TECHNICAL NOTE

Cross-species amplification and development of microsatellitesfor six species of European Coenagrionid damselflies

Helena Johansson • Par K. Ingvarsson •

Frank Johansson

Received: 9 August 2011 / Accepted: 27 August 2011 / Published online: 30 September 2011

� Springer Science+Business Media B.V. 2011

Abstract We describe the cross-amplification and

development of new loci for six species of closely

related European damselflies. First, twenty-nine published

microsatellites for the damselflies Coenagrion puella and

C. mercuriale were multiplexed using M13-tagged

primers, tested on 23 individuals, and then cross-species

amplified on 21–26 individuals of C. armatum, C. johans-

soni, C. pulchellum and C. scitulum. Second, sixteen new

primers were developed for use in C. armatum, C. johans-

soni and C. scitulum, and screened on 21 individuals.

Values for observed heterozygosities and number of alleles

ranged between 0.00–0.87 and 2–19 respectively (over all

loci and species). For all species the tested loci provide a

minimum of 1–8 usable markers for population genetic

studies.

Keywords Coenagrion � Damselfly � Microsatellite �Cross-species amplification

Several European Odonates have extended their ranges

polewards in response to climate change (Sahlen et al.

2006). To assess adaptation to these changes, large-scale

patterns of neutral genetic variation can be compared with

reaction norms in ecologically relevant traits of congeneric

species. Microsatellite markers were developed for Coe-

nagrion puella (Lowe et al. 2007) and C. mercuriale

(Watts et al. 2004a) from British populations (Table 1),

and the latter set cross-amplified on 3–8 individuals in

other Zygoptera (Watts et al. 2004b). To assess genetic

variation cost-effectively in six Coenagrionid species we:

(1) used 29 M13-tailed primers (Oetting et al. 1995) to

assess these loci in European populations of the original

species (C. puella and C. mercuriale) (2) multiplexed

amplifying loci, and (3) evaluated rates of amplification

and polymorphism in closely related species (C. pulchel-

lum, C. armatum, C. johanssoni and C. scitulum) sampled

from the centre of their respective distribution ranges. To

increase the number of loci we developed new microsat-

ellites for use in C. armatum, C. johanssoni and C. scitulum

using a DNA pooling protocol (Keever et al. 2008) and 454

sequencing technology.

For the cross-amplification tests forward primers were

modified with M13-tags (Table 1); of which three were

dye-specific and one universal (Oetting et al. 1995).

Reverse primers were modified with a 50 end PIG tail

(GTTT) to improve reliability of allele scoring (Brownstein

et al. 1996).

For the development of new primers DNA was extracted

from the thorax muscle of two individuals from each of

C. armatum, C. johanssoni and C. scitulum using Qiagen

DNeasy Blood and Tissue kit. DNA extractions were pooled

in equal amounts for a final concentration of 50 ng/ll.

Enrichment of microsatellite tetranucleotide motifs and 454-

sequencing were performed by Savannah River Ecology

H. Johansson (&)

Department of Biosciences, Helsinki University,

P.O. Box 65, 00014 Helsinki, Finland

e-mail: helena.z.johansson@helsinki.fi

P. K. Ingvarsson

Department of Ecology and Environmental Science,

Umea University, 901 87 Umea, Sweden

P. K. Ingvarsson

Umea Plant Science Centre, Umea University,

901 87 Umea, Sweden

F. Johansson

Department of Ecology and Genetics, Uppsala University,

Norbyvagen 18 D, 752 36 Uppsala, Sweden

123

Conservation Genet Resour (2012) 4:191–196

DOI 10.1007/s12686-011-9506-4

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CA

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CT

CT

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TA

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GG

GC

AC

GT

GC

GT

CT

AA

TA

CC

BV

695837

(AC

) 5…

(AT

) 7(A

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157–167

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T21-0

3a,d

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AA

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GT

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V695838

(CA

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154–226

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T21-0

4a

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GA

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TG

TA

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TG

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AG

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9(C

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4125–154

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3249–291

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M13

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T4-0

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CA

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AA

AT

BV

096720

(CA

) 8…

(CA

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(CA

) 2…

(CA

) 2…

(CA

) 9181–187

?19bs

LIS

T4-0

67

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CA

AG

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CG

AA

CT

GT

GC

CT

TT

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TG

CA

GC

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V079670

(GC

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103–135

M13

LIS

T4-0

71

bA

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CC

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V0

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7(C

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475–105

Bhg-r

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T4-0

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96

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286–292

M13

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V0

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5(C

AT

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AT

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T4023

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AA

TT

TG

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TA

GA

AA

CA

CT

GA

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TC

GT

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V0

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2(A

AC

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V0

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68

3(A

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?19bs

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bC

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GA

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CC

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CA

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Dev

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of

new

loci

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323–334

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C2_1708

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TT

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(AC

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Bhg-r

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GA

CC

CA

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TG

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(AC

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GG

GT

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GA

GG

AG

CG

TA

AT

GT

CJN

65

76

99

(AA

TG

) 5197–204

M13R

192 Conservation Genet Resour (2012) 4:191–196

123

Ta

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50

Modifi

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C_2146

AC

CG

GC

AG

GT

TC

TG

TA

TC

CT

TT

GC

CA

GG

AG

TA

TG

TC

GC

JN657700

(AC

TT

) 7215–246

Bhg-r

C3_976

TC

CA

AG

TC

GT

CG

AA

AT

CA

GC

CC

GT

TG

CA

CT

GT

TG

TG

AA

GJN

657701

(AC

TG

) 5233–254

Bhg-r

C2_1519

CA

AA

TA

AC

GC

AA

CG

CC

CA

AC

GC

AC

CT

GT

TC

TG

AT

GC

TC

AC

JN657702

(AG

) 11

260–268

Bhg-r

C3_1875

AA

TT

TC

AC

GG

TT

CT

AT

CA

CC

AG

GG

AA

AT

CA

AC

CG

GT

AA

CT

CG

AC

JN657703

(AC

TG

) 4293–305

M13F

C2_2304

CT

GC

TC

AC

CA

GC

CA

AT

CG

AC

GG

AT

AC

CT

TC

CA

CT

GC

JN657704

(AT

CC

) 7233–245

CA

G

C2_2355

TC

CG

CC

AT

GT

TC

AA

CC

TG

CC

TT

CC

GT

GG

TT

CC

AG

TT

TG

JN657705

(AC

) 8280–284

M13R

C3_1513

AT

CG

GC

GA

GG

GA

TC

TA

AC

GC

CG

CA

AC

GT

GA

AA

CC

TG

JN657706

(AA

TC

) 10

388–444

Bhg-r

C3_857

TA

CA

GT

TC

TC

GT

TC

CG

CC

TC

CA

AT

AT

GT

GC

AG

AG

TC

GC

CC

JN657707

(AG

) 10

296–318

Bhg-r

C3_2029

TC

CG

TT

GT

GT

GA

AC

TC

GT

AA

AG

CA

CT

GC

CT

CG

AT

AA

GT

TG

GC

JN657708

(AG

AT

) 6205–227

Bhg-r

Cro

ssam

pli

fica

tion

New

pri

mer

dev

elopm

ent

Tag

sequen

ces

and

dye

s

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GC

CG

CT

CT

AG

AA

CT

AG

TG

FA

MM

13F

TG

TA

AA

AC

GA

CG

GC

CA

GT

D3

M13R

-pU

CG

CA

GG

AA

AC

AG

CT

AT

GA

CN

ED

Bhg-r

TA

GA

AG

GC

AC

AG

TC

GA

GG

D4

Bhg-r

TA

GA

AG

GC

AC

AG

TC

GA

GG

VIC

CA

GC

AG

TC

GG

GC

GT

CA

TC

A

D2,D

3,D

4M

13

TG

TA

AA

AC

GA

CG

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CA

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PE

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(2007)

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ci

Conservation Genet Resour (2012) 4:191–196 193

123

Table 2 Multiplexed loci for six species

Species Locus Allele range NA HO/HE [F] (lM) [R] (mM)

C. puella (N = 23) LIST4002 136–150 5 0.26/0.24 1 0.050 0.500

Multiplex 1 LIST4034 276–278 3 0.39/0.55 1 0.050 0.500

LIST4063 219–227 7 0.74/0.82 1 0.250 1.000

LIST21-06 130–172 9 0.87/0.83 0.100 0.250

LIST21-04 174–215 7 0.70/0.73 0.075 0.750

LIST21-01 158–174 7 0.70/0.77 1 0.075 0.750

Multiplex 2 LIST21-05 182–253 19 0.30/0.96* 1 0.038 0.375

LIST4024 228–239 8 0.57/0.85* 0.063 0.625

C. mercuriale (N = 23) LIST4002 145–148 2 0.09/0.13 0.050 0.500

Multiplex 1 LIST4034 276–285 2 0.04/0.09* 0.025 0.250

LIST4037 230–236 4 0.48/0.52 0.013 0.125

LIST4063 226–268 6 0.65/0.66 0.100 1.000

LIST4062 195–199 4 0.09/0.31* 0.050 0.500

LIST4024 226–229 4 0.83/0.53* 1 0.038 0.375

Multiplex 2 LIST4031 271–275 2 0.48/0.481 0.025 0.250

TA1 = 57� 9 10 cycles LIST4030 259–294 5 0.22/0.58* 0.025 0.250

TA2 = 52� 9 28 cycles LIST4023 220–288 5 0.43/0.68 0.025 0.250

LIST4059 302–309 3 0.04/0.43* 0.025 0.250

LIST4060 230–240 4 0.52/0.71 0.013 0.125

LIST4066 198–243 8 0.48/0.68 0.013 0.125

LIST40-71 79–111 5 0.33/0.48 0.025 0.250

C. pulchellum (N = 26) LIST4024 233–237 5 0.12/0.81* 0.025 0.500

Multiplex 1 LIST4002 144–148 2 0.23/0.55 0.038 0.375

TA1 = 60� 9 10 cycles LIST4037 238–244 5 0.58/0.47 0.038 0.375

TA2 = 53� 9 28 cycles LIST40-67 106–115 5 0.65/0.73 0.025 0.500

LIST4066 200–204 3 0.31/0.41 0.025 0.500

LIST4030 267–279 4 0.54/0.61 0.038 0.375

LIST4034 277–278 2 0.00/0.25* 0.038 0.375

C. johanssoni (N = 21) LIST4034 264–275 5 0.48/0.77 0.050 0.500

Multiplex 1 LIST4037 235–261 7 0.14/0.71* 0.013 0.875

LIST4062 194–199 3 0.54/0.55 0.125 1.250

LIST4063 225–240 6 0.31/0.36 0.050 0.500

LIST4067 98–104 3 0.35/0.38 0.050 0.500

Multiplex 2 C2_1050 323–334 7 0.77/0.82 0.060 0.600

C2_1708 206–219 5 0.24/0.53* 0.060 0.600

C3_2109 258–265 3 0.50/0.56 0.060 0.600

Multiplex 3 C3_1503 284–386 3 0.07/0.37* 0.050 0.500

C3_1472 197–204 3 0.62/0.59 0.040 0.400

C. armatum (N = 21) LIST4034 276–289 7 0.62/0.83 0.038 0.375

Multiplex 1 LIST4037 233–235 3 0.48/0.67 0.038 0.375

LIST4042 145–160 6 0.35/0.46 1 0.038 0.375

LIST4071 217–219 3 0.17/0.63* 0.038 0.375

Multiplex 2 LIST4060 259–261 3 0.13/0.52* 4 0.113 0.563

TA = 53� LIST4072 259–263 5 0.22/0.58* 3 0.113 0.563

Multiplex 3 C_2146 215–246 6 0.38/0.55 0.070 0.700

C3_976 233–254 2 0.14/0.32 1 0.030 0.300

C2_1519 260–268 4 0.13/0.74* 0.050 0.500

C3_1875 293–305 4 0.26/0.45 2 0.050 0.500

C2_2304 293–305 4 0.74/0.63 2 0.050 0.500

C2_2355 280–284 3 0.79/0.59 0.050 0.500

C3_1513 388–444 10 0.43/0.87* 4,5 0.050 0.500

194 Conservation Genet Resour (2012) 4:191–196

123

Laboratories (USA). The obtained sequences were aligned

and 2,281 unique contigs created using SeaView 4.2

(Galtier et al. 1996). MicrosatelliteCommander 1.0.0 (Rozen

and Skaletsky 2000; Faircloth 2008) was used to identify

microsatellite repeats and design primers with an annealing

temperature of 60�C. A total of 192 primer pairs for unique

microsatellite repeats were selected, of which 26 were di-, six

were tri- and 160 were tetra- nucleotide repeats. Primers were

M13-tagged (Table 1), and PIG-tailed as above.

DNA for screening loci was extracted by placing a

single leg in 100 ll of 6% WV Chelex (Bio-Rad) sus-

pended in Buffer EB (Qiagen), incubated overnight with

3 ll Proteinase K (Fermentas) at 56�C; followed by

enzyme inactivation at 95� for 10 min, and centrifugation.

Initial amplification for all loci was carried out in 5 ll

reactions containing Type-It Mastermix (Qiagen), 1 ll

DNA-Chelex solution, 0.5 mM tailmix, 0.05 lM forward

and 5 lM reverse primer. For the cross-amplification tests

primers were first tested in triples (one dye for each loci) on

four individuals from the original species. Reliably

amplifying loci were multiplexed according to annealing

temperature (and primer concentrations adjusted) into two

multiplexes each for C. puella and C. mercuriale (Table 2),

before screening for polymorphism on 23 individuals. The

multiplexes were used for primer testing on the remaining

four species. Loci that were non-amplifying and non-

polymorphic in the species of origin were tested separately.

Loci were screened on 23–26 individuals following

multiplexing and primer concentration adjustment. Ther-

mal cycling followed the manufacturer’s instructions at

annealing temperatures of 60, 55 and 53�C in a PTC 200

thermal cycler (MJ-Research). Genotyping was performed

on a Beckman CEQ 8000, using GenomeLabTM DNA Size

Standard Kit–400 as internal size standard, and analysed

with the Beckman CEQ Fragment Analysis Software

(Beckman Coulter).

For initial screening of the new loci, four loci were

screened together as above, with 1 ll DNA from (a) one

individual (b) DNA pooled from eight individuals, for each

species, at an annealing temperature 60� (128 primer pairs

were tested at 53 and 56�C, yielding no loci). Genotyping

was carried out on a 3720xl Genetic Analyzer with

LIZ-500 as an internal size standard, and scored using

GeneMapper 4.0 (Applied Biosystems). Amplifying loci

were organized into new multiplexes using Multiplex-

Manager 1.0 (Holleley and Geerts 2009) and screened on

21 individuals.

Number of alleles (NA) was calculated for each locus,

and Arlequin version 3.01 (Schneider et al. 2000) was used

to test for departures from Hardy–Weinberg equilibrium

(HWE) and linkage disequilibrium (LD; Table 2) with a

sequential Bonferroni correction applied to control for

multiple comparisons.

Of the 29 loci tested for cross-amplification, nine did not

amplify in any species, and one locus (LIST4034) ampli-

fied in all (Table 2). Values for HO (0.04–0.87) and

NA [2–19 alleles, mean = 5.6 (2–9 and mean = 4.67

respectively, excluding LIST21-05)] for C. puella and

C. mercuriale were consistent with results from British

populations (Lowe et al. 2007; Watts et al. 2004a). Several

loci developed for C. mercuriale cross-amplified, and

showed similar NA (2–7, mean = 4) and HO (0.00–0.65).

Of the 192 new loci sixty-four primer pairs amplified

product in the right range on one or two species. Ampli-

fication in C. johanssoni and C. armatum were most suc-

cessful, yielding 25 and 30 amplifying loci respectively,

whereas only nine loci amplified in C. scitulum. After

removing loci that were inconsistent or monomorphic,

fifteen primer pairs remained (Tables 1, 2). Values of HO:

0.07–0.79 and NA: 2–10 (mean = 5.2) were similar to the

cross-amplifying loci. Patterns of significant HWE and LD

varied, with most occurrences in C. armatum.

Table 2 continued

Species Locus Allele range NA HO/HE [F] (lM) [R] (mM)

Multiplex 4 C3_857 296–318 8 0.30/0.79* 3 0.030 0.300

C2_1708 217–231 8 0.48/0.83 5 0.070 0.700

C3_2029 205–227 8 0.78/0.80* 0.070 0.700

C. scitulum (N = 21) LIST4034 281–285 2 0.04/0.51* 0.038 0.375

Multiplex 1 LIST4037 242–244 2 0.04/0.51* 0.038 0.375

LIST4063 222–225 3 0.00/0.63* 0.038 0.375

Multiplex 2 C2_1050 318–325 5 0.21/0.26 0.050 0.500

C2_2355 278–282 2 0.00/0.06* 0.050 0.500

C3_2109 380–386 4 0.13/0.53* 0.050 0.500

N number of individuals, TA thermal cycling conditions (28 cycles at 60�C unless indicated), NA number of alleles, HO observed heterozygosities,

HE expected heterozygosities, [F] = forward primer concentration (lM), [R] = reverse primer concentration (mM)

* Significant deviation from HWE, 1–5 = significant linkage disequilibrium (offending loci in bold for C. puella)

Conservation Genet Resour (2012) 4:191–196 195

123

Acknowledgments This work was funded by Stiftelsen J Gust

Richert, Carl Tryggers Foundation and FORMAS. We thank Kenyon

Mobley, Maria Lindgren, Owen Rowe and Nathaniel Street for their

assistance.

References

Brownstein M, Carpten J, Smith J (1996) Modulation of nontemplated

nucleotide addition of Taq DNA polymerase: primer modifica-

tions that facilitate genotyping. BioTechniques 20:1004–1010

Faircloth BC (2008) MSATCOMMANDER: detection of microsat-

ellite repeat arrays and automated, locus-specific primer design.

Mol Ecol Res 8:92–94

Galtier N, Gouy M, Gaultier C (1996) SEAVIEW and PHYLO_WIN:

two graphic tools for sequence alignment and molecular

phylogeny. Comput Appl Biosci 12:543–548

Holleley CE, Geerts PG (2009) Multiplex manager 1.0: a crossplat-

form computer program that plans and optimizes multiplex PCR.

BioTechniques 46:511–517

Keever CC, Sunday J, Wood C, Byrne M, Hart MW (2008) Discovery

and cross-amplification of microsatellite polymorphisms in

Asterinid Sea Stars. Biol Bull 215:164–172

Lowe CD, Kemp SJ, Harvey IF, Thompson DJ, Watts PC (2007)

Variable microsatellite loci isolated from the azure damselfly,

Coenagrion puella (L.) (Zygoptera; Coenagrionidae). Mol Ecol

Notes 7:880–882

Oetting WS, Lee HK, Flanders GL et al (1995) Linkage analysis with

multiplexed short tandem repeat polymorphisms using infrared

fluorescence and M13-tailed primers. Genomics 30:450–458

Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users

and for biologist programmers. In: Krawetz S, Misener S (eds)

Bioinformatics methods protocols: methods in molecular biol-

ogy. Humana Press, Totowa

Sahlen G, Bernard R, Cordero Rivera A, Ketelaar R, Suhling F (2006)

Critical species of Odonata in Europe. Int J Odonatol 7:385–398

Schneider S, Roessli D, Excoffier L (2000) Arlequin: a software for

population genetics data analysis. V3.01. Genetics and Biometry

Lab, Dept. of Anthropology, University of Geneva

Watts PC, Wu JH, Westgarth DJ, Thompson DJ, Kemp SJ (2004a) A

panel of microsatellite loci for the Southern Damselfly, Coe-nagrion mercuriale (Odonata: Coenagrionidae). Conserv Genet

8:117–119

Watts PC, Thompson DH, Kemp SJ (2004b) Cross-species amplifi-

cation of microsatellite loci in some European zygopteran

species (Odonata: Coenagrionidae). Int J Odonatol 7:87–96

196 Conservation Genet Resour (2012) 4:191–196

123