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.