Differences in ectomycorrhizal community assembly between native and exotic pines are reflected in their enzymatic functional capacities

  • Chen NingEmail author
  • Wenhua Xiang
  • Gregory M. Mueller
  • Louise M. Egerton-Warburton
  • Wende Yan
  • Shuguang Liu
Regular Article


Background and Aims

Introducing exotic tree species for afforestation out of their natural range may alter the local ectomycorrhizal (ECM) fungal communities. The potential functional consequences shaped by exotic trees with recruited local ECM fungi rather than native trees remain unclear. This study examined (a) whether the composition and extracellular enzyme function of ECM fungal communities differed between native masson pine (Pinus massoniana) and exotic slash pine (Pinus elliottii) during seedling establishment; and (b) how differences in enzyme functioning were linked to the growth pattern of the host plants.


Native (masson) and exotic (slash) pine seedlings were planted into soil cores collected from each study site. At three months growth, root tips were collected from seedlings and assayed for ECM fungal community composition using high-throughput sequencing, and functioning using single root tip assays for enzymes associated with N, P and C acquisition.


ECM fungi on masson pines showed higher activities of nitrogen- (N-acetylglucosaminidase, 280–300%), phosphorus- (acid phosphatase, 105–152%), and cellulose (β-glucosidase, 204–235%; cellobiohydrolase, 142–255%) compound degrading enzymes compared to those on slash pines. Those differences was attributed to the host-specific performance of certain ECM fungal taxa, such as Rhizopogon spp. Information theory model selection showed that plant nutrient status in masson pines was correlated with the enzymatic contribution of Rhizopogon spp., whereas slash pines depended on a diverse enzyme palette from multiple ECM fungal taxa.


Host identity strongly influenced ECM fungal community composition and extracellular enzymatic functions of specific ECM fungal taxa, which could feedback to host establishment and nutrient cycling processes of restored ecosystem. Therefore, the origin of afforestation tree species should be an important factor when selecting tree species for restoration of degraded lands.


Community structure and function Ectomycorrhizal fungi Extracellular enzyme Exotic pine Rhizopogon Russula 



We thank the graduate students Xiaowei Ni, Zhizhou Liu, Yi Chen, Xinghao Huang, and Juan Cao for their assistance in the field and laboratory manipulations. We thank the Forest Administration of of Longli Forest Farm for the permission to sample and carry out this field investigation. We also thank Dr. Peter Avis and Dr. Andrew Wilson for their comments on an earlier version of the paper. This study was supported by National Science Foundation of China (31570447, awarded to WX), the collaborative program of Plant Biology and Conservation at Northwestern University and Chicago Botanic Garden, and China Scholarship Council (CSC201408430072, awarded to CN). We gratefully acknowledge Dr. Martin Nuñez and two anonymous reviewers for providing invaluable comments during the review of this manuscript.

Author’s contributions

CN, GMM and LEW conceived the ideas and designed methodology; CN collected the data; CN analyzed the data; CN, WX, LEW, and GMM led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

Supplementary material

11104_2019_4355_MOESM1_ESM.docx (339 kb)
ESM 1 (DOCX 339 kb)


  1. Amend AS, Seifert KA, Bruns TD (2010) Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol 19:5555–5565PubMedCrossRefGoogle Scholar
  2. Akroume E, Maillard F, Bach C, Hossann C, Brechet C, Angeli N, Zeller B, Saint-André L, Buée M (2019) First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass. Environ Microbiol 21:197–208PubMedCrossRefGoogle Scholar
  3. Avis PG, Charvat I (2005) The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply. Mycologia 97:329–337PubMedCrossRefGoogle Scholar
  4. Bahram M, Köljalg U, Kohout P, Mirshahvaladi S, Tedersoo L (2013) Ectomycorrhizal fungi of exotic pine plantations in relation to native;host trees in Iran: evidence of host range expansion by local symbionts;to distantly related host taxa. Mycorrhiza 23:11–19PubMedCrossRefGoogle Scholar
  5. Bartoń K (2013) MuMIn: Multi-model inference. R core teamGoogle Scholar
  6. Bever JD, Richardson SC, Lawrence BM, Holmes J, Watson M (2009) Preferential allocation to beneficial symbiont with spatial structure maintains mycorrhizal mutualism. Ecol Lett 12:13–21PubMedCrossRefGoogle Scholar
  7. Cairney JWG (2012) Extramatrical mycelia of ectomycorrhizal fungi as moderators of carbon dynamics in forest soil. Soil Biol Biochem 47:198–208CrossRefGoogle Scholar
  8. Certano AD, Fernandez CW, Heckman KA, Kennedy PG (2018) The after life effects of fungal morphology: contrasting decomposition rates between diffuse and rhizomorphic necromass. Soil Biol Biochem 126:76–81CrossRefGoogle Scholar
  9. Chen QJ, Shu ZZ, Pan W, Long YZ, Wei BS (1995) The cultivation zoning and base layout of slash pine in China. For Sci Tech Guangdong Prov 11:1–6 (in Chinese with English abstract)Google Scholar
  10. Chen YL, Kang LH, Malajczuk N, Dell B (2006) Selecting ectomycorrhizal fungi for inoculating plantations in South China: effect of scleroderma, on colonization and growth of exotic Eucalyptus globulus, E. urophylla, Pinus elliottii, and P. radiata. Mycorrhiza 16:251–259PubMedCrossRefPubMedCentralGoogle Scholar
  11. Chen FL, Zheng H, Yang BS, Ouyang ZY, Zhang K, Tu NM (2011) Effects of exotic species slash pine (Pinus elliottii) litter on the structure and function of the soil microbial community. Acta Ecol Sin 31:3543–3550 (in Chinese with English abstract)Google Scholar
  12. Corrêa A, Gurevitch J, Martins-Loucão MA, Cruz C (2012) C allocation to the fungus is not a cost to the plant in ectomycorrhizae. Oikos 121:449–463CrossRefGoogle Scholar
  13. Courty PE, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005) Activity profiling of ectomycorrhizal communities in two forest soils using multiple enzymatic tests. New Phytol 167:309–319PubMedCrossRefPubMedCentralGoogle Scholar
  14. Courty PE, Buée M, Diedhiou AG, Frey-Klett P, Tacon FL, Rineau F, Turpault MP, Uroz S, Garbaye J (2010a) The role of ectomycorrhizal communities in forest ecosystem processes: new perspectives and emerging concepts. Soil Biol Biochem 42:679–698CrossRefGoogle Scholar
  15. Courty PE, Franc A, Garbaye J (2010b) Temporal and functional pattern of secreted enzyme activities in an ectomycorrhizal community. Soil Biol Biochem 42:2022–2025CrossRefGoogle Scholar
  16. Craig A, Woods S, Hoeksema JD (2016) Influences of host plant identity and disturbance on spatial structure and community composition of ectomycorrhizal fungi in a northern Mississippi uplands ecosystem. Fungal Ecol 24:7–14CrossRefGoogle Scholar
  17. Dickie IA, Bolstridge N, Cooper JA, Peltzer DA (2010) Co-invasion by pinus and its mycorrhizal fungi. New Phytol 187:475–484PubMedCrossRefGoogle Scholar
  18. Dodet M, Collet C (2012) When should exotic forest plantation tree species be considered as an invasive threat and how should we treat them? Biol Invasions 14:1765–1778CrossRefGoogle Scholar
  19. Fernandez CW, Langley JA, Chapman S, McCormack ML, Koide RT (2016) The decomposition of ectomycorrhizal fungal necromass. Soil Biol Biochem 93:38–49CrossRefGoogle Scholar
  20. Gehring CA, Sthultz CM, Flores-Rentería L, Whipple AV, Whitham TG (2017) Tree genetics defines fungal partner communities that may confer drought tolerance. Proc Natl Acad Sci 114:11169–11174PubMedCrossRefPubMedCentralGoogle Scholar
  21. Hawkins BJ, Jones MD, Kranabetter JM (2015) Ectomycorrhizae and tree seedling nitrogen nutrition in forest restoration. New For 46:747–771CrossRefGoogle Scholar
  22. Hayward J, Horton TR, Pauchard A, Nuñez MA (2015) A single ectomycorrhizal fungal species can enable a Pinus invasion. Ecology 96:1438–1444PubMedCrossRefPubMedCentralGoogle Scholar
  23. Hoeksema JD, Hernandez JV, Rogers DL, Mendoza LL, Thompson JN (2012) Geographic divergence in a species-rich symbiosis: interactions between Monterey pines and ectomycorrhizal fungi. Ecology 93:2274–2285PubMedCrossRefPubMedCentralGoogle Scholar
  24. Hortal S, Plett KL, Plett JM, Cresswell T, Johansen M, Pendall E, Anderson IC (2017) Role of plant-fungal nutrient trading and hosts control in determining the competitive success of ectomycorrhizal fungi. ISME J 11:2666–2676PubMedPubMedCentralCrossRefGoogle Scholar
  25. Hupperts SF, Karst J, Pritsch K, Landhäusser SM (2017) Host phenology and potential saprotrophism of ectomycorrhizal fungi in the boreal forest. Funct Ecol 31:116–126CrossRefGoogle Scholar
  26. Ishida TA, Nara K, Hogetsu T (2007) Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests. New Phytol 174:430–440PubMedCrossRefPubMedCentralGoogle Scholar
  27. Johnson D, Martin F, Cairney JWG, Anderson IC (2012) The importance of individuals: intraspecific diversity of mycorrhizal plants and fungi in ecosystems. New Phytol 194:614–628PubMedCrossRefPubMedCentralGoogle Scholar
  28. Jones MD, Phillips LA, Treu R, Ward V, Berch SM (2012) Functional responses of ectomycorrhizal fungal communities to long-term fertilization of lodgepole pine (Pinus contorta Dougl. Ex loud. Var. latifolia Engelm.) stands in Central British Columbia. Appl Soil Ecol 60:29–40CrossRefGoogle Scholar
  29. Kennedy PG, Peay KG, Bruns TD (2009) Root tip competition among ectomycorrhizal fungi: are priority effects a rule or an exception? Ecology 90:2098–2107PubMedCrossRefPubMedCentralGoogle Scholar
  30. Kennedy PG, Higgins LM, Rogers RH, Weber MG (2011) Colonization-competition tradeoffs as a mechanism driving successional dynamics in ectomycorrhizal fungal communities. PLoS One 6:e25126PubMedPubMedCentralCrossRefGoogle Scholar
  31. Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E, Fellbaum CR, Kowalchuk GA, Hart MM, Bago A, Palmer TM, West SA, Vandenkoornhuyse P, Jansa J, Bucking H (2011) Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science 333:880–882PubMedCrossRefGoogle Scholar
  32. Kipfer T, Wohlgemuth T, van der Heijden MGA, Ghazoul J, Egli S (2012) Growth response of drought-stressed Pinus sylvestris seedlings to single- and multi-species inoculation with ectomycorrhizal fungi. PLoS One 7:e35275PubMedPubMedCentralCrossRefGoogle Scholar
  33. Konopka A (2009) What is microbial community ecology? ISME J 3:1223–1230PubMedCrossRefGoogle Scholar
  34. Kohout P, Sýkorová Z, Bahram M, Hadincová V, Albrechtová J, Tedersoo L, Vohník M (2011) Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment. Mycorrhiza 21:403–412PubMedCrossRefGoogle Scholar
  35. Koide RT, Fernandez C, Malcolm G (2014) Determining place and process: functional traits of ectomycorrhizal fungi that affect both community structure and ecosystem function. New Phytol 201:433–439PubMedCrossRefGoogle Scholar
  36. Lang C, Finkeldey R, Polle A (2013) Spatial patterns of ectomycorrhizal assemblages in a monospecific forest in relation to host tree genotype. Front Plant Sci 4:1–9CrossRefGoogle Scholar
  37. Leski T, Aučina A, Skridaila A, Pietras M, Riepšas E, Rudawska M (2010) Ectomycorrhizal community structure of different genotypes of scots pine under forest nursery conditions. Mycorrhiza 20:473–481PubMedCrossRefGoogle Scholar
  38. Liao HL, Chen Y, Vilgalys R (2016) Metatranscriptomic study of common and host-specific patterns of gene expression between pines and their symbiotic ectomycorrhizal fungi in the genus Suillus. PLoS Genet 12:e1006348PubMedPubMedCentralCrossRefGoogle Scholar
  39. Lilleskov EA, Hobbie EA, Horton TR (2011) Conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic N deposition. Fungal Ecol 4:174–183CrossRefGoogle Scholar
  40. Livne-Luzon S, Ovadia O, Weber G, Avidan Y, Migael H, Glassman SI, Bruns TD, Shemesh H (2017) Small-scale spatial variability in the distribution of ectomycorrhizal fungi affects plant performance and fungal diversity. Ecol Lett 20:1192–1202PubMedCrossRefGoogle Scholar
  41. Ma Z, Hartmann H, Wang H, Li Q, Wang Y, Li S (2014) Carbon dynamics and stability between native masson pine and exotic slash pine plantations in subtropical China. Eur J For Res 133:307–321CrossRefGoogle Scholar
  42. Maillard F, Didion M, Fauchery L, Bach C, Buée M (2018) N-Acetylglucosaminidase activity, a functional trait of chitin degradation, is regulated differentially within two orders of ectomycorrhizal fungi: Boletales and Agaricales. Mycorrhiza 28:391–397PubMedCrossRefGoogle Scholar
  43. McCormack ML, Pritchard SG, Breland S, Davis MA, Prior SA, Runion GB, Mitchel RJ, Rogers HH (2010) Soil fungi respond more strongly than fine roots to elevated CO2 in a model regenerating longleaf pine-wiregrass ecosystem. Ecosystems 13:901–916CrossRefGoogle Scholar
  44. McMurdie PJ, Holmes S (2014) Waste not, want not: why rarefying microbiome data is inadmissible. PLoS Comput Biol 10:e1003531PubMedPubMedCentralCrossRefGoogle Scholar
  45. Moeller HV, Peay KG (2016) Competition-function tradeoffs in ectomycorrhizal fungi. Peer J 4:e2270PubMedCrossRefGoogle Scholar
  46. Nicolás C, Martin-Bertelsen T, Floudas D, Bentzer J, Smits M, Johansson T, Troein C, Persson P, Tunlid A (2019) The soil organic matter decomposition mechanisms in ectomycorrhizal fungi are tuned for liberating soil organic nitrogen. ISME J 13:977–988PubMedCrossRefGoogle Scholar
  47. Ning C, Mueller GM, Egerton-Warburton LM, Wilson AW, Yan W, Xiang W (2018) Diversity and enzyme activity of ectomycorrhizal fungal communities following nitrogen fertilization in an urban-adjacent pine plantation. Forests 9:99CrossRefGoogle Scholar
  48. Ning C, Mueller GM, Egerton-Warburton LM, Wilson AW, Yan W, Xiang W (2019) Host phylogenetic relatedness and soil nutrients shape ectomycorrhizal community composition in native and exotic pine plantations. Forests 10:263CrossRefGoogle Scholar
  49. Nguyen NH, Williams LJ, Vincent JB, Stefanski A, Cavender-Bares J, Messier C, Paquette A, Gravel D, Reich PB, Kennedy PC (2016) Ectomycorrhizal fungal diversity and saprotrophic fungal diversity are linked to different tree community attributes in a field-based tree experiment. Mol Ecol 25:4032–4046PubMedCrossRefGoogle Scholar
  50. Nuñez MA, Chiuffo MC, Torres A, Paul T, Dimarco RD, Raal P, Policelli N, Moyano J. García RA, van Wilgen BW, Pauchard A, Richardson DM (2017) Ecology and management of invasive Pinaceae around the world: progress and challenges. Biol Invasions 19: 3099–3120CrossRefGoogle Scholar
  51. Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O'Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H (2017) Vegan: community ecology package. R package version 2:4–3 Google Scholar
  52. Paulson JN, Stine OC, Bravo HC, Pop M (2013) Differential abundance analysis for microbial marker-gene surveys. Nat Methods 10:1200–1202PubMedPubMedCentralCrossRefGoogle Scholar
  53. Peay KG, Russo SE, McGuire KL, Lim Z, Chan JP, Tan S, Davies SJ (2015) Lack of host specificity leads to independent assortment of dipterocarps and ectomycorrhizal fungi across a soil fertility gradient. Ecol Lett 18:807–816PubMedCrossRefPubMedCentralGoogle Scholar
  54. Peay KG (2016) The mutualistic niche: mycorrhizal symbiosis and community dynamics. Ann Rev Ecol Evol Syst 47:143–164CrossRefGoogle Scholar
  55. Pickles B, Genney DR, Potts JM, Lennon JJ, Anderson IC, Alexander IJ (2010) Spatial and temporal ecology of scots pine ectomycorrhizas. New Phytol 186:755–768PubMedCrossRefPubMedCentralGoogle Scholar
  56. Plett JM, Tisserant E, Brun A, Morin E, Grigoriev IV, Kuo A, Martin F, Kohler A (2015) The mutualist Laccaria bicolor expresses a core gene regulon during the colonization of diverse host plants and a variable regulon to counteract host-specific defenses. Mol Plant-Microbe Interact 28:261–273PubMedCrossRefPubMedCentralGoogle Scholar
  57. Policelli N, ,Bruns TD, Vilgalys R, Nuñez MA (2019) Suilloid fungi as global drivers of pine invasions. New Phytol, 222: 714–725PubMedCrossRefPubMedCentralGoogle Scholar
  58. Pringle A, Bever JD, Gardes M, Parrent JL, Rillig MC, Klironomos JN (2009) Mycorrhizal symbioses and plant invasions. Ann Rev Ecol Evol Syst 40:699–715CrossRefGoogle Scholar
  59. Pritsch K, Courty PE, Churin JL, Coutier-Hurteau B, Arif Ali M, Damon C, Duchemin M, Egli S, Ernst J, Fraissinet-Tachet L, Kuhar F, Legname E, Marmeisse R, Müller A, Nikolova P, Peter M, Plassard C, Richard F, Schloter M, Selosse MA, Franc A, Garbaye J (2011) Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae. Mycorrhiza 21:589–600PubMedCrossRefPubMedCentralGoogle Scholar
  60. Richardson DM (1998) Pines as invaders in the southern hemisphere. In: Richardson DM (ed) Ecology and biogeography of Pinus. University Press, Cambridge, UK, pp 450–473Google Scholar
  61. Rineau F, Courty PE (2011) Secreted enzymatic activities of ectomycorrhizal fungi as a case study of functional diversity and functional redundancy. Ann For Sci 68:69–80CrossRefGoogle Scholar
  62. Rusca TA, Kennedy PG, Bruns TD (2006) The effect of different pine hosts on the sampling of Rhizopogon spore banks in five eastern Sierra Nevada forests. New Phytol 170:551–560PubMedCrossRefPubMedCentralGoogle Scholar
  63. SFA (State Forestry Administration PR China) (2014) National Forest Resources Statistics (2009-2013). NFI 8th PR state forestry administration, Beijing, ChinaGoogle Scholar
  64. Schaefer VH (2011) Remembering our roots: a possible connection between loss of ecological memory, alien invasions and ecological restoration. Urban Ecosyst 14:35–44CrossRefGoogle Scholar
  65. Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic Press, Cambridge, UKGoogle Scholar
  66. Smith DP, Peay KG (2014) Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS One 9:e90234PubMedPubMedCentralCrossRefGoogle Scholar
  67. Talbot JM, Allison SD, Treseder KK (2008) Decomposers in disguise: mycorrhizal fungi as regulators of soil C dynamics in ecosystems under global change. Funct Ecol 22:955–963CrossRefGoogle Scholar
  68. Taylor DL, Walters WA, Lennon NJ, Bochicchio J, Krohn A, Caporaso JG, Pennanen T (2016) Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing. Appl Environ Micro 82:7217–7226CrossRefGoogle Scholar
  69. Tedersoo L, Suvi T, Beaver K, Köljalg U (2007) Ectomycorrhizal fungi of the Seychelles: diversity patterns and host shifts from the native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia bijuga (Caesalpiniaceae) to the introduced Eucalyptus robusta (Myrtaceae), but not Pinus caribea (Pinaceae). New Phytol 175:321–333PubMedCrossRefPubMedCentralGoogle Scholar
  70. Tian D, Xiang W, Yan W (2004) Comparison of biomass dynamics and nutrient cycling between Pinus massoniana plantation and Pinus elliottii planatation. Acta Ecol Sin 24:2207–2210 (in Chinese with English abstract)Google Scholar
  71. Treseder KK, Allen MF, Ruess RW, Pregitzer KS, Hendrick RL (2005) Lifespans of fungal rhizomorphs under nitrogen fertilization in a pinyon-juniper wood-land. Plant Soil 270:249–255CrossRefGoogle Scholar
  72. Trocha LK, Kalucka I, Stasińska M, Nowak W, Dabert M, Leski T, Rudawska M, Oleksyn J (2012) Ectomycorrhizal fungal communities of native and non-native Pinus and Quercus species in a common garden of 35-year-old trees. Mycorrhiza 22:121–134PubMedCrossRefGoogle Scholar
  73. Van der Linde S, Suz LM, Orme CDL, Cox F, Andreae H, Asi E, Atkinson B, Benham S, Carroll C, Cools N, De Vos B, Dietrich H, Eichhorn J, Gehrmann J, Grebenc T, Gweon HS HS, Hansen K, Jacob F, Kristöfel F, Lech P, Manninger M, Martin J, Meesenburg H, Merilä P, Nicolas M, Pavlenda P, Rautio P, Schaub M, Schröck H, Seidling W, Šrámek V, Thimonier A, Thomsen IM, Titeux H, Vanguelova E, Verstraeten A, Vesterdal L, Waldner P, Wijk S, Zhang Y, Žlindra D, Bidartondo MJ (2018) Environment and host as large-scale controls of ectomycorrhizal fungi. Nature 558:243–248PubMedCrossRefGoogle Scholar
  74. Velmala SM, Rajala T, Heinonsalo J, Taylor AFS, Pennanen T (2014) Profiling functions of ectomycorrhizal diversity and root structuring in seedlings of Norway spruce (Picea abies) with fast- and slow-growing phenotypes. New Phytol 201:610–622PubMedCrossRefGoogle Scholar
  75. Walker JKM, Cohen H, Higgins LM, Kennedy PG (2014) Testing the link between community structure and function for ectomycorrhizal fungi involved in a global tripartite symbiosis. New Phytol 202:287–296PubMedCrossRefGoogle Scholar
  76. Wang XH, Kent M, Fang XF (2007) Evergreen broad-leaved forest in eastern China: its ecology and conservation and the importance of resprouting in forest restoration. For Ecol Manag 245:76–87CrossRefGoogle Scholar
  77. Wang Y, Wang H, Xu M, Ma Z, Wang Z (2015) Soil organic carbon stocks and CO2 effluxes of native and exotic pine plantations in subtropical China. Catena 128:167–173CrossRefGoogle Scholar
  78. Weight RB, Raidl S, Verma R, Agerer R (2012) Exploration type-specific standard values of extramatrical mycelium - a step towards quantifying ectomycorrhizal space occupation and biomass in natural soil. Mycol Progr 11:287–297CrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Faculty of Life Science and TechnologyCentral South University of Forestry and TechnologyChangshaChina
  2. 2.Program in Plant Biology and ConservationNorthwestern UniversityEvanstonUSA
  3. 3.Chicago Botanic GardenGlencoeUSA

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