Diversity and Host Relationships of the Mycoparasite Sepedonium (Hypocreales, Ascomycota) in Temperate Central Chile

We present the first major survey of regional diversity, distribution and host-association of Sepedonium. Whereas the rather scarce worldwide records of this mycoparasitic fungus suggested no specific distribution pattern of most species before, we provide new evidence of endemic and specific host-parasite guilds of Sepedonium in Southern South America, including the description of a new species. The corresponding inventory was performed in temperate central Chile. The regional landscape, a mosaic of exotic timber plantations and remnants of native Nothofagus forests, facilitates a unique combination of endemic and adventitious Boletales hosts. During a two-year survey, 35 Sepedonium strains were isolated and cultured from infected basidiomata of allochthonous Chalciporus piperatus, Paxillus involutus, Rhizopogon spp. and Suillus spp., as well as from the native Boletus loyita, B. loyo, B. putidus and Gastroboletus valdivianus. Taxonomic diagnosis included morphology of conidia and conidiophores, sequences of ITS, RPB2 and EF1 molecular markers and characteristics of in vitro cultures. Phylogenetic reconstructions were performed using Bayesian methods. Four Sepedonium species could be identified and characterized, viz.: S. ampullosporum, S. chrysospermum, S. laevigatum and the newly described species S. loyorum. The most frequent species on introduced Boletales was S. ampullosporum, followed by S. chrysospermum and S. laevigatum. S. loyorum sp. nov. was found exclusively on native boletacean hosts, separated from its closest relative S. chalcipori by micromorphological and molecular attributes. Species descriptions and identification keys are provided. Ecological and biogeographical aspects of endemic and allochthonous symbiotic units consisting of mycoparasite, ectomycorrhizal fungal host and respective mycorrhizal tree are discussed.


Introduction
Mycophilic fungi belonging to the anamorphic genus Sepedonium Link (Hypocreaceae, Ascomycota), teleomorph Hypomyces (Fr.) Tul. & C. Tul., are known as highly specialized parasites on basidiomata of various genera of Boletales E.-J. Gilbert [1]. Once infected and specificity patterns of the mycoparasite and its hosts in a relatively small geographic area.
As the result of a two-year inventory between 2017 and 2018, we provide the first comprehensive study of diversity of Sepedonium spp. in temperate central Chile, combining morphological diagnostic attributes, culture characteristics, host diversity and molecular phylogeny based on three DNA markers, viz.: ITS, EF1-α and RPB2. A total of four species was recorded, characterized and identified along with their hosts, one of them new to science.

Study Area and Material Collection
Basidiomata infected by Sepedonium were collected during opportunistic forays in temperate central Chile in the administrative regions of Biobío, Ñuble and Araucanía ( Figure 1, Table S1), approximately between 36 • 15 and 38 • 45 S.L, during the complete mushroom growing season 2017 (April to October) and autumn 2018 (February to May).
Microorganisms 2021, 9, 2261 3 of 19 habitats are supporting a unique combination of autochthonous and adventitious Boletales [22][23][24][25], many of them compatible with mycophilic Sepedonium spp., conditions which allowed us a broad approach towards novel knowledge of diversity, distribution and specificity patterns of the mycoparasite and its hosts in a relatively small geographic area.
As the result of a two-year inventory between 2017 and 2018, we provide the first comprehensive study of diversity of Sepedonium spp. in temperate central Chile, combining morphological diagnostic attributes, culture characteristics, host diversity and molecular phylogeny based on three DNA markers, viz.: ITS, EF1-α and RPB2. A total of four species was recorded, characterized and identified along with their hosts, one of them new to science.

Study Area and Material Collection
Basidiomata infected by Sepedonium were collected during opportunistic forays in temperate central Chile in the administrative regions of Biobío, Ñ uble and Araucanía (Figure 1, Table S1), approximately between 36°15′ and 38°45′ S.L, during the complete mushroom growing season 2017 (April to October) and autumn 2018 (February to May).  basidiomata with visible symptoms of infection by Sepedonium were documented in situ, extracted, stored in plastic containers and transferred to the laboratory within the same day where they were refrigerated at 4 • C for further processing.

Host Species
Basidiomata of Boletales hosts were identified in the field and in the laboratory by macroscopic and microscopic examination of diagnostic morphological attributes, according to keys and reference descriptions [13,22,26] for native Chilean species; for adventitious taxa we consulted [27][28][29], as well as various electronic resources. Dehydrated voucher specimens were deposited at CONC-F (Fungarium G. Palfner, Universidad de Concepción, Concepción, Chile).

Strain Cultivation
Scrapings of Sepedonium mycelium and conidia were taken from fresh, infected basidiomata under sterile conditions and placed on Petri dishes with malt peptone extract agar [3]. Three replicates of each strain were incubated at 25 • C and purified from contamination, if necessary, until obtaining axenic cultures. Strains are kept at Laboratorio de Investigación en Agentes Antibacterianos, Universidad de Concepción (Concepción, Chile) and at the Kulturen Sammlung Halle (KSH), Institute for Plant Biochemistry (Halle (Saale), Germany).

Optical Microscopy
Pieces of mycelium and conidia scrapings were mounted on glass slides with a drop of distilled water without staining. Specimens were observed at 1000× magnification under an Olympus CX31 compound microscope (Olympus, Tokyo, Japan) equipped with an attached digital camera and drawing device. Diagnostic structures such as conidiophores, phialoconidia and aleurioconidia were examined, measured and documented according to [6].

Scanning Electron Microscopy
Dehydrated samples of Sepedonium on infected hosts and from axenic cultures were fixed on SEM mounts with patches of double-sided adhesive tape, metallized with gold and observed in a JSM-6380LV scanning electron microscope (JEOL, Tokyo, Japan), in order to obtain high resolution images of aleurioconidia wall structure.

DNA Extraction
With a sterile inoculation loop, mycelium samples were scraped with or without agar from culture plates and suspended in a buffer solution provided by FastDNA™ SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA), suitable for the extraction of fungal genomic DNA. The manufacturer's instructions for DNA extraction and purification were followed, including the mechanical lysis step consisting of two 40 s to 6 m/s cycles on a FastPrep-24™ Classic Instrument high-speed homogenizer (MP Biomedicals, Solon, OH, USA). The purified DNA was stored at −20 • C before further processing.

Sequence Processing and Phylogenetic Analysis
The sequences obtained from Macrogen for each molecular marker (ITS1, EF1-α, RPB2) were edited through the Codon Code Aligner v. 3.0.3 program (CodonCode Corporation, www.codoncode.com, accessed on 1 June 2021). The sequences were aligned using the MUSCLE program [33] also integrating Sepedonium sequences available from the GenBank database (Table S2). The saturation test was performed in the DAMBE v. 5.2 program [34], to evaluate the usefulness of the sequences for phylogenetic analyses. The proportion of invariant sites, a key parameter for the saturation test, was obtained with jModeltest 2 [35]; the same software was used to identify the best fit nucleotide replacement model for each gene which turned out T93 + G for ITS1, EF1-α and RPB2 under Akaike's reporting criteria.
Two species trees were recovered in *BEAST, a component of BEAST v. 2.3.2 [36], one performed at the individual level using the ITS 1 molecular marker (68 strains) and the other performed at the species level using 25 individuals (eight strains of S. ampullosporum; four strains of S. chrysospermum; seven strains of S. loyorum; four strains of S. chalcipori and two strains of S. laevigatum) for which the ITS1, EF1-α and RPB2 sequences were available (Table S2). This second phylogenetic tree was built under the coalescent multispecies model [37,38]. We used the same nucleotide substitution and model configuration for each dataset. Considering that T93 + G is a simple evolutionary model, we evaluated the effect of more complex models (GTR + G + I), obtaining identical topologies. For each dataset, 100 million iterations were performed and sampled every 1000 steps, the first 25% of the results being discarded by burn-in. The convergence of MCMC analysis was visually examined in Tracer v1.6 [39] [40]. We also calculated uncorrected pairwise genetic distances based on ITS in MEGA 5.0.

Taxonomy of Sepedonium Strains and Host Taxa
The 35 Sepedonium strains sucessfully retrieved from different locations and hosts ( Figure 1, Table S1), apart from molecular analysis, were characterized and identified based on diagnostic micromorphological attributes, viz.: phialoconidiophore branching, phialid length, size and shape of phialoconidia, size and ornamentation of aleurioconidia.

Phylogenetic Characterization of Sepedonium Strains
We obtained 64 sequences with 517 base pairs for the ITS1 marker, 25 sequences with 659 base pairs for the RPB2 gene and 25 sequences with 596 base pairs for the EF1-α gene (Table S2). All three markers showed low saturation with p < 0.05 (ITS1: 0.33971; EF1-α: For the tree represented by the ITS molecular marker (Figure 2), the genus Sepedonium was recovered as monophyletic (1.0 Posterior Probability) by Bayesian inference analysis. The nine different taxa, including the four species known from Chile, are divided in three main clades: The first clade is represented by Sepedonium chlorinum which is supported by 0.71 PP (posteriori probability). The second clade is composed of S. loyorum sp. nov. and S. chalcipori and is supported by 0.98 PP. The third clade includes S. ampullosporum, S. chrysospermum, S. microspermum, S. tulasneanum, S. laevigatum and S. brunneum, which is supported by 1.0 PP.

Phylogenetic Characterization of Sepedonium Strains
We obtained 64 sequences with 517 base pairs for the ITS1 marker, 25 sequences with 659 base pairs for the RPB2 gene and 25 sequences with 596 base pairs for the EF1-α gene (Table S2). All three markers showed low saturation with p < 0.05 (ITS1: 0.33971; EF1-α: 0.00188; RPB2: 0.54632). The polymorphic sites for the ITS1 marker are 70, 224 for the EF1α gene and 156 for the RPB2 gene.
For the tree represented by the ITS molecular marker (Figure 2), the genus Sepedonium was recovered as monophyletic (1.0 Posterior Probability) by Bayesian inference analysis. The nine different taxa, including the four species known from Chile, are divided in three main clades: The first clade is represented by Sepedonium chlorinum which is supported by 0.71 PP (posteriori probability). The second clade is composed of S. loyorum sp. nov. and S. chalcipori and is supported by 0.98 PP. The third clade includes S. ampullosporum, S. chrysospermum, S. microspermum, S. tulasneanum, S. laevigatum and S. brunneum, which is supported by 1.0 PP.  The multilocus species tree (ITS, EF1 and RPB2), including the four taxa reported from Chile and S. chalcipori as neighbor taxon of S. loyorum (Figure 3), recovered the Sepedonium species cluster as monophyletic (PP = 1.0). The species fall into two major clades (PP = 0.7), one represented by S. loyorum sp. nov. and S. chalcipori with a PP support = 1.0, the second clade including S. ampullosporum, S. chrysospermum and S. laevigatum supported with PP = 1.0 ( Figure 3).
The uncorrected pairwise genetic distances based on ITS showed that S. chalcipori has 3% of genetic distance with respect to S. loyorum sp. nov. Trichoderma aerugineum as external group, inferred from ITS, RPB2 and EF1-α -multigene sequence alignment; node values express posterior probability; aleurioconidia micrographs obtained from this study except Sepedonium chalcipori micrograph extracted and modified from [8].
The uncorrected pairwise genetic distances based on ITS showed that S. chalcipori has 3% of genetic distance with respect to S. loyorum sp nov.    Trichoderma aerugineum as external group, inferred from ITS, RPB2 and EF1-α -multigene sequence alignment; node values express posterior probability; aleurioconidia micrographs obtained from this study except Sepedonium chalcipori micrograph extracted and modified from [8].

Morphological Characterization of Sepedonium spp.
The uncorrected pairwise genetic distances based on ITS showed that S. chalcipori has 3% of genetic distance with respect to S. loyorum sp nov.        Etymology: loyorum: latinized plural of loyo, referring to the main host species Boletus loyo and Boletus loyita; both epithets are derived from the corresponding native Chilean (mapuzungún) names which are "Loyo" for B. loyo and "Pichiloyo" (small loyo) for B. loyita.
Mycelium forming a tomentose to pubescent layer or mat on carpophores of native Chilean Boletaceae, white in the beginning, at maturity golden yellow (Figure 6a Figures 6f, 7a and 8a).

Host Specificity and Frequency
Endemic Boletaceae so far have been found to be exclusively colonized by S. loyorum sp. nov. and, correspondingly, the latter has not been registered on an allochtonous host which indicates a highly specific parasite-host association. Among the three widely distributed species, S. chrysospermum shows a marked preference for Paxillus involutus, with nine out of ten records. The relatively high number of records of S. ampullosporum on Suillus luteus (six records) is rather owed to the high frequency of the host species in the visited habitats (pine plantations).

Keys for Sepedonium spp.
The following dichotomous key, apart from the newly described S. loyorum, includes all currently accepted Sepedonium species treated by [6][7][8] and for which DNA sequences could be retrieved; species recorded in Chile to date are marked with bold letters.

Phylogenetic Kinship of S. loyorum sp. nov.
Our data indicate that Sepedonium loyorum sp. nov. is probably co-endemic with its hosts; it can be distinguished morphologically from its closest phylogenetic relative S. chalcipori by architecture and dimensions of phialoconidiophores. Our Bayesian phylogenetic tree based on ITS gene recovered S. loyorum sp. nov. as sister species of S. chalcipori with high posterior probability (1.0). Unsurprisingly, our analyses resulted in a topology not congruent with the neighbor-joining tree of [8]. The most obvious rearrangement involves S. brunneum as sister species of a clade composed by S. chrysospermum, S. microspermum, S. laevigatum and S. tulasneanum, with S. tulasneanum as sister species of S. laevigatum. This pattern is largely coherent with the ITS phylogeny obtained by [41] for seven species of Sepedonium in order to elucidate the kinship of S. microspermum from Iran.
Our multilocus species tree (based on ITS, RPB2 and EF1-α) also recovered S. loyorum sp. nov. as sister species of S. chalcipori with high posterior probability (1.0), this result supports the concept of S. loyorum sp. nov. being a phylogenetic species different from S. chalcipori following the phylogenetic species concept criteria [42].

Biogeographical Aspects of Sepedonium in Chile
This study represents, to our knowledge, the first survey of Sepedonium in a major coherent geographical area. Although S. chrysospermum has been historically reported rather frequently from several countries on both hemispheres including Chile, not all records may have been correctly identified, especially those dating before the comprehensive studies by [6][7][8]: It should be considered that S. chrysospermum was one of only about three species described until the second half of the 20th century, also that aleuroconidia on infected host basidiomata often were the principal or even only diagnostic attribute to be examined, and finally that molecular tools have been applied for species delimitation for only about 30 years before date.
Published worldwide records of other Sepedonium spp. are still too scarce and scattered to allow assertions about their biogeography. However, the apparently endemic S. loyorum seems to be much more specifically associated to its regional environment and hosts than has been previously assumed for other species of the genus. Due to its evident affiliation to endemic Boletaceae from South American Nothofagus forests, a Gondwanean origin of S. loyorum appears likely. This suggestion is strengthened by the observation that its closest phylogenetic neighbour, S. chalcipori, seems to have a geographical connection to New Zealand [8], although it may have been introduced in Australasia from the northern hemisphere together with its host Chalciporus associated with exotic pine species [43]. In any case it would be highly interesting to investigate co-evolution of both species with their known hosts which may also shed new light on diversification and mycogeographical aspects of Boletales on the Southern hemisphere. None of the other three detected Sepedonium species (S. ampullosporum, S. chrysospermum, S. laevigatum) could be found on endemic Chilean boletes during our study which allows the conclusion that those taxa, like their respective hosts and associated trees, are adventitious in Chile. The report of S. chrysospermum on B. bresinskyanus Garrido, an insufficiently known Chilean bolete [13], may be the result of a misidentification based on the historic taxonomic limitations mentioned above.

Sepedonium-Resistant Boletales
An interesting Sepedonium host group due to contrasting infection patterns in the studied area is the Paxillus-Austropaxillus complex: Austropaxillus boletinoides (Singer) Bresinsky & Jarosch and A. statuum (Speg.) Bresinsky & Jarosch are common native ectomycorrhizal fungi in Chilean Nothofagus forests but, contrasting our expectations, were never found to be infected by Sepedonium during our surveys, whereas the adventitious Paxillus involutus which commonly grows at disturbed sites like gardens, parks and timber plantations under introduced trees, such as chestnut, oak or eucalypt, but also under native Nothofagus, was regularly infected, preferentially by S. chrysospermum. Phylogenetic distance between both genera within the Boletales, may be decisive for this difference in susceptibility to the mycoparasite: the genus Austropaxillus was separated from Paxillus by [44], mainly based on molecular evidence, and convincingly placed in the family Serpulaceae together with Gymnopaxillus and Serpula [45,46]. As to our knowledge, none of the three genera has so far been reported to be parasitized by Sepedonium, another example that host-parasite relationships can be important indicators of phylogeny.

Trophic Connections and Specificity within Parasite-Host Guilds: Invasive vs. Endemic Taxa
Considering that almost all detected Boletales hosts of Sepedonium are ectomycorrhizal symbionts, we can contemplate tripartite trophic associations between mycoparasite, mycorrhizal fungal host and mycorrhizal tree which may even share their status of endemism as in the case of S. loyorum and its endemic hosts which again are forming mycorrhiza with Nothofagus species autochthonous in Southern South America. In the special case of Chalciporus piperatus which is supposed to parasitize mycelium of Amanita muscaria [43] and which we found infected by S. ampullosporum, this association would be still further extended to the epiparasitic level, viz.: Sepedonium-Chalciporus-Amanita and hence to a quadripartite symbiosis when carbon flux from the mycorrhizal tree is taken into account.
Looking at these rather complex nutrient pathways towards Sepedonium in forest ecosystems, it is of importance to estimate the invasive potential of allochthonous parasitehost associations in endemic communities, especially in a mosaic landscape consisting of exotic timber plantations and native forest remnants which characterizes not only our study area in central Chile, but nowadays exists globally in many regions [25,43,47]. Amanita muscaria accompanied by Chalciporus piperatus was originally adventitious with introduced Monterrey Pine in Southern Chile, but is now observed in pure Nothofagus forest with increasing frequency [24,25]; it can therefore safely be assumed that a host-tree switch has taken place from Pinus to Nothofagus which has the capacity to promote penetration of Amanita into native forest as a precursor or vector, as well for Chalciporus as for exotic Sepedonium spp., where competition with, or even replacement of, endemic fungi on both, mycorrhizal and mycoparasitic level may occur. Time will reveal whether resilience of the endemic taxa is strong enough to integrate this co-invasive force in a stable community, without erosion or even loss of native fungal diversity.

Conservation Criteria for Sepedonium on Threatened Hosts
A documented and pressing threat to endemic Chilean Boletaceae and associated S. loyorum is the decrease and deterioration of their native Nothofagus forest habitats as a result of direct and indirect effects of land use change in central and Southern Chile [48][49][50]. The conservation category of all four identified hosts has been classified between 2014 and 2019 by IUCN criteria: Boletus loyo and Gastroboletus valdivianus as threatened (EN), B. loyita as vulnerable (VU) and B. putidus as nearly threatened (NT) (https://clasificacionespecies. mma.gob.cl/, accessed on 1 June 2021). In their global proposal of conservation strategies for metazoan parasites [51], the authors state that "parasites face a double threat: they are directly vulnerable to extinction due to anthropogenic factors like climate change or invasive species, and indirectly vulnerable through coextinction with hosts, especially in changing environments". This is certainly also true for mycophilic fungi and, consequently, conservation concepts should be applied for species with endemic status and specific association to threatened hosts and habitats, such as Sepedonium loyorum.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9112261/s1, Table S1: Locations of the Sepedonium strains collected in Central Southern Chile with their respective hosts, Table S2: Sepedonium species/strains (in ascending alphabetical/numerical order) included in molecular phylogeny with their respective molecular markers and GenBank access numbers.