Advertisement

Microbial Ecology

, Volume 57, Issue 4, pp 728–739 | Cite as

Are Basidiomycete Laccase Gene Abundance and Composition Related to Reduced Lignolytic Activity Under Elevated Atmospheric NO3 Deposition in a Northern Hardwood Forest?

  • John E. HassettEmail author
  • Donald R. Zak
  • Christopher B. Blackwood
  • Kurt S. Pregitzer
Original Article

Abstract

Anthropogenic release of biologically available N has increased atmospheric N deposition in forest ecosystems, which may slow decomposition by reducing the lignolytic activity of white-rot fungi. We investigated the potential for atmospheric N deposition to reduce the abundance and alter the composition of lignolytic basidiomycetes in a regional network of four northern hardwood forest stands receiving experimental NO3 deposition (30 kg NO3 −N ha−1 year−1) for a decade. To estimate the abundance of basidiomycetes with lignolytic potential, we used PCR primers targeting laccase (polyphenol oxidase) and quantitative fluorescence PCR to estimate gene copy number. Natural variation in laccase gene size permitted use of length heterogeneity PCR to profile basidiomycete community composition across two sampling dates in forest floor and mineral soil. Although past work has identified significant and consistent negative effects of NO3 deposition on lignolytic enzyme activity, microbial biomass, soil respiration, and decomposition rate, we found no consistent effect of NO3 deposition on basidiomycete laccase gene abundance or community profile. Rather, laccase abundance under NO3 deposition was lower (−52%), higher (+223%), or unchanged, depending on stand. Only a single stand exhibited a significant change in basidiomycete laccase gene profile. Basidiomycete laccase genes occurring in mineral soil were a subset of the genes observed in the forest floor. Moreover, significant effects on laccase abundance were confined to the forest floor, suggesting that species composition plays some role in determining how lignolytic basidiomycetes are affected by N deposition. Community profiles differed between July and October sampling dates, and basidiomycete communities sampled in October had lower laccase gene abundance in the forest floor, but higher laccase abundance in mineral soil. Although experimental N deposition significantly suppresses lignolytic activity in these forests, this change is not related to the abundance or community composition of basidiomycete fungi with laccase genes. Understanding the expression of laccases and other lignolytic enzymes by basidiomycete fungi and other lignin-decaying organisms appears to hold promise for explaining the consistent decline in lignolytic activity elicited by experimental N deposition.

Keywords

Mineral Soil Forest Floor Laccase Gene Basidiomycete Fungus Quantitative Fluorescence Polymerase Chain Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

This work was supported by grants from the NSF Ecosystem Studies Program (DEB 9629842, DEB 0315138, DEB 0614422), and by NSF-IGERT support to J.E. Hassett through the Biosphere Atmosphere Research Training program (DGE 0504552). Site access was made possible by the Manistee National Forest and the Michigan Department of Natural Resources. We thank the numerous contributors and collaborators who have supported this effort, especially Terrestrial Ecosystems Laboratory personnel at the University of Michigan. Nicole Seleno and Kirsten Hofmockel provided invaluable technical and analytical support. Editorial suggestions by Ivan Edwards were highly beneficial to manuscript development.

Supplementary material

248_2008_9440_MOESM1_ESM.pdf (10 kb)
Supplemental Table S1 Examples of length heterogeneity in published laccase sequences. Rows contain sequence information for specific laccase sequences presented in published reports, retrievable by GenBank accession number. For each sequence, columns indicate which of 18 identified sequence features is present between the Cu1F and Cu2R primer sites, as well as the total length of the expected PCR amplicon. Positions of sequence features are given in base pair, as measured 5′ to 3′ from the 5′ end of primer Cu1F on a consensus laccase sequence 142 bases long. For each sequence and feature, a numeric value indicates the length of a predicted intron in the sequence, (+) indicates a codon insertion/deletion, and (−) indicates no feature at the given position (PDF 13.6 KB)
248_2008_9440_MOESM2_ESM.pdf (29 kb)
Supplemental Figure S1 Intron map of partial basidiomycete laccase sequence, representing the 66% majority consensus amino acid sequence encoded by exons of 36 published laccase genes (see Supplemental Table 1). Sequence proceeds 5′→3′, enclosed areas represent PCR priming sites for primers Cu1F (left) and Cu2R (right). Tilde (~) indicates codon position without consensus. Open triangles (s) above sequence indicate the positions of 16 predicted introns; closed triangle (p) below sequence indicates position of a single codon insertion. Bar (−) spans the site of a single-codon deletion (PDF 28.5 KB)
248_2008_9440_MOESM3_ESM.pdf (191 kb)
Supplemental Figure S2 Examples of laccase LH-PCR profiles. Chromatograms were generated by capillary gel separation of PCR products amplified using basidiomycete laccase primers Cu1FFAM and Cu2R [S4]. Profiles display the results for single samples derived from the forest floor (A) and mineral soil (B) of stand A (PDF 191 KB)
248_2008_9440_MOESM4_ESM.pdf (13 kb)
Supplemental References (PDF 13.1 KB)

References

  1. 1.
    Abe Y (1989) Effect of moisture on decay of wood by xylariaceous and diatrypaceous fungi and quantitative changes in the chemical components of decayed woods. Trans Mycol Soc Jpn 30:169–181Google Scholar
  2. 2.
    Arnolds E (1991) Decline of ectomycorrhizal fungi in Europe. Agric Ecosyst Environ 25:209–244CrossRefGoogle Scholar
  3. 3.
    Baek JM, Kenerley CM (1998) Detection and enumeration of a genetically modified fungus in soil environments by quantitative competitive polymerase chain reaction. FEMS Microbiol Ecol 25:419–428CrossRefGoogle Scholar
  4. 4.
    Baldrian P (2006) Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30:215–242PubMedCrossRefGoogle Scholar
  5. 5.
    Berg B (1986) Nutrient release from litter and humus in coniferous forest soils—a mini review. Scand J For Res 1:359–369CrossRefGoogle Scholar
  6. 6.
    Berg B, Matzner E (1997) Effect of N deposition on decomposition of plant litter and soil organic matter in forest systems. Environ Rev 5:1–25CrossRefGoogle Scholar
  7. 7.
    Biederbeck VO, Campbell CA, Ukrainetz H, Curtin D, Bouman OT (1996) Soil microbial and biochemical properties after ten years of fertilization with urea and anhydrous ammonia. Can J Soil Sci 76:7–14Google Scholar
  8. 8.
    Blackwood CB, Marsh T, Kim S, Paul EA (2003) Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Appl Environ Microbiol 69:926–932PubMedCrossRefGoogle Scholar
  9. 9.
    Blackwood CB, Waldrop MP, Zak DR, Sinsabaugh RL (2007) Molecular analysis of fungal communities and laccase genes in decomposing litter reveal differences among forest types but no impact of N deposition. Environ Microbiol 9:1306–1316PubMedCrossRefGoogle Scholar
  10. 10.
    Boyle D (1998) Nutritional factors limiting the growth of Lentinula edodes and other white-rot fungi in wood. Soil Biol Biochem 30:817–823CrossRefGoogle Scholar
  11. 11.
    Burton AJ, Pregitzer KS, MacDonald NW (1993) Foliar nutrients in sugar maple forests along a regional pollution-climate gradient. Soil Sci Soc Am. J 57:1619–162Google Scholar
  12. 12.
    Burton AJ, Pregitzer KS, Hendrick RL (2000) Relationships between fine root dynamics and nitrogen availability in Michigan northern hardwood forests. Oecologia 125:389–399CrossRefGoogle Scholar
  13. 13.
    Burton AJ, Pregitzer KS, Crawford JN, Zogg GP, Zak DR (2004) Simulated chronic NO3 deposition reduces soil respiration in northern hardwood forests. Global Change Biol 10:1080–1091CrossRefGoogle Scholar
  14. 14.
    Carreiro MM, Sinsabaugh RL, Rupert DA, Parkhurst DF (2000) Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition. Ecology 81:2359–2365Google Scholar
  15. 15.
    Chen DM, Bastias BA, Taylor AFS, Cairney JWG (2003) Identification of laccase-like genes in ectomycorrhizal basidiomycetes and transcriptional regulation by nitrogen in Piloderma byssinum. New Phytol 157:547–554CrossRefGoogle Scholar
  16. 16.
    Colpaert JV, van Laere A (1996) A comparison of the extracellular enzyme activities of two ectomycorrhizal and a leaf-saprotrophic basidiomycete colonizing beech leaf litter. New Phytol 134:133–141CrossRefGoogle Scholar
  17. 17.
    Courty PE, Pouysegur R, Buée M, Garbaye J (2006) Laccase and phosphatase activities of the dominant ectomycorrhizal types in a lowland oak forest. Soil Biol Biochem 38:1219–1222CrossRefGoogle Scholar
  18. 18.
    DeForest J, Zak DR, Pregitzer KS, Burton AJ (2004) Atmospheric nitrate deposition, microbial community composition, and enzyme activity in northern hardwood forests. Soil Sci Soc Am J 68:132–138Google Scholar
  19. 19.
    Dijkstra FA, Hobbie SE, Knops JMH, Reich PB (2004) Nitrogen deposition and plant species interact to influence soil carbon stabilization. Ecol Lett 7:1192–119CrossRefGoogle Scholar
  20. 20.
    Dix NJ, Webster J (1995) Fungal ecology. Chapman & Hall, LondonGoogle Scholar
  21. 21.
    Frankland JC (1998) Fungal succession—unraveling the unpredictable. Mycol Res 102:1–1CrossRefGoogle Scholar
  22. 22.
    Franklin O, Högberg P, Ekblad A, Ågren GI (2003) Pine forest floor carbon accumulation in response to N and PK additions: bomb 14C modelling and respiration studies. Ecosystems 6:644–665CrossRefGoogle Scholar
  23. 23.
    Fredricks DN, Smith C, Meier A (2005) Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol 43:5122–5128PubMedCrossRefGoogle Scholar
  24. 24.
    Fog K (1988) The effect of added nitrogen on the rate of decomposition of organic matter. Biol Rev 63:433–462CrossRefGoogle Scholar
  25. 25.
    Frey SD, Knorr M, Parrent JL, Simpson RT (2004) Chronic nitrogen enrichment affects the structure and function of the soil microbial community in temperate hardwood and pine forests. For Ecol Manag 196:159–171CrossRefGoogle Scholar
  26. 26.
    Frostegård Å, Courtois S, Ramisse V, Clerc S, Bernillon D, Le Gall F, Jeannin P, Nesme X, Simonet P (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 65:5409–5420PubMedGoogle Scholar
  27. 27.
    Garćia C, Hernández T (1996) Influence of salinity on the biological and biochemical activity of a calciorthird soil. Plant Soil 178:255–263CrossRefGoogle Scholar
  28. 28.
    Hatakka A (1994) Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol Rev 13:125–135CrossRefGoogle Scholar
  29. 29.
    Hagedorn F, Spinnler D, Siegwolf R (2003) Increased N deposition retards mineralization of old soil organic matter. Soil Biol Biochem 35:1683–1692CrossRefGoogle Scholar
  30. 30.
    Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994PubMedCrossRefGoogle Scholar
  31. 31.
    Hendrick RL, Pregitzer KS (1993) The dynamics of fine root length, biomass, and nitrogen content in two northern hardwood ecosystems. Can J For Res 23:2507–2520CrossRefGoogle Scholar
  32. 32.
    Keyser P, Kirk TK, Zeikus JG (1976) Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J Bacteriol 135:790–797Google Scholar
  33. 33.
    Kilaru S, Hoegger P, Kües U (2006) The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies. Curr Genet 50:45–60PubMedCrossRefGoogle Scholar
  34. 34.
    Kirby R (2005) Actinomycetes and lignin degradation. Adv Appl Microbiol 58:125–168CrossRefGoogle Scholar
  35. 35.
    Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H, Trevors JT (2004) Methods of studying soil microbial diversity. J Microbiol Methods 58:169–188PubMedCrossRefGoogle Scholar
  36. 36.
    Kontainis EJ, Reed FA (2006) Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors. J Forensic Sci 51:795–804CrossRefGoogle Scholar
  37. 37.
    Kuyper TW, DeVries BWL (1990) Effects of fertilization on the mycoflora of a pine forest. Agric Univ Wageningen Pap 90:102–111Google Scholar
  38. 38.
    Kuyper TW, Bokeloh DJ (1994) Ligninolysis and nitrification in vitro by a nitrotolerant and a nitrophobic decomposer basidiomycete. Oikos 70:417–420CrossRefGoogle Scholar
  39. 39.
    Lakay FM, Botha A, Prior AB (2007) Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. J Appl Microbiol 102:265–273PubMedCrossRefGoogle Scholar
  40. 40.
    Larrondo LF, González BL, Cullen D, Vicuña R (2004) Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts. Microbiology 150:2775–2783PubMedCrossRefGoogle Scholar
  41. 41.
    Leatham GF, Kirk TK (1983) Regulation of ligninolytic activity by nutrient nitrogen in white-rot basidiomycetes. FEMS Microbiol Lett 16:65–6CrossRefGoogle Scholar
  42. 42.
    Li X, Li F, Ma Q, Cui Z (2006) Interactions of NaCl and Na2SO4 on soil organic C mineralization after addition of maize straw. Soil Biol Biochem 38:2328–2335CrossRefGoogle Scholar
  43. 43.
    Lilleskov EA, Fahey TJ, Horton TR, Lovett GM (2002) Belowground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. Ecology 83:104–11CrossRefGoogle Scholar
  44. 44.
    Lindahl BO, Taylor AFS, Finlay RD (2002) Defining nutritional constraints on carbon cycling in boreal forests—towards a less “phytocentric” perspective. Plant Soil 242:123–135CrossRefGoogle Scholar
  45. 45.
    Liu W, Saint DA (2002) Validation of a quantitative method for real time PCR kinetics. Biochem Biophys Res Comm 294:347–35PubMedCrossRefGoogle Scholar
  46. 46.
    Luis P, Walther G, Kellner H, Martin F, Buscot F (2004) Diversity of laccase genes from basidiomycetes in a forest soil. Soil Biol Biochem 36:1025–1036CrossRefGoogle Scholar
  47. 47.
    Luis P, Kellner H, Zimdars B, Langer U, Martin F, Buscot F (2005) Patchiness and spatial distribution of laccase genes of ectomycorrhizal, saprotrophic, and unknown basidiomycetes in the upper horizons of a mixed forest cambisol. Microb Ecol 50:570–579PubMedCrossRefGoogle Scholar
  48. 48.
    Luis P, Kellner H, Martin F, Buscot F (2005b) A molecular method to evaluate basidiomycete laccase gene expression in forest soils. Geoderma 128:18–27CrossRefGoogle Scholar
  49. 49.
    MacDonald NW, Zak DR, Pregitzer KS (1995) Temperature effects on kinetics of microbial respiration and net nitrogen and sulfur mineralization. Soil Sci Soc Am J 59:223–240Google Scholar
  50. 50.
    McDowell WH, Currie WS, Aber JD, Yano Y (1998) Effects of chronic nitrogen amendments on production of dissolved organic carbon and nitrogen in forest soils. Water Air Soil Pollut 105:175–182CrossRefGoogle Scholar
  51. 51.
    Mumy KL, Finlay RH (2004) Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. J Microbiol Meth 57:259–268CrossRefGoogle Scholar
  52. 52.
    National Atmospheric Deposition Program (2005) National Atmospheric Deposition Program 2004 annual summary. NADP Data Report 2005-01. Illinois State Water Survey, Champaign, ILGoogle Scholar
  53. 53.
    National Oceanic and Atmospheric Administration (2005) Climatological data annual summary Michigan 2004 (vol 119, no 13). National Climatic Data Center, Asheville, NCGoogle Scholar
  54. 54.
    Neff JC, Townsend AR, Gleixner G, Lehman SJ, Turnbull J, Bowman WD (2002) Variable effects of nitrogen additions on the stability and turnover of soil carbon. Nature 419:915–917PubMedCrossRefGoogle Scholar
  55. 55.
    Osono T (2007) Ecology of ligninolytic fungi associated with leaf litter decomposition. Ecol Res 22:955–974CrossRefGoogle Scholar
  56. 56.
    Peter M, Ayer F, Egli S (2001) Nitrogen addition in a Norway spruce stand altered macromycete sporocarp production and below-ground ectomycorrhizal species composition. New Phytol 149:311–325CrossRefGoogle Scholar
  57. 57.
    Peter M, Ayer F, Egli S, Honegger R (2001b) Above- and below-ground community structure of ectomycorrhizal fungi in three Norway spruce (Picea abies) stands in Switzerland. Can J Bot 79:1134–1151CrossRefGoogle Scholar
  58. 58.
    Pointing SB, Parungao MM, Hyde KD (2003) Production of wood-decay enzymes, mass loss and lignin solubilization in wood by tropical Xylariaceae. Mycol Res 107:231–235PubMedCrossRefGoogle Scholar
  59. 59.
    Pregitzer KS, Burton AJ, Zak DR, Talhelm AF (2008) Simulated chronic nitrogen deposition increases carbon storage in northern temperate forests. Glob Chang Biol 14:142–153Google Scholar
  60. 60.
    Pregitzer KS, Zak DR, Burton AJ, Ashby JA, MacDonald NW (2004) Chronic nitrate additions dramatically increase the export of carbon and nitrogen from northern hardwood ecosystems. Biogeochemistry 68:179–197CrossRefGoogle Scholar
  61. 61.
    Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66PubMedCrossRefGoogle Scholar
  62. 62.
    Read DJ, Perez-Moreno J (2003) Mycorrhizas and nutrient cycling in ecosystems—a journey towards relevance? New Phytol 157:475–492CrossRefGoogle Scholar
  63. 63.
    Reeleder RD, Capell BB, Tomlinson LD, Hickey WJ (2003) The extraction of fungal DNA from multiple large soil samples. Can J Plant Pathol 25:182–191Google Scholar
  64. 64.
    Rietz DN, Haynes RJ (2003) Effects of irrigation-induced salinity and sodicity on soil microbial activity. Soil Biol Biochem 35:845–854CrossRefGoogle Scholar
  65. 65.
    Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160PubMedCrossRefGoogle Scholar
  66. 66.
    Ritchie NJ, Schutter ME, Dick RP, Myrold DD (2000) Use of length heterogeneity PCR and fatty acid methyl ester profiles to characterize microbial communities in soil. Appl Environ Microbiol 66:1668–1675PubMedCrossRefGoogle Scholar
  67. 67.
    Rühling Å, Tyler G (1991) Effects of simulated nitrogen deposition to the forest floor on the macrofungal flora of a beech forest. Ambio 20:261–263Google Scholar
  68. 68.
    Saiya-Cork KR, Sinsabaugh RL, Zak DR (2002) The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil. Soil Biol Biochem 34:1309–1315CrossRefGoogle Scholar
  69. 69.
    Sagova-Mareckova M, Cermak L, Novotna J, Plhackova K, Forstova J, Kopecky J (2008) Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl Environ Microbiol 74:2902–2907PubMedCrossRefGoogle Scholar
  70. 70.
    Saleh-Lakha S, Miller M, Campbell GR, Schneider K, Elahimanesh P, Hart MM, Trevors JT (2005) Microbial gene expression in soil: methods, applications and challenges. J Microbiol Methods 63:1–19PubMedCrossRefGoogle Scholar
  71. 71.
    Sinsabaugh RL, Gallo ME, Lauber C, Waldrop MO, Zak DR (2005) Extracellular enzyme activities and soil organic matter dynamics for northern hardwood forests receiving simulated nitrogen deposition. Biogeochemistry 75:201–215CrossRefGoogle Scholar
  72. 72.
    Suzuki MT, Rappé MS, Giovannoni SJ (1998) Kinetic bias in estimates of costal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity. Appl Environ Microbiol 64:4522–4529PubMedGoogle Scholar
  73. 73.
    Tarvainen O, Markkola AM, Strömmer R (2003) Diversity of macrofungi and plants in Scots pine forests along an urban pollution gradient. Basic Appl Ecol 4:547–556CrossRefGoogle Scholar
  74. 74.
    Varma A (1999) Hydrolytic enzymes from arbuscular mycorrhizae: the current status. In: Varma A, Hock B (eds) Mycorrhiza. 2nd edn. Springer, Berlin Heidelberg New York, pp 373–389Google Scholar
  75. 75.
    Waldrop MP, Zak DR, Sinsabaugh RL, Gallo M, Lauber C (2004) Nitrogen deposition modifies soil carbon storage through changes in microbial enzyme activity. Ecol Appl 14:1172–1177CrossRefGoogle Scholar
  76. 76.
    Waldrop MP, Zak DR (2006) Response of oxidative enzyme activities to nitrogen deposition affects soil concentrations of dissolved organic carbon. Ecosystems 9:921–933CrossRefGoogle Scholar
  77. 77.
    Wallander H (1995) A new hypothesis to explain allocation of dry matter between mycorrhizal fungi and pine seedlings in relation to nutrient supply. Plant Soil 168–169:243–248CrossRefGoogle Scholar
  78. 78.
    Wallenda T, Kottke I (1998) Nitrogen deposition and ectomycorrhizas. New Phytol 139:169–187CrossRefGoogle Scholar
  79. 79.
    Zadrazil F, Brunnert H (1980) The influence of ammonium nitrate supplementation on degradation and in vitro digestibility of straw colonized by higher fungi. Eur J Appl Microbiol Biotechnol 9:37–44CrossRefGoogle Scholar
  80. 80.
    Zak DR, Holmes WE, Burton AJ, Pregitzer KS, Talhelm AF (2008) Simulated atmospheric NO3 deposition increases soil organic matter by slowing decomposition. Ecol Appl (in press)Google Scholar
  81. 81.
    Zak DR, Holmes WE, Tomlinson MJ, Pregitzer KS, Burton AJ (2006) Microbial cycling of C and N in northern hardwood forests receiving chronic atmospheric NO3 deposition. Ecosystems 9:242–253CrossRefGoogle Scholar
  82. 82.
    Zak DR, Pregitzer KS, Holmes WE, Burton AJ, Zogg GP (2004) Anthropogenic N deposition and the fate of 15NO3 in a northern hardwood ecosystem. Biogeochemistry 69:143–57CrossRefGoogle Scholar
  83. 83.
    Zak DR, Holmes WE, MacDonald NW, Pregitzer KS (1999) Soil temperature, matric potential, and the kinetics of microbial respiration and nitrogen mineralization. Soil Sci Soc Am J 63:575–584Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • John E. Hassett
    • 1
    Email author
  • Donald R. Zak
    • 1
  • Christopher B. Blackwood
    • 2
  • Kurt S. Pregitzer
    • 3
  1. 1.School of Natural Resources & EnvironmentUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biological SciencesKent State UniversityKentUSA
  3. 3.Department of Natural Resources and Environmental ScienceUniversity of NevadaRenoUSA

Personalised recommendations