f u n g a l e c o l o g y 6 ( 2 0 1 3 ) 9 2 e1 0 1

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/funeco

Distribution patterns of Dikarya in arid and semiarid soils of

Baja California, Mexico

l C. BAPTISTA-ROSASa,b, Ana E. ESCALANTEc,1,
Adriana L. ROMERO-OLIVARESa, Rau
Stephen H. BULLOCKc, Meritxell RIQUELMEd,*
a

Molecular Ecology & Biotechnology Graduate Program, Universidad Autonoma de Baja California (UABC), Ensenada, 22800 Baja California,
Mexico
b
Molecular Epidemiology Laboratory, School of Health Sciences UABC, Ensenada, 22890 Baja California, Mexico
c
Department of Conservation Biology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, 22860 Baja
California, Mexico
d
Department of Microbiology, CICESE, Ensenada, 22860 Baja California, Mexico

article info

abstract

Article history:

Approximately four-fifths of the land area of Baja California (BC) in Mexico are occupied by

Received 18 April 2012

arid and semiarid soils, the mycobiota of which is virtually uncharacterized. In the first

Revision received 19 August 2012

culture-independent study of the mycobiota of BC, we collected soil from five different

Accepted 13 September 2012

locations in the region and constructed a Dikarya-specific gene library for the ITS region of

Available online 27 November 2012

nuclear ribosomal DNA. Clones were analyzed by RFLP, were sequenced for phylogenetic

Corresponding editor:

analyses, and diversity and similarity indices were calculated. The ascomycete Penicillium

Kevin K. Newsham

dipodomyicola was the most frequent fungus found in soil at the most arid location studied,
and the basidiomycete Coprinellus radians was the most frequent at the location receiving

Keywords:

the highest rainfall. Other frequent members of the soil mycobiota were identified as

Baja California

Alternaria spp., Ceratobasidium sp., Coniozyma leucospermi, Nematoctonus robustus, Penicillium

Dikarya

griseofulvum, Tulostoma kotlabae and uncultured members of the Dikarya. Several sequences

Fungal diversity

were identified as those of uncultured fungi, one of which was previously reported from

ITS region

other hot deserts. Arid soils and the transitional zones between arid and semiarid soils had

Molecular ecology

the most similar fungal diversity, with the former soils having a community from which

Mycobiota

basidiomycetes were absent, and the soil receiving the highest precipitation having

RFLPs

a community dominated by basidiomycetes.

Semiarid ecosystems

2012 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction
As in other biomes, fungi in arid and semiarid ecosystems
(defined as areas receiving <254 mm total annual precipitation)
participate in mineralization, organic matter decomposition

and mobilization of nutrients (Fragoso & Rojas 2010). Despite

this, in comparison with boreal and tropical ecosystems, little
is known of fungal diversity in arid and semiarid soils, even
though deserts occupy more than one-third of the Earths land
surface (Collins et al. 2008). The majority of studies on fungal

* Corresponding author. Tel.: 52 646 1750500; fax: 52 646 1750595.

E-mail address: riquelme@cicese.mx (M. Riquelme).
1
Present address: Ecology of Biodiversity Department, Institute of Ecology, Universidad Nacional Autonoma de Mexico, Mexico D.F.
04510, Mexico.
1754-5048/$ e see front matter 2012 Elsevier Ltd and The British Mycological Society. All rights reserved.
http://dx.doi.org/10.1016/j.funeco.2012.09.004

Distribution patterns of Dikarya in arid and semiarid soils

diversity in desert soils have been culture-based, and have

identified ascomycetes as frequent members of the mycobiota
of the soils (Mouchacca 2005). For example, species of Penicillium and Aspergillus e xerotolerant and xerophilic fungi that
can grow at water potentials below 22.4 MPa (Magan & Lacey
1984) e have been isolated from desert soils in Mexico, Chile,
Argentina, Saudi Arabia, Iraq and Israel (Abdel-Hafez 1981;
Abdullah et al. 1986; Giusiano et al. 2002; Piontelli et al. 2002;
Samaniego-Gaxiola & Chew-Madinaveitia 2007; Grishkan &
Nevo 2008). Melanized dematiaceous fungi such as Alternaria
and Exophiala have also been isolated from desert soils in these
countries, as well as in Libya and the USA (Bates et al. 2006;
El-Said & Saleem 2008). These genera, along with other
uncultured and unclassified fungi, have also been found in
culture-independent studies (Bates et al. 2006). Some taxa have
been reported exclusively from desert soils in the Middle East
(e.g. Aspergillus carneus) (Abdullah et al. 1986; Ali-Shtayeh &
Jamous 2000; El-Said & Saleem 2008), whilst some others
have been reported from desert soils worldwide (e.g. A. flavus
and A. fumigatus) (Abdel-Hafez 1981; Giusiano et al. 2002;
Piontelli et al. 2002; El-Said & Saleem 2008).
Approximately four-fifths of the land area of Baja California (BC) in Mexico is occupied by arid and semiarid soils
(Peinado et al. 2006) that receive 100e200 mm total annual
precipitation (INEGI 2011). BC is classified as having a Mediterranean climate with latitudinal transitions from arid to
semiarid, and temperature extremes lessened by cool sea
breezes, along with exceptionally low humidity (Meigs 1966).
The region exhibits latitudinal and longitudinal macroclimatic gradients owing to the peninsulas location and
physiography (Markham 1972), which, along with soil chemistry and geomorphology, influence plant species composition
(Franco-Vizcaino et al. 1993; Ward & Olsvig-Whittaker 1993;
Peinado et al. 1994a). Much is known of plant diversity in
the region (Delgadillo 1992; Peinado et al. 1995, 2006), but
knowledge of soil fungi is much more limited, with the
macromycetes Geastrum triplex, Calvatia sculpta, Astraeus
hygrometricus, as well as the micromycetes Coccidioides spp.,
Aphanoascus keratinophilus and A. canadensis being the only
fungi reported from BC (Ayala & Ochoa 1991; Baptista-Rosas
et al. 2012). Therefore, in order to enhance knowledge of soil
mycology in BC, we conducted the first culture-independent
analysis of the fungi present in the soils of the region.

93

Fig 1 e Map of the State of Baja California (Google Earth

V5.1 2009) showing the location of the five sites, where soil
samples were collected (LS, Laguna Salada; VDP, Valle de
las Palmas; SC, Santa Catarina; ER, El Rosario; CA,
~ a). The ecosystem type, based on the vegetation
Catavin
composition of each of the locations, can also be observed.
Worth noting, the ecosystems transcend from North to
South bound, ending with the same ecosystem type at the
South and the North of the state.

Methods
Site descriptions
We selected five representative inland habitats, ranging from
arid to semiarid and transitional ecosystems (Fig 1). The
selected locations were Laguna Salada (LS), Valle de las Pal~a
mas (VDP), Santa Catarina (SC), El Rosario (ER) and Catavin
(CA). Details of these locations, and their climates, soil types
and vegetation, are shown in Table 1.

Sampling and soil analyses

Five soil samples were collected from each of the five locations
between May and Jul 2009. Sampling points, established

randomly, were located at least 1 km from paved road in

undisturbed areas that were >50 m apart. All soil samples
were from areas that lacked a plant canopy (i.e., interspaces)
and a conspicuous litter layer. Bulk soil (c. 100 g), lacking roots
or visible organic matter, was collected from between 10 and
15 cm below the soil surface with a clean disposable spoon
into sterile containers and were transported to the laboratory.
The soil was stored at room temperature (c. 23  C) in the dark
for 1 week before DNA extraction. Measurements of soil
moisture, total organic matter, pH and water retention
capacity (WRC) were obtained for each of the samples. Soil
moisture and total organic matter were measured gravimetrically (110  C for 48 hr and 380  C for 4 hr, respectively) and pH
was measured in a slurry of soil (10 g) and water (20 ml). WRC

94

A.L. Romero-Olivares et al.

Table 1 e Characteristics of the sampling locations.

Minimum
and
maximum
annual air
temperatures
( C)a

Total annual
precipitation
(mm)a

Soil typeb

Ecosystem type
and vegetationc

30

7.2, 42.3

64

29 110 56.3400 W,

114 170 16.9700 W

545

6.6, 36.5

118

Lacustrine solonchak with

silty, evaporite and clay
sediments that are subject
to rare inundation from
the Gulf of California and
the Colorado River
Shallow and coarse
residual yermosol on
tonalite

El Rosario
(ER)

29 420 22.1700 N,

115 220 50.9900 W

300

8.2, 29.3

177

Weakly developed regosol

Valle de
las
Palmas
(VDP)

32 240 3500 N,

116 400 4500 W

280

4.0, 35.2

209

Rendzina rich in humus

and developed over
granites and
conglomerates

Santa
Catarina
(SC)

31 370 000 N,

115 480 000 W

1150

2.5, 31.8

250

Residual lithosol

Hyperarid and saline

arid desert shrubland
with virtually no native
vegetation, although the
invasive Tamarix petandra
is locally frequent
Arid, xerophytic desert
shrubland with evergreen
(Larrea tridentata,
Simmondsia chinensis),
deciduous (Ambrosia and
Viguiera spp.) and
succulent (Agave cerulata
and Cylindropuntia spp.)
elements
Mediterranean desert,
transitional between
California coastal and
Sonoran desert
shrublands, with
Viguiera laciniata, Agave
shawii, Lycium sp. and
Cylindropuntia rosarica
Desert chaparral, with
Simmondsia chinensis,
Ephedra californica,
Adenostoma fasciculatum,
Yucca schidigera,
Mammillaria dioica,
Acacia greggii, Eriogonum
fasciculatum and
Artemisia californica
Chaparral, with Juniperus
californicae - Pinetum
monophyllae association
and Pinus monophylla and
P. quadrifolia along the
crest and eastern margin
of Sierra Juarez

Location

Latitude and
longitude

Laguna
Salada
(LS)

32 330 5000 N,

115 470 0000 W

~a
Catavin
(CA)

Elevation
(m s l)

a Climatic data are from the nearest available meteorological station.

b Soil names follow the nomenclature and maps of INEGI (2011).
c Plant community data are from Delgadillo (1992) and Peinado et al. (1994b).

was analyzed by measuring the volume of 20 ml of water that

passed through 20 g of soil.

DNA extraction and nested PCR

Total DNA was extracted from each soil sample (1 g) using an
Ultraclean Soil DNA Isolation kit (MoBio Laboratories Inc.,
Carlsbad, CA, USA). DNA was quantified by spectrophotometry at 260/280 nm and verified by 1 % agarose gel electrophoresis (BioRad, Hercules, CA, USA). Nested PCR
amplifications were carried out using PCR primers specific for
the subkingdom Dikarya (Martin & Rygiewicz 2005). The first
amplification was performed in a 20 ml reaction, consisting of
1 ml of template DNA, 2.5 mM MgCl2, 0.2 mM each dNTP,

1 GoTaq Flexi polymerase 5 buffer (Promega, Madison,

WI, USA), 1U GoTaq Flexi polymerase (Promega) and
0.6 mM of each primer (forward primer NSA3 [50 -AAACTCT
GTCGTGCTGGGGATA-30 ] and reverse primer NLC2 [50 -GAGCT
GCATTCCCAAACAACTC-30 ]). The resulting amplicons were
pooled by each of the five locations. The second round of
amplification was performed in 50 ml reactions, consisting of
1 ml of each pooled reaction as the template, 2.5 mM MgCl2,
0.2 mM each dNTP, 1 GoTaq Flexi polymerase 5 buffer
(Promega), 2U GoTaq Flexi polymerase (Promega) and 0.6 mM
each primer (forward primer NSI1 [50 -GATTGAATGGCTTAG
TGAGG-30 ] and reverse primer NLB4 [50 GGATTCTCACCCTC
TATGAC-30 ]). DNA of Neurospora crassa strain N1 (Fungal
Genetics Stock Center #988) and water were used as positive

Distribution patterns of Dikarya in arid and semiarid soils

95

Table 2 e List of ITS sequences found in arid and semiarid soils of Baja California, Mexico.
Groupeda samples
LS2
VDP8
VDP10
VDP11
LS3
VDP19
LS4
LS6
LS8
LS7
LS10
ER14
CA14
LS11
LS12
CA10
VDP1
VDP2
VDP3
VDP4
VDP9
VDP16
VDP5
VDP6
VDP7
VDP12
VDP13
VDP15
VDP17
VDP20
VDP18
VDP22
SC1
VDP23
VDP24
VDP25
SC2
SC4
SC6
SC7
SC8
SC10
SC11
SC12
SC14
SC9
SC13
ER2
ER3
ER7
ER4
ER5
ER6
ER8
ER9
CA16
ER10
ER11
ER12
ER13
CA4
CA7
CA2

GenBank acc. no.

JQ247361
JQ247390
JQ247392
JQ247393
JQ247362
JQ247401
JQ247363
JQ247365
JQ247367
JQ247366
JQ247368
JQ247359
JQ247344
JQ247369
JQ247370
JQ247340
JQ247383
JQ247384
JQ247385
JQ247386
JQ247391
JQ247398
JQ247387
JQ247388
JQ247389
JQ247394
JQ247395
JQ247397
JQ247399
JQ247402
JQ247400
JQ247404
JQ247371
JQ247405
JQ247406
JQ247407
JQ247372
JQ247373
JQ247374
JQ247375
JQ247376
JQ247378
JQ247379
JQ247380
JQ247382
JQ247377
JQ247381
JQ247347
JQ247348
JQ247352
JQ247349
JQ247350
JQ247351
JQ247353
JQ247354
JQ247346
JQ247355
JQ247356
JQ247357
JQ247358
JQ247335
JQ247337
JQ247333

ITS sequenceb alignment

Uncultured Ascomycete

Uncultured Dikarya
Penicillium clavigerum
Penicillium expansum
Uncultured Phoma sp.
Penicillium dipodomyicola

Alternaria tenuissima
Alternaria alternata
Uncultured Dikarya
Phoma medicaginis
Geastrum campestre
Uncultured Ascomycete

Uncultured Ascomycete
Penicillium griseofulvum
Ulocladium atrum
Geastrum triplex
Uncultured basidiomycete
Ceratobasidium sp.
Uncultured Ascomycete
Uncultured Dikarya
Tulostoma kotlabae
Phoma pinodella
Didymella fabae
Phialophora hyalina
Rhizosphaera kalkhoffii
Tulostoma brumale
Uncultured basidiomycete
Uncultured Dikarya
Coprinellus radians

Coniozyma leucospermi
Uncultured basidiomycete
Coprinellus vosoustii
Uncultured Dikarya
Sphaerobolus iowensis
Coprinus sterquilinus
Uncultured basidiomycete
Uncultured Oidiodendron
Psathyrella candoleana
Uncultured Dikarya
Pleosporales sp.
Pyrenochaeta lycopersici
Uncultured Dikarya

Emmonsia parva

No. of occurrences of patternc

OTUidd

13
13
1
1
4
2
1
6
5
2
38
3
36
13
6
6
5
4
1
12
1
36
5
10
4
1
12
17
2
1
2
1
15
1
2
1
7
1
12
3
3
28
6
9
4
22
1
1
3
1
11
8
3
7
7
1
36
7
1
1
3
3
1

P1

P2
P3
P4
P5
P6

P7
P8
P9
P10
P11
P12

P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
P25
P26
P27
P28
P29

P30
P31
P32
P33
P34
P35
P36
P37
P38
P39
P40
P41
P42

P43
(continued on next page)

96

A.L. Romero-Olivares et al.

Table 2 e (continued )
Groupeda samples
CA3
CA5
CA8
CA9
CA12
CA1
CA11
CA13
CA15
LS1

GenBank acc. no.

ITS sequenceb alignment

No. of occurrences of patternc

OTUidd

JQ247334
JQ247336
JQ247338
JQ247339
JQ247342
JQ247332
JQ247341
JQ247343
JQ247345
JQ247360

Myxotrichum sp.
Uncultured basidiomycete
Uncultured Dikarya

3
8
4
20
10
4
1
3
12
9

P44
P45
P46

Nematoctonus robustus

Phoma nebulosa
Ilyonectria sp.
Paracoccidioides brasiliensis

P47

P48
P49
P50

a Samples with different restriction pattern that aligned to the same ITS sequence after BLAST. LS, Laguna Salada; VDP, Valle de las Palmas; SC,
~ a.
Santa Catarina; ER, El Rosario; CA, Catavin
b Phylotype alignment based on BLAST searches of the rDNA of fungi in GenBank.
c Number of times the restriction pattern was repeated in the RFLP analysis.
d OTU identification (id.) (Supplementary Fig 1).

and negative controls, respectively. All reactions were carried

out in a Multigene II thermal cycler (Labnet International Inc.,
Edison, NJ, USA) equipped with a heated lid. The PCR amplification programs consisted of denaturation at 95  C for 2 min,
followed by 35 cycles of denaturation at 95  C for 30 s,
annealing for 40 s at 55  C for the first PCR and 60  C for the
nested PCR and elongation at 72  C for 1 min, followed by
a final elongation step at 72  C for 5 min for the first PCR and at
72  C for 10 min for the nested PCR.

deposited in GenBank (NCBI acc. nos. JQ247332 e JQ247407 )

(Table 2). The DNA sequences that aligned with the same ITS
sequence and shared at least 97 % similarity were grouped
into Operational Taxonomic Units (OTUs). A representative
sequence of each OTU and reference sequences recovered
from NCBI GenBank were compared in a multiple alignment
file using the ITS region of Chytridium confervae (NCBI acc. no.
AY349119) as an outgroup. The resulting alignment was used
to construct a neighbor-joining phylogram with MEGA 5.05
(Tamura et al. 2011).

Clone libraries and RFLP analyses

UniFrac analyses
Amplicons, resolved by electrophoresis in 1 % agarose gels,
were purified (Gel extraction kit Qiagen, Germantown, MD,
USA) and cloned into the pCR2.1 vector of the TOPO TA
Cloning kit (Invitrogen Co. Carlsbad, CA, USA). One hundred
and fifty positive clones per location were selected and verified by colony PCR.
Plasmid DNA was extracted by alkaline lysis and subjected
to restriction analysis (RFLP) using RsaI (New England Biolabs
Inc. Ipswich, MA, USA), an enzyme with a recognition
sequence frequently found in fungal ITS regions (Alvarado &
Manjon 2009). An additional restriction analysis using EcoRI
(Promega), which also has frequent recognition sequences in
fungal ITS regions, and particularly those of basidiomycetes
(Vilgalys 1996), was carried out for plasmids that did not
contain an ITS region sequence with a recognition sequence
for RsaI. After resolving with 1 % agarose gel electrophoresis
(Biorad), identical RFLP band patterns were grouped and at
least two clones from each restriction pattern were selected
for sequencing. High quality plasmid DNA was obtained using
the QIAprep spin miniprep kit (QIAGEN) and sequencing was
carried out by MACROGEN Inc. (Seoul, Korea).

The weighted UniFrac algorithm (Lozupone et al. 2006) was

used to compare the phylogenetic diversity of fungi between
the five locations. The multiple alignment file (without the
reference sequence) was used as the input data. A UniFrac
standard matrix, with all of the OTUs and their abundances in
each sample, was then constructed (Table 2). The input data
were used to compute a UniFrac environmental distance
matrix, which calculates the distance between the fungal
communities at each pair of sites. This matrix was subsequently used as input data for the calculation of the UniFrac
principal coordinates analysis (PCA).

Diversity and similarity analyses

Diversity values using the Shannon (H0 ) and Simpson (D0 )
indices were calculated based on OTU patterns. For the
similitude analyses, the Jaccard (J) and Sorensen (QS) coefficients were calculated. All analyses were carried out using
EstimateS v8.2 (Colwell 2009). Rarefaction curves were also
constructed from individual-based values calculated using
Ecosim v7.72 (Gotelli & Entsminger 2000).

Sequence analyses
A total of 83 ITS amplicons were sequenced. The sequences
were edited manually using Mega 5.05 (Tamura et al. 2011) and
forward and reverse sequences were assembled in a single file
for each sample and submitted to nucleotide BLAST in Genbank. BLAST hits were selected depending on the levels of
coverage, percentage identities and E-values. Sequences were

Results and discussion

Soil analyses
Moisture content was low in all soils and varied between
0.086 % (LS) and 0.722 % (ER). Moisture content in soils from SC,

Distribution patterns of Dikarya in arid and semiarid soils

VDP and CA was 0.512 %, 0.340 % and 0.260 %, respectively.

Total organic matter content varied between 0.101 % (LS) and
0.674 % (SC), with values of 0.103 %, 0.301 % and 0.428 % in soils
from ER, CA and VDP, respectively. Soil pH values had
a tendency to be more alkaline in arid ecosystems, with values
of 7.05 at LS and CA, and 7.09, 6.85 and 6.78 at ER, VDP and SC,
respectively. WRC varied between 21 % (LS) and 32 % (CA),
with capacities of 23 %, 28 % and 31 % at ER, VDP and SC,
respectively. Other hot desert soils, such as the Negev desert,
have similar characteristics to LS soils with pH values of 7.14,
moisture content of 0.05 % and total organic matter of 0.105 %
(Pen-Mouratov et al. 2010). Other semiarid ecosystems, such as
the Chihuahuan desert, have similar characteristics to SC
soils, with pH, moisture and total organic matter content of
6.28, 0.460 % and 0.743 % respectively (Bell et al. 2009).

RFLP and cloning analyses

Despite low total DNA concentrations (100e360 ng
DNA g1 dwt soil), nested PCR amplifications were successful,
indicating that the DNA of Dikarya was present in soil at each
location. RFLP analyses showed a wide variety of patterns (50
OTUs; Supplementary Fig 1). One restriction pattern that was
common to all five locations after digestion with RsaI (i.e., P12
and P18 shown in Supplementary Fig 1), was shown to
produce different patterns after digestion with EcoRI
(Supplementary Fig 2). Rarefaction analyses showed that
much of the fungal diversity present in soil was accounted for
by the RFLP and subsequent cloning analyses, with the curves
from all sites except VDP almost reaching asymptotes
(Supplementary Fig 3). Of the 83 ITS amplicons that were
sequenced, 10 were excluded from the analysis because the
sequences were identified as cloning vector or were of bad
quality. Of the remaining 73 sequences, 39 could be identified
to the species level, five to the genus level, one to the order
level and 15 to the phylum level (Table 2). The remaining 13
were designated as uncultured Dikarya (Table 2).
Each location sampled had a different fungal OTU
composition (Table 2). At LS, the hottest and most arid location sampled (Table 1), sequences matching Alternaria tenuissima and Penicillium spp., and particularly Penicillium
dipodomyicola, were frequently recovered from soil (Table 2). In
support of previous data from deserts in the Middle East
(Mouchacca 2005), all of the OTUs at this location were identified as ascomycetes (Table 2, Fig 2). Sequences of basidiomycetes, which are typically found in moister and more
temperate soils (Heilmann-Clausen & Boddy 2008), were not
recovered from soil at this location.
The sequences found in soil from CA, which was the next
most arid location (Table 1), corresponded mostly to the
phylum Ascomycota, although a few unclassified basidiomycetes were also found at this location (Table 2, Fig 2).
Sequences matching with P. dipodomyicola and Alternaria
alternata were frequently recovered from this soil, although
Nematoctonus robustus and Ilyonectria sp. were also present in
soil at CA (Table 2). N. robustus and Emmonsia parva were
exclusively found in soil at this location. Several sequences
recovered from soil at CA were also found at LS and ER
(P. dipodomyicola, A. alternata and Psathyrella candoleana), with
the similitude analyses further indicating the similarities

97

between the soil fungal communities at these locations

(Supplementary Fig 4).
According to the environmental data available (Table 1)
and our analyses, soils from ER, although having a low WRC
and organic matter content, had higher moisture content,
possibly reflecting the stronger maritime influence at this site
than at the other locations (Fig 1). ER has previously been
identified as a climatic, floral and faunal boundary zone
(Peinado et al. 1995). Accordingly, ascomycetes and basidiomycetes were found in equal numbers in soil from this location (Table 2, Fig 2). A single OTU, assigned to an uncultured
member of the Dikarya (ER10), which has previously been
reported from a semiarid grassland (Porras-Alfaro et al. 2011),
was frequent in soil at ER. Also frequent in soil at this location
were OTUs with sequences matching Sphaerobolus iowensis,
Coprinus sterquilinus and P. candoleana (Table 2). Soil at this
location shared OTUs with soils in the north and south of BC,
viz., P. dipodomyicola (LS and CA), P. candoleana and an uncultured Dikarya (CA).
Soil from VDP, which receives more rainfall than all locations other than SC, contained the highest number of OTUs (18;
Table 2, Fig 2). Most of these OTUs were assigned to the ascomycetes (e.g. Penicillium griseofulvum), although some basidiomycetes (e.g. Ceratobasidium sp.) were also frequently present.
Of these 18 different OTUs, eight were classified only at the
phylum or subkingdom level and most of them were exclusive
to VDP, except for three OTUs, one shared with SC (Tulostoma
kotlabae), and the remaining two with LS (an uncultured
ascomycete and an uncultured member of the Dikarya).
The OTU composition of soil from SC, which receives the
highest annual precipitation of all of the five locations studied
(Table 1), was dissimilar to the other sites (Supplementary
Fig 4). In contrast with the other locations, and in accordance with previous studies (Heilmann-Clausen & Boddy
2008), the majority of the OTUs in soil from SC were identified as basidiomycetes, with the most frequent matching with
Coprinellus radians (Table 2). Two OTUs belonging to the
phylum Ascomycota were identified from soil at this location
(Rhizosphaera kalkhoffii and Coniozyma leucospermi; Table 2,
Fig 2). There was almost no overlap between the OTUs obtained from soil from SC and the other locations, with only
one OTU, that for Tulastoma kotlabae, being recorded at
another site (VDP; Table 2).
In order to gain a broad view of fungal diversity within and
among locations, OTUs were grouped according to orders
(Fig 3). Uncultured fungi that could not be identified to order
level were abundant in soil at all localities. LS and SC had the
lowest number of orders (four each), with the Eurotiales and
Agaricales being the predominant orders at each location,
respectively. The Eurotiales were the most common order in
soil from CA, which contained fungi in seven orders. ER was
again evident as a transitional location, with seven of the eight
orders present in soil at this site being shared with other
localities. Soil from VDP contained the highest number of
orders (12), with a predominance of Pleosporales (Fig 3).

UniFrac analyses
The UniFrac environmental distance matrix suggested that the
ecosystems sharing the least similarities were LS and SC (0.9273,

98

A.L. Romero-Olivares et al.

Distribution patterns of Dikarya in arid and semiarid soils

99

~ a, (ER)
Fig 3 e Relative abundance of fungal ITS sequences at the taxonomic level of order in (LS) Laguna Salada, (CA) Catavin
El Rosario, (VDP) Valle de las Palmas and (SC) Santa Catarina.

P 0.03). The UniFrac PCA showed clustering of localities

apparently based on climatic characteristics. The semiarid
Mediterranean ecosystems (SC and VDP), were on the left hand
side of the plot, whereas the arid sites (LS and CA) were on the
right hand side, with the transitional site at ER being located
between the arid and semiarid habitats (Fig 4). However, the
neighbor-joining tree showed no apparent structure associated
with the distribution of Dikarya present in the soils of BC (Fig 2).

diversity present in the soils studied (Avis et al. 2006). The use
of taxa-specific primers in our study would also have underestimated true diversity, since culture-based analyses of our
soil samples have shown the presence of fungi formerly
classified as zygomycetes in soils from ER (data not shown).
Similarly, the fungal pathogen Coccidioides spp. was detected
in soil from VDP using a nested PCR approach specific for the
detection of this fungus (Baptista-Rosas et al. 2012), but was
not identified in our gene library.

Diversity and similarity analyses

The richness indices suggested that soil fungal diversity was
lowest at SC and highest at VDP (Supplementary Table). Even
though VDP had the highest apparent diversity, these observations may be biased by the frequent occurrence of
a restriction pattern belonging to an uncultured ascomycete
in soil at this site (VDP16). The same occurred at ER, where
there was a dominance of a single restriction pattern identified as an uncultured member of the Dikarya (ER10). It is
important to emphasize that the diversity of fungi present in
soil may have been underestimated in our study. It is recognized that the use of diversity indices based on RFLP analysis
of clone libraries may not provide accurate estimates of true
diversity in microbial communities (Blackwood et al. 2007) and
that diversity and similarity indices obtained from RFLP
analyses could either under- or over-estimate the true fungal

Conclusions
The diversity of fungi belonging to the subkingdom Dikarya
was found to vary between the arid and semiarid soils sampled
from BC. In accordance with previous data (Mouchacca 2005;
Heilmann-Clausen & Boddy 2008), our results indicate that
ascomycetes are frequent in arid desert soils, and that basidiomycetes become more frequent in soils that receive higher
levels of precipitation. The frequent occurrence in arid desert
soils of dematiaceous fungi (e.g. Harutyunyan et al. 2008), which
possess melanized cell walls that are thought to confer desiccation and radiation tolerance (Sterflinger et al. 2012), are
confirmed by our analyses, which indicate the presence in the
soils of BC of fungi such as A. alternata, A. tenuissima, Ulocladium
atrum, Penicillium expansum and P. griseofulvum. Each of these

Fig 2 e Neighbor-joining tree showing the phylogenetic relationships between the representative OTUs of the soilinhabiting Dikarya fungi of the sampled areas in the state of Baja California. Next to each representative OTU, colored circles
~ a; red: ER, El Rosario; purple: VDP, Valle de las Palmas; blue: SC, Santa Catarina;)
(yellow: LS, La Salada; green: CA, Catavin
indicate the locality where the OTUs were found. Bold letter ITS sequences were used as reference, and were all downloaded
from the GenBank; the rest of the sequences belong to the present study. Before each OTU, GenBank accession numbers are
indicated and in parenthesis the OTU id is shown. Sequences were aligned using the Clustal W algorithm and the
phylogenetic tree was constructed using MEGA 5.05 (Tamura et al. 2011) and condensed for values >50. The ITS region of
Chytridium confervae was used as outgroup.

100

A.L. Romero-Olivares et al.

Appendix A. Supplementary data

Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.funeco.2012.09.004.

references

Fig 4 e UniFrac (Lozupone et al. 2006) principal coordinates

analysis (PCA) of plotted environments showing a clear
distribution of the localities that could be based on the
climatic characteristics and fungal diversity. A division
between arid (right rectangle: LS, Laguna Salada; CA,
~ a) and semiarid (left rectangle: VDP, Valle de las
Catavin
Palmas; SC, Santa Catarina) environments can be
observed. ER is in a transitional zone between arid and
semiarid environments, correspondingly to its
intermediate position on P3.

fungi have been previously reported from desert soils of

Argentina, Saudi Arabia, Chile, Israel, the USA, Egypt and
Palestine (Abdel-Hafez 1981; Abdel-Hafez et al. 1990; AliShtayeh & Jamous 2000; Giusiano et al. 2002; Piontelli et al.
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et al. 1994b). Based upon the data from the UniFrac PCA reported here, we broadly conclude that climate exerts an influence on soil fungal diversity in BC. Our work offers a glimpse of
the high fungal diversity that may be present in soils of hot
desert ecosystems, and points to the importance of further
research on fungal diversity in deserts worldwide.

Acknowledgments
This research was supported by a grant from CONACyTSEMARNAT S0010-FONSEC SEMARNAT 23479. We would like
to thank Dr Luis Enriquez from the School of Marine Sciences

n-Dibene and
at UABC for providing reagents, to Jovani Catala

lez for technical field assistance, and to
Guillermo Gonza
CONACyT for studentship 19704 to ALRO. Three anonymous
referees supplied very helpful comments on the manuscript.

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