THE GENETIC BASIS OF SEXUAL ISOLATION BETWEEN DROSOPHILA MELANOGASTER AND D. SIMULANS

The genetic analysis of sexual isolation between the closely-related species Drosophila melanogaster and Drosophila simulans involved two experiments with no-choice tests. The efficiency of sexual isolation was measured by the frequency of courtship initiation and interspecific mating. We first surveyed the variation in sexual isolation between D. melanogaster strains and D. simulans strains of different geographic origin. Then, to investigate variation in sexual isolation within strains, we made F₁ diallel sets of reciprocal crosses within strains of D. melanogaster and D. simulans. The F₁ diallel progeny of one sex were paired with the opposite sex of the other species. The first experiment showed significant differences in the frequency of interspecific mating between geographic strains. There were more matings between D. simulans females and D. melanogaster males than between D. melanogaster females and D. simulans males. The second experiment uncovered that the male genotypes in the D. melanogaster diallel significantly differed in interspecific mating frequency, but not in courtship initiation frequency. The female genotypes in the D. simulans diallel were not significantly different in courtship initiation and interspecific mating frequency. Genetic analysis reveals that in D. melanogaster males sexual isolation was not affected by either maternal cytoplasmic effects, sex-linked effects, or epistatic interaction. The main genetic components were directional dominance and overdominance. The F₁ males achieved more matings with D. simulans females than the inbred males. The genetic architecture of sexual isolation in D. melanogaster males argues for a history of weak or no selection for lower interspecific mating propensity. The behavioral causes of variation in sexual isolation between the two species are discussed.     

Optimal Strategy for Competence Differentiation in Bacteria

A phylogenetically diverse subset of bacterial species are naturally competent for transformation by DNA. Transformation entails recombination of genes between different lineages, representing a form of bacterial sex that increases standing genetic variation. We first assess whether homologous recombination by transformation is favored by evolution. Using stochastic population genetic computer simulations in which beneficial and deleterious mutations occur at many loci throughout the whole genome, we find that transformation can increase both the rate of adaptive evolution and the equilibrium level of fitness. Secondly, motivated by experimental observations of Bacillus subtilis, we assume that competence additionally entails a weak persister phenotype, i.e., the rates of birth and death are reduced for these cells. Consequently, persisters evolve more slowly than non-persisters. We show via simulation that strains which stochastically switch into and out of the competent phenotype are evolutionarily favored over strains that express only a single phenotype. Our model''s simplicity enables us to derive and numerically solve a system of finite- deterministic equations that describe the evolutionary dynamics. The observed tradeoff between the benefit of recombination and the cost of persistence may explain the previously mysterious observation that only a fractional subpopulation of B. subtilis cells express competence. More generally, this work demonstrates that population genetic forces can give rise to phenotypic diversity even in an unchanging and homogeneous environment.     

Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells.

Lysogenic strains of Bacillus subtilis 168 were reduced in their level of transformation as compared to non-lysogenic strains. The level of transformation decreased even further if the competent lysogenic cells were allowed to incubate in growth media prior to selection on minimal agar. This reduction in the frequency of transformation was attributable to the selective elimination of transformed lysogenic cells from the competent population. Concurrent with the decrease in the number of transformants from a lysogenic competent population was the release of bacteriophage by these cells. The lysogenic bacteria demonstrated this dramatic release of bacteriophage only if the cells were grown to competence. Both the selective elimination of transformed lysogens and the induction of prophage was prevented by the inhibition of protein synthesis. Additionally, competent lysogenic cells released significantly higher amounts of exogenous donor transforming deoxyribonucleic acid than did competent non-lysogenic cells or competent lysogenic cells incubated with erythromycin. These data establish that the induction of the prophage from the competent lysogenic cells was responsible for the selective elmination of the lysogenic transformants. A model is presented that accounts for the induction of the prophage from competent lysogenic bacteria via the induction of a repair system. It is postulated that a repair system is induced or derepressed by the accumulation of gaps in the chromosomes of competent bacteria. This hypothetical enzyme(s) is ultimately responsible for the induction of the prophage and the selective elimination of transformants.     

Can Ca2+-dependent competence be repeatedly induced in the same Escherichia coli cells?

Summary With the help of devised multicycle consecutive transformation (MCT) it is shown that Ca²⁺-dependent competence can be repeatedly induced in the same population of Escherichia coli cells. The same fraction of cells is induced to competence and transformed during MCT. In contrast to the results on classical transformation with mixed DNA preparations, no double transformants are observed in MCT. The competent cells and transformants are found to be more fragile than nontransformed cells. The latter are represented presumably by the cells that have not absorbed exogenous plasmid DNA. The results suggest that there is strong interference between plasmid DNAs during MCT, and that the presence of exogenous DNA makes the cells more sensitive to the apparently harmful procedure of repeated competence induction.     

Transformation and transduction of a large deletion mutation in Bacillus subtilis

Summary Large arsenate sensitive deletion mutations of B. subtilis 168 are transformed to wild type at least 100 times less efficiently than they are transduced by PBS1 phage. The lower transformation efficiency is apparently due to the degradation of the donor DNA by competent cells to fragments ineffective for repair of long deletions. The efficiency of transduction of deletion mutations is also reduced when competent cells, rather than logarithmically growing cells are used as recipients. An endonuclease activity specifically associated with the physiological state of competence is proposed to explain these observation.     

Phylogeography of the ground squirrel subgenus Xerospermophilus and assembly of the Mojave Desert biota

Aim The Mohave ground squirrel (Xerospermophilus mohavensis) is one of a few endemic species of the Mojave Desert of south‐western North America. We describe phylogeographic patterns within this species and its sister taxon (Xerospermophilus tereticaudus) and test hypotheses concerning their biogeographical history using genetic signatures of stable versus expanding populations. We compare these patterns with those of other Mojave species to evaluate the role of vicariance in producing phylogeographic structure during the assembly of the Mojave Desert biota. Location The Mojave Desert and adjacent desert regions of south‐western North America. Methods Complete cytochrome b gene sequences of X. mohavensis (46 individuals representing 11 localities) and X. tereticaudus (38 individuals representing 14 localities) were analysed using Bayesian methods to infer phylogenetic relationships. Genetic signals of stable or expanding populations were examined based on the distribution of recent mutations and pairwise differences, as well as with a coalescent‐based approach. Results The two species are reciprocally monophyletic and may have diverged in response to the late Pliocene–early Pleistocene uplift of the Transverse Ranges and Mojave block. Little phylogeographic structure is evident within X. mohavensis, but there is a signature of northern expansion from a presumably full‐pluvial refugium in the Mojave River basin. Four geographic subgroups are evident within X. tereticaudus, and there is a signature of northern expansion from a presumably full‐pluvial refugium in the Sonoran coastal plains. Roughly congruent phylogeographic patterns are found within five arid‐adapted taxa, indicating a strong element of vicariance during the assembly of the generally transitional Mojave Desert biota. Main conclusions We present a preliminary model for the historical assembly of the Mojave Desert biota that indicates a strong vicariant element producing autochthonous lineages (including X. mohavensis) that diverged during the major geological and climatic events of the last 5 Myr. Phylogeographic partitioning within the Mojave Desert underscores the necessity of immediate conservation measures for this unique and fragile arid ecosystem that is locked between two large metropolitan population centres and is the target of continued adverse environmental impact.     

Evidence for no sexual isolation between Drosophila albomicans and D. nasuta

Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species‐specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple‐choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.     

Patterns of reproductive isolation within and between two Lygaeus species characterized by sexual conflicts over mating

Theory suggests that, under some circumstances, sexual conflict over mating can lead to divergent sexually antagonistic coevolution among populations for traits associated with mating, and that this can promote reproductive isolation and hence speciation. However, sexual conflict over mating may also select for traits (e.g. male willingness to mate) that enhance gene flow between populations, limiting population divergence. In the present study, we compare pre‐ and post‐mating isolation within and between two species characterized by male–female conflict over mating rate. We quantify sexual isolation among five populations of the seed bug Lygaeus equestris collected from Italy and Sweden, and two replicates of a population of the sister‐species Lygaeus simulans, also collected from Italy. We find no evidence of reproductive isolation amongst populations of L. equestris, suggesting that sexual conflict over mating has not led to population divergence in relevant mating traits in L. equestris. However, there was strong asymmetric pre‐mating isolation between L. equestris and L. simulans: male L. simulans were able to mate successfully with female L. equestris, whereas male L. equestris were largely unable to mate with female L. simulans. We found little evidence for strong post‐mating isolation between the two species, however, with hybrid F₂ offspring being produced. Our results suggest that sexual conflict over mating has not led to population divergence, and indeed perhaps supports the contrary theoretical prediction that male willingness to mate may retard speciation by promoting gene flow.     

How isolated are Pleistocene refugia? Results from a study on a relict woodrat population from the Mojave Desert,California

Pleistocene vicariance is often invoked to explain the disjunct populations of animals in habitat refugia throughout the southwestern United States. The combined effects of small population size and isolation from the rest of the contiguous range are thought to result in genetic differentiation of relict organisms. Here, we describe a relict population of dusky‐footed woodrats (Neotoma fuscipes Baird) found in a pinyon‐juniper‐oak community in a small mountain range within the Mojave Desert. We compare morphological and genetic data for these individuals with two populations within the contiguous range, and with another species of woodrat (Neotoma lepida). We also examine the distributional overlap between contemporary oak species and dusky‐footed woodrats, and estimate the potential oak woodland habitat available during the late Quaternary. As expected, both the morphological and genetic analysis confirm that the relict population is N. fuscipes. Within the limitations of our data, we detect no evidence of differentiation. Instead, the relict population forms a paraphyletic group with the nearest population within the contiguous range. This may be explained by the combined influences of a shorter period of isolation and a greater effective population size than was originally expected. The linkage between contemporary oak and dusky‐footed woodrat distributions is very tight, reinforcing the idea of an obligate relationship between the two. We estimate that at ~8000 ybp, pinyon‐juniper‐oak woodlands may have covered ~53% of the central Mojave, forming large contiguous areas of habitat. Although considerably more fragmented, at present ~12% of the area consists of relict woodlands. Our results suggest that there may be numerous other woodrat refugia, with a relatively high degree of connectiveness between the larger ones. Animals within them may effectively function as a single metapopulation, buffering against occasional stochastic extinction events.     

Characterization of a novel South American population of the astaxanthin producing yeast <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> (<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>)

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Inheritance genetics of the trait vector competence in Frankliniella occidentalis (Western flower thrips) in the transmission of Tomato spotted wilt virus

The complexity of tospovirus–vector–host plant interaction is linked to a range of factors influencing vector's efficacy in virus transmission, leading to high variability in the transmission efficiency within vector populations. Main shortcomings of most studies are the missing information on the intrinsic potential of individual insects to serve as efficient vectors, both at phenotypic and at genotypic levels. Moreover, detailed analysis of vector competence heredity and monitoring the splitting of both genotypes and phenotypes in filial generations has not been reported. In this study, using the model system Frankliniella occidentalis and Tomato spotted wilt virus, we evaluated the inheritance and stability of the trait vector competence in a population through basic crossings of individually characterized partners, as well as virgin reproduction. We hypothesized that the trait is heritable in F. occidentalis and is controlled by a recessive allele. From the results, 83% and 94% of competent and noncompetent males respectively, inherited their status from their mothers. The trait was only expressed when females were homozygous for the corresponding allele. Furthermore, the allele frequencies were different between males and females, and the competent allele had the highest frequency in the population. These suggest that the trait vector competence is inherited in single recessive gene in F. occidentalis, for which the phenotype is determined by the haplodiploid mechanism. These findings are fundamental for our understanding of the temporal and spatial variability within vector populations with respect to the trait vector competence and at the same time offer an essential basis for further molecular studies.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

Sex at the origin: an Asian population of the rice blast fungus Magnaporthe oryzae reproduces sexually

Sexual reproduction may be cryptic or facultative in fungi and therefore difficult to detect. Magnaporthe oryzae, which causes blast, the most damaging fungal disease of rice, is thought to originate from southeast Asia. It reproduces asexually in all rice‐growing regions. Sexual reproduction has been suspected in limited areas of southeast Asia, but has never been demonstrated in contemporary populations. We characterized several M. oryzae populations worldwide both biologically and genetically, to identify candidate populations for sexual reproduction. The sexual cycle of M. oryzae requires two strains of opposite mating types, at least one of which is female‐fertile, to come into contact. In one Chinese population, the two mating types were found to be present at similar frequencies and almost all strains were female‐fertile. Compatible strains from this population completed the sexual cycle in vitro and produced viable progenies. Genotypic richness and linkage disequilibrium data also supported the existence of sexual reproduction in this population. We resampled this population the following year, and the data obtained confirmed the presence of all the biological and genetic characteristics of sexual reproduction. In particular, a considerable genetic reshuffling of alleles was observed between the 2 years. Computer simulations confirmed that the observed genetic characteristics were unlikely to have arisen in the absence of recombination. We therefore concluded that a contemporary population of M. oryzae, pathogenic on rice, reproduces sexually in natura in southeast Asia. Our findings provide evidence for the loss of sexual reproduction by a fungal plant pathogen outside its centre of origin.     

A simple method for the production of highly competent cells ofAgrobacterium for transformation via electroporation

The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations ofAgrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformedAgrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of threeArgobacterium tumefaciens (EHA101, LBA4404, C58) and twoArgobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin^res, kanamycin^res, and gentamycin^res).     

Speciation,population structure,and demographic history of the Mojave Fringe‐toed Lizard (Uma scoparia), a species of conservation concern

The North American deserts were impacted by both Neogene plate tectonics and Quaternary climatic fluctuations, yet it remains unclear how these events influenced speciation in this region. We tested published hypotheses regarding the timing and mode of speciation, population structure, and demographic history of the Mojave Fringe‐toed Lizard (Uma scoparia), a sand dune specialist endemic to the Mojave Desert of California and Arizona. We sampled 109 individual lizards representing 22 insular dune localities, obtained DNA sequences for 14 nuclear loci, and found that U. scoparia has low genetic diversity relative to the U. notata species complex, comparable to that of chimpanzees and southern elephant seals. Analyses of genotypes using Bayesian clustering algorithms did not identify discrete populations within U. scoparia. Using isolation‐with‐migration (IM) models and a novel coalescent‐based hypothesis testing approach, we estimated that U. scoparia diverged from U. notata in the Pleistocene epoch. The likelihood ratio test and the Akaike Information Criterion consistently rejected nested speciation models that included parameters for migration and population growth of U. scoparia. We reject the Neogene vicariance hypothesis for the speciation of U. scoparia and define this species as a single evolutionarily significant unit for conservation purposes.     

Structural features of DNA in competent Bacillus subtilis

Summary For efficient transformation with B. subtilis, recipient cells must be grown to the state termed competence. Previous findings indicated that such competent cells contained DNA which exhibited about 5% single-strandedness. In this work, the physico-chemical properties of this DNA are compared to artificially nicked DNA. Evidence is presented that breakdown of the host DNA occurs during growth to competence. Inhibition of this breakdown also prevents the formation of partially single-stranded chromosomes within the competent cells. Use of this DNA as donor in transformation studies indicated a deficiency in biological activity within specific genes. Of three models considered, it is concluded that the results are best explained by the occurrence of single-stranded gaps within the chromosomes of competent cells.     

Spontaneous Transformation and Its Use for Genetic Mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.     

Sexual isolation with and without ecological isolation in marine isopods Jaera albifrons and J. praehirsuta

Sexual barriers associated with mate choice are often found to be associated with some level of ecological isolation between species. The independence and relative strength of sexual isolation are thus difficult to assess. Here, we take advantage of a pair of marine isopod species (Jaera albifrons and J. praehirsuta) that show sexual isolation and coexist in populations where they share the same microhabitat or not (i.e. without or with ecological isolation). We estimated the strength of sexual isolation between J. albifrons and J. praehirsuta using no‐choice trials and a multiple‐choice experimental population. We found that sexual isolation is strong in both the presence and the absence of ecological isolation, but that it is asymmetric and fails to prevent interspecific gene flow entirely. First‐generation intrinsic post‐zygotic barriers were low, and there was no sexual isolation within J. praehirsuta across habitats. The J. albifrons/J. praehirsuta species pair thus provides an example where the role of sexual isolation as a barrier to gene flow (a) does not depend upon current ecological isolation, (b) seems to have evolved independently of local ecological conditions, but (c) is insufficient to complete speciation entirely on its own.