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

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.


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.

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 as primary fungal barcode marker for fungi 3,4 . 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.

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  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.

Phylogenetic analysis
Sequence data were aligned with Geneious R8 and later manually adjusted with Mesquite version 3.04 7 . 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 PhyML 8 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 Ecuador 13 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 study 13 , 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 genera 14 , but the real number remains unknown 15 . 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 Xylaria 13 . In fact, new fungal species in SP, belonging to the Agaricales, have recently been described 16 .
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 genera 13 , but the real number remains unknown 15 . 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 PS, belonging to the Agaricales, have recently been described 16 .

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: 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 (volatile organic compounds) I believe that I could analyze the reported information data with an appropriate, conceptual and methodological level to confirm that this report devoted to mycological exploration of Ascomycota-Xylariales in tropical Andean region has a quite recognized scientific standards for being published in F1000Research.
In this context, please, I have some considerations regarding the submitted article: -it could be of great significant and relevance to evaluate some biogeochemical parameters of eco-geographical collecting zones for potential determining the zonal distribution of Xylariales in these areas, including any potential correlation with tree diversity.
It would have been highly didactic, and recommended, the inclusion, in the proposal submitted, of some photographs in real time, taken during the collection phase in the national parks, and some, comparative ones, taken during the preparation phase in the lab (for morphological purposes only!)

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

Are the conclusions drawn adequately supported by the results?
is not clear why this point was not addressed in revisions, though it would require renumbering of the unidentified species in tables and figures and text. Also, Xylaria sp. 4 appears to be X. fossils based on the phylogeny. The abstract says there were 4 unidentified Xylaria species, but if sp. 3 is X. curta and sp. 4 is X. fossils, there are only two suspected undescribed species -sp. 1 and sp. 2.
The conclusions say there were four unnamed species also, but X. fissilis is among the named species based on one collection, but sp. 4 has an identical sequence to the named one, so that is also X. fissilis and not a new species.
The manuscript should not be indexed until the authors reduce the number of undescribed species from 4 to 2 and make the corresponding changes to the text throughout.
No competing interests were disclosed.

Competing Interests:
I have read this submission. I 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. 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 KP133352 ECU. I would expect to be reasonably common X. curta X. curta 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 1,2 1 2 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
Is the study design appropriate and is the work technically sound? Yes

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

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I 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. The article "Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador " provides internal transcribed spacer sequences from isolates collected from Xylaria 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.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? 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

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Referee Expertise: Phylogenetics I have read this submission. I 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.
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