Evolution of the WANCY region in amniote mitochondrial DNA

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.      

Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes

Several different groups of birds have been proposed as being the oldest or earliest diverging extant lineage within the avian phylogenetic tree, particularly ratites (Struthioniformes), waterfowl (Anseriformes), and shorebirds (Charadriiformes). Difficulty in resolving this issue stems from several factors, including the relatively rapid radiation of primary (ordinal) bird lineages and the lack of characters from an extant outgroup for birds that is closely related to them by measure of time. To help resolve this question, we have sequenced entire mitochondrial genomes for five birds (a rhea, a duck, a falcon, and two perching birds), one crocodilian, and one turtle. Maximum parsimony and maximum likelihood analyses of these new sequences together with published sequences (18 taxa total) yield the same optimal tree topology, in which a perching bird (Passeriformes) is sister to all the other bird taxa. A basal position for waterfowl among the bird study taxa is rejected by maximum likelihood analyses. However, neither the conventional view, in which ratites (including rhea) are basal to other birds, nor tree topologies with falcon or chicken basal among birds could be rejected in the same manner. In likelihood analyses of a subset of seven birds, alligator, and turtle (9 taxa total), we find that increasing the number of parameters in the model shifts the optimal topology from one with a perching bird basal among birds to the conventional view with ratites diverging basally; moreover, likelihood scores for the two trees are not significantly different. Thus, although our largest set of taxa and characters supports a tree with perching birds diverging basally among birds, the position of this earliest divergence among birds appears unstable. Our analyses indicate a sister relationship between a waterfowl/chicken clade and ratites, relative to perching birds and falcon. We find support for a sister relationship between turtles and a bird/crocodilian clade, and for rejecting both the Haemothermia hypothesis (birds and mammals as sister taxa) and the placement of turtles as basal within the phylogenetic tree for amniote animals.     

A Phylogenomic Approach to Vertebrate Phylogeny Supports a Turtle-Archosaur Affinity and a Possible Paraphyletic Lissamphibia

In resolving the vertebrate tree of life, two fundamental questions remain: 1) what is the phylogenetic position of turtles within amniotes, and 2) what are the relationships between the three major lissamphibian (extant amphibian) groups? These relationships have historically been difficult to resolve, with five different hypotheses proposed for turtle placement, and four proposed branching patterns within Lissamphibia. We compiled a large cDNA/EST dataset for vertebrates (75 genes for 129 taxa) to address these outstanding questions. Gene-specific phylogenetic analyses revealed a great deal of variation in preferred topology, resulting in topologically ambiguous conclusions from the combined dataset. Due to consistent preferences for the same divergent topologies across genes, we suspected systematic phylogenetic error as a cause of some variation. Accordingly, we developed and tested a novel statistical method that identifies sites that have a high probability of containing biased signal for a specific phylogenetic relationship. After removing putatively biased sites, support emerged for a sister relationship between turtles and either crocodilians or archosaurs, as well as for a caecilian-salamander sister relationship within Lissamphibia, with Lissamphibia potentially paraphyletic.     

Integration of morphological data sets for phylogenetic analysis of Amniota: the importance of integumentary characters and increased taxonomic sampling

Several mutually exclusive hypotheses have been advanced to explain the phylogenetic position of turtles among amniotes. Traditional morphology-based analyses place turtles among extinct anapsids (reptiles with a solid skull roof), whereas more recent studies of both morphological and molecular data support an origin of turtles from within Diapsida (reptiles with a doubly fenestrated skull roof). Evaluation of these conflicting hypotheses has been hampered by nonoverlapping taxonomic samples and the exclusion of significant taxa from published analyses. Furthermore, although data from soft tissues and anatomical systems such as the integument may be particularly relevant to this problem, they are often excluded from large-scale analyses of morphological systematics. Here, conflicting hypotheses of turtle relationships are tested by (1) combining published data into a supermatrix of morphological characters to address issues of character conflict and missing data; (2) increasing taxonomic sampling by more than doubling the number of operational taxonomic units to test internal relationships within suprageneric ingroup taxa; and (3) increasing character sampling by approximately 25% by adding new data on the osteology and histology of the integument, an anatomical system that has been historically underrepresented in morphological systematics. The morphological data set assembled here represents the largest yet compiled for Amniota. Reevaluation of character data from prior studies of amniote phylogeny favors the hypothesis that turtles indeed have diapsid affinities. Addition of new ingroup taxa alone leads to a decrease in overall phylogenetic resolution, indicating that existing characters used for amniote phylogeny are insufficient to explain the evolution of more highly nested taxa. Incorporation of new data from the soft and osseous components of the integument, however, helps resolve relationships among both basal and highly nested amniote taxa. Analysis of a data set compiled from published sources and data original to this study supports monophyly of Amniota, Synapsida, Reptilia, Parareptilia, Eureptilia, Eosuchia, Diapsida, Neodiapsida, Sauria, Lepidosauria, and Archosauriformes, as well as several more highly nested divisions within the latter two clades. Turtles are here resolved as the sister taxon to a monophyletic Lepidosauria (squamates + Sphenodon), a novel phylogenetic position that nevertheless is consistent with recent molecular and morphological studies that have hypothesized diapsid affinities for this clade.     

MicroRNAs support a turtle + lizard clade

Despite much interest in amniote systematics, the origin of turtles remains elusive. Traditional morphological phylogenetic analyses place turtles outside Diapsida-amniotes whose ancestor had two fenestrae in the temporal region of the skull (among the living forms the tuatara, lizards, birds and crocodilians)-and allied with some unfenestrate-skulled (anapsid) taxa. Nonetheless, some morphological analyses place turtles within Diapsida, allied with Lepidosauria (tuatara and lizards). Most molecular studies agree that turtles are diapsids, but rather than allying them with lepidosaurs, instead place turtles near or within Archosauria (crocodilians and birds). Thus, three basic phylogenetic positions for turtles with respect to extant Diapsida are currently debated: (i) sister to Diapsida, (ii) sister to Lepidosauria, or (iii) sister to, or within, Archosauria. Interestingly, although these three alternatives are consistent with a single unrooted four-taxon tree for extant reptiles, they differ with respect to the position of the root. Here, we apply a novel molecular dataset, the presence versus absence of specific microRNAs, to the problem of the phylogenetic position of turtles and the root of the reptilian tree, and find that this dataset unambiguously supports a turtle + lepidosaur group. We find that turtles and lizards share four unique miRNA gene families that are not found in any other organisms' genome or small RNA library, and no miRNAs are found in all diapsids but not turtles, or in turtles and archosaurs but not in lizards. The concordance between our result and some morphological analyses suggests that there have been numerous morphological convergences and reversals in reptile phylogeny, including the loss of temporal fenestrae.     

More taxa,more characters: the hoatzin problem is still unresolved

The apparently rapid and ancient diversification of many avian orders complicates the resolution of their relationships using molecular data. Recent studies based on complete mitochondrial DNA (mtDNA) sequences or shorter lengths of nuclear sequence have helped corroborate the basic structure of the avian tree (e.g., a basal split between Paleognathae and Neognathae) but have made relatively little progress in resolving relationships among the many orders within Neoaves. We explored the potential of a moderately sized mtDNA data set ( approximately 5000 bp for each of 41 taxa), supplemented with data from a nuclear intron ( approximately 700 bp per taxon), to resolve relationships among avian orders. Our sampling of taxa addresses two issues: (1). the sister relationship and monophyly, respectively, of Anseriformes and Galliformes and (2). relationships of the enigmatic hoatzin Opisthocomus hoazin. Our analyses support a basal split between Galloanserae and Neoaves within Neognathae and monophyly of both Galliformes and Anseriformes. Within Galliformes, megapodes and then cracids branch basally. Within Anseriformes, mitochondrial data support a screamer (Anhimidae) plus magpie goose (Anseranatidae) clade. This result, however, may be an artifact of divergent base composition in one of the two anatids we sampled. With deletion of the latter taxon, Anseranas is sister to anatids as in traditional arrangements and recent morphological studies. Although our data provide limited resolution of relationships within Neoaves, we find no support for a sister relationship between either cuckoos (Cuculiformes) or turacos (Musophagiformes) and hoatzin. Both mitochondrial and nuclear data are consistent with a relationship between hoatzin and doves (Columbiformes), although this result is weakly supported. We also show that mtDNA sequences reported in another recent study included pervasive errors that biased the analysis towards finding a sister relationship between hoatzin and turacos.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

Phylogenetic relationships of salangid fishes (Osmeridae, Salanginae) with comments on phylogenetic placement of the salangids based on mitochondrial DNA sequences

We used partial DNA sequences of cytochrome b and 16S mitochondrial genes to determine the phylogenetic placement of salangid fishes and the generic relationships within the salangids. Our molecular data strongly support the monophyly of salangid fishes, the inclusion of salangids in the Osmeridae, and the sister group relationship between salangids and osmerids. Our analyses suggest that Plecoglossus can be separated from all the other salangids and osmerids. Mallotus and Hypomesus are clustered within Osmerinae, rather than allied with Salanginae. As regards the relationships within the salangids, our analyses are incongruent with all previous classification hypotheses. Our phylogenetic analyses support the sister group relationships between Protosalanx and Neosalanx, and between Salanx and Hemisalanx. More evidences show that Leucosoma is more closely related to the Salanx-Hemisalanx clade, while Salangichthys forms part of an unresolved basal polytomy.     

The mitochondrial genomes of the iguana (Iguana iguana) and the caiman (Caiman crocodylus): implications for amniote phylogeny

The complete mitochondrial genomes of two reptiles, the common iguana (Iguana iguana) and the caiman (Caiman crocodylus), were sequenced in order to investigate phylogenetic questions of tetrapod evolution. The addition of the two species allows analysis of reptilian relationships using data sets other than those including only fast-evolving species. The crocodilian mitochondrial genomes seem to have evolved generally at a higher rate than those of other vertebrates. Phylogenetic analyses of 2889 amino-acid sites from 35 mitochondrial genomes supported the bird-crocodile relationship, lending no support to the Haematotherma hypothesis (with birds and mammals representing sister groups). The analyses corroborated the view that turtles are at the base of the bird-crocodile branch. This position of the turtles makes Diapsida paraphyletic. The origin of the squamates was estimated at 294 million years (Myr) ago and that of the turtles at 278 Myr ago. Phylogenetic analysis of mammalian relationships using the additional outgroups corroborated the Marsupionta hypothesis, which joins the monotremes and the marsupials to the exclusion of the eutherians.     

More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs

We present the first genomic-scale analysis addressing the phylogenetic position of turtles, using over 1000 loci from representatives of all major reptile lineages including tuatara. Previously, studies of morphological traits positioned turtles either at the base of the reptile tree or with lizards, snakes and tuatara (lepidosaurs), whereas molecular analyses typically allied turtles with crocodiles and birds (archosaurs). A recent analysis of shared microRNA families found that turtles are more closely related to lepidosaurs. To test this hypothesis with data from many single-copy nuclear loci dispersed throughout the genome, we used sequence capture, high-throughput sequencing and published genomes to obtain sequences from 1145 ultraconserved elements (UCEs) and their variable flanking DNA. The resulting phylogeny provides overwhelming support for the hypothesis that turtles evolved from a common ancestor of birds and crocodilians, rejecting the hypothesized relationship between turtles and lepidosaurs.     

Phylogenetic relationships and divergence dates of softshell turtles (Testudines: Trionychidae) inferred from complete mitochondrial genomes

The softshell turtles (Trionychidae) are one of the most widely distributed reptile groups in the world, and fossils have been found on all continents except Antarctica. The phylogenetic relationships among members of this group have been previously studied; however, disagreements regarding its taxonomy, its phylogeography and divergence times are still poorly understood as well. Here, we present a comprehensive mitogenomic study of softshell turtles. We sequenced the complete mitochondrial genomes of 10 softshell turtles, in addition to the GenBank sequence of Dogania subplana, Lissemys punctata, Trionyx triunguis, which cover all extant genera within Trionychidae except for Cyclanorbis and Cycloderma. These data were combined with other mitogenomes of turtles for phylogenetic analyses. Divergence time calibration and ancestral reconstruction were calculated using BEAST and RASP software, respectively. Our phylogenetic analyses indicate that Trionychidae is the sister taxon of Carettochelyidae, and support the monophyly of Trionychinae and Cyclanorbinae, which is consistent with morphological data and molecular analysis. Our phylogenetic analyses have established a sister taxon relationship between the Asian Rafetus and the Asian Palea + Pelodiscus + Dogania + Nilssonia + Amyda, whereas a previous study grouped the Asian Rafetus with the American Apalone. The results of divergence time estimates and area ancestral reconstruction show that extant Trionychidae originated in Asia at around 108 million years ago (MA), and radiations mainly occurred during two warm periods, namely Late Cretaceous–Early Eocene and Oligocene. By combining the estimated divergence time and the reconstructed ancestral area of softshell turtles, we determined that the dispersal of softshell turtles out of Asia may have taken three routes. Furthermore, the times of dispersal seem to be in agreement with the time of the India–Asia collision and opening of the Bering Strait, which provide evidence for the accuracy of our estimation of divergence time. Overall, the mitogenomes of this group were used to explore the origin and dispersal route of Trionychidae and have provided new insights on the evolution of this group.     

Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae)

The blood flukes of turtles (Digenea: Spirorchiidae) and the blood flukes of crocodilians, birds and mammals (Digenea: Schistosomatidae) have long been considered as closely related, but distinct evolutionary lineages. Recent morphological and molecular studies have considered these families as sister taxa within the Schistosomatoidea. Representatives of both families have similar furcocercous cercariae and similar two-host life cycles, but have different definitive hosts, distinct reproductive patterns and different morphologies. Sequences including approximately 1800 bases of the small subunit ribosomal DNA and 1200 bases of the large subunit ribosomal DNA were generated from representatives of eight spirorchiid genera. These sequences were aligned with pre-existing sequences of Schistosomatidae and other representatives of the Diplostomida and analysed for phylogenetic signal using maximum parsimony and Bayesian inference. These analyses revealed that the Spirorchiidae is paraphyletic and that the turtle blood flukes are basal to the highly derived schistosomatids. Three genera of spirorchiids from marine turtles form a sister group to the Schistosomatidae and five genera of spirorchiids from freshwater turtles occupy basal positions in the phylogeny of tetrapod blood flukes. Marine turtles are considered to be derived from freshwater turtles and the results of the current study indicate that the spirorchiid parasites of marine turtles are similarly derived from a freshwater ancestor. The close relationship of the marine spirorchiids to schistosomatids and the basal position of the marine transmitted Austrobilharzia and Ornithobilharzia in the schistosomatid clade suggests that schistosomatids arose after a marine turtle blood fluke ancestor successfully colonised birds.     

The evolution of island gigantism and body size variation in tortoises and turtles

Extant chelonians (turtles and tortoises) span almost four orders of magnitude of body size, including the startling examples of gigantism seen in the tortoises of the Galapagos and Seychelles islands. However, the evolutionary determinants of size diversity in chelonians are poorly understood. We present a comparative analysis of body size evolution in turtles and tortoises within a phylogenetic framework. Our results reveal a pronounced relationship between habitat and optimal body size in chelonians. We found strong evidence for separate, larger optimal body sizes for sea turtles and island tortoises, the latter showing support for the rule of island gigantism in non-mammalian amniotes. Optimal sizes for freshwater and mainland terrestrial turtles are similar and smaller, although the range of body size variation in these forms is qualitatively greater. The greater number of potential niches in freshwater and terrestrial environments may mean that body size relationships are more complicated in these habitats.     

Resolution of the laurasiatherian phylogeny: evidence from genomic data

Despite great progress over the past decade, some portions of the mammalian tree of life remain unresolved. In particular, relationships among the different orders included within the supraordinal group Laurasiatheria have been proven difficult to determine, and have received poor support in the vast majority of phylogenomic studies of mammalian systematics. We estimated interordinal relationships within Laurasiatheria using sequence data from 3733 protein-coding genes. Our study included data from from 11 placental mammals, corresponding to five of the six orders of Laurasiatheria, plus five outgroup species. Ingroup and outgroup species were chosen to maximize the number single-copy ortholog genes for which sequence data was available for all species in our study. Phylogenetic analyses of the concatenated dataset using maximum likelihood and Bayesian methods resulted on an identical and well supported topology in all alignment strategies compared. Our analyses provide high support for the sister relationship between Chiroptera and Cetartiodactyla and also provide support for placing Perissodactyla as sister to Carnivora. We obtained maximal estimates of bootstrap support (100%) and posterior probability (1.00) for all nodes within Laurasiatheria. Our study provides a further demonstration of the utility of very large and conserved genomic dataset to clarify our understanding of the evolutionary relationships among mammals.     

Pike and salmon as sister taxa: Detailed intraclade resolution and divergence time estimation of Esociformes + Salmoniformes based on whole mitochondrial genome sequences

The increasing number of taxa and loci in molecular phylogenetic studies of basal euteleosts has brought stability in a controversial area. A key emerging aspect to these studies is a sister Esociformes (pike) and Salmoniformes (salmon) relationship. We evaluate mitochondrial genome support for a sister Esociformes and Salmoniformes hypothesis by surveying many potential outgroups for these taxa, employing multiple phylogenetic approaches, and utilizing a thorough sampling scheme. Secondly, we conduct a simultaneous divergence time estimation and phylogenetic inference in a Bayesian framework with fossil calibrations focusing on relationships within Esociformes + Salmoniformes. Our dataset supports a sister relationship between Esociformes and Salmoniformes; however the nearest relatives of Esociformes + Salmoniformes are inconsistent among analyses. Within the order Esociformes, we advocate for a single family, Esocidae. Subfamily relationships within Salmonidae are poorly supported as Salmoninae sister to Thymallinae + Coregoninae.     

Molecular phylogenetics and historical biogeography among salamandrids of the "true" salamander clade: rapid branching of numerous highly divergent lineages in Mertensiella luschani associated with the rise of Anatolia

Phylogenetic relationships among salamandrids of the "true" salamander clade are investigated using 2019 aligned base positions (713 parsimony informative) of 20 mitochondrial DNA sequences from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase), plus the origin for light-strand replication (O(L)) between the tRNA(Asn) and the tRNA(Cys) genes. Parsimony analysis produces a robust phylogenetic estimate for the relationships of the major groups of "true" salamanders. Strong support is provided for the sister taxon relationship of Chioglossa and Mertensiella caucasica and for the placement of Salamandra and Mertensiella luschani as sister taxa. These relationships suggest two vicariant events between Europe and Anatolia caused by the formation of seaways in the Mediterranean Basin. Molecular divergence indicates an Early Miocene separation of Chioglossa and M. caucasica and a Late Miocene separation of Salamandra and M. luschani. The traditional phylogenetic hypothesis of a monophyletic Mertensiella is statistically rejected, indicating that southwestern and northeastern Anatolian populations have separate historical biogeographic origins. Therefore, we recommend placement of M. luschani in the genus Salamandra. Within M. luschani, six highly divergent lineages showing 7.6 to 10.1% pairwise sequence divergence are identified. Tests using four-taxon subsamples suggest that these lineages diverged nearly simultaneously in the Late Miocene, approximately 6 to 8 million years ago, when extensive uplifting of Anatolia occurred in response to the Arabian collision.     

Patterns of clade support across the major lineages of moss phylogeny

Relationships among the major branches of moss phylogeny are understudied compared with other major land‐plant groups. We addressed this by surveying 14–17 plastid genes from taxa representing the major lineages, using different phylogenetic methods (parsimony, likelihood) and codon‐ and gene‐based data partitioning schemes (likelihood). Our phylogenetic inferences generally corroborated the best supported clades across multiple recent studies, with comparable or higher levels of clade support here. We resolved persistent ambiguities with strong to moderate support across analyses, including several early nodes in subclass Dicranidae, and relationships among other subclasses of peristomate mosses. In particular, we resolved a sister‐group relationship between Bryidae and Dicranidae, between these subclasses and Timiidae, and between this entire clade and Funariidae. We consistently recovered Tetraphidopsida (a nematodontous class) as the sister group of arthrodontous mosses (Bryopsida), although with only weak support. Strongly conflicting arrangements at the base of moss phylogeny concerning Takakiopsida and Sphagnopsida, two non‐peristomate moss lineages, were inferred in parsimony and likelihood analysis, but this depended on how base‐frequency parameters were estimated and how data were partitioned in likelihood analysis. Relationships inferred for the remaining peristomate and non‐peristomate moss clades, and their associated support values, were otherwise broadly congruent across analyses.     

Molecular phylogenetics and evolution of turtles

Turtles are one of Earth's most instantly recognizable life forms, distinguished for over 200 million years in the fossil record. Even so, key nodes in the phylogeny of turtles remain uncertain. To address this issue, we sequenced >90% of the nuclear recombination activase gene 1 (RAG-1) for 24 species representing all modern turtle families. RAG-1 exhibited negligible saturation and base composition bias, and extensive base composition homogeneity. Most of the relationships suggested by prior phylogenetic analyses were also supported by RAG-1 and, for at least two critical nodes, with a much higher level of support. RAG-1 also indicates that the enigmatic Platysternidae and Chelydridae, often considered sister taxa based on morphological evidence, are not closely related, although their precise phylogenetic placement in the turtle tree is still unresolved. Although RAG-1 is phylogenetically informative, our research revealed fundamental conflicts among analytical methods for estimating phylogenetic hypotheses. Maximum parsimony analyses of RAG-1 alone and in combination with two mitochondrial genes suggest the earliest phylogenetic splits separating into three basal branches, the pig-nosed turtles (Carettochelyidae), the softshell turtles (Trionychidae), and a clade comprising all remaining extant turtles. Maximum likelihood and Bayesian analyses group Carettochelyidae and Trionychidae (=Trionychoidae) in their more traditional location as the sister taxon to all other hidden-necked turtles, collectively forming the Cryptodira. Our research highlights the utility of molecular data in identifying issues of character homology in morphological datasets, while shedding valuable light on the biodiversity of a globally imperiled taxon.