Affinities between Asian non-human Schistosoma species,the S. indicum group,and the African human schistosomes

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.     

Molecular phylogenetic position of Schistosoma sinensium in the genus Schistosoma

The status of Schistosoma sinensium (samples from Thailand and from Sichuan, China) relative to other species of the genus Schistosoma was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer 2 (ITS2). Trees inferred from these sequences place S. sinensium as sister to the S. japonicum group and suggest a basal position in the clade utilizing snails of the family Pomatiopsidae. The sequence differences between specimens of S. sinensium from China and Thailand are at least as great as between S. malayensis and S. mekongi. Schistosoma sinensium is probably best regarded as a species complex.     

Identities of two Paragonimus species from Sri Lanka inferred from molecular sequences

Metacercariae of Paragonimus spp. were obtained from field-collected freshwater crabs in Sri Lanka. Genomic DNA was extracted from single metacercariae. Two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified using the polymerase chain reaction. Two differing sequences were obtained for each of these gene regions. Phylogenetic analyses placed the type 1 sequences as sister to a clade containing P. westermani and P. siamensis whereas the type 2 sequences were close to published sequences of P. siamensis from Thailand. The possible taxonomic status of these two types are discussed. This is the first report of molecular data about Paragonimus from Sri Lanka.     

DNA barcoding in the rust genus Chrysomyxa and its implications for the phylogeny of the genus

Chrysomyxa rusts are fungal pathogens widely present in the boreal forest. Taxonomic delimitation and precise species identification are difficult within this genus because several species display similar morphological features. We applied a DNA barcode system based on the ribosomal internal transcribed spacer region (ITS), large subunit (28S) ribosomal RNA gene, mitochondrial cytochrome oxidase 1 (CO1) and mitochondrial NADH dehydrogenase subunit 6 (NAD6) in 86 strains from 16 different Chrysomyxa species, including members of the Chrysomyxa ledi species complex. The nuclear ITS and 28S loci revealed higher resolving power than the mitochondrial genes. Amplification of the full CO1 barcode region failed due to the presence of introns limiting the dataset obtained with this barcode. In most cases the ITS barcodes were in agreement with taxonomic species based on phenotypic characters. Nevertheless we observed genetically distinct (different DNA barcodes) lineages within Chrysomyxa pyrolae and Chrysomyxa rhododendri, providing some evidence for allopatric speciation within these morphologically defined species. This finding, together with the observed pattern of host specificities of the studied rust fungi, suggest that species diversification within the C. ledi species complex might be governed by a set of factors such as specialisation to certain Ericaceae species as telial hosts and to a lesser extent specialization to different spruce species as aecial hosts. Moreover allopatric speciation by geographic disruption of species also seems to take place. When our data were integrated into a broader phylogenetic framework the Chrysomyxa genus unexpectedly was not resolved as a monophyletic group. Indeed the spruce cone rusts C. pyrolae and C. monesis coalesced with the pine needle rusts belonging to the genus Coleosporium, whereas the microcyclic species Chrysomyxa weirii was embedded within a clade comprising the genus Melampsora.     

The taxonomic status of Digramma (Pseudophyllidea: Ligulidae) inferred from DNA sequences

The traditional classification of the ligulid tapeworms into 2 genera, Ligula Bloch, 1782 and Digramma Cholodkovsky, 1914, remains controversial. Molecular data of sequences for the 5' end of the nuclear 28S ribosomal ribonucleic acid (rRNA) gene, the mitochondrial cytochrome c oxidase subunit I (COI) gene, and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) gene, as well as the first internal transcribed spacer (ITS1) of the nuclear ribosomal deoxyribonucleic acid (DNA), were used to characterize Digramma and to investigate its relationship with Ligula. Digramma spp. exhibited identical sequences with Ligula intestinalis both in the 28S rRNA and the COI gene and differed from L. intestinalis by 0.7% in the ITS1 region and 7.4% in the ND1 gene, respectively. A high degree of genetic conservation within 28S ribosomal DNA, COI, ITS1, and even ND1 genes, was found in Ligula and Digramma. The low genetic divergence in the 4 genes between Ligula and Digramma indicates that Digramma is probably not an independent genus. Therefore, it is proposed that Ligula and Digramma should be considered as 2 species within the genus Ligula and the tapeworms of Digramma collected from diverse localities in China belong to the same species. The present study also suggests that ITS1 and ND1 sequences can act as useful genetic markers to distinguish Ligula and Digramma.     

Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.     

Comparison of ribosomal DNA sequences of Lamellodiscus spp. (monogenea, diplectanidae) parasitising Pagellus (sparidae, teleostei) in the North Mediterranean Sea: species divergence and coevolutionary interactions

We sequenced DNA fragments from four monogenean species of the genus Lamellodiscus and their three fish host species from the genus Pagellus in the North Mediterranean Sea, in order to estimate the molecular divergence and the coevolutionary interactions in this association. By comparing the ITS1 sequences of the parasites, we assessed their level of interspecific differences and tested the phylogenetic status of Lamellodiscus virgula and Lamellodiscus obeliae, formerly described as two different species. Moreover, we wanted to know if closely related parasites used closely related hosts, to investigate the coevolutionary interactions in this complex. Phylogenetic relationships among Lamellodiscus species were estimated with partial 18S ribosomal DNA sequences while mitochondrial cytochrome-b DNA sequences were used for their fish hosts. The ITS1 sequences appear to be highly variable among Lamellodiscus species, except L.virgula and L.obeliae, suggesting an old divergence time or a rapid molecular evolution within this genus. This fish-parasite association seems to exhibit coevolutionary interactions. L.virgula and L.obeliae are proposed to be a single species on the basis of their almost identical ITS1 sequences.     

A molecular perspective on the genera Paragonimus Braun, Euparagonimus Chen and Pagumogonimus Chen

The status of the genera Euparagonimus Chen, 1963 and Pagumogonimus Chen, 1963 relative to Paragonimus Braun, 1899 was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit I (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). In the phylogenetic trees constructed, the genus Pagumogonimus is clearly not monophyletic and therefore not a natural taxon. Indeed, the type species of Pagumogonimus, P. skrjabini from China, is very closely related to Paragonimus miyazakii from Japan. The status of Euparagonimus is less obvious. Euparagonimus cenocopiosus lies distant from other lungflukes included in the analysis. It can be placed as sister to Paragonimus in some analyses and falls within the genus in others. A recently published morphological study placed E. cenocopiosus within the genus Paragonimus and probably this is where it should remain.     

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

Abstract The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree‐species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species‐specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well‐supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co‐occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4–1.0 million years, depending on molecular clock calibrations.     

Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.     

直突摇蚊亚科部分属28S rDNA D1序列的分子系统发育关系(双翅目,摇蚊科)

利用28S rDNA D1部分基因序列对直突摇蚊亚科代表性属级阶元进行了分子系统学研究。测定了12个内群属和2种外群的28SrDNAD1片段,并结合GenBank中3个同亚科种类该基因的同源序列进行了分析。采用2种建树方法(距离邻近法NJ和最大俭约法MP)分析了直突摇蚊亚科内属级分类单元的分子系统发育关系。结果表明,滨海摇蚊属Clunio位于系统发育树的基部,与该属营海洋生活的特殊性一致。心突摇蚊属和真开氏摇蚊属互为姐妹群,流环足摇蚊属和刀突摇蚊属互为姐妹群,此结果与基于形态学的系统发育研究相结果一致。其它属间的系统发育关系因尚无前人研究而有待做进一步研究。本研究同时证明28S rDNA D1基因片段在分析摇蚊科昆虫属级及属内阶元关系上具有一定的指导意义。     

A revision of the interrelationships of Schistosoma including the recently described Schistosoma guineensis

In light of the recently described human schistosome Schistosoma guineensis and recent phylogenetic studies of the genus Schistosoma, a revision of the interrelationships of the members of this genus is needed. This paper adds to previous phylogenetic studies on the family Schistosomatidae and offers the most up to date and robust phylogeny of the group based on complete small and large nuclear subunit rRNA genes and partial mitochondrial cox1, incorporating most of the 21 species of Schistosoma. Our findings show that the group retains the same topology as that resolved in previous studies except Schistosoma margrebowiei was resolved as the sister taxon to all others in the Schistosoma haematobium species group and S. guineensis was placed as sister species to both Schistosoma bovis and Schistosoma curassoni. The S. haematobium species group contains eight species of which many are of significant medical and veterinary importance. Additionally, many of these species have been shown to hybridise both in the wild and experimentally, making the correct identification and recognition of species very important. A pairwise comparison of cox1 among Schistosoma species suggests this gene alone would fail as a reliable barcode for species identification. Phylogenetic results clearly treat Schistosoma intercalatum and S. guineensis as separate taxa with each more closely related evolutionarily to S. haematobium than to each other. The study also highlights the problems associated with wrongly attributed sequences on public databases such as GenBank.     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

Interspecific variation within the 'hypervariable' region of the 18S ribosomal RNA gene among species of Schistosoma Weinland, 1858 (Digenea)

The 'hypervariable' region of the 18S ribosomal DNA gene, comprising 344 base pairs, was sequenced for five species of the genus Schistosoma (S. intercalatum, S. margrebowiei, S. mattheei, S. malayensis and S. rodhaini), representing three of the schistosome species groups. Heterobilharzia americana was also included as an outgroup. The sequences were compared with previously published sequences (S. haematobium, S. japonicum, S. mansoni and S. spindale). The analysis confirmed that the Asian schistosomes were distantly related to the other groups of schistosomes and showed that the amount of variation within the S. japonicum group (1.49%) was greater than that detected within the S. mansoni and S. haematobium groups (0% in each case).     

A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador

A molecular phylogeographic study of Paragonimus mexicanus collected from Guatemala and Ecuador was performed. Genomic DNA was extracted from individual metacercariae, and two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified by the polymerase chain reaction (PCR). Sequences segregated in a phylogenetic tree according to their geographic origins. ITS2 sequences from Ecuador and Guatemala differed at only one site. Pairwise distances among CO1 sequences within a country were always lower than between countries. Nevertheless, genetic distances between countries were less than between geographical forms of P. westermani that have been suggested to be distinct species. This result suggests that populations from Guatemala and Ecuador are genetically differentiated perhaps at the level of subspecies.     

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

衣藻属的系统发育分析——基于形态形状和nrDNA ITS序列

通过实验分析莱茵衣藻 ( Chlamydomonas reinhardtii) 1个种和互连网获得衣藻属 1 5个种及丝藻属 1个种 ( Ulothrix zonata) ,共 1 7个种的 nr DNA ITS序列 ,并以 U.zonata为外类群 ,采用计算机分析软件包对其进行分析及构建分子系统发育树图。同时以 1 2个传统分类性状 ,对此 1 6种衣藻构建数据矩阵 ;以 U.zonata动孢子的相应性状为外类群原始性状 ,用Wagner法在计算机上对其进行分枝分析 ;然后比较并分析分子系统树和表征性状分支分析树的异同。初步尝试以 ITS分子序列系统发育分析作为传统性状分析的补充来研究衣藻种间的亲缘关系。