Computational protocol: Across the Indian Ocean: A remarkable example of trans-oceanic dispersal in an austral mygalomorph spider

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Protocol publication

[…] Five non-migid outgroups were sourced from []: the scorpion Urodacus planimanus Pocock, 1893, the red-back spider Latrodectus hasseltii Thorell, 1870, the curtain-web mygalomorph spider Cethegus fugax Simon, 1908, and the idiopid trapdoor spiders Aganippe sp. O. P.-Cambridge, 1877 and Euoplos sp. (). All newly obtained sequences were edited with reference to chromatograms using Geneious []. Forward and reverse sequences were assembled, and the resulting consensus sequences were then aligned using the ‘Geneious Alignment’ function of Geneious. PartitionFinder [] was used to select the model that best fit each gene, with the protein coding genes being divided into three codon positions. For COI, the General Time Reversible (GTR) [] + gamma (G) [] model was selected for the first codon position, the Felstein 81 (F81) [] + invariant (I) I+G for the second codon position, and the Hasegawa, Kishino and Yano (HKY) [] +I+G for the third. For ITS1, ITS2 and 18S, the model Kimura 80 (K80) [] +G was chosen. For 5.8S and 28S, the GTR+I+G model was chosen. For H3, the GTR+I+G model was chosen for codon position one and the K80+G model was chosen for positions two and three. For XPNPEP3, the HKY+G model was chosen for all positions.Phylogenetic reconstruction was undertaken using MrBayes 3.2.6 [] employing the CIPRES Science Gateway []. In the Bayesian analysis, each codon position was modelled separately using the models listed above. All parameters were unlinked and the rates were allowed to vary over the partitions. For all reconstructions, two runs with four chains each were run simultaneously for 100 million generations, with every 1,000th tree sampled. A burnin of 1,000, chosen using the program Tracer 1.6 [], was set for building the maximum clade credibility tree. The resulting tree was viewed using FigTree v1.3.1 [] (). A maximum likelihood analysis was also undertaken using RAxML [] on the BlackBox server [] with COI, H3 and XPNPEP3 partitioned by codons and ITS1, 5.8S, ITS2 and 28S partitioned individually, with the GTR + G model used for all genes. [...] Divergence dating analysis was performed using BEAST 1.8.0 [] to determine the time of divergence of M. rainbowi from its African relatives. The program BEAUti 1.8.0 (part of the BEAST software package) was used to create.xml files to input into BEAST. Given the robustness of phylogenetic analyses placing M. rainbowi within the African Moggridgea clade (see ), we focused on the Moggridgea taxa only for our molecular clock analyses. Exclusion of distantly related taxa, such as Bertmainius, avoided potential issues with saturation of the third codon positions of COI. This still enabled us to effectively date the nodes of most interest, i.e. the divergence time between the closest African sister to M. rainbowi, M. intermedia (see []), and the divergence between the two KI M. rainbowi populations. We included only the specimens for which we had a complete set of sequence data; this allowed us to link the trees and resulted in a single tree for analysis. The gene H3 had a larger proportion of missing data than the other genes, so was not included in the dating analysis. 28S was also not included as it could not be sequenced for M. intermedia, which was found to be the closest relative to M. rainbowi.Six separate BEAST analyses were carried out using different clock models, including a strict clock, uncorrelated lognormal clock and exponential relaxed clock, and both the GTR + G + I and HKY nucleotide substitution modes. Each analysis was run for 20 million generations with a burnin of 1 million generations (i.e. 10%), and the program Tracer 1.6 [] was used to analyse the parameter distributions estimated from BEAST and check for convergence of the chains. Stationarity was checked for, and no evidence of non-stationarity was found in all BEAST runs. As fossil calibrations were unavailable to date nodes of the Moggridgea phylogeny, the mean COI substitution rate was fixed at 0.02 substitutions per site per million years, based on the estimates of 4% divergence between lineages per million years [by ] for the trapdoor spider Aptostichus simus Chamberlin 1917. Rates for all other genes were estimated. Site models and clock models were unlinked and trees were linked. The tree priors selected for separate analyses were Speciation: Yule Process and Birth-Death Process, as both are suitable for inter-species relationships. Priors on the ucld.mean for each gene were defined as uniform with an initial value of 0.00115, an upper value of 0.0115 and a minimum value of 0.0001. The universal substitution rate estimated for arthropod mtDNA [] was used to define the upper value. Due to the average slower pace of nuclear genes compared with mitochondrial ones, the initial value was one order of magnitude slower (as per []). TreeAnnotator [] was used to produce a single “target” tree which was then visualised using FigTree v1.3.1 []. […]

Pipeline specifications

Software tools Geneious, PartitionFinder, MrBayes, FigTree, RAxML, BEAST
Application Phylogenetics
Organisms Homo sapiens