Molecular phylogeny of the subgenus Sophophora of Drosophila derived from large subunit of ribosomal RNA sequences

RNA sequencing has been used to assess the relationships among species of the subgenus Sophophora of the genus Drosophila. Two divergent domains, D1 and D2, of the large ribosomal RNA (28S), totalling 550 nucleotides have been sequenced using the rRNA direct sequencing method. A tree has been reconstructed from the neighbor-joining algorithm and the confidence intervals were evaluated by the bootstrap procedure. Results have shown that the branching of the willistoni and saltans groups of the subgenus Sophophora is very ancient and probably predates that of the subgenus Drosophila. The other groups and subgroups of Sophophora are clustered in three main lineages: 1) the melanogaster and oriental subgroups; 2) the montium subgroup; 3) the ananassae subgroup of the melanogaster group clustered with the fima and obscura groups. Thus, in comparison with our results, several taxa of various ranks appear paraphyletic (the genus Drosophila, the subgenus Sophophora and the melanogaster group). Our biochemical phylogeny is only in partial agreement with the pattern of Throckmorton's radiations as well as with classical taxonomy, both based on morphological data.     

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.     

Post-integration behavior of a Minos transposon in the malaria mosquito Anopheles stephensi

Transposable elements represent important tools to perform functional studies in insects. In Drosophila melanogaster, the remobilization properties of transposable elements have been utilized for enhancer-trapping and insertional mutagenesis experiments, which have considerably helped in the functional characterization of the fruitfly genome. In Anopheles mosquitoes, the sole vectors of human malaria, as well as in other mosquito vectors of disease, the use of transposons has also been advocated to achieve the spread of anti-parasitic genes throughout field populations. Here we report on the post-integration behavior of the Minos transposon in both the germ-line and somatic tissues of Anopheles mosquitoes. Transgenic An. stephensi lines developed using the piggyBac transposon and expressing the Minos transposase were tested for their ability to remobilize an X-linked Minos element. Germ-line remobilization events were not detected, while somatic excisions and transpositions were consistently recovered. The analysis of these events showed that Minos activity in Anopheles cells is characterized by unconventional functionality of the transposon. In the two cases analyzed, re-integration of the transposon occurred onto the same X chromosome, suggesting a tendency for local hopping of Minos in the mosquito genome. This is the first report of the post-integration behavior of a transposable element in a human malaria vector. Christina Scali and Tony Nolan contributed equally to the work.     

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.     

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     

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     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

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     

Search for Canonical <Emphasis Type="Italic">P</Emphasis> Element in Genomes of Drosophilinae Subfamily Species

Using the FISH method and PCR analysis, the presence of canonical P element was studied in the genomes of a number of laboratory lines isolated from nature in different years from the Zaprionus genus (Z. indianus) and Drosophila genus, Sophophora subgenus (D. ananassae, D. eugracilis, D. simulans, D. immigrans), Drosophila subgenus (D. virilis, D. mercatorum, D. hydei, D. funebris, D. pseudoobscura), Lordiphosa subgenus (L. magnipectinata), and Dorsilopha subgenus (D. busckii) in a search for new cases of horizontal transfer. According to our data, the L. magnipectinata genome contains sequences homologous to terminal regions of canonical P element, as well as the sequence with a weak homology from the central part of canonical P element. The P-element hybridization sites adjacent to the chromocenter were found in the D. pseudoobscura genome; this can indicate an ancient origin of the sequences homologous to the P element. The P element is absent in old D. simulans lines, except for the line isolated from nature in 2014 (in which the P element was found); this confirms data of other researchers about recent cases of horizontal P-element transfer in this species. No new cases of horizontal transfer were detected in the analyzed lines.     

ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10^–9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.     

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.     

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.     

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     

Phylogenetic relationships in Peniocereus (Cactaceae) inferred from plastid DNA sequence data

The phylogenetic relationships of Peniocereus (Cactaceae) species were studied using parsimony analyses of DNA sequence data. The plastid rpl16 and trnL-F regions were sequenced for 98 taxa including 17 species of Peniocereus, representatives from all genera of tribe Pachycereeae, four genera of tribe Hylocereeae, as well as from three additional outgroup genera of tribes Calymmantheae, Notocacteae, and Trichocereeae. Phylogenetic analyses support neither the monophyly of Peniocereus as currently circumscribed, nor the monophyly of tribe Pachycereeae since species of Peniocereus subgenus Pseudoacanthocereus are embedded within tribe Hylocereeae. Furthermore, these results show that the eight species of Peniocereus subgenus Peniocereus (Peniocereus sensu stricto) form a well-supported clade within subtribe Pachycereinae; P. serpentinus is also a member of this subtribe, but is sister to Bergerocactus. Moreover, Nyctocereus should be resurrected as a monotypic genus. Species of Peniocereus subgenus Pseudoacanthocereus are positioned among species of Acanthocereus within tribe Hylocereeae, indicating that they may be better classified within that genus. A number of morphological and anatomical characters, especially related to the presence or absence of dimorphic branches, are discussed to support these relationships.     

Subdivision of the genus Pleuroxus (Anomopoda,Chydoridae) into subgenera worldwide

Examination of many species of Pleuroxus from the entire World has revealed a major dichotomy in morphology, one line related to aduncus: the other to laevis both from Europe. Pleuroxus trigonellus (the type species of the genus) and Peracantha truncata are in two other distinct lines. The new subgenus Tylopleuroxus is proposed for the species related to aduncus, and the subgenus Picripleuroxus for those related to laevis. Tylopleuroxus is primarily Southern Hemisphere in distribution, Picripleuroxus primarily Northern Hemisphere. Evidence thus far suggests that probably most, perhaps all, of the taxa are confined to single continents or land masses. Deceased     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

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.     

Stable expression of human growth hormone over 50 generations in transgenic insect larvae

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.