Phylogeny of the genus Lophozia (Dumort.) Dumort. s. str. inferred from nuclear and chloroplast sequences ITS1-2 and TRNL-F

Abstract

Maximum parsimony and maximum likelihood phylogenetic trees were constructed for 21 taxa of Lophozia s. str. and the related genera, Schistochilopsis (5 species), Protolophozia elongate, and Obtusifolium obtusum based on pooled nuclear ITS 1-2 and chloroplast trnL-F DNA sequences. The trees were characterized by similar topology. It was demonstrated that the genus Lophozia s. str. was monophyletic, excluding L. sudetica, which deserved isolation into a distinct cryptic genus. The species distribution among the clades disagreed with the sections distinguished based on anatomical and morphological data. The relationships within the genus Schistochilopsis were consistent with the sectioning of the genus, based on morphological characters. Analysis of molecular data provided more precise definition of the systematic position of a number of taxa. Small genetic divergence of geographically distant forms was demonstrated.

Full text

ISSN 1022-7954, Russian Journal of Genetics, 2007, Vol. 43, No. 11, pp. 1306–1313. © Pleiades Publishing, Inc., 2007.
Original Russian Text © A.A. Vilnet, I.A. Milyutina, N.A. Konstantinova, M.S. Ignatov, A.V. Troitsky, 2007, published in Genetika, 2007, Vol. 43, No. 11, pp. 1556–1564.
1306
INTRODUCTION
Liverworts represent the most ancient group of land
plants. Natural classification of this group is extremely
complicated due to the low number of anatomical and
morphological characters available for comparative
analysis.
Lophozia
(Dumort.) Dumort. s. str. is one of
most polymorphic and problematic for identification
liverwort genus, especially in Arctic. The concept of
some species and intraspecific taxa is controversial.
Many taxa were described relatively recently. Further-
more, they are often known only from the type locali-
ties, or several more localities [1–4]. As a result, liver-
wort variation and phylogenetic relationships are
scarcely studied. At present, a gradual transition
towards more narrow concept of the genus
Lophozia
s. 1.
is observed. Contrary to the ideas of Schuster on the
genus size [1], many modern authors distinguish other
distinct genera,
Leiocolea
(Muell. Frib.) H. Buch and
Barbilophozia
Loeske [5–8]. Most Russian authors
share the ideas of Schljakov [3], who, following the
narrow concept of the genera suggested by Scandina-
vian researchers [9, 10], in addition to the above genera,
also recognizes
Isopaches
H. Buch,
Obtusifollium
(H.
Buch) S. Arnell, and
Orthocaulis
H. Buch as isolated
genera. Furthermore, this author raises the subgenera
Massula
K. Müll. emend R. M. Schust. and
Protolo-
phozia
R. M. Schust. to the genus rank. Potemkin [11]
supports the recognition of
Schistochilopsis
as a genus,
but with inclusion of the
Obtusifolium
into it with the
status of a section.
The arguments of the followers of either broad or
narrow concept of the genera, based on anatomical and
morphological characters, are briefly discussed by
Bakalin [4]. The existing arguments, however, are not
sufficiently conclusive to accept one or another concept
of the genera in
Lophozia
s. 1. Earlier, based on the
sequence data for the chloroplast DNA (cpDNA)
trnL-F
locus from the 47 species of the family Lophoziaceae,
it was demonstrated that the “narrow” concept of the
genera in this family was, probably, more correct [12].
In the present study, we tried to evaluate phylogenetic
relationships within the
Lophozia
s. str. and in related
taxa based on sequence analysis of the internal tran-
scribed spacer of nuclear rDNA (ITS1-2) and the
trnL-F
locus of cpDNA of a higher number of taxa of
Lophozia
s. str., as well as its close relatives,
Schistochilopsis
,
Obtusifolium
, and
Protholophozia
.
MATERIALS AND METHODS
Taxon sampling.
DNA was extracted from the plants
selected from the herbarium specimens of Polar–
Alpine Botanical Garden–Institute (KPABG) of Kola
Research Center, Russian Academy of Sciences (table).
The analysis included 21 out of 33 taxa of the genus
Lophozia
s. str., indicated for the world [4], as well as
Schistochiopsis
(5 species),
Protolophozia elongata
,
Phylogeny of the Genus
Lophozia
(Dumort.) Dumort. s. str.
Inferred from Nuclear and Chloroplast Sequences
ITS1-2 and TRNL-F
A. A. Vilnet
a
, I. A. Milyutina
b
, N. A. Konstantinova
a
, M. S. Ignatov
c
, and A. V. Troitsky
b
a
Polar–Alpine Botanical Garden–Institute of Kola Research Center, Russian Academy of Sciences,
Kirovsk, 184236, Russia; e-mail: anya_v@list.ru; nadya_k@aprec.ru
b
Belozersky Institute of Physicochemical Biology, Moscow State University, Moscow 119992, Russia;
e-mail: tav@genebee.msu.su
c
Main Botanical Garden of Russian Academy of Sciences, Moscow, 127276 Russia; e-mail: misha_ignatov@list.ru
Received October 6, 2006
Abstract
—Maximum parsimony and maximum likelihood phylogenetic trees were constructed for 21 taxa of
Lophozia
s. str. and the related genera,
Schistochilopsis
(5 species),
Protolophozia elongata
, and
Obtusifolium
obtusum
based on combined nuclear ITS1-2 and chloroplast
trnL-F
DNA sequences. The trees were character-
ized by similar topology. It was demonstrated that the genus
Lophozia
s. str. was monophyletic, excluding
L. sudetica
, which deserved isolation into a distinct cryptic genus. The species distribution among the clades
disagreed with the sections distinguished based on anatomical and morphological data. The relationships within
the genus
Schistochilopsis
were consistent with the sectioning of the genus, based on morphological characters.
Analysis of molecular data provided more precise definition of the systematic position of a number of taxa.
A low level of genetic divergence of geographically distant forms was demonstrated.
DOI:
10.1134/S1022795407110142
PLANT
GENETICS

RUSSIAN JOURNAL OF GENETICS
Vol. 43
No. 11
2007
PHYLOGENY OF THE GENUS
LOPHOZIA
(DUMORT.) DUMORT. S. STR. 1307
and
Obtusifolium obtusum
. The species as
Lophozia
sudetica
,
L. silvicoides
,
L. excisa
,
Schistochilopsis
incisa
and
S. opacifolia
were represented by several
samples collected in different regions.
Anastrepta
orcadensis
,
Anastrophyllum michauxii
, and
Sphenolo-
bus saxicola
were used as an outgroup.
DNA isolation and sequencing.
DNA was isolated
using either CTAB-based method [13], or the kit for
extraction of plant DNA, NucleoSpin Plant Kit (Mach-
erey-Nagel, Germany).
Amplification of ITS1-2 and 5.8S rDNA was done
using external and internal primers [14]. Amplification
of the
trnL-F
region was performed using a pair of
primers, which allowed production of the full-sized
trnL
intron and 3' exon sequences, as well as the
sequence of the
trnL–trnF
intergenic spacer [15].
The amplification profile was 3 min at 94
°
C, fol-
lowed by 30 cycles (94
°
C for 30 s, 58
°
C for 40 s, and
72
°
C for 60 s) with a final elongation at 72
°
C for 2 min.
The produced DNA fragments were analyzed by elec-
trophoresis in 1% agarose gel in 1
×
TAE buffer with
ethidium bromide. The samples were purified using the
GFX(PCR DNA kit and Gel Band Purification Kit
(Amersham Bioscience, United States).
DNA was sequenced using the method of cyclic
sequencing with the ABI PRISM (BigDye (Terminator
v. 3.1 reagent kit. The reactions were performed in the
Genome Center of Joint Use (Engelhardt Institute of
Molecular Biology, Russian Academy of Sciences,
Moscow) with further analysis of the reaction products
on the ABI PRISM 3100-Avant DNA automated
sequencer. The sequences determined were submitted
to the GenBank (table).
Phylogenetic analysis.
Nucleotide sequences
were aligned manually using the BioEdit software [16].
The trees were constructed using the methods of
maximum parsimony (MP) and maximum likelihood
(ML) as implemented in the TNT [17] and PHYML [18],
respectively.
In the work with the TNT program the New Tech-
nology Search algorithms were used with the fivefold
reiterated minimal tree search and 500 jackknife repli-
cations with the removal 36% of the positions. Other
parameters were set as defaults. To calculate the
Bremer support index, the trees that were 20 steps
longer than the minimum tree were used.
To work with the PHYML program, an evolutionary
model of the GTR + G + I nucleotide substitutions cho-
sen by the ModelGenerator program [19] was selected.
Eight gamma density categories and 100 bootstrap rep-
lications were used. (Our previous experience of treat-
ing the sequences of similar length showed that the
bootstrap support values obtained at the increase of the
replications number up to 1000, differed from those at
100 replications no more than the bootstrap index val-
ues obtained in case of the independent tree construc-
tion.)
RESULTS
The ITS1-2 and
trnL-F
sequences were determined
in 36 samples. The
trnL-F
sequences for
Lophozia sil-
vicola, L. sudetica, Schistochilopsis incisa
, and
Obtusi-
folium obtusum
, used in our previous investigations
[12], were also included in the analysis.
For phylogenetic analysis, the ITS1-2 and
trnL-F
sequences were combined. The combined ITS1-2 and
trnL-F
sequences for
Lophozia polaris, L. silvicola,
L. silvicoloides
(Spitsbergen), and
Obtusifolium obtusum
were constructed by uniting sequence data from differ-
ent samples (table). Both
Schistochilopsis incisa
sam-
ples were represented only by the sequences of the
trnL-F
locus, while
Lophozia sudetica
was represented
by the ITS1-2 sequence (K41-5-04).
The overall alignment for 39 samples consisted of
1416 nucleotide pairs (bp), among which 57% posi-
tions were conservative, 39% were variable, and 27.5%
were parsimoniously informative. The
trnL-F
sequence
contains larger number of conservative sites, compared
to the ITS1-2 (69 and 51%, respectively), along with
the low number of variable (28 versus 46%) and parsi-
mony informative positions (20 versus 32%). Nucle-
otide composition is 24.2% (T), 24.5% (C), 23.8% (A),
and 27.5% (G).
Among the
Schistochilopsis
, the shortest ITS1-2
sequence was found in
S. capitata
(787 bp), while
S. opacifolia
(832 bp) had the longest ITS1-2. Among
the
Lophozia
s. str., the shortest ITS1-2 was in
L. wen-
zelii
var.
groelandica
(795 bp), and the longest ITS1-2
was in
L. sudetica
(June 4, 1998) (821 bp).Taken
together, the length of the ITS1-2 fragment was longer in
the representatives of the clade, formed by
L. excisa,
L. propagulifera, L. polaris, L. polaris
var.
sphagnorum,
L. longidens
, compared to other species of
Lophozia
s.
str. (the Excisa clade, distinguished on phylogenetic
trees, see below).
The length of the
trnL-F
sequence varied from
453 bp in
Schistochilopsis grandiretis
to 498 bp in
S. laxa
due to the insertion in the hairpin P8 of the
trnL
intron. An analogous situation was observed in the
Lophozia
s. str. species: two insertions within the hair-
pin P8 increased the length of the
trnL-F
sequence up
to 497 bp in
Lophozia longidens, L. polaris
, and
L. polaris
var.
shagnorum
, compared to the length of
this sequence in
L. austro-sibirica, L.
cf.
lacerata,
L. ventricosa
var.
guttulata, L.
cf.
wenzelii
var.
groen-
landica, L. wenzelii
var.
groenlandica, L. wenzelii
var.
litoralis,
and
L. wenzelii var. massularioides (463 bp).
Thus, the species forming the Excisa clade differed
from the other Lophozia s. str. by longer genomic
regions examined, which were formed due to the inser-
tions into ITS2 and P8.

1308
RUSSIAN JOURNAL OF GENETICS Vol. 43 No. 11 2007
VILNET et al.
A list of taxa used in the study with the indication of the herbarium vouchers and GenBank accession numbers
Taxon
Sampling locality, collector,
KPABG herbarium number
GenBank accession number
trnL ITS
Anastrepta orcadensis (Hook.) Schiffn. Russia, Bury-atia, N. A. Konstantinova,
59-1-01
DQ875088 DQ875126
Anastrophyllum michauxii (F. Weber)
H. Buch
Russia, Bury-atia, N. A. Konstantinova,
17-1-02
DQ875087 DQ875125
Lophozia ascendens (Warnst.)
R. M. Schust.
Russia, Bury-atia, N. A. Konstantinova,
109-3-01
DQ875054 DQ875089
Lophozia austro-sibirica Bakalin Russia, Buryatia, V. A. Bakalin, B 15-9-99 DQ875069 DQ875105
Lophozia excisa (Dicks.) Dumort Spitsbergen, N. A. Konstantinova, 104-1-04 DQ875056 DQ875091
Lophozia excisa (Dicks.) Dumort. Murmansk region, N. A. Konstantinova,
41-2-97
DQ875057 DQ875092
Lophozia excisa (Dicks.) Dumort. Spitsbergen, N. A. Konstantinova, K-21-2-05 DQ875058 DQ875093
Lophozia heteromorpha R. M. Schust. Russia, Kamchatka region, V. A. Bakalin,
K-47-3-02
DQ875068 DQ875104
Lophozia cf. lacerata N. Kitag. Russia, Commander Islands, V. A. Bakalin,
K-3-2-02-VB
DQ875071 DQ875107
Lophozia lantratoviae Bakalin Russia, Buryatia, V. A. Bakalin, 76-7-01 DQ875055 DQ875090
Lophozia longidens (Lindb.) Macoun Russia, Murmansk region, N. A. Konstantinova,
360-2-00
DQ875059 DQ875094
Lophozia polaris (R. M. Schust.)
R. M. Schust. et Damsh.
Russia, Kamchatka region, V. A. Bakalin,
30-01-02
DQ875060 No data
Lophozia polaris (R. M. Schust.)
R. M. Schust. et Damsh.
Spitsbergen, N. A. Konstantinova, K-9-2-05 No data DQ875095
Lophozia polaris (R. M. Schust.)
R. M. Schust. et Damsh. var. sphagnorum
(R. M. Schust.) R. M. Schust. et Damsh.
Russia, Yakutia, V. A. Bakalin, 23-11-00 DQ875061 DQ875096
Lophozia propagulifera (Gottsche) Steph. Russia, Kamchatka region, V. A. Bakalin,
K-53-6-02-VBD
Q875062 DQ875097
Lophozia schusteriana Schljakov Russia, Murmansk region, V. A. Bakalin, G9331 DQ875067 DQ875103
Lophozia silvicola H. Buch Russia, Karelia, V. A. Bakalin, August 2, 1998 AF519197
[12]
No data
Lophozia silvicola H. Buch Russia, Nizhni Novgorod region,
N. A. Konstantinova, 124-1-03
No data DQ875102
Lophozia silvicoloides N. Kitag. Russia, Murmansk region, N. A. Konstantinova,
356-4-00
DQ875064 DQ875099
Lophozia silvicoloides N. Kitag. Russia, Kamchatka region, V. A. Bakalin,
K-57-23-02-VB
DQ875063 DQ875098
Lophozia silvicoloides N. Kitag. Spitsbergen, N. A. Konstantinova, 150-6-04 DQ875065 No data
Lophozia silvicoloides N. Kitag. Spitsbergen, N. A. Konstantinova, 150-2-04 No data DQ875100
Lophozia sudetica (Nees ex Huebener)
Grolle
Russia, Murmansk region, V. A. Bakalin,
June 4, 1998
AF519195
[12]
DQ875113
Lophozia sudetica (Nees ex Huebener)
Grolle
Russia, Commander Islands, V. A. Bakalin,
K-41-5-04
No data DQ875115
Lophozia sudetica (Nees ex Huebener)
Grolle
Russia, Kemerovo region, N. A. Konstantinova,
90-7-00
DQ875077 DQ875114
Lophozia cf. wenzelii (Nees)
Steph. var. groenlandica (Nees) Bakalin
Russia, Kemerovo region, N. A. Konstantinova,
67-3-00
DQ875070 DQ875106
Lophozia ventricosa (Dicks.) Dumort. var.
guttulata (Lindb. et S.W. Arnell) Bakalin
Russia, Buryatia, N. A. Konstantinova,
81-1-01
DQ875072 DQ875108

RUSSIAN JOURNAL OF GENETICS Vol. 43 No. 11 2007
PHYLOGENY OF THE GENUS LOPHOZIA (DUMORT.) DUMORT. S. STR. 1309
The ML phylogenetic tree is presented in the Fig. 1.
In MP analysis, a single tree with the length of 1587
steps was found. It is presented in Fig. 2 with the indi-
cation of Bremer support index, jackknife support val-
ues, and the branch lengths. The ML and MP tree topol-
ogies were identical, except for relative positions of
Lophozia cf. wenzelii var. groenlandica, and L. wenzelii
var. lapponica, as well as of Schistochilopsis incisa
(Murmansk region) within the clades.
DISCUSSION
Judging by the trees constructed, the genus Lopho-
zia s. str. is monophyletic. At the same time, all samples
of Lophozia sudetica formed a clade, which was sepa-
rated from the other species belonging to Lophozia
s. str. by the species of the genus Schistochilopsis,
along with Protolophozi elongata and Obtusifolium
obtusum (Figs. 1 and 2). Similar results were reported
in the previous work of our laboratories on the analysis
of the trnL-F region using another samples of taxa [12].
The ITS1-2 sequence variation among the Lophozia
sudetica samples constituted 1 to 2%, and variation of
the trnL-F in the same sample was 0.5%.
In phylogenetic reconstructions, Obtusifolium obtusum
formed a clade, which was separated from the Schis-
tochilopsis by the branch of Protolophozia elongata.
A number of morphological characters (the leaf shape
and insertion, the presence of amphigastria, the struc-
ture of the capsule wall, etc.), which differed Obtusifo-
Table (Contd.)
Taxon
Sampling locality, collector,
KPABG herbarium number
GenBank accession number
trnL ITS
Lophozia ventricosa (Dicks.) Dumort.
var. longiflora (Nees) Macoun
Russia, Chita region, N. A. Konstantinova,
11-5-00
DQ875066 DQ875101
Lophozia wenzelii (Nees) Steph.
var. groenlandica (Nees) Bakalin
Russia, Murmansk region,
N. A. Konstantinova, 9329
DQ875073 DQ875109
Lophozia wenzelii (Nees) Steph.
var. lapponica H. Buch et S.W. Arnell
Spitsbergen, N. A. Konstantinova,124-2-04 DQ875076 DQ875112
Lophozia wenzelii (Nees) Steph.
var. litoralis (S.W. Arnell) Bakalin
Russia, Murmansk region, V. A. Bakalin,
12-3-02
DQ875074 DQ875110
Lophozia wenzelii (Nees) Steph.
var. massularioides Bakalin
Russia, the Caucasus, V. A. Onipchenko,
August 31, 1983
DQ875075 DQ875111
Schistochilopsis capitata (Hook.) Macoun Russia, Nizhni Novgorod region,
N. A. Konstantinova, 132-03
DQ875080 DQ875119
Schistochilopsis incisa (Schrad.)
Konstantinova
Russia, the Caucasus, K. O. Korotkov,
August 23, 1999
AY327784
[12]
No data
Schistochilopsis incisa (Schrad.)
Konstantinova
Russia, Murmansk region,
N. A. Konstantinova,187-1-02
DQ875083 No data
Schistochilopsis grandiretis
(Lindb. Ex Kaal.) Schiffn.
Russia, Kamchatka region, V. A. Bakalin,
99-5-01-VB
DQ875081 DQ875120
Schistochilopsis laxa (Lindb.) Grolle Russia, Murmansk region,
N. A. Konstantinova, 40-6-94
DQ875084 DQ875053
(5,8SpDNA,
ITS2)
DQ875122
(ITS1)
Schistochilopsis opacifolia (Meyl.)
Konstantinova
Spitsbergen, N. A. Konstantinova,
K-43-2-05
DQ875082 DQ875121
Schistochilopsis opacifolia (Meyl.)
Konstantinova
Russia, Murmansk region,
N. A. Konstantinova, 315-4-00
DQ875085 DQ875123
Sphenolobus saxicola (Schrad.) Steph. Russia, Buryatia, N. A. Konstantinova,
123-3-02
DQ875086 DQ875124
Protolophozia elongata (Steph.) Schljakov Russia, Murmansk region, V. A. Bakalin,
3-1-02
DQ875078 DQ875116
Obtusifolium obtusum (Lindb.) S.W. Arnell Russia, Murmansk region, V. A. Bakalin,
July 1, 2001
AY327769
[12]
No data
Obtusifolium obtusum
(Lindb.) S.W. Arnell Russia, Perm region, N. A. Konstantinova,
K-315-1-04
No data DQ875118

1310
RUSSIAN JOURNAL OF GENETICS Vol. 43 No. 11 2007
VILNET et al.
lium obtusum from all the other species of the genus
Schistochilopsis were supported by the presence of a
number of substitutions and indels, along with specific
deletion within the P8 hairpin of the trnL intron. These
data provided additional basis for treatment of the
Obtusifolium rather as an individual genus [3, 4, 10, 20],
than as the section of the genus Schistochilopsis [1, 11].
All the species of Schistochilopsis examined formed
a clade, however, with weak support (Figs. 1 and 2). Tree
positions of Schistochilopsis laxa and S. capitata were
consistent with their grouping into the section Heter-
ogemma (Joerg.) Potemkin (Marshicae R. M. Schust).
Similarly, tree positions of S. opacifolia, S. incisa, and
S. grandiretis were consistent with their grouping into the
section Incisae (C.E.O. Jensen) Potemkin [8, 11, 21, 22].
Sequence differences between Schistochilopsis opaci-
folia and S. incisa were low (3 to 4%) and almost the
same as between the two samples of S. opacifolia from
Spitsbergen and Murmansk region. Morphologically
studied samples of S. opacifolia and S. incisa from
Murmansk region were very similar, differing only in
the toothing patterns of the perianth mouth and leaves.
These findings support of the viewpoint of Bisang [22]
and Schuster [1], who recognized Schistochilopsis
opacifolia as a subspecies of S. incisa.
Schistochilopsis grandiretis demonstrated the pres-
ence of substantial morphological differences from
S. opacifolia and S. incisa (cell network pattern, stem
coloration, and others), which characterized it as fairly
distinct species. In the trees constructed (Figs. 1 and 2),
it was a sister species to the S. opacifolia + incisa.
Comparison of the minimum numbers of nucleotide
substitutions occurred during the evolution in accor-
dance with the MP tree, suggests a relative molecular
criterion of the taxa ranks within the genus Schis-
tochilopsis. If we assume the number of 1 to 12 nucle-
otide substitutions as typical to the subspecies of Schis-
tochilopsis, then the species status of S. grandiretis,
S. incisa + opacifolia, as well as of S. laxa and S. capi-
tata will be determined by 40 to 75 substitutions, while
the sections of Incisae and Heterogemma will be deter-
mined by 128 to 170 substitutions.
0.02
94
95
80
70
68
76
100
51
86
100
82
80
93
100
100
100
90
92
89
96
99
44
100
78
100
68
100
Anastrepta orcadensis
Sphenolobus saxicola
Anastrophyllum michauxii
Lophozia sudetica Commander Islands
Lophozia sudetica Kemerovo region
Lophozia sudetica Murmansk region
Obtusifolium obtusum
Protolophozia elongata
Schistochilopsis laxa
100
Schistochilopsis capitata
Schistochilopsis grandiretis
Schistochilopsis opacifolia Murmansk region
Schistochilopsis incisa Murmansk region
Schistochilopsis opacifolia Spitsbergen
86
57
100
Schistochilopsis incisa Caucasus
Lophozia polaris
Lophozia polaris var. sphagnorum
Lophozia longidens
Lophozia excisa Spitsbergen K-21-2-05
Lophozi excisa Spitsbergen 104-1-04
Lophozia excisa Murmansk region
Lophozia propagulifera
Lophozia ventricosa var. longiflora
Lophozia silvicoloides Spitsbergen
Lophozia silvicoloides Murmansk region
Lophozia silvicoloides Kamchatka region
Lophozia ascendens
Lophozia lantratoviae
Lophozia silvicola
Lophozia cf. wenzelii var. groenlandica
Lophozia schusteriana
Lophozia wenzelii var. lapponica
Lophozia wenzelii var. massularioides
Lophozia wenzelii var. groenlandica
Lophozia cf. lacerata
Lophozia wenzelii var. litoralis
Lophozia heteromorpha
Lophozia ventricosa var. guttulata
Lophozia austro-sibirica
“Excisa”
Fig. 1. Phylogenetic tree for the genus Lophozia s. str. and relative genera, based on combined nucleotide sequences of nuclear DNA
ITS1-2 and cpDNA trnL-F locus by the method of maximum likelihood (loglk = –7761.38761). The bootstrap supports are indi-
cated.

RUSSIAN JOURNAL OF GENETICS Vol. 43 No. 11 2007
PHYLOGENY OF THE GENUS LOPHOZIA (DUMORT.) DUMORT. S. STR. 1311
In all trees constructed, the species belonging to
Lophozia s. str. (except for Lophozia sudetica, see
above) were subdivided into two main clusters.
Except for the Lophozia lantratoviae, the species of
the genus Lophozia s. str., having either reddish or
brown-red gemma, formed a monophyletic group. This
group was in general consistent with the volume of the
section Excisae, according to Bakalin [4]. However,
Lophozia longidens also falls into this section. Schuster
[1] treated the latter species as an independent section,
Longidenatae R. M. Schust. Schljakov [3] placed this
species into the section Guttulatae Schljakov, while
Bakalin [4] placed L. longidens into the section Lopho-
zia. Thus, differently treated morphological characters
(stem structure, growing pattern, leaf shape, bulbil col-
oration, and others) do not provide determination of the
relationships of Lophozia longidens. Based on molecu-
lar data obtained, the species was placed into the sec-
tion Excisa.
In modern classification systems, position of the lit-
tle-known, predominantly Arctic species, Lophozia
polaris, remains uncertain. In different works, the spe-
cies was attributed to the sections Lophozia [1], Hetero-
morphae R. M. Schust. [3, 6], and finally, to Excisa [4].
Our data favor the latter viewpoint. Sequence differences
between the two varieties of this species from two far dis-
tant regions (Spitsbergen and Yakutia) (26 substitutions
in the MP tree) are comparable with those between the
three forms of Lophozia excisa from the Spitsbergen
and Murmansk region, as well as between L. excisa and
L. propagulifera (32 to 46 substitutions).
Three forms of Lophozia excisa and L. propagu-
lifera (L. latifolia) formed a separate subclade within
the clade Excisa with the strongest support. Lophozia
excisa is one of widely distributed and polymorphic
species of the genus. This situation is reflected in the
variation of the obtained genome loci in the species
samples from different regions and ecological condi-
tions (table). The difference between the two large-cell
forms of L. excisa from Spitsbergen and Murmansk
region (K21-2-05 and 41-2-97, respectively) is close to
that between the small-cell form of L. excisa (104-1-04)
and L. propagulifera (32 and 46 substitutions, respec-
tively). Sequence differences between the large-cell
forms of L. excisa from Murmansk region and Spitsber-
gen are comparable to those revealed upon comparison
“Excisa”
3/95
0
1
3
0
5
8
5
6
4/94
4
1/62
1
4/86
4
3
4
3/89
4
3
17/100
23
8
>20/100
27
>4/29
9
38
59
>20/100
23
20
4/29
2
3/41
5
5/97
6
11/100
11
24
10
5
5
4/95
13
>20/100
44
27
12
15
7/92
8
17/100
19
12/98
9
17/99
14
20/18
>20/100
>20/100
>20/100
>20/100
1
9
34
2
38
6
101
56
38
26
45
>20/100
50
6
>20/99
21
117
4
1
>20/82
15
47
>20/99
5
31
>20/100
18
>20/100
21
>20/25
5
42
>20/27
3
21
>20/100
Anastrepta orcadensis
Sphenolobus saxicola
Anastrophyllum michauxii
Lophozia sudetica Commander Islands
Lophozia sudetica Kemerovo region
Lophozia sudetica Murmansk region
Obtusifolium obtusum
Protolophozia elongata
Schistochilopsis laxa
Schistochilopsis capitata
Schistochilopsis grandiretis
Schistochilopsis opacifolia Murmansk region
Schistochilopsis incisa Murmansk region
Schistochilopsis opacifolia Spitsbergen
Schistochilopsis incisa Caucasus
Lophozia polaris
Lophozia polaris var. sphagnorum
Lophozia longidens
Lophozia excisa Spitsbergen K-21-2-05
Lophozi excisa Spitsbergen 104-1-04
Lophozia excisa Murmansk region
Lophozia propagulifera
Lophozia ventricosa var. longiflora
Lophozia silvicoloides Spitsbergen
Lophozia silvicoloides Murmansk region
Lophozia silvicoloides Kamchatka region
Lophozia ascendens
Lophozia lantratoviae
Lophozia silvicola
Lophozia cf. wenzelii var. groenlandica
Lophozia schusteriana
Lophozia wenzelii var. lapponica
Lophozia wenzelii var. massularioides
Lophozia wenzelii var. groenlandica
Lophozia cf. lacerata
Lophozia wenzelii var. litorais
Lophozia heteromorpha
Lophozia ventricosa var. guttulata
Lophozia austro-sibirica
Fig. 2. Phylogenetic tree for the genus Lophozia s. str. and relative genera, based on combined nucleotide sequences of nuclear DNA
ITS1-2 and cpDNA trnL-F locus by the method of maximum parsimony. The tree length is 1587. Numbers above branches indicate
Bremer and jackknife support indices, and numbers below branches indicate branch lengths.

1312
RUSSIAN JOURNAL OF GENETICS Vol. 43 No. 11 2007
VILNET et al.
of large- and small-cell specimens from Spitsbergen.
Morphological differences between the specimens
mentioned were also rather high (leaf thickness at the
basement, leaf shape, plant size, and others). Further
investigations will show whether these characters are
stable, and whether the forms described deserve award-
ing a certain taxonomic rank.
In the group of Lophozia with green gemma, a clade
with strong support is formed by Lophozia silvicoloides
and L. ventricosa var. longiflora. Furthermore, the
sequences of the Lophozia silvicoloides specimens
from the Commander Islands, Murmansk region, and
Spitsbergen are identical and remarkably different form
the examined sequences of L. ventricosa var. longiflora
from Chita region (by 5% at the ITS1-2 and by 2% at
the trnL). Substantial morphological differences of
these species (perianth mouth structure, oil bodies' pat-
tern, and others), along with molecular data confirm the
species status of Lophozia silvicoloides and demon-
strate the absence of close relationships with L. silvi-
cola (Figs. 1 and 2), which were earlier suggested by
Schuster [1].
Lophozia ascendens and L. lantratovie group into
one clade, albeit their ITS1-2 and trnL-F sequences in
the MP tree are separated by 97 steps. L. ascendens is
placed in the sections: Longidentatae R. M. Schust. [1],
Guttulatae Schljakov. [3], and Lophozia [4]. However,
according to Bakalin [4], the last section includes the
two former ones. Concerning L. lantratoviae, it should
be noted that this recently described species is also
included by its author into the section Lophozia. The
two species discussed are substantially different rela-
tive to their morphology, and they occupy different eco-
logical niches. Specifically, L. ascendens is obligate
epixylous, while L. lantratoviae is found on the fine
soil at the banks of the mountain rivers.
Lophozia ventricosa–Lophozia wenzelii represent
the most complicated and intricate complex. Many
varieties, subspecies, and species were included into
these taxa as different ranks and variants. As shown in
the Figs. 1 and 2, genetic differences between them are
low. Nevertheless, in the trees constructed, the samples
belonging to different taxa studied and attributed to
L. wenzelii, were united into two groups. One group con-
sisted of L. wenzelii var. groenlandica and L. wenzelii var.
massularioides. L. wenzelii var. lapponica appeared to
be close to them. However, L. wenzelii var. litoralis fell
into another clade. In any case, attribution of L. wenzelii
and L. schusteriana to the section Sudeticae, suggested
by Schljakov [3], and then by Bakalin [4], was not con-
firmed. The trees constructed in the present and previ-
ous studies [12] showed that Lophozia sudetica formed
an independent clade, which was far distant from the
other species of Lophozia s. str.
The tree position of recently described Lophozia
austro-sibirica
, which is known only from the type
locality in Southern Siberia and from one site in West-
ern Siberia [4], should be discussed in more detail. This
species formed an isolated clade together with L. ven-
tricosa var. guttulata with very strong support. In these
taxa, the genomic sequences examined differed only by
one substitution in the trnF gene. Morphological differ-
ences were very small, and mostly constituted in the
fact that L. austro-sibirica was monoecious, while
L. ventricosa var. guttulata was dioecious. It seems
likely that L. austro-sibirica and L. ventricosa var. gut-
tulata represent one species (the presence of monoe-
cious and dioecious individuals was described in some
Holarctic species of Lophozia, Isopaches, and some
others).
We would like to note the low genetic divergence
between the geographically distant populations of some
taxa shown for the loci examined in this study.
Thus, based on sequence analysis of nuclear DNA
ITS1-2 and cpDNA trnL-F loci in a great number of
Lophozia s. str. species, including the poorly studied
and recently described taxa, it was demonstrated that
the genus of interest was monophyletic, except for
Lophozia sudetica, which deserved isolation into a
genus. None of the existing sectioning of Lophozia
s. str. was supported by the tree topology. However, one
out of two clearly distinguished groups was consistent
with volume of the section Excisae, according to Baka-
lin [4]. This group contained all the species with red or
brownish gemma examined, excluding Lophozia lant-
ratoviae. Among the species with either colorless or
green gemma the clade Lophozia silvicoloides + L. ven-
tricosa var. longiflora was distinguished. The relation-
ships within the complex Lophozia ventricosa–Lopho-
zia wenzelii remain unclear. The recently described
species, L. austro-sibirica, practically does not differ
from L. ventricosa var. guttulata in the sequences of the
genomic regions examined, and probably can not be
treated as a distinct species. Species rank of L. lantra-
toviae, described in 2003, is confirmed by substantial
genetic differences from the other species of the genus.
The tree relationships within the Schistochilopsis
were consistent with the sectioning of the genus based
on morphological characters. The remarkable genetic
differences between S. laxa and S. capitata confirmed
their species status. The idea on the inclusion of Obtusi-
folium obtusum into Schistochilopsis was not sup-
ported.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research (grant nos. 06-04-48225 and 06-04-
49493).
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