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).

Figure 1. 

Location of new Xanthoparmelia recollection sites from arid regions from central part of Mexico. Oaxaca (pink), Puebla (green), Mexico City (red), Estado de México (blue), Querétaro (purple), Guanajuato (brown), Hidalgo (grey), Aguas Calientes (yellow), San Luis Potosí (black), Zacatecas (orange).

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) and ITS4 (White et al. 1990); MCM7 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 primers AL2R (Mangold et al. 2008) and LR6 (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 (ITSAY581063, nuLSUHM125760, MCM7HM579689, mtSSUKR995373) using the mapping feature implemented in Geneious v11.0.3 (http://www.geneious.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.

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 (Castresana 2000). SequenceMatrix software (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 8.2.10 (Stamatakis 2014) on the Cipres Science Gateway (Miller et al. 2010) using GTR+G+I substitution model with 1000 bootstrap pseudoreplicates. For the BA, substitution models for each locus were estimated using jModelTest-2.1.9 (Guindon and 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 (Huelsenbeck 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 (Rambaut 2014). The ITS, MCM7, mtSSU and nuLSU sequences are deposited in GenBank (Table 1).

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.

Figure 2. 

Phylogenetic relationships of the Xanthoparmelia mexicana group based on a concatenated data set of ITS, mtSSU, nuLSU and MCM7. Topology based on maximum likelihood (ML) analyses. Bootstrap values above 75 and 0.95 posterior probability are indicated on each branch. The clades I, II and III are highlighted in blue, yellow and pink, respectively. Selected specimens representing clades (habit and isidia): I, X. mexicana s. lat. (A, B); II, X.pedregalensis (C, D) and III, X. mexicana s. str. (E, F), a picture of the X. mexicana type specimen from BP is included (G).

Clade ‘I’ (=X. ‘isidiomontana’ nom prov, ‘D2’ in Leavitt et al. 2013) included 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 (Hale 1990). 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. str. (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 groups (Pawar 2003, Chiarucci et al. 2011, Lücking 2012, Coleman 2015, Troia and McManamay 2016, Troudet et al. 2017), which is particularly evident in lichenised fungi (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.

Xanthoparmelia ajoensis (Nash) Egan, 1975: 217.

Parmelia ajoensis Nash, 1974: 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 low elevations (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).

Xanthoparmelia moctezumensis Nash in C. Culberson, Nash & Johnson, 1979: 155. [Type collection: 28 km E of Moctezuma, Sonora, Mexico, Nash 12548 (ASU, holotype; DUKE, US, isotypes).]

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 (Nash 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 habitats (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, Barcenas-Peña 7356 (all MEXU).