Molecular phylogenetics and ecological diversification of the transisthmian fish genus Centropomus (Perciformes: Centropomidae).

Phylogenetic relationships among the 12 recognized fish species in the New World genus Centropomus (Pisces, Centropomidae) were analyzed using allozyme electrophoresis and 618 bp of the mitochondrial DNA 16S ribosomal RNA (rRNA) gene. Molecular phylogenetic trees were generally consistent with previously published partial hypotheses based on morphological evidence. However, previously undefined sister group relationships between major species groups were resolved using molecular data, and phylogenetic hypotheses for Centropomus based on 16S rRNA sequences were better supported than were allozyme-based hypotheses. The high level of congruence among the trees inferred from the nuclear and mitochondrial characters provided a firm phylogenetic basis for analysis of ecological diversification and molecular evolution in the genus. Compared to basal Centropomus species, members of the most nested species group were significantly larger in body size and occupied a marine niche only peripherally utilized by their congeners. We also observed substitution rate heterogeneity among 16S rRNA lineages; in contrast to expectations based on "metabolic rate" and "generation interval" models, relative substitution rates were faster than expected for the group of large-bodied snooks. Using the Pliocene rise of the Central American isthmian marine barrier to calibrate rates of 16S ribosomal gene evolution in Centropomus, we found that the rates for the genus were similar to those reported for higher vertebrates. Analysis of the three sets of transisthmian geminate taxa in Centropomus indicated that two of the pairs were probably formed during the Pliocene rise of the isthmus while the third pair diverged several million years earlier.     

Complete sequence and gene organization of the Nosema heliothidis ribosomal RNA gene region

By sequencing the entire ribosomal RNA (rRNA) gene region of Nosema heliothidis isolated from cotton bollworm (Helicoverpa armigera), we showed that its gene organization is similar to the type species, Nosema bombycis: the 5'-large subunit rRNA (2,490 bp)-internal transcribed spacer (192 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (274 bp)-5S rRNA (115 bp)-3'. We constructed two phylogenetic trees, analyzed phylogenetic relationships, examined rRNA organization of microsporidia, and compared the secondary structure of small subunit rRNA with closely related microsporidia. The latter two features may provide important information for the classification and phylogenetic analysis of microsporidia.     

A phylogenetic analysis of relationships among genera of Conopidae (Diptera) based on molecular and morphological data

Members of the family Conopidae (Diptera) have been the focus of little targeted phylogenetic research. The most comprehensive test of phylogenetic support for the present subfamily classification of Conopidae is presented here using 66 specimens, including 59 species of Conopidae and seven outgroup taxa. Relationships among subfamily clades are also explored. A total of 6824 bp of DNA sequence data from five gene regions (12S ribosomal DNA, cytochrome c oxidase subunit I, cytochrome b, 28S ribosomal DNA and alanyl‐tRNA synthetase) are combined with 111 morphological characters in a combined analysis using both parsimony and Bayesian methods. Parsimony analysis recovers three shortest trees. Bayesian analysis recovers a nearly identical tree. Five monophyletic subfamilies of Conopidae are recovered. The rarely acknowledged Zodioninae is restored, including the genera Zodion and Parazodion. The genus Sicus is removed from Myopinae. Morphological synapomorphies are discussed for each subfamily and inter‐subfamily clade, including a comprehensive review of the character interpretaions of previous authors. Included are detailed comparative illustrations of male and female genitalia of representatives of all five subfamilies with new morphological interpretation.     

Molecular phylogenetic analysis among bryophytes and tracheophytes based on combined data of plastid coded genes and the 18S rRNA gene.

The basal relationship of bryophytes and tracheophytes is problematic in land plant phylogeny. In addition to cladistic analyses of morphological data, molecular phylogenetic analyses of the nuclear small-subunit ribosomal RNA gene and the plastic gene rbcL have been performed, but no confident conclusions have been reached. Using the maximum-likelihood (ML) method, we analyzed 4,563 bp of aligned sequences from plastid protein-coding genes and 1,680 bp from the nuclear 18S rRNA gene. In the ML tree of deduced amino acid sequences of the plastid genes, hornworts were basal among the land plants, while mosses and liverworts each formed a clade and were sister to each other. Total-evidence evaluation of rRNA data and plastid protein-coding genes by TOTALML had an almost identical result.     

The mitochondrial 16 s rRNA reveals high anthropogenic influence on land snail diversity in a preliminary island survey

A total of 30 specimens belonging to five species, namely; Cryptozona siamensis, Sarika resplendens and Sarika sp. from the family Ariophantidae as well as Quantula striata and Quantula sp. from the family Dyakiidae were collected from the Langkawi Island in Northern Peninsular Malaysia. All specimens were identified through comparisons of shell morphology and amplification of a 500 bp segment of the 16S rRNA mtDNA gene. To assess phylogenetic insights, the sequences were aligned using ClustalW and phylogenetic trees were constructed. The analyses showed two major lineages in both Maximum Parsimony and Neighbour Joining phylogenetic trees. Each putative taxonomic group formed a monophyletic cluster. Our study revealed low species and intraspecies genetic diversities based on the 16S rRNA gene sequences. Thus, this study has provided an insight of land snail diversity in populations of an island highly influenced by anthropogenic activities through complementary use of shell morphological and molecular data.     

Complete mitochondrial genome of a forensically important muscid, Hydrotaea chalcogaster (Diptera: Muscidae), with notes on its phylogenetic position

Muscidae are a dipteran family which is important for forensic investigations. However, it has received limited attention in forensic entomological experiments as a reason of identification issues. It is hard to identify specimens by morphological methods, especially in developmental stages. Therefore, complete mitochondrial genome sequences can be important tool in forensic entomology for identifying species. In this study we sequenced and analyzed the first complete mitochondrial genome from a forensically important Muscidae species Hydrotaea (=Ophyra) chalcogaster by next-generation sequencing. The mitochondrial genome of the sequenced species is circular molecules of 17,076?bp which have the typical mitochondrial genome complement of 13 protein-coding genes, 22 tRNAs, two ribosomal RNA genes and a control region. Rearrangements of gene positions are identical with the ancestral insect genome. Furthermore, phylogenetic relationships of the family Muscidae were evaluated in regard to mitochondrial protein coding genes. The inferred trees indicate that the Muscidae is a paraphyletic family. These data provide additional information for molecular identification of muscid species.     

基于18S rRNA和线粒体16S rRNA基因序列的柳蚕进化分析

为探讨柳蚕Actias selene Hübner与鳞翅目昆虫的系统发育关系,本研究利用PCR扩增获得了柳蚕核糖体18S rRNA和线粒体16S rRNA基因的部分序列,长度分别为391bp和428bp。并采用邻近距离法(NJ)、最大简约法(MP)、类平均聚类法(UPGMA)构建系统进化树。结果表明,柳蚕线粒体16SrRNA基因序列与大蚕蛾科昆虫的16SrRNA基因序列均表现出偏好于碱基AT的倾向。柳蚕与所研究的其它蚕类的遗传距离介于0.016至0.140之间,其中与温带柞蚕Antheraea roylii的遗传距离最小,与野桑蚕Bombyx mandarina的遗传距离最大。而基于鳞翅目昆虫18S rRNA基因部分序列的进化分析显示,柳蚕与柞蚕Antheraea pernyi之间的遗传距离最小(0.010),与蓖麻蚕Samia ricini的遗传距离最大(0.017)。     

球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析

用粗糙脉孢菌（Neurospora crassa）XP_322380和赤霉菌（Gibberella zeag）PH-1（EAA76971）的60S核糖体蛋白L10a基因（60S ribosomal protein L10a,RPL10a）蛋白序列对球毛壳菌（Chaetomium globosum）ESTs序列数据库进行tBlastn检索,获得了球毛壳菌RPL10a cDNA序列。cDNA序列长765bp,开放阅读框654bp,编码217个氨基酸组成的多肽,蛋白分了量为23．9kD。BlastP分析表明该基因氨基酸序列与粗糙脉胞菌相似最高为89％;与玉蜀黍黑粉菌（Ustilago maydis）相似性最低为78％。cDNA序列及推测的氨基酸序列在GenBank登录（登录号分别为AY669070,AAT74578）。     

Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.     

基于28S rDNA D2基因片段与形态特征的优茧蜂亚科系统发育研究

本研究选取优茧蜂亚科Euphorinae(膜翅目Hymenoptera:茧蜂科Braconidae)的8族19属23种作为内群,茧蜂其它6个亚科的8属8种作外群,首次结合同源核糖体28S rDNA D2基因序列片段和41个形态学特征对该亚科进行了系统发育学研究。利用"圆口类"的内茧蜂亚科Rogadinae、茧蜂亚科Braconinae、矛茧蜂亚科Doryctinae的3个亚科为根,以PAUP*4.0和MrBayes3.0B4软件分别应用最大简约法(MP)和贝叶斯法对优茧蜂亚科的分子数据和分子数据与非分子数据的结合体进行了分析;并以PAUP*4.0对优茧蜂亚科的28S rDNA D2基因序列的片段的碱基组成与碱基替代情况进行了分析。结果表明:优茧蜂亚科的28S rDNA D2基因序列片段的GC%含量在40.00%~49.25%之间变动,而对于碱基替代情况来讲,优茧蜂亚科各个成员间序列变异位点上颠换(transversion)大于转换(transition);不同的分析和算法所产生的系统发育树都表明目前根据形态定义出的优茧蜂亚科Euphorinae不是一个单系群,而是一个与蚁茧蜂亚科Neoneurinae和高腹茧蜂亚科Cenocoelinae混杂在一起的并系群;在优茧蜂亚科内部,悬茧蜂族Meterorini和食甲茧蜂族Microctonini(排除猎户茧蜂属Orionis)为单系群,而宽鞘茧蜂族Centistini、大颚茧蜂族Cosmophorini、优茧蜂族Euphorini、瓢虫茧蜂族Dinocampini为并系群;悬茧蜂族Meterorini在优茧蜂亚科Euphorinae内位于基部位置的观点得到部分的支持,同时食甲茧蜂族Microctonini被判定为相对进化的类群。此外对于优茧蜂亚科内各属之间的相互亲缘关系,不同算法所得到的系统发育属的结果不完全一致,这表明优茧蜂亚科内(属及族)的系统发育关系还有待于进一步研究。     

5S ribosomal RNA genes of the newt Notophthalmus viridescens.

The genes which code for the 5S ribosomal RNA in the newt, Notophthalmus viridescens have been cloned and analyzed. Two types of repeating unit were detected: a major type consisting of a 120 bp coding region with a 111 bp spacer, and a minor type composed of a coding region, a pseudogene, and a 113 bp spacer. The pseudogene is a 36 bp segment which corresponds to the 3' terminal third of the 5S RNA gene, and is situated immediately 3' to the gene, being separated from it by 2 bp. Two recombinant plasmids were obtained in which the major and minor units were arranged in an interspersed pattern.     

Pattern of morphological diversification in the Leptocarabus ground beetles (Coleoptera: Carabidae) as deduced from mitochondrial ND5 gene and nuclear 28S rDNA sequences

Most of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene and a part of nuclear 28S ribosomal RNA gene were sequenced for 14 species of ground beetles belonging to the genus Leptocarabus. In both the ND5 and the 28S rDNA phylogenetic trees of Leptocarabus, three major lineages were recognized: (1) L. marcilhaci/L. yokoael/Leptocarabus sp. from China, (2) L. koreanus/L. truncaticollis/L. seishinensis/L. semiopacus/L. canaliculatus/L. kurilensis from the northern Eurasian continent including Korea and Hokkaido, Japan, and (3) all of the Japanese species except L. kurilensis. Clustering of the species in the trees is largely linked to their geographic distribution and does not correlate with morphological characters. The species belonging to different species groups are clustered in the same lineages, and those in the same species group are scattered among the different lineages. One of the possible interpretations of the present results would be that morphological transformations independently took place in the different lineages, sometimes with accompanying parallel morphological evolution, resulting in the occurrence of the morphological species belonging to the same species group (= type) in the different lineages.     

Phylogeny of ground beetles subgenus Nialoe (s. lat.) Tanaka (Coleoptera: Carabidae; genus Pterostichus): A molecular phylogenetic approach

We constructed a phylogeny of the ground beetle subgenus Nialoe ( s. lat. ), genus Pterostichus (Coleoptera: Carabidae) based on two mitochondrial (cytochrome oxidase I and 16S ribosomal DNA) and one nuclear (28S ribosomal DNA) gene sequences. Thirty-three representative species of the group and three outgroup species were analyzed. The resultant trees (maximum parsimonious, maximum likelihood and Bayesian trees of the combined data of the three gene sequences) indicated that there are two large and three small lineages in the group, some of which were supported by a previous morphology-based phylogeny. In all the analyses, the small lineage composed of two Korean species is sister to the rest of the subgenus, but relationships of other four lineages differed among the analyses and remained unresolved. The implications of the present results are discussed in terms of taxonomy and biogeography of the group.     

Affinities among anostracan (Crustacea: Branchiopoda) families inferred from phylogenetic analyses of multiple gene sequences

To gain insights into the relationships among anostracan families, molecular phylogenetic analyses were performed on nuclear (28S D1-D3 ribosomal DNA) and mitochondrial (16S rDNA, COI) gene regions for representatives of seven families and an outgroup. Data matrices used in the analyses included 951 base pairs (bp) of aligned sequences for 28S, 465 bp for 16S, and 658 bp (219 amino acids) for COI. Maximum-parsimony and maximum-likelihood methods were used to construct phylogenetic trees, enabling the evaluation of both previous hypotheses of taxonomic relationships among families based on morphology, and of the relative merits of independent versus simultaneous analyses of multiple data sets for phylogeny construction. Data from various combinations of the gene regions produced relatively congruent patterns of phylogenetic affinity. In most analyses, two monophyletic groups were resolved: one cluster included the families Polyartemiidae, Chirocephalidae, Branchinectidae, Streptocephalidae, and Thamnocephalidae, while the other contained the Artemiidae and Branchipodidae. Comparative analyses showed that combining gene regions in a single matrix generally resulted in increased resolution and support for each cluster relative to those obtained from single-gene analyses. Statistical tests demonstrated that morphology-based hypotheses of relationships among families had poorer support than those determined from molecular data, reflecting the homoplasy in characters used to differentiate families.     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

Aetiology of Opuntia ficus-indica malformations and stunting disease

The phytoplasma aetiology of epidemic stunting of cladodes and stunted growth observed in a cactus pear plantation in Carlentini (Sicily, Italy) was investigated with graft inoculation trials and PCR/restriction fragment length polymorphism analyses of 16S ribosomal RNA (rRNA) gene and S3 ribosomal protein gene. After sequencing of the 1525‐bp from 16S rRNA gene of both naturally infected and graft‐inoculated symptomatic cactus pear samples, phytoplasma TS belonging to ribosomal subgroup 16SrII‐C were identified as aetiological agents of this worldwide spread disease.     

1株抗菌植物内生菌EJH-2菌株的分离和鉴定

目的从中药植物金银花的组织中分离到1株具有抗菌活性的植物内生菌EJH-2菌株,并对其进行了分子生物学鉴定。方法通过总DNA提取,PCR扩增,得到1300bp的16S rRNA序列。PCR产物序列通过BLAST软件在NCBI网站中进行同源性比较。通过Bioedit7．0和Treedrawing软件绘制系统发育树。结果EJH-2的16SrRNA序列和数据库中的类多粘芽胞杆菌KCTC 1663菌株的序列的同源性为99．76％。在系统发育树中,EJH-2菌株和多粘类芽胞杆菌在同一分支。结论EJH-2菌株应归属于多粘类芽胞杆菌（Paenibacillus polymyxa）。     

Molecular phylogenetic analysis of the dragonfly genera Libellula, Ladona, and Plathemis (Odonata: Libellulidae) based on mitochondrial cytochrome oxidase I and 16S rRNA sequence data

Molecular phylogenetic relationships among members of the odonate genus Libellula (Odonata: Anisoptera: Libellulidae) were examined using 735 bp of mitochondrial COI and 416 bp of 16S ribosomal RNA gene sequences. Considerable debate exists over several relationships within Libellula, as well over the status of two putative genera often placed as subgenera within Libellula: Ladona and Plathemis. Parsimony and maximum-likelihood analyses of the separate and combined data sets indicate that Plathemis is basal and monophyletic and that Ladona is the sister clade to the remainder of Libellula sensu stricto (s.s.) (all species within the genus Libellula, excluding Plathemis and Ladona). Moreover, two European taxa, Libellula fulva and L. depressa, were found to occupy a sister group relationship within the Ladona clade. Relationships within Libellula s.s. are less well resolved. However, monophyletic lineages within the genus are largely consistent with morphologically based subgeneric classifications. Although tree topologies from each analysis differed in some details, the differences were in no case statistically significant. The analysis of the combined COI and 16S data yielded trees with overall stronger support than analyses of either gene alone. Several analyses failed to support the monophyly of Libellula sensu lato due to the inclusion of one or more outgroup species. However, statistical comparisons of topologies produced by unconstrained analyses and analyses in which the monophyly of Libellula was constrained indicate that any differences are nonsignificant. Based on morphological data, we therefore reject the paraphyly of Libellula and accept the outgroup status of Orthemis ferruginea and Pachydiplax longipennis.     

Evolutionary relationships among the Scarabaeini (Coleoptera: Scarabaeidae) based on combined molecular and morphological data

The Scarabaeini is an old world tribe of ball-rolling dung beetles that have origins dating back to at least the mid-upper Miocene (19-8 million years ago). The tribe has received little to no attention in morphological or molecular phylogenetics. We obtained sequence data from the mitochondrial cytochrome oxidase subunit I (1,197 bp) and 16S ribosomal RNA (461 bp) genes for 25 species of the Scarabaeini in an attempt to further resolve broad phylogenetic relationships within this tribe. Sequence data from both markers along with 216 morphological and 3 biological characters were analysed separately and combined. Independent analyses showed poorly resolved trees with many of the intermediate and basal nodes collapsed by low bootstrap values. Many sites in both genes exhibited strong A+T nucleotide bias and high interlineage divergences. The combined analysis revealed a number of well supported relationships such as the monophyly of the nocturnal species Scarabaeus satyrus, S. [Neateuchus] proboscideus, and S. zambesianus. Furthermore, the total evidence tree suggested to elevate S. (Pachysoma) to the status of an independent genus, Pachysoma, as a sister taxon to a clade containing Pachylomerus femoralis and Scarabaeus sensu lato. Within the latter, the following subgenera were maintained by the combination of data sets: S. (Scarabaeolus), S. (Sceliages), and S. (Kheper). Both, feeding specialisation and food relocation behaviour, were inferred to be polyphyletic in the Scarabaeini. Total evidence analysis found no support for common ancestry of Scarabaeini and Eucraniini.