High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae).

Several hypotheses have been proposed to explain the rich floristic diversity in regions characterised by nutrient-impoverished soils; however, none of these hypotheses have been able to explain the rapid diversification over a relatively short evolutionary time period of Grevillea, an Australian plant genus with 452 recognised species/subspecies and only 11 million years of evolutionary history. Here, we hypothesise that the apparent evolutionary success of Grevillea might have been triggered by the highly efficient use of key nutrients. The nutrient content in the seeds and nutrient-use efficiency during early seedling growth of 12 species of Grevillea were compared with those of 24 species of Hakea, a closely related genus. Compared with Hakea, the Grevillea species achieved similar growth rates (root and shoot length) during the early stages of seedling growth but contained only approximately half of the seed nutrient content. We conclude that the high nutrient-use efficiency observed in Grevillea might have provided a selective advantage in nutrient-poor ecosystems during evolution and that this property likely contributed to the evolutionary success in Grevillea.

Mediterranean climate regions, such as those in southwest Western Australia (SWA), the South African Cape region, and the Mediterranean Basin, have particularly diverse species and endemic rich flora and are conpan class="Disease">sidered globally significant12. Numerous hypn>otheses have been propn>osed to expn>lain the floristic diversity of these biodiversity hotspn>ots3. For example, Hopn>per propn>osed that landscape age contributed to the high diversity in SWA4, and Lengyel et al. repn>orted higher global diversification rates in myrmecochorous lineages relative to their sister groups and spn>eculated that myrmecochory contributed to the high diversification of SWA flora5. However, none of the current hypn>otheses can expn>lain the extreme diversity of many spn>ecies-rich genera in Australia, such as n>an class="Species">Grevillea in the family Proteaceae.

Grevillea is a predominantly Australian genus containing 362 currently recognised species or 452 taxa, which includes subspecies, with five species present in New Guinea, New Caledonia, and Sulawesi6. The evolutionary time period for species accumulation has emerged as a significant expn>lanatory variable for biodiversity34, and this expn>lanation is supported by fossil evidence7. Grevillea is one of the youngest genera, with only c. 11.2 million years of evolutionary history8, and it is the most species-rich genus in the Proteaceae family; thus, the greater-time-for-speciation theory does not explain the high numbers of species in this genus. However, Hopper proposed that taxa in old and climate-buffered landscapes, including Grevillea and other genera, such as the closely related genus Hakea, typically have a low extinction rate4. Grevillea species are generally dispersed by ants, which has been hypothesised to be a key evolutionary novelty that may account for the high diversification rate in this genus5. However, many other plant lineages in SWA, typically species that co-occur with Grevillea and are also dispersed by ants, have markedly fewer species within their genera. For example, ant-dispersed Daviesia (Fabaceae) diversified into 127 species over c. 26 million years, whereas ant-dispersed Bossiaea (Fabaceae) diversified into 80 species within c. 10 million years of evolutionary history3. Therefore, myrmecochory alone is unlikely to contribute to the high diversification rate in Grevillea. With a relatively short evolutionary history of 11.2 million years and as the third largest plant genus in Australia after Acacia and Eucalyptus, Grevillea probably has one of the highest net speciation rates of all large Australian plant clades.

Species in the family pan class="Chemical">Proteaceae are highly adapted to and became diversified on the most nutrient-impn>overished soils observed worldwide. Highly infertile soils have long been demonstrated to influence biological and physiological responses and speciation patterns49. Spn>ecies have developn>ed diverse morpn>hological and physiological adaptations to limited nutrient availability in order to successfully establish in nutrient-limited soils10111213. These species typically exhibit highly efficient photosynthetic pan class="Chemical">phosphorus use14.

In southwestern Australia, seed germination and successful seedling establishment predominantly occurs following winter rains after summer fires. Seed-stored nutrients, particularly pan class="Chemical">nitrogen (N) and n>an class="Chemical">phosphorus (P), are crucial for the emergence and early growth of seedlings, and the efficient use of these nutrients is critical for the successful establishment of seedlings in severely nutrient-poor and water-limited environments. Nutrient limitation has recently been proposed as a potential driver for variation in species diversity15. Lambers et al. suggested that high plant species diversity on infertile soils, particularly in species-rich biomes, is associated with functional diversity for nutritional strategies, including nutrient-use efficiency16. Despite extensive research on plant species richness in relation to nutrient availability, few studies have attempted to explore the relationship between nutrient-use efficiency and speciation patterns. Here, we compare the nutrient concentration and content in seeds and nutrient-use efficiency during early seedling growth of Grevillea with those of the closely related genus Hakea, another Australian genus with 149 species that efficiently use seed nutrients during early seedling growth16. Our study aimed to provide unique insights into the potential mechanisms underlying the evolutionary success (i.e., high diversification rate) of Grevillea.

Results

Seed mass varies markedly in both Grevillea (2.8–244 mg, 116 species/subspecies surveyed) and Hakea (2.6–501 mg, 153 species/subspecies surveyed), and Grevillea has a slightly higher median seed mass than Hakea (24.2 vs. 18.7 mg). The average seed masses of the 30 Grevillea species and 29 Hakea species used in the present study were statistically similar (Table 1). Nutrient assays on the 30 Grevillea species and 29 Hakea species revealed significantly higher concentrations of N and P in the Hakea seeds than in the Grevillea seeds (Table 1; Table S1). The total N and P contents per seed in Hakea were also much higher than in Grevillea (N: 2.66 vs. 0.56 mg, P = 0.002; P: 0.47 vs. 0.17 mg, P = 0.008; Fig. 1).

Table 1

Seed mass and nutrient concentration (N and P), seed generation, and seedling growth in Hakea and Grevillea.

	Hakea	Grevillea	t-test, P value
Number of species	149a	362b	–
Age (million years)c	15.8	11	–
Seed mass (mg)d	30.4 (2.6–501.0)	34.9 (2.8–244.0)	–
Seed mass (mg)e	33.3 ± 29.2	39.5 ± 15.5	t = −0.40, P = 0.703
Seed N (%)	7.61 ± 1.36	3.53 ± 0.79	t = −1.09, P < 0.001
Seed P (%)	1.36 ± 0.38	0.63 ± 0.23	t = −1.48, P < 0.001
Germination rate	0.82 ± 0.12	0.32 ± 0.15	t = −5.07, P < 0.001
Days to first true leaf	32 ± 6	31 ± 5	t = 0.02, P = 0.942
Number of leaves	16.7 ± 5.4	8.3 ± 1.2	t = −3.57, P = 0.002
Root/shoot biomass	1.67 ± 0.71	1.34 ± 0.55	t = −1.27, P = 0.218
Total dry biomass (g)	0.54 ± 0.39	0.28 ± 0.13	t = −1.09, P = 0.093
Root length (mm)	468 ± 198	580 ± 181	t = −1.48, P = 0.152

a6;

b6;

c8;

d28;

e:species examined in this study.

Figure 1

Comparison of nitrogen (N) and phosphorus (P) content per seed of the test species of Hakea and Grevillea.

The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile.

The seeds of the 25 Hakea species that were tested readily germinated without requiring pre-treatment, and they had an overall germination rate of 82%. pan class="Species">Grevillea seeds (29 species) pre-treated with smoke pan class="Chemical">water exhibited a significantly lower germination rate of 32% (Table 1). However, for the 24 Hakea and 12 Grevillea species that yielded a sufficient number of germinants for growth comparisons, the time period to expand the first true leaves was similar, although Grevillea plants had significantly fewer leaves after growing for 100 days. Both Hakea and Grevillea diverted more resources to the roots than to the shoots, with a statistically similar root:shoot ratio. Grevillea and Hakea species accumulated similar amounts of dry biomass and had similar root depths in the soil after 100 days of growth (Table 1).

Further analysis of the seed nutrient content and seedling growth rate revealed that pan class="Species">Grevillea species had significantly higher nutrient-use efficiencies (Table 1; Fig. 2). Compn>ared with Hakea, for each unit of pan class="Chemical">P in the seed, Grevillea accumulated 1.6-fold more dry biomass and had roots that descended 2.9 times deeper into the soil, whereas for each unit of N in the seed, Grevillea accumulated 1.4-fold more dry biomass and had taproots that descended 3.1 times deeper into the soil (Table 1, Fig. 2).

Figure 2

The nutrient-use efficiency (N and P) of the test species of Hakea and Grevillea.

The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile.

Discussion

The seed nutrient concentration in pan class="Species">Grevillea repn>orted in the present study, particularly with respn>ect to n>an class="Chemical">P, was only half of that reported in previous scattered studies. For example, Hocking reported an average of 11.4 ± 2.6 mg.g−1 P for ten Grevillea species17. The results of the present study on 30 species reported an average of 6.3 ± 2.3 mg.g−1. This discrepancy is unlikely to be a consequence of methodological errors in our analysis because the 29 Hakea species were assayed in the same batch in the present study, and they yielded an average P content of 13.6 ± 3.8 mg.g−1, which is similar to the 15.1 ± 4.2 mg.g−1 P concentration reported for 13 species from ten previous studies (except Hakea pycnoneura, which was reported to have an unusually high P concentration of 36 mg.g−1)18. Interestingly, although the N and P concentrations in the seeds were lower in Grevillea than in Hakea, the N:P ratios in both taxa groups were similar. Notably, the lower seed N and P concentrations in Grevillea relative to those in Hakea did not reflect dilution by seed mass because the tested species in the two groups had similar seed masses.

With only half the seed nutrient content compared with species from the closely related pan class="Disease">genus Hakea, the species in pan class="Species">Grevillea achieved similar levels of seedling growth during the early stages. Indeed, both groups of plants expanded their first true leaves within a similar time, accumulated similar amounts of dry biomass, and diverted similar proportions of resources to the shoots and roots. Consequently, the results of this analysis suggest that compared with Hakea, Grevillea species use nutrients significantly more efficiently than do Hakea. This efficiency represents a clear ecological advantage over Hakea because Grevillea species can accumulate twice as much dry biomass and descend their roots three times deeper into the soil for every unit of nutrient in their seeds.

Seed-stored nutrients (N and pan class="Chemical">P) are crucial for the successful establishment of seedlings in nutrient-impn>overished landscapes. The rapid descent of the taproots to reach more reliable soil moisture reserves prior to the onset of summer droughts is critical for the survival of seedlings, particularly in Mediterranean climates with dry summer conditions19. Using fewer nutrients, pan class="Species">Grevillea species can grow roots equal in length to those of Hakea and survive dry Mediterranean-type summers equally as well, and these characteristics provide a selective advantage to Grevillea compared with other organisms that might require additional nutrients in nutrient-impoverished soils.

Seed production is inversely proportional to the per-seed reserve of nutrients, particularly pan class="Chemical">P, in nutrient-impoverished soils20. In the fire-prone environments of SWA, which are generally characterised by poor soil, respn>routers (popn>ulations that predominantly regenerate through spn>routs from the trunk or underground organs) typn>ically produce fewer seeds than nonspn>routers (popn>ulations that predominantly regenerate through seeds)21. However, no significant differences were observed in the n>an class="Chemical">P content between nonsprouting and resprouting species growing on P-impoverished soil in a survey of 41 species in SWA18. The lower nutrient requirements for seedling growth in Grevillea relative to Hakea, which has much higher nutrient (N and P) concentrations, might allow Grevillea species to produce more seeds regardless of the post-fire regeneration mode. Because of challenges in gauging the size of the soil seedbank, systematic surveys of Grevillea seed production are rare, although Pickup et al. reported a large seedbank for Grevillea rivularis at 193 ± 73 seeds m–2 22. Because of its capacity to grow to 2.5 m tall and 3 m wide, this plant species has the potential to produce up to 2000 seeds per plant, which is much higher than that of most Hakea species23. Levin24 proposed that propagule-rich lineages are likely to have high speciation rates because the increased number of seeds increases the opportunities for ecological and geographical speciation, and this hypothesis could also be true for Grevillea. Interestingly, high propagule pressure has also been proposed as one of the mechanisms facilitating successful invasion25.

Species in the pan class="Chemical">Proteaceae family grow well in the world’s most nutrient-impoverished landscapes, particularly pan class="Chemical">P-impoverished soils. Apart from diverse and efficient nutrient-acquisition strategies26, high nutrient-use efficiency is likely another key adaptation. Recent studies have observed high photosynthetic P-use efficiency in SWA Proteaceae species10. Lambers et al. reported that Proteaceae species such as Hakea species (Grevillea species were not included) from severely P-impoverished soils extensively replace phospholipids with galactolipids and sulpholipids during leaf development to achieve a high photosynthetic P-use efficiency, and proposed the “sulphur-for-phosphorus” hypothesis16. Sulpice et al.27 observed that Proteaceae species grow with very low levels of ribosomes, particularly plastidic ribosomes, at early stages of leaf development. Many of the above studies involved Hakea species, although they rarely included Grevillea species; thus, additional research is required to test whether Grevillea have developed peak nutrient-use efficiency.

It can be speculated that once pan class="Species">Grevillea overcome the barriers impn>osed by the two most limiting factors to its developn>ment, low soil nutrient availability and periodic pan class="Chemical">water availability, these plants can invade most habitats. Indeed, species of Grevillea have been observed throughout Australia. High nutrient-use efficiency has interacted with multiple factors, including myrmecochory, a predominantly sexual reproduction strategy, and a high seed output, to allow the Grevillea genus to diversify into a group with 452 species/subspecies within only 11 million years.

Methods

The seeds of pan class="Species">Grevillea and Hakea used for the nutrient analyses and germination and seedling growth compn>arisons were acquired from Nindethana Seed Service (Albany, Western Australia, Australia). Genus-wide seed mass information was extracted from the Seed Information Database28. The nutrient analysis was conducted on seeds from 59 species (29 Hakea and 30 pan class="Species">Grevillea, Table S1). These species cover the major intra-generic taxonomy sections and provenances; therefore, they represent a wide diversity of the respective genera. For each species, up to 20 g of dry seeds were analysed for N and P concentrations at the ChemCentre (Perth, Western Australia, Australia) using the combustion method for N and inductively coupled plasma spectrometry for P.

A total of 52 species (25 Hakea and 27 pan class="Species">Grevillea species, Table S1) were used in the germination trials. Each species had three repn>licates, and there were upn> to 30 seeds per repn>licate (depn>ending on seed size). Each repn>licate was contained within a 120-mm sterile pan class="Chemical">Petri dish with 2 layers of sterilized Whatman Grade 1 filter paper and sealed with Parafilm. Petri dishes with Hakea seeds were filled with 5 mL autoclaved deionised water, whereas those with Grevillea seeds (because of inherent dormancy) were filled with 5 mL 10% smoke water solution (Regen 2000 Smokemaster); previous studies have suggested that Grevillea seed germination is enhanced by smoke stimulation29. To reduce the risk of fungal growth throughout the germination experiment, the smoke water was passed through a 0.20-μm filter. Germination was conducted in an environmental chamber at a constant 15 °C with a 12 hour light/dark cycle. The germinants (seeds with radicle emergence ≥1 mm) were counted and recorded every two days for a period of up to 60 days. Seeds that did not germinate were assessed for viability using a cut test to visually assess the health of the embryo. The Petri dishes were randomised within the environmental chamber in each experiment.

For each of the species germinated in the above-described germination phase (12 pan class="Species">Grevillea and 24 Hakea species, with no or minimal germination recorded in some species), ten germinants (or fewer when available) were individually planted in pan class="Chemical">PVC tubes (100 cm in length and 5 cm in diameter) with a substrate of washed white sand (with no nutrients) and placed in a hoop house. The seedlings were watered (20 mL) every 2 to 3 days and grown for 100 days (from late winter to spring 2014) prior to harvesting. The measurements taken at harvest were leaf number, shoot length, root length and fresh and dry shoot and root mass. The dry biomass of the shoot and root was recorded after the samples were oven dried at 70 °C for 48 hours.

The nutrient-use efficiency (N and P) was defined as the amount of biomass produced or root length gained per unit of nutrient (mg) in the seeds. The nutrient-use efficiencies of Hakea and Grevillea were compared. Comparisons of the parameter values between Grevillea and Hakea were analysed with a t-test and implemented in PAST30. P < 0.05 was considered to represent statistical significance.

Additional Information

How to cite this article: He, T. et al. High nutrient-use efficiency during early seedling growth in diverse pan class="Species">Grevillea species (pan class="Chemical">Proteaceae). Sci. Rep. 5, 17132; doi: 10.1038/srep17132 (2015).