Phylogenetic study and taxonomic revision of the Xanthoparmelia mexicana group, including the description of a new species (Parmeliaceae, Ascomycota)
Phylogenetic study and taxonomic revision of the Xanthoparmelia mexicana group, including the description of a new species (Parmeliaceae, Ascomycota)
Alejandrina Barcenas-Peña 1
Steven D. Leavitt 0
Jen-Pan Huang 1
Felix Grewe 1
H. Thorsten Lumbsch 1
0 Department of Biology and M.L. Bean Life Science Museum, Brigham Young University , 4102 Life Science Building, Provo, UT 84602 , USA
1 Science & Education, The Field Museum , 1400 South Lake Shore Drive, Chicago, IL 60605-2496 , USA
Xanthoparmelia (Parmeliaceae, Ascomycota) is the most species-rich genus of lichen-forming fungi. Species boundaries are based on morphological and chemical features, varying reproductive strategies and, more recently, molecular sequence data. The isidiate Xanthoparmelia mexicana group is common in arid regions of North and Central America and includes a range of morphological variation and variable secondary metabolites - salazinic or stictic acids mainly. In order to better understand the evolutionary history of this group and potential taxonomic implications, a molecular phylogeny representing 58 ingroup samples was reconstructed using four loci, including ITS, mtSSU, nuLSU rDNA and MCM7. Results indicate the existence of multiple, distinct lineages phenotypically agreeing with X. mexicana. One of these isidiate, salazinic acid-containing lineages is described here as a new species, X. pedregalensis sp. nov., including populations from xerophytic scrub vegetation in Pedregal de San Angel, Mexico City. X. mexicana s. str. is less isidiate than X. pedregalensis and has salazinic and consalazinic acid, occasionally with norstictic acid; whereas X. pedregalensis contains salazinic and norstictic acids and an unknown substance. Samples from the Old World, morphologically agreeing with X. mexicana, are only distantly related to X. mexicana s. str. Our results indicate that X. mexicana is likely less common than previously assumed and ongoing taxonomic revisions are required for isidiate Xanthoparmelia species.
The family Parmeliaceae is the largest family of lichenised fungi
(Jaklitsch et al. 2016)
currently classified in approximately 70 genera with almost 2,800 species
and Huhndorf 2010, Divakar et al. 2017)
. Xanthoparmelia, with about 800 described
species, is the largest genus of lichen-forming fungi (Lücking et al. 2016), with two
centres of distribution in Australia and southern Africa; a smaller number of species
occur in the Holarctic
(Blanco et al. 2004, Eriksson et al. 2004, Crespo et al. 2010, Thell
et al. 2012, Leavitt et al. 2018)
. To date, 74 species have been reported from Mexico,
amongst these species, 27 are isidiate
(Nash et al. 2016)
Isidiate Xanthoparmelia species are distributed in boreal, temperate and tropical
regions. However, they commonly occur in semi-arid to arid regions worldwide
especially on siliceous rocks, such as granite and sandstone. In North and Central America,
Xanthoparmelia mexicana (Gyelnik) Hale ranks amongst the most common isidiate
species. This taxon is widely distributed and has been reported from western USA,
Mexico, Dominican Republic, Argentina, Kenya, Australia, New Zealand, Japan,
China and Nepal
(Hale 1990, Elix 1994, Nash and Elix 2004)
. X. mexicana is part of
a complex of morphologically similar species, with adnate to slightly attached thalli,
cylindrical isidia and a brown lower side of the thalli, which are primarily separated
by their secondary metabolites. The species complex also includes X. ajoensis (T. H.
Nash) Egan (diffractaic acid), X. dierythra (Hale) Hale (norstictic acid), X. joranadia
(T. H. Nash) Hale (lecanoric acid), X. maricopensis T. H. Nash & Elix (norstictic and
hyposalazinic acids), X. moctezumensis T. H. Nash (3-α-hydroxybarbatic acid), X. plittii
(Gyelnik) Hale (stictic acid), X. schmidtii Hale (barbatic, norstictic and salazinic acids),
X. subramigera (Gyelnik) Hale (fumarprotocetraric acid) and X. weberi (Hale) Hale
(Hale 1990, Nash et al. 2016)
. However, previous studies
indicate that current interpretations of morphological features and secondary
metabolites likely fail to accurately characterise species-level diversity in isidiate
(Leavitt et al. 2011, 2013)
To better understand the evolutionary history of the Xanthoparmelia mexicana
complex and potential taxonomic implications, isidiate Xanthoparmelia specimens
were collected from different locations throughout arid regions of Mexico and
supplemented with previously available sequence data. The new samples came from
xerophytic scrublands in the states Puebla, Oaxaca, San Luis Potosí, Querétaro, Estado de
México, Mexico City, Guanajuato, Zacatecas and Hidalgo, all in the central part of Mexico.
We focused on sampling Xanthoparmelia populations that were phenotypically similar
to X. mexicana, e.g. isidiate specimens containing salazinic acid. X. mexicana was
originally described by
as Parmelia mexicana and was later combined into
. The type specimen was collected from San Jerónimo,
in Pedregal de San Angel, Mexico City. The syntype in the Bouly de Lesdain herbarium
was destroyed during World War II, whereas the lectotype in the Budapest herbarium
(BP) was not available for molecular study. Therefore, we attempted to recollect
material at the type locality of X. mexicana and other regions throughout Mexico. Based on
the results of this study, we formally describe a previously unrecognised species-level
lineage comprised of isidiate specimens as new to science.
Material and methods
Specimens were studied from the herbaria ASU, BRY, F, MAF and new collections
from different localities throughout arid regions from the central part of Mexico
(Table 1, Fig. 1). A total of 83 specimens, representing 43 species were included,
with an emphasis on isidiate species/populations from Central and North America
(all epithets are validly published, with the exception of X. isidiomontana nom prov
assigned to the clade ‘D2’ from Leavitt et al. 2013)
. New sequences were generated
from 25 specimens and supplemented with 34 sequences from a previous analysis
(Leavitt et al. 2018)
and 24 additional sequences from GenBank (Table 1). Four
species in the genus Xanthoparmelia that have previously been shown to be distantly
related to X. mexicana were used as outgroup – X. beatricea, X. austroafricana, X.
subramigera and X. aff. subramigera
(Leavitt et al. 2018)
8837 USA:(W02e0st86V9ir4g6inNiaY;)Streets MG695550 MG695717 MG695803 MG695651
GenBank USA: Le(aBviRttYe-Cta)l. 55463 HM578915 HM579708 – HM579323
GenBank USA: Le(aBviRttYe-Cta)l. 55354 HM578805 HM579620 – HM579216
9904 USA: Ariz(oBnRaY;-LCe)avitt 9904 MG695555 MG695720 MG695809 MG695656
GenBank Spain: 9956 (MAF-Lich) AY581094 JX974718 AY582330 AY578960
GenBank USA: Le(aBviRttYe-Cta)l. 55230 HM578685 HM579500 – –
15489 Mexico4:6M26o1re(lWos;ISN)ash III MH580227 MH686131 – MH699920
14894 Mexico: Pu5e8b5la7; B(Fa)rcenas-Peña MH580223 MH686127 MH699896 MH699916
14905 Mexico: Pu5e8b8la4; B(Fa)rcenas-Peña MH580224 MH686128 MH699897 MH699917
14906 Mexico: Oa5x9a0ca5; (BFa)rcenas-Peña MH580225 MH686129 MH699898 MH699918
14910 Mexico: Pu5e8b8la8; B(Fa)rcenas-Peña MH580226 MH686130 MH699899 MH699919
245f US3A1–:2A5ri9zo(5n5a;3E06ABcoRlYle-cCti)on MG695556 MG695721 MG695810 MG695657
6698 USA: Ariz(oBnRa;YJ-.CL)eavitt 001 MG695558 MG695723 MG695812 MG695659
291f USA: Le(aBviRttYe-Cta)l. 55328 HM578780 HM579596 – HM579192
786f USA: Le(aBviRttYe-Cta)l. 55462 HM578914 HM579707 – HM579322
097f Mexico: L(eBaRviYtt-Cet)al. 55233 HM578688 HM579503 - HM579098
GenBank South Korea(:KJoaLnRgIe)t al. 005486 KM250123 – – –
15479 MBaerxciecnoa:sS-PaneñLau7is31P6ot(oFsí); MH580231 MH686135 MH699904 MH699923
15472 MBaerxciecnoa:sS-PaneñLau7is40P8ot(oFsí); MH580229 MH699932 – MH699922
15466 MBaerxciecnoa:sS-PaneñLau7is44P1ot(oFsí); MH686404 MH686133 MH699902 –
15461 Mexico:PQeñuae7ré1t7ar8o;(FB)arcenas- MH686401 MH699930 MH699901 –
15485 MexiPceoñ:aQ7u2e0r9éta(Mro;EBXaUrc)enas- MH686402 MH686136 MH699905 –
15471 MBaerxciecnoa:sS-PaneñLau7is27P3ot(oFsí); MH686403 MH699931 MH699903 –
15473 Mexico4:5H12id6a(lgWo;ISN)ash III MH580230 MH686134 – –
156f USA: Le(aBviRttYe-Cta)l. 55267 HM578721 HM579536 – HM579132
15487 MexicoP:eHñaid7a4lg7o0; (BFa)rcenas- MH580232 MH686137 MH699906 –
14899 Mexico: Oa5x9a1ca8; (BFa)rcenas-Peña MH580228 MH686132 MH699900 MH699921
14897 Mexico: Pu5eb8l9a1;(BFa)rcenas-Peña MH580233 MH686138 MH699907 MH699924
Morphology and chemistry
Morphological characters were observed using a Zeiss Stemi 2000-C stereoscope.
Ascomatal anatomy, ascospore in addition to conidia shape and size were observed using
a Zeiss Axioscope. Secondary metabolites were identified using spot test KOH 10%,
KC, C, PD and high-performance thin layer chromatography (HPTLC), using solvent
systems C following established methods
(Culberson and Johnson 1982, Arup et al.
1993, Lumbsch 2002, Orange et al. 2010)
Total genomic DNA was extracted from thallus fragments following the
manufacturers’ instructions using the ZR Fungal/Bacterial DNA Miniprep Kit (Zymo Research
Corp., Irvine, CA). DNA sequences were generated for four markers using
polymerase chain reaction (PCR): the nuclear ribosomal internal transcribed spacer region
(ITS), a fragment of nuclear large subunit rDNA (nuLSU), the nuclear protein-coding
marker minichromosome maintenance complex component 7 (MCM7) and a
fragment of the mitochondrial small subunit rDNA (mtSSU). PCR reactions contained
6.25 ml of MyTaq Mix, 25 ml H2O, 0.25 ml forward and reverse primer and 0.5 ml
template DNA, for a total reaction volume of 12.5 ml. The ITS region was amplified
using primers ITS1F
(Gardes and Bruns 1993)
(White et al. 1990)
using primers MCM7-709f and Mcm7-1348r
(Schmitt et al. 2009)
, mtSSU using
primers mrSSU1 and mrSSU3R
(Zoller et al. 1999)
and nuLSU rDNA using
(Mangold et al. 2008)
(Vilgalys and Hester 1990)
. PCR products
were sequenced using an ABI PRISM 3730 DNA Analyser (Applied Biosystems) at the
Pritzker Laboratory for Molecular Systematics and Evolution at The Field Museum,
Chicago, Illinois, USA. Nine specimens were obtained previously for a global
phylogenetic study of the genus and sequenced using next generation sequencing technology
as described in
Leavitt et al. (2018)
(Table 1). In short, metagenomic Nextera libraries
(prepared from total DNA extraction) were sequenced on the Nextseq platform at the
Core Genomics Facility at the University of Illinois at Chicago, USA. Illumina reads
of each specimen were mapped to reference marker sequences downloaded from
Genbank (ITS AY581063, nuLSU HM125760, MCM7 HM579689, mtSSU KR995373)
using the mapping feature implemented in Geneious v11.0.3
com, Kearse et al. 2012)
. The consensus sequence of each locus was extracted and
added to the data set of Sanger sequences to build a combined alignment.
Sequence alignment and phylogenetic analysis
Sanger sequences, consensus Illumina reads and sequences available on GenBank were
added to an alignment published in
Leavitt et al. (2018)
using Mafft v7 with the option
‘add sequence’ (Table 1). ITS, MCM7, mtSSU and nuLSU sequences were aligned
independently using the ‘automatic’ option in Mafft v7, with the remaining parameters
set to default values. Ambiguous positions of each one-locus alignment were removed
using options for a “less stringent” selection on Gblocks 0.91b
(Vaidya et al. 2011)
was used for the alignment
concatenation. Phylogenetic analyses were performed using Maximum Likelihood (ML) and
Bayesian Analysis (BA). ML trees were calculated with RAxML-HPC2 on XSEDE
on the Cipres Science Gateway
(Miller et al. 2010)
GTR+G+I substitution model with 1000 bootstrap pseudoreplicates. For the BA,
substitution models for each locus were estimated using jModelTest-2.1.9
Gascuel 2003, Darriba et al. 2012)
: in ITS the TIM2ef+I+G, in MCM7 the K80+G,
in mtSSU the TPM2uf+I and in nuLSU the TrN+I were used. Two parallel Markov
chain Monte Carlo (MCMC) runs were performed in MrBayes 3.2.6
and Ronquist 2001, Ronquist and Huelsenbeck 2003)
, each using 10,000,000
generations which were sampled every 100 steps. A 50% majority rule consensus tree was
generated from the combined sampled trees of both runs after discarding the first 25%
as burn-in. Tree files were visualised with FigTree 1.4.2
. The ITS,
MCM7, mtSSU and nuLSU sequences are deposited in GenBank (Table 1).
Results and Discussion
Results from phylogenetic analyses presented here clearly indicate that the taxonomy
in the Xanthoparmelia mexicana group requires revision because different samples
assigned to the same species based on phenotypical characters may not form a
monophyletic group. Specimens identified as X. mexicana from Asia (Pakistan and South Korea)
were distantly related to samples of the species collected in North America and Europe
(included in X. isidiomontana nom prov) (Fig. 2). The latter specimens fell into three
distinct and well supported clades (clade I-III in Fig. 2). Note that the three distinct
and well supported clades did not form a monophyletic group.
(=X. ‘isidiomontana’ nom prov, ‘D2’ in Leavitt et al. 2013)
isidiate specimens from North America and Europe and samples identified as X. dierythra,
X. mexicana (Figs. 2A and B) and X. plittii, in addition to a number of non-isidiate,
fertile specimens. Additional studies will be necessary to better understand the
delimitation of X. dierythra, which is also polyphyletic and is currently accommodating
specimens with norstictic acid and lacking salazinic acid
. This clade likely
represents another species-level lineage lacking formal taxonomic recognition and a
formal description of this lineage will be proposed once the phylogenetic placement of
X. dierythra s. str. is ascertained.
Clade ‘II’ included specimens collected in the Pedregal, south of Mexico City, which
is also the type locality of X. mexicana. However, the new material does not correspond
phenotypically with the type specimen of X. mexicana in BP (Fig. 2G). These specimens
are different from X. mexicana specimens (represented by Clade III in phylogenetic
analysis) in having less contiguous lobes, densely isidiate thallus, presence of salazinic
acid, norstictic acid and an unknown substance. Since clade ‘II’ differs phylogenetically
and phenotypically from clade ‘III’ (representing X. mexicana s. str. – see below), we
describe clade ‘II’ as a species new to science, X. pedregalensis (Figs. 2C and D).
Clade ‘III’ includes the majority of samples identified as X. mexicana collected in
different localities of Mexico (Oaxaca, Puebla, San Luis Potosí, Querétaro, Hidalgo).
Specimens recovered in this clade were morphologically and chemically similar to the lectotype
of X. mexicana in BP (Fig. 2G). Therefore, clade ‘III’ is here recognised as X. mexicana s.
(Gyelnik 1931, Hale 1974)
(Figs. 2E and F). So far, we have only been able to
confirm the presence of X. mexicana s.str. in Mexico. Specimens collected in other areas and
previously identified as X. mexicana likely represent different species. For example, none
of the samples from Asia or those reported in Leavitt et al. (2013) from western United
States belongs to X. mexicana s. str. Further studies are needed to evaluate the occurrence
of this species in other parts of the world, including North America and Europe.
Underestimates of species diversity is common amongst under-studied organismal
(Pawar 2003, Chiarucci et al. 2011, Lücking 2012, Coleman 2015, Troia and
McManamay 2016, Troudet et al. 2017)
, which is particularly evident in lichenised
(Crespo and Perez-Ortega 2009, Crespo and Lumbsch 2010, Leavitt et al. 2011,
Lumbsch and Leavitt 2011, Leavitt et al. 2013, Leavitt et al. 2016, Lücking et al.
2016, Leavitt et al. 2018)
. Previous studies concluded that the species delimitation
in lichenised ascomycetes with traditional morphological and chemical characters are
apparently misleading with respect to species diversity. In the study of Leavitt et al.
(2016), several new taxa were described primarily based on evidence from genetic
data, but it does not preclude the possibility that additional studies investigating
morphological and chemical characters may identify additional independent characters
or combinations of characters, supporting the species circumscribed using molecular
data. Our results corroborate findings from the previous studies by showing the need
of an integrative approach using not only conventional (i.e. morphology and TLC
data), but also new sets of data (e.g. DNA sequence data) to better understand the
pattern of species diversity. Our study shows that, by incorporating molecular data,
the taxonomic status of a conventionally difficult group based on morphology can be
resolved: the three main clades belonging to the X. mexicana complex do not form
a monophyletic group based on our newly reconstructed phylogeny (Fig. 1). In this
context, the species diversity in the X. mexicana complex is likely under-estimated and
morphologically cryptic species may be identified in the future.
Type. MEXICO. Ciudad de México: Coyoacán, Pedregal de San Angel,
19°19'8.3"N, 99°11'25.93"W, 2321 m elev., xerophytic scrub, on rocks, November,
2017, Ruiz Cazares 1553 (MEXU-holotype), same locality and date Ruiz Cazares
Diagnosis. Thallus moderately adnate to adnate, imbricate, upper surface
yellowgreen, lower surface tan-brown, abundant isidia subglobose to cylindrical, simple to
branched and medulla containing salazinic and norstictic acids as major compounds
and an unknown substance. Differing from the phenotypically similar X. mexicana by
nucleotide position characters in the ITS sequence as shown in Table 2.
Etymology. The taxon name is based on its occurrence in the Pedregal de San
Angel region of Mexico.
Description. Thallus foliose, moderately adnate to adnate, 2–7 cm in diam.,
irregularly lobate; lobes subirregular, elongate, plane to subconvex, 1.5–3 mm
wide, not lobulate; apices subrotund, smooth, eciliate. Upper surface yellow-green,
smooth, shiny, epruinose and emaculate, densely isidiate; isidia initially subglobose,
becoming cylindrical to coralloid branched with age, 0.1–0.2 mm in diam., 0.1–
0.9 mm tall; tips syncorticate, brown to dark brown, sometimes weakly erumpent;
soralia and pustulae absent. Medulla white, with continuous algal layer. Lower
surface tan to brown, plane, moderately rhizinate; rhizines pale to dark brown,
simple, 0.5–0.9 mm long. Apothecia rare, sessile, 1–2 mm wide, laminal on thallus;
disc cinnamon-brown to dark brown; margin smooth, pruina absent; asci: clavate,
8-spored; ascospores hyaline, simple, ellipsoid, 9–10 × 4–5 µm. Pycnidia rare,
immersed conidia bifusiform, 5–7 × 1 µm.
Secondary metabolites. Upper cortex K–, C–, KC–, P–; medulla K+ yellow then
dark red, KC–, C–, P+ yellow-orange. Upper cortex with usnic acid (major); medulla
with salazinic (major) and norstictic acids (submajor) and an unknown substance
(minor) (Rf 28–30, brown in daylight after heating, UV brown-dark, yellow halo
Distribution and ecology. The new species was found in xerophytic scrub
vegetation, in Pedregal de San Angel south of Mexico City, growing on volcanic rocks. It is
currently known only from the type locality.
Notes. Xanthoparmelia pedregalensis is morphological and chemically similar to X.
mexicana. However, the latter has more contiguous lobes and is less isidiate than X.
pedregalensis. In addition X. mexicana has salazinic (major) and consalazinic acid (minor)
and usually norstictic and protocetraric acids (trace) in the medulla, whereas X.
pedregalensis contains salazinic (major) and norstictic acids (submajor) and an unknown
substance. Distinguishing the two species is supported by molecular data.
Additional specimens examined. Mexico. Ciudad de México: Coyoacán, Pedregal
de San Angel, 19°19'8.3"N, 99°11'25.93"W, 2321 m elev., xerophytic scrub, on rocks,
November, 2017, Ruiz Cazares 1552 (MEXU); 19°19'15.19"N, 99°11'15.22"W,
2311 m, Ruiz Cazares 1555, 1557 (F).
New state records
Xanthoparmelia ajoensis (Nash)
: 234. [Type collection: Organ Pipe Cactus National
Monument, Pima Co., Arizona, USA, Nash 5999 (ASU, holotype; DUKE, US,
isotypes).] New to Oaxaca, X. ajoensis is distributed across western USA and Mexico
where it has previously been reported from Baja California Sur, Durango, Morelos,
Puebla, Sinaloa and Sonora on acidic rocks, often in open, arid habitats at relatively
(Hale 1990, Nash and Elix 2004, Nash et al. 2016)
Specimens Examined: Mexico. Oaxaca: Quiotepec, 17°54'18.9"N, 96°58'01.8"W,
696 m elev., xerophytic scrub, on rock, October, 2016, Barcenas-Peña 5906, 5908,
5913, 5915 (MEXU).
New to Puebla. Xanthoparmelia moctezumensis is distributed throughout
south-western USA and Mexico where it has been reported from Baja California Sur, Durango,
Sinaloa and Sonora on acidic rocks, often in open, arid to woodland habitats
and Elix 2004, Nash et al. 2016)
Specimens Examined: Mexico. Puebla: San Rafael Coxcatlán, 18°13'16.6"N,
97°07'22.4"W, 1148 m elev., xerophytic scrub, on rock, October, 2016, Barcenas-Peña
5887, 5890, 5891, 5893 (MEXU).
Xanthoparmelia mexicana (Gyelnik) Hale, 1974: 488.
New to Querétaro, San Luis Potosí and Zacatecas. Xanthoparmelia mexicana has been
reported from Baja California, Baja California Sur, Chihuahua, Coahuila, Distrito
Federal, Durango, Guanajuato, Hidalgo, Jalisco, Michoacán, Nuevo León, Oaxaca,
Puebla, Sonora and Veracruz, on acidic rocks, often on soil near the coast in open, arid
(Nash et al. 2004, 2016)
Specimens Examined: Mexico: Querétaro. Tequisquiapan, Rancho Las Fuentes,
20°33'51.0"N, 100°01'54.6"W W, 1942 m elev., xerophytic scrub, on rock,
August, 2017, Barcenas-Peña 7516; San Luis Potosí, Mexquitic de Carmona, La
Campana, 22°15'28.9"N, 101°05'26.8"W, 2012 m elev., xerophytic scrub, on rock,
August, 2017, Barcenas-Peña 7441; Zacatecas, Fresnillo, El Poleo, 23°06'16.4"N,
102°54'24.3"W, 2227 m elev., xerophytic scrub, on rock, August, 2017,
BarcenasPeña 7356 (all MEXU).
The first author thanks the National Council of Science and Technology
(CONACYT) by grants for supporting her research stay to the Field Museum. We are grateful
to Dr. Tom Nash III, Biol. Alin Ruiz and José Vladimir Rodríguez for sending us the
specimens. We are grateful to Dr. Armando Burgos and Biol. Maricarmen Altamirano
for their assistance in the field work. We are grateful to Dra. Silke Cram for logistical
support at Reserva Ecológica del Pedregal de San Angel. The authors are thankful to
the Pritzker Laboratory for Molecular Systematics at the Field Museum. We thank to
Negaunee Foundation for financial support.
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