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Research Note

Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador

[version 1; peer review: 2 approved with reservations]
María-Fernanda Guevara
https://orcid.org/0000-0001-5141-8464
1,2, Paula Salazar3, Bence Mátyás
https://orcid.org/0000-0003-2694-1848
4, María-Eugenia Ordoñez5
María-Fernanda Guevara
https://orcid.org/0000-0001-5141-8464
1,2, Paula Salazar3, Bence Mátyás
https://orcid.org/0000-0003-2694-1848
4, María-Eugenia Ordoñez5
PUBLISHED 23 Feb 2018
Author details Author details
OPEN PEER REVIEW
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This article is included in the Ecology and Global Change gateway.

Abstract

In the present study fungi collections were sampled in the Sangay (SP) and Llanganates (LP) National Parks, from which sequences of the regions of the internal transcribable spacer (ITS1-5.8S-ITS2) of the ribosomal DNA were obtained (RDNA).
The taxonomic identification of fungi of the order Xylariales was achieved with the bioinformatic tools, to further study the phylogenetic relationships among the collected individuals and thus contribute with base information on their biological diversity, necessary to design and implement measures for the conservation of fungi.
All records belong to the genus Xylaria, of these eight belong to PL and two to SP. A record was not identified at the species level, suggesting that it could be a new species. A phylogenetic tree of Maximum Likelihood was built.

Keywords

Diversity, ITS, Llanganates, National Park, Sangay, Xylarial

Corresponding author: Bence Mátyás
Competing interests: No competing interests were disclosed.
Grant information: The Secretaria de Educación Superior, Ciencia, Tecnología e Innovación del Ecuador (SENESCYT), Arca de Noé Initiative and the QCAM Fungarium supported the project.
Copyright:  © 2018 Guevara MF et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Guevara MF, Salazar P, Mátyás B and Ordoñez ME. Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.1) First published: 23 Feb 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.1) Latest published: 17 May 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.2)

Introduction

Sangay (SP) and Llanganates (LP) National Parks in Ecuador are considered as high priority conservation units in the Tropical Andes, due to their high biodiversity and high endemism1,2. However, their mycological diversity is still unknown. This study aims to contribute to the conservation of fungi, showing the results of their diversity, based on molecular taxonomy, by analyzing the ITS (internal transcribed spacer) regions. ITS is the accepted ’barcode’ for fungi3. For this, the DNA sequence of specimens of exploratory fungal collections were analyzed within the aforementioned parks. Here we present results exclusively for the Xylariales order.

Methods

Obtaining the sequences and molecular identification

Sample collection was carried out during the months of January and February 2015. The fruiting bodies collected were deposited in the QCAM Fungarium (Catholic University Mycology Collection, Quito). Table 1 displays the collection codes, as stored at the QCAM. The ITS1-5.8S-ITS2 region was amplified by PCR with primers (provided by Invitrogen Co., Carlsbad, CA, USA) ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’)4 and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)5. The amplified fragments were sent for sequencing to Macrogen Inc. (Seoul, South Korea). The obtained sequences were edited in Geneious R8 (Biomatter Ltd. 2005–2012) by selecting the “de novo assemble” tool and then trimming the ends. The consensus sequence was manually edited, and submitted to GenBank. Sequence data were analyzed by comparison with sequences available in GenBank. An assignment to the lower taxonomic level was made by direct homology of the consensus sequences with the search results in BLASTn (NCBI) optimized for highly similar sequences (megablast), alignments that presented 100

Table 1. Fungi of the order Xylariales collected in the Sangay and Llanganates National Parks in Ecuador.

QCAM
Fungarium
Code		GenBank
Accession
Number		IdentificationSampling location
National
Park		Altitude
QCAM4663MG768840Xylaria enterogenaLlangantes1370
QCAM4551MG768839Xylaria enterogenaLlangantes1387
QCAM4537MG768834Xylaria fissilisLlangantes1377
QCAM4540MG768836Xylaria schweinitziiLlangantes1377
QCAM4232AMG768832ÂXylaria telfairiiSangay2885
QCAM4550MG768838Xylaria sp. 1Llangantes1387
QCAM4666MG768841Xylaria sp. 1Llangantes1379
QCAM4306AMG768833Xylaria sp. 2Sangay2885
QCAM4545MG768837Xylaria sp. 3Llangantes1373
QCAM4539MG768835Xylaria sp. 4Llangantes1377

Phylogenetic analysis

Sequence data were aligned with Geneious R8 and later manually adjusted with Mesquite version 3.046. Public sequences are available in GenBank that corresponded to specimens that gave the greatest homology in BLASTn with the sequences of the collected specimens were included in the analyses. Phylogenetic trees were constructed in Geneious R8 using the PhyML7 plugin for Maximum Likelihood (ML) with a Custom (010230) substitution model determined by jModelTest 2.1.4.8,9, according to Corrected Akaike Information Criterion (AICc)10,11. A bootstrap of 1000 replicas was used.

Results

All the specimens collected were of the genus Xylaria. The eight specimens from Llanganates National Park were identified as X. enterogena, X. fissilis, X. schweinitzii, X. telfairii and three unidentified species. For the two samples from Sangay National Park, one was X. telfairi and the other was an unidentified species. Differences in the number of samples found at each park could be due to the sampling effort that was different in each park, however, this is a sample of the high biodiversity in LP and SP. The unidentified species were different in each park. The analysis shows that there are no shared species of Xylaria at the two parks (Table 1), this is important for conservation decisions. The phylogenetic relationships recovered from the analysis of the ITS sequences (Figure 1) shows two major groups. The first major group, composed by clades A and B, is well supported (bootstrap > 95) includes specimens from LP and PS. Clade A includes all X. entogena specimens and is well supported (bootstrap > 95). Clade B includes all X. telfairii specimens and Xylarya sp.1 specimens (bootstrap < 50), it could be supposed that Xylarya sp.1 belongs to the X. telfairii species, but because the high difference among the sequences it is considered a different species. In the second major group (bootstrap > 75), clade C is sister to clades D, E and F; this major group includes specimens from LP and SP, too. Clade C includes all X. schweinitzii specimens (bootstrap > 95). Clade D (bootstrap > 80) includes Xylaria sp. 2, the closest sequence to Xylaria sp. 2 from SP was a previously reported collection also from Ecuador12 in a cloud forest in the province of Imbabura, that was also identified only at the genus level. Clades E (bootstrap > 95) shows Xylaria sp. 3, the closest sequence to this individuals belongs to the same previously reported collection12, identified only at the genus level. Clade F (bootstrap = 100) includes Xylaria fissilis sequences from LP and one from 12; clade F also includes Xylaria sp. 4 an unidentified specimen.

559129af-0df6-407c-a698-b49d39435f62_figure1.gif

Figure 1. Maximum likelihood phylogenetic tree of specimens of the Xylariales order obtained in Sangay and Llangantes National Parks, based on sequences of the ITS (internal transcribed spacer) region.

These unidentified specimens might represent new species. Additional loci and more detailed morphological analyses are needed to determine this. The genus Xylaria is probably the largest in the family Xylariaceae, with 35 estimated genera13, but the real number remains unknown14. Studies in relation to the biological diversity of this order in the National Parks of Ecuador are scarce, more systematic field studies would surely reveal a greater diversity of families, genera and species within the Xylariales in SP and LP, as well as other regions and protected areas of Ecuador, especially if we take into account the cosmopolitan distribution of Xylaria13. In fact, new fungal species in SP, belonging to the Agaricales, have recently been described15.

Conclusions

The results obtained allow us to establish a baseline for the biological diversity of the Xylariales in SP and LP, an important step to the conservation of fungi. This is the main contribution of this study. We found four species of Xylaria: X. enterogena, X. telfairii, X. schweinitzii, and X. fissilis, and four potential new species; the species found in LP are different from those found in SP. However, there is much more to discover. A huge and complex task is pending. To advance our understanding of this Kingdom we must start by deciphering the diversity of fungi present in these sites.

Data availability

The sequencing data are available on the NCBI Genbank webpage:

Xylaria enterogena: https://www.ncbi.nlm.nih.gov/nuccore/MG768840

https://www.ncbi.nlm.nih.gov/nuccore/MG768839

Xylaria fissilis: https://www.ncbi.nlm.nih.gov/nuccore/MG768834

Xylaria schweinitzii: https://www.ncbi.nlm.nih.gov/nuccore/MG768836

Xylaria telfairii: https://www.ncbi.nlm.nih.gov/nuccore/MG768832

Xylaria sp. 1: https://www.ncbi.nlm.nih.gov/nuccore/MG768838

https://www.ncbi.nlm.nih.gov/nuccore/MG768841

Xylaria sp. 2: https://www.ncbi.nlm.nih.gov/nuccore/MG768833

Xylaria sp. 3: https://www.ncbi.nlm.nih.gov/nuccore/MG768837

Xylaria sp. 4: https://www.ncbi.nlm.nih.gov/nuccore/MG768835

Competing interests

No competing interests were disclosed.

Grant information

The Secretaria de Educación Superior, Ciencia, Tecnología e Innovación del Ecuador (SENESCYT), Arca de Noé Initiative and the QCAM Fungarium supported the project.

Acknowledgements

We are grateful to Marcel A. Caminer, Charles W. Barnes and Cristina E. Toapanta for their invaluable contribution to the development of the present research.

References

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  • 2.  Ministerio del Ambiente del Ecuador: Plan de Manejo Parque Nacional Llanganates. Quito-Ecuador, 2013. Reference Source
  • 3.  Schoch CL, Robbertse B, Roberts V, et al.: Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database (Oxford). 2014; 2014; pii: bau061. PubMed Abstract | Publisher Full Text | Free Full Text
  • 4.  Gardes M, Bruns TD: ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts. Mol Ecol. 1993; 2(2): 113–118. PubMed Abstract | Publisher Full Text
  • 5.  White TJ, Bruns T, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. Mycologia. 2013; 64(1). Reference Source
  • 6.  Maddison WP, Maddison DR: Mesquite: a modular system for evolutionary analysis. Version 3.04. 2015. Reference Source
  • 7.  Guindon S, Dufayard JF, Lefort V, et al.: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010; 59(3): 307–321. PubMed Abstract | Publisher Full Text
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  • 9.  Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2013; 52(5): 696–704. PubMed Abstract | Publisher Full Text
  • 10.  Akaike H: Information theory and an extension of the maximum likelihood principle. In: Petrov BN, Csaki F. (Eds.), 2nd International Symposium on Information Theory. Budapest: Akademiai Kiado, Budapest, Hungary, 1973; 267–281. Reference Source
  • 11.  Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008; 25(7): 1253–1256. PubMed Abstract | Publisher Full Text
  • 12.  Thomas DC, Vandegrift R, Ludden A, et al.: Spatial Ecology of the Fungal Genus Xylaria in a Tropical Cloud Forest. Biotropica. 2016; 48(3): 381–393. Publisher Full Text
  • 13.  Lee JS, Ko KS, Jung HS: Phylogenetic analysis of Xylaria based on nuclear ribosomal ITS1-5. 8S-ITS2 sequences. FEMS Microbiol Lett. 2000; 187(1): 89–93. PubMed Abstract | Publisher Full Text
  • 14.  Hsieh HM, Lin CR, Fang MJ, et al.: Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Mol Phylogenet Evol. 2010; 54(3): 957–969. PubMed Abstract | Publisher Full Text
  • 15.  Crous PW, Wingfield MJ, Burgess TI, et al.: Fungal Planet description sheets: 558–624. Persoonia. 2017; 38(1): 240–384. PubMed Abstract | Publisher Full Text | Free Full Text

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Guevara MF, Salazar P, Mátyás B and Ordoñez ME. Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.1)
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Reviewer Report 19 Apr 2018
D. Jean Lodge, Department of Agriculture, USDA Forest Service, Luquillo, PR, USA;  Department of Plant Biology, University of Georgia, Athens, GA, USA 
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https://doi.org/10.5256/f1000research.14800.r32603
Introduction. Because the aim is to compare these two parks in Ecuador, it is important that the elevation ranges of each are included, as well as the distance between the two parks.
 
Methods, p. 3, last line ... Continue reading
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Lodge DJ. Reviewer Report For: Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.5256/f1000research.14800.r32603)
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Reviewer Report 01 Mar 2018
Francisco J. Flores, Departamento de Ciencias de la vida y la Agricultura, Universidad de las Fuerzas Armadas–ESPE, Sangolquí, Ecuador 
Approved with Reservations
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https://doi.org/10.5256/f1000research.14800.r31232
The article "Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador " provides internal transcribed spacer sequences from Xylaria isolates collected from Ecuadorian national parks. Information about fungal diversity in Ecuador is still scarce, which makes this manuscript relevant. ... Continue reading
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Flores F. Reviewer Report For: Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.5256/f1000research.14800.r31232)
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NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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  1. Francisco J. Flores, Universidad de las Fuerzas Armadas–ESPE, Sangolquí, Ecuador
  2. D. Jean Lodge, USDA Forest Service, Luquillo, USA; University of Georgia, Athens, USA
  3. Juan Enrique Tacoronte Morales, Central University of Ecuador, Quito, Ecuador

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    Alongside their report, reviewers assign a status to the article:
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