全 文 ：Mycosystema
菌 物 学 报 15 September 2009, 28(5): 705-711

jwxt@im.ac.cn
ISSN1672-6472 CN11-5180Q
©2009 Institute of Microbiology, CAS, all rights reserved.






Supported by the Talent Grant of Southwest University of Science and Technology and the National Natural Science Foundation of China (No.
30770012)
*Corresponding author. E-mail: chaoylll@sohu.com
Received: 14-10-2008, accepted: 17-03-2009

Comparative analysis of secondary structure of
5.8S-ITS2 rRNA in the genus Usnea
LIU Chao-Yang1* GUO Shou-Yu2
1Southwest University of Science and Technology, Mianyang 621010, China
2Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China



Abstract: The secondary structure of 5.8S rRNA and ITS2 of Usnea (Parmeliaceae) was investigated and a secondary structure
model for this group of lichens was proposed. Structure variations among species were mainly located in the helixes of ITS2 and
different types of helixes in ITS2 were recognized. Length variations of these domains seemed to be species-specific. The
phylogenetic relationships among species of Usnea based on nucleotide comparison was identical with those inferred from secondary
structure characters, which suggests that the structure information could be used as an additional taxonomic character for species
identification and phylogenetic implication of Usnea.
Key words: identification, model, phylogenetic relationship

松萝属的5.8S-ITS2 二级结构比较分析
刘超洋 1* 郭守玉 2
1西南科技大学生命科学与工程学院 绵阳 621010
2中国科学院微生物研究所 北京 100101
摘 要：本研究以松萝属为研究对象，构建了该属核糖体 5.8S 和第二转录区间（ITS2）的二级结构模型，并对种间的结构
差异进行了比较。结果显示，松萝属的结构与先前发表的真核生物 ITS2 二级结构模型非常相似。属内种间的结构差异主要
集中在第二转录区间的臂上。核酸序列分析所产生的系统树与结构特征的比较结果相一致，表明 ITS2 的二级结构信息可以
作为一种辅助的分类手段，适用于松萝属的种的鉴定和亲缘关系研究。
关键词：鉴定，模型，亲缘关系


INTRODUCTION
The genus Usnea (Parmeliaceae, Lecanorales,
Ascomycota) is recognized by fruticose (“hair-like”)
thallus with a radial organization. An axis consisting of a
cartilaginous strand of longitudinally arranged hyphae
DOI:10.13346/j.mycosystema.2009.05.018
706 Mycosystema
gives rise to many branches. Delimitation of Usnea has
been discussed by different authors, and the generic
concept changed from time to time (Clerc &
Herrera-Campos 1997; Krog 1982; Walker 1985). Many
taxonomic uncertainties remain at species level. Species
identification of Usnea is thought to be exceptionally
difficult by most lichenologists because they are
extremely variable on morphology and the
ecophenotypes of the same species often look radically
different.
Along with the development and application of
molecular techniques, the internal transcribed spacer
(ITS) of rRNA has been frequently used for investigation
of phylogenetic relationships of Usnea. Significant
progress in taxonomy of Neuropogon and some other
subgenera or sections of Usnea has been made based on
beta-tubulin and nuclear ribosomal (ITS and LSU) DNA
(Articus 2004; Ohmura 2002; Ohmura & Kanda 2004;
Wirtz et al. 2006). However, due to the high evolution
rate existing as size variation and sequence divergences
in ITS region, it became somewhat difficult to reliably
construct alignments reflecting speciation relationships
(Marquez et al. 2003; Vollmer & Palumbi 2004). In
addition, the accuracy of an alignment depending on the
substitution matrix while the gap opening or extension
penalties frequently causes unreliable phylogenetic
inference (Tyson 1992; Wheeler et al. 1995).
In recent years, there has been continuous
recognition that the higher order structure is fundamental
for establishing important structure-function
relationships among biological macromolecules
(Vukmirovic & Tilghman 2001; Mittl & Grütter 2001;
Collins et al. 2000; Caetano-anollés 2002). The reports
have suggested that the secondary structures of the
spacer regions of rRNA play an important role in
ribosome assembly (Lalev & Nazar 1999, 2001). Albeit
divergent in nucleotide sequence, the structure of internal
transcribed spacers 2 (ITS2) retains a highly conserved
configuration (Jörg et al. 2005; Annette 2007).
To date, applications of secondary structure in
fungal taxonomy are mostly indirect. Instead, the
structure information was mainly used to improve the
alignment qualities of highly divergent regions or to
describe position of introns in rDNA (Gottschling et al.
2001; Guindon & Gascuel 2003; Gargas et al. 1995).
Kruger & Gargas (2004, 2008) once defined the
consensus structures of the ITS2 rRNA in Lycoperdaceae
as taxonomic characters for basidiomycetes. The
secondary structure information of various domains of
the mitochondrial small-subunit ribosomal DNA within
the genera Ganoderma and Pleurotus were shown to be a
useful marker in delineation of phylogenetic grouping
(Hong & Jung 2004; Gonzalez & Labarère 2000). In this
study, the secondary structures of ITS2 together with
5.8S rRNA of Usnea were comprehensively investigated.
The structure model of the genus was developed. The
infraspecific structure variations were compared and the
structural characters were tested as a tool for species
identification.
1 MATERIALS AND METHODS
The 5.8S and ITS2 sequences were obtained from
the GenBank. To avoid the influences of species
misidentification, 57 sequences used in this paper came
from previously important molecular studies of Usnea
and Parmeliaceae. In addition, all sequences were subject
to BLAST searches for further verification of their
identities. The sequences were aligned with conserved
motif 5′-GCATCGATGAAGAACGCAGCG versus
GACCTCGGATCAGGTA-3′ to improve the accuracy of
secondary structure prediction. Thus, a total of 24
species of the genus Usnea were analyzed, including U.
aciculifera AB051049, U. arizonica AF297732, U.
articulata AJ457139, AJ457140, U. aurantiacoatra
DQ534488, U. baileyi AB051050, AB051051, U.
barbata AJ457138, U. ceratina AB051053, AB051052,
AB051054, AB051055, U. chaetophora AJ748106, U.
dasaea AB051056, U. diffracta AB051057, AB051058,
AB051061, AB051062, AB051063, AB051064,
DQ232663, DQ394374, U. filipendula AJ457149,
AJ457150, U. florida AJ457144, AJ457147, AJ457148,
U. fragilescens AJ748104, AJ748105, U. glabrescens
AB051639, U. hirta AJ457151, U. igniaria DQ219307,
U. longissima AB051642, AB051643, AB051644,
AB051647, AB051648, DQ001304, U. AB051645,
AB051646, DQ383647, U. merrillii AB051649, U.
pectinata AB051655, U. rigida AJ457152, U.
rubrotincta DQ232664, U. subantarctica EF179805,
EF179806, U. subfloridana AJ457154, AJ457155,
AJ457156, AJ457157, U. trichodeoides AB051665,
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AB051667, AB051668, AB051671, AB051666, U.
wasmuthii AB051674. Multiple alignments were
performed with the program ClustalX (Thompson et al.
1997). A Neighbor joining tree was constructed with
Kimura 2-parameter model and the bootstrap proportions
(BP) were determined from 1000 replications with the
program MEGA 4.0 (Tamura et al. 2007). The secondary
structures were folded using the mfold web server (Zuker
2003) by reference to the model secondary structure of
Saccharomyces cerevisiae (Cannone et al. 2002). The
paired regions were identified by mutual comparison of
sequences (phylogenetic comparative method) and by
complementary base substitutions.
2 RESULTS AND DISCUSSIONS
2.1 5.8S-ITS2 secondary structure of Usnea
All species examined in this study shared a common
secondary structure model (Fig. 1). It consisted of an
open multi-branched loop with several paired regions.
The structure of 5.8S, including five helixes (H1-H5) and
a single-stranded region, was conserved among species.
Structural differences were present in ITS2 (H7-H10).
Numerical and statistical descriptions of the deviations in
helixes are given in Table 1.


Fig. 1 The 5.8S-ITS2 secondary structure of Usnea.
2.2 Comparison of secondary structures of H7, H8,
H9 and H10
The helix 7 was 23bp long and of two structure
types (H7A and H7B) (Fig. 2-A). It appeared that the last
G-C pair in the stem of H7A was transformed into two
unpaired bases, G and U, in hairpin loop of H7B. H7A
was characterized by most species of Usnea, except for
U. aurantiacoatra and U. subantarctica which are of
H7B (Fig. 2-A).
The helix 8 was 31－33bp long and recognized as 3
types: H8A (U. aciculifera, U. ceratina, U. dasaea, U.
glabrescens, U. merrillii, U. pangiana, U. pygmoidea, U.
wasmuthii, U. barbata, U. articulata, U. florida, U.
filipendula, U. hirta, U. rigida, U. subfloridana, U.
fragilescens, U. chaetophora, U. igniaria and U.
rubrotincta), H8B (U. arizonica, U. aurantiacoatra, U.
diffracta, U. longissima, U. pectinata, U. subantarctica
and U. trichodeoides) and H8C (U. baileyi). The
structural variations were found in size of bugle loop and
hairpin loop (Fig. 2-B).
The helix 9 was the most variable and longest
regions with 73 － 76bp. H9 showed significant
differences in structures even at species level (Fig. 3).
This helix was divided into 9 types based on position of
bugle loop and length of paired bases: H9A, H9B (U.
ceratina), H9C (U. subantarctica, U. aurantiacoatra, U.
igniaria and U. hirta), H9D, H9E, H9F (U. diffracta),
H9G (U. trichodeoides), H9H (U. longissima) and H9I
(U. aciculifera and U. dasaea). Within the same type,
such as H9A, H9D and H9E, variations were evident in
number of bulge loop, size of bulge loop and hairpin
loop: H9A1 (U. subfloridana and U. florida), H9A2 (U.
filipendula, U. wasmuthii, U. barbata, U. chaetophora,
U. rigida, U. arizonica and U. glabrescens), H9D1 (U.
baileyi), H9D2 (U. pectinata), H9E1 (U. articulata),
H9E2 (U. merrillii and U. rubrotincta), H9E3 (U.
fragilescens) (Fig. 3).
Most species do not have helix 10, except for U.
aciculifera, U. arizonica, U. filipendula and U.
aurantiacoatra. The region was 17－21bp long, and 4
structural types existed: H10A (U. aciculifera), H10B (U.
arizonica), H10C (U. filipendula) and H10D (U.
aurantiacoatra). The main differences were shown in
size of hairpin loop and of the single stranded area. In
addition, a new bugle loop of stem was shown in H10D
(Fig. 2-C).
2.3 Phylogenetic analysis based on primary
sequences and structure characters
There were 301bp in the alignment for 5.8S-ITS2
region, in which 233 were constant, 68 were variable, 50
were parsimony-informative sites and 18 were
parsimony singleton sites in the phylogenetic tree (Fig.
3).Three main groups (Clades I, II, III) were recognized.
708 Mycosystema
Clade I corresponding to secondary structure (SS) of
H9D was monophyletic with a 100% bootstrap value
support. It included U. baileyi and U. pectinata, both
belonging to the subgenus Eumitria, which is
distinguished from subgenera Usnea and Dolichousnea
by the fistulose axis.
Table 1 Numerical and statistical values of the secondary structures of
H7, H8, H9 and H10
Loop length Structure
types
Sequence
length
Length
of
paired
bases Hairpin Bugle
Number
of
bugle
loop
H7 A 23 6 3
B 23 5 5
H8 A 31 11 3 3 1
B 32 11 3 4 1
C 33 11 4 4 1
H9 A1 75 26 7 11 6
A2 76 26 8 11 6
B 76 27 8 9 5
C 76 25 8 13 6
D1 73 23 13 9 5
D2 74 23 14 9 5
E1 76 26 8 11 6
E2 76 27 8 9 5
E3 75 28 5 9 5
F 75 27 7 9 6
G 75 26 7 11 6
H 75 26 7 11 7
I 76 27 8 9 5
H10 A 17 4 6
B 21 5 7
C 20 4 9
D 21 6 4 2 1
Clade II corresponding to SS H9F, H9G and H9H
was composed of three monophyletic species: U.
diffracta, U. trichodeoides and U. longissima with a high
support (BP = 81%). They belong to the subgenus
Dolichousnea which is characterized by the presence of
annular-pseudocyphellae and a somewhat thick
hypothecium (Ohmura 2001). A sister relationship
between U. trichodeoides and U. longissima was found
with 84% bootstrap proportion. They resemble each
other by sharing an iso-dichotomous branching thallus
with elongated terminal branches. However, they were
different in the branching types and the secondary
medullary substances. They can be readily distinguished
from U. diffracta by the fragile cortex.
The remaining members formed Clade III
corresponding to SS H9A, H9B, H9C, H9E and H9I,
which are different from the subgenus Dolichousnea by
the absence of anular-pseudocyphellae, thinner
hypothecium and I− reaction. Within this clade, three
subgroups were observed. The subgroup A,
corresponding to section Usena, is characterized by
leptodermatous cortical hyphae, was composed of U. ari
zonica, U. barbata, U. chaetophora, U. filipendula, U.
florida, U. glabrescens, U. rigida, U. subfloridana and U.
wasmuthii with a low support (BP = 54%). Other taxa
formed subgroup B is characterized by the
pachydermatous hyphae in cortex. Rest of the species, U.
ceratina (SS: H9B), U. articulate (SS: H9E1), U.
merrillii (SS: H9E2), U. rubrotincta (SS: H9E2), U.
fragilescens (SS: H9E3), U. aciculifera (SS: H9I) and U.
dasaea (SS: H9I), grouped together and formed
subgroup C with very weak support.

Fig. 2 Secondary structures of helixes 7, 8 and 10 in Usnea. The number “0” represented unpaired base and the number “1” represented paired base. A:
Secondary structure of helix 7; B: Secondary structure of helix 8; C: Secondary structure of helix 10.
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Fig. 3 A consensus neighbor joining pylogenetic tree of Usnea grouping in association with the secondary structures of ITS2 helixe 9. Bootstrap
proportion is shown near each branch.

3 DISCUSSIONS
Species classification based exclusively on
phenotypic characters (e.g. morphology and secondary
metabolites) is often difficult. Recently, the quantitative
comparison among nucleotide sequences becames a
useful tool to facilitate species identification (Hebert et
al. 2003; Tautz et al. 2003). However, several factor,
such as variable evolution rates, divergent pseudogene
copies, highly complex evolution and the ways in
constructing phylogenetic tree, may confound the
reconstruction of evolutionary relationships. Similar to
other phenotypic characters, the secondary structure of
rRNA has been proposed as a molecular marker for
identification, classification and phylogeny of fungi
(Caetano-Annolés 2002; Smith et al. 2004; Kruger &
Gargas 2004, 2008; Billoud et al. 2000).
In our analyses, the secondary structure of
5.8S-ITS2 in Usnea is similar to the previously described
structures of eukaryotes. In comparison with ITS2 region,
the secondary structure of 5.8S is more conserved.
Structure variations among species are mainly located in
710 Mycosystema
H7, H8, H9 and H10 of ITS2 region. Length variations
of these domains seemed to be species-specific. Our
study suggests that the regions should be treated as
homologous if bugle loop and hairpin loop share a
common position. Species with homologous structure are
closely related. Position of structural elements is the first
criterion for distinguishing a special group or species.
Also, the size of structural elements is a useful character.
Differences among homologous structure may be
distinguished by number of bases within a loop.
Previous molecular studies of Usnea mainly
focused on taxonomic status of some subgenera. The
phylogenetic relationships among different species were
also explored. The nucleotide sequence comparison is
used as main analysis method in these studies (Articus et
al. 2002; Ohmura 2001, 2002). Here, our work
concentrated on application of morpho-molecular
features to delineating phylogenetic groups and species
determination. The results of structure comparison
support the previous phylogenetic studies based on
ribosomal DNA and beta-tubulin data. Species with close
relationship share similar secondary structures, which
indicate that secondary structure of ITS2 is a useful
character in taxonomy and phylogeny of Usnea. The
differences of secondary structure appear relatively easy
to be identified in contrast to nucleotide base comparison.
Similar to morphological characters, to use the molecular
structure as a classifier should be further explored in
fungal systematics.

Acknowledgements: The authors thank Prof. Zhuang
Wen-Ying, Institute of Microbiology for critical review of the
manuscript and valuable suggestions.

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