© 2017 J. Cramer in Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, www.borntraeger-cramer.de
Germany. DOI: 10.1127/nova_hedwigia/2017/0419 0029-5035/2017/0419 $ 3.50
Nova Hedwigia Vol. 105 (2017) Issue 3–4, 397–410
published online April 24, 2017; published in print November 2017 Article
A new family, Pervetustaceae with a new genus,
Pervetustus, and P. simplex sp. nov. (Paraglomerales),
and a new genus, Innospora with I. majewskii comb. nov.
(Paraglomeraceae) in the Glomeromycotina
Janusz Błaszkowski1*, Anna Kozłowska1, Thomas Crossay2, Sarah
Symanczik3 and Mohamed N. Al-Yahya’ei4
1 Department of Ecology, Protection and Shaping of Environment, West Pomeranian
Abstract: A new arbuscular mycorrhizal, Paraglomus-like, fungal species (Glomeromycotina) was
found and propagated in single-species cultures established from spores coming from Oman, Greece,
Tunisia, and New Caledonia. The hyaline spores of the fungus are small, 17–67 µm diam. when
globose. Their spore wall consists of an evanescent, 1.0–2.3 µm thick, short-lived outer layer and a
laminate, smooth, 3.8–7.3 µm thick inner layer, of which none shows amyloid or dextrinoid reaction
in Melzer’s reagent. Mycorrhizal structures (arbuscules and hyphae without vesicles) of the fungus
stained slightly in Trypan blue, thus like those of the formerly described Paraglomus spp., in which
the histochemical feature has been recognized. However, in the phylogenies gained from analyses
of nrDNA sequences, the fungus formed a distinct lineage in a basal position relative to and highly
diverged from that with P. majewskii and that with the other Paraglomus spp. of known molecular
phylogeny. Comparisons of similarity of sequences and the spore wall structure of the new fungus
with those of P. majewskii and the other Paraglomus spp. and the positions of clades of the three taxa
relative to those of the other taxa of the Glomeromycotina suggested transferring P. majewskii to a
new genus in the Paraglomeraceae and describing the new fungus as a new species of a new genus
in a new family of the order Paraglomerales.
Key words: arbuscular mycorrhizal fungi, molecular phylogeny, mycorrhiza, new taxa.
*Corresponding author: firstname.lastname@example.org
Based on rRNA gene sequence data, Redecker et al. (2000) and Sawaki et al. (1998)
recognized the ancient origin of ﬁve species of arbuscular mycorrhizal fungi (AMF)
that have been classiﬁed in the phylum Zygomycota. Three of them, Acaulospora
gerdemannii N.C.Schenck & T.H.Nicolson, A. trappei R.N.Ames & Linderman and
Glomus gerdemannii S.L.Rose, B.A.Daniels & Trappe, were dimorphic organisms
(Morton & Redecker 2001, Spain 2003, Spain et al. 2006). Spores of one of the
morphs arose laterally from the neck of a sporiferous saccule like in Acaulospora
laevis Gerd. & Trappe, the type species of the genus Acaulospora Gerd. & Trappe
(Gerdemann & Trappe 1974), but differed in subcellular structure and the phenotypic
and histochemical characters of components of their spore walls. In addition, the spores
usually developed from a short branch of the sporiferous saccule neck rather than
directly from it as sessile acaulosporoid spores of Acaulospora spp. The second morph
produced spores blastically at the tip of a sporogenous hypha (called glomoid spores)
like G. microcarpum Tul. & C. Tul., which at that time was considered the type species
of the genus (Gerdemann & Trappe 1974); now it is G. macrocarpum Tul. & C.Tul.
(Schüßler & Walker 2010). Glomus brasilianum Spain & J. Miranda and G. occultum
C. Walker formed only glomoid spores (Błaszkowski 2012, Morton and Redecker
2001). In addition, the fungi were distinguished by the following (i) their mycorrhizal
structures usually lacked vesicles and the other structures (arbuscules, intraradical and
extraradical hyphae) stained much lighter than those of other AMF or did not stain at all
in mycological stains and (ii) G. leptotichum Schenck & Smith (the synanamorph of A.
gerdemannii) and G. occultum contained a unique fatty acid, C16:1 ω7 cis (Redecker
et al. 2000, Spain et al. 2006). However, all of the morphological, histochemical and
biochemical characters did not contain enough information to determine the position
of the species among other AMF in a phylogenetic tree. Therefore, Morton & Redecker
(2001), based on sequences of the 18S ribosomal RNA gene, transferred G. brasilianum
and G. occultum to a newly erected genus, Paraglomus J.B.Morton & D.Redecker, in a
new family, Paraglomeraceae J.B.Morton & D.Redecker. The other three species were
accommodated in the genus Archaeospora J.B.Morton & D.Redecker of the family
Archaeosporaceae J.B.Morton & D.Redecker.
Currently AMF are grouped in the subphylum Glomeromycotina (C.Walker
& A.Schüßler) Spatafora & Stajichthe of the phylum Mucoromycota Doweld
(Spatafora et al. 2016) and the species mentioned above are in three families:
Ambisporaceae C.Walker, Vestberg & A.Schüßler, Archaeosporaceae representing
the order Archaeosporales C.Walker & A.Schüßler, and Paraglomeraceae belonging
to Paraglomerales Walker & A.Schüßler (Bills & Morton 2015, Oehl et al. 2011a, b,
Redecker et al. 2013, Schüßler & Walker 2010), of which the last family is the focus
of this study.
Apart from the two Paraglomus spp. mentioned above, currently the genus Paraglomus
still comprises four other species with known molecular phylogenies, i.e. P. bolivianum
(Sieverd. & Oehl) Oehl & G.A.Silva, P. laccatum (Błaszk.) Renker, Błaszk. & Buscot,
P. majewskii Błaszk. & Kovács, and P. pernambucanum Oehl et al. (Błaszkowski 2012,
Mello et al. 2013). In addition, Oehl et al. (2011a) proposed two new combinations
in the Paraglomus, i.e. P. albidum (C.Walker & L.H.Rhodes) Oehl et al. (formerly
G. albidum C.Walker & L.H.Rhodes) and P. lacteum (S.L.Rose & Trappe) Oehl et
al. (formerly G. lacteum S.L.Rose & Trappe) only based on morphological similarity
of their spores to those of most Paraglomus spp. mentioned above. However, given
that Paraglomus spp. represent the most ancient lineage in the Glomeromycotina,
the number of named species of the genus compared to that of the other clades of the
subphylum is low and probably concerns a small part of species existing in the world,
as Krüger et al. (2012) suggested.
Except for P. bolivianum and P. pernambucanum found to have 2-walled spores (Mello
et al. 2013), the other Paraglomus spp. were originally described as forming spores with
one spore wall, as all species of the Glomeromycotina with glomoid spores. However,
Mello et al. (2013) suggested that only P. majewskii produces one-walled spores and
this corresponds with its clearly diverged and basal molecular phylogenetic position
relative to the other Paraglomus spp. of recognized natural phylogeny (Błaszkowski et
al. 2012). In addition, the currently named Paraglomus spp. are either uncharacterized
or heterogeneous in respect of histochemical features and pigmentation of their spores.
Spores of six Paraglomus spp. lack amyloid or dextrinoid reaction in Melzer’s reagent,
young P. albidum spores turn pink to orange in this reagent, and this property has not
been recognized at all in P. lacteum (Błaszkowski 2012, Mello et al. 2013, Rose &
Trappe 1980, Walker & Rhodes 1981). Except for P. bolivianum producing yellow
brown to brown spores (Mello et al. 2013), spores of the other Paraglomus spp. are
hyaline to light-coloured at most (Błaszkowski 2012, Mello et al. 2013, Morton &
We found and grew in single-species cultures a Paraglomus-like fungus with small,
hyaline spores with a simple, 2-layered spore wall, whose mycorrhizal structures stain
slightly in Trypan blue, thus like those of, for example, P. occultum, the type species
of the genus Paraglomus (Morton & Redecker 2001). However, phylogenetic analyses
of nrDNA sequences of this Paraglomus-like fungus placed it in its own clade located
basally relative to but highly diverged from the clade of P. majewskii and that of the
other Paraglomus spp. of known molecular phylogeny. Comparisons of similarity of
sequences and the spore wall structure of the new fungus with those of P. majewskii
and the other Paraglomus spp. and the positions of clades of the three taxa relative to
those containing the other taxa of the Glomeromycotina strengthened our conﬁdence
that P. majewskii represents a separate genus in the Paraglomeraceae, and the new
fungus forming the basal lineage is an undescribed species of a new family in the
order Paraglomerales. Therefore, below we transferred P. majewskii to a new genus
in the Paraglomeraceae and erected a new family with a new genus and a new species
in the Paraglomerales.
Materials and methods
Origin Of the study material, establishment and grOwth Of trap and single-species cultures,
extract iOn O f spOr es, and staining Of mycOrrhizal structures: The study material used in
morphological and molecular analyses of the new species described below came from Oman, Greece,
and Tunisia (North Africa). The presence of the fungus in New Caledonia was conﬁrmed on the
basis of sequences and microscopic pictures provided by T. Crossay. Spores were ﬁrst extracted
from trap cultures that were established from ﬁeld-collected rhizosphere soils and roots of sampled
plants mixed with autoclaved coarse grained sand and then grown in conditions described previously
(Błaszkowski et al. 2012, Al-Yahya’ei et al. 2011). The sampled plants were Salvadora persica Wall.
and Tetraena qatarensis Beier & Thulin. growing at AlKamel, the area located between Al-Sharqiya
Sands and Oman Mountains in southern Arabia in the Sultanate of Oman (Al-Yahya’ei et al. 2011),
and Ammophila arenaria (L.) Link colonizing sand dunes of the Mediterranean Sea located near
Thessalonica, Greece, and Tunis, Tunisia.
Single-species cultures of the fungus coming from Oman were initiated by positioning a single spore
close to a leek (Allium porrum L.) seed and growing in conditions described by Symanczik et al.
(2014), and those with the Greek and Tunisian isolates by inoculating roots of Plantago lanceolata
L. with 10–20 spores per pot and growing according to Błaszkowski et al. (2012). Spores for
morphological and molecular analyses and roots for studies of mycorrhizal structures were collected
from four- to ﬁve-month-old cultures. Spores were extracted from trap and single-species cultures by
wet sieving and sucrose density gradient centrifugation (Daniels & Skipper 1982) and by the method
described by Błaszkowski et al. (2015). Roots were stained as described in Błaszkowski (2012).
micrOscOpy and nOmenclature: Morphological features of spores and the phenotypic and
histochemical characters of spore wall layers were determined after examination of at least 100
spores mounted in water, lactic acid, polyvinyl alcohol/lactic acid/glycerol (PVLG, Omar et al.
1979) and a mixture of PVLG and Melzer’s reagent (1:1, v/v). The preparation of spores for studies,
determination of colour, and photographing of spores and mycorrhizal structures were as those
described previously (Błaszkowski 2012, Błaszkowski et al. 2012). Types of spore wall layers are
those deﬁned by Błaszkowski (2012), Stürmer & Morton (1997), and Walker (1983). Colour names
are from Kornerup & Wanscher (1983). Nomenclature of fungi and the authors of fungal names are
from the Index Fungorum website http://www.indexfungorum.org/AuthorsOfFungalNames.htm.
Voucher specimens were mounted in PVLG and a mixture of PVLG and Melzer’s reagent (1:1, v/v)
on slides and deposited at the common mycological herbarium of the ETH and University of Zurich,
Switzerland (Z+ZT, holotype), the Department of Ecology, Protection and Shaping of Environment
(DEPSE), West Pomeranian University of Technology in Szczecin, Poland and in the herbarium at
Oregon State University (OSC) in Corvallis, Oregon, USA (isotypes).
mOlecular phylOgeny, dna extractiOn, pOlymerase chain reactiOn, clOning, and dna sequencing:
Molecular analyses of spores from Oman were performed at Zurich Basel Plant Science Center,
Institute of Botany, University of Basel, Switzerland. The Greek and Tunisian isolates were studied
at J. Błaszkowski’s lab. Crude DNA was extracted from eight single spores. The treatment of the
spores prior to polymerase chain reactions (PCRs), the conditions and primers used in the PCRs to
obtain sequences of the small subunit (SSU) rRNA gene, the internal transcribed spacer 1 (ITS1),
the 5.8S rRNA gene, ITS2 rDNA (hereinafter named ITS), and the large subunit (LSU) rRNA gene
(together named SSU–ITS–LSU nrDNA), as well as cloning and sequencing were as those described
in Błaszkowski et al. (2013) and Symanczik et al. (2014). The sequences were deposited in GenBank
sequence alignment and phylOgenetic analyses: Aligning SSU‒ITS–LSU nrDNA sequences of
AMF belonging to distant clades may be impossible or may led to erroneous conclusions due to the
highly variable ITS1 and ITS2 regions (Krüger et al. 2012, pers. observ.). Therefore, to know the
position of the AMF discussed here among other taxa of the Glomeromycotina, we performed pilot
phylogenetic analyses separately with SSU-only and LSU-only sequences of the fungus and species
representing all named genera of AMF with glomoid spores. The analyses placed the fungus basally
relative to the other AMF and indicated that it is most closely related to Paraglomus spp. (data not
shown). Consequently, we assembled a set with sequences covering the SSU–ITS–LSU region
or part thereof. Apart from 17 sequences of our fungus, the set contained 60 other sequences that
characterized seven species originally described in the genus Paraglomus and one to three species
each of the families Ambisporaceae, Archaeosporaceae and Geosiphonaceae. The outgroup was
represented by sequences of species of the families Endogonaceae Paol. and Mortierellaceae A.Fisch.
The pilot and ﬁnal sequence sets were aligned with MAFFT v. 7 using the auto option (http://mafft.
cbrc.jp/alignment/server/). In the SSU–ITS–LSU set, indels were coded by means of the simple indel
coding algorithm (Simmons et al. 2001) as implemented in GapCoder (Young & Healy 2003) and this
binary character set was added to the nucleotide alignment, as described and justiﬁed in Błaszkowski
et al. (2014). Bayesian (BI) analysis was conducted with MrBayes 3.1.2 (Huelsenbeck & Ronquist
2001) using SSU–ITS–LSU sequences plus indel analysis divided into four partitions. GTR+G and
two-parameter Markov (Mk2 Lewis) models were applied for the nucleotide partitions and indel
matrices, respectively. The GTR+G nucleotide substitution model was selected in jModelTest (Posada
2008) considering the Akaike criterion. Four Markov chains were run for 5 000 000 generations,
sampling every 100 steps, with a burn-in at 7500 sampled trees. Maximum likelihood (ML) analysis
was carried out with the raxmlGUI (Silvestro & Michalak 2012) implementation of RAxML
(Stamatakis 2014) with the GTRGAMMA algorithm. Rapid bootstrap analysis with 1000 replicates
was used to test the support for the branches. The generated phylogenetic trees were visualized and
edited in MEGA6 (Tamura et al. 2013).
phylOgeny: The phylogenies of the fungi studied here were determined based on a set
of SSU–ITS–LSU nrDNA sequences. The set was 2377 characters in length, had 2167
(91.17%) informative sites and comprised 77 sequences, of which 28 characterized
six Paraglomus spp. (including the fungus originally described as P. majewskii), and
17 sequences represented our new AMF. Bayesian and ML phylogenetic analyses of
the sequence set proved that P. majewskii represents a new basal genus in the family
Paraglomeraceae and our undescribed species belongs to a new genus in a new basal
family of the order Paraglomerales (Fig. 1). The clade with P. majewskii sequences
and the node connecting it with the clade with sequences of the other Paraglomus spp.
obtained full BI (1.0) and ML (100%) support values. The clade with the new taxon
was also fully supported, and the BI and ML support values of the node connecting it
with the clade representing the family Paraglomeraceae were 1.0 and 99%, respectively.
The eight P. majewskii and the four P. laccatum sequences used in the analyses showed
83.6% similarity. The similarity between P. majewskii sequences and those of our new
species was 77.8%. The intraspeciﬁc variability of sequences of P. majewskii and the
new species were 0.9% and 1.3%, respectively. No environmental sequences were
found matching closer than 85% with the sequences of the new species. The taxa are
erected and deﬁned below.
Erection of a new genus in the Paraglomeraceae
Innospora Błaszk., Kovács, Chwat & Kozłowska, gen. nov.
MycoBank MB 820124.
Spores single, glomoid, hyaline with one spore wall and a subtending hypha with
an open pore. Mycorrhiza with arbuscules and vesicles staining variable in Try-
pan blue. nrDNA sequences highly divergent from those of Paraglomus spp., from
which and other genera of the Glomeromycotina and other fungi differing in the
SSU nrDNA sequences GATAGAGGCCTACCATGGTAGTAACGGGTAACGGG
Fig. 1. A 50% majority rule consensus phylogram inferred from a Bayesian analysis of SSU–ITS–
LSU nrDNA sequences of Innospora majewskii comb. nov. and Pervetustus simplex gen. and sp.
nov., ﬁve known Paraglomus spp. and six species of the families Ambisporaceae, Archaeosporaceae
and Geosiphonaceae of the Glomeromycotina with seven species of the families Endogonaceae and
Mortierellaceae as outgroup. The two new taxa are in boldface and their names are followed by
GenBank accession numbers. The Bayesian posterior probabilities ≥0.50 and ML bootstrap values
≥50% are shown near the branches, respectively. Bar indicates 0.1 expected change per site per branch.
TTTGTTGGTTCTAGGACCGTTGTAATGATTAATAGGGATAGT and GGGGGC
CATGGGAAACCAAAGTGTTTGGGT, the ITS2‒LSU nrDNA signature sequence
GAAGAAGTCTGTCGCAG and the LSU nrDNA sequences TTGTGAAATTTTTC
GCATGGCAGGTCAGCATCAGTTTC and GCTCCCCGTGCTCAACAGCATGC
etymOlOgy: Latin, Innospora, inno = ﬂoat, spora = spore, referring to spores of this
fungus ﬂoating in water.
type species: Innospora majewskii (Błaszk. & Kovács) Błaszk., Kovács, Chwat & Kozłowska, comb.
nov. MycoBank No. MB 820125.
basiOnym: Paraglomus majewskii Błaszk. & Kovács. Mycologia 104: 149, 2012.
nOtes: Other data on the morphology, phylogeny, and distribution of the species are
in Błaszkowski (2012) and Błaszkowski et al. (2012).
Innospora majewskii does not contain any particular morphological trait conclusively
distinguishing the species from those of other genera of the Glomeromycotina with
glomoid spores. The formation of one-walled spores clearly separates I. majewskii
from Paraglomus spp. having two-walled spores, as Mello et al. (2013) concluded.
However, one-walled glomoid spores are also produced in all members of the orders
Archaeosporales and Glomerales and many species of the order Diversisporales
(Redecker et al. 2013, Bills & Morton 2015), as well as a fungus representing a new
family described below. Thus, only a large phylogenetic distance, unique molecular
properties included in the signature sequences mentioned above, and the position
of the clade with the sequences relative to other clades in the Glomeromycotina
unambiguously indicate that the species should represent a new genus (Fig. 1).
Erection of a new family and a new genus in the Paraglomerales
Pervetustaceae Błaszk., Chwat, Kozłowska, Symanczik & Al-Yahya’ei, fam. nov.
MycoBank MB 820126.
Spores single, glomoid, hyaline. Mycorrhiza with arbuscules staining variably in Trypan
blue. nrDNA sequences highly divergent from those of Innospora and Paraglomus spp.,
from which and other genera of the Glomeromycotina and other fungi differing in the SSU
nrDNA signature sequences CTTGGTGTCCGCGGGAACCAGGACCTTTACCTT
CGACTGGGAATCGGGCGATGTTA, the ITS2–LSU nrDNA sequence ATTTCCT
AGC and the LSU nrDNA sequences GTCCACGGCCGGCGCACCGGATGCG
GGC,TGCGTTCCGAGCTTGCTCGGGCGTGGGTGACCGTTCGCTCAA and C
etymOlOgy: Latin, from pervetusta = very old, referring to the molecular phylogeny
of the type species of the family, suggesting its very old origin.
type genus: Pervetustus Błaszk., Chwat, Kozłowska, Symanczik & Al-Yahya’ei.
Pervetustus Błaszk., Chwat, Kozłowska, Symanczik & Al-Yahya’ei, gen. nov.
MycoBank MB 820127.
Diagnostic characters as for Pervetustaceae.
type species: Pervetustus simplex Błaszk., Chwat, Kozłowska, Crossay, Symanczik & Al-Yahya’ei,
etymOlOgy: Latin, Pervetustus, as for Pervetustaceae.
nOtes: The formation of single, colourless, very small spores with one spore wall
(Figs 2–7) by the new fungus characterized below suggested that it is most closely
related to I. majewskii. However, the unique nrDNA sequences of the fungus, which
differ from those of I. majewskii by an average of 22.2%, and the basal position of the
clade with the sequences relative to the clades with sequences of the other species of
the Glomeromycotina (Fig. 1) proved that the fungus is a new species of a previously
unrecognized new genus and a new family.
According to Oehl et al. (2011a), likely all Paraglomus spp. germinate through the
spore wall instead of through the subtending hypha as in species of most other genera
with glomoid spores. Unfortunately, the mode of spore germination in I. majewskii
and Pe. simplex has not been recognized to date.
Pervetustus simplex Błaszk., Chwat, Kozłowska, Crossay, Symanczik & Al-Yahya’ei,
sp. nov. Figs 2–11
MycoBank MB 820128.
cOllectiOns examined: hOlOtype: ZT Myc 57795 (Z+ZT), isotypes: 3258–3274, 3276–3280
(DEPSE), and OSC 156399, OSC 156400 (OSC).
Figs 2–7. Pervetustus simplex. 2. Intact spores. 3, 4. Spore wall layers (swl) 1 and 2 and subtending
hyphal wall layer (shwl) 2; shwl1 is completely sloughed. 5. Spore wall layers (swl) 1 and 2. 6.
Spore wall layers (swl) 1 and 2 and subtending hyphal wall layers (shwl) 1 and 2. 7. Spore wall
layers (swl) 1 and 2 and subtending hyphal wall layer (shwl) 2; shwl1 is completely sloughed; note
the septum (s) in the lumen of the subtending hypha. Figs 3, 4, 6, 7. Spores in PVLG. Figs 2, 5.
Spores in PVLG+Melzer’s reagent. Figs 2–7. Differential interference microscopy. Bars: Fig. 2 =
20 µm, Figs 3–7 = 10 µm.
etymOlOgy: Latin, simplex, referring to the morphological simplicity of the fungus.
spOrOcarps unknown. Spores formed singly in soil (Figs 2–7) and blastically at the
tip of sporogenous hyphae developed from mycorrhizal extraradical hyphae. spOres
hyaline; globose to subglobose; (17–)51(–67) µm diam.; very rarely ovoid; 52–58 ×
58–63 µm; with one subtending hypha (Figs 2–7). spOre wall consists of two hyaline
layers (Figs 3–7). Layer 1, forming the spore surface, evanescent, (1.0–)1.5(–2.3) µm
thick, short-lived, usually highly deteriorated or completely sloughed in most spores
(Figs 3–7). Layer 2 laminate, smooth, (3.8–)5.1(–7.3) µm thick, consisting of very thin,
<0.5 µm thick, laminae, tightly adherent to each other (Figs 3–7). Layers 1 and 2 do
not show amyloid or dextrinoid reaction in Melzer’s reagent (Figs 2, 5). subtending
hypha hyaline; straight or recurved, cylindrical to slightly funnel-shaped, sometimes
slightly constricted at the spore base; (3.0–)4.2(–5.8) µm wide at the spore base (Figs
3, 4, 6, 7). wall Of subtending hypha hyaline; (1.0–)1.3(–1.8) µm thick at the spore
base; continuous with spore wall layers 1 and 2; layer 1 usually highly deteriorated
and present only near the spore base or completely sloughed in most spores (Figs 3,
4, 6, 7). pOre (0.8–)1.8(–2.8) µm diam, open (Figs 3, 4, 6) or occluded by a straight or
curved septum connecting the inner surfaces of subtending hyphal wall layer 2 (Fig.
7). germinatiOn unknown.
mycOrrhizal assOciatiOns: In the ﬁeld, Pe. simplex was likely associated with roots of
S. persica and T. qatarensis (growing in undisturbed natural ﬁeld at Al-Kamel in Al-
Sharqyia region of Oman), Am. arenaria (colonizing sand dunes of the Mediterranean
Sea near Thessalonica, Greece and Tunis, Tunisia) and Alphitonia neocaledonica
(Schltr.) Guillaumin (an endemic plant of New Caledonia that grew in soil of a nickel
mine). However, the presence of the fungus inside roots of the plant species was not
examined using molecular methods.
In single-species cultures with A. porrum and P. lanceolata as host plants, Pe. simplex
formed mycorrhiza with arbuscules and intraradical and extraradical hyphae (Figs
8–11). No vesicles were found. Arbuscules were generally widely dispersed along
the root fragments examined (Figs 8, 9). Intraradical hyphae grew along the root axis,
were (2.0–)3.8(–8.8) µm wide, straight or slightly recurved, and occasionally formed
Y-shaped branches and coils (Fig. 9). The coils were ellipsoidal; 22.8–28.8 × 36.8–
62.5 µm; when seen in a plan view. Extraradical hyphae were (1.8–)3.1(–4.5) µm wide
and occurred infrequently or abundantly, depending on the root fragment examined
(Figs 10, 11). In 0.1% Trypan blue, arbuscules stained pale violet (17A3) to greyish
violet (17B3), intraradical hyphae violet white (17A2) to light violet (17A5), coils
pale violet (17A3) to light violet (17A5), and extraradical hyphae pastel violet (17A4)
to greyish violet (17C6, Figs 8–11).
phylOgenetic pOsitiOn: As that of Pervetustaceae fam. nov. (see above and Fig. 1).
distributiOn and habitat: As mentioned in "Mycorrhizal associations", Pe. simplex
has so far been found only in an undisturbed natural ﬁeld located in Oman, in two
maritime sand dune sites belonging to Greece and Tunisia, and in soil of a nickel mine
of New Caledonia. The Oman ﬁeld is situated in a sandy plain area (22°14'11"N,
59°10'30"E) characterized by its hyperaridity based on the aridity index deﬁned by
the United Nations Environmental Program (UNEP 2006). Annual rainfall does not
exceed 100 mm and summer temperatures exceed 48 °C (Symanczik et al. 2014). The
Greek (40°33'59"E, 22°57'26"E) and Tunisian (36°48'19"N, 10°12'04"E) sites were
located ca. 50 m and 20 m, respectively, from the bank of the Mediterranean Sea. The
New Caledonian nickel mine is situated in the great south ultramaﬁc massif of the
archipelago, near Plum (22°16'35"S, 166°36'55"E). The annual rainfall on the study
site ranges from 900 to 2000 mm with variations between and within years (Enright
et al. 2001). Sampling in Oman and Tunisia took place in August 2006, in Greece in
September 2008, and in New Caledonia in September 2014. Chemical and physical
properties of the habitat soil from Oman are presented in Al-Yahya’ei et al. (2011). The
soil from New Caledonia is a colluvial lateritic soil with the following characteristics:
coarse sand, 39.4%; ﬁne sand, 22.1%; silt-clay, 37.2%; pH H20, 5.9; pH KCl, 5.6; total
C, 42.1 g kg−1; total N, 2.2 g kg−1; total P, 147 mg kg−1; available P (Mehlich), 3 mg
kg−1; and total Ni, 4.78 g kg−1.
Figs 8–11. Mycorrhizal structures of Pervetustus simplex in roots of Plantago lanceolata stained in
0.1% Trypan blue. 8. Arbuscules (a) and arbuscular trunk (t) developed from parent hypha (ph). 9.
Arbuscules (a), Y-shaped branch (Yb), straight hypha (sh), and coil (c). 10. Extraradical hyphae (eh)
on the root surface. 11. Extraradical hyphae (eh) and spore (sp). Figs 8–11. In PVLG. Figs 8–11.
Differential interference microscopy. Bars: Figs 8–10 = 10 µm, Fig. 11 = 20 µm.
BLAST queries did not show any sequence from environmental samples with at least
97% similarity to those of Pe. simplex. The fungus has not also been found in ca.
3000 ﬁeld-collected soil samples and ca. 2900 trap cultures that represented different
cultivated and uncultivated sites located in Africa, Asia, Brazil, Europe, and USA
(Błaszkowski, pers. observ.). Thus, Pe. simplex is likely to be rare in the world.
nOtes: Morphologically Pe. simplex is conspicuous because of its very small and
colourless spores that are produced singly in soil and have a simple spore wall structure
consisting of an evanescent, short-lived, thin outer layer and a much thicker, permanent,
smooth, laminate inner layer. None of these layers shows amyloid or dextrinoid reaction
in Melzer’s reagent (Figs 2–7).
Spores of Pe. simplex and I. majewskii are similar in colour (are hyaline), size, width of
their subtending hypha, thickness of its wall and in diameter of the subtending hyphal
pore at the spore base (Błaszkowski et al. 2012). However, in I. majewskii the spore
wall is 1.4–1.6-fold thicker and consists of three layers (vs. two layers in Pe. simplex,
Figs 3–7), and its subtending hyphal pore is open (vs. open or occluded by a septum,
Figs 3, 4, 6, 7). Finally, the subtending hypha of I. majewskii spores is more regular
in shape (cylindrical to ﬂared vs. cylindrical to slightly funnel-shaped in Pe. simplex).
Most importantly, Pe. simplex is unique due to its molecular properties expressed
among others in the signature sequences given above.
We thank Dr. Roland Kirschner for managing the review process of the paper. This study was supported
in part by the Polish National Centre of Science, grants no. 2012/05/B/NZ8/00498 and 2012/07/N/
NZ8/02363, Oman’s Ministry of Agriculture and Fisheries, the Universities of Basel and of UAE
which are gratefully acknowledged.
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Manuscript submitted November 10, 2016: accepted March 31, 2017.