4.1 The heavy metal specialist: Scopelophila cataractae
The unique ecological specialization of S. cataractae for heavy metals has inspired some research on its phenotypic variation in the past. In studies across the broad and discontinuous geographical range of this species, Shaw (1993b, 1995) found high levels of morphological variation in several leaf and cell traits of the gametophores; most of this variation (up to 78%) was found among individuals within populations, even though he included populations from five widely separated geographical regions (Shaw 1993b). Allozyme analyses on a subset of these populations showed high genetic variation at the species level that was mostly accounted for by variation among populations, and many populations were genetically uniform (Shaw, 1995). Some of these populations never showed evidence of sexual reproduction (by presence of sporophytes) and were mainly comprised of sterile plants and plants from only one sex (Shaw 1993a; 1995), suggesting that they were clones. Therefore, Shaw (1995) attributed the high levels of morphological variation within populations of S. cataractae to phenotypic plasticity rather than genetic variation.
Our field populations also consisted almost entirely of non-sexual plants (except for Sc3 where 20% of the plants bore male gametangia and 80% did not express sex) and no sporophytes were observed in this, or previous studies of these populations (e.g. Shaw, 1993a). Although we cannot rule out some genetic differentiation among our populations, we expected some gene flow among populations through the movement of asexual propagules (e.g. gemma) or fragments of gametophores from one population to the other populations (especially down the slope from Sc1 towards Sc4 passing through Sc2 and Sc3) due to their geographical proximity (<500 m away from each other). Under these circumstances, we expected limited levels of phenotypic differentiation among our populations. Still, we demonstrated, for the first time, the existence of intraspecific differentiation for heavy metal accumulation and tolerance in S. cataractae on a very limited geographical scale. This finding suggests high phenotypic variation among populations with potentially limited genetic diversity.
The differences that we found for this species contrast to some extent with the results of previous experimental work on S. cataractae . Shaw (1987, 1993a) propagated gametophytic tissue of eight and five North American populations of this species respectively growing under different levels of heavy metal pollution in the field on different soil-types: polluted soil (mine soil enriched in Cu and Zn), unpolluted soil, and mixtures of these two. He found a strong effect of soil-type on plant growth, and all populations grew better in polluted than unpolluted soils in both studies. All populations performed equally well in polluted soil regardless of their original environment, but some populations grew better than others in control and mixed soils (Shaw, 1993a). In our study, populations responded differently to Cd and Cu, and this response was related to the levels of metals in their origin environments, i.e. those exposed to higher levels of metals in the field were the most tolerant. This discrepancy could be explained partly by the differences in the way that the treatments were applied. We applied Cd and Cu in solution to a commercial soil, so both metals were readily and directly available for the plants, which was not true in the mine soil in Shaw’s studies (1987, 1993a). Because the amount of phenotypic variation expressed by one species can vary between environments (e.g. Pigliucci, 2001; Richards et al., 2006, 2010; Shaw 1993a), our experimental conditions could reveal novel differences in metal tolerance in S. cataractae , similar to how unpolluted and mixed soils revealed phenotypic differences that were not evident in polluted soil (Shaw 1993a) Interestingly, the phenotypic differentiation found in our study resulted after only 30 days of treatment of adult gametophores. We thus predict that differences among populations would be even greater if the treatments had been applied from the beginning of the experiment.
Aside from the metal treatments, plants from Sc2, collected from the site with an outstanding concentration of Cu, were always smaller than plants from the other three populations when grown in the laboratory under control conditions. This pattern suggests that greater metal tolerance may come with a biological cost, at least in Sc2, a phenomenon previously reported for other bryophytes (Jules and Shaw, 1994). Metal tolerance entails a metabolic cost due to the allocation of energetic resources to counteracting the potentially toxic effect of metals (e.g. synthesis of chelating agents, upregulation of the ROS scavenging machinery, metal transportation; see Maestri, Marmiroli, Visioli, & Marmiroli, 2010). Other adverse environmental conditions that characterize polluted soils, i.e. intense sun exposure, poor nutrient supply, or low water retention capacity, also contribute to the slower growth rates, lower biomass production, and lower reproductive output of metal-adapted plants (Baker, 1987; Bothe and Slomka, 2017; Ernst 2006). In this study, plants were sampled in microhabitats that clearly differed between the center and the edges of the mine. In particular, the center of the mine was devoid of any vegetation except for the mats of S. cataractae so plants from Sc2 and Sc3 surely experienced higher light and temperatures, and lower water availability. On the contrary, plants from Sc1 and Sc4 were under vascular plant cover and Sc4 was alongside a seasonal streamlet that had abundant water during sample collection. All these microhabitat differences, including their different metal levels (Fig. 1A, D), are reflected in the smaller size of plants from Sc2 and Sc3 in the field (Fig. 1G-I), and were maintained to some extent in Sc2 in the common garden experiment under control conditions, suggesting that this population maintained the “metal-tolerant phenotype” beyond the field.
All populations of S. cataractae in this study behaved as Cd- and Cu-hyperaccumulators according to Maestri et al. (2010), as their total concentrations reached ~1% d.w. of Cu (1.1% in Sc1 and Sc3, 0.96% in Sc2, and 1.0% in Sc4) and between 0.21 – 0.45% d.w. of Cd (0.21, 0.19, 0.30, 0.45% in Sc1 to Sc4 respectively) (Fig. 2A, D). Although we did not find differences in the total concentrations of Cu among populations, i.e. no differences in their capacity to accumulate Cu, the relative concentrations in leaves of plants from central mine populations, Sc2 and Sc3, were lower than those in leaves of plants from the mine edges, Sc1 and Sc4. Also, relative levels of Cu in stems of Sc2, Sc3 and Sc4 exceeded those in leaves. Therefore, we speculate that relocation of absorbed Cu towards the stem could protect leaves and their photosynthetic activity and explain to some extent the higher tolerance observed in Sc2 and Sc3. Some studies have already shown evidence for acropetal and basipetal transport of elements, including metals, within moss gametophores (Brümelis & Brown, 1997; Sidhu & Brown, 1996; Wells & Brown, 1996), as well as differences in preferential accumulation of Cu in specific parts of the gametophores between tolerant and non-tolerant moss species (Antreich, Sassmann, & Lang, 2016). Moss leaves are important functional organs whose structure allows maximization of light and nutrient capture, and gas exchange, making them very efficient photosynthetic structures (Renzaglia, Duff, Nickrent, & Garbary, 2000; Renzaglia et al., 2007). Thus, leaf protection seems a plausible explanation for the tissue-specific relative levels of Cu found here.
The high affinity of S. cataractae for Cu has been demonstrated by its repeated appearance in Cu-enriched substrates worldwide (see Shaw, 1993b), experimentally by Shaw (1987, 1993a) and more recently by Nomura & Hasezawa (2011). Nomura & Hasezawa (2011), however, showed that the beneficial effect of Cu on protonemal growth was reversed at 1 mM CuSO4. At this concentration, the size of the protonemal mats growing axenically in agar plates decreased slightly compared to the controls. Thus, even though this species constitutively needed high concentrations of Cu to perform at its best, too high concentrations such as those used in the present study and in Nomura & Hasezawa (2011) may result in toxicity (e.g. increase in oxidative damage shown in Fig. 3D), and reveal differences in tolerance and accumulation patterns that would otherwise have been hidden. On the contrary, Cd is non-essential, highly toxic, and there is no evidence ofS. cataractae showing high affinity for this metal which might explain the intraspecific differences observed in the capacity ofS. cataractae to accumulate Cd (Fig. 2D) and the lack of preferential relocation towards the stem.