New Phytologist Supporting Information Article Title: Are there geographic mosaics of mycorrhizal specificity and partial mycoheterotrophy? A case study in Moneses uniflora (Ericaceae) Authors: Nicole A. Hynson1, Martin I. Bidartondo2 and David J. Read3 1 Department of Botany, University of Hawaii Manoa, 3190 Maile Way Room 101, Honolulu, HI 96822, USA; 2Department of Biological Sciences, Imperial College London and Royal Botanic Gardens, Kew TW9 3DS, UK; 3Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK Article acceptance date: 2 July 2015 The following Supporting Information is available for this article: Table S1 Sampling site locations including latitude and longitude, species collected and replicates (n) from each site, and group. Table S2 Mean 13C or 15N isotope enrichment factors by group or species with replicates (n), and locality. Methods S1 Enrichment factor calculation equation and statistical analyses. 1 Table S1 Sampling site locations including latitude and longitude, species collected and replicates (n) from each site, and group. Locality and site Olympic Peninsula, WA, USA Sol Duc 1 Sol Duc 2 Latitude/longitude 47º57’29.70”N 123º49’97.53”W 47º57’27.70”N 123º49’95.67”W Species (n) Moneses uniflora (1) Tsuga heterophylla (3) Vaccinium parvifolium (3) Target species Moneses uniflora (1) Listera cordata (2) Corallorhiza striata (3) Target species Tsuga heterophylla (3) Vaccinium parvifolium (3) Sol Duc 3 Sol Duc 4 47º57’27.70”N 123º49’95.67”W 47º57’26.73”N 123º49’98.36”W Group Autotroph Autotroph Green orchid Fully mycoheterotrophic orchid Autotroph Autotroph Moneses uniflora (1) Listera cordata (1) Clintonia uniflora (3) Tsuga heterophylla (3) Target species Moneses uniflora (1) Listera cordata (3) Tsuga heterophylla (2) Vaccinium Target species Green orchid Autotroph Autotroph Green orchid Autotroph Autotroph 2 parvifolium (4) Sol Duc 5 Hoh 1 Hoh 2 Västernorrland County, Sweden Near Gideabergs bog 48º03’94.36”N 124º06’764.0”W 47º49’77.79”N 123º59’50.07”W 47º49’79.94”N 123º59’52.20”W 63°17’59.02”N 16°33’18.08”E Moneses uniflora (1) Menziesia ferruginea (3) Picea sitchensis (2) Tsuga heterophylla (3) Target species Moneses uniflora (1) Blechnum spicant (2) Tsuga heterophylla (2) Vaccinium parvifolium (3) Target species Moneses uniflora (1) Hypopitys monotropa (1) Picea sitchensis (3) Tsuga heterophylla (5) Target species Moneses uniflora (8) Epipogium aphyllum (3) Target species Autotroph Autotroph Autotroph Autotroph Autotroph Autotroph Fully mycoheterotrophic Ericaceae Autotroph Autotroph Fully mycoheterotrophic orchid Oxalis sp. (3) Autotroph Convallaria Autotroph majalis (3) Paris Autotroph 3 quadrifolia (3) Solidago virgaurea (3) East Sutherland Scotland, UK Bonar Bridge Ardgay Golf Club Balblair Woods 57°53’34.98”N 4°19’42.89”W 57º91’52.57”N 4º36’82.38”W Autotroph Moneses uniflora (3) Deschampsia flexuosa (3) Luzula campestris (3) Veronica chamaedrys (3) Target species Moneses uniflora (3) Calluna vulgaris (3) Empetrum nigrum (3) Fagus sylvatica (3) Target species Autotroph Autotroph Autotroph Autotroph Autotroph Autotroph 4 Table S2 Mean 13C or 15N isotope enrichment factors by group or species with replicates (n), and locality. Locality Olympic Peninsula, WA, USA Västernorrland County, Sweden East Sutherland Scotland, UK Moneses uniflora (7) Mean 13C by locality (SE) ‰ 1.33(0.45) Mean 15N by locality (SE) ‰ 3.09(0.47) Neottia cordata (6) Corallorhiza striata (3) Hypopitys monotropa (1) Autotrophs (47) 1.1(0.6) 9.57(0.59) 7.34 0(0.11) 1.18(0.51) 10.38(0.38) 15.29 0(0.17) Moneses uniflora (8) -0.41(0.35) 2.52(0.29) Epipogium aphyllum(3) 9.56(0.04) 9.88(1.5) Autotrophs (12) 0(0.43) 0(0.35) Moneses uniflora (6) -2.17(0.32) 2.88(0.17) Species or group (n) Autotrophs (21) 0(0.33) In parentheses is one standard error (SE) of the mean. 0(0.32) 5 Methods S1 Enrichment factor calculation Because leaf stable isotope compositions are influenced by local environmental conditions, to make comparisons of these values across our study sites we used a data normalizing calculation know as an isotope enrichment factor () approach (Preiss & Gebauer, 2008). Enrichment factors were calculated on a site-by-site basis in the following way: = δXsample-δXref where δXsample is the δ15N or δ13C of an individual sample (Moneses uniflora, autotrophic understory species, green orchid or fully mycoheterotrophic species) and δXref is the mean δ15N or δ13C of all autotrophic plants from a given site (Table S2). Enrichment factors per locality (northwest USA, north eastern Scotland or northern Sweden – here after referred to as Olympic Peninsula, Scotland or Sweden) and species (or group in the case of autotrophs) were then averaged (Fig. 1). All values are shown 1 standard error (SE). Statistical analyses Shapiro-Wilk tests revealed that all enrichment factors for all localities were normally distributed except for the 13C values for M. uniflora from Scotland, thus we used a non-parametric MannWhitney U’ tests for comparisons of the Scottish data. For the remaining localities (Olympic Peninsula or Sweden) statistical comparisons between the enrichment factors for each species by locality and their associated autotrophs were made using one-way ANOVAs with post hoc Tukey HSD tests for multiple comparisons. These tests incorporated the spread of the data not only for M. uniflora and fully mycoheterotrophic species, but the autotrophic references as well since individual replicates of reference species are also subjected to the enrichment factor calculation (i.e. statistical comparisons of M. uniflora are not to zero, but the variation of the references and M. uniflora from zero). Also, we chose to analyze our data by locality rather than site, thus increasing the total number of M. uniflora replicates and reference species for the Olympic Peninsula and Scotland (Table S1). All statistics were carried out in SPSS v.22 (IMB Armonk, NY, USA) and considered significant at ≤0.05. Reference Preiss K, Gebauer G. 2008. A methodological approach to improve estimates of nutrient gains by partially myco-heterotrophic plants. Isotopes in Environmental and Health Studies 44: 393– 401. 6
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