DOI: 10.5061/dryad.s630c
title: Data from: Nuclear introns outperform mitochondrial DNA in inter-specific phylogenetic reconstruction: Lessons from horseshoe bats (Rhinolophidae: Chiroptera)

description: Despite many studies illustrating the perils of utilising mitochondrial
 DNA in phylogenetic studies, it remains one of the most widely used
 genetic markers for this purpose. Over the last decade, nuclear introns
 have been proposed as alternative markers for phylogenetic reconstruction.
 However, the resolution capabilities of mtDNA and nuclear introns have
 rarely been quantified and compared. In the current study we generated a
 novel ∼5 kb dataset comprising six nuclear introns and a mtDNA fragment.
 We assessed the relative resolution capabilities of the six intronic
 fragments with respect to each other, when used in various combinations
 together, and when compared to the traditionally used mtDNA. We focused on
 a major clade in the horseshoe bat family (Afro-Palaearctic clade;
 Rhinolophidae) as our case study. This old, widely distributed and
 speciose group contains a high level of conserved morphology. This
 morphological stasis renders the reconstruction of the phylogeny of this
 group with traditional morphological characters complex. We sampled
 multiple individuals per species to represent their geographic
 distributions as best as possible (122 individuals, 24 species, 68
 localities). We reconstructed the species phylogeny using several
 complementary methods (partitioned Maximum Likelihood and Bayesian and
 Bayesian multispecies-coalescent) and made inferences based on consensus
 across these methods. We computed pairwise comparisons based on
 Robinson–Foulds tree distance metric between all Bayesian topologies
 generated (27,000) for every gene(s) and visualised the tree space using
 multidimensional scaling (MDS) plots. Using our supported species
 phylogeny we estimated the ancestral state of key traits of interest
 within this group, e.g. echolocation peak frequency which has been
 implicated in speciation. Our results revealed many potential cryptic
 species within this group, even in taxa where this was not suspected a
 priori and also found evidence for mtDNA introgression. We demonstrated
 that by using just two introns one can recover a better supported species
 tree than when using the mtDNA alone, despite the shorter overall length
 of the combined introns. Additionally, when combining any single intron
 with mtDNA, we showed that the result is highly similar to the mtDNA gene
 tree and far from the true species tree and therefore this approach should
 be avoided. We caution against the indiscriminate use of mtDNA in
 phylogenetic studies and advocate for pilot studies to select nuclear
 introns. The selection of marker type and number is a crucial step that is
 best based on critical examination of preliminary or previously published
 data. Based on our findings and previous publications, we recommend the
 following markers to recover phylogenetic relationships between recently
 diverged taxa (&lt;20 My) in bats and other mammals: ACOX2, COPS7A,
 BGN, ROGDI and STAT5A.
description: Fasta alignment files
 for all genetic loci usedFASTA alignment files of
 the sequences for each molecular marker used in the current study. The
 number of individuals is indicated in the file name (e.g. N=122).
 Information concerning choice of loci, primers used and alignment
 optimisation is described in section 2.2 of the paper (2.2 DNA extraction
 & PCR), in Table S2 and section 2.3 Sequencing & alignments. The
 alignment files provided (and used in the paper) are the optimised
 alignments (i.e. sites in the alignment which consisted of one or a few
 individuals having a 1bp insertion were typically highlighted a bad
 following T-COFFEE analysis and were removed). The original sequences are
 available on GenBank (see Table S1).1_Fasta_alignment_files.zipMaximum likelihood tree filesMaximum likelihood tree files generated using RAXML for each nuclear intron and mtDNA and 6-nuclear intron partition. Please see the ReadME file for the full data package for further information.3_ML_tree_files.zipPAML MCMCTREE dated phylogenyPAML MCMCTREE dated phylogeny based on the Bayesian 6-nuclear topology generated in BEAST. Please see the ReadME file for the full data package for further information.5_PAML.zipBayesian tree files - Gene TreesTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information1_Individual_gene_LogC_TAn_files.zipBayesian tree files - 6-intron partitioned treeTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information2_6NucIntronTopology_LogC_TAn_files.zipBayesian tree files - 5-intron partitioned topologiesTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information3_5NucTopologies_LogC_TAn_files.zipBayesian tree files - Combinations-of-two-introns topologiesTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information4_Intron_combinations_LogC_TAn_files.zipBayesian tree files - Combinations-of-mtDNA+each intron in turnTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information5_mtDNA_with_each_intron_LogC_TAn_files.zipBayesian tree files - Subsets of the mtDNA locus topologiesTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information6_parts_of_mtDNA_LogC_TAn_files.zipBayesian tree files - 6-intron+mtDNA partitioned topologyTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information7_6NucIntronPlusMtDNATopology_LogC_TAn_files.zipBayesian tree files - Rhinolophus capensis clade, mtDNA topologyTree files for the Bayesian analysis (NEXUS format). All Bayesian analysis were conducted in BEAST v. 1.8. This is described in section 2.4 of the paper (2.4 Phylogenetic reconstruction). The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.4. The LogCombiner files contain all trees generated from 3 independent runs with burn-in removed for each analysis (i.e. 27,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 27,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files. ‘P’ in the file name simply stands for ‘partitioned’; however all phylogenetic analyses with more than one locus were partitioned whether this is stated in the file name or not. See Readme file section 3 for more information8_CapensisCladeOnly_mtDNA_LogC_TAn_files.zipTree files for the Bayesian multi-species coalescent (*BEAST; NEXUS format).Tree files for the Bayesian multi-species coalescent (*BEAST; NEXUS format). This analysis is described in section 2.5 of the paper, Species delimitation & divergence time estimation, and was conducted in BEAST v. 1.8. The files were generated using the software associated with BEAST: LogCombiner and TreeAnnotator as described in section 2.5. The LogCombiner files contain all trees generated from 5 independent runs with burn-in removed for each analysis (i.e. 180,000 trees); here called ‘LogC’ files. The TreeAnnotator files contain a single consensus topology generated from these 180,000 trees (a maximum clade credibility tree, keeping target node heights); here called ‘TAn’ files.4_StarBEAST.zip