ORIGINAL ARTICLE
Testing DNA barcoding in Usnea (Parmeliaceae) in Colombia using the internal transcribed spacer (ITS)
Bibiana Moncada 1, 2
,   Harrie Sipman 3,   Robert Lücking 3, 2  
 
More details
Hide details
1
Licenciatura en Biología, Universidad Distrital Francisco José de Caldas, Cra. 4 No. 26D-54, Torre de Laboratorios, Herbario, Bogotá D.C., Colombia
2
Research Associate, Integrative Research Center, The Field Museum, 1400 S Lake Shore Drive, Chicago, IL 60605, USA
3
Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Straße 6–8, 14195 Berlin, Germany
Online publication date: 2020-12-29
Publication date: 2020-12-29
 
Plant and Fungal Systematics 2020; 65(2): 358–385
 
KEYWORDS
ABSTRACT
We tested the functionality of ITS-based DNA barcoding in lichen fungi using Colombian samples of the genus Usnea as an example. New ITS sequences were generated for 15 samples from five localities in two different ecoregions, representing varying morphologies and medullary chemistries. We employed five strategies to identify the samples: (1) BLASTn on the NCBI BLAST site with the original identifications of the best matching reference sequences; (2) as previous, but with revised identifications of the reference sequences based on a separately published revision of ITS sequences published for the genus; (3) local BLASTn in BioEdit using a separately published, revised and curated set of ITS reference sequences for the genus; (4) multiple alignment based phylogenetic analysis within the framework of all available ITS sequences for Usnea s.str.; and (5) integrative taxonomy, combining molecular phylogeny and comparative analysis of phenotype and chemical data. Using the latter approach as reference, we found that NCBI BLASTn with original identifications performed poorly, resulting in an identification success rate of only 7% (a single sample). NCBI BLASTn with revised identifications more than tripled identification success (23%), but was still unsatisfactory. Local BLASTn in BioEdit using the revised, curated reference data further doubled identification success (47%), but remained inadequate. Multiple alignment-based phylogenetic analysis achieved an identification success rate of 80% compared to the result from integrative taxonomy. Based on these results, we conclude that ITS-based DNA barcoding of the genus Usnea under the current circumstances performs poorly, but can be substantially improved using three strategies: (1) update identifications of reference sequences in primary repositories such as GenBank or alternatively use a curated reference data set; (2) perform local BLAST with a curated reference data set focusing on the target genus only, combined with multiple alignment-based phylogenetic analysis as a verification step; and (3) close substantial geographic and taxonomic gaps in the existing reference data. Our analyses suggest that if a near-complete reference data set with correct identifications existed for the genus, then standard BLAST approaches could achieve high levels of identification success close to 100%. As part of our DNA barcoding exercise, which generated the first 15 ITS sequences for Colombian samples of the genus Usnea, we confirm the presence of U. aranea and U. wasmuthii in Colombia and we report for the first time U. tenuicorticata for the country.
 
REFERENCES (92)
1.
Abarenkov, K., Henrik Nilsson, R., Larsson, K. H., Alexander, I. J., Eberhardt, U., Erland, S., Høiland, K., Kjøller, R., Larsson, E., Pennanen, T. & Sen, R. 2010. The UNITE database for molecular identification of fungi – recent updates and future perspectives. New Phytologist 186: 281–285. https://doi.org/10.1111/j.1469....
 
2.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403–410. https://doi.org/10.1016/s0022-....
 
3.
Begerow, D., Nilsson, H., Unterseher, M. & Maier, W. 2010. Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Applied Microbiology and Biotechnology 87: 99–108. https://doi.org/10.1007/s00253....
 
4.
Bengtsson-Palme, J., Ryberg, M., Hartmann, M., Branco, S., Wang, Z., Godhe, A., De Wit, P., Sánchez-García, M., Ebersberger, I., de Sousa, F. & Amend, A. 2013. Improved software detection and extraction of ITS1 and ITS 2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods in Ecology and Evolution 4: 914–919. https://doi.org/10.1111/2041-2....
 
5.
Bidartondo, M. 2008 Preserving accuracy in GenBank. Science 319: 1616.
 
6.
Bungartz, F., Truong, C., Herrera-Campos, M. A. & Clerc, P. 2018. The genus Usnea (Parmeliaceae, Lecanoromycetes) in the Galapagos Islands. Herzogia 31: 571–629. https://doi.org/10.13158/heia.....
 
7.
Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. & Madden, T. L. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10: 421. https://doi.org/10.1186/1471-2....
 
8.
Clerc, P. 1987. Systematics of the Usnea fragilescens aggregate and its distribution in Scandinavia. Nordic Journal of Botany 7: 479–495. https://doi.org/10.1111/j.1756....
 
9.
Clerc, P. 2004. Notes on the genus Usnea Adanson. II. Bibliotheca Lichenologica 88: 79–90.
 
10.
Clerc, P. 2006. Synopsis of Usnea (lichenized Ascomycetes) from the Azores with additional information on the species in Macaronesia. The Lichenologist 38: 191–212. https://doi.org/10.1017/s00242....
 
11.
Clerc, P. 2007. Usnea. In: Nash, T. H. III, Gries, C. & Bungartz, F. (eds), Lichen Flora of the Greater Sonoran Desert Region, Vol. 3, pp. 302–335. Lichens Unlimited, Arizona State University, Tempe.
 
12.
Clerc, P. 2016. Notes on the genus Usnea (lichenized Ascomycota, Parmeliaceae) IV. Herzogia 29: 403–411. https://doi.org/10.13158/heia.....
 
13.
Clerc, P. & Otte, V. 2018. Usnea viktoriana (Ascomycota, Parmeliaceae), a new European taxon of the Usnea barbata-dasopoga group, with a key to the shrubby-subpendulous sorediate Usnea species in Europe. The Lichenologist 50: 513–527. https://doi.org/10.1017/s00242....
 
14.
Díaz-Escandón, D., Soto- Medina, E., Lücking, R. & Silverstone-Sopkin, P. A. 2016: Corticolous lichens as environmental indicators of natural sulphur emissions near the sulphur mine El Vinagre (Cauca, Colombia). The Lichenologist 48: 147–159. https://doi.org/10.1017/s00242....
 
15.
Dorey, J. E., Hoffman, J. R., Martino, J. L., Lendemer, J. C. & Allen, J. L. 2019. First record of Usnea (Parmeliaceae) growing in New York City in nearly 200 years. The Journal of the Torrey Botanical Society 146: 69–77. https://doi.org/10.3159/torrey....
 
16.
Elix, J. A., Wirtz, N. & Lumbsch, H. T. 2007. Studies on the chemistry of some Usnea species of the Neuropogon group (Lecanorales, Ascomycota). Nova Hedwigia 85: 491–501. https://doi.org/10.1127/0029-5....
 
17.
Edgar, R. C. 2018. Accuracy of taxonomy prediction for 16S rRNA and fungal ITS sequences. PeerJ 6: e4652. https://doi.org/10.7717/peerj.....
 
18.
Esslinger, T. L. 2019. A cumulative checklist for the lichen-forming, lichenicolous and allied fungi of the continental United States and Canada, version 23. Opuscula Philolichenum 18: 102–378.
 
19.
Galinato, M. G. M., Baguinon, J. R. C. & Santiago, K. A. A. 2018. Review of the lichen genus Usnea in the Philippines. Studies in Fungi 3: 39–48.
 
20.
Gams, W. 2004. Report of the Committee for Fungi: 11. Taxon 53: 1067–1069.
 
21.
Gardes, M. & Bruns, T. D. 1993. ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118. https://doi.org/10.1111/j.1365....
 
22.
Gerlach, A. C. L., Clerc, P. & Silveira, R. M. B. 2017. Taxonomy of the corticolous, shrubby, esorediate, neotropical species of Usnea Adans. (Parmeliaceae) with an emphasis on southern Brazil. The Lichenologist 49: 199–238. https://doi.org/10.1017/s00242....
 
23.
Gerlach, A. C. L., Toprak, Z., Naciri, Y., Caviró, E. A., Silveira, R. M. B. & Clerc, P. 2019. New insights into the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota): Molecular analysis reveals high genetic diversity correlated with chemistry. Molecular Phylogenetics and Evolution 131: 125–137. [First published online 30 October 2018] https://doi.org/10.1016/j.ympe....
 
24.
Gerlach, A, Silveira, R. M. B., Rojas, C. & Clerc, P. 2020. Naming and describing the diversity in the Usnea cornuta aggregate (lichenized Ascomycota, Parmeliaceae) occurring in Brazil. Plant and Fungal Systematics 65: 272–302. https://doi.org/10.35535/pfsys....
 
25.
Grewe, F., Lagostina, E., Wu, H., Printzen, C. & Lumbsch, H. T. 2018. Population genomic analyses of RAD sequences resolves the phylogenetic relationship of the lichen-forming fungal species Usnea antarctica and Usnea aurantiacoatra. MycoKeys 43: 91–113. https://doi.org/10.3897/mycoke....
 
26.
Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
 
27.
Hall, T. 2011. BioEdit: an important software for molecular biology. GERF Bulletin Biosci 2: 60–61.
 
28.
Halonen, P. 2000. Usnea pacificana, sp. nov. and U. wasmuthii (lichenized Ascomycetes) in Pacific North America. The Bryologist 103: 38–43. https://doi.org/10.1639/0007-2....
 
29.
Halonen, P. & Ahti, T. 2002. (1516) Proposal to conserve the name Usnea fulvoreagens (lichenized Ascomycota, Parmeliaceae) with a conserved type. Taxon 51: 183–183. https://doi.org/10.2307/155498....
 
30.
Halonen, P., Clerc, P., Goward, T., Brodo, I. M. & Wulff, K. 1998. Synopsis of the genus Usnea (lichenized Ascomycetes) in British Columbia, Canada. The Bryologist 101: 36–60. https://doi.org/10.2307/324407....
 
31.
Halonen, P., Myllys, L., Ahti, T. & Petrova, O. V. 1999. The lichen genus Usnea in East Fennoscandia. III. The shrubby species. Annales Botanici Fennici 36: 235–256.
 
32.
Herrera-Campos, M. A. 2016. Usnea in Mexico. Bibliotheca Lichenologica 110: 505–620.
 
33.
Herrera-Campos, M. A., Nash III, T. H. & Garcia, A. Z. 2001. Preliminary study of the Usnea fragilescens aggregate in Mexico. The Bryologist 104: 235–259. https://doi.org/10.1639/0007-2....
 
34.
Hofstetter, V., Buyck, B., Eyssartier, G., Schnee, S. & Gindro, K. 2019. The unbearable lightness of sequenced-based identification. Fungal Diversity 96: 243–284. https://doi.org/10.1007/s13225....
 
35.
Huneck, S. & Yoshimura, I. 1996. Identification of Lichen Substances. Springer, Berlin, Heidelberg.
 
36.
Irinyi, L., Serena, C., Garcia-Hermoso, D., Arabatzis, M., Desnos-Ollivier, M., Vu, D., Cardinali, G., Arthur, I., Normand, A. C., Giraldo, A., da Cunha, K. C., Sandoval-Denis, M., Hendrickx, M., Nishikaku, A. S., de Azevedo Melo, A. S., Merseguel, K. B., Khan, A., Parente Rocha, J. A., Sampaio, P., da Silva Briones, M. R., Ferreira, R. C., de Medeiros Muniz, M., Castañón-Olivares, L. R., Estrada-Barcenas, D., Cassagne, C., Mary. C., Duan, S. Y., Kong, F., Sun, A. Y., Zeng, X., Zhao, Z., Gantois, N., Botterel, F., Robbertse, B., Schoch. C. L., Gams, W., Ellis, D., Halliday, C., Chen, S., Sorrell, T. C., Piarroux, R., Colombo, A. L., Pais, C., de Hoog, S., Zancopé-Oliveira, R. M., Taylor, M. L., Toriello, C., de Almeida Soares, C. M., Delhaes, L., Stubbe, D., Dromer, F., Ranque, S., Guarro, J., Cano-Lira, J. F., Robert, V., Velegraki, A. & Meyer W. 2015. International Society of Human and Animal Mycology (ISHAM) – ITS reference DNA barcoding database – The quality controlled standard tool for routine identification of human and animal pathogenic fungi. Medical Mycology 53: 313–337. https://doi.org/10.1093/mmy/my....
 
37.
Jeewon, R. & Hyde, K. D. 2016. Establishing species boundaries and new taxa among fungi: recommendations to resolve taxonomic ambiguities. Mycosphere 7: 1669–1677.
 
38.
Jørgensen, P. M., James, P. W. & Jarvis, C. E. 1994. Linnaean lichen names and their typification. Botanical Journal of the Linnean Society 115: 261–405. https://doi.org/10.1111/j.1095....
 
39.
Kelly, L. J., Hollingsworth, P. M., Coppins, B. J., Ellis, C. J., Harrold, P., Tosh, J. & Yahr, R. 2011. DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context. New Phytologist 191: 288–300. https://doi.org/10.1111/j.1469....
 
40.
Kõljalg, U., Nilsson, R. H., Abarenkov, K., Tedersoo, L., Taylor, A. F. S., Bahram, M., Bates, S. T., Bruns, T. D., Bengtsson-Palme, J., Callaghan, T. M., Douglas, B., Drenkhan, T., Eberhardt, U., Dueñas, M., Grebenc, T., Griffith, G. W., Hartmann, M., Kirk, P. M., Kohout, P., Larsson, E., Lindahl, B. D., Lücking, R., Martín, M. P., Matheny, B., Nguyen, N. H., Niskanen, T., Oja, J., Peay, K. G., Peintner, U., Peterson, M., Oldmaa, K. P., Saag, L., Saar, R., Schüssler, A., Scott, J. A., Senés, C., Smith, M. E., Suija, A., Taylor, D. L., Telleria, M. T., Weiss, M. & Larsson, K. H. 2013. Towards a unified paradigm for sequence‐based identification of Fungi. Molecular Ecology 22: 5271–5277. https://doi.org/10.1111/mec.12....
 
41.
Kõljalg, U., Abarenkov, K., Nilsson, R. H., Larsson, K. H. & Taylor, A. F. 2019. The UNITE Database for Molecular Identification and for Communicating Fungal Species. Biodiversity Information Science and Standards 3: e37402. https://doi.org/10.3897/biss.3....
 
42.
Lagostina, E., Dal Grande, F., Andreev, M. & Printzen, C. 2018. The use of microsatellite markers for species delimitation in Antarctic Usnea subgenus Neuropogon. Mycologia 110: 1047–1057. https://doi.org/10.1080/002755....
 
43.
Lücking, R., Hodkinson, B. P. & Leavitt, S. D. 2017a. The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota – Approaching one thousand genera. The Bryologist 119: 361–416. https://doi.org/10.1639/0007-2....
 
44.
Lücking, R., Dal Forno, M., Moncada, B., Coca, L. F., Vargas-Mendoza, l. Y., Aptroot, A., Arias, L. J., Besal, B., Bungartz, F., Cabrera-Amaya, D. M., Cáceres, M. E. S., Chaves, J. L., Eliasaro, S., Gutiérrez, M. C., Hernández-M., J. E., Herrera-Campos, M. A., Holgado- Rojas, M. E., Jonitz, H., Kukwa, M., Lucheta, F., Madriñán, S., Marcelli, M. P., Martins, S. M. A., Mercado-Díaz, J. A., Molina, J. A., Morales, E. A., Nelson, P. R., Nugra, F., Ortega, F., Paredes, T., Patiño, A. L., Peláez-Pulido, R. N., Pérez-Pérez, R. E., Perlmutter, G. B., Rivas-Plata, M. E., Robayo, J., Rodríguez, C., Simijaca, D. F., Soto-Medina, E., Spielmann, A. A., Suárez-Corredor, A., Torres, J. M., Vargas, C. A., Yánez-Ayabaca, A., Weerakoon, G., Wilk. K., Celis-Pacheco, M., Diazgranados, M., Brokamp, G., Borsch, T., Gillevet, P. M., Sikaroodi, M. & Lawrey, J. D. 2017b. Turbo-taxonomy to assemble a megadiverse lichen genus: seventy new species of Cora (Basidiomycota: Agaricales: Hygrophoraceae), honouring David Leslie Hawksworth’s seventieth birthday. Fungal Diversity 84: 139–207. https://doi.org/10.1007/s13225....
 
45.
Lücking, R., Aime, M. C., Robberts, B., Miller, A. N., Ariyawansa, H. A., Aoki, T., Cardinali, G., Crous, P. W., Druzhinina, I. S., Geiser, D. M., Hawksworth, D. L., Hyde, K. D., Irinyi, L., Jeewon, R., Johnston, P. R., Kirk, P. M., Malosso, E., May, T. W., Meyer, W., Öpik, M., Robert, V., Stadler, M., Thines, M., Vu, D., Yurkov, A. M., Zhang, N., Schoch, C. L. 2020a. Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal barcoding? IMA Fungus 11: 14. https://doi.org/10.1186/s43008....
 
46.
Lücking, R., Truong, B. V., Huong, D. T. T., Le, N. H., Nguyen, Q. D., Nguyen, V. D., Von Raab-Straube, E., Bollendorff, S., Govers. K. & Di Vincenzo, V. 2020b. Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations. Willdenowia 50: 383–403. https://doi.org/10.3372/wi.50.....
 
47.
Lücking, R., Nadel, M. R. A., Araujo-Caviró, E. & Gerlach, A. C. L. 2020c. Two decades of DNA barcoding in the genus Usnea (Parmeliaceae): how useful and reliable is the ITS? Plant and Fungal Systematics 65: 303–357. https://doi.org/10.35535/pfsys....
 
48.
Lücking, R., Moncada, B., Soto-Medina, E., Simijaca, D. & Sipman, H. J. M. 2020d. Actualización nomenclatural y taxonómica al Catálogo de Líquenes de Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales (in press). https://doi.org/10.18257/racce....
 
49.
Lumbsch, H. T. & Wirtz, N. 2011. Phylogenetic relationships of the neuropogonoid core group in the genus Usnea (Ascomycota: Parmeliaceae). The Lichenologist 43: 553–559. https://doi.org/10.1017/s00242....
 
50.
Mark, K., Saag, L., Leavitt, S. D., Will-Wolf, S., Nelsen, M. P., Tõrra, T., Saag, A., Randlane, T. & Lumbsch, H. T. 2016. Evaluation of traditionally circumscribed species in the lichen-forming genus Usnea, section Usnea (Parmeliaceae, Ascomycota) using a six-locus dataset. Organisms Diversity & Evolution 16: 497–524. https://doi.org/10.1007/s13127....
 
51.
Menlove, K. J., Clement, M. & Crandall, K. A. 2009. Similarity searching using BLAST. In: Posada, D. (ed.), Bioinformatics for DNA Sequence Analysis, pp. 1–22. Humana Press, New York. https://doi.org/10.1007/978-1-....
 
52.
Miller, M. A., Pfeiffer, W. & Schwartz, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), pp. 1–8. New Orleans.
 
53.
Moncada, B., Lücking, R. & Suárez, A. 2014. Molecular phylogeny of the genus Sticta (lichenized Ascomycota: Lobariaceae) in Colombia. Fungal Diversity 64: 205–231. https://doi.org/10.1007/s13225....
 
54.
Motyka, J. 1938. Lichenum Generis Usnea Studium Monographicum. Pars Systematica, Volumen Secundum. Leopoli, Lublin.
 
55.
Nilsson, R. H., Ryberg, M., Kristiansson, E., Abarenkov, K., Larsson, K. H. & Kõljalg, U. 2006. Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective. PloS One 1(1): e59. https://doi.org/10.1371/journa....
 
56.
Nilsson, R. H., Tedersoo, L., Abarenkov, K., Ryberg, M., Kristiansson, E., Hartmann, M., Schoch, C. L., Nylander, J. A., Bergsten, J., Porter, T. M., Jumpponen, A., Vaishampayan, P., Ovaskainen, O., Hallenberg, N., Bengtsson-Palme, J., Eriksson, K. M., Larsson, K.-H., Larsson, E. & Kõljalg, U. 2012. Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences. MycoKeys 4: 37–63. https://doi.org/10.3897/mycoke....
 
57.
Nilsson, R. H, Larsson, K.-H., Taylor, A. F. S., Bengtsson-Palme, J., Jeppesen, T. S., Schigel, D., Kennedy, P., Picard, K., Glöckner, F. O., Tedersoo, L., Saar, I., Kõljalg, U. & Abarenkov, K. 2019. The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Research 47(D1): D259–D264. https://doi.org/10.1093/nar/gk....
 
58.
Ohmura, Y. 2001. Taxonomic study of the genus Usnea (lichenized Ascomycetes) in Japan and Taiwan. Journal of the Hattori Botanical Laboratory 90: 1–96.
 
59.
Ohmura, Y. 2012. A synopsis of the lichen genus Usnea (Parmeliaceae, Ascomycota) in Taiwan. Memories of the National Science Museum Tokyo 48: 91–137.
 
60.
Orange, A., James, P. W. & White, F. J. 2001. Microchemical Methods for the Identification of Lichens. 2nd Edition. British Lichen Society, London.
 
61.
Pearson, W. R. 2013. An introduction to sequence similarity (‘homology’) searching. Current Protocols in Bioinformatics 42: 3.1.1–3.18. https://doi.org/10.1002/047125....
 
62.
Pulido-Herrera, K. & Ramos-Montaño, C. 2016. Efecto de borde en la distribución de líquenes y el contenido de clorofilas en fragmentos de Polylepis quadrijuga (Rosaceae) en el páramo de La Rusia (Boyacá-Colombia). Revista de Biología Tropical 64: 1683–1697. https://doi.org/10.15517/rbt.v....
 
63.
Ramírez-Morán, N. A., León-Gómez, M. & Lücking, R. 2015. Uso de biotipos de líquenes como indicadores de perturbación en dos fragmentos de bosque andino, Reserva Biológica ‘Encenillo’, Colombia. Caldasia 38: 31–52. https://doi.org/10.15446/calda....
 
64.
Randlane, T., Tõrra, T., Saag, A. & Saag, L. 2009. Key to European Usnea species. Bibliotheca Lichenologica 100: 419–462.
 
65.
Räsänen, V. 1931. Die Flechten Estlands. 1. Annales Academiae Scientiarum Fennicae, Ser. A, 34: 1–163.
 
66.
Räsänen, V. 1935. Lichenes Fenniae Exsiccati, a Museo Botanico Universitatis Helsinkiensis Edita. Fasc. I (nos. 1–50), curavit V. Räsänen: no. 13.
 
67.
Räsänen, V. 1936. Collationes ad lichenologiam Chilensem pertinentes. Revista Universitaria Santiago 21: 137–148.
 
68.
Räsänen, V. 1939. Die Flechtenflora der Nordlichen Kustengegend am Laatokka-See. Annales Botanici Societatis Zoologicae-Botanicae Fennicae Vanamo 12: 1–240.
 
69.
Ryan, D. 2018. Global Plants: A model of international collaboration. Biodiversity Information Science and Standards 2: e28233. https://doi.org/10.3897/biss.2....
 
70.
Saag, L., Tõrra, T., Saag, A., Del-Prado, R. & Randlane, T. 2011. Phylogenetic relations of European shrubby taxa of the genus Usnea. The Lichenologist 43: 427–444. https://doi.org/10.1017/s00242....
 
71.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W. & Fungal Barcoding Consortium. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 6241–6246. https://doi.org/10.1073/pnas.1....
 
72.
Seymour, F. A., Crittenden, P. D., Wirtz, N., Øvstedal, D. O., Dyer, P. S. & Lumbsch, H. T. 2007. Phylogenetic and morphological analysis of Antarctic lichen-forming Usnea species in the group Neuropogon. Antarctic Science 19: 71–82. https://doi.org/10.1017/s09541....
 
73.
Shen, Y. M., Hsieh, H. J., Yeh, R. Y. & Hung, T. H. 2012. Five apothecium- producing lichenized fungi of the genus Usnea in Taiwan. Fungal Science 27: 31–44.
 
74.
Simijaca, D., Moncada, B. & Lücking, R. 2018. Bosque de roble o plantación de coníferas, ¿qué prefieren los líquenes epífitos? Colombia Forestal 21: 123–141. https://doi.org/10.14483/22562....
 
75.
Sipman, H. J. M. & Aguirre-C., J. 2016. Líquenes. In: Bernal, R., Gradstein, S. R. & Celis, M. (eds), Catálogo de Plantas y Líquenes de Colombia. Volumen 1, pp. 159–281. Universidad Nacional de.
 
76.
Stirton, J. 1881. On the genus Usnea and a new genus allied to it. Scottish Naturalist 6: 100–107.
 
77.
Tavares, I. I. 2002. Notes on lectotypification of Usnea fulvoreagens, U. gigantea, U. sulcata, and U. cavernosa. Constancea 83: 1–5.
 
78.
Tedersoo, L., Abarenkov, K., Nilsson, R. H., Schüssler, A., Grelet, G. A., Kohout, P., Oja, J., Bonito, G. M., Veldre, V., Jairus, T., Ryberg, M., Larsson, K.-H. & Kõljalg, U. 2011. Tidying up international nucleotide sequence databases: ecological, geographical and sequence quality annotation of ITS sequences of mycorrhizal fungi. PLoS One 6(9): e24940. https://doi.org/10.1371/journa....
 
79.
Tedersoo, L., Anslan, S., Bahram, M., Põlme, S., Riit, T., Liiv, I., Kõljalg, U., Kisand, V., Nilsson, H., Hildebrand, F., Bork, P. & Abarenkov, K. 2015. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi. MycoKeys 10: 1–43. https://doi.org/10.3897/mycoke....
 
80.
Temu, S. G., Clerc, P., Tibell, L., Tibuhwa, D. D., Tibell, S. 2019. Phylogeny of the subgenus Eumitria in Tanzania. Mycology 10: 250–260. https://doi.org/10.1080/215012....
 
81.
Truong, C. & Clerc, P. 2012. The lichen genus Usnea (Parmeliaceae) in tropical South America: species with a pigmented medulla, reacting C+ yellow. The Lichenologist 44: 625–637. https://doi.org/10.1017/s00242....
 
82.
Truong, C. & Clerc, P. 2016. New species and new records in the genus Usnea (Parmeliaceae, lichenized Ascomycota) from tropical South America. The Lichenologist 48: 71–93. https://doi.org/10.1017/s00242....
 
83.
Truong, C., Bungartz, F. & Clerc, P. 2011. The lichen genus Usnea (Parmeliaceae) in the tropical Andes and the Galapagos: species with a red-orange cortical or subcortical pigmentation. The Bryologist 114: 477–503. https://doi.org/10.1639/0007-2....
 
84.
Truong, C., Divakar, P. K., Yahr, R., Crespo, A. & Clerc, P. 2013a. Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of the lichen genus Usnea (Parmeliaceae, Ascomycota). Molecular Phylogenetics and Evolution 68: 357–372. https://doi.org/10.1016/j.ympe....
 
85.
Truong, C., Rodridguez, J. M. & Clerc, P. 2013b. Pendulous Usnea species (Parmeliaceae, lichenized Ascomycota) in tropical South America and the Galapagos. The Lichenologist 45: 505–542. https://doi.org/10.1017/s00242....
 
86.
Truong, C., Mujic, A. B., Healy, R., Kuhar, F., Furci, G., Torres, D., Niskanen, T., Sandoval‐Leiva, P. A., Fernández, N., Escobar, J. M. & Moretto, A. 2017. How to know the fungi: combining field inventories and DNA‐barcoding to document fungal diversity. New Phytologist 214: 913–919. https://doi.org/10.1111/nph.14....
 
87.
Vareschi, V. 2001. El genero Usnea en Venezuela. Boletín de la Academia de Ciencias Físicas, Matemáticas y Naturales 61: 9–63.
 
88.
Vilgalys, R. 2003. Taxonomic misidentification in public DNA databases. New Phytologist 160: 4–5. https://doi.org/10.1046/j.1469....
 
89.
White, T. J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of ribosomal RNA genes and the internal transcribed spacer in fungi. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. H. (eds), PCR-Protocols and Applications – A Laboratory Manual, pp. 315–322. Academic Press, New York. https://doi.org/10.1016/b978-0....
 
90.
Wirtz, N., Printzen, C. & Lumbsch, H. T. 2008. The delimitation of Antarctic and bipolar species of neuropogonoid Usnea (Ascomycota, Lecanorales): a cohesion approach of species recognition for the Usnea perpusilla complex. Mycological Research 112: 472–484. https://doi.org/10.1016/j.mycr....
 
91.
Wirtz, N., Printzen, C. & Lumbsch, H. T. 2012. Using haplotype networks, estimation of gene flow and phenotypic characters to understand species delimitation in fungi of a predominantly Antarctic Usnea group (Ascomycota, Parmeliaceae). Organisms Diversity & Evolution 12: 17–37. https://doi.org/10.1007/s13127....
 
92.
Xu, J. 2016. Fungal DNA barcoding. Genome 59: 913–932.
 
eISSN:2657-5000
ISSN:2544-7459