A 1 LTI- 'lETIIOD ,.\ PPRO II TO .\ ~. E ~ .. I G (' RBO L , T R TE IES A 0 RE, PO SE TO DI ST TRITIO 1)' O II ETEROT ROPlriC ERI C CE E hy Rebecca L. Bo\\ ler B c . l ' 111\ cr~1 tv... of'\; on hcn1 Bn ll ~ h Col UJnhw. 20 10 THE I EM ITTED IN PARTIAL FULF ILLM [: T OJ~ THE REQ IREME T FOR THE DEGREe OF M STER OF SC IENCE • I ATURAL RE Ol'RCES A D ENV IRONMf: TAL STUD IE. UN IVERS ITY OF NORTHERN BRITISH COLUMB IA Dcccn1 her 20 15 (C Rebecca Bo\\ ler. 20 15 Ab tract Myco heterotrophy i a nutn tiona] ~ tra tegy \\ he1e pl a nt ~ obtain ~o n1 c po111 on of thetr rcquu·ed carbon through Inycon·hi?al fungt Th1 ~ fo u1 -ycar <;tudy assessed 11 C and 1c; N stabl e tso topcs, ga -exchange data and populati on d c n ~ Jtt cs o f' putatJ\ c partl al (PM I I) and full n1 ycohctcrotrophic (MH ) e ri cacco u ~ ~ p cc t c~ relati ve to asso11ed aut o tro ph ~ across various habttats and d1 turbancc le\ cL I \\ o of the putatJ \ c PMH Pyrolcac ~p cc 1 c~ cxhJhitcd 11 approx1n1atcly 30° o MH carbon ga tns. h O\\ C\ cr. c.;o n1 e data JndJ ca ted th at C cnn chtncnt in Pyrolcac specie may re ult from umque autotrophic phy tology or rn c ta ho lt ~ rn rath er th an fun gal ca rbon acqui ition. Rega rdl ess or nutntt onal ~ta tu , the Pyro lcac appea red sensiti ve to hi gh in·adi ance. Long-term ex posure to exce~s li ght tnay have contributed to photosynth eti c impairment and population declines observed in c learcuts, where residual vegetati on appeared to promote resilience by providing shade and nitrogen obtained through mycon·hizal fun gi. This study pro" ide \ aluable insight into phys Jologtcal and environmentallitnitations of plant pecies that are pat1iall y to full y mycohctcrotrophic . .. II Table of C ontent . Abstr·act ... ................................................................... ................................................................ Jl ... Table of Contents .......................................................................................... ........................... 111 . List of Tabl e ........................................................................................... .................................. Vl Li t of Figure .. • • • ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 0 • " •••••• • •••••• • ••••••••••••••••••••••••• Vll Glo sary and abbrev 1atio n........................................................................................................ x Ackno\\ lcdgcn1 ent . · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · .... · . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X l J1 1. Introduction .......................................................................................................................... 1 1.1 Myco rrhi1a and n1ycoheterotrophy ................................................... ............................. 1 1.2 Approaches to tud y1ng the ecoph y Jology of mycotroph1c pl ants ................................. 4 1.3 Stud y pecie ............................ ...... ................................................................................ 11 1.4 Thesis objective ........ .................................................................................................... J 3 2. A e ing partial myco heterotrophic carbon nutrition of three Pyrol cac species und er drought and shade treatment : a co tnbi ned stabl e 13 C isotopic and gas-exchange approach.. 15 Ab tract .................................................................. .................................................................. J5 2. l Introduction ........................................................................................................... .. ...... 16 Drivers of partial mycoheterotrophy ................................................................................ 18 2.2 Methods .......................................................................................................................... 22 . d esct·1pt1on . . ... ......... .......................................................................................... 72 S 1te 0 ••••••• • •• ._ Experimental setup and data collection ............................................................................ 23 Calculations to assess total seasonal photosynthetic ca rbon exchange and net carbon gains ........................ .......................................................................................................... 27 Statisti cal analyses .... .... ....................................... ............................................................. 28 2.3 Results ... ........................ .......... ................................................ ....................................... 29 Light and temperature environn1ent ........... ......... ............................................................. 29 Photosynthetic rates by n1onth and treatn1ent. ........... ...... ............ ... ... ..... .. ... ... . .. .... .. .... . .. 32 Bu lk leaf tissue 8 13 C values by treattnent and over titn e ........................................... ...... 34 Light response curves and seasonal carbon exchange e titnate .................................... 35 2.4 Discussion o····················o··········o·············· ·············o·············o········································· 38 Seasona l and treatm ent e fTec ts on PS and 8 13 C ............................ ......................... ....... .. 39 Litnitations in detecting mycohetcrotrophic ca rbon gains and options for future research 44 ........................... 0 0 ••• 0 ••• 0 • 0 •••••• • •• 0 0 • 0 ••• 0 ••••• 0 •••••• 0 ••••••••••••••••••••• Ill 0 ••••••••• 0 •••••••••••••••••••••••••••••••••••••• Concluding retnark ................ .. .... .............. .............................................. ........ ............... 46 2.5 LitCJ'"'ature Cited .............................................................................................................. 48 3. Effect of alvage harve ttng of lodgepole pine-don1inatcd forests follow ing tnountain pine beetl e disturbance on dens tty and stab le carbon and nitrogen isotopes of part1al mycohctcrotrophic Pyrolcae ~pec t c (En caccac) in central B1 itish Co ltunbi a ........................ 55 Ab tr·act ...................................................................................................................................... 55 3.1 Intt·oducti on ............................................................................................................... ...... 56 Myco nhi1al ymb1o t5 and tnycohcterotrophy .............................. ............ .................... 56 Effect of d1 turbance on mycohetcrotroph populations .................................................. 59 3.2 Materia I and Method .................................................................................................. 62 ite dcscri pttor1s ...................................... ......................................................................... 62 urvey data and satnple collection ...... ............................................................................ 65 tati tical analysi ............................................................................................................ 67 3.3 Results ............................... ............................................................................................... 68 Sur,,ey data .. ..................................................................................................................... 68 Isotope data ....................................................................................................................... 70 3.4 Discussion ............................................. ......................................................................... 73 13 1 Impacts of di sturbance on P) roleae abunda nce, 8 C and 8 'iN ....................................... 73 Additi onal fac tors influencing persistence following di sturbance ................................... 78 Concluding re111arks .............. .......................... ................................................................. 82 3.5 Literature Cited ....................................................................... .. ..................................... 84 4. Assessing mycoheterotrophy in eticaceou species using two-so urce linear n1ixing n1odels based on photosynthetic rates and stable isotopes o [ ca rbon ( 13 C) and nitrogen ( 15 .......................... 0 •••••••• 0 ................................. 0 .......... 0 ) ..................................................... ........ 90 Abstract .................... ... ........................... ... .. ............................................................................ 90 4.1 lntroducti on .................................................................................................................... 9l 4.2 Methods .......................................................................................................................... 97 Site descripti ons all years .......................................... ....................................................... 97 Satnp le selection ............................................................................................. ................ l 01 Gas Exchange Mcasurcrnents ............. ......................... .................................................. 102 Calculations to estitn ate% photosynthetic-deri ved carbon ( 0 oCnP) ............................. 104 Statistical Analyses .... ........................... ............................................... ........................... 105 4.3 Rcsults....... ................................................................................................................... l07 . 1 Specie differences in photosy nthc<; is rates and 8 1'c .................................................... 107 Estitnate of 0 oC or via photosynthesis and isotope n1 ethods ........................................ 11 2 pcc ies differen ces in 8 1 <; , 0 oC and 0 o ......... ................. .. ........................................ I I 5 4.4 Di cu sion .. .................................................................................................................. I J 7 Low photo ynthctic rates masking degree o [ ca rbon tnycohctcrotrophy? ..................... 11 9 Con iderati on for future research ...................................................... ....................... .. J 26 Final remat·ks .................................................................................................................. 12 9 4.5 Litci·atu t·e cited ................................................................................................................ 112 5. Conclusion ......... ..................... ... .... .... ... .. .. ... . .. ...... .. ........... ...... ....... ........... ... ....... .... ....... 144 5.1 Degree of carbon and njtrogcn 1nycoheterotrophy in Pyrolcac species ....................... 144 5.2 Sen itivi ty to di sturbance ............................................................................................. 14 7 5.3 Study li1nitations and future directions ........................................................................ 149 5.4 Additional literature cited ............................................................................................ 15 1 v List of Table Table 2- 1. Treatment n1eans (± I . . F) o f 811 (0 oo) for Jun e. , epten1 ber and the difference over titne (Septetnbcr-Junc) for each spec tcs Trca ttnents arc li ght dry (LD), light watered (LW), shad e dry ( D) and shade watered ( W) ignificant trea tlnent differences between eptetn ber 8 J3C \ aluc by pccie<; arc indi ca ted b; di fferen t lcttcrcs \\ ithin the app ropriate column. Listed p-va luc arc result fro1n paired t-tests dcten111ning di fferences between Jun e and eptcmber 8 13 \ aluc . /\I I result<; \\Cre concs idcrcd ()ignifi ca nt at a .-- 0. 10 . ...... ........ ... 35 upplen1entary Table 2- 1. Mean(± I E) seasonal gas-exchange parmnetcrs for autotrophic reference pccies and three putative parti al naycohetcro trophi c Pyrolcae species und er field manipulation treatinents (n = 36)~ as well as fu ll y mycohetcrotrophic reference species (n 4, not included in treattnent ).......... ........................................... ............. .... ... ....... .......... .. ..... ..... 53 11 Suppl ementary Table 2-2. GrO\\ ing season 6 C. o/oC. 8 1c; and o/oN and sampl e size (n) of each species of autotrophic reference .................................................. .................................. 54 Table 4- 1. General locati on , ecological descripti on and target parttal mycoheterotrophic Pyroleae and full tnycoheterotrophic (MH) Monotropoidcae species presence for all six study sites aero s all stud y year (20 10 through 20 13 growing seasons)....................................... .. 98 Suppl ementary Table 4-1. Mean (± 1 E) natura l abund ances of 8 13C and 815N (%o) , and C and N concentrations(%) of autotrophic pecies, six putati ve partia l mycohetcrotrophic Pyroleae species (in italics) and full n1ycoheterotrop hs (Fu ll MHs). Spec ies shari ng the sa1ne letter within a co lumn are not signifi ca ntl y diffe rent at a < 0. 1. ........................................... 138 Suppl ementary Table 4-2 . Mean seaso nal gas-exchange data including net photosynthetic (PS) rates and addition al paran1eters 1neasured und er a1nbient conditi ons and during light response curve measuretnents. Average PS rates under aJnbient conditions are estimated marginalJn eans ± 1 SE (population-level) resulting fro1n linear n1ixed 1nodels. Species sharing the sa1ne letter within a column are not signifi cantl y different at a < 0. 1. All othe r gas-exchange parameters and measure1ncnts are descriptive n1 ea ns ± 1 SO ........................ 141 . Vl List of Figure Figure 1- J. chetnati c diagrarn illustrating fra ctionation effects agai nst heavy isotopes. The increa ed tnas of extra neutron result 1n lower kin etic reactions during physiologica l and biogeochen1i cal proees cs, resulting in the source substrate having a different relatt vc rati o of heavy to light i.. otopc cotnpared to the produ ct. R e publi ~h cd with pennis Ion of Springer Science and Bu Medt a B V, fron1 table I otopc Ecology, Fry, B., 2006~ pcn11ission conveyed through opyri ght learance Center. Jn c ................... .. ......... ...... ........ .... ... .. ... ... ....... 6 Figure 1-2. A sc he1natic diagran1 of carbon exchange fro1n autotrophs to th eir ecton1ycoiThizal fungi and frorn fungi to fu ll or partialinycohctcrotrophs, and the known range of 8 11 C values for the vari ou carbon pool . Italic11ed processes arc those whi ch cause isotope di . crirnination, and th e weight of arrows indtca tes the degree o f en ri chment or depletion bet\veen source and sink ( ce Hyn on et al. 20 12 for ful l detaib). Republished with penni ssion of pringer cience and Bus Medi a B V, fro1n Hynson, NA, Mambelli S, Atnend AS, Daw on TE, Oecologia 169. 20 II : pennission conveyed through Copyrj ght Clearance Ce11ter, Inc ............................................................... .................................................. 8 Figure 1-3. Photos of tudy pecies a) Clurnaplula Lunhellata, b) Ortlnha secunda , c) Mon ese, uniflora, d) Pyrola asarifolia, e) Py rola chlorantha, f) Pyrola n1inor, g) Pterospora andromedea, h) Monotropa hypopitys, i ) Mon otropa un~flora . Photo credits for image c) and i) belong to H ugues Massicottes/Roy Rea ....................................... ........................................ 12 Figure 2- 1. Mea n photo yntheticall y active rad iation (PAR : jlmo lm-2 s- 1) and ten1peraturc (°C) for light treatments (solid lines) and shad e treatments (dashed Jines) ac ross the experimental period of May 29 to Sept 22, 20 12. Panels a) and c) are daily averages, with PAR averaged for only dayli ght hours, and panels b) and d) represent hourl y average ensetnbles fo r all da ys ............................................................................................... ...... ......... 30 Figure 2-2. Mean (± 1 SE) instantaneous soiltnoisture (% by volutne) as 1nea ured in th e earl y (June), mid (Jul y to early Au gust), and late (n1id -August to n1id-Septen1ber) sun1mer in 20 12 for dry (LD and SD) and watered (LW and SW) trea tments at two depth ................... 32 Figure 2-3 . Estimated n1arginalmeans (± 90% Cl) of net photosynth etic rates (jlmol m 2 s- 1) of autotrophic reference plants and the three Pyrolcae species by a) tnonth (all treatn1ent combined) and b) treatment (all n1onths cotnbined) . Trea t1n ents are light dry (LD), light watered (LW), shade dry (SD) and shade watered ( W). Non-overlappin g error bars \vithin species represent signifi cant differences between tn onth or trea tn1 ent at a < 0. 10..... ....... 33 Figure 2-4. Mea n (± 90% 1) net C0 2 exc hange (jlmoltn -2 1) as a non-linear regress ton function of photosyntheti call y act ive radiati on (light response curve ) for reference autotrophic plants and the three target Pyrolcae species under fi eld n1anipul at1on treatrncnts .. V ll Treattn ents arc li ght dry (LD ), hght watered (LW), shnd e dry (S O) and shade watered (SW) ......................................... ........................ ..... ........................................................... .................. 36 Ftgure 2-5 F~ttn1 atcd n1 ea n (-t 90°o CI) total ~cn~on C c" changc (g C 111 ) dcrl\ eel rron1 P rc. pon c~ to lt ght and sca~o n d lli ght lc\ cl" fo r auto tt oph tc tcfetcncc pl a nt ~ and the tht cc Pyrolcac spcctes frorn 1ay 29 through cpt 14 Da1k grc; b a r~ 1cpt c~c nt net sea~o nal photo ynthctt c gat n , and lt ght grey bars rcpt c~c nt total rc~ pu a tory C losses at ni ght No noverl apping error bar indi ca te ~ig niri c a nt difTe rc ncc" at CJ. < 0. 1 ........... ........... .............. .... 37 2 Ftgure 3- l chcn1atl c d tagran1 o r 111) cohctcroll oph field ~ U J\ C)~ a) 'I I anc;cct<:, or 90 111 ran approxi tnntcl; pcrpendtcul at to han e"t bou nda r;.\\ tth each tJ cl n ~e c l ~pl1t1nto tht ce 10 111 segm e nt~ o f fo rest. edge and clea rcut. tx ha nd ~ per pot1ton v.- ere c~ ta bli s h cd at 5 n1 Interva ls along trn n ~cc t , fo r a total 18 b a nd ~ per tra n~ee t h) l:ach band wa ~ con1pnsed of three adjacent I n1 2 plot fo r atnpling Pyrolcae .......................................................................... 66 "\ Figure 3-2 Mean {± 1 D) d c n ~ tt)' ( tnd i\ 1du a l ~ 111 ) of fo ur Pyroleae ~ p ec 1 c~ counted 1n forest, edge and clcarcut segn1c nt ~ along tra n ~ec t~ at three c,ite ~ tn central 8 o error bar tninin1un1s arc shown at <' 0 as populat ions canno t be negatl\ e Stgntfi ca nt dtffc rcn cc~ bet\\ een segtn cnts \\ithin eac h site nre represented h; di fferent l e tt e r~ at u. <" 0. 1.................. 69 • Figure 3-3. Mea n (::1: I D \\hen n > 3) 8 13 C \a lu c~ fro tn bulk l ca ftt ~c,u e of four Pyrolcae specie in forest, edge and clca rcut egtnent along tran ects at three ~ tt c s in central BC Significant dtffcrences bctv.'ccn locations \\Jere onl y tested fo r all ~ Jt c~ cotn bined, and arc represe nted by different letters at CJ. < 0.1. ........................................................................ ... .... 7 1 Figure 3-4. Mea n (± I D \\ hen n -> 3) 81'iN values fi·on1 bulk lcaf ti ~~u c of four Pyrolcae species in forest edge and clcarcut segments along tran cct at three ~ tt cs 1n central BC . Significant differences bet\vecn locations\\ ere onl y tested for all "1 te" cotnb incd, and are represented by di ffe rent letterc, at u < 0.1 . ............................................................................... 73 Figure 4- 1. D istri but ion of a' cragc seaso na I net photosynth eti c ( PS) rntcs ( ~tm o I n1 2 s 1) 11 under an1bi cnt conditions in 20 I 0 and ?O 12, and 20 I 0- 20 12 average 6 C (0 oo ) \ a l ue~ for .... several autotrophic plant , putative pat1ialtnycohctcrotrophic Pyrolcne s pec i e ~ and fu ll 1nycoheterotrophs (MHs) at three site in central BC haded h o ' c~ represent the JnterquartiJ c range (IQR) with the line at the 1ncdi nn and the dwn1 ond representing the n1cc1n . Whiskers represent data within 1.5*IQR and points arc outli ers beyond the 1 5* IQR... pccies codes are: Auto Autotrophs (va rious spec i es ) ~ Pyrolcae: Cu Ch unap/11 Ia Lunhc/la ta. 0" Ortlulia secl uula , Pa - P rrola a wtn(olia, Pc P chlorantha, Pn1 P nunor: ful l i\11 1: Monotropa unt/lora ( U BC and WR 20 10), .\1 /n ·popt/_1'\' (CR 20 12) and Pte! o'pora andromcdca (C R 2010/ 12).................................................................................................... ! 08 Figure 4-2. Mean {..L J SE) net CO.., exchan ge (pmo lm 2 s 1 ) as a non-ltnear rcgJe~'"-tOn function ofphoto"iynthcttcall y nc tt vc radiation (lt ght response c u ne~o., l RC) fot rc kr~ n cc . v tll autotrophic plant and three Pyroleae pecie at three site mea ured in June 20 1I. The 2 1 upper 4 panels are the fu II l RC to 1500 ~un o I n1 s ; the lower 4 panels are zooJned in to low light le\ el of 0 to l 00 ~m o l 1n 2 s 1 The three s ites arc· BC - niversity of Northern BC · WR - Wi llow Rivec CR rooked Ri\ er ................................................................... I 10 Figure 4-3. Mea n (± 90° 0 c I) e tin1ated photosyntheti c c gains ( 0 oC J)P ) 0 r autotrophs and target Pyrolcae ·pecie u<; ing 8 13 (' va lue and a\ eragc seaso nal photosynthesis (P ) rates ( 0/o C nP(J~Ol and 0 oC DPcP~)· re pecti" ely) for cornb1n cd data at BC. WR and R over two yea r in central BC. Different letter aero s panel s ind1cate signifi cant species differences at a 0. 1: upper ca e letters are pcc1es ddTerence 111 °o 1 w 11 ~ 0 l cstitnatcs and lower case letter are specie difference for 01 o DPrP(\) esti1n ates ........................ ................................... 11 3 lX Glo arv and abbreviation • chlorophyllous Plants '' ht ch lack chl oroph yll and arc Incapabl e of pho t o~ynthcs 1 s t;v e of tn yco rrh11a ''here 'a cui a r r Iant hoc;ts · root cc lis arc penetrated by fun gi of the ph; lun1 Jl on1 e1otnycota. fon111ng at bu ~c ul es and ' e~ tc l c~ rbu ~c u lar n1yco rThi1a - Arbutoid Inonotropoid tnyco n hiz a - ll o~ t- n1 edwtc d va riants of r~ M in .. n cncco us p lants where intracellular penetrati on of root cptdennal cell " occu1 ~ A1butoid rn yccHThi; a~ fonn e xte n ~ I \ e hypha I cod In cptdennal cell" 1n the genet a 1rctrJ\Iapln lo5 • .- 11 hutu\ and th e tnbc Pyroleae, whereas n1onotropotd 1nyco11ht za l fungi onl y fon11 sJn all fungal pegs tn cptclenn al cells o f plants o r achloroph yllous MonotropoidcOn 20 14) Mycoheterotroph1 c pl ant5 arc charactcn scd a~ Initial. pnrtial or full M ils InttJal Mils arc plant with ltn11tcd seed re~ en es and rely on fu nga l ~ymbtont!) for the gennu1at1 on or bclO\\ ground stage 111 ord er to reach adul thood. and may be autottoph . 0 1 par1ial or full Mils a adults (Leake 1994, B1dm1ondo 2005. Merckx et al 2009) l--ull rnycoheterotroph y ic;, kno\v n 111 about 400 spec1c of plant 111 87 genera and 10 faJntltc~. whi ch ha ve lost all photo ynthetic capac it y and therefore rely solely upon n1yco1Thi/al fungi for their C nutntton . • The e plants exhtbit tnorphologtcal tratt s ~ u c h a lack of chloroph ylL reduced leaf structures, few to no stotnata. du t seed which lack endo penn, and'or roots with very low surface area or tuberous fom1 that are heav il y colonu cd by n1 yco n·hi zal fun g1 (Leake 1994 ). Pa11tal MHs (PMHs). somctin1es termed tnixotrophs, arc chlorophyll ous species capab le or photosynthesis at adult stage that are thought to maintain a degree of MH C acqutsi tion (Jul ou et al. 2005. Abadie et al. 2006~ Selosse et al. 2006: Merckx et al. 2009). They share a nun1ber of morphological trait with full MHs such as redu ced lea r area, du ~ t seeds and reduced root systcrn s. While various n1odes of mixotrophy have been known to occur in son1c alga l species (Havs kutn and Rien1ann 1996) and also in plant tissue culture ysten1~ (Hctfcl/ ct cd. 2000), partial rn ycoheterotroph y was only recently docun1 cnted in tctTestn al green plant~ (e g., Gebauer and Meyer 2003 ). Though no t restricted to the e speciiic groups, two of the don11nant l~undt es exhrb1ting sotn c level of tn ycoheterotrophy are the Orchtd accae and Encdcc,tc It 1~ thought 3 that 1nost if not all orchids are at least initially Ml ls, with up to several hundred species rernai ning part ial or full MH at n1 atun ty (Bidarto ndo 2005; JIynson et al. 20 13a). Much of the re earch into parti al and full MHs has fo cused on green orchids wi th either closely related fan1il y n1e1nber that are full MHs, or that have albino indl\ iclua Is w1th1n a populati on (e.g., Gebauer and Meyer 2003: badie et al. 2006, Ltebel and Gebauer 20 11 ). The PMII strategy in the e green orchids eetn to have evolved pnor to fu ll MH nutriti on as an adaptation to low light levels in haded ecosystetn <; (Tedersoo et al. 2007, Preiss et al. 20 10). Based on th e theo ry of ~ irnilar adaptations bet\\ ccn fu ll MHs and closely related chl orophyllous species, Tedersoo et al. (2007) hypothesi7ed that 1nany green forest understory species in th e tribe Pyroleae (pyroloid ) may be PMH , as a i ter tn be to the full MHs tn the Monotropeae and Pterosporeae tribes (subfa1nily Monotropoideae, family Ericaceae; often refen ed to as tnonotropes). Many recent studie have provided insight into th e ecophysiology of MH plants, uch as identificati on of the phylogeneti c and functi onal diversity of fungi assoc iated with MHs and qu antification of the extent to whi ch these plants gain C and from their tnycorrhizal networks (e.g., Bidar1ondo and Bruns 2002~ Julou et al. 2005 ~ Zitntner et al. 2007~ Hynson et al. 20 12). 1.2 Approaches to studyin g th e ecoph ys iol ogy of n1ycotrophic plants Two advanced techniques have been widely used in studies on MH plants and their fun gal syn1bionts, often in cotnbination : molecular DNA sequ encing to identify fungal species (or broader ph ylogenies) and functional groups (i.e., endophytic or tn ycorrhi£al) colonizing MH roots, and the analysis of the natural abundance of stable C and N isotopes (e.g., Gebauer and Meye r 2 003~ Abadie et al. 2 006 ~ Tedersoo et al. 2007~ Hyn on and Bruns 2009). Though there are exceptions (see Hynso n et al. 20 13b ), identificati on of fungal 4 syn1bionts ha .. rc\ calcd that full y Mil plant u uall y lul\ c high ~c kc l t \ 1ty for narrow Iangcs of fun gal fa1ni llcs. genera or C\ en "Pecic" (B1dartondo and Bruns 2002) These fun gt in tu rn can be qu 1tc spec tfic to parttcu lar ph) t o bt o nt ~ . \\ h JL h ha" un pltcatt on" for d 1.,tu rhance C\ cnt ~ and con en atton stratcg1e (l laeu""ler ct al. 2002, Biclm1ondo and Bruns 2005 ). The tnonotropcs shO\\ .. on1e of the n1n" t (ungal-"pec tfic d""nc tat ion ~. \\ 1th eac h ltncage be1ng spcc tfic to onl y one of fj , c dt \ta nt cltldc" of oblt ga te ec ton1ycorrh11 al ha" Idtotn ycetc" Rl11::opoe,on. I nc holcnna. Gauff( na, Ru\ 'uh1ccac 0 1 I l rdnellum ( Btdartondo and Brun<; 2002. 2005). In rnost ca .. es. PM I I spcc tcs tend to a"c:;oc iate with a broad er range o r fun gi (Teder oo et al 2007, Zin11ncr ct c1 l 2007. c l o~"c and Roy 2009) It ha" been ~ h o\\. n. ho\\ ever. that Mll plant may ha\ e tnore l1mtted funga l syn1b1 ontc:; du ttng pat1tcul ar Ide tages. potenti ally litniting recruittncnt (Bidat1ond o and Bruns 2005, I I a~ hitn oto ct al 20 1 2~ Hynson et al. 20 13b). For exan1plc. a variety of fun ga l assoctatc<, tn th e order Scbac tn alec:; have been idcnti fi cd fron1 three Pyroleae pecies gcnn1n ants. 'N 1th \.\ell-developed <.,ecd lings across two separate studies sharing associations with clade B Scbac inalcs (Hashi1noto et al. 20 12: Hyn on et al. 20 13b). Hynson ct al. (20 13b ) suggested that thc<.,c fungi may be cntl cal at developmental stages beyo nd initial ge1mination Son1e putati ve PMHs n1ay also fo rn1 unusual syn1b ioscs with atypi cal functional groups, such as green orchids assoc iating with ECM rather than the common saprotrophic. endophytic or pathogeni c fungi (although fu ll MH orchids usuall y do associate with ECM fung i ~ Bidationd o et al. 2004 ). Beca u ~c of the hi gh variability in potential fun gal syt11bionts, dctcrn1ini ng sources and qu anti fyi ng funga l nutrient ga ins in Mils, moreso in PMIIs, is very challenging. Stable isotope analyses prov td e a relativ ely sin1ple and non-destructi\ e '' cl] to evaluate these biological interactions. The techn ique is based on the rnct that 1110St unportant biological elen1cnts occur naturall y in two or n1orc stab le i otopes due to dtfTcrent nutnbct~ 5 of neutron 111 the nucleu . \v tth one occun·tng 1n greater abundance tha n the other (Dawso n ct al. 2002, Fry 2006) Btologtcal and ph) tologtca l processes fra ctionate (dtscritnin atc) agatn t the heel\ ) l ~O topc . becaU'-.C of thctr ht gher 111cl '-.~. \\ htch lead'-. to ~ 111 a ll but cli ~ ttn c t n1 ea urable co nccntratJ on ddTcrcnccs \v 1thtn ah1 otJ c and btott c n1ate1wb and subs tan ce~ (try 2006: J o h a n s~o n 20 14 ). SOMETIMES THE EXTRA NEUTRON MAKES A DIFFERENCE. IT'S HARDER TO PUSH THE HEAVY MOLECULES UP AN ENERGY HILL ... rf( : 1 • . .. SO THAT PRODUCTS HAVE MORE OF THE LIGHT ISOTOPE AND LESS OF THE HEAV Y ISOTOPE . Figure 1- 1. chetnatic diagratn Jllu ~ trat 1ng fracti onati on effect~ aga 1nst hea\ y i so to pe~ T he increased 111ass of extra neutron result in slower k1netic reacti ons clunng physiological an d biogeochen1i ca l processes, resulting in the source substrate hav ing a different relatt\ e rati o of heavy to li ght isotopes con1parcd to the produ ct. Republished with penni ssion of Springer Science and Bus Medi a BY, fron1 Stable Isotope Ecology, Fry, B , 2006. penn iss1on conveyed through Copyright Clearance Center, Inc. Figure 1- 1 provides an Il lustrati on of the general concept or k111ett c fractlonat ton, a un idirecti onal process with a rn ass-dcpendcnt reac tion rate, e.g. in the Ji "yro/a ch/orantha. f) J>l 'rolu IJ/IJIOI'. g) J>tt.'I'O\f}()ru ~ lono/ ropa hrpoJJi I\'\. i) 'lono/ I'OjJU unifloru. Photo c I cd I h rot i llla gl~ c) and i) belong to ll uguc~ MassicottC'·JRo) Rca . (IJ]({f(JI11edeu. h) 12 1.4 T he i obj ccti \'C The O\ erall goa l oftht s thesis \\ a~ to tn\c5tt gatc and quanllf'y the degree of xnycohetcrotroph y tn se\ eral putatl\ e P ~ I J ~pec i c5 111 the tribe Pyroleac (~ uhf~1n1i l y Monotropotdcac. fan1tl y En caccae), to asses~ potentia l f ac to r ~ co ntttbuttng to C and N nutntton in Mil<;, and to dctcnntn e ho'' po pul a ti o n ~ and nutntio nal n1odc arc affected by e\ crc dt turbancc C\C nL To achi e\ c t h1 ~. a cotnbm atl on ofga~-cxc h an gc tncasurcmcnts, table i otopc analyses and populallon ~un C)~ \\ ere pcrfo nn ed dunng the gro'A tng seasons of 20 l 0 through 20 13 at e' era! ~ tte under both experuncntal dnd un-rnan1pul ated ficJd eond iti on . In tti all y data \\'ere collected in 20 l 0 for a ~ 1n1pl e prel1n1tnary study a~~e<:,s tn g only ga -exchange and table i otope<; of all Pyrolcae pcc i e~ comn1on to the central tnterior or • BC at a ubset of the ite . In 20 11 , pre-cx perin1 ent data were collected at th e same <:, Jtes and sa1nples were harve ted for a co ntrolled greenhouse experiment th at was un s u cce~sfuL in pari because of equipment fa ilure. A fi eld tn anipul ati on ex pcnment \v ith a 2 x 2 factori al d e~ 1 gn of light and water levels was perform ed in 20 12 at only one site with a subset of the target spec1es, again measuring gas-exchange and stable isotopes along\\ tth conttnuous rn easuretn ents of the prevalent light environtn ent and instantaneo us n1easurcs of so iltn o i ~ ture under each trea trn ent. In 20 13, PMH populati on sur\ cy data along\\ ith their ~tab le I soto pe~ were detenn ined at a different subset of sites that had been heavil y itnpac tcd by n1ountain pine beetle (Dendroctonus p onderosae I Iopkin ) and subsequent clcarcut han c~ ttng 8d"ed on the nature o f each growing seasons' da ta, this thesis is presented non-chronologicall y, with the 2012 cx peritnent results presented fi rst. fo lio\\ eel by the popul atton \ tUd) 111 disturbed habitats, and fi nall y a con1pilation of related da ta o cr all rour yea rs to assess 13 trend tn the degree of mycohetcrotrophy across species. years and ~ rtes The ~ p cc ifi c go ale; of th1 s the 1 "'ere 1 l) To as~e~s gas-e\.change tn easurcrnents and ~ tclhl e I (' and I~ 1~otope signatures o f three Pyt oleae .. p ec t e~ under light and \\ atc1 Jnantpulatl;d tr catn1e nt ~. and to exa n1111 c other gas-exc hange \ anabJes to a~~es~ phy~to lo g r ca J c hara cten ~tJ C~ of the target ~pcc ie rn co n1panso n to fully autotrophtc and n1 ycohete1ott ophic rererencc sp ec i e~ (C hapter 2) 2) To a ess populations and ~table 11 C and I '\ ISOtopes or Pyroleae species across a di sturbance gradient fron1 \\ rthrn rntact res tdual forest<, following n1ountain pine beetle attack out into sa h age harv ested e learc ut~ (Chapter 1). 3) To characterize the <,table isotope ignaturcs and ga~-cxc hange character] tics of all Pyrolcae species sa n1pl ed through the four co nsecuti vc growing seasons and at each study site. to assess trends at the species Je, cl (Chapter 4 ). 4) To develop a novel photosynthe is-based two-source n1ixing n1oclel derived fron1 and compared to the stab le isotop tc t\vo-so urce 1nixing tnodcl to as ess the proportional contributi ons of photosynthesis and n1ycohetcrotrophy to overall carbon budgets in several Pyro leae species, and deten11ine \vhether there arc lunitations to the application of linear-tnixing n1odels to gas-exchange data (Chapter 4 ). 14 2. e ing partial nl) cohcterotrophic carbon nutrition of three P) rolca e pecics under drou ght and hade treatn1ent : a co mbim1ed stable "C isotopic and ga -exchange approach. Ab tract Plant.. ca pable o f gm ntng cat hon ( ) tht ough hoth ph o t oc;,ynth e~ t ~ and tn yco n h1 /a l fun ga l yrnb1ont are tenncd parti al n1 ycoheterotrophc;; (PM 1b ) Recent 1 e~ca rc h into PMH C budget~ ha pnn1anl y focu5ed on ana l y~ t ~ o f ~tabl e C t ~otopc ra tJ o cnric hn1 cnt (8 C) to 11 qu antify the pr0p011i0n 0 f fun ga l ga tn In target ~ p ec t C~. partJ CU] arl y und er }ight -I itnt ted condition . The unprecedented n1ounta1n p1ne beetl e (IJendroctonu\ pondero\ae) epJdCJTil C in British ColUinbia ha altered \ ast ex p a n~e of forests. \A. ith elevated li ght le\ cls du e to needle-drop and 1n1plication to oil m o i ~ ture regin1es. both of \J\, h1ch may Infl uence PM II ecophys iology. Owing to these habitat change , durin g th e 20 12 growing sea~o n . light and soil n1oi ture were experitnentally manipulated in the fie ld to tn ea~ ure the eff ects on net photo<.;ynthesis ( P, ) and 811 C signature of autotrophs and three putati ve P MH Pyro leae sp ec 1 e~ ( Ch imaplula unzbellata. Pyrola ch loran! ha and Ort/11 ha \ec undo) grO\\ 1ng 10 p1 ne- doininated forests of central Bntish Colun1bi a, Ca nada. Ch unaplu la Lunhellata sho\A. ed ev tdence of autotrophic C nutrition, with significant response .. to ~ h a d e treattnent in P and 8 'c signatures, and estimated net seasonal C uptake exceeding that of autotrophs V\ hen 1 cotnb1ning all treatn1ents. For all treatn1 ents, P ra te~ in P. chlorantha and () \Ccundu \\ ~re generall y 30% to 50% lower than autotrophs, indicati ve of consistentl y hi gh lc' els or Mil nutntl on. Signi ricant effec ts or watering on 111 tantaneous P onl y occurred for P. chlorantha and only under the unshad cd trcattn ent. but e titnated net seasonal C uptak.e w a~ ~ I gniii ca nt l y higher undet the sa n1e treatincnt l o1 both C tunhcllata dtH.I J>. cltlorantlta. 15 Orthiha secunda P rates vaned llttle across trca tn1 ents but were hi ghest und er the shade trcatrnent, and both 0 \·ccunda and P chlorantha 8 1'c \a lues abo\ a1 1Cd little and nonsignrfi cantl y ac1 0<-,5 treatment<; Along \\ 1th P. rate<.,, other ga<;-e\.change van able" 1nd 1ca ted a low tol erance of 1ncrca ed l1 ght le\ cis 111 0 \('( unda and to a l e~~e r degree P chlorantha . This tna y increase photoprotectl\ c adaptatio n<., <., uch a~ hi gher photoJ e!-,plrati on rates, reducin g net photo ynth e~ ' " under tt e<.,~rul co n cbt1 o n~ signature indicate any hot1-tcnn change I he lo\\ \artabdJt\. 111 bulk l ca ru ~~uc <') 1'C 111 PMI I nutntton due to drought stress were not detectabl e: therefore. as<;ec;sment ol so luble ', Uga r 6 1'c tn ay be rn orc <.,u ttab lc 111 future rc earch There i aL o a need to understand tf and ho\\ the P}roleac ddTcr in pho t o~ynthc ti c 13 C discrim1nat1 on compared to autotroph , \\ h1ch n1 ay alter interpretation of PMJ I st1atcg1cs. 2.1 Introducti o n Mycoheterotrophy is a nutntional strategy know n in O\ er 400 plant species, where orgarue C is obtained through tn ycorrhizal fun gal ~yrnbi o nts in pl ace of, or in addition to, photosynthetic CO:! fixation (Leake 1994 ). Thi . trategy \v a fir ·t rccogntzed O\ er a century ago in the non-photosynthetic, achlorophyllous pccics ~fonotropa ln·pop1(vs L.. \\here Jt \\as di scovered that the plants were not directly parasitic on tree roots. Instead, nutrients arc passed fron1 host trees to fungi to plant in what is cuncntl y considered d tnpa11itc (or tnore accurately, n1ultipartite) relationship (Leake 1994, Julou et al. 2005 ). pecte hke M. hypopi~vs that arc incapable of photosynthesis (P ) are tenncd fullJn yco h etc rotroph~ (MH ) and rely ~o lcJ y upon n1yco1ThiLal fungi for thei r C acquisition through the1r entue hi~ cycle (Leake 1994 ~ Trudell et al. 2003~ Bidartondo 2005, Mass1cotte et t1l 20 12) ._ on1c plants are only M H at the gcrn1ination and below ground life tagcs (e.g., tnost Orch1daceac), becoming autotrophic as adults (Bichu1ondo 2005 ). ()nly recently ha~ 1t been found that 16 sorne chloroph yllou pecies clo. ely related to non-photo ynthet1 c full y Ml I plants can obta1n a suppletn ent of heterotrophic C 111 addttion to photosynth etic C and ha ve been clas di ed a partial MH (PMII Leake 1994, Gebauer and Meyer 2003 , S clo ~~c ct al. 2006: Mcrckx ct al. 2009) The fmntl y En caccac IS one o f the ~e\ eral plant hncage~ known to exhibit full and part ial tnycohetcrotroph y, ~ p cc di ca ll y \\ 1thtn the v. cll-stud1 ed subfatn dy Monotropotdeae Th1 group 1nclude. full y \t1H pl a nt ~ tn the tnbes Monotropcae and Pterosporeae (n1onotrope ). and clo ely related green plants tn the tnbe Pyroleae (Teder()OO et al. ?007; Hyn on et al. 20 12) Over the past coupl e of decade . analy i of ~ta bl e Isotopes 11 1 C and " 1n plants and fungt has becotn e a primary \\ ay to detenntne charac teristi cs such a<; troph1c <:, tatu (e.g., MI I ver ·u autotrophic plant ). ecological ni che (e.g., EC M versus sa pro trophic fungi), or portt on of C budget deri\ ed from funga l source In PMH (Gebauer and Meyer 2003: Trud ell ct al. 2004: Preiss and Gebauer 2008). The stable i otope rncthod n1 easures the rati o o f naturall y occurring heavy to Iight i otope (i.e., 13C. 12 C and 1"N. 14 : sec Secti on 1-2 for full details), and is based on the fact that n1ctabolic and ph ysica l processes generally di scrin1inate aga inst heavy Isotopes ( Daw on et al. 2002: Fry 2006 ). W 1thin the foo d chatn, hi gher trophic le"' els accu1nu latc heavy isotope based on access to nutriti onal ources enri ched in the heavy isotope(s) and the differenti al fra ctionation of heavy or hght n1olcc ul e in n1ctabo lic acll\ 1t 1 c~ (DcN iro and Epstein 1978: Gleixner et al. 1993~ Farquhar et al. 1989). In general. full MH plants and their tn ycorrhizal fu ngi are sitni larly ennchcd in isotopic abundance of 11C dnd I 'iN, autotrophs arc depleted in these isotopes, and PM II species have been round to ha\ c isotope levels intcnn edia te to autotrophs and MH (Figure 1-2, .cbauer and l\1eyer 200~, .T u lou ct a I. 2005, Tcdersoo et rd. 2007: Ztn11n cr ct al 2007: Preiss ct al 20 10, II vnson ct al. 20 12) 17 Drn ·ers of partuil Jnycoheterotrophy [t 1 thou ght that pa111al n1 yco heterotroph), at least in green orchtds and Pyrol eae, evolved prior to full tn ycohetct otrophy a~ an adaptation to l o v~ li ght levels tn ~ had ed eco ysten1 s (Tcdcr~oo et al 2007, Pre 1 ~ ct al 20 l 0) Light a' allablltty has been found a~ th e priJnary detcrn1lnant of the dcgt ee or PM JI nutntl on in l\\ 0 green orchtd ~p ec t e s (PreiSS ct al 201 0) The reaso ntng behtnd tht s concl u ~ t on ts ba<.,cd on the theo1ett caltnodel by Farquhar et 1 1 al ( 1982, 19 9) All thing betngcqual.anyp hoto<..,ynth eticC ~ plant\\illha vc hi gher 6 Cin higher' er u been detnon trated In autnt1n ph ~ dunng m vcs ti gat1on ~ lO\\ er lt ght. "htch ha 11 on PMH orchid (Gebauer and Meyer 2001, Abadie et al 2006) F1xa tion of C dunng PS is dri' en by the interplay of btochcn11 cal dcn1and for co, by Rubt <.,co as well a<.., <.., uppl y of C02 through the stomata into leaf tnterccllular spaces and fl.u1her diffus1on through tn esophyll tissues to the ite of carboxylation (Farquhar et al. 1989) In ht gh light. in the ab. encc of limiting condition (e.g., water or all tress) C0 2 is fi xed at a relati vely high rate co1npared to under low light condition . This lowers the intercellular to atn1ospheric CO, concentration ratio (C.:Ca. in tctms of partial pressures), and any 13C0 2 present 1s 1norc likely assirnilated due to lln1ited C02 availability and high den1and by Rub1 sco. Con\ ersely, under loVv light conditions C. :Ca is higher and Rubisco ca n preferenti all y di scrin1inate against 11C, resulttng in lower 8 1~C va lue~ (Farquhar ct al. 1989: Dawson et al. 2002 ). The findings of enriched 11 C levels in understory plants relati ve to surrounding autotrophs provided evidence that the tudy specie were indeed accessing 11(' -cnnchcd mycorrhiza l C sources (Gebauer and Meyer 2003~ Preiss et al. 20 I 0). Several P) rolcae specie~ have also shown enri ched 13 C relative to urrounchng au totrophs. or tndt\ tdutlb tn shaded versus sunny conditio ns, inclucli ng C!J imaplu Ia uJnhe//ata ( l ) W P C' Barton, Orth ilia secunda (L.) II ousc, P vro/a ch/orantlia Sw, P\·ro/a pte Ia, Pyro/a JOponica t1nd I~vro/a 18 rotund1{o ha (Teder oo ct al 2007, Zunn1er et a! 2 0 07~ Hyn on et al 20 1 2~ Matsud a et al. 20 12). In a recent expcrin1ent by Hynson et al (20 12 ). C l(Jnhcl/ata responded to ~ ha d i n g itntlar to autotroph , '' tth ~o lubl c <)ugar 81 ~(' lc' cl~ hetng depleted O\ er ttn1 e fh c oth er test speete , P. pte! a. shov. ed rnt xcd results of p os~ 1hl e PM I-I conclu i' e nutnll on hut were not entu ely easo nal changel.i tn the ex tent of rn yco hctcrotrophy were l 0 111 he1ght). which tnd udccl about ~ 0 o subt1lptnc fir (Ah/(, la.\'locarpa (H ook.) N utt. ), as well as -:. 5000 ste1ns ha 1 or pine, lir and 0). Daily averages ranged frotn a minimu1n of - 20 J..tm ol m-2 s- 1 in the shade treatn1 ents to - 260 J..tmOIJn-2 s- 1 in the light treatments (Figure 2- 1a). with n1aximu1n levels often reaching 1600 Jlnl ol m-2 s- 1 or tnore. Hourly averages across all days showed peak light levels of 150 and 350 Jlrnol 1n·2 s- 1 in shad e and light treatments, respecti vely, and typicall y exceeded 100 ~un o! 111-2 s- 1 bet\\ een 9 am and 4 pm Pacifi c Daylight Savings Titne ( PDST~ (Figure 2- 1b). 29 400 b a L1ght t rea tment (/) 300 N ' E 0 - §. 200 S had ~ trea tment 0:: •• < Q_ •• ~ 100 ....' . . .: " . ..:: . . :. ... ! • •• • \ ."' ' .. ' ,,·. • • .. . . : ' f :- :.~{ ' ·; . '•. Q) 2 • ~ • • • •• 0~ I I .l .L J I .L 3J- d c - () c......... 25 I . . . .\ Q) '- :::J ro • 'Q) a. E Q) 1- 15- c ro . ' .. •. Q) ~ 10 ' I ..... -. .. ' .• • 51 0~-29 I 00-23 07-24 08-21 I t 00-18 co 02 04 Date I I I 00 10 .. 2 14 16 18 20 22 Hour of Day Figure 2- 1 Mea n photosyntheti ca ll) acti ve rad iati on ( P!\ R: ~tm o l 111-2 s 1) and tern perature ('"C) for li ght treatn1ents ( olid lines) and shade treatrnents (dashed li nes) across the experi1nental period of May 29 to Sept 22, 20 12. Panels a) and c) are da ll y averages, \\ ith PAR averaged for onl y dayli ght hours, and panels b) and d) represent hou rl y average ensen1bl es for all days. The total season averages for ternperature were very sitnilar for both shade and light treattnents at 14.1 °C and 15.0 °C, respecti \ cl y, with dall y ten1peratures ranging frorn only 2 to 3 oc in Septen1hcr to over 20 °C on rnany days bet\\ een earl y Jul y and August ( Ftgurc 2- 1c). Below free~ in g ten1peratures occ uncd once in ea rl y June and n1ost ntghts after Scpten1bct l L reachin g a reco rd ed It)\\ of -2 1 C Ilourl y a\ c1age tetn peraturcs pe,lked around the smne ti rn c as li ght levels. reaching appro'\ una tel} ?2 oc and 27 ( C 111 the "'hade 10 and li ght treatn1cnt, re pectncl y (Ftgure 2- ld ) The typtcal trend tn sun1n1 erts fo t the hottest part of the day to be delayed ~o nl C\\ hat fron1 ~o lar noo n. hut the data and logge1 fat lure tndt cated th at the loggers\\ ere s u~c~ pttbl e to O\crh eattng du1 tng pea k lt ght tntcnstty Cotnpansons were rn ade to abo\ e canop y ciCita fi on1 a nearby rec::,carch Site (11 111 tower w1th then110COupl es) to detcrn'll ne I r the cia ta \\ Cl e gt o~c::, l y 0\ eresttnla ted. The greatest di , crepancy tn dail) a\erage tcn1peratu t c~ (8 C tn th e plotc;; \Cr'.u5 16 C aho\C canopy) occutTed tn epten1ber Most othc1 dad) conlpan<:,on5 \\ere\\ tthtn a fev. C, and the sea~o na l a\ eragc~ for both light and hade pl o t~\\ erc aln1ost tdcnttcal to the tov., cr data. otl 11101 ture le\'el \\ere tn ca~urecl pcn ochca ll y aeros5 the growtn g ~cason in co njuncti on w1th field rn ea uren1ents in ea rl y (Jun e), n11d (Jul y to ea rl y August) and late (tnid-August to September) ·u1nn1er. In June, rainf'all totalled 109 3 rntn at the nearby re earch installation, v.'hil e dry and \\a tercel trea tn1ents differed littl e 1n so tl n1ot sture at either depth (Figure 2-2). There wa a clear dry-dov. nO\ cr the course of the gro\\ rng ~caso n \v tth successi\ ely less rai n (42, 27 .7 and 2 1 4 111111 O\Cr the nex t three n1onth ':>, rc~ p cc tJ \c l y), a~ shown in the unwatered treatn1 ents ( LD and D) \\here sod tnoisturc v., as as lo\v as 50%> of watered trea ttn cnts at the 10 ctn depth. The watered treatn1ents (LW and SW) Inatntained sin1i lar levels across the season. The soil n1oisturc was very low at 4 cn1, often ha ving\ a lues of only 2%, and was typi call y half th e an1ou nt as the I 0 ern depth (Figure 2-2) 11 4 em 10 em Dry - Wet 0 > 10 >- .D - '$. Q) '- -- ::J en 0 2 o s- (f) c ro Q) 2 0I I I I I I Early M1d Late Early Md La te Sampling Penod of Grow1ng Season Figure 2-2. Mean(± I E) tn ~ta ntan cous soil n1otsturc (0 o by vo iUJnc) as tn casurcd 1n th e earl y (June), n11d (July to early August). and late (tntd-Auguc;,t to n1Jd -Scptcn1bcr) ~un1m c 1 1n 2012 for dry (LD and D) and \\ atercd (l \V and W) trca trncntc;, at t~o depths Photosrnthet1c rate.\ hr . . month and treutn1ent Photo ynthc i rate of each ()pccic by tnonth a\ eragcd O\ er all trcatrncnh ex hibt ted slightl y different seasonal patterns (Figure 2-3a). In all ~ p ecies, Septcn1hcr PS was ~ 1 gnifi cantl y lower than PS 111 June through Augu ~ t (p c 0 00 I for all but June\ e r~us Scpternbcr for autotrophs, withp = 0 017) . No other n1onthl y differences \\ere ~ignifi ca nt Autotrophs had tnod crate PS in June and but increased over the sun1rn er to reach a peak 111 August. There was 'cry little seasonal chan ge bet\veen June and August in the P rates of th e three P yro l eac~ C. umhellata tnaintaincd allnost exactly the san1 c n1ean rates, 0 ,\ ecunda had sitnilar rates in June and August but dropped sotncw hat in July, and P chlorantha had sli ghtly increasing ra tes fro rn June throu gh Augu t (Figure 2-3a) (a) Month 6- ----- - I (/) - --...... - --- - - - N --- E 0 ~ -- ---- 2- ... ... .. ... ... ... .. ... ... .. ... ' ... E ' Spec1es .::1. ..........., Autotrophs (/) ·- ( /) I I I I .L June July Aug Sept c. (b) Treatment ~ >(/) 0 6...... • Ch lmaphlla umbellata -• Orthtlla secunda -+ Pyrola chlorantha 0 ..c 0... ....... z 4c ro - - -· - s • --- -- --- ... --- ---- ~ I ! LD LW SO SW Figure 2-3 . E timated margi nalrneans (± 90°o Cl ) of net ph oto~)ntheti c rates (~trn o l m"2 s 1) of autotrophtc reference plants and the three Pyroleae . pec1es by a) month (all trcatrnents cornbined) and b) treatrnent (a ll n1onth con1b1ncd ). Treatn1ents are light dry ( LD ), light watered (LW), shade dry (S O) and hade watered (SW) Non-o\crl apping CtTOr barl) within specie repre ent significant dtfTerences bet\vcen month~ or treatrncnts at u ~ 0. 10. Trcatn1ent e ffects on P averaged over all months indicated two di sttnct groupings: C. umhellata was imilar to autotroph , with both d1splay1ng a clear drop In P under <.; hade conditions, while 0 . .\ ecunda and P . chlorantlza were s1n1dar and dtd not ddTer greatly in P ~ across the lo ur trea trn ents (Figure 2-3 b). For C. uJubellata, both li ght trcatlnents \VCre significantl y different than both hade treatlncnts (p <' 0 082). For autotrophs, plants 1n the LD treatrn cnt differed frotn tho e in both of the shade trca trnents, and plants 1n the . \V trcattnent difTered f'ron1 those in both of th e li ght trea ttncnts (p co n1pariso n~ were not ~ i gnifi c ant (Ftgurc 2-3 b) significa nt for 0 . secunda (uni vari ate F , 22 22 0 035), all other The O\c rall effects ofttctltlnent \\ etc 2 626, p ll 0 076 ), hO\\ e\ et . no po"t hoc igntfi cance ·were detected. th ough the ell fTcrence bet\\ ccn the S D and W trcattncnts had p 0. 10 1 For p ch lorantha, th ere appeared to be an effec t o r\\Cltell ng Vvht ch also tnciJ catccl a partial re ponse to shading. '' 1th the L\V but not the l D trcattncnt plants ha\ tng signtfi cantl y hi gher p than both ~ had e ll ca ttn cnt<, (p () 028 and 0 066, re<.,pcc tJ ve ly, r~ Jgurc 2-3 b) Bulk lea( II.\ .\ lie (> 1 \C ,·ahte\ In · 11 catn1ent and o1 ·er tune V\'ithin-spcc1e5 ~ ta bl e ca rbon 1so tope cotnpo<., ttt on<, fo r t1 eat n1cnt co n1p a n so n ~ arc hown 1n Table 2- 1 With rc~ pcc t to trea tn1cnt effec t5 on Scptctnher 6 1 'C, only C umhellata h O\\ cd signi ficant treatment effec ts. \\ tth both shade tJ catment<., bct ng s1gnt ficantl y more depleted than th e LD treattn cnt (p .,... 0.036 ). Autotrophs and C. um hellata had very sitnd ar Septetnber 8 13 C value under the hade trcat n1 ents but the latter v. as 5lightl y more ennchcd under the li ght treatments. Both 0. ~ecunda and P. chlorantha v.- e1e consistentl y enri ched co mpared to the autotroph in cnch trea tm ent b] - I 0/oo. 0" er the ~ca~on , 6 1~C \ alue becan1 e more depleted fo r the Pyroleae species (Tab le 2- 1). Both C. Lunhellata and 0 \ecunda showed the greatest depletion in the SW treatment. ha\ ing significantl y lO\\ cr n1 can 8 13 C in Septctnbcr versus June, but C. Lunhellata also had signifi cant depletion under th e L W and SO treatrncnts (Tabl e 2- 1). For P. c.h loranlha , only the SO trcatin ent bccatne significantl y depleted over titn e, but there was a trend of greater depleti on in both dry treatn1cnts compared to the watered trcattn cnts. 14 Table 2- 1. Treatment n1eans (± I ... E) of8 l3C (%o) for Jun e. eptetnber and th e difference over time ( eptembcr-June) for each pecic Treatm ents are light dry (LD ), light wa tered (LW), hade dry ( D ) and hade \Ya tered ( W ). ignifi cant trca tn1 ent differences between , eptetn bcr 8 13C value b) pec ie arc indicated by diffe rent letter~ within th e appropriate colutnn. Listed p-value arc result fron1 patrcd t- test~ dctcnnining differences between Jun e _._I_O._ _ _ _ __ and eptcn1 ber 8 13 va lue . II rc ult ~:i \\ere considered significa nt at_a_<'_O . Jun e (0oo) 8 11 C . ept (%o) LD LW -30.09a ± 0.29 -29 95a ± 0 30 D -3 ll 3 b~ 0 30 sw -3 0 39a ± 0 24 -3 0.98± 031 -30.80 ± 0.3 0 -3 1.0 ± 0.32 -30.95 ± 0.29 n/a n/a n/a n/a n/a n/a Ch in1aph 1/a LD -3 0. 1 ± 0.2 1 -30.26 ± 0. 14'1 -0.08 ± 0. 15 0.589 umbel/ala LW -30.14 ± 022 -30.54 ± 0.21 200 j.!l110ltn-2 s- 1, which is con i tent with PS show n in Figure 2-3 b. Conversely, LRC n1easuren1 ents of C. umhcllata exhibited the hi ghest PS under th e SO rather than the LD trea t111ent. Orthilia secunda also s h O\\ ed th e highest LRC rates under the SO treattnent (Figure 2-4 ), and was consistent \\ ith PS under natura l light (Figure 2-3 b). Both 0 . s ecunda and especially P. ch/orantha had lo\V \ ariabtht] 35 rn LRC xnea urcn1et1ts bctv.'ccn treatn1cnt"'. When co rnpanng June <1 nd August LRC hade l1 ea tn1cnts and C unthcllata 111 dt y trca tn1 ents had hi gher tncasuretncnts, autotroph 10 P abo' e I 00 ~uno I 111 2 1 to J unc and dropped b; ,\ugu~t (not hO\\ n) utotroph~ al~o had the htghe t Rd, \\ 1th progrc Sl\ cl) decrca<;,1ng rntc~ f o 1 C undnllata, () \CC unda and P. chlorantha. in that ord er. but all t.., p cc te~ <;hO\\ecl \Cry low \anabtlJty a c t o s~ treatJn ents (F tgure 2-4) undc1r to photo~) nthct 1c rate<;, au tot1 oph1c t e~ p1 ration 1ctte~ for the ~ h ad e treatment \\ere co n ~ Jd crab l ) ht ghcl ( n1orc ncga tl\ e) in June (not ~ h O\\ n) Lt ght C0111pCn ation potnts (where photo ~ynthes i s rc~ptratlon), \VCre qutte low f'or aJJ ~peC J CS ancJ trcatrnent . being htghcst for autoll oph~ ( 15 ~tmol rn . . ~ 1) and IO\\ c~ t fo r 0 \ecunda and P chlorantha( 10~Hn o lm " 2 1 ). Chimaphila umbellata Autotrophs • 7 5- -;- C/) .. .. -- -------- -- - --' ---- : --- -- =~--::. - ~- 50- "'I I E 2 5o E 3 0 0 ___,. = - - - - - - - - - - - - - - Treatment (/) (/) (1) .c ...... Orth1lia secunda c >(/) 7 5o ...... .... LD A LW Pyrola ch lorantha --- so • sw 0 .c 0.. 50 - +-' (1) --- - -- - -- z c 2 5co ·. • - -- -- - -- -- ~ • • • •• • ~ ----=--------: ... . ~ .:."'"" - - ~ r a'""'- r • = • e • " ' ~ ' (1) 2 0.0 I I I I 0 200 400 600 1 I 800 0 I I I I 200 400 600 800 Photosynthebcally Acttve Radtatton (~mol m 7 s 1 ) r igurc 2-4. Mean (~ 90° o C l) net C0 2 CAc hange (~uno l n1 2 -I) as a non-linear regrc~~1on fun cti on of photosyntheti cally active radiation (light rcspon .. e cun cs) fo1 1cfcrcncc autotrophic plants and the three target Pyro lcae species und er fi eld n1antpulatton treatn1cnts T rcallncnts arc li ght dry (LD) , light wa tered (LW). shade dry (SO) and shad e'' atercd (S\\) 36 Estimates of n1ean net sea .. onal C ga1ns do not exac tly refl ec t light response curves, ba ed on th e actu al atnount of li ght in each plot For each sp ec t c~. a!Jno t all tJ ca trnents were highl y tgndicantl y ddTcrcnt (p < 0.002). \\Jth onl ) t\\ O trea tm e nt ~ per <5 p ec J e~ that did not significa ntl y dtffer frorn the oth ers (FigUJ c 2-5) The ~ h ade trcCl trne nt~ dtcl not ddTer ignifi ca ntl y fo r both auto tro ph ~ and P chlrn antha, v. b erea~ fot C umhellatc, the dry treatn1ents \\ ere stattsttcall) not di fferent For 0 \C c unda, the I Wand D trca tn1 ents clJd not di ffer. Autotrophs Ch1maph1/a umbel/ala Orthilia secunda Pyrola chlorantha 200150- - 100 - E 50- N • - 0> Q) g' ro 0 ..c (.) X w -500 c 0 C/) 200 - ro Q) (f) -r- ro 150 - 0 c 100ro Q) :2 500 -50 T I I 1 I l I 1 LD LW SO SW LD LW SO SW Treatment Figure 2-5. Estin1 ated tn ca n ( 1 90% Cl) total seaso n C exchange (g C 111 2 ) dcri\ cd frorn P responses to li ght and seasonal light levels for autotroph ic reference plants and the three Pyrolcac species frorn May 29 through cpt 14. Dark grey bars rc p re~cnt net ~cd~onal photosyntheti c C ga ins, and li ght grey b ar~ rcpre ent total re piratory C lo~sc~ ctt nt ght ~on ­ overl apping error bars indicate signifi cant difTcrcnces at ex <.. 0. 1. 37 Contrary to atnbient photosyntheti C Inca urcJn cnts (Fi gure 2-1 b), patterns o r net ea onal C gain bet\\ ec n treatn1 cnt fo r eac h specie<; dtf fcrccl so n1C\\ hat '' 1th J> cJllorantha appearing rnore autotrophtc and tc<;ponsJ\ C to '-lhadtng than Ftgut c 2-1 b, and C umhella ta appeanng tnore respons1' e to '' atcnng and <;hading. but not b0th Only 0 .\ eutnda sho'V\ s relati\ ely con 1.. tent p a tt e rn~ 0 r net c uptake COJllpat cd to actua I rs Iate<; bet\\ cen trcatrn cn t ~ (Ftgurc 2-5) Trend s bet\\ CCn <;pCC JC al<;O diffe red fl on1 r n1ea'-IUI CI11 Cnt(). the net sea<;onal C ga ins for C. lllnhellata and P ch lorantha \\ ere htghcr than auto trophs for all trcattn cnts except the LD, and 0 'ecunda '-lhO\\ ed higher C ga111'-l than autot rophs undct the SO treattnent (Figure 2-5 ). For e tin1atcs of RJ C lo. ses, all spec 1cs shO\\ eel stgntficant cit ffcrencc'-1 hetwcen all treatn1ent (p < 0.00 1 for all ) except 0. \ecunda. with no difference hct\Veen LD and S W treatme nt~, and P . clllorantha . v. tth no ddTerence bel\\ ccn I ight trcatn1ents Based on th e similar length of clark peri od fo r each trca ttncnt and no tctnperaturc correc ti ons, cstitnatcs had very low variability around n1eans (Fi gure 2-5). Ortlulu1 .\ ccunda and P ch/orantha had consistentl y lower total R d C losses than autotrophs and C. unzhellata. The I O\\ cr net C ga in ~ of the autotrophs under the LW and shade trcattnents relates to the htgh relatt\ e Rd con1parcd to the Pyroleae, as total C gains be fore ~1 losses (i ndt ca ted by total colutn n length ) was\ cry similar between all species in at least the shade treatn1 e nt ~ (Figure 2-5). 2.4 Di scu sion The MPB epidcn1ic in ce ntral British Colutnbia has greatl y 11npacted ptnc-donunatcd st a nd ~. with loss of ca nopy pine tree exceeding 90° o 111 forest ~ at our ~t u d) <11 Ctl Gl\ en a diverse assemb lage of und erstory species rangin g fi·on1 rull autotroph to full Mil c1 long \\ tth (l host of putati ve PM II Pyrolcac specie. , \\ c h to 70% of autott oph1c 1a te~o.,, ~upp oJ1 t ng po tulatt on by H yn ~o n ct al (20 11) of 1nhcrcnt IO\\ PS 1ate" 111 t h e~c " p ec 1 e~ (I~ t gurcs 2-1 b and 2-4 ). T here\\ a~ a pat1J al autotrophic re~p on e to ~ h achn g 1n P chloran tha PS ratcc;;, but corresponding 8 11 C \ ctlue \a ri ed littl e ae ro~~ trcatnlcnt'-1, potenti all y 1ndica t1ng 1ncrca~cd M il c gain under hade. Ort In lui \('(l l llda d I rr cred fron1 the other Pyroleae 111 th at j t pari tall y showed a po itive respon e to shade. wtth the hi ghe~t PS under the SD treatn1ent that exceeded both light treatinents (Ftgure 2-3 b. Table 2- 1). Though treatrnent responses to light \\ ere n1in1n1 al with regards to PS rates of 0 . ~ecunda and P . chlorantha. th ere are t\VO <;easonal a~p ec ts of ll ght that 111ay have influenced photosynthetic productivity and PMH C ga tns. First. hi gh producti vity 1n spring such as shown by Isogai et al. (2003) may have occuned, with the n1ajonty of organ grov. th and developtnent occurring prior to overstory leaf flu sh and ca nopy closure. The l RCs measured in 1une ~howed hi gher Rd in autotrophs cotnpared to the August n1ca<:,uren1ent ~ for all trcatn1ents, whil e there was littl e change over tin1c in all three Pyroleae Rd (not sho,vn) It is assutn ed that the higher Rd for autotrophs in June refl ect in c rca~ed gro\\ th rc~ ptration during lea f fl ush, which ca n exceed n1a intenance rcspirntion by thn:e to ten tunes (Larc hcr 1995 as cited by lsogni et al 2001 ). The lac k of change 111 Rtt bel\\ cen June ,1nd \ugu~t 1n the Pyroleae indicates son1e earl y season growth did occur, though crncrg1ng le;n cs \\ere observed in the Pyroleac s pec 1 e~ dunng the san1e pc110d as autotrophs The higher Rd in 40 autotroph tn June rna y ha' e aL o re fl ected ~ ttnpl y produ cti on of much greateJ an1ounts of bton1as econcL ht gh lt ght tntcn. tty 111 n1Jd -~un1n1 c t in nddJtt on to ht gh tetnperntu1 e~ can he detnrnen tnl to p lnnts, causing ph o t o re~ p1 1 nt1on. photo1nht b1 tt on and photo-ox ida t1vc dan1 agc. Thi fa cto r appeared to ha\C a negalt \e c rrcc t on () \('(UJlda PS rates Ill parttculm , with th e htghcst PS (Ftgures 2-3 b and 2-4 ) a I 00% and nea rl y 97° o of autotrophic estirnate for C. u Jnhellata and P chlorantha, rc~p cc ti \e l y (Figure 2-5). Addttt onall y, all data for 0 ,·ec unda ho,ved the grea te<;t le\ c l ~ of au totroph y in hade, stgnt fy1ng that low PS rates under higher itTadi ance 1nay ha\ e been a res ult of high levels of photo1c~ pu·a tt o n 111 these specie . It is also possible the Pyroleac cxpen cnced a do\\ n-regul at1on, or photoinhibi ti on, of photosystem IL where effi ciency in the harve ttng or ll ght energy to drive C0 2 assitnil ati on is reduced and instead the excess energy is released as heCl t (C haves ct aJ. 200 2~ Sou£a et al. 2004 ~ Xu et al. 20 I 0). This cCln occur a a response to ltght energ) 111 excess of that of biochemical de1n ands, as \\ ell as that of stomatal closure to pre~en c \\ ater (Chaves et al. 2002). Und er the LO treatrn cnt. there is hi gh potenttal for both light and drought stresses in the Pyrolcae. The lack of response to shading 111 0 . secunda indicates it is n1ore ~ h ade adapted than the other species (Fi gure 2-3 b). ince PS rates would be saturatc<.l at l o v~ er !Jght k\ cis than light adapted species, under idcnt1cal condit tons 0 ,·ecunda should C'< penence ,\ g1e,1ter 41 exce s of light energy to be di ssipated through non-photoc hemi cal n1 ea ns such as chlorophyll fluorescence or hea t relea"e. \\ 1th greater potentt al for photo-ox tcl att\ e datnagc than the other t\VO spec1cs (Krieden1ann 1999) In a "tud y by I lunt and ll o p c-S JJ11f> ~O n ( 1990), P. rotuncl!(oha gro\\ tng 111 sand dun es 111 I ngland \\ere fou nd to have chlorottc, less luxut tant leaves 111 habitats\\ tth tnin1n1al ~h ad e. and during hot ~ unn y p e n o d ~. htghet n1ortality occuned tn the e tndt \dduab co n1pared to 11101e protected. shaded co ndt tio n ~ C'o mpanng \\ atercd to dt) trea tm e n t~ ~ howed fatrly unex pected result ~ for PS rates (except 1n June \\ hen rat n \\a~ pl entiful) and little to no rec;pon~c to \\ atenng 1n 6"C va lues (Figure 2-3 b. Table 2- 1). T he expectati on of lO\\'Cr PS ra te~ undct the dry ver~ u ~ watered treatment for a gl\ en ltght le\ cl \\ as not n1ct in most ea~es, v. tth only P. chlorantha show tng signifi cantl y hi gher P rates under the L W treattnent con1pared to both shade treat1ncnts (Figure 2-3 b). In fact, exclud 1ng P ch lora nt ha. only 25° o of th e p o~c; t ble con1pan c;ons between watered and dry treatn1 ents (monthl y averages) shoVved hi gher PS rates when watered, including those of autotrophs (data not show n). It is uncet1ain Vv hy th1 s occurred. In several studies, response to rehydration following drought showed a lag period between the watcnng and reco' ery of P rates, wht ch took several hours to days and d1d not ah\ ays reach pre-drought rates (Willis and Balasubra1nani an1 1 968~ Sou;.a et al. 2004 ~ Miyashita ct al. 2005 ~ Morales et al. 20 13 ). S to1natal clo ure and reduced tnesophyll conductance triggered by low leaf water potent ial and abscisic ac id (ABA) produced in roo t~ have been attributed as the n1ain driver for decreased P und er n1tld to n1oderate drought condition~. ~ whi ch usually recover fairly qui ckly (Chaves et al. 2002~ Souza et al. 2004: Miyashita et al. 2005: Xu et al. 20 I 0). Although PS rates v. ere generall y IO\\ er under the "atcnng trellltncnt. transptration rates and co nductance were altn os t always hi gher than the dry treattncnts. cspcc rall y for C. uiJlhellata and P chlorantha (a nd to a lesser e\.tcnt 0 ,.( ( unda in Juh"' ). 42 indicating ston1atal and or n1esophyll co ndu ctance \\ ere reco\ cnng fol lowing rehydration ( uppl etnentary Table 2- 1. hctca fter referred to as Table . 2- 1) d<.lltionall y, 1ncreascd tran p1rat10n rnay ha\ e caused lO\\ er P due to n1 olcc ul ar 1ntcrfercncc. i c. CO-, ddTuston into the lea f 1 slO\\ ed by mtcrfe t encc \\ tth II"l o dtffu"ton out ( l·a1quh ar and Shark ey 1982 ). It i posstbl e th ere was also a lag tn rccO\CJ} of" ton1a tal guard cel l tUJ gor th at was slo\vcr than regular eprdcn11 al cell and n1orc pt onc to clo~UJ C upon fut1hcr strcsse~ ( Willi ~ and Balasubratnani arn 196 ). \\ tth our ga -e"\changc n1 ea"u1 crn ent<; taktn g place so n1 ewhat pno1 to fu 11 reco' cry As ·es n1ent of C 1:C a rati os, though not specdica ll y tested. 1e\ ca led th at all s p ec 1 c~ had sli ghtly hi gher cl Ca ratiOS tn the shade t rea tln ent ~ cornparcd to at least the LD treatn1 ent, though not neces .. aril y the L W trea tment (Tabl e S2- I). Beca use hi gher CI.Ca rat1 os • contribute to greater photo yntheti c 13 C dJ "Crtl11lnattOI1. the relati onship of th1 ~ van able 11 ben.veen treatments likely contributed to the sli ghtl y n1 ore depleted c) C va lu es fo r th e Pyroleac under the shade treatn1cnts. Whdc 1t coul d be expected that plants under the LW treatlncnt should also have lower CI:Ca rati os due to hi gher PS rates and thu s bioc hetnt cal de1nand for C0 2 than the shade treatments, it see1ns that 1ncreased sto1natal co nductance followin g watering n1ay have resulted in 1nore C0 2 chffusing into sub-ston1atal spaces. T hi s could contribute to the watered plants 1naintaining a relatively hi gh C 1:C a ratto under the light compared to unw atered sampl es, and may have been a factor in the 1nini1n al di fferences in the respecti ve treattn cnt 8 C va lucs (Table 2- 1). Due to the e trends, the lac k of ~tgndicc.1nt 11 13 differences in Sc pternbcr 8 C values for 0 . secunda and P. chlorontha, and low variabtlit ..v across all trea tm ent~ 111 all three Pyroleae {a 1Tia'X ilTIU111 di rrcrencc of 0.8°oo: .I dble 2- 1). no conclusion cou ld be n1ad e whether the degree of rnycohcterotroph y increased dUJ 1ng drou ght peri od ~. Genera II y the pattern~ of 8 C "alucs seetncd cotnparablc to P rtltc' 1n tno-.,t c ,he'. 11 43 \vhere treatn1 ents ha' tng hi gher P rat es u ua II) had corre, pondi ng h ighcr o C (Fi gut c 2-1 ~ 11 Table 2- l ) Thts itnpltes that for tht ~ study, l ~O tO pl C Signatures 111 0 1C strongly re ncc t I~ photo )11thettc effects on C rather than P~1H C gm n ~ Lunitai/Oil.\ 111 de tee ling mycohcrcrotroplnc ca1 hon ~alit.\ and opt loll.\ (or fut ure re.H•orch There arc ~C\ cral a~pcct~ of ga tns that n1 a; red uce the apparent degree o f n1 yco- heterotroph} 111 P; tolcae. tncluclJng lc)\\ Inherent PS t a t e~. ~ea~o n a l c han gc~ tn C nutJtllonal strategies and the mtegratl\ e nature of 61~C stgna ture~ tn bulk leaf ll~sucs (c g. Matsuda ct al. 20 12: Hyn on et al 20 12: Morale~ et al 2013) Ftr~t. lov.' Inherent PS rcltcs of Pyrolcae n1 ay be rn a king the le\ cl of mycohctero trophtc ga tn ~. a~.., ~uggested by II ynson et al. (20 13 ), e1thcr 11 due to relati\ ely greater u e of C-dcpleted rcsptrcd C0 2 com pared to spccJcs wtth ht gher C0 2 needs. or itnpro\ ed equtltbrati on inC, to C,, (1 c . C,·Ca ratio~). resulting in grea ter di scrimination of 11 C Light response curve and est1n1ated net seaso nal C ga1 ns dtd show considerabl y 1o"Yver Rd in the Pyrol eae, par1icul arl y for 0 . secunda and P. chlorantha, indicating the first theory could be va lid (Fi gures 2-4 and 2-5 ): however, a detailed discussion of respiratory effects on 11 C dt criminati on is provided 111 Chapter 4. The potential effects of CI :Ca ratios on isotopic di scrin1inat1 on are also va ltd, but tnay not have long-term effects on differential di se ritninati on between the Pyro leae and reference autotrophs. The is because, although und er arnbient conditi on C, c . ratio~\\ ere generally sli ghtl y hi gher in the Pyrol eae cotnpared to autotrophs (Table 2-1 ). dunng li ght response curves th ey we re lowe r than or eq ual to tho e of autotrophs at I ight lc\ ~I<; < 400 prnol m . , s 1 (not shown) and should theoreti call y balance out\\ tth dtun1 al c h a n ge~ tn tiTcH.hance I het e arc also unknown errects of tnesoph y1l conductance rates and subsequent C0 2 concentration~ 44 at the ite o f carbox ylati on ( (. ). \\ l11ch could hen ca n 1nnuencc on 11 C dtscntninatt on as well a. photorc p1rat1 on (c g , Martin ~ ct al. 20 14) econd. photo yntheti c li ght le\·cls ( L ogat et al 2001) gat n ~ rn ay be h1 g.hcr 111 ~ pnng to take ach antagc of optltnal T ht ~ ~ tJ ate g) n1 ay occur 1n the C\- crg1ccn Pyroleac spcc1es ~ h ich could he Ip C\. pia tn the o m ettn1e~ signIfica nt clec1ca~c tn 6 11 \ al u e~ between June and eptetnber (Table 2- l ) For C\.an1pl c. \ 1atsud a ct al. (20 12) found funga l colont?atton \.\of total C) in 0 . .\ecunda and P chlorantha. placi ng both species in the range of c. 30°o to 36°o fungal C (i.e. 70°o to 64~o PS C). rnatching \cry \\Cll to correspondtng PS rates compared to autotrophs under the satne trcatn1ents. Thi could he a happy coincidence, but also alludes to the possibility that repea ted n1 ea~u res of PS O\ et titn e can revea l PMH strategies in species of interest. Analysis of Isotopes ts quite co~th. especiall y to obta in data for large sa n1ple sizes. and therefore n1ea~u1 cn1cnh of photosynthetic properti es tnay prov tdc a chea per alterna ti ve to estnna te PMH C gm n~ Fu tiher, gas-exc hange data n1ay help assess whether there nrc other spectcs pre\ tou~l\ 46 thought to be autotrophtc that actually obtain sorn c po111 on ofC' \ 1a Jn yconhl/as, or even the other\\ ay around W1th re pect to en\ 1romncntal factor" that Influence myco heterotrophtc C gmns, rc ult .. tnchcatcd that at least 0 'cc unda 1equ1rc . o1ne lc\ el of "hade as a n1 ca n ~ of protec tion from hi gh light Jnt c n ~ tty 111 adcl1ti on to pt O\ 1d1ng nece""a ry C fro n1 O\ crstory ho!-,t trees In \Veil-Itt forest , tn c rea~cd tnycoheterotroph) n1a) re~u It I ron1 n1ore light rathe1 than less to co n1pcn5ate for l os~ of photosyntheti c capacity via datnagc Though the data <;bowed hi gh variabtlJty lD p rate. and C11 C \ alue , there \\as photo"ynthellc e\ 1dencc of water lin1 1tatJOn ~ on autotrophic C nutriti on 10 the Pyroleac at least in the "hort ten11 , \v hi ch rnay hcl\ e increased PMH C ga 1n , but unfortunately could not be\ crified O\ cr the longer tenn wtth 11 bulk leaf tiss ue 8 C. It is uncertain whether the changes associated v. 1th over"tory pine tree loss significantl y in1pacted populati on at tht s stte, s1nce th e InttJal attack took place over a decade ago. Based on the een1ing sensi ti vity to light, particularly in 0. secunda, and possible unknown effects on 1nyconhiza l fungal comn1unities, 1t '"likely th at there v. ere negative effects on the Pyroleae species at thi s site. 47 2.5 Literature Cited .. Abadie, J-C, Ptittsepp U, Gebauer G, Facc1o , B o nf~111t c P, e l oc.;~c M-A 2006 Cephalan thera lon f!.l/oha ( eotll eae. Orchid aceae) ~ ~ n1Ixotrophtc a cornparatl\ c ~ tud y bet\veen green and nonphoto<;ynth ettc Jndl\ tdual Canadian Journal or Botany 84 I462 1477 dot. 10 1139 806- 101 Azc6n-Bicto, J. Os rn ond CB . 1981 Relati on hip betw een photosynthe~1s and resp1n1tion . 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"JY Spnngcr Sc tencc +- Bu ines Med1a, p 297-342 dor 10 1007 978- 1-46 14-5209-6 8 • I ogai, N, Yan1an1ura Y. Manko . akano T. 2003 Seasonal pattern of photosynthetic production in a ubalpine evergreen herb. l~lTola unarnata. Joun1al of Plant Resea rch 11 6 . 199- ?06. doi · l 0 1007 10265-003-00 -4 Johan on, VA. 20 14. Recruitment ecology and funga l interactlons tn rnycohcterotroph1c Ericaceae [Doctoral d isseriation co rnprchcnsive c.,u rnn1 ary]. S tockholin, Sweden: Stoc khol111 Uni versity. 46 p. Available at http:/lsu.diva-pot1al.org/srn ash/record .js f'?pid diva2%3A 767181 &dswid=3570. Johansson, VA, Mikusinska A, Ekblad A, Etiksso n 0 2015. Patilal n1ycoheterotrophy 111 Pyro leae: nitrogen and ca rbon tab le isotope ignatures during devclopn1cnt fro1n seedl tng to adult. Oecologia 177: 203-2 11. doi : l 0. 1007/s00442-0 14-3 137-x Julou. T, Burghardt B, Gebauer G, Bcveiller D, D a1n e~in C, Se l os~c M-A. 2005. Mixotroph) in orchids: insight~ frotn a cotnparati\ c tud y of green indi\ idual and nonphotos)nthettc individuals of Cephalanthera d(llllU.\oniuni. ew Phytologi t 166: 639 653. doi : 10.1111 /j .1469-8 137.2005.0 1364.x Kri ede1nann PE, editor. 1999. Chapter 12: Sunli ght: an all pervasive source of energy. In. Atwell , BJ, Krieden1ann PE, Turnbull CGN, editors. Plants in Action Adaptation to ature. Perfonnancc in Cul ti vation. 1sl Ed ition. Melbourne. Australia: Mactn tll an Education Austra lia Pty Ltd. Accessed 23 .Jan 201 5 fron1 : http.//plantsinact ion.science uq.cdu .au edition 1 '? q content. chapter- 12 ~unlight-all. pervasive-source-energy Leake, .JR 1994. The biology of n1 yco-heterotrophtc ('saprophytic') plant~ Fan, /c.\' R( l'iew No 69. New Phytologist 127: I 7 1 2 16. doi : 10.111 L.J 1469- 137 1994 tb042 72 "' 50 Martin , V. Gahrtc, J. Cavatte PC. Pereira L F. Ventrcll a M . DaMatta FM 20 14 nderstandin g th e low photosynth ett c rate of un and ~ lu1d c coffee lea ves bridg1ng th e gap on the relati ve roles of hydraul1 c. ddTu ~ l\ c and btochen11cal co nstra1nt~ to ph o tosynth e~ J s PLo E 9· e9557 1 doi. I 0 137 1 JO Utl1al pone 009)57 I Mas 1cotte. HB. Peter. on RL. \!1 eh til e Lll. uotn a DI 20 12 Chapter 5 Btology o f mycohetcrotrophtc and n1txotropluc plant In ou th \\ orth. D. edt tor Bt ocon1plex tty of Plant-Fungal Interacti on 1' 1 Fdttton John Wd e) & So n~ Inc. p 109 110. doi: 10 1002 978 111 83 14364 MaL uda. 't'. htn1t/ U . ~1 o n \1. Ito -I. elo~~e \1 - \ 2012 ea~o n al and en\ 1ronn1ental change of n1ycorrh11al as octa t to n ~ and heterotroph) le\cb tn n1P,otr ophtc P1·ro/a JOf UJ il lca (Eri caceae) gro\\ tng und er dtfferent light ern rro n n1c nt ~ Arncn can Journ al of Botany 99 1177- 1 l . do i · l 0.3732 aj b. l 100 54 6 Merck-x. V, Btdartondo M L H) n ~o n ,\ 2009 M yeo- heterotroph y \\ hen fung1 h o~ t pl a nt~ nnal of Botany 104 125 ~-126 1 dot: 10. I 09 3 aoh n1cp2 35 Mikkelson, KM. Maxv. ell RM. Fc rgu ~o n I. Stcd nt ck .J D, \1cCray J E. Sharp JO . 20 11 Mountain pine beetle infestation itnpact · modeling water and energy budgets at th e hdl slope cale. Eco hydrology 6: 64-72. doi: 10. 1002 ceo 278 • Miya hita. K, Tanakamaru S. Ma1tani T. Ki1n ura K 2005 . Reco\ ery re~ p o n scs of photo ynthe i , tran piration, and ton1ata l conductance tn kidney bea n fo llowing drought stress. Enviromnental and Experitnental Botany 53· 205-2 14. doL 10 1016/j .envex pbot 2004.03.0 15 Morales, CG, Pi no MT. del PoLo A. 20 13. Phenologica l and physiological rcspon~c~ to drought tress and ub equent rehydratio n cycles in t\\ o ra pben-y cultt \ ars. Scientia Horticulturae 162 : 234-241. doi: 10.1016j . cienta 20 13.07.025 Preiss, K, Gebauer G. 2008. A n1ethodological approach to improve cstin1ates of nu tn cnt ga ins by partiall y rn yco-heterotrophic plants. Isotopes in Environm ental and Health Studt es 44: 393-40 l . doi: I 0.1080/ 102560 10802507458 Preiss, K, Adarn IKU, Gebauer G. 20 10. Irradi ance go' erns exploitati on of fungi · fine-tuning of ca rbon gain by two partially n1yco-heterotrophic orchids. Procce d t n g~ of the Ro;al octet)' 8 : Biological Sciences 277: 1333- 1336. doi: 10. 1098/rspb.2009.1966 Prioul, JL, Charti er P. 1977. Pat1itioning of tran fer or ca rboxylati on C0111ponents or tntrace llular rcs i ~ ta nce to photosynt hetic C0 2 fi xa ti on· a critical analyst" or the n1cthod~ u"ed Annals of Botany 4 1· 789- 800. Rex, J, Dubc S. Foord V. 20 13. Mountain pine beetl es, sa h age loggtng. and hyclrologtc change: predicting wet ground areas. Water 5: 443-46 1. dot: I 0 3390/\\ 5020443 51 Selos c, M-A, Ri chard F. He X. unard \V 2006 Mycorrhi1al net\\ ork<; de\ luuson,· dan~ereu\e5? Trends tn Eco logy and E' olutton 21 621 628 dot l 0 I 016 J tree 2006 07 001 Souza, RP. Machado EC, dva JAB, Lag6a AMMA., tl vetra lACi 2004 Photosyntheti c gas exchange, chlorophyll Ouore"ccncc and ~o n1 c assoctated rnetabolt c ch8ngcc;; in cow pea (l 'l ~lutun ~Luculata) dunng \\aler " trcs~ and reco,eJ} l·n,tronn1 ental and [ \per11nental Botany 51 45- 56 do1. I 0 10 I 6 , 009 -R472( 03 )000)9- ') Tedcrsoo, L, Pellet P. K6lj8lg L'. Se l os~e M- \ 2007 Pat allel C \ olutionary paths to myco heterotroph y tn und er tot C} Encaccac and Ot cl11daceae ecologtcal e' tdencc for n1i~otrophy tn r, I olcac. Occologta 151 206 2 17 dot 10 1007 s00442-006-058 l-2 Te ~ itcl, J. Leps J. Yn1blo' a M. Can1cron DO 20 11. 'I he role of hcterotrophtc carbon acqut tlton by the hen11parastttc plant Rlun(llllhu' ah(/orolophu' 111 ~eedltn g e~ tabli s hn1 cnt tn natural con1muntt1cs a ph y. tologtcal pc r~pec tt\ e , e\\ Ph yto l og t ~ t 192. 188 199 dot 10.1111 j . J469-8 13 7 2011.03777 .x ttrogcn and ca rbon <;table 1"otope TrudelL A. Ryg te\\ tel PT. Edrnond RL 2003 abundance up port th e tnyco-hcterotroph1c nature and host-spec t fi e tty of cet1atn achlorophyllou plants. ew Phytologist 160: 39 1-401 . doi: I 0. 1046 j . l469813 7.2003 .00876.x • TrudelL A. Rygtcwicz PT. Edrnond RL 2004. P attcn1 ~ of nitrogen and ca rbon stabl e isotope ratios in n1acrofun gi, plant and so ib tn two old-growth conifer forests. New Phytologist 164 :3 17- 335. doi : 10.1111 j .l469-8 137 2004.01J62 .x Willis, AJ, Bala ubramanian1 . 1968. Stornatal beha" tour in relation to rates of photosynthe is and transpiration in Pelargonnun. New Phytojo gi~ t 67 : 265 285 . Xu. Z. Zhou G and himizu H. 2010. Plant respon es to drought and re\\ atenng Pl ant Signaling and Behavior 5-6. 649-654. do1 : I 0.4161 psb 5.6.11398 Zitntncr, K, Hynson NA, Gebauer G. Allen EB, Allen MF, Read OJ 2007. Wide geographi caJ and ecological di ~ tribution of nitrogen and ca rbon gmns frotn fungt in pyrolotd~ and tnonotropoids (Ericaceae) and in orchids. ew Phytologist 17 5 166- 17 5 dot : 10. llll /j . l469-8 13 7.2007.02065 .x 52 upplcrncntary Table 2- 1 1ean (+ I [~ ) ~caso n a l gas-exchange parmnctcrs for autotrophtc refc1cncc spcc tcs and three putatn c part wl 111 ) cohctcrotrophic Pyrolcac ~ rcc J cs undct field Jn alllpulatt on trcat1nent (n 36). a~ \\ e ll as full ) n1yco hctcrotrophtc tcfcrcncc ~ p cc t cs (n 4 . not tncludcd tn trca trnenL) ----~---------------------------------------------- pcc tcs Treatn1cnt P, fra n ~ ptt a tJ o n c.. Co nducta nce .., il D ( 1111110 l II 20 "' ' , ( ~.un o I 111 s ) 111 s I ) 5.50 ± 0 14 2 41 1 0 18 5 27 ± 0 54 1 X1 ~ 0 28 2 92 1 () ) 8 4 08 + 0 42 V..' 3 13 !- 010 2 9 1 4 : 0 24 0 16 + 00 1 0 85 l 0 02 Ch unaplnla LD 4 70 ± 0 41 27 1 ± 019 0. 14 . 0 0 I 08 1 ± 00 1 umhellata LW 4 6 + 0 18 3 44 ± 0 28 0 18 ± 0 02 0 82 1 0 02 0 3.23 ± 0 36 2 52 ± 0 I 6 013 ± 00 1 () 84 ± 0.0 1 w 323 ± 037 3 26 J: 0.34 0. 18 :L 0 02 0 87 l 0 03 LD LW I .79 ± 0.15 D 2.48 .1. 0.26 2.27 ± 0.27 2.54 ± 0 28 2 28 ± 0.20 2 08 + 0 12 0. 10 ± 0.0 1 0.12 ± 0.0 1 0.12 ± 00 1 0 82 ± 0.02 0 87 J:: 0.0 1 0 85 1: () 02 sw 1.6? ± 0.26 2 24 ± 0.2 1 0.12 ± 001 089 J: 00 1 P1ro/a LD chlorantha LW sw 3.06 :I: 0.33 2.94 ± 0.28 1.68 ± 0.23 2.26 ± 0 32 2 11 ± 0 15 287 ± 0 16 2.26 ± 0.23 3.02 ± 0.27 0.12 ± 0 0 1 0.16 ± 0 0 1 0. 15 ± 0 03 0.17 ± 0.02 0 83 + 0 02 0 87 ± 0 01 0 87 ± 0.02 088 -.- 00 J NA -0.80 ± 0.09 0.76 ± 0.37 0.03 ± 0.0 I 1.1 4 ± 0.05 I Autotrophs LD LW Ortlulia secunda SD Mycohctcrotrophs 51 (ongu1 al units: (11101 1120 111 s ) ? ~t111 o l 111 S I) 0 17 ± 0 0) () 75 1 0.02 0 81 1 () 02 0 2 1 :t 0 02 0 82 ~ () 02 0 18 + 0 01 I Supplcinentary Tabl e 2-2. Growing sea on 8 each species of autotrophic references 13 ,% ' 8 I c; 8 1'c M ea n so Mean Ah1es lasiocaJ]Ja A mela ncln er a hnfo!Ja AjJO() '11 u 111 androsaemJfol!unl -30.4 -3 1 2 0.6 49.4 45 0 -30 9 -30 7 -31 6 -29. -29 2 -30 8 -29.5 02 10 -30.8 -30.5 -29.9 -30.4 -32.0 -28.8 -32 .9 -30.8 0.4 0.7 Arctostaphylos ll\'a-ur,\1 Epi loh1u 111 angustJ(oh un1 Geocaulon ln ·idum 1-fleracnun alhzflorum Loniccra inl'olucrata Melampyrum lineare Picea glauca x. engehna nn ii • Pinus cantor/a Rosa aciculari Smilacina racen1osa 8 I c;N o1>C Species and %N and san1p lc size (n) of 0 1. 2 45 8 51 9 45 5 44 8 44 6 45 2 4L6 06 15 48.7 49.7 45 .3 42.9 44.1 45.5 47.7 48 .8 l.l 0.5 %N Mean so Mean so ll -4 3 -2.7 0.9 10 1.3 0.2 17 -2 .6 -1.5 -3 I -3 8 -3 .8 -6 5 -3 .3 03 1] 30 0.2 0. 1 -3 7 -4.6 -2.9 -3 5 -4.8 -2.7 -0. 6 - 1. 8 0.6 1.0 I I ] 2.3 2.4 2.8 1.8 2.0 0.6 l.O 2.6 1.4 2.6 29 2. 1 1.6 4 29 2 l 2 2 l 0.2 0.2 5 10 I 2 5 Solidago spathulata 1.2 0.9 1.0 0.4 ] Sorbus . copulina Vaccinium caespitosznn 1 Vaccinium rnyrti l!oides I .0 1.0 0.9 0.4 17 Sampl es were collected primaril y in June and Septetnber for analysis and con1parison to Pyroleae spec ies for each treatment. but included some sa1nples coll ected in July and August. 54 3. Effect of alvage harve. ting of lodgepole pine-don1inated fore t foliO\\'ing mountain pine beetle di turbance on den ity and stable carbon and nitrogen i s otope ~ of partial m) cohcterotrophic Pyroleae pecie ( ricaceae) in central Briti h olun1bia. Ab tract J u t O\ er a decade ago, an unpt eceden ted moun tat n ptnc beetl e (f)uulrrn tonu\ pondenH a e ) outbreak began that affected O\er 18 n1tllion ha ofp tne-leading fo re~ t~ tn Bnlish Colutnb.a . Increased al\agc han e ting ac tt vtlles have resu lted 111 tnany clearcu t areas where forests ~ ould othcn\ i ~e ha\ e rema1ncd rela tl\ ely 1ntac t M ycoheterotropht c. ( MI I) plant c.,, Vv ht ch gai n all or on1 e ca rbon (C) tndtrectly frotn ho t autotrophic pl ants v1a ~ h a red rn ycotThl/al networks, may be negati vely affected by uch large-sca le di sturbances. This study assessed putati\'e parttal MH Pyroleae pecie (Ericaceae) populations and ~ta hl e 13 C and I" ISOtopes between fore t Interi ors, edge and sa h age h a rvc~ ted clearcuts along transects perpcndt cul ar to the tree line at three sites in central BC. Average Pyroleae de n s iti e~ for all sites and species con1bined ~ ere highe t in forest at more 4 3 tndi\ idual 2 111 - , \\ Ith ahnost 9 1 o of that ° along edges, whereas clearcuts only had approxin1ately 30°o of forest density. D e~ptt c lo\ver densities in clcarcuts, increased 11 C enrichment along forest edges and/or clea rcuts co tnpared to forests in many cases ind icated po itive autotrophi c re pon es to tncrca ed light The exception was P1ro/a chlorantha, found only at the dry nutri ent poor ~ itc, \\ ith lo\\ cr 6 C b; 11 1.8%o in the clca rcut COinparcd to the forest. This tnay be a sign or a loss or MH c nutntton in c l earcuts~ how ever, limited Salnpl es precluded anal y~ i s or cJea rcut I SO tOp e for thl\ species. /~pro/a chlorantha showed cons1 tent I; hi gh 6 1" \a lues a\ ct aging at lcd\t ") 79oo higher than the other three species, but cll l spec tes had at least one 1ndt\ tdu al wtth htgh l} enriched 11 N. Also at the dry stte, Clu maplula u JJihella Ia and Ortlu /ia H 'Ulltda had 55 ignificantl) hi gher 8 1 ~ in clcarcuts. tndtcatlng a hr gh reliance on rnycorrhvas for N nutrition, or potentially change" 111 source or fo rn1s of Though ntunerou s C. uiJihcllata and 0 . . ecunda chd occur tn clearcuts at all (\ lt c~~ tho\e 1ndl\ rdu a l ~ ''er e \ In alL chi or ollc and usually loca ted nca r rcsrdual trees or shrubs. Th e al!nost con1plcte dtsa ppea rance of P ch/orantha and lack of\ rgour and abundance of the other Pyroleae tn clcarcut<; unplt es that the e .. pcc 1es suffer fron1 llght-tnduccd <:;tre~~ an d potentially lo\\ or altered nutntt on. Thi gudd of planb doc not respond \\ell to clea1cut harvesllng. but n1a y be bettct su1tecl to 111anagement strateg1e uch as parti al harvesting and preservati on of ~t ru ct ura l di verSity, rc idual -vegetation. and connectJ\ 1ty to the tTI )CCll al net\\ ork or re~tdual plants 3.1 Introdu cti on • Mrcorrlu::.a/ vvmh1osJ and 111) cohclcrotrophy Myconhi7al symbioses, association bet\\ een terrestrial plant roob and fungal symbionts, have long been ob erved to be ubiquitous and critical to the ecology of a n1aJOiity of plant species globally (S mith and Read 2008). Generally. these ) ll1b i o~es are thought or as tnutualisti c~ plants ga in 1nineral nutri ents and water through fungal pa11ners, \.\. hrch tn tun1 ga in carbon (C) throu gh transfer or photosynthatcs fron1 the host plant (Molina et al. 1992~ Merckx 20 13) There are a nun1bcr of plant fan1ilies with Inetnbcrs kno\\ n to unden111ne this tnutualism, ex ploiting the relationship without rec iprocating the nutn cnt exchange. 'I he\c plants, tenncd rn ycoheterotrophs (M H ), ga in all or pat1 of their C requirctncnt~ 'ia organic fungal sources, originating fron1 ovcrstory host trees and sutToundtng autott oph tc pl,H1h \11 Mils obtain organic C in their genntnant stage, and tnay either becon1e full) autotrophtc ,h adults, or contin ue to gain son1c or all of th e C rcquiren1 ents throughout thctr It\ es 111 the sa n1c fash ton (Leake 1994) The f~Hntl y Ericaceae contatns fully Mil specte\ 1n the 56 ubfan1i ly Monotropo1dcac that con1plctcl y lack chloroph yll and ren1a1n dependent on 1nycorrhual C gm ns throu ghout thctr 11\ c~. a well as putatl\ c partial rn yco hetcrotrophs (PMH ) 1n the tnbc Pyro lcac that are pho to ~ynth cti c at n1atunty but arc thou ght to obtai n a pot1ton ofrequ1rcd C fron1 111]COb1onts ( l ca ke 1994 . Tcdcr5oo ct al 2007) In addJtlon to C, high rcla t1 \ c nttrogcn ( J) con tent (0 o) and 1. unpll c"> these fun g1 arc extre1nely 1111portant In th e ecology o f MHs. EY1dcnce for MH nutntt onal strategic<:, tn pl anh has pnn1anl y heen show n th rough th e use of natural abu nd ance of . table 1"\C and 1c;N t<:,otope<:, Oi <:,cnn1inat1on aga inst heavy i otope is con1n1on in biolog1cal and physiological processes, and generally show 1n c rca~ed level of heavy isotope (enrichn1ent) wtth 1nc rea~ in g troph1c level (Fa rquhar et al. 19X9: • Da\\ on et al. 2002). The itndant1 c betv.. een ECM ~p orocarp t<:,otopc ~ i gnatures and full MHs have prov 1ded e1npirical ev1dcnce that the. e pl ants ga in nutri ent<:, via tnyco rThi /.a l networks (Trudell et al. 2003 ). Typicall y autotrophic (includi ng ECM) plant species arc hi ghl y depleted in 813 C, full MH species are the rn o~t enriched, and PMH specie fall sotnewhere in between and indicate the tnixed acquiSi tion of C through both autotroph y and 1nycoheterotrophy (see Figure 1-2 for the range o r repo rted 8 11 C va lues in each trophtc group. as per Hynso n ct al. 20 12) . . irnilar trend~ occ ur for 8 1<; \\ ith autotrophic ~pcc1e~ having the lowest values and full Ml Is being n1orc cnn ched by as rnuch a~ 10°oo or n1 ore 11 (e.g., Trudell et al. 2003 ). Conlra ry to 8 C va lues. 8 1c;N va lues vary by sitc and soi l nu trient ~ ta t us. and likely plant and funga l N n1etaboli sn1 and fra cti ona tion (c g .. Taylor et al 2001. Hogberg et al. 1999). Additi ona l! ). 8 "N va lues ofautotroph1c and P 111 plant tl~~ue~ n1<1y 1 be either positive or nega tive con1parcd to internati onal stand ards rat her than ah\ av~ e . ne!.!,att\ .... 58 1 like 8 13C, with our Pyroleae tudy species arc being signifi cantl y enri ched in "N cotnparcd to ECM autotroph in th e arne enviromnent (e g., Tedcrsoo et al. 2007: Zitntncr ct al. 2007). Effect of disturhance on nzycoheterotroph populations Natural di turbance regin1es of the interior region of British Colun1bia have histori cally been cau ·cd by t\\0 prcvatling sources.\\ ildfirc and beetl e tnfestati ons (Safranyik and Wil on 2006). In~ cct di turbancc . \\hi le known to a ffcct a n1uch grea ter area than fires at the regional or bion1e level. have different characteri stics that generali y result in lower impact on ecosy tern a a whole b eca u ~ c onl y elect species or age classes are affec ted, therefore leaving more biological legacies and intact ecological fu nctions in a given forest stand (CCFM 2006). The current rnountain pine beetl e (MPB : Dendroctonus ponderosae) epidemic has affected approxitnately 18 rnilli on ha of land and killed approxitnately 710 million m3 of titnber in BC (BC MFL RO 20 13). The surge of increased salvage harvestin g in response to the epidemic is of greatest concern. exacerbating the generall y lower impacts of insect di sturbances and potentially creating cascading effects that co uld lead to regional declines of MH plants. There are a nurnber of different factors related to MPB disturbance and sub equcnt silviculture and harvesting activi ties that could influence MH populations, including change to nutrient status and fluxes, soil di sturbance levels, or suitab le n1icrosites related to tnoi ture and light environments. In most cases, where stands are tnixed or canopy n1011ality is not 100% (as in rnost MPB-a ttackcd stand s), ecologica l linkage can rctnain relatively intact Yet Haeussler et al. (2007) found total MH species richness declined significantly C\ en with only 113 partial cut harves ting in boreal Jnixedwood forests in not1hen1 Ontano and Quebec, 59 with total species riclme and frequency positl\ ely a ociated with lower levels of large conifers and coarse woody debris (presumably related to suitable tni crosites). Loss of ca nopy trees n1ay also redu ce C nuxcs to le' cJs unabl e to suppori ceria in ECM funga l species. For example, Hobbie and Agerer (20 10) found that fungi with high biorna s exploration type \Vere n1ore pre\ alent at t\VO nutn cnt- (nitrogen) lunited sites, whi le lower biornass exploration-type fung1 dotnlnated at th e tVvo less lirnited s1tes. The authors attributed the fun ga l explorat1 on types, and thus biomass requ iretn cnts, to their need to explore long di tance for patchy nutrient resources: often th e fungi fonn specialized hydrophobic rhizornorphs for effictent nutri ent transport (Taylor et al 2003, Hobbie ct al. 2009: Hobbie and Agerer 20 l 0). A greater proporti on of pri1nary productivi ty is th erefore allocated below ground under nutrient poor conditions (Hobbie and Agerer 201 0), and in central BC, MPB-attacked pine-dominated stands frequently occup y nutrient poor sites (DeLong et al. 1993, and see Section 3.2 for site descriptions). High biomass fu ngi are known to facilitate organic N release by proteolytic enzytnes, al lowing direct uptake of amino acids and oligopeptides that are assumed to be preferentially assi1nilated by MHs (Hobbie et al. 2009~ Kranabetter and MacKenzie 20 10~ Hobbie and Hogberg 2012 ). The ful l MHs, M onotropa hypopitys L. and Pterospora andro1nedea Nutt., are both assoc iated w1th high biomass fungi in the genera Tricho/oma and Rhi::opogon, respectively (Bidartondo and Bruns 2005 ; Hobbie and Agerer 201 0). As urnin g that MPB-associated rno11ali ty would reduce primary productivity at the stand level, we would expect to see decline In C allocation to associated funga l symbionts that could ulti1nately restri ct the presence of at least full MI-ls. Even if adequate C flow s arc maintained following n1ortahty, ECM spec1es composition may chan ge from pine-specific hosts to other spec ies not cotnrnonly as . . octatcd 60 with MH . In one tud y, artificial defoliati on of lodgepole pine (Pinus contort a var. lati(olia) in a tnixed canopy with Engelmann pruce (P1cea enge!Jnannn) caused a decline 111 the dotninant pine-a ociated lnocvhc. and a dcc rea e 111 the rati o of p1ne to spruce ECM sytnbi ont (Cullings et al. 200 I ). lnocThe has been found to assoc iate with Pvrola chlorantha \V .• eedhng (Hyn on et al 20 13), vduch means the potential i1npact o f shifts in funga l pecics on full Ml l di cussed abo\ e tnay also i1npact Pyroleae species to a certain degree. imil arly, Johans on (20 14) fo und the gctminat1on and recru itrnent of Chin1aph ila umbel/ala (L.) W.P.C Barton and P. chloranlha tn a dry Scots pine (Pinus sylvestns) forest in Sweden were po iti vely affected by ECM divers1ty, and was negati vely affected by increased soil nutri ent tatu . The latter has been attributed to the tendency for EC M fungi and plants as well as arbutoid and cricoid pl ant species to decline and arbu scular tn yco rrhiza to ri se in prevalence with increasing so il prod uctivity (i.e. levels), as shown by Kranabetter and MacKenzie (20 10). Increasing disturbance levels by clearcutting and slashburning n1ay have 1nore substantial and long-term effects on MH populatjons. Three years after di stu rbance, only chl orotic individuals of Pyrola [syn: Orth ilia ] secunda occurred on unburned clearcuts, and no species were found in burned clearcuts in the study by Haeussler ct al. (2007). During continuous surveys over 24 years following clearcuttin g with and without burn1ng in the West Cascade Mountains of Oregon, Halpen1 and Spies ( 1995) ob erved local extinctions of C. umbel/a/a , Py rola asari(olia Mi chx. and Pyrola pic/a, though eac h species dtd hO\\t surviva l or reestablishn1ent in at lea tone treattnent/site con1bination, n1ainl y unbun1ed In the satne region, Schoonmaker and McKee ( 1988) observed the eradi cati on of two full MHs, Corallorhi=a mertensiana and P. andron1cdea dunng the first 40 years after logging and burning. Ne ither of these studi es speciall y addressed MH population or causes for declines, 61 but a nun1bcr of possible influence on the lack of rccc)\ cry\\ ere c!J ~c u sscd These include ensitt vtty of C umhc /lata to fire and ltn1ttallon to sun tval and rec nllttncnt \\hen di turbanccs arc not as patchy a~ natural fi 1e~. Intolerance to }ugh I ight level<; (llalpcrn and p1 c~ 1995). and dependence on rccolonllatJon of the ~ tl e by co tnpat1ble n1yco rrht za l fun gt ( choon1naker and McKee 198R) Any one of the afo rcn1 ent1oncd factot ~ tnay co ntn butc to dcclt ncs or eve n ex tirpation of fully MH p ccic~. howe, cr. it 15 l cs~ cc11atn '"' hat the effec t of the current MPB outbreak will be on putatl' c PMH sp cctc~ of Pyrolcac. particularly \\hen the ·natural· di~turbancc i\ follow ed by clearcut harvesting and or s l a~h or site burning. It ~ ~especia ll y 1111portant regionally due to the tncrea e tn han e t ac tt\ 1t1es (to rnttigatc ccono1ntc l o~ses), and th e in1plica tion of uch a large and co1npound cd tncrcascs In disturbance across the landscape Thi chapter focu ed on a e sing populat tons of putatt\ e PMH Pyro lcae spcc tcs in sites that had varying level of rnortality fo llowing MPB attack While full MH indi vi duals were also of interest if obsen, ed during surveys, the pri1nary target species \\ere the Pyrolcae. Specifi cally, populations were co1npared between forest, edge and clearcut habi tats to detennine whether there \Vere Significant changes in abundance due to han esting practices Additionall y, sa1nplcs were collected fo r isotopic analys is to detennine ho'v C and N nutrition were affected fo llowing disturbances. 3.2 Materials and Methods Site descnplions The stud y took place 111 n1id-Jul y to n11d- ugust of 2013 at three "llc~ locdtcd ncdr the comn1unity of Bea r Lake, Bntish C'ol un1bia. approx1n1atdy 70 kn1 not1h of Pnncc George The ~ tud y sttes were fo rest stand s that had all been hnn c~ted bct\\ccn 2005 tttH.l 200X. t\\ o to 62 five years after MPB attack. The MPB eptdetntc first started 111 the general area in 2001 , ultitnatel y resulting in up to 95°o lo()S of O\erstory lodgepole p1nc (Punt\ contorta Douglas ex Loudon\ ar. lafl/olia Engc hn C'\ \Vatson) 1 he <;., Jtcs were abo c ho ~c n for the pre~en ce of Pyroleae ~ p ec 1 e~ in the Intact fot ec;.,ts sunoundtn g the clea rcut harv es ted areas All three stte \vere ~ 1thu1 the n101St coo l sub;onc of ub-Botea l Spruce ;one (SBStnkl) acco1din g to the biogeocl unatt c ceo ) stern c l<.l c;.,~ dl ca tl o n ( B I ·) c;.,: "tern fo r Bnt ish C o Iurn h ia (Me 1d 111gc r and Pojar 199 L BC MFL RO 20 14 ). The O\ e1all area contatntng the ~ Jtes is characten zed by n1ainl y gra\'ell y glacio ilu\ 1al parent matenal c, tn a\ an ety of landfonns (e.g. eskers, ten·ace : BC MOE 19 9) . H arve~lln g h1 tory. tand con1po ition and ~1 t c sen es tnfon11at1on regardin g the stud y ite were provided by Lee Evan , planning fore ster of BC Tin1ber Sa les (pers. cointn ). The • fir t ite (DM Moi t) \\a located at approxima tely 54 26 ' 15 11 II • 122 18 · ') 1 W on the Davie-Muskeg Forest Serv ice Road Though the tnaJonty of the clea rcut area was Vv tthtn the SBSn1k 1-05 site seri es, the surveyed area around the cutblock boundary bordered either a stnall wetland or tran itioned into the Crooked Rt\ Cr ripatian zone, possibl y at the n101st end of the 06 site seri es (nutri ent poor, ubhygric to hygn c: DeLong et al. 1993 ). The pre~e n cc of standing water along the deacti va ted road indica ted so ds of the Stellako a~sociation rather than the Ra1nsay. whi ch are flu vial regosols w1th thin orga nic layer~ or gleysolic so d ~\\ tth hi gh water tabl es (BC MOE 1989). The clearcut area \va~ han e~tecl in 2007, on ginall) having about 65% lodgepole pine and 30% hybrid \Vhite spruce (P1cea glauco (Moench) Voss X cnge/nut/11111 Parry ex. Engcln1. ). The res tdual forest had a grea ter COI11poncnt or black spruce (P1 cea manana Bntton, terns & Poggenh ) and subalpine fir (Ahzc, fa,,o, arpa (l I ook.) N utt. ), with n1any sn1a II suppressed firs also rcn1 a ining in the clcarcut 111 c1d d tt ton to planted lodgepole pine and hybtl<.l whtte ~pru cc 61 The seco nd 5ite (DL Mcsrc) ~a located at about 54°3 1' . 122°42' W along an unnarned access road to Dav1e l akc JU t und er 5 kn1 fron1 the Bear l ake store. No ~ 1t c seri es Information \\as a\ at! able for th1 "' <.; rte but based on the presence of Douglas fir (P..,eudot\·uga 1nen:::1esu (M1rb) Franco ssp ~lauca ( B e1 n) A E MurTay) along v. 1th a van ety of <.; hrubs typi cal of ubn1es1c to rnesic. tte~. the nHIJOnty o f the area fell w1thm dry to rncsic s1tc seri es of medium to n ch nutnent ( e g . . _ B n1k 1-0 l. 04 or 0~. DeLong ct al. 1991 ). So d ~ -were probably brun1soltc or hu1no-ferne pod;ols (Paul Sanborn. pers cornrn ) The sJtc was con1posed of 2°o lodgepole ptnc and I 0°o D o u g la ~ fir pn or to harvc~ t rn 2006, but si rnil ar to the fir t Ite. the forest left out~ 1d c the cutbl oc k \s.. as co n1pn ed of a ~lightl y less prnedorninated pcc1e n11x, includ1ng a large con1ponent of ~ ub alpin e fir and hybrid wh1tc pruce. Tran ects were spaced the farthe t apart at th1 s Site rangi ng fi·oJn a dry ridge with a very thick hrub layer, a n1id- lope area \\'ith a dark, ~ p ar e undcr<.;tory to a lo-wer lytng mo1ster area. The third site (PM Dry) wa located at c. 54°30'37" N. 1'2°4 1'30" W, about 500 tn from the north we t co rner of the Polar Milllurnber ya rd. The srte \\as \cry distinctly of the glaciofluvial Bear Lake as ociation, characterized by rapidl y drained loatny sand or fine 5and and often occu rTing in rare dune cotnpl exes (BC MO E 1989). The site was likely in the SBSrnk l -03 site cries, subxeric and nutrient poor (DeLong et al. 1993 ). The poor nutncnt status suppor1ed litnited vegetation, primarily don1inatcd by lod gepol e prnc (up to 95° o killed) with a cornponent of young to rnature subalpi ne fir and srnal l hybrid whtte sp ru ce. The site was harvested sometin1c bet\veen 2005 and 200 , though ~orne portton of the southern clcarcut area n1ay have been harvested pno t to 2002 a part of the sa\\ rnill operati ons, as there was a srn all c1 In1atc stat ion set up on route to the first tr tlnsect. R,lther than th e clcarcut bctng centered tnside rc idual forests a~ at the other "Ite~. this "tte had 64 clearcuts bord ering a re idual stand. One transect was sarnplcd to the no11h rather than the south of the fore t, with a greater hrub con1ponent and a cluster of trernbling aspen (Populus trenndoide · Michx.) ju t beyond th e end of the transect. The under tory tree at all 1tes v. ere reOceti ve of the rn ix of the overstory species present in adj acent re idual forest , \Vith a lower p1ne co rn poncnt at DM Moist based on the wetter, black pruce don1inant areas. , malL suppressed non-eco notnic subalpine fir trees were oft en left wi thin clea rcuts, espec tally at DM Mo ist. and at OL Mesic where a small scattered population of mature Douglas fir \Vere also retained. Cornma n shrubs and forbs species for each ite can be found in th e Land Management Handbooks fo r th e site seri es listed above (DeLong et al. 1993 ), but all sites tended to be do1ni nated by ericaceous shrubs and herbs (e.g., Vaccinium spp. ), alder (Alnus spp. ), wi llow (Salix spp.) and prickly rose (Rosa acicularis Lindl. ). The hrub layer was often in dense patches at OM Moist and DL Mesic, while cant at PM Dry: the latter site had a substantial reindeer lichen layer (i.e., Cladonia/Cladina ). 0 f the target Pyroleae, on] y Ch inutpl11la znnhella Ia and Ortln Ita .\ ecunda (L.) House occurred consistently at alJ three sites. At PM Dry, the only other species were Py ro/a chlorantha and the full y MH Pterospora andron1edea, whil e at OL Mesic, Pyrola asar~(olia and the fully MH Monotropa hJ??Opitys were found, and both Pyrola specie were found at DM Moist (though P. chloran tha did not occur within the sa n1pling plots). Jvfoneses uniflora A. Gray was also fo und at both OM Moist and DL Mesic. The t\VO fu ll MHs were sparse and did not fall within satnp le plots. Survey data and san1ple collection At each site, once Pyroleac were detec ted in the un-han ested forest, three transects per site were selected as per the fo lJ owing three criteria: a) forest int eriors that C\.tcnded n1orc 65 than 50 m a a unifotm fore t type (i.e., not tncrging into a riparian zone or drainage channel): b) tree lines that \\ ere abrupt enough to be co ns idered a di stinct ~e d ge' fo r vegetation transitions, and : c) a\ oided highl y disturbed areas (i e, access roads or skid trail s). Survey 1n ethods were xnodifi ed fron1 el on and Halpern (2005 ). Transects of 90 tn were laid out approximately perpendtcul ar to th e harve t boundary, with the 0 n1 start point inside the forest so the re ul ting tnidpoi nt of 45 111 lay at the bound ary (F igu re 3- I ). Each transect was stratified into three egn1 ent con tdered trea tm ent groups of forest (0 m to 30 m), edge (30 tn to 60 m) and clearcut (60 1n to 90 111). Eighteen bands were esta bl ished at 5 111 interval along each tran ect (not includi ng th e 0 m start point to keep a balanced design), with six band per egn1ent and eac h band consisting of three 1 n1 2 plots placed adjacentl y and perpendicular to the transect length. --- .._T]~ Forest 30m Sm - lm Edge 30m Clearcut a} 30m b) Figure 3-1. Schen1atic di agratn of n1ycoheterotroph fi eld surveys. a) Transects of 90 111 ran approximately perpend icular to harvest bound ary, with each transect split into three 30 n1 segtnents of forest, edge and clearcut. Six bands per portion were establ ished at 5 111 intcr. al along transect, for a total 18 bands per transect. b) Each band wa cotnpri cd of tlu·ee 2 adjacent 1 1n plots fo r san1pling Pyroleae. Indi vidual steins were taJlied for each Pyroleae species encountered in plots Satnples were counted as an indi vidual where the aboveg round po t1 ion was connected to a single sten1 leading to the underground rhizo n1e. At each site, at least three indi\ tduals of eac h spec tcs per scgn1ent type (i.e., forest, edge and clca rcut) were collected fo r natural abundance or 66 stable C and N iso tope analysis at the Stable Iso tope Facility at th e University of Saskatchewan, a katoon, Canada. In areas where few indi viduals fel l wi thin plots, satnpl cs 11 \),/ere collected frotn an area closest to the plot. To deri c 8 C and 8 1"N values fo r each foliar an1ple, elec ted mate1ials v. ere prepared and analy7cd accord ing to n1 cthods in Section 1.2 and 2.2 Statistical aJ7alvsis The two data et . urvey count data (# tndJ\'Jduals tn ~) and 6 C and o1"N va lues (%o) 11 of the Pyroleac pecics, were te ted differentl y due to th eir different characteri sti cs. The survey count data were highl y non-normal and th erefore non-paratnetric Friedm an tests for replica ted randon1ized complete block designs \Vith post hoc testing were used to detenninc difference in each transect segn1ent (forest, edge and clearcut) at the three sites for each species separately and all species cotnbined. Co unt data were analysed using th e averages of the three 1 m 2 plots at each distance, effectively reducing the vari ability around the tneans for graphical purposes but not altering the significance of the tests. The isotope data rnet normality and homogeneity assumptions in almost all cases so were analyzed usi ng parametric linear tnixed models. However, the isotope data san1pl e size was quite 1in1ited so were onl y tested for each species using data fron1 all sites co n1bined. For P. chlorantha, insufficient replicates in clearcuts allowed only tests between forest and edge segrnents. In all tests, segn1 ents were the grouping/trea hnent fa ctor, with transect (for count data) or transect within site (for isotope data) as the blocking/randon1 factors. The count data we re anal ysed using pac kage "agri co lnc" v. 1.2- 1 (de Mcndiburu 20 13) in R statiStical software (v. 3. 1.0, R Dcvelop1ncnt Core Tcatn 2008). Iso tope data \verc ana ly7ed ustng SPSS Versions 2 1 or 22 (SPSS Inc., Chicago, IL, USA). For count dnta. p-\ alues refer to the 67 overall tnodel r '-stati tJ c, whereas for ISOtope data, p-values refer to S tdak-adjUSted post hoc n1ultiple cornpanso ns, with allrn oclel p-\ alu c<; co n ~ id c rc d signtfi cant at (J.. < 0. 1. 3.3 Re ult Sun 'er . da ta The count data generall ) "hO\\ cd that fo n.:"l 0 1 edge c:,cgtn e nt ~ had greater nun1bers of Pyro leae than clearcuts, dependtng on "t te {FtguJ c 1-2) At DM Mot"t. C tllnhellota had significantl y ht gher den ity at th e edge than in th e clearcut (p \\ ere ob ef\ ed at all. \\ h1l e 0 \<.'C unda and P 0 05 1), where no indt viduals a\an(olia had c; tgnt fi cantl y higher dens1ty tn th e forest con1pared to edge and or c]carcut (p 0 001 and 0 028, respecti vely). At DL Mesic, C. umhellata and 0. secunda follovvcd the ~ atne trend as OM Moist but with no • significant di fference , \vhcrea for P. a\an/oha . . the edge had a grea ter density th an the cleareut, \vhere no individual were ob erved (p 0 08~ Ftgure 3-2) One transect at DL Mesic had very high counts of the three Pyroleae species, C. zunhcllata tn particul ar, whi ch do1ninated the site averages. Only one transect<;. forest segn1ent co ntatned P. chlorantha at these sites, but samples did not fall within plots o were not used 111 th1 s analysis. The lowest density of all species occutTed at PM Dry, and co ntrary to the other sttes, it was the clearcut that had the highest populati on of C unzhellata. \\ hd e only one or a handful of individual were counted in edge and fo rc~t scginents, rc~ p cctn ely (Figure 1-2 ). Though the forest had the highest density of 0 . .H'cunda at thi s s1te, the clca rcut denst ty exceeded that of the edge For both aforcn1enti oned spec tes, clearcut d en ~ t t) \\ a~ don11n,1ted by one transect that had a co nsiderable populatt on o f tndt \ tduab a~ oppo,ed to none an the other two transects. 6X Chimaphila umbellata 40 - 30- 20- a 10 - ab I .....:L. 025- - b Orthilia secunda a 20- N E 15- -(/) ro 10:J "0 -> b b 5- I "0 -c: 0Pyrola asarifolia £ 20- a c: 0 15- a Q) ab Ol ro 10 'Q) > • .... ) 8 1 ~C \a]ue~ frorn bulk l eaf ll~~u c of four Pyroleac specie in fore t. edge and clearcut segment alo ng transect at three si tes in central BC Signjficant difference between location were onl y tested for all sttcs cornbin ed. and arc represented by different letter at u. < 0. 1. Looki ng at the ite-speci fi c summary data, the PM Dry site tended to be n1o~t 11 enriched for C. unzhellata and 0 . ,\ ccunda, \\ ith uniform 8 C \a lues onl y varytng by a rnax imum of 0.48%o between loca ti ons (Figure 3-3 ). The thick. dense forest segtncnts at DL Mesic site tended to have the lowe t 8 13 C \ alue fo r the three species occurring there. \\ Ith edge and clcarcut 8 13 C values being very in1ilar, pa r1icul arl y for () \eLunda and P. asari(o!Ja. Additionally, the site had the la rgest di screpancies between forest and clcarcut 8 13 C va lues for C umbellata, \vith the clearcut <;; ampl cs being.., 6°oo n1ore enricheJ than 11 forest sarnplcs. S i rn i lar large d rfTcrcnccs in 8 C "c1lues between for e!:-tt and clca rcut ...,,1n1ple also occurred ror (). secunda and P. asan/(J!Ja at that site and even ~ lr ghtl y rnorc !:-tO at the OM Moist site (Ftgure 3-1 ) At the OM Moist ~ 1l e, edge satnplcs \\ere . . ltghtl) n1o1 c <.kplcted 71 than forest atnpl es for C. umbella ta , and were enriched to a lesser degree for P. asari(olia compared to th e DL Me ic site, po tbl y owing to the aspect of the treeline where sa1npl es were obtained. I c; arn pie o a lues were quite va ri ab le, ~ ith few stte/species co1nbinati ons showing any imilaritics to trends in va lues between transect seg1nents. The onl y signifi cant dtfference for th e combtncd s1tc data occ urTed fo r C. uJnhe/lata . with the sa1ne trends as o13C of being significantl y enriched in 1c; Again, edge o I c; corn pared to forests (p - 0 . 088~ Figure 3-4 ). value fell bet\\ ecn fo rest and clearc ut va lues and did not signifi cantl y di ffer fron1 either. The other pecies showed compl etely d1ffercnt relati onships for all data combined. Edge samples of 0. secunda were the most depleted and both fo rest and clearcut sarnpl es were about two times as enri ched. Sampl es of P. a.~anfo!Ja had the lowest overaJl • 15 N enrichment, with values decreasing sJightl y from the forests out to the clearcuts, whil e all samples of P. chlorantha were hi ghly enri ched compared to the other species and showed the opposite trend as P. asar~(olia at its primary site (Figure 3-4 ). The inconsistency in trends betw een transect segments was clearest at DL Mesic, wi th C. umbellata being n1ost enriched in the clearcu t and tnost depleted in the forest, with the opposite occulTing for 0 . secunda, and P. asar[fo!ia having the 1nost enriched va lues 1n edge (Figure 3-4 ). Perhaps tnost interesting was at PM Dry, where the trend of increasing cnriclunent fron1 forest to clearcut observed in P. chlorantha also occu rTed fo r C. umhellata 15 and 0 . secunda. The 8 N values increased between fo rests to clea rcuts by 3.4%o and 5.3°'oo for C. tnnhellata and 0 . secunda, respecti vely, vv' hich were the two large t di fferences between any scgtnents for al1 species and sites (Figure 3-4 ). 72 Orth;Jia secunda Chimaphi/a umbe/Jata 7 5ab a 50 b 2 50 0........... 0 , o _.., 5 - ...__., 0 ' Location .... z Forest Edge Clearcut Pyrola ch/orantha Pyrola asarffolia I() 7 5- 5.02.5 0 0-2 5- I I OM rv1o1st Dl Mes1c Pf\1 Dry I All Sites I OM ~"loist Ol l\lesic PM Dry A ll Sites S1te Figure 3-4. Mean (± I D 'vhen n > 3) 6 1'i N values f'rotn bulk leaf tissue or four Pyrolca c species in fore~ t. edge and clearcut ~egn1 e nt along transect at three sitcC) 111 central BC Significant differences bet\\ cen locations were onl y tc ted for all 1tes con1b1ncd, and m c repre c;cnted by different l e tte r~ at a <' 0. 1. 3.4 Di cuss ion 11npacts old1sturhance on Pyroleoe ahundance. r) 1 ?(' and c) !'iN In this ~tudy, Pyroleae den ity and table C and i otopcs ~ere C\ alu ated to dcten11ine how populations and nutritional statu of Pyroleae specie (and any, fu ll MJ b 111 the area) differed in several forests w1th 'ariable le' els of MPB-kllled p1nc ttnd sub~cqucnt clearcut harve~ tin g. Regarding full MJ Is, a few indi\ iduals of A/ !trpopllr' and P andromedea were observed 1n forested scgn1cnt~ at DL Mcstc and Pl\1 Dt) , 1c~pcclt\ cl; I Iowcver, the scarcity or these plants In cant that no rul l MH s Cel l within plot~, so "tllnplc'-1 73 were only collected for isotope data, pre ented in Chapter 4 (Table 4- 1). The Pyroleae species howed definite trends of dec rea ed abundance in clearcuts compared to forest or edge areas, though hi gh variability did not ahvay result in significa nt differences for those cotnpari ons (Fi gure 3-2). AI o, within edge seg1nents. more satnples we re found in the interior 15 tn (i nside the treeline) than the cxtenor 15 tn. The hi gher den tti e of C. un1hel/ata and P. asanfolia in edge segtn ents cotnpared to forest at DL Mesic (Figure 3-2) provided evidence of a favourab le autotrophic response to increa ed light in the e spec tes. At D L Mesic, the forest transects were relatively dark and, in one transect, the under tory shrub component vvas particul arl y thi ck, and therefore the increased in·adiance at the forest edge would protn ote increased photosynthetic C ga ins. In Chapter 2, C. znnhe/lata had increased 13 C enrichm ent coJTesponding to hi gher PS rates und er the un-shaded trea tments con1pared to shaded treatments (Figure 2-3b ). If this relationship is consistent for this species, and possibly fo r P. asarifolia, it would be reaso nabl e to assum e the increasing enrichtnent of 13 C of sa1nples progressively fro1n the forest to the clearcut are indicative of higher PS in edge and/or clearcut segments co1npared to fo rests at this si te (Figure 3-3). Similar results occurred at DM Mo ist for both species, though with sotn e variations, likely due in part to transect aspect where satnpl es occuned (i.e., C. znnhe/!ata samples from shaded, not1h aspect edges resulted in minin1al differences 1n forest versus edge 8 C values~ Figure 3-3). At all three sites, 0. secunda had the highe t abundance in the 13 forests con1pared to clearcuts, though there was a greater abundance in the clearcut than edge at PM Dry, and similarly P . cldorant!Ja was also signifi cantl y n1ore abundant in the forest at PM Dry, with no indi viduals occurring in clearcuts (Figure 3-2). The hi gher densities in forest seg1nents for these two species indica tes grea ter relwnce on fun ga l C gain or a lo\\ cr tolerance to hi gh inadiance, Or a CO tnbination 0 f both. 74 Desp1te the general trend of 1ncrea 1ng e nnchm ent of 13 (' w1 th 1ncrcastng li ght at OM Moist and DL Mes1c for C umhcllota and P a.,anfoha, a well as () .\ ccunda, the tend ency for al l Pyroleae to ha\ e I O\\ cr dcn ~ ttt cs tn c l cn rc ut ~ co n1pared to fore~ ts or edge~ 1nclt ca ted that li ght le\ cls \\ere not opt1 n1alunder full c:.; unlt ght For exa rnpl e, the lack of P o.Htrl/ol!a 11 individual <; in clcn rcut pl ots."" \\ell as imilnrit) in edge and clcnrcut 8 C valu es for both P a. an(oha and 0 "ec.unda at DL \1 e<;tc and DM Mot~t fo1 the lattc1 ~pec t ec:.;, 1nay have rc ulted fron1 li ght aturatton hetng reached at the edge (F i gu re~ 1-2 and '3-3 ). Thts 1s supported by data 1n Chapters 2 and 4. ~ ith li ght re~pon~e curves ror 0 secunda show ing photo ynthct1 c aturation at ~ 400 pn1ol 111.:.! <; 1. ahout I 5 of full1n1d-~un1n1er irradtance, whil e P. asan(olu1 reached sa turati on at a ran ge of around 400 to I 000 prno l rn 2 1 s , dependin g on site canopy cover (Figures 2-4 and 4-2). Contrad ictory to the majority of the • data for 0. 5.cc undo indicating ensi tl\ ity to hi gh li ght ( 1n all three data Chapterc;,), the ~ p cc i es had the highest den. ities of all the Pyroleae 1n clca rcuts at all three c;,ttes (Figure 3-2), hut it is uncertain why thi s is the case. The tno t interesting re ult regarding C nutntion occurred ror P. chlorantha. While the decrease in abundance fron1 forest to clearcut ~as not unexpected. this was the on Iy species to show decreased 8 11 C va lues in clearcuts co n1pared to fo rest and edge scgn1 cnts (Figure 3-3) Unfottunately, only one satnpl e was found in the clca rcut even\\ Ith considerable search effort, so the clearcut segtncnt could not be Included 1n sta tt ~ tl cal analysis for thi s species. If the trend were to hold with increas ing san1pl es, h O\\ e\ cr. tt could be convincing evidence of so n1e level of obl tga tc heterotrophic C ga tn'-1 ~i rnil a r to tho"e suggested by l lynson et al. (20 12) ror Pyrola /)/( Ia Though p J71Cia l~ not a ~pecte" lound 111 our region, con1parisons of photographs or the two spcc tes show s un ilantics 111 leaf n1orphoJogy and co lout. They arc ph ylogencttcnlly closely related, and tlrc thought to htl\ e 75 originated in colder, darker boreal clirnates cotnpared to Clun zaphila and Ortln!Ja lineages, possibly indicating a greater tend ency to retain heterotrophi c nutriti on (L1u et al. 2014 ). Nitrogen isotope signatures are so1newhat tnore difficult to interpret du e to potential difference in fun gal pec tes presence, syn1biotic associations with Pyroleae, and forms of N 15 available to plants and fungi across the stud y sites. This is evident by the variability in 8 N bet,veen 1te , tran ect and pec1cs, espec tall y at DM Motst and DL Mesic (Fi gure 3-4). Onl y at PM Dry \vere the pattern the san1 e fo r all species. with increasing eru·iclunent fron1 forest to clearcut. significantly o for C Lonhellata and 0 . .\ecunda. The pattern for 15N along 15 productivity gradients is generall y that. Vv here N JS abundant 1n soils, foliar N in EC M plants is greater due to direct uptake frotn root , a opposed to transfer through mycorrhi zas, whi le in N-poor sites, organic cycling dornin ates and pl ants become depleted in the isotope whil e fungal sporocarps become eru·iched (Lilleskov ct al. 2002~ Craine et al. 2009; Kranabetter and MacKenzie 2010). This is bcca u e within the fungal tissues. biocherni caJ reactions create 1 N eruiched pools (i.e. proteins) and 1'iN depleted pools (i.e. chitin, atnin o 5 acids, ammonium), the former predominantly retained by the fungi and the latter (excluding chitin) In ore commonly passed to the plant (Lilleskov et al. 2002~ Hobbie and Colpaert 2003 ~ 15 Hobbie and Hogberg 20 12). This could contribute to the increasing 8 N in clearcuts (or edge) compared to forests for C. umhellata at all sites, and for 0. secunda and P. chlorantha at PM Dry (Figure 3-4 ). If there was greater tnycotThi zal fungal bio1nass upported wi thin the forest canopy, more sources of 15 N-depleted N may have tran ferred to the plant , resulting in low valu es in the forest and edge (Hobbie and Hobbie 2008 ). In the clca rcut. lack of trees could have lin1ited C fl ow to the cos tl y n1ycorThi zcd fung1, causing a dcc llne in overal l fungal bi otnass~ fewer sources of 15N-dep lctcd N \vould res ult in higher 8 1"N signatures in the plants. Alternatively, the Pyrolcae n1ay hcl\ c shift ed to ddTerent fungal 76 species that provided different sources o f N. such a rnore enri ched an1ino acids (Johansson et al. 20 15). The consistentl y much hi gher 81c; of P. chlorantha con1pared to the other Pyroleae (Figure 3-4) indicates thi species, hke full MHs. nwy preferentiall y access fungal proteins which are - 7%o to 1s<~oo tn ore enri ched th an chitin (Trud ell ct al. 2003: I Iobbic and ll bgberg 20 12), or po ibl y even dige t fun ga l s ttuc ture~. th ough no lys1 of fun gal cells has been observed (Teder oo et al. 2007, Ytncenot ct al. 2008). In addttion, th e lack of individual 15 P. chlorantha in clearcut plots PM Dry (Figure 3-2) and minirnal differe nces in 8 N across transect locati ons cotnpared to the other two species (Figure 3-4) supports th e idea that th e specie ma y al o have a higher le' el of fun ga l specifictty stmilar to full MHs, though studi es have shown that they can associate with div erse funga l species (Tedcrsoo ct al. 2007). Altern ati ve Iy or additi onall y. hi gh 8 15 N in P. chI orant ha 1nay indicate an association wi th high bion1ass. hi gh 81 c;N fungi at our site uch as Tricholon1a , which has been fou nd with high frequency in P. chlorantha roots and is the sole n1ycobiont genus forM. hypop11ys (Bidartondo and Bruns 2005~ Tedersoo et al. 2007), which was found in the study area (D L Mesic and see Chapters 2 and 4 for CR site description). Some species of these fungi seen1 to be patiicularly adapted to dry, nutri ent poor sites (e.g .. Trudell ct al. 2003, 2004: Johansson 20 14 ). It ma y also be that co1npatible funga l sy1nbionts, \v hatever the species, were unable to be supported once overstory trees were retnoved, or persisted only as \ ery small ren1nants. The general decrease in 0 . secunda and P . asanf'o/ia 8 15 N fron1 forest to clearcut at DM Moist and DL Mesic (Figure 3-4) possibly resulted rro1n lufts to different fungal symbi onts outside the tree line. Based on the relatively hi gh levels of 1c;N cnn chn1cnt 111 Pyrol eae cotnpared to full autotrophs (see Chapters 2 and 4 ). potenti ally all the In\ cstignted 77 pecie preferentially as itnilate 1~ -ennched orga ni c N to so1ne degree or other (Hobbie and Hogberg 20 12). If the fun gal species at these sites arc hi gh bio1na 'S species with high C co t , the loss of C fl ow from ovcrstory trees could ca use a shift to lower btoinass, generalist species such as Russula, Corttnan11s and Lactanus, all of which ha\ e been found on Pyroleae pecie (Hyn on and Brun 2009: Hobb ie and Agercr 20 10: Hashi1noto et al. 20 12). Additionall y. if shi ft ing of funga l SYJnbi onts docs occur, tt is quite po~s1bl c theN transfen·ed to plants could be in dtfferent form , such as the n1 orc depleted an11noniu1n rather th an proteinaceous fotm . At DM Moi t and DL Mesic, hi gher 1" enrichrnen t in edge smnpl es co-occun·ed with greater abundance of C. umhel!ata and P. asan(olza, respectively (Figures 3-2 and 3-4 ). These results may bee idence of better trade-offs bet'Neen the pl ants and fun gi, where the increased photosynthetic C activity along the fore t edge tnay increase allocati on to fungal symbionts and thus promote greater allocation of back to the plant. T hi s bi-directional C Oow has been observed in the pa11ial MH orchid Goodyera repens and its n1ycobiont (Cameron et al. 2006), and may have contributed to the increased 1"N enrichJn ent of C. umbellata at the PM D ry clearcut. A dditiona/factors i1~(luencing persistence (ollo1\ 'ing disturbance As 1nentioned, a few individual M. !typopitys and P. andromedl!a plants were observed in the forests at DL Mesic and PM Dry, respecti vely. Because of their con1pletc reliance On host autotrophic C Oow via 1nycorrhizal net"''Orks, the Jack of indi\ iduals in clearcut transects indicated that residual vegetation, or connectivi ty to res1d unl vegetation. was insufficient to suppl y the necessa ry C requiren1ents to these plants. Based on the presence Of large trees along the inner porti on of the edge transect up to the tn;e line, it IS 78 po, tblc full MH could ex1st ncar cl ecu c ut~ . but ac;; ll aeus ler et al (2002) po1nt out. these pcc1cs rarel y tolerate full unlight. Int c rc~ tln g l }. a nun1ber of P and1 omcdca Jnd l\ idual() have been ob en. cd 111 htghly C\.pO<;ed areas 1 ight along the tree l1nc at the stud y ~ 1te for Chapter 2, as well as in the clcarcut at ca bet\\ een transec t'-, at DL M e~ J e (albe1t tu cked under a nlodcratc-s11cd ptnc ), dcmonstrat1ng ~o n1 c 1c~ d 1cncc to htgh Iight 111 sotne co nd i lions Cun1n11ngs and \Vel chtnc) cr ( 1997) pet Jonn ed pt gn1cnt an a l y<; t ~ on a nun1ber or Ml I ~ rec i es acros dl\ cr5C taxa. and P andro111<.ch a had the hi ghe~ t lc\ e l ~ o f em otenotd <; related to xanthophylls cycles. \\hJ ch arc tn1po11ant lo1 photoprotection (furthcJ dt <;c u ~~ • o n below ) The other MI Is\\ ht ch had noticeabl y grea ter ca rotenoid pt gn1c nt ~ \\ere t h o~e with red colourati on and can be found under n1 odcrately high lt ghl co ndttion (1.e .. Sarcode\ \angz unea, the snow plant which cn1erges in spring und er open ca nopi es 111 southwes t USA) L1kely the few fungal specie as ociated with the full MI-ls would be di1n1ni hed followi ng clearcutt ing, lun1ting h o~ many individual could be ~ upp o rt ed \V ith limited rc<:,o urce~ along forest edges. With the exception of a fe\\ indi \ idual P a\an(oiLa. almo<;t allJndt viduals found 111 clearcuts were due to the pre ence of C. Lnnhellata and 0 .~ccunda ( Ftgure 3-2 ). Wi th these generall y occurring in high nun1bers in one transec t that \Vas contrary to the general trends. The results are similar to those of Haeussler ct al. (2007), where they found onl y (J. secunda present in clearcuts and onl y those not additi onally slash burned. Microclirnatc changes. specificall y increased light and ten1peraturc, secn1 to he \ ery itnportant 111 detern11ning surviva l of the Pyroleae follo\ving di sturbance T here are a coupl e ltn c~ or C\ td cnce fo r th ts from thi s stud y. First, when atnp les were found in clcarcuts or e' en JU5t o ut ~tde the t1ec hne in tnany cases, they were associated wtth fea tures that would pro' 1de thcn1 \\ tth son1c ~ h clter froJn direct sunli ght. In n1ost cases, th is was nca r sn1all trees or shrubs that could pnn tde thcn1 with additional access to tn ycorrhi ;,al lungi, but a few indi\ idunls at DM Motst \\ere 79 C\ en found c lu ~ tcrcd around a sturnp that could pro\ tdc orne degree of ~ had e t PM Dry~ the presence of Pyroleae 111 clearc ut ~ onl) occurred on the one not1h-fCictng cleat cut. \\ hich co ntrasted the sou th-fac tng clcarcu t 111 that it had a tnu ch greater ~h rub co n1pon ent resctnbling the other si te!>. prcsu n1 ably allo\\ mg persistence of ECM fung1 I he south- fa cing clcarcut \\as pritnanly tnhnb1ted by rcmdccr lichens Cl nd blueberries. whtch incllcatcs a poss tblc greater proportion of en co tel fungi. \vhich arc not knO\\ n to tnfcct Pyrolcac (Mac:,~tcotte ct al 200 . but cc Lat gent ct al. 1980 ). cconcl. although these tndt\ tdudb '' crc p1 c~cnt. they\\ ere ft cquc ntl y very chlorotic, a condition also observ ed by Ha cu~'-.lcr ct al. (2007). As Hunt and Jlopc-S IIllpc:,o n ( 1990) point out. the C\ crgrecn leaves of Pyrolcac ~pcctcs are an ada ptation to shad ed ecosystcn1s that arc slo\v to adapt their 1nesoph yll anaton1y\ plastid apparatus and p1gn1ent concentration , which tnay inc rca e chance of photo-ox idatt \ c dan1agc under high ltght co nell tton ~ Photoox idati' c dan1age occur when exec . ltght energy not uc;ed for photo'-.ynthcs ts cau~c~ dan1age to the reacti\ e photo ystem II protcJns at a rate exceeding the abdJty to rcpatr those proteins, resulting in decreased photo ynthctic capacity and bleaching of the remaining auxiliary chlorophyll antenna tnol ecul es ( Pon1pelli et al. 20 I 0~ Turan 20 12 ). Because photosyntheti c saturation occurs at lower li ght levels in shade plant~ con1parcd to sun plants, for a given li ght level. the shade-adapted plant absorbs a greater propo11 ion of energy not used for photosynthesis (Krieden1aru1 1999). One key protective m echant ~Jn to pre\ ent photo-oxidatn e dmnage 1s through xanthophylls cycles, primarily the productton of zeaxanthin and anthcrcr\anthtn frotn violaxanth1n (a ll carotenoids), wtth the fonn er two bctng impot1ant for sdfc dtsstpatton of excess Iight energy as heat (V crhocvcn ct a l. I 997 ~ PotnpelJ i et al. 20 I0~ JUt an 20 I~) Nitrogen nutrition is in1portant fcH both the fun ctioning of chlorophyll and \.,uHhoph)lls xo Under low N condition , chlorophyll concentrati on ca n decrease along with photosynthetic capacity, while rati os of 7eaxanthin and antheraxanthin to total xanthophyll s increases, both of which have been ob en ed 1n spinac h (Sp111ac1a olcracea) and coffee ( Cof(ea arabica; Verhoeven ct al. 1997: Potnpelli et al 20 I 0) With decreased photosynthetic capac tty, the potential for photo-oxidation Increases e'en more. Under low N conditions, coffee trees showed up-regulation of photop rotec ti on \ ta xanthophylls as vvell a antioxidant capacity, but were not suffi cient to prevent photo-oxidati ve datnagc in low N treatn1 ents (Pon1pelli ct al. 20 10). With adequate , produ cti on of protective p1grnentc:; to prevent photo-oxidative da1nage i enhanced and help di si pate excesc:; heat, rn ai ntaining photosynthetic capacity. The retn oval of ovcrstory trees and potenti al shifts in EC M fungi could very well be crea ting condition negatively synergistic for the Pyroleac. S1nce the Pyroleae arc clearly capable of photosynthe izing at rates suffi cient for growth and reproduction under low to relatively high light levels (see Chapters 2 and 4 ), it is unlikely that loss of any heterotrophic C gains resulting from disturbance are the main cause for extirpation or minin1 al persistence in clearcuts as it is for the full MHs. Nitrogen nutrition may be a n1ore important determinant in Pyroleae survival, and photosynthetic C nutrition may be influ enced negatively wi th low N to a point unabl e to sustain the plants. A possible scenario explaining inabili ty to adapt to disturbance is that following harvesting, hi gh light intensity increases photo-oxidative dmnage whil e photosynthetic capacity is decreased, especially in a species like 0. secunda with low inherent rate and presutnably low chlorophyll concentration (based on the n1ore yellow lea\ cs con1pared to most other Pyrol eae). The lack of P. chlorantha at PM Dry clea rcuts and the\ er} high o1-; signatures (Figures 3-2 and 3-4) tnay have been due to a hi gher reliance on spccdic mycorrhiza for N nutrition at this site; therefore, 1ninin1al N uptake could ha\ e reduced 81 photoprotective pign1ents to the point of effecti vely extirpating the species fron1 harvested areas ~ \V hi ch generally appeared to be the case. The chl orotic state of C. u1J1hellata and 0 . secunda in clcarcuts ugge ts they are n1ai nta1ning Inycorrhr7.:al connections folJowing harvestin g, but the fung1 were less capable of providing adequate for photoprotection and tnaintcnance of photo ynthcttc capacity. Over the long tcnn , the effect of reduced C uptak e 1nay cau e the eventual dea th of these indl\ iduals, espec1ally in nutri ent poor sites. Additional strc es as ociated w1th drought and h1gh tcn1perature 1nay acce lerate the decline of these pecies in heavil y di sturbed areas. Con eluding remarks It is evident fron1 this data and other studi es ( choo nm aker and McKee 1988 ~ H alpe111 and Spies 1 995~ Haeu ler et al., 2002, 2007) that mature Pyroleae are able to resist hi gher • levels of disturbances co1npared to full MHs, whi ch are rnuch tnorc sensiti ve due to th eir mycorrhizal specificity and high nutritional detnands from rnycobionts and autotrophic hosts (e.g., Bidartondo and Bruns 2002~ Bruns et al. 2002). Yet at PM Dry, even wi th approxi1nately 95 % n1ortality of pine ho ts following MPB attack, P. anc/r()l77edea is ca pable of surviving. Therefore, the greatest in1pact on full MH persistence is compl ete removal of overstory trees, and therefore C supp ly to fun gal sy1nbiont and the MHs. The pre ence. but lack of vigour, of Pyroleae plants in clearcuts (Figure 3-2) indicates that rernovaJ of overstory trees is not the actual cause of Pyroleae species decl ines following disturbance per se . Instead, the cotnbined effects of detri1nental n1icroclitnate conditi ons, concunent declines or shifts in cotnpatiblc 1n yconhizal fun gi, and lack of su11icient N (and possibl y C) nutnti on, reduce the abiJity of these species to survive extrcn1e di sturbance. 82 Ecosy tetn di sturbances are becotning tnore and 1nore anthropogenic, with many resource sectors di turbing the landscape at n1any scales through oil and gas ex ploration, mining, forestry. road acccs . and all the biogcochctni cal alterations associated with these activities. Sensitive tnaturc forest species arc n1ore likely to becotne species of concern. Cornn1on pecie here are at risk due to di sturbance and cli1natc change elsewhere. In Sweden. C. umhellata is rcd-hsted. v.:ith Afonotropa unT/lora and P clllorantha also in decline (Johan on et al. 20 14 ). There are at least seven different 1nonotropcs found in British Columbia, three of which are co1nmon in the Prince George region and the others found only in the lower tn atnl and , Vancou\ cr Island or the central coast region. The full MHs and Pyrola minor L. fall under provinc ial conservati on status classes S3S4 or S4 in British Columbia, due to their su ceptibility to extirpation, and one full MH , Pleuncospora (imbriolata A. Gray, i red-listed (BC MOE 20 14 ). It is becotning increasingly recognized that understory and fungal species may hi ghl y influence fo rest succession and species competition, and thus protecting th ese species is in1p011ant for reasons other than simply maintaining cryptic herbaceous species (Hynson et al. 20 13 ). Certain management strategies can help protect these ensitive species during extensive salvage operations, including partial-harvest teclmiques or, in cases where econotnic gains are n1arginal, discourage salvage logging. If harvesting is to occu r, effo11s should be tnade to pro1note structural and tnicroclimate diversity as tnuch as possible. This can be done tlu·ough activities such as avoiding unnecessary destruction of residual shrubs, suppressed or young trees, and preserving itnpot1ant features such as coarse woody debris. 83 3.5 Literature ited Bellgard E, Willi an1s E. 20 11. Re ponse of tnyconhizal diversity to current clin1 ati c changes. Di ver ity 3: 8- 90. doi: 10.3 390/d30 10008 Bidationd o, MI. Bruns TD. 2002 Fine-level n1yconh1 za l specificity in the Monotropoicleae (Ericaceae) : pecifi city fo r funga l species groups Molecular Ecology 11 : 557- 569. doi: 10.1046/j .0962- 10 3.200 1.0 1443 .X Bidartond o, ML Brun TO 2005. On the o n gin of cx trctn e 111 yco nh i ~:a l specifi city in the Monotropoideae (Eti caceae): perfonnance trade-offs dun ng seed gem1ination and seedlin g development Molecul ar Eco logy 14. 1549- 1560. do1: 10. 11 J l/j . I365-294X.2005 .02503.x [BC MOE] Briti h Colun1bia Ministry of Envtrontn cnt. l 989 oils of th e Prince George McLeod Lake Area. MOE Techni cal Repori 29. Bri t1sh olurnb1a Soil Survey Report No. 23, British Columbia Ministry of Agri culture and Fi herics Victon a. BC. 2 19 p. [BC MOE] British Columbia Ministry of Enviromn ent. 20 14. BC Conservati on Data Centre· BC Specie and Eco y tern Explorer. Victoria, B C. Accessed 26 Mar 20 14 fro1n: http://a 1OO.gov. bc.catpub/eswp ' [BC MFLNRO ] Briti h Columbia Ministry of Forests, Lands and Natural Resource Operati ons. [Internet]. 20 13. Facts about B.C.'s moun ta in pin e beetl e. http://www .fo r.gov.bc.ca/hfphn ountain_p ine_beetl e/Updated-BeetJ e-F acts April 20 13. pdf. [BC MFLNRO] British Colutnbia Ministry of Forest , Lands, and Natural Resource Operations. [Internet]. 20 14. BEC WEB: Products and Resources- Biogeoclitnate subzone/variant wa ll maps. Research Branch, British Columbia Ministry o f Forest , Vi ctoria, B.C. 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Acce sed 23 Jan 20 15 fron1 http://plantsinacti on. cience.uq edu au edtll on i '?q contcntJ chaptcr- 12-sunl ight-all. pervas1ve- ourcc-energy Largent, DL, ugihara , Wishner C 19 0 ccurrence of tn ycorrh i1.ae on eri caccous and pyrolaceou plant in not1hem Ca1I fon11a. Canadia n Joun1 al of Botany 58: 2274-2279. doi: 10. 11 39/b 0-262 Leake, JR. 1994. The biology of tn yco-hetcrotrophic ('saprophytic') plants. Tan.\ ley Review N o. 69. ew Phytologi t 127 : 17 1- 2 16. doi 10. 1111 j. l469-8 137 1994 tb04272.x Lille kov, EA, Hobbie EA, Fahey TJ. 2002 . Ectotn ycorrhi Lal fu ngal taxa differing in response to nitrogen deposition also diffe r In pure culture organi c ni trogen use and natural abundance of nitrogen isotopes. cw Phytologist 154 . 2 19- 23 1. doi: l 0.1046/j . 14698137.2002 .00367 . X Liu, Z-W , Jolle DD, Zhou J, Peng H, Milne Rl. 20 14. Mul tip le origins of circum boreal taxa in Pyro la (E1i caceae), a group with a Tertiary reli ct distribution. Ann als of Botany 114: 170 1- 1709. doi: l 0.1093/aob/mcu 198 Massicot1e, HB, Melville LH, Tackaberry LE, Peterson RL. 2008. A cotn parative study of mycorrhizas in several genera of Pyroleae (Eri caceae) fron1 westen1 Canada. Botany 86: 610-622. doi: I 0.11 39/B08-027 Matsuda, Y, Shimizu S, Mon M, Ito S-L Selosse M-A. 2012. Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in tnixotrophic Py rola japontca (Ericaceae) growing under different li ght cnvirorun ents. A1neri can Journal of Botany 99 : 1177- 11 88. doi: 10.3732/aj b.ll 00546 Meidinger, D, Pojar J. 1991. Ecosysten1s of British Columbia. Research Branch, Ministry of Forests, 31 Bastion Square, Victoria BC, 330 p. Merckx, VSFT. 201 3. Mycoheterotrophy: an introducti on. In: Merckx, V FT (Ed). Mycoheterotrophy: The Biology of Plants Living on Fungi. New York, NY : pringer c1ence + Business Media. p. 1- 17. doi: 10.1007/978- 1-461 4-5209-6 1 Molina, R, Massicotte I L Trappe JM. 1992 . Specificity phenomena in n1ycorrhizal symbiosis: community-ecological consequ ences and practical itnplicattons. ln. MJ Allen, editor. MycotThizal Functioning: An Integrati ve Plant-Funga l Process. London, Y: Chapman and Hall p. 357-423 . ISBN 0-41 2-0 189 1-8 87 Nel on, CR, Halpern CB. 2005. ho11-ten11 e ffect of titnber harvest and forest edges on ground-layer mos e and livervvot1s. anadian Jo urnal of Bo tany 83 : 610 620. doi: 10. 1139/B05-036 Potnpelli, MF, M artrns CV. ntunes \V , Chaves ARM. DaMa tta FM. Pho tosynthesis and photopro tecti on in coffee leave is affec ted by nitrogen and light availabilities in w inter condition . .J outnal of Pl ant Phys iology 167: 1052 1060 dot : 10. 10 16/j .jplph.20 l 0.03 .00 I afranyik, L, WJI o n B. 2006 The tnountatn ptne beetle: a synthes is of b1ology, In anagetnent, and itnpacts on lodgepole pine atura l Rec;; ources Ca nada, Ca nadi an Forest Service, Pac ific Forestry Centre, V1ctona. 8 . 304 p ISBN 0-662-42623- 1 Schoontnaker, P, McKee . 19 pccies co n1pos1tion and dtvers1ty dunng seco ndary ucce io n of co ni fe rou fore t in the Western Cascade Mo untatns of Oregon. Forest Science 34: 960-979 . Selo e, M-A, Richard F. He . unard W . 2006. Myco rThi La l networks. des liaisons dangereu. e. ? Trends in Ecology and Evolution 2 1: 621-628 dot : 10.1016/j .trcc.2006.07 .003 Stnith, SE, Read DJ . 2008 . Mycorrhizal UK. 605 p. ISBN : 978-0- 12-370526-6 ytnbiosis. 3 rd Edition. Acaden1i c Press, London, Taylor, AFS, Fransson PM, Hogberg P, Hogberg MN, Pl amboeck A H . 2003 . Species level 15 13 patterns in C and N abundance of ecto1n yconhizal and saprotrophi c fu ngal sporocarps. New Phytologist 159: 757- 774 . doi : 10.1046 1j . l469-8137.2003 .00838 .x Tedersoo, L, Pellet P . Ko ljalg U, Selo se M-A. 2007 . Parallel evo lutionary p aths to m ycoheterotro phy in und erstorey Ericaceae and O rchidaceae: ecologi cal evidence for mixotrophy in Pyt·oleae. Oeco1ogia 15 1: 206- 217. doi : l 0.1007/s00442-006-0581-2 Trudell, SA, R ygiew icz PT, Ed1nonds RL. 2003. itrogen and carbon stab le isotope abundances support the 1nyco-h eterotrophic nature and host-specificity of ce11ain achlorophyllous plants. New Phytologist 160 : 391-40 1. doi: 10. 1046/j .14698 137.2003 .00876.x Trudell, SA, R ygiewicz PT, Edn1ond RL. 2004. Patte1ns o f nitrogen and carbon stable isotope ratios in n1acrofungi, plants and soils in two old -gro·wth conifer forests. cw Phytologist 164: 3 17- 335. doi: IO. lllllj . l469-8 l37 .2004.0ll62.x Turan, S. 201 2. Light acclitnation in pl ants: photoinhibitin and photoprotectlon . Advances 111 Bioresearch 3: 90- 94. Verhoeven, AS , Demn1i g-Adan1s B, Adams Ill WW. 1997 . Enhanced etnpl oyn1cnt of the xanthophylls cycle and thcnnal energy di sipation in spinach exposed to high li ght and stress. Plant Phys io logy 113: 8 17- 824. doi: 1O. ll 04/pp. l1 3.3.8 17 88 Vincenot, L, Teder oo L, Richard F, 1-Iorcinc H, K6ljalg U, elos e M-A. 2008 . Fungal as ociate of Pyrola rotun(h(o/ia, a Jni xotrophic Ericaceae, fro1n two E toni an boreal forests. MycotThiza 19: 15- 25 . doi: 10. I 007 s00572-008-0 199-9 Zimtner, K, Hynson N . Gebauer G, Allen EB. )] en MF, Read OJ. 2007. Wide geographical and ecological distnbutio n of nt trogcn and carbon ga ins fro111 fun gi in pyroloids and n1onotropoids (Ericaccae) and in orchtds. ew Phyto logist I 75: 166- 175 . doi : 10.1111 j . l 469-8 137.2007.02065.'< 89 4. A e in g n1ycoheterotrophy in cri caccou pecie u in g two- ource lin ea r mixin g models ba ed on photo yntheti c rate and tabl e i otope of carbon ( 11C) and nitrogen ( 15N) A b tract The tud y ofp a11i al n1ycohctcrotrophs (PMH s) has ga ined considerabJ e attenti on in the la t decade, primaril y through assc stng the natural abund ance of stable carbon and nitrogen ( 13 C and 1" ) i otope . The aitn of this tudy '.vas to assess the cotnbtned dataset for six putatively PMH specie and three full tn ycoheterotrop hs (MHs) in th e fatniJ y Ericaceae to: a) te t for pecie difference in the natural abund ance of 11 C an d 1~N, C and N concentrations a \.vell as photosynth etic (P ) rat es and other gas-exchange paratneters, with particul ar emphasi on C nutrition, and ~ b) to qu anti fy the degree of PMH C nutrition in the Pyroleae by applying an end -rnember mix ing tnodel to both 13 C and PS data, deriving an estimate of %C ga ined via photosynthesis (%C DP ). All Pyrolcae and full y MH species were significantly enriched in 1~ compared to autotrophs, indicating organi c N gain . Photosynthetic rates were often lower in Pyroleae species compared to autotrophs. with 13 C being consequ entl y enriched, especiall y Ortlzi!Ja .\·ecunda and Pyrola clzlorantha. The exception was Mon eses un(/lora, which was the most depleted in 13 C, indicating autotrophy. Based on the input data, %Cor estirnates indicated primarily autotrophic C nutrition fo r Chimaphila umhellata , while 0 . secunda and P. ch/orantha gained only 70~o Cnr. Prro/a asa r~/olia and P. minor showed sotne levels of PMI I C gain but sn1all satnpl e izes and or lack of agreement between %Cor estimates 1nade conclusion unccrtmn. Photosynthetic capacity may be inherently lin1ited in these shade adapted species, resulting in increased levels of photoi nhibition and photorcspiration. Their physiological rc.. ponse to h1gh ltght 90 lc' cis n1ay influence photo ynthet1 c 11 (' d1 ~cnn1Inat ion , leading to eJTonco u" concl us tons on the apparent degree of PMH C acqu1 ~ 1tt o n Fur1hcr c;tudt ec; on b1 ochcn1t cal co n~ tra n1ts to autotrophy us1ng addttional 1ncthocb tnay t cso h c uncertmnt1 es 111 th c~c ~ p ec 1 cs, all ow ing rnorc accurate under tand ing of th e con1pl cx nutntl onal 1node of these pl ants. 4.1 Introducti on table 1 otope rati o tn a"~ "pcctro rnctr) ha" hccornc a pO\vCrfu] too l rn eco logical tud1c that aiJ O\\ re ea rchcL a n on-dc~ tru c t l\C, tntcgrati Yc \ I C\\ tnto pl ant- or anirnalcn\ ironn1 cnt tnterac ti on , both \\ 1th ah tott c rc~o urcc and other orga n 1 ~111~ ( D aw~o n ct al. 2002). T he n1ethod i ba cd on the fac t th at n1ctahol1 c and phys ical proccssc~ generall y di scri1ninatc aga in t heavy i otopcs. leadin g to d ist1nct isotopic Signatures or between di fferent o rgani m or even bet\\ ccn d tffercnt orga ns of a single o rga nt ~ nl (Farquh ar et al 1989: G lctxncr et al. 1993: Teder oo ct al 2007). W1thin the food chatn. hr ghcr trophic levels accu1nul ate heavy i otope . re ultJng 1n uniq ue i otopic s i gnature~ acros~ trophtc groups (Da\VSOn et al. 2002). Thi ha all owed researchers to d1fterentra te bct'A-ccn nutritional strategies of pl ants and fungi (e.g., Hogberg et al. 1 999~ Geba uer and Meyer 2003: Trudell ct al. 20 04 ~ Preiss and Gebauer 2008). In par1i cular, analysis of natura l abundance of 11 stable carbon and nitrogen ( C and 1"N) isotopes 111 ce11ain groups of plants. as \\el l as fungal sytnbi onts, has provid ed strong ctnptn cal and quantitative e\ idence en ttcal to understanding the unique nutritionaltnodc or n1ycoheterotrophy In a setninal paper, Leake ( 1994) first adequ ately de en bed the nutnttontll ~tt atcgy of rn ycohcterotrophic (MH) plants. Earl y research tnto full MI Is, plants that cornpletel} lack chl orophyll and arc not ca pnble o r photosyntheti c C ga ins, led to the beli ef tha t these plants were directly paras itlc on host autotrophtc plants xylcn1 and/ot ph loern by rnean" of 91 hau to rial (root-hkc) connections (Leake 1994) ll o\v e\ cr, 1t was eventuall y rccognt /cd that tnycoheterotrophs arc indtrcc tl y parasttt c (epi paras I tcs) on host au totrophs, gain1 ng organtc C through expl o1tatton of bared l11 )Con·htzal net\\ ot k ~ (Lea ke 1994, Pretf3 2009) In the ~an1 c review, Leake ( 1994) recogn1/cd that there \\ ere probabl y son1e n1 ycohctcrotrophs that al so contained chlorophyll and ga tned so tnc C '1Cl pho t o~y nth es is ( P ) 1 hcsc plants arc a type of n1ixotroph, n1ost accurately d c~c nhcd a part ta l tn y c o h c t c ro tro ph ~ ( PMI Is) Mycoheterotrophs are thu s 111\ oh eel 1n a tn partttc 1clatt on"h1p hct \\ ccn n1yconht La l l un g1 associate that arc in tum as~o cwtcd \\ ith sunound1n g host autotrophic pl ants wht ch arc th e ultimate C ource 111 the 5)' . tcn1 ( 81dationdo 2005) In ten1pcratc and borea l ccosy terns, ecton1ycorrh1La ( EC M) arc the dotntnant tn ycorrhi7al clas associated \\ ith Pinaceae. Bctul accac and Fagaceae, fannin g cxtcnstve network -vvtthin and bet\veen tnany different plant taxa (Moltna ct al.. 1992, Se l os~c ct al, 2006). The. c di ver e taxa incl ude a \ ari ety of MH plants in the fatn tiies Orchid accac and Ericaceae. The latter famil y contain the full y MII sub family Monotropoideae, as well as recentl y discovered photo ynthcttc PMH spec tes tn the tn be Pyroleae (Tcdersoo et al 2007 ), which are the focus of thi s paper (with emphasi on PMH Pyrolcac) Spec ificall y, 111 central interior British ColUJnbia, Pyroleae species incl ude Clnmaph ila umhella Ia ( L.) W.P.C Barton, Ortlu lia secunda (L.) House, Pyro/a asanfolut Michx .. P1 rola chlorantha \\ , Py rola nun or L ., and M oneses unr flora (L .) A . Gray \\ 1th the latter t\\ o being the lca~t common. Full Mll Monotropoidcac species include /vfonotropa touflora L , AI !trpopli1 'S L. and Pterosp ora andro1nedca N utt. While the lllYCOIThtzas rorn1ing on Pyroleac and Monotropotd cac dttTer fron1 l·C \ 1 111 that di stinct rnorphological structures penetrate th e root cpidcnnnl cells totally (at hutotd rnycorrh1Las ) or pat1ially (rnonotropo1d 1nycorrh uas), respect1\ ely ( Mo hna ct al. 1992 ), 92 1noden1 advances in tnol ecular ecology ha c revealed that these plants do in fact associate with ba idiomycetc fu ngi that are ECM on co-occuning autotrophic plants, often w1th hi gh pecificity with pat1icular fungal genera and or spec1es (Bidartondo and Btuns 20 02 ~ Lea ke 2004 and ources theretn). Around the san1e ti1ne a 1nany fun gal identi {ication advances were being made, the applicati on of stab le isotope analys is becan1 c a prin1ary way o f determining the sources, pathways. ecology and stgnatures of target species. otnc key findings are as follow . First, in photo ynth eti c plants. overs tory trees arc typicall y n1 ore enri ched in 13 C th an und erstory plant due to exposure to greater inadiancc, resulting 1n hi gher PS rates, whi ch reduce di crimination against 13 C during the carboxylation reacti on of Rubisco (Fa rquhar et al. 19 89~ Hogberg et al. 1 999~ Courty et al. 2011 ). This is primaril y attributed to a drawd own of intercellular C02 o that concentrations relative to atnbi ent C0 2 (C, and Ca, respecti vely, expressed as the ratio of C,:Ca) are lower and a greater proportion of 13 C will be assimilated compared to plants with higher C1:Ca (Farquh ar et al. 1982: Farquhar and Sharkey 1982). Understory plants also show lower 13 C enrichment due to the incorporation of greater 13 atnounts of C-depleted C0 2 originating from soil respiration (Farquhar et al. 1989; Cou t1y et al. 20 11 ; Hynson et al. 20 13). Based on these differences, Hogberg ct al. (1999) showed that overstory host trees provide a greater propotti on of C to ECM fungi than understory autotrophs, supporting the idea that overstory trees are of greater itnpottance in n1ycorrhi?al and MH C nutrition than understory plants. Second, sin1ple carbohydrates transfetTed to ECM fungi arc less enriched in 11 C co1npared to n1ore complex molecules such as cellulose and lignin decotnposcd by saprotrophic fungi (G leixner et al. 1993: Badcck et al. 2005). Along with ddTcrences 111 N 1netabo li sn1, di stinct dua l iso tope signatures result between ECM and saprotrophtc fungt, 93 with ECM fungi tnore enriched in 13 C and 15N co rnpared to their host trees, and saprotrophic fun gi more enriched in 11 C than their woody substrates (Hogberg et a!. 1999; Kohzu et al. 1999: Henn and Chapela 200 l : Taylor et al. 2003) This cnri clunent is tnirrored by full y MH plant pecie relative to thetr funga l syn1b1onts (typ tcall y E M, but also sa protroph1c fun gi ~ see Ogura-T UJita et a] 2009), provid ing key evidence that EC M fun g1 are the food source for MH plants (Trudell et al 2003 ). It wa the 1nten11edJate enn chtnent of 13C isotopes in green plant relative to full MH spcices and surrounding au totorphs provided evidence that the e photosynthetic plant were gaining rnu ch o f thei r C through fun ga l pathways (e.g., Gebauer and Meyer, 2003: Jul ou et al. , 2005: Tedcrsoo et al., 2007). A coinmon applicati on of i otopes in ecologica l studi es is that of end-member 1nixing models, which enables quantification of the source contributions to a 1n ixture (Dawson et al. 2002). Since there are only two potenti al C ource (photosynthesis and fu ngal symbionts) contributing to the 8 13 C signature of PMH species, sin1ple two-source linear tnixing models are used to estimate the proportion of fungal-deri ved C (%C 0 1-) in these plants (Gebauer and Meyer 2003; Tedersoo et al. 2007~ Zi1nmer et al. 2007: Preiss and Gebauer 2008). The 1nethod assumes a linear correlation between fungal derived C and the enrichment in plant 13 C, with the endpoints of the model defmed as the n1ean o13 C values of autotrop hic reference plants (0% fungal derived C) and full MH references ( 100% funga l derived C). Fu11hen11ore, the model also makes the assutnption that the reference plant fu ll y represent the source isotope signatures (Hynson et a1. 20 13 ). Since photosynthetic C ga ins are a key cotn ponent in isotopic n1ixing n1odel, 1t is somewhat surpri sing how few studies actu ally tneasure PS rates in full and parttal MH plants. Of the lin1ited studies considering PS rates, we onl y know of three pa rticular studies that directl y 1neasured gas-exchange in Mil plants, all with orchids, wi th one ustng a 1~C0 2 trdcer 94 method (Julou et al. 2005; Girlanda et al. 2006: Ca1ncron et al. 2009). In all these cases, gasexchange showed net respiration rate du e to either a) a low-light cnvironn1ent where net C0 2 uptake could not con1pensate for net respiratory C0 2 losses in Cephalanth era dan1asontun1 (i.e., below light-compensa tion potnt: Jul ou et al. 2005), or b) inefficient photosynthetic capa city in Lunodonun ahorfn'll/11 (Gtrlanda et al. 2006), Corallnrlu::a tn(ida and Neot11a nidu, -av1s (t he latter essenti all y lack ing photosyntheti c ca pacity; Cmneron et al. 2009). In one other tudy, gas-exchange data wa5 used to e~titn a te seasonal productivity in a Pyroleae species, Pyrola 1ncanzata, but onl y via respiration rn easurctnents coupled with changes in plant biomass (l ogai et al. 2003). This study also did not focus on the species in the context of mycoheterotrophy. All other tudi es that di scuss aspects of photosynthesis or gas-exchange in full or PMH species used data such as stabl e 13C data, fungal identification through mol ecular barcoding, and/or chl orophyll co ntent and fluorescence analys is (e.g., Abadie et al. 2006; Tedersoo et al. 2007: Preiss et al. 20 10; Hynson et al. 20 12: Matsud a et al. 2012), often in conj unction with envirorunental co nditi ons (i.e., light levels; Preiss et al. 20 10). Due to the lack of any data on photosyntheti c rates of mycoheterotrophi c species in the famil y Ericaceae, the purpose of this study was to characteri ze the PS rates and several gas-exchange para1neters influential to 13 C di scrirnination of putative PMH Pyroleae and fu ll MH Monotropoideae species in relation to a variety of autotrophic reference plants across diverse taxa at several sites and over n1ultiple yea rs in central British ColUJnbi a. To COlnplement the gas-exchange data, natural abundance of stab le 13C and 1"'N data \Vere obta ined for sarnples measured for gas-exchange as well as additional san1plcs obtained Cor a population study in 20 13 (see chapter 3 ). Finally, we wa nted to assess \vhethcr an cndn1 etnber n1ixing n1odeJ could be applied toPS data similar to isotope-based n1odcls to 95 e tin1ate % photosyn thesi -derived C' (%Cor). Only data fron1 the tvvo years where repea ted measurement of PS rate were tnade on individual over the season were used. itnilar as urnption \vere made for thi novel approach, narncly that plant photosynthesis-derived C lies on a spectrum betv.een autotrophs (100°o Crw) and fu ll Mils (0% Cor). This alternati ve model wa u ed to co rroborate 'Nith 11 C isotopic model results regardin g extent of mycoheterotrophy in each species. Ba ed on the unique ituati on of u ing a no el approach to an end-me1nbcr mixing model method, \Ve wtll briefly di scuss some co nceptual considerati ons. The most obvious difference between an i otoptc ver us photosynthetic two-source rni xi ng model is that the 11 i otope n1odel pertain to a phy ical amount of C, of which there arc two possibl e sources - PS and fungal C- and are req uired to have distinctly different 13 C signatures. Ideall y there is negligible fractionation or rnixing of ources during the transfer of the resource from the source to the plant tissues (Dawson et al. 2002: Fry 2006). The photosynthesis 1nodel, on the other hand, consists of two processes, photo yntbetic C0 2 uptake and respiratory C0 2 losses, which together are one of the sources contributing to nc enrichment. Theoreticall y, the models are similar in that both versions include end-metnbcrs that do not receive/carry out one of the sources/processes (i.e., autotrophs do not gain fungal C and full MHs do not obtain C via photosynthesis). However, the assurnption of a linear cotTelation bct\veen fungal C and 13 C enrichment has no analogous context for the PS rnodel, and plants have non-linear photosyntheti c responses to light. Additionall y, there can be other physiological factors that influence not only PS rates but differential discritnination of 13C, as stated by Hynson ct al. (20 13), wh ich tn ay be v iolating the assurnpti on of linea rity of nutotrophic and PMH 11 photosyntheti c C discritnination. We co uld consider thi s instend as a sin1plc ratio tnodel, where the inclusio n of full Mi l gas-exchange va lues essentia lly acts as addtng a constant to 96 the P . This eli1ninates tn athetnatical probletns a ocia ted with potentially fracti onal or negative C0 2 exchange rate , si1ntlar to 1c; data and funga l derived N (%N 01 ) calculations (e.g., Gebauer and Meyer 2003: Zi1n1ner ct al. 2007). By eva luating these tnodcls w ith reference to input 81"c and P rate and other unpo11ant gas-exchan ge data, we Jntend to detennine how well the two n1od els agree and \\hat ph ysiological fa ctor n1 ay contribute to discrepancies betvveen n1odel e tJn1ate of ~oCDr Beca use of these un ceiiaintics in both this novel approach and in cotrunon iso topic tnixi ng Inodcls. sin1d ar cauti on n1ust be taken when interpreting e timated 0 oCoP a i generall y suggested for 0 1oCnt estirn ates (e.g., Hynson et aJ. 2013), e pecially for th e 20 10 data whi ch had \ ery s1nall sa mpl e size and Jacked plot specifi c reference . 4.2 Methods Site descriptions all years The six study sites were sampl ed over the growing sea ons from 2010 through 20 13, located within 70 km of the city of Prince George, Briti sh Colutnbia (Table 4- 1). Three sites were selected for preliminary data collecti on in 20 10 and 20 1 I . The first was located approximately 500 n1 west of the University of Northern BC catnpus grounds in what is known as ' Forests for the World' (UNBC). The second was near Willow Ri ver, approximately 30 km east of Prince George on Hi ghwa y 16 at a vehi cle pull-out to for a wildlife observation tower (WR) . The third site was located across froJn Crooked River Provincial Park on Highway 97, approximately 70 kt11 north of Prince George near the comJnunity of Bear Lake (CR). In 20 J 2, CR was the site of a field cxperi1nent ( cc Chapter 2 for full details). 97 Table 4- l. General locations. ecological desc ripti ons and target partialrn]coheterotrophic P]rolcae and fullrn]c oheterotrophic (M il ) \1onotropoideae c;pec ic c:; presence for all six stud; sites acrose:; a II stud; :ears (20 I 0 through 20 I 3 grO\\ ing seasons). Site L'NBC WRa CR Dl\1 Moi~th DL \r1esicc Pl\1 Dr) Year 20 I 0 - 20 II '0 I 0 - 20 I I 2010 - 2012 2013 2013 20 13 Latitude 53°53'35" N 53°54'26" N 54°28'58" N 54°26 '15" N 54 31' ~ 54°30'37" N Longitude 122°49" 12" w 122° 19 . 5 I II w I 22°4 0 . 26 II 'A' 122°38'53" 'A 122°42' 'v\ 122 41 '30" w Elevation BECd 800 111 765 rn 723 rn 703 m 710 - 730 m 720 rn ~BSd\\ 3/0 I (04) S8 S\v k I I 0 I ( 03.05 ) SB Sm k I I 03 S8Sn1k I 06-09 SBc..;rnkl '01(04, 05) SBSn1klf03 \r1AP (n1m) 494 931 727 (644) 727(644 ) 727(644) 727(644) \t1A T (°C ) 2.6 Subn1csic . 2.6 1.5 (3.5) Sub'\eric I 5 (3 5) 15(35) ~ubrncstc - ~ub'\cric - 15(15) <.,ubxcrrc <..ubh \Jnc \ u bh~c\·(l rt c SoilrnoistureJ lllC~IC Subrnesic subh\ oric 5oil nutrient0 Poor- rich Poor- rich \' cr: poor poor \' cr) poor - rrch Poor - rrch \cry poor poor Predominant sot! association Don1inion fabor Lake (PineY ie\\ -8 0\\ ron) Bear Lake Stcllako Unn1appcd. "'"1riab lc Bcdr L'"1ke Parent materialt' Mcdiun1 tc:\tured basal till C oa rse/mcd ium (tine) tc'\ture. nu' iaI a llu \ i aI !~111 ~ (glaciolacustrine) Sand\"' ....glacio!lu\ ial out\\ a\h terrace~ (sand clune C0111plc'\C\) Coar~e, rncJiun1 Coar5e le'\Lured !.!.lactal OUl\\USh .... Sand\" ....glacroflu\ rnl out\\ a~h terrace~ (sdnd June corn pi~,~~) Gra\CII] ~and) loarn-gra\ ell) loarn Gra\elh sand\ loam - gra\ ell\., loam\ sa nd (~i It' loan1) L oarn' sand - Variable - sand\ Ioa n1 to s tit .,\ cl '"l \ loam (son1c organic ) .... GrJ\CII\. ~and\" loan1- ....gr~l\elh loatn\ !)and Lonrn\ ~and tine sands Sot! texture ~c- ~ ~ - ~ line sands ~ 98 tc:\ture flu' ia I terrace\ ~ ~ ~ ~ ~ Table -t- 1 continued Soil t)pee l lun1o-ferric pod1ols Degraded d\"' stric bruniso ls (lu\ isol<)) Degraded .... d\"' stric bruni sols Orth ic and glc: cd regosoh ./ ./ ./ ./ ./ .../ ./ ./ ./ ,; ../ ../ ./ .../ ../ ./ ~ ll um o-ferric podzols (Degraded d) ~ tric brunisols) Degraded ._ d\"' stric bruni so I<) ../ ./ Pvroleae ~ ~111111£1j?l7ill7 umbel!t1ta Orth!l ia yecunda Afoneses zuuflvra P~rola asarifolla . Pvrola chlortlntha P~rola nunvr ~ v' Full MH l\:l ono tro po idca e .\fonotropa h_~popTIJ .s ../ ./ Jfonotropa untf/ora ../ Pterospora andron1edea ()recn Green Green. f\1111 Green.l\111 Grcen.l\ 11 1 Green Orchid s J\\ R: \1 oistu re n1ainl) drier than range. soi l texture most li~d) coar~c (gra\ els and ')a nd ~ ob,e n cd) \\ 1th 1111nor an1ounts o r sdt clay "'D\ 1 \It o 1st. 8 I.:C (biogeoc Iimati c ccos\- sten1 c la!>si lication) ,j tc "'e rie ~ 111a in h., 06 but nearer ri \ er 09 occu r~. l1 ~e h- ~orne organ 1c ~o ti s : tnain part ot s1te relati\·cly rn oi~..,t (o..,eepage. hi gh \\ater table) and ~ee tnin gl ) nutrient poor-n1edturn l \l o!>t l) dry (ridge\\ ith e\.posed rot~) to n1es1c n1ot sturc. but ')O rne\\ hat n1oister in lo \\C St I) ing t1rca. soil t) pe est1n1ates frorn Paul ~an born (pe rs. con1n1.). J ~ource tnatcrial for BI::.C and "'o il rn o i ~turc nutrient regnncs dcri\cd rron1 DeLong et al. ( 1993) anJ Del ong (2003). though e~ti1nated via indicator plant ">pcc Jc <> and pcr<)onal ob~e n ations. ~ ou rce n1atenal lor <>oil a')')OCtations. parent n1atcri als. tc\.turc~ and L) pes fron1 BC ~dOL ( 1989) and PJul Sanborn (pers. con1n1 .). \1AT (tncan annual tcrnpcraturc) and MAP (mean annual precipitation) for SBSn1h..l has rnultiple so urces. first \alues frotn DeLong et al. ( 1993 ). seco nd \a lues 111 brackets fron1 Canadian Carbon Prog ra rn (20 I 0) '-' ~ 99 Three additional sites within 7 ktn of CR were included for population surveys in 201 3 (See Chapter 3 for full details). The e were located along the Davie-Muskeg FSR (DM Moist), an un-natned access road to Davie Lake (DL Me ic) and nca r th e Polar Milllutnber yard (PM D ry). Choice of each ite wa dependent on the presence and abundance of tnultiple Pyroleae specie , the pre ence of full MHs, ease of access for gas-exchange equipment (20 10 through 20 12), and harvested si te~ with Pyrolcae species present in adj acent intac t forest (20 13 ). ite de cription for all yea r are suJn tnari7ed in Ta bl e 4- 1. The BEC va ri ants and soil characteri stic in Table 4- l were esti1n ated based on so iJ rnapping, techni ca l reports and personal comtnun1 ca ti on (BC MOE 1989; Paul anborn, pers. com1n.), indica tor plant species (e.g. DeLong ct al. 1993 ), and personal observati ons. All sites were co nifer-don1inated and located in the Sub-Boreal Spruce biogeocli1natic zone (BC MFLNRO 20 14; Meidinger and Pojar. 199 1). UN BC. WR, DM Moist and DL Mesic had the most diverse mix of species including Doug]as fi r (Pseudotsuga n1en:=ze.vii (Mirb.) Franco ssp. gla uca (Beissn.) A.E. Munay), hybrid white spruce (Picea glauca (Moench) Vo s x. engel!nannii Parry ex. Engelm .), and subalpine fir (A hies lasiocarpa (Hook) N utt. ). Black spruce (Picea nu triana Britton, Ste111s & Poggenb.) occun·ed at DM Moist, while at the other aforernentioned sites, tren1bl ing aspen (Populu.\' trenut!oides Michx.) and paper birch (Betula papy rifera Marshall ) were also present. Prior to mountain pine beetle (MPB : D endroctonus p ond erosae Hopkins) attack tarting around 2003, lodgepole pine (Pinus contorta Douglas ex Louden var. lat{(o!Ja Engelln. ex . Watson)\\ a also a con11non or dominant component at eac h site, with all but UNBC (selectively harvested for safety) containing residunl standing dead and rcgenerattng or sun i\ ing trees At CR and PM Dry sites, pine was the don1inant canopy specie and suffered 95° o n10t1ality, though n1any live seedlings to intennediate sized tnaturc pine and subalpine fir\\ ere present. 100 as well as a few small hybrid white pruce. A wide variety of con11non shtub and forb pecies were found at each ite, w1th tnost site havi ng hi gh diversit y of taxa except at CR and PM Dry, \vhcrc eri caceou vegetation do1ninatcd the understory. pecics details can be found in Land Managetnent Handbooks for the specdic sub7ones in Table 4- 1 (DeLong et al. 1993 ~ DeLo ng 2003). Onl y C. zonhcl/ata and 0 'ecunda ~ere present at all sites, while each ite vari ed in add itional Pyroleac as umtn an Led in Table 4- 1. Sample selection Each year of data coll ection had different purposes and there fore di fferent species elected for analy i of ga -exchange and/or natural abundance stable isotope ratios for 13 C and 1~. A a preli1ninary tudy, target pecics in 2010 included all Pyroleae species found at each site, as well as reference autotrophs and full MHs (see Table 4- l for site specific Pyroleae and MH specie ). Autotroph were all co nifers, ei ther subalpin e fir, hybrid white spruce or lodgepole pine. ln 20 11 , only P. asarifolia, C. un1bellata and 0. secunda were measured with the intent to perform a transplant greenhouse experi1nent. Reference autotrophs for this year were all eri caceous, and tncluded Vaccinium 111e1nhranaceum Douglas ex Torr. (all three sites), Arctostaphylos U1'a-ursi (L.) Spreng. (at WR and CR) and Va cciniznn myrtilloides Michx. (CR only); no MHs were n1easured since they were unlikely to survive transplanting. In 20 12, once n1ore a subset of species was chosen for an 111 .Htu field experiment, including C. urnbellata, 0 . secunda and P. chlorantha (see Chapter 2 for fuJI details). Again, full MHs were Jn easured for con1parati ve purposes, and autotrophic reference species were frotn a vari ety of shrubs and forb s over tnultiplc gui lds (e g., contfers. annuals, woody perenni als, va rious Jnycorrhiza l classes, etc.). ln 20 13. satnple for the population/disturbance stud y included any Pyrolcac and Monotropoidcae found at each of the l 0l three tudy areas. The coininon data across all years was natural abundan ce stabl e isotopes, whi le gas-exchange data were collected for three of the four year (20 l 0 through 20 12). Gas Exchange A1easurenzcnts All foliar gas-e"Xehange tneasuretnents were pcrfonncd non-destructi vely (with the exception of otne autotroph sa tnplc . c g. co nt fer~) us1ng a portable gas-exchange system (model LI-6400, L1 Co r Inc., Lincoln, E. U A) In 20 I 0, a n1inimurn of three san1ples per pecie \V ere measured 111 situ und er a broad range of atn btent conditions approxi1nately once per month during the growtng ea on (May - Septc1nber) U!:> in g a transparent coni fer cha1nber (tnodel LI-6400-05), while controlling fo r C0 2 co ncentrati on (400 J.Un ol mol 1 air) and now rate (500 J.tn1 ol -1). et photo ) nth eti c rates ( P : J.tln ol C0 2 m 2 leaf areas 1) and PAR (photosynthetically active radiation betw een 400-700 nm : ~uno I photons m 2 s· 1), using an external quantum en or (Ll-6400 990 1-0 13 ), were measured, as well as a vari ety of other important physiological and environtnental parameters (e.g., leaf and air tc1npcrature, transpiration rates, intercellular C0 2 levels, etc.). Hereafter PS and PAR units will use standard notation of J.tnlol m-2 s-1. Fully MH species were also measured once they had emerged in late July or August by enclosing stems within the chamber. Sampl e leaves were collected for isotope analysis foll owing final September tneasureinents. In 2011 , four plots per species were selected for a tran plant experin1ent. Photosynthetic responses to light (light response curves: LRC) were first developed in s1 tu in 1nid-J une by 1neasuring gas exchange rates at light levels of 0, 10, 25. I 00, 400, I 000 and 1500 J.tm ol m 2 s 1 using a leaf chan1ber with LED li ght sources (n1odcl LI-6400-028) . Leaves were taken at that time for earl y season iso topic analys is. Sarnpl cs 'A ere transplanted into pots and placed in co ntrolled growth chan1bers, which unfortunately had an unrorcsccn 102 progratntning probletn undiscovered until the tnajori ty of the plants had di ed; therefore, only the June field data are considered reliabl e. In 20 12, both an1bient gas-exchan ge and LRC n1easurcmcnt ~ occurred. Though ambi en t co nditi on \V ere modifi ed for expenn1ental purposes (see chapter 2), the treatments were well within the natural range of va riabllity and data for all trcattnents for each species were con1bincd. Ga -exchange n1easuretn ents were ahnost identi cal to those of 2010 and 20 ll except that ambient n1easuren1ents stm1ed 1n June rather than May, LRC dcveJopm ent used different light levels of 0. 10. 25, 100, 400 and 800 ~un o I m s 1, and were perfonned in 2 both June and August of 20 12. Satnple ize for 20 12 well exceeded previous years since replica te were required for the different treatments. ine plots with four sampl es (one of each treatment) v.'ere measured for a total of 36 indi viduals per species, including the wide • variety of autotrophs. Leaf satnples for isotope analysis were co ll ected in both June and September. Full detail s are available in Chapter 2. All photosynthetic rates were expressed on a hen1i -surface area basis (HSA: cm2). For san1ples measured under aJnbient conditions. HSA was determined for broadl eavcs optically, by scanning leaves or traced leaf outlines using a flat bed scanner (Epson Expression 1640 XL), and image analyzing software (Winfolia, v. Pro 2003d, Regent Instrutnent Inc., Quebec, Canada). Conifer needle surface areas were detennined using a volutne di splacen1ent method (Chen et aJ., 1997). Additional details on this method of surface area calculations for MH species are avai lable in Chapter 2. All species H A\ alue were re-entered into th e Li-6400 and final photosynthetic rates were recalc ul ated by the instrutnent prior to anal ysis. For LRC sa rnpJ es, th e chan1ber is exactly 6 cn1 2 and only san1pJes that were too small to entirely cover the chmnber area were rncasured optically as above. 103 Calculations to estunate 0 o p hoto.\:rnthetlc-den vcd carbon (%Cop) Plant ampl es co ll ected for tsotope analysis were prepared according to the 1nethods of Secti on 2.2, and sent to the table Isotope FacJltty at the nive rsity of Saskatchewan, Sa katoon. Canada for determination of natura l abundances of C and N concentrati ons (% 13 1 and %N) and stable i otopes. C and ' as per Eq l - 1. E tirnate of 0 oC or were calcul ated ba cd on both 1sotope and net photosynthesis d a ta~ therefore, \ve use the subscript ( I ~Ol fo r the iso tope rnethod and subscript rP~l for our photo ynthesi method. The fo llowing equations were used to determine satnpl e enri chrnents (E Af\tru-) u ing the 13C i otope [Eq. 4- 1a] and photosynthesis [ Eq 4- 1b] rncthods: 13 [Eq . 4- 1a] E~Af\. IPL H ,~o) - &' C~ \f\IPLb- 8 CRl , Au ro [Eq. 4- lb] r ~t\M Pl L( P~) = P S ~ \~1PI I - PSRI r !\Ill 3 where 8 C~ ·'\t-.tPLL and 8 13 CRrT \lJl 0 are the 8 13 C for any plant and the rnean of autotrophic 13 reference plants, respectively, and ~ P S ~At-. tPLt and PSRrr M il arc average seasonal net photosynthetic rates for any plant and the tncan of full MHs, respectively. Finall y, the% fungal- or photosynthesis-derived C in PMHs were calculated using the follow ing equations: [Eq. 4-2a] %CoH JSO) = (ErMII(JSOl/ E r-.111 ( 1 ~<))) x 100% [Eq. 4-2b] o/oCDrtPS) = (EPMII(PSl / EAlJfO(P~) ) X 100% where ErM II OSO) and ErMJHPS) are individual PMH plant i otopic and photosynthetic enri chn1 ents, respectively, EMII(ISO) is the n1can isotopic enrichn1ent fo r M H references and EAu 1O(PS) is the tn ean photosyntheti c enrichment fo r autotrophic references. To be able to directl y cotnpare estimates frotn both 1nethods, the total C budget of a plant ca n be represented by: [Eq. 4-3 ] 100% %C J)J + %C DP 104 where a plants' total Cis deri ved frotn fungal associates (C[))) and/or photosynth esis (CDr). By sin1ply ubtracting %CD 1 ( l~O) frotn 1OOOJC>, \Ve obtain a 0/o CnP( I ~o) es tim ate that can be co1npared to the %C DP(P. l estin1ates. With regard to 0 oCnr calcul ati ons, the fo llowing 1nethods were used. The 2010 estimates were ba cd on site-specific reference only as per Gebauer and Meyer (2003) since a a preliminary tud y, peci fi e plot Vv ere not e tabiJshed. econd, du e to th e experim ental nature of the 20 12 data (a randomiled bloc k design). plot- as well as trea tinent-specif1c autotrophic references were u ed to n1 ainta1n spatt al resolution and to account for the mi croenvironmental conditions in1po ed by the experimenta l treatincnts (Preiss and Gebauer 2008), resulting in only one value fo r autotrophs per plotltreatn1cnt. The treatJn ents were all well within the natural range of vari ability at the site so were not considered uncharacteri sti c, but did cause significant differences in PS rates (see chapter 2 for trea tm ent conditi on deta il s and PS results). Third, du e to the scarcity of fu ll MH references and occurrence onl y outside of plots (20 12 data), few reference san1ples were used and only at the site level. Lastl y, isotope values per plot and treatm ent for all autotrophs and Pyroleae satn ples were the average of sampl es collected in June and September 20 12, and in some cases July and/or August autotrophs were included since species va ri ed at each 1neasuren1cnt peri od. This covered a greater range of possible autotophic references as well as a]lowing fo r changes 111 both isotopes and PS rates over the season. Statistica l A na6Jses All data collected during th e fo ur years of thi study were tested to dctcrn11nc species differences in 8 13 C and 8 15 N, and C and N concentrations. Gas-exchange \ anables tnea~urcd in 20 10 and 20 12 were also tested fo r species differences in photo ynthet1c, co nductance, c1nd 105 tran ptratton rates, an d C , Ca ratio Til ..;E,. c~D ~C.. and us:l" and each yea r separately. \\herem, C and . eparately, all four C 1 te t were penrorn1C(I •ror cac )1 s1te co ncentrati ons were onl; te~ t ed for eac h year vanabl e ''ere abo t e~ ted for ~pcc t e<> dtffcrencec;;, fot all Sites and yea rs con1bined (grand mea ns) The gas-c'< change data were only te5tcd for each site and year separately. Linear tntxeclrnodels '' 1th tdak-con cctcd post hoc con1parisons were pcrfonncd \\ hene\ er randon1 e fTech ''etc tncluded or non- tnd cpend et data \\' ere t c~ t ed and assurnptt on of normalny and h o n1 o~ceda\lH..tt) '' et e n1 et ba<;ed on Shapu o Wdk ~ and [ C\ cnc · te<;t. respccti\ el) Rcle\ ant c;;, uhJ ec t grouptng \ ariablec;;, and or randotn f~1 c tors Incl uded grouping variable of plot and transect for the 2012 data and 1ndi vIdual <:, Jte tests in 20 13, re. pectively, as \ve il a random ~ l o p e~ for the 20 12 treatrn ents. Onl y th e 1nc1Ividual site te ts for the 2013 8 11 and 8 1" data and 6 1'c data for 20 12 met th e hotnogcncJty of vari ance as umption. Additionally, onl y sotne of the 2012 gas-exchange data had hon1ogencous van ance . Therefore. one-V\'ay A OV As assu tning unequal variances \\ere pcrfonncd to evaluate the pecies difference tn eac h van able \\hen hornogencity ~ as notrnet \Vtth the Welch F-rallo reported. All other data that had hotnogeneous vananccs v. ere tested u in g one-way ANOVAs with unadjusted F -rati os. Non-hotnogeneous data used Dunnett's r 3 post hoc pairwise comparison to tn aintain co ntrol over fatnil y-w ise error rate whi le being rnost appropriate for small san1ple sizes and pau-v. isc cotnpari sons (rather than con1pari ons to a control) ~ othern ise, hotno cedasti c data u<5ed I uk.~) 's H.._ 0 post hoc co1npan ~o n ~. This is the first known exan1plc of us111g pho to~ynthe is data to cst unatc 0 /oC DP (and by default 0 oCDJ ), so in order to assess how \\ell the cstin1ate of 0 oCnp agreed \\tth each other, paired t-tests were pcrforn1 ed to deten11111 c whether 0 oC nPtP'- l cl IfTc1eel ~ 1 gnt1i cp c~t un atcs were then tested first by site and yen1 separately, nnd then con1htncd Ilon1ogcnctt) or 106 variance assumpti ons were only n1 et for WR, CR (both variables) and %CoPci~O> for UNBC. Post hoc anal sis for th ese te ts used Tuke y' If 0 test. The 20 12 data and %Cr)PcP~> data for UNBC in 20 10 used the Welch con cct1on factor and ]~~- rati os. wi th Dunnett 's T3 post hoc cornpari on . AIJ anal yse 'Nere perfonned using P Vers ions 2 J or 22 ( P S Inc., Chicago, IL, U A) and all p-va lue \\ere co nsidered signifi ca nt at a <' 0. 1 . 4.3 Res ults Specie differences 111 photo.\) 11/hesi\ rates and () 1 ~(' 1 The expectation for thi study wa th at along the autotrophic-n1ycohcterotrophic continuum, average P rates should have a negati ve relationship to 8 11 C values such that 11 autotrophic species wou ld have the hi ghe t P rates and lowest 8 C, MH species wo uld have the opposite, and the PMH Pyroleae should show a grad ient of values in between. Con1parisons of ave rage sea onal P rates with 8 13 C values for 2010 and 20 12 showed the clearest exan1ple of the expected relationship at CR, parti cularly in 20 12 (Figure 4- 1). The 20 12 PS rates of 0 . secunda and P. chlorantha were significantly lower than autotrophs and C. Lunbellata , with corresponding significant enrichrnent in 13 C (p < 0.00 l for all significant differences; Supplementary Tables S4-1 and S4-2). The full MHs always had net C0 2 etn issions (respiration) regardless of light levels, and were hi ghly enriched in 13 C (Figure 4-1). As a result, the full MRs were significantly di fferent than all the other species for both 13 PS rates and 8 C values fo r all tlu·ee sites and both years (p < 0.086 except at U BC \\. hen no MHs were tested for 8 13 C). No oth er species difference were detected at WR and CR 1n 20 10 for PS rates (Tables S4-2). 107 CR 201 0 WR 201 0 UNBC 2010 CR 201 2 • ·-to Cl) Q)- ..r: ":" ..... tfJ 5- e N ~. - ~ E 0 ..!: a_ 0 E -5- - • -26- 0 0 ~ $ • ..... • R :::1. z do ·~ . 0- • - ..... Q) .... ~ .... 0 ~ • • ~~ <> -28- • u ('") .- LO -30 ~ <> ~ -32 -34 - WR 2011 UNBC 2011 - CR 2011 -30 - 0 0~ u -32 - .- • • • ('") LO 0 -34 - T r I I f I ITT J f ~I I I I I I I I I I I I AutoCu Os Pa Pc MH AutoCu Os Pa Pm MH AutoCu Os Pa Pc MH AutoCu Os Pa Pc MH Spectes Figure 4-1. Distributi on of average seasonal net photosyntheti c ( P ) rates (Jlmo l m -2 s- 1) under ambient conditions in 20 l 0 and 2012, and 2010--20 1? average 8 13 C (%o) va Iues for several autotrophic plants, putative partial mycoheterotrophic Pyroleae species and full mycoheterotrophs (MHs) at three sites in central BC. Shaded boxes represent the interquartile range (IQR) with the line at the n1edian and the diarnond repre enting the n1ean. Whiskers represent data within 1.5* IQR and points are outli ers beyond the l .5*IQR. Specie codes are: Auto = Autotrophs (various spec i es)~ Pyroleae: Cu - Chimaphila umhcllata, OsOrthilia secunda , Pa = Pyrola asari(olia, Pc = P. chlorantlza, Pn1 = P. nzinor~ full MH: Mon otropa uniflora (UNBC and WR 201 0), M . hypopi(vs (CR 20 12) and Pterospora andromedea (CR 20 10/ 12). At UN BC, 0 . secunda and P. asar((olia also had significantl y lovver PS rates than autotrophs (p - 0.01 and 0.002, respectively), but it wa P. chlorantha that wa ~ significantl y more enri ched in 11 C than autotrophs, C. umhellata and P. asan(oha (p <.. 0.076: rablc S..t--1 ). This tnay have been a result of one outlier, however, which had <.~ 1 ~ Cor 4°/oo greater than the other samples but did not exhibit substanti al ly lower P rates as wou ld be C'\.pccted based 108 on its isotopic signature. The 20 10 811 C va lues for 0 . secunda at WR, and P. chlorantha and 0 . secunda at CR were al o signifi cantl y hi gher than autotrophic references (p 0.068, 0.00 I and 0.065, respectively): 0 . secunda also had signjfi cantly higher o1 C values compared to 1 P. asarifolia at CR (p - 0 002: TClble 4-1 ). 11 The data for 20 I I sho\\ a si1n i Jar story for both o C valucs (Figure 4-1 ~ Table S4- l ) and LRC (Figure 4-2: Table 4-2 ). thou gh stat1stic8l testc;; were not perforn1ed for th e LRCs. Simil ar to 20 10, autotrophs \vere generall y th e 111ost depl eted in 11C, C. Lunhellata was sin1ilar or lightly 1nore enn ched (though h1ghl y variable at U BC). and 0 . secunda was consi tcntly n1ore enriched (Fi gure 4-1: Table S4-l ). Prrola asarifo!ia was n1orc enriched than autotrophs at UN BC and WR, but was actua ll y more depleted than autotrop hs at CR. Only CR had significant pecies differences, wi th 0 . secunda being 111ore enri ched than autotrophs a well asP. asarifo/ia (p = 0.0 15 and 0.0 11, respecti vely) . Overall 813C values were more depleted for this year co1npared to 20 l 0 (Figure 4-1: Table S4- l ), but sampl es were collected in June rather than Septetnber (or both months) and the weather was n1uch cooler and rainier than 20 10 and 20 12. The 20 11 LRC PS rates at li ght levels of 400 j.lmol 111 -2 s-1 or greater showed the expected relationships, with autotrophs usually havi ng the highest rates in general, C. urnhe/lata having similar or only slightly lower rates, whil e 0 . secunda and P. asan(olza had the lowest rates (Fi gure 4-2: Table S4-2). At WR, however, the PS rates of P. asanfoha exceeded those of autotrophs at the higher light levels. Autotrophs usuall y also had the hi ghest respiration rates and light cotnpensa ti on points (the level of inad iance ~here photosynthetic C0 2 uptake is equal to respiratory C0 2 lo ses), except at CR in 2011 \\hen C. umhellata exceeded autotrophs in those paran1eters (F igure 4-2: Table 109 4-2) Chimaphila umbel/ata Autotrophs 10 0 7 5I N 50- V) , _ ... , ,, ------ ----- --------- ,en.::~.~~.-.::.:.-.=:~ 1 ., ............. .-..... ······-··· . ., ·... , ·~,~~----+---• I E 2 5- 0 ~ , -~ --------- was hi gher than %CDPC P~l· Significant species differences occuned fo r all 4 individual site/year con1binations and the con1bined dataset (both estimati on methods) with the exception of 0 oC nr(P\l at WR in 20 I 0, where no species e ffects occurred (F4 10 2.074. p = 0.159). The con1bined . . ite year data showed C. tunhel/ata was pritnaril y autotrophic, with both e ti1na tes ind1cat1ng 94° o C via photosynthesis and neither differing from autotrophs (p > 0.3 05 ~ Figure 4-1 ). \\ hich 1 12 were not always significantly different at the indi vidual site level (data not shown) . At UNBC in 2010, one P. chlorantha sample (tnenti oned in the previous secti on) was hi ghly enriched at -25.04%o. es enti all y the san1e as full y Ml I M onotropa uniflora sa mpl es. This resulted in a site average of only 3 8° o C nr 11 ~ 0 l for the species. significant ly differin g frotn all other pecie (p < 0.076) except 0 . ~ccunda (p - 0 422) and was the lowest of all %Cnp estimates (data not shown). Th1s wa also the one case \vhere %C oro<;ol was much lower than %Coro><;), with the latter e tin1ate at 78o/o. In contrast, P. asanfolia had tnuch higher e titnates of %CDP!ISO) co tnpared to <}oCoPW"l at U BC and CR in 20 10 (not included in 20 12 sa1npling). In both ca e , 0 ~Cnro~nl estitnates were 100%, whereas %CDP(P<;) estirnates were onl y 69%. Becau e of the e differences, the con1bined data for P. as an(olia onl y differed significantly frorn autotrophs and C. urnhellata for %Crwrr<;J estin1ates (p < 0.067~ Figure • 4-3). The %Cor(PSl estimate fo r P. n1inor wa also sotn ew hat less than %CDrrt<;O) at 78% and 86%, respectively, but did not differ from any other species due to its occutTcnce only at WR and thu s limited sample size. Based on both methods, ranking species along the autotroph -MH continuum shows C. urnbellata as the most autotrophic at --95% Cor. Pn·o/a minor showed sotne PMH nutrition with just over 80% autotrophic C, while 0 . secunda and P. chlorantha showed considerabl e PMH nutriti on with both methods averaging to about 70°1o Cor. Averaging the Jnethods for P. asar((olia results in slightl y more autotrophic C ga ins than P. 1ninor (86°o) but the difference between the two n1ethods n1akes this ranking uncet1ain. SJnall san1pl e ize may have played a rol e in the di screpancy of values but the data 1nay reflect other itnpo11ant ecological and ph ys iological fa ctors. 11 4 15 Species differences in 6 N. %C and %N Though this study was mainl y focused towards carbon heterotroph y, results were also obtained for 81" . 1 Autotrophs aln1o t alv. ay had nega ti ve (depleted) 8 "N va lues except at UNBC and WR in 20 l L in which they were posi tive (ennched; Tab le S4- J) This may have occutTed due to the species sampled for eac h yea r. In 20 10 onl y conifers were sarnpl ed and were depleted in 1"N, whereas in 20 11 cri caccous species (1nai nl y Vacc111ium spp.) were the only autotroph , re u]tlng in enri ched 1" 1 at the aforetnentioned sites. The negati ve 8 "N values at CR in 20 11 tnay haYe been due to the inc]uston of A. u1·a-ursi, an evergreen rather than deciduou specie like T'a ccinium species. General1y the Pyroleae were tnore enriched than autotroph , u ually with po iti e values, but aga in a few exceptions occulTed where the Pyroleae were either less enri ched than autotrophs or had negative va lues, or both (Table • S4-l ). Full MH were always highly enriched compared to autotrophs and the Pyroleac and always significantly differed from other specie when included in analysis (p < 0.084 ), with the exception of P. chlorantha in 2012 (p = 0.77 4 ~ Table S4-1 ). Aside from UNBC in 20 11 and DL Mesic in 20 13, all tests resulted in at least one Pyroleae species being significantly more enriched in 1"N tha n autotroph or another Pyroleae species. Similar to full MHs. P. chlorantha was always significantly n1ore enriched than autotrophs and the other Pyroleae species when included in analysis (p <' 0.0 19) except at CR in 20 l 0, where it did not differ signifi cantly fron1 P. asanfolia (p = 0.133~ Table S4-l ). Aside fro tn M oneses uniflora, the four other Pyroleae species \\ ere significantly ennched in 15 N compared to autotrophs in multiple individual ite/yea r tests, so1netin1es ddiering fron1 each other as we ll w ith 0 . secunda usuall y hav ing hi gher enri chn1ent (Table S-1--1 ). Though individ ual site/yea r tests did not always result in signifi ca nt differences or consistent spcc tcs differences, the co n1bined dataset revealed the foll owi ng: au totrophs were significantly l 15 depleted in 15 compared to all other pec ies (a ll p < 0.003): M. uniflora was slightl y depleted and significantly so in relation to P. asan(o ha and 0 . secunda (p - 0.088 and 0.04 7, respectively); C. tunhe/lata and P. nun or were enri ched at intcJmediate levels and did not differ from each other or fron1 the three aforen1entioned Pyroleae, and finall y~ P. chlorantha and fu ll MHs were aga in significa ntl y enri ched co1npared to all other species (all p <" 0.00 I ), with full MH al o diffenng frotn P. ch/orantha (p - 0.00 l: Tabl e S4- l ). Annual species tnean for C and co ncentrati ons showed nu1n crous instances o r signifi cant pecie difference , probabl y du e to the in c rea~e in sa1nple size by con1bining sites. Both eletnent bowed con iderable vari abdity between species and yea rs, with little consistency in pattern (Table 4-1 ). For C concentrati ons. onl y P. asan fo lia and P. chlorantha showed similar trends across years, with P. asarifolia usuall y having the highest average concentration and P. ch/orantha the lowest. Both differed significantl y frotn the other Pyroleae species except P. 1nin or~ P. chlorantha, along with M. uniflora, also differed significantly fro1n autotrophs for the grand means (p < 0.001 ). Chimapln /a umbellata had slightly greater C concentratons than 0 . sec.:unda, significantl y di ffering from each other for the grand 1neans (p = 0.073 ), but not autotrophs (Table S4-1). Small sa1nple size and/or high variability resulted in P. rninor and full MHs not di ffering fron1 any other species for the grand tneans of C concentration, though the latter was signifi cantl y lower than autotrophs in 2010 (p < 0.001 ). TheN concentration data was sitnilar to C concentratons in the lack of con i tent trends but differed in the ranking of species. Though AI wnflora was the onl y spec ies exceeding 2% N, s1nall sampl e sizes resulted in fewer significant dilTcrenccs than would be expected, such that it only differed fron1 autotroph and C. t11nhel/ata (p = 0.065 and 0.033, respectively; Table S4- l ). Instead, the full MHs and P. ch/oran tha \\ ith shghtly lo\\ cr N 1 16 concentrations ignifi cantly differed frorn autotrophs, C. u!nhellata , 0 . secunda and P . asari(olia (p < 0.025). In tnost cases C. lnnhellata had the lowest. often significantly, N concentrations of only -- 1°/o. with the grand 1nea n being signifi ca ntl y lower than all species except P. n1inor (p < 0.043 ). Again, sn1 all smnple size was a fac tor in the lack of di fferences between P. nu nor and other spec re for concentrations, even though tt had relati vely higher % than everal pecies (Tab le 4- 1). For the hi gher n species, low variability in the data resulted in differences a maJl as 0 1° o bei ng signtfi cant. 4.4 Di scu ssion Thi stud y aimed to characten ze the stable 11 C and 1"N isotopes and especially photosynthetic rates of each of the prevalent Pyroleae species fo und in the central interi or of • BC. Based on the lack of photosynthesis data for these species in the li terature, we chose to focus pri1naril y on C nutriti on. Over four years of data coJlection, three of whi ch included gas-exchange measuren1ents and all of whi ch included isotope analysis, the resul ts strongly support previous studies oftnainly autotrophic C gains in C. umhellata. Si1nilarly, though frequently showing high variability in isotopic signatures and photosynthetic rates, P. chlorantha and especially 0 . secunda indicated more or less con i tent reliance on mycoheterotrophic C gains (Figures 4- l through 4-3: Tables S4- l and S4-2: see Chapters 2 and 3 also). Data from previous literature on all Pyroleae species found in thi s study (e,cept P. asari(olia) were presented in a di agran1 with the general pl acern ent along the autotrophic- tnycohctcrotrophic continuu1n (see Figure 7 in Johansson 20 14) The three tnost pre\ alent species in this study showed highl y consistent agrecn1cnt with that figure. Look.tng nuunly at the 2010 and 20 12 data with both PS and iso tope tn casurcinents, C. umhcllata had 117 photo ynthetic rate and 813C values indicating about 95% autotrophic C gains (Figures 4-1 through 4-3 ). The P rates, 813 C va lue . C conce ntration and %Cor data for thi s species were not found to be ignifi ca ntly different than autotrophs for any year in any cotnparative tests, with the exception of the 01oC or(l O) at CR in 20 10 (not sho,vn). Both 0 . secunda and P. chlorantha were shown as PMH spcclCs by Johansson (20 14 ). gaining - 63 ° o and 68° o, re pecti\ ely (based on \ 1sual assessment as no nutn erical values were pre ented). In our case, 8 13 C data resu lted 111 slightl y hi gher estimates of %CnP(JSO) at 78°o and 75°o fo r 0 . secunda and P. chlorantha, respectively, but were offset by usually lower PS rates and 0 oCoPW\ ) (Tables S4- l an d S 4- 2~ Figure 4-3). Quite remarkabl y, our combined ite/year 0 ~Cor(PSl va lues were nearly identi cal at just over 62% and 67% over the sites and years (Figure 4-3 ), though individual site averages ranged frotn as low as 38.2% • Cor(tSO) for P. chlorantha at UN BC up to 95% Corer 1 for 0 . secunda at WR in 2010 (data not shown). The lesser studied P. minor and M. uniflora are shown as autotrophs in the diagratn by Johansson (20 14 ). The highly depleted 13C of M . un(flora (Table S4- l ) indicates it does not receive fungal C and fits under the autotroph status as reported by Johansson et al. (20 15). However, our study had few satnpJ es and lacked PS data, and Johansson et al. (20 15) suggest that their conclusion of adult autotrophy be considered tentative since theirs was the first and only comprehensive study evaluating this species. They propo e that since M. uniflora is comtnon ly found in 1noister, richer habitats than the other Pyroleae species 1t tnay assi1niJate C differently. Thi s n1ay be important in und e rstandin g'~ hy it was the only Pyroleae to be signifi ca ntly depleted cOin pared to autotrophs, rather than enriched (fable S4- l ), but is beyond the scope of thi s stud y. The final putative PMH pcctcs P. nun or has also been reported as autotrophic in the adult stage (Z1n1n1er et al. 2007, Johansson ct al. 11 8 20 15) Whtl c tlu 0 1 o rwestunate due to the Yery stnall n, P, rates were about half that o fautotro phs~ 8 '\ va lues \\ ere 0 pecies did not tatl sti call y dt ffcr fron1 autotrophs tn any Jneasurctnents or 1°/oo more enri ched th an autotroph <; (I 1gure 4- 1: Tables S4- J and S4-2), and oCnp cstunates we re only 78°o and 86°o Cnp (Figure 4-3), posstbl y du e to PM I I nutritton in orne cases. The cstin1ate for P a\anfoha \\ ere the n1 ost contradictory, the 1sotope data provided e\ 1dence of autotrophic C nutn t1on tn n1o~ t ca~e~. \\ 1th t\\'O ~ 1t e ',pecdic cases or I 00° o CnP(I\0) and a\eragtng to 95°o c()P( I\0) ()\Crall (F tgurc 4-3) On the other h a nd ~ p, rates were u ually con iderab ly lO\\ er than autotrophr., except at WR 1n 20 J 0 (Table S4-2) resulting tn an overall c titn ate o f only 75°o CrwiP">(Fi gure 4-3) Interestingly, l1 ght tesponse curves in 20 11 indicated sufficient photosyntheti c capac ity und er most li ght levels th at were cotnparable to autotroph in general (Figure 4-2) The fi eld condttio ns du ring those mea urcn1ent were cool and mot L indtca ttng photosynthetic li rnt tatto ns under ht gh hght, temperature and or drought condition in later uJntn er. Sitn ilar to P m1nor, oth er studies on this pcc1es are lacking and as uch it is dd1icult to deten11ine ~ h ere tt fits 111 the host-parasite spectrutn, though Hashimoto et al. (20 12) l11Cntion unpublished 13 C da ta indicatJVC or PMH strategies. LoH photosrnthetic rates 1nasku1g dee,ree of carhon n1ycohcterotropln? It is ac tu ally the discrepancies between the t~ o tnethods of 0 oC DP e~t 1 n1atton 111 the~e s p ec i es~ particularl y those showing SOI11 e IC\ el of 111ycoheterotroph), \\ htch 111d) fC\ cal tn1p011ant ph ys iological characteristics of the Pyro lcac species. ln a recent book chl1pter on Jn ycoheterotroph y, Hynson ct al. (20 J 1) raises son1 c co nsidcratt ons or applvtng ltnetlt nlt\.tng rnodels to Mil food webs. One or the issues di SCUSsed is"' hcther the as~U111pttOil'-1 or 1l 9 I inearity betVveen the 8 11C ignatures of reference autotrophic end-tn cinbcrs and those of the target pecies arc vio lated. This could occur bcca u e of potential differences across environ1ncntaJ gradients and/or differing rates of photosynthesis, which may actually 1nask the apparent degree of n1yco heterotrophy 111 PMH spec tes by essentia lly dduting th e 13Cenriched C via n1 yco1Th17a with more 11 C -depleted photoc:;ynthcti c (relative to autotrophic photo yntheti c C). By follo\ving co mn1only used smnpling procedures (e.g., Hynson et al. 20 12), we a un1e env 1ronJncntal vari ability \\las minimi£ed and l1kcly did not play a large role in differential 8 1~C values between Individuals and species at a given site. It 1s clear from the re ults, however. that in many case the Pyroleae tudied do have lower PS rates than autotroph , often signifi cantly so (Figures 4-1 and 4-2: Table S4-2). In the sitnplified tnodel by Farquhar et al. ( 1982, 1989) describing photosyntheti c fra ctionation of 13 C. the tnain eli criminating teps are diffusion of C0 2 into th e leaf through sto1nata and net carboxylation by Rubisco. Stomatal diffusion is regulated by stomatal and boundary layer conductance, and in conjunction with effects of transpi rati on, results in differences in C 1:Ca ratios (Farquhar et al. 1 982~ Farquhar and Sharkey 1982). The full rnodel includes a variety of other paran1eters that are often assu1ned to have negligible effects on 13 C di sctiJnination, including n1esophyll condu ctance and (photo )respiratory discrimination relative to photosynth etic products used in respiratory processes (Gha hghaie et al. 2003; Werner et al. 2012). Hynson et al. (2013) identify two of thee factors that could cause differentialleve1s of 13C discri1nination and assin1ilation between autotrophs and MH plants, that of higher CI:Ca ratios due to lower PS rates and grea ter use of respired co:! in the latter grou p. The first aspect of low PS and hi gher Cl in PMl ls would result in better equdibrat1on between C02 concentra tions of the intercellular spaces and atinosphcre ( \ ia stotnatal 120 diffu ion), resulting in a higher C,:Ca ratio. This essentiall y means that there wou ld be a greater arnount of C0 2 tnolecul es in the intercellular space con1pared to a plant with lower C,:Ca ratio. Assu tning the arne 13 C: 12 C rati o in the source C0 2, a greater absolute a1nount of 12 C-C0 2 hould be present when C, i hi gher, diffusing to th e s1te of carboxylation quicker and increa ing di critninati on agai nst 11 C (Farquh ar ct al 1989). In 20 I 0, the only significant species di fferences occurTed [or MH , which had co nsistentl y h1gh C, C'a ratios that were not unexpected since they only respire C0 2 (Table 4-2). have few ston1ata (Leake 1994) and presumabl y have considerably IO\\'er diffusion rates than photosyntheti c pl ants. De pite a lack of signifi cant difference in the 20 10 data, the ratios for P. asanfolia and P. n1inor at CR and WR, re pectively, were both 0.09 hi gher than autotrophs. Simil arl y, at UNBC autotroph had C,:Ca ratios of 0.69, while all the Pyroleac were considerably higher at 0.88-0.91 (Table S4-2). In 2012, the much larger sa1nple size led to significantly higher C,:Ca ratios in 0 . secunda and P. chlorantlza cotnpared to autotrophs (p = 0.048 and 0.009, respectively), all examples which lend support to the theory In almost every case where C,:Ca ratios of the Pyroleae were at least 0.05 higher than autotrophs, PS rates were considerabl y lower (Table S4-2). Based on this evidence, it is very plausible that low PS rates and consequently hi gh C, concentrations could contribute to increased 13 C discri1nination and therefore may be lowering the apparent! level of tnycoheterotrophy in PMH Pyroleae. Nevertheless, there was a trend of lower C,:Ca ratio in Pyroleae at lower light levels (e.g., < 100 ~m o l m -2 s-1) but hi gher C,:Ca rati os once exposed to higher light levels. The opposite occuned in autotrophs, possibly refl ecting differences in shade \ er us sun adapted species. Und er Jow light conditions of earl y tnorning and e\ cntng or 111 deeply shaded habitats, C,:Ca ratios tn ay be n1ore equ atable between autotrophs and Pyr oleac than in thi s study where gas-exchange tncasuretnents took place in peak daytirn e hours and 12 1 frequently not in the most shaded areas (e.g., see Figure 2- 1), and n1ay balance out 13C di crirnination over the long tenn. Since C,:Ca ratios and P rates are parti all y d1iven by sto1natal conductance, it could be ex pected that there are ton1atal liinitattons to P rates 111 the Pyrol eae, th ough lower stomatal conductance 1s known to occur in shad e adapted plants whereas high conductance in un plants fa cilitate rapid C0 2 uptake to fulfi ll b1ochen1t cal demand (Kriedetnann 1999). According to Farquhar ct al. ( 1982). \\hen assin1il ati on rates arc reduced due to low stomatal conductance, c. should actuall y dec rea e and 813 C increase, whereas when leaf tnetabolisJn limit assimilation, C, hould increa e and 8 13 C decrease. The only instance where PS, conductance and C, :Ca rati os were cornparativcly low and indicati ve of stomatal limitations toPS wa for 0 . ·ecunda at CR in 20 10 (Table S4-2), although all species including ' autotroph exhibited some tomatal closure durin g the hottest dri est part of su1n1ner in 20 I 0 at all sites (data not shown). Similarly, the effects of mesophyll conductance on assimi lation rates have been found to be controlled pritnarily by biochemical C0 2 den1and, influencin g the C0 2 concentrati ons at the site of carboxylation (Cc) and therefore 13 C discriminati on. GeneralJ y, findin gs have shown in fairly consistent positive correlations between ston1atal and tnesophyll conductances such that C, and Cc are maintained at appropriate levels for biochetnical C0 2 optimization. Plants exhibiting low PS and stornatal conductance rates tend to ha\ e low mesophyll conductance as well , though tnesophyll conductance can increase to n1aintain positive carbon balance if stoJnatal conductance is low to consen e water (Lauten et al. 1 997~ Piel ct al. 2002; Vrabl et al. 2 009 ~ Martins ct al 2014 ). It secn1s likely the Pyroleae would have low mesophyll conductance if this relationship holds tru e. based on thetr ~to rn a tal condu ctance rates. It is uncer1ain whether C ~ and 11 C di sc rir11ination wou ld be substantiall y 122 different in these plants co mpared to the reference autotrophs, but Lauteri et al. ( 1997) found low tnesophyll conductance and lower th an predi cted 11 di scriminati on in chestnut leaves. 11 High mesophyll re istancc to di ffusion (low conductance) could increa e 8 C signatures and thus influence interpretation of PMH C gains These pat arnctcrs would need fu1iher stud y in the Pyroleae to determine their inDuence on ass irnilati on rates as welJ as isotope signatures, and possible itnpacts of Jow c(. on photoresptratton. This leads to the other aspect potentt ally mask ing the apparent degree of heterotrophy di scussed by Hyn on et al. (20 13 ), that of hi gher usc of plant-respired C0 2 in PM! I pl ants than in pecie with higher C0 2 need (Wetner et al. 20 12: ll ynson et al. 20 13 ), though the authors do not indicate whether this applies to mitochondri al respiration (often referred to as dark re piration, ~, s in ce it is inhibited though not absent in the light) or photorcspirati on, or both. Fractionation effects of Rd can result in C0 2 that is enriched or depleted in 13 C, and are dependent on many complex factors primaril y related to: a) the specific position of 11C atotns in hexose sugars, usually in the C-3 and C-4 positions, which in turn arc dependent on fractionation differences during the Calvin cycl e~ b) different source pools utilized for respiration and related metabolic pathways, and; c) enzyrnatic effects of decarboxylation reactions (Gleixner et al. 1993; Ghashghaie et al. 2003; Badeck et al. 2005~ Werner et al. 20 12). These factors alone could result in substantial differences in 13C signatures between Pyroleae and autotrophic species regardless of re-assimilation of respired C0 2 , but arc highly cotnplicated processes and fu r1her study on the pat1icular n1etabo lisn1 of Pyrolcae as well as funga l symbionts is needed. Photorcspi ration, on the other hand , can exceed Rd by 3- to 5-fold in C1 crop species. (Zelitch 1973 ) and Rd in the li ght has been estitnated at ~ or less of Rd 111 the dark 111 co1Tec plants under sun and shade trca ttncnts (Martins et al. 20 14) Add itionally. dtscnn11natton 123 against 11 C dunng photore ptratt on depends n1atnl y on decarboxylation processes, and though mtxed findtngs ha' e been repo11cd. tends to result In 11 C depletion of resptred C0 1 and subsequent ennchn1ent of the. ou rcc sub<;tratc pools (Ghashgha1e ct al 2001 ). Therefore we as un1 e the potential usc of resptred CO , Indica ted by Ilynson et al (20 11) pertains tnainl y to photorespirati on. wi th th e apparent le\ el of dt sc rimtnat1 on in plant ti ssue being dependent on stomata l co nductance and the extent of rc fixatt on of the resp11 eel ('0., (G ha hghaic et al 2003, \Verner ct al 20 12) Photorcsptration Increase~ under condttt ons of hi gh li ght. tc n1peratu re~. and 0 ? concentration , often a a re ult of ston1C1tal clo ure to preserv e watet undet drought ~tress conditi on , effec tively reducing net PS rates (Chav es et al. 2002~ Gha~hg h atc et al. 2003 ). Since shade adapted species reach maxitnu1n P rates at lower li ght levels thCln plants ada pted to hi gher light. at equivalent irradiancc, shade species such as PM H plClnl<;, should be rnorc prone to photoinhibition than rnany autotroph . As a result, they ~h o uld tncrease photorespiration as an energy di sper<;a l or uttli;Cltion n1echan1s1n to reduce phototnhtbitton and photo-oxidative damage of the PSII reaction centres t Gcrbaud and Andre 1980, Kri cdetn ann 1 999~ Takahashi et al. 2007). Low N content has Cllso been round to reduce P rates (Lauteri ct al. 1997; Verhoeven et Cll. 1 997~ Piel et al. 2002), but based on the generally signi fi ca ntl y hi gher %N and 8 1"N in the Pyrolcae con1pared to autotrophs (Tahl e 4- 1), lo\\ N is probably not a fa ctor in this stud y (but sec Chapter 3 for discus~ton or the unpact of di sturbance on EC M com1nunities and N nutntion). While we ca nnot kno'A the level of ph o tore~ptrClti o n our smnple\ c\.penenced '' tthout adequate Jncasurc1ncnts, the n1ulttpl e ohsen at ions or each sa tnple (data not ~htn\ n) tndtca ted possible photorespiration in the Pyro lcac as we ll ClS the rciCltively shade to let ant ~uhalptnc fir and hybrid spruce sampl es in late Jul y to Jnici -Augusl or 20 10 and 20 I I 124 \Vhcn \,llnplc\ \\ere fi rst enclosed in the chatnber, net C0 2 uptake was observed or in so1n e cases net respiration wa already occuning (though no cer1ainty could be tn ade on contributions of each respiratory proces ). As the leaf temperatures increa ed in the transparent chan1bcr, C02 uptake decl ined or re pi rati on increa ed, or san1ples changed fro1n net P to net respiration. These dropping PS rates \\'ere accon1panied by very low conductance rates and often increa ing cl le cl (da ta not show n). The tncreascd cl Inay have been the result of photorespiratory C0 2 evolutton. Low condu c t ance~ in these sa tnplcs indicated stotnatal clo ure, potentiall y leading tore-as imilation of photorcspired C0 2 at hi gher cotnparati ve rates than plant that did not exhibit th e e trends. A nun1ber of sa1nples ex hibitin g the afore1nentioned trends did have lower 6 13 C va lues than others of the same species th at did not, lending support to the theory posed by Hyn on et aJ (20 13) of increased re-assi1nil ati on • of 13 C-depleted C0 2 . As 813C signatures reflect all 13 C sources and losses over the 1ife span of the lea f, however, appropriate experimentation would be needed to deten11ine if this was the case. Leading back to the topic of potential low PS rates in Pyroleae rnasking the apparent level of mycoheterotrophy, there are two opposing situations that could occur in relation to photorespiration that may ex plain sorne of the differences betw een estin1atcs of 0 oC DP!P~l and 11 o/oCoroSOl· First is the aforernentioned scenario that re-assitnilatlon of the C -depleted photorespired C0 2 dilutes more 13 C-enriched inputs so th at o/oCnr(I~Ol und erestin1ates MH nutrition. The second is photorespiration is n1asking the apparent lc\ el of autotroph}· during measure1n ents and that %C D P(P~l is overe titnating Mil C acquisition. The seco nd si tu ation seems quite likely in n1any cases, especially at C R, since gas-exchange n1casurctncnts \\ere instantaneous; occurred infrequentl y in relation to the life span or th e lea' ~s n1casured. especially over-representing periods of n1id -sun1n1 er drought and heat: were t)'VlCtllly 125 performed over daytin1e hour "''hen light levels were highest (generall y between about 9 aJn and 4 pm), a nd ~ no n1easurcments occun·ed prior to the end of May fo r any year (usually stat1ed in Jun e) such that early spring producti vity was not assessed. Though li ght response curve of June 20 11 (Figure 2) and 20 12 (Chapter 2~ Figu re 2-4, con1bined data for June and Augu t) show that even under rnorc favo urab le conditions the Pyrolcae usuall y had lower overall P rate at moderate to htgh light level cotnpared to autotrophs, at low light levels the opposite was tru e. 0 er the long tern1, estimated total seaso nal C ga ins in 20 12 (g tn-2 ) re ulted in C. urnbcllata having greater C ga1 ns than reference autotrophs over all trcattnents, P. chlorantha had 96% and even 0 . secunda exceeded autotrophs under the shade dry treatinent. This supports the second theory that our %C DP(P~J estin1ates may actuall y have rna ked autotrophic levels of the forn1er two pecies und er inh ibi ting light levels, whereas the possible underesti1nation of %Coro~Ol du e to lirn itations to PS tnay be son1ewhat valid for 0 . secunda. Considering there are no other accounts of a simi lar method of esti1nati ng %Cor using PS data, however, we cannot discount possible fl aws in the n1odel itself, nor ca n we di scount the potential usefulness of including other gas-exchange parameters such as conductance to modify the sin1plisti c approach. Considerations for future research To evaluate how the physiological factors discussed above could alter the overall 8 13 C signatures in PMH species and influence interpretation of the degree of MH C gains using isotopic 1nixing n1odels, a nun1ber of techniques are currentl y available that could relati\ ely sin1pl y esti tnate these paran1eters. Lo ng and Bernacchi (2003) di cuss a' ariety of biochetnical and biophys icalli1ni tations toPS that can be tnea urcd by the sin1ultancous usc of both gas-exchange tnethods and chlorophyll il uorcsccncc tncasuretnents These include 126 n1esophylJ conductance, Cc maximun1 carboxylation rate of Rubisco, electron transport rates that drive regenerati on of RuBP , and photorespiratory rates. While all of these, except photorespiration, have been indirectl y estu11ated using onJ y gas-exchange 1neas uren1ents of photo ynthetic respon e to C0 2 (as A C, curve mcasurcn1ents), they each require a va ri ety of n1athetn ati cal fratn c,.vorks and a un1pt1on that are bcco1ning Increasingly recogni zed as invalid or at least va ri abl e aero s species and enviromnental conditio ns (Long and Bernacchi 2003; Manter and Kerrigan 2004: Vrab l et al. 2009). By using both methods in tanden1, these variables could be asses ed in relation to PMH species. AdclitionalJ y, chlorophyll and/or Rubisco content could be n1easured to determine wheth er any of these species have substantiall y redu ced biochen1ical ca pacity for C0 2 fi xation (La uteri et al. 1997; Piel et al. 2002). The yellow-green colour of 0. secunda coupl ed with consistentl y low PS rates (Figures 1 and 2~ Table S2) ind icates at least this species could have substanti all y lower chlorophyll content than autotrophs. Once the most litn iting factors to PS rates are identified in the Pyroleae or other putati ve PMH plants, fu11her studies could he lp resolve the discrepancies between P rates. 8 13C values and subsequen t estin1ates of %C DP· This could lead to more accurate assessments of v., hether observed 8 11 C va lues refl ect PMH C gai ns or whether P rates signifi cantl y modi fy interpretation of 8 13C va lues. With tnixing tnodels being a prin1ar-y focus of this study, there are a nu n1ber of different strategies to improve %Cor (or o/oCn 1 ) e tin1ations using both isotopic and photosynthetic data. First is the use of lea f solubl e ugar rather than bulk leaf ti sue 8 nc values, though this data could be highly va luable with concunent gas-exchange n1casurements regardl ess of whether mixing tnodels arc applied to the data. A n1cnt1oned. 11 bu lk leaf tissue 8 C refl ec t the integrated C inp uts over the life pa n of the leaf, posstbly tn asking short-term MH C ga ins, whereas olu blc suga rs arc the initial products of 127 photosynthesis and are the dominant fonn of tnetabo lites passed from autotrophs to fun gi (Hogberg et al. 1999; Hynson et al. 20 12). Assessing leaf soluble suga r 8 1 "c and PS rates on a tnonthly or enviroruncntal condition basis, coupled' ith esti1n ates of %C nr for each measurement period, could elucidate ,.vhcn and under \vhat conditi ons the Pyro lcae in this region tend to obtain fungal C. econdl y, cstunatcs of 0 oCnr could be calcul ated using light response data to take into account the non-linear P response to light. Alten1at1vely, light response data could be used in conjunction with continuou light data to detcnnin e continuous seaso nal PS rates or estin1ate total C uptake (as sho·wn in Figure 2-5) could give an estimate of longer tern1 levels of and limitations to, autotrophy. Lastly, the hi gh variability of isotopic enricrunent as well as PS rates of autotrophic and fu ll MH end -rnembers used in th e models need to be considered (Hynson et al. 2009). It is logical that no individual can gain greater than 100% C frotn any source but in several cases either method resulted in C. un1hellata or P. asan(olia having more than 100% Cor gains. This result fron1 the end-tne1nbers of the calculations not representing the full spectrum of possible values for a given vari able (Hynson et al. 2009). To address this, Hynson et al. (20 13) recon1mend using MH end-m embers fron1 the same plant family when perfotming calculations, which we did and so applied the co ncept to autotrophic end-members by recalculating 20 12 %C DP estimates u ing either only ericaceous species or only evergreen autotrophs. The results still end ed with C. umhellata gaining > 100% Cor in a couple of cases: therefore it appears that even references fron1 irnilar fan1ili es and life forms tnay not provide the full spectnun of autotrophic P capabili ties or isotopic signatures at any given site. Using a wider vnri ety of species of different tnycorrhizal classes and fun ctional groups tnay help unpro e estin1ations, but assessrnents of limitations to autotrophy should also be considered. 128 Final remarks The gas-exc hange tnea uretn ents coupled with iso tope analysis in thi s study revealed that, over rnultiple site and years, P. cltlorantha and pa11icularl y 0. secunda ex hibited significant PMH C ga m at relatiYcly consistent levels of 8pproxitnately 70% Cnr on average (Tables S4- l and 4-2: Figure 4- 1 and 4-3 ), agreei ng well \Vith prev ious fjndings of oth er tudi e (e.g., Tedersoo et al. 2007: Johansso n et al 20 15). Chi1naphila tunhcflata showed mainly autotrophic C nutrition at 94°~ Cop, also agreeing wi th other research (Zi mm er et al. 2007; Hyn on et al. 20 12: Johan on et al. 20 15), though the slightl y hi gher average 13 C enriclunent compared to autotrophs (Tab le S4- l ) and low 0 oC DP( 1<;,oJ esti1nates in 20 10 (not shown) were pos ibly due to low level of PMH nu trition in some cases. The PS rates and 13 8 C values for P. asari(olia vve re qu ite vatiab le (Tables S4- l and S4-2), resuJting in different interpretati ons of PMH or autotrophic C gains. Based on the 8 13 C data, the species was autotrophic at 95% Cor but photosyntheticall y exhibi ted an average of only 76% Cnr (Figure 4-3), limiting the ability to make a soli d co nclusion about the degree of PMH strategies. The lack of data on this species also does not assist in determining how the species fi ts the autotrophic-MH continuum, but the 8 13C analysis fro n1 the overall dataset provided evidence that it is probably 1nore autotrophic than PS rates wou ld ind icate, pa11icul arly since many P rneasurements occuned during hot dry peri ods when the plants were stressed. The extremely small sampl e size for P . 1ninor lin1ited any detecti on of significant differences fro1n autotrophs rega rding any vari able, but in some cases, the data indicated low levels of PMH C acquisition (Figure 4-3~ Tables S4- l and S4-2). Interestingly, P. minor sho,ved considerably hi gher conductance and transpirati on rates than 8ll ot her species at WR, a well as higher C,:Ca ratios, indi ca ting different physiologica l or anaton1ic8l traits that rnay be wo r1h\\ hdc investi gating. The inclusion of the less con1n1on Moncses l1111/lora indicated autotrophic 129 1 nutriti on a \\ell. but e\ en \\ith a \ Cr) c;, rnall sampl e size frotn onl) one ye ar. 8 ' C va lues \\ ere , tgntfi cantl y depleted con1pared to the oth er Pyrolcae, igndica ntl y hi gh 0 oN was also found , indtca ttng thi s s p ec 1 e~ has rath et chfTerent nutnttonal strategtes co rnparcd to the other speCieS. With rega rds to other gas-exc hange \ a n a hl e~ obtained durin g n1 easurcrnents and the possibtltt} o r lO\\ p rate J11asktng the lc\ el of MII c gat ns a~ proposed hy Hynson et al (20 13), the data 111d1Ca te that C, C,1 ra ti O~\\ ere generall y COnSIStent With autotroph <, despite the lO\\ conductance and tra n ~pu atJo n ra te~ . c~p ecta ll y tn 0 . c..C!utnda (Table S4-2) This indica tes that any non-linearity of PS di ~cruni na ti on of 1'c acros<; spec1es i~ ltkely not due to better equil ibration to an1bi cnt C0 2 • though the posstbl e effects of 1nesoph yll condu ctance on Cc could not be a essed. The data dtd indica te oth er li kely physiolog1ca l and hi ochen1ical lin1itati ons such a low stoJnatal conductance and photosyntheti c capac tty (hy Rubi sco) and higher photore piration rates in the Pyrolcac, cspectall y 0 . secunda. \\htch could resu lt in a greater relative incorporation of 13 C -depleted respired C0 2 1n these plant<, cotnpared to autotrophs. This may explain the disc repancies bct\\een 8 13 C and PS ra te~ fo r P. aw1n{oha. Conversely, in son1e cases it appeared that th e levels of autotroph y were poss1bly rnasked due to hi gher levels of photorespiration and photoinhibition in these shade adapted pl ants. with 8 'c valu es either accurately refl ecting MH nutriti on or e'.- en bein g soJTIC\\ hat cnnched fro1n 1 photoc;,ynthctic processes. As these paran1 ctcrs could not be asse~ scd adcq untel) due to lack of appropri ate tneasurements, additionnl \ tudt cs should consider u~ 1ng ga~-c'\c h nnge tn additt on to other tnethods such as chloroph yll flu orescence Usc or n1ult1plc rncthod"' could help identify any critical ph y~ t o l og t ca l and biocherni ca l litnitat ions to PS 111 these pl,lnts onl y wou ld a vari ety of n1 cthod s better es t11natc the actu al level or M II nutrition tn putdtl\ c PMII species, phys iological data could mdi ca tc what additi onal en\ trontncntal conthtion"' 110 ot aside frotn light could increase the reliance on fungal-mediated nutrients, whether C gains are prin1arily a ide-produ ct of and P gains. and pos ibl y identify this fascinatin g strategy in other plant species. 13 I 4.5 Literature ci ted .. Abadie J-C, Putt epp U, Gebauer G, Facc io A, Bo nfante P, Selosse M -A . 2006. 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Mean(± l SE) natural abundances of 8 13 C and 8 15N (%o). and C and N co nce ntrati ons (0/o) of autotrophic species. six putati" e partial tnycoheterotroph ic Pyroleac spec ies (in ita Iics) and fu 11 111 yco heterotrophs (Full Mils). Species sharing th e same letter \r\ ith in a column are not significantl y differe nt at a < 0. 1. Estin1ated Marginal Means± Standard Error 8 13 C (%o) 20 10 Year Site Autotrophs Ch11naph 1/a umbellata 20 I I 20 12 tUNBC t WR tCR tUN BC t WR tC R tC R -32.4 ( l. 1)a -31.7 (0.8)a -32.6 (0. 3 )a -30.4 -33.5 (0. 7) -33.2 ( I. I) -33.5 (0. 9) -3 1.8 (0.6) -30.9 (0.3)J -30.4 (0.5tb -30.7 (0. I )a . ., 0 . ).. - j . . , I .J. ., - j (0 .4)ab co. 1 rl -29.6 (0 .4 )abc (0. I )a 1\foneses zauflora Ortlu!Ja secunda Pvrofa asar1(ofia J Prrola chlorantha ~ -30.1 (0.8)ab -32.4 (0.4 )a -27.7 ( 1.3 )h P1trofa nunor - Full MHs *-24.8 Fixed effect of 5pecies F-ratlo 4.59 p-value 0.023 Of 4. I 0 -31.0 (0.2 )b . . , 1 . ., - j .J (0 .6)ab -33.2 -28.6 (0.2t -30.4 (0.2tb -29.4 (0.2 )bL -31.6 (0.2)ab -25.4 (0.2)( -25.8 (0.4 )J 80.57 49.5 I < 0.00 I < 0.001 5, 18 • 5. 14 -32.6 (0.6) -32.3 (0.8) -3? .0 (0 .4) -3 I . I (0.6) -29 . I (0.2)h -3 I .2 (0 ...+)a -19.8 (O. l )h -29.7 (0. 1/' 2013 tDt\1 tDL :!: PM -30.7 (0.5)b -34.0 (0. 2 )a -30.7 (0.6)h -3 1.0 (O..t)b -31.0 (0.4) -30.4 (0.2) • 2.3 1 0.128 5.36 0.0 1 3. 12 3. 12 3. 16 138 -31 .4 (0.5) -3 I. 1 (0.6) -26.2 •-25. 1 0.57 0.571 I. 23.26 1.74 155.42 0.2 14 2. 12.75 <0.00 l 7,29.43 "' 4 1 .03 7 . -I...,.J < 0.00 I 4.93. 15 0.00 I 3.27 -19.8 (0.4) -30.6 (O..t) -32.0 -26.2 (0. l )( 0..+3 0.734 "- ,, 8 j _ , §Grand mean -31.2 (0.2)b -30.8 (0.1 )be -3-t.O (0.2 ):t -30.4 (0.2 )cd -31.4 (0.2 )b -2 9.8 (O.l)d -31.6 (0.2)bc -25.6 (0.2 )e Supplementar: Table 4-1 continued I~ 8 "N (0/oo) '{ear Site Autotrophs Ch1Jnaph11a umbellata 201 1 20 10 tUN BC -'-·') (0.3)il ') ') -·- (0.6)' 2012 t ~' R i"CR tUN BC i" WR tCR §C R -2.0 (0.7)a 2.7 (0.6)h -2.5 0.7 (0.6) 0.5 (0.5) I .9 -1.9 (0.5)a -1. 1 (0.3)tlh -3.4 (O A)a -0.3 (0.9th (0.6)'1 ') ..., ~ ._) (0.8)'1 (0.2t -0.5 (0.2)h Aloneses un(flora Orth;/ia secunda 1.0 (0. I )be Pvrofa • asari(ofza Prrola . chlorantha -0.6 (0.3 ) .tb 7.5 (0.6 )J ') -'· - ( 1.1 )b 1.6 ( 0.4 )b ,. 0. I (0. 9)b -- ') _ ,) (0.4 )bL 1.6 (0.4) -0.0 I (0.5) 4.8 (0 .5 )h 1.3 co.5 r• -0.5 .6 rth 0.4 (0.6)h co -0.02 (0 ·' )b 5.0 (0.8t 6.8 (O A t (0.2) 11 88 (0.2t 8.3 ( 0. 7 )0 8.9 ( 1.4 6.5 tDi\ 1 t DL +Prv1 + 3.7 (0.5)b -0.5 (0 1 ) 1 49 (0 6) '\ 0g 1 (0.2) 1.7 (0.3) 0.8 ( 0. 9 )tl I • 0.3 1.4 (0 4) 24 (0.9) -0 ') (1. 1/ 7. I (0 .4)b 9.0 1.0 Prrola nunor , Full ~1H s ..., 2013 8I .. t I 3. 7 • 8.5 §G rand n1 ca n -2.5 (0.2 Y' 0.6 (0.2 )bl -0.3 (0.3 )b 1.2 (0.3 )c 1.1 (0.2)c 6.8 (0.3 )d 1.0 (0.2 )bc 9.8 (0.6 )e Fixed effect of ~peuc\ F-ratto 71.3 p -va lue < 0.00 I 41 12 40.75 2. I I 6.38 _), . ., 6...,.) 148.7 1 24 37 0 85 26. 1 3 139.54 < 0.00 I < 0.00 I 0.008 0.036 < 0.001 5. 18 3. 12 3. 16 0 439 1 ,..., -4 _, _ ..).) < 0.00 I 5. 14 < 0.00 I 4. 14.25 < 0.00 I 4. I 0 0.153 . ., I'.... ..), 2. 14 7' 35.66 139 . ., '7 ..), _ , Suppletnentar) Table .f- 1 continued 0 Year Autotrophs Ch1n1aphiia umbellata §2010 §20 1 1 §20 12 51.5 (0.4)'49.7 (0.6)ilbl: 49.3 (0.8)Jb .f9 .I (0.3 )b 50.6 (O A Y1b -+8.8 (O.l)b Jloneses· zouflora OrtJuiLa "iec.unda Pvrola . a~ar~(olia Pvrola , chlorantha P~rola rninor , t Full i\1Hs 49.0 (0.2) 51.4 (0.3 t 47.9 (0.2)J .f9 .5 (0. 9 ).IlA 48.4 (0 .3 )at· 49.3 (0.4 )J 50.7 (0.3 t 0 oC -+8.6 (0.1 )b 4 7 .l ( 0. 1)a 48.4 (0.3)ab S?Q 1" s_, ' §Grand n1 ca n 49.6 (0. 3 )bcd 49.5 {0. I )be 49.4 (0.1 )'' .f 7.4 1 (0.2r .f9 .0 (O. l )b 50.0 (0.2)" 47.0 are estimated rnarginal n1ea ns :t: I SE (population-le\ el) resulting frorn li near n1 ixed models. Spec ies ~ harin g the satn e letter\\ ithin a column are not significantl y diffe rent at a.:::; 0. 1. All other gas-c:\.c hange parameter() and rn ea~ urc tn c nt c; arc desc ripti \ e means± I <;D. Gas-e\.chan gc n1 casurcrnent n1ean s ± standard errore; Ambient mcasurcn1cnts seasonal averages Average photosynthesis ()..unol m -:! s- 1) Co nductance rate (rno l rn- 2 s- 1) Year Site Autotrophs Chln7aphiia unzbella!a Orthilia ~ecunda 201 0 P1.rola , chlorantha 20 10 s~UN B C 1 3.6 (0.5r 4.5 (0.3)a 0.0-t (0.0 I tb 0.05 (0.00-t ) 0.1 1 (0.03) 0. 18 (0.0 i t 1 '"(0 ·-c;)a -·) 3.3 (O 1r1 4.0 (0.2)l 0. 06 ( 0.0 I yl 0 06 (0.0 1) 0.07 (0.0 1 ) 0 16 (0.0 I t 0.6 (0.2)t 2.0 (0.3 )J 1.7(0.4/ 2.2(0. 1)h 0.02 (0.00-t )h 0.05 (0.01) 0.06 (0.02) 0. 11 (O.O I)b 0.6 (0.1 )h 2.4 (0.5r' t. 9 (0.3 rl 0.03 (0.0 I t b 0.07 (0.0 I ) 0 09 (0.0 1) 2. 1 (0.2) a o. 9 co 5rtb 1.3 (0.4 r'h .. 1.06 §C R ~ 2.9 (O..t rl 1 _ .)- (0.')h ._ 0.05 (0.0 I )ab Prrola m1nor • 1.0 (0.3)J Full I'v1Hs -2.9 (0.7)'Fixed effect of 5pcc ics F-rat1o 22 ..t-6 p -value < 0.00 I Of 5. 7. 79 -2. 1 (0.7)h -2. 1 (0.6)b -0. 8 (0. 1( 0. I .t ( 0. I 0 )"b I IA I < 0.00 I 5. 21 19.33 < 0.00 I -'0-). _' I 3.86 0.0--t- 5 5. 7.9-t Year ,.., '" ) . _ .) 20 10 ·n NBC ~~ WR SVv' R ~ • SC R SCR ~ ~ 0.07 (0.02) 0. 15 (O.O I)a 0 03 (0.004) 0.04 (0.0 I ) 0.03 (0.0 I y: ' -o 1.86 0. 199 5. 8. 86 19.69 < 0 00 1 4. 23 . 10 0.05 0. I I ( 0.04) < 0.00 I --t- . 4708 ·~' C R -) 0 135 5. 6.76 ., c .Ca ratios ( fronl units of ~Lm o l C02 mor 1) ~tte 20 12 SCR t WR §UN BC 2.5 (0. 1)a P1., rola a~an(ofta 201 2 Transprratio n rate ( ~Ln1ol H20 m-- ~- ) 20 12 sSC R 1-t I §UN BC 20 10 s~ \\ R 20 12 §CR ~~C R , Supplementar~ Table .f-2 continued Autotrophs Chnnaphzla unzbellata Orthil1a secunda Prrola .. asarifolia Pvrola , chlorantha 0.69 (0.07).1 0.73 (0.02}a 0.77 (0.0-f)J 0. 8 I ( 0. 0 I )a 0.67 (0.1 O)ab 0. 79 (0 .06 )J'l I .-+0 (0.34) 3.02(0.18).1 0 0.85 (0.0-+)" 0.99 (0.20)"b I . I I (0. I 6) 2.98 (0 16).1 0.89 (0.06)a 0.75 (0.07)a 0.73 (0.02).1 0.86 (0.0 I )h 0.38 (0.08)b 0.84 (0. 12tb 0.89 (0.'2) 2. I I ( 0. I I )bt 0.82 (0.04}a 0.77 (0.02)a 0.86 (0.06)'' 0.45 (0.1 O)b 1.06(0. 14)a 1.07(0. 11) 0.88 (0.06)a 0.75 (0.0-f)" 0.77 (0.09) 1 0.8-f (0.0 I }" 0.91 (O.IOt ~0.76 1 0.74 (0.05)' 0.86 (0.0 I) h o.69 co. 14 r'b 0.79 I. I I (0. I 6) 1 56 (0. 14tb 1. 18 (o. 36r'b 0.82 (0.07)a Pvrola mznor , • 1.17 (0.05 )b 1.50 (0.18)h 1.30 (0.09)b t Full :VlHs Fixed effect of species . . . .0'"".) .) I 1.99 F-ratio 5 6-+ p -value 0.003 0.092 < 0.00 I 5. _.) ,.., 5, 6.88 df 5. 18 1.13 (0.05)'- I. 71 (0. 97th 0.)_ -") ( 0.06-h ) 0.67(0.17) 0 76 (037)c I 0.00 < 0.00 I 4. 20.32 5.56 0.018 5. 7.67 1 -+6 0.072 5. 6.64 1.-+6 0.2-+0 I I. 98 < 0 001 4. 21.24 -.....,.., ) .) 2 s· 1) Lig ht res po nse curve m cas urern ents Dark respiration (~Lmol m· 2 s· 1) Year ':>1te Autotroph~ Chinu.tph1/a umhellata Onhilia secunda Pvrola .a.w.trij(Jl ia 20 II , \nla\. (~unol n1· 20 12 ( (l 800 P. \ R) l ~BC \\ R CR -0.7 (0.1) -0.9 (0.2) -0.9 (0.2) 2012 CR -0.9 (0.1) - o.)- (·a'_) -0.7(0.1) -1.5 (0.5) -0.7 (0. 1) _). _) . . . . . . ( 0 ..)-) 5.3 (0.4) 6.-+ (0. 8) 5.3 (0.3) -0.2 (0.0)5 -0.4 (0.1) -0.5 (0.1) -0.5 (0.0-f) 2.9 (0.2) 3.7 (0.6) 5.3 (0.3) 3.0 (0.2) -0 .6 (0.3) -0.5(0.1) -0.8 (0.3) 3.3 (0.3) 5.9 (0.6) 6.-+ ( 1.0) 142 2011 (a 1500 P \R ) U"\JBC \\ R CR CR 3.8( 1.0) 55 (0.6) 7.6('.6) 5.9 (0 5) Supplementar: Table 4 -2 continued Pvrola . chlorantha -0.4 (0.04) 4.4 (0.2) Ligh t con1pensation point ( PA R: ~ltl1 0 I m-2 s-I) Year Site Autotrophs CJu maplui a umhellata Ortlulia \CCltllda 20 I I UN BC WR CR 11.0( 1.7) 18.6(8.2) 19.0(4. 1) 20 12 CR 15. 8 ( 1.3) 8.9 (3 .4) 13.8( 1.3) 46.7( 17.2) 13.0 (2.2) 4.2(1.1) 8.8 (2.4) 11.7 (3.0) l I .3 ( 1.0) 9.3 (4.2) 8. 7 (2.0) 19.5 (8.8) Pvrula . cnanfolla Pvrola • 11.5(1.4) ch/orantha ~ - 5tatistical te<;t. one-\\ a) ANOY A assun1ing unequa l va riance,\\ ith ~ elch F-ra tio and Dunne tt's I 3 po-.,t hoc pain,\ ise co n1pansons. t - Statistical test. onc-\\ay ANOVA assutning equal variance. I u"-.c: ·s I lS D post hoc pain\ tse con1(1artc;ons. * - Not included in ~tatistical tests. n - I. :......__. 143 5. Conclusion The overall goal of this thesi wa to evaluate and quantify th e degree of mycoheterotrophy in several putative PMH specie in the tribe Pyrolcac in relation to full autotroph and clo ely related fully MH Monotropoideae species in the central interior of Briti h Colurnbia. Additionall y. in light of th e vast area irnpactcd by the recent n1 ountain pine beetl e epidemic, ub equent clea rcut sah age logging and alterations to forest habitats, we ought to und er tand how populations, 1 otopic signatu res and phys iology would change under variabl e di turbance level , particul arl y under increased exposure to li ght and drought. While tnany studi e have used natural abund ance of 13 C and 1c;N isotopes to assess or quantify various attribute of parti al and/or fullin ycohcterotrophs, thi s is the first know n stud y to evaluate the gas-exchange physiology of Pyroleae species and how those attributes • may contribute to isotopic signatures. particul arl y 6 13 C, and interpretation of nutritional modes of the Pyroleae specie . 5.1 Degree of ca rbon and nitrogen m ycoheterotrophy in Pyroleae pecies The co llective data for P rates and 8 13 C values frotn Chapters 2 and 4 were generally in agreement with findings in the literature, that two of the six Pyro leae species studi es, 0 . secunda and P. chlorantha, may gain as much as 30% of their C from mycoheterotrophy (Tedersoo et al. 2007; Zinm1er et aJ. 2007 ~ Johansson 20 14 ~ Johansson et al. 20 15 ). Resul ts for the other Species indicated primarily autotrophic C gains, with the high an1ple S l /'C of C. Lunhel/ata showing the most consistent trends and strongly agreeing wi th pre\ tou s resea rch (e.g., Zi1n1ner ct al. 2007; Hyn on ct al. 2009, 20 12). Data for Afonescs ton/lora IS limited but our data of highl y depleted 8 C va lues agrees ~~ ith findings f'ron1 Johansson et al. 11 (20 15) of adult autotrophy, though the C assin11lation strategies tna y ddTer fron1 its~ ~ tet ta\.d 144 based on differences in each pecies Isotopic signature . Both P. asanfo ha and P. minor had mixed results, with generall y stati "'ti cal support of adult autotrophy but so tn e indication o f 13 PM JI nutriti on based on both P rates and 8 C signatures. There wa little Indication that drought had any in1pact on the degree of PM I I nutrition in 0. secunda or P. chlorantha, nor d1d the P rates s1gni fy the plants were particularly en itive to dry periods (C hapter 2), though it n1ay be that in 20 12, the length of tin1e between rain event was not pro longed enough to inh1 bit autotrophic growth in these species, which are presurnably well-adapted to tn id-su1n n1cr conditi ons. The effect of increa ed irradiance resulting frotn mo1iality fo ll ow ing tno un tai n pine beetle attack in both residual forests and in salvage-harvested clearcu ts did appear to have some impacts on th e physiology and persi tence of not onl y the Pyroleac but also the full MHs included in this stud y (Chapter 3 ). The two apparently PMH species showed the greatest indication of sensitivity to changes in habitat, with 0. secunda exhibi ting pho tosyntheti c li1n ita ti ons at high irradiance and P. chlorantlza unable to persist in clearcut conditions. While stable isotope signatures of the Pyrolcae in thi s study were very cotnparable to 11 frndings in the literature, and there was reasonable agreetnent between PS rates and 8 C values (particularly for C. umbellata, 0. secunda and P. c/zlorantha), some aspects of the 13 data were co ntradi ctory regardin g the interpretati on of 8 C val ues and the degree of mycoheterotrophy in these plants. There is a possibility that low inherent PS rates, phys iological, anatomi cal and biochetnical adaptations to relatively shaded habitats co ntribute to incorporation of 13C -depleted C0 2 in leaf tissues, which has been proposed to 13 possibly dilute C -eru·ichcd MI I C gains and therefore lower the apparent le\ cl of PMH nutrition in the Pyro leae (Hynson ct al. 20 13a). T here is sotne evidence that these shade adapted plants may have increased photorcspiratio n 111 ~ell - It t ccosystcn1s (Chapters 2 and 4) 145 and this may be exacerbated by other phy iological characteristi cs such as low rn esophyll co nductance rate and sub equent low C c· resulting in a feedba ck cycle. Interestingly, th e data provided evidence that th ese sarne characteristi cs n1ay have the opposite effect on P rates and 8 13 C signatures, such that photorespiration is lowering the apparent level of autotrophy during gas-exchange rn easurements, as well as increasing photosyn thetic 1~C enrich1nent. Thi leads to the possibility th at interpretation of PMH nutrition in the Pyroleae is an arti fac t of the pa11icular Jnetabo lic and biochetni caJ pathways of the species, which tn ay differ considerably fro rn other functional groups such as overstory trees (e.g., conifer , regardl ess of maturity) or broadleaf/deciduous trees and shrubs. Though quite specul ative at thi point, we pose the theory for the foll owing reasons. First, sampl es often had much higher PS rates than the presented averages, often comparable to or exceeding reference autotrophic rates. Second, li ght response curve tneasuretn ents indicated high photosynthetic effi ciency at low li ght levels common to PyroJeae habitats, as well as often high PS rates at high er li ght levels (depending on species: Chapters 2 and 4 ). Lastly, and perhaps most interesting, frotn light response curve and continuous light data in 20 12, 2 estimated total seasonal C uptake (g m- ) resulted in C. zunhel/ata and P. chlorantha gaining > 100% and nearly 97% as tnu ch C as autotrophs, respectively, over all treatinents. Even 0 . secunda was estimated to obtain 130% that of autotrophic C und er one shad e treatn1 ent. The discrepancies betw een the types of gas-exchange n1easure1nents, as well as between isotopic signatures and PS rates n1erit fut1h er study. To conclude on the nature of C and N tnycoheterotrophy in the Pyrolcac as a group. four of the six species showed h1 gher tend cncie to autotrophic C ga ins over the tn aJority of the dataset. Fut1her, in spite of the consistency of the pnn1ary data (PS rates and 8 1 ~C signatures) indicating 0 . secunda and P. chorantha ga in as 1nuch as one thit d of then· C fron1 146 mycorrhizal networks. the discrepancic in the data di cu cd above point to the possibi li ty of these two pecic being n1ore autotrophic than 8" values alone can elucidate. In contra t, all Pyroleae species were ignificantly enri ched in 1~N c01npared to autotrophs, with P. chlorantha being almo t as enriched as full Ml Is. The scenarios presented in Chapter 3 and below indicate that there i a hi gh reliance on Inyco tThizal fun gi for N acquisition in these plants. Therefore. we conclude that O\era ll the Pyro leae tend towa rds N rather than C mycoheterotrophy. 5.2 en itivity to di turban cc Both the Pyroleae and full MH species altnost universall y showed intolerance to substantial disturbances (Chapter 3 ). The lack of any full MHs observed outside of forest canopies indicates they do not receive adequate C when overstory trees are re1noved, but interestingly, there were several individuals of the two red-orange full MHs, P. androtnedea and M. hypopitys at the 20 12 stud y site that occuned along th e forest edge, i1nplying some tolerance to high light due to protective carotenoid pig1nents (as opposed to the colourless Mon otropa uniflora; Cutnmings and Welscluneyer 1997). The Pyroleae had lower densities in clea rcuts compared to forest interiors and/or edges (Chapter 3 ). with edges along outh or western aspects tending to show lower populations than not1h or easten1 a pects Indi-vidual that were found in clearcuts showed reduced growth and bleachin g of chl orophylL and \\ere ahnost always associated with son1e larger vegetation. The occurrence of Pyrol eae individuals near residual trees and slu-ubs in clearcuts would be an obvious on gn1 or C for those species exhibiting signifi cant PMH nutriti on~ however, since all the Pyroleae studt ed appeared to ga in the n1aj ority or their C through autotrophy (Chapter 4 ). effec ts of excess light n1ay have greater itnplications to sensitivi ty to disturbance. 147 ln species adapted to shade, not only i there potential for photorespiration and photoinhibition under full sunli ght, but long-teJm exposure can lead to photo-ox idative datna ge to and down-regulation of photosynthetic n1achinery (V crhocven et al. 1997; Pon1pelli et al. 20 10), reductng the already low P capacity o f the Pyroleae (Chapters 2 and 4 ). The low density and \ igour of the Pyro lcae in clearcuts suggest th e e mechani sms are quite in1portant and arc an underl ying reason for sensiti vi ty to di sturbance, especiall y in li ght of the fa ct that P. chlorantha did not occur in clearcuts with th e exception o f one individual though in tnany cases it exhibited relati vely high levels of autotrophy, whereas the n1ore consistently PMH 0 . secunda had co nsiderabl y hi gh nun1bcrs of indivi duals in clearcuts at 15 all three sites (Chapter 3). All the P) roleae had signifi cantl y hi gher 8 N than autotrophs on average. as \Vell a often hi gher % . Since N is an impot1ant ele1nent for photoprotective molecules in add ition to chlorophyll and critical enzymes like Rubi sco (Verhoeven et al. 1 997~ Potnpelli et aJ. 20 10), obtaining sufficient N may be the key fa ctor for persistence in stressful conditions. In nutrient poor soils cotnmon to our study areas (e.g., British Columbia Ministry of Environment 1 989~ DeLong 2003 ), th e fungal sy1nbi onts of the Pyroleac n1ay be sotnewhat speciali zed in obtaining N fro1n reca lcitrant sources or providing access to organic at our study sites (e.g., Hobbie and llogberg 20 12). There is potential for eco logica lly signifi cant shifts in fungal species con1position following canopy los (e.g., Cullings et al. 200 I), especiall y during clea rcutting, wh1 ch n1ay have re ulted in a decrea e or los of suitable symbionts for the Pyrol cae. Even though the plants 1nay obtain sufficient C \ ia autotroph}'. substantial reduction inN acquisition could reduce photoprotection and repatr of datnageu photosystetns. This tnay lead to eventual in1painncnt or photosystcn1S to the potnt or incapacity in n1aintaining a positt e C balance and thus cause the plants to die Further 148 lin1itations as ociated with n1icrosi tc pccificity tnay also prevent these species from succe ful reproduction (Johansson 20 14 ). Given th e appreciable differences in 81-;N across the Pyrolae as well as frotn forest to clcarcut scgtncnts, fu1ihcr stud y is clearl y wan·anted to detennine the ignificancc, if any, of ecological co nditi ons including ECM and/or MH status on nitrogen acquisition 1n this system. 5.3 tudv limitations and future direction s ~ Li1nitations to the stud y included son1e cotnn1on logistt cal constraints, such determining and acce ing suitable , ites, particularl y for the 20 J 3 survey, fi eld and lab titne limits due to equipment needs, ~' C ather conditi ons and satnple processing time, and financial lirnitations for sa1nple processing and travel. There was also no way to determine pre- and • post-disturbance effects foll owing tnountain pine beetle attack since the initial attack occutTed over a decade ago, but it would have been interesting to assess the immediate effects of canopy mortality, particularl y on P. andromedea. In thi s study, an additional limitation was actually the inclusion of such a wealth of data, which required focus on key variab l es~ p1i1narily photosynthetic rates and stable isotopes. While measurernents of PS rates have provided valuable insight into the target species, it was not until 20 13 when a book on mycoheterotrophy wa published that the idea of 8 13 C signatures not accurately refl ecting PMH nutrition was pre cnted, due to inherent physiological differences in PMH versus autotrophic species. As such, son1e of the potentially in1po11ant variables di scussed in Chapter 4 capnblc of being n1easured using gasexchange systetns were unknown at the tin1e, and therefore di scu ion of the e topics\\ as litnited to logic and speculation. Many detail ed strategies arc presented 111 Chapter -+ to tnore adequately address S0111C of these potentiall y in1p0rtant phys iologicnl differences. as\\ dl cl\ 149 other techniques that 1n ay help dctennine soluti ons to unanswered questions of Chapters 2 and 3. Other methods such as radi o-active isotope tracer studies or pigtn ent analysis n1ay also help elucidate unknovvn charac teristics of Pyroleae or other PMH species. One of the n1ost fund aJn ental questi on to be answered wi th regards to MI I nutrition, not only in the Pyrolcac add res ed in this study but in other potenti ally PMH green plants (e.g., orchids) a well as full MHs. is what metabolic substances are transfen·ed between plants and their fun gal syrnbionts, and what isotope fractionation effects those transfers have. Add itionally, with some of the data indicating possible photosyntheti c effects on 8 13 C that may confo und interpretati on of PMH C gains, it wdl be crit1cal to determin e what and how particular aspects of the plants· phy iology and biochen1istry may vary and infl uence that interpretation. If th ere are particular n1etabolic pathways that result in substantiall y enri ched or depleted 13 C or 15 N pools in certain pl ants or fu ngi, we n1ay be ab le to better und erstand nutritional and energy budgets of species of interest. Upon understanding these factors. we can then more full y evaluate how anthropogeni c effects such as increased disturbance or climate change may influence the abundance and distribution of populations of sensitive species, whether n1ycoheterotrophic or not, and provide adequate conservation and rn anagement strategies when necessary. 150 5.4 dditional literature cited cbaucr G. r·aecto A.. Bonfante P, c lo ~sc M- 2006 Cephalanthera lon~r(oha ( eotllcac. Orchtdaccae) 1 1nixotroph1c a co n1parat1vc ~ tud y bet\\ een green and nonphotosynthctt c tndn tduals Canadian Journal of Botany 84: 14621477. doi . 10.11 39 806- 101 Abadte. J-C. Putt epp . Badcck, F-W, Tcherkez G. ogucs . Ptcl C. Ghashghaie J. 2005. Pos t-photosyntheti c fractionation of stabl e ca rbon 1 otopcs between plant organs a wtdcspr cad phcno1n enon. Rapid otntnunications in Ma ~s pcc tron1ctry 19. 118 1- 119 1 doc I 0 J 002/rctn . 1912 Bell gard. 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