Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Fertile triploid males: an uncommon case among hybrid vertebrates

The endemic Iberian minnow Squalius alburnoides is a complex of fishes of hybrid origin including both males and females with distinct ploidy levels and varying proportions of the parental genomes. In this paper we demonstrated that in contrast to many vertebrate hybrid lineages the sperm of triploid hybrid males of S. alburnoides is viable and fully functional. Flow cytometry and analysis of sequences of a fragment of the beta-actin nuclear gene applied to progenitors and offspring evidenced that these males produced their sperm clonally, as already described for diploid hybrids. The presence of different types of fertile males (nonhybrid diploids with normal meiosis and both diploid and triploid hybrids) coupled with hybridogenetic meiosis in females endows this vertebrate complex with a high level of independence from other species and contributes to maintain its genetic variability.     

Erratum to Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Cytological Diversity and Triploid Frequency in a Complex Population of Ranunculus ficaria L.

An extensive mixed population of Ranunculus ficaria polyplotypeswas mapped, sampled, and examined cytologically. The presenceand relative abundance of different polyplotypes was as follows:31 per cent diploid (2n = 16), 40 per cent tetraploid (2n =32), and 29 per cent presumed intermediate triploid (2n = 24).It was demonstrated that there is no simple character for distinguishingall polyplotypes in the field and chromosome counts must beused for this purpose. The action of winter flood water on bulbildissemination and differences in ecological preference of thepolyplotypes is proposed to account for differences in distributionover the population area. There is some indication that triploidseeds may be produced under a situation of excessive numbersof tetraploid plants relative to diploids which reduces diploid/tetraploidpollen competition on diploid stigmas. Diploid plants with 1–8B-chromosomes were found to occur frequently but no consistenteffect of these B's on vigour, fertility, and meiosis was evident.B-chromosomes were entirely absent from triploid and tetraploidkaryotypes.     

The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.     

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring - if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus - or in siblings with microchromosomes - if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.     

A Preliminary Study on Cytology of Chinese Pteris

Cytological and biosystematic studies on the genus Pteris have made a great contribution to the theory of speciation and evolution in ferns. Sino-Japanese area is one of the speciation centers of this genus. But only a few Chinese species have been known cytologically. As a preliminary study, ten species of the genus Pteris and two species of the related genera Pteridium and Histiopteris were observed. All the materials were collected from southwest and south China. Their young sori were fixed in ethanol and glacial acetic acid (3:1), in the field. The preparations were made with acetocarmine squash method. The vouchers are deposited in PE. The results of observation are summarized in Table 1. Of Pteris, four species are agamosporous, eight are polyploid, six are actually the members of species complexes. Only two species are purely sexual diploids. Pteris cretica L. var. nervosa (Thunb.) Ching et S. H. Wu has 58 autobivalents at meiosis of spore mother cell, and usually produces 32 diplospores per sporangium. It is obviously an agamosporous diploid. Apart from 32-spored sporangium, some 4-, 8-, 16-, 64-, 13-, 34-, 36-spored, completely abortive or partly abortive sporangia were also found in the fixed material of a single individual. For explaining these unusual types of sporangia at least nine sporogenesis routes can be inferred. It may not be impossible that besides mainly producing functional diplospores, P. cretica var. nervosa also gives a few functional giant spores with a doubled or multiplied somatic chromosome number, which means that polyploids may be simply derived from the spores produced by their diploid parent. The spores in a sporangium are usually tetrahedral, but a few bilateral ones are also found in some sporangia. Sometimes, the bilateral spores are even more than the tetrahedral ones in a sporangium. Pteris vittata L. from the type locality shows 58 bivalents at diakinesis of meiosis. Without doubt, it is a sexual tetraploid, which was also found in south Guangdong and south Yunnan. However, Its natural ancestral diploid has been widely found in the subtropical regions of China, such as Sichuan, Guizhou, Yunnan and Hubei provinces. A sterile triploid with the chromosome number of n=201Ⅰ+26Ⅱ+5Ⅲ and a sterile tetraploid hybrid with the chromosome number of n=9Ⅰ+45Ⅱ+3Ⅲ+21Ⅴ were found in south Yunnan and south Guangdong respec tively. It is evident that P. vittata L. sensu lato is a species complex which includes several cy totypes. Its hexaploid form was reported from south India by Abraham et al. The distribu tion pattern of different cytotypes in P. vittata complex may indicate that the tropics is more favourable to formation and surviving of polyploid than the subtropics. However, it is most possible that Chinese subtropics is the origin place of the ancestral member in the P. vittata complex. In gross morphology, the tetraploid and triploid forms are only bigger and stronger than the diploid form. But the diploid can also grow rather big in cultivation. They can har dly be distinguished from each other. Therefore, they are not given formal names here. The author fully agree with Prof. T. Reichstein when he said in 1983 that it was hoped that a new nomenclature could be adopted for the cytotypes of species complex in future. The special chromosome number of Pteris deltodon Bak .was counted in this work for the first time. It has 55 bivalents at meiosis of SMC. This number is the only exception in the genus Pteris, and shows that aneuploidy may have taken place in this genus. However, the pos sibility can not be ruled out that P. deltodon is an allotetraploid came from two diploid species respectively with the chromosome number 26 and 29. Pteris gallinopes described by Prof. Ching in i983 is an agamosporous tetraploid with 116 autobivalents at meiosis, different from the related triploid species P. dactylina Hook. and P. henryi Christ. P. ensiformis Burm^。 P. muftifida Poir. and P. semipinnata L. are sexual tetraploids with n=58. P. excelsa Gaud. and P. oshimensis Hieron. var. paraemeiensis Ching are agamosporous triploids with n = 87 autobivalents. P. wallichiana Agardh is a sexual diploid with n=29. The high frequency of polyploids and agamospory among these random sampled Chinese species futher confirms the conclusion that both polyploidy and apomixis have played an important role in speciation and evolution of the genus Pteris. Pteridium aquilimum (L.) Kunh var. latiusculum (Desv.) Underw. ex Heller and Histiopteris incisa (Thunb.) J. Sm. have n=52 and n=96 respectively. They are both sexual tetraploids. Their chromosome numbers show again that the genera Pteridium and Histiopteris are distinquished from the genus Pteris by different basic numbers. This project is supported by the National Natural Science Foundation of China.     

Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behaviour

Background and Aims

Polyploidy is a dominant feature of flowering-plant genomes, including those of many important crop species. Arachis is a largely diploid genus with just four polyploid species. Two of them are economically important: the cultivated peanut and A. glabrata, a tropical forage crop. Even though it is usually accepted that polyploids within papilionoid legumes have arisen via hybridization and further chromosome doubling, it has been recently suggested that peanut arose through bilateral sexual polyploidization. In this paper, the polyploid nature of the recent, spontaneously originated triploid cytotype of the tropical lucerne, A. pintoi, was analysed, and thereby the mechanism by which polyploids may arise in the genus.

Methods

Chromosome morphology of 2x and 3x A. pintoi was determined by the Feulgeńs technique and the rDNA sites were mapped by FISH. To investigate whether polyploidization occurred by means of unreduced gametes, a detailed analysis of the microsporogenesis and pollen grains was made.

Key Results

The 2x and 3x plants presented 9m + 1sm and a satellited chromosome type 2 in each haploid genome. Physical mapping revealed a cluster of 18S–26S rDNA, proximally located on chromosome 6, and two 5S rDNA loci on chromosomes 3 and 5. Diploid plants presented 10II in meiosis while trivalents were observed in all triploids, with a maximum of 10III by cell. Diploid A. pintoi produced normal tetrads, but also triads, dyads and monads. Two types of pollen grains were detected: (1) normal-sized with a prolate shape and (2) large ones with a tetrahedral morphology.

Conclusions

Karyotype and meiotic analysis demonstrate that the 3x clone of A. pintoi arose by autopolyploidy. The occurrence of unreduced gametes strongly supports unilateral sexual polyploidization as the most probable mechanism that could have led to the origin of the triploid cytotype. This mechanism of polyploidization would probably be one of the most important mechanisms involved in the origin of economically important species of Arachis, either by triploid bridge or bilateral sexual polyploidization.     

Cytogenetics of diploid and triploid salamanders of the Ambystoma jeffersonianum complex

Analysis of C-band heterochromatin (CBH) and cold-induced secondary constrictions (CICs) indicates that gynogenetic triploidy in the Ambystoma jeffersonianum complex is a cytogenetic consequence of hybridization between the two diploid species, A. jeffersonianum and A. laterale. The key feature in the history of this complex was the apparent proclivity for germ-line chromosome reduplication, and incidental production of chromosomally unreduced ova, allowing the circumvention of meiotic difficulties in diploid hybrid females. Chromosome structure, in terms of CBH and CICs, the mechanism of sex determination (dominant W, female heterogametic), and a recognizable WZ female/ZZ male sex chromosome heteromorphism in the diploid species A. laterale, are cytogenetic factors that allow reconstruction of the probable evolutionary history of the complex. The constitution of the triploid karyotypes suggests that the putative ancestral hybrid diploid females resulted from a mating between female A. jeffersonianum and male A. laterale, and that when such a hybrid female backcrossed to normal males of A. jeffersonianum and A. laterale, both kinds of allotriploids, A. platineum and A. tremblayi respectively, were produced. Karyological differentiation in each triploid species suggests that their origin was relatively recent and virtually simultaneous. It is conceivable that only one such hybrid female gave rise to both kinds of allotriploids in just one or two breeding seasons, and that present geographic distributions are due to persistent post-glaciation migrations of the resulting triploid clones. All offspring from such a back-cross carried a jeffersonianum W-chromosome and hence were female as well as triploid, and probably continued to produce chromosomally unreduced (triploid) ova by the same mechanism that operated in their hybrid mother. Sperm rejection resulting in gynogenesis in the allotriploids can be viewed as a physiological response to pseudopolyspermy by the chromosomally unreduced triploid ova. Evidence is presented that one of the triploid species, A. platineum, may produce a high percentage of diploid offspring with karyotypes identical to A. jeffersonianum. Diploids have not been detected among the offspring of A. tremblayi, but tetraploids are occasionally produced.Dedicated to my mentor and valued friend, James Kezer     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Cytology of meiosis in the triploid gynogenetic salamander Ambystoma tremblayi

Female meiosis was analyzed in the triploid gynogenetic salamander Ambystoma tremblayi to determine the mechanism by which a stable chromosome number is maintained in this unisexual species. Gross details of the reproductive cycle and the cytology of meiosis were analyzed in 20 specimens and 320 oocytes involving all stages from early diplotene to the beginning of anaphase II Ovulation apparently continues progressively involving a few oocytes at a time. Oocytes from the ovary contained chromosomes in diplotene, and diakinesis. The first metaphase was not observed since this stage occurs swiftly either immediately prior to or during ovulation. Oocytes in the most anterior region of the oviduct were in metaphase II, and those in the most posterior region were undergoing the beginning of anaphase II. Telophase II was not observed. Chromosome numbers obtained at all stages of prophase gave counts of approximately 42 bivalents, equivalent to the triploid somatic number known for this species. Similar numbers of dyads were obtained from metaphase II plates. This analysis supports earlier evidence suggesting that the triploid number of chromosomes in oocytes of A. tremblayi is doubled prior to meiosis, and the somatic number is later restored by two normal meiotic divisions.     

Triploidy and mosaicism in Liolaemus chiliensis (Sauria: Tropiduridae).

A remarkable diversity of ploidy, i.e., diploidy, triploidy, and diploid-triploid mosaicism, was found among the somatic and testicular cells in a natural population of the lizard Liolaemus chiliensis from central Chile. Intra pop ulation, intersexual, and intraindividual ploidy variation is reported. In contrast with other species of polyploid reptiles, 86% of L. chiliensis males were mosaics (2n/3n) and 14% were diploids; 33% of females were triploid, 57.1% were mosaics, and 9.5% were diploid. Among 21 specimens, no triploid males were found in this sample. In the mosaic males, the diploid and triploid spermatogonia both enter meiosis, producing both reduced and unreduced metaphase II spermatocytes, most of them euploids. We discuss the origin for this ploidy in this iguanid lizard.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

Contrasting patterns of spatial genetic structure of diploid and triploid populations of the clonal aquatic species,<Emphasis Type="Italic">Butomus umbellatus (Butomaceae)</Emphasis>, in Central Europe

Genetic diversity in a sample of an aquatic plantButomus umbellatus from 37 localities in Czechia and Slovakia was studied by analyzing six polymorphic loci in three enzymatic systems (SKDH, PGD and AAT). Diversity among ramets was low in eight populations with relatively extensive sampling (only one population possessed more than one multilocus genotype), suggesting high clonality of reproduction in these populations. However, among-population diversity was high: G = 0.782 and 0.881 for the samples of diploid and triploid populations, respectively. Heterozygosity of individual plants averaged over variable loci was also high: H = 0.554 for diploids and 0.453 for triploids. Genetic differentiation among populations was additionally studied using cluster analysis. Several populations of diploids clustered separately from all other populations, whereas another group of diploid populations clustered with some triploid populations, indicating the possibility of relatively recent, probably multiple origin of these triploid populations from their diploid progenitors. Association between matrices of Nei’s genetic distances among populations from different localities and matrices of geographic distances among these localities revealed highly significant correlation for the sample of diploid populations (r = 0.60,P < 0.001) but no significant correlation for the sample of triploid populations (r = 0.02,P = 0.593). These results indicate a spatial structure of diploid populations in accordance with the isolation by distance model, and a random distribution of genotypes among triploid populations ofB. umbellatus.     

Mutations in AtPS1 (Arabidopsis thaliana Parallel Spindle 1) Lead to the Production of Diploid Pollen Grains

Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most—if not all—angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes) in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1), implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parent''s heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.     

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.     

Male meiosis,morphometric analysis and natural propagation in the 2× and 3× cytotypes of Tordyliopsis brunonis (Apiaceae) from northwest Himalayas (India)

Tordyliopsis brunonis (Apiaceae) is cytologically investigated here for the first time from India. The chromosome count of 2n = 33, ascertained here, represents a new intraspecific triploid cytotype in the species, supplementing the earlier report of a diploid cytotype with 2n = 22 from Nepal Himalayas. The diploid chromosome count (n = 11) has also been found in some of the presently investigated individuals which showed perfectly normal meiosis with 100 % pollen fertility and normal seed set. However, the individuals with triploid chromosome count showed irregular meiotic behaviour and abnormal microsporogenesis resulting in high pollen sterility (56.26 %) and no seed set. The irregular meiotic behaviour in the triploid individuals is attributed to the occurrence of variable number of univalents (1–7) at diakinesis and metaphase-I. In the subsequent meiotic stages, these univalents lagged at anaphases and constituted micronuclei in sporads. The triploid plants were also observed for natural propagation and it was noticed that no seeds were set. These plants were noticed to propagate vegetatively by rootstocks. Chromosomal pairing in triploid cytotype is typical of an allopolyploid. Based on the characterization of chromosomal pairing during meiosis, we assumed that the triploid individuals are probably alloploid in nature. Hypotheses concerning the possible origin of allotriploid in T. brunonis are also discussed.