Ecological Entomology (2005) 30, 541–547
Conservation implications of genetic variation
between spatially and temporally distinct colonies of
the endangered damselfly Coenagrion mercuriale
P H I L L I P C . W A T T S , S T E P H E N J . K E M P , I L I K J . S A C C H E R I and D A V I D
J . T H O M P S O N The Biosciences Building, School of Biological Sciences, Liverpool University, U.K.
Abstract. 1. Good conservation management is underpinned by a thorough
understanding of species’ historical and contemporary dispersal capabilities
along with the possible adaptive or neutral processes behind any spatio-temporal
genetic structuring. These issues are investigated with respect to the rare damselfly
Coenagrion mercuriale (Charpentier) – the only odonate species currently listed in
the U.K.’s Biodiversity Action Plan – in east Devon where its distribution has
become fragmented.
2. The two east Devon C. mercuriale populations, only 3.5 km apart, have
accumulated strong differences in frequencies of alleles at 14 microsatellite loci
as a consequence of poor adult dispersal and drift. There is no contemporary
migration between sites.
3. A genetic signature of population decline at both sites corresponds with
known demographic reductions. Coenagrion mercuriale in east Devon are now
significantly genetically less diverse than those from a population stronghold in
the Itchen Valley.
4. Colonies would benefit from improved connectivity between areas and
possibly by a transfer of individuals from other ecologically similar areas.
5. Because C. mercuriale has a semivoltine life cycle throughout the U.K., the
possibility that alternate-year cohorts are reproductively isolated is explored.
Genetic differentiation among cohorts is an order of magnitude less than between
sites, suggesting that some larvae delay their development into adults for a year
and recruit to a different cohort.
6. To our knowledge, this is the first study to document migration and gene
flow between alternate-year cohorts in a species of odonate. From a conservation
standpoint, the cohorts do not require separate management.
Key words. Bottleneck, conservation, gene flow, genetic diversity, microsatellite, Odonata, voltinism.
Introduction
Defining intraspecific management units can be a problematic and controversial issue in conservation biology
Correspondence: Phill Watts, Animal Genomics Laboratory,
The Biosciences Building, School of Biological Sciences,
Liverpool University, Crown Street, Liverpool L69 7ZB, U.K.
E-mail: [email protected]
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2005 The Royal Entomological Society
(Frankham et al., 2002). The frequently employed concept
of an evolutionary significant unit (ESU; Moritz, 1994), for
example, has been criticised on several grounds (e.g. see
Crandall et al., 2000), but principally because the recent
adherence to delineating ESUs on the basis of the spatial
distribution of neutral genetic markers can fail to recognise
important population-specific adaptations. Rather than
separately managing divergent units or promoting gene
flow among all populations, Crandall et al. (2000) argued
for a more holistic approach to defining population distinctiveness that should accommodate the concepts of
541
542 Phillip C. Watts et al.
ecological and genetic exchangeability. Where possible,
maintaining natural levels of connectivity throughout a
habitat network is encouraged because this preserves evolutionary patterns and processes rather than distinct intraspecific phenotypes (see Moritz, 1999). Good conservation
practice therefore is underpinned by a thorough understanding of species’ historical and contemporary dispersal
capabilities along with the possible adaptive or neutral
processes behind any spatio-temporal genetic structure.
These issues are addressed in respect of the damselfly
Coenagrion mercuriale (Charpentier) – the only odonate
species currently listed in the U.K.’s Biodiversity Action
Plan – in a part of its range in which its distribution has
become increasingly fragmented. Coenagrion mercuriale has
been declining in its U.K. distribution for 50 years
(Thompson et al., 2003) and is protected under the
Wildlife & Countryside Act (1981) within the U.K. It is
now limited to strongholds in the New Forest, Itchen
Valley (Hampshire, southern England) and on the Preseli
Hills (Pembrokeshire, Wales) and small populations at sites
in Anglesey, Gower, Oxfordshire, Dorset and Devon
(Purse, 2001). Because C. mercuriale adults typically do
not move more than 100 m (Hunger & Röske, 2001;
Purse et al., 2003; Watts et al., 2004a), the majority of
colonies are considered to be vulnerable because they are
unlikely to regularly receive immigrants from even apparently local sites. There is specific concern for C. mercuriale
on the East Devon Pebblebeds because the last sightings of
this species at Southey Moor (ST 192110), Luppitt (ST
164065), Hense Moor (ST 175080), and Venn Ottery
Common (SY 063922) were in 1959, 1963, 1965, and 1990
respectively. This paper characterises the genetic structure
of the two remaining C. mercuriale colonies known to exist
in east Devon, at Aylesbeare (SY 054906) and Colaton
Raleigh (SY 050868) Commons. Coenagrion mercuriale
has been recorded in low numbers from Aylesbeare since
1956 but this population has increased substantially following the implementation of an enlightened grazing regime in
1989 (Kerry, 2001). Population sizes at Colaton Raleigh
Common, some 3.5 km to the south of Aylesbeare, were
substantially higher than at Aylesbeare during the mid
1980s, but this population crashed during the late 1990s
and is presently being managed to aid population recovery;
it is not known whether there is any inter-site movement.
An interesting facet to the management of this species is
that it has a semivoltine life cycle through most of its
European range and certainly throughout the U.K. (Purse
& Thompson, 2002). Kerry (2001) reported larger numbers
of C. mercuriale in the odd year class (i.e. 1993, 1995, 1999,
2001) at Aylesbeare. That population demography varies
between cohorts implies that they may be reproductively
isolated; for example, Coates et al. (2004) reported genetic
differences between sympatric uni- and bivoltine ecotypes
of the corn borer Ostrinia nubilalis in North America. On
the other hand, odonate larval developmental period is
probably a plastic trait; larvae of C. mercuriale can accelerate their developmental time to 1 year (Thielen, 1992)
while those of other congeners switch between semi- and
#
univoltinism depending upon the environment (Corbet,
1999). This raises the possibility of ‘leakage’ between
C. mercuriale cohorts brought about by some individuals
taking 1 or 3 years to complete their life cycle. Understanding
the dynamics of possible links between cohorts is clearly important for conservation as this affects their management as either a
single entity or two distinct units.
The aims of this study were (1) to relate contemporary
levels of genetic diversity and gene flow within and among
the remaining colonies of C. mercuriale in east Devon with
their recorded demographic histories, and (2) to assess the
extent of any genetic differentiation between alternate year
cohorts at Aylesbeare Common. These results are compared with published data for this species from a large,
continuously distributed population in the Itchen Valley,
Hampshire, U.K. to provide context to contemporary
levels of gene diversity and genetic exchange in the remaining east Devon populations.
Methods
Site description
Coenagrion mercuriale occurs in small areas of southern
heathland on Aylesbeare and Colaton Raleigh Commons.
The breeding habitat consists of shallow, narrow, slowflowing, spring-fed flushes that run over small pebbles
overlaid with peat. There are approximately 100 m of suitable flushes at each site. Details of the five C. mercuriale
sites within the Lower Itchen Complex in the Itchen Valley
are provided by Watts et al. (2004a).
DNA extraction and PCR
We obtained DNA non-lethally by removing a hind leg
(stored in individual 1.5 ml tubes containing 100% ethanol)
from 20 to 48 animals from Aylesbeare Common and
Colaton Raleigh Common in the summer of 2002, while a
second sample from Aylesbeare Common was obtained in
2003 also. Removal of a single damselfly leg does not
measurably affect fitness (Fincke & Hadrys, 2001) and no
significant effect of sampling upon recapture rate was
observed elsewhere (D. J. Thompson, unpubl. data).
Genomic DNA was extracted from legs using a high salt
protocol (Sunnucks & Hales, 1996). Allelic variation in 14
microsatellite loci were examined (LIST4-002, LIST4-023,
LIST4-024, LIST4-030, LIST4-031, LIST4-034, LIST4035, LIST4-037, LIST4-042, LIST4-060, LIST4-062,
LIST4-063, LIST4-066, LIST4-067) characterised by
Watts et al. (2004b, c). Approximately 5 ng of DNA was
used for PCR in a 10 ml final reaction volume 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 Taq
polymerase (ABgene, Epson, U.K.). Thermal cycling conditions are described by Watts et al. (2004b, c). PCR
2005 The Royal Entomological Society, Ecological Entomology, 30, 541–547
Spatio-temporal genetic variation in a rare odonate 543
products were pooled into one of two genotyping pools,
determined by allelic size range and the 50 fluorescent dye,
along with a GENESCAN-500 LIZ size standard (Applied
Biosystems, Warrington, U.K.) 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.
Statistical 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
GENEPOP (Raymond & Rousset, 1995). Genetic diversity
within each sample was measured by the allelic richness
(AR, based on 19 individuals) and gene diversity (He)
using FSTAT v.2.9.3 (Goudet, 1995). The significance of
any differences in AR and He among sites from Devon
with those from the Itchen Valley (see Watts et al., 2004a
for further details) was made by making 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. All permutation procedures were executed by FSTAT (Goudet, 1995).
Population bottleneck. Evidence for a bottleneck within
each sample was examined using BOTTLENECK v.1.2.02 software (Piry et al., 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 a sample had a significant heterozygote
excess (compared with that expected given the sample’s allelic diversity) that is characteristic of a decline in population
size (Cornuet & Luikart, 1996; Luikart & Cornuet, 1998).
Population structure. Heterogeneity of genotype frequencies among all pairs of populations was characterised using
the exact test employed by FSTAT v.2.9.3 (Goudet, 1995);
HWE within samples was assumed and alleles were permuted 5000 times among samples. Genetic differentiation
between pairs of samples was also determined by calculating Weir and Cockerham’s (1984) estimator of Wright’s
(1951) FST (y in Weir & Cockerham’s terminology) using
ARLEQUIN v.2.001 (Schneider et al., 2000). The significance
of the estimates of y from zero was assessed by making
1000 permutations of genotypes between populations.
Hierarchical analysis of molecular variance (AMOVA)
(Excoffier et al., 1992; Schneider et al., 2000) was used to
simultaneously partition the contribution to genetic diversity arising from differences (i) between Aylesbeare and
Colaton Raleigh, (ii) among cohorts within Aylesbeare,
and (iii) among individuals within sample sites. The significance of the fixation indices was tested using 10 100
permutations.
#
Results
As only two loci demonstrated significant linkage disequilibrium within any sample (LIST4-002 and LIST4-035 in
Aylesbeare 2002, P < 0.05, k ¼ 66), all genotype data
were retained for subsequent analyses. All but three sample-locus comparisons were in expected HWE conditions:
LIST4-031 and LIST4-060 had a heterozygote deficit in
Aylesbeare 2003 and Colaton Raleigh respectively, while
there was a significant excess of heterozygotes in
Aylesbeare 2003 at LIST4-042 (data not shown).
Individual sample-locus data for allelic richness (AR) and
expected heterozygosity (He) are presented in Table 1.
Genetic diversity was similar between the two separate
cohorts at Aylesbeare (He ¼ 0.26–0.28, AR ¼ 2.3) but
lower than in the colony at Colaton Raleigh (He ¼ 0.44,
AR ¼ 2.7); it is notable that two loci were monomorphic in
the former site while all loci were polymorphic at the latter
area (Table 1). Average He and AR was 0.49 and 3.5 respectively in the Itchen Valley. There was a significant difference
in genetic diversity between sites from Devon and the
Itchen Valley (P < 0.02 in both He and AR), with genetic
diversity lower in the former region (Fig. 1a,b, see also
Watts et al., 2004a).
Neither cohort from Aylesbeare had a significant excess
of heterozygotes (P > 0.05 for 2002 and 2003, and both
IAM and SMM). The sample from Colaton Raleigh
showed evidence for a bottleneck with significant heterozygote excess under the IAM (P < 0.01) but not the SMM
(P ¼ 0.13).
There were significant (P < 0.05, k ¼ 3) genetic differences between all samples, although these differences were
greater between sites than among cohorts (Table 2).
Genetic differentiation between Aylesbeare and Colaton
Raleigh (y ¼ 0.25) but not among the cohorts (y ¼ 0.015)
is more than an order of magnitude greater than that
observed between sites within the Lower Itchen Valley
Complex (average y ¼ 0.013; Watts et al., 2004a)
(Table 2). By partitioning genetic variation using a nested
AMOVA design, it is evident that almost all of the genetic
variation occurred among individuals within sites or
between Aylesbeare and Colaton Raleigh (73% and 25%
respectively); less than 1% of total genetic variation among
the sampled east Devon colonies could be attributed to
differences between the Aylesbeare cohorts (Table 3).
Discussion
This paper characterises levels of genetic diversity and
population differentiation within and among isolated colonies of the endangered damselfly C. mercuriale from east
Devon. The site-specific pattern of genetic variation corresponds with the timing of demographic reductions: thus a
recent reduction at Colaton Raleigh left a characteristic
heterozygosity excess, while the older and more extensive
population decline at Aylesbeare has allowed the cohorts to
approach their new mutation-drift equilibrium conditions
2005 The Royal Entomological Society, Ecological Entomology, 30, 541–547
544 Phillip C. Watts et al.
Table 1. Genetic diversity at 14 microsatellite loci for samples of Coenagrion mercuriale from east Devon (AYL, Aylesbeare; CRC, Colaton
Raleigh). Sample size (n), expected heterozygosity (He), and allelic richness based on 19 individuals (AR) are given.
Population
Locus
AYL 2002
(n ¼ 48)
AYL 2003
(n ¼ 48)
CRC 2002
(n ¼ 20)
All samples
(n ¼ 116)
4-002
He
AR
0.294
2.000
0.061
1.784
0.471
2.000
0.275
1.999
4-023
He
AR
0.279
2.413
0.385
2.396
0.466
2.000
0.377
3.234
4-024
He
AR
0.322
2.924
0.371
2.648
0.142
2.000
0.278
2.722
4-030
He
AR
0.154
1.986
0.221
1.999
0.482
2.000
0.286
1.999
4-031
He
AR
0.237
1.999
0.101
1.925
0.409
3.000
0.249
2.408
4-034
He
AR
0.504
2.000
0.474
2.000
0.329
2.000
0.436
2.000
4-035
He
AR
0.711
3.989
0.633
3.396
0.427
3.000
0.590
5.100
4-037
He
AR
0.000
1.000
0.000
1.000
0.358
2.000
0.119
1.806
4-042
He
AR
0.476
2.000
0.502
2.000
0.326
2.000
0.435
2.000
4-060
He
AR
0.213
1.998
0.269
2.395
0.500
2.000
0.327
2.168
4-062
He
AR
0.000
1.000
0.000
1.000
0.638
3.000
0.213
2.656
4-063
He
AR
0.237
1.999
0.102
1.930
0.553
3.000
0.297
2.517
4-066
He
AR
0.041
1.792
0.042
1.648
0.240
3.000
0.108
2.207
4-067
He
AR
0.428
5.174
0.446
6.208
0.799
6.899
0.558
8.236
Average
He
AR
0.278
2.305
0.258
2.309
0.439
2.707
0.325
2.932
(Luikart & Cornuet, 1998). Nevertheless, all colonies in east
Devon are genetically less diverse than the larger
C. mercuriale population in the Itchen Valley (Fig. 1a,b).
Genetic diversity is associated with population viability
(Saccheri et al., 1998; Madsen et al., 1999; Bijlsma et al.,
2000) and the evolutionary potential of a species to respond
to environmental change (Reed & Frankham, 2003; Schmitt
& Hewitt, 2004). However, there is some controversy about
the relative importance of genetic variation for the persistence
of species/populations because other factors are thought to
be more prevalent in driving extinctions (e.g. habitat loss,
pollution or invasive organisms). Certainly the present habitat restoration at Aylesbeare has led to population enlargement (Kerry, 2001); however, given Spielman et al.’s (2004)
#
suggestion that low genetic variability does impact upon a
species before it goes extinct, future management of
C. mercuriale in Devon should perhaps consider genetic augmentation. The number of individuals that should be transferred is unclear. For example, just a few individuals may
have a large impact on improving the viability of vertebrate
populations (Madsen et al., 1999). It may seem reasonable to
assume that a larger sample should be transferred to counteract stochastic effects in demographically unstable insect
populations; however, there are not enough data to make
general recommendations for insect populations at present.
In addition, before any managed transfer of individuals is
undertaken the genetic and ecological exchangeability among
populations needs to be considered (Crandall et al., 2000).
2005 The Royal Entomological Society, Ecological Entomology, 30, 541–547
Spatio-temporal genetic variation in a rare odonate 545
Table 2. Genetic differentiation among samples of Coenagrion
mercuriale from east Devon (AYL, Aylesbeare; CRC, Colaton
Raleigh). Exact probability of genotypic differentiation between
samples (above diagonal) and pairwise estimates of genetic differentiation (y) (below diagonal) are given.
(a)
0.6
AYL 2002
AYL 2003
CRC 2002
Gene diversity (He)
0.5
0.3
0.1
(b)
5.0
4.0
Allelic richness (AR)
AYL 2003
CRC 2002
–
0.0142*
0.2512*
0.0333*
–
0.2556*
0.01667*
0.01667*
–
*Significant (P < 0.05, k ¼ 3) genetic difference between populations.
0.4
0.2
3.0
2.0
East Devon
Itchen Valley (lower)
Itchen Valley (mid)
Itchen Valley (lower)
Allington Manor
West Horton
Colaton Raleigh
Common
Aylesbeare (2003)
Aylesbeare (2002)
1.0
Itchen Valley
Fig. 1. Variation (mean 95% CI) in (a) gene diversity (He) and
(b) allelic richness (AR) for colonies of Coenagrion mercuriale from
east Devon and the Itchen Valley, Hampshire, U.K.
#
AYL 2002
Strong genetic differentiation between Aylesbeare and
Colaton Raleigh (Table 2), much greater than that between
equivalently distant sites within the southern Itchen Valley
(Watts et al., 2004a), indicates that contemporary migration between sites is negligible. This is concordant with our
understanding of C. mercuriale’s dispersal ability as the
distance between Aylesbeare and Colaton Raleigh is almost
twice the maximum movement made by marked adult
C. mercuriale (Watts et al., 2004a) and the sites are presently separated by inhospitable habitat (farmland, roads).
It is likely that there was historical gene flow between
Colaton Raleigh and Aylesbeare because they mostly differ
in their allele frequencies rather than having different alleles
(data not shown). From a conservation standpoint we need
to understand whether localised variation at neutral genetic
markers reflects adaptive divergence. Certainly the genomic
architecture of insects can evolve rapidly in small and/or
fragmented habitats (Singer & Thomas, 1996; Van Dyck &
Matthysen, 1999; Kuussaari et al., 2000) and a reduced
tendency for dispersal has been observed in an isolated
C. mercuriale colony (Watts et al., 2004a). Coenagrion
mercuriale does have specific habitat requirements (Purse
et al., 2003) but neither the genetic basis of this species’
response to changing environmental conditions nor the
extent of relevant environmental differences among sites
are understood at present. The latter point is important
because local specialisation depends not only upon spatial
genetic structuring but also the strength of local selection
(Slatkin, 1985). Because the east Devon habitats are
broadly similar, both are basic flush systems dominated
by Schoenus nigricans (Kerry, 1994), they are probably
ecologically comparable; these sites are considered genetically exchangeable because the genetic differences are more
easily explained by genetic drift in small populations (see
also Hedrick, 1999; Hedrick et al., 2001) than directional
selection. Therefore, the recommendation here is to treat
the east Devon sites as a single network that would benefit
from improved connectivity to reduce individual site extinction risk (e.g. see Newman & Tallmon, 2001) and possibly
also by a transfer of individuals from other heathland areas
in the U.K., the most appropriate of which would be the
New Forest heaths.
Given the emerging pattern of localised genetic differentiation in this species (Watts et al., 2004a and Tables 2 and
2005 The Royal Entomological Society, Ecological Entomology, 30, 541–547
546 Phillip C. Watts et al.
Table 3. Hierarchical analysis of molecular variation (AMOVA) of microsatellite loci among samples of Coenagrion mercuriale from east
Devon. P, probability of a more extreme variance component than that observed.
Source of
variation
d.f.
SS
Variance
components
%
variation
Fixation
index
P
Among sites
Among cohorts
Within populations
1
1
229
50.187
4.292
459.892
0.710
0.024
2.008
25.89
0.87
73.24
0.268
0.012
0.259
0.000
0.003
0.000
3 in the present study), significant genetic differences would
be expected to accumulate rapidly between cohorts if they
were reproductively isolated. Similar to results obtained for
populations of the plectopterans Pteronarcys proteus and
Peltoperla tarteri (White, 1989; Schultheis et al., 2002) and
the lepidopteran Xestia tecta (Kankare et al. 2002), low
levels of genetic differences were present between alternate-year cohorts. There are two alternate hypotheses to
explain this pattern: (i) the cohorts are separate but have
had insufficient time to diverge since they split from a
common, ancestral lineage and (ii) the cohorts are coupled
by contemporary gene flow. The latter hypothesis is presently favoured for two reasons. First, allele frequencies
among the local east Devon populations have rapidly
drifted apart while genetic differences between cohorts
remained relatively small during the same period. Second,
developmental plasticity is a common feature of many
insect species (e.g. Nylin & Gotthard, 1998), including
voltinism in odonates (Thielen, 1992; Corbet, 1999). The
relatively cool temperatures experienced by C. mercuriale in
the U.K. (at the northern limit of its range) most likely lead
to larvae postponing their development to 3 years and
recruiting to a different cohort. Inter-cohort migration presumably prevents a difference in genetic diversity accumulating between cohorts of contrasting sizes (see Kerry,
2001). These results indicate that larval development is
not fixed even in natural habitats and to our knowledge
the presence of multiple developmental pathways within the
same population is a biological feature that has not been
considered for the management of this species or indeed
any odonate species.
To conclude, this study lends further support to the growing
body of evidence that C. mercuriale populations are liable to
accumulate localised genetic differences through poor dispersal
and drift in small populations. Severe population decline at
Aylesbeare and Colaton Raleigh has led to a loss of genetic
diversity suggesting that the colonies would benefit from
improved connectivity between sites. Although the populations
are recovering, a managed transfer of individuals from other
heathland areas may further improve growth rates. Leakage
between alternate-year cohorts within a site acts to increase
effective population size and buffer against genetic erosion.
Acknowledgements
Coenagrion mercuriale is protected under the 1981 Wildlife
and Countryside Act. This work was carried out under
#
licence from English Nature. We thank Clinton Devon
Estates and the RSPB for permission to work on Colaton
Raleigh and Aylesbeare Commons respectively. Pete
Gotham and Lesley Kerry provided wonderful hospitality
and shared their knowledge of the east Devon sites with us.
We are grateful to the NERC (grant no. NER/A/S/2000/
01322) for provision of funds.
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Accepted 7 April 2005
2005 The Royal Entomological Society, Ecological Entomology, 30, 541–547