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 (12,988 bp) 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.

Convergent intron loss of MRP1 in Drosophila and mosquito species.

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.

Optimal Formula of Angelica sinensis Ameliorates Memory Deficits in ß-amyloid Protein-induced Alzheimer's Disease Rat Model.

OBJECTIVE: Angelica (A.) sinensis is used as a traditional medical herb for the treatment of neurodegeneration, aging, and inflammation in Asia. A. sinensis optimal formula (AOF) is the best combination in A. sinensis that has been screened to rescue the cognitive ability in ß-amyloid peptide (Aß25-35)-treated Alzheimer's disease (AD) rats. The objective of this study was to investigate the effect of AOF on the learning and memory of AD rats as well as to explore the underlying mechanisms. METHODS: Male Wistar rats were infused with Aß25-35 for AD model induction or saline (negative control). Five groups of AD rats were fed on AOF at 20, 40, or 80 mL/kg every day, donepezil at 0.9 mg/kg every day (positive control), or an equal volume of water (AD model) intragastrically once a day for 4 weeks, while the negative control rats were fed on water. The Morris water maze test was used to evaluate the cognitive function of the rats. The Aß accumulation, cholinergic levels, and antioxidative ability were detected by ELISA. Additionally, the candidate mechanism was determined by gene sequencing and quantitative real-time polymerase chain reaction. RESULTS: The results showed that AOF administration significantly ameliorated Aß25-35-induced memory impairment. AOF decreased the levels of amyloid-ß precursor protein and Aß in the hippocampus, rescued the cholinergic levels, increased the activity of superoxide dismutase, and decreased the malondialdehyde level. In addition, AOF inhibited the expression of IL1b, Mpo, and Prkcg in the hippocampus. CONCLUSION: These experimental findings illustrate that AOF prevents the decrease in cognitive function and Aß deposits in Aß25-35-treated rats via modulating neuroinflammation and oxidative stress, thus highlighting a potential therapeutic avenue to promote the co-administration of formulas that act on different nodes to maximize beneficial effects and minimize negative side effects.

An intron loss of Dfak gene in species of the Drosophila melanogaster subgroup and phylogenetic analysis.

Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.

[Sequence analysis of the DM domain of three Dmrt genes in loach, Paramisgurnus dabryanus].

We amplified genomic DNA of the Paramisgurnus dabryanus using the DM degenerate primers and detected a band, approximately 150 bp. After cloned into pMD18-T vector and sequenced, three sequences showed high homology with the DM domain. They were named as PdDmrt1, PdDmrt3, and PdDmrt5. Based on similarities of amino acid sequences of the DM domain, Dmrt gene protein sequences from six vertebrates species were included in a phylogenetic tree. It confirmed the identity of the three PCR products. Our results reveal that Dmrt gene is highly conservative in phylogeny.

[Karyotypes of five species in the Drosophila melanogaster species group].

Based on the traditional knocking-slides and Giemsa-staining methods,this paper firstly analyzed the metaphase chromosomes of five species (D. constricta, D. ohnishii, D. ogumai, D. pseudobaimaii and D. tani) of Drosophila melanogaster species group. The results show that all of the five species share the same chromosomal numbers (2n=8) but with different typical shapes. D. pseudobaimaii and D. tani share the shape 2V, 1R, 1D; D. constricta shows 2V,1R,1D with unclear recognized dot chromosomes; D. ohnishii and D. ogumai share the same chromosomal shapes 2V2R. And that between karyotype and phylogenetic relationships are found to be correspond.

Hsp27 gene in Drosophila ananassae subgroup was split by a recently acquired intron.

In Drosophila, heat shock protein 27 (Hsp27) is a critical single-copy intron-free nuclear gene involved in the defense response against fungi and bacteria, and is a regulator of adult lifespan. In the present study, 33 homologous Hsp27 nucleotide sequences from different Drosophila species were amplified by PCR and reverse transcription PCR, and the phylogenetic relationships were analysed using neighbour-joining, maximum-likelihood and Bayesian methods. The phylogenetic topologies from analysis with different algorithms were similar, suggesting that the Hsp27 gene was split by a recently acquired intron during the evolution of the Drosophila ananassae subgroup.

[Random amplified polymorphic DNA (RAPD) analysis of Drosophila simulans in the mainland of China].

The widespread distribution of Drosophila simulans in the mainland of China was found recently. Random amplified polymorphic DNA (RAPD) was used to analyzed DNA polymorphisms of 38 Drosophila simulans populations collected from the mainland of China. The origins of D. simulans in China were also discussed. Using 40 arbitary primers (10 bp), we made PCR amplifications under the optimized reaction conditions for RAPD that had been established in our laboratory. Our molecular phylogenetic tree constructed by UPGMA method showed as follows: (1) 38 populations are apparently classified into Northern cluster and Southern cluster, where NJ(Nanjing) can be considered as the boundary according to latitude. The populations in the Northern cluster can be further grouped into two sub-clusters, where BJ(Beijing) can be considered as the boundary according to latitude. The Northeast one consists of 10 populations, including Mohe(MH), Hailaer(HLR), Heihe(HH), Jiamusi(JMS), Haerbin(HRB), Changchun(CC), Shenyang(SY), Dandong(DD), Yanji(YJ), Tumen(TM); the other consists of 12 populations, including Beijing (BJ), Datong(DT), Huhehaote(HHHT), Yinchun(YC), Xinin(XN), Lanzhou(LZ), Taiyuan(TY), Shijiazhuang(SJZ), Yantai (YT), Jinan(JN), Xuzhou(XZ), Lianyungang(LYG). The southern cluster consists of the left 16 populations, including Zhengzhou (ZZ), Wuhan(WH), Shanghai(SH), Nanjing(NJ), Hangzhou(HZ), Chongqing(CQ), Nanchang(NC), Wenzhou(WZ), Changsha (CS), Guiyang(GY), Fuzhou(FZ), Kunming(KM), Xiamen(XM), Guangzhou(GZ), Nanning(NN), Hainan(HN). (2) The genetic distances among populations relate to the collection sites to a great extent, though there are some exceptions. According to the facts that we know, (1) D. simulans is an exotic species; (2) The invasion of D. simulans in the mainland of China took place in recent 30 years or so. (3) The widespread distribution of D. simulans in the mainland of China started in recent 10 years or so. Therefore, the results above might not be due to geographical differentiation, but due to genetic differences of initial founder individual(s) between geographical populations. Some of geographically adjacent populations, however, have common initial founder individual(s), which results in the classification of D. simulans populations according to their geographical relationship. We suggest that there are two possible reasons which result in the genetic differences of initial founder individual(s) between geographical populations, one is the D. simulans in the mainland of China had multiple origins, the other is founder effect and/or bottleneck effect that the D. simulans have experienced in the course of expansion.

[Molecular phylogenetic relationships of Drosophila immigrans species group based on H2a-H2b spacer and Cyt b sequences].

In order to investigate molecular phylogenetic relationships of Drosophila immigrans species group, nucleotide sequences of H2a-H2b spacer regions and partial nucleotide sequences of Cytochrome b gene for five specieses subgroup in Drosophila immigrans species group and D. hydei which was selected as outgroup were acquired. Phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood, neighbor joining and Bayesian methods based on H2a-H2b and Cyt b DNA sequences. The results indicated that Drosophila curviceps species subgroup is located at the base of molecular trees, diverged first in this group; the second clade is Drosophila quadrilineata species subgroup; the third is Drosophila hypocausta species subgroup; the fourth is Drosophila immigrans species subgroup; Drosophila nasuta species subgroup is the last diverged clade. Moreover, the phylogentic relationships of seven specieses in Drosophila nasuta subgroup was investigated.

[The evolutionary traits of mitocondrial ND4-ND4L in Drosophila melanogaster species group].

The sequences of the mitochondrial ND4-ND4L were determined for all the 33 extant taxaes of the Drosophila melanogaster species group, so evolutionary traits of ND4 gene sequences were analyzed. We found a strong transition bias in closed related species and saturation of transitional substitutions in distant related species. Moreover, we calculated the diverge time of eight subgroup according to the Tamura-Nei distance between D. melanogaster and D. yakuba. Our result suggested that ananassae subgroup branched off firstly, and montium, melanogaster, ficsphila, eugracilis, elegans, suzukii-takahashii branched off sequentially.

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B.

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.

[The distribution and origin of P elements in Drosophila melanogaster strains from northeast China].

Eukaryotic transposable elements, especially P transposable elements of Drosophla, are very important to study the evolution of biology. We collected 130 single--female lines of D. melanogaster from 13 places in Northeast China and 3 places nearby which are Beijing(BC), Yantai (YT), Huhehaote(HHT) in 1999 and 2000. We amplified the fragments from ORF2 to ORF3 of P elements using PCR and calculated the defective frequencies and defective parameters of P elements from different places. Basing on the distribution of defective P elements, we suggested the invasion route of P elements of D. melanogaster in Northeast China. The results indicate that the defective frequencies decrease from the frontier to the inland, and in the isolated places the frequencies are lower. It is proposed that P elements of D. melanogaster in Northeast China originate from Korea and Russia to the frontiers of P.R. China, then expand into the inland.