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 | https://doi.org/10.22541/au.160315210.03739090/v1 | This a preprint and has not been peer reviewed.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. n hmsr xs tdffrn pta n eprlsae Bl ta. 93 iluc,20) naddition, In 2001). Pigliucci, 1993; al., et (Bell composition scales soil temporal and availability, and water ubiq- spatial radiation, is different temperature, heterogeneity at Environmental in exist variation chemistry organisms. spatial and living and all gradients to as challenges uitous, continuous and poses environment research Lichtscheidl The 2020 Irene Horizon Union’s Dr. at European thank analyses Introduction the we soil 1. University from and Sk funding for Research, Marie (VLSI) received infrastructure Ultrastructure the Instruments has under & and Life-Science research programme Imaging This Vienna support innovation sample Cell the providing support. Facility with of her for Core member for help Lewis the – their B. at Vienna David for performed of Aguero was Dr. populations Blanka SEM-EDX lab-maintained and USF. from Atlas Dr. material critically D. providing for and for Zachary and Shaw McDaniel project F. the A. Stuart of Jonathan of development Dr. the collection; throughout and Dr. advice localization his manuscript; for the Herrera M. reviewing Carlos Dr. in thank tolerance We metal heavy in differences lack Acknowledgements sex-specific and metal-dependent, variation in for genetic variation evidence of high levels for as low support well provide potentially as results despite stress, Our challenge bryophytes. to environmental males). to exposure than preferential respond tolerant previous dimorphism to sexual more to of associated for bryophytes being evidence be of (females first could the purpureus capacity Cu report C. the also of Scopelophila We in case specialist organs. tolerance the photosynthetic metal in Cd main their which metals. heavy for protect accumulation these the relatively to to in stem laboratory, yet tolerance differences the the towards resilient showed in stress relocation differences in specialist most metal intraspecific metal Cu the showed heavy heavy and of purpureus, the to Cd Ceratodon one Only response metallophyte to bryophytes, facultative plant exposed in the intraspe- to mosses and tolerance of cataractae related mechanisms terrestrial and and Two traits accumulation patterns metal in the as taxa. investigate heavy variation such We understudied for survival. of conditions variation population’s environmental study a phenotypic challenging of the cific likelihood to Hence, the adjustment into insight rapid pollution. valuable for provides metal crucial heavy is to populations exposure natural in variation Phenotypic Abstract 2020 20, October 4 3 2 1 Alonso Conchita Boquete Teresa M. habitat contrasting with species specialization moss terrestrial and two accumulation in metal tolerance heavy of mechanisms and Patterns nvriyo ot Florida South of University Sciences Vienna Life of Universitiy of Faculty Vienna of University Estaci´on Do˜nana Biol´ogica de .purpureus C. uz iv iosadOii ataofrhl uiglbrtr oka S;Dr. USF; at work laboratory during help for Santiago Olivia and Simoes Silva Luiza ; 1 1 neogLang Ingeborg , 2 l ailieWeidinger Marieluise , dwk-ui rn gemn o704141-BryOmics. No agreement grant odowska-Curie 1 3 hitn Richards Christina , 4 and , 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. iceia epne ohaymtlepsr ne otoldlbrtr odtoswtotacerfocus and clear physiological a their without or conditions 2020), laboratory controlled 2014; Raaba, 2006; under Nakamura, al., & exposure & on Jonczakb, et metal Yamashita, heavy focused Sut-Lohmanna, R., Ares to Kofuji mostly 2018; in: responses Kobayashi, has Greger, Sabovljevi´c,biochemical (reviewed 2017; field & al., Stankovi´c, & Sabovljevi´c, pollution et this Landberg, 2001; metal Itouga in Sandhi, Onianwa, heavy and (e.g. research phytoremediation 2015; of 1970s Recent and biomonitors Aboal, late species. 2018) on as the target & work bryophytes from few the Carballeira, (e.g. of dates a of Fern´andez, Shaw, Boquete, value bulk on populations species 1994; the bryophyte applied focused Shaw, few However, natural was the 1991). & a in and Beer, Jules in accumulation 1990s, & 2017; metal and Jules, plasticity early Medina, heavy Shaw, tolerance inherent Est´ebanez, & tolerate 1987; metal broad Cogolludo, Anderson, to heavy & as 1978; bryophytes Antonovics, of House, well Shaw, capacity & 1988; as the Brown differentiation, in 1972; ecotypic variation Briggs, found intraspecific deal have and to inter- pollution pathways of metabolic insights studies important unique provide Several of responses. could poikilohydric variety these study their of a their to evolution 2017a,b), use the due Glime, they into 2009; regulation Since (Cuming mya, self-internal morpho- challenges 2009). ~500 of environmental of plants capacity Goffinet, with degree vascular low low & of a of relatively (Vanderpoorten a group and concentrations nature complexity, with sister high anatomical plants their accumulate (haploid) and from & and gametophyte-dominant logical diverged Hermans, non-vascular, tolerate Verbruggen, plants are to 2017; they These capacity espe- al., and 1994). the tracheophytes, et (Shaw, shown from Reeves pollutants also derived 2006; these have largely Ernst, extent underlying Bryophytes is 2014; mechanisms the in- 2009). Pilon-Smits, significance the of Schat, and & evolutionary plants, knowledge (Cappa inter- and in current angiosperms ecological of tolerance and our cially their and levels Accumulation However, accumulation and high 1981,). 2015). metal traits, while show heavy Baker, Mason, these metals that for 2016; and variation of traits (Goolsby al., intraspecific uptake quantitative plants of et avoid distinct in (Adlassnig to genetically variation levels mechanisms metals complex traspecific different have heavy thus substrates at to are enriched them exposure tolerance metal accumulate of heavy presence can tolerance the in others develop in living Such to fitness plants 2003). populations maintain Jain, Most plant to & of capacity Budhiraja response the Pandey, rapid S., or 2000). similarly Labana (Simms, pollution, a Singh, metal requires 2005; production heavy toxicity energy Stoffella, deposition to soil as surface & well in of Yang, as in result increase activities, He, a increase mining rapid 2002; as and dramatic metals al., agricultural, a in industrial, et enriched caused from naturally (Bradl have derived not particles activities are and anthropogenic that dust soils Kruckeberg decades of in (Whiting 1985; last rise metals value Malaisse, the of giving conservation and concentrations during run, significant the Brooks However, long of Beraza´ın, 1996) the 2004). (e.g. & endemicity in al., Borhidi, of adapted Baker, et have level Reeves, species considerable 1990; weathering plant a Kruckeberg, Pauwels, natural some with and The which (e.g. flora 2006). to metallophyte species al., soils a plant and et generated to Wright many plants, has 2017; in on al., rocks at ecotypes pressures et even metal-rich hyperaccumulator selective Reeves toxic of intense and/or 2006; be exert tolerant Saumitou-Laprade, can & can of and concentra- toxicity Bonnin, evolution functions high Frerot, Such physiological the at known toxic Hg). to no Pb, be led have 2006). can Cd, has others Scherson, they while (e.g. & but Zn), concentrations plants Stanton, Mn, low Wright, all Fe, very in 2004; Cu, functioning Co, Boyd, normal 1990; (e.g. for Bradshaw, Shaw, tions essential g/cm (Antonovics, 1987; are 5 implications metals Macnair, evolutionary > heavy 2006; and Some density Ernst, ecological specific 1971; important without a Turner, with respond with & plants elements to for of ability pressure (i.e. probability the lective the metals increase requires heavy and to it ses- reproduction, Exposure as The 2009). and Hoffmann, survival demanding, lifespan. their & more their compromise (Willi even throughout would extinction so task organisms population do these by this to with faced makes Failure Coping challenges plants moving. Mitchell- 2001;). main of & Lehman, the nature and Willis, of Tilman sile (Anderson, one 1997; ecosystems therefore, Melillo, major is, the & the Lubchenco, cond 2 3 au,20)i oeflse- powerful a is 2003) Jarup, ; 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. aeilwssprtdaddidt eemn oa dadC otns(eto .) nte lqo was the aliquot In another dark. 2.5), the (section contents in Cu cooler and a Cd of total inside aliquots determine stored Various to nitrogen. and dried soil. liquid and bags -80 in the separated at plastic frozen from was stored and zip-lock date, material moss were tubes into same Eppendorf of samples put the ml frozen clumps was 1.5 On laboratory, several in material (Sc4). stored separate edge remaining were to clumps NW The through knife these (Sc1), the from a edge in tissue SE used finishing gametophytic the we and in was occasion, Sc3), beginning mine and sampling slope The (Sc2 m). the ~300 down mine to sampled purpureus the (~20 were of short populations by was center (separated and patches the species these slope we this between a Here of distance patches 1987). on the scattered Shaw, situated though and 1984; even unconnected (Wickland physically soil), to 1950s bare refer the mostly to in of population discontinued term populations was the activity four use whose from al., (USA), plants Carolina et collected North (Shaw we areas 2016, metallophyte September unpolluted facultative In or in pseudometallophyte reproduction a sexual considered be undergoes thus areas. frequently (1987). could disturbed it highly species and to This preserved sexes 1991). well separate from ranging has that substrates, also suggesting of variety study a this on in occurs used resulting populations purpureus cycle clonal. the Ceratodon life mainly in gametangia the or of observed female exclusively phase and are been diploid copper- male populations never the these i.e. have of are dioecious, reproduction, distribution which is mosses, sexual Sporophytes, the gametophores. species from copper matches different The roughly in other 1995). developed that 1993a,b, to are dry 1987, worldwide Similar plant’s Fujimoto, (Shaw, distribution the & substrates 1988). of geographical enriched Otake, Fuwa, 3% disjunct Itoh, to & but Nakajima, up Nishikawa, reach 1999; broad can Shibata, Matsumoto, Cu Satake, & and Nishiyama, hyperaccumulator 2011; Sakamoto, Cu a Nagano, (Aikawa, is It weight 1993a). (Shaw, habitats enriched cataractae Scopelophila sampling. field and species settings Study 2017). environmental 2.1 Moore, different 2016; in Methods Mcletchie, species & and other Burton, Material for Marks, 2. shown 2000; as Mishler, the males & whereas McLetchie, than Stark, levels, exposure (Bowker, metal accumulation heavy similar to under tolerant specialist tolerance metal ( increased the accumulation, decreased has that i.e. that predict strategy aspect We an populations tolerator” knowledge. non-specialist metals, female “stress tolerate our populations, and and to a among accumulate male previously show to differences with capacity addressed phenotypic it their evaluated been in compared We differed not and conditions. range sexes field axenic whether a examined under the in and in laboratory growing collected the populations tolerant in population among metal grown one tolerance but studied and non-specialist, we accumulation the ceae), For for variation soils. metal-rich was metal a of there ac- in if growth of determine vegetative patterns to maintain their to ( examined ability environment the and ractae control as treatments, phenotypic a tolerance metal define vs. intraspecific different spe- we stressed heavy of under Here, habitat for extent laboratory tolerance. contrasting affinities the contrasting the and to with cumulation in detail relation species them in more moss bryophytes grew terrestrial in different in metals, explore ecologically & tolerance two Kov´acik, to Dresler, and selected 2018; We research accumulation cialization. al., metal classic et heavy the Esposito for on variation 2020; builds al., et study Bellini This 2018). al., (e.g. et traits Liang these 2020; of Babula, variation natural the on Mt. rt Ptica) esuidfu edppltoscletdwti omrcpe mine copper former a within collected populations field four studied we (Pottiaceae), Broth (Mitt.) C1 a olce ihna ra rai uhm ot aoia(S) teeyst and site every At (USA). Carolina North Durham, in area urban an within collected was (Cp1) .purpureus C. soeo h otcsooia roht pce ihabodeooia ag.It range. ecological broad a with species bryophyte cosmopolitan most the of one is soeo h ocle cpe oss u oishg ffiiyfrhaymetal heavy for affinity high its to due mosses” “copper so-called the of one is sensu ol hwa“tesaodne taey ..icesdtlrnersligfrom resulting tolerance increased i.e. strategy, avoidance” “stress a show would ae,18) lo epeitta eae of females that predict we Also, 1981). Baker, sensu im,22) o h ev ea specialist metal heavy the For 2020). Simms, º o irsoyaayi scin252,a lqo ffresh of aliquot an 2.5.2), (section analysis microscopy for C 3 eaoo purpureus Ceratodon .cataractae S. .purpureus C. namn iei ivrHill, Silver in site mine a in Hd. rd (Ditricha- Brid. (Hedw.) eaoo purpureus Ceratodon .cataractae S. .cataractae S. cplpiacata- Scopelophila ol emore be would sensu Ceratodon would Baker a a has 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. .. rwho .pruesadmrhlg fS cataractae S. of morphology and purpureus C. of Growth 2.4.1 4 performance at plant stored of 50 Measurements was at -80 2.4 aliquot oven at the last stored in one and kept determination; N in order was liquid Cu (only in aliquot in and treatment one frozen and Cd analyses; plants, immediately population total microscopy the were each and harvested minimize from aliquots the we peroxidation to aliquots several of experiment, lipid week several analyses: every end garden separated different chamber and common the perform the paper, each to at within filter of and replicates with end the them beginning the replicate). of blotted At the per position The effects. mats at the tolerance. microenvironmental (n=5 changed replicate in local mat we each differences protonemal experiments, obvious each of both of their pictures In growth to levels took individual due The the we treatments: treatment). species measure and following and between to population differed days the experiment per treatments 21 replicates under the (n=7 lasted metals in (Cd) treatment Cd with Cu (n=4-5 mM and enriched 0.01 (Cd) Cd (Cu), medium Cd of Cu mM BCD mM 0.1 contained 0.02 and (C), plate control (Cu), Each Cu every discs. mM plants 1 lophane the (control), watering treatments). water by 3 of: treatments watered For of ml following and each 3 20 the chamber, in x growth with applied population populations days the We per (4 in 30 water. replicates pots months exactly DI 60 3 for with of previously for days days a total treatment) two containing two a and pots every cultured cm population We 4x4 pots per in soil. the them replicates potting spread 5 and and (Turface) water, x DI clay treatments of of ml mixture 2 2:1 with each autoclaved trials mixed blade, culturing razor and a with 2010), Katoh, & Nakashima, For Konno, Itouga, laboratory. Ares, 2004; (e.g. our al., literature in available et out the Carginale to carried 2018; according Sakakibara, death, its & causing Kato, without performance moss in CdCl effects (as Cd of concentrations The 2009). the al., treatments from et metal McDaniel (Cove Heavy Stuart agar 2.3 Dr. by with USA). donated of solidified (Gainesville, spreading respectively), medium population Florida and Cp2.f growth additional minute, of and BCD University 1 (Cp2.m one separately of for propagated growing shaking ml we plants with 30 female conditions, water containing sterile same dishes in the petri tissue Under cm cm the 4x4 rinsing 9 into min, into population 1-2 it each for spread shaking with and soil. bleach blade, commercial razor and (Turface) a clay with For of it envi- mixture cut of 2:1 carryover brush, a i.e. a containing plants, and pots the water of of (DI) history tissue deionized gametophytic stress using cleaned physiological carefully the of We eliminate populations effects. to ronmental processing. field months further all several until propagated for dark chamber we the treatments, in the environments. cooler origin Before a their in the laboratory in kept underneath the exposed right and in were soil bags Culture mosses of plastic which 2.2 cm zip-lock to 2-5 in metals upper soil stored heavy the surface were of the of levels samples of consisting the These composition samples assess chemical soil to the collected by clumps we influenced moss site mostly each are at they Thus, roots, layer. growth real a no have in mosses experiments. maintained Because the was of material duration remaining the the throughout and maintained 2.4.1), (section 22 at plants chamber the phenotype to separated .cataractae, S. .purpureus, C. .purpureus C. .cataractae S. º n 6 ih/hdr opromtecmo adneprmns hs odtoswere conditions These experiments. garden common the perform to dark light/8h 16h and C eseiie aeohtctsu ftefil olce ouain p,uig0.2-1% using Cp1, population, collected field the of tissue gametophytic sterilized we , ectapoiaey5-0cengmtpoe rmec ouainit ml pieces small into population each from gametophores clean 50-70 approximately cut we etaslne -a l rtnm nonwptidse vradwt trlzdcel- sterilized with overlaid dishes petri new into protonema old 7-day transplanted we ). 2 n u(sCuCl (as Cu and ) 4 2 sdi hssuywr eetdt nuesignificant induce to selected were study this in used ) .cataractae S. .cataractae S. º ntefig opeoyeteplants the phenotype to fridge the in C .purpureus C. ne h iscinmicroscope dissection the under and .purpureus C. º npatctbsfor tubes plastic in C pi noml and male into split nagrowth a in º for C 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. hmt n odri iselsr(= elctsprsml) hn edgse ewe -7m of mg 1-17 between digested we Then, HNO sample). of per ml replicates 2 (n=3 with 175 50 lyser vessels at at Teflon tissue plate small treatments a in in and tissue powder populations plant fine all a from to soil them tissue and moss moss dried in of We content interference Cu possible and the Cd eliminate Total in 2.5.1 to nm metals nm 600 1992). heavy 532 Bramlage, and of at & Contents 532 that (Du 2.5 calculated at samples from was the absorbance nm MDA in its 600 of present measured concentration at sugars The and soluble absorbance Biotek). supernatant, the (Epoch subtracting the 5 reader recovered after by microplate we followed spectrophotometric Finally, acid 96-well tube, min. trichloroacetic a each 15 0.1% to 500 for centrifuged T5500-25G) of recovered We 95 g Aldrich, ml at and (Sigma 1 them. incubated minutes TBA were vortexed added 20 tubes 0.5% immersed and then for were containing standard, We Samples g TCA and sec. 10,000 20% sample melting. 30 at each of tissue tubes to rounds prevent moss all in ICN19605780) to frozen min, Scientific, of 2-4 rounds Fisher mg during between (TCA; 75.8 II) N and TissueLyser 3.6 liquid (Qiagen between lyser malonaldehyde in homogenized tissue 20, precursor, we a 10, MDA sample, in 5, each the tissue For 0, of of ICN10116290). solution standards Scientific, stock Fisher built in mM and damage 2 T9889) 40 oxidative a Aldrich, (1968) and of Packer of prepared Sigma amount & (1,1,3,3-Tetraethoxypropane; level first the Heath acetal) We by measuring the bis(diethyl by (2010). described of assay sample) al. proxy (TBA) per Catal´a et acid a replicates and thiobarbituric (n=3 as the treatments following membrane different (MDA) plasma the malondialdehyde under the grown of tissues plant peroxidation lipid evaluated We peroxidation of Lipid taken 2.4.2 photos the processing replicate. by each made purpureus for were C. mosses measurements mats growth in 5 and of production morphological growth All biomass the averaged for (mm and proxy mat experiment, suitable protonemal the for each a of of 2020), considered Carey, area is the of & the mat tracting population McDaniel, of protonemal field Payton, mm the each List, in per from of (Burtscher, width leaves gametophores average area three 50 the the of as in mm Because width, measured in leaf also length iii) were average and the traits cataractae gametophore; as same length S. the length, These the of leaf leaves. as part ii) three length, gametophyte; most same plant the apical i) of the treatment: part from and green shoot population the For each of fitness. from mm for gametophores in proxy 10 a in as traits growth morphological vegetative plant measured We rcin n rudte oafiepwe eoeaayi.Bten93 go re olwr ietdin digested were soil dried of mg 9-30 mm Between 2 the analysis. HNO separate of before to ml powder them 2 sieved fine with sample), a vessels per Teflon to analytical replicates small them with (n=3 ground field along contamination. the and it and from fraction, analyzed precision samples instrumental and soil way, for dried same we control Similarly, the to in samples 12 1997) every Makinen, after & blanks Lippo, Ruhling, Steinnes, tissue, 175 at 2h for oa otnso dadC eedtrie nbt arcsa h olg fMrn cecs(Univer- Sciences Marine of College the at matrices both in the determined were as National Cu way PACS-2, and sediment same Cd (marine the of precision in contents instrumental prepared Total for material (Melo, control reference metals Canada). to heavy certified of of samples, Council one fraction Research 12 used labile/reactive every We most once 2016). the samples, Rate, mainly recovering & soil Gilkes, the Batista, in minerals structural of μ D ydltn h tc ouini 0 tao ih2 uhltdhdotoun (BHT; hydroytholuene buthylated 2% with ethanol 80% in solution stock the diluting by MDA M º º .Atr2h eadd08m fH of ml 0.8 added we h, 2 After C. .W lopeae n apeo etfidrfrnemtra (M2, material reference certified of sample one prepared also We C. . ihIae 15 Shedr abn,&Eier,2012). Eliceiri, & Rasband, (Schneider, v1.51 ImageJ with º nahtpaefr3 iue,coe ucl nie n etiue t10,000 at centrifuged and ice, on quickly cooled minutes, 30 for plate hot a in C 3 10)fr8 t175 at 8h for (100%) 2 2 ttebgnigo h xeiett h raa h end the at area the to experiment the of beginning the at ) O 2 ASgae oec apeadicbtdte again them incubated and sample each to grade) (ACS 5 3 10,Taeea rd,Fse hmcl nahot a in Chemical) Fisher Grade, TraceMetal (100%, μ .purpureus C. fspraat eadda qa oueof volume equal an added We supernatant. of l º .Ti cddsovsol eysalfraction small very a only dissolves acid This C. .cataractae S. º ,icuigfil ape,adground and samples, field including C, ecluae oa rwhb sub- by growth total calculated we emaue h following the measured we , luoimschreberi Pleurozium .cataractae S. μ fBT All BHT. of l moss and 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. ie3soe h ihs ocnrto olwdb ie ,1ad4(hc i o ie rmec other). each from differ not did (which Cd, 4 For (edge). and 4 1 ultimately 2, and sites (center), by 3 followed and concentration (edge) highest 1 the sites showed by 3 followed Cu, site ( of sites concentrations across highest significantly differed the levels) (total field soils the in from Cu and and cataractae Cd S. of concentrations of The morphology and and levels Cd metal Heavy of 3.1 concentrations total the on Results treatment 3. the of effect the assess replicates. to method test in Wallis (1995) Cu Kruskal Hochberg a & Benjamini used the We (FDR). using rates adjusted discovery used were false we p-values obtain Then all to Finally, 1). Table significant. in were (summarized transfor- effects models function BoxCox the the applied of with using fit or gaussian the comparisons 1), improve and = to link, deviation order function log in standard the with 1964) 0, gaussian Cox, = (inverse & (mean homoscedas- (Box response variables mations and the standardized normality model used for to link), tested distributions inverse and family trends, other any tried we for functions species. models the each the using for of ticity conditions residuals control inspected the graphically to We enriched) Cu width and leaf enriched and (Cd length, four treatments leaf in the length, width and plant leaf samples and and Cu, field Cd and The of Cd concentrations in details). of MDA total concentrations more relative samples: for and Field distribution 1 total i) in Table gamma soil; see datasets: the in following (but using Cu the cases and experiments comprised most garden experiments in common garden function common the link measured during log variables the species the with both on in levels) measured (4 variables population for of of levels effect samples 4 the field-collected evaluate the to in 2019) Team, RStudio v.1.2.5019, ( models linear sample). generalized the used for We in except present ( elements structures, percent all analyses weight the of Statistical weight as of 2.6 the expressed each by are two for divided concentrations For interest plant: measurements Relative of per (n=12 stems). element treatment. measurements for stem and 4 6 the population performed = on per and n two measured), which and be in could leaves, Sc1 leaves different the two software only on which and 35 For in detector) conditions: time. samples, acquisition drift dead following field 30% out (silicon the carried used energy SDD a We We with by and kV. charge. combination time, 20 analyses surface at in prevent elemental operating (SEM) to for V.4.3, JSM-IT300 020) TEAM (EDX) JEOL MED microanalysis EM microscope X-ray (Leica electron dispersive layer scanning carbon with nm measurements 10-20 all sticky a double with with stubs them aluminum Tabscoated on PELCO treatment foils, and (SEM-carbon for disks population each carbon 131% from and samples moss freeze-dried 102% in mounted and We content respectively, Cu soil and and Cd The plant Relative 7500cx). 2.5.2 in Agilent respectively. Cu (ICP-MS, soil for and Spectrometry plant 86% Mass in and Plasma Cd 82% Coupled averaged Inductively recovery by percentage Florida) South of sity .cataractae S. χ ² .purpureus C. .6 p 4.36, = .cataractae S. .cataractae S. anova i omngre:ttladrltv ocnrtoso dadC,adcnetainof concentration and Cu, and Cd of concentrations relative and total garden: Common ii) ; < .cataractae S. ots o h infiac ftemi ffcso h oesfloe ymlil pairwise multiple by followed models the of effects main the of significance the for test to ntelbrtr ihu conigfrtepplto ffc u otelwnme of number low the to due effect population the for accounting without laboratory the in 2.2e and -16 ,tetet( ees oto sC;cnrlv u n hi neato nthe on interaction their and Cu) vs control Cd; vs control levels: (2 treatment ), shapiro.test .purpureus C. o dadC epciey iue1.St i h etro h ie showed mine) the of center the (in 2 Site 1). Figure respectively; Cu and Cd for glht ntecmo adn ecmae h epne oec ftespecific the of each to responses the compared we garden, common the In . .cataractae S. .cataractae S. glm mlcm akg,Hton rt,&Wsfl,20)we h main the when 2008) Westfall, & Bretz, Hothorn, package, (multcomp .purpureus C. and ucinwti ...,RCr em 08 unn ne Studio R under running 2018, Team, Core R v.3.5.1, R within function rtnmlgot in growth protonemal ; leveneTest TM eslce lnsprpplto n ramn (including treatment and population per plants 6 selected we , n h ffcso ouain( eesfor levels (3 population of effects the and , abnCnutv as obeCae)adte carbon then and Coated) Double Tabs, Conductive Carbon rtnm,w efre oa fn1 measurements n=10 of total a performed we protonema, 6 epciey hnteeasmtoswr o met, not were assumptions these When respectively. .purpureus C. n ln egh eflength, leaf length, plant and , º aeo nl,a3 live s 30 a angle, off take χ ² .4 p 0.64, = i.e. .purpureus C. egto the of weight < 2.2e -16 ; 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. eeaie iermdl ngot eeldasgicn Pplto yTetet neato nthe in interaction ( p Treatment” width -4.384, leaf by and = “Population length p (z significant leaf Sc4 0.57, length, a for = plant revealed only in Cu stems growth what of in to on effect populations. those contrary three models Finally, exceeded other linear leaves stems. the the Generalized the among in in differ Cd the Cd Sc4 not of and and of did Sc2 levels levels 2D), they from the relative (Fig. significantly and p Cu, differ similar Sc2 5.23, not for reached and did found = populations Sc1 Sc3 we (z that four than except Sc4 All concentrations pattern, and 2E). total similar Sc3, (Fig. a higher showed Sc2, significantly leaves in reached in p levels leaves Sc4 Sc3; relative vs the and vs Sc1 in Sc4 Sc3 for for those Cd, -6.82 For 5.51 exceeded p = Sc2, stems 5.31, (z vs = the center Sc4 z the in for from Sc2; Cu 5.45 Sc3, for of Sc3, and levels vs Sc2 Sc1 interaction the than Treatment” for treatment that by -6.81 after “Population = leaves populations stems significant z in among the the Cu Sc2; the differed by of in of however, indicated levels end Cu relative as effect, the more higher leaves treatment at significantly in the Cu stems accumulated of of the that ( levels magnitude on Sc4 relative The Cu treated the of populations. for for levels other except relative the C), similar and than as 2A well (Fig. as to experiment concentrations compared total plants similar treated reached all leaves; leaves; in in in Cd Cu substantially relative ( increased for 2 Cu 40.88 Figure = and in Cd shown experiment as of garden controls common concentrations cataractae: relative S. and in in Total tolerance width and leaf uptake except metal studied Heavy traits I). all 3.2 to for 1G Sc4) Fig. the (Sc1, width; of edges leaf center the for the from from those plants than ( significantly that smaller revealed Sc2 differ were samples show not Sc3) field not did (Sc2, the did however, on among mine and tissue, performed significantly Sc1 plant measurements differ in in morphological Cd not lowest The of did was levels ( and relative Cd populations The Total Sc3 among 1D). in 1C). differences (Fig. highest significant and populations were three 1B other Cu (Fig. the relative among populations and three Total other soils. the in found as pattern in Cu p and 1.28, Cd of levels Total ial,C asdasgicn n ossetices ntecnetain fMAi l populations all in MDA of concentrations the in increase consistent and significant plants a ( treated and caused control Cu of means Finally, trait overall, and, p Sc3 close. -3.01, affect pretty = significantly (z are not Sc4 did in Treatments plants 3B,F). between Cu-treated p Fig. and length -3.36, Cd- plant and = for control p (z differences between significant -2.47, Sc1 leaf yielded = in Tests width and (z Cu-treated plants. leaf length treated Cd-treated and leaf in length and than and leaf length, control controls control length, plant between in plant length means for p for plant trait 2.88, plants for plants only = Cd-treated Cu-treated significant (z in statistically in Cu-treated 11% were compared differences 11% and plants to the treated 14, seem 14, although in 2, metals respectively), traits 50, by Both all by and in conditions. respectively, means control increased higher width in towards (means grown tendency controls when the by populations to shown other as growth, all ( Sc2 from enhance leaves those the than of smaller length were the for significant only nC-nihdmda hseetwsntsgicn o derce utrsta xiie idand mild exhibited that cultures Cd-enriched for significant not was effect This media. Cu-enriched in χ χ ² ² pop:treat treatment χ ² < 48,p 24.80, = .0 o dadC epciey,hwvr h ieecsfudi lnsddntrflc h same the reflect not did plants in found differences the however, respectively), Cu and Cd for 0.001 < .1frla egh and length; leaf for 0.01 .1 p 8.61, = .5 p 0.35, = χ ² treatment < < < 2.2e .01frS3 .0 p 2.50, = z Sc3; for 0.0001 < .01.I atclr ouain c n c,fo h de ftemn,showed mine, the of edges the from Sc4, and Sc1 populations particular, In 0.0001). < 0.6frrltv ui tm p stem; in Cu relative for 102.36 = 2.2e .5.PpltosS1adS4soe h poieted ihgnrlyhigher generally with trend, opposite the showed Sc4 and Sc1 Populations 0.05). -16 .cataractae S. o ln length; plant for -16 χ i.3)ta vrgdoeal14.2 overall averaged that 3D) Fig. ; < ² treatment χ .1adz=-.4 p -2.94, = z and 0.01 ² treatment χ < ² pop:treat .5 i.3) n eflnt n efwdhbtencnrland control between width leaf and length leaf and 3E), Fig. 0.05; .7frttlCd; total for 4.57 = χ lodffrdaogppltos( populations among differed also ² χ 88 o eaieC nstem; in Cd relative for 28.89 = .2 =.1;Fg 1F). Fig. p=0.615; 0.02, = ² χ pop:treat .7 p 0.37, = ² .5 p 2.75, = < .5frS4 u o nS1( 10,p=0.405). = p -1.09, = (z Sc1 in not but Sc4) for 0.05 7 χ .9 p 0.49, = < ² < pop:treat .5rsetvl;Fg BC,adfrla length leaf for and 3B,C), Fig. respectively; 0.05 .5frla it) o d hsitrcinwas interaction this Cd, For width). leaf for 0.05 < < 2.2e χ 2.2e ² < .0 p 0.80, = treatment .5.I eea,gmtpoe rmSc2 from gametophores general, In 0.05). -16 < -16 .5adz=-.9 p -2.79, = z and 0.05 o eflength; leaf for μ nalcss.FrC,alpopulations all Cu, For cases). all in < ncnrlclue n 80.5 and cultures control in M .01i l ae) eas found also We cases). all in 0.0001 4.7frttlCu; total for 143.07 = < .5frpatlength; plant for 0.05 χ χ ² ² treatment .0 p 3.00, = χ ² 21 o relative for 52.15 = .9 p 3.39, = < < .5respectively; 0.05 .01and 0.0001 χ χ < ² < < ² treatment pop:treat 0.001), 2.2e 0.0001 χ ² μ -16 M = 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. easicesdsgicnl nams l ouain sidctdb h infiateeto h treatment the of effect significant the by indicated as populations all ( almost in significantly increased metals ( aeohrs oto hsvrain(pt 8)wsfudaogidvdaswti ouain,even populations, within individuals among the of found traits was this cell 78%) of and leaf range to several geographical (up in discontinuous variation variation and morphological this of broad of levels the most high across found gametophores; studies 1995) In (1993b, Shaw past. species, the in of response variation specialization whose phenotypic ecological but unique stress cataractae environmental The Scopelophila to specialist: patterns metal tolerant and heavy highly extent The the plants 4.1 on of knowledge group understudied. current relatively accumulation a to Hanley mechanistic are metal adds in the mechanisms & heavy discuss investigation variation Parmesan and for present phenotypic metals 2010; variation The heavy of al. for intraspecific differences. affinity et such of contrasting Nicotra for with patterns species basis 2019; the bryophyte two Nicotra, investigate in we & tolerance environmental context, and Kruuk, specific species this (Arnold, to and/or response In taxa in population variation 2015). plant phenotypic individual, different of 2015). to levels Hanley in the contributes & understand drivers plasticity Parmesan better phenotypic to 2010; need whether al. species we ultimately, survival, understand et Bossdorf, Williams, and, (Nicotra to Richards, population 2015; change order of 2007; global Rieseberg, In component Fr´eville, al. of & critical context Turner, et a current habitats 2008; be Geng the new could al., 2012; under of plasticity et invasion al., persistence Hence, the Richards 1995). et Black, during 2006; Drenovsky & or Pigliucci, (e.g. Mack, plants & limited envi- clonal heterogeneous Gurevitch, in be highly particularly Muth, can McIntyre in 2010;), diversity 2015; plants al. genetic al., L´azaro-Nogal of et et 2018; where survival Richards al., the 2014; et to Henn Strauss, (e.g. contribute & expectation conditions may classic environmental 1965, now traits changing a (Bradshaw and functional Indeed, ronments conditions of 2000). environmental 1987, plasticity Sultan, of diverse that to 1986; ability Schlichting, under envi- is the plants 2012; challenging phenotypes i.e. Stoehr, of different to & 2010), Panhuis, capacity express al., Kelly, accommodate 2006; et to the can Nicotra genotype challenges Organisms 2006; single (Bradshaw, conditions. 2019), a plasticity environmental toxic new Rypdal, phenotypic in highly a through & of shifts conditions define (Vallack emission ronmental rapid to epoch global the proposed continuous this to scientists The during that respond 2015). point place Maslin, & the taking (Lewis to pollutants Anthropocene Earth the the epoch, reshaping geological profoundly are activities Human ( Discussion populations 4. across differed effect (Cp2.f). this population MDA. same and of concentrations effect the by significant p measured respectively. the a as 3.60, Cp2.f of showed damage and oxidative size treatment Cp2.m in mean effect Cp1, Cd the significant from However, Cd, a plants For have respectively. Cd-treated not did in Cp2.f treatment 26% and Cu and Cp2.m 74 in 53, 59% by and decreased 47 mats by protonemal decreased it 17%, by decreased ( p interaction Treatment” 8.21, by = decrease “Population significant significant a the caused ( by metals populations these three to Exposure all < Cp1. in for growth plants protonemal control ( in than Cu Cu for more only accumulate not significant was interaction Treatment” by 0.39 were Cp1 collected we where soil the in Cu experiment and garden Cd common μ of purpureus: concentrations C. total in The tolerance and uptake metal Heavy 3H). 3.3 (Fig. concentrations MDA in changes non-consistent χ χ g g ² ² 2.2e treatment treatment -1 -16 epciey oa ocnrtoso dadC nrae infiatyi d n utetdplants Cu-treated and Cd- in significantly increased Cu and Cd of concentrations Total respectively. < o d i.4,G lhuhtemgiueo h ffc iee mn ouain sindicated as populations among differed effect the of magnitude the although G) 4C, Fig. Cd; for < .01 ihmlsfo p C2m xeinigmr aaeta p n eae rmthe from females and Cp1 than damage more experiencing (Cp2.m) Cp2 from males with 0.0001) .,p 7.5, = .6 p 2.06, = .01frC) hs hra h ensz ftepooea a fC-rae lnso Cp1 of plants Cu-treated of mat protonemal the of size mean the whereas Thus, Cd). for 0.0001 < < .1frCd; for 0.01 .0 o Cu; for 0.001 χ ² treatment χ ² .cataractae S. treatment .1 p 8.31, = χ ² .8 p 6.88, = treatment 8 o ev eashsisie oersac nits on research some inspired has metals heavy for < χ ² .1frC;Fg A ) h eaielvl fboth of levels relative The E). 4A, Fig. Cu; for 0.01 pop:treat .1 p 2.31, = χ ² < pop:treat .01frC;Fg B ) h “Population The F). 4B, Fig. Cd; for 0.0001 .7 p 1.07, = < 32,p 33.20, = .01frCu; for 0.0001 < .5 sC-rae lnsdid plants Cu-treated as 0.05) < .01frCu; for 0.0001 ± χ ² .7ad15.9 and 0.07 treatment χ ² 85,p 98.50, = pop:treat χ ² pop:treat ± 4.7 = Posted on Authorea 20 Oct 2020 — The copyright holder is the author/funder. 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Data may be preliminary. c n c eeudrvsua ln oe n c a lnsd esnlsraltta a abundant had that streamlet seasonal a alongside was from Sc4 plants and contrary, cover of the plant On mats vascular availability. center water under the the lower were particular, for and Sc4 In except temperatures, and mine. vegetation and the Sc1 any light of higher of edges sampled experienced the devoid were surely and was plants center study, of mine this the output the between In reproductive 2006). differed of lower Ernst capa- clearly and 2017; retention that production, Slomka, water microhabitats biomass and low in lower Bothe or metal rates, 1987; supply, machinery, (Baker, growth nutrient plants slower scavenging poor metal-adapted toxic the ROS exposure, conditions to potentially sun environmental the contribute intense the adverse also of i.e. Other city, counteracting soils, 2010). upregulation to Marmiroli, polluted agents, resources & characterize chelating Visioli, that energetic Marmiroli, of at of Maestri, synthesis cost, tolerance allocation see Metal (e.g. biological transportation; the 1994). a metals Shaw, to and with of due (Jules come bryophytes effect cost under may other laboratory metabolic for tolerance the reported a in metal previously entails grown phenomenon greater when concentration a that populations Sc2, outstanding three suggests in an other least with pattern the site This from experiment. the plants conditions. the from than of control collected smaller beginning Sc2, always the were from from Cu, plants of treatments, applied among metal been differences the had that from treatments predict thus Aside the our We if in gametophores. greater found adult even differentiation of phenotypic treatment be the of would Interestingly, days populations 1993a) 30 in and (Shaw only differences soil after readily novel resulted polluted reveal study were in could evident metals Pigliucci, conditions not (e.g. experimental were both Because environments our that 1993a). so between 1993a), (1987, vary in Shaw soil, studies can tolerance 2010; Shaw’s commercial species 2006, metal in one al., soil a by et mine to Richards expressed treatments the variation 2001; in the solution phenotypic true that in not of the way was amount were the Cu which field the in plants, and the the differences the in to Cd for the metals available related by of applied directly was partly levels We response explained higher to applied. this be exposed and could (Shaw, were those of discrepancy Cu, soils i.e. This regardless mixed and environments, tolerant. and Cd soil origin most control their to of polluted polluted in in differently mixtures in metals in others responded well of and better than populations levels equally grew better study, soil, populations grew performed our unpolluted all populations In populations and Zn), some 1993a). All growth, but field and studies. plant environment, the on Cu both original in soil-type their in in of pollution soils enriched effect metal unpolluted strong soil heavy a than (mine of found levels He soil two. different polluted these under soil-types: growing different respectively experimental on previous species of this results the of with extent populations some to contrast genetic species on limited this work for potentially found we with that differences populations The among variation phenotypic high intraspecific in suggests diversity. of tolerance existence finding the and This time, accumulation first scale. pro- metal the geographical heavy for their demonstrated, for to we differentiation due Still, Sc3) populations. and of our Sc2 movement among populations through differentiation other the passing the or through Sc4 to towards this, population populations ( Sc1 one in ximity from among from observed slope differen- flow gametophores the genetic were of gene down some fragments sporophytes (especially some out the or no rule gemma) expected of (e.g. cannot and we 20% we propagules sex) where asexual Although populations, 1993a). express Sc3 our Shaw, for not among (e.g. (except did tiation populations plants these 80% of non-sexual and studies of gametangia previous entirely male almost bore consisted plants also populations populations within field variation Our morphological they and that of suggesting sporophytes) levels 1995), high of 1993a; the (Shaw presence attributed sex (by of one (1995) only Shaw reproduction from mostly Therefore, sexual plants was clones. and of were plants that evidence sterile 1995). level of (Shaw, showed species comprised uniform Allozyme never mainly the genetically were were 1993b). populations at populations variation these (Shaw many genetic of and regions high populations, Some showed geographical among variation populations separated by these for widely of accounted five subset a from on populations analyses included he though .cataractae S. < .cataractae S. 0 wyfo ahohr.Udrteecrusacs eepce iie eeso phenotypic of levels limited expected we circumstances, these Under other). each from away m 500 opeoyi lsiiyrte hngntcvariation. genetic than rather plasticity phenotypic to .cataractae S. hw(97 93)poaae aeohtctsu fegtadfieNrhAmerican North five and eight of tissue gametophytic propagated 1993a) (1987, Shaw . iia ohwuplue n ie ol eeldpeoyi differences phenotypic revealed soils mixed and unpolluted how to similar , 9 .cataractae S. .cataractae S. navr iie geographical limited very a on opat rmS2adSc3 and Sc2 from plants so Posted on Authorea 20 Oct 2020 — The copyright holder is the author/funder. 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Data may be preliminary. teswihcudtasaet eea testlrne hra p a engoigi h laboratory the in some growing experienced been likely Cp1 had it of Cp2 where in tolerance field whereas higher Cu the tolerance, times in higher stress collected 3.5 the was general and that this to 2.8 argue as was translate could effect, decline could One hardiness this general which 4C). a whereas this stress (Fig. to treatment, reduced of due respectively was Cu magnitude growth be Cp2 under Protonemal the could from Cp1 but measured. females in trait study, and the 17% this on males only from and metal by populations the average all on on depended negatively field. population affected the each tolerance negatively in on when support metal effect metals Cd further differentiation heavy of and provides in levels ecotypic differences study Cu high intraspecific undergo This Both of experienced evidence never also 1994). showing had Shaw by can that & species it populations this (Jules between of pressure but resilience selective high 1991) enough the for al., strong a 1991), tolerance et metal to al., (Shaw heavy subjected et on environments studies Shaw Previous enriched 1982; that tolerance. metal Ireland, showed ecological broad species 1951; a this Dunham, has degrees in species 2010; different with moss Lawley, and this substrates canopies), Bosanquet, that rocky well-developed suggesting Atherton, to or (see soil woody, bare sandy, disturbance (from soils, of vegetation calcareous surrounding to of amounts acid varying characteristics: environmental diverse with no purpureus is purpureusCeratodon Ceratodon there metallophyte: and facultative toxic, The highly 4.2 non-essential, is stem. increase Cd that the of patterns contrary, towards (e.g. capacity accumulation toxicity the the and in in On tolerance result observed this in may differences though hidden. (2011) reveal of Hasezawa even been such and evidence & concentrations 3D), have Thus, Nomura high Fig. otherwise in too on in and controls. would best, Cu shown study its the damage present of at oxidative to the perform effect in in to compared beneficial Cu used slightly of those the concentrations decreased as that high plates needed showed constitutively agar CuSO however, species mM in (2011), 1 axenically Hasezawa at growing reversed & Nomura by was Nomura recently growth more and protonemal 1993a) (2011). (1987, Hasezawa Shaw by experimentally & 1993b), Shaw, here. (see found worldwide seems substrates Cu protection of photosynthetic of leaf affinity efficient levels Thus, high relative very 2007). The tissue-specific them al., allows the et making structure for Renzaglia exchange, species whose explanation 2000; gas moss Garbary, organs plausible & a non-tolerant functional and Nickrent, important and capture, Duff, tolerant are nutrient (Renzaglia, preferential leaves between structures and shown in Moss gametophores gametophores light differences already as the of moss 2016). have well of maximization Lang, within as studies parts 1996), & metals, Some Brown, specific Sassmann, & including Sc3. (Antreich, in Wells speculate elements, and 1996; Cu we Brown, of Sc2 of Therefore, & and accumulation Sidhu in transport leaves. activity 1997; observed in basipetal photosynthetic Brown, those their & tolerance and Sc4. (Brumelis and exceeded higher and acropetal leaves Sc4 Sc1 the protect for and edges, could extent evidence mine Sc3 stem some the central the Sc2, from towards no to from of plants Cu i.e. plants explain stems of absorbed of populations, leaves in of (Fig. leaves among in Cu respectively) relocation in those Cu Sc4 that of than concentrations of to levels lower relative concentrations and Sc1 were relative the Sc2, in total Sc3, Also, in Cu, 0.45% the and 0.96% 0.30, accumulate Sc2 in Sc3, 0.19, populations, to differences and (0.21, mine capacity find Sc1 Cd their not in of in (1.1% did d.w. differences Cu we 0.45% of – Although d.w. 0.21 D). ˜1% between 2A, reached and concentrations Sc4) main- total that in were their 1.0% suggesting and as conditions, 1G-I), (2010), control (Fig. al. under field et the experiment field. in of the garden populations Sc3 beyond common All and phenotype” the “metal-tolerant Sc2 in the from Sc2 maintained plants population in of (Fig. this size extent levels smaller some metal different the to their in tained including reflected differences, are microhabitat D), these 1A, All collection. sample during water .cataractae S. .cataractae S. .cataractae S. a iegorpia ag Cu nesn 91 n cur nhabitats in occurrs and 1981) Anderson, & (Crum range geographical wide a has hwn ihant o hsmtlwihmgtepanteitapcfi differences intraspecific the explain might which metal this for affinity high showing .cataractae S. .purpureus C. nti td eae sC-adC-yeacmltr codn oMaestri to according Cu-hyperaccumulators and Cd- as behaved study this in o uhsbe eosrtdb t eetdapaac nCu-enriched in appearance repeated its by demonstrated been has Cu for oacmlt d(i.2)adtelc fpeeeta relocation preferential of lack the and 2D) (Fig. Cd accumulate to abrdacntttv aaiyt oeaeadsriei heavy in survive and tolerate to capacity constitutive a harbored 4 tti ocnrto,tesz ftepooea mats protonemal the of size the concentration, this At . 10 Posted on Authorea 20 Oct 2020 — The copyright holder is the author/funder. 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Data may be preliminary. o oeac,(i)lwacmlto n ihtlrne n i)lwacmlto n o oeac.Given tolerance. and low accumulation and high accumulation (ii) low (iv) traits tolerance, and two high tolerance, these and high between accumulation and continuous interaction high accumulation a (i) the low in (iii) that plants: vary tolerance, proposed of low can categories (2015) that four Mason traits about & quantitative brings Goolsby different this, two Considering are tolerance manner. and accumulation to metal populations Heavy sexual plant of of frequency have capacity the comparison can the Interspecies limiting sex) 4.3 restrict ratios one turn sex changes. in in environmental population fitness might the in individual to which bias 2000; reduced dimorphism adapt recombination, as Sexual al., genetic (e.g. such et 2104;). level and level Mcletchie, (Bowker individual been reproduction population & the desiccation has the Trott, at to at Stieha, Slate, stress environment response Middleton, consequences environmental the 1993; Stieha, the to to Gaughan, 2017; on response response Moore, & in focused 2016; in Shaw research al., dimorphism 1999; most et sexual Marks and Beer, However, frequently, & less Shaw 2017). addressed 2012; Eppley, Eppley, & Rosenstiel, & 2016; Rosenstiel, (Balkan, Pankow, composition community organic Melnychenko, fungal activity, associated fact, photosynthetic Shortlidge, even morphology, and of in production, differences matter compound sex-specific volatile PˆortO, a exhibiting & Mcletchie, bryophytes As Alvarenga, Stark, in Santos, 2011; Mcletchie, dimorphism Dos & 2001). (e.g. Stark, times Horsley, Mishler, multiple 2014; , & reported Laaka-Lindberg (Bisang been & sexes Tesitel, has Vesalainen, separate bryophytes Hola, having has in 2018; species traits Dioecy the history data. of life damage 50% and more oxidative than significantly and more were growth to Heden females protonemal leading & but both bryophytes, by Cu in heavy evidenced times to in as multiple tolerant evolved differences males, similarly than sex-specific were Cd metal-dependent, sexes to for Both tolerant evidence and bryophytes. time, field in first our tolerance the total among metal for differed tolerance. showed, our systems results in in scavenging and our differences ROS Finally, for observed metals the oxidative accounted the in of bind to increase capacity being to contributing the population-specific still capacity that a populations, while caused its possible lab-reared toxicity, that is in Cd it their differences for the (MDA), limiting especially the in damage Second, or cells, explain to concentrations. wall, the could relative response the and inside exposure in of entrance metal change composition heavy their original can Mellerow- prevent to the bryophytes Lenartowska, Krzeslowska, in of responds the 2010; differences wall of it al., Therefore, cell way composition et 2009). the (Konno heavy the Wozny, exposure and of on Samardakiewicz, nutrient metal composition icz, depends in heavy bryophytes and role as and such major structure First, 1989), cues that a Dainty, the environmental plays hypothesize and Further, mechanisms. that we (Richter complementary walls in wall. Thus, regulation cell two cell Cu and their metals. to in binding, relative up observed due (CEC) uptake, take differences for capacity be metal to exchange the (except could cation capacity that controls high their tolerance indicating relative a their in in have populations and differences to differences all total to compared observed in in related plants the similar not increase treated were be significant below tolerance to all the magnitude in seemed in Despite of accumulation order Cu an metal 0.1%). were and Cp1), concentrations Cd ([?] Cu total hyperaccumulators of whereas Cu concentrations 2010), al., for et threshold (Maestri the 0.01% [?] plants i.e. the tors, oxidative in less Cp2.m. MDA of had and of populations (Cp2.f) concentration Cp1 All females the any than suggesting on in Cd-tolerant growth, MDA Cd more protonemal in of were we the increase effect and field by significant which an damage shown the a was more well, pattern cause from times there Cp1 the as not However, derived 2.9 paralleled did and 4D). tolerant of individuals which Cu, 2.0 than Cd plants (Fig. with dropped Treatment Cd populations more growth First, 4G). average the to be whose of tolerant (Fig. Cp2.m, to reasons. Cp2.f more strain, in Cp1 was two lab than Cp2.f, male expect for respectively the strain, would the and case lab in we Cp1, the female generations plants, case collected clonal The is the several find. this were propagating not by this that experiments did if think the before Second, not degree do laboratory. certain a we to However, dehardened were years. several for s 05 cail tod ulih 02.Sxa iopimi opooia,physiological, morphological, in dimorphism Sexual 2012). Burleigh, & Atwood, McDaniel, 2005; ¨ as, .purpureus C. nti td ece h hehl ocnrtosfrC hyperaccumula- Cd for concentrations threshold the reached study this in .purpureus C. 11 a fe enue samdlsse o sexual for system model a as used been often has 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. nrihS,Ssmn . agI 21) iie cuuaino opri ev ea dpe mosses. adapted Res metal heavy Ecol in Adv copper of 141-148. accumulation plants. 101: Limited Biochemistry, in (2016). and I. tolerance Physiology Lang Plant metal & S., Heavy Sassmann S., Antreich (1971). R.G. Turner Trends & adaptation. A.D., plant 7:1–85. of Bradshaw genetics J., Evolutionary metal Antonovics heavy (2011). T. of 258-266. Mitchell-Olds Contents 27(7): & Genet., J.H., (1999). 835–842 Willis S. 45: J.T., Nutr., Anderson Matsumoto Plant & Sci. M., Soil Nishiyama substrates. their S., mosses: Sakamoto copper in I., substrate elements Vegetation, 1037-1049 Nagano – habitats 563–564: Y., metal Environment, for Aikawa Total Lang model The a I.K.,& of Lichtscheidl as Austria, N., Science Knappenberg, Baumann contamination. heap F., and spoil Hofhansl copper G., The Steinhauser (2016). S., I. Sassmann mi- Y.S., Weiss to draft W., contributed first Adlassnig manuscript. wrote M.G. the and and edited analysis I.L. and and reviewed C.A.; References collection critically and data authors C.L.R. performed all manuscript; M.T.B. of the analysis; advice of and the collection with data experiments croscopy the designed M.T.B. declare. to contributions that interest Author of idea conflict the no have as con- authors well current The as the in stress, populations interest to plant of exposure natural Conflict of previous po- fate of change. despite the environmental lack challenge for of environmental and important text to is variation Our variation heavy respond genetic differentiation. phenotypic for to of such intraspecific bryophytes differentiation levels underlying of intraspecific mechanisms low capacity of the tentially unparalleled patterns and the the bryophytes support into in results insights tolerance novel strategies. and generalist provide accumulation varied, results metal vs. are from bryophytes our specialist Nevertheless, between of of conclusion, differences patterns gametophore). concentrations In of tolerance-accumulation vs actual predictions the protonema the with that - applied, congruent showed assessed were are results stages and treatments experimental our developmental the the perspective, the how in broad and a differences used, (e.g. above. inevitable Cu categories species and the to study of Cd two due any our comparisons to between between-species them design assign populations further as all “high cannot avoided and ii we behaved we Cu category metal, hyperaccumulate Finally, Cp2.m, as this not behaved did and of Cp2.m species concentrations Cp1 and this final Cp1 Because to similar while Cd. “high reached relative Cd, for i for tolerator” tolerance tolerator” category low greater high and a accumulator and showed as accumulator category and and For “high a leaves) i concentrations). Cd category as in total metal-dependent hyperaccumulated accumulation behaved Cu (higher and that (limited for Cd Sc3 different tolerator” Cu for Cp2.f, whereas high tolerator” for have concentrations), and high tolerator” Sc3, total accumulator and high “low accumulator (lower and and iii Cd accumulator Sc2 category “low and a leaves) iii as i.e. behaved in Sc2 species, accumulation innate other. (limited this seemingly each but of to specialization relative for populations of strategies found polluted lack patterns whereas tolerance-accumulation its heavily metals, The to heavy that pollutants. due for these iii) affinity Thus, tolerate (category high to metals. its strategy capacity we heavy stress-avoidance to metallophyte, with due a facultative i) cope follow vs. (category would to obligate strategy strategies stress-tolerator i.e. different a follow species, follow would study two they our that of predicted characteristics ecological distinct the cplpialigulata Scopelophila , cplpiacataractae Scopelophila 12 and , .purpureus C. ilchfrajaponica Mielichhoferia .cataractae S. .cataractae S. eseuaethat speculate we , .purpureus C. suggested would and 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. 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Gaughan purpureus & A.J. Shaw oooemtenerum Monosoleum Dtihca) m .Bt,8:512–521 86: Bot., J. Am. (Ditrichaceae). 584-591. 80(5): Botany, of Journal American . eaoo purpureus Ceratodon Ptica) ytmtcBtn,1() 525-537 18(3): Botany, Systematic (Pottiaceae). .EooiooyadEvrnetlSft,3:109844. 30: Safety, Environmental and Ecotoxicology ). ytihacaninervis Syntrichia Dcaae) n o. 2() 845-854. 120(5): Bot., Ann (Dicranales). ln clg,172:183-196. 157(2): Ecology, Plant . 18 cplpiacataractae Scopelophila cplpiacataractae Scopelophila acatainflexa Marchantia (Marchantiaceae). Ptica) Pl. (Pottiaceae). mJBot J Am . Ceratodon Ceratodon 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. olfil ape Mcnet oa u( Cu Total Cd garden Common contents HM Transformation (link) family glm Cu garden Common variable Dependent samples field Soil Measurement species Moss samples Field population the considering a without Experiment instead, plants replicates), treated of vs. number control low compare the to to used performed, (due effect. was treatments performed test garden-Cd. the glm (KW) common no of in wallis plants NA(KW): each kruskal Cd-treated model; with vs common linear controls the controls and For generalized study. garden-Cu, the glm: this common comparing in in datasets plants by Cu-treated the done vs of controls each were for soils tests out carried non-serpentine experiments, analyses and garden statistical the serpentine of to Summary 1: adaptation Table Local (2006) R. Scherson introduced & in 1569–1580. of M.L., 76: Stanton Ecophysiology Ecology J.W., ticity. (1995). Wright plas- R.A. phenotypic Black of role & the R.N., Hawaii: Mack 124-133. D.G., 22: persistence Biology, Williams population Evolutionary influence of variation genetic Journal and change. factors environmental Demographic (2009). under A.A. Hoffmann Hill. & Piedmont. Y. Chapel Carolina Willi metallophyte North Carolina, the North of in sites of 106-116. conservation mine University 12: metal for dissertation, Ecology, heavy Salt, derelict Ph.D. priorities Restoration in Smith, O.W., remediation. patterns Research Vegetation Purvis, A., site (1984). (2004), D.E. and T., Paton, Wickland A.J.M. restoration McIntyre, F., Baker, for R., Malaisse, potential & Johns, their J.A., F.J. Jaffr´e, T., and Zhao, Cooke, R., biodiversity H., Ginocchio, M.S., Schat, R.L., Johnson, moss Chaney, D.E., D., the J.S., Richards, of Angle, R.D., shoots J.A.C., Reeves, within S.N., recycling Whiting, nutrient Mineral (1996). D.H. Brown squarrosus & J.M., ecosystems. Wells Earth’s of domination Human (1997). 494-499. J.M. Melillo 277: & Science, J., Lubchenco H.A., Mooney plants. P.M., in Vitousek hyperaccumulation metal of 759–776. mechanisms Molecular 181: (2009). Phytologist, H. New Schat & C., Hermans N., Verbruggen Collinsiasparsiflora nrlto ogot.J ro.1:1-17. 19: Bryol. J. growth. to relation in vlEo e 8:1–21 Res Ecol Evol . ceohl cataractae Scoelophila purpureus Ceratodon cataractae Scoelophila cataractae Scoelophila ln opooyPatlnt m)gma(o)No No (log) gamma (standardized) (log) Yes gamma No inverse.gaussian(log) (inverse) gausian (mm) length ( Plant Total morphology Plant levels No MDA contents HM (mm2) Growth damage Oxidative levels MDA gowth Protonemal ( Cu Total damage Oxidative (log) gamma contents HM (mm) length Plant No morphology Plant ( Cu Total (log) gamma contents HM (mm) length Plant morphology Plant 19 eaieC nse % am lg No No No No (log) gamma (log) gamma (log) (BoxCox) (%) gamma Yes stem (log) in gamma Cu (BoxCox) (%) Yes Relative leaves in Cu Relative (mm) width (log) Leaf gamma No (mm) length (log) Leaf gamma No ( Cd Total (log) gamma (%) Cd Relative (log) (%) gamma Cu Relative ( Cd Total (mm) width Leaf (mm) length Leaf eaie()gma(o)No (log) gamma (%) Relative μ -)N(W - NA(KW) g-1) g μ μ μ μ μ -)gma(o)No (log) gamma No No (1/muˆ2) No g-1) gamma g (log) No gamma (log) g-1) gamma g (log) g-1) gamma g g-1) g g-1) g enstmsetaceum Pennisetum Rhytidiadelphus i.e. on 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. etrntdffrn infiaty re n e oo ersn oto n rae ape respectively. samples treated and control represent sharing color means red with values. and comparisons, mean pairwise represent Green multiple boxplots significantly. the the differing of (mean inside not results Cu- stars plots the and letter Black represent Bar control a boxes plants. in plants. graph (H) (MDA) the G) Cd-treated malondialdehyde inside and and of Letters control concentration (mm (F in the growth Cd-treated and of protonemal and (D) replicates) control treated the 10 in with of deviation and and standard C) %) + and weight of (B by plants Cu-treated (n=10; (B) and concentrations Cd-treated relative and the control of in and Cp1 (A) for treated except replicates respectively. 2 mine with represent deviation the colors of red center multiple and (D), the 4: the Green Cu-treated Figure of significantly. and results and differing edges the control not the represent letter in at boxes a (MDA) graph sampled the sharing malondialdehyde populations inside means of Letters with G) concentration plants. comparisons, F, (H) the pairwise Cd-treated (E, of and Cd-treated Sc1) control and in for control and n=1 in and and Sc4 C), and B, (A, (mean Cu-treated plots and Bar control in of (mm) plants width respectively. leaf mine and the the length, of color of center red Black results and the the 3: Green respectively). and represent Figure significantly. F edges (B, differing boxes (E, the plants not graph plants at do Cu-treated the letter sampled Cd-treated inside and a populations and sharing Letters control represent control means values. of with of mean comparisons, stems stems pairwise represent and and multiple boxplots and leaves leaves the (A), in in inside Cu-treated Cd Cu stars and of of of control and plants in %) respectively), (D) Cu weight C Cd-treated of (n=12; concentrations and concentrations total control relative the in of Cd replicates) the of 3 and with edges deviation the standard at sampled morphological populations 2: the represent Figure color depict sharing means respectively. red I with mine and the comparisons, and the Green of pairwise of H, multiple each significantly. center the G, from differing of plants not Panels results n=50 the letter represent in a weight). boxes performed graph by mm) the % inside in Letters width (n=12; leaf where F) and of E) (C, (B, length, populations of samples leaf plants length, and moss D) (plant in (A, ( measurements soil concentrations Cd in mean Cd and represent and Cu Cu bars of the concentrations total inside the numbers of replicates) 3 with deviation 1: Figure Transformation captions Figure (link) family glm variable Dependent Measurement species Moss Experiment eaoo purpureus Ceratodon .catatactae S. cplpiacatatactae Scopelophila cplpiacatatactae Scopelophila cplpiacatatactae Scopelophila .cataractae S. μ indicated M eaoo purpureus Ceratodon utrdi h aoaoy lc tr nietebxlt ersn envalues. mean represent boxplots the inside stars Black laboratory. the in cultured olce ntefil.Baksasisd h oposrpeetma values. mean represent boxplots the inside stars Black field. the in collected epnet uadC ramn.Brpos(mean plots Bar treatment. Cd and Cu to response ± tnaddvainwt elctsecp o otoswoen2frSc3 for n=2 whose controls for except replicates 3 with deviation standard epnet uadC ramn.Bxlt n=1)o ln egh leaf length, plant of 10) = (n Boxplots treatment. Cd and Cu to response n olcletdwti h oprmn.Brpos(en+standard + (mean plots Bar mine. copper the within collected soil and epnet uadC ramn.Brpos(mean plots Bar treatment. Cd and Cu to response dwt =)o h oa ocnrtoso ui oto n Cu- and control in Cu of concentrations total the of n=1) with Cd xdtv aaeMAlvl nes.asinlg No inverse.gaussian(log) levels MDA ( Cd Total damage Oxidative contents HM xdtv aaeMAlvl am lg No (standardized) (log) Yes gamma (inverse) gausian ( Total levels MDA contents HM (mm2) Growth damage Oxidative gowth Protonemal .catatactae S. 20 .purpureus C. μ g g -1 utrdi h aoaoy oposo the of Boxplots laboratory. the in cultured .Bxlt fterltv ocnrtosof concentrations relative the of Boxplots ). eaieC nse % am lg No No No No (log) gamma (log) gamma (log) (%) gamma stem (log) in gamma Cd (%) Relative leaves in Cd Relative (mm) width Leaf length(mm) Leaf eaie()gma(o)No (log) gamma (%) Relative utrdi h aoaoy Boxplots laboratory. the in cultured μ -)N(W - NA(KW) g-1) g μ μ -)gma(o)Ys(BoxCox) Yes (log) gamma g-1) g g g -1 niae standard + indicated 2 μ =5 ncontrol in n=35) ; g g -1 μ indicated M niae + indicated 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. D A G Plant length (mm) Total Cd in soil (µg g- 1) Total Cu in soil (µg g- 1) 1000 1500 500 18 36 54 72 10 0 2 4 6 8 0 0 6.36 818 Sc1 a a a 1 1 12.69 1569 Sc2 b b b 2 2 28.18 806 Sc3 a c 3 3 c 3.91 259 Sc4 c d d 4 4 E H - 1 B Leaf length (mm) Total Cd in plants (µg g ) Total Cu in plants (µg g- 1) 1000 1500 500 0.0 0.7 1.4 2.1 2.8 18 36 54 72 0 0 1 520 Sc1 Sc1 Sc1 1.7 a a a 21 608 Sc2 Sc2 Sc2 49 a b b 1 40.3 Sc3 Sc3 Sc3 135 b b c 33.1 553 Sc4 Sc4 Sc4 a b d I F C Leaf width (mm) Relative Cd in plants (%) Relative Cu in plants (%) 0.0 0.7 1.4 2.1 2.8 0.0 0.2 0.4 0.6 0.8 0.0 0.1 0.2 0.3 0.4 Sc1 Sc1 Sc1 a a a Sc2 Sc2 Sc2 a a a Sc3 Sc3 Sc3 a b b Sc4 Sc4 Sc4 a a a 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. C B A Stem relative Cu (wt %) Leaf relative Cu (wt %) Total Cu (µg g- 1) 12000 3000 6000 9000 0 1 2 3 4 0 1 2 3 4 0 12.9 a,c C a a Sc1 1 1099 Cu b b b 4.8 C a a c Sc2 9577 Cu c b b 4.8 C a a c Sc3 1 1292 Cu c b b 6.1 C a c d Sc4 10188 Cu d d b 22 F E D Stem relative Cd (wt %) Leaf relative Cd (wt %) Total Cd (µg g- 1) 1250 2500 3750 5000 0.0 0.1 0.2 0.3 0.4 0.0 0.2 0.4 0.6 0.8 0 0.2 C a a a Sc1 2139 Cd b b b 0.5 C c a a Sc2 1905 Cd b b b 0.2 C d a a Sc3 3002 Cd b e b ,c 1.4 C a a f Sc4 4470 Cd c b g 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.

Concentration D Leaf width (mm) C Leaf length (mm) B Plant length (mm) A

of MDA (µM)100 150 0.6 0.0 0.5 1.0 1.5 2.0 0.0 0.2 0.4 50 12 0 0 3 6 9 a,c 15.9 a,c a C a ,d Sc1 64.3 b Cu b a b ,e ,c 16.1 C b a b c Sc2 1 16.4 Cu a,c b a ,c b 1 a,c 1.5 a C c c Sc3 66.2 Cu a,c a b c 13.2 a,c a a d C Sc4 75.1 a,b c Cu a b ,e 23 H F E Concentration Leaf width (mm) G Leaf length (mm) Plant length (mm)

of MDA (µM) 0.0 0.2 0.4 0.6 0.0 0.5 1.0 1.5 2.0 10 20 12 0 3 6 9 0 a,b 15.9 a,c C a a ,c Sc1 17.2 Cd a,b a,c a b a,b 16.1 b a C b ,c Sc2 10.7 a,b Cd a b b a,b 1 c 1.5 a C a ,b ,c Sc3 a,b 14.7 a,b Cd a a ,c a,b 13.2 a C a c ,c Sc4 19.1 a,b Cd a a a 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. B D C A Concentration Protonemal Relative Cu (wt %) Total Cu (µg g-1) of MDA (µM) 2

200 400 600 800 growth100 (mm150 )200 250 126 189 252 0.2 0.3 0.0 0.1 50 63 0 0 0 4.94 151 C a a a Cp1 219 187 a,b Cu a b 5.08 281 b C b b ,c Cp2.f 425 220 Cu c a c 6.63 478 C c b b Cp2.m 156 450 Cu c a c 24 F E H Concentration Protonemal G -1 of MDA (µM) 2 Relative Cd (wt %) Total Cd (µg g ) 1000 1500 growth (mm ) 500 100 150 200 250 126 189 252 0.0 0.1 0.2 0.3 63 50 0 0 0 151 0.13 C a a a Cp1 156 502 b Cd b b ,c 0.06 281 C b c c Cp2.f 1406 151 c Cd d ,d d 0.12 478 C a c c Cp2.m 208 224 a,b Cd d b