Deep Ancestral Introgression Shapes Evolutionary History of Dragonflies and Damselflies.

Introgression is an important biological process affecting at least 10% of the extant species in the animal kingdom. Introgression significantly impacts inference of phylogenetic species relationships where a strictly binary tree model cannot adequately explain reticulate net-like species relationships. Here, we use phylogenomic approaches to understand patterns of introgression along the evolutionary history of a unique, nonmodel insect system: dragonflies and damselflies (Odonata). We demonstrate that introgression is a pervasive evolutionary force across various taxonomic levels within Odonata. In particular, we show that the morphologically "intermediate" species of Anisozygoptera (one of the three primary suborders within Odonata besides Zygoptera and Anisoptera), which retain phenotypic characteristics of the other two suborders, experienced high levels of introgression likely coming from zygopteran genomes. Additionally, we find evidence for multiple cases of deep inter-superfamilial ancestral introgression. [Gene flow; Odonata; phylogenomics; reticulate evolution.].

Codon use and aversion is largely phylogenetically conserved across the tree of life.

Using parsimony, we analyzed codon usages across 12,337 species and 25,727 orthologous genes to rank specific genes and codons according to their phylogenetic signal. We examined each codon within each ortholog to determine the codon usage for each species. In total, 890,814 codons were parsimony informative. Next, we compared species that used a codon with species that did not use the codon. We assessed each codon's congruence with species relationships provided in the Open Tree of Life (OTL) and determined the statistical probability of observing these results by random chance. We determined that 25,771 codons had no parallelisms or reversals when mapped to the OTL. Codon usages from orthologous genes spanning many species were 1109× more likely to be congruent with species relationships in the OTL than would be expected by random chance. Using the OTL as a reference, we show that codon usage is phylogenetically conserved within orthologous genes in archaea, bacteria, plants, mammals, and other vertebrates. We also show how to use our provided framework to test different tree hypotheses by confirming the placement of turtles as sister taxa to archosaurs.

Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae).

Fireflies are some of the most captivating organisms on the planet. They have a rich history as subjects of scientific study, especially in relation to their bioluminescent behavior. Yet, the phylogenetic relationships of fireflies are still poorly understood. Here, we present the first total evidence approach to reconstruct lampyrid phylogeny using both a molecular matrix from six loci and an extensive morphological matrix. Using this phylogeny we test the hypothesis that adult bioluminescence evolved after the origin of the firefly clade. The ancestral state of adult bioluminescence is recovered as non-bioluminescent with one to six gains and five to ten subsequent losses. The monophyly of the family, as well as the subfamilies is also tested. Ototretinae, Cyphonocerinae, Luciolinae (incl. Pristolycus), Amydetinae, "cheguevarinae" sensu Jeng 2008, and Photurinae are highly supported as monophyletic. With the exception of four taxa, Lampyrinae is also recovered as monophyletic with high support. Based on phylogenetic and morphological data Lamprohiza, Phausis, and Lamprigera are transferred to Lampyridae incertae sedis.

Missing something? Codon aversion as a new character system in phylogenetics.

Although many studies have documented codon usage bias in different species, the importance of codon usage in a phylogenetic framework remains largely unknown. We demonstrate that a phylogenetic signal is present in the codon usage and non-usage biases of 17 717 orthologues evaluated across 72 tetrapod species using a simple parsimony analysis of a binary matrix of codon characters. Phylogenies estimated using stop codons were more congruent with previous hypotheses than phylogenies based on any other single codon or a combination of codons. Although each codon is present in every species, specific genes have different codon preferences and may or may not use every possible codon. This observation allowed us to map the pattern of codon usage and non-usage across the topology. These results suggest that codon usage is phylogenetically conserved across shallow and deep levels within tetrapods.

On the probability of dinosaur fleas.

Recently, a set of publications described flea fossils from Jurassic and Early Cretaceous geological strata in northeastern China, which were suggested to have parasitized feathered dinosaurs, pterosaurs, and early birds or mammals. In support of these fossils being fleas, a recent publication in BMC Evolutionary Biology described the extended abdomen of a female fossil specimen as due to blood feeding.We here comment on these findings, and conclude that the current interpretation of the evolutionary trajectory and ecology of these putative dinosaur fleas is based on appeal to probability, rather than evidence. Hence, their taxonomic positioning as fleas, or stem fleas, as well as their ecological classification as ectoparasites and blood feeders is not supported by currently available data.

Reassessing the phylogenetic position of the epizoic earwigs (Insecta: Dermaptera).

Dermaptera is a relatively small order of free-living insects that typically feed on detritus and other plant material. However, two earwig lineages - Arixeniidae and Hemimeridae - are epizoic on Cheiromeles bats and Beamys and Cricetomys rats respectively. Both of these epizoic families are comprised of viviparous species. The monophyly of these epizoic lineages and their placement within dermapteran phylogeny has remained unclear. A phylogenetic analyses was performed on a diverse sample of 47 earwig taxa for five loci (18S rDNA, 28S rDNA, COI, Histone 3, and Tubulin Alpha I). Our results support two independent origins of the epizoic lifestyle within Dermaptera, with Hemimeridae and Arixeniidae each derived from a different lineage of Spongiphoridae. Our analyses places Marava, a genus of spongiphorids that includes free-living but viviparous earwigs, as sister group to Arixeniidae, suggesting that viviparity evolved prior to the shift to the epizoic lifestyle. Additionally, our results support the monophyly of Forficulidae and Chelisochidae and the paraphyly of Labiduridae, Pygidicranidae, Spongiphoridae, and Anisolabididae.

Fleas (Siphonaptera) are Cretaceous, and evolved with Theria.

Fleas (order Siphonaptera) are highly-specialized, diverse blood-feeding ectoparasites of mammals and birds with an enigmatic evolutionary history and obscure origin. We here present a molecular phylogenetic study based on a comprehensive taxon sampling of 259 flea taxa, representing 16 of the 18 extant families of this order. A Bayesian phylogenetic tree with strong nodal support was recovered, consisting of seven sequentially derived lineages with Macropsyllidae as the earliest divergence, followed by Stephanocircidae. Divergence times of flea lineages were estimated based on fossil records and host specific associations to bats (Chiroptera), suggesting that the common ancestor of extant Siphonaptera diversified during the Cretaceous. However, most of the intraordinal divergence into extant lineages took place after the K-Pg boundary. Ancestral states of host association and biogeographical distribution were reconstructed, suggesting with high likelihood that fleas originated in the southern continents (Gondwana) and migrated from South America to their extant distributions in a relatively short time frame. Theria (placental mammals and marsupials) represent the most likely ancestral host group of extant Siphonaptera, with marsupials occupying a more important role than previously assumed. Major extant flea families evolved in connection to post K-Pg diversification of Placentalia. The association of fleas with monotremes and birds is likely due to later secondary host association. These results suggest caution in casually interpreting recently discovered Mesozoic fossil "dinosaur fleas" of Northeast Asia as part of what we currently consider Siphonaptera.

300 million years of diversification: elucidating the patterns of orthopteran evolution based on comprehensive taxon and gene sampling.

Orthoptera is the most diverse order among the polyneopteran groups and includes familiar insects, such as grasshoppers, crickets, katydids, and their kin. Due to a long history of conflicting classification schemes based on different interpretations of morphological characters, the phylogenetic relationships within Orthoptera are poorly understood and its higher classification has remained unstable. In this study, we establish a robust phylogeny of Orthoptera including 36 of 40 families representing all 15 currently recognized superfamilies and based on complete mitochondrial genomes and four nuclear loci, in order to test previous phylogenetic hypotheses and to provide a framework for a natural classification and a reference for studying the pattern of divergence and diversification. We find strong support for monophyletic suborders (Ensifera and Caelifera) as well as major superfamilies. Our results corroborate most of the higher-level relationships previously proposed for Caelifera, but suggest some novel relationships for Ensifera. Using fossil calibrations, we provide divergence time estimates for major orthopteran lineages and show that the current diversity has been shaped by dynamic shifts of diversification rates at different geological times across different lineages. We also show that mitochondrial tRNA gene orders have been relatively stable throughout the evolutionary history of Orthoptera, but a major tRNA gene rearrangement occurred in the common ancestor of Tetrigoidea and Acridomorpha, thereby representing a robust molecular synapomorphy, which has persisted for 250 Myr.

Phylogenetic analyses of termite post-embryonic sequences illuminate caste and developmental pathway evolution.

Termites are highly eusocial insects with a caste polyphenism (i.e., discontinuous morphological differences between castes) and elaborated behaviors. While the developmental pathways leading to caste occurrence are well-known in many species, the evolutionary origin of these pathways is still obscure. Recent molecular phylogenetic studies suggest multiple independent origins of sterile castes in termites, reviving a 30 years old debate. We demonstrate here that diploid sterile castes ("true" workers) evolved several times independently in this group and that this caste was lost at least once in a lineage with developmentally more flexible workers called pseudergates or "false" workers. We also infer that flexibility in post-embryonic development was acquired multiple times independently during termite evolution. We suggest that focusing on detailed developmental pathways in phylogenetic analyses is essential for elucidating the origin of caste polyphenism in termites.

A century of paraphyly: a molecular phylogeny of katydids (Orthoptera: Tettigoniidae) supports multiple origins of leaf-like wings.

The phylogenetic relationships of Tettigoniidae (katydids and bush-crickets) were inferred using molecular sequence data. Six genes (18S rDNA, 28S rDNA, Cytochrome Oxidase II, Histone 3, Tubulin Alpha I, and Wingless) were sequenced for 135 ingroup taxa representing 16 of the 19 extant katydid subfamilies. Five subfamilies (Tettigoniinae, Pseudophyllinae, Mecopodinae, Meconematinae, and Listroscelidinae) were found to be paraphyletic under various tree reconstruction methods (Maximum Likelihood, Bayesisan Inference and Maximum Parsimony). Seven subfamilies - Conocephalinae, Hetrodinae, Hexacentrinae, Saginae, Phaneropterinae, Phyllophorinae, and Lipotactinae - were each recovered as well-supported monophyletic groups. We mapped the small and exposed thoracic auditory spiracle (a defining character of the subfamily Pseudophyllinae) and found it to be homoplasious. We also found the leaf-like wings of katydids have been derived independently in at least six lineages.

Searching for the optimal data partitioning strategy in mitochondrial phylogenomics: a phylogeny of Acridoidea (Insecta: Orthoptera: Caelifera) as a case study.

One of the main challenges in analyzing multi-locus phylogenomic data is to find an optimal data partitioning strategy to account for variable evolutionary histories of different loci for any given dataset. Although a number of studies have addressed the issue of data partitioning in a Bayesian phylogenetic framework, such studies in a maximum likelihood framework are comparatively lacking. Furthermore, a rigorous statistical exploration of possible data partitioning schemes has not been applied to mitochondrial genome (mtgenome) data, which provide a complex, but manageable platform for addressing various challenges in analyzing phylogenomic data. In this study, we investigate the issue of data partitioning in the maximum likelihood framework in the context of the mitochondrial phylogenomics of an orthopteran superfamily Acridoidea (Orthoptera: Caelifera). The present study analyzes 34 terminals representing all 8 superfamilies within Caelifera, which includes newly sequenced partial or complete mtgenomes for 11 families. Using a new partition-selection method implemented in the software PartitionFinder, we compare a large number of data partitioning schemes in an attempt to identify the most effective method of analyzing the mtgenome data. We find that the best-fit partitioning scheme selected by PartitionFinder is superior to any a priori schemes commonly utilized in mitochondrial phylogenomics. We also show that over-partitioning is often detrimental to phylogenetic reconstruction. A comparative analysis of mtgenome structures finds that the tRNA gene rearrangement between cytochrome c oxidase subunit II and ATP synthase protein 8 does not occur in the most basal caeliferan lineage Tridactyloidea, suggesting that this gene rearrangement must have evolved at least in the common ancestor of Tetrigoidea and Acridomorpha. We find that mtgenome data contain sufficient phylogenetic information to broadly resolve the relationships across Acridomorpha and Acridoidea.

Uncovering historical signature of mitochondrial DNA hidden in the nuclear genome: the biogeography of Schistocerca revisited.

Inadvertent coamplification of nuclear mitochondrial pseudogenes (numts) is a serious problem in mitochondrial systematics, but numts can also be a valuable source of information because they represent ancient forms of mtDNA. We present a conceptual framework of numt accumulation, which states that in a given species there can be two types of numts, synaponumts and autaponumts, resulting from integration occurring respectively before and after a speciation event. In a given clade, a species that diverged early can only have its own autaponumts as well as synaponumts that were already present in the genome of the last common ancestor. A species that diverged more recently may, however, have many different synaponumts integrated at each different divergence as well as its own autaponumts. Therefore it is possible to decipher the evolutionary history of a species based on the phylogenetic distribution of numts in a simultaneous analysis of numts and extant mtDNA. In this study, we test this idea empirically in the context of addressing a controversial question regarding the biogeography of the grasshopper genus Schistocerca Stål (Orthoptera: Acrididae), based on numts of the cytochrome c oxidase subunit I (COI) gene. We find that our empirical data can be explained adequately by our conceptual framework, and that the phylogenetic distribution of COI numts reveals intricate evolutionary histories about past speciation events that are otherwise difficult to detect using conventional markers. Our study strongly favours the Old World origin of the desert locust, Schistocerca gregaria and the New World Schistocerca species are descendants from an ancestral gregaria-like species that colonized the New World via westward transatlantic flight. However, the phylogenetic distribution of S. gregaria numts raises a distinct possibility that there might have been multiple founding events from Africa to America to give rise to the present-day diversity of the genus. This is a case study for a creative use of numts as molecular fossils, and we demonstrate that numts provide an interesting and powerful phylogenetic signal, much more than what extant mtDNA or nuclear gene sequences might be able to provide.

On seven undescribed leaf insect species revealed within the recent "Tree of Leaves" (Phasmatodea, Phylliidae).

With the recent advance in molecular phylogenetics focused on the leaf insects (Phasmatodea, Phylliidae), gaps in knowledge are beginning to be filled. Yet, shortcomings are also being highlighted, for instance, the unveiling of numerous undescribed phylliid species. Here, some of these taxa are described, including Phylliumiyadaonsp. nov. from Mindoro Island, Philippines; Phylliumsamarensesp. nov. from Samar Island, Philippines; Phylliumortizisp. nov. from Mindanao Island, Philippines; Pulchriphylliumheraclessp. nov. from Vietnam; Pulchriphylliumdelisleisp. nov. from South Kalimantan, Indonesia; and Pulchriphylliumbhaskaraisp. nov. from Java, Indonesia. Several additional specimens of these species together with a seventh species described herein, Pulchriphylliumanangusp. nov. from southwestern India, were incorporated into a newly constructed phylogenetic tree. Additionally, two taxa that were originally described as species, but in recent decades have been treated as subspecies, are elevated back to species status to reflect their unique morphology and geographic isolation, creating the following new combinations: Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. from Bangladesh and northeastern India, and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from the Seychelles islands. Lectotype specimens are also designated for Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from original type material.

Mitochondrial genome deletions and minicircles are common in lice (Insecta: Phthiraptera).

BACKGROUND: The gene composition, gene order and structure of the mitochondrial genome are remarkably stable across bilaterian animals. Lice (Insecta: Phthiraptera) are a major exception to this genomic stability in that the canonical single chromosome with 37 genes found in almost all other bilaterians has been lost in multiple lineages in favour of multiple, minicircular chromosomes with less than 37 genes on each chromosome. RESULTS: Minicircular mt genomes are found in six of the ten louse species examined to date and three types of minicircles were identified: heteroplasmic minicircles which coexist with full sized mt genomes (type 1); multigene chromosomes with short, simple control regions, we infer that the genome consists of several such chromosomes (type 2); and multiple, single to three gene chromosomes with large, complex control regions (type 3). Mapping minicircle types onto a phylogenetic tree of lice fails to show a pattern of their occurrence consistent with an evolutionary series of minicircle types. Analysis of the nuclear-encoded, mitochondrially-targetted genes inferred from the body louse, Pediculus, suggests that the loss of mitochondrial single-stranded binding protein (mtSSB) may be responsible for the presence of minicircles in at least species with the most derived type 3 minicircles (Pediculus, Damalinia). CONCLUSIONS: Minicircular mt genomes are common in lice and appear to have arisen multiple times within the group. Life history adaptive explanations which attribute minicircular mt genomes in lice to the adoption of blood-feeding in the Anoplura are not supported by this expanded data set as minicircles are found in multiple non-blood feeding louse groups but are not found in the blood-feeding genus Heterodoxus. In contrast, a mechanist explanation based on the loss of mtSSB suggests that minicircles may be selectively favoured due to the incapacity of the mt replisome to synthesize long replicative products without mtSSB and thus the loss of this gene lead to the formation of minicircles in lice.

Morphological and molecular evidence converge upon a robust phylogeny of the megadiverse Holometabola.

We present the largest morphological character set ever compiled for Holometabola. This was made possible through an optimized acquisition of data. Based on our analyses and recently published hypotheses based on molecular data, we discuss higher-level phylogeny and evolutionary changes. We comment on the information content of different character systems and discuss the role of morphology in the age of phylogenomics. Microcomputer tomography in combination with other techniques proved highly efficient for acquiring and documenting morphological data. Detailed anatomical information (356 characters) is now available for 30 representatives of all holometabolan orders. A combination of traditional and novel techniques complemented each other and rapidly provided reliable data. In addition, our approach facilitates documenting the anatomy of model organisms. Our results show little congruence with studies based on rRNA, but confirm most clades retrieved in a recent study based on nuclear genes: Holometabola excluding Hymenoptera, Coleopterida (= Strepsiptera + Coleoptera), Neuropterida excl. Neuroptera, and Mecoptera. Mecopterida (= Antliophora + Amphiesmenoptera) was retrieved only in Bayesian analyses. All orders except Megaloptera are monophyletic. Problems in the analyses are caused by taxa with numerous autapomorphies and/or inapplicable character states due to the loss of major structures (such as wings). Different factors have contributed to the evolutionary success of various holometabolan lineages. It is likely that good flying performance, the ability to occupy different habitats as larvae and adults, parasitism, liquid feeding, and co-evolution with flowering plants have played important roles. We argue that even in the "age of phylogenomics", comparative morphology will still play a vital role. In addition, morphology is essential for reconstructing major evolutionary transformations at the phenotypic level, for testing evolutionary scenarios, and for placing fossil taxa. © The Willi Hennig Society 2010.

Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified.

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.

A comprehensive analysis of the phylogenetic signal in ramp sequences in 211 vertebrates.

Ramp sequences increase translational speed and accuracy when rare, slowly-translated codons are found at the beginnings of genes. Here, the results of the first analysis of ramp sequences in a phylogenetic construct are presented. Ramp sequences were compared from 247 vertebrates (114 Mammalian and 133 non-mammalian), where the presence and absence of ramp sequences was analyzed as a binary character in a parsimony and maximum likelihood framework. Additionally, ramp sequences were mapped to the Open Tree of Life synthetic tree to determine the number of parallelisms and reversals that occurred, and those results were compared to random permutations. Parsimony and maximum likelihood analyses of the presence and absence of ramp sequences recovered phylogenies that are highly congruent with established phylogenies. Additionally, 81% of vertebrate mammalian ramps and 81.2% of other vertebrate ramps had less parallelisms and reversals than the mean from 1000 randomly permuted trees. A chi-square analysis of completely orthologous ramp sequences resulted in a p-value < 0.001 as compared to random chance. Ramp sequences recover comparable phylogenies as other phylogenomic methods. Although not all ramp sequences appear to have a phylogenetic signal, more ramp sequences track speciation than expected by random chance. Therefore, ramp sequences may be used in conjunction with other phylogenomic approaches if many orthologs are taken into account. However, phylogenomic methods utilizing few orthologs should be cautious in incorporating ramp sequences because individual ramp sequences may provide conflicting signals.

Characterization of 67 mitochondrial tRNA gene rearrangements in the Hymenoptera suggests that mitochondrial tRNA gene position is selectively neutral.

We present entire sequences of two hymenopteran mitochondrial genomes and the major portion of three others. We combined these data with nine previously sequenced hymenopteran mitochondrial genomes. This allowed us to infer and analyze the evolution of the 67 mitochondrial gene rearrangements so far found in this order. All of these involve tRNA genes, whereas four also involve larger (protein-coding or ribosomal RNA) genes. We find that the vast majority of mitochondrial gene rearrangements are independently derived. A maximum of four of these rearrangements represent shared, derived organizations, whereas three are convergently derived. The remaining mitochondrial gene rearrangements represent new mitochondrial genome organizations. These data are consistent with the proposal that there are an enormous number of alternative mitochondrial genome organizations possible and that mitochondrial genome organization is, for the most part, selectively neutral. Nevertheless, some mitochondrial genes appear less mobile than others. Genes close to the noncoding region are generally more mobile but only marginally so. Some mitochondrial genes rearrange in a pattern consistent with the duplication/random loss model, but more mitochondrial genes move in a pattern inconsistent with this model. An increased rate of mitochondrial gene rearrangement is not tightly associated with the evolution of parasitism. Although parasitic lineages tend to have more mitochondrial gene rearrangements than nonparasitic lineages, there are exceptions (e.g., Orussus and Schlettererius). It is likely that only a small proportion of the total number of mitochondrial gene rearrangements that have occurred during the evolution of the Hymenoptera have been sampled in the present study.

Nonstationary evolution and compositional heterogeneity in beetle mitochondrial phylogenomics.

Many published phylogenies are based on methods that assume equal nucleotide composition among taxa. Studies have shown, however, that this assumption is often not accurate, particularly in divergent lineages. Nonstationary sequence evolution, when taxa in different lineages evolve in different ways, can lead to unequal nucleotide composition. This can cause inference methods to fail and phylogenies to be inaccurate. Recent advancements in phylogenetic theory have proposed new models of nonstationary sequence evolution; these models often outperform equivalent stationary models. A variety of new phylogenetic software implementing such models has been developed, but the studies employing the new methodology are still few. We discovered convergence of nucleotide composition within mitochondrial genomes of the insect order Coleoptera (beetles). We found variation in base content both among species and among genes in the genome. To this data set, we have applied a broad range of phylogenetic methods, including some traditional stationary models of evolution and all the more recent nonstationary models. We compare 8 inference methods applied to the same data set. Although the more commonly used methods universally fail to recover established clades, we find that some of the newer software packages are more appropriate for data of this nature. The software packages p4, PHASE, and nhPhyML were able to overcome the systematic bias in our data set, but parsimony, MrBayes, NJ, LogDet, and PhyloBayes were not.

Codon Pairs are Phylogenetically Conserved: A comprehensive analysis of codon pairing conservation across the Tree of Life.

Identical codon pairing and co-tRNA codon pairing increase translational efficiency within genes when two codons that encode the same amino acid are translated by the same tRNA before it diffuses from the ribosome. We examine the phylogenetic signal in both identical and co-tRNA codon pairing across 23 428 species using alignment-free and parsimony methods. We determined that conserved codon pairing typically has a smaller window size than the length of a ribosome, and codon pairing tracks phylogenies across various taxonomic groups. We report a comprehensive analysis of codon pairing, including the extent to which each codon pairs. Our parsimony method generally recovers phylogenies that are more congruent with the established phylogenies than our alignment-free method. However, four of the ten taxonomic groups did not have sufficient orthologous codon pairings and were therefore analyzed using only the alignment-free methods. Since the recovered phylogenies using only codon pairing largely match phylogenies from the Open Tree of Life and the NCBI taxonomy, and are comparable to trees recovered by other algorithms, we propose that codon pairing biases are phylogenetically conserved and should be considered in conjunction with other phylogenomic techniques.