An integrative study of Pannaria hookeri (Ascomycota lichenized) from Argentina and the update of the taxon circumscription based on specimens from the Northern and Southern Hemispheres

Pannaria hookeri (Borrer ex Sm.) Nyl. is reported as a bipolar cyanolichen that occurs in more than twenty countries in both Hemispheres. In the Northern Hemisphere, a minor variation is attributed to the species, whereas larger morphological and chemical variations have been reported in specimens from the Southern Hemisphere. Six P. hookeri DNA sequences are available in GenBank, and only one (from Australia) is associated with a published morphological description. In this study, we performed a preliminary phylogenetic analysis including seven nuITS and mtSSU sequences from eight specimens collected in south Argentina. In addition, we provide an update of the taxon circumscription based on 50 specimens from 13 countries. Here, the molecular analyses revealed a well-supported monophyletic group formed by the P. hookeri sequences from Argentina, Australia, Norway and USA (Alaska). Regarding the morphological and anatomical characteristics, the Southern Hemisphere specimens did not present the historical reported variations, resulting in the proposition of a concise species description that accomplishes most of P. hookeri known geographical distribution.


Introduction
Pannaria Delise ex Bory is one of the largest genera within Pannariaceae (lichenized Ascomycota), in which the lichen-forming fungi can be associated with different photobiont partners (Ekman et al. 2014;Magain & Sérusiaux 2014).Usually, the thalli are bipartite and the mycobiont is associated with Nostoc cyanobacteria.Still, tripartite thalli are also found within the genus where the mycobiont is associated with green algae, as the main photobiont, and the cyanobacteria are confined in structures called cephalodia, Marcos Junji Kitaura, Mayara Camila Scur, Adriano Afonso Spielmann and Aline Pedroso Lorenz for example in Pannaria durietzii (P.James & Henssen) Elvebakk & D.J. Galloway (Elvebakk et al. 2008).The Pannaria species are characterized by squamulose or foliose thalli, apothecia with thalline margins, an amyloid hymenium (I+ blue), asci without internal amyloid apical structures, secondary metabolism with pannarin and related substances, and occurrence in tropical, subtropical, temperate and polar regions from both Hemispheres (Ekman et al. 2014).
Regarding the morphological descriptions, a minor variation is reported to P. hookeri from Europe (Jørgensen 1978), while a larger variation has been described to P. hookeri from the Southern Hemisphere (Jørgensen 2000a).In the same way, the chemical profile differs according to the region studied.Traces of atranorin and ergosterol peroxide was found in specimens from Robert Island (Quilhot et al. 1989), the absence of secondary substances to Bouvetøya specimens (Jørgensen 1986), and pannarin or related substances were reported to specimens from Argentina (Passo et al. 2020), Australia (Kantvilas & Gueidan 2018), and Europe (Jørgensen 1978;2007), inferring cryptic lineages and taxonomical problems in the morphological and chemical studies.
Despite the observed inconsistencies, the wide geographical distribution of P. hookeri was confirmed when the sequences of the nuITS and mtSSU regions were generated from an Australian specimen and clustered with P. hookeri sequences from Norway (Kantvilas & Gueidan 2018).
Nowadays, only six specimens have available sequences in the GenBank: one from Australia, three from Norway, one from USA (Alaska) and one of unknown origin (Ekman & Jørgensen 2002;Spribille & Muggia 2013;Kantvilas & Gueidan 2018;Park et al. 2018;Marthinsen et al. 2019).Therefore, better knowledge is still needed for the circumscription characters that belong to P. hookeri, including genetic analysis with Southern Hemisphere specimens.
The aim of this study was to a) perform a phylogenetic analysis of P. hookeri from Argentina and sequences available on GenBank, generating the first molecular sequences from the South America; and b) to update the species circumscription based on specimens from the Northern (including topotypes) and Southern Hemispheres, establishing a unified morphological description.

Sampling
Fresh samples were collected in the National Park of Tierra del Fuego, Ushuaia, extreme south of the Argentina, during the summer of 2016-2017 (Proyecto Número 105-CPA-2016, supported by CONICET and PROANTAR), and specimens were found on rock and saxicolous mosses.

Phylogenetic analysis
Eight fresh-collected specimens were used in the molecular analysis.DNA extraction, amplification, and sequencing of the nuITS (nuclear internal transcribed spacer) and mtSSU (mitochondrial small subunit) regions were performed according to Kitaura et al. (2018).However, the specimens requested as loans have more than 10 years, and the DNA extraction was not performed.In addition, four sequences of P. hookeri of each nuITS and mtSSU regions were retrieved from GenBank.These sequences were generated from Australian, Norwegian, U.S.A., and unknown origin specimens (Tab.1).
The alignments were performed in Geneious v9.1.2(Kearse et al. 2012) with the MAFFT v7.308 algorithm (Katoh et al. 2002) using default settings, and the Gblocks web server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) was used to exclude unreliable aligned sites using the less stringent options.The alignments were used to infer trees from nuITS, mtSSU, and concatenated regions.Phylogenetic trees were estimated using the Bayesian (BA) and Maximum Likelihood (ML) approaches, and analyses for each region separately and for a combined data matrix were performed.
An integrative study of Pannaria hookeri (Ascomycota lichenized) from Argentina and the update of the taxon circumscription based on specimens from the Northern and Southern Hemispheres Marcos Junji Kitaura, Mayara Camila Scur, Adriano Afonso Spielmann and Aline Pedroso Lorenz The nucleotide substitution and site heterogeneity models were inferred following the Bayesian Inference Criterion in jModelTest2 on XSEDE, CIPRES Science Gateway V.3.3 (Guindon & Gascuel 2003;Miller et al. 2010;Darriba et al. 2012).The BA was performed in MrBayes v.3.2.7a on XSEDE, available in the CIPRES Science Gateway V.3.3 (Huelsenbeck & Ronquist 2001;Ronquist & Huelsenbeck 2003;Miller et al. 2010) using TrNef+I+G as substitution model for nuITS, TPM2uf+I+G for mtSSU regions and TIM1+I+G for the concatenated dataset, and their respective base frequencies were settled.The settings were: two independent runs with four chains (length of 10,000,000 generations), with trees sampled every 1,000 steps.The first 25 % of the generated trees were discarded as burn-in, and a 50 %-majority-rule tree was built.The ML trees were built with the RaxML-HPC2 v.8.2.12 on XSEDE available in the CIPRES Science Gateway V.3.3 (Miller et al. 2010;Stamatakis 2014), using GTR+G+I as nucleotide substitution model and 1,000 bootstrap replicates.FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) was used to edit the trees.Branches with posterior probability values above 0.95 (BA) and bootstrap above 75 (ML) were considered significantly supported.

Morphological and anatomical studies
The AAS, C, COLO, E, H, M, O, and S herbaria sent 35 specimens from the Northern Hemisphere and seven from the Southern Hemisphere.The specimens received as loan were morphologically examined, and the determinations were confirmed (Text S1), when compared with eight fresh P. hookeri specimens.
The P. hookeri description was built according to Jørgensen (1978).Then, our description was compared with other available in the literature (Lindsay 1974;Redón 1985;Jørgensen 1986Jørgensen , 2007;;Øvstedal & Lewis Smith 2001;Kantvilas & Gueidan 2018;Passo et al. 2020), and the variable characters were highlighted in Table 2 and discussed here.The specimens were tested chemically by applying PD spot tests, and orange reactions were interpreted as indicating the presence of pannarin.Therefore, TLC and HPLC analyses are necessary to confirm the substances.

Molecular analysis
The dataset of the present study was composed of sequences of the nuITS and mtSSU regions generated from eight specimens of Pannaria hookeri collected in southern Argentina.Furthermore, six P. hookeri sequences retrieved from GenBank composed the dataset together with other Pannaria species sequences and the selected outgroups (Tab.1).The final dataset comprised 31 sequences and an alignment 1105 base long, with 423 of the nuITS and 682 of the mtSSU regions.
Trees of both BA and ML approaches, among genes and concatenated datasets, showed congruent topology, so only the concatenated tree is presented, with the values of the posterior probabilities and bootstrap values shown (Fig. 1).The sequences of the specimens identified as P. hookeri, newly produced and those obtained from GenBank, presented 0.89/95 of posterior probability and bootstrap values, respectively, grouping the specimens from Argentina, Australia, Norway, and the USA in a wellsupported monophyletic group.The divergence in the nuITS region, considered the universal barcode DNA of fungi (Schoch et al. 2012), revealed a low variation, with the mean percentage of identity of 99.34 % (98.25 % to 100 %) among the sequences, without deeply divergent populations or cryptic lineages.
The morphological descriptions of Pannaria hookeri from the literature were gathered and used for the circumscription of the species and for the observation of inconsistencies in the species descriptions and identifications, with the differences found highlighted in the sections "Description of Species" and "Discussion" below (Tab.2).
We examined specimens are from Austria (4 specimens), Canada (2), Denmark (Faroe Islands, 1), Greenland (1), Iceland (1), Italy (1), Norway (16), Russia (1), Scotland (4), and Sweden (4) from the Northern Hemisphere; and Argentina (11), Chile (1), Kerguelen Island (2), and New Zealand (1) from the Southern Hemisphere.The determination of the specimens received on loan from herbaria was confirmed only through morphological approach.Unfortunately, most of the received material was collected more than 10 years ago (Text S1), and we were not able to generate genetic sequences.When the P. hookeri characters were not observed in the material, the specimen was excluded of the study.

Discussion
In this study, we re-confirmed the wide geographical distribution of Pannaria hookeri through phylogenetic and morphological evidence, now including the first P. hookeri sequences from the southern South America, Argentina.The phylogenetic analysis resulted in a well-supported clade with P. hookeri sequences from Argentina, Australia, Norway, and the USA, whereas the morphological analysis included specimens from Argentina, Austria, Canada, Chile, Denmark (Faroe Islands), Greenland, Iceland, Italy, Kerguelen Island, New Zealand, Norway, Russia, Scotland and Sweden, generating a concise morphological description.The materials from Scotland are topotypes (Text S1) and were compared with fresh specimens.
The bipolar distribution has been concepted as species that are disjunctly distributed in both hemispheres, mostly in polar and subpolar regions, and are largely absent in the tropics.However, many lichens reported as bipolar are also distributed in temperate and tropical latitudes -usually in high mountains, where their ecological requirements are met (Garrido-Benavent & Pérez-Ortega 2017), which is the case of P. hookeri, and should be carefully revised and possibly considered as (sub)cosmopolitan.Nowadays, the species is mentioned in high altitudes of Ecuador and Kenya (material not revised in the present study) (Frisch & Hertel 1998;Jørgensen & Palice 2010), but efforts are still needed to confirm or not the species distribution through the tropical latitudes.Studies including DNA analysis of species reported as bipolar has confirmed this remarkable distribution, for example in Austroplaca, Cetraria and Cladonia (Myllys et al. 2003;Søchting & Castello 2012;Fernández-Mendoza & Printzen 2013), while others has resulted in the discovery of cryptic lineages or restricted distribution, such as in Parmelia, Sphaerophorus and Rusavskia (Crespo et al. 2002;Högnabba & Wedin 2003;Scur et al. 2022), indicating that phylogeographical studies should be performed to revise the distribution stated to P. hookeri.
Historically, Pannaria hookeri specimens from the Southern Hemisphere were reported with a larger morphological plasticity than specimens from Northern Hemisphere (Jørgensen 2000a), and the taxonomic problems has invalited other results, e.g. the chemical study (Quilhot et al. 1989).The variations in the color of the thallus, branching, diameter of apothecia, and color of the apothecium discs, which were reported in the literature from Antarctic specimens (Lindsay 1974;Redón 1985;Jørgensen 1986;2000a;Øvstedal & Lewis Smith 2001), were not observed in the material confirmed by us and can be characters of other cryptic species.For example, apothecia up to 5.0 mm diam.were reported to the specimens from South Georgia and South Shetland Islands (Lindsay 1974;Redón 1985), apothecia up to 3.0 mm diam. in specimens from Bouvetøya Island (Jørgensen 1986), but only apothecia up to 2 mm diam.were observed during the present study.
In the same way, Øvstedal & Lewis Smith (2001) reported the species to South Georgia, Bouvetøya, South Orkney, South Shetland Islands, and Antarctic Peninsula, but posteriorly transferred the most of examined specimens within P. caespitosa P.M. Jørg.(Jorgensen 2000a).Only two specimens, SJA 168 and RILS 8347, were kept within P. hookeri, but they have the presence of pale brown discs in the apothecia (Øvstedal & Lewis Smith 2001) which do not match with the dark brown to blackish discs of P. hookeri (Tab.2).Therefore, a revision is still required to the both specimens.
The material received as loan that were not confirmed as P. hookeri are from Antarctic and subantarctic Islands (Antarctica Peninsula and Kerguelen, Marion, South Georgia, South Orkney and King George islands -Text S1), highlighting the taxonomic problem in the region.In addition, fresh specimens collected during four Brazilian Antarctic Expeditions in the Antarctic Peninsula (near to Esperanza and Primavera stations), James Ross Island, Marambio Island, and South Shetland Islands (Deception, Greenwich, King George, Livingston, Nelson, Robert, and Snow Islands) did not cluster with the P. hookeri sequences and they were also excluded from our analysis.An integrative revision of the Psoroma-complex is also required to Antarctica as well as a complete identification key of the group; P. hookeri in Antarctica can be more restricted than previously thought.
An integrative study of Pannaria hookeri (Ascomycota lichenized) from Argentina and the update of the taxon circumscription based on specimens from the Northern and Southern Hemispheres Previously, P. hookeri was considered as strictly saxicolous (Jørgensen 2000a).However, we observed in Argentina that the species can grow on the rock (Fig. 1A), and also on saxicolous moss (Fig. 1B), expanding the species' known ecology.Furthermore, the species were usually found on the mountains in Argentina, Australia and Chile (Jørgensen 2000a;Kantvilas & Gueidan 2018).Calvelo & Fryday (2006) reported P. hookeri on rock outcrops along a stream the sea-level in Argentina, but the material has not been reviewed by us.
Our description was based on 50 specimens from 13 countries, corroborating with the descriptions provided by Jørgensen (1978;2000a;2007), Kantvilas & Gueidan (2018) and Passo et al. (2020).Therefore, the large morphological variations reported for specimens from the Southern Hemisphere are potentially characters of different Psoroma species.Studies including more genetic sequences, chemical and morphological variation analysis should be performed to dept the evolutionary history and concept of Pannaria hookeri.
Figure 1.Bayesian Maximum Clade Credibility tree based on nuITS and mtSSU datasets showing the phylogenetic relationships within the Pannaria and P. hookeri sequences from Argentina, Australia, Norway and USA (Alaska).Posterior probabilities and Bootstrap values are above branches, respectively.Bold branches have posterior probabilities (PP) >0.95 % or bootstrap values >75.Sequences obtained in this study are marked in bold.Text after species names corresponds to voucher information (Tab.1).

Table 1 .
Dataset used in the present study for the phylogenetic analysis.Species, geographical origin, voucher information, nuITS and mtSSU GenBank accession codes and references.Sequences generated in this study are marked in bold.

Table 2 .
Morphological descriptions of Pannaria hookeri reported in the literature and in the present study.