Cytological evidence of spontaneous androgenesis in the freshwater clam Corbicula leana Prime

 Cytological observations and DNA microfluorometry of the hermaphrodite freshwater triploid clam Corbicula leana revealed unusual androgenetic development as follows: (1) the maternal genome of zygotes was extruded as two polar bodies just after karyokinesis of the first meiosis, (2) only chromosomes derived from one male pronucleus constituted the metaphase of the first cleavage of zygotes, (3) DNA content of 7-day-old veliger larvae was identical to the somatic cells of the parent. This spontaneous androgenetic process in C. leana zygotes is the first case in the phylum Mollusca and may be related to the specialized mode of reproduction; i.e. hermaphroditism and self-fertilization. Received: 22 September 1997/Accepted: 7 December 1997     

Origin and possible role of males in hermaphroditic androgenetic Corbicula clams

Hermaphroditic Corbicula leana clams reproduce by androgenesis and have been regarded as simultaneous hermaphrodites. To date, there has been no report on the occurrence of male clams in hermaphroditic Corbicula. In an irrigation ditch in Shiga Prefecture, we found that 78.2% of C. leana specimens were males and 21.8% were hermaphrodites. Microfluorometric analysis revealed that males were diploids and hermaphrodites were triploids. All males produced nonreductional and biflagellate spermatozoa. The sequence analysis of mitochondrial DNA (cytochrome b, 621 bp) for 31 specimens of C. leana showed that four male and nine hermaphrodites shared the same H2 mtDNA haplotype; H1 was detected from 17 males and H3 was detected from one hermaphrodite. Coexisting C. fluminea clams also have haplotypes H1 and H2. Phylogenetic tree by a neighborjoining method based on the partial sequence of cytochrome b revealed that the haplotypes (H1- 3) of C. leana were evidently different from those of dioecious C. sandai (S1 and S2) and C. japonica (J1 and J2). These results suggest that males may be derived from hermaphrodite C. leana clams. The role of males in hermaphroditic populations is unknown. However, if the spermatozoon from a male is able to fertilize an egg from a hermaphrodite and the nuclear genome of the egg is expelled as polar bodies, the sperm nucleus could form a zygote nucleus. This mode of reproduction would allow the replacement of the nuclear genome.     

The second meiosis occurs in cytochalasin D-treated eggs of Corbicula leana even though it is not observed in control androgenetic eggs because the maternal chromosomes and centrosomes are extruded at first meiosis

The hermaphroditic freshwater clam Corbicula leana reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as the two first polar bodies, and subsequently, second meiosis did not occur. But, in C. leana eggs treated with cytochalasin D (CD) to inhibit polar body extrusion, the second meiosis occurred. At metaphase-I, the spindle showed the typical bipolar structure and two spheroid centrosomes were located at its poles. All the maternal chromosomes were divided at anaphase-I, but they were not extruded as polar bodies due to the effects of CD. After completion of first meiosis, the maternal centrosomes split into four. At the second meiosis, twin or tetrapolar spindles were formed and two groups of maternal chromosomes divided into four sets of chromosomes. After the second meiosis, the spindle disassociated and the four maternal centrosomes disappeared. Four groups of maternal chromosomes transformed into the four female pronuclei. Male and female pronuclei became metaphase chromosomes of the first mitosis. The present study clearly indicates that typical meiosis systems still proceed in androgenetic triploid C. leana. We conclude that the androgenetic form may have arisen from the meiotic form.     

A hypothesis of ploidy elevation by formation of a female pronucleus in the androgenetic clam Corbicula fluminea in the Tone River estuary, Japan

We propose a hypothesis of ploidy elevation in the androgenetic clam Corbicula fluminea, based on an abnormal process of fertilization in clams collected at the Tone River, Ibaraki Prefecture, Japan. Most eggs showed androgenesis, that is, extrusion of all maternal chromosomes as two polar bodies during the first meiotic division. Most eggs did not form a female pronucleus, but only a male pronucleus. However, some eggs proceeded to the second meiosis and formed both a female and a male pronucleus. The formation of the female pronucleus suggests the hypothesis that ploidy elevation in androgenetic clams may have occurred by aberrant meiosis due to an altered orientation of the meiotic spindle.     

Phylogeny of Hesionidae (Aciculata, Polychaeta), assessed from morphology, 18S rDNA, 28S rDNA, 16S rDNA and COI

We assess phylogenetic relationships within the polychaete family Hesionidae from morphological data combined with nucleotide data from 18S rDNA, 28S rDNA, 16S rDNA and COI. Parsimony and Bayesian analyses were performed on two data sets; the first was based on a more restricted set of terminals with both morphological and molecular data (17 ingroup terminals), while the second included additional taxa with morphological data only (25 ingroup terminals). The different sets of terminals yielded fully congruent results, as did the parsimony and the Bayesian analyses. Our results indicate high levels of homoplasy in traditionally used morphological characters in the group, and that Hesioninae, Gyptini and Gyptis are nonmonophyletic. Hesionini (mainly Hesione and Leocrates ), Psamathini (mainly Hesiospina , Micropodarke , Nereimyra , Psamathe and Syllidia ), Ophiodrominae (Gyptini and Ophiodromini) and Ophiodromini (mainly Heteropodarke , Ophiodromus and Podarkeopsis ) are monophyletic and agree with previous classifications, and Hesionini is probably the sister to all other hesionids. The placements of the small hesionids capricornia and Lizardia , the hydrothermal vent taxa Hesiodeira and Hesiolyra , and the newly described Hesiobranchia , remain uncertain.     

Phylogeny of the Trichostrongylina (Nematoda) inferred from 28S rDNA sequences

We produced a molecular phylogeny of species within the order Strongylida (bursate nematodes) using the D1 and D2 domains of 28S rDNA, with 23 new sequences for each domain. A first analysis using Caenorhabditis elegans as an outgroup produced a tree with low resolution in which three taxa (Dictyocaulus filaria, Dictyocaulus noerneri, and Metastrongylus pudendotectus) showed highly divergent sequences. In a second analysis, these three species and C. elegans were removed and an Ancylostomatina, Bunostomum trigonocephalum, was chosen (on the basis of previous morphological analyses) as the outgroup for an analysis of the phylogenetic relationships between and within the Strongylina (strongyles) and Trichostrongylina (trichostrongyles). A very robust tree was obtained. The Trichostrongylina were monophyletic, but the Strongylina were paraphyletic, though this requires confirmation. Within the Trichostrongylina, the three superfamilies defined from morphological characters are confirmed, with the Trichostrongyloidea sister group to a clade including the Molineoidea and Heligmosomoidea. Within the Trichostrongyloidea, the Cooperiidae, Trichostrongylidae, and Haemonchidae were polytomous, the Haemonchinae were monophyletic, but the Ostertagiinae were paraphyletic. The sister-group relationships between Molineoidea and Heligmosomoidea were unsuspected from previous morphological analysis. No unequivocal morphological synapomorphy could be found for the grouping Molineoidea + Heligmosomoidea, but none was found which contradicted it.     

RFLP analysis of the PCR-amplified 28S rDNA inRhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.     

杉科、柏科的系统发生分析——来自28S rDNA序列分析的证据

对杉科（Taxodiaceae）与柏科（Cupressaceae s.s.）的28S rRNA基因的部分序列（约630 bp）进行PCR扩增、序列测定和系统发生关系分析,用简约法和邻接法构建的系统发生树基本一致。结果表明,杉科与柏科构成一个单系群,支持将杉科、柏科（Sciadopitys除外）合并为一个科——广义柏科（Cupressaceae sensu lato）的观点。在广义柏科中,Taiwania、Athrotaxis分别形成一支系;Metasequoia、Sequoia、Sequoiadendron关系较近,聚成一支系; Taxodium、Glyptostrobus、Cryptomeria聚成一支系;柏科聚成一支系。这一分析结果与叶绿体基因序列的分析结果相吻合,但是由于28S rRNA基因的进化速率较慢,尚不能分辨上述各个支系之间的系统演化关系。 Abstract:DNA sequences from 28S rDNA were used to assess relationships between and within traditional Taxodiaceae and Cupressaceae s.s.The MP tree and NJ tree generally are similar to one another.The results show that Taxodiaceae and Cupressaceae s.s.form a monophyletic conifer lineage excluding Sciadopitys.In the Taxodiaceae-Cupressaceae s.s.monophyletic group,the Taxodiaceae is paraphyletic.Taxodium,Glyptostrobus and Cryptomeria forming a clade(Taxodioideae),in which Glyptostrobus and Taxodium are closely related and sister to Cryptomeria;Sequoia,Sequoiadendron and Metasequoia are closely related to each other,forming another clade (Sequoioideae),in which Sequoia and Sequoiadendron are closely related and sister to Metasequoia;the seven genera of Cupressaceae s.s.are found to be closely related to form a monophyletic lineage (Cupressoideae).These results are basically similar to analyses from chloroplast gene data.But the relationships among Taiwania,Sequoioideae,Taxodioideae,and Cupressoideae remain unclear because of the slow evolution rate of 28S rDNA,which might best be answered by sequencing more rapidly evolving nuclear genes.     

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed, differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided.     

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed, differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided.     

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster. Received: 29 September 1999 / Accepted: 24 November 1999     

Detection of Raillietina saudiae from the domestic pigeon in Saudi Arabia through 18S and 28S rDNA genes

Raillietina saudiae is a well-studied avian gastrointestinal parasite belonging to the family Davaineidae and is the most prevalent cyclophyllid tapeworm infecting pigeon in Saudi Arabia. The present study considered as a complementary analysis of Al-Quraishy et al. (2019; Parasitol Int 71 , 59–72) with molecular studies for two ribosomal DNA genes employed for precise recognition of this Raillietina species. The annotated partial 18S and 28S rDNA gene regions were found to be 888 and 900 bp long that utilized further to elucidate their genetic relationships at species level using maximum likelihood method. The query sequence of R. saudiae is well aligned and placed within the Davaineidae family, with the same clade of all species of Raillietina that well separated from other cyclophyllidean cestodes especially taeniid and hymenolepid species. Sequence data recorded the monophyly of Raillietina species. The current phylogeny supports the usage of the partial 18S and 28S rDNA genes as reliable markers for phylogenetic reconstructions.     

Phylogeny of the monopisthocotylea and Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences

This study focuses on the phylogenetic relationships within the Polyopisthocotylea and Monopisthocotylea, two groups that are often grouped within the monogeneans, a group of disputed paraphyly. Phylogenetic analyses were conducted with multiple outgroups chosen according to two hypotheses, a paraphyletic Monogenea or a monophyletic Monogenea, and with three methods, namely maximum parsimony, neighbour joining and maximum likelihood. Sequences used were from the partial domain C1, full domain D1, and partial domain C2 (550 nucleotides, 209 unambiguously aligned sites) from the 28S ribosomal RNA gene for 16 species of monopisthocotyleans, 26 polyopisthocotyleans including six polystomatids, and other Platyhelminthes (61 species in total, 27 new sequences). Results were similar with outgroups corresponding to the two hypotheses. Within the Monopisthocotylea, relationships were: ?[(Udonella, capsalids), monocotylids], (diplectanids, ancyrocephalids)?; each of these families was found to be monophyletic and their monophyly was supported by high bootstrap values in neighbour joining and maximum parsimony. Within the Polyopisthocotylea, the polystomatids were the sister-group of all others. Among the latter, Hexabothrium, parasite of chondrichthyans, was the most basal, and the mazocraeids, mainly parasites of clupeomorph teleosts, were the sister-groups of all other studied polyopisthocotyleans, these, mainly parasites of euteleosts, being polytomous.     

Primed in situ labelling detection of 5S rDNA sequences in normal and androgenetic rainbow trout

Sex genotypes in mature androgenetic and control rainbow trout Oncorhynchus mykiss were determined using primed in situ labelling (PRINS) detection of 5S rDNA sequences on the X chromosomes. Three sex genotypes, corresponding to phenotypic sex, were revealed: female XX, male XY and supermale YY.     

28S rDNA evolution in the Eumalacostraca and the phylogenetic position of krill

The Malacostraca are an ancient and morphologically diverse class of Crustacea. The phylogenetic position of one order within this class, the Euphausiacea ("krill," subclass Eumalacostraca) was investigated using 28S rDNA sequences from representatives of several malacostracan orders. Phylogenies for these sequences were estimated by maximum-likelihood and maximum-parsimony analysis. The results of these analyses produced a new scheme for evolution within the Eumalacostraca. The new phylogenies suggested that Euphausiacea are most closely related to the Mysida and not the Decapoda, as is generally thought. Furthermore, the Mysida were found not to be closely related to the Lophogastrida, which are often considered their sister taxon. These hypotheses were tested against the hypotheses of monophyly for the Eucarida, Mysidacea, and Peracarida and found to be significantly better on the basis of the 28S rDNA data.     

Phylogenetic relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA, 28S rDNA D2, 18S rDNA gene sequences and morphological characters

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA, 28S rDNA D2 region, and 18S rDNA gene sequences and morphological data using both PAUP* 4.0 and MRBAYES 3.0B4 from 88 in-group taxa representing 35 subfamilies. The monophyletic nature of almost all subfamilies, of which multiple representatives are present in this study, is well-supported except for two subfamilies, Cenocoelinae and Neoneurinae that should probably be treated as tribal rank taxa in the subfamily Euphorinae. The topology of the trees generated in the present study supported the existence of three large generally accepted lineage or groupings of subfamilies: two main entirely endoparasitic lineages of this family, referred to as the "helconoid complex" and the "microgastroid complex," and the third "the cyclostome." The Aphidiinae was recovered as a member of the non-cyclostomes, probably a sister group of Euphorinae or Euphorinae-complex. The basal position of the microgastroid complex among the non-cyclostomes has been found in all our analyses. The cyclostomes were resolved as a monophyletic group in all analyses if two putatively misplaced groups (Mesostoa and Aspilodemon) were excluded from them. Certain well-supported relationships evident in this family from the previous analyses were recovered, such as a sister-group relationships of Alysiinae+Opiinae, of Braconinae+Doryctinae, and a close relationship between Macrocentrinae, Xiphozelinae, Homolobinae, and Charmontinae. The relationships of "Ichneutinae + ((Adeliinae + Cheloninae) + (Miracinae + (Cardiochilinae + Microgastrinae)))" was confirmed within the microgastroid complex. The position of Acampsohelconinae, Blacinae, and Trachypetinae is problematic.     

Reproduction and development of the freshwater clam Corbicula australis in southeast Australia

The freshwater clam Corbicula australis is an important component of the macrobiota of the river systems of southeast Australia. Reproduction of two populations of this clam in the Nepean River at Douglas Park and Menangle was investigated to document the gametogenic cycle, larval morphology and to determine when they incubate embryos. C. australis is a simultaneous hermaphrodite and broods its young in the inner demibranchs. The gonads are ovotestes with oogenic and spermatogenic regions in each ascinus. The sperm are biflagellate, a condition unique in the Bivalvia to clonal corbiculids. Gametogenesis was continuous and did not exhibit a seasonal pattern. In contrast, spawning and incubation of embryos was limited to the warmer months of the year. Embryos were present in the gills from October to May. C. australis develops through a modified veliger larva with a vestigially ciliated velum which is not used for swimming or particle capture. The velum is covered by microvilli and it is suggested that the velar epithelium may be specialised for nutrient uptake in the marsupial environment. C. australis produces several clutches each year and the young are released as advanced juveniles with a well-developed foot. Reproductive output differed between the two populations. This was in part due to the larger size of the clams from Menangle and may also reflect the enhanced productivity at this site. The suite of life history traits exhibited by C. australis: hermaphroditism, potential for self-fertilization/androgenesis, brooding progeny to the crawl-away juvenile stage and a high reproductive output, provide for rapid colonization and population growth in this clam which typically inhabits disturbance prone sandy lotic habitats.     

基于部分18S rDNA, 28S rDNA和COI基因序列的索科线虫亲缘关系

通过PCR扩增获得我国常见昆虫病原索科线虫6属10种18S rDNA、28S rDNA(D3区)和COI基因序列,结合来自GenBank中6属10种索科线虫的18S rDNA同源序列,用邻接法和最大简约法构建系统进化树。结果显示:12属索科线虫分为三大类群,第一大类群是三种罗索属线虫(Romanomermis)先聚在一起,再与两索属(Amphimermis)和蛛索属(Aranimermis)线虫聚为一支;在第二大类群中,六索属(Hexamermis)、卵索属线虫(Ovomermis)和多索属(Agamermis)亲缘关系最近,先聚在一起,再与八腱索属(Octomyomermis)和Thaumamermis线虫聚为一支。第三大类群由索属(Mermis)和异索属(Allomermis)线虫以显著水平的置信度先聚在一起,再与蠓索属(Heleidomermis)和施特克尔霍夫索属(Strelkovimermis)线虫聚为一支。从遗传距离看,基于3个基因的数据集均显示索科线虫属内种间差异明显小于属间差异,武昌罗索线虫(R.wuchangensis)和食蚊罗索线虫(R.culicivorax)同属蚊幼寄生罗索属线虫,其种间的遗传距离最小。