Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.     

Sequence capture and next‐generation sequencing of ultraconserved elements in a large‐genome salamander

Amidst the rapid advancement in next‐generation sequencing (NGS) technology over the last few years, salamanders have been left behind. Salamanders have enormous genomes—up to 40 times the size of the human genome—and this poses challenges to generating NGS data sets of quality and quantity similar to those of other vertebrates. However, optimization of laboratory protocols is time‐consuming and often cost prohibitive, and continued omission of salamanders from novel phylogeographic research is detrimental to species facing decline. Here, we use a salamander endemic to the southeastern United States, Plethodon serratus, to test the utility of an established protocol for sequence capture of ultraconserved elements (UCEs) in resolving intraspecific phylogeographic relationships and delimiting cryptic species. Without modifying the standard laboratory protocol, we generated a data set consisting of over 600 million reads for 85 P. serratus samples. Species delimitation analyses support recognition of seven species within P. serratus sensu lato, and all phylogenetic relationships among the seven species are fully resolved under a coalescent model. Results also corroborate previous data suggesting nonmonophyly of the Ouachita and Louisiana regions. Our results demonstrate that established UCE protocols can successfully be used in phylogeographic studies of salamander species, providing a powerful tool for future research on evolutionary history of amphibians and other organisms with large genomes.     

A genomic assessment of species boundaries and hybridization in a group of highly polymorphic anoles (distichus species complex)

Delimiting young species is one of the great challenges of systematic biology, particularly when the species in question exhibit little morphological divergence. Anolis distichus, a trunk anole with more than a dozen subspecies that are defined primarily by dewlap color, may actually represent several independent evolutionary lineages. To test this, we utilized amplified fragment length polymorphisms (AFLP) genome scans and genetic clustering analyses in conjunction with a coalescent‐based species delimitation method. We examined a geographically widespread set of samples and two heavily sampled hybrid zones. We find that genetic divergence is associated with a major biogeographic barrier, the Hispaniolan paleo‐island boundary, but not with dewlap color. Additionally, we find support for hypotheses regarding colonization of two Hispaniolan satellite islands and the Bahamas from mainland Hispaniola. Our results show that A. distichus is composed of seven distinct evolutionary lineages still experiencing a limited degree of gene flow. We suggest that A. distichus merits taxonomic revision, but that dewlap color cannot be relied upon as the primary diagnostic character.     

Considering gene flow when using coalescent methods to delimit lineages of North American pitvipers of the genus Agkistrodon

Examining species diversity and mechanisms of speciation using coalescent models provides a framework for how regional diversity is accrued, even in well‐studied areas such as the Nearctic. It is likely, that gene flow among closely‐related species with adjacent distributions may be common. However, the absence of gene flow is a primary assumption of many phylogeographical methods that produce species trees and delimit species using Bayesian or likelihood functions in a coalescent framework. In the present study, we examine delimitation when gene flow between species is present using empirical datasets from two species of North American pitvipers of the genus Agkistrodon. We also use niche modelling to determine whether these young lineages occur in distinct environmental niches. To manage the problem of gene flow between species, we first identify admixed individuals, demonstrate that gene flow has occurred, and then identify the impact of alternative population assignments of admixed individuals on delimitation posterior probabilities. In addition, we examine the influence of mitochondrial genes relative to other loci combined in coalescent analyses that delimit species. Here, we find that the copperheads (Agkistrodon contortrix) and the cottonmouths (Agkistrodon piscivorus) are each composed of two distinct species, with each occupying different niches. Importantly, we find that species can be delimited when the amount of gene flow between lineages is low, although the methods are acutely sensitive to population assignment of individuals. © 2014 The Linnean Society of London     

Sky island diversification meets the multispecies coalescent – divergence in the spruce‐fir moss spider (Microhexura montivaga,Araneae, Mygalomorphae) on the highest peaks of southern Appalachia

Microhexura montivaga is a miniature tarantula‐like spider endemic to the highest peaks of the southern Appalachian mountains and is known only from six allopatric, highly disjunct montane populations. Because of severe declines in spruce‐fir forest in the late 20th century, M. montivaga was formally listed as a US federally endangered species in 1995. Using DNA sequence data from one mitochondrial and seven nuclear genes, patterns of multigenic genetic divergence were assessed for six montane populations. Independent mitochondrial and nuclear discovery analyses reveal obvious genetic fragmentation both within and among montane populations, with five to seven primary genetic lineages recovered. Multispecies coalescent validation analyses [guide tree and unguided Bayesian Phylogenetics and Phylogeography (BPP), Bayes factor delimitation (BFD)] using nuclear‐only data congruently recover six or seven distinct lineages; BFD analyses using combined nuclear plus mitochondrial data favour seven or eight lineages. In stark contrast to this clear genetic fragmentation, a survey of secondary sexual features for available males indicates morphological conservatism across montane populations. While it is certainly possible that morphologically cryptic speciation has occurred in this taxon, this system may alternatively represent a case where extreme population genetic structuring (but not speciation) leads to an oversplitting of lineage diversity by multispecies coalescent methods. Our results have clear conservation implications for this federally endangered taxon and illustrate a methodological issue expected to become more common as genomic‐scale data sets are gathered for taxa found in naturally fragmented habitats.     

A genomic evaluation of taxonomic trends through time in coast horned lizards (genus Phrynosoma)

Determining the boundaries between species and deciding when to describe new species are challenging practices that are particularly difficult in groups with high levels of geographic variation. The coast horned lizards (Phrynosoma blainvillii, Phrynosoma cerroense and P. coronatum) have an extensive geographic distribution spanning many distinctive ecological regions ranging from northern California to the Cape Region of Baja California, Mexico, and populations differ substantially with respect to external morphology across much of this range. The number of taxa recognized in the group has been reevaluated by herpetologists over 20 times during the last 180 years, and typically without the aid of explicit species delimitation methods, resulting in a turbulent taxonomy containing anywhere from one to seven taxa. In this study, we evaluate taxonomic trends through time by ranking 15 of these species delimitation models (SDMs) using coalescent analyses of nuclear loci and SNPs in a Bayesian model comparison framework. Species delimitation models containing more species were generally favoured by Bayesian model selection; however, several three‐species models outperformed some four‐ and five‐species SDMs, and the top‐ranked model, which contained five species, outperformed all SDMs containing six species. Model performance peaked in the 1950s based on marginal likelihoods estimated from nuclear loci and SNPs. Not surprisingly, SDMs based on genetic data outperformed morphological taxonomies when using genetic data alone to evaluate models. The de novo estimation of population structure favours a three‐population model that matches the currently recognized integrative taxonomy containing three species. We discuss why Bayesian model selection might favour models containing more species, and why recognizing more than three species might be warranted.     

Species‐delimitation and phylogenetic analyses of some cosmopolitan species of Hypnea (Rhodophyta) reveal synonyms and misapplied names to H. cervicornis,including a new species from Brazil

Hypnea has an intricate nomenclatural history due to a wide pantropical distribution and considerable morphological variation. Recent molecular studies have provided further clarification on the systematics of the genus; however, species of uncertain affinities remain due to flawed taxonomic identification. Detailed analyses coupled with literature review indicated a strong relationship among H. aspera, H. cervicornis, H. flexicaulis, and H. tenuis, suggesting a need for further taxonomic studies. Here, we analyzed sequences from two molecular markers (COI‐5P and rbcL) and performed several DNA‐based delimitation methods (mBGD, ABGD, SPN, PTP and GMYC). These molecular approaches were contrasted with morphological and phylogenetic evidence from type specimens and/or topotype collections of related species under a conservative approach. Our results demonstrate that H. aspera and H. flexicaulis represent heterotypic synonyms of H. cervicornis and indicate the existence of a misidentified Hypnea species, widely distributed on the Brazilian coast, described here as a new species: H. brasiliensis. Finally, inconsistencies observed among our results based on six different species delimitation methods evidence the need for adequate sampling and marker choice for different methods.     

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.     

Toward an inordinate fondness for stars,beetles and Lobophora? Species diversity of the genus Lobophora (Dictyotales,Phaeophyceae) in New Caledonia

Until the recent use of molecular markers, species diversity of Lobophora, an ecologically important brown algal genus with a worldwide distribution in temperate and tropical seas, has been critically underestimated. Using a DNA‐based taxonomic approach, we re‐examined diversity of the genus from New Caledonia in the Southwest Pacific Ocean. First, species were delineated using general mixed Yule coalescent‐based and barcoding gap approaches applied to a mitochondrial cox3 data set. Results were subsequently confirmed using chloroplast psbA and rbcL data sets. Species delimitation analyses agreed well across markers and delimitation algorithms, with the barcoding gap approach being slightly more conservative. Analyses of the cox3 data set resulted in 31–39 molecular operational taxonomic units (MOTUs), four of which are previously described species (L. asiatica, L. crassa, L. nigrescens s.l., L. pachyventera). Of the remaining MOTUs for which we obtained a representative number of sequences and results are corroborated across analyses and genes, we described 10 species de novo: L. abaculusa, L. abscondita, L. densa, L. dimorpha, L. gibbera, L. hederacea, L. monticola, L. petila, L. rosacea, and L. undulata. Our study presents an excellent case of how a traditional morphology‐based taxonomy fails to provide accurate estimates of algal diversity. Furthermore, the level of Lobophora diversity unveiled from a single locality in the Pacific Ocean raises important questions with respect to the global diversity of the genus, the distributions and range sizes of the individual species, as well as the mechanisms facilitating coexistence.     

Recent rapid speciation and ecomorph divergence in Indo‐Australian sea snakes

The viviparous sea snakes (Hydrophiinae) are a young radiation of at least 62 species that display spectacular morphological diversity and high levels of local sympatry. To shed light on the mechanisms underlying sea snake diversification, we investigated recent speciation and eco‐morphological differentiation in a clade of four nominal species with overlapping ranges in Southeast Asia and Australia. Analyses of morphology and stomach contents identified the presence of two distinct ecomorphs: a ‘macrocephalic’ ecomorph that reaches >2 m in length, has a large head and feeds on crevice‐dwelling eels and gobies; and a ‘microcephalic’ ecomorph that rarely exceeds 1 m in length, has a small head and narrow fore‐body and hunts snake eels in burrows. Mitochondrial sequences show a lack of reciprocal monophyly between ecomorphs and among putative species. However, individual assignment based on newly developed microsatellites separated co‐distributed specimens into four significantly differentiated clusters corresponding to morphological species designations, indicating limited recent gene flow and progress towards speciation. A coalescent species tree (based on mitochondrial and nuclear sequences) and isolation‐migration model (mitochondrial and microsatellite markers) suggest between one and three transitions between ecomorphs within the last approximately 1.2 million to approximately 840 000 years. In particular, the macrocephalic ‘eastern’ population of Hydrophis cyanocinctus and microcephalic H. melanocephalus appear to have diverged very recently and rapidly, resulting in major phenotypic differences and restriction of gene flow in sympatry. These results highlight the viviparous sea snakes as a promising system for speciation studies in the marine environment.     

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by‐product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data‐limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.     

Genome‐wide sequence information reveals recurrent hybridization among diploid wheat wild relatives

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome‐wide sequence information, independent data were obtained from genotyping‐by‐sequencing and a target‐enrichment experiment that returned 244 low‐copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent‐based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat‐group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat‐group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.     

An assessment of species limits of the South American mouse genus Oligoryzomys (Rodentia,Cricetidae) using unilocus delimitation methods

Oligoryzomys, as currently understood is formed by 25 living species, is the most diverse genus of the tribe Oryzomyini of the New World subfamily Sigmodontinae of cricetid rodents. Nonetheless, the species richness of Oligoryzomys seems to be an underestimate, given some species complex has been proposed in previous studies, at the time that large geographic areas remain to be sampled, and several taxonomic forms have not been assessed with contemporary approaches. In this study, we present a new assessment of the species diversity of Oligoryzomys based on multiple unilocus species delimitation methods (ABGD, BPP, PTP, GMYC and b GMYC), using 665 cytb gene sequences as evidence (532 gathered from Genbank and 133 obtained in this study). We sampled representatives of almost all currently known species of Oligoryzomys, at the time that extending the geographic coverage to the Central Andes, a large area that was largely unrepresented in previous studies. Phylogenetic relationships, based on a non‐redundant alignment, were inferred via maximum likelihood and Bayesian inference; an ultrametric tree, used in species delimitation analyses, was obtained using multiple secondary calibration points. Results of species delimitation methods are discussed at the light of previous knowledge (e.g., taxonomic history and geographic provenance of samples in relation to type localities) and the morphological assessments of some specimens. Results of the distinct delimitation methods are mostly congruent, being BPP and PTP the most sensible to estimate species delimitation, allowing us to suggest that Oligoryzomys is composed of 30 lineages of species level. Of these, 22 correspond to forms currently considered species; some of these include in their synonymy some forms currently considered valid species (e.g., yatesi would be a synonym of longicaudatus). The remaining eight lineages are candidate species that need to be further evaluated. This study, by advancing taxonomic hypothesis that should be further tested in future studies, constitutes a stepping‐stone for upcoming taxonomic and biogeographic studies centred on Oligoryzomys.     

Contrasting support for alternative models of genomic variation based on microhabitat preference: species‐specific effects of climate change in alpine sedges

Deterministic processes may uniquely affect codistributed species’ phylogeographic patterns such that discordant genetic variation among taxa is predicted. Yet, explicitly testing expectations of genomic discordance in a statistical framework remains challenging. Here, we construct spatially and temporally dynamic models to investigate the hypothesized effect of microhabitat preferences on the permeability of glaciated regions to gene flow in two closely related montane species. Utilizing environmental niche models from the Last Glacial Maximum and the present to inform demographic models of changes in habitat suitability over time, we evaluate the relative probabilities of two alternative models using approximate Bayesian computation (ABC) in which glaciated regions are either (i) permeable or (ii) a barrier to gene flow. Results based on the fit of the empirical data to data sets simulated using a spatially explicit coalescent under alternative models indicate that genomic data are consistent with predictions about the hypothesized role of microhabitat in generating discordant patterns of genetic variation among the taxa. Specifically, a model in which glaciated areas acted as a barrier was much more probable based on patterns of genomic variation in Carex nova, a wet‐adapted species. However, in the dry‐adapted Carex chalciolepis, the permeable model was more probable, although the difference in the support of the models was small. This work highlights how statistical inferences can be used to distinguish deterministic processes that are expected to result in discordant genomic patterns among species, including species‐specific responses to climate change.     

Bayesian Poisson tree processes and multispecies coalescent models shed new light on the diversification of Nawab butterflies in the Solomon Islands (Nymphalidae,Charaxinae, Polyura)

Butterflies of the genus Polyura form a widespread tropical group distributed from Pakistan to Fiji. The rare endemic Polyura epigenes Godman & Salvin, 1888 from the Solomon Islands archipelago represents a case of marked island polymorphism. We sequenced museum specimens of this species across its geographic range to study the phylogeography and genetic differentiation of populations in the archipelago. We used the Bayesian Poisson tree processes and multispecies coalescent models, to study species boundaries. We also estimated divergence times to investigate the biogeographic history of populations. Our molecular species delimitation and nuclear DNA network analyses unambiguously indicate that Malaita populations form an independent metapopulation lineage, as defined in the generalized lineage concept. This lineage, previously ranked as a subspecies, is raised to species rank under the name Polyura bicolor Turlin & Sato, 1995  stat. nov. Divergence time estimates suggest that this lineage split from its sister taxon in the late Pleistocene. At this time, the bathymetric isolation of Malaita from the rest of the archipelago probably prevented gene flow during periods of lower sea level, thereby fostering allopatric speciation. The combination of molecular species delimitation methods, morphological comparisons, and divergence time estimation is useful to study lineage diversification across intricate geographic regions.     

Blood parasite infection differentially relates to carotenoid‐based plumage and bill color in the American goldfinch

Male and female American goldfinches (Spinus tristis) express condition‐dependent carotenoid‐based plumage and bill coloration. Plumage color is relatively static, as pigments incorporated into feathers during the spring molt cannot be mobilized thereafter. In contrast, bill color is dynamic, reflecting changes in condition over short time periods. Previous studies have shown that male and female ornaments, though similar in expression, are differentially related to measures of immunocompetence, suggesting that the relationship between ornamentation and parasite infection may differ between the sexes. In this study, we evaluate the relationship between condition‐dependent ornamentation (plumage and bill color) and blood parasite infection in male and female American goldfinches. We captured goldfinches after completion of the pre‐alternate molt and prior to the onset of nesting and assessed prevalence of Trypanosoma parasites via blood smears. Plumage color strongly predicted trypanosome infection: Birds with more colorful plumage were less likely to present infections. In contrast, we detected no relationship between infection and bill color, which in other studies has been shown to dynamically reflect current condition. Sex did not affect the relationship between infection status and either ornament. Together, these results suggest that physiological pathways linking carotenoid ornamentation and infection may vary even within a single species.