4.2 The facultative metallophyte: Ceratodon purpureus
Ceratodon purpureus has a wide geographical range (Crum & Anderson, 1981) and occurrs in habitats with diverse environmental characteristics: acid to calcareous soils, sandy, woody, or rocky substrates with varying amounts of surrounding vegetation (from bare soil to well-developed canopies), and different degrees of disturbance (see Atherton, Bosanquet, Lawley, 2010; Dunham, 1951; Ireland, 1982; Shaw et al., 1991), suggesting that this moss species has a broad ecological tolerance. Previous studies on heavy metal tolerance in this species showed that C. purpureus harbored a constitutive capacity to tolerate and survive in heavy metal enriched environments (Shaw et al., 1991) but it can also undergo ecotypic differentiation when subjected to a strong enough selective pressure (Jules & Shaw 1994). This study provides further support for the high resilience of this species by showing evidence of intraspecific differences in heavy metal tolerance between populations that had never experienced high levels of metals in the field.
Both Cu and Cd negatively affected negatively all populations from this study, but the magnitude of this effect on each population depended on the metal and on the trait measured. Protonemal growth was reduced on average by only 17% in Cp1 under Cu treatment, whereas this decline was 2.8 and 3.5 times higher in males and females from Cp2 respectively (Fig. 4C). One could argue that the higher Cu tolerance of Cp1 could be due to a general hardiness effect, as this was collected in the field where it likely experienced some stress which could translate to general stress tolerance, whereas Cp2 had been growing in the laboratory for several years. However, we do not think that this is the case for two reasons. First, plants of Cp1 were dehardened to a certain degree before the experiments by propagating several clonal generations in the laboratory. Second, if this were the case we would expect Cp1 to be more Cd tolerant as well, which we did not find. The female lab strain, Cp2.f, was more tolerant to Cd than individuals derived from the field collected plants, Cp1, and the male lab strain, Cp2.m, whose average growth dropped 2.0 and 2.9 times more respectively than in Cp2.f (Fig. 4G). Treatment with Cu, did not cause a significant increase in MDA in any of the populations (Fig. 4D). However, there was an effect of Cd on the concentration of MDA in the plants which paralleled the pattern shown by the protonemal growth, suggesting females (Cp2.f) had less oxidative damage and were more Cd-tolerant than Cp1 and Cp2.m.
All populations of C. purpureus in this study reached the threshold concentrations for Cd hyperaccumulators, i.e. ≥ 0.01% (Maestri et al., 2010), whereas total Cu concentrations were an order of magnitude below the threshold for Cu hyperaccumulators (≥ 0.1%). Despite the significant increase in total and relative concentrations of Cd and Cu in all treated plants compared to their controls (except for relative Cu in Cp1), metal accumulation seemed to be similar in all populations indicating that the differences observed in tolerance were not related to differences in their capacity to take up metals. Thus, we hypothesize that the observed differences in tolerance could be due to two complementary mechanisms. First, bryophytes have a high cation exchange capacity (CEC) in their cell walls that plays a major role in nutrient and heavy metal uptake, binding, and regulation (Richter and Dainty, 1989), and depends on the composition of the cell wall. Further, the structure and composition of the cell wall of bryophytes can change in response to environmental cues such as heavy metal exposure (Konno et al., 2010; Krzeslowska, Lenartowska, Mellerowicz, Samardakiewicz, Wozny, 2009). Therefore, differences in the original composition of the wall, or in the way it responds to heavy metal exposure could explain the differences in its capacity to bind metals and prevent their entrance inside the cells, limiting their toxicity, while still being accounted for in our total and relative concentrations. Second, especially for Cd that caused a population-specific increase in oxidative damage (MDA), it is possible that the capacity of the ROS scavenging systems differed among our field and lab-reared populations, contributing to the observed differences in tolerance.
Finally, our results showed, for the first time, evidence for metal-dependent, sex-specific differences in heavy metal tolerance in bryophytes. Both sexes were similarly tolerant to Cu but females were significantly more tolerant to Cd than males, as evidenced by both protonemal growth and oxidative damage data. Dioecy has evolved multiple times in bryophytes, leading to more than 50% of the species having separate sexes (Bisang & Hedenäs, 2005; McDaniel, Atwood, & Burleigh, 2012). Sexual dimorphism in morphological, physiological, and life history traits in bryophytes has been reported multiple times (e.g. Dos Santos, Alvarenga, & PôrtO, 2018; Holá, Vesalainen, Těšitel, & Laaka-Lindberg , 2014; Horsley, Stark, & Mcletchie, 2011; Stark, Mcletchie, & Mishler, 2001). As a matter of fact, C. purpureushas often been used as a model system for sexual dimorphism in bryophytes exhibiting sex-specific differences in morphology, photosynthetic activity, organic volatile compound production, and even associated fungal community composition (Balkan, 2016; Rosenstiel, Shortlidge, Melnychenko, Pankow, & Eppley, 2012; Shaw & Beer, 1999; Shaw & Gaughan, 1993; Slate, Rosenstiel, & Eppley, 2017). However, sexual dimorphism in response to environmental stress has been addressed less frequently, and most research focused on the response to desiccation (Bowker et al., 2000; Marks et al., 2016; Moore, 2017; Stieha, Middleton, Stieha, Trott, & Mcletchie, 2104;). Sexual dimorphism in response to the environment at the individual level (e.g. reduced individual fitness in one sex) can have consequences at the population level such as bias in population sex ratios limiting the frequency of sexual reproduction and genetic recombination, which might in turn restrict the capacity of plant populations to adapt to the environmental changes.