The members of Bryoria sect. Implexae are pendent, copiously branched lichens with circumboreal distribution (Brodo and Hawksworth, 1977; Myllys et al., 2011a). They are an important component of the boreal forests (Glavich et al., 2005), and their frequency depends on forest fragmentation (Hilmo and Holien, 2002). These lichen-forming fungi are haploid and disperse with vegetative propagules; sexual reproduction with ascospores is uncommon (Brodo and Hawksworth, 1977). Bryoria sect. Implexae includes seven morphologically and chemically recognized species in Europe (Myllys et al., 2011a), which have different frequency across longitudinal and altitudinal gradients (Hawksworth, 1972; Myllys et al., 2011a). Molecular data confirm the monophyly of the section, although the relationships among the currently recognized species remain poorly understood because phylogenetic analyses suggest that several species are conspecific (Myllys et al., 2011b). Highly variable microsatellite markers of the fungal partner of lichen symbioses (Widmer et al., 2010; Devkota et al., 2014) will be used to study the genetic diversity and differentiation in Bryoria sect. Implexae, to determine the gene flow across and within the currently recognized species, and to assess the impact of land use and habitat fragmentation on population structure of these locally rare and threatened, boreal forest-associated lichens.
METHODS AND RESULTS
Eighty-two specimens representing the three morphologically and chemically characterized species, Bryoria capillaris (Ach.) Brodo & D. Hawksw., B. fuscescens (Gyeln.) Brodo & D. Hawksw., and B. implexa (Hoffm.) Brodo & D. Hawksw., were collected in three regions (Spain, Switzerland, and Finland; Appendix 1). All specimens are deposited in the Lichens Herbarium of the Universidad Complutense de Madrid (MAF-Lich), and duplicates are stored at the Swiss Federal Research Institute WSL at −20°C. A subset of 30 specimens was used for total DNA extraction with the MoBio PowerPlant Pro DNA Isolation Kit (MO BIO Laboratories, Carlsbad, California, USA). The pooled DNA was used to create a shotgun multiplex identifier library using the GS FLX Titanium Rapid Library Preparation Kit (Roche Diagnostics, Basel, Switzerland), and Microsynth AG (Balgach, Switzerland) provided the barcode adapters. The library was sequenced on 1/4th of a plate on a Roche 454 Genome Sequencer FLX at Microsynth. We obtained 533,962 reads of an average length of 812 bp (National Center for Biotechnology Information [NCBI] Sequence Read Archive [SRA] accession no. SRR1283191; http://www.ncbi.nlm.nih.gov/sra). The unassembled sequences were screened for di-, tri-, tetra-, and pentanucleotide microsatellites using MSATCOMMANDER 1.0.2 alpha (Rozen and Skaletsky, 1999; Faircloth, 2008), ensuring a minimum repeat length of 8 bp for dinucleotides and 6 bp for all others.
MSATCOMMANDER recovered 6329 primer pairs that fulfilled the default primer parameters among all reads. Of those, 5932 pairs were discarded from further studies because they contained unfavorable secondary structure, primer-dimer formation, monorepeats in the flanking region, or because they were duplicates, which we detected after alignment using CLC Main Work-bench 6 (CLC bio, Aarhus, Denmark). Putative sequences of algae, plants, animals, or microorganisms, which are often present in epiphytic samples, were identified and removed using the ntBLAST search on http://www.ncbi.nlm.gov. This inspection resulted in 58 primer pairs used for further analysis, i.e., to test for amplification with the symbiotic partner of these lichen-forming fungi. We used DNA from five axenic cultures of Trebouxia spp., which are hypothesized to be the photobionts of Bryoria sect. Implexae (Lindgren et al., 2014): T. angustilobata Beck (SAG2204), T. asymmetrica Friedl & Gärtner (SAG48.88), T. arboricola Puymaly (SAG219-1a), T. jamesii (Hildreth & Ahmadjian) Gärtner (SAG2103), and T. simplex Tschermak-Woess (SAG101.80). Forward primers were labeled with an M13 tag (5′-TGTAAAACGACGGCCAGT-3′) for PCR amplification (Schuelke, 2000). All PCR runs were performed on Veriti Thermal Cyclers (Life Technologies, Carlsbad, California, USA). The PCR reactions were evaluated in a temperature gradient with one-degree steps from 56–61°C, performed with the JumpStart REDTaq ReadyMix (Sigma-Aldrich, St. Louis, Missouri, USA) according to the manufacturer's protocol, with the following conditions: denaturation for 2 min at 94°C, followed by 30 cycles of 30 s at 94°C, 45 s at 56–61°C, and 45 s at 72°C; then for the M13-tag binding additional eight cycles of 30 s at 94°C, 45 s at 53°C, and 45 s at 72°C, with a final extension of 30 min at 72°C. In total, 14 primer pairs produced positive PCR reactions with at least one of the five Trebouxia species, and were excluded from further analyses because they were considered alga-specific.
Overview of the microsatellite loci developed for the group of lichen-forming fungi Bryoria sect. Implexae.
The amplification of the fungal component of Bryoria sect. Implexae was tested with the 44 remaining loci under the same conditions as mentioned above. There were 14 loci that produced specific single products at an annealing temperature of 56°C, 12 at 57°C, six at 58°C, six at 60°C, and six at 61°C. Polymorphism of the 44 microsatellite loci was initially tested on a subset of 12 individuals (four individuals from each of three countries: Spain, Switzerland, and Finland), resulting in 18 polymorphic loci with satisfactory amplification. All PCR products obtained were multiplexed (Table 1). PCR reactions were performed in a total volume of 10 µL containing 1 µL of ∼5 ng genomic DNA, 1 µL each of forward and reverse primers of varying concentration (Table 1), and 5 µL of Type-it Multiplex PCR Master Mix (QIAGEN, Hilden, Germany). The PCR protocol used fluorescent forward primers and the reaction was adjusted to: 5 min at 95°C; followed by 30 cycles of 30 s at 95°C, 90 s at 56, 58, or 60°C (Table 1), and 30 s at 72°C; with a final extension of 60 min at 60°C. PCR products were run on a 3130xl DNA Analyzer with GeneScan 500 LIZ as the size standard for fragment analysis (both by Life Technologies).
The 18 polymorphic microsatellite markers were tested for locus variability and marker consistency on three populations (Table 2). Alleles were sized using GeneMapper 5.0 (Life Technologies). The linkage disequilibrium (LD) between microsatellite loci and their variability were measured by counting the number of alleles and calculating Nei's unbiased gene diversity using Arlequin 3.11 (Excoffier et al., 2005). Dinucleotide microsatellites (n = 13) were the most common microsatellite motifs among the 18 loci (Table 1). The microsatellite loci revealed significant LD based on 999 permutations (P < 0.001). They show two to 13 alleles per locus with a mean of 4.6, and average gene diversities varied from 0.38 to 0.52 over three populations (Table 2).
Results of microsatellite screening in 82 individuals of lichen-forming fungi of Bryoria sect. Implexae between species of Bryoria sect. Implexae, and between compared regions.
Cross-species amplifications within three congeneric species were analyzed with the chi-square test; B. capillaris was shown to not amplify consistently, while B. fuscescens and B. implexa amplified more regularly (Appendix 2). Most markers amplified with all three species. However, the microsatellite marker Bi 15 only amplified with B. fuscescens, Bi05 with B. fuscescens and B. implexa, and Bi18 with B. capillaris and B. fuscescens.
The fungus-specific markers developed here will facilitate studies on genetic diversity and differentiation in Bryoria sect. Implexae throughout its geographic distribution, and on effects of forest management on genetic diversity of populations in this species group. Furthermore, putative phylogenetic signal within the flanking regions of the microsatellite sequences might help to delimit closely related species and to assess the taxonomic value of the morphological and chemical characters of these regionally rare and threatened lichens.
 The authors thank the Genetic Diversity Centre, ETH Zurich, for technical assistance; David L. Hawksworth (London-Madrid) for organizing the mini-symposium on the Bryoria implexa group; and Christine Keller (WSL) for helping with thin-layer chromatography analyses. Funding was received from the Swiss National Science Foundation (grant 31003A_ 1276346/1 to C.S.), the Federal Office for the Environment (FOEN, grant to C.S.), the Ministerio de Ciencia e Innovación de España (project CGL201125003 to A.C., V.J.R., and C.G.B.), and the Academy of Finland (grant 1133858 to L.M.).