Seahorses (Syngnathidae, Gasterosteiformes) are emblematic and threatened fish with remarkable morphology and biology, including male pregnancy (Avise et al. 2002; CITES 2002). Only two of the 32 species in the world live in the Northeast Atlantic: Hippocampus guttulatus and H. hippocampus. No accurate biological and distribution data are available for these species, further research being needed to assess their conservation status (IUCN 2006). In order to address population genetic analysis of the long-snouted seahorse H. guttulatus, we have developed primers for 12 polymorphic microsatellite loci from enriched genomic libraries in this species. Microsatellites have proven to be powerful markers for population genetics and its application to conservation biology (Ellegren 2004). These loci are also useful for parentage analysis to support reproduction in captivity (Castro et al. 2004) and to study genetic mating systems (Avise et al. 2002).

The genetic data presented here represent the first population analysis in this European seahorse. H. guttulatus was collected in Galician coasts (NW Spain), using non-invasive sampling of dorsal fin in live seahorses, and tissues stored in 100% ethanol. Genomic DNA was isolated by Chelex resin (Estoup et al. 1996) and standard phenol–chloroform methods.

Microsatellites were isolated using a partial enriched genomic library from muscle of a single dead seahorse according to the FIASCO protocol (Fast Isolation by AFLP of Sequences Containing repeats; Zane et al. 2002). Linked genomic fragments were enriched using a (AC)17 biotinylated probe as described by Pardo et al. (2006).

Polymorphism was preliminarily evaluated in four individuals using 2.5% agarose gels dyed with ethidium bromide using unlabelled primers. Allelic variation of putative polymorphic markers was confirmed in 32 wild seahorses using an ABI 3100 automated sequencer (Applied Biosystems). The forward primer of each pair was 5′ fluorescently labelled (Applied Biosystems). Genotyping was carried out using the genemapper 3.7 software (Applied Biosystems), with GenScan 500LIZ as internal size standard.

One hundred and fifty six clones out of 263 sequenced, revealed characteristic short tandem repeats of microsatellite loci. Primers pairs were designed for 52 sequences, where enough large flanking regions existed. Nineteen of these were successfully amplified and 12 of them result polymorphic (Table 1). Ten out of the variable loci contained dinucleotide repeats and the remaining ones presented tetranucleotide motifs. Eight microsatellites were perfect, three imperfect and one compound. The final yield of variable microsatellite markers rendered by this enrichment protocol was 4.6%, higher than in other fish studies using the same procedure (2.3%, Zane et al. 2002; 0.6%, Carreras-Carbonell et al. 2004).

Table 1 Characteristics of isolated microsatellite loci in the long-snouted seahorse H. guttulatus and cross-amplification in H. hippocampus
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Population genetic parameters were estimated using the program cervus 3.0 (Marshall et al. 1998) in 32 seahorses from NW Spain (Table 2). Number of alleles ranged from 2 (Hgu-USC10 and Hgu-USC11) to 15 (Hgu-USC7). Expected heterozygosity and PIC ranged from 0.031 to 0.912 and from 0.030 to 0.889, respectively, at loci Hgu-USC11 and Hgu-USC7. All loci conformed to Hardy–Weinberg and genotype linkage equilibrium expectations (genepop 3.1 program; Raymond and Rousset 1995), after sequential Bonferroni correction. These data are compatible with random mating, and suggest no close linkage between loci. However, null alleles might be present at two loci (Hgu-USC1 and Hgu-USC7; Table 2) as revealed by micro-checker 2.2.3 program (van Oosterhout et al. 2004), suggesting caution in their application for kinship analysis. The probability of exclusion of a false parent for this set of loci when the other parent is unknown or known was 0.973 and 0.998, respectively, using cervus program (Table 2). All these data support the usefulness of this set of loci for population genetic studies and parentage analysis. They will permit to evaluate wild and cultured genetic resources, to address genetic mating system in this species and to support the development of its reproduction in captivity.

Table 2 Genetic diversity estimates of isolated microsatellite loci in the long-snouted seahorse H. guttulatus
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We also evaluated the cross-amplification of these loci in two individuals of the short-snouted seahorse H. hippocampus, using a range of MgCl2 (1.5–2 mM) and temperature (48–60°C) PCR conditions. All loci were successfully amplified, some of them showing interspecific size differences (Table 1). This set of markers are potentially useful to obtain appropriate population data for conservation and management of genetic resources of the two threatened European seahorses.