Evidence for stasis and not genetic piracy in developmental expression patterns of Branchiostoma lanceolatum and Branchiostoma floridae, two amphioxus species that have evolved independently over the course of 200 Myr

Cephalochordates, the most basal extant group in the phylum Chordata, are represented chiefly by about 20 species of the genus Branchiostoma, commonly called amphioxus or lancelets. In recent years, insights into the evolutionary origin of the vertebrates have been gained from molecular genetic studies during the development of three of these amphioxus species (Branchiostoma floridae in North America, Branchiostoma lanceolatum in Europe, and Branchiostoma belcheri in East Asia). In spite of an estimated divergence time of 100-200 Myr among these species, all three are remarkably similar morphologically, and students of amphioxus have tacitly assumed that such resemblances arise during ontogeny from nearly identical networks of developmental genes. We felt that this assumption needed to be reexamined because instances are known--even in comparisons of closely related species--where characters seeming homologous on the basis of morphology actually develop under the control of conspicuously divergent genetic programs (a phenomenon termed "genetic piracy"). In the present work, we tested the hypothesis that morphological similarities reflect strict conservation of developmentally important genes' expression patterns in order to assess whether the developmental genetics of different amphioxus species show evidence of genetic piracy. To these ends, we cloned 18 genes implicated in different developmental functions in B. lanceolatum and compared their gene expression patterns with the known expression patterns of their orthologous genes in B. floridae. We show that, for the most part, conservation of gene expression parallels that of morphology in these two species. We also identified some differences in gene expression, likely reflecting experimental sensitivity, with the exception of Pax1/9, which may result from true developmental specificities in each amphioxus species. Our results demonstrate that morphological conservation reflects stasis in developmental gene expression patterns and find no evidence for genetic piracy. Thus, different species of amphioxus appear to be very similar, not only morphologically, but also in the genetic programs directing the development of their structural features. Moreover, we provide the first catalogue of gene expression data for the European species, B. lanceolatum.     

Comparative analyses of the complete mitochondrial genomes of the two ruminant hookworms Bunostomum trigonocephalum and Bunostomum phlebotomum

Bunostomum trigonocephalum and Bunostomum phlebotomum are blood-feeding hookworms of sheep and cattle, causing considerable economic losses to the live stock industries. Studying genetic variability within and among hookworm populations is critical to addressing epidemiological and ecological questions. Mitochondrial (mt) DNA is known to provide useful markers for investigations of population genetics of hookworms, but mt genome sequence data are scant. In the present study, the complete mitochondrial DNA (mtDNA) sequences of the sheep and goat hookworm B. trigonocephalum were determined for the first time, and the mt genome of B. phlebotomum from yak in China was also sequenced for comparative analyses of their gene contents and genome organizations. The lengths of mt DNA sequences of B. trigonocephalum sheep isolate, B.trigonocephalum goat isolate and B. phlebotomum China yak isolate were 13,764 bp, 13,771 bp and 13,803 bp in size, respectively. The identity of the mt genomes was 99.7% between B. trigonocephalum sheep isolate and B. trigonocephalum goat isolate. The identity of B. phlebotomum China yak isolate mt genomes was 85.3% with B. trigonocephalum sheep isolate, and 85.2% with B. trigonocephalum goat isolate. All the mt genes of the two hookworms were transcribed in the same direction and gene arrangements were consistent with those of the GA3 type, including 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes, but lacking ATP synthetase subunit 8 gene. The mt genomes of B. trigonocephalum and B. phlebotomum were similar to prefer bases A and T, the contents of A + T are 76.5% (sheep isolate), 76.4% (goat isolate) and 76.9% (China yak isolate), respectively. Phylogenetic relationships reconstructed using concatenated amino acid sequences of 12 protein-coding genes with three methods (maximum likelihood, Bayesian inference and neighbor joining) revealed that the B. trigonocephalum and B. phlebotomum represent distinct but closely-related species. These data provide novel and useful genetic markers for studying the systematics, and population genetics of the two ruminant hookworms.     

Mitochondrial genomic comparisons of the subterranean termites from the Genus Reticulitermes (Insecta: Isoptera: Rhinotermitidae).

Termites of the genus Reticulitermes are some of the most significant pests of structural timber and tree farming in the northern hemisphere, causing losses in the billions of dollars annually because of direct damage and termite control costs. This group has been frequently targeted for population genetic, phylogenetic, and species limit studies, most of which use mitochondrial (mt) genes; however, only a small fraction of the genome has been sequenced. The entire mt genome was sequenced for the eastern North American members of Reticulitermes: R. flavipes, R. santonensis, R. virginicus, and R. hageni. The mt genome has the same gene content and organization as that found in most insect species; however, the nucleotide composition and skew are highly biased (AT% low, strong A- and C-skew). Both the protein-coding and transfer RNA genes show high absolute levels of nucleotide substitution, suggesting that the high rates of mutation within Reticulitermes inferred from analyses of single mt genes are a general characteristic of the entire mt genome. The AT-rich or control region has a remarkable structure not previously observed in insect mt genomes. The majority of the control region is made up of 2 sets of repeat units, typically with 2 full and 1 partial copies of both the A (or small; 186 bp) and B (or large; 552 bp) repeats. The partial repeat units overlap by 36 bp. The size, location, and degree of overlap for the partial repeat units correspond to highly conserved stem/loop structures within the repeat units, suggesting that these structures are involved in the replication-mediated processes that govern repeat-unit evolution within mt genomes. Finally, molecular variation within the mt gene regions was compared with previous regions used in molecular diagnostics or phylogenetics of Reticulitermes. High numbers of single nucleotide polymorphisms were found in each of the mt genes, and some of the highest variability was found in gene regions that have not previously been investigated in this group. The whole mt genome sequence can thus be used to predict useful regions for future investigation.     

Ancient phylogenetic separation between Pacific and Atlantic cephalochordates as revealed by mitochondrial genome analysis

The subphylum Cephalochordata (lancelets) is a relatively small taxonomic group in contrast to the subphyla Urochordata and Vertebrata. As an initial step to determine whether lancelets exhibit small genetic divergence in keeping with their conservative body organization or large genetic variation, four Branchiostoma species from the Pacific (B. belcheri and B. malayanum) and Atlantic (B. floridae and B. lanceolatum) Oceans were genetically compared using partial mitochondrial DNA sequences of the cytochrome oxidase c subunit I (COI) and 16S ribosomal RNA (16S rRNA) genes. In both genes, large genetic differences were revealed between the Pacific and Atlantic species, as well as within the former. Two maximum-likelihood trees from the COI and 16S rRNA genes showed that the Pacific and Atlantic lancelets were reciprocally clustered into different clades. Furthermore, both gene trees consistently exhibited deep phylogenetic separation between the two oceans. The estimated divergence time suggested that differentiation may have followed the migration of ancestral lancelets from the Pacific to the Atlantic Oceans via the Tethys Sea.     

Genomic structure of the amphioxus calcium vector protein.

Calcium vector protein (CaVP) is an EF-hand Ca(2+)-binding protein, which is unique to the protochordate, amphioxus. CaVP is supposed to act as a Ca(2+) signal transductor, but its exact function remains unknown. Not only its function but also its exact evolutionary relationship to other Ca(2+)-binding proteins is unclear. To investigate the evolution of CaVP, we have determined the complete sequences of CaVP cDNAs from two amphioxus species, Branchiostoma lanceolatum and B. floridae, whose open reading frame cDNA and amino acid sequences show 96.5 and 98.2% identity, respectively. We have also elucidated the structure of the gene of B. floridae CaVP, which is made up of seven exons and six introns. The positions of four of the six introns (introns 1, 2, 3, and 5) are identical with those of calmodulin, troponin C, and the Spec protein of the sea urchin. These latter proteins belong to the so-called troponin C superfamily (TnC superfamily) and thus CaVP likely also belongs to this family. Intron 6 is positioned in the 3' noncoding region and is unique to CaVP, so it may represent a landmark of the CaVP lineage only. The position of intron 4 is not conserved in the genes of the TnC superfamily or CaVP, and seems to result from either intron sliding or the addition of an intron (randomly inserted into or close to domain III) to the genes of the TnC superfamily during their evolution.     

Complete nucleotide sequence of the mitochondrial genome of a Malagasy poison frog Mantella madagascariensis: evolutionary implications on mitochondrial genomes of higher anuran groups

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of a Malagasy poison frog, Mantella madagascariensis (family Mantellidae), and partial sequences of two Mantella (M. baroni and M. bernhardi) and two additional mantellid species (Boophis madagascariensis and Mantidactylus cf. ulcerosus). The M. madagascariensis genome was shown to be the largest (23kbp) of all vertebrate mtDNAs investigated so far. Furthermore, the following unique features were revealed: (1) the positions of some genes and gene regions were rearranged compared to mitochondrial genomes typical for vertebrates and other anuran groups, (2) two distinct genes and a pseudogene corresponding to transfer RNA gene for methionine (tRNA-Met) were encoded, and (3) two control regions with very high sequence homology were present. These features were shared by the two other Mantella species but not the other mantellid species, indicating dynamic genome reorganization in a common ancestor linage before divergence of the Mantella genus. The reorganization pathway could be explained by a model of gene duplication and deletion. Duplication and deletion events also seem to have been responsible for concerted sequence evolution of the control regions in Mantella mt genomes. It is also suggested that the pseudo tRNA-Met gene sustained for a long time in Mantella mt genomes possibly functions as a punctuation marker for NADH dehydrogenase subunit (ND) 2 mRNA processing. Phylogenetic analyses employing a large sequence data set of mt genes supported the monophyly of Mantellidae and Rhacophoridae and other recent phylogenetic views for ranoid frogs. The resultant phylogenetic relationship also suggested parallel occurrence of two tRNA-Met genes, duplicated control regions, and ND5 gene translocation in independent ranoid lineages.     

Unexpectedly large number of conserved noncoding regions within the ancestral chordate Hox cluster

The single amphioxus Hox cluster contains 15 genes and may well resemble the ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of the European amphioxus Branchiostoma lanceolatum and compared it to the American species, Branchiostoma floridae, by phylogenetic footprinting to gain insights into the evolution of Hox gene regulation in chordates. We found that Hox intergenic regions are largely conserved between the two amphioxus species, especially in the case of genes located at the 3' of the cluster, a trend previously observed in vertebrates. We further compared the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus.     

Highly conserved gene arrangement of the mitochondrial genomes of 23 Plasmodium species

Mitochondrial (mt) genomes from diverse phylogenetic groups vary considerably in size, structure and organization. The genus Plasmodium, the causative agent of malaria, has the smallest mt genome in the form of a tandemly repeated, linear element of 6 kb. The Plasmodium mt genome encodes only three protein genes (cox1, cox3 and cob) and large- and small-subunit ribosomal RNA (rRNA) genes, which are highly fragmented with 19 identified rRNA pieces. The complete mt genome sequences of 21 Plasmodium species have been published but a thorough investigation of the arrangement of rRNA gene fragments has been undertaken for only Plasmodium falciparum, the human malaria parasite. In this study, we determined the arrangement of mt rRNA gene fragments in 23 Plasmodium species, including two newly determined mt genome sequences from P. gallinaceum and P. vinckei vinckei, as well as Leucocytozoon caulleryi, an outgroup of Plasmodium. Comparative analysis reveals complete conservation of the arrangement of rRNA gene fragments in the mt genomes of all the 23 Plasmodium species and L. caulleryi. Surveys for a new rRNA gene fragment using hidden Markov models enriched with recent mt genome sequences led us to suggest the mtR-26 sequence as a novel candidate LSU rRNA fragment in the mt genomes of the 24 species. Additionally, we found 22-25 bp-inverted repeat sequences, which may be involved in the generation of lineage-specific mt genome arrangements after divergence from a common ancestor of the genera Eimeria and Plasmodium/Leucocytozoon.     

Rates of gene rearrangement and nucleotide substitution are correlated in the mitochondrial genomes of insects

A number of studies indicated that lineages of animals with high rates of mitochondrial (mt) gene rearrangement might have high rates of mt nucleotide substitution. We chose the hemipteroid assemblage and the Insecta to test the idea that rates of mt gene rearrangement and mt nucleotide substitution are correlated. For this purpose, we sequenced the mt genome of a lepidopsocid from the Psocoptera, the only order of hemipteroid insects for which an entire mtDNA sequence is not available. The mt genome of this lepidopsocid is circular, 16,924 bp long, and contains 37 genes and a putative control region; seven tRNA genes and a protein-coding gene in this genome have changed positions relative to the ancestral arrangement of mt genes of insects. We then compared the relative rates of nucleotide substitution among species from each of the four orders of hemipteroid insects and among the 20 insects whose mt genomes have been sequenced entirely. All comparisons among the hemipteroid insects showed that species with higher rates of gene rearrangement also had significantly higher rates of nucleotide substitution statistically than did species with lower rates of gene rearrangement. In comparisons among the 20 insects, where the mt genomes of the two species differed by more than five breakpoints, the more rearranged species always had a significantly higher rate of nucleotide substitution than the less rearranged species. However, in comparisons where the mt genomes of two species differed by five or less breakpoints, the more rearranged species did not always have a significantly higher rate of nucleotide substitution than the less rearranged species. We tested the statistical significance of the correlation between the rates of mt gene rearrangement and mt nucleotide substitution with nine pairs of insects that were phylogenetically independent from one another. We found that the correlation was positive and statistically significant (R2 = 0.73, P = 0.01; Rs = 0.67, P < 0.05). We propose that increased rates of nucleotide substitution may lead to increased rates of gene rearrangement in the mt genomes of insects.     

Phylogenetic relationships of discoglossid frogs (Amphibia:Anura:Discoglossidae) based on complete mitochondrial genomes and nuclear genes

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for three species of discoglossid frogs (Amphibia:Anura:Discoglossidae), representing three of the four recognized genera: Alytes obstetricans, Bombina orientalis, and Discoglossus galganoi. The organization and size of these newly determined mt genomes are similar to those previously reported for other vertebrates. Phylogenetic analyses (maximum likelihood, Bayesian inference, minimum evolution, and maximum parsimony) of mt protein-coding genes at the amino acid level were performed in combination with already published mt genome sequence data of three species of Neobatrachia, one of Pipoidea, and four of Caudata. Phylogenetic analyses based on the deduced amino acid sequences of all mt protein-coding genes arrived at the same topology. The monophyly of Discoglossidae is strongly supported. Within the Discoglossidae, Alytes is consistently recovered as sister group of Discoglossus, to the exclusion of Bombina. The three species representing Neobatrachia exhibited extremely long branches irrespective of the phylogenetic inference method used, and hence their relative position with respect to Discoglossidae and Xenopus may be artefactual due to a severe long branch attraction effect. To further investigate the phylogenetic intrarelationships of discoglossids, nucleotide sequences of four nuclear protein-coding genes (CXCR4, RAG1, RAG2, and Rhodopsin) with sequences available for the three discoglossid genera and Xenopus were retrieved from GenBank, and together with a concatenated nucleotide sequence data set containing all mt protein-coding genes except ND6 were subjected to separate and combined phylogenetic analyses. In all cases, a sister group relationship between Alytes and Discoglossus was recovered with high statistical support.     

Flanking genes of an essential gene give information about the evolution of metazoa

We collected as much information as possible on new lamin genes and their flanking genes. The number of lamin genes varies from 1 to 4 depending more or less on the phylogenetic position of the species. Strong genome drift is recognised by fewer and unusually placed introns and a change in flanking genes. This applies to the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the urochordate Ciona intestinalis, the annelid Capitella teleta and the planaria Schmidtea mediterranea. In contrast stable genomes show astonishing conservation of the flanking genes. These are identical in the sea anemone Nematostella vectensis and the cephalochordate Branchiostoma floridae lamin B1 gene. Even in the lamin B1 genes from Xenopus tropicalis and man one of the flanking genes is conserved. Finally our analysis forms the basis for a molecular analysis of metazoan phylogeny.     

强壮粗体虫的线粒体基因组及棘头虫的系统发育研究

通过长距离PCR方法,克隆了鳜（Siniperca chuatsi Basilewsky）肠道内寄生虫强壮粗体虫（Hebesoma violentum Van Cleave）线粒体基因组全长序列,共13393 bp （GenBank登录号:KC415004）,有36个基因,其中蛋白编码基因12个,核糖体基因2个,tRNA22个。所有基因均由线粒体基因组同一条链按同一个方向转录。利用该线粒体基因组和已经报道的一些轮虫纲种类的线粒体基因组序列,构建了棘头虫和轮虫的系统发育树。系统发育研究表明:包括强壮粗体虫、隐藏新棘虫Pallisentis celatus（Van Cleave）和Paratenuisentis ambiguous（Van Cleave）在内的始新棘头虫纲（Eoacanthocephala）与古棘头虫纲（Palaeacanthocephala）亲缘关系较近,聚为一枝后再与原棘头虫纲（Archiacanthocephala）聚在一起;棘头虫与双巢类轮虫（Bdelloid）亲缘关系最近,聚为一枝,然后再与单巢类轮虫（Monogonont）聚在一起,表明棘头虫和轮虫具有较近的亲缘关系。       

Mitochondrial genomes of the genus Ephydatia Lamouroux, 1816: can palindromic elements be used in species-level studies?

Poriferan mitochondrial DNA (mtDNA), especially large intergenic regions, is a target for the insertion of repetitive hairpin-forming elements. These elements are responsible for the large mt genome size differences observed even among closely related sponge taxa. In this study, we present the new, nearly complete, mt genome sequence of Ephydatia fluviatilis and compare it with previously published mt genomes of freshwater sponges. Special emphasis was placed on comparison with the closely related species Ephydatia muelleri, thereby comparing the only two species of the genus Ephydatia on the western Balkan Peninsula. In particular, we analyzed repetitive palindromic elements within the mitochondrial intergenic regions. The genomic distribution of these repetitive elements was analyzed and their potential role in the evolution of mt genomes discussed. We show here that palindromic elements are widespread through the whole mt genome, including the protein coding genes, thus introducing genetic variability into mt genomes.     

Metal dealing at the origin of the chordata phylum: the metallothionein system and metal overload response in amphioxus

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.