Freshwater Biology (2006) 51, 193–205
doi:10.1111/j.1365-2427.2005.01478.x
Population structure and the impact of regional and local
habitat isolation upon levels of genetic diversity of the
endangered damselfly Coenagrion mercuriale (Odonata:
Zygoptera)
PHILLIP C. WATTS, ILIK J. SACCHERI, STEPHEN J. KEMP AND DAVID J. THOMPSON
School of Biological Sciences, Liverpool University, Crown Street, Liverpool, L69 7ZB, U.K.
SUMMARY
1. Coenagrion mercuriale is one of Europe’s most threatened damselflies. There is concern for
the long-term persistence of many of its U.K. colonies because adult lifetime movement is
limited, making isolated populations susceptible to extinction.
2. Using 14 microsatellite loci we characterised levels of genetic diversity, evidence for a
recent decline and the spatial genetic structure for C. mercuriale population in Wales, U.K.
3. Spatial isolation is not an absolute predictor of low genetic diversity at either local or
regional scales.
4. One population inhabiting a remote, edge of range site is genetically impoverished with
levels of variability (at microsatellite loci) among the lowest reported for any insect species.
5. Agricultural land and high ground are physical barriers to dispersal by adults.
6. Consistent with work from elsewhere, movement by mature C. mercuriale in
Pembrokeshire is sufficient to prevent significant genetic differentiation throughout a
habitat matrix of some 3–4 km if the suitable habitat sites are <2 km apart and lack barriers
to movement. Even within a good habitat matrix, however, genetic isolation by distance
develops within 10 km.
Keywords: biodiversity, bottleneck, conservation, dispersal, population structure
Introduction
Destruction and fragmentation of habitat are major
causes of biodiversity loss [World Conservation
Monitoring Centre (WCMC), 1992]. Organisms sensitive to the effects of habitat fragmentation are likely to
have some combination of low natural abundance/
high area requirement, large population fluctuations,
low intrinsic growth rate, specialised habitat requirements and/or poor dispersal capability (Henle et al.,
2004). Particularly by impacting upon the capability of
individuals to disperse, habitat fragmentation can
Correspondence: Phill Watts, Marine and Freshwater Biology
Research Group, The Biosciences Building, School of Biological
Sciences, Liverpool University, Crown Street, Liverpool,
L69 7ZB, U.K.
E-mail: p.c.watts@liv.ac.uk
lead to a reduction in population size that will
heighten extinction risk by increasing vulnerability
to stochastic demographic changes or reducing genetic diversity (Frankham, 1995; Frankham, Ballou &
Briscoe, 2002). Maintaining genetic diversity is important as it is associated with population viability
(Saccheri et al., 1998; Madsen et al., 1999; Spielman
et al., 2004a; Spielman, Brook & Frankham, 2004b) and
the evolutionary potential of a species to respond to
environmental change (Frankham et al., 2002; Reed &
Frankham, 2003; Schmitt & Hewitt, 2004). European
wetlands, which are inherently discontinuous, are
becoming increasingly patchy, largely because of
pollution, eutrophication or changes in land use. A
decline in movement among habitat patches may be
expected for many freshwater species, with the
expected consequences of genetic erosion and population decline.
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193
194
P.C. Watts et al.
As successful conservation of insect taxa is largely
achieved by maintaining areas of suitable habitat it is
imperative that species’ dispersal capabilities are
characterised so that an appropriate level of connectivity within a habitat network can be established in
order to ameliorate the detrimental effects of fragmentation. Characterising dispersal can be problematic for freshwater insects as many can disperse as
aquatic larvae and/or terrestrial adults. Nevertheless,
a number of studies have directly tracked dispersal of
aquatic insects (e.g. Miller, Blinn & Keim, 2002; Elliott,
2003; Petersen et al., 2004; Watts et al., 2004a). Such
direct observations, however, are likely to overlook
rare, long-distance movement events and, importantly, fail to inform on the successful transfer of
gametes that affect levels of diversity within populations (Slatkin, 1985). Many studies, therefore, have
used the spatial distribution of neutral genetic markers to make indirect appraisals of the dispersal
characteristics of a variety of freshwater insect taxa
(e.g. Geenen et al., 2000; Smith & Collier, 2001; Kelly,
Rundle & Bilton, 2002; Miller et al., 2002; Hughes,
Hillyer & Bunn, 2003a; Hughes et al., 2003b; Wilcock,
Nichols & Hildrew, 2003; Wishart & Hughes, 2003;
Watts et al., 2004a).
Odonates (damselflies and dragonflies) are a key
component of many freshwater ecosystems that are
generally perceived to be good fliers, potentially
capable of wide dispersal and possibly less sensitive
to habitat fragmentation than other freshwater taxa.
Numerous odonate species are declining, however,
with nearly 40% of indigenous European odonates
classified as endangered, vulnerable or rare under
IUCN red book categories (Van Tol & Verdonk, 1988).
The axiom of wide dispersal may hold for many
anisopterans (Corbet, 1999; Freeland et al., 2003) but
zygopterans are weaker fliers, with many species not
dispersing beyond several kilometres (Conrad et al.,
1999; Geenen et al., 2000; Purse et al., 2003; Watts et al.,
2004a). Thus, it is somewhat surprising that genetic
differentiation has been reported among zygopteran
populations separated by up to 100 km (Andrés,
_ & Cordero Rivera, 2000, 2002) but
Sanchez-Guillen
not shorter distances (Geenen et al., 2000; Wong,
Smith & Forbes, 2003; but cf. Watts et al., 2004a).
This paper presents an analysis of genetic variability and population structure of an endangered
damselfly, Coenagrion mercuriale (Charpentier, 1840)
(Odonata: Zygoptera). Coenagrion mercuriale, restricted
to the south and west of Europe, is considered extinct
or on the edge of extinction in seven European
countries (Grand, 1996) and is listed in Appendix II
of the Berne Convention of 1979 (Grand, 1996), in
Appendix II of the European Community Habitat and
Species Directive of 1992 (Jackson & McLeod, 2000)
and as rare in the British Red Data Book. This species
is protected by the Wildlife and Countryside Act
(TSO, 1981) within the U.K. where, largely because of
decline/fragmentation of suitable habitat, it has suffered a 30% reduction in distribution during the last
100 years (Thompson, Rouquette & Purse, 2003).
Coenagrion mercuriale is now limited to a few populations in southern England and Wales, with core sites
in the New Forest and along the River Itchen (both in
England) and in Pembrokeshire (Wales). There is
concern for the long-term persistence of C. mercuriale
because adult lifetime movement rarely exceeds 2 km
and is typically <100 m (Hunger & Röske, 2001; Purse
et al., 2003; Watts et al., 2004a), restricting (re-) colonisation of suitable habitat to localities in close
proximity to extant sites. A previous study in the
Itchen Valley demonstrated that limited movement by
C. mercuriale leads to significant, local spatial genetic
structuring (Watts et al., 2004a). Here, we extend our
investigation into the population structure of C.
mercuriale to include all remaining population centres
in Wales (U.K.) that contrast with sites along the
Itchen Valley by exhibiting a greater level of spatial
isolation. In addition to exploring the pattern of
spatial genetic structure, we ask:
(i) What is the effect of habitat isolation upon
genetic diversity at local and regional scales? and
(ii) Do populations of C. mercuriale show a genetic
signature of demographic decline?
These questions are discussed with respect to the
general conservation of this species.
Methods
Description of study sites
We studied the remaining regions in Wales (Pembrokeshire, Gower and Anglesey) that host populations of C. mercuriale. This species does not inhabit
areas above 300 m in the U.K. (Purse, 2001) but the
Pembrokeshire populations (situated >150 m above
sea level) are at higher altitudes than other sites in
Wales (60–70 m; S. Coker, personal communication).
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
Genetic variation in an endangered damselfly 195
Fig. 1 Map of the location of three remaining population centres of C. mercuriale in Wales, U.K. Expanded section highlights geographic location of sampled (filled circles) and unsampled (open circles) sites occupied by C. mercuriale in Pembrokeshire: northern
[Brynberian (BRY), Clun Maen (CLM)], eastern (not sampled), southern [Cors Tewgyll (COT), Dolau Isaf (DOI), Gors Fawr (GOF),
Pantithel (PAN), Waun Isaf North (WAN)], Waun Maes (WAM) and Waun Fawr (WAF); light grey shading indicates land >300 m
high.
In Pembrokeshire, populations distributed in springs
on the Preseli Hills (Fig. 1) are undergoing a reduction
in size and distribution but still thought to be large
and extensive enough to be self-sustaining. Within
this area we collected samples from nine sites (Fig. 1)
that represent four of the five population centres that
have some separation by improved agricultural land;
northern [Brynberian (BRY), Clun Maen (CLM)],
eastern (not sampled), southern [Cors Tewgyll
(COT), Dolau Isaf (DOI), Gors Fawr (GOF), Pantithel
(PAN), Waun Isaf North (WAN)] and two colonies at
Waun Maes (WAM) and Waun Fawr (WAF). The
latter two sites are separated from the core areas by
relatively large distances (approximately 3 and 6 km,
respectively) and high (>300 m) ground. At 290 m in
altitude, WAM is 80–90 m higher than the other
Pembrokeshire sites and at the environmental limit of
this species (Purse, 2001). Although presently vigorous, the WAF population is believed to be susceptible
to even subtle environmental changes because it is too
remote to receive migrants (Coker, 2001). All colonies
(except DOI and PAN which are on private land) in
Pembrokeshire are on common grazing land. Coenagrion mercuriale was presumably more widespread on
Gower but is now found at only two sites (Cefn Bryn
and Rhossili) that are separated by 7.5 km of farmland. At the extreme north of C. mercuriale’s U.K. (and
European) distribution and 150 km from the nearest
populations in Pembrokeshire, Nant Isaf on the island
of Anglesey represents an extremely remote site
(Fig. 1). There is concern for the small C. mercuriale
population at Anglesey because changes in the
grazing regime on this privately owned farmland
might have reduced habitat quality.
DNA extraction and polymerase chain reaction
Genomic DNA was extracted using a high salt
protocol (Sunnucks & Hales, 1996) from a tibia (stored
in a 1.5 mL tube containing 100% ethanol) for about
50 damselflies per site (Appendix). Loss of a leg does
not measurably affect fitness in damselflies (Fincke &
Hadrys, 2001) and we observed no significant effect of
sampling upon recapture rate elsewhere (D.J. Thompson, unpublished data). We examined allelic variation
in 14 microsatellite loci (Appendix) characterised by
Watts, Thompson & Kemp (2004b) and Watts et al.
(2004c). Approximately 5 ng of DNA was used for a
10 lL polymerase chain reaction (PCR) containing
75 mM Tris-HCl pH 8.9, 20 mM (NH4)2SO4, 0.01% v/
v Tween-20, 0.2 mM each dNTP, 3.0 mM MgCl2,
20 pmol forward primer, 30 pmol reverse primer
and 0.25 U T A Q polymerase (ABgene, Epsom, U.K.).
Thermal cycling conditions are described by Watts
et al. (2004b,c). PCR products were pooled into one of
two genotyping pools, determined by allelic size
range and the 5¢ fluorescent dye, along with a G E N E S C A N -500 L I Z size standard (Applied Biosystems,
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
196
P.C. Watts et al.
Foster City, CA, U.S.A.) and separated by capillary
electrophoresis through a denaturing acrylamide gel
on an ABI3100 automated sequencer (Applied Biosystems). Alleles were sized using the cubic model
of analysis in the GeneMapper analysis software
(Applied Biosystems).
Data analysis
Genetic variability. Genotypic linkage equilibrium
among all locus-pair combinations was assessed for
each population using the Fisher’s exact test implemented by the online version (3.1c, http://
wbiomed.curtin.edu.au/genepop/) of G E N E P O P (Raymond & Rousset, 1995). Genetic diversity within each
sample was measured by the allelic richness (AR),
expected heterozygosity (He) and Wright’s (1951)
inbreeding coefficient (f) using F S T A T v.2.9.3 (Goudet,
1995); AR was standardised at 31 individuals to account
for missing genotypes at one locus-sample combination (LIST4-002, CLM). We used F S T A T to test the
significance of any differences in AR, He and f among
sites at Pembrokeshire, Gower and Anglesey (the latter
site was randomly divided into two) by a permutation
procedure (5000 permutations of samples among
groups). The significance of any deviation from expected Hardy–Weinberg equilibrium (HWE) conditions
was estimated by making 5000 permutations of alleles
among individuals within samples using F S T A T .
Population bottleneck. We examined each sample for
evidence of a population decline (Thompson et al.,
2003) using two approaches. First, we used B O T T L E N E C K v.1.2.02 software (Piry, Luikart & Cornuet,
1999) to estimate (assuming populations are in
mutation-drift equilibrium) the expected distribution
of heterozygosity from the observed number of alleles
under the infinite allele (IAM) and stepwise mutation
(SMM) models of mutation. A Wilcoxon signed-rank
test was used to test whether there was a significant
heterozygote excess (compared with that expected
given the sample’s allelic diversity) that is characteristic of a population bottleneck (Cornuet & Luikart,
1996; Luikart & Cornuet, 1998). Second, for each
sample we calculated the average (over all loci) ratio
(M) of allelic size range (number of microsatellite
repeats) against the total number of alleles; values of
M above 0.82 indicate a stable population (Garza &
Williamson, 2001).
Population structure. We tested for heterogeneity of
genotype frequencies among all pairs of populations
using the exact test employed by F S T A T v.2.9.3
(Goudet, 1995); HWE within samples was not
assumed and genotypes were permuted 5000 times
among samples. Genetic differentiation between all
pairs of samples was also determined by calculating
Weir & Cockerham’s (1984) estimator of Wright’s
(1951) FST (h in Weir & Cockerham’s terminology)
using A R L E Q U I N v.2.001 (Schneider, Roessli &
Excoffier, 2000). The significance of the estimates of
h from zero was assessed by making 1000 permutations of genotypes between populations. Heirarchical
analysis of molecular variance (A M O V A ; Schneider
et al., 2000) was used to partition the contribution to
genetic diversity arising from differences (i) between
regions (Pembrokeshire, Gower and Anglesey), (ii)
among sample sites within regions and (iii) among
individuals within sites. The significance of the
fixation indices was tested using 10 000 permutations.
Isolation by distance genetic structure (Wright,
1943) was examined for (i) all populations and (ii)
the Pembrokeshire samples separately by a regression
of pairwise estimates of genetic differentiation
[h/(1 ) h)] against the corresponding natural logarithm of the geographic distance (m) separating the
populations (Rousset, 1997). For simplicity we calculated the Euclidian distance among pairs of samples.
Some sites in Pembrokeshire are separated by high
ground that may limit dispersal more than expected
from their linear separation (Fig. 1); therefore, we also
measured the shortest routes that were restricted to
terrain <300 m high (i.e. that circumvented the central
ridge) between pairs of Pembrokeshire sites. A Mantel
test (1000 permutations of population locations
among all locations) was used to assess the significance of any correlation between pairwise genetic
differentiation and geographic distance using the
online version (3.1c) of G E N E P O P (Raymond &
Rousset, 1995). Mantel tests were applied to all pairs
of samples as well as the colonies from Pembrokeshire
only; for the latter analysis we considered both
Euclidian distance among samples as well as the
routes that avoid high ground.
Multiple testing. Where appropriate a sequential Bonferroni correction (Rice, 1989) was applied to adjust
the significance of (k) multiple tests.
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
Genetic variation in an endangered damselfly 197
Results
(a)
5.0
Genetic differentiation between samples
Sites at Anglesey and Gower had significantly different (P < 0.05, k ¼ 45) genotype frequencies from all
2.0
(b)
Expected heterozygosity (He)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(c)
0.4
0.3
0.2
0.1
0.0
–0.1
Rhossili
Anglesey
WAF
Cefn Bryn
BRY
WAM
DOI
CLM
COT
WAN
–0.2
GOF
Only C. mercuriale from Gower and Pembrokeshire
(five samples) demonstrated a significant (P < 0.05)
heterozygote excess from that expected under an IAM
of mutation [but none did under the SMM – see Luikart
& Cornuet (1998) for discussion on mutational models]. This heterozygote excess remained significant
(P < 0.05, k ¼ 12) after a sequential Bonferroni correction (Rice, 1989) for CEB and RHO only. The ratio M
was 0.72 or less for all samples, with the lowest value
(M ¼ 0.61) at RHO (Table 1); M was 0.753 (variance
0.052) for the combined Pembrokeshire population.
3.0
PAN
Bottleneck analysis
4.0
1.0
Wright's (1951) inbreeding coefficient (f)
Because only two loci demonstrated significant linkage disequilibrium within any sample (LIST4-035LIST4-066 in GOF, P < 0.05, k ¼ 91) all loci were
retained for subsequent genetic analyses. Individual
sample-locus results for measures of genetic diversity
are presented in Appendix. Almost all samples met
expected HWE conditions with four and one samplelocus combination(s) having a significant (P < 0.05,
k ¼ 14) heterozygote deficit or excess respectively
(Appendix).
Raw values of allelic richness (AR), expected heterozygosity (He) and Wright’s (1951) inbreeding coefficient (f) are provided in Appendix. Particularly
notable is that five loci (36%) were monomorphic at
Anglesey, while only one locus (LIST4-037) was
invariable in two of the samples elsewhere (WAM &
WAF). Average levels of genetic diversity are generally similar among samples from Pembrokeshire
and Gower, but substantially lower in Anglesey
(Fig. 2a–c). Permutation analyses demonstrated that
AR, He or f did not significantly differ between groups
of samples from Pembrokeshire and Gower (P ¼
0.234, 0.954 and 0.108, respectively), although when
Anglesey was included there were significant differences in AR (P ¼ 0.001) and He (P ¼ 0.001) but not f
(P ¼ 0.396) among groups.
Allelic richness (AR)
Genetic diversity
Fig. 2 Variation (mean ± 95% CI) in (a) allelic richness (AR)
standardised for a sample size of 31 individuals (b) expected
heterozygosity (He) and (c) Wright’s (1951) inbreeding coefficient (f) among samples of C. mercuriale from the Pembrokeshire
(PAN-WAF), Gower and Anglesey. See Methods and Fig. 1 for
further description of site names and locations.
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198
P.C. Watts et al.
Table 1 Results of bottleneck analysis for samples of C. mercuriale from Pembrokeshire, Gower and Anglesey (see Methods
and Fig. 1 for full sample site names and their location)
Bottleneck
Region
Population IAM
Pembrokeshire PAN
GOF
COT
WAN
DOI
CLM
BRY
WAM
WAF
Gower
Cefn Bryn
Rhossili
Anglesey
0.2508
0.0209
0.0338
0.0595
0.0863
0.0101
0.0209
0.0090
0.1082
0.0003*
0.0017*
0.0645
M
SMM
Mean
Variance
0.9406
0.8794
0.4758
0.8917
0.9031
0.5961
0.7869
0.2367
0.4197
0.1955
0.3129
0.1250
0.6549
0.6760
0.6694
0.6542
0.6404
0.6695
0.6328
0.6182
0.6378
0.7245
0.6120
0.7000
0.0733
0.0630
0.0758
0.0653
0.0545
0.0578
0.0564
0.0837
0.0733
0.0820
0.0862
0.0620
Bottleneck: *indicates a significant (P < 0.05, k ¼ 12) heterozygote excess for microsatellite loci under infinite allele (IAM) and
stepwise (SMM) models of mutation.
M: ratio of allelic size range (number of microsatellite repeats)
against the total number of alleles.
other samples (Table 1). Among populations from
Pembrokeshire, there was significant (P < 0.05, k ¼
66) heterogeneity in genotype frequency between
samples separated by the central hill ridge (except
between GOF and CLM) and also among all four
northern sites. The southern Pembrokeshire samples
were mostly similar, with significant (P < 0.05, k ¼
66) genotype differences between PAN and GOF, and
PAN and WAN only (Table 1).
All pairwise estimates of genetic differentiation (h)
between either Anglesey and the Gower sites and all
other samples were significantly (P < 0.05, k ¼ 66)
different from zero, with comparisons involving
Anglesey notably high (h ¼ 0.35–0.44). Genetic differentiation among the southern Pembrokeshire samples
was either low (h ¼ 0.016 and 0.018, PAN-COT and
PAN-WAN, respectively, P < 0.05, k ¼ 66) or nonsignificant (P > 0.05, k ¼ 66). With the exception of
two comparisons involving CLM, all northern Pembrokeshire samples were significantly dissimilar to all
other samples from Pembrokeshire irrespective of
their geographic location. Estimates of h between
WAM and other samples from Pembrokeshire are
relatively large compared with h between other pairs
of samples from this area. Likewise, h between the
Gower colonies is also greater than that generally
observed between similarly spaced sites in Pembrokeshire (Table 2). Hierarchical A M O V A revealed significant (P > 0.05) genetic structure at all levels but with
almost all variance (>97%) attributed to differences
within samples and among regions (Table 3).
There is a significant pattern of genetic isolation by
distance at a broad scale between all of the samples
(y ¼ 0.120x ) 0.974, P ¼ 0.0002, r2 ¼ 0.63) and also
locally among the Pembrokeshire samples (Fig. 3). For
the latter samples, although there is a positive and
significant regression between genetic differentiation
[h/(1 ) h)] and Euclidian distance (ln m) between
samples (y ¼ 0.013x ) 0.077, P ¼ 0.016, r2 ¼ 0.24) the
fit of this relationship (y ¼ 0.014x ) 0.087, P ¼ 0.003,
Table 2 Genetic differentiation among samples of C. mercuriale from Pembrokeshire, Gower and Anglesey; see Methods and Fig. 1 for
full sample site names and their location. Exact probability of genotypic differentiation between samples (below diagonal) and
pairwise estimates of genetic differentiation (h) (above diagonal).
Pembrokeshire
PAN
PAN
GOF
COT
WAN
DOI
CLM
BRY
WAM
WAF
Cefn Bryn
Rhossili
Anglesey
0.00076*
0.00379
0.00076*
0.00152
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
Gower
GOF
COT
WAN
DOI
CLM
BRY
WAM
WAF
Cefn Bryn Rhossili Anglesey
0.0112
0.0158*
0.0031
0.0178*
0.0022
0.0076
0.0144
0.0070
0.0096
0.0059
0.0302*
0.0146
0.0140
0.0165*
0.0155*
0.0410*
0.0229*
0.0293*
0.0250*
0.0191*
0.0180*
0.0764*
0.0678*
0.0657*
0.0653*
0.0456*
0.0604*
0.0336*
0.0349*
0.0212*
0.0282*
0.0339*
0.0224*
0.0105
0.0187*
0.0558*
0.1521*
0.1137*
0.1511*
0.1383*
0.1270*
0.1523*
0.1489*
0.1834*
0.1383*
0.18485
0.02121
0.01136
0.00152
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.02348
0.02500
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.02803
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076*
0.00076* 0.00076*
0.00076* 0.00076* 0.00076*
0.00076* 0.00076* 0.00076*
0.1531*
0.1293*
0.1564*
0.1514*
0.1462*
0.1662*
0.1491*
0.1913*
0.1521*
0.0844*
0.4014*
0.4055*
0.4149*
0.4262*
0.4037*
0.4244*
0.4076*
0.3482*
0.4123*
0.4844*
0.4425*
0.00076*
*Indicates a significant (P < 0.05, k ¼ 45) genetic difference between populations.
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
Genetic variation in an endangered damselfly 199
(Couvet, 2002; Frankham et al., 2002). In this study the
large Pembrokeshire populations are the most genetically diverse, although somewhat surprisingly the
smaller, separate Gower colonies have resisted substantial genetic erosion. The contrast in levels of
diversity at these sites with that observed at Anglesey
(Fig. 2a,b) may be attributed to one or a combination
of several causes.
Table 3 Hierarchical analysis of molecular variance (A M O V A )
of microsatellite loci among samples of C. mercuriale from three
regions in Wales, U.K.: Pembrokeshire, Gower and Anglesey
Source of
variation
Fixation
index
P-value
Percentage
variation
Among regions
Among samples
within regions
Within samples
0.2343
0.0312
0.0012
0.0000
20.96
2.47
0.2097
0.0000
76.57
P ¼ probability of a more extreme variance component than that
observed.
Impact of habitat isolation and population decline
Anglesey, some 150 km north from Pembrokeshire
(Fig. 1) and probably a remnant from when C.
mercuriale extended along the lowland coastal areas
of Wales, meets several (not mutually exclusive)
criteria associated with genetically depauperate insect
populations: isolated (Keller & Largiadèr, 2003; Williams, Brawn & Paige, 2003; Krauss et al., 2004), small
size (Schmitt & Seitz, 2002; Harper, MacLean &
Goulson, 2003; Joyce & Pullin, 2003) and/or at the
edge of a species’ range (Schmitt & Hewitt, 2004). The
contrast in diversity at the isolated sites of Anglesey
and Gower underlines the point that genetic impoverishment is not simply an unavoidable consequence
of habitat fragmentation but rather reflects the effect
of population size upon the rate of genetic drift (see
Frankham, 1996; Frankham et al., 2002). For example,
using the relationship:
r2 ¼ 0.36) is improved if the distance measured
between sites is restricted to a route below 300 m
altitude.
Discussion
Molecular genetic studies have provided detailed
insights into factors affecting the spatial genetic
structure of aquatic insects (e.g. Geenen et al., 2000;
Smith & Collier, 2001; Kelly et al., 2002; Miller et al.,
2002; Hughes et al., 2003a,b; Wilcock et al., 2003;
Wishart & Hughes, 2003; Watts et al., 2004a). However, most studies focus on dispersal rather than
processes affecting levels of genetic variability, despite an increasing recognition that conservation
strategies should prevent or minimise genetic erosion
and if possible restore background levels of variability
Ht ¼ Ho ð1 # 1=2Ne Þt
1.0
Fig. 3 Relationship between geographic
distance (ln m) separating samples of C.
mercuriale from Pembrokeshire, Gower
and Anglesey and the corresponding
estimate of pairwise genetic differentiation as defined by [h/(1 ) h)]. Expanded
plot of the Pembrokeshire samples in the
dashed frame highlights the isolation by
distance relationship at this site.
Genetic differentiation [q/(1–q)]
Pembrokeshire samples only
0.8
0.6
0.4
0.2
0.0
6
7
8
9
10
11
Geographic distance (m)
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
12
13
200
P.C. Watts et al.
where Ht ¼ expected heterozygosity after t generations, Ho ¼ initial expected heterozygosity and Ne ¼
effective population size (Crow & Kimura, 1970), the
present level of expected heterozygosity at Anglesey
(from an initial Ht ¼ 0.5) is equivalent to Ne ¼ 10 or
1000 for about 20 or 1900 generations, respectively. It
may seem remarkable that there is no evidence for a
bottleneck at Anglesey but this is consistent with the
poor statistical power of (i) the ratio test when the
starting population size is small and (ii) both tests
with increasing time following the bottleneck (Luikart
& Cornuet, 1998; Garza & Williamson, 2001). A useful
feature of the ratio test, however, is that allelic
diversity remains low in permanently reduced populations, although the value of M partially recovers
(Garza & Williamson, 2001). The Anglesey colony
evidently suffered a severe reduction in size. While
the colony at Anglesey may have been founded by
one/few gravid females (see e.g. Joyce & Pullin, 2003
for genetic diversity in an introduced, isolated
population of Euphydryas aurinia Rott.) this is unlikely
given this species’ poor dispersal capability (Hunger
& Röske, 2001; Purse et al., 2003; Watts et al., 2004a).
Adaptation to an extreme, edge of range environment
may further contribute to the low genetic diversity at
Anglesey, but the consistency across loci suggests that
genome-wide drift is the principal cause.
Also in contrast to expectations were the similar
levels of genetic diversity at Pembrokeshire and
Gower. Similarly, Monaghan et al. (2002) found no
effect of fragmentation on the genetic diversity of
stream insects because population sizes were large
enough to minimise the rate of genetic drift and/or
there is gene flow among populations. The Gower sites,
50 km south of the Pembrokeshire colonies, are likely to
have been isolated for a shorter duration than Anglesey
and may have experienced more recent immigration
(contemporary gene flow to the Gower is unlikely, see
below) and/or historically maintained larger effective
population sizes. The genetic signature of recent
population decline corresponds with qualitative observations of a reduction in distribution of C. mercuriale at
Gower (D.J. Thompson, personal observations) that
will accelerate a reduction in gene diversity.
Within Pembrokeshire, localised differences in genetic variability are indicative of fine-scale variation in
demography that is typical of many insect populations
(e.g. Schmitt & Seitz, 2002; Wynne et al., 2003). Of note
is the likely recent decline in size at WAM (cf. Table 1)
and concurrent reduction in AR (it is possible that low
diversity reflects a selective response to altitude); any
loss of variability at WAM will be sustained by
restricted immigration (see below). By contrast, the
level of variability at WAF further highlights the point
that the rate of genetic drift in large populations is
generally low, such that substantial levels of diversity
may be retained even when a population is isolated
(see also Keller, Nentwig & Largiadèr, 2004). Elsewhere in Pembrokeshire, connectivity between the
southern, eastern and northern populations may
buffer individual colonies against genetic erosion
associated with demographic fluctuations.
Population genetic structure and movement among sites
The substantial level of genetic differentiation that may
be attributed to differences among regions (Table 3;
Fig. 3) undoubtedly corresponds to breaks in distribution that far outreach C. mercuriale’s dispersal capability
(Hunger & Röske, 2001; Purse et al., 2003; Watts et al.,
2004a). The pattern of genetic differentiation among
populations with distance, however, provides more
insight into processes affecting population structure
(Miller et al., 2002). Weak and/or variable regional
genetic differences and absence of isolation by distance
that have been reported for some aquatic insects are
indicative of, for example, wide dispersal, genetic
disequilibrium, balancing selection or poor marker
resolution (e.g. Smith & Collier, 2001; Miller et al., 2002;
Monaghan et al., 2002; Hughes et al., 2003b). In contrast,
an apparent pattern of isolation by distance (Fig. 3) is a
feature of restricted dispersal and relatively small
populations that facilitates rapid population-differentiation through genetic drift (see also Miller et al., 2002;
Wilcock et al., 2003).
At a local scale, genetic differentiation between
the Gower sites is greater than that observed
between similarly spaced sites in continuously
distributed C. mercuriale populations (Table 2; Watts
et al., 2004a) indicating that dispersal is disrupted by
agricultural improvement. Indeed, restricted gene
flow has been associated with anthropogenic changes to habitat for several terrestrial (Keller &
Largiadèr, 2003; Williams et al., 2003; Keller et al.,
2004) and aquatic insects (Smith & Collier, 2001;
Watts et al., 2004a). Similarly, the improved fit of the
regression based on movement among sites within
Pembrokeshire that avoids high ground corresponds
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
Genetic variation in an endangered damselfly 201
with observations that, like many freshwater insects
(e.g. Petersen et al., 2004), adult C. mercuriale move
exclusively between areas of suitable habitat (Purse
et al., 2003); the hill ridge is a natural barrier to
dispersal (see also Wishart & Hughes, 2003). Low or
non-significant genetic differences among adjacent
areas in Pembrokeshire (Table 2) indicate dispersal
by C. mercuriale over 1–2 km, although this does not
prevent genetic isolation by distance (see Fig. 3,
insert). Increased genetic differentiation at WAM
(Table 2) is a likely consequence of isolation by high
ground, but directional selection or drift during the
recent demographic reduction may also accentuate
genetic differences between this site and elsewhere
(see Hedrick, Gutierrez-Espeleta & Lee, 2001).
Variable but often low genetic differences between
the central sites and WAF do not indicate connectivity, but reflect a historical genetic signature
with the large WAF population not yet achieving
migration-drift equilibrium since its isolation.
Neither Geenen et al. (2000) or Wong et al. (2003)
identified fine-scale genetic structure in other species of zygopteran but the pattern and magnitude of
local genetic differences in Pembrokeshire parallels
that observed for this species in the Itchen Valley
(Watts et al., 2004a).
Implications for conservation
Population size is an important correlate of extinction
risk (O’Grady et al., 2004) and, accordingly, the
current U.K. strategy for C. mercuriale conservation is
based upon habitat protection to maintain large
populations. However, even apparently healthy populations can be at long-term risk if they lack genetic
diversity (Saccheri et al., 1998; Frankham et al., 2002;
Keller & Waller, 2002; Reed & Frankham, 2003;
Schmitt & Hewitt, 2004; Spielman et al., 2004a,b).
Improving connectivity among fragmented populations will help maintain/restore genetic diversity and
preserve evolutionary patterns and processes (Moritz,
1999): such a strategy is not possible between regions
but is generally appropriate within each area, especially the Gower sites. Somewhat ironically, the historical lack of management (agricultural improvement)
imposed by its common land status has helped limit
genetic erosion in the Pembrokeshire, although a
recent decline in sheep grazing has unfortunately
reduced habitat quality. It is relevant, therefore, that
M < 0.68, characteristic of each Pembrokeshire sample, is symptomatic of a recent reduction in population size (Garza & Williamson, 2001); even when
genotype data for all Pembrokeshire sites are combined to constitute a single population the calculated
value of M ¼ 0.75 is still below that observed in
demographically stable populations (M ¼ 0.82, Garza
& Williamson, 2001). Data on levels of genetic
variability in C. mercuriale populations towards the
centre of its range would provide further context to
the variation observed in Pembrokeshire and Gower.
Some combination of habitat isolation, small population size and/or directional selection has lead to
localised reduction in genetic variability at WAM and
a striking level of genetic impoverishment at Anglesey
that is more typical of threatened mammal (e.g.
Hedrick et al., 2001; Whitehouse & Harley, 2001; Jones
et al., 2004) than fragmented insect (e.g. Keller &
Largiadèr, 2003; Williams et al., 2003; Keller et al.,
2004) populations. There is particular concern for
C. mercuriale at Anglesey which may be (i) locally
adapted and/or (ii) likely to suffer as a result of no
immigration (see Couvet, 2002; Keller & Waller, 2002),
for example, from inbreeding depression (Saccheri
et al., 1998) or increased susceptibility to disease
(Spielman et al., 2004b). The former implies that there
should be focus on habitat management while the latter
suggests that the population may benefit from augmentation (e.g. Madsen et al., 1999). Because we have
not yet addressed either scenario it would be unwise to
suggest augmentation (see Moritz, 1999; Stockwell,
Hendry & Kinnison, 2003). It is interesting to note that,
if the decision was taken for management plans to
prioritise the conservation of genetic diversity per se, for
example to allow future evolutionary processes, rather
than current adaptive novelties (see Moritz, 1999), then
any effort directed towards managing the Anglesey site
should perhaps be diminished and efforts focused on
the larger populations in Pembrokeshire and
Hampshire.
To conclude, populations of C. mercuriale exhibit
local and regional variation in levels of genetic
diversity that is associated with their population
demographics. Rather than directly eliciting a loss of
variation, habitat fragmentation reinforces genetic
impoverishment in small populations by preventing
augmentation via gene flow. Consistent with the
pattern of spatial genetic structure from elsewhere in
the U.K., dispersal by mature C. mercuriale in Pem-
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
202
P.C. Watts et al.
brokeshire is sufficient to prevent significant genetic
differentiation throughout a habitat matrix of about
3–4 km if the sites are <2 km apart and lack
intervening barriers. Nevertheless, dispersal by
C. mercuriale is sufficiently restricted so that isolation
by distance genetic structure develops within 10 km.
Acknowledgments
Coenagrion mercuriale is a protected species under
Schedule 5 of the Wildlife and Countryside Act (1981).
All work was carried out under licence from the
Countryside Council for Wales. We are grateful to the
NERC (grant no. NER/A/S/2000/01322) for provision of funds and the landowners for allowing us
access to their land. Stephen Coker and Adrian
Fowles provided helpful comments on the manuscript. Jac Pearson, Sarah Perry, Carri Westgarth and
Kerry Woodbine were excellent field assistants.
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Appendix Genetic diversity at 14 microsatellite loci for samples of C. mercuriale from the Pembrokeshire (PAN–WAF), Gower and
Anglesey (see Methods and Fig. 1 for full sample site names and their location
Pembrokeshire
Gower
PAN
GOF
COT
WAN
DOI
CLM
BRY
WAM
WAF
Cefn Bryn Rhossili Anglesey
(n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 47) (n ¼ 48) (n ¼ 48) (n ¼ 48)
(n ¼ 48) (n ¼ 52)
4–002
AR
2.000
He
0.479
f
)0.001
4–023
AR
4.272
He
0.555
f
0.078
2.000
0.491
)0.018
2.000
0.443
0.295
2.000
0.505
0.052
2.000
0.506
0.126
2.000
0.508
0.224
2.000
0.437
0.054
2.000
0.389
)0.017
2.000
0.502
0.103
2.000
0.506
0.012
2.000
0.505
)0.053
1.000
0.000
–
3.494
0.573
0.128
2.997
0.584
)0.263
2.992
0.573
0.009
3.955
0.615
0.081
3.649
0.573
)0.113
3.401
0.546
0.061
2.438
0.514
)0.136
2.880
0.540
0.074
3.437
0.564
)0.035
3.000
0.627
)0.097
1.656
0.039
1.000
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205
Genetic variation in an endangered damselfly 205
Appendix (Continued)
Pembrokeshire
Gower
PAN
GOF
COT
WAN
DOI
CLM
BRY
WAM
WAF
Cefn Bryn Rhossili Anglesey
(n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 48) (n ¼ 47) (n ¼ 48) (n ¼ 48) (n ¼ 48)
(n ¼ 48) (n ¼ 52)
4–024
AR
He
f
4–030
AR
He
f
4–031
AR
He
f
4–034
AR
He
f
4–035
AR
He
f
4–037
AR
He
f
4–042
AR
He
f
4–060
AR
He
f
4–062
AR
He
f
4–063
AR
He
f
4–066
AR
He
f
4–067
AR
He
f
3.989
0.287
0.149
2.447
0.419
)0.271
2.994
0.341
0.083
2.972
0.283
)0.127
2.994
0.393
)0.091
2.987
0.455
)0.098
2.730
0.434
0.057
2.992
0.368
)0.076
2.000
0.446
)0.259
2.553
0.423
)0.245
3.000
0.648
0.049
1.000
0.000
–
2.000
0.501
)0.462
2.000
0.504
)0.112
2.871
0.527
)0.103
2.000
0.490
)0.065
2.000
0.499
)0.570†
2.000
0.391
0.011
2.000
0.494
)0.235
2.000
0.498
)0.266
2.000
0.463
)0.338
2.000
0.368
0.039
2.000
0.426
)0.027
1.000
0.000
–
3.753
0.643
)0.185
3.500
0.661
0.244
3.871
0.684
0.048
3.447
0.672
0.304*
3.707
0.680
)0.215
3.707
0.684
0.014
3.512
0.646
0.169
3.000
0.646
)0.135
3.512
0.630
0.187
3.437
0.680
0.142
3.000
0.618
0.090
1.000
0.000
–
1.993
0.157
)0.082
2.000
0.241
0.029
2.000
0.307
)0.221
1.999
0.221
0.058
2.000
0.216
)0.125
2.000
0.241
0.029
1.994
0.160
)0.084
2.000
0.369
0.153
2.000
0.327
)0.073
2.000
0.234
0.118
2.000
0.271
)0.020
1.420
0.020
0.000
7.241
0.735
)0.162
8.611
0.798
0.046
9.138
0.809
)0.158
7.668
0.765
)0.023
8.306
0.771
)0.091
7.108
0.779
)0.113
8.503
0.781
)0.086
6.642
0.561
)0.077
8.523
0.772
0.059
6.482
0.768
)0.069
4.385
0.398
0.092
2.000
0.499
0.121
2.826
0.304
)0.029
2.864
0.179
)0.067
2.693
0.213
)0.100
2.853
0.177
0.292
1.871
0.068
)0.024
2.000
0.276
0.135
1.457
0.022
0.000
1.000
0.000
–
1.000
0.000
–
2.000
0.451
0.211
3.000
0.565
0.097
2.000
0.389
)0.125
2.000
0.367
)0.306
2.000
0.410
)0.061
2.000
0.345
)0.026
2.000
0.438
)0.165
2.477
0.361
0.118
1.999
0.205
)0.119
2.000
0.369
0.174
1.992
0.155
0.192
1.996
0.167
)0.089
2.000
0.401
)0.246
2.000
0.507
0.343
2.000
0.465
)0.119
2.877
0.237
)0.105
2.500
0.331
)0.006
3.357
0.271
0.141
2.835
0.323
)0.054
2.456
0.279
)0.012
2.000
0.261
0.000
2.953
0.355
0.015
2.985
0.461
0.186
2.500
0.420
0.036
2.000
0.479
0.701*
2.477
0.454
)0.051
2.000
0.470
0.044
2.992
0.499
)0.085
2.844
0.446
)0.024
2.985
0.518
)0.086
2.997
0.482
)0.014
2.961
0.552
)0.006
2.948
0.530
0.096
3.000
0.614
0.292
3.000
0.670
)0.039
2.753
0.482
0.210
2.686
0.438
0.001
2.000
0.439
0.032
2.000
0.498
)0.045
3.853
0.546
)0.059
3.731
0.463
0.009
2.972
0.499
0.040
3.648
0.333
)0.023
3.951
0.466
)0.001
3.590
0.488
0.042
3.688
0.537
)0.133
2.438
0.515
)0.092
2.999
0.580
0.224
1.888
0.071
)0.026
2.447
0.303
)0.053
1.447
0.021
0.000
5.240
0.421
)0.187
6.598
0.551
0.050
6.898
0.559
0.002
6.086
0.501
0.045
6.160
0.421
)0.158
6.538
0.357
0.004
6.776
0.613
)0.074
3.993
0.610
)0.069
4.678
0.254
)0.082
2.000
0.320
)0.105
4.757
0.535
0.204
2.000
0.278
0.559*
7.243
0.617
0.020
6.961
0.738
0.019
8.951
0.769
)0.098
7.551
0.727
)0.107
8.009
0.721
0.029
6.084
0.719
)0.061
6.729
0.691
)0.184
4.590
0.592
)0.222
9.154
0.665
)0.165
9.026
0.810
0.021
7.186
0.692
0.278*
1.000
0.000
–
Sample size (n), allelic richness (AR), expected heterozygosity (He) and Wright’s (1951) inbreeding coefficient (f ).
* and † indicate a significant (P < 0.05, k ¼ 9–14) heterozygote deficit or excess, respectively – indicates monomorphic locus, so test not
carried out.
! 2005 The Authors, Journal compilation ! 2005 Blackwell Publishing Ltd, Freshwater Biology, 51, 193–205