Parapatric distribution of the lizards Plestiodon (formerly Eumeces) latiscutatus and P. japonicus (Reptilia: Scincidae) around the Izu Peninsula, Central Japan, and its biogeographic implications

The scincid lizard Plestiodon latiscutatus is found in the Izu Islands and Izu Peninsula of central Japan, whereas P. japonicus, a close relative, is found over the entire main island group of Japan, except the Izu Peninsula. The precise area of occupancy of these species was surveyed around the Izu Peninsula. Species identification was made through comparison of mitochondrial DNA partial sequences of specimens from the Izu Peninsula with those from the other regions, since morphological differences between these species have not yet been characterized. This study determined that these species are deeply diverged from each other in mitochondrial DNA sequence, and that the ranges of these species overlap only in a narrow zone. The results imply that gene flow between these species, if any, is restricted to a low level, without physical barriers. The boundary between the geographic ranges of these species was established as occurring along the lower Fuji River, Mt. Fuji, and the Sakawa River. This region is concordant with that of the old sea that is assumed to have separated the Izu Peninsula from other parts of the Japanese main island group until the middle Pleistocene. This pattern suggests that P. latiscutatus and P. japonicus were differentiated allopatrically before the connection of land areas of the Izu Peninsula and Honshu, the main island of Japan, and come into secondary contact through this connection. Thus, the species boundary is likely to have been maintained in situ, without physical barriers, since the secondary contact in the middle Pleistocene.     

Estimating absolute rates of molecular evolution and divergence times: a penalized likelihood approach.

Rates of molecular evolution vary widely between lineages, but quantification of how rates change has proven difficult. Recently proposed estimation procedures have mainly adopted highly parametric approaches that model rate evolution explicitly. In this study, a semiparametric smoothing method is developed using penalized likelihood. A saturated model in which every lineage has a separate rate is combined with a roughness penalty that discourages rates from varying too much across a phylogeny. A data-driven cross-validation criterion is then used to determine an optimal level of smoothing. This criterion is based on an estimate of the average prediction error associated with pruning lineages from the tree. The methods are applied to three data sets of six genes across a sample of land plants. Optimally smoothed estimates of absolute rates entailed 2- to 10-fold variation across lineages.     

Taxon sampling effects in molecular clock dating: an example from the African Restionaceae

Three commonly used molecular dating methods for correction of variable rates (non-parametric rate smoothing, penalized likelihood, and Bayesian rate correction) as well as the assumption of a global molecular clock were tested for sensitivity to taxon sampling. The test dataset of 6854 basepairs for 300 terminals includes a nearly complete sample of the Restio-clade of the African Restionaceae (272 of the 288 species), as well as 26 outgroup species. Of this, nested subsets of 35, 51, 80, 120, 150, and the full 300 species were used. Molecular dating experiments with these datasets showed that all methods are sensitive to undersampling, but that this effect is more severe in analyses that use more extreme rate smoothing. Additionally, the undersampling effect is positively related to distance from the calibration node. The combined effect of undersampling and distance from the calibration node resulted in up to threefold differences in the age estimation of nodes from the same dataset with the same calibration point. We suggest that the most suitable methods are penalized likelihood and Bayesian when a global clock assumption has been rejected, as these methods are more successful at finding optimal levels of smoothing to correct for rate heterogeneity, and are less sensitive to undersampling.     

r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock

SUMMARY: Estimating divergence times and rates of substitution from sequence data is plagued by the problem of rate variation between lineages. R8s version 1.5 is a program which uses parametric, nonparametric and semiparametric methods to relax the assumption of constant rates of evolution to obtain better estimates of rates and times. Unlike most programs for rate inference or phylogenetics, r8s permits users to convert results to absolute rates and ages by constraining one or more node times to be fixed, minimum or maximum ages (using fossil or other evidence). Version 1.5 uses truncated Newton nonlinear optimization code with bound constraints, offering superior performance over previous versions. AVAILABILITY: The linux executable, C source code, sample data sets and user manual are available free at http://ginger.ucdavis.edu/r8s.     

Comparative methods for the analysis of gene-expression evolution: an example using yeast functional genomic data

Understanding the evolution of gene function is a primary challenge of modern evolutionary biology. Despite an expanding database from genomic and developmental studies, we are lacking quantitative methods for analyzing the evolution of some important measures of gene function, such as gene-expression patterns. Here, we introduce phylogenetic comparative methods to compare different models of gene-expression evolution in a maximum-likelihood framework. We find that expression of duplicated genes has evolved according to a nonphylogenetic model, where closely related genes are no more likely than more distantly related genes to share common expression patterns. These results are consistent with previous studies that found rapid evolution of gene expression during the history of yeast. The comparative methods presented here are general enough to test a wide range of evolutionary hypotheses using genomic-scale data from any organism.     

Evaluating fossil calibrations for dating phylogenies in light of rates of molecular evolution: a comparison of three approaches

Evolutionary and biogeographic studies increasingly rely on calibrated molecular clocks to date key events. Although there has been significant recent progress in development of the techniques used for molecular dating, many issues remain. In particular, controversies abound over the appropriate use and placement of fossils for calibrating molecular clocks. Several methods have been proposed for evaluating candidate fossils; however, few studies have compared the results obtained by different approaches. Moreover, no previous study has incorporated the effects of nucleotide saturation from different data types in the evaluation of candidate fossils. In order to address these issues, we compared three approaches for evaluating fossil calibrations: the single-fossil cross-validation method of Near, Meylan, and Shaffer (2005. Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles. Am. Nat. 165:137-146), the empirical fossil coverage method of Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibration points. Am. Nat. 171:726-742), and the Bayesian multicalibration method of Sanders and Lee (2007. Evaluating molecular clock calibrations using Bayesian analyses with soft and hard bounds. Biol. Lett. 3:275-279) and explicitly incorporate the effects of data type (nuclear vs. mitochondrial DNA) for identifying the most reliable or congruent fossil calibrations. We used advanced (Caenophidian) snakes as a case study; however, our results are applicable to any taxonomic group with multiple candidate fossils, provided appropriate taxon sampling and sufficient molecular sequence data are available. We found that data type strongly influenced which fossil calibrations were identified as outliers, regardless of which method was used. Despite the use of complex partitioned models of sequence evolution and multiple calibrations throughout the tree, saturation severely compressed basal branch lengths obtained from mitochondrial DNA compared with nuclear DNA. The effects of mitochondrial saturation were not ameliorated by analyzing a combined nuclear and mitochondrial data set. Although removing the third codon positions from the mitochondrial coding regions did not ameliorate saturation effects in the single-fossil cross-validations, it did in the Bayesian multicalibration analyses. Saturation significantly influenced the fossils that were selected as most reliable for all three methods evaluated. Our findings highlight the need to critically evaluate the fossils selected by data with different rates of nucleotide substitution and how data with different evolutionary rates affect the results of each method for evaluating fossils. Our empirical evaluation demonstrates that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages.     

Genetic divergence and larval dispersal in two spider crabs (Crustacea: Decapoda)

The spider crabs Inachus dorsettensis (Pennant) and Hyas coarctatus Leach are widespread in subtidal areas of muddy sand or gravel around western Europe. Both species have a life cycle with an obligatory planktonic larval phase of several weeks, which might be expected to cause widespread larval dispersal and consequent genetic homogeneity over considerable distances. However, earlier work on both taxa has indicated differences in growth pattern between populations separated by tens of kilometres. This study was undertaken to determine whether these differences were purely environmental or whether, despite the short distances involved, differences may have a genetic basis. A study of gene frequencies, as indicated by allozymes in samples of adults collected off the Isle of Man (northern Irish Sea), indicates significant genetic differentiation between populations over a geographical distance of only about 40 km in both Inachus dorsettensis ( = 0.086 ± 0.048) and Hyas coarctatus ( = 0.023 ± 0.017). Variability measures differed between species, showing I. dorsettensis to have a mean number of alleles per locus of 2.5–2.6 and a range of gene diversity of 0.216–0.241, while H. coarctatus showed lower values of mean number of alleles (1.9–2.0) and a range of gene diversity from 0.122 to 0.124. Given the high expected larval mobility of the two species the results are most surprising. Possible explanations are discussed in relation to population discontinuities and patterns of larval drift.     

Phylogeny of hoplocercine lizards (Squamata: Iguania) with estimates of relative divergence times

Hoplocercine lizards form a clade of 11 currently recognized species traditionally placed in three genera (Enyalioides, Hoplocercus, and Morunasaurus) that occur in the lowlands on both sides of the Andes between Panama and the Brazilian Cerrado. We analyze 11 mitochondrial and two nuclear loci using probabilistic methods and different partitioning strategies to (1) infer the phylogenetic relationships among species of Hoplocercinae, (2) examine amounts of inter- and intraspecific sequence divergence, (3) address monophyly of four species, (4) test previous phylogenetic hypotheses, and (5) estimate divergence times. Our preferred hypothesis places H. spinosus as the sister taxon to all other species of hoplocercines, with M. annularis nested within Enyalioides. Species with multiple samples are monophyletic except for Enyalioides oshaughnessyi, which is paraphyletic relative to an undescribed species of Enyalioides. All previously published phylogenetic hypotheses for hoplocercines are rejected. Monophyly of Enyalioides cannot be rejected and, consequently, the position of Morunasaurus remains unclear. The most recent common ancestor of Hoplocercinae probably occurred east of the Andes; western taxa included in our analyses originated from at least two separate colonizations whether pre- or post-dating vicariance resulting from uplift of the Andes.     

Inferences from protein and nucleic acid sequences: Early molecular evolution,divergence of kingdoms and rates of change

Presently the sequences of more than 150 different kinds of proteins and nucleic acids are known from the many thousands thought to exist in all living creatures. Some few of these have occpied much the same functional niche within the living cell from near the beginning of life. In three of these latter, sequence evidence pointing to duplications of genetic material in a primitive ancestor is available and in the fourth other evidence suggests it. Such a duplication, shared by the many descendant species, permits us to locate the point of earliest time on an evolutionary tree and to infer the actual order of subsequent evolutionary events. The amounts of change which have occurred in each descendant line can be estimated with good confidence. Some inferences can be made of the structure of the ancestral duplicated sequence, the evolutionary mechanisms which have been operative on it, and the functional capacity of the organism in which it originated. We will describe new, sensitive, objective methods for establishing the probable common ancestry of very distantly related sequences and the quantitative evolutionary change which has taken place. These methods will be applied to the four families, and evolutionary trees will be derived where possible. Of the three families containing duplications of genetic material, two are nucleic acids: transfer RNA and 5S ribosomal RNA. Both of these structures are functional in the synthesis of coded proteins, and prototypes must have been present in the cell at the inception of the fundamental coding process that all living things share. There are many types of tRNA which recognise the various nucleotide triplets and the 20 amino acids. These types are thought to have arisen as a result of many gene duplications. Relationships among these types will be discussed. The 5S ribosomal RNA, presently functional in both eukaryotes and prokaryotes, is very likely descended from an early form incorporating almost a complete duplication of genetic material. The amount of evolution in the various lines can again be compared. The other two families containing duplications are proteins: ferredoxin and cytochrome c. Ferredoxin from photosynthetic and nonphotosynthetic bacteria shows clear evidence of a duplication of genetic material. This duplication is very possibly shared by the ferredoxin from plant plastids and the related adrenodoxin from mammalian mitochondria. If so, a chronology of the detalls of evolution of these groups can be inferred. From these examples of protein and nucleic acid sequence, we conclude that the amount of change in the bacterial lines is less than that in the eukaryote lines. Even though mutant bacteria are easily produced in the laboratory, though their evolutionary adaptation to new drugs is very rapid, and though new virulent strains often appear spontaneously, nevertheless the sequences of ancient structures in the wild types have changed less than those in the eukaryote lines. Cytochrome c sequences from many eukaryotes and the closely related cytochrome c₂ fromRhodospirillum rubrum are known. Other types of cytochrome, such as c₅₅₁ and c₅₅₃, are probably related to these through gene duplication. Knowledge of enough of these structures to establish an early duplication will provide a time orientation for the cytochrome c evolutionary tree. This quantitative tree now contains sequences from animals, fungi, green plants, protozoa, and bacteria, examples from all five biological kingdoms.     

Shifting sands and shifty lizards: molecular phylogeny and biogeography of African flat lizards (Platysaurus)

The African flat lizard genus Platysaurus is widely distributed on rock outcrops in southern Africa and is found both east and west of the Kalahari Desert and between major river drainage systems. We assembled a molecular phylogeny for the genus in order to test several biogeographic hypotheses. Sequence data were obtained from 29 specimens representing 14 taxa of Platysaurus that span the geographic range of the genus. We targeted a fragment of the mitochondrial genome comprising the 3' half of the ND4 gene and most of the flanking tRNA-HSL cluster. The edited alignment comprised 864 characters, of which 479 (55%) were variable and 461 (96%) parsimony informative. Overall, the phylogeny was well resolved and supported by high bootstrap values. Four major clades were identified comprising two to seven species: P. mitchelli and P. maculatus maculatus from the north-eastern range of the genus; P. broadleyi and P. capensis from the western range; P. imperator, P. torquatus, and P. intermedius rhodesianus; P. i. intermedius, P. monotropis, P. minor, P. i. nigrescens, P. lebomboensis, P. i. wilhelmi, and P. o. orientalis. Platysaurus has been suggested to represent a recent adaptive radiation where rapid speciation was fuelled by population fragmentation brought on by vicariant events and possibly divergent sexual selection. The traditional explanation for the radiation of the genus is that the eastern migration of the Kalahari sands fragmented populations in the Plio-Pleistocene, resulting in conditions favorable for speciation. Our genetic data strongly suggests that many of the speciation events in Platysaurus already had occurred prior to the Plio-Pleistocene. Moreover, vicariant events associated with the formation of the major river systems played an additional role in the evolution and distribution of Platysaurus species. Our topology displays long internodes and long terminal branches, suggesting that the radiation is much older than previously believed.     

Evolutionary divergence of the ribosomal internal transcribed spacer 2 (ITS2) in lizards

During the pre-rRNA cleavage pathway, the excision of ITS2, a eukaryotic specific insertion, remains the most elusive processing step, even in yeast. Comparison of the ITS2 sequences in different organisms permits to reveal conservative, presumably functionally important elements as well as obtain new information about ITS2 divergence in evolution. We have cloned and sequenced the ITS2 of three lizard species, Agama caucasia (Agamidae), Darevskia armeniaca and Lacerta strigata (Lacertidae) and detected in them a set of specific and conservative structural elements employing secondary structure consensus for vertebrate ITS2. Furthermore, we have performed an alignment and comparative analysis of the ITS2 sequences for the two lizards families. It enables us to propose that modern lizard species formation in evolution was accompanied by ITS2 duplication in the rDNA of their common progenitors.     

Molecular divergence and phylogeny: rates and patterns of cytochrome b evolution in cranes

Analyses of complete cytochrome b sequences from all species of cranes (Aves: Gruidae) reveal aspects of sequence evolution in the early stages of divergence. These DNA sequences are > or = 89% identical, but expected departures from random substitution are evident. Silent, third- position pyrimidine transitions are the dominant substitution type, with transversion comprising only a small fraction of sequence differences. Substitution patterns are not clearly manifested until divergence has reached a moderate level (> 3%), as expected for a stochastic process. Variation in the frequency of mismatch types among lineages decreases at larger divergences, but the level of bias does not decay. Divergence varies up to fivefold among gene regions but is not correlated with structural domain. All protein structural domains except extramembrane 4 display < 20% variable residues. Regions corresponding to putative functional domains show the excepted conservation of amino acids, although the C-terminal portion of the Q0 reaction center displays several nonconservative replacements. Phylogenetic analyses incorporating substitution asymmetries produced mixed results. Distances estimated with multiple parameters (transition, codon-position, composition, and pyrimidine-transition biases) yielded identical additive tree topologies with comparable bootstrap values, all consistent with uncontroversial species relationships. Maximum likelihood analysis incorporating these biases, as well as equally weighted parsimony analysis, produced similar results. Static, differential weighting for parsimony did not improve the phylogenetic signal but produced unusual trees with low bootstraps. The overall rate of nucleotide substitution varies slightly but significantly among cranes, and calibration of distances against fossil dates suggests divergence rates of 0.7%-1.7% per million years.      

Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods

Among Polystomatidae (Monogenea), the genus Polystoma, which mainly infests neobatrachian hosts, is the most diverse and occurs principally in Africa, from where half the species have been reported. Previous molecular phylogenetic studies have shown that this genus originated in South America, and later colonised Eurasia and Africa. No mention was made on dispersal corridors between Europe and Africa or of the origin of the African Polystoma radiation. Therefore, a molecular phylogeny was inferred from ITS1 sequences of 21 taxa comprising two species from America, seven representatives from Europe and 12 from Africa. The topology of the phylogenetic tree reveals that a single event of colonisation took place from Europe to Africa and that the putative host carrying along the ancestral polystome is to be found among ancestral pelobatids. Percentage divergences estimates suggest that some presumably distinct vesicular species in unrelated South African anurans and some neotenic forms found in several distinct hosts in Ivory Coast, could, in fact, belong to two single polystome species parasitising divergent hosts. Two main factors are identified that may explain the diversity of African polystomes: (i), we propose that following some degree of generalism, at least during the juvenile stages of both hosts and parasites, distinctive larval behaviour of polystomes engenders isolation between parasite populations that precludes sympatric speciations; (ii), cospeciation events between Ptychadena hosts and their parasites are another factor of diversification of Polystoma on the African continent. Finally, we discuss the systematic status of the Madagascan parasite Metapolystoma, as well as the colonisation of Madagascar by the host Ptychadena mascareniensis.     

The effects of fossil placement and calibration on divergence times and rates: An example from the termites (Insecta: Isoptera)

Among insects, eusocial behavior occurs in termites, ants, some bees and wasps. Isoptera and Hymenoptera convergently share social behavior, and for both taxa its evolution remains poorly understood. While dating analyses provide researchers with the opportunity to date the origin of eusociality, fossil calibration methodology may mislead subsequent ecological interpretations. Using a comprehensive termite dataset, we explored the effect of fossil placement and calibration methodology. A combined molecular and morphological dataset for 42 extant termite lineages was used, and a second dataset including these 42 taxa, plus an additional 39 fossil lineages for which we had only morphological data. MrBayes doublet-model analyses recovered similar topologies, with one minor exception (Stolotermitidae is sister to the Hodotermitidae, s.s., in the 42-taxon analysis but is in a polytomy with Hodotermitidae and (Kalotermitidae + Neoisoptera) in the 81-taxon analysis). Analyses using the r8s program on these topologies were run with either minimum/maximum constraints (analysis a = 42-taxon and analysis c = 81-taxon analyses) or with the fossil taxon ages fixed (ages fixed to be the geological age of the deposit from which they came, analysis b = 81-taxon analysis). Confidence intervals were determined for the resulting ultrametric trees, and for most major clades there was significant overlap between dates recovered for analyses A and C (with exceptions, such as the nodes Neoisoptera, and Euisoptera). With the exception of isopteran and eusiopteran node ages, however, none of the major clade ages overlapped when analysis B is compared with either analysis A or C. Future studies on Dictyoptera should note that the age of Kalotermitidae was underestimated in absence of kalotermitid fossils with fixed ages.     

Cranial kinesis in lizards (Lacertilia): Origin,biomechanics, and evolution

Various methods of investigation of cranial kinesis are compared. The biomechanical model of the amphikinetic cranial mechanism of lizards developed by Frazzetta (1962) corresponds to the skulls of species with the dependent streptostyly. A new modification of this model is proposed for species with the independent streptostyly, which conforms to the results of experimental investigations of cranial kinesis in living lizards. The lacertilian amphikinesis has developed on the basis of the pleurokinesis inherited from fish ancestors of tetrapods. Movable connections of the maxillo-buccal segments with the axial skull persisted in the amphikinetic skull and were completed by the transversal flexible connections in the dermatocranial roof and loose connections of dermatocranium with the braincase. The development of new movable intracranial connections could have been preceded by transformations in the jaw musculature (formation of the pterygoideus muscle and inclined position of the external jaw adductor), which caused longitudinal jaw movements. Development of new movable connections within the skull was triggered by paedomorphosis processes. In various lacertilian groups, the cranial kinesis was improved by the development of various forms of streptostyly and flexipalatality.     

Arrival of an Australian anguillid eel in New Zealand: an example of transoceanic dispersal

Anguilla reinhardtii, hitherto known from eastern Australia, New Caledonia, Norfolk Island, and Lord Howe Island, has recently been discovered in rivers of northern New Zealand. Identification, based on morphological and genetic characteristics, is unequivocal; eight consecutive year classes have been found. The only reasonable explanation of this occurrence is transoceanic dispersal to New Zealand, probably from subtropical oceanic spawning grounds north of New Zealand. This corroborates past hypotheses that the strongly diadromous freshwater fish fauna of New Zealand is derived by transoceanic dispersal of known marine life intervals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles

Although still controversial, estimation of divergence times using molecular data has emerged as a powerful tool to examine the tempo and mode of evolutionary change. Two primary obstacles in improving the accuracy of molecular dating are heterogeneity in DNA substitution rates and accuracy of the fossil record as calibration points. Recent methodological advances have provided powerful methods that estimate relative divergence times in the face of heterogeneity of nucleotide substitution rates among lineages. However, relatively little attention has focused on the accuracy of fossil calibration points that allow one to translate relative divergence times into absolute time. We present a new cross-validation method that identifies inconsistent fossils when multiple fossil calibrations are available for a clade and apply our method to a molecular phylogeny of living turtles with fossil calibration times for 17 of the 22 internal nodes in the tree. Our cross-validation procedure identified seven inconsistent fossils. Using the consistent fossils as calibration points, we found that despite their overall antiquity as a lineage, the most species-rich clades of turtles diversified well within the Cenozoic. Many of the truly ancient lineages of turtles are currently represented by a few, often endangered species that deserve high priority as conservation targets.     

The evolution of age-specific mortality rates in Drosophila melanogaster: genetic divergence among unselected lines.

Age-specific effects of spontaneous mutations on mortality rates in Drosophila are inferred from three large demographic experiments. Data were collected from inbred lines that were allowed to accumulate spontaneous mutations for 10, 19, and 47 generations. Estimates of age-specific mutational variance for mortality were based on data from all three experiments, totalling approximately 225,000 flies, using a model developed for genetic analysis of age-dependent traits (the character process model). Both within- and among-generation analyses suggest that the input of genetic variance is greater for early life mortality rates than for mortality at older ages. In females, age-specific mutational variances ranged over an order of magnitude from 5.96 x 10(-3) at 2 wk posteclosion to 0.02 x 10(-3) at 7 wk. The male data show a similar pattern. Age-specific genetic variances were substantially less at generation 47 than at generation 19-an unexplained observation that is likely due to block effects. Mutational correlations among mortality rates at different ages tend to increase with the accumulation of new mutations. Comparison of the mutation-accumulation lines at generations 19 and 47 with their respective control lines suggests little age-specific mutational bias.