bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeographical structure of the Dover soleMicrostomus pacificus: the larval retention hypothesis and genetic divergence along the deep continental slope of the northeastern Pacific Ocean
Authors: Stepien C. A.
Published: March 2003

Population genetic variability and phylogeographical structure of the Dover sole,Microstomus pacificus(Teleostei: Pleuronectidae), were examined along the continental slope of the northeastern Pacific Ocean using sequences of the left domain of the mitochondrial DNA (mtDNA) control region. Six sites were sampled in three primary biogeographical provinces (based on surface water temperatures), ranging from the subarctic to the warm temperate zones. Many haplotypes were discerned, totalling 90 for 110 individuals. Neighbour joining and parsimony analyses of the entire data set showed that some groups of haplotypes of adult Dover sole were widely distributed, suggesting either panmixia or homoplasy and reversals. However, a substantial number of groups of related haplotypes were geographically circumscribed, and there were significant differences among sites in the distribution of restricted haplotypes (based on a reduced number of characters), supporting geographical structure. Geographical differentiation of populations appeared to be consistent with the member‐vagrant hypothesis for retention of the pelagic larvae in currents and recruitment to adult habitats, reducing long‐distance dispersal. Results of this study indicate phylogeographical structure among some populations, despite extended pelagic larval periods, and apparent environmental homogeneity in the adult habitats along the continental slope.
Molecular phylogeography ofDryas integrifolia: glacial refugia and postglacial recolonization
Authors: Tremblay N. O. , Schoen D. J.
Published: March 2003

Chloroplast DNA variation in the Arctic plant speciesDryas integrifolia(Rosaceae) was analysed in relation to both the present‐day geographical distribution of populations and to Pleistocene fossil records of this species. The phylogeographical structure was weak but the analysis of haplotype diversity revealed several groups of haplotypes having present‐day geographical ranges that overlap locations postulated from geographical and fossil evidence to have been glacial refugia. Based on this information we infer that two important refugial sources of Arctic recolonization by this species were Beringia and the High Arctic. Two other putative refugia, located southeast of the ice sheet and along coastal regions of the eastern Arctic may have served as sources for recolonization of smaller portions of the Arctic. The genetic substructure in the species is mostly due to variation among populations regardless of the ecogeographical region in which they are found. Spatial autocorrelation at the regional scale was also detected. High levels of diversity both within populations and ecogeographical regions are probably indicative of population establishment from several sources possibly combined with recent gene flow.
Phylogeography of a post‐glacial colonizer:Microtus longicaudus(Rodentia: Muridae)
Authors: Conroy C. J. , Cook J. A.
Published: March 2003

The molecular phylogeography ofMicrotus longicauduswas investigated with DNA sequences of the mitochondrial cytochromebgene. We used phylogenetic and pairwise distance methods to reconstruct the history of the species with particular emphasis on the Pacific Northwest. Genetic variation across the species was consistent with vicariant events during the Pleistocene and subsequent northern postglacial expansion following the receding Laurentide and Cordilleran ice sheets. The largest break (> 6% uncorrected sequence divergence) was found to exist between populations found southeast of the Colorado River (eastern Arizona, Colorado, Wyoming and New Mexico) and all other western populations. Other well‐supported subclades were composed of samples from: (i) the islands and north coast of southeast Alaska; (ii) eastern Alaska, British Columbia, Washington and Oregon; and (iii) northern California, Idaho and Montana. Within subclades, divergence was low. Our results suggest that the close relationships among haplotypes within northern subclades are a result of recent colonization, whereas higher among‐subclade divergence is caused by genetic differentiation during prolonged periods of isolation, possibly as a result of mid‐Pleistocene climatic events.
Genetic distinctness of isolated populations of an endangered marsupial, the mountain pygmy‐possum, Burramys parvus
Authors: Osborne M. J. , Norman J. A. , Christidis L. , Murray N. D.
Published: March 2003

The mountain pygmy‐possum, Burramys parvus, exists in isolated and fragmented populations in the Australian alps. To examine the degree of interpopulation divergence, mitochondrial cytochrome b and NADH dehydrogenase subunit 2 (NADH2) sequences were obtained from samples representing all populations of B. parvus. Three divergent mitochondrial DNA (mtDNA) lineages were identified which exhibited strong phylogeographical structure. This indicates the presence of three maternal clades corresponding to populations in the northern, central and southern Australian alps. Molecular clock estimates suggest that the mtDNA lineages diverged from one another 420–680 thousand years ago. On this basis it is argued that B. parvus populations have probably been isolated since the mid‐Pleistocene, and that management should focus on maintaining viable B. parvus populations in each of the three regional localities.
Effects of natural selection on patterns of DNA sequence variation at the transferrin, somatolactin, and p53 genes within and among chinook salmon (Oncorhynchus tshawytscha) populations
Authors: Ford Michael J.
Published: March 2003

This paper describes DNA sequence variation within and among four populations of chinook salmon (Oncorhynchus tshawytscha) at the transferrin, somatolactin and p53 genes. Patterns of variation among salmon species at the transferrin gene have been hypothesized to be shaped by positive natural selection for new alleles because the rate of nonsynonymous substitution is significantly greater than the rate of synonymous substitution. The twin goals of this study were to determine if the history of selection among salmon species at the transferrin gene is also reflected in patterns of intraspecific variation in chinook salmon, and to look for evidence of local adaptation at the transferrin gene by comparing patterns of nonsynonymous and synonymous variation among chinook salmon populations. The analyses presented here show that unlike patterns of variation between species, there is no evidence of greater differentiation among chinook salmon populations at nonsynonymous compared to synonymous sites. There is also no evidence of a reduction of within‐species variation due to the hitchhiking effect at the transferrin gene, although in some populations nonsynonymous and synonymous derived mutations are both at higher frequencies than expected under a simple neutral model. Population size weighted selection coefficients (4Ns) that are consistent with both the inter and intraspecific data range from ~10 to ~235, and imply that between 1 and 40% of new nonsynonymous mutations at the transferrin gene have been beneficial.
Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans
Authors: Xu Jianping , Vilgalys Rytas , Mitchell Thomas G.
Published: March 2003

Cryptococcus neoformans (= Filobasidiella neoformans) is a significant emerging fungal pathogen of humans. To understand the evolution of this pathogen, 34 strains were obtained from various locations around the world and fragments of four genes were sequenced from each. These strains represented all three varieties and five serotypes. The four sequenced genes are: (i) the mitochondrial large ribosomal subunit RNA; (ii) the internal transcribed spacer region of the nuclear rRNA, including ITS1, 5.8S rRNA subunit and ITS2; (iii) orotidine monophosphate pyrophosphorylase; and (iv) diphenol oxidase. Phylogenetic analyses indicated considerable divergence among lineages, which corresponded to the current classification of C. neoformans into three varieties. However, there is no apparent phylogeographic pattern. Significant incongruences were observed among gene genealogies. The analyses indicated that the major lineages in C. neoformans diverged tens of millions of years ago but have undergone recent dispersion and hybridization.
Dispersal barriers in tropical oceans and speciation in Atlantic and eastern Pacific sea urchins of the genus Echinometra
Authors: Mccartney M. A. , Keller G. , Lessios H. A.
Published: March 2003

Echinometra is a pantropical sea urchin made famous through studies of phylogeny, speciation, and genetic structure of the Indo‐West Pacific (IWP) species. We sequenced 630 bp of the cytochrome oxidase I (COI) mitochondrial gene to provide comparable information on the eastern Pacific and Atlantic species, using divergence between those separated by closure of the Isthmus of Panama 3.1 million years ago (Ma) to estimate dates for cladogenic events. Most recently (1.27–1.62 Ma), the Atlantic species E. lucunter and E. viridis diverged from each other, at a time in the Pleistocene that sea levels fell and Caribbean coral speciation and extinction rates were high. An earlier split, assumed to have been coincident with the completion of the Isthmus of Panama, separated the eastern Pacific E. vanbrunti from the Atlantic common ancestor. Transisthmian COI divergence similar to that in the sea urchin genus Eucidaris supports this assumption. The most ancient split in Echinometra occurred between the IWP and the neotropical clades, due to cessation of larval exchange around South Africa or across the Eastern Pacific Barrier. Gene flow within species is generally high; however, there are restrictions to genetic exchange between E. lucunter populations from the Caribbean and those from the rest of the Atlantic. Correlation between cladogenic and vicariant events supports E. Mayr’s contention that marine species, despite their high dispersal potential, form by means of geographical separation. That sympatric, nonhybridizing E. lucunter and E. viridis were split so recently suggests, however, that perfection of reproductive barriers between marine species with large populations can occur in less than 1.6 million years (Myr).
Evidence for predominant clones in a cyclically parthenogenetic organism provided by combined demographic and genetic analyses
Authors: Haack L. , Simon J.‐C. , Gauthier J.‐P. , Plantegenest M. , Dedryver C.‐A.
Published: March 2003

Aphids are particularly interesting models in the study of genetic and demographic components of plant adaptation because of their breeding system which combines parthenogenesis and sexual reproduction (i.e. cyclical parthenogenesis), and the frequent emergence of host‐adapted races reported in this group. In this paper, patterns of host adaptation were assessed on local populations of the aphid Sitobion avenae by following their demographic and genetic structure in a maize field for two consecutive years. The existence of putative generalist (polyphagous) or specialized (host‐adapted) genotypes was also investigated by comparing the genotypic distribution of this aphid on maize and other cultivated host plants, using five microsatellite loci. Although population dynamics revealed strong variation in aphid abundance during the colonization period on maize, two genotypes identified at seven additional microsatellite loci were predominant and exhibited stable frequencies over cropping season and between years. Based on present and earlier studies, these two prevalent genotypes were shown to survive on different host plants other than maize, to colonize large geographical zones and to persist parthenogenetically for several years. All these data strongly suggest that these two genotypes are asexual generalist clones that could have been favoured by agricultural practices encountered in western Europe. Besides these two clones, a continual replacement of rare genotypes was observed on maize in both years. Hypotheses involving selection via aphid–plant interactions and natural enemies were proposed for explaining the disappearance of these genotypes on maize.
Temporal and spatial genetic variation in spawning grounds of European hake (Merluccius merluccius) in the Bay of Biscay
Authors: Lundy Caroline J. , Rico Ciro , Hewitt Godfrey M.
Published: March 2003

Polymorphism at five microsatellite loci were screened to determine the genetic variability and the temporal stability of population structure in natural populations of European hake (Merluccius merluccius, L.) within the Bay of Biscay. In addition, the control region (900 bp) and two protein coding genes (ATPase, subunits 6 and 8, 842 bp and a partial sequence of the ND1, 800 bp) of the mitochondrial DNA (mtDNA) were sequenced from geographically distant populations from the extremes of the species range. One hundred individuals from either side of a supposed stock boundary within the bay were collected in autumn 1997. This sampling strategy was repeated during hake spawning seasons in late spring of 1998 and 1999. Low levels of population subdivision were found between putative populations within years. Similarly, low levels of differentiation were found between autumn 1997 northern samples and spring 1998 southern samples which were collected 7 months later on spawning grounds. These results are discussed in relation to ecological, behavioural and oceanographic information. Sampling effects, which may influence these results, are also discussed. Theta (θ) estimates were significantly different from zero in every other pairwise comparison between geographical areas (north and south of the Bay of Biscay) and between years within the same area (P < 0.05). Hierarchical analysis of molecular variance (amova) does not confirm the temporal persistence of population structure. These results are discussed in relation to variance in reproductive success, and temporal spawning patterns, which may exist within the bay. mtDNA variability was very low between geographically distant samples from Norway and the Mediterranean Sea with only 10 variable sites found in a total of 2542 bp of mtDNA, these differences being exclusively in the d‐loop.
Phylogeography, population history and conservation genetics of jaguars (Panthera onca, Mammalia, Felidae)
Authors: Eizirik Eduardo , Kim Jae‐Heup , Menotti‐Raymond Marilyn , Crawshaw JR. Peter G. , O’Brien Stephen J. , Johnson Warren E.
Published: March 2003

The jaguar (Panthera onca), the largest felid in the American Continent, is currently threatened by habitat loss, fragmentation and human persecution. We have investigated the genetic diversity, population structure and demographic history of jaguars across their geographical range by analysing 715 base pairs of the mitochondrial DNA (mtDNA) control region and 29 microsatellite loci in ≈40 individuals sampled from Mexico to southern Brazil. Jaguars display low to moderate levels of mtDNA diversity and medium to high levels of microsatellite size variation, and show evidence of a recent demographic expansion. We estimate that extant jaguar mtDNA lineages arose 280 000–510 000 years ago (95% CI 137 000–830 000 years ago), a younger date than suggested by available fossil data. No strong geographical structure was observed, in contrast to previously proposed subspecific partitions. However, major geographical barriers such as the Amazon river and the Darien straits between northern South America and Central America appear to have restricted historical gene flow in this species, producing measurable genetic differentiation. Jaguars could be divided into four incompletely isolated phylogeographic groups, and further sampling may reveal a finer pattern of subdivision or isolation by distance on a regional level. Operational conservation units for this species can be defined on a biome or ecosystem scale, but should take into account the historical barriers to dispersal identified here. Conservation strategies for jaguars should aim to maintain high levels of gene flow over broad geographical areas, possibly through active management of disconnected populations on a regional scale.
Mating asymmetry and the direction of evolution in the Hawaiian cricket genus Laupala
Authors: Shaw Kerry L. , Lugo Ezequiel
Published: March 2003

Based on studies from native Hawaiian Drosophila, a model was proposed to explain sexual isolation and mating asymmetry, from which one could potentially infer the ‘direction of evolution’. We examined sexual isolation between allopatric cricket species of the genus Laupala, another endemic Hawaiian insect with an elaborate mating system, to begin to explore the nature of sexual isolation and mating asymmetry in closely related Hawaiian organisms. We studied sexual isolation and mating asymmetry in two contrasts. First, an inter‐island comparison, including L. makaio from the older island of Maui and L. paranigra from the younger island of Hawaii, and second, an intra‐island (Hawaii) comparison, including L. nigra from the older volcano of Mauna Kea and L. paranigra with a primary distribution on the younger volcanoes of Mauna Loa and Kilauea. We used a ‘no‐choice’ experimental design, pairing individual males and females in homospecific or heterospecific combinations. Several behavioural aspects of courtship (proportion of male singing, latency to male singing, production of spermatophores and courtship initiation speed) were quantified as well as the success or failure of matings. We demonstrate asymmetry in sexual isolation between reciprocal combinations of L. makaio and L. paranigra. This result is examined in light of the differences in courtship behaviour manifest in the experiments with these two species. We did not find evidence of asymmetry in sexual isolation between L. nigra and L. paranigra, although differences in courtship initiation speed were evident between reciprocal combinations of these two species. In addition to the geological argument that species on older islands and older volcanoes give rise to species on younger islands and younger volcanoes, we discuss phylogenetic evidence consistent with these biogeographic hypotheses of relationships among the focal taxa. The patterns of asymmetrical sexual isolation and mating asymmetry are consistent with those found in the native Hawaiian Drosophila.
The evolution of geographic parthenogenesis in Timema walking‐sticks
Authors: Law Jennifer H. , Crespi Bernard J.
Published: March 2003

Phylogenetic studies of asexual lineages and their sexual progenitors are useful for inferring the causes of geographical parthenogenesis and testing hypotheses regarding the evolution of sex. With five known parthenogens and well‐studied ecology, Timema walking‐sticks are a useful system for studying these questions. Timema are mainly endemic to California and they exhibit the common pattern of geographical parthenogenesis, with asexuals exhibiting more‐northerly distributions. Neighbour‐joining and maximum‐parsimony analyses of 416 bp of mitochondrial cytochrome oxidase I (COI) from 168 individuals were used to infer general phylogenetic relationships, resulting in three major phylogeographical subdivisions: a Northern clade; a Santa Barbara clade; and a Southern clade. A nested cladistic analysis, comparing intra‐ and interspecific haplotypic variation on a geographical scale, revealed that the overall pattern of geographical parthenogenesis in Timema could be attributed to historical range expansion. These results suggest that geographical parthenogenesis is the result of more‐extensive northerly dispersal of asexuals than sexuals.
Selection and gene flow between microenvironments: the case of Drosophila at Lower Nahal Oren, Mount Carmel, Israel
Authors: Colson I.
Published: March 2003

Drosophila melanogaster and D. simulans from contrasted microenvironments (south‐ and north‐facing slopes of Lower Nahal Oren Canyon in Israel) were tested for genetic differentiation at microsatellite loci, which might be linked to differential adaptation to local ecological factors. No overall genetic differentiation was observed in either species. This indicates that the contrasted selective pressures on the two sides of the valley are not strong enough to cause population subdivision in highly mobile species such as Drosophila. Significant differences in allele frequencies were observed at two microsatellite loci, but on the whole the level of divergence we observed is far lower than has been reported previously.
The incidence and diversity ofWolbachiain gallwasps (Hymenoptera; Cynipidae) on oak
Authors: Rokas A. , Atkinson R. J. , Nieves‐Aldrey J.‐L. , West S. A. , Stone G. N.
Published: March 2003

Wolbachiabacteria infect ≈ 20% of all insect species, and cause a range of alterations to host reproduction, including imposition of thelytoky. The incidence and phenotypic impact ofWolbachiaremains to be established in many insect taxa, and considerable research effort is currently focused on its association with particular reproductive modes and the relative importance of the various pathways via which infection occurs. Gallwasps represent an attractive system for addressing these issues for two reasons. First, they show a diversity of reproductive modes (including arrhenotoky, thelytoky and cyclical parthenogenesis) in which the impact ofWolbachiainfection can be examined. Second, they occupy two intimately linked trophic niches (gall‐inducers and inquilines) between which there is potential for the horizontal exchange ofWolbachiainfection. In the arrhenotokous gallwasp lineages screened to date (the herb‐galling ‘Aylacini’ and the rose‐galling Diplolepidini),Wolbachiainfection always induces thelytoky. The impact ofWolbachiain other arrhenotokous clades, and in the cyclically parthenogenetic clades remains unknown. Here we use polymerase chain reaction (PCR) screening and sequence data for twoWolbachiagenes (wspandftsZ) to examine the prevalence and incidence ofWolbachiainfection in 64 species (a total of 609 individuals) in two further tribes: the arrhenotokous inquilines (tribe Synergini), and the cyclically parthenogenetic oak gallwasps (tribe Cynipini). We ask: (i) whetherWolbachiainfection has any apparent impact on host reproduction in the two tribes and (ii) whether there is any correlation betweenWolbachiainfection and the apparent lack of an arrhenotokous generation in many oak gallwasp life cycles. We show: (i) thatWolbachiainfection is rare in the Cynipini. Infected species show no deviation from cyclical parthenogenesis, and infection is no more common in species known only from a thelytokous generation; (ii) that there is a higher incidence of infection within the arrhenotokous inquilines, and generally in gallwasp tribes without cyclical parthenogensis; (iii) allWolbachia‐positive inquiline species are known to possess males, implying either thatWolbachiainfection does not result in loss of sex in this tribe or, more probably, that (as for some rose gallwasps)Wolbachiainfection leads to loss of sex in specific populations; and (iv) although we find some inquilines and gall inducers to be infected withWolbachiahaving the samewspsequence, these hosts are not members of the same gall communities, arguing against frequent horizontal transmission between these two trophic groups. We suggest that exchange may be mediated by the generalist parasitoids common in oak galls.
Capercaillie in the Alps: genetic evidence of metapopulation structure and population decline
Authors: Segelbacher Gernot , Storch Ilse
Published: March 2003

In the Alps, the capercaillie is distributed in a metapopulation pattern with local populations on mountain ranges separated by farmland valleys. Habitat deterioration, primarily related to human land use, resulted in population declines and range contractions became obvious. At the edge of a species’ range, lower connectivity and less gene flow may render populations more susceptible to decline and extinction than in the core of the range. If this were true for the capercaillie in the Alps, edge populations should be subject to limited gene flow and should show genetic signs of a more severe population decline than core populations. To test this hypothesis, we used microsatellite DNA typing techniques. We assessed genetic variation within and among 18 local capercaillie populations across the Alps in relation to geographical distribution within the metapopulation system. All populations showed high levels of genetic variation in terms of average number of alleles, allelic richness and heterozygosity. Excess heterozygosity suggested a recent population decline, that was more pronounced in edge than core populations. We found high gene flow, but also significant differentiation among populations. Differentiation among edge populations was related to geographical distance, and appeared to be a recent process, most probably caused by reduced gene flow after population decline. In the core group, the high mountains of the central Alps seem to limit dispersal, and genetic drift was the most likely explanation for the observed differentiation among populations. We conclude that maintaining connectivity within the metapopulation system is vital for capercaillie conservation in the Alps.
Phylogeographical variation of chloroplast DNA in holm oak (Quercus ilex L.)
Authors: Lumaret R. , Mir C. , Michaud H. , Raynal V.
Published: March 2003

Variation in the lengths of restriction fragments (RFLPs) of the whole chloroplast DNA molecule was studied in 174 populations of Quercus ilex L. sampled over the entire distribution of this evergreen and mainly Mediterranean oak species. By using five endonucleases, 323 distinct fragments were obtained. From the 29 and 17 cpDNA changes identified as site and length mutations, respectively, 25 distinct chlorotypes were distinguished, mapped and treated cladistically with a parsimony analysis, using as an outgroup Q. alnifolia Poech, a closely related evergreen oak species endemic to Cyprus where Q. ilex does not grow. The predominant role of Q. ilex as maternal parent in hybridization with other species was reflected by the occurrence of a single very specific lineage of related chlorotypes, the most ancestral and recent ones being located in the southeastern and in the northwestern parts of the species’ geographical distribution, respectively. The lineage was constituted of two clusters of chlorotypes observed in the ‘ilex’ morphotyped populations of the Balkan and Italian Peninsulas (including the contiguous French Riviera), respectively. A third cluster was divided into two subclusters identified in the ‘rotundifolia’ morphotyped populations of North Africa, and of Iberia and the adjacent French regions, respectively. Postglacial colonization probably started from three distinct southerly refugia located in each of the three European peninsulas, and a contact area between the Italian and the Iberian migration routes was identified in the Rhône valley (France). Chlorotypes identical or related to those of the Iberian cluster were identified in the populations from Catalonia and the French Languedoc region, which showed intermediate morphotypes, and in the French Atlantic populations which possessed the ‘ilex’ morphotype, suggesting the occurrence of adaptive morphological changes in the northern part of the species’ distribution.
A DNA‐based method for identification of krill species and its application to analysing the diet of marine vertebrate predators
Authors: Jarman S. N. , Gales N. J. , Tierney M. , Gill P. C. , Elliott N. G.
Published: March 2003

Accurate identification of species that are consumed by vertebrate predators is necessary for understanding marine food webs. Morphological methods for identifying prey components after consumption often fail to make accurate identifications of invertebrates because prey morphology becomes damaged during capture, ingestion and digestion. Another disadvantage of morphological methods for prey identification is that they often involve sampling procedures that are disruptive for the predator, such as stomach flushing or lethal collection. We have developed a DNA‐based method for identifying species of krill (Crustacea: Malacostraca), an enormously abundant group of invertebrates that are directly consumed by many groups of marine vertebrates. The DNA‐based approach allows identification of krill species present in samples of vertebrate stomach contents, vomit, and, more importantly, faeces. Utilizing samples of faeces from vertebrate predators minimizes the impact of dietary studies on the subject animals. We demonstrate our method first on samples of Adelie penguin (Pygoscelis adeliae) stomach contents, where DNA‐based species identification can be confirmed by prey morphology. We then apply the method to faeces of Adelie penguins and to faeces of the endangered pygmy blue whale (Balaenoptera musculus brevicauda). In each of these cases, krill species consumed by the predators could be identified from their DNA present in faeces or stomach contents.
Introgression patterns in the mosaic hybrid zone betweenMytilus edulisandM. galloprovincialis
Authors: Bierne N. , Borsa P. , Daguin C. , Jollivet D. , Viard F. , Bonhomme F. , David P.
Published: March 2003

Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine musselsMytilus edulisandM. galloprovincialis, using three length‐polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient fromM. galloprovincialison the Iberian Peninsula toM. edulispopulations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical ofM. galloprovincialisfirst decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantlyM. edulis‐like andM. galloprovincialis‐like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre‐ or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory ‘short‐cut’ connectingM. edulis‐like populations of the Charente patch to an externalM. edulispopulation in Normandy and thought to reflect artificial transfer of spat for aquaculture.
Phylogeography and molecular rates of subterranean aquatic Stenasellid Isopods with a peri‐Tyrrhenian distribution
Authors: Ketmaier V. , Argano R. , Caccone A.
Published: March 2003

The subterranean Isopods belonging to the genus Stenasellus have an interesting disjunct distribution in the peri‐Tyrrhenian area with morphologically closely related taxa occurring in Sardinia, Corsica, Tuscany and in the Pyrenees phreatic and interstitial waters. Because the dispersal capacities of these organisms are limited, their distribution has been associated traditionally with the tectonic events leading to the separation of the Sardinia–Corsica microplate from the Pyrenees and its subsequent movement towards the Italian peninsula. We sequenced a fragment of the mtDNA cytochrome oxidase I gene (COI) for multiple populations of the S. racovitzai species‐group (Corsica, Sardinia, Tuscany) and S. virei (Pyrenees). We found that multiple phylogenetic analyses always gave the same topology, which is consistent with the genetic relations found using allozyme data, and with the palaeogeography of the area. The molecular data suggest that a combination of vicariance and dispersal events explain most effectively the present distribution pattern of these organisms. We also calculated COI rates and calibrated them against absolute time, taking advantage of the availability of two geologically based time estimates. Rates on all substitutions are similar to those published for other crustaceans for the same COI fragment, including taxonomically and ecologically distant groups. Rates on third codon positions or on transversions are generally lower than those found in other crustaceans.
Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli
Authors: Bouveret Emmanuelle , Rigal Alain , Lazdunski Claude , Bénédetti Hélène
Published: March 2003

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N‐terminal domain of colicins is involved in the import process. The N‐terminal domains of colicins A and E1 have been shown to interact with TolA, and the N‐terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N‐terminal domain of all group A colicins, the ‘TolA box’, was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N‐terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N‐terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild‐type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N‐terminal domain interact with TolA and TolB. The colicin A N‐terminal domain was also shown to form a trimeric complex with TolA and TolB.
Octopine‐type Ti plasmids code for a mannopine‐inducible dominant‐negative allele of traR, the quorum‐sensing activator that regulates Ti plasmid conjugal transfer
Authors: Oger Philippe , Kim Kun‐Soo , Sackett Rebecca L. , Piper Kevin R. , Farrand Stephen K.
Published: March 2003

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchal signalling systems. Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium. Induction also requires an acyl‐homoserine lactone signal, called AAI, that is produced by the bacteria themselves. AAI is the co‐inducer for TraR, the transcriptional activator required for expression of the tra regulon. Octopine induces conjugation of the octopine–mannityl opine‐type Ti plasmids by regulating the expression of traR via OccR, the octopine‐dependent activator of the opine regulon. We have discovered a second traR‐like gene, trlR, on the octopine–mannityl opine‐type Ti plasmids pTi15955 and pTiR10. This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon. Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy‐terminal domain thought to constitute the DNA‐binding region of TraR. Expression of trlR inhibited octopine‐induced conjugation of pTi15955 and pTiR10 by suppressing the TraR‐mediated transcription of the tra and trb operons. Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR. Southern hybridizations indicated that several other Ti and opine‐catabolic plasmids contain more than one copy of genes homologous to traR. We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.
Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins
Authors: Das Anath , Xie Yong‐Hong
Published: March 2003

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T‐DNA transfer proteins, we constructed a transposon, Tn3phoA. The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty‐eight unique phoA‐positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N‐terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T‐DNA transport pore is presented.
Heat shock inducibility of an archaeal TATA‐like promoter is controlled by adjacent sequence elements
Authors: Thompson Dorothea K. , Daniels Charles J.
Published: March 2003

The expression of a heat‐inducible cct1 (chaperonin‐containing Tcp‐1) family member gene is regulated at the transcription level in the archaeon Haloferax volcanii. Transcriptional fusions of the cct1 promoter region with a yeast proline tRNA reporter gene were constructed to analyse the functional domains of this archaeal heat shock promoter. Both basal and heat‐induced transcription of the reporter gene was directed by an archaeal consensus TATA element (5′‐TTTATA‐3′) centred 25 bp upstream of the transcription start site. Deletion mutagenesis indicated that the 5′ boundary of the cct1 regulatory region mapped to position − 37. Nucleotide alignment with the 5′ flanking regions of two additional cct‐related genes identified in H. volcanii showed a high degree of sequence conservation between positions +1 and − 37, especially in and immediately surrounding the TATA element of the putative core promoter. Mutational analysis of conserved sequences demonstrated that basal and heat‐induced transcription required sequence elements located upstream and downstream of the TATA‐box. These findings indicate that the regulatory sequences involved in heat‐induced transcription lie within the core promoter region and suggest that the mechanism controlling heat shock gene expression in H. volcanii differs from the bacterial and eukaryal strategies.
Molecular characterization of LbpB, the second lactoferrin‐binding protein of Neisseria meningitidis
Authors: Pettersson Annika , Prinz Thorsten , Umar Arzu , Van Der Biezen Jenny , Tommassen Jan
Published: March 2003

The lbpA gene of Neisseria meningitidis encodes an outer membrane lactoferrin‐binding protein and shows homology to the transferrin‐binding protein, TbpA. Previously, we have detected part of an open reading frame upstream of lbpA. The putative product of this open reading frame, tentatively designated lbpBshowed homology to the transferrin‐binding protein TbpB, suggesting that the lactoferrrin receptor, like the transferrin receptor, consists of two proteins. The complete nucleotide sequence of lbpB was determined. The gene encodes a 77.5 kDa protein, probably a lipoprotein, with homology, 33% identity to the TbpB of N. meningitidis. A unique feature of LbpB is the presence of two stretches of negatively charged residues, which might be involved in lactoferrin binding. Antisera were raised against synthetic peptides corresponding to the C‐terminal part of the putative protein and used to demonstrate that the gene is indeed expressed. Consistent with the presence of a putative Fur binding site upstream of the lbpB gene, expression of both LbpA and LbpB was proved to be iron regulated in Western blot experiments. The LbpB protein appeared to be less stable than TbpB in SDS‐containing sample buffer. Isogenic mutants lacking either LbpA or LbpB exhibited a reduced ability to bind lactoferrin. In contrast to the lbpB mutant, the lbpA mutant was completely unable to use lactoferrin as a sole source of iron.
The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE‐independent manner
Authors: Fawcett Paul , Melnikov Alexandre , Youngman Philip
Published: March 2003

The process of bacterial cell division involves the assembly of a complex of proteins at the site of septation that probably provides both the structural and the cytokinetic functions required for elaboration and closure of the septal annulus. During sporulation in Bacillus subtilis, this complex of proteins is modified by the inclusion of a sporulation‐specific protein, SpoIIE, which plays a direct role in gene regulation and also has a genetically separable role in determining the gross structural properties of the specialized sporulation septum. We demonstrate by both green fluorescent protein (GFP) fusions and indirect immunofluorescence microscopy that SpoIIGA, a protein required for proteolytic cleavage of pro‐σE, is also targeted to the sporulation septum. Septal localization of SpoIIGA–GFP occurred even in the structurally abnormal septum formed by a SpoIIE null mutant. We also report the isolation of a spoIIGA homologue from Bacillus megaterium, a species in which the cells are significantly larger than those of B. subtilis. We have exploited the physical dimensions of the B. megaterium sporangium, in conjunction with wide‐field deconvolution microscopy, to construct three‐dimensional projections of sporulating cells. These projections indicate that SpoIIGA–GFP is initially localized in an annulus at the septal periphery and is only later localized uniformly throughout the septa. Localization was also detected in a B. subtilis spo0H null strain that fails to construct a spore septum. We propose that SpoIIGA is sequestered in the septum by an interaction with components of the septation machinery and that this interaction begins before the construction of the asymmetric septum.
Identification of the Rhodobacter sphaeroides SOS box
Authors: Fernández de Henestrosa Antonio R. , Rivera Eusebi , Tapias Angels , Barbé Jordi
Published: March 2003

Gel‐mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA–protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions −21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA–protein complexes in vitro as well as for the DNA damage‐mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild‐type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage‐mediated expression of this gene. The fact that the recA and uvrA wild‐type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.
In vitro interaction between components of the inner membrane complex of the maltose ABC transporter of Escherichia coli : modulation by ATP
Authors: Mourez Michaël , Jéhanno Muguette , Schneider Erwin , Dassa Elie
Published: March 2003

Interactions between domains of ATP‐binding cassette (ABC) transporters are of great functional importance and yet are poorly understood. To gain further knowledge of these protein–protein interactions, we studied the inner membrane complex of the maltose transporter of Escherichia coli. We focused on interactions between the nucleotide‐binding protein, MalK, and the transmembrane proteins, MalF and MalG. We incubated purified MalK with inverted membrane vesicles containing MalF and MalG. MalK bound specifically to MalF and MalG and reconstituted a functional complex. We used this approach and limited proteolysis with trypsin to show that binding and hydrolysis of ATP, inducing conformational changes in MalK, modulate its interaction with MalF and MalG. MalK in the reconstituted complex was less sensitive to protease added from the cytoplasmic side of the membrane, and one proteolytic cleavage site located in the middle of a putative helical domain of MalK was protected. These results suggest that the putative helical domain of the nucleotide‐binding domains is involved, through its conformational changes, in the coupling between the transmembrane domains and ATP binding/hydrolysis at the nucleotide‐binding domains.
Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine‐mediated iron uptake
Authors: Gaballa Ahmed , Baysse Christine , Koedam Nico , Muyldermans Serge , Cornelis Pierre
Published: March 2003

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC‐deficient mutant is also compromised in the production and utilization of pyoverdine, the high‐affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane‐spanning regions with three periplasmic loops. Site‐directed mutagenesis was used in order to assess the importance of some periplasm‐exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His‐61, Val‐62 and Pro‐63 in the first periplasmic loop, and Leu‐184, His‐185 and Gln‐186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp‐115 and Gly‐116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp‐127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp‐126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.