The shapes of neutral gene genealogies in two species: probabilities of monophyly,paraphyly, and polyphyly in a coalescent model

Abstract.— The genealogies of samples of orthologous regions from multiple species can be classified by their shapes. Using a neutral coalescent model of two species, I give exact probabilities of each of four possible genealogical shapes: reciprocal monophyly, two types of paraphyly, and polyphyly. After the divergence that forms two species, each of which has population size N , polyphyly is the most likely genealogical shape for the lineages of the two species. At ∼ 1.300 N generations after divergence, paraphyly becomes most likely, and reciprocal monophyly becomes most likely at ∼1.665 N generations. For a given species, the time at which 99% of its loci acquire monophyletic genealogies is ∼5.298 N generations, assuming all loci in its sister species are monophyletic. The probability that all lineages of two species are reciprocally monophyletic given that a sample from the two species has a reciprocally monophyletic genealogy increases rapidly with sample size, as does the probability that the most recent common ancestor (MRCA) for a sample is also the MRCA for all lineages from the two species. The results have potential applications for the testing of evolutionary hypotheses.     

How many species are in the genus Batrachuperus? A phylogeographical analysis of the stream salamanders (family Hynobiidae) from southwestern China

Phylogeographical analysis of DNA sequence data has been routinely used to test species boundaries using the monophyly criterion; however, a complementary criterion, reproductive isolation, is often ignored. We used a combination of phylogenetic and population genetic approaches to determine species boundaries among stream salamanders in the genus Batrachuperus . First, cytochrome b sequence data from 174 Batrachuperus individuals, sampled from 78 populations, were used to reconstruct historical relationships within the genus. Second, allozyme data for 14 presumptive nuclear loci, from 463 individuals sampled from 60 populations, were collected and analysed to assess population similarity or disparity, as well as potential reproductive isolation. The DNA sequence data grouped all populations into seven major monophyletic groups, and the allozyme data provided evidence for reproductive isolation among four of the seven groups, thereby supporting the species status of these groups. The allozyme data suggested that two of the other groups share the same gene pool, and therefore belong to a single species. Finally, the allozyme data revealed two reproductively isolated units within the seventh group, which we suggest represents a case of 'budding speciation' based on the DNA gene tree. In total, seven species of the genus Batrachuperus were defined, two of which were previously unknown. The phylogeographical analysis also revealed that vicariance events might have dominated the evolutionary history of this group, but the speciation events might precede the formation of the existing mountain topology. This study demonstrates the importance of including frequency data from multiple nuclear gene loci in determining species boundaries.     

A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora

To critically examine the relationship between species recognized by phylogenetic and reproductive compatibility criteria, we applied phylogenetic species recognition (PSR) to the fungus in which biological species recognition (BSR) has been most comprehensively applied, the well-studied genus Neurospora. Four independent anonymous nuclear loci were characterized and sequenced from 147 individuals that were representative of all described outbreeding species of Neurospora. We developed a consensus-tree approach that identified monophyletic genealogical groups that were concordantly supported by the majority of the loci, or were well supported by at least one locus but not contradicted by any other locus. We recognized a total of eight phylogenetic species, five of which corresponded with the five traditional biological species, and three of which were newly discovered. Not only were phylogenetic criteria superior to traditional reproductive compatibility criteria in revealing the full species diversity of Neurospora, but also significant phylogenetic subdivisions were detected within some species. Despite previous suggestions of hybridization between N. crassa and N. intermedia in nature, and the fact that several putative hybrid individuals were included in this study, no molecular evidence in support of recent interspecific gene flow or the existence of true hybrids was observed. The sequence data from the four loci were combined and used to clarify how the species discovered by PSR were related. Although species-level clades were strongly supported, the phylogenetic relationships among species remained difficult to resolve, perhaps due to conflicting signals resulting from differential lineage sorting.     

PREDICTING NUCLEAR GENE COALESCENCE FROM MITOCHONDRIAL DATA: THE THREE-TIMES RULE

Abstract.— Coalescence theory predicts when genetic drift at nuclear loci will result in fixation of sequence differences to produce monophyletic gene trees. However, the theory is difficult to apply to particular taxa because it hinges on genetically effective population size, which is generally unknown. Neutral theory also predicts that evolution of monophyly will be four times slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. Variation in mitochondrial DNA (mtDNA) within and between species has been studied extensively, but can these mtDNA data be used to predict coalescence in nuclear loci? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The “three‐times rule” states that, on average, most nuclear loci will be monophyletic when the branch length leading to the mtDNA sequences of a species is three times longer than the average mtDNA sequence diversity observed within that species. A test using mitochondrial and nuclear intron data from seven species of whales and dolphins suggests general agreement with predictions of the three‐times rule. We define the coalescence ratio as the mitochondrial branch length for a species divided by intraspecific mtDNA diversity. We show that species with high coalescence ratios show nuclear monophyly, whereas species with low ratios have polyphyletic nuclear gene trees. As expected, species with intermediate coalescence ratios show a variety of patterns. Especially at very high or low coalescence ratios, the three‐times rule predicts nuclear gene patterns that can help detect the action of selection. The three‐times rule may be useful as an empirical benchmark for evaluating evolutionary processes occurring at multiple loci.     

Perspective: gene divergence, population divergence, and the variance in coalescence time in phylogeographic studies

Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (theta) to T and the effect of uncertainty in theta on estimates of population divergence (tau) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on tau decrease when the range over which ancestral population size theta is assumed to be distributed decreases and when tau increases; they generally exclude zero when tau/(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding tau = 0 is roughly that l tau/(4Ne) > 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time.     

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

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

Multi‐gene phylogenetic analysis of south‐east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy

Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south‐east Asia and the Pacific. Identifying molecular diagnostics has proven problematic for these four taxa, a situation that cofounds biosecurity and quarantine efforts and which may be the result of at least some of these taxa representing the same biological species. We therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide geographic range; sequencing six loci (cox1, nad4‐3′, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic frameworks for individual loci and concatenated data sets for which we applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenberg's reciprocal monophyly measure, P(AB), Rodrigo's (P(RD)) and the genealogical sorting index, gsi. We specifically tested whether there was phylogenetic support for the four ‘ingroup’ pest species using a data set of multiple individuals sampled from a number of populations. Based on our combined data set, Bactrocera carambolae emerges as a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are unresolved. These data add to the growing body of evidence that B. dorsalis s.s., B. papayae and B. philippinensis are the same biological species, which poses consequences for quarantine, trade and pest management.     

Species discovery in marine planktonic invertebrates through global molecular screening

Species discovery through large‐scale sampling of mitochondrial diversity, as advocated under DNA barcoding, has been widely criticized. Two of the primary weaknesses of this approach, the use of a single gene marker for species delineation and the possible co‐amplification of nuclear pseudogenes, can be circumvented through incorporation of multiple data sources. Here I show that for taxonomic groups with poorly characterized systematics, large‐scale genetic screening using a mitochondrial DNA marker can be a very effective approach to species discovery. Global sampling (120 locations) of 1295 individuals of 22 described species of eucalanid copepods identified 15 novel evolutionarily significant units (ESUs) within this marine holoplanktonic family. Species limits were tested under reciprocal monophyly at the mitochondrial (mt) gene 16S rRNA, and 13 of 15 lineages were reciprocally monophyletic under three phylogenetic inference methods. Five of these mitochondrial ESUs also received moderate support for reciprocal monophyly at the independently‐inherited nuclear gene, internal transcribed spacer 2 (ITS2). Additional support for the utility of mt DNA as a proxy for species boundaries in this taxon is discussed, including results from related morphological and biogeographic studies. Minimal overlap of intra‐ESU and inter‐ESU 16S rRNA genetic distances was observed, suggesting that this mt marker performs well for species discovery via molecular screening. Sampling coverage required for the discovery of new ESUs was found to be in the range of >50 individuals/species, well above the sampling intensity of most current DNA Barcoding studies. Large‐scale genetic screening can provide critical first data on the presence of cryptic species, and should be used as an approach to generate systematic hypotheses in groups with incomplete taxonomies.     

Polyphyly and species delimitation of Picea brachytyla (Pinaceae) based on population genetic data

Accurate species delimitation is fundamental to biodiversity conservation. The endangered spruce Picea brachytyla (Franch.) E. Pritz. was suggested to be polyphyletic based on a limited number of samples in previous studies. To evaluate polyphyly of P. brachytyla, we sampled 139 individuals from 16 populations across most of its distributional range, plus representatives of two related species, Picea likiangensis (Franch.) E. Pritz. and Picea wilsonii Mast. We sequenced 13 nuclear loci and three chloroplast and two mitochondrial loci for the following species delimitation. Phylogenetic analyses of nuclear loci grouped all individuals of P. brachytyla from Sichuan and Chongqing into one distinct lineage and those from Yunnan and Tibet (southern distribution) nested within the P. likiangensis species complex. Structure analyses confirmed this result. Networks of chloroplast DNA haplotypes similarly showed that P. brachytyla from the southern distribution nested within the P. likiangensis species complex, whereas haplotypes for the northern distribution comprised a separate and well-supported lineage. These results suggest that P. brachytyla from the southern distribution is a part of the P. likiangensis species complex and should be removed from P. brachytyla. Our study highlights the utility of population genetic evidence in delimitating endangered species and understanding the conservation status of such species.     

Multiple nuclear gene sequences identify phylogenetic species boundaries in the rapidly radiating clade of Mexican ambystomatid salamanders

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.     

Population structure in the American oyster as inferred by nuclear gene genealogies

Multiple haplotypes from each of three nuclear loci were isolated and sequenced from geographic populations of the American oyster, Crassostrea virginica. In tests of alternative phylogeographic hypotheses for this species, nuclear gene genealogies constructed for these haplotypes were compared to one another, to a mitochondrial gene tree, and to patterns of allele frequency variation in nuclear restriction site polymorphisms (RFLPs) and allozymes. Oyster populations from the Atlantic versus the Gulf of Mexico are not reciprocally monophyletic in any of the nuclear gene trees, despite considerable genetic variation and despite large allele frequency differences previously reported in several other genetic assays. If these populations were separated vicariantly in the past, either insufficient time has elapsed for neutral lineage sorting to have achieved monophyly at most nuclear loci, or balancing selection may have inhibited lineage extinction, or secondary gene flow may have moved haplotypes between regions. These and other possibilities are examined in light of available genetic evidence, and it is concluded that no simple explanation can account for the great variety of population genetic patterns across loci displayed by American oysters. Regardless of the source of this heterogeneity, this study provides an empirical demonstration that different sequences of DNA within the same organismal pedigree can have quite different phylogeographic histories.      

A genealogical view of chromosomal evolution and species delimitation in the Drosophila virilis species subgroup

Chromosomal arrangement was a historically important character used for defining taxonomic boundaries. The Drosophila virilis species group exhibits a series of chromosomal rearrangements, and the resulting differences among karyotypes were primary characters originally used to define taxa within the group. However, some chromosomally divergent forms have not been sufficiently resolved in phylogenetic reconstructions of DNA sequences from several nuclear genes. Sequences of mitochondrial regions have the potential for finer-scale resolution of closely related taxa; therefore, sequences of two mitochondrial genes were used to examine phylogenetic relationships within the chromosomally variable virilis subgroup. Sequences were obtained from multiple strains of the Palearctic species, D. virilis and D. lummei, and the Nearctic species, D. novamexicana and two chromosomal forms of D. americana. Analyses support the recent emergence of the different chromosomal forms in North America. However, none of these chromosomally divergent forms exhibit reciprocal monophyly of their mtDNA sequences, which is the requirement for attaining genealogical species status.     

Genealogical exclusivity in geographically proximate populations of Hypochilus thorelli Marx (Araneae,Hypochilidae) on the Cumberland Plateau of North America

The issue of sampling sufficiency is too infrequently explored in phylogeographical analysis, despite both theoretical work and analytical methods that stress the importance of sampling effort. Regarding the evolutionary pattern of reciprocal monophyly, both the probability of recovering this pattern and the possible inferences derived from this pattern, are highly contingent upon the density and geographical scale of sampling. Here, we present an empirical example that relates directly to this issue. We analyse genetic structure in the southern Appalachian spider Hypochilus thorelli, using an average sample of 5 mitochondrial DNA (mtDNA) sequences per location for 19 locations. All sampled sites are reciprocally monophyletic for mtDNA variation, even when separated by geographical distances as small as 5 km. For populations separated by greater geographical distances of 20-50 km, mtDNA sequences are not only exclusive, but are also highly divergent (uncorrected p-distances exceeding 5%). Although these extreme genealogical patterns are most seemingly consistent with a complete isolation model, both a coalescent method and nested cladistic analysis suggest that other restricted, but nonzero, gene flow models may also apply. Hypochilus thorelli appears to have maintained morphological cohesion despite this limited female-based gene flow, suggesting a pattern of stasis similar to that observed at higher taxonomic levels in Hypochilus.     

Contrasting patterns of genetic structure in two species of the coral trout Plectropomus (Serranidae) from east and west Australia: introgressive hybridisation or ancestral polymorphisms

Inter-specific genetic relationships among regional populations of two species of grouper (Plectropomus maculatus and Plectropomus leopardus) were examined using mitochondrial and nuclear markers. mtDNA revealed contrasting regional inter-specific patterns whilst nuclear markers revealed contrasting patterns among markers, irrespective of region. In eastern Australia (EA) the species form a single mtDNA lineage, but the two species are reciprocally monophyletic in Western Australia (WA). This supports previous evidence for hybridisation between these species on the east coast. WA P. leopardus forms a sister relationship with the EA P. leopardus-maculatus clade while WA P. maculatus is more basal and sister to the P. leopardus lineages, indicating mtDNA does not suffer from incomplete lineage sorting for these species. In contrast, one of three nuclear markers (locus 7-90TG) differentiated the species into two reciprocally monophyletic clades, with no evidence of hybridisation or ancestral polymorphism. The remaining two nuclear markers (2-22 and ETS-2) did not separate these two species, while distinguishing other plectropomid species, suggesting incomplete lineage sorting at these nuclear loci. These results together with coalescence analyses suggest that P. leopardus females have hybridised historically with P. maculatus males and that P. maculatus mitochondria were displaced through introgressive hybridisation and fixation in the P. maculatus founder population on the Great Barrier Reef. The contrasting regional patterns of mtDNA structure may be attributed to Quaternary sea-level changes and shelf width differences driving different reef configurations on each coast. These reef configurations have provided opportunities for local scale interaction and reproduction among species on the narrower EA continental shelves, but not on the broader WA continental shelves.     

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.     

Estimating species phylogeny from gene-tree probabilities despite incomplete lineage sorting: an example from Melanoplus grasshoppers

Estimating phylogenetic relationships among closely related species can be extremely difficult when there is incongruence among gene trees and between the gene trees and the species tree. Here we show that incorporating a model of the stochastic loss of gene lineages by genetic drift into the phylogenetic estimation procedure can provide a robust estimate of species relationships, despite widespread incomplete sorting of ancestral polymorphism. This approach is applied to a group of montane Melanoplus grasshoppers for which genealogical discordance among loci and incomplete lineage sorting obscures any obvious phylogenetic relationships among species. Unlike traditional treatments where gene trees estimated using standard phylogenetic methods are implicitly equated with the species tree, with the coalescent-based approach the species tree is modeled probabilistically from the estimated gene trees. The estimated species phylogeny (the ESP) is calculated for the grasshoppers from multiple gene trees reconstructed for nuclear loci and a mitochondrial gene. This empirical application is coupled with a simulation study to explore the performance of the coalescent-based approach. Specifically, we test the accuracy of the ESP given the data based on analyses of simulated data matching the multilocus data collected in Melanoplus (i.e., data were simulated for each locus with the same number of base pairs and locus-specific mutational models). The results of the study show that ESPs can be computed using the coalescent-based approach long before reciprocal monophyly has been achieved, and that these statistical estimates are accurate. This contrasts with analyses of the empirical data collected in Melanoplus and simulated data based on concatenation of multiple loci, for which the incomplete lineage sorting of recently diverged species posed significant problems. The strengths and potential challenges associated with incorporating an explicit model of gene-lineage coalescence into the phylogenetic procedure to obtain an ESP, as illustrated by application to Melanoplus, versus concatenation and consensus approaches are discussed. This study represents a fundamental shift in how species relationships are estimated - the relationship between the gene trees and the species phylogeny is modeled probabilistically rather than equating gene trees with a species tree.     

Genealogical use of chloroplast DNA variation for intraspecific studies of Aegilops tauschii Coss.

Intraspecific patterns of chloroplast DNA variation was studied in Aegilops tauschii Coss., the D-genome progenitor of bread wheat. Nucleotide sequences of ten chloroplast microsatellite loci were analyzed for 63 accessions that cover the central part of the species distribution. As is often the case with nuclear microsatellites, those of chloroplasts of Ae. tauschii bear complex mutations. Several types of mutations other than change in the microsatellite repeat number were found, including base substitutions and length mutations in flanking regions. In total, eight mutations were present in the flanking regions of four loci. Most mutations in the flanking regions of microsatellite repeats are associated with biallelic polymorphisms. Phylogeographic analyses showed that such biallelic polymorphisms are useful to investigate intraspecific patterns of monophyletic lineage divergence. In contrast, most microsatellite repeat sites are multiallelic, variable within intraspecific lineages, and useful to compare degrees of genetic diversity between lineages. These findings show that the chloroplast genome harbors evolutionary variations informative for intraspecific studies of Ae. tauschii and can be analyzed by genealogical approaches.Electronic Supplementary Material Supplementary material is available for this article at     

Delimiting species without monophyletic gene trees

Genetic data are frequently used to delimit species, where species status is determined on the basis of an exclusivity criterium, such as reciprocal monophyly. Not only are there numerous empirical examples of incongruence between the boundaries inferred from such data compared to other sources like morphology -- especially with recently derived species, but population genetic theory also clearly shows that an inevitable bias in species status results because genetic thresholds do not explicitly take into account how the timing of speciation influences patterns of genetic differentiation. This study represents a fundamental shift in how genetic data might be used to delimit species. Rather than equating gene trees with a species tree or basing species status on some genetic threshold, the relationship between the gene trees and the species history is modeled probabilistically. Here we show that the same theory that is used to calculate the probability of reciprocal monophyly can also be used to delimit species despite widespread incomplete lineage sorting. The results from a preliminary simulation study suggest that very recently derived species can be accurately identified long before the requisite time for reciprocal monophyly to be achieved following speciation. The study also indicates the importance of sampling, both with regards to loci and individuals. Withstanding a thorough investigation into the conditions under which the coalescent-based approach will be effective, namely how the timing of divergence relative to the effective population size of species affects accurate species delimitation, the results are nevertheless consistent with other recent studies (aimed at inferring species relationships), showing that despite the lack of monophyletic gene trees, a signal of species divergence persists and can be extracted. Using an explicit model-based approach also avoids two primary problems with species delimitation that result when genetic thresholds are applied with genetic data -- the inherent biases in species detection arising from when and how speciation occurred, and failure to take into account the high stochastic variance of genetic processes. Both the utility and sensitivities of the coalescent-based approach outlined here are discussed; most notably, a model-based approach is essential for determining whether incompletely sorted gene lineages are (or are not) consistent with separate species lineages, and such inferences require accurate model parameterization (i.e., a range of realistic effective population sizes relative to potential times of divergence for the purported species). It is the goal (and motivation of this study) that genetic data might be used effectively as a source of complementation to other sources of data for diagnosing species, as opposed to the exclusion of other evidence for species delimitation, which will require an explicit consideration of the effects of the temporal dynamic of lineage splitting on genetic data.     

Phylogenetics of New World Astragalus: Screening of novel nuclear loci for the reconstruction of phylogenies at low taxonomic levels

This study explores methods to use information gathered from genomics technology to understand evolutionary relationships in the hyperdiverse legume group Neo-Astragalus. These species inhabit deserts and mountains of North and South America, and even though the monophyly of the group is well established, relationships within it are still poorly understood. Plastid genes, commonly used to infer phylogenies in plants, are usually not useful for closely related taxa because of low levels of genetic variation. TheMedicago truncatula genome project provided a suite of candidate nuclear loci with high levels of variation that might prove suitable for low-level phylogenetics. This paper reports the development of methods for screening a large number of these nuclear loci, and detailed analysis of four of them. Four different patterns of phylogenetic diversification occur in the loci sampled from these genomes ofAstragalus species. One locus (CNGC4) was single copy and could be directly used in phylogenetic analyses. Two loci (ARG10 and FENR) showed patterns strongly suggestive of duplication events in some taxa, and one locus (tRALS) has apparently undergone a cryptic duplication, making it very difficult to diagnose. Potential methods for using the information provided by these loci are discussed.     

Species monophyly

In biological systematics, as well as in the philosophy of biology, species and higher taxa are individuated through their unique evolutionary origin. This is taken by some authors to mean that monophyly is a (relational) property not only of higher taxa, but also of species. A species is said to originate through speciation, and to go extinct when it splits into two daughter species (or through terminal extinction). Its unique evolutionary origin is said to bestow identity on a species through time and change, and to render species names rigid designators. Species names are thus believed to function just like names of supraspecific taxa. However, large parts of the Web of Life are composed of species that do not have a unique evolutionary origin from a single population, lineage or stem-species. Further, monophyly is an ambiguous concept if it is defined simply in terms of 'unique evolutionary origin'. Disambiguating the concept by defining a monophyletic taxon as 'a taxon that includes the ancestor and all, and only, its descendant' renders monophyly inapplicable to species. At the heart of the problem lies a fundamental distinction between species and monophyletic taxa, where species form mutually exclusive reticulated systems, while higher taxa form inclusive hierarchical systems. Examples are given both at the species level and below to illustrate the problems that result from the application of the monophyly criterion to species. The conclusion is that the concepts of exclusivity and monophyly should be treated as non-overlapping: exclusivity marks out a species synchronistically, i.e. in the present time. Monophyly marks out clades (groups of species) diachronistically, i.e. within an historical dimension.