Posted on Authorea 20 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160315210.03739090/v1 | This a preprint and has not been peer reviewed. Data may be preliminary.
Patterns and mechanisms of heavy metal accumulation and
tolerance in two terrestrial moss species with contrasting habitat
specialization
M. Teresa Boquete
1
, Ingeborg Lang
2
, Marieluise Weidinger
3
, Christina Richards
4
, and
Conchita Alonso
1
1
Estaci´on Biol´ogica de Do˜nana
2
University of Vienna Faculty of Life Sciences
3
Universitiy of Vienna
4
University of South Florida
October 20, 2020
Abstract
Phenotypic variation in natural populations is crucial for rapid adjustment to challenging environmental conditions such as
exposure to heavy metal pollution. Hence, the study of variation in traits related to plant response to heavy metal stress
provides valuable insight into the likelihood of a population’s survival. We investigate the patterns and mechanisms of intraspe-
cific phenotypic variation for heavy metal accumulation and tolerance in bryophytes, one of the most resilient yet relatively
understudied taxa. Two terrestrial mosses exposed to Cd and Cu in the laboratory, the heavy metal specialist Scopelophila
cataractae and the facultative metallophyte Ceratodon purpureus, showed intraspecific differences in tolerance to these metals.
Only the heavy metal specialist showed differences in accumulation which in the case of Cu could be associated to preferential
relocation towards the stem to protect their main photosynthetic organs. We also report the first evidence for sexual dimorphism
for Cd tolerance in C. purpureus (females being more tolerant than males). Our results provide support for high variation in
the capacity of bryophytes to respond to environmental challenge despite potentially low levels of genetic variation and lack
of previous exposure to stress, as well as evidence for metal-dependent, sex-specific differences in heavy metal tolerance in
bryophytes.
Acknowledgements
We thank Dr. Carlos M. Herrera for his advice throughout the development of the project and for critically
reviewing the manuscript; Dr. Jonathan A. Shaw and Dr. Blanka Aguero for their help with sample
localization and collection; Dr. Stuart F. McDaniel for providing material from lab-maintained populations
of C. purpureus ; Luiza Silva Simoes and Olivia Santiago for help during laboratory work at USF; Dr.
Zachary D. Atlas and Dr. David B. Lewis for providing support and infrastructure for soil analyses at
USF. SEM-EDX was performed at the Core Facility Cell Imaging & Ultrastructure Research, University
of Vienna – member of the Vienna Life-Science Instruments (VLSI) and we thank Dr. Irene Lichtscheidl
for her support. This research has received funding from the European Union’s Horizon 2020 research and
innovation programme under the Marie Sk lodowska-Curie grant agreement No 704141-BryOmics.
1. Introduction
The environment poses continuous challenges to all living organisms. Environmental heterogeneity is ubiq-
uitous, 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,
1
Posted on Authorea 20 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160315210.03739090/v1 | This a preprint and has not been peer reviewed. Data may be preliminary.
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 ses-
sile 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/cm
3
; Jarup, 2003) is a powerful se-
lective 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 concentra-
tions (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,
Frerot, 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, & Beraza´ın, 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 in-
traspecific 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, espe-
cially 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 morpho-
logical 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, Est´ebanez, & 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;
Fern´andez, Boquete, Carballeira, & Aboal, 2015; Onianwa, 2001; Stankovi´c, Sabovljevi´c, & Sabovljevi´c,
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
2
Posted on Authorea 20 Oct 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160315210.03739090/v1 | This a preprint and has not been peer reviewed. Data may be preliminary.
on the natural variation of these traits (e.g. Bellini et al., 2020; Esposito et al., 2018; Kov´acik, 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 spe-
cialization. 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 ac-
cumulation 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 cata-
ractae (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. (Ditricha-
ceae), 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).
2. Material and Methods
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
3
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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.
2.2 Culture in the laboratory
Before the treatments, we propagated all field populations of S. cataractae and C. purpureus in a growth
chamber for several months to eliminate the physiological stress history of the plants, i.e. carryover of envi-
ronmental effects. We carefully cleaned gametophytic tissue of S. cataractae under the dissection microscope
using deionized (DI) water and a brush, cut it with a razor blade, and spread each population into 4x4 cm
pots containing a 2:1 mixture of clay (Turface) and commercial soil.
For C. purpureus , we sterilized gametophytic tissue of the field collected population, Cp1, using 0.2-1%
bleach with shaking for 1-2 min, rinsing the tissue in sterile water with shaking for 1 minute, and spreading
it into 9 cm petri dishes containing 30 ml of BCD growth medium solidified with agar (Cove et al., 2009).
Under the same conditions, we propagated one additional population of C. purpureus split into male and
female plants growing separately (Cp2.m and Cp2.f respectively), donated by Dr. Stuart McDaniel from the
University of Florida (Gainesville, USA).
2.3 Heavy metal treatments
The concentrations of Cd (as CdCl
2
) and Cu (as CuCl
2
) used in this study were selected to induce significant
effects in moss performance without causing its death, according to the available literature (e.g. Ares, Itouga,
Kato, & Sakakibara, 2018; Carginale et al., 2004; Konno, Nakashima, & Katoh, 2010), and culturing trials
carried out in our laboratory.
For S. cataractae, we cut approximately 50-70 clean gametophores from each population into small pieces
with a razor blade, mixed each with 2 ml of DI water, and spread them in 4x4 cm pots containing a previously
autoclaved 2:1 mixture of clay (Turface) and potting soil. We cultured a total of 60 pots (4 populations x 3
treatments x 5 replicates per population and treatment) for 3 months in the growth chamber, and watered
the pots every two days with DI water. We applied the following treatments by watering the plants every
two days for exactly 30 days with 20 ml of: water (control), 1 mM Cu (Cu), and 0.1 mM Cd (Cd) (n=4-5
replicates per population in each of 3 treatments).
For C. purpureus, we transplanted 7-day old protonema into new petri dishes overlaid with sterilized cel-
lophane discs. Each plate contained BCD medium enriched with metals under the following treatments:
control (C), 0.02 mM Cu (Cu), 0.01 mM Cd (Cd) (n=7 replicates per population and treatment). The levels
of Cd and Cu in the treatments differed between species due to their obvious differences in tolerance. The
treatment lasted 21 days and we took pictures of each replicate at the beginning and at the end of the
experiment to measure the individual growth of each protonemal mat (n=5 mats per replicate).
In both experiments, we changed the position of the replicates within the chamber every week to minimize
local microenvironmental effects. At the end of each common garden experiment, we harvested the plants,
blotted them with filter paper, and separated several aliquots from each population and treatment in order
to perform different analyses: several aliquots were immediately frozen in liquid N and stored at -80ºC for
lipid peroxidation and microscopy analyses; one aliquot was kept in the oven at 50 ºC in plastic tubes for
total Cd and Cu determination; one last aliquot was stored at 4ºC in the fridge to phenotype the plants
(only in S. cataractae ).
2.4 Measurements of plant performance
2.4.1 Growth of C. purpureus and morphology of S. cataractae
4
Posted on Authorea 20 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160315210.03739090/v1 — This a preprint and has not been peer reviewed. Data may be preliminary.
We measured plant vegetative growth as a proxy for fitness. For S. cataractae , we measured the following
morphological traits in 10 gametophores from each population and treatment: i) plant length, as the length
in mm of the green part of the gametophyte; ii) leaf length, as the average length in mm of three leaves per
shoot from the apical most part of the gametophore; and iii) leaf width, as the average width in mm of the
same three leaves. These same traits were also measured in 50 gametophores from each field population of
S. cataractae .
Because the area of the protonemal mat is considered a suitable proxy for biomass production in mosses
(Burtscher, List, Payton, McDaniel, & Carey, 2020), for C. purpureus we calculated total growth by sub-
tracting the area of each protonemal mat (mm
2
) at the beginning of the experiment to the area at the end
of the experiment, and averaged the growth of 5 mats for each replicate.
All morphological and growth measurements were made by processing the photos taken of S. cataractae and
C. purpureus with ImageJ v1.51 (Schneider, Rasband, & Eliceiri, 2012).
2.4.2 Lipid peroxidation
We evaluated lipid peroxidation of the plasma membrane as a proxy of the level of oxidative damage in
plant tissues grown under the different treatments (n=3 replicates per sample) by measuring the amount of
malondialdehyde (MDA) following the thiobarbituric acid (TBA) assay described by Heath & Packer (1968)
and Catal´a et al. (2010). We first prepared a 2 mM stock solution of the MDA precursor, malonaldehyde
bis(diethyl acetal) (1,1,3,3-Tetraethoxypropane; Sigma Aldrich, T9889) and built standards of 0, 5, 10, 20,
and 40 μM MDA by diluting the stock solution in 80% ethanol with 2% buthylated hydroytholuene (BHT;
Fisher Scientific, ICN10116290). For each sample, we homogenized between 3.6 and 75.8 mg of frozen moss
tissue in a tissue lyser (Qiagen TissueLyser II) during 2-4 min, in rounds of 30 sec. Samples were immersed
in liquid N between rounds to prevent tissue melting. We then added 1 ml of 0.1% trichloroacetic acid
(TCA; Fisher Scientific, ICN19605780) to each sample and standard, and vortexed them. We centrifuged
all tubes at 10,000 g for 20 minutes and recovered 500 μl of supernatant. We added an equal volume of
20% TCA containing 0.5% TBA (Sigma Aldrich, T5500-25G) to each tube, followed by 5 μl of BHT. All
tubes were incubated at 95ºC in a hot plate for 30 minutes, cooled quickly on ice, and centrifuged at 10,000
g for 15 min. Finally, we recovered the supernatant, and measured its absorbance at 532 and 600 nm in
a 96-well spectrophotometric microplate reader (Epoch Biotek). The concentration of MDA was calculated
after subtracting the absorbance at 600 nm from that at 532 nm to eliminate the possible interference of
soluble sugars present in the samples (Du & Bramlage, 1992).
2.5 Contents of heavy metals
2.5.1 Total Cd and Cu content in moss and soil
We dried moss tissue from all populations and treatments at 50 ºC, including field samples, and ground
them to a fine powder in a tissue lyser (n=3 replicates per sample). Then, we digested between 1-17 mg of
plant tissue in small Teflon vessels with 2 ml of HNO
3
(100%, TraceMetal Grade, Fisher Chemical) in a hot
plate at 175ºC. After 2 h, we added 0.8 ml of H
2
O
2
(ACS grade) to each sample and incubated them again
for 2h at 175ºC. We also prepared one sample of certified reference material (M2,Pleurozium schreberi moss
tissue, Steinnes, Ruhling, Lippo, & Makinen, 1997) in the same way, and analyzed it along with analytical
blanks after every 12 samples to control for instrumental precision and contamination.
Similarly, we dried soil samples from the field (n=3 replicates per sample), sieved them to separate the 2 mm
fraction, and ground them to a fine powder before analysis. Between 9-30 mg of dried soil were digested in
small Teflon vessels with 2 ml of HNO
3
(100%) for 8h at 175ºC. This acid dissolves only a very small fraction
of structural minerals in the soil recovering mainly the most labile/reactive fraction of heavy metals (Melo,
Batista, Gilkes, & Rate, 2016). We used one certified reference material prepared in the same way as the
samples, once every 12 samples, to control for instrumental precision (marine sediment PACS-2, National
Research Council of Canada).
Total contents of Cd and Cu were determined in both matrices at the College of Marine Sciences (Univer-
5