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.     

Species status and phylogeography of two closely related Coptolabrus species (Coleoptera: Carabidae) in South Korea inferred from mitochondrial and nuclear gene sequences

We investigated the species status and intraspecific phylogeography in South Korea of two ground beetle species, Coptolabrus jankowskii and Coptolabrus smaragdinus (Coleoptera: Carabidae), using statistical parsimony networks and nested clade analyses based on sequences from the mitochondrial cytochrome oxidase subunit I (COI) and nuclear phosphoenolpyruvate carboxykinase (PepCK) and wingless (Wg) genes. Although traditional parsimony tree construction generally failed to resolve interspecific relationships and construct biologically meaningful genealogies, analysis using statistical parsimony networks yielded statistically significant inter- and intraspecific genealogical structures. We found that although these two species represent a notable case of trans-species polymorphisms in both mitochondrial and nuclear gene sequences, their status as separate species was evidenced by the nonrandom association between species and nested clades at various nesting levels. The exceptional occurrence of shared identical or very similar COI sequences was considered to be the result of introgressive hybridization. In addition, range expansion and fragmentation events across the Korean Peninsula and adjacent islands were inferred from nested clade phylogeographical analyses. The COI gene revealed the geographical divergence of major eastern and western clades and historical biogeographical events within each major clade, whereas the nuclear PepCK gene, which did not reveal corresponding east-west clades, indicated past fragmentation and range expansion across wide areas that may have been the result of older biogeographical events. Thus, phylogeographical inferences drawn from analyses of mitochondrial and nuclear genes can reveal different and potentially complementary information about phylogeographical processes.     

Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history

Since the 1920s, population geneticists have had measures that describe how genetic variation is distributed spatially within a species' geographical range. Modern genetic survey techniques frequently yield information on the evolutionary relationships among the alleles or haplotypes as well as information on allele frequencies and their spatial distributions. This evolutionary information is often expressed in the form of an estimated haplotype or allele tree. Traditional statistics of population structure, such as F statistics, do not make use of evolutionary genealogical information, so it is necessary to develop new statistical estimators and tests that explicitly incorporate information from the haplotype tree. One such technique is to use the haplotype tree to define a nested series of branches (clades), thereby allowing an evolutionary nested analysis of the spatial distribution of genetic variation. Such a nested analysis can be performed regarding the geographical sampling locations either as categorical or continuous variables (i.e. some measure of spatial distance). It is shown that such nested phylogeographical analyses have more power to detect geographical associations than traditional, nonhistorical analyses and, as a consequence, allow a broader range of gene-flow parameters to be estimated in a precise fashion. More importantly, such nested analyses can discriminate between phylogeographical associations due to recurrent but restricted gene flow vs. historical events operating at the population level (e.g. past fragmentation, colonization, or range expansion events). Restricted gene flow and historical events can be intertwined, and the cladistic analyses can reconstruct their temporal juxtapositions, thereby yielding great insight into both the evolutionary history and population structure of the species. Examples are given that illustrate these properties, concentrating on the detection of range expansion events.     

The highly cross-fertile coral species,Acropora hyacinthus and Acropora cytherea,constitute statistically distinguishable lineages

A major challenge for understanding the evolutionary genetics of mass-spawning corals is to explain the maintenance of discrete morphospecies in view of high rates of interspecific fertilization in vitro and nonmonophyletic patterns in molecular phylogenies. In this study, we focused on Acropora cytherea and A. hyacinthus, which have one of the highest potentials for interspecific fertilization. Using sequences of a nuclear intron, we performed phylogenetic and nested clade analyses (NCA). Both species were polyphyletic in molecular phylogenies, but the NCA indicated that they constitute statistically distinguishable lineages. Phylogenetic analysis using an intergenic region of the mitochondrial DNA (mtDNA), was inconclusive because of low levels of variability in this marker. The position of these two species differed between the nuclear DNA (nDNA) and mtDNA phylogenies and was also at odds with a cladistic analysis based on morphology. We conclude that despite the potential for high levels of hybridization and introgression, A. cytherea and A. hyacinthus constitute statistically distinguishable lineages and their taxonomic status is consistent with the cohesion species concept.     

The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation

Phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA (mtDNA) variation were used to infer the temporal dynamics of distributional and demographic history of brown trout (Salmo trutta). Both new and previously published data were analyzed for 1,794 trout from 174 populations. This combined analysis improved our knowledge of the complex evolutionary history of brown trout throughout its native Eurasian and North African range of distribution in many ways. It confirmed the existence of five major evolutionary lineages that evolved in geographic isolation during the Pleistocene and have remained largely allopatric since then. These should be recognized as the basic evolutionarily significant units within brown trout. Finer phylogeographic structuring was also resolved within major lineages. Contrasting temporal juxtaposition of different evolutionary factors and timing of major demographic expansions were observed among lineages. These unique evolutionary histories have been shaped both by the differential latitudinal impact of glaciations on habitat loss and potential for dispersal, as well as climatic impacts and landscape heterogeneity that translated in a longitudinal pattern of genetic diversity and population structuring at more southern latitudes. This study also provided evidence for the role of biological factors in addition to that of physical isolation in limiting introgressive hybridization among major trout lineages.     

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.     

Testing phylogeographic predictions on an active volcanic island: Brachyderes rugatus (Coleoptera: Curculionidae) on La Palma (Canary Islands)

Volcanic islands with well-characterized geological histories can provide ideal templates for generating and testing phylogeographic predictions. Many studies have sought to utilize these to investigate patterns of colonization and speciation within groups of closely related species across a number of islands. Here we focus attention within a single volcanic island with a well-characterized geological history to develop and test phylogeographic predictions. We develop phylogeographic predictions within the island of La Palma of the Canary Islands and test these using 69 haplotypes from 570 base pairs of mitochondrial DNA cytochrome oxidase II sequence data for 138 individuals of Brachyderes rugatus rugatus, a local endemic subspecies of curculionid beetle occurring throughout the island in the forests of Pinus canariensis. Although geological data do provide some explanatory power for the phylogeographic patterns found, our network-based analyses reveal a more complicated phylogeographic history than initial predictions generated from data on the geological history of the island. Reciprocal illumination of geological and phylogeographic history is also demonstrated with previous geological speculation gaining phylogeographic corroboration from our analyses.     

Statistical phylogeography: methods of evaluating and minimizing inference errors

Nested clade phylogeographical analysis (NCPA) has become a common tool in intraspecific phylogeography. To evaluate the validity of its inferences, NCPA was applied to actual data sets with 150 strong a priori expectations, the majority of which had not been analysed previously by NCPA. NCPA did well overall, but it sometimes failed to detect an expected event and less commonly resulted in a false positive. An examination of these errors suggested some alterations in the NCPA inference key, and these modifications reduce the incidence of false positives at the cost of a slight reduction in power. Moreover, NCPA does equally well in inferring events regardless of the presence or absence of other, unrelated events. A reanalysis of some recent computer simulations that are seemingly discordant with these results revealed that NCPA performed appropriately in these simulated samples and was not prone to a high rate of false positives under sampling assumptions that typify real data sets. NCPA makes a posteriori use of an explicit inference key for biological interpretation after statistical hypothesis testing. Alternatives to NCPA that claim that biological inference emerges directly from statistical testing are shown in fact to use an a priori inference key, albeit implicitly. It is argued that the a priori and a posteriori approaches to intraspecific phylogeography are complementary, not contradictory. Finally, cross-validation using multiple DNA regions is shown to be a powerful method of minimizing inference errors. A likelihood ratio hypothesis testing framework has been developed that allows testing of phylogeographical hypotheses, extends NCPA to testing specific hypotheses not within the formal inference key (such as the out-of-Africa replacement hypothesis of recent human evolution) and integrates intra- and interspecific phylogeographical inference.     

autoinfer1.0: a computer program to infer biogeographical events automatically

autoinfer is a computer program for biogeographical inference based on nested clade analysis. To reduce the obscurity caused by manual inference, we defined geographically concordant clades and intermediate geographical areas between two clades. The program will perform most of the inferences automatically with a minimum of input from the user. We believe that autoinfer will save much time for the user compared with using the inference key by hand and, furthermore, will reduce the errors of inference resulting from different criteria in deduction.     

Phylogeography of the Vosges mountains populations of Gonioctena pallida (Coleoptera: Chrysomelidae): a nested clade analysis of mitochondrial DNA haplotypes

The pattern of genetic variation in the leaf beetle Gonioctena pallida was investigated inside the Vosges mountains using a highly variable 363 bp DNA fragment of the mitochondrial control region. Sequencing of 242 individuals, sampled in a geographical area of 100 x 40 km, identified 61 haplotypes whose genealogy was inferred. The resulting haplotype network exhibits four star-like phylogenies, two of which may be indicative of a population having recently expanded in size from a small number of founders. Nested clade analysis suggested multiple past expansion events, but also isolation by distance and possibly past fragmentation events, as the causes of the detected geographical associations of haplotypes. These results indicate the existence of effective barriers to gene flow inside the investigated area. Because the oldest demographic events inferred in the nested clade analysis were identified as expansion events, we hypothesize that a small population of founders have expanded not only in size, but also in geographical range from the south towards the north and east of the Vosges.     

Population genetic structure of the toad Bufo woodhousii: an empirical assessment of the effects of haplotype extinction on nested cladistic analysis

Nested cladistic analysis (NCA) is increasingly being used to infer historical population-level processes, including population fragmentation, range expansion and long-distance colonization. However, the effects on interpretation of NCA inferences of stochastic extinction of haplotypes due to genetic drift (lineage sorting), or of haplotype loss via localized biotic or climatic influences, have not been thoroughly explored. We provide empirical evidence suggesting that NCA may misinterpret population history when haplotypes or haplotype groups from one clade are replaced by those of another clade. We do so by using NCA to analyse mitochondrial sequences from the toad Bufo woodhousii from 45 locations spanning the Great Plains and southwestern USA. Portions of this region were glaciated and/or desertified in the late Pleistocene and early Holocene, and hence uninhabitable for plains-dwelling organisms. Although NCA inferences of isolation-by-distance and gradual range expansion in B. woodhousii are compatible with expectations based on climatic data and toad biology, NCA also detected several instances of long-distance movement. Such movement seems unlikely, given the low vagility of this species. We conclude that inferences of long-distance colonization likely result from extinction of haplotypes in intervening areas. We suggest using additional methods to look for congruent inferences, and amending the NCA inference key, to help avoid misinterpretations resulting from haplotype extinction.     

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.     

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.     

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.     

Population differentiation and phylogeographic pattern of a relict species, Conandron ramondioides (Gesneriaceae), revealed from sequence polymorphism and haplotypes of the CYCLOIDEA gene

Historical events are expected to affect population genetic differentiation and DNA molecular evolution,but the impact of these effects remains a matter of debate.Here,for Conandron ramondioides (Gesne...     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.     

Phylogeography of Cavernularia hultenii: evidence of slow genetic drift in a widely disjunct lichen

Population structure and history is poorly known in most lichenized ascomycetes. Many species display large-scale infraspecific disjunctions, which have been explained alternately by range fragmentation in species of high age and widespread long-distance dispersal. Using the lichen Cavernularia hultenii, which is widely disjunct across North America and Europe, Pleistocene and Holocene population history was inferred. The internal transcribed spacer (ITS) and part of the the intergenic spacer (IGS) region of the nuclear ribosomal DNA were sequenced in 300 individuals representing 62 populations across the range of the species. While four ancestral haplotypes are found in all areas, none of the observed tip haplotypes is present in more than one of the three part ranges. Although this is evidence for a past fragmentation event, nested clade analysis (NCA) remains equivocal in the choice between allopatric fragmentation and long-distance dispersal. Mismatch distributions indicate exponential population growth, probably during postglacial invasion of C. hultenii into formerly glaciated areas of western North America. The presence of one southern and at least one northern glacial refugium in South Central Alaska is inferred. Evidence for another refugium in the Queen Charlotte Islands or Alexander Archipelago is inconclusive because of sparse sampling. However, a range expansion was not confirmed unambiguously by NCA. The limited power of NCA to infer past range fragmentations and expansions is due apparently to the shallow haplotype network and widespread ancestral haplotypes. This can be explained by slow genetic drift causing incomplete removal of ancestral haplotypes from the postfragmentation and postexpansion areas.