DOI: 10.5061/dryad.612jm643j
title: Development of tools to rapidly identify cryptic species and characterize their genetic diversity in different European kelp species

description: Marine ecosystems formed by kelp forests are severely threatened by global
 change and local coastline disturbances in many regions. In order to take
 appropriate conservation, mitigation and restoration actions, it is
 crucial to identify the most diverse populations which could serve as a
 “reservoir” of genetic diversity. This requires the development of
 specific tools, such as microsatellite markers to investigate the level
 and spatial distribution of genetic diversity.  Here, we
 tested new polymorphic microsatellite loci from the genome of the
 kelp, Laminaria digitata, and tested them for
 cross-amplification and polymorphism in four closely related
 congeneric species (Laminaria hyperborea, Laminaria
 ochroleuca, Laminaria rodriguezii and Laminaria
 pallida). Adding these 20 new microsatellite loci to the ten L.
 digitata loci previously developed by Billot et al. (1998) and
 Brenan et al. (2014) and to the ten L. ochroleuca loci
 previously developed by Coelho et al. (2014), we retained a total of 30
 polymorphic loci for L. digitata, 19 for L. hyperborea,
 16 for L ochroleuca, 19 for L. rodriguezii and
 12 for L. pallida. These markers have been tested for the first
 time in the last two species. As predicted, the proportion of
 markers that cross-amplified between species
 decreased with increasing genetic distance. In addition, as
 problems of species identification were reported in this genus, mainly
 between L. digitata and Hedophyllum
 nigripes, but also between L. digitata, L.
 hyperborea and L. ochroleuca in areas where
 their range distributions overlap, we report a rapid PCR
 identification method based on species-specific cox1 mitochondrial primers
 that allows these four species of kelp to be rapidly distinguished.
description: Contigs available in the repository come from the draft genome of
 a male gametophyte of <em>Laminaria digitata</em>. A total of
 23 contigs contained previously developed microsatellite loci
 for <i>L. digitata</i> (Billot et al. 1998; Brennan et al.
 2014) and <i>L. ochroleuca</i> (Coelho et al. 2014). A total
 of 17 contigs contained monomorphic microsatellite loci and 20 contigs
 contained polymorphic microsatellite loci developed in this study. The new
 microsatellite loci were found into this draft genome using the SPUTNIK
 program (http://abajian.net/sputnik/), which uses a recursive algorithm to
 identify repeated patterns of nucleotides with a length ranging between
 two and five. Two types of genotype tables are
 available in the repository, first for the "wide scale sampling"
 dataset for which we used one to several individuals sampled from
 different populations over the entire range distribution of the different
 species (15 <i>L. digitata</i>; 13 <i>L.
 hyperborea</i>; 11 <i>L. ochroleuca</i>;
 8 <i>L. rodriguezii </i>and 11 <i>L.
 pallida). </i>Second one for the “single population sampling” for
 which we used individuals coming from a unique population to characterize
 new microsatellite markers (32 <i>L. digitata</i>;
 32 <i>L. hyperborea</i>; 16 <i>L. ochroleuca</i>;
 16 <i>L. rodriguezii </i>and 11 <i>L.
 pallida).</i> Genotype tables were obtained using an ABI 3130 XL
 capillary sequencer (Applied Biosystems, USA) and scored manually
 in Genemapper version 4.0 (Applied Biosystems).
 Sequences available in the repository come from fragments of the
 mitochondrial COI gene (COI-5P) was obtained for <i>L. digitata, L.
 hyperborea, L. ochroleuca </i>and<i> H.
 nigripes</i> using the primers GAZ_F2 (5’- CCAACCAYAAAGATATWGGTAC
 -3’) and GAZ_R2 (5’- GGATGACCAAARAACCAAAA -3’, Lane et al. 2007) and
 sequenced using an ABI 3130 XL capillary sequencer (Applied Biosystems,
 USA). A total of 47 individuals (15 <i>L. digitata</i>,
 13 <i>L. hyperborea</i>, 11 <i>L.
 ochroleuca</i> and 8 <i>H. nigripes</i>) were sequenced
 and four consensus sequences (one per species) were generated using
 CODONCODEALIGNER
 (https://www.codoncode.com/).