Long-term fungal inventory in Cusuco National Park, Honduras

The Kingdom Fungi includes a highly diverse group of organisms, in terms of both species richness as well as the ecological roles they deliver. Their unique biological characteristics such as polarized hyphal growth, external enzymatic digestion of macromolecular nutrients, and the production of large amounts of reproductive mito- and meiospores have made the fungi one of the evolutionarily most successful groups on the planet, colonizing almost every available habitat and substratum. Fungi fulfil essential roles in many ecosystems as (i) saprotrophs (‘recyclers’ of the natural environment, decaying dead organic material and releasing its carbon and nutrient content back into the ecosystem); (ii) mutualists (deriving their nutrients from living organisms while at the same time providing benefits); and (iii) parasites and pathogens (‘regulators’ of the natural environment, obtaining nutrients from other living organisms).

Apart from Arthropoda, fungi constitute the most diverse group of eukaryotes on the planet. Since the start of modern fungal taxonomy with Micheli’s (1729) Nova Plantarum Genera, 148,000 species have been formally described. This number may seem startling but is only a fraction of the estimated number of species, which ranges from a conservative 1.5 to 6.0 million species, or more. While we are getting close to knowing all species in groups such as the flowering plants and birds, the gap between numbers of described and estimated species is huge for fungi and insects, where the bulk of species is still undescribed as of today. Alliances like the International Barcode of Life try to fill this gap, e.g., with multi-million-dollar funded programs aimed at generating DNA barcodes for hundreds of thousands of species.

So where are these missing fungi? Well, there are cryptic taxa, those that are morphologically indistinguishable. The more data that are being generated, the more it becomes obvious that certain species names “hide” multiple species. Second, it is estimated that 75% of the planet’s diversity is already in collections—fungal collections, fungaria, may contain new species hidden under current names or not currently identified to species level. Other natural history collections are also sources for fungal discoveries, e.g., plant herbaria for plant-associated rust fungi and insect collections for Laboulbeniales fungi. There is the issue of lack of sequence data. Some numbers:

• As much as 74.4% of newly described fungal species in Index Fungorum are not represented by sequence data in the National Center for Biotechnology Information GenBank database.
• 70% of the fungal taxonomic diversity in herbarium collections are not represented in International Nucleotide Sequence Databases such as GenBank and the European Nucleotide Archive.
• Of 14,000,000 sequences of fungi submitted to GenBank only 500,000 (= 3.5%) are verified, that is, they were generated from holotype material.

And last but not least, many areas that are often hotspots for fungal diversity, remain underexplored. And it is here that tropical mycology comes in the picture. In tropical and subtropical areas, the diversity of biotic and abiotic factors, including high density and diversity of potential host species, high humidity, and the availability of a plethora of microhabitats, is expected to promote high fungal diversity.

Mycology would be nothing without the tropics

E.J.H. Corner (1946)

To this day, the vast majority of fungi have been described by European and North American mycologists. This geographic bias is reflected in type localities of known species. Of 1,882 fungal species described in 2019, only 9.5% originated from the Neotropics and 9% from the entire African continent, whereas Europe and North America collectively accounted for 33%. However, knowledge of tropical diversity is necessary to gain a complete picture of phylogeny and evolutionary history. The exploration of tropical biodiversity and including tropical sampling in our dataset has often drastically changed our perspective on relationships. Let’s illustrate this with the Russulaceae, one of the dominant ectomycorrhizal groups in all ecosystems worldwide. Only once tropical data were included in phylogenetic analyses, it was revealed that Lactarius and Russula consisted of more than the two historical, “well-defined” clades. In reality, there are four genus-level clades, one of them—Multifurca—consisting of some species of Lactarius and a small number of species of the former Russula. The fourth genus is Lactifluus with 200 described species of milkcaps. It is mostly distributed in understudied regions in tropical Africa, which explains why it was historically least studied. Understanding the evolution of Russulaceae in function of climate and available host trees, is only possible if tropical representatives are included. Many taxa in tropical areas (at the level of species, but also at higher taxonomic levels) are endemic and represent key factors in evolutionary reconstructions.

Central American mycology

There is no such thing as an inventory of tropical fungi. Only a few historic initiatives were undertaken documenting neotropical fungal diversity. These generally covered either specific groups of fungi or limited geographic ranges. A proposal for an All-Taxa Biodiversity Inventory (ATBI) of fungi at the Area de Conservación Guanacaste in Costa Rica, sparked by a ten-day workshop, demonstrates the enormous effort necessary just to compile a complete inventory of recorded species (Rossman et al. 1998). The authors reported a total of 1,200 known fungal taxa in Costa Rica (data from the U.S. National Fungus Collections).

Some of the most extensive fungal documentation in Central America in the last decade has occurred in Panama. In 2007, Dr. Meike Piepenbring compiled an annotated checklist of 1,807 species of fungi from Panama based on literature data. A large effort for long-term monitoring of fungal diversity in Panama was subsequently initiated, with monthly sampling along a 500-m transect in secondary vegetation in lowland of western Panama. Piepenbring et al. (2012) reported 567 species of fungi and fungus-like organisms, of which two were undescribed and 19 represented new country records. None of the simulated species estimators approached an asymptote. In 2020, an updated online checklist of Panamanian fungi was published, this time presenting 3,103 species.

Fungal inventory in Cusuco National Park, Honduras

In 2019, we conducted an opportunistic fungal survey in Cusuco National Park, a 23,440-ha protected area in the Merendón range in northwestern Honduras, under the umbrella of Operation Wallacea. Starting in 2022, a formal fungal ATBI project will be initiated at Cusuco. The goals of this monitoring initiative are to describe the diversity of fungi encountered in the park based on morphological, ecological, and molecular information; and to assess potential changes in fungal abundance and fungal species diversity over multiple years. This project aims to lay out a roadmap towards an enduring fungal inventory in this area as part of Operation Wallacea’s multi-year, multi-taxa biodiversity monitoring and surveillance programme in Honduras (Martin et al. 2020).

This formal survey work consists of systematic searches on the existing network of transects and habitat plots in Cusuco. Two transects per camp are surveyed, with four randomly selected 10×10m survey plots that are located on each transect. On each survey plot, two random subplots of 2×2m are chosen with 30 minutes of intensive sampling per subplot. Each subplot sampling event is followed by processing of collections, which includes taking notes, sorting and labeling specimens, taking tissue for DNA, et cetera. Subplots are located adjacent to the existing multi-taxa composite habitat plots along the transects, which are being monitored for environmental variables to characterize the soil, epiphyte density, number of samplings, and overall vegetation density. In this way, our survey methods allow the collection of not only distribution data, but comparative data on fungal diversity variation among habitats, which environmental variables define these variations, how fungal diversity changes over time, and how patterns in fungal species richness correlate with those in other surveyed taxa.

In 2019, 116 collections were made, of which 91 have been identified to order-level, 65 to genus, and 25 to species. Of these, 34 collections were identified as Agaricales, the mushroom-formers, followed by 11 collections of both Helotiales and Hypocreales. A total of 37 genera were identified; best represented were Marasmius (10 collections), Xylaria (7), and Amanita (4). To date, ITS sequences have been generated and analyzed for 17 collections. When comparing to the NCBI GenBank sequence database, ten of our ITS sequences shared between 89.05 and maximum 97.60% identity with known sequences. Using an identity threshold of 98.5%, these results imply that those collections represent either undescribed species or species that are currently unsequenced for this barcode.

Trechispora hondurensis, a crust-like fungus, is the first fungus that was formally described from Cusuco National Park (Haelewaters et al. 2020). These days we are working on an undescribed species in Lactifluus (with Dr. Eske De Crop) and one in Mycocitrus (with Dr. Patricia Kaishian). More genera with potentially undescribed species are: Calostoma (Boletales), Chlorociboria, Chlorosplenium, Ionomidotis (Helotiales), Cyathus, Gymnopus, Mycena, Pterula (Agaricales), and Xylaria (Xylariales). It seems like we’ve found ourselves a fungal treasure trove in Cusuco National Park. The Mesoamerican biodiversity hotspot holds many cloud forest ecosystems, with each cloud forest ‘patch’ supporting its own unique community compositions. Given the presence of micro-endemic species within other taxa at CNP, it is likely that even more numerous new fungal species await discovery. Any site-restricted fungi may be highly threatened with extinction given the alarming rates of habitat destruction in Cusuco, which provides us with the urgent need to collect and document these species before they disappear.

Sources

Cheek M, Lughadha EN, Kirk P, Lindon H, Carretero J, Looney B, Douglas B, Haelewaters D, Gaya E, Llewellyn T, Ainsworth M, Gafforov Y, Hyde K, Crous P, Hughes M, Walker BE, Forzza RC, Meng WK, Niskanen T. 2020. New scientific discoveries: Plants and fungi. Plants, People, Planet 2(5): 371-388. https://doi.org/10.1002/ppp3.10148

De Crop E, Delgat L, Nuytinck J, Halling RE, Verbeken A. 2021. A short story of nearly everything in Lactifluus (Russulaceae). Fungal Systematics and Evolution 7: 133-164. https://doi.org/10.3114/fuse.2021.07.07

Haelewaters D, Dima B, Abdel-Hafiz BII, Abdel-Wahab MA, Abul-Ezz SR, Acar I, Aguirre-Acosta E, Aime MC, Aldemir S, Ali M, Ayala-Vásquez O, Bakhit MS, Bashir H, Battistin E, Bendiksen E, Castro-Rivera R, Çolak ÖF, De Kesel A, de la Fuente JI, Dizkırıcı A, Hussain S, Jansen GM, Kaygusuz O, Khalid AN, Khan J, Kiyashko AA, Larsson E, Martínez-González CR, Morozova OV, Niazi AR, Noordeloos ME, Pham THG, Popov ES, Psurtseva NV, Schoutteten N, Sher H, Türkekul İ, Verbeken A, Ahmad H, Afshan NS, Christe P, Fiaz M, Glaizot O, Liu J, Majeed J, Markotter W, Nagy A, Nawaz H, Papp V, Péter Á, Pfliegler WP, Qasim T, Riaz M, Sándor AD, Szentiványi T, Voglmayr H, Yousaf N, Krisai-Greilhuber I. 2020. Fungal Systematics and Evolution 6. Sydowia 72: 231-356. https://doi.org/10.12905/0380.sydowia72-2020-0231 [pdf]

Haelewaters D, Schoutteten N, Medina-van Berkum P, Martin TE, Verbeken A, Aime MC. 2021. Pioneering a fungal inventory at Cusuco National Park, Honduras. Journal of Mesoamerican Biology 1(1): 111-131. [pdf]

Hawksworth DL, Lücking R. 2017. Fungal diversity revisited: 2.2 to 3.8 million species. Microbiology Spectrum 5(4): FUNK-0052–2016. https://doi.org/10.1128/9781555819583.ch4

Lücking R, Aime MC, Robbertse B, Miller AN, Ariyawansa HA, Aoki T, Cardinali G, Crous PW, Druzhinina IS, Geiser DM, Hawksworth DL, Hyde KD, Irinyi L, Jeewon R, Johnston PR, Kirk PM, Malosso E, May TW, Meyer W, Öpik M, Robert V, Stadler M, Thines M, Vu D, Yurkov AM, Zhang N, Schoch CL. 2020. Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding? IMA Fungus 11: 14. https://doi.org/10.1186/s43008-020-00033-z

Martin TE, Jones SEI, Creedy TJ, Hoskins HMJ, McCann NP, Batke SP, Kelly DL, Kolby JE, Downing R, Zelaya SMS, Green SEW, Lonsdale G, Brown T, Waters S, Rodríguez-Vásquez F, McCravy KW, D’Souza ML, Crace D, Nuñez-Mino JM, Haelewaters D, Medina-van Berkum P, Phipps CD, Barker RJ, Castañeda F, Reid N, Jocqué M. 2021. A review of the ecological value of Cusuco National Park: an urgent call for conservation action in a highly threatened Mesoamerican cloud forest. Journal of Mesoamerican Biology 1(1): 6-50. [pdf]

Piepenbring M. 2007. Inventoring the fungi of Panama. Biodiversity and Conservation 16(1): 73-84. https://doi.org/10.1007/s10531-006-9051-8

Piepenbring M, Hofmann TA, Unterseher M, Kost G. 2012. Species richness of plants and fungi in western Panama: towards a fungal inventory in the tropics. Biodiversity and Conservation 21(9): 2181-2193. https://doi.org/10.1007/s10531-011-0213-y

Rossman AY, Tulloss RE, O’Dell TE, Thorn RG. 1998. Protocols for an all taxa biodiversity inventory of fungi in a Costa Rican conservation area. Parkway Publishers, Boone, North Carolina. [pdf]