1. Introduction
The environment poses continuous challenges to all living organisms.
Environmental heterogeneity is ubiquitous, as gradients and spatial
variation in temperature, radiation, water availability, and soil
composition and chemistry exist at different spatial and temporal scales
(Bell et al., 1993; Pigliucci, 2001). In addition, anthropogenic
activities change the environment altering climate, the structure of the
landscape, the major biogeochemical cycles, and also introducing
pollutants into the ecosystems (Anderson, Willis, & Mitchell-Olds,
2011; Vitousek, Mooney, Lubchenco, & Melillo, 1997; Tilman and Lehman,
2001;). Coping with these conditions is, therefore, one of the main
challenges faced by organisms throughout their lifespan. The sessile
nature of plants makes this task even more demanding, as it requires the
ability to respond without moving. Failure to do so would compromise
their survival and reproduction, and increase the probability of
population extinction (Willi & Hoffmann, 2009).
Exposure to heavy metals (i.e. elements with a specific density
> 5 g/cm3; Jarup, 2003) is a powerful
selective pressure for plants with important ecological and evolutionary
implications (Antonovics, Bradshaw, & Turner, 1971; Ernst, 2006;
Macnair, 1987; Shaw, 1990; Boyd, 2004; Wright, Stanton, & Scherson,
2006). Some heavy metals are essential for normal functioning in all
plants but they can be toxic at high concentrations (e.g. Co, Cu, Fe,
Mn, Zn), while others have no known physiological functions and can be
toxic even at very low concentrations (e.g. Cd, Pb, Hg). Such toxicity
can exert intense selective pressures on plants, and has led to the
evolution of tolerant and/or hyperaccumulator ecotypes in many plant
species (e.g. Pauwels, Frérot, Bonnin, & Saumitou-Laprade, 2006; Reeves
et al., 2017; Wright et al., 2006). The natural weathering of metal-rich
rocks has generated soils to which some plant species have adapted in
the long run, giving rise to a metallophyte flora with a considerable
level of endemicity (e.g. Brooks and Malaisse, 1985; Kruckeberg and
Kruckeberg, 1990; Reeves, Baker, Borhidi, & Berazaín, 1996) of
significant conservation value (Whiting et al., 2004). However, during
the last decades anthropogenic activities have caused a dramatic
increase in the concentrations of metals in soils that are not naturally
enriched in metals as a result of surface deposition of dust and
particles derived from industrial, agricultural, and mining activities,
as well as energy production (Bradl et al., 2002; He, Yang, &
Stoffella, 2005; Singh, Labana S., Pandey, Budhiraja & Jain, 2003).
Such rapid increase in soil toxicity requires a similarly rapid response
of plant populations to develop tolerance to heavy metal pollution, or
the capacity to maintain fitness in the presence of exposure to heavy
metals (Simms, 2000).
Most plants living in heavy metal enriched substrates have mechanisms to
avoid uptake of metals while others can accumulate them at different
levels (Adlassnig et al., 2016; Baker, 1981,). Accumulation and
tolerance are thus complex genetically distinct quantitative traits that
show high levels of inter- and intraspecific variation in plants
(Goolsby and Mason, 2015). However, our current knowledge of the extent
of intraspecific variation for heavy metal accumulation and tolerance in
plants, the mechanisms underlying these traits, and their ecological and
evolutionary significance is largely derived from tracheophytes,
especially angiosperms (Cappa & Pilon-Smits, 2014; Ernst, 2006; Reeves
et al., 2017; Verbruggen, Hermans, & Schat, 2009). Bryophytes have also
shown the capacity to tolerate and accumulate high concentrations of
these pollutants (Shaw, 1994). These plants diverged from their sister
group of vascular plants ~500 mya, and they are
non-vascular, gametophyte-dominant (haploid) plants with a relatively
low degree of morphological and anatomical complexity, and a low
capacity of self-internal regulation due to their poikilohydric nature
(Vanderpoorten & Goffinet, 2009). Since they use a variety of unique
metabolic pathways to deal with environmental challenges (Cuming 2009;
Glime, 2017a,b), their study could provide important insights into the
evolution of these responses.
Several studies of inter- and intraspecific variation in the capacity of
bryophytes to tolerate heavy metal pollution have found ecotypic
differentiation, as well as broad inherent plasticity in a few species
(e.g. Briggs, 1972; Brown & House, 1978; Cogolludo, Estébanez, &
Medina, 2017; Jules & Shaw, 1994; Shaw, 1988; Shaw, Antonovics, &
Anderson, 1987; Shaw, Jules, & Beer, 1991). However, the bulk of the
work on heavy metal tolerance and accumulation in natural bryophyte
populations dates from the late 1970s and early 1990s, and was focused
on a few target species. Recent research in this field has mostly
focused on the applied value of bryophytes as biomonitors of heavy metal
pollution (reviewed in: Ares et al., 2014; Fernández, Boquete,
Carballeira, & Aboal, 2015; Onianwa, 2001; Stanković, Sabovljević, &
Sabovljević, 2018) and phytoremediation (e.g. Itouga et al., 2017;
Kobayashi, Kofuji R., Yamashita, & Nakamura, 2006; Sandhi, Landberg, &
Greger, 2018; Sut-Lohmanna, Jonczakb, & Raaba, 2020), or their
physiological and biochemical responses to heavy metal exposure under
controlled laboratory conditions without a clear focus on the natural
variation of these traits (e.g. Bellini et al., 2020; Esposito et al.,
2018; Kovácik, Dresler, & Babula, 2020; Liang et al., 2018).
This study builds on the classic research to explore in more detail the
extent of intraspecific phenotypic variation for heavy metal
accumulation and tolerance in bryophytes in relation to contrasting
habitat specialization. We selected two ecologically different
terrestrial moss species with contrasting affinities for heavy metals,
grew them in the laboratory under different metal treatments, and
examined their patterns of accumulation and tolerance. Here, we define
tolerance as the ability to maintain vegetative growth in a metal
stressed vs. a control environment (sensu Simms, 2020). For the
heavy metal specialist Scopelophila cataractae (Mitt.) Broth
(Pottiaceae), we studied four field populations collected within a
former copper mine to determine if there was variation for accumulation
and tolerance among populations growing in a range of metal-rich soils.
For the non-specialist, but metal tolerant Ceratodon purpureus(Hedw.) Brid. (Ditrichaceae), we studied one population collected in the
field and compared it with male and female populations grown in the
laboratory under axenic conditions. We evaluated phenotypic differences
among populations, and examined whether sexes differed in their capacity
to accumulate and tolerate metals, an aspect that has not been addressed
previously to our knowledge. We predict that the metal specialistS. cataractae would show a “stress tolerator” strategy i.e.
increased tolerance under similar accumulation levels, whereas the
non-specialist C. purpureus would show a “stress avoidance”
strategy, i.e. increased tolerance resulting from decreased
accumulation, (sensu Baker, 1981). Also, we predict that females
of C. purpureus would be more tolerant to heavy metal exposure
than males as shown for other species in different environmental
settings (Bowker, Stark, McLetchie, & Mishler, 2000; Marks, Burton, &
Mcletchie, 2016; Moore, 2017).