Freshwater Biology (2009) 54, 283–295
doi:10.1111/j.1365-2427.2008.02107.x
Contrasting patterns of population structure in the
montane caddisflies Hydropsyche tenuis and Drusus
discolor in the Central European highlands
S T E P H A N I E L E H R I A N * , †, S T E F F E N U . P A U L S * , 1 A N D P E T E R H A A S E *
*Department of Limnology and Conservation, Research Institute and Natural History Museum Senckenberg, Gelnhausen,
Germany
†
Department Aquatic Ecotoxicology, Johann Wolfgang Goethe University Frankfurt am Main, Frankfurt, Germany
SUMMARY
1. In this study, we compared mitochondrial sequence data (cytochrome oxidase I) to infer
the population structure of the two montane caddisflies Hydropsyche tenuis and Drusus
discolor. The two species are contrasting examples of montane aquatic insects with insular
distributions: D. discolor is restricted to altitudes above 600 m, H. tenuis is limited to the
same mountain ranges in Central Europe but inhabits lower altitudes.
2. In particular, we ask whether these two species with similar regional distributions show
similar patterns of population structure and haplotype diversity, and whether any
differences can be attributed to population history and ⁄ or autecology.
3. To determine the population structure of both species, we applied conventional
population genetics analyses to mitochondrial sequence data. We collected and sampled
121 specimens of H. tenuis from 29 sites in 10 different regions of the Central European
highlands and 138 individuals of D. discolor from 40 sites in 11 different regions.
4. Nine unique haplotypes were identified for H. tenuis and 34 for D. discolor. There were
eight variable positions in H. tenuis and 41 in D. discolor. The maximum difference between
haplotypes was 0.8% (4 bp) for H. tenuis and 4.2% (21 bp) for D. discolor. We observed
haplotype overlap between geographic regions for both species. Analysis of molecular
variance showed that two-thirds of the total variance in H. tenuis was found among regions
while in D. discolor, a larger portion of variance was found within regions and populations
due to a higher number of haplotypes observed within regions. Mantel test showed a
significant relationship between genetic and geographic distance in D. discolor, but no
significant relationship in H. tenuis.
5. Our analyses show that, despite their very similar overall distribution pattern in
Europe, the two species exhibit distinct population structures, which may reflect
differences in phylogeographic history, dispersal capabilities, habitat specifity or withinregion geographic occurrence.
Keywords: cytochrome oxidase I, mitochondrial DNA, montane, population structure, Trichoptera
Correspondence: Stephanie Lehrian, Department of Limnology and Conservation Senckenberg, Research Institute and Natural History
Museum, Clamecystrasse 12, 63571 Gelnhausen, Germany. E-mail: [email protected]
1
Present address: Department of Entomology, University of Minnesota, 1980 Folwell Ave, St Paul, MN 55108, U.S.A.
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd
283
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S. Lehrian et al.
Introduction
Genetic population structuring results from limited
gene flow between subpopulations at different temporal or spatial scales. In the northern hemisphere,
especially in Central Europe, climatic shifts in the
Pleistocene may have been responsible for structuring
populations. The effects of climatic shifts on the
European biota have been widely studied and much
debated (e.g. Hewitt, 1999, 2000, 2004a). Pleistocene
climate oscillations drove numerous range expansions
and contractions, which had especially severe consequences in Europe where the Alps form a major
barrier for populations moving south or north (Thienemann, 1950; Holdhaus, 1954; De Lattin, 1967;
Taberlet et al., 1998; Hewitt, 2000). Retreat into stable
isolated refugial populations can isolate populations
that subsequently accumulate genetic differences.
These genetic differences may be maintained or
enforced if a species continues to have an insular or
discontinuous distribution or has limited powers
of dispersal (Bennett, Tzedakis & Willis, 1991; Taberlet
et al., 1998; Hewitt, 1999; Conroy & Cook, 2000;
Schmitt & Seitz, 2001; Whorley, Alvarez-Castaneda
& Kenagy, 2004; Schmitt, 2007). Thus, genetic markers
can be used to reconstruct fragmentation events and
secondary contact and ⁄ or hybridization between
lineages previously isolated in independent refugia
(Taberlet et al., 1998; Hewitt, 2000; Barton, 2001;
Pfenninger & Posada, 2002). Many studies have
focussed on examining population genetic structure
using a variety of fingerprinting methods to infer
the Pleistocene history of European species (see
Hewitt, 2004a,b; Schmitt, 2007 for recent reviews).
Although the patterns of species response to
climatic cooling are currently only understood for
some species, most workers agree that the majority of
temperate species currently occurring in Central
Europe survived glaciations in refugia with more
favourable climates in Southern Europe. During ice
free periods, such as interstadials, interglacials or in
postglacial times, the recolonization of Central Europe
started from these refugia (e.g. Zwick, 1981, 1982;
Hewitt, 1996, 2000, 2004a; Bernatchez & Wilson, 1998;
Schmitt & Seitz, 2001; Hänfling et al., 2002; Bunje,
2005) .
To date, several studies have examined the genetic
population structure of aquatic organisms in Europe
(e.g. Kelly, Bilton & Rundle, 2001; Wilcock, Hildrew &
Nichols, 2001; Kelly, Rundle & Bilton, 2002; Monaghan et al., 2002; Wilcock, Nichols & Hildrew, 2003;
Wilcock et al., 2005, 2007; Pauls, Lumbsch & Haase,
2006). In Europe, aquatic and terrestrial species may
have reacted differently to historic climate change due
to the comparatively buffered nature of the thermal
regime in aquatic ecosystems compared to that of
terrestrial systems (Malicky, 1983; Pauls et al., 2006).
Several studies of European aquatic insects have
found some novel and unexpected patterns. For
example, Monaghan et al. (2002) found differentiation
within and among streams in the mayfly Baetis alpinus
(Pictet, 1843), but not among major drainages of the
Alps. Cryptic genetic diversity was found in sympatric populations of the mayfly Baetis rhodani (Pictet,
1843) (Williams, Ormerod & Bruford, 2006) and the
flatworm Crenobia alpina (Dana, 1766) (Brändle et al.,
2007). In contrast, Pauls et al. (2006) observed Central
European Pleistocene persistence and high levels of
geographically associated population divergence in
the caddisfly Drusus discolor (Rambur, 1842). Together,
these studies show that a variety of different patterns
of population structure exist in the diverse European
aquatic insect fauna.
Generally, variation in patterns of population structure depend on the life history and evolution of the
taxa examined (Avise, 2000, 2007; Wilcock et al., 2007).
We thus propose that closely related taxa and those
occupying the same ecological niche or geographic
distribution may show similarities in genetic population structure. While Wilcock et al. (2007) compared
the genetic population structure of two caddisfly
species in the U.K. in great detail, no studies have
addressed this hypothesis in a range-wide, directly
comparative framework. Here, we compare the
population structure of two aquatic insects using
homologous mitochondrial cytochrome oxidase I
(mtCOI) sequence data. We examined the same
498 bp for both species to allow direct comparison
between the two species and avoid potential issues of
rate heterogeneity within mtCOI, as shown by Roe &
Sperling (2007). Two montane caddisflies, Hydropsyche
tenuis Navas, 1932 (Trichoptera: Hydropsychidae,
Hydropsychinae) and Drusus discolor (Trichoptera:
Limnephilidae, Drusinae) were assayed. Both species
are montane, extending to altitudes above 800 m
above sea level (asl; Haase, 1999). H. tenuis is distributed across the Central European mountain ranges
and the Apennines of Italy; D. discolor occurs in the
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 54, 283–295
Patterns of population structure in montane caddisflies
same mountain ranges in Central Europe, but its range
extends further east and west (Pitsch, 1993; Fauna
Europaea Web Service, 2007; Pauls, 2004). Both species
are cold-tolerant (Lavandier, 1992; Pitsch, 1993) and
geographically limited to these montane regions. Thus
they exhibit insular distribution patterns. However,
within montane regions, D. discolor is restricted to
altitudes above 600 m asl, whereas H. tenuis occupies a
wider altitudinal range from 200 m asl to above 1200 m
asl (Haase,1999; Voigt et al., 2006).
Our comparative study had two main aims. First,
we wanted to examine whether H. tenuis exhibits
population structure and if this is comparable to that
of D. discolor. Second, we wanted to elucidate how the
contrasting ecology and local distribution of these two
species affects their population structure. In particular, we asked if the regionally co-distributed species
show similar patterns of population structure and
haplotype diversity, and whether similarities and
differences can be attributed to population history
and ⁄ or autecology. To address these questions, we
applied conventional population genetics analyses to
mitochondrial sequence data for both species, as these
approaches are ideally suited to elucidating subdivision among such isolated populations (Knowles, 2000;
Finn & Adler, 2006).
285
(a)
(b)
Methods
Study material
We collected and sampled 121 specimens of H. tenuis
(23 adults) from 29 sites in 10 different regions of the
Central European highlands (Appendix S1, Fig. 1a).
We defined a ‘population’ as all specimens collected
from a single stream location. Specimens were collected using light traps or hand nets and preserved in
70–96% EtOH until DNA extraction. We identified
specimens to species using the study of Pitsch (1993),
Waringer & Graf (1997) and Neu & Tobias (2004) for
larvae and Malicky (2004) for adults. Voucher
specimens were deposited in the collections of the
Senckenberg Natural History Museum, Frankfurt,
Germany or in the private collection of Ralf Küttner
(Appendix S1). To compare the population structure
of H. tenuis with that of D. discolor, we included a
subset of previously published data on D. discolor
(Pauls et al., 2006). This subset was selected to cover
approximately the same geographic range that we
Fig. 1 Distribution in Central Europe and sampling sites for
Hydropsyche tenuis (a) and Drusus discolor (b). Hatched markings
indicate the distribution area in Central Europe, dots sampling
sites of H. tenuis, and boxes sampling sites of D. discolor. Hz, Harz;
RH, Rothaargebirge; ES, Elbsandsteingebirge; EZ, Erzgebirge;
TF, Thuringian Forest; RO, Rhoen; FG, Fichtelgebirge; BM,
Bohemian Massif; BF, Black Forest; VM, Vosges Mountains;
JM, Jura Mountains; AL, Alps; AM, Apennine Mountains.
sampled for H. tenuis. The analysed subset for
D. discolor comprised 138 individuals (14 adults) of
D. discolor from 40 sites in 11 different regions in
Central Europe (Appendix S2, Fig. 1b).
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Laboratory procedures
We extracted total genomic DNA from the thorax,
abdomen or legs, using DNEasy Blood & Tissue Kit or
QIAmp Microkit (both QIAGEN, Hilden, Germany)
following the protocol for purification of total DNA
from insects or tissue samples. A homologous fragment of mitochondrial DNA encoding for COI
(mtCOI) was amplified by PCR. PCR primers were
Dave
(5¢-AGTTTTAGCAGGAGCAATTACTAT-3¢,
position 2059 at 3¢ end relative to Drosophila yakuba
Burla, 1954: Zhang & Hewitt, 1996) and Inger
(5¢-AAAAATGTTGAGGGAAAAATGTTA-3¢, position 2735 at 3¢ end relative to D. yakuba: Zhang &
Hewitt, 1996) for H. tenuis. PCR amplification was
performed on a Biometra T-Gradient thermal cycler in
25 lL reactions containing one Ready-To-Go PCR
Bead (GE Healthcare, München, Germany), 0.8 lM of
each primer and 2 lL to 5 lL DNA template. PCR
cycling conditions were: 5 min at 95 C, 36 cycles of
1 min at 95 C, 1 min at 50 C, 2 min at 72 C and a
final extension of 7 min at 72 C. Laboratory procedures for D. discolor were almost the same as for
H. tenuis. PCR primers were Jerry (5¢-CAACATT
TATTTTGATTTTTTGG-3¢, position 2183 at 3¢ end
relative to D. yakuba: Simon et al., 1994) and S20
(5¢-GGGAAAAAGGTTAAATTTACTCC-3¢, position
2724 at 3¢ end relative to D. yakuba: Pauls, Lumbsch
& Haase, 2003) following the protocol published in
Pauls et al. (2006).
Sequences were generated by two commercial
sequencing companies, GATC Ltd., Konstanz,
Germany, and Nano+BioCenter, Kaiserslautern,
Germany, using the PCR primers. Sequences were
aligned, manually checked and edited in Seqman 4.0
(DNAStar, Madison, WI, U.S.A.). Only unambiguous
sequences without double peaks were included in the
study. The identity of sequences was verified using a
BLAST search (Altschul, Madden & Schäffer, 1997).
Sequences were aligned using Clustal W (Thompson,
Higgins & Gibson, 1994) as implemented in Bioedit
7.0.0 (Hall, 1999). Alignment parameters were default.
Population genetic analyses
To allow a direct comparison between the two species,
we analysed the same homologous 498 bp long
segment of mtCOI sequence data. We generated a
haplotype matrix from 121 sequences of H. tenuis and
138 sequences of D. discolor using DNAsp 4.10.9
(Universitat de Barcelona, Spain) (Rozas et al., 2003).
Median-joining (MJ) networks (Bandelt, Forster &
Röhl, 1999) were calculated in Network 4.2.0.1 Fluxus
Technology Ltd (2004–2007, Suffolk U.K.) using the
default settings. We calculated haplotype frequencies
and mean pairwise differences between geographical
regions and each population as well as genetic
differentiation with pairwise FST and exact test of
population differentiation (ETPD) (Raymond & Rousset, 1995) using the default parameters in Arlequin
3.11 (Excoffier, Laval & Schneider, 2005). Analysis of
molecular variance (A M O V A , Excoffier, Smouse &
Quattro, 1992) was performed in Arlequin 3.11 to
examine the distribution of total genetic variation at
different geographic hierarchies (within populations,
between populations within groups and among
groups). The a priori grouping design was based on
major mountain ranges and sampled populations
were grouped accordingly. This approach is nonrandom, but reflects the natural insular distribution
pattern of both species. This procedure resulted in 10
groups for H. tenuis and 11 groups for D. discolor.
A Mantel test was applied to the matrices of pairwise
FST and geographical distance between each mountain
range to assess isolation-by-distance (Mantel, 1967).
Geographic distances were based on the geographic
centre of populations within each mountain range.
Ten thousand permutations were run. For mountain
ranges and the total data set, we calculated Tajima’s D
(Tajima, 1989) and Fu’s F (Fu, 1997). Neutrality tests,
gene diversity (Nei, 1987) and pairwise uncorrected
distances within mountain ranges were calculated in
Arlequin 3.11 (Excoffier et al., 2005) using default
settings.
Results
We generated a 498-bp aligned mtCOI sequence fragment of 121 H. tenuis specimens. A homologous
sequence fragment was generated for 138 D. discolor
specimens from a comparable geographic range (Fig. 1)
using previously published data. The data set comprised all specimens published by Pauls et al. (2006)
from 11 regions: the Harz, Rothaargebirge, Thuringian
Forest, Rhoen, Erzgebirge, Fichtelgebirge, Bohemian
Massif, Black Forest, Vosges Mountains, Jura Mountains and Alps (Appendix S2). The alignments did not
contain ambiguous sites or gaps. Eight variable
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 54, 283–295
Patterns of population structure in montane caddisflies
positions were found for H. tenuis and 41 variable
positions for D. discolor. Nine unique haplotypes were
identified for H. tenuis (Fig. 2; GenBank Accession
numbers EU884268–EU884276). Across a comparable
Table 1 Summary of population differentiation values within
populations and mountain ranges for both species
n ⁄ % of
significant
pairwise FST
Populations
H. tenuis
D. discolor
Regions
H. tenuis
D. discolor
n ⁄ % of
significant
ETPD results
18 ⁄ 62
37 ⁄ 92.5
9 ⁄ 90
11 ⁄ 100
22 ⁄ 75.9
37 ⁄ 92.5
9 ⁄ 90
11 ⁄ 100
ETPD, exact test of population differentiation.
Detailed pairwise tests are shown in Appendix S3.
287
geographic range, 34 haplotypes were identified in the
data set of D. discolor (Fig. 2; GenBank Accessions: AY954396, AY954397, DQ351157–DQ351162,
DQ351164–DQ351166, DQ351168–DQ351170, DQ351
171, DQ351173–DQ351175, DQ351181, DQ351183,
DQ351184–DQ351188, DQ351190–DQ351194, DQ351
196, DQ351206, DQ351211).
Population genetic analyses and haplotype distribution
A MJ network was calculated for each species
(Fig. 2). For H. tenuis eight mutational steps were
sufficient to combine all haplotypes, while for
D. discolor 55 steps were necessary. We observed
haplotype overlap between geographic regions for
both species. The numbers of haplotypes per region
ranged from one in the Rhoen to four in the Alps
Fig. 2 Upper: median-joining network of Hydropsyche tenuis; lower: median-joining network of Drusus discolor. Haplotype diameter
reflects the number of individuals carrying a given haplotype. Missing intermediates are added to the network and called median
vectors (Posada & Crandall, 2001). Median vectors are illustrated with boxes in the network. Colour coding refers to geographic origin
of specimens.
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S. Lehrian et al.
for H. tenuis and one to 13 in D. discolor (Jura
Mountains and Alps).
In H. tenuis, the most common haplotype (H5)
was found in the Erzgebirge, Bohemian Massif,
Elbsandsteingebirge, Black Forest, the Alps and in
the Jura Mountains (Fig. 2 upper). The second most
common haplotype (H4) was found in the Alps,
Apennine Mountains and the Bohemian Massif. In the
northern populations (Harz, Rhoen and Thuringian
Forest), four haplotypes (H1, H2, H3, H8) occurred
that showed no overlap with the southern populations. Private haplotypes were found in four localities:
one (H2) in the Rhoen, two (H3, H8) in Thuringian
Forest, two (H6, H7) in the Alps and one (H9) in the
Jura Mountains. In total, six haplotypes (67%) were
private to a single mountain range, while the remaining haplotypes were either restricted to the northern
or southern populations.
In D. discolor, we found four haplotype groups (‡3
mutational steps divergence) (Fig. 2 lower). Group 1
(left) consisted of 13 haplotypes and individuals from
the Alps, Bohemian Massif, Erzgebirge and Fichtelgebirge. Group 2 (top) represents a second Alps
lineage, which was quite diverged (‡11 mutational
steps). Group 3 (central) comprised ten haplotypes
and individuals from the Erzgebirge, Fichtelgebirge,
Harz, Rhoen, Rothaargebirge and Thuringian Forest.
Group 4 (right) consisted of seven haplotypes from
individuals from the Black Forest, Jura and Vosges
Mountains. Haplotype overlap between geographic
regions was observed in the most common and central
haplotypes of groups 1 and 3 (H6 and H12 respectively) and within group 4. In total, 30 D. discolor
haplotypes (88%) were private to a single region.
Twelve private haplotypes occurred in the Alps; four
in the Thuringian Forest; three in the Black Forest and
Bohemian Massif; two each in the Erzgebirge, Rothaargebirge and Vosges Mountains; and one in both
the Harz and Fichtelgebirge. Potentially the private
haplotypes of H. tenuis and D. discolor are endemic.
However, due to small sampling size in some regions
with private haplotypes (i.e. two individuals in Jura
Mountains for H. tenuis and four individuals in
Rothaargebirge for D. discolor), their restricted
occurrence could be a sampling effect for some of
these haplotypes (H9 in H. tenuis, and H05 and H07 in
D. discolor).
Exact tests of population differentiation showed
that 22 of 29 populations of H. tenuis and 37 of 40
populations of D. discolor were significantly differentiated. The ETPD revealed significant differentiation
in nine of 10 regions for H. tenuis and all 11 regions
for D. discolor (Table 1, Appendix S3). The region
Elbsandsteingebirge, which did not show significant
difference in H. tenuis, comprised only one individual
with the common haplotype H5. FST-values supported
the ETPD analyses. Significant differentiation
occurred between 18 of 29 populations of H. tenuis
and between 37 of 40 populations of D. discolor. When
regions were compared, nine of 10 regions of H. tenuis
(all except Elbsandsteingebirge) and all 11 regions of
D. discolor were significantly differentiated based on
FST-values (Table 1, Appendix S3).
The results of the A M O V A from both species are
summarized in Table 2. The highest variance (58.15%)
for H. tenuis was found among mountain ranges.
Among populations within mountain ranges and
within populations variation is about equal (c. 20%).
For D. discolor, similar levels of genetic variance were
found among populations within mountain ranges
(39%) and among mountain ranges (38%). In
D. discolor, within population variation explained
23% of the total variation.
Within mountain ranges, gene diversity ranged
from 0.295 ± 0.156 to 1 ± 0.500 in H. tenuis and from
0.333 ± 0.215 to 0.833 ± 0.222 in D. discolor (Table 3).
Pairwise uncorrected distances between haplotypes
within mountain ranges ranged in H. tenuis from 0%
to 1.0% and in D. discolor from 0% to 6.31%. Mantel
test did not demonstrate a significant isolation by
% of
variation
P-value
Source of variation
Ht
Dd
Ht
Dd
Ht
Dd
Among regions
Among populations within regions
Within populations
58.15
22.12
19.73
37.60
39.31
23.10
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.582
0.529
0.803
0.376
0.630
0.769
/-statistics
Table 2 Analysis of molecular variance
for the groupings of Hydropsyche tenuis
and Drusus discolor populations
Ht, Hydropsyche tenuis; Dd, Drusus discolor.
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Patterns of population structure in montane caddisflies
289
Table 3 Gene diversity (Nei, 1987) and pairwise uncorrected distances within mountain ranges for Hydropsyche tenuis and Drusus
discolor
n
Uncorrected pairwise distances
Gene diversity
Mountain range
Ht
Dd
Harz
Rothaargebirge
Elbsandsteingebirge
Erzgebirge
Thuringian Forest
Rhoen
Fichtelgebirge
Bohemian Massif
Black Forest
Vosges Mountains
Jura Mountains
Alps
Apennine Mountains
all populations
11
n.d.
1
4
13
5
n.d.
2
9
n.d.
2
63
9
121
6
4
n.d.
16
16
3
12
8
9
11
5
48
n.d.
138
Ht
Dd
–
n.d.
–
–
0.295 ±
–
n.d.
1.000 ±
–
n.d.
1.000 ±
0.573 ±
–
0.708 ±
0.156
0.500
0.500
0.044
0.027
0.333 ±
0.833 ±
n.d.
0.717 ±
0.450 ±
–
0.546 ±
0.786 ±
0.694 ±
0.746 ±
–
0.751 ±
n.d.
0.875 ±
0.215
0.222
0.071
0.151
0.062
0.113
0.147
0.098
0.061
0.018
Ht
Dd
0
n.d.
0
0
0.31
0
n.d.
1.00
0
n.d.
1.00
0.70
0
1.03
0.33
1.17
n.d.
3.03
0.50
0
3.27
1.07
1.83
1.60
0
6.31
n.d.
6.09
Ht, Hydropsyche tenuis; Dd, Drusus discolor.
‘n’ refers to the number of specimens studied from each mountain range; ‘–’ indicates there was only one haplotype; ‘n.d.’ means there
were no data available.
distance effect in H. tenuis (r = 0.106, P = 0.31), while
isolation by distance was inferred for D. discolor
(r = 0.574, P = 0.00).
Neutrality tests showed no evidence that either
species experienced departures from selective neutrality when we examined the entire populations. This
suggested that neither species has recently experienced recent demographic expansion as a whole.
However, in particular regions, there was evidence for
demographic expansion based on significantly negative Tajima’s D and Fu’s F (Table 4). This was the case
in the Thuringian Forest populations for both species,
although only Fu’s F was significantly negative ()1.40,
P < 0.02) for H. tenuis. In D. discolor, there was also
evidence for demographic expansion in the Eastern
Alps.
Discussion
Contrasting patterns of population structure
Our ETPD, A M O V A and FST analyses all indicated that
both species have significant population structure.
However, these structures are different. Both species
carry numerous regionally private haplotypes (six in
H. tenuis, 30 in D. discolor). Three of these (H9 in
H. tenuis, and H05 and H07 in D. discolor) could result
from small sample size within some regions (two
individuals in Jura Mountains for H. tenuis and four
individuals in Rothaargebirge for D. discolor). However, the consistent occurring of private haplotypes
across several regions in both species (Rhoen, Thuringian Forest, Jura Mountains, Alps in H. tenuis; all
regions except Jura Mountains and Rhoen in
D. discolor) suggests that these could be regionally
endemic, in particular those that are carried by two or
more individuals. Most populations and mountain
ranges are significantly differentiated from one
another in both species.
In contrast, stark differences occur with respect to
haplotype diversity and variability. Drusus discolor has
more than three times as many haplotypes as H. tenuis
across a similar geographic range and with comparable
sample sizes. Also, D. discolor only has four haplotypes
shared between neighbouring regions (H06, H12, H13,
H36), whereas H. tenuis has two widespread haplotypes (H4, H5). Also gene diversity within D. discolor
(H = 0.875) is higher than in H. tenuis (H = 0.708).
These results clearly show that D. discolor is genetically more diversified in the Central European
highlands than H. tenuis. A major difference between
D. discolor and H. tenuis is the within- and amongmountain range divergence between haplotypes and
haplotype clades. Within H. tenuis all haplotypes can
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290
S. Lehrian et al.
Table 4 Tests of selective neutrality for Hydropsyche tenuis and Drusus discolor
Ht
Dd
Ht
Dd
Mountain range
Tajima’s D
P
Tajima’s D
P
Fu’s Fs
P
Fu’s Fs
P
Harz
Rothaargebirge
Elbsandsteingebirge
Erzgebirge
Fichtelgebirge
Rhoen
Thuringian Forest
Bohemian Massif
Black Forest
Vosges Mountains
Jura Mountains
Alps
Apennine Mountains
Eastern Alps (Haplogroup 1 in Dd)
Western Alps (Haplogroup 2 in Dd)
all populations
–
n.d.
–
–
n.d.
–
)1.40
–
–
n.d.
–
0.19
–
n.d.
n.d.
)0.73
–
n.d.
–
–
n.d.
–
0.064
–
–
n.d.
–
0.640
–
n.d.
n.d.
0.225
)0.93
0.59
n.d.
1.53
2.47
–
)1.83*
)0.30
)0.74
0.63
–
0.54
n.d.
)2.13*
0.72
)0.56
0.256
0.817
n.d.
0.945
0.999
–
0.014*
0.386
0.248
0.739
–
0.758
n.d.
0.002*
0.773
0.334
–
n.d.
–
–
n.d.
–
)1.40*
–
–
n.d.
–
0.13
–
n.d.
n.d.
)2.60
–
n.d.
–
–
n.d.
–
0.007*
–
–
n.d.
–
0.504
–
n.d.
n.d.
0.138
)0.003
)0.66
n.d.
2.69
6.27
–
)3.31*
)1.1
0.21
0.27
–
0.85
n.d.
)10.37*
0.59
)8.34
0.256
0.154
n.d.
0.905
0.992
–
0.001*
0.082
0.510
0.550
–
0.671
n.d.
<0.001*
0.634
0.036
Ht, Hydropsyche tenuis; Dd, Drusus discolor.
‘–‘ indicates there was only one haplotype; ‘n.d.’ means there were no data available.
*Significant values at P < 0.05 (Tajima’s D) and P < 0.02 (Fu’s F) (Excoffier et al., 2005).
be connected by a single base pair change and the MJ
network spans eight nucleotide changes. In D. discolor,
the MJ network spans 55 nucleotide changes. The
maximum difference between haplotypes in H. tenuis
is four basepairs (0.8%) compared to 21 basepairs
(4.2%) in D. discolor. The larger genetic differences
between haplotypes show that isolation mechanisms
between populations and mountain ranges seem to
have been stronger and ⁄ or in place for a longer time in
D. discolor than in H. tenuis.
Reasons for differences in divergence and diversification could lie in the species’ ecology and lifehistory traits (Avise, 2000, 2007; Wilcock et al., 2007).
These species’ different altitude preferences may have
affected their lateral dispersal among mountain
ranges. H. tenuis tends to occupy lower altitudes
(>200 m asl) than D. discolor (>600 m asl) within the
same montane mountain ranges. Therefore, the average distance between suitable habitats among mountain ranges is shorter for H. tenuis than for D. discolor.
The shorter distances could allow more frequent
dispersal events between geographic isolates. As with
most caddisflies, larval movement of H. tenuis and
D. discolor is limited to the upstream or downstream
movement in the water course. Therefore, lateral
dispersal between different courses of running water
is limited to the winged adult stage. Although our
knowledge of dispersal capabilities of caddisflies is
limited, most existing studies show that adult aquatic
insects generally tend to stay close to the stream from
which they emerge (Sode & Wiberg-Larsen, 1993;
Kovats, Ciborowski & Corkum, 1996; Collier & Smith,
1998; Petersen et al., 2004). On the other hand,
Hydropsychidae are considered relatively good fliers.
Malicky (1987) showed that the flight range of
Hydropsyche saxonica McLachlan, 1884 extends beyond
3 km. A combination of better flight dispersal and
shorter distances between populations could favour
gene flow between H. tenuis populations within and
between mountain ranges. However, in a scenario of
more frequent dispersal between populations, we
would also expect less population differentiation in
H. tenuis. Our FST-values and ETPD indicate that most
populations and regions in H. tenuis are significantly
differentiated, thus inferring limited or no gene flow.
Therefore, we find no evidence that differences in
niche distribution and adult dispersal play an important role for the distinct present-day patterns of
population differentiation.
If adult dispersal within regions was frequent, we
would expect an isolation-by-distance pattern, which
was rejected for H. tenuis by the Mantel test. In
contrast, isolation-by-distance was observed in
D. discolor in Central Europe. This result is somewhat
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 54, 283–295
Patterns of population structure in montane caddisflies
unexpected based on findings across the whole
distribution range of D. discolor, where neighbouring
mountain ranges in western Europe (Western Alps,
Massif Central, Pyrenees) are among the most
diverged (Pauls et al., 2006). However, the clustering
of only minimally diverged haplotypes into regional
clades leads to a pattern of isolation-by-distance in the
data analysed. The different genetic structure between
northern and southern populations suggests that
processes structuring the Central European and
Southern European populations of D. discolor may be
different.
While they do not seem to be the primary source for
differences in population structure, the differences in
life history of both species may have a pronounced
and varying effect on their respective population
histories. For D. discolor, a pattern of Pleistocene
persistence in mountain ranges in Central Europe has
been inferred based on the species distribution
(Malicky, 1983), its extreme cold tolerance (Lavandier,
1992) and its strong genetic population structure
(Pauls et al., 2006; Pauls, Lumbsch & Haase, in press).
Lower tolerance of extremely cold habitats and
tolerance of much warmer streams may have driven
H. tenuis into milder Pleistocene refugia in southern
Europe. The most common haplotype in H. tenuis,
(H5), is widespread across the central distribution
area and is missing only in the most northern (Rhoen,
Thuringian Forest and Harz) and most southern
(Apennine Mountains) populations. Its geographic
distribution and central position in the network
indicate that H5 is an ancestral haplotype (Posada &
Crandall, 2001) and that the other eight haplotypes
were derived from it. Also, with only single basepair
changes and so few haplotypes, time since divergence
in H. tenuis appears limited compared to D. discolor.
Although divergence times cannot be calculated with
much credibility based on single basepair changes, the
pattern observed in H. tenuis is consistent with
isolation occurring after last glacial maximum
(LGM) c. 12 000 years ago. In contrast, the pattern in
D. discolor is dated to well before the LGM (Pauls
et al., 2006). Further analyses of more variable markers
are needed to obtain more robust estimates of intraspecific divergence times for H. tenuis.
The population structure and intraspecific divergence and diversity of H. tenuis are not typical of
species like D. discolor that show northern Pleistocene
persistence. Rather, the pattern is consistent with a
291
warm-tolerant aquatic species that postglacially
expanded northward from a southern refugia. The
data collected for H. tenuis has characteristics of two
patterns that are consistent with such a recolonization
event: reduced genetic variation (Hewitt, 1999; Muster
& Berendonk, 2006; Pabijan & Babik, 2006), either as a
consequence of bottlenecks in the refugial population,
and ⁄ or founder effects during the recolonization
(Freeland, 2005), and a haplotype gradient along the
recolonization route(s). Very little haplotype diversity
exists, and the northern populations (Harz, Thuringian Forest, Rhoen) only have regionally private,
‘northern’ haplotypes. These data support a scenario
of postglacial recolonization of H. tenuis from one or a
few refugia, presumably in Southern Europe. After
expansion into other montane regions, gene flow
could have become limited between populations from
different mountain ranges, allowing endemic haplotypes to evolve. However, with only three H. tenuis
samples from two localities (Bohemian Massif and
Elbsandsteingebirge), our sampling is limited in
the transitional area between the ‘northern’ and
‘southern’ clade. We also do not see an isolation-bydistance effect in H. tenuis, or significant results in
neutrality tests, which could indicate recent demographic expansion of the transitional populations or
the entire species (Tajima, 1989; Fedorov & Stenseth,
2002; Lopes, Miño & Del Lama, 2007). Thus, we
cannot confidently resolve whether there is a north–
south haplotype gradient with our data. More
detailed sampling of the entire distribution range
combined with detailed phylogeographic analyses of
mtCOI and other independent nuclear markers (e.g.
microsatellites) are needed to test our hypothesis of
southern refugia, expansion and subsequent isolation
in H. tenuis.
General patterns of population structure in caddisflies
Clear patterns of genetic population structure due to
limited gene flow and high levels of microendemic
diversity are common in insects inhabiting high
montane regions, or ‘sky islands’ (Knoll & RowellRahier, 1998; Knowles, 2001; Mardulyn, 2001; Finn
et al., 2006; Pauls et al., 2008; W. Graf, J. Waringer &
S. U. Pauls, unpubl. data) and several studies show
that aquatic insects with insular distribution patterns
in Europe exhibit population structure. Kelly et al.
(2001, 2002) found that two caddisfly species endemic
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 54, 283–295
292
S. Lehrian et al.
to the Canary Islands show high levels of population
structure, although this is not hierarchically structured. A similar pattern is presented for D. discolor
across mountains in Europe (Pauls et al., 2006). However, in D. discolor, the degree of population differentiation is very high. Each region may maintain
independent evolutionary entities (e.g. cryptic species) that have not accumulated sufficient morphological characters to allow easy distinction. Similarly,
in their study of Orthopsyche fimbriata (McLachlan,
1862), a North Island endemic of New Zealand, Smith,
McVeagh & Collier (2006) observed up to 5.2% mtCOI
sequence divergence between different regional haplotype clades. The overall pattern of population
structure showed population differentiation among
more distant populations and between catchments
and regions, while closer populations were not
significantly differentiated. However, in contrast to
D. discolor, O. fimbriata does not exhibit an insular
distribution pattern. Another caddisfly with insular
distribution pattern is Rhyacophila pubescens Pictet,
1834. This species is restricted to limestone tufa
streams in mountain regions across Central Europe.
The species exhibits strong population structure but
only little differences (0.84%) between haplotypes
across a shorter but homologous fragment of mtCOI
(Engelhardt, Pauls & Haase, in press). Haplotype
differences are almost the same in H. tenuis (0.80%)
and R. pubescens.
Varying patterns of population differentiation have
also been observed in species with less insular distribution patterns. Wilcock et al. (2003, 2007) found
contrasting patterns of population genetic structure
between two cofamilial caddisflies Plectrocnemia
conspersa (Curtis, 1834) and Polycentropus flavomaculatus (Pictet, 1834), which have less pronounced insular
distribution patterns than H. tenuis and D. discolor. In
two regions in south-east and north-west England,
P. conspersa showed varying patterns of population
structure. While there was no relationship of increasing genetic differentiation and geographic distance
below 100 km in the south-east, such a pattern was
observed at different levels below 40 km in the northwest. Polycentropus flavomaculatus exhibited much
stronger genetic structure in south-east England than
P. conspersa in either region (Wilcock et al., 2007).
Although the number of studies on European
caddisfly population genetics is limited to date,
different patterns of gene flow and population
structure exist across different geographic scales.
Most of the studies observe different, and often
unexpected and contrasting patterns between different species, even when they occupy a similar ecological niche and have comparable geographic
distributions. Our study, for example, shows that
H. tenuis and D. discolor may have similar geographic
distribution patterns, but that their population structure is very different. It would appear that the
differences in local niche occupancy and local spatial
distribution are not responsible for these differences.
Rather, the phylogeographic history of the two species, related to their different ecologies, may have
played a major role in shaping contemporary patterns.
The reported diversity in patterns of population
genetic structure in caddisflies suggests that many
more interesting and unique patterns will be found by
studying the population genetics and phylogeography
of caddisflies and other aquatic insects. These patterns
can then serve as an important basis for addressing
more general questions on their recruitment, dispersal, diversification and evolution.
Acknowledgments
We wish to thank all our colleagues who supported
this study by providing material and information or
assisting in collecting H. tenuis and D. discolor (see
Appendices S1 and S2) and Michel Sartori and Ralf
Küttner who sent individuals from their collections.
We thank Susan J. Weller, Alan G. Hildrew and two
anonymous reviewers for providing helpful comments on an earlier version of this manuscript. This
study was financially supported by the German
Research Foundation (DFG) (HA3431 ⁄ 2-1) to PH and
SUP and a scholarship from German National Merit
Foundation (Studienstiftung des deutschen Volkes) to
SL.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article.
Appendix S1. Material of Hydropsyche tenuis used in
this study.
Appendix S2. Material of Drusus discolor used in
this study.
Appendix S3. Results of exact tests of regional
differentiation (ETPD) and pairwise regional FSTvalues for Hydropsyche tenuis (Ht) and Drusus discolor
(Dd).
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(Manuscript accepted 7 August 2008)
2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 54, 283–295