Joining forces in Ochnaceae phylogenomics: a tale of two targeted sequencing probe kits.

PREMISE: Both universal and family-specific targeted sequencing probe kits are becoming widely used for reconstruction of phylogenetic relationships in angiosperms. Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits. Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships. METHODS: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support. RESULTS: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree. CONCLUSIONS: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.

Plastome phylogenomic study of Gentianeae (Gentianaceae): widespread gene tree discordance and its association with evolutionary rate heterogeneity of plastid genes.

BACKGROUND: Plastome-scale data have been prevalent in reconstructing the plant Tree of Life. However, phylogenomic studies currently based on plastomes rely primarily on maximum likelihood inference of concatenated alignments of plastid genes, and thus phylogenetic discordance produced by individual plastid genes has generally been ignored. Moreover, structural and functional characteristics of plastomes indicate that plastid genes may not evolve as a single locus and are experiencing different evolutionary forces, yet the genetic characteristics of plastid genes within a lineage remain poorly studied. RESULTS: We sequenced and annotated 10 plastome sequences of Gentianeae. Phylogenomic analyses yielded robust relationships among genera within Gentianeae. We detected great variation of gene tree topologies and revealed that more than half of the genes, including one (atpB) of the three widely used plastid markers (rbcL, atpB and matK) in phylogenetic inference of Gentianeae, are likely contributing to phylogenetic ambiguity of Gentianeae. Estimation of nucleotide substitution rates showed extensive rate heterogeneity among different plastid genes and among different functional groups of genes. Comparative analysis suggested that the ribosomal protein (RPL and RPS) genes and the RNA polymerase (RPO) genes have higher substitution rates and genetic variations among plastid genes in Gentianeae. Our study revealed that just one (matK) of the three (matK, ndhB and rbcL) widely used markers show high phylogenetic informativeness (PI) value. Due to the high PI and lowest gene-tree discordance, rpoC2 is advocated as a promising plastid DNA barcode for taxonomic studies of Gentianeae. Furthermore, our analyses revealed a positive correlation of evolutionary rates with genetic variation of plastid genes, but a negative correlation with gene-tree discordance under purifying selection. CONCLUSIONS: Overall, our results demonstrate the heterogeneity of nucleotide substitution rates and genetic characteristics among plastid genes providing new insights into plastome evolution, while highlighting the necessity of considering gene-tree discordance into phylogenomic studies based on plastome-scale data.

Optimal markers for the identification of Colletotrichum species.

Colletotrichum is among the most important genera of fungal plant pathogens. Molecular phylogenetic studies over the last decade have resulted in a much better understanding of the evolutionary relationships and species boundaries within the genus. There are now approximately 200 species accepted, most of which are distributed among 13 species complexes. Given their prominence on agricultural crops around the world, rapid identification of a large collection of Colletotrichum isolates is routinely needed by plant pathologists, regulatory officials, and fungal biologists. However, there is no agreement on the best molecular markers to discriminate species in each species complex. Here we calculate the barcode gap distance and intra/inter-specific distance overlap to evaluate each of the most commonly applied molecular markers for their utility as a barcode for species identification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone-3 (HIS3), DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), and intergenic spacer between GAPDH and a hypothetical protein (GAP2-IGS) have the properties of good barcodes, whereas sequences of actin (ACT), chitin synthase (CHS-1) and nuclear rDNA internal transcribed spacers (nrITS) are not able to distinguish most species. Finally, we assessed the utility of these markers for phylogenetic studies using phylogenetic informativeness profiling, the genealogical sorting index (GSI), and Bayesian concordance analyses (BCA). Although GAPDH, HIS3 and ß-tubulin (TUB2) were frequently among the best markers, there was not a single set of markers that were best for all species complexes. Eliminating markers with low phylogenetic signal tends to decrease uncertainty in the topology, regardless of species complex, and leads to a larger proportion of markers that support each lineage in the Bayesian concordance analyses. Finally, we reconstruct the phylogeny of each species complex using a minimal set of phylogenetic markers with the strongest phylogenetic signal and find the majority of species are strongly supported as monophyletic.

Utility of targeted sequence capture for phylogenomics in rapid, recent angiosperm radiations: Neotropical Burmeistera bellflowers as a case study.

Targeted sequence capture is a promising approach for large-scale phylogenomics. However, rapid evolutionary radiations pose significant challenges for phylogenetic inference (e.g. incomplete lineages sorting (ILS), phylogenetic noise), and the ability of targeted nuclear loci to resolve species trees despite such issues remains poorly studied. We test the utility of targeted sequence capture for inferring phylogenetic relationships in rapid, recent angiosperm radiations, focusing on Burmeistera bellflowers (Campanulaceae), which diversified into ~130 species over less than 3 million years. We compared phylogenies estimated from supercontig (exons plus flanking sequences), exon-only, and flanking-only datasets with 506-546 loci (~4.7 million bases) for 46 Burmeistera species/lineages and 10 outgroup taxa. Nuclear loci resolved backbone nodes and many congruent internal relationships with high support in concatenation and coalescent-based species tree analyses, and inferences were largely robust to effects of missing taxa and base composition biases. Nevertheless, species trees were incongruent between datasets, and gene trees exhibited remarkably high levels of conflict (~4-60% congruence, ~40-99% conflict) not simply driven by poor gene tree resolution. Higher gene tree heterogeneity at shorter branches suggests an important role of ILS, as expected for rapid radiations. Phylogenetic informativeness analyses also suggest this incongruence has resulted from low resolving power at short internal branches, consistent with ILS, and homoplasy at deeper nodes, with exons exhibiting much greater risk of incorrect topologies due to homoplasy than other datasets. Our findings suggest that targeted sequence capture is feasible for resolving rapid, recent angiosperm radiations, and that results based on supercontig alignments containing nuclear exons and flanking sequences have higher phylogenetic utility and accuracy than either alone. We use our results to make practical recommendations for future target capture-based studies of Burmeistera and other rapid angiosperm radiations, including that such studies should analyze supercontigs to maximize the phylogenetic information content of loci.

Identification and assessment of variable single-copy orthologous (SCO) nuclear loci for low-level phylogenomics: a case study in the genus Rosa (Rosaceae).

BACKGROUND: With an ever-growing number of published genomes, many low levels of the Tree of Life now contain several species with enough molecular data to perform shallow-scale phylogenomic studies. Moving away from using just a few universal phylogenetic markers, we can now target thousands of other loci to decipher taxa relationships. Making the best possible selection of informative sequences regarding the taxa studied has emerged as a new issue. Here, we developed a general procedure to mine genomic data, looking for orthologous single-copy loci capable of deciphering phylogenetic relationships below the generic rank. To develop our strategy, we chose the genus Rosa, a rapid-evolving lineage of the Rosaceae family in which several species genomes have recently been sequenced. We also compared our loci to conventional plastid markers, commonly used for phylogenetic inference in this genus. RESULTS: We generated 1856 sequence tags in putative single-copy orthologous nuclear loci. Associated in silico primer pairs can potentially amplify fragments able to resolve a wide range of speciation events within the genus Rosa. Analysis of parsimony-informative site content showed the value of non-coding genomic regions to obtain variable sequences despite the fact that they may be more difficult to target in less related species. Dozens of nuclear loci outperform the conventional plastid phylogenetic markers in terms of phylogenetic informativeness, for both recent and ancient evolutionary divergences. However, conflicting phylogenetic signals were found between nuclear gene tree topologies and the species-tree topology, shedding light on the many patterns of hybridization and/or incomplete lineage sorting that occur in the genus Rosa. CONCLUSIONS: With recently published genome sequence data, we developed a set of single-copy orthologous nuclear loci to resolve species-level phylogenomics in the genus Rosa. This genome-wide scale dataset contains hundreds of highly variable loci which phylogenetic interest was assessed in terms of phylogenetic informativeness and topological conflict. Our target identification procedure can easily be reproduced to identify new highly informative loci for other taxonomic groups and ranks.

Assessing phylogenetic information to reveal uncertainty in historical data: An example using Goodeinae (Teleostei: Cyprinodontiformes: Goodeidae).

A major emerging challenge to resolution of a stable phylogenetic Tree of Life has been incongruent inference among studies. Given the increasing ubiquity of incongruent studies, analyzing the predicted phylogenetic utility and quantitative evidence regarding contributions toward resolution of commonly-used markers in historical studies over the last decade represents an important, yet neglected, component of phylogenetics. Here we examine the phylogenetic utility of two sets of commonly-used legacy markers for understanding the evolutionary relationships among goodeines, a group of viviparous freshwater fishes endemic to central Mexico. Our analyses reveal that the validity of existing inferences is compromised by both lack of information and substantially biased patterns of nucleotide substitution. Our analyses demonstrate that many of the evolutionary relationships of goodeines remain uncertain - despite over a century of work. Our results provide an updated baseline of critically needed areas of investigation for the group and underscore the importance of quantifying phylogenetic information content as a fundamental step towards eroding false confidence in results based on weak and biased evidence.

First mitochondrial genomes of five hoverfly species of the genus Eristalinus (Diptera: Syrphidae).

The hoverfly genus Eristalinus (Diptera, Syrphidae) contains many widespread pollinators. The majority of the species of Eristalinus occur in the Afrotropics and their molecular systematics still needs to be investigated. This study presents the first complete and annotated mitochondrial genomes for five species of Eristalinus. They were obtained by high-throughput sequencing of total genomic DNA. The total length of the mitogenomes varied between 15 757 and 16 245 base pairs. Gene composition, positions, and orientation were shared across species, and were identical to those observed for other Diptera. Phylogenetic analyses (maximum likelihood and Bayesian inference) based on the 13 protein coding and both rRNA genes suggested that the subgenus Eristalinus was paraphyletic with respect to the subgenus Eristalodes. An analysis of the phylogenetic informativeness of all protein coding and rRNA genes suggested that NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit 1, nad4, nad2, cytochrome b, and 16S rRNA genes are the most promising mitochondrial molecular markers to result in supported phylogenetic hypotheses of the genus. In addition to the five complete mitogenomes currently available for hoverflies, the five mitogenomes published here will be useful for broader molecular phylogenetic analyses among hoverflies.

Phylogenetic analysis of Antarctic notothenioids illuminates the utility of RADseq for resolving Cenozoic adaptive radiations.

Notothenioids are a clade of ∼120 species of marine fishes distributed in extreme southern hemisphere temperate near-shore habitats and in the Southern Ocean surrounding Antarctica. Over the past 25 years, molecular and morphological approaches have redefined hypotheses of relationships among notothenioid lineages as well as their relationships among major lineages of percomorph teleosts. These phylogenies provide a basis for investigation of mechanisms of evolutionary diversification within the clade and have enhanced our understanding of the notothenioid adaptive radiation. Despite extensive efforts, there remain several questions concerning the phylogeny of notothenioids. In this study, we deploy DNA sequences of ∼100,000 loci obtained using RADseq to investigate the phylogenetic relationships of notothenioids and to assess the utility of RADseq loci for lineages that exhibit divergence times ranging from the Paleogene to the Quaternary. The notothenioid phylogenies inferred from the RADseq loci provide unparalleled resolution and node support for several long-standing problems including, (1) relationships among species of Trematomus, (2) resolution of Indonotothenia cyanobrancha as the sister lineage of Trematomus, (3) the deep paraphyly of Nototheniidae, (4) the paraphyly of Lepidonotothen s.l., (5) paraphyly of Artedidraco, and 6) the monophyly of the Bathydraconidae. Assessment of site rates demonstrates that RADseq loci are similar to mtDNA protein coding genes and exhibit peak phylogenetic informativeness at the time interval during which the major Antarctic notothenioid lineages originated and diversified. In addition to providing a well-resolved phylogenetic hypothesis for notothenioids, our analyses quantify the predicted utility of RADseq loci for Cenozoic phylogenetic inferences.

Explosive radiation or uninformative genes? Origin and early diversification of tachinid flies (Diptera: Tachinidae).

Molecular phylogenetic studies at all taxonomic levels often infer rapid radiation events based on short, poorly resolved internodes. While such rapid episodes of diversification are an important and widespread evolutionary phenomenon, much of this poor phylogenetic resolution may be attributed to the continuing widespread use of "traditional" markers (mitochondrial, ribosomal, and some nuclear protein-coding genes) that are often poorly suited to resolve difficult, higher-level phylogenetic problems. Here we reconstruct phylogenetic relationships among a representative set of taxa of the parasitoid fly family Tachinidae and related outgroups of the superfamily Oestroidea. The Tachinidae are one of the most species rich, yet evolutionarily recent families of Diptera, providing an ideal case study for examining the differential performance of loci in resolving phylogenetic relationships and the benefits of adding more loci to phylogenetic analyses. We assess the phylogenetic utility of nine genes including both traditional genes (e.g., CO1 mtDNA, 28S rDNA) and nuclear protein-coding genes newly developed for phylogenetic analysis. Our phylogenetic findings, based on a limited set of taxa, include: a close relationship between Tachinidae and the calliphorid subfamily Polleninae, monophyly of Tachinidae and the subfamilies Exoristinae and Dexiinae, subfamily groupings of Dexiinae+Phasiinae and Tachininae+Exoristinae, and robust phylogenetic placement of the somewhat enigmatic genera Strongygaster, Euthera, and Ceracia. In contrast to poor resolution and phylogenetic incongruence of "traditional genes," we find that a more selective set of highly informative genes is able to more precisely identify regions of the phylogeny that experienced rapid radiation of lineages, while more accurately depicting their phylogenetic context. Although much expanded taxon sampling is necessary to effectively assess the monophyly of and relationships among major tachinid lineages and their relatives, we show that a small number of well-chosen nuclear protein-coding genes can successfully resolve even difficult phylogenetic problems.

Genome-wide ultraconserved elements exhibit higher phylogenetic informativeness than traditional gene markers in percomorph fishes.

Ultraconserved elements (UCEs) have become popular markers in phylogenomic studies because of their cost effectiveness and their potential to resolve problematic phylogenetic relationships. Although UCE datasets typically contain a much larger number of loci and sites than more traditional datasets of PCR-amplified, single-copy, protein coding genes, a fraction of UCE sites are expected to be part of a nearly invariant core, and the relative performance of UCE datasets versus protein coding gene datasets is poorly understood. Here we use phylogenetic informativeness (PI) to compare the resolving power of multi-locus and UCE datasets in a sample of percomorph fishes with sequenced genomes (genome-enabled). We compare three data sets: UCE core regions, flanking sequence adjacent to the UCE core and a set of ten protein coding genes commonly used in fish systematics. We found the net informativeness of UCE core and flank regions to be roughly ten-fold and 100-fold more informative than that of the protein coding genes. On a per locus basis UCEs and protein coding genes exhibited similar levels of phylogenetic informativeness. Our results suggest that UCEs offer enormous potential for resolving relationships across the percomorph tree of life.

On the distribution of interspecies correlation for Markov models of character evolution on Yule trees.

Efforts to reconstruct phylogenetic trees and understand evolutionary processes depend fundamentally on stochastic models of speciation and mutation. The simplest continuous-time model for speciation in phylogenetic trees is the Yule process, in which new species are "born" from existing lineages at a constant rate. Recent work has illuminated some of the structural properties of Yule trees, but it remains mostly unknown how these properties affect sequence and trait patterns observed at the tips of the phylogenetic tree. Understanding the interplay between speciation and mutation under simple models of evolution is essential for deriving valid phylogenetic inference methods and gives insight into the optimal design of phylogenetic studies. In this work, we derive the probability distribution of interspecies covariance under Brownian motion and Ornstein-Uhlenbeck models of phenotypic change on a Yule tree. We compute the probability distribution of the number of mutations shared between two randomly chosen taxa in a Yule tree under discrete Markov mutation models. Our results suggest summary measures of phylogenetic information content, illuminate the correlation between site patterns in sequences or traits of related organisms, and provide heuristics for experimental design and reconstruction of phylogenetic trees.

HiMAP2: Identifying phylogenetically informative genetic markers from diverse genomic resources.

Multiplexed amplicon sequencing offers a cost-effective and rapid solution for phylogenomic studies that include a large number of individuals. Selecting informative genetic markers is a critical initial step in designing such multiplexed amplicon panels, but screening various genomic data and selecting markers that are informative for the question at hand can be laborious. Here, we present a flexible and user-friendly tool, HiMAP2, for identifying, visualizing and filtering phylogenetically informative loci from diverse genomic and transcriptomic resources. This bioinformatics pipeline includes orthology prediction, exon extraction and filtering of aligned exon sequences according to user-defined specifications. Additionally, HiMAP2 facilitates exploration of the final filtered exons by incorporating phylogenetic inference of individual exon trees with raxml-ng as well as the estimation of a species tree using astral. Finally, results of the marker selection can be visualized and refined with an interactive Bokeh application that can be used to generate publication-quality figures. Source code and user instructions for HiMAP2 are available at https://github.com/popphylotools/HiMAP_v2.

New plastome structural rearrangements discovered in core Tillandsioideae (Bromeliaceae) support recently adopted taxonomy.

Full plastome sequences for land plants have become readily accessible thanks to the development of Next Generation Sequencing (NGS) techniques and powerful bioinformatic tools. Despite this vast amount of genomic data, some lineages remain understudied. Full plastome sequences from the highly diverse (>1,500 spp.) subfamily Tillandsioideae (Bromeliaceae, Poales) have been published for only three (i.e., Guzmania, Tillandsia, and Vriesea) out of 22 currently recognized genera. Here, we focus on core Tillandsioideae, a clade within subfamily Tillandsioideae, and explore the contribution of individual plastid markers and data categories to inform deep divergences of a plastome phylogeny. We generated 37 high quality plastome assemblies and performed a comparative analysis in terms of plastome structure, size, gene content and order, GC content, as well as number and type of repeat motifs. Using the obtained phylogenetic context, we reconstructed the evolution of these plastome attributes and assessed if significant shifts on the evolutionary traits' rates have occurred in the evolution of the core Tillandsioideae. Our results agree with previously published phylogenetic hypotheses based on plastid data, providing stronger statistical support for some recalcitrant nodes. However, phylogenetic discordance with previously published nuclear marker-based hypotheses was found. Several plastid markers that have been consistently used to address phylogenetic relationships within Tillandsioideae were highly informative for the retrieved plastome phylogeny and further loci are here identified as promising additional markers for future studies. New lineage-specific plastome rearrangements were found to support recently adopted taxonomic groups, including large inversions, as well as expansions and contractions of the inverted repeats. Evolutionary trait rate shifts associated with changes in size and GC content of the plastome regions were found across the phylogeny of core Tillandsioideae.

Comparing Ultraconserved Elements and Exons for Phylogenomic Analyses of Middle American Cichlids: When Data Agree to Disagree.

Choosing among types of genomic markers to be used in a phylogenomic study can have a major influence on the cost, design, and results of a study. Yet few attempts have been made to compare categories of next-generation sequence markers limiting our ability to compare the suitability of these different genomic fragment types. Here, we explore properties of different genomic markers to find if they vary in the accuracy of component phylogenetic trees and to clarify the causes of conflict obtained from different data sets or inference methods. As a test case, we explore the causes of discordance between phylogenetic hypotheses obtained using a novel data set of ultraconserved elements (UCEs) and a recently published exon data set of the cichlid tribe Heroini. Resolving relationships among heroine cichlids has historically been difficult, and the processes of colonization and diversification in Middle America and the Greater Antilles are not yet well understood. Despite differences in informativeness and levels of gene tree discordance between UCEs and exons, the resulting phylogenomic hypotheses generally agree on most relationships. The independent data sets disagreed in areas with low phylogenetic signal that were overwhelmed by incomplete lineage sorting and nonphylogenetic signals. For UCEs, high levels of incomplete lineage sorting were found to be the major cause of gene tree discordance, whereas, for exons, nonphylogenetic signal is most likely caused by a reduced number of highly informative loci. This paucity of informative loci in exons might be due to heterogeneous substitution rates that are problematic to model (i.e., computationally restrictive) resulting in systematic errors that UCEs (being less informative individually but more uniform) are less prone to. These results generally demonstrate the robustness of phylogenomic methods to accommodate genomic markers with different biological and phylogenetic properties. However, we identify common and unique pitfalls of different categories of genomic fragments when inferring enigmatic phylogenetic relationships.

Optimizing the widely used nuclear protein-coding gene primers in beetle phylogenies and their application in the genus Sasajiscymnus Vandenberg (Coleoptera: Coccinellidae).

Advances in genomic biology and the increasing availability of genomic resources allow developing hundreds of nuclear protein-coding (NPC) markers, which can be used in phylogenetic research. However, for low taxonomic levels, it may be more practical to select a handful of suitable molecular loci for phylogenetic inference. Unfortunately, the presence of degenerate primers of NPC markers can be a major impediment, as the amplification success rate is low and they tend to amplify nontargeted regions. In this study, we optimized five NPC fragments widely used in beetle phylogenetics (i.e., two parts of carbamoyl-phosphate synthetase: CADXM and CADMC, Topoisomerase, Wingless and Pepck) by reducing the degenerate site of primers and the length of target genes slightly. These five NPC fragments and 6 other molecular loci were amplified to test the monophyly of the coccinellid genus Sasajiscymnus Vandenberg. The analysis of our molecular data set clearly supported the genus Sasajiscymnus may be monophyletic but confirmation with an extended sampling is required. A fossil-calibrated chronogram was generated by BEAST, indicating an origin of the genus at the end of the Cretaceous (77.87 Myr). Furthermore, a phylogenetic informativeness profile was generated to compare the phylogenetic properties of each gene more explicitly. The results showed that COI provides the strongest phylogenetic signal among all the genes, but Pepck, Topoisomerase, CADXM and CADMC are also relatively informative. Our results provide insight into the evolution of the genus Sasajiscymnus, and also enrich the molecular data resources for further study.

Structure, evolution and phylogenetic informativeness of eelpouts (Cottoidei: Zoarcales) mitochondrial control region sequences.

Control region (CR) is a major non-coding domain of mitochondrial DNA in vertebrates which contains the promoters for replication and transcription of mitochondrial genome along with the binding sites for metabolic machinery and, hence, is a vital element for the integrity of mitochondrial genome as a biological replicator. The origin and diversity of structural elements within CR have been intensively studied in recent years with the involvement of new diverse taxa. In this paper, we provide new data on the nucleotide and structural patterns of CR evolution and phylogenetic suitability among eelpouts (Cottoidei: Zoarcales). To achieve this, we carried out a comparative phylogenetic and structural analysis of 29 CR sequences belonging to the long shanny Stichaeus grigorjewi together with nine sequences of other eelpouts taxa representing four families in contrast to mitochondrial protein-coding fragments. The CR organization within S. grigorjewi, as well as in all other eelpouts, is consistent with the common three-domain structure known from most vertebrates. We found a hidden CR variation constrains on the landscape level and a lack of nucleotide saturation. Finally, our results demonstrate the advantage of the length variation in CR sequences for phylogenetic reconstructions among eelpouts.

Targeted Capture of Hundreds of Nuclear Genes Unravels Phylogenetic Relationships of the Diverse Neotropical Palm Tribe Geonomateae.

The tribe Geonomateae is a widely distributed group of 103 species of Neotropical palms which contains six ecologically important understory or subcanopy genera. Although it has been the focus of many studies, our understanding of the evolutionary history of this group, and in particular of the taxonomically complex genus Geonoma, is far from complete due to a lack of molecular data. Specifically, the previous Sanger sequencing-based studies used a few informative characters and partial sampling. To overcome these limitations, we used a recently developed Arecaceae-specific target capture bait set to undertake a phylogenomic analysis of the tribe Geonomateae. We sequenced 3,988 genomic regions for 85% of the species of the tribe, including 84% of the species of the largest genus, Geonoma. Phylogenetic relationships were inferred using both concatenation and coalescent methods. Overall, our phylogenetic tree is highly supported and congruent with taxonomic delimitations although several morphological taxa were revealed to be non-monophyletic. It is the first time that such a large genomic dataset is provided for an entire tribe within the Arecaceae. Our study lays the groundwork not only for detailed macro- and micro-evolutionary studies within the group, but also sets a workflow for understanding other species complexes across the tree of life.

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species.

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies.

Nearly neutral evolution in IFNL3 gene retains the immune function to detect and clear the viral infection in HCV.

IFNL3 gene plays a crucial role in immune defense against viruses. It induces the interferon stimulated genes (ISGs) with antiviral properties by activating the JAK-STAT pathway. In this study, we investigated the evolutionary force involved in shaping the IFNL3 gene to perform its downstream function as a regulatory gene in HCV clearance. We have selected 25 IFNL3 coding sequences with human gene as a reference sequence and constructed a phylogeny. Furthermore, rate of variation, substitution saturation test, phylogenetic informativeness and differential selection were also analysed. The codon evolution result suggests that nearly neutral mutation is the key pattern in shaping the IFNL3 evolution. The results were validated by subjecting the human IFNL3 protein variants to that of the native through a molecular dynamics simulation study. The molecular dynamics simulation clearly depicts the negative impact on the reported variants in human IFNL3 protein. However, these detrimental mutations (R157Q and R157W) were shown to be negatively selected in the evolutionary study of the mammals. Hence, the variation revealed a mild impact on the IFNL3 function and may be removed from the population through negative selection due to its high functional constraints. In a nutshell, our study may contribute the overall evidence in phylotyping and structural transformation that takes place in the non-synonymous substitutions of IFNL3 protein. Substantially, our obtained theoretical knowledge will lay the path to extend the experimental validation in HCV clearance.