Computational protocol: Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii

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Protocol publication

[…] We used standard protocols for DNA extraction, amplification, and sequencing for a 657 bp segment near the 5′-terminus of the mitochondrial COI gene [] for all individuals. We generated sequences from the Dby 7th intron region of the y-chromosome from DNA extracts of 80 male specimens using the primers in Lim et al.[]. For 70 male individuals we recovered 767 bp from the 5’ end of the nuclear exonic region of the RAG2 gene (region F1B to R1) using the primers and regions described by Baker et al.[]. All PCR and sequencing primers are given in Additional file and references therein. We edited COI sequences using SeqScape v.2.1.1 (Applied Biosystems) and Dby 7th intron and the RAG2 region sequences in Sequencher v.4.5 (Gene Codes), and manually aligned all sequences in BioEdit v.7.0.9 (Ibis BioSciences). [...] We constructed a 95% confidence limit haplotype network of all COI sequences using statistical parsimony in TCS v.1.13 []. Following Clare [], in all further analyses we define putative cryptic species or “groups” based on the presence of independent (unconnected) COI networks at the 95% confidence level.We selected appropriate models of sequence evolution in MODELTEST [] executed in Phylemon v.2.0 [] using the Akaike information criterion (AIC) for each set of sequences (COI, Dby 7th intron and RAG2 region). We reduced all datasets to unique haplotypes for phylogenetic reconstruction. For COI sequences and a combined COI + Dby 7th intron + RAG2 sequence dataset we constructed a maximum likelihood phylogeny using PhyML v.3.0 [] as implemented by the ATGC Montpellier Bioinformatics Platform ( using the best fit model of sequence evolution. Branch support was calculated using the non-parametric Shimodaira-Hasegawa-like (SH-like) approximate likelihood ratio test (aLRT) []. For the same datasets we constructed a Bayesian phylogeny partitioned by gene and codon position using alternate models of sequence evolution in MrBayes v.3.1.2 []. The analyses were performed for 1,000,000 generations for every 10 specimens in the analysis (e.g. for 70 specimens + 3 outgroups in the concatenated tree, runs ran for 7.3 million generations to reach stationarity) and sampled every 50 generations with a burnin of 10,000 generations using multiple outgroups (Noctilio albiventris and Saccopteryx bilineata from South America and Rousettus aegyptiacus from the Old World). Convergence and stationarity were compared between multiple runs. We also evaluated species trees against this concatenated gene tree with the same parameters using the *BEAST option in BEAST v.1.7.4 [] including input files generated in BEAUTi v.1.7.4 []. [...] We estimated divergence times among genetic groups with RAG2 and COI sequences using Bayesian MCMC, executed in BEAST (as above). Since no estimates of COI mutation rate are available for bats, we did not use a fixed substitution rate but estimated minimum and maximum divergence times using two substitution models, 2% and 5% per million years, based on estimates for cyt b in small-bodied mammals []. Similar estimates of 2.6% for phyllostomid bats [], 2.3–5% in Carollia[], and 4% from fossil calibrations [] have been suggested. We used a fixed clock of 0.194% per million years for the RAG2 sequences []. The nucleotide substitution model was the same as that used for phylogenetic analysis. Branching structure of the groups was fixed based on the topology supported by the multi-gene reconstruction. The chain ran for 10,000,000 generations using the 28 unique haplotypes + outgroups with a burnin of 10,000. We estimated the mean and 95% confidence interval (CI) of the divergence times using Tracer v.1.4.1 [] and summarized the trees using TreeAnnotator v.1.4.8 []. We visualized the trees with FigTree v.1.2.1 (A. Rambaut 2009 […]

Pipeline specifications

Software tools SeqScape, Sequencher, BioEdit, ModelTest-NG, Phylemon, PhyML, MrBayes, BEAST, FigTree
Databases ATGC
Applications Phylogenetics, Sanger sequencing
Organisms Pteronotus parnellii