Species delimitation with abc and other coalescent-based methods: a test of accuracy with simulations and an empirical example with lizards of the liolaemus darwinii complex (squamata: liolaemidae)

Species delimitation is a major research focus in evolutionary biology because accurate species boundaries are a prerequisite for the study of speciation. New species delimitation methods (SDMs) can accommodate nonmonophyletic species and gene tree discordance as a result of incomplete lineage sorting via the coalescent model, but do not explicitly accommodate gene flow after divergence. Approximate Bayesian computation (ABC) can incorporate gene flow and estimate other relevant parameters of the speciation process while testing alternative species delimitation hypotheses. We evaluated the accuracy of BPP, SpeDeSTEM, and ABC for delimiting species using simulated data and applied these methods to empirical data from lizards of the Liolaemus darwinii complex. Overall, BPP was the most accurate, ABC showed an intermediate accuracy, and SpeDeSTEM was the least accurate under most simulated conditions. All three SDMs showed lower accuracy when speciation occurred despite gene flow, as found in previous studies, but ABC was the method with the smallest decrease in accuracy. All three SDMs consistently supported the distinctness of southern and northern lineages within L. darwinii. These SDMs based on genetic data should be complemented with novel SDMs based on morphological and ecological data to achieve truly integrative and statistically robust approaches to species discovery.     

QUANTITATIVE GENETICS OF BRYOZOAN PHENOTYPIC EVOLUTION. I. RATE TESTS FOR RANDOM CHANGE VERSUS SELECTION IN DIFFERENTIATION OF LIVING SPECIES

The possible roles of random genetic change and natural selection in bryozoan speciation were analyzed using quantitative genetic methods on breeding data for traits of skeletal morphology in two closely related species of the cheilostome Stylopoma. The hypothesis that morphologic differences between the species are caused entirely by mutation and genetic drift could not be rejected for reasonable rates of mutation maintained for as few as 10³ to 10⁴ generations. Divergence times this short or shorter are consistent with the abrupt appearances of many invertebrate species in the fossil record, commonly followed by millions of years of morphologic stasis. To produce these differences over 10³ generations or fewer, directional selection acting alone would require unrealistically high levels of minimum selective mortality throughout divergence. Thus, selection is unnecessary to explain the divergence of these species, except as a means of accelerating the effects of random genetic change on shorter time scales (directional selection), or decelerating them over longer ones (stabilizing selection). These results are consistent with a variety of models of phenotypic evolution involving random shifts between multiple adaptive peaks. Similar results were obtained by substituting trait heritabilities and genetic covariances reconstructed by partitioning within- and among-colony phenotypic variance in place of the values based on breeding data. Quantitative genetic analysis of speciation in fossil bryozoan lineages is thus justified.     

Methods for delimiting species via population genetics and phylogenetics using genotype data

In species delimitation, a formidable goal in the discipline of systematic biology, we identify and describe species morphologically and ecologically based on phenotypic data. Efficient genotyping technologies produce genetic and genomic data with relative ease, which promotes species discovery and validation using genotype data. For the last two decades, we have seen the development of species delimitation methods based on genetic distances and phylogenetic trees using genotype data. However, speciation processes via evolutionary relationship among species were mostly divorced from species delimitation. Recent approaches to drawing species boundaries use multi-locus sequence data to account for evolutionary processes including speciation and gene flow. They allow us to learn of jointly speciation and species delimitation, leveraging computational and statistical techniques developed in population genetics and phylogenetics. Here, we review the recent progress in the development of species delimitation using genotype data and discuss the future outlook for the research of developing species delimitation methods.     

Species detection and individual assignment in species delimitation: can integrative data increase efficacy?

Statistical species delimitation usually relies on singular data, primarily genetic, for detecting putative species and individual assignment to putative species. Given the variety of speciation mechanisms, singular data may not adequately represent the genetic, morphological and ecological diversity relevant to species delimitation. We describe a methodological framework combining multivariate and clustering techniques that uses genetic, morphological and ecological data to detect and assign individuals to putative species. Our approach recovers a similar number of species recognized using traditional, qualitative taxonomic approaches that are not detected when using purely genetic methods. Furthermore, our approach detects groupings that traditional, qualitative taxonomic approaches do not. This empirical test suggests that our approach to detecting and assigning individuals to putative species could be useful in species delimitation despite varying levels of differentiation across genetic, phenotypic and ecological axes. This work highlights a critical, and often overlooked, aspect of the process of statistical species delimitation—species detection and individual assignment. Irrespective of the species delimitation approach used, all downstream processing relies on how individuals are initially assigned, and the practices and statistical issues surrounding individual assignment warrant careful consideration.     

Genomic data reveal deep genetic structure but no support for current taxonomic designation in a grasshopper species complex

Taxonomy has traditionally relied on morphological and ecological traits to interpret and classify biological diversity. Over the last decade, technological advances and conceptual developments in the field of molecular ecology and systematics have eased the generation of genomic data and changed the paradigm of biodiversity analysis. Here we illustrate how traditional taxonomy has led to species designations that are supported neither by high throughput sequencing data nor by the quantitative integration of genomic information with other sources of evidence. Specifically, we focus on Omocestus antigai and Omocestus navasi, two montane grasshoppers from the Pyrenean region that were originally described based on quantitative phenotypic differences and distinct habitat associations (alpine vs. Mediterranean‐montane habitats). To validate current taxonomic designations, test species boundaries, and understand the factors that have contributed to genetic divergence, we obtained phenotypic (geometric morphometrics) and genome‐wide SNP data (ddRADSeq) from populations covering the entire known distribution of the two taxa. Coalescent‐based phylogenetic reconstructions, integrative Bayesian model‐based species delimitation, and landscape genetic analyses revealed that populations assigned to the two taxa show a spatial distribution of genetic variation that do not match with current taxonomic designations and is incompatible with ecological/environmental speciation. Our results support little phenotypic variation among populations and a marked genetic structure that is mostly explained by geographic distances and limited population connectivity across the abrupt landscapes characterizing the study region. Overall, this study highlights the importance of integrative approaches to identify taxonomic units and elucidate the evolutionary history of species.     

Incorporating the speciation process into species delimitation

The “multispecies” coalescent (MSC) model that underlies many genomic species-delimitation approaches is problematic because it does not distinguish between genetic structure associated with species versus that of populations within species. Consequently, as both the genomic and spatial resolution of data increases, a proliferation of artifactual species results as within-species population lineages, detected due to restrictions in gene flow, are identified as distinct species. The toll of this extends beyond systematic studies, getting magnified across the many disciplines that rely upon an accurate framework of identified species. Here we present the first of a new class of approaches that addresses this issue by incorporating an extended speciation process for species delimitation. We model the formation of population lineages and their subsequent development into independent species as separate processes and provide for a way to incorporate current understanding of the species boundaries in the system through specification of species identities of a subset of population lineages. As a result, species boundaries and within-species lineages boundaries can be discriminated across the entire system, and species identities can be assigned to the remaining lineages of unknown affinities with quantified probabilities. In addition to the identification of species units in nature, the primary goal of species delimitation, the incorporation of a speciation model also allows us insights into the links between population and species-level processes. By explicitly accounting for restrictions in gene flow not only between, but also within, species, we also address the limits of genetic data for delimiting species. Specifically, while genetic data alone is not sufficient for accurate delimitation, when considered in conjunction with other information we are able to not only learn about species boundaries, but also about the tempo of the speciation process itself.     

Ecological niche dimensionality and the evolutionary diversification of stick insects

The degree of phenotypic divergence and reproductive isolation between taxon pairs can vary quantitatively, and often increases as evolutionary divergence proceeds through various stages, from polymorphism to population differentiation, ecotype and race formation, speciation, and post-speciational divergence. Although divergent natural selection promotes divergence, it does not always result in strong differentiation. For example, divergent selection can fail to complete speciation, and distinct species pairs sometimes collapse ('speciation in reverse'). Widely-discussed explanations for this variability concern genetic architecture, and the geographic arrangement of populations. A less-explored possibility is that the degree of phenotypic and reproductive divergence between taxon pairs is positively related to the number of ecological niche dimensions (i.e., traits) subject to divergent selection. Some data supporting this idea stem from laboratory experimental evolution studies using Drosophila, but tests from nature are lacking. Here we report results from manipulative field experiments in natural populations of herbivorous Timema stick insects that are consistent with this 'niche dimensionality' hypothesis. In such insects, divergent selection between host plants might occur for cryptic colouration (camouflage to evade visual predation), physiology (to detoxify plant chemicals), or both of these niche dimensions. We show that divergent selection on the single niche dimension of cryptic colouration can result in ecotype formation and intermediate levels of phenotypic and reproductive divergence between populations feeding on different hosts. However, greater divergence between a species pair involved divergent selection on both niche dimensions. Although further replication of the trends reported here is required, the results suggest that dimensionality of selection may complement genetic and geographic explanations for the degree of diversification in nature.     

Discord reigns among nuclear, mitochondrial and phenotypic estimates of divergence in nine lineages of trans-Beringian birds

Proposals for genetic thresholds for species delimitation assume that simple genetic data sets (e.g. mitochondrial sequence data) are correlated with speciation; i.e. such data sets accurately reflect organismal lineage divergence. We used taxonomically stratified phenotypic levels of differentiation (populations, subspecies and species) among nine avian lineages using paired, trans-Beringian samples from three lineages each in three orders (Anseriformes, Charadriiformes, and Passeriformes) to test this assumption. Using mitochondrial DNA sequence data and nuclear genomic data (amplified fragment length polymorphisms), we found a lack of concordance between these two genomes in their respective estimates of divergence and little or no relationship between phenotype (taxonomic relatedness) and genetic differentiation between taxon pairs. There are several possible reasons for the discord observed (e.g. selection on one of the genomes or perhaps lineage sorting), but the implications are that genetic estimates of lineage divergence may not be correlated with estimates from other parts of the genome, are not well correlated with the speciation process and are thus not reliable indicators of species limits.     

Extraordinarily rapid life-history divergence between Cryptasterina sea star species

Life history plays a critical role in governing microevolutionary processes such as gene flow and adaptation, as well as macroevolutionary processes such speciation. Here, we use multilocus phylogeographic analyses to examine a speciation event involving spectacular life-history differences between sister species of sea stars. Cryptasterina hystera has evolved a suite of derived life-history traits (including internal self-fertilization and brood protection) that differ from its sister species Cryptasterina pentagona, a gonochoric broadcast spawner. We show that these species have only been reproductively isolated for approximately 6000 years (95% highest posterior density of 905-22 628), and that this life-history change may be responsible for dramatic genetic consequences, including low nucleotide diversity, zero heterozygosity and no gene flow. The rapid divergence of these species rules out some mechanisms of isolation such as adaptation to microhabitats in sympatry, or slow divergence by genetic drift during prolonged isolation. We hypothesize that the large phenotypic differences between species relative to the short divergence time suggests that the life-history differences observed may be direct responses to disruptive selection between populations. We speculate that local environmental or demographic differences at the southern range margin are possible mechanisms of selection driving one of the fastest known marine speciation events.     

Changes in sexual signals are greater than changes in ecological traits in a dichromatic group of fishes

Understanding the mechanisms by which phenotypic divergence occurs is central to speciation research. These mechanisms can be revealed by measuring differences in traits that are subject to different selection pressures; greater influence of different types of selection can be inferred from greater divergence in associated traits. Here, we address the potential roles of natural and sexual selection in promoting phenotypic divergence between species of snubnose darters by comparing differences in body shape, an ecologically relevant trait, and male color, a sexual signal. Body shape was measured using geometric morphometrics, and male color was measured using digital photography and visual system‐dependent color values. Differences in male color are larger than differences in body shape across eight allopatric, phylogenetically independent species pairs. While this does not exclude the action of divergent natural selection, our results suggest a relatively more important role for sexual selection in promoting recent divergence in darters. Variation in the relative differences between male color and body shape across species pairs reflects the continuous nature of speciation mechanisms, ranging from ecological speciation to speciation by sexual selection alone.     

Reconstructing the history of selection during homoploid hybrid speciation

This study aims to identify selection pressures during the historical process of homoploid hybrid speciation in three Helianthus (sunflower) hybrid species. If selection against intrinsic genetic incompatibilities (fertility selection) or for important morphological/ecological traits (phenotypic selection) were important in hybrid speciation, we would expect this selection to have influenced the parentage of molecular markers or chromosomal segments in the hybrid species' genomes. To infer past selection, we compared the parentage of molecular markers in high-density maps of the three hybrid species with predicted marker parentage from an analysis of fertility selection in artificial hybrids and from the directions of quantitative trait loci effects with respect to the phenotypes of the hybrid species. Multiple logistic regression models were consistent with both fertility and phenotypic selection in all three species. To further investigate traits under selection, we used a permutation test to determine whether marker parentage predicted from groups of functionally related traits differed from neutral expectations. Our results suggest that trait groups associated with ecological divergence were under selection during hybrid speciation. This study presents a new method to test for selection and supports earlier claims that fertility selection and phenotypic selection on ecologically relevant traits have operated simultaneously during sunflower hybrid speciation.     

The influence of locus number and information content on species delimitation: an empirical test case in an endangered Mexican salamander

Perhaps the most important recent advance in species delimitation has been the development of model‐based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next‐generation sequence data sets provides powerful insights into genome‐wide patterns of divergence during speciation. However, applying complex models to large data sets is time‐consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight‐locus, 137‐individual data set and an 89‐locus, seven‐individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow‐scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range.     

Panmixia supports divergence with gene flow in Darwin's small ground finch, Geospiza fuliginosa, on Santa Cruz, Galápagos Islands

The divergence‐with‐gene‐flow model of speciation has a strong theoretical basis with a growing number of plausible examples in nature, but remains hotly debated. Darwin’s finches of the Galápagos Archipelago have played an important role in our understanding of speciation processes. Recent studies suggest that this group may also provide insights into speciation via divergence with gene flow. On the island of Santa Cruz, recent studies found evidence for adaptive divergence in Darwin’s small ground finch, Geospiza fuliginosa, between ecologically contrasting arid and humid zones. Despite the short geographical distance between these zones, strong disruptive selection during low rainfall periods is expected to generate and maintain adaptive divergence. Conversely, during high rainfall periods, when disruptive selection is predicted to be weakened, population divergence in adaptive traits is expected to break down. Because periods of low and high rainfall irregularly alternate, the geographical pattern of adaptive divergence can be assumed to break down and, importantly, regenerate in situ. Here, we use microsatellite allele frequency data to assess the genetic population structure of G. fuliginosa on Santa Cruz. We sample 21 sites and four ecological zones across the island. We reject hypotheses of population substructure linked to ecological and geographical differences among sites in favour of a single panmictic population. Panmixia implies high levels of gene flow within Santa Cruz, which favours selection over genetic drift as a valid process generating phenotypic divergence in G. fuliginosa on Santa Cruz. We discuss how our findings may support classic adaptation, phenotypic plasticity, matching habitat choice or any combination of these three processes.     

Local adaptation within a hybrid species

Ecological divergence among populations may be strongly influenced by their genetic background. For instance, genetic admixture through introgressive hybridization or hybrid speciation is likely to affect the genetic variation and evolvability of phenotypic traits. We studied geographic variation in two beak dimensions and three other phenotypic traits of the Italian sparrow (Passer italiae), a young hybrid species formed through interbreeding between house sparrows (P. domesticus) and Spanish sparrows (P. hispaniolensis). We found that beak morphology was strongly influenced by precipitation regimes and that it appeared to be the target of divergent selection within Italian sparrows. Interestingly, however, the degree of parental genetic contribution in the hybrid species had no effect on phenotypic beak variation. Moreover, beak height divergence may mediate genetic differentiation between populations, consistent with isolation-by-adaptation within this hybrid species. The study illustrates how hybrid species may be relatively unconstrained by their admixed genetic background, allowing them to adapt rapidly to environmental variation.     

Weak disruptive selection and incomplete phenotypic divergence in two classic examples of sympatric speciation: cameroon crater lake cichlids

Abstract Recent documentation of a few compelling examples of sympatric speciation led to a proliferation of theoretical models. Unfortunately, plausible examples from nature have rarely been used to test model predictions, such as the initial presence of strong disruptive selection. Here I estimated the form and strength of selection in two classic examples of sympatric speciation: radiations of Cameroon cichlids restricted to Lakes Barombi Mbo and Ejagham. I measured five functional traits and relative growth rates in over 500 individuals within incipient species complexes from each lake. Disruptive selection was prevalent in both groups on single and multivariate trait axes but weak relative to stabilizing selection on other traits and most published estimates of disruptive selection. Furthermore, despite genetic structure, assortative mating, and bimodal species-diagnostic coloration, trait distributions were unimodal in both species complexes, indicating the earliest stages of speciation. Long waiting times or incomplete sympatric speciation may result when disruptive selection is initially weak. Alternatively, I present evidence of additional constraints in both species complexes, including weak linkage between coloration and morphology, reduced morphological variance aligned with nonlinear selection surfaces, and minimal ecological divergence. While other species within these radiations show complete phenotypic separation, morphological and ecological divergence in these species complexes may be slow or incomplete outside optimal parameter ranges, in contrast to rapid divergence of their sexual coloration.     

Sexual selection and population divergence II. Divergence in different sexual traits and signal modalities in field crickets (Teleogryllus oceanicus)

Sexual selection can target many different types of traits. However, the relative influence of different sexually selected traits during evolutionary divergence is poorly understood. We used the field cricket Teleogryllus oceanicus to quantify and compare how five traits from each of three sexual signal modalities and components diverge among allopatric populations: male advertisement song, cuticular hydrocarbon (CHC) profiles and forewing morphology. Population divergence was unexpectedly consistent: we estimated the among‐population (genetic) variance‐covariance matrix, D , for all 15 traits, and D_max explained nearly two‐thirds of its variation. CHC and wing traits were most tightly integrated, whereas song varied more independently. We modeled the dependence of among‐population trait divergence on genetic distance estimated from neutral markers to test for signatures of selection versus neutral divergence. For all three sexual trait types, phenotypic variation among populations was largely explained by a neutral model of divergence. Our findings illustrate how phenotypic integration across different types of sexual traits might impose constraints on the evolution of mating isolation and divergence via sexual selection.     

Divergent selection and phenotypic plasticity during incipient speciation in Lake Victoria cichlid fish

Divergent selection acting on several different traits that cause multidimensional shifts are supposed to promote speciation, but the outcome of this process is highly dependent on the balance between the strength of selection vs. gene flow. Here, we studied a pair of sister species of Lake Victoria cichlids at a location where they hybridize and tested the hypothesis that divergent selection acting on several traits can maintain phenotypic differentiation despite gene flow. To explore the possible role of selection we tested for correlations between phenotypes and environment and compared phenotypic divergence (P_ST) with that based on neutral markers (F_ST). We found indications for disruptive selection acting on male breeding colour and divergent selection acting on several morphological traits. By performing common garden experiments we also separated the environmental and heritable components of divergence and found evidence for phenotypic plasticity in some morphological traits contributing to species differences.     

Rapid song divergence leads to discordance between genetic distance and phenotypic characters important in reproductive isolation

The criteria for species delimitation in birds have long been debated, and several recent studies have proposed new methods for such delimitation. On one side, there is a large consensus of investigators who believe that the only evidence that can be used to delimit species is molecular phylogenetics, and with increasing numbers of markers to gain better support, whereas on the other, there are investigators adopting alternative approaches based largely on phenotypic differences, including in morphology and communication signals. Yet, these methods have little to say about rapid differentiation in specific traits shown to be important in reproductive isolation. Here, we examine variation in phenotypic (morphology, plumage, and song) and genotypic (mitochondrial and nuclear DNA) traits among populations of yellow‐rumped tinkerbird Pogoniulus bilineatus in East Africa. Strikingly, song divergence between the P. b. fischeri subspecies from Kenya and Zanzibar and P. b. bilineatus from Tanzania is discordant with genetic distance, having occurred over a short time frame, and playback experiments show that adjacent populations of P. b. bilineatus and P. b. fischeri do not recognize one another's songs. While such rapid divergence might suggest a founder effect following invasion of Zanzibar, molecular evidence suggests otherwise, with insular P. b. fischeri nested within mainland P. b. fischeri. Populations from the Eastern Arc Mountains are genetically more distant, yet share the same song with P. b. bilineatus from Coastal Tanzania and Southern Africa, suggesting they would interbreed. We believe investigators ought to examine potentially rapid divergence in traits important in species recognition and sexual selection when delimiting species, rather than relying entirely on arbitrary quantitative characters or molecular markers.     

Evolution of mate choice and the so‐called magic traits in ecological speciation

Non‐random mating provides multiple evolutionary benefits and can result in speciation. Biological organisms are characterised by a myriad of different traits, many of which can serve as mating cues. We consider multiple mechanisms of non‐random mating simultaneously within a unified modelling framework in an attempt to understand better which are more likely to evolve in natural populations going through the process of local adaptation and ecological speciation. We show that certain traits that are under direct natural selection are more likely to be co‐opted as mating cues, leading to the appearance of magic traits (i.e. phenotypic traits involved in both local adaptation and mating decisions). Multiple mechanisms of non‐random mating can interact so that trait co‐evolution enables the evolution of non‐random mating mechanisms that would not evolve alone. The presence of magic traits may suggest that ecological selection was acting during the origin of new species.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.