Molecular Ecology (2004)
doi: 10.1111/j.1365-294X.2004.02423.x
Microsatellites reveal regional population differentiation
and isolation in Lobaria pulmonaria, an epiphytic lichen
Blackwell Publishing, Ltd.
JEAN-CLAUDE WALSER, ROLF HOLDEREGGER, FELIX GUGERLI, SUSAN EVA HOEBEE
and C H R I S T O P H S C H E I D E G G E R
Division of Ecological Genetics, Swiss Federal Research Institute WSL, Züercherstrasse 111, CH-8903 Birmensdorf, Switzerland
Abstract
Many lichen species produce both sexual and asexual propagules, but, aside from being
minute, these diaspores lack special adaptations for long-distance dispersal. So far, molecular studies have not directly addressed isolation and genetic differentiation of lichen
populations, both being affected by gene flow, at a regional scale. We used six mycobiontspecific microsatellite loci to investigate the population genetic structure of the epiphytic
lichen Lobaria pulmonaria in two regions that strongly differed with respect to anthropogenic impact. In British Columbia, L. pulmonaria grows in continuous old-growth forests,
while its populations in the old cultural landscape of Switzerland are comparably small
and fragmented. Populations from both British Columbia and Switzerland were genetically diverse at the loci. Geographically restricted alleles, low historical gene flow, and
analyses of genetic distance (UPGMA tree) and of differentiation (AMOVA) indicated that
populations from Vancouver Island and from the Canadian mainland were separated from
each other, except for one, geographically intermediate population. This differentiation
was attributed to different glacial and postglacial histories of coastal and inland populations
in British Columbia. In contrast to expectations, the three investigated Swiss populations
were genetically neither isolated nor differentiated from each other despite the long-lasting
negative human impact on the lichen’s range size in Central Europe. We propose that
detailed studies integrating local landscape and regional scales are now needed to understand the processes of dispersal and gene flow in lichens.
Keywords: dispersal, glaciation, genetic diversity, isolation by distance, lichen-forming fungi,
population history
Received 13 July 2004; revision received 27 October 2004; accepted 2 November 2004
Introduction
Approximately 50% of all fungi obtain nutrients by living
in close association with other organisms (Honegger 1996;
Tunlid & Talbot 2002). One conspicuous example is lichenization, defined as a mutualistic symbiosis between a
fungus (mycobiont) and at least one algal and /or cyanobacterial species (photobiont; Hawksworth & Honegger
1994). Lichens dominate approximately 8% of the world’s
terrestrial ecosystems, and more than 20% of all fungal
species are lichenized (Hawksworth et al. 1995). A recent
study of evolutionary relationships in fungi revealed that
several nonlichenized fungi, including plant or human
Correspondence: J.-C. Walser, Tel.: (773) 834 0467; Fax: (773)
702 0037; E-mail: [email protected]
© 2004 Blackwell Publishing Ltd
pathogens, have lichen-forming ancestors (Lutzoni et al.
2001). This suggests that mutualistic, coevolutionary systems are not necessarily evolutionary dead ends, and that
lichenization is probably an ancient nutritional strategy.
Many regional lichen floras include endemic taxa, but
most lichen species have much broader, though often
scattered, geographical distributional ranges than vascular
plants (Galloway 1996). It is not clear, however, whether
such biogeographical patterns reflect long-distance dispersal or historic fragmentation. Except for being minute,
sexually and asexually produced lichen propagules lack
special morphological adaptations for long-distance
dispersal (Heinken 1999; Dettki et al. 2000; Sillett et al.
2000). Therefore, it has been assumed that lichens are
often dispersal-limited (e.g. Bailey 1976; Armstrong 1990).
Kärnefelt (1990), suggested that a slow evolutionary or
2 J.-C. WALSER ET AL.
speciation rate, rather than a vast dispersal capacity, could
explain why lichens exhibit morphological uniformity over
large geographical areas. Recent molecular studies (Walser
et al. 2001; Printzen et al. 2003; Walser 2004) imply that the
dispersal of at least vegetative propagules could well be
limited at a landscape level, but little is known about the
effectiveness of lichen dispersal and, thus, gene flow by
means of either sexual or asexual propagules over larger
geographical distances within regions. Molecular studies
could help to understand population history, genetic differentiation or isolation, and gene exchange among lichen
populations. The paucity of this type of information in the
literature can be attributed to a previous lack of suitable
genetic markers, but Walser et al. (2003) recently illustrated
the use of mycobiont-specific microsatellites in population
genetic analyses of lichens at different spatial scales. They
also showed that the within-population diversity was much
higher than had been suggested by earlier studies mainly
based on nuclear ribosomal DNA (Bridge & Hawksworth
1998; Zoller et al. 1999; Kroken & Taylor 2001).
We investigated the genetic variation at six microsatellite
loci in the mycobiont of the epiphytic lichen Lobaria pulmonaria from two regions, namely, British Columbia (Canada)
and Switzerland. These regions are drastically different
with respect to the availability of suitable habitats for L.
pulmonaria, owing to anthropogenic modification of the
landscape. Populations of L. pulmonaria in British Columbia
grow in undisturbed, continuous old-growth forests, whereas
the species’ habitats in Switzerland are fragmented, and
populations are small due to long-term forest management
and dramatic environmental changes during the 20th century
(Wirth et al. 1996; Zoller et al. 1999). As a consequence, patterns of population differentiation and of gene exchange
among populations in British Columbia should differ from
those in Switzerland. We hypothesize that populations of
L. pulmonaria from British Columbia are characterized by
high genetic variation and low differentiation indicating
abundant historical gene flow among populations. However,
because human impact has likely led to substantially higher
genetic differentiation of populations in Switzerland, owing
to smaller population sizes and random sampling effects
such as genetic drift (Hartl & Clark 1997), we also hypothesize that Swiss populations are genetically less diverse
than those from British Columbia.
Materials and methods
ascomycete of the order Lecanorales, suborder Peltigerineae
(Tehler 1996). Its primary photosymbiotic partner is the
eukaryotic green alga Dictyochloropsis reticulata (Geitler 1966),
and its second partner is the nitrogen-fixing cyanobacterium Nostoc sp. ( Jordan 1970). The species is known to
reproduce both sexually and asexually ( Jordan 1973; Denison
2003). However, the sexual cycle of the photobiont is suppressed, and only the mycobiont goes through sexual reproduction forming ascospores (Malachowski et al. 1980).
A recent study found evidence for recombination, which
indicates that L. pulmonaria is an outcrossing lichen (Walser
et al. 2004). In addition to sexually derived propagules, L.
pulmonaria also forms different types of asexual (symbiotic)
dispersal units such as soredia, isidioid soredia, and thallus
fragments (Scheidegger 1995; Büdel & Scheidegger 1996).
While L. pulmonaria has a large distribution and is still
widespread and locally common in boreal North America
(Brodo et al. 2001), it is considered endangered in many
parts of Central Europe (Wirth et al. 1996). For example,
L. pulmonaria has almost completely vanished from the
Swiss Plateau, and the remaining populations in the Swiss
Pre-Alps and Jura Mountains have become increasingly
small and fragmented (Scheidegger et al. 2002).
Sampling
We sampled populations of L. pulmonaria in two regions:
in British Columbia, Canada, where the species is common,
and in Switzerland, where it is rare. A population was
considered as a spatially distinct patch of trees colonized
by L. pulmonaria. Beyond the patch perimeter, L. pulmonaria
did locally not occur. In total, 565 thalli were sampled from
nine populations in British Columbia and from three
populations in Switzerland (Table 1). British Columbia is
an ecologically diverse region with several biotic zones.
Goward (1999) divided the province into four ‘life zones’.
Assuming that genetic differentiation might be more pronounced between populations from different geoclimatic
regions (i.e. life zones), we collected the samples from
British Columbia along a transect from the south coast
of Vancouver Island to the interior of the province within
three of these life zones: hypermaritime, maritime, and intermontane (Table 1, Fig. 1a). The samples from Switzerland
were collected from relatively large populations in the
Pre-Alps and the Jura Mountains harbouring at least 30
colonized trees (Table 1). One thallus per tree was randomly
taken across each population.
Species
The foliose macrolichen Lobaria pulmonaria (L.) Hoffm.
predominately grows epiphytically (Brodo et al. 2001) and
has an estimated generation time of more than 30 years
(Scheidegger et al. 1998). It is known to be a tripartite lichen
species, with the fungal component (mycobiont) being an
Microsatellite analysis
Owing to its tripartite nature, as well as the difficulty in
obtaining algal-free thallus material, anonymous DNA
fingerprinting, such as RAPDs, is not applicable to population genetic questions in L. pulmonaria. Instead, we used
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
ISOLATION AND DIFFERENTIATION OF LICHEN POPULATIONS 3
Table 1 Locations and codes of 12 investigated populations of Lobaria pulmonaria from British Columbia (Canada) and Switzerland, and
their grouping in a set of five hierarchical amovas (Table 4). Populations sharing the same letter were grouped together
Grouping of populations†
Location
Code
Biogeoclimatic
zones
British Columbia
Ayum Creek, Vancouver Island
Chesterman Beach, Tofino
Cape Scott Provincial Park
Lakelse Lake Provincial Park
Date Creek, Kispiox
Clayton Falls, Bella Coola
Carp Lake Provincial Park
Bowron Lake Provincial Park
Oregana Creek, Tumtum Lake
AY
TO
CS
PR
DC
BC
CL
BL
OC
hypermaritime
hypermaritime
hypermaritime
maritime
maritime
maritime
intermontane
intermontane
intermontane
Switzerland
Taaren Wald, Toggenburg
Murgtal, Walensee
Marchairuz, Jura Mountains
TW
MT
UZ
Pre-Alps
Pre-Alps
Jura Mountains
Altitude
a.s.l.* (m)
Latitude
Longitude
(a)
(b)
(c)
(d)
(e)
15
5
5
105
550
5
860
910
723
48°23’29’’ N
49°06’47’’ N
50°40’27’’ N
54°22’54’’ N
55°24’52’’ N
52°22’12’’ N
54°52’08’’ N
53°15’19’’ N
51°59’08’’ N
123°39’37’‘ W
125°53’31’‘ W
128°16’32’‘ W
128°31’52’‘ W
127°48’52’‘ W
126°48’49’‘ W
123°15’39’‘ W
121°21’03’‘ W
119°05’21’‘ W
A
A
A
B
B
B
C
C
C
D
D
D
E
E
D
E
E
E
F
F
F
G
G
G
G
G
G
H
H
H
I
I
K
I
I
I
—
—
—
—
—
—
—
—
—
1350
1280
1200
47°10’50’’ N
47°03’52’’ N
46°29’57’’ N
9°18’15’‘ E
9°11’51’‘ E
6°10’21’‘ E
—
—
—
—
—
—
—
—
—
—
—
—
L
L
L
*a.s.l.: above sea level; †grouping of populations: (a) among three life zones in British Columbia; (b) British Columbia coast vs. interior;
(c) Vancouver Island vs. mainland; (d) Vancouver Island vs. population BC against inland; (e) within Switzerland.
Fig. 1 Estimated number of migrants (Nm) and genetic relationship among populations of the epiphytic lichen species Lobaria pulmonaria
from British Columbia (Canada) and Switzerland. (a) Location of the investigated populations in British Columbia. Different symbols refer
to different life zones (circles: hypermaritime; squares: maritime; triangles intermontane). Populations with altitude < 50 m above sea level
(a.s.l.) are given in white (coast) and populations > 50 m a.s.l. in black (interior). The number of migrants among populations based on
private alleles is symbolized by the style of the lines (Nm-values < 1 are not given). For population codes see Table 1. (b) upgma cluster of
the Canadian and Swiss populations, based on genetic distance DCE of six microsatellite loci. The values beside the nodes represent
bootstrap support for genetic distance DCE and DB, respectively, over 1000 permutations. The vertical bars refer to genetic lineages from
Switzerland (grey), British Columbia coast (see above; white) and British Columbia interior ( black).
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
4 J.-C. WALSER ET AL.
six microsatellite markers specific to the mycobiont of
L. pulmonaria (Walser et al. 2003).
DNA was isolated according to Walser et al. (2003) from
approximately 20 mg of apical thallus material. The multilocus genotypes were based on data obtained from five
dinucleotide (LPu03, LPu15, LPu16, LPu20, LPu27) and one
nine-base-pair repeat (LPu09) microsatellite loci, which
were amplified in multiplex-PCR following the protocols
of Walser et al. (2004). The fungal partner of the lichen is
haploid and thus has only one allele per locus. In this and
in an earlier study, no evidence for intrathalline variation
was found (Walser et al. 2003).
Data analysis
Recurrent multilocus genotypes can either be the result
of vegetative reproduction or selfing in homothallic lichen
species or chance products of sexual reproduction. If asexual
reproduction is abundant, shared multilocus genotypes
among populations could be interpreted as long-distance
dispersal of asexual propagules. On the other hand, individuals within or among populations with the same multilocus genotype could also be generated by independent
sexual events. Within populations, the probability of detecting sexually produced individuals with the same multilocus
genotype (Pse) was calculated as the product of the given
genotype’s allele frequencies according to Parks & Werth
(1993) and Wang et al. (1997).
The proportion of different multilocus genotypes (M),
genotypic diversity (number of observed genotypes divided
by the number of samples, Go /N; Stoddart & Taylor 1988),
the mean number of alleles (A), and the mean effective
number of alleles (A e) were estimated using the free
statistics software r version 1.8.1 (R Development Core Team
2003; http://www.R-project.org). Two distance estimators
were used to assess different evolutionary assumptions. First,
genetic distances among populations (without recurring
genotypes) were estimated using the chord distance DCD
(Cavalli-Sforza & Edwards 1967). Additional to this measure based on the infinite allele model (IAM), we also applied
an estimate based on the stepwise mutation model (SMM),
namely, DB (Bowcock et al. 1994), i.e. the proportion of
shared allele distances. Genetic distance estimations were
carried out using microsatellite analyser 2.65 (Dieringer
& Schlötterer 2003) and msatbootstrap 1.1 (Landry et al.
2002). The resulting genetic distance matrices were compared
by creating an unweighted pair– group method with arithmetic mean (upgma) tree with confidence estimates assigned
to its topology based on 1000 bootstrap replicates. The upgma
dendrograms were constructed using the neighbour and
consense components of phylip 3.57c (Felsenstein 1989).
Differentiation among populations (without recurring
genotypes) was estimated with hierarchical analysis of
molecular variance (amova) using both RST (SMM) and FST
(IAM) values in arlequin version 2.000 (Schneider et al.
2000). The populations were assigned to different groups:
(a) three life zones in British Columbia, (b) British Columbia
coast (altitude < 50 m above sea level) vs. interior (all others),
(c) Vancouver Island against mainland, (d) Vancouver
Island vs. population B C vs. interior, and (e) within Switzerland (Table 1).
Mantel tests (Mantel 1967), with 1000 permutations, were
conducted using the statistics software r 1.8.1 (R Development Core Team 2003) to determine the correlation between
geographical distance and genetic differentiation for populations from British Columbia. According to Nybom et al.
(2004), we used both RST and FST estimates. As Raybould
et al. (1998) suggested, separate Mantel tests for populations
less than 500 km apart were additionally performed.
Pairwise multilocus estimates of the effective number
of migrants (Nm) for populations from British Columbia
and from Switzerland based on private alleles (Slatkin 1985;
Barton & Slatkin 1986) were computed using genepop 3.1c
(http://wbiomed.curtin.edu.au/genepop/), and are, thus,
independent from FST estimates. The results were adjusted
since there are only half the numbers of migrant genes
for haploid data. We conducted a second Mantel test with
1000 permutations to determine the relationship between
geographical distances and number of migrants (Nm) in
British Columbia populations using r (R Development Core
Team 2003).
Results
Genetic and genotypic diversity
Three thalli were excluded from the data set because of
incomplete genotype assessment. The number of alleles per
locus within populations ranged from one to a maximum
of 22, and, on average, between 2.3 and 12.4 alleles were
found per locus across the 12 populations (Table 2). The
total number of different alleles per locus ranged from
three to 31 in populations from British Columbia and from
four to 20 in Swiss populations (Table 2). In populations
from British Columbia, mean allele sizes at loci LPu20 and
LPu27 were generally shorter than in Swiss populations,
but longer at locus LPu09 and LPu15 (Fig. 2). Furthermore,
and in contrast to all the other loci, the allele size range at
locus LPu27 did not overlap between samples from the
two continents (Fig. 2). Some alleles were geographically
confined to a single or to only few populations within
British Columbia (Fig. 2). At locus LPu03, which showed
little variation (Table 2), the shortest allele of 187 bp was
exclusively found in the coastal populations AY (Ayum
Creek, Vancouver Island), CS (Cape Scott Provincial Park),
and BC (Clayton Falls, Bella Coola). Populations PR
(Lakelse Lake Provincial Park), DC (Date Creek, Kispiox),
CL (Carp Lake Provincial Park), BL (Bowron Lake
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
ISOLATION AND DIFFERENTIATION OF LICHEN POPULATIONS 5
British Columbia
Switzerland
Loci
Min
Max
Mean ± SE
Total
Min
Max
Mean ± SE
Total
LPu03
LPu09
LPu15
LPu16
LPu20
LPu27
2
7
6
9
5
1
3
17
9
15
22
4
2.3 ± 0.1
11.3 ± 1.1
7.2 ± 0.4
11.6 ± 0.7
12.4 ± 1.7
2.7 ± 0.3
3
28
14
23
31
7
3
5
5
4
11
2
4
9
9
9
14
6
3.3 ± 0.3
7.0 ± 1.2
6.7 ± 1.2
6.7 ± 1.5
12.0 ± 1.0
4.3 ± 1.2
4
13
10
10
20
7
Table 2 Allele numbers at six microsatellite loci in Lobaria pulmonaria from British
Columbia (Canada) and Switzerland. Minimum (Min) and maximum number (Max),
mean (Mean), standard error (SE) of number
of alleles per population and the total number
of alleles (Total) over all populations
from British Columbia and Switzerland,
respectively
Fig. 2 Box-plots of the allele size distributions of five microsatellite loci (LPu09, LPu15,
LPu16, LPu20, LPu27) in Lobaria pulmonaria
populations from British Columbia (Canada)
and Switzerland. Locus LPu03 had a maximum of only four different alleles and is not
included in the figure. Circles denote outliers. For population codes see Table 1.
Pronvincial Park), and OC (Oregana Creek, Tumtum Lake)
from the interior of British Columbia exhibited high
frequencies of the 167 bp allele at locus LPu20. This allele
was rare in the coastal population BC and absent in the
populations from Vancouver Island AY, TO, and CS
(Fig. 2). At the same locus, alleles equal to or shorter than
165 bp were only found in the populations from Vancouver
Island and in the coastal population BC. Similarly, populations originating from the coast (AY, TO, CS, BC) displayed an allele of 180 bp at locus LPu27, which was absent
from the interior populations (Fig. 2). In addition, there
were no alleles specific to any of the three life zones
investigated. A high number of those alleles that were only
found once in single individuals were detected at the nine-
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
6 J.-C. WALSER ET AL.
Population
N
M
A
Ae
Go / N
Pse
British Columbia
AY
TO
CS
PR
DC
BC
CL
BL
OC
39
47
50
50
52
50
51
49
52
0.79
0.62
0.52
0.90
0.92
0.90
0.53
0.90
0.92
7.8
7.2
7.7
8.7
7.3
10.5
6.3
7.2
8.7
5.1
4.3
4.8
4.4
3.4
5.7
3.1
3.3
4.2
0.62
0.26
0.26
0.83
0.87
0.83
0.28
0.78
0.84
96.8%
96.6%
100.0%
80.0%
60.4%
97.8%
48.1%
47.7%
75.0%
Switzerland
TW
MT
UZ
52
38
32
0.52
0.47
0.53
7.8
5.3
6.8
4.4
3.6
3.8
0.26
0.26
0.30
96.3%
77.8%
88.2%
British Columbia mean
Switzerland mean
Total mean
49 ± 1.3
41 ± 5.9
47 ± 1.9
0.78 ± 0.06
0.51 ± 0.02
0.71 ± 0.06
7.9 ± 0.4
6.6 ± 0.7
7.6 ± 0.4
4.3 ± 0.3
3.9 ± 0.2
4.2 ± 0.2
0.62 ± 0.09
0.27 ± 0.01
0.53 ± 0.08
base-pair repeat locus LPu09 (Fig. 2), while locus LPu03
had no such unique alleles.
The proportion of different multilocus genotypes sampled
per population (M) varied between 0.52 and 0.92 in British
Columbia with a mean (± SE) of 0.78 ± 0.06 (Table 3). The
three Swiss populations revealed lower values between
0.47 and 0.53, similar to populations CL and CS from
British Columbia, and a mean of 0.51 ± 0.02. The Canadian
population BC harboured the highest mean and mean
effective number of alleles ( Table 3). It also shared characteristic alleles with populations from Vancouver Island
and the interior of British Columbia. In general, populations
from Switzerland and British Columbia exhibited similar
values for the mean and the mean effective number of alleles
( Table 3), while size-corrected genotypic diversity (Go /N)
was comparatively low in the Swiss populations. Only three
populations from British Columbia exhibited similarly low
values (Go /N < 0.3; Table 3). Most populations from the maritime zone and the intermontane zone of British Columbia, as
well as population AY from Vancouver Island, had substantially higher values of genotypic diversity (Go /N > 0.6; Table 3).
The 440 samples from British Columbia revealed 343
different multilocus genotypes (78%) and 296 singleoccurrence genotypes. For most genotypes, the probability
of a second encounter as a result of sexual reproduction
within a population was smaller than 5% (Table 3). This
indicates that recurrent genotypes at a given location were
most likely the product of asexual propagation.
The 122 samples from the three Swiss populations
harboured 62 distinct multilocus genotypes (51%) of which
41 occurred only once. In accordance to the results presented
for British Columbia, the probability of a second encounter of
a given genotype resulting from sexual reproduction within
Table 3 Number of samples (N), proportion of different multilocus genotypes (M),
mean number of alleles (A), mean effective
number of alleles (Ae), sample size-corrected
genotypic diversity (Go / N), and the proportion of multilocus genotypes which had a
lower probability than Pse < 0.05 to be found
twice as the result of sexual reproduction
within a given population of Lobaria
pulmonaria from British Columbia (Canada)
and Switzerland. Means and standard errors
(SE) are given for British Columbia, Switzerland and the total data set. For population
codes see Table 1
—
—
—
Table 4 Correlation between geographical distances and genetic
differentiation (RST, FST) among populations of Lobaria pulmonaria
from British Columbia (Canada) using Mantel tests. ns = not
significant
Mantel test
Geographic
distance
Genetic
differentiation*
rm
P-value
All
All
< 500 km
< 500 km
RST
FST
RST
FST
0.572
− 0.400
0.197
0.392
< 0.001
ns
0.003
< 0.001
*RST: Slatkin (1995), FST: Wright (1951).
the Swiss population was mostly less than 5% (Table 3).
The Swiss populations did not share a common genotype.
Genetic differentiation
There was substantial genetic differentiation among the
populations based on RST and on FST estimates for both
British Columbia and Switzerland. Most of the pairwise
comparisons of populations were significantly different
from zero; only nine of the RST estimates (14%) and two of
the FST estimates (3%) were not significant. RST estimates
were almost twice as high as FST values (data not shown).
Mantel tests between pairwise geographical distance and
genetic differentiation for populations from British Columbia
(Table 4) only showed a significantly positive relationship
when based on RST values (P < 0.001). For FST values, such
a positive relationship was only found at shorter distances
of less than 500 km (Table 4). Hence, the two measurements
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
ISOLATION AND DIFFERENTIATION OF LICHEN POPULATIONS 7
Table 5 Results of analyses of molecular variance (amovas) with six microsatellite loci of Lobaria pulmonaria from nine populations from
British Columbia (Canada) and three populations from Switzerland based on RST- and FST-values. Recurring genotypes were excluded from
the analysis. For different groupings of populations see Table 1. Significance levels were based on 1000 permutations. Variance components
values: among groups = RCT/FCT, among populations within groups = RSC/FSC, among populations = RST/FST
RST
Grouping of populations
Source of variation
d.f.
(a) Among life zones
in British Columbia
Among groups
Among populations within groups
Within populations
Among populations
2
6
334
Among groups
Among populations within groups
Within populations
Among populations
1
7
334
Among groups
Among populations within groups
Within populations
Among populations
1
7
334
Among groups
Among populations within groups
Within populations
Among populations
2
6
334
(b) British Columbia coast vs.
British Columbia interior
(c) Vancouver Island vs.
mainland
(d) Vancouver Island vs.
population BC vs. interior
(e) Within Switzerland
Among populations
Within populations
of genetic differentiation led to conflicting results when
geographical distances were greater than 500 km.
The estimates of the effective number of migrants per
generation (Nm) based on private alleles ranged between
0.42 and 2.28. Within British Columbia, Nm values among
populations from the maritime and intermontane zone
were higher compared with those among populations from
Vancouver Island and the mainland (Fig. 1a). Particularly
high numbers of migrants were estimated for population
BC and all other populations (Fig. 1a). The Mantel test
showed a significant negative correlation (rm = −0.64, P <
0.001) between geographical distance and Nm values indicating increased population isolation with increasing
distance in British Columbia. The mean Nm value for the
three Swiss populations was below 0.9 (data not shown).
The upgma dendrogram based on DCE clearly separated
the Lobaria pulmonaria populations from British Columbia
from those of Switzerland (Fig. 1b). The upgma tree based
on DB had an identical topology (Fig. 1b). In British Columbia,
there was also a well-supported genetic divergence between
populations from the coast and the interior (Fig. 1b).
When the populations from British Columbia were
assigned to three different life zones ( Table 1, Fig. 1a), most
of the total variance resided within populations, and a
comparably small proportion of the genetic variance was
found among the life zones (RCT: 7%, P = 0.081; FCT: 4%, P =
2
59
Variance
component
6.74%
8.81%
84.45%
15.55%
15.49%
4.76%
79.75%
20.25%
11.84%
8.06%
80.10%
19.90%
13.34%
5.19%
81.46%
18.53%
−3.94%
103.94%
FST
Variance
component
P
0.081
< 0.001
< 0.001
0.007
< 0.001
< 0.001
0.023
< 0.001
< 0.001
0.015
< 0.001
< 0.001
0.943
4.17%
3.80%
92.03%
7.97%
6.94%
3.03%
90.03%
9.97%
7.29%
3.57%
89.14%
10.86%
7.06%
2.47%
90.47%
9.53%
1.24%
98.76%
P
< 0.021
< 0.001
< 0.001
0.007
< 0.001
< 0.001
0.016
< 0.001
< 0.001
0.008
< 0.001
< 0.001
0.121
0.021). When the coastal populations were grouped separately from the interior populations, 15% (RCT, P = 0.007)
and 7% (FCT, P = 0.007) of the total genetic variance resided
between the two groups (Table 5). The genetic variance
between groups was again smaller when populations
from Vancouver Island (hypermaritime) were compared
with mainland populations (maritime and intermontane;
RCT = 12%, P = 0.023; FCT = 7%, P = 0.016) or when they
were tested against population BC and against all other
populations from British Columbia (RCT = 13%, P = 0.015;
FCT = 7%, P = 0.008), respectively. The amova for populations from Switzerland revealed that the total genetic
variation residing among the three populations was very
small and not significantly different from zero (both for RST
and FST; Table 5).
Discussion
The use of microsatellites is now routine in plant and animal
population genetics (Goldstein & Schlötterer 1999). In lichenforming fungi, microsatellite loci have only recently been
introduced, and, consequently, few studies are yet available
(Walser et al. 2003). We used six microsatellite loci to investigate population differentiation and isolation by distance
in the epiphytic lichen Lobaria pulmonaria, and to draw conclusions on its history at regional scales. Genetic analysis
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
8 J.-C. WALSER ET AL.
of nine populations of L. pulmonaria from British Columbia
and three populations from Switzerland revealed high levels
of genetic variation within populations. The microsatellite
loci used were sufficient to detect 405 different multilocus
genotypes among the 562 analysed lichen thalli. This
confirms the high resolving power of microsatellites for
determining genotypic diversity in lichen species and their
subsequent use in analyses of genetic structure. In this
study, populations of L. pulmonaria from British Columbia
were found to harbour substantially greater genotypic diversity than Swiss populations.
Genetic patterns in British Columbia
Our first premise was that populations of L. pulmonaria
in the continuous old-growth forests of British Columbia
should be genetically diverse and show low differentiation
because of abundant gene flow among them. The Canadian
populations indeed exhibited high genetic variation but, in
contrast to our expectations, low historical gene flow and
the results of the analyses of genetic distance (upgma tree)
and differentiation (amova) indicated a distinct genetic geographical structure of L. pulmonaria populations. Specifically,
populations from Vancouver Island were clearly separated
from the mainland populations, and the mainland population BC, close to the coast, showed an intermediate
position. This geographical structure is possibly not caused
by anthropogenic factors or geoclimatic zonation, but may
be due to postglacial population history (Fig. 1a). In this
context, molecular data suggest that the ice ages profoundly influenced the genetic architecture of the flora
and fauna of the Pacific Northwest (Soltis et al. 1997). Most
plants and animals of British Columbia are descendants of
immigrants that colonized the province after the retreat
of the Pleistocene ice sheet 10 000 –13 000 bp (Cannings &
Cannings 1996). Glaciation, however, did not thoroughly
deplete the diversity of the British Columbia biota. Many
of the extant taxa survived the ice ages either in one or
more ice-free but isolated refugia to the north or south
of the province or alternatively on a few peninsulas and
offshore islands including the Brooks Peninsula on the
northwestern coast of Vancouver Island (Soltis et al. 1997).
After glaciation, migration and the mixing of once isolated
and possibly genetically differentiated glacial populations
(owing to restricted gene flow) resulted in the formation of
continuous geographical distributions (Soltis et al. 1997). It
has been suggested that the different refugial source areas
and the different migration corridors east and west of the
Coastal Mountains have led to present-day genetic
differentiation of populations from the coast and from the
mainland of British Columbia (Cannings & Cannings 1996).
Because the sea level was lower (Josenhans et al. 1997) and
the coastline elevated by upwarping during glaciation
(Benson et al. 1999; Clague & James 2002), the coastal refugia
were probably larger than would be apparent today. In accordance with this Vancouver Island refugium hypothesis,
our data revealed that populations from Vancouver Island
were genetically differentiated from inland populations,
suggesting that populations of L. pulmonaria from the two
areas may well have had different glacial and postglacial
histories. It seems possible that L. pulmonaria populations
from Vancouver Island originating from in-situ surviving
populations or from genetic lineages migrating to Vancouver
Island from southern coastal refugia, have left their genetic
footprints on the current genetic structure.
The affiliation of population BC either to populations from
Vancouver Island or to populations from the mainland was
unclear. The geographically intermediate position of this
population could also explain its apparent genetic position
midway between island and mainland populations. For
example, the relative values of estimated historical gene
flow for population BC were particularly high when compared with those of most other populations (Fig. 1a). This
result has to be interpreted with caution because of the
possible effects of homoplasy. Furthermore, population
BC shared geographically restricted alleles with both the
coastal and the inland populations and, consequently,
showed the highest mean number of alleles per locus of all
investigated populations (Table 3). The results suggest that
populations from Vancouver Island and from the interior
of the continent have influenced the genetic structure of
population BC and that this population is situated in the
area of a contact zone of postglacial migration routes of L.
pulmonaria (cf. Petit et al. 2003).
The comparison of genetic and geographical distances in
isolation-by-distance tests resulted in conflicting results
when populations more than 500 km apart were taken into
consideration (Table 4). Whereas the overall Mantel test
was only significant for RST values, it was not so for FST
values. On the other hand, there was significant isolation
by distance for FST values at distances of less than 500 km.
This is to be expected in microsatellite analyses (Raybould
et al. 1998), reflecting the sensitivity to large genetic distances
of the IAM-based FST values where the effect of isolation by
distance and that of mutation overlap (Raybould et al.
1998; Balloux & Lugon-Moulin 2002). Distances greater
than 500 km mostly occurred between populations from
Vancouver Island and the inland regions, where historical
gene flow rates were comparatively low (Fig. 1a) so perhaps
one can have more confidence, at least in this instance, in
the RST estimates of overall genetic differentiation.
Population differentiation values are often lower when
based on the assumption of the IAM as compared to the
SMM, particularly if polymorphism is high and rates of
migration are low (Bossart & Prowell 1998; Raybould et al.
1998; Reusch et al. 2000; Collevatti et al. 2001; Balloux &
Lugon-Moulin 2002). The IAM-based estimates indicate
lower differentiation because they do not distinguish among
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
ISOLATION AND DIFFERENTIATION OF LICHEN POPULATIONS 9
shared alleles in different populations that are not identical
by descent (i.e. they arose by independent mutations and
are not shared by gene exchange). Similar results are only
to be expected when mutation rates are negligible in
comparison to migration and drift. Conversely, when
stepwise-like mutations contribute to population differentiation, RST values should be larger than FST values (Hardy
et al. 2003), except for the case of parallel evolution.
Genetic patterns in Switzerland
Our second hypothesis stated that, owing to long-lasting
human impact in Central Europe, Swiss populations of
L. pulmonaria should be clearly differentiated because of
the effects of smaller population sizes and enhanced
random sampling (cf. populations from British Columbia).
Given that L. pulmonaria suffered a significant decline in
Switzerland during the last century and is now considered
endangered in lowland regions (Scheidegger et al. 2002),
the remaining populations in the Swiss Pre-Alps and Jura
Mountains are geographically strongly isolated from each
other (Walser 2004). Apart from the fact that the geographical distances among the studied Swiss populations were
relatively small (15–244 km) it is likely that high mountains
represent substantial natural barriers to dispersal and
that current genetic exchange therefore appears improbable.
Despite a high degree of clonal reproduction within the
Swiss L. pulmonaria populations (Walser et al. 2004), and
contrary to our expectations, population diversity was still
high and the estimates of pairwise genetic differentiation
among populations were insignificant (Table 5). While this
might point to a common postglacial history of Swiss populations of L. pulmonaria, it could also indicate substantial
among-population gene exchange at least in the past.
Concordantly, the low differentiation found in this study
suggests that, historically, the populations of L. pulmonaria
were connected. That the recent dramatic demographic
changes that the species has suffered have not yet result in
detectable alterations of its genetic structure in Switzerland
might be explained by the longevity of L. pulmonaria
individuals.
Gene flow between continents
Among the many evolutionary processes acting at the species
level, it is still not clear to what extent modern disjunct
distribution patterns represent long-distance colonization
events (Högberg et al. 2002) and/or contiguous population
expansion followed by range contraction and fragmentation (i.e. vicariance; Kärnefelt 1990; Galloway 1996). In
lichenized fungi, Kärnefelt (1990) hypothesized that the
broad, but often scattered, global distributions characteristic
of many lichen species are caused not by long-distance
dispersal, but by historical fragmentation of formerly more
continuous distribution areas. The nonoverlapping allele
size distribution at locus LPu27 (Fig. 2) might be taken
as evidence against recent gene flow between the Swiss
and the Canadian populations and could point towards
past fragmentation. However, further data from the east of
North America, eastern Eurasia and western Europe may
provide more information on historical gene flow between
continents. Furthermore, this is only one level (i.e. the
fungal) of investigating diversity and differentiation in
lichen populations. Subsequent research on either the algal
or cyanobacterial photobiont might then be overlaid to give
a complete representation of lichen population structure(s).
Conclusions
Earlier studies on genetic differentiation and isolation of
lichen populations at regional scales are sparse and limited
to investigations of nuclear ribosomal DNA, which permit
the detection of only rather low levels of genetic diversity.
Our research has extended these studies by using more
variable microsatellite markers in the epiphytic lichen species
Lobaria pulmonaria. The results of our study showed that
populations both from British Columbia and Switzerland
were genetically diverse, but that the Swiss populations
showed a higher degree of vegetative propagation; and
that populations from mainland British Columbia were
substantially differentiated from coastal populations, but
not genetically isolated. This latter result may be attributable
to different glacial and postglacial histories of the populations studied. Finally Swiss populations, in contrast to
our expectations, were not genetically isolated from
each other despite the strong negative human impact on
the lichen’s range size in this Central European region.
We suggest that regional population genetic investigations should now be extended by in-depth studies of the
dispersal or gene flow processes of lichens integrating local,
landscape, regional and intercontinental scales.
Acknowledgements
We would like to thank Trevor Goward for accommodation, lively
discussions, and field assistance and Andy MacKinnon for accommodation and field assistance. We are also grateful to Phil LePage,
Davide Cuzner, and Traci Leys-Schirok from the British Columbia
Forest Service for their support. We are indebted to Trevor
Goward, Gary Walker and the anonymous reviewers for constructive comments on the manuscript. This research was funded by
the Swiss National Science Foundation (SNF 31–59241.99) and is
associated with the research program NCCR Plant Survival PS6.
References
Armstrong RA (1990) Dispersal, establishment and survival of
soredia and fragments of the lichen Hypogymnia physodes (L.)
Nyl. New Phytologist, 114, 239–245.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
10 J . - C . W A L S E R E T A L .
Bailey RH (1976) Ecological aspects of dispersal and establishment
in lichens. In: Lichenology: Progress and Problems (eds Brown DH,
Hawksworth DL, Bailey RH), pp. 215 –247. Academic Press,
London.
Balloux F, Lugon-Moulin N (2002) The estimation of population
differentiation with microsatellite markers. Molecular Ecology,
11, 155–165.
Barton NH, Slatkin M (1986) A quasi-equilibrium theory of the
distribution of rare alleles in a subdivided population. Heredity,
56, 409–415.
Benson BE, Clague JJ, Grimm KA (1999) Relative sea-level change
inferred from intertidal sediments beneath marshes on Vancouver
Island, British Columbia. Quaternary International, 60, 49–54.
Bossart JL, Prowell DP (1998) Genetic estimates of population
structure and gene flow: limitations, lessons and new directions.
Trends in Ecology and Evolution, 13, 202 – 206.
Bowcock AM, Ruizlinares A, Tomfohrde J et al. (1994) High
resolution of human evolutionary trees with polymorphic
microsatellites. Nature, 368, 455 – 457.
Bridge PD, Hawksworth DL (1998) What molecular biology has to
tell us at the species level in lichenized fungi. Lichenologist, 30,
307–320.
Brodo IM, Duran Sharnoff S, Sharnoff S (2001) Lichens of North
America. Yale University Press, London.
Cannings R, Cannings S (1996) British Columbia — A Natural History.
Greystone Books, Vancouver.
Cavalli-Sforza LL, Edwards AWF (1967) Phylogenetic analysis:
models and estimation procedures. American Journal of Human
Genetics, 19, 233–257.
Clague JJ, James TS (2002) History and isostatic effects of the last ice
sheet in southern British Columbia. Quaternary Science Reviews,
21, 71–87.
Collevatti RG, Grattapaglia D, Hay JD (2001) Population genetic
structure of the endangered tropical tree species Caryocar brasiliense,
based on variability at microsatellite loci. Molecular Ecology, 10,
349–356.
Büdel B, Scheidegger C (1996) Thallus morphology and anatomy.
In: Lichen Biology (ed. Nash TH III), pp. 37– 64. University Press,
Cambridge.
Denison WC (2003) Apothecia and ascospores of Lobaria oregana
and Lobaria pulmonaria. Mycologia, 95, 513 – 518.
Dettki H, Klintberg P, Esseen PA (2000) Are epiphytic lichens in
young forests limited by local dispersal? Ecoscience, 7, 317–325.
Dieringer D, Schlötterer C (2003) microsatellite analyser (msa):
a platform-independent analysis tool for large microsatellite
data sets. Molecular Ecology Notes, 3, 167–169.
Felsenstein J (1989) phylip — phylogeny inference package. Cladistics,
5, 164–166.
Galloway DJ (1996) Lichen biogeography. In: Lichen Biology (ed.
Nash TH III), pp. 199 –216. University Press, Cambridge.
Geitler L (1966) Die Chlorococcalen Dictyochloris und Dictyochloropsis, nov. Gen. Österreichische Botanische Zeitschrift, 113, 155–
164.
Goldstein DB, Schlötterer C (1999) Microsatellites: Evolution and
Applications. Oxford University Press, London.
Goward T (1999) The Lichens of British Columbia. British Colombia
Ministry of Forests, Victoria.
Hardy OJ, Charbonnel N, Fréville H, Heuertz M (2003) Microsatellite allele size: a simple test to assess their significance on
genetic differentiation. Genetics, 163, 1467–1482.
Hartl DL, Clark AG (1997) Principles of Population Genetics. Sinauer,
Sunderland.
Hawksworth DL, Honegger R (1994) The lichen thallus: a symbiotic
phenotype of nutritionally specialized fungi and its response to
gall producers. In: Plant Galls (ed. Williams MAJ), pp. 77– 98.
Clarendon Press, Oxford.
Hawksworth DL, Kirk PM, Sutton BC, Pegler DN (1995) Ainsworth
and Bisby’s Dictionary of the Fungi. Cambridge University Press,
Cambridge.
Heinken T (1999) Dispersal patterns of terricolous lichens by
thallus fragments. Lichenologist, 31, 603–612.
Högberg N, Kroken S, Thor G, Taylor JW (2002) Reproductive
mode and genetic variation suggest a North American origin of
European Letharia vulpina. Molecular Ecology, 11, 1191–1196.
Honegger R (1996) Mycobionts. In: Lichen Biology (ed. Nash TH
III), pp. 24–36. Cambridge University Press, Cambridge.
Jordan W (1970) The internal cephalodia of the genus Lobaria.
Bryologist, 73, 669–681.
Jordan W (1973) The genus Lobaria in North America north of
Mexico. Bryologist, 76, 225–251.
Josenhans H, Fedje D, Pienitz R, Southon J (1997) Early humans
and rapidly changing Holocene sea levels in the Queen
Charlotte Islands Hecate Strait, British Columbia, Canada.
Science, 277, 71–74.
Kärnefelt I (1990) Isidiate taxa in the Teloschistales and their
ecological and evolutionary significance. Lichenologist, 22, 307–
320.
Kroken S, Taylor JW (2001) Outcrossing and recombination in the
lichenized fungus Letharia. Fungal Genetics and Biology, 34, 83 –
92.
Landry PA, Koskinen MT, Primmer CR (2002) Deriving evolutionary
relationships among populations using microsatellites and (δµ)2:
all loci are equal, but some are more equal than others. Genetics,
161, 1339–1347.
Lutzoni F, Pagel M, Reeb V (2001) Major fungal lineages are
derived from lichen symbiotic ancestors. Nature, 411, 937– 940.
Malachowski J, Baker K, Hooper G (1980) Anatomy and the
algal–fungal interactions in the lichen Usnea cavernosa. Journal of
Phycology, 16, 346–354.
Mantel N (1967) Detection of disease clustering and a generalized
regression approach. Cancer Research, 27, 209–220.
Nybom H, Esselink GD, Werlemark G, Vosman B (2004) Microsatellite DNA marker inheritance indicates preferential pairing
between two highly homologous genomes in polyploid and
hemisexual dog-roses, Rosa L. Sect. Caninae DC. Heredity, 92,
139–150.
Parks JC, Werth CR (1993) A study of spatial features of clones in
a population of Bracken Fern, Pteridium aquilinum (Dennstaedtiaceae). American Journal of Botany, 80, 537–544.
Petit RJ, Aquinagalde I, de Beaulieu et al. (2003) Glacial refugia:
hotspots but not melting pots of genetic diversity. Science, 300,
1563–1565.
Printzen C, Ekman S, Tønsberg T (2003) Phylogeography of
Cavernularia hultenii: evidence of slow genetic drift in a widely
disjunct lichen. Molecular Ecology, 12, 1473–1486.
R Development Core Team (2003) r: a language and environment
for statistical computing. R Foundation for Statistical Computing.
Vienna, Austria.
Raybould AF, Mogg RJ, Aldam C et al. (1998) The genetic structure
of sea beet (Beta vulgaris ssp. maritima) populations. III. Detection of isolation by distance at microsatellite loci. Heredity, 80,
127–132.
Reusch TBH, Stam WT, Olsen JL (2000) A microsatellite-based
estimation of clonal diversity and population subdivision in
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x
I S O L A T I O N A N D D I F F E R E N T I A T I O N O F L I C H E N P O P U L A T I O N S 11
Zostera marina, a marine flowering plant. Molecular Ecology, 9,
127–140.
Scheidegger C (1995) Early development of transplanted isidioid
soredia of Lobaria pulmonaria in an endangered population.
Lichenologist, 27, 361– 374.
Scheidegger C, Clerc P, Dietrich M et al. (2002) Rote Liste der
Gefährdeten Arten der Schweiz: Baum- und Erdbewohnende Flechten.
BUWAL, Bern.
Scheidegger C, Frey B, Walser J-C (1998) Reintroduction and
augmentation of populations of the endangered Lobaria pulmonaria: methods and concepts. In: Lobarion Lichens as Indicators of
the Primeval Forests of the Eastern Carpathians (eds Kondratyuk SY
Coppins BJ), pp. 33 – 52. Ukrainian Phytosociological Centre,
Kiev.
Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN Version 2.000:
A Software for Population Genetic Analysis. Genetics and Biometry
Laboratory University of Geneva, Geneva.
Sillett SC, McCune B, Peck JE, Rambo TR, Ruchty A (2000) Dispersal
limitations of epiphytic lichens result in species dependent on
old-growth forests. Ecological Applications, 10, 789 –799.
Slatkin M (1985) Rare alleles as indicators of gene flow. Evolution,
39, 53–65.
Slatkin M (1995) A measure of population subdivision based on
microsatellite allele frequencies. Genetics, 159, 457– 462.
Soltis DE, Gitzendanner MA, Strenge DD, Soltis PS (1997) Chloroplast
DNA intraspecific phylogeography of plants from the Pacific
Northwest of North America. Plant Systematics and Evolution,
206, 353–373.
Stoddart JA, Taylor JF (1988) Genotypic diversity — estimation
and prediction in samples. Genetics, 118, 705 –711.
Tehler A (1996) Systematics, phylogeny and classification. In:
Lichen Biology (ed. Nash TH III), pp. 217– 239. University Press,
Cambridge.
Tunlid A, Talbot NJ (2002) Genomics of parasitic and symbiotic
fungi. Current Opinion in Microbiology, 5, 513 – 519.
Walser J-C (2004) Molecular evidence for limited dispersal of
vegetative propagules in the epiphytic lichen Lobaria pulmonaria.
American Journal of Botany, 91, 1273 –1276.
Walser J-C, Gugerli F, Holderegger R, Kuonen D, Scheidegger C
(2004) Recombination and clonal propagation in different populations of the lichen Lobaria pulmonaria. Heredity, 93, 322–329.
Walser J-C, Sperisen C, Soliva M, Scheidegger C (2003) Fungusspecific microsatellite primers of lichens: application for the
assessment of genetic variation on different spatial scales in
Lobaria pulmonaria. Fungal Genetics and Biology, 40, 72–82.
Walser J-C, Zoller S, Büchler U, Scheidegger C (2001) Speciesspecific detection of Lobaria pulmonaria (lichenized ascomycetes)
diaspores in litter samples trapped in snow cover. Molecular
Ecology, 10, 2139–2138.
Wang X, Ennos R, Szmidt A, Hansson P (1997) Genetic variability
in the canker pathogen fungus, Gremmeniella abietina. 2. Fine
scale investigation of the population genetic structure. Canadian
Journal of Botany, 75, 1460–1469.
Wirth V, Scholler H, Scholz P et al. (1996) Rote Liste der Flechten
(Lichenes) der Bundesrepublik Deutschland. Schriftenreihe für
Vegetationskunde, 28, 307–368.
Wright S (1951) The genetical structure of populations. Annals of
Eugenics, 15, 323–354.
Zoller S, Lutzoni F, Scheidegger C (1999) Genetic variation within
and among populations of the threatened lichen Lobaria pulmonaria in Switzerland and implications for its conservation.
Molecular Ecology, 8, 2049–2059.
This study was part of the PhD thesis of Jean-Claude Walser
on population genetic processes and ecological adaptation in
lichenized fungi. Rolf Holderegger studies the population and
landscape genetics of plants and their application in conservation
biology. Felix Gugerli’s research focuses on the application of
molecular markers for elucidating population processes in
space and time. Susan Hoebee is a postdoctoral fellow whose
primary research interest includes population genetics, ecology,
and conservation of threatened flora. Christoph Scheidegger
is head of the Ecological Genetics Division at the Swiss
Federal Research Institute WSL. His research interests are the
morphology, physiology, and conservation biology of lichenized
fungi.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 10.1111/j.1365-294X.2004.02423.x