Molecular phylogeny of Drosophila based on ribosomal RNA sequences

Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.Correspondence to: M. Pelandakis     

Distribution of the Transposable Element Minos in the Genus Drosophila

We analyzed 28 species of the genus Drosophilafor the presence of the Tc1-like transposable element Minosusing Southern blot hybridization under high stringency conditions. The Minostransposon was found in members of both the Drosophilaand the Sophophorasubgenus showing a distribution that is wider if compared to other well-studied Drosophilatransposons such as the Pelement, hoboand mariner. The presence of Minos-hybridizing sequences was discontinuous in the Sophophorasubgenus, especially in the melanogasterspecies group. Using the Polymerase Chain Reaction we amplified a portion corresponding to the putative Minostransposase from different Drosophilaspecies. Cloning and sequence analysis of randomly selected Minoscopies from D. mojavensisis, D. saltansand D. willistonisupports the idea that event(s) of horizontal transfer may have contributed to the spreading of this transposon in the Drosophilagenus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

Relationships within the Melanogaster subgroup species of the genus Drosophila (Sophophora)

Five members of the melanogaster species subgroup of the subgenus Sophophora have been studied cytologically, their mitotic chromosomes analysed after Giemsa, C-banding and quinacrine staining. In all five species (D. yakuba, D. teissieri, D. erecta, D. orena and D. mauritiana) n=4 and all of the species except D. orena have a typical melanogaster like mitotic karyotype though there are clear differences between species in the distribution of both C⁺ and Q⁺ material. D. orena has large metacentric X and Y chromosomes due to the accumulation of intensively fluorescing material on these elements with respect to their homologues in melanogaster. This extra heterochromatin of D. orena correlates with a very high proportion of satellite DNA in its nuclear genome (S. Barnes, unpublished). The polytene chromosomes of these species were studied after quinacrine staining and Q⁺ material found to be restricted to the polytene fourth chromosomes, with the exception of D. orena which possesses considerable Q⁺ material in its chromocentre. These findings are discussed in the light of other studies of karyotype evolution in the genus Drosophila.     

Identification of One Intron Loss and Phylogenetic Evolution of Dfak Gene in the Drosophila melanogaster Species Group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.     

Evolutionary History and Mode of the <Emphasis Type="Italic">amylase</Emphasis> Multigene Family in <Emphasis Type="Italic">Drosophila</Emphasis>

Previous studies indicate that the tandemly repeated members of the amylase (Amy) gene family evolved in a concerted manner in the melanogaster subgroup and in some other species. In this paper, we analyzed all of the 49 active and complete Amy gene sequences in Drosophila, mostly from subgenus Sophophora. Phylogenetic analysis indicated that the two types of diverged Amy genes in the Drosophila montium subgroup and Drosophila ananassae, which are located in distant chromosomal regions from each other, originated independently in different evolutionary lineages of the melanogaster group after the split of the obscura and melanogaster groups. One of the two clusters was lost after duplication in the melanogaster subgroup. Given the time, 24.9 mya, of divergence between the obscura and the melanogaster groups (Russo et al. 1995), the two duplication events were estimated to occur at about 13.96 ± 1.93 and 12.38 ± 1.76 mya in the montium subgroup and D. ananassae, respectively. An accelerated rate of amino acid changes was not observed in either lineage after these gene duplications. However, the G+C contents at the third codon positions (GC3) decreased significantly along one of the two Amy clusters both in the montium subgroup and in D. ananassae right after gene duplication. Furthermore, one of the two types of the Amy genes with a lower GC3 content has lost a specific regulatory element within the montium subgroup species and D. ananassae. While the tandemly repeated members evolved in a concerted manner, the two types of diverged Amy genes in Drosophila experienced frequent gene duplication, gene loss, and divergent evolution following the model of a birth-and-death process.     

Phylogenetic analysis and a time tree for a large drosophilid data set (Diptera: Drosophilidae)

Drosophila is the genus responsible for the birth of experimental genetics, but the taxonomy of drosophilids is difficult because of the overwhelming diversity of the group. In this study, we assembled sequences for 358 species (14 genera, eight subgenera, 57 species groups, and 65 subgroups) to generate a maximum‐likelihood topology and a Bayesian timescale. In addition to sampling an unprecedented diversity of Drosophila lineages, our analyses incorporated a geographical perspective because of the high levels of endemism. In our topology, Drosophila funebris (Fabricius, 1787) (the type species of Drosophila) is tightly clustered with the pinicola subgroup in a North American clade within subgenus Drosophila. The type species of other drosophilid genera fall within the Drosophila radiation, presenting interesting prospects for the phylogenetic taxonomy of the group. Our timescale suggests that a few drosophilid lineages survived the Cretaceous–Palaeogene (K‐Pg) extinction. The drosophilid diversification began during the Palaeocene in Eurasia, but peaked during the Miocene, an epoch of drastic climatic changes. The most recent common ancestor of the clades corresponding to subgenera Sophophora and Drosophila lived approximately 56 Mya. Additionally, Hawaiian drosophilids diverged from an East Asian lineage approximately 26 Mya, which is similar to the age of the oldest emerging atoll in the Hawaiian–Emperor Chain. Interestingly, the time estimates for major geographical splits (New World versus Asia and Africa versus Asia) were highly similar for independent lineages. These results suggest that vicariance played a significant role in the radiation of fruit flies. © 2013 The Linnean Society of London     

Evolutionary changes in non-histone chromosomal proteins within the Drosophila melanogaster group revealed by monoclonal antibodies

Monoclonal antibodies directed against nonhistone chromosomal proteins of D. melanogaster were tested for crossreactivity with the homologous antigens of various Drosophila species. — By indirect immunofluorescence it could be shown that three antibodies react only with polytene chromosomes of species of the D. melanogaster subgroup, and only much less with chromosomes of other species of Drosophila. — With chromosomes of various other species of the Sophophora or Drosophila radiations only a reaction at background level could be observed. — The results suggest that the three antibodies react with different antigenic determinants of a single protein whose conformation changed rather fast during evolution of the Drosophilidae.     

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.     

Comparative mapping of cosmids and gene clones from a 1.6 Mb chromosomal region of Drosophila melanogaster in three species of the distantly related subgenus Drosophila

The successful hybridization of cosmid clones from Drosophila melanogaster (Sophophora subgenus) to the salivary gland chromosomes of other species as distantly related as those in the Drosophila subgenus attests their great potential for unravelling genome evolution. We have carried out, using 28 cosmids and 13 gene clones, a study of the organization of the D. melanogaster 95A-96A chromosomal region in three Drosophila subgenus species: D. repleta, D. buzzattii and D. virilis. These clones were first used to built an accurate map of this 1.6 Mb region of D. melanogaster chromosome 3R (Muller’s element E). Then, they were hybridized and mapped to the homologous chromosome 2 of the other three distantly related species. The studied region is disseminated over 13 different sites of chromosome 2 in the Drosophila subgenus species, which implies a minimum of 12 inversion breakpoints fixed between the two subgenera. Extrapolation to the entire chromosome gives 90 fixed inversions. The D. melanogaster Pp1-96A-Acr96Aa segment conserved in D. repleta and D. buzzatii is longer than previously thought and is also conserved in D. virilis. In addition, three other D. melanogaster segments conserved in the three Drosophila subgenus species were found. Finally, our data indicate significant statistical differences in the evolution rate of Muller’s element E among lineages, a result that agrees well with the previous cytogenetic data. Received: 22 July 1998; in revised form: 11 November 1998 / Accepted: 12 November 1998     

Characterization of the shsp genes in Drosophila buzzatii and association between the frequency of Valine mutations in hsp23 and climatic variables along a longitudinal gradient in Australia

The small heat shock gene (shsp) cluster of Drosophila buzzatii was sequenced and the gene order and DNA sequence were compared with those of the shsps in Drosophila melanogaster. The D. buzzatii shsp cluster contains an inversion and a duplication of hsp26. A phylogenetic tree was constructed based on hsp26 genes from several Drosophila species of the Sophophora and Drosophila subgenera. The tree shows first a separation of the Sophophora and the Drosophila subgenera and then the Drosophila subgenus is divided into the Hawaiian Drosophila and the repleta/virilis groups. Only the latter contain a duplicated hsp26. Comparing the gene organisation of the shsp cluster shows that all the Drosophila subgenus species contain the inversion. Putative heat shock elements (HSE) were found in the promoters of all the shsp and putative regulator elements for tissue specific expression were found in the promoter of hsp23, hsp27 and one of the hsp26 genes. hsp23 was found to be polymorphic for four non-synonymous changes that all lead to exchange of a Valine. The duplicated hsp26 gene in D. buzzatii (phsp26) was polymorphic for two non-synonymous changes. The allele frequencies of these variants were determined in nine D. buzzatii populations covering most of its distribution in Australia using high-resolution melting curves. The allele frequencies of one of the hsp23 variants showed a significant linear regression with longitude and the pooled frequency of the four Valine changes of hsp23 in the nine populations showed a significant linear regression with longitude and with a composite measure of climatic variables.     

Karyotype variation in Drosophila birchii

Drosophila birchii, a member of the melanogaster species group of the subgenus Sophophora, is common in the tropical rain forests of the Australia-New Guinea areas. Chromosome squashes are easily prepared from the larval ganglion cells and the sex chromosomes are readily recognizable. The species exhibits a remarkable karyotype variation. The metaphase plate figures, in general, show two pairs of V's, one pair of dots and one pair of sex chromosomes. Variations in metaphase chromosome morphology are found in the X (with four types), the Y (with three types) and chromosome IV (with two types). Chromosomal interchanges between X- and Y-chromosomes Type I are postulated to be involved in the differentiation of sex chromosome morphology while the modification of chromosome IV seems likely to be a result of the acquisition of extra heterochromatin. These chromosome types form seven distinct metaphase plate figures, all encountered in wild populations, thus giving D. birchii the most variable karyotype in the genus Drosophila.     

Sex-specific methylation in <Emphasis Type="Italic">Drosophila</Emphasis>: an investigation of the <Emphasis Type="Italic">Sophophora</Emphasis> subgenus

Epigenetic phenomena have been widely characterized in the genomes of vertebrates and DNA methylation is a key mechanism of epigenetic regulation. The DNA methylation systems of invertebrates and vertebrates show several notable differences. However, the evolutionary implications of those differences only recently began to be revealed. Our study investigated the recurrence of sex-specific methylation, as previously described for the species Drosophila willistoni, in other species of the Sophophora subgenus that present close evolutionary relationship. The MSRE and Southern blot techniques were used to analyze rDNA of some species of the willistoni, melanogaster, saltans and obscura groups of Drosophila and the results suggested that differential DNA methylation between sexes only occurs in Drosophila tropicalis and D. insularis, two sibling species of the willistoni subgroup. However, only using the MSRE technique we could detect sex-specific patterns of DNA methylation in all species of willistoni subgroup. These results indicate that DNA methylation may present important differences, even between closely related species, shedding new light on this Neotropical species complex.     

On the Evolution of Dopa decarboxylase (Ddc) and Drosophila Systematics

We have sequenced most of the coding region of the gene Dopa decarboxylase (Ddc) in 24 fruitfly species. The Ddc gene is quite informative about Drosophila phylogeny. Several outstanding issues in Drosophila phylogeny are resolved by analysis of the Ddc sequences alone or in combination with three other genes, Sod, Adh, and Gpdh. The three species groups, melanogaster, obscura, and willistoni, are each monophyletic and all three combined form a monophyletic group, which corresponds to the subgenus Sophophora. The Sophophora subgenus is the sister group to all other Drosophila subgenera (including some named genera, previously considered outside the Drosophila genus, namely, Scaptomyza and Zaprionus, which are therefore downgraded to the category of subgenus). The Hawaiian Drosophila and Scaptomyza are a monophyletic group, which is the sister clade to the virilis and repleta groups of the subgenus Drosophila. The subgenus Drosophila appears to be paraphyletic, although this is not definitely resolved. The two genera Scaptodrosophila and Chymomyza are older than the genus Drosophila. The data favor the hypothesis that Chymomyza is older than Scaptodrosophila, although this issue is not definitely resolved. Molecular evolution is erratic. The rates of nucleotide substitution in 3rd codon position relative to positions 1 + 2 vary from one species lineage to another and from gene to gene. Received: 2 June 1998 / Accepted: 3 September 1998     

Polyphyly of Lordiphosa and its relationships in Drosophilinae (Diptera: Drosophilidae)

The phylogenetic relationships of Lordiphosa and some taxa in Drosophilinae were analysed on the basis of a total of forty‐one selected drosophilid species. These included eighteen species of five Lordiphosa species‐groups as the main target, twenty‐three species representative of the major drosophiline ingroup taxa and four species of Steganinae as outgroup. Sixty‐eight morphological characters of adults were subjected to cladistic analysis. From the results it is concluded that Lordiphosa is polyphyletic; the Lo. tenuicauda species‐group and genus Nesiodrosophila form a single monophyletic group; Lordiphosa proper (i.e. Lordiphosa spp. minus the tenuicauda group) comprises another monophyletic group; within Lordiphosa proper the fenestrarum, nigricolor and denticeps groups are all monophyletic, but monophyly of the miki group is not strongly supported; genera Hirtodrosophila and Scaptomyza and subgenus Sophophora are all monophyletic; and within Drosophilinae, genus Scaptodrosophila is the first to have split from the main lineage, but the branching order of other clades, Chymomyza, Lordiphosa proper, Sophophora, Hirtodrosophila, Nesiodrosophila+ Lo. tenuicauda group, Scaptomyza, Dorsilopha and subgenus Drosophila, remains unresolved. The topology of maximum parsimony cladograms suggests that Lordiphosa proper lies close to Sophophora as proposed previously, although its phylogenetic position could not be determined conclusively. By contrast, bootstrap values tended to contradict another hypothesis that Lordiphosa and Scaptomyza are sister groups.     

Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene

The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific “bush” that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421–469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197:1–139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. Correspondence requests to: F. J. Ayala     

Mise à jour taxonomique et répartition des puces du genre Ctenophthalmus Kolenati 1856 en région paléarctique occidentale (Insecta : Siphonaptera : Ctenophthalmidae)

Summary. Taxonomic update and geographic distribution of fleas of the genus Ctenophthalmus Kolenati 1856 in the Western Palearctic Region (Insecta: Siphonaptera: Ctenophthalmidae). Among fleas (Siphonaptera), the genus Ctenophthalmus is the one that comprises the largest number of taxa and is also characterized by a large geographical range. Here, we present a taxonomic revision of the Western Paleartic subgenera, groups, species and subspecies. We recognized a total of 143 taxa (57 species and 86 subspecies). These taxa are clustered into 23 groups of species, which fall into seven of the 16 subgenera of the genus Ctenophthalmus. According to Hopkins & Rothschild (1966), the subgenus Ctenophthalmus would only include the agyrtes group, itself divided into subgroups. We decided to raise these subgroups to group status to clarify taxonomic relationships within the subgenus Ctenophthalmus. Within this subgenus, the arvernus group is renamed baeticus, the fransmiti group is confirmed, and the egregius group is created. For each taxon, we provided information on geographical distribution, mammalian hosts, and host specificity.     

Evolution of the transposable element mariner in the Drosophila melanogaster species group

The population biology and molecular evolution of the transposable element mariner has been studied in the eight species of the melanogaster subgroup of the Drosophila subgenus Sophophora. The element occurs in D. simulans, D. mauritiana, D. sechellia, D. teissieri, and D. yakuba, but is not found in D. melanogaster, D. erecta, or D. orena. Sequence comparisons suggest that the mariner element was present in the ancestor of the species subgroup and was lost in some of the lineages. Most species contain both active and inactive mariner elements. A deletion of most of the 3 end characterizes many elements in D. teissieri, but in other species the inactive elements differ from active ones only by simple nucleotide substitutions or small additions/deletions. Active mariner elements from all species are quite similar in nucleotide sequence, although there are some-species-specific differences. Many, but not all, of the inactive elements are also quite closely related. The genome of D. mauritiana contains 20–30 copies of mariner, that of D. simulans 0–10, and that of D. sechellia only two copies (at fixed positions in the genome). The mariner situation in D. sechellia may reflect a reduced effective population size owing to the restricted geographical range of this species and its ecological specialization to the fruit of Morinda citrifolia.     

ALCOHOL TOLERANCE,ADH ACTIVITY,AND ECOLOGICAL NICHE OF DROSOPHILA SPECIES

In vitro alcohol dehydrogenase (ADH) activity was measured in adults of species belonging to Drosophila and to the related genus Zaprionus. Data were analyzed according to the known breeding sites and the level of ethanol tolerance of these species. Alcohol dehydrogenase activity was assayed with both ethanol (E) and isopropanol (I). Our results show a very broad range of activities among the 71 species investigated, the ratio of the highest value observed (D. melanogaster) to the lowest (D. pruinosa) being 65:1. A general positive correlation was found between the level of ADH activity and the capacity to detoxify ethanol. Nevertheless, many species show exceptions to this rule. Contrary to a logical expectation, adaptation to high alcoholic resources, which has been a recurrent evolutionary event, was not mediated by a more efficient use of ethanol, that is, an increase of the E/I ratio. This ratio seems to be quite variable according to the phylogeny and is especially low in the subgenus Sophophora as well as in Zaprionus. Alcohol tolerance clearly is related to the larval habitat of the species and shows that adaptation to alcoholic resources has been a major evolutionary challenge in drosophilids. This adaptation is not related to phylogeny, having occurred independently several times during the evolution of the group. Finally, it should be borne in mind that, besides metabolization and detoxification, other physiological processes such as nervous-system tolerance or ethanol excretion may be involved in ethanol tolerance, and such functions also should be investigated. Environmental ethanol, which is certainly a major ecological parameter for many drosophilids, has selected a diversity of physiological adaptations, all related to the Adh locus, but presumably much more complicated than was previously believed.