Outcrossing rates in two self‐compatible,hybridising Rhinanthus species: implications for hybrid formation

The congeners Rhinanthus angustifolius and Rhinanthus minor, two annual hemiparasites pollinated by bumblebees, are known to hybridise in the wild. Both species are self‐compatible, but the capacity for autonomous selfing is higher in R. minor. This suggests a difference in realized outcrossing rates, which have not been determined before in these species. Using microsatellites, both species turned out to have mixed mating systems, but with a much lower multilocus outcrossing rate (0.13) for R. minor compared to R. angustifolius (0.76). We hypothesised that a higher outcrossing rate should lead to a higher chance of heterospecific pollination, and we therefore determined the rate of hybrid formation on each species in an artificial mixed population. Hybrid seeds were produced at low frequency (4.5%), and no significant difference was found between the species. It is therefore likely that post‐pollination processes influence hybrid seed formation to counteract the expected difference in heterospecific pollen deposition. We checked fruit set, seed set and the rate of autonomous selfing in controlled crosses in the greenhouse in 2 years, and found that fruit set (2003) or seed set (2010) were lower in R. angustifolius × R. minor crosses relative to the reciprocal cross. Hybrid seeds produced on R. angustifolius also had a much lower germination rate, so most of the established F₁ hybrid plants have the R. minor cytoplasm. The formation of advanced hybrids depends on pollinator preference, which is biased towards R. angustifolius if present in sufficient numbers, because it offers more rewards.     

Population genetic diversity and species relationships in the genus Rhinanthus L. based on microsatellite markers

The genus Rhinanthus L. is complex, containing many taxonomically unresolved taxa. In this paper we studied genetic variation and species relationships in 15 populations of six Rhinanthus species from three sections. For this purpose, we developed new microsatellite primers for R. osiliensis and used them to investigate genetic variation in two narrow endemics (R. osiliensis, R. javorkae) and in four widespread species (R. rumelicus R. wagneri, R. angustifolius and R. minor). Species‐specific private alleles were found in all species except R. osiliensis and R. angustifolius. The Bulgarian endemic R. javorkae showed the lowest genetic variation, followed by widespread R. minor and Estonian endemic R. osiliensis. Rhinanthus javorkae and R. minor were genetically most differentiated. Section Cleistolemus is weakly structured genetically, indicating close affinity between R. osiliensis, R. rumelicus, R. wagneri and R. angustifolius.     

Shared pollinators and pollen transfer dynamics in two hybridizing species, Rhinanthus minor and R. angustifolius

Gene flow between hybridizing plant species depends strongly on pollinator behaviour, which affects pollen transfer among floral types and reproductive isolation. We examined bumblebee behaviour and pollen transfer between two hybridizing Rhinanthus species that are very similar in ecology and floral traits. The two species, Rhinanthus minor and R. angustifolius, shared similar pollinator guilds and assemblages, but pollinator recruitment and flower visitation rates were higher in R. angustifolius sites, probably because of its higher reward levels and better visibility. When presented with Rhinanthus flowers, bumblebees that previously foraged on R. angustifolius were less prone to visit R. minor inflorescences, while R. minor foragers accepted both species in similar proportions. Although Rhinanthus has been cited as a case of mechanical isolation resulting from interactions between bee behaviour and differences in stigma and anther placement, we found no support for efficient mechanical reproductive isolation. Bumblebees that foraged on R. minor flowers carried more pollen, but pollen placement on their bodies was similar to that of bees that visited R. angustifolius, and cross-specific stigmatic pollen deposition was similar in both directions. However, the asymmetry in pollinator handling time between the two species, due to dissimilar pollen rewards, may have lowered relative heterospecific pollen receipt on R. angustifolius, suggesting that net gene flow resulting from pollen transfer dynamics is more likely towards R. minor, although this effect remains weak and will be most likely counterbalanced by context-based labile pollinator preference.     

Genetic diversity and differentiation in six species of the genus Rhinanthus (Orobanchaceae)

The intricate genus Rhinanthus L. contains many taxonomically unresolved taxa. The assessment of genetic diversity may clarify the differentiation and taxonomic relationships between species and help to determine their mating system, which is important for understanding the future prospects of populations and species. Isozymes were used to assess the mating system, genetic diversity, and differentiation in R. rumelicus, R. osiliensis, R. wagneri, R. alectorolophus, and R. angustifolius from sect. Cleistolemus and R. minor from sect. Rhinanthus. Genetic diversity at species level was found to be higher in widespread R. angustifolius and R. rumelicus compared with insular endemic R. osiliensis. Positive inbreeding coefficient revealed dominant inbreeding in endemic R. osiliensis and R. wagneri. We also detected a significant departure from Hardy–Weinberg equilibrium in some populations of R. rumelicus and R. angustifolius. The common R. alectorolophus and R. minor and several populations of R. rumelicus and R. angustifolius displayed a mixed mating mode. Six studied species divided into three distinct clusters. Rhinanthus alectorolophus was the most differentiated taxon among the investigated species. The species of sect. Cleistolemus formed two distinct groups. The first cluster consisted only of R. alectorolophus while another comprised R. rumelicus, R. osiliensis, R. wagneri, and R. angustifolius, indicating their close affinity. Thus, our isozyme results call into question the monophyly of section Cleistolemus and the taxonomic position of R. alectorolophus.     

Fitness of reciprocal F1 hybrids between Rhinanthus minor and Rhinanthus major under controlled conditions and in the field

The performance of first‐generation hybrids determines to a large extent the long‐term outcome of hybridization in natural populations. F₁ hybrids can facilitate further gene flow between the two parental species, especially in animal‐pollinated flowering plants. We studied the performance of reciprocal F₁ hybrids between Rhinanthus minor and R. major, two hemiparasitic, annual, self‐compatible plant species, from seed germination to seed production under controlled conditions and in the field. We sowed seeds with known ancestry outdoors before winter and followed the complete life cycle until plant death in July the following season. Germination under laboratory conditions was much lower for the F₁ hybrid formed on R. major compared with the reciprocal hybrid formed on R. minor, and this confirmed previous results from similar experiments. However, this difference was not found under field conditions, which seems to indicate that the experimental conditions used for germination in the laboratory are not representative for the germination behaviour of the hybrids under more natural conditions. The earlier interpretation that F₁ hybrid seeds formed on R. major face intrinsic genetic incompatibilities therefore appears to be incorrect. Both F₁ hybrids performed at least as well as and sometimes better than R. minor, which had a higher fitness than R. major in one of the two years in the greenhouse and in the field transplant experiment. The high fitness of the F₁ hybrids confirms findings from naturally mixed populations, where F₁ hybrids appear in the first year after the two species meet, which leads to extensive advanced‐hybrid formation and introgression in subsequent generations.     

Rhinanthus plants found in calcareous fens on Gotland (Sweden): Are they related to Rhinanthus osiliensis from Saaremaa (Estonia)?

Rhinanthus osiliensis is a narrow endemic growing in calcareous spring fens on the island Saaremaa, Estonia. Plants very similar to R. osiliensis have been found in Sweden on the island of Gotland. Fifteen microsatellite markers were used to characterize genetic variation within R. osiliensis and the unknown taxon from Gotland. Results were compared with common Rhinanthus minor and Rhinanthus angstifolius. Our results showed that genetic diversity was remarkably lower in the Rhinanthus sp. from Gotland compared to Estonian endemic R. osiliensis. The microsatellite loci we used distinguished four studied species. In total, 46 species-specific alleles were found. The Neighbor-Joining dendrogram based on Nei's genetic distances, the Bayesian clustering analysis and the principle coordinate analysis showed that R. osiliensis and Rhinanthus sp. from Gotland are genetically differentiated and could not be the same species. In addition, endemic R. osiliensis was closer to widespread R. angustifolius. Further genetic comparison between unknown Rhinanthus sp. and local R. angustifolius and R. minor from Gotland is necessary for determine the species status and checking the possible alternative hypothesis about the origin of the unclear taxon.     

Hybridization in Annual Plants: Patterns and DynamicsDuring a Four-Year Study in Mixed Rhinanthus Populations

Hybridization in annual plants is rare, but their short life cycle provides an excellent opportunity to study the dynamics of hybridization. Hybridization occurs between the annual hemiparasites Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae). Using flower morphology, Kwak (1980) found a prevalence of hybrids close to R. angustifolius in a single population. We aim to find whether this pattern is also found using genetic markers, whether it is generally occurring in mixed populations, and whether these populations are stable over time. We used species-specific genetic markers to determine the number of individuals in a range of hybrid classes in three mixed populations of different ages during four consecutive years. In the young population, F₁ hybrids were found in the first year and mostly hybrids between R. minor and these F₁s in the second year, but in the years after that, hybrids close to R. angustifolius became more abundant. We also found this in the two older populations, where hybrids close to R. angustifolius always occurred in higher frequencies than hybrids close to R. minor. Over time, R. angustifolius strongly increased in frequency in two populations. Patterns of marker presence and absence suggested that advanced-generation hybrids are mainly formed by backcrossing with one of the parents, predominantly R. angustifolius whenever its frequency in the population is higher than 15%. The dynamics of mixed populations depend on the ecological conditions that regulate the presence of the two parental species, and introgression into R. angustifolius seems prevalent.     

Germination characteristics of Rhinanthus minor influence field emergence,competitiveness and potential use in restoration projects

• The facultative root hemi‐parasite Rhinanthus minor is often used in grassland habitat restoration projects to regulate ecosystem structure and function. Its impact on community productivity and diversity as a function of resource supply, sward composition and management has been widely investigated. However, there is a lack of information about the possible influence of seed quality on the efficacy of the hemi‐parasite.
• Ten seed lots from commercial sources were sown in the field and their germination characteristics investigated in the laboratory. Seeds from four lots were also germinated and sown in pots alongside plants of two host species, Lotus corniculatus and Holcus lanatus. Plant establishment, height and flowering density were evaluated for the hemi‐parasite, while plant biomass was measured for both R. minor and its host.
• Two aspects of seed quality influenced the field emergence of seed lots of R. minor, the radicle emergence (%) and the length of the lag period from the beginning of imbibition to germination (mean germination time), which indicates seed vigour. A longer lag period (lower vigour) was associated with higher levels of seedling mortality and lower plant vigour, in terms of plant height and biomass accumulation and was also reflected in the parasitic impact of the seed lots.
• Seed quality, specifically germination and vigour, can influence the establishment, survival, subsequent plant productivity and parasitic impact of R. minor in vegetation restoration projects. Seed quality is discussed as a key factor to consider when predicting the impact of the hemi‐parasite on community productivity and diversity.

     

Dormancy and germination of sixRhinanthus species in relation to climate

The genusRhinanthus (Orobanchaceae) consists of annual hemiparasites that occur in a wide range of climates. Patterns of dormancy and germination were studied for six species sampled in areas ranging from the Pyrenees to Northern Scandinavia, and from sea level up to about 2500 m altitude in the Alpine region. Dormancy was broken by a 2 to 6 months period of cold stratification. Optimal temperature and length of the stratification period appeared to vary between and within species. Two patterns of dormancy and germination were distinguished. Seeds of the first group, including the widely distributedR. minor andR. angustifolius, further referred to as the LW group, require a relatively long period of cold stratification. Moreover, their germination is accelerated if they are subjected to a widening range of higher temperatures in the last weeks of the stratification period. In the other species (R. alectorolophus, R. glacialis, R. mediterraneus and perhapsR. antiquus) the release of dormancy is completed in a rather short period. Higher temperatures in the last weeks of the stratification period hardly affect the germination process of this SN-group, with short dormancy and no accelerated germination at higher temperatures. In both groups, temperatures above the low values during stratification reduce germination percentages and induce secondary dormancy in non-germinated seeds. The effects strongly vary with timing and temperature. The patterns have a genetic basis and seem to be species-specific. They do not vary with climate conditions, since samplesR. minor, whether collected in sub-arctic or sub-alpine areas or at sea level, generally react according to the LW pattern, and samples ofR. alectorolophus from areas at sea level up to montane regions according to the SN one. The ecological significance of the various stratification requirements and effects of higher temperatures on germination is discussed in relation to the local climate of the species and the evolutionary history of the genus.     

The significance of pollination time and frequency and of purity of pollen loads for seed set in Rhinanthus angustifolius (Scrophulariaceae) and Viscaria vulgaris (Caryophyllaceae)

Summary The effects of time of the day and frequency of application and of purity of artificially deposited pollen loads on fruiting and seed set were studied in Rhinanthus angustifolius (diurnally visited by bumblebees) and Viscaria vulgaris (diurnally visited by bumblebees, nocturnally by sphingid moths).Time of the day did not influence pollination success in either species. Increase of pollination frequency increased fruiting and seed set in Rhinanthus but had no affect on Viscaria. Five successive artificial pollinations were needed to achieve seed set equal to that observed naturally in Rhinanthus while a single artificial pollination was sufficient in Viscaria. Mixing Lupinus and Viscaria pollen did not reduce fruiting and seed set in Viscaria. The results are discussed in relation to observed seed sets in early and late flowers, and small and large patches of Viscaria vulgaris and among Rhinanthus flowers in populations of different densities.     

Germination patterns throughout an insular altitudinal gradient: The case of the Macaronesian endemic plant Rubia fruticosa Ait. (Rubiaceae) in El Hierro (Canary Islands)

Seed germination percentage and rate of Rubia fruticosa, an endemic Macaronesian shrub was assessed with seed lots collected at three levels of an altitudinal gradient (200–800 m asl) located in El Hierro Island (Canarian Archipelago). Both seeds collected from control plants and those found in lizard droppings were studied. A significant decrease in the germination percentage was observed with control seeds and those seeds which are previously eaten by lizards if collected at 800 m asl. This indicates that R. fruticosa is a plant whose optimal germination environment is located between 200 and 500 m asl, occurring in higher altitudes only under sub-optimal conditions. The effect of lizard gut on R. fruticosa seeds was variable in the two study years: while germination percentage of seeds having passed through the lizards was not significantly different to that of control seeds in the first year, an increased germination was found for the animal-treated seeds in all three altitudinal zones in the second year. In general, germination rate of control seeds was rather variable between years. However, improvement of R. fruticosa seed germination caused by lizards may be important for its fitness; and a co-evolution may have occurred, since lizards and seeds of this endemic plant have been intensively interacting for millions of years in the lower zones of the Canary Islands.     

Maintenance of species integrity in sympatrically occurring Rhinanthus minor and R. serotinus in The Netherlands

Summary In certain localities, R. minor and R. serotinus grow sympatrically and the flowering-periods overlap. The species hybridize but can still be recognized as distinct taxonomic entities. In the field s x m crosses can be expected to occur more frequently than the reverse, on the basis of flower morphology and pollinator (bumblebee) efficiency. Observation of pollen germination, pollen tube growth, seed set, and seed germination in artificial, reciprocal crosses permits the conclusion that a single m x s pollination leads to more offspring than a single s x m pollination. The two species are isolated from each other by a series of mechanisms none of which is 100% effective by itself, but their combined action comes close to that figure. The leakages in the ethological barrier against hybridization are closed, partly, by physiological mechanisms.     

Ecotypic differentiation reveals seed colour‐related alkaloid content in a crop wild relative

• Crop wild relatives can be a useful source of genotypes that maximise crop survival and yield in specific habitats. Lupinus angustifolius is an annual forb with crop varieties derived from a narrow genetic basis but that are cultivated worldwide. Its seeds have high nutritional value, but they naturally contain alkaloids with anti‐nutritive factors. The study of its wild populations can be useful to find genotypes that contribute to higher climate resilience and greater yield under stressing environmental conditions.
• Using a common garden, we evaluated ecotypic differentiation in four natural populations from two contrasting latitudes in terms of plant biomass, seed mass and number, alkaloid content in seeds for the three main alkaloids present in the plant and seed colour, including its possible influence on post‐dispersal predation. Correlations among traits were also assessed.
• We found differences among populations for all traits except final biomass. Northern populations had lighter seeds and a tendency to yield more seeds when they produced white seeds, compared to southern populations and variegated seeds. Regardless of latitude, populations showed differences in alkaloid concentration, with all three alkaloids found generally in high or low concentrations in each population. Proportion of white seed morphs varied in each population. Seed colour did not influence predator preference. In addition, white seed colour was related to a low alkaloid content.
• Our results evidence the existence of natural ecotypic differentiation in L. angustifolius not only due to latitudinal range, but also to local environmental factors. White seed coat colour could be used as a visual clue for identification of low‐alkaloid genotypes, a priority trait in L. angustifolius breeding programmes.

     

Does gall midge larvae cause pre-dispersal seed mortality and limit cornflower population growth?

Many kinds of pests can reduce seed production. Some directly attack seeds before they are released, and some are hosted by the fruit and impact seed ripening and viability indirectly. Pre-dispersal seed mortality may have strong effects on plant population dynamics and evolution. Our goals were to determine to what extent insect-mediated pre-dispersal seed mortality contributes to population-level declines of cornflower, Centaurea cyanus L. We recorded occurrence and abundance of seed-feeding insects on flower heads in twelve cornflower populations. We measured flower head size, number of disc florets, seed production, and seed viability and germination. Larger flower heads had proportionally fewer healthy seeds. Although we observed no visible damage to the C. cyanus seed, the presence of gall midge (Cecidomyiidae) larvae inside the flower head correlated with four times fewer viable seeds. It seems that gall midges could have a significant impact on ovule fertilization, seed abortion and viability of fully developed cornflower seeds. The higher rate of aborted seeds in the presence of gall midge larvae could have been because the larvae extracted resources from the seeds, or because the larvae repelled pollinators. The viability of apparently healthy seeds was 40% lower in flower heads that contained larvae and/or aborted seed. Insect-mediated pre-dispersal mortality could select against evolution toward larger flower head, and have detrimental consequences on seed number, viability and germination, all of which could limit the spread of C. cyanus populations.     

Detecting hybridization in mixed populations ofRhinanthus minor andRhinanthus angustifolius

Rhinanthus minor andRhinanthus angustifolius are known to hybridize in mixed populations in nature. These hybridization events can have important evolutionary consequences. The development and use of species-specific RAPD and ISSR markers allowed the detection of hybrid individuals not always distinguishable with morphological characters. Two mixed populations of different ages were studied. In a young mixed 2-year-old population, both individuals of the two parental species and F₁ hybrids were found using genetic analysis, showing that hybridization occurred rapidly. Flower morphology of F₁ hybrids was too variable to distinguish all these hybrids from the parental species. This morphological variability of F₁ hybrids was also confirmed in artificial crosses in the greenhouse. In an old and no longer mixed 30-year-old population, onlyR. angustifolius plants and a few genetically introgressed individuals close toR. angustifolius were present. Genetic markers showed traces of past hybridization and introgression. Unidirectional introgression ofR. minor intoR. angustifolius with the complete disappearance ofR. minor from this population was observed.     

Dependency of seed dormancy types on embryo traits and environmental conditions in Ribes species

The hypothesis that seed dormancy may be dependent on environmental conditions and seed morphological traits was tested for six Ribes species, across an altitudinal gradient of 1300 m and a longitudinal separation of 120°. Embryo measurements and seed germination experiments were conducted for R. alpinum L., R. hudsonianum Richardson var. petiolare (Douglas) Jancz., R. nevadaense Kellogg, R. roezlii Regel var. cruentum (Greene) Rehder and R. speciosum Pursh, and data taken from the literature for R. multiflorum Kit. ex Schult. ssp. sandalioticum Arrigoni. Germination was compared with seed viability to reveal proportional seed dormancy, which was then correlated to seed/embryo morphological traits and these traits related to the seed provenance environment. The embryos of all the investigated species are linear underdeveloped and all had a morphological component of seed dormancy (MD). Seeds of R. roezlii, R. hudsonianum and R. nevadaense required a temperature and/or hormone pre‐treatment in order to germinate, highlighting morphophysiological seed dormancy (MPD). Seed dormancy was found to be strongly negatively correlated with embryo length, but not with embryo to seed (E:S) ratio or seed mass. Initial embryo length was positively related to mean annual temperature. Seed dormancy in the investigated Ribes species could be quantified and predicted by the interaction of embryo traits and environmental conditions. This approach may be helpful in assessing and predicting seed dormancy in the Ribes genus and in other genera and families with underdeveloped embryos.     

Impact of hemiparasitic Rhinanthus angustifolius and R. minor on nitrogen availability in grasslands

Root hemiparasites like Rhinanthus angustifolius C.C. Gmel and R. minor L. have a potential to accelerate the restoration of semi-natural grasslands because they may decrease above-ground biomass of the vegetation. This, in turn, may be beneficial for species diversity. It is known that hemiparasites often accumulate high nutrient concentrations in their above-ground parts, resulting in high quality litter. Because of the short life cycle of many parasitic plants, litter is released early in the season and the main part is not removed from the grassland by hay-making. This has been shown to yield an increased nutrient availability locally. We performed an introduction experiment with R. angustifolius and R. minor in three semi-natural grasslands in Flanders (Belgium). In the second year after sowing, the above-ground nitrogen (N) content of the grasses and of the potential host vegetation (excluding the hemiparasite), was increased in the parasitized plots. The reduction of grass (and legume) above-ground biomass in parasitized plots resulted in a decrease in the total above-ground N uptake of grasses, host and total vegetation (ex- and including the parasite, respectively) of the parasitized plots compared to the control. Furthermore, with a tracer experiment (¹⁵N), we demonstrated that the N from the added tracer was relatively less available in parasitized plots, suggesting larger soil N pools in these treatments. This is probably the consequence of increased mineralization, resulting from the high-quality, parasitic litter. Further experiments should be conducted to investigate the impact of hemiparasitic Rhinanthus spp., e.g. on the availability of other nutrients such as phosphorus.     

Size hierarchy and mineral status of Rhinanthus angustifolius populations under different grassland management regimes

The size hierarchy and mineral concentrations of adult plants in flower and their seed crop have been investigated in three populations of Rhinanthus angustifolius under a grassland management regime consisting of haying without fertilization. The effect of this type of management on the nutrient budgets of soil or Rhinanthus plants was shown to be small initially, i.e. up to the twelfth year of changed management. Although the mean biomass of plants significantly diminished by 30% in this period, their nutrient concentration was not strongly affected. Small plants consistently had lower concentrations of N and Na than large plants. Prolongation of this management regime beyond twelve years let to a general reduction in the phosphorus concentration of the plants. An observed increase in the concentrations of K, Mn, and Cu, especially in small plants, is tentatively ascribed to an imbalance in mineral nutrition.Nomenclature of vascular plants follows Van der Meijden et al. (1983). Flora van Nederland.     

Seed bank of Littorella uniflora (L.) Asch. in the Czech Republic,Central Europe: does burial depth and sediment type influence seed germination?

Littorella uniflora (Plantaginaceae) is a scarce and declining aquatic plant in Europe. Its population has been strongly reduced by changes in fishpond management (eutrophication) in the Czech Republic since the 1950s. We studied its seed bank in both recent (n = 8) and historical localities (the last found from 1972 to 2000; n = 10) and tested the effects of sediment type and burial depth on seed germination using extracted seeds from two recent populations. The seeds were found in 60% of the historical localities, mostly in low densities (≤6 seeds per 3.75 l of sediment), and also in 100% of the recent localities in various densities (8–1390 seeds per 3.75 l of sediment); however, low germination rates (0–13.3%) were estimated. The seeds germinated best on wet filter paper, followed by nutrient-rich fishpond sediment, but poorly on sand mixed with different substrates. Burial depth significantly affected seed germination. The seeds germinated only on the soil surface and at the depth of 1 cm but no seed germinated at the depth of 3 and 5 cm. Besides low water transparency, both high rate of sediment accumulation and the absence of summer drainage may endanger L. uniflora populations in fishponds.

     

Heat increases germination of water-permeable seeds of obligate-seeding <Emphasis Type="Italic">Darwinia</Emphasis> species (Myrtaceae)

We examined the response of seeds to heat in four geographically restricted and one widespread species of shrubby Darwinia from the fire-prone region of southeastern Australia. These shrubs are killed by fire and rely on seed germination after a fire to maintain populations. We replicated the germination trials across several sites and several fruiting seasons for most species. Seeds had a high level of viability and were largely dispersed in a dormant state, except in D. glaucophylla, where seed dormancy varied significantly across fruiting seasons. The indehiscent fruit of all species readily imbibes moisture when wet and seeds are not considered to be ‘hard-seeded’. All species had increased seed germination in response to a limited range of heating temperatures (generally 80–100°C). Higher temperatures killed increasing proportions of seeds. This pattern was broadly consistent across species, population and seasons, although the proportion of seeds whose germination was promoted by heat varied from high (D. diminuta, D. fascicularis, D. glaucophylla) to moderate (D. biflora, D. procera). Our work highlights the importance of heat as a mechanism for influencing germination in species that are not hard-seeded. Consequently, soil temperatures during a fire should strongly influence post-fire germination levels in Darwinia. The roles of other cues that promote germination, i.e. smoke, seasonal temperatures and their interactions with heat, remain to be investigated.