Characterization of microsatellite markers in the cosmopolitan lichen-forming fungus Rhizoplaca melanophthalma ( Lecanoraceae )

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, populationand landscape-level processes in the cosmopolitan taxon Rhizoplaca melanophthalma s. str.


Rhizoplaca melanophthalma (Lecanoraceae) Introduction
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(Leavitt et al. , 2013(Leavitt et al. , 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.

Materials and methods
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).
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.

Table 1 .
Voucher information for Rhizoplaca specimens used in this study.Herbaria codes are provided for each specimen in parentheses following voucher number.

Table 3 .
Sample size, number of alleles (A) and Nei's unbiased genetic diversity (H e ) of ten microsatellite loci developed for Rhizoplaca melanophthalma s. str.