Lobaria pulmonaria (L.) Hoffm. (Lobariaceae, Peltigerales) is a widely distributed lichen in the northern hemisphere and afro-temperate forests in South Africa. In central Europe, the species has faced a severe decline in the past decades, and the species is therefore of conservation concern. In the past decade, L. pulmonaria has become a model species for the population biology and conservation biology of lichens (Scheidegger and Werth, 2009). Eight microsatellite markers have thus far been published for the lichen fungus L. pulmonaria, a species that mainly reproduces clonally (Dal Grande et al., 2012; Werth and Scheidegger, 2012). Here, we develop 14 additional microsatellite markers to increase the genetic resolution for detailed studies of population subdivision and the reproductive system of this species, and we test for cross-amplification with two Macaronesian endemics closely related to L. pulmonaria—L. immixta Vain. and L. macaronesica C. Cornejo & Scheid.
METHODS AND RESULTS
We collected thallus fragments from 190 individuals from São Miguel Island, Azores (SM1: 37.85132°N, 25.78249°W), and from El Hierro, Canary Islands (SH2: 27.74317°N, 17.98651999°W; SH3: 27.73292°N, 18.01134°W) (see Appendix 1 for voucher information). Samples were air-dried after collection and stored at -20°C until DNA extraction using the DNeasy Plant Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The fungus L. pulmonaria was grown in axenic culture from ascospores (voucher: GB1-10; 36.69658°N, 5.02322°W), and total genomic DNA isolated from the axenic culture was used for 454 pyrosequencing following standard protocols on a GS FLX instrument (Roche, Schlieren, Switzerland) performed at Microsynth (Balgach, Switzerland). The data amounted to 233260 reads of an average length of 313.8 bases, in total 73 171 881 bases.
MSATCOMMANDER version 0.8.2 (Faircloth, 2008) was used to find di-, tri-, tetra-, penta-, and hexanucleotide repeats and design primers with Primer3 (Rozen and Skaletsky, 2000) using default values (annealing temperature [Ta] = 60°C, GC content 35–75%, primer length 19 bp) based on the 454 database. A total of 478 contigs contained microsatellite repeats (43 di-, 359 tri-, 37 tetra-, 11 penta-, and 28 hexanucleotides). For 304 of these contigs, flanking regions allowed primer design. Subsequently, 16 loci were tested, each with more than 11 repeats. Fourteen of these loci amplified successfully, and polymorphism was assessed based on 99 thalli of L. pulmonaria, 59 of L. macaronesica, and 32 of L. immixta. Multiplex PCR reactions were carried out in 5 µL reaction volumes using the M13 method (Schuelke, 2000), adding 1 µL of primer mix containing all loci to be labeled with the same fluorescent dye (primer without M13-tail: 0.15 µM, M13-tailed primer: 0.01 µM, dye-labeled M13 primer: n × 0.15 µM, n = number of loci in multiplex), 2.5 µL Jump-Start Taq ReadyMix (Sigma-Aldrich, Buchs, Switzerland), 0.5 µL of genomic DNA, and 1 µL of ddH2O. For primer sequences, see Table 1. PCR amplifications used an initial denaturation at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 45 s at the annealing temperature of 60°C, 45 s at 72°C; followed by eight cycles of 30 s at 94°C, 45 s at 53°C, 45 s at 72°C to incorporate the dye-labeled M13 primer (5′-TGTAAAACGACGGCCAGT-3′); and a final extension at 72°C for 60 min.
Fragment analysis was performed after pooling PCR products labeled with four dyes, using GeneScan-500 LIZ (Life Technologies, Rotkreuz, Switzerland) as an internal size standard on a 3130xl Genetic Analyzer (Life Technologies). Genotyping was performed with GeneMapper version 3.7 (Life Technologies). Polymorphism was determined using our own code (available upon request) in R (R Development Core Team, 2011).
Of the 14 loci assessed, 13 amplified and were polymorphic in L. pulmonaria, eight in L. macaronesica, and seven in L. immixta. None of the loci amplified within a culture of Dictyochloropsis reticulata, the green-algal photobiont of L. pulmonaria. The number of alleles per locus ranged from two to 23, and the maximum gene diversity was 0.846 (Table 2). Eight of the new markers and five of the previously published markers had 10 or more alleles in the studied individuals. Hence, the resolution for future studies will be increased. Moreover, with seven and eight loci working for L. macaronesica and L. immixta, the new markers appear to be useful for population genetic studies of closely related species in Lobaria sect. Lobaria.
By increasing the marker resolution, the newly developed polymorphic microsatellite loci will allow us to perform detailed studies of the reproductive system of L. pulmonaria (and its close relatives), and hence aid our understanding of the population biology of a fascinating lichen symbiosis.
Overview of the microsatellite loci designed for the lichen fungus Lobaria pulmonaria.
Polymorphism in 14 new and eight previously published microsatellite loci developed for the tree lungwort Lobaria pulmonaria, and cross-amplified for its close relatives L. immixta and L. macaronesica.a