Reproductive isolating barriers between colour‐differentiated populations of an African annual killifish,Nothobranchius korthausae (Cyprinodontiformes)

Allopatric populations separated by vicariance events are expected to evolve reproductive isolating mechanisms as a result of disparate selection pressures and genetic drift. The appearance of reproductive isolating mechanisms may vary across taxa with differences in the opportunity for mate choice, and may be asymmetrical. In addition, premating barriers may be affected by individual mating experience. We used choice and no‐choice experiments to investigate reproductive isolation between two allopatric (island and mainland) and colour‐differentiated populations of an African annual fish, Nothobranchius korthausae. Assortative mating under experimental conditions was limited and asymmetrical. Preference for sympatric males was only expressed in nonvirgin females from one population. Virgin fish from both populations mated indiscriminately. No difference in the number of eggs laid, fertilization rate and hatching success was detected in no‐choice experiments. All mating combinations produced viable offspring and no postmating barriers were detected in terms of the performance and fertility of F1 hybrids. Overall, we found little evidence for significant reproductive isolation, which is in contrast with the related killifish taxa in which assortative mating can be strong, even among allopatric populations with no colour differentiation. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 62–72.     

Female choice in Drosophila: evidence from Hawaii and implications for evolutionary biology

Details of female choice of mate in Drosophila silvestris of Hawaii strikingly parallels epigamic behavioral systems in many other animals and may be common in other species of Drosophilidae. Females respond selectively to male circling, wing displays, songs and tactile stimulation with foreleg cilia, a quantitative character that is highly variable in some populations. I hypothesize that the female can exert choice based on these cues from individual males that differ genetically by quantitative trait loci. Laboratory tests show that one third of courting males are repeatedly rejected in favor of a minority of alpha males. This result imposes non-random mating at the local population level. Past multiple-choice lab tests, widely used to measure isolation between pairs of populations or species of Drosophila may be flawed, since random mating has been assumed in the interpretation of results. Pre-mating sexual selection is clearly a powerful intrapopulation force in population biology. This view creates difficulties for discerning any proposed simultaneous interpopulation selective events in the presence of strong female choice. The long-held theory assuming that there is significant selection for pre-mating isolation between groups is questionable.     

Evaluation of pre‐ and post‐zygotic mating barriers,hybrid fitness and phylogenetic relationship between Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (Cyprinodontiformes,Teleostei)

The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l^?1, but incapable of long‐term survival in freshwater (< 2 mM Na⁺). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4–1 mM Na⁺) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na⁺ uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre‐ and post‐zygotic isolation, Na⁺ transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con‐specific males in choice mating experiments. Partial post‐zygotic isolation was also observed with significant (59–89%) reductions in hatching success of hybrid embryos compared with con‐specific embryos. Na⁺ uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short‐term (96 h) survival of juveniles acutely transferred from 7 mM Na⁺ to a range of more dilute (0.1–2 mM Na⁺) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.     

Effects of population size on genetic diversity and seed production in the rare <Emphasis Type="Italic">Dictamnus albus</Emphasis>(Rutaceae) in central Germany

It is widely assumed that population size significantly affects the dynamics of plant populations. Smaller populations are threatened by genetic drift and inbreeding depression, both of which may result in a decrease of genetic variation and a resulting negative impact on plant fitness. In our study we analysed the patterns of random amplified polymorphic DNA (RAPD) variation among 10 Dictamnus albuspopulations of varying size. The aim was to examine local differentiation in relation to spatial isolation resulting from limited population size and geographical distancing between populations. Significant correlations were noted between population size and both percentage of polymorphic loci (P <0.01) and genetic diversity (P<0.01). The matrix correlation between genetic and geographical distances revealed that geographical differentiation was reflected in the RAPD profile (Mantel test: r²=0.34, P<0.001). We found the highest level of molecular variance of RAPD patterns among individuals within the populations (72.6%), whereas among-population variation accounted for only 21.6% of variation. These results were highly significant in that they indicated a restricted population differentiation, as would be expected from outcrossing species. An additional analysis of seed production showed that there was significant variation among populations in terms of mean seed number per flower and mean seed mass per population which could be attributed to differences in population size as well as levels of genetic variation.     

Geographic contrasts between pre‐ and postzygotic barriers are consistent with reinforcement in Heliconius butterflies

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.     

Postglacial loss of microsatellite variation in the landlocked Lake Saimaa ringed seal

The Lake Saimaa ringed sealPhoca hispida saimensis has lived as anisolated landlocked population in easternFinland since the early post-glacial. In thelast century, the population crashed down to c.200 individuals, and is under a constant threatof extinction. We evaluated the genetic historyof the Saimaa population through a comparisonwith the conspecific sister populations in theArctic Ocean and the Baltic Sea, which haveretained high levels of variation since thedeglaciation. At eight microsatellite loci, thecurrent gene diversity (heterozygosity) of theSaimaa seal was 69% lower than in thereference populations. Allowing reasonablemutation rates ( = 10^–4), thisimplies a long-term post-glacial effectivepopulation size of N _e 350,and a slow average rate of inbreeding F 0.15% per generation during the c.860 generations (9 500 years) of isolation. Thecurrent N _e is an order of magnitudesmaller and F correspondinglylarger. Whereas the additional loss of markervariation in the short term will not be highrelative to that already taken place, it seemsunwarranted to suppose that the past, slowinbreeding would have effectively purged thepopulation of genetic load and reduced thegenetic risks from small population size.Although the population is now clearlygeographically subdivided in the complex lakesystem, we found little genetic differentiationbetween main breeding areas (F _ST =0.02). However, at the current low populationdensities, the subdivision may markedly furtherincrease the future rate of inbreeding.     

Chromosome replacement in mixed populations of compound-2L; free-2R and standard strains of Drosophila melanogaster

Summary Crosses between compound-2L; free-2R (free-arm) and standard strains of Drosophila melanogaster produce two classes of inviable aneuploid hybrids in equal proportions: monosomic 2L and trisomic 2L. The lethal period for monosomics occurs during embryogenesis while the trisomics survive to late pupae. Since the hybrids are inviable, standard and free-arm strains within a mixed population remain genetically isolated. Genetic isolation in the absence of mating isolation offers an extreme example of unstable equilibrium. Relative fitness data indicate that an unstable equilibrium will be established between free-arm and standard strains at a ratio of 2.51. Indeed, in three cage experiments established at initial ratios of 31, free arms to standards, laboratory (Oregon R) or native (Okanagan S) standard strains were completely replaced in approximately 100 days by free-arm lines derived either from laboratory or from native genetic background. In contrast, one cage established at an initial ratio of 41 failed to show replacement and for 92 days remained at approximately the initial ratio. Subsequent genetic analysis of flies removed from this cage identified the presence of an anomalous strain through which genetic information was transferred reciprocally between the free-arm and standard lines. The second chromosomes carried by this strain consisted of a free-2R and a standard second on the right arm of which was attached a duplication for all of 2L. While the origin of the 2L·2R+2L chromosome was uncertain, genetic and cytological examinations revealed that it represented the reciprocal crossover product expected from an exchange that generated a F(2R). Additional crosses disclosed that the transmission frequency of the asymmetrical pair of second chromosomes, as well as their right-arm crossover products, was disproportionately in favor of the short arm. Since unequal transmission was invariably greater from female parents, this phenomenon was viewed as further evidence in support of the drag hypothesis.Supported by research grant A5853 from the National Science and Engineering Research council of Canada to D.G.H.     

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.     

Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population “A” (Fusarium moniliforme)

We examined 25 strains of Fusarium moniliforme from eight states known to be associated with equine leukoencephalomalacia, a disease caused by the mycotoxin fumonisin B₁. We determined the mating population, mating type, and vegetative compatibility group to which each of these strains belonged. All 25 strains were in the A mating population; 12 were A⁺ and 13 were A^–. Seventeen of the 25 strains were female fertile; these strains also averaged higher levels of fumonisin B₁ production than did the strains that were female sterile. Nitrate non-utilizing (nit) mutants were generated in all 25 strains and each strain was assigned to a unique vegetative compatibility group based on the inability of the derived nit mutants to form a prototrophic heterokaryon with complementary nit mutants derived from any of the other strains examined. From these data, we concluded that the production of fumonisin B₁ is a general characteristic of strains from the A mating population of Gibberella fujikuroi associated with equine leukoencephalomalacia, since all 25 of the isolates that we examined were genetically distinct individuals.     

The context dependence of assortative mating: a demonstration with conspecific salmonid populations

Assortative mating is thought to play a key role in reproductive isolation. However, most experimental studies of assortative mating do not take place in multiple natural environments, and hence, they ignore its potential context dependence. We implemented an experiment in which two populations of brown trout (Salmo trutta) with different natural flow regimes were placed into semi‐natural stream channels under two different artificial flow regimes. Natural reproduction was allowed, and reproductive isolation was measured by means of parentage assignment to compare within‐population vs. between‐population male–female mating and relative offspring production. For both metrics, reproductive isolation was highly context dependent: no isolation was evident under one flow regime, but strong isolation was evident under the other flow regime. These patterns were fully driven by variance in the mating success of males from one of the two populations. Our results highlight how reproductive isolation through assortative mating can be strongly context dependent, which could have dramatic consequences for patterns of gene flow and speciation under environmental change.     

Hybridisation and lack of prezygotic barriers between Phymata pennsylvanica and americana

1. The persistence of both geographical and reproductive boundaries between related species poses a fundamental puzzle in biology. Reproductive interactions between species can have a substantial impact on the maintenance of a boundary, potentially contributing to its collapse (e.g. via hybridisation) or facilitating reproductive isolation (e.g. via reinforcement). 2. The degree to which two parapatric insect species in the genus Phymata are reproductively isolated was evaluated and several mechanisms that could contribute to the maintenance of species boundaries were assessed. 3. Behavioural assays showed no indication of species‐assortative mating, nor any fecundity costs associated with heterospecific mating. Thus, there was no evidence of prezygotic mechanisms of reproductive isolation between the two species. 4.In laboratory crosses, it was found that the two species were indeed capable of producing viable F1 hybrids. Morphologically, these hybrids were phenotypically intermediate to the two parental species, and similar to the phenotypes seen in natural populations thought to occur in a hybrid zone. F1 hybrids did not show reduced viability, although there was some suggestion of ‘hybrid breakdown’, evident from the lower viability observed for progeny of ‘natural hybrids’. 5. Collectively, we show that despite genetically based morphological differences between species, P. americana and pennsylvanica can, and probably do hybridise. More studies are needed to understand the mechanisms that maintain the distinct phenotypes and geographical ranges of these species, despite the considerable potential for introgression.     

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura

Reinforcement occurs when hybridization between closely related lineages produces low‐fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male–female interactions than interactions with heterospecifics.     

Impact of experimental design on Drosophila sexual isolation studies: direct effects and comparison to field hybridization data

Abstract Many studies of speciation rely critically on estimates of sexual isolation obtained in the laboratory. Here we examine the sensitivity of sexual isolation to alterations in experimental design and mating environment in two sister species of Drosophila, D. santomea and D. yakuba. We use a newly devised measure of mating frequencies that is able to disentangle sexual isolation from species differences in mating propensity. Variation in fly density, presence or absence of a quasi‐natural environment, degree of starvation, and relative frequency of species had little or no effect on sexual isolation, but one factor did have a significant effect: the possibility of choice. Designs that allowed flies to choose between conspecific and heterospecific mates showed significantly more sexual isolation than other designs that did not allow choice. These experiments suggest that sexual isolation between these species (whose ranges overlap on the island of STo Tomé) is due largely to discrimination against D. yakuba males by D. santomea females. This suggestion was confirmed by direct observations of mating behavior. Drosophila santomea males also court D. yakuba females less ardently than conspecific females, whereas neither males nor females of D. yakuba show strong mate discrimination. Thus, sexual isolation appears to be a result of evolutionary changes in the derived island endemic D. santomea. Surprisingly, as reported in a companion paper (Llopart et al. 2005), the genotypes of hybrids found in nature do not accord with expectations from these laboratory studies: all F₁ hybrids in nature come from matings between D. santomea females and D. yakuba males, matings that occur only rarely in the laboratory.     

Outbreeding depression in the common frog, <Emphasis Type="Italic">Rana temporaria</Emphasis>

Theory suggests that parental relatedness is a continuous variable with a fitness optimum that we heretoforth will refer to as optimal outbreeding. In the present paper, we test this proposition from a conservation (translocation) perspective. Amphibians are facing a global decline and many amphibian populations are today small and threatened by extinction. Because genetic differentiation is often high between amphibian populations, they could be particularly sensitive to outbreeding depression, e.g. due to breakdown of locally adapted gene complexes. We tested if outbreeding would reduce fitness in common frogs, Rana temporaria, crossed from a large and an isolated, small population, separated by 130km, using artificial fertilization. For females from the large population, tadpoles were significantly smaller and more malformed in crosses with males from the small population, than with males from the large population. For the small population, however, no significant paternal genetic effects could be found. The difference in response to outbreeding between populations was accompanied with significant differences in the importance of maternal effects. We conclude that care should be taken when translocating frogs between distantly related populations to avoid outbreeding depression.     

Hybridization and sequential components of reproductive isolation between parapatric walnut‐infesting sister species Rhagoletis completa and Rhagoletis zoqui

Hybridization can provide a window into how populations diverge to form new species. Here, we confirm hybridization between Rhagoletis completa Cresson, 1929 and Rhagoletis zoqui Bush, 1966, two species of walnut husk‐infesting flies that geographically overlap in a narrow area of parapatry in Northeastern Mexico. Rhagoletis completa and R. zoqui are members of a species group (Rhagoletis suavis) that has been hypothesized to speciate in allopatry. Sexual selection has been argued to be a potentially important factor for generating pre‐mating isolation among walnut husk flies, because of the differences in wing morphology and body coloration, and the existence of sexual dimorphism within species. However, there was no evidence for pre‐mating isolation between R. completa and R. zoqui, based on choice and no‐choice mating experiments conducted on adults of fly populations outside the contact zone. There was also no support for reduced fertility of hybrid matings or for F₁ inviability; however, F₁ hybrids appeared to have lower fertility and F₂ offspring have reduced survivorship. Postzygotic isolation in later generation hybrids of mixed ancestry therefore appears to be the first intrinsic barrier to gene flow evolving between R. completa and R. zoqui. We discuss the implications of our results for allopatric speciation in walnut flies and the potential evolutionary fate of R. zoqui and R. completa if they were to come into broad geographic contact in the future. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting

Amentotaxus, a genus of the Taxaceae, represents an ancient lineage that has long existed in Eurasia. All Amentotaxus species experienced frequent population expansion and contraction over periodical glaciations in Tertiary and Quaternary. Among them, Amentotaxus argotaenia complex consists of three morphologically alike species, A. argotaenia, Amentotaxus yunnanensis, and Amentotaxus formosana. This complex is distributed in the subtropical region of mainland China and Taiwan where many Pleistocene refugia have been documented. In this study, genetic diversity and population structuring within and between species were investigated based on the inter-simple sequence repeats (ISSR) fingerprinting. Mean genetic diversity within populations was estimated in three ways: (1) the percentage of polymorphic loci out of all loci (P) (2) Neis unbiased expected heterozygosity (He), and (3) Shannons index of phenotypic diversity. For a total of 310 individuals of 15 populations sampled from the three species, low levels of ISSR genetic variation within populations were detected, with P=4.66–16.58%, He=0.0176–0.0645 and Hpop=0.0263–0.0939, agreeing with their seriously threatened status. AMOVA analyses revealed that the differences between species only accounted for 27.38% of the total variation, whereas differences among populations and within populations were 57.70 and 14.92%, respectively, indicating substantial isolation between the patch-like populations. A neighbor-joining tree identified a close affinity between A. yunnanensis and A. formosana. Genetic drift due to small population size, plus limited current gene flow, resulted in significant genetic structuring. Low levels of intrapopulational genetic variation and considerable interpopulational divergence were also attributable to demographic bottlenecks during and/or after the Pleistocene glaciations.     

Hybridization as a source of evolutionary novelty: leaf shape in a Hawaiian composite

Hybridization is increasingly recognized as a significant creative force in evolution. Interbreeding among species can lead to the creation of novel genotypes and morphologies that lead to adaptation. On the Hawaiian island of Oahu, populations of two species of plants in the endemic genus Lipochaeta grow at similar elevations in the northern Waianae Mountains. These two species represent extremes of the phenotypic distribution of leaf shape: the leaves of Lipochaeta tenuifolia individuals are compound and highly dissected while leaves of L. tenuis are simple. Based primarily on leaf shape morphology, a putative hybrid population of Lipochaeta located at Puu Kawiwi was identified. Individuals in this population exhibit a range of leaf shapes intermediate in varying degrees between the leaf shapes of the putative parental species. We analyzed individuals from pure populations of L. tenuifolia, L. tenuis and the putative hybrids using 133 AFLP markers. Genetic analysis of these neutral markers provided support for the hybrid origin of this population. The correlation between genetic background and leaf morphology in the hybrids suggested that the genome of the parental species with simple leaves might have significantly contributed to the evolution of a novel, compound leaf morphology.     

Increased inbreeding and inter-species gene flow in remnant populations of the rare <Emphasis Type="Italic">Eucalyptus benthamii</Emphasis>

Eucalyptus benthamii Maiden & Cambage is a forest tree of interest for conservation and plantation forestry. It is vulnerable to extinction, occurring on the alluvial floodplains of the Nepean River and its tributaries, south-west of Sydney, Australia. These floodplains were largely cleared of native vegetation for agriculture by the mid-1800s. Flooding of the Cox Valley for Sydneys water supply further decreased the species distribution. The species is now confined to one population of approximately 6500 trees in the Kedumba valley and three remnant populations on the Nepean River at Bents Basin (about 300 trees), Wallacia (nine trees) and Camden (about 30 trees). Genetic analysis of the four populations using microsatellite markers revealed significant divergence among all populations, despite the Bents Basin, Wallacia and Camden remnants being separated by distances of only a few kilometres. Trees in these populations have been estimated to range from 35 to 200 years old, suggesting genetic divergence among populations occurred prior to land clearing. To investigate the impact of fragmentation on the next generation, outcrossing rates were estimated from 41 families. While no direct relationship was found between population size and outcrossing rates, fragmentation and the isolation of trees appears to have resulted in higher levels of selfing and biparental inbreeding in seed collected from the Camden and Wallacia remnants. There was also evidence from seedling morphology that inter-species gene flow increased with fragmentation since 20% of the progeny from Camden and 30% of the progeny from Wallacia were hybrids. Seed viability and germination rates were significantly lower in the remnant populations, reducing their value as seed sources for regeneration and plantation forestry. To maintain the genetic integrity of the remnant populations, germplasm should be sourced from the local area. Outcrossed, non-hybrid seed could be produced by controlled pollination in ex-situ conservation stands or by using seedling morphology and microsatellites to screen seedlings from the remnant populations.     

<Emphasis Type="Italic">Fusarium</Emphasis> species of the <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis> complex and fumonisin contamination of pearl millet and corn in Georgia,USA

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and ODonnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB₁ and FB₂ were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 g/g.     

A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures

Isochrysis galbana, one of the most widely usedmarine microalgae in the rearing of finfish and shellfish larvae, is masscultured frequently in outdoor tanks. Under prolonged and repeated culture,severe contamination occurs. Axenic isolation of I.galbanafrom such cultures was best achieved by using a ternary procedure involvingpercoll-gradient centrifugation, treatment with antibiotics, and growth on agarmedium. Protozoa and other algae were removed most effectively by isolation ofI. galbana at the 30–40% density layer on apercoll-gradient. Removal of bacteria was accomplished using a mixture of 5antibiotics (250 g mL^–1 ampicillin, 50g mL^–1 gentamycin, 100 gmL^–1 kanamycin, 500 gmL^–1 neomycin, 50 gmL^–1 streptomycin). Axenic colonies were isolated fromasolid medium prepared from 1% purified agar. The ternary procedure isconsideredapplicable to the isolation of other axenic single-celled microalgae fromheavily contaminated cultures.