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Abstract

We describe the development based on 454 pyrosequencing technology of thirteen microsatellite markers for two closely related species of lamprey: Lampetra fluviatilis and L. planeri. The number of alleles per locus ranged from 2 to 5 in L. fluviatilis and from 2 to 6 in L. planeri. Gene diversity ranged from 0.062 to 0.718 in L. fluviatilis and from 0.322 to 0.677 in L. planeri. These markers will be helpful to study population genetic structure of both species and resolve their taxonomic status as separate species or ecotypes of a single species.
TECHNICAL NOTE
Characterization of thirteen microsatellite markers in river
and brook lampreys (Lampetra fluviatilis and L. planeri)
Arnaud Gaigher
Sophie Launey
Emilien Lasne
Anne-Laure Besnard
Guillaume Evanno
Received: 8 August 2012 / Accepted: 13 August 2012 / Published online: 24 August 2012
Ó Springer Science+Business Media B.V. 2012
Abstract We describe the development based on 454
pyrosequencing technology of thirteen microsatellite
markers for two closely related species of lamprey: Lam-
petra fluviatilis and L. planeri. The number of alleles per
locus ranged from 2 to 5 in L. fluviatilis and from 2 to 6 in
L. planeri. Gene diversity ranged from 0.062 to 0.718 in L.
fluviatilis and from 0.322 to 0.677 in L. planeri. These
markers will be helpful to study population genetic struc-
ture of both species and resolve their taxonomic status as
separate species or ecotypes of a single species.
Keywords Lampetra fluviatilis Lampetra planeri
Microsatellites Gene flow Genetic structure
The river lamprey Lampetra fluviatilis and brook lamprey
L. planeri are two aquatic agnathans distributed throughout
Europe. L. fluviatilis is anadromous with a parasite feeding
style, while L. planeri is sedentary in freshwater and non-
parasitic. These species have undergone a significant
decline throughout Europe (Renaud 1997) and are listed in
Appendix III of the Bern convention. In France the river
lamprey is listed as vulnerable while the brook lamprey is
classified as least concern (UICN 2010). At the adult stage
both species are morphologically very similar and mostly
differ by their size with L. fluviatilis being larger than L.
planeri. In addition, they often coexist on the same
spawning sites (Lasne et al. 2010) and they cannot be
discriminated at the larval stage. They were classified as
distinct species due to their distinct life histories and
morphological differences (Hardisty and Potter 1971).
However, some studies based on mitochondrial DNA or
allozymes failed to reveal a strong divergence between
them, hence the hypothesis that they may in fact be two
ecotypes of a single species (Schreiber and Engelhorn
1998; Espanhol et al. 2007). Here we describe the devel-
opment of thirteen microsatellite markers that will be
useful to infer gene flow and disentangle two hypotheses:
(1) a recent divergence resulting in two closely related
species or (2) the existence of two ecotypes within a single
species. The distinction between both hypotheses may have
important consequences for the management of L. fluvia-
tilis and L. planeri populations.
Genomic DNA was extracted using the DNeasy Blood
and Tissue Kit (Qiagen) on tissues of 3 L. fluviatilis and 3 L.
planeri adults collected in France. The six samples were
pooled to make a microsatellite library common to both
species. We used a procedure combining DNA enrichment
and high throughput pyrosequencing (GS-FLX, Roche
Diagnostics) recently developed by Malausa et al. (2011).
The program QDD was used to identify and select micro-
satellites sequences and to design primer pairs (Megle
´
cz
et al. 2010). A total of 208 microsatellites were identified by
the Genoscreen company (Lille, France) and 48 loci were
selected based on their relatively high number of repetitions.
A. Gaigher S. Launey A.-L. Besnard G. Evanno (&)
INRA, UMR 985 Ecologie et Sante
´
des Ecosyste
`
mes,
35042 Rennes, France
e-mail: guillaume.evanno@rennes.inra.fr
A. Gaigher S. Launey A.-L. Besnard G. Evanno
Agrocampus Ouest, UMR ESE, 65 rue de Saint-Brieuc,
35042 Rennes, France
A. Gaigher
Department of Ecology and Evolution, University of Lausanne,
1015 Lausanne, Switzerland
E. Lasne
Muse
´
um National d’Histoire Naturelle, CRESCO, 35800 Dinard,
France
123
Conservation Genet Resour (2013) 5:141–143
DOI 10.1007/s12686-012-9753-z
Table 1 Characteristics of thirteen microsatellites developed for Lampetra fluviatilis and Lampetra planeri
Locus/GenBank accession Primer sequence (5
0
–3
0
) Repeat motif Fluorescent label/
multiplex panel
Allele size
range (bp)
L. fluviatilis (n = 32) L. planeri (n = 35)
Na Ho He Na Ho He
LP-003/ F: M13-TCACGTACGCGTTAACTCCA (CA)
8
6-FAM/1 111–115 2 0.406 0.448 3 0.371 0.322
JX468083 R: TTCCTTAATTGGTCTGCCTCA
LP-006/ F: M13-TGCCCACACGTGATAGACAT (AG)
8
PET/1 147–151 3 0.469 0.523 2 0.543 0.501
JX468084 R: GGCGATCGTCATAAATAGCC
LP-009/ F: M13-AACTCCCACGTGCAAAATTC (TG)
7
NED/4 206–210 3 0.563 0.597 3 0.400 0.341
JX468085 R: AGGCATCACTCCTAACGACG
LP-018/ F: M13-TTAAAAGTGCGGCGAAATCT (TG)
8
PET/3 245–251 5 0.594 0.675 6 0.629 0.637
JX468086 R: TGTTCCATAACCACTGCTCG
LP-022/ F: M13-GACAGCTCGCTCAAGGCTAC (AGG)
7
PET/1 232–238 3 0.313 0.304 2 0.457 0.504
JX468087 R: TCGTCGTGGTCACAGTCATC
LP-027/ F: M13-ACAGTCAACCTCCGACATCC (ATC)
7
PET/2 212–224 5 0.563 0.540 4 0.382 0.476
JX468088 R: AGCCCATGATGATTCCATTC
LP-028/ F: M13-AGAACTCTGTGGACGTTCCG (ATC)
5
6-FAM/2 250–259 4 0.781 0.718 4 0.571 0.646
JX468089 R: TCTCAAGAAATGAGTTCTCAATCG
LP-030/ F: M13-TGAGGGGAAAATGGAAACAG (CAT)
5
NED/4 259–268 4 0.563 0.626 3 0.543 0.560
JX468090 R: TTCAGGATGATAGCACTGCC
LP-037/ F: M13-GGATAATCGTCGCTGGTGTT (CA)
7
VIC/2 156–160 2 0.063 0.062 2 0.400 0.437
JX468091 R: GCACAAGCTTGATGTGACAAA
LP-039/ F: M13-GCACTTCCAACAAAGCCAGT (AC)
6
PET/4 159–178 5 0.375 0.484 4 0.743 0.677
JX468092 R: TATTTCAGCCACTTGGGCAT
LP-043/ F: M13-CTCCATTATACACGGGGACAG (TA)
6
NED/1 164–166 2 0.375 0.381 2 0.343 0.388
JX468093 R: TGAACCTTGGTGCTGAGATG
LP-045/ F: M13-AGAGGTGTTTCGCGTGCTAT (GTT)
6
VIC/3 190–196 3 0.531 0.558 2 0.429 0.466
JX468094 R: AAGGAGAGAGGAGGTTTCGG
LP-046/ F: M13-ACCGCAAACTCATCAGGAAC (CAC)
6
PET/3 154–166 3 0.688 0.670 4 0.457 0.487
JX468095 R: AAGCGGATTTAGAAGCGACA
M13 sequence: 5
0
-CACGACGTTGTAAAACGAC-3
0
The annealing temperature is the same for all loci (see the main text)
n number of samples, Na number of alleles, Ho observed heterozygosity, He expected heterozygosity
142 Conservation Genet Resour (2013) 5:141–143
123
PCR conditions were optimized for these loci using 17 L.
fluviatilis and 12 L. planeri. Genomic DNA was extracted
from tissue in a 10 % Chelex
Ò
solution (Sigma-Aldrich)
with 1 mg/ml Proteinase K and 1X TE (Tris/EDTA), heated
at 55 °C for 2 h followed by 99 °C for 10 min (modified
from Estoup et al. 1996). The amplification followed the
M13 method developed by Schuelke (2000). PCR was per-
formed in a 6 ll total volume containing approximately
15 ng DNA, 0.06 U Taq DNA polymerase, 5X PCR buffer
(GoTaq, Promega), 208 lM each dNTP, 2.17 mM MgCl
2
,
0.055 lM forward primer with a tail extented M13, 0.55 lM
reverse primer and 0.57 lM M13 primer labeled with a
fluorochrome (6-FAM, VIC, NED or PET). A Touchdown
PCR program was applied as follows: initial denaturation of
4 min at 94 °C, then 20 cycles including denaturation of 30 s
at 94 °C, annealing of 30 s starting at 65 °C then decreasing
of 0.5 °C per cycle and extension of 1 min at 72 °C; then, 10
cycles with denaturation of 30 s at 94 °C, annealing of 30 s
at 55 °C and extension of 1 min at 72 °C; and a final
extension step of 5 min at 72 °C. PCR products from dif-
ferent markers were pooled into four multiplexes before
migration (Table 1). Fragments were size fractionated on an
ABI Prism 3130xl Genetic Analyzer using the size standard
GeneScan 500 LIZ. Allele sizes were determined with
GeneMapper 3.7 (Applied Biosystems).
Thirteen markers were chosen based on successful
amplification and we assessed their polymorphism on 32 L.
fluviatilis from the Loire River (France) and 35 L. planeri
from the Oir River (France) (Table 1). The number of
alleles per locus (Na) and observed and expected hetero-
zygosities (Ho and He) were calculated using G
ENETIX
4.05.2 (Belkhir et al. 1996–2004). All loci were polymor-
phic (Table 1) and the number of alleles per locus ranged
from 2 to 5 for L. fluviatilis (mean 3.4) and 2 to 6 for L.
planeri (mean 3.2). The expected heterozygosity ranged
from 0.062 to 0.718 for L. fluviatilis and 0.322 to 0.677 for
L. planeri.
No null alleles or genotyping errors were found using
M
ICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004). Devi-
ations from Hardy–Weinberg equilibrium (HWE) and
linkage disequilibrium between loci were tested using
G
ENEPOP 4.1 (Rousset 2008). No linkage disequilibrium
was found and no deviation from HWE was detected after
sequential Bonferroni correction (Rice 1989).
These loci can be amplified in both species and will thus
be useful to assess the distribution of genetic diversity
within and between species and to clarify their taxonomic
status.
Acknowledgments We thank our colleagues from the INRA ‘U3E’
unit who helped us with samples’ collection. This study was funded
by the European Regional Development Fund (Transnational program
Interreg IV, Atlantic Aquatic Resource Conservation Project) and by
the French Ministry of Ecology and Sustainable Development (project
‘Amphihalins, cohe
´
rence du re
´
seau N2000 en mer’).
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PhD Thesis: Lampreys (Order Petromyzontiformes) have existed for over 365 million years and are considered the most ancient group of living vertebrates. Given the socioeconomic, cultural, and ecological consequences of declining lamprey populations, it is imperative to address declines by implementing effective conservation management. This thesis explores the conservation issues affecting the European lamprey species pair Lampetra fluviatilis and Lampetra planeri and offers a holistic approach to their management and conservation in relation to anthropogenic impacts. The rapid development of smallscale hydropower provides substantial risk to migrating biota. At the site of an Archimedes screw turbine, damage rates to lampreys that passed through the screw were low (1.5%) and distinct seasonal, and diel, patterns of migration were exhibited by recently transformed juvenile and larval lampreys. Results indicated longer periods of impingement risk than expected. Cumulative potential impacts of multiple hydropower sites on downstream fish passage (including lampreys) should, however, be considered by regulatory agencies when planning hydropower development within catchments. Anthropogenic barriers were also found to intensify differentiation between L. planeri populations and anadromous L. fluviatilis populations. Gene flow was consequently found to be asymmetric due to the barriers allowing downstream movement, whilst obstructing active upstream migration. Samples of 543 European river lamprey Lampetra fluviatilis and European brook lamprey Lampetra planeri from across 15 sites, primarily in the British Isles, were investigated for 829bp mtDNA sequence and 13 polymorphic microsatellite DNA loci. Contrasting patterns of population structure were found for mtDNA (which revealed no differentiation between species) and microsatellite DNA markers. Microsatellite markers revealed strong differentiation among freshwater-resident L. planeri populations, and between L. fluviatilis and L. planeri in most cases, but little structure was evident among anadromous L. fluviatilis populations. There is also evidence that there has been some degree of gene flow between L. fluviatilis and L. planeri since these populations were established. There is much debate as to whether lamprey paired-species constitute distinct species or are divergent ecotypes of a single polymorphic species. Overall, these findings are suggestive of multiple independent divergences of L. planeri from an anadromous ancestor (i.e. L. planeri are polyphyletic). Focus of conservation and management efforts, therefore, needs to be directed towards ensuring the longitudinal connectivity within rivers, and the continued existence of the specific habitats necessitated within lamprey life-cycles. Molecular techniques should be applied to identify genetically differentiated populations of freshwater-resident lampreys. Appropriate measures, such as, the designation of a network of Special Areas of Conservation (SACs), and recognising these populations as distinct Evolutionarily Significant Units, should also be implemented to ensure the survival of these populations.
... Thirteen recently developed polymorphic microsatellite loci were used to examine genetic differentiation among and between all L. fluviatilis and L. planeri populations. Eight microsatellite primers developed for European Lampetra (Lp-003, Lp-006, Lp-009, Lp-018, Lp-027, Lp-028, Lp-046 and Lp-045; Gaigher et al. 2013), one primer set developed for Lampetra richardsoni (Lri-5; Luzier et al. 2010), and four microsatellite primers developed in this study (using the protocol described in White et al. 2010) and optimized for European Lampetra species (Lamper_1, Lamper_2, Lamper_3, Lamper_4) were included (Table S3, Supporting information). ...
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