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.