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

[version 2; peer review: 1 approved, 2 approved with reservations]
María-Fernanda Guevara
https://orcid.org/0000-0001-5141-8464
1,2, Bence Mátyás
https://orcid.org/0000-0003-2694-1848
3,4, María-Eugenia Ordoñez5
María-Fernanda Guevara
https://orcid.org/0000-0001-5141-8464
1,2, Bence Mátyás
https://orcid.org/0000-0003-2694-1848
3,4, María-Eugenia Ordoñez5
PUBLISHED 17 May 2018
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This article is included in the Ecology and Global Change gateway.

Abstract

Fungal samples were collected in the Sangay (SP) and Llanganates (LP) National Parks in Ecuador. Sequences of the internal transcribed spacer regions (ITS1-5.8S-ITS2) of the ribosomal DNA of the samples were analyzed.Taxonomic identification of fungi of the order Xylariales was done through phylogenetic analysis using a Maximun Likelihood method. All analyzed collections presented here belong to the genus Xylaria, of these eight belong to PL and two to SP. Four samples were not identified at the species level, suggesting it could be a new species. This data contributes with base information on the biodiversity of the Parks, necessary to design and implement measures for the conservation of fungi in Ecuador.

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, Mátyás B and Ordoñez ME. Xylariales: First results of mycological exploration in the Sangay and Llanganates National Parks, Ecuador [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.2) 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)

Revised Amendments from Version 1

According to the referee's suggestions we have made the following changes in the manuscript:

  • The abstract, introduction and methods were re-written for ease of comprehension.
  • The results are explained objectively
  • The table and figure legends are also modified for ease of comprehension of the content.
  • In addition, we rephrased 2 sentences in Conclusions.
  • We have removed one of the authors from the second version of the article as an error was made when she was included in the moment of submitting the first version of the article. We were working on several articles at the same time and because of confusion, her name was included in this article. Paula Salazar is a valuable person for the Universidad Politécnica Salesiana and she has collaborated in several projects, but unfortunately she did not collaborate in this research done in association with Pontificia Universidad Católica.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

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 as primary fungal barcode marker for fungi3,4. 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, other fungal orders were also collected but are not shown here.

Methods

Sequencing 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 ITS1-5.8SITS2 region was amplified by PCR with primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’)5 and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)6. The amplified fragments were sent for sequencing to Macrogen Inc. (Seoul, South Korea). The forward and reverse sequences obtained for each isolate were assembled in Geneious R8 (Biomatter Ltd. 2005–2012), 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% coverage and at least 99% identity with a sequence previously reported in GenBank were considered. The results were compared with the previously made morphological identification at the QCAM Fungarium, to check the taxonomic designation.

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

*SNPs: Single nucleotide polymorphisms found between the sample analyzed and the closest hit on the BLASTn search.

QCAM
Fungarium
Code		GenBank
Accession
Number		IdentificationSampling location
National ParkAltitude (m)
QCAM4663MG768840Xylaria
enterogena		Llangantes1370
QCAM4551MG768839Xylaria
enterogena		Llangantes1387
QCAM4537MG768834Xylaria
fissilis		Llangantes1377
QCAM4540MG768836Xylaria
schweinitzii		Llangantes1377
QCAM4232AMG768832ÂXylaria
telfairii		Sangay2885
QCAM4550MG768838Xylaria
sp. 1		Llangantes1387
QCAM4666MG768841Xylaria
sp. 1		Llangantes1379
QCAM4306AMG768833Xylaria
sp. 2		Sangay2885
QCAM4545MG768837Xylaria
sp. 3		Llangantes1373
QCAM4539MG768835Xylaria
sp. 4		Llangantes1377

Phylogenetic analysis

Sequence data were aligned with Geneious R8 and later manually adjusted with Mesquite version 3.047. Public sequences available in GenBank that corresponded to specimens that gave the greatest homology in BLASTn with the sequences of the collected specimens were included. Phylogenetic trees were constructed in Geneious R8 using the PhyML8 plugin for Maximum Likelihood (ML) with a custom substitution model (010230), determined by jModelTest 2.1.4.9,10, according to Corrected Akaike Information Criterion (AICc)11,12. A bootstrap of 1000 replica was used.

Results

All the specimens analyzed 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, and not necessarily to the richness of the Xylareales in the Parks. The unidentified species were different in each park. The analysis shows that there are no shared species of Xylaria at the two sampled sites (Table 1), this is important for conservation decision making. 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, it includes specimens from LP and PS. Clade A includes all X. entogena specimens and Clade B includes all X. telfairii specimens, and Xylarya sp.1 specimens. It is possible that Xylarya sp.1 might belong to the X. telfairii group, but due to the differences among the sequences it is likely a different species. In the second major group, clade C is sister to clades D, E and F. This major group includes specimens from both LP and SP. Clade C includes all X. schweinitzii specimens. Clade D includes Xylaria sp. 2. The closest sequence to Xylaria sp. 2 from SP was a previously reported collection also from Ecuador13 in a cloud forest in the province of Imbabura, that was also identified only at the genus level. Clade E shows Xylaria sp. 3, the closest sequence to this individual belongs to the same previously reported study13, identified only at the genus level. Clade F includes Xylaria fissilis sequences from LP and one from 13. Clade F also includes Xylaria sp. 4, an unidentified specimen. The number of nucleotide differences (SNPs) between the sample and the closest hit on the BLASTn search suggests that these specimens may belong to new species (Table 1). 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 genera14, but the real number remains unknown15. Studies related 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 described16.

3bbaa47b-040d-4b76-98c8-8b40053bb6fd_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 entire 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 unknown15. 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 PS, belonging to the Agaricales, have recently been described16.

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 based on ITS sequences divergence; 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 the Kingdom Fungi we must start by deciphering the diversity of species 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 from "Pontificia Universidad Católica del Ecuador" supported the project.

Acknowledgements

We are grateful to Marcel A. Caminer, Charles W. Barnes and Cristina E. Toapanta for their contribution to the development of the present research. This research has been carried out according to the MAE-DNB-ARRGG-CM-2014-002 "Contrato Marco de Acceso a Recursos Genéticos".

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, Seifert KA, Huhndorf S, et al.: Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A. 2012; 109(16): 6241–6. PubMed Abstract | Publisher Full Text | Free Full Text
  • 4.  Schoch CL, Robbertse B, Robert 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
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  • 7.  Maddison WP, Maddison DR: Mesquite: a modular system for evolutionary analysis. Version 3.04. 2015. Reference Source
  • 8.  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
  • 9.  Darriba D, Taboada GL, Doallo R, et al.: jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8): 772. PubMed Abstract | Publisher Full Text | Free Full Text
  • 10.  Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003; 52(5): 696–704. PubMed Abstract | Publisher Full Text
  • 11.  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
  • 12.  Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008; 25(7): 1253–1256. PubMed Abstract | Publisher Full Text
  • 13.  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
  • 14.  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
  • 15.  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
  • 16.  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, Mátyás B and Ordoñez ME. Xylariales: First results of mycological exploration in the Sangay and Llanganates National Parks, Ecuador [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.12688/f1000research.13623.2)
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Reviewer Report 16 Jul 2018
Juan Enrique Tacoronte Morales, Central University of Ecuador, Quito, Ecuador 
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https://doi.org/10.5256/f1000research.16220.r35790
I have read this submission proposal and all related information reported in (introduction, methodology, results, discussion and cited references). Considering my expertise in secondary metabolites and bioactive natural products isolated from invertebrates (defensive secretions and applied chemical ecology) and fungi ... Continue reading
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Francisco J. Flores, Departamento de Ciencias de la vida y la Agricultura, Universidad de las Fuerzas Armadas–ESPE, Sangolquí, Ecuador 
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https://doi.org/10.5256/f1000research.16220.r34114
There are still several issues that need to be addressed before indexing. Please see the ... Continue reading
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Flores F. Reviewer Report For: Xylariales: First results of mycological exploration in the Sangay and Llanganates National Parks, Ecuador [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.5256/f1000research.16220.r34114)
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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.16220.r34113
Abstract:
4th line from top - change 'done' to 'inferred' because you can't be sure of an identification based on ITS blast.
 
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Lodge DJ. Reviewer Report For: Xylariales: First results of mycological exploration in the Sangay and Llanganates National Parks, Ecuador [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.5256/f1000research.16220.r34113)
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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.
 
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Lodge DJ. Reviewer Report For: Xylariales: First results of mycological exploration in the Sangay and Llanganates National Parks, Ecuador [version 2; peer review: 1 approved, 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 
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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 Parks, Ecuador [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:222 (https://doi.org/10.5256/f1000research.14800.r31232)
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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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