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Bryological Note
Genetic structure of mosses Pleurozium
schreberi (Willd. ex Brid.) Mitt. and
Racomitrium lanuginosum (Hedw.) Brid. along
altitude gradients in Hokkaido, Japan
Helena Korpelainen
1
, Annika Ja
¨gerbrand
2
, Maria von Cra
¨utlein
1
1
University of Helsinki, Finland,
2
Swedish National Road and Transport Research Institute, Sweden
Dispersal and gene flow are two fundamental
interlinked events, which are largely dependent on
the reproductive mode, the efficiency of reproduction,
and the mobility of the gametes and other propagules
produced. Studies on bryophytes have shown that
sexually produced spores are able to disperse further
from the source than generally larger asexual
propagules or shoot fragments (e.g. Sundberg, 2005;
Pohjamo et al., 2006; Korpelainen et al., 2011), while
asexual propagules may have higher establishment
rates (Lo¨ bel et al., 2009). Even within a single plant
species, rates of dispersal and gene flow can vary
considerably depending on the environmental condi-
tions (e.g. Pohjamo et al., 2006). In the case of
restricted dispersal, gene flow is reduced, resulting in
great genetic differentiation (Pohjamo et al., 2008;
Korpelainen et al., 2011).
Mountain ecosystems are unique, as the physical
distances among high altitude and low altitude
populations are short but environmental conditions
and topography may differ greatly. Such varying
conditions along altitudinal gradients can markedly
affect genetic variation patterns of plants, as shown
in many studies (e.g. Chen et al., 2008), while only
little or no differences with respect to altitude have
also been reported (e.g. Zhang et al., 2006; see also,
Ohsawa & Ide, 2008). Effective dispersal is a key
factor that prevents the development of altitudinal
structuring in genetic variation patterns.
The aim of the present study was to investigate and
compare the amount of genetic diversity and differ-
entiation in two common, widely distributed moss
species, Pleurozium schreberi (Willd. ex Brid.) Mitt. and
Racomitrium lanuginosum (Hedw.) Brid., occurring at a
wide range of altitudes in the study area in Hokkaido,
Japan. Both species are dioecious with rare sporophyte
production (Ulvinen et al., 2002), although in some
regions sporophyte production is observed (Hill et al.,
2007). Yet, vegetative propagation is typically the main
mode of reproduction, and dispersal is expected to be
reduced even within the same mountain slope. We
hypothesized that differentiation among populations is
great even on a local scale. Also, as high altitudes
represent marginal habitats for the studied mosses, we
hypothesized that the level of genetic diversity is lower
at high altitudes.
Pleurozium schreberi was collected from 21 popula-
tions from six localities (pairwise distances among
localities 12–115 km), altitudes ranging from 460 to
1910 m, and R. lanuginosum was sampled from eight
populations, all from one locality, altitudes ranging
from 550 to 1000 m (Table 1). Sampling took place in
June 2006 and 2007. Each population sampling area
was about 10615 m. No sporophytes were observed.
The study region has cold winters and relatively
warm summers. For instance, population sites for P.
schreberi on Mt. Oakan range from forest vegetation
at 460 m to alpine vegetation at 1350 m and reflect a
lowering temperature gradient of approximately 4.6–
6.0uC, based on the altitudinal temperature lapse
rates in Japan (Tada & Ishida, 1956).
Microsatellite markers were used as a tool to
investigate the genetic structures of P. schreberi and R.
lanuginosum. Total DNA was extracted from dry plant
material using DNeasy Plant Mini Kit (Qiagen, Inc.,
Valencia, CA, USA). Ten microsatellite markers avail-
able for P. schreberi and 15 microsatellite markers
previously developed for R. microcarpon (Hedw.) Brid.
(Korpelainen et al., 2008) were tested for the samples of
P. schreberi and R. lanuginosum used in the present
study. Genotyping was conducted as described in
Korpelainen et al. (2011). The data analysis included
the determination of genetic diversity (Nei, 1987) for
each population, the analysis of molecular variance with
1000 permutations, and Mantel teststodiscoverpossible
relationships between genetic and vertical distances, all
Correspondence to: Helena Korpelainen, Department of Agricultural
Sciences, PO Box 27, FI-00014 University of Helsinki, Finland. Email:
helena.korpelainen@helsinki.fi
DOI 10.1179/1743282012Y.0000000031
Bryological notes
Journal of Bryology 2012 VOL.34 NO.4 309
using the ARLEQUIN 3.01 software (Excoffier et al.,
2005). Additionally, correlations between genetic diver-
sities and altitudes were calculated.
Out of 10 microsatellites tested for in P. schreberi,
seven markers amplified well and were polymorphic,
while out of 15 microsatellites tested for in R.
lanuginosum, seven markers amplified well and four
of them were polymorphic (Table 2). A few plants
sharing the same multilocus genotypes were detected
but still included in data analyses. Among P.
schreberi populations, genetic diversities ranged from
0.210 to 0.556 (Table 1), while means across 21
populations and six localities equalled 0.447 and
0.414, respectively, and the diversity including all
samples was 0.552. In R. lanuginosum, diversities
among populations ranged from 0.143 to 0.410
(Table 1), while the mean across eight populations
equalled 0.336, and the diversity including all samples
was 0.390. The numbers of alleles per polymorphic
locus ranged from 3 to 15, and the mean numbers
were 6.3 in P. schreberi and 3.8 in R. lanuginosum
(Table 2). Relationships between the level of genetic
diversity and altitude were conflicting: P. schreberi
populations from different altitudes on Mt. Oakan
showed a positive correlation (r50.755; P,0.031),
while the correlation coefficient calculated using all P.
schreberi samples showed an opposite tendency
(r520.324; P50.122). R. lanuginosum populations,
all originating from Koma, had a nearly significant
negative relationship (r520.686; P,0.067). In pre-
vious studies on bryophytes, widely varying levels of
genetic diversities have been observed, e.g. a micro-
satellite variation range of 0.356–0.744 in six
Sphagnum species (Shaw et al., 2008) but only 0.010
in a microsatellite study on Sphagnum wulfianum
Girg. on a global scale (Kyrkjeeide et al., 2012).
Differentiation levels, overall F
ST
equalling 0.144
in P. schreberi and 0.052 in R. lanuginosum, were
significant. The detected difference between the
species may reflect different amounts of gene flow
or different colonization histories, e.g. R. lanugino-
sum populations may have separated from common
ancestral populations relatively recently. Based on a
previous review of population genetic investigations
in terrestrial moss species, the average value of F
ST
or
comparable differentiation measures equalled 0.234,
but there was great variation in the values, range
0–0.864 even in geographically local studies
(Korpelainen et al., 2005). However, it is important
to notice that comparisons of F
ST
values are truly
meaningful only if similar markers are used and
populations with similar levels of polymorphisms are
studied, as otherwise differences in total genetic
variation affect the values. Besides gene flow, low
F
ST
values can also reflect recent separation from
common ancestral populations. The hierarchical
analysis of molecular variance conducted for P.
schreberi showed that variation among localities,
Table 1 Populations and genetic diversities (Nei, 1987) of Pleurozium schreberi and Racomitrium laguninosum from
Hokkaido, Japan surveyed using microsatellite markers
Species Population Locality Altitude (m) Latitude Longitude Sample size Genetic diversity
Pleurozium schreberi Te1 Teshio 1400 43u979N 142u899E 10 0.433
Hi1 Hirayama 1740 43u769N 143u009E 10 0.210
Ha1 Hakutou 590 43u429N 144u089E 5 0.482
Ha2 645 9 0.388
Ha3 700 10 0.523
Ha4 820 5 0.610
Ma1 Maeyama 700 43u159N 142u229E 5 0.330
Ta1 Taisetsu 1440 43u569N 142u869E 5 0.457
Ta2 1700 5 0.381
Ta3 1850 5 0.470
Ta4 1910 5 0.296
Oa1 Mt. Oakan 460 43u459N 144u169E 10 0.433
Oa2 550 8 0.424
Oa3 670 10 0.409
Oa4 760 10 0.526
Oa5 865 9 0.500
Oa6 965 10 0.481
Oa7 1055 10 0.553
Oa8 1185 10 0.556
Oa9 1255 5 0.482
Oa10 1350 10 0.553
Racomitrium lanuginosum Ko1 Koma 550 42u079N 140u689E 10 0.384
Ko2 600 10 0.347
Ko3 650 10 0.332
Ko4 700 10 0.410
Ko5 750 10 0.395
Ko6 850 10 0.368
Ko7 900 10 0.310
Ko8 1000 4 0.143
Bryological notes
310 Journal of Bryology 2012 VOL.34 NO.4
among populations within localities, and among
individuals within populations equalled 11.7, 8.4,
and 79.9%, respectively. Mantel tests showed that
there is a significant association between genetic and
vertical distances in P. schreberi in the Mt. Oakan
region (r50.519; P50.000), while Hakutou and
Taisetsu regions, with only four population samples
each, possessed a significant positive correlation
(r50.880; P50.039) and a non-significant correlation
(r50.124; P50.346), respectively. The correlation
detected in R. lanuginosum in Koma was non-
significant (r50.327; P50.123).
In mountain ecosystems, although there may be
significant environmental differences between sites
within the same locality, the physical distance
between high altitude and low altitude populations
is short. Although our sample sizes were small, which
may have affected the outcome of the genetic
analyses, we were able to provide tentative informa-
tion indicating that there is a fair amount of gene flow
among populations along the same slope, especially
in R. lanuginosum, and even among populations
originating from different slopes (localities). These
results proved our hypothesis of great differentiation
at least partly wrong. Yet, in P. schreberi, signifi-
cantly positive correlations between genetic and
vertical distances were detected. Thus, altitude affects
the genetic differentiation pattern of the studied
mosses, but more strongly in P. schreberi than in R.
lanuginosum, perhaps as a result of differences in
dispersal efficiency. The relationships between genetic
diversity and altitude remained inconclusive, which
may at least partly relate to variable dispersal rates
among populations. Previously, Ohsawa & Ide (2008)
Table 2 Characteristics and primer sequences of microsatellite loci developed by Korpelainen et al. (2008) and found
polymorphic in the studied populations of Pleurozium schreberi and Racomitrium lanuginosum
Species Locus Repeat T
a
(uC) Primer sequences (59–39)
Allele size
range (bp) A
tot
Pleurozium
schreberi
PLSC1 [TCT]
4
[TC]
8
54 F: GTACAGCCAGTC
CTATGTTCGA
89–93 3
R: AAACAGAGCCTGCTTG
TTTCAG
PLSC3 [KC]
11
54 F: CAGACGCAAAC
AAGAATCGAGA
62–78 8
R: GGACAGGACGATA
TGATGCCTC
PLSC4 [GM]
13
55 F: CGATGTGGATG
ATGGCCTTTCA
68–78 6
R: CACATCGCCGACA
TACTCGAG
PLSC5 [CK]
13
56 F: ATACTCGAGC
TCCTCCTCTGAA
64–72 4
R: GACGATGTGGATG
ATGGCCTTTC
PLSC6 [TG]
10
55 F: AAGCTTGCCA
GTGTGATCCGTA
139–181 5
R: TCACCGCTGCATT
ACTCAAACAC
PLSC7 [CA]
14
53 F: CACACGCAAA
CACATTCACACAC
68–72 3
R: AGAGTTTTTGCGT
GCGTGTGA
PLSC12 [GA]
25
55 F: GCCTGCGATTC
CTACAGATGATAA
122–158 15
R: AGCATTGATCGTA
TGAGAGACAAC
Racomitrium
lanuginosum
RAMI6 [TTCATTCACTA]
21
57 F: CGGCCACCTG
CGTTCATATTC
175–181 4
R: CCTCCTCCCCTT
GATCTGTGGA
RAMI10 [GAN]
18
56 F: GCCGCGGAC
CATATCATAGAG
107–113 3
R: CTATGTCCTCTG
TTTCTTGGTCTG
RAMI12 [TG]
6
56 F: CCTCTCTGATA
TCGGTTTTGCTC
69–75 4
R: AGTGACTCCAA
AACCCCAAACCCA
RAMI14 [AG]
14
53 F: CGTTCGTTGTG
ATTGAGTTTGC
104–110 4
R: GGAACAAGCAA
CAATTGGATCGA
Note: T
a
, annealing temperature; A
tot
, number of alleles detected.
Bryological notes
Journal of Bryology 2012 VOL.34 NO.4 311
showed in their comprehensive review that plant
populations do not possess a single consistent pattern
of variation along altitudinal gradients. It appears that
there are several factors that influence genetic varia-
tion, and geographically core populations are not
always the most diverse ones. Future studies incorpor-
ating larger-scale sampling and spatialgenetic analyses
will be able to provide more comprehensive answers to
questions concerning dispersal and gene flow, and
diversity and differentiation along altitude in P.
schreberi and R. lanuginosum.
We acknowledge the financial support from Maj and
Tor Nessling Foundation to HK and the Japan
Society for Promotion of Science (P07727). We thank
the officer at the Akan National Park for our research
permission to work in the area of Mt. Oakan.
Taxonomic Additions and Changes: Nil.
References
Chen, G., Wang, Y., Zhao, C., Korpelainen, H. & Li, C. 2008.
Genetic diversity of Hippophae rhamnoides populations at
varying altitudes in the Wolong Natural Reserve of China as
revealed by ISSR markers. Silvae Genetica, 57: 29–36.
Excoffier, L., Guillaume, L. & Schneider, S. 2005. Arlequin (version
3.0): an integrated software package for population genetics
data analysis. Evolutionary Bioinformatics Online, 1: 47–50.
Hill, M.O., Preston, C.D., Bosanquet, S.D.S. & Roy, D.B. 2007.
BRYOATT. Attributes of British and Irish mosses, liverworts
and hornworts. Cambridgeshire: NERC Centre for Ecology and
Hydrology and Countryside Council for Wales.
Korpelainen, H., Kostamo, K., Karttunen, H., Virtanen, V. &
Pohjamo, M. 2008. Isolation and characterization of micro-
satellite loci for twenty common bryophyte species. In: H.
Mohamed, B.B. Baki, A. Nasrulhaq-Boyce & P.K.Y. Lee, eds.
Bryology in the new millennium. Kuala Lumpur: University of
Malaya, pp. 241–57.
Korpelainen, H., Pohjamo, M. & Laaka-Lindberg, S. 2005. How
efficiently does bryophyte dispersal lead to gene flow? Journal
of the Hattori Botanical Laboratory, 97:195–205.
Korpelainen, H., von Cra¨utlein, M., Laaka-Lindberg, S. &
Huttunen, S. 2011. Fine-scale spatial genetic structure of a
liverwort (Barbilophozia attenuata) within a network of ant
trails. Evolutionary Ecology, 25: 45–57.
Kyrkjeeide, M.O., Hassel, K., Flatberg, K.I. & Stenøien, H.K. 2012.
The rare peat moss Sphagnum wulfianum (Sphagnaceae) did
not survive the last glacial period in northern European
refugia. American Journal of Botany, 99: 677–89.
Lo¨bel, S., Sna¨ll, T. & Rydin, H. 2009. Mating system, reproduction
mode and diaspore size affect metacommunity diversity.
Journal of Ecology, 97: 176–85.
Nei, M. 1987. Molecular evolutionary genetics. New York:
Columbia University Press.
Ohsawa, T. & Ide, Y. 2008. Global patterns of genetic variation
in plant species along vertical and horizontal gradients
on mountains Global Ecology and Biogeography, 17: 152–
63.
Pohjamo, M., Korpelainen, H. & Kalinauskaite´, N. 2008. Restricted
gene flow in the clonal hepatic Trichocolea tomentella in
fragmented landscapes. Biological Conservation, 141: 1204–17.
Pohjamo, M., Laaka-Lindberg, S., Ovaskainen, O. & Korpelainen,
H. 2006. Dispersal potential of spores and asexual propagules
in the epixylic hepatic Anastrophyllum hellerianum.
Evolutionary Ecology, 20: 415–30.
Shaw, A.J., Pokorny, L., Shaw, B., Ricca, M., Boles, S. & Szo¨ve´nyi,
P. 2008. Genetic structure and genealogy in the Sphagnum
subsecundum complex (Sphagnaceae: Bryophyta). Molecular
Phylogenetics and Evolution, 49: 304–17.
Sundberg, S. 2005. Larger capsules enhance short-range dispersal in
Sphagnum, but what happens further on? Oikos, 108: 115–24.
Tada, F. & Ishida, R. (eds). 1956. The mountain geography. Tokyo:
Kawade Shobo.
Ulvinen, T., Syrja¨nen, K. & Anttila, S. (eds). 2002. Bryophytes in
Finland: distribution, ecology and Red List status. Helsinki: The
Finnish Environment Institute.
Zhang, X., Korpelainen, H. & Li, C. 2006. Microsatellite variation
of Quercus aquifolioides populations at varying altitudes in the
Wolong Natural Reserve of China. Silva Fennica, 40: 407–15.
Bryological notes
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