Rhizoplaca melanophthalma s.l. is a group of morphologically distinct and chemically diverse species that commonly occur in desert, steppe and montane habitats worldwide. In this study, we developed microsatellite markers to facilitate studies of genetic diversity, population structure, and gene flow in the nominal taxon of this group, Rhizoplaca melanophthalma. We characterized 10 microsatellite markers using a draft genome of R. melanophthalma s. str. assembled from Illumina reads. These loci were tested for 21 R. melanophthalma s. str. specimens and also with a subset of 18 specimens representing six additional species in the R. melanophthalma complex. The number of alleles per locus in R. melanophthalma s. str. ranged from 3 to 11 with an average of 6.7. Nei’s unbiased gene diversity ranged from 0.35 to 0.91. Amplifications of the microsatellite loci were largely successful in the other six species, although only three markers were found to be polymorphic. The new markers will provide an additional resource for studying genetic, population- and landscape-level processes in the cosmopolitan taxon Rhizoplaca melanophthalma s. str.

Ascomycetes , gene flow, landscape genetics, lichen-forming fungi, microsatellites, Rhizoplaca melanophthalma

Rhizoplaca melanophthalma (DC.) Leuckert & Poelt s.l. represents a group of morphologically distinct and chemically diverse species of lichen-forming fungi with broad ecological and geographical distributions. Species in this group occur all over the world in disjunct populations in continental climates, although species in this complex are notably absent from Australia. Rhizoplaca melanophthalma s.l. commonly grows on siliceous or calcareous rock in arid climates, but can also be found in montane coniferous forests, alpine tundra habitats, and bi-polar populations in the Arctic and Antarctica (McCune 1987). Members of this group are commonly used in air-quality biomonitoring research, making it an important species for conservation (Aslan et al. 2004; Dillman 1996). The species complex belongs to the recently re-circumscribed monophyletic genus Rhizoplaca in Lecanoraceae (Zhao et al. 2016).

Previous multi-locus and phylogenomic studies support the circumscription of multiple species within R. melanophthalma s.l. (Leavitt et al. 2011, 2013, 2016b), many of which occur in sympatry in Western North America. In Western North America the distribution area of these species extends from the northern boreal zone to Mexico along the Rocky Mountains with a center of diversity in the Great Basin region (Leavitt et al. 2011). Rhizoplaca melanophthalma s. str. has the broadest ecological and geographic distribution of all known species within this complex, with populations occurring in desert, montane and steppe ecosystems in Antarctica, Central Asia, Europe, and North and South America (Leavitt et al. 2013).

The Rhizoplaca melanophthalma group provides an interesting system for assessing genetic diversity, population structure and gene flow in symbiotic fungal species with broad ecological and geographic distributions. To facilitate additional research into population- and landscape-level processes, 10 microsatellite markers were developed for R. melanophthalma s.str.

A total of 42 specimens representing seven different species in the Rhizoplaca melanophthalma species complex were included in this study. Twenty-one of these represented R. melanophthalma s. str., three R. haydenii, four R. novomexicana, two R. parilis, four R. polymorpha, six R. porteri and two R. shushanii (Table 1). DNA was extracted from these specimens as described previously (Leavitt et al. 2011).

A draft genome of an axenic culture of R. melanophthalma was obtained from a previous study (Leavitt et al. 2016a). The program MSATCOMMANDER 1.0.8 (Faircloth 2008) was used to search for di-, tri-, tetra-, penta-, and hexanucleotide microsatellite repeats in contigs >5 kb from the draft assembly. Only repeats with a minimum length of 8 bp for dinucleotide repeats and 6 bp for the rest were accepted. A total of 244 scaffolds contained microsatellite repeats (87 di-, 127 tri-, 11 tetra-, 5 penta-, and 14 hexanucleotides). For 25 of these repeats, primers were designed with Primer3 (Rozen and Skaletsky 2000) as implemented in MSATCOMMANDER. An M13 tag (5’-TGTAAAACGACGGCCAGT-3’) was appended to forward primers and 5’ ends of the reverse primers were tailed with 5’-GTGTCTT-3’ tag.

Singleplex PCR reactions were performed in 10 µl reaction volumes consisting of 5.89 µl H₂O, 1 µl 10x buffer (Roche Diagnostics, Indianapolis, USA), 0.6 µl 8 mM dNTP, 1 µl BSA, 0.15 µl Taq (Roche Diagnostics, Indianapolis, USA), 0.16 µl 6-FAM labeled M13 primer, 0.04 µl 10 µM M13 tailed forward primer, 0.16 µl 10 µM reverse primer, and 1 µl of genomic DNA. DNA amplification was performed using a touchdown PCR with initial denaturation at 95 °C for 5 min; followed by first 11 cycles of 30 s at 95 °C, 30 s at 60–50 °C, 1 min. at 72 °C, and then 35 cycles of 30 s at 95 °C, 30 s at 50 °C, 1.5 min. at 72 °C, and a final extension of 10 min. at 72 °C.

Fragment analysis was performed on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan-500 LIZ (Life Technologies, Warrington, UK) as an internal size standard. Genotyping was performed utilizing the microsatellite plugin in Geneious 9.1.2 (Biomatters Limited). Polymorphism within the microsatellites was tested in GenAlEx 6.5 (Peakall and Smouse 2012) by calculating Nei’s unbiased genetic diversity.

Of the 25 microsatellites assessed, 18 amplified successfully and 10 were polymorphic in all 21 R. melanophthalma s. str. specimens (Table 2). The number of alleles per locus ranged from three to 11 with an average of 6.7. Nei’s unbiased genetic diversity varied between 0.353 and 0.919 with the average genetic diversity being 0.765 (Table 3). The same 18 microsatellites that amplified successfully with R. melanophthalma s. str. also amplified in R. haydenii, R. novomexicana, R. parilis, R. polymorpha, R. porteri, and R. shushanii, but only three loci were polymorphic in all these species. For these three loci (Rmel1, Rmel, 4, and Rmel8) the number of alleles ranged from 11 to 15 with the average of 13 and Nei’s unbiased genetic diversity varied between 0.892 and 0.905 with average of 0.900.

The 10 polymorphic microsatellite markers for the lichen-forming fungus R. melanophthalma will help elucidate population processes that have led to the observed distribution patterns in this widespread species.

The authors would like to thank the Negaunee Foundation for providing funding for this study, and Kevin Feldheim (Pritzker Laboratory for Molecular Systematics) for assistance in the lab.