Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 573–584.     

Population genetic study of allozyme variation in natural populations of Drosophila antonietae (Insecta, Diptera)

Drosophila antonietae is an endemic South American cactophilic species found in relictual xerophytic vegetation, mostly associated with Cereus hildmaniannus cactus. Low differentiation among populations of this species has been detected using several markers. In this work, we performed an allozyme genetic variability analysis of 11 natural populations of D. antonietae and included a discussion about the possible influences of several evolutionary processes that might be acting to maintain the pattern observed. The genetic variability of 14 isoenzyme loci was analysed and showed a high genetic diversity (average observed heterozygosity = 0.319) and a moderate genetic differentiation among populations ( F statistics = 0.0723). A correlation between genetic and geographical and ecological distances was detected among pairs of populations and the regional equilibrium analysis was thus applied. This analysis resulted in Nm (number of migrants) of approximately 3.21, indicating that moderate levels of both gene flow and genetic drift occur in this species, with gene flow overlapping genetic drift. However, considering ecological features of drosophilids, we propose a hypothesis to explain the moderate differentiation encountered as a result of three different processes, or a combination of them: (1) gene flow; (2) a short period of differentiation, i.e. maintenance of ancestral polymorphism; and (3) action of natural selection. Moreover, if gene flow is present, the high genetic diversity compared with other cactophilic and non-cactophilic species could be due to differential selection in different populations followed by gene exchange among them. These factors are discussed in the light of D. antonietae 's historical and evolutionary association with the host cactus.     

Temporal and Spatial Allozyme Variation in the South American Cactophilic Drosophila antonietae (Diptera; Drosophilidae)

Drosophila antonietae is an endemic South American cactophilic species that uses Cereus hildmaniannus rotting cladodes as breeding sites. We assessed temporal and spatial intrapopulational allozyme variation of two natural populations. Our results suggest that environmental variation (rain precipitation) is probably influencing allozyme temporal variation. Moreover, it seems that D. antonietae does not have intrapopulation structure and has N _ev (variance effective size) 83 and N _ec (number of adult flies that colonize each rotting cladode) = 21. The deficiency of heterozygotes found must be due to null alleles, a temporal Wahlund effect, or selection against heterozygotes. Assortative mating and inbreeding are discarded. This is the first report on allozyme variation in D. antonietae. It gives some insight on intrapopulational genetics through space and time for this species. This is important to understand its general genetic variability and will be essential to future works on the natural history and evolution of this species.     

Isolation and characterization of 30 polymorphic microsatellite loci from the mycophagous fly Drosophila innubila

Drosophila innubila is a mushroom-feeding member of the quinaria group, found in the woodlands and forests of the 'sky islands' in Arizona and New Mexico and extending south into central Mexico. Here, we describe and characterize 30 polymorphic microsatellite loci from D. innubila collected in the Chiricahua Mountains in Arizona. The number of alleles ranged from three to 21, and observed heterozygosity ranged from 0.0513 to 0.9737. Six loci were putatively X-linked, six departed from Hardy-Weinberg equilibrium, seven had evidence of null alleles, and six showed evidence of linkage disequilibrium. These markers will be useful for examining population structure of D. innubila and its association with male-killing Wolbachia.     

Wing morphology is related to host plants in cactophilic Drosophila gouveai and Drosophila antonietae (Diptera,Drosophilidae)

A central issue in evolutionary biology is to understand the mechanisms promoting morphological evolution during speciation. In a previous study, we showed that the Neotropical cactophilic sibling species Drosophila gouveai and Drosophila antonietae can be reared in media prepared with their presumptive natural host plants (Pilosocereus machrisis and Cereus hildmaniannus) and that egg to adult viability is not independent of the cactus host. In the present study, we investigate the effects of ecological and genetic factors on interspecific divergence in wing morphology, in relation to the pattern of wing venation and phenotypic plasticity in D. gouveai and D. antonietae, by means of the comparative analysis of isofemale lines reared in the two cactus hosts. The species differed significantly in wing size and shape, although specific differences were mainly localized in a particular portion of the wing. We detected significant variation in form among lines, which was not independent of the breeding cactus, suggesting the presence of genetic variation for phenotypic plasticity and wing shape variation in both species. We discuss the results considering the plausible role of host plant use in the evolutionary history of cactophilic Drosophila inhabiting the arid zones of South America. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 655–665.     

Identification and characterization of 21 polymorphic microsatellite loci from the mycophagous fly Drosophila neotestacea

Drosophila neotestacea is a mushroom‐feeding fly that is common in the temperate and boreal forests of North America. Here I describe the isolation and characterization of 21 polymorphic microsatellite loci from the D. neotestacea genome. In a sample of flies from Rochester, New York, the expected and observed heterozygosities ranged from 0.344 to 1.000 and from 0.384 to 0.923, respectively. Of the 21 markers, six were likely X‐linked, seven showed a departure from Hardy–Weinberg equilibrium, and none were found to be in linkage disequilibrium. These loci will facilitate future ecological and population genetic studies of D. neotestacea.     

Geographic variation of reproductive diapause in the Drosophila auraria complex (Diptera: Drosophilidae)

ABSTRACT. In the Drosophila auraria complex (D. auraria Peng, D. biauraria Bock & Wheeler, D. triauraria Bock & Wheeler, D. quadraria Bock & Wheeler and D. subauraria Kimura), D. triauraria from the island of Yaku (30.3N in latitude) and D. quadraria from Taiwan (24.1N) had no or weak diapause, while strains of all the four species from the main islands of Japan had a firm reproductive diapause. These strains entered diapause at short daylengths and the critical daylength was longer and the incidence of diapause higher at a low rather than high temperature. In each species, the critical daylength was longer in strains from higher latitudes. Among sympatric populations of different species, D. subauraria always had the longest critical daylength, while D. auraria had the shortest one. The populations of D. biauraria and D. triauraria had longer critical daylengths than the sympatric populations of D. auraria , but this difference became smaller northwards. Since a longer critical daylength induces diapause earlier in autumn, D. subauraria would enter diapause earliest among these species, and D. auraria would do so latest. The late start of diapause in D. auraria may be related to its adaptation to domestic environments. On the other hand, the adaptive significance of the late start of diapause in northern populations of D. biauraria and D. triauraria is not clear.     

Microsatellite markers for the relictual dove tree, Davidia involucrata (Cornaceae)

? Premise of the study: Microsatellite markers were developed for the dove tree, Davidia involucrata (Cornaceae), a Tertiary relict currently endemic to China, to investigate its population genetics and phylogeography. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, nine polymorphic microsatellite loci were identified and screened in 44 individuals from three wild populations of D. involucrata. The number of alleles per locus ranged from two to three, while the observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.6000 and from 0.0000 to 0.6323, respectively. ? Conclusions: These new microsatellite loci will facilitate further studies of the population genetics and phylogeography of D. involucrata, as well as of the evolutionary history of the plant and other Tertiary relicts endemic to East Asia.     

Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae)

Eight microsatellite primers were developed from ISSR (intersimple sequence repeats) markers for the stingless bee Melipona rufiventris. These primers were tested in 20 M. rufiventris workers, representing a single population from Minas Gerais state. The number of alleles per locus ranged from 2 to 5 (mean = 2.63) and the observed and expected heterozygosity values ranged from 0.00 to 0.44 (mean = 0.20) and from 0.05 to 0.68 (mean = 0.31), respectively. Several loci were also polymorphic in M. quadrifasciata, M. bicolor, M. mandacaia and Partamona helleri and should prove useful in population studies of other stingless bees.     

Variation of wing shape in the Drosophila virilis species group (Diptera: Drosophilidae)

Wing shape variation was analysed with geometric morphometric methods in 17 laboratory strains, representing 11 closely related species (including two subspecies) of the Drosophila virilis group: D. virilis, D. lummei, D. novamexicana, D. americana americana, D. americana texana, D. montana, D. lacicola, D. flavomontana, D. borealis, D. littoralis, D. ezoana and D. kanekoi. Overall shape estimated using Procrustes coordinates of 14 landmarks was highly variable among strains and very similar in females and males. The landmarks in the distal part of the wing showed higher variation across strains than those in the proximal part. Procrustes distances between species were not consistent with phylogenetic distances previously suggested for the virilis group. Moreover, Procrustes distances between strains within species and within two major phylads (virilis and montana) were comparable with those between species and between phylads, respectively. The most different from other members of the group was the endemic D. kanekoi species, currently viewed as separate subphylad within the montana phylad. Allometric effects were found to be partly responsible for shape differences between the strains. Three most significant shape transformations were considered using the relative warp analysis and the strains were ordinated in accordance with transformation values. The pattern of relative warp scores could be easily interpreted only for the third warp explaining about 13% of shape variation. It separated the largest species, D. montana, D. ezoana and D. kanekoi, from other ones and was mainly associated with shape changes in the proximal region of the wing. The results of the present work suggest that wing shape in the virilis species group is not related to the speciation process. The observed proximal‐distal contrasts and allometric effects are in agreement with data of other studies, in which wing shape variation was analysed within Drosophila species.     

Phenotypic plasticity in Drosophila cactophilic species: the effect of competition,density, and breeding sites

Changes in the environmental conditions experienced by naturally occurring populations are frequently accompanied by changes in adaptive traits allowing the organism to cope with environmental unpredictability. Phenotypic plasticity is a major aspect of adaptation and it has been involved in population dynamics of interacting species. In this study, phenotypic plasticity (i.e., environmental sensitivity) of morphological adaptive traits were analyzed in the cactophilic species Drosophila buzzatii and Drosophila koepferae (Diptera: Drosophilidae) considering the effect of crowding conditions (low and high density), type of competition (intraspecific and interspecific competition) and cacti hosts (Opuntia and Columnar cacti). All traits (wing length, wing width, thorax length, wing loading and wing aspect) showed significant variation for each environmental factor considered in both Drosophila species. The phenotypic plasticity pattern observed for each trait was different within and between these cactophilic Drosophila species depending on the environmental factor analyzed suggesting that body size‐related traits respond almost independently to environmental heterogeneity. The effects of ecological factors analyzed in this study are discussed in order to elucidate the causal factors investigated (type of competition, crowding conditions and alternative host) affecting the election of the breeding site and/or the range of distribution of these cactophilic species.     

Drosophila subpulchrella, a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: Drosophilidae)

Drosophila (Sophophora) subpulchrella Takamori and Watabe, sp. nov., of the D. suzukii subgroup in the D. melanogaster species group, is described from Japan and southern China, and compared with its sibling species, D. pulchrella Tan et al. distributed in the Yun‐Gui Highland, south‐western China. The results of cross‐experiments show a complete pre‐mating isolation between D. subpulchrella and D. pulchrella.     

Microsatellite markers developed for Corallodiscus lanuginosus (Gesneriaceae) and their cross-species transferability

• Premise of the study: Corallodiscus is widespread across China and in adjacent countries to the west and south, with C. lanuginosus covering the entire range of the genus. Microsatellite markers will be useful to address within-complex taxonomic and biogeographic structures of this species. • Methods and Results: Fourteen markers were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol. Polymorphism was assessed in 12 individuals each from two populations from West Yunnan, China. Nine primers displayed polymorphisms. The number of alleles per locus ranged from one to six (mean: 2.7). The observed and expected heterozygosities ranged from 0 to 1 and from 0 to 0.772 (average: 0.487 and 0.448). Only one of the polymorphic loci deviated from Hardy–Weinberg equilibrium. Among the four congeneric species, cross-amplification success ranged from two to eight markers. • Conclusions: The markers will be useful for population genetic and evolutionary history studies for C. lanuginosus and the allied species C. bhutanicus, C. cooperi, and C. kingianus.     

Microsatellite analysis of genetic structure in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

The level of genetic variation throughout the entire worldwide range of the mangrove species Avicennia marina (Forsk.) Vierh. was examined using microsatellite markers. Three microsatellite loci detected high levels of allelic diversity (70 alleles in total), essential for an accurate estimation of population genetic parameters. The informativeness of the microsatellite loci tended to increase with increasing average number of repeats. The levels of heterozygosity detected for each population, over all loci, ranged from 0.0 to 0.8, with an average of 0.407, indicating that some populations had little or no genetic variation, whereas others had a large amount. Populations at the extremes of the distribution range showed reduced levels of heterozygosity, and significant levels of inbreeding. This is not unexpected as these populations may be subject to founder effects and environmental constraints. The presence of genetic structure was tested in A. marina populations using three models: (i) a single panmictic model; (ii) the discrete subpopulation model; and (iii) the isolation by distance model. The discrete subpopulations model was supported by the overall measures of population differentiation based on the infinite alleles model (F-statistics), and the stepwise mutation model (R statistics). In addition, an analysis of molecular variance (AMOVA), using both theoretical models, found that most of the variation was between populations (41-71%), and within individuals in the total population (31-49%). There was little variation among individuals within populations (0-10%). There was no significant isolation by distance. The high levels of genetic differentiation observed among populations of A. marina may be due to environmental and ecological factors, particularly past sea level and climatic changes.     

Remating, sperm transfer, and sperm displacement in the cactophilic species Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae)

The mating system of Drosophila buzzatii is characterized by short copulation duration, frequent remating in both males and females, and male ejaculate partitioning. Additional features of the system are strong sperm displacement and a high frequency of sterile matings. Remating frequencies and the effects of remating on various mating parameters were studied. In order to characterize variation, five isofemale lines from geographically distant localities in Australia (three localities), Brazil and the Canary Islands were used. Mating parameters studied were: premating time, copulation duration, interval between successive matings, and progeny number as a measure of sperm transfer. Variation for sperm displacement was studied in crosses between laboratory stocks and a number of isofemale lines from Australia. There were significant between‐line differences in female remating frequencies, premating time, copulation duration, interval between successive matings, and progeny numbers, indicating genetic variation for these traits. Females from the five lines mated on average 1.6 to 3.1 times in 4 h, with a maximum of eight matings for one female. The males were given a maximum of ten virgin females in sequence and more than one‐third of the males mated all ten females in the 2 h observation period. Copulation duration decreased and interval between matings increased with copulation number in multiply mated males. Mean copulation duration was c. 2 min. Sperm transfer, measured as the average number of progeny from a single mating, was low (c. 25) and multiply mated females gave more progeny than single mated females, although with much lower progeny numbers than observed in wild‐caught non‐virgin females. A surprisingly high proportion of observed matings gave no progeny, i.e. they were sterile matings. Sperm displacement was strong in most crosses and remained strong in multiply mated females. The results are discussed in relation to the evolution of mating patterns in Drosophila.     

Isolation and characterization of microsatellite loci in Penstemon rostriflorus (Plantaginaceae) and cross‐species amplification

Eight novel microsatellite primer pairs are presented for Penstemon rostriflorus, representing the first microsatellite markers available for this genus. Loci were characterized for 20 individuals from two populations in the Great Basin, USA. All loci are polymorphic within P. rostriflorus (seven to 13 alleles per locus; observed heterozygosity between 0.40 and 0.95), and therefore useful for population genetic studies within the species. Cross‐species transferability was tested on 40 additional species of Penstemon, and results indicate that these primers pairs will likely be useful for population genetic studies on many Penstemon species.     

中国特有的弯头果蝇种亚组及二新种记述(双翅目，果蝇科)

本文记述弯头果蝇种亚组的二新种,施氏果蝇 Drosophila (Drosophila) shi sp. nov.,短乳突果蝇Drosophila (Drosophila) brevipapilla sp.nov.,并对中国特有的弯头果蝇种亚组中11 个种的分布进行了分析。     

A comparative study of competitive ability between two cactophilic species in their natural hosts

Abstract Competition is a major aspect of the ecology of insect communities exploiting ephemeral and fragmented resources. We analysed the effect of intraspecific (single species culture) and interspecific (mixed species culture) competition on larval viability, developmental time and wing length in the cactophilic Drosophila buzzatii and Drosophila koepferae (Diptera: Drosophilidae) reared in cultured media prepared with fermenting tissues of three common natural cactus hosts in nature at different densities. Our results show that all traits measured were affected by both intra‐ and interspecifc competition, although the effect of competition depended on the Drosophila species and the rearing cactus. In fact, flies tended to have a lower viability, shorter wing size and longer developmental time as a function of increasing density in single species culture in both D. buzzatii and D. koepferae (intraespecific competition). Besides, the performance of both species was seriously affected (shorter body size, slower developmental times, lower viability) by the presence of heterospecific competitors except in the case of D. koepferae reared in its primary host plant, Trichocereus terschekii. We also show that D. koepferae successfully utilized Opuntia quimilo, which is absent in most parts of its distribution range. We discuss the roles of intra‐ and interspecific competition as determinants of the relative abundance of these two species in the arid zones of Southern South America.     

Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.