ELECTROPHORESIS IS MODIFYING OUR CONCEPTS OF EVOLUTION IN HOMOSPOROUS PTERIDOPHYTES

Analyses of electrophoretically detectable enzyme variants in homosporous pteridophytes are facilitating the development of new insights into their genetics and evolution. The number of isozymes per enzyme indicates that homosporous pteridophytes are genetic diploids, in spite of the fact that they have high chromosome numbers. High levels of heterozygosity and genetic variability in sporophytic populations indicate that many diploid species are outcrossing with inbreeding representing a derived character state. Because the congeneric homosporous pteridophyte species analyzed to date have low genetic identities, allozymic characters are also proving to be useful as genomic markers for elucidating patterns of reticulate evolution. The accumulated data suggest that the genetic system of homosporous pteridophytes differs fundamentally from that of seed plants. The present genomic constitution of extant taxa may be the result of repeated cycles of allopolyploidy followed by gene silencing and extinction of progenitor taxa. Alternatively, the original homosporous pteridophytes may have had high chromosome numbers. Although current species probably evolved recently, their phylogenetic roots may be difficult to trace because even closely related pteridophytes are genetically distant and extinction has obliterated the ancestral intermediates between lineages. These hypotheses can and should be tested using a combination of molecular, phylogenetic, and population biology methods.     

Theory for why dioecious plants have equal length sex chromosomes

Dioecy and sex chromosomes almost certainly evolved from ancestral hermaphrodites that only possessed autosomes. There is a growing body of evidence that genes for female or male function were then epigenetically suppressed in some of these hermaphrodites, creating the first males or females and nascent sex chromosomes. The incipient sex-determining epigenetic signals, such as cytosine methylation, then drove Muller's ratchet in many animals, resulting in shorter Y chromosomes. Based on this theory of sex chromosome evolution and limited data on gametophyte gene expression, I argue that plants should be largely immune from Muller's ratchet and therefore retain their ancestral state of equal length sex chromosomes, unless they incur chromosomal rearrangements or large-scale insertions of duplicated genomes. Usually heteromorphic sex chromosomes canalize dioecy, but extensive polyploidy or polysomy can provide an escape from this canalized dioecy. This theory implies that dioecy due to heteromorphic sex chromosomes should be evolutionarily ephemeral in bryophytes and homosporous pteridophytes because of their extraordinarily high incidences of polyploidy. And, if anything, these very high incidences of polyploidy are responsible for translocation or gradual addition of beneficial genes, rather than gradual reduction in the length of a sex chromosome.     

Do plants adjust their sex allocation and secondary sexual morphology in response to their neighbours?

Background and aims

Changes in the sex allocation (i.e. in pollen versus seed production) of hermaphroditic plants often occur in response to the environment. In some homosporous ferns, gametophytes choose their gender in response to chemical cues sent by neighbours, such that spores develop as male gametophytes if they perceive a female or hermaphrodite nearby. Here it is considered whether a similar process might occur in the androdioecious angiosperm species Mercurialis annua, in which males co-occur with hermaphrodites; previous work on a Spanish population of M. annua found that individuals were more likely to develop as males at high density.

Methods

Using a novel approach to treat plants with leachate from pots containing males or hermaphrodites of M. annua, the hypothesis that individuals assess their mating opportunities, and adjust their sex expression accordingly, was tested through an exchange of chemical cues through the soil.

Key Results

For the population under study, from Morocco, no evidence was found for soil-signal-dependent sex expression: neither sex ratios nor sex allocation differed among experimental treatments.

Conclusions

The results imply either that the Moroccan population under study behaves differently from that previously studied in Spain (pointing to potential geographical variation in plasticity for sex expression), or that our method failed to capture the signals used by M. annua for adjustment of sex expression.     

'Inconstant males' and the maintenance of labile sex expression in subdioecious plants

* Here, we evaluate the role of pollen limitation and selfing in the maintenance of labile sex expression in subdioecious plant species. * We used a literature survey to explore which factors correlated with a significant occurrence of hermaphrodites in dioecious species. We developed models to explore the selective maintenance of labile sex expression. The models had similar ecological assumptions but differed in the genetic basis of sex lability. * We found that a significant frequency of hermaphrodites was associated with animal pollination, and that hermaphrodites were 'inconstant' males with perfect flowers, suggesting evolution through the gynodioecious pathway. Models showed that a modifier converting pure males into inconstant males could be maintained under a wide range of reduction in both male and female fitness. Pollen limitation and self-fertilization facilitated invasion of the modifier. Depending on the genetics of sex determination, we found pure dioecy, stable subdioecy (trioecy), and situations where inconstant males coexisted with either pure females or pure males. Under selfing and pollen limitation, certain conditions selected for inconstant males which will drive populations to extinction. * We discuss our results in relation to the evolution towards, and the breakdown of, dioecy, and the ecological and evolutionary implications of labile sex expression.     

Trends for Inbreeding in Polyploid Pteridophytes

Abstract Previous studies on the sequence of the gametangium formation in homosporous fern gametophytes (Masuyama, 1975a, b) suggested that diploid and polyploid taxa may favor gametophytic crossing and gametophytic selfing, respectively. In this view, intraspecific polyploids of three fern species ( Phegopteris decursive-pinnata, Lepisorus thunbergianus and Pteris dispar ) have been investigated for the amount of genetic load or the rate of gametophytic selfing. As expected, a marked contrast of mating systems is found between diploids and tetraploids; the former predominantly undergo gametophytic crossing and the latter predominantly undergo gametophytic selfing. It seems likely that diploid homosporous pteridophytes favor gametophytic crossing with some exceptions favoring gametophytic selfing as a derived condition, whereas polyploid homosporous pteridophytes favor gametophytic selfing predominantly.     

Pteridophytic evolution: an often underappreciated phytological success story

The role and success of pteridophytes in the vascular land flora from the Silurian to Recent are examined. Success is defined as the ability of plants to occupy physical and ecological space through time, and evaluated in terms of species richness and percent cover. These factors are examined for vascular plants of successively more specialized grades of life cycles. Four grades, including homospory, heterospory, extreme heterospory, and angiospermy are recognized, with angiospermy providing for the greatest species richness and percent cover in the modern flora. In terms of percent cover, gymnosperms are also relatively successful, but in terms of species richness homosporous pteridophytes are more successful than all other non-angiospermous grades combined. The diversity of growth forms and life history patterns that have helped pteridophytes become successful throughout time is examined, and evidence for rhizomatous herbs, vines, epiphytes, trees of diverse architectures, and specialized modes of growth and vegetative propagation are reviewed. Comparison of several analyses of land plant diversity through the Phanerozoic suggests that plants with pteridophytic reproduction were dominant in most floras long after the origin of seed plants, and that pteridophytes probably have continued to increase in species richness up to the present. The loss of pteridophytic dominance in terms of percent cover at the end of the Paleozoic was associated with species turnover in which arborescent pteridophytes of several clades became extinct, but some herbaceous pteridophytes may have retained dominance in many floras into the Paleogene.     

ECOLOGICAL ASPECTS OF SEX EXPRESSION IN SUBDIOECIOUS SCHIEDEA GLOBOSA (CARYOPHYLLACEAE)

The importance of ecological factors such as sex lability, spatial segregation, and resource allocation in the evolution of dioecy were examined in Schiedea globosa. S. globosa is a subdioecious species with equal numbers of plants possessing strictly male or female function and a small proportion of hermaphrodites. The propensity for labile sex expression was under both environmental and genetic control; some plants with male function became hermaphroditic (by producing female flowers) under better growing conditions in the field and in the greenhouse. There was some spatial segregation of the sexes. Because of sex lability, more hermaphrodites than males occurred on moister slopes. Although there were not measurable sex-related differences in mortality within or between two flowering seasons, more females than males and hermaphrodites occurred at the bottom of slopes. Males and females produced the same number of ramets and inflorescences, but females had a greater number of flowers per inflorescence. Males and females had the same number of ovules (vestigial in males), but females had larger ovules and longer stigmas. Hermaphrodites and males had the same amount of pollen per flower despite the production of fruit by the hermaphrodites. In hermaphrodites, there was no apparent tradeoff within flowers between pollen production and ovule production. These results indicate that spatial segregation, sex lability, and environmental conditions influence allocation patterns of S. globosa, and in combination with high inbreeding depression and selling rates, may promote the further evolution of dioecy in S. globosa.     

Antheridogen activity in the fern Ceratopteris thalictroides (L.) Brongn.

Spores of the homosporous fern Ceratopteris thalictroides , in multispore culture, initially produce spatulate gametophytes bearing only antheridia (males) and cordate gametophytes bearing both antheridia and archegonia (hermaphrodites). When multispore cultures are sampled, the ratio of male to hermaphroditic gametophytes is a constant for each population examined. Four possible causes of such a sex ratio (cytoplasmic inheritance, nuclear inheritance, incipient heterospory and an antheridogen) are investigated. Evidence presented indicates that an antheridogen causes the existence of two gametophyte types, while one or more cytoplasmic units are the probable cause of the sex ratio. The activity of the antheridogen is to cause potentially hermaphroditic plants to become male. This activity was elucidated in monospore culture. Populational differences in antheridogen activity are also demonstrated. The significance of antheridogens is discussed in relation to the mating system of these plants.     

Sex allocation of females and hermaphrodites in the gynodioecious Geranium sylvaticum

Seed production and patterns of sex allocation were studied in female and hermaphroditic plants in two gynodioecious populations of Geranium sylvaticum (Geraniaceae). Females produced more flower buds and seeds than hermaphrodites in one of the two study populations. The other female traits measured (pistil biomass, seed number per fruit, individual seed mass) did not differ between the gender morphs. The relative seed fitness of hermaphrodites differed between the study populations, with hermaphrodites gaining less of their fitness through female function in the population with a high frequency of females. However, the amount and size of pollen produced by hermaphrodites did not differ between populations. The number of flower buds was positively correlated with seed production in females, whereas in hermaphrodites a positive correlation between number of buds and seed production was found in only one of the two study populations. These results suggest that fitness gain through female function is labile in hermaphrodites of this species, and is probably affected by environmental factors such as the sex ratio of the population.     

Do antheridiogens act via gametophyte size? A study of Woodwardia radicans (Blechnaceae)

For many plants, sex is not fixed by genotype but determined by environmental conditions during development. In homosporous pteridophytes, sex is environmentally determined by the presence or absence of antheridiogens, maleness-inducing pheromones. It has been proposed that antheridiogens primarily reduce growth rate, with small gametophyte size responsible for maleness. To test this hypothesis, the effects of antheridiogen and intergametophytic competition on gender expression and gametophyte size were studied in a culture experiment with Woodwardia radicans. We found that (1) antheridiogen inhibited growth of gametophytes; and (2) slow growth favored maleness, whereas fast growth favored femaleness, irrespective of the presence or absence of antheridiogen. Both conclusions are consistent with the hypothesis that, in W. radicans, antheridiogen effect is mediated by size. They also agree with the "size-advantage" hypothesis in which energetic limitations associated with relatively small individual size impose a less severe limitation for male reproductive success than for female reproductive success. The results are also discussed with regard to a genetic sex-determining pathway that has recently been identified.     

Evolution of Inbreeding and Outcrossing in Ferns and Fern-Allies

Abstract Mating systems of 18 species of homosporous ferns follow a bimodal distribution, similar to that observed for seed plants (Schemske and Lande, 1985). Most species are highly outcrossing, a few are inbreeding, and two species examined to date have mixed mating systems. Equisetum arvense and several species of lycopods are also highly outcrossing. Several mechanisms, including inbreeding depression, antheridiogen, and ontogenetic sequences that result in effectively unisexual gametophytes, promote outcrossing in homosporous ferns and perhaps other homosporous pteridophytes as well. In some species of homosporous ferns, selection has favored the evolution of inbreeding as an adaptation for colonization. High levels of intra- and interpopulational gene flow via spore dispersal, coupled with high levels of intergametophytic crossing, generally lead to genetically homogeneous populations and species of homosporous ferns. However, rock-dwelling ferns and ferns from xeric habitats may exhibit significant population genetic structure due to physically patchy habitats. Reticulate evolution in homosporous ferns may be enhanced by high levels of intergametophytic crossing.     

ARE LYCOPODS WITH HIGH CHROMOSOME NUMBERS ANCIENT POLYPLOIDS?

It has been established that the number of isozymes (different forms of an enzyme encoded by different gene loci) is highly conserved in diploid angiosperms and gymnosperms. In contrast, allopolyploid angiosperms display an increase in isozyme number due to the addition of divergent genomes. Lycopods (Microphyllophyta) are an ancient lineage of vascular plants having very high chromosome numbers. It has been maintained that lycopods acquired these high chromosome numbers through repeated episodes of polyploidy. Despite high chromosome numbers, however, lycopod species having the lowest chromosome numbers within genera possess the number of isozymes typical of diploid seed plants for all enzymes examined except triosephosphate isomerase. There is, therefore, no genetic evidence from enzyme electrophoresis for polyploidy in these plants. These results are comparable to findings for other homosporous pteridophytes including the ferns (Pteridophyta) and horsetails (Arthrophyta). Alternative hypotheses for widespread genetic diploidy in homosporous pteridophytes are 1) repeated cycles of allopolyploidy followed by gene silencing; 2) repeated cycles of autopolyploidy, which would result in duplicated, but not divergent genes for isozymes; 3) initiation of these lineages with relatively high chromosome numbers.     

Density-dependent regulation of the sex ratio in an annual plant

Sex ratios are subject to strong frequency-dependent selection regulated by the mating system and the relative male versus female investment. In androdioecious plant populations, where males co-occur with hermaphrodites, the sex ratio depends on the rate of self-fertilization by hermaphrodites and on the relative pollen production of males versus hermaphrodites. Here, we report evolutionary changes in the sex ratio from experimental mating arrays of the androdioecious plant Mercurialis annua. We found that the progeny sex ratio depended strongly on density, with fewer males in the progeny of plants grown under low density. This occurred in part because of a plastic adjustment in pollen production by hermaphrodites, which produced more pollen when grown at low density than at high density. Our results provide support for the prediction that environmental conditions govern sex ratios through their effects on the relative fertility of unisexual versus hermaphrodite individuals.     

Determinants of sex allocation in a gynodioecious wild strawberry: implications for the evolution of dioecy and sexual dimorphism

One evolutionary pathway from plants with combined male and female functions (hermaphroditism) to those with separate sexes (dioecy) involves females coexisting with hermaphrodites (gynodioecy). The research presented here explores sex allocation in Fragaria virginiana (a gynodioecious wild strawberry), within the context of theory on the gynodioecy–dioecy transition. By growing clonally replicated plants in the greenhouse and surveying six populations in situ, I evaluated the effects of plant size, genotype, sexual identity, population of origin and female frequency on sex allocation. I found significant positive effects of plant size on most sex allocation traits studied. In addition to strong sex-specific allocation patterns, I found significant broad-sense heritabilities for all traits, suggesting that plants could respond to selection. Moreover, there was a negative genetic correlation between pollen production and fruit set per flower within hermaphrodites, lending support to a basic assumption of sex allocation theory. On the other hand, several sex allocation traits, namely pollen and ovules per flower in hermaphrodites, were positively genetically correlated, suggesting that they may act to constrain the evolution of sexual dimorphism. Populations differed in the frequency of females, and females were more prevalent on sites with lower soil moisture and where hermaphrodites were least likely to produce fruit, suggesting that females’ seed fitness relative to that of hermaphrodites may be strongly environment-dependent in this species.     

ELECTROPHORETIC EVIDENCE FOR GENETIC DIPLOIDY IN PSILOTUM NUDUM

Psilotum nudum (2n = 104) has been considered an ancient polyploid, having resulted from repeated cycles of hybridization and allopolyploidy. However, electrophoretic analysis indicates that this species is genetically diploid despite its high chromosome number. Sixteen enzymes, encoded by 28 loci, revealed in P. nudum the number of isozymes typical of diploid seed plants. There is, therefore, no evidence of polyploid gene expression for the enzymes analyzed. These results for Psilotophyta are similar to those obtained for other lineages of homosporous pteridophytes, i.e., Arthrophyta and homosporous Microphyllophyta and Pteridophyta, all of which should be considered genetically diploid. Several hypotheses have been proposed to explain these results, most notably 1) cycles of allopolyploidy followed by massive gene silencing, and 2) initiation of these lineages with high chromosome numbers, possibly via chromosomal fission. Discrimination between these hypotheses awaits testing with molecular genetic techniques.     

ESTIMATES OF INTRAGAMETOPHYTIC SELFING AND INTERPOPULATIONAL GENE FLOW IN HOMOSPOROUS FERNS

Relatively little information is available on mating systems and interpopulational gene flow in species of homosporous pteridophytes. Because of the proximity of antheridia and archegonia on the same thallus, it has long been maintained that intragametophytic selling is the predominant mode of reproduction in natural populations of homosporous ferns and other homosporous plants. Furthermore, quantitative estimates of interpopulational gene flow via spore dispersal are lacking. In this paper, we examine five species of homosporous ferns (Botrychium virginianum, Polystichum munitum, P. imbricans, Blechnum spicant, and Dryopteris expansa) and present estimates of 1) rates of intragametophytic selling, 2) levels of interpopulational gene flow, and 3) interpopulational genetic differentiation (F_ST). Our data demonstrate that mating systems vary among species of ferns, just as they do among species of seed plants. The data also suggest that levels of interpopulational gene flow are generally high. The F_ST values indicate little genetic divergence among populations for all species except Dryopteris expansa, which exhibits significant levels of interpopulational genetic differentiation. Patterns of genetic diversity in the five species examined are related to the mating system and rate of interpopulational gene flow in each species. The F_ST values for all species except Botrychium virginianum are in close agreement with those predicted for an island model of population structure.     

Variation in restorer genes and primary sexual investment in gynodioecious Plantago coronopus: the trade-off between male and female function

In many gynodioecious species the nuclear inheritance of male fertility is complex and involves multiple (restorer) genes. In addition to restoring plants from the female (male sterile) to the hermaphrodite (male fertile) state, these genes are also thought to play a role in the determination of the quantity of pollen produced by hermaphrodites. The more restorer alleles a hermaphroditic plant possesses, the higher the pollen production. To test this hypothesis I combined the results of crossing studies of the genetics of male sterility with phenotypic data on investment in stamens and ovules among the progeny of plants involved in these studies. The sex ratio (i.e. the frequency of hermaphrodites among the progeny), being a measure of the number of restorer alleles of the maternal plant, was positively related to the investment in pollen (male function), but negatively related to the investment in ovules (female function), in both field and greenhouse experiments. Consequently, a negative correlation between male and female function was observed (trade-off) and it is suggested that antagonistic pleiotropic effects of restorer genes might be the cause. Phenotypic gender, a measure combining investment in both pollen and ovules, was highly repeatable between field and greenhouse, indicating genetic determination of a more male- or female-biased allocation pattern among the studied plants.     

An experimental test of the effects of resources and sex ratio on maternal fitness and phenotypic selection in gynodioecious Fragaria virginiana

Resources, sex ratio, and seed production by hermaphrodites covary among natural populations of many gynodioecious plant species, such that they are functionally "more dioecious" as resources become more limiting. Strong correlations among these three factors confound our understanding of their relative roles in maintaining polymorphic sexual systems. We manipulated resource availability and sex ratio and measured their effects on relative fertility and phenotypic selection through the maternal fitness of females and hermaphrodites of Fragaria virginiana. Two results were particularly surprising. First, hermaphrodites showed little variability in fecundity across resource treatments and showed strong positive and context-dependent selection for fruit set. This suggests that variation in hermaphrodite seed production along resource gradients in nature may result from adaptation rather than plasticity. Second, although females increased their fecundity with higher resources, their fertility was unaffected by sex ratio, which is predicted to mediate pollen limitation of females in natural populations where they are common. Selection on petal size of females was also weak, indicating a minimal effect of pollinator attraction on variation in the fertility of female plants. Hence, we found no mechanistic explanation for the complete absence of high-resource high female populations in nature. Despite strong selection for increased fruit set of hermaphrodites, both the strength of selection and its contribution to the maintenance of gynodioecy are severely reduced under conditions where females have high relative fecundity (i.e., low resources and high-female sex ratios). High relative fertility plus high female frequency means that the evolution of phenotypic traits in hermaphrodites (i.e., response to selection via seed function) should be manifested through females because most hermaphrodites will have female mothers. Fruit set was never under strong selection in females; hence, selection to increase fruit set hermaphrodites will be less effective in maintaining their fruiting ability in natural populations with low resources and high female frequency. In sum, both sex ratio and resource availability influence trait evolution indirectly-through their effects on relative fertility of the sexes and patterns of selection. Sex ratio did not impose strong pollen limitation on females but did directly moderate the outcome of natural selection by biasing the maternal sex of the next generation. This direct effect of sex ratio on the manifestation of natural selection is expected to have far greater impact on the evolution of traits, such as seed-producing ability in hermaphrodites and the maintenance of sexual polymorphisms in nature, compared to indirect effects of sex ratio on relative fertility of the sexes.     

AN ELECTROPHORETIC INVESTIGATION OF INTRAGAMETOPHYTIC SELFING IN EQUISETUM ARVENSE

Because homosporous pteridophytes (Psilotophyta, Arthrophyta, most Microphyllophyta and Pteridophyta) produce bisexual gametophytes, it was maintained that high levels of inbreeding would characterize these plants. Electrophoretic evidence was used to estimate the frequency of intragametophytic selfing in Equisetum arvense (Arthrophyta). A total of 669 samples from 17 populations was examined from western North America. Although some populations exhibited as many as seven or eight genotypes, 10 populations were each characterized by only a single genotype; eight of these populations were heterozygous for one or more loci. For most populations, estimates of intragametophytic self-fertilization are 0.000, indicating that virtually all matings involve different gametophytes. Genetic data corroborate predictions based on earlier field and laboratory investigations of Equisetum gametophytes. These detailed studies demonstrated that in many species, including E. arvense, gametophytes are initially either male or female; only later and in the absence of fertilization do some gametophytes become bisexual. Our findings join a growing electrophoretic data base which demonstrates that homosporous pteridophytes are not highly inbreeding as previously suggested.     

Wise flies: a pre‐dispersal seed predator prefers hermaphrodites over females in the gynodioecious Polemonium foliosissimum

1. Oviposition choices can profoundly affect offspring performance. Oviposition decisions of the dipteran pre‐dispersal seed predator, Hylemya sp. (Diptera: Anthomyiidae), when choosing among sex morphs of their host‐plant—Polemonium foliosissimum Hook—were evaluated. Polemonium foliosissimum is gynodioecious, with female and hermaphrodite sex morphs that differ in flower size. 2. It was asked: Do female flies preferentially oviposit on hermaphrodite plants and, if so, are oviposition choices correlated with flower size? Is larval survivorship higher on hermaphrodite plants and, if so, is larval success correlated with flower size? Can the differences in oviposition and/or larval success be explained by slight differences in flowering phenology between the sexes? 3. Hermaphrodite flowers received 45% more Hylemya eggs than females. Although hermaphrodites had larger petals and sepals than females, egg loads were not correlated with petal or sepal size. Larval survival was 30% greater on hermaphrodites than females and higher on plants with larger sepals. However, the difference in sepal area between genders did not fully explain larval survival differences. Egg numbers declined over weeks, but differences in egg loads between the sex morphs were not attributable to a slight phenological delay of females. Larval survival declined over the season; however, larval survival differences between sex morphs were consistent. 4. Hylemya preferentially oviposited on hermaphrodites where their larvae had a significantly greater survival rate compared with females. The present results add to the knowledge that intra‐specific choices have consequences for phytophagous insects and that the relationship between antagonists and their gynodioecious host plants is complex.