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.     

Genealogical portraits of speciation in montane grasshoppers (genus Melanoplus) from the sky islands of the Rocky Mountains

Grasshoppers in the genus Melanoplus have undergone a radiation in the 'sky islands' of western North America, with many species originating during the Pleistocene. Despite their recent origins, phylogenetic analyses indicate that all the species exhibit monophyletic or paraphyletic gene trees. The objectives of this study were to determine whether the monophyletic genealogies are the result of a bottleneck at speciation and to investigate the extent to which the different phylogenetic states of eight species (i.e. monophyletic versus paraphyletic gene trees) can be ascribed to the effects of speciation. A coalescent simulation was used to test for a bottleneck at speciation in each species. The effective population sizes and demographic histories of species were compared across taxa to evaluate the possibility that the paraphyly versus monophyly of the species reflects differential rates of lineage loss rather than speciation mode. While coalescent analyses indicate that the monophyly of Melanoplus species might not be indicative of bottlenecks at speciation, the results suggest that the paraphyletic gene trees may reflect the demography of speciation, involving localized divergences in the ancestral species. With respect to different models of Pleistocene divergence, the data do not support a model of founder-effect speciation but are compatible with divergence in allopatric refugia.     

Quaternary diversification in a sexual Holarctic zooplankter, Daphnia galeata

The effects of Quaternary glacial range partitioning on the diversification of Holarctic biota remain unclear. Glacial refugial lineages may form vicariant species, hybrid products, or merge after secondary contact. Here, we assess the effects of Quaternary glaciation on a Holarctic sexual zooplankter, Daphnia galeata, with apparently marked dispersal capacity and a widespread hybrid lineage in the New World. We collected samples of this species from 148 Holarctic lakes, analysed the nuclear and mitochondrial gene sequences, and tested predictions for hypotheses that account for the origin and spread of the New World D. galeata. We detected five nuclear phylogroups and four mitochondrial phylogroups, most of which were restricted to either the New World or the Old World. The oldest mitochondrial phylogroup was restricted to Japan. One major mitochondrial clade was distributed throughout the Holarctic, but only four haplotypes were shared among continents, and analysis of molecular variance indicated significant structure at the continental level. Haplotype sharing among continents could largely be attributed to anthropogenic introductions. Mismatch distributions, haplotype networks, phylogenetic trees, longitudinal haplotype diversity erosion and coalescence analyses are consistent with colonization from an Old World and a New World refugium. Our nuclear and mitochondrial DNA sequence evidence supports the hypothesis that the New World D. galeata underwent introgression with Daphnia dentifera, with dispersal being enhanced by glaciation. We conclude that Quaternary glaciation had a pronounced effect on the diversification of a Holarctic sexual zooplankter.     

Phylogeny, biogeography and taxonomy of the African wattle-eyes (Aves: Passeriformes: Platysteiridae)

The African wattle-eyes (genera Platysteira and Dyaphorophyia) comprise 10 species endemic to Africa. We analyzed both mitochondrial and nuclear DNA sequence data to test the monophyly of this group and its two genera, provide a preliminary assessment of species limits, and gain insight into the phylogeographic history of the wattle-eye radiation. Analyses based on mitochondrial ND2 sequences failed to recover wattle-eye monophyly, but the alternatives were not well-supported. In contrast, analyses of two nuclear introns (myoglobin intron-2 and beta-fibrinogen intron-5) recovered wattle-eye monophyly, as did combined analyses of mitochondrial and nuclear data. These analyses, however, did not support reciprocal monophyly of the two wattle-eye genera typically recognized, suggesting instead that Platysteira is nested within a paraphyletic Dyaphorophyia. The diversification of most wattle-eye species and many subspecies occurred through the divergence of allopatric populations well before the Pleistocene. Species and subspecies with disjunct distributions are typically characterized by deep genetic divergences, suggesting that many of these populations are evolutionary independent and could be recognized as additional phylogenetic species. In D. castanea and D. chalybea, for example, divergent haplotypes from geographically disjunct populations were paraphyletic with respect to those of D. tonsa and D. jamesoni, respectively. Similarly, Platysteira laticincta is highly divergent from its sister taxon P. peltata ( approximately 9.5% ND2 sequence divergence), consistent with species level recognition of this endangered species. In contrast, more broadly distributed taxa inhabiting a greater diversity of habitats (e.g., P. peltata and P. cyannea) show evidence of gene flow and connectivity among regions, suggesting that previously isolated populations expanded and fused into one another. Our study provides a framework for additional analyses of intraspecific phylogeography and species limits in these colorful birds.     

Mito‐nuclear discord in six congeneric lineages of Holarctic ducks (genus Anas)

Many species have Holarctic distributions that extend across Europe, Asia and North America. Most genetics research on these species has examined only mitochondrial (mt) DNA, which has revealed wide variance in divergence between Old World (OW) and New World (NW) populations, ranging from shallow, unstructured genealogies to deeply divergent lineages. In this study, we sequenced 20 nuclear introns to test for concordant patterns of OW–NW differentiation between mtDNA and nuclear (nu) DNA for six lineages of Holarctic ducks (genus Anas). Genetic differentiation for both marker types varied widely among these lineages (idiosyncratic population histories), but mtDNA and nuDNA divergence within lineages was not significantly correlated. Moreover, compared with the association between mtDNA and nuDNA divergence observed among different species, OW–NW nuDNA differentiation was generally lower than mtDNA divergence, at least for lineages with deeply divergent mtDNA. Furthermore, coalescent estimates indicated significantly higher rates of gene flow for nuDNA than mtDNA for four of the six lineages. Thus, Holarctic ducks show prominent mito‐nuclear discord between OW and NW populations, and we reject differences in sorting rates as the sole cause of the within‐species discord. Male‐mediated intercontinental gene flow is likely a leading contributor to this discord, although selection could also cause increased mtDNA divergence relative to weak nuDNA differentiation. The population genetics of these ducks contribute to growing evidence that mtDNA can be an unreliable indicator of stage of speciation and that more holistic approaches are needed for species delimitation.     

The effects of subdivision on the genetic divergence of populations and species

Abstract. An island model of migration is used to study the effects of subdivision within populations and species on sample genealogies and on between-population or between-species measures of genetic variation. The model assumes that the number of demes within each population or species is large. When populations (or species), connected either by gene flow or historical association, are themselves subdivided into demes, changes in the migration rate among demes alter both the structure of genealogies and the time scale of the coalescent process. The time scale of the coalescent is related to the effective size of the population, which depends on the migration rate among demes. When the migration rate among demes within populations is low, isolation (or speciation) events seem more recent and migration rates among populations seem higher because the effective size of each population is increased. This affects the probability of reciprocal monophyly of two samples, the chance that a gene tree of a sample matches the species tree, and relative likelihoods of different types of polymorphic sites. It can also have a profound effect on the estimation of divergence times.     

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.     

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.     

Biogeography of Speciation of Two Sister Species of Neotropical Amazona (Aves,Psittaciformes) Based on Mitochondrial Sequence Data

Coalescent theory provides powerful models for population genetic inference and is now increasingly important in estimates of divergence times and speciation research. We use molecular data and methods based on coalescent theory to investigate whether genetic evidence supports the hypothesis of A. pretrei and A. tucumana as separate species and whether genetic data allow us to assess which allopatric model seems to better explain the diversification process in these taxa. We sampled 13 A. tucumana from two provinces in northern Argentina and 28 A. pretrei from nine localities of Rio Grande do Sul, Brazil. A 491 bp segment of the mitochondrial gene cytochrome c oxidase I was evaluated using the haplotype network and phylogenetic methods. The divergence time and other demographic quantities were estimated using the isolation and migration model based on coalescent theory. The network and phylogenetic reconstructions showed similar results, supporting reciprocal monophyly for these two taxa. The divergence time of lineage separation was estimated to be approximately 1.3 million years ago, which corresponds to the lower Pleistocene. Our results enforce the current taxonomic status for these two Amazon species. They also support that A. pretrei and A. tucumana diverged with little or no gene flow approximately 1.3 million years ago, most likely after the establishment of a small population in the Southern Yungas forest by dispersion of a few founders from the A. pretrei ancestral population. This process may have been favored by habitat corridors formed in hot and humid periods of the Quaternary. Considering that these two species are considered threatened, the results were evaluated for their implications for the conservation of these two species.     

Population genetics and recent colonization history of the invasive drosophilid Zaprionus indianus in Mexico and Central America

Zaprionus indianus, also known as the African fig fly, is an invasive pest of a variety of commercial and native fruit. The species was first reported in Brazil in 1999, but has established itself in much of the New World within the last 10–15 years. We used nucleotide sequences from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene to examine haplotype relationships, population structure, and infer the colonization history of Z. indianus in Mexico and Panama. Construction of a haplotype network showed that six COI haplotypes, obtained from flies collected at six localities in Mexico and one in Panama, clustered into three distinct clades. Clade composition was generally consistent in flies from Panama to northwestern Mexico, and analysis of molecular variance indicated no significant structure among populations. Three of the six haplotypes from Mexico and Panama were identical to previously reported haplotypes from Brazil. None of the six haplotypes, however, were shared with previously reported haplotypes from potential source populations in the Old World. The results of our genetic analysis suggest that the invasion of Z. indianus into Central America and Mexico most probably includes a northward migration of individuals from Brazil, with the possibility of at least one additional introduction of Z. indianus to the New World. Additional sequence data from potential source populations in the Old World will be required to confidently determine the number of introductions of Z. indianus into the New World, and to identify the geographic source.     

Phylogeography of Biomphalaria glabrata and B. pfeifferi, important intermediate hosts of Schistosoma mansoni in the New and Old World tropics

The historical phylogeography of the two most important intermediate host species of the human blood fluke Schistosoma mansoni, B. glabrata in the New World, and B. pfeifferi in the Old World, was investigated using partial 16S and ND1 sequences from the mitochondrial genome. Nuclear sequences of an actin intron and internal transcribed spacer (ITS)-1 were also obtained, but they were uninformative for the relationships among populations. Phylogenetic analyses based on mtDNA revealed six well-differentiated clades within B. glabrata: the Greater Antilles, Venezuela and the Lesser Antilles, and four geographically overlapping Brazilian clades. Application of a Biomphalaria-specific mutation rate gives an estimate of the early Pleistocene for their divergence. The Brazilian clades were inferred to be the result of fragmentation, due possibly to climate oscillations, with subsequent range expansion producing the overlapping ranges. Within the Venezuela and Lesser Antilles clade, lineages from each of these areas were estimated to have separated approximately 740 000 years ago. Compared to B. glabrata, mitochondrial sequences of B. pfeifferi are about 4x lower in diversity, reflecting a much younger age for the species, with the most recent common ancestor of all haplotypes estimated to have existed 880 000 years ago. The oldest B. pfeifferi haplotypes occurred in southern Africa, suggesting it may have been a refugium during dry periods. A recent range expansion was inferred for eastern Africa less than 100 000 years ago. Several putative species and subspecies, B. arabica, B. gaudi, B. rhodesiensis and B. stanleyi, are shown to be undifferentiated from other B. pfeifferi populations.     

Multiple merger gene genealogies in two species: Monophyly, paraphyly, and polyphyly for two examples of Lambda coalescents

Probabilities of monophyly, paraphyly, and polyphyly of two-species gene genealogies are computed for modest sample sizes and compared for two different Λ coalescent processes. Coalescent processes belonging to the Λ coalescent family admit asynchronous multiple mergers of active ancestral lineages. Assigning a timescale to the time of divergence becomes a central issue when different populations have different coalescent processes running on different timescales. Clade probabilities in single populations are also computed, which can be useful for testing for taxonomic distinctiveness of an observed set of monophyletic lineages. The coalescence rates of multiple merger coalescent processes are functions of coalescent parameters. The effect of coalescent parameters on the probabilities studied depends on the coalescent process, and if the population is ancestral or derived. The probability of reciprocal monophyly tends to be somewhat lower, when associated with a Λ coalescent, under the null hypothesis that two groups come from the same population. However, even for fairly recent divergence times, the probability of monophyly tends to be higher as a function of the number of generations for coalescent processes that admit multiple mergers, and is sensitive to the parameter of one of the example processes.     

Ancestral Asian source(s) of new world Y-chromosome founder haplotypes

Haplotypes constructed from Y-chromosome markers were used to trace the origins of Native Americans. Our sample consisted of 2,198 males from 60 global populations, including 19 Native American and 15 indigenous North Asian groups. A set of 12 biallelic polymorphisms gave rise to 14 unique Y-chromosome haplotypes that were unevenly distributed among the populations. Combining multiallelic variation at two Y-linked microsatellites (DYS19 and DXYS156Y) with the unique haplotypes results in a total of 95 combination haplotypes. Contra previous findings based on Y- chromosome data, our new results suggest the possibility of more than one Native American paternal founder haplotype. We postulate that, of the nine unique haplotypes found in Native Americans, haplotypes 1C and 1F are the best candidates for major New World founder haplotypes, whereas haplotypes 1B, 1I, and 1U may either be founder haplotypes and/or have arrived in the New World via recent admixture. Two of the other four haplotypes (YAP+ haplotypes 4 and 5) are probably present because of post-Columbian admixture, whereas haplotype 1G may have originated in the New World, and the Old World source of the final New World haplotype (1D) remains unresolved. The contrasting distribution patterns of the two major candidate founder haplotypes in Asia and the New World, as well as the results of a nested cladistic analysis, suggest the possibility of more than one paternal migration from the general region of Lake Baikal to the Americas.     

Diversification of plant species in a subtropical region of eastern South American highlands: a phylogeographic perspective on native Petunia (Solanaceae)

In the Southern and Southeastern Brazilian highlands, a clade of seven species of Petunia that are endemic to the region (P. altiplana, P. bonjardinensis, P. guarapuavensis, P. mantiqueirensis, P. reitzii, P. saxicola and P. scheideana) exists in association with grassland formations. These formations are isolated in high-altitude regions, being surrounded by forested areas, and experienced contraction-expansion cycles associated with the glacial cycles of the Pleistocene. To understand the evolutionary history of this group, the divergence of which is probably linked to these past shifts in habitat, we analysed the sequences of the plastidial intergenic spacers trnH-psbA and trnS-trnG from populations throughout the known distributions of all seven species. The common ancestor of this highland clade started to differentiate ～0.9 million years (Myr) ago, which corresponds to a high diversification rate of 2.06 species per Myr in the intervening period. The high level of haplotype sharing among several species in the clade and the absence of reciprocal monophyly suggest the persistence of ancestral polymorphisms during speciation events and/or past hybridization, because no hybrid was found. Four of the five species displayed very low genetic diversity and possessed either one or two haplotypes, which is consistent with long-term isolation in restricted areas. The three more diverse species displayed significant population structure, and P. altiplana showed a clear signs of population growth during the last glacial period. These results suggest that diversification occurred as a result of expansion of the ancestral species of the clade during glacial periods followed by fragmentation and isolation during retraction in interglacial periods.     

Cyclic habitat displacements during Pleistocene glaciations have induced independent evolution of Tasimia palpata populations (Trichoptera: Tasimiidae) in isolated subtropical rain forest patches

Aim Mechanisms generating biodiversity and endemism are influenced by both historical and ecological patterns, and the relative roles of history vs. ecological interactions are still being debated. The phylogeography of one rain forest‐restricted caddisfly species, Tasimia palpata, thought to have good dispersal abilities, is used to address questions about shifts of highland rain forest habitat during Pleistocene glaciations and about their consequences for haplotype composition and distribution. Location Tasimia palpata occurs in highland subtropical rain forest patches, which are separated from one another by lowland dry bush, in south‐eastern Queensland, Australia. Methods We sequenced 375 base pairs of the mitochondrial cytochrome oxidase I gene from 169 individuals (20 populations) of T. palpata, mainly from three fragmented subtropical rain forest blocks, revealing 46 haplotypes. Analysis of molecular variance (amova ), genetic divergence between populations, nested clade analyses and tests based on coalescent theory were used to analyse phylogeographical relationships among T. palpata populations. Results amova indicates spatial genetic structure between isolated subtropical rain forest patches, with an isolation‐by‐distance effect. Tests based on coalescent theory suggest a repeated process of population reductions and divergence between isolated rain forests during Pleistocene glaciations as a consequence of habitat constrictions followed by population expansions during interglacial periods when subtropical rain forest expanded. In addition, these results suggest that, prior to the Pleistocene, rain forest and T. palpata had more widespread distributions in this region. Main conclusions Historical rain forest expansion and contraction during the Pleistocene resulted in changes in demography and genetic diversity of T. palpata, as well as in an increase in genetic divergence between populations from different patches of subtropical rain forest. Despite the fact that this caddisfly species was isolated in separate highland rain forest patches at various times during the Pleistocene, there is no evidence of allopatric speciation during the Quaternary, which contrasts with other examples of endemism and high diversity in rain forest highlands.     

Phylogeography and conservation genetics of the hellbender salamander (Cryptobranchus alleganiensis)

We investigated hellbender phylogeography through phylogenetic analyses of individuals sampled from 16 locations throughout their range in the eastern United States. Analyses were conducted on concatenated cytochrome-oxidase I (COI), cytochrome-b (Cytb) and NADH dehydrogenase subunit 4 (ND4) mtDNA sequence, totaling 2160 nucleotides. Hellbender haplotypes differed by 0.1 to 5.8% maximum likelihood (ML) corrected sequence divergence. Phylogenetic analyses reveal that hellbenders are separated into 8 reciprocally monophyletic populations or clades differentiated by a minimum of 0.7 to 5.4% sequence divergence, each of which constitutes a separate Management Unit (MU). High among population divergence and reciprocal monophyly suggest that female-mediated gene flow is severely restricted or non-existent among each MU. Hellbenders are currently divided into two subspecies, Cryptobranchus alleganiensis alleganiensis and C. a. bishopi based on morphological characters. The phylogenetic analyses presented here strongly indicate that these subspecies are paraphyletic. Management priorities for the hellbender should be reconsidered in light of these new molecular data. Results from Bayesian rooting indicate the root of the hellbender mtDNA tree lies on the branch leading to hellbender haplotypes from the Current, Eleven Point and New Rivers. The rooted tree suggests that a common ancestor in the southern Ozarks and/or southern Appalachians gave rise to northern hellbender populations, consistent with a Pleistocene refuge hypothesis.     

THE F1 HYBRIDS OF SOME SAPOGENIN-BEARING DIOSCOREA SPECIES

Fifteen species of Dioscorea and one of Rajania were cross-pollinated in 60 different combinations. Crosses among New World species were partially successful, whereas crosses between New and Old World or among Old World species usually failed entirely. Viable hybrids were obtained from 13 combinations, including all 12 possible combinations among 4 sapogenin-bearing species. Hybrids were intermediate to their parents with respect to vegetative characteristics, but exhibited wider variation or poor physiological control of critical growth phases, including length of the juvenile phase, dry season dormancy, and season of flowering. The flowers of hybrids were also intermediate to those of parents with no clear expressions of dominance. Chromosomal behavior of hybrids was essentially normal but reduction in fertility was manifested by high percentages of abnormal pollen, low crossability and poor seed germination. The significance of these findings with reference to speciation, reproductive barriers, and improvement by breeding is discussed.     

Australasian sky islands act as a diversity pump facilitating peripheral speciation and complex reversal from narrow endemic to widespread ecological supertramp

The Australasian archipelago is biologically extremely diverse as a result of a highly puzzling geological and biological evolution. Unveiling the underlying mechanisms has never been more attainable as molecular phylogenetic and geological methods improve, and has become a research priority considering increasing human‐mediated loss of biodiversity. However, studies of finer scaled evolutionary patterns remain rare particularly for megadiverse Melanesian biota. While oceanic islands have received some attention in the region, likewise insular mountain blocks that serve as species pumps remain understudied, even though Australasia, for example, features some of the most spectacular tropical alpine habitats in the World. Here, we sequenced almost 2 kb of mitochondrial DNA from the widespread diving beetle Rhantus suturalis from across Australasia and the Indomalayan Archipelago, including remote New Guinean highlands. Based on expert taxonomy with a multigene phylogenetic backbone study, and combining molecular phylogenetics, phylogeography, divergence time estimation, and historical demography, we recover comparably low geographic signal, but complex phylogenetic relationships and population structure within R. suturalis. Four narrowly endemic New Guinea highland species are subordinated and two populations (New Guinea, New Zealand) seem to constitute cases of ongoing speciation. We reveal repeated colonization of remote mountain chains where haplotypes out of a core clade of very widespread haplotypes syntopically might occur with well‐isolated ones. These results are corroborated by a Pleistocene origin approximately 2.4 Ma ago, followed by a sudden demographic expansion 600,000 years ago that may have been initiated through climatic adaptations. This study is a snapshot of the early stages of lineage diversification by peripatric speciation in Australasia, and supports New Guinea sky islands as cradles of evolution, in line with geological evidence suggesting very recent origin of high altitudes in the region.     

Species delimitation with abc and other coalescent-based methods: a test of accuracy with simulations and an empirical example with lizards of the liolaemus darwinii complex (squamata: liolaemidae)

Species delimitation is a major research focus in evolutionary biology because accurate species boundaries are a prerequisite for the study of speciation. New species delimitation methods (SDMs) can accommodate nonmonophyletic species and gene tree discordance as a result of incomplete lineage sorting via the coalescent model, but do not explicitly accommodate gene flow after divergence. Approximate Bayesian computation (ABC) can incorporate gene flow and estimate other relevant parameters of the speciation process while testing alternative species delimitation hypotheses. We evaluated the accuracy of BPP, SpeDeSTEM, and ABC for delimiting species using simulated data and applied these methods to empirical data from lizards of the Liolaemus darwinii complex. Overall, BPP was the most accurate, ABC showed an intermediate accuracy, and SpeDeSTEM was the least accurate under most simulated conditions. All three SDMs showed lower accuracy when speciation occurred despite gene flow, as found in previous studies, but ABC was the method with the smallest decrease in accuracy. All three SDMs consistently supported the distinctness of southern and northern lineages within L. darwinii. These SDMs based on genetic data should be complemented with novel SDMs based on morphological and ecological data to achieve truly integrative and statistically robust approaches to species discovery.     

Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionize phylogeography.