Gynodioecy in mountain thyme (Thymus praecox agg.) : why more females at higher altitudes?

Zusammenfassung Die Untersuchung der Frage, warum beim Bergthymian in höher gelegenen Lagen Weibchen häufiger vorkommen, kann zum Verständnis der Erhaltung des gynodiözischen Fortpflanzungssystems (Nebeneinander von Weibchen und Zwittern) beitragen. Zuerst wurden die genetische Vielfalt und räumliche Verteilung der nukleären und cytoplasmatischen geschlechtsbestimmenden Gene anhand von Geschlechterhäufigkeiten in kontrollierten Kreuzungen untersucht. Die Ergebnisse wiesen darauf hin, dass die geschlechtsbestimmenden Gene natürlicher Selektion unterlagen (lokale Anpassung/Kosten der Wiederherstellung der männlichen Funktion) und dass Umwelteinflüsse, welche die relative Fitness von Zwittern und Weibchen beeinflussen, die Variation der Geschlechterhäufigkeiten entlang von Höhengradienten verursachen. Die Bedeutung der obligaten Kreuzbestäubung von Weibchen (Fremdbefruchtungsvorteil gegenüber Zwittern) für die relative Häufigkeit der beiden Geschlechter in natürlichen Populationen wurde danach genauer untersucht. Zu diesem Zweck wurden genetische Marker (Mikrosatelliten) zur Schätzung von Heterozygotie unter tetrasomer Vererbung entwickelt. Die anschliessenden molekulargenetischen Untersuchungen zeigten, dass Nachkommen aus Selbstbestäubung das Erwachsenenalter kaum erreichen können (starke Inzuchtdepression) und dass Selbstbestäubung auf Zwittern häufig ist. Allerdings war der daraus resultierende Fremdbefruchtungsvorteil der Weibchen in subalpinen und in alpinen Populationen konstant gross. Somit wurde die Hypothese verworfen, dass beim Bergthymian Selektion auf Vermeidung von Inzucht die beobachteten Geschlechterhäufigkeiten entlang von Höhengradienten verursachen könnte. Diese Resultate sind auch von allgemeiner Bedeutung, da sie aufzeigen, dass effiziente Kreuzbestäubung durch Insekten unter alpinen Bedingungen möglich ist. Summary An explanation for why females are more frequent at higher altitudes in Mountain Thyme may help with understanding the evolutionary maintenance of the gynodioecious breeding system, i.e. the coexistence of females and hermaphrodites. The genetic diversity and spatial dynamics of nuclear and cytoplasmic sex-determining genes were first inferred from sex ratio variation among controlled crosses. Results suggested that sex-determining genes were subject to selective processes (local adaptation/cost of restoration of the male function) and that variation in the relative seed fitness and/or survival rates of females and hermaphrodites governs sex ratio variation along subalpine to alpine elevation gradients. The role of the outcrossing advantage of females (i.e. avoidance of inbreeding by obligately outcrossed females) for their relative frequency within populations was further explored. For this purpose, molecular genetic (microsatellite) markers applicable to assess heterozygosity under tetrasomic inheritance were developed. Subsequent microsatellite analyses revealed severe costs of self-fertilisation (inbreeding depression) and considerable rates of geitonogamous selfing in hermaphrodites, thus pointing to a substantial outcrossing advantage of females. However, both the rates and costs of selfing were constant across altitudes, hence rejecting the hypothesis that selection to avoid inbreeding was the driving force maintaining the observed sex ratio variation in Mountain Thyme. More generally, these findings also demonstrate that efficient cross-pollination by insects occurs in alpine environments

Allelic configuration and polysomic inheritance of highly variable microsatellites in tetraploid gynodioecious Thymus praecox agg

Polyploidy plays a pivotal role in plant evolution. However, polyploids with polysomic inheritance have hitherto been severely underrepresented in plant population genetic studies, mainly due to a lack of appropriate molecular genetic markers. Here we report the establishment and experimental validation of six fully informative microsatellite markers in tetraploid gynodioecious Thymus praecox agg. Sequence data of 150 microsatellite alleles and their flanking regions revealed high variation, which may be characteristic for polyploids with a reticulate evolutionary history. Understanding the patterns of mutation (indels and substitutions) in microsatellite flanking-sequences was a prerequisite for the development of co-dominant markers for fragment analyses. Allelic segregation patterns among progeny arrays from ten test crosses revealed tetrasomic inheritance in T. praecox agg. No evidence of frequent double reduction was detected. Polymerase chain reaction (PCR) based dosage effects allowed for precise assignment of allelic configuration at all six microsatellite loci. The quantification of allele copy numbers in PCR was verified by comparisons of observed and expected gametic allele frequencies and heterozygosities in test crosses. Our study illustrates how PCR based markers can provide reliable estimates of heterozygosity and, thus, powerful tools for breeding system and population genetic analyses in polyploid organism

Between-year variation in seed weights across altitudes in the high-alpine plant Eritrichium nanum

Seed weight is a prominent life history trait of plants affecting dispersal, establishment, and survival. In alpine environments, the few studies investigating the effect of elevation on seed weight within species have mainly detected a decrease in seed weight with increasing elevation. This relationship is generally attributed to the adverse climate at high elevations. In order to test this hypothesis, we analyzed seed weight variation across altitudes (2,435-3,055m a.s.l.) in two consecutive years that differed in weather conditions in the high-alpine cushion plant Eritrichium nanum. We found a significant reduction in seed weight with increasing elevation in both years, but in the growing season with more adverse weather conditions, the reduction was more substantial than in the more favorable year. We conclude that alpine plants may be able to produce well-developed seeds at low elevations in almost all years, independent of weather conditions, whereas reproduction through seeds is potentially limited to years of favorable weather at high elevatio

Effects of floral neighborhood on seed set and degree of outbreeding in a high-alpine cushion plant

Plants flowering together may influence each other's pollination and fecundity over a range of physical distances. Their effects on one another can be competitive, neutral, or facilitative. We manipulated the floral neighborhood of the high-alpine cushion plant Eritrichium nanum in the Swiss Alps and measured the effects of co-flowering neighbors on both the number of seeds produced and the degree of inbreeding and outbreeding in the offspring, as deduced from nuclear microsatellite markers. Seed set of E. nanum did not vary significantly with the presence or absence of two Saxifraga species growing as near neighbors, but it was higher in E. nanum cushions growing at low conspecific density than in those growing at high density. In addition, floral neighborhood had no detectable effect on the degree of selfing of E. nanum, but seeds from cushions growing at low conspecific density were more highly outbred than seeds from cushions at high density. Thus, there was no evidence of either competition or facilitation between E. nanum and Saxifraga spp. as mediated by pollinators at the spatial scale of our experimental manipulation. In contrast, the greater fecundity of E. nanum cushions at low density was consistent with reduced intraspecific competition for pollinators and might also represent a beneficial effect of highly outbred seeds as brought about by more long-distance pollinator flights under low-density condition

Allelic configuration and polysomic inheritance of highly variable microsatellites in tetraploid gynodioecious Thymus praecox agg

Polyploidy plays a pivotal role in plant evolution. However, polyploids with polysomic inheritance have hitherto been severely underrepresented in plant population genetic studies, mainly due to a lack of appropriate molecular genetic markers. Here we report the establishment and experimental validation of six fully informative microsatellite markers in tetraploid gynodioecious Thymus praecox agg. Sequence data of 150 microsatellite alleles and their flanking regions revealed high variation, which may be characteristic for polyploids with a reticulate evolutionary history. Understanding the patterns of mutation (indels and substitutions) in microsatellite flanking-sequences was a prerequisite for the development of co-dominant markers for fragment analyses. Allelic segregation patterns among progeny arrays from ten test crosses revealed tetrasomic inheritance in T. praecox agg. No evidence of frequent double reduction was detected. Polymerase chain reaction (PCR) based dosage effects allowed for precise assignment of allelic configuration at all six microsatellite loci. The quantification of allele copy numbers in PCR was verified by comparisons of observed and expected gametic allele frequencies and heterozygosities in test crosses. Our study illustrates how PCR based markers can provide reliable estimates of heterozygosity and, thus, powerful tools for breeding system and population genetic analyses in polyploid organism

Sex-ratio variation and spatial distribution of nuclear and cytoplasmic sex-determining genes in gynodioecious Thymus praecox across altitudinal gradients

ABSTRACT Background: Females and hermaphrodites co-exist in populations of gynodioecious plant species. Gynodioecy often depends on persistent polymorphisms for both cytoplasmic male-sterility (CMS) genes and nuclear restorers of male fertility. Questions: How do ecological gradients affect population sex ratio? Is sex-ratio variation attributable to variation in the nuclear-cytoplasmic genetic composition of populations? Is there spatial covariation between CMS types and nuclear restorer alleles among populations? Study system: Late-successional populations of gynodioecious Thymus praecox, a species widespread in the European Alps and distributed from subalpine to alpine altitudes. Methods: We surveyed sex-ratio variation of adults along altitudinal gradients. We used offspring sex ratios from open pollination in natural populations and from controlled crosses within and among populations to estimate the diversity and spatial distribution of sex-determining alleles. Results: The proportion of hermaphrodites decreased with increasing altitude. However, offspring sex ratios were constant across altitudes, indicating similar cytonuclear genetic diversity at contrasting adult sex ratios. Sex-determining alleles were geographically widespread, but significantly higher proportions of hermaphrodites produced by withinpopulation crosses compared with those among populations indicated locally adjusted restorer frequencies. Balancing selection may act on sex-determining genes. Our findings further point to a role for environment-dependent selection via the relative maternal fitness of females and hermaphrodites in maintaining the altitudinal sex-ratio variation

Effects of floral neighborhood on seed set and degree of outbreeding in a high-alpine cushion plant

Plants flowering together may influence each other’s pollination and fecundity over a range of physical distances. Their effects on one another can be competitive, neutral, or facilitative. We manipulated the floral neighborhood of the high-alpine cushion plant Eritrichium nanum in the Swiss Alps and measured the effects of coflowering neighbors on both the number of seeds produced and the degree of inbreeding and outbreeding in the offspring, as deduced from nuclear microsatellite markers. Seed set of E. nanum did not vary significantly with the presence or absence of two Saxifraga species growing as near neighbors, but it was higher in E. nanum cushions growing at low conspecific density than in those growing at high density. In addition, floral neighborhood had no detectable effect on the degree of selfing of E. nanum, but seeds from cushions growing at low conspecific density were more highly outbred than seeds from cushions at high density. Thus, there was no evidence of either competition or facilitation between E. nanum and Saxifraga spp. as mediated by pollinators at the spatial scale of our experimental manipulation. In contrast, the greater fecundity of E. nanum cushions at low density was consistent with reduced intraspecific competition for pollinators and might also represent a beneficial effect of highly outbred seeds as brought about by more long-distance pollinator flights under low-density conditions