Genomes of the Orestias pupfish from the Andean Altiplano shed light on their evolutionary history and phylogenetic relationships within Cyprinodontiformes.

BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

Skeleton phylogeny reconstructed with transcriptomes for the tribe Drosophilini (Diptera: Drosophilidae).

The family Drosophilidae is one of the most important model systems in evolutionary biology. Thanks to advances in high-throughput sequencing technology, a number of molecular phylogenetic analyses have been undertaken by using large data sets of many genes and many species sampled across this family. Especially, recent analyses using genome sequences have depicted the family-wide skeleton phylogeny with high confidence. However, the taxon sampling is still insufficient for minor lineages and non-Drosophila genera. In this study, we carried out phylogenetic analyses using a large number of transcriptome-based nucleotide sequences, focusing on the largest, core tribe Drosophilini in the Drosophilidae. In our analyses, some noise factors against phylogenetic reconstruction were taken into account by removing putative paralogy from the datasets and examining the effects of missing data, i.e. gene occupancy and site coverage, and incomplete lineage sorting. The inferred phylogeny has newly resolved the following phylogenetic positions/relationships at the genomic scale: (i) the monophyly of the subgenus Siphlodora including Zaprionus flavofasciatus to be transferred therein; (ii) the paraphyly of the robusta and melanica species groups within a clade comprised of the robusta, melanica and quadrisetata groups and Z. flavofasciatus; (iii) Drosophila curviceps (representing the curviceps group), D. annulipes (the quadrilineata subgroup of the immigrans group) and D. maculinotata clustered into a clade sister to the Idiomyia + Scaptomyza clade, forming together the expanded Hawaiian drosophilid lineage; (iv) Dichaetophora tenuicauda (representing the lineage comprised of the Zygothrica genus group and Dichaetophora) placed as the sister to the clade of the expanded Hawaiian drosophilid lineage and Siphlodora; and (v) relationships of the subgenus Drosophila and the genus Zaprionus as follows: (Zaprionus, (the quadrilineata subgroup, ((D. sternopleuralis, the immigrans group proper), (the quinaria radiation, the tripunctata radiation)))). These results are to be incorporated into the so-far published phylogenomic tree as a backbone (constraint) tree for grafting much more species based on sequences of a limited number of genes. Such a comprehensive, highly confident phylogenetic tree with extensive and dense taxon sampling will provide an essential framework for comparative studies of the Drosophilidae.

Molecular phylogeny of Hesperiidae (Lepidoptera) with an emphasis on Asian and African genera.

Despite considerable research efforts in recent years, the deeper phylogenetic relationships among skipper butterflies (Hesperiidae) remain unresolved. This is primarily because of limited sampling, especially within Asian and African lineages. In this study, we consolidated previous data and extensively sampled Asian and African taxa to elucidate the phylogenetic relationships within Hesperiidae. The molecular dataset comprised sequences from two mitochondrial and two nuclear gene regions from 563 species that represented 353 genera. Our analyses revealed seven subfamilies within Hesperiidae: Coeliadinae, Euschemoninae, Eudaminae, Pyrginae, Heteropterinae, Trapezitinae, and Hesperiinae. The systematics of most tribes and genera aligned with those of prior studies. However, notable differences were observed in several tribes and genera. Overall, the position of taxa assigned to incertae sedis in Hesperiinae is largely clarified in this study. Our results strongly support the monophyly of the tribe Tagiadini (Pyrginae), and the systematics of some genera are clarified with comprehensive discussion. We recognize 15 tribes within the subfamily Hesperiinae. Of these, nine tribes are discussed in detail: Aeromachini, Astictopterini, Erionotini, Unkanini (new status), Ancistroidini, Ismini (confirmed status), Plastingini (new status), Gretnini (confirmed status), and Eetionini (confirmed status). We propose four subtribes within Astictopterini: Hypoleucina subtrib.n., Aclerosina, Cupithina, and Astictopterina. Furthermore, we describe a new genus (Hyarotoidesgen.n.) and reinstate two genera (Zeareinst.stat. and Separeinst.stat.) as valid. Additionally, we propose several new combinations: Zea mythecacomb.n.,Sepa bononiacomb.n. & reinst.stat., and Sepa umbrosacomb.n. This study, with extensive sampling of Asian and African taxa, greatly enhances the understanding of the knowledge of the skipper tree of life.

Robustness of Felsenstein's Versus Transfer Bootstrap Supports With Respect to Taxon Sampling.

The bootstrap method is based on resampling sequence alignments and re-estimating trees. Felsenstein's bootstrap proportions (FBP) are the most common approach to assess the reliability and robustness of sequence-based phylogenies. However, when increasing taxon sampling (i.e., the number of sequences) to hundreds or thousands of taxa, FBP tend to return low support for deep branches. The transfer bootstrap expectation (TBE) has been recently suggested as an alternative to FBP. TBE is measured using a continuous transfer index in [0,1] for each bootstrap tree, instead of the binary {0,1} index used in FBP to measure the presence/absence of the branch of interest. TBE has been shown to yield higher and more informative supports while inducing a very low number of falsely supported branches. Nonetheless, it has been argued that TBE must be used with care due to sampling issues, especially in datasets with a high number of closely related taxa. In this study, we conduct multiple experiments by varying taxon sampling and comparing FBP and TBE support values on different phylogenetic depths, using empirical datasets. Our results show that the main critique of TBE stands in extreme cases with shallow branches and highly unbalanced sampling among clades, but that TBE is still robust in most cases, while FBP is inescapably negatively impacted by high taxon sampling. We suggest guidelines and good practices in TBE (and FBP) computing and interpretation.

Phylogenetic Signal, Congruence, and Uncertainty across Bacteria and Archaea.

Reconstruction of the Tree of Life is a central goal in biology. Although numerous novel phyla of bacteria and archaea have recently been discovered, inconsistent phylogenetic relationships are routinely reported, and many inter-phylum and inter-domain evolutionary relationships remain unclear. Here, we benchmark different marker genes often used in constructing multidomain phylogenetic trees of bacteria and archaea and present a set of marker genes that perform best for multidomain trees constructed from concatenated alignments. We use recently-developed Tree Certainty metrics to assess the confidence of our results and to obviate the complications of traditional bootstrap-based metrics. Given the vastly disparate number of genomes available for different phyla of bacteria and archaea, we also assessed the impact of taxon sampling on multidomain tree construction. Our results demonstrate that biases between the representation of different taxonomic groups can dramatically impact the topology of resulting trees. Inspection of our highest-quality tree supports the division of most bacteria into Terrabacteria and Gracilicutes, with Thermatogota and Synergistota branching earlier from these superphyla. This tree also supports the inclusion of the Patescibacteria within the Terrabacteria as a sister group to the Chloroflexota instead of as a basal-branching lineage. For the Archaea, our tree supports three monophyletic lineages (DPANN, Euryarchaeota, and TACK/Asgard), although we note the basal placement of the DPANN may still represent an artifact caused by biased sequence composition. Our findings provide a robust and standardized framework for multidomain phylogenetic reconstruction that can be used to evaluate inter-phylum relationships and assess uncertainty in conflicting topologies of the Tree of Life.

Revisiting metazoan phylogeny with genomic sampling of all phyla.

Proper biological interpretation of a phylogeny can sometimes hinge on the placement of key taxa-or fail when such key taxa are not sampled. In this light, we here present the first attempt to investigate (though not conclusively resolve) animal relationships using genome-scale data from all phyla. Results from the site-heterogeneous CAT + GTR model recapitulate many established major clades, and strongly confirm some recent discoveries, such as a monophyletic Lophophorata, and a sister group relationship between Gnathifera and Chaetognatha, raising continued questions on the nature of the spiralian ancestor. We also explore matrix construction with an eye towards testing specific relationships; this approach uniquely recovers support for Panarthropoda, and shows that Lophotrochozoa (a subclade of Spiralia) can be constructed in strongly conflicting ways using different taxon- and/or orthologue sets. Dayhoff-6 recoding sacrifices information, but can also reveal surprising outcomes, e.g. full support for a clade of Lophophorata and Entoprocta + Cycliophora, a clade of Placozoa + Cnidaria, and raising support for Ctenophora as sister group to the remaining Metazoa, in a manner dependent on the gene and/or taxon sampling of the matrix in question. Future work should test the hypothesis that the few remaining uncertainties in animal phylogeny might reflect violations of the various stationarity assumptions used in contemporary inference methods.

Taxon sampling effects on the quantification and comparison of community phylogenetic diversity.

Ecologists are increasingly making use of molecular phylogenies, especially in the fields of community ecology and conservation. However, these phylogenies are often used without full appreciation of their underlying assumptions and uncertainties. A frequent practice in ecological studies is inferring a phylogeny with molecular data from taxa only within the community of interest. These "inferred community phylogenies" are inherently biased in their taxon sampling. Despite the importance of comprehensive sampling in constructing phylogenies, the implications of using inferred community phylogenies in ecological studies have not been examined. Here, we evaluate how taxon sampling affects the quantification and comparison of community phylogenetic diversity using both simulated and empirical data sets. We demonstrate that inferred community trees greatly underestimate phylogenetic diversity and that the probability of incorrectly ranking community diversity can reach up to 25%, depending on the dating methods employed. We argue that to reach reliable conclusions, ecological studies must improve their taxon sampling and generate the best phylogeny possible.

Phylogenetic relationships and classification of the Holarctic family Leuciscidae (Cypriniformes: Cyprinoidei).

The phylogenetic relationships and classification of the freshwater fish order Cypriniformes, like many other species-rich groups of vertebrates, has evolved over time with some consistency and inconsistencies of relationships across various studies. Within Cypriniformes, the Holarctic family Leuciscidae is one of the most widely distributed and highly diverse monophyletic groups of cyprinoids. Despite several studies conducted on this group, alternative hypotheses exist as to the composition and relationships within Leuciscidae. Here we assess the extent, composition, phylogenetic relationships, and taxonomy of this highly diverse group of fishes, using multiple mitochondrial and nuclear loci and a comprehensive and dense taxonomic sampling. Analyses of 418 specimens (410 species) resolve a well-supported Leuciscidae including 362 specimens (358 taxa) in six well-supported subfamilies/major clades: Pseudaspininae/Far East Asian clade (FEA); Laviniinae/North American Western clade (WC); Plagopterinae/North American Creek Chub-Plagopterin clade (CC-P); Leuciscinae/Eurasian Old World clade (OW) (minus Phoxinus) plus North American Notemigonus; Phoxininae/Eurasian Phoxinus clade (PHX); and Pogonichthyinae/North American clade (NA) including all remaining leuciscids. Within Leuciscidae, neither the traditional phoxinins (Phoxinus, FEA, Nearctic genera) nor all Nearctic genera (minus Notemigonus) are resolved as monophyletic; whereas the WC and CC-P form two independent lineages from remaining North American cyprinoids. A close relationship exists between Eurasian Phoxinus, NA, and OW clades, while FEA is the sister group to all remaining Leuciscidae. Major lineages resolved within these six subfamilies are mostly congruent with some previous studies. Our results suggests a complex evolutionary history of this diverse and widespread group of fishes.

Hidden diversity within the depauperate genera of the snake tribe Lampropeltini (Serpentes, Colubridae).

Accurate representation of lineage diversity through complete taxon sampling is crucial to understanding the evolution of biodiversity, particularly when using molecular phylogenetics to estimate evolutionary relationships. In this interest, taxonomic diversity is often used as a proxy for lineage diversity even though the two concepts are not synonymous. We explore this within the snake tribe Lampropeltini which includes some of the most conspicuous and heavily studied snakes in North America. Both the taxonomy and hypothesized relationships within this tribe have been in flux. The number of species has increased from 23 to 51 over the last thirty years, predominately within three of the nine genera (Lampropeltis, Pantherophis, Pituophis). The remaining six depauperate genera (Arizona, Bogertophis, Cemophora, Pseudelaphe, Rhinocheilus, and Senticolis) have been poorly represented in phylogenetic studies. To estimate evolutionary relationships and determine if the dichotomy in depauperate and speciose genera within Lampropeltini is a function of taxon sampling or truly represents the lineage diversity, we estimated the phylogeny of this group using nuclear and mitochondrial loci in a concatenated and coalescent framework with the largest sampling of the six depauperate genera to date. In addition, we estimated the divergence dates among the genera to assess whether the instability of Lampropeltini phylogenetic relationships is due to an adaptive radiation. While some nodes still remain unresolved, the generic-level relationships we recovered agree with those of a recent next-generation study that used a much larger set of loci for fewer individuals. We also tested two putative species, Arizona pacata and Pseudelaphe phaescens, for the first time phylogenetically and find evidence that they are distinct lineages. Overall, we find that the taxonomic and genetic diversity are not correlated in Lampropeltini and that representing putative diversity in phylogenies will lead to a better estimate of evolutionary histories, especially in groups with complex radiations.

On the importance of geographic and taxonomic sampling in phylogeography: A reevaluation of diversification and species limits in a Neotropical thrush (Aves, Turdidae).

Phylogeographic studies seeking to describe biogeographic patterns, infer evolutionary processes, and revise species-level classification should properly characterize the distribution ranges of study species, and thoroughly sample genetic variation across taxa and geography. This is particularly necessary for widely distributed organisms occurring in complex landscapes, such as the Neotropical region. Here, we clarify the geographic range and revisit the phylogeography of the Black-billed Thrush (Turdus ignobilis), a common passerine bird from lowland tropical South America, whose evolutionary relationships and species limits were recently evaluated employing phylogeographic analyses based on partial knowledge of its distribution and incomplete sampling of populations. Our work employing mitochondrial and nuclear DNA sequences sampled all named subspecies and multiple populations across northern South America, and uncovered patterns not apparent in earlier work, including a biogeographic interplay between the Amazon and Orinoco basins and the occurrence of distinct lineages with seemingly different habitat affinities in regional sympatry in the Colombian Amazon. In addition, we found that previous inferences about the affinities and taxonomic status of Andean populations assumed to be allied to populations from the Pantepui region were incorrect, implying that inferred biogeographic and taxonomic scenarios need re-evaluation. We propose a new taxonomic treatment, which recognizes two distinct biological species in the group. Our findings illustrate the importance of sufficient taxon and geographic sampling to reconstruct evolutionary history and to evaluate species limits among Neotropical organisms. Considering the scope of the questions asked, advances in Neotropical phylogeography will often require substantial cross-country scientific collaboration.

How Should Genes and Taxa be Sampled for Phylogenomic Analyses with Missing Data? An Empirical Study in Iguanian Lizards.

Targeted sequence capture is becoming a widespread tool for generating large phylogenomic data sets to address difficult phylogenetic problems. However, this methodology often generates data sets in which increasing the number of taxa and loci increases amounts of missing data. Thus, a fundamental (but still unresolved) question is whether sampling should be designed to maximize sampling of taxa or genes, or to minimize the inclusion of missing data cells. Here, we explore this question for an ancient, rapid radiation of lizards, the pleurodont iguanians. Pleurodonts include many well-known clades (e.g., anoles, basilisks, iguanas, and spiny lizards) but relationships among families have proven difficult to resolve strongly and consistently using traditional sequencing approaches. We generated up to 4921 ultraconserved elements with sampling strategies including 16, 29, and 44 taxa, from 1179 to approximately 2.4 million characters per matrix and approximately 30% to 60% total missing data. We then compared mean branch support for interfamilial relationships under these 15 different sampling strategies for both concatenated (maximum likelihood) and species tree (NJst) approaches (after showing that mean branch support appears to be related to accuracy). We found that both approaches had the highest support when including loci with up to 50% missing taxa (matrices with ~40-55% missing data overall). Thus, our results show that simply excluding all missing data may be highly problematic as the primary guiding principle for the inclusion or exclusion of taxa and genes. The optimal strategy was somewhat different for each approach, a pattern that has not been shown previously. For concatenated analyses, branch support was maximized when including many taxa (44) but fewer characters (1.1 million). For species-tree analyses, branch support was maximized with minimal taxon sampling (16) but many loci (4789 of 4921). We also show that the choice of these sampling strategies can be critically important for phylogenomic analyses, since some strategies lead to demonstrably incorrect inferences (using the same method) that have strong statistical support. Our preferred estimate provides strong support for most interfamilial relationships in this important but phylogenetically challenging group.

Nomenclatural notes on Nadsoniella and the human opportunist black yeast genus Exophiala.

The opportunistic black yeast are particularly known through the genus Exophiala, characterised by annellidic budding cells. However, this phenotype is polyphyletic within the order Chaetothyriales. Seventeen generic names are available in the family Herpotrichiellaceae, one of which is Exophiala. Future taxonomy will be based on molecular phylogeny; each multi-species clade may qualify for one of these names. This paper focuses on the genus Nadsoniella, which is the oldest valid name in the Herpotrichiellaceae. Despite its exophiala-like phenotype, the type species of Nadsoniella clusters in the jeanselmei-clade, competing with the sympodial genus Rhinocladiella. In contrast, Exophiala competes with morphologically pronounced genera Thysanorea and Veronaea. Replacing the current phenotypic system for phylogenetic nomenclature requires highly stable phylogenies, which currently are not available.

Split-specific bootstrap measures for quantifying phylogenetic stability and the influence of taxon selection.

Assessing the robustness of an inferred phylogeny is an important element of phylogenetics. This is typically done with measures of stabilities at the internal branches and the variation of the positions of the leaf nodes. The bootstrap support for branches in maximum parsimony, distance and maximum likelihood estimation, or posterior probabilities in Bayesian inference, measure the uncertainty about a branch due to the sampling of the sites from genes or sampling genes from genomes. However, these measures do not reveal how taxon sampling affects branch support and the effects of taxon sampling on the estimated phylogeny. An internal branch in a phylogenetic tree can be viewed as a split that separates the taxa into two nonempty complementary subsets. We develop several split-specific measures of stability determined from bootstrap support for quartets. These include BPtaxon_split (average bootstrap percentage [BP] for all quartets involving a taxon within a split), BPsplit (BPtaxon_split averaged over taxa), BPtaxon (BPtaxon_split averaged over splits) and RBIC-taxon (average BP over all splits after removing a taxon). We also develop a pruned-tree distance metric. Application of our measures to empirical and simulated data illustrate that existing measures of overall stability can fail to detect taxa that are the primary source of a split-specific instability. Moreover, we show that the use of many reduced sets of quartets is important in being able to detect the influence of joint sets of taxa rather than individual taxa. These new measures are valuable diagnostic tools to guide taxon sampling in phylogenetic experimental design.

Comprehensive phylogeny, biogeography and new classification of the diverse bee tribe Megachilini: Can we use DNA barcodes in phylogenies of large genera?

Classification and evolutionary studies of particularly speciose clades pose important challenges, as phylogenetic analyses typically sample a small proportion of the existing diversity. We examine here one of the largest bee genera, the genus Megachile - the dauber and leafcutting bees. Besides presenting a phylogeny based on five nuclear genes (5480 aligned nucleotide positions), we attempt to use the phylogenetic signal of mitochondrial DNA barcodes, which are rapidly accumulating and already include a substantial proportion of the known species diversity in the genus. We used barcodes in two ways: first, to identify particularly divergent lineages and thus to guide taxon sampling in our nuclear phylogeny; second, to augment taxon sampling by combining nuclear markers (as backbone for ancient divergences) with DNA barcodes. Our results indicate that DNA barcodes bear phylogenetic signal limited to very recent divergences (3-4 my before present). Sampling within clades of very closely related species may be augmented using this technique, but our results also suggest statistically supported, but incongruent placements of some taxa. However, the addition of one single nuclear gene (LW-rhodopsin) to the DNA barcode data was enough to recover meaningful placement with high clade support values for nodes up to 15 million years old. We discuss different proposals for the generic classification of the tribe Megachilini. Finding a classification that is both in agreement with our phylogenetic hypotheses and practical in terms of diagnosability is particularly challenging as our analyses recover several well-supported clades that include morphologically heterogeneous lineages. We favour a classification that recognizes seven morphologically well-delimited genera in Megachilini: Coelioxys, Gronoceras, Heriadopsis, Matangapis, Megachile, Noteriades and Radoszkowskiana. Our results also lead to the following classification changes: the groups known as Dinavis, Neglectella, Eurymella and Phaenosarus are reestablished as valid subgenera of the genus Megachile, while the subgenus Alocanthedon is placed in synonymy with M. (Callomegachile), the subgenera Parachalicodoma and Largella with M. (Pseudomegachile), Anodonteutricharaea with M. (Paracella), Platysta with M. (Eurymella), and Grosapis and Eumegachile with M. (Megachile) (new synonymies). In addition, we use maximum likelihood reconstructions of ancestral geographic ranges to infer the origin of the tribe and reconstruct the main dispersal routes explaining the current, cosmopolitan distribution of this genus.

When do species-tree and concatenated estimates disagree? An empirical analysis with higher-level scincid lizard phylogeny.

Simulation studies suggest that coalescent-based species-tree methods are generally more accurate than concatenated analyses. However, these species-tree methods remain impractical for many large datasets. Thus, a critical but unresolved issue is when and why concatenated and coalescent species-tree estimates will differ. We predict such differences for branches in concatenated trees that are short, weakly supported, and have conflicting gene trees. We test these predictions in Scincidae, the largest lizard family, with data from 10 nuclear genes for 17 ingroup taxa and 44 genes for 12 taxa. We support our initial predictions, andsuggest that simply considering uncertainty in concatenated trees may sometimes encompass the differences between these methods. We also found that relaxed-clock concatenated trees can be surprisingly similar to the species-tree estimate. Remarkably, the coalescent species-tree estimates had slightly lower support values when based on many more genes (44 vs. 10) and a small (∼30%) reduction in taxon sampling. Thus, taxon sampling may be more important than gene sampling when applying species-tree methods to deep phylogenetic questions. Finally, our coalescent species-tree estimates tentatively support division of Scincidae into three monophyletic subfamilies, a result otherwise found only in concatenated analyses with extensive species sampling.

The time-dependent reconstructed evolutionary process with a key-role for mass-extinction events.

The homogeneous reconstructed evolutionary process is a birth-death process without observed extinct lineages. Each species evolves independently with the same diversification rate-speciation rate, λ(t), and extinction rate, µ(t)-that may change over time. The process is commonly applied to model species diversification where the data are reconstructed phylogenies, e.g. trees estimated from present-day molecular data, and used to infer diversification rates. In the present paper I develop the general probability density of a reconstructed tree under any homogeneous, time-dependent birth-death process. I demonstrate how to adapt this probability density when conditioning on the survival of one or two initial lineages, or on the process realizing n species, and also how to transform between the probability density of a reconstructed tree and the probability density of the speciation times. I demonstrate the use of the general time-dependent probability density functions by deriving the probability density of a reconstructed tree under a birth-death-shift model with explicit mass-extinction events. I extend these functions to several special cases, including the pure-birth process, the pure-death process, the birth-death process, and the critical-branching process. Thus, I specify equations for the most commonly used birth-death models in a unified framework (e.g. same condition and same data) using a common notation.

Diagnostics for a troubled backbone: testing topological hypotheses of trapelioid lichenized fungi in a large-scale phylogeny of Ostropomycetidae (Lecanoromycetes).

Trapelioid fungi constitute a widespread group of mostly crust-forming lichen mycobionts that are key to understanding the early evolutionary splits in the Ostropomycetidae, the second-most species-rich subclass of lichenized Ascomycota. The uncertain phylogenetic resolution of the approximately 170 species referred to this group contributes to a poorly resolved backbone for the entire subclass. Based on a data set including 657 newly generated sequences from four ribosomal and four protein-coding gene loci, we tested a series of a priori and new evolutionary hypotheses regarding the relationships of trapelioid clades within Ostropomycetidae. We found strong support for a monophyletic group of nine core trapelioid genera but no statistical support to reject the long-standing hypothesis that trapelioid genera are sister to Baeomycetaceae or Hymeneliaceae. However, we can reject a sister group relationship to Ostropales with high confidence. Our data also shed light on several long-standing questions, recovering Anamylopsoraceae nested within Baeomycetaceae, elucidating two major monophyletic groups within trapelioids (recognized here as Trapeliaceae and Xylographaceae), and rejecting the monophyly of the genus Rimularia. We transfer eleven species of the latter genus to Lambiella and describe the genus Parainoa to accommodate a previously misunderstood species of Trapeliopsis. Past phylogenetic studies in Ostropomycetidae have invoked "divergence order" for drawing taxonomic conclusions on higher level taxa. Our data show that if backbone support is lacking, contrasting solutions may be recovered with different or added data. We accordingly urge caution in concluding evolutionary relationships from unresolved phylogenies.

Incongruence among different mitochondrial regions: a case study using complete mitogenomes.

Mitochondrial sequences have long been used to examine vertebrate phylogenetic relationships. The extensive use of mitochondrial data reflects the ease of obtaining mitochondrial sequences and its relatively rapid coalescence time. Mitochondrial genomes typically do not undergo recombination, so the entire mitogenome should have the same underlying gene tree. Thus, given appropriate analyses, conflict among estimates of phylogeny from different mitochondrial regions should not exist. However, estimates of phylogeny based upon different mitochondrial regions can exhibit incongruence. Conflict in phylogenetic signal among mitochondrial regions has been observed in galliform birds for the position of the Odontophoridae (New World quail). To explore this, we expanded sampling to 47 galliform mitogenomes, adding six new mitogenomes, which included representatives of two previously unsampled families. Analyses of complete mitogenomes recovered a well-supported topology that was congruent with expectations from multi-locus studies. However, when analyzing individual regions, we found conflicting positions for the Odontophoridae and several other relationships at multiple taxonomic levels. We tested multiple analytical strategies to reduce incongruence among regions, including partitioning by codon position, using mixture and codon-based models, RY coding, and excluding potentially misleading sites. No approach consistently reduced the conflict among mitochondrial regions at any taxonomic level. The biological attributes of both strongly misleading and non-misleading sites were essentially identical. Increasing taxa actually appeared to increase conflicting signal, even when taxa were selected to break up long branches. Collectively, our results indicate that analyzing mitochondrial data remains difficult, although analyzing complete mitogenomes resulted in a good estimate of the mitochondrial gene tree.

The impact of automated filtering of BLAST-determined homologs in the phylogenetic detection of horizontal gene transfer from a transcriptome assembly.

Phylomes (comprehensive sets of gene phylogenies for organisms) are built to investigate fundamental questions in genomics and evolutionary biology, such as those pertaining to the detection and characterization of horizontal gene transfer in microbes. To address these questions, phylome construction demands rigorous yet efficient phylogenetic methods. Currently, many sequence alignment and tree-building models can analyze several thousands of genes in a high-throughput manner. However, the phylogenetics is complicated by variability in sequence divergence and different taxon sampling among genes. In addition, homolog selection for automated approaches often relies on arbitrary sequence similarity thresholds that are likely inappropriate for all genes in a genome. To investigate the effects of automated homolog selection on the detection of horizontal gene transfer using phylogenomics, we constructed the phylome of a transcriptome assembly of Alexandrium tamarense, a microbial eukaryote with a history of horizontal and endosymbiotic gene transfer, using seven sequence similarity thresholds for selecting putative homologs to be included in phylogenetic analyses. We show that no single threshold recovered informative trees for the majority of A. tamarense unigenes compared to the pooled results from all pipeline iterations. As much as 29% of trees built could have misleading phylogenetic relationships that appear biased in favor of those otherwise indicative of horizontal gene transfer. Perhaps worse, nearly half of the unigenes were represented by a single tree built at just one threshold, making it difficult to assess the validity of phylogenetic relationships recovered in these cases. However, combining the results from several pipeline iterations maximizes the number of informative phylogenies. Moreover, when the same phylogenetic relationship for a given unigene is recovered in multiple pipeline iterations, conclusions regarding gene origin are more robust to methodological artifact. Using these methods, the majority of A. tamarense unigenes showed evolutionary relationships indicative of vertical inheritance. Nevertheless, many other unigenes revealed diverse phylogenetic associations, suggestive of possible gene transfer. This analysis suggests that caution should be used when interpreting the results from phylogenetic pipelines implementing a single similarity threshold. Our approach is a practical method to mitigate the problems associated with automated sequence selection in phylogenomics.