Organisation of the nervous system in the Acoela: an immunocytochemical study

In order to broaden the information about the organisation of the nervous system in taxon Acoela, an immunocytochemical study of an undetermined Acoela from Cape Kartesh, Faerlea glomerata, Avagina incola and Paraphanostoma crassum has been performed. Antibodies to 5-HT and the native flatworm neuropeptide GYIRFamide were used. As in earlier studies, the pattern of 5-HT immunoreactivity revealed an anterior structure composed mainly of commissures, a so-called commissural brain. Three types of brain shapes were observed. No regular orthogon was visualised. GYIRFamide immunoreactive cell clusters were observed peripherally to the 5-HT immunoreactive commissural brain. Staining with anti-GYIRFamide revealed more nerve processes than did staining with anti-FMRFamide. As no synapomorphies were found in the organisation of the nervous system of the Acoela and that of the Platyhelminthes, the results support the view that the Acoela is not a member of the Platyhelminthes.     

Elongation factor 1-alpha sequences do not support an early divergence of the Acoela

The phylogenetic position of the Acoela is a key problem in the understanding of metazoan evolution. Recent studies based on 18S ribosomal DNA (rDNA) sequences have placed the Acoela in an extremely basal position as the sister group to all other extant triploblastic animals, suggesting that the phylum Platyhelminthes is polyphyletic. In order to test the results obtained with 18S rDNA, we sequenced elongation factor 1-alpha (EF1a) for the acoel Convoluta roscoffensis and five species of Turbellaria (two polyclads, Leptoplana tremellaris, and Prostheceraeus vittatus, and three triclads, Crenobia alpina, Schmidtea polychroa, and Girardia tigrina). Phylogenetic analyses of EF1a sequences show that the acoel sequences branch within the Platyhelminthes, in opposition to the 18S rDNA data. Moreover, comparison of the central variable region of EF1a shows similar sequence signatures between C. roscoffensis and the three triclad species. Although EF1a sequences fail to prove the monophyly of the phylum Platyhelminthes, they do not confirm the early divergence of the Acoela.     

Molecular systematics of the Acoela (Acoelomorpha,Platyhelminthes) and its concordance with morphology

The phylogenetic relationships of the lower worm group Acoela were investigated using newly obtained nuclear 18S rDNA sequences from 16 acoels in combination with 16 acoel sequences available on GenBank from other laboratories. Parsimony and maximum likelihood analyses of the molecular data supported the concept that the Acoela is monophyletic; however, the gene tree produced by these analyses conflicts with the current taxonomic system for the Acoela in several family-level groupings. Most notable is the apparent polyphyly of the largest family of acoels, the Convolutidae. DNA analysis grouped together species of small-bodied convolutids in one clade, while large-bodied convolutids grouped in a separate clade with other large-bodied acoels. Despite such conflicts, the branching pattern in the gene tree is well supported by morphological characters of sperm and body-wall musculature.     

Flatworm phylogeny from partial 18S rDNA sequences

Partial 18S rDNA sequences from 29 flatworms and 2 outgroup taxa were used in a cladistic analysis of the Platyhelminthes. Support for the clades in the resulting single most parsimonious tree was estimated through bootstrap analysis, jack-knife analysis and decay indices. The Acoelomorpha (Acoela and Nemertodermatida) were absent from the most parsimonious tree. The Acoela and the Fecampiidae form a strongly supported clade, the sister group of which may be the Tricladida. There is some support for monophyly of the rhabdocoel taxon Dalyellioida, previously regarded as paraphyletic. The sister group of the Neodermata remains unresolved. This revised version was published online in July 2006 with corrections to the Cover Date.     

田中碘泡虫的重描述及系统发育分析

基于形态学和分子数据(18S rDNA)重新描述了寄生于重庆江津地区金鱼 (Carassius auratus auratus)鳃丝的田中碘泡虫 (Myxobolus tanakai Kato, et al., 2017), 并结合系统发育分析对其近缘种进行了比较研究。形态学特征: 该虫种孢子为后端钝圆的长梨形, 大小为(14.58±0.54) μm×(6.09±0.37) μm; 两个极囊等大; 极丝与极囊长轴垂直缠绕8—10圈; 囊间突起位于极囊前端呈三角形; 具有嗜碘泡。分子特征: 共获得3个分离样本的18S rDNA序列, 其一级结构序列与日本黑川地区鲤 (Cyprinus carpio) 鳃丝寄生的田中碘泡虫的一致性达99.3%—99.6%, 且在系统发生树中聚为一支。结果表明: 本研究种与日本的田中碘泡虫为同种, 此为中国新记录; 金鱼为田中碘泡虫的宿主新记录。18S rDNA的V2、V4和V7区二级结构的比较结果表明: 田中碘泡虫已发生了明显的种内变异, 推测可能因宿主差异和地理隔离同时所致。此外, GenBank中报道的数个野鲤碘泡虫的序列是否为同种有待进一步厘清。     

The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes

Recent hypotheses on metazoan phylogeny have recognized three main clades of bilaterian animals: Deuterostomia, Ecdysozoa and Lophotrochozoa. The acoelomate and 'pseudocoelomate' metazoans, including the Platyhelminthes, long considered basal bilaterians, have been referred to positions within these clades by many authors. However, a recent study based on ribosomal DNA placed the flatworm group Acoela as the sister group of all other extant bilaterian lineages. Unexpectedly, the nemertodermatid flatworms, usually considered the sister group of the Acoela together forming the Acoelomorpha, were grouped separately from the Acoela with the rest of the Platyhelminthes (the Rhabditophora) within the Lophotrochozoa. To re-evaluate and clarify the phylogenetic position of the Nemertodermatida, new sequence data from 18S ribosomal DNA and mitochondrial genes of nemertodermatid and other bilaterian species were analysed with parsimony and maximum likelihood methods. The analyses strongly support a basal position within the Bilateria for the Nemertodermatida as a sister group to all other bilaterian taxa except the Acoela. Despite the basal position of both Nemertodermatida and Acoela, the clade Acoelomorpha was not retrieved. These results imply that the last common ancestor of bilaterian metazoans was a small, benthic, direct developer without segments, coelomic cavities, nephrida or a true brain. The name Nephrozoa is proposed for the ancestor of all bilaterians excluding the Nemertodermatida and the Acoela, and its descendants.     

DISCRIMINATIVE POWER OF NUCLEAR rDNA SEQUENCES FOR THE DNA TAXONOMY OF THE DINOFLAGELLATE GENUS PERIDINIUM (DINOPHYCEAE)1

The genus Peridinium Ehrenb. comprises a group of highly diversified dinoflagellates. Their morphological taxonomy has been established over the last century. Here, we examined relationships within the genus Peridinium, including Peridinium bipes F. Stein sensu lato, based on a molecular phylogeny derived from nuclear rDNA sequences. Extensive rDNA analyses of nine selected Peridinium species showed that intraspecies genetic variation was considerably low, but interspecies genetic divergence was high (>1.5% dissimilarity in the nearly complete 18S sequence; >4.4% in the 28S rDNA D1/D2). The 18S and 28S rDNA Bayesian tree topologies showed that Peridinium species grouped according to their taxonomic positions and certain morphological characters (e.g., epithecal plate formula). Of these groups, the quinquecorne group (plate formula of 3′, 2a, 7″) diverged first, followed by the umbonatum group (4′, 2a, 7″) and polonicum group (4′, 1a, 7″). Peridinium species with a plate formula of 4′, 3a, 7″ diverged last. Thus, 18S and 28S rDNA D1/D2 sequences are informative about relationships among Peridinium species. Statistical analyses revealed that the 28S rDNA D1/D2 region had a significantly higher genetic divergence than the 18S rDNA region, suggesting that the former as DNA markers may be more suitable for sequence‐based delimitation of Peridinium. The rDNA sequences had sufficient discriminative power to separate P. bipes f. occultaum (Er. Lindem.) M. Lefèvre and P. bipes f. globosum Er. Lindem. into two distinct species, even though these taxa are morphologically only marginally discriminated by spines on antapical plates and the shape of red bodies during the generation of cysts. Our results suggest that 28S rDNA can be used for all Peridinium species to make species‐level taxonomic distinctions, allowing improved taxonomic classification of Peridinium.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

Genomic organization of rDNA loci in natural populations of Medicago truncatula Gaertn

Medicago truncatula Gaertn. is an annual self-pollinating species characterized by a diploid complement 2n = 16 and low DNA content. It responds very well to transformation methods so it is used as a model species for Leguminosae. In contrast with the advanced studies in molecular biology, cytogenetic research has remained limited even though it is an extremely valuable approach to the analysis of the genome structure. In the present study we examined the chromosomal distribution of rDNA sequences in five natural populations of M. truncatula, explored the genomic diversity of this species and found markers for chromosome identification. FISH experiments revealed three distribution patterns of rDNA sequences, distinguished by one, two and three loci of 5S genes; 18S-5.8S-25S genes were always localized at a single locus. The results add information to the genome structure of M. truncatula, revealing a pattern of distribution of rDNA genes unobserved previously, which consists of 5S genes clustered at a single locus. The physical mapping of rDNA sequences is a first contribution towards the construction of a detailed molecular karyotype of M. truncatula.     

Nuclear DNA content,base composition,heterochromatin and rDNA in Picea omorika and Picea abies

Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species. Received: 18 October 2000 / 14 June 2001     

Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis

The heterochromatin composition and loca- tion in the genome of the fish Astyanax janeiroensis was investigated using Chromomycin A(3) and DAPI fluorochromes and fluorescence in situ hybridization (FISH) with 18S rDNA and As51 satellite DNA probes, respectively. Distinct repetitive DNA classes were found, namely: (1) C-positive centromeric/telomeric heterochromatin, (2) NOR-associated GC-rich heterochromatin (18S(+)/GC(+)) and (3) As51(+)/18S(+) heterochromatin colocalized on 14 distinct heterochromatic domains with attenuated fluorescence of DAPI staining (As51(+)/18S(+)/DAPI attenuated signal). Besides these fourteen associated repetitive DNAs, another eight sites with only 18S rDNA were also found, comprising altogether 22 18S rDNA sites in the genome of the species under study. Up to seven 18S rDNA sites were found to be active, i.e., were characterized as positive after silver staining (Ag-NORs). It was noteworthy that in all As51(+)/18S(+) domains the 18S rDNA were not found to be active sites due to the silencing of these genes when associated with the As51 satellite DNA in the same heterochromatic domain. The dispersion of the As51 sites in the genome of the species is hypothesized to probably originate from a transposable element. Several chromosomal and karyotype markers are similar between A. janeiroensis and A. scabripinnis, indicating a close relationship between these species.     

EVALUATING THE MORPHOSPECIES CONCEPT IN THE SELENASTRACEAE (CHLOROPHYCEAE,CHLOROPHYTA)1

Isolates of the genera Monoraphidium Kom.‐Legn., Ankistrodesmus Corda and Raphidocelis Hindák emend. Marvan et al. were cultured from two areas in Minnesota and North Dakota, USA. These isolates were identified to species level (when possible), using light microscopy and standard monographs and then characterized by 18S rDNA sequence analysis. Phylogenetic analyses indicated that in some cases, 18S rDNA sequences from these isolates were very similar, but not identical to the sequences of other isolates of the same morphospecies from different parts of the world. However, some isolates that were identified as the same species actually belong to different lineages within the Selenastraceae, whereas other isolates with identical or nearly identical 18S rDNA sequences possessed rather different morphologies. Overall, our data suggest that the application of a broad morphospecies concept to the Selenastraceae has resulted in an underestimation of the species diversity of this family and probably erroneous conclusions about the distribution of species.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae)

All Aloe taxa (～400 species) share a conserved bimodal karyotype with a basic genome of four large and three small submetacentric/acrocentric chromosomes. We investigated the physical organization of 18S-5.8S-26S and 5S ribosomal DNA (rDNA) using fluorescent in situ hybridization (FISH) to 13 Aloe species. The organization was compared with a phylogenetic tree of 28 species (including the 13 used for FISH) constructed by sequence analysis of the internal transcribed spacer (ITS) of 18S-5.8S-26S rDNA. The phylogeny showed little divergence within Aloe, although distinct, well-supported clades were found. FISH analysis of 5S rDNA distribution showed a similar interstitial location on a large chromosome in all species examined. In contrast, the distribution of 18S-5.8S-26S rDNA was variable, with differences in number, location, and size of loci found between species. Nevertheless, within well-supported clades, all species had the same organizational patterns. Thus, despite the striking stability of karyotype structure and location of 5S rDNA, the distribution of 18S-5.8S-26S rDNA is not so constrained and has clearly changed during Aloe speciation.     

Phylogenetic analysis of a group of palaeonemerteans (Nemertea) including two new species from Queensland and the Great Barrier Reef, Australia

Sundberg, P., Gibson, R. & Olsson, U. (2003). Phylogenetic analysis of a group of palaeonemerteans (Nemertea) including two new species from Queensland and the Great Barrier Reef, Australia. — Zoologica Scripta, 32, 279–296.
Based on 18S rDNA nucleotide sequences and morphological characters, we reconstruct the phylogeny for a group of palaeonemerteans estimated to be monophyletic. Two new palaeonemertean species from Queensland and the Great Barrier Reef, Australia are included in the phylogenetic analysis. The results confirm that one of the species, Cephalothrix queenslandica sp. n., is part of the Cephalothrix–Cephalotrichella–Procephalothrix group. These genera are redefined phylogenetically under the name Cephalothrix based on the cladistic analysis. The other species, Balionemertes australiensis gen. et sp. n., is placed in a new genus which forms a sister taxon to Cephalothrix . The morphology of both new species is described in detail.     

A commissural brain! The pattern of 5-HT immunoreactivity in Acoela (Plathelminthes)

 In order to solve the question whether Plathelminthes belonging to the taxon Acoela have a brain and an orthogon of the common flatworm type, an immunocytochemical study of the pattern of serotonin (5-HT) in four species was performed. In all species the 5-HT immunoreactivity revealed no ganglionic cell mass typical for other Plathelminthes, only a symmetrical brain-like structure composed of commissural fibres associated with a few cell bodies. 5-HT immunoreactivity was detected in three to five pairs of longitudinal nerve cords, connected by an irregular network of immunoreactive transverse fibres. No regular orthogon was visualised. All the surface sensilla were strongly immunoreactive. The unique commissural brain and cordal nervous system found in Acoela support the view of a deep gap lying between Acoela and other Plathelminthes. Accepted: 15 December 1997     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers

Cytologically, the species of Passiflora with known chromosome number can be divided into four groups: (1) 2n = 12, 24, 36; (2) 2n = 24; (3) 2n = 18, 72; and (4) 2n = 20. The base chromosome number proposed for the genus is x = 6, with x = 9, x = 10 and x = 12 being considered secondary base numbers. In the present study, variability of 5S and 45S rDNA sites was investigated in 20 species of these four groups to check the reliability of this hypothesis. In the group with x = 6, five diploid species (2n = 12) exhibit two 5S rDNA sites and two (P. capsularis, P. morifolia and P. rubra) or four (P. misera 2x and P. tricuspis) 45S rDNA sites. The hexaploid cytotype of P. misera had 12 45S rDNA sites and six 5S rDNA. A tetraploid species, P. suberosa, had ten 45S rDNA sites and four 5S rDNA sites, both in the same chromosomes as the 45S rDNA sites. In the group with x = 9, P. actinia, P. amethystina, P. edmundoi, P. elegans, P. galbana, P. glandulosa and P. mucronata displayed six 45S rDNA sites, whereas P. alata, P. cincinnata, P. edulis f. flavicarpa, P. edulis var. roxo and P. laurifolia had four sites. In this group, all species were diploid (2n = 18) and had only two 5S rDNA sites. Passiflora foetida, the only species with 2n = 20, had six 45S rDNA sites and four 5S rDNA sites. The species with x = 12 (2n = 24), P. haematostigma and P. pentagona, showed four 45S rDNA sites and two 5S rDNA. In general, the number and location of 5S and 45S rDNA sites were consistent with the hypothesis of x = 6 as the probable ancestral genome for the genus, while the groups of species with x = 9, x = 10 and x = 12 were considered to be of tetraploid origin with descending dysploidy and gene silencing of some redundant gene sites, mainly those of 5S rDNA.