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. 2012 Jun 20;7(6):e39683. doi: 10.1371/journal.pone.0039683

Transoceanic Dispersal and Subsequent Diversification on Separate Continents Shaped Diversity of the Xanthoparmelia pulla Group (Ascomycota)

Guillermo Amo de Paz 1, Paloma Cubas 1, Ana Crespo 1, John A Elix 2, H Thorsten Lumbsch 3,*
Editor: Zhengguang Zhang4
PMCID: PMC3379998  PMID: 22745810

Abstract

In traditional morphology-based concepts many species of lichenized fungi have world-wide distributions. Molecular data have revolutionized the species delimitation in lichens and have demonstrated that we underestimated the diversity of these organisms. The aim of this study is to explore the phylogeography and the evolutionary patterns of the Xanthoparmelia pulla group, a widespread group of one of largest genera of macrolichens. We used a dated phylogeny based on nuITS and nuLSU rDNA sequences and performed an ancestral range reconstruction to understand the processes and explain their current distribution, dating the divergence of the major lineages in the group. An inferred age of radiation of parmelioid lichens and the age of a Parmelia fossil were used as the calibration points for the phylogeny. The results show that many species of the X. pulla group as currently delimited are polyphyletic and five major lineages correlate with their geographical distribution and the biosynthetic pathways of secondary metabolites. South Africa is the area where the X. pulla group radiated during the Miocene times, and currently is the region with the highest genetic, morphological and chemical diversity. From this center of radiation the different lineages migrated by long-distance dispersal to others areas, where secondary radiations developed. The ancestral range reconstruction also detected that a secondary lineage migrated from Australia to South America via long-distance dispersal and subsequent continental radiation.

Introduction

Methods for delimiting species, the fundamental taxonomic unit, have always fascinated evolutionary biologists [1]–[3]. Understanding the circumscription of species is important for biological and ecological studies and for conservation issues. However, the main challenge is to recognize species in organisms with relatively simple morphologies. In lichenized fungi traditional species circumscriptions are based on phenotypic characters, such as thallus and ascomatal morphology or chemical characters. However, there is a growing body of evidence from molecular studies that the traditional morphology-based species circumscriptions are insufficient to represent the diversity in lichenized ascomycetes [4]–[26]. A number of DNA sequence-based phylogenetic studies revealed the presence of distinct lineages within currently delimited species. Subsequent, detailed studies often revealed previously overlooked morphological subtleties or chemical differences among those clades and authors often refer to these as “semi-cryptic” species [8].

On a par with the phenotypic-based species circumscription, researchers often accepted wide distribution ranges for species occurring on different continents. This was at least partially due to a common belief by lichenologists in the “everything is everywhere” hypothesis [27], [28] applied to fungi, as discussed elsewhere [8], [29]. In several cases molecular data assisted in a better understanding of the biogeography of lichen-forming fungi where taxa were shown to represent different species on different continents, e.g. the Leptogium furfuraceum aggr. on different continents [18], Melanelixia glabra s. lat. in Europe and North America [14], Parmelina quercina s. lat. on different continents [4], Physcia aipolia aggr. in Europe and Australia [9], Xanthoparmelia spp. in North America and Australia [15], [16]. In the Leptogium furfuraceum aggr. complex sister-group relationships were found between populations from the same hemispheres which were incongruent with previous classifications based on morphological differences [18], and the dated phylogeny indicated that the species had migrated via transoceanic dispersal to different continents.

Here we report another case of a group of lichenized fungi where transoceanic dispersal to different continents is correlated with the phylogenetic lineages. The group studied here is the Xanthoparmelia pulla group which belongs to the family Parmeliaceae. This family represents one of the largest families of lichenized fungi [30], [31]. The main clade of the family is the parmelioid clade with almost 2000 species [32] currently classified in 27 genera, with Xanthoparmelia being the largest with over 800 accepted species [33]. The species in this genus characteristically occur on siliceous rocks or soil, predominantly in arid to subarid regions, with a center of distribution in the southern hemisphere. The genus is characterized by having cell wall polysaccharides of the Xanthoparmelia-type, small ascospores with an arachiform vacuolar body [34], and the presence of a pored epicortex [33], [35]. It has been hypothesized that the genus diversified in a rapid radiation following a shift towards drier habitats at the base of the Xanthoparmelia clade [36] leading to the high current diversity.

Previously, the Xanthoparmelia pulla group has been classified within the separate genus Neofuscelia based on the different cortical chemistry (having melanoid pigments and lacking usnic acid or atranorin, characteristic of the majority of Xanthoparmelia species) [37], [38]. A subsequent molecular study showed that the genus Neofuscelia was polyphyletic, with its clades scattered within Xanthoparmelia [33]. Consequently, the genus Neofuscelia was reduced to synonymy with Xanthoparmelia, as have other genera previously distinguished by cortical chemistry or growth form [33], [39]–[43]. The Xanthoparmelia pulla group is a monophyletic clade within the complete Xanthoparmelia clade, that includes the former Esslinger's Xanthoparmelia pulla species and other related species [44].

Although the clades were largely incongruent with the current species circumscription, we found a correlation of the main clades of X. pulla group with their geographical distribution and chemical profile (Fig. 1, 2). Clade 1 includes specimens from California, Macaronesia and areas around the Mediterranean basin, all of which contain depsides and depsidones derived from the orcinol pathway or with aliphatic acids; clade 2 includes specimens from Australia (subclade 2.1) and South America (subclade 2.2) with depsides and depsidones derived from the orcinol pathway; clade 3 derives from South African specimens containing olivetoric acid; clade 4 specimens with hypostictic acid from California (subclade 4.1) and South America (subclade 4.2); and clade 5 specimens with physodic acid (orcinol depsidones) from South Africa.

Figure 1. Phylogenetic relationships of the Xanthoparmelia pulla group based on nuITS and nuLSU rDNA sequences.

Figure 1

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Topology based on maximum-likelihood (ML) analyses. Posterior probabilities and bootstrap values are indicated on each branch. Branches with posterior probabilities under Bayesian analysis equal or above 0.95 and/or bootstrap values equal or above 70% under MP are in bold. Medullary compounds, and results of the divergence time estimation and ancestral range reconstruction analyses are shown. Black arrow head indicates the polyphyletic species.

Figure 2. Schematic map showing the relationships between phylogeny, medullary compounds, ancestral range and divergence times estimation for the Xanthoparmelia pulla group.

Figure 2

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The species delimitation within the Xanthoparmelia pulla group is currently based on a combination of morphological and chemical characters (Table 1). The morphological characters include the color of the lower surface, shape of the lobes, attachment to the substrate, and presence of vegetative propagules while the chemical differences pertain to upper cortical and medullary secondary metabolites. A number of the currently accepted species have a wide distribution spanning several continents. To address the species delimitation in this group and to test the hypothesis of widely distributed species we have generated a data set using two loci (nuITS rDNA, nuLSU rDNA) from specimens collected on different continents. The molecular data were used to perform phylogenetic reconstructions in a maximum likelihood (ML) and Bayesian (B/MCMC) framework. We have also estimated the timing of the diversification events leading to the main clades found in our study to discriminate between vicariance and long-distance dispersal as possible explanations for the current distribution patterns. A Bayesian-based approach of ancestral range reconstruction was used to identify potential areas in which the group and major clades within the group originated.

Table 1. Main differences of the species of Xanthoparmelia pulla group studied in this paper [38], [44], [47], [59], [60].

Characters and distribution
Species Morphology of lobes Isidia Lower surface Chemistry Distribution
X. atroviridis 1–2 mm broad, subirregular, contiguous to imbricate Absent Black, moderately rhizinate, rhizines concolorous, up to 0.4 mm long. Medulla: Hypoconstictic, hypostictic, hyposalazinic acids; Cortex: HNO3 + violet South Africa
X. caliginosa 1–2.5 (−3.5) mm broad, subirregular, contiguous to imbricate Sparse to crowded and areolate. Isidia erumpent, globose to cylindrical, 0.1–0.6 (−0.8) mm tall. Dark brown to black, moderately rhizinate, rhizines more or less concolorous, up to 0.6 mm long. Medulla: Olivetoric acid. Cortex: HNO3 + blue-green South Africa
X. delisei 1–4 mm broad, sublinear to irregular, flat to slightly concave or convex, becoming laciniate, often imbricate and entangled Absent Dark brown to black, often paler near the apices, moderately to densely rhizinate, the rhizines simple and concolorous with the lower surface, to 1 mm long Medulla: glomelliferic, glomellic, perlatolic acids; ± gyrophoric acid. Cortex: HNO3 + blue-green Europe, Asia, Africa, Australia, Macaronesia, South America
X. fissurina 1–3 mm broad, contiguous to imbricate or entangled Absent Pale tan to pale brown, moderately rhizinate, rhizines concolorous, to 1 mm long Medulla: hypostictic, hypoconstictic, hyposalazinic acids, unknown compounds. Cortex: HNO3 + blue-green South Africa and South America
X. glabrans 0.5–3.0 mm broad, sublinear to linear-elongate, imbricate to loosely entangled. Absent black, dull, slightly rugulose, moderately rhizinate; rhizines black, simple or fasciculate, to 1 mm long Medulla: alectoronic acid; ± a-collatolic and gyrophoric acids. Cortex: HNO3 + blue-green Australia, Europe, Africa, South America, New Zealand
X. imitatrix 0.5–3.0 mm broad, sublinear to linear-elongate, imbricate to laciniate entangled, rarely developing subfruticose branches Absent Dark brown to black, sparsely to moderately rhizinate, rhizines simple, to 1.5 mm long Medulla: physodic acid; ±4-O-methylphysodic and alectoronic acids. Cortex: HNO3 + blue-green Australia, Africa, South America, New Zealand
X. lineella 0.1–0.5mm broad, linear and dichotomously branched and entangled Absent Black, sparsely rhizinate, rhizines concolorous, to 1 mm long Medulla: physodic acid; Cortex: HNO3 + blue-green South Africa
X. loxodes (0.5-)1–3(−5) mm broad, subirregular to sublinear, contiguous to entangled. Sparsely to densely isidiate, isidia more or less spherical and distinctly pustular, erumpent Dark brown to black, smooth to somewhat rugulose, moderately rhizinate, rhizines concolorous, to 1 mm long Medulla: glomelliferic, glomellic and perlatolic acids; ±gyrophoric acid. Cortex: HNO3 + blue-green Europe, North Africa, Asia, North America, Macaronesia
X. luteonotata (0.5-)1–3 mm broad, sublinear to irregular, discrete to imbricate, rarely developing subfruticose branches Absent Pale tan to pale brown, moderately to densely rhizinate, rhizines simple, to 0.5 mm long Medulla: ± divaricatic and stenosporic acids; ± gyrophoric acid. Cortex: HNO3 + blue-green Australia, Europe, Africa, New Zealand
X. pokornyi 1–2 mm broad, sublinear to linear, discrete to loosely imbricate or entangled Absent Pale tan to brown, moderately to sparsely rhizinate, rhizines concolorous or darkening, to 1 (−1.5) mm long Medulla: stenosporic acid; ± gyrophoric and divaricatic acids. Cortex: HNO3 + blue-green Europe, Asia
X. perrugata 1–3 (−5) mm broad, sublinear to linear-elongate, discrete to imbricate or entangled. Absent Dark brown to black, moderately to densely rhizinate, rhizines simple, to 1.5 mm long. Medulla: divaricatic acid; ± stenosporic, oxostenosporic, gyrophoric, lecanoric acids. Cortex: HNO3 + blue-green Europe, North Africa, Australia, Asia
X. pseudoglabrans 1–2.5 mm broad, subirregular to sublinear, imbricate to entangled Absent Black; moderately rhizinate or rhizines rather parse, concolorous with the lower surface Medulla: alectoronic acid; ± a-collatolic acid. Cortex: HNO3 - South Africa
X. pulla 1–3 (−5) mm broad, sublinear to linear-elongate, discrete to imbricate or entangled Absent Dark brown to black, moderately to densely rhizinate, rhizines simple, to 1.5 mm long Medulla: stenosporic acid; ± divaricatic, gyrophoric, perlatolic, 4-O-demethylstenosporic acid, oxostenosporic acids. Cortex: HNO3 + blue-green Europe, Australia, New Zealand, Africa
X. pulloides 1–2 mm broad, subirregular to sublinear, contiguous to subimbricate Absent Black, moderately rhizinate, rhizines concolorous, to 0.5 mm long Medulla: constipatic and protoconstipatic acids; ± gyrophoric acid. Cortex: HNO3 + blue-green Macaronesia, Asia
X. quintarioides 1–2.5 (−3) mm broad, strongly convex and short-flabellate, discrete but close to more or less contiguous Absent Tan to pale brown, sparsely to moderately rhizinate, the rhizines short and hapterate Medulla: hypostictic, hypoconstictic, cryptostictic acids; ± hyposalazinic acid. Cortex: HNO3 + blue-green South Africa
X. ryssolea 1–3 mm broad, linear elongate, subterete, convex. Absent Pale yellow-brown to red-brown, canaliculate, sparsely rhizinate, rhizines concolorous, to 0.6 mm long. Medulla: stenosporic acid; ± gyrophoric, oxostenosporic, divaricatic acids. HNO3 + blue-green Europe, Asia
X. squamans 1–2 mm broad, sublinear, imbricate to contiguous Absent Dark brown to black, moderately to sparsely rhizinate, rhizines concolorous, to 1 mm long Medulla: hypostictic, hypoconstictic, hyposalazinic acids. HNO3 + blue-green South Africa, South America, New Zealand
X. subhosseana 1–2 mm broad, subirregular, contiguous to slightly imbricate Sparsely to densely isidiate. Isidia pustular, erumpent Dark brown to black, moderately rhizinate, rhizines concolorous, to 0.6 mm long Medulla: hypostictic, hyposalazinic, hypoconstictic acids. Cortex: HNO3 + blue-green South Africa, North America, South America, New Zealand
X. subimitatrix 0.5–2.0 mm broad, sublinear to subirregular, discrete to subimbricate Absent Pale tan to brown, moderately rhizinate, rhizines simple, brown or often blackened, to 0.8 mm long Medulla: physodic acid and alectoronic acids. Cortex: HNO3 + blue-green South Africa, Australia.
X. subincerta 0.5–1 mm broad, flat, sublinear, more or less imbricate Isidia cylindrical, simple or densely branched, 0.08–0.5 mm tall. Apices syncorticate Black, moderately rhizinate, rhizines simple, black, to 0.3 mm long Medulla: glomelliferonic acid; ± loxodellonic and glomellonic acids. Cortex: HNO3 + blue-green Australia, South Africa
X. subprolixa 1–3 (−5) mm broad, sublinear to linear-elongate, discrete to imbricate or entangled. Absent Dark brown to black, often paler at apices, moderately to densely rhizinate, rhizines simple, to 1.5 mm long Medulla: divaricatic acid; ± stenosporic, nordivaricatic acids. Cortex: HNO3 + blue-green Australia, New Zealand
X. torulosa 1.0–3.5 mm broad, sublinear to sublirregular, imbricate; laciniae at periphery and within thallus, ± subfruticose, sublinear to elongate, 0.3–1.0 mm broad. Absent Black, moderately to densely rhizinate; rhizines simple or occasionally tufted, slender. Medulla: divaricatic acid; ± nordivaricatic, stenosporic acids. Cortex: HNO3 + blue-green Australia
X. verisidiosa 1–3 mm broad, irregular to sublinear, flat, short and rounded, contiguous to imbricate Sparsely to densely isidiate. Isidia cylindrical, simple or becoming densely branched, 0.2–1 mm tall. Apices syncorticate Black, moderately to sparsely rhizinate, rhizines simple, black to 0.4 mm long Medulla: alectoronic and a-collatolic acids. Cortex: HNO3 + blue-green Australia, New Zealand, South Africa
X. verrucella 0.5–2 mm wide, irregular to sublinear, flat, imbricate to entangled. Moderate to densely isidiate. Isidia cylindrical, simple or becoming branched, to 1 mm tall. Apices syncorticate Black, sparsely to moderately rhizinate, rhizines simple, simple, black, to 0.4 mm long. Medulla: divaricatic acid; ± stenosporic acid. Cortex: HNO3 + blue-green Australia, New Zealand, South Africa
X. verruculifera 1–2 mm broad, subirregular to sublinear, contiguous to imbricate Sparcely to densely isidiate. Isidia pustular, erumpent. Dark brown to black, moderately rhizinate, rhizines concolorous, to 0.8 mm long Medulla: divaricatic acid; ± stenosporic and gyrophoric acids. Cortex: HNO3 + blue-green North Africa, Europe, North America
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Results

Phylogenetic analyses

One hundred sixty-eight DNA sequences of ITS and nuLSU rDNA of 88 representative specimens of Xanthoparmelia were assembled. One hundred forty of these sequences were newly generated in this study. The specimens included 25 currently accepted species in the Xanthoparmelia pulla group, four unassigned specimens, and six samples of four species as outgroup. A data matrix of 1283 unambiguously aligned characters, with 454 characters in the ITS and 829 characters in the nuLSU rDNA was used for phylogenetic analyses. The data set included 1081 constant characters. The general time-reversible model with a gamma distribution and invariant model of rate heterogeneity (GTR+I+G) was employed for analyses of the single-loci and concatenated data sets. Since no strongly supported conflicts between the two single-locus ML phylogenetic trees were detected, a combined data set was analyzed. In the B/MCMC analysis of the combined data set, the likelihood parameters in the sample had the following averaged values for the partitioned data set (± standard deviation): base frequencies π(A)  = 0.25 (±1.54E-4), π(C)  = 0.24 (±1.42E-4), π(G)  = 0.28 (±1.58E-4), π(T)  = 0.23 (±1.51E-4); rate matrix r(AC)  = 4.42 (±1.43E-4), r(AG)  = 0.23 (±8.35E-4), r(AT)  = 9.53 (±2.19E-4), r(CG)  = 4.82 (±1.45E-4), r(CT)  = 0.54 (±9.26E-4), r(GT)  = 2.98 (±1.11E-4) and the gamma shape parameter α  = 0.21 (±3.86E-4). The likelihood parameters in the sample had a mean likelihood of LnL  = −4608.25 (±0.49), while the ML tree had a likelihood of LnL = −4163.64.

The phylogenetic estimates of the ML and B/MCMC analyses were congruent, hence only the ML tree (Fig. 1) is shown here. Specimens of the Xanthoparmelia pulla group form a strongly supported monophyletic group with five main, mostly well-supported, clades (Fig. 1). The clades do not agree with the current species circumscription, with 11 species being polyphyletic, five of them with specimens from different continents entering different clades. For example, all the Northern Hemisphere specimens identified as X. luteonotata, X. pulla, X. delisei or X. glabrans belong to clade 1 while all the Australian specimens of the same species belong to clade 2.1. Similarly, specimens of X. imitatrix from South America belong to clade 2.2 and are not directly related to the South African specimen.

Estimates of divergence times and ancestral range reconstructions

A Bayesian phylogenetic tree was dated to estimate the age of the X. pulla group and its main clades. The results of the divergence time analysis are summarized in Fig. 2, and the whole parmelioid tree is shown as supp. mat. (Fig. S1). The Xanthoparmelia pulla group started to diversify around 11.61 Ma (7.61 – 16.50 Ma), the age of the crown node of clade 1 was estimated at 5.31 Ma (3.01 – 8.38 Ma), the ancestor of clade 2 around 8.10 Ma (5.13 – 11.74 Ma), and the crown of clade 4 around 3.44 Ma (1.56 – 6.07 Ma).

The results of the ancestral range reconstruction analyses are summarized in Figs. 1 and 2. This established that South Africa was the most likely origin of the X. pulla group, with a marginal probability of 0.745, indicating localized uncertainty. The four other areas explored (South America, Australia, California and the Mediterranean basin) were rejected with probabilities below 0.05. For the base of clade 1, the Mediterranean basin was reconstructed as the most likely ancestral range with a marginal probability of 0.555, but California could not be rejected (probability of 0.104). For clade 2, Australia was recovered as the most likely ancestral area with marginal probability of 0.672; while South America, the other area from which specimens of this clade occur, was rejected as potential ancestral area (p<0.05); similarly, California, the Mediterranean basin and South Africa were also rejected as ancestral areas. South America was found to be the most likely origin for clade 4 (which also includes specimens from California and South America) with a marginal probability of 0.48, although neither California nor South Africa were rejected (probabilities of 0.106 and 0.083, respectively). Australia and the Mediterranean basin were rejected as ancestral ranges for clade 4.

Discussion

Understanding the diversity and delimiting species in lichenized fungi has been a long standing challenge and current studies using molecular data have dramatically changed our ability to distinguish species in this group [8], [23], [45]. The Xanthoparmelia pulla group is a good example for illustrating the difficulties in in distinguishing species by morphology due to the remarkable plasticity of morphological characters in this group. Consequently, secondary metabolites have played an important role in delimiting species in this group [44], [46], [47]. Following the current classification using a combination of vegetative morphology and secondary chemistry, a number of species have broad geographical distributions spanning several continents.

Here we have used molecular data to investigate the current classification within the group and attempt to explain their distribution. We used likelihood-based and Bayesian approaches to investigate the evolutionary origin of the group and timing of speciation events. Hopefully such data will reveal evolutionary patterns so we may develop a framework for their taxonomic classification which better reflects the phylogenetic relationships in the X. pulla group. Our results clearly indicate that the species as currently delimited are polyphyletic (Fig. 1). This is consistent with results from other studies of Xanthoparmelia species believed to occur on different continents which were subsequently found to represent distinct lineages [15], [16]. Further, similar patterns have been found in other groups of lichenized fungi [4], [14], [18], [48].

The ancestral range reconstruction points to South Africa as the most likely origin of the X. pulla group. Although there is a certain degree of uncertainty in the reconstruction (marginal probability of 0.745), the analysis rejected other areas as potential ancestral areas for the group. Interestingly, South Africa has the highest morphological and chemical diversity within the group and the specimens studied here belong to different, unrelated lineages (Fig. 1). South African specimens containing olivetoric acid cluster in clade 3 and those with physodic acid in clade 5. The phylogenetic relationships of other specimens from South Africa with hypostictic acid, physodic acid or other orcinol depsides and depsidones are still unresolved. The South African specimens show remarkable morphological variability, including subcrustose and foliose species. Further, many Xanthoparmelia species occur in arid climates and the diversification of X. pulla group occurred around 11.61 Ma (7.61 – 16.50). At this time the Cape Region underwent a major aridification [49], which may be responsible for the rapid radiation and current richness of the Cape flora. Thus, it is likely that the X. pulla group originated in South Africa around the same time. Unlike most Cape region elements in flowering plants, the species of the X. pulla group subsequently extended their distribution by transoceanic dispersal.

Within the X. pulla group, the five lineages identified are characterized by the presence of different substance classes and in some cases they diverged secondarily in different geographical areas. The correlation between chemical pathways and the lineages found in molecular studies has also been found in Pertusariaceae among lichenized fungi [50]–[52].

Clade 1 includes specimens containing orcinol depsides and depsidones that occur in California and the Mediterranean basin. Neither internally supported subclades nor a geographical pattern was found within this clade, and specimens with different phenotypical characters from different geographical areas are intermingled. In fact, shifting between orcinol depsides and depsidones can occur by one-step transformations [46]. By contrast, in other genera (e.g. Melanelixia, Parmelina, Leptogium) with similar disjunct distributions (North America and the Mediterranean basin), the geographical distribution correlates with the clades found in the molecular study. In the X. pulla group this pattern was not found, possibly due to insufficient sampling or absence of a phylogenetic signal in the markers used. This might be due to the slower evolutionary rates of lichenized fungi from arid and subarid regions compared to oceanic parmelioid lichens [36]. The diversification age of clade 1 was estimated at 5.31 Ma (3.01 – 8.38 Ma), at the end of the Miocene, which was a geological period when numerous groups radiated in arid conditions. The Mediterranean region was suggested as the ancestral area of this clade by the ancestral range analysis, but the result was poorly supported.

Clade 2 also contains species with orcinol depsides and depsidones and comprises two disjunct lineages, one occurring in Australia (clade 2.1) and the other in South America (2.2). The estimated age for clade 2 (8.10 Ma; 5.13 – 11.74 Ma) rules out the possibility of vicariance, since the breakup of Australia, Antarctica and South America occurred between 35–52 Ma ago [53]. The ancestral range reconstruction points to Australia as the ancestral area of clade 2. This would be consistent with long distance dispersal from Australia to South America, a phenomenon frequently found in many plants groups [54]. Within the Australian clade several strongly supported lineages are not consistent with the current species delimitation of the group, indicating that the phenotypical characters used to distinguish species in the group have limited phylogenetic validity. Similar disparities between phylogenetic relationships and current species delimitations were found within the yellow Xanthoparmelia species from western North American [55], [56].

Clade 4 includes specimens containing hypostictic acid from California and South America. Here again all the South American specimens form a monophyletic group. Specimens of X. subhosseana occurring in different continents are not closely related. The most likely ancestral origin of clade 4 is South America (marginal probability of 0.48), although neither California nor South Africa could be rejected.

Our study indicates that the X. pulla group started to radiate during the Miocene in South Africa, where the highest diversity of this group is found. From this region, different lineages with distinct secondary metabolites belonging to different chemical pathways were dispersed to other regions, where they experienced rapid and more recent radiations. In some cases our results showed that the sympatric species of the X. pulla group in an area belong to distantly related groups. For example, the Californian X. pulla flora includes species from clade 1 and clade 4.1, the latter most probably having migrated from South America. Indeed our study indicated that the current taxonomic circumscription of species in the group does not agree with the evolutionary hypotheses inferred by molecular markers. The incongruence of phenotype-based classification and molecular phylogeny is a challenge for the classification of these fungi. Additional studies will be needed to determine whether the lineages found here represent cryptic species or whether new phenotypical characters can be found to distinguish these distinct lineages (as has been found in some other Parmeliaceae [10], [57]). Future research should address how such parallel evolution of phenotypical characters in lichenized fungi could be explained in order to provide a better framework to test the adaptive value of these characters [58]. Our results here have important implications for conservation and ecological issues, since species were found to have much more restricted distribution than previously thought.

Materials and Methods

Taxon sampling

Eighty two specimens of the Xanthoparmelia pulla group from California, the Mediterranean basin, Macaronesia, South America, Australia and South Africa were used for the phylogenetic study. The specimens were identified following the current species delimitations [38], [44], [47], [59], [60]. Chemical constituents were identified using thin layer chromatography (TLC) [61]–[64], and gradient-elution high performance liquid chromatography (HPLC) [65]. The major medullary compounds were classified into four major groups based on their chemical structure: 1) Orcinol depsides: olivetoric, divaricatic, stenosporic and glomelliferic acids. 2) Orcinol depsidones, physodic, alectoronic and glomelliferonic acids. 3) β-Orcinol depsidones: stictic acid (only present in the outgroup) and hypostictic acid. 4) aliphatic acids: constipatic acid. All necessary permits were obtained for the described field studies. Collecting permits in Australia were all obtained by J.A. Elix (ca. 50 permit numbers for each states and over several years) and in Chile by W. Quilhot. For European locations specific permission was not required, since the locations were neither in privately-owned or protected areas. The field studies did not involve endangered or protected species.

Molecular study

Total DNA was extracted from frozen lobes of thalli crushed with sterile glass pestles, using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions and modifications of Crespo et al. [66]. The following primers were used: ITS1-LM [67] and ITS2-KL [68] for nuITS rDNA, and LR0R and LR5 [69] for nuLSU rDNA.

For each amplification we used a reaction mixture of 25 μL, containing: 2.5 μL of 10x DNA polymerase buffer (including MgCl2 2mM) (Biotools), 1.25 μL of each primer, 0.75 μL of DNA polymerase (1U/μL), 0.5 μL of dNTPs containing 10 mM of each base (Biotools), 5 μL of DNA (third elution of DNA extraction) and 13.5 μL dH2O. Amplifications were carried out in an automatic thermocycler (Techne Progene 3000) with the following steps: an initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 1 min, 58°C (nuITS rDNA) or 56°C (nuLSU rDNA) for 1 min, and 72°C for 1.5 min; a final extension at 72°C for 5 min. PCR products were cleaned with DNA Purification Kit (Flavorgen) and sequenced with the same primers using the ABI Prism Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems) with the following program: initial denaturation at 94°C for 3 min, 25 cycles at 96°C for 10s, 50°C for 5s and 60°C for 4 min. Sequencing reactions were electrophoresed on a 3730 DNA analyzer (Applied Biosystems). The sequence fragments were assembled with Bioedit v. 7.0 [70] and manually adjusted.

Sequence alignment and selection of the substitution model

We used a dataset of 2 loci of 82 specimens representing 25 species of the Xanthoparmelia pulla group and 6 specimens as outgroup. The sequences were mainly generated in this study (140 sequences) and the others taken from our previous studies [33], [41]. The outgroup selection was based on previous phylogenetic studies [41]. GenBank accession numbers and sources of the specimens are listed in Table 2.

Table 2. Specimens used in this study with country of collection, voucher information and GenBank accession numbers.

GenBank accession no.
Species Country Herbarium acc. no. nuITS nuLSU
Xanthoparmelia adhaerens 1 South Africa MAF-Lich 16212 HM125744 HM125766
X. adhaerens 2 South Africa MAF-Lich 16213 HM125746 HM125768
X. atroviridis 1 South Africa MAF-Lich 17163 JQ912329 JQ912425
X. atroviridis 1 South Africa MAF-Lich 17168 JQ912351 JQ912448
X. atroviridis 2 South Africa MAF-Lich 17158 JQ912314 JQ912415
X. atroviridis 2 South Africa MAF-Lich 17154 JQ912320 JQ912419
X. atroviridis 3 South Africa MAF-Lich 17153 JQ912349 JQ912446
X. caliginosa 1 South Africa MAF-Lich 17157 JQ912350 JQ912447
X. caliginosa 2 South Africa MAF-Lich 17150 JQ912315 -
X. caliginosa 3 South Africa MAF-Lich 17156 JQ912333 JQ912430
X. caliginosa 4 South Africa MAF-Lich 17152 JQ912317 -
X. caliginosa 5 South Africa MAF-Lich 17186 JQ912348 JQ912445
X. delisei 1 Turkey MAF-Lich 17139 JQ912307 JQ912408
X. delisei 2 Australia MAF-Lich 7432 AY581067 AY578930
X. delisei 3 Spain MAF-Lich 7659 AY581068 AY578931
X. delisei 4 Turkey MAF-Lich 17134 JQ912308 JQ912409
X. delisei 5 Turkey MAF-Lich 17135 JQ912305 JQ912406
X. fissurina South Africa MAF-Lich 17162 JQ912353 JQ912450
X. glabrans 1 Australia CANB 746334 JQ912291 JQ912393
X. glabrans 2 Australia CANB 746340 JQ912289 JQ912391
X. glabrans 3 Australia MAF-Lich 7665 AY581069 AY578932
X. glabrans 4 Australia CANB 681875.1 JQ912290 JQ912392
X. glabrans 5 Spain MAF-Lich 9912 AY581072 AY578935
X. glabrans 6 Turkey MAF-Lich 17137 JQ912306 JQ912407
X. glabrans 7 Morocco MAF-Lich 17144 JQ912286 JQ912388
X. imitatrix 1 Chile MAF-Lich 17132 JQ912344 JQ912441
X. imitatrix 2 Chile MAF-Lich 17126 JQ912288 JQ912390
X. imitatrix 3 Chile MAF-Lich 17123 JQ912287 JQ912389
X. imitatrix 4 Chile MAF-Lich 17127 JQ912342 JQ912439
X. imitatrix 5 Chile MAF-Lich 17122 JQ912285 JQ912387
X. imitatrix 6 Chile MAF-Lich 17124 JQ912326 JQ912422
X. imitatrix 7 South Africa MAF-Lich 17155 JQ912352 JQ912449
X. lineella South Africa MAF-Lich 17160 JQ912319 JQ912418
X. loxodes 1 Spain MAF-Lich 7072 AY581076 AY578940
X. loxodes 2 Spain MAF-Lich 6206 AY581070 AY578933
X. luteonotata 1 Spain MAF-Lich 17120 JQ912341 JQ912438
X. luteonotata 2 Australia CANB 746358 JQ912293 -
X. luteonotata 3 Australia CANB 746366.1 JQ912292 JQ912394
X. luteonotata 4 Spain MAF-Lich 17119 JQ912340 JQ912437
X. mougeotii 1 Spain MAF-Lich 6802 AY37006 AY578966
X. mougeotii 2 Spain MAF-Lich 9916 AY581100 AY578967
X. perrugata Spain MAF-Lich 17118 JQ912324 -
X. pokornyi 1 Spain MAF-Lich 6052 AY037005 AY578934
X. pokornyi 2 Spain MAF-Lich 9908 AY581075 AY578939
X. pokornyi 3 Turkey MAF-Lich 17140 JQ912310 JQ912411
X. pokornyi 4 Spain MAF-Lich 17117 JQ912323 -
X. pokornyi 5 Turkey MAF-Lich 17143 JQ912313 JQ912414
X. pokornyi 6 Turkey MAF-Lich 17142 JQ912312 JQ912413
X. pokornyi 7 Turkey MAF-Lich 17136 JQ912304 JQ912405
X. pseudoglabrans South Africa MAF-Lich 17161 JQ912316 JQ912416
X. pulla 1 Spain MAF-Lich 17115 - JQ912420
X. pulla 2 Australia CANB 739130.1 JQ912294 JQ912395
X. pulla 3 Australia CBG 9810185 JQ912295 JQ912396
X. pulla 5 Spain MAF-Lich 6794 AY581071 AJ 421433
X. pulloides 1 Spain MAF-Lich 17121 JQ912347 JQ912444
X. pulloides 2 Spain MAF-Lich 6784 AY037004 AY578936
X. quintarioides South Africa MAF-Lich 17159 JQ912318 JQ912417
X. ryssolea 1 Turkey MAF-Lich 17141 JQ912311 JQ912412
X. ryssolea 2 Turkey MAF-Lich 17138 JQ912309 JQ912410
X. ryssolea 3 Spain MAF-Lich 17116 JQ912322 -
X. sp. 1 South Africa MAF-Lich 17166 JQ912330 JQ912426
X. sp. 2 South Africa MAF-Lich 17167 JQ912331 JQ912427
X. sp. 3 South Africa MAF-Lich 17165 - JQ912429
X. sp. 4 South Africa MAF-Lich 17164 JQ912339 JQ912436
X. squamans 1 Chile MAF-Lich 17128 JQ912325 JQ912421
X. squamans 2 Chile MAF-Lich 17129 JQ912327 JQ912423
X. squamans 3 Chile MAF-Lich 17131 JQ912343 JQ912440
X. subhosseana 1 USA MAF-Lich 17149 JQ912337 JQ912434
X. subhosseana 2 Chile MAF-Lich 17133 JQ912345 JQ912442
X. subimitatrix Chile MAF-Lich 17130 JQ912328 JQ912424
X. subincerta 1 Australia CANB 746346.1 JQ912296 JQ912397
X. subincerta 2 Australia MAF-Lich 7494 AY581073 AY578937
X. subprolixa 1 Australia MAF-Lich 7667 AY581074 AY578938
X. subprolixa 2 Australia CANB 746355 JQ912297 JQ912398
X. tegeta Australia MAF-Lich 7523 AY581107 AY578975
X. torulosa 1 Australia CANB 746363.1 JQ912299 JQ912400
X. torulosa 2 Australia CANB 746351 JQ912298 JQ912399
X. verisidiosa 1 Australia CANB 746341.1 JQ912301 JQ912402
X. verisidiosa 2 Australia CANB 746345.1 JQ912300 JQ912401
X. verrucella 1 Australia CANB 746353 JQ912303 JQ912404
X. verrucella 2 Australia CANB 746349 JQ912302 JQ912403
X. verrucella 3 South Africa MAF-Lich 17151 JQ912332 JQ912428
X. verruculifera 1 USA MAF-Lich 17146 JQ912334 JQ912431
X. verruculifera 2 USA MAF-Lich 17147 JQ912336 JQ912433
X. verruculifera 3 USA MAF-Lich 17145 JQ912338 JQ912435
X. verruculifera 4 USA MAF-Lich 17148 JQ912335 JQ912432
X. verruculifera 5 Spain MAF-Lich 17114 JQ912321 -
X. xanthomelaena Australia MAF-Lich 16447 HM125740 HM125761
Open in a new tab

New sequences are in bold.

The two loci were aligned separately with Muscle 3.6 [71] and the ambiguous positions were removed manually. The general time reversible model including estimation of invariant sites (GTR+I+G) was selected by jModelTest v 0.1.1 [72] as the most appropriate nucleotide substitution model for both loci.

Phylogenetic Analyses

Potential conflict between the two loci was assessed by comparison of the ML analyses obtained with Garli 0.96 [73] for each locus, using 100 pseudoreplicates for the bootstrap analyses. The phylogenetic analyses of the combined matrix were done using maximum likelihood (ML) and a Bayesian approach. ML analysis was performed using Garli 0.96 [73] with default settings and 100 replicates for the bootstrap analyses. The Bayesian analysis was performed using MrBayes 3.1.1 [74] using the GTR+I+G model, and the data set partitioned into nu ITS and nu LSU. Each partition was allowed to have its own parameter values [75]. Heating of chains was set to 0.2, with 5 million generations sampled every 500th tree. The first 1000 trees were discarded as burn in. We used AWTY [76] to compare splits frequencies in the different runs and to plot cumulative split frequencies to insure that stationarity was reached. Of the remaining 18000 trees (9000 from each of the parallel runs) a majority rule consensus tree with average branch lengths and posterior probabilities was calculated using the sumt option of MrBayes. Clades with bootstrap support equal or above 70 % under ML and/or posterior probabilities ≥0.95 in the Bayesian analysis were considered as strongly supported. Phylogenetic trees were visualized using the program FigTree [77].

Calibration of nodes and dating analysis

The ages of the X. pulla group and its major clades were estimated by a divergence time analysis based on a calibrated phylogeny of the parmelioid lichens [78]. We used a matrix of two loci (nu ITS and LSU) with a proportional number of samples of each parmelioid clade to have a representative tree and trend in speciation through time [79]. The matrix included 299 specimens of parmelioid lichens and 3 specimens of the genus Usnea (as outgroup); 62 new sequences of Xanthoparmelia species outside the X. pulla group were included. GenBank accession numbers with the specimens of the dating analysis are listed in Table S1. The sequence alignment, selection of the nucleotide substitution model, and phylogenetic analyses were done using the same procedures used for the Xanthoparmelia pulla dataset (see above).

The divergence time analyses were performed using BEAST v.1.6.1 [80]. We used a starting tree obtained from a ML analysis using Garli 0. 96 [73] of the concatenated dataset, calculated an ultrametric tree using nonparametric rate smoothing (NPRS) implemented in TreeEdit v.10a10 [81]. The age of the crown node of the parmelioid lichens was calibrated at 60 Ma, following Amo de Paz et al. [78]. The starting tree was topologically congruent with the parmelioid phylogeny presented in Crespo et al. [32].

For the divergence time analyses we used two points of calibration: the age of the crown node of the parmelioid lichens set at 60.28 Ma (49.81 – 73.55 Ma) [78], and the age of the crown node of the genus Parmelia (dated from the fossil Parmelia ambra from the Dominican amber, 15–45 Ma, [82] as discussed previously) [78].

The BEAST analysis was performed using the GTR+I+G substitution model, a Birth-Death process tree prior, and a relaxed clock model (uncorrelated lognormal) for the concatenated dataset. Calibration points were defined as prior distribution, minimal ages and calibrated with a lognormal distributions: 1) the parmelioid crown node at uniform distribution between 49 – 73 Ma; 2) the Parmelia crown node at log-normal mean  = 2.77, offset  = 14, lognormal standard deviation  = 0.5. The analysis was run for 10 million generations, with parameter values sampled every 1000 generation. We checked the stationary plateau with Tracer v. 1.5 [83]. We discarded 10% of the initial trees as burn in and the consensus tree was calculated using Tree Annotator v 1.6.1 [80]. The results were visualized with FigTree v. 1.3.1 [84]. Ages of the X. pulla clades were estimated for nodes with more than 0.95 of posterior probability in the BEAST runs and in the previous Bayesian analysis.

Ancestral range reconstructions

The biogeographical analysis to reconstruct the ancestral area was performed using an indirect Bayesian approach to character state reconstruction [85] implemented in SIMMAP v1.5 [86] following [87]. This analysis integrates the combination of the uncertainty in the tree, branch lengths and the substitution models using Markov chain Monte Carlo. We treated the biogeographic regions as discrete characters. The major areas in which X. pulla species are distributed were categorised broadly into five areas: California, Mediterranean basin (including Macaronesia), South America, South Africa, and Australia. Presence/absence was coded as binary states and each area was given equal probability. We performed the ancestral state reconstruction analysis on a sub-sample of 1000 trees derived from the MrBayes tree sampling of the Xanthoparmelia pulla group.

We also performed ancestral range reconstruction analysis using dispersal-extinction-cladogenesis (DEC) implemented in Lagrange program [88]. The results were inconclusive due to the lack of confidence in parts of the X. pulla phylogeny and hence the results are not included in this paper.

Supporting Information

Figure S1

Chronogram of parmelioid lichens focusing in Xanthoparmelia pulla group. Calibration points: A, inferred age of radiation of parmelioid lichens and B, age of the Parmelia fossil. The Xanthoparmelia pulla group is highlighted by a box and the dated clades are indicated by a branch in bold.

(TIF)

Click here for additional data file. (2.7MB, tif)
Table S1

GenBank accession numbers of parmelioid lichens (except X. pulla group, see Table 2 ) used for divergence time analysis. New sequences are in bold.

(DOC)

Click here for additional data file. (187KB, doc)

Acknowledgments

We thank L. Mucina (Perth) for his help in the organization of the field work in South Africa. GAP thanks W. Quilot (Valparaiso) and C. Rubio (Valparaiso) and C. Cargill (Canberra) who kindly facilitated the visits to the Universidad de Valparaiso (Chile) and ANU and CANBR (Australia).

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (CGL2010-21646) and a grant by the National Science Foundation (USA) (DEB-0949147). The Universidad Complutense de Madrid is thanked for providing all complementary facilities and funds (SYSTEMOL research group UCM-BSCH GR35/10A). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Chronogram of parmelioid lichens focusing in Xanthoparmelia pulla group. Calibration points: A, inferred age of radiation of parmelioid lichens and B, age of the Parmelia fossil. The Xanthoparmelia pulla group is highlighted by a box and the dated clades are indicated by a branch in bold.

(TIF)

Click here for additional data file. (2.7MB, tif)
Table S1

GenBank accession numbers of parmelioid lichens (except X. pulla group, see Table 2 ) used for divergence time analysis. New sequences are in bold.

(DOC)

Click here for additional data file. (187KB, doc)

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