Abstract
The study of processes driving diversification requires a fully sampled and well resolved phylogeny, although a lack of phylogenetic markers remains a limitation for many non-model groups. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (to the Canaries, respectively Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining genomics, coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex.
Highlights
Any inference about the ecological and evolutionary processes driving diversification requires a well sampled and fully resolved phylogeny upon which traits can be mapped.Molecular phylogenetic studies historically have been limited to a small number of loci
We detected 918 putative orthologous gene sequences for C. dipterum from the contigs derived from the shotgun library, 1,298 putative orthologous gene sequences from the contigs of the combined assembly, and 416 for Baetis sp. (Supplementary Table 6)
Based on preliminary laboratory testing, 59 markers were selected that amplified consistently and had similar annealing temperatures in order to simplify the large number of PCR reactions (Supplementary Table 3)
Summary
Any inference about the ecological and evolutionary processes driving diversification requires a well sampled and fully resolved phylogeny upon which traits can be mapped.Molecular phylogenetic studies historically have been limited to a small number of loci. The majority of studies are based largely on mitochondrial DNA (mtDNA) loci (Avise et al., 2000; Garrick et al, 2015) which have the benefit of small population size and high levels of polymorphism but suffer from several characteristics that can limit their suitability to reconstruct the evolutionary process These include an inability to detect processes that confound gene trees and species trees such as hybridization and introgression, the inference of oversimplified or unresolved evolutionary relationships based on their matrilineal history, underestimated genetic diversity (Zhang and Hewitt 2003), and overestimation of divergence times (Zheng et al, 2011). Phylogenetics has begun to benefit from more widespread use of single-copy nuclear DNA (nDNA) loci, and several recent studies have applied greater numbers of nDNA loci with success at the species (e.g. Ambystoma tigrinum (O'Neill et al, 2013); Triturus cristatus (Wielstra et al, 2014)), genus (e.g. Takydromus (Tseng et al, 2014); Heliconius (Kozak et al, 2015)), and higher taxonomic levels
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