AN EVALUATION OF METHODS USED TO ASSESS INTERGENERIC HYBRIDIZATION IN KELP USING PACIFIC LAMINARIALES (PHAEOPHYCEAE)1

Kelp intergeneric laminarialean hybridizations and hybridization protocol were assessed using seven northeast Pacific kelp species: Alaria marginata Postels and Ruprecht, Costaria costata (C. A. Agardh) Saunders, Eisenia arborea Areschoug, Laminaria saccharina (L) Lamouroux, Lessoniopsis littoralis (Tilden) Reinke, Macrocystis integrifolia Bory, and Nereocystis leutkeana (Mertens) Postels and Ruprecht. Survival and development of sporophyte morphologies derived from selfings, separate males and females, and reciprocal crosses were evaluated over 30 weeks of cultivation. All cultures were initiated from cloned gametophytes. Two closely related species, Laminaria angustata Kjellman and L. japonica Areschoug, demonstrated the efficacy of long‐term (up to 30 years) cloned gametophytes in hybridization studies. Sporophyte morphologies appeared in 34%–69% of control and hybridization trials, and 6%–16% of all trials produced sporophytes in control and hybridization conditions that persisted through 30 weeks of cultivation. Sporophytes in control and hybridization conditions could appear normal or abnormal. Usually, the morphology of sporophytes in hybridizations and female controls resembled the female parent, whereas the sporophytes in male controls often had an abbreviated morphology, lacking definitive generic features. Species‐specific rDNA internal transcribed spacer molecular primers were used to determine the parentage of five putative hybrids. Only the L. japonica♀/L. angustata♂ hybrid bore both parental genomes. That negative controls could produce persistent and normal‐appearing sporophytes negates their value and emphasizes the importance of molecular confirmation in hybridization studies. These findings were applied to critique the only known wild intergeneric hybrid, Pelagophycus/Macrocystis.     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between <Emphasis Type="Italic">Pinus sylvestris</Emphasis> and <Emphasis Type="Italic">Pinus mugo</Emphasis> and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.     

GENETIC STRUCTURE VARIABILITY OF KELP ALARIA MARGINATA OVER SPACE AND TIME

The genetic variability of Alaria marginata Postels & Ruprecht was investigated spatially and seasonally using the finger printing technique of amplified fragment length polymorphism (AFLP). Using 206 scoreable bands generated by one primer pair, individual plants that were separated by as little as a few decimeters to> 100 km could be distinguished, and followed an isolation-by-distance model. Genetic similarity ranged from 76% for patches (a few decimeters in diameter), to 71% for individual kelp stands (15 m across) and 67% for a group of stands separated by 185 km. Greater genetic similarity of patches occurred at the wave-sheltered site than at wave-exposed site. The influence of wave to genetic variability and the ability to predict gene flow on small stretch of beach were discussed. In one stand, genetic similarities were markedly different between seasons. This seasonal pattern may be the result of different age groups dominating the sampled stands over time. The genetic structure of A. marginata comprises local scale (patch and within stand) heterogeneity and larger scale (between stands) homogeneity.     

The cost of realized sexual reproduction: assessing patterns of reproductive allocation and sporophyte abortion in a desert moss

The desert moss Syntrichia caninervis exhibits one of the most skewed sex ratios in the plant kingdom, with female individuals far outnumbering male individuals (exceeding 14♀:1♂). The "cost of sex hypothesis" derives from allocational theory and predicts that the sex which is most expensive should be the rarer sex. This hypothesis, which, as considered here represents the realized cost of sexual reproduction, is contingent upon two assumptions that are explored: (1) that male sex expression is more expensive than female sex expression, and (2) that sexual reproduction is resource limited. Using inflorescence biomass and discounting sperm, male sex expression was found to be in the neighborhood of one order of magnitude more expensive than female sex expression, and this difference is reflected in higher numbers of gametangia per male inflorescence, presence of paraphyses in male inflorescences, and a much longer developmental time for male inflorescences. The realized cost of female reproduction from two communities dominated by S. caninervis was found to be lower than the realized cost of male sexual reproduction. Resource-limited reproduction was assessed by determining the frequency of sporophyte abortion, the age distribution of sporophyte abortions, and patterns of sporophyte abortion that may be density dependent. Among ten sexually reproducing populations, abortive sporophytes occurred at a frequency of 0.64. Abortive sporophytes averaged 8% the mass of mature sporophytes, and cohort sporophytes from the same individual female were found to abort in a density-dependent pattern. We conclude that the two assumptions, upon which the cost of sex hypothesis depends, are supported.     

IAA-induced apogamy in Platycerium coronarium (Koenig) Desv. gametophytes cultured in vitro

Apogamous sporophytes were produced on Platycerium coronarium gametophytes cultured in the presence of indole-3-acetic acid (IAA). The percentage of apogamy as well as the total number of apogamous sporophytes produced per gametophyte clump were highest in the presence of 40 M IAA. When ethylene was allowed to accumulate in the culture vessel in the presence of an optimum level of IAA, the percentage and total number of apogamous sporophyte production decreased significantly. Using light microscope and confocal laser scanning microscope we have shown that nuclear size can be used as a quick parameter to estimate the ploidy level of P. coronarium.Abbreviations CLSM confocal laser scanning microscope - IAA Indole-3-acetic acid - MS Murashige and Skoog     

Enhancement of Apospory in Liquid Culture of Matteuccia struthioptens

Apospory in the fern Matteuccia struthiopteris, which is reportedhere for the first time, was induced more readily in liquidculture than on agar medium. It occurred more frequently fromdetached meristems than from juvenile leaves. Gametophytic outgrowthsbearing rhizoids, but lacking sex organs were found occasionallyon roots. Aposporous gametophytes isolated from liquid culturewere induced to form sporophytes either sexually or apogamously.Both types of sporophyte also behaved aposporously in liquidculture. Matteuccia, fern, apospory, apogamy, liquid culture, detached meristem     

Hybridization involving independent gametophytes in the Vandenboschia radicans complex (Hymenophyllaceae): a new perspective on the distribution of fern hybrids

To test our hypothesis of hybrid formation involving the ‘independent gametophyte’ phenomenon in ferns, we identified the genomic formulae and ploidy level of gametophytes of the Vandenboschia radicans complex at the periphery of a sporophyte population. We identified haploid gametophytes of V. kalamocarpa (one of the two putative parents of V.×stenosiphon) in a hybrid sporophyte population in Japan that lacks fertile non‐hybrid individuals. Furthermore, diploid sporophytes of the species were not found within a 50‐km radius. This finding supports a hypothesis of hybridization involving the ‘independent gametophyte’ phenomenon and provides a new perspective on the geographical distribution of fern hybrids.     

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500–1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

A method to establish an “immortalized F2” sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae)

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an “immortalized F₂” (IF₂) population of Undaria pinnatifida. Doubled-haploid “female” and “male” sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a “monoicous” gametophyte clone (originally male), respectively, and they were used as the parents. The F₁ hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F₂ gametophyte clones, consisting of 260 females and 260 males, were established from an F₁ hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF₂ population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF₂ population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.     

Correlation in the tolerance to ozone between sporophytes and male gametophytes of several fruit and nut tree species (Rosaceae)

We analyzed relative sensitivities to ozone partial pressure for male gametophytes (pollen and pollen tubes) of five tree-crop species in the family Rosaceae (almond, apple, apricot, nectarine/peach, pear) and of four cultivars of almond. Relative sensitivities to ozone partial pressure were previously described using field-based, whole-plant, physiological measurements for the sporophytes of each of the species and cultivars used in this study. Our objective was to determine the extent of correlation between sporophyte and gametophyte responses to ozone among and within these tree species. If the relative sensitivities prove to be similar for the two generations, two research strategies become possible for breeders interested in developing new cultivars with improved tolerance to atmospheric ozone: First, because screening large numbers of sporophytes for their responses to ozone partial pressure is a formidable task, exploiting the much more easily manipulated male gametophyte generation for large-scale screening of individuals for ozone tolerance could greatly facilitate identification and selection of tolerant genotypes. Second, it opens the possibility of applying selection pressure to the gametophyte generation (as during pollen germination or pollen tube growth) to favor production of ozonetolerant progeny. Results indicate that, among and within species, relative responses of pollen tube growth to ozone partial pressure are highly correlated with that of the sporophytes. Pollen germination was inhibited by ozone treatments for all but one species (pear), but differences among and within species were neither significant nor (with the exception of pear) did they correlate with relative sporophyte sensitivities.     

NUCLEOTIDE SEQUENCES OF THE SMALL-SUBUNIT RIBOSOMAL RNA GENES FROM SELECTED LAMINARIALES (PHAEOPHYTA): IMPLICATIONS FOR KELP EVOLUTION1

The entire small-subunit (SSU) ribosomal ribonucleic acid sequence was inferred for kelp representing seven genera: Alaria marginata Postels and Ruprecht (1824 bp), Egregia menziesii (Turner) Areschoug (1825 bp), Lessoniopsis littoralis (Tilden) Reinke (1825 bp), Macrocystis integrifolia Bory (1825 bp), Nereocystis leutkeana (Mertens) Postels and Ruprecht (1824 bp), Postelsia palmaeformis Ruprecht (1826 bp), and Pterygophora californica Ruprecht (1825 bp). We obtained a partial sequence for Eisenia arborea Areschoug (1669 bp) from a single clone of polymerase chain reaction-amplified product. The SSU sequence was too conserved among these morphologically distinct taxa to permit phylogenetic analysis. The divergence between the most distant taxa was only 0.66%. This value was used in a SSU molecular clock to suggest that the most distantly related kelp investigated in this study diverged between 16 and 30 (more probably 16 and 20) million years ago.     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

Mate limited reproductive success in two dioicous mosses

We tested whether mate availability in the pleurocarpous dioicous mosses Rhytidiadelphus triquetrus and Abietinella abietina affects fertilization success by transplanting individual male shoots into non-sporophytic female colonies. Fertilization success in both species was limited by the availability of mates and distance-dependent, and the number of sporophytes produced was higher in R. triquetrus than in A. abietina . Our estimate of a maximum fertilization distance of 34 cm in R. triquetrus is an order of magnitude larger than previous estimates that have been based on non-manipulative approaches. In R. triquetrus the number of female reproductive branches and plot inclination (horizontal vs sloping) had significant effects on the number of sporophytes produced. In A. abietina , the number of female reproductive branches per plot did not affect sporophyte production. Within sloping experimental plots, sporophyte number was significantly higher, and the distance of sporophytes from the spermatozoid source was larger below the male transplants than above. We conclude that mate availability may be important for reproductive success in sessile dioecious organisms, and that the spatial structure at various scales is essential to understand their dynamics.     

Effects of temperature and nutrients on microscopic stages of the bull kelp (Nereocystis luetkeana,Phaeophyceae)

Warming ocean temperatures have been linked to kelp forest declines worldwide, and elevated temperatures can act synergistically with other local stressors to exacerbate kelp loss. The bull kelp Nereocystis luetkeana is the primary canopy-forming kelp species in the Salish Sea, where it is declining in areas with elevated summer water temperatures and low nutrient concentrations. To determine the interactive effects of these two stressors on microscopic stages of N. luetkeana, we cultured gametophytes and microscopic sporophytes from seven different Salish Sea populations across seven different temperatures (10–22°C) and two nitrogen concentrations. The thermal tolerance of microscopic gametophytes and sporophytes was similar across populations, and high temperatures were more stressful than low nitrogen levels. Additional nitrogen did not improve gametophyte or sporophyte survival at high temperatures. Gametophyte densities were highest between 10 and 16°C and declined sharply at 18°C, and temperatures of 20 and 22°C were lethal. The window for successful sporophyte production was narrower, peaking at 10–14°C. Across all populations, the warmest temperature at which sporophytes were produced was 16 or 18°C, but sporophyte densities were 78% lower at 16°C and 95% lower at 18°C compared to cooler temperatures. In the field, bottom temperatures revealed that the thermal limits of gametophyte growth (18°C) and sporophyte production (16–18°C) were reached during the summer at multiple sites. Prolonged exposure of bull kelp gametophytes to temperatures of 16°C and above could limit reproduction, and therefore recruitment, of adult kelp sporophytes.     

Arbuscular mycorrhiza and water and nutrient supply differently impact seedling performance of dry woodland species with different acquisition strategies

Background: We recently demonstrated that dwarf male plants are much more common among mosses than hitherto thought. Dwarf plants, producing functional male sexual organs occur in 10–20% of the moss species worldwide.

Aims: We investigated how an inadvertent omission of dwarf males affects estimates of male sex expression rates and sex ratios.

Methods: We recorded frequencies of specimens with female-expressing plants, male-expressing normal-sized and/or dwarf plants, and sporophytes in herbarium material of five model species from Macaronesia. We analysed how frequency estimates of male occurrence and sex ratios were affected by excluding dwarf males.

Results: Occurrence of dwarf males was positively associated with sporophyte formation. When dwarf males were excluded, male mate availability was missed in 51–61% of specimens (in 74–76% of sporophyte-containing specimens). Specimen-level sex ratio was balanced in material with sporophytes in the three species exhibiting male dwarfism, but strongly female biased if dwarf males were omitted.

Conclusions: We provide the first quantitative evidence that sex-expressing dwarf males are essential for sexual reproduction and sporophyte production in mosses that form such males. Considering its abundance, male dwarfism therefore deserves more attention among plant biologists. Innovative approaches are required to further study the minute male plants.     

Biological and nutritional aspects involved in fern multiplication

Gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes. Differences in the time period from spore culture to sporophyte development were perceivable between species. For those species with a fast life cycle and high sporophyte production such as Woodwardia virginica and Dryopteris affinis sp. affinis, homogenization of gametophytes can be considered to be excellent method for propagation, yielding hundreds of sporophytes in a short period of time. Sporophyte formation was inhibited in O. regalis by the succesive application of homogenization to gametophytes regenerated by this technique. The effect of the culture medium composition on fern production was also studied in O. regalis and P. ensiformis gametophytes. In these species, sporophyte formation increased when the gametophytes were cultured in a medium containing water+0.7% agar. Addition of sucrose inhibited gametophyte development and induced their necrosis. The 1/2 dilution of Murashige and Skoog basal medium, without sucrose, favoured leaf expansion in P. ensiformis sporophytes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.