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A new species of stalked puffball, Tulostoma rufescens, was observed and collected from subtropical scrub vegetation within the Sonoran Sky Islands, Mexico, and was characterized morphologically and molecularly. The new fungus is characterized by small to medium sized spore-sacs, a thinly membranous exoperidium persisting in patches in the pinkish endoperidium, a tortuous stem with a basal bulb strongly intermixed with sand and debris, subhyaline capillitia with swollen and pigmented septa, and strongly echinulate basidiospores with spines that occasionally coalesce to form a subreticulum. Maximum likelihood and neighbor-joining phylogenetic analyses of full ITS1-5.8S-ITS2 and D1-D2 LSU DNA regions placed our collection within the monophyletic genus Tulostoma but separate from all of the available sequenced species.
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MYCOTAXON
ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2018
July–September 2018—Volume 133, pp. 459–471
https://doi.org/10.5248/133.459
Tulostoma rufescens sp. nov. from Sonora, Mexico
E H-N, A G,
J H. R-P, F S-T, M E*
1 Centro de Investigación en Alimentación y Desarrollo A.C.
Km 0.6 Carretera a La Victoria, Hermosillo, Sonora 83304, México
2 Centro de Investigación Cientíca de Yucatán, A.C.
Calle 43 No. 130, Colonia Chuburná de Hidalgo, Mérida, Yucatán 97200, México
* C : esqueda@ciad.mx
A—A new species of stalked puall, Tulostoma rufescens, was observed and
collected from subtropical scrub vegetation within the Sonoran Sky Islands, Mexico, and was
characterized morphologically and molecularly. e new fungus is characterized by small
to medium sized spore-sacs, a thinly membranous exoperidium persisting in patches in
the pinkish endoperidium, a tortuous stem with a basal bulb strongly intermixed with sand
and debris, subhyaline capillitia with swollen and pigmented septa, and strongly echinulate
basidiospores with spines that occasionally coalesce to form a subreticulum. Maximum
likelihood and neighbor-joining phylogenetic analyses of full ITS1-5.8S-ITS2 and D1-D2
LSU DNA regions placed our collection within the monophyletic genus Tulostoma but
separate from all of the available sequenced species.
K —Agaricaceae, Agaricales, chorology, gasteroid fungi, molecular systematics
Introduction
e Madrean Sky Islands (or Madrean Archipelago) are a set of approximately
40 mountains in southern and southeastern Arizona, southwestern New
Mexico, and northwestern Mexico. ey combine temperate pine-oak forests
at the highest elevations with arid to semiarid vegetation in the lowlands,
the latter forming part of the Sonoran and Chihuahuan deserts. ese
environments preserve a remarkable richness, complexity, unusual neo- and
archaeo-endemics, and an exceptional mixture of Nearctic and Neotropical
460 ... Hernández-Navarro & al.
species (Warshall 1995). e biodiversity of the Madrean Sky Islands has been
studied extensively, totaling close to two thousand species including 1380
plants, 358 birds, 104 reptiles and amphibians, 76 mammals, and 39 sh species
(Van Devender & al. 2013), but information on fungal diversity is scant.
Sonora is the second largest Mexican state (179,355 km²) and ranks h of
the 32 states of Mexico in fungal diversity with more than 618 morphospecies
(Aguirre-Acosta & al. 2014), of which around 210 represent gasteroid and
sequestrate Agaricomycetes (GSA), with Tulostoma Pers. (with 30 spp.) being
the most diverse and representative genus (Hernández-Navarro & al. 2017).
Some Sonoran GSA species, including the stalked pualls, are rare worldwide;
e.g., T. portoricense is known only from its type locality (Puerto Rico) and
Sonora (Esqueda-Valle & al. 1998), and T. gracilipes only from its type locality
(South Africa) and Sonora (Piña & al. 2010).
Tulostoma was proposed and sanctioned by Persoon (1794, 1801) and is
characterized by gastrocarps composed of a hollow stalk inserted in a socket of
a spore-sac with an apical ostiole. e genus has a cosmopolitan distribution but
is especially richly diverse and abundant in arid and semiarid areas. According
to Wright (1987), most species exhibit a terrestrial habit, with the remarkable
exception of T. exasperatum, which grows in decaying wood. Tulostoma species
can be classied based on their habitat as psammophilous (sandy soils in arid
regions), terricolous (clay-loving species, in pastures, roadsides), and “forest
soil-loving species” (in tropical or temperate zones with a high content of
organic matter). In his Tulostoma world monograph, Wright (1987) included
139 species; at the present time, Species Fungorum accepts c 155 taxa (www.
speciesfungorum.org).
ere is scant molecular information on Tulostoma species. From the
Americas, only T. domingueziae from Argentina has been described based
on molecular data (Hernández Caot & al. 2011); and from Asia only
T. ahmadii from Pakistan (Hussain & al. 2016). Jeppson & al. (2017) made a
major contribution, sequencing European species of Tulostoma, including
34 holotypes and some specimens from other continents. As a result, they
characterized 30 known species, proposed ve new species, and identied
at least 27 new undescribed species; they also conrmed Tulostoma as
monophyletic, found an unexpectedly large cryptic diversity, and cited areas
of steppe vegetation in Hungary and Spain as hot spots for Tulostoma species
diversity. e complete ITS1-5.8S-ITS2 nrDNA region is considered as the
barcode for fungal species recognition. To reconstruct a better phylogeny, it has
been suggested to complement this information with other ribosomal regions
Tulostoma rufescens sp. nov. (Mexico) ... 461
such as D1-D2 LSU, and/or protein coding genes such as atp6, EF1-α, or RPB1,
among others (Stielow & al. 2015).
As part of a major research, we studied some unidentied Tulostoma
collections from the Sonoran Sky Islands whose morphological characterization
did not match with any of the currently known morphospecies; their molecular
characterization led us to propose T. rufescens as a new species.
Materials & methods
e studied material is preserved in the Collection of Macromycetes of Sonoran State
University, Hermosillo, Mexico (UES). It was collected in sandy soil, in a subtropical
scrub vegetation, located at 29°53¢44¢N 109°27¢21¢W at 1214 m asl. Basidiomes were
characterized and conserved following conventional mycological techniques. Codes
in parentheses aer colors in basidiomata descriptions follow Kornerup & Wanscher
(1978). Microscopic features were measured by examining gleba sections mounted in
10% KOH preparations using an Olympus BX-51 light microscope (LM). Fiy spores
from each basidiome (including ornamentation, capillitia, and septa) were randomly
measured and the mean and standard deviation were calculated. All measurements
were made using the Innity analyze Soware v. 6.5.4 (Lumenera Corp.). Scanning
electron microscope (SEM) micrographs were produced with a JEOL-JSM 600 LB
microscope using critical point drying and sputtered with gold-palladium according
to Moreno & al. (1995).
Genomic DNA was extracted following a standard CTAB 2% protocol (Cubero &
al. 1999) with some modications. Aer rst grinding stipe tissue in liquid nitrogen
and placing ~100 mg of dusted tissue into a 2 mL tube, 1 mL CTAB 2% + 100 µL of
β-mercaptoethanol were added, and the tubes were incubated at 55°C for 30 min,
mixing every 10 min. en, 600 µL of chloroform : isoamylic alcohol (24:1) was added
and mixed by inversion for 10 min. e mix was then centrifuged for 15 min at 12,000
× g; the supernatant was transferred to a 1.5 mL tube and 500 µL of isopropanol +
50 µL of 3M sodium acetate was added, mixed by inversion, and stored at –20°C for
1 h. e mix was centrifuged 10 min at 12,000 × g and the supernatant discarded. e
remaining pellet was washed twice with EtOH 70%, dried at room temperature, and
resuspended in 50 µL of ultrapure water. e gDNA was then treated with 1 µL of 10
mg/mL of RNAsa and stored for 30 min at 37°C, quantied in a NanoDrop

2000,
and its integrity veried by visualization on a 1% agarose gel stained with Ethidium
Bromide (EtBr).
e gDNA was diluted to 10 ng/µL to amplify nuclear ribosomal RNA genes (the
full ITS1-5.8S-ITS2 and D1-D2 LSU regions); for this, we used the primer pairs ITS1/
ITS4 and LR0R/LR5. PCR reactions were carried out in a volume of 20 µL, with 20 ng
gDNA, using the mix content and thermal cycler conditions described by Schoch &
al. (2012). PCR amplicons were then visualized in a 1% agarose gel stained with EtBr.
Amplicons were puried from the gel using the kit Wizard® SV Gel and PCR Clean-
Up System and cloned using pGEM®-T-Easy following the manufacturer’s instructions
462 ... Hernández-Navarro & al.
using Escherichia coli DB10B chemically competent cells. e recombinant clones
were grown in LB medium with IPTG (100 mM), ampicillin (50 µg/mL), and X-gal
(50 mg/mL) as selection markers. e positive colonies were grown overnight in LB
broth with ampicillin (50 µg/mL) and the pDNA was extracted using the alkaline lysis
method (Sambrook & al. 1989). e presence of the inserts was veried by visualizing
EcoRI enzymatic digestion of 1µg of the pDNA, and also by PCR using the primers
M13F and M13R in 1% agarose gels stained with EtBr. e pDNA was sequenced in
triplicate by Macrogen Korea.
e obtained sequences were manually curated by inspecting their chromatograms
on the SequencherSoware

v. 5.2.3 and the clean assembled sequence was used
for BLASTN query at NCBI’s GenBank. en, a MegaBlast was performed, and 95
highly similar Tulostoma sequences and two outgroup sequences were downloaded
from GenBank, aligned using the MUSCLE algorithm with default parameters (Edgar
2004), and manually edited using MEGA 6.0 soware suite (Tamura & al. 2013). We
performed two dierent molecular phylogenetic analyses for the 98 complete ITS1-
5.8S-ITS2 and D1-D2 LSU rDNA gene sequences. One tree consisted of a Maximum
likelihood (ML) phylogenetic analysis, with the GTR+G+I model (Nei & Kumar 2000)
with gaps treated as partial deletions with a 95% of coverage, using an NNI heuristic
method for topology improvement; the other consisted of a Neighbour-Joining (NJ)
distance analysis, with gaps treated as pairwise deletions (Saitou & Nei 1987); both
with 1000 bootstrap replicates. e trees were rooted using Mycenastrum corium as
ingroup for Agaricaceae and Psathyrella secotioides as outgroup for Psathyrellaceae
(Matheny & al. 2006, Larsson & Jeppson 2008, Moreno & al. 2015).
Results
Tulostoma rufescens Hern.-Nav. & Esqueda, sp. nov. F –
MB MB 
Diers from Tulostoma adhaerens by its small to medium size spore-sacs, thinly
membranous exoperidium persisting in patches in the pinkish endoperidium, and
strongly echinulate basidiospores with the spines sometimes coalescing to form a
subreticulum.
T: Mexico, Sonora, municipality of Moctezuma, “La Madera,” 29°5344N
109°2721W, alt. 1214 m, sandy soil, in a subtropical scrub vegetation, 2 August 2010,
leg. E. Hernández-Navarro, C. Piña, R. Maldonado & A. Gutiérrez (Holotype, UES
10528) (two complete basidiocarps); GenBank MF319226).
E: e name refers to the pinkish tone of the endoperidium.
S-S 7.5–8.4 mm diam. × 5.7–5.9 mm high. E thinly
membranous, easily removable in big scales when dissected, dark because of
sand and particles on the outside and whitish in the inside. Persistent in parts
of the endoperidium like clay or scales, especially at the basal part of the spore-
sac. E grayish pink (11B3-12B3), mottled with dark spots that
Tulostoma rufescens sp. nov. (Mexico) ... 463
F. 1–6. Tulostoma rufescens (holotype, UES 10528): 1–3. Basidiocarps; 4. Capillitium and
spores (LM); 5, 6. Spore ornamentation (SEM). Scale bars: 2 = 10 mm; 3 = 5 mm; 4 = 5 µm;
5, 6 = 1 µm.
simulate warts due to the eect of the persistent exoperidium. M round
to elliptic, 1 × 1.2 mm diam. and with a projection up to 0.4 mm in height.
S conspicuous, deep, quite separate from the endoperidium, with
a denticulated to lacerated membrane. G dark ferruginous to brown
(8D4-8E8). S partially buried in the sand, woody-stulose, tortuous,
27–28 × 1.7–1.8 mm, light brown to reddish brown (6C4-7E8),
464 ... Hernández-Navarro & al.
F. 7. Molecular phylogenetic analysis of Tulostoma species based on the Maximum Likelihood
method using the General Time Reversible model. e tree with the highest log likelihood
(–8278.3120) is shown. e percentage of trees in which the associated taxa clustered together is
shown next to the branches, based on 1000 bootstrap replicates. e numbers aer “Tulostoma
sp.” refer to Jeppson & al. (2017). Our new sequence is set in bold.
surface rugose-scaly, with a conspicuous basal bulb with hyphae
strongly mixed with grains of sand and debris. S globose to
subglobose, reddish brown, echinulate, some spines slightly curved,
some appearing subreticulate, 4.2–6.7 µm including ornamentation
[mean = 5.1 µm, Qm = 0.99 n = 100]; under SEM the ornamentation is
formed by conspicuous conic structures, spines commonly fused in the
apex, but some coalescing irregularly to a subreticulate pattern, with
a pedicel variable in size. C subhyaline to slightly colored,
2–7 µm diam. [mean = 3.9 µm, n = 100], septa concolor with the spores,
somewhat swollen 3–9.9 µm [mean = 5.5 µm n = 100].
C—Initial sequence homology tests by BLASTN of ITS region
showed that the closest species is Tulostoma sp. 17 (Jeppson & al. 2017), an
undescribed species from Hungary and Spain whose ITS barcode diers from
our collection by 4% (data not shown). ML analyses of the complete ITS-5.8S-
ITS and D1-D2 LSU regions placed our collection within the monophyletic
genus Tulostoma (F. 7) and support its separation from similar morphospecies
(e.g., T. squamosum ).
When using distances in an NJ tree, the results are similar (F. 8). Some
changes can be observed, especially concerning unnamed taxa and topology of
lower branches. Tulostoma sp. 17 has not been described and its morphological
traits are unknown; nonetheless, molecular analysis suggests that our collection
is dierent from any sequenced Tulostoma species to date. It was also remarkable
how the treatment of the gaps gave a better topology for NJ analysis than ML.
is same result was observed with other algorithms (UPGMA, Minimum
Evolution, Maximum Parsimony) when gaps were treated as pairwise deletions
(data not shown).
Discussion
Tulostoma rufescens is distinguished by combination of the following
characters: small to medium size spore-sac (<9 mm), pinkish endoperidium
(a very uncommon color in the genus), tortuous stem with a conspicuous
basal bulb, and the strongly echinulate spores that may coalesce to form a
subreticulum in some spores.
Tulostoma rufescens sp. nov. (Mexico) ... 465
466 ... Hernández-Navarro & al.
F. 8. Molecular phylogenetic analysis of Tulostoma species based on the Neighbor-Joining
method. e optimal tree is shown (with the sum of branch length = 0.89290951). e percentage
of trees in which the associated taxa clustered together is shown next to the branches, based
on 1000 bootstrap replicates. e numbers aer “Tulostoma sp.” refer to Jeppson & al. (2017).
Our new sequence is set in bold.
e closest morphospecies is possibly T. adhaerens Lloyd recorded from
South Africa, Madagascar, Australia, Malaysia, and Japan, which diers by
its indistinct exoperidium, more robust sporocarp, bigger spores (5.0–7.5 µm
diam.), and epispore comprising independent spines, some of which are at
the apex. Wright (1987) described the Australian holotype and isotype of
T. adhaerens as medium sized (10–20 mm) with an indistinct exoperidum
and subreticulate spores under LM but echinulate with independent spines
in SEM. e Japanese material is close to 15 mm diam., with a spore
ornamentation comprising several spines in coherent fascicles and each
spine isolated at the base (Asai & Asai 2008). Wright (1987) designated
T. adhaerens as the type of Tulostoma sect. Hyphales J.E. Wright based on
its exoperidium with a thick layer of hyphae and a tubular or compressed
cylindrical ostiole. Of the 37 species included in this section, 35 are accepted
in Species Fungorum.
Tulostoma rufescens can also be confused with T. s q u a m o sum (J.F. Gmel.)
Pers., which diers by its true verrucose exoperidium formed by irregular
dark cells with a thick wall (sphaerocysts) and a squamous reddish brown
stipe. A similar case would be T. subsquamosum Long & S. Ahmad, which
also has spines but truly subreticulate spores in both LM and SEM, and an
uncolored to ochraceous endoperidium. Tulostoma rufescens could also be
confused with T. beccarianum Bres. and allied species such as T. simulans
Lloyd, two taxa once synonymized based on morphology, but recently
supported as independent by molecular data. e two species are separated
morphologically from T. rufescens by their uncolored endoperidium and
smaller spores with verrucae, not spines (Altés & Moreno 1993, Altés & al.
1996, Jeppson & al. 2017).
Tulostoma rufescens might be confused with T. calcareum Jeppson & al.,
which diers by its hyphal-verrucose exoperidium that is deciduous (but
sometimes persisting as whitish scattered verrucae); its brownish-ochraceous
endoperidium, initially rather dark colored but with age fading to greyish
white; its greyish to brownish peristome; and its slightly smaller verrucose-
echinate spores (4–6 µm, mean = 4.7–5.0 µm; Jeppson & al. 2017).
ere is a debate about gap management in phylogenetic analyses.
It has been reported that treating indels as missing data in both Bayesian
Tulostoma rufescens sp. nov. (Mexico) ... 467
468 ... Hernández-Navarro & al.
and maximum likelihood phylogenetic estimations can be statistically
inconsistent for determining a general and rather simple model of sequence
evolution. Resulting priors on branch lengths and rate heterogeneity
parameters may exacerbate the eects of ambiguous data, producing strongly
misleading bipartition posterior probabilities, even while showing the true
alignment (Nagy & al. 2012).
Some authors use a 3% threshold of sequence identity to determine
conspecicity (Begerow & al. 2010); nevertheless, this value is not generally
accepted for all fungi, since it has been shown to vary between groups, being
too high for some taxa and too low for others (Nilsson & al. 2008). In both
our analyses, bootstrap values were strong (≥70%) in the higher branches,
while some lower branches were not well supported (≤70%). Low bootstrap
values are related to evolutionary processes like incomplete lineage sorting
and introgression of alleles across species boundaries (e.g. incomplete
reproductive isolation) due to dierent selective processes (Morando & al.
2004).
Balasundaram & al. (2015) have suggested that the right DNA marker (or
a particular combination of markers) and its intraspecic distances must be
evaluated in order to reconstruct the accurate phylogeny of each group. e
interspecic genetic distances must also be evaluated, as such distances are
higher in geographically widely distributed species, stressing the importance
of sampling more specimens from wider geographical ranges to determine
intraspecic variation. In the absence of this information, the proposal of
new names might not reect cryptic diversication but rather taxonomic
ination, leading to a changed species concept rather than to new discoveries.
is directly inuences macroecology and conservation analyses, most of
which are based on species lists (Isaac & al. 2004).
In addition, many researchers admit that the ITS region does not provide
a precise species recognition for some fungal groups, such as for yeast,
arbuscular mycorrhizas, and lichens. e two spacer regions (ITS1, ITS2)
do not evolve independently because the variation of both regions is highly
correlated, and 5.8S rRNA region is well conserved within all fungal species.
However, the complete ITS meets the criterion for a good barcode marker:
it is short (<1000 bp), and there are several copies in the genome, making
it easily amplied, even from degraded, environmental or old herbarium
samples (Schoch & al. 2012). Other protein-coding genes might give a better
species resolution but lack the many practical applications of ITS (Kõljalg &
al. 2013). e ITS region is repeatedly criticized for indel-induced alignment
Tulostoma rufescens sp. nov. (Mexico) ... 469
problems and the lack of phylogenetic resolution above the species level, one
reason why some authors chose to delete ambiguous characters from ITS and
thereby losing potentially valuable information. However, it has been argued
that ITS indels are slightly more conserved than nucleotide substitutions and
when included in phylogenetic analyses improved the resolution and branch
support, thus extending the resolving power of ITS (Nagy & al. 2012).
From a barcoding perspective and considering only the complete ITS
region, currently fewer than 40 Tulostoma species have been properly
sequenced, representing approximately 25% of known morphospecies.
Despite the unusual characteristics of our collection, there are several examples
of convergent evolution in fungi, where the molecular characterization of
specimens has revealed cryptic species with shared morphological traits
(Nguyen & al. 2013, 2016). Tulostoma is no exception, since common species
such as T. mbriatum are polyphyletic, supporting the idea that many of the
morphological characters used for segregation of taxa are plesiomorphic or
homoplastic due to convergent evolution or parallelism (Jeppson & al. 2017).
Nevertheless, further molecular analysis of the holotype of T. adhaerens,
other Tulostoma holotypes, and Tulostoma specimens from Sonora and
elsewhere are needed so as to verify the authenticity of names based on
morphological species concepts, estimate the actual number of species,
determine fully which morphological traits are taxonomically and
phylogenetically informative, and understand the boundaries between
closely-related species and genera.
Acknowledgments
EHN thanks CONACyT (Mexico) for the fellowship to carry out his Ph.D. studies.
We thank I.Q. Silvia Andrade and Biol. Felipe Barredo (CICY) for processing SEM
samples and images and M. en C. Carolina Piña and Biol. Vanessa Parra for reviewing
an earlier English version of manuscript. We also would like to express our gratitude
to Dr. Alberto Altés and Dra. María M. Dios for reviewing the manuscript.
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... The most studied groups in Sonora are gasteroid and sequestrate Agaricomycetes (GSA), with more than 120 cited species. Of these, 37 correspond with tulostomataceous stalked-puffballs (TSPs), which are the most diverse and abundant group in Sonora (Esqueda, Pérez-Silva, Herrera, Altés et al., 1998;Esqueda et al., 2004Esqueda et al., , 2010Hernández-Navarro et al., 2015, 2018Piña-Páez et al., 2010). ...
... These 3 species also differ in their mouth, which is fibrillose in T. cretaceum and circular with an entire lip in T. albocretaceum and T. obesum, even though it tends toward indefinite in the 3 species. During its immature stage, S. laceratum can be confused with any of the aforementioned taxa but, in its mature stage, the dehiscence is substelliform with larger spores (Esqueda et al., 2004;Hernández-Navarro et al., 2018;Wright, 1987). ...
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