Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: an example from squamate reptiles

Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., 700bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates.     

Concordance analysis in mitogenomic phylogenetics

Here I advocate the utility of Bayesian concordance analysis as a mechanism for exploring the magnitude and source of phylogenetic signal in concatenated mitogenomic phylogenetic studies. While typically applied to the study of independently evolving gene trees, Bayesian concordance analysis can also be applied to linked, but individually analyzed, gene regions using a prior probability that reflects the expectation of similar phylogenetic reconstructions. For true branches in the mitogenomic tree, concordance factors should represent the number of gene regions that contain phylogenetic signal for a particular clade. As a demonstration of the application of Bayesian concordance analysis to empirical data, I analyzed two different salamander (Hynobiidae and Plethodontidae) mitogenomic data sets using a gene-based partitioning strategy. The results revealed many strongly supported clades in the concatenated trees that have high concordance factors, permitting the inference that these are robustly resolved through phylogenetic signal distributed across the mitogenome. In contrast, a number of strongly supported clades in the concatenated tree received low concordance factors, indicating that their reconstruction is either driven primarily by phylogenetic signal in a small number of gene regions, or that they are inconsistent reconstructions influenced by properties of the data that can produce inaccurate trees (e.g., compositional bias, selection, etc.). Exploration of the Bayesian joint posterior distribution of trees highlighted partitions that contribute phylogenetic information to similar clade reconstructions. This approach was particularly insightful in the hynobiid data, where different combinations of genes were identified that support alternative tree reconstructions. Concatenated analysis of these different subsets of genes highlighted through Bayesian concordance analysis produced strongly supported and contrasting trees, demonstrating the potential for inconsistency in concatenated mitogenomic phylogenetics. The overall results presented here suggest that Bayesian concordance analysis can serve as an effective exploration of the influence of different gene regions in mitogenomic (and other organellar genomic) phylogenetic studies.     

Turtle origins: insights from phylogenetic retrofitting and molecular scaffolds

Adding new taxa to morphological phylogenetic analyses without substantially revising the set of included characters is a common practice, with drawbacks (undersampling of relevant characters) and potential benefits (character selection is not biased by preconceptions over the affinities of the ‘retrofitted’ taxon). Retrofitting turtles (Testudines) and other taxa to recent reptile phylogenies consistently places turtles with anapsid‐grade parareptiles (especially Eunotosaurus and/or pareiasauromorphs), under both Bayesian and parsimony analyses. This morphological evidence for turtle–parareptile affinities appears to contradict the robust genomic evidence that extant (living) turtles are nested within diapsids as sister to extant archosaurs (birds and crocodilians). However, the morphological data are almost equally consistent with a turtle–archosaur clade: enforcing this molecular scaffold onto the morphological data does not greatly increase tree length (parsimony) or reduce likelihood (Bayesian inference). Moreover, under certain analytic conditions, Eunotosaurus groups with turtles and thus also falls within the turtle–archosaur clade. This result raises the possibility that turtles could simultaneously be most closely related to a taxon traditionally considered a parareptile (Eunotosaurus) and still have archosaurs as their closest extant sister group.     

Phylogeny of Bacterial and Archaeal Genomes Using Conserved Genes: Supertrees and Supermatrices

Over 3000 microbial (bacterial and archaeal) genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML) tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA), as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a “primary concordance” tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy) tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.     

Calculating the evolutionary rates of different genes: a fast, accurate estimator with applications to maximum likelihood phylogenetic analysis

In phylogenetic analyses with combined multigene or multiprotein data sets, accounting for differing evolutionary dynamics at different loci is essential for accurate tree prediction. Existing maximum likelihood (ML) and Bayesian approaches are computationally intensive. We present an alternative approach that is orders of magnitude faster. The method, Distance Rates (DistR), estimates rates based upon distances derived from gene/protein sequence data. Simulation studies indicate that this technique is accurate compared with other methods and robust to missing sequence data. The DistR method was applied to a fungal mitochondrial data set, and the rate estimates compared well to those obtained using existing ML and Bayesian approaches. Inclusion of the protein rates estimated from the DistR method into the ML calculation of trees as a branch length multiplier resulted in a significantly improved fit as measured by the Akaike Information Criterion (AIC). Furthermore, bootstrap support for the ML topology was significantly greater when protein rates were used, and some evident errors in the concatenated ML tree topology (i.e., without protein rates) were corrected. [Bayesian credible intervals; DistR method; multigene phylogeny; PHYML; rate heterogeneity.].     

Phylogeny of iguanian lizards inferred from 29 nuclear loci, and a comparison of concatenated and species-tree approaches for an ancient, rapid radiation

Iguanian lizards form a diverse clade whose members have been the focus of many comparative studies of ecology, behavior, and evolution. Despite the importance of phylogeny to such studies, interrelationships among many iguanian clades remain uncertain. Within the Old World clade Acrodonta, Agamidae is sometimes found to be paraphyletic with respect to Chamaeleonidae, and recent molecular studies have produced conflicting results for many major clades. Within the largely New World clade Pleurodonta, relationships among the 12 currently recognized major subclades (mostly ranked as families) have been largely unresolved or poorly supported in previous studies. To clarify iguanian evolutionary history, we first infer phylogenies using concatenated maximum-likelihood (ML) and Bayesian analyses of DNA sequence data from 29 nuclear protein-coding genes for 47 iguanian and 29 outgroup taxa. We then estimate a relaxed-clock Bayesian chronogram for iguanians using BEAST. All three methods produce identical topologies. Within Acrodonta, we find strong support for monophyly of Agamidae with respect to Chamaeleonidae, and for almost all relationships within agamids. Within Pleurodonta, we find strong Bayesian support for almost all relationships, and strong ML support for some interfamilial relationships and for monophyly of almost all families (excepting Polychrotidae). Our phylogenetic results suggest a non-traditional biogeographic scenario in which pleurodonts originated in the Northern Hemisphere and subsequently spread southward into South America. The pleurodont portion of the tree is characterized by several very short, deep branches, raising the possibility of deep coalescences that may confound concatenated analyses. We therefore also use 27 of these genes to implement a coalescent-based species-tree approach for pleurodonts. Although this analysis strongly supports monophyly of the pleurodont families, interfamilial relationships are generally different from those in the concatenated tree, and support is uniformly poor. However, a species-tree analysis using only the seven most variable loci yields higher support and more congruence with the concatenated tree. This suggests that low support in the 27-gene species-tree analysis may be an artifact of the many loci that are uninformative for very short branches. This may be a general problem for the application of species-tree methods to rapid radiations, even with phylogenomic data sets. Finally, we correct the non-monophyly of Polychrotidae by recognizing the pleurodont genus Anolis (sensu lato) as a separate family (Dactyloidae), and we correct the non-monophyly of the agamid genus Physignathus by resurrection of the genus Istiurus for the former Physignathus lesueurii.     

Combined nuclear and mitochondrial DNA sequences resolve generic relationships within the Cracidae (Galliformes,Aves)

The Cracidae is one of the most endangered and distinctive bird families in the Neotropics, yet the higher relationships among taxa remain uncertain. The molecular phylogeny of its 11 genera was inferred using 10,678 analyzable sites (5,412 from seven different mitochondrial segments and 5,266 sites from four nuclear genes). We performed combinability tests to check conflicts in phylogenetic signals of separate genes and genomes. Phylogenetic analysis showed that the unrooted tree of ((curassows, horned guan) (guans, chachalacas)) was favored by most data partitions and that different data partitions provided support for different parts of the tree. In particular, the concatenated mitochondrial DNA (mtDNA) genes resolved shallower nodes, whereas the combined nuclear sequences resolved the basal connections among the major clades of curassows, horned guan, chachalacas, and guans. Therefore, we decided that for the Cracidae all data should be combined for phylogenetic analysis. Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses of this large data set produced similar trees. The MP tree indicated that guans are the sister group to (horned guan, (curassows, chachalacas)), whereas the ML and Bayesian analysis recovered a tree where the horned guan is a sister clade to curassows, and these two clades had the chachalacas as a sister group. Parametric bootstrapping showed that alternative trees previously proposed for the cracid genera are significantly less likely than our estimate of their relationships. A likelihood ratio test of the hypothesis of a molecular clock for cracid mtDNA sequences using the optimal ML topology did not reject rate constancy of substitutions through time. We estimated cracids to have originated between 64 and 90 million years ago (MYA), with a mean estimate of 76 MYA. Diversification of the genera occurred approximately 41-3 MYA, corresponding with periods of global climate change and other Earth history events that likely promoted divergences of higher level taxa.     

Toward the resolution of an explosive radiation--a multilocus phylogeny of oceanic dolphins (Delphinidae)

Oceanic dolphins (Delphinidae) are the product of a rapid radiation that yielded ～36 extant species of small to medium-sized cetaceans that first emerged in the Late Miocene. Although they are a charismatic group of organisms that have become poster children for marine conservation, many phylogenetic relationships within Delphinidae remain elusive due to the slow molecular evolution of the group and the difficulty of resolving short branches from successive cladogenic events. Here I combine existing and newly generated sequences from four mitochondrial (mt) genes and 20 nuclear (nu) genes to reconstruct a well-supported phylogenetic hypothesis for Delphinidae. This study compares maximum-likelihood and Bayesian inference methods of several data sets including mtDNA, combined nuDNA, gene trees of individual nuDNA loci, and concatenated mtDNA+nuDNA. In addition, I contrast these standard phylogenetic analyses with the species tree reconstruction method of Bayesian concordance analysis (BCA). Despite finding discordance between mtDNA and individual nuDNA loci, the concatenated matrix recovers a completely resolved and robustly supported phylogeny that is also broadly congruent with BCA trees. This study strongly supports groupings such as Delphininae, Lissodelphininae, Globicephalinae, Sotalia+Delphininae, Steno+Orcaella+Globicephalinae, and Leucopleurus acutus, Lagenorhynchus albirostris, and Orcinus orca as basal delphinid taxa.     

Using Genes as Characters and a Parsimony Analysis to Explore the Phylogenetic Position of Turtles

The phylogenetic position of turtles within the vertebrate tree of life remains controversial. Conflicting conclusions from different studies are likely a consequence of systematic error in the tree construction process, rather than random error from small amounts of data. Using genomic data, we evaluate the phylogenetic position of turtles with both conventional concatenated data analysis and a “genes as characters” approach. Two datasets were constructed, one with seven species (human, opossum, zebra finch, chicken, green anole, Chinese pond turtle, and western clawed frog) and 4584 orthologous genes, and the second with four additional species (soft-shelled turtle, Nile crocodile, royal python, and tuatara) but only 1638 genes. Our concatenated data analysis strongly supported turtle as the sister-group to archosaurs (the archosaur hypothesis), similar to several recent genomic data based studies using similar methods. When using genes as characters and gene trees as character-state trees with equal weighting for each gene, however, our parsimony analysis suggested that turtles are possibly sister-group to diapsids, archosaurs, or lepidosaurs. None of these resolutions were strongly supported by bootstraps. Furthermore, our incongruence analysis clearly demonstrated that there is a large amount of inconsistency among genes and most of the conflict relates to the placement of turtles. We conclude that the uncertain placement of turtles is a reflection of the true state of nature. Concatenated data analysis of large and heterogeneous datasets likely suffers from systematic error and over-estimates of confidence as a consequence of a large number of characters. Using genes as characters offers an alternative for phylogenomic analysis. It has potential to reduce systematic error, such as data heterogeneity and long-branch attraction, and it can also avoid problems associated with computation time and model selection. Finally, treating genes as characters provides a convenient method for examining gene and genome evolution.     

Molecular systematics of Middle American harvest mice Reithrodontomys (Muridae), estimated from mitochondrial cytochrome b gene sequences

We estimated phylogenetic relationships among 16 species of harvest mice using sequences from the mitochondrial cytochrome b (cyt b) gene. Gene phylogenies constructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) optimality criteria were largely congruent and arranged taxa into two groups corresponding to the two recognized subgenera (Aporodon and Reithrodontomys). All analyses also recovered R. mexicanus and R. microdon as polyphyletic, although greater resolution was obtained using ML and BI approaches. Within R. mexicanus, three clades were identified with high nodal support (MP and ML bootstrap, Bremer decay and Bayesian posterior probabilities). One represented a subspecies of R. mexicanus from Costa Rica (R. m. cherrii) and a second was distributed in the Sierra Madre Oriental of Mexico. The third R. mexicanus clade consisted of mice from southern Mexico southward to South America. Polyphyly between the two moieties of R. microdon corresponded to the Isthmus of Tehuantepec in southern Mexico. Populations of R. microdon microdon to the east of the isthmus (Chiapas, Mexico) grouped with R. tenuirostris, whereas samples of R. m. albilabris to the west in Oaxaca, Mexico, formed a clade with R. bakeri. Within the subgenus Reithrodontomys, all analyses recovered R. montanus and R. raviventris as sister taxa, a finding consistent with earlier studies based on allozymes and cyt b data. There was also strong support (ML and BI criteria) for a clade consisting of ((R. megalotis, R. zacatecae) (R. sumichrasti)). In addition, cytb gene phylogenies (MP, ML, and BI) recovered R. fulvescens and R. hirsutus (ML and BI) as basal taxa within the subgenus Reithrodontomys. Constraint analyses demonstrated that tree topologies treating the two subgenera (Aporodon and Reithrodontomys) as monophyletic (ML criterion) was significantly better (p>0.036) and supported polyphyly of R. mexicanus (both ML and MP criteria - p>0.013) and R. microdon (MP criterion only for certain topologies; p>0.02). Although several species-level taxa were identified based on multiple, independent data sets, we recommended a conservative approach which will involve thorough analyses of museum specimens including material from type localities together with additional sampling and data from multiple, nuclear gene markers.     

Delimiting species in recent radiations

Despite considerable effort from the systematics community, delimiting species boundaries in recent radiations remains a daunting challenge. We argue that genealogical approaches, although sometimes useful, may not solve this important problem, because recently derived species often have not had sufficient time to achieve monophyly. Instead, we suggest that population genetic approaches that rely on large sets of informative markers like single nucleotide polymorphisms (SNPs) provide an alternative framework for delimiting very recently derived species. We address two major challenges in applying such markers to species delimitation: discovering markers in nonmodel systems and using them to delimit recently derived species. Using turtles as a test case, we explore the utility of a single, relatively low-coverage genomic resource as an aid in gene and marker discovery. We exploit an end-sequenced bacterial artificial chromosome (BAC) library from an individual painted turtle (Chrysemys picta) and outline a novel protocol that efficiently identifies primer pairs that amplify homologous sequences across the tree of living turtles. Preliminary data using this library to discover SNPs in Emydura macquarii, a species that diverged from C. picta approximately 210 million years ago, indicate that sequences identified from the Chrysemys BAC library provide useful SNPs even in this very distantly related taxon. Several recent methods in wide use in the population genetics literature allow one to discover potential species, or test existing species hypotheses, with SNP data and may be particularly informative for very recently derived species. As BAC and other genomic resources become increasingly available for scattered taxa across the tree of life, we are optimistic that these resources will provide abundant, inexpensive markers that will help delimit boundaries in problematic, recent species radiations.     

Resolving an ancient, rapid radiation in Saxifragales

Despite the prior use of approximately 9000 bp, deep-level relationships within the angiosperm clade, Saxifragales remain enigmatic, due to an ancient, rapid radiation (89.5 to 110 Ma based on the fossil record). To resolve these deep relationships, we constructed several new data sets: (1) 16 genes representing the three genomic compartments within plant cells (2 nuclear, 10 plastid, 4 mitochondrial; aligned, analyzed length = 21,460 bp) for 28 taxa; (2) the entire plastid inverted repeat (IR; 26,625 bp) for 17 taxa; (3) "total evidence" (50,845 bp) for both 17 and 28 taxa (the latter missing the IR). Bayesian and ML methods yielded identical topologies across partitions with most clades receiving high posterior probability (pp = 1.0) and bootstrap (95% to 100%) values, suggesting that with sufficient data, rapid radiations can be resolved. In contrast, parsimony analyses of different partitions yielded conflicting topologies, particularly with respect to the placement of Paeoniaceae, a clade characterized by a long branch. In agreement with published simulations, the addition of characters increased bootstrap support for the putatively erroneous placement of Paeoniaceae. Although having far fewer parsimony-informative sites, slowly evolving plastid genes provided higher resolution and support for deep-level relationships than rapidly evolving plastid genes, yielding a topology close to the Bayesian and ML total evidence tree. The plastid IR region may be an ideal source of slowly evolving genes for resolution of deep-level angiosperm divergences that date to 90 My or more. Rapidly evolving genes provided support for tip relationships not recovered with slowly evolving genes, indicating some complementarity. Age estimates using penalized likelihood with and without age constraints for the 28-taxon, total evidence data set are comparable to fossil dates, whereas estimates based on the 17-taxon data are much older than implied by the fossil record. Hence, sufficient taxon density, and not simply numerous base pairs, is important in reliably estimating ages. Age estimates indicate that the early diversification of Saxifragales occurred rapidly, over a time span as short as 6 million years. Between 25,000 and 50,000 bp were needed to resolve this radiation with high support values. Extrapolating from Saxifragales, a similar number of base pairs may be needed to resolve the many other deep-level radiations of comparable age in angiosperms.     

Genetic Algorithm-Based Maximum-Likelihood Analysis for Molecular Phylogeny

A heuristic approach to search for the maximum-likelihood (ML) phylogenetic tree based on a genetic algorithm (GA) has been developed. It outputs the best tree as well as multiple alternative trees that are not significantly worse than the best one on the basis of the likelihood criterion. These near-optimum trees are subjected to further statistical tests. This approach enables ones to infer phylogenetic trees of over 20 taxa taking account of the rate heterogeneity among sites on practical time scales on a PC cluster. Computer simulations were conducted to compare the efficiency of the present approach with that of several likelihood-based methods and distance-based methods, using amino acid sequence data of relatively large (5–24) taxa. The superiority of the ML method over distance-based methods increases as the condition of simulations becomes more realistic (an incorrect model is assumed or many taxa are involved). This approach was applied to the inference of the universal tree based on the concatenated amino acid sequences of vertically descendent genes that are shared among all genomes whose complete sequences have been reported. The inferred tree strongly supports that Archaea is paraphyletic and Eukarya is specifically related to Crenarchaeota. Apart from the paraphyly of Archaea and some minor disagreements, the universal tree based on these genes is largely consistent with the universal tree based on SSU rRNA. Received: 4 January 2001 / Accepted: 16 May 2001     

Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the association of rhizaria with chromalveolates

Here we use phylogenomics with expressed sequence tag (EST) data from the ecologically important coccolithophore-forming alga Emiliania huxleyi and the plastid-lacking cryptophyte Goniomonas cf. pacifica to establish their phylogenetic positions in the eukaryotic tree. Haptophytes and cryptophytes are members of the putative eukaryotic supergroup Chromalveolata (chromists [cryptophytes, haptophytes, stramenopiles] and alveolates [apicomplexans, ciliates, and dinoflagellates]). The chromalveolates are postulated to be monophyletic on the basis of plastid pigmentation in photosynthetic members, plastid gene and genome relationships, nuclear "host" phylogenies of some chromalveolate lineages, unique gene duplication and replacements shared by these taxa, and the evolutionary history of components of the plastid import and translocation systems. However the phylogenetic position of cryptophytes and haptophytes and the monophyly of chromalveolates as a whole remain to be substantiated. Here we assess chromalveolate monophyly using a multigene dataset of nuclear genes that includes members of all 6 eukaryotic supergroups. An automated phylogenomics pipeline followed by targeted database searches was used to assemble a 16-protein dataset (6,735 aa) from 46 taxa for tree inference. Maximum likelihood and Bayesian analyses of these data support the monophyly of haptophytes and cryptophytes. This relationship is consistent with a gene replacement via horizontal gene transfer of plastid-encoded rpl36 that is uniquely shared by these taxa. The haptophytes + cryptophytes are sister to a clade that includes all other chromalveolates and, surprisingly, two members of the Rhizaria, Reticulomyxa filosa and Bigelowiella natans. The association of the two Rhizaria with chromalveolates is supported by the approximately unbiased (AU)-test and when the fastest evolving amino acid sites are removed from the 16-protein alignment.     

Phylogenetic placement of diverse amoebae inferred from multigene analyses and assessment of clade stability within 'Amoebozoa' upon removal of varying rate classes of SSU-rDNA

Placing amoeboid lineages on the eukaryotic tree of life is difficult due to the paucity of comparable morphological characters and the limited molecular data available for many groups. This situation has led to the lumping of distantly related lineages into large inclusive groups, such as Sarcodina, that do not reflect evolutionary relationships. Previous analyses of molecular markers with limited taxon sampling reveal members of Sarcodina are scattered in five of the six proposed supergroups. We have used multigene analyses to place seven diverse amoeboid lineages-two Nolandella spp., Rhizamoeba sp., Pessonella sp., Arcella hemisphaerica, Arachnula sp. and Trichosphaerium sp.-on the eukaryotic tree of life. Bayesian analysis of the concatenated data of the four genes sequenced (SSU-rDNA, actin, alpha-tubulin and beta-tubulin), including diverse representatives of eukaryotes, indicates that all seven taxa group within the 'Amoebozoa' supergroup. We further performed separate analyses of the well-sampled SSU-rDNA and actin genes using Bayesian and Maximum Likelihood analyses to assess the positions of our newly characterized taxa. In the case of SSU-rDNA, we performed extensive analyses with removal of the fastest rates classes to evaluate the stability and resolution of various taxonomic hypotheses within 'Amoebozoa'. Five of our seven amoeboid lineages fall within well-supported clades that are corroborated by morphology. In contrast, the positions of Arachnula sp. and Trichosphaerium sp. in the SSU-rDNA gene trees are unstable and vary by analyses. Placement of these taxa will require additional data from slowly evolving genes combined with taxon-rich phylogenetic analyses. Finally, the analyses without the fastest rate classes demonstrate that SSU-rDNA has a limited signal for deep relationships within the 'Amoebozoa'.     

Species tree estimation and the historical biogeography of heroine cichlids

Heroine cichlids are major components of the fish faunas in both Central America and the Caribbean. To examine the evolutionary patterns of how cichlids colonized both of these regions, we reconstructed the phylogenetic relationships among 23 cichlid lineages. We used three phylogenetically novel nuclear markers (Dystropin b, Myomesin1, and Wnt7b) in combination with sequence data from seven other gene regions (Nd2, Rag1, Enc1, Sreb2, Ptr, Plagl2, and Zic1) to elucidate the species tree of these cichlids. The species examined represent major heroine lineages in South America, Central America, and the Greater Antilles. The individual gene trees of these groups were topologically quite discordant. Therefore, we combined the genetic partitions and inferred the species tree using both concatenation and a coalescent-based Bayesian method. The two resulting phylogenetic topologies were largely concordant but differed in two fundamental ways. First, more nodes in the concatenated tree were supported with substantial or 100% Bayesian posterior support than in the coalescent-based tree. Second, there was a minor, but biogeographically critical, topological difference between the concatenated and coalescent-based trees. Nevertheless, both analyses recovered topologies consistent with the Greater Antillean heroines being phylogenetically nested within the largely Central American heroine radiation. This study suggests that reconstructions of cichlid phylogeny and historical biogeography should account for the vagaries of individual gene histories.     

Phylogenetic hypotheses for the turtle family Geoemydidae

The turtle family Geoemydidae represents the largest, most diverse, and most poorly understood family of turtles. Little is known about this group, including intrafamilial systematics. The only complete phylogenetic hypothesis for this family positions geoemydids as paraphyletic with respect to tortoises, but this arrangement has not been accepted by many workers. We compiled a 79-taxon mitochondrial and nuclear DNA data set to reconstruct phylogenetic relationships for 65 species and subspecies representing all 23 genera of the Geoemydidae. Maximum parsimony (MP) and maximum-likelihood (ML) analyses and Bayesian analysis produced similar, well-resolved trees. Our analyses identified three main clades comprising the tortoises (Testudinidae), the old-world Geoemydidae, and the South American geoemydid genus Rhinoclemmys. Within Geoemydidae, many nodes were strongly supported, particularly based on Bayesian posterior probabilities of the combined three-gene dataset. We found that adding data for a subset of taxa improved resolution of some deeper nodes in the tree. Several strongly supported groupings within the Geoemydidae demonstrate non-monophyly of some genera and possible interspecific hybrids, and we recommend several taxonomic revisions based on available evidence.     

Molecular phylogeny of the higher taxa of Odonata (Insecta) inferred from COI, 16S rRNA, 28S rRNA,and EF1‐α sequences

In this study, we sequenced both two mitochondrial genes (COI and 16S rRNA) and nuclear genes (28S rRNA and elongation factor‐1α) from 71 species of Odonata that represent 7 superfamilies in 3 suborders. Phylogenetic testing for each two concatenated gene sequences based on function (ribosomal vs protein‐coding genes) and origin (mitochondrial vs nuclear genes) proved limited resolution. Thus, four concatenated sequences were utilized to test the previous phylogenetic hypotheses of higher taxa of Odonata via Bayesian inference (BI) and maximum likelihood (ML) algorithms, along with the data partition by the BI method. As a result, three slightly different topologies were obtained, but the BI tree without partition was slightly better supported by the topological test. This topology supported the suborders Anisoptera and Zygoptera each being a monophyly, and the close relationship of Anisozygoptera to Anisoptera. All the families represented by multiple taxa in both Anisoptera and Zygoptera were consistently revealed to each be a monophyly with the highest nodal support. Unlike consistent and robust familial relationships in Zygoptera those of Anisoptera were partially unresolved, presenting the following relationships: ((((Libellulidae + Corduliidae) + Macromiidae) + Gomphidae + Aeshnidae) + Anisozygoptera) + (((Coenagrionidae + Platycnemdidae) + Calopterygidae) + Lestidae). The subfamily Sympetrinae, represented by three genera in the anisopteran family Libellulidae, was not monophyletic, dividing Crocothemis and Deielia in one group together with other subfamilies and Sympetrum in another independent group.     

Optimal data partitioning and a test case for ray-finned fishes (Actinopterygii) based on ten nuclear loci

Data partitioning, the combined phylogenetic analysis of homogeneous blocks of data, is a common strategy used to accommodate heterogeneities in complex multilocus data sets. Variation in evolutionary rates and substitution patterns among sites are typically addressed by partitioning data by gene, codon position, or both. Excessive partitioning of the data, however, could lead to overparameterization; therefore, it seems critical to define the minimum numbers of partitions necessary to improve the overall fit of the model. We propose a new method, based on cluster analysis, to find an optimal partitioning strategy for multilocus protein-coding data sets. A heuristic exploration of alternative partitioning schemes, based on Bayesian and maximum likelihood (ML) criteria, is shown here to produce an optimal number of partitions. We tested this method using sequence data of 10 nuclear genes collected from 52 ray-finned fish (Actinopterygii) and four tetrapods. The concatenated sequences included 7995 nucleotide sites maximally split into 30 partitions defined a priori based on gene and codon position. Our results show that a model based on only 10 partitions defined by cluster analysis performed better than partitioning by both gene and codon position. Alternative data partitioning schemes also are shown to affect the topologies resulting from phylogenetic analysis, especially when Bayesian methods are used, suggesting that overpartitioning may be of major concern. The phylogenetic relationships among the major clades of ray-finned fish were assessed using the best data-partitioning schemes under ML and Bayesian methods. Some significant results include the monophyly of "Holostei" (Amia and Lepisosteus), the sister-group relationships between (1) esociforms and salmoniforms and (2) osmeriforms and stomiiforms, the polyphyly of Perciformes, and a close relationship of cichlids and atherinomorphs.