F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.13623.1 Research Note Articles Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador

[version 1; peer review: 2 approved with reservations]

 Guevara María-Fernanda Conceptualization Investigation Methodology Writing – Original Draft Preparation https://orcid.org/0000-0001-5141-8464 1 2 Salazar Paula Supervision Writing – Original Draft Preparation 3 Mátyás Bence Project Administration Writing – Review & Editing https://orcid.org/0000-0003-2694-1848 a 4 Ordoñez María-Eugenia Investigation Methodology Writing – Original Draft Preparation 5 Biotechnology of Natural Resources, Universidad Politécnica Salesiana, Quito, 170525, Ecuador Environmental Research Group, Secondary Metabolites and Animal Biotechnology NUNKUY-WAKAN, Universidad Politécnica Salesiana, Quito, 170525, Ecuador Carrera de Biotecnología de los Recursos Naturales, Universidad Politécnica Salesiana, Quito, 170525, Ecuador Grupo de Investigación Mentoria y Gestión del Cambio, Universidad Politécnica Salesiana, Cuenca, 010102, Ecuador School of Biological Sciences, Pontifical Catholic University of Ecuador, Quito, 170143, Ecuador bmatyas@ups.edu.ec

No competing interests were disclosed.

 23 2 2018 2018 7 222 20 2 2018 Copyright: © 2018 Guevara MF et al. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the present study fungal collections were obtained from the Sangay and Llanganates National Parks in Ecuador. For taxonomic identification of the samples, the internal transcribable spacer (ITS) DNA sequence was analyzed. Fungi of the Xylariales order were subjected to phylogenetic analysis using the maximum likelihood method. All samples belonged to the genus Xylaria. Four species were identified: Xylariales enterogena, X. fissilis, X. schweinitzii and X telfairii. Three collections were not identified to species based on ITS sequence, suggesting they could be new species. Further analysis of these samples is necessary. This work aims to contribute with baseline information on the fungal biological diversity in Ecuador, necessary to design and implement measures for the conservation of fungi.

 Diversity ITS Llanganates National Park Sangay Xylarial Secretaría de Educación Superior, Ciencia, Tecnología e Innovación Arca de Noe Catholic University Mycology Collection, Fungarium The Secretaria de Educación Superior, Ciencia, Tecnología e Innovación del Ecuador (SENESCYT), Arca de Noé Initiative and the QCAM Fungarium supported the project. 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 endemism 1, 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 fungi 3 . 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

Fungi of the order Xylariales collected in the Sangay and Llanganates National Parks in Ecuador.
QCAM Fungarium Code GenBank Accession Number Identification Sampling location
National Park Altitude
QCAM4663 MG768840 Xylaria enterogena Llangantes 1370
QCAM4551 MG768839 Xylaria enterogena Llangantes 1387
QCAM4537 MG768834 Xylaria fissilis Llangantes 1377
QCAM4540 MG768836 Xylaria schweinitzii Llangantes 1377
QCAM4232A MG768832Â Xylaria telfairii Sangay 2885
QCAM4550 MG768838 Xylaria sp. 1 Llangantes 1387
QCAM4666 MG768841 Xylaria sp. 1 Llangantes 1379
QCAM4306A MG768833 Xylaria sp. 2 Sangay 2885
QCAM4545 MG768837 Xylaria sp. 3 Llangantes 1373
QCAM4539 MG768835 Xylaria sp. 4 Llangantes 1377
Phylogenetic analysis

Sequence data were aligned with Geneious R8 and later manually adjusted with Mesquite version 3.04 6 . 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 PhyML 7 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 Ecuador 12 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 collection 12 , 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.

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 genera 13 , but the real number remains unknown 14 . 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 Xylaria 13 . In fact, new fungal species in SP, belonging to the Agaricales, have recently been described 15 .

 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

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.

 Bajaña F Rivas J Sánchez D : Informe de la evaluación inicial del Parque Nacional Sangay como Sitio de Patrimonio Natural de la Humanidad. Quito-Ecuador: Ministerio del Ambiente, Fundación Natura, EcoCiencia y UICN-Sur. 2002. Ministerio del Ambiente del Ecuador: Plan de Manejo Parque Nacional Llanganates. Quito-Ecuador,2013. Reference Source Schoch CL Robbertse B Roberts V : Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database (Oxford). 2014;2014; pii: bau061. 24980130 10.1093/database/bau061 4075928 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. 8180733 10.1111/j.1365-294X.1993.tb00005.x White TJ Bruns T Taylor J : Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. Mycologia. 2013;64(1). Reference Source Maddison WP Maddison DR : Mesquite: a modular system for evolutionary analysis. Version 3.04.2015. Reference Source Guindon S Dufayard JF Lefort V : New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–321. 20525638 10.1093/sysbio/syq010 Darriba D Taboada GL Doallo R : jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9(8):772. 22847109 10.1038/nmeth.2109 4594756 Guindon S Gascuel O : A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2013;52(5):696–704. 14530136 10.1080/10635150390235520 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 Posada D : jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008;25(7):1253–1256. 18397919 10.1093/molbev/msn083 Thomas DC Vandegrift R Ludden A : Spatial Ecology of the Fungal Genus Xylaria in a Tropical Cloud Forest. Biotropica. 2016;48(3):381–393. 10.1111/btp.12273 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. 10828406 10.1016/S0378-1097(00)00181-6 Hsieh HM Lin CR Fang MJ : 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. 20035889 10.1016/j.ympev.2009.12.015 Crous PW Wingfield MJ Burgess TI : Fungal Planet description sheets: 558–624. Persoonia. 2017;38(1):240–384. 29151634 10.3767/003158517X698941 5645186 10.5256/f1000research.14800.r32603 Reviewer response for version 1 Lodge D. Jean 1 2 Referee Department of Plant Biology, University of Georgia, Athens, GA, USA Department of Agriculture, USDA Forest Service, Luquillo, PR, USA

Competing interests: No competing interests were disclosed.

 19 4 2018 Copyright: © 2018 Lodge DJ 2018 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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 seems to be an incomplete sentence. It ends with ‘BLASTn’ (NCBI) optimized for highly similar sequences (megablast), alignments that presented 100’

It is probably meant to say ‘overlapped 100%.’

Results: Text for Clade E does not match the phylogram. The text says that sp. 3 was related to another collection identified only at the genus level. However, the phylogram shows a well-supported clade (98% BS) comprised of sp. 3 and X. curta KP133352 ECU. I would expect X. curta to be reasonably common there. There is either an error in identification of KP133352 ECU or the text in resutls needs to be changed, and the abstract and first line of results and conclusions also need to be changed to include X. curta.

That also means that the unidentified species need to be renumbered in the text, table, and phylogram, with sp. 4 becoming sp. 3.

There are likely 3 undescribed species included in this study. The journal allows for photographs, and photos should be added to the manuscript. Even if photos were not taken of the fresh specimens, photos of the dried specimens can be used, and are very helpful. If photos of the asci, especially the ascus plug stained with iodine/Melzer’s reagent, and photos of the ascospores showing the germ slits, that would be very helpful also for future work. The ascospore length and width ranges, the shape and extent of the germ slit, and the shape and size of the ascus plug would be helpful additions.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.14800.r31232 Reviewer response for version 1 Flores Francisco J. 1 Referee https://orcid.org/0000-0001-8092-3750 Departamento de Ciencias de la vida y la Agricultura, Universidad de las Fuerzas Armadas–ESPE, Sangolquí, Ecuador

Competing interests: No competing interests were disclosed.

 1 3 2018 Copyright: © 2018 Flores F 2018 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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. Nevertheless, I have several concerns, mainly:

Phylogenetic analysis using only the ITS sequence is very superficial, and, most likely, it won´t show an accurate representation of the real evolutionary relationships between isolates.

There is no mention about the morphological characteristics of isolates. Is the morphology congruent with the genotype?

 The manuscript needs major revision to improve clarity.

Several suggestions are made in the file linked below.

https://f1000researchdata.s3.amazonaws.com/linked/196501.Bence_Matyas_rev.pdf

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Phylogenetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.