Computational protocol: Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe

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Protocol publication

[…] Invasive specimens were euthanized with a lethal injection of sodium pentobarbital. Muscle tissue was dissected from invasive specimens, while native wild caught specimens were toe-clipped to obtain tissue samples. Genomic DNA was extracted by means of a NucleoSpin® tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Five genomic DNA fragments (circa (ca.) 2,040 bp) representing two mitochondrial and three nuclear gene fragments were amplified using PCR. These genes were selected in order to enable comparison with previously published work on native (; ; ; ) and invasive X. laevis specimens (; ). Fragments of the mitochondrial cytochrome b gene (Cytb; ca. 280 bp) and 16S ribosomal DNA (16S; ca. 800 bp) were amplified and sequenced with the primer pairs Cytb I /Cytb II and 16Sc-L/16Sd-H (; ). Fragments of the nuclear protein coding genes arginine methyltransferase 6 (Prmt6; ca. 666 bp), androgen receptor isoform α (AR; ca. 402 bp) and microtubule associated serine/threonine kinase-like protein (Mastl; ca. 539 bp) were amplified and sequenced with the primer pairs Exon4_for1/Exon4_rev2, XLAR_for_40/XLAR_rev_431 and Exon13_fora/Exon13_reva (). The nuclear primer pairs are assumed to be paralog-specific, hence only amplifying one pair of alleles (see ). PCR amplifications were run with the conditions reported in Online Resource 3. PCR products were purified with FastAP TM thermosensitive alkaline phosphatase in combination with exonuclease I and subsequently sequenced in both directions. Nucleotide sequences were assembled and edited in CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA). They were aligned together with corresponding well-documented sequences of South African X. laevis available from GenBank using the ClustalW algorithm () in MEGA v. 6 (). For comparison, the two Cytb haplotypes found by in Chilean (CL) invasive X. laevis populations were included in the Cytb alignment. Cytb and 16S gene fragments obtained by were not included in the current alignments as they only partially overlapped due to the use of different primers. Nuclear sequences were converted into haplotypes using the PHASE algorithm (; ) implemented in DnaSP v. 5 (). All new sequence data were deposited in Genbank (accession numbers in Online Resource 1: [...] Phylogenetic relationships among invasive and native X. laevis mtDNA sequences were reconstructed with Bayesian inference (BI) and Maximum Parsimony (MP). Three alignments were created: two separate Cytb and 16S alignments including only unique alleles and a concatenated Cytb-16S alignment including only unique haplotypes. In all analyses, X. gilli was used as outgroup. Parsimony informative sites were calculated with DnaSP v. 5 (). BI analyses were performed using MrBayes v. 3.2.4 (). Cytb, 16S and Cytb-16S alignments were analysed under a general time reversible (GTR) model with all model parameters estimated from the data and a proportion of invariant sites (+I) as selected by jModeltest v. 2.1.7 (). BI analyses were run with two different Metropolis-coupled Markov chains for 10 million generations with sampling every 1,000th generation. The average standard deviations of split frequencies, the potential scale reduction factors and the plots of likelihood versus generation were evaluated to ensure convergence based on the log file and using Tracer v1.6 (). A total of 25% of the trees were discarded as burn-in and posterior probabilities were calculated for each split from the remaining set of trees. MP trees were estimated with a neighbour joining tree as starting tree using the Phangorn package () in R 3.1.1 (). A set of most-parsimonious trees was generated using the parsimony ratchet () with nearest neighbour interchange rearrangement, 10,000 ratchet iterations and up to maximum 10 rounds. Parsimony bootstrap values were obtained with 1,000 bootstrap replicates using the bootstrap.phyDat function in R 3.1.1. All topologies were visualized and edited in respectively Figtree v. 1.4.2 () and TreeGraph2 v. 2.4.0-456 beta (). Differentiation between native geographical areas and invaded regions was quantified using pairwise Fst values computed based upon nucleotide pairwise distances and tested for significance with 999 permutations using Arlequin v. (). The population comparison was done solely for the 16S alignment as this dataset included sequences of most individuals (n = 179) and representatives of all populations. [...] The minimum number of recombination events within alleles were estimated in DnaSP v.5 (; ). Given putative recombination, median-joining networks (MJN) () were constructed in PopART v.1.7.2 (PopART, 2015) for each nuclear locus separately to illustrate the mutational differences between alleles and their distribution among invasive and native populations (). Pairwise Fst values were computed based on the allele frequencies to show to what extent alleles are differently sorted among populations (e.g., where the same alleles are found in different populations but with different frequencies). Native populations were defined based upon the geographical regions mentioned in ‘Taxon sampling.’ Individuals from France and Portugal respectively were treated as two populations. Pairwise Fst values were computed from the allele frequencies with 999 permutations to generate the probability that a random value would be greater than or equal to the observed data using GenAlEx v. 6.5 (. Individuals with missing data for more than one gene were excluded from the analysis. In order to visualize population differentiation, the pairwise Fst matrix was subsequently used as a distance matrix for a principal coordinates analysis (PCoA) in GenAlEx v.6.5. […]

Pipeline specifications

Software tools CodonCode Aligner, Clustal W, MEGA-V, DnaSP, MrBayes, jModelTest, Phangorn, FigTree, TreeGraph, Arlequin, PopART, GenAlEx
Applications Phylogenetics, Population genetic analysis, GWAS
Organisms Xenopus laevis
Diseases Genetic Diseases, X-Linked