Lichenized fungi form mutualistic relationships with photoautotrophic organisms (photobionts), mainly green algae (Trebouxiophyceae and Ulvophyceae) and/or cyanobacteria. The lichen symbiosis has been highly successful within fungi, especially Ascomycota, with more than 18,000 currently accepted species  and an estimated diversity of greater than 28,000 species . Due to the availability of genetic data and analytical improvements, DNA-based approaches play an increasing role in the recognition of diversity in lichenized fungi that would otherwise be impossible to recognize using classical phenotypic characters due to morphological convergence or parallelism [3–11].
In spite of the recent advancements in recognizing diversity in lichen-forming fungi, assessing diversification within a temporal context remains largely unexplored in nearly all groups of these important fungal symbionts, (exceptions include [12–14]). This is largely due to the poor fossil record for lichenized fungi, and also fungi in general, and uncertainties in the interpretation of the few known fossil records [15–17]. However, the timing of speciation events plays a valuable role, complementary to discovering and describing diversity, by addressing biogeographical, climatic, ecological, and other factors associated with diversification and extinction within a temporal context, (e.g. [12, 18–20]).
In spite of difficulties in obtaining accurate estimates of divergence times [21, 22], recent analytical advances in using relaxed molecular clocks, the inclusion of multiple fossil calibrations, and informative priors on substitution rates have improved accuracy in molecular dating [23–26]. However, a recent study also suggested that methods that fail to incorporate the process of gene lineage coalescence may provide inaccurate estimates of divergence dates . While gene trees, including phylogenies estimated from concatenated sequence data, can overestimate divergence times because they do not correct for genetic divergence that predates speciation, species tree methods incorporating the process of gene lineage coalescence likely provide a more biologically realistic framework for dating divergence events .
Within lichen-forming ascomycetes, Parmeliaceae (Lecanorales) constitutes one of the largest and best-studied families [27–32]. Although some lichen-forming fungal lineages have geographically restricted distributions, (e.g. [10, 14]), there is mounting evidence that transoceanic dispersal has commonly occurred within some lichenized fungi and has played a major role in diversification within Parmeliaceae [12, 33, 34].
Unrecognized diversity is common in many phenotype-based species circumscriptions in the family, confounding the current interpretation of ecological and biogeographical patterns [7, 9, 10, 35, 36]. Additionally, interpreting biogeographical patterns and factors driving diversification is further complicated by a common pattern of long-distance dispersal in many taxa in Parmeliaceae, (e.g. [12, 33, 34, 37]). Therefore, accurate estimates of divergence times are especially critical for identifying major factors driving diversification. A recent study investigating the origin and diversification in the largest clade of macrolichens, the parmelioid lichens (Parmeliaceae, Ascomycota), provides valuable taxon-specific estimates of substitution rates for three genetic markers based on three calibration points, including two dated fossils from Parmeliaceae .
Within Parmeliaceae, the genus Melanohalea O. Blanco et al. includes 22 species based on traditional taxonomy, most of which occur primarily on bark and wood throughout the Holarctic [38–44]. Only four known taxa occur in the southern Hemisphere [45–47]. Many Melanohalea species display broad geographic and ecological distributions, although a limited number of taxa appear to have more restricted ranges . Otte et al.  suggested that distribution patterns in Melanohalea are largely determined by contemporary ecogeographical factors, and most species have largely filled their potential distributions in the northern Hemisphere. Furthermore, the distributions of some Melanohalea taxa, including M. elegantula (Zahlbr.) O. Blanco et al. and M. exasperatula (Nyl.) O. Blanco et al., have been linked to eutrophication, air pollution, and other anthropogenic factors [48–52].
While Pleistocene events have been shown to have been important factors driving diversification and affecting distributions in many groups, (e.g. [18, 53–55]), recent estimates suggest that the Melanohalea radiation vastly predates Pleistocene glacial cycles . Within many parmelioid genera, major radiations occurred from the Late Oligocene to the early Pliocene, before the climate became cooler, drier, and more seasonal at the end of the Pliocene [12, 56]. However, the overall importance of Pleistocene glacial cycles in diversification within Melanohalea is unknown.
Reproductive strategies vary among Melanohalea taxa. Sexual reproduction is restricted to characteristic fungal fruiting bodies (ascomata) producing meiospores (=ascospores), and is common in at least 13 of the 22 described species. Ascospores are dispersed independent of the photosynthesizing partner (photobiont) and require reacquisition of the appropriate photobiont partner in order to re-establish the lichenized condition. In contrast, other species within Melanohalea commonly propagate asexually by means of vegetative diaspores, either isidia or soredia. These specialized vegetative reproductive propagules contain both fungal and algal symbionts, eliminating the need for independent acquisition of the appropriate photobiont partner. The isidiate taxa M. elegantula and M. exasperatula show a remarkable potential for dispersal and may be spreading in some areas [43, 48, 49]. Poelt  hypothesized that lichenized fungi reproducing asexually (via soredia and isidia) are generally more successful in pioneering formerly glaciated areas than forms that reproduce sexually. In contrast, Nimis  argued that the distribution of Holarctic lichens is more likely determined by general ecology than by their reproductive strategy alone. The latter argument supports the assumption that the distribution of Melanohalea within the Holarctic today widely reflects their biological constitution, rather than their geographic origin or dispersal strategy . However, population structure and history is poorly understood in most lichen-forming ascomycetes and the role of their reproductive strategy in response to climate fluctuation remains unclear.
Cryptic lineages within phenotypically circumscribed taxa are common in Parmeliaceae , and previously unrecognized species-level lineages have now been recognized within six of the phenotype-based Melanohalea species from a broad sampling of populations in the northern Hemisphere . Diversity within this genus is now well-characterized, at least in the northern Hemisphere, providing an excellent study system with which to test the relative influence of Miocene orogeny and climatic conditions and Pleistocene glacial cycles on common lichen-forming fungi.
In this study our goals are twofold: (1) we aim to estimate divergence times in the lichenized genus Melanohalea using both concatenated gene tree and coalescent-based multilocus species tree approaches; and (2) we evaluate the impact of Pleistocene glacial cycles on the population demography among four common sexually reproducing lichen-forming fungal species and two taxa reproducing largely via vegetative diaspores. With estimates of divergence times, we examine the relative roles of Miocene orogeny and climate change and Pleistocene glacial cycles on diversification in the lichenized fungal genus Melanohalea. We are also interested in population demographic changes in common Melanohalea species after the last glacial maximum (LGM), including anthropogenic factors. Here we present estimates of divergence times within Melanohalea and assess population demographic histories in relation to the LGM.