96℃ for 30 seconds ,
annealing at 50℃ for 15 seconds , and extension at
60℃ for 4 minutes.The DNA was cleaned through Se-
quenced fragments were analyzed using an ABI 377
DNA automatic sequencer(Perkin Elmer).
Data Analyses.Boundaries of the ITS , ITS1 ,
5.8S , and ITS2 regions were identified in comparison
with other available sequences[ 9] .Sequences were
aligned with DNAMAN4.0(Lynnon Biosoft)and trans-
ferred to PAUP＊ 4.0b8a[ 10] .The most parsimony tree
(MPT)was obtained using the heuristic search with
tree-bisection-reconnection (TBR) branch swapping
and up to 1 000 random-addition sequence replica-
tions.Clade stability of most parsimonious trees was
estimated by 1 000 bootstrap replicates[ 11] using the
same parameter set up in the initial heuristic search.
MEGA5[ 12] was applied to construct the neighbor-join-
ing (NJ)and ML trees for the data sets according to
Kimura′s two-parameter model[ 13] .The genetic dis-
tance was also calculated with MEGA5 based on num-
ber of nucleotide difference , positions with gaps and
missing data were excluded.
Molecular dating.In the absence of fossil evi-
dence in lichens , the molecular evolution rates for ITS
(2.52 × 10-9) reported for a group of As-
comycetes[ 14] , were used to estimate the time to the
most recent common ancestor (MRCA)for all clades
resulting from the Bayesian phylogeny of the ITS data
set.
Estimated ages of divergence were obtained using
the MCMC algorithm as implemented in the MCMCtree
program in the PAML package[ 15] following the ap-
proach described in Rannala and Yang
[ 16]
under three
clock models.The variable clock=1 , 2 , 3 represents
the global clock , independent rates , and correlated
rates models , respectively.We use the HKY85 +Γ
substitution model , with a discrete gamma model of
variable rates among sites , with five rate categories
used.The MCMC was run for 200 000 iterations , after
a burn-in of 10 000 iterations.For each analysis , the
MCMC algorithm was run at least twice using different
starting values to confirm convergence to the same pos-
terior.
2　Results
2.1　Sequences variation
The entire ITS region was sequences for a total of
10 collections including 2 in the genus Lasallia and 8
in Umbilicaria (Table 1).The length of the whole ITS
region for the collections varies from 485 to 496 bp.
All samples have a conserved length of 5.8S coding re-
gion (157 bp).Combined with 5 sequences directly
downloaded from GenBank , 15 samples of Umbilicari-
aceae were aligned to 509 bp in length.
2.2　Sequence divergence
Sequence divergence of ITS1 , ITS2 , and the
whole ITS was calculated , respectively , and that of the
whole ITS is shown in Table 2.The lowest sequence
divergence was found for putative disjunct species in
every special region.For example , In the whole ITS
region , the pairwise distance between two Lasallia
pennsylvanica collections , which are from China and
North America , respectively is the lowest (Number of
Nucleotide differences=4 , in the same continent ,
N=2).A little greater divergence of every subregion
or the whole region was observed for allospecific dis-
junct species than conspecific disjunct species , howev-
er , obviously less than different species.
149
　第 10卷　第 3 期　　GUO Shou-yu et al.:Assessing Biogeographic Relationships of Some Species in the Lichen Family
Umbilicariaceae Based on Nuclear ITS rDNA Sequence
Table 2.Sequence divergence of ITS among selected species in Lasallia and Umbilicaria
ITS/No.of Sites:509 bp (1)a (2) (3) (4) (5) (6) (7) (8) (9)
(1)AF284447 U.esculenta(CN)
(2)DQ394397 U.esculenta 9b
(3)AF284448 U.mammulata (US) 11 16
(4)DQ782851 U.mammulata 19 13 10
(5)AF096204 U.muehlenbergii 39 39 40 40
(6)AF297666 U.muehlenbergii(CN) 40 40 41 43 5
(7)AF297667 U.muehlenbergii(US) 39 39 40 40 1 6
(8)AF297674 L.pennsylvanica (CN) 48 53 52 55 41 46 41
(9)AF297675 L.pennsylvanica (US) 51 56 55 58 43 46 43 4
(10)HM161513 L.pennsylvanica 50 55 54 57 42 45 42 4 2
　　Note:“ a” Symbols(1)to(9)in row 1 correspond to those samples with the same symbol in column 1;“ b” Numbers in“ 　” represent the nu-
cleotide difference between the samples with disjunct or vicarious patterns
2.3　Phylogenet ic analysis
The final alignment consisted of 509 sites for the
whole ITS (ITS1:177 bp , 5.8s 157 bp and ITS2:
175 bp).The maximum pasimony analyses generated
only 1 most parsimony tree (tree length=151steps ,
CI=0.834 457 , RI=0.754 902 , RC =0.629 918)
(not shown).This tree was almost the same as the ML
tree(Fig.1)and NJ tree (not shown)in topology , a
little difference was the bootstrap value.All these trees
have the same topological structures for the putative al-
lospecific and conspecific disjunct species , they formed
the distinct , well supported clades , respectively.The
bootstrap values for L .pennsylvanica , U.muehlen-
bergii , and U.esculenta vs.U.mammulata were all
100%.The same bootstrap values were also observed
in the combining ITS and LSU tree (not shown).The
hypotheses that L.pennsylvanica and U .muehlen-
bergii are distinct taxa with nearly disjunct distributions
were well supported.Three samples for each of these
two species formed the monophyletic clades , respec-
tively.
Note:Support is indicated for branches characterized by bootstrap frequencies exceeding 70%(below the branches and with thickened lines);T1—
T13 above the branches stand for the node numbers corresponding to those in Table 3;The branches are drawn to show posterior means of divergence
times estimated under autocorrelated-rates model(Clock 3 in Table 3)
Fig.1.The ML tree of nine species with 15 samples for ITS data set
150 菌　物　研　究 2012 年
2.4　Divergence dates for disjunct distributions
Posterior mean time (Mya)estimates under the
model clock 3 for each node within and among the
species of Umbilicaria and Lasallia are shown in Fig.2
(Plots for clock 1 and clock 2 not shown.).The con-
siderable scatter near the regression line indicates that
the sequence data may be suitable for the estimates.
Posterior mean time and 95% CIs of divergence times
(Mya)under different clock models are listed in
Table 3.
The split between the Asian and North American
lineages for U.muehlenbergii clade and for L.penn-
sylvanica was estimated at 8.7 (4.2 , 16.1)and 6.1
(2.8 , 12.9)million years ago (Mya), respectively.
The divergence between the eastern Asian U.esculen-
ta and the eastern North American U .mammulata was
estimated at 17.8(10.2 , 24.0)Mya.
Note:Basing on the analysis of ITS data for some species inUmbili-
cariaceae under clock 3(correlated rates model)with HKY85+Γ5
model.See Table 3 for more details
Fig.2.The 95% posterior CI widths plotted against the
posterior means of divergence times(Mya)
Table 3.Posterior mean times and 95% CIs of divergence times(Mya)estimated under different clock models for ITS data
Node Clock 1 Clock 2 Clock 3
T0(Umbi licaria-Lasallia)
T1
T2
T3
T4
T5
T6
T7
T8
T9(U.esculenta-mammulata)
T10
T11
T12
T13
69.5(41.9, 97.2)
60.4(35.9, 88.2)
41.9(23.7, 64.0)
35.1(19.5, 54.1)
32.1(17.5, 50.2)
17.4(8.5 , 30.9)
8.4(2.9 , 16.8)
1.5(0 , 5.0)
54.4(31.9, 80.5)
17.3(9.5 , 24.0)
10.4(4.5 , 18.1)
7.8(3.3 , 14.0)
6.0(1.9 , 12.8)
1.7(0.2 , 5.0)
68.9(41.9 , 97.1)
60.1(35.7 , 88.7)
42.7(23.3 , 67.0)
35.2(18.4 , 56.4)
31.5(16.1 , 51.5)
17.1(7.8 , 32.0)
8.4(2.7 , 17.6)
1.5(0 , 5.7)
53.0(30.7 , 79.8)
17.3(9.4 , 24.0)
10.5(4.3 , 18.6)
7.9(3.2 , 14.4)
6.4(2.0 , 14.2)
1.9(0.2 , 5.4)
68.7(41.8 , 96.7)
60.6(36.8 , 87.6)
37.8(22.1 , 57.0)
31.6(19.0 , 45.8)
30.5(18.4 , 44.0)
17.2(9.1 , 27.7)
8.7(4.2 , 16.1)
2.9(1.1 , 6.6)
55.7(32.9 , 83.3)
17.8(10.2 , 24.0)
10.7(5.1 , 18.4)
7.9(3.6 , 13.5)
6.1(2.8 , 12.9)
2.0(0.8 , 4.6)
151
　第 10卷　第 3 期　　GUO Shou-yu et al.:Assessing Biogeographic Relationships of Some Species in the Lichen Family
Umbilicariaceae Based on Nuclear ITS rDNA Sequence
3　Discussions
3.1　Putative eastern North American-eastern
Asian disjunct and vicarious taxa recognized
in Umbilicaria and Lasallia
As predicted , material of U.muehlenbergii from
China and United States formed monophyletic group on
the ML trees from ITS1 , ITS2 and the Whole ITS data
sets(100%bootstrap , not shown).The disjunct dis-
tribution of L.pennsylvanica was also supported by
these analyses.Morphological similar material of U.
esculenta from eastern Asia(China and South Korea),
and U.mammulata from eastern United States formed
a monophyletic group , which is sister to U.lobope-
ripherica.These two species share a most recent com-
mon ancestor.The ancestor may occur in some areas
near the Arctic region and Bering strait[ 5] .After the
isolation of Eurasian North American continents , diver-
gence following geographical isolation from a common
ancestor followed different paths , with changing in
morphology and ITS sequence.In Eurasia , it dispersed
along mountains in eastern Asia to south , and in North
America , it spread to south along the Appalachia
Mountain.
3.2　Biogeographic pattern is evident in Umbili-
cariaceae
Phylogenetic analyses have provided data consis-
tent with the current hypothesis that some species of
macrolichens display discrete distribution patterns that
mirror those observed for species of plants and animals ,
although these special fungi , just like the non-lich-
enized macrofungi[ 1-2 , 17] have spores that may be dis-
persed great distances and result in many lichen species
distributing widespread.
Two hypotheses can be used to explain the low
level of ITS sequence divergence observed among the
putative disjunct lichen taxa , one is consistent with the
hypothesis that these disjunct populations represent for-
merly broadly distributed taxa that were relatively re-
cently isolated through vicariance-promoted extinction.
The other one is about the gene flow , though widely
separated , gene flow still occurs in these disjunct pop-
ulations by accident.
The low levels of conspecific ITS sequence diver-
gence found in Umbilicaria muehlenbergii (5—6 varia-
tion positions)and Lasallia pennsylvanica (4).Just as
that of intraspecfic (0—2)found in Ramalina [ 18-19]
and Letharia[ 20] .The allospecific difference between
the East Asian endemics Umbilcaria esculenta and the
North American endemics U.mammulata (11—16)
are a little higher than those of the intraspecific , but is
lower than those of ordinary different species.These
data show that the biogeographic pattern is evident in
Umbilicariaceae and can be assessed using nuclear ITS
Sequences.However , the observed relatively close re-
lationship between the macrolichens of eastern North
America and eastern Asia is not the result of a single
event , each of the putative disjuncts is affected by
complicate climatic and geological events and has indi-
vidual histories.
Our results further confirm that the lichen-forming
fungi speciate slowly.Slowevolutionary rates in part
might be ascribed to the long generation of many
lichens.A study on growth and reproduction in several
alpine Umbilicaria species by Hestmark et al.[ 21]
demonstrated generation times much longer than that of
most vascular plants:50—80 y from establishment to
first reproduction.For Umbilicaria lichens the plastici-
ty and possible morphological and structural changes al-
so seem restricted.However , genetic difference be-
tween geographic isolated populations of species in Um-
bilicariaceae , e.g.from China and North America can
be demonstrated by using sequencing data of ITS region
in the nuclear ribosome RNA gene.
Acknowledgement:The authors are very much
grateful to Prof.Jiangchun Wei(IMCAS , Beijing)for
guiding in the study , and to Drs Qiuxin Wu andGrego-
ry M.Mueller (FMNH , Chicago)for arranging and
generously sponsoring work in USA , Lee Weigt for
technical assistance in DNA sequencing.DNA se-
quences were generated in the Pritzker Laboratory for
Molecular Systematics and Evolution at the FieldMuse-
um , Chicago.
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　第 10卷　第 3 期　　GUO Shou-yu et al.:Assessing Biogeographic Relationships of Some Species in the Lichen Family
Umbilicariaceae Based on Nuclear ITS rDNA Sequence