Montium Subgroup Research Articles

Pupation site preference selection in Drosophila jambulina

Larvae of three latitudinally different populations of Drosophila jambulina belonging to the montium subgroup were tested for pupation site preference in relation to temperature. At 30 °C, larvae preferred to pupate on food whereas at 21 °C pupation occurred on the cotton. Genetic basis of larval pupation behavior was studied by conducting reciprocal crosses for 30 generations on food-selected and on cotton-selected pupae. Results from genetic analysis between food-selected and cotton-selected strains indicated a single gene responsible for the pupation site preference, with F1 progeny pupated on cotton and F2 (F1 × F1) larvae pupated on both food as well as on cotton. Although we found no change in morphological traits in food vs cotton selected populations, significantly different growth rate (body weight) between the two strains was observed. These results suggest that pupation site preferences can affect life-history traits in D. jambulina.

An investigation of Y chromosome incorporations in 400 species of Drosophila and related genera.

Y chromosomes are widely believed to evolve from a normal autosome through a process of massive gene loss (with preservation of some male genes), shaped by sex-antagonistic selection and complemented by occasional gains of male-related genes. The net result of these processes is a male-specialized chromosome. This might be expected to be an irreversible process, but it was found in 2005 that the Drosophila pseudoobscura Y chromosome was incorporated into an autosome. Y chromosome incorporations have important consequences: a formerly male-restricted chromosome reverts to autosomal inheritance, and the species may shift from an XY/XX to X0/XX sex-chromosome system. In order to assess the frequency and causes of this phenomenon we searched for Y chromosome incorporations in 400 species from Drosophila and related genera. We found one additional large scale event of Y chromosome incorporation, affecting the whole montium subgroup (40 species in our sample); overall 13% of the sampled species (52/400) have Y incorporations. While previous data indicated that after the Y incorporation the ancestral Y disappeared as a free chromosome, the much larger data set analyzed here indicates that a copy of the Y survived as a free chromosome both in montium and pseudoobscura species, and that the current Y of the pseudoobscura lineage results from a fusion between this free Y and the neoY. The 400 species sample also showed that the previously suggested causal connection between X-autosome fusions and Y incorporations is, at best, weak: the new case of Y incorporation (montium) does not have X-autosome fusion, whereas nine independent cases of X-autosome fusions were not followed by Y incorporations. Y incorporation is an underappreciated mechanism affecting Y chromosome evolution; our results show that at least in Drosophila it plays a relevant role and highlight the need of similar studies in other groups.

Single-Molecule Sequencing of the Drosophila serrata Genome.

Long-read sequencing technology promises to greatly enhance de novo assembly of genomes for nonmodel species. Although the error rates of long reads have been a stumbling block, sequencing at high coverage permits the self-correction of many errors. Here, we sequence and de novo assemble the genome of Drosophila serrata, a species from the montium subgroup that has been well-studied for latitudinal clines, sexual selection, and gene expression, but which lacks a reference genome. Using 11 PacBio single-molecule real-time (SMRT cells), we generated 12 Gbp of raw sequence data comprising ∼65 × whole-genome coverage. Read lengths averaged 8940 bp (NRead50 12,200) with the longest read at 53 kbp. We self-corrected reads using the PBDagCon algorithm and assembled the genome using the MHAP algorithm within the PBcR assembler. Total genome length was 198 Mbp with an N50 just under 1 Mbp. Contigs displayed a high degree of chromosome arm-level conservation with the D. melanogaster genome and many could be sensibly placed on the D. serrata physical map. We also provide an initial annotation for this genome using in silico gene predictions that were supported by RNA-seq data.

The pdm3 Locus Is a Hotspot for Recurrent Evolution of Female-Limited Color Dimorphism in Drosophila

Sex-limited polymorphisms are an intriguing form of sexual dimorphism that offer unique opportunities to reconstruct the evolutionary changes that decouple male and female traits encoded by a shared genome. We investigated the genetic basis of a Mendelian female-limited color dimorphism (FLCD) that segregates in natural populations of more than 20 species of the Drosophila montium subgroup. In these species, females have alternative abdominal color morphs, light and dark, whereas males have only one color morph in each species. A comprehensive molecular phylogeny of the montium subgroup supports multiple origins of FLCD. Despite this, we mapped FLCD to the same locus in four distantly related species-the transcription factor POU domain motif3 (pdm3), which acts as a repressor of abdominal pigmentation in D.melanogaster. In D.serrata, FLCD maps to a structural variant in the first intron of pdm3; however, this variant is not found in the three other species-D.kikkawai, D.leontia, and D.burlai-and sequence analysis strongly suggests the pdm3 alleles responsible for FLCD originated independently at least three times. We propose that cis-regulatory changes in pdm3 form sexually dimorphic and monomorphic alleles that segregate within species and are preserved, at least in one species, by structural variation. Surprisingly, pdm3 has not been implicated in the evolution of sex-specific pigmentation outside the montium subgroup, suggesting that the genetic paths to sexual dimorphism may be constrained within a clade but variable across clades.

Variations in the esterase expression pattern with respect to different light regimes in Drosophila agumbensis and Drosophila nagarholensis

Circadian clock enables organism’s to adapt under fluctuating environmental conditions by coupling of behavioral, physiological and molecular processes in a wide variety of organisms including bacteria, fungi, plants, birds and mammals. The endogenous circadian system functions to organize behavior and physiology to adapt to and anticipate environment changes in light and temperature. The present study is an attempt to understand enzyme profiles (alpha- and beta-esterases) of Drosophila agumbensis and Drosophila nagarholensis under light/dark (LD), constant dark (DD) constant light (LL), conditions over twenty generations. A polyacrylamide gel electrophoresis (7.5% – Native gel) was used to study the esterase expression patterns in two species of the montium subgroup of Drosophila. Alpha- and beta-esterase expression was significantly decreased in LL when compared to LD and DD at both the generations and species. In all the light regimes, females were found to have significantly higher level of α- and β-esterase expression than males. Flies were maintained under different light regimes showed difference in their expression patterns with respect to alpha- and beta-esterases. The present study showed that constant light conditions affect the expression of esterases in D. agumbensis and D. nagarholensis.

Effect of different light regimes on fitness of two species of Drosophila montium subgroup

Most organisms possess “biological chronometers” in the form of circadian clocks. Organism possessing circadian clock gains fitness advantage in two ways, by synchronizing its behavior through physiological process and secondly by coordinating its internal metabolic process. Environmental manipulations of circadian clocks have been shown to affect many life-history-related traits. Life-history traits are important components of fitness. To enhance individual fitness, organism has to synchronize the physiology with the surrounding environment. The present investigations were made to understand whether rhythm changes affect fitness of two co-existing species of montium a subgroup of Drosophila. The stocks were maintained at 20 ± 1 °C with 75% RH. Fitness such as fecundity, male lifetime fertility, female lifetime fertility, and longevity was assessed in LD (light/dark), LL (continuous light), and DD (continuous dark) for 15 and 30th generations. Fecundity was assessed in 25 pairs of flies for 20 days, and fertility and longevity was assessed in 10 pairs of flies until lifetime. The result revealed differential effect of light regimes on the two different species of Drosophila. Although the two species are related, effect of the three light regimes, LD, LL, and DD on them was different. It is evident that these two species although genetically related exhibit different responses to different light regimes.

An Evidence of Enhanced Fitness by Male Remounting during the Courtship in three Species of Montium Subgroup of Drosophila

Male mating a second female after first mate (male remating) is a common feature in Drosophila, particularly in mass culture. On the other hand, female remating is not as frequent as that of male remating because the sperms introduced along with the semen into the spermatheca are stored there. However, the phenomena of female remating have been reported in few species. During sexual activity, the males and females are involved an elaborate courtship at the end of which the male mounts on to the female followed by mating. During each mating, the mounted male through its ejaculate, transmits the sperms into the females' genital tract where it is stored. The sperms are stored in the spermatheca. Usually, with one mount maximum sperms are transferred to female hence second mounting does not occur. In most Drosophila, when a male mounts a female after an elaborate courtship, the pairs remain in cupola for a fixed period then dismounts. No more courtship or mounting is performed by the male towards the same female. In D.jambulina, D.nagarholensis and D.gangotrii remounting occurs two or three times immediately after the first mount. The reason for remounting has been analyzed in the present study through measuring the width of the female reproductive tract and sperm count after each mount. The female reproductive tract was dissected out and width of uterus was measured. The result showed significant difference in the width after each mount. The sperm count of these species after each mount also showed significant variation. Remounting perhaps enhances the fitness of the mating pairs.

Courtship Patterns in theDrosophila montiumSpecies Subgroup: Repeated Loss of Precopulatory Courtship?

During precopulatory courtship, male Drosophila typically produce wing vibration to generate species-specific songs before mounting females. Three species in the lini clade of the montium species subgroup have been found to produce species-specific sine song only after mounting and during copulation. Here we investigated and analyzed the courtship behavior of 29 species in the montium subgroup from video and song recordings and measured the duration of wing vibration. We describe a great diversity of courtship behavior in the montium subgroup. The courtship patterns can be categorized into four types in the montium subgroup: 1) type P/C, species with both precopulatory and copulatory courtship, such as D. parvula and D. nikananu, 2) type P-/C, species with sporadic precopulatory and mainly copulatory courtship, such as D. auraria and D. triauraria. 3) type C, species with only copulatory courtship, such as D. tani and D. pectinifera, 4) type C-, species with only very brief copulatory courtship, such as D. rufa and D. asahinai. According to a phylogenetic tree based on sequences of mitochondrial COI and COII, and the nuclear Adh, both precopulatory courtship and copulatory courtship were present in the most basal species D. parvula. Each of two branches in the montium subgroup contains four types of courtship behavior. Type C is present in each sub-branch. These results suggest that the courtship behavior initially involved both precopulatory and copulatory courtship, but that subsequently precopulatory courtship has gradually been lost in the montium subgroup. We suggest reasons why precopulatory behavior might come to be lost in the montium subgroup.

Divergent strategy for adaptation to drought stress in two sibling species of montium species subgroup: Drosophila kikkawai and Drosophila leontia

Drosophila leontia (warm adapted) has been considered as a sister species of Drosophila kikkawai (sub-cosmopolitan) with a very similar morphology. We found divergent strategies for coping with desiccation stress in these two species of montium subgroup. Interestingly, in contrast to clinal variation for body melanization in D. kikkawai, cuticular lipid mass showed a positive cline in D. leontia across a latitudinal transect. On the basis of isofemale line analysis, within population trait variability in cuticular lipid mass per fly is positively correlated with desiccation resistance and negatively correlated with cuticular water loss in D. leontia. A comparative analysis of water budget of these two species showed that higher abdominal melanization, reduced rate of water loss and greater dehydration tolerance confer higher desiccation resistance in D. kikkawai while the reduced rate of water loss is the only possible mechanism to enhance desiccation tolerance in D. leontia. The use of organic solvents has supported water proofing role of cuticular lipids in D. leontia but not in D. kikkawai. Thus, we may suggest that body melanization and cuticular lipids may represent alternative strategies for coping with dehydration stress in melanic versus non-melanic drosophilids. In both these species, carbohydrates were utilized under desiccation stress but a higher level of stored carbohydrates was evident in D. kikkawai. Further, we found increase desiccation resistance in D. kikkawai through acclimation while D. leontia lacks such a response. Thus, species specific divergence in water balance related traits in these species are consistent with their adaptations to wet and dry habitats.

Copulatory song in three species of the Drosophila montium subgroup extends copulation and shows unusual genetic control

Most Drosophila species produce species-specific male courtship song as part of their precopulatory courtship repertoire. Females usually mate most quickly when stimulated with appropriate song. Unusually, males of Drosophila lini and its sibling species sing sine song only during copulation. Sine song frequency is high in D. lini and Drosophila ogumai and lower in Drosophila ohnishii. Drosophila ogumai and D. lini are allopatric, although the range of D. ohnishii may meet or overlap with that of D. ogumai or D. lini. We conducted playback experiments with each species to examine the role of sine song frequency. Copulation duration increased for females of D. lini or D. ogumai when stimulated with high-frequency (240–250 Hz) artificial song and for females of D. ohnishii when stimulated with low-frequency (170–180 Hz) artificial song. This suggests that sine song frequency extends copulation duration in the three species and that species-typical song is more effective. We also examined song frequency in crosses between D. lini and D. ohnishii to assess its genetic control. Analysis of F1 and backcross generations between D. lini and D. ohnishii showed that sine song frequency is mainly due to additive autosomal variation, although a transgressive effect of the X chromosome and maternal or Y-linked effects were also found.

Increasing the data size to accurately reconstruct the phylogenetic relationships between nine subgroups of the Drosophila melanogaster species group (Drosophilidae, Diptera)

Previous phylogenetic analyses of the melanogaster species group have led to conflicting hypotheses concerning their relationship; therefore the addition of new sequence data is necessary to discover the phylogeny of this species group. Here we present new data derived from 17 genes and representing 48 species to reconstruct the phylogeny of the melanogaster group. A variety of statistical tests, as well as maximum likelihood mapping analysis, were performed to estimate data quality, suggesting that all genes had a high degree of contribution to resolve the phylogeny.Individual locus was analyzed using maximum likelihood (ML), and the concatenated dataset (12,988bp) were analyzed using partitioned maximum likelihood (ML) and Bayesian analyses. Separated analysis produced various phylogenetic relationships, however, phylogenetic topologies from ML and Bayesian analysis based on concatenated dataset, at the subgroup level, were completely identical to each other with high levels of support. Our results recovered three major clades: the ananassae subgroup, followed by the montium subgroup, the melanogaster subgroup and the oriental subgroups form the third monophyletic clade, in which melanogaster (takahashii, suzukii) forms one subclade and ficusphila [eugracilis (elegans, rhopaloa)] forms another. However, more data are necessary to determine the phylogenetic position of Drosophila lucipennis which proved difficult to place.

Horizontal transmission versus vertical inheritance of P elements in Drosophila and Scaptomyza: has the M-type subfamily spread from East Asia?

Polymerase chain reaction (PCR) screening for P elements was carried out in 77 species with a primer set highly specific for the M-type subfamily. In the course of this search M-type elements were detected in 29 species: In the melanogaster (subgroups montium and rhopaloa) and obscura species groups of Drosophila (25 out of 71 species examined), and in the genus Scaptomyza (four out of six species). M-type elements are present in all species of the montium subgroup investigated so far (21), but occur only sporadically in other groups. Within the montium subgroup 20 species possess only incomplete copies, only one species (D. lacteicornis) harbours apparently full-sized elements. In contrast, outside the montium subgroup almost all species with M-type elements carry full-sized copies suggesting transpositional activity, at least in the recent past. The interior section of the full-sized M-type element of D. lacteicornis was partially sequenced (936 bp). In addition, the complete sequences of four internally deleted M-type elements of D. lacteicornis, D. rufa, D. quadraria, and D. triauraria were determined. Sequence comparisons (including sequence data from previous investigations) revealed striking discrepancies between P element phylogeny and the cladogenesis of their host species. Among several possible pathways for interspecific transfers of M-type elements, we favour the hypothesis assuming the invasion of Scaptomyza as well as the obscura group species of Drosophila via independent transmission routes originating from Asian species of the montium subgroup of Drosophila. The logical geographic scenario for these events would be East-Asia.

Developmental constraints and convergent evolution inDrosophilasex comb formation

The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii, where the SCs remain approximately transverse, and Drosophila biarmipes, where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species (Drosophila nikananu) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster, forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac), is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies.

BackgroundThe native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup.ResultsA normalized cDNA library was constructed from whole fly bodies at several developmental stages, including larvae and adults. Assembly of 11,616 clones sequenced from the 3' end allowed us to identify 6,607 unique contigs, of which at least 90% encoded peptides. Partial transcripts were discovered from a variety of genes of evolutionary interest by BLASTing contigs against the 12 Drosophila genomes currently sequenced. By incorporating into the cDNA library multiple individuals from populations spanning a large portion of the geographical range of D. serrata, we were able to identify 11,057 putative single nucleotide polymorphisms (SNPs), with 278 different contigs having at least one "double hit" SNP that is highly likely to be a real polymorphism. At least 394 EST-associated microsatellite markers, representing 355 different contigs, were also found, providing an additional set of genetic markers. The assembled EST library is available online at .ConclusionWe have provided the first gene collection and largest set of polymorphic genetic markers, to date, for the fly D. serrata. The EST collection will provide much needed genomic resources for this model species and facilitate comparative evolutionary studies within the montium subgroup of the D. melanogaster lineage.

Research on the Karyotype and Evolution of Drosophila melanogaster Species Group

Mitotic metaphase chromosomes of 34 species of Drosophila melanogaster species group were examined. Certain new karyotypes were described for the first time, and their evolutionary and interspecific genetic relationships among 8 subgroups of D. melanogaster species group were analyzed systematically. The results were as follows. The basic karyotype of elegans subgroup was type A. The karyotypes of eugracilis subgroup, melanogaster subgroup, and ficusphila subgroup were all type C. The karyotypes of takahashii subgroup and suzukii subgroup were both type C and type D. The montium subgroup had six kinds of karyotypes: types B, C, C', D, D', and E. The ananassae subgroup had three kinds of karyotypes: types F, G, and H. Thus, the melanogaster species group was classified into five pedigrees based on the diversity of these karyotypes: 1) elegans; 2) eugracilis-melanogaster-ficusphila; 3) takkahashii-suzukii; 4) montium; 5) ananassae. The above-mentioned results in karyotypic evolution were consistent with those of DNA sequence analysis reported by Yang except for the elegans subgroup and this subgroup was considered as the ancestral subgroup. Karyotype analysis of the same drosophila from different isofemale lines indicated that the same Drosophila from different places showed karyotypic variation which might be due to different geographical environment and evolutionary degree or interaction between the two factors.

Localization and Characterization of Heterochromatin among Four Species of the Montium Subgroup of Drosophila

The karyotype of 4 species namely D. agumbensis, D. anomelani, D. truncata and D. cauverii have been analysed. The heterochromatin content has been characterized by using C- and Q-profiles. The Y- and 4th chromosome have shown a great degree of divergence in their form and size. The heterochromatin has played a very important role in bringing about this high degree of differentiation within the limit of 2n=8 among the members of the montium subgroup of Drosophila.

Drosophila bunnanda— a new species from northern Australia with notes on other Australian members of the montium subgroup (Diptera: Drosophilidae)

Drosophila bunnanda— a new species from northern Australia with notes on other Australian members of the montium subgroup (Diptera: Drosophilidae)

Drosophila bunnanda-a new species from northern Australia with notes on other Australian members of the montium subgroup (Diptera: Drosophilidae)

An unknown Drosophila montium subgroup species was collected on Cape York Peninsula in 1992, a live culture of the same species was established from flies collected in the vicinity of Lake Placid near Cairns in 2001. From these specimens we now have sufficient information to describe a new species—Drosophila bunnanda. It differs morphologically from the four other montium subgroup species already known from northern Queensland—D. serrata, D. birchii, D. kikkawai, and D. sp. cf. jambulina, and from one very similar species—D. dominicana—known from Papua New Guinea. Molecular data support the taxonomic findings. Additional information and a key for all Australian montium subgroup species is provided to allow clear differentiation between them and D. bunnanda.

Regulation of Body Pigmentation by the Abdominal-B Hox Protein and Its Gain and Loss in Drosophila Evolution

Hox genes have been implicated in the evolution of many animal body patterns, but the molecular events underlying trait modification have not been elucidated. Pigmentation of the posterior male abdomen is a recently acquired trait in the Drosophila melanogaster lineage. Here, we show that the Abdominal-B (ABD-B) Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. ABD-B regulation of pigmentation evolved through the gain of ABD-B binding sites in a specific cis-regulatory element of the yellow gene of a common ancestor of sexually dimorphic species. Within the melanogaster species group, male-specific pigmentation has subsequently been lost by at least three different mechanisms, including the mutational inactivation of a key ABD-B binding site in one lineage. These results demonstrate how Hox regulation of traits and target genes is gained and lost at the species level and have general implications for the evolution of body form at higher taxonomic levels.

Basal relationships in the Drosophila melanogaster species group

The Drosophila melanogaster species group is a popular model for evolutionary studies due to its morphological and ecological diversity and its inclusion of the model species D. melanogaster. However, phylogenetic relationships among major lineages within this species group remain controversial. In this report, the phylogeny of 10 species representing each of the well-supported monophyletic clades in the melanogaster group was studied using the sequences of 14 loci that together comprise 9493 nucleotide positions. Combined Bayesian analysis using gene-specific substitution models produced a 100% credible set of two trees. In the strict consensus of these trees, the ananassae subgroup branches first in the melanogaster species group, followed by the montium subgroup. The remaining lineages form a monophyletic clade in which D. ficusphila and D. elegans branch first, followed by D. biarmipes, D. eugracilis, and the melanogaster subgroup. This strongly supported phylogeny resolves most basal relationships in the melanogaster species group, and provides a framework that can be extended in the future to encompass more species.