Molecular and morphological divergence in the butterfly genus Lycaeides (Lepidoptera: Lycaenidae) in North America: evidence of recent speciation

Male genital morphology, allozyme allele frequencies and mtDNA sequence variation were surveyed in the butterfly species Lycaeides idas and L. melissa from across much of their range in North America. Despite clear differences in male genital morphology, wing colour patterns and habitat characteristics, genetic variation was not taxonomically or geographically structured and the species were not identifiable by either genetic data set. Genetic distances (Nei's D=0.002–0.078, calculated from allozyme data) between all populations of both species were within the range commonly observed for conspecific populations of other butterflies. The most frequent mtDNA haplotype was present in individuals of both species in populations from southern California to Wisconsin. We conclude that speciation has probably happened recently and the lack of genetic differentiation between the species is the product of either (1) recent or ongoing gene flow at neutral loci, and/or (2) an insufficiency of time for lineage sorting. The evolution of male genital morphology, wing colour patterns and ecological characteristics has proceeded more rapidly than allozyme or mtDNA evolution.     

Phylogeography of shy and white-capped albatrosses inferred from mitochondrial DNA sequences: implications for population history and taxonomy

The evolutionary relationship between shy (Thalassarche cauta) and white-capped (T. steadi) albatrosses was examined using mitochondrial control region sequences. Results were interpreted in the context of a recent and contentious taxonomic revision that recommended full species status for shy and white-capped albatrosses. Low sequence divergence between shy and white-capped albatrosses (1.8%) and between their close relatives, Salvin's and Chatham albatrosses (2.9%), was observed. Much higher sequence divergence was found between the shy/white-capped pair and the Salvin's/Chatham pair (7.0%). Phylogenetic analyses confirmed the separation of the shy/white-capped pair from the Salvin's/Chatham pair but did not provide species-level resolution. Phylogeographic analyses, including a nested clade analysis, FST estimates and an analysis of molecular variance, indicated unambiguous genetic structuring between shy and white-capped albatrosses, thus confirming the demographic isolation of the species, but showed little to no structuring within each species. The geographical distribution of mtDNA haplotypes and other evidence suggest that shy albatrosses arose through range expansion by white-capped albatrosses.     

Cryptic Neogene vicariance and Quaternary dispersal of the red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas

We define the geographical distributions of mitochondrial DNA (mtDNA) lineages embedded within a broadly distributed, arid-dwelling toad, Bufo punctatus. These patterns were evaluated as they relate to hypothesized vicariant events leading to the formation of desert biotas within western North America. We assessed mtDNA sequence variation among 191 samples from 82 sites located throughout much of the species' range. Parsimony-based haplotype networks of major identified lineages were used in nested clade analysis (NCA) to further elucidate and evaluate shallow phylogeographic patterns potentially associated with Quaternary (Pleistocene-Holocene) vicariance and dispersal. Phylogenetic analyses provided strong support for three monophyletic lineages (clades) within B. punctatus. The geographical distributions of the clades showed little overlap and corresponded to the general boundaries of the Peninsular Desert, and two continental desert regions, Eastern (Chihuahuan Desert-Colorado Plateau) and Western (Mojave-Sonoran deserts), geographically separated along the Rocky Mountains and Sierra Madre Occidental. The observed divergence levels and congruence with postulated events in earth history implicate a late Neogene (latest Miocene-early Pliocene) time frame for separation of the major mtDNA lineages. Evaluation of nucleotide and haplotype diversity and interpretations from NCA reveal that populations on the Colorado Plateau resulted from a recent, likely post-Pleistocene, range expansion from the Chihuahuan Desert. Dispersal across historical barriers separating major continental clades appear to be recent, resulting in secondary contacts in at least two areas. Given the observed contact between major clades, we speculated as to why the observed deep phylogeographic structure has not been eroded during the multiple previous interglacials of the Pleistocene.     

Cryptic speciation in the southern African vlei rat Otomys irroratus complex: evidence derived from mitochondrial cyt b and niche modelling

The vlei rat Otomys irroratus has a wide distribution in southern Africa with several datasets indicating the presence of two putative species (O. irroratus and O. auratus). In the present study we use mitochrondrial cyt b data (～950 bp) from 98 specimens (including museum material) collected throughout the range of the species to determine the geographical limits of the two recognized species, and we link this to niche modelling to validate these species. Phylogenetic analysis of the DNA sequence data, using maximum parsimony, neighbour joining and Bayesian inference, retrieved two divergent statistically well‐supported clades. Clade A occurs in the Western and Eastern Cape while Clade B occurs in the Free State, KwaZulu‐Natal, Northern Cape and Mpumalanga provinces of South Africa and Zimbabwe. Mean sequence divergence between the two clades (A and B) was 7.0% and between sub‐clades comprising clade B it was 4.8%; the two clades diverged during the Pleistocene. Within Clade A the mean sequence divergence among specimens was 1.91%. Niche modelling revealed that the incipient species occupy distinct bioclimatic niches associated with seasonality of precipitation. Our data allow insightful analysis into the factors that could have led to cladogenesis within this rodent. More significantly, the new data enable us to pinpoint the Eastern Cape province as a contact zone for the divergent species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 192–206.     

Phylogeographic history of the land snail Candidula unifasciata (Helicellinae,Stylommatophora): fragmentation,corridor migration,and secondary contact

Abstract.— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.     

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae)

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A. drummondii as a monophyletic taxon, but found A. holboellii to bear chloroplast haplotypes from highly diverged evolutionary lineages. This raised the question of a possible polyphyletic origin of A. holboellii. Arabis x divaricarpa was found to be of recent and polytopic origin, a result consistent with its presumed hybrid origin. One hundred and three chloroplast haplotypes were detected within 719 Arabis accessions investigated. The majority of chloroplast-types were estimated to have arisen prior to the Wisconsin glaciation. Phylogeographical analysis using nested clade analysis, suggested for A. holboellii (i). past fragmentation events, partitioning genetic variation in several instances between the Sierra Nevada, the Southern Rocky Mountains and the Colorado Plateau on the one hand and the Central to Northern Rockies of the United States and adjacent Cascades on the other; and for both parental species (ii). recolonization of major areas formerly covered by the Wisconsin glaciation by three haplotypes; and (iii). restricted gene flow indicating isolation by distance in areas south of the last glacial maximum. Arabis x divaricarpa is closely codistributed with its parental species and resampled their haplotypes. The highest genetic diversity was found in the Rocky Mountains from Idaho and Montana south to Utah and Colorado. This area was further hypothesized to have played a major role in the origin of both parental species and probably represented an important glacial refugium. However, evidence for glacial refugia was also found in arctic and boreal regions of Alaska and near the Great Lakes. In comparison to nuclear ribosomal internal transcribed spacer data, chloroplast DNA divergence was very high and evidently predated the origin of A. drummondii and possibly A. holboellii. Divergence of major chloroplast lineages dates back to the middle of the Pleistocene at least. Extensive hybridization is the most likely evolutionary factor working on A. holboellii to explain the revealed discrepancy in nuclear DNA and chloroplast DNA diversification.     

Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences

The nature of gene flow in plants -- including the propensity for interspecific introgression -- makes them interesting candidates for phylogeographical analysis. Plant phylogeography studies have been limited, however, by the availability of suitable intraspecific variation. In this study, DNA sequence variation from a nuclear gene [Glyceraldehyde 3-phosphate dehydrogenase; (G3pdh)] was used to examine the population history of Manihot esculenta ssp. flabellifolia and a potentially hybridizing species, M. pruinosa. These species occur in the rainforest-savanna ecotone adjoining the Amazon basin, a region believed to have undergone major habitat shifts since the Pleistocene. Geographical distributions of the G3pdh haplotypes indicate genetic isolation-by-distance across the range of M. esculenta ssp. flabellifolia. However, there is greater genetic similarity between northeastern and western populations than would be expected given the present species distribution. A nested clade analysis suggests that northeastern and western populations were connected by gene flow until relatively recently, when they became fragmented. This inferred fragmentation event is consistent with post-Pleistocene habitat shifts proposed for the Amazon basin. At the interspecific level, haplotype sharing with M. pruinosa may reflect either recent interspecific introgression or incomplete lineage sorting between these closely related species.     

Gene flow, dispersal, and nested clade analysis among populations of the stonefly Peltoperla tarteri in the southern Appalachians

We examined the genetic structure and phylogeography of populations of the stonefly Peltoperla tarteri in the Southern Appalachians to determine the extent and likely mechanism for dispersal of this stream insect. A 454-base-pair (bp) portion of the mitochondrial control region was sequenced from a minimum of 20 individuals from eight populations. Pairwise FST and exact tests showed high levels of differentiation among almost all populations except those on the same stream. amova analysis detected significant genetic differentiation between streams within drainages (phi(SD) = 0.14, P < 0.001), and there was a slight positive correlation between aquatic distance and genetic distance (r = 0.295, P = 0.03). According to nested clade analysis, the present day pattern of genetic variation in P. tarteri is the result of a historical range expansion coupled with restricted gene flow with isolation by distance. Together, these analyses suggest that adult dispersal is limited and that movement by larvae is the primary dispersal mechanism for P. tarteri.     

Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.     

The evolution of geographic parthenogenesis in Timema walking-sticks

Phylogenetic studies of asexual lineages and their sexual progenitors are useful for inferring the causes of geographical parthenogenesis and testing hypotheses regarding the evolution of sex. With five known parthenogens and well-studied ecology, Timema walking-sticks are a useful system for studying these questions. Timema are mainly endemic to California and they exhibit the common pattern of geographical parthenogenesis, with asexuals exhibiting more-northerly distributions. Neighbour-joining and maximum-parsimony analyses of 416 bp of mitochondrial cytochrome oxidase I (COI) from 168 individuals were used to infer general phylogenetic relationships, resulting in three major phylogeographical subdivisions: a Northern clade; a Santa Barbara clade; and a Southern clade. A nested cladistic analysis, comparing intra- and interspecific haplotypic variation on a geographical scale, revealed that the overall pattern of geographical parthenogenesis in Timema could be attributed to historical range expansion. These results suggest that geographical parthenogenesis is the result of more-extensive northerly dispersal of asexuals than sexuals.     

Reconciling geography and genealogy: phylogeography of giant freshwater prawns from the Lake Carpentaria region

There is convincing geological evidence for the historical existence of an ancient lake on the Australian-New Guinea continental shelf during the late Pleistocene. Lake Carpentaria was a vast fresh- to brackishwater lake that would presumably have provided habitat for, and facilitated gene flow among, aquatic taxa that tolerate low to moderate salinities in this region. Moreover, it has been argued that the outflow of Papua New Guinea's Fly River was diverted westward into Lake Carpentaria during this period, although this hypothesis is controversial. We predicted that these events, if a true history, would have promoted gene flow and population growth via range-expansion events in the giant freshwater prawn (Macrobrachium rosenbergii) and restricted gene flow subsequently by way of a vicariant event as sea levels rose during the late Pleistocene, and a marine environment replaced Lake Carpentaria. We tested these hypotheses using phylogeographical and phylogenetic analyses of mitochondrial DNA variation in M. rosenbergii populations sampled from the Lake Carpentaria region. Our results support the hypothesis that Lake Carpentaria facilitated gene flow among populations of M. rosenbergii that are today isolated, but contest claims of a westward diversion of the Fly River. We inferred the timing of initial expansion in the 'Lake Carpentaria lineage' and found the timing of this event to be broadly concordant with geological dating of the formation of Lake Carpentaria. Reconciling geological and molecular data, as presented here, provides a powerful framework for investigating the influence of historical earth history events on the distribution of biological (i.e. molecular) diversity.     

Phylogeographical patterns in chloroplast DNA variation within the Acacia acuminata (Leguminosae: Mimosoideae) complex in Western Australia

Abstract The Acacia acuminata complex includes three taxa, A. acuminata ssp. acuminata, A. acuminata ssp. burkittii and A. oldfieldii, along with several informal variants of A. acuminata. It is widespread throughout southern Australia with the centre of diversity in south‐west Western Australia. Phylogeographical patterns in the complex were investigated using a nested clade analysis of cpDNA RFLPs from 25 populations in Western Australia. Except for A. oldfieldii that was clearly identified as a distinct entity, haplotypes were not restricted to sub‐specific taxa or variants within A. acuminata. There was significant association between phylogenetic position of many haplotypes and their geographical distribution. The fine‐scale phylogeographical patterns were complex but at deeper levels in the phylogeny there was evidence of divergence between two lineages. The pattern of shared haplotypes between lineages suggests retention of ancestral polymorphism as a result of incomplete lineage sorting. The divergence of these lineages is consistent with fragmentation caused by climatic instability during the Pleistocene.     

Phylogeographic analysis of the nocturnal velvet ant genus Dilophotopsis (Hymenoptera: Mutillidae) provides insights into diversification in the Nearctic deserts

Understanding the history of diversification in the North American deserts has long been a goal of biogeographers and evolutionary biologists. Although it appears that a consensus is forming regarding the patterns of diversification in the Nearctic deserts in vertebrate taxa, little work has been done exploring the historical biogeography of widespread invertebrate taxa. Before a robust model of geobiotic change in the North American deserts can be proposed, it needs to be determined whether the same historical events affected vertebrate and invertebrate taxa in the same way. We explore the phylogeographic patterns in a widespread nocturnal wasp genus Dilophotopsis using two rDNA loci, the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2). We use Bayesian phylogenetic analysis and haplotype network analysis to determine whether a consistent geographic pattern exists among species and populations within Dilophotopsis. We also used molecular dating techniques to estimate divergence dates of the major phylogenetic clades. Our analyses indicates that the species‐level divergences in Dilophotopsis occurred in the Neogene, and likely were driven by mountain building during the Miocene–Pliocene boundary (approximately 5 Mya) similar to the divergences in many vertebrate taxa. The population‐level divergences within species occurred during the Pleistocene (0.1–1.8 Mya). The present study shows that similar patterns of diversification exist in vertebrate and invertebrate taxa. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 360–375.     

Multiple causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan

In order to assess the utility of nested clade analysis, both standard phylogenetic algorithms and nested clade analysis were performed on a geographically widespread survey of mitochondrial DNA haplotypes of the bamboo viper, Trimeresurus stejnegeri, within Taiwan. Gross tree topologies were congruent for all analyses and indicated the presence of two geographically overlapping clades within Taiwan. The smaller lineage was restricted to the north and east coasts, whereas the larger lineage occupied all but the northern range of the species within Taiwan including the Pacific offshore populations of Green and Orchid Islands. The phylogeographical pattern supports the existence of at least one colonization event from the continent since the initial isolation of Taiwan from the mainland in the Pliocene. However, determining the exact number of colonization events was not possible due to the simultaneous vicariant forces of hypothesized continental landbridge connections and the occurrence of dramatic in situ orogenesis throughout the Pleistocene. Nested clade analysis provided multiple temporal and spatial population historical inferences that are not possible with standard analyses and therefore should become widely applied to future phylogeographical studies.     

Impact of Plio-Pleistocene arid cycling on the population history of a southwestern Australian frog

Southwestern Australia is regarded as a global biodiversity hotspot. The region contains a high number of endemic species, ranging from Gondwanan relicts to much more recently evolved plant and animal species. Myobatrachid frogs are diverse in southwestern Australia, and while we know they have speciated in situ in the southwest, we know little about the temporal and geographical patterning of speciation events. Crinia georgiana is an ideal subject to test hypotheses concerning the effect of climatic history on southwestern Australian anurans, as it is an old lineage with a broad distribution covering the entire region. We compiled an extensive phylogeographical data set based on 1085 bp of the mitochondrial gene ND2 for 68 individuals from 18 sites across the species' range. Two major genetic clades were identified which were largely confined to the high rainfall and southeast coastal biogeographical zones, respectively. The clades appear to have diverged around the Plio-Pleistocene border (1.26-1.72 million years ago), concordant with increasing intensity and frequency of arid climate cycles. Subsequent phylogeographical structure appears to have developed primarily during the Pleistocene climatic fluctuations that also have been integral in generating species diversity in the endemic southwestern Australian flora. Phylogeographical analyses identified several dispersal routes, possible refugial areas within the range of the species and also regions of secondary contact. Dispersal routes identified may now be closed to the species because of habitat destruction and salinity problems in inland regions, posing concerns about the evolutionary potential of the species in light of predicted climate change.     

Evolution of rattlesnakes (Viperidae; Crotalus) in the warm deserts of western North America shaped by Neogene vicariance and Quaternary climate change

During Pleistocene, the Laurentide ice sheet rearranged and diversified biotic distributions in eastern North America, yet had minimal physical impact in western North America where lineage diversification is instead hypothesized to result from climatic changes. If Pleistocene climatic fluctuations impacted desert species, the latter would reflect patterns of restricted gene flow concomitant with indications of demographic bottlenecks. Accordingly, molecular evidence for refugia should be present within these distributions and for subsequent range expansions as conditions improved. We sought answers to these questions by evaluating mitochondrial DNA (mtDNA) sequences from four species of rattlesnakes [Crotalus mitchellii (speckled rattlesnake), Crotalus cerastes (sidewinder), Crotalus tigris (tiger rattlesnake), Crotalus ruber (red diamond rattlesnake)] with distributions restricted to desert regions of southwestern North America. We inferred relationships using parsimony and maximum likelihood, tested intraspecific clades for population expansions, applied an isolation-with-migration model to determine bi-directional migration rates (m) among regions, and inferred divergence times for species and clades by applying a semiparametric penalized likelihood approach to our molecular data. Evidence for significant range expansion was present in two of eight regions in two species (Crotalus mitchellii pyrrhus, C. tigris region north). Two species (C. cerastes, C. mitchellii) showed a distribution concomitant with northward displacement of Baja California from mainland México, followed by vicariant separation into subclades. Effects of Pleistocene climate fluctuations were found in the distributions of all four species. Three regional diversification patterns were identified: (i) shallow genetic diversity that resulted from Pleistocene climatic events (C. tigris, C. ruber); (ii) deep Pleistocene divisions indicating allopatric segregation of subclades within refugia (C. mitchellii, C. cerastes); and (iii) lineage diversifications that extended to Pliocene or Late Miocene (C. mitchellii, C. cerastes). Clade-diversifying and clade-constraining effects impacted the four species of rattlesnakes unequally. We found relatively high levels of molecular diversification in the two most broadly distributed species (C. mitchellii, C. cerastes), and lower levels of genetic diversification in the two species (C. tigris, C. ruber) whose ranges are relatively more restricted. Furthermore, in several cases, the distributions of subspecies were not congruent with our molecular information. We suggest regional conservation perspectives for southwestern deserts cannot rely upon subspecies as biodiversity surrogates, but must instead employ a molecular and deep historical perspective as a primary mechanism to frame biodiversity reserves within this region.     

A phylogeographic analysis of populations of the wood turtle (Glyptemys insculpta) throughout its range

We investigated the phylogeography of wood turtles, Glyptemys insculpta, in North America using 750 bp of the mitochondrial control region from 117 individuals sampled at 29 localities across the species' range. A total of 21 haplotypes were identified and little genetic variation was found. The highest pairwise difference was 2%. From nested clade analysis (NCA), one main postglacial dispersal route was inferred along the east coast, with subsequent westward dispersal. NCA further revealed some patterns of restricted gene flow/dispersal. We propose that wood turtles experienced a combined effect of bottleneck during the Pleistocene as well as of selective sweep, which produced the low level of variation observed. Following the Pleistocene, wood turtles would have undergone a rapid northward expansion from a common southern refugium as glaciers retreated. These findings shed light on where to direct conservation priorities, on conservation strategies needed, and on the potential effects of interpopulation transfers for this vulnerable species.     

Comparative phylogeography of the two pink salmon broodlines: an analysis based on a mitochondrial DNA genealogy

Over most of their natural northern Pacific Ocean range, pink salmon (Oncorhynchus gorbuscha) spawn in a habitat that was repeatedly and profoundly affected by Pleistocene glacial advances. A strictly two-year life cycle of pink salmon has resulted in two reproductively isolated broodlines, which spawn in alternating years and evolved as temporal replicates of the same species. To study the influence of historical events on phylogeographical and population genetic structure of the two broodlines, we first reconstructed a fine-scale mtDNA haplotype genealogy from a sample of 80 individuals and then determined the geographical distribution of the major genealogical assemblages for 718 individuals sampled from nine Alaskan and eastern Asian even- and nine odd-year pink salmon populations. Analysis of restriction site states in seven polymerase chain reaction (PCR)-amplified mtDNA regions (comprising 97% of the mitochondrial genome) using 13 endonucleases resolved 38 haplotypes, which clustered into five genealogical lineages that differed from 0.065 to 0.225% in net sequence divergence. The lineage sorting between broodlines was incomplete, which suggests a recent common ancestry. Within each lineage, haplotypes exhibited star-like genealogies indicating recent population growth. The depth of the haplotype genealogy is shallow ( approximately 0.5% of nucleotide sequence divergence) and probably reflects repeated decreases in population size due to Pleistocene glacial advances. Nested clade analysis (NCA) of geographical distances showed that the geographical distribution observed for mitochondrial DNA (mtDNA) haplotypes resulted from alternating influences of historical range expansions and episodes of restricted dispersal. Analyses of molecular variance showed weak geographical structuring of mtDNA variation, except for the strong subdivision between Asian and Alaskan populations within the even-year broodline. The genetic similarities observed among and within geographical regions probably originated from postglacial recolonizations from common sources rather than extensive gene flow. The phylogeographical and population genetic structures differ substantally between broodlines. This can be explained by stochastic lineage sorting in glacial refugia and perhaps different recolonization routes in even- and odd-year broodlines.     

The automation and evaluation of nested clade phylogeographic analysis

Nested clade phylogeographic analysis (NCPA) is a popular method for reconstructing the demographic history of spatially distributed populations from genetic data. Although some parts of the analysis are automated, there is no unique and widely followed algorithm for doing this in its entirety, beginning with the data, and ending with the inferences drawn from the data. This article describes a method that automates NCPA, thereby providing a framework for replicating analyses in an objective way. To do so, a number of decisions need to be made so that the automated implementation is representative of previous analyses. We review how the NCPA procedure has evolved since its inception and conclude that there is scope for some variability in the manual application of NCPA. We apply the automated software to three published datasets previously analyzed manually and replicate many details of the manual analyses, suggesting that the current algorithm is representative of how a typical user will perform NCPA. We simulate a large number of replicate datasets for geographically distributed, but entirely random-mating, populations. These are then analyzed using the automated NCPA algorithm. Results indicate that NCPA tends to give a high frequency of false positives. In our simulations we observe that 14% of the clades give a conclusive inference that a demographic event has occurred, and that 75% of the datasets have at least one clade that gives such an inference. This is mainly due to the generation of multiple statistics per clade, of which only one is required to be significant to apply the inference key. We survey the inferences that have been made in recent publications and show that the most commonly inferred processes (restricted gene flow with isolation by distance and contiguous range expansion) are those that are commonly inferred in our simulations. However, published datasets typically yield a richer set of inferences with NCPA than obtained in our random-mating simulations, and further testing of NCPA with models of structured populations is necessary to examine its accuracy.