The genus Xanthoparmelia is the largest genus of lichen- forming fungi with about 800 species worldwide. Xanthoparmelia is also common in the deserts of central Mexico, but only a few molecular studies exist on its species’ diversity in this region. In this study, we sampled 38 Xanthoparmelia species from around the world including species from the xerophytic scrubs of central Mexico to assess the diversity using an integrative approach. Molecular phylogenetic analyses were performed using a combination of the ITS, mtSSU and nuLSU genetic markers. We evaluated our phylogenetic results in a context of traditional morphological and chemical characters. The combined evidence of molecular, morphological, and chemical data identified a total of 18 Xanthoparmelia species-level lineages occurring in central Mexico. However, numerous traditionally circumscribed species did not form monophyletic groups in the molecular phylogenetic reconstructions. This conflict indicates that taxonomy and species delimitation in the genus Xanthoparmelia requires revision and emphasizes the importance of molecular evidence for more robust species delimitations in this genus.
Resumen
Xanthoparmelia es el género más grande de hongos liquenizados, con alrededor de 800 especies en todo el mundo. Xanthoparmelia es común en los desiertos del centro de México, pero existen pocos estudios moleculares sobre la diversidad de especies en esta región. En este estudio, muestreamos 38 especies de Xanthoparmelia de diferentes partes del mundo, incluidas especies de los matorrales xerófilos del centro de México, para evaluar la diversidad usando una aproximación integrativa. Los análisis filogenéticos moleculares se realizaron combinando los marcadores genéticos ITS, mtSSU y nuLSU. Además, evaluamos nuestros resultados filogenéticos en un contexto de caracteres morfológicos y químicos usados en la taxonomía tradicional. Teniendo en cuenta las evidencias obtenidas a partir de caracteres moleculares, morfológicos y químicos se identificaron un total de 18 linajes de Xanthoparmelia con categoría de especie que aparecen en el centro de México. Sin embargo, muchas especies tradicionalmente circunscritas no formaron grupos monofiléticos. Este conflicto indica que la taxonomía y delimitación de especies en el género Xanthoparmelia requiere revisión y enfatiza la importancia de los datos moleculares para una delimitación más robusta de especies en este género.
Cryptic speciesbiodiversitysecondary metaboliteslichensMexicoEspecies crípticasbiodiversidadmetabolitos secundarioslíquenesMéxicoNational Council of Science and Technology (CONACYT)The first author thanks the National Council of Science and Technology (CONACYT) for support through grants to allow for a research visit to the Field Museum. We are grateful to Dr. Armando Burgos, Biol. Maricarmen Altamirano and Aline Cazares for their assistance in the field and the elaboration of the map. Sequencing was done at the Pritzker Laboratory for Molecular Systematics at the Field Museum. Introduction
The diversity of organisms is distributed unequally in the world. Species distributions are influenced by a wide range of factors, including biogeographic history, topography, or climate (Fischer 1960; Brown 2001; Rozzi & al. 2008). These factors can result in species-rich regions, including hotspots of biodiversity where rare and endemic species occur. One of the 25 biodiversity hotspots of the world is the Mesoamerican region that extends from northern Costa Rica and Nicaragua to central Mexico (Myers & al. 2000). The high levels of biodiversity in Mexico have been explained by the country’s geographic position between tropical and temperate regions and its importance as a refugial area for temperate species during Pleistocene glaciations (Mittermeier 1988; Ramamoorthy & al. 1993; Myers & al. 2000). Mexico has a wide range of ecosystems from rainforest to dry deserts. We focus on the xerophytic scrub, which is among the most widespread ecosystems in Mexico, covering about 40% of the country and harbouring high biodiversity. This vegetation is adapted to face aridity and mainly consists of low trees, shrubs and succulent plants (Rzedowski 1978). Lichen communities in xerophytic scrub frequently consist of crustose lichens, as well as, members of the lichen-forming fungi family Parmeliaceae F.Berchtold & J.Presl. In Parmeliaceae, species of the genera Usnea Dill. ex Adans., Parmotrema A.Massal., Hypotrachyna (Vain.) Hale, and Xanthoparmelia (Vain.) Hale are particularly common (Lücking & al. 2016).
The genus Xanthoparmelia is the most diverse genus of lichen-forming fungi with about 800 species worldwide (Blanco & al. 2004; Thell & al. 2012; Jaklitsch & al. 2016). Xanthoparmelia is chemically diverse and the presence or absence of secondary metabolites has been widely used, in conjunction with morphological characters, to delimit species (Hale 1990; Elix 1994; Nash & al. 2016). Xanthoparmelia diversified dramatically during the Miocene (Kraichak & al. 2015). Recent studies show that Xanthoparmelia could have originated in the African continent during the early Miocene, eventually spreading to Australia and South Africa -both current centers of diversity for this genus. In contrast, the Holarctic has been more recently colonized and diversification happened mainly during the late Miocene and early Pliocene, resulting in a lower diversity of Xanthoparmelia in this region (Leavitt & al. 2018). However, in Mexico there exists a high Xanthoparmelia diversity and a high chemosyndromic variation has been recognized inside of the genus (Culberson & al. 1979). Due to this, deep studies are needed to clarify many of the phylogenetic clades within the group (Nash & al. 2016). Nevertheless, at the date, there only exists a single molecular study of Xanthoparmelia that included five taxa from xerophytic scrub (Barcenas-Peña & al. 2018), despite the fact that this is the most extensive ecosystem in the country and the preferred habitat for Xanthoparmelia species. Consequently, the xerophytic scrub is an ecosystem that requires a deep molecular study of Xanthoparmelia species (Barcenas-Peña & al. 2018).
In Mexico, Xanthoparmelia species are very abundant due to the presence of numerous exposed rocky substrates, and c. 75 species are currently accepted based mainly on phenotypical characters (Nash & al. 2016; Barcenas-Peña & al. 2018). Forty six Xanthoparmelia species are recorded from xerophytic scrub vegetation in the country (Lücking & al. 2016; Nash & al. 2016; Barcenas-Peña & al. 2018) mainly from the Sonoran Desert in the north of the country, one of the most studied taxonomically regions (morphological and chemically) (Nash & Elix 2004; Nash & al. 2016). However, the central area of the country still requires attention. In addition, in Mexico there are currently few studies of lichen fungi at the molecular level. For instance, only a work about Xanthoparmelia mexicana (Gyeln.) Hale group from xerophytic scrub in Mexico has been done (Barcenas-Peña & al 2018). Here we studied the diversity of Xanthoparmelia species in xerophytic scrub vegetation of the central part of Mexico using morphology, secondary chemistry, and molecular sequence data to understand the phylogenetic diversity and relationship among the species.
Material and methodsStudy area
All Xanthoparmelia specimens were collected on rocks from different localities throughout arid regions of xerophytic scrub in the central part of Mexico: Zacatecas, Aguas Calientes, San Luis Potosi, Jalisco, Guanajuato, Querétaro, Hidalgo, Estado de Mexico and Mexico City (Fig. 1). Xerophytic scrub occupies approximately 40% of the country’s surface and is distributed from the Baja California Peninsula, the Coastal Plain and the lower Sierra de Sonora. Likewise, it is characteristic of the Altiplano from Chihuahua and Coahuila to Jalisco, Guanajuato, Hidalgo and Estado de Mexico, extending to Puebla and Oaxaca. It also constitutes the vegetation of a part of the northeastern Coastal Plain of Coahuila and Tamaulipas. These areas show average temperatures between 12°C and 26°C and an average annual rainfall of 100 ml to 400 ml (Rzedowski 1978).
Location of <italic>Xanthoparmelia</italic> (Vain.) Hale collection sites from arid regions of central Mexico
Mexico City (MX CITY), Estado de México (EDO MEX), Querétaro (QRO), Guanajuato (GTO), Hidalgo (HGO), Aguas Calientes (AGS), Jalisco (JAL), San Luis Potosí (SLP) and Zacatecas (ZAC)
Anatomical studies
For all specimens collected (about 440, deposited at F and MEXU; Appendix 1) the morphology and chemistry were assessed. Morphological characters, such as, shape and size of the thallus and lobules, isidia shape, lower surface and medulla color were studied according to Hale (1990) and Nash & al. (2016) using a Zeiss Stemi 2000-C stereoscope. Ascomatal anatomy, ascospore, conidia shape and size were studied using a Zeiss Axioscope. Secondary metabolites were identified using spot test with 10% KOH, KC, C, PD and high-performance thin layer chromatography (HPTLC) using solvent system C following established methods (Culberson & Johnson 1982; Arup & al. 1993; Lumbsch 2002; Orange & al. 2010).
Taxon sampling
Since a large number of specimens had lichenicolous fungi, only selected specimens of each species were used to obtain DNA. A total of 29 specimens of 18 species were selected for molecular analyses from xerophytic scrub vegetation representing the range of morphological and chemical variation, and supplemented with sequences of 20 specimens of 4 species from Puebla and Oaxaca (Mexico) of a previous analysis (Barcenas-Peña & al. 2018) (Appendix 2). In addition, sequences of 138 specimens of 27 species from different parts of the world were downloaded from GenBank (Appendix 2). Four species that have previously been shown to be distantly related to the Xanthoparmelia species from North America were used as outgroups, including X. crespoae Elix, Louwhoff & M.C. Molina, X. filarszkyana (Gyeln.) Hale, X. substrigosa (Hale) Hale and X. lithophiloides (Kurok.) Elix (Leavitt & al. 2018). Altogether, a total of 187 specimens of 38 species were included in this study (Appendix 2).
Molecular methods
From the 29 specimens sampled from Mexico, 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 three markers using the polymerase chain reaction (PCR): the nuclear ribosomal internal transcribed spacer region (ITS), a region of the mitochondrial small subunit rDNA (mtSSU), and a region of the nuclear large subunit rDNA (nuLSU). PCR reactions contained 6.25 µl of MyTaq™ Red DNA Polymerase (Bioline, Taunton, MA, USA), 5.25 µl of H2O, 0.25 µl of forward and reverse primers (10 μM), and 0.5 µl of template DNA (10X), for a total reaction volume of 12.5 µl. The ITS region was amplified using primers ITS1F (Gardes & Bruns 1993) and ITS4 (White & al. 1990); mtSSU using primers mrSSU1 and mrSSU3R (Zoller & al. 1999), and nuLSU rDNA using primers AL2R (Mangold & al. 2008) and LR6 (Vilgalys & Hester 1990). PCR products were sequenced using the same primers used for amplification and ABI PRISM 3730 DNA Analyzer (Applied Biosystems) at the Pritzker Laboratory for Molecular Systematics and Evolution at The Field Museum, Chicago, Illinois, USA.
Sequence alignment and phylogenetic analysis
ITS, mtSSU and nuLSU sequences were aligned independently using the ‘auto’ option with FFT-NS-i algorithm, in Mafft v7 (Katoh & Standley 2013), with the remaining parameters set to default values. Ambiguous positions of each alignment were removed using options for a “less stringent” selection on Gblocks 0.91b (Castresana 2000). SequenceMatrix software (Vaidya & al. 2011) was used to concatenate all three alignments. Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian analyses (BA). ML trees were calculated with RAxML-HPC2 on XSEDE 8.2.10 (Stamatakis 2014) on the Cipres Science Gateway (Miller & al. 2010) using GTR+G+I substitution model with 1000 bootstrap pseudoreplicates, with the data partitioned by loci. For the BA, substitution models for each locus were estimated using jModelTest-2.1.9 (Guindon & Gascuel 2003; Darriba & al. 2012), which recommended for ITS locus the TIM2ef+I+G model, for mtSSU locus the F81+I model and for the nuLSU locus the TIM2ef+I+G model. Due the TIM2ef substitution models are not implemented in MrBayes were replaced by the GTR model (Ronquist & Huelsenbeck 2003). The proportion of invariable sites (I) and gamma distributed rates (G) defined in jModeltest were conserved in both cases. Two parallel Markov chain Monte Carlo (MCMC) runs were performed in MrBayes 3.2.6 (Huelsenbeck & Ronquist 2001; Ronquist & 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 (149,965) of both runs after discarding the first 25% trees as burn-in. Convergence diagnostic as PSRF was reasonably close to 1.0 for all parameters and average deviation of split frequencies was below 0.01 (Gelman & Rubin 1992). Tree files were visualised with FigTree 1.4.2 (Rambaut 2014). The ITS, mtSSU and nuLSU sequences are deposited in GenBank (Appendix 2).
Results and discussionPhylogeny
Our phylogenetic analyses recovered nine well supported clades of Xanthoparmelia (Fig. 2). Seven of them (clades 1 to 7) include Mexican specimens of the same species forming monophyletic clusters, the rest of the Mexican specimens are distributed all over the phylogenetic tree. In total our phylogenetic analysis revealed 18 species-level lineages from xerophytic scrublands in Mexico (Fig. 2, Appendix 2). Clades 8 and 9 are X. mexicana groups from USA-Spain and USA respectively. The seven major lineages of Mexican Xanthoparmelia samples from xerophytic scrublands included: 1) X. mexicana s. str., 2) the X. moctezumensis T.H.Nash group, 3) the X. ajoensis (T.H.Nash) Egan group, 4) the X. lavicola (Gyeln.) Hale group, 5) the X. pedregalensis Barcenas Peña, Lumbsch & S.D.Leav. group, 6) the X. hypomelaena (Hale) Hale group, and 7) the X. subramigera (Gyeln.) Hale group.
The 18 species recognized in this analysis have been previously reported in Mexico in morphological and chemical studies (Nash & Elix 2004; Nash & al. 2004; Nash & al. 2016; Barcenas-Peña & al. 2018). However, many of these species did not have sequences available in Genbank including Xanthoparmelia hypomelaena, X. joranadia (T.H.Nash) Egan, X. neotaractica Hale, X. planilobata (Gyeln.) Hale, X. subtasmanica Elix & T.H.Nash, X. tuckeriana Elix & T.H.Nash and X. tucsonensis (T.H. Nash) Egan (Appendix 2). The ML and BA phylogeny (Fig. 2) show that the phenotype-based taxonomy in the genus requires revision, with numerous species identified using morphological and chemical characters not forming monophyletic groups. In addition, with exception of X. pedregalensis none of these species are type materials from Mexico. In the X. mexicana group from the USA and Spain (clade 8) there are not samples from Mexico. As well, within clade 8 samples of X. dierythra (Hale) Hale, X. lineola (E.C.Berry) Hale, X. mexicana, and X. plittii (Gyeln.) Hale are included and do not form monophyletic clades. These species are currently distinguished based on their reproduction and secondary chemistry (Hale 1990). However, the substances used as diagnostic characters (norstictic, salazinic, and stictic acids) belong to the same chemosyndrome, which has, in some cases, been interpreted as intraspecific variation (Lumbsch 1998a, 1998b). Further, one sample agreeing with the current circumscription of the vagrant X. chlorochroa (Tuck.) Hale clustered in this group. In fact, all phenotypes (=currently accepted species) in this clade also clustered in other clades in the phylogenetic tree. This strongly indicates that the characters used for the circumscription of species in this clade need re-evaluation, considering that a recent study from Australian Xanthoparmelia species has observed that closely related samples may exhibit distinct chemical profiles. Additionally, that in the evolution of secondary metabolite composition can be rapid, which may result in convergence between distantly related samples (Autumn & al. 2020). A similar pattern is found in the clade of Mexican X. mexicana specimens (clade 1 of Mexican specimens in Fig. 2), in which samples identified as X. chlorochroa, X. dierythra, X. lineola, and X. mexicana are found and none of them form a monophyletic group. Even though clade 1 is a little below the supported values (bootstrap values above 75%), we still considered it a good example that reflects the need of re-evaluation of species. Additionally, the presence of several well-supported clades within clade 1 suggests that even after the segregation of X. pedregalensis (Barcenas-Peña & al. 2018), X. mexicana in Mexico is not well understood. However, given that the species was described from central Mexico, we regard clade 1 as X. mexicana whereas specimens currently accepted as X. mexicana from other parts of the world could belong to other taxa.
Phylogenetic relationships of the <italic>Xanthoparmelia</italic> (Vain.) Hale species from Mexican xerophytic scrub based on a concatenated data set of ITS, mtSSU and nuLSU. Topology is based on maximum likelihood (ML) analyses. ML bootstrap values above 75% and Bayesian posterior probability values above 0.95 are indicated on each branch. The 29 specimens samples generated in this study are indicated with a black dot. Nine lineages, including seven major groups of Mexican samples, are indicated with color boxes. A red asterisk indicates Mexican samples falling outside the seven major clades including Mexican specimens (clades 1-7). Collapsed nodes coI-coVII.
The Xanthoparmelia mexicana group from the USA (clade 9) includes samples of the nonisidiate X. cumberlandia (Gyeln.) Hale, X. maricopensis T.H.Nash & Elix (also containing hyposalazinic acid), and the chemically different X. psoromifera (Hale) Hale (with psoromic acid). Psoromic acid has been found to be inconsistent with monophyletic groups in the distantly related genus Cladonia (Pino-Bodas & al. 2012).
The Xanthoparmelia moctezumensis clade also includes a sample of X. ajoensis. However, the two species contain the closely-related depsides 3-a-hydroxybarbatic or diffractaic acids, respectively. This is consistent with the hypothesis that morphologically identical specimens with closely-related substances often represent variation within a single species (Feige & Lumbsch 1995). However, the majority of specimens phenotypically identified as X. ajoensis are found in a separate clade, the X. ajoensis group (clade 3) with unresolved relationships to the X. moctezumensis group (clade 2). Additional studies with an extended sampling of specimens and genetic markers of these two groups is required to better understand the phylogenetic relationships in this part of the phylogenetic tree.
The Xanthoparmelia lavicola group (clade 4) is represented by three samples of this species that was originally described from central Mexico. It was strongly supported as a monophyletic group but its phylogenetic relationships remain unresolved. Another strongly supported clade in the ML analysis was the X. pedregalensis group (clade 5) that is closely related to X. neotaractica, X. sublaevis (Cout.) Hale, X. lineola and X. coloradoensis (Gyeln.) Hale. X. pedregalensis has been treated in detail elsewhere (Barcenas & al. 2018). The X. hypomelaena group (clade 6) included two Mexican specimens-here again the relationships remain unresolved. All studied samples of X. subramigera from Mexico and Kenya clustered together (clade 7), indicating that this species indeed has a larger distributional range and is a well-delimited taxon.
A number of Mexican samples did not cluster in one of the seven clades discussed above including Xanthoparmelia californica Hale, X. conspersa (Ehrh. ex Ach) Hale, X. coloradoensis, X. cumberlandia, X. joranadia, X. planilobata, X. tuckeriana, and X. tucsonensis, as well as samples of X. lineola, X. neotaractica, and X. subtasmanica, none of them forming monophyletic groups. Samples of some of these species from other localities out of Mexico did not cluster with the Mexican specimens, indicating that the delimitation of these species is poorly understood. Since none of these species’ names are based on type materials from Mexico, they might represent distinct lineages that might require formal description. This, however, has to wait until we have gained a better understanding of delimitation of species in Holarctic species of Xanthoparmelia. Since the Holarctic species of the genus are hypothesized to have originated and diversified relatively recently, species delimitation with multi-gene data sets have been shown to be difficult (Leavitt & al. 2011, 2013) and hence reduced genomic data sets, such as RADseq might help elucidate species delimitations (Grewe & al. 2017, 2018). In addition, our collection sampling in Mexico will need to be extended, since the described diversity of Xanthoparmelia species based on phenotypical characters in the Mexican xerophytic scrub vegetation is higher (44 species) (Nash & al. 2016; Lücking & al. 2009) than found in this work (23 species, 18 included in the phylogenetic analyses).
Taxonomy
We identified 23 species from the Mexican xerophytic scrub based on their morphology, mainly presence of isidia and lower surface colour, as well as secondary substances (Table 1, Fig. 3). Both isidiate and not isidiate species were found in almost the same proportion, while species with pale to brown lower surface were more frequent. Additionally, we see a high variety of secondary substances in species morphologically similar (Table 1). Nevertheless, nearly 44 Xanthoparmelia species were found in the Mexican xerophytic scrub mainly from the northern part of Mexico (Nash & Elix 2004; Nash & al. 2004; Nash & al. 2016). While in the present study we collected in the central part of Mexico and the diversity of species found is high (23 putative species-level lineages). Additional studies that can include all of the diversity of the country are necessary. Incorporation of molecular analyses using different genetic markers to the morphological and chemical study towards a better species delimitation is also necessary.
Summary of <italic>Xanthoparmelia</italic> (Vain.) Hale species identified in this study, indicating chemistry substances, isidia presence (+) /absence (-) and lower surface colour.
Species
Chemistry
Isidiate
Lower surface color
X. ajoensis (T.H.Nash) Egan [G3]
Diffractaic and barbatic
+
Pale tan or brown
X. californica Hale *
Norstictic and connorstictic
-
Pale to medium brown
X. coloradoensis (Gyeln.) Hale *
Consalazinic, norstictic, protocetraric and salazinic
-
Brown
X. conspersa (Ehrh. ex Ach.) Hale *
Stictic, constictic, cryptostictic and norstictic
+
Black
X. cumberlandia (Gyeln.) Hale *
Stictic and constictic and norstictic
-
Pale brown or brown
X. hypomelaena (Hale) Hale [G6]
Fumarprotocetraric
-
Black
X. jornadia (T.H.Nash) Hale *
Lecanoric
+
Pale brown
X. lavicola (Gyeln.) Hale [G4]
Psoromic
+
Pale to medium brown
X. lineola (E.C.Berry) Hale *
Salazinic and consalazinic
-
Pale to medium brown
X. mexicana (Gyeln.) Hale s. str. [G1]
Salazinic, consalazinic and norstictic
+
Pale to medium brown
X. moctezumensis T.H.Nash [G2]
3-α-hydroxybarbatic, barbatic, baeomycesic and squamatic
Succinprotocetraric, fumarprotocetraric and protocetraric
+
Pale to medium brown
X. subtasmanica Elix & T.H.Nash *
Salazinic and consalazinic
-
Black
X. tinctina (Maheu & A.Gillet) Hale
Salazinic and consalazinic
+
Black
X. tuckeriana Elix & T.H.Nash *
Fumarprotocetraric and protocetraric
-
Ivory to pale brown
X. tucsonensis (T.H.Nash) Egan *
Diffractaic and barbatic
-
Pale brown
Some species of <italic>Xanthoparmelia</italic> (Vain.) Hale included in this study: <bold>a,</bold>
<italic>Xanthoparmelia ajoensis</italic> (T.H.Nash.) Egan (Barcenas-Peña 5900, F); <bold>b,</bold>
<italic>Xanthoparmelia lavicola</italic> (Gyeln.) Hale (Barcenas-Peña 5905, F); <bold>c,</bold>
<italic>Xanthoparmelia moctezumensis</italic> T.H.Nash (Barcenas-Peña 5891, F); <bold>d,</bold>
<italic>Xanthoparmelia subramigera</italic> (Gyeln.) Hale (Ruiz-Cazares 1620, F)
Acknowledgements
The first author thanks the National Council of Science and Technology (CONACYT) for support through grants to allow for a research visit to the Field Museum. We are grateful to Dr. Armando Burgos, Biol. Maricarmen Altamirano and Aline Cazares for their assistance in the field and the elaboration of the map. Sequencing was done at the Pritzker Laboratory for Molecular Systematics at the Field Museum.
ReferencesArupU.EkmanS.LindblomL.MattssonJ-E.1993High performance thin layer chromatography (HPTLC), an improved technique for screening lichen substancesLichenologist256171AutumnK.Barcenas-PeñaA.Kish-LevineS.HuangJ-PLumbschT.H.2020Repeated colonization between arid and seasonal wet habitats, frequent transition among substrate preferences, and chemical diversity in Western Australian Xanthoparmelia lichensFrontiers in Ecology and Evolution8129Barcenas-PeñaA.LeavittS.D.HuangJ.P.GreweF.LumbschH.T.2018Phylogenetic study and taxonomic revision of the Xanthoparmelia mexicana group, including the description of a new species (Parmeliaceae, Ascomycota)MycoKeys401328BlancoO.CrespoA.ElixJ.A.HawksworthD.L.LumbschH.T.2004A molecular phylogeny and a new classification of parmelioid lichens containing Xanthoparmelia-type lichenan (Ascomycota: Lecanorales)Taxon53959975BrownJ.2001Mammals on mountainsides: elevational pattern of diversityGlobal Ecology and Biogeography10101109CastresanaJ.2000Selection of conserved blocks from multiple alignments for their use in phylogenetic analysisMolecular Biology and Evolution17540552CulbersonC.JohnsonA.1982Substitution of methyl tert.-butyl ether for diethyl ether in standardized thin layer chromatographic method for lichen productsJournal of Chromatography B238438487CulbersonC.NashT.H.IIIJohnsonA.19793-α-hydroxibarbatic acid, a new depside in chemosyndrome of some Xanthoparmeliae with α-orcinol depsidesBryologist82154161DarribaD.TaboadaG.L.DoalloR.PosadaD.2012jModelTest 2: more models, new heuristics and parallel computingNature Methods9772ElixJ.A.1994XanthoparmeliaFlora of Australia55201308FeigeG.B.LumbschH.T.1995Some types of chemical variation in lichensCryptogamic Botany53135FischerA.G.1960Latitudinal variations in organic diversityEvolution146481GardesM.BrunsT.1993ITS primers with enhanced specificity for basidiomycetes-Application to the identification of mycorrhizae and rustsMolecular Ecology2113118GelmanA.RubinD.B.1992Inference from iterative simulation using multiple sequencesStatistical Science7457511GreweF.HuangJ.P.LeavittS.D.LumbschH.T.2017Reference-based RADseq resolves robust relationships among closely related species of lichen-forming fungi using metagenomic DNAScientific Reports79884GreweF.LagostinaE.WuH.PrintzenC.LumbschH.T.2018Population genomic analyses of RAD sequences resolves the phylogenetic relationship of the lichen-forming fungal species Usnea antarctica and Usnea aurantiacoatraMycokeys4391113GuindonS.GascuelO.2003A simple, fast and accurate method to estimate large phylogenies by maximum-likelihoodSystematic Biology52696704HaleM.E.Jr.1990A synopsis of the lichen genus Xanthoparmelia (Vainio) Hale (Ascomycotina, Parmeliaceae)Smithsonian Contributions to Botany 741250HuelsenbeckJ.P.RonquistF.2001MRBAYES: Bayesian inference of phylogenetic treesBioinformatics17754755JaklitschW.M.BaralH.O.LückingR.LumbschH.T.2016AscomycotaFreyW.Syllabus of Plant Families - Adolf Engler’s Syllabus der Pflanzenfamilien1150StuttgartGebr. Borntraeger VerlagsbuchhandlungKatohK.StandleyD.M.2013MAFFT multiple sequence alignment software version 7: improvements in performance and usabilityMolecular Biology and Evolution30772780KraichakE.DivakarP.K.CrespoA.LeavittS.D.NelsenM.P.LückingR.LumbschH.T.2015A tale of two hyper-diversities: diversification dynamics of the two largest families of lichenized fungiScientific Reports5e10028LeavittS.D.JohnsonL.A.GowardT.St.ClairL.L.2011Species delimitation in taxonomically difficult lichen-forming fungi: an example from morphologically and chemically diverse Xanthoparmelia (Parmeliaceae) in North AmericaMolecular Phylogenetics and Evolution60317332LeavittS.D.KirikaP.M.Amo de PazG.HuangJ.P.HurJ.S.ElixJ.A.GreweF.DivakarP.K.LumbschH.T.2018Assessing phylogeny and historical biogeography of the largest genus in lichen-forming fungi, Xanthoparmelia (Parmeliaceae, Ascomycota)Lichenologist50299312LeavittS.D.LumbschH.T.StenroosS.St ClairL.L.2013Pleistocene speciation in North American lichenized fungi and the impact of alternative species circumscriptions and rates of molecular evolution on divergence estimatesPlos One8e85240LückingR.Rivas PlataE.ChavesJ.L.UmañaL.SipmanH.J.M.2009How many tropical lichens are there. . . really?Bibliotheca Lichenologica100399418LückingR.VillaseñorJ.L.Herrera-CamposM.A.Pérez-PérezR.M.EganS.R.EsslingerH.L.Nash IIIT.H.2016Phylogenetic structure of metacommunities in Mexican Parmeliaceae (lichenized Ascomycota: Lecanorales)Bibliotheca Lichenologica1102754LumbschH.T.1998aThe use of metabolic data in lichenology at the species and subspecific levelsLichenologist30357367LumbschH.T.1998bThe taxonomic use of metabolic data in lichen-forming fungiFrisvadJ.C.BridgeP.D.AroraD.K.Chemical Fungal Taxonomy345387Marcel DekkerNew YorkLumbschH.T.2002Analysis of phenolic products in lichens for identification and taxonomyKrannerI.BeckettR.VarmaA.Protocols in Lichenology Culturing, biochemistry, ecophysiology and use in biomonitoring281295SpringerBerlinMangoldA.MartínM.P.LückingR.LumbschH.T.2008Molecular phylogeny suggests synonymy of Thelotremataceae within Graphidaceae (Ascomycota: Ostropales)Taxon57476486MillerM.A.PfeiferW.SchwartzT.2010CIPRES Science Gateway surveyhttp://www.phylo.org24 Mar. 2020MittermeierR.1988Primate diversity and the tropical forest. Case studies from Brazil and Madagascar and the importance of the megadiversity countriesWilsonE.O.Biodiversity145154National Academy PressWashingtonMyersN.MittermeierR.A.MittermeierC.G.da FonsecaG.A.B.KentJ.2000Biodiversity hotspots for conservation prioritiesNature403853858Nash IIIT.H.ElixJ.A.2004XanthoparmeliaNash IIIT.H.RyanB.D.DiederichP.GriesC.BungartzF.Lichen Flora of the Greater Sonoran Desert Region2566605Lichens UnlimitedArizona State UniversityTempeNash IIIT.H.Herrera-CamposM.A.ElixJ.A.2004Preliminary treatment of Xanthoparmelia for MexicoSymbolae Botanicae Upsaliensis34289326Nash IIIT.H.Herrera-CamposM.A.EsslingerT.L.ElixJ.A.2016Xanthoparmelia in MexicoBibliotheca Lichenologica110621641OrangeA.JamesP.W.WhiteF.J.2010Microchemical methods for the identification of lichens, second edition with additions and correctionsBritish Lichen SocietyLondonPino-BodasR.Rosa BurgazA.MartinM.P.LumbschH.T.2012Species delimitations in the Cladonia cariosa group (Cladoniaceae, Ascomycota)Lichenologist44121135RamamoorthyT.P.ByeR.LotA.FaJ.1993Biological Diversity of Mexico: Origins and DistributionOxford University PressOxfordRambautA.2014FigTree1.2.2.http://tree.bio.ed.ac.uk/software/fgtree/24 Mar. 2020RonquistF.HuelsenbeckJ.P.2003MrBayes 3: Bayesian phylogenetic inference under mixed modelsBioinformatics1915721574RozziR.ArmestoJ.J.GoffinetB.BuckW.MassardoF.SilanderJ.ArroyoM.K.T.RussellS.AndersonC.B.CavieresL.A.CallicottJ.B.2008Changing lenses to assess biodiversity: patterns of species richness in sub-Antarctic plants and implications for global conservationFrontiers in Ecology and the Environment6131137RzedowskiJ.1978Vegetación de MéxicoEditoral LimusaMéxicoStamatakisA.2014RAxML Version 8: A tool for phylogenetic analysis and post-analysis of large phylogeniesBioinformatics3013121313ThellA.CrespoA.DivakarP.K.KärnefeltI.LeavittS.D.LumbschH.T.SeawardM.R.D.2012A review of the lichen family Parmeliaceae - history, phylogeny and current taxonomyNordic Journal of Botany30641664VaidyaG.LohmanD.J.MeierR.2011SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon informationCladistics27171180VilgalysR.HesterM.1990Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus speciesJournal of Bacteriology17242384246WhiteT.J.BrunsT.D.LeeS.TaylorJ.1990Amplification and direct sequencing of fungal ribosomal RNA genes for phylogeneticsInnisM.A.GelfandD.H.SninskyJ.J.WhiteT.J.PCR Protocols315322Academic PressSan DiegoZollerS.ScheideggerC.SperisenC.1999PCR primers for the amplication of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetesLichenologist31511516List of <italic>Xanthoparmelia</italic> specimens collected in Mexico and deposited in the F and MEXU herbaria as indicated.
Specimens of <italic>Xanthoparmelia</italic> included in the molecular study: species, country/voucher information, GenBank accession numbers for ITS, mtSSU and nuLSU sequences, respectively. New generated sequences are indicated by an asterisk (*), missing sequences are indicated by a dash (-), and specimens of collapsed nodes for <xref ref-type="fig" rid="f2">Fig. 2</xref> are marked with their respective clade labels (coI-coVII).