Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora

Symbiosis between insects and fungi arose multiple times during the evolution of both groups, and some of the most biologically diverse and economically important are mutualisms in which the insects cultivate and feed on fungi. Among these are bark beetles, whose ascomycetous cultivars are better known and studied than their frequently-overlooked and poorly understood basidiomycetous partners. In this study, we propose five new species of Entomocorticium, fungal mutualists in the Russulales (Basidiomycota) that are mutualistic symbionts of scolytine beetles. We have isolated these fungi from the beetle mycangia, which are structures adapted for the selective storage and transportation of fungal mutualists. Herein, we present the most complete phylogeny of the closely related genera Entomocorticium and Peniophora and provide insights into how an insect-associated taxon (Entomocorticium) evolved from within a wood-decaying, wind-dispersed lineage (Peniophora). Our results indicate that following a transition from angiosperms to gymnosperms, fungal domestication by beetles facilitated the evolution and diversification of Entomocorticium. We additionally propose four new species: Entomocorticium fibulatum Araújo, Li & Hulcr, sp. nov.; E. belizense Araújo, Li & Hulcr, sp. nov.; E. perryae Araújo, Li & Hulcr, sp. nov.; and E. macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov. Our findings highlight the fact that insect-fungi associations remain an understudied field and that these associations harbor a large reservoir of novel fungal species.


Introduction
Several insect groups within ants, termites, wasps, and beetles have independently evolved mutualisms with a variety of fungal lineages that help them extract nutrients from wood, an otherwise intractable substrate [1][2][3]. In many of these associations, the derived from Peniophora, a genus of resupinate wood decay fungi that colonize several plant families and that rely exclusively on the wind to disperse their spores. A more recent study described seven of Hsiau & Harrington's nine putative species of Entomocorticium based on morphology and molecular data from the ITS and 28S rDNA [15]. Unfortunately, no studies to date have addressed the broader evolutionary picture regarding the ecological relationships between the genera Peniophora and Entomocorticium as well as the context in which their associations with the beetle vectors and host trees might have occurred.
In this study, we propose five new species belonging to the genus Entomocorticium and explore the diversity and evolutionary relationships of this fungal lineage with their beetle vectors and tree hosts. In order to investigate possible evolutionary scenarios, we have built a comprehensive phylogeny based on all available data from the genera Peniophora (54 species) and Entomocorticium (13 named species, including those proposed herein) and three putative species. We tested whether Entomocorticium is a distinct, monophyletic genus within the order Russulales and what factors promoted its differentiation from the genus Peniophora. In terms of ecology and evolution, we investigated the beetle host spectrum across the Entomocorticium phylogeny and provide a hypothesis on how the association between gymnosperms, angiosperms and beetles influenced the rise of these fungal mutualists.

Fungus Isolation
The fungi used in this study were isolated from pronotal mycangia of adult bark beetles Dendroctonus brevicomis, D. frontalis, and Pityoborus comatus, in the USA (California, Colorado, Florida, Louisiana, Montana, Michigan, New Mexico, South Carolina, Texas and Utah) (see [25]) and Belize ( Table 1). Isolates of Entomocorticium fibulatum and E. belizense were conducted for this study, while the isolation of E. perryae and E. macrovesiculatum was previously conducted by Bracewell and Six [25]. Beetles were identified using external morphology with identification keys and images [26][27][28]. Whole beetles were surfacewashed by vortexing for 1 min in 1 mL of sterile distilled water with one small drop of Tween detergent. Pronota of adult beetles were removed and crushed in a 500 µL of sterile phosphate buffer saline and vortexed for 30 s. The resulting solutions were diluted to 1:100 and 1:1000 concentrations, and each dilution was used to inoculate potato dextrose agar (PDA; Becton, Dickinson and Company, MD, USA) plates. Fungi were allowed to grow at 25 • C for 5-10 d. Representative isolates of different fungal morphotypes were placed onto new 2% potato dextrose agar (PDA) plates to obtain pure cultures and these were retained for molecular identification. In addition, we attempted to induce the production of the sexual stage by plating the isolates in Malt agar and also inoculating them in pinewood chips, but these efforts failed to promote the production of the sexual stage in all our isolates. Axenic cultures of the fungi are deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and in the Westerdijk Fungal Biodiversity Institute collections (CBS). Beetle remains of specimens collected in Belize or Florida were vouchered the UF Forest Entomology (UFFE) cryo-collection.

Morphological Observations
To access the micro-morphological features, we collected small samples of each isolate in 3-5 parts across the plate, i.e., edge, intermediate portion and center. These fungal pieces were mounted in 4% lactic acid or lacto-fuchsin and observed under an optical microscope (Zeiss Axioscope 5). Measurement of taxonomically relevant structures, e.g., vesicles and chlamydospores, were performed using the Zen software (Zeiss, Jena, Germany). The semi-permanent slides were sealed with nail polished by direct application of at least 3 layers around the cover slip edges and stored in a slide box for further observation.

Taxa Sampling and Sources
In order to test the relationship of Entomocorticium species with other genera within the order Russulales, we built a comprehensive phylogeny based on LSU and ITS sequences from [29,30] (Supplementary Figure S1 and Table S1). Once we established the relationship between Entomocorticium and Peniophora, we performed a second analysis including five loci, (SSU, LSU, TEF, ITS and IGS) consisting of 129 taxa from Peniophora and Entomocorticium species and four outgroup taxa (Dichostereum spp.). Sequences in the analysis included those from our isolates as well as Peniophora and Entomocorticium sequences archived in GenBank. However, the majority of taxa of our dataset (78 out of 138) were composed of only ITS and LSU rDNA due to limited data availability in GenBank for this fungal group ( Table 2). As a quality control approach to confirm the identity of sequences used in this study, we subjected all sequences, including newly generated sequences of Entomocorticium from beetle mycangia ( Table 2) to a BLAST comparison with reliable ex-types.

Phylogenetic Analyses
Sequence alignment was performed with MAFFT 1.4.0 [35]) separately for each marker. The alignment for each individual locus was improved manually by trimming the longer unique ends and removing gaps. The sequences were then annotated and concatenated into a single combined dataset using Geneious v. 11.1.5 [34]. Ambiguously aligned regions were excluded from phylogenetic analysis and gaps were treated as missing data. The final alignment is available in Treebase.org (http://purl.org/phylo/treebase/phylows/study/ TB2:S29025). The first analysis of the order Russulales was composed of 145 sequences divided into four partitions: ITS1, and 28S rDNA (Supplementary Table S1). The final alignment length was 1942 bp, 683 for ITS (ITS1, 5.8S and ITS2) and 1259 bp for 28S rDNA. For the second analysis of Peniophora and Entomocorticium (Table 2), the final alignment length was 4662 bp: 1259 bp for 18S rDNA, 951 bp for 28S rDNA, 1040 bp for TEF, 1004 bp for ITS and 408 bp for mt-lsu. Maximum likelihood (ML) analyses were performed with RAxML v. 8.2.4 [36] on a concatenated dataset. The dataset consisted of seven data partitions, including one each for SSU, LSU, TEF, mt-lsu and three for ITS (ITS1, 5.8S and ITS2). The GTRGAMMA model of nucleotide substitution was employed during the generation of 1000 bootstrap replicates.

Ancestral Character State Reconstruction
To understand the evolutionary history of Peniophora and Entomocorticium and their associations with beetle vectors and tree hosts, we conducted ancestral character state reconstruction (ACSR) in Mesquite [37], using the best-scoring ML tree produced in RAxML. To interpret host association evolution, each taxon was coded as associated with either angiosperms or gymnosperms (Pinaceae). Additionally, in order to understand the evolution of the association with beetle vectors, we performed a second analysis of the association between Entomocorticium and six vector categories: Dendroctonus brevicomis, D. frontalis, D. ponderosae, Pityoborus comatus and Ips avulsus. We used maximum likelihood model MK1, as implemented in Mesquite v. 3.61 [37]. Only nodes presenting > 50% probability were displayed and used to color-code the branches on the figures.

Post-Analyses Graphical Display
Following the phylogenetic and ancestral character state reconstruction analyses, we used tools available in Geneious v. 11.1.5 [34] and Dendroscope [38] to optimize the tree layout. Further graphic treatment was performed in Adobe Illustrator and Procreate software in iPad Pro.

Results
To understand the species diversity and the evolutionary and ecological processes that led to the domestication of a wood-decaying fungal lineage by bark beetles, we built the most comprehensive phylogeny of the genera Peniophora (54 spp.) and Entomocorticium (17 spp.) to date. Our phylogenetic reconstruction corroborates previous studies connecting both fungal genera [15,16] (Figure 1).

Results
To understand the species diversity and the evolutionary and ecological processes that led to the domestication of a wood-decaying fungal lineage by bark beetles, we built the most comprehensive phylogeny of the genera Peniophora (54 spp.) and Entomocorticium (17 spp.) to date. Our phylogenetic reconstruction corroborates previous studies connecting both fungal genera [15,16] (Figure 1).  We describe an evolutionary switch from fungi with relatively complex basidiocarps that are strictly wind-dispersed (Peniophora) to fungi with minimal or unknown reproductive structures that are actively dispersed within beetle mycangia (Entomocorticium). Our ancestral character state reconstruction (ACSR) indicates that Peniophora is ancestrally associated with angiosperms but has transitioned to gymnosperms at least five times. Among the 54 species of Peniophora included in this study, only nine are associated with gymnosperms, i.e., Peniophora duplex, P. exima, P. parvocistidiata, P. piceae, P. pini, P. pseudonuda, P. pseudo-pini, P. pithya and P. septentrionalis (Figure 1, green branches). Our results indicate that following one of these transitions from angiosperms to gymnosperms (Figure 1, Node A), fungal domestication by bark beetles facilitated the evolution of Entomocorticium (Figure 1, Node B). Our data suggest that the domestication of these fungi by beetles might have promoted speciation and dissemination of this new fungal lineage across at least five beetle lineages. Currently, we have records for six beetle species associated with Entomocorticium (five shown in Figure 2), which might represent at least three independent origins (beetle genera) of Entomocorticium farming and multiple vector switches within those beetle groups.
Character State Reconstructions (ACSR) analyses based on fungal association with their plant hosts. Black branches mean association with angiosperms, green indicate an association with gymnosperms and no association with beetles, brown indicates association with gymnosperms and beetles. Pinecones indicate a transition from angiosperms to gymnosperms. Node A indicates the transition from angiosperms to gymnosperms and the origin of Entomocorticium, node B indicates fungal radiation following the association of Entomocorticium with bark beetles. Photos by Patrick Harvey, Jerzy Opioła, Eva Skific and Andrew Johnson.
We describe an evolutionary switch from fungi with relatively complex basidiocarps that are strictly wind-dispersed (Peniophora) to fungi with minimal or unknown reproductive structures that are actively dispersed within beetle mycangia (Entomocorticium). Our ancestral character state reconstruction (ACSR) indicates that Peniophora is ancestrally associated with angiosperms but has transitioned to gymnosperms at least five times. Among the 54 species of Peniophora included in this study, only nine are associated with gymnosperms, i.e., Peniophora duplex, P. exima, P. parvocistidiata, P. piceae, P. pini, P. pseudonuda, P. pseudo-pini, P. pithya and P. septentrionalis (Figure 1, green branches). Our results indicate that following one of these transitions from angiosperms to gymnosperms (Figure 1, Node A), fungal domestication by bark beetles facilitated the evolution of Entomocorticium (Figure 1, Node B). Our data suggest that the domestication of these fungi by beetles might have promoted speciation and dissemination of this new fungal lineage across at least five beetle lineages. Currently, we have records for six beetle species associated with Entomocorticium (five shown in Figure 2), which might represent at least three independent origins (beetle genera) of Entomocorticium farming and multiple vector switches within those beetle groups. With the current state of sampling of Entomocorticium we investigated the radiation of the genus with its beetle vectors. Our analysis, considering the beetle vector associations, suggests that the first beetle lineage to have domesticated an ancestor of the genus Entomocorticium was likely the twig beetles in Pityoborus (ACSR = 58%; Figure 2). After With the current state of sampling of Entomocorticium we investigated the radiation of the genus with its beetle vectors. Our analysis, considering the beetle vector associations, suggests that the first beetle lineage to have domesticated an ancestor of the genus Entomocorticium was likely the twig beetles in Pityoborus (ACSR = 58%; Figure 2). After that, a transition from twig beetles to D. ponderosae appears to have occurred relatively soon after the initial domestication. Interestingly, Entomocorticium spp. found with D. ponderosae are not consistent, never carried in mycangia, and any association with the beetle is, therefore, most likely facultative and co-evolution is not expected. There were at least four switches after acquisition by D. ponderosae to other beetles, including D. frontalis (Figure 2 node B, ACSR = 95%) and D. brevicomis (Figure 2 node C, ACSR = 94%) and to other beetle genera, i.e., Ips avulsus (Figure 2, node D. ACSR = 88%), and a re-association with Pityoborus ( Figure 2, node E, ACSR = 99%).

Taxonomy
Prior to this work, the genus Entomocorticium was comprised of eight species: E. dendroctoni, E. cobbii, E. kirisitsii, E. parmeteri, E. oberwinkleri, E. whitneyi, E. sullivanii and E. portiae [14,15]. Distinct lineages in Entomocorticium can be recognized using a combination of morphology, distribution, vector-host associations and molecular markers (see Supplementary Table S2 showing inter and intraspecific genetic variation across in Entomocorticium). The topology of our multi-loci phylogenetic analyses revealed distinct fungal lineages associated with distinct beetle vectors and Pinus (Figure 2). We propose five new species of Entomocorticium based on all these traits combined. These new species were isolated from mycangia of D. brevicomis, D. frontalis and Pityoborus comatus inhabiting Pinus ponderosa, P. caribaea, P. taeda and P. elliottii in several USA states and Belize. Several additional lineages were found which are likely to be new taxa but were not described because we were unable to revive live cultures for obtaining morphology and depositing type material. Diagnosis. Fungus associated within Pityoborus comatus mycangium, inhabiting Pinus elliottii. Sterile hyphae exhibit abundant clamp connections throughout the mycelium.
Sexual morph not observed. Asexual morph is composed of sterile mycelium, simple or sparsely branched hyphae that are 2.1-5.8 µm wide, septate, with anastomosing hyphae and abundant clamp connections. Hyphae cylindrical, hyaline, sub-hyaline, forming thin-walled chlamydospore structures averaging 8 × 6 µm. Aleurioconidia not observed. Mycelial mat in culture regular, circular, pale brown becoming darker brown with age, slightly fimbriate, velvety, growing within and on the media. Etymology. Named after Thelma Perry, a pioneering African American female mycology technician responsible for the first description of mycangia in Dendroctonus frontalis and the first to report a basidiomycete from a scolytine mycangium.
Sexual morph not observed. Asexual morph is composed of sterile, simple, or sparsely branched hyphae that are 1.5-5 µm wide and regular or irregularly septate, clamp connections rare. Hyphae cylindrical and uniform, forming thin-walled chlamydospores of 6-11 × 8.2-13.5 µm. Aleurioconidia absent. Cultures floccose to dense and felty, circular, white becoming light grey to brown with age, fimbriate margin, growing within and on the media. Etymology. Named after Thelma Perry, a pioneering African American female mycology technician responsible for the first description of mycangia in Dendroctonus frontalis and the first to report a basidiomycete from a scolytine mycangium.
Sexual morph not observed. Asexual morph is composed of branched hyphae that are 2-6 µm wide and regularly septate, clamp connections present but rare. Hyphae cylindrical, often swollen, monilioid, sparsely forming abundant thin-walled vesicles 13 × 37 µm, commonly bursting when mounted for light microscopy. Aleurioconidia terminal or intercalary within hyphae, apparently produced by the enlargement of single cells, capitate to ovoid, abundant, 5.5-11 × 7-15 µm. Cultures irregular, white to light cream to tan, cottony center with lacunose and viscous margins.

Discussion
In order to understand the evolution of symbiotic relationships, it is important to consider what factors have been involved in the acquisition of new hosts and vectors [39]. Host shifts by microbial symbionts are often associated with species diversification driven by the exploitation of new adaptive zones [40]. In the case of Entomocorticium and bark beetles, our results indicate a considerable diversity of fungal lineages within Entomocorticium with each species consistently associated with a particular taxon of bark beetles and their host pines.
Our phylogenetic results agree with the previously published phylogeny of Entomocorticium [15,16]. However, our study aimed to be more inclusive and provide further clarification regarding the evolutionary pathways that might have facilitated the origin of the genus Entomocorticium and promoted its further speciation. We propose a hypothesis of an evolutionary transition from a strictly wood-decaying, wind-dispersed fungal lineage (Peniophora) to a beetle-associated lineage engaged in highly selective vertical transmission through mycangia (Entomocorticium). We also provide new hypotheses on how beetle species involved in these symbiotic relationships likely played a crucial role in promoting diversity within this fungal group.
Our findings support Entomocorticium as a monophyletic fungal lineage that exhibits common morphological, molecular and ecological traits. Therefore, we are convinced that Entomocorticium should be treated as a separate genus from Peniophora, although that renders Peniophora a polyphyletic group. We hope that this study encourages further efforts to elucidate the relationships within Peniophora, which would ultimately result in a new taxonomic arrangement for the genus.

How Did Such Relationships Arise?
Our results indicate that most species within Peniophora, the genus from which Entomocorticium is derived, are broadly associated with angiosperms with at least five transitions to gymnosperms, particularly Pinus (Figure 1). Following one of these transitions (Figure 1 node A), the ancestor of Entomocorticium (related to Peniophora pithya) encountered bark beetles and transitioned to dissemination via beetle vectors. Given that Peniophora is a group of wood-rotting fungi that colonize and degrade dead wood, initial encounters between a member(s) of this group and bark beetles likely occurred in recently killed or moribund tree tissues. While Entomocorticum is likely undersampled in our analysis, our results indicate that twig beetles that exploit moribund phloem on shaded-out pine twigs (e.g., Pityoborus) were among the earliest vectors of these fungi ( Figure 2).
The subsequent switches to new beetle vectors were likely facilitated by co-colonization of pine phloem by multiple species of bark beetles, resulting in exposure of the fungus to a diverse vector pool. Co-colonization of trees, i.e., niche overlap, is common in bark beetles and can result in exposure to a diverse pool of potential symbionts [41]. Shifts to new hosts may have driven both symbiont and beetle diversification in at least some cases by allowing the exploitation of new adaptive zones. Host-shift events driven by niche overlap are relatively common in fungi, especially within Hypocreales [39,[42][43][44][45]. In the case of Entomocorticium and bark beetles, our results indicate a considerable diversity of lineages of these fungi, with each species consistently associated with a particular taxon of scolytine beetles in Pinus.

Distinct Associations across Bark Beetles and Entomocorticium
Not all symbioses between Entomocorticium and bark beetles are the same. There is a range of dependencies varying from loose and facultative (e.g., D. ponderosae) to obligate (e.g., D. frontalis and D. brevicomis) [46]. Likewise, the effects of the fungi on beetle fitness are not clear. For example, several species of Entomocorticium have been isolated from the pupal chambers of D. ponderosae and these have been suggested to be nutritional mutualists [15,16]. However, these fungal species have sporadic distributions with D. ponderosae [47,48] and have never been isolated from their mycangia [8,49], despite numerous isolations from beetles collected in many locations. Additionally, these fungi have been only rarely isolated from the beetle's exoskeleton, suggesting the beetle may be an inefficient vector and the beneficial aspects of this symbiosis to the beetle, if any, is unreliable.
In contrast, D. frontalis and D. brevicomis are obligately associated with Entomocorticum species and these fungi provide crucial nutrients for the development of beetle larvae. The association of D. brevicomis with Entomocorticium is ancient and highly coevolved with the fungi co-speciating along with the host beetle in response to a period of isolation during glaciation [49]. Vertical transmission via highly selective mycangia enforces fidelity and reduces the potential for invasion by new lineages [46,[50][51][52][53][54][55].
Regarding Pityoborus comatus and Ips avulsus, both species have been studied much less than Dendroctonus, but observational evidence of larval development suggests that they are completely mycophagous, at least in the larval stage. Some Ips species appear to be dependent on Ophiostoma species for nutrition [53] and this may also be the case for those that associate with Entomocorticium. However, most aspects of this association have not been investigated, especially regarding mycangia or other structures that facilitate fungal dissemination and little is known about specificity and nutritional effects.

Distinct Functional Traits in Basidiomycota and Ascomycota Associated with Bark Beetles
The association of Entomocorticium (Basidiomycota) with conifer-colonizing bark beetles is clearly limited compared to conifer-colonizing bark beetles occurring with Ophiostomatales (Ascomycota), which are ubiquitous worldwide [15]. For many bark beetles, Ophiostomatales fungal symbionts are facultative or obligate nutritional mutualists. The necrotrophic nature of many of these fungi allows them to survive and grow in a dying tree host during the early colonization phase of a tree and then to exploit dead tree tissues over the longer period of larval and fungal mycelial development [5]. Ophiostomatales do not degrade cellulose and lignin, which limits them to foraging for amino acids and simple carbohydrates [53].
On the other hand, the Basidiomycota symbiont species, such as those in the genera Entomocorticium and Peniophora, can actively decay the structural components of wood. Both genera are saprobic and do not invade living tissues, as demonstrated for the Entomocorticium species associated with D. brevicomis [53]. While they also consume amino acids and simple carbohydrates for energy and growth, they use these resources to support the degradation of cellulose and lignin, resulting in greater access to resources within the tree. These different qualities between the Ascomycota and Basidiomycota associates of bark beetles are not trivial and are critical to understanding the development and maintenance of such novel symbioses within bark beetles as a whole. Differences in growth within trees and the ability to access and acquire nutrients indicate different pathways to exploit wood as a niche and potentially to reduce niche overlap and competition [53].

Domestication of Entomocorticium by Beetles Facilitated the Loss of Morphological Traits
The transition from free-living (Peniophora) to beetle-associated (Entomocorticium) coincided with a transition to moribund phloem: a resource that presents benefits, as well as costs. Tree parts, such as moribund phloem are relatively free of competition and are more nutritious than dead wood or woody debris. However, moribund phloem is still alive and chemically defended and is also spatially patchy and intermittently available. Therefore, exploitation of such a resource is greatly facilitated by association with an agile insect vector. The optimal resource for the vector and the fungus are hence similar.
The overall loss of morphological complexity from Peniophora to Entomocorticium species is consistent with the loss of morphological features in other beetle-associated fungi [54]. Likewise, a reduction in sexual reproduction is consistent with predictions for microbes involved in mutualisms [51][52][53]. Peniophora are corticoid fungi that reproduce sexually and exhibit a broad diversity of basidiome morphologies (e.g., resupinate, effused, membranaceous, ceraceous, etc.), colors (e.g., reddish, orange, pink, violaceous, greyish, yellow, lilac, etc.) and colonize wood of a broad variety of plant hosts [56,57]. In contrast, Entomocorticium are restricted to beetle-colonized Pinus and only form simple whitish mycelial mats, often supporting the production of large numbers of asexual spores (chlamydospores, aleurioconidia) and with sexual spores (basidiospores) formed only rarely or not at all [14][15][16]. Basidia, when they do form, have been described as lacking Buller's drops reflecting their production inside the tree with no potential for wind dispersal. However, as with other putative asexual mutualists, evidence of rare recombination events can be found, potentially maintained to reduce the effects of Muller's ratchet predicted for fully asexual species [58,59].
Bark beetles are tremendously important evolutionarily, ecologically, and economically, and their complex relationships with trees and fungi are beginning to be better understood [60,61]. The descriptions of new species we provide as well as their relationships are noteworthy. They expand upon recent descriptions from Harrington et al. [15], indicating greater complexity and diversity of fungal associates of Dendroctonus and other bark beetle species. This work also furthers understanding of the players in this group of model organisms for the study of symbiosis.

Conclusions
The genus Entomocorticium provides an interesting insight into the origins of insect microbial mutualisms. This lineage of Basidiomycota has arisen quite successfully from a wood-decaying ancestor (Peniophora) within a matrix of pre-existing symbioses between several lineages of Ascomycota fungi and their beetle vectors [5]. Targeted sampling for Entomocorticium across a variety of bark beetles with various tree colonization strategies, careful investigation of fungal vectoring capacity and specialized structures of beetles, and studies on the effects of the fungi on beetle fitness via nutrient provisioning should be a focus of future investigations into beetle-fungus symbioses. This is particularly true for Entomocorticium associated with P. comatus, a beetle which has not yet been found to associate with fungi in Ophiostomatales (Ascomycota), and also with D. ponderosae, a beetle which has not yet been shown to harbor Entomocorticium symbionts within the mycangia, only from its galleries. Genetic and morphological descriptions of the fungi can provide additional information on symbiosis type and strength, as well as provide a better understanding of the functional morphology of these fungal lineages and how they evolved. Furthermore, the diversity of fungi with bark beetles in Pinus in Mexico and Central America, which are almost completely unsampled, should be specially targeted. These regions exhibit amazing diversity of pines and bark beetles, and most likely fungal symbionts as well. For example, Mexico alone has 43 species of Pinus with a myriad of unknown beetle-fungus associations [62] and these diverse pine forests most likely harbor the largest reservoirs of these intriguing, fascinating and ecologically important fungi.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7121043/s1, Figure S1. Maximum likelihood tree showing Russulales clade obtained from RAxML analyses with a concatenated dataset of 2-loci (LSU and ITS). Table S1. Species used in the Russulales analyses and their GenBank accession numbers.