We have shown previously that B. ilicifolia demonstrates preferential outcrossing and strong early acting inbreeding depression, where outcrossed seedlings are more vigorous than selfed seedlings on native soils (Heliyanto et al., 2005). In the present study, the intensity of inbreeding depression was found to be as high as 62% for seed germination and 37% for projected leaf area. These measures underestimate the true strength of inbreeding depression in B. ilicifolia. For example, seed set following self-pollination by hand is <30% that following cross-pollination by hand, and self-seedling survival following fungal attack was less than half that of outcrossed seeds (Heliyanto et al., 2005). Ultimately, survival to 16 weeks of age for selfed progeny under glasshouse conditions was approximately 7% of that of outcrossed progeny, equating to an inbreeding depression of 93% (Heliyanto et al., 2005). Inbreeding depression is almost certainly more severe under natural field conditions than in the glasshouse conditions of the current experiment (Dudash, 1990). Therefore, few, if any, selfed offspring are expected to survive to reproductive age.
Consequently, elevated inbreeding may severely impact on the short-term viability, and long-term evolutionary potential, of small fragmented populations of B. ilicifolia. Although realized outcrossing rates are not yet known for B. ilicifolia, other species in the genus are typically completely, or highly, outcrossed (Goldingay & Carthew, 1998). In the closely related species B. cuneata, outcrossing rates varied between 0.65 and 0.95, with increased inbreeding detected in small, highly disturbed populations (Coates & Sokolowski, 1992). As a result, high outcrossing rates might be expected in B. ilicifolia populations, but small, and particularly recently fragmented, populations may show higher selfing rates and increased inbreeding (Sampson et al., 1989; Coates & Sokolowski, 1992; England et al., 2001).
We also demonstrate that inbreeding effects extend beyond the comparison of self vs. outcross progeny. Although not significantly different across all individual measures, we found a consistent tendency of superior performance of seedlings produced by wide outcrossing compared with seedlings produced by outcrossing between plants within the study population. Wide outcrossing can result in heterosis and/or outbreeding depression, and these effects can vary from F1 to F2 generations, and beyond (Waser, 1993; Hufford & Mazer, 2003). Our study was restricted to the F1 generation, and heterotic effects observed could be maintained, or hybrid breakdown may occur, in F2 or even F3 generations (Fenster & Galloway, 2000; Keller et al., 2000). Heterosis and outbreeding depression are obviously relative terms, influenced by the extent of inbreeding depression within populations (Fenster & Galloway, 2000). The relative performance of narrow outcross seedlings to wide outcross seedlings suggests that the study population of B. ilicifolia is inbred, with heterosis a consequence of wide outcrossing in F1 seedlings. Outbreeding depression has not been detected, suggesting that an optimal outcrossing distance (Price & Waser, 1979; Waddington, 1983; Waser, 1993), at least for the B. ilicifolia study population, lies at or beyond the genetic distance to the pollen source population 30 km away.
Increased ecological amplitude following wide-outcrossing
In the current study, we have extended the assessment of inbreeding depression on a common or local substrate to also assess the relative performance of the products of wide outcrossing to narrow outcrossing on nonlocal substrates and stress conditions. Here, we demonstrate that the outcrossed products of mating between individuals within a typically small and relatively isolated population of B. ilicifolia show a significant decline in vigour when grown on non-native lateritic soils, with biomass approximately half that of seedlings grown on their native sandy soils. In contrast, the outcrossed products of mating between individuals from populations 30 km apart showed no decline in biomass when grown on non-native lateritic soils compared with their performance on native sandy soils. This is despite the observation that the growth of all seedlings in laterite, irrespective of pollen source, was inhibited when younger, possibly due to reduced root growth due to high soil bulk density. By harvest though (when seedlings were 46 weeks old), wide-outcrossed seedlings had recovered, possibly due to increased rhizosphere carboxylate exudation, whereas narrow outcrossed seedlings had not.
These results suggest that even complete outcrossing between individuals within a typical population of B. ilicifolia, reinforces narrow ecological amplitude, whereas heterosis, following wide outcrossing between individuals from genetically differentiated populations, can increase ecological amplitude. Our results suggest that, in the absence of wide outcrossing, there is an interaction between narrow ecological amplitude, small relatively isolated populations and mating that acts to prevent the wider distribution of this species, and may contribute to local endemism more generally. Although wide outcrossing has been shown to play an important part in the ‘genetic rescue’ of locally inbred populations (Richards, 2000; Ingvarsson, 2001; Tallmon et al., 2004), we suggest that wide outcrossing, facilitated by inter-population dispersal, may provide a natural mechanism for increasing ecological amplitude, and extending the distribution beyond current environmental constraints, of species. An ability to increase the range or ecological amplitude of a species will depend on a multitude of factors, including the frequency of wide outcrossing, the strength of heterosis in the products of wide outcrossing and a balance between population genetic differentiation and inbreeding within small fragmented populations. A capacity for long-distance dispersal of pollen and seed, thereby facilitating wide outcrossing, has been demonstrated in some Banksia species (Coates & Sokolowski, 1992; He et al., 2004)
Although widely outcrossed seedlings tended to outperform narrowly outcrossed seedlings, there was no effect of native and non-native sandy soil on the performance of seedlings from within the same progeny class. This result, the measured physical and chemical soil properties, and similar patterns of rhizosphere carboxylate exudation, suggest that, in terms of plant performance, the native and non-native sandy soils are similar, and reinforces the observation that depth to groundwater is the key limiting factor affecting the distribution of B. ilicifolia (Groom et al., 2001; Groom, 2004). Declining B. ilicifolia populations have been linked to groundwater drawdown and/or below-average annual rainfall (Groom et al., 2000). For example, up to 80% reduction in B. ilicifolia tree numbers has been observed when groundwater levels fell by 2 m between two consecutive summers, in conjunction with extremes in summer temperature (Groom et al., 2000). Thus, heterosis following wide outcrossing is unlikely to translate to significant range expansion where access to groundwater is limiting. Genetic rescue, though, may play an important role in increasing the resilience of inbred populations to harsh summer conditions. Field trials over many years with relatively inbred and outbred progeny are required to assess this suggestion. However, the ideal situation of conducting these experiments over two or three generations (Fenster & Galloway, 2000) is unlikely to be achievable in this long-lived, slow-growing species.
In contrast to B. ilicifolia, two congeneric species, both widespread co-dominant overstorey trees, Banksia attenuata and B. menziesii, occur in very large, more or less continuous populations, showing wide ecological amplitude by inhabiting a wide range of topographical positions within the landscape from dune crest to low-lying areas. The impact of groundwater drawdown and harsh summer conditions on these two species is far less than that for B. ilicifolia (Groom et al., 2001). Although further testing is needed, these observations suggest that these robust plants are possibly relatively outbred, with large populations avoiding the genetic problems associated with small isolated populations, emphasizing the relationship between genetic variation, inbreeding, population size and isolation, and ecological amplitude. Some populations of B. attenuata and B. menziesii are known to be completely outcrossing (Scott, 1980), and B. menziesii is self-incompatible (Ramsey & Vaughton, 1991). Although we do not yet have data on genetic variation for B. ilicifolia and its more widespread congeners, widespread plant species generally show higher genetic variation than their rare congeners (Gitzendanner & Soltis, 2000). However, many exceptions to this trend are known, requiring empirical studies of individual taxa to be sure about population genetic architecture.
In the present study, seedling growth differed in response to soils of different texture and chemistry. Decreased growth of local outcrossed seedlings in lateritic soil compared with native sandy soils, was associated with reduced size of the root system, possibly associated with a high soil bulk density or aluminium toxicity. Although the seedlings on lateritic soil had a lower shoot P concentrations than those on sandy soils, it is unlikely that this caused the decrease in growth, because these P concentrations are in the range considered normal for Proteaceae (Foulds, 1993). Also, local outcrossed seedlings had similar P concentrations as nonlocal outcrossed plants, but showed less growth. Interestingly, roots of nonlocal outcrossed plants produced much larger amounts of carboxylates than local-outcrossed seedlings, but this was not in response to low leaf P concentrations and did not lead to increased P uptake, because local-outcrossed plants had the same P concentration at much lower carboxylate exudation. Increased carboxylate exudation may have been a response to high Al concentrations in the rhizosphere (Delhaize et al., 1993; Lambers et al., 2002; Shane et al., 2003). The detoxification of Al by outcrossed plants may have allowed for better growth compared with that of selfed plants (Delhaize et al., 1993; Lambers et al., 2002; Shane et al., 2003).
Waterlogging and water stress had relatively small effects on growth, despite relatively large effects on shoot P concentration. This supports the conclusion that for the duration of the experiment, decreased P uptake was not the most important growth-limiting factor. The 6-week experiment did not reveal significant differences between local and nonlocal outcrossed plants in terms of their tolerance to waterlogging or water stress, but differences might become apparent during longer-term experiments or at different stress levels.