Phylogeny of Drosophila saltans group (Diptera: Drosophilidae) based on morphological and molecular evidence.

Drosophila saltans group belongs to the subgenus Sophophora (family Drosophilidae), and it is subdivided into five subgroups, with 23 species. The species in this group are widely distributed in the Americas, primarily in the Neotropics. In the literature, the phylogenetic reconstruction of this group has been performed with various markers, but many inconsistencies remain. Here, we present a phylogenetic reconstruction of the saltans group with a greater number of species, 16 species, which is the most complete to date for the saltans group and includes all subgroups, in a combined analysis with morphological and molecular markers. We incorporated 48 morphological characters of male terminalia, the highest number used to date, and molecular markers based on mitochondrial genes COI and COII. Based on the results, which have recovered the five subgroups as distinct lineages, we propose a new hypothesis regarding the phylogenetic relationships among the subgroups of the saltans group. The relationships of the species within the sturtevanti and elliptica subgroups were well supported. The saltans subgroup showed several polytomies, but the relationship between the sibling species D. austrosaltans and D. saltans and their close relation with D. nigrosaltans were well supported in the molecular and total evidence analyses. The morphological analysis additionally supported the formation of the clade D. nigrosaltans-D. pseudosaltans. The observed polytomies may represent synchronous radiations or have resulted from speciation rates that have been too fast relative to the pace of substitution accumulation.

Male terminalia morphology of sixteen species of the Drosophila saltans group Sturtevant (Diptera, Drosophilidae).

Male terminalia in insects with internal fertilization evolve more rapidly than other structures. The aedeagus is the most variable structure, making it a valuable diagnostic feature to distinguish species. The saltans group Sturtevant of Drosophila Fall contains sibling species, that can be distinguished by their aedeagi. Here, we revised and illustrated the morphology of the male terminalia of the following species: Drosophila prosaltans Duda, 1927; D. saltans Sturtevant, 1916; D. lusaltans Magalhes, 1962; D. austrosaltans Spassky, 1957; D. septentriosaltans Magalhes, 1962; D. nigrosaltans Magalhes, 1962; D. pseudosaltans Magalhes, 1956; D. sturtevanti Duda, 1927; D. lehrmanae Madi-Ravazzi et al., 2021; D. dacunhai Mouro Bicudo, 1967; D. milleri Magalhes, 1962; D. parasaltans Magalhes, 1956; D. emarginata Sturtevant, 1942; D. neoelliptica Pavan Magalhes in Pavan, 1950; D. neosaltans Pavan Magalhes in Pavan, 1950 and D. neocordata Magalhes, 1956. We found that phallic structures (e.g., the aedeagus) evolve more rapidly than periphallic structures (e.g., epandrium), being completely different among the subgroups and within them. This rapid evolution may be due to the action of sexual selection or to the potential role of those structures in speciation.

Integrative taxonomy and a new species description in the sturtevanti subgroup of the Drosophila saltans group (Diptera: Drosophilidae).

Although the biological concept of species is well established in animals, sometimes the decision about the specific status of a new species is difficult and hence requires support of an integrative analysis of several character sets. To date, the species Drosophila sturtevanti, D. magalhaesi, D. milleri and D. dacunhai, belonging to the sturtevanti subgroup of the Neotropical saltans species group, are identified mainly by the aedeagus morphology, but also present some differences in spot coloration and patterning of the female sixth tergite and in the shape and size of the spermathecae, parallel to a pattern of reproductive isolation. In the present study, we describe a novel saltans group species from French Guiana belonging to the sturtevanti subgroup. Our species designation is based on an integrative approach covering (i) aedeagi and spermathecae morphology by scanning electron microscopy, (ii) analysis of female sixth-tergite color, (iii) morphometrical analysis of aedeagi and wings, (iv) analysis of partial sequence of the COI, COII and ND4 mitochondrial genes as well as (v) intercrosses for analysis of reproductive isolation. The comparative analysis of the results on these markers with those of D. sturtevanti, D. milleri and D. dacunhai supports that this line belongs to a new species of the sturtevanti subgroup that we name Drosophila lehrmanae sp. nov. in honor of Prof. Lee Ehrman´s 85th birthday.

Moderate Population Structure in Drosophila sturtevanti from the South American Atlantic Forest Biome.

Drosophila sturtevanti is a widely distributed Neotropical species. In South America, it is abundant and adapted to different phytophysiognomies of the Atlantic Forest biome. Reproductive, chromosomal and enzymatic studies have indicated the existence of a differentiation among D. sturtevanti populations. In this work, the level of genetic diversity and the population genetic structure were analyzed using four population groupings. One hundred and twenty-six D. sturtevanti males collected from nine forest fragments were analyzed for 11 species-specific microsatellite loci. A total of 109 alleles, ranging from 2 to 16 alleles per locus, were detected. The highest mean observed heterozygosity -H O was estimated in samples from the largest collection areas, and the lowest H O was from a population where fire events are common. A low molecular variation, around 3% among populations and negative among groups, an absence of genetic and geographic correlations and a moderate genetic differentiation -F ST = 0.0663 -indicated that D. sturtevanti is not strongly structured. Besides no overall genetic and geographic distance correlation, the pair of closest geographically populations Matão and Nova Granada showed the lower differentiation through F ST, DC and a Neighbor Joining tree. Ribeirão da Ilha -RDI, an isolated insular population, was the most differentiated according to F ST, DC and a cluster-based Bayesian analysis. The isolation of RDI that resulted in significant divergence could be ancient, because of sea level regressions/transgressions, or more recently via founder effect/genetic drift by anthropic action carrying D. sturtevanti hosts from continent to island. This work is important for understanding the genetic variability distribution of a Neotropical forest-dwelling Drosophila species using for the first time, a wide population distribution approach.

Signature of climatic differentiation on mitochondrial DNA of Drosophila sturtevanti.

Pleistocene climatic changes have played a major role in the evolution of Brazilian Atlantic Forest and South America biodiversity but their impacts on the genetic structure of widely distributed species remain unclear. Here, we investigate mitochondrial DNA (mtDNA) diversity in 21 geographical populations of Drosophila sturtevanti, Nucleotide sequences of the cytochrome C oxidase subunits I and II genes (COI and COII, respectively) from 163 individuals, showed a significant north-south structure, in spite of an overall low level of variation. The haplotypes clustered in three groups that showed strong correlations with geographical and climatic variables, suggesting that local adaptations might have contributed to differentiation within the species. Coalescent-based analyses indicated that the three clusters have differentiated nearly ∼17.000 years ago, suggesting a major role for Pleistocene changes in shaping current day distributions and differentiation of widespread Neotropical species.

Fluctuating asymmetry of meristic traits: an isofemale line analysis in an invasive drosophilid, Zaprionus indianus.

Metric (e.g., body size) and meristic (e.g., bristle number) traits are of general use in quantitative genetic studies, and the phenotypic variance is subdivided into a genetic and a non-genetic environmental component. The non-genetic variance may have two origins: a common garden effect between individuals and a developmental instability within the same individual. Developmental instability may be studied by considering the fluctuating asymmetry (FA) between the two sides of the body. The isofemale line technique is a convenient method for investigating the architecture of natural populations but has been rarely implemented for investigating FA. In this paper, we use this experimental design for analyzing four meristic traits in eight populations of the cosmopolitan Zaprionus indianus. A study of the correlation between left and right side of each line revealed that almost 90% of the variability was due to a developmental noise, while a much higher correlation among the means of the lines from the same population was observed. A slight trend toward a directional asymmetry was observed: more thoracic bristles on the left side. Four kinds of indices, scaled or non-scaled to the mean were used for comparing the different traits. Unscaled values (mean absolute values or standard deviation of each line) revealed a linear increase with the means. Interestingly the results of ovariole number were included in the same regression. With the scaled indices (mean absolute divided by each individual value or stadard deviation devided by the mean), the differences among traits were considerably decreased, but still remained significant. The mean FA of the various traits were not correlated, suggesting that each trait harbors its own developmental stability. The CVs of FA were high with a magnitude similar to those of the trait themselves, slightly less than 10%. Finally, even with the isofemale line design, which is a powerful means for unravelling slight genetic variations, we did not to find any clear indication of a genetic component of FA under the optimal environmental conditions used in this study.

Ultrastructural features of spermatozoa and their phylogenetic application in Zaprionus (Diptera, Drosophilidae).

The genus Zaprionus consists of approximately 60 species of drosophilids that are native to the Afrotropical region. The phylogenetic position of Zaprionus within the Drosophilidae family is still unresolved. In the present study, ultrastructural features of spermatozoa of 6 species of Zaprionus as well as the species Drosophila willistoni and Scaptodrosophila latifasciaeformis were analyzed. The ultrastructure revealed that the species have the same flagellar ultrastructure. Two mitochondrial derivatives, one larger than the other, close to the axoneme were present, primarily in D. willistoni (subgenus Sophophora). Except for Z. davidi and Z. tuberculatus, the analyzed species had paracrystalline material in both mitochondrial derivatives. Moreover, the testes showed 64 spermatozoa per bundle in all of the species. In the cluster analysis, 6 Zaprionus species were grouped closely, but there were some incongruent positions in the cladogram. The results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects.

Scanning electron microscopy of male terminalia and its application to species recognition and phylogenetic reconstruction in the Drosophila saltans group.

The Drosophila saltans group consists of five subgroups and 21 species, most of which have been identified only by morphological aspects of the male terminalia revealed by drawings using a camera lucida and a bright-field microscope. However, several species in the group, mainly those included in the saltans subgroup, are difficult to differentiate using only these characteristics. In this study, we used scanning electron microscopy (SEM) to analyze 19 structures of the male terminalia in 10 species from the five saltans subgroups. Among these structures, nine could be identified only through SEM analysis. We aimed to find other characteristics useful for morphological recognition of these species and to use these characteristics for phylogenetic reconstruction. These morphological differences enabled us to effectively distinguish among sibling species. These findings confirmed the monophyly of this group as previously determined in evolutionary studies based on other markers. The single most parsimonious tree (CI = 87 and RI = 90) indicated that the cordata subgroup is the most basal lineage and the saltans subgroup is the most apical lineage, as shown in earlier studies based on morphological data. However, our findings differed somewhat from these studies with respect to the phylogenetic relationships of species in the saltans group indicating that this group is still a puzzle that remains to be deciphered.

Edge-interior differences in the species richness and abundance of drosophilids in a semideciduous forest fragment.

Habitat fragmentation is the main cause of biodiversity loss, as remnant fragments are exposed to negative influences that include edge effects, prevention of migration, declines in effective population sizes, loss of genetic variability and invasion of exotic species. The Drosophilidae (Diptera), especially species of the genus Drosophila, which are highly sensitive to environmental variation, have been used as bioindicators. A twelve-month field study was conducted to evaluate the abundance and richness of drosophilids in an edge-interior transect in a fragment of semideciduous forest in São Paulo State, Brazil. One objective of the study was to evaluate the applied methodology with respect to its potential use in future studies addressing the monitoring and conservation of threatened areas. The species abundance along the transect showed a clear gradient, with species associated with disturbed environments, such as Drosophila simulans, Scaptodrosophila latifasciaeformis and Zaprionus indianus, being collected at the fragment edge and the species D. willistoni and D. mediostriata being found in the fragment's interior. Replacement of these species occurred at approximately 60 meters from the edge, which may be a reflection of edge effects on species abundance and richness because the species found within the habitat fragment are more sensitive to variations in temperature and humidity than those sampled near the edge. The results support the use of this methodology in studies on environmental impacts.

Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera, Drosophilidae): Cytochemical, structural and ultrastructural characterization.

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1-3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.

Polyphyly of the Zaprionus genus group (Diptera: Drosophilidae).

The Zaprionus genus group comprises three drosophilid genera (Zaprionus, Phorticella and Samoaia) that are thought to be related to the Drosophila immigrans species group. We revised the phylogenetic relationships among the three genera and their placement within the subfamily Drosophilinae using one mitochondrial (COII) and one nuclear (Amyrel) gene. The Bayesian tree inferred from concatenated amino acid sequences of the two genes strongly suggests the polyphyly of the Zaprionus genus group and of each of the genera Zaprionus and Phorticella. Paraphyly of the D.immigrans species group was also shown here; the quadrilineata subgroup formed the sister clade to the genus Samoaia. These results suggest the necessity of taxonomic revisions for some relevant genera and species groups included within the genus Drosophila.

On the use of open or closed traps in the capture of drosophilids / Sobre o uso de armadilhas abertas ou fechadas na captura de drosofilídeos

The use of open or closed traps in order to standardize the drosophilid collection is still a matter of discussion among researchers. In this paper, comparative data of drosophilid collections, using open and closed traps are presented. The collections were made monthly, during one year, in one fragment of a semidecidual forest located in the São Paulo State, Brazil. The efficiency of the traps was evaluated by the use of community descriptors: population abundance and richness and diversity of species. Closed traps were more efficient than the open ones in relation to the population abundance and the species richness, indicating their adequacy for standardization of drosophilid collection in studies of biodiversity and seasonality.

Uma discussão ainda em pauta refere-se ao uso de armadilhas abertas ou fechadas na padronização das coletas de drosofilideos para alguns estudos específicos. Neste trabalho são apresentados dados comparativos das coletas de drosofilídeos utilizando armadilhas abertas e fechadas em um período de 12 meses, em um fragmento de floresta semidecidual, localizada no Estado de São Paulo, Brasil. A eficiência das armadilhas foi avaliada usando os seguintes descritores da comunidade: abundância populacional, riqueza e diversidade de espécies. As armadilhas fechadas foram mais eficientes em relação à abundância populacional e à riqueza de espécies. Os resultados indicam que as armadilhas fechadas constituem o tipo mais adequado para padronização de coleta de drosofilídeos, em estudos de biodiversidade e sazonalidade.

DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus.

Comparing introduced to ancestral populations within a phylogeographical context is crucial in any study aiming to understand the ecological genetics of an invasive species. Zaprionus indianus is a cosmopolitan drosophilid that has recently succeeded to expand its geographical range upon three continents (Africa, Asia and the Americas). We studied the distribution of mitochondrial DNA (mtDNA) haplotypes for two genes (CO-I and CO-II) among 23 geographical populations. mtDNA revealed the presence of two well-supported phylogenetic lineages (phylads), with bootstrap value of 100%. Phylad I included three African populations, reinforcing the African-origin hypothesis of the species. Within phylad II, a distinct phylogeographical pattern was discovered: Atlantic populations (from the Americas and Madeira) were closer to the ancestral African populations than to Eastern ones (from Madagascar, Middle East and India). This means that during its passage from endemism to cosmopolitanism, Z. indianus exhibited two independent radiations, the older (the Eastern) to the East, and the younger (the Atlantic) to the West. Discriminant function analysis using 13 morphometrical characters was also able to discriminate between the two molecular phylads (93.34 ± 1.67%), although detailed morphological analysis of male genitalia using scanning electron microscopy showed no significant differences. Finally, crossing experiments revealed the presence of reproductive barrier between populations from the two phylads, and further between populations within phylad I. Hence, a bona species status was assigned to two new, cryptic species: Zaprionus africanus and Zaprionus gabonicus, and both were encompassed along with Z. indianus and Zaprionus megalorchis into the indianus complex. The ecology of these two species reveals that they are forest dwellers, which explains their restricted endemic distribution, in contrast to their relative cosmopolitan Z. indianus, known to be a human-commensal. Our results reconfirm the great utility of mtDNA at both inter- and intraspecific analyses within the frame of an integrated taxonomical project.