PHYLOGENETIC ANALYSIS OF BREEDING SITE USE AND α-AMANITIN TOLERANCE WITHIN THE DROSOPHILA QUINARIA SPECIES GROUP

The Drosophila quinaria group is unusual within the genus in that it comprises both mycophagous and nonmycophagous species. DNA sequence data from three regions of the mitochondrial genome were used to infer relationships among four mycophagous species and three that breed on decaying water plants. Phylogenetic analysis of these species show that breeding in mushrooms and tolerance of high levels of α-amanitin were the ancestral states within the group. Thus, breeding in decaying water plants and intolerance of α-amanitin are derived conditions. We also found that the D. quinaria species group does not comprise separate mycophagous and nonmycophagous clades, but rather that (1) the shift from mushrooms to decaying plants occurred on at least two occasions; or (2) mycophagy reevolved within a lineage that had previously shifted to breeding on plants. The correlation between mycophagy and α-amanitin tolerance is perfect across the species we have examined, indicating that there is no detectable time lag between an ecological shift to a new breeding site and correlated changes in biochemical adaptation. The genetic distance between the mycophagous D. recens and the nonmycophagous D. quinaria indicates that these species split only about 1 M.Y.B.P. In terms of α-amanitin tolerance, D. recens and D. quinaria are typical of other ecologically similar species within the group. Thus, evolutionary changes in α-amanitin tolerance can evidently occur on the order of about 1 million yr. Our data also indicate that, in comparison to other groups of Drosophila, the quinaria species group may be undergoing an adaptive radiation.     

Nested subset structure of larval mycophagous fly assemblages: nestedness in a non-island system

Nested subset structure has been studied in archipelagoes and fragmented habitats, and has been attributed to differential colonization and extinction rates among species and nested environmental tolerances. In this experiment, we tested for nestedness in assemblages of mycophagous fly larvae. Twenty mushrooms in each of three size classes (4.8–6.0 g, 10–15 g, 21–32 g) were placed on moist potting soil in experimental cups. The cups were placed in oak and pine forests in Greenville, S.C., USA for 5 days, where they were available to ovipositing flies. Upon collection, the mushrooms were incubated in the laboratory for 3 weeks and all emerging flies were sorted by species, counted, and weighed. A random placement analysis was conducted to determine whether the species richness pattern was a sampling artifact of the species abundance distributions. The actual species richness pattern did not conform to the random placement model; most mushrooms contained significantly fewer species than predicted by random sampling. The communities were strongly nested as measured by two different indices, and the nestedness pattern was related to mushroom size. Small mushrooms usually produced no flies or a single species, Dohrniphora sp. (Phoridae). Medium and large mushrooms typically produced more species-rich communities that usually contained the phorid and Drosophila putrida, D. tripunctata, and Leucophenga varia. This core guild was nested within a more diverse assemblage that included D. falleni, Mycodrosophila dimidiata, a muscid, and two Leptocera sp. (sphaeroceridae). These patterns are tentatively explained in the context of nested desiccation tolerances, mediated by differences in mushroom size.     

Diversity and host associations of parasitoids attacking mycophagous drosophilids (Diptera: Drosophilidae) in northern and central Japan

The diversity and host associations of parasitoids attacking mycophagous drosophilids were studied in Tokyo (a warm‐temperate region) and Sapporo (a cool‐temperate region) in Japan. Field collections were carried out using traps baited with mushrooms in May, June, September and October 2009 in Tokyo and in July and August 2010 in Sapporo. The major drosophilid species that emerged from mushroom baits was Drosophila bizonata in Tokyo and D. orientacea in Sapporo. In total, 13 parasitoid species emerged from drosophilids occurring in mushroom baits, and 11 of them were larval parasitoids belonging to Braconidae and Figitidae. Among the 11 larval parasitoids, 10 were collected in Tokyo, while only two were collected in Sapporo. It is not known why their diversity differed so much between these two regions. Four of the 11 larval parasitoids have also been recorded from drosophilid larvae occurring in fruit (banana). The use of these two habitats (mushrooms and fruit) by these four species seems to reflect the occurrence (i.e. resource use) of their suitable hosts. On the other hand, most larval parasitoids from Tokyo attacked D. bizonata, and two larval parasitoids from Sapporo attacked D. orientacea, suggesting that the abundance of potential hosts is one of the important factors affecting their host use.     

The mushroom habitat as an ecological arena for global exchange of Wolbachia

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non‐mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non‐mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia‐host co‐speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.     

Inter‐ and intraspecific variation in mycotoxin tolerance: A study of four Drosophila species

Many mycophagous Drosophila species have adapted to tolerate high concentrations of mycotoxins, an ability not reported in any other eukaryotes. Although an association between mycophagy and mycotoxin tolerance has been established in many Drosophila species, the genetic mechanisms of the tolerance are unknown. This study presents the inter‐ and intraspecific variation in the mycotoxin tolerance trait. We studied the mycotoxin tolerance in four Drosophila species from four separate clades within the immigrans‐tripunctata radiation from two distinct locations. The effect of mycotoxin treatment on 20 isofemale lines per species was studied using seven gross phenotypes: survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity. We observed interspecific variation among four species, with D. falleni being the most tolerant, followed by D. recens, D. neotestacea, and D. tripunctata, in that order. The results also revealed geographical variation and intraspecific genetic variation in mycotoxin tolerance. This report provides the foundation for further delineating the genetic mechanisms of the mycotoxin tolerance trait.     

Distribution and mechanism of α-amanitin tolerance in mycophagous Drosophila (Diptera: Drosophilidae)

Many mycophagous species of Drosophila can tolerate the mushroom poison α-amanitin in wild mushrooms and in artificial diet. We conducted feeding assays with sixteen Drosophila species and α-amanitin in artificial diet to better determine the phylogenetic distribution of this tolerance. For eight tolerant and one related susceptible species, we sequenced the gene encoding the large subunit of RNA Polymerase II, which is the target site of α-amanitin. We found no differences in the gene that could account for differences in susceptibility to the toxin. We also conducted feeding assays in which α-amanitin was combined with chemical inhibitors of cytochrome P450s or glutathione S-transferases (GSTs) in artificial diet to determine if either of these enzyme families is involved in tolerance to α-amanitin. We found that an inhibitor of GSTs did not reduce tolerance to α-amanitin, but that an inhibitor of cytochrome P450s reduced tolerance in several species. It is possible that the same cytochrome P450 activity that produces tolerance of α-amanitin might produce tolerance of other mushroom toxins as well. If so, a general detoxification mechanism based on cytochrome P450s might answer the question of how tolerance to α-amanitin arose in mycophagous Drosophila when this toxin is found in relatively few mushrooms.     

Food preferences and nematode parasitism in mycophagousDrosophila

Food preferences and nematode parasitism were studied in natural populations of mycophagousDrosophila in and near Sapporo, northern Japan. Species which preferred fresh mushrooms showed species-specific responses toPleurotus mushrooms:D. pirka bred only onPleurotus cornucopiae, D. trivitata onP. cornucopiae andP. ostreatus, D. trilineata on these twoPleurotus mushrooms and some other mushrooms, whileD. sexvittata bred on a wide variety of mushrooms but seldom onPleurotus mushrooms. Species which preferred decayed mushrooms (D. quadrivittata, D. histrioides, D. testacea and species of thequinaria species-group) showed host preferences different from those of the above species. The rate of parasitism by nematodes was generally higher in species which prefer decayed mushrooms than in species which prefer fresh mushrooms. Among species which prefer fresh mushrooms, onlyD. trilineata was parasitized frequently by nematodes. It was not clear what factors determine the rate of parasitism in these mycophagousDrosophila. D. pirka, D. trivittata andD. trilineata passed through three or four generations per year and entered reproductive diapause in early September in and near Sapporo. Bionomics of Drosophilidae (Diptera) in Hokkaido, IX.     

Life history adaptations and stress tolerance of four domestic species of Drosophila

Life history traits and stress tolerance were studied in four domestic species of Drosophila–D. melanogaster, D. simulans, D. auraria and D. immigrans– to understand how they adapt to their environments. In all species, larval weight approximately doubled in 1 day. The relative egg weight (egg weight : pupal weight) was smaller and the larval period was longer in D. immigrans than in the other three species. The pupal period was the longest in D. auraria. However, the adaptive significance of these differences in larval and pupal periods was not clear. The pupal case was generally thicker in the larger species, probably to support the larger pupal body. The start of oviposition was earliest and reproductive effort was greatest in female D. simulans, followed by female D. melanogaster. In contrast, starvation tolerance and the increase in bodyweight after eclosion was greater in D. immigrans and D. auraria than in the other two species. Pupal desiccation tolerance was greatest in D. melanogaster and lowest in D. auraria, and the less tolerant species seemed to select more humid sites for pupation. Adult tolerance to desiccation was greatest in D. melanogaster and lowest in D. simulans. In contrast, adult cold tolerance was greater in D. auraria and adult heat tolerance was lower in D. immigrans than in the other species. These differences in life history traits and stress tolerance represent the Drosophila species differential adaptations, and are assumed to allow coexistence of the species.     

No evidence for behavioural adaptations to nematode parasitism by the fly Drosophila putrida

Behavioural adaptations of hosts to their parasites form an important component of the evolutionary dynamics of host–parasite interactions. As mushroom‐feeding Drosophila can tolerate deadly mycotoxins, but their Howardula nematode parasites cannot, we asked how consuming the potent mycotoxin α‐amanitin has affected this host–parasite interaction. We used the fly D. putrida and its parasite H. aoronymphium, which is both highly virulent and at high prevalence in some populations, and investigated whether adult flies utilize food with toxin to prevent infection in the next generation or consume the toxin to reduce the virulence of an already established infection. First, we found that uninfected females did not prefer to eat or lay their eggs on toxic food, indicating that selection has not acted on the flies to alter their behaviour towards α‐amanitin to prevent their offspring from becoming infected by Howardula. However, we cannot rule out that flies use an alternate cue that is associated with toxin presence in the wild. Second, we found that infected females did not prefer to eat food with α‐amanitin and that consuming α‐amanitin did not cure or reduce the virulence of the parasite in adults that were already infected. In sum, our results indicate there are no direct effects of eating α‐amanitin on this host–parasite interaction, and we suggest that toxin tolerance is more likely maintained by selection due to competition for resources than as a mechanism to avoid parasite infection or to reduce the virulence of infection.     

Divergence in wing morphology among sibling species of the Drosophila buzzatii cluster

The Drosophila buzzatii species cluster consists of the sibling species D. buzzatii, D. koepferae, D. serido, D. borborema, D. seriema, D. antonietae and D. gouveai, all of which breed exclusively in decaying cactus tissue and, except for D. buzzatii (a colonizing subcosmopolitan species), are endemic to South America. Using a morphometric approach and multivariate analysis of 17 wing parameters, we investigated the degree of divergence in wing morphology among the sibling species of this cluster. Significant differences were obtained among the species and discriminant analysis showed that wing morphology was sufficiently different to allow the correct classification of 98.6% of the 70 individuals analysed. The phenetic relationships among the species inferred from UPGMA cluster analysis based on squared Mahalanobis distances (D²) were generally compatible with previously published phylogenetic relationships. These results suggest that wing morphology within D. buzzatii cluster is of phylogenetic importance.     

INFECTIOUS ADAPTATION: POTENTIAL HOST RANGE OF A DEFENSIVE ENDOSYMBIONT IN DROSOPHILA

Maternally transmitted symbionts persist over macroevolutionary timescales by undergoing occasional lateral transfer to new host species. To invade a new species, a symbiont must survive and reproduce in the new host, undergo maternal transmission, and confer a selective benefit sufficient to overcome losses due to imperfect maternal transmission. Drosophila neotestacea is naturally infected with a strain of Spiroplasma that restores fertility to nematode‐parasitized females, which are otherwise sterilized by parasitism. We experimentally transferred Spiroplasma from D. neotestacea to four other species of mycophagous Drosophila that vary in their ability to resist and/or tolerate nematode parasitism. In all four species, Spiroplasma achieved within‐host densities and experienced rates of maternal transmission similar to that in D. neotestacea. Spiroplasma restored fertility to nematode‐parasitized females in one of these novel host species. Based on estimates of maternal transmission fidelity and the expected benefit of Spiroplasma infection in the wild, we conclude that Spiroplasma has the potential to spread and become abundant within Drosophila putrida, which is broadly sympatric with D. neotestacea and in which females are rendered completely sterile by nematode parasitism. Thus, a major adaptation within D. putrida could arise via lateral transmission of a heritable symbiont from D. neotestacea.     

Mycophagy in small mammals: A comparison of the occurrence and diversity of hypogeal fungi in the diet of the long-nosed potoroo Potorous tridactylus and the bush rat Rattus fuscipes from southwestern Victoria,Australia

Abstract Three broad dietary categories—fungus, plant and arthropod—were identified from faecal samples of two species of small terrestrial mammal in forest vegetation in southwestern Victoria. Fungal material formed the major component of the diet of the long-nosed potoroo Potorous tridactylus throughout the year and of the bush rat Rattus fuscipes during autumn and winter. Fungal material was most abundant for both species during autumn and winter and significantly less common in spring and summer. These results confirm previous studies which found P. tridactylus to be highly mycophagous throughout the year and R. fuscipes to be strongly mycophagous seasonally. Particular consideration was given to the composition of fungi in the diet. Fungal spores in faecal material were assigned to spore classes, which represent one or more fungal species that have similar spore morphology. Twenty-four fungal spore classes were recorded, but for both animal species most of the fungi consumed were from seven major spore classes. The proportions of major spore classes in the diet of both animals were generally similar, even though the composition of spore classes differed markedly across seasons. Minor differences between species in the fungi consumed may be related to differences in selectivity, foraging, or microhabitat use. If fungal resources are limiting, competition for such resources may be important in this and other small mammal communities. The amount and diversity of hypogeal fungi consumed by the two animal species makes them both important spore dispersal agents in forest ecosystems. The capacity of R. fuscipes and other seasonally mycophagous mammals in this role may be more important than previously recognized, especially in habitats where species of the Potoroidae are absent.     

Comparisons of yeast florae from natural substrates and larval guts of southwestern Drosophila

Summary The yeast florae in the natural substrates of four desert and three non-desert Drosophila species were compared both qualitatively and quantatively to the yeast present in the guts of Drosophila larvae living in those substrates. The desert species breed in rotting cacti and the other Drosophila were found breeding in necrotic oranges. Larvae of one cactophilic species, D. mojavensis, and larvae of all of the species utilizing oranges (D. melanogaster, D. pseudoobscura, and D. arizonensis) were found to contain non-random samples of the yeasts available in their respective substrates. Larval preference behavior is most likely responsible for these differences. The other cactophilic Drosophila (D. nigrospiracula, D. mettleri, and D. pachea) did not exhibit significant differences when the yeast florae of their larvae and substrates were compared. Selective feeding by larvae appears to be related to the degree of polyphagy in that only larvae of polyphagous species are selective. Trade-off between generalism and specialism at two biological levels is discussed.     

Identification of the sibling species of the Drosophila willistoni subgroup through the electrophoretical mobility of acid phosphatase-1

The Drosophila willistoni group consists of 23 species of which six are sibling species and belong to the D. willistoni subgroup: D. willistoni, Drosophila equinoxialis, Drosophila tropicalis, Drosophila insularis, Drosophila pavlovskiana and Drosophila paulistorum. These sibling species are abundant in the Neotropical region and can hardly be differentiated by the usual taxonomic traits. Four of them (D. willistoni, D. equinoxialis, D. tropicalis and D. paulistorum) cover extensive geographic distribution areas overlapping in places while two of them are endemic (D. insularis and D. pavlovskiana). In this study, we presented a method for the identification of five sibling species of the D. willistoni subgroup based on the allozyme variation of acid phosphatase‐1 (Acph‐1) in acrylamide gel electrophoresis. Our work showed that Acph‐1 allozyme differences can be used for species‐diagnostic characterization. This method was shown to be a more efficient tool for species identification than others because it is both quicker and produces reliable results.     

GENETIC POPULATION STRUCTURE OF DROSOPHILA TRIPUNCTATA: PATTERNS OF VARIATION AND COVARIATION OF TRAITS AFFECTING RESOURCE USE

Drosophila tripunctata is an ecological generalist, using both fruits and mushrooms as breeding sites. Isofemale strains of this species were established from seven populations over a wide part of its range and assayed for electrophoretic variability, oviposition-site preference, and larval performance on several types of substrates. Significant variation among strains within populations was found for oviposition-site preference, larval development time on tomatoes versus mushrooms, and tolerance (as measured by development time) of the mushroom toxin α-amanitin. Even populations at the periphery of the range, which electrophoretic data suggest have been through bottlenecks, harbored levels of variation for oviposition-site preference approximately equal to that found in central populations. All correlations between preference and various measures of larval performance were close to zero. Thus, there is no evidence for sympatric divergence of host races or for coadapted complexes of genes related to host specificity. Strains with higher-than-average amanitin tolerance tended to develop more rapidly on tomatoes than on nontoxic mushrooms, whereas the less-tolerant strains had slower development on tomatoes. This suggests that there may be genetically based correlations and trade-offs in larval performance on different breeding sites. No geographic differentiation among populations was found for either oviposition-site preference or any measure of larval performance. There is also very little electrophoretic variation among populations. Thus, the species as a whole, rather than local populations, appears to be the unit of evolution with respect to resource use in D. tripunctata.     

A comparison of behavioural change in Drosophila during exposure to thermal stress

In order to understand how adaptive tolerance to stress has evolved, we compared related species and populations of Drosophila for a variety of fitness relevant traits while flies directly experienced the stress. Two main questions were addressed. First, how much variation exists in the frequency of both courtship and mating among D. melanogaster, D. simulans, and D. mojavensis when each are exposed to a range of temperatures? Second, how does variation in these same behaviours compare among four geographically isolated populations of D. mojavensis, a desert species with a well defined ecology? Our hierarchical study demonstrated that mating success under stress can vary as much between related species, such as D. melanogaster and D. simulans, as between the ecologically disparate pair, D. melanogaster and D. mojavensis. Strains of this latter desert species likewise varied in tolerance, with differences approaching the levels observed among species. The consequences of stress on male courtship differed markedly from those on female receptivity to courtship, as mating behaviours among species and among strains of D. mojavensis varied in subtle but significant ways. Finally, a comparison of variation in thermotolerance of F₁ hybrids between the two most extreme D. mojavensis populations confirmed that genetic variation underlying traits such as survival or the ability to fly after heat stress is completely different. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 197–205.     

Molecular identification of dipteran pests (Diptera: Sciaroidea) from shiitake mushroom

On shiitake farms, mycophagous maggots can cause serious damage by preventing formation of the fruiting body. Recently, these pests have significantly reduced shiitake production in Korea. However, larvae and female adults cannot be identified due to their lack of morphological characteristics. Therefore, farmers and applied entomologists are unable to determine which species is the primary cause of the shiitake damage. In this study, mycophagous flies (colonized larvae) were collected from damaged shiitake farms and subsequently identified by matching identified males with the cytochrome c oxidase subunit I (COI) sequences from the larvae. Divergences of the COI sequences among the species discriminated the clusters clearly, and the mycophagous pests were identified as Camptomyia corticalis and C. heterobia. Interestingly, these two species coexisted under the bark of shiitake oak bed logs.     

The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.