GAMETOPHYTE ONTOGENY,SEX EXPRESSION,AND GENETIC LOAD AS MEASURES OF POPULATION DIVERGENCE IN BLECHNUM SPICANT

Early gametophyte ontogeny was quantitatively distinct for Olympic Peninsula, Alaskan, and disjunct Idaho populations of the homosporous fern Blechnum spicant (L.) J. Sm. Although variable, gametophyte sex expression was shown to have a genetic component. Statistically different patterns of sex expression characterize each population. The Olympic Peninsula populations were distinct from each other but consistent in having a predominantly unisexual pattern. The disjunct Idaho population was predominantly bisexual at the time when comparable field collected gametophytes bear sporophytes. Preliminary experiments suggest that an antheridogen operates in this species. Increased sowing density favors maleness, and an extract from soil cultures of gametophytes shifts cultures to an exclusively male pattern after a dramatic suppression of growth. Mating experiments revealed that all populations are interfertile, although fertility was highest when the test Idaho population underwent intergametophytic-selfing. The Idaho population evidenced a low level of genetic load consistent with predictions based on its sex expression. Although Olympic Peninsula populations evidenced apparent high genetic load in some experiments, failure to produce abundant sporophytes in other experiments suggested that additional cultural factors operated to reduce sporophyte formation. Moderate density mating experiments produced single sporophytes that were comparable to field collections. Isolated gametophytes underwent polyembryony after a time delay and gametophyte proliferation. Cultural conditions which allow sporophyte formation on isolated gametophytes without this delay or proliferation must be sought before further genetic analysis is undertaken.     

Inbreeding depression in Vriesea gigantea,a perennial bromeliad from southern Brazil

Inbreeding depression is a reduction of fitness in the progeny of closely related individuals and its effects are assigned to selfing or biparental inbreeding. Vriesea gigantea is a self‐compatible bromeliad species distributed in the Brazilian Atlantic rainforest and habitat destruction and fragmentation and collection have decreased the natural populations. We aim to describe the occurrence of inbreeding depression (δ) in three natural populations of V. gigantea and to correlate this phenomenon with previous studies of fertility, genetic diversity, population genetic structure, gene flow, mating system and seed dispersal in this species. Fifty‐four adult plants were sampled and 108 flowers were used for pollination treatments (selfing, outcrossing and control). For adult plants, we analysed plant and inflorescence height, flower numbers and seed set. In the progenies, evaluated parameters included seed germination and seedling survival rate. The results indicated low to moderate levels of inbreeding depression in V. gigantea (δ = 0.02 to 0.39), in agreement with molecular data from a previous study. Vriesea gigantea populations tolerate some degree of inbreeding, which is consistent with previous results on fertility, mating system, genetic diversity and gene flow. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 312–319.     

Potential for parthenogenesis of virgin females in a bisexual population of the geographically parthenogenetic mayfly Ephoron shigae (Insecta: Ephemeroptera,Polymitarcyidae)

The burrowing polymitarcyid mayfly Ephoron shigae is a geographically parthenogenetic species. Interestingly, the distributions of the bisexual and unisexual populations overlap broadly in their respective geographic ranges. In this mayfly, obligatory diploid thelytoky appears within unisexual populations. In the present study, we examined the potential for parthenogenesis or the parthenogenetic ability of females in a bisexual population aiming to understand the emergence of unisexual populations. The results obtained revealed that females in the examined bisexual populations showed a potential for diploid thelytoky as also seen in the unisexual populations, although, in females from bisexual populations, the development success rates of their unfertilized eggs were considerably lower than those of virgin females from unisexual populations. In the three bisexual reproducing species (Ephemera japonica, Ephemera strigata, and Ephemera orientalis) in the closely‐related family Ephemeridae, diploid thelytoky (i.e. tychoparthenogenesis; < 3%) was also observed. However, in this case, the parthenogenetic development success rates of unfertilized eggs were significantly lower than those of virgin females in the bisexual (Hino‐yosui Irrigation Canal) population of E. shigae. Accordingly, we suggest that parthenogenetic ability (i.e. tychoparthenogenesis or facultative parthenogenesis) in bisexual populations of E. shigae may facilitate the evolutionary transition to unisexual populations with fully obligatory parthenogenesis. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 326–334.     

Sporophyte formation in experimentally-induced unisexual female and bisexual gametophytes ofLygodium japonicum

Unisexual female and male and bisexual gametophytes were experimentally induced inLygodium japonicum. A single bisexual gametophyte was isolated in a dish and a female gametophyte was paired with a male one to allow intragametophytic selfing and intergametophytic mating, respectively. About 30% of the females formed sporophytes but no bisexual gametophytes formed them.     

POPULATIONAL AND GENETIC STUDIES OF A HOMOSPOROUS FERN—OSMUNDA REGALIS

The mating system and the genetic system of the homosporous fern Osmunda regalis were investigated. Seven populations from western Massachusetts were sampled. All the sporophytes investigated were found to be heterozygous for zygotic lethals. Morphological studies of the gametophytes indicated an intergametophytic mating system when the gametophytes were spatially and chronologically situated to exchange male gametes. Genetic studies evidenced a genetic system based upon duplicate loci.     

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.     

On the sexual reproduction ofDictyosiphon foeniculaceus (Phaeophyceae,Dictyosiphonales)

Dictyosiphon foeniculaceus from Sweden and Newfoundland was studied in laboratory culture. Zoids from unilocular sporangia developed into dioecious microscopic filamentous gametophytes which produced uniseriate plurilocular gametangia in low temperatures (0 to 8 °C). Zygotes and unfused isogametes gave rise to filamentous protonemata on which parenchymatous macroscopic sporophytes were formed. Isolates from Sweden and Newfoundland were interfertile. Although formed in culture, genetically unisexual sporophytes were not detected in nature. Female gametes ofD. foeniculaceus produced a sexual pheromone. It was identified as finavarrene, which is also known as the sperm attractant inAscophyllum nodosum.     

Population genetics of bisexual and unisexual populations of the scaly-winged bark louse Echmepteryx hageni (Insecta: Psocoptera)

The scaly-winged bark louse, Echmepteryx hageni, exhibits a unique pattern of co-existence of apparently differnt reproductive modes. Unisexuality is widespread in eastern North America, while sexual populations are restricted to isolated rock out-croppings in southern Illinois and eastern Kentucky. Three of the four nuclear loci examined show greater genetic diversity in the unisexual form compared to the sexual form of E. hageni, in accordance with the pattern previously shown in mitochondrial genetic data. Neutrality tests of the nuclear loci indicate a consistent signal of demographic expansion in asexual populations, but not in sexual populations. There was evidence of inbreeding in the isolated sexual populations at three of the nuclear loci, and one locus had signs of gene specific balancing selection. However, there is no significant genetic differentiation between bisexual and unisexual populations, possibly due to the greater effective population size of nuclear loci relative to mitochondrial loci. The mitochondrial differentiation of E. hageni populations in the northwestern part of their range (Minnesota and Wisconsin) was also not reflected in the nuclear data. We present three hypotheses that may explain the disparity in observed nuclear and mitochondrial genetic diversity between the reproductive forms of E. hageni.     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

Density affects gametophyte growth and sexual expression of Osmunda cinnamomea (Osmundaceae: Pteridophyta)

BACKGROUND AND AIMS: To understand how gametophyte densities affect the sexual expression and sizes of Osmunda cinnamomea and to provide information on the density of growth needed to favour successful reproduction, fresh spores were sown at various densities and subsequent gametophyte growth was studied. METHODS: Spores were sown and cultured in the laboratory. Subsequent gameophytes at different population densities were sampled and their sexual expression and sizes were recorded. KEY RESULTS: One-year-old multispore cultures of the fern O. cinnamomea demonstrated that population density affected gametophyte growth and sexual expression. As density increased, gametophytes became significantly smaller and more slender. Female and asexual gametophytes dominated in populations of low and high densities, respectively. At intermediate population densities, hermaphroditic and male gametophytes were dominant. Female gametophytes were larger than gametophytes of all other types. Hermaphroditic gametophytes were larger than male gametophytes, which were larger than asexual gametophytes. Large gametophytes were wide-cordate, whereas smaller ones tended to be narrow-spathulate. CONCLUSIONS: Gametophyte size of O. cinnamomea is negatively related to the population density, which significantly affects gametophytes' sexual expression. The presence of unisexual and bisexual gametophytes at intermediate densities indicates that both intergametophytic and intragametophytic selfing may occur.     

Long‐distance pollen and seed dispersal and inbreeding depression in Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) in the Brazilian savannah

Hymenaea stigonocarpa is a neotropical tree that is economically important due to its high‐quality wood; however, because it has been exploited extensively, it is currently considered threatened. Microsatellite loci were used to investigate the pollen and seed dispersal, mating patterns, spatial genetic structure (SGS), genetic diversity, and inbreeding depression in H. stigonocarpa adults, juveniles, and open‐pollinated seeds, which were sampled from isolated trees in a pasture and trees within a forest fragment in the Brazilian savannah. We found that the species presented a mixed mating system, with population and individual variations in the outcrossing rate (0.53–1.0). The studied populations were not genetically isolated due to pollen and seed flow between the studied populations and between the populations and individuals located outside of the study area. Pollen and seed dispersal occurred over long distances (>8 km); however, the dispersal patterns were isolated by distance, with a high frequency of mating occurring between near‐neighbor trees and seeds dispersed near the parent trees. The correlated mating for individual seed trees was higher within than among fruits, indicating that fruits present a high proportion of full‐sibs. Genetic diversity and SGS were similar among the populations, but offspring showed evidence of inbreeding, mainly originating from mating among related trees, which suggests inbreeding depression between the seed and adult stages. Selfing resulted in a higher inbreeding depression than mating among relatives, as assessed through survival and height. As the populations are not genetically isolated, both are important targets for in situ conservation to maintain their genetic diversity; for ex situ conservation, seeds can be collected from at least 78 trees in both populations separated by at least 250 m.     

Conservation genetics of the rare Iberian endemic Cheirolophus uliginosus (Asteraceae)

Cheirolophus uliginosus is a rare species, endemic to the south‐western Iberian Peninsula, and listed as a characteristic taxon from the temperate Atlantic wet heaths, a priority habitat for conservation by the European Union. The conservation status of this species in most of its distribution area is poorly known, but, in recent times, some populations have disappeared and there has been a reduction in the number of individuals in others. In this context, we analysed the effects of population size on genetic diversity, revealing that genetic erosion and inbreeding depression could be having a significant impact on smaller populations. Furthermore, we studied the patterns of genetic structure and variability at the species level, finding a strikingly low within‐population diversity and high among‐population genetic differentiation. Finally, the genetic structure analyses suggested a long and complex phylogeographical history of C. uliginosus in the region, in agreement with the climate relict status proposed for this species. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 157–171.     

OBLIGATE OUTCROSSING IN A HOMOSPOROUS FERN: FIELD CONFIRMATION OF A LABORATORY PREDICTION

While homosporous ferns are potentially capable of producing totally homozygous sporophytes in one generation via selfing of their bisexual gametophytes, laboratory analyses indicate that a variety of mechanisms promote gametophytic outcrossing. The operation of these mechanisms in natural sporophyte populations, however, has not been previously demonstrated. Laboratory analyses of gametophyte ontogeny show that Bommeria hispida is obligately outcrossing. Electrophoretic data presented here indicate that individuals from natural sporophyte populations of this species are highly heterozygous. Electrophoretic data, therefore, corroborate evidence from the in vitro analysis of gametophyte development and demonstrate that sporophytes of B. hispida in nature typically are products of outcrossing between genetically different gametophytes. Extrapolations from the literature, together with our findings, indicate that outcrossing mechanisms may operate frequently in ferns, thereby maintaining genetic variability between individuals within populations. This evidence questions whether most ferns are highly inbred and therefore predominantly homozygous.     

Is allometry of sexual traits adaptive? A field test with territorial damselflies

Recent studies have linked static allometry of sexual traits to selective advantages, in terms of sexual selection. An underlying, yet untested, assumption is that the allometry of sexual traits confers higher mating success and/or survival. Here, we investigated whether the allometry of two sexual traits is related to male mating success and survival in two species of damselflies: wing size in Paraphlebia zoe and the red‐pigmented wing spot in Hetaerina americana. We used large field‐based data sets of marked‐recaptured animals, in which we recorded male mating success and survival. Both sexual traits exhibited hyperallometric patterns; however, allometry was not linked to either mating success or survival. These results indicate that, at least during the period of sexual competition, allometry does not seem to be adaptive. Although our results may only apply to our damselfly study subjects (which nevertheless would require further tests in different seasons and/or study sites), our findings should encourage researchers to evaluate at least whether the assumed adaptiveness of sexual trait allometry holds for their study animals. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 327–334.     

WHAT USES ARE MATING TYPES? THE “DEVELOPMENTAL SWITCH” MODEL

Why mating types exist at all is subject to much debate. Among hypotheses, mating types evolved to control organelle transmission during sexual reproduction, or to prevent inbreeding or same-clone mating. Here I review data from a diversity of taxa (including ciliates, algae, slime molds, ascomycetes, and basidiomycetes) to show that the structure and function of mating types run counter the above hypotheses. I argue instead for a key role in triggering developmental switches. Genomes must fulfill a diversity of alternative programs along the sexual cycle. As a haploid gametophyte, an individual may grow vegetatively (through haploid mitoses), or initiate gametogenesis and mating. As a diploid sporophyte, similarly, it may grow vegetatively (through diploid mitoses) or initiate meiosis and sporulation. Only diploid sporophytes (and not haploid gametophytes) should switch on the meiotic program. Similarly, only haploid gametophytes (not sporophytes) should switch on gametogenesis and mating. And they should only do so when other gametophytes are ready to do the same in the neighborhood. As argued here, mating types have evolved primarily to switch on the right program at the right moment.     

INBREEDING AND THE RATE OF SELF-FERTILIZATION IN A GRAPE FERN,BOTRYCHIUM DISSECTUM

Botrychium dissectum is a homosporous fern with bisexual, subterranean gametophytes. Because of these features, B. dissectum would be suspected of displaying a very high frequency of self-fertilization. Sporophytes collected from three populations of this species were assayed for heterozygosity by determining the electrophoretic mobility patterns displayed by two polymorphic enzymes. Extreme deviations from Hardy-Weinberg expectations were observed in each population and analyzed by means of F-statistics. The average inbreeding coefficient was found to be 0.951. A population genetic model is derived that demonstrates that the rate of intragametophytic self-fertilization in homosporous ferns is equal to the inbreeding coefficient calculated from deviations from Hardy-Weinberg expectations. It is therefore concluded that B. dissectum outcrosses about 5% of the time.     

Balance between inbreeding and outcrossing in a nannandrous species,the moss Homalothecium lutescens

Epiphytic dwarf males on the females present a possible solution to the problem of short fertilization distances in mosses. However, leptokurtic spore dispersal makes dwarf males likely to be closely related to their host shoot, with an accompanying risk of inbreeding. The capacity of a female to harbour a high number of different dwarf males suggests that there may be mechanisms in place that counteract inbreeding, such as polyandry and post-fertilization selection. We have genotyped sporophytes, female host shoots and dwarf males in four populations of the moss Homalothecium lutescens. We found no evidence of selective sporophyte abortion based on level of heterozygosity. The occurrence of entirely homozygous sporophytes together with significantly positive inbreeding coefficients in three of the populations (mean F_IS between 0.48 and 0.64) suggest frequent mother–son mating events. However, 23% of all sampled sporophytes had a higher level of heterozygosity compared with the mean expected heterozygosity at the population level. Polyandry was frequent, on average 59% of the sporophytes on a female shoot were sired by distinct fathers. In conclusion, sporadic fertilizations by dwarf males originating from nonhost female shoots appear to counteract strong inbreeding.     

Coexistence of bisexual and unisexual populations of Nysius groenlandicus in the Zackenberg Valley,Northeast Greenland

The seed‐feeding true bug Nysius groenlandicus (Zetterstedt) (Heteroptera: Lygaeidae) has temperature and humidity preferences similar to many desert‐inhabiting insects. Nevertheless, it is widely distributed in Greenland and occurs even in high arctic areas. This seeming paradox implies that the species has developed physiological, ecological, and behavioral mechanisms that enable it to cope with the extreme arctic environment. One of these adaptations is its ability to reproduce both sexually and asexually. Sexual reproduction is most common in coastal populations, whereas asexual reproduction (parthenogenesis) occurs mainly in inland populations. Preliminary data from the Zackenberg Valley in Northeast Greenland indicated that both reproductive strategies occurred within the same area. We therefore decided to focus on this area to find evidence of (1) coexistence of unisexual and bisexual populations, (2) factors that correlate with the distribution of the two reproductive types, and (3) morphological differences between females from sexual and asexual populations. We sampled N. groenlandicus at 51 sites within the Zackenberg area. Males and females were found in almost equal numbers in only four of the samples, and only in samples taken close to the coast line, whereas samples from the inner part of the valley consisted almost entirely of females. Thus, the distribution of uni‐ and bisexual populations at the local scale agreed with the pattern previously found for Greenland as a whole. Using data obtained from two climate stations operated in the area, we showed that average summer temperatures tend to be more favorable for N. groenlandicus in the inner part of the valley than at the coast. The inland populations inhabited small isolated patches, whereas the sexual populations were found in the more homogeneous areas along the coast. Data support the hypothesis that coexistence of the two reproductive types is maintained by a combination of environmental variability, which gives sexual reproduction an advantage, and metapopulation dynamics characterized by frequent extinctions and re‐colonizations, which favors asexual reproduction. Head width of females from unisexual populations was on average 7% larger than that of females from bisexual populations. The mechanism behind this morphological difference needs further investigation.     

Development of microsatellites for the peat moss Sphagnum capillifolium using ISSR cloning

Sphagnum mosses are major components of peat bogs but populations of many species are under threat due to habitat fragmentation resulting from the cutting of peat for fuel. We have used an intersimple sequence repeat (ISSR)‐based cloning method to develop nine polymorphic nuclear microsatellites for the peat moss species Sphagnum capillifolium. Between three and seven alleles per locus were detected in a sample of 48 haploid gametophytes and levels of gene diversity ranged from 0.5391 to 0.7960. These represent the first microsatellite markers developed for this important genus and most also exhibited cross‐species amplification across a range of common Sphagnum species.