Host‐dependent phenotypic plasticity of aedeagus morphology in a pair of cactophilic sibling Drosophila species of the repleta group (Diptera,Drosophilidae)

The rapid evolution of male genital morphology is a characteristic feature of several animal groups. Such rapid divergence makes this trait a useful key for species identification. The aedeagus, the intromittent organ of male genitalia, is considered the main diagnostic trait in the Drosophila repleta group. In this study we analysed phenotypic plasticity and genetic variations associated with aedeagus size and shape in the cactophilic sibling species Drosophila gouveai Tidon‐Sklorz and Sene, 2001 and Drosophila antonietae Tidon‐Sklorz and Sene, 2001. Phenotypic plasticity in aedeagus morphology was evaluated in terms of the response to rearing media prepared with each species’ natural host plant, Pilosocereus machrisii Dawson, 1957 and Cereus hildmannianus Schum, 1890 respectively. Our results show that aedeagal shape differed significantly between species and that both shape and size presented host‐related phenotypic plasticity in both species. Flies reared on P. machrisii had, on average, larger aedeagi than those grown in C. hildmannianus. The general shape of aedeagus also differed significantly between flies that emerged in different host cactus. Patterns of variation in aedeagus morphology are discussed in the light of the current knowledge of evolutionary relationships and host plant use, in the D. buzzatii cluster, an assemblage of species in active cladogenesis.     

Host use and developmental instability in the cactophilic sibling species Drosophila gouveai and D. antonietae

The Drosophila repleta group encompasses an ensemble of species that inhabit desertic areas that are inhospitable to other drosophilids. These species have a tractable ecology, as they breed and feed on necrotic tissues of a wide diversity of species of Cactaceae, with a certain degree of host specificity, which makes them suitable models to investigate the role of host plant shifts in diversification. Most species have their own primary host plant, which may be shared with a closely related species. However, the consequences of host plant shifts from primary to secondary hosts have not been thoroughly studied so far. We investigated the effects of the cactus host on developmental instability and performance of D. gouveai Tidon‐Sklorz & Sene and D. antonietae Tidon‐Sklorz & Sene (Diptera: Drosophilidae), a pair of closely related sibling species, and of their F1 interspecific hybrids reared in primary and secondary host plants. Fluctuating asymmetry (FA) for wing size was significantly greater in flies of both species reared in their respective secondary cactus host than in those grown in the primary host. Interspecific hybrids also exhibited host‐dependent levels of FA. However, hybrids did not show greater FA than the parental species, suggesting that hybridization per se did not affect developmental stability. Even though cactus host shifts affected different measures of fitness (larval viability and adult size) both in D. antonietae and D. gouveai, we did not detect a consistent relationship between FA and fitness. Our results suggest that FA cannot be used as a sensitive indicator of genetic and phenotypic quality and we discuss its application as a fitness predictor.     

Wing morphology and fluctuating asymmetry depend on the host plant in cactophilic Drosophila

As in most insect groups, host plant shifts in cactophilic Drosophila represent environmental challenges as flies must adjust their developmental programme to the presence of different chemical compounds and/or to a microflora that may differ in the diversity and abundance of yeasts and bacteria. In this context, wing morphology provides an excellent opportunity to investigate the factors that may induce changes during development. In this work, we investigated phenotypic plasticity and developmental instability of wing morphology in flies on the cactophilic Drosophila buzzatii and Drosophila koepferae raised on alternative breeding substrates. We detected significant differences in wing size between and within species, and between flies reared on different cactus hosts. However, differences in wing shape between flies emerged from different cactus hosts were not significant either in D. buzzatii or in D. koepferae. Our results also showed that morphological responses involved the entire organ, as variation in size and shape correlated between different portions of the wing. Finally, we studied the effect of the rearing cactus host on developmental instability as measured by the degree of fluctuating asymmetry (FA). Levels of FA in wing size were significantly greater in flies of both species reared in non-preferred when compared with those reared in preferred host cacti. Our results are discussed in the framework of an integrative view aimed at investigating the relevance of host plant shifts in the evolution of the guild of cactophilic Drosophila species that diversified in South America.     

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.     

Breeding structure of an isolated cactophilic Drosophila population on a sandstone table hill

The effect of host cacti on the breeding structure of an isolated population of cactophilic Drosophila gouveai was studied. A comparison was made, using F statistics, of the allozyme frequencies at 11 loci among temporal samples of the adult population and six samples of progeny obtained from individual rotting cactus cladodes. The population appears to be structured by the cacti, forming breeding groups, and approximately four individuals contribute gametes to the progeny of each cladode. This D. gouveai population had a low degree of heterozygosity, compared with mean values for most Drosophila species, however it was within the range of values reported for other cactophilic Drosophila.     

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.     

An alkaloid fraction extracted from the cactus Trichocereus terscheckii affects fitness in the cactophilic fly Drosophila buzzatii (Diptera: Drosophilidae)

The host‐plant environment of phytophagous insects directly affects various aspects of an insect's life cycle. Interestingly, relatively few insect groups have specialized in the exploitation of plants in the Cactaceae family, potentially because of the chemical and ecological challenges imposed by these plants. The cactophilic Drosophila buzzatii Patterson & Wheeler, 1942 is a well‐studied model in evolutionary ecology, partially because of its ability to exploit toxic cactus hosts. Previous studies have shown a negative effect on performance when flies are reared in an alternative columnar cactus host of the genus Trichocereus, relative to its primary cactus host, Opuntia. These observations were attributed to the presence of alkaloids in Trichocereus tissues, a chemical deterrent to herbivores that indirectly affects Drosophila larvae; however, the putative toxic effect of alkaloids has never been tested directly in D. buzzatii. The present study is the first attempt to relate chemical extracts in Trichocereus terscheckii Britton & Rose, 1920 with detrimental effects on D. buzzatii. We assessed the effects of a crude alkaloid extract, rich in phenylethylamines, and a ‘non‐alkaloid fraction’ on viability and adult wing morphology. Our results indicate that rearing larvae on an artificial diet containing different concentrations of the crude alkaloid extract decreased pupal viability and adult size in a concentration‐dependent manner. We discuss the role of cactus alkaloids in the evolution of host‐plant use in cactophilic flies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 342–353.     

Host alkaloids differentially affect developmental stability and wing vein canalization in cactophilic Drosophila buzzatii

Host shifts cause drastic consequences on fitness in cactophilic species of Drosophila. It has been argued that changes in the nutritional values accompanying host shifts may elicit these fitness responses, but they may also reflect the presence of potentially toxic secondary compounds that affect resource quality. Recent studies reported that alkaloids extracted from the columnar cactus Trichocereus terscheckii are toxic for the developing larvae of Drosophila buzzatii. In this study, we tested the effect of artificial diets including increasing doses of host alkaloids on developmental stability and wing morphology in D. buzzatii. We found that alkaloids disrupt normal wing venation patterning and affect viability, wing size and fluctuating asymmetry, suggesting the involvement of stress–response mechanisms. Theoretical implications are discussed in the context of developmental stability, stress, fitness and their relationship with robustness, canalization and phenotypic plasticity.     

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.     

Genetic differentiation of island populations: geographical barrier or a host switch?

In the Sonoran desert, there exists a diverse community of cactophilic drosophilids that exploit toxic, rotting cactus tissue as a food resource. The chemistry of the necrotic cactus tissue varies among species, and several drosphilid species have evolved specialized detoxification mechanisms and a preference for certain cactus types. In the present study, we compared the genetic structure of two columnar cactus species, Drosophila mettleri and Drosophila mojavensis, and two prickly pear species, Drosophila mainlandi and Drosophila hamatofila, which have all recently colonized Catalina Island off the coast of southern California. Because there are no columnar cactus species on Catalina Island, the two columnar specialists underwent a host switch to prickly pear cactus, the only cactus present on the island. Previous genetic studies of D. mettleri and D. mojavensis showed significant genetic differentiation between mainland and island populations, which could result from restricted gene flow as a result of the San Pedro Channel, or because of a host switch to prickly pear. To distinguish between these possibilities, we analyzed the genetic structure of the prickly pear species aiming to isolate the effects of geography versus host switching. The results obtained show little to no genetic differentiation for the prickly pear species, supporting the hypothesis that the genetic differentiation of the two columnar species is a result of a host switch from columnar cacti to prickly pear. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Allozymatic divergence between border populations of two cryptic species of the Drosophila buzzatii cluster species (Diptera: Drosophilidae)

Drosophila antonietae and Drosophila gouveai are allopatric, cactophilic, cryptic and endemic of South America species, which aedeagus morphology is considered the main diagnostic character. In this work, single close populations from the edge distributions of each species, located in an “introgressive corridor”, were analyzed regarding temporal isozenzymatic genetic variability. Isocitrate dehydrogenase (Idh) appeared as a diagnostic locus between D. antonieate and D. gouveai because each population was fixed for different alleles. Moreover, several polymorphic loci showed accentuated divergence in the allele frequency, as evidenced by Nei’s I (0.3188) and D (1.1432), and also by Reynolds’ genetic distance and identity (1.3207 and 0.7331, respectively). Our results showed that, in spite of the very similar external morphology, related evolutionary histories, close distributions, and events of introgression in the studied area, these cryptic species have high allozymatic differentiation, and this is discussed here.     

Intra‐ and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X‐linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.     

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.     

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.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

The vectoring of cactophilic yeasts by Drosophila

Summary At two locations in the Sonoran Desert, yeasts were sampled from species of Drosophila, the flies' cactus hosts, and other neighboring sources of cactophilic yeasts to determine the relation between the yeasts vectored by the fly and the yeasts found in their breeding sites. D. mojavensis, D. nigrospiracula, and D. mettleri vectored yeast assemblages significantly more similar to the yeast species found on the rot from which the flies were collected than to the yeasts found on other rots from the flies host cactus or other rotting cactus at the same site. Rots with Drosophila had fewer yeast species than those without flies, suggesting that flies were associated with younger rots. Rots with flies and the Drosophila also had more yeast species with the capability to produce ethyl acetate than rots without flies. The results support the contention that cactophilic Drosophila feed on a subset of the yeasts available in an area, and may act to maintain differences among the yeast communities found on different species of cactus.     

Phenotypic plasticity across 50 MY of evolution: Drosophila wing size and temperature

We studied the response in wing size to rearing at different temperatures of nine strains of Drosophila representing six species. The species varied in their natural habitats from tropical to temperate and one cosmopolitan. The evolutionary divergence of the species spans 50 million years. While some quantitative differences were found, all species responded to temperature very similarly: females increased an average of ∼11% and males ∼14% when reared at 19 °C compared to 25 °C. The phenotypic plasticity in wing size in response to temperature appears to be a fixed trait in Drosophila across long evolutionary time and diverse ecological settings. This likely reflects the close relationship between wing area (and thus wing loading) and insect body mass that is a crucial factor for flight regardless of ecology and is, thus, maintained across long evolutionary time.     

Patterns of variation in wing morphology in the cactophilic Drosophila buzzatii and its sibling D. koepferae

Drosophila buzzatii and D. koepferae are two sibling species that breed on the necrotic tissues of several cactus species and show a certain degree of niche overlap. Also, they show differences in several life history traits, such as body size and developmental time, which probably evolved as a consequence of adaptation to different host plants. In this work we investigate the ecological and genetic factors affecting wing morphology variation both within and between species. Three wing traits were scored, distal and proximal wing length and width in isofemale lines reared in two of the most important host cacti: Opuntia sulphurea and Trichocereus terschekii. Our results revealed that differences between species and sexes in wing size and shape were significant, whereas the cactus factor was only significant for wing size. Intraspecific analyses showed that differences among isofemale lines were highly significant for both size and shape in both species, suggesting that an important fraction of variation in wing morphology has a genetic basis. Moreover, the line by cactus interaction, which can be interpreted as a genotype by environment interaction, also accounted for a significant proportion of variation. In summary, our study shows that wing size is phenotypically plastic and that populations of D. buzzatii and D. koepferae harbour substantial amounts of genetic variation for wing size and shape. Interspecific differences in wing size and shape are interpreted in terms of spatial predictability of the different host plants in nature.