Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas

• Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex – ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes.
• Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios.
• We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae.
• Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

     

Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae‐Bombacoideae)

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.     

Polyembryony and apomixis in Eriotheca pubescens (Malvaceae - Bombacoideae)

Apomixis and adventitious polyembryony have been reported for several species of Bombacoideae, including Eriotheca pubescens, a tree species of the Neotropical savanna (Cerrado) areas in Brazil. However, the origin of polyembryonic seeds and their importance for the reproduction of the species remained to be shown. Here, we analyzed the early embryology of this species to establish the apomictic origin of extranumerary embryos. We also observed the geographic distribution of polyembryony in E. pubescens, and tested if apomixis was related to the source of pollen (self or cross) and population density. Moreover, we tested if polyembryonic apomictic embryos would develop normally into seedlings. In the observed seed primordia, after a relatively long quiescent period, the zygote developed into a sexual embryo concurrently with adventitious apomictic embryos which developed from nucellus cells. Adventitious embryos develop faster than sexual ones and are morphologically similar, so that 44 days after anthesis it was virtually impossible to distinguish and trace the fate of the sexual embryo. Polyembryony is widely distributed in populations some 400 km distant, and only one strictly monoembryonic individual was observed during the study. The number of embryos per seed varied between fruits and individuals but was significantly higher in seeds from cross-pollinations than from selfs, although fruit and seed set after crosses were much lower than after selfs. Embryo development into seedlings depended on their weight at germination, but polyembryonic seeds germinated and produced up to seven seedlings per seed in greenhouse conditions. Adventitious embryony and apomictic seedlings would explain the mostly clonal populations suggested by molecular studies.     

Sexual,apomictic and mixed populations in <Emphasis Type="Italic">Handroanthus ochraceus</Emphasis> (Bignoniaceae) polyploid complex

Some tropical Bignoniaceae form sporophytic apomictic polyploid complexes are similar to better studied temperate plants. Handroanthus ochraceus is a widely distributed Neotropical savanna tree with both monoembryonic/self-sterile, and polyembryonic/apomictic and self-fertile populations, but lacking chromosome number and morphological comparative studies. We tested if monoembryonic/non-apomictic and polyembryonic/apomictic populations differed in ploidy and morphological features, as a basis to understand evolution and biogeography of these plants. Chromosome number and embryo number per seed were investigated, and uni- and multivariate analyses of flower and leaf morphology were done for five populations of H. ochraceus. We found two pure monoembryonic diploid (2n?=?40), and one pure polyembryonic (62–94% of polyembryonic seeds) tetraploid population (2n?=?80). One of the diploid populations presented only one individual with 3.2% polyembryonic seeds and was considered a non-apomictic population. Another population showed predominantly polyembryonic (27–66% of polyembryonic seeds) tetraploid individuals, but one diploid individual with 2% of polyembryonic seeds, and was considered a mixed apomictic and non-apomictic population. Morphological analyses confirmed breeding system clusters, and that stigma width, as well as pollen grain area, was consistently larger in polyembryonic populations. Polyploid plants showed larger cells, as well as larger organs and other distinctive features, which will be useful to identify apomictic populations and to future taxonomic discussions. The species can be considered an agamic complex with apomixis associated with neopolyploidy. This trend is also found in other tropical sporophytic apomictics, contrasting with the usual reports linking diploidy or paleopolyploidy to this kind of apomictics.     

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis<Emphasis Type="Italic">)</Emphasis> in <Emphasis Type="Italic">Citrus maxima</Emphasis> × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony.     

Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula

Apomicts tend to have larger geographical distributional ranges and to occur in ecologically more extreme environments than their sexual progenitors. However, the expression of apomixis is typically linked to polyploidy. Thus, it is a priori not clear whether intrinsic effects related to the change in the reproductive mode or rather in the ploidy drive ecological differentiation. We used sympatric sexual and apomictic populations of Potentilla puberula to test for ecological differentiation. To distinguish the effects of reproductive mode and ploidy on the ecology of cytotypes, we compared the niches (a) of sexuals (tetraploids) and autopolyploid apomicts (penta‐, hepta‐, and octoploids) and (b) of the three apomictic cytotypes. We based comparisons on a ploidy screen of 238 populations along a latitudinal transect through the Eastern European Alps and associated bioclimatic, and soil and topographic data. Sexual tetraploids preferred primary habitats at drier, steeper, more south‐oriented slopes, while apomicts mostly occurred in human‐made habitats with higher water availability. Contrariwise, we found no or only marginal ecological differentiation among the apomictic higher ploids. Based on the pronounced ecological differences found between sexuals and apomicts, in addition to the lack of niche differentiation among cytotypes of the same reproductive mode, we conclude that reproductive mode rather than ploidy is the main driver of the observed differences. Moreover, we compared our system with others from the literature, to stress the importance of identifying alternative confounding effects (such as hybrid origin). Finally, we underline the relevance of studying ecological parthenogenesis in sympatry, to minimize the effects of differential migration abilities.     

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.     

The Detection,Rate and Manifestation of Residual Sexuality in Apomictic Populations of Pilosella (Asteraceae,Lactuceae)

The effect of maternal, facultatively apomictic plants on population diversity was evaluated in seven hybridizing polyploid Pilosella populations, where apomictic (P. bauhini or P. aurantiaca) and sexual (P. officinarum) biotypes coexist. The ploidy level, reproductive system, morphology, clonal structure and chloroplast DNA haplotypes were used to characterize these plants and their hybrids. The reproductive origins of the progeny were assessed through either a flow cytometric seed screen and/or a comparison between the ploidy level of progeny embryos/seedlings and the maternal ploidy level. The cultivated progeny derived from residual sexuality in maternal apomicts were also identified based on their morphology and reproductive behaviour. The progeny different from their maternal parents (0.6?92.3 % of progeny embryos and 0?100 % of progeny seedlings) originated either sexually or via haploid parthenogenesis. Comparing the facultatively apomictic and sexual mothers, the progeny arrays generated in the field showed that apomictic mothers produce progeny that is more variable in ploidy level. This effect was demonstrated at both the embryonic and seedling stages of progeny development. Residual sexuality in apomicts was also effective in experimental crosses, generating progeny similar to spontaneous hybrids in the field. The 2n + n hybrids produced from an apomictic and a sexual parent displayed similar reproductive behaviour, producing polyhaploid, sexual and apomictic progeny in variable ratios. Repeated hybridizations between parental species and/or multi-step crosses can result in hybrid swarms rich in cytotypes and morphotypes. The variation recorded in these populations suggests prevailing introgressive hybridization towards the sexual species P. officinarum.     

RAPD Evidence for Apomixis and Clonal Populations in Eriotheca (Bombacaceae)

Abstract: Two woody species of Eriotheca (Bombacaceae) of the Central Brazilian Cerrados were submitted to RAPD analyses. Both species are bee pollinated and have a similar flower structure, but E. pubescens presents adventitious embryony and apomixis, while E. gracilipes is self-incompatible. The RAPD screening reflects these differences in breeding systems, with very low genetic variation in the apomictic species, while the sexual species presented much higher variability with no similar genotypes among the sampled trees. The results suggest that adventitious embryony in E. pubescens effectively results in clonal populations or population mosaics of clonal individuals. Since recent studies have indicated poly-embryony and possibly apomixis in a number of Cerrado woody species otherwise considered obligatorily allogamous, the RAPD results presented here indicate the technique will be a useful tool to detect clonal populations of apomictic origin among Cerrado woody species with mixed mating systems and will help to assess the importance of apomixis as a breeding system for the Cerrado flora.     

The best of both worlds: Apomixis and sexuality co-occur in species of Microlicia,Melastomataceae

Apomixis, the asexual formation of embryos and seeds, occurs in approximately 18% of angiosperm families. Melastomataceae exhibits a remarkable number of apomictic species, distributed among different tribes. This mode of reproduction has been elucidated in Miconieae, but remains unclarified for other groups, such as Microlicieae. Although apomixis has been previously described for Microlicieae species, the cytological basis for this phenomenon is entirely unknown in this group. Thus, populations of Microlicia fasciculata and M. polystemma were used in order to (a) investigate the presence of autonomous apomixis; (b) verify if this mode of reproduction leads to polyembryony; and (c) investigate whether apomixis may occur in parallel with the sexual process. We tested these species for autonomous fruit set and polyembryony, and pollen viability, and analyzed pollen tube growth. Anatomical techniques were used to elucidate the micro- and megasporogenesis and gametogenesis. The species showed autonomous fruit and seed formation and exhibited polyembryony. Apospory and adventitious embryony were the developmental mechanisms of apomixis in M. fasciculata and M. polystemma, respectively. Both species exhibited low pollen viability. However, some viable pollen, reduced embryo sac formation, natural pollination and pollen tube growth enable sexual reproduction and characterize these species as facultative apomicts. The independence of pollinators for fruit set, uniparental reproduction and the possibility of sexual reproduction, confer reproductive assurance and flexibility, bringing together advantages of sexual and asexual reproduction. In this sense, apomixis may have played an important role in the evolution and diversification of Microlicia, a widely distributed genus in the Brazilian Cerrado.     

Evolutionary constraints on polyembryony in parasitic wasps: a simulation model

Polyembryony involves the production of several genetically identical progeny from a single egg through clonal division. Although polyembryonic development allows highly efficient reproduction, especially in some parasitoid wasps, it is far less common than monoembryony (development of one embryo per egg). To understand what might constrain the evolutionary success of polyembryony in parasitoids, we developed Monte Carlo models that simulate the competition between polyembryonic females and their monoembryonic counterparts. We investigated which simulated life‐history traits of the females allow the monoembryonic mode of development to succeed. Published empirical studies were surveyed to explore whether these traits indeed differ between polyembryonic parasitoids and related monoembryonic species. The simulations predict an advantage to monoembryony in parasitoids whose reproduction is limited by host availability rather than by egg supply, and that parasitize small‐bodied hosts. Comparative data on the parasitoid families Encyrtidae and (to a lesser extent) Braconidae, but not the data from Platygastridae, circumstantially support these predictions. The model also predicts monoembryony to outcompete polyembryony when: 1) hosts vary considerably in quality, 2) polyembryonic development carries high physiological costs, and 3) monoembryonic females make optimal clutch size decisions upon attacking hosts. These multiple constraints may account for the rarity of polyembryony among parasitoid species.     

Sporophytic apomixis in polyploid Anemopaegma species (Bignoniaceae) from central Brazil

Apomixis and polyploidy have been important in the evolution of the angiosperms, and sporophytic apomixis has been associated with polyembryony and polyploidy in tropical floras. We studied the occurrence of polyembryony in populations of tetraploid Anemopaegma acutifolium, A. arvense and A. glaucum from the Brazilian cerrados, and histological features of sexual and apomictic processes were investigated in A. acutifolium. All populations and species were polyembryonic (68.9–98.4% of seeds). Normal double fertilization occurred in most ovules, with exceptions being that 3% of ovules were penetrated but not fertilized and in 4% of ovules both synergids were penetrated. The penetration of both synergids suggests a continuous attraction of pollen tubes and polyspermy. Adventitious embryo precursor cells (AEPs) arose from nucellar and integumental cells of the ovule in pollinated and unpollinated A. acutifolium, indicating sporophytic apomixis. However, further embryo and endosperm development required pollination and fertilization. This pseudogamy also allows concurrent sexual embryo development. Similar polyembryony rates and polyploidy indicated that A. arvense and A. glaucum are also apomictic, forming an agamic complex similar to that observed for some species of confamilial, but not closely related Handroanthus. The co‐occurrence of apomixis and polyploidy in different groups of Bignoniaceae indicates homoplasious origin of these agamic complexes. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 77–91.     

Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae)

Background and Aims

Asexual organisms are more widespread in previously glaciated areas than their sexual relatives (‘geographical parthenogenesis’). In plants, this pattern is probably dependent on reproductive isolation and stability of cytotypes within their respective distribution areas. Both partial apomixis and introgressive hybridization potentially destabilize the spatial separation of sexual and apomictic populations. The wide distribution of apomicts may be further enhanced by uniparental reproduction which is advantageous for colonization. These factors are studied in the alpine species Ranunculus kuepferi.

Methods

Geographical distribution, diversity and mode of reproduction of cytotypes were assessed using flow cytometry and flow cytometric seed screening on samples from 59 natural populations of Ranunculus kuepferi. Seed set of cytotypes was compared in the wild.

Key Results

Diploid sexuals are confined to the south-western parts of the Alps, while tetraploid apomicts dominate in previously glaciated and in geographically isolated areas despite a significantly lower fertility. Other cytotypes (3x, 5x and 6x) occur mainly in the sympatric zone, but without establishing populations. The tetraploids are predominantly apomictic, but also show a partial apomixis via an uncoupling of apomeiosis and parthenogenesis in the seed material. Both pseudogamy and autonomous endosperm formation are observed which may enhance uniparental reproduction.

Conclusions

Diploids occupy a glacial relic area and resist introgression of apomixis, probably because of a significantly higher seed set. Among the polyploids, only apomictic tetraploids form stable populations; the other cytotypes arising from partial apomixis fail to establish, probably because of minority cytotype disadvantages. Tetraploid apomicts colonize previously devastated and also distant areas via long-distance dispersal, confirming Baker''s law of an advantage of uniparental reproduction. It is concluded that stability of cytotypes and of modes of reproduction are important factors for establishing a pattern of geographical parthenogenesis.     

Can obligate apomixis and more stable reproductive assurance explain the distributional successes of asexual triploids in Hieracium alpinum (Asteraceae)?

• Although reproductive assurance has been suggested to be one of the most important factors shaping the differential distributional patterns between sexuals and asexuals (geographic parthenogenesis), it has only rarely been studied in natural populations of vascular plants with autonomous apomixis. Moreover, there are almost no data concerning the putative relationship between the level of apomictic versus sexual plant reproduction on one hand, and reproductive assurance on the other.
• We assessed the level of sexual versus apomictic reproduction in diploid and triploid plants of Hieracium alpinum across its distributional range using flow cytometric analyses of seeds, and compared the level of potential and realized seed set, i.e. reproductive assurance, between the two cytotypes under field and greenhouse conditions.
• Flow cytometric screening of embryos and endosperms of more than 4,100 seeds showed that diploids produced solely diploid progeny sexually, while triploids produced triploid progeny by obligate apomixis. Potential fruit set was much the same in diploids and triploids from the field and the greenhouse experiment. While in the pollination‐limited environment in the greenhouse apomictic triploids had considerably higher realized fruit set than sexual diploids, there was no significant difference between cytotypes under natural conditions. In addition, sexuals varied to a significantly larger extent in realized fruit set than asexuals under both natural and greenhouse conditions.
• Our results indicate that triploid plants reproduce by obligate apomixis, assuring more stable and predictable fruit reproduction when compared to sexual diploids. This advantage could provide apomictic triploids with a superior colonisation ability, mirrored in a strong geographic parthenogenesis pattern observed in this species.

     

Asynchronous expression of duplicate genes in angiosperms may cause apomixis, bispory, tetraspory, and polyembryony

Apomicts that produce unreduced parthenogenetic eggs are generally polyploid and occur in at least 33 of 460 families of angiosperms. Embryo sacs of these apomicts form precociously from ameiotic megaspore mother cells (diplospory) or adjacent somatic cells (apospory). Polysporic species (bisporic and tetrasporic) are sexual and occur in at least 88 families. Their embryo sacs also form precociously, but only non-critical portions of meiosis are affected. It is hypothesized that (i) the partial to complete replacement of meiosis by embryo sac formation in apomictic and polysporic species results from asynchronously-expressed duplicate genes that control female development, (ii) duplicate genes result from polyploidy or paleopolyploidy (diploidized polyploidy with chromatin from multiple genomes), (iii) apomixis results from competition between nearly complete sets of asynchronously-expressed duplicate genes, and (iv) polyspory and polyembryony result from competition between incomplete sets of asynchronously-expressed duplicate genes. Phylogenetic and genomic studies were conducted to evaluate this hypothesis. Apomictic, polysporic, and polyembryonic species tended to occur together in cosmopolitan families in which temporal variation in female development is expected, apomicts were generally polyploid with few chromosomes per genome (X = 9.6pL0.4 SE), and polysporic and polyembryonic species were paleopolyploid with many chromosomes per genome (x= 15.7pL0.6 and 13.2pL0.4, respectively). These findings support the proposed duplicate-gene asynchrony hypothesis and further suggest asexual reproduction in apomicts preserves primary genomes, sexual reproduction in polysporic and polyembryonic polyploids accelerates paleopolyploidization, and pa-leopolyploidization may sometimes eliminate gene duplications required for apomixis while retaining duplications required for polyspory or polyembryony. Hence, apomixis, with its long-term reproductive stability, may occasionally serve as an evolutionary springboard in the evolution of normal and developmentally-novel paleopolyploid sexual species and genera.     

Morphological characteristics of leaf epidermis and size variation of leaf, flower and fruit in different ploidy levels in Buddleja macrostachya (Buddlejaceae)

Buddleja macrostachya (Buddlejaceae) is a widespread shrub native to the Sino-Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n=12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations of B. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length increased with an increase at ploidy level (P<0.01), whereas adaxial cell and stomatal density decreased with an increase at ploidy level (P<0.01). We found no conspicuous differences in relative leaf size (P>0.05) in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaploid from dodecaploid cytotypes either in the field or in herbarium specimens.     

Morphological differentiation within the Ranunculus cassubicus group compared to variation of isozymes, ploidy levels, and reproductive systems: implications for taxonomy

 In the partly apomictic Ranunculus cassubicus group, a subgroup of the R. auricomus complex, two species were studied by morphometric analyses: R. cassubicifolius W. Koch (with three diploid and two autotetraploid sexual populations), and R. carpaticola Soó (with three diploid sexual populations and a hexaploid apomictic one). Multidimensional scaling analyses (MDS) of individuals, boxplots and cluster analyses of populations revealed a differentiation of R. cassubicifolius and R. carpaticola, whereby in MDS the hexaploid apomictic individuals are partly intermediate between R. cassubicifolius and R. carpaticola. The cytodemes of R. cassubicifolius showed no morphological and only a weak genetic differentiation. A comparison of morphology, isozymes, reproductive system and ploidy levels showed only partly congruence of data sets in respect of grouping populations, thus illustrating the problem to find criteria for a taxonomic concept. A treatment of the apomictic population as a separate group is indicated by all data sets, afterwards R. cassubicifolius and diploid R. carpaticola represent two other well-defined groups. Canonical variate analysis including all characters confirmed the three suggested groups as significantly different and showed that a total of 89.3% of individuals are correctly classified; number of teeth of stem leaf segments and number of petals are the most discriminating characters. Herbarium studies confirm the morphological differentiation yielded from population samples. The three population groups are even better separated in a canonical variate analysis of isozyme data (presence/absence of 25 alleles) of the same material, here 92.6% of individuals are correctly classified. Morphology and isozyme data suggest that the hexaploid apomict originated from hybrids of R. cassubicifolius and diploid R. carpaticola and must be excluded from the sexual taxa; the final classification and naming of the apomicts must be left for further studies on a larger material. The sexual taxa should be classified as separate species. Herbarium studies indicate that R. carpaticola s.str. is widespread over the Carpathians and might include other populations hitherto ascribed to other microspecies as well. Received November 20, 2001; accepted May 10, 2002 Published online: September 13, 2002     

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     

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations ('clone mates') within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.     

Diploid and autotetraploid sexuals and their relationships to apomicts in the Ranunculus cassubicus group: insights from DNA content and isozyme variation

 From the predominantly aposporous Ranunculus cassubicus group, a subgroup of the R. auricomus complex, two species with both diploid and tetraploid cytodemes (R. cassubicifolius W. Koch and R. carpaticola Soó) were known. Nine population samples of both species have been analyzed for variation of ploidy levels and isozymes. DNA image analysis showed that three populations of R. cassubicifolius from Bavaria and Salzburg are diploid, two from Lower Austria tetraploid. In R. carpaticola, two populations from Central Slovakia and one from Romania are diploid, one from northern Slovakia is hexaploid. The polyploid populations had somewhat smaller C-values than expected from diploids. Ploidy levels are consistent within populations, contradicting previous hypotheses that cycles of diploid-tetra-ploid-dihaploid apomictic individuals occur in natural populations of goldilocks. Isozyme-allozyme analysis of nine polymorphic loci showed that individual variation and genetic measures of di-ploid and tetraploid populations are equivalent to those of sexual taxa. Multiple allelism and unbalanced gene dosages in tetraploid R. cassubicifolius give evidence for autopolyploidy that is most probably of multiple origin. The observed excess of homozygotes and the high diversity between populations in the diploid populations of R. cassubicifolius are most probably due to geographical isolation of population groups, that might have happened during the Würm glaciation and might have promoted the separation of autotetraploids in the easternmost part of the distribution range. Genetic distance values analyzed by UPGMA of all sexual populations separated the two taxa and the cytodemes within R. cassubicifolius. Multidimensional scaling of individuals (including the apomicts) based on a presence/absence matrix of alleles confirmed this differentiation with an overlapping zone between R. cassubicifolius and R. carpaticola. The hexaploid R. carpaticola population showed reduced genotypic diversity, an increased number of heterozygotes and fixed heterozygosity as typical for apomictic mode of reproduction. This population shared alleles of both species without any specific ones, multidimensional scaling placed genotypes among R. cassubicifolius. Thus, the hexa-ploid apomicts might have originated from hybrids of R. cassubicifolius and R. carpaticola. In general, evolution of agamic lineages in the R. cassubicus complex might have been facilitated by autopoly-ploids, because they can provide a bridge between the reproductive systems that are otherwise isolated by ploidy levels, and also a starting point for spontaneous origin of apomixis. Received October 8, 2001; accepted May 10, 2002 Published online: November 7, 2002 Addresses of the authors: Elvia H?randl (E-mail: elvira.hoerandl@univie.ac.at), Department of Systematics and Evolution of Higher Plants; Johann Greilhuber (E-mail: Johann.Greilhuber@univie. ac.at), Department of Systematic Karyology and Embryology of Plants; both: Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.