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...31, Z41293, and NE49 (37, 40). MLSA and phylogeny. MLSA and phylogenetic analyses were performed as described previously (30). MEGA 4 (21) was used for sequence alignments, and MrBayes 3.1.2 software =-=(19)-=- was used for phylogenetic analyses. Only the phylogenetic trees generated using MLSA were rooted. B. lusitaniae sequences were omitted from ospA and ospC analyses because of insufficient sequence ove...

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...al haplotype genealogies were estimated from complete ND2 and ND3 sequences with maximum likelihood (PAUP 4.0b10: Swofford, 2003) and Bayesian algorithms (MRBAYES 3.1.2: Ronquist & Huelsenbeck, 2003; =-=Huelsenbeck & Ronquist, 2005-=-). For both maximum-likelihood and Bayesian inferences, the general time reversible model with corrections for invariant characters and gamma-distributed rate heterogeneity (GTR + I + G) was applied a...

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... model of sequence evolution for the concatenated ND2 and ND3 sequences was the Tamura– Nei (TrN) model (Tamura & Nei, 1993) with a gamma Phylogeographic patterns of genetic diversity in water frogs Journal of Biogeography 37, 2111–2124 2113 ª 2010 Blackwell Publishing Ltd distribution (G) for site mutation rate (shape parameter = 0.2). All distance calculations were performed with mega 4.1 (Beta 2). A haplotype phylogeny based on the 83 unique concatenated ND2 and ND3 sequences (Appendix S2) was estimated using Bayesian inference as implemented in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003; Huelsenbeck & Ronquist, 2005). For the analysis, the general time-reversible model, with corrections for invariant characters and gamma-distributed rate heterogeneity (GTR + I + G), was applied as recommended by Huelsenbeck & Rannala (2004). Mutation model parameters were estimated from the dataset. Posterior probabilities of phylogenetic trees were calculated by employing a 107-iteration Metropolis-coupled Markov chain Monte Carlo procedure (four chains, chain temperature parameter = 0.2). Trees were sampled every 1000 generations; the first 1000 recorded trees were discarded; 9000 recorded trees were examined. The autoc...

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...e performed with all closest relatives according to BLAST results (data not shown). For clarity, not all of these sequences were included in Figure 2. Phylogeny was inferred using MrBayes version 3.1 =-=[58,59]-=-, assuming the GTR (general time reversible) phylogenetic model with 6 substitution rate parameters, a gamma-shaped rate variation with a proportion of invariable sites and default priors of the progr...

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...m likelihood (ML) analyses were conducted on static alignments. Sequences were aligned using MUSCLE ver. 3.6 (Edgar 2004) with default parameters. BI analyses were performed using MrBayes ver. 3.1.2 (=-=Huelsenbeck & Ronquist 2005-=-) with a unique GTR model of sequence evolution (Tavare 1986) with corrections for a discrete gamma distribution and a proportion of invariant sites (GTR + Γ + I; Yang 1996) specified for each partit...

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...nt posterior distribution of trees and models, so no specific sequence evolution model was specified for MrBayes beyond the generic GTR+I+G with all parameters to be estimated during the run. MrBayes =-=[55,56]-=-, available at http:// mrbayes.csit.fsu.edu/, was used to conduct the phylogenetic analysis. Default parameters for estimation under a GTR+I+G model were used, with uniform priors set on the base freq...

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...fied to code the new specimen and additional fossils (Eophalangium sheari andMesobunus dunlopi), containing 158 characters and 46 taxa. The morphological data matrix is available in the public database Morphobank (Project 793). The morphological data matrix was analyzed in combination with the five-gene molecular data set of Giribet et al. [6], modified by treatment with GBlocks v.0.91b [48] to cull positions of ambiguous homology. The combined matrix of 4,043 characters is available at http://dx.doi.org/10.5061/dryad.r32p3. These data were analyzed under Bayesian inference in MrBayes v.3.1.2 [49], with the morphological partition assigned a discrete equal ratesmodel [50] and each gene assigned a unique GTR + I + G model, as selected in jModeltest v.0.1.1 [51, 52]. Four runs each with four chains, and a default distribution of hot and cold chains, were conducted for 107 generations. Convergence diagnostics were assessed in Tracer v.1.5 [53], and 25% of all 16 runs were discarded as Current Biology Vol 24 No 9 6 Please cite this article in press as: Garwood et al., A Paleozoic Stem Group to Mite Harvestmen Revealed through Integration of Phylogenetics and Development, Current Biology (2...

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...appropriate evolutionary model by the Akaike corrected information criterion (AICc), the program jModeltest 0.1.1 was used (Posada, 2008). Phylogenetic relationships among all the RHDV strains were inferred using Bayesian Inference (BI) in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003), simulating four simultaneous Markov chains (MCMC) for 4 106 generations each, and using a sampling frequency of 100 generations. Convergence between run parameters from paired simultaneous runs was adjusted considering an adequate sampling based on average standard deviation of split frequencies being <0.01 (Huelsenbeck and Ronquist, 2005). The program Tracer v1.5 (Rambaut and Drummond, 2007) was used to determine burn-in length considering the set of trees saved prior to log likelihood stabilization and convergence. 3. Results 3.1. Detection of RHDV in mice and rabbits All sampled rodents appeared healthy. RHDV positivity was detected by molecular analysis in the liver of 3 mice (5.8%): 2 A. sylvaticus (As15 and As26) and 1 M. spretus (Ms8). The rabbits analyzed also appeared healthy, showing no sign of the disease. Out of the 31 rabbits caught, the virus was detected in 7 (23.5%), of which 6 provided analysable sequences (Oc1...