全 文 ：第 13卷第 2期 菌 物 研 究 Vol. 13 No. 2
2015年 6月 Journal of Fungal Ｒesearch June 2015
! Foundation item:National Natural Science Foundation of China (31370067，30770012)
Biography:QIN Zheng-nan，male，graduate candidate for Master of Science Degree，majoring in lichen and endolichenic fungal diversity．
Ｒeceive date:2015-03-27
!! Corresponding author:GUO Shou-yu，E-mail:guosy@ im．ac．cn
A Species of Cladonia New to China and Its
Divergence with Closely Ｒelated Lineage!
QIN Zheng-nan1，2，HAN Liu-fu1，GUO Shou-yu2!!
(1．College of Life Science，Hebei Normal University，Shijiazhuang 050024，China;2． State Key La-
boratory of Mycology，Institute of Microbiology，Chinese Academy of Sciences，Beijing 100101，Chi-
na)
Abstract:In the investigation of Cladonia species in China，C． rudis collected from Hainan island
was identified as new to China based on morphological characteristics and nrDNA ITS sequence data．
Descriptions with illustration are presented． The divergence times with its closely related lineages
were estimated based on the sequence data．
Key words:Taxonomy;phylogeny;divergence time;Cladoniaceae;nrITS;tropical region;new
recorded species
CLC Number:Q949．325 Document:A Article ID:1672-3538(2015)02-0068-05
DOI:10．13341 / j．jfr．2014．2022
中国石蕊属地衣新记录种及其与近缘谱系的分化
秦正男1，2，韩留福1，郭守玉2!!
( 1． 河北师范大学生命科学院，石家庄 050024; 2． 中国科学院微生物研究所真菌学国家重点
实验室，北京 100101)
摘 要:在中国石蕊属种类的研究中，依据形态特征及 nrDNA ITS 序列数据，鉴定出采自海南的一新记录
种———粗糙石蕊(Cladonia rudis)。提供了描述及图片，依据序列数据估计了其与近缘谱系的分化时间。
关键词:分类学;系统发育;分化时间;石蕊科;nrITS;热带地区;新记录种
中图分类号:Q949．325 文献标识码:A 文章编号:1672-3538(2015)02-0068-05
DOI:10．13341 / j．jfr．2014．2022
引文格式:秦正男，韩留福，郭守玉．中国石蕊属地衣新记录种及其与近缘谱系的分化［J］．菌物研究，2015，13
(2):68-72．
1 Introduction
The genus Cladonia P．Browne was established
in 1756． It typically has a medium to large fruticose
thallus and a worldwide distribution，and includes
520 species in the Cladoniaceae，Lecanorales［1］． In
modern taxonomy of lichens，molecular techniques
were usually used to delimit species within the ge-
nus［2-3］． Stenroos et al．［2］carried out a phylogentic a-
nalysis by combining morphological and chemical
第 13卷 第 2期
QIN Zheng-nan et al．:A Species of Cladonia New to China and Its
Divergence with Closely Ｒelated Lineage
characters and nrDNA ITS sequence data． Their re-
sults presented an infrageneric reorganization of the
whole genus［2-3］．
Cladonia has been relatively well studied in the
Northern Hemisphere． In China，about 120 species
have been recorded［4-6］，among them，42 species
were recorded in the tropical region［5-8］． In the ongo-
ing study of Cladonia species in China，one species
collected from Hainan island was identified as new to
China based on morphological characteristics and
nrDNA ITS sequence data，it is described here as C．
rudis． We further assess the monophyletic nature and
phylogenetic afnities of the new species． The diver-
gence times with its closely related lineages were es-
timated based on the sequence data．
2 Materials and methods
2．1 Specimens，morphology and chemistry
The present paper is based on collections from
the Herbarium Mycologicum Academiae Sinicae-Li-
chenes (HMAS-L)． All the specimens were examined
and measured under a dissecting microscope (Zeiss
Stemi SV11)and a compound microscope (Zeiss
Imager A2)． Thin layer chromatography (TLC)was
performed for all specimens following the method of
Orange et al．［9］ with solvent system C．
2．2 DNA extraction，PCＲ amplification and
sequencing
The samples of thallus fragments were removed
from 3 selected specimens for DNA extraction． DNA
was extracted using the DNA secure Plant DNA Kit
(Tiangen，China)following the manufacturer s pro-
tocol． Amplification of the ITS region followed the
methods described in Han et al．［10］． The whole
nrITS region (ITS1，5. 8S and ITS2)of the nrDNA
repeat tandem was targeted with PCＲ using the
primers ITS1 with ITS4 directly． The amplification
reaction was performed in a 25 μL volume containing
0. 75 units of TransStart Taq Polymerase (Tiangen，
China) ，2. 5 μL of its buffer，0. 5 μL of a 5 μmol /L
solution of the primers，2 μL of 2. 5 mol /L for each
dNTP solution，and 1 μL of genomic DNA． The con-
ditions for thermocycling of nrITS were the
following:95℃ for 3 min linked to 35 cycles at
94℃ for 30 s，54℃ for 30 s，and 72℃ for 1 min
with a final extension of 72℃ for 10 min． PCＲ prod-
ucts were screened on 1% agarose gels stained with
ethidium bromide． The PCＲ products were sequenced
by the Genewiz Inc． (Beijing)．
Three newly obtained sequences were submitted
to GenBank (see Fig． 2 for accession numbers)． Se-
quences for specimens collected in China were
aligned with representatives of taxa sharing most sim-
ilarity with them，for which nrITS sequences were a-
vailable from GenBank;their accession numbers are
also provided in the tree (Fig． 2)． The representatives
of taxa were selected mainly according to the mor-
phological characters，Blast results of sequence data，
and the references［2-3，11］．
2．3 Phylogenetic analysis and divergence
time dating
The whole ITS sequences of 3 samples examined
and the 15 representatives selected(including 2 as
outgroup) were aligned by both ClustalW and
Muscle implemented in MEGA6［12］． The final matrix
can be obtained from the corresponding author．
The evolutionary history was inferred using the
Maximum Likelihood method based on the Kimura 2-
parameter model in MEGA6． The divergence times
were estimated by MCMC tree method implemented
in PAML package［13-14］ according to the following
protocol．
Estimated ages of divergence were obtained
using the MCMC algorithm under two clock models．
The variable clock = 2，3 represents the 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 40 000 iterations． For
each analysis，the MCMC algorithm was analyzed at
least twice using different starting values to confirm
convergence to the same posterior．
In the absence of relevant fossil evidence for-
Cladonia，we used molecular evolution rates from re-
cent reports for Parmeliaceae［15-17］，which also
belongs to Lecanorales． To estimate the time to the
most recent common ancestor (MＲCA)for all linea-
ges，we used the nrITS (including 5. 8 S)rate of
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菌 物 研 究 2015年
3. 50× 10－9 s /(s· yr)estimated from Parmeliaceae
(2. 10－4. 50×10－9for CI 95%)［15-17］．
3 Ｒesults and discussion
3．1 New record
Cladonia rudis Ahti ＆ Parnmen，in Ahti，Parn-
men ＆ Mongkolsuk，Sauteria 15:17，2008(Fig．1)．
Fig． 1． Thallus of Cladonia rudis (Guo 14032526，
HMAS－L)
Primary thallus:usually disappearing;or eva-
nescent，consisting of very small，whitish squamules;
squamules:up to 2 mm long and 1 mm wide，0. 2－
0. 5 mm thick，brownish-grey，irregularly lobate to
crenate-lobate． Podetia: (8－)10－15 (－18)mm
tall，0. 5－1. 0 mm wide，whitish-grey，somewhat mel-
anotic at base;forming dense tufts，branched by ir-
regular，anisotomic dichotomy，poorly differentiated
main stems，closed axils，tips subulate，ascyphose，
occasionally with scyphoid，with longitudinal fissures，
ultimate tips pale to dark brown;surface discontinu-
ously corticate，esorediate，highly uneven，scabrid
throughout，almost esquamose but scabrosities may de-
velop into very small microsquamules，larger squam-
ules very sparingly produced near base or middle
part of the podetia，up to 3 mm long and 1. 5 mm
wide，0. 2 － 0. 6 mm thick． Podetial wall 180 －
210 μm，cortex 12－20 μm，medulla (with algal lay-
er)70－100 μm stereome hard，90－110 μm central
canal papillate． Conidiomata frequent at tips of pode-
tia，black，shortly cylindrical，conidia not seen．
Apothecia not seen．
Chemistry:K－，PD+ red;contains fumarpro-
tocetraric acid (major) ，protocetraric acid (minor) ，
and confumarprotocetraric acid (minor) (TLC)．
Habitat and ecology:on soil along the road，
sometimes among mosses．
Specimens examined:China，Hainan，Lingshui，
Mt． Diaoluo (吊罗山) ，alt． 800 m，2014-03-25，S．
Y．Guo 14032525，14032526;Qiongzhong，Mt． Limu
(黎母山)，alt． 700 m，2014-04-16，S．Y．Guo 14040416
(HMAS－L)．
Comments:In the traditional classification C．
rudis would be placed in the group called section As-
cyphiferae by Ahti ［11，18］，i． e．，the Cladonia furcata
group． In our molecular analysis (Fig． 2) ，it was
placed near C． corymbescens Nyl． ex Leight． in the
same clade as C． scabriuscula (Delise) Nyl．
Stenroos et al．［2］ the last two species and Cladonia
furcata (Huds． )Schrad． Was placed in the“super-
group” Cladonia． C． scabriuscula as the new
recorded species is distinguished by the brownish-
grey tint and very rough，sometimes mini-squamulose
surface without soredia in upper part of the podetia．
C． corymbescens can be distinguished from C． rudis
by containing atranorin and slender，longer podetia．
3．2 Phylogenetic analysis and the divergence
times among closely related lineages
The whole ITS region was successfully
sequenced for three samples of the collections from
Hainanisland，China． The ITS sequences of the new
samples，15 reference taxa of Cladonia were included
in the phylogenetic analyses． In the alignment of the
18 taxa，the data matrix comprised 582 characters，of
which 479 (82. 3%)characters were constant and
81 (13. 9%)were parsimony informative．
07
第 13卷 第 2期
QIN Zheng-nan et al．:A Species of Cladonia New to China and Its
Divergence with Closely Ｒelated Lineage
Fig． 2． Phylogenetic relationshipsinferred from nrITS sequence data of Cladonia rudis and its related lineages in
Cladonia (with C． caroliniana as outgroup)． Support is indicated for branches characterized by bootstrap
frequencies with Maximum Likelihood method． The tree is drawn to scale，with branch lengths measured
in the number of substitutions per site． T1 to T10 refer to the nodes estimated the divergence times in Ta-
ble 1． GenBank accession numbers and the voucher specimen collectors and collecting numbers followed
by the herbarium abbreviation are provided．
The maximum parsimony (MP)analysis of the
alignment dataset was subjected to a heuristic search
for the most parsimonious trees and a tree was ob-
tained (not shown，Treelength = 130，CI = 0. 877，
ＲI= 0. 923，ＲC = 0. 810，HI = 0. 151)． The same a-
lignment dataset was also performed using MEGA6
for Maxium Likilihood analysis，and the same
topology as the most parsimony tree were obtained．
Therefore，the ML tree with bootstrap values (1 000
replications)at branches is shown in Fig． 2． In the
phylogenetic tree，the new samples from China
formed a clade with Cladonia rudis from Thailand
with relatively strong support (ML 86%)． The dif-
ferent samples of C． rudis and morphologically
similar species C． corymbescens formed a clade with
87% support of ML．
Posterior mean time and 95% CIs of divergence
times (Mya)under different clock models are listed
in Table 1． The split between the Asian endemic
species Cladonia rudis and cosmopolitan C． corymb-
escens clade was estimated at 8. 16 (4. 89，11. 87)
and 8. 14 (4. 90，11. 89)million years ago (Mya) ，
under clock2 and clock3 model，respectively． The di-
vergence between the clade of C． rudis and C．
corymbescences，and C． scabriuscula was estimated at
25－27 (10． 1，47． 5)Mya．
Basing on the present nrDNA sequence data，we
estimated the pruned nrITS (excluding 5. 8 S，about
410 nt)rate should be 4. 60 × 10－9 s /(s· yr)for
Cladoniaceae (2. 80－5. 90×10－9for CI 95%)．
Our results demonstrate that the terricolous and
corticolous lichens speciate faster than the saxicolous
lichens (e． g． Umbilcariaceae［19］vs． Parmeliaceae［15-17］
and Leptogium［20］) ，perhaps due to the better
growing conditions and richer nutrition．
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菌 物 研 究 2015年
Table 1． Posterior mean times and 95% CIs of divergence times (Mya)estimated under different clock models for
pruned nrITS sequence data (410 nt)
Item Clock2 Clock3
T1 (lineage split with C． scabriuscula)
T2 (C． rudis vs． C． corymbescens)
T3 (withinC． rudis)
T4
T5
T6 (withinC． corymbescens)
T7 (withinC． scabriuscula)
T8
T9
T10
Mu (μ)
Parameter Sigma2
Kappa
lnL
26. 80 (10. 1，47. 5)
8. 16 (4. 89，11. 87)
2. 81 (0. 55，5. 65)
1. 86 (0. 25，4. 01)
1. 06 (0. 02，2. 58)
0. 58 (0. 00，1. 79)
8. 27 (3. 00，15. 91)
5. 56 (2. 01，9. 74)
3. 14 (0. 77，6. 09)
1. 56 (0. 13，5. 52)
0. 198 (0. 081，0. 342)
0. 077 (0. 000，0. 234)
4. 731 (2. 876，6. 824)
－891. 735(－896. 435，－887. 520)
24. 70 (10. 4，41. 5)
8. 14 (4. 90，11. 89)
2. 68 (0. 60，5. 35)
1. 78 (0. 25，3. 79 )
1. 02 (0. 02，2. 47)
0. 52 (0. 00，1. 59)
7. 87 (2. 96，14. 17)
5. 38 (2. 05，9. 46)
3. 02 (0. 76，5. 81)
1. 51 (0. 13，3. 35)
0. 205 (0. 087，0. 352)
0. 094 (0. 000，0. 288)
4. 728 (2. 817，6. 787)
－891. 699(－896. 267，－887. 714)
Acknowledgements: The authors are very
much grateful to Prof． Jiang-chun Wei ( IMCAS，
Beijing)for guiding in the study，and to Dr Qiong
Ding and her graduate student Xi Yuan (Hainan U-
niversity，Haikou)for arranging the field work．
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