CALIBRATING DIVERGENCE TIMES ON SPECIES TREES VERSUS GENE TREES: IMPLICATIONS FOR SPECIATION HISTORY OF APHELOCOMA JAYS

Estimates of the timing of divergence are central to testing the underlying causes of speciation. Relaxed molecular clocks and fossil calibration have improved these estimates; however, these advances are implemented in the context of gene trees, which can overestimate divergence times. Here we couple recent innovations for dating speciation events with the analytical power of species trees, where multilocus data are considered in a coalescent context. Divergence times are estimated in the bird genus Aphelocoma to test whether speciation in these jays coincided with mountain uplift or glacial cycles. Gene trees and species trees show general agreement that diversification began in the Miocene amid mountain uplift. However, dates from the multilocus species tree are more recent, occurring predominately in the Pleistocene, consistent with theory that divergence times can be significantly overestimated with gene‐tree based approaches that do not correct for genetic divergence that predates speciation. In addition to coalescent stochasticity, Haldane's rule could account for some differences in timing estimates between mitochondrial DNA and nuclear genes. By incorporating a fossil calibration applied to the species tree, in addition to the process of gene lineage coalescence, the present approach provides a more biologically realistic framework for dating speciation events, and hence for testing the links between diversification and specific biogeographic and geologic events.     

Vagility: The Neglected Component in Historical Biogeography

The conceptual gap between ecological and historical biogeography is wide, although both disciplines are concerned with explaining how distributions have been shaped. A central aim of modern historical biogeography is to use a phylogenetic framework to reconstruct the geographic history of a group in terms of dispersals and vicariant events, and a number of analytical methods have been developed to do so. To date the most popular analytical methods in historical biogeography have been parsimony-based. Such methods can be classified into two groups based on the assumptions used. The first group assumes that vicariance between two areas creates common patterns of disjunct distributions across several taxa whereas dispersals and extinctions generate clade specific patterns. The second group of methods assumes that passive vicariance and within-area speciation have a higher probability of occurrence than active dispersal events and extinction. Typically, none of these methods takes into account the ecology of the taxa in question. I discuss why these methods can be potentially misleading if the ecology of the taxon is ignored. In particular, the vagility or dispersal ability of taxa plays a pivotal role in shaping the distributions and modes of speciation. I argue that the vagility of taxa should be explicitly incorporated in biogeographic analyses. Likelihood-based methods with models in which more realistic probabilities of dispersal and modes of speciation can be specified are arguably the way ahead. Although objective quantification will pose a challenge, the complete ignorance of this vital aspect, as has been done in many historical biogeographic analyses, can be dangerous. I use worked examples to show a simple way of utilizing such information, but better methods need to be developed to more effectively use ecological knowledge in historical biogeography.     

Speciation in North American black basses,Micropterus (Actinopterygii: Centrarchidae)

Abstract.— The Pleistocene Epoch has been frequently cited as a period of intense speciation for a significant portion of temperate continental biotas. To critically assess the role of Pleistocene glaciations on the evolution of the freshwater fish clade Micropterus , we use a phylogenetic analysis of complete gene sequences from two mitochondrial genes (cytochrome b and ND2), and a fossil calibration of the molecular clock to estimate ages of speciation events and rates of diversification. The absence of substantial morphological and ecological divergence together with endemism of five of the eight species in North American tributaries of the Gulf of Mexico may be interpreted as the result of a recent Pleistocene origin for these species. Speciation dates in Micropterus range from 1.01 ± 0.32 to 11.17 ± 1.02 million years ago. Only one speciation event is dated to the Pleistocene, and rates of diversification are not significantly variable in Micropterus. The premise that the Pleistocene was an exceptional period of speciation in Micropterus is not supported. Instead, a Gulf Coast allopatric speciation model is proposed, and predicts periods of dynamic speciation driven by sea level fluctuations in the Late Miocene and Pliocene. The Pleistocene, however, was a period of significant intraspecific mitochondrial lineage diversification. The application of the Gulf Coast allopatric speciation model to the remaining aquatic fauna of the Gulf of Mexico coast in North America will rely on robust phylogenetic hypotheses and accurate age estimations of speciation events.     

Evolutionary rate variation among vertebrate beta globin genes: implications for dating gene family duplication events

A comprehensive dataset of 62 beta globin gene sequences from various vertebrates was compiled to test the molecular clock and to estimate dates of gene duplications. We found that evolution of the beta globin family of genes is not clock-like, a result that is at odds with the common use of this family as an example of a constant rate of evolution over time. Divergence dates were estimated either with or without assuming the molecular clock, and both analyses produced similar date estimates, which are also in general agreement with estimates reported previously. In addition we report date estimates for seven previously unexamined duplication events within the beta globin family. Despite multiple sources of rate variation, the average rate across the beta globin phylogeny yielded reasonable estimates of divergence dates in most cases. Exceptions were cases of gene conversion, where it appears to have led to underestimates of divergence dates. Our results suggest (i) the major duplications giving rise to the paralogous beta globin genes are associated with significant evolutionary rate variation among gene lineages; and (ii) genes arising from more recent gene duplications (e.g., tandem duplications within lineages) do not appear to differ greatly in rate. We believe this pattern reflects a complex interplay of evolutionary forces where natural selection for diversifying paralogous functions and lineage-specific effects contribute to rate variation on a long-term basis, while gene conversion tends to increase sequence similarity. Gene conversion effects appear to be stronger on recent gene duplicates, as their sequences are highly similar. Lastly, phylogenetic analyses do not support a previous report that avian globins are members of a relic lineage of omega globins.     

Nuclear locus divergence at the early stages of speciation in the Orchard Oriole complex

As two lineages diverge from one another, mitochondrial DNA should evolve fixed differences more rapidly than nuclear DNA due to its smaller effective population size and faster mutation rate. As a consequence, molecular systematists have focused on the criteria of reciprocal monophyly in mitochondrial DNA for delimiting species boundaries. However, mitochondrial gene trees do not necessarily reflect the evolutionary history of the taxa in question, and even mitochondrial loci are not expected to be reciprocally monophyletic when the speciation event happened very recently. The goal of this study was to examine mitochondrial paraphyly within the Orchard Oriole complex, which is composed of Icterus spurius (Orchard Oriole) and Icterus fuertesi (Fuertes' Oriole). We increased the geographic sampling, added four nuclear loci, and used a range of population genetic and coalescent methods to examine the divergence between the taxa. With increased taxon sampling, we found evidence of clear structure between the taxa for mitochondrial DNA. However, nuclear loci showed little evidence of population structure, indicating a very recent divergence between I. spurius and I. fuertesi. Another goal was to examine the genetic variation within each taxon to look for evidence of a past founder event within the I. fuertesi lineage. Based on the high amounts of genetic variation for all nuclear loci, we found no evidence of such an event – thus, we found no support for the possible founding of I. fuertesi through a change in migratory behavior, followed by peripheral isolates speciation. Our results demonstrate that these two taxa are in the earliest stages of speciation, at a point when they have fixed differences in plumage color that are not reflected in monophyly of the mitochondrial or nuclear DNA markers in this study. This very recent divergence makes them ideal for continued studies of species boundaries and the earliest stages of speciation.     

Recent and rapid speciation with limited morphological disparity in the genus Rattus

Recent and rapid radiations provide rich material to examine the factors that drive speciation. Most recent and rapid radiations that have been well-characterized involve species that exhibit overt ecomorphological differences associated with clear partitioning of ecological niches in sympatry. The most diverse genus of rodents, Rattus (66 species), evolved fairly recently, but without overt ecomorphological divergence among species. We used multilocus molecular phylogenetic data and five fossil calibrations to estimate the tempo of diversification in Rattus, and their radiation on Australia and New Guinea (Sahul, 24 species). Based on our analyses, the genus Rattus originated at a date centered on the Pliocene-Pleistocene boundary (1.84-3.17 Ma) with a subsequent colonization of Sahul in the middle Pleistocene (0.85-1.28 Ma). Given these dates, the per lineage diversification rates in Rattus and Sahulian Rattus are among the highest reported for vertebrates (1.1-1.9 and 1.6-3.0 species per lineage per million years, respectively). Despite their rapid diversification, Rattus display little ecomorphological divergence among species and do not fit clearly into current models of adaptive radiations. Lineage through time plots and ancestral state reconstruction of ecological characters suggest that diversification of Sahulian Rattus was most rapid early on as they expanded into novel ecological conditions. However, rapid lineage accumulation occurred even when morphological disparity within lineages was low suggesting that future studies consider other phenotypes in the diversification of Rattus.     

Lineage divergence and speciation in the Web‐toed Salamanders (Plethodontidae: Hydromantes) of the Sierra Nevada,California

Peripatric speciation and the importance of founder effects have long been controversial, and multilocus sequence data and coalescent methods now allow hypotheses of peripatric speciation to be tested in a rigorous manner. Using a multilocus phylogeographical data set for two species of salamanders (genus Hydromantes) from the Sierra Nevada of California, hypotheses of recent divergence by peripatric speciation and older, allopatric divergence were tested. Phylogeographical analysis revealed two divergent lineages within Hydromantes platycephalus, which were estimated to have diverged in the Pliocene. By contrast, a low‐elevation species, Hydromantes brunus, diverged from within the northern lineage of H. platycephalus much more recently (mid‐Pleistocene), during a time of major climatic change in the Sierra Nevada. Multilocus species tree estimation and coalescent estimates of divergence time, migration rate, and growth rate reject a scenario of ancient speciation of H. brunus with subsequent gene flow and introgression from H. platycephalus, instead supporting a more recent divergence with population expansion. Although the small, peripheral distribution of H. brunus suggests the possibility of peripatric speciation, the estimated founding population size of the species was too large to have allowed founder effects to be important in its divergence. These results provide evidence for both recent speciation, most likely tied to the climatic changes of the Pleistocene, and older lineage divergence, possibly due to geological events, and add to evidence that Pleistocene glacial cycles were an important driver of diversification in the Sierra Nevada.     

Estimation of primate speciation dates using local molecular clocks

Protein-coding genes of the mitochondrial genomes from 31 mammalian species were analyzed to estimate the speciation dates within primates and also between rats and mice. Three calibration points were used based on paleontological data: one at 20-25 MYA for the hominoid/cercopithecoid divergence, one at 53-57 MYA for the cetacean/artiodactyl divergence, and the third at 110-130 MYA for the metatherian/eutherian divergence. Both the nucleotide and the amino acid sequences were analyzed, producing conflicting results. The global molecular clock was clearly violated for both the nucleotide and the amino acid data. Models of local clocks were implemented using maximum likelihood, allowing different evolutionary rates for some lineages while assuming rate constancy in others. Surprisingly, the highly divergent third codon positions appeared to contain phylogenetic information and produced more sensible estimates of primate divergence dates than did the amino acid sequences. Estimated dates varied considerably depending on the data type, the calibration point, and the substitution model but differed little among the four tree topologies used. We conclude that the calibration derived from the primate fossil record is too recent to be reliable; we also point out a number of problems in date estimation when the molecular clock does not hold. Despite these obstacles, we derived estimates of primate divergence dates that were well supported by the data and were generally consistent with the paleontological record. Estimation of the mouse-rat divergence date, however, was problematic.     

Environmental harshness is positively correlated with intraspecific divergence in mammals and birds

Life on Earth is conspicuously more diverse in the tropics. Although this intriguing geographical pattern has been linked to many biotic and abiotic factors, their relative importance and potential interactions are still poorly understood. The way in which latitudinal changes in ecological conditions influence evolutionary processes is particularly controversial, as there is evidence for both a positive and a negative latitudinal gradient in speciation rates. Here, we identify and address some methodological issues (how patterns are analysed and how latitude is quantified) that could lead to such conflicting results. To address these issues, we assemble a comprehensive data set of the environmental correlates of latitude (including climate, net primary productivity and habitat heterogeneity) and combine it with biological, historical and molecular data to explore global patterns in recent divergence events (subspeciation). Surprisingly, we find that the harsher conditions that typify temperate habitats (lower primary productivity, decreased rainfall and more variable and unpredictable temperatures) are positively correlated with greater subspecies richness in terrestrial mammals and birds. Thus, our findings indicate that intraspecific divergence is greater in regions with lower biodiversity, a pattern that is robust to both sampling variation and latitudinal biases in taxonomic knowledge. We discuss possible causal mechanisms for the link between environmental harshness and subspecies richness (faster rates of evolution, greater likelihood of range discontinuities and more opportunities for divergence) and conclude that this pattern supports recent indications that latitudinal gradients of diversity are maintained by simultaneously higher potentials for both speciation and extinction in temperate than tropical regions.     

Detecting shifts in diversification rates without fossils

Studies of shifts in diversification rates and adaptive radiations are difficult when there are no fossils because past events cannot be inferred. The phylogenies of recent species, however, allow one to infer the patterns of past diversifications. I present a new method for estimating the diversification rate of a lineage, provided that a phylogeny of recent species, constructed, for instance, with molecular data, is available. This method was inspired by survival models and takes into account species that are not included in detailed phylogenetic data, provided that approximate dates of origin of these species are known. Likelihood ratio tests and Akaike Information Criterion make it possible to test for differences in diversification among lineages or groups of lineages and, thus, to evaluate adaptive radiation hypotheses. The present modeling approach can easily be extended to include temporal variations in diversification rates. A simulation study showed that the method is statistically consistent, avoiding Type I and Type II errors, and that it is robust to periodic or random fluctuations in the speciation rate. An example is presented with a composite phylogeny of primates.     

Comparative phylogeography of pitvipers suggests a consensus of ancient Middle American highland biogeography

Aim  We used inferences of phylogenetic relationships and divergence times for three lineages of highland pitvipers to identify broad-scale historical events that have shaped the evolutionary history of Middle American highland taxa, and to test previous hypotheses of Neotropical speciation.
Location  Middle America (Central America and Mexico).
Methods  We used 2306 base pairs of mitochondrial gene sequences from 178 individuals to estimate the phylogeny and divergence times of New World pitviper lineages, focusing on three genera ( Atropoides , Bothriechis and Cerrophidion ) that are broadly co-distributed across Middle American highlands.
Results  We found strong correspondence across three highland lineages for temporally and geographically coincident divergences in the Miocene and Pliocene, and further identified widespread within-species divergences across multiple lineages that occurred in the early–middle Pleistocene.
Main conclusions  Available data suggest that there were at least three major historical events in Middle America that had broad impacts on species divergence and lineage diversification among highland taxa. In addition, we find widespread within-species genetic structure that may be attributable to the climatic changes that affected gene flow among highland taxa during the middle–late Pleistocene.     

Hard and soft allopatry: physically and ecologically mediated modes of geographic speciation

Aim Three common patterns have emerged in comparative phylogeographic analyses at many barriers: (1) a potential geographic pseudocongruence of lineage divergences; (2) a disconnect between the inference of temporally clustered, relatively recent timing for observed speciation events, and dates spanning a broader, apparently random time‐scale; and (3) an apparent prevalence of speciation with recent or continuing gene flow. It is unclear if there is a unifying explanation for these phenomena. We argue that the interaction between geographic barriers to dispersal and ecological limits on the distribution of species can explain these patterns. We suggest that these patterns can be explained by the presence of a continuum between two underlying processes, here termed ‘hard’ and ‘soft’ allopatric divergence, which result from the interplay between organismal ecology and the physioecological nature of geographic barriers. Location Examples from North America. Methods We examine comparative phylogeographic divergences in 18 groups of terrestrial vertebrates at two major biogeographic features in North America – the Mississippi River Embayment and the Cochise Filter Barrier – to test predictions made by this hypothesis. Results We find support for the two distinct processes of hard and soft allopatry, and note several examples exhibiting characteristics of both. Hard allopatry is caused by physical barriers promoting divergence as a function of consistent geographic isolation. Soft allopatry is caused by ecological processes that isolate populations geographically in allopatric refugia through niche conservatism, or across ecological transition zones through niche divergence, but which may be periodic or inconsistent through time. Main conclusions Viewing geographic speciation as a continuum between hard and soft allopatry can explain all three patterns as a consequence of the physical and ecological mechanisms that isolate populations, and provides an alternative perspective on the impact of ecological factors and physical barriers on lineage formation.     

Molecular evidence of a peripatric origin for two sympatric species of field crickets (Gryllus rubens and G. texensis) revealed from coalescent simulations and population genetic tests

Species pairs that differ primarily in characters involved in mating interactions and are largely sympatric raise intriguing questions about the mode of speciation. When species divergence is relatively recent, the footprint of the demographic history during speciation might be preserved and used to reconstruct the biogeography of species divergence. In this study, patterns of genetic variation were examined throughout the geographical range of two cryptic sister taxa of field crickets, Gryllus texensis and G. rubens; mitochondrial cytochrome oxidase I (COI) was sequenced in 365 individuals sampled from 48 localities. Despite significant molecular divergence between the species, they were not reciprocally monophyletic. We devised several analyses to statistically explore what historical processes might have given rise to this genealogical structure. The analyses indicated that the biogeographical pattern of genetic variation does not support a model of recent gene flow between species. Instead, coalescent simulations suggested that the genealogical structure within G. texensis, namely a deep split between two geographically overlapping clades, reflects historical substructure within G. texensis. Additional tests that consider the concentration of G. rubens haplotypes in one of the two G. texensis genetic clusters suggest a model of speciation in which G. rubens was derived from one lineage of a geographically subdivided ancestor. These results indicate that, despite the contemporary sympatry of G. texensis and G. rubens, the data are indicative of an peripatric origin in which G. rubens was derived from one of the two historical partitions in the species currently recognized as G. texensis. This proposed model of species divergence suggests how the interplay of geography and selection may give rise to new species, although this requires testing with multilocus data. Specifically, the model highlights how that geographical partitioning of ancestral variation in the past may augment the selectively driven divergence of characters involved in the reproductive isolation of the species today.     

A likelihood framework for inferring the evolution of geographic range on phylogenetic trees

Abstract At a time when historical biogeography appears to be again expanding its scope after a period of focusing primarily on discerning area relationships using cladograms, new inference methods are needed to bring more kinds of data to bear on questions about the geographic history of lineages. Here we describe a likelihood framework for inferring the evolution of geographic range on phylogenies that models lineage dispersal and local extinction in a set of discrete areas as stochastic events in continuous time. Unlike existing methods for estimating ancestral areas, such as dispersal‐vicariance analysis, this approach incorporates information on the timing of both lineage divergences and the availability of connections between areas (dispersal routes). Monte Carlo methods are used to estimate branch‐specific transition probabilities for geographic ranges, enabling the likelihood of the data (observed species distributions) to be evaluated for a given phylogeny and parameterized paleogeographic model. We demonstrate how the method can be used to address two biogeographic questions: What were the ancestral geographic ranges on a phylogenetic tree? How were those ancestral ranges affected by speciation and inherited by the daughter lineages at cladogenesis events? For illustration we use hypothetical examples and an analysis of a Northern Hemisphere plant clade (Cercis), comparing and contrasting inferences to those obtained from dispersal‐vicariance analysis. Although the particular model we implement is somewhat simplistic, the framework itself is flexible and could readily be modified to incorporate additional sources of information and also be extended to address other aspects of historical biogeography.     

Playing chicken (Gallus gallus): methodological inconsistencies of molecular divergence date estimates due to secondary calibration points

For any given taxonomic divergence event, one may find in the literature a wide range of time estimates. Many factors contribute to the variation in molecular date estimates for the same evolutionary event. High on the list is the choice of calibration points for converting genetic distances into evolutionary rates and, subsequently, into dates of divergence. In this study, we investigate one critical source of error in estimating divergence times, i.e. the use of secondary calibration points, which are divergence time estimates that have been derived from one molecular dataset on the basis of a primary external calibration point, and which are used again independently of the original external calibration point on a second dataset. Unless particular care is exercised, this practice leads to internal inconsistencies, and the inferred dates of divergence are by necessity unreliable. We present a consistency test for assessing the reliability of divergence time estimates based on secondary calibration points. As a case study, we examine recent estimates of divergence times among phyla and kingdoms based on multiple nuclear protein-coding genes, and show that they fail the consistency test.     

Lineages,splits and divergence challenge whether the terms anagenesis and cladogenesis are necessary

Using the framework of evolutionary lineages to separate the process of evolution and classification of species, we observe that ‘anagenesis’ and ‘cladogenesis’ are unnecessary terms. The terms have changed significantly in meaning over time, and current usage is inconsistent and vague across many different disciplines. The most popular definition of cladogenesis is the splitting of evolutionary lineages (cessation of gene flow), whereas anagenesis is evolutionary change between splits. Cladogenesis (and lineage‐splitting) is also regularly made synonymous with speciation. This definition is misleading as lineage‐splitting is prolific during evolution and because palaeontological studies provide no direct estimate of gene flow. The terms also fail to incorporate speciation without being arbitrary or relative, and the focus upon lineage‐splitting ignores the importance of divergence, hybridization, extinction and informative value (i.e. what is helpful to describe as a taxon) for species classification. We conclude and demonstrate that evolution and species diversity can be considered with greater clarity using simpler, more transparent terms than anagenesis and cladogenesis. Describing evolution and taxonomic classification can be straightforward, and there is no need to ‘make words mean so many different things’.     

Geographic distribution,evolution, and disease importance of species within the Neotropical Anopheles albitarsis Group (Diptera,Culicidae)

The Anopheles albitarsis group of mosquitoes comprises eight recognized species and one mitochondrial lineage. Our knowledge of malaria vectorial importance and the distribution and evolution of these taxa is incomplete. We constructed ecological niche models (ENMs) for these taxa and used hypothesized phylogenetic relationships and ENMs to investigate environmental and ecological divergence associated with speciation events. Two major clades were identified, one north (Clade 1) and one south (Clade 2) of the Amazon River that likely is or was a barrier to mosquito movement. Clade 1 species occur more often in higher average temperature locations than Clade 2 species, and taxon splits within Clade 1 corresponded with a greater divergence of variables related to precipitation than was the case within Clade 2. Comparison of the ecological profiles of sympatric species and sister species support the idea that phylogenetic proximity is related to ecological similarity. Anopheles albitarsis I, An. janconnae, and An. marajoara ENMs had the highest percentage of their predicted suitable habitat overlapping distribution models of Plasmodium falciparum and P. vivax, and warrant additional studies of the transmission potential of these species. Phylogenetic proximity may be related to malaria vectorial importance within the Albitarsis Group.     

Time to the most recent common ancestor and divergence times of populations of common chaffinches (Fringilla coelebs) in Europe and North Africa: insights into Pleistocene refugia and current levels of migration

We analyzed sequences from a 275-bp hypervariable region in the 5' end of the mitochondrial DNA control region in 190 common chaffinches (Fringilla coelebs) from 19 populations in Europe and North Africa, including new samples from Greece and Morocco. Coalescent techniques were applied to estimate the time to the most recent common ancestor (TMRCA) and divergence times of these populations. The first objective of this study was to infer the locations of refugia where chaffinches survived the last glacial episode, and this was achieved by estimating the TMRCA of populations in regions surrounding the Mediterranean that were unglaciated in the late Pleistocene. Although extant populations in Iberia, Corsica, Greece, and North Africa harbor haplotypes that are basal in a phylogenetic tree, this information alone cannot be used to infer that these localities served as refugia, because it is impossible to infer the ages of populations and their divergence times without also considering the population genetic processes of mutation, migration, and drift. Provided we assume the TMRCAs of populations are a reasonable estimate of a population's age, coalescent-based methods place resident populations in Iberia, Corsica, Greece, and North Africa during the time of the last glacial maximum, suggesting these regions served as refugia for the common chaffinch. The second objective was to determine when populations began diverging from each other and to use this as a baseline to estimate current levels of gene flow. Divergence time estimates suggest that European populations began diverging about 60,000 years before present. The relatively recent divergence of populations in North Africa, Italy, and Iberia may explain why classic migration estimates based on equilibrium assumptions are high for these populations. We compare these estimates with nonequilibrium-based estimates and show that the nonequilibrium estimates are consistently lower than the equilibrium estimates.     

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.