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Development of tools to rapidly identify cryptic species and characterize their genetic diversity in different European kelp species

Citation

Mauger, Stéphane; Fouqueau, Louise; Valero, Myriam (2021), Development of tools to rapidly identify cryptic species and characterize their genetic diversity in different European kelp species, Dryad, Dataset, https://doi.org/10.5061/dryad.612jm643j

Abstract

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.

Methods

Contigs available in the repository come from the draft genome of a male gametophyte of Laminaria digitata. A total of 23 contigs contained previously developed microsatellite loci for L. digitata (Billot et al. 1998; Brennan et al. 2014) and L. ochroleuca (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 L. digitata; 13 L. hyperborea; 11 L. ochroleuca; 8 L. rodriguezii and 11 L. pallida). Second one for the “single population sampling” for which we used individuals coming from a unique population to characterize new microsatellite markers (32 L. digitata; 32 L. hyperborea; 16 L. ochroleuca; 16 L. rodriguezii and 11 L. pallida). 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 L. digitata, L. hyperborea, L. ochroleuca and H. nigripes 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 L. digitata, 13 L. hyperborea, 11 L. ochroleuca and 8 H. nigripes) were sequenced and four consensus sequences (one per species) were generated using CODONCODEALIGNER (https://www.codoncode.com/). 

Funding

IDEALG, Award: ANR-10-BTBR-04

Phaeoexplorer, Award: ANR-10-INBS-09

MARFOR, Award: Biodiversa/004/2015

Région Bretagne, Award: ARED 2017 REEALG

Foundation for Science and Technology (Portugal), Award: UIDB/04326/2020, DL57/2016/CP1361/CT0010 SFRH/BSAB/150485/2019

EU Horizon GENIALG, Award: Grant Agreement No 727892

IDEALG, Award: ANR-10-BTBR-04

Phaeoexplorer, Award: ANR-10-INBS-09

MARFOR, Award: Biodiversa/004/2015

EU Horizon GENIALG, Award: Grant Agreement No 727892