Markers For Phylogenetic Studies Research Articles

Claw asymmetry in crabs: approaching an old issue from a new point of view

Abstract Crabs are considered exceptional examples of antisymmetry resulting from the phenomenon of heterochely. Here we investigate morphometrically both the size and the shape of heterochely in 28 crab species, distributed unequally along a brachyuran phylogeny. We address the importance of investigating claw size and shape for interspecific comparisons by linking geometric morphometric outputs to phylogenetic data for 134 brachyuran species. New indices introduced as new sexual dimorphic characters of size and shape, namely heterometry (right chela size/left chela size) and heteromorphy (Procrustes distance between right and left chelae shape), revealed sexually dimorphic differences in diverse crab species. We demonstrate that both size and shape heterochely occur amongst the examined species, but there are no ecological correlations. Our study demonstrates that claw similarity between two or more species was due mainly to phylogenetic relatedness rather than ecological convergence, suggesting that claw morphological features could be useful morphological markers in phylogenetic studies. Although further investigation is needed, this study represents one of the first to thoroughly analyse the origin and evolution of heterochely within the Brachyura clade.

Determination of the evolutionary pressure on Camellia oleifera on Hainan Island using the complete chloroplast genome sequence.

Camellia oleifera is one of the four largest woody edible oil plants in the world with high ecological and medicinal values. Due to frequent interspecific hybridization, it was difficult to study its genetics and evolutionary history. This study used C. oleifera that was collected on Hainan Island to conduct our research. The unique island environment makes the quality of tea oil higher than that of other species grown in the mainland. Moreover, a long-term geographic isolation might affect gene structure. In order to better understand the molecular biology of this species, protect excellent germplasm resources, and promote the population genetics and phylogenetic studies of Camellia plants, high-throughput sequencing technology was used to obtain the chloroplast genome sequence of Hainan C. oleifera. The results showed that the whole chloroplast genome of C. oleifera in Hainan was 156,995 bp in length, with a typical quadripartite structure of a large single copy (LSC) region of 86,648 bp, a small single copy (SSC) region of 18,297 bp, and a pair of inverted repeats (IRs) of 26,025 bp. The whole genome encoded a total of 141 genes (115 different genes), including 88 protein-coding genes, 45 tRNA genes, and eight rRNA genes. Among these genes, nine genes contained one intron, two genes contained two introns, and four overlapping genes were also detected. The total GC content of Hainan C. oleifera’s chloroplast genome was 37.29%. The chloroplast genome structure characteristics of Hainan C. oleifera were compared with mainland C. oleifera and those of the other eight closely related Theaceae species; it was found that the contractions and expansions of the IR/LSC and IR/SSC regions affected the length of chloroplast genome. The chloroplast genome sequences of these Theaceae species were highly similar. A comparative analysis indicated that the Theaceae species were conserved in structure and evolution. A total of 51 simple sequence repeat (SSR) loci were detected in the chloroplast genome of Hainan C. oleifera, and all Camellia plants did not have pentanucleotide repeats, which could be used as a good marker in phylogenetic studies. We also detected seven long repeats, the base composition of all repeats was biased toward A/T, which was consistent with the codon bias. It was found that Hainan C. oleifera had a similar evolutionary relationship with C. crapnelliana, through the use of codons and phylogenetic analysis. This study can provide an effective genomic resource for the evolutionary history of Theaceae family.

Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids

BackgroundIn Metazoa, mitochondrial markers are the most commonly used targets for inferring species-level molecular phylogenies due to their extremely low rate of recombination, maternal inheritance, ease of use and fast substitution rate in comparison to nuclear DNA. The mitochondrial control region (CR) is the main non-coding area of the mitochondrial genome and contains the mitochondrial origin of replication and transcription.While sequences of the cytochrome oxidase subunit 1 (COI) and 16S rRNA genes are the prime mitochondrial markers in phylogenetic studies, the highly variable CR is typically ignored and not targeted in such analyses. However, the higher substitution rate of the CR can be harnessed to infer the phylogeny of closely related species, and the use of a non-coding region alleviates biases resulting from both directional and purifying selection. Additionally, complete mitochondrial genome assemblies utilizing next generation sequencing (NGS) data often show exceptionally low coverage at specific regions, including the CR. This can only be resolved by targeted sequencing of this region.ResultsHere we provide novel sequence data for the echinoid mitochondrial control region in over 40 species across the echinoid phylogenetic tree. We demonstrate the advantages of directly targeting the CR and adjacent tRNAs to facilitate complementing low coverage NGS data from complete mitochondrial genome assemblies. Finally, we test the performance of this region as a phylogenetic marker both in the lab and in phylogenetic analyses, and demonstrate its superior performance over the other available mitochondrial markers in echinoids.ConclusionsOur target region of the mitochondrial CR (1) facilitates the first thorough investigation of this region across a wide range of echinoid taxa, (2) provides a tool for complementing missing data in NGS experiments, and (3) identifies the CR as a powerful, novel marker for phylogenetic inference in echinoids due to its high variability, lack of selection, and high compatibility across the entire class, outperforming conventional mitochondrial markers.

Convergent herbivory on conifers by Choristoneura moths after boreal forest formation

Mitogenomes are useful markers for phylogenetic studies across a range of taxonomic levels. Here, we focus on mitogenome variation across the tortricid moth genus Choristoneura and particularly the spruce budworm (Choristoneura fumiferana) species complex, a notorious pest group of North American conifer forests. Phylogenetic relationships of Tortricidae, representing two subfamilies, four tribes and nine genera, were analyzed using 21 mitogenomes. These included six newly-sequenced mitogenomes for species in the spruce budworm complex plus three additional Choristoneura species and 12 previously published mitogenomes from other tortricids and one from the Cossidae. We evaluated the phylogenetic informativeness of the mitogenomes and reconstructed a time-calibrated tree with fossil and secondary calibrations. We found that tortricid mitogenomes had conserved protein and ribosomal regions, and analysis of all protein-coding plus ribosomal genes together provided an efficient marker at any taxonomic rank. The time-calibrated phylogeny showed evolutionary convergence of conifer feeding within Choristoneura, with two independent lineages, the Nearctic spruce budworm complex and the Palearctic species Choristoneura murinana, both shifting onto conifers about 11 million years ago from angiosperms. These two host-plant shifts both occurred after the formation of boreal forest in the late Miocene. Haplotype diversification within the spruce budworm complex occurred in the last 4 million years, and is probably linked to the initial cooling cycles of the Northern Hemisphere in the Pliocene.

Genetic Diversity and Molecular Phylogeny of Iranian Goats Based on Cytochrome Oxidase I (COXI) Gene Sequences (KERAGAMAN GENETIK DAN FILOGENI MOLEKULER KAMBING-KAMBING IRAN BERDASARKAN SEKUENS GEN CYTOCHROME OXIDASE I (COXI))

Mitochondrial DNA has been one of the most widely used molecular markers for phylogenetic studies in animals because of its simple genomic structure. This study examines the genetic characteristic of domestic goat using sequence analysis of mitochondrial DNA Cytochrome oxidase subunit I (COXI) to identify and differentiate among three common breeds (Adani, Najdi and Markhoz) of Iran. The genomic DNA was isolated by salting out method and amplified cytochrome oxidase I gene using Polymerase Chain Reaction (PCR) method with a pair of primer. Phylogenetic trees and pairwise calculations were obtained by using Mega 6 software. A partial sequence of cytochrome oxidase I gene of Iranian goats is 1286 bp and contained four variable sites and three haplotypes. Phylogenetic analysis of haplotype in the combination with the goat from GenBank showed that Iranian goatclustered in a separate lineage. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

Sequencing and analysis of the complete mitochondrial genome in Anopheles sinensis (Diptera: Culicidae)

BackgroundAnopheles sinensis (Diptera: Culicidae) is a primary vector of Plasmodium vivax and Brugia malayi in most regions of China. In addition, its phylogenetic relationship with the cryptic species of the Hyrcanus Group is complex and remains unresolved. Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, of which mitochondrial genome data of An. sinensis is not available.MethodsAn. sinensis samples was collected from Shandong, China, and identified by molecular marker. Genomic DNA was extracted, followed by the Illumina sequencing. Two complete mitochondrial genomes were assembled and annotated using the mitochondrial genome of An. gambiae as reference. The mitochondrial genomes sequences of the 28 known Anopheles species were aligned and reconstructed phylogenetic tree by Maximum Likelihood (ML) method.FindingsThe length of complete mitochondrial genomes of An. sinensis was 15,076 bp and 15,138 bp, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and an AT-rich control region. As in other insects, most mitochondrial genes are encoded on the J strand, except for ND5, ND4, ND4L, ND1, two rRNA and eight tRNA genes, which are encoded on the N strand. The bootstrap value was set as 1000 in ML analyses. The topologies restored phylogenetic affinity within subfamily Anophelinae. The ML tree showed four major clades, corresponding to the subgenera Cellia, Anopheles, Nyssorhynchus and Kerteszia of the genus Anopheles.ConclusionsThe complete mitochondrial genomes of An. sinensis were obtained. The number, order and transcription direction of An. sinensis mitochondrial genes were the same as in other species of family Culicidae.

Venom Gland Transcriptomic and Proteomic Analyses of the Enigmatic Scorpion Superstitionia donensis (Scorpiones: Superstitioniidae), with Insights on the Evolution of Its Venom Components.

Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.

Phylogenetic informativeness of plastid regions in inferring the species relationships amongCucumisspecies

Molecular approaches are playing major role in phylogeny reconstruction and species relationship studies. The uniparent inheritance, small size, non recombinant nature and extremely low rate of evolution of chloroplast genome make it an appropriate marker for phylogenetic studies. In the present study, phylogenetic informativeness of two coding regions, rbcL and rpoC1 and two non-coding intergenic spacers psbA-trnH and trnL-F of plastid DNA were analysed for inferring relationships among Cucumis species. Maximum evolutionary divergence was shown by trnL-F followed by psbA-trnH region. The non-coding spacers evolved 1.62 to 9 times faster than coding regions of rbcL and rpoC1. In the phylogenetic analyses, all species of Asian origin showed clear divergence from Cucumis dipsaceus and Cucumis prophetarum, which were African in origin. Mukia maderaspatana and Dicaelospermum ritchiei showed very low evolutionary divergence and are reported close to each other. C. setosus had been confirmed as distinct species and C. callosus was found to be a wild progenitor of C. melo.

NEW DATA OF CRYPHONECTRIA PARASITICA (MURR.) BARR POPULATION IN CARPATHIAN-BASIN

The ascomycete fungus, Cryphonectria parasitica (Murr.) Barr, is an important fungal pathogen of chestnut in Europe and North America. The fungus grows into the bark up to the cambium, causing typical diffuse cancers and the death of distal parts of the trunks or branches. The pathogen kills the infected tree branches and the rapid death of the entire tree take place which is causing high environmental and economic concerns. In this study we employed a part of the translation elongation factor 1 subunit alpha (EF-1α=tef1) and ITS region, as a potential genetic markers to infer phylogenetic relationships among Cryphonectria parasitica isolates. The translation elongation factor 1 subunit alpha (EF-1α=tef1) containing both introns and exons and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA, as a potential genetic markers to infer phylogenetic relationships among Cryphonectria parasitica isolates. Translation elongation factor 1 subunit alpha (EF1α=tef1) is part of the cytosolic EF1 complex, whose primary function is to promote the binding of aminoacyl-tRNA to the ribosome in a GTP-dependent process. Simultaneously, elongation factor 1α (EF-1α) is a highly conserved ubiquitous protein that has been suggested to have desirable properties for phylogenetic inference and population genetic studies. Ribosomal DNA (rDNA) has long been used as a potential marker for phylogenetic studies. rRNA genes are organized in clusters of tandemly repeated units, each of which consists of coding regions (18S, 5.8S, and 28S) and 2 internal transcribed spacers (ITS) and intergenic spacer (formerly called the Non-Transcibed Spacer, NTS) region. While the coding regions are evolutionarily conserved and have been utilized for phylogenetic inferences for major phyla, the 2 ITS regions are appropriate for detecting differences between co-specific individuals and are hence potentially useful markers to study the relationships of populations and closely related species in fungal, plant, and animal taxa due to their relatively rapid evolutionary rates.

Non-concerted ITS evolution in fungi, as revealed from the important medicinal fungus Ophiocordyceps sinensis

The internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) has been widely used as a molecular marker in phylogenetic studies and has been selected as a DNA barcode for fungi. It is generally believed that nrDNA conforms to concerted evolution in most eukaryotes; however, intraindividual–intraspecific polymorphisms of this region were reported in various organisms, suggesting a non-concerted evolutionary process. In Ophiocordyceps sinensis, one of the most valuable medicinal fungi, a remarkable variation of the ITS region has been revealed. Some highly divergent sequences were thought to represent cryptic species, different species or genotypes in previous studies. To clarify the unusual ITS polymorphisms observed in O. sinensis, specific primers were designed to amplify ITS paralogs from pure cultures of both single-ascospore and tissue isolates in this study. All of the available ITS sequences, including those generated by this group and those in GenBank, were analyzed. Several AT-biased ITS paralogs were classified as pseudogenes based on their nucleotide compositions, secondary structures and minimum free energies of their 5.8S rRNAs, substitution rates, phylogenetic positions and gene expression analyses. Furthermore, ITS pseudogenes were amplified with specific primers from 10 of the 28 strains tested, including eight single-ascospore and two tissue isolates. Divergent ITS paralogs were proved to coexist in individual genomes, suggesting a non-concerted mechanism of evolution in the ITS region of O. sinensis. The hypotheses that divergent ITS paralogs represent cryptic or other species or different genotypes were thus rejected.

Molecular Characterization of the Asian Tiger Mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) in Northern Italy

Polymorphism of the gene COI encoding cytochrome C oxidase subunit I and of the rRNA second internal transcribed spacer (ITS2) was measured in Aedes albopictus collected in three locations in Piedmont region and one location in Liguria region in Italy. According to our data, besides the middle part of the COI gene usually used for analysis, there are also SNPs in the 5′ half of the gene, which can provide additional information about the polymorphism of A. albopictus populations. Based on COI gene, three various mitochondrial haplotypes were identified in A. albopictus from four north Italian sampling sites. Different sources of introductions can be considered. The variability between COI sequences of the north Italian A. albopictus is 0.001 while the variability between them based on the pairwise analysis of ITS2 sequences is 10 time more—0.011. For the first time, a 4% divergence between the populations of A. albopictus of tropical and subtropical origins was found on the basis of analyzing the ITS2. A relatively high level of variability of ITS2 between tropical and subtropical populations can be used to develop new markers for phylogenetic studies. No individuals with mitochondrial and nuclear haplotypes specific to tropical populations were found in the four north Italian sampling sites.

Genetic variation among Clonorchis sinensis isolates from different hosts and geographical locations revealed by sequence analysis of mitochondrial and ribosomal DNA regions

The present study examined genetic variability among Clonorchis sinensis isolates from four different geographical localities (Guangzhou, Nanning, Jiamusi and Daqing) and host species (cats, dogs, human and rabbits) in Mainland China by sequence analyses of two mitochondrial DNA (mtDNA) genes, namely NADH dehydrogenase subunits 2, 5 (nad2 and nad5) and ribosomal internal transcribed spacer 1 (ITS1). A portion of the ITS1, nad2 (pnad2) and nad5 (pnad5) was amplified by polymerase chain reaction separately from adult C. sinensis individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of ITS1, pnad2 and pnad5 was 643, 666 and 771 bp, respectively. The intraspecific sequence variations within C. sinensis were 0–1.7% for ITS1, 0–1.4% for pnad2 and 0–0.9% for pnad5. The interspecific sequence variations within other zoonotic trematodes, which were published previously, were 4.5–84.9% for ITS1, 21.9–43.6% for pnad2 and 19.2–48.9% for pnad5. The A+T contents of the sequences were 45.26–45.88% (ITS1), 62.91–63.51% (pnad2) and 58.24–58.63% (pnad5). Phylogenetic analyses using ribosomal and mitochondrial sequence data set, with three different computational algorithms (Bayesian inference, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support. These findings demonstrated the existence of low-level intraspecific variations in ribosomal DNA (rDNA) and mtDNA sequences among C. sinensis isolates from four different regions and hosts in China and elucidated that mtDNA sequences and rDNA sequences provided reliable genetic markers for phylogenetic studies of zoonotic trematodes.

Molecular markers for plant species identification and phylogenetics

In this review we summarized the information on application of molecular markers for plant species identification and phylogenetics: positive sides and limitations of main markers, representing sequencing data of taxonomically important chloroplast and nuclear DNA regions. Markers, based on polymorphism of PCR and restriction products, are also discussed as accessorial markers in phylogenetic studies.

Characteristics analysis of complete ribosomal DNA sequence in Chinese mitten crab (<I>Eriocheir sinensis</I>)

Ribosomal DNA is usually selected as molecular marker for phylogenetic study and the sequence variances especially in IGS region can affect the growth rate of organisms to certain extent. A complete rDNA of Chinese mitten crab (Eriocheir sinensis) was isolated in this study. The rDNA is 11 660 bp in size and consists of 18S(1 873 bp),ITS-1(317 bp),5. 8S(163 bp),ITS-2(614 bp),28S(4 461 bp) and IGS(4 240bp). The (AT) contents of different regions range from 40. 1% to 48. 6% ,which are lower than those in Drosophila melanogaster (55. 8% - 80. 0% ) and higher than Cyprinus carpio (22. 0% - 43. 7% ). The SSR loci per 100 nucleotides in the sequence of descending are ITS-2(0. 98% ),IGS(0. 49% ),28S(0. 38% ), ITS-1(0. 32% ),18S(0. 21% ),5. 8S(0% ). The results of ITS2 intra-variances analysis showed that two types of ITS-2 existed in Chinese mitten crab,with a 12 bp(CGCAGGACCACC) insertion at 405 nt. The IGS region is 4 240 bp in size and there are 136 - 139 bp species-specific repeats that contain an Xho I restriction site. And a stem-loop structure (182 pairs stem and 21 nucleotides loop) is located following the specific repeats. The sequence of rDNA in the Chinese mitten crab will provide the foundation of studying the variances among different geographical populations and exploring the molecular marker associated with the fast growth rate of the Chinese mitten crab.

Molecular Cloning and Sequence Analysis of Bile Salt Hydrolase Gene (bsh) from Lactobacillus plantarum MBUL90 Strain of Human Origin

This study provided genetic information on a bile salt hydrolase (bsh) of a Lactobacillus plantarum strain of Indian origin, MBUL90. L. plantarum strains were screened by PCR for the determination of the bsh locus in their genome using specific primers. None of the lactobacilli strains produced the expected size of amplicon (∼ 1.0 kb) except L. plantarum strains, which proved the specificity of the primers. The bsh amplicon of L. plantarum MBUL90 was cloned into pDrive vector, and nucleotide sequences were determined. Sequence analysis of bsh genes revealed a high level of similarity within the species of L. plantarum as well as with other species of Lactobacillus. The resulting nucleotide sequence of an ORF of 975 bp encoded a predicted protein of 324 amino acids representing a theoretical molecular mass of 37 kDa with a pI of 4.92. The protein deduced from the complete ORF had high similarity with other Bsh proteins, and four highly conserved amino acid motifs (YFGRNXD, NEXGLXXAGLNF, VXVLTNNPXF, and SXSRFVRXAF) were located around the active site. Genetic data presented in this paper provide a sound foundation for better understanding the genetic diversity of bsh in Lactobacillus genus and may provide a new genetic marker for phylogenetic study.

Sequence variation of the internal transcribed spacer (ITS) region of ribosomal DNA in Cerastoderma species (Bivalvia: Cardiidae)

The internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rRNA gene and ITS2) constitutes one of the most widely applied molecular markers in phylogenetic studies and species differentiation. Here, the ITS region of the cockles Cerastoderma edule from NW Spain and C. glaucum from the Mediterranean and Baltic Seas was characterized and its variation examined. The length of the ITS was 762–783 bp (ITS1, 226–251 bp; 5.8S rRNA gene, 158–161 bp; ITS2, 305–325 bp) and the GC content ranged between 57% and 60% (ITS1, 52–62%; 5.8S rRNA gene, 56–57%; ITS2, 61–63%). All individuals showed variation among ITS repeats, mostly in the spacers, with nucleotide diversity values of 0.0005–0.07. Among the C. glaucum sequences, two types of ITS (ITSI and ITSII) were distinguished, based on the percentage of differences observed in the spacers, sequence of the 5.8S gene, GC content of the ITS1 and clustering in phylogenetic trees. A PCR assay using specific primers for each ITS type demonstrated that both types coexist in all C. glaucum individuals. While type I seems to contain the bona fide 5.8S rRNA gene, type II could contain a pseudogene. Cerastoderma edule sequences grouped separately on phylogenetic trees, as expected for a differentiated species, but those of C. glaucum from the Mediterranean and Baltic Seas were intermixed in the two clades defining the ITS types, which supports the absence of systematic division previously inferred from other molecular markers.

ITS2 secondary structure improves phylogeny estimation in a radiation of blue butterflies of the subgenus Agrodiaetus (Lepidoptera: Lycaenidae: Polyommatus ).

BackgroundCurrent molecular phylogenetic studies of Lepidoptera and most other arthropods are predominantly based on mitochondrial genes and a limited number of nuclear genes. The nuclear genes, however, generally do not provide sufficient information for young radiations. ITS2 , which has proven to be an excellent nuclear marker for similarly aged radiations in other organisms like fungi and plants, is only rarely used for phylogeny estimation in arthropods, although universal primers exist. This is partly due to difficulties in the alignment of ITS2 sequences in more distant taxa. The present study uses ITS2 secondary structure information to elucidate the phylogeny of a species-rich young radiation of arthropods, the butterfly subgenus Agrodiaetus. One aim is to evaluate the efficiency of ITS2 to resolve the phylogeny of the subgenus in comparison with COI , the most important mitochondrial marker in arthropods. Furthermore, we assess the use of compensatory base changes in ITS2 for the delimitation of species and discuss the prospects of ITS2 as a nuclear marker for barcoding studies.ResultsIn the butterfly family Lycaenidae, ITS2 secondary structure enabled us to successfully align sequences of different subtribes in Polyommatini and produce a Profile Neighbour Joining tree of this tribe, the resolution of which is comparable to phylogenetic trees obtained with COI+COII . The subgenus Agrodiaetus comprises 6 major clades which are in agreement with COI analyses. A dispersal-vicariance analysis (DIVA) traced the origin of most Agrodiaetus clades to separate biogeographical areas in the region encompassing Eastern Anatolia, Transcaucasia and Iran.ConclusionsWith the inclusion of secondary structure information, ITS2 appears to be a suitable nuclear marker to infer the phylogeny of young radiations, as well as more distantly related genera within a diverse arthropod family. Its phylogenetic signal is comparable to the mitochondrial marker COI . Compensatory base changes are very rare within Polyommatini and cannot be used for species delimitation. The implementation of secondary structure information into character-based phylogenetic methods is suggested to further improve the versatility of this marker in phylogenetic studies.

Phylogenetic comparison of local length plasticity of the small subunit of nuclear rDNAs among all Hexapoda orders and the impact of hyper-length-variation on alignment

The SSU nrDNA (18S), is one of the most frequently sequenced molecular markers in phylogenetic studies. However, the length-hyper-variation at multiple positions of this gene can affect the accuracy of alignment greatly and this length variation makes alignment across arthropod orders a serious problem. The analyses of Hexapoda phylogeny is such a case. A more clear recognition of the distribution of the length-variable-regions is needed. In this study, the secondary structure of some length-variable-regions in the SSU nrRNA of Arthropoda was adjusted by the principle of co-variation. It is found that the extent of plasticity of some length-variable-region can extraordinarily be higher than 600 bases in hexapods. And the numbers of hyper length-variable-regions are largest in Strepsiptera and Sternorrhyncha (Hemiptera). Our study shows that some length-variable-regions can serve as synapomorphies for some groups. The phylogenetic comparison also suggested that the expansion of a lateral bulge could be the origin of a helix.

Phylogenetic Studies of Gordonia Species Based on gyrB and secA1 Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.

Molecular cloning and sequencing of the Na+/K+-ATPase alpha-subunit of the medical leech Hirudo medicinalis (Annelida) - implications for modelling protostomian evolution

Within the past few years, the phylogenetic tree is discussed controversially regarding the position of the different bilaterian groups. There are two varying views of evolution: the classical one based on morphological structures where the annelids and arthropods are combined in the group of Articulata, and a new hypothesis based on molecular data sets, which divides the protostomian groups in Ecdysozoa (e.g. arthropods, and nematodes) and Lophotrochozoa (e.g. annelids, molluscs, and platyhelminthes). The Na+/K+-ATPase is a highly conserved protein and fulfils a very important role in physiology and maintaining the homeostasis of cells and can be found in almost all eukaryotic animals. Due to its similar molecular structure throughout the animal kingdom the Na+/K+-ATPase is an excellent marker for phylogenetic studies. Here we report the molecular cloning, sequencing and phylogenetic analysis of Na+/K+-ATPase complementary deoxyribonucleic acid (cDNA) of the medical leech Hirudo medicinalis. The cloned cDNA codes for a polypeptide of 1022 amino acids and possesses a predicted molecular mass of 113.33 kDa. Phylogenetic analysis of the complete Na+/K+-ATPase α-subunit of H. medicinalis and sequence data from other Na+/K+-ATPases supports the previously developed ‘Ecdysozoa concept’ with high posterior probabilities. A common clade comprising annelids and platyhelminthes can be defined, whereas nematodes are in a basal position at the arthropod stem line.