Hostname: page-component-76fb5796d-zzh7m Total loading time: 0 Render date: 2024-04-29T21:01:48.080Z Has data issue: false hasContentIssue false
  • English
  • Français

Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

Published online by Cambridge University Press:  22 July 2021

Micheline Carvalho-Silva Open the ORCID record for Micheline Carvalho-Silva [Opens in a new window] ,
Luiz Henrique Rosa Open the ORCID record for Luiz Henrique Rosa [Opens in a new window] ,
Otávio H.B. Pinto Open the ORCID record for Otávio H.B. Pinto [Opens in a new window] ,
Thamar Holanda Da Silva Open the ORCID record for Thamar Holanda Da Silva [Opens in a new window] ,
Diego Knop Henriques Open the ORCID record for Diego Knop Henriques [Opens in a new window] ,
Peter Convey Open the ORCID record for Peter Convey [Opens in a new window]  and
Paulo E.A.S. Câmara Open the ORCID record for Paulo E.A.S. Câmara [Opens in a new window]
Micheline Carvalho-Silva*
Affiliation:
Departamento de Botânica, Universidade de Brasília (UnB), Brasília, Brazil
Luiz Henrique Rosa
Affiliation:
Departamento de Microbiologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
Otávio H.B. Pinto
Affiliation:
Departamento deBiologia celular e Molecular, Universidade de Brasília (UnB), Brasília, Brazil
Thamar Holanda Da Silva
Affiliation:
Departamento de Microbiologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
Diego Knop Henriques
Affiliation:
Departamento de Botânica, Universidade de Brasília (UnB), Brasília, Brazil
Peter Convey
Affiliation:
British Antarctic Survey, Cambridge, UK
Paulo E.A.S. Câmara
Affiliation:
Departamento de Botânica, Universidade de Brasília (UnB), Brasília, Brazil
*
silvamicheline@gmail.com
Get access Rights & Permissions [Opens in a new window]

Abstract

The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

Keywords

Crater LakeeDNAHTSITS2next-generation sequencingWhalers Bay
Type
Biological Sciences
Information
Antarctic Science , Volume 33 , Issue 5 , October 2021 , pp. 469 - 478
Check for updates
Copyright
Copyright © Antarctic Science Ltd 2021

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Agostini, K.M., Rodrigues, L.A.C, Alencar, A.S., Mendonça, C.B.F. & Gonçalves-Esteves, V. 2017. Analysis of exotic pollen grains and spores from thawing lakes of King George Island, Antarctic Peninsula. Review of Palaeobotany and Palynology, 245, 10.1016/j.revpalbo.2017.05.006.Google Scholar
Amesbury, M.J., Roland, T.P., Royles, J., Hodgson, D.A., Convey, P., Griffiths, H., Charman, D.J. 2017. Widespread biological response to rapid warming on the Antarctic Peninsula. Current Biology, 27, 1616–1622.10.1016/j.cub.2017.04.034CrossRefGoogle ScholarPubMed
Banchi, E., Ametrano, C.G. Greco, S., Stankovic´, D., Muggia, L. & Pallavicini, A. 2020. PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding. Database, 2020, 10.1093/database/baz155.10.1093/database/baz155CrossRefGoogle ScholarPubMed
Barnes, M.A. & Turner, C.R. 2015. The ecology of environmental DNA and implications for conservation genetics. Conservation Genetics, 17, 10.1007/s10592-015-0775-4.Google Scholar
Bednarek-Ochyra, H., Vana, J., Ochyra, R. & Smith, R.I.L. 2000. The liverwort flora of Antarctica. Krakow: Polish Academy of Sciences, 236 pp.Google Scholar
Biersma, E.M., Jackson, J.A. Bracegirdle, T.J., Griffiths, H., Linse, K. & Convey, P. 2018a. Low genetic variation between South American and Antarctic populations of the bank-forming moss Chorisodontium aciphyllum (Dicranaceae). Polar Biology, 41, 599–610.10.1007/s00300-017-2221-1CrossRefGoogle Scholar
Biersma, E.M., Jackson, J.A., Stech, M., Griffiths, H., Linse, K. & Convey, P. 2018b. Long-term in situ Antarctic persistence within Antarctica's most speciose plant genus, Schistidium. Frontiers in Ecology and Evolution, 6, 10.3389/fevo.2018.00077.10.3389/fevo.2018.00077CrossRefGoogle Scholar
Biersma, E.M., Torres-Díaz, C., Molina-Montenegro, M.A., Newsham, K.K, Vidal, M.A., Collado, G.A., et al. 2020. Multiple late-Pleistocene colonisation events of the Antarctic pearwort Colobanthus quitensis (Caryophyllaceae) revel the recent arrival of native Antarctic vascular flora. Journal of Biogeography, 47, 1663–1673.10.1111/jbi.13843CrossRefGoogle Scholar
Biersma, E.M., Convey, P., Wyber, R., Robinson, S.A., Dowton, M., van de Vijver, B., et al. 2021. Latitudinal biogeographic structuring in the globally distributed moss Ceratodon purpureus. Frontiers in Plant Science, 11, 10.3389/fpls.2020.502359.Google Scholar
Bokulich, N.A., Subramanian, S., Faith, J.J., Gevers, D., Gordon, J.I., Knight, R., et al. 2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature Methods, 10, 10.1038/nmeth.2276.10.1038/nmeth.2276CrossRefGoogle ScholarPubMed
Bolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Abnet, C.C., Al-Ghalith, A., et al. 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnolog y, 37, 10.1038/s41587-019-0209-9.Google ScholarPubMed
Cakil, Z.V., Garlasché, G., Iakovenko, N., Di Cesare, A., Eckert, E.M., Guidetti, R., et al. 2021. Comparative phylogeography reveals consistently shallow genetic diversity in a mitochondrial marker in Antarctic bdelloid rotifers. Journal of Biogeography, 10.1111/jbi.14116.10.1111/jbi.14116CrossRefGoogle Scholar
Câmara, P.E.A.S., Soares, A.E.R., Hernriques, D.K., Peralta, D.F., Bordin, J., Carvalho-Silva, M. & Stech, M. 2019. New insights into the species diversity of Bartramia Hedw. (Bryophyta) in Antarctica from a morpho-molecular approach. Antarctic Science, 31, 208–215.10.1017/S0954102019000257CrossRefGoogle Scholar
Câmara, P.E.A.S., Carvalho-Silva, M., Pinto, O.H.B., Amorim, E.T., Henriques, D.K., Silva, T.H., et al. 2021. Diversity and ecology of Chlorophyta (Viridiplantae) assemblages in protected and non-protected sites in Deception Island (Antarctica, South Shetland Islands) assessed using an NGS approach. Microbial Ecology, 81, 10.1007/s00248-020-01584-9.10.1007/s00248-020-01584-9CrossRefGoogle ScholarPubMed
Cannone, N., Corinti, T., Malfasi, F., Vianelli, A., Vanetti, I., Zaccara, S., Convey, P. & Guglielmin, M. 2017. Moss survival through in situ cryptobiosis after six centuries of glacier burial. Scientific Reports, 7, 10.1038/s41598-017-04848-6.10.1038/s41598-017-04848-6CrossRefGoogle ScholarPubMed
Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., et al. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One, 5, e8613.10.1371/journal.pone.0008613CrossRefGoogle ScholarPubMed
Chong, C.W., Pearce, D.A. & Convey, P. 2015. Emerging spatial patterns in Antarctic prokaryotes. Frontiers in Microbiology, 6, 1058.10.3389/fmicb.2015.01058CrossRefGoogle ScholarPubMed
Chown, S.L., Huiskes, A.H.L., Gremmen, N.J.M., Lee, J.E., Terauds, A., Crosbie, K., et al. 2012. Continent-wide risk assessment for the establishment of nonindigenous species in Antarctica. Proceedings of the National Academy of Sciences of the United States of America, 109, 4938–4943.10.1073/pnas.1119787109CrossRefGoogle ScholarPubMed
Chua, C.Y., Yong, S.T., Gonzalez, M.A., Lavin, P., Cheah, Y.K., Tan, G.Y.A. & Wong, C.M.V. L. 2018. Analysis of bacterial communities of King George and Deception Islands, Antarctica using high-throughput sequencing. Current Science, 115, 10.18520/cs/v115/i9/1701-1705.10.18520/cs/v115/i9/1701-1705CrossRefGoogle Scholar
Collins, N.J. 1969. The effects of volcanic activity on the vegetation of Deception I. BAS Bulletin, No. 21, 79–94.Google Scholar
Convey, P., Lewis Smith, R.I., Hodgson, D.A. & Pear, H.J. 2020. The flora of the South Sandwich Islands, with particular reference to the influence geothermal heating. Journal of Biogeography, 27, 1279–1295.10.1046/j.1365-2699.2000.00512.xCrossRefGoogle Scholar
Cowan, D.A., Chown, S.L., Convey, P., Tuffin, M., Hughes, K., Pointing, S. & Vincent, W. 2011. Non-indigenous microorganisms in the Antarctic: assessing the risks. Trends in Microbiology, 19, 540–548.10.1016/j.tim.2011.07.008CrossRefGoogle ScholarPubMed
Crosby, M.R. & Magill, R.E. 1981. Dict. Mosses (3rd edn). St Louis, MO: Missouri Botanical Garden, 43 pp.Google Scholar
Cuba-Díaz, M., Troncoso, J.M., Cordero, C., Finot, V.L. & Rondanelli-Reyes, M. 2012. Juncus bufonius L., a new alien vascular plant in King George Island, South Shetland Archipelago. Antarctic Science, 25, 10.1017/S0954102012000958.Google Scholar
Deiner, K., Bik, H.M., Mächler, E., Seymour, M., Lacoursière-Roussel, A., Altermatt, F., et al. 2017. Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26, 5872–5895.10.1111/mec.14350CrossRefGoogle ScholarPubMed
Fahner, N.A., Shokralla, S., Baird, D.J. & Hajibabaei, M. 2016. Large-scale monitoring of plants through environmental DNA metabarcoding of soil: recovery, resolution, and annotation of four DNA markers. PLoS One, 11, 10.1371/journal.pone.0157505.10.1371/journal.pone.0157505CrossRefGoogle ScholarPubMed
Fraser, C.I., Connell, L., Lee, C.K. & Cary, S.C. 2018. Evidence of plant and animal communities at exposed and subglacial (cave) geothermal sites in Antarctica. Polar Biology, 41, 10.1007/s00300-017-2198-9.10.1007/s00300-017-2198-9CrossRefGoogle Scholar
Fraser, C.I., Nikula, R., Ruzzante, D.E. & Waters, J.M. 2012. Poleward bound: biological impacts of Southern Hemisphere glaciation. Trends in Ecology and Evolution, 27, 462–471.10.1016/j.tree.2012.04.011CrossRefGoogle ScholarPubMed
Frenot, Y., Chown, S.L., Whinam, J., Selkirk, P.M., Convey, P., Stoknicki, M. & Bergstrom, D.M. 2005. Biological invasions in the Antarctic: extent, impacts and implications. Biological Reviews, 80, 45–72.10.1017/S1464793104006542CrossRefGoogle ScholarPubMed
Galera, H., Rudak, A., Czyż, E.A., Chwedorzewska, K.J., Znój, A. & Wódkiewicz, M. 2019. The role of the soil seed store in the survival of an invasive population of Poa annua at Point Thomas Oasis, King George Island, maritime Antarctica. Global Ecology and Conservation, 19, e00679.10.1016/j.gecco.2019.e00679CrossRefGoogle Scholar
Garrido-Benavent, I, Pérez-Ortega, S., Durán, J., Ascaso, C., Pointing, S.B., Rodríguez-Cielos, R., et al. 2020. Differential colonization and succession of microbial communities in rock and soil substrates on a Maritime Antarctic glacier forefield. Frontiers in Microbiology, 11, 10.3389/fmicb.2020.00126.10.3389/fmicb.2020.00126CrossRefGoogle ScholarPubMed
Giner, C.R., Forn, I., Romac, S., Logares, R.C. & Massana, R. 2016. Environmental sequencing provides reasonable estimates of the relative abundance of specific picoeukaryotes. Applied Environmental Microbiology, 82, 4757–4766.10.1128/AEM.00560-16CrossRefGoogle ScholarPubMed
Greenslade, P., Potapov, M., Russel, R. & Convey, P. 2012. Global Collembola on Deception Island. Journal of Insect Science, 12, 10.1673/031.012.11101.10.1673/031.012.11101CrossRefGoogle ScholarPubMed
Hammer, Ø., Harper, D.A.T. & Ryan, P.D. 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 9.Google Scholar
Hart, I.B. 2006. Whaling in the Falkland Islands dependencies, 1904–1931. Newton St. Margarets: Pequena, 363 pp.Google Scholar
Hughes, K.A. & Convey, P. 2010. The protection of Antarctic terrestrial ecosystems from inter and intra-continental transfer of non-indigenous species by human activities: a review of current systems and practices. Global Environmental Change - Human and Policy Dimensions, 20, 96–112.10.1016/j.gloenvcha.2009.09.005CrossRefGoogle Scholar
Hughes, K.A. & Convey, P. 2012. Determining the native/non-native status of newly discovered terrestrial and freshwater species in Antarctica - current knowledge, methodology and management action. Journal of Environmental Management, 93, 52–66.10.1016/j.jenvman.2011.08.017CrossRefGoogle ScholarPubMed
Hughes, K.A., Ott, S., Bolter, M. & Convey, P. 2006. Colonization processes. In Bergstrom, D., Convey, P. & Huiskes, A.H.L., eds. Trends in Antarctic terrestrial and limnetic ecosystems. Dordrecht: Springer, 35–54.10.1007/1-4020-5277-4_3CrossRefGoogle Scholar
Hughes, K.A., Pertierra, L.R., Molina-Montenegro, M.A. et al. 2015. Biological invasions in terrestrial Antarctica: what is the current status and can we respond? Biodiversity and Conservation, 24, 10.1007/s10531-015-0896-6.10.1007/s10531-015-0896-6CrossRefGoogle Scholar
Iakovenko, N.S., Smykla, J., Convey, P., Kašparová, E., Kozeretska, I.A., Trokhymets, V., et al. 2015. Antarctic bdelloid rotifers: diversity, endemism and evolution. Hydrobiologia, 761, 5–43.10.1007/s10750-015-2463-2CrossRefGoogle Scholar
Jain, R., Rivera, M.C., & Lake, J.A. 1999. Horizontal gene transfer among genomes: the complexity hypothesis. Proceedings of the National Academy of Science of the United States of America, 96, 3801–3806.10.1073/pnas.96.7.3801CrossRefGoogle ScholarPubMed
Jung, C.M., Crocker, F.H., Eberly, J.O. & Indest, K.J. 2011. Horizontal gene transfer (HGT) as a mechanism of disseminating RDX-degrading activity among Actinomycete bacteria. Journal of Applied Microbiology, 110, 1449–1459.10.1111/j.1365-2672.2011.04995.xCrossRefGoogle ScholarPubMed
Kappen, L. & Straka, H. 1998. Pollen and spores transport into the Antarctic. Polar Biology, 8, 10.1007/BF00443450.Google Scholar
La Farge, C., Williams, K.H. & England, J. H. 2013. Regeneration of Little Ice Age bryophytes emerging from a polar glacier with implications of totipotency in extreme environments. Proceedings of the National Academy of Science of the United States of America, 110, 10.1073/pnas.1304199110.10.1073/pnas.1304199110CrossRefGoogle ScholarPubMed
Lewis Smith, R.I. & Richardson, M. 2011. Fuegian plants in Antarctica: natural or anthropogenically assisted immigrants? Biological Invasions, 13, 10.1007/s10530-010-9784-x.10.1007/s10530-010-9784-xCrossRefGoogle Scholar
Longton, R.E. 1988. The biology of polar bryophytes and lichens. Cambridge: Cambridge University Press, viii + 391 pp.10.1017/CBO9780511565212CrossRefGoogle Scholar
Ma, Y., Wang, L. & Shao, S. 2006. Pseudomonas, the dominant polycyclic aromatic hydrocarbon-degrading bacteria isolated from Antarctic soils and the role of large plasmids in horizontal gene transfer. Environmental Microbiology, 8, 455–465.10.1111/j.1462-2920.2005.00911.xCrossRefGoogle ScholarPubMed
Malfasi, F., Convey, P. & Cannone, N. 2020. Establishment and eradication of an alien plant species in Antarctica: Poa annua at Signy Island. Biodiversity and Conservation, 29, 10.1007/s10531-019-01877-7.10.1007/s10531-019-01877-7CrossRefGoogle Scholar
Molina-Montenegro, M.A., Carrasco-Urra, F., Rodrigo, C., Convey, P., Valladares, F. & Gianoli, E. 2012. Occurrence of the non-native annual bluegrass on the Antarctic mainland and its negative effects on native plants. Conservation Biology, 26, 10.1111/j.1523-1739.2012.01865.x.10.1111/j.1523-1739.2012.01865.xCrossRefGoogle ScholarPubMed
Molina-Montenegro, M.A., Oses, R., Atala, C., Torres-Díaz, C., Bolados, G. & León-Lobos, P. 2016. Nurse effect and soil microorganisms are key to improve the establishment of native plants in a semiarid community. Journal of Arid Environments, 126, 10.1016/j.jaridenv.2015.10.016.10.1016/j.jaridenv.2015.10.016CrossRefGoogle Scholar
Newsham, K.K., Davey, M.L., Hopkins, D.W. & Dennis, P.G. 2021. Regional diversity of maritime Antarctic soil fungi and predicted responses of guilds and growth forms to climate change. Frontiers in Microbiology, 11, 10.3389/fmicb.2020.615659.10.3389/fmicb.2020.615659CrossRefGoogle ScholarPubMed
Nowell, R.W., Almeida, P., Wilson, C.G., Smith, T.P., Fontaneto, D., Crisp, A., et al. 2018. Comparative genomics of bdelloid rotifers: insights from desiccating and nondesiccating species. PLoS Biology, 16, e2004830.10.1371/journal.pbio.2004830CrossRefGoogle ScholarPubMed
Ochyra, R., Lewis Smith, R.I. & Bednarek-Ochyra, H. 2008. The illustrated moss flora of Antarctica. Cambridge: Cambridge University Press, 704 pp.Google Scholar
Parnikoza, I., Dykyy, I., Ivanets, V., Kozeretska, I., Kunakh, V., Rozhok, A., et al. 2012. Use of Deschampsia antarctica for nest building by the kelp gull in the Argentine Islands area (maritime Antarctica) and its possible role in plant dispersal. Polar Biology, 35, 1753–1758.10.1007/s00300-012-1212-5CrossRefGoogle Scholar
Pertierra, L.R., Hughes, K.A., Vega, G.C. & Olalla-Tárraga, M.Á. 2017a. Correction: High resolution spatial mapping of human footprint across Antarctica and its implications for the strategic conservation of avifauna. PLoS One, 12, e0173649.10.1371/journal.pone.0173649CrossRefGoogle Scholar
Pertierra, L.R., Hughes, K.A., Vega, G.C. & Olalla-Tárraga, M.Á. 2017b. High resolution spatial mapping of human footprint across Antarctica and its implications for the strategic conservation of avifauna. PLoS ONE, 12, 10.1371/journal.pone.0168280.Google Scholar
Roads, E., Longton, R.E. & Convey, P. 2014. Millennial timescale regeneration in a moss from Antarctica. Current Biology, 24, R222–R223.10.1016/j.cub.2014.01.053CrossRefGoogle Scholar
Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahé, F. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ, 2016, 10.7717/peerj.2584.Google Scholar
Rosa, L.H., da Silva, T.H., Ogaki, M.B., Pinto, O.H.B., Stech, M., Convey, P., et al. 2020a. DNA metabarcoding uncovers fungal diversity in soils of protected and non-protected areas on Deception Island, Antarctica. Scientific Reports, 10, 10.1038/s41598-020-78934-7.10.1038/s41598-020-78934-7CrossRefGoogle Scholar
Rosa, L.H., Pinto, O.H.B., Šantl-Temkiv, T., Convey, P., Carvalho-Silva, M., Rosa, C.A., et al. 2020b. DNA metabarcoding of fungal diversity in air and snow of Livingston Island, South Shetland Islands, Antarctica. Scientific Report, 10, 10.1038/s41598-020-78630-6.Google Scholar
Roura, M. 2012. Being there: examining the behaviour of Antarctic tourists through their blogs Ricardo M. Polar Research, 201, 1–23.Google Scholar
Smith, R.I.L. 2005. The thermophilic bryoflora of Deception Island: unique plant communities as a criterion for designating an Antarctic Specially Protected Area. Antarctic Science, 17, 17–27.10.1017/S0954102005002385CrossRefGoogle Scholar
Smith, R.I.L. 2013. A fern cultured from Antarctic glacier detritus. Antarctic Science, 26, 341–344.10.1017/S0954102013000606CrossRefGoogle Scholar
Song, J.M. et al. 2010. Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail, Cryptopygus antarcticus, horizontally acquired from bacteria. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 155: 403–412.10.1016/j.cbpb.2010.01.003CrossRefGoogle ScholarPubMed
Taberlet, P., Coissac, E., Pompanon, F., Brochmann, C. & Willerslev, E. 2012. Towards next-generation biodiversity assessment using DNA metabarcoding. Molecular Ecology, 21, 2045–2050.10.1111/j.1365-294X.2012.05470.xCrossRefGoogle ScholarPubMed
Tejedo, P., Gutiérrez, B., Pertierra, L. & Benayas, J. 2015. Analysis of published scientific research from Deception Island, South Shetland Islands. Antarctic Science, 27, 10.1017/S0954102014000455.10.1017/S0954102014000455CrossRefGoogle Scholar
Verleyen, E., Van de Vijver, B., Tytgat, B., Pinseel, E., Hodgson, D.A., Kopalová, K., et al. 2021. Diatoms define a novel freshwater biogeography of the Antarctic. Ecography, 44, 10.1111/ecog.05374.10.1111/ecog.05374CrossRefGoogle Scholar
Weber, A.A. & Pawlowski, J. 2013. Can abundance of protists be inferred from sequence data: a case study of Foraminifera. PLoS One, 8, 10.1371/journal.pone.0056739.10.1371/journal.pone.0056739CrossRefGoogle ScholarPubMed
White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J., eds. PCR protocols: a guide to methods and applications. New York: Academic Press, 315–322.Google Scholar