Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae)

We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.     

Intraspecific cpDNA variations of diploid and tetraploid perennial buckwheat, Fagopyrum cymosum (Polygonaceae)

We investigated the phylogenetic and biogeographic relationships of natural populations of diploid and tetraploid Fagopyrum cymosum (Polygonaceae). Intraspecific variation of chloroplast DNA sequences was detected in three regions approximately 5 kb long in total: the 3' end of rbcL, accD and associated intergenic spacer region, the trnC (GCA)-rpoB spacer region, the trnK (UUU) intron, and the matK region. The accessions of F. cymosum were divided into two major groups, a Tibet-Himalayan clade and a Yunnan-Sichuan clade, with a high bootstrap probability. It was estimated that these two clades diverged about 0.7 million years ago. The geographical and climatic interruption by the Hengduanshan mountains might have caused the genetic divergence in F. cymosum. Autotetraploid populations of F. cymosum have arisen allopatrically from a diploid progenitor at least twice, once in the Tibet-Himalayan area and once in the Yunnan-Sichuan area. This conclusion reinforces a previous study based on allozyme variation. We also found that F. tataricum, a close relative of F. cymosum, was completely included within the Tibet-Himalayan clade in the phylogenetic tree. This suggests that F. tataricum speciated from F. cymosum in the Tibet-Himalayan area.     

Nucleotide sequence variation of the chloroplast trnK/matK region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice

Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intraspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (pi), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

A search for the phylogenetic position of the seven-son flower (Heptacodium, Dipsacales): Combining molecular and morphological evidence

A first report on the problematic phylogenetic position ofHeptacodium (2 spp.; China) using molecular data from chloroplast DNA is presented. Amplification of ORF2280 homolog region was executed in a number of representative taxa in order to determine ifHeptacodium shows similar structural rearrangements as other Dipsacales. DNA sequences ofndhF were generated to clarify the phylogenetic position ofHeptacodium among Caprifoliaceae (s.l.). Six outgroup taxa and fifteen representatives of Dipsacales were sampled and more than 2100 basepairs ofndhF sequence were used in a cladistic analysis. Parsimony analysis produced two shortest trees and showedHeptacodium as sister to all members of Caprifoliaceae (s.str.), although weakly supported. Additionally, trees were constructed withndhF data supplemented with availablerbcL sequences and a morphological data set. Results of all analyses support an unresolved basal position forHeptacodium among Caprifoliaceae (s.l.), which in part explains the difficulty experienced previously in classifying the genus.     

Convergent evolution of strigiform and caprimulgiform dark-activity is supported by phylogenetic analysis using the arylalkylamine N-acetyltransferase (Aanat) gene

Alternative hypotheses propose the sister order of owls (Strigiformes) to be either day-active raptors (Falconiformes) or dark-active nightjars and allies (Caprimulgiformes). In an effort to identify molecular characters distinguishing between these hypotheses we examined a gene, arylalkylamine N-acetyltransferase (Aanat), potentially associated with the evolution of avian dark-activity. Partial Aanat coding sequences, and two introns, were obtained from the genomic DNA of 16 species: Strigiformes (four species), Falconiformes (four species), Caprimulgiformes (five species), with outgroups: Ciconiiformes (one species), Passeriformes (one species), and Apterygiformes (one species). Phylogenetic trees derived from aligned, evolutionarily conserved Aanat regions did not consistently recover clades corresponding to orders Strigiformes and Falconiformes but did place a caprimulgiform clade more distant from the strigiform and falconiform species than the latter two groups are to each other. This finding was supported by spectral analysis. The taxonomic distribution of seven intronic indels is consistent with the Aanat derived phylogenetic trees and supports conventional family-level groupings within both Strigiformes and Caprimulgiformes. The phylogenetic analyses also indicate that Caprimulgiformes is a polyphyletic grouping. In conclusion the data support, but do not conclusively prove, the proposal that Falconiformes is the sister order to Strigiformes and therefore, that the dark-activity characteristic of Strigiformes and Caprimulgiformes arose by convergent evolution.     

Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.     

基于ITS序列探讨荻属及其近缘植物的系统发育关系

荻属是禾本科的一个小属,其分类地位存在争议。通过测定和从Gen Bank中提取荻属Triarrhena及其近缘植物的ITS序列,以芦苇Phragmites australis为外类群,采用MP(maximum parsimony)和NJ(neighbor-joining)分析方法进行了系统发育分析,这两种方法得到的系统发育树基本相同。荻属和芒属Miscauthus的种类形成一个单系类群;河八王Narenga porphyrocoma与芒属和荻属的植物聚在一起并构成姐妹群;白茅Imperata cylindrica与斑茅Saccharum arundinaceum聚成一小支并构成姐妹群,白茅与荻属的关系较远;荻属植物归并到芒属更为合理,不支持将荻属置入白茅属或另立一属的观点。斑茅与白茅、河八王及蔗茅Erianthus fulvus系统发育关系较近,而与滇蔗茅E.rockii及甘蔗属的竹蔗Saccharum sinense、甘蔗S.officinarum、细秆甘蔗S.barberi和甜根子草S.spontaneum的关系较远。甘蔗属、河八王属及蔗茅属关系复杂,还需要进一步研究。     

Molecular comparison of the negative-acting nitrogen control gene,nmr, inNeurospora crassa and otherNeurospora and fungal species

In Neurospora crassa, the expression of unlinked structural genes which encode nitrogen catabolic enzymes is subject to genetic and metabolic regulation. The negative-acting nmr regulatory gene appears to play a role in nitrogen catabolite repression. Using the N. crassa nmr gene as a probe, homologous sequences were identified in a variety of other filamentous fungi. The polymerase chain reaction was used to isolate the nmr-like gene from the exotic Mauriceville strain of N. crassa and from the two related species, N. intermedia and N. sitophila. Sequence comparisons were carried out with a 1.7-kb DNA segment which includes the entire coding region of nmr plus 5' and 3' noncoding sequences. The size of the nmr coding region was identical in all three Neurospora species. Approximately 30 nucleotide base substitutions were found in the coding region of the nmr gene of each of the sister species when compared to the standard N. crassa sequence. However, most of the base changes occurred in third codon positions and were silent. The NMR proteins of N. sitophila and of N. intermedia display only three and four amino acid substitutions, respectively, from the N. crassa protein. Two regions of high variability, which include deletions and insertions of bases, were found in the 5' and 3' noncoding regions of the gene.     

Molecular systematics of Trilliaceae II. Phylogenetic analyses of Trillium and its allies using sequences of rbcL and matK genes of cpDNA and internal transcribed spacers of 18S–26S nrDNA

Coding regions of the rbcL and matK genes of cp DNA and internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced to study phylogenetic relationships within and among all four genera of Trilliaceae: Trillium, Paris, Daiswa and Kinugasa . The rbcL gene has evolved much slower than matK and in particular ITS; hence the phylogenetic trees based on the rbcL gene show a much lower resolution than trees based on either matK or ITS. The general topology of phylogenetic trees resulting from separate parsimony analyses of the matK and ITS sequences are relatively congruent, with the exception of the placement of T. pusillum . Both matK and ITS phylogenies reveal that T. rivale diverges at the base of the trees. In both trees, Paris, Daiswa and Kinugasa form a relatively weakly supported group. Within this group, the allo-octaploid Kinugasa japonica is the sister group of Daiswa species. The Paris–Daiswa – Kinugasa group, the major Trillium group, and T. undulatum and T. govanianum showed a loosely related topology, but their affinities are not evident according to these two molecular markers. However, phylogenetic analysis of amino acid sequences derived from matK shows that T. rivale together with clades T. undulatum–T. govanianum, Daiswa–Kinugasa and Paris is basally diverged as a sister group to the remainder of Trillium .     

甜果螨全线粒体基因组测序与分析

【目的】测定和分析甜果螨Carpoglyphus lactis线粒体基因组全序列,并在线粒体基因组水平探讨其在真螨总目(Acariformes)中的系统发育地位,为真螨总目分类及果螨科线粒体基因组研究提供科学依据。【方法】挑取实验室饲养的甜果螨成螨,用传统的酚氯仿抽提法和试剂盒提取法提取甜果螨基因组DNA。然后采用节肢动物或螨类线粒体基因的通用引物PCR扩增出甜果螨线粒体基因cox1,cob,rrnS和nad4-nad5的部分序列;再设计种特异性引物进行Long-PCR扩增和步移法测序,测出甜果螨线粒体基因组全序列。应用SeqMan, SEQUIN 9.0和tRNAscan等生物信息学软件,对甜果螨线粒体基因组的基因结构等进行生物信息学分析。最后基于17种真螨总目螨类的蛋白质编码基因,采用最大似然法构建系统发育树。【结果】甜果螨线粒体全基因组总长为14 060 bp(GenBank登录号:MN073839),为典型的闭合双链DNA分子,共由37个基因组成,包括13个蛋白质编码基因(PCGs)、22个tRNA基因和2个rRNA基因;甜果螨线粒体基因组还包括1个大的非编码区(large n...     

Phylogenetic relationships amongTetrahymena species determined using the polymerase chain reaction

Summary The species of theTetrahymena pyriformis complex present a conundrum with regard to their highly conservative morphology and widely divergent molecular characteristics. We have investigated the phylogenetic relationships among these species using the nucleotide sequences from the histone H3II/H4II region of the genome. This region includes portions of the two histone coding sequences, as well as the intergenic region. The DNA sequences of these regions were amplified by the polymerase chain reaction (PCR) and the sequence of each was determined. Nucleotide substitutions and insertions/deletions within this set of sequences were compared to determine the phylogenetic relationships among the species of the complex. These data yield phylogenetic trees with identical topologies when different tree-building routines are used, indicating that the data are very robust.Glaucoma chattoni was used as an outgroup to root the trees for this analysis. The genome organization ofG. chattoni and the divergence of its histone H3II/H4II region sequence relative to those of the complex clearly indicate that this species has diverged considerably from the complex. These results show that PCR amplification analysis is feasible over considerable evolutionary distances. However, DNA-DNA hybridization may be more useful than sequence analysis in resolving the relationships among the closely related species in the complex.     

Macroevolutionary relationships of species of Drosophila melanogaster group based on mtDNA sequences

The phylogenetic relationships among the Drosophila melanogaster group species were analyzed using approximately 1700 nucleotide-long sequences of the mitochondrial DNA. Phylogenetic analysis was performed using this region consisting of a part of the cytochrome b (cytb) coding gene, the entire coding sequences of tRNA-Leu, tRNA-Ser and the first subunit of NADH dehydrogenase (NADH1), and a part of the 16S-rRNA gene. The study of these sequences showed that this region of mtDNA is very invariable, as regards with the type of the genes that it contains, as well as the order that they are located on it. The resulting phylogenetic trees reveal a topology that separates the species into three main ancestral lines, leading to the following subgroups: (a) ananassae subgroup, (b) montium subgroup, and (c) melanogaster and Oriental subgroups. The inferred topology complements and generally agrees with previously proposed classifications based on morphological and molecular data.     

Molecular evidence for a Mediterranean origin of the Macaronesian endemic genus Argyranthemum (Asteraceae)

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.     

基于孕激素受体基因的类人猿亚目的分子系统学研究

通过对类人猿亚目中部分种类的孕激素受体基因进行分析,重建类人猿亚目的 系统发育关系.扩增并测定了来源于14个属的类人猿亚目物种的孕激素受体编码区序列,并基于这一序列数据,分别采用邻接法、最大简约法和最大似然法重建了系统发育关系.除了阔鼻下目,3种方法构建的系统发生树的拓扑结构类似且各节点支持率高.重建的人猿超科和猴超科内部亲缘关系支持多数人所认可的分类系统.本研究为黑猩猩和人的姐妹群关系提供了证据,提示黑猩猩比大猩猩或其他猿猴更接近人类.阔鼻下目中蜘蛛猴科、卷尾猴科和僧面猴科三者之间的系统发育关系在本研究中未得到很好辨析.     

Phylogenetic Analysis of Heterothallic Neurospora Species

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.     

Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer

The 5S rDNA coding region and its spacer have been successfully utilized in phylogenetic studies of plants. However, it has not been utilized in the phylogenetic analysis of Cucurbitaceae. Here, we obtained the 5S rDNA sequences of 12 Cucurbitaceae species by direct PCR or cloning. The 5S rDNA sequences ranged from 275 to 359 bp, and the coding regions of all species were 120 bp long, except for that of Cucurbita, which was 119 bp. Some genus-specific SNPs were observed in the coding regions of Cucurbita, Lagenaria, Melothria, and Tricosanthes. The GC content of the coding regions was generally higher than that of the NTS regions, and the difference in GC content between the coding and NTS regions varied among species, with Gynostemma pentaphyllum having the greatest difference of 20.3. The phylogenetic trees generated using maximum parsimony and maximum likelihood were congruent and well supported by the recently published classification of Cucurbitaceae. These results demonstrated the utility of the 5S rDNA sequence in inferring phylogenetic relationships among 12 Cucurbitaceae species, and its utility could be extended by using a greater number of species in future studies.     

Structure and patterns of sequence variation in the mitochondrial DNA control region of the great cats

Mitochondrial DNA control region structure and variation were determined in the five species of the genus Panthera. Comparative analyses revealed two hypervariable segments, a central conserved region, and the occurrence of size and sequence heteroplasmy. As observed in the domestic cat, but not commonly seen in other animals, two repetitive sequence arrays (RS-2 with an 80-bp motif and RS-3 with a 6-10-bp motif) were identified. The 3' ends of RS-2 and RS-3 were highly conserved among species, suggesting that these motifs have different functional constraints. Control region sequences provided improved phylogenetic resolution grouping the sister taxa lion (Panthera leo) and leopard (Panthera pardus), with the jaguar (Panthera onca).     

Phylogenetic Relationships of Bears (the Ursidae) Inferred from Mitochondrial DNA Sequences

The phylogenetic relationships among some bear species are still open questions. We present here mitochondrial DNA sequences of D-loop region, cytochrome b, 12S rRNA, tRNA^Pro, and tRNA^Thr genes from all bear species and the giant panda. A series of evolutionary trees with concordant topology has been derived based on the combined data set of all of the mitochondrial DNA sequences, which may have resolved the evolutionary relationships of all bear species: the ancestor of the spectacled bear diverged first, followed by the sloth bear; the brown bear and polar bear are sister taxa relative to the Asiatic black bear; the closest relative of the American black bear is the sun bear. Primers for forensic identification of the giant panda and bears are proposed. Analysis of these data, in combination with data from primates and antelopes, suggests that relative substitutional rates between different mitochondrial DNA regions may vary greatly among different taxa of the vertebrates.     

Familial placement and relations of Rehmannia and Triaenophora (Scrophulariaceae s.l.) inferred from five gene regions

Accurate classification systems based on evolution are imperative for biological investigations. The recent explosion of molecular phylogenetics has resulted in a much improved classification of angiosperms. More than five phylogenetic lineages have been recognized from Scrophulariaceae sensu lato since the family was determined to be polyphyletic; however, questions remain about the genera that have not been assigned to one of the segregate families of Scrophulariaceae s.l. Rehmannia Liboschitz and Triaenophora Solereder are such genera with uncertain familial placement. There also is debate whether Triaenophora should be segregated from Rehmannia. To evaluate the phylogenetic relations between Rehmannia and Triaenophora, to find their closest relatives, and to verify their familial placement, we conducted phylogenetic analyses of the sequences of one nuclear DNA (ITS) region and four chloroplast DNA gene regions (trnL-F, rps16, rbcL, and rps2) individually and combined. The analyses showed that Rehmannia and Triaenophora are each strongly supported as monophyletic and together are sister to Orobanchaceae. This relation was corroborated by phytochemical and morphological data. Based on these data, we suggest that Rehmannia and Triaenophora represent the second nonparasitic branch sister to the remainder of Orobanchaceae (including Lindenbergia).