Luna (2020) noted that most ungerminated seeds that had been given a heat treatment after one month at 50/20°C were soft (they imbibed water when immersed) in contrast to those that were heated before the summer treatment. As a result, they suggested that summer temperatures prime (‘sensitize’) the seeds to soften more readily in the presence of fire-type heat and thus serve to promote germination. The grounds for the sensitizing effect by the summer pretreatment was the heat-then-summer treatment gave 32.6 % germination and the summer-then-heat treatment 80.7 % germination (P < 0.0001, paired t-test, recalculated from Table S4 in Luna 2020). However, this pairwise comparison confounds two treatments given simultaneously: a summer treatment at time 1 and 2, and a heat treatment at time 2 and 1 that lacks controls. Other treatments (undertaken under the same conditions with the same batches of seeds but considered apart by Luna) included a control, summer treatment, heat treatment, and a summer-then-heat treatment (Table 1). Together, these show that germination was negligible in both the control and summer treatment, i.e., high summer temperatures did not promote germination. Fire-type heat caused a 7−8 times increase in germination, whether or not the seeds received a summer treatment. That is, the seeds did not need to be ‘sensitized’ to germinate fully in response to the heat pulse. A similar comparison can be made by replacing the heat-then-summer treatment by the summer-then-heat treatment (Table 2). Here, both sets of heated seeds germinated much more than the unheated (72% of variance), but it is the interaction effect that is of most interest: the heat-then-summer sequence yielded 55% fewer germinated seeds than the summer-then-heat sequence. Luna (2020) interpreted this as failure of most of the heat-then-summer-treated seeds to be softened by the heat in contrast to the summer-then-heat-treated. But Table 1 makes it clear that summer priming was not required in the presence of the same heat treatment.
Table 1
2-way ANOVA of ± summer treatment (50/20°C for 1 mo) and ± heat treatment (100°C for 10 min) that followed the summer treatment for mean germination ± SD (%) of 12 Cistaceae species at 20°C. Calculated from Tables S3 and S4 in Luna1 (2020). Different superscript letters indicate significant differences at P < 0.01 according to Tukey’s HSD test.
Germination
|
Control
|
Summer (time 1)
|
Source of variation
|
df
|
P
|
No heat
|
9.9 ±7.5b
|
4.3 ± 6.5b
|
± Summer
|
1
|
0.7656
|
Heat (time 2)
|
73.0 ± 17.4a
|
80.7 ± 15.1a
|
± Heat
|
1
|
< 0.0001
|
|
|
|
Interaction
|
1
|
0.0732
|
|
|
|
Error
|
44
|
|
Table 2
2-way ANOVA of ± summer treatment (50/20°C for 1 mo) and ± heat treatment (100°C for 10 min) that preceded the summer treatment for mean germination ± SD (%) of 12 Cistaceae species at 20°C. Calculated from Tables S3 and S4 in Luna1 (2020). Different superscript letters indicate significant differences at P < 0.01 according to Tukey’s HSD test.
Germination
|
Control
|
Summer (time 2)
|
Source of variation
|
df
|
P
|
Contribution to variance (%)
|
No heat
|
9.9 ±7.5c
|
4.3 ± 6.5c
|
± Heat
|
1
|
< 0.0001
|
71.5
|
Heat (time 1)
|
73.0 ± 17.4a
|
32.6 ± 27.7b
|
± Summer
|
1
|
< 0.0001
|
18.1
|
|
|
|
Interaction
|
1
|
0.0001
|
10.4
|
|
|
|
Error
|
44
|
|
|
A more likely explanation is that 40% of the presummer heated seeds had gone back into dormancy by becoming hard-seeded again as they dried out at 50/20°C over the ensuing month, a case of secondary physical dormancy. Micrographs and diagrams of the region in Cistaceae (Corral et al 1989, Nandi 1998, Gama-Arachchige et al. 2013) show that it is feasible for the retracted chalazal plug to be pushed back into the pore as the seed continues to desiccate. In support, Hagon and Ballard (1970) showed that scarified seeds of Trifolium subterraneum became hard again when stored at 5% humidity and 20°C, not unlike summer drought conditions although at lower temperatures. They further noted that the strophiole resealed under these conditions, a point missed by those who consider secondary dormancy is not possible among hard-seeded species. Luna (2020) was aware of this reference but chose the ‘sensitizing’ view because they believed that secondary dormancy was not possible among seeds with physical dormancy, and felt it fitted better with their data (personal communication). However, it is difficult to see why seeds would need ‘sensitizing’ before responding to such an extreme treatment as dry heat at 100°C for 10 min, and species used by Luna have produced as high levels of germination in the past, sometimes at lower temperatures for shorter exposure times, without the need for any priming (Thanos et al. 1992, Moreira et al. 2010, Luna et al. 2019).
Fluctuating dormancy has also been recorded among other Cistaceae: Fumana ericoides seeds were shown to go from soft to hard and soft again over time (Llorens et al. 2008). Most Cistus clusii seeds from two populations did not germinate after storing them air dry at 4°C, in contrast to those kept at room temperature (Castro and Romero-García 1999). Most Helianthemum salicifolium seeds would no longer germinate after storing under “cold, dry” conditions in contrast to those kept at room temperature (Sánchez et al. 2014). We interpret both these cases as drying having imposed physical dormancy but note that Castro and Romero-García (1999) thought it was secondary physiological dormancy without checking the seeds for hardness nor appreciating that cold stratification requires moist conditions. Both these cases are consistent with the dry summer treatment of Luna (2020) ‘desensitizing’ some seeds by imposing dormancy on them (Table 2).
If low humidity is the key, then a month at low humidity might be as effective in restoring physical dormancy in Cistaceae as the 50/20°C used here. The heat-then-summer-treated seeds did remain viable according to Luna’s ‘cut’ test and so these would be available to germinate following a subsequent fire. Thus, the response can be considered adaptive. This also can explain the few soft ungerminated seeds among those in the summer-then-heat-treated seeds: they had not yet had time to harden again. Assuming that the cut test is unreliable under these circumstances, it is also possible that the seeds which failed to germinate had actually died through the additional desiccation that would now occur if the pore remained unplugged over the extended period at 50°C. [One of us, GB, observed considerable additional water loss by Acacia seeds once the lens had popped though they did not die nor become hard again]. We agree with Luna that the relative location of the pore and plug needs to be examined by electron microscopy after various drying/heating treatments to resolve these alternative interpretations (personal communication).
Bastida and Talavera (2002) showed that most seeds of two Cistus species were stored beneath the plant crown. Tuberaria guttata seeds are stored < 10 cm from the parent plant (Guarino, Ferrario and Mossa 2005). Further, Cistaceae seeds are tiny (< 2 mg) and spheroid (Bastida et al. 2009) so will easily penetrate the litter to reach the soil. These observations indicate that invariably Cistaceae seeds will be stored in litter-rich parts of the soil surface. Seeds of some species are dispersed by harvester ants but these are unlikely to account for more than 20% of their final location (Bastida et al. 2009). Some seeds can be ingested without harm by ruminants – here the seeds remain in the dung and are not exposed to sunlight (Ramos, Robles & Castro 2006). Seeds of various Cistaceae stored beneath the plant crown to a depth of 50 mm averaged 1400−4000 m− 2 (Herranz et al. 1999, Bastida and Talavera 2002). Dias et al. (2019) noted that the hypocotyl of C. ladanifer could extend to 22 mm and suggested this represented the maximum depth from which seeds could emerge unless surface soil was removed postfire (as may occur, Lamont et al. 1993).
What temperatures then are reached at soil locations where Cistaceae seeds are likely to be stored? Brits (1986) studied the likely temperature fluctuations in pre- and post-burn surface soil in detail. For a site matching parts of the Mediterranean Basin, this work showed for the hottest month of the year that, at a soil depth of 5 mm, the warmest 12-h period of the day averaged 41°C and the coolest 22°C under light shade, and 28°C and 22°C under heavy shade (Table 3, see Moreira and Pausas 2012 for similar results). Thus, a summer pretreatment temperature at 50°C continuously for 1−2 months (Luna 2020) appears extremely high compared with expected summer temperatures at a depth of 5 mm even for mineral soil exposed after fire (47/28°C, Table 3), while that at 50/20°C 12-h cycles seems a reasonable simulation of unburnt sites under light shade. However, the data reviewed above indicate that heavily shaded conditions at greater (cooler) depths are more likely for stored Cistaceae seeds in the Mediterranean Basin.
Table 3
Mean daily temperatures (to nearest 0.5°C) at a depth of 5 mm for the warmest month of the year at the ¾ position (midway between the mean and maximum for the warmest 12 h) and ¼ position (midway between mean and minimum for the coolest 12 h) of the temperature range for three possible locations in shrubland. Compiled from Brits (1986) for a mild Mediterranean-type climate in South Africa. These provide a guide as to suitable temperatures to set on a 12/12-h cycle if attempting to simulate temperature conditions at the hottest time of year where stored seeds are located at 5 mm depth.
Part of temperature range
|
Exposed gap (postburn)
|
Light shade (unburnt)
|
Heavy shade (unburnt)
|
¾ position
|
46.5
|
41
|
27.5
|
¼ position
|
27.5
|
22
|
22.5
|
The fire season in the Mediterranean Basin is considered to cover the period June (early summer) to September (early autumn) (Moriondo et al. 2006, Arca et al. 2012). This is supported by the abrupt but broad peak in fire ‘danger’ days at this time (Dimitrakopoulos et al. 2011) (Fig. 1). The moisture content of living fine foliage of Cistus species is used as an index of fire-proneness (Pellizzaro et al. 2007, Chuvieco et al. 2009) and low levels cover this period. Mean air temperatures also peak during the fire season. Monthly rainfall is at a minimum but does rise sharply in September, at least on the E side of the Iberian Peninsula (the source of Cistaceae studied by Luna 2020). As a suitable ignition source, the incidence of lightning shows that there is a sharp peak in September to December (Rivas Soriano and de Pablo 2002, Price and Federmesser 2006). At a depth of 5 mm, means of the monthly soil temperatures at the extremes of postfire bare ground and prefire dense shade peak in July (Brits (1986). We note that the deep-shade pattern is almost the same as that for the Stevenson-screen air temperatures for Valencia (www.climatestotravel.com/climate/spain/valencia) and indicates this could be used as a reasonable surrogate for shaded surface soils.
Putting these data together shows that, when the fire regime is controlled by lightning, fires are most likely to occur at the end of the fireprone season, September (Fig. 1). This is about six weeks away (October-November) from soil conditions optimal for germination of Cistaceae seeds (15−20°C, moist soil) (Herranz et al. 1999, Moreira et al. 2010, Dias et al. 2019, Luna et al. 2019, Luna 2020). Thus, the period between breaking dormancy and germination opportunity is short, soil temperatures are 7°C less than the summer peak, and autumn rains are already substantial. This means that any return to dormancy as suggested here during the remaining postfire, summer-autumn period will be short so that it has little role in reducing the extent to which previously heated seeds will germinate. More important will be the type of fire (He, Lamont and Pausas 2019) and properties of the wet season (Céspedes et al. 2012). We agree with Luna (personal communication) that seed burial under different field conditions is required to ensure future studies mimic what occurs in nature.
In conclusion, the simulated summer treatments need to be compatible with where seeds are actually stored over summer. It is clear that a continuous 50°C for 1−2 months has no equivalent in nature and its only use might be in the context of exploring pre-adaptations (Lamont and He 2017). Most buried seeds will be well insulated from sunlight and mean temperatures in the order of 20–40°C, as obtained under shade over the hottest summer month under a mediterranean climate by Brits (1986), appear more reasonable. Any differential effect of varying the sequence of heat and ‘summer’ treatments is of ecological and management relevance. If the interest is in the effect of long periods of severe summer heat following fire on seed viability and germination, then a fire-type heat treatment followed by 1−2 months at 50/20°C realistically simulates conditions that Brits (1986) determined for postfire bare areas at a depth of 5 mm (Table 3). It is well established that late season fires result in greater seedling establishment than early season fires (Enright and Lamont 1989). The usual explanation is in terms of greater dormancy-breaking efficiency (or seed release by serotinous species) by more intense fires rather than early fires resulting in a return to dormancy before winter rains begin as our preferred interpretation of Luna’s data. Only studies that overtly studied the presence of dormancy cycling would be in a position to distinguish these two possibilities. The results could be added to guidelines on the most suitable time for management fires, bearing in mind that Enright and Lamont (1989) showed that recruitment success of early and late fires tended to merge with time.