Resolved phylogeny and biogeography of the root pathogen Armillaria and its gasteroid relative, Guyanagaster

Abstract

Background
Armillaria is a globally distributed mushroom-forming genus composed primarily of plant pathogens. Species in this genus are prolific producers of rhizomorphs, or vegetative structures, which, when found, are often associated with infection. Because of their importance as plant pathogens, understanding the evolutionary origins of this genus and how it gained a worldwide distribution is of interest. The first gasteroid fungus with close affinities to Armillaria—Guyanagaster necrorhizus—was described from the Neotropical rainforests of Guyana. In this study, we conducted phylogenetic analyses to fully resolve the relationship of G. necrorhizus with Armillaria. Data sets containing Guyanagaster from two collecting localities, along with a global sampling of 21 Armillaria species—including newly collected specimens from Guyana and Africa—at six loci (28S, EF1α, RPB2, TUB, actin-1 and gpd) were used. Three loci—28S, EF1α and RPB2—were analyzed in a partitioned nucleotide data set to infer divergence dates and ancestral range estimations for well-supported, monophyletic lineages.

Results
The six-locus phylogenetic analysis resolves Guyanagaster as the earliest diverging lineage in the armillarioid clade. The next lineage to diverge is that composed of species in Armillaria subgenus Desarmillaria. This subgenus is elevated to genus level to accommodate the exannulate mushroom-forming armillarioid species. The final lineage to diverge is that composed of annulate mushroom-forming armillarioid species, in what is now Armillaria sensu stricto. The molecular clock analysis and ancestral range estimation suggest the most recent common ancestor to the armillarioid lineage arose 51 million years ago in Eurasia. A new species, Guyanagaster lucianii sp. nov. from Guyana, is described.

Conclusions
The armillarioid lineage evolved in Eurasia during the height of tropical rainforest expansion about 51 million years ago, a time marked by a warm and wet global climate. Species of Guyanagaster and Desarmillaria represent extant taxa of these early diverging lineages. Desarmillaria represents an armillarioid lineage that was likely much more widespread in the past. Guyanagaster likely evolved from a gilled mushroom ancestor and could represent a highly specialized endemic in the Guiana Shield. Armillaria species represent those that evolved after the shift in climate from warm and tropical to cool and arid during the late Eocene. No species in either Desarmillaria or Guyanagaster are known to produce melanized rhizomorphs in nature, whereas almost all Armillaria species are known to produce them. The production of rhizomorphs is an adaptation to harsh environments, and could be a driver of diversification in Armillaria by conferring a competitive advantage to the species that produce them.

Electronic supplementary material
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Full text

R E S E A R C H A R T I C L E Open Access
Resolved phylogeny and biogeography of
the root pathogen Armillaria and its
gasteroid relative, Guyanagaster
Rachel A. Koch
1
, Andrew W. Wilson
2
, Olivier Séné
3
, Terry W. Henkel
4
and M. Catherine Aime
1*
Abstract
Background: Armillaria is a globally distributed mushroom-forming genus composed primarily of plant pathogens.
Species in this genus are prolific producers of rhizomorphs, or vegetative structures, which, when found, are often
associated with infection. Because of their importance as plant pathogens, understanding the evolutionary origins
of this genus and how it gained a worldwide distribution is of interest. The first gasteroid fungus with close affinities
to Armillaria—Guyanagaster necrorhizus—was described from the Neotropical rainforests of Guyana.In this study, we
conducted phylogenetic analyses to fully resolve the relationship of G. necrorhizus with Armillaria. Data sets containing
Guyanagaster from two collecting localities, along with a global sampling of 21 Armillaria species—including newly
collected specimens from Guyana and Africa—at six loci (28S, EF1α,RPB2,TUB,actin-1and gpd)wereused.Three
loci—28S, EF1αand RPB2—were analyzed in a partitioned nucleotide data set to infer divergence dates and ancestral
range estimations for well-supported, monophyletic lineages.
Results: The six-locus phylogenetic analysis resolves Guyanagaster as the earliest diverging lineage in the armillarioid
clade. The next lineage to diverge is that composed of species in Armillaria subgenus Desarmillaria. This subgenus is
elevated to genus level to accommodate the exannulate mushroom-forming armillarioid species. The final lineage to
diverge is that composed of annulate mushroom-forming armillarioid species, in what is now Armillaria sensu stricto. The
molecular clock analysis and ancestral range estimation suggest the most recent common ancestor to the armillarioid
lineage arose 51 million years ago in Eurasia. A new species, Guyanagaster lucianii sp. nov. from Guyana, is described.
Conclusions: The armillarioid lineage evolved in Eurasia during the height of tropical rainforest expansion about 51
million years ago, a time marked by a warm and wet global climate. Species of Guyanagaster and Desarmillaria represent
extant taxa of these early diverging lineages. Desarmillaria represents an armillarioid lineage that was likely much more
widespread in the past. Guyanagaster likely evolved from a gilled mushroom ancestor and could represent a highly
specialized endemic in the Guiana Shield. Armillaria species represent those that evolved after the shift in climate from
warm and tropical to cool and arid during the late Eocene. No species in either Desarmillaria or Guyanagaster are known
to produce melanized rhizomorphs in nature, whereas almost all Armillaria speciesareknowntoproducethem.The
production of rhizomorphs is an adaptation to harsh environments, and could be a driver of diversification in Armillaria by
conferring a competitive advantage to the species that produce them.
Keywords: Armillaria root rot, Cameroon, Eocene, Fungal taxonomy, Gasteromycetation, Guiana Shield, Melanin,
Mushroom evolution, Physalacriaceae, Systematics
* Correspondence: maime@purdue.edu
1
Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN 47907, USA
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Koch et al. BMC Evolutionary Biology (2017) 17:33
DOI 10.1186/s12862-017-0877-3

Background
The globally distributed mushroom-forming genus Armil-
laria contains species that are frequently encountered as
tree pathogens in natural forests [1, 2] as well as silvicul-
tural and agronomic systems [3, 4], and are the causal agent
of the root disease Armillaria root rot [5]. Besides being
pathogens, Armillaria species also play a critical role as
decomposers. Extensive infection observed in stumps and
roots and long possession of the substrate suggest that
Armillaria species contribute significantly to decompos-
ition and mineral cycling within many forests [6].
Armillaria species form basidiomes—in this case,
mushrooms—which serve as reproductive structures
that fruit seasonally when conditions are optimal. Each
of these basidiomes produces their reproductive propa-
gules, or basidiospores, on an exposed spore-bearing
surface, or hymenium. At maturity, the basidiospores are
propelled into the air column via a mechanism of
forcible spore discharge known as ballistospory. Basidio-
spores of other fungi capable of ballistospory have been
shown to disperse between continents [7–9]. Based on
molecular clock analyses, the origin of Armillaria post-
dates the Gondwana break-up, leading to the hypothesis
that several such long distance basidiospore dispersal
events led to the global distribution of Armillaria [10].
One change in basidiome form that has occurred re-
peatedly within mushroom-forming lineages is the shift
from producing basidiospores on an exposed hymenium
to producing them within an enclosed hymenium. This
loss of an exposed hymenium has resulted in the loss of
ballistospory [11]. Fungi that undergo this change will
hereinafter be referred to as gasteroid fungi as they go
through a process called gasteromycetation. A suite of
changes in basidiome morphology, including the devel-
opment of a fully enclosed basidiospore-bearing mass
(termed a gleba) that is encased in a specialized covering
(termed a peridium), typically take place during gastero-
mycetation. Gasteromycetation is believed to be a uni-
directional process, as gasteroid fungi have never been
observed to regain the mechanism of ballistospory [11].
In order to persist, gasteroid fungi have evolved a diverse
array of dispersal mechanisms that engage exogenous
forces. Examples include animal mycophagy for volatile-
producing underground truffle-like basidiomycetes [12]
and the “bellows”mechanism of puffballs [13].
Gasteroid fungi have evolved independently many
times from ballistosporic ancestors within the Agarico-
mycetidae (e.g. [14–20]). These ancestors of gasteroid
fungi include agaricoid lineages (i.e. those with lamellate
hymenophores in the Agaricales and Russulales) and
boletoid lineages (i.e. those with porose-tubulose hyme-
nophores in the Boletales). To date, gasteroid fungi have
only been documented as ectomycorrhizal (ECM) or
saprotrophic in their ecology (e.g. [11, 21–26]).
The first known gasteroid fungus closely related to
Armillaria was described in 2010 [27]. The monotypic
genus, Guyanagaster T. W. Henkel, Aime & M. E. Sm.
and the species G. necrorhizus T. W. Henkel, Aime & M.
E. Sm. (Basidiomycota; Agaricales; Physalacriaceae) are
only known from the Pakaraima Mountains of Guyana
in the Guiana Shield region of South America [27]. This
fungus produces subhypogeous basidiomes that are often
attached to decaying tree roots, has a thick black peridium
with pyramidal warts, a reduced stipe, and a tough, gelat-
inous gleba that changes from white to pink to brick red
with maturation [27]. This species is remarkable in the
sense that it does not possess the morphological hallmarks
of gasteroid fungi adapted for any of the aforementioned
dispersal mechanisms (e.g. mammal or mechanical
dispersal).
To our knowledge, until the discovery of Guyanagaster,
no known gasteroid fungus has been demonstrated to
have evolved from a pathogenic lineage. Guyanagaster
necrorhizus basidiomes are almost always found fruiting
from dead and decaying tree roots, in this case primarily
of the forest-dominating trees in the genus Dicymbe
Spruce ex Benth. (Fabaceae subfam. Caesalpinioideae).
Roots to which G. necrorhizus are attached display signs
of white rot, indicating a wood decay capability for this
fungus. Given the association of G. necrorhizus to dead
and decaying roots and its close relationship to Armillaria
species, it is conjectured that it is also has pathogenic cap-
abilities, but no experimental evidence exists to confirm
this.
Prior multi-gene phylogenetic analyses by Henkel et al.
were equivocal in that some loci suggested G. necrorhizus
was sister to Armillaria, while other loci showed G.
necrorhizus derived from within Armillaria [27]. Resolving
the phylogenetic placement of G. necrorhizus in relation
to Armillaria will create a more complete picture of
armillariod (a term to denote both Armillaria and
Guyanagaster species) evolution through time and
allow us to understand traits that led to the success of
this group. A resolved phylogeny will also give us a ro-
bust framework in which to understand the evolution
of Guyanagaster.
Here we use molecular data from G. necrorhizus and
Armillaria species from every major infrageneric lineage,
including Armillaria puiggarii Speg.—the only Armil-
laria species to be collected in the same region as G.
necrorhizus—to provide a fully resolved phylogenetic
hypothesis for armillarioid fungi.We also erect a new
genus, Desarmillaria, composed of the exannulate
mushroom-forming armillarioid species, to accommodate
those species formerly placed in Armillaria subgenus
Desarmillaria. A second species of Guyanagaster,Guya-
nagaster lucianii, is described as new to science. Using
these data we produce a time-calibrated phylogeny, which
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 2 of 16

we analyze in combination with ancestral range esti-
mations, in order to examine the morphological and
biogeographic evolution of armillarioid fungi.
Methods
Collecting and morphological analyses
Collecting expeditions to the Upper Potaro River Basin
(hereafter referred to as the Potaro) in the west-central
Pakaraima Mountains of Guyana were conducted dur-
ing the rainy seasons of May–July of 2002–2013. Fungi
were collected within a 15-km radius of a previously
established base camp (5°18'04.80"N, 59°54'40.40"W) in
forests dominated by Dicymbe corymbosa Spruce ex
Benth. and Dicymbe altsonii Sandw. (Fabaceae subfam.
Caesalpinioideae) [28, 29]. In December 2013–January
2014, a collecting expedition to the Mabura Ecological
Reserve (hereafter referred to as Mabura) in Mabura
Hill, Guyana—approximately 125 km from the Potar-
o—was conducted. Fungi were collected within a 3-km
radius of the field station (5°10'09.26"N, 58°42'14.40"W)
in forests with high densities of D. altsonii [30]. New
collections of African Armillaria species were made in
theDjaBiosphereReserveintheEastProvinceof
Cameroon during the rainy season of August–September
2014 within a 2-km radius of the Dja base camp (3°
21'29.80"N, 12°43'46.90"W).
Fresh morphological characteristics and substratum
relationships were described in the field. Color was de-
scribed subjectively and coded according to Kornerup and
Wanscher [31], with color plates noted in parentheses.
The descriptions of the collected Armillaria specimens
were compared to the descriptions of validly published
species. Additionally, attempts to obtain specimen cul-
tures were made in the field by placing small pieces of
unexposed basidiome tissue on Potato Dextrose Agar
(PDA) (BD Difco™, Franklin Lakes, New Jersey, USA) and
grown for one month at room temperature. Specimens
were then field-dried with silica gel.
Micromorphological features on dried specimens were
examined in the laboratory using an Olympus BH2-
RFCA compound microscope. Two dried Armillaria
specimens from Africa, TH 9926 and THDJA 91, and
four dried Guyanagaster specimens, MCA 4424, RAK
84, RAK 88 and RAK 89, were rehydrated in 70% etha-
nol and then sectioned by hand and mounted in water,
Melzer’s reagent and 1% aqueous Congo red. Twenty
randomly selected basidiospores were measured per col-
lection under a 100× objective. Length/width Q values
for basidiospores are reported as Q
r
(range of Q values
over 20 basidiospores measured) and Q
m
(mean of Q
values ± SD). Specimens were deposited in the following
herbaria: PUL (Kriebel Herbarium), BRG (Guyana
National Herbarium), HSC (Humboldt State University)
and YA (National Herbarium of Cameroon).
Molecular methods
DNA was extracted from basidiome tissue using the
Wizard® Genomic DNA Purification kit (Promega Co.,
Madison, Wisconsin, USA). Specimens housed in the
Kriebel Herbarium at Purdue University were used to
obtain DNA if sequence data for equivalent taxa were
not available on the NCBI Nucleotide database. PCR re-
actions included 12.5 μL of MeanGreen 2× Taq DNA
Polymerase PCR Master Mix (Syzygy Biotech, Grand
Rapids, Michigan, USA), 1.25 μL of each primer (at
10 μM) and approximately 100 ng of DNA. The final
PCR reaction volume was 25 μL. The recommended
cycling conditions for each primer pair we used were
followed.
To determine the identity of the recently collected
Armillaria specimens from Guyana and Africa, as well
as to confirm the identity of the recently collected
Guyanagaster specimens, PCR was performed to ac-
quiresequencedatafromtheinternaltranscribedspa-
cer (ITS) region (inclusive of ITS1, 5.8S and ITS2
regions), using the primer pair ITS1F/ITS4B [32]. To
analyze the phylogenetic relationship of the armillarioid
fungi, PCR was performed on all specimens at the three
following loci: nuclear ribosomal large subunit DNA
(28S), Elongation Factor 1-α(EF1α)andRNA polymer-
ase II (RPB2) genes using the following primer pairs,
respectively: LROR/LR6 [33, 34], 987 F/2218R [35] and
bRPB2-6 F/bRPB2-7.1R [36].
Uncleaned PCR products were sent to Beckman
Coulter, Inc. (Danvers, Massachusetts, USA) for sequen-
cing. Sequences were manually edited using Sequencher
5.2.3 (Gene Codes Corporation, Ann Arbor, Michigan,
USA). The ITS sequences we generated were used as
queries in the sequence similarity search tool, NCBI
BLAST, to search the NCBI Nucleotide database.
Phylogenetic analyses
In order to resolve the phylogeny of Guyanagaster and
Armillaria, we compiled a dataset composed of
sequence data from both Armillaria and Guyanagaster
specimens. Two closely related species in the Physala-
criaceae, Oudemansiella mucida and Strobilurus esculen-
tus, served as outgroup taxa. Six loci were used (28S,
EF1α,RPB2,Actin-1 (actin-1), Glyceraldehyde-3-Phos-
phate Dehydrogenase (gpd) and Beta-Tubulin (TUB)) to
assess Guyanagaster and Armillaria systematic relation-
ships through phylogenetic analysis. Not all loci were
available for all specimens. DNA sequences that were
not generated in this study were obtained from Gen-
Bank or published whole genome sequences. Collection
information for all specimens and GenBank accession
numbers for the included sequences are compiled in
Additional file 1.
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 3 of 16

Sequences were aligned in Mega 5.0 [37] using the
MUSCLE algorithm [38] with refinements to the align-
ment done manually. The introns of EF1αwere alignable
across the Armillaria/Guyanagaster dataset and were
therefore included. Phylogenies were reconstructed
using maximum likelihood (ML) and Bayesian methods.
The GTR + G model of molecular evolution was selected
for all data sets as determined by PartitionFinder v1.1.0
[39]. Maximum likelihood bootstrap analysis for phyl-
ogeny and assessment of the branch support by boot-
strap percentages (BS%) was performed using RAxML
v2.2.3 [40]. One thousand bootstrap replicates were
produced. Each locus was analyzed separately as well as
all together in a supermatrix data set using ML. Bayesian
analyses for the reporting of Bayesian posterior prob-
ability (BPP) support for branches was conducted on
individual loci as well as the six-gene data set using
the program Mr. Bayes v3.2.2 [41]. Four simultan-
eous, independent runs each with four Markov chain
Monte Carlo (MCMC) chains, were initiated and run
at a temperature of 0.15 for 20 million generations,
sampling trees every 1000 generations until the stand-
ard deviation of the split frequencies reached a final
stop value of 0.01. We discarded the initial 10% of
trees as burn in and produced a maximum clade
credibility tree from the remaining trees; 0.95 BPP
represents a well-supported lineage.
Divergence time estimation
Divergence ages within the armillarioid clade were esti-
mated using the fossil calibration approach described
and implemented by [24, 42–44]. Molecular clock ana-
lysis was performed using BEAST v1.8.3 [45] with XML
files containing BEAST commands and priors assembled
in BEAUTi v1.8.3. These files included the following
analytical settings: GTR + G model of evolution, uncor-
related relaxed clock with lognormal rate distribution;
tree prior was set to speciation birth-death process, run-
ning 100 million generations, sampling every 1000
th
tree.
The analysis was run three times. The first 10% of trees
were removed as burn-in after ensuring a minimum
effective sample size (ESS) of 200 was reached for all of
the parameters. The remaining trees—representing the
posterior distribution from all Bayesian analyses—were
combined into a single file using LogCombiner v1.8.3.
This file was used to produce a summary tree using Tree
Annotator v1.8.3. The mean ages of supported nodes, as
well as the corresponding 95% highest posterior dens-
ities (HPDs), were examined from BEAST logfiles using
Tracer v1.6 [46].
Taxa used in this analysis are in bold in Additional file
1. It includes 21 Armillaria species (one representative
specimen from each of the species in the first analysis),
two Guyanagaster specimens (one representative from
each collecting locality), nine Physalacriaceae species
(one representative from nine different genera that en-
compass the major lineages within this family fide [47]),
while six species from Agaricales families closely related
to the Physalacriaceae fide [48] served as outgroup taxa.
The alignment was analyzed using five partitions: 28S
was analyzed in a single partition, while two codon parti-
tions ((1 + 2), 3) were utilized for both EF1αand RPB2.
The marasmioid fungi (Marasmius alliaceus,Marasmius
rotula and Mycena amabilissima) were calibrated based
on a 90-Ma fossil Archaeomarasmius leggetti from mid-
Cretaceous amber [49], following the parameter settings
of [24]. In BEAUTi, this prior was set as a lognormal
distribution with a mean of 10, log standard deviation of
1 and offset of 90, with mean in real space, truncated on
the lower end at 90 and at the upper end at 200.
Ancestral range estimation
To estimate the ancestral range of the armillarioid clade
and lineages within, we used dispersal-extinction-
cladogenesis (DEC) analysis in the package Lagrange
[50] and implemented in the program RASP [51]. Six
areas were defined: North America, Europe + Asia (here-
inafter referred to as Eurasia), Africa, tropical South
America (composed of the area covered by tropical rain-
forest in South America), Australasia (composed of
Australia, New Zealand and southeast Asia) and temper-
ate South America (composed of the area not covered
by tropical rainforest in South America). Each taxon in
our data set was assigned to areas based on its current
known range. In the scenario we tested, movement be-
tween any area at any time was unconstrained. Under
this model, all areas are treated as equally probable an-
cestral ranges. This model was tested under both two
and three area constraints. The consensus tree produced
during the divergence time analysis was used for all
Lagrange calculations and the root node of the armillar-
ioid clade was calibrated to 51 Ma.
Results
Sequences, collections and cultures
Eight ITS, eleven 28S, twelve EF1αand five RPB2 se-
quences were generated during this study (Additional
file 1). The size of the sequences ranged from 514–713,
705–1031, 796–1233, and 553–704 bp, respectively.
After the ends of the individual alignments were
trimmed, the size of the aligned datasets were as follows:
28S was 906 bp; EF1αwas 906 bp; RPB2 was 638 bp;
actin-1 was 607 bp; gpd was 499 bp; TUB was 911 bp.
The six-gene dataset was composed of a total of 52
Armillaria specimens, representing 21 species, as well as
four Guyanagaster specimens, representing two species.
The number of taxa in each of the single-locus phyloge-
nies is as follows: 28S had 42 taxa; EF1αhad 57 taxa;
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 4 of 16

RPB2 had 32 taxa; actin-1 had19 taxa; gpd had 19 taxa;
TUB had 12 taxa. Phylogenies for each locus, as well as
a combination of loci, are available in Additional file 2.
Two Armillaria specimens were collected from Guya-
na—MCA 3111 and TH 9751. ITS sequences from both
shared 99% identity with Armillaria puiggarii (GenBank:
FJ664608). No significant morphological differences
were found when comparing our field descriptions to
the species description by [52]. Two Armillaria speci-
mens were collected from Cameroon—TH 9926 and TH
DJA 91. ITS sequences from both shared 99% identity
with an unidentified Armillaria species collected from
Zimbabwe (GenBank: AY882982). Additionally, there
was variation between the two sequences. No significant
morphological differences were found when comparing TH
9926 to the species description of Armillaria camerunensis
[53, 54], but there was some morphological variation when
comparing TH DJA 91. For now, we conservatively
hypothesize that both of these specimens represent Armil-
laria camerunensis. Morphological descriptions of TH
9926 and TH DJA 91 are available in Additional file 3.
Pure vegetative cultures were obtained from Guyanaga-
ster specimens MCA 3950 and RAK 88, as well as Armil-
laria puiggarii specimen TH 9751, with rhizomorphs
produced in each (Fig. 1). After one month, differences in
growth and branching pattern of the rhizomorphs can be
observed between the cultures of the two Guyanagaster
specimens, but the rhizomorphs of neither were observed
to become melanized. In contrast, the rhizomorphs in the
culture of A. puiggarii became melanized almost com-
pletely. Strain MCA 3950 is deposited in Centraalbureau
voor Schimmelcultures with the strain accession number
CBS 138623, while strains RAK 88 and TH 9751 are avail-
able from the authors upon request.
Resolved Guyanagaster phylogenetic hypothesis
No single locus was sufficient to obtain a well-supported
armillarioid phylogeny (see Additional file 2), but through
the use of multiple loci, we were able to obtain a well-
supported armillarioid phylogeny (Fig. 2). The six-gene
analyses recovered a strongly supported armillarioid clade
(100% BS and 1.00 BPP) (Fig. 2), which is composed of all
known Armillaria and Guyanagaster species. The armil-
larioid clade is further composed of three well-supported
lineages that were recovered from both analyses (Fig. 2):
(1) the annulate lineage (100% BS and 1.00 BPP), which
includes annulate mushroom-forming armillarioid species,
(2) the exannulate lineage (91% BS and 1.00 BPP), com-
posed of exannulate mushroom-forming armillarioid spe-
cies formerly placed in Armillaria subgen. Desarmillaria
and (3) the gasteroid lineage (100% BS and 1.00 BPP),
composed of Guyanagaster species. The gasteroid lineage
consists of two distinct species: G. necrorhizus from the
Potaro region and an undescribed Guyanagaster species
from the Mabura region of Guyana. These two species are
geographically separated by mountains of elevations over
1000 m, as well as Kaieteur Falls and the Essequibo River,
all within a distance of 125 km.
The annulate lineage is further composed of four well-
supported lineages (Fig. 2), with species compositions as
follows: (1) African lineage composed of Armillaria fus-
cipes and A. camerunensis; (2) melleioid lineage com-
posed of A. mellea; (3) northern hemisphere lineage
composed of A. altimontana, A. borealis,A. calvescens,
A. cepistipes,A. gallica, A. gemina,A. sinapina and A.
solidipes; and (4) Australasian + temperate South Ameri-
can lineage composed of A. affinis, A. fumosa,A. hinnu-
lea,A. limonea, A. luteobubalina, A. novae-zelandiae,A.
pallidula and A. puiggarii.
The most glaring conflict between single gene and
multi-gene analyses is the placement of the African
lineage: in gpd,actin-1 and EF1αthis lineage is nested
within or sister to the Australasian + temperate South
American lineage; in TUB and RPB2, it is sister to A.
mellea; in 28S it is sister to Guyanagaster. In the multi-
gene analyses, it is consistently recovered as diverging
earlier than the A. mellea lineage. In all of the single
gene analyses except for 28S, a close relationship
between the exannulate and the gasteroid lineages was
recovered.
Fig. 1 aVegetative culture of Guyanagaster necrorhizus MCA 3950 showing unmelanized rhizomorphs. bVegetative culture of Guyanagaster lucianii
RAK 88 showing unmelanized rhizomorphs. cVegetative culture of Armillaria puiggarii TH 9751 showing melanized rhizomorphs. Bar = one cm
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 5 of 16

Age and ancestral range of Armillaria and Guyanagaster
Table1hasacompletelistofthedivergencedatesfor
the well-supported nodes recovered in the phylogen-
etic analysis. Figure 3 is the time-calibrated phylogeny
for the group. The time to most recent common an-
cestor (tMRCA) of the armillarioid clade estimated in
the BEAST analysis was 51 Ma (node 1; 95% HPD
30–73 Ma). The tMRCA of the exannulate lineage
was estimated at 41 Ma (node 4; 95% HPD 24–59 Ma).
ThetMRCAoftheannulatearmillarioidlineagewas
estimated at 33 Ma (node 5; 95% HPD 19–47 Ma).
The date of the divergence between the two Guyana-
gaster species was estimated at 8 Ma (node 2; 95%
HPD 3–14 Ma).
a
b
d
e
f
hi j
g
c
Fig. 2 Phylogram generated from the analysis of six gene regions (28S, EF1α, RPB2, TUB, gpd and actin-1) from 58 taxa. Guyanagaster is the earliest
diverging lineage and is sister to the mushroom-forming armillarioid species. The exannulate armillarioid species are the next to diverge, and compose
Desarmillaria. The annulate armillarioid species form a monophyletic lineage and compose Armillaria sensu stricto. The major lineages within Armillaria are
indicated in bold text at the corresponding node. The exannulate armillarioid species are sister to the annulate armillarioid species. Strobilurus esculentus
and Oudemansiella mucida were selected as outgroup taxa. Black circles represent support of 90% (maximum likelihood bootstrap values, shown as
percentages) and 0.95 BPP (Bayesian posterior probabilities) or greater, grey circles represent support of 0.95 BPP or greater and white circles represent
support of 75% or greater. Images: (a)Armillaria sinapina;(b)Armillaria hinnulea;(c)Armillaria puiggarii;(d)Armillaria mellea;(e)Armillaria camerunensis;(f)
Desarmillaria tabescens;(g)Desarmillaria ectypa,(h–j)Guyanagaster necrorhizus. (Photo credits: (a) Christian Schwarz; (b)J.J.Harrison;(c) Todd F. Elliott; (d,
h–j) Rachel A. Koch; (e) Terry W. Henkel; (f) Stephen D. Russell; (g) Tatyana Svetasheva)
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 6 of 16

Table 1 Ages for nodes, confidence intervals and posterior probability on the phylogenetic tree presented in Fig. 3
Node Lineage Mean Age 95% HPD [min, max] Posterior Probability
1 Armillarioid 50.81 30.03, 72.32 1.00
2Guyanagaster 7.88 2.70, 13.91 1.00
3Desarmillaria +Armillaria 41.32 24.47, 59.07 0.90
4Desarmillaria 31.74 15.83, 47.71 1.00
5Armillaria 33.00 18.90, 46.98 1.00
6 African lineage 15.49 6.84, 24.76 1.00
7 Melleioid lineage 30.12 17.73, 43.43 0.90
8 Northern hemisphere + Australasian + temperate South American lineage 27.52 17.13, 37.60 0.94
9 Northern hemisphere 12.07 6.12, 18.87 1.00
10 A. borealis + A. solidipes + A. gemina 6.66 2.41, 11.51 1.00
11 A. altimontana + A. calvescens + A. gallica 8.58 3.80, 14.02 1.00
12 A. cepistipes + A. sinapina 6.44 2.22, 11.24 0.52
13 Australasian + temperate South American 19.27 11.18, 27.99 0.98
14 A. novae-zelandiae + A. affinis + A. puiggarii 8.96 4.04, 14.47 1.00
15 A. hinnulea + A. pallidula + A. fumosa + A. limonea + A. luteobubalina 18.13 10.30, 26.29 –
Fig. 3 Time-calibrated phylogeny generated from Bayesian analysis of three gene regions (28S, EF1α, RPB2)from19Armillaria species, two
Desarmillaria species and two Guyanagaster species. Geographic origin of each specimen is indicated by the box to the left of the name:
red =Neotropics, blue = Australasia, brown = temperate South America, lime green = Eurasia, purple = North America, and turquoise =Africa.
Boxes at ancestral nodes correspond to the most probable ancestral range at that node as presented in Additional file 4. Geologic epochs are noted
above the time scale. Numbers at nodes correspond to lineages in Table 1 and Additional file 4. Dark grey bars correspond to the 95% HPD and
correspond to the values in Table 1. The map in the lower left hand corner represents the proposed dispersal pattern
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 7 of 16

We estimated the ancestral ranges for 15 well-
supported clades. The most probable area was the same
under range constraints of ≤2and≤3 for all nodes. The
most probable ancestral range for the most recent com-
mon ancestor (MRCA) of the armillarioid clade is Eurasia.
Eurasia is also the most probable ancestral range for all of
the annulate mushroom-forming lineages containing
northern hemisphere taxa. Migration to both Africa and
Australasia from Eurasia is the most probable scenario as
estimated from this analysis. Additional file 4 provides the
probabilities for each area being the ancestral range under
both area constraints; numbers in bold represent the most
probable region at that node.
Discussion
Our phylogenetic analyses, coupled with morphological
data, support a three-genus composition of the armillar-
ioid clade: Armillaria contains the annulate mushroom-
forming species, Desarmillaria contains the exannulate
mushroom-forming species, and Guyanagaster contains
the known gasteroid species. Our estimates of the
tMRCA of the armillarioid clade suggest it arose 51 Ma,
which is in accord with a previous estimate [10], even
though a different set of taxa, loci and calibration
methods were used. During this time, Earth was experi-
encing one of the warmest intervals of the past 65 mil-
lion years known as the Paleocene-Eocene Thermal
Maximum (PETM) [55]. This warm and humid climate
led to the widespread expansion of area covered in trop-
ical rainforests. The ancestral range estimation suggests
the most probable area for the origin of the MRCA is
the Eurasian subcontinent (Additional file 4), much of
which contained tropical rainforests at that time [56].
Biogeography and evolution of Guyanagaster and
Desarmillaria
Our phylogenetic reconstruction suggests that Guyanaga-
ster species evolved within the earliest diverging lineage in
the armillarioid clade. The process of gasteromycetation is
believed to be unidirectional; gasteroid fungi evolve from a
ballistosporic ancestor, and there is no evidence that this
transformation has ever been reversed [11]. With this in
mind, the ancestor to the armillarioid lineage was likely a
gilled mushroom. According to our estimations, between
51 and 8 Ma the mushroom progenitor to Guyanagaster
underwent the gasteromycetation process, resulting in the
evolution of Guyanagaster. At one point in time, this
ancient lineage was composed of mushroom-forming spe-
cies—all of which likely went extinct—as the only known
extant members of this lineage are Guyanagaster species.
The next lineage to diverge is composed of the two
described Desarmillaria species: D. ectypa and D.
tabescens. Neither of the species in this genus have
an annulus at maturity, in contrast to all other known
mushroom-forming armillarioid species. Desarmillaria
ectypa is different from all other armillarioid species
by virtue of its ecology: whereas Armillaria species
are pathogens and wood decomposers, D. ectypa is
restricted to peat bogs and associated with Sphagnum
[57]. The ecology of D. tabescens is much more typ-
ical of Armillaria species. It is a primary pathogen
towards introduced Eucalyptus species in France, as
well as a secondary pathogen to Quercus species, but
in many cases it is a saprotroph, colonizing stumps of
Quercus species [58].
Both Guyanagaster and Desarmillaria evolved within
the oldest lineages in the armillarioid clade and both
genera contain two known species. Armillaria repre-
sents the most recently diverged genus-level lineage
within the armillarioid clade, yet has over 35 described
species (see [59] for species counts). The older ages of
the lineages from which Guyanagaster and Desarmil-
laria evolved, coupled with the fact that they contain
far fewer extant species, suggest these two genera are
on a different evolutionary trajectory compared to
Armillaria. As stated above, our ancestral range estima-
tion suggests the most probable ancestral range for the
MRCA to the armillarioid clade is Eurasia, whereas spe-
cies of Guyanagaster have only so far been found in
Guyana. One question is how the mushroom progeni-
tor to Guyanagaster dispersed from Eurasia to Guyana.
One possibility is that the mushroom progenitor to
Guyanagaster was widely dispersed within the humid,
tropical rainforest habitat that predominated at the
time it arose. It is possible that Guyanagaster evolved
withintheGuianaShieldandrepresentsahighlyspe-
cialized endemic to this region.
The two species of Desarmillaria are thought to be
more thermophilic than their sympatric Armillaria
species [57, 60]. This temperature adaptation could be
a residual characteristic of their origin during the
PETM and could also explain the paucity of species
in this genus. As the climate changed, species adapted
for a cool and arid climate could have competitively
displaced the species adapted for a warm and humid
climate, in this case, species of Desarmillaria and the
mushroom progenitor to Guyanagaster.Thenovel
morphology of Guyanagaster species suggests that
they are highly specialized for their current habitat,
and as stated above, the restriction of D. ectypa to
peat bogs is a specialized habitat for armillarioid
fungi. Habitat specialization has been shown to act as
buffer from extinction [61], and could be what has
allowed species in these early diverging lineages to
avoid competitive displacement by better-adapted
armillarioid fungi. Whether D. tabescens occupies a
specialized niche that has aided in its persistence to
the present remains unknown.
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 8 of 16

Biogeography and spread of Armillaria
According to our analyses, Armillaria sensu stricto
started diversifying approximately 33 Ma in Eurasia, at
which time the Earth’s climate shifted from the relatively
ice-free world to one with glacial conditions; the tropical
climate during which the MRCA of the armillarioid
clade arose was replaced by a period of severe cooling and
drying as well as an increase in seasonality [55]. This led
to a contraction in the area covered by tropical rainforests
[62] and the geographic expansion of species-poor decidu-
ous vegetation [63]. Therefore, we hypothesize that Armil-
laria species represent annulate mushrooms that
survived, diversified and radiated with their hosts as they
adapted to the drier, cooler and seasonal climate (i.e. tem-
perate) of the late Eocene and beyond.
Armillaria species in Africa form a monophyletic
lineage (100% and 1.00 BPP). Single gene analyses and
multi-gene analyses were ambiguous as to where this
lineage diverged within the armillarioid clade (Additional
file 2). In all multi-gene combinations, the African
Armillaria lineage is resolved as the earliest diverging
within Armillaria sensu stricto, which is similar to previ-
ous work that has also suggested that this lineage is
divergent compared to the rest of the armillarioid
lineage [10, 64]. Our ancestral area estimation suggests
that the ancestral region of the African lineage is Eur-
asia. During the Eocene and Oligocene, a shallow seaway
separated Africa from Eurasia, acting as a barrier to dis-
persal. In the early Miocene (19–12 Ma), the seaway
drained, effectively linking the biotas in these two
regions [65]. Our molecular clock analysis suggests that
extant species in Africa started diversifying approxi-
mately 15 Ma, which fits a migration from Eurasia
during this time.
Many northern hemisphere Armillaria species are
found in both North America and Eurasia (see [58, 59]).
In a study of A. mellea, which is a species distributed
throughout the northern hemisphere, four distinct line-
ages were identified, corresponding to their locality: 1)
western North America, 2) eastern North America, 3)
western Eurasia and 4) eastern Eurasia [66]. They sug-
gest that this species was once widespread and is now in
the process of speciation [66]. Within this backdrop, it is
possible that Armillaria species facilitated their dispersal
as pathogens on the diverse flora that existed during this
time. As available pathways disappeared, ultimately ceas-
ing migration, the species started to diverge. From our
ancestral range estimation, we can see that there were
multiple armillarioid introductions into North America
(Fig. 3), suggesting a dynamic process of dispersal across
the northern hemisphere. A more comprehensive study
on northern hemisphere Armillaria species is necessary
to determine when and how they migrated between the
major landmasses.
Armillaria species started diversifying in the temperate
southern hemisphere (outside of Africa) about 19 Ma
(Fig. 3, node 13). Our results suggest that the most prob-
able dispersal pathway was from Eurasia to Australasia.
In a plant biogeographic meta-analysis of the Australa-
sian region, it was found that after 25 Ma, there was an
increase of flora inputs to Australia from the IndoMala-
yan region, representing the time when the continental
shelf containing Southeast Asia collided with that con-
taining New Guinea and Australia [67]. The authors of
this study hypothesized that habitat instability in the re-
gion at that time generated vacant niches and increased
the probability of successful establishment of dispersed
lineages compared with the relatively stable, saturated
states existing prior to approximately 25 Ma [67]. In this
scenario both the greater dispersal distance and relative
difficulty in establishing viable populations together con-
tributed to the infrequency of successful exchanges prior
to 25 Ma. This too follows our hypothesis that Armil-
laria species dispersed as pathogens on flora that existed
during this time.
Many of the same Armillaria species are found in both
Australasia and temperate South America [68]. This pat-
tern fits with an ancient Gondwanan distribution, but
the lineage is far too young for this to be the case. Alter-
natively, the discovery of fossilized Nothofagus wood in
Antarctic deposits from the Pliocene [69] suggests this
could have been an overland dispersal route until 2 Ma.
Many of the fungi in this Australasian + temperate South
American lineage are thought to be close associates with
Nothofagus [68],suggesting again that they could have
dispersed with their plant associates through Antarctica.
Alternatively, long distance basidiospore dispersal events
could account for the geographic disjunction in closely
related Armillaria species. Because appropriate hos-
ts—species of Nothofagus—
occur in both Australasia and
temperate South America [70], establishment after a long-
distance basidiospore dispersal event could be more
probable.
The rise of rhizomorphs: morphological developments in
the armillarioid clade
Our phylogenetic hypothesis sheds light on the evolution
of rhizomorphs in the armillarioid clade. Rhizomorphs are
discrete, filamentous aggregations that extend from a re-
source base into substrates that may not support their
growth, foraging for new resource bases [71]. Individuals
that produce rhizomorphs can occupy huge swaths of
habitat and prevent other organisms from establishing, as
is the case with the “humungous fungus”[72]. All armil-
larioid species have the capacity to form rhizomorphs in
culture, but only the later diverging lineages have been ob-
served to form them in nature (see Table 2, Fig. 1).
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 9 of 16

Table 2 Trophic strategy, rhizomorph production in nature and known geographic range of species used in this study
Species Facultative
Necrotroph?
Characterization Rhizomorphs
in nature?
Hosts Known range References
Armillaria
affinis
Unknown Unknown Central America [59]
Armillaria
altimontana
Unknown Yes Hardwoods and
conifers
Higher elevation forests
of Western interior of
North America
[83]
Armillaria
borealis
Yes Weakly pathogenic,
opportunistic
Yes Birch, wild cherry Europe [58,84]
Armillaria
calvescens
Yes Opportunistic pathogen
on stressed trees
Yes Hardwoods, particularly
sugar maple
Eastern North America [85,86]
Armillaria
camerunensis
Unknown Observed in a disease center,
but unknown if it is the causal
agent
None observed Unknown Africa This study
Armillaria
cepistipes
Yes Weakly pathogenic Yes Conifers Europe, North America [58,87]
Armillaria
fumosa
Unknown Unknown Australia [59]
Armillaria
fuscipes
Yes Particularly pathogenic
to exotic species
None observed Pine forest plantations,
Acacia and Cordia species
Africa, India [74,75,88]
Armillaria
gallica
Yes Weakly or secondarily
pathogenic
Yes Hardwoods Europe, North America,
Asia
[58]
Armillaria
gemina
Yes Primary pathogen Yes Maples, beech Birch Eastern North America [86]
Armillaria
hinnulea
Yes Secondary pathogen Yes In wet sclerophyll forests Australia, New Zealand [89,90]
Armillaria
limonea
Yes Pathogenic to pine seedlings
(introduced tree)
Yes Pine Argentina, Chile,
New Zealand
[68,91,92]
Armillaria
luteobubalina
Yes Primary pathogen in native
forests
Yes Eucalyptus Australia, Tasmania [68,93–95]
Armillaria
mellea
Yes Highly pathogenic Yes Over 600 ornamentals,
hardwood and orchard
trees
Europe, North America,
Asia
[58,96]
Armillaria
novae-zelandiae
Yes Pathogenic to pine seedlings
(introduced tree)
Yes Pine Argentina,
Australia, Chile, New
Zealand
[91,92,95]
Armillaria
pallidula
Unknown Unknown Australia [59]
Armillaria
puiggarii
Unknown Observed in a disease center,
but unknown if it is the causal
agent
Melanized
rhizomorphs
observed in the field
Dicymbe spp. Argentina, Bolivia
Caribbean, Guyana
[95] and
this study
Armillaria
sinapina
Yes Weakly pathogenic Yes Conifers North America, Japan [97]
Armillaria
solidipes
Yes Highly pathogenic Yes Conifers Cooler regions of
North America, Europe,
China
[58,98]
Desarmillaria
ectypa
Unknown Saprotrophic on decaying
peat moss
No Sphagnum moss Europe, Russia, Japan,
China
[57]
Desarmillaria
tabescens
Yes Highly pathogenic No Eucalyptus, Quercus Asia, Europe,
North America
[58,73]
Guyanagaster
lucianii
Unknown Only saprotrophic stage
observed
No Eperua spp. Guyana This study
Guyanagaster
necrorhizus
Unknown Only saprotrophic stage
observed
No, but short non-
melanized hyphal
cords may be
produced
Dicymbe spp. Guyana [27]
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 10 of 16

Species in the earliest diverging genera in the armillar-
ioid clade, Desarmillaria and Guyanagaster, have not
been observed to form rhizomorphs in nature, but they
do produce unmelanized rhizomorphs in culture [27, 57,
58, 73] (Fig. 1). Rhizomorph production of Armillaria
species from Africa, the next lineage to diverge, has been
scantily observed in nature [74], but the production of
melanized rhizomorphs does occur in culture [75]. The
next lineage to diverge is that composed of A. mellea,
which produces short-lived melanized rhizomorphs with
limited growth in the soil [58]. The most species rich armil-
larioid lineage, the northern hemisphere and Australasian/
temperate South America lineage, produce melanized rhi-
zomorphs in nature (see Table 1).
The capacity of Armillaria species to produce mela-
nized rhizomorphs in nature is thought to be an adapta-
tion to harsh environments [71]. Melanized rhizomorphs
can confer advantages like protection against other mi-
crobial competitors, translocation of resources, growth
from a suitable resource base into an environment that
may not support growth, as well as enhancement of in-
oculum potential [71]. Additionally, melanin is thought
to promote longevity and survival of rhizomorphs within
the soil [76], and has also been found to help other fungi
survive in extreme environments [77]. Although all
species in the armillarioid clade appear to retain the trait
of rhizomorph production, only the most recently
diverged appear to be adapted for melanized rhizomorph
production in the current environment. In a controlled en-
vironment, it was found that only under high oxygen avail-
ability and near saturated moisture would D. tabescens
produce melanized rhizomorphs [78]. It was concluded that
these environmental conditions are sufficiently stringent in
the climate of today, and could explain why D. tabescens
has not been observed to form rhizomorphs in nature. It is
possible that the production of melanized rhizomorphs
could be a driver of diversification in Armillaria by confer-
ring a competitive advantage to the species that produce
them.
Desarmillaria species lack an annulus at maturity,
whereas Armillaria species have a robust annulus at ma-
turity. An annulus is the remnant of a partial veil that
remains after the pileus expands. Partial veils protect the
immature hymenium [79]. The development of a more
protected hymenium could be an adaptation of Armil-
laria to drier, more unpredictable habitats that were
common when this lineage began to diversify.
Conclusions
Our analyses suggest that the armillarioid clade arose in
Eurasia during the PETM, a time marked by a warm,
tropical climate. Two lineages arose during this time: the
earliest diverging lineage—which eventually led to the
gasteroid genus Guyanagaster—and the exannulate
mushroom-forming genus Desarmillaria. Besides being
the oldest lineages in the armillarioid clade, they are also
depauperate compared to Armillaria. Armillaria diverged
after the shift to a cooler and more arid climate at the
Eocene-Oligocene boundary. The production of mela-
nized rhizomorphs in nature and the development of a
protective partial veil could be adaptations that led to the
subsequent dispersal and diversification of Armillaria in
the much harsher, temperate climate. The success of
Armillaria could have displaced now-extinct species in
the exannulate and gasteroid lineages. Guyanagaster and
Desarmillaria species have likely persisted to the present
because they are highly specialized for their habitat.
Formal taxonomic descriptions
Desarmillaria (Herink) R. A. Koch & Aime gen. et
stat. nov.
Basionym—Armillaria ss. Fries subgenus Desarmillaria
Herink, Sympozium o václavce obecné (J. Hasek). 1972
September. Lesnicka fakulta VSZ Brno: 44. 1973.
Type—Armillaria socialis (DC. ex Fr.) Herink, Sympo-
zium o václavce obecné (J. Hasek). 1972 September.
Lesnicka fakulta VSZ Brno: 44. 1973.
MycoBank number—MB 819124 (genus).
Description—Basidiomata stipitate, usually in caespi-
tose clusters on wood. Pileus convex to applanate, usu-
ally some shade of brown. Lamellae pallid, adnexed to
adnate to subdecurrent with lamellulae. Stipe central,
various colorations but often concolorous with cap, lon-
gitudinally striate, usually with fibrils on upper third.
Annulus absent. Basidiospores ellipsoid to spherical,
smooth, hyaline, inamyloid. Basidia clavate, four-
sterigmate, hyaline. Cheilocystidia clavate, usually resem-
bling basidioles. Pileipellis suprapellis composed of
round, ellipsoid, cylindric, utriform, brown to hyaline,
verrucose cells; subpellis composed of a layer of
compact, shortened hyphae. Clamp connections absent.
Rhizomorph production not observed in nature, while
unmelanized production in culture has been observed.
Saprotrophic to parasitic. Known only from the northern
hemisphere.
Commentary—Desarmillaria includes mushroom-
forming armillarioid species that lack an annulus. This
difference in morphology led Singer [80, 81] to divide
Armillaria (as Armillariella) into two sections based on
the presence or absence of an annulus at maturity.
Herink [82], then, recognized Armillaria as an annulate
subgenus and Desarmillaria as an exannulate subgenus.
Additionally, members of this genus have not been ob-
served producing rhizomorphs in the field [57, 58], which
is in contrast to most Armillaria species, most of which
do produce rhizomorphs in the field (see Table 2). Rhizo-
morph production in nature appears to be a lost trait in
this genus, as species are still able to form them in culture.
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 11 of 16

We have opted to elevate Desarmillaria to genus-level
for two reasons. First, the absence of an annulus in
Desarmillaria species and the presence of one in Armil-
laria species is a reliable characteristic to differentiate
the two genera. Second, our phylogenetic and molecular
clock analyses show Desarmillaria is on a separate evo-
lutionary trajectory compared to Armillaria, meriting a
separate genus.
Desarmillaria tabescens (Scop.) R. A. Koch & Aime
comb. nov.
Basionym—Agaricus tabescens Scop., Scopoli 1772, Flora
Carniolica Plantas Corniolae Indigenas no. 1537: 446.
MycoBank number—MB 819125 (species).
Desarmillaria ectypa (Fr.) R. A. Koch & Aime
comb. nov.
Basionym—Agaricus ectypus Fr., Fries 1821, Syst.
Mycol. 1: 108.
MycoBank number—MB 819126 (species).
Guyanagaster lucianii R. A. Koch & Aime sp.
nov.—Holotype—BRG 41292, isotype—PUL F2891
(Figs. 1 and 3b–h). Mabura Ecological Reserve, (5°
10'09.26"N, 58°42'14.40"W), 25 December 2013. R. A.
Koch 89.
MycoBank number—MB 815807 (species).
Representative DNA barcode—RAK 89 (holotype),
ITS—GenBank KU170950, 28S—GenBank KU170940,
EF1α—GenBank KU289110.
Etymology—Lucianii = in honor of “Lucian”Edmund, a
Patamona fungal parataxonomist, who discovered the
type locality of G. lucianii and has since been invaluable
in collecting Guyanagaster specimens in the field.
Description—Figures 2 and 4a–g. Basidiomata gaster-
oid, subhypogeous to hypogeous, scattered, or in linear
troops, attached directly to woody roots of Eperua
falcata and Dicymbe altsonii trees; 12–34 mm broad, 8–
25 mm tall, globose to subglobose to ovoid and irregu-
larly broadly lobate, dense, base with smooth, sterile
concolorous stipe, 5–44 × 2–5 mm, attached directly to
the substratum. Peridium dark brown (8 F4-8 F3) to
black (8 F1) during all stages of development, moist,
tough, covered in 4–6 sided polygonal pyramidal warts
that come to a shallow point, these 0.8–3.1 mm broad,
0.5 mm tall, sharply differentiated from endoperidium;
endoperidium white (8A1) to light pink (8A2), tough,
0.5–1 mm thick, composed of matted hyphae transition-
ing evenly to sterile, white hyphal veins between the gle-
bal locules; gleba composed of well-defined locules and
intervening veins of hyphae; locules globose to ovate to
subangular, 0.2–2 mm broad, initially white (8A1), ma-
turing in stages to gold (3A2-4A5), light orange-pink
(7B6), to deep orange (8B8), often evenly, but rarely the
locules that border the peridium and columella take on
the darker shade first; hyphae separating locules initially
white (8A1), hyaline and waterlogged in mature speci-
mens; columella well-defined to not, base continuous
with stipe, 6–13 × 3–6 mm, initially white (8A1), turning
to brown (6D7), taking on the color of the gleba with
age, cottony to gelatinous. Exoperidium individual
hyphae of outer layer dark brown, individual hyphae
curved to tortuously curved, thick-walled, non-gelatinous,
18–99 × 4–11 μm. Endoperidium 700–800 μmthick,well-
differentiated from exoperidium and glebal locules, hyphae
Fig. 4 a–gGuyanagaster lucianii (BRG 41292 HOLOTYPE).aExterior showing dark brown peridium and irregularly lobed shape. bSubhypogeous
habit. cLongitudinal section of basidioma showing immature gleba, developed columella and elongated stipe. dMaturing gleba. eBasidiospores
of immature basidioma. fBasidiospores of mature basidioma. gMature gleba. Bar = one cm in a,b,c,dand g. Bar = 20 μmineand f
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 12 of 16

hyaline, 3–11 μm. Sterile hyphae separating locules similar
to endoperidial hyphae, hyaline, thin-walled, branching, 3–
5 um wide, organized in a parallel manner. Basidia not
observed. Basidiospores globose, dextrinoid; 21.4–30.4 ×
21.4–29.3 μm (mean = 25.1 ± 1.5 × 24.8 ± 1.5 μm, Q
r
=
0.95–1.10, Q
m
= 1.01 ± 0.04; n = 20); immature basidio-
spores hyaline when immature, then darkening to light pink
and then rusty brown with maturity. Columella hyphae
hyaline, thin-walled, not as tightly packed, some randomly
inflated at septa, 4–13 μm wide, random apical cells
inflated, 13–26 × 37–57 μm. Clamp connections absent in
all tissues. Tas t e not tested. Odor not tested.
Habit, habitat and distribution—Attached to decaying
woody roots of Dicymbe altsonii and Eperua falcata in
tropical rainforests. Known only from the type locality in
the Mabura Ecological Reserve of Guyana.
Specimens examined—Guyana. Region 10 Upper
Demerara-Berbice—Mabura Hill, elevation 161 m; vicinity
of basecamp, Eduba stand 4, four basidiomata, 11 June
2011, MCA 4424 (BRG 41294, PUL F3439); vicinity of
basecamp, patch 32, one basidioma found at the base of a
dead Eperua falcata, 24 December 2013, RAK 84 (BRG
41230, PUL F3440); vicinity of basecamp, patch 35, four
basidiomata found at the base of a dead Eperua falcata,
24 December 2013, RAK 88 (BRG 41293, PUL F2892);
2.5 km north of basecamp, patch 31, nine basidiomata
found along the decaying roots of Eperua falcata,25
December 2013, RAK 89 (BRG 41292, PUL F2891).
Commentary—Phylogenetic evidence suggests that
the two lineages of Guyanagaster represent distinct spe-
cies that diverged approximately 8 Ma. The sister species
share 87% (777/893 bp) nucleotide identity at the ITS
region and 96% (705/731 bp) nucleotide identity at LSU.
The drastic morphological changes that occur during
the maturation of Guyanagaster basidiomata (i.e. gleba
coloration) as well as the morphological plasticity between
individual basidiomata (e.g. basidioma size and shape,
columella dimensions) make delimiting species using
macromorphological characters unreliable. However, G.
lucianii can be distinguished from G. necrorhizus by its
consistently larger basidiospores. During all stages of mat-
uration, the basidiospores of G. lucianii measure between
21–30 × 21–29 μm, compared to those of G. necrorhizus,
which measure 15–19 × 15–18 μm. Additionally, the ba-
sidiospores of G. lucianii lack the pedicel that is readily ap-
parent on the basidiospores of G. necrorhizus (Fig. 4e–f).
Ecologically, G. lucianii appears to occupy the same
niche as G. necrorhizus. Basidiomata are hypogeous to
subhypogeous and growing from the roots of dead and
decaying trees. Both Guyanagaster species are white rot-
ters as the decaying roots to which they are attached are
all light-colored, spongy and sometimes gelatinous. The
known habitat for G. necrorhizus and G. lucianii are sep-
arated by only 125 km, so elucidation of their dispersal
strategy is needed to understand the evolutionary forces
that led to their speciation.
Additional files
Additional file 1: Collection data for the specimens used in the
phylogenetic analyses and GenBank accession numbers for the
corresponding sequence data, both generated for this study and
obtained from previous studies [10, 64, 83, 99–114]. (XLSX 54 kb)
Additional file 2: Single-locus and multi-gene phylogenies for the six
loci used. (PDF 3262 kb)
Additional file 3: Morphological description of Armillaria camerunensis
TH DJA 91 and TH 9926. (DOCX 108 kb)
Additional file 4: Results from the ancestral range estimation analysis.
(XLSX 38 kb)
Acknowledgements
The authors wish to thank the following for assisting with this study: Dillon
Husbands functioned as the Guyanese local counterpart and assisted with field
collecting, descriptions and specimen processing. Todd F. Elliott, J. J. Harrison,
Stephen D. Russell, Christian Schwarz and Tatyana Svetasheva provided
photographs for Fig. 2. Additional field assistance in Guyana was provided by C.
Andrew,V.Joseph,P.Joseph,F.Edmond,L.EdmondandL.Williams.In
Cameroon, Dr. Jean Michel Onana, Head of The National Herbarium of Cameroon
(Institute of Agricultural Research for Development, IRAD), provided much
logistical assistance. The Conservator of the Dja Biosphere Reserve, Mr.
Mengamenya Goue Achille, and his staff greatly assisted the fieldwork in the Dja.
Field assistance in Cameroon was provided by Alamane Gabriel (a.k.a. Sikiro),
Abate Jackson, Mamane Jean-Pierre, Mei Lin Chin, Todd Elliott, and Camille
Truong. Finally, the authors wish to thank Brandon Matheny and four anonymous
reviewers for their insightful comments on an earlier version of this manuscript.
Research permits were granted by the Guyana Environmental Protection Agency
and by the Cameroon Ministry of Research and Scientific Innovation. This paper is
number 217 in the Smithsonian Institution’s Biological Diversity of the Guiana
Shield Program publication series.
Funding
The authors wish to thank the following for financial support: National
Science Foundation DEB-0918591, NSF DEB-0732968, NSF DEB-1556412
and Linnaean Society Systematics Research grant to MCA, the National
Geographic Society’s Committee for Research and Exploration grant 9235–13
to TWH, Explorer’s Club Exploration Fund, Mycological Society of America Fun-
gal Forest Ecology grant, American Philosophical Society Lewis and Clark Fund
for Exploration and the T. Woods Thomas award to RAK.
Availability of data and materials
The alignment and tree files are available in TreeBase and can be accessed
at http://purl.org/phylo/treebase/phylows/study/TB2:S20469. Sequences
generated in this study were deposited in GenBank and accession numbers
for those sequences are available in Additional file 1.
Authors’contributions
RAK, AWW and MCA conceived and developed the study. RAK, OS, TWH and
MCA were involved in specimen collection and field descriptions. RAK performed
all of the molecular work and RAK and AWW performed the analyses. RAK, AWW,
and MCA wrote the paper. All authors read, advised on revisions and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Botany and Plant Pathology, Purdue University, West
Lafayette, IN 47907, USA.
2
Sam Mitchel Herbarium of Fungi, Denver Botanic
Gardens, Denver, CO 80206, USA.
3
Institute of Agricultural Research for
Development (IRAD), National Herbarium of Cameroon (MINRESI), PO Box
1601, Yaoundé, Cameroon.
4
Department of Biological Sciences, Humboldt
State University, Arcata, CA 95521, USA.
Koch et al. BMC Evolutionary Biology (2017) 17:33 Page 13 of 16

Received: 8 June 2016 Accepted: 10 January 2017
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