Clonal Propagules Research Articles

Clonal diversity and spatial genetic structure of Potamogeton pectinatus in managed pond and river populations

Higher levels of genetic diversity of river macrophytes are expected in downstream parts because of potential accumulation of various genotypes from upstream sites. We assessed the clonal diversity and spatial genetic structure of fennel pondweed (Potamogeton pectinatus or Stuckenia pectinata) populations with emphasis on the estimation of dispersal via clonal propagules along a river in connection to upstream ponds. We analysed genetic diversity of 354 plant shoots sampled in 2005 and 2006 at three pond and five river sites in the Woluwe river catchment (Belgium). Nine microsatellite DNA markers revealed 88 genets of which 89% occurred in only one site. Clonal propagule dispersal was detected up to 10 km along the river. Few multilocus genotypes were repeatedly present along a major part of the river indicating vegetative spread. Populations of ponds contained a higher amount of clonal diversity, indicating the importance of local seed recruitment. A fine-scaled spatial genetic structure indicated that most seedling recruitment occurred at a distance <5 m in pond populations whereas clones in river sites were unrelated and showed no spatial autocorrelation. The clonal diversity decreased along the river from upstream to downstream due to establishment of few large clones.

Trade‐offs between clonal and sexual reproduction in Sagittaria latifolia (Alismataceae) scale up to affect the fitness of entire clones

Many plants combine sexual reproduction with vegetative propagation, but how trade-offs between these reproductive modes affect fitness is poorly understood. Although such trade-offs have been demonstrated at the level of individual shoots (ramets), there is little evidence that they scale up to affect genet fitness. For hermaphrodites, reproductive investment is further divided between female and male sexual functions. Female function should generally incur greater carbon costs than male function, which might involve greater nitrogen (N) costs. Using a common garden experiment with diclinous, clonal Sagittaria latifolia we manipulated investment in reproduction through female and male sex functions of 412 plants from monoecious and dioecious populations. We detected a 1:1 trade-off between biomass investment in female function and clonal reproduction. For male function, there was no apparent trade-off between clonal and sexual reproduction in terms of biomass investment. Instead, male function incurred a substantially higher N cost. Our results indicate that: trade-offs between investment in clonal propagation and sexual reproduction occur at the genet level in S. latifolia; and sexual reproduction interferes with clonal expansion, with investment in female function limiting the quantity of clonal propagules produced, and investment in male function limiting the nutrient content of clonal propagules.

Novel micropropagation and weaning methods for the integrated conservation of a critically endangered tree species, Medusagyne oppositifolia

Medusagyne oppositifolia is a critically endangered monotypic species with only a few trees left in the wild. Due to invasion and habitat clearance, genetic diversity of this species has been reduced to an alarmingly low level. Long-term in vitro cultures of M. oppositifolia were slow-growing and eventually lost the ability to multiply and root. Because of the lack of new seed and vegetative materials, efforts were made to rejuvenate the existing cultures. This was attempted by a series of steps involving serial pruning of the shoot tips and growing on a modified MS medium containing silver nitrate and thidiazuron. Rapid cycling clonal propagules were produced this way from cultured nodes. Isolated shoots were rooted on Florialite™, and rooted plants were weaned in a transition stage using a Nutrient Film Technique (NFT) hydroponic system before being completely weaned under glasshouse conditions. The importance of screening cultures for multiplication and rooting efficiency during long-term maintenance is discussed in the context of stress metabolite production and its influence on growth in culture. Factors contributing to rejuvenation of old cultures, application of unconventional supporting systems for rooting and application of the hydroponic system as a weaning tool are also discussed.

The evolutionary ecology of clonally propagated domesticated plants

While seed-propagated crops have contributed many evolutionary insights, evolutionary biologists have often neglected clonally propagated crops. We argue that widespread notions about their evolution under domestication are oversimplified, and that they offer rich material for evolutionary studies. The diversity of their wild ancestors, the diverse ecologies of the crop populations themselves, and the intricate mix of selection pressures, acting not only on the parts harvested but also on the parts used by humans to make clonal propagules, result in complex and diverse evolutionary trajectories under domestication. We examine why farmers propagate some plants clonally, and discuss the evolutionary dynamics of sexual reproduction in clonal crops. We explore how their mixed clonal/sexual reproductive systems function, based on the sole example studied in detail, cassava (Manihot esculenta). Biotechnology is now expanding the number of clonal crops, continuing the 10 000-yr-old trend to increase crop yields by propagating elite genotypes. In an era of rapid global change, it is more important than ever to understand how the adaptive potential of clonal crops can be maintained. A key component of strategies for preserving this adaptive potential is the maintenance of mixed clonal/sexual systems, which can be achieved by encouraging and valuing farmer knowledge about the sexual reproductive biology of their clonal crops.

The production of sexual and asexual larvae within single broods of the scleractinian coral, Pocillopora damicornis

The genotypic compositions of two populations of the brooding coral, Pocillopora damicornis, were studied by exhaustive sampling of adult colonies on a fine scale in southern Taiwan. In addition, 100 larvae were randomly selected among more than 1,000 brooded larvae collected from a single broodparent within each population. Using 7 polymorphic microsatellite markers, both populations were found to be highly clonal, and 7 clonal lineages were characterized. One clonal lineage (C1) dominated both study areas and comprises 54.9% of all colonies sampled, while any of the other 6 clonal lineages represented no more than 5%. Among the 100 larvae randomly selected for genotyping from each broodparent, the extent of clonal reproduction was high, and only 29 and 6 larvae, respectively, were found to be genotypically different from their broodparent. Among the 35 genotypically distinct larvae, 33 were thought to be derived from sexual reproduction, and 2 were assumed to be clonal propagules that had undergone somatic mutations. Two genotypically identical larvae were also found in one of the 2 sexually derived larva arrays, indicating the possible existence of polyembryony. The high proportions of clonality in both adult colonies and brooded larvae suggest that asexually produced larvae might significantly contribute to the recruitment of local populations. The dense clonal population of P. damicornis in the study area favors the quick recolonization view of clonal propagules after disturbances.

Influence of potential growth factors on the production of proembryogenic masses of Cyclamen persicum Mill. in bioreactors

Propagation in liquid culture, especially bioreactors, is one possible way to produce clonal propagules of Cyclamen persicum Mill. at a low cost. The current propagation method for C. persicum is from expensive hybrid seeds. This paper presents models of the potential effects of oxygen concentration, daily mean temperature, the difference between day and night temperature (DIF), and daily light integral on the development of proembryogenic masses of C. persicum Mill. in bioreactors. Each of the four growth factors was observed at three levels; oxygen concentration (50, 100 and 150% of fully oxygen saturated medium without cells), daily mean temperature (15, 20 and 25 °C), DIF (+10, 0 and −10) and daily light integral (0, 1.3 and 2.6 mol m −2 day −1). Two response variables, biomass growth and cell viability, were measured at day 0, 7, 14, 21 and 28 after start-up. The optimal values for biomass growth were 150% oxygen, 25 °C, 1.11 mol m −2 day −1 and DIF + 10. There was a stable positive linear effect from temperature. Oxygen showed a similar, but less stable effect. DIF gave maximum effect at the outer levels, and its optimum was the upper level +10. The optimal values for light were between 1.03 and 1.19 mol m −2 day −1. The optimal time for cell viability in the bioreactors was 10–11 days. Temperature and daily light integral had stable optima of 20.8 °C and 1.10 mol m −2 day −1. For oxygen the optimum was more unstable, but in most cases it was below 100%. There was no significant effect of DIF on cell viability.

The effectiveness of somatic embryogenesis in eliminating the cocoa swollen shoot virus from infected cocoa trees

Investigations were undertaken on the use of somatic embryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis.

Modelling forest recolonization by an epiphytic lichen using a landscape genetic approach

The process of recolonization after disturbance is crucial for the persistence and dynamics of patch-tracking metapopulations. We developed a model to compare the spatial distribution and spatial genetic structure of the epiphytic lichen Lobaria pulmonaria within the perimeter of two reconstructed 19th century disturbances with a nearby reference area without stand-level disturbance. Population genetic data suggested that after stand-replacing disturbance, each plot was colonized by one or a few genotypes only, which subsequently spread clonally within a local neighborhood. The model (cellular automaton) aimed at testing the validity of this interpretation and at assessing the relative importance of local dispersal of clonal propagules vs. long-distance dispersal of clonal and/or sexual diaspores. A reasonable model fit was reached for the empirical data on host tree distribution, lichen distribution, and tree- and plot-level genotype diversity of the lichen in the reference area. Although model calibration suggested a predominance of local dispersal of clonal propagules, a substantial contribution of immigration of non-local genotypes by long-distance dispersal was needed to reach the observed levels of genotype diversity. The model could not fully explain the high degree of clonality after stand-replacing disturbance, suggesting that the dispersal process itself may not be stationary but depend on the ecological conditions related to disturbance.

Effect of disturbances on the genetic diversity of an old‐forest associated lichen

Lichens associated with old forest are commonly assumed to be negatively affected by tree logging or natural disturbances. However, in this study performed in a spruce-dominated sylvopastoral landscape in the Swiss Jura Mountains, we found that genetic diversity of the epiphytic old-forest lichen Lobaria pulmonaria depends on the type of disturbance. We collected 923 thalli from 41 sampling plots of 1 ha corresponding to the categories stand-replacing disturbance (burnt), intensive logging (logged) and uneven-aged forestry (uneven-aged), and analysed the thalli at six mycobiont-specific microsatellite loci. We found evidence for multiple independent immigrations into demes located in burnt and logged areas. Using spatial autocorrelation methods, the spatial scale of the genetic structure caused by the clonal and recombinant component of genetic variation was determined. Spatial autocorrelation of genotype diversity was strong at short distances up to 50 m in logged demes, up to 100 m in uneven-aged demes, with the strongest autocorrelation up to 150 m for burnt demes. The spatial autocorrelation was predominantly attributed to clonal dispersal of vegetative propagules. After accounting for the clonal component, we did not find significant spatial autocorrelation in gene diversity. This pattern may indicate low dispersal ranges of clonal propagules, but random dispersal of sexual ascospores. Genetic diversity was highest in logged demes, and lowest in burnt demes. Our results suggest that genetic diversity of epiphytic lichen demes may not necessarily be impacted by stand-level disturbances for extended time periods.

Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae)

The effects of competition on the genetic composition of natural populations are not well understood. We combined demography and molecular genetics to study how intraspecific competition affects microevolution in cohorts of volunteer plants of cassava (Manihot esculenta) originating from seeds in slash-and-burn fields of Palikur Amerindians in French Guiana. In this clonally propagated crop, genotypic diversity is enhanced by the incorporation of volunteer plants into farmers' stocks of clonal propagules. Mortality of volunteer plants was density-dependent. Furthermore, the size asymmetry of intraspecific competition increased with local clustering of plants. Size of plants was correlated with their multilocus heterozygosity, and stronger size-dependence of survival in clusters of plants, compared with solitary plants, increased the magnitude of inbreeding depression when competition was severe. The density-dependence of inbreeding depression of volunteer plants helps explain the high heterozygosity of volunteers that survive to harvest time and thus become candidates for clonal propagation. This effect could help favour the maintenance of sex in this 'vegetatively' propagated crop plant.

Internal brooding of clonal propagules by a sea anemone, Anthopleura sp.

Abstract. We investigated the seasonal prevalence of reproductive activities and of the development of brooded propagules in an intertidal sea anemone, Anthopleura sp., on the rocky shore of Mutsu Bay, in northern Japan. A monthly examination of anemones, by dissection and histological techniques, revealed no sign of gonad development, but did reveal that they produce and internally brood propagules throughout the year. Release of propagules was observed in the field. This anemone population appears to be entirely asexual and agametic, and may persist solely through clonal propagation.

Branch propagation, not cladoptosis, permits dispersive, clonal reproduction of riparian cottonwoods

In riparian (floodplain) zones, physical disturbances of floods and ice-scour undercut and topple shrubs and trees into the river where branches are sheared off by tumbling and turbulence. These branch fragments are deposited downstream along bars with sediments that retain moisture and promote adventitious rooting to produce vigorous clonal saplings. We observed such ‘branch propagation’ along rivers of western North America for Tacamahaca cottonwoods, being common for P. balsamifera (balsam poplar) and P. balsamifera subsp. trichocarpa (black cottonwood) but less common for Populus angustifolia (narrowleaf cottonwood). It was rare for the Aigeiros cottonwoods, P. deltoides (prairie cottonwood) or P. fremontii (Fremont cottonwood). Branch propagation was sparse downstream from dams due to the trapping of branch fragments and sediments by the reservoirs combined with unfavorable patterns of flow regulation. Conversely, branch propagation was abundant after major floods such as for black cottonwoods along the Elk River, British Columbia, following the flood-of-record in 1995. In that case, excavation of saplings from branch fragments in 1997 revealed that 91% originated from sheared branches, 7% from beaver browse and only 1% included the basal enlargement characteristic of physiological abscission. Consistent with this minimal representation, only 3% of abscised branches from narrowleaf, prairie and black cottonwoods successfully propagated under favorable greenhouse conditions in moist sand, in contrast to 96% success of winter shoot cuttings from these species. These studies indicate that branch propagation enables dispersive clonal reproduction for riparian cottonwoods. It provides vigorous saplings and is promoted by geomorphic disturbance and beaver browse. These observations reject the concept of ‘cladoptosis’ which proposed that physiologically abscised cottonwood branches commonly serve as clonal propagules.

Evaluation of Argentine and Peruvian Prosopis germplasm for growth at seawater salinities

The objectives of this study were (a) to identify individual plants of Prosopis alba and P. pallida that could grow at near seawater salinities to use directly as clonal propagules and to establish seed orchard and (b) to analyse the population structure to aid in future germplasm collections for salinity tolerance in Prosopis. This hydroponic greenhouse study compared the growth and survival of 27 Prosopis families (seed from single mother trees) as a function of salinity from 10 to 45 dS m −1. Nine P. alba families from one saline area (provenance) near Santiago del Estero, Argentina were compared to 14 P. pallida families from six provenances in Peru and to one family each of P. alba/ flexuosa from Chile, P. juliflora from Senegal, P. flexuosa from Argentina and P. ruscifolia from Argentina. The mean of the P. pallida families had a significantly greater survival (61.1% vs. 41.7%) and percentage of seedlings that grew (37.4% vs. 23%) at seawater salinities of 45 dS m −1 than P. alba. Families from P. pallida had the greatest mean growth at the highest salinity but P. alba families were ranked third and sixth from the top in growth. There were little differences in maximum height growth of the individual seedlings from these two species. For P. alba, the percentage of seedlings which survived at the highest salinity level had a lower correlation ( r 2=0.46) with the number of seedlings which grew at that salinity level than for P. pallida ( r 2=0.70). The correlation between mean family growth and maximum individual growth was lower for P. alba ( r 2=0.24) than for P. pallida ( r 2=0.49) suggesting that within P. alba, with a sufficiently high number of seedlings, it would be possible to find individuals with rapid growth rates in most families. Approximately 50 of the most rapidly growing seedlings at the highest salinity level have been repotted to be multiplied by rooting of cuttings and to serve as seed orchards for salt tolerance. Given the success in identifying rapidly growing P. alba individuals from only one provenance in Argentina, more extensive germplasm collections and testing are warranted to locate individuals with superior growth rates. Due to the previously demonstrated erect form, high biomass productivity and sweet pods in these P. pallida families in Haiti, Cape Verde, India and Peru, extensive recollections and field testing of P. pallida are warranted.

Resistance to locust leafminer (Coleoptera: Chrysomelidae) in black locust.

Locust leafminer, Odontota dorsalis (Thunberg), activity was observed for 2 yr on 70 black locust, Robinia pseudoacacia L., trees grown from seed collected from its native range. Four black locust seedlings from Tennessee had low numbers of eggs, larvae, pupae, and mines, and were selected for clonal propagation. These clonal propagules were resistant to locust leafminers collected from three different counties in Maryland. This resistance was also observed in controlled choice and no-choice tests in outdoor screen houses. Adults showed different ovipositional preferences for black locust trees from different seed sources, which were reflected in lower numbers of larvae, pupae, and mines in resistant clones. It was concluded that antixenosis and antibiosis were involved in the mechanisms of resistance to the locust leafminer in black locust.

Comparison of Seedling and Cuttings for Evaluating Winter Hardiness in Alfalfa

Studies of the genetic control of winter hardiness (WH) in alfalfa (Medicago sativa L.) require the replication of specific genotypes in the field. Clonal propagules would provide worthwhile information in replicated trials if seedlings and cuttings give similar genotype rankings. In this study, we compared seedlings with genetically identical cuttings for WH, fall dormancy (FD),and freezing tolerance (FT), and evaluated the relationships among these traits. Seventy‐two plants each from 15 genotypes, covering a broad range of FD, were clonally propagated and transplanted to the field as seedlings and cuttings in a randomized complete block design. For all traits, genotype was a highly significant (P &lt; 0.001) source of variation, but the genotype × establishment method (seedlings vs. cuttings) interaction was not significant (P &gt; 0.05). Winter survival was significantly correlated with freezing sensitivity (r = −0.92, P &lt; 0.01) and FD (r = −0.66). Our results suggest that alfalfa plants established from cuttings will faithfully represent seedling‐established plants for the purpose of evaluating winter hardiness.

Isozyme and quantitative traits polymorphisms in European provenances of perennial ryegrass (Lolium perenne L)

Twenty populations of Lolium perenne originating from a range of habitats in Europe were compared for isoenzyme polymorphisms and agronomically important quantitative traits in order to establish relationships of the levels of diversity with the origin of each population and to assess their suitability to be included in the European core collection of Lolium germplasm. Forty genotypes from each of the twenty populations and each genotype represented by three clonal propagules, were field planted in a fully randomized spaced plant design and fifteen quantitative characters including yield, persistency, reproductive and disease resistence characters were evaluated over a period of two years. Seven putative isozyme loci were assayed to compare the allozyme divergence of populations. The results of the isozyme survey indicate that 71–100% of the loci were polymorphic, 2.3–3.0 alleles/locus and the gene diversity was varying from 0.234–0.410. Of the total allelic diversity 94% remained within populations (Hs) whilst only 6% was distributed among populations (Dst). The differences between populations were determined on the basis of allele frequencies and multivariate analyses of quantitative characters. Populations significantly differed in their allele frequencies at all loci analysed. Random mating was predominant in all populations at most of the loci. The study revealed that the German accession BA 10998 was clearly distinct from the rest both in quantitative characters and allele frequencies. German accession BA 11015 with the lowest gene diversity showed the highest genetic variation for quantitative characters. However, no strict relationship was found between the genetic distance and the geograpical distribution of the populations. Among the quantitative characters, flowering time showed a strong relationship with the type of management practised at the collection site than their place of origin.

Variation in white clover for root‐knot nematode resistance

Abstract Root‐knot nematode (Meloidogyne sp.) reduces growth and nutrition of white clover (Trifolium repens L.). Breeding resistant cultivars is the preferred control method. A range of overseas and New Zealand white clover lines was screened for resistance, measured as a reduction in the number of galls per root size. Highly significant differences between white clover lines were found, although no plants were immune. There was variation in both gall numbers and root size, and root size was a major factor determining the galls/ root ratio. The most resistant genotypes generally were from larger‐leaved lines, whereas the most suceptible genotypes generally were from smaller‐leaved lines. Resistant genotypes (and some susceptible genotypes) were re‐screened, some twice, using five clonal propagules per genotype. While there were significant correlations between the re‐screens for galls, roots, and galls/root, the values showed that repeatability was low. Comparisons with other nematode and clover species ...

組織培養によるフキ（Ｐｅｔａｓｉｔｅｓ ｊａｐｏｎｉｃｕｓ Ｆｒ．Ｓｃｈｍｉｄｔ）ウイルスフリー株の大量増殖

The vegetative propagation rate of Japanese butterbur plants is low, it often permits four viruses to be transmitted to the next generation through the rhizomes as in other vegetatively propagated crops. All of the butterbur plants in the field are eventually inflicted with viruses. The purpose of this study is to establish an efficient masspropagation system of virus-free plants by tissue culture.Shoots from flower-heads were induced on Murashige and Skoog's (MS) solid medium containing 0.1 mg•liter-1 NAA and 1.0mg•liter-1 BA. After 12 weeks of culture, 5.2 shoots per explant were obtained from 80% of the flower-heads.Rapid clonal propagules were obtained when 2-cm shoots were transferred to MS liquid medium supplemented with 0.01mg•liter-1 NAA and 3.0mg•liter-1 BA and placed on a gyrating, rotary shaker set at 100 rpm. After 8 weeks of culture, 80.9 shoots from axillary buds were obtained. These shoots rooted remarkably well in half-strength B 5 liquid medium without phytohormones while being gyrated.All regenerated plants became acclimated readily when grown in a sandy loam at 25°C.Although no cucumber mosaic virus (CMV) or butterbur mosaic virus (BuMV) was detected in the plants, 8.1% were infected with arabis mosaic virus (ArMV). Thus, 91.9% of the regenerated plants were virus-free.These results indicate that this micropropagation system of using flower-heads and axillary buds seems adaptable for a large-scale system of supplying virus-free Japanese butterbur plants.

COMPARISON OF ROOTS OF THREE MICROPROPAGATED CLONES OF PRUNUS SEROTINA VAR. VIRENS GROWN IN TWO SIZES OF CONTAINERS USING IMAGE ANALYSIS

Three different lines of Chisos cherry obtained from trees with profuse or scant root systems were grown in either 2 or 3 liter containers to determine if the effects of container volume could be detected as early as 12 weeks. The root characteristics (profuse or scant root development) seen in the parent material were not shared by the clonal propagules from those lines. There was a significant effect of the clone on surface area of fine roots, coarse to fine root ratio, and root dry weight (P&lt;0.05) and on surface area of coarse roots (P&lt;0.1). The smaller container reduced root dry weight significantly (P&lt;0.1) and coarse to fine root ratio (P&lt;0.05). Using an image capture and analysis system (ICAS) to classify and quantify the roots of the microcuttings, an effect of container volume could be detected even though the experiment was terminated only 3 weeks after roots had encountered the container walls and began exhibiting a coiling response.

The adaptive significance of clonal reproduction in angiosperms: an aquatic perspective

Traditionally, clonal reproduction has been viewed as an alternative to sexual reproduction and adaptive explanations have sought to compare both modes of ‘reproduction’. General theoretical discussions have included a very narrow range of the variation in forms of clonal reproduction and have tended to exclude many of the more common forms amongst aquatic angiosperms. More recent attempts to explain the adaptive significance of cloning in plants have focused on the dichotomy of compact versus diffusely spreading types and are still too narrow to provide a broad understanding. In this paper the case for the existence of six major categories of selective forces operating on clonal propagules is presented: numerical increase, dispersal, resource acquisition, storage, protection, and anchorage. It is shown that different methods of clonal reproduction differ in terms of these six characteristics. Further, there exists a partial correlation between the syndrome of functional attributes and the habitat relations for a given method of clonal reproduction. In order to develop a more preproductive relationship between plant form and habitat affinities it will be necessary to consider the entire growth form and life history. In doing this it will be necessary to recognize the numerous functions performed during clonal reproduction.