A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution, and phylogenetic implications

Restriction endonuclease fragments produced by EcoRI/AvaI or KpnI digestion and containing the small (12S) ribosomal RNA (rRNA) genes from the mitochondrial DNAs (mtDNAs) of the common chimpanzee, pygmy chimpanzee, gorilla, and orangutan were inserted into the plasmids pBR322 or pADD1. After species verification the inserted fragments were digested with SauIIIA, subcloned into M13mp7 vectors, and sequenced. The small rRNA gene sequences were compared with each other and with the published human sequence (Anderson et al. 1981). Substitutions were detected at 118 of the 955 nucleotide positions compared. Pairwise, the sequence differences ranged from 1% (between the chimpanzee species) to 9% (comparisons involving the orangutan); the proportion that were transitions ranged from 87% to 100%. Deletions and/or additions were noted at seven locations. With respect to evolutionary sequence lability, kinetic analysis indicated the presence of at least two classes of nucleotide positions; the more labile class occurs in sequences thought to form self-complementary duplexes (stems) in the mature rRNA. The high frequency of compensating substitutions, which maintain base-pairing within these sequences, corroborates their inferred structure. Phylogenetic inferences drawn from the sequence comparisons support the notion of an approximately equidistant relationship among chimpanzees, gorilla, and man, with the orangutan much less closely related. However, inference from a shared deletion suggests that the gorilla and the chimpanzees may be more closely related to one another than they are to man.     

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.     

Morphology and molecular phylogeny of a new brackish pleurostomatid ciliate,Loxophyllum paludosum sp. n. (Ciliophora,Litostomatea, Haptoria), from a mangrove wetland in China

A new ciliate species of the genus Loxophyllum Dujardin, 1841, Loxophyllum paludosum sp. n., is described from a mangrove wetland near Daya Bay in Guangdong Province, southern China, based on morphological and molecular analyses. The new species can be distinguished from its congeners by a combination of the following characters: (1) 12–14 right kineties and 4–6 left kineties; (2) two macronuclear nodules and one micronucleus; (3) a single contractile vacuole located terminally; (4) extrusomes bar-shaped, evenly spaced along entire ventral margin, and clustered to form 5–7 warts along dorsal margin; and (5) presence of three ridges on the left side of cell. The new species is divergent from its congeners from 0.4% to 6.7% (5–104 nucleotide sites) based on the small subunit (SSU) rRNA gene sequence data. The validity of the new species is also supported by molecular phylogenetic trees inferred from SSU rRNA gene sequences.     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Nucleotide sequence of Halobacterium cutirubrum ribosomal 5 S ribonucleic acid. An altered secondary structure in halophilic organisms

The nucleotide sequence of ribosomal 5 S RNA from a halophilic bacterium, Halobacterium cutirubrum, grown in 4 M sodium chloride is U-U-A-A-G-G-C-G-G-C-C-A-U-A-G-C-G-G-U-G-G-G-G-U-U-A-C-U-C-C-C-G-U-A-C-C-C-A-U-C-C-C-G-A-A-C-A-C-G-G-A-A-G-A-U-A-A-G-C-C-C-G-C-C-U-G-C-G-U-U-C-C-G-G-U-C-A-G-U-A-C-U-G-G-A-G-U-G-C-G-A-G-C-C-U-C-U-G-G-G-A-A-A-U-C-C-G-G-U-U-C-G-C-C-G-C-C-U-A-C-U. This nucleotide sequence is the longest prokaryotic 5 S rRNA to be reported and unlike other 5 S species does not contain a terminal mononucleoside diphosphate residue at its 5'-end. When compared to other 5 S rRNA's, the sequence homology is greatest (about 68%) with Bacillus subtilis; there is a lower but similar degree of homology (about 58%) with either Escherichia coli or human 5 S RNA. The comparisons further indicate that among 5 S RNA's, eleven of the nucleotide residues are unique to H. cutirubrum. Estimates of the secondary structure of the H. cutirubrum 5 S RNA molecule contain one additional stable hairpin loop which is not found in other 5 S rRNA species; this unusual structure is probably an adaptation to the high salt environment within H. cutirubrum cells.     

Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Molecular Evolution of the Small Subunit of Ribulose Bisphosphate Carboxylase: Nucleotide Substitution and Gene Conversion

The nucleotide sequences encoding the mature portion of 31 ribulose 1.5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is approximately 6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species.     

Detection of Babesia caballi in Amblyomma variegatum ticks (Acari: Ixodidae) collected from cattle in the Republic of Guinea

A reverse line blot hybridisation (RLB) assay was applied to screen Amblyomma variegatum adult ticks (n = 504) collected from N'Dama cattle in the Republic of Guinea. In a PCR, the V1 hypervariable region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for species of the genera Anaplasma and Ehrlichia, and the V4 hypervariable region of the 18S rRNA gene was amplified with primers specific for members of the genera Theileria and Babesia. Amplified PCR products from A. variegatum ticks were hybridised onto a membrane, to which oligonucleotide probes species-specific for Ehrlichia/Anaplasma and Theileria/Babesia parasites were covalently linked. No pathogens belonging to Ehrlichia/Anaplasma species were found, while 10 DNA samples resulted positive for Babesia caballi and 5 samples for Theileria velifera. This is the first report of B. caballi in A. variegatum ticks. One of the B. caballi positive samples was sequenced. This new strain (BcabGuinea) showed a 97% similarity to the Z15104 B. caballi GenBank sequence.     

Description of a new species of Myxobolus (Myxozoa: myxobolidae) based on morphological and molecular data

Myxobolus ampullicapsulatus n. sp. was isolated from the gills of Carassius auratus auratus (L., 1758) in Chongqing, China. Myxospores were pyriform, measuring 16.5-19.5 microm long x 8.5-10.0 microm wide x 7.0 microm thick. Two equal polar capsules were ampullaceous, measuring 7.0-10.0 microm long x 2.5-4.0 microm wide, containing polar filaments coiled 9-10 turns. Spore length of this species exceeds that of the majority of other Myxobolus spp., and those overlapping in this dimension can be differentially diagnosed by other characters. Furthermore, the small subunit ribosomal DNA (SSU rDNA) of M. ampullicapsulatus n. sp. is unique among myxozoans sequenced to date. Phylogenetic analyses of the SSU rDNA gene sequence placed this species in a clade composed exclusively of gill parasites, most closely related to Myxobolus longisporus, which also infects the gills of cyprinid fishes in China.     

Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: Strongylidae)

The nucleotide sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA were determined for adults of Cylicostephanus minutus from different geographical origins. The lengths of first and second internal transcribed spacer sequences ranged from 370 to 372bp and 215 to 216bp, respectively. Pairwise sequence comparisons revealed that some individuals of C. minutus had identical first and second internal transcribed spacer sequences, whereas others differed by 3.0% and 7.4% in their first and second transcribed spacers, respectively. Some individuals with sequence differences originated from the same host. The levels of difference within C. minutus were higher than that between the morphologically distinct species, Cylicostephanus goldi and Cylicostephanus longibursatus (0.8% for the first internal transcribed spacer and 3.8% for the second internal transcribed spacer). The data provide support for the proposal that C. minutus represents a complex of at least two species. In order to study the population genetic structure of C. minutus, a PCR-linked single-strand conformation polymorphism technique was also established.     

Cyathostoma (Cyathostoma) phenisci Baudet, 1937 (Nematoda: Syngamidae), a parasite of respiratory tract of African penguin Spheniscus demersus: Morphological and molecular characterisation with some ecological and veterinary notes

Here we provide a morphological and molecular analysis of the taxonomic status of Cyathostoma (Cyathostoma) phenisci Baudet, 1937, a rare nematode parasite of African penguin Spheniscus demersus. Taxonomical evaluation is supplemented wi th ecological and epidemiological analysis of the nematode's occurrence in the African penguin's population. Tracheae and air sacs of 13 among the 94 necropsied birds (overall prevalence 13.8%) contained a total of 33 nematode specimens (20 females, 13 males). The highest prevalence was observed in juveniles (6 infected, 25%) and “blues” (6 infected, 14.3%), followed by nestlings (1 infected, 7.7%); no nematodes were found in adults. Our morphological and morphometric analysis shows that C. phenisci is closely related to another species, Cyathostoma (Cyathostoma) verrucosum (Hovorka & Macko, 1959). The doubtful status of the latter species was confirmed by molecular data: comparison of ITS2 sequence of C. phenisci with previously deposited sequences of C. verrucosum showed 96.3% similarity in this region. On this basis, we recognized Cyathostoma (Cyathostoma) verrucosum (Hovorka & Macko, 1959) as a synonym of Cyathostoma (Cyathostoma) phenisci Baudet, 1937.     

Establishment of Liebermannia dichroplusae n. comb. on the basis of molecular characterization of Perezia dichroplusae Lange, 1987 (Microsporidia)

Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania-Orthosomella branch forms a clade with the Endoreticulatus-Cystosporogenus-Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.     

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho‐species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.     

裳卷蛾变形孢虫SSU rRNA核心序列的克隆与系统发育分析

[目的]利用分子生物学手段探索裳卷蛾变形孢虫的遗传发育地位.[方法]微孢子虫的SSUrRNA序列是构建系统发育进化树的重要工具.试验通过T-A克隆法对裳卷蛾变形孢虫(Vairimorphaceraces)SSU rRNA核心序列进行了克隆,并采用近邻法构建了系统发育进化树.[结果]克隆得到了长为1228bp的核苷酸序列(GenBank EU267796).系统发育分析结果表明:裳卷蛾变形孢虫与分离于小菜蛾(Plutella xylostella)的Vairimorpha sp.Germany(GenBank AF124331)和Vairimorphaimperyecta(GenBank AJ131645)相似性最高,它们在系统发育进化树中与寄主为鳞翅目昆虫的Nosema属聚为一类,与纳卡变形孢虫(Vairimorpha necatrix)为代表的Vairimorpha属为相邻集.[结论]结合其生物学特征,裳卷蛾变形孢虫确实为Vairimorph a属的成员,但根据系统发育分析归入Nosematidae科可能更为合适.     

Scanning electron microscopy and molecular characterization of a new Haplosporidium species (Haplosporidia), a parasite of the marine gastropod Siphonaria pectinata (Mollusca: Gastropoda: Siphonariidae) in the Gulf of Mexico

Based on scanning electron microscopy and the small subunit ribosomal RNA (SSU rRNA), Haplosporidium tuxtlensis n. sp. (Haplosporidia), a parasite found in the visceral tissues of the false limpet Siphonaria pectinata (Linnaeus, 1758), is described. The spores are ellipsoidal (3.61 ± 0.15 μm × 2.69 ± 0.19 μm), with a circular lid (2.94 ± 0.5 μm) representing the operculum. The spore wall bears filaments occurring singly, or in clusters, of 2 to 8, fusing distally. Phylogenetic relationships of H. tuxtlensis n. sp. were assessed with other described species using the SSU rRNA sequence. Haplosporidium tuxtlensis n. sp. is sister taxon to Haplosporidium pickfordi Barrow, 1961. The morphological characteristics (spore wall structure, shape, size, and filament structure) and the unique host identity corroborate it as a new species. Additionally, this is the first record of Haplosporidia infecting striped false limpets in the Gulf of Mexico.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.     

A new deepwater species of Stauromedusae, Lucernaria janetae (Cnidaria, Staurozoa, Lucernariidae), and a preliminary investigation of stauromedusan phylogeny based on nuclear and mitochondrial rDNA data

The deepwater stauromedusan Lucernaria janetae n. sp is described from adult and juvenile specimens collected from the East Pacific Rise. Lucernaria janetae is the first species in the genus recorded from the Pacific Ocean, and differs from its congeners in size and morphology. Mitochondrial (16S) and nuclear (SSU) ribosomal gene sequences from L. janetae were analyzed with those of representative stauromedusan taxa to evaluate stauromedusan monophyly. Both genes recovered a strongly monophyletic Stauromedusae that is the sister group to all other medusozoans. Support of these hypotheses is robust to method of phylogenetic reconstruction and to outgroup selection, buttressing the argument that Stauromedusae should be recognized as the class Staurozoa. The molecular markers used here favor the same topology of relationships among our samples and clearly distinguished between two species, Haliclystus sanjuanensis and H. octoradiatus, that have been considered synonymous by many workers. A stable systematic framework for Stauromedusae appears achievable through comprehensive study of both morphological and sequence data.