Genetic panmixia within a narrow contact zone between chromosomally and ecologically distinct black fly sibling species (Diptera: Simuliidae).

Hybrid zones are windows into the speciation process, and their study can give clues into the maintenance and breakdown of species boundaries. Using both genetic and ecological tools, we investigate lineage diversification across a contact zone characterized by chromosome rearrangements. We show that black fly sibling species, Simulium arcticum sensu stricto (s.s.) and Simulium saxosum, lack genetic differentiation at both microsatellite and mtDNA loci in allopatry and sympatry, as well as exhibit high levels of gene flow and continuous chromosome variation in sympatry. Furthermore, hybrid frequencies at the contact zone are similar to those seen between races, rather than species. In contrast, S. arcticum s.s. and S. saxosum maintain ecological differences and distinct habitat associations - the contact zone situated at the margin of suitable habitat for each sibling species. Moreover, gene flow occurs only in a narrow band along an ecological transition. Except for the contact zone, S. arcticum s.s. and S. saxosum hybrids do not occur elsewhere within the sibling species' ranges. Although S. arcticum s.s. and S. saxosum maintain the potential to interbreed freely, we conclude that habitat associations and, perhaps, chromosome systems prevent expansion of ranges and assimilation of lineages.

Allopolyploidization is not so simple: evidence from the origin of the tribe Cyprinini (Teleostei: Cypriniformes).

The identification of allopolyploidization events benefits from molecular dating and divergence assessments of progenitor genomes. Information on gene duplications only, either orthologs or paralogs, provides incomplete information. Analyses of mitochondrial DNA yield insights into matrilineal history, which may differ from patrilineal evolution. Two important food and pet fishes, the common carp (Cyprinus carpio) and goldfish (Carassius sp.), appear to have experienced allotetraploidization sometime from 12 to 20 million years ago (Ma). However, much work is necessary to detail the initial polyploidization event. Herein, we use this group of fishes as a model system to investigate competing scenarios for allopolyploidization. We analyze both the nuclear genes encoding growth hormone (GH), recombination activating protein 1 (RAG1) and HOXA2B gene, and the maternal heredited 12 concatenated mitochondrial protein-coding gene in 19 species of cyprinids and use two species in Balitoridae as outgroup taxa. Our analyses clarify the phylogenetic position of the paternal and maternal ancestors for the common carp and goldfish. The estimation of matrilineal divergence (10.71-12.42 Ma) is significantly younger than the dates of the parental ancestor divergedthat obtained by nuclear genes (16.62-19.64 Ma). Analyses of both genomes date the allopolyploidization event of the common ancestor of Cy. carpio and Ca sp. to about 10.71-12.42 Ma, which is most likely represented by maternal divergent time. The divergence of the two copies of the nuclear genes which was more ancient than the maternal markers might have been included the divergence of the progenitors' genome divergence when the allopolyploidization event occurred. Thus, the scenarios of allopolyploidzation for this group of fish can be suggested as the following: the matrilineal common ancestor of species in tribe Cyprinini might have doubled its genome by mating with a paternal ancestor in the subfamily Cyprininae, which was a sister-group that diverged around 4.20-8.93 Ma. Our work provides new evidence for the divergence dates of allopolyploidization within the Cyprinini, and documents the necessity of considering both matrilineal and patrilineal histories when investigating allopolyploidization.

Tempo and mode of recurrent polyploidization in the Carassius auratus species complex (Cypriniformes, Cyprinidae).

Polyploidization is an evolutionarily rare but important mechanism in both plants and animals because it increases genetic diversity. Goldfish of the Carassius auratus species complex can be tetraploids, hexaploids and octaploids. Polyploidization events have occurred repeatedly in goldfish, yet the extent of this phenomenon and its phyletic history are poorly understood. We explore the origin, tempo and frequency of polyploidization in Chinese and Japanese goldfish using both mitochondrial (mtDNA) and nuclear DNA sequences from up to 1202 individuals including the outgroup taxon, Cyprinus carpio. Analyses of de novo nuclear gene data resolve two clusters of alleles and the pattern supports the prior hypothesis of an ancient allotetraploidization for Carassius. Alleles shared by tetraploid and hexaploid individuals indicate recent autoploidizations within the C. auratus complex. Sympatric tetraploids and hexaploids share mtDNA haplotypes and these frequently occur independently within six well-supported lineages and sublineages on a small spatial scale. Gene flow estimates (Fst values) indicate that hexaploids differ only slightly from sympatric tetraploids, if at all. In contrast, allopatric populations of tetraploids and hexaploids differ from one another to a far greater extent. Gene flow between sampled localities appears to be limited. Coalescence-based time estimations for hexaploids reveal that the oldest lineage within any sampled locality is around one million years old, which is very young. Sympatric, recurrent autoploidization occurs in all sampled populations of the C. auratus complex. Goldfish experience polyploidization events more frequently than any other vertebrate.

Multiple ancient horizontal gene transfers and duplications in lepidopteran species.

Eukaryotic horizontal gene transfer (HGT) events are increasingly being discovered yet few reports have summarized multiple occurrences in a wide range of species. We systematically investigated HGT events in the order Lepidoptera by employing a series of filters. Bombyx mori, Danaus plexippus and Heliconius melpomene had 13, 12 and 12 HGTs, respectively, from bacteria and fungi. These HGTs contributed a total of 64 predicted genes: 22 to B. mori, 22 to D. plexippus and 20 to H. melpomene. Several new genes were generated by post-transfer duplications. Post-transfer duplication of a suite of functional HGTs has rarely been reported in higher organisms. The distributional patterns of paralogues for certain genes differed in the three species, indicating potential independent duplication or loss events. All of these HGTs had homologues expressed in some other lepidopterans, indicating ancient transfer events. Most HGTs were involved in the metabolism of sugar and amino acids. These HGTs appeared to have experienced amelioration, purifying selection and accelerated evolution to adapt to the background genome of the recipient. The discovery of ancient, massive HGTs and duplications in lepidopterans and their adaptive evolution provides further insights into the evolutionary significance of the events from donors to multicellular host recipients.

Chicken domestication: an updated perspective based on mitochondrial genomes.

Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A-I and W-Z. Common haplogroups A-G were shared by domestic chickens and red junglefowl. Rare haplogroups H-I and W-Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.

The Coachella valley fringe-toed lizard (Uma inornata): genetic diversity and phylogenetic relationships of an endangered species.

A phylogeny was reconstructed for 23 populations of fringe-toed lizards (genus Uma) from the three most northern species of the genus, including the Mojave fringe-toed lizard U. scoparia, the Colorado Desert fringe-toed lizard U. notata, and the endangered Coachella Valley fringe-toed lizard U. inornata. The outgroup taxa were the zebra-tailed lizard, Callisaurus draconoides; the lesser earless lizard, Holbrookia maculata; and the greater earless lizard, Cophosaurus texanus. Evaluation of 1630 combined nucleotide sequence from the mitochondrial genes ATPase 6 and cytochrome b yielded 10 most parsimonious trees. Reweighting the characters using the rescaled consistency index eliminated eight of these trees. The remaining two trees differ only in the placement of two individuals from the Superstition Mountains which either formed a monophlyetic unit or grouped with one individual from the Anza-Borrego population. The preferred phylogeny, one more consistent with geography, had two primary clades: one consisting of U. scoparia and the other placing U. inornata inside the clade containing U. notata. Uma inornata was most closely related to nearby U. notata notata, as opposed to more distant U. notata rufopunctata.

Genetic variability among endangered Chinese giant salamanders, Andrias davidianus.

The endangered Chinese giant salamander (Andrias davidianus) is endemic to mainland China. Genetic divergence among six populations of the species was investigated by means of isozyme electrophoresis and mitochondrial DNA (mtDNA) sequences. Forty allozyme loci were resolved for all populations; the amount of genetic divergence among populations was comparable to that in other amphibians. mtDNA sequences showed a similar level of divergence. The population from Huangshan is distinct from other populations, indicating the existence of localized divergence. Both allozyme and mtDNA data failed to associate the populations into a pattern corresponding to the three Chinese river systems, which may be the consequence of human relocation. Conservation policies should emphasize the protection of localized populations and cessation of human-facilitated introductions. Future studies should focus on investigating the divergence among localized populations from isolated mountain regions, particularly using more fine-grained techniques such as microsatellite DNA.

Phylogeny of east asian bufonids inferred from mitochondrial DNA sequences (Anura: amphibia).

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei, B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B. bankorensis from Taiwan is evaluated.

Allozyme variation patterns and multiple hybridization origins: clonal variation among four sibling parthenogenetic Caucasian rock lizards.

Allozyme electrophoresis of four sibling parthenogenetic Caucasian rock lizards Darevskia unisexualis, D. uzzelli, D. sapphirina, and D. bendimahiensis found seven clones and five variable loci. The data supported the hypothesis that D. raddei and D. valentini are the parental species of all four parthenogens. Variation patterns in Darevskia were summarized. Species that originated from a single F1 typically consisted of one widespread clone with a few rare clones. Species with multiple origins displayed variation only slightly higher than species with a single origin. This is contrary to other genera of parthenogenetic lizards, in which cases massive clonal variations were observed.

Discriminating and locating character covariance: an application of permutation tail probability (PTP) analyses.

The ability of permutation tail probability (PTP) analyses to discriminate between character covariance and noise is investigated with both hypothetical and published data sets. PTP is shown to be a powerful tool, not only for detecting character covariance, but also for locating that covariance on trees. PTP is especially useful for evaluating DNA sequence data that may have a high level of homoplasy. A three-step PTP procedure for locating covaried characters is presented.

Limited genetic variation in Lacerta mixta and its parthenogenetic daughter species: evidence from cytochrome b and ATPase 6 gene DNA sequences.

Little mtDNA variation was observed among populations of the bisexual Caucasian rock lizard Lacerta mixta and unisexual L. dahli and L. armeniaca. Three haplotypes were detected in L. mixta and the maximum pairwise difference among the samples was 0.67%. No intra- and interspecific variation was found among populations of either L. armeniaca or L. dahli. Moreover, both unisexual species were identical to one of the three haplotypes of L. mixta. The limited variation in L. mixta is likely the result of bottleneck effect, although the small sample size may also be responsible. The lack of variation in the unisexual was attributed to the restricted variation among the maternal parents, limited involvement of females in the hybridization, and recent origin.

Phylogeny of the side-blotched lizards (Phrynosomatidae:Uta) based on mtDNA sequences: support for midpeninsular seaway in Baja California.

We investigated the phylogenetic relationships of western North American Side-blotched lizards, genus Uta, using mitochondrial DNA partial gene sequences from cytochorme b and ATPase 6. Uta stejnegeri appeared basal in our tree followed by U. palmeri. Uta stansburiana from the islands of Angel de la Guarda, Mejia, and Raza formed the next clade, followed by U. antiqua and other populations of U. stansburiana. These relationships suggest that the populations on the Angel de la Guarda island block should be recognized as a distinct species. Remaining populations of U. stansburiana formed two clades, corresponding to the northern and southern Baja California peninsula. Uta stellata and U. squamata occurred within the northern and southern clades, respectively. The discontinuity requires a long-lasting isolation, the only reasonable explanation being a former midpeninsular seaway. Correlations between our cladogram and magnetic anomalies in the Gulf of California date formation of the seaway at 1 million years ago.

Disjunct habitats as islands: genetic variability in the Caucasian rock lizard Lacerta portschinskii.

Genetic diversity at 37 allozyme loci was surveyed in Lacerta portschinskii from contiguous populations and from a disjunct population. Indices of genetic diversity (heterozygosity, number of alleles per locus, and percentage of loci polymorphic) were greater in contiguous populations than in the smaller disjunct population. In this regard, disjunct populations appear to be similar to island populations. Indices of genetic diversity in Caucasian Lacerta are less than those reported from vagile lizard taxa and more similar to those of sit-and-wait predators.

Old age, multiple formations or genetic plasticity? Clonal diversity in the uniparental Caucasian rock lizard, Lacerta dahli.

Allozyme variation at 35 gene loci is investigated in 161 specimens of the uniparental Caucasian lizard Lacerta dahli from several locations in Armenia and Georgia. All individuals are heterozygotic at 12 loci, and homozygotic at 21 loci. Variation at two loci results in five uniparental clones. One clone is widespread whereas four are geographically restricted and are represented by only one or two individuals. Because successful formation of uniparental clones is rare, and because the biparental species forming them are now allopatric, the most probable explanation for the origin of the observed clonal diversity is either mutation or recombination within the common clone. The rare clones have lower levels of enzyme activity at four loci, suggesting that these organisms may be genetically deficient. Although the evidence points to change in a pre-existing clone, the possibility of multiple origins cannot be ruled out.

Recombinant human bone morphogenetic proteins-2 and -4 induce several mesenchymal phenotypes in culture.

Bone morphogenetic protein has previously been shown to induce the formation of cartilage and bone in vivo. We have isolated a population of mesenchymal stem cells from rat skeletal muscle capable of forming multiple mesodermal morphologies in vitro. These cells were treated with recombinant human bone morphogenetic proteins-2 and -4 to determine the differentiation-inducing activities of bone morphogenetic protein on these cells. The mesenchymal stem cells were cultured in medium with 10% preselected horse serum containing 0 to 100 ng/ml recombinant human bone morphogenetic proteins-2 or -4 for a maximum of 4 weeks. Control cultures maintained the stellate morphology of mesenchymal stem cells. Cultures treated with recombinant human bone morphogenetic protein-2 exhibited discrete cartilage nodules and mineralized bone nodules. The first increase in chondrogenesis was seen at 0.5 ng/ml. Cultures treated with recombinant human bone morphogenetic protein-4 also exhibited an increase in chondrogenesis at the higher concentration of 2 ng/ml. Skeletal myotubes and adipocytes also appeared in cultures treated with either bone morphogenetic protein. Mesenchymal stem cells do respond to inductive factors, but bone morphogenetic proteins-2 and -4 were not specific for the induction of cartilage and bone.

Clonal variation in the parthenogenetic rock lizard Lacerta armeniaca.

Genetic diversity at 35 allozyme loci was surveyed in seven populations of Lacerta armeniaca. Fixed heterozygotes were present at 16 loci, with homozygotes at 17 loci. Variation occurred at two loci, one in each of two populations, indicating one widespread clone, one restricted clone, and one apparently restricted clone. The low level of variation in this species suggests a recent restricted origin, involving few parental individuals.

A comparison of controlled negative pressure and aerosol quantitative respirator fit test systems by using human subjects.

A quantitative respirator fit test system based on controlled negative pressure was evaluated by comparison testing with a computerized aerosol fit test system. Experiments ranged from multiple sequential tests of a single subject wearing a respirator equipped with a series of fixed leaks to sequential fit tests of 125 U.S. Air Force personnel using both systems. Throughout each test phase, measured negative pressure fit factors were consistently more conservative and less variable than aerosol fit factors. Comparison of subject and fixed leak fit factors indicated significant loss of aerosol during subject fit tests. Negative pressure system results did not show any effect from subject-related losses.

A comparison of controlled negative pressure and aerosol quantitative respirator fit test systems by using fixed leaks.

An automated version of a new method for quantitative respirator fit testing by controlled negative pressure was compared with a computerized aerosol fit test system. The controlled negative pressure technique eliminates many of the problems associated with aerosol and pressure decay fit test methods. A series of fixed leaks was used to compare the leak measurement capabilities of the controlled negative pressure system against a standard computerized aerosol fit test system. Negative pressure and aerosol fit factors determined for a series of fixed leaks through hypodermic needles were highly correlated with each other (r = 0.998) and with the cross-sectional areas of the leak needles (r greater than 0.995).

Use of nicotinamide adenine dinucleotide (NAD)-dependent glucose-6-phosphate dehydrogenase in enzyme staining procedures.

Substitution of nicotinamide adenine dinucleotide dependent glucose-6-phosphate dehydrogenase for the nicotinamide adenine dinucleotide phosphate dependent enzyme has produced identical results in a number of enzyme-linked electrophoretic staining procedures. This substitution significantly reduces the cost of staining for adenylate kinase, creatine kinase, glucosephosphate isomerase, mannosephosphate isomerase, phosphoglucomutase, and pyruvate kinase activity by utilizing NAD rather than the more expensive NADP.