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Article
Phylogeny and taxonomy of the genus Amphichorda (Bionectri-
aceae): an update on beauveria-like strains and description of a
novel species from marine sediments
Daniel Guerra-Mateo 1, Josepa Gené 1,2* Vladimir Baulin 2,3 and José F. Cano-Lira 1,2
1 Unitat de Micologia i Microbiologia Ambiental, Facultat de Medicina i Ciències de la Salut, Universitat
Rovira i Virgili, Reus, Spain
2 Institut Universitari de Recerca en Sostenibilitat, Canvi Climàtic i Transició Energètica (IU-RESCAT), Uni-
versitat Rovira i Virgili, Tarragona, Spain
3 Física i Cristal·lografia de Materials, Escola Tècnica Superior d'Enginyeria Química, Universitat Rovira i
Virgili, Tarragona, Spain.
* Correspondence: josepa.gene@urv.cat; Tel.: +34-977759359
Abstract: The genus Amphichorda has been recently re-erected as an independent linage from Beau-
veria, circumscribed within Cordycipitaceae. However, its phylogenetic relationships with other
members of this family remain obscure. In our on-going efforts to expand the knowledge on the
diversity of culturable ascomycetes from the Mediterranean Sea, we isolated several specimens of
Amphichorda. Preliminary sequence analyses revealed great phylogenetic distance with accepted
Amphichorda species and a close relation to Onychophora coprophila. Onychophora is a monotypic ge-
nus of enteroblastic conidiogenous cells, presumably related to Acremonium (Bionectriaceae); while
Amphichorda exhibits holoblastic conidiogenesis. Here, we examine representative strains of Amphi-
chorda species to resolve the taxonomy of the genus and the above-mentioned fungi combining mor-
phological, ultrastructure and multi-locus phylogenetic analyses (ITS, LSU, tef1, BenA). The results
revealed Amphichorda as a member of the Bionectriaceae, where its asexual morphs represent a tran-
sition between enteroblastic and holoblastic conidiogenesis for this group of fungi. We also describe
and illustrate Amphichorda littoralis sp. nov., and propose the new combination Amphichorda coproph-
ila. In addition, we stablish key phenotypic features to distinguish Amphichorda species and demon-
strate the higher salt tolerance degree of A. littoralis, consistent with its marine origin. This work
provides a comprehensive framework for future studies in the genus.
Keywords: Ascomycota; asexual fungi; marine fungi; multi-locus phylogeny; new taxa; taxonomy;
ultrastructure
1. Introduction
The order Hypocreales (Sordariomycetes, Pezizomycotina, Ascomycota) currently com-
prises around 300 genera distributed across 17 families. Their species inhabit a wide range
of substrates in terrestrial and aquatic (marine and freshwater) environments, and they
show a great variety of lifestyles, such as saprobic, endophytic, and pathogenic fungi for
plants and animals, including humans [1,2]. The Cordycipitaceae is one of the most complex
families in the order due to the pathogenic behavior of most of its species, which includes
a wide range of invertebrate hosts and results in a variety of morphological features of the
sexual morph, primarily associated with its ascomata (stroma and perithecia). The asexual
morph, however, are very similar, most frequently showing phialidic conidiogenous cells.
Therefore, genera like Amphichorda, Beauveria, Cordyceps or Isaria have been difficult to cir-
cumscribe and recent works have dealt with numerous taxonomical problems [3,4]. In
particular, the genus Amphichorda has been traditionally accepted as a member of
Cordycipitaceae based on the taxonomical history of its type species, Amphichorda felina (=
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© 2023 by the author(s). Distributed under a Creative Commons CC BY license.
Beauveria felina). Despite this, the most recent multi-locus phylogeny of the family has re-
solved Amphichorda as a sister linage to the main Cordycipitaceae clade [4]. Indeed, the tax-
onomic status of Amphichorda has been controversial since its original description.
The genus Amphichorda was described by Fries in 1825 and typified by A. felina, a
fungus isolated from cat dung in France and previously classified in the genus Clavaria
[5,6]. The morphological description stated the white farinaceous color of the colonies and
the production of filiform conidiogenous cells, which inspired the name of the genus. In
1832, A. felina was transferred to the genus Isaria, a genus that at this time lacked a type
species and, therefore, comprised morphologically heterogenous fungi [7,8]. In 1972, de
Hoog re-defined Isaria based on the production of synnemata and, following von Arx,
accepted Isaria felina as the lectotype of the genus [9]. This circumscription, however, was
rejected due to the previous lectotypification with the species Isaria farinosa [8,10]. Isaria
felina was then transferred to the genus Beauveria based on the morphological resemblance
of their holoblastic conidiogenous cells. Although, early phylogenetic analyses suggested
great dissimilarity between B. felina and other Beauveria species [8]. Recently, the genus
Amphichorda has been re-erected for the description of two novel species, Amphichorda cav-
ernicola and Amphichorda guana, by Zhang et al. [11,12]. Zhang’s studies represent the first
phylogenetic backbone for the genus Amphichorda and demonstrate the great phylogenetic
distance between Amphichorda and Beauveria. Despite this, morphological differences be-
tween Amphichorda species are confusing and the phylogenetic relationships between Am-
phichorda and other genera remain obscure. Moreover, the type material of A. felina seems
to be lost. Zhang et al. considered the strain CBS 250.34, the type of Isaria cretaceae, as the
type strain of A. felina [12]. Isaria cretaceae was synonymized with A. felina by de Hoog [9],
however, so far this strain has not been designated as the epitype of the species. Never-
theless, the type strain of I. cretacea was isolated from a package of moldy pressed yeast
from Epsom, England [13] and, according to the criteria of substrate and geographical
similarity required for fungal epityfication [14], the strain CBS 250.34 could not represent
A. felina on the basis of its coprophilous origin from France [5,6]. Thus, the taxonomical
status and the phylogenetic relationships of Amphichorda need to be revised.
In our latest efforts to expand the knowledge on the diversity of culturable ascomy-
cetes from the Mediterranean Sea, we isolated several interesting specimens of an amphi-
chorda-like fungus. A preliminary sequence analysis of the nuclear ribosomal operon (i.e.,
the 28S large ribosomal subunit-LSU, and the internal transcribed spacer -ITS, including
the 5.8S rDNA gene) revealed that these specimens belong to the genus Amphichorda, but
they did not fit into any of the described species. This preliminary sequence analysis also
revealed that the marine strains were closely related to Onychophora coprophila. Ony-
chophora is a monotypic genus, which conidiogenous apparatus morphologically resem-
bles Amphichorda species. However, the conidiogenous cells of the former were described
as enteroblastic (phialidic), while Amphichorda exhibits holoblastic conidiogenesis
[11,12,15]. In addition, according to Mycobank and Index Fungorum databases, O.
coprophila is a fungus of uncertain position among Ascomycota, although, the original au-
thors suggested a possible relation to Acremonium (Bionectriaceae) [15].
The aim of the present study was, therefore, to clarify the taxonomy of the above-
mentioned fungi based on morphological features, including ultrastructure, and multi-
locus phylogenetic analyses inferred with sequences of the nuclear markers available for
Amphichorda species. These are the ITS and LSU regions of the rDNA and partial fragments
of the translation elongation factor 1-α (tef1) and the β-tubulin (BenA) regions. Then, we
used these nuclear markers to determine the strains phylogenetically related to our strains
and examined their available living cultures in order to assess the diversity within Amphi-
chorda. In this work, we provide and update on the morphological and molecular diversity
of Amphichorda, determine its phylogenetic relationships with other genera in Hypocreales
and discuss the type strain for A. felina.
2. Materials and Methods
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2.1. Sampling and strains
Sediment samples were collected from the Mediterranean Sea in two points of the
Tarragona coast, the Miracle and Arrabassada beaches, through 2021 and 2022. Each beach
was sampled twice, the Miracle beach on June and October 2021 and the Arrabassada
beach on February and June 2022. These beaches are located in the southern part of Cata-
lonia, right next to the port of Tarragona, the fifth most important harbor in Spain and an
important stop for tourism cruise ships [16].
Marine sediments were collected following the same methodology at both locations.
We stablished four collection points based on the sediment grain size and depth in the
column of water. The first point was at 6 meters of depth (sand sediment), the second at
13 m (sand sediment), the third at 20 m (transition between sand and silt sediment) and
the last point was at 30 m of depth (silt sediment). Four sub-samples were collected at
each point ca 15 cm below the surface of the sea bed, using 50 mL sterile plastic containers,
which were transported in a refrigerated container to the laboratory and processed imme-
diately. For each sampling point, sediment sub-samples were mixed and vigorously
shaken in a container; then, after 1 min at rest, the water was decanted and the sediment
was poured into plastic trays with several layers of sterile filter paper to remove excess
water.
Three agar media were used to achieve a greater ascomycetous diversity in culture
and restrict the growth of certain fungal groups: dichloran rose-bengal-chloramphenicol
agar (DRBC; 5 g peptone, 10 g glucose, 1 g KH2PO4, 0.5 g MgSO4, 25 mg rose-bengal, 200
mg chloramphenicol, 2 mg dichloran, 15 g agar, 1 L distilled water); 3% malt extract agar
supplemented with sea water (SWMEA3%; 30 g malt extract, 5 g mycological peptone, 15
g agar, 1L sea water) was used as a suitable medium for the isolation of marine fungi [17]
and potato dextrose agar (PDA; Condalab, Madrid, Spain) supplemented with 2 g/L of
cycloheximide (PDA+A) was used to isolate strains resistant to this protein synthesis in-
hibitor, a frequent trait among fungi [18]. Both SWMEA3% and PDA+A culture media
were supplemented with 5 mL of chloramphenicol (15 g/L ethanol) to prevent bacterial
growth.
The culture methodology was as follows: 1 g of sediment from each sampling point
was distributed across two Petri dishes and mixed with melted SWMEA3% at 45 °C; the
same procedure was used to mix the sediment with PDA+A. In the case of the sediment
mixed with DRBC, only 0.5 g of sediment from each sampling point was distributed across
two Petri dishes to deal with fast growing fungi. A set of the plates of the different culture
media was incubated at 22–24 °C and the other set at 15 °C to enable the detection of slow-
growing fungi. Plates were stored in darkness and examined weekly by stereomicroscope
for 5–8 weeks. Pure cultures were obtained from colony fragments or conidia of the fungi
growing on primary plate cultures using a sterile dissection needle. These fragments were
cultured on PDA and incubated at 25 °C in darkness. These PDA cultures were used for a
preliminary morphological identification before DNA extraction.
Living cultures of putative novel or rare fungi were preserved and deposited in the
culture collection of the Faculty of Medicine in Reus (FMR, Spain) for further studies. Tax-
onomic information and nomenclature for the new species were deposited in MycoBank.
Cultures from ex-type strains and holotypes, which consisted of dry colonies on the most
appropriate media for their sporulation, were also deposited at the Westerdijk Fungal Bi-
odiversity Institute in Utrecht (CBS, The Netherlands).
In addition to the strains from marine sediments, we revived another amphichorda-
like fungus from our fungal culture collection (FMR 17952). This strain was isolated from
a fragment of a rubber tire floating in the seawater of the Miracle beach in July 2020. The
tire fragment was washed three times with 10% NaClO (bleach) for 30 s, cut in small pieces
and cultured on DRBC at 25 °C in darkness. We also examined several strains of A. felina,
available in the CBS culture collection and labelled as Beauveria felina (i.e., CBS 110.08, CBS
250.34, CBS 312.50, CBS 648.66 and CBS 173.71), to study the morphological and molecular
variability of this species and for epityfication purposes based on the lack of a type strain
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for this species. Furthermore, the ex-type strain and a reference strain of O. coprophila (i.e.,
CBS 247.82 and CBS 424.88) were also added to the study due to the above-mentioned
reasons (Table 1).
2.2. Phenotypic analysis
Microscopic characterization was performed from strains growing on oatmeal agar
(OA; 30 g oatmeal, 15 g agar, 1 L distilled water) after 14 days at 25 °C in darkness. Micro-
scopic slides were mounted with lactic acid and observed with an Olympus BH-2 bright
field microscope (Olympus Corporation, Tokyo, Japan). In species descriptions, size
ranges of relevant structures derived from at least 30 measurements. Photomicrographs
were obtained using a Zeiss Axio-Imager M1 light microscope (Zeiss, Oberkochen, Ger-
many) with a DeltaPix Infinity digital camera. Due to the difficulties in observing the co-
nidiogenic patterns in Amphichorda and Onychophora under light microscope, representa-
tive strains of species of both genera were examined under Scanning Electron Microscopy
(SEM) using the Quanta 600 FEG Scanning Electron Microscope (Thermo Fisher Scientific,
Waltham, Massachusetts, USA). The specimens were processed in accordance to Figueras
& Guarro [19].
Macroscopic characterization of the colonies was made on PDA, OA and synthetic
nutrient-poor agar (SNA; 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO47·H2O, 0.5 g KCl, 0.2 g
glucose, 0.2 g sucrose, 15 g agar, 1 L distilled water) after 14 days at 25 °C in darkness.
Color notations in descriptions followed Kornerup and Wanscher [20]. Photoplates were
assembled using GIMP v.2.10.34 (GNU Image Manipulation Program).
In addition, we assessed the ability of Amphichorda species to grow at different tem-
peratures by culturing the strains on PDA from 5 to 40 °C, at intervals of 5 °C. Colony
diameter was measured after 14 days in darkness. Moreover, the marine strains were com-
pared with their phylogenetically related taxa to test a possible adaptation to the marine
environment (salt tolerance). Cultures were carried out on solid agar plates of malt extract
agar (MEA; 20 g malt extract, 15 g agar, 1 L distilled water) and MEA supplemented with
3.5% (35 ppt, the salt concentration in the marine environment), 5%, 10% and 15% NaCl.
We used the strain Aspergillus chevalieri FMR 19829 (obtained from the marine sediments
of this study) as a positive control of the ability to grow at high NaCl concentrations. Col-
ony diameter was measured after 14 days (data not shown) and after 28 days at 25 °C. All
tests were performed in duplicate, and the results represent the mean of the colony diam-
eter between duplicate plates.
2.3. DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted through the modified protocol of Müller et al. [21]
and quantified using Nanodrop 2000 (Thermo Scientific, Madrid, Spain). Four loci were
used and amplified with the following primer pairs: ITS and LSU barcodes of the nrDNA
with ITS5/LR5 [22,23], partial fragments of the BenA gene with T10/Bt2b [24] and the tef1
gene with EF-983F/EF-2218R [25], respectively. Briefly, PCR conditions for ITS, LSU, BenA
and tef1 were set as follows: an initial denaturation at 95 °C for 5 min, followed by 35
cycles of 30 s at 95 °C, 45 s at 56 °C, and 1 min at 72 °C, and a final extension step at 72 °C
for 10 min. PCR products were purified and sequenced at Macrogen Corp. Europe (Ma-
drid, Spain) with the same primers used for amplification. Consensus sequences were as-
sembled using SeqMan v. 7.0.0 (DNAStar Lasergene, Madison, Wisconsin, USA).
The preliminary species identification of the strains was performed by comparing
their ITS regions with those available at the National Center for Biotechnology Infor-
mation (NCBI) using the Basic Local Alignment Search Tool (BLAST;
https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 October 2022). A maximum similar-
ity level of ≥ 99% with ≥ 90% of sequence cover was used for species-level identification.
Lower similarity values were considered as putative unknown fungi, and their taxonomic
position was assessed with the loci mentioned above.
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The sequences used for species-level identification were obtained from GenBank. In
Table 1 are listed the strains of Amphichorda and Onychophora and their GenBank accession
numbers. In Table S1 (supplementary material) is included the information about repre-
sentative strains of Bionectriaceae, Cordycipitaceae and outgroups used in the phylogenetic
analyses.
2.4. Phylogenetic analyses
The phylogenetic relationships of the strains examined in the present study were as-
sessed using the ITS, LSU and tef1 regions. Sequences from each gene region were aligned
individually in MEGA (Molecular Evolutionary Genetics Analysis) software v.6.0 [26], us-
ing the ClustalW algorithm [27] and, when necessary, refined with MUSCLE [28] or ad-
justed manually. Before combining the regions, the phylogenetic concordance between
each individual phylogeny was tested through visual comparison to assess incongruent
results among clades with high statistical support. When the concordance was confirmed,
individual alignments were concatenated into a single data matrix. The partial fragments
of the BenA gene were excluded from the phylogenetic analyses due to the limited avail-
ability of this region within the Bionectriaceae and most notably on taxa phylogenetically
close to Amphichorda. However, BenA sequences were used to assess the similarity be-
tween Amphichorda species.
Maximum-likelihood (ML) and Bayesian analysis (BA) were used for phylogenetic
inference of individual sequence alignments and the concatenated alignment (ITS-LSU
and ITS-tef1). ML analyses were conducted using the CIPRES Science Gateway portal v.
3.3 (https://www.phylo.org/; [29]) and RAxML-HPC2 on XSEDE v. 8.2.12 [30] with default
GTR substitution matrix and 1000 rapid bootstrap replications. Additional ML analyses
were performed using IQ-TREE v. 2.1.2 [31,32] with ultrafast bootstrapping for the esti-
mation of branch support [33]. The most suitable evolutionary model for each partition
was estimated using ModelFinder [34,35], implemented in IQ-TREE. Bootstrap support
(bs) ≥ 70 was considered significant [36]. Bayesian analyses were performed using
MrBayes v. 3.2.6 [37]. The best substitution model for each locus was estimated using
jModelTest v.2.1.3 following the Akaike criterion [38,39]. Markov Chain Monte Carlo sam-
pling (MCMC) was performed for 10 million generations using four simultaneous chains
(one cold chain and three heated chains) starting from a random tree topology. Trees were
sampled every 1000 generation or until the run was stopped automatically when the av-
erage standard deviation of split frequencies fell below 0.01. The first 25 % of the trees
were discarded as the burn-in phase of each analysis, and the remaining trees were used
to calculate posterior probabilities (pp). A pp value of ≥ 0.95 was considered significant
[40]. The resulting trees were plotted using FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/soft-
ware/figtree/, accessed on 5 December 2022). The DNA sequences generated in this study
were deposited in GenBank (Table 1) and the alignments were submitted to Zenodo
(https://doi.org/10.5281/zenodo.7937438).
3. Results
Among the fungi detected from marine sediments collected at different depths, we
recovered four strains (FMR 19404, FMR 19611, FMR 20067 and FMR 20149) exclusively
from samples collected at 20 m of depth in both the Miracle and Arrabassada beaches,
using SWMEA3% and DRBC culture media. These strains and FMR 17952, the latter iso-
lated from a rubber tire floating in seawater, were morphologically identified as Amphi-
chorda sp. However, although they showed the typical morphological features of the ge-
nus (i.e. synnematous and mononematous conidiophores, flask-shaped with a strongly
bent neck conidiogeous cells and solitary conidia that remain attached to the apex of the
conidiogenous cell), they exhibited some morphological traits that did not exactly fit into
any of the accepted species of Amphichorda.
Table 1. GenBank accesions of the Amphichorda strains included in the present study.
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Species
Strain number
Substrate (country)
GenBank accesion numbers1
Citation
ITS
LSU
tef1
BenA
A. cavernicola
CGMCC3.19571T
Bird faeces (China)
MK329056
MK328961
MK335997
NA
[12]
LC 12560
Animal faeces (China)
MK329061
MK328966
MK336002
NA
[12]
LC 12674
Plant debris (China)
MK329065
MK328970
MK336006
NA
[12]
A. coprophila
CBS 247.82T (ex-type
of O. coprophila)
Rabbit dung (England)
MH861494
MH873238
OQ954487
pending
[41]; this
study
CBS 424.88 (received
as O. coprophila)
Chipmunk dung
(Canada)
OQ942929
OQ943166
OQ954488
pending
This
study
CBS 173.71 (received
as B. felina)
Porcupine dung
(Canada)
AY261368
MH871833
OQ954489
pending
[41]; this
study
A. felina
CBS 250.34 (ex-type of
I. cretacea)
Pressed yeast (England)
MH855498
OQ943167
OQ954490
pending
[41]; this
study
CBS 648.66 (received
as B. felina)
Unknown, (Argentina)
OQ942930
MH870575
OQ954491
pending
[41]; this
study
CBS 110.08 (received
as B. felina)
Unknown
MH854578
OQ943168
OQ954492
pending
[41]; this
study
A. guana
CGMCC3.17908T
Bat guano (China)
KU746665
KU746711
KX855211
NA
[11]
CGMCC3.17909
Bat guano (China)
KU746666
KU746712
KX855212
NA
[11]
CBS 312.50 (received
as B. felina)
Rabbit dung (Unknown)
MH856641
MH868150
OQ954493
pending
[41]; this
study
A. littoralis
FMR 17952
Floating rubber tire
(Spain)
OQ942925
OQ943162
OQ954483
pending
This
study
FMR 19404T
Marine sediment (Spain)
OQ942924
OQ943161
OQ954482
pending
This
study
FMR 19611
Marine sediment (Spain)
OQ942926
OQ943163
OQ954484
pending
This
study
FMR 20067
Marine sediment (Spain)
OQ942927
OQ943164
OQ954485
pending
This
study
FMR 20149
Marine sediment (Spain)
OQ942928
OQ943165
OQ954486
pending
This
study
CBS: Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands;
CGMCC: China General Microbiological Culture Collection Center, China; FMR: Facultat de Me-
dicina i Ciències de la Salut, Reus, Spain. T indicates ex-type strains. “NA” indicates sequences not
used in this study. 1ITS: Internal transcribed spacer region of the rDNA and 5.8S gene; LSU: 28S
large ribosomal subunit; tef 1: translation elongation factor 1α; BenA: tubulin. Sequences generated
in this study are highlighted in bold.
3.1. Phylogeny
The molecular identification based on the BLAST search of our five unidentified
strains revealed a high percentage of similarity with species of the genus Amphichorda us-
ing ITS sequences. Specifically, the percentage of identity was of 98% with A. cavernicola
(CGMCC 3.19571) and between 95-97% with other species of this genus. The molecular
comparison using the LSU region revealed a 99% of similarity to O. coprophila (CBS
247.82), A. cavernicola (CGMCC 3.19571) and between 98-99% of similarity with other spe-
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 May 2023 doi:10.20944/preprints202305.1524.v1
cies of the genus Amphichorda. Other taxa closely related to our strains with a 97% of iden-
tity with this locus were Nigrosabulum globosum (CBS 512.70), Acremonium (Ac.) curvum
(GZUIFR 22.035) and Ac. alternatum (CBS 407.66). Members of the genera Beauveria and
Cordyceps did not match with our sequences in the BLAST results. Despite this, we in-
cluded them in the phylogenetic analysis due to the traditional placement of Amphichorda
as a member of Cordycipitaceae. In addition, sequence analyses of these two gene markers
allowed us to confirm or reidentify the CBS reference strains of A. felina as following: CBS
110.08 and CBS 648.66 as A. felina, CBS 312.50 as A. guana, and CBS 173.71 as O. coprophila.
Based on the BLAST results, we assessed the phylogenetic relationships among gen-
era phylogenetically related to Amphichorda with the ITS and LSU regions. The resulting
tree topologies from the individual analyses of these two gene markers were similar and
did not show incongruences. Therefore, both alignments were concatenated into a single
matrix. The final alignment of the concatenated ITS and LSU regions comprised 65 taxa
that included two representative strains from each Amphichorda species, as well as, two
representative strains from O. coprophila and the strains recovered from the marine envi-
ronment to prevent branch imbalance; together with representative species belonging to
the families Bionectriaceae and Cordycipitaceae. The tree was rooted with Pochonia chlamyd-
ospora (CBS 504.66) and Metapochonia suchlasporia (CBS 251.83) as outgroup. The total
length comprised 1435 characters including gaps (ITS: 624, LSU: 811 characters). Among
these, 906 characters were conserved sites (ITS: 255, LSU: 651), 529 characters were varia-
ble sites (ITS: 369, LSU: 160) and 417 characters were parsimony informative (ITS: 290,
LSU: 127). For the ML analyses, the best fit models were TIM2+F+I+G4 for the ITS region
and TIM2e+I+G4 for the LSU region. For the BI analysis, the best fit models were GTR+I+G
for both the ITS and LSU region. Here, we represented the Maximum Likelihood (RAxML)
tree with the bootstraps support values of the ML analyses (RAxML and IQ-TREE) and
Bayesian posterior probabilities at the nodes. The resulting phylogenetic tree resolved the
genus Amphichorda as a monophyletic linage within the family Bionectriaceae (Fig. 1), being
closely related with a well-supported clade that comprised two accepted genera in the
family, Nigrosabulum and Hapsidospora, together with a recently described Acremonium
species, Ac. curvum [42]. This latter species was, however, placed very distantly from the
genus Acremonium s. str. The concatenated analysis defined five terminal clades within
Amphichorda, where two marine strains (FMR 19404 and FMR 17952), representatives of
our unidentified Amphichorda species, and those strains of O. coprophila (CBS 173.71 and
CBS 247.82) represented two independent Amphichorda linages. However, these molecular
markers lacked resolution to determine the phylogenetic relationships among Amphi-
chorda species. Therefore, we performed a phylogenetic analysis combining the ITS and
LSU regions and the elongation factor (tef1) gene in order to delineate Amphichorda species
with precision.
The individual ITS, LSU and tef1 alignments were concatenated into a single matrix,
because the resulting individual trees represented similar topologies. The final ITS, LSU
and tef1 alignment comprised the five unidentified Amphichorda strains, nine strains rep-
resentatives of the known Amphichorda species, and three strains identified as O. coproph-
ila. Acremonium curvum (GZUIFR 22.035), Ac. globosisporium (GZUIFR 22.036) and Ac. scle-
rotigenum (A101) were used as outgroup. The total length comprised 2168 characters in-
cluding gaps (ITS: 506, LSU: 779, tef1: 883 characters). Among these, 1865 characters were
conserved sites (ITS: 393, LSU: 727, tef1: 745), 303 characters were variable sites (ITS: 113,
LSU: 52, tef1: 138), and 166 characters were parsimony informative (ITS: 55, LSU: 30, tef1:
81). For the ML analyses, the best fit models were TNe+G4 for the ITS region, TNe+I for
the LSU region and TN+F+G4 for the tef1 region. For the BI analysis the best fit models
were K80+I for both the ITS and LSU regions and GTR+G for the tef1 region. Here, we
represented the Maximum Likelihood (RAxML) tree with the bootstraps support values
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Figure 1. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on
a concatenated alignment of ITS and LSU sequences of 65 strains representing Bionectriaceae,
Cordycipitaceae and outgroups. Numbers at the branches indicate support values (RAxML-BS / IQ-
TREE-BS / BI-PP) above 70 % / 90 % / 0.95. The genus Amphichorda is printed in bold and the other
strains are collapsed based on their genera. Bold branches indicate full support values (100/100/1).
T indicates ex-type strains. The tree is rooted to Metapochonia suchlasporia CBS 251.83 and Pochonia
chlamydosporia CBS 504.66. Quote marks indicate strains with unresolved taxonomy. The scale bar
represents the expected number of changes per site.
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of the ML analyses (RAxML and IQ-TREE) and Bayesian posterior probabilities at the
nodes. The resulting phylogenetic tree resolved the three species currently accepted in
Amphichorda (A. cavernicola, A. felina and A. guana) as independent linages (Fig. 2). The five
marine strains delineated an undescribed linage within Amphichorda, closely related to the
clade representative of O. coprophila. The marine strains are proposed below as Amphi-
chorda littoralis and O. coprophila is accepted as an Amphichorda species. A detailed mor-
phological characterization of the novel fungi is provided in the taxonomy section.
Figure 2. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on
a concatenated alignment of ITS, LSU and tef1 sequences of 20 strains representing Amphichorda and
outgroups. Numbers at the branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP)
above 70 % / 90 % / 0.95. Bold branches indicate full support values (100/100/1). The novel species
and combination are printed in bold. T indicates ex-type strains. The tree is rooted to Acremonium
curvum (GZUIFR 22.035), Acremonium globosisporium GZUIFR 22.036 and Acremonium sclerotigenum
A101. The scale bar represents the expected number of changes per site.
3.2. Morphological analysis
In order to perform a morphological comparison of our strains, we reviewed the ex-
isting literature on Amphichorda and examined living cultures of the following species: A.
coprophila (CBS 247.82, CBS 424.88 and CBS 173.71), A. felina (CBS 250.34, CBS 110.08 and
CBS 648.66) and A. guana (CBS 312.50). Unfortunately, strains of A. cavernicola were not
available for comparison. The colony color displayed across PDA, OA and SNA culture
media represented the most accurate character to distinguish species. Microscopically, the
reproductive structures were almost similar between species and consisting in flask-
shaped conidiogenous cells that produced subglobose and smooth conidia. Although,
there were subtle differences on the size range of these structures, they overlapped be-
tween species. In Table 2 we provide a synopsis of the key morphological characters that
allow discrimination among species of Amphichorda.
Table 2. Synopsis of the morphological characters defining Amphichorda species.
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Species
Colony on PDA*
Colony on OA/SNA*
Microscopic features
Citation
Color
Diffusible
pigment
Color
Conidiogenous
cells size (µm)
Conidia size (µm)
A. cavernicola
Cream yellow
to sea shell
Not
observed
White
4.5-8 x 2-3
2.5-4 x 2-3.5
[12]; this
study
A. coprophila
Orange to
brownish
orange
Greyish
orange
Light yellow
6-10 x 2-2.5
3.5-5.5 x 2-2.5
[15]; this
study
A. felina
White
Greyish
orange
White
3-8.5 x 2-2.5
2.5-4.5 x 2-3.5
[9]; this
study
A. guana
White to
yellowish
Yellowish
White
7-10 x 2-3
4.5-5.5 x 3.5-5
[11]; this
study
A. littoralis
Greenish
yellow
Light
yellow
Greenish yellow
5.5-11.5 x 1.5-2.5
2.5-4 x 2.5-3
This study
*Obverse side of the colony.
However, the pattern of conidiogenesis could not be properly determined under light
microscopy. Therefore, we selected representative strains of A. coprophila, A. felina, A.
guana and A. littoralis for examination under SEM (Fig. 3).
Figure 3. Scanning electron microscopy (SEM) of the reproductive structures from representa-
tive Amphichorda species. (A) Conidiophores, holoblastic conidiogenous cells and conidia in Amphi-
chorda felina (CBS 250.34). (B) Holoblastic conidiogenous cell in Amphichorda guana (CBS 312.50). (C)
Conidiophores, holoblastic conidiogenous cells and conidia in Amphichorda littoralis (FMR 20067).
(D) Conidiophores, holoblastic conidiogenous cells and conidia from Amphichorda coprophila (ex-
type CBS 247.82). (E, F) Enteroblastic (phialidic−arrows) and holoblastic conidiogenous cells in Am-
phichorda coprophila (CBS 424.88), respectively. The black arrow points at the enteroblastic collarette.
Scale bars: (A, C, D) = 5 µm; (B, E, F) = 2.5 µm.
Almost all strains of Amphichorda species mentioned above showed a holoblastic pat-
tern of conidiogenesis, producing few conidia in an apparent sympodial proliferation.
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However, the strains of A. coprophila differed in that, while the ex-type strain CBS 247.82
showed exclusively holoblastic conidiogenesis (Fig. 3D), CBS 424.88 exhibited both holo-
blastic (Fig. 3F) and enteroblastic (phialidic) conidiogenous cells even showing a small
collaret in the apex (Fig. 3E). Another difference observed in these latter strains was the
presence of both smooth or roughened conidiogenous cells. In the rest of the species ex-
amined the conidiogenous cells were smooth.
3.3. Salt tolerance test
Considering the marine origin of A. littoralis, we compared its ability to grow under
different NaCl concentrations with other species of the genus, predominantly isolated
from terrestrial environments, to ascertain a possible preference for the marine environ-
ment. Although, all Amphichorda strains managed to grow up to 10% NaCl, each species
showed different colony diameters across media. The colony diameter of A. coprophila, A.
felina and A. guana decreased in an inverse proportion to the addition of NaCl. The only
exception was the strain CBS 247.82 of A. coprophila, which growth was restricted and
nearly similar across media with different salt concentrations. Amphichorda littoralis
achieved similar maximum colony diameters across MEA and MEA supplemented with
3.5% and 5% NaCl. In particular, the strains FMR 19404, FMR 19611 and FMR 20067
reached the maximum colony diameter in MEA supplemented with 3.5% and 5% NaCl
(Fig. 4).
Figure 4. Mean colony diameter achieved by representative Amphichorda strains on malt extract
agar (MEA) supplemented with different concentrations of NaCl after 4 weeks at 25 °C. The strain
Aspergillus chevalieri (FMR 19829) was used as a positive control of growth in the different culture
media. On the right side, culture plates on MEA5% and MEA10% are represented for representative
strains from each species.
3.3. Taxonomy
Amphichorda Fries, Systema Orbis vegetalis 1:170 (1825)
= Onychophora W. Gams, P.J. Fisher & J. Webster, Transactions of the British Myco-
logical Society 82 (1): 174 (1984)
Type species. Amphichorda felina (DC) Fries, Systema Orbis vegetalis 1: 170 (1825).
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For synonyms of the species see Mycoblank database (https://www.mycobank.org/).
Emended description
Asexual morph with conidiophores synnematous or mononematous, semi-macronem-
atous, erect, straight or flexuous, bearing lateral or terminal conidiogenous cells, arranged
single or in whorls, sometimes micronematous and reduced to conidiogenous cells grow-
ing directly from vegetative hyphae. Conidiogenous cells flask-shaped, usually with a
strongly bent neck, holoblastic, rarely enteroblastic, phialidic, hyaline, smooth-walled or
roughened. Conidia solitary, often remaining attached to the apex of the conidiogenous
cell, subglobose, hyaline, smooth-walled. Sexual morph not observed.
Amphichorda coprophila (W. Gams, P.J. Fisher & J. Webster) Guerra-Mateo, Cano &
Gené, comb. nov.
Mycobank: MB848789.
Basionym. Onychophora coprophila W. Gams, P.J. Fisher & J. Webster, Transactions of
the British Mycological Society 82 (1): 174 (1984)
Type. ENGLAND, Devon, Dawlish Warren, from rabbit dung incubated at relative
humidity of 95% for several weeks, Dec. 1981, J. Webster (holotype CBS H-1740 = IMI
275663, ex-type culture CBS 247.82).
Asexual morph described in Gams et al. [15].
Culture characteristics (after 14 days at 25 °C). Colonies on PDA attaining 22-24 mm
diam., slightly raised, irregularly sulcated, glabrous and brownish orange (7C5) at center
(CBS 424.88 and CBS 173.71 orange (5B5)), velvety and white at periphery, margin crenate;
reverse brownish orange at center and white at periphery; diffusible pigment greyish or-
ange (6B5). On OA, colonies reaching 34–40 mm diam., flat, velvety, pale yellow (4A3) at
center to white at periphery, margin entire and slightly lobated; reverse pale yellow. On
SNA, colonies reaching 5–10 mm diam., glabrous, pale yellow, margin slightly lobated;
reverse pale yellow.
Additional specimens examined. CANADA, Ontario, Landmark County, along Clyde
River, from chipmunk dung, K.A. Seifert (CBS 424.88); ibid., Stoneleigh, from porcupine
dung, Sep. 1969, R.F. Cain and D.W. Malloch (CBS 173.71).
Notes. Amphichorda coprophila is a well-supported species that represents a distant in-
dependent linage in the genus Amphichorda (Figs 1, 2). It can be morphologically distin-
guished by its orange to brownish orange colonies on PDA (Table 2), the production of
conidia through both holoblastic and phialidic conidiogenous cells (Fig. 3 D-F) and the
occasional rough ornamentation of the conidiogenous cells under SEM.
Amphichorda littoralis Guerra-Mateo, Torres-Garcia, Cano & Gené, sp. nov. Fig. 5.
Mycobank: MB 848035.
Etymology. Name refers to the area where this species was isolated, Mediterranean
coast (Tarragona, Spain).
Type. SPAIN, Catalonia, Mediterranean coast, Tarragona, Platja del Miracle, N
41º6´19´´, E 1º15´37´´, from sediments at 20 m of depth, Jun. 2021, G. Quiroga-Jofre and D.
Guerra-Mateo (holotype CBS H-25254, ex-type culture FMR 19404, CBS 149935).
Asexual morph on OA. Mycelium composed of smooth-walled, branched, septate, hy-
aline, 1–1.5 µm wide hyphae. Conidiophores monomematous, rarely synnematous, arising
directly from superficial mycelium, micronematous and reduced to conidiogenous cells
growing directly or on a short lateral protrusion from vegetative hyphae, or semi-mac-
ronematous, erect, straight or flexuous, commonly unbranched, bearing lateral or termi-
nal conidiogenous cells, arranged single or in whorls of 2–4, hyaline and smooth-walled;
synnematous conidiophores only observed in FMR 20067 on PDA at the margin of the
colony, yellowish white, cylindrical with tomentose apex. Conidiogenous cells flask-shaped,
usually with a strongly bent neck, 6–10(–11.5) × 1.5–2 µm, hyaline, smooth-walled. Conidia
solitary, often remaining attached to the apex of the conidiogenous cell, subglobose, 3–4 x
2.5–3 µm, hyaline, smooth-walled. Sexual morph not observed.
Culture characteristics (after 14 days at 25 °C). Colonies on PDA attaining 20 mm
diam., slightly raised, irregularly sulcated, glabrous and greenish yellow (1A7) at center,
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velvety (fasciculate in FMR 20067) and white at periphery, margin crenate; reverse green-
ish yellow (1A7) at center and white at periphery; diffusible pigment light yellow (4A4)
produced after 21 days. On OA, colonies reaching 30–32 mm diam., flat, velvety, greenish
yellow at center to greyish yellow at periphery, margin entire and slightly lobated; reverse
greenish yellow (1A7). On SNA, colonies reaching 9–14 mm diam., glabrous, greenish yel-
low, margin slightly lobated; reverse greenish yellow (1A7).
Cardinal temperatures for growth. Minimum at 10 °C, optimum at 25 °C, maximum at
30 °C.
Figure 5. Amphichorda littoralis (ex-type FMR 19404). (A) Colony on PDA. (B) Colony on OA.
(C) Colony on SNA, after 14 d. at 25 °C. (D) Synnema from strain FMR 20067. (E) Semi-macronem-
atous conidiophore. (F, G) Micronematous conidiophores with attached conidia. (H, I) Conidioge-
nous cells growing directly from hyphae with attached conidia. (J) Conidia. Scale bars: 10 µm.
Additional specimens examined. SPAIN, Catalonia, Mediterranean coast, Tarragona,
Platja del Miracle, N 41°6´19´´, E 1°15´37´´, from sediments at 20 m of depth, Oct. 2021, G.
Quiroga-Jofre and D. Guerra-Mateo (FMR 19611); ibid., Platja de la Arrabassada, N 41°6´53´´,
E 1°16´48´´, from sediments at 20 m of depth, Jun. 2022, G. Quiroga-Jofre and D. Guerra-
Mateo (FMR 20149); ibid., from sediments at 20 m of depth, Jun. 2022, G. Quiroga-Jofre and
D. Guerra-Mateo (FMR 20067); ibid., Mediterranean coast, Tarragona, from a fragment of
floating rubber tire, Jul. 2020, D. Torres-García (FMR 17952).
Notes. Amphichorda littoralis is phylogenetically related to A. coprophila (Figs 1, 2). Mac-
roscopically, they can be distinguished by the color of the colony (Table 2). In the novel
species, colonies are consistently greenish yellow across PDA, OA and SNA, while in A.
coprophila, colony color ranges from brown orange to pale yellow. Microscopically, the
conidiogenous cells of A. littoralis are consistently smooth, while A. coprophila can show a
rough ornamentation. Moreover, the phylogenetic distance between this novel species
and other members of Amphichorda is around 96% for the tef1 region and 95% for the BenA
region.
4. Discussion
In previous morphological and phylogenetic studies, the taxonomic circumscription
of the genus Amphichorda has been controversial and its phylogenetic relationships with
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other taxa have remained obscure. The most recent multi-locus phylogenetic tree as-
sessing the diversity within Cordycipitaceae, included the type strain of A. guana, analysed
its LSU and tef1 sequences and resolved Amphichorda as a distant independent linage sis-
ter to Cordycipitaceae [4]. Our phylogenetic tree combining the ITS and LSU regions deter-
mined the close phylogenetic relationships of Amphichorda with the genera Hapsidospora
and Nigrosabulum (Fig. 1), two accepted members of the family Bionectriaceae. These results
allow to recognize Amphichorda as a member of this family, despite it is the only repre-
sentative genus with members producing conidia holoblastically. In the most recent re-
view of the family 41 genera were accepted, composed exclusively of fungi showing phi-
alidic conidiogenous cells [2]. However, the order Hypocreales comprises members with
asexual morphs producing both enteroblastic (phialidic) and holoblastic conidiogenous
cells. Although most of the families accepted in the order, such as Clavicipitaceae, Ijuhya-
ceae, Myrotheciomycetaceae, Nectriaceae, Niessliaceae, Ophiocordycipitaceae, Sarocladiaceae,
Stachybotriaceae, Stromatonectriaceae, Tilachlidiaceae and Xanthonectriaceae only show phia-
lidic conidiogenesis, other families like Calcarisporiaceae, Cordycipitaceae and Hypocreaceae
show both types of conidiogenesis[1,2,4,43–47]. Only genera like Beauveria and Calcarispo-
rium exhibit holoblastic conidiogenous cells [2,48]. Thus, it seems that phialides represent
the ancestral way of asexual reproduction in Hypocreales. In this sense, blastic conidiogen-
esis would have appeared independently as a secondary trait across different families. In
particular, Amphichorda seems to represent a transition between both types of conidiogen-
esis for the Bionectriaceae. The type species of the genus, A. felina, and the rest of the species
accepted show holoblastic conidiogenesis. The exception is A. coprophila, which can pro-
duce both types of condiogenous cells depending on the strain studied, but in particular
in the strain CBS 424.88. Here, we propose this species as a novel combination of the genus
Amphichorda. However, as mentioned before, the original authors of the species already
suggested the possible relation of this species with Acremonium (Bionectriaceae) [15]. They
observed the conidiogenous cells of the species and concluded a phialidic conidiogenous
pattern. Based on the close phylogenetic relationship of A. coprophila with holoblastic spe-
cies, we can conclude with confidence that it can produce both types of conidiogenous
cells. This trait, although odd, has already been described in Bionectriaceae with the holo-
blastic mesoconidia described on some Fusarium species [49,50].
Amphichorda represents a group of morphologically cryptic species. The microscopic
reproductive structures show subtle variations on their size range (Table 2). Thus, the best
morphological character to distinguish species is the colony color across different culture
media. We have found this trait consistent across several strains on PDA, OA and SNA.
However, colony color may be of little use in culture media like MEA (Fig. 4). For this
reason, phylogenetic analyses represent the most accurate way to identify Amphichorda
species. In particular, the ITS region, the fungal barcode, is able to distinguish species of
Amphichorda with precision, but other structural genes like tef1 and BenA can be used as
secondary barcodes with similar results.
Correct identification of species based on phylogenetic analyses needs for DNA se-
quences obtained from type strains. This is frequently a limitation when working with
fungi described before the development of DNA sequencing techniques. In our particular
case, the type material of A. felina seems to be lost. We therefore selected representative
strains identified as A. felina from the CBS culture collection, including some of coproph-
ilous origin like the protologue of this species, in order to determine a suitable candidate
for epitypification and study the morphological and genetic variability of the species.
However, the former goal was not feasible because the strains of coprophilous origin rep-
resented A. guana and A. coprophila (Table 1), and the strains that phylogenetically
matched with A. felina did not correspond with the origin of the protologue, preventing
the epitypification [14]. Despite this, the strain CBS 250.34 fits the morphological descrip-
tion of A. felina and it has been extensively used to characterize the species in phylogenetic
analyses. Therefore, we accept the strain CBS 250.34 as reference to stabilize the nomen-
clature of A. felina and, consequently, the genus Amphichorda, but its representation as type
strain should be avoided.
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Finally, we propose the novel species A. littoralis, the first species of the genus de-
scribed from the marine environment. Marine fungi are described as those that can grow
and sporulate under marine-like conditions [51]. All the strains that represent this species
have been isolated from the marine habitat and all strains managed to grow and sporulate
up to MEA 10% NaCl. We understand this as an indicative for a preference for the marine
environment and consider this species may represent a marine fungus. This species up-
dates the ecological range of the genus. Amphichorda was previously recognized as a group
of parasitic and coprophilous fungi. Although, a pathogenic behavior has been described
for A. felina [52], we conclude that it is predominantly composed of saprotrophic fungi
with preference for substrates with abundant organic matter like dung or marine sedi-
ments.
5. Concluding remarks
Our phylogenetic analyses combining the ITS and LSU regions revealed the genus
Amphichorda as a member of the family Bionectriaceae, where it represents the only holo-
blastic group. The combination of morphological and phylogenetic analyses determined
our marine strains as a novel species, A. littoralis, and resolved the taxonomic position of
O. coprophila as a new member of the genus Amphichorda. The current study is the largest
sampling of Amphichorda ever subjected to multi-locus sequence analyses, provides a com-
prehensive phylogenetic backbone and represents a framework for future studies on the
genus.
Supplementary Materials: TThe following supporting information can be downloaded at the web-
site of this paper posted on Preprints.org., Figure S1: GenBank accessions of representative taxa
from Bionectriaceae, Cordycipitaceae and outgroups included in the phylogenetic analyses.
Author Contributions: Conceptualization, D.G.-M., J.F.C.-L. and J.G.; methodology, D.G.-M., J.G.
and J.F.C.-L.; software, D.G.-M. and J.F.C.-L.; validation, J.F.C.-L. and J.G.; formal analysis, D.G.-M.,
J.F.C.-L and J.G.; investigation, D.G.-M., J.F.C.-L and J.G.; resources, V.B., J.F.C.-L and J.G.; data cu-
ration, J.F.C.-L and J.G.; writing original draft preparation, D.G.-M. and J.G.; writing review and
editing, D.G.-M., J.F.C.-L. and J.G.; visualization, J.F.C.-L and J.G.; supervision, J.F.C.-L and J.G.;
project administration, V.B. and J.G.; funding acquisition, J.G. All authors have read and agreed to
the published version of the manuscript.
Funding: This study was supported by the grant PID2021-128068NB-100 funded by MCIN/AEI/10.
13039/501100011033/ and by “ERDF A way of making Europe”.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank to the CBS culture collection (The Netherlands) and its cu-
rators for providing some fungal strain included in the study and to Gabriel Quiroga-Jofre (Tarraco
Diving Center) for the services in the collection of the samples.
Conflicts of Interest: The authors declare no conflict of interest.
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