2.1 Study species and field sampling.
Scopelophila cataractae is one of the so-called “copper mosses”
due to its high affinity for heavy metal enriched habitats (Shaw,
1993a). It is a Cu hyperaccumulator and Cu can reach up to 3% of the
plant’s dry weight (Aikawa, Nagano, Sakamoto, Nishiyama, & Matsumoto,
1999; Nakajima, Itoh, Otake, & Fujimoto, 2011; Satake, Shibata,
Nishikawa, & Fuwa, 1988). Similar to other copper mosses, S.
cataractae has a broad but disjunct geographical distribution worldwide
that roughly matches the distribution of copper-enriched substrates
(Shaw, 1987, 1993a,b, 1995). The species is dioecious, i.e. male and
female gametangia are developed in different gametophores. Sporophytes,
which are the diploid phase of the life cycle resulting from sexual
reproduction, have never been observed in the populations used in this
study suggesting that these populations are exclusively or mainly
clonal.
Ceratodon purpureus is one of the most cosmopolitan bryophyte
species with a broad ecological range. It occurs on a variety of
substrates, ranging from well preserved to highly disturbed areas.Ceratodon purpureus also has separate sexes and it frequently
undergoes sexual reproduction in unpolluted areas (Shaw et al., 1991).
This species could thus be considered a pseudometallophyte or
facultative metallophyte sensu Baker (1987).
In September 2016, we collected plants from four populations of S.
cataractae in a mine site in Silver Hill, North Carolina (USA), whose
activity was discontinued in the 1950s (Wickland 1984; Shaw, 1987). Here
we use the term population to refer to physically unconnected and
scattered patches of this species (separated by mostly bare soil), even
though the distance between these patches was short (~20
to ~300 m). The mine was situated on a slope and
populations were sampled down the slope beginning in the SE edge (Sc1),
through the center of the mine (Sc2 and Sc3), and finishing in the NW
edge (Sc4). On the same date, Ceratodon purpureus (Cp1) was
collected within an urban area in Durham, North Carolina (USA). At every
site and sampling occasion, we used a knife to separate several clumps
of moss from the soil. Various aliquots of gametophytic tissue from
these clumps were stored in 1.5 ml Eppendorf tubes and frozen in liquid
nitrogen. The remaining material was put into zip-lock plastic bags and
stored inside a cooler in the dark. In the laboratory, frozen samples
were stored at -80ºC for microscopy analysis (section 2.5.2), an aliquot
of fresh material was separated and dried to determine total Cd and Cu
contents (section 2.5), another aliquot was separated to phenotype the
plants (section 2.4.1), and the remaining material was maintained in a
growth chamber at 22 ºC and 16h light/8h dark to perform the common
garden experiments. These conditions were maintained throughout the
duration of the experiments.
Because mosses have no real roots, they are mostly influenced by the
chemical composition of the surface soil layer. Thus, at each site we
collected soil samples consisting of the upper 2-5 cm of soil right
underneath the moss clumps to assess the levels of heavy metals to which
mosses were exposed in their origin environments. These samples were
stored in zip-lock plastic bags and kept in a cooler in the dark until
further processing.