Incomplete lineage sorting impacts the inference of macroevolutionary regimes from molecular phylogenies when concatenation is employed: An analysis based on Cetacea

Interest in methods that estimate speciation and extinction rates from molecular phylogenies has increased over the last decade. The application of such methods requires reliable estimates of tree topology and node ages, which are frequently obtained using standard phylogenetic inference combining concatenated loci and molecular dating. However, this practice disregards population‐level processes that generate gene tree/species tree discordance. We evaluated the impact of employing concatenation and coalescent‐based phylogeny inference in recovering the correct macroevolutionary regime using simulated data based on the well‐established diversification rate shift of delphinids in Cetacea. We found that under scenarios of strong incomplete lineage sorting, macroevolutionary analysis of phylogenies inferred by concatenating loci failed to recover the delphinid diversification shift, while the coalescent‐based tree consistently retrieved the correct rate regime. We suggest that ignoring microevolutionary processes reduces the power of methods that estimate macroevolutionary regimes from molecular data.     

Phylogenetic analysis of RAD‐seq data: examining the influence of gene genealogy conflict on analysis of concatenated data

One of the major issues in phylogenetic analysis is that gene genealogies from different gene regions may not reflect the true species tree or history of speciation. This has led to considerable debate about whether concatenation of loci is the best approach for phylogenetic analysis. The application of Next‐generation sequencing techniques such as RAD‐seq generates thousands of relatively short sequence reads from across the genomes of the sampled taxa. These data sets are typically concatenated for phylogenetic analysis leading to data sets that contain millions of base pairs per taxon. The influence of gene region conflict among so many loci in determining the phylogenetic relationships among taxa is unclear. We simulated RAD‐seq data by sampling 100 and 500 base pairs from alignments of over 6000 coding regions that each produce one of three highly supported alternative phylogenies of seven species of Drosophila. We conducted phylogenetic analyses on different sets of these regions to vary the sampling of loci with alternative gene trees to examine the effect on detecting the species tree. Irrespective of sequence length sampled per region and which subset of regions was used, phylogenetic analyses of the concatenated data always recovered the species tree. The results suggest that concatenated alignments of Next‐generation data that consist of many short sequences are robust to gene tree/species tree conflict when the goal is to determine the phylogenetic relationships among taxa.     

Ultraconserved elements show utility in phylogenetic inference of Adephaga (Coleoptera) and suggest paraphyly of ‘Hydradephaga’

The beetle suborder Adephaga has been the subject of many phylogenetic reconstructions utilizing a variety of data sources and inference methods. However, no strong consensus has yet emerged on the relationships among major adephagan lineages. Ultraconserved elements (UCEs) have proved useful for inferring difficult or unresolved phylogenies at varying timescales in vertebrates, arachnids and Hymenoptera. Recently, a UCE bait set was developed for Coleoptera using polyphagan genomes and a member of the order Strepsiptera as an outgroup. Here, we examine the utility of UCEs for reconstructing the phylogeny of adephagan families, in the first in vitro application a UCE bait set in Coleoptera. Our final dataset included 305 UCE loci for 18 representatives of all adephagan families except Aspidytidae, and two polyphagan outgroups, with a total concatenated length of 83 547 bp. We inferred trees using maximum likelihood analyses of the concatenated UCE alignment and coalescent species tree methods (astral ii , ASTRID, svdquartets ). Although the coalescent species tree methods had poor resolution and weak support, concatenated analyses produced well‐resolved, highly supported trees. Hydradephaga was recovered as paraphyletic, with Gyrinidae sister to Geadephaga and all other adephagans. Haliplidae was recovered as sister to Dytiscoidea, with Hygrobiidae and Amphizoidae successive sisters to Dytiscidae. Finally, Noteridae was recovered as monophyletic and sister to Meruidae. Given the success of UCE data for resolving phylogenetic relationships within Adephaga, we suggest the potential for further resolution of relationships within Adephaga using UCEs with improved taxon sampling, and by developing Adephaga‐specific probes.     

Picking holes in traditional species delimitations: an integrative taxonomic reassessment of the Parmotrema perforatum group (Parmeliaceae,Ascomycota)

Accurate species delimitation is important as species are a fundamental unit in ecological, evolutionary and conservation biology research. In lichenized fungi, species delimitation has been difficult due to a lack of taxonomically important characteristics and due to the limits of traditional, morphology‐based species concepts. In this study we reassess the current taxonomy of the Parmotrema perforatum group, which recognizes six closely related species divided into three species pairs, each pair comprising one apotheciate (sexual) and one sorediate (asexual) species. Each pair is further characterized by a distinct combination of secondary metabolites. It was hypothesized that the three apotheciate species are reproductively isolated sibling species and that each sorediate species evolved once from the chemically identical apotheciate species. In this study, species boundaries were re‐examined using an integrative approach incorporating morphological, chemical and molecular sequence data to delimit species boundaries. Phylogenetic trees were inferred from a seven‐locus DNA sequence dataset using concatenated gene tree and coalescent‐based species‐tree inference methods. Furthermore, we employed a multi‐species coalescent method to validate candidate species. Micromorphological measurements of conidia were found to be congruent with phylogenetic clusters. Each approach that we applied to the P. perforatum group consistently recovered four of the currently circumscribed species (P. perforatum, P. hypotropum, P. subrigidum and P. louisianae), whereas P. preperforatum and P. hypoleucinum were consistently combined and are thus interpreted as conspecific.     

Multilocus phylogenetics of a rapid radiation in the genus Thomomys (Rodentia: Geomyidae)

Species complexes undergoing rapid radiation present a challenge in molecular systematics because of the possibility that ancestral polymorphism is retained in component gene trees. Coalescent theory has demonstrated that gene trees often fail to match lineage trees when taxon divergence times are less than the ancestral effective population sizes. Suggestions to increase the number of loci and the number of individuals per taxon have been proposed; however, phylogenetic methods to adequately analyze these data in a coalescent framework are scarce. We compare two approaches to estimating lineage (species) trees using multiple individuals and multiple loci: the commonly used partitioned Bayesian analysis of concatenated sequences and a modification of a newly developed hierarchical Bayesian method (BEST) that simultaneously estimates gene trees and species trees from multilocus data. We test these approaches on a phylogeny of rapidly radiating species wherein divergence times are likely to be smaller than effective population sizes, and incomplete lineage sorting is known, in the rodent genus, Thomomys. We use seven independent noncoding nuclear sequence loci (total approximately 4300 bp) and between 1 and 12 individuals per taxon to construct a phylogenetic hypothesis for eight Thomomys species. The majority-rule consensus tree from the partitioned concatenated analysis included 14 strongly supported bipartitions, corroborating monophyletic species status of five of the eight named species. The BEST tree strongly supported only the split between the two subgenera and showed very low support for any other clade. Comparison of both lineage trees to individual gene trees revealed that the concatenation method appears to ignore conflicting signals among gene trees, whereas the BEST tree considers conflicting signals and downweights support for those nodes. Bayes factor analysis of posterior tree distributions from both analyses strongly favor the model underlying the BEST analysis. This comparison underscores the risks of overreliance on results from concatenation, and ignoring the properties of coalescence, especially in cases of recent, rapid radiations.     

THE INFLUENCE OF SAMPLING DESIGN ON SPECIES TREE INFERENCE: A NEW RELATIONSHIP FOR THE NEW WORLD CHICKADEES (AVES: POECILE)

In this study, we explore the long‐standing issue of how many loci are needed to infer accurate phylogenetic relationships, and whether loci with particular attributes (e.g., parsimony informativeness, variability, gene tree resolution) outperform others. To do so, we use an empirical data set consisting of the seven species of chickadees (Aves: Paridae), an analytically tractable, recently diverged group, and well‐studied ecologically but lacking a nuclear phylogeny. We estimate relationships using 40 nuclear loci and mitochondrial DNA using four coalescent‐based species tree inference methods (BEST, *BEAST, STEM, STELLS). Collectively, our analyses contrast with previous studies and support a sister relationship between the Black‐capped and Carolina Chickadee, two superficially similar species that hybridize along a long zone of contact. Gene flow is a potential source of conflict between nuclear and mitochondrial gene trees, yet we find a significant, albeit low, signal of gene flow. Our results suggest that relatively few loci with high information content may be sufficient for estimating an accurate species tree, but that substantially more loci are necessary for accurate parameter estimation. We provide an empirical reference point for researchers designing sampling protocols with the purpose of inferring phylogenies and population parameters of closely related taxa.     

Ultraconserved elements resolve the phylogeny of potoos (Aves: Nyctibiidae)

In this study, we apply a genome‐scale set of molecular markers, ultraconserved elements, to fully resolve the phylogeny of a family of secretive, nocturnal birds, the potoos (Nyctibiidae). This dataset provides an opportunity to explore some challenges of phylogenetic analyses of genome‐scale datasets, which we address in several ways. We generate data matrices ranging between 2610–4175 loci (1 477 319–3 848 295 aligned base pairs) that represent versions of the data differing in whether or not alignments were trimmed prior to concatenation, and whether 100 or 75% of all taxa were required to be represented by data for inclusion of a given locus. These matrices are analyzed with both maximum likelihood and coalescent algorithms, to check for artifacts of concatenation. Then, we subsample our data matrix by locus into randomly‐selected replicates of 125–1000 loci, and compare the topologies and statistical support of the resulting trees to look for evidence of systematic error. In analyses of complete matrices, we find strong statistical support for all ingroup nodes of the tree with no evidence for systematic error introduced by alignment trimming, missing data, or concatenation. We find further support for that topology in our subsampling analyses and statistical topology tests. The earliest branch of the tree separates Nyctibius bracteatus from the rest of the potoos, followed successively by N. grandis and N. aethereus. Two pairs of species, N. jamaicensis plus N. griseus, and N. leucopterus plus N. aethereus comprise the distal tips of the tree. Finally, we compare our strongly supported topology to those of previous studies, and use the phylogeny to examine the evolutionary history of potoos.     

Species tree inference in a recent radiation of orioles (Genus Icterus): multiple markers and methods reveal cytonuclear discordance in the northern oriole group

Recent computational advances provide novel opportunities to infer species trees based on multiple independent loci. Thus, single gene trees no longer need suffice as proxies for species phylogenies. Several methods have been developed to deal with the challenges posed by incomplete and stochastic lineage sorting. In this study, we employed four Bayesian methods to infer the phylogeny of a clade of 11 recently diverged oriole species within the genus Icterus. We obtained well-resolved and mostly congruent phylogenies using a set of seven unlinked nuclear intron loci and sampling multiple individuals per species. Most notably, Bayesian concordance analysis generally agreed well with concatenation; the two methods agreed fully on eight of nine nodes. The coalescent-based method ^∗BEAST further supported six of these eight nodes. The fourth method used, BEST, failed to converge despite exhaustive efforts to optimize the tree search. Overall, the results obtained by new species tree methods and concatenation generally corroborate our findings from previous analyses and data sets. However, we found striking disagreement between mitochondrial and nuclear DNA involving relationships within the northern oriole group. Our results highlight the danger of reliance on mtDNA alone for phylogenetic inference. We demonstrate that in spite of low variability and incomplete lineage sorting, multiple nuclear loci can produce largely congruent phylogenies based on multiple species tree methods, even for very closely-related species.     

Phylogenetic and mixed Yule‐coalescent analyses reveal cryptic lineages within two South American marine snails of the genus Crepipatella (Gastropoda: Calyptraeidae)

The presence of sibling species within the marine gastropod genus Crepipatella has complicated the taxonomy of members of the group. Since the establishment of the genus, 15 species have been described, but recent studies have indicated that there are only five valid species, two of which inhabit the coasts of Chile, namely C. dilatata and C. fecunda. The two species are morphologically indistinguishable as adults, but can be differentiated on the basis of their encapsulated developmental stages. The primary aim of this study was to reconstruct phylogeny within the genus, and to establish species limits of C. dilatata and C. fecunda, using mitochondrial DNA data. To this end, we used maximum parsimony, maximum likelihood, and Bayesian inference to reconstruct phylogenies using 589 bp of the cytochrome oxidase I (COI) gene. The mtDNA phylogenies were then used as input in a general mixed Yule‐coalescent (GMYC) analysis to estimate species boundaries. In addition, quarter likelihood mapping was used to test a posteriori the confidence of inner branch patterns in the phylogenetic tree. Both DNA tree‐based and GMYC methods provide support for five isolated lineages within this species complex. Our data also suggest that Late Pleistocene and Holocene fragmentation and subsequent range expansion events may have shaped contemporary genetic patterns of Crepipatella in South America.     

Discordance of species trees with their most likely gene trees

Because of the stochastic way in which lineages sort during speciation, gene trees may differ in topology from each other and from species trees. Surprisingly, assuming that genetic lineages follow a coalescent model of within-species evolution, we find that for any species tree topology with five or more species, there exist branch lengths for which gene tree discordance is so common that the most likely gene tree topology to evolve along the branches of a species tree differs from the species phylogeny. This counterintuitive result implies that in combining data on multiple loci, the straightforward procedure of using the most frequently observed gene tree topology as an estimate of the species tree topology can be asymptotically guaranteed to produce an incorrect estimate. We conclude with suggestions that can aid in overcoming this new obstacle to accurate genomic inference of species phylogenies.     

Phylogeny and species delimitation of North European Lumbricillus (Clitellata,Enchytraeidae)

The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of non‐tropical marine and brackish waters world‐wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non‐monophyletic group. In this study, species boundaries and the phylogeny of this genus is re‐assessed using more than 300 DNA‐barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non‐monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister‐group to the genus Grania or as sister‐group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.     

Evolutionary drivers of phylogeographical diversity in the highlands of Mexico: a case study of the Crotalus triseriatus species group of montane rattlesnakes

Aim To assess the genealogical relationships of widespread montane rattlesnakes in the Crotalus triseriatus species group and to clarify the role of Late Neogene mountain building and Pleistocene pine–oak forest fragmentation in driving the diversification of Mexican highland taxa. Location Highlands of mainland Mexico and the south‐western United States (Texas, New Mexico, and Arizona). Methods A synthesis of inferences was used to address several associated questions about the biogeography of the Mexican highlands and the evolutionary drivers of phylogeographical diversity in co‐distributed taxa. We combined extensive range‐wide sampling (130 individuals representing five putative species) and mixed‐model phylogenetic analyses of 2408 base pairs of mitochondrial DNA to estimate genealogical relationships and divergence times within the C. triseriatus species group. We then assessed the tempo of diversification using a maximum likelihood framework based on the birth–death process. Estimated times of divergences provided a probabilistic temporal component and questioned whether diversification rates have remained constant or varied over time. Finally, we looked for phylogeographical patterns in other co‐distributed taxa. Results We identified eight major lineages within the C. triseriatus group, and inferred strong correspondence between maternal and geographic history within most lineages. At least one cryptic species was detected. Relationships among lineages were generally congruent with previous molecular studies, with differences largely attributable to our expanded taxonomic and geographic sampling. Estimated divergences between most major lineages occurred in the Late Miocene and Pliocene. Phylogeographical structure within each lineage appeared to have been generated primarily during the Pleistocene. Although the scale of genetic diversity recognized affected estimated rates of diversification, rates appeared to have been constant through time. Main conclusions The biogeographical history of the C. triseriatus group implies a dynamic history for the highlands of Mexico. The Neogene formation of the Transvolcanic Belt appears responsible for structuring geographic diversity among major lineages. Pleistocene glacial–interglacial climatic cycles and resultant expansions and contractions of the Mexican pine–oak forest appear to have driven widespread divergences within lineages. Climatic change, paired with the complex topography of Mexico, probably produced a myriad of species‐specific responses in co‐distributed Mexican highland taxa. The high degree of genetic differentiation recovered in our study and others suggests that the Mexican highlands may contain considerably more diversity than currently recognized.     

How well do multispecies coalescent methods perform with mitochondrial genomic data? A case study of butterflies and moths (Insecta: Lepidoptera)

Despite the broad adoption of multispecies coalescent (MSC) methods for nuclear phylogenomics, they have yet to be applied to mitochondrial (mt) genomic data. As the potential sources of phylogenomic bias that MSC methods can address, such as incomplete lineage sorting, horizontal gene transfer and gene tree heterogeneity, have been found in mt genomic data, these approaches may improve the accuracy of phylogenetic inference with these data. In the present study, we examined the behaviour of MSC methods in reconstructing the phylogeny of Lepidoptera (butterflies and moths), a group for which mt genomic data are known to have strong resolving power. Traditional concatenation methods of analysing mt genomes for Lepidoptera infer topologies highly congruent with those generated from independent nuclear datasets. Individual mt gene trees performed poorly in recovering consensus relationships at deep levels (i.e. superfamily monophyly and inter-relationships) and only moderately well for shallow relationships (i.e. within Papilionoidea). In contrast, MSC analyses with ASTRAL performed strongly with almost complete concordance to both concatenated mt genome analyses and independent nuclear analyses at both deep and shallow phylogenetic scales. Outgroup choice had a limited impact on tree accuracy, with even phylogenetically distant outgroups still resulting in topologies highly congruent with results from nuclear datasets, although MSC analyses appeared to be marginally more affected by outgroup choice than concatenation analyses. In general, discordance between concatenation and MSC analyses was found at nodes whose resolution varied between previous nuclear phylogenomic studies. The sensitivity of individual relationships to analysis with MSC vs concatenation can thus be used to test the robustness of phylogenetic hypotheses. For insect phylogenetics, MSC is a reliable inference method for mt genomic data and is thus a useful complement to the already widely used concatenation approaches.     

Model‐based approach to test hard polytomies in the Eulaemus clade of the most diverse South American lizard genus Liolaemus (Liolaemini,Squamata)

Lack of resolution in a phylogenetic tree is usually represented as a polytomy, and often adding more data (loci and taxa) resolves the species tree. These are the ‘soft’ polytomies, but in other cases additional data fail to resolve relationships; these are the ‘hard’ polytomies. This latter case is often interpreted as a simultaneous radiation of lineages in the history of a clade. Although hard polytomies are difficult to address, model‐based approaches provide new tools to test these hypotheses. Here, we used a clade of 144 species of the South American lizard clade Eulaemus to estimate phylogenies using a traditional concatenated matrix and three species tree methods: *BEAST, BEST, and minimizing deep coalescences (MDC). The different species tree methods recovered largely discordant results, but all resolved the same polytomy (e.g. very short internodes amongst lineages and low nodal support in Bayesian methods). We simulated data sets under eight explicit evolutionary models (including hard polytomies), tested these against empirical data (a total of 14 loci), and found support for two polytomies as the most plausible hypothesis for diversification of this clade. We discuss the performance of these methods and their limitations under the challenging scenario of hard polytomies. © 2015 The Linnean Society of London     

PHYLOGENETIC INFERENCE OF NUPTIAL TRAIT EVOLUTION IN THE CONTEXT OF ASYMMETRICAL INTROGRESSION IN NORTH AMERICAN DARTERS (TELEOSTEI)

Introgressive hybridization and incomplete lineage sorting complicate the inference of phylogeny, and available species‐tree methods do not simultaneously account for these processes. Both hybridization and ancestral polymorphism have been invoked to explain divergent phylogenies inferred from different datasets for Stigmacerca, a clade of 11 North American darter species. Species of Stigmacerca are characterized by a mating system involving parental care with males guarding nesting territories and fertilized eggs. Males of four species of Stigmacerca develop egg‐mimic nuptial structures on their second dorsal fins during the breeding season. Previous phylogenies suggest contrasting scenarios for the evolution of this nuptial trait. Using a combination of coalescent‐based methods, we analyzed a dataset comprising a mitochondrial gene and 15 nuclear loci to estimate relationships and simultaneously test for introgressive hybridization. Our analyses identified several instances of interspecific gene flow involving both cytoplamsmic haplotypes and nuclear alleles. The new phylogeny was used to infer a single origin and recent loss of egg‐mimic structures in Stigmacerca and led to the discovery of a phylogenetically distinct species. Our results highlight the limited strategies available to account for introgressive hybridization in the inference of species relationships and the likely effects of this process on reconstructing trait evolution.     

The phylogenetic systematics of blue‐tailed skinks (Plestiodon) and the family Scincidae

Blue‐tailed skinks (genus Plestiodon) are a common component of the terrestrial herpetofauna throughout their range in eastern Eurasia and North and Middle America. Plestiodon species are also frequent subjects of ecological and evolutionary research, yet a comprehensive, well‐supported phylogenetic framework does not yet exist for this genus. We construct a comprehensive molecular phylogeny of Plestiodon using Bayesian phylogenetic analyses of a nine‐locus data set comprising 8308 base pairs of DNA, sampled from 38 of the 43 species in the genus. We evaluate potential gene tree/species tree discordance by conducting phylogenetic analyses of the concatenated and individual locus data sets, as well as employing coalescent‐based methods. Specifically, we address the placement of Plestiodon within the evolutionary tree of Scincidae, as well as the phylogenetic relationships between Plestiodon species, and their taxonomy. Given our sampling of major Scincidae lineages, we also re‐evaluate ‘deep’ relationships within the family, with the goal of resolving relationships that have been ambiguous in recent molecular phylogenetic analyses. We infer strong support for several scincid relationships, including a major clade of ‘scincines’ and the inter‐relationships of major Mediterranean and southern African genera. Although we could not estimate the precise phylogenetic affinities of Plestiodon with statistically significant support, we nonetheless infer significant support for its inclusion in a large ‘scincine’ clade exclusive of Acontinae, Lygosominae, Brachymeles, and Ophiomorus. Plestiodon comprises three major geographically cohesive clades. One of these clades is composed of mostly large‐bodied species inhabiting northern Indochina, south‐eastern China (including Taiwan), and the southern Ryukyu Islands of Japan. The second clade comprises species inhabiting central China (including Taiwan) and the entire Japanese archipelago. The third clade exclusively inhabits North and Middle America and the island of Bermuda. A vast majority of interspecific relationships are strongly supported in the concatenated data analysis, but there is nonetheless significant conflict amongst the individual gene trees. Coalescent‐based gene tree/species tree analyses indicate that incongruence amongst the nuclear loci may severely obscure the phylogenetic inter‐relationships of the primarily small‐bodied Plestiodon species that inhabit the central Mexican highlands. These same analyses do support the sister relationship between Plestiodon marginatus Hallowell, 1861 and Plestiodon stimpsonii (Thompson, 1912), and differ with the mitochondrial DNA analysis that supports Plestiodon elegans (Boulenger, 1887) + P. stimpsonii. Finally, because the existing Plestiodon taxonomy is a poor representation of evolutionary relationships, we replace the existing supraspecific taxonomy with one congruent with our phylogenetic results. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 163–189.