Robust Phylogenetic Tree Research Articles

Magnetic beads, a particularly effective novel method for extraction of NGS-ready DNA from macroalgae

Next Generation Sequencing (NGS) technologies allow for the generation of robust information on the genetic diversity of organisms at the individual, species and higher taxon levels. Indeed, the number of single nucleotide polymorphisms (SNPs) detected using next-generation sequencing is order of magnitudes higher than SNPs and/or alleles detected using traditional barcoding or microsatellites. However, the amount and quality of DNA required for next-generation sequencing is greater as compared with PCR-based methods. Such high DNA amount and quality requirements can be a hindrance when working on species that are rich in polyphenols and polysaccharides, such as macroalgae (seaweeds). Various protocols, based on column-based DNA extraction kits and CTAB/Phenol:Chloroform, are available to tackle this issue. However, those protocols are usually costly and/or time-consuming and may not be reliable due to variations in the polyphenols/polysaccharide content between individuals of the same species. Here, we report the successful use of a magnetic-beads-based protocol for efficient, reliable, fast and simultaneous DNA extraction of several macroalgae species. DNA extracted from macroalgae using this method is of high quality and purity, allowing successful library preparation for next generation sequencing. We generated Genotype-By-Sequencing (GBS) data for 12 Ulva spp. (Sea Lettuce; Ulvophyceae, Ulvaceae) individuals and were able to generate a robust phylogenetic tree that we compared with traditional barcoding methods.

Gene arrangement and sequence of mitochondrial genomes yield insights into the phylogeny and evolution of bees and sphecid wasps (Hymenoptera: Apoidea)

The Apoidea represent a large and common superfamily of the Hymenoptera including the bees and sphecid wasps. A robust phylogenetic tree is essential to understanding the diversity, taxonomy and evolution of the Apoidea. In this study, features of apoid mitochondrial genomes were used to reconstruct phylogenetic relationships. Twelve apoid mitochondrial genomes were newly sequenced, representing six families and nine subfamilies. Gene rearrangement events have occurred in all apoid mitochondrial genomes sequenced to date. Sphecid wasps have both tRNA and protein-coding gene rearrangements in 5 of 8 species. In bees, the only rearranged genes are tRNAs; long-tongued bees (Apidae + Megachilidae) are characterized by movement of trnA to the trnI-trnQ-trnM tRNA cluster. Phylogenetic analyses of mitochondrial gene sequences support the known paraphyly of sphecid wasps, with bees nested within this clade. The Ampulicidae is sister to the remaining Apoidea. Crabronidae is paraphyletic, split into Crabronidae s.s. and Philanthidae, with the latter group a sister clade to bees. The monophyletic bees are either classified into two clades, long-tongued bees (Apidae + Megachilidae) and short-tongued bees (Andrenidae + Halictidae + Colletidae + Melitidae), or three groups with the Melitidae sister to the other bees. Our study showed that both gene sequences and arrangements provide information on the phylogeny of apoid families.

Revision, phylogeny and phylogeography of the cicada genus Auritibicen (Hemiptera: Cicadidae), with descriptions of ten new species

We review the cicada genus Auritibicen Lee, 2015 based on the description of ten new species: A. aethus sp. n., A. daoxianensis sp. n., A. pallidus sp. n., A. rotundus sp. n., A. curvatus sp. n., A. purus sp. n., A. parvus sp. n., A. gracilis sp. n., A. septatus sp. n. and A. lijiangensis sp. n. Auritibicen shikokuanus (Kato, 1959) is confirmed to be a synonym of Auritibicen kyushyuensis (Kato, 1926). Diagnoses and descriptions, along with illustrations of the structure of male genitalia, are provided for all Auritibicen species. The systematics of Auritibicen is elucidated using both morphological and molecular characterization. Thirty-five morphological characters of the 24 species of Auritibicen and one outgroup taxon, Chremistica ochracea (Walker, 1850), were scored. Morphological phylogenetic analyses reveal the relationships among related species of Auritibicen, which are supported by a number of morphological characters. The mitochondrial gene fragments of Cytochrome Oxidase I (COI) of 11 species of Auritibicen and two outgroup Lyristes species were analyzed and yielded identical robust phylogenetic trees. The phylogram based on a Bayesian analysis of both morphological and molecular data is similar to the ML/BI topologies based only on the molecular data. The molecular phylogenetic analysis indicates that species of Auritibicen are structured phylogeographically, with related species clustered into three lineages. The divergence time estimated based on molecular data indicates that the divergence of Auritibicen from Lyristes occurred during the Miocene, and the most recent common ancestor (tMRCA) of Auritibicen evolved during the Pliocene. However, the time when the main divergence events of species of Auritibicen occurred was the Pleistocene. From the combination of the phylogeny and updated geographical distributions, we infer that the center of distribution of Auritibicen could be Southwest China (e.g., Sichuan and Yunnan Provinces), from where species of this genus spreaded northeastwards to Shaanxi, Hubei and other provinces along the Qinling and Daba Mountains, then further northeastwards to Hebei Province in China and also to Far East Russia, the Korean Penisula, and Japan.

Comparative Flower and Inflorescence Organogenesis among Genera of Betulaceae: Implications for Phylogenetic Relationships

Betulaceae have simple flowers but complex inflorescences. Recent phylogenetic analyses using molecular data have produced robust phylogenetic trees of Betulaceae. In this study, we evaluated the phylogenetic value of comparative organogenetic data of reproductive organs in the context of molecular phylogenies. Flower and inflorescence developmental processes of 21 species from all six genera in Betulaceae were documented with scanning electron microscopy. In each pistillate cyme, there are one primary bract, two secondary bracts, and two or three flowers in the six genera; only in Alnus are there two tertiary bracts on the abaxial side. The pistillate flower of all genera but Alnus has tepal primordia. Two tepals stop developing early on, resulting in the lack of tepals in mature flowers of Betula; while the tepals are initiated from a common circular primordium at the base of pistil in Corylus, Ostryopsis, Carpinus, and Ostrya, and the developed tepals with irregular shape and unstable number of lobes are adnate to the top of the pistil. In staminate organogenesis, each cyme has one primary bract and three flowers in all genera; two secondary bracts are only present in Alnus, Betula, and Corylus. Staminate flowers have no tepals except in Alnus and Betula, and exhibit high variation in number of stamens among genera. The number of secondary and tertiary bracts in each pistillate and staminate cyme, as well as the presence of tepals in pistillate and staminate flowers was clarified in all genera. Micro-morphological characters were used to infer the phylogenetic relationships of genera and sections of Betulaceae. Our analyses support the division of two subfamilies: Betuloideae (Alnus and Betula) and Coryloideae (Corylus, Carpinus, Ostrya, and Ostryopsis), and three tribes: Betuleae (Alnus and Betula), Coryleae (Corylus), and Carpineae (Carpinus, Ostrya, and Ostryopsis). The results agree with those from molecular phylogenetic studies, and suggest that micro-morphological characters are phylogenetically informative in Betulaceae, and reproductive organs of Betulaceae have evolved in the direction of reduction in bracts and tepals.

Size homoplasy and mutational behavior of chloroplast simple sequence repeats (cpSSRs) inferred from intra- and interspecific variations in four chloroplast regions of diploid and polyploid Triticum and Aegilops species

Chloroplast simple sequence repeats (cpSSRs) are widely distributed in the chloroplast genomes of all plant species, and are frequently employed for genotypic and phylogenetic analysis. However, information on intra- and interspecies variation in cpSSRs is lacking. In this study, we sequenced four intergenic (non-coding) chloroplast DNA regions in 57 accessions of 12 tetraploid, and 16 accessions of 4 hexaploid species of Triticum and Aegilops. These sequence data added to our previous data for diploid species in the same chloroplast regions. Intra- and interspecific genetic variation was analyzed for a total of 189 accessions of 13 diploid, 12 tetraploid, and 4 hexaploid species of Triticum and Aegilops, such that all species were represented by multiple accessions. The data were used to infer phylogenetic relationships within and among Triticum and Aegilops species. Based on this robust phylogenetic tree, seven of eight cpSSR loci clearly exhibited “size homoplasy,” referring to the fact that cpSSRs of identical size and DNA sequence can arise even if the alleles are not descended from a common ancestor. These data indicate that cpSSRs should be used with caution in phylogenetic analyzes. Interestingly, as observed from several previous studies, our data also suggest that observed mutation rates may increase significantly when mononucleotide (homopolymer) repeat numbers reach or exceed 9 bp. In the present report, using this sequence data set involving cpSSRs, 81 unique haplotypes among 189 accessions were detected, and five tetraploid Triticum and Aegilops species were successfully identified and genotyped. Our results indicate that combinations of nucleotide substitutions, indels and SSRs of chloroplast nucleotide sequences are available for genotyping at the species accession level.

Multilocus and SSU rRNA gene phylogenetic analyses of available cyanobacterial genomes, and their relation to the current taxonomic system

Despite the global significance of cyanobacteria, their taxonomy is still heavily lagging behind the progress in genome sequencing. Besides nomenclatural issues and difficulties in species identification, this problem is largely a consequence of missing reference strains and sequences for many cyanobacterial taxa. In this study, a robust multilocus phylogenetic tree based on 23 conserved proteins from the current set of available genomes is presented and compared to a respective 16S rRNA gene tree. To facilitate further progress in achieving a stable system of monophyletic taxa, the phylogenetic status of eight cyanobacterial orders recently proposed in the botanical system was evaluated. While several orders addressed by previous studies (Gloeobacterales, Spirulinales, Chroococcidiopsidales, Nostocales) retain their monophyly, the remaining big groups (Synechococcales, Pleurocapsales, Chroococcales, Oscillatoriales) are polyphyletic and require thorough revision. Comparison of the 16S rRNA tree to the multilocus tree supports the careful use of this gene in distinguishing cyanobacterial genera, whereas its application at other taxonomic levels is limited.

The complete mitochondrial genomes of three geographical lineages in short ninespine stickleback (Pungitius kaibarae) complex and their phylogenetic implication

The short ninespine stickleback, Pungitius kaibarae, is a small gasterosteid species complex containing three geographical lineages: ND, NE and SE. Here, complete mitochondrial genomes of these three lineages were analyzed to estimate the genetic differentiation among them and to identify their phylogenetic placement in genus Pungitius. Although the overall genome structure was identical among those three lineages, the genome sizes were slightly different from each other, ranging from 16,489 to 16,500 bp. Upon robust phylogenetic tree inferred by Bayesian algorithm, ND and SE showed relatively higher affinity, and those three lineages formed a monophyletic group with Russian P. tymensis, clearly supporting previous studies

Resolving phylogenetic relationships of Delphacini and Tropidocephalini (Hemiptera: Delphacidae: Delphacinae) as inferred from four genetic loci

This paper explores the phylogeny of the delphacid subfamily Delphacinae based on nuclear ribosomal and mitochondrial DNA sequences of four genetic loci (16S rDNA, 28S rDNA, Cytochrome oxidase I and Cytochrome b). Maximum likelihood and Bayesian analyses yield robust phylogenetic trees. The topologies support the monophyly of Delphacinae and its basal split into three tribes, and provisionally support subdividing Delphacini into three clades, including a more broadly defined Numatina. The tribe Tropidocephalini is divided into two clades. In addition, Paranectopia is transferred from Tropidocephalini to Delphacini and Harmalia syn. nov. is regarded as a junior synonym of Opiconsiva. The genera Bambusiphaga, Megadelphax and Muirodelphax are found to be paraphyletic. The estimated time to the most recent common ancestor of Delphacinae is roughly at 90 million years ago in Late Cretaceous.

Molecular phylogeny of Glossodoris (Ehrenberg, 1831) nudibranchs and related genera reveals cryptic and pseudocryptic species complexes.

Chromodorid nudibranchs (Chromodorididae) are brightly coloured sea slugs that live in some of the most biodiverse and threatened coral reefs on the planet. However, the evolutionary relationships within this family have not been well understood, especially in the genus Glossodoris. Members of Glossodoris have experienced large-scale taxonomic instability over the last century and have been the subject of repeated taxonomic changes, in part due to morphological characters being the sole traditional taxonomic sources of data. Changing concepts of traditional generic boundaries based on morphology also have contributed to this instability. Despite recent advances in molecular systematics, many aspects of chromodorid taxonomy remain poorly understood, particularly at the traditional species and generic levels. In this study, 77 individuals comprising 32 previously defined species were used to build the most robust phylogenetic tree of Glossodoris and related genera using mitochondrial genes cytochrome c oxidase subunit I and 16S, and the nuclear gene 28S. Bayesian inference, maximum likelihood, and maximum parsimony analyses verify the most recent hypothesized evolutionary relationships within Glossodoris. Additionally, a pseudocryptic and cryptic species complex within Glossodoris cincta and a pseudocryptic complex within Glossodoris pallida emerged, and three new species of Doriprismatica are identified.

Phylogenomic Analyses and Comparative Studies on Genomes of the Bifidobacteriales: Identification of Molecular Signatures Specific for the Order Bifidobacteriales and Its Different Subclades.

The order Bifidobacteriales comprises a diverse variety of species found in the gastrointestinal tract of humans and other animals, some of which are opportunistic pathogens, whereas a number of others exhibit health-promoting effects. However, currently very few biochemical or molecular characteristics are known which are specific for the order Bifidobacteriales, or specific clades within this order, which distinguish them from other bacteria. This study reports the results of detailed comparative genomic and phylogenetic studies on 62 genome-sequenced species/strains from the order Bifidobacteriales. In a robust phylogenetic tree for the Bifidobacteriales constructed based on 614 core proteins, a number of well-resolved clades were observed including a clade separating the Scarodvia-related genera (Scardovia clade) from the genera Bifidobacterium and Gardnerella, as well as a number of previously reported clusters of Bifidobacterium spp. In parallel, our comparative analyses of protein sequences from the Bifidobacteriales genomes have identified numerous molecular markers that are specific for this group of bacteria. Of these markers, 32 conserved signature indels (CSIs) in widely distributed proteins and 10 signature proteins are distinctive characteristics of all sequenced Bifidobacteriales species and provide novel and highly specific means for distinguishing these bacteria. In addition, multiple other molecular signatures are specific for the following clades of Bifidobacteriales: (i) 5 CSIs specific for a clade comprising of the Scardovia-related genera; (ii) 3 CSIs and 2 CSPs specific for a clade consisting of the Bifidobacterium and Gardnerella spp.; (iii) multiple other signatures demarcating a number of clusters of the B. asteroides-and B. longum- related species. The described molecular markers provide novel and reliable means for distinguishing the Bifidobacteriales and a number of their clades in molecular terms and for the classification of these bacteria. The Bifidobacteriales-specific CSIs, found in important proteins, are predicted to play important roles in modifying the cellular functions of the affected proteins. Hence, biochemical studies on the cellular functions of these CSIs could lead to discovery of novel characteristics of either all Bifidobacteriales, or specific groups of bacteria within this order. Some of the functions affected/modified by these genetic changes could also be important for the probiotic/pathogenic activities of the bifidobacteria.

Reconsidering Animal Phylogeny: Tree-Thinking

New phylogenomic methods have made it possible to obtain a robust phylogenetic tree of the animal kingdom. The resulting tree confirms much of what was already known but also contains some nice surprises.

A phylogenomic reappraisal of family-level divisions within the class Halobacteria: proposal to divide the order Halobacteriales into the families Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov., and the order Haloferacales into the families, Haloferacaceae and Halorubraceae fam nov.

The evolutionary interrelationships between the archaeal organisms which comprise the class Halobacteria have proven difficult to elucidate using traditional phylogenetic tools. The class currently contains three orders. However, little is known about the family level relationships within these orders. In this work, we have completed a comprehensive comparative analysis of 129 sequenced genomes from members of the class Halobacteria in order to identify shared molecular characteristics, in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can provide reliable evidence, independent of phylogenetic trees, that the species from the groups in which they are found are specifically related to each other due to common ancestry. Here we present 20 CSIs and 31 CSPs which are unique characteristics of infra-order level groups of genera within the class Halobacteria. We also present 40 CSIs and 234 CSPs which are characteristic of Haloarcula, Halococcus, Haloferax, or Halorubrum. Importantly, the CSIs and CSPs identified here provide evidence that the order Haloferacales contains two main groups, one consisting of Haloferax and related genera supported by four CSIs and five CSPs and the other consisting of Halorubrum and related genera supported by four CSPs. We have also identified molecular characteristics that suggest that the polyphyletic order Halobacteriales contains at least two large monophyletic clusters of organisms in addition to the polyphyletic members of the order, one cluster consisting of Haloarcula and related genera supported by ten CSIs and nineteen CSPs and the other group consisting of the members of the genus Halococcus supported by nine CSIs and 23 CSPs. We have also produced a highly robust phylogenetic tree based on the concatenated sequences of 766 proteins which provide additional support for the relationships identified by the CSIs and CSPs. On the basis of the phylogenetic analyses and the identified conserved molecular characteristics presented here, we propose a division of the order Haloferacales into two families, an emended family Haloferacaceae and Halorubraceae fam. nov. and a division of the order Halobacteriales into three families, an emended family Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov.

Family-Level Sampling of Mitochondrial Genomes in Coleoptera: Compositional Heterogeneity and Phylogenetics.

Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML (“nonhomogeneous”) algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees.

ITS2 and 18S rDNA sequence-structure phylogeny of Chlorella and allies (Chlorophyta, Trebouxiophyceae, Chlorellaceae)

In the last decade, the evolutionary diversity of Chlorella and allies has been discussed in a huge number of publications using internal transcribed spacer 2 (ITS2) and/or 18S ribosomal RNA gene sequences to infer the phylogenies. However, sister-group relations between different genera classified within the Chlorellaceae remained provisional, due to a lack of bootstrap support. In this study, using more than four hundred sequences, a comprehensive phylogenetic portrait of Chlorella and allies is presented and discussed; sixty key taxa are reconsidered by an analysis using primary sequences and their individual secondary structures simultaneously in inferring neighbor-joining, maximum parsimony and maximum likelihood trees, an approach most recently reviewed, with increasing robustness and accuracy of reconstructed phylogenies. While neighbor-joining and maximum parsimony analyses failed in inferring a robust phylogenetic tree, the maximum likelihood tree (in particular on a concatenated data set) provides a supported phylogeny preceding any taxonomic discussion.

Using Mollusk Phylogeny to Teach Evolution

New phylogenomic tools have made it possible to construct a robust phylogenetic tree of mollusks. This tree can be used to teach important evolutionary principles.

Application of RAD-based phylogenetics to complex relationships among variously related taxa in a species flock

Restriction site-associated DNA (RAD) sequences from entire genomes can be used to resolve complex phylogenetic problems. However, the processed data matrix varies depending on the strategies used to determine orthologous loci and to filter loci according to the number of taxa with sequence data for the loci, and often contains plenty of missing data. To explore the utility of RAD sequences for elucidating the phylogenetics of variously related species, we conducted RAD sequencing for the Ohomopterus ground beetles and attempted maximum-likelihood phylogenetic analyses using 42 data matrices ranging from 1.6×104 to 8.1×106 base pairs, with 11–72% missing data. We demonstrate that robust phylogenetic trees, in terms of bootstrap values, do not necessarily result from larger data matrices, as previously suggested. Robust trees for distantly related and closely related taxa resulted from different data matrices, and topologically different robust trees for distantly related taxa resulted from various data matrices. For closely related taxa, moderately large data matrices strongly supported a topology that is incompatible with morphological evidence, possibly due to the effect of introgressive hybridization. Practically, exploring variously prepared data matrices is an effective way to propose important alternative phylogenetic hypotheses for this study group.

Turning points in the evolution of peroxidase-catalase superfamily: molecular phylogeny of hybrid heme peroxidases.

Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase–catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase–catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.

Phylogenetic and molecular dating analysis of Taiwan Blue Pheasant (Lophura swinhoii)

The Swinhoe's Pheasant (Lophura swinhoii) is an endemic and most endangered species to Taiwan, China. It belongs to the genus Lophura, family Phasianidae. To further investigate the evolutionary history of L. swinhoii, we determined its complete mitochondrial genome and reconstructed a single, robust phylogenetic tree. Our results showed that L. swinhoii is clustered with Lophura nycthemera and forms a sister group of Lophura ignita. The genus Lophura is strongly supported as the sister taxon of the genus Crossoptilon. The molecular clock analysis showed that the genetic divergence of L. swinhoii occurred in 2.71 (1.31–4.22)Mya. The most common ancestor of L. swinhoii might have migrated from mainland of South East Asia to Taiwan Island by the land bridge at 2.71Mya ago. Taiwan Island is separated from the mainland by the sea (Taiwan Strait) and formed a separate island at around 2.5Mya because of the transgression and regression. Therefore, geographical isolation and climate change may accelerate the evolution of L. swinhoii. In this study, we propose a biogeographic hypothesis for speciation of the L. swinhoii based on known events of the geographic and geological history of South East Asia and southeast China, which would benefit the understanding of evolutionary history of L. swinhoii as well as other galliform birds.

Snp-search: simple processing, manipulation and searching of SNPs from high-throughput sequencing

BackgroundA typical bacterial pathogen genome mapping project can identify thousands of single nucleotide polymorphisms (SNP). Interpreting SNP data is complex and it is difficult to conceptualise the data contained within the large flat files that are the typical output from most SNP calling algorithms. One solution to this problem is to construct a database that can be queried using simple commands so that SNP interrogation and output is both easy and comprehensible.ResultsHere we present snp-search, a tool that manages SNP data and allows for manipulation and searching of SNP data. After creation of a SNP database from a VCF file, snp-search can be used to convert the selected SNP data into FASTA sequences, construct phylogenies, look for unique SNPs, and output contextual information about each SNP. The FASTA output from snp-search is particularly useful for the generation of robust phylogenetic trees that are based on SNP differences across the conserved positions in whole genomes. Queries can be designed to answer critical genomic questions such as the association of SNPs with particular phenotypes.Conclusionssnp-search is a tool that manages SNP data and outputs useful information which can be used to test important biological hypotheses.

Systematic Identification of Gene Families for Use as “Markers” for Phylogenetic and Phylogeny-Driven Ecological Studies of Bacteria and Archaea and Their Major Subgroups

With the astonishing rate that genomic and metagenomic sequence data sets are accumulating, there are many reasons to constrain the data analyses. One approach to such constrained analyses is to focus on select subsets of gene families that are particularly well suited for the tasks at hand. Such gene families have generally been referred to as “marker” genes. We are particularly interested in identifying and using such marker genes for phylogenetic and phylogeny-driven ecological studies of microbes and their communities (e.g., construction of species trees, phylogenetic based assignment of metagenomic sequence reads to taxonomic groups, phylogeny-based assessment of alpha- and beta-diversity of microbial communities from metagenomic data). We therefore refer to these as PhyEco (for phylogenetic and phylogenetic ecology) markers. The dual use of these PhyEco markers means that we needed to develop and apply a set of somewhat novel criteria for identification of the best candidates for such markers. The criteria we focused on included universality across the taxa of interest, ability to be used to produce robust phylogenetic trees that reflect as much as possible the evolution of the species from which the genes come, and low variation in copy number across taxa.We describe here an automated protocol for identifying potential PhyEco markers from a set of complete genome sequences. The protocol combines rapid searching, clustering and phylogenetic tree building algorithms to generate protein families that meet the criteria listed above. We report here the identification of PhyEco markers for different taxonomic levels including 40 for “all bacteria and archaea”, 114 for “all bacteria (greatly expanding on the ∼30 commonly used), and 100 s to 1000 s for some of the individual phyla of bacteria. This new list of PhyEco markers should allow much more detailed automated phylogenetic and phylogenetic ecology analyses of these groups than possible previously.