Phylogeographic inference using Bayesian model comparison across a fragmented chorus frog species complex

Fragmented species complexes provide an interesting system for investigating biogeographic history and the present distribution of genetic variation. Recent advances in sequencing technology and statistical phylogeography enable the collection and rigorous analysis of large multilocus data sets, but designing studies that produce meaningful phylogeographic inferences remains challenging. We implemented a Bayesian model comparison approach to investigate previous biogeographic hypotheses while simultaneously inferring the presence of genetic structure in a chorus frog species complex. The Illinois chorus frog (Pseudacris illinoensis), originally described as a subspecies of the broadly distributed Strecker's chorus frog (Pseudacris streckeri), occurs in small, disjunct regions associated with scarce sand prairie habitats that have been impacted by human development. We used high‐throughput sequencing to develop and collect a multitiered genetic data set comprised of three different marker types (23 anonymous nuclear sequence loci, four mitochondrial genes and 14 microsatellite loci) designed to address questions across different evolutionary timescales. Phylogenetic analyses uncovered a deep divergence between populations in the Edwards Plateau of central Texas and all other P. streckeri/P. illinoensis populations, but suggest the disjunct distribution of P. illinoensis occurred more recently. Our best‐supported migration model is consistent with the hypothesis that central Texas represented a refugium from which populations expanded via multiple routes. This model also indicates that disjunct northern and southern regions of P. illinoensis should be considered genetically distinct management units. Our study provides an evolutionary context for future studies and conservation efforts in P. illinoensis and demonstrates the utility of model‐based approaches for phylogeographic inference.     

Applications of next-generation sequencing to phylogeography and phylogenetics

This is a time of unprecedented transition in DNA sequencing technologies. Next-generation sequencing (NGS) clearly holds promise for fast and cost-effective generation of multilocus sequence data for phylogeography and phylogenetics. However, the focus on non-model organisms, in addition to uncertainty about which sample preparation methods and analyses are appropriate for different research questions and evolutionary timescales, have contributed to a lag in the application of NGS to these fields. Here, we outline some of the major obstacles specific to the application of NGS to phylogeography and phylogenetics, including the focus on non-model organisms, the necessity of obtaining orthologous loci in a cost-effective manner, and the predominate use of gene trees in these fields. We describe the most promising methods of sample preparation that address these challenges. Methods that reduce the genome by restriction digest and manual size selection are most appropriate for studies at the intraspecific level, whereas methods that target specific genomic regions (i.e., target enrichment or sequence capture) have wider applicability from the population level to deep-level phylogenomics. Additionally, we give an overview of how to analyze NGS data to arrive at data sets applicable to the standard toolkit of phylogeography and phylogenetics, including initial data processing to alignment and genotype calling (both SNPs and loci involving many SNPs). Even though whole-genome sequencing is likely to become affordable rather soon, because phylogeography and phylogenetics rely on analysis of hundreds of individuals in many cases, methods that reduce the genome to a subset of loci should remain more cost-effective for some time to come.     

Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata)

We describe the cloning and characterization of eight novel tetranucleotide microsatellite loci in the ornate chorus frog (Pseudacris ornata). We also screened 26 loci from GenBank that were isolated from other Pseudacris species and obtained consistent product from five of these dinucleotide loci. All loci are polymorphic. In our sample of 26 frogs from a natural population, polymorphism ranged from 1 to 22 alleles per locus with expected heterozygosities ranging from 0 to 0.958. These loci enable high-resolution studies of P. ornata. Moreover, cross-species amplification success suggests they will also be useful for other chorus frog species.     

Phylogenetics of modern birds in the era of genomics

In the 14 years since the first higher-level bird phylogenies based on DNA sequence data, avian phylogenetics has witnessed the advent and maturation of the genomics era, the completion of the chicken genome and a suite of technologies that promise to add considerably to the agenda of avian phylogenetics. In this review, we summarize current approaches and data characteristics of recent higher-level bird studies and suggest a number of as yet untested molecular and analytical approaches for the unfolding tree of life for birds. A variety of comparative genomics strategies, including adoption of objective quality scores for sequence data, analysis of contiguous DNA sequences provided by large-insert genomic libraries, and the systematic use of retroposon insertions and other rare genomic changes all promise an integrated phylogenetics that is solidly grounded in genome evolution. The avian genome is an excellent testing ground for such approaches because of the more balanced representation of single-copy and repetitive DNA regions than in mammals. Although comparative genomics has a number of obvious uses in avian phylogenetics, its application to large numbers of taxa poses a number of methodological and infrastructural challenges, and can be greatly facilitated by a 'community genomics' approach in which the modest sequencing throughputs of single PI laboratories are pooled to produce larger, complementary datasets. Although the polymerase chain reaction era of avian phylogenetics is far from complete, the comparative genomics era-with its ability to vastly increase the number and type of molecular characters and to provide a genomic context for these characters-will usher in a host of new perspectives and opportunities for integrating genome evolution and avian phylogenetics.     

The perils of single gene trees — mitochondrial versus single-copy nuclear DNA variation in white-eyes (Aves: Zosteropidae)

Phylogenetic relationships among animal populations and species commonly have been inferred from patterns of variation observed within a single gene system, most often the mitochondrial genome. Analysis of restriction site variation in the mitochondrial DNA of two species of white-eye ( Zosterops lateralis and Z. lutea ) in Australia produced a single gene tree that does not accurately represent the organismal tree. In contrast, patterns of variation at two anonymous, single-copy nuclear DNA loci revealed a phylogeography consistent with traditional classification of the species. Discordance between mitochondrial DNA and single-copy nuclear DNA variation is probably the result of past hybridization between Z. lateralis and Z. lutea , evidence of which has been lost from the nuclear genome by recombination. This study provides a clear empirical demonstration that single gene genealogies cannot be assumed to accurately represent the true phylogenies, and emphasizes the need for composite genetic analyses.     

A fair fight between molecular marker types in a seascape genetics setting

From its inception, population genetics has been nearly as concerned with the genetic data type—to which analyses are brought to bear—as it is with the analysis methods themselves. The field has traversed allozymes, microsatellites, segregating sites in multilocus alignments and, currently, single nucleotide polymorphisms (SNPs) generated by high‐throughput genomic sequencing methods, primarily whole genome sequencing and reduced representation library (RRL) sequencing. As each emerging data type has gained traction, it has been compared to existing methods, based on its relative ability to discern population structural complexity at increasing levels of resolution. However, this is usually done by comparing the gold standard in one data type to the gold standard in the new data type. These gold standards frequently differ in power and in sampling density, both across a genome and throughout a spatial range. In this issue of Molecular Ecology, D’Aloia et al. apply the high‐throughput approach as fully as possible to microsatellites, nuclear loci and SNPs genotyped through an RRL method; this is coupled with a spatially dense sampling scheme. Completing a battery of population genetics analyses across data types (including a series of down‐sampled data sets), the authors find that SNP data are slightly more sensitive to fine‐scale genetic structure, and the results are more resilient to down‐sampling than microsatellites and nonrepetitive nuclear loci. However, their results are far from an unqualified victory for RRL SNP data over all previous data types: the authors note that modest additions to the microsatellites and nuclear loci data sets may provide the necessary analytical power to delineate the fine‐scale genetic structuring identified by SNPs. As always, as the field begins to fully embrace the newest thing, good science reminds us that traditional data types are far from useless, especially when combined with a well‐designed sampling scheme.     

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics

? Premise of the study: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. ? Methods: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). ? Key results: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. ? Conclusions: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.     

Nuclear gene genealogies reveal historical,demographic and selective factors associated with speciation in field crickets

Population genetics theory predicts that genetic drift should eliminate shared polymorphism, leading to monophyly or exclusivity of populations, when the elapsed time between lineage-splitting events is large relative to effective population size. We examined patterns of nucleotide variation in introns at four nuclear loci to relate processes affecting the history of genes to patterns of divergence among natural populations and species. Ancestral polymorphisms were shared among three recognized species, Gryllus firmus, G. pennsylvanicus, and G. ovisopis, and genealogical patterns suggest that successive speciation events occurred recently and rapidly relative to effective population size. High levels of shared polymorphism among these morphologically, behaviorally, and ecologically distinct species indicate that only a small fraction of the genome needs to become differentiated for speciation to occur. Among the four nuclear gene loci there was a 10-fold range in nucleotide diversity, and patterns of polymorphism and divergence suggest that natural selection has acted to maintain or eliminate variation at some loci. While nuclear gene genealogies may have limited applications in phylogeography or other approaches dependent on population monophyly, they provide important insights into the historical, demographic, and selective forces that shape speciation.     

Rapid,economical single‐nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non‐model organism: a case study of Atlantic cod Gadus morhua

By combining next‐generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single‐nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non‐model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small‐scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small‐scale next‐generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions.     

Next‐generation phylogenetics takes root

It has been a tumultuous 5 years in phylogeography and phylogenetics during which both fields have struggled to harness the power of next‐generation sequencing (NGS) (Ekblom & Galindo 2010 ; McCormack et al. 2012a ). Fortunately, several methodological approaches appear to be taking root. In this issue of Molecular Ecology, O'Neill et al. 2013 ) employ one such method – parallel tagged sequencing (PTS) – to elucidate the phylogeography of a tiger salamander (Ambystoma tigrinum) species complex. This study demonstrates a practical application of NGS on a scale appropriate (and not overkill) for most biologists interested in phylogeography (~100 loci for ~100 individuals), and their results highlight several analytical challenges that lie ahead for researchers employing NGS techniques.     

Combined next‐generation sequencing and morphology reveal fine‐scale speciation in Crocodile Skinks (Squamata: Scincidae: Tribolonotus)

Next‐generation sequencing has vast potential to revolutionize the fields of phylogenetics and population genetics through its ability to collect genomic scale data sets of thousands of orthologous loci. Despite this potential, other types of data (e.g. morphology, ecology) remain important, particularly for studies endeavouring to delimit species. Here, we integrate next‐generation sequencing with morphology to examine divergence between populations of Tribolonotus pseudoponceleti on the islands of Buka and Bougainville in the Solomon Archipelago. We used the Ion Torrent PGM to collect over 648 Mbp of sequence data for 12 samples, representing 1526 loci recovered from all samples, and 3342 were recovered from at least six samples. Genetic structure analyses strongly support the distinctiveness of these two populations, and Bayes factor delimitations decisively select speciation between Buka and Bougainville. Principal components and discriminant function analyses reveal concordant morphological divergence. Finally, demographic analyses via diffusion approximation and approximate Bayesian computation prefer a complex model of mid‐Pleistocene divergence with migration, and a later decrease or cessation of migration and population size shift, suggesting a scenario in which migration was enabled by Pleistocene merging of these two islands, and limited when isolated by higher sea levels. Further analysis of four Sanger sequenced loci in IMa2 had limited power to distinguish among models including and excluding migration, but resulted in similar population size and divergence time estimates, although with much broader confidence intervals. This study represents a framework for how next‐generation sequencing and morphological data can be combined and leveraged towards validating putative species and testing demographic scenarios for speciation.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

When vicars meet: a narrow contact zone between morphologically cryptic phylogeographic lineages of the rainforest skink, Carlia rubrigularis

Phylogeographic analyses of the fauna of the Australian wet tropics rainforest have provided strong evidence for long-term isolation of populations among allopatric refugia, yet typically there is no corresponding divergence in morphology. This system provides an opportunity to examine the consequences of geographic isolation, independent of morphological divergence, and thus to assess the broader significance of historical subdivisions revealed through mitochondrial DNA phylogeography. We have located and characterized a zone of secondary contact between two long isolated (mtDNA divergence > 15%) lineages of the skink Carlia rubrigularis using one mitochondrial and eight nuclear (two intron, six microsatellite) markers. This revealed a remarkably narrow (width < 3 km) hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes at two of three nuclear loci with diagnostic alleles. Cline centers were coincident across loci. Using a novel form of likelihood analysis, we were unable to distinguish between sigmoidal and stepped cline shapes except at one nuclear locus for which the latter was inferred. Given estimated dispersal rates of 90-133 m x gen(-1/2) and assuming equilibrium, the observed cline widths suggest effective selection against heterozygotes of at least 22-49% and possibly as high as 70%. These observations reveal substantial postmating isolation, although the absence of consistent deviations from Hardy-Weinberg equilibrium at diagnostic loci suggests that there is little accompanying premating isolation. The tight geographic correspondence between transitions in mtDNA and those for nuclear genes and corresponding evidence for selection against hybrids indicates that these morphologically cryptic phylogroups could be considered as incipient species. Nonetheless, we caution against the use of mtDNA phylogeography as a sole criterion for defining species boundaries.     

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis.     

Geological and climatic forces driving speciation in the continentally distributed trilling chorus frogs (Pseudacris)

Tertiary geological events and Quaternary climatic fluctuations have been proposed as important factors of speciation in the North American flora and fauna. Few studies, however, have rigorously tested hypotheses regarding the specific factors driving divergence of taxa. Here, we test explicit speciation hypotheses by correlating geologic events with divergence times among species in the continentally distributed trilling chorus frogs (Pseudacris). In particular, we ask whether marine inundation of the Mississippi Embayment, uplift of the Appalachian Mountains, or modification of the ancient Teays-Mahomet River system contributed to speciation. To examine the plausibility of ancient rivers causing divergence, we tested whether modern river systems inhibit gene flow. Additionally, we compared the effects of Quaternary climatic factors (glaciation and aridification) on levels of genetic variation. Divergence time estimates using penalized likelihood and coalescent approaches indicate that the major lineages of chorus frogs diversified during the Tertiary, and also exclude Quaternary climate change as a factor in speciation of chorus frogs. We show the first evidence that inundation of the Mississippi Embayment contributed to speciation. We reject the hypotheses that Cenozoic uplift of the Appalachians and that diversion of the Teays-Mahomet River contributed to speciation in this clade. We find that by reducing gene flow, rivers have the potential to cause divergence of lineages. Finally, we demonstrate that populations in areas affected by Quaternary glaciation and aridification have reduced levels of genetic variation compared to those from more equable regions, suggesting recent colonization.     

Extreme mtDNA divergences in a terrestrial slug (Gastropoda, Pulmonata, Arionidae): accelerated evolution, allopatric divergence and secondary contact

Extremely high levels of intraspecific mtDNA differences in pulmonate gastropods have been reported repeatedly and several hypotheses to explain them have been postulated. We studied the phylogeny and phylogeography of 51 populations (n = 843) of the highly polymorphic terrestrial slug Arion subfuscus (Draparnaud, 1805) across its native distribution range in Western Europe. By combining the analysis of single stranded conformation polymorphisms (SSCP) and nucleotide sequencing, we obtained individual sequence data for a fragment of the mitochondrial 16S rDNA and a fragment of the nuclear ITS1. Additionally, five polymorphic allozyme loci were scored. Based on the 16S rDNA phylogeny, five monophyletic haplotype groups with sequence divergences of 9-21% were found. Despite this deep mitochondrial divergence, the haplotype groups were not monophyletic for the nuclear ITS1 fragment and haplotype group-specific allozyme alleles were not found. Although there is evidence for an accelerated mtDNA clock, the divergence among the haplotype groups is older than the Pleistocene and their current allopatric ranges probably reflect allopatric divergence and glacial survival in separate refugia from which different post-glacial colonization routes were established. A range-overlap of two mtDNA groups (S1 and S2, 21% sequence divergence) stretched from Central France and Belgium up to the North of the British Isles. The nuclear data suggest that this secondary contact resulted in hybridization between the allopatrically diverged groups. Therefore, it seems that, at least for two of the groups, the deep mtDNA divergence was only partially accompanied by the formation of reproductive isolation.     

Characterizing restriction enzyme‐associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom‐designed RNA probes

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom‐designed RNA probes could enrich for RRL loci (Restriction Enzyme‐Associated Loci baits, or REALbaits). Starting with genotyping‐by‐sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well‐characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19‐ to 151‐fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.     

Multilocus phylogeography and phylogenetics using sequence-based markers

We review recent trends in phylogeography and phylogenetics and argue that these two fields stand to be reunited by the common yardstick provided by sequence and SNP data and by new multilocus methods for phylogenetic analysis. Whereas the modern incarnation of both fields was spawned by PCR approaches applied to mitochondrial DNA in the late 1980s, the two fields diverged during the 1990s largely due to the adoption by phylogeographers of microsatellites, in contrast to the adoption of nuclear sequence data by phylogeneticists. Sequence-based markers possess a number of advantages over microsatellites, even on the recent time scales that are the purview of phylogeography. Using examples primarily from vertebrates, we trace the maturation of nuclear gene phylogeography and phylogenetics and suggest that the abundant instances of gene tree heterogeneity beckon a new generation of phylogenetic methods that focus on estimating species trees as distinct from gene trees. Whole genomes provide a powerful common yardstick on which both phylogeography and phylogenetics can assume their proper place as ends of a continuum.     

Consistent geographic structure among multiple nuclear sequences and cpDNA polymorphisms of Cardamine nipponica Franch. et Savat. (Brassicaceae)

Molecular phylogeography has inferred the history of differentiation between regions and/or among populations following the Pleistocene climatic oscillations, mostly based on the genetic structure of organelle DNA. However, such genetic structure only reflects the history of a single gene, and studies based on single-copy genes of nuclear DNA (nDNA) are required for phylogeography, although their efficiency remains unclear. To examine the utility of nDNA loci, the genetic structures of three genes from Cardamine nipponica, which is closely related to the model species Arabidopsis thaliana, were elucidated: the nDNA genes DET1, PHYA, PHYE, as well as chloroplast DNA (cpDNA). In 279 individuals collected from throughout the range of the species, strong genetic differentiation between northern and central Japan was found for all loci. This result suggested that populations in central Japan experienced a different history from those in northern Japan during the Pleistocene climatic oscillations. In addition, the evidence of refugia at the edges of the distribution, where the genetic structure was less influenced by colonization following range expansion, was shown for several loci. The specific genetic structure within the southernmost populations of northern Japan suggested that this region was also isolated during range expansion. Hence, the consistent history among loci and a more detailed history from several loci indicated that cpDNA can represent the history of vicariance and demonstrated the efficiency of single-copy nuclear genes in phylogeography.