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The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

The monophyly of European newts of the genus Triturus within the family Salamandridae has for decades rested on presumably homologous behavioral and morphological characters. Molecular data challenge this hypothesis, but the phylogenetic position of Triturus within the Salamandridae has not yet been convincingly resolved. We addressed this issue and the temporal divergence of Triturus within the Salamandridae with novel Bayesian approaches applied to DNA sequence data from three mitochondrial genes (12S, 16S and cytb). We included 38 salamandrid species comprising all 13 recognized species of Triturus and 16 out of 17 salamandrid genera. A clade comprising all the “Newts” can be separated from the “True Salamanders” and Salamandrina clades. Within the “Newts” well‐supported clades are: Tylototriton–Pleurodeles, the “New World Newts” (Notophthalmus–Taricha), and the “Modern Eurasian Newts” (Cynops, Pachytriton, Paramesotriton=together the “Modern Asian Newts”, Calotriton, Euproctus, Neurergus and Triturus species). We found that Triturus is a non‐monophyletic species assemblage, which includes four groups that are themselves monophyletic: (i) the “Large‐Bodied Triturus” (six species), (ii) the “Small‐Bodied Triturus” (five species), (iii) T. alpestris and (iv) T. vittatus. We estimated that the last common ancestor of Triturus existed around 64 million years ago (mya) while the root of the Salamandridae dates back to 95 mya. This was estimated using a fossil‐based molecular dating approach and an explicit framework to select calibration points that least underestimated their corresponding nodes. Using the molecular phylogeny we mapped the evolution of life history and courtship traits in Triturus and found that several Triturus‐specific courtship traits evolved independently. J. Exp. Zool. (Mol. Dev. Evol.) 306B, 2006. © 2006 Wiley‐Liss, Inc.

During the Pleistocene pygmy elephantids, some only a quarter of their ancestors' size, were present on Mediterranean islands until about 10000 years ago (y.a.). Using a new methodology for ancient DNA (aDNA) studies, the whole genomic multiple displacement amplification method, we were able to retrieve cytochrome b(cytb) DNA fragments from 4200 to 800000 y.a. specimens from island and mainland samples, including pygmy and normal-sized forms. The short DNA sequence (43bp) retrieved from the 800000 y.a. sample is one of the oldest DNA fragment ever retrieved. Duplication of the experiments in two laboratories, the occurrence of three diagnostic sites and the results of the phylogenetic analyses strongly support its authenticity. Our results challenge the prevailing view that pygmy elephantids of the eastern Mediterranean originated exclusively from Elephas, suggesting independent histories of dwarfism and the presence of both pygmy mammoths and elephant-like taxa on these islands. Based on our molecular data, the origin of the Tilos and Cyprus elephantids from a lineage within the genus Elephasis confirmed, while the DNA sequence from the Cretan sample falls clearly within the mammoth clade. Thus, the name Mammuthus creticusrather than Elephas creticus, seems to be justified for this form. Our findings also suggest a need to re-evaluate the evolutionary history of the Sicilian/Maltese species, traditionally included in the genus Elephas.

A cryptic taxon of Galápagos tortoise in conservation peril Michael A Russello1,*Scott Glaberman1James P Gibbs3Cruz Marquez4Jeffrey R Powell1Adalgisa Caccone1,21Department of Ecology and Evolutionary Biology, Yale UniversityNew Haven, CT 06520, USA 2Yale Institute for Biospheric Studies, Yale UniversityNew Haven, CT 06520, USA 3College of Environmental Sciences and Forestry, State University of New YorkSyracuse, NY 13210, USA 4Charles Darwin Research StationPuerto Ayora, Santa Cruz, Galápagos Islands, Ecuador *Author for correspondence (michael.russello@yale.edu) As once boldly stated, bad taxonomy can kill,highlighting the critical importance of accurate taxonomy forthe conservation of endangered taxa. The concept continues toevolve almost 15 years later largely because most legal protectionsaimed at preserving biological diversity are based on formaltaxonomic designations. In this paper we report unrecognizedgenetic divisions within the giant tortoises of the Galápagos.We found three distinct lineages among populations formerlyconsidered a single taxon on the most populous and accessibleisland of Santa Cruz; their diagnosability, degree of geneticdivergence and phylogenetic placement merit the recognitionof at least one new taxon. These results demonstrate the fundamentalimportance of continuing taxonomic investigations to recognizebiological diversity and designate units of conservation, evenwithin long-studied organisms such as Galápagos tortoises,whose evolutionary heritage and contribution to human intellectualhistory warrant them special attention.Geochelone nigra (elephantopus)giant tortoisesmicrosatellitesconservation geneticsphylogeographyhistorical DNA

Twenty years ago, the field of ancient DNA was launched with the publication of two short mitochondrial (mt) DNA sequences from a single quagga (Equus quagga) museum skin, an extinct South African equid (Higuchi et al. 1984Nature312, 282–284). This was the first extinct species from which genetic information was retrieved. The DNA sequences of the quagga showed that it was more closely related to zebras than to horses. However, quagga evolutionary history is far from clear. We have isolated DNA from eight quaggas and a plains zebra (subspecies or phenotype Equus burchelli burchelli). We show that the quagga displayed little genetic diversity and very recently diverged from the plains zebra, probably during the penultimate glacial maximum. This emphasizes the importance of Pleistocene climate changes for phylogeographic patterns in African as well as Holarctic fauna.

Four of the five tortoise species in Madagascar,Pyxis arachnoides, P. planicauda, Geochelone radiata,andG. yniphora,are endemic and on the verge of extinction. Their phylogenetic relationships remain controversial and unresolved. Here we address the phylogeny of this group using DNA sequences for the 12S and 16S rDNA and cytbgenes in mitochondrial DNA. As outgroups we used two species ofGeochelone, pardalis(mainland Africa) andnigra(Galápagos), as well as a more distant North American tortoise,Gopherus polyphemus.We conclude that the twoPyxisspecies are sister taxa and are imbedded in the genusGeochelone,rendering this latter genus paraphyletic. There is moderate support for the sister status of the two MadagascarGeocheloneand for the monophyletic origin of all four endemics, suggesting a single colonization of the island. The separation of Madagascar from other land masses (90–165 mya) predates the origin of the endemic tortoises (estimated to be 14–22 mya). This suggests founding by rafting, a process known to have occurred with other tortoises. The derived morphological divergence of thePyxisspecies in a relatively short period of time (13–20 my) stands in contrast to the notoriously slow rate of morphological evolution in most lineages of Chelonia.

Summary DNA-DNA hybridization studies of insects, more specificallyDrosophila and cave crickets, have revealed interesting patterns of genome evolution that contrast markedly with what has been seen in other taxa, especially mammals and birds. Insect genomes are composed of sections of single-copy DNA with extreme variation in rates of evolutionary change. This variation is more extreme than between introns and exons; introns fall into the relatively conserved fraction of the genome. Attempts to calculate absolute rates of change inDrosophila DNA have all led to estimates some 5–10 times faster than those found in most vertebrates; this is true even for the more conservative part of the nuclear genome. Finally we point out that morphological similarity, chromosomal similarity, and/or ability to form interspecific hybrids is often associated with quite high levels of single-copy DNA divergence in insects as compared to mammals and birds.

Summary This paper emanated from a conference concerning the value, accuracy, and technical considerations of DNA-DNA hybridization for evolutionary studies. Our laboratory has been performing the so-called TEACL (tetraethylammonium chloride) method, and we have amassed sufficient data to indicate that this method is very powerful if performed properly with correct analyses. Here we address five technical considerations: (1) We present empirical data that size correction for tracer length is legitimate and accurate. (2) We show that the error of ΔTm measurement does not significantly increase with increasing distance up to at least 10°C. (3) The error distribution for ΔTm does not deviate from the expected normal distribution indicating parametric statistics are probably legitimate for analyses. (4) Using a known phylogeny we examined the resolving power of the technique by showing that at least five taxa can be correctly placed in phylogenies with a maximum ΔTm of 2.5°C. (5) To data, all our data sets based on DNA-DNA hybridization are very robust with respect to analytical procedures in that every algorithm used on the data sets has yielded identical trees with nearly identical branch lengths. Nevertheless, we point out that theoretical analyses of distance data (as generated by DNA-DNA hybridization) are lacking, especially with regard to tests of the molecular clock hypothesis.

One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (ΔTm) to base pair mismatch have led to conflicting results. We have studied the ΔTm between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of ΔTm and percent base pair mismatch is remarkably linear over this range with a correlation coefficient >0.98. A ΔTm of 1°C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.

In this paper we attempt to investigate relationships between the amount of genetic divergence in nuclear genes and the degree of morphological differentiation for different sets of characters in Dolichopodacave crickets.Six populations representing five Dolichopodaspecies from Central and Southern Italy have been studied. The overall genetic divergence at nuclear genes was estimated both by single copy DNA-DNA hybridization and allozyme frequencies at 26 loci. Euclidean distances for two multivariate sets of morphometric variables: one describing body and appendage morphology, the other male epiphallus shape. Results showed a close agreement between the branching patterns of ?Tmvalues from DNA hybridization and Nei's allozyme distance values. On the other hand, patterns of morphological divergence revealed independent trends, although the branching pattern based on epiphallus morphology matched to some extent the phylogenies inferred from molecular data. The relative value of molecular and morphological characters as reliable phylogenetic tracers was evaluated in relation to their dependence on evolutionary factors.Implications of these findings on the calibration of molecular clocks are also discussed. The absolute rate of molecular change based on scDNA was estimated to be at least 0.98% divergence/my/lineage. This result is in agreement with calibrations attempted on other insects. Estimates of time of divergence based on allozymes (Nei's D) were highly consistent with the estimate from geological data.

Using DNA–DNA hybridization, we have determined the degree of single‐copy DNA (scDNA) divergence among eight species of the Drosophila obscuragroup. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinissubgroup are more closely related to American members of the obscurasubgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔTmof 6.5°C can form hybrids whereas species separated by a ΔTmof 2.5°C cannot. As with other groups of Drosophila, the obscuragroup has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscuraand the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔTmof 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscuragroup may be evolving considerably slower than in the melanogastersubgroup.

The problem of the phylogenetic relationships of the extant members of Hominoidea is a long-standing one in evolution and anthropology. Morphological, fossil, and other more traditional data led to a variety of proposed trees with no clear consensus. It was hoped that molecular data, which should be an independent source of information, would shed light on the problem. Until recently these hopes have not been realized. However, two sources of molecular data have recently been obtained which appear to solve the problem with reasonable assurance. These data involve total measurement of DNA divergence among taxa using DNA-DNA hybridization and more detailed sequence data of a globin pseudogene. The theory behind the use of each kind of data for solving the Hominoidea problem is discussed as well as the problem of species phylogenies and gene phylogenies. The main phylogenetic conclusion is that man and chimpanzees are the most closely related extant primates, followed by gorillas, orangutans, and gibbons. Old World monkeys, the Cercophethecines, are the sister taxon to man and African apes.

We have determined the degree of single‐copy DNA divergence among the extant members of the Hominoidea employing the technique of DNA‐DNA hybridization. The species studied include humans, two species of chimpanzees, gorillas, two subspecies of orangutans, and two species of gibbons; as an outgroup we have used a member of the Old World monkeys (Cercopithecidae), the baboon. Our methods are different from those previously used and allow us to control for two factors other than base‐pair mismatch that can affect the thermal stability of DNA duplexes: the base composition and duplex length. In addition, we have studied more than one individual for most species and thus are able to assess the effect of intraspecific variation on phylogenetic conclusions. The results indicate that the closest extant relatives of humans are the chimpanzees. Gorillas are the next closest, followed by orangutans and gibbons. This result is strongly supported statistically, as there is virtually no overlap in measurements between different taxa. Our conclusions are in agreement with a growing amount of molecular evidence supporting this pattern of relatedness. The data behave as a reasonably good molecular clock, and we do not see an indication of slowdown in molecular evolution in the clade containing humans and African apes, contrary to what has been documented for protein‐coding regions. Because of the clocklike nature of the results, we have estimated that the divergence of humans and chimpanzees occurred about 6–8 million years ago. Results from orangutans indicate that the Borneo and Sumatra populations are genetically distinct, about as different as the named species of chimpanzees.

Single‐copy DNA divergence among 23 populations of cave crickets belonging to two genera (Euhadenoecusand Hadenoecus) has been determined by DNA‐DNA hybridization employing the TEACL method. These same populations have been studied for allozyme variation (Caccone and Sbordoni, 1987). In addition, a European relative (Dolichopoda laetitiae) has been included as an outgroup for rooting the phylogeny. One of the most remarkable findings is the large degree of DNA divergence among these species and populations. A ΔTmof up to 5°C has been found between populations of the same species; even further divergence is indicated by a lowered normalized percentage of reassociation. A phylogeny was constructed and tested for synchrony of rates, i.e., a molecular clock. Statistically, we could not reject the clock hypothesis. Attempts to calibrate the clock led to the conclusion that these insects are among the fastest evolving (with respect to single‐copy DNA) groups yet studied—at least as fast as Drosophilaand sea urchins—where a ΔTmof 1°C indicates 0.5 to 1.5 MY since the last common ancestor. In general, the phylogeny derived from the DNA data agrees with that derived from isozymes. Nei's Dand ΔTmare correlated; in this group a Dof 0.1 corresponds to a ΔTmof about 1.5°C. This indicates that, relative to total single‐copy DNA, the protein‐coding regions of the genome are slowly evolving.

Forty‐nine populations of nine species of North American cave crickets (genera Euhadenoecusand Hadenoecus) have been studied for genetic variation at 41 loci by electrophoresis. Wright's FST, Slatkin's Nm*gene‐flow estimator, and Nei's genetic distances (D) have been used to compare closely related species that have different ecological requirements (cave vs. forest species), distribution patterns, and/or different degrees of geographic isolation among populations. Cave and epigean (noncave) species differ greatly in their levels of genetic differentiation. Cave species have lower rates of gene exchange (low Nm, high D, and FST) than epigean species. Within cave species the degree of genetic differentiation among populations is correlated with the limestone structure of the area where the species occur. Species or groups of populations inhabiting areas where the limestone is continuous and highly fissured (e.g., H. subterraneuspopulations in the Mammoth Cave region) are genetically less differentiated than are populations occurring in regions where the limestone distribution is more fragmented, such as the Appalachian Ridge where E. fragilisoccurs; this effect is more extreme in Central Tennessee where genetically differentiated E. insolituspopulations occur only a few kilometers apart. This suggests that epigean dispersal through forest habitat in cave‐dwelling species is negligable. For forest species, the data indicate relatively recent radiation with ongoing gene exchange among populations. For cave species, the distribution of protein polymorphisms is apparently more a function of historical patterns of gene exchange rather than current gene exchange. Phylogenetic relationships were studied using cluster analyses (UPGMA and Wagner algorithms) of Nei's and Edwards' genetic distances and multivariate analysis (correspondence analysis) of the raw allele frequencies. Different algorithms result in branching patterns that are similar but not entirely concordant with one another or with the phylogeny based on morphology.

Slatkin's method (1981) for analyzing gene flow levels is applied to eleven species of cave arthropods. This provides insights into the strength of gene flow as a force affecting the evolution of cave organisms, while serving as a test of Slatkin's method. The results show that patterns of gene flow are consistent with the dispersal abilities and the ecological requirements of each species. Troglobites in general have lower gene flow values than troglophiles or trogloxenes. However, the geology of the area, its vegetation profile, and the geographic distance among the populations considered are also important in determining the gene flow levels. Gene flow patterns in three cave species, Ptomaphagus hirtus, Neaphaenops tellkampfii tellkampfiiand Hadenoecus subterraneus, are compared in detail. These species inhabit the same highly interconnected karst area in south‐central Kentucky but differ in their ecological requirements. The results suggest that gene flow levels are more dependent upon the intrinsic characteristics of each species than upon the availability of routes for dispersal. Overall estimates of gene flow were coupled with a more detailed population‐structure analysis in four terrestrial cave arthropods: Speonomus delarouzeei, Hadenoecus cumberlandicus, Hadenoecus subterraneusand Euhadenoecus puteanus. The results stress the need for this type of analysis for subdivided species, in which estimates of the average gene flow can produce misleading results. Moreover, they show how these types of measures are more relevant in describing historical patterns of gene exchange than in indicating current levels of gene flow.

Mitochondrial DNA (mtDNA) sequence variation was examined in the three species belonging to the newt genus Euproctus: E. asper, E. montanus, and E. platycephalus, and in three other species belonging to the same family: Triturus carnifex, T. vulgarisand Pleurodeles waltl. The Euproctusspecies inhabit mountain streams in the Pyrenean region, Corsica, and Sardinia, respectively. This vicariant distribution is believed to be a result of the disjunction and rotation of the Sardinia-Corsica microplate from the Pyrenean region and suggested dates for each cladogenetic event are available. A total of 915 bp from 12S and 16S ribosomal rRNA genes were compared for each taxon. These are the first mt-rDNA sequence data for salamanders. Sequences were used to reconstruct phylogenetic trees, investigate evolutionary rates for these genes, calibrate them with absolute time since divergence, and compare rates with published ones.Using P. waltlas the outgroup, all phylogenetic methods used (parsimony, maximum likelihood, and Neighbor Joining) produced trees with identical topologies and similar bootstrap values associated with each node. These sequence data cannot unambiguously resolve the splitting events leading to the main radiation of the genus Triturusand the origin of the genus Euproctus. These events may well have occurred very close in time, consistent with other sorts of data. Although it is unlikely strict linearity holds for all kinds of substitutions, relative rate tests of the molecular clock hypothesis could not reject clock-like behavior of sequence changes along Euproctuslineages. Estimates of absolute rates of base changes are 0.35% per Myr since divergence for all substitutions and 0.14% per Myr for transversions; these estimates are similar to other vertebrate estimates. A comparison with distance measures from allozyme studies agrees quite well with regard to relative divergences of the three Euproctusspecies.

We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.

Background: The Asian tiger mosquito Aedes albopictusis globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictusgenome is essential to develop new approaches that involve genetic manipulation of mosquitoes. Results: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictusgenome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictuspopulations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. Conclusion: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictusgenome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

Allozymic variation in proteins encoded by 14 loci was analyzed in 3 cavernicolousMonolistraand in 2 marineSphaeromaspecies. Genetic distance data, high levels of heterozygosity and the divergence time calculations support the hypothesis thatMonolistradiverged from its Sphaeroma-like marine ancestor during the Messinian, in connection with the Mediterranean salinity critis.

Evidence of hybridization between 2 pairs of allopatricDolichopodaspecies is provided by electrophoretic analysis. Occurrence of hybrids was revealed by laboratory crosses and in nature both by occasional co-existence due to passive dispersal and by transplantation experiments.

Sequence divergence for segments of three mitochondrial DNA (mtDNA) genes encoding the 12S and 16S ribosomal RNA and cytochrome b was examined in newts belonging to the genus Euproctus (E. asper, E. montanus, E. platycephalus) and in three other species belonging to the same family (Salamandridae), Triturus carnifex, T. vulgaris, and Pleurodeles waltl. The three Euproctus species occur (one species each) in Corsica, Sardinia, and the Pyrenees. This vicariant distribution is believed to have been determined by the disjunction and rotation of the Sardinia–Corsica microplate from the Pyreneean region. Because time estimates are available for the tectonic events that led to the separation of the three landmasses, we used sequence data to estimate rates of evolution for the three gene fragments and investigated whether they conform to the rate-constancy hypothesis. By the Tajima (1993, Genetics 135:599–607) test, we could not detect rate heterogeneities for the ribosomal genes and for transversions in the cytochrome b gene. Assuming that these sites are evolving linearly over time and that cessation of gene flow occurred simultaneously with vicariant events, we compared the time of divergence estimated by molecular distances with the divergence times based on the geological estimates. Because we have two estimates of divergence time from the geological record, the split of Corsica/Sardinia from the Pyrenees and the split of Corsica from Sardinia, we could compare ratios of molecular divergence with the ratio of geological time divergence. The ratios are very similar, indicating that the molecular clock hypothesis cannot be rejected. These geological events also allowed us to calculate absolute rates of evolution for ribosomal and cytochrome b genes and compare them to rates for the same regions in other salamandrids and other vertebrates. Ribosomal mtDNA rates are comparable to those reported for other vertebrates, but cytochrome b rates are 3–7 times lower in salamanders than in other ectotherms. From a phylogenetic perspective, our data suggest that the cladogenic events leading to species formation in Euproctus and Triturus occurred very closely in time, indicating that the two genera may not be monophyletic. A duplication of the cytochrome b gene in T. carnifex was found, and the implications of this finding for mtDNA phylogenetic studies are discussed.