Interspecific hybridization among Hieracium species in New Zealand: evidence from flow cytometry

Hieracium pilosella: (Asteraceae) was accidentally introduced to New Zealand about 100 years ago. Since then it has become an aggressive weed, and an unexpected degree of genetic and genome size variation has been detected; features that might result from interspecies hybridization. We investigated the possibility that H. pilosella has hybridized with related taxa. Of the four other subgenus Pilosella species introduced to New Zealand, H. praealtum is the most abundant and, on morphological and distributional evidence, most likely to be the other parent. Flow cytometry was used to estimate relative genome size for 156 Hieracium plants collected from the wild. Plants assigned to either parental or hybrid morphotypes were found to comprise tetraploid and pentaploid individuals using genome size measurements, and this was confirmed with direct mitotic chromosome counts for a subset of plants. The haploid DNA content of H. praealtum was approximately 22% larger than that of H. pilosella. Putative hybrids that were tetraploid had mean genome sizes equivalent to two H. pilosella and two H. praealtum haploid chromosome sets, implying they were hybrids arising from the fertilization of two reduced gametes. Similar results were obtained from tetraploid hybrids produced by controlled pollination. However, the majority of field hybrids were pentaploid with a genome size equivalent to four H. pilosella and one H. praealtum haploid chromosome sets. We infer that these are not first-generation hybrids but represent successful backcrossing with H. pilosella and/or hybrid-hybrid crossing, and that sexual tetraploid hybrids have been the parents. We note that populations putatively of H. pilosella often comprise apomictic pentaploid hybrids. Significantly, our data indicate the emergence of sexual hybrids that provide further opportunity for gene flow among taxa in this complex.     

Chromosomes carrying meiotic avoidance loci in three apomictic eudicot Hieracium subgenus Pilosella species share structural features with two monocot apomicts

The LOSS OF APOMEIOSIS (LOA) locus is one of two dominant loci known to control apomixis in the eudicot Hieracium praealtum. LOA stimulates the differentiation of somatic aposporous initial cells after the initiation of meiosis in ovules. Aposporous initial cells undergo nuclear proliferation close to sexual megaspores, forming unreduced aposporous embryo sacs, and the sexual program ceases. LOA-linked genetic markers were used to isolate 1.2 Mb of LOA-associated DNAs from H. praealtum. Physical mapping defined the genomic region essential for LOA function between two markers, flanking 400 kb of identified sequence and central unknown sequences. Cytogenetic and sequence analyses revealed that the LOA locus is located on a single chromosome near the tip of the long arm and surrounded by extensive, abundant complex repeat and transposon sequences. Chromosomal features and LOA-linked markers are conserved in aposporous Hieracium caespitosum and Hieracium piloselloides but absent in sexual Hieracium pilosella. Their absence in apomictic Hieracium aurantiacum suggests that meiotic avoidance may have evolved independently in aposporous subgenus Pilosella species. The structure of the hemizygous chromosomal region containing the LOA locus in the three Hieracium subgenus Pilosella species resembles that of the hemizygous apospory-specific genomic regions in monocot Pennisetum squamulatum and Cenchrus ciliaris. Analyses of partial DNA sequences at these loci show no obvious conservation, indicating that they are unlikely to share a common ancestral origin. This suggests convergent evolution of repeat-rich hemizygous chromosomal regions containing apospory loci in these monocot and eudicot species, which may be required for the function and maintenance of the trait.     

Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium

Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.     

Sexual and apomictic seed formation in Hieracium requires the plant polycomb-group gene FERTILIZATION INDEPENDENT ENDOSPERM

A Polycomb-Group (PcG) complex, FERTILIZATION INDEPENDENT SEED (FIS), represses endosperm development in Arabidopsis thaliana until fertilization occurs. The Hieracium genus contains apomictic species that form viable seeds asexually. To investigate FIS function during apomictic seed formation, FERTILIZATION INDEPENDENT ENDOSPERM (FIE), encoding a WD-repeat member of the FIS complex, was isolated and downregulated in sexual and apomictic Hieracium species. General downregulation led to defects in leaf and seed development, consistent with a role in developmental transitions and cell fate. PcG-like activity of Hieracium FIE was also supported by its interaction in vitro with the Arabidopsis CURLY LEAF PcG protein. By contrast, specific downregulation of FIE in developing seeds of sexual Hieracium did not result in autonomous endosperm proliferation but led to seed abortion after cross-pollination. Furthermore, in apomictic Hieracium, specific FIE downregulation inhibited autonomous embryo and endosperm initiation, and most autonomous seeds displayed defective embryo and endosperm growth. Therefore, FIE is required for both apomictic and fertilization-induced seed initiation in Hieracium. Since Hieracium FIE failed to interact with FIS class proteins in vitro, its partner proteins might differ from those in the FIS complex of Arabidopsis. These differences in protein interaction were attributed to structural modifications predicted from comparisons of Arabidopsis and Hieracium FIE molecular models.     

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops. Received: 21 June 1999 / Revision accepted: 17 November 1999     

Changes in the Structure of Tall Tussock Grasslands and Infestation by Species of

A plant sociological survey of tall-tussock grasslands in the Mackenzie country was repeated after an interval of 26-28 years. Changes in physiognomy of the grasslands which have been inferred from earlier studies have been found to be continuing on many sites. A noteworthy feature of most sites has been a reduction in number of indigenous species found. An increase in abundance of Hieracium pilosella or H. praealtum has occurred at most sites. About 140 species and 9 variables from 53 sites were interrelated in a direct unimodal ordination (canonical correspondence analysis). Ordination results of the 1960s and the 1989 data were compared and interpreted with respect to spread of Hieracium pilosella and H. praealtum. The dynamics of Hieracium infestation was studied within changing community structure. A distinct trend is demonstrated of increasing infestation with increasing grassland degradation.     

Enriching Ploidy Level Diversity: the Role of Apomictic and Sexual Biotypes of Hieracium subgen. Pilosella (Asteraceae) that Coexist in Polyploid Populations

The capacity to generate variation in ploidy and reproductive mode was compared in facultatively apomictic versus sexual maternal plants that coexist in two model populations. The population structure was studied in polyploid hybrid swarms comprised of Hieracium pilosella (usually sexual, less commonly apomictic), H. bauhini (apomictic), and their hybrids (sexual, apomictic, or sterile). Relationships among established biotypes were proposed on the basis of their DNA ploidy level/chromosome number, reproductive mode and morphology. Isozyme phenotypes and chloroplast DNA haplotypes were assayed in the population that was richer in hybrids. The reproductive origin of seed progeny was identified in both sexual and apomictic mothers, using alternative methods: the karyological, morphological and reproductive characters of the cultivated progeny were compared with those of respective mothers, or flow cytometric seed screening was used. In both populations, the progeny of sexual mothers mainly retained a rather narrow range of ploidy level/chromosome number, while the progeny of facultatively apomictic mothers was more variable. The high-polyploid hybrids, which had arisen from the fertilization of unreduced egg cells of apomicts, mainly produced aberrant non-maternal progeny (either sexually and/or via haploid parthenogenesis). Apparently, such versatile reproduction resulted in genomic instability of the recently formed high-polyploid hybrids. While the progeny produced by both true apomictic and sexual mothers mostly maintained the maternal reproductive mode, the progeny of those ‘versatile’ mothers was mainly sexual. Herein, we argue that polyploid facultative apomicts can considerably increase population diversity.     

Chloroplast DNA diversity of Hieracium Pilosella (Asteraceae) introduced to New Zealand: reticulation, hybridization, and invasion

The European hawkweed Hieracium pilosella is a successful invader and a troublesome weed in New Zealand. The systematics of the genus Hieracium is extremely complex and contentious, probably due to recent speciation, hybridization, polyploidy, and diverse reproductive strategies. In the first chloroplast DNA survey of the group, we sequenced 285 plants (including H. pilosella and 12 other species of subgenus Pilosella) from New Zealand and Europe for 900 bp of trnL-trnF. Eleven haplotypes were identified with much sharing among species. Three haplotypes (A, D, G) were found in seven, three, and four species, respectively, but two species (H. lactucella and H. auricula) had single, private haplotypes. Our cpDNA data for subgenus Pilosella are consistent with the group's having incomplete lineage sorting and/or recent reticulate evolution. Six haplotypes were identified in H. pilosella, four of these unique to this taxon in our sample. In New Zealand, haplotype A was common and occurred in plants of different ploidy (i.e., 4×, 5×, 6×), whereas haplotypes C, B, and M were restricted to 4×, 5×, and 6× plants, respectively. The distribution of haplotype variation suggests that some or all of the H. pilosella seeds accidentally introduced into New Zealand probably came from east Europe rather than the United Kingdom and that a minimum of four lineages were introduced. Within New Zealand, hybridization of H. pilosella with a related taxon (probably H. praealtum) has occurred at least three times, involving both obligate sexual tetraploids and facultative apomictic pentaploids of H. pilosella.     

Modes of speciation and evolution of the sections inTaraxacum

Modes of evolution of species classified within different sections inTaraxacum involve diverse processes, viz. primary divergence of an ancestral sexual diploid, hybridization between a tetraploid apomict and a diploid sexual hybrid, differentiation of an advanced apomictic taxon at one ploidy level, hybridization between a sexual tetraploid and a sexual diploid, formation of a polyploid series from an apomictic ancestor of a lower polyploidy level, and remote hybridization between an autumn-flowering ancestral diploid and a spring-flowering derivative diploid or apomict. Various reproduction systems of the plants involved, different environments and different timing of the processes contribute to a very varied nature of the species groups.     

Cytogeography and reproduction of the Paspalum simplex polyploid complex

 Paspalum simplex is a grass distributed throughout the phytogeographic Chaco region in South America from which sexual diploid and apomictic tetraploid races have been reported. We analysed native populations to determine their homogeneity of ploidy level, and the relationship between geographic distribution, ploidy levels, and reproductive systems. The ploidy level was established for 379 plants from 32 wild populations. Tetraploidy and apomixis constitute the most common combination for this species all over the Chaco region. Apomictic hexaploid plants were found associated with 4x populations. Diploids were confined to a small sector of the region. One sexual triploid plant arose from seed harvested in a pure 2x population, and one apomictic 3x plant was found in a mixed 2x-4x population. The results suggest that P. simplex is a core agamic complex characteristic of the Chaco region from which other apomictic polyploid species of the subgenus Anachyris could have evolved. Received July 24, 2002; accepted September 12, 2002 Published online: December 11, 2002     

Cytotaxonomic study of genusHymenasplenium (Aspleniaceae) in Xishuangbanna, Southwestern China

The gametic chromosome numbers of sevenHymenasplenium (Aspleniaceae) species from Xishuangbanna, Yunnan Prov., China, were investigated. All the examined individuals ofH. obscurum, H. cheilosorum andH. latipinnum were sexual diploids with n=39 chromosomes. Intraspecific cytological variation was found inH. excisum, which has a sexual diploid (n=39) and a tetraploid (n=78). Only a triploid apogamous cytotype (n=ca.117) was found inH. laterepens. Hymenasplenium apogamum showed the most complicated intraspecific variation and included a sexual diploid (n=39), a sexual tetraploid (n=78) and an apogamous triploid (n=ca.117). This work reports for the first time the sexual diploids ofH. cheilosorum andH. apogamum, which are only apogamous elsewhere in east Asia, Himalayas and Indochina. These results may indicate that this area is one of the diversity centers ofHymenasplenium. Most of the above species have chromosome numbers based on x=39. In contrast,H. costarisorum contains a sexual diploid (n=36) and a sexual tetraploid (n=72), indicating that its basic number is x=36.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.     

Rapid short-tussock grassland decline with and without grazing, Marlborough, New Zealand

Species abundance, species richness, and ground cover were measured over 10 years on nine paired grazed and exclosure plots in short-tussock grassland in the early stages of invasion by Hieracium species. With and without grazing, H. pilosella and H. caespitosum increased markedly and H. lepidulum increased locally. In contrast, 50% of all other common species and species groups, and total, native, and exotic species richness declined significantly. Exclusion increased or had no effect on rates of increase in Hieracium species and rates of decline in short tussocks, and did not reduce rates of decline in other species. Exclusion had no effect on decline in native species richness, but mainly accelerated declines in total and exotic richness. Declines in 13 key vegetation variables were significantly predicted by increase in Hieracium abundance, suggesting competitive exclusion. With or without grazing, Hieracium species will become more dominant and other species will continue to decline. The effects of large herbivores on plant species diversity can often be predicted from site productivity. Our results indicate the need also to account for species origin, spatial scale, time, and exotic invasion.     

Deciphering the origins of apomictic polyploids in the Cheilanthes yavapensis complex (Pteridaceae)

Deciphering species relationships and hybrid origins in polyploid agamic species complexes is notoriously difficult. In this study of cheilanthoid ferns, we demonstrate increased resolving power for clarifying the origins of polyploid lineages by integrating evidence from a diverse selection of biosystematic methods. The prevalence of polyploidy, hybridization, and apomixis in ferns suggests that these processes play a significant role in their evolution and diversification. Using a combination of systematic approaches, we investigated the origins of apomictic polyploids belonging to the Cheilanthes yavapensis complex. Spore studies allowed us to assess ploidy levels; plastid and nuclear DNA sequencing revealed evolutionary relationships and confirmed the putative progenitors (both maternal and paternal) of taxa of hybrid origin; enzyme electrophoretic evidence provided information on genome dosage in allopolyploids. We find here that the widespread apomictic triploid, Cheilanthes lindheimeri, is an autopolyploid derived from a rare, previously undetected sexual diploid. The apomictic triploid Cheilanthes wootonii is shown to be an interspecific hybrid between C. fendleri and C. lindheimeri, whereas the apomictic tetraploid C. yavapensis is comprised of two cryptic and geographically distinct lineages. We show that earlier morphology-based hypotheses of species relationships, while not altogether incorrect, only partially explain the complicated evolutionary history of these ferns.     

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.     

<Emphasis Type="Italic">Hieracium</Emphasis> subgen. <Emphasis Type="Italic">Pilosella</Emphasis>: pollen stainability in sexual,apomictic and sterile plants

Two pollen stainability tests (Alexander’s stain and acetocarmine) were used to detect differences in pollen viability of the sexual, apomictic and sterile plants of Hieracium subgen. Pilosella. In sexual taxa (Hieracium bauhini and H. densiflorum), the average stainability was 93.7–98.4%. Similarly high stainability (92.2–97.2%) was found in the apomictic Hieracium pilosellinum and in the majority of the apomictic populations (or plants) of the pentaploid and hexaploid H. bauhini. In some apomictic plants of Hieracium bauhini the average pollen stainability was 49.0–75.4%. The lowest pollen stainability was found in the sterile plants, i.e. the triploid H. pistoriense (33.6%) and the pentaploid H. brachiatum (29.6%).     

Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae)

Microsporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed.     

Mendelian segregation for two-factor apomixis in Erigeron annuus (Asteraceae)

The inheritance of asexual seed development (apomixis) in Erigeron annuus (Asteraceae) was evaluated in a triploid (2n=3x=27) population resulting from a cross between an apomictic tetraploid (2n=4x=36) pollen parent and a sexual diploid (2n=2x=18) seed parent. Diplospory (unreduced female gametophyte formation) and autonomous development (embryo and endosperm together) segregated independently in the population yielding four distinct phenotype classes: (1) apomictic plants combining diplospory and autonomous development, (2) diplosporous plants lacking autonomous development, (3) meiotic plants with autonomous (though abortive) development and (4) meiotic plants lacking autonomous development. Each class was represented by approximately one-quarter of the population (n=117), thus corresponding to a two-factor genetic model with no linkage (chi(2)=2.59, P=0.11). Observations demonstrate that autonomous embryo and endosperm development (jointly) may occur in either reduced or unreduced egg cells. The cosegregation of the traits is attributed to tight linkage or pleiotropy. The data are consistent with the hypothesis that autonomous development in E. annuus is regulated by a single fertilization factor, F, which initiates development of both the embryo and the endosperm in the absence of fertilization.