Molecular phylogeny of the Drosophila virilis section (Diptera : Drosophilidae) based on mitochondrial and nuclear sequences

Regardless of the well-documented virilis species group, most groups of the Drosophila virilis section have not been completely studied at molecular level since it was suggested. Therefore, phylogenetic relationships among and within species groups of the

果蝇 virilis section 的起源和系统发育研究

果蝇 virilis section(Hsu 1949)自建立以来其合理性在果蝇系统发育中还没有得到 检验。本文以线粒体烟酰胺腺嘌呤二核苷酸脱氢酶第二亚单位基因(ND2)全序列和细 胞色素氧化酶I 基因(COI)部分片段以及核中乙醇脱氢酶基因Adh 的编码区为遗传标 记，对virilis section 内61 个物种(13 个未发表新种),共计117 个个体进行了序列测 定。通过对单个基因和合并数据集进行简约分析，以及对合并数据集进行邻接法分析 和贝叶斯分析，本研究试图对以下问题进行探讨：(1)果蝇virilis section 的合理性。 (2)各种组间的系统关系。(3)种组内部各物种间的系统关系。(4)果蝇virilis section 的进化历史。现将主要结果总结如下： 1)分子系统学研究显示果蝇virilis section 为一个紧密相关的进化簇，为果蝇virilis section 的合理性从分子遗传学的角度提供了证据。 2)系统发育分析支持果蝇polychaeta 种组为单系群，该种组与未归类物种D. fluvialis 具有较近的亲缘关系。它们形成virilis section 中早期分化出的谱系。robusta 种组、melanica 种组、quadrisetata 种组以及新种组间关系较近。其中，robusta 种亚 组和melanica 种组关系较近；quadrisetata 种组和新种组形成姐妹群，它们形成的谱 系与robusta 种组内的okadai 种亚组关系紧密。 3) polychaeta 种组内部分为两枝，姐妹种D. polychaeta 和D. asper 组成其中一枝， 杂交实验显示它们具有非对称的交配前生殖隔离。另一枝中D. latifshahi 和D. daruma 显示了较近的亲缘关系。非洲物种D. hirtipes 与采自西双版纳的D. polychaeta X 关系 较近，提示它们可能有共同的起源。 4)合并数据集支持D. angor 种组为单系群，其中D. angor A 和D. velox 的姐妹种 关系得到较高的支持。但是该种组内部的其它物种间关系还需要进一步研究。 5) 分子系统发育分析结果清楚地表明robusta 种组属于多系发生。该种组被分为 三个种亚组：okadai 种亚组，robusta 种亚组和lacertosa 种亚组。在lacertosa 种亚组 内，D. bai 与其它成员相对远缘。okadai 种亚组的物种由于具有2n = 12 条染色体，且X 染色体为棒状，因而被认为是robusta 种组中较早分化出来的类群。但是我们的 分子数据并不支持这种观点。D. moriwakii 最初被描述为robusta 种组物种，后被订正 至melanica 种组。我们的系统发育分析显示该种与robusta 种亚组具有较近的关系， 而与melanica 种组物种的关系相对较远。除D. moriwakii 外，melanica 种组新旧大陆 物种各自形成单系。在系统树上，中国品系D. tsigana 与D. longiserrata 关系较近， 而与日本品系D. tsigana 的关系较远。杂交实验结果显示D. tsigana 中国品系和日本 品系间存在不对称的交配倾向，表明该种不同的地理群体间可能正处于分化阶段，而 中国品系则有可能已经演化为不同的物种。 6)果蝇quadrisetata 种组为单系发生，其内部分为两个明显的亚世系。第一个亚 世系包括D. sp T，D. barutani，D. potamophila 和D. spIZU；另一个亚世系包括D. beppui，D. karakasa，D. quadrisetata，D. multidentata，D. perlucida 和D. pilosa，其 中D. beppui 为该谱系中最早分化出的物种。 通过估计谱系间的分歧时间，本文推测果蝇virilis section 的祖先大约于中新世早 期起源于热带地区，virilis section 内许多物种可能栖息在旧大陆的低纬度地区，然后 通过适应性辐射扩散到各地。几乎所有的新大陆virilis section 物种是旧大陆物种通过 白令陆桥迁移到新大陆而演化形成的。

果蝇virillis section的起源和系统发育研究

果蝇vlfrlflisseetion（Hsu1949）自建立以来其合理性在果蝇系统发育中还没有得到检验。本文以线粒体烟酞胺腺嘌吟二核昔酸脱氢酶第二亚单位基因（ND2）全序列和细胞色素氧化酶I基因（COl）部分片段以及核中乙醇脱氢酶基因Adh的编码区为遗传标记，对viriltsseetion内61个物种（13个未发表新种），共计117个个体进行了序列测定。通过对单个基因和合并数据集进行简约分析，以及对合并数据集进行邻接法分析和贝叶斯分析，本研究试图对以下问题进行探讨：（1）果蝇virilisseetion的合理性。（2）各种组间的系统关系。（3）种组内部各物种间的系统关系。（4）果蝇virilissection的进化历史。现将主要结果总结如下：1）分子系统学研究显示果蝇virilissection为一个紧密相关的进化簇，为果蝇virilissection的合理性从分子遗传学的角度提供了证据。2）系统发育分析支持果蝇polychaeta种组为单系群，该种组与未归类物种D．Uvl'alis具有较近的亲缘关系。它们形成virilissection中早期分化出的谱系。robusta种组、melanica种组、quadrisetata种组以及新种组间关系较近。其中，robusta种亚组和melanica种组关系较近；quadrisetata种组和新种组形成姐妹群，它们形成的谱系与robusta种组内的。加dai种亚组关系紧密。3）polychaeta种组内部分为两枝，姐妹种D．polychaeta和D.asPer组成其中一枝，杂交实验显示它们具有非对称的交配前生殖隔离。另一枝中D.Iatshahi和D.daruma显示了较近的亲缘关系。非洲物种D．hirtiPes与采自西双版纳的D．polychaetaX关系较近，提示它们可能有共同的起源。4）合并数据集支持D．angor种组为单系群，其中D．angorA和D.velox的姐妹种关系得到较高的支持。但是该种组内部的其它物种间关系还需要进一步研究。5）分子系统发育分析结果清楚地表明robusta种组属于多系发生。该种组被分为三个种亚组：okadai种亚组，robsta种亚组和lacertosa种亚组。在laCertosa种亚组内，D.bai与其它成员相对远缘。okadai种亚组的物种由于具有2n=12条染色体，且X染色体为棒状，因而被认为是robusta种组中较早分化出来的类群。但是我们的分子数据并不支持这种观点。D.moriwakii最初被描述为robusta种组物种，后被订正至melanica种组。我们的系统发育分析显示该种与robsta种亚组具有较近的关系，而与melanica种组物种的关系相对较远。除D．moriwakii外，melanica种组新旧大陆物种各自形成单系。在系统树上，中国品系D．tsigana与D．longiserrata关系较近，而与日本品系D．tsigana的关系较远。杂交实验结果显示D．tsigana中国品系和日本品系间存在不对称的交配倾向，表明该种不同的地理群体间可能正处于分化阶段，而中国品系则有可能已经演化为不同的物种。6）果蝇quadrisetata种组为单系发生，其内部分为两个明显的亚世系。第一个亚世系包括；另，，个亚世系包括D．，其中D．beppui为该谱系中最早分化出韵物种。通过估计谱系间的分歧时间，本文推测果蝇virilissection的祖先大约于中新世早期起源于热带地区，virilissection内许多物种可能栖息在旧大陆的低纬度地区，然后通过适应性辐射扩散到各地。几乎所有的新大陆virilissection物种是旧大陆物种通过自令陆桥迁移到新大陆而演化形成的。

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

Diverse transposable elements are mobilized in hybrid dysgenesis in Drosophila virilis.

We describe a system of hybrid dysgenesis in Drosophila virilis in which at least four unrelated transposable elements are all mobilized following a dysgenic cross. The data are largely consistent with the superposition of at least three different systems of hybrid dysgenesis, each repressing a different transposable element, which break down following the hybrid cross, possibly because they share a common pathway in the host. The data are also consistent with a mechanism in which mobilization of a single element triggers that of others, perhaps through chromosome breakage. The mobilization of multiple, unrelated elements in hybrid dysgenesis is reminiscent of McClintock's evidence [McClintock, B. (1955) Brookhaven Symp. Biol. 8, 58-74] for simultaneous mobilization of different transposable elements in maize.

A screen for fast evolving genes from Drosophila

In an attempt to quantify the rates of protein sequence divergence in Drosophila, we have devised a screen to differentiate between slow and fast evolving genes. We find that over one-third of randomly drawn cDNAs from a Drosophila melanogaster library do not cross-hybridize with Drosophila virilis DNA, indicating that they evolve with a very high rate. To determine the evolutionary characteristics of such protein sequences, we sequenced their homologs from a more closely related species (Drosophila yakuba). The amino acid substitution rates among these cDNAs are among the fastest known and several are only about 2-fold lower than the corresponding values for silent substitutions. An analysis of within-species polymorphisms for one of these sequences reveals an exceptionally high number of polymorphic amino acid positions, indicating that the protein is not under strong negative selection. We conclude that the Drosophila genome harbors a substantial proportion of genes with a very high divergence rate.

Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.

Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Conservation and function of the transcriptional regulatory protein Runt.

A phylogenetic approach was used to identify conserved regions of the transcriptional regulator Runt. Alignment of the deduced protein sequences from Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virilis revealed eight blocks of high sequence homology separated by regions with little or no homology. The largest conserved block contains the Runt domain, a DNA and protein binding domain conserved in a small family of mammalian transcription factors. The functional properties of the Runt domain from the D. melanogaster gene and the human AML1 (acute myeloid leukemia 1) gene were compared in vitro and in vivo. Electrophoretic mobility-shift assays with Runt/AML1 chimeras demonstrated that the different DNA binding properties of Runt and AML1 are due to differences within their respective Runt domains. Ectopic expression experiments indicated that proteins containing the AML1 Runt domain function in Drosophila embryos and that sequences outside of this domain are important in vivo.

Genetic differentiation and cryptic speciation in natural populations of Drosophila lacertosa

Drosophila lacertosa, an Oriental member of the robusta species group in the virilis-repleta radiation, has a wide distribution from northern India throughout China to the Far East. Phylogenetic analyses of mitochondrial ND2 gene sequences revealed two ge

The mode of evolution of aggregation pheromones in Drosophila species

Aggregation pheromones are used by fruit flies of the genus Drosophila to assemble on breeding substrates, where they feed, mate and oviposit communally. These pheromones consist of species-specific blends of chemicals. Here, using a phylogenetic framework, we examine how differences among species in these pheromone blends have evolved. Theoretical predictions, genetic evidence, and previous empirical analysis of bark beetle species, suggest that aggregation pheromones do not evolve gradually, but via major, saltational shifts in chemical composition. Using pheromone data for 28 species of Drosophila we show that, unlike with bark beetles, the distribution of chemical components among species is highly congruent with their phylogeny, with closely related species being more similar in their pheromone blends than are distantly related species. This pattern is also strong within the melanogaster species group, but less so within the virilis species group. Our analysis strongly suggests that the aggregation pheromones of Drosophila exhibit a gradual, not saltational, mode of evolution. We propose that these findings reflect the function of the pheromones in the ecology of Drosophila, which does not hinge on species specificity of aggregation pheromones as signals.

A regulação do elemento transponível Helena em Drosophila

Pós-graduação em Genética - IBILCE