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

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

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.     

The impact of intragenic recombination on phylogenetic reconstruction at the sectional level in Eucalyptus when using a single copy nuclear gene (cinnamoyl CoA reductase)

Low copy number nuclear genes have been found to be useful for phylogenetic reconstruction at different taxonomic levels. This study investigated the utility of a single copy gene, cinnamoyl CoA reductase (CCR), for resolving phylogenetic relationships at the sectional level within Eucalyptus. The monophyly of sections Exsertaria and Latoangulatae was explored, using section Maidenaria as an outgroup, and the impact of intragenic recombination on phylogenetic reconstruction examined. Phylogenetic analysis did not resolve monophyletic groups. Latoangulatae and Maidenaria were polyphyletic or paraphyletic. Exsertaria species formed a clade but included a single Latoangulatae species (E. major). Recombination analysis identified two intragenic recombination events that involved species from different sections, which have probably been facilitated by inter-sectional hybridisation. One of the events most likely occurred prior to speciation, with several Latoangulatae species having the recombinant allele. The other event may have occurred after speciation, since only one of two E. globulus samples possessed the recombinant allele. This is the first detailed report of intragenic recombination in both CCR and Eucalyptus, and between species of different sections of a plant genus. The occurrence of intragenic recombination may explain the anomalous positions of some species within the phylogenetic tree, and indicates that phylogenetic analysis of Eucalyptus using nuclear genes will be problematic unless recombination is taken into account.     

A Case of Rapid Diversification in the Neotropics: Phylogenetic Relationships among Cranioleuca Spinetails (Aves, Furnariidae)

Relationships among the 18-19 species of spinetails of the genus Cranioleuca are difficult to establish. Attempts based on traditional taxonomic characters have failed because of a high degree of homoplasy. Most morphological characters vary independently, producing leap-frog patterns of variation along the Eastern Brazilian Andean track, and behavior and vocalizations vary little. We use mtDNA sequence data from the cyt b and ND2 genes in an attempt to clarify relationships within the genus. We show (i) that Cranioleuca represents a recent burst of speciation and (ii) that a set of species thought by Maijer and Fjelds? (1997) to form a natural group is in fact a paraphyletic assemblage which also includes humid forest species with different pigmentations and vocalizations. However, synapomorphic variation in the sequences is not sufficient to unambiguously resolve the relationships within the genus. Several species (C. baroni, C. antisiensis, C. pyrrhophia, C. albiceps) show more than one haplotype, without any obvious correlation between genetic and geographic or morphological variation, and the different species do not always show reciprocal monophyly in haplotype diversity. Nevertheless, low genetic differentiation characterizes not only allopatric taxa but also some forms which are essentially sympatric, supporting species rank for the former. Our data suggest a recent diversification and proliferation possibly linked to Pleistocene climatic variation and its consequent vegetational shifts, at least in the Andean species.     

Genetic signatures of intermediate divergence: population history of Old and New World Holarctic ravens (Corvus corax)

Many studies of phylogeography, speciation, and species limits restrict their focus to a narrow issue: gene tree monophyly. However, reciprocal monophyly does not provide an ideal touchstone criterion of any aspect of evolutionary divergence. There is a continuum of divergence stages as isolated populations go from initial allele frequency differences to well-differentiated species. Studying intermediate stages of divergence will increase our understanding of geographical speciation, species limits, and conservation priorities. We develop a conceptual framework and terminology for thinking about the stages of 'intermediate polyphyly'. The Holarctic clade of common ravens (Corvus corax), found throughout much of Eurasia and North America, provides a case study of these stages of intermediate divergence. We used coalescent, phylogenetic, and population genetic methods to investigate the history and current status of this Old World-New World distribution using 107 mitochondrial control region sequences. Phylogenetically, New World and Old World samples are intermixed. However, most samples are grouped into small subclades that are restricted to either the New World or the Old World, and only one haplotype is shared between the hemispheres. Analysis of moleculalr variance (amova) results reflect this low haplotype sharing between hemispheres (Phi(ST) = 0.13, P < 0.01). Isolation with Migration (im) coalescent results suggest a sustained period of divergence between the hemispheres and low levels of maternal gene flow. Although there has not been sufficient time to evolve reciprocal monophyly and some gene flow may occur, New World and Old World ravens are genetically quite distinct. We use this example to demonstrate these early stages of divergence as populations go from sharing only internal haplotypes, to sharing no haplotypes, to having population specific subclades. Studies of phylogeography, speciation and systematics will benefit from increased attention to these stages of intermediate polyphyly.     

Phylogeography and alpha taxonomy of the common dolphin (Delphinus sp.)

The resolution of taxonomic classifications for delphinid cetaceans has been problematic, especially for species in the genera Delphinus, Tursiops and Stenella. The frequent lack of correspondence between morphological and genetic differentiation in these species raises questions about the mechanisms responsible for their evolution. In this study we focus on the genus Delphinus, and use molecular markers to address questions about speciation and the evolution of population structure. Delphinus species have a worldwide distribution and show a high degree of morphological variation. Two distinct morphotypes, long-beaked and short-beaked, have been considered different species named D. capensis and D. delphis, respectively. However, genetic differentiation between these two forms has only been demonstrated in the Pacific. We analysed samples from eight different geographical regions, including two morphologically defined long-beaked form populations, and compared these with the eastern North Pacific populations. We found high differentiation among the populations described as long-beaked instead of the expected monophyly, suggesting that these populations may have evolved from independent events converging on the same morphotype. We observed low genetic differentiation among the short-beaked populations across a large geographical scale. We interpret these phylogeographical patterns in the context of life history and population structure in related species.     

Mathematical consequences of the genealogical species concept

A genealogical species is defined as a basal group of organisms whose members are all more closely related to each other than they are to any organisms outside the group ("exclusivity"), and which contains no exclusive group within it. In practice, a pair of species is so defined when phylogenies of alleles from a sample of loci shows them to be reciprocally monophyletic at all or some specified fraction of the loci. We investigate the length of time it takes to attain this status when an ancestral population divides into two descendant populations of equal size with no gene exchange, and when genetic drift and mutation are the only evolutionary forces operating. The number of loci used has a substantial effect on the probability of observing reciprocal monophyly at different times after population separation, with very long times needed to observe complete reciprocal monophyly for a large number of loci. In contrast, the number of alleles sampled per locus has a relatively small effect on the probability of reciprocal monophyly. Because a single mitochondrial or chloroplast locus becomes reciprocally monophyletic much faster than does a single nuclear locus, it is not advisable to use mitochondrial and chloroplast DNA to recognize genealogical species for long periods after population divergence. Using a weaker criterion of assigning genealogical species status when more than 50% of sampled nuclear loci show reciprocal monophyly, genealogical species status depends much less on the number of sampled loci, and is attained at roughly 4-7 N generations after populations are isolated, where N is the historically effective population size of each descendant. If genealogical species status is defined as more than 95% of sampled nuclear loci showing reciprocal monophyly, this status is attained after roughly 9-12 N generations.     

Phylogeny of Philippine slender skinks (Scincidae: Brachymeles) reveals underestimated species diversity, complex biogeographical relationships, and cryptic patterns of lineage diversification

The spectacular, virtually endemic radiation of Philippine semi-fossorial skinks of the genus Brachymeles represent one of the few radiations of scincid lizards to possess both fully limbed and limbless species. And yet, nothing is known of the phylogenetic relationships of this exceptional group. Morphologically similar body plans have made it difficult to assess species-level diversity, and the genus has long been recognized as one of the more modest radiations of southeast Asian lizards. However, recent large-scale survey efforts have resulted in the discovery of numerous new species, and taxonomic studies indicate that the diversity within the genus Brachymeles is grossly underestimated. In this study we provide the first robust estimate of phylogenetic relationships within the genus Brachymeles using a multi-locus dataset and nearly complete taxonomic sampling. We provide statistical tests of monophyly for all polytypic species and two widespread limb-reduced species and our results indicate wholesale deviations from past summaries and taxonomic evaluations of the genus. With few exceptions, we are able to reject the monophyly of all polytypic and widespread species, thereby validating the need for large-scale taxonomic revisions. Our results reveal that the limbless, monotypic, genus Davewakeum is nested within Brachymeles. Mapping of body form on our preferred phylogenetic tree suggests that limb-reduction and digit loss has occurred on multiple occasions in the history of the genus. A Bayesian reconstruction of ancestral areas indicates strong statistical support for a minimum of five major dispersal events that have given rise to a major component of the observed species diversity on separate Pleistocene aggregate island platforms of the archipelago.     

Pleistocene effects on North American songbird evolution

Recent studies have used comparisons of mitochondrial DNA (mtDNA) sequence divergence among populations and species to test existing hypotheses about avian evolution during the Pleistocene epoch. In 1998, Avise and Walker concluded that the Pleistocene was an important time for avian evolution, including the initiation of phylogeographic separations and the completion of speciation events that began in the Pliocene. The study implied that these conclusions conflicted with the study, in 1997, by Klicka and Zink, which concluded that most species pairs previously thought to have originated in the past 250 000 years were much older. The two studies are complementary in the sense that Avise and Walker dealt primarily with phylogeographic (intraspecific) separations. Furthermore, Klicka and Zink concentrated on the inception of divergences whereas the Avise and Walker focused on the timing of the completion of speciation. To accomplish this, Avise and Walker analysed ''phylogroups'', geographically coherent subsets of biological species in which mtDNA haplotypes exhibit reciprocal monophyly. The study used the average interphylogroup mtDNA distance (0.027), calibrated at 2% per million years, to conclude that speciation required on average one million years to complete. Hence, speciation events begun in the Late Pliocene would have been completed in the mid- to late Pleistocene. Although we appreciate the extended nature of the speciation process and Avise and Walker''s insightful attempt to estimate its duration, we conclude that their value was an overestimate by a factor of two. In particular we question whether phylogroups can be used in the novel evolutionary role that Avise and Walker envisioned, because of the vagaries of taxonomic practices and lack of consensus regarding species concepts. To extend their analysis of intraspecific, phylogeographic separations, we compiled previously analysed and newly available data for divergence times for North American songbird (order Passeriformes) phylogroups. More than 80% were initiated at least one million years ago, which is inconsistent with the late Pleistocene origins model previously rejected by Klicka and Zink mentioned above. Although some divergence events can be traced to the late Pleistocene, the significance of the distribution must be judged with reference to a null model. Whether the Pleistocene was a profound time for avian phylogeographic differentiation is at present unknown.     

Tempo and mode of speciation in the Baja California disjunct fish species Anisotremus davidsonii

The Baja California region provides a natural setting for studying the early mechanisms of allopatric speciation in marine systems. Disjunct fish populations from several species that occur in the northern Gulf of California and northern Pacific coast of Baja California, but are absent from its southern shores, were previously shown to be genetically isolated, making them excellent candidates for studying allopatry. In addition, one of these species, the sargo Anisotremus davidsonii, has two pairs of congeneric Panamic trans-isthmian geminate species that allow for internal molecular clock calibration. Phylogeographic and demographic approaches based on mitochondrial (cytochrome b) and nuclear (S7 ribosomal protein) sequences showed that A. davidsonii entered the gulf from the south, and later colonized the Pacific coast, approximately 0.6-0.16 million years ago. Pacific coast colonization may have used a route either around the southern cape of Baja California or across the peninsula through a natural seaway. However, while several seaways have been described from different geological times, none matches the dates of population disjunction, yet much geological work remains to be done in that area. At the present time, there is no evidence for dispersal around the southern tip of the Baja California Peninsula. Signatures of incipient allopatric speciation were observed, such as the reciprocal monophyly of disjunct populations for the mitochondrial marker. However, other characteristics were lacking, such as a strong difference in divergence and coalescence times. Taken together, these results suggest that disjunct populations of A. davidsonii may be consistent with the earliest stages of allopatric speciation.     

Phylogeographic structure and cryptic speciation in the trans-Antarctic moss Pyrrhobryum mnioides

Abstract Many bryophyte species have distributions that span multiple continents. The hypotheses historically advanced to explain such distributions rely on either long-distance spore dispersal or slow rates of morphological evolution following ancient continental vicariance events. We use phylogenetic analyses of DNA sequence variation at three chloroplast loci ( atpB-rbcL spacer, rps4 gene, and trnL intron and 3'spacer) to examine these two hypotheses in the trans-Antarctic moss Pyrrhobryum mnioides. We find: (1) reciprocal monophyly of Australasian and South American populations, indicating a lack of intercontinental dispersal; (2) shared haplotypes between Australia and New Zealand, suggesting recent or ongoing migration across the Tasman Sea; and (3) reciprocal monophyly among Patagonian and neotropical populations, suggesting no recent migration along the Andes. These results corroborate experimental work suggesting that spore features may be critical determinants of species range. We use the mid-Miocene development of the Atacama Desert, 14 million years ago, to calibrate a molecular clock for the tree. The age of the trans-Antarctic disjunction is estimated to be 80 million years ago, consistent with Gondwanan vicariance, making it among the most ancient documented cases of cryptic speciation. These data are in accord with niche conservatism, but whether the morphological stasis is a product of stabilizing selection or phylogenetic constraint is unknown.     

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.     

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.     

Incipient speciation in a neotropical Gesneriaceae: <Emphasis Type="Italic">Columnea kucyniakii</Emphasis> is nested within <Emphasis Type="Italic">C. strigosa</Emphasis>

Speciation is an ongoing process. Many recognized species are fully divergent from each other and their ancestors, whereas others are in earlier stages in the diversification process. Such incipient speciation may create patterns when one or a few populations are phenotypically distinct, but lack genomic level coalescence from each other or from their ancestral species. As a result, such progenitor-derivative species pairs are likely to lack reciprocal monophyly or generate paraphyletic ancestral species. Here we examine phylogenetic patterns in the Columnea strigosa (Gesneriaceae) complex to evaluate whether populations that have been named C. kucyniakii are reciprocally monophyletic with C. strigosa, its presumed ancestral species. Molecular phylogenetic results do not support reciprocal monophyly of the two species, implying that incipient speciation is occurring within the C. strigosa complex. We hereby recommend that C. kucyniakii be recognized at the specific rank despite the fact that it creates a paraphyletic C. strigosa. These findings bear importance in taxonomic decisions about paraphyletic taxa and recognizing evolutionary and morphologically distinct lineages.     

Species designation of the Bruneau Dune tiger beetle (Cicindela waynei) is supported by phylogenetic analysis of mitochondrial DNA sequence data

Beetles comprise nearly one quarter of described species and show high levels of morphological and ecological diversification. Because of their wide distribution, ease of detection, and correlation of species richness patterns with other taxa, tiger beetles have been recommended for use as a global indicator of regional biodiversity, requiring accurate taxonomic designations. The Bruneau Dune tiger beetle (Cicindela waynei), whose habitat consists of an isolated dune field in southern Idaho, was recently described as a distinct species from the St. Anthony Dunes tiger beetle (C. arenicola) based on morphological characteristics. While these characteristics include distinct differences in genital morphology that could indicate intrinsic reproductive isolation, morphological characteristics have sometimes been misleading in tiger beetle taxonomy. To evaluate genetic support for this species designation, we analyzed 1,751 base pairs of mitochondrial DNA sequence from 147 tiger beetles collected throughout the range of both C. arenicola and C. waynei. Maximum-likelihood and Bayesian phylogenetic analyses indicated monophyly for C. waynei on a well-supported, short branch nested within C. arenicola. Bayesian species delimitation analyses strongly supported C. waynei as a distinct species (speciation probability = 1.0) with the estimated time of divergence ca. 14,500–67,000 years ago. This lack of reciprocal monophyly and recency of speciation is consistent with C. waynei as a member of an evolutionary front where speciation has occurred at a rapid rate. Mitochondrial sequence data supports the species designation of C. waynei, emphasizing the need to determine appropriate management for this species and its restricted habitat.     

Molecular phylogenetics and evolutionary analysis of body shape in the genus Cyrtonus (Coleoptera, Chrysomelidae)

The leaf beetle genus Cyrtonus Latreille, 1829 (Chrysomelidae, Chrysomelinae) is a species-rich genus subendemic to the Iberian Peninsula, with only a few species in the neighbouring France and Morocco. All the species are wingless and preferentially inhabiting mountainous areas. The taxonomic knowledge of this group is extremely poor and its systematics almost inexistent. Here, we analyse and characterize with a morphometric analysis one promising systematic trait, the adult body profile, distinguishing between elongated and rounded shapes. Although the monophyly of the genus is not contentious, we test for it using mitochondrial rrnl sequences and the chrysomeline homologous sequences available in GenBank. In addition, four genetic markers, two mitochondrial and two nuclear are used to produce a phylogenetic hypothesis for half the species within the genus and to analyse the evolution of shape, summarized as two continuous variables, length and width, and their ratio. These traits covary significantly with the phylogeny, showing a strong phylogenetic association: elongated species appear to constitute a clade within a paraphyletic assemblage of rounded species. In addition, the mitochondrial DNA tree is used to test for constant rate of evolution in this marker and is calibrated using both biogeographical evidence and the standard insect mitochondrial average substitution rate. This molecular clock hypothesis is used to date the age of speciation events on the phylogeny, reconstructing the origin of the genus in the Middle Miocene, with a relatively constant speciation rate until the end of the Pliocene and an apparent increase in this rate in the Pleistocene, possibly associated with the effect of dramatic climatic changes in this period. Finally, the high systematic value of shape profile in Cyrtonus is discussed, arguing the absence of evidence relating it to adaptation.     

Rapid radiation and cryptic speciation in squat lobsters of the genus Munida (Crustacea, Decapoda) and related genera in the South West Pacific: molecular and morphological evidence

Squat lobsters (genus Munida and related genera) are among the most diverse taxa of western Pacific crustaceans, though several features of their biology and phylogenetic relationships are unknown. This paper reports an extensive phylogenetic analysis based on mitochondrial DNA sequences (cytochrome c oxidase subunit I and 16S rRNA) and the morphology of 72 species of 12 genera of western Pacific squat lobsters. Our phylogenetic reconstruction using molecular data supports the recent taxonomic splitting of the genus Munida into several genera. Excluding one species (M. callista), the monophyly of the genus Munida was supported by Bayesian analysis of the molecular data. Three moderately diverse genera (Onconida, Paramunida, and Raymunida) also appeared monophyletic, both according to morphological and molecular data, always with high support. However, other genera (Crosnierita and Agononida) seem to be para- or polyphyletic. Three new cryptic species were identified in the course of this study. It would appear that the evolution of this group was marked by rapid speciation and stasis, or certain constraints, in its morphological evolution.