Materials and Methods

Multi-locus DNA sequence data for A. muscaria specimens were generated in previous studies (Geml et al. 2006, 2008, and Oda et al. 2004). Geml et al. (2006) reported that there are at least three phylogenetic species clades within the A. muscaria species complex that occur in Alaska (referred to as Clades I, II, and III). To our knowledge, Clade I is restricted to North America, while Clades II and III occur in Eurasia and in Alaska. For our tests in this study, 114 ITS rDNA sequences were chosen, which represent the three phylogenetic species. Because the phylogenetic species mentioned are non-interbreeding entities, population-level analyses were conducted separately for each species clade. Multiple sequence alignments were made using Clustal W (Thompson et al. 1997) and subsequently were corrected manually. Identical sequences were collapsed into haplotypes using SNAP Map (Aylor and Carbone 2003) after recoding insertion or deletions (indels) and excluding infinite-sites violations. The analyses presented here assume an infinite sites model, under which a polymorphic site is caused by exactly one mutation and there can be no more than two bases segregating. Base substitutions were categorised as phylogenetically uninformative or informative, and as transitions or transversions. Site compatibility matrices were generated from each haplotype dataset using SNAP Clade and SNAP Matrix (Markwordt et al. 2003) to examine compatibility/ incompatibility among all variable sites, with four resultant incompatible sites removed from the data set. This was important as subsequent coalescent analyses assume that all variable sites are fully compatible. Two migration models were used. First, MDIV (Nielsen and Wakeley 2001) was used to determine whether there was any evidence of migration between Alaskan and non-Alaskan populations in each species clade, i.e. to test whether Alaskan populations could have survived the LGM in local forest refugia. For this purpose, specimens were assigned either to the 'Alaskan' or the 'non-Alaskan' group based on their localities. In Clades I and II, the 'non-Alaskan' groups consisted of specimens collected in the contiguous states of the US or in Eurasia, respectively. These represented populations that survived the LGM in Southern refugia. MDIV implements both likelihood and Bayesian methods using Markov chain Monte Carlo (MCMC) coalescent simulations to estimate the migration rate (M), population mean mutation rate (Theta), divergence time (T) and the time since the most recent common ancestor (TMRCA). This approach assumes that all populations descended from one panmictic population that may or may not have been followed by migration. For each dataset, the data was simulated assuming an infinite sites model with uniform prior. We used 2,000,000 steps in the chain for estimating the posterior probability distribution and an initial 500,000 steps to ensure that enough genealogies were simulated before approximating the posterior distribution. Second, if MDIV showed evidence of migration, MIGRATE was used to estimate migration rates assuming equilibrium migration rates (symmetrical or asymmetrical) in the history of the populations (Beerli and Felsenstein 2001). We applied the following specifications for the MIGRATE maximum-likelihood analyses: M and Theta generated from the FST calculation, migration model with variable Theta and constant mutation rate. Subsequently, we reconstructed the genealogy with the highest root probability, the ages of mutations and the TMRCA of the sample using coalescent simulations in Genetree v. 9.0 (Griffiths and Tavaré 1994). Ages were measured in coalescent units of 2N, where N is the population size.

Was this article helpful?

0 0

Post a comment