Species Delimitation: A Decade After the Renaissance

A decade ago Sites and Marshall [1] described the empirical practice of species delimitation as “a Renaissance issue in systematic biology”. At the time there was an odd disconnect be‐ tween the two frequently stated empirical goals systematic biology: the discovery of: (1) monophyletic groups (clades) and relationships within these at all hierarchical levels above species; and (2) lineages (species); compared to the actual practice of the discipline. While much of systematic biology had been devoted to the first goal, the second goal had until re‐ cently been largely ignored [2], despite the fact that species are routinely used as the basic units of analysis in biogeography, ecology, evolutionary biology, and conservation biology [3,4]. However, Sites and Marshall [1] noted “signs of a Renaissance” at the time of their re‐ view, which was precipitated in part by others emphasizing the need to distinguish between a non-operational, ontological definition of species, versus the empirical (operational) data needed to test their reality [5-7]. De Queiroz [7] (p. 60) noted that “All modern species defi‐ nitions either explicitly or implicitly equate species with segments of population level evolu‐ tionary lineages.” De Queiroz also noted that this was a revised version of Simpson’s “evolutionary species concept”, which defines a species as “a lineage (an ancestraldescend‐ ent sequence of populations) evolving separately from others and with its own evolutionary role and tendencies” ([8], p. 153), and called this a General Lineage Concept (GLC) of species ([7], p. 65). De Queiroz [9] further emphasized that the multiple empirical criteria simply re‐ flect the many contingent properties (differences in genetic or morphological features, adap‐ tive zones or ecological niches, mate-recognition systems, reproductive compatibility, monophyly, etc.) of diverging populations associated with different evolutionary processes operating in various geographic contexts [10,11]. Sites and Marshall [1] noted that the emerging consensus among systematists and evolutionary biologists was based on the utili‐ ty of this distinction (ontological definition vs. empirical species delimitation [SDL] meth‐


Introduction
A decade ago Sites and Marshall [1] described the empirical practice of species delimitation as "a Renaissance issue in systematic biology".At the time there was an odd disconnect between the two frequently stated empirical goals systematic biology: the discovery of: (1) monophyletic groups (clades) and relationships within these at all hierarchical levels above species; and (2) lineages (species); compared to the actual practice of the discipline.While much of systematic biology had been devoted to the first goal, the second goal had until recently been largely ignored [2], despite the fact that species are routinely used as the basic units of analysis in biogeography, ecology, evolutionary biology, and conservation biology [3,4].However, Sites and Marshall [1] noted "signs of a Renaissance" at the time of their review, which was precipitated in part by others emphasizing the need to distinguish between a non-operational, ontological definition of species, versus the empirical (operational) data needed to test their reality [5][6][7].De Queiroz [7] (p.60) noted that "All modern species definitions either explicitly or implicitly equate species with segments of population level evolutionary lineages."De Queiroz also noted that this was a revised version of Simpson's "evolutionary species concept", which defines a species as "a lineage (an ancestral-descendent sequence of populations) evolving separately from others and with its own evolutionary role and tendencies" ( [8], p. 153), and called this a General Lineage Concept (GLC) of species ( [7], p. 65).De Queiroz [9] further emphasized that the multiple empirical criteria simply reflect the many contingent properties (differences in genetic or morphological features, adaptive zones or ecological niches, mate-recognition systems, reproductive compatibility, monophyly, etc.) of diverging populations associated with different evolutionary processes operating in various geographic contexts [10,11].Sites and Marshall [1] noted that the emerging consensus among systematists and evolutionary biologists was based on the utility of this distinction (ontological definition vs. empirical species delimitation [SDL] meth-ods), and as also noted by de Queiroz [12], due to the contingencies of speciation processes, any single criterion or data set will artificially reduce the complexity of evolving lineages.
The subject matter of these and other reviews [12,13] focused strictly on methods of detecting various lines of evidence for lineage independence (reproductive isolation, ecological distinctiveness, diagnosability, monophyly, etc.), and since then new methods continue to be described [14], as do studies comparing the performance of some of these [14,15].In 2006, the Society of Systematic Biologists (SSB'06) organized the first symposium dedicated to the topic of species delimitation [2]; 11 papers were presented and six of those published, including an update by referenced de Queiroz [16], which emphasized the distinction between the GLC as "separately evolving metapopulation lineages, or more specifically, with segments of such lineages", versus secondary biological attributes or properties of organisms that can be quantified to empirically test for species status.This is a crucial distinction because it clearly separates the conceptual issue of defining the species category from the methodological issues of delimiting species; previously these had been conflated with the result that properties used to infer species boundaries (the empirical test) were also sometimes regarded as necessary for defining a species (a conceptualization issue).The advantage of the unified GLC is that no specific biological attributes of a species are considered necessary properties -species may exist as segments of metapopulations lineages regardless of our ability to empirically delimit them.Prior to this clarification and the realization that many different properties are relevant to the issue of species delimitation [17], the alternative species "concepts" in which various biological attributes had accumulated in diverging lineages required these same attributes to be necessary properties of species.This led to a confusing situation in which a different property was considered necessary under each alternative concept (22 such "concepts" were identified by Mayden [6]), and a long and ultimately non-productive debate about species definitions.Now most of these earlier "concepts" can be viewed as secondary species criteria that provide evidence of lineage separation.
Recently, Hausdorf [17] argued for an up-dated ontological species concept, based in new insights into speciation processes, particularly evidence that reproductive barriers are semipermeable to some gene flow, and that speciation may occur despite ongoing gene flow between diverging populations [18][19][20][21][22][23].Two other lines of evidence are relevant to the point of re-visiting the GLC: (1) findings of polyphyletic species of animals, due to parallel speciation in which similar traits conferring reproductive isolation arise separately in closely related populations [24,25], or in plants, due to recurrent polyploidization in different populations of the ancestral species [26,27]; and (2) discoveries of uniparental organisms that can be characterized as distinct units resembling species of biparental organisms [28].We cannot resolve all of these larger issues here, but we return to some of the general points raised by Hausdorf [17] in the discussion.
Empirically, species delimitation continues to be a topic of increasing interest in evolutionary biology.A reference search in the ISI Web of Science with the keyword 'species delimitation' retrieved 227 articles published since 2000, of which 60% were published after 2008.Less than 10 articles per year were published between 2000 and 2005; subse-  1A).These increases include papers describing new SDL methods, or using existing methods with novel data sets and/or applications to new taxa.Because new SDL methods apply the same coalescent models developed for species tree estimation and usually lead to the discovery of morphologically 'cryptic' species, we also searched for references with the keywords 'species tree' and 'cryptic species'.During the same period of time, papers about 'species trees' were few until 2007, increased between 2008 and 2010 to 5-10 articles per year, and nearly doubled to >20 papers last year (Figure 1B).Publications referring to 'cryptic species' show a constant increase from 20 papers/year in 2000 to 90 papers/year in 2011 with the larger annual increase between 2010 and 2011 (Figure 1C).These publication trends suggest that the recent paradigm shift in phylogenetic systematics to incorporate species trees (29) is having a positive impact on the development of new SDL methods, which are gradually being incorporated into integrative taxonomic practices for the discovery of cryptic species diversity [30].

Short history of some early methods
Sites and Marshall [1,13] separated SDL methods into non-tree and tree-based approaches, and included among the former (1) pairwise genetic distances that could be tested for either correlations with reproductive isolation [31,32], morphological distances [33], or geographic distances [34]; (2) gene flow statistics to estimate the extent of gene flow across hybrid zones [35]; (3) fixed alternative character states as an indicator of no gene flow in a "population aggregation analysis" (PAA; [36]); (4) the presence of heterozygous genotypes as an indicator of a "field for recombination" [37]; and (5) genotypic clusters [38].
Early tree-based methods included: (1) three versions of the phylogenetic species "concept" based on apomorphy, or lineage splitting, or node-based criteria, following the terminology of Brooks and McLennan [39]; (2) cladistic haplotype aggregation [40]; (3) molecular-morphological assessments using dichotomous flow charts [41]; (4) genealogical exclusivity [42]; and (5) an extension of the nested clade analysis [43] that includes tests of species boundaries [44].The data sets in these early studies most often included genotypes resolved from multilocus isozymes [15], morphological (usually meristic) characters, and with few exceptions [45,46], mitochondrial DNA (mtDNA) sequences.An innovative phylogenetic method described by Pons et al. [14] was based on a likelihood analysis of the mtDNA gene tree that estimates the inflection point between species-level (speciation-extinction) and populationlevel (coalescent) evolutionary processes, and demonstrated that groups delimited by this approach were generally concordant with geographic distributions and morphologically recognized species.This was one of a small number of early studies comparing the performance of multiple SDL methods (see also [15,40,45,46]).The published contributions of SSB'06 symposium [2] included several novel SDL methods, the first method [47] described a coalescent approach to estimating species boundaries based on multiple unlinked gene trees, and that does not require species to be characterized by reciprocal monophyly.This is an explicitly model-based approach that accommodates stochastic variance of the gene sorting process by linking estimates of two key parameters, a range of estimates of effective population sizes relative to possible divergence times.This type of gene tree-coalescence approach also directly links population genetic SDL methods to phylogenetic inference at deeper levels of divergence, which has been identified as a "new paradigm" in systematics [29].In this same issue, Shaffer and Thomson [48] introduced a population genetic SDL based on large sets of single nucleotide polymorphisms (SNPs), which would be most suited to delimiting very young species.Finally, this volume included two more novel SDL methods, both in this case using ecological and distributional data in novel ways to model "niche envelopes" that can augment molecular or morphological data in species delimitation [49][50][51].

New methods & new theory
New empirical SDL methods continue to be developed, based on multiple lines of evidence and multiple statistical methods.Among some of these is the approach of Bond and Stockman [52] that is especially relevant to highly geographically-structured populations in which traditional sequence-only data sets are likely to recover large numbers of well-defined, well-supported, and geographically concordant/genetically divergent-but-morphologically cryptic populations (species).These authors describe a framework for testing potential genetic and ecological exchangeability as a means of delimiting cohesion species [53], and present an example in trapdoor spiders of the Aptostichus atomarius complex.A completely different approach [54] is based on statistical tests of both population structure [48] and genealogical exclusivity [54] of nuclear loci, to test species provisionally identified from wellsupported mtDNA haploclades; the focal taxa in this study were Malagasy mouse lemurs (55; genus Microcebus).As a third example, Puillandre et al. [56] described a four-step approach to "generating robust speciation hypotheses" in mollusk family Turridae (genus Gemmula) based on: (a) collection of the COI DNA barcode gene for GMYC [14] and ABGD (Automatic Barcode Gap Discovery; [57]) analyses; coupled with (b) nuclear gene (rRNA 28S), morphological, geographical and bathymetrical data, to redefine species boundaries in this clade.This protocol more than doubled the previously known species diversity in Gemmula, and may be useful for large-scale SDL in hyperdiverse groups.A few additional examples include genotype-based methods for dominant and co-dominant multi-locus markers [58], combined estimates of divergence times and gene flow to discriminate intraspecific from interspecific patterns [59], and an extension of the R package GENELAND to include genetic, phenotypic (morphometric), and geographic data for delimitation of populations and species [60].
The recent merge of coalescent theory with phylogenetics has driven a new generation of SDL methods and a new paradigm in systematics [29].This new theoretical framework, and its derived analytical applications, was in part required as a solution for accommodating the observed conflict among genealogies from multiple loci (gene trees) with the underlying population-level genealogies (species trees) [61].A multi-species or 'censored' model was formulated to account for this discordance by considering each branch of the species tree as a separate coalescent model and by connecting them into a population-level genealogy following the topology of the species tree [62,63].Under this new approach, two major key innovations over the classic phylogenetic methods were achieved.First, multiple individual samples can be assigned to a single species and the estimated phylogeny represents the speciation history of ancestral and descendant species-level lineages, in contrast to the gene genealogies estimated with individual samples.Second, because the coalescent process of each gene tree is dependent upon parameters of its containing species tree, this approach can coestimate gene and species tree simultaneously, by-passing the task of calculating a consensus tree or estimating a phylogeny from a concatenated dataset.This new theoretical framework allows prediction of the probability distribution of gene trees given the species tree, and consequently, several methods were developed for estimating species trees from a collection of multiple gene trees under different algorithms [64,65].Based on these new methods, a generation of fully-coalescent SDL methods was introduced that consisted of selecting the best species-tree model from a set of alternative models that represent different hypotheses of species limits.For instance, one approach finds the maximum-likelihood for the full species tree (all species are hypothesized as separate lineages) and for alternative species trees (two or more species at a given node are collapsed into one), and then selects the best model using Akaike information criteria, assuming fixed gene trees and constant population sizes along the species tree (SpeDeSTEM; [66]).
Another SDL method consists of sampling from the Bayesian posterior distribution of species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC) with the program BP&P 2.1 [67].This approach accomodates gene tree uncertainty and variable population sizes, but a "known" species tree must be provided a priori.In addition, heuristic and/or semi-parametric approaches have been developed for: resolving the boundary between coalescent and speciation processes using single gene trees (generalized mixed Yulecoalescent, [14]), finding both the optimum species tree and species limits via minimization of gene tree conflict and intraspecific structure (Brownie; [68]), and selection of SDL models using approximate Bayesian computation (ABC) [69].Other tree-based [54] and non-treebased [58] SDL methods that can handle multiple loci with limited variation have been applied with success.In addition, there has been also a resurgence of morphology-based SDL using multivariate techniques in a hypothesis-driven statistical framework [60,70].

New kinds of data
The development of new multi-species/multi-locus SDL methods was also in part due to the demand of efficient analytical tools to handle the rapidly increasing amounts of molecular data collected with modern techniques.New SDL methods should be able to handle tens of loci for multiple individuals derived from the development and screening of anonymous nuclear loci (ANL), introns, and protein-coding loci using genomic resources [71][72][73].However, these new SDL methods are inadequate to analyze the influx of whole-genome data that have started to be collected for non-model organisms via next-generation sequencing (NGS) technologies ( [74][75][76]; e.g, genome of the lizard Anolis carolinensis; [77]).NGS technologies have been recently applied to development of thousands of gene regions spanning multiple divergence times [78], or loci targeted for "shallow-scale" phylogenetic/phylogeographic studies [79], and microsatellites [80] or SNPs [81] for extremely shallow phylogeo- The Species Problem -Ongoing Issues graphic histories [82].The microsatellite or SNP data should be useful for genotyping individuals for SDL studies of very young species [48].
More efficient and less costly whole-genome sequencing is becoming available on a regular basis, a trend that started with the first-generation technology (Sanger capillary-sequencing), followed by the second-generation (i.e., SOLiD 454, Illumina, Solexa, etc; [83]), and continuing today with the recently introduced third-generation 'nanopore' sequencing [84,85].A significant by-product of these single-molecule sequencing methods is their ability to automatically resolve the allelic phases of heterozygotes, in contrast to the time-consuming phase estimation and/or cloning required after direct dideoxy-sequencing [86].In addition, the uniform sampling of hundreds of loci across the genome can help identifying "outlier" loci via genome scans, which can represent candidate genes with fitness value, subject to selection and linked to processes such as ecological speciation [87].

Advantages of Multi-Species Coalescent-Based Methods (MSCM)
Model-based.-Becausethese SDL are based in the multi-species coalescent model, the likelihood of the data can be evaluated to find maximum-likelihood and posterior probability estimates of parameters and testing alternative SDL models under different criteria (e.g., likelihood-ratio test, Akaike information criterion, Bayes factors [46,88]).More importantly, these methods implement SDL in a hypothesis-testing framework, and taking into account uncertainty due to genetic processes and insufficient sampling [89,90].In addition, coalescent simulations generated under a null hypothesis of no-speciation and the alternative hypothesis of speciation can be used for evaluating the performance of these methods based on estimations of inferential errors (type I and II errors, see [91]).For example, the accuracy of three coalescent-based SDL (SpeDeSTEM, BP&P, and ABC) has been compared using simulations under a model of speciation for variable sampling densities and parameter values to estimate type II error (i.e., failing to reject no-speciation when it is false) across a range of conditions [66,69,92].When there is no migration, SpeDeSTEM can delimit species that have diverged as recently as 0.5N e generations ago using only 5 loci and 5 alleles per species [66] while BP&P could detect speciation at shorter divergence times (0.4N e generations ago) with the same sampling design [67,92].In agreement with these results, a comparison under identical simulation conditions showed that BPP outperformed SpeDeSTEM (and also ABC) when speciation takes place with or without gene flow [69].In spite of these simulations covering different speciation scenarios, sampling designs, and SDLs, the practical question of the appropriate balance between number of loci and number of alleles sequenced has not been explicitly explored until now.Below, in the last section of this chapter, we performed some simulations for a preliminary evaluation of the relative benefits of sampling more alleles vs. loci for accurate species delimitation.
Neutral loci.-Thesemarkers should be insensitive to 'phenotypic plasticity', the phenotypic response to environmental variation that is not genetically-based (in contrast to adaptive variation), which could bias morphological-based taxonomy.Environmental variation in different parts of the range can lead to a plastic phenotypic response, which can be revealed and distinguished from local adaptation via reciprocal transplant or 'common garden' ex-periments [93].In these cases, morphological variation as a result of this plastic response could be used as a criterion to delimit species, while neutral markers would indicate that there is no genetic differentiation [94,95].In contrast, in cases of morphologically-cryptic species due to for example to niche conservatism [96], genetic divergence and lineage sorting is expected to occur in neutral markers due to independent evolution in isolation, and those markers with higher mutation rates and smaller effective population size (e.g.mtDNA) should be ideal for species delimitation [97,98].Moreover, it has been suggested that neutral loci will also differ in their usefulness for species delimitation since those with higher rates of intra-specific gene flow will be less sensitive to the effect of inter-specific introgression [99].However, the mitochondrial locus does not always meet assumptions of neutrality [100], and it frequently introgresses across species boundaries [101], so in our view it should be used to identify "candidate species" [102], which can then be verified with independent lines of evidence [103].
Repeatability.-The results of a SDL analysis can be replicated exactly when using the same data and the same analytical methods, which eliminates much of the subjectivity and/or investigator bias for/against certain kinds of data (morphology vs. molecular, etc.).Because these methods rely on explicit predictions about genealogical patterns under alternative models of lineage divergence, it is possible to carry out species delimitation in a more objective and bias-free fashion compared to diagnosability-based SDL methods [90].In addition, because inferences are dependent upon a specific sampling design and the method used, one can make explicit statements about how robust a given species delimitation method is to variation in these parameters, and to violations of the method's assumptions.
Universality.-Thesame SDL method and the same kind of data (i.e,DNA sequences or gene trees from homologous regions of the genome) can be used for SDL across different taxa, making these approaches comparable across all parts of the Tree of Life, as long as the assumptions of the method are reasonable for the taxon under study (see below).Another advantage associated with the use of neutral markers in coalescent-based SDLs is related to the standard criterion used for assigning species status across a variety of taxa when using the same markers and analyses [90], assuming that these markers offer similar resolving power.This is a desirable property for a SDL method since a uniform criterion implies that the species level could be compared readily among different higher-level taxa, thereby allowing meaningful analyses of species diversity among communities typical of ecological studies [91].

Disadvantages of MSCM
Many of the advantages listed above also impose some limitations of MSCM and other SDL methods for different reasons.First, these are model-based methods, and any violations of assumptions of the standard coalescent are expected to introduce inference errors.For instance, and most relevant to the SDL problem, while the standard coalescent assumes panmixis within populations, it is clear that in most natural populations, there is almost always some degree of population structure (i.e, demes connected by limited gene flow).In fact, a recent study using the Brownie's SDL method found that more dense sampling in- The Species Problem -Ongoing Issues creased the chances of detecting population structure, supporting more species boundaries, and consequently, inflating estimates of the number of species [104].Thus, MSCM could be more prone to split a single real species into multiple lineages due to intra-specific population structure alone, increasing type I error (i.e., rejecting a true hypothesis of a single species), and leading to 'taxonomic inflation' [91].Fortunately, some flexible MSCM methods allow incorporating population structure within species via coalescent simulation of island, stepping-stone, and other potential models, and subsequent comparison of SDL hypotheses with ABC approaches [69].
Another frequent assumption of most MSCM is that species have diverged from a common ancestral species without gene flow even though speciation with gene flow seems to be rather common in nature, especially in cases of ecological speciation [22,95,105].While these methods ignore the effects of gene flow, simulation testing has shown that some of them are relatively robust to low levels of gene flow [66,92], and that its impact on delimitation accuracy is ameliorated when gene flow is explicitly incorporated in the speciation model [69].This result supports the suggestion that, in order to distinguish between species-and population-level differentiation, it is necessary to jointly consider the two components of the divergence process: time since splitting and gene flow after divergence [59].
Second, sampling effort is well known to strongly impact coalescent-based and other SDL methods.A number of studies evaluating the accuracy of several MSCM methods suggest that limited sampling of loci and sequences will decrease the probability of detecting speciation when this hypothesis is correct [66,69,92] and consequently, increasing type II error [91].In addition, these simulation results also support the intuitive idea that the problem of insufficient sampling becomes more serious when SDL is more difficult: shorter divergence times, larger population size, and increasing inter-specific gene flow.However, more simulations are necessary to evaluate the appropriate balance between sampling intensity and design (e.g., geographic vs. genealogical dimensions, [91]) for different parameter configurations, in a power analysis context to provide further guidance to empirical studies [106].In addition to limited geographic sampling, the collected sequence data also impose a limit to the amount of genetic data available for analysis.In the next section, we explore how accuracy in species delimitation responds to variable sampling of loci and alleles for a fixed sequencing effort.
Third, coalescent-based SDL approaches assume selective neutrality of gene regions used, but divergent selection on ecological traits, across habitats or along an environmental gradient, can lead to local adaptation and correlated reproductive isolation in a process of ecological speciation [95,107].Phenotypic divergence can be so fast that mutation rates could produce little or no differentiation at all in neutral markers used in SDL approaches.Only those "outlier loci" under selection revealed by genomic scans, which are potentially associated with the selected traits, would be appropriate markers under these scenarios [87].
Fourth, as in other methods data conflict may be evident when multiple data sets are used.These SDL methods are not expected to resolve the discordance among different kinds of data sets (i.e., morphological, behavioral, ecological, molecular, etc.) since they typically use sequence data or gene trees from presumably neutral loci.However, Bayesian approaches Species Delimitation: A Decade After the Renaissance http://dx.doi.org/10.5772/52664 have the potential of incorporating previous information about species limits derived from non-molecular data into prior distributions of genetic-based analyses [67].
Fifth, there may be conflicts with traditional taxonomic practices.The discovery of new cryptic species with coalescent-based SDL in a statistical framework, is still insufficient for formal taxonomic descriptions, since nomenclatural rules still require traditional morphologybased diagnoses [108,109].While these methods will help diagnosing new cryptic diversity, many taxonomists will be reluctant to formally describe new species based on molecular-data alone, which ultimately will further expand the 'taxonomy-phylogeny' gap [91].While the description of cryptic species is complicated by the lack of morphological diagnostic characters, another difficulty relies in the inability of MSCM to assign newly collected specimens to species (i.e., taxonomic determination) unless new analyses are carried out to reevaluate species limits.

Future directions
Statistical testing of SDL.-The ongoing surge in the new generation of SDL methods will probably encourage many taxonomists to apply these methods empirically, especially for recently evolved, cryptic taxa that cannot be delimited with other data.The ability to frame species limits as statistical hypotheses that can be tested objectively with multi-locus and multi-species analyses make these new SDL methods very appealing for empirical systematists in the context of an 'integrative taxonomy' [4,110,111].In addition to empirical application to real data sets, we also expect that more simulation studies will be carried out to compare the performance of different data sets, under different methods/assumptions, and for variable sampling designs, using statistical power analyses.Previous studies have compared methods for a limited set of parameter conditions (e.g., usually population size has been assumed to remain constant) and have examined the effect of increased sampling effort for loci or sequences separately.However, performance of these SDL methods has not been evaluated for a variable sampling design and a fixed sampling effort; in other words, what should be the optimal balance between number of loci and number of sequences when the total number of sequenced base pairs (bp) is the same?
In order to provide a preliminary evaluation of the impact of sampling design on performance of new SDM, we simulated coalescent genealogies with the program ms [112] and sequence data with the program Seq-Gen [113] for a speciation model between species A and B for three increasing divergence times: 0.25, 0.5, and 1N e (Figure 2A).We assumed a constant θ per site = 0.01, 500 bp per locus, and ~50 variable sites per locus.For each divergence time, we simulated 5 combinations of number of loci (1, 2, 4, 10, and 20) and number of sequences per species (1, 2, 5, 10, and 20) while keeping the total sequencing effort constant (20 sequences per species).We simulated 100 replicates for each sampling treatment which were analyzed with BP&P to calculate the mean speciation probability between species A and B across replicates, which represents the accuracy of the method (i.e., the probability of detecting speciation when it is the true hypothesis).We also simulated a no-speciation model The Species Problem -Ongoing Issues where sequences from species A and B were collapsed into a single lineage, and repeated the same sampling and analytical procedure to examine the performance of the method based on a plot of true positive and false positives rates (i.e., ROC plot; [114]).
The results show that under the conditions examined, more sequences per species is better than more loci at least in the range of 1-20 loci and sequences per species (Figure 2B).The ROC plots for the 5 sampling treatments at a divergence time of 0.5N e show that performance is higher (i.e., area under ROC curve is larger) when sampling 20 sequences for 1 locus or 10 sequences for 2 loci, but performance gradually decreased with more loci and fewer sequences (Figure 2C).These results are congruent with the impact of sampling design on the accuracy of species-tree methods (STM) at shallow divergence times [115,116], which is an expected outcome because both STM and SDL methods share the same basic multispecies coalescent model [67,117].However, our results are contingent upon the conditions simulated, in particular the assumptions of panmixia within species, and a constant θ across the species tree.This second assumption is a critical parameter of coalescent models, which can be estimated more accurately with a larger sample of loci [118].Our attempt with this simulation example was to show how we can evaluate the performance of a SDL method under a variety of sampling conditions based on a power analysis, and that this same approach can be applied for comparisons across different SDL methods and more complex speciation scenarios than those that have been examined so far.
Population and species delimitation.-Theapplication of coalescent-based SDM, which can delimit species at very shallow levels of divergence [66,69,92] should reduce the 'taxonomyphylogeny' gap and help decrease the type I error of biological-species criteria that often fail to detect species, when reproductive isolation is not yet complete [91].Thus, coalescentbased SDL methods will probably help to delimit entities, name taxonomic units, and give appropriate conservation priority to the increasing amounts of cryptic diversity being discovered in nature [91].On the other hand, MLCM should be used with caution to avoid confusing species-level divergence with intra-specific population structure and therefore, oversplitting lineages, with serious consequences for conservation science since limited resources would be potentially wasted due to bad taxonomy [91].
A potential protocol for an informed species delimitation approach that takes into account population structure, could consist of first applying a clustering/population aggregation method to identify the smaller clusters of individuals under a population genetics criterion based on genotype or allele frequency data ('e.g., Structure 48,58,60).Subsequently, a SDL method can be applied to test if these clusters also represent independent evolutionary lineages based on the pattern of allele coalescence in gene genealogies (e.g.BP&P).Because initial population divergence starts with differentiation in allele frequencies and secondly, with random lineage sorting and mutation that further differentiates lineages during speciation [59], population genetics approaches are expected to detect lineages earlier than SDL approaches.For example, an empirical analysis of West African forest geckos (Hemidactylus fasciatus) found ~10 populations with Structure, which were considered as 'candidate' species in a subsequent BPP analysis that collapsed them into 4 species [119].This two-stage approach would provide a consistent and standard criterion for distinguishing between popu-lation-and species-level divergence, a threshold that has been difficult to resolve with genetic parameters measuring amounts of evolutionary differentiation [59].The next generation of SDL methods.-Wehave emphasized that species delimitation should take into account the speciation processes that have shaped the patterns of trait divergence in genetic, morphological, and ecological data [89].In a process-oriented classification of modes of speciation, we can distinguish between 'passive' modes driven by random divergence associated with the classic allopatric models, and the 'adaptive' modes of speciation.The formulation of a null hypothesis of speciation due to stochastic forces (i.e., 'passive divergence' or 'drift-only' model) should facilitate testing this mode of speciation, because rejecting this hypothesis is probably easier than demonstrating 'adaptive' speciation due to deterministic processes [120].In nature, both speciation models appear to interact and work in concert during diversification of closely related lineages [121,122].Adaptive speciation in turn can be subdivided into 'ecological' speciation, reproductive isolation due to disruptive natural selection operating on ecological traits [95], and speciation due to sexual selection that results in divergent mating prefer-ences and assortative mating [123].In theory, both kinds of selection seem to be necessary to drive speciation to completion [124], and limited empirical data supports the role of this interaction during diversification [125].Due to this variety in speciation processes, we should expect different patterns of trait divergence, and consequently, different kinds of data would be more appropriate for species delimitation under each speciation scenario.Therefore, relying on any single kind of trait could potentially miss the detection of a speciation event, for example using exclusively morphological data will fail to recognize cryptic species.Similarly, if we use only the typical neutral genetic markers of phylogeography and population genetics, we could miss many instances of ecological speciation that takes place in contemporary time scales [126], and/or without divergence in neutral loci [127].

Conclusion
There is an ongoing genomics revolution for the study of adaptation in ecological and evolutionary non-model organisms derived from (NGS) technologies [76,128].Decreasing sequencing costs and new protocols for discovering and screening thousands of markers scattered throughout the genome [79], is now allowing application of population genomics approaches to identifying the candidate loci underlying adaptive traits with ecological significance [87].In fact, recent studies have found genomic regions and/or specific loci related to repeated local adaptation, population divergence, and reproductive isolation between ecotypes in different habitats or hosts [129,130].We anticipate that these 'speciation genomics' approaches will become more common in non-model organisms and will provide a basis for species delimitation in scenarios of adaptive speciation SDL methods, complementing current SDL methods.Moreover, this plurality of criteria for species delimitation based on multiple kinds of traits is consistent with the GLC of species that views these organismal traits as evolving in different temporal order depending on how speciation has actually taken place [9,12].In addition, it is also compatible with the more recent 'differential fitness' concept, which is based on those organismal features of one species that have negative fitness effects in other species and cannot be exchanged upon contact [17].

Figure 2 .
Figure 2. Simulation-based testing of the accuracy of BP&P to detect speciation between species A and B using five alternative sampling designs with the same sequencing effort and at three increasing divergence times (0.25, 0.5, and 1Ne) (A).Plot of accuracy and divergence time for each sampling design (B).ROC plot for each sampling design when divergence time = 0.5Ne (C).
The Species Problem -Ongoing Issues quently 10-20 articles per year between 2006 and 2008, and after 2008 the publication rate reached ~ 40 articles (Figure