Understanding phylogenetic incongruence: lessons from phyllostomid bats

All characters and trait systems in an organism share a common evolutionary history that can be estimated using phylogenetic methods. However, differential rates of change and the evolutionary mechanisms driving those rates result in pervasive phylogenetic conflict. These drivers need to be uncovered because mismatches between evolutionary processes and phylogenetic models can lead to high confidence in incorrect hypotheses. Incongruence between phylogenies derived from morphological versus molecular analyses, and between trees based on different subsets of molecular sequences has become pervasive as datasets have expanded rapidly in both characters and species. For more than a decade, evolutionary relationships among members of the New World bat family Phyllostomidae inferred from morphological and molecular data have been in conflict. Here, we develop and apply methods to minimize systematic biases, uncover the biological mechanisms underlying phylogenetic conflict, and outline data requirements for future phylogenomic and morphological data collection. We introduce new morphological data for phyllostomids and outgroups and expand previous molecular analyses to eliminate methodological sources of phylogenetic conflict such as taxonomic sampling, sparse character sampling, or use of different algorithms to estimate the phylogeny. We also evaluate the impact of biological sources of conflict: saturation in morphological changes and molecular substitutions, and other processes that result in incongruent trees, including convergent morphological and molecular evolution. Methodological sources of incongruence play some role in generating phylogenetic conflict, and are relatively easy to eliminate by matching taxa, collecting more characters, and applying the same algorithms to optimize phylogeny. The evolutionary patterns uncovered are consistent with multiple biological sources of conflict, including saturation in morphological and molecular changes, adaptive morphological convergence among nectar‐feeding lineages, and incongruent gene trees. Applying methods to account for nucleotide sequence saturation reduces, but does not completely eliminate, phylogenetic conflict. We ruled out paralogy, lateral gene transfer, and poor taxon sampling and outgroup choices among the processes leading to incongruent gene trees in phyllostomid bats. Uncovering and countering the possible effects of introgression and lineage sorting of ancestral polymorphism on gene trees will require great leaps in genomic and allelic sequencing in this species‐rich mammalian family. We also found evidence for adaptive molecular evolution leading to convergence in mitochondrial proteins among nectar‐feeding lineages. In conclusion, the biological processes that generate phylogenetic conflict are ubiquitous, and overcoming incongruence requires better models and more data than have been collected even in well‐studied organisms such as phyllostomid bats.     

Partitioned Gene-Tree Analyses and Gene-Based Topology Testing Help Resolve Incongruence in a Phylogenomic Study of Host-Specialist Bees (Apidae: Eucerinae)

Incongruence among phylogenetic results has become a common occurrence in analyses of genome-scale data sets. Incongruence originates from uncertainty in underlying evolutionary processes (e.g., incomplete lineage sorting) and from difficulties in determining the best analytical approaches for each situation. To overcome these difficulties, more studies are needed that identify incongruences and demonstrate practical ways to confidently resolve them. Here, we present results of a phylogenomic study based on the analysis 197 taxa and 2,526 ultraconserved element (UCE) loci. We investigate evolutionary relationships of Eucerinae, a diverse subfamily of apid bees (relatives of honey bees and bumble bees) with >1,200 species. We sampled representatives of all tribes within the group and >80% of genera, including two mysterious South American genera, Chilimalopsis and Teratognatha. Initial analysis of the UCE data revealed two conflicting hypotheses for relationships among tribes. To resolve the incongruence, we tested concatenation and species tree approaches and used a variety of additional strategies including locus filtering, partitioned gene-trees searches, and gene-based topological tests. We show that within-locus partitioning improves gene tree and subsequent species-tree estimation, and that this approach, confidently resolves the incongruence observed in our data set. After exploring our proposed analytical strategy on eucerine bees, we validated its efficacy to resolve hard phylogenetic problems by implementing it on a published UCE data set of Adephaga (Insecta: Coleoptera). Our results provide a robust phylogenetic hypothesis for Eucerinae and demonstrate a practical strategy for resolving incongruence in other phylogenomic data sets.     

An HMM-Based Comparative Genomic Framework for Detecting Introgression in Eukaryotes

One outcome of interspecific hybridization and subsequent effects of evolutionary forces is introgression, which is the integration of genetic material from one species into the genome of an individual in another species. The evolution of several groups of eukaryotic species has involved hybridization, and cases of adaptation through introgression have been already established. In this work, we report on PhyloNet-HMM—a new comparative genomic framework for detecting introgression in genomes. PhyloNet-HMM combines phylogenetic networks with hidden Markov models (HMMs) to simultaneously capture the (potentially reticulate) evolutionary history of the genomes and dependencies within genomes. A novel aspect of our work is that it also accounts for incomplete lineage sorting and dependence across loci. Application of our model to variation data from chromosome 7 in the mouse (Mus musculus domesticus) genome detected a recently reported adaptive introgression event involving the rodent poison resistance gene Vkorc1, in addition to other newly detected introgressed genomic regions. Based on our analysis, it is estimated that about 9% of all sites within chromosome 7 are of introgressive origin (these cover about 13 Mbp of chromosome 7, and over 300 genes). Further, our model detected no introgression in a negative control data set. We also found that our model accurately detected introgression and other evolutionary processes from synthetic data sets simulated under the coalescent model with recombination, isolation, and migration. Our work provides a powerful framework for systematic analysis of introgression while simultaneously accounting for dependence across sites, point mutations, recombination, and ancestral polymorphism.     

Are our phylomorphospace plots so terribly tangled? An investigation of disorder in data simulated under adaptive and nonadaptive models

Contemporary methods for visualizing phenotypic evolution, such as phylomorphospaces, often reveal patterns which depart strongly from a naïve expectation of consistently divergent branching and expansion. Instead, branches regularly crisscross as convergence, reversals, or other forms of homoplasy occur, forming patterns described as “birds’ nests”, “flies in vials”, or less elegantly, “a mess”. In other words, the phenotypic tree of life often appears highly tangled. Various explanations are given for this, such as differential degrees of developmental constraint, adaptation, or lack of adaptation. However, null expectations for the magnitude of disorder or “tangling” have never been established, so it is unclear which or even whether various evolutionary factors are required to explain messy patterns of evolution. I simulated evolution along phylogenies under a number of varying parameters (number of taxa and number of traits) and models (Brownian motion, Ornstein–Uhlenbeck (OU)-based, early burst, and character displacement (CD)] and quantified disorder using 2 measures. All models produce substantial amounts of disorder. Disorder increases with tree size and the number of phenotypic traits. OU models produced the largest amounts of disorder—adaptive peaks influence lineages to evolve within restricted areas, with concomitant increases in crossing of branches and density of evolution. Large early changes in trait values can be important in minimizing disorder. CD consistently produced trees with low (but not absent) disorder. Overall, neither constraints nor a lack of adaptation is required to explain messy phylomorphospaces—both stochastic and deterministic processes can act to produce the tantalizingly tangled phenotypic tree of life.     

The role of mitochondrial introgression in illuminating the evolutionary history of Nearctic treefrogs

Inferring the evolutionary history of lineages often becomes difficult when gene histories are in conflict with each other. Introgression, for example, can cause DNA sequences from one species to be more similar to sequences of a different species and lead to incongruence amongst gene trees. However, incorporating congruent and incongruent locus‐specific phylogenetic estimates with the geographical distribution of lineages may provide valuable insight into evolutionary processes important to speciation. In this study, we investigated mitochondrial introgression within the Hyla eximia group to better understand its role in illuminating the evolutionary history and phylogeography of these treefrogs. We reconstructed and compared the matrilineal history of the Hyla eximia group with estimates of evolutionary history inferred from nuclear genes. We tested for introgression within the mitochondrial and nuclear genes using a posterior predictive checking approach. Reconstructions of the species tree based on the mitochondrial DNA (mtDNA) and nuclear DNA data were strongly discordant. Introgression between lineages was widespread in the mtDNA data set (145 occurrences amongst 11 of the 16 lineages), but uncommon in the nuclear genes (12 occurrences amongst four of the 16 lineages). Nonetheless, the geographical structuring of mtDNA within species provides valuable information on biogeographical areas, ancient areas of hybridization, and unique histories of lineages within the H. eximia group. These results suggest that the combination of nuclear, mitochondrial, and spatial information can provide a more complete picture of ‘how evolutionary history played out’, particularly in cases where mitochondrial introgression is known to occur. © 2014 The Linnean Society of London     

Replicated divergence in cichlid radiations mirrors a major vertebrate innovation

Decoupling of the upper jaw bones—jaw kinesis—is a distinctive feature of the ray-finned fishes, but it is not clear how the innovation is related to the extraordinary diversity of feeding behaviours and feeding ecology in this group. We address this issue in a lineage of ray-finned fishes that is well known for its ecological and functional diversity—African rift lake cichlids. We sequenced ultraconserved elements to generate a phylogenomic tree of the Lake Tanganyika and Lake Malawi cichlid radiations. We filmed a diverse array of over 50 cichlid species capturing live prey and quantified the extent of jaw kinesis in the premaxillary and maxillary bones. Our combination of phylogenomic and kinematic data reveals a strong association between biting modes of feeding and reduced jaw kinesis, suggesting that the contrasting demands of biting and suction feeding have strongly influenced cranial evolution in both cichlid radiations.     

Tempo and mode of morphological evolution are decoupled from latitude in birds

The latitudinal diversity gradient is one of the most striking patterns in nature, yet its implications for morphological evolution are poorly understood. In particular, it has been proposed that an increased intensity of species interactions in tropical biota may either promote or constrain trait evolution, but which of these outcomes predominates remains uncertain. Here, we develop tools for fitting phylogenetic models of phenotypic evolution in which the impact of species interactions—namely, competition—can vary across lineages. Deploying these models on a global avian trait dataset to explore differences in trait divergence between tropical and temperate lineages, we find that the effect of latitude on the mode and tempo of morphological evolution is weak and clade- or trait dependent. Our results indicate that species interactions do not disproportionately impact morphological evolution in tropical bird families and question the validity of previously reported patterns of slower trait evolution in the tropics.

Analyses of morphological measurements from over 9400 bird species reveal that interactions between species have impacted evolution, but fail to support the longstanding hypothesis that competition is stronger in the tropics.     

Gene and species trees reveal mitochondrial and nuclear discordance in the Drosophila cardini group (Diptera: Drosophilidae)

Abstract. The Drosophila cardini group includes 15 species, which are subdivided into the D. cardini and D. dunni subgroups. Although many phylogenetic hypotheses have been proposed for this group during the last five decades (based on patterns of reproductive isolation, morphology of male genitalia, chromosomal inversions, isozyme variation, or molecular sequence data), these are mostly discordant with each other. We aimed to clarify some of the evolutionary patterns related to the origin of this incongruence, while also attempting to provide a better-supported phylogenetic hypothesis for the D. cardini subgroup. For this purpose, sequences from three mitochondrial and three nuclear loci were gathered for at least eight species, and both individual gene trees and joint species tree estimates were evaluated. Although there was concordance among gene trees within each of the nuclear and mitochondrial sets, considerable incongruence was revealed in the comparisons between these two data sets. The branching position of D. neocardini was the main source of incongruence, and species trees reconstructed using different approaches with and without this species were particularly incongruent. In addition to providing a better approximation of the evolutionary history of the D. cardini group, this study suggests that incomplete lineage sorting or introgression may be biasing previous species tree estimates. More generally, the results also suggest that the use of supermatrix methods can lead to an overestimation of support for the inferred relationships, and highlight the potential effects of different taxon sampling strategies in phylogenetic reconstruction.     

Convergence in pigmentation at multiple levels: mutations,genes and function

Convergence—the independent evolution of the same trait by two or more taxa—has long been of interest to evolutionary biologists, but only recently has the molecular basis of phenotypic convergence been identified. Here, we highlight studies of rapid evolution of cryptic coloration in vertebrates to demonstrate that phenotypic convergence can occur at multiple levels: mutations, genes and gene function. We first show that different genes can be responsible for convergent phenotypes even among closely related populations, for example, in the pale beach mice inhabiting Florida''s Gulf and Atlantic coasts. By contrast, the exact same mutation can create similar phenotypes in distantly related species such as mice and mammoths. Next, we show that different mutations in the same gene need not be functionally equivalent to produce similar phenotypes. For example, separate mutations produce divergent protein function but convergent pale coloration in two lizard species. Similarly, mutations that alter the expression of a gene in different ways can, nevertheless, result in similar phenotypes, as demonstrated by sister species of deer mice. Together these studies underscore the importance of identifying not only the genes, but also the precise mutations and their effects on protein function, that contribute to adaptation and highlight how convergence can occur at different genetic levels.     

Variant evolutionary trees under phenotypic variance

Evolutionary branching, which is a coevolutionary phenomenon of the development of two or more distinctive traits from a single trait in a population, is the issue of recent studies on adaptive dynamics. In previous studies, it was revealed that trait variance is a minimum requirement for evolutionary branching, and that it does not play an important role in the formation of an evolutionary pattern of branching. Here we demonstrate that the trait evolution exhibits various evolutionary branching paths starting from an identical initial trait to different evolutional terminus traits as determined by only changing the assumption of trait variance. The key feature of this phenomenon is the topological configuration of equilibria and the initial point in the manifold of dimorphism from which dimorphic branches develop. This suggests that the existing monomorphic or polymorphic set in a population is not an unique inevitable consequence of an identical initial phenotype.     

A model of horizontal gene transfer and the bacterial phylogeny problem

How much horizontal gene transfer (HGT) between species influences bacterial phylogenomics is a controversial issue. This debate, however, lacks any quantitative assessment of the impact of HGT on phylogenies and of the ability of tree-building methods to cope with such events. I introduce a Markov model of genome evolution with HGT, accounting for the constraints on time -- an HGT event can only occur between concomitantly living species. This model is used to simulate multigene sequence data sets with or without HGT. The consequences of HGT on phylogenomic inference are analyzed and compared to other well-known phylogenetic artefacts. It is found that supertree methods are quite robust to HGT, keeping high levels of performance even when gene trees are largely incongruent with each other. Gene tree incongruence per se is not indicative of HGT. HGT, however, removes the (otherwise observed) positive relationship between sequence length and gene tree congruence to the estimated species tree. Surprisingly, when applied to a bacterial and a eukaryotic multigene data set, this criterion rejects the HGT hypothesis for the former, but not the latter data set.     

Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe)

Inferences about past processes of adaptation and speciation require a gene-scale and genome-wide understanding of the evolutionary history of diverging taxa. In this study, we use genome-wide capture of nuclear gene sequences, plus skimming of organellar sequences, to investigate the phylogenomics of monkeyflowers in Mimulus section Erythranthe (27 accessions from seven species). Taxa within Erythranthe, particularly the parapatric and putatively sister species M. lewisii (bee-pollinated) and M. cardinalis (hummingbird-pollinated), have been a model system for investigating the ecological genetics of speciation and adaptation for over five decades. Across >8000 nuclear loci, multiple methods resolve a predominant species tree in which M. cardinalis groups with other hummingbird-pollinated taxa (37% of gene trees), rather than being sister to M. lewisii (32% of gene trees). We independently corroborate a single evolution of hummingbird pollination syndrome in Erythranthe by demonstrating functional redundancy in genetic complementation tests of floral traits in hybrids; together, these analyses overturn a textbook case of pollination-syndrome convergence. Strong asymmetries in allele sharing (Patterson’s D-statistic and related tests) indicate that gene tree discordance reflects ancient and recent introgression rather than incomplete lineage sorting. Consistent with abundant introgression blurring the history of divergence, low-recombination and adaptation-associated regions support the new species tree, while high-recombination regions generate phylogenetic evidence for sister status for M. lewisii and M. cardinalis. Population-level sampling of core taxa also revealed two instances of chloroplast capture, with Sierran M. lewisii and Southern Californian M. parishii each carrying organelle genomes nested within respective sympatric M. cardinalis clades. A recent organellar transfer from M. cardinalis, an outcrosser where selfish cytonuclear dynamics are more likely, may account for the unexpected cytoplasmic male sterility effects of selfer M. parishii organelles in hybrids with M. lewisii. Overall, our phylogenomic results reveal extensive reticulation throughout the evolutionary history of a classic monkeyflower radiation, suggesting that natural selection (re-)assembles and maintains species-diagnostic traits and barriers in the face of gene flow. Our findings further underline the challenges, even in reproductively isolated species, in distinguishing re-use of adaptive alleles from true convergence and emphasize the value of a phylogenomic framework for reconstructing the evolutionary genetics of adaptation and speciation.     

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo’s law of irreversible loss

Dollo’s law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.     

Bayesian Inference of Reticulate Phylogenies under the Multispecies Network Coalescent

The multispecies coalescent (MSC) is a statistical framework that models how gene genealogies grow within the branches of a species tree. The field of computational phylogenetics has witnessed an explosion in the development of methods for species tree inference under MSC, owing mainly to the accumulating evidence of incomplete lineage sorting in phylogenomic analyses. However, the evolutionary history of a set of genomes, or species, could be reticulate due to the occurrence of evolutionary processes such as hybridization or horizontal gene transfer. We report on a novel method for Bayesian inference of genome and species phylogenies under the multispecies network coalescent (MSNC). This framework models gene evolution within the branches of a phylogenetic network, thus incorporating reticulate evolutionary processes, such as hybridization, in addition to incomplete lineage sorting. As phylogenetic networks with different numbers of reticulation events correspond to points of different dimensions in the space of models, we devise a reversible-jump Markov chain Monte Carlo (RJMCMC) technique for sampling the posterior distribution of phylogenetic networks under MSNC. We implemented the methods in the publicly available, open-source software package PhyloNet and studied their performance on simulated and biological data. The work extends the reach of Bayesian inference to phylogenetic networks and enables new evolutionary analyses that account for reticulation.     

Footprints in time: comparative quantitative trait loci mapping of the pitcher-plant mosquito,Wyeomyia smithii

Identifying regions of the genome contributing to phenotypic evolution often involves genetic mapping of quantitative traits. The focus then turns to identifying regions of ‘major’ effect, overlooking the observation that traits of ecological or evolutionary relevance usually involve many genes whose individual effects are small but whose cumulative effect is large. Herein, we use the power of fully interfertile natural populations of a single species of mosquito to develop three quantitative trait loci (QTL) maps: one between two post-glacially diverged populations and two between a more ancient and a post-glacial population. All demonstrate that photoperiodic response is genetically a highly complex trait. Furthermore, we show that marker regressions identify apparently ‘non-significant’ regions of the genome not identified by composite interval mapping, that the perception of the genetic basis of adaptive evolution is crucially dependent upon genetic background and that the genetic basis for adaptive evolution of photoperiodic response is highly variable within contemporary populations as well as between anciently diverged populations.     

Phylogeny of γ‐proteobacteria: resolution of one branch of the universal tree?

The reconstruction of bacterial evolutionary relationships has proven to be a daunting task because variable mutation rates and horizontal gene transfer (HGT) among species can cause grave incongruities between phylogenetic trees based on single genes. Recently, a highly robust phylogenetic tree was constructed for 13 gamma-proteobacteria using the combined alignments of 205 conserved orthologous proteins.1 Only two proteins had incongruent tree topologies, which were attributed to HGT between Pseudomonas species and Vibrio cholerae or enterics. While the evolutionary relationships among these species appears to be resolved, further analysis suggests that HGT events with other bacterial partners likely occurred; this alters the implicit assumption of gamma-proteobacteria monophyly. Thus, any thorough reconstruction of bacterial evolution must not only choose a suitable set of molecular markers but also strive to reduce potential bias in the selection of species.     

Evidence for canalization of Distal-less function in the leg of Drosophila melanogaster

A considerable body of theory pertaining to the evolution of canalization has emerged recently, yet there have been few empirical investigations of their predictions. To address this, patterns of canalization and trait correlation were investigated under the individual and joint effects of the introgression of a loss-of-function allele of the Distal-less gene and high-temperature stress on a panel of iso-female lines. Variation was examined for number of sex comb teeth and the length of the basi-tarsus on the pro-thoracic leg of male Drosophila melanogaster. I demonstrate that whereas there is evidence for trait canalization, there is no evidence to support the hypothesis of the evolution of genetic canalization as a response to microenvironmental canalization. Furthermore, I demonstrate that although there are genetic correlations between these traits, there is no association between their measures of canalization. I discuss the prospects of the evolutionary lability of the Distal-less gene within the context of changes in genetic variation and covariation.