C-banding analysis of six species of lung flukes, Paragonimus spp. (Trematoda: Platyhelminthes), from Japan and Korea

We examined C-banded karyotypes of six species of lung flukes from Japan and Korea; diploid and triploid Paragonimus westermani, P. miyazakii, P. ohirai, P. iloktsuenensis and P. sadoensis, with special reference to their karyotypic diversification. C-band analysis between the diploid and the triploid westermani revealed that two of three homologues of the triploid resembled those of the diploid in C-band pattern, while the remaining chromosome showed a different pattern from any species examined here. This karyological evidence indicates that the triploid is allotriploid probably induced by interspecific hybridization between the diploid westermani and an unknown species; we, therefore, suggest that the triploid westermani is an independent species and synonymous with P. pulmonalis (Miyazaki 1978). As the morphologically similar three species, ohirai, iloktsuenensis and sadoensis, had the same C-band polymorphism in chromosome No. 4, these species are classified as the local races of P. ohirai. Paragonimus miyazakii has one common C-band (5q) with the diploid westermani, but other bands (1q, 4q, 6q, 7p and 7q) are different. From these observations, the six species examined are phylogenetically divided into three groups: (1) westermani group containing diploid and triploid (= pulmonalis) species, (2) miyazakii and (3) ohirai including two geographic races, iloktsuenensis and sadoensis.     

Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tag analyses

Expressed sequence tag (EST) analysis of the diploid and triploid Paragonimus westermani genes was done to have a rapid and informative outlook of the gene-expression profiles of the parasites. Totals of 506 and 505 ESTs were generated from the diploid and triploid P. westermani cDNA libraries. Based on the BLASTx search results of the diploid P. westermani ESTs, 308 (60.9%) matched significantly with formerly identified genes and 198 (39.1%) showed no significant homology in the GenBank database. A similar homology pattern was shown from the triploid EST BLASTx search results with 346 (68.5%) sharing homology with previously identified genes and 159 (31.5%) showing no significant homology. The EST data from both libraries were analyzed and grouped into 9 categories. Comparison of the 2 EST pools revealed high similarities among the categories of the significantly matched genes. Single genes matched repeatedly were also observed in the 2 EST data. Some genes were found that are not yet characterized in P. westermani; these genes were matched by both the diploid and triploid ESTs. Further study of these genes may provide us with more understanding on the parasite's biology and their specific functions in the 2 strains.     

The maternal origins of the triploid ginbuna (Carassius auratus langsdorfi): phylogenetic relationships within the C. auratus taxa by partial mitochondrial D-loop sequencing

The hyper-variable segments (323-327 bp) of the mitochondrial D-loop for 169 Carassius auratus fishes in Japan were amplified by the polymerase chain reaction and the amplified products were sequenced directly and compared. A dendrogram showing three major clusters was generated with the sequence data for 37 haplotypes at 66 polymorphic sites. One cluster (cluster I) exclusively consisted of the gengorobuna, which was regarded as an independent (sub) species. The triploid ginbuna belonged to two remaining clusters, mainly in the diploid ginbuna cluster (cluster III) and partially in the goldfish cluster (cluster II). This finding suggests that the triploid ginbuna has been derived from two different maternal lineages. The triploid ginbuna was considered to have come into existence during the last ice age on the basis of this phylogenetic data. No geographic differentiation was observed with respect to the triploid ginbuna sampled at three different localities in Japan; the Shibuta River in Kanagawa, Lake Imba in Chiba and Lake Biwa in Shiga. The phylogenetic tree also demonstrated a monophyletic relationship amongst the nigorobuna, the nagabuna and the ginbuna, sharing cluster III. The nigorobuna and nagabuna populations have most likely arisen from geographic and temporal variations within the ginbuna populations. We also discuss the evolutionary origin of the triploid in view of its paternal ancestors.     

Phylogenetic relationship of ribosomal ITS2 and mitochondrial COI among diploid and triploid Paragonimus westermani isolates

We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.     

Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation

Reconstruction of the evolutionary history of asexual lineages undermines their suitability as models for the studies of evolutionary consequences of sexual reproduction. Using molecular tools we addressed the origin, age and maternal ancestry of diploid and triploid asexual lineages arisen through the hybridization between spiny loaches Cobitis elongatoides, C. taenia and C. tanaitica. Reconstructions of the phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes, revealed by sequence analyses, suggest that both hybrid complexes (C. elongatoides-taenia and C. elongatoides-tanaitica) contained several asexual lineages of independent origin. Cobitis elongatoides was the exclusive maternal ancestor of all the C. elongatoides-tanaitica hybrids, whereas within the C. elongatoides-taenia complex, hybridization was reciprocal. In both complexes the low haplotype divergences were consistent with a recent origin of asexual lineages. Combined mtDNA and allozyme data suggest that the triploids arose through the incorporation of a haploid sperm genome into unreduced ova produced by diploid hybrids.     

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.     

Triploidy in Paragonimus westermani

The finding of triploid and diploid forms of Paragonimus westermani in Japan has caused tremendous controversy over the identity and speciation of this parasite. Should these two genetically different forms be given different specific names? Hirohiso Hirai and Takeshi Agatsuma have studied the lung flukes by using cytogenetic techniques and zymodeme analysis. In this article, they briefly compare triploid P. westermani with the diploid form and develop a hypothesis for the mechanism and origin of triploidy.     

我国松阳和宽甸地区卫氏并殖吸虫的同工酶分析

作者采用Disc-PAGE电泳对我国浙江松阳和辽宁宽甸夹皮沟二倍体型和三倍体型卫氏并殖吸虫的乳酸脱氨酶(LDH)、苹果酸脱氢酶(MDH)、酯酶(EST)同工酶进行了比较。结果显示两型卫氏并殖吸虫的LDH、MDH和EST同工酶酶带的数目、Rf值、优势同工酶带的位置存在明显的差异。这些差异是否属种的特征尚待进一步研究。     

Inferring genome trees by using a filter to eliminate phylogenetically discordant sequences and a distance matrix based on mean normalized BLASTP scores

Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree). Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used. We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome. PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae). In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees. Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs. Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree.     

On the origin and evolution of apomixis in Boechera

The genetic mechanisms causing seed development by gametophytic apomixis in plants are predominantly unknown. As apomixis is consistently associated with hybridity and polyploidy, these confounding factors may either (a) be the underlying mechanism for the expression of apomixis, or (b) obscure the genetic factors which cause apomixis. To distinguish between these hypotheses, we analyzed the population genetic patterns of diploid and triploid apomictic lineages and their sexual progenitors in the genus Boechera (Brassicaceae). We find that while triploid apomixis is associated with hybridization, the majority of diploid apomictic lineages are likely the product of intra-specific crosses. We then show that these diploid apomicts are more likely to sire triploid apomictic lineages than conspecific sexuals. Combined with flow cytometric seed screen phenotyping for male and female components of apomixis, our analyses demonstrate that hybridization is an indirect correlate of apomixis in Boechera.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (<Emphasis Type="Italic">Cobitis</Emphasis>: Teleostei), and their ability to establish successful clonal lineages—on the evolution of polyploidy in asexual vertebrates

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides × C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.     

EVALUATION OF ELEVATED PLOIDY AND ASEXUAL REPRODUCTION AS ALTERNATIVE EXPLANATIONS FOR GEOGRAPHIC PARTHENOGENESIS IN EUCYPRIS VIRENS OSTRACODS

Transitions from sexual to asexual reproduction are often coupled with elevations in ploidy. As a consequence, the importance of ploidy per se for the maintenance and spread of asexual populations is unclear. To examine the effects of ploidy and asexual reproduction as independent determinants of the success of asexual lineages, we sampled diploid sexual, diploid asexual, and triploid asexual Eucypris virens ostracods across a European wide range. Applying nuclear and mitochondrial markers, we found that E. virens consists of genetically highly differentiated diploid sexual populations, to the extent that these sexual clades could be considered as cryptic species. All sexual populations were found in southern Europe and North Africa and we found that both diploid asexual and triploid asexual lineages have originated multiple times from several sexual lineages. Therefore, the asexual lineages show a wide variety of genetic backgrounds and very strong population genetic structure across the wide geographic range. Finally, we found that triploid, but not diploid, asexual clones dominate habitats in northern Europe. The limited distribution of diploid asexual lineages, despite their shared ancestry with triploid asexual lineages, strongly suggests that the wider geographic distribution of triploids is due to elevated ploidy rather than to asexuality.     

Evolution of clonality and polyploidy in a weevil system

The increased interest in asexual organisms calls for in-depth studies of asexual complexes that actively give rise to new clones. We present an extensive molecular study of the Otiorhynchus scaber (Coleoptera, Curculionidae) weevil system. Three forms have traditionally been recognized: diploid sexuals, triploid, and tetraploid parthenogens. All forms coexist in a small central area, but only the polyploid parthenogens have colonized marginal areas. Analyzing the phylogenetic relationship, based on three partial mitochondrial genes, of 95 individuals from 19 populations, we find that parthenogenesis and polyploidy have originated at least three times from different diploid lineages. We observe two major mitochondrial lineages, with over 2.5% sequence divergence between the most basal groups within them, and find that current distribution and phylogenetic relationships are weakly correlated. Quite unexpectedly, we also discover diploid clones that coexist with, and are morphologically indistinguishable from, the diploid sexual females. Our results support that these diploid clones are derived directly from the diploid sexuals. We also find that it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

二倍体型与三倍体型卫氏并殖吸虫同工酶谱的发育变异及其分类学意义

采用生趣板状聚丙烯酰胺胶是民泳法分析比较了二倍体型与三倍体型卫氏并殖吸虫童虫、成虫的醛缩酶、已糖激酶和葡萄糖－６－磷酸脱氢酶的同工酶谱,结果显示２ｎ型与３ｎ型卫氏并殖吸虫在不同发育期之间及两型吸虫相应各发育期之间ＡＬＤ、ＨＫ及Ｇ６ＰＤ同工酶谱均显著变异,变异表现为两种方式,一种是在同一个基因座位区的同工酶的酶带数目     

Intra- and Intergenomic variation of Ploidy and Clonality characterize <Emphasis Type="Italic">Phytophthora capsici</Emphasis> on <Emphasis Type="Italic">Capsicum</Emphasis> sp. in Taiwan

Phytophthora capsici is a devastating disease of pepper (Capsicum sp.) in Taiwan causing complete loss of commercial fields. The objective of this study was to characterize genetic diversity for 38 newly collected isolates and three historical isolates. Analysis of data includes whole genome sequence for two new isolates and for two isolates collected previously in 1987 and 1995. In addition, 63 single nucleotide polymorphism loci were genotyped using targeted-sequencing, revealing 27 unique genotypes. Genotypes fell into three genetic groups: two of the groups contain 90% (n = 33) of the 2016 isolates, are triploid (or higher), are exclusively the A2 mating type and appear to be two distinct clonal lineages. The isolates from 2016 that grouped with the historical isolates are diploid and the A1 mating type. Whole genome sequence revealed that ploidy varies by linkage group, and it appears the A2 clonal lineages may have switched mating type due to increased ploidy. Most of the isolates were recently race-typed on a set of differential C. annuum, and although there was no direct correlation between virulence and ploidy, many of the triploid isolates were less virulent as compared to the historical diploid isolates. The implications for breeding resistant pepper and conducting population analyses are discussed.     

Biodiversity of flukes

As many others parasites, speciation of flukes depends on the genetic characteristics and on ploidia. Ploidia of flukes can be different in a same species. In Asia, diploid, triploid and hybrid (2n/3n) populations are encountered. The comparison of morphological parameters between diploid and triploid flukes showed that they were morphologically different. Nevertheless, a genetic relationship between parthenogenetic organisms would exist regardless of their ploidia. In the Fasciola genus, the main consequence of the high level of diversity is the frequent probability of development of resistance to anthelmintics and fast adaptation to climatic changes. In the Paragonimus genus, diversity can enhance different forms of pathogenicity, can also be related to the species of intermediate hosts, and to the definitive host. The strain of flukes plays a part in the visceral localization of P. westermani adults.     

Biogeographic distribution of polyploidy and B chromosomes in the apomictic Boechera holboellii complex

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploid individuals, whereas apomictic individuals are diploid or triploid and may additionally have B chromosomes. Using flow cytometry and karyotype analysis, we have shown that B chromosomes (a) occur in both diploid and triploid apomictic B. holboellii, (b) may occur in triploid B. drummondii, and (c) are dispensable for the plant. Both diploid and triploid karyotypes are found in multiple chloroplast haplotypes of both species, suggesting that triploid forms have originated multiple times during the evolution of this complex. B chromosome carriers are found in geographically and genetically distinct populations, but it is unknown whether the extra chromosomes are shared by common descent (single origin) or have originated via introgressive hybridization and repeated transitions from diploidy to triploidy. Diploid plants containing the Bs reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. Finally, our analyses of pollen size and viability suggest that irregular chromosome segregation in some triploid lineages may lead to the generation of diploid individuals which carry the B chromosomes.     

PHYLOGENY AND EVOLUTION OF PARTHENOGENETIC WEEVILS OF THE ARAMIGUS TESSELLATUS SPECIES COMPLEX (COLEOPTERA: CURCULIONIDAE: NAUPACTINI): EVIDENCE FROM MITOCHONDRIAL DNA SEQUENCES

Molecular-phylogenetic studies of parthenogenetic animals have been a valuable recent addition to the literature on the evolutionary biology of sex. By illuminating the origins and ages of parthenogenetic lineages, such studies can help to define the temporal scale at which selection acts against parthenogenetic lineages, as well as provide an essential framework for further study. Although parthenogenetic weevils have played an important role in cytogenetic and protein-electrophoretic studies of parthenogenesis, they have not previously been subjects of DNA-based molecular-phylogenetic study. A mitochondrial DNA study of Aramigus tessellatus, a species complex of weevils native to South America, indentified 12 distinct (1–9% divergent) maternal lineages, of which 2 represent sexual populations, while at least 9 represent parthenogenetic lineages. These lineages partially correspond to lineages previously recognized by morphological differences. Phylogenetic analysis found 14 most parsimonious trees, according to which parthenogenesis appears to have arisen 3–7 times. There is a monophyletic group of lineages (the “brown clade”), having up to 4.5% sequence divergence within it, which may be primitively parthenogenetic and over 2 million years old.