Experimental

For library preparation, ten gametophytic fronds of P. umbilicalis were collected from Punta Herminia, Coruña, Spain, in April 2010 (43°23′23″N, 8°24′01″W). Specimens from this population were placed at the herbarium of the University of Santiago de Compostela, Spain (SANT-Algae No: 24072). Genomic DNA was isolated using a nuclei isolation protocol (Varela-Álvarez et al., Reference Varela-Álvarez, Andreakis, Lago-Lestón, Pearson, Serrão, Procaccini, Duarte and Marbá2006) followed by a LiCl extraction protocol described by Hong et al. (Reference Hong, Coury, Polne-Fuller and Gibor1992) as modified by van Oppen et al. (Reference van Oppen, Olsen and Stam1995). Four microsatellite enriched libraries were constructed following Varela-Álvarez et al. (Reference Varela-Álvarez, Paulino and Serrão2017), based on Glenn and Schable (Reference Glenn, Schable, Zimmer and Roalson2005) and Billote et al. (Reference Billote, Lagoda, Risterucci and Baurens1999). We also used nucleotide repeats that were complementary to those in other Porphyra spp. microsatellites: P. haitanensis (Zuo et al., Reference Zuo, Wang, Cao, Su, Liao and Chen2007) and P. yezoensis (Kong et al., Reference Kong, Mao, Yang, Qu, Yan and Wang2009) (recently transferred to the genus Pyropia (Sutherland et al., Reference Sutherland, Lindstrom, Nelson, Brodie, Lynch, Hwang, Choi, Miyata, Kikuchi, Oliveira, Farr, Neefus, Mols-Mortensen, Milstein and Müller2011)). From the libraries (development details in supplementary materials), 146 sequences were recovered, of which, 97 contained a total number of 241 repeats identified using MSATCOMMANDER (Faircloth, Reference Faircloth2008). Using these, 68 primer pairs were designed with PRIMER3 (Rozen and Skaletsky, Reference Rozen, Skaletsky, Krawetz and Misener2000). Primers were tested under two different strategies. A subset of 38 primers pairs were tested with a labelled tail, where one primer in each pair was modified on the 5′ end with a CAG sequence tail (5′-CAGTCGGGCGTCATCA-3′) to allow the use of a fluorescently labelled third primer in the PCR. The CAG tail was removed after primer testing and optimization. The other 30 primers were designed without any tail. For primer optimization we used first six individuals from the same population used for the library (Punta Herminia, Coruña, Spain) and two positive clones (the plasmid isolated from the library and the DNA template used to build the library). We selected seven primers to test for polymorphism in a panel of 44 individuals from four populations (11 individuals per population). These populations covered were: St Ives and Polzeath, UK (50°12′56″N, 5°28′56″W; 50°34′34″N, 4°55′12″W), Pta. Herminia, Coruña (43°23′23″N, 8°24′01″W) and Sanxenxo, Pontevedra, Spain (42°23′58″N, 8°48′54″W). Cross amplification on other species was tested, using on five individuals of Porphyra dioica Brodie and L.M. Irvine from Oeiras, Portugal (38°41′08″N, 9°18′33″W) and four individuals of Porphyra linearis Greville from São Bartolomeu do Mar, Portugal (41°34′27″N, 8°48′00″W).

Polymerase chain reactions (PCR) for all markers were performed separately in a 20 µl reaction volume containing 5–50 ng genomic DNA. For PCR using non-tailed primers, PCR reactions contained 0.25 mM of each primer, 0.2 mM of dNTPs, 2–3 mM of MgCl₂ (see Table 1 for loci optimizations), 1× GoTaq Flexi Buffer and 1 U GoTaq DNA polymerase (Promega). Forward primers were labelled either with FAM or HEX dye. For PCR including a third labelled primer, we used 0.25 µM unlabelled primer, 0.025 µM CAG tail-primer, and 0.225 µM universal dye-labelled primer. Amplifications using an Applied Biosystems thermal cycler (GeneAmp 2720) were conducted with the following three programs: (a) Program 1: 95°C for 5 min followed by 35 cycles at 95°C for 20 s, annealing at a specific temperature for each locus (see Table 1 for details) for 20 s (except for Por_021 and Por_042, only 15 s annealing), 72°C for 30 s, and a final extension step at 72°C for 10 min (b) Program 2: Program 1 but with 30 cycles (c) Program 3: Touchdown thermal cycling program consisting of an initial denaturing step of 3 min at 95°C, followed by 14 cycles of ‘touchdown’ PCR consisting of 15 s at 95°C, 15 s at 62°C (reduced by 0.5°C each subsequent cycle), and 15 s at 72°C, followed by 24 cycles consisting of 15 s at 95°C, 15 s at 55°C and 15 s at 72°C; with a final elongation step at 72°C for 10 min. PCR products were run on an ABI PRISM 3130xl sequencer (Applied Biosystems) and sized with GeneScan-350ROX size standard. Results were analysed using Genemapper version 4.0 (Applied Biosystems) and Genodive (Meirmans and Van Tienderen, Reference Meirmans and Van Tienderen2004). Binning and allele rounding were also checked with TANDEM (Matschiner and Salzburger, Reference Matschiner and Salzburger2009).

Table 1.

Characterization of polymorphic microsatellite loci for the red alga Porphyra umbilicalis: Locus name, GenBank accession number, primer sequences, repeat motif from the original clone, PCR conditions (Tª (C): annealing temperature/PCR program: P1, P2, P3/MgCl₂ (mM): Magnesium concentration), number of individuals amplified (N), size range, total number of alleles/ number of alleles per individual (Na/Ni), and Cross amplification (L: P. linearis, D: P. dioica). (For details on the PCR program: P1, P2 and P3, see text)

Null alleles were detected in three loci (below in the table).