Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora
1
School of Forest, Fisheries and Geomatics Sciences, University of Florida, Gainesville, FL 32611, USA
2
Institute of Systematic Botany, The New York Botanical Garden, Bronx, NY 10458, USA
3
Department of Ecosystem and Conservation Sciences, The University of Montana, Missoula, MT 59812, USA
4
National Scientific and Technical Research Council (CONICET), Buenos Aires C1425FQB, Argentina
5
Patagonian Andes Forest Research and Extension Centre (CIEFAP), Esquel 9200, Argentina
6
Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA
7
Joseph W. Jones Ecological Research Center, Odum School of Ecology, University of Georgia, Newton, GA 30602, USA
8
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, The Netherlands
9
Section for Ecology and Evolution, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark
10
Biology Department, The College of William and Mary, Williamsburg, VA 23185, USA
*
Author to whom correspondence should be addressed.
J. Fungi 2021, 7(12), 1043; https://doi.org/10.3390/jof7121043
Received: 21 October 2021
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Revised: 30 November 2021
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Accepted: 3 December 2021
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Published: 6 December 2021
(This article belongs to the Special Issue Dimensions of Tropical Fungal Diversity)
Abstract
: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.
1. 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 insects are true fungus farmers, i.e., they inoculate their fungal symbionts into the substrate and cultivate them to feed their progeny, and therefore, the fungal symbionts have become domesticated crops [4,5]. Many of these insect-cultivated fungi have evolved nutrient-provisioning adaptations and have become dependent on their partner insects for dispersal [4]. Many of these insects, including beetles, have evolved specific, highly evolved organs (mycangia) to maintain and transport their symbiotic fungi [6,7,8]. Unfortunately, fungal mutualists have thus far been studied in fewer than 5% of all bark beetles [5].
The most well-known fungi found living mutualistically with Scolytinae beetles are species within Ophiostomatales (Ascomycota). However, because these are often targeted in surveys of bark beetle fungi, a dearth of knowledge exists on other potential fungal mutualists. Surprisingly, this even includes important pest species. For example, the southern pine beetle (SPB), Dendroctonus frontalis (Curculionidae, Scolytinae, Hylurgini) is the most economically important pest in pine plantations across the Southern USA and Central America [9]. The biology, ecology and management of this beetle have been extensively investigated [9,10,11]. The southern pine beetle has also served as a model system for understanding symbiotic interactions between beetles and fungi [12,13]. Despite these previous investigations of symbiotic associations with D. frontalis, the diversity and evolutionary history of its most beneficial fungal associate—an Entomocorticium species (Russulaceae, Russulales, Basidiomycota)—remains obscure.
The genus Entomocorticium is currently comprised of eight species, all associated with Scolytinae beetles [14,15]. Entomocorticium dendroctoni, the type species of Entomocorticium [14], was described based exclusively on morphological features to accommodate a cryptic fungus that was observed growing intermingled with a blue stain fungus [14]. According to the original description, the fungus produced abundant sessile basidiospores in the galleries and pupal chambers of the mountain pine bark beetle D. ponderosae (mountain pine beetle) in Pinus contorta (lodgepole pine) [14].
The nutritional symbioses with Entomocorticium are only known for a few beetle species thus far [15,16]. Some of these beetles use mycangia to carry their Entomocorticium partners from tree to tree. Within the genus Dendroctonus (Curculionidae, Scolytinae, Hylurgini), some of the main documented vectors of Entomocorticium fungi, there are two clades of beetles with independent origins of mycangia [17,18]. One clade contains D. ponderosae and D. jeffreyi, which possess maxillary mycangia (located in the maxillae, the segmented mouthparts) wherein they carry obligate Ophiostomatales mutualist fungi [19]. These beetles appear to have loose associations with multiple Entomocorticum species that have not previously been found to be transported in mycangia. They are occasionally found in the beetle’s pupal chambers but how the fungi are disseminated is unknown [20]. The second clade of beetles, which includes D. frontalis, D. brevicomis, and several other species, all possess prothoracic mycangia (a tube in the inner wall of the pronotum) [21,22]. Some of these bark beetles carry Entomocorticium in their mycangia and for at least two, D. frontalis and D. brevicomis, the fungi are obligate nutritional mutualists. However, the fungal symbionts are unknown for many of these beetles.
Two other genera of pine beetles, only distantly related to Dendroctonus, are also known to be associated with Entomocorticium. A twig beetle, Pityoborus comatus (Curculionidae, Scolytinae, Corthylini), carries an Entomocorticium sp. in large, pubescent impressions on the sides of its prothorax that function as a mycangium [23]. Ips avulsus (Curculionidae, Scolytinae, Ipini) is also commonly found with an Entomocorticium species but how it is disseminated is not known and the presence of mycangia or other spore carrying-structures have not been investigated for this beetle [24].
Remarkably, the diversity of this important group of fungi remained uncharacterized until the early 2000s. Hsiau & Harrington (2003) were the first to show that Entomocorticium was a diverse fungal lineage associated solely with a group of phloem-inhabiting bark beetles that feed heavily on fungi. In addition to E. dendroctoni, they identified nine putative species based on their mt-SSU, ITS and IGS-1 analyses. Hsiau & Harrington [16] also suggested that the Entomocorticium clade was relatively young, likely having been recently 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.
2. Material and Methods
2.1. 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 surface-washed 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.
2.2. 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.
2.3. 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.
2.4. DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from fungal cultures of the new Entomocorticium isolates grown on PDA using the Extract-N-Amp Plant PCR kit (Sigma-Aldrich, St. Louis, MO, USA) with the modification of using 3% bovine serum albumin (BSA) as a replacement for a dilution solution. Primer combinations used for PCR amplifications were: (1) LR0R/LR5 [31] for nuclear large subunit (28S rDNA) ribosomal DNA; (2) NS1/NS4 [32] for nuclear small subunit (18S rDNA) ribosomal DNA (rDNA); (3) 983F/2218R for Translation elongation factor 1-α (TEF); (4) ITS1/ITS4 for the Internal Transcribed Spacer rDNA (ITS1-5.8S-ITS2, hereafter referred to as ITS) [33] and (5) IGS (P1/5SRNA) (Hsiau & Harrington 2003). The sequencing was performed at Eurofins. As a quality control procedure, we inspected electropherograms of each sequence individually and performed de novo assembling in Geneious v. 11.1.5 [34].
2.5. 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.
2.6. 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.
2.7. Post-Analyses Graphical Display
3. 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.
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.
Etymology. The species epithet is derived from fibula (L. adj. f., with clamp) and refers to the abundant presence of clamp connections throughout the mycelium.
Typus. USA, Miami-Dade-FL, from Pityoborus comatus mycangium, 15 July 2015, J. Skelton, Y. Li & J. Hulcr (holotype FLAS-F-68307 (dried culture), ex-type CBS 148418 (live culture)).
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.
Vector–Pityoborus comatus (Coleoptera, Curculionidae), Voucher UFFE: 28951.
Host–Pinus elliottii (Pinales, Pinaceae)
Distribution–Only recorded from Miami-Dade, FL (USA).
Entomocorticium perryae Araújo, Li, Six & Hulcr, sp. nov.–MycoBank MB 839834; Figure 4.
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.
Typus. USA, Tropic-UT, from Dendroctonus brevicomis mycangium, 5 July 2015, D. Six (holotype FLAS-F-68308, ex-type CBS 148419).
Diagnosis. The fungus associated with Dendroctonus brevicomis inhabiting Pinus ponderosa. Chlamydospores av. 6–11 × 8–13 µm.
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.
Vector–Dendroctonus brevicomis (Coleoptera, Curculionidae)
Host–Pinus ponderosa (Pinales, Pinaceae)
Distribution–Only recorded from Tropic, UT (USA).
Additional specimen examined: USA, Gainesville-FL, from Dendroctonus frontalis mycangium, 15 July 2019, J. Skelton, (FLAS-F-68306, CBS 148417 (live culture)) (as E. cf. perryae 17783): Fungus associated within Dendroctonus frontalis mycangium, inhabiting Pinus taeda. Sterile hyphae exhibit swollen hyphae, morphologically resembling those of ambrosial fungi by its clavate to globose cells that are usually irregular in size. Asexual morph composed of sterile, simple or branched, irregularly swollen, irregularly swollen hyphae, av. 2–5 µm width, regularly septate, clamp connections present but rare, chlamydospores absent. Aleurioconidia absent. Mycelial mat homogeneous, circular, light brown becoming darker with age, effuse, aerial hyphae scarce, with hyphae growing within the media.
Vector. Dendroctonus frontalis (Coleoptera, Curculionidae), Voucher UFFE:29184.
Host. Pinus taeda (Pinales, Pinaceae).
Distribution. Only recorded from Gainesville, FL (USA).
Note: Although we suspect that Entomocorticium cf. perryae (17783–Figure 2) is a distinct species, based on the host and vector association, we decided to take a conservative approach and include it within E. perryae in this study due to the very high genetic similarity (see Supplementary Table S2) and lack of morphological features. Future studies including more E. perryae specimens will elucidate this question.
Entomocorticium belizense Araújo, Li & Hulcr, sp. nov.–MycoBank MB 839835; Figure 5.
Etymology. Named after the place of origin where it was collected, Belize.
Typus. Belize, Mountain Pine Ridge, from Dendroctonus frontalis mycangium, 21 January 2019, J. Skelton, Y. Li & J. Hulcr (holotype FLAS-F-68309 (dried culture), ex-type CBS 148420 (live culture)).
Diagnosis. The fungus associated within Dendroctonus frontalis mycangium, inhabiting Pinus caribaea, exhibits characteristic papillate aleurioconidia.
Sexual morph not observed. Asexual morph composed of simple or sparsely branched hyphae that are 1.5–4 µm wide and irregularly septate, clamp connections not observed. Hyphae cylindrical and uniform, sparsely forming thin-walled chlamydospores av. 12 × 5 µm. Aleurioconidia is produced at the tips of some hyphae, thick-walled, spherical to ovoid, commonly papillate, 6.5–9 × 8–17 µm. Cultures irregular, light cream to tan, center cottony with scarce hyphae and adpressed edges.
Vector. Dendroctonus frontalis (Coleoptera, Curculionidae). Voucher UFFE:30866, GenBank accession number: OL631193.
Host. Pinus caribaea (Pinales, Pinaceae).
Distribution. Only recorded from Belize.
Additional specimens examined: Belize, Mountain Pine Ridge, from Dendroctonus frontalis (Voucher UFFE:30867) mycangium, 21 January 2019, J. Skelton, Y. Li & J. Hulcr (18051).
Entomocorticium macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov.–MycoBank MB 839837; Figure 6.
Etymology. The name refers to the large vesicles commonly seen in this species.
Typus. USA, McCloud-CA, from Dendroctonus brevicomis mycangium, July 2014, D. Six & R. Bracewell (holotype FLAS-F-68310 (dried culture), ex-type CBS 148421 (live culture)).
Diagnosis. The fungus associated within Dendroctonus brevicomis mycangium, inhabiting Pinus ponderosa, exhibiting abundant large vesicles.
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.
Vector. Dendroctonus brevicomis (Coleoptera, Curculionidae)
Host. Pinus ponderosa (Pinales, Pinaceae)
Distribution. Recorded from several sites across the Western USA: Chiloquim (OR), Greenough (MT), McCloud (CA), Missoula (MT), Placerville (CA), Ruisoso (NM) and San Bernardino Mountains (CA).
Additional specimens examined: USA, Missoula-MT, from Dendroctonus brevicomis mycangium, 17 January 2019, D. Six & R. Bracewell (MI17); USA, Placerville-CA, from Dendroctonus brevicomis mycangium, 20 February 2019, D. Six & R. Bracewell (PL6). USA, Ruisoso-NM, from Dendroctonus brevicomis mycangium, 10 January 2019, D. Six & R. Bracewell (RO10).
4. 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.
4.1. 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.
4.2. 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.
4.3. 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].
4.4. 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.
5. 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. Table S2. Heatmap showing the genetic similarities within Entomocorticium species.
Author Contributions
Conceptualization: J.P.M.A., J.H.; Methodology: J.P.M.A., Y.L., S.N.A.; Formal analysis: J.P.M.A.; Investigation: J.P.M.A., Y.L., D.S., K.D.K., J.H.; Data curation: J.P.M.A., Y.L., D.S., A.J.J., P.W.C., C.A.L.-D., S.N.A., J.H. and J.S.; writing-original draft preparation: J.P.M.A., D.S., M.R., M.E.S., A.J.J., K.D.K., J.H.; Supervision: D.S., M.R., K.D.K., J.H.; Funding acquisition: K.D.K., J.H. All authors have read and agrees to be published version of the manuscript.
Funding
This research was funded through an agreement with The Jones Center at Ichauway and NSF project 6046-201-2200-G000170.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data presented in this study is available at Treebase at http://purl.org/phylo/treebase/phylows/study/TB2:S29025.
Acknowledgments
This project was supported by the National Science Foundation, the Jones Center at Ichauway and the USDA Forest Services. We also would like to thank our collaborators in Belize for the support along this work, the Belize Forest Department, The Friends of Conservation and Development–Succotz–Belize, and Bull Ridge Ltd.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Maximum likelihood tree showing Peniophora/Entomocorticium clade obtained from RAxML analyses with a concatenated dataset of 5-loci (SSU, LSU, TEF, ITS and IGS). Ancestral 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.
Figure 1.
Maximum likelihood tree showing Peniophora/Entomocorticium clade obtained from RAxML analyses with a concatenated dataset of 5-loci (SSU, LSU, TEF, ITS and IGS). Ancestral 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.
Figure 2.
Close-up of the Entomocorticium clade showed in Figure 1. Character-state reconstruction of the association of Entomocorticium spp. with beetle vectors. Branch and boxes color mean: Yellow = Pityoborus comatus; Red = Dendroctonus frontalis; Blue = D. brevicomis; Green = D. ponderosae; Purple = Ips avulsus. Node A indicates transition from Pityoborus comatus to Dendroctonus ponderosae, node B from D. ponderosa to D. frontalis, node C from D. ponderosae to D. brevicomis, node D from D. ponderosae to Ips avulsus and node E from D. ponderosae back to P. comatus. Scale bar is in relation to the beetle sizes = 5 mm. Beetle photos by Andrew Johnson.
Figure 2.
Close-up of the Entomocorticium clade showed in Figure 1. Character-state reconstruction of the association of Entomocorticium spp. with beetle vectors. Branch and boxes color mean: Yellow = Pityoborus comatus; Red = Dendroctonus frontalis; Blue = D. brevicomis; Green = D. ponderosae; Purple = Ips avulsus. Node A indicates transition from Pityoborus comatus to Dendroctonus ponderosae, node B from D. ponderosa to D. frontalis, node C from D. ponderosae to D. brevicomis, node D from D. ponderosae to Ips avulsus and node E from D. ponderosae back to P. comatus. Scale bar is in relation to the beetle sizes = 5 mm. Beetle photos by Andrew Johnson.
Figure 3.
Entomocorticium fibulatum. (a) Culture aspect on PDA plate; (b) The beetle vector Pityoborus comatus; (c,d) Clamp connections; (e) Early stage of chlamydospores formed by a clamp connection (arrows); (f) Clamp connections; (g) Hyphae anastomosing (arrow); (h) Hypha exhibiting regular clamp connections. Scale bars: (a) = 2 cm; (b) = 0.5 cm; (c,d) = 4 µm; (e–h) = 5 µm.
Figure 3.
Entomocorticium fibulatum. (a) Culture aspect on PDA plate; (b) The beetle vector Pityoborus comatus; (c,d) Clamp connections; (e) Early stage of chlamydospores formed by a clamp connection (arrows); (f) Clamp connections; (g) Hyphae anastomosing (arrow); (h) Hypha exhibiting regular clamp connections. Scale bars: (a) = 2 cm; (b) = 0.5 cm; (c,d) = 4 µm; (e–h) = 5 µm.
Figure 4.
Entomocorticium perryae. (a) Culture aspect on PDA plate; (b) Chlamydospore in formation; (c) Dendroctonus brevicomis (vector); (d) Apical hyphae; (e) Chlamydospore. Scale bars: (a) = 1 cm; (b) = 5 µm; (c) = 2 mm; (d,e) = 5 µm.
Figure 4.
Entomocorticium perryae. (a) Culture aspect on PDA plate; (b) Chlamydospore in formation; (c) Dendroctonus brevicomis (vector); (d) Apical hyphae; (e) Chlamydospore. Scale bars: (a) = 1 cm; (b) = 5 µm; (c) = 2 mm; (d,e) = 5 µm.
Figure 5.
Entomocorticium belizense. (a) Culture aspect on PDA plate; (b) Dendroctonus frontalis; (c–f) Early stages of aleurioconidia; (g) Fully developed aleurioconidia. Scale bars: (a) = 1 cm; (b) = 0.5 cm; (c–g) = 5 µm.
Figure 5.
Entomocorticium belizense. (a) Culture aspect on PDA plate; (b) Dendroctonus frontalis; (c–f) Early stages of aleurioconidia; (g) Fully developed aleurioconidia. Scale bars: (a) = 1 cm; (b) = 0.5 cm; (c–g) = 5 µm.
Figure 6.
Entomocorticium macrovesiculatum. (a) Culture aspect, including two pieces of pine wood. Black line on the underside of the Petri dish indicates culture diameter on 22 December 2020, photo taken on 21 January 2021; (b,c) Chlamydospores; (d) Dendroctonus brevicomis, the vector; (e,f) Vegetative hyphae and chlamydospore-like/vesicles. Scale bars: (a) = 1 cm; (b) = 30 µm; (c) = 20 µm; (d) = 2 mm; (e) = 15 µm; (f) = 30 µm.
Figure 6.
Entomocorticium macrovesiculatum. (a) Culture aspect, including two pieces of pine wood. Black line on the underside of the Petri dish indicates culture diameter on 22 December 2020, photo taken on 21 January 2021; (b,c) Chlamydospores; (d) Dendroctonus brevicomis, the vector; (e,f) Vegetative hyphae and chlamydospore-like/vesicles. Scale bars: (a) = 1 cm; (b) = 30 µm; (c) = 20 µm; (d) = 2 mm; (e) = 15 µm; (f) = 30 µm.
Table 1.
List of isolates obtained in this study and other Entomocorticium isolates included in the analyses.
Table 1.
List of isolates obtained in this study and other Entomocorticium isolates included in the analyses.
| Species | Voucher (Extype) | Beetle Vector | Tree Host | Isolate Origin | Material Source | Reference |
|---|---|---|---|---|---|---|
| Entomocorticium belizense | 18050 (CBS 148421) | Dendroctonus frontalis | Pinus caribaea | Belize | Mycangium | This study |
| 18051 | Dendroctonus frontalis | Pinus caribaea | Belize | Mycangium | This study | |
| Entomocorticium cobbii | B720 | Dendroctonus frontalis | Pinus taeda | Rapides Parish, LO, USA | Mycangium | Harrington et al. (2021) |
| Entomocorticium dendroctoni | DAVFP 23165 | Dendroctonus ponderosae | Pinus ponderosa | British Columbia, Canada | Pupal Chamber | Whitney et al. (1987) |
| Entomocorticium fibulatum | 17762 (CBS 148418) | Pityoborus comatus | Pinus elliotii | Miami-Dade, FL, USA | Mycangium | This study |
| Entomocorticium perryae | UT16 (CBS 148419) | Dendroctonus brevicomis | Pinus ponderosa | Tropic, UT, USA | Mycangium | This study |
| Entomocorticium kirisitsii | B1065 | Dendroctonus ponderosae | Pinus ponderosa | Estes Park, CO, USA | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium macrovesiculatum | PL6 | Dendroctonus brevicomis | Pinus ponderosa | Placerville, CA, USA | Mycangium | This study |
| LF21 | Dendroctonus brevicomis | Pinus ponderosa | Greenough, MT, USA | Mycangium | This study, Bracewell and Six (2014) | |
| CQ11 | Dendroctonus brevicomis | Pinus ponderosa | Chiloquim, OR, USA | Mycangium | This study, Bracewell and Six (2014) | |
| MI17 | Dendroctonus brevicomis | Pinus ponderosa | Missoula, MT, USA | Mycangium | This study, Bracewell and Six (2014) | |
| Ro10 | Dendroctonus brevicomis | Pinus ponderosa | Ruisoso, NM, USA | Mycangium | This study, Bracewell and Six (2014) | |
| SB13 | Dendroctonus brevicomis | Pinus ponderosa | San Bernardino Mtns, CA, USA | Mycangium | This study, Bracewell and Six (2014) | |
| MC16 (CBS 148421) | Dendroctonus brevicomis | Pinus ponderosa | McCloud, CA, USA | Mycangium | This study, Bracewell and Six (2014) | |
| Entomocorticium oberwinkleri | B1053 | Dendroctonus ponderosae | Pinus contorta | Pilot Springs, CA, USA | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium parmeteri | B1503 | Dendroctonus brevicomis | Pinus ponderosa | Tuolumme County, CA, USA | Gallery | Harrington et al. (2021) |
| Entomocorticium cf. perryae | 17783 (CBS 148417) | Dendroctonus frontalis | Pinus taeda | Gainesville, FL, USA | Mycangium | This study |
| Entomocorticium portiae | B1039 | Dendroctonus ponderosae | Pinus lambertiana | Blodgett Res. Forest, CA, USA | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium portiae | B1060 | Dendroctonus ponderosae | Pinus contorta | San Bernardino Mts., CA, USA | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium sp. | MMF-4485 | Pitioborus comatus | Pinus ponderosa | Florida | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium sp. | 9470 | Pityoborus comatus | unknown | Gainesville, FL, USA | Mycangium | This study |
| Entomocorticium sp. | 9576 | Pityoborus comatus | Pinus taeda | Gainesville, FL, USA | Mycangium | This study |
| Entomocorticium sullivanii | B1252 | Ips avulsus | Pinus taeda | Athens, GA, USA | Pupal Chamber | Harrington et al. (2021) |
| Entomocorticium whitneyi | B1069 | Dendroctonus ponderosae | Pinus ponderosa | Estes Park, CO, USA | Pupal Chamber | Harrington et al. (2021) |
Table 2.
Species used in the Russulales analyses and their GenBank accession numbers.
| Species | Host | Voucher | SSU | ITS | LSU | TEF | Citation |
|---|---|---|---|---|---|---|---|
| Entomocorticium belizense | Pinus caribaea | 18050 | – | MZ098132 | MZ098117 | – | This study |
| 18051 | – | MZ098133 | MZ098116 | – | This study | ||
| Entomocorticium cobbii | Pinus taeda | B720 | – | MT741707 | MT741692 | – | Harrington et al. (2021) |
| Entomocorticium fibulatum | Pinus elliottii | 17762 | MZ098147 | MZ098135 | MZ098120 | – | This study |
| Entomocorticium perryae | Pinus ponderosa | UT16 | MZ098145 | MZ098123 | MZ098118 | MZ144591 | This study, Bracewell and Six (2014) |
| Entomocorticium kirisitsii | Pinus ponderosa | B1065 | – | MT741714 | MT741699 | – | Harrington et al. (2021) |
| Entomocorticium macrovesiculatum | Pinus ponderosa | MI17 | MZ098143 | MZ098129 | – | MZ144589 | This study, Bracewell and Six (2014) |
| RO10 | MZ098149 | MZ098130 | MZ098108 | MZ144590 | This study, Bracewell and Six (2014) | ||
| SB13 | MZ098141 | MZ098125 | MZ098110 | MZ144586 | This study, Bracewell and Six (2014) | ||
| B1037 | MZ098138 | MZ098124 | MZ098109 | MZ144585 | This study, Bracewell and Six (2014) | ||
| LF21 | MZ098139 | – | MZ098113 | MZ144587 | This study, Bracewell and Six (2014) | ||
| PL6 | MZ098140 | MZ098126 | MZ098114 | MZ144588 | This study | ||
| MC16 | MZ098144 | MZ098128 | MZ098112 | – | This study, Bracewell and Six (2014) | ||
| CQ11 | MZ098142 | MZ098127 | MZ098111 | – | This study, Bracewell and Six (2014) | ||
| Entomocorticium oberwinkleri | Pinus contorta | B1053 | – | MT741712 | MT741697 | – | Harrington et al. (2021) |
| Entomocorticium parmeteri | Pinus ponderosa | B1503 | – | MT741709 | MT741694 | – | Harrington et al. (2021) |
| Entomocorticium cf. perryae | Pinus taeda | 17783 | MZ098146 | MZ098131 | MZ098115 | MZ144592 | This study |
| Entomocorticium portiae | Pinus lambertiana | B1039 | – | MT741710 | MT741695 | – | Harrington et al. (2021) |
| Pinus contorta | B1045 | – | MT741711 | MT741696 | – | Harrington et al. (2021) | |
| Entomocorticium sp. | Pinus taeda | 9576 | MZ098148 | MZ098134 | – | MZ144593 | This study |
| Entomocorticium sp. | Pinus ponderosa | TSpCB896 | – | AF119510 | – | – | Harrington et al. (2021) |
| Entomocorticium sullivanii | Pinus taeda | CBS 146270 | – | MT741715 | MT741700 | – | Harrington et al. (2021) |
| Entomocorticum dendroctoni | Pinus contorta | DAVFP 23165 | – | AF119506 | – | – | Hsiau & Harrington (2003) |
| Entomocorticum whitneyi | Pinus ponderosa | B1069 | – | MT741713 | MT741698 | – | Harrington et al. (2021) |
| Peniophora albobadia | Angiosperms | CBS 329.66 | – | MH858809 | MH870448 | – | Andreasen & Hellenberg (2009) |
| Peniophora aurantiaca | Alnus (Betulaceae) | – | – | Boidin (1994) | |||
| Peniophora bicornis | Pentaclethra (Fabaceae), Musanga (Urticaceae), Anthocleista (Gentianaceae), Casuarina (Casuarinaceae), Acacia (Fabaceae), Acanthophoenyx (Areceae) | He4767 | – | MK588764 | MK588804 | – | Boidin et al. (1991) |
| He3609 | – | MK588763 | MK588803 | – | Boidin et al. (1991) | ||
| Peniophora borbonica | Hypericum (Hypericaceae), Acacia (Fabaceae), Fuchsia (Onagraceae) | He4597 | – | MK588766 | MK588806 | – | Boidin et al. (1991) |
| He4606 | – | MK588765 | MK588805 | – | Boidin et al. (1991) | ||
| Peniophora cinerea | “Angiosperms and Gymnosperms” | B1020 | – | MN475151 | MN475818 | – | Andreasen & Hellenberg (2009) |
| Peniophora crassitunicata | Morinda (Rubiaceae), Schinus (Anacardiaceae), Casuarina (Casuarinaceae), Lantana (Verbenaceae), Tylophora (Apocynaceae), Acanthophoenyx (Arecaceae), Scaevola (Goodeniaceae) | CBS 663.91 | – | MH862292 | MH873972 | – | Boidin et al. (1991) |
| Peniophora duplex | Gymnosperm “similar to P. pini/pseudo-pini” | CBS 286.58 | – | MH857787 | MH869321 | – | Andreasen & Hellenberg (2009) |
| B1022 | – | MN475153 | MN475820 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora eriksonii | Alnus glutinosa (Betulaceae) | CBS 287.58 | – | MH857788 | MH869322 | – | Boidin (1994) |
| Cui11871 | – | MK588771 | MK588811 | – | Boidin (1994) | ||
| Peniophora exima | Abies (Pinaceae) | B1012 | – | MN475159 | MN475826 | – | Boidin (1994) |
| B1011 | – | MN475155 | MN475821 | – | Boidin (1994) | ||
| T523 | – | MK588772 | MK588812 | – | Boidin (1994) | ||
| Peniophora fasticata | Angiosperms | CBS 942.96 | – | MH862624 | – | – | Andreasen & Hellenberg (2009) |
| Peniophora fissilis | Cryptomeria (Cupressaceae), Lantana (Verbenaceae) | CBS 681.91 | MZ233430 | MH862298 | MH873975 | – | Boidin et al. (1991) |
| CBS 684.91 | MZ233431 | MH862299 | MH873976 | – | Boidin et al. (1991) | ||
| Peniophora gabonensis | Pandanus (Pandanaceae) | CBS 673.91 | – | MH862293 | – | – | Andreasen & Hellenberg (2009) |
| Peniophora gilbertsonii | Prosopis juriflora (Fabaceae), Baccharis (Asteraceae), Cercidium (Fabaceae), Condalia (Rhamnaceae), Fouquieria (Fouquieraceae) | CBS 357.95 | – | MH862528 | MH874164 | – | Boidin et al. (1991) |
| CBS 360.95 | – | MH862530 | MH874165 | – | Boidin et al. (1991) | ||
| Peniophora guadelupensis | Leguminosae | CBS 715.91 | – | MH862304 | MH873977 | – | Andreasen & Hellenberg (2009) |
| Peniophora halimi | Atriplex (Amaranthaceae) | CBS 862.84 | – | MH861843 | MH873531 | – | Andreasen & Hellenberg (2009) |
| CBS 860.84 | – | MH861842 | MH873530 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora incarnata | On angiosperms, rarely on Gymnosperms | B1016 | – | MN475156 | MN475822 | – | Andreasen & Hellenberg (2009) |
| CBS 430.72 | – | MH860518 | MH872230 | – | Andreasen & Hellenberg (2009) | ||
| AF506425 | – | AF506425 | – | – | Andreasen & Hellenberg (2009) | ||
| NH10271 | – | AF506425 | – | – | Andreasen & Hellenberg (2009) | ||
| Peniophora junipericola | Juniperus | He2462 | – | MK588773 | MK588813 | – | Boidin (1994) |
| Peniophora laeta | Carpinus (Betulaceae), Ostrya (Betulaceae) | CBS 256.56 | – | MH857617 | MH869165 | – | Andreasen & Hellenberg (2009) |
| CBS 255.56 | – | MH857616 | MH869164 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora laurentii | Populus (Salicaceae), Betula (Betulaceae), Salix (Salicaceae) | CBS 325.73 | – | – | MH872397 | – | Boidin (1994) |
| Peniophora laxitexta | Angiosperms | BAFC 3309 | – | FJ882040 | – | – | Andreasen & Hellenberg (2009) |
| LGMF1159 | – | JX559580 | – | – | Andreasen & Hellenberg (2009) | ||
| BAFC 4687 | – | MN518328 | – | – | Andreasen & Hellenberg (2009) | ||
| Peniophora lilacea | Celtis (Cannabaceae), Staphylea (Staphyleaceae), Alnus (Betulaceae), Gleditsia (Fabaceae), Fraxinus (Olaceae) | CBS 337.66 | – | MH858813 | MH870452 | – | Boidin (1994) |
| CBS 337.66 | – | MH858813 | MH870452 | – | Boidin (1994) | ||
| Peniophora limitata | Fraximus, Syringa, Ligustrum, Phillyrea | CLZhao 5716 | – | MK269148 | – | – | Boidin (1994) |
| Peniophora lycii | Unkonwn | CBS 264.56 | – | MH857624 | MH869169 | – | Andreasen & Hellenberg (2009) |
| CBS 261.56 | – | MH857621 | MH869167 | – | Andreasen & Hellenberg (2009) | ||
| CBS 352.54 | – | MH857357 | MH868899 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora malaiensis | Calophyllum (Calophyllaceae) | CBS 679.91 | – | MH862297 | MH873974 | – | Andreasen & Hellenberg (2009) |
| He4870 | – | MK588775 | MK588815 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora manshurica | Quercus (Fagaceae) | He2956 | – | MK588776 | MK588816 | – | Andreasen and Hellenberg (2009) |
| He3729 | – | MK588777 | MK588817 | – | |||
| Peniophora meridionalis | Quercus, Cistus (Cistaceae), Lentiscus, Eucalyptus (Myrtaceae), Erica (Ericaceae) | CBS 289.58 | – | MH857789 | MH869323 | – | Boidin et al. (1991) |
| Peniophora molesta | Unknown | CBS 678.91 | – | MH862296 | – | – | Andreasen & Hellenberg (2009) |
| CBS 677.91 | – | MH862295 | – | – | Andreasen & Hellenberg (2009) | ||
| CBS 676.91 | – | MH862294 | MH873973 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora monticola | Hypericum (Hypericaceae), Dombeya (Malvaceae) | CBS 649.91 | – | MH862289 | MH873970 | – | Boidin et al. (1991) |
| Peniophora nuda | Angiosperms, rarely Gymnosperms | AFTOL_ID_660 | – | DQ411533 | DQ435788 | – | Andreasen & Hellenberg (2009) |
| Peniophora ovalispora | Acacia (Acaciae), Cryptomeria (Cupressaceae), Fuchsia (Onagraceae), Solanum (Solanaceae), Cyathea (Fern) | CBS 653.91 | – | MH862290 | MH873971 | – | Boidin et al. (1991) |
| Peniophora parvocystidiata | Pinus (Pinaceae) | CBS 716.91 | – | MH862305 | MH873978 | – | Andreasen & Hellenberg (2009) |
| CBS 717.91 | – | MH862306 | MH873979 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora piceae | Abies, Pseudotsuga (Pinaceae) | B1010 | – | MN475158 | MN475825 | – | Boidin (1994) |
| B1009 | – | MN475157 | MN475824 | – | Boidin (1994) | ||
| Peniophora pilatiana | Quercus, Cistus, Nerium, Vitis, Prunus, Pistacia, Olea, Rhammus, Salix, Eucalyptus, Ilex | CBS 269.56 | – | MH857627 | MH869172 | – | Boidin (1994) |
| CBS 265.56 | – | MH857625 | MH869170 | – | Boidin (1994) | ||
| CBS 266.56 | – | MH857626 | MH869171 | – | Boidin (1994) | ||
| Peniophora pini | Pinus sylvestris (Pinaceae) | CBS 272.56 | – | CBS 272.56 | MH869175 | – | Gibson (1960) |
| CBS 273.56 | – | MH857631 | MH869176 | – | Gibson (1960) | ||
| CBS 270.56 | – | MH857628 | MH869173 | – | Gibson (1960) | ||
| CBS 274.56 | – | MH857632 | MH869177 | – | Gibson (1960) | ||
| CBS 414.34 | – | MH855589 | MH867099 | – | Gibson (1960) | ||
| Peniophora pithya | On Gymnosperms (Pinaceae), rarely on Salix | CBS 276.56 | MZ233428 | MH857634 | MH869179 | – | Boidin et al. (1991) |
| B1013 | – | MN475160 | MN475827 | – | |||
| CBS 275.56 | MZ233427 | MH857633 | MH869178 | – | |||
| Peniophora polygonia | Populus (Salicaceae) | He3668 | – | MH669233 | MH669237 | – | Boidin (1994) |
| CBS 404.50 | – | MH856684 | MH868201 | – | Boidin (1994) | ||
| Peniophora proxima | Buxus (Buxaceae) | CBS 406.50 | – | MH856686 | MH868203 | – | Boidin (1994) |
| CBS 405.50 | – | MH856685 | MH868202 | – | |||
| Peniophora pseudo-pini | Pinus, Abies, Pseudotsuga | B1025 | – | MN475164 | MN475830 | – | Gibson (1960) |
| DAOM-30124 | – | MK588784 | MK588824 | – | Gibson (1960) | ||
| B1024 | – | MN475163 | MN475829 | – | Gibson (1960) | ||
| B1007 | – | MN475162 | MN475828 | – | Gibson (1960) | ||
| Peniophora pseudonuda | Quercus, Fagus (Fagaceae) | FCUG 2384 | – | GU322866 | – | – | Boidin (1994) |
| FCUG 2390 | – | GU322865 | – | – | Boidin (1994) | ||
| FCUG 86 | – | GU322867 | – | – | Boidin (1994) | ||
| Peniophora pseudoversicolor | Quercus (Fagaceae) | CBS 125881 | – | MH864303 | MH875753 | – | Boidin (1994) |
| CBS 338.66 | – | MH858814 | MH870453 | – | Boidin (1994) | ||
| Peniophora quercina | Betula, Castanea, Fagus, Salix | CBS 409.50 | – | MH856689 | MH868206 | – | Boidin (1994) |
| CBS 408.50 | – | MH856688 | MH868205 | – | Boidin (1994) | ||
| CBS 407.50 | – | MH856687 | MH868204 | – | Boidin (1994) | ||
| Peniophora reidii | Quercus (Fagaceae), Laurus, Betula, Salix, Fagus, Eucalyptus | CBS 397.83 | – | MH861616 | MH873334 | – | Boidin (1994) |
| Peniophora rufa | Populus tremuloides (Salicaceae) | CBS 351.59 | – | MH857891 | MH869432 | – | Chamuris & Falk (198) |
| B1014 | – | MN475165 | MN475831 | – | Chamuris & Falk (198) | ||
| Peniophora rufomarginata | Quercus, Populus, Tilia and Arbutrus (Ericaceae) | CBS 282.56 | – | MH857640 | MH869184 | – | Andreasen & Hellenberg (2009) |
| CBS 281.56 | – | MH857639 | MH869183 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora septentrionalis | Picea, Abies (Pinaceae) | CBS 294.58 | MZ233429 | MH857791 | MH869325 | – | Andreasen & Hellenberg (2009) |
| Peniophora simulans | Fagus | CBS 875.84 | – | MH861850 | MH873538 | – | Reid (1969) |
| CBS 874.84 | – | MH861849 | MH873537 | – | Reid (1969) | ||
| Peniophora subsalmonea | Mimosaceae | CBS 697.91 | – | MH862303 | – | – | Andreasen & Hellenberg (2009) |
| CBS 696.91 | – | MH862302 | – | – | Andreasen & Hellenberg (2009) | ||
| Peniophora taiwanensis | Angisosperms | Wu 9206 28 | – | MK588793 | MK588833 | – | Andreasen & Hellenberg (2009) |
| Wu 9209 14 | – | MK588794 | MK588834 | – | Andreasen & Hellenberg (2009) | ||
| Peniophora tamaricicola | Tamarix (Tamaricaceae) | CBS 439.62 | – | MH858204 | MH869803 | – | Gilbertson (1975) |
| CBS 441.62 | – | MH858205 | MH869804 | – | Gilbertson (1975) | ||
| CBS 438.62 | – | MH858203 | MH869802 | – | Gilbertson (1975) | ||
| Peniophora versicolor | Salix (Salicaceae), Acer (Sapindaceae), Ostrya (Betulaceae), Celtis (Cannabaceae), Robinia (Fabaceae) and Ceratonia (Fabaceae) | CBS 358.61 | – | MH858082 | MH869651 | – | Boidin (1994) |
| Peniophora violaceolivida | Salicaceae, rarely on “Gymnosperms” | CBS 348.52 | – | MH857077 | MH868613 | – | Andreasen & Hellenberg (2009) |
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Araújo, J.P.M.; Li, Y.; Six, D.; Rajchenberg, M.; Smith, M.E.; Johnson, A.J.; Klepzig, K.D.; Crous, P.W.; Leal-Dutra, C.A.; Skelton, J.; et al. Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora. J. Fungi 2021, 7, 1043. https://doi.org/10.3390/jof7121043
AMA Style
Araújo JPM, Li Y, Six D, Rajchenberg M, Smith ME, Johnson AJ, Klepzig KD, Crous PW, Leal-Dutra CA, Skelton J, et al. Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora. Journal of Fungi. 2021; 7(12):1043. https://doi.org/10.3390/jof7121043
Chicago/Turabian StyleAraújo, João P. M., You Li, Diana Six, Mario Rajchenberg, Matthew E. Smith, Andrew J. Johnson, Kier D. Klepzig, Pedro W. Crous, Caio A. Leal-Dutra, James Skelton, and et al. 2021. "Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora" Journal of Fungi 7, no. 12: 1043. https://doi.org/10.3390/jof7121043
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