Corroboration among Data Sets in Simultaneous Analysis: Hidden Support for Phylogenetic Relationships among Higher Level Artiodactyl Taxa

In the taxonomic congruence approach to systematics, data sets are analyzed separately, and corroboration among data sets is indicated by replicated components in topologies derived from the separate analyses. By contrast, in the total evidence and conditional combination approaches, characters from different data sets are mixed in combined phylogenetic analyses. In optimal topologies derived from such simultaneous analyses, support for a particular node may be attributed to one, some, or all of the individual data sets. Partitioned branch support (PBS) is one technique for describing the distribution of character support and conflict among data sets in simultaneous analysis. PBS is analogous to branch support (BS), but recognizes hidden support and conflicts that emerge with the combination of characters from different data sets. For both BS and PBS, support for a particular node is interpreted as the difference in cost between optimal and suboptimal topologies. A different measure, the clade stability index (CSI), assesses the robustness of a particular node through the successive removal of characters. Here, we introduce variations of the CSI, the data set removal index (DRI) and nodal data set influence (NDI), that indicate the stability of a particular node to the removal of entire data sets. Like PBS, the DRI and NDI summarize the influence of different data sets in simultaneous analysis. However, because these new methods and PBS use different perturbations to assess stability, DRI and NDI scores do not always predict PBS scores and vice versa. In this report, the DRI and NDI are compared to PBS and taxonomic congruence in a cladistic analysis of 17 data sets for Artiodactyla (Mammalia). Five indices of hidden support and conflict are defined and applied to the combined artiodactyl character set. These measures identify substantial hidden support for controversial relationships within Artiodactyla. Hidden character support is ignored in the taxonomic congruence approach to systematics, but the DRI, NDI, and PBS utilize this cryptic information in estimates of support among data sets for a given node.     

The interpretation of hidden support in combined data phylogenetics

In phylogenetic analysis, support for a given clade is ‘hidden’ when isolated partitions support that clade less than in the analysis of combined data sets. In such simultaneous analyses, signal common to the majority of partitions dominates the topology at the expense of any signal idiosyncratic to each partition. This process is often referred to as synergy and is commonly used to validate the combination of disparate data partitions. We investigate the behaviour of hidden branch support (HBS), partitioned branch support (PBS) and hidden synapomorphy (HS) as measures of hidden support using artificial, real and experimentally manipulated phylogenetic data sets. Our analyses demonstrate that high levels of both HBS and HS can be obtained by combining data with little shared phylogenetic signal. This finding is in agreement with the original intent of hidden support metrics, which essentially quantify the extent of data set interaction, both through the dispersion of homoplasy and revelation of underlying shared signal (positive data synergy). High levels of HBS alone are insufficient to justify data combination. We advocate the use of multiple hidden support measures to distinguish between the dispersion of homoplasy and positive data synergy, and to better interpret data interactions. Furthermore, we suggest two criteria that help identify hidden support resulting from homoplasy dispersion: first, when total support decreases with the addition of a data partition and second, when total HBS per unit total support (TS) per node is similar to that derived from randomized data.     

Partitioned likelihood support and the evaluation of data set conflict

In simultaneous analyses of multiple data partitions, the trees relevant when measuring support for a clade are the optimal tree, and the best tree lacking the clade (i.e., the most reasonable alternative). The parsimony-based method of partitioned branch support (PBS) "forces" each data set to arbitrate between the two relevant trees. This value is the amount each data set contributes to clade support in the combined analysis, and can be very different to support apparent in separate analyses. The approach used in PBS can also be employed in likelihood: a simultaneous analysis of all data retrieves the maximum likelihood tree, and the best tree without the clade of interest is also found. Each data set is fitted to the two trees and the log-likelihood difference calculated, giving "partitioned likelihood support" (PLS) for each data set. These calculations can be performed regardless of the complexity of the ML model adopted. The significance of PLS can be evaluated using a variety of resampling methods, such as the Kishino-Hasegawa test, the Shimodiara-Hasegawa test, or likelihood weights, although the appropriateness and assumptions of these tests remains debated.     

Morphology versus molecules: the phylogenetic relationships of Sepsidae (Diptera: Cyclorrhapha) based on morphology and DNA sequence data from ten genes

The Sepsidae is, with approximately 300 described species, a relatively small family of cyclorrhaphan flies whose behaviour, morphology, and development have been extensively studied. However, currently the only available tree for Sepsidae is more than 10 years old and was based entirely on morphological characters. Here, we present the results of parsimony and Bayesian analyses based on 75 species, ten genes, and morphology. Parsimony and Bayesian analyses produce largely congruent and well‐supported topologies regardless of whether indels are coded as 5th character states, as missing values, or all sites with indels are removed. The tree confirms the monophyly of Sepsidae and identifies the Ropalomeridae as its sister group. With regard to higher‐level relationships, we identify widespread conflict between the morphological and the DNA sequence data. The proposed hypothesis based on both partitions largely reflects the signal in the molecular data. Particularly surprising is the rejection of two relationship hypotheses with strong morphological support, namely the sister group relationship between Orygma and the remaining Sepsidae and the monophyly of the Sepsis species group. Our partitioned Bremer support (PBS) analyses imply that indel coding has a stronger effect on the relative performance of individual gene partitions than the exclusion of alignment‐ambiguous sequences or the location of a gene on the mitochondrial or nuclear genome. However, these analyses also reveal unexpectedly strong fluctuations in PBS values given that indel treatment has only a minor effect on tree topology and jacknife support. These unexpected fluctuations highlight the need for a comparative study across multiple data sets that investigates the influence of conflict and indel treatment on PBS values. © The Willi Hennig Society 2008.     

Partitioned Bremer support and multiple trees

Partitioned Bremer support (PBS) is a valuable means of assessing congruence in combined data sets, but some aspects require clarification. When more than one equally parsimonious tree is found during the constrained search for trees lacking the node of interest, averaging PBS for each data set across these trees can conceal conflict, and PBS should ideally be examined for each constrained tree. Similarly, when multiple most parsimonious trees (MPTs) are generated during analysis of the combined data, PBS is usually calculated on the consensus tree. However, extra information can be obtained if PBS is calculated on each of the MPTs or even suboptimal trees.     

Missing data,clade support and “reticulation”: the molecular systematics of Heliconius and related genera (Lepidoptera: Nymphalidae) re‐examined

Kozak et al. (2015, Syst. Biol., 64: 505) portrayed the inference of evolutionary history among Heliconius and allied butterfly genera as a particularly difficult problem for systematics due to prevalent gene conflict caused by interspecific reticulation. To control for this, Kozak et al. conducted a series of multispecies coalescent phylogenetic analyses that they claimed revealed pervasive conflict among markers, but ultimately chose as their preferred hypothesis a phylogenetic tree generated by the traditional supermatrix approach. Intrigued by this seemingly contradictory set of conclusions, we conducted further analyses focusing on two prevalent aspects of the data set: missing data and the uneven contribution of phylogenetic signal among markers. Here, we demonstrate that Kozak et al. overstated their findings of reticulation and that evidence of gene‐tree conflict is largely lacking. The distribution of intrinsic homoplasy and incongruence homoplasy in their data set does not follow the pattern expected if phylogenetic history had been obscured by pervasive horizontal gene flow; in fact, noise within individual gene partitions is ten times higher than the incongruence among gene partitions. We show that the patterns explained by Kozak et al. as a result of reticulation can be accounted for by missing data and homoplasy. We also find that although the preferred topology is resilient to missing data, measures of support are sensitive to, and strongly eroded by too many empty cells in the data matrix. Perhaps more importantly, we show that when some taxa are missing almost all characters, adding more genes to the data set provides little or no increase in support for the tree.     

Exploring data interaction and nucleotide alignment in a multiple gene analysis of Ips (Coleoptera: Scolytinae)

The possibility of gene tree incongruence in a species-level phylogenetic analysis of the genus Ips (Coleoptera: Scolytidae) was investigated based on mitochondrial 16S rRNA (16S) and nuclear elongation factor-1 alpha (EF-1 alpha) sequences, and existing cytochrome oxidase I (COI) and nonmolecular data sets. Separate cladistic analyses of the data partitions resulted in partially discordant most-parsimonious trees but revealed only low conflict of the phylogenetic signal. Interactions among data partitions, which differed in the extent of sequence divergence (COI > 16S > EF-1 alpha), base composition, and homoplasy, revealed that much of the branch support emerges only in the simultaneous analysis, particularly for deeper nodes in the tree, which are almost entirely supported through "hidden support" (sensu Gatesy et al., Cladistics 15:271-313, 1999). Apparent incongruence between data partitions is in part due to suboptimal alignments and bias of character transformations, but little evidence supports invoking incongruent phylogenetic histories of genetic loci. There is also no justification for eliminating or downweighting gene partitions on the basis of their apparent homoplasy or incongruence with other partitions, because the signal emerges only in the interaction of all data. In comparison with traditional taxonomy, the pini, plastographus, and perturbatus groups are polyphyletic, whereas the grandicollis group is monophyletic except for inclusion of the (monophyletic) calligraphus group. The latidens group and some European species are distantly related and closer to other genera within Ipini. Our robust cladogram was used to revise the classification of Ips. We provide new diagnoses for Ips and four subgeneric taxa.     

The devil in the details: interactions between the branch-length prior and likelihood model affect node support and branch lengths in the phylogeny of the Psoraceae

In popular use of Bayesian phylogenetics, a default branch-length prior is almost universally applied without knowing how a different prior would have affected the outcome. We performed Bayesian and maximum likelihood (ML) inference of phylogeny based on empirical nucleotide sequence data from a family of lichenized ascomycetes, the Psoraceae, the morphological delimitation of which has been controversial. We specifically assessed the influence of the combination of Bayesian branch-length prior and likelihood model on the properties of the Markov chain Monte Carlo tree sample, including node support, branch lengths, and taxon stability. Data included two regions of the mitochondrial ribosomal RNA gene, the internal transcribed spacer region of the nuclear ribosomal RNA gene, and the protein-coding largest subunit of RNA polymerase II. Data partitioning was performed using Bayes' factors, whereas the best-fitting model of each partition was selected using the Bayesian information criterion (BIC). Given the data and model, short Bayesian branch-length priors generate higher numbers of strongly supported nodes as well as short and topologically similar trees sampled from parts of tree space that are largely unexplored by the ML bootstrap. Long branch-length priors generate fewer strongly supported nodes and longer and more dissimilar trees that are sampled mostly from inside the range of tree space sampled by the ML bootstrap. Priors near the ML distribution of branch lengths generate the best marginal likelihood and the highest frequency of "rogue" (unstable) taxa. The branch-length prior was shown to interact with the likelihood model. Trees inferred under complex partitioned models are more affected by the stretching effect of the branch-length prior. Fewer nodes are strongly supported under a complex model given the same branch-length prior. Irrespective of model, internal branches make up a larger proportion of total tree length under the shortest branch-length priors compared with longer priors. Relative effects on branch lengths caused by the branch-length prior can be problematic to downstream phylogenetic comparative methods making use of the branch lengths. Furthermore, given the same branch-length prior, trees are on average more dissimilar under a simple unpartitioned model compared with a more complex partitioned models. The distribution of ML branch lengths was shown to better fit a gamma or Pareto distribution than an exponential one. Model adequacy tests indicate that the best-fitting model selected by the BIC is insufficient for describing data patterns in 5 of 8 partitions. More general substitution models are required to explain the data in three of these partitions, one of which also requires nonstationarity. The two mitochondrial ribosomal RNA gene partitions need heterotachous models. We found no significant correlations between, on the one hand, the amount of ambiguous data or the smallest branch-length distance to another taxon and, on the other hand, the topological stability of individual taxa. Integrating over several exponentially distributed means under the best-fitting model, node support for the family Psoraceae, including Psora, Protoblastenia, and the Micarea sylvicola group, is approximately 0.96. Support for the genus Psora is distinctly lower, but we found no evidence to contradict the current classification.     

Branch support via resampling: an empirical study

The success of resampling approaches to branch support depends on the effectiveness of the underlying tree searches. Two primary factors are identified as key: the depth of tree search and the number of trees saved per resampling replicate. Two datasets were explored for a range of search parameters using jackknifing. Greater depth of tree search tends to increase support values because shorter trees conflict less with each other, while increasing numbers of trees saved tends to reduce support values because of conflict that reduces structure in the replicate consensus. Although a relatively small amount of branch swapping will achieve near‐accurate values for a majority of clades, some clades do not yield accurate values until more extensive searches are performed. This means that in order to maximize the accuracy of resampling analyses, one should employ as extensive a search strategy as possible, and save as many trees per replicate as possible. Strict consensus summary of resampling replicates is preferable to frequency‐within‐replicates summary because it is a more conservative approach to the reporting of replicate results. Jackknife analysis is preferable to bootstrap because of its closer relationship to the original data.© The Willi Hennig Society 2010.     

Phylogenetic relationships and generic delimitation in Inuleae subtribe Inulinae (Asteraceae) based on ITS and cpDNA sequence data

Phylogenetic relationships in Inuleae subtribe Inulinae (Asteraceae) were investigated. DNA sequence data from three chloroplast regions ( ndhF , trnL–F and psbA–trnH ) and the nuclear ribosomal internal transcribed spacer (ITS) region were analysed separately and in combination using parsimony and Bayesian inference. A total of 163 ingroup taxa were included, of which 60 were sampled for all four markers. Conflicts between chloroplast and nuclear data were assessed using partitioned Bremer support (PBS). Rather than averaging PBS over several trees from constrained searches, individual trees were considered by evaluating PBS ranges. Criteria to be used in the detection of a significant conflict between data partitions are proposed. Three nodes in the total data tree were found to encompass significant conflict that could result from ancient hybridization. Neither of the large, heterogeneous and widespread genera Inula and Pulicaria is monophyletic. A monophyletic group ("the Inula complex") that comprises all species of Inula includes also Telekia , Carpesium , Chrysophthalmum , Rhanteriopsis , Amblyocarpum , and Pentanema sensu stricto . Two species of Pentanema were found to be closer to Blumea (including Blumeopsis and Merrittia ) and Caesulia . The monophyletic " Pulicaria complex" includes all taxa with heteromorphic pappus. Within this group, Francoeuria is distinct from Pulicaria and merits recognition as a separate genus.
 © The Willi Hennig Society 2009.     

Sources of character conflict in a clade of water striders (Heteroptera: Gerridae)

Incongruence among trees reconstructed with different data may stem from historical (gene tree‐species tree conflict) or process (character change biases) phenomena. Regardless of the source, incongruent data, as determined with “global” measures of homoplasy, have often been excluded from parsimony analysis of the combined data. Recent studies suggest that these homoplasy measures do not predict the contribution of each character to overall tree structure. Branch support measures identify, on a character to node basis, sources of support and conflict resulting from a simultaneous analysis of the data. We implement these branch support measures to identify sources of character conflict in a clade of water striders consisting of Gerris Fabricius, Aquarius Schellenberg, and Limnoporus Stål species. Separate analyses of morphology, mitochondrial cytochrome oxidase I (COI), large mitochondrial ribosomal subunit (16SrRNA), and elongation factor‐1α (EF‐1α) data resulted in cladograms that varied in resolution and topological concordance. Simultaneous analysis of the data resulted in two trees that were unresolved for one node in a strict consensus. The topology agreed with current classification except for the placements of Aquarius chilensis and the Aquarius remigis species group closer to Gerris than to congeneric species. Branch support measures indicated that support derived from each data set varied among nodes, but COI had an overall negative effect on branch support. However, Spearman rank correlation of partitioned branch support values indicated no negative associations of branch support between any data sets and a positive association between EF‐1α and 16SrRNA. Thus incongruence among data sets was not drastic and the gene‐tree versus species tree phenomenon was not implicated. Biases in character change were a more likely reason for incongruence, although saturation curves and incongruence length difference for COI indicated little potential for homoplasy. However, a posteriori inspection of COI nucleotide change with reference to the simultaneous analysis tree revealed AT and codon biases. These biases were not associated with branch support measures. Therefore, it is difficult to predict incongruence or identify its cause. Exclusion of data is ill advised because every character is potentially parsimony informative.     

A molecular perspective on the evolution of microteiid lizards (Squamata, Gymnophthalmidae), and a new classification for the family

A molecular phylogeny was reconstructed for 26 recognized genera of the Gymnophthalmidae using a total of 2379 bp of mitochondrial (12S, 16S and ND4) and nuclear (18S and c-mos) DNA sequences. We performed maximum parsimony (MP) and maximum likelihood (ML) analyses, and data partitions were analysed separately and in combination under MP. ML analyses were carried out only on the combined sequences for computational simplicity. Robustness for the recovered nodes was assessed with bootstrap and partitioned Bremer support (PBS) analyses. The total molecular evidence provided a better-resolved hypothesis than did separate analysis of individual partitions, and the PBS analysis indicates congruence among independent partitions for support of some internal nodes. Based on this hypothesis, a new classification for the family is proposed. Alopoglossus , the sister group of all the other Gymnophthalmidae was allocated to a new subfamily Alopoglossinae, and Rhachisaurus (a new genus for Anotosaura brachylepis) to the new Rhachisaurinae. Two tribes are recognized within the subfamily Gymnophthalminae: Heterodactylini and Gymnophthalmini, and two others within Cercosaurinae (Ecpleopini and Cercosaurini). Some ecological and evolutionary implications of the phylogenetic hypothesis are considered, including the independent occurrence of limb reduction, body elongation, and other characters associated with fossoriality.     

Phylogeny of Drosophilinae (Diptera: Drosophilidae), with comments on combined analysis and character support

Drosophilidae (Diptera) is a diverse, cosmopolitan family of flies. Here, we present a combined analysis phylogeny of Drosophilinae, one of the two subfamilies of Drosophilidae, based on data from six different data partitions, including both molecular and morphological characters. Although our data show support for the monophyly of the Hawaiian Drosophilidae, and the subgenus Sophophora, neither the genus Drosophila nor the subgenus Drosophila is monophyletic. Partitioned Bremer support (PBS) indicates that morphological data taken from Grimaldi's monograph (Grimaldi, 1990a), as well as sequences from the mitochondrial (mt) 16S rDNA and the nuclear Adh gene, lend much support to our tree's topology. This is particularly interesting in the case of Grimaldi's data, since his published hypothesis conflicts with ours in significant ways. Our combined analysis cladogram phylogeny reflects the catch-all designation that the name Drosophila has become, in that the cladogram does not support the monophyly of either the genus or subgenus Drosophila.     

From kissing to belly stridulation: comparative analysis reveals surprising diversity,rapid evolution,and much homoplasy in the mating behaviour of 27 species of sepsid flies (Diptera: Sepsidae)

Our understanding of how fast mating behaviour evolves in insects is rather poor due to a lack of comparative studies among insect groups for which phylogenetic relationships are known. Here, we present a detailed study of the mating behaviour of 27 species of Sepsidae (Diptera) for which a well‐resolved and supported phylogeny is available. We demonstrate that mating behaviour is extremely diverse in sepsids with each species having its own mating profile. We define 32 behavioural characters and document them with video clips. Based on sister species comparisons, we provide several examples where mating behaviour evolves faster than all sexually dimorphic morphological traits. Mapping the behaviours onto the molecular tree reveals much homoplasy, comparable to that observed for third positions of mitochondrial protein‐encoding genes. A partitioned Bremer support (PBS) analysis reveals conflict between the molecular and behavioural data, but behavioural characters have higher PBS values per parsimony‐informative character than DNA sequence characters.     

Tail paradox, partial identifiability, and influential priors in Bayesian branch length inference

Recent studies have observed that Bayesian analyses of sequence data sets using the program MrBayes sometimes generate extremely large branch lengths, with posterior credibility intervals for the tree length (sum of branch lengths) excluding the maximum likelihood estimates. Suggested explanations for this phenomenon include the existence of multiple local peaks in the posterior, lack of convergence of the chain in the tail of the posterior, mixing problems, and misspecified priors on branch lengths. Here, we analyze the behavior of Bayesian Markov chain Monte Carlo algorithms when the chain is in the tail of the posterior distribution and note that all these phenomena can occur. In Bayesian phylogenetics, the likelihood function approaches a constant instead of zero when the branch lengths increase to infinity. The flat tail of the likelihood can cause poor mixing and undue influence of the prior. We suggest that the main cause of the extreme branch length estimates produced in many Bayesian analyses is the poor choice of a default prior on branch lengths in current Bayesian phylogenetic programs. The default prior in MrBayes assigns independent and identical distributions to branch lengths, imposing strong (and unreasonable) assumptions about the tree length. The problem is exacerbated by the strong correlation between the branch lengths and parameters in models of variable rates among sites or among site partitions. To resolve the problem, we suggest two multivariate priors for the branch lengths (called compound Dirichlet priors) that are fairly diffuse and demonstrate their utility in the special case of branch length estimation on a star phylogeny. Our analysis highlights the need for careful thought in the specification of high-dimensional priors in Bayesian analyses.     

Misleading results of likelihood‐based phylogenetic analyses in the presence of missing data

The amount of missing data in many contemporary phylogenetic analyses has substantially increased relative to previous norms, particularly in supermatrix studies that compile characters from multiple previous analyses. In such cases the missing data are non‐randomly distributed and usually present in all partitions (i.e. groups of characters) sampled. Parametric methods often provide greater resolution and support than parsimony in such cases, yet this may be caused by extrapolation of branch lengths from one partition to another. In this study I use contrived and simulated examples to demonstrate that likelihood, even when applied to simple matrices with little or no homoplasy, homogeneous evolution across groups of characters, perfect model fit, and hundreds or thousands of variable characters, can provide strong support for incorrect topologies when the matrices have non‐random distributions of missing data distributed across all partitions. I do so using a systematic exploration of alternative seven‐taxon tree topologies and distributions of missing data in two partitions to demonstrate that these likelihood‐based artefacts may occur frequently and are not shared by parsimony. I also demonstrate that Bayesian Markov chain Monte Carlo analysis is more robust to these artefacts than is likelihood. © The Willi Hennig Society 2011.     

The deterministic effects of alignment bias in phylogenetic inference

Alignment of nucleotide and/or amino acid sequences is a fundamental component of sequence‐based molecular phylogenetic studies. Here we examined how different alignment methods affect the phylogenetic trees that are inferred from the alignments. We used simulations to determine how alignment errors can lead to systematic biases that affect phylogenetic inference from those sequences. We compared four approaches to sequence alignment: progressive pairwise alignment, simultaneous multiple alignment of sequence fragments, local pairwise alignment and direct optimization. When taking into account branch support, implied alignments produced by direct optimization were found to show the most extreme behaviour (based on the alignment programs for which nearly equivalent alignment parameters could be set) in that they provided the strongest support for the correct tree in the simulations in which it was easy to resolve the correct tree and the strongest support for the incorrect tree in our long‐branch‐attraction simulations. When applied to alignment‐sensitive process partitions with different histories, direct optimization showed the strongest mutual influence between the process partitions when they were aligned and phylogenetically analysed together, which makes detecting recombination more difficult. Simultaneous alignment performed well relative to direct optimization and progressive pairwise alignment across all simulations. Rather than relying upon methods that integrate alignment and tree search into a single step without accounting for alignment uncertainty, as with implied alignments, we suggest that simultaneous alignment using the similarity criterion, within the context of information available on biological processes and function, be applied whenever possible for sequence‐based phylogenetic analyses.     

Evaluating the clade size effect in alternative measures of branch support

The clade size effect refers to a bias that causes middle‐sized clades to be less supported than small or large‐sized clades. This bias is present in resampling measures of support calculated under maximum likelihood and maximum parsimony and in Bayesian posterior probabilities. Previous analyses indicated that the clade size effect is worst in maximum parsimony, followed by maximum likelihood, while Bayesian inference is the least affected. Homoplasy was interpreted as the main cause of the effect. In this study, we explored the presence of the clade size effect in alternative measures of branch support under maximum parsimony: Bremer support and symmetric resampling, expressed as absolute frequencies and frequency differences. Analyses were performed using 50 molecular and morphological matrices. Symmetric resampling showed the same tendency that bootstrap and jackknife did for maximum parsimony and maximum likelihood. Few matrices showed a significant bias using Bremer support, presenting a better performance than resampling measures of support and comparable to Bayesian posterior probabilities. Our results indicate that the problem is not maximum parsimony, but resampling measures of support. We corroborated the role of homoplasy as a possible cause of the clade size effect, increasing the number of random trees during the resampling, which together with the higher chances that medium‐sized clades have of being contradicted generates the bias during the perturbation of the original matrix, making it stronger in resampling measures of support.     

A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): identification of localized incongruence using a combinational bootstrap approach

Multi-gene phylogenetic analyses were conducted to address the evolution of Clavicipitaceae (Ascomycota). Data are presented here for approximately 5900 base pairs from portions of seven loci: the nuclear ribosomal small and large subunit DNA (nrSSU and nrLSU), beta-tubulin, elongation factor 1alpha (EF-1alpha), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), and mitochondrial ATP Synthase subunit 6 (mtATP6). These data were analyzed in a complete 66-taxon matrix and 91-taxon supermatrix that included some missing data. Separate phylogenetic analyses, with data partitioned according to genes, produced some conflicting results. The results of separate analyses from RPB1 and RPB2 are in agreement with the combined analyses that resolve a paraphyletic Clavicipitaceae comprising three well-supported clades (i.e., Clavicipitaceae clade A, B, and C), whereas the tree obtained from mtATP6 is in strong conflict with the monophyly of Clavicipitaceae clade B and the sister-group relationship of Hypocreaceae and Clavicipitaceae clade C. The distribution of relative contribution of nodal support for each gene partition was assessed using both partitioned Bremer support (PBS) values and combinational bootstrap (CB) analyses, the latter of which analyzed bootstrap proportions from all possible combinations of the seven gene partitions. These results suggest that CB analyses provide a more consistent estimate of nodal support than PBS and that combining heterogeneous gene partitions, which individually support a limited number of nodes, results in increased support for overall tree topology. Analyses of the 91-taxa supermatrix data sets revealed that some nodes were more strongly supported by increased taxon sampling. Identifying the localized incongruence of mtATP6 and analyses of complete and supermatrix data sets strengthen the evidence for rejecting the monophyly of Clavicipitaceae and much of the current subfamilial classification of the family. Although the monophyly of the grass-associated subfamily Clavicipitoideae (e.g., Claviceps, Balansia, and Epichlo?) is strongly supported, the subfamily Cordycipitoideae (e.g., Cordyceps and Torrubiella) is not monophyletic. In particular, species of the genus Cordyceps, which are pathogens of arthropods and truffles, are found in all three clavicipitaceous clades. These results imply that most characters used in the current familial classification of Clavicipitaceae are not diagnostic of monophyly.     

Catching the phylogenic history through the ontogenic hourglass: a phylogenomic analysis of Drosophila body segmentation genes

SUMMARY The phylogenetic information content of different developmental stages is a long‐standing issue in the study of development and evolution. We performed phylogenetic analyses of 51 body segmentation genes in 12 species of Drosophila in order to investigate the impact of the mode of evolution of development on phylogeny inference. Previous studies of these genes in Drosophila using pairwise phenetic comparisons at the species group level revealed the presence of an “hourglass model” (HG), wherein mid‐embryonic stages are the most evolutionarily constrained. We utilized two character‐based approaches: taxonomic congruence using the relative consensus fork index (RCFI), in which phylogenies are inferred from each gene separately and compared with a total evidence tree (TET), and partitioned simultaneous analysis using several indices such as branch support (BS) and localized incongruence length difference (LILD) test. We also proposed a new index, the recapitulatory index (R), which divides the number of synapomorphies on the total number of informative characters in a data set. Polynomial adjustment of both BS and R indices showed strong support for the hourglass model regardless of the taxonomic level (species subgroup vs. subgenera), showing less phylogenetic information content for mid‐developmental stages (mainly the zygotic segment polarity stage). Significant LILD scores were randomly distributed among developmental stages revealing the absence of differential selective constraints, but were significantly related to chromosomal location showing physical (linkage) impact on phylogenetic incongruence. RCFI was the most sensitive measure to taxonomic level, having a convex parabola at the species subgroup level in support of the hourglass model and a concave parabola at the subgeneric level in support of the adaptive penetrance model. This time‐dependent discrepancy of best fit developmental model parallels previous conflicting results from the vertebrates. Because of the quasi‐phenetic nature of this index, we argue that the discrepancy is due to the evolutionary rate heterogeneity of developmental genes rather than to fundamental differences among organisms. We suggest that simultaneous character‐based analyses give better macroevolutionary support to the hourglass model of the developmental constraints on genome evolution than pairwise phenetic comparisons.