Delimiting species without monophyletic gene trees

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

Delimiting species using DNA and morphological variation and discordant species limits in spiny lizards (Sceloporus)

Haplotype phylogenies based on DNA sequence data are increasingly being used to test traditional species-level taxonomies based on morphology. However, few studies have critically compared species limits based on morphological and DNA data, and the methods used to delimit species using either type of data are only rarely explained. In this paper, we review three approaches for species delimitation (tree-based with DNA data and tree-based and character-based with morphological data) and propose explicit protocols for each. We then compare species limits inferred from these approaches, using morphological and mtDNA data for the Yarrow's spiny lizard (Sceloporus jarrovii), a traditionally polytypic species from the southwestern United States and Mexico. All three approaches support division of S. jarrovii into five species, but only two species are the same among the three approaches. We find the greatest support for the five species that are delimited based on mtDNA data, and we argue that mtDNA data may have important (and previously unappreciated) advantages for species delimitation. Because different data and approaches can disagree so extensively, our results demonstrate that the methodology of species delimitation is a critical issue in systematics.     

Delimiting cohesion species: extreme population structuring and the role of ecological interchangeability

Species exhibiting morphological homogeneity and strong population structuring present challenging taxonomic problems: morphology-based approaches infer few species, whereas genetic approaches often indicate more. Morphologically cryptic, yet genetically divergent species groups require alternative approaches to delimiting species that assess adaptive divergence and ecological interchangeability of lineages. We apply such an approach to Promyrmekiaphila, a small genus (three nominal taxa) of trapdoor spiders endemic to northern California to define cohesion species (lineages that are genetically exchangeable and ecologically interchangeable). Genetic exchangeability is evaluated using standard phylogeographical techniques (e.g. nested clade analysis); ecological interchangeability is assessed using two GIS-based approaches. First, climatic values are extracted from layer data for each locality point and utilized in a principal components analysis followed by MANOVA. Second, niche-based distribution models of genetically divergent lineages are created using a maximum-entropy modelling approach; the amount of overlap among lineages is calculated and evaluated against a probability distribution of null overlap. Lineages that have significant amounts of predicted overlap are considered ecologically interchangeable. Based on a synthetic evaluation of ecological interchangeability, geographical concordance, and morphological differentiation, we conclude that Promyrmekiaphila comprises six cohesion species, five of which are cryptic (i.e. undetectable by conventional means).     

Delimiting species boundaries for endangered Canary Island grasshoppers based on DNA sequence data

Mitochondrial DNA cytochrome oxidase subunit I and nuclear ITS2 sequences were surveyed from Canary Islands threatened species of the genera Purpuraria and Acrostira (Orthoptera: Pamphagidae). Phylogenetic and population analyses show that the two previously recognized Purpuraria erna subspecies are not valid as conservation units, and that there is a new unrecognized species of Purpuraria, coincident with recently discovered morphological variation within the genus. In addition, mitochondrial introgression seems to occur between the two Purpuraria species in southwest Lanzarote. Species-delimitation based on the morphological taxonomy of Acrostira, which recognizes four single-island endemics, is only partially supported by the genetic data. It shows that currently admitted species from the central and western islands of Tenerife, La Gomera and La Palma are closely related, with evidence of recent gene flow between the Tenerife and La Gomera populations. MtDNA variation also showed that A. euphorbiae, currently considered as the most critically endangered grasshopper species in the Canaries, has lower population diversity than its close relatives.     

Patterns of speciation in the Yucca moths: parallel species radiations within the Tegeticula yuccasella species complex

The interaction between yuccas and yucca moths has been central to understanding the origin and loss of obligate mutualism and mutualism reversal. Previous systematic research using mtDNA sequence data and characters associated with genitalic morphology revealed that a widespread pollinator species in the genus Tegeticula was in fact a complex of pollinator species that differed in host use and the placement of eggs into yucca flowers. Within this mutualistic clade two nonpollinating "cheater" species evolved. Cheaters feed on yucca seeds but lack the tentacular mouthparts necessary for yucca pollination. Previous work suggested that the species complex formed via a rapid radiation within the last several million years. In this study, we use an expanded mtDNA sequence data set and AFLP markers to examine the phylogenetic relationships among this rapidly diverging clade of moths and compare these relationships to patterns in genitalic morphology. Topologies obtained from analyses of the mtDNA and AFLP data differed significantly. Both data sets, however, corroborated the hypothesis of a rapid species radiation and suggested that there were likely two independent species radiations. Morphological analyses based on oviposition habit produced species groupings more similar to the AFLP topology than the mtDNA topology and suggested the two radiations coincided with differences in oviposition habit. The evolution of cheating was reaffirmed to have evolved twice and the closest pollinating relative for one cheater species was identified by both mtDNA and AFLP markers. For the other cheater species, however, the closest pollinating relative remains ambiguous, and mtDNA, AFLP, and morphological data suggest this cheater species may be diverged based on host use. Much of the divergence in the species complex can be explained by geographic isolation associated with the evolution of two oviposition habits.     

Fossil calibration of molecular divergence infers a moderate mutation rate and recent radiations for pinus

Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31x10(-9) and 0.22-0.42x10(-9).site-1.year-1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.     

Molecular phylogeny and biogeography of the bipolar Euphrasia (Orobanchaceae): recent radiations in an old genus

Euphrasia includes perennial or annual green parasitic plants, and has a disjunct bipolar distribution except for one transtropical connection across the high mountains of Oceania. The disjunction is coupled with strikingly contrasting patterns of morphological diversity between the southern and northern hemispheres, making it an exciting model to study processes of evolutionary diversification which shaped present floras. We inferred the relationships among 51 species representing 14 of the 15 sections of the genus based on nrDNA ITS and cpDNA trnL intron, trnL-trnF and atpB-rbcL intergenic spacers. Maximum parsimony and Bayesian inference support monophyly of the genus and of several intrageneric groups characterized by morphology, ploidy level, and geographic range. Molecular phylogenetic dating using Bayesian “relaxed” clock methods suggests that the earliest Euphrasia radiations occurred minimum 11–8 Mya with bipolarity being achieved 7–5 Mya. Biogeographic analyses using explicit model-based approach inferred Eurasia as an ancestral area for the genus. The most parsimonious reconstruction found by a dispersal-vicariance analysis requires 17 dispersals to account for the current biogeographic pattern and supports Eurasian origin for Euphrasia. Both long-distance dispersal and across land vicariance can be invoked to explain the diversification in the genus, which experienced rapid radiations driven by new ecological opportunities of the late Pliocene and Pleistocene but also retained a set of local endemic or relict species of an earlier origin.     

Delimiting species boundaries and the conservation genetics of the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough)

Species delimitation is a difficult problem that has implications across organismal biology, yet no single method has proved wholly satisfactory. We tested the utility of combining species-delimitation methods based on phylogeny and gene flow statistics using two parapatric members of the Coenonympha tullia group as an example: the endangered maritime ringlet butterfly (Coenonympha nipisiquit McDunnough) and the common inornate ringlet butterfly (Coenonympha inornata Edwards). We reconstructed the phylogeny of the nearctic C. tullia-group taxa from mitochondrial DNA (mtDNA) sequences (cytochrome oxidase I and mitochondrial control region) to explore the ancestry of the C. nipisiquit lineage within the group. We investigated the extent of gene flow between the two taxa with F-statistics using 587 nuclear amplified fragment length polymorphism markers, accounting for the effect of potential scoring 'collisions' where a marker may represent more than one DNA fragment. Combining species-delimitation methods was especially effective because it uncovered both historical and recent evolutionary patterns. Phylogenetic analysis of mtDNA revealed the early divergence of C. nipisiquit from other C. tullia-group taxa, including the morphologically similar C. inornata. F-statistics and gene-by-gene introgression profiles demonstrated clear isolation between the two taxa and revealed strong population structure within C. nipisiquit. C. nipisiquit is the first taxon in the nearctic C. tullia group showing strong evidence of genetic isolation. The methods we used are relatively inexpensive and can be widely used to delimit taxonomic boundaries near the species level, both generally and in particular for taxa that may be targets of conservation efforts.     

Alien species as a driver of recent extinctions

We assessed the prevalence of alien species as a driver of recent extinctions in five major taxa (plants, amphibians, reptiles, birds and mammals), using data from the IUCN Red List. Our results show that alien species are the second most common threat associated with species that have gone completely extinct from these taxa since AD 1500. Aliens are the most common threat associated with extinctions in three of the five taxa analysed, and for vertebrate extinctions overall.     

Delimiting Species without Nuclear Monophyly in Madagascar's Mouse Lemurs

Background

Speciation begins when populations become genetically separated through a substantial reduction in gene flow, and it is at this point that a genetically cohesive set of populations attain the sole property of species: the independent evolution of a population-level lineage. The comprehensive delimitation of species within biodiversity hotspots, regardless of their level of divergence, is important for understanding the factors that drive the diversification of biota and for identifying them as targets for conservation. However, delimiting recently diverged species is challenging due to insufficient time for the differential evolution of characters—including morphological differences, reproductive isolation, and gene tree monophyly—that are typically used as evidence for separately evolving lineages.

Methodology

In this study, we assembled multiple lines of evidence from the analysis of mtDNA and nDNA sequence data for the delimitation of a high diversity of cryptically diverged population-level mouse lemur lineages across the island of Madagascar. Our study uses a multi-faceted approach that applies phylogenetic, population genetic, and genealogical analysis for recognizing lineage diversity and presents the most thoroughly sampled species delimitation of mouse lemur ever performed.

Conclusions

The resolution of a large number of geographically defined clades in the mtDNA gene tree provides strong initial evidence for recognizing a high diversity of population-level lineages in mouse lemurs. We find additional support for lineage recognition in the striking concordance between mtDNA clades and patterns of nuclear population structure. Lineages identified using these two sources of evidence also exhibit patterns of population divergence according to genealogical exclusivity estimates. Mouse lemur lineage diversity is reflected in both a geographically fine-scaled pattern of population divergence within established and geographically widespread taxa, as well as newly resolved patterns of micro-endemism revealed through expanded field sampling into previously poorly and well-sampled regions.     

Resolution of recent radiations within three evolutionary lineages of felidae using mitochondrial restriction fragment length polymorphism variation

Patterns of mitochondrial restriction fragment length polymorphism (RFLP) variation were used to resolve more recent relationships among the species of the Felidae ocelot lineage, domestic cat lineage, and pantherine lineage. Twenty-five of 28 restriction enzymes revealed site variation in at least 1 of 21 cat species. The ocelot lineage was resolved into three separate sistertaxa groups: Geoffroy's cat (Oncifelis geoffroyi) and kodkod (O. guigna), ocelot (Leopardus pardalis) and margay (L. wiedii), and pampas cat (Lynchailurus colocolo) and most of the tigrina samples (Leopardus tigrina). Within the domestic cat lineage, domestic cat (Felis catus), European wild cat (F. silvestris), and African wild cat (F. libyca) formed a monophyletic trichotomy, which was joined with sand cat (F. margarita) to a common ancestor. Jungle cat (F. chaus) and black-footed cat (F. nigripes) mtDNAs diverged earlier than those of the other domestic cat lineage species and are less closely related. Within the pantherine lineage, phylogenetic analysis identified two distinct groups, uniting lion (P. leo) with leopard (P. pardus) and tiger (P. tigris) with snow leopard (P. uncia).To whom correspondence should be addressed.     

DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus

Comparing introduced to ancestral populations within a phylogeographical context is crucial in any study aiming to understand the ecological genetics of an invasive species. Zaprionus indianus is a cosmopolitan drosophilid that has recently succeeded to expand its geographical range upon three continents (Africa, Asia and the Americas). We studied the distribution of mitochondrial DNA (mtDNA) haplotypes for two genes (CO‐I and CO‐II) among 23 geographical populations. mtDNA revealed the presence of two well‐supported phylogenetic lineages (phylads), with bootstrap value of 100%. Phylad I included three African populations, reinforcing the African‐origin hypothesis of the species. Within phylad II, a distinct phylogeographical pattern was discovered: Atlantic populations (from the Americas and Madeira) were closer to the ancestral African populations than to Eastern ones (from Madagascar, Middle East and India). This means that during its passage from endemism to cosmopolitanism, Z. indianus exhibited two independent radiations, the older (the Eastern) to the East, and the younger (the Atlantic) to the West. Discriminant function analysis using 13 morphometrical characters was also able to discriminate between the two molecular phylads (93.34 ± 1.67%), although detailed morphological analysis of male genitalia using scanning electron microscopy showed no significant differences. Finally, crossing experiments revealed the presence of reproductive barrier between populations from the two phylads, and further between populations within phylad I. Hence, a bona species status was assigned to two new, cryptic species: Zaprionus africanus and Zaprionus gabonicus, and both were encompassed along with Z. indianus and Zaprionus megalorchis into the indianus complex. The ecology of these two species reveals that they are forest dwellers, which explains their restricted endemic distribution, in contrast to their relative cosmopolitan Z. indianus, known to be a human‐commensal. Our results reconfirm the great utility of mtDNA at both inter‐ and intraspecific analyses within the frame of an integrated taxonomical project.     

Ancestral polymorphism and recent invasion of transposable elements in Drosophila species

ABSTRACT: BACKGROUND: During the evolutionary history of transposable elements, some processes, such as ancestral polymorphisms and horizontal transfer of sequences between species, can produce incongruences in phylogenies. We investigated the evolutionary history of the transposable elements Bari and 412 in the sequenced genomes of the Drosophila melanogaster group and in the sibling species D. melanogaster and D. simulans using traditional phylogenetic and network approaches. RESULTS: The maximum likelihood (ML) phylogenetic analyses revealed incongruences and unresolved relationships for both the Bari and 412 elements. The DNA transposon Bari within the D. ananassae genome is more closely related to the element of the melanogaster complex than to the sequence in D. erecta, which is inconsistent with the species phylogeny. Divergence analysis and the comparison of the rate of synonymous substitutions per synonymous site of the Bari and host gene sequences explain the incongruence as an ancestral polymorphism inherited stochastically by the derived species. Unresolved relationships were observed in the ML phylogeny of both elements involving D. melanogaster, D. simulans and D. sechellia. A network approach was used to attempt to resolve these relationships. The resulting tree suggests recent transfers of both elements between D. melanogaster and D. simulans. The divergence values of the elements between these species support this conclusion. CONCLUSIONS: We showed that an ancestral polymorphism and recent invasion of genomes due to introgression or horizontal transfer between species occurred during the evolutionary history of the Bari and 412 elements in the melanogaster group. These invasions likely occurred in Africa during the Pleistocene, before the worldwide expansion of D. melanogaster and D. simulans.     

The uneven phylogeny and biogeography of Erodium (Geraniaceae): radiations in the Mediterranean and recent recurrent intercontinental colonization

Background and Aims

The genus Erodium is a common feature of Mediterranean-type climates throughout the world, but the Mediterranean Basin has significantly higher diversity than other areas. The aim here is to reveal the biogeographical history of the genus and the causes behind the evolution of the uneven distribution.

Methods

Seventy-eight new nrITS sequences were incorporated with existing plastid data to explore the phylogenetic relationships and biogeography of Erodium using several reconstruction methods. Divergence times for major clades were calculated and contrasted with other previously published information. Furthermore, topological and temporal diversification rate shift analyses were employed using these data.

Key Results

Phylogenetic relationships among species are widely congruent with previous plastid reconstructions, which refute the classical taxonomical classification. Biogeographical reconstructions point to Asia as the ancestral area of Erodium, arising approx. 18 MYA. Four incidences of intercontinental dispersal from the Mediterranean Basin to similar climates are demonstrated. Increases in diversification were present in two independent Erodium lineages concurrently. Two bursts of diversification (3 MYA and 0·69 MYA) were detected only in the Mediterranean flora.

Conclusions

Two lineages diverged early in the evolution of the genus Erodium: (1) subgenus Erodium plus subgenus Barbata subsection Absinthioidea and (2) the remainder of subgenus Barbata. Dispersal across major water bodies, although uncommon, has had a major influence on the distribution of this genus and is likely to have played as significant role as in other, more easily dispersed, genera. Establishment of Mediterranean climates has facilitated the spread of the genus and been crucial in its diversification. Two, independent, rapid radiations in response to the onset of drought and glacial climate change indicate putative adaptive radiations in the genus.     

A population genomic approach to map recent positive selection in model species

Based on nearly complete genome sequences from a variety of organisms data on naturally occurring genetic variation on the scale of hundreds of loci to entire genomes have been collected in recent years. In parallel, new statistical tests have been developed to infer evidence of recent positive selection from these data and to localize the target regions of selection in the genome. These methods have now been successfully applied to Drosophila melanogaster , humans, mice and a few plant species. In genomic regions of normal recombination rates, the targets of positive selection have been mapped down to the level of individual genes.     

Landscape genetics of a recent population extirpation in a burnet moth species

The intensification of agricultural land use over wide parts of Europe has led to the decline of semi-natural habitats, such as extensively used meadows, with those that remain often being small and isolated. These rapid changes in land use during recent decades have strongly affected populations inhabiting these ecosystems. Increasing habitat deterioration and declining permeability of the surrounding landscape matrix disrupt the gene flow within metapopulations. The burnet moth species Zygaena loti has suffered strongly from recent habitat fragmentation, as reflected by its declining abundance. We have studied its population genetic structure and found a high level of genetic diversity in some of the populations analysed, while others display low genetic diversity and a lack of heterozygosity. Zygaena loti was formerly highly abundant in meadows and along the skirts of forests. However, the species is currently restricted to isolated habitat remnants, which is reflected by the high genetic divergence among populations (F _ST: 0.136). Species distribution modelling as well as the spatial examination of panmictic clusters within the study area strongly support a scattered population structure for this species. We suggest that populations with a high level of genetic diversity still represent the former genetic structure of interconnected populations, while populations with low numbers of alleles, high F _IS values, and a lack of heterozygosity display the negative effects of reduced interconnectivity. A continuous exchange of individuals is necessary to maintain high genetic variability. Based on these results, we draw the general conclusion that more common taxa with originally large population networks and high genetic diversity suffer stronger from sudden habitat fragmentation than highly specialised species with lower genetic diversity which have persisted in isolated patches for long periods of time.