Lobaria pindarensis Räsänen (Lobariaceae, Peltigerales) is a foliose lichen species known from mountain forests and open woodlands in the Himalayas of Bhutan, India, and Nepal. The lichen thallus is haploid and it mainly disperses with vegetative propagules, but sexual reproduction with ascospores can also occur (Scheidegger et al., 2010). The lichen disperses locally, thus sharing ecological traits with L. pulmonaria (L.) Hoffm. (Scheidegger and Werth, 2009; Scheidegger et al., 2012). Although microsatellite markers are available for L. pulmonaria (Dal Grande et al., 2010; Werth et al., 2013), only three markers (LPu32425, LPu40211, and LPu34888) published by Werth et al. (2013) reveal small, multiple bands when amplified with L. pindarensis. All other published markers do not amplify with L. pindarensis. Here, we develop microsatellite markers to study the impact of land use and habitat fragmentation on gene flow of this dispersal-limited lichen (Scheidegger et al., 2010).
METHODS AND RESULTS
Ten specimens of L. pindarensis, collected in two valleys in Nepal (Table 1; Manaslu Conservation Area [MCA] and Sagarmatha National Park [SNP]), were used for total DNA extraction with the QIAGEN Plant Mini Kit (QIAGEN, Hilden, Germany). Subsequently, whole genome 454 pyrosequencing of pooled DNA samples was performed using a Roche GS FLX sequencer to generate a sufficient number of microsatellite loci. Library preparation and sequencing were performed by Microsynth (Balgach, Switzerland). Shotgun libraries were prepared using the GS FLX Titanium Rapid Library Preparation Kit (Roche Diagnostics, Basel, Switzerland), while Microsynth provided barcode adapters. Out of a 1/4th run, we obtained 233,260 reads of an average length of 314 bases for a total of 73,191,881 bases. The unassembled sequences were screened for all possible sequence motifs of di-, tri-, tetra-, and pentanucleotide microsatellites using Primer3, as implemented in MSATCOMMANDER version 1.0.2 alpha (Rozen and Skaletsky, 2000; Faircloth, 2008). Microsatellites with motifs repeated at least eight times (for dinucleotides) or six (for all others) were chosen. For each locus, primer pairs were developed with MSATCOMMANDER using the default parameters.
Using all reads, MSATCOMMANDER found 1021 primer pairs that fulfilled the default primer parameters. Subsequently, 656 pairs were discarded either because they contained unfavorable secondary structure, primer dimer formation, or mononucleotide repeats in the flanking region, or because they were duplicates, which were detected after alignment using CLC DNA Workbench 5 (CLC bio, Aarhus, Denmark). The remaining 365 sequences were verified one by one using ntBLAST with the megablast option ( http://www.ncbi.nlm.nih.gov/blast) to exclude those that were highly similar to algae, plants, or microorganisms that are often present in environmental samples. To test for cross-amplification with the photobiont of L. pindarensis, Dictyochloropsis reticulata (Tschermak-Woess) Tschermak-Woess, PCRs of the remaining 116 primer pairs (including 44 di-, 65 tri-, and 7 tetranucleotides) were run using DNA from an axenic culture of D. reticulata (Dal Grande et al., 2010, 2012; Widmer et al., 2010, 2012). The 56 loci that produced positive PCR reactions were excluded from further analyses because they were considered alga-specific rather than fungus-specific. For PCR amplification, forward primers were labeled with an M13 tag (5′-TGTAAAACGACGGCCAGT-3′) (Schuelke, 2000). PCR reactions were performed in a total volume of 10 µL containing 1 µL of ∼1–5 ng genomic DNA, 0.5 µL of 5 µM forward and reverse primers, and 1× Type-it Multiplex PCR Master Mix (QIAGEN). All PCRs were performed on Veriti Thermal Cyclers (Life Technologies, Carlsbad, California, USA). The PCR reactions were assessed in a temperature gradient increasing by one-degree increments from 56–61°C, performed with the Type-it Microsatellite PCR Kit (QIAGEN) according to the manufacturer's protocol, and under the following conditions: denaturation for 5 min at 95°C; followed by 33 cycles of 30 s at 95°C, 90 s at 56–61°C, and 30 s at 72°C; then for the M13-tag binding an additional eight cycles of 30 s at 95°C, 90 s at 53°C, and 30 s at 72°C; with a final extension of 30 min at 60°C.
Characteristics of 18 polymorphic microsatellite loci developed for Lobaria pindarensisa and screened in 109 individuals.
The 60 remaining loci were assessed for amplification for the fungal component of L. pindarensis under the same conditions as above and using the total DNA of L. pindarensis. Out of these 60 loci, 48 produced specific single products, all at an annealing temperature of 57°C. Polymorphism of the 48 microsatellite loci was initially tested on a subset of seven individuals from three valleys, including three individuals from MCA, three from SNP, and two from Kanchenjunga Conservation Area (KCA), resulting in the detection of 25 polymorphic loci. These 25 loci were then tested in one population of 48 specimens from MCA, resulting in 18 loci with satisfactory amplification.
To characterize these 18 polymorphic L. pindarensis loci, PCRs of 109 individuals from three valleys were conducted (Table 1). The PCR protocol used fluorescent forward primers and the reaction was adjusted to the following conditions: 5 min at 95°C; followed by 25 cycles of 30 s at 95°C, 90 s at 57°C, and 30 s at 72°C; with a final extension of 60 min at 60°C. All PCR products obtained were multiplexed (Table 2) and run on a 3130xl DNA Analyzer with a GeneScan 500 LIZ Size Standard (G5 dye set) for fragment analysis (both by Life Technologies). Alleles were sized using GeneMapper version 3.7 (Life Technologies), and the variability of each microsatellite locus was measured by counting the number of alleles and calculating gene diversity using Arlequin version 3.11 (Excoffier et al., 2005). Trinucleotide microsatellites (n = 15) were the most common loci detected among the 18 microsatellite motifs (Table 2). The microsatellite loci produced 3–11 alleles per locus with an average of 6.9, and mean gene diversities over three populations varied from 0.514 to 0.685 (Table 1).
Cross-species amplification of seven closely related species of Lobaria (Schreb.) Hoffm. was tested on one specimen of each species (Appendix 1), applying the same PCR conditions established for L. pindarensis. All fragments were sequenced according to Cornejo and Scheidegger (2010) except Lpi01 and Lpi05, which were verified on an agarose gel but not sequenced. The transferability was high. Only one locus (Lpi05) did not amplify in L. chinensis Yoshim. However, several loci contained insertions within the flanking regions, and in others the microsatellite was disrupted (imperfect or interrupted microsatellite). In some loci, the microsatellite sequences were reduced or disappeared completely, as in Lpi10 in L. isidiophora Yoshim. and Lpi16 in L. gyrophorica Yoshim., L. pulmonaria, L. spathulata (Inum.) Yoshim., and Lobaria sp. (Fig. 1). In general, the cross-amplification success of Lpi markers decreased with decreasing relationship to L. pindarensis, being lowest in L. pulmonaria and Lobaria sp. (with four and six loci not amplifying, respectively).
Fungi, algae, and/or cyanobacteria live in close contact within the lichen thallus and hence the manual separation of symbionts for later molecular analyses is technically unfeasible. Therefore, symbiont-specific genetic markers have to be used in population genetic studies of lichens (Widmer et al., 2010). The newly developed, highly variable fungus-specific markers reported here will allow detailed studies on regional genetic differentiation, effects of forest disturbance on genetic diversity, and the contributions of clonal and sexual reproduction in this lichen species. Moreover, the flanking regions of the microsatellites will be used for sequence analyses in future phylogenetic studies of related taxa of the genus Lobaria.
Overview of the microsatellite loci developed for the lichen fungus Lobaria pindarensis.
 The authors thank the Genetic Diversity Centre, ETH Zurich, for technical assistance; the Swiss National Science Foundation (grant JRP IZ70Z0_131338/1 to C.S.); the European Commission (Marie Curie Actions “Lichenomics” to S.W.); and the Department of National Parks and Wildlife Conservation, Ministry of Forest and Soil Conservation, Government of Nepal, and the National Trust for Nature Conservation, Nepal, for providing collecting permits in the study areas.