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.     

Multiple data sets, high homoplasy, and the phylogeny of softshell turtles (Testudines: Trionychidae)

We present a phylogenetic hypothesis and novel, rank-free classification for all extant species of softshell turtles (Testudines:Trionychidae). Our data set included DNA sequence data from two mitochondrial protein-coding genes and a approximately 1-kb nuclear intron for 23 of 26 recognized species, and 59 previously published morphological characters for a complimentary set of 24 species. The combined data set provided complete taxonomic coverage for this globally distributed clade of turtles, with incomplete data for a few taxa. Although our taxonomic sampling is complete, most of the modern taxa are representatives of old and very divergent lineages. Thus, due to biological realities, our sampling consists of one or a few representatives of several ancient lineages across a relatively deep phylogenetic tree. Our analyses of the combined data set converge on a set of well-supported relationships, which is in accord with many aspects of traditional softshell systematics including the monophyly of the Cyclanorbinae and Trionychinae. However, our results conflict with other aspects of current taxonomy and indicate that most of the currently recognized tribes are not monophyletic. We use this strong estimate of the phylogeny of softshell turtles for two purposes: (1) as the basis for a novel rank-free classification, and (2) to retrospectively examine strategies for analyzing highly homoplasious mtDNA data in deep phylogenetic problems where increased taxon sampling is not an option. Weeded and weighted parsimony, and model-based techniques, generally improved the phylogenetic performance of highly homoplasious mtDNA sequences, but no single strategy completely mitigated the problems of associated with these highly homoplasious data. Many deep nodes in the softshell turtle phylogeny were confidently recovered only after the addition of largely nonhomoplasious data from the nuclear intron.     

Molecular evidence for a clade of turtles.

Although turtles have been generally grouped with the most primitive reptile species, the origin and phylogenetic relationships of turtles have remained unresolved to date. To confirm the phylogenetic position of turtles in amniotes, we have cloned and determined the cDNA sequences encoding for skink lactate dehydrogenase (LDH)-A and LDH-B, snake LDH-A, and African clawed frog LDH-A; four alpha-enolase cDNA sequences from turtle, alligator, skink, and snake were also cloned and determined. All of these eight cDNA sequences, as well as the previously published LDH-A, LDH-B, and alpha-enolase of mammals, birds, reptiles, and African clawed frog, were analyzed by the phylogenetic tree reconstruction methods of neighbor-joining, maximum parsimony, and maximum likelihood. In the phylogenetic analyses, the turtle was found to be closely related to the alligator. Also, we found that the turtle had diverged after the divergence of squamates and birds. This departs from previous hypotheses of turtle evolution and further suggests that turtles are the latest of divergent reptiles, having been derived from an ancestor of crocodilian lineage within the last 200 million years.     

Phylogenetic utility of 141 low-copy nuclear regions in taxa at different taxonomic levels in two distantly related families of rosids

Angiosperm systematics has progressed to the point where it is now expected that multiple, independent markers be used in phylogenetic studies. Universal primers for amplifying informative regions of the chloroplast genome are readily available, but in the faster-evolving nuclear genome it is challenging to discover priming sites that are conserved across distantly related taxa. With goals including the identification of informative markers in rosids, and perhaps other angiosperms, we screened 141 nuclear primer combinations for phylogenetic utility in two distinct groups of rosids at different taxonomic levels-Psiguria (Cucurbitaceae) and Geraniaceae. We discovered three phylogenetically informative regions in Psiguria and two in Geraniaceae, but none that were useful in both groups. Extending beyond rosids, we combined our findings with those of another recent effort testing these primer pairs in Asteraceae, Brassicaceae, and Orchidaceae. From this comparison, we identified 32 primer combinations that amplified regions in representative species of at least two of the five distantly related angiosperm families, giving some prior indication about phylogenetic usefulness of these markers in other flowering plants. This reduced set of primer pairs for amplifying low-copy nuclear markers along with a recommended experimental strategy provide a framework for identifying phylogenetically informative regions in angiosperms.     

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.     

Marine turtle mitogenome phylogenetics and evolution

The sea turtles are a group of cretaceous origin containing seven recognized living species: leatherback, hawksbill, Kemp's ridley, olive ridley, loggerhead, green, and flatback. The leatherback is the single member of the Dermochelidae family, whereas all other sea turtles belong in Cheloniidae. Analyses of partial mitochondrial sequences and some nuclear markers have revealed phylogenetic inconsistencies within Cheloniidae, especially regarding the placement of the flatback. Population genetic studies based on D-Loop sequences have shown considerable structuring in species with broad geographic distributions, shedding light on complex migration patterns and possible geographic or climatic events as driving forces of sea-turtle distribution. We have sequenced complete mitogenomes for all sea-turtle species, including samples from their geographic range extremes, and performed phylogenetic analyses to assess sea-turtle evolution with a large molecular dataset. We found variation in the length of the ATP8 gene and a highly variable site in ND4 near a proton translocation channel in the resulting protein. Complete mitogenomes show strong support and resolution for phylogenetic relationships among all sea turtles, and reveal phylogeographic patterns within globally-distributed species. Although there was clear concordance between phylogenies and geographic origin of samples in most taxa, we found evidence of more recent dispersal events in the loggerhead and olive ridley turtles, suggesting more recent migrations (<1Myr) in these species. Overall, our results demonstrate the complexity of sea-turtle diversity, and indicate the need for further research in phylogeography and molecular evolution.     

Turtle phylogeny: insights from a novel nuclear intron

Introns have gained considerable popularity as markers for molecular phylogenetics. However, no primers exist for a nuclear intron that amplifies across all turtles. Available data from morphology and mitochondrial DNA have not unambiguously resolved relationships within the superfamily Trionychoidea and the family Chelidae, which together form a large portion of extant turtle diversity. We tested the phylogenetic utility of a novel intron from the RNA fingerprint protein 35 (R35) as applied to these two areas of turtle systematics. We found the intron to be a single-copy locus that provides excellent resolving power for lineages among turtles, though problems with alignment made it impossible to infer deeper amniote relationships. Maximum parsimony and maximum likelihood both demonstrated the polyphyly of Trionychoidea and the reciprocal monophyly of Australian/New Guinea and South American chelid turtles. This is the first study to resolve such relationships with strong statistical support, and we suggest that R35 holds great promise for resolving additional persistent problems in the phylogeny of living turtles.     

Relationships among turtles of the genus Clemmys (Reptilia, Testudines, Emydidae) as suggested by plastron scute morphology

The Conservative morphology of hardshelled turtles has fostered the use of size relationships between epidermal scutes (scales) on the shell to differentiate between species and subspecies of many taxa. The size relationship of the six major pairs of plastral scutes were used to compare the four currently recognized species of the genus Clemmys with each other. as well as with the distantly related Graptemys barbouri using Jaccard Coefficients. Shannon-Weiner diversity indices, and multivariate analysis. Results were concordant among the three techniques used and confirm our prediction that plastral morphology varies little among closely related species and widely among distantly related taxa. Clemmys muhlenbergii appears to he more different from Clemmys guttata than previously suggested. Analysis of plastral morphology shows promise as a taxonomic tool for turtle systematists.     

New primers for single-copy nuclear-encoded chloroplast-expressed glutamine synthetase (ncpGS) in Oxalidaceae

? Premise of the study: New primers were developed for the nuclear marker glutamine synthetase (ncpGS) in Oxalidaceae. ? Methods and Results: New forward and reverse primers were designed and tested across a wide range of Oxalidaceae. Selected taxa were sequenced to confirm homology. Potential for phylogenetic study was assessed by comparing sequenced taxa with commonly used nuclear and plastid markers. ? Conclusions: Four out of five Oxalidaceae genera and all tested Oxalis spp. amplified successfully. Sequencing confirmed homology of the amplicon. Parsimony analysis of ncpGS showed that it is a promising candidate for future phylogenetic work in Oxalidaceae.     

Development of eight mitochondrial markers for Funariaceae (Musci) and their amplification success in other mosses

? Premise of the study: In comparison to the wide use of chloroplast markers, few mitochondrial markers are available for phylogenetic studies in bryophytes. We investigated the phylogenetic suitability of several mtDNA markers within the Funariaceae and across mosses. ? Methods and Results: By comparing mitochondrial genomes of two mosses, eight regions with higher substitution rates were identified and sequenced for three species in the Funariaceae and one outgroup taxon. Variations in the substitution rate of these new loci were compared to previously used markers. Thirty-four samples representing all major moss lineages were targeted to assess the universality of the newly designed primers. ? Conclusions: The new markers provided similar or more sequence variations in Funariaceae compared to previously developed mtDNA markers. Five out of eight loci were amplified in 70% of other taxa, indicating that these markers may be suitable for phylogenetic studies in other moss lineages.     

The complete mitochondrial genome of the Korean soft-shelled turtle Pelodiscus sinensis (Testudines, Trionychidae).

We isolated Korean soft-shelled turtle, Pelodiscus sinensis, mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitochondrial genome (mitogenome) with primer walking using flanking sequences. The P. sinensis mitochondrial DNA has 17,042 bp and its structural organization is conserved compared to those of other reptiles and mammals. To unveil the phylogenetic relationship of the turtles, we used the NJ, MP, and ML analysis methods after inferring those sequences from the mitochondrial 16S rRNA gene. We also compared two P. sinensis variants from Korea and China using the mitochondrial genome. In this study, we report the basic characteristics of the P. sinensis mitochondrial genome, including structural organization and base composition of the rRNAs, tRNAs and protein-coding genes, as well as characteristics of tRNAs. These features are applicable for the study of phylogenetic relationships in turtles.     

Isolation and characterization of twelve polymorphic microsatellite markers in bottlenose dolphins (Tursiops truncatus)

We developed polymerase chain reaction primers for 12 dinucleotide microsatellite loci in the bottlenose dolphin, Tursiops truncatus. Seven markers were obtained after hybridization screening, and five following random genome sequencing. Orthologous positions were computed for nine markers on the bovine genome and for seven on the human genome. The markers are distributed across chromosomes and found in different types of DNA regions. All 12 loci are polymorphic for Tursiops. Five loci were also polymorphic in the related species Stenella frontalis and the more distantly related river dolphin, Inia geoffrensis, indicating these markers will be informative across the Delphinidae and other cetacean taxa.     

Multiple genome alignments facilitate development of NPCL markers: a case study of tetrapod phylogeny focusing on the position of turtles

In recent years, the increasing availability of genomic resources has provided an opportunity to develop phylogenetic markers for phylogenomics. Efficient methods to search for candidate markers from the huge number of genes within genomic data are particularly needed in the era of phylogenomics. Here, rather than using the traditional approach of comparing genomes of two distantly related taxa to develop conserved primers, we take advantage of the multiple genome alignment resources from the the University of California-San Cruz Genome Browser and present a simple and straightforward bioinformatic approach to automatically screen for candidate nuclear protein-coding locus (NPCL) markers. We tested our protocol in tetrapods and successfully obtained 21 new NPCL markers with high success rates of polymerase chain reaction amplification (mostly over 80%) in 16 diverse tetrapod taxa. These 21 newly developed markers together with two reference genes (RAG1 and mitochondrial 12S-16S) are used to infer the higher level relationships of tetrapods, with emphasis on the debated position of turtles. Both maximum likelihood (ML) and Bayesian analyses on the concatenated data combining the 23 markers (21,137 bp) yield the same tree, with ML bootstrap values over 95% and Bayesian posterior probability equaling 1.0 for most nodes. Species tree estimation using the program BEST without data concatenation produces similar results. In all analyses, turtles are robustly recovered as the sister group of Archosauria (birds and crocodilians). The jackknife analysis on the concatenated data showed that the minimum sequence length needed to robustly resolve the position of turtles is 13-14 kb. Based on the large 23-gene data set and the well-resolved tree, we also estimated evolutionary timescales for tetrapods with the popular Bayesian method MultiDivTime. Most of the estimated ages among tetrapods are similar to the average estimates of the previous dating studies summarized by the book The Timetree of Life.     

Pareiasaur phylogeny and the origin of turtles

The evolutionary relationship of all the valid species (and thus genera) of pareiasaurs are assessed through a phylogcnctic analysis of these taxa together with turtles, Owenetta, Barasaurus, Sclerosaurus, procolophonids, lanthanosuchids, nyctiphruretids, and nycterolctcrids. 128 os-teological characters were used, and almost all relevant taxa were examined. The results confirm that among these taxa, pareiasaurs and turtles form a robust clade, to the exclusion of all other taxa including procolophonids. However, pareiasaurs might not be the mono-phyletic sister group of turtles, as previously suggested. Rather, there is some evidence that pareiasaurs are paraphyletic with respect to (i.e. ‘ancestral to’) turtles. Among pareiasaurs, the early, large, heavily ossified forms such as Brady.saurus are most distantly related to turtles. These forms are characterized by rather smooth skulls, and dermal armour restricted to the dorsal midline. More closely related to turtles are forms such as Scutosaurus, Pareiasuchus, and Elginia. These taxa form a distinct clade of pareiasaurs, characterized by a very ‘mammallike’ pelvis, elaborate cranial ornamentation and a loose covering of osteoderms over the entire dorsum. The late, dwarf pareiasaurs Nanoparia, Anthodon, and Pumiliopareia are the nearest relatives of turtles. These forms exhibit otherwise uniquely turtle features such as a rigid covering of dermal armour over the entire dorsal region, expanded flattened ribs, cylindrical scapula blade, great reduction of humeral torsion (to 25^o), greatly developed trochanter major, offset femoral head, and reduced cnemial crest of the tibia. Thus, many features thought to be restricted to turtles (and thus to have evolved simultaneously with the turtle shell) actually arose earlier, at various points along the pareiasaurian stem lineage. The identification of the nature and sequence of anatomical changes leading to the origin of turtles, and the possibility that turtles are derived from dwarf pareiasaurs, should have important implications for speculations on the evolutionary biology of turtle origins.     

A Paedomorphic parasite associated with a neotenic amphibian host: phylogenetic evidence suggests a revised systematic position for Sphyranuridae within anuran and turtle Polystomatoineans

The phylogenetic relationships of the families Polystomatidae and Sphyranuridae (subclass Polystomatoinea) within tetrapod monogenean parasites were investigated using partial 18S rDNA sequences. About 600 nucleotides of 11 species were sequenced, including 7 species of the most common subfamilies of Polystomatidae found in anurans and turtles, 1 species of the family Sphyranuridae parasitizing exclusively urodelans, and 3 species of the subclass Oligonchoinea infesting teleostean fishes. The phylogenetic analyses were performed using three reconstruction methods: neighbor-joining, maximum-parsimony, and maximum-likelihood. Polystomatoineans but not polystomatids were shown to be monophyletic. Within the polystomatoineans there are two clades: one includes the amphibian monogeneans (anuran polystomatids and urodelan sphyranurids) and the other includes the turtle polystomatids. Polystomatoineans may have coevolved with amphibian hosts, and an ancestral "polystome" dispersed at least 200 million years ago, either from the basal stem of lissamphibians or from an anuran ancestral stock, to freshwater turtles. Furthermore, the urodelan genus Sphyranura, initially assigned to the family Sphyranuridae on the basis of morphological and ontogenetic evidence, is clearly nested within polystomatids, suggesting that its systematic status must be revised. This supports recent findings which argue that species of the family Sphyranuridae may be paedomorphic parasites exclusively infesting neotenic mudpuppies.     

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.