Next-generation phylogeography of the banded newts (Ommatotriton): A phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure

Isolde Van Riemsdijk,Jan W Arntzen,Wiesław Babik,Sergé Bogaerts,Michael Franzen,Konstantinos Kalaentzis,Spartak N Litvinchuk,Kurtuluş Olgun,Jan Willem P.M Wijnands,Ben Wielstra

doi:10.1016/j.ympev.2021.107361

Abstract

Technological developments now make it possible to employ many markers for many individuals in a phylogeographic setting, even for taxa with large and complex genomes such as salamanders. The banded newt (genus Ommatotriton) from the Near East has been proposed to contain three species (O. nesterovi, O. ophryticus and O. vittatus) with unclear phylogenetic relationships, apparently limited interspecific gene flow and deep intraspecific geographic mtDNA structure. We use parallel tagged amplicon sequencing to obtain 177 nuclear DNA markers for 35 banded newts sampled throughout the range. We determine population structure (with Bayesian clustering and principal component analysis), interspecific gene flow (by determining the distribution of species-diagnostic alleles) and phylogenetic relationships (by maximum likelihood inference of concatenated sequence data and based on a summary-coalescent approach). We confirm that the three proposed species are genetically distinct. A sister relationship between O. nesterovi and O. ophryticus is suggested. We find evidence for introgression between O. nesterovi and O. ophryticus, but this is geographically limited. Intraspecific structuring is extensive, with the only recognized banded newt subspecies, O. vittatus cilicensis, representing the most distinct lineage below the species level. While mtDNA mostly mirrors the pattern observed in nuclear DNA, all banded newt species show mito-nuclear discordance as well.

Highlights

The vast size and complexity of the genomes of certain taxa such as amphibians hampers whole genome sequencing (Calboli et al, 2011; Gregory, 2019; Mohlhenrich and Mueller, 2016)
Sequence data are ob tained via multiplex PCR and Ion Torrent next-generation sequencing for 35 individuals, from the same number of localities, that span the complete banded newt distribution range (Fig. 1). With this dataset we address four questions: 1) Is there support for three banded newt species? 2) What are the phylogenetic relationships among banded newt species? 3) Is there geographically extensive, asymmetric introgression be tween banded newt species? 4) Is deep geographical mtDNA substructuring in banded newts mirrored in the nuclear genome?
Structure under k = 3, the optimal k value based on the Δk criterion, separates the three banded newt species (Fig. 1, Q scores in Appendix 1)

Summary

Introduction

The vast size and complexity of the genomes of certain taxa such as amphibians hampers whole genome sequencing (Calboli et al, 2011; Gregory, 2019; Mohlhenrich and Mueller, 2016). A popular method to obtain a large number of loci randomly distributed throughout the genome is restriction-site associated DNA (RAD) sequencing (Baird et al, 2008). A downside of this technique is that the number of shared restriction sites decreases relatively rapidly with increasing genetic divergence, mean ing that the number of homologous markers obtained across increas ingly divergent species quickly drops off, therewith representing a considerable limitation of RAD-sequencing data for phylogenetic infer ence (Eaton et al, 2017; Lemmon and Lemmon, 2013). Transcriptome sequencing ensures that a large number of markers with a known function are obtained (Ekblom and Galindo, 2011; Everett et al, 2011). If the aim is to obtain dozens to hundreds of homologous markers, primer-based amplifica tion by multiplex PCR is an efficient and economic approach (Wielstra et al, 2014; Zielinski et al, 2014)

Methods

Discussion

Conclusion