Molecular Phylogenetics And Biogeography Of Living Anurans

Anurans are one of the most diverse groups of vertebrates and comprise 88% of living amphibian species.Their world distribution and diverse biology,such as habitat,life history and Morphology,make them well-suited for assessing fundamental questions in evolution,ecology and conservation.Yet,despite their scientific importance,the evolutionary history and tempo diversification of anurans remain poorly understand.The main reason is insufficient phylogenetic signals resulting from limited available molecular markers.Therefore,it is still challenging to resolve anurans’ evolution history.Shen et al.recently developed a vertebrate toolkit including 102 nuclear markers,which may be used to resolve anuran evolution.However,these markers are amplified by PCR.It means that tens of thousands of PCR products need to be sequenced,if we use these markers.It will be very expensive if sequencing these PCR products using traditional Sanger method.Hence,another challenge is exploring a cost effective approach to sequence these PCR products.To deal with these two challenges,here we start in two sides.The main contents and findings are as follows.(1)Development of method for sequencing large number of PCR productsIn phylogenetics,multiple loci are often used to resolve species relationships.Normally,these loci are enriched by PCR and sequenced via Sanger sequencing method,which is expensive when the number of amplicons is large.In the past ten years,Next generation sequencing(NGS)technologies have revolutionized sequences collection in biology.Tremendous progress has been made in reducing sequencing cost.Researchers tried to develop NGS methods for parallel amplicon sequencing.For most current NGS methods,amplicons need to be purified and quantified before pooled together and their lengths are also restricted(normally <700 bp).More over,these methods mainly depend on NGS platforms with longer read length,smaller amount of output data.These requirement limited application of these methods.From a point,these PCR products can be approximated as transcripts.In transcriptome sequencing projects,these transcripts are not processed with different treatments,but are sequenced in a mixture.Here,basing on transcriptme sequencing strategies,we describe an approach to parallelly sequence amplicons of any length using the Illumina HiSeq 2000 platform which is more cost-effective and more widely used.Using our method,PCR products of a sample are pooled at equal volume rather than at equal concentration,thus eliminating the laborious steps for purifying and quantifying every PCR product.These pooled PCR products are then sheared into smaller fragments and linked with sample specific barcode linkers.Finally,these fragments of all samples are pooled together prior to Illumina sequencing library preparation.Sequencing data is sorted into relative files baed on barcode linkers and then assembled using the transcriptome assembly program TRINITY.Final sequences were recovered after BLAST and following filters.We demonstrated the utility of our approach by recovering 93.5% of the target amplicons(size up to 1650 bp)in full length for a 16 taxa 101 loci project,using ~2.0 GB of Illumina HiSeq paired-end 90-bp data.Overall,we validate a rapid,costeffective and scalable approach to sequence a large number of targeted loci from many samples.Our method is particularly suitable for both phylogenetics and population genetics studies that require a modest scale of data.(2)Phylogenetic and biogeographic analysis of anuransAnurans are the most diverse groups of amphiba(88% of living amphibia),comprise 6741 living species,446 genera and 54 families.The evolutionary history of anuans has attracted considerable attention of evolutionary biologists.However,with limited molecular data,previous studies yeild conflicted results.To resolve anuran evoluton history,here we produced a novel and so far largest molecular dataset using above method.This dataset consists of 95 nuclear genes in 156 frog species and 8 outgroup species.Total alignment length is 88302 bp,wichi is at least 7 larger than previous studies.Using Maximum likelihood(ML),Bayes Inference(BI)and ASTRAL species tree methods,a strongly supported phylogeny was constructed.All the analyses are concordant for most nodes and clades.This phylogeny resolved conflict in anuran tree,such as Heleophrynidae as the basal position of Neobatrachia,Sooglossidae as the sister lineage of Ranoidea.In conjunction with 20 fossil-based calibrations,our time analysis provides a novel timescale of frog evolution,suggesting more recent divergence times among major neobatrachian lineages.The last common ancestor of crown-group Anura had lived during the Upper Triassic at 210.0 Mya,and the age of crown Neobatrachia is in the Lower Cretaceous at 142.1 Mya.Unexpectedly,our divergence-time analyses show that three species-rich clades(Hyloidea,Microhylidae,and Natatanura),which together comprise ~87% of extant anuran species,simultaneously underwent rapid diversification at the Cretaceous-Paleogene(K-Pg)boundary.These three clades was preceded by a long branch(> 32 My)before K-Pg and followedd by very short brach(< 4.6 My),indicating three extinctions followed by rapid divergence.This suggests that the K-Pg mass extinction may have triggered explosive radiations of anurans through creating new ecological opportunities.Biogeographical analyses show that the ancestral area of modern frogs is Africa and their current distribution is largely associated with the breakup of Pangaea and subsequent Gondwanan fragmentation.