Plant and Fungal Systematics 65(2): 303–357, 2020 ISSN 2544-7459 (print) DOI: https://doi.org/10.35535/pfsyst-2020-0025 ISSN 2657-5000 (online)

Two decades of DNA barcoding in the genus Usnea (Parmeliaceae): how useful and reliable is the ITS?

Robert Lücking1*, Miko Rain Abraham Nadel2, Elena Araujo3 & Alice Gerlach4

Abstract. We present an exhaustive analysis of the ITS barcoding marker in the genus Article info Usnea s.lat., separated into Dolichousnea, Eumitria, and Usnea including the subgenus Received: 7 Aug. 2020 Neuropogon, analyzing 1,751 accessions. We found only a few low-quality accessions, Revision received: 11 Nov. 2020 whereas information on voucher specimens and accuracy and precision of identifications Accepted: 11 Nov. 2020 was of subpar quality for many accessions. We provide an updated voucher table, align- Published: 29 Dec. 2020 ment and phylogenetic tree to facilitate DNA barcoding of Usnea, either locally or through Associate Editor curated databases such as UNITE. Taxonomic and geographic coverage was moderate: while María de los Angeles Herrera Dolichousnea and subgenus Neuropogon were well-represented among ITS data, sampling Campos for Eumitria and Usnea s.str. was sparse and biased towards certain lineages and geographic regions, such as Antarctica, Europe, and South America. North America, Africa, Asia and Oceania were undersampled. A peculiar situation arose with New Zealand, represented by a large amount of ITS accessions from across both major islands, but most of them left unidentified. The species pair Usnea antarctica vs. U. aurantiacoatra was the most sampled clade, including numerous ITS accessions from taxonomic and ecological studies. However, published analyses of highly resolved microsatellite and RADseq markers showed that ITS was not able to properly resolve the two species present in this complex. While lack of resolution appears to be an issue with ITS in recently evolving species complexes, we did not find evidence for gene duplication (paralogs) or hybridization for this marker. Comparison with other markers demonstrated that particularly IGS and RPB1 are useful to complement ITS-based phylogenies. Both IGS and RPB1 provided better backbone resolution and support than ITS; while IGS also showed better resolution and support at species level, RPB1 was less resolved and delineated for larger species complexes. The nuLSU was of limited use, providing neither resolution nor backbone support. The other three commonly employed protein-coding markers, TUB2, RPB2, and MCM7, showed variable evidence of possible gene duplication and paralog formation, particularly in the MCM7, and these markers should be used with care, especially in multimarker coalescence approaches. A substantial challenge was provided by difficult morphospecies that did not form coherent clades with ITS or other markers, suggesting various levels of cryptic speciation, the most notorious example being the U. cornuta complex. In these cases, the available data suggest that multimarker approaches using ITS, IGS and RPB1 help to assess distinct lineages. Overall, ITS was found to be a good first approximation to assess species delimitation and recognition in Usnea s.lat., as long as the data are carefully analyzed, and reference sequences are critically assessed and not taken at face value. In difficult groups, we recommend IGS as a secondary barcode marker, with the option to employ more resource-intensive approaches, such as RADseq, in species complexes involving so-called species pairs or other cases of disparate morphology not reflected in the ITS or IGS. Attempts should be made to close taxonomic and geographic gaps especially for the latter two markers, in particular in Eumitria and Usnea s.str. and in the highly diverse areas of North America and Central America, Africa, Asia, and Oceania. Key words: accuracy in GenBank, incorrect sequence labeling, paralogs, species delimi- tation; species richness

1 Botanischer Garten und Botanisches Museum, Freie Universität Berlin, 3 Departamento de Biología Vegetal II, Facultad de Farmacia, Univer- Königin-Luise-Straße 6–8, 14195 Berlin, Germany sidad Complutense de Madrid, 28040 Madrid, Spain 2 San Francisco State University, Department of Biology, 1600 Holloway 4 Conservatoire et Jardin botaniques de la Ville de Genève, 1 ch. de Ave., San Francisco, CA 94132, USA l’Impératrice, 1292 Chambésy/GE, Switzerland * Corresponding author e-mail: [email protected]

© 2020 W. Szafer Institute of Botany, Polish Academy of Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/) 304 Plant and Fungal Systematics 65(2): 303–357, 2020

Introduction habitats with high UV radiation (Lumbsch & Wirtz 2011). Likewise, the phenotypic distinction of Dolichousnea DNA barcoding refers to the use of a particular marker and Eumitria from Usnea s.str. is not straightforward to identify species relative to a set of identified reference (see below). sequences. DNA-based delimitation and identification of Species identification in Usnea s.lat. is notoriously fungi was discussed already in the mid-1980s (Kurtz- difficult. Usnea was therefore among the first genera man 1985), and molecular phylogenetic work on fungi where DNA-based identification via the ITS was tested. including lichens took off in the 1990s (Bruns et al. The first ITS sequence generated in this genus was for 1991; Gargas & Taylor 1995; Gargas et al. 1995). The a specimen of U. florida (L.) F.H. Wigg from Sweden internal transcribed spacer (ITS) of the ribosomal DNA (AF117996; Mattson 4001, UPS), although the sequence cistron was one of the first markers used to reconstruct was not published at the time because it was considered ‘... fungal phylogenies (White et al. 1990; Lee & Taylor too preliminary to be submitted to GenBank ...’ (Mattson 1992; Rehner & Uecker 1994; Feibelman et al. 1994; & Wedin 1998: 465). It was published one year later Kusaba & Tsuge 1995; Goffinet & Bayer 1997), includ- (Wedin et al. 1999), thus losing the race for the first ITS ing the pioneering work on lichen photosymbiodemes by sequence of Usnea registered with GenBank to U. strigosa Daniele Armaleo and Philippe Clerc (Armaleo & Clerc (Ach.) Pers. (AF112990), used as outgroup in a study on 1991). Molecular identification of fungi including lichens Ramalina (LaGreca 1999). using the ITS also emerged in the early 1990s (Kasuga Shortly after the turn of the millennium, the first stud- et al. 1993; Tisserat et al. 1994; Groner & LaGreca 1997; ies emerged that used the ITS to assess species delim- Lohtander et al. 1998), and ITS was employed for popu- itation in Usnea s.lat. Initially, focus was laid on the lation genetics in conservation assessments in the model taxonomic status of so-called species pairs, forms with taxon Lobaria pulmonaria (L.) Hoffm. (Zoller et al. identical morphology, but differing reproductive strat- 1999). However, only less than a decade ago, the ITS egy, such as the apotheciate U. florida vs. the sorediate was proposed as the universal DNA barcoding marker U. subfloridana Stirt. (Articus et al. 2002). At the time, for fungi (Schoch et al. 2012). the authors concluded that the two morphs represented Usnea Dill. ex Adans. is one of the largest genus- a single species. Another objective was the delimitation level clades of lichenized fungi, currently listed among of natural groupings within Usnea s.lat. based on the ITS the ten most speciose genera, with approximately 350 (Ohmura 2002; Articus 2004; Ohmura & Kanda 2004; taxa (Thell et al. 2012; Lücking et al. 2017a). The clas- Wirtz et al. 2006). Based on Japanese taxa, Ohmura sification of usneoid lichens has been in flux, besides (2002) resolved Dolichousnea as sister to a clade with Protousnea (Motyka) Krog either recognizing a single Eumitria sister Usnea s.str., recognizing the three clades genus Usnea s.lat., with several subgenera and sections at subgenus level. Articus (2004) recovered Dolichousnea (Motyka 1936, 1938; Ohmura 2002; Ohmura & Kanda plus Eumitria as sister to Neuropogon plus Usnea and pro- 2004; Wirtz et al. 2006; Truong & Clerc 2013; Ohmura posed to recognize the four groups at genus level, although & Kashiwadani 2018; Temu et al. 2019) or separating both ITS alone and a three-locus dataset (ITS, nuLSU, Dolichousnea (Y. Ohmura) Articus, Eumitria Stirt., and β-tubulin = TUB2) rendered Usnea s.str. paraphyletic with Neuropogon Nees & Flot. (Fig. 1), as smaller genera from respect to Neuropogon. Ohmura & Kanda (2004), again Usnea s.str. (Articus 2004). Given the underlying data, we based on ITS, recovered Eumitria as sister to the other follow Divakar et al. (2017) and Kraichak et al. (2017) to three groups and Dolichousnea as sister to Neuropogon recognize Dolichousnea and Eumitria as separate genera, plus Usnea, also with the latter forming a paraphyletic while retaining Neuropogon within Usnea at the level of grade relative to Neuropogon. Wirtz et al. (2006) found subgenus. Besides considerations of evolutionary age, an ITS-based topology similar to that of Articus (2004), this reflects the fact that bothDolichousnea and Eumitria with Neuropogon polyphyletic and nested within Usnea have consistently been recovered as monophyletic groups s.str., and proposed to recognize a single, large genus in all phylogenetic studies, whereas neuropogonoid taxa Usnea s.lat., also subsuming Dolichousnea and Eumitria are polyphyletic and the neuropogonoid core group was within the latter. mostly found nested within Usnea s.str., rendering the Most subsequent works utilizing the ITS, without latter paraphyletic (Ohmura 2002; Articus 2004; Ohmura or with additional markers, focused on species taxon- & Kanda 2004; Wirtz et al. 2006). The only study ren- omy, with emphasis on neuropogonoid taxa (Seymour dering Neuropogon s.str. and Usnea s.str. reciprocally et al. 2007; Wirtz et al. 2008, 2012; Lumbsch & Wirtz monophyletic, based on six markers, had a limited taxon 2011). Several studies worked on other groups within sampling (Divakar et al. 2017; Kraichak et al. 2017). Since Usnea s.lat., such as Eumitria (Temu et al. 2019), Usnea neuropogonoid taxa such as U. acanthella (I.M. Lamb) s.str. with pigmented cortex (Ohmura 2008), and the F.J. Walker and U. durietzii Motyka are nested within U. cornuta Körb. aggregate (Gerlach et al. 2019a). Usnea s.str. (Wirtz et al. 2006), there are also no clear- Geographic focal studies included Usnea from North cut morphological characters to support Neuropogon as America and Europe (Mark et al. 2016a; Clerc & Naciri a separate genus. Rather, the production of melanoid sub- 2021), European shrubby species (Saag et al. 2011), the stances in the thalline cortex and the apothecial discs has Iberian Peninsula (Araujo 2016), Taiwan (Shen et al. been considered an adaptation to harsh environmental 2012), Africa (Nadel 2016), South America (Truong conditions, as neuropogonoid taxa are typically found in et al. 2013a; Truong & Clerc 2016; Gerlach et al. 2017, R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 305

2019a, b), and urban New York (Dorey et al. 2019). The thesis works by E. Araujo (Araujo 2016) and M. Nadel (Nadel 2016) have not been published and the under- lying sequences have not been deposited in GenBank previously, but are included in the present study with newly issued accession numbers. Broader DNA barcoding studies also included data on Usnea or in part focused on the latter, particularly in New Zealand (Kelly et al. 2011), but also Switzerland (Mark et al. 2016b), Norway (Marthinsen et al. 2019), and Cameroon (Orock et al. 2012). A large amount of ITS data was generated for analyses of mycobiont-photobiont associations of Usnea lichens and their corresponding ecology and morphology in New Zealand and Antarctica (Rafat 2014; Buckley et al. 2014; Cao et al. 2015, 2018; Park et al. 2015; Rafat et al. 2015). Another broad study in the Northern Hemisphere focused on genetic variation in Dolichousnea longissima (Ach.) Articus (Rolstad et al. 2013). These data were subsequently used to employ niche distribution modeling to predict geographic outliers not representing that species using a novel approach to visu- alize ecogeographic barriers (Smith et al. 2016). Millanes et al. (2014) analyzed relationships between lichenicolous Biatoropsis Räsanen species and their Usnea hosts. The level of resolution of the ITS was tested in two studies on the species pair U. antarctica Du Rietz and U. aurantia- coatra (Jacq.) Bory using newly developed microsatellite markers (Lagostina et al. 2018) and RADseq (Grewe et al. 2018). Both studies supported the two taxa as separate species, although they were not resolved by ITS. Using DNA sequences for the identification of fungi has been criticized early on (Seifert et al. 1995), but has since become established as an indispensable component of integrative taxonomy of fungi (Lücking et al. 2020a). However, the application of ITS as a universal fungal bar- coding marker (Schoch et al. 2012) has been challenged by lack of resolution in certain fungal lineages and by potential artifacts due to intragenomic variation reflecting either hybridization and introgression or gene duplication (Nilsson et al. 2008; Linder & Banik 2011; Pino-Bodas et al. 2013; Stielow et al. 2015; Li et al. 2017; Bado- tti et al. 2018; Lücking et al. 2020a). Another notorious problem pertains to data quality, both the quality of the underlying sequence data and the quality of metadata, in particular sequence identifications (Nilsson et al. 2006, 2018; Bidartondo 2008; Kozlov et al. 2016; Schoch et al. 2017; Meiklejohn et al. 2019; Lücking et al. 2020a, b; Clerc & Naciri 2021). Here, we use the example of the genus Usnea s.lat. to quantitatively assess these issues, by analyzing all 1,610 ITS sequences deposited under the label Usnea in GenBank between 1999 and 2020 (cut-off date: 15 June 2020), plus the 147 previously unregistered sequences from Araujo (2016) and Nadel (2016). We want to emphasize that this work is not to be understood as a phylogenetic revision of Usnea s.lat. The latter would require careful examination of all underlying vouchers in the context of the historic taxonomy of this genus. Figure 1. Representatives of the four main groups of Usnea s.lat. A – Dolichousnea (D. longissima); B – Eumitria (E. baileyi); C – subgenus Rather, we attempted to establish a vetted phylogenetic Neuropogon (U. sphacelata); D – Usnea s.str. (U. erinacea). Photo- framework for DNA barcoding approaches, given the data graphs by RL except (A), kindly provided by Y. Ohmura. at hand, which are still far from complete. 306 Plant and Fungal Systematics 65(2): 303–357, 2020

We dedicate this work to our esteemed colleague, nuLSU), the data set was automatically aligned using mentor and friend, Philippe Clerc, on the occasion of MAFFT 7.244 with the [--auto] and the [--reorder] his retirement from active duty as Head of the Herbar- function (Katoh & Standley 2013). The entire block ium at the Conservatoire et Jardin Botaniques de la Ville of ITS sequences (1,757 total) was extracted from this de Genève. Since his first papers on the topic 36 years data set, automatically realigned again in MAFFT, and ago (Clerc 1984a, b), Philippe has been instrumental in manually inspected and adjusted in BioEdit 7.2.5 (Hall shaping the taxonomy of Usnea s.lat., one of the largest 1999, 2011). The alignment was subsequently refined and taxonomically most difficult genera of lichenized by first removing ambiguously aligned regions, recon- fungi, through his own, numerous contributions, through structing a tree, and then resorting the original sequences fruitful international collaborations, and through train- based on the tree so that more variable regions could ing of various generations of usneologists around the be directly compared between closely related species. world. In these modern times, where taxonomy is moving Sequence names were then batch-edited to reflect the towards a misguided concept of automatism, Philippe’s format >Genus_species_Accession_Country_sampleID, work demonstrates the requirement for individual dedi- by saving the FASTA file as a text file, formatting it for cation and the motivation to learn, gain experience, and manipulation in MICROSOFT Excel 10, extracting the transfer this knowledge, to achieve long-lasting impact names block and filtering and editing the corresponding in taxonomy and in cataloguing our planet’s biodiver- name elements. Three sequences of Protousnea dusenii sity. We wish Philippe our best for his future endeavors, were used as outgroup. which hopefully will include a good deal of ‘usneology’. After reconstructing a maximum likelihood tree (see We humbly recognize that he is the one person miss- below), the sequences were resorted following their ing as expert co-author in this study dedicated to him, arrangement in the tree. This was accomplished by and we hope he will forgive the errors we may have extracting the names in corresponding order from the made in ­trying to make sense of our findings in this PDF-format tree file, opening them in MICROSOFT ­complex genus. Excel 10 and numbering them consecutively. The under- lying FASTA alignment was also opened in MICROSOFT Material and methods Excel 10 after preformatting in MICROSOFT Word 10 to achieve placement of elements in separate columns, and Species concept the sequences were resorted using the numbers from the tree file. After this step, which sorted sequences accord- The present study focuses on molecular data used to ing to their phylogenetic relationships instead of simi- formulate species hypotheses. As far as phenotype data larity, the alignment was again manually inspected and (including secondary chemistry) were available, we inte- adjusted. This process was repeated three times to obtain grated these into the assessment of species delimitations the final alignment with a length of 636 bases (File S1). on an ad hoc basis in the discussions under each clade. The sequence metadata were then edited in table form in However, our primary approach was to delimit clades MICROSOFT Excel 10 and the corresponding original using a combination of branch support and branch length. publications, where available, were tracked down for each We thereby established phylogenetic species hypothe- sequence (Table S1). ses using either reciprocal monophyly or, in exceptional In order to assess the performance of the ITS in terms cases, nested relationships leading to the recognition of of resolution and clade fidelity, we analyzed six addi- paraphyletic species. Paraphyletic species are a reality tional markers for comparison for which sufficient data of evolution (Crisp & Chandler 1996; Funk & Omland were available, namely nuLSU, IGS, β-tubulin (TUB2), 2003; Kuchta et al. 2018) and we accepted such instances RPB1, RPB2, and MCM7, including unpublished RPB1 under the following conditions: (1) the nested clade had and MCM7 sequences from the thesis work of Araujo a substantially longer stem branch than other terminal (2016) and from the study by Gerlach et al. (2019a). branches in the including clade; (2) the stem branch of The data sets were extracted from the originally down- the nested clade was strongly supported; (3) phenotypic loaded data set and treated in the same way as the ITS and/or geographic differences were associated with the (see above), resulting in six additional alignments (Files nested clade vs. its paraphyletic residual. S2–S7), with the following sizes and lengths: nuLSU = We should also emphasize that while accurate species 154 terminals, 872 bases; IGS = 488 terminals, 410 bases; hypotheses are critical to the DNA barcoding approach, TUB2 = 204 terminals, 342 bases; RPB1 = 723 terminals, the performance of barcoding markers is first and foremost 710 bases; RPB2 = 154 terminals, 776 bases; MCM7 = lineage-based, regardless at what taxonomic level these 398 terminals, 541 bases. These markers were analysed lineages are recognized. without outgroup and rooted with the Dolichousnea-Eu- mitria clade (e.g., Divakar et al. 2017). Molecular data analyses Overall, ITS was best represented among the seven Data assembly. We downloaded all available sequence markers with more than two times as many terminals as data for the genus Usnea from GenBank and added the second most frequently sequenced marker (RPB1). unpublished sequences from the thesis works of Araujo The length of the ITS alignment was in the median range, (2016) and Nadel (2016). After separating sequences surpassed by nuLSU, RPB1, and RPB2, but substantially that combined several markers (e.g., nuSSU, ITS, longer than MCM7, IGS, and TUB2. The total size of the R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 307 characters × terminals matrix over all markers was nearly Results and discussion 2.4 million data points. Data quality Phylogenetic analysis. For the ITS and each of the other . six markers, a best-scoring maximum likelihood tree was Sequence quality Among the 1,757 ITS sequences reconstructed in RAxML 8 (Stamatakis 2014), in case of initially compiled, four had to be removed as they did the ITS and RPB1 with RAxML-HPC Blackbox 8.2.12 not represent species of that genus, but other Parme- in the CIPRES Science Gateway (Miller et al. 2010) and liaceae. All four are from an unpublished study from for the other markers locally with RAxML 8.2.0. For South Korea submitted by S.-H. Jang et al. in 2018 all markers, we used the universal GTR-Gamma model (MH258910, MH258911, MH258912, MH258913). All with either 1,000 bootstrap replicates (locally) or with other sequences were confirmed as belonging to Usnea an automatically determined number of non-parametric s.lat., resulting in an accuracy of 99.8% in terms of cor- bootstrap replicates (456 in case of the ITS) using a sat- rect labeling at genus level (Table 1). Two further, also uration criterion (CIPRES). apparently unpublished sequences deposited by C. R. In the assessment of the resulting phylogenies, we A. Stewart et al. in 2018 (MH887539, MH887540) had used the term clade for monophyletic and the term grade to be removed, as they originated from combined nuS- for paraphyletic groupings. SU-ITS-nuLSU tandem repeats, but did not contain the variable ITS1 and ITS2 portions. This left a total of Pairwise identity threshold analysis. For selected 1,751 ingroup sequences. species complexes, we performed an assessment of phy- Twelve sequences were determined as limited to low logenetic resolution in the ITS by computing the pair- quality, due to a higher number of ambiguous base calls wise BLAST identity matrix in BioEdit 7.2.5 (Hall 1999, (>3), often associated with additional odd base calls 2011). The symmetrical matrices were then shaded using (File S1; Table S1), including Usnea sp. (KM369422: 15 98.5% as threshold, following the common use of this ambiguous sites), U. florida (AF117996: 14), U. inter- value in reference databases (Irinyi et al. 2015; Jeewon media (A. Massal.) Jatta (JN009731: 8), U. cladocarpa & Hyde 2016; Kõljalg et al. 2019; Nilsson et al. 2019), Fée (KY021905: 4), U. silesiaca Motyka (KU352630: 4), as well as evidence from empirical studies (Lücking et al. and U. subparvula A. Gerlach & P. Clerc (KY021928: 4). 2014; Garnica et al. 2016; Vu et al. 2016, 2019; Moncada Particularly notable were four sequences of U. articulata et al. 2020a). (L.) Hoffm. (JN086277: 6; JN086278: 19; JN086279: While there are no fixed similarity thresholds between 13; JN086280: 14) and two labeled U. dasaea Stirt. species, the 98.5% level is useful to assess potential struc- (JN086283: 16; JN086284: 15). In general, low qual- ture in the sequence data of a complex clade. Thus, phy- ity did not affect the correct placement of the affected logenetically defined lineages within species complexes, sequences, but often considerably reduced support for including nested clades opposed to paraphyletic residu- the containing clades (Fig. S1). In the case of the two als (see above), were considered to potentially represent sequences labeled U. dasaea from Portugal, the ambigu- distinct species when pairwise identity values between ous base calls occurred principally in the second portion members of different lineages were consistently 98.5% of the ITS2 and were associated with numerous odd or lower. base calls, resulting in an extremely long stem branch (see below). These were the only two instances where Metadata assessment the underlying topology was considered unreliable due to potential issues with sequence quality. Using the ITS voucher table (Table S1), we assessed var- ious categories of metadata, including geographic origin (country or region), original paper where sequences were Table 1. Quality assessment for ITS sequences and their metadata in first published, year of publication, alternative identifica- Usnea s.lat. We used the following grading system: excellent (≥90% and ≤100%); very good (≥80% and <90%); good (≥70% and <80%); satis- tions when differing between GenBank submission and factory (≥50% and <70%); moderate (≥30% and <50%); poor (≥10% original paper, and either the revised identification after and <30%); bad (≥0% and <10%). studying the voucher material or the likely identification based on phylogenetic context and published data. Category Value Assessment In order to assess the taxonomic and geographic rep- Genus identification 99.8% excellent resentativity of the known ITS sequences, we assembled Sequence quality 95.9% excellent a global checklist for species of Usnea s.lat., mostly based Voucher information 76.0% good on recent publications (after 1970), including monographic Precision of identifications 77.5% good revisions and biotic surveys and in plausible cases also Taxonomic diversity 26.4% poor non-taxonomic studies, such as biochemical analyses of Weighted taxonomic diversity 55.1% satisfactory selected Usnea species. All names were corrected for established synonymies following most recent studies. Geographic coverage 41.7% moderate The final list had 5,066 entries (Table S2), correspond- Taxon-region records 18.4% poor ing to a total of 735 names including currently accepted Group-region representativity 34.4% moderate synonyms, based on 260 references. Topical diversity – excellent Identification success 70.6% good 308 Plant and Fungal Systematics 65(2): 303–357, 2020

Occasionally, odd base calls in otherwise conserved 474, table 1), with detailed voucher information, the hap- regions within a clade were observed in 34 sequences, lotypes were indicated, but not the isolate numbers, so in including two of low quality (File S1; Table S1). These some cases, the GenBank accessions could not be unam- also did not seem to affect phylogenetic placement as biguously linked to voucher information. For instance, indicated by the underlying identifications and other meta- haplotype 17 corresponded to five accessions (EF492152, data, but in some instances clade support. A total of 103 EF492158, EF492181, EF492198, EF492202), whereas in sequences exhibited conspicuous short gaps particularly the table this haplotype was indicated for six samples from in the second portion of the ITS1, including two of low four localities, one from Ecuador, three from Argentina, quality (File S1; Table S1). The gaps appeared in unrelated and two from two different places in Antarctica. The same clades in the same positions and were not lineage-specific. applied to haplotype 8. For six accessions (EF492146, Their causes are unknown, but likely represent artifacts EF492147, EF492153, EF492161, EF492213, EF492216), of the corresponding sequencing approach. Except for no haplotypes were indicated. These were given in the a lack of potentially synapomorphic signal in the gappy tree figure (Wirtz et al. 2008: 478, fig. 1) as ‘potential regions, the phylogenetic position of the sequences was recombinants’ (see above). Their geographic origin could not affected by these gaps. The same applied to ten short be inferred by comparing the isolate numbers with those sequences missing larger portions at the 3’ or 5’ end of haplotypes indicated in the table. Two further ‘potential (File S1; Table S1). recombinants’ given in the tree figure (Wirtz et al. 2008: Using a scoring scheme of 3 = high quality (1,598 478, fig. 1), with the isolate numbers 208-19 and 221-1, sequences), 2 = gappy or short (103), 1 = odd base calls were apparently not included in the GenBank submis- (34), and 0 = low quality (12), with a given sequence sions. Upon request, we received the complete voucher receiving the lowest possible score when two or more table from the original author (N. Wirtz, pers. comm. July limiting criteria applied, the 1,751 sequences thus received 2020), which enabled us to fill in most of the missing an overall quality score of 5,054 out of a maximum of data, except geographic origin in 16 cases (Table S1). 5,271, or 95.9%. Therefore, sequence quality of available Ohmura (2008) generated 24 new ITS sequences from ITS data in the genus Usnea was considered very high 24 specimens for a study of Usnea rubicunda Stirt. and overall (Table 1). U. rubrotincta Stirt. However, only six unique sequences Wirtz et al. (2008) gave eight ITS sequences in sub- were submitted to GenBank, presumably subsuming spec- genus Neuropogon as potential recombinants (Table S1). imens with sequences representing identical haplotypes This suggests they were composed of portions representing under a single accession. However, each GenBank acces- at least two distinct lineages in a chimeric combination, sion only included a single voucher and not all specimens e.g. in the ITS1 vs. ITS2. However, we could not detect corresponding to that haplotype. A similar approach was issues with these eight sequences that would indicate them taken by Ohmura & Clerc (2019), with three accessions as potentially chimeric. Chimeric sequences composed of (LC479120, LC479122, LC479123) representing a total of portions of two different taxa would be expected to fall 15 specimens of U. cornuta, but in GenBank only citing on long, yet unsupported branches, which was not the one specimen each. For taxonomic studies, this practice case for any of these eight sequences (Table S1; Fig. S1). is not recommended. Ideally, specimen-based Sanger Rather, they appeared to represent variants comparable sequences should relate to voucher specimens in a 1:1 to those in other instances. ratio. If multiple specimens share the same haplotype and are represented in a single accession, all specimens should Voucher information. Several studies had issues with be cited with that accession in GenBank. The current incomplete or erroneous voucher information or lacked submission tools do not actually allow this for structured voucher information altogether. In Thell et al. (2002: data, but the additional specimens could be listed in the 341–342, Table 2), two specimens had the same accession title of the representative sequence. number (AF451739) for the ITS, namely Flavocetraria The voucher table submitted as a supplementary file cucullata (Bellardi) Kärnefelt & A. Thell (DNA-932) and by Millanes et al. (2014) contained detailed voucher Usnea florida (DNA-840). The latter corresponded to the information, but in the final version the authors missed deposition in GenBank, whereas the erroneous accession to replace the ‘X’ with the actual accession numbers of number for the Flavocetraria was a lapsus for AF451793. the newly generated sequences, including 26 of the ITS. Twelve accessions of Neuropogon (EF179795–EF179806) In that case, it was possible to associate the accession were indicated in GenBank to correspond to the study numbers with the vouchers using the isolate numbers. In by Wirtz et al. (2006), but were not used or cited in that their broad study on mycobiont-photobiont associations of work; therefore, their geographic origin could mostly not New Zealand Usnea, Buckley et al. (2014) did not provide be ascertained. a voucher table for the 265 samples. The GenBank acces- Wirtz et al. (2008) generated 71 new ITS sequences for sion numbers were subsequently added in a correction, but subgenus Neuropogon, but instead of a detailed voucher only indicating the ranges, without any specific locality table, the accession numbers were cited as a single text information associated with each sequence. string: ‘All sequences, including the haplotype infor- A series of 52 accessions (JN943506 through JN943562 mation, were submitted to GenBank [accession nos: p.p.) was indicated in GenBank as corresponding to the EF492146–EF492216 (ITS) ...’ (Wirtz et al. 2008: 477). In barcoding studies by Kelly et al. (2011) and Schoch et al. the morphological species assignment (Wirtz et al. 2008: (2012). The accessions were not cited in either work, but R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 309 the biomaterial numbers indicate that these were dupli- studies (Truong et al. 2013a, 2016; Gerlach et al. 2017, cate submissions of sequences already submitted in Kelly 2019a), in anticipation of further work to resolve the tax- et al. (2011) under another number series (e.g., JN943506 onomic status of this material (e.g., Gerlach et al. 2020). = FR799053, JN943562 = FR799085, etc.). Four addi- Of the 353 unidentified sequences, 33 were also from tional accessions (JN943507, JN943508, JN943512, taxonomic studies (Thell et al. 2004; Wirtz et al. 2006, JN943514) were published twice as newly generated 2008, 2012; Shen et al. 2012; Gerlach et al. 2019a). Three sequences. In the Swiss barcoding study by Mark et al. further sequences were from a study on lichenicolous (2016b), seven accessions, corresponding to the genus Biatoropsis species on Usnea, with the host lichens left Usnea (KX132919, KX132920, KX132921, KX132928, unidentified (Millanes et al. 2014), and another seven KX132929, KX132930, KX132932), were published with from environmental or general barcoding studies (Kelly incorrect voucher information as the entries were appar- et al. 2011; Jaouen et al. 2019; Canini et al. 2020). A total ently shifted in the voucher table (Mark et al. 2016b: of 32 unidentified sequences deposited in GenBank have 689, table 1). apparently not been published, their dates of deposition Excluding the 147 ITS sequences provided by the sec- ranging between 2010 and 2020 (Table S1). We assume ond and third author of this paper (MRAN, EAC), which that the more recently deposited sequences are currently were submitted to GenBank as part of this study, as well in the process of being published in peer-reviewed stud- as 143 unpublished sequences, 438 out of the remaining ies. The bulk of the unidentified depositions originated 1,461 accessions (30.0%) had issues with proper voucher from studies on mycobiont-photobiont associations of information. For 99 of these, the information could be New Zealand Usnea (Rafat 2014; Buckley et al. 2014; inferred by cross-comparison with metadata, whereas Rafat et al. 2015). for 339 (77.4% of the problematic cases and 23.2% of For 112 accessions, the identification given with the all accessions), important voucher information such as accession in GenBank did not match the identification locality data could not be resolved from published infor- in the corresponding original publication (Table S1). In mation alone. 24 cases of accessions labeled Usnea sp., precise identi- Using a scoring scheme of 3 = voucher informa- fications were provided in the actual papers (Shen et al. tion complete in original publication (1,023 sequences), 2012; Buckley et al. 2014; Rafat et al. 2015), but not 2 = incomplete or erroneous but could be reconstructed updated in the GenBank accessions. In six instances, the from GenBank metadata or other sources (99), 1 = incom- label Usnea sp. was either specified as Usnea sp. 1 or as plete or erroneous and could not be reconstructed (62), and Usnea lineage 14A in the corresponding publication, i.e., 0 = absent (277), the 1,461 sequences originally published denoting specific taxa without a formal name awaiting fur- in corresponding scientific papers thus received a total ther work (Wirtz et al. 2006; Gerlach et al. 2019a, 2020). score of 3,329 out of a maximum of 4,383, or 76.0%. In three cases (MF669881, MF669883, MN080241), the Therefore, the quality of published voucher information full identification provided in the publication deviated for these sequences could overall be considered good from that of the GenBank accession (Gerlach et al. 2019a; when using a standard grading scheme (Table 1). While Temu et al. 2019). For 65 accessions, the labels given in ‘good’ might seem a desirable result for basic voucher the principal components of the published work (voucher information, such as sample ID and geographic origin, specimens, phylogenetic trees) differed from those sub- one would, however, expect no less than 100%, so in mitted with the accessions. In this case, the labels on the relative terms 76% is a poor result. accessions were the correct ones, but the corresponding A very positive aspect in some studies was that the identifications were published in the paper in somewhat secondary chemistry of the underlying vouchers was cryptic form at the end of the discussion (Wirtz et al. listed, either in the voucher table (Truong et al. 2013a; 2008: 482): ‘The following species could be identified Gerlach et al. 2017) or in other sources of information within a collection of specimens, which were a priori (e.g., Nadel 2016; Mark et al. 2016a: 510–511, fig. 1; assigned to two species, U. perpusilla and U. sphacelata: Gerlach et al. 2019a: 131, table 1; Temu et al. 2019: 256, (1) Usnea sp. 1, (2) Usnea sp. 3, (3) Usnea ushuaiensis, fig. 3). Given that current taxonomy in Usnea for many (4) U. perpusilla, and (5) U. lambii (Usnea sp. 2).’ Most taxa assumes chemical variability and so the applied name of these issues have been rectified in the updated voucher is not necessarily a reflection of the underlying chemistry, table assembled for this study (Table S1), in part with the inclusion of the chemotype in the voucher informa- assistance by the original authors. Overall, these issues tion is strongly recommended for phylogenetic and DNA resulted in a conflict between submitted and published barcoding studies. label information, or in reduced precision in GenBank accessions compared to published voucher identifications Identifications. Of the 1,604 Usnea sequences depos- for 41 entries. ited in GenBank (excluding those provided by EAC and Excluding the 147 ITS sequences provided by EAC MRAN submitted as part of this study), 1,168 were identi- and MRAN that were submitted to GenBank as part of fied to species, 64 provisionally to species using the prefix this study, we used a scoring scheme of 3 = precise identi- ‘aff.’ (60) or ‘cf.’ (4), 19 provisionally to species using fication to species, identical in submission and publication the designation ‘sp.’ plus a number, and 353 were only (1,133 sequences); 2 = imprecise identification to species identified to genus. The 19 provisionally identified spe- (aff., cf., sp. 1, etc.), identical in submission and publica- cies using ‘sp.’ plus a number were from four taxonomic tion (83); 1 = identification different in submission and 310 Plant and Fungal Systematics 65(2): 303–357, 2020

publication (41); and 0 = no identification to species (sp.), formally associated with this genus. However, seven of identical in submission and publication (306). Applying these names (besides several others under Usnea) are this scheme, the total for all 1,604 sequences was 3,729, considered synonyms of E. baileyi Stirt.: E. antillarum out of a maximum of 4,812, or 77.5%, resulting in an Vain., E. asperrima (Müll. Arg.) Vain., E. endochroa assessment of ‘good’ for the quality of the precision of the Vain., E. endorhodina Vain., E. formosa Stirt., E. implicita identifications (Table 1). It should be noted that this does Stirt., E. perrubescens Vain., and E. tasmanica (Müll. not extend to their implied accuracy, which was assessed Arg.) Vain. (Rogers & Stevens 1988; Truong & Clerc in detail a phylogenetic context (see below). 2013; Buaruang et al. 2017). At least four names rep- Overall, the Usnea data set underlined the common resent distinct species in Eumitria, namely E. firmula problem of mismatch between identification labels on Stirt., E. liechtensteinii (J. Steiner) Vain., E. pectinata submitted accessions versus published information, both (Taylor) Articus, and E. trullifera (Nyl.) Vain. (Swinscow in the original papers or regarding subsequently published & Krog 1974, 1986; Ohmura 2002; Articus 2004; Ohmura taxonomic or nomenclatural changes, with original sub- & Kanda 2004; Nadel 2016; Buaruang et al. 2017; Temu mitters often not updating their records (Schoch et al. et al. 2019). 2017), although in the present case, the proportion of More than a dozen additional names have been asso- such cases was low. ciated with Eumitria, but without formal combinations into that genus, mostly from Africa, but also from North Metadata diversity America and Asia, including Usnea antigua Swinscow & Krog, U. brunnescens C.W. Dodge, U. cervicornis C.W. Taxonomic diversity and coverage. The 1,751 ITS Dodge, U. congdonii Krog, U. cristata Motyka, U. elata accessions corresponded to a total of 118 different names, Motyka, U. flaveola Motyka, U. perplectata Motyka, 105 precise names in the accession labels, four addi- U. pulvinulata C.W. Dodge, U. recurvata J.D. Chao, L.W. tional names as components of imprecise identifications Hsu & Z.M. Sun, U. subcristata C.W. Dodge, U. subfla- in the accession labels, and nine further names in the veola Truong & P. Clerc, U. subrectangulata J.D. Chao, corresponding publications based on subsequently pub- L.W. Hsu & Z.M. Sun, U. uluguruensis Krog, U. vainioi lished corrections compared to the original accessions Motyka, U. welwitschiana Motyka, and U. zombensis (Table S1). While Thell et al. (2012) and Lücking et al. Krog (Chao et al. 1975; Swinscow & Krog 1986; Rogers (2017a) gave the total number of accepted species for & Stevens 1988; Krog 1994; Truong & Clerc 2013; Temu Usnea s.lat. as 350, our global survey revealed a total et al. 2019). The Eumitria pectinata complex consists of of 447 currently accepted names, a figure much higher pendulous species with an almost solid, brown-pigmented than previously estimated. Of these, three corresponded axis that only partially becomes fistulate, including also to Dolichousnea, 21 to Eumitria, 24 to subgenus Neu- U. mexicana Vain. and its relatives or putative synonyms, ropogon, and 399 to Usnea s.str. excluding subgenus U. chloreoides Motyka, U. duriuscula Motyka, U. gigas Neuropogon (Table S2). Thus, overall the names orig- Motyka, and U. himantodes Stirt. (Swinscow & Krog inally applied to the ITS sequences represented 26.4% 1988; Herrera-Campos et al. 1998; Ohmura 2001; Truong of the currently recognized species richness in Usnea et al. 2013b; Nadel 2016; Temu et al. 2019). Our global s.lat. (Table 1, Fig. 2). Dolichousnea was the only group survey indicates that Eumitria currently contains about represented by all (three) currently recognized species two dozen named species, only two of which had ITS (Ohmura 2002; Articus 2004). sequence accessions in GenBank, namely E. baileyi and The number of species corresponding to Eumitria E. pectinata (Ohmura 2002; Orock et al. 2012; Jaouen is difficult to assess. A total of 14 names have been et al. 2019; Temu et al. 2019). One unidentified species from China had GenBank accessions that have not yet been published. Whether this species corresponds to a new or a known species is unclear. In addition, the unpublished thesis work on Usnea from São Tomé and Príncipe (Nadel 2016) included ITS sequences for E. firmula (Table S1). Thus, as a whole, only around 12% of the species of this genus currently have ITS sequence data (Fig. 2). Walker (1985) accepted 15 species under the concept of subgenus Neuropogon. Of these, U. acanthella and U. durietzii do not belong to Neuropogon (Wirtz et al. 2006). Eight species represent the core clade of subgenus Neuropogon, including U. acromelana Stirt., U. antarc- tica, U. aurantiacoatra, U. patagonica F.J. Walker, U. per- pusilla (I.M. Lamb) F.J. Walker, U. sphacelata R. Br., U. subantarctica F.J. Walker, and U. trachycarpa (Stirt.) Müll. Arg., to which the recently recognized U. lambii (Imshaug) Wirtz & Lumbsch and U. ushuaiensis (I.M. Figure 2. Number of species per genus or genus group in Usnea s.lat. Lamb) Wirtz, Printzen & Lumbsch can be added (Arti- (orange) and number of species sequenced by comparison (purple). cus 2004; Wirtz et al. 2006, 2008, 2012; Seymour et al. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 311

Figure 3. Geographic coverage of available ITS sequences in Usnea s.lat. The area of the circles is proportional to the number of sequences. Purple = well-sampled, lilac = moderately well-sampled; orange = moderately sampled; beige = poorly sampled (including geographically biased sampling and/or high proportion of unidentified samples). Base map taken from Wikimedia Commons [https://commons.wikimedia.org/wiki/ File:BlankMap-World-v2.png].

2007; Cao et al. 2015, 2018; Lagostina et al. 2018). Two Neuropogon, Usnea s.str. retained 399 taxa in our global species treated in Walker (1985) under subgenus Neuro- checklist (Table S2), representing the bulk of the known pogon clustered in a sister clade, namely Usnea ciliata species richness in Usnea s.lat. Of these, 91 names (23.5%) (Müll. Arg.) Vain. and U. subcapillaris (D.J. Galloway) were represented among ITS accessions (Fig. 2). Over- F.J. Walker (see also Wirtz et al. 2006). This clade of 148 all, taxonomic diversity among available ITS sequences terminals was almost exclusively formed by samples from was biased in favor of Dolichousnea (100% of known New Zealand, with the exception of one specimen from species sequenced) and subgenus Neuropogon (82.6%), Antarctica and another from Chile (Fig. S1). Most of the whereas relatively few of the known species have been samples were not identified (Buckley et al. 2014), but sequenced in Usnea s.str. (23.5%) and Eumitria (14.2%). the few identified specimens (Rafat et al. 2015) include Giving each group equal weight, the weighted average two names also associated with subgenus Neuropogon by of the taxonomic diversity represented in sequence data Walker (1985), namely U. inermis Motyka and U. torulosa amounted to 55.1% (Table 1). (Müll. Arg.) Zahlbr. Three further identifications in this clade, U. ciliifera Motyka, U. pusilla (Räsänen) Räsanen Geographic coverage. ITS sequence data for Usnea and U. xanthopoga Nyl. (Rafat et al. 2015), have not been were available from all major geographic regions, but associated with Neuropogon by Walker (1985). Another with strong bias towards Antarctica, Europe, and South three taxa accepted by Walker (1985), U. neuropogonoides America (Fig. 3). Compared to area size, by far most Motyka, U. pseudocapillaris F.J. Walker, and U. taylorii sequences have been generated for Oceania; however, Hook. f., have not yet been sequenced. Thus, the core this figure is misleading for two reasons: (1) all available group of Neuropogon was found to be rather well rep- sequences were from New Zealand only, none for other resented by ITS sequence data (Fig. 2), both in terms of parts of Oceania; and (2) only a few of these sequences species and in the number of terminals, with a total of were actually identified. Overall, best represented was 446 (27.8% of all Usnea ITS accessions). In contrast, the Antarctica, both regarding the amount of sequence data New Zealand sister clade, while also containing a large and the level of identifications. Many sequence data were number of samples (9.2% of all accessions), will require also available for Northern Europe and South America, substantial reassessment of the missing taxonomic iden- whereas North America, the rest of Europe, and Asia tifications to allow for a reliable interpretation of its tax- had a limited amount of data, in the latter case biased onomic composition. towards Japan. Central America including Mexico and After the removal of species in Dolichousnea and the Caribbean, as well as Africa, were strongly under- Eumitria, and separating species in subgenus or sect. represented (Fig. 3). 312 Plant and Fungal Systematics 65(2): 303–357, 2020

We established the following scoring system, taking into account total number of sequences and within-re- gion bias, as well as taxonomic resolution: 3 = well-rep- resented (Antarctica), 2 = moderately well-represented (all of Europe, South America), 1 = limited data (North America, Asia, Oceania), and 0 = poorly represented or no data (Central America, Africa), we computed an overall score of 10 out of 24 maximum, or 41.7%, for geographic coverage (Table 1). Considering the eight regions, we also counted the number of unique taxon-region records, both for taxonomic records (946 total; Table S2) and for ITS sequence data (174 total; Table S1), resulting in a pro- portion of 18.4% (Table 1). We further subdivided the unique taxon-region records by geographic region and taxonomic group (genus or sub- genus) to compute a group-dependent weighted proportion of available sequence data (Fig. 4). North America and Europe were best represented regarding Dolichousnea and subgenus Neuropogon, whereas South America was biased towards Neuropogon and Usnea, Africa towards Figure 5. Topical diversity of studies producing ITS sequences in Usnea s.lat., based in the proportion of accessions pertaining to each topic. Eumitria, and Asia towards Dolichousnea and Eumitria. Dolichousnea was best represented in terms of sequence data vs. recorded species richness in Asia, Europe, and (e.g., North and Central America, Africa, continental and North America, Eumitria best in Asia, Oceania, and tropical Asia) and taxonomic groups in those regions (e.g., Africa, subgenus Neuropogon best in Antarctica, North Eumitria) should be preferentially targeted for further America, Europe, and South America, and Usnea s.str. DNA-based inventories. best in Europe, followed by North and South America and Asia (Fig. 4). Overall, there was strong taxon- and region- Topical diversity. The 1,751 ITS accessions for the genus based bias with the average taxon-region representativity Usnea were generated in 80 studies, 52 published, two in ITS sequence data resulting in 34.4% (Table 1). These corresponding to thesis works, and 26 representing (yet) figures give guidelines for which geographic regions unpublished submissions (Fig. 5). More than half (56%) of the accessions stem from taxonomic studies focus- ing on Usnea s.lat. (Articus et al. 2002; Seymour et al. 2007; Ohmura 2008; Wirtz et al. 2008, 2012; Lumbsch & Wirtz 2011; Saag et al. 2011; Shen et al. 2012; Truong et al. 2013a; Araujo 2016; Nadel 2016; Truong & Clerc 2016; Mark et al. 2016a; Gerlach et al. 2017, 2019a, b; Lagostina et al. 2018; Ohmura & Clerc 2019; Temu et al. 2019). A few additional papers focused on generic delim- itation either within Usnea s.lat. or within Parmeliaceae (Wedin et al. 1999; Ohmura 2002; Articus 2004; Ohmura & Kanda 2004; Wirtz et al. 2006; Arup et al. 2007). Usnea sequences were also generated as outgroups for other taxa (LaGreca 1999; Thell et al. 2002; Schmull et al. 2011; Myllys et al. 2014). Another large block (16%) of ITS sequences for Usnea were generated for studies on mycobiont-photobiont asso- ciations (Buckley et al. 2014; Park et al. 2014, 2015; Rafat et al. 2015), including one accession for a taxonomically diverse culturing survey (McDonald et al. 2013). The mycobiont-photobiont studies focused exclusively on New Zealand (Buckley et al. 2014; Rafat et al. 2015) and Antarctica (Park et al. 2014, 2015). The third major group of accessions (11%) originated from floristic and (meta-)barcoding surveys (Hur et al. 2005; Kim et al. 2006; Kelly et al. 2011; Orock et al. 2012; Schoch et al. 2012; Šoun et al. 2015; Mark et al. 2016b; Jaouen et al. Figure 4. Group-by-region representativity of ITS sequence data for Usnea s.lat., in terms of sequenced vs. reported names per group and 2019; Lendemer et al. 2019; Marthinsen et al. 2019). region. Orange = reported names (set to 100%), purple = sequenced Other diverse topics included lichenicolous fungi names (as proportion), grey = no names reported for group and region. (Biatoropsis) on Usnea hosts (Millanes et al. 2014), R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 313 morphological variation and population genetics (Rol- of the two genera was strongly supported on long stem stad et al. 2013; Cao et al. 2018), genome sequencing branches (94–96%), matching other findings that recov- (as yet unpublished), ecology (Cao et al. 2015; Green- ered both genera as supported clades, but with varying wood et al. 2016; Canini et al. 2020), and monitoring and positions as early diverging lineages within Usnea s.lat. conservation (Ohmura 2011; Dorey et al. 2019). Each (Ohmura 2002; Articus 2004; Ohmura & Kanda 2004; of these accounted for 1–3% of the accessions (Fig. 5). Wirtz et al. 2006). As other markers agree in these pat- Considering that voucher-based fungal ITS sequences are terns, in particular the long stem branches leading to both almost exclusively generated for taxonomic purposes, the groups (see below), we therefore follow Articus (2004) diversity of topics leading to ITS accessions in Usnea and Divakar et al. (2017) to recognize Dolichousnea and s.lat., and consequently the broad application of ITS bar- Eumitria as separate genera. Dolichousnea is thereby coding also for ecological and other studies, was here characterized by annular pseudocyphellae and an amyloid considered excellent (Table 1). central axis, whereas Eumitria generally features a hol- low, often fistulate central axis (Swinscow & Krog 1974; Years published. As mentioned above, the first ITS Ohmura 2001, 2002; Articus 2004; Temu et al. 2019). sequence for Usnea s.lat. was published in 1999, with Given that these features are not always easily discernable a marked increase in the generation of sequence data and are not fully consistent with the recognized genera from 2002 onwards. However, by far, most of the avail- (e.g., Eumitria pectinata complex, Usnea durietzii, see able sequence data were generated in the past decade, below), one could also defend the recognition of a sin- particularly in the first half (Fig. 6). gle genus Usnea, maintaining the subdivision into three subgenera (e.g., Ohmura 2002; Ohmura & Kanda 2004; Wirtz et al. 2006; Truong & Clerc 2013; Truong et al. 2013b; Ohmura & Kashiwadani 2018; Temu et al. 2019). However, we consider a three-genus solution a good com- promise between limited phenotypic distinctiveness and the long history of independent evolution undergone by these three clades (Divakar et al. 2017). The Usnea-Neuropogon clade was supported in our ITS-based tree, albeit at low level (71%). The species originally included in subgenus Neuropogon by Walker (1985) were found in three positions. U. acanthella and U. durietzii formed two separate clades within Usnea s.str., whereas all other species clustered in an unsupported, ter- minal clade. Articus (2004) had recovered Neuropogon as one of four supported clades within Usnea s.lat. based on ITS data, but without backbone resolution, proposing to separate Neuropogon at genus level, although it was not reciprocally monophyletic in relation to Usnea s.str. Ohmura & Kanda (2004), also using ITS data, obtained a more resolved backbone, recognizing three subgenera: Dolichousnea, Eumitria, and Usnea, with Neuropogon nested within the latter, and therefore proposed to treat Neuropogon as a section within subgenus Usnea. Wirtz et al. (2006), again based on ITS, found Neuropogon to be polyphyletic, the two species U. acanthella and Figure 6. Temporal pattern of the generation of ITS sequence data in Usnea s.lat. Purple = published sequences (with date corresponding to U. durietzii, which had not been sampled in earlier studies, publication date), orange = unpublished sequences (with date corre- falling outside the monophyletic core clade. Their topol- sponding to submission date). Grey dots and line denote 3-year moving ogy was congruent with ours. Overall, this would support average. the formal inclusion of neuropogonoid species within Usnea s.str., although Divakar et al. (2017), based on ITS-based phylogeny a six-marker data set (ITS, nuLSU, mtSSU, RPB1, MCM7, TSR1), found the Neuropogon core clade to form an early Overall topology and genus concept. The ITS-based diverging lineage with supported reciprocal monophyly maximum likelihood tree of 1,751 ingroup sequences mir- in relation to Usnea s.str. These authors did not recognize rored the topology obtained in other recent studies (Nadel Neuropogon as a separate genus, because the time of 2016; Divakar et al. 2017; Kraichak et al. 2017), with divergence was too recent under their temporal band- Dolichousnea and Eumitria forming a monophyletic clade ing scheme (see also Kraichak et al. 2017, 2018). While sister to Usnea including Neuropogon, and Neuropogon this may not be considered a sole reason to define taxa s.str. forming a monophyletic clade nested within Usnea (Lücking 2019), here we agree to maintain Neuropogon s.str. (Fig. 7; Fig. S1). The sister group relationship of within Usnea, as the sampling in the study by Divakar Dolichousnea and Eumitria was not supported, but each et al. (2017) was limited and it cannot be assumed that 314 Plant and Fungal Systematics 65(2): 303–357, 2020

reciprocal monophyly of the Neuropogon would be main- Räsänen, U. glabrescens (Nyl. ex Vain.) Vain., U. sub- tained in an expanded taxon set using multiple markers. floridana, and U. wasmuthii Räsänen, whereas sect. Cer- To simplify our taxonomic approach, we consider Neu- atinae contained U. aciculifera Vain., U. ceratina Ach., ropogon a subgenus within Usnea s.str., although the U. dasaea, U. himalayana C. Bab., U. intumescens Asa- precise rank to be applied to this taxon, whether subgenus hina, U. merrillii Motyka, U. mutabilis Stirt., U. nippar- or section, remains open to interpretation, and henceforth, ensis Asahina, U. pangiana Stirt., U. pygmoidea (Asahina) we use the name Neuropogon when referring to subgenus Y. Ohmura, U. rubicunda, and U. rubrotincta. Based on Neuropogon. Species of this group mostly have a patchy a smaller and partly different sampling, Articus (2004) black pigmentation in the cortex and a mostly brown to recovered sect. Usnea by inclusion of U. wasmuthii, but black apothecial disc (Ohmura 2001, 2002; Articus 2004; also found additional taxa clustering in that clade, namely Ohmura & Kanda 2004; Wirtz et al. 2006, 2008, 2012; U. barbata (L.) F.H. Wigg., U. chaetophora Stirt. (now Lumbsch & Wirtz 2011). included in U. dasopoga), U. dasopoga (Ach.) Nyl. (as In our global ITS phylogeny, the Neuropogon clade U. filipendula Stirt.), U. florida, and U. rigida (Ach.) Röhl. was divided into two unsupported subclades: one was ex Zahlbr. (nom. illeg.; = U. intermedia). Sect. Ceratinae almost exclusively from New Zealand (147 out of 149 was only represented by U. ceratina. A third clade was samples), plus one specimen from Antarctica and another formed by U. articulata, U. fragilescens Hav. ex Lynge, from Chile, both forming early diverging lineages in that and U. hirta (L.) Weber, species not included in Ohmura subclade, whereas the other, considered the Neuropogon (2002). Wirtz et al. (2006) resolved sect. Usnea, with the core clade, with 446 terminals, included samples from same species as in Ohmura (2002), Ohmura & Kanda Antarctica, South America, North America (U. lambii, (2004) and Articus (2004), as well-supported clade. How- U. sphacelata), and Europe (U. sphacelata), but not ever, sect. Ceratinae was not recovered, forming an unre- a single one from New Zealand. This topology was also solved paraphyletic residual of early diverging lineages recovered in other studies (Wirtz et al. 2006), but the within subgenus Usnea. In our own global ITS-based impressive, albeit unsupported division of a New Zealand phylogeny, neither section was recovered, although one vs. a non-New Zealand subclade had apparently not been large, unsupported subclade largely corresponded to sect. noticed before. Rafat et al. (2015) obtained a different Usnea. Overall, Usnea s.str. was divided into numerous, topology, with the Neuropogon core clade nested within mostly unsupported subclades with smaller, supported the New Zealand clade, but this topology was not sup- clades within, which are outlined and discussed in detail ported and likely biased by the unusual outgroup selec- below. tion (Ramalina; Ramalinaceae) that may have resulted in alignment artifacts. The division of Neuropogon into two Comparison between ITS and other markers. The large, geographically separated clades was also remark- nuclear large subunit of the ribosomal DNA (nuLSU) was able because one species in the Neuropogon core clade, available for three of the four groups (Eumitria, subgenus U. acromelana, was originally described from New Zea- Neuropogon, Usnea s.str.). As with ITS, Eumitria (data land, and two further species, U. antarctica and U. sphace- mostly from Temu et al. 2019) was supported as early lata, were reported from there (Walker 1985; Galloway diverging lineage, whereas Neuropogon (data mostly from 2007). Yet, although both New Zealand and Antarctica Lee et al. 2008) was nested within Usnea s.str. (Fig. S2). have been extensively sampled for molecular data (Wirtz The Usnea s.str. backbone (data mostly from Schoch et al. et al. 2006, 2008, 2012; Seymour et al. 2007; Buckley 2012 and Millanes et al. 2014) was largely unresolved et al. 2014; Park et al. 2015; Rafat et al. 2015; Cao et al. with even less structure than in the ITS. A substantial 2015, 2018), these three species have not been demon- number of nuLSU sequences exhibited quality issues, strated to be present in New Zealand based on molecular particularly the occurrence of random single and contig- data, but have been frequently identified in sequenced uous gaps (File S2). samples from Antarctica and/or southern South Amer- The second ribosomal DNA marker, the intergenic ica (Table S1). If this pattern is confirmed, it provides spacer (IGS), included sequences of Dolichousnea, Neu- a taxonomic challenge, as the numerous samples from ropogon, and Usnea s.str. As with ITS, Dolichousnea Argentina identified asU. acromelana (Wirtz et al. 2006, (data from Rolstad et al. 2013) was supported as an early 2012; Seymour et al. 2007) may not belong to that species diverging clade and Neuropogon (data from Lumbsch (see also below). On the other hand, given the confirmed & Wirtz 2011 and Wirtz et al. 2012) was nested within wide distribution of U. sphacelata with sequence data Usnea s.str. (Fig. S3). In contrast to ITS and nuLSU, IGS from Antarctica, throughout South and North America offered a surprisingly high level of supported resolution and northern Europe (Table S1, Fig. S1), its occurrence in the Usnea s.str. backbone: while U. ceratina was sup- in New Zealand appears likely. ported as an early diverging clade, the remainder of Usnea The backbone of the paraphyletic Usnea s.str. grade s.str. formed a supported sister group relationship with was not supported in the ITS, although some larger sub- Neuropogon, and sect. Usnea was resolved as a large, clades received support. Ohmura (2002) and Ohmura well-supported clade. Terminal resolution also surpassed & Kanda (2004) proposed to divide Usnea s.str. into two that found in ITS for the same clades and taxa (see details sections, sect. Usnea and sect. Ceratinae, neither of the below). While the observed backbone support for sect. two supported in that work. In the limited sampling of that Usnea may in part be due to most of the data focusing study, sect. Usnea included U. fulvoreagens (Räsänen) on this particular group (Mark et al. 2016a), it appears R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 315 that IGS surpasses ITS in performance both at deep and seen with ITS and RPB1 for these species (see more terminal nodes in this genus. details below). In addition to the two rDNA markers, four pro- Overall, the ITS-based topology was largely congru- tein-coding markers were analyzed. Of these, β-tubulin ent with that of IGS and RPB1 in the delimitation of (TUB2) is a classic marker used in many fungal studies, terminal clades, while backbone resolution and support but is known to cause potential problems with paralogs were superior in the IGS and RPB1. With few exceptions (Landvik et al. 2001; Zhao et al. 2014). The available data, detailed below, we did not observe aberrant patterns in the mostly covering Usnea s.str. and few for Dolichousnea ITS topology, these being more frequent in TUB2, RPB2, and Neuropogon, recovered Dolichousnea (data from Arti- and particularly MCM7. We therefore consider the ITS cus 2004) as strongly supported early diverging lineage a suitable marker for a first approach to molecular delim- and Neuropogon (data from Articus 2004) nested within itation and identification ofUsnea species with additional Usnea s.str., albeit not in a terminal position, as in the terminal (and backbone) resolution provided by IGS and three ribosomal markers. Usnea s.str. (data mostly from RPB1. Regarding generic delimitation within Usnea s.lat., Saag et al. 2011 and Mark et al. 2016a) was divided all markers invariably resolved Dolichousnea and Eumi- into five strongly supported clades, with Neuropogon tria as early diverging lineages, although only one marker situated in between (Fig. S4). Notably, sect. Usnea was besides ITS (MCM7) had data for both, lending strong recovered, while sect. Ceratinae sensu Ohmura (2002) support to their separation at genus level (see above). All and Ohmura & Kanda (2004) was not, with U. ceratina markers except RPB1 gave Neuropogon as nested within clearly separated from the remainder of this group. There Usnea s.str. The reciprocal monophyly of the two groups was no indication of artifactual topologies due to potential in the RPB1 tree was not an artifact of taxon sampling and paralogs in TUB2 in the backbone, but terminal positions so is presumed to be genuine to this marker. Inclusion of particularly in sect. Usnea showed some aberrant place- RPB1 was therefore likely responsible for the reciprocal ments (see below for details) and the terminal resolution monophyly between Usnea s.str. and Neuropogon in the was lower than in the ITS. multimarker study by Divakar et al. (2017). Data for RPB1 were available for Eumitria, Neoro- Given the observed patterns, we interpret the aberrant pogon and Usnea s.str., again resolving Eumitria (data topologies for particular taxa in TUB2, RPB2, and MCM7, from Temu et al. 2019) as supported, early diverging as potential paralogs. An alternative explanation for topo- clade (Fig. S5). For Neuropogon (data from Lumbsch logical conflict between markers would be hybridization & Wirtz 2011 and Wirtz et al. 2012), backbone solution and introgression through complex reticulate evolution in was overall lower than for ITS, and reciprocal monophyly the recent past (Abbott et al. 2016; Widhelm et al. 2019). of the largely New Zealand-based clade was not recov- However, in such cases, we would expect the aberrant ered, as this clade was nested within the Neuropogon copies at least in part to correspond to potential ‘par- core clade. Terminal resolution in RPB1 was lower than ent’ lineages (e.g., Boluda et al. 2019), which we did in ITS. Within Neuropogon, we observed numerous aber- not observe here (see details below). Rather, the copies rant terminal placements with regard to species otherwise appear to explore new phylospace, which would be in well-delimited in the ITS and IGS (see details below). accordance with gene duplication. Alternatively, one could Notably, Neuropogon and Usnea s.str. were recovered in imagine that these copies correspond to not yet sampled supported reciprocal monophyly, with Usnea s.str. includ- or extinct lineages (Bradshaw et al. 2020); however, we ing all major groupings (data mostly from Schoch et al. do not consider this likely as the best documented cases 2012, Wirtz et al. 2012, and Mark et al. 2016a). Both sect. were detected in well-sampled species complexes. Gene Ceratinae and sect. Usnea were recovered in supported duplication does not necessarily mean that orthologs and clades, together with some smaller groups. Only limited paralogs are actually present in the genome, because para- data for Neuropogon (data from Spatafora et al. 2006) logs may eventually replace orthologs as functional genes, and Usnea s.str. (data largely from Mark et al. 2016a) leading to complex situations of deviating single-copy were available for RPB2, and backbone resolution was orthologs and paralogs in different lineages of a species much lower than for RPB1. There was also indication (Linder & Rieseberg 2004). of paralogs, such as the split of U. cavernosa Tuck. into Potential paralogs should not be confused with the two separate clades, compared to single, homogeneous phenomenon of incomplete lineage sorting. The latter clades in ITS and RPB1 (Fig. S6; see more details below). refers to instances where, within a given clade, different Data for MCM7 were generated in a number of studies markers diverge at different speed and even in different (e.g., Truong et al. 2013a; Mark et al. 2016a; Gerlach patterns, so there might be a lack of congruence between et al. 2017, 2019a). Dolichousnea and Eumitria were markers when it comes to delimit lineages within a clade. resolved as early diverging lineages in a supported sister However, all markers should still delimit the same larger group relationship (Fig. S7), whereas data for Neuropogon clade, i.e., there should be a deeper node of congruent were not available. Usnea s.str. formed a highly dissected, ancestry. In contrast, non-homologous copies will cluster unsupported backbone and there was indication of paralog in separate clades, typically with counterparts representing formation, as for instance in the split of U. parafloridana the homologous copies of related clades after gene dupli- K. Mark, S. Will-Wolf & T. Randlane [= U. praetervisa cation occurred. Some examples are illustrated below. (Asahina) P. Clerc; see below] and U. wasmuthii into Given the potential presence of paralogs in TUB2, RPB2 separate, unrelated clades, quite different from patterns and MCM7, these markers are not recommended to assess 316 Plant and Fungal Systematics 65(2): 303–357, 2020

species delimitation in Usnea s.lat. The best markers for Ohmura & Kashiwadani 2018; Bungartz et al. 2018; Ess- this purpose appear to be IGS and RPB1 in addition to linger 2019; Temu et al. 2019; Lücking et al. 2020c). In ITS. We have not provided a multimarker phylogeny, as our analysis, the first subclade included specimens from this was not the scope of this study and unfortunately, Africa and Asia/Oceania, the latter clustering in a sup- the different markers were largely generated in different ported subclade. The second clade exclusively comprised studies for different purposes and taxa, and so there was African samples. The presence of geographic signal and overall little overlap between samples. However, broader supported phylogenetic structure within a single area, sequencing of the IGS and RPB1 as a routine approach for together with the notion that most of its geographic range the phylogeny of Usnea s.lat. seems a promising approach, has not yet been sampled for molecular data, indicates that including for multispecies coalescence approaches as in E. baileyi represents a complex of several species. This Mark et al. (2016a) and Gerlach et al. (2019a). is also underlined by the existence of unique phenotypes, In the following, the various clades and groups are such as an unusual specimen from the Colombian Amazon discussed in detail in terms of species delimitation and the with a brightly orange inner axis (Fig. 1A). The latter may usefulness of ITS-based DNA barcoding, also including morphologically correspond to E. baileyi f. endocrocea comparison between ITS and other markers. Zahlbr. described from Taiwan (Zahlbruckner 1933: 60), although it is unclear whether the author referred to the ITS-based species delimitation and DNA barcoding medulla in the proper sense or the fistulate inner axis with ‘Medulla ... crocea’. The secondary chemistry of Eumitria. This strongly supported genus, which is for- E. baileyi s.lat. appears to be rather uniform, containing mally accepted here following Divakar et al. (2017), norstictic plus mostly salazinic and sometimes diffrac- formed four supported subclades based on the ITS (Fig. 7; tatic acids, as well as eumitrins throughout its reported Fig. S1), corresponding to one as yet unnamed species range (Ohmura 2001; Truong & Clerc 2013; Nadel 2016; composed of unpublished sequences from China, depos- Temu et al. 2019). Some aberrant chemotypes have been ited by S. Guo et al. in 2019 and L. Han et al. in 2020, reported, such as thamnolic acid, corresponding to the as well as E. firmula, E. pectinata s.lat., and E. baileyi type of Usnea eizanensis Asahina (Ohmura 2001), and s.lat. (Ohmura 2002; Nadel 2016; Jaouen et al. 2019; protocetraric acid in certain varieties (Swinscow & Krog Temu et al. 2019). The E. pectinata clade contained one 1974, 1988). These latter are candidates possibly repre- sample (MN080241) deposited under the name E. baileyi, senting distinct species, although these chemotypes do but correctly labeled E. pectinata in the corresponding not appear to have been sequenced yet. publication (Temu et al. 2019). The unnamed species Regarding the other markers for which data were from China was internally homogeneous. The supported available for Eumitria baileyi (nuLSU and RPB1, MCM7) subclade corresponded to two consistent substitutions, all from Tanzania; Temu et al. 2019), the three available resulting in a similarity of 99.6% with the other three RPB1 sequences (SGT110, SGT120, SGT156) were uni- terminals. Eumitria firmula, based on sequenced mate- form (Fig. S5), although in the ITS tree the corresponding rial from São Tomé and Príncipe (Nadel 2016) was also samples appeared in the two different subclades, indicat- homogeneous, with minor internal variation (File S1). ing lack of resolution in the RPB1. With nuLSU, E. baileyi The Eumitria baileyi clade formed two rather well-sup- also formed two supported clades, but the clades were ported (88% and 89%), reciprocally monophyletic sub- partially in supported conflict with those formed with ITS clades, indicating the presence of at least two species (Fig. S2). Thus, specimens SGT63 and SGT65 formed (Fig. 7; Fig. S1). This was further supported by the iden- one of the two subclades in the nuLSU, but were found in tity matrix (JQ673442 excluded due to reduced length), the two separate subclades in the ITS. Likewise, the other which resulted in values below 98.5% for all cross-com- subclade in the nuLSU contained eight samples (SGT110, parisons between the two clades (Fig. 8). In addition, the SGT112, SGT118, SGT119, SGT120, SGT122, SGT156, early diverging singleton sequence (MW267138) in the SGT157), which were found in the ITS either in the first first subclade was revealed as an additional candidate (SGT110, SGT112, SGT119) or the second subclade species based on the identity matrix, whereas a strongly (SGT118, SGT120, SGT122, SGT156, SGT157). Few supported (97%) subclade in the second subclade, com- data were available for MCM7 (from Temu et al. 2019), prising five terminals, exhibited partial similarity overlap with one supported conflict for sample SGT65 (Fig. S7). with the remainder of the second subclade (Fig. 8) and The Eumitria pectinata clade exhibited internal struc- could at best be considered a candidate infraspecies. ture, but no distinct division into reciprocally monophy- Eumitria baileyi was originally described from Aus- letic clades. The backbone was structured into various tralia (Stirton 1881), but has since been reported from all singletons and several smaller, partly supported subclades, tropical and subtropical regions (Fig. 9; Swinscow & Krog including one subclade with the three samples from South- 1988; Mies 1989; Elix & McCarthy 1998, 2008; Stevens east Asia (Ohmura 2002), but without separation in the 1999; Aptroot & Seaward 1999; Ohmura 2001, 2002; identity matrix at the 98.5% level (Fig. 10). However, Galloway 2007; Mercado-Díaz 2009; Schumm & Aptroot three nested, well-supported subclades on longer branches 2010; Singh & Sinha 2010; Gumboski & Eliasaro 2011; appeared as candidate species in the identity matrix, Orock et al. 2012; Truong & Clerc 2013; Aptroot 2016; including samples from São Tomé and Príncipe and/or Herrera-Campos 2016; Nadel 2016; Sipman & Aguirre-C. Tanzania (Nadel 2016; Temu et al. 2019). The largely 2016; Rodríguez-Flakus et al. 2016; Galinato et al. 2017; unresolved backbone had a variable chemistry including R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 317

Usnea_sp

Usnea_spec-uncultured_KM369410_New-Zealand_ID-FNV13

Usn

Usnea_spec-uncultured_K Usnea_spec-unculture Usnea_spec-unculture Us Us Usnea_ Usnea_spec-uncultu Usnea_spec-uncultured_KM369342_New-Zeal U Usnea_spec-uncultured_KM369248_

Usn

Usnea_spec-uncultu

Usnea_spec-unculture Us Usnea_spec-uncultured_KM369179_Ne snea e U nea_s Usnea_spec-u n Usn U a_spe

s ea_sp

Usnea_spec-un s ne ne ea 019a Usnea_spec-un nea_sp Usnea_spec-uncu Usnea_spec-unc 911 Usnea_spec-uncultu Usnea_ushuaiensis_

Usnea_s 4 Usnea_ushuaiensis_EF492168_ Usn

Usnea_ushuaiensis_EF4 Usnea_spec-uncultured Usnea_us 74021a Usnea_ushuaiensis_ ea _spec Usnea_spe a_spec e

a_s _ s 3 1

c-unc

Usnea_spec-unc pec a Usnea sp pec U 17

c-un Usnea_spec-uncultured_KM369188_New-Zealand_ID-F526 _spec-uncultured_KM3 N0067

UN1914 Usnea_spec-unc ec-uncu Usnea_spec UN1 Usnea_spec-uncultured_KM369405_New-Zealand_ID-FNV8 Usnea_spec- sn

e -1 P

pec-u e 266 Usnea_spe 2661 F-1174022a

a_

-uncul S

c- - Usnea_spec- 3b

-F ea_spec- N SD Usnea_spec-uncultured_KM369308_New-Zealand_ID-F619 e -uncultu U uncultured_KM3

c

u -1- Usnea_spec-uncultured_KM369309_New-Zealand_ID-F620 c-uncu u ushuaie -unc CN Usnea_spec-uncultured_KM369194_New-Zealand_ID-FAK4 sn Usnea_spec-uncultured_KM369407_New-Zealand_ID-FNV10Usnea_spec-uncultured_KM369303_New-Zealand_ID-F614 -2 u Usnea_spec-uncultured_KM36934 _spe US pec lture 174023 nc Usne SC

l Usnea_spec-uncultured_KM369330_New-Zealand_ID-FLEW 1 ncultured_KM36 tu Usnea_spec_JX144650 hu Usnea_spec-uncultured_KM36 ncul ea ID-U

Usnea_spec-uncultured_KM369403_New-Zealand_ID-FNV5 Feuerer-sn _ID-USSCN1951 u ltur c-uncul D-192-21

tured_KM369305_New-Zeala - -uncultured_KM36 ulture red Usnea_spec-uncultured_KM369239_New-Zealand_ID-F210 D-221- Usnea_spec-uncultured_KM369406_New-Zealand_ID-FNV9 _ID- a lture _sp ai 1 1 -1174024a D-221 c-uncu d -2 c

or_ID-63-F-1174020a 66-2 1-2 r F D-USS a ltured_KM36 ID-266 _K cult ensis_EF492182_ ultur ture red tina_ID-191-3-F-1174018a ed_KM ina_ a_ID-222-10 - Usnea_spec-uncultured_KM369256_New-Zealand_ID-F545 ed_KM369408_New-Zealand_ID-FNV11 ID-US 1 uncu tina_ID-USDUN1912 a_I _KM _s _ nsis_ 15 -un d_KM re entina_ID-179-3 Us c-uncult ultured_KM36940 d_ 29- 2 _I 4

ad

ec e_ID Usnea_spec-uncultured_KM369261_New-Zealand_ID-F550 unc n in -21 2 -

l lture ador en 216- Usnea_spe d C2 u na_I Usnea_s t ina_ID-227-1 d_KM369243_N p _ 7 Usne Usnea_spec-uncultur d d_KM r u KM369306 Usnea_spec-uncultured_KM369371_New-Zealand_ID-FNO16 ne ntina_I Us u _KM36 ti uncultured_KM369250_Ne d_KM3 _JX 2 Usnea_subcapillaris_DQ235478_New-Zealand_ c D- ica_ID-269 -1 tured KM3693 ed_K ec_JX144647_New-Zealan 2 _K eru_ nt red_ cu I M369 ntina_ID-218-2-F-1 t Usnea_ l en -208-19-F- u 3 9 u r rgentina_ID-USD ltur rgent 08-1 ulture ltured_KM369242_New-Zealand_ID-F217ture EF4 3 4 a e 67-2

EF492210_Arge 692 2500_New-Zea en Usnea_spec-uncultured_KM369196_New-Zealand_ID-FAK6 l E nea_spec_KR091 l _Ecu 67 369401_New-Zealand_ID-FNV2 A d_KM369 22- 22 6 tured_ 3 tured_KM36919 a_ Usnea_spec-unc _spec-uncult d_ M3 _P 1-2 1 g D-178-1 14 -208-2 F49 tica_ID- - rge 2 69304_New-Zealand_ID-F615 tica_ID-166-3 ID-22 Us 6 5_Pe a r n 5 e 3_Argentin D- Usnea_ciliata_DQ235 Usnea_spec-uncultured_KM369200_New-Zealand_ID-FCILI ured_KM3691 369409_New-Z 34_Chi 250-3 KM369323 _KM3 _ ID-2 F9 _spec-uncultured_KM3 9301_New-Zealand_ID-F612 _Arge d _KM36 M369193_New-Zealand_ID-FAK3KM 9 d_ 0 rc - Usnea_ciliata_DQ235479_ 8 D-1 464 - 436_New-Zea 69326 9 rc _Argentin 2211_Arg _Arge a 9310_Ne 17_Antarctica_ID-AFTOL-ID-816 6 2 684 _I nea _KM3692 688_E D-F1104 Usnea_spec_JX144649_New-Zealand_ID-F207 3 p 69 _ID d 2167_Argentina_ID-258-1 ina_ID Usnea_spec-unculture 22 680_A Usnea_spec_ 918 ca_I _I 93 146_unk ID _ID- subc 9302_N _N ec-u KM36942 40_ Us _K _I 369430_New-Zealand_ID-F35 arctica_ID-2 06 ca na_ID -3d c-uncultu KM 201 4_Ne

Usnea_spec-uncultured_KM369231_New-Zealand_ID-FPhees _ 13_Ar na_ID-227-7 a 165 rgenti 0 ntin ti _c 314 i 489_A 3a 69427_New-Zealand_ID-F25 ew-Z U New-Zealand_ID-F617 nea_subcapi Usnea_ciliata_JF283511_New-Zealand_ID-USCINN5 M36 0_New-Ze 66 7_ 5488_A ctica_ID-167-6 9 N nti 2 _New-Zeal New 1 9425_New Ant nt 1- snea_ iliata_DQ235475_New-Zealand_ ncultured_KM369366_New-Zealand_I 369173_New-Zealand_ID-F410 Q314 api 402_ _Ne A 0_Anta 02 Ar ew- 492150 rge 2 Ne 23 arctica 492215_Argentina_ID-218-1 Argent Usnea_c tar ge D-42 4_N _New e w-Z _JQ3146 4-1 rgentina_ID-258-2 ge Usnea_spec-uncultured_KM369311_New-Zealand_I w 917 _New-Ze ntarc w 19 _ g ew-Zeala gentina_ID-179-7 r Usnea_spec_JX144646_New-Zealand_ID-F204 ealand_ Usnea_s lla u 45_New w a_J

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7_Brazil_ID-2 Usnea_dasaea_JN086283_Portugal_ID-das-01 37_Bra a D-11 Usnea_articulata_MN0571_SaoTome-PrincipeID-MN0571 hispid z Usnea_dasaea_JQ837306_Ecuador_ID-81u il_ID-118BRID Usnea_articulata_MN0211_SaoTome-PrincipeID-MN0211 ta-3_MF6698 -1 2B Usnea da 1 Usnea_articulata_MN0551_SaoTome-PrincipeID-MN0551 ella zil_ID-115 4BR R Us sae s ance pidella_Mella_M Usnea_perhispidella_MF66nea_ _MF669904_Brazil_ID-178BR _spec-6_ a_ 9B Usnea_perhispidella_MF669905_Brazil_ID-179BR Usnea_spec-A-Nadel_MN0553_SaoTome-PrincipeID-MN0553 JN _ID-PC2FR R Usnea_perhispidella_MF669898_Brazil_ID-82BRspec_HQ671307_unknown_ID-Hur-TW090007 Usnea_spec-A-Nadel_MN0552_SaoTome-PrincipeID-MN0552 0 F669879_ 63_Brazi BR 86284_Portugal_ID- Usnea_aciculife F6699 Usnea_spec-A-Nadel_MN0253_SaoTome-PrincipeID-MN0253 razil_ID-sp-26BR Us MF669 Usnea_spec-A-Nadel_MN0116_SaoTome-PrincipeID-MN0116 Usnea_aciculifera_MT259216_China_ID-U3-2018ne Usnea_spec-A-Nadel_MN01 Usnea_aciculifera_MT259217_China_ID-U4-2018120540a 02_ Br l_ 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Usnea_spec_FJ494952_Taiwan d_ a_ iana_ J ID-4913 79B Usnea_spec-4_MF669 Q8373 Usnea_ do MF6 alan Usnea_strigosa_MH3 R d _ID-109 -36 Usnea_strigosa_AF112990_USA_unknown MF669895 KM369298_New-Zealan Usnea_strigosa_MN038162_USA_ID-EAT6069 ge 69893_Brazil_ID-84BR Usnea_spec ance_ID-130 D 3 24 r Usnea_endochrysea_MH310883_USA_ID-NY1506 _M _ 7 i_MF669886_Brazil_ID-54BR 7323_Boli eineri-97BR 8BR Usnea_spec-uncultured_KM369300_New-Zealand_ID-F611 Usnea_e JQ ew-Ze 125 s _P Usnea_spec-uncultured_KM369183_New-Zealand_ID-F518 - 6 Usnea_strigos p F6 ID-4924 dor_I Usnea_ e 837314_Peru_I eru Usnea_spec-uncultured_KM3 a 69923_Brazil_ID-133BR c-uncultured_KM369351_New-Zea 7_N Usne c-4_MF669939_Brazil_ID-sp4-135BR Usnea_spec-uncultured_KM369299_New-Zealand_ID-F610 Usnea_strigosa_KC592273_USA_ID-CBS-132745 _ _I Usnea_mut Br D-80 Usnea_nidifica_FJ494933_Taiwan_ID-Li207 Usnea_mutabilis_AB051651_Japan_ID-Ohmura-4493Andoch v Usnea_spec_FJ494947_Taiwan_ID-Li328 Usnea_mutabilis a a ia_ID-34 Usnea_nidifica_FJ494941_Taiwan_ID-Li190 _mu strigos Usnea_spec-uncultured_KM369414_New-Zealand_ID-FRob2 Usnea_spec_FJ494948_Taiwan_ID-Li336 Usnea_mu zil_ Usnea_spec-uncultured_KM369417_New-Zealand_ID-FTAD1 U Usnea_mutabilis_JN086321_Portugal_ID-mut-03 93 Usnea_spec-uncultured_KM369418_New-Zealand_ID-FTAD2 sn I Usnea_spe Usnea_mutabilis_MH310886_USA_ID-NY3905 t r D-102 a 10890_USA_ 8_Br Usnea_spec-uncultured_KM369270_New-Zealand_ID-F575 Usnea_mutabilis_JN086319_Portugal_ID-mut-01e a_HQ y b D Usnea_aff-rubicunda_MF669912_Brazil_ID-184BRa a se Usnea_spec-uncultured_KM369297_New-Zealand_ID-F608 56BR ilis_JN0863 nea_spec-uncultured_ Usnea_crocata_JQ837303_Peru_ID-35 - Usnea_rubicunda_AB244613_USA_ID-Lendemer-562_ abi _MH310889_USA_ID-NY1504 2 - BR a_MH310884_USA_I _Braz 16 s _JQ837326_Ecu Usnea_rub mutabilis_MH310887_USA_ID-NY3908 azil_ BR Usnea_spec-uncultured_KM369329_New-Zealand_ID-FLeesUsnea_himalayana_FJ494934_Taiwan_ID-Li Usnea_rubicunda_E _I U tabilis_ lis_AB 6 Usnea_himalayana_FJ494935_Taiwan_ID-Li348Usnea_crocata_JQ837304_Bolivia_ID-59 Usnea 506 D Usnea_pennsylvanica_K 8967_Galapagos_ID-12 -Li ID-66 Usnea_rubicund I rnuta Q837311_Galapagos_ID 69807_Brazil_ID-16BR ID-sp5 D-sp4-134BR i _ Usnea_ l_ID-5246 _ 87 Usnea_rubicunda_FJ494949_Tai 252 F6 MH31088 Usnea_rubicunda_AB368487_Japan_ID-TNS-YO5826B 051650_Japan I 5_J _rubicu M _U D-NY173 Usnea_himalayana_AB051640_Malaysia_ID-Ohmura-4374 KP66 M Usnea_rubicunda_AB051659_Japan_ID-Ohmura-3114 2 Usnea_subcornuta_JQ837325_F Usnea_rub icu H310885_USA_ 0_Portugal_ID-mut-02SA_ID-AFTOL-ID-5 Usnea_crocata_MF669877_Brazil_ID-49BR0264a_SaoTome-PrincipeID-MN0264aec- Usnea_rubi N 595 9 Usnea_rubicunda_MF669908_Braz Usnea_rubicunda_KY021924_Brazil_ID-4891 rub nd_ID-FRob3 nda_EAC4447 Usnea_spec-uncultured_KM369226_New-Zealand_ID-FNIDI Usnea_rubicunda_MF669909_Brazil_ID Usnea_subcornuta_EAC4888_SpainID-EAC4888 Usn D-F F59 nda_EA Usnea_subcornuta_JQ837327_Ecuador_ID-45snea_subcoa_M Usnea_rubicunda_FR799095_England_I icunda_AB244 8_ Usnea_spec-uncultured_KM36931 Usnea_subcornuta_JN086325_Portugal_ID-subc-01 1 U _I Usnea_rubi nea_sp nd_ID-FN10 Usnea_rubicunda_MF USA_ID-NY4159 d D Usnea_rubicunda_JQ837374 ea_rub F669933_Brazil R2 -NY1549 Us Usnea_rubicunda_JN086322_Engl i a AC461 Usnea_aff-flammea_MN0579_SaoTome-PrincipeID-MN0579 Usn cunda_JN086324_Portugal_ID-rub-04 Usnea_clerciana_KP668966_Galapagos_ID-124 A _ _ID-Ohmura 5 MF669873_Brazil_ Usne c liensis-2_MF669808_Brazil_ID-204BR JQ837319_Madeira_ID-75 nea_clerciana_ Usnea_rubicunda_JQ837315_Gal unda_ C4887_SpainID-E Usnea_clerciana_KP668965_Galapagos_ID-127 Usnea_erinac si _New-Zeala I -FM Usnea_rubicun e D-JED261 Us aff-brasiliensis-2_ Usn i 5 a_rubicunda_JN0 DEN Usnea_rubicunda_ c _SpainI a_ru Y 3 F Usnea_rubicunda_JN943518_Scotland unda_JN943547_England_ID-EDNA09-02083 _PortugalID-EAC4613 Usne cunda 114 ornuta_ 941 d_ID U A nea_ ea 612_USA_ID- Usnea_merrillii_AB051649_Japan_ID-Ohmura-4397 Usnea_r flammea_M Usnea_rubicunda_FRsnea B244611_Japan_I bicun Usnea_sanguine an Usnea_spec_H _rubicunda_JN 892_USA_ID-Lendemer-sn Us Usn -4 Usnea_ a D-EAC4 Usnea_aff-brasiliensis-2_MF669812_Brazil_ID-13BR Usn 407 _rubi _ U Usnea_aff-brasiliensis-2_MF669806_Brazil_ID-15BR _ KY021923_Bra Usnea_aspera_MF669805_Brazil_ID-140BR _New-Zealand_ID-FNO8 Zeal U Usnea_rubrotincta_LC479125_Japan_ID-TNS-YO10300s ea_spec-u 529 rubicun da_ 5 Usnea_spec-uncultured_KM369291_New-Zealand_ID-F598snea_r ea_J Usnea_aff-braUsnea_aff-cornuta_MF669876_Brazil_ID-sp5-193BRUsnea_aff-cornuta_MF669829_Brazil_ID-32BR n ed_KM36 Us ea ubi wa Usnea_aff-cornuta_MF669875_Brazil_ID-sp5-172BR 9260_New-Zealand_ID-F549 Usnea_spec-uncultured ea_r Usnea_spec-uncultured_KM369350_New-Z c 669 AC48 7 d Usnea_aff-cornuta_MF669874_Brazil_ID-sp5-132BR Usnea_spec-unc Usnea_aff-c ltured_KM369221_New-Zealand_ID-FMAR6 _rub JQ8 n_ID 36 New- Usnea_spec-uncultured_KM369374_New-Zeal nea_sp s unda_ a_JQ83 Usnea_sub cun 4 Usnea_spec-uncul pec-uncultu Q83 U 47 Usnea_subflammea_MF669932_Brazil_ID-65BR 911_Canary-Is L 34 Us ubrotincta 86 Usnea_spec endemer-478 ncu KM sn ub da_ Usn 37318_Bolivia_ID- 87 217_ U icu d F -Li325 U nea_spec-un 7307 323_En i Usne l_ID-60 sne e roti Q671306 a R799098_No 021 Usnea_spec-uncultured_KM369187_New- ncul FR 94 snea_spec-uncultu _Galapagos Usnea_spec- a_s _FR79 D ec nda_ JN943517_Scotland_ID-EDNA09-02358 ew-Zealand_ID-F593 Usnea_spec ea_spec 731 Usnea_spec Usnea_spec- 3516 -Ohmura-4864 N Usnea_spec-u 799097_Scotland_ID-EDNA09-02358 z a_spec- ncta_DQ232 M369 Usnea_spec-unculture -unculture _ il_ID-4890 Usnea p tured_KM369396_New- Usnea_s a_ 6_Bolivia_IDG Us -6 ec-un 799 Usnea_spec-uncultured_ KJ406275_New-Zealand_ID-AM295 g B D-EAC4491 Usnea_spec-uncultur alap Usnea_subdasaea_JQ837328_Peru_ID-8 _AB051660_Japan_ID-Ohmura a spec-uncultured_KM369343_New-Zea red_KM36 9 _Northe 1BR Usnea_subflammea_MF669935_Canary-Islands_ID-239 land_ID-rub-02 R Usne n nd_ID-r Usnea_spec-uncultured_KM369195_New-Zealand_ID-FAK5 Us _unknown_ID-Hu 096 D inI U 094_Scotland_ID-EDNA09-02357 a Usnea_spec-uncultured_KM369345 ea_spec-unculture - -uncultured_ Usnea_spec -E 6 Usnea_spec-uncultured_KM369357_New-Zealand_ID-FN23 -uncu uncultured_KM369218_New-Zealand_ID-FMAR3 pagos_ID-17 l s Us ul R _s agos_ID-104 and Us nea_spec-uncu Usnea_spec-u Usnea_spec-uncultured_KM369237_New-Z cultu n u Usnea_spec-uncultur U _Wales_ID-EDNA10-0074 DNA Usnea_spec-uncultured_KM369321_New-Zealand_ID-FAK2 Usne c rthern-I _I 92B ture Usnea_spec-uncultured_KM369189_New-Zealand_ID-F528 Usnea_spec-uncultured Usnea_spec-uncultured_KM36 ea_s ncultured_K Usnea_spec-u sn n a pec-uncultured_KM369400_New-Zealand_I ultured Us nea_spec-unc pe t Usnea_s - D-18 Usnea_spec-unculture u d m_ID-AM110 Usnea_spe _ ured_KM369253 s_ ea_spe - 24B Usnea_s uncultur Usnea_spec-uncultured_KM369176_New-Zealand_ID-F502 Usnea_spec-uncultured_KM3 r Usnea_spec-u uncultured_KM369238_New-Zealand_ID-FWH2ncultured_ _K 40 ub-01 Usnea_ 66 Usnea_spec-uncultured_KM369391_New-Zealand_ID-FNO41 _spec 1BR s Usnea_ e unc ltur n 98BR 299 Usnea_esperantiana_E _K Us ne c- d_ 09 Usnea_spec-uncultured_KM369347_New-Zeala 97BR Usne red_KM369397_New-Zealand_ID-FNO48 YO737 Usnea_esperantia ID-235TEN Usnea_e pec-u EN Usnea_esperantiana_FR799089_England_ID-EDNA09-02076 a_ - Usn 9380 Usnea_esperantiana_FR79 a_sp - Usnea_esperantiana_JN94351 - Usnea_spec-uncultured_KM369349_New-Zealand_ID-FN13 Us pe nc Usnea_es 4_South-Korea_ID-Hur Ireland_ID-EDNA09-02359 ngland_ID-EDNA09-0112 Usnea_glabrata_FJ Us M -29 Usnea_aff-glabrata_MF669864_Fr uncul Usnea_ 38 M369190_New-Zealand_ID-F Us Usnea_subflammea_MF669934_Canary-Islands_ID-238TEN -1 U Usnea_spec-uncultured_KM369288_New-Zealan Usnea_spec-uncultured_KM369362_New-Zealand_ID-FNO5 Usnea_spec-uncultured_KM369364_New-Zealand_ID-FNO7 Usn Us KM369372_New-Zealand_I -02083 a_sp M3693 _ID-EDNA0 n spec-uncultured_KM369385_New-Zealand_ID-FNO33 ed_K re 3T D ul K l_ID-23BR 44 re 369295_New-Zealand_ID-F605 ID-1 -AM snea_halei_MG c-uncult I e ultured_KM369320_New-Zealand_ID-F5425 nea_esperan _KM36 Usnea_spec-uncultured_KM369292_New-Zealand_ID- 0BR _ 1BR nea_spec_MT2618 nea_

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_JN086293_England_ID-flam-01 ed_KM36 na tugalID-EAC4616 36 669860_ a_ n _co 20 2 e ID-E az D-EDNA09-02131 190 r a_E Usnea_spec-uncultured_KM369361_New-Zealand_ID-FNO3 MF6 D KM369 aland_ sta-R M 7 66 _New-Zealand_ID-FL 7 9-023 Usnea_spec-uncultured_KM369411_New-Zealand_ID-FOran3 819_Brazil_ I _KM3 aland_ID-FN14 8 52375_USA_ID-46374 d_KM3692 a 36 -2 d_KM -Zealand_ID-FMAR5 Usnea_spec-uncultured_KM369352_New-Zealand_ID-FN16 JQ 92 _ID-EDNA09-02081 _FR799090_England_ 22_Japan_ID- 494932_Ta _ID-EDNA09

Islands_ID-230TEN JN08 Usnea_spec-uncultured_KM369413_New-Zealand_ID-FRob1 7294_Madeira_ID- K81 a 04_C a Usnea_spec-uncultured_KM369285_New-Zealand_ID-F592 _F 1_ d_ d_KM 6_N 1 - _K 406272_ JN943551_England_ID-EDNA09-02076 910 Usnea_spec-uncultured_KM369286_ 69 Co _KM3 _ Usne 69 _JN0862 _Br 6_ AC 9257_New-Zealand_ID-F 669852_Spain_ID-4486ES 31_Ch Usnea_spec-uncultured_KM369287_New-Zealand_ID-F594 AC4452_Sp 93 ew-Zeala Usnea_cornuta_JN943562_Ireland_ID-EDNA09-01124 _MF6 es_I an - a- nd land_ID-F533 Usnea_cornuta_MF669856_Spain_ID-4491ES 6 JN086290_England_ID-esp R Usnea_spec-uncultured_KM369282_New-Zealand_ID-F588 Usnea_cornuta_JN943532_Wales_ID-EDNA09-02134 t 50347 Usnea_cornuta_MF669861_Brazil_ID-19BR and_ID lan Usnea_cornuta_JQ837301_France_ID-42 M36 59 Usnea_spec-uncultured_KM369294_New-Zealand_ID-F604 l 60 New- Usnea Usnea_cornuta_MF669828_Brazil_ID-30BR 123_Japan_ID-TNS-YO10417 1847_N Zea Usnea_cornuta_EAC4489_SpainID-EAC4489 Usnea_cornuta_JQ837302_Madeira_ID-43 Usnea_cornuta_JQ837300_USA_ID-32 u 799093_England_I 99 _New-Zealand_ID-F6 ew-Ze w-Z J406274_United-Kingdom_ID-AA10 tland_ID-ED 6938 d Usnea_cornuta_MF669859_Brazil_ID-17BR _Canary-Islands_ID-23 4451_SpainID- M ID-FNO18 _KJ 69920_Brazil_ID-sp1-80BR tland 381_New-Zea Usnea_cornuta_FR799084_Scotland_ID-EDNA09-02345 8 6291_E 369395_New-Zealand_ JQ83 uta MF6 398_ Usnea_cornuta_FR799085_Ireland_ID-EDNA09-01124 Usnea_cornuta_EAC4487_PortugalID-EAC4487 616_Po 69845_Braz _ID 9355_ 369387_New-Zealand_ID-FNO3 27 Usnea_cornuta_FR799088_Ireland_ID-EDNA09-01125 Y0219 o Usnea_spec-uncultured rn 369393_Ne 9416_Ne ew-Zealand_ Usnea_cornuta_JN943526_Scotland_ID-EDNA09-02345 _New-Zealand_ID-FNO50 rnuta-1_MF6698 4 9 69280_N D 9809_ 62 356 6 lan _K a_MF _New 479 s_ o 2_ 925 la t K ealand_ Usnea_spec_MN006799_USA_ID-sample50 091 Usnea_cornuta F6 6 ina_ID 9378_N -FNO17 Usnea_cornuta_JN943561_Ireland_ID-EDNA09-01125Usnea_cornuta_FR799087_Wales_ID-EDNA09- 1_MF669811_Brazil_ID c Zea MF6 - Usnea_cornuta_FR799086_England_ID-EDNA09-01127 - 4_New 34_Wales_ID-EDNA09- 34_New- nd_

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5

Figure 7. Global tree (best scoring maximum likelihood tree) of Usnea s.lat. based on the fungal ITS DNA barcoding marker. Figure can be enlarged to discern labels. For separate phylogram including bootstrap support values, see File S10. constictic, salazinic, protocetraric, and/or diffractaic acids Eumitria pectinata has been described from Southeast in various combinations, but never with salazinic acid and Asia, the type containing norstictic, menegazziaic, stictic, very rarely with protocetraric acid as major compounds. and constictic acids (Ohmura 2001). The chemistry of The three nested, supported clades on longer branches had eastern paleotropical material is uniform and consistent relatively uniform chemistries deviating from the varia- with that of the type (Ohmura 2001; Nadel 2016), which tion in the backbone: the clade composed of MN080235, means that possibly only the small clade formed by the MW267152, MW267153, MW267154, and MW267155, three Southeast Asian samples represents E. pectinata from São Tomé and Príncipe and Tanzania (relabeled s.str. and the other terminals in the backbone were there- E. aff. pecinata 1; Table S1) had protocetraric acid as fore relabeled E. cf. pectinata (Table S1). The small clade major compound; the clade containing MW267156, relabeled E. aff. pectinata 3 has a chemistry matching MW267157, MW267158, MW267159, MN080233, and that of Usnea mexicana, which presumably only occurs MW267160, from São Tomé and Príncipe and Tanzania in the Neotropics (Herrera-Campos et al. 1998; Truong (relabeled E. aff. pectinata 2) featured mostly salazinic et al. 2013b), but has not yet been sequenced from there. acid as major substance (with one exception), and the In addition, at least five specimens in the backbone fea- clade formed by MN080238, MN080237, and MN080239, ture that chemistry: three from São Tomé and Príncipe from Tanzania (relabeled E. aff.pectinata 3), had constic- (MN0063, MN0583, MN0585) and two from Tanzania tic and diffractaic acid (Nadel 2016; Temu et al. 2019). (MN080240, MN080241). Although Nadel (2016) and 318 Plant and Fungal Systematics 65(2): 303–357, 2020

Figure 8. Pairwise identity matrix based on the ITS for the Eumitria baileyi clade. Dark grey = identity below 98.5%; light grey = identity exactly 98.5%; white, blue and beige = identity above 98.5%.

Figure 9. World distribution of Eumitria baileyi s.lat. according to the Global Biodiversity Information Facility, GBIF [https://www.gbif.org/ species/7247545].

Temu et al. (2019) do not discuss the name U. mexicana (Ohmura 2002, 2012; Singh & Sinha 2010; Nadel 2016; for their African material, either the small clade or part of Ohmura & Kashiwadani 2018; Temu et al. 2019). Unfor- the backbone may represent that species, implying a con- tunately, the chemistry of this material is not known, but siderable range extension. Notably, GBIF has one record given the fact that its ITS is identical to MW267148 from of U. mexicana from Cameroon, identified by P. Clerc São Tomé and Príncipe and the latter has a mixed chem- (https://www.gbif.org/occurrence/1144796085), which istry of protocetraric, salazinic, constictic, and diffractaic would support our interpretation. A potential name for acids (Nadel 2016), whereas the specimens matching the specimens containing protocetraric acid is U. duriuscula, chemistry of U. mexicana are separate (see above), we can described from Brazil (Truong et al. 2013b). Besides the exclude the possibility that the French Guianan specimen larger subclade relabeled E. aff.pectinata 1, this substance represents U. mexicana. It is also distinct from the clade was also detected in one specimen of the other subclade representing E. pectinata s.str., so it is possible that this (E. aff. pectinata 2) from Tanzania and a singleton in the is another unrecognized candidate species. backbone (MN080236) from Tanzania (Temu et al. 2019), Data available from the nuLSU (from Temu et al. 2019) so the possible application of this name remains unclear. were fully congruent with the ITS (Fig. S2), whereas the A sample from French Guiana (MK547011), originally RPB1 (from Temu et al. 2019) provided less resolution identified asUsnea sp. as part of a broad fungal barcoding and one supported conflict for sample SGT87, which clus- study (Jaouen et al. 2019), clustered with two specimens tered with a specimen (SGT117) representing subclade from São Tomé and Príncipe in an early diverging, sup- E. aff. pectinata 3 (Fig. S5). ported lineage, its sequence identical to one of the two Overall, Eumitria is in need of a much broader molec- samples from São Tomé and Príncipe. Eumitria pecti- ular sample of phenotypically defined species (mostly nata has so far only been reported from Africa and Asia from Africa and Asia, but including species currently R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 319

Figure 10. Pairwise identity matrix based on the ITS for the Eumitria pectinata clade. Dark grey = identity below 98.5%; grey = identity exactly 98.5%; white, blue and beige = identity above 98.5%. placed in Usnea from the Neotropics, in particular U. mex- subclades on long stem branches, suggesting the presence icana), as well as a detailed phylogenetic revision of spe- of several lineages even within a single country (Japan; cies complexes such as E. baileyi s.lat. and E. pectinata Ohmura 2002; Articus 2004; Truong et al. 2013a). This is s.lat., to obtain a better understanding of this genus and another species that has only been reported from Africa, its species. The latter two do not seem to represent sin- Asia and Oceania (Ahti et al. 2016; Aptroot 2016; Arti- gle species, but rather recently diversifying clades that cus 2004; Galloway 2007; Jayalal et al. 2013; Ohmura can only be resolved with broad sampling using several 2002; Schumm & Aptroot 2010; Stevens 1999; Swin- markers. One may also argue that the observed ITS var- scow & Krog 1978, 1988; Truong et al. 2013a; Ohmura iation in these clades is overinterpreted, but given that in & Kashiwadani 2018), but one unpublished accession other instances, taxa that have identical ITS were shown (MN006813), deposited by M. L. Hale and J. Taylor in to represent different species with approaches such as 2019, appears to be from California, although the geo- RADseq (Grewe et al. 2018), it seems inconsistent to graphic origin was not precisely indicated. That sequence assume that structured differences in the ITS, as observed formed part of a small, strongly supported subclade also in E. baileyi s.lat. and E. pectinata s.lat., may just be including sequences from Japan and Taiwan (Fig. 7; infraspecific variation in these cases. Fig. S1). This taxon thus appears to represent an unre- solved species complex, the various lineages rather well Dolichousnea. Dolichousnea is by far the best sampled separated in the identity matrix (Fig. 11). Ohmura (2001, group in Usnea s.lat., with all three currently recognized 2012) reported three chemotypes for D. trichodeoides, taxa represented by ample sequence data (Figs 2, 7; including salazinic or fumarprotocetraric acids or atra- Table S1; Fig. S1). The three taxa were all recovered in norin as major compounds. For one sequenced accession monophyletic clades, but exhibited different topological patterns. Dolichousnea diffracta (Vain.) Articus formed a strongly supported clade on a very long stem branch with little internal variation across its sampled range including, Japan, South Korea, and Taiwan (Articus 2004; Ohmura 2002; Park et al. 2014; and several unpublished accessions). The chemistry of this taxon appears to be complex, but rather uniform with regard to major com- pounds in most of the studied material with the exception of some extremely rare, aberrant chemotypes (Ohmura 2001, 2012). In contrast, Dolichousnea trichodeoides (Vain. ex Figure 11. Pairwise identity matrix based on the ITS for the Doli- Motyka) Articus was not supported and exhibited sub- chousnea trichodeoides clade. Dark grey = identity below 98.5%; grey stantial internal variation, with some strongly supported = identity exactly 98.5%; blue and beige = identity above 98.5%. 320 Plant and Fungal Systematics 65(2): 303–357, 2020

from Japan (AB051668), the chemotype (fumarprotoce- chemistry has been reported, AB051643 (barbatic acid traric acid) was given by Ohmura (2001), corresponding major) and AB051645 (diffractaic acid major), cluster to the first diverging, larger subclade. Chemical data for in distant portions of the clade. the other specimens have apparently not been published, Few data from other markers were available for but should be available from the voucher material, so Dolichousnea. IGS data for D. longissima (from Rol- testing of a potential correlation between phylogeny and stad et al. 2013) also showed a high level of internal chemotypes should give further insight into this clade. structure for that taxon (Fig. S3). Unfortunately, there Considerable internal variation was also observed was little overlap between the ITS and IGS data from in the supported Dolichousnea longissima clade with the same study (Rolstad et al. 2013). Of the 48 samples samples from across the Northern Hemisphere (Figs 2, 7; with either ITS or IGS data, only ten had both markers. Table S1; Fig. S1). The internal structure was largely Based on these, the internal topology for D. longissima reflected in a cascading backbone, without clearly dis- was largely congruent between both markers and did not cernable larger lineages. The corresponding identity exhibit supported conflict, although IGS provided a more matrix identified several subgroups at the 98.5% level, resolved topology (Figs S1, S3). Due to a limited amount but pairwise comparison between samples across all or lack of data, the other markers could not be compared subgroups also revealed partially high identity levels with ITS. above 98.5% (Fig. 12). Thus, D. longissima apparently cannot currently be subdivided into more than one spe- Usnea subgenus Neuropogon New Zealand clade. cies based on ITS data, even if it exhibited consider- This large, unsupported clade of 150 terminals was almost able internal variation. Secondary chemistry may help exclusively composed of samples from New Zealand, with to discern lineages, although it has not been reported two exceptions: one unpublished sequence labeled U. aff. for most of the sequenced specimens. The three most igniaria Motyka from Antarctica (DQ219307), deposited common chemotypes produce diffractaic, barbatic, or by K.-M. Kim and J.-S. Hur in 2005, and one unidenti- evernic acid as major compounds (Halonen et al. 1998; fied sequence from Chile (KJ406284) from the study by Ohmura 2001, 2012; Randlane et al. 2009). The dif- Millanes et al. (2014). Both formed separate singletons fractaic acid chemotype corresponds to D. longissima likely representing separate species. Of the remaining s.str. according to the type material (Ohmura 2001, 148 sequences, only nine were identified as GenBank 2012). Two sequenced Japanese samples for which the deposits, representing U. ciliata and U. subcapillaris

Figure 12. Pairwise identity matrix based on the ITS for the Dolichousnea longissima clade. Dark grey = identity below 98.5%; grey = identity exactly 98.5%; white and shades of green = identity above 98.5%. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 321

(Wirtz et al. 2006, 2012; Lumbsch & Wirtz 2011). The U. pusilla, and U. xanthopoga. Twenty accessions remaining 139 terminals almost exclusively stem from (JX144648 through KM369173) formed an unsupported two studies (Buckley et al. 2014; Rafat et al. 2015). Of clade containing five terminals subsequently labeled these, 18 specimens were identified in the corresponding U. ciliifera. These contained a well-unsupported sub- papers, but left unidentified in the GenBank accessions. clade (88%) of eight accessions (KR091723 through Given this information, the entire clade can currently KM369173), two with odd base calls (see above) on only be outlined provisionally (see Fig. 7; Fig. S1). long branches (Fig. 7; Fig. S1). This latter subclade had Following three singletons as early diverging lineages, two consistent substitutions and one indel (99.4% simi- a paraphyletic grade of ten sequences (KM369335 larity) and so the two groups were labeled U. ciliifera 1 through KM369241) included two subsequently iden- and U. ciliifera 2. The Usnea pusilla clade (KM369188 tified as U. torulosa (Buckley et al. 2014; Rafat et al. through JX144650), including a single, subsequently iden- 2015) and was here interpreted to represent that taxon. tified accession (Rafat et al. 2015), was well-supported The grade contained an unsupported subclade of five and internally variable, partly due to accessions with odd sequences (KM369431 through KM369241), differing base calls, but did not exhibit supported internal struc- consistently in two substitutions in the ITS2, i.e. about ture. The large U. xanthopoga clade (KM369401 through 99.6% similarity with the other sequences and not suf- KM369408) included two sequences subsequently iden- ficient to consider this a separate species. The first of tified with that name (Rafat et al. 2015). This clade was these sequences (KM369431) included odd base calls (see more or less supported (74%) and included some partly above), accounting for the long branch. Another, strongly supported subclades, in particular one consisting of three supported clade of five terminals (KM369197 through accessions (KM369248, KM369436, JX144644) with KM369435), containing one accession subsequently three consistent substitutions, and another of nine acces- identified as U. torulosa (Buckley et al. 2014), differed sions (KM369409 through KM369408) with two consist- consistently in five substitutions (99% similarity) and was ent substitutions, all in the ITS1 (File S1). In addition to here considered a separate species (U. aff. torulosa), as these subsequently identified or provisionally identified it fell elsewhere. A similar situation was found with five clades, the New Zealand clade of subgenus Neuropogon specimens identified as U. inermis (Buckley et al. 2014; contained four separate lineages that remained unidenti- Rafat et al. 2015), which fell into two larger, unsupported fied (Fig. 7; Table S1; Fig. S1). clades (Fig. 7; Fig. S1), the first considered U. inermis Unfortunately, few data are available on other markers s.str. and the second U. aff. inermis. The first clade of 27 for this clade. Only IGS and RPB1 covered specimens of accessions (KM369276 through KM369332) was inter- this clade, whereas data on other markers all corresponded nally rather uniform, but contained several sequences with to the Neuropogon core clade (see below). Both IGS and odd base calls (see above; Table S1). The second clade RPB1 data (Lumbsch & Wirtz 2011; Wirtz et al. 2012) formed two subclades, each with one sequence subse- placed accessions identified as U. ciliata and U. subcap- quently identified as U. inermis. The first, smaller sub- illaris in a strongly to fully supported clade within the clade (KM369369 through KM369249) was sister to two supported Neuropogon core clade (Fig. S3), thus sup- specimens subsequently identified asU. pusilla (Buckley porting the close relationship of the New Zealand clade et al. 2014; Rafat et al. 2015), here labeled U. aff.pusilla with the species in the core clade, but leaving open the (see below). They differed from the second, larger, rather question whether both groups indeed form sister clades, uniform subclade (KM369279 through JX144643) con- as suggested by the unsupported ITS topology. sistently in six substitutions (98.8% similarity) and so Walker (1985) reported six species corresponding to the two subclades were here labeled U. aff. inermis 1 subgenus Neuropogon for New Zealand: U. acromelana, and U. aff. inermis 2. U. antarctica, U. ciliata, U. pseudocapillaris, U. sphace- Three specimens of Usnea subcapillaris (Wirtz et al. lata, and U. subcapillaris. Three of these (U. acromelana, 2006, 2012; Lumbsch & Wirtz 2011) formed a strongly U. antarctica, U. sphacelata) belong to the Neuropogon supported clade, whereas a fourth accession fell separate core clade (see below) and have not yet been confirmed (U. aff. subcapillaris). The same applied to U. ciliata to occur in New Zealand based on sequence data. Thus, (Wirtz et al. 2006, 2012; Lumbsch & Wirtz 2011), where of the seven names used to identify lineages in this clade a single accession originally identified as U. cf. ciliata (U. ciliata, U. ciliifera, U. inermis, U. pusilla, U. sub- did not cluster with the remaining samples, which formed capillaris, U. torulosa, U. xanthopoga), only two agreed a paraphyletic grade basal to U. subcapillaris (Fig. 7; with Walker (1985) and one (U. pseudocapillaris) was Fig. S1), including also one originally unidentified sample missing, possibly corresponding to one of the unnamed or (Buckley et al. 2014). Four accessions (JX144646 through provisionally named clades here. This suggests that sub- KM369371) formed a fully supported clade on a long genus Neuropogon in New Zealand requires a thorough branch; one was subsequently identified as U. articulata phylogenetic revision. Both U. inermis and U. torulosa (Rafat et al. 2015) and was here labeled U. aff. articu- were treated by Walker (1985) as somewhat similar to lata 4, although this lineage was obviously not related to subgenus Neuropogon, but not related to the latter. If the U. articulata (see below). identifications given by Buckley et al. (2014) and Rafat The remaining accessions largely represented three et al. (2015) are correct, then these taxa belong in subge- names subsequently identified in the corresponding stud- nus Neuropogon. Usnea xanthopoga was mentioned by ies (Buckley et al. 2014; Rafat et al. 2015): U. ciliifera, Walker (1985), but referred to subgenus Usnea and, based 322 Plant and Fungal Systematics 65(2): 303–357, 2020

on the available data, it also belongs in subgenus Neuro- results from other works, such as the classic study by pogon. Usnea ciliifera and U. pusilla were not treated by Kroken & Taylor (2001) on the Letharia columbiana / Walker (1985) and not related to the Neuropogon group L. vulpina species pair, which demonstrated the exist- by Galloway (2007). Overall, the New Zealand clade ence of six lineages, the sorediate ones sometimes also of subgenus Neuropogon thus appears to include some producing apothecia. species that do not share the typical characteristics of the Wirtz et al. (2008) and Lumbsch & Wirtz (2011) Neuropogon core clade (see below). studied the Usnea perpusilla complex, resolving it into five species, besides U. perpusilla also distinguishing Usnea subgenus Neuropogon core clade. Although U. lambii, U. messutiae Wirtz & Lumbsch, U. pal- containing a much larger number of terminals (446), lidocarpa Wirtz & Lumbsch and U. ushuaiensis. The this clade was genetically more uniform compared to five taxa formed three groups in the ITS-based tree, the New Zealand clade (Fig. 7; File S1; Fig. 10). Spe- one consisting of U. ushuaiensis, one representing the cies in this clade are generally characterized by annulate U. perpusilla-lambii aggregate, and one the U. messu- black pigmentation along the branches (Fig. 1C) and black tiae-pallidocarpa aggregate. The U. ushuaiensis clade apothecial discs (Walker 1985; Ohmura 2002; Ohmura was not supported, likely due to an unidentified singleton & Kanda 2004; Articus 2004; Wirtz et al. 2006, 2008, sequence (AY251434) on a long branch nested within 2012; Seymour et al. 2007). Notably, this clade did not (Thell et al. 2004). A supported subclade included three contain a single accession from New Zealand and was accessions (EF492213, EF492167, EF492182), differing largely restricted to Antarctica and South America, with from the other sequences in three consistent substitutions. the exception of the more widely distributed U. lambii One of these accessions was regarded as ‘potential recom- and U. sphacelata. binant’ by Wirtz et al. (2008), but the isolated positions More than half of the Neuropogon core clade (247 of the three substitutions (99.4% similarity) and the good accessions) was comprised of the species pair U. antarc- support for this clade did not support this assumption; tica (sorediate) and U. aurantiacoatra (apotheciate), both rather, it was here considered an infraspecific lineage. with norstictic and salazinic acids as major compounds Medullary secondary chemistry in this clade is compara- (Walker 1985). These accessions stem from a large num- tively simple, lacking substances or containing psoromic ber of studies (Articus 2004; Ohmura & Kanda 2004; acid as major and/or the dibenzofurane hypostrepsilic acid Kim et al. 2006; Seymour et al. 2007; Lumbsch & Wirtz chemosyndrome as minor (Walker 1985; Elix et al. 2007; 2011; Schmull et al. 2011; Wirtz et al. 2006, 2012; Park Lumbsch & Wirtz 2011; Lumbsch et al. 2011). Wirtz et al. 2015; Cao et al. 2015, 2018; Lagostina et al. 2018; et al. (2008) found a lack of correlation between clades Canini et al. 2020), including numerous apparently unpub- and secondary chemistry, but unfortunately did not report lished sequences deposited by K.-M. Lim & J.-S. Hur in the exact chemistry for each terminal. 2005, N. Wirtz in 2006, H. Li & Q.-M. Zhou in 2012, Usnea perpusilla and U. lambii were not resolved as and S. Cao in 2015. Among Usnea s.lat., this is by far reciprocally monophyletic clades in the ITS and instead the best studied species-level clade in terms of taxon- formed several supported and unsupported subclades omy, phylogeny, ecology, and distribution. The strongly (Fig. 7; Fig. S1). The same clades were also recovered in supported clade was comparatively uniform regarding the IGS, although with higher support (Fig. S3). The two the ITS, with some unsupported internal structure and taxa are distinguished by their reproductive mode, U. per- no resolution into clades corresponding to either name, pusilla producing apothecia and U. lambii soredia. Wirtz suggesting this species pair to represent a single taxon et al. (2008) resolved U. perpusilla as paraphyletic relative (Seymour et al. 2007; Wirtz et al. 2012). Data from the to U. lambii, but this topology was likely caused by the IGS (Wirtz et al. 2012) did not include sorediate forms inclusion of RPB1, in addition to ITS and IGS. Our analy- (U. antarctica), but the apotheciate forms (U. aurantia- sis showed that RPB1 behaved erratically for this complex coatra) formed several smaller, partly supported, nested and other species in the Neuropogon core clade, mixing subclades with U. acromelana nested within on a long, accessions of various taxa in various shallow grades and strongly supported branch (Fig. S3). RPB1 data (Sey- clades, an indication of the formation of paralogs (Fig. 13; mour et al. 2007; Wirtz et al. 2012) showed a similar Fig. S5). Lumbsch & Wirtz (2011) recovered U. perpusilla pattern as with ITS with U. antarctica samples intermin- and U. lambii as reciprocally monophyletic, but this was gled with U. aurantiacoatra (Fig. S5). Using a RADseq an artifact of limited sampling, representing one subclade approach, Grewe et al. (2018) demonstrated the separation each of the complex assemblage evident when joining all of U. antarctica and U. aurantiacoatra into two strongly available data. Based on both ITS and IGS data, the situ- supported clades, suggesting the presence of two closely ation in U. perpusilla versus U. lambii appears similarly related species that ITS, IGS and RPB1 were not able to complex as in Letharia columbiana (Nutt.) J.W. Thomson resolve. This confirmed findings by Lagostina et al. (2018) versus L. vulpina (L.) Hue (Kroken & Taylor 2001; see using microsatellite markers, although in the latter study, above). Based on the identity matrix from the ITS data some apotheciate specimens clustered with the sorediate (not shown), the differences between the various subclades U. antarctica, suggesting a more complex situation in were very shallow with most pairwise similarity values at which two species can be distinguished, one persistently or above 98.5%. However, considering the results from fertile (apotheciate) and one usually sterile (sorediate) microsatellite and RADseq markers in the much shallower and sometimes fertile (apotheciate). This would mirror U. antarctica-aurantiacoatra complex (see above), it is R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 323

patagonica patagonica patagonica ushuaiensis sphacelata sphacelata perpusilla subantarctica sp. trachycarpa lambii trachycarpa aff. perpusilla perpusilla perpusilla trachycarpa ushuaiensis lambii subantarctica lambii acromelana perpusilla sp. aff. perpusilla

aurantiacoatra lambii

perpusilla sphacelata ushuaiensis subantarctica lambii trachycarpa perpusilla

ushuaiensis sp. perpusilla aurantiacoatra

subantarctica aurantiacoatra trachycarpa acromelana aff. perpusilla aurantiacoatra acromelana

aurantiacoatra sphacelata ITS IGS RPB1 Figure 13. Comparison of topologies in the Neuropogon core clade between the ITS, IGS and RPB1 (cladograms). Each color corresponds to an accepted species. RPB1 provided limited resolution or coherence for some species, but accessions of others were distributed across the two main clades. possible that each of the clades formed in the U. perpu- paraphyletic and the U. subantarctica clade was not sup- silla-lambii complex represents a separate species. ported; U. acromelana formed a strongly supported clade, Usnea messutiae formed two unsupported subclades but was nested within the U. antarctica-aurantiaca com- in the ITS, associated with a strongly supported clade plex (Fig. S3). As mentioned, RPB1 exhibited an erratic representing U. pallidocarpa (Fig. 7; Table S1; Fig. S1), topology for taxa in this and the previous group, joining the latter including additional accessions from Wirtz et al. U. sphacelata, U. subantarctica, and U. trachycarpa in (2006). IGS data exhibited a partly conflicting topology. a homogeneous grade also including isolated accessions The second U. messutiae subclade was mirrored by of U. pallidocarpa, U. perpusilla, and U. ushuaiensis a strongly supported subclade in the IGS, whereas the first (Fig. 13). Another, unsupported shallow clade was com- subclade was not only dissected into a grade, a strongly posed of accessions of U. aurantiacoatra, U. lambii, supported subclade, and one singleton in the IGS, but U. perpusilla, U. sphacelata, and U. ushuaiensis. A third the U. messutiae grade also included two accessions of group formed a shallow grade including U. acromelana, U. pallidocarpa with identical sequences (Fig. S3), one of the U. antarctica-aurantiacoatra complex, and a single the rare instances of supported conflict between ITS and accession of U. sphacelata. Only U. patagonia formed IGS data in Usnea. In contrast, RPB1 reflected both species a fully supported clade (Fig. S5). Given particularly the well in two unsupported, homogeneous clades (Fig. S5). data of the ITS and IGS, there was thus at least partial The remaining five species in the Neuropogon core support for U. acromelana, U. patagonica, U. sphacelata, clade formed comparatively homogeneous groupings: U. subantarctica, and U. trachycarpa to form well-de- U. patagonica and U. subantarctica were strongly sup- limited species. ported each in monophyletic clades, U. sphacelata clus- We detected only two instances of potentially misi- tered in an unsupported clade, and U. trachycarpa and dentified accessions in the entire Neuropogon core clade U. acromelana in paraphyletic grades, one basal to U. sub- (Table S1), both identified asU. subantarctica, but falling antarctica, that relationship without support, and the other within U. trachycarpa. Five further accessions labeled basal to the U. antarctica-aurantiacoatra complex, that U. sphacelata (Ohmura & Kanda 2004; Seymour et al. relationship being strongly supported (Fig. 7; Fig. S1). 2007; Schmull et al. 2011) belonged in the U. perpusil- With IGS, U. patagonica and U. sphacelata formed sup- la-lambii complex which was only subsequently defined ported clades, whereas U. trachycarpa was again rendered by Wirtz et al. (2008) and Lumbsch & Wirtz (2011). 324 Plant and Fungal Systematics 65(2): 303–357, 2020

Two singleton sequences identified as U. trachycarpa species is U. shimadae, reported from Mexico and Tai- (AJ748103, JQ314761; Articus 2004; Wirtz et al. 2012) wan (Clerc 2007; Ohmura 2001, 2012; Shen et al. 2012; fell at the base of the U. sphacelata clade and one of Herrera-Campos 2016). The only available sequence, that these (AJ748103) had odd base calls. clustered with a supported, unidentified sequence from China (MT261826), was from Taiwan and corresponds Usnea s.str. – Singletons. A total of 14 names were rep- to the taxon geographically, whereas the reports from resented in the ITS-based tree by singleton clades only. Mexico need to be further investigated. These are considered putatively correct identifications Five species are either widespread in the Americas or representing separate species. They include U. angulata found across North or South America: U. aspera, U. cir- Ach. (JQ837291), U. aspera (Eschw.) Vain. (MF669805), rosa, U. halei, U. meridionalis, and U. subrubicunda U. aurantiaciparvula A. Gerlach & P. Clerc (KY021902), (Hekking & Sipman 1988; Marcano et al. 1996; Clerc U. cirrosa Motyka (KY021903), U. ghattensis G. Awasthi & Herrera-Campos 1997; Herrera-Campos et al. 2001; (KY021914), U. halei P. Clerc (MG252375), U. intumescens Calvelo & Liberatore 2002; Clerc 2007; Truong et al. (AB051641), U. meridionalis Zahlbr. (KY021919), U. mer- 2011, 2013a; Truong 2012; Herrera-Campos 2016; Truong rillii (AB051649), U. aff. sanguinea Swinscow & Krog & Clerc 2016; Gerlach et al. 2017, 2019a; Rodríguez-R. (MW267168), U. shimadae Asahina (FJ494952; deposited et al. 2017; Esslinger 2019; Lücking et al. 2020c). For as Usnea sp.), U. sinensis Motyka (FJ494953, deposited as these taxa, the paucity of sequence data was surprising. Usnea sp.), U. subglabrata Truong & P. Clerc (JQ837312, Usnea aspera clustered with U. aff. cornuta (not repre- deposited as Usnea sp. 6), and U. subrubicunda P. Clerc senting that species, see below) from Brazil; U. cirrosa (JQ837332; Ohmura 2002; Shen et al. 2012; Truong et al. fell in the vicinity of U. cladocarpa and U. halei close 2013a, 2016; Nadel 2016; Gerlach et al. 2017, 2019a). to U. flammea Stirt., both relationships without sup- Among these, two taxa are common and presumably port; U. meridionalis formed an unsupported clade with widespread, namely U. angulata (Fig. 14) and U. merrillii U. durietzii and U. glabrata; and U. subrubicunda was (Marcano et al. 1996; Herrera-Campos et al. 1998; Elix found, without support, basal to the U. barbata-dasopo- & McCarthy 1998, 2008; Stevens 1999; Ohmura 2002; ga-wasmuthii clade (Fig. 7; Table S1; Fig. S1). Galloway 2007; Lin 2007; Singh & Sinha 2010; Truong The remaining six species included two recently et al. 2013b; Herrera-Campos 2016; Sipman & Aguirre-C. described taxa, U. subglabrata from Bolivia (Truong 2016; Truong & Clerc 2016; Rodríguez-Flakus et al. 2016; & Clerc 2016) and U. aurantiaciparvula from Brazil (Ger- Ohmura & Kashiwadani 2018; Bungartz et al. 2018; Ess- lach et al. 2017), and four species with more restricted linger 2019; Lücking 2020c), and hence their sparse rep- distribution in Africa and Asia, namely U. ghattensis, resentation by ITS sequence data was surprising. Usnea U. intumenscens, U. aff. sanguinea, and U. sinensis angulata, which is particularly common in the Americas (Ohmura 2002; Lin 2007; Singh & Sinha 2010; Shen (Fig. 14), formed an early diverging lineage in our phy- et al. 2012; Gerlach et al. 2017; Ohmura & Kashiwadani logeny, although this topology was not supported (Fig. 7; 2018). Usnea ghattensis fell close to U. articulata, while Table S1; Fig. S1). Usnea merrillii formed a separate U. intumescens clustered with support with U. bismol- lineage near U. clerciana Truong, but that relationship liuscula and U. sinensis with U. nipparensis (Fig. 7; was also not supported. Another presumably widespread Table S1; Fig. S1). The accession originally identified

Figure 14. World distribution of Usnea angulata according to the Global Biodiversity Information Facility, GBIF [https://www.gbif.org/spe- cies/2606124], fitting well with published records in taxonomic revisions. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 325 as U. aff. sanguinea (Nadel 2016) formed a singleton onset (Truong & Clerc 2016). The four ITS accessions clade unrelated to U. erinacea with which it has been were rather uniform, but the case of U. antarctica vs. synonymized (Clerc 2004, 2007, 2011a; see below). How- U. aurantiacoatra (see above) shows that species pairs ever, the voucher specimen, while lacking soredia, does may not necessarily be resolved with the ITS marker. The not produce apothecia and has a deviating chemistry, so saxicolous, apotheciate species U. densirostra Taylor, this identification is preliminary. described from Uruguay, was represented by three acces- No IGS data were available for any of the singletons. sions from nearby southern Brazil (Gerlach et al. 2017, In RPB1, two taxa, U. aurantiaciparvula and U. ghat- 2019a). Its sorediate counterpart, U. amblyoclada (Müll. tensis, also formed singletons, whereas two accessions Arg.) Zahlbr, also saxicolous and known from the same of U. cirrosa formed a paraphyletic grade at the base of area (Rodriguez et al. 2011a), has not yet been sequenced. U. cornuta s.str. (Fig. S5). Usnea dodgei and U. durietzii represent cases similar to U. chilensis and U. cladocarpa, whereas U. dodgei was Usnea s.str. – Monophyletic species-level clades. described from Costa Rica (Motyka 1938), with three A total of 30 taxa were recovered as monophyletic and accessions from Brazil (Gerlach et al. 2019a), U. durietzii internally more or less homogeneous and could be consid- represents a neuropogonoid taxon originally described ered well-defined species. These areU. acanthella, U. ara- from Chile (Motyka 1938), with two ITS accessions from nea Truong & P. Clerc, U. cavernosa, U. chilensis Motyka, Peru (Wirtz et al. 2006). U. cladocarpa, U. clerciana, U. densirostra Taylor, Phylogenetic inference for Usnea flammea, forming U. dodgei Motyka, U. durietzii, U. flammea, U. flavocardia a well-supported clade of several accessions from Mac- Räsänen, U. fleigiae A. Gerlach & P. Clerc, U. grandisora aronesia and western Europe (Kelly et al. 2011; Saag Truong & P. Clerc, U. grandispora A. Gerlach & P. Clerc, et al. 2011; Schoch et al. 2012; Gerlach et al. 2019a; U. hirta, U. macaronesica P. Clerc, U. malmei Motyka, Marthinsen et al. 2019), was well in line with its type U. masudana Asahina, U. nidifica Taylor, U. nipparensis, originating from Portugal (Stirton 1881). The situation U. oreophila A. Gerlach & P. Clerc, U. pangiana, U. par- was the opposite for U. flavocardia, described from Chile vula Motyka, U. pseudogatae, U. rubriglabrata Truong (Räsänen 1936), but all ITS accessions originating from & P. Clerc, U. silesiaca, U. subaranea Truong & P. Clerc, Europe and North America (Kelly et al. 2011; Saag et al. U. subdasaea Truong & P. Clerc, U. subparvula, and 2011; Schoch et al. 2014; Millanes et al. 2014; Araujo U. wasmuthii (Fig. 7, Table S1, Fig. S1). 2016). The two presumed North American accessions Usnea acanthella, described from Peru (Lamb 1939) are unpublished, deposited by M. L. Hale and J. Taylor and represented by various accessions from Ecuador and in 2019, and did not have specific voucher information, Peru (Wirtz et al. 2006, 2012), was one of two neuro- so their exact origin remains unclear. A further accession pogonoid species falling outside the Neoropogon clade from New Zealand (KJ406280) identified with this name (Wirtz et al. 2006). Usnea aranea was recently described (Millanes et al. 2014) fell outside the flavocardia clade, based on a non-sequenced specimen from Bolivia as type, but clustered with an unidentified lineage from New Zea- whereas the sequenced specimens originated from Ecua- land (Buckley et al. 2014). Usnea fleigiae, U. grandis- dor and Peru (Truong et al. 2013a; Truong & Clerc 2016). ora, and U. grandispora are recently established species Given the complexity of species delimitations in the genus from South America (Truong & Clerc 2016; Gerlach et al. Usnea, this could cause problems unless a sequenced epi- 2017). Usnea grandisora was described from Galapa- type is being designated. Usnea cavernosa was originally gos and also reported from Venezuela (Truong & Clerc described from North America (Tuckerman in Agassiz 2016), whereas the available ITS sequences are from 1850) and the fully supported clade, including specimens Brazil (Gerlach et al. 2017), so the status of this taxon from the USA and Switzerland (Mark et al. 2016a), was remains unclear. consistent with the inferred distribution of the species (see Usnea hirta is one of the presumably most widespread below). As the epithet implies, U. chilensis was originally species in the genus with reports from all major areas in established based on material from Chile (Motyka 1938), North and South America, Europe, Africa, Asia, and Oce- but was here represented by two sequences from southern ania (Mies 1989; Halonen et al. 1998; Elix & McCarthy Brazil (Gerlach et al. 2019a). A similar situation applied to 1998, 2008; Fos & Clerc 2000; Calvelo & Liberatore U. cladocarpa, a taxon described from Brazil (Fée 1825), 2002; Articus et al. 2002; Bjerke et al. 2006; Clerc 2007; with two sequences from Costa Rica (Gerlach et al. 2017). Randlane et al. 2009; Kelly et al. 2011; Saag et al. 2011; Given that presumably widespread neotropical or Amer- Shrestha et al. 2012; Noer et al. 2013; Santiago et al. ican species in other genera have recently been shown 2013; Myllys et al. 2014; Burkin & Kononenko 2015; to represent complexes of species with more restricted Shukla et al. 2015; Herrera-Campos 2016; Paliya et al. distribution, as in the case of Neoprotoparmelia multifera 2016; Gagarina et al. 2017; Galinato et al. 2017, 2018; (Nyl.) Garima Singh, Lumbsch & I. Schmitt (Santos et al. Bungartz et al. 2018; Esslinger 2019). All accessions in 2019). Thus, such cases in the genus Usnea need to be this strongly supported clade were from Europe (Articus further explored by attempting to collect samples from et al. 2002; Kelly et al. 2011; Saag et al. 2011; Millanes the type regions. et al. 2014; Araujo 2016). It is unclear whether this taxon The recently established Usnea clerciana, a putative has simply not been sequenced from other areas, but given Galapagos endemic, is an example of a taxon including the geographic coverage of ITS accessions for the genus sexual and asexual morphs in a single species from the and the documented main distribution of U. hirta in North 326 Plant and Fungal Systematics 65(2): 303–357, 2020

America and Europe (Fig. 15), a possible explanation is & Clerc 2016). Usnea subdasaea is one example of that U. hirta is a holarctic taxon, absent from the tropics a species originally sequenced only from Galapagos, but and the Southern Hemisphere, and has simply not yet with additional material reported from continental South been sequenced in its North American and temperate America (Truong et al. 2011, 2013a), and later confirmed Asian range. through sequence data from Brazil (Gerlach et al. 2019a). Of the remaining species, six were also recently estab- A deviating terpenoid chemotype originally identified as lished, namely U. macaronesica from Portugal (Clerc U. subdasaea (Truong et al. 2013a) clustered elsewhere in 2006), with two accessions from that country (Millanes our ITS-based tree (JQ837328) and represents a separate et al. 2014), the saxicolous U. oreophila from Brazil (Ger- lineage, as already stated by Truong et al. (2013a). lach et al. 2019b), U. rubriglabrata from Peru (Truong The identity of sequenced specimens identified as et al. 2013a; Truong & Clerc 2016), U. subaranea from Usnea malmei is most certainly incorrect. The pendu- Colombia (Truong & Clerc 2016), U. subdasaea from lous, sorediate species with conspicuous annular cracks Galapagos (Truong et al. 2011, 2013a), and U. subparvula and elongate pseudocyphellae, producing norstictic and from Brazil (Gerlach et al. 2017). Usnea macaronesica salazinic acid and the unknown substances UP1 and clustered with the singleton U. subglabrata from Bolivia UP2, was described from Brazil (Motyka 1938) and (see above) in a fully supported clade, but both were sep- has since been reported from Mexico (Herrera-Campos arated by seven consistent substitutions (File S3). When et al. 1998; Herrera-Campos 2016), the northern Andes describing U. subglabrata, Truong & Clerc (2016) did not (Truong et al. 2013b) and Argentina (Calvelo & Liber- discuss the new species with U. macaronesica, a species atore 2002). Yet, the four available sequences all stem established only ten years prior (Clerc 2006), although from Taiwan. Two unpublished sequences (FJ494937, both are remarkably similar. One would be tempted to FJ494938) were deposited by Y.-M. Shen et al. in 2008 subsume U. subglabrata as synonym under U. macarone- and two additional accessions (KU863002, KU863003) sica, but the notable differences in the ITS, together with originated from a DNA barcoding approach (Greenwood the geographic distance of the samples, suggest that this et al. 2016), who apparently took up the identification as is a clade undergoing cryptic speciation. U. malmei based on these previously deposited, unpub- Usnea rubriglabrata is another case where a species lished and almost certainly mislabeled sequences. This may have been based on a non-sequenced type spec- is an exemplar case of a ‘snowballing effect’ in DNA imen with sequences possibly originating from other barcoding, where initially misidentified sequences caused specimens. Truong & Clerc (2016) gave Truong 1877 as subsequently matching depositions to perpetuate this error holotype and Truong 1625 and Truong 1894 as paratypes, (Gilks et al. 2002). The sequenced samples in this case whereas the sequenced vouchers were identified as isolate appear to represent an Asian endemic, as suggested by 2 (JQ837320) and isolate 135 (KP668968), so there is no their nested placement within a larger, unsupported clade way of knowing whether one of these corresponded to of Asian lineages (Fig. 7; Fig. S1). the holotype or not. In the case of U. subaranea, based In contrast, the following five Asian species were all on type material from Colombia (Truong & Clerc 2016), represented by ITS accessions either from the type region the two ITS accessions were definitely non-type material or at least near the type region: U. masudana, described from Ecuador and Peru (Truong et al. 2013a; Truong from Taiwan (Asahina 1970) and with sequences from that

Figure 15. World distribution of Usnea hirta according to the Global Biodiversity Information Facility, GBIF [https://www.gbif.org/species/2606030]; the tropical and Southern Hemisphere records are not that species. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 327 region (Shen et al. 2012); U. nidifica, described from Oce- 34 accessions, including several from the type region, but ania (Norfolk Island; Taylor 1847) and with two unpub- was akin to a species complex, with considerable internal lished accessions from Taiwan; U. nipparensis, described structure (Fig. 7; Fig. S1). from Japan (Asahina 1956), with accessions from Japan Data for other markers were available for some of (Ohmura 2002, 2011) and unpublished sequences from these species, mostly RPB1 and MCM7, but also IGS, China; U. pangiana, described from India (Stirton 1883), TUB2, and RPB2 (Lumbsch & Wirtz 2011; Saag et al. with sequences from Japan (Ohmura 2002) and China 2011; Wirtz et al. 2012; Mark et al. 2016a; Araujo 2016; (unpubl.), and U. pseudogatae Asahina, described from Gerlach et al. 2017, 2019a). IGS data of four species Taiwan (Asahina 1970) and with sequences from the same rendered three of these monophyletic, although with area (Shen et al. 2012). In the cases of U. masudana and internal structure, namely U. acanthella, U. cavernosa, U. pseudogatae, the sequences were only identified in the and U. silesiaca. Usnea wasmuthii formed a paraphy- corresponding paper (Shen et al. 2012), but not in the letic grade and had one conflicting sequence (was-03) original depositions (see above). Usnea pangiana fell in clustering with U. subfloridana (Fig. 16; Fig. S3). Data a clade with several other sequences on long branches, of TUB2 were available for six taxa, with two singletons including three additional unpublished accessions, depos- (U. dasaea, U. flavocardia), two monophyletic clades ited by S. Guo et al. in 2019 and one sequence under lacking support and with considerable internal variation the name U. pangiana (AB051653; Ohmura 2002), but (U. cavernosa, U. hirta), and two polyphyletic assem- apparently not representing that species in the strict sense. blages, suggesting the potential formation of paralogs This clade requires further study. (U. silesiaca, U. wasmuthii; Fig. 16; Fig. S4). The remaining three species were historically described RPB1 offered data for 13 taxa, six of them single- from the Americas or Europe: U. parvula from Argentina tons (U. cladocarpa, U. densirostra, U. fleigiae, U. gran- (Motyka 1938), with accessions from southern Brazil dispora, U. parvula, U. subparvula). Five further taxa (Gerlach et al. 2017); U. silesiaca, from Poland (Motyka resulted monophyletic and internally homogeneous, 1930), with sequences from Portugal and North America namely U. acanthella, U. erinacea, U. flammea, U. hirta, (Araujo 2016; Mark et al. 2016a) and an apparently mis- and U. silesiaca (Fig. S5). Usnea cavernosa exhibited identified outlier from Ecuador (Truong et al. 2013a); and some internal variation, whereas U. wasmuthii formed U. wasmuthii Räsänen, described from Estonia (Räsänen a paraphyletic grade with internal variation (Fig. S5). The 1931). The latter formed a more or less coherent clade of situation for RPB2 was different; of the three taxa with

Figure 16. Comparison of the phylogenetic structure in six markers of Usnea sect. Usnea including the two species U. cavernosa (beige) and U. wasmuthii (purple). Both in TUB2 and MCM7 the formation of paralogs was evident. Selected corresponding specimens are marked with dots, including an outlier in the IGS (large dot) and one of the paralog clades in MCM7 (small dots). For detailed trees with labels, see Figs S1, S3–7. 328 Plant and Fungal Systematics 65(2): 303–357, 2020

available data, only U. silesiaca was rendered monophy- a monophyletic, unsupported clade next to U. ghattensis letic, although with internal variation (Fig. S6). Usnea and two unidentified, unpublished sequences of unknown cavernosa resulted polyphyletic, suggesting the formation origin, but likely from South Korea, deposited by J.-S. Hur of paralogs, and U. wasmuthii formed a grade plus one in 2011. Additional sequences from São Tomé and Prín- outlier (was-08; Fig. 16). cipe clustered in an unsupported clade in the vicinity with For a total of 18 taxa, data for the MCM7 were avail- two basally emerging lineages representing the unnamed able. One represented a singleton (U. flammea) and only Usnea sp. Afrom the same area (Nadel 2016). one was recovered monophyletic and internally homoge- Thus, Usnea articulata exhibited considerable phy- neous (U. oreophila). Seven taxa exhibited considerably logenetic variation. Seven practically identical sequences internal variation, often more so than with other markers, from Europe formed the backbone of the main clade and namely U. densirostra, U. dodgei, U. fleigiae, U. grandi- four additional sequences fell on long branches. These sora, U. hirta, U. parvula, and U. subparvula (Fig. S7). four sequences were among those determined as of low Two further taxa that were rendered monophyletic with quality (see above), apparently the reason for this aberrant the ITS, U. cladocarpa and U. dasaea, formed paraphy- topology and the overall low clade support. We therefore letic, heterogeneous grades with long internal branches. considered this an artifact, perhaps caused by a licheni- Finally, seven taxa appeared polyphyletic with MCM7, colous fungus specific to this taxon, and as a consequence, suggesting the formation of paralogs, including U. cav- regarded U. articulata s.str. as represented by the group ernosa, U. chilensis, U. erinacea, U. grandispora, U. sile- formed by the seven homogeneous, European accessions, siaca, U. subdasaea, and U. wasmuthii (Fig. 16; Fig. S7). is in line with the origin of the type material. This is an example how sequence quality could affect topology Usnea s.str. – Polyphyletic or highly variable species and hence taxonomic interpretation if sequence data are complexes. A large number of names representing com- not inspected carefully. The African samples, including mon and widespread species were either polyphyletic or 13 from São Tomé and Príncipe (Nadel 2016) and one resulted in highly variable clades in the ITS (and other from Cameroon (Orock et al. 2012) formed four separate, markers), suggesting that potentially more than one spe- partly supported clades. Unfortunately, the accession from cies were involved in these cases. They are outlined and Cameroon was too short to be reliably assessed, miss- discussed in detail as follows. ing the entire ITS1 portion (File S1). The ITS identity matrix indicated the largest clade from São Tomé and Usnea articulata aggregate. A total of 12 sequences Príncipe to represent a different species, whereas the two were deposited under this name in GenBank, mostly from smaller clades closer to U. articulata s.str. can be best Europe including Russia and one from Cameroon, plus interpreted as infraspecific lineages (Fig. 17). According an additional sequence from New Zealand (JX144646), to Nadel (2016), specimens in the large, separate clade submitted as Usnea sp., but in the corresponding paper differed from U. articulata s.str. in the pseudocyphellate identified as U. articulata (Rafat et al. 2015). Further cortex and the barbatic acid chemistry (protocetraric and sequences were reported from Africa (São Tomé and Prín- fumarprotocetraric acid in U. articulata s.str.; Swinscow cipe) by Nadel (2016). The European sequences, together & Krog 1976a; Randlane et al. 2009) and may correspond with five accessions from São Tomé and Príncipe, formed to U. speciosa Motyka described from São Tomé and

Figure 17. Pairwise identity matrix based on the ITS for the Usnea articulata aggregate after removing four low quality accessions and the short sequence from Cameroon. Dark grey = identity below 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate infraspecific lineages. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 329

Figure 18. World distribution of Usnea articulata according to the Global Biodiversity Information Facility, GBIF [https://www.gbif.org/spe- cies/2606030]; based on available sequence data, many of the extra-European records may not represent that species.

Príncipe (Motyka 1936). This clade somehow intergrades but the only available sequence from that region identified with the unnamed Usnea sp. A, although the latter differs with that name was not even related to the European core considerably in morphology and chemistry (Nadel. 2016). clade or any of the African lineages. The two smaller clades closer to U. articulata s.str. lacked pseudocyphellae and differed in medullary Usnea barbata-intermedia-lapponica-substerilis agg- chemistry, both containing psoromic acid (Nadel 2016). regate. A large, unsupported cluster comprising 59 acces- According to Swinscow & Krog (1976a), African mate- sions from North America and Europe contained several rial of U. articulata represents five chemotypes, whereas identifications in largely unresolved patterns (Fig. 7, only one is known from Europe (Randlane et al. 2009). Table S1, Fig. S1), including U. arizonica (1), U. barbata Given the topology, this supports the notion that African (14), U. chaetophora (2), U. dasopoga (3), U. diplotypus U. articulata represents various distinct species, mainly (3), U. cf. glabrescens (1), U. intermedia (11), U. lap- characterized by secondary chemistry and the frequency ponica (17), U. rigida (1), and U. substerilis (5). Most and nature of the pseudocyphellae. Swinscow & Krog of these are from the studies by Saag et al. (2011) and (1976a) listed several names with variable chemistry as Mark et al. (2016a). Usnea arizonica, corresponding to an synonyms of U. articulata, but did not include U. spe- apparently unpublished sequence (AF297732), was estab- ciosa among them. lished as a synonym of U. intermedia by Clerc (2007). The Three other markers had more than one sequence for use of the name U. rigida [as U. rigida (Ach.) Motyka], Usnea articulata, namely nuLSU, TUB2, and RPB1, all corresponding to a sequence (AJ457152) generated by corresponding to the European core clade. In all three Articus et al. (2002), was clarified by Lendemer & Tavares instances, U. articulata s.str. was resolved as supported, (2003a), who established that Motyka (1936) technically monophyletic clade, thus congruent with the ITS data, described a new species to be cited as U. rigida Motyka, but with better resolution (Figs S2, S4–5). thus creating an illegitimate later homonym of U. rigida It must be noted that, while the available sequence data Vain., a species of Eumitria for which Motyka (1936) originated from Europe and Africa, Usnea articulata is had established the replacement name U. welwitschiana regarded as a cosmopolitan species (Fig. 18; Swinscow Motyka. For this reason, Lendemer & Tavares (2003a) & Krog 1976a, 1978, 1988; Elix & McCarthy 1998, 2008; introduced U. quasirigida Lendemer & I. I. Tav. as the Stevens 1999; Clerc 2007, 2011; Galloway 2007; Rand- replacement name for Motyka’s U. rigida. The latter was lane et al. 2009; Schumm & Aptroot 2010; Gumboski listed as a synonym of U. intermedia by Clerc (2007), & Eliasaro 2011; Kelly et al. 2011; Saag et al. 2011; who did not mention the name U. quasirigida, but by Ohmura 2012; Orock et al. 2012; Schoch et al. 2012; extension both U. rigida sensu Motyka and U. quasi- Truong et al. 2013a, b; Millanes et al. 2014; Rafat et al. rigida are to be considered synonyms of U. intermedia. 2015; Nadel 2016; Galinato et al. 2017; Lücking et al. Usnea cf. glabrescens (KU352668; Mark et al. 2016a) 2020c). The sequenced African samples already indicated was apparently a misidentification, as this taxon clusters that U. articulata s.lat. represents a complex of several in another clade. species. A geographically broader sample is therefore nec- This left seven names as identifications in this assem- essary to assess its status, particularly since the species has blage. All of these share a similar chemistry with salazinic also been reported from New Zealand (Galloway 2007), acid as the main compound and often with protocetraric 330 Plant and Fungal Systematics 65(2): 303–357, 2020

as an accessory compound, with a pendent to subpendent provide clear resolution at the species level for U. bar- (barbata, chaetophora, dasopoga, intermedia) or subpe- bata-intermedia vs. U. lapponica-substerilis (Fig. 19). ndent to shrubby (diplotypus, lapponica, substerilis) habit IGS data for this clade were largely congruent with ITS and with soredia and/or isidiomorphs except U. inter- (Fig. S3). RPB1 did not resolve the barbata-intermedia and media, which produces apothecia (Halonen et al. 1998; lapponica-substerilis subgroups, but place one sample in an Clerc 2007, 2011b; Randlane et al. 2009). Given that entirely different, supported clade with U. florida/U. sub- U. dasopoga formed the bulk of accessions in the next floridana (Fig. S5), an indication of a possible paralog. clade (see below), we consider the three identifications The complex situation in this group was also discussed in this clade as incorrect (see also Clerc & Naciri 2021). by Mark et al. (2016a), who, based on a six-marker anal- Two of them clustered with three specimens identified ysis found that this entire assemblage, compared to the as U. diplotypus, one as U. barbata, one as U. chae- dasopoga aggregate (see below), exhibited a thinner cortex tophora, and one unidentified, in a strongly supported and a thicker, lax medulla on average. These authors sug- subclade. This subclade may be considered to represent gested that some of the names established in this complex U. diplotypus s.str., a species characterized by relatively may merely represent morphological variants, but also long isidiomorphs (Halonen et al. 1998; Randlane et al. found a separation of the pendent U. barbata and U. inter- 2009). However, five of the eight samples in this clade media from the subpendent to shrubby U. lapponica and were placed with strong support in the U. dasopoga core U. substerilis, proposing synonymy of U. substerilis under clade in the RPB1 tree, thus providing an unresolved, U. lapponica. We tend to agree with the latter, although the supported conflict (see below). example of U. antarctica vs. U. aurantiacoatra (see below) The remaining accessions formed five clusters: one shows that ITS and even multiple markers may not resolve supported subclade (83%) consisting of four specimens very closely related, recently evolved lineages, which could labeled U. barbata and U. intermedia, one singleton clade then also apply to the two clades formed by mixed samples on a long branch representing U. arizonica, one supported identified asU. barbata and U. intermedia, i.e., each clade subclade (72%) including U. barbata and U. intermedia, potentially representing two species. The importance of one unsupported, homogeneous grade including U. bar- correct phenotypical assessments of sequenced material bata, U. chaetophora, U. intermedia (with one low quality has been shown for this group by Clerc & Naciri (2021). accession: JN009731), U. lapponica, and U. substerilis, Several accessions of fertile material identified asU. inter- and one unsupported clade including U. lapponica and media clustered with U. lapponica, suggesting that the U. substerilis. This topology suggested that U. arizonica latter may have an apotheciate counterpart. may indeed be a good species, although more material needs to be studied and sequenced. The remaining acces- Usnea bismolliuscula-intumescens aggregate. Usnea sions formed three groups that may be interpreted as sepa- bismolliuscula is a sorediate species characterized by rate species, but the correct nomenclature remains unclear. a shrubby to subpendent habit with small perforations The first two clades consisted of mixed assemblies of the on the branch surface and predominantly producing pendent taxa U. barbata (vegetative) and U. intermedia compounds of the stictic acid chemosyndrome (Ohmura (fertile), suggesting that the mode of reproduction may 2001). Usnea intumescens agrees in gross morphology, not be a diagnostic feature in this complex, but that indeed but lacks perforations and produces salazinic or psoromic two otherwise characterized, separate species are present. acid (Ohmura 2001). It was represented by a singleton This situation is similar to what has been found in the sequence (Ohmura 2002). ‘species pair’ Letharia columbiana vs. L. vulpina (Kroken The four sequences of U. bismolliuscula have appar- & Taylor 2001) or in the case of U. florida vs. U. subflor- ently not been published and so no precise voucher idana (see below). The remaining accessions seemed to information was available. The singleton sequence was indicate the separation of a shrubby taxon centered around deposited by J.-S. Hur in 2010 and so is possibly from U. lapponica, so the morphology of accessions identi- South Korea, and the other three sequences, forming fied as U. barbata and U. intermedia in this assemblage a strongly supported clade on a long branch, originated needs to be revised. However, there was no evidence for from Taiwan, deposited by Y.-M. Shen et al. in 2008. It a separation of U. substerilis from U. lapponica. The two was not possible to determine which of the two lineages, taxa are indeed difficult to distinguish, largely based on if any, corresponded to U. bismolliuscula s.str. Usnea the shape of the soralia (Halonen et al. 1998; Randlane bismolliuscula was originally described from Japan, as et al. 2009; Clerc 2011b). The status of U. chaetophora U. molliuscula Vain., an illegitimate later homonym of also remains doubtful. The species is distinguished from U. molliuscula Stirt. (Ohmura 2001, 2012). Thus, it is U. dasopoga mainly by the formation of annular cracks more likely that the singleton presumably from South (Halonen et al. 1998; Randlane et al. 2009), but the acces- Korea represents this taxon, whereas the Taiwanese clade sions identified with that name fell into three different would be a different species. Ohmura (2002) distinguished positions in the tree. Usnea chaetophora has also been two chemotypes, one with the stictic acid complex that considered a morphological variation of U. barbata or was shown to be widespread in Australasia including U. dasopoga and has been synonymized with the latter Japan and Taiwan, and one with thamnolic acid, only (Clerc 2011b, 2016). found in Japan. Types of all examined synonyms rep- The ITS identity matrix separated Usnea diplotypus resented the first chemotype, thus offering no potential from the U. barbata-lapponica aggregate, but did not name for a deviating tropical lineage present in Taiwan. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 331

Figure 19. Pairwise identity matrix based on the ITS for the Usnea barbata-intermedia-lapponica-substerilis aggregate after removing four low quality accessions and the short sequence from Cameroon. Dark grey = identity below 98.5%; light grey = identity exactly 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate insufficiently separated lineages, each with internal identity levels above 98.5%.

Usnea brasiliensis-cornuta aggregate. The Usnea cor- Galinato et al. 2017, 2018; Ohmura & Kashiwadani 2018; nuta aggregate exhibited the most complex situation in the Bungartz et al. 2018; Esslinger 2019; Ohmura & Clerc genus. No less than 100 accessions bear the names cornuta, 2019; Gerlach et al. 2019a; Lendemer et al. 2019; Lücking aff.cornuta , or subcornuta, distributed in six, partly sep- et al. 2020c). Truong et al. (2013a) first demonstrated that arate clades containing a total of 208 successions (Fig. 7, U. cornuta s.lat. was polyphyletic. A much more detailed Fig. S1). Usnea cornuta s.lat. is defined as a shrubby, sore- study of this aggregate, using a multispecies coalescent diate taxon with inflated branches constricted at their point approach, showed that the U. cornuta aggregate (including of attachment, minute soralia producing isidiomorphs U. brasiliensis and U. dasaea) formed 14 different, in along the terminal branches, but the taxon was described part strongly supported lineages with striking correlation as highly variable both morphologically and in the medul- with medullary chemistry (Gerlach et al. 2019a). Several lary chemistry (Clerc 2007; Randlane et al. 2009; Gerlach of these are newly described in this issue (Gerlach et al. et al. 2019a, 2020). It has been reported from all major 2020) and there are also several synonyms available to regions and is considered presumably subcosmopolitan cover potentially distinct lineages (Clerc 2007; Randlane (Awasthi 1986; Clerc 1987, 2004, 2006, 2007, 2011b; et al. 2009). Marcano et al. 1996; Clerc & Herrera-Campos 1997; The main question is which clade corresponds to Halonen et al. 1998; Elix & McCarthy 1998, 2008; Fos Usnea cornuta s.str. The lectotype is from Central Europe & Clerc 2000; Herrera-Campos et al. 2001; Galloway (Germany; Clerc 1987, 2004). Within the six clades in the 2007; Hinds & Hinds 2007; Randlane et al. 2009; Smith ITS tree containing accessions labeled U. cornuta, only et al. 2009; Kelly et al. 2011; Rodriguez et al. 2011a; Saag three contained subclades with specimens from north- et al. 2011; Ohmura 2012; Schoch et al. 2012; Noer et al. ern and central Europe. One subclade corresponded to 2013; Santiago et al. 2013; Truong et al. 2013a; Millanes U. esperantiana s.str. (see below) and a second one to et al. 2014; Araujo 2016, as Usnea sp. 1; Chaker 2016; U. flammea. This pointed to a third clade with accessions Herrera-Campos 2016; Truong & Clerc 2016; Ohmura from Great Britain, France, Spain, Madeira, and Brazil et al. 2017; Rodríguez-R. et al. 2017; Timbreza et al. 2017; (Kelly et al. 2011; Truong et al. 2013a; Gerlach et al. 332 Plant and Fungal Systematics 65(2): 303–357, 2020

2019a), as representing U. cornuta s.str. (Fig. 7, Fig. S1). This assessment agrees with Gerlach et al. (2019a), who considered U. cornuta s.str. to form part of their line- age 5, and the clade here defined as U. cornuta s.str. corresponded to most of their lineage 5A. Gerlach et al. (2019a) also combined other small lin- eages in their lineage 5, largely based on the formation of a single clade with RPB1 (Fig. S5), but the ITS data suggest that these may not belong to U. cornuta s.str. They were here tentatively labeled U. aff. cornuta 1 (Northern Hemisphere), U. aff. cornuta 2 (Peru), U. aff. cornuta 3 (Great Britain), U. aff.cornuta 4–7 (all Brazil), and U. aff. Figure 20. Pairwise identity matrix based on the ITS for the Usnea ar- cornuta 8–9 (USA). A further clade of two unpublished ianae clade. Dark grey = identity below 98.5%; the blue vs. beige areas sequences identified as U. cornuta from Taiwan, depos- indicate separate species, each with internal identity levels above 98.5%. ited by Y.-M. Shen et al. in 2008, was labeled U. aff. cornuta 10. This clade requires further examination as U. arianae remained heterogeneous (Fig. 7, Fig. S1), as the two sequences include numerous odd base calls (see also shown in the ITS identity matrix (Fig. 20), whereas above) and U. cornuta has not been reported from Taiwan RPB1 resolved these samples in a single clade (Fig. S5). (Ohmura 2001). A similar species described from Japan Also, the ITS revealed a subclade of two unidentified and also reported from Taiwan, U. confusa, had been sequences from New Zealand (Buckley et al. 2014) nested synonymized with U. cornuta by Clerc (2004). ITS data within U. arianae based on the long branch not repre- suggested that U. confusa s.lat. formed two distinct clades senting that species in the strict sense. Thus, U. arianae with unresolved affinities to U. cornuta s.str. (Ohmura appears to be an evolving, subcosmopolitan species com- & Clerc 2019), although the authors of that study did not plex. A large, unsupported clade with several supported arrive at a conclusion of whether U. confusa was separate subclades contained accessions labeled U. aff. cornuta from U. cornuta. In our expanded data set, the speci- from South America (Truong et al. 2013a; Gerlach et al. mens labeled U. confusa in Ohmura & Clerc (2019) also 2019a) and unidentified accessions from New Zealand formed two clades, clustering in the immediate vicinity (Buckley et al. 2014). The South American subclades of U. cornuta s.str. The first clade also included spec- correspond to lineages 14A–C in Gerlach et al. (2019a) imens from Brazil and Macaronesia and was relabeled and may represent at least four taxa, here labeled U. aff. U. aff. cornuta 1, whereas the second was considered to arianae 1–4. represent U. confusa s.str. Overall, the large containing A singleton sequence from Ecuador (JQ837326) labe- clade of 116 accessions represented the following species led U. subcornuta (Truong et al. 2013a) apparently does and subclades: U. subflammea, U. halei, U. flammea, the not represent that species (see below) and remained uni- newly recognized U. tenuicorticata and U. trachyclada dentified. Two sequences labeledU. cornuta (JN086281, (Gerlach et al. 2020), U. cornuta s.str., U. aff. cornuta JN086282) from Portugal (Saag et al. 2011) clustered out- 1–10, U. cirrosa, U. cladocarpa, the newly recognized side the larger clades representing the U. cornuta aggre- U. stipitata (Gerlach et al. 2020), and a large, homogene- gate and instead in the vicinity of U. subscabrosa Nyl. ous clade representing an unidentified species from New ex Motyka. Their identifications also remained unclear. Zealand (Fig. 7, Table S1, Fig. S1). Finally, a clade of 17 accessions from Central and South While this complex situation only covered the largest America, deposited under U. cornuta and U. aff. cor- of the six clades, the second largest clade with 44 acces- nuta 3, the latter corresponding to lineage 9 in Gerlach sions corresponded mostly to the U. esperantiana aggre- et al. (2019a), represent the U. subpectinata aggregate gate (see below). Four of the five accessions deposited (see below). under the name U. aff. cornuta in this clade represented Usnea brasiliensis was considered a subspecies of a newly recognized species, U. kriegeriana ad int. (Ger- U. cornuta by Clerc (2007), but later resurrected based on lach et al., in prep.), which had been labeled sp. 5 or morphological, chemical and molecular data (Pérez-Var- lineage 18 by Gerlach et al. (2019a) and is characterized gas et al. 2010a; Truong et al. 2013a; Bungartz et al. 2018; by a protocetraric acid chemistry. The remaining accession Gerlach et al. 2019a). In the ITS-based phylogeny, eight (MF669829) corresponded to lineage 6 in Gerlach et al. specimens were labeled U. brasiliensis, one from Madeira (2019a) and was here labeled U. aff. kriegeriana. (Truong et al. 2013a) and seven from Brazil and Costa The third largest clade included 28 accessions rep- Rica (Gerlach et al. 2019a). These formed three separate, resenting South America, the Mediterranean, and New unrelated clades: one strongly supported clade from Brazil Zealand. Named species in this clade included U. crocata was identified as U. brasiliensis s.str., a singleton from (South America: Andes), U. grandispora (Brazil), U. mac- Brazil (MF669810) was described as U. flabelliformis, aronesica (Azores), and U. subglabrata (Bolivia). Two and another, unsupported clade from Costa Rica, Bra- newly recognized species were U. arianae from South zil and Madeira was named U. tenuicorticata (Gerlach America and Europe and U. rubropallens from Brazil et al. 2020). (Gerlach et al. 2020), corresponding to lineages 11 and No data were available for most of this complex in the 13 in Gerlach et al. (2019a). However, based on ITS data, IGS, TUB2 and RPB2. RPB1 did provide less resolution R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 333 overall, mostly not discriminating between the lineages above), but in the latter case, studies with other markers affine to Usnea cornuta s.str., but distinct in the ITS showed that both morphs are indeed phylogenetically (Fig. S5). A similar picture was found for MCM7 that different (Grewe et al. 2018; Lagostina et al. 2018). There- separated the now segregated tropical species (Gerlach fore, caution should be applied before synonymizing taxa et al. 2020) rather well. However, one accession of U. ten- based on lack of resolution in the ITS. uicorticata did not cluster with the other two (Fig. S7). In The other three clades with specimens labeled Usnea summary, the highly complex Usnea cornuta aggregate ceratina formed two strongly supported clades from Japan corresponds to numerous different species, largely with and Taiwan and one unsupported clade from North Amer- a strong correlation with medullary chemistry, as already ica, Brazil, and Portugal. These clades were not clearly outlined by Gerlach et al. (2019a). separated in our identity matrix and could conservatively be considered a single species, although up to three spe- Usnea ceratina aggregate. Usnea ceratina is a (sub-) cies could be distinguished if a more progressive concept pendulous, sorediate species characterized by a pink was applied. The two Asian clades are well separated medulla and the production of diffractaic and barbatic from each other in their identity values, but both overlap acids (Truong & Clerc 2012). It is presumably cosmopo- with the American-Mediterranean clade (Fig. 21). In any litan (Marcano et al. 1996; Herrera-Campos et al. 1998; case, the members of this clade are not conspecific with Elix & McCarthy 1998, 2008; Ohmura 2001, 2002, 2012; U. ceratina s.str. and cannot be identified with that name. Articus et al. 2002; Calvelo & Liberatore 2002; Clerc For the American-Mediterranean clade, three names are 2007; Randlane et al. 2009; Kelly et al. 2011; Truong potentially available, U. calicornica Herre, U. subco- & Clerc 2012; Millanes et al. 2014; Herrera-Campos mosa Vain., U. pachyclada Motyka, and U. solida Motyka 2016; Sipman & Aguirre-C. 2016; Mark et al. 2016a; (Herrera-Campos et al. 1998; Halonen et al. 1999; Clerc Rodríguez-Flakus et al. 2016; Galinato et al. 2017; Ess- 2007; Truong & Clerc 2012), U. california having priority. linger 2019; Gerlach et al. 2019a; Lücking et al. 2020c). Ohmura (2001) listed two species-level synonyms from Our ITS-based phylogeny divided Usnea ceratina into Japan under U. ceratina, namely U. roseola Vain. and four clades, three smaller clades in a supported, monophy- U. subroseola Asahina, which would then be available letic group and one large, separate clade (Fig. 7, Table S1, for one or both of the Asian clades. Here, we tentatively Fig. S1). The large clade, comprised of 14 specimens from used the name roseola (Table S1). Notably, the chemis- Central Europe, Great Britain, and North America, was try of most Asian samples of U. ceratina appears to be clearly distinct from the three smaller clades (Fig. 21). It different, in addition to diffractaic and barbatic acids also had nine fully consistent and several partial substitutions including baeomycesic and squamatic acid, which would compared to the smaller clades (File S1). Given that the lend support for the division of the smaller clade into three lectotype of U. ceratina is from Poland (Ohmura 2001; entities. The name U. roseola is actually in use, having Truong & Clerc 2012), this clade was here interpreted been applied to material from North America (Esslinger as U. ceratina s.str. This clade also included a fertile 2019), Africa (Swinscow & Krog 1979, 1988), and Aus- specimen from Spain identified as U. cristatula (Araujo tralasia (Stevens 1999; Singh & Sinha 2010). However, 2016), which is considered the apotheciate counterpart of it has not been associated with any sequenced specimen U. ceratina (Clerc 2011a; Truong & Clerc 2012; Gerlach so far and hence its use must be revised. With the data et al. 2017). ITS did not resolve these as separate lineages, at hand, the situation cannot be fully resolved, but for a similar case as for U. florida versus U. subfloridana (see the American-Mediterranean clade, one could adopt the below) and U. antarctica versus U. aurantiacoatra (see name U. californica.

Figure 21. Pairwise identity matrix based on the ITS for the Usnea ceratina aggregate. Dark grey = identity below 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate insufficiently separated lineages. 334 Plant and Fungal Systematics 65(2): 303–357, 2020

There is also a rare chemotype with barbatic acid as a smaller number of specimens (not sequenced) produce only major compound, reported from both the Americas salazinic and barbatic acids (Clerc & Otte 2018). Usnea and Asia (Ohmura 2001; Truong & Clerc 2012). In Asia, wasmuthii is characterized by salazinic and barbatic acids the name U. roseola subsp. pseudoroseola Asahina cor- as major compounds (Ohmura 2001; Randlane et al. 2009; responds to this chemotype (Ohmura 2001). Clerc & Otte 2018). This taxon clustered in a subsequent clade (Fig. 7, Fig. S1) and hence the two identifications Usnea dasaea aggregate. Usnea dasaea is a shrubby in the U. dasopoga aggregate apparently do not represent to subpendent species with branches constricted at their that species. The small clade including these accessions insertion and with numerous fibrils, producing soralia with was here considered as U. aff.viktoriana , as the sequences isidiomorphs, and with galbinic, norstictic and salazinic in this portion all share a characteristic ITS sequence acids as major substances. It presumably has a cosmopol- motif at the end of the ITS2 (File S1). The singleton itan distribution (Clerc 2007; Randlane et al. 2009) and sequence labeled U. silesiaca in this aggregate from Ecua- a large number of listed synonyms, including U. dolosa dor (JQ837331; Truong et al. 2013a) contained odd base Motyka, U. galbinifera Asahina, U. kinkiensis Asahina, calls and its correct identification is uncertain.U. silesiaca U. kyotoensis Asahina, U. spinigera Asahina, U. spinulif- s.str. clustered in another part of the tree. era (Vain.) Motyka and U. undulata Stirt. (Clerc & Her- The remaining accessions formed a supported clade rera-Campos 1997; Ohmura 2001). (74%) with three clusters: a basal grade of four accessions A total of 13 ITS accessions were deposited under this identified as U. barbata and U. cf. cylindrica, a strongly name in GenBank. Given that U. dasaea was described supported subclade of six accessions identified asU. bar- from Portugal (Stirton 1881), the clade of two accessions bata, U. dasopoga, and U. substerilis, and a remaining from that country (JN086283, JN086284; Saag et al. 2011) shallow grade including the bulk of U. dasopoga iden- likely represents that species. The remaining 11 acces- tifications (Fig. 7, Fig. S1). The entire clade originated sions represented several other taxa, such as U. gran- from the Northern Hemisphere, mostly from Europe disora (MF669881), U. aff. perhispidella (MF669883), (Table S1). Usnea cylindrica was only recently described U. subdasaea (four accessions), and U. subpectinata from Sweden (Clerc 2011b), differing in branching pattern (MF669884), all from Brazil (Gerlach et al. 2019a). from U. dasopoga, and the only available sequence ten- Another Brazilian accession (MF669878; Gerlach et al. tatively identified with that name by Mark et al. (2016a) 2019a) might represent genuine U. spinulifera, whereas an is probably not that species, a suspicion confirmed by accession from Ecuador (JQ837306; Truong et al. 2013a) Clerc & Naciri (2021). Usnea barbata may be simi- belongs to an as of yet undescribed species labeled Usnea lar to U. dasopoga and best separated by the thickness sp. 6 in Gerlach et al. (2019a). Two further accessions from of the cortex and the extension and consistency of the Japan (AB051056; Ohmura 2002) and Peru (JQ837305; medulla (Randlane et al. 2009; Mark et al. 2016a), and Truong et al. 2013a) represent unnamed lineages. so the identifications as U. barbata in this clade refer to U. dasopoga (see also Clerc & Naciri 2021). As a conse- Usnea dasopoga-viktoriana aggregate. This aggregate quence, we consider this clade to represent U. dasopoga formed an unsupported clade following the U. barba- s.str., with the nested supported subclade requiring fur- ta-intermedia-lapponica aggregate (see above). It was ther study. Apotheciate specimens of U. dasopoga were also found associated with the latter in the six-marker reported by Lukáč (2010), and so the samples identified analysis by Mark et al. (2016a), who described the two as U. barbata in this clade were presumed to represent clusters to differ in the thickness of the cortex and the fertile U. dasopoga. extension and consistency of the medulla, while largely The Usnea dasopoga core clade was well supported in agreeing in chemistry. The aggregate contained 43 sam- both the nuLSU (few data) and the RPB1 (Fig. S5). How- ples labeled with the following identifications:U. barbata ever, the ITS subclade was not obvious from RPB1 data (7), U. cf. cylindrica (1), U. chaetophora (2), U. dasopoga and all specimens were relatively uniform, a pattern also (5), U. diplotypus (2), U. filipendula (17), U. lapponica seen with IGS (Fig. S3). Notably, the dasopoga clade in (2), U. silesiaca (1), U. substerilis (3), U. viktoriana (as RPB1 contained at least two samples (BAR-08, CHE-09) U. praetervisa; 3), U. wasmuthii (2), and Usnea sp. (1). which based on ITS clustered in the strongly supported Usnea filipendula has been established as a synonym of diplotypus clade in the barbata-intermedia-lapponica U. dasopoga and the latter as the correct spelling of what aggregate (see above). This supported conflict needs to has often been named U. dasypoga (Arcadia 2013). Thus, be examined further. Mark et al. (2016a) detected similar the bulk of identifications in this aggregate corresponded conflicts, but did not further elaborate on these. to U. dasopoga (22). Overall, this aggregate was subdivided into several Usnea endochrysea-mutabilis-strigosa aggregate. This clusters. A singleton and a doubleton clade were located at aggregate was strongly supported on a long branch in our the base, including the two likely erroneous identifications ITS-based phylogeny (Fig. 7, Fig. S1). Few data were as U. wasmuthii, close to an unsupported clade containing available from other markers, with U. endochrysea and a strongly supported subclade of three sequences origi- U. mutabilis forming a monophyletic clade with MCM7 nally identified as U. praetervisa (Mark et al. 2016a), (Fig. S7) and U. mutabilis a monophyletic clade in TUB2 but correctly named U. viktoriana (Clerc & Otte 2018). (Fig. S4). With nuLSU, this clade was not recovered as The latter is characterized by alectorialic acid, although monophyletic (Fig. S2). R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 335

This clade contains species usually having a (pink-) currently not possible to resolve this complex taxonom- red-pigmented medulla (pigment identified as eumitrin ically or to determine which clade, if at all, represented A2 in U. mutabilis) and norstictic acid as the major com- U. strigosa and U. endochrysea s.str. Broader sampling pound, although U. strigosa s.lat. has also been reported and detailed documentation of potentially important char- as having a white medulla and producing diffractaic, acters, such as chemistry and ascospore sizes, is required, psoromic or thamnolic acid, and U. mutabilis as lacking as well as reinvestigation of currently listed synonyms. norstictic acid and producing fatty acids of the murolic acid complex (Hale 1962; Ohmura 2001; Clerc 2007; Usnea erinacea-moreliana-steineri aggregate. This Randlane et al. 2009; Lendemer et al. 2016). Usnea stri- unsupported clade of 19 accessions contained four names gosa and U. endochrysea produce apothecia and differ that were not resolved in reciprocal monophyly by ITS. chiefly in ascospore size (smaller vs. larger than 9 µm), All have cortical or subcortical reddish pigments. While although there are conflicting reports in the literature U. erinacea and U. steineri are apotheciate, U. more- (Clerc 2007; Lendemer et al. 2016). Usnea mutabilis liana (including U. rubricornuta) is sorediate. Chemi- is sorediate. All three species were originally described cally, terpenoides such as UT6 are common (diagnostic in from North America (Acharius 1803; Stirton 1881), but U. moreliana) and U. erinacea also contains protocetraric, U. strigosa is considered widespread in the Americas and norstictic, salazinic and/or stictic acid (Truong et al. 2011; U. mutabilis subcosmopolitan (Clerc & Herrera-Campos Truong & Clerc 2016; Gerlach et al. 2017). 1997; LaGreca 1999; Fos & Clerc 2000; Vareschi 2001; Usnea rubricornuta was established by Truong et al. Ohmura 2001, 2002; Calvelo & Liberatore 2002; Clerc (2011) for a neotropical taxon, but this name was later 2007; Mercado-Díaz 2009; Randlane et al. 2009; Saag synonymized with U. moreliana (Truong & Clerc 2016). et al. 2011; Schmull et al. 2011; McDonald et al. 2013; Usnea moreliana (as U. rubricornuta) formed a paraphyl- Herrera-Campos 2016; Ohmura & Kashiwadani 2018; etic grade relative to U. steineri in the analysis by Truong Funk et al. 2018; Esslinger 2019; Dorey et al. 2019; Len- et al. (2013a), but the inclusion of additional samples demer et al. 2019; Lücking et al. 2020c). (Gerlach et al. 2017, 2019a) has rendered the picture more ITS data did not resolve the three taxa as reciprocally complex. Thus, while U. moreliana s.str. in our tree clus- monophyletic and the entire clade exhibited substantial tered in a well-supported clade including one accession structure. Thus, the six specimens identified asU. strigosa of U. rubricornuta from Bolivia (JQ837323), three other appeared on four clades, two clades clustering on a sup- sequences originally identified as U. rubricornuta fell ported, early diverging branch, one clade clustering with outside this clade, one from Brazil clustering with U. eri- specimens identified asU. endochrysea, and one specimen nacea (JQ837322) and another from Peru (JQ837324) nested within the strongly supported U. mutabilis clade, with a sequence apparently misidentified asU. rubicunda. suggesting an apotheciate morph of the latter. Usnea muta- It is unclear whether the Bolivian accession actually cor- bilis formed five supported subclades, but based on the responds to the type of U. rubricornuta, as in the various identity matrix (Fig. 22), only the two specimens from publications, no link was given between the DNA voucher Japan could be potentially considered a separate taxon. number of the Bolivian accession (34) and the collec- Specimens identified as U. strigosa and U. endochry- tion number of the type (Truong 3132) and more than sea could be interpreted as representing potentially four one collection from Bolivia was cited in the protologue. lineages, two singletons each corresponding to U. stri- The synonymy of U. rubricornuta with U. moreliana is gosa (MH310890) and U. endochrysea (MH310883), therefore not definitely established. one subclade containing two specimens of U. strigosa Usnea steineri formed three distinct lineages on long (AF112990, MN038162), and one with U. endochrysea branches. Samples identified as U. erinacea formed an (MH310884) and U. strigosa (HQ650687, MH310884) unsupported clade including one accession of U. rubri- intermingled. Unfortunately, with the data at hand it is cornuta. Sequences labeled U. erinacea were also found outside this aggregate, variously clustering with sequences identified as U. rubicunda s.lat. (see below), confirming the notion that U. erinacea as currently understood is polyphyletic (Truong et al. 2013a; Gerlach et al. 2017). Another pigmented, apotheciate taxon, Usnea san- guinea, was described from Africa and used to name African and Australian samples corresponding to this morphology (Swinscow & Krog 1979, 1988; Stevens 1999; Nadel 2016). Clerc (2004, 2007) synonymized U. sanguinea with U. erinacea. A sequence from São Tomé and Principe labeled U. aff. sanguinea (Nadel 2016) fell far from the U. erinacea complex (Fig. 7; Fig. S1), initially supporting the hypothesis that U. san- guinea represents a separate species. This interpretation Figure 22. Pairwise identity matrix based on the ITS for the Usnea endochrysea-mutabilis-strigosa aggregate. Dark grey = identity below is, however, tentative, as the single specimen from São 98.5%; the blue vs. beige areas indicate separate species, whereas the Tomé and Principe differs in medullary chemistry from differently shaded beige areas indicate insufficiently separated lineages. the type of U. sanguinea (barbatic vs. norstictic and 336 Plant and Fungal Systematics 65(2): 303–357, 2020

salazinic acid) and was sterile, lacking both apothecia New Zealand, a paraphyletic grade of three specimens, and soredia (Nadel. 2016). two unidentified from New Zealand and one identified Data from the RPB1 clustered the accessions labeled from Great Britain, and a remaining, more or less sup- U. rubicunda within U. moreliana, which in turn clus- ported (72%) clade of 14 specimens. That larger clade tered with support with U. steineri, whereas U. erinacea contained mostly samples from Europe identified as formed an unsupported clade next to this clade (Fig. S5). U. esperantiana (Kelly et al. 2011; Schoch et al. 2012), Overall, this suggests largely a distinction of three species but also one sample identified as U. aff. glabrata from complexes, U. erinacea, U. moreliana, and U. steineri, but France (Gerlach et al. 2019a), one unpublished sequence this aggregate requires further study based on a broader from Taiwan, deposited under the name U. glabrata by sample. Notably, the MCM7 did not resolve these taxa Y.-M. Shen et al. in 2008, and one unpublished and uni- as clusters, but exhibited a confusing pattern suggesting dentified sequence from China, deposited by L. Han et al. the formation of paralogs (Fig. S7). in 2020. The identity matrix (Fig. 23) suggested that the Usnea esperantiana aggregate. Usnea esperantiana first clade, exclusively from New Zealand, represented is a shrubby species forming large soralia and produc- a separate species, its identification pending further study ing salazinic and bourgeanic acids (Clerc 1992, 2007). It based on authentic material. The status of the remaining appears to be a widespread Northern Hemisphere species accessions from New Zealand cannot be assessed. They reported from North America, Europe, Africa, and Asia formed a paraphyletic grade with high similarity to Usnea (Mies 1989; Clerc 2007; Randlane et al. 2009; Kelly et al. esperantiana s.str. (Fig. 23), the latter also including two 2011; Saag et al. 2011; Herrera-Campos 2016; Galinato samples from New Zealand, implying that this species also et al. 2017; Esslinger 2019). occurs there. Unfortunately, since no voucher material In our global phylogeny, sequences deposited under was preserved for these sequences (see above), verifica- that name formed a monophyletic clade, but with clear tion will only be possible with newly collected, properly internal structure and associated with numerous uniden- vouchered and sequenced specimens from that region. tified sequences from New Zealand (Fig. 7, Table S1, Fig. S1). The entire clade was strongly supported (90%) Usnea florida-parafloridana-subfloridana aggregate. and contained five clusters: a fully supported subclade of Usnea florida and U. subfloridana have been considered 11 unidentified specimens from New Zealand (Buckley a classic example of a ‘species pair’ (Clerc 1984a) and et al. 2014), a more or less supported clade (70%) of were the first complex to be studied with molecular data another seven unidentified specimens from New Zealand (Articus et al. 2002). Using ITS, nuLSU and TUB2, from that same study, a further unidentified singleton from these authors found that the species pair concept was

Figure 23. Pairwise identity matrix based on the ITS for the Usnea esperantiana aggregate. Dark grey = identity below 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate insufficiently separated lineages. R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 337

Figure 24. Pairwise identity matrix based on the ITS for the Usnea floridana-subfloridana aggregate. Dark grey = identity below 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate insufficiently separated lineages. not supported and that instead the studied specimens resolved U. praetervisa as to separate, unrelated clades, formed various clades uncorrelated with reproductive suggesting a paralog (Fig. S7). mode or chemistry. Both taxa have mostly been reported Mark et al. (2016a) suggested that U. florida and to contain squamatic and/or thamnolic acid, sometimes U. subfloridana may be synonyms, but this was not in in combination with alectorialic acid (Articus et al. 2002; accordance with the complex topology in the ITS data, Randlane et al. 2009), but for U. florida also norstictic which suggested that several lineages are involved, in acid has been listed (Clerc 2007). In their six-marker part with mixed reproductive strategies, similar to the study, Mark et al. (2016a) also found these taxa to form situation in Letharia colombiana and L. vulpina (Kroken several clades, including one characterized by a norstictic & Taylor 2001). Indeed, specimens identified as Usnea acid chemistry newly described as U. parafloridana. The florida or U. subfloridana clustered in regions in the tree: authors thereby overlooked that the name U. praetervisa one supported clade (77%) containing seven accessions was available for this clade (Clerc & Otte 2018). Most of bearing either name (plus an apparently misidentified the ITS sequences for U. praetervisa (as U. parafloridana) U. wasmuthii) from across the Northern Hemisphere; formed a homogeneous, weakly supported clade (66%), one strongly supported clade (99%) containing five with one sequence (KU352670; WW-002) clustering at specimens from North America and Europe identified as the base of that clade and another (KU352708; WW-151) U. subfloridana; a weakly supported clade (69%) with clustering elsewhere. seven specimens of U. subfloridana from North America; IGS resolved U. praetervisa (as U. parafloridana) as another weakly supported clade (68%) with 14 accessions a fully supported clade, with the exception of one acces- bearing either name from North America and Europe; sion (KU352588; WW-151), i.e., the same that did not and a large, rather homogeneous assembly of 50 samples cluster with the others in the ITS (Fig. S3). In contrast, also bearing both names from across the Northern Hemi- with RPB1, U. praetervisa (as U. parafloridana) formed sphere (Fig. 7, Fig. S1). The latter included a low-quality one grade and one weakly supported clade (72%), the sequence (AF117996) on a long branch, explaining the latter also including the two outliers (Fig. S5). Thus, while lack of support for this clade. These clades were separated ITS and IGS were largely in line, RPB1 provided a sup- by three other species, namely U. wasmuthii, U. fulvore- ported conflict and also offered less resolution. MCM7 agens and U. glabrescens. The ITS identity matrix only 338 Plant and Fungal Systematics 65(2): 303–357, 2020

supported the consideration of the strongly supported (Articus 2004) were not available, but their phylogenetic second clade as a potentially distinct species (Fig. 24). placement suggests a more complex situation of deviating With RPB1, the five clades were not resolved, the chemotypes in relation to the U. cornuta aggregate. corresponding specimens forming a single, supported clade (75%) that included a nested, strongly supported Usnea fulvoreagens-glabrescens-pacificana aggregate. (95%) subclade (Fig. S5). The distribution of specimens Usnea fulvoreagens and U. glabrescens are shrubby, sore- in the RPB1 subclades was not correlated with the for- diate species forming large soralia and with a variable mation of subclades in the ITS, providing a minor, yet chemistry, mostly norstictic acid, but usually in variable supported conflict between ITS andRPB1 when it comes combinations with other main or accessory compounds, to species delimitation (see above). In this case, MCM7 such as stictic, salazinic, protocetraric, diffractaic, or also resolved the U. florida-subfloridana complex in two squamatic acids (Clerc 2007; Randlane et al. 2009). The separate, unrelated clades, suggesting a paralog situa- main difference is in the shape of the soralia: ring-shaped, tion, particularly as in both cases the U. praetervisa and becoming excavate and lacking isidiomorphs in U. fulvore- U. florida/U. subfloridana haplotypes clustered together agens vs. punctiform to maculiform, remaining discrete (Fig. 25; Fig. S7). and producing isidiomorphs in U. glabrescens (Randlane It therefore remains unclear how many species are et al. 2009). In Far East Russia and Asia, U. fulvoreagens to be distinguished in this complex, what the diagnostic contains also zeorin, whereas U. glabrescens does not characters would be and whether the reproductive mode (Ohmura 2001, 2012; Ohmura et al. 2017). According played any role in that, and which clade corresponds to the Halonen et al. (1999), the delimitation of U. fulvoreagens taxa in the strict sense. Particularly, the largest florida-sub- and U. glabrescens can be difficult and U. fulvoreagens floridana clade was reminiscent of the situation in another was considered a synonym of U. glabrescens by Clerc species pair, Usnea antarctica vs. U. aurantiacoatra, in (2011b). Clerc & Otte (2018) opted for the recognition which microsatellite markers and a RADseq approach of two varieties: var. glabrescens, with three chemotypes, demonstrated that the sexual and asexual morphs largely occurring in boreal and montane temperate areas, and formed separate taxa (Lagostina et al. 2018; Grewe et al. var. fulvoreagens Räsänen, also with three chemotypes, 2018). Tõrra et al. (2014) and Degtjarenko et al. (2018, with a boreal-montane to sub-Mediterranean distribution. 2019) developed microsatellite markers for the U. sub- In the ITS-based phylogeny, the two taxa were not floridana complex, but did not report on their potential resolved in reciprocal monophyly, but there was also use to separate U. florida from U. subfloridana. no evidence that they represent a single species. Usnea fulvoreagens formed one well-supported subclade from Usnea fragilescens aggregate. Usnea fragilescens is southern Europe and Japan, one grade from North Amer- a shrubby to subpendent species producing large sora- ica, and one larger, strongly supported clade from north- lia and with a variable chemistry of either stictic and ern and northwestern Europe and North America. This norstictic acids (Europe) or psoromic or salazinic acid separation was also reflected in the IGS and RPB1 data (Americas; Clerc 2007; Randlane et al. 2009). Specimens (Figs S3, S5). We considered the larger subclade to rep- identified with this name mostly formed a homogeneous, resent U. fulvoreagens s.str. and the small subclade and strongly supported clade entirely comprised of European the grade two potentially separate species. Usnea gla- accessions (Kelly et al. 2011; Marthinsen et al. 2019), brescens formed a small grade including two samples which can be considered U. fragilescens s.str. Two spec- originally identified as U. diplotypus and U. substerilis imens from Bolivia, both with salazinic acid (Truong (Saag et al. 2011), plus a larger, unsupported clade from et al. 2013a) clustered at the base of this clade, together across the Northern Hemisphere, including two specimens forming a supported clade (84%). Three further sequences originally identified as U. pacificana and U. substerilis identified asU. fragilescens (AJ748104, AJ748105: Arti- (Fig. 7; Fig. S1). cus 2004; FR799083: Kelly et al. 2011) clustered in the Usnea pacificana was rather recently described from U. cornuta aggregate (see above), a similar species (see North America (Halonen 2000), morphologically similar Clerc & Otte 2018 for differences). to U. glabrescens, but with a chemistry of baeomycesic For nuLSU, data were available for the Bolivian and and squamatic acid. In the protologue, it was not compared European samples (Kelly et al. 2011; Truong et al. 2013a) to U. glabrescens. The species was subsequently also with a similar topology as with ITS (Fig. S2). With RPB1, reported from Turkey (Senkardesler & Clerc 2017). Mark the two available sequences of U. fragilescens, represent- et al. (2016a) listed two ITS accessions under U. pacifi- ing specimens of the European core clade (Kelly et al. cana in their voucher table, one (KU352651) originally 2001) formed a fully supported clade (Fig. S5). No IGS deposited under that name and the other (JN086329) data were available for the species. For MCM7, only the originally named U. substerilis (Saag et al. 2011). Only two Bolivian specimens were sequenced (Truong et al. the second was used in the species delimitation analysis 2013a), also forming a fully supported clade (Fig. S7). by Mark et al. (2016a) and tentatively accepted as repre- These data suggest that Usnea fragilescens is a phy- senting U. pacificana. It came out on a separate, longer logenetically well-defined European species, characterized branch, but this was likely caused by three aberrant base by a stictic acid chemistry, and that the South American calls that may represent reading errors, as they appear in salazinic acid lineage forms a closely related, but separate sites otherwise constant for all other sequences in this species. Chemical data for the two Canadian specimens portion of the tree. Apart from these three base calls, the R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 339

Figure 25. Circle phylogram of Usnea sect. Usnea based on MCM7, highlighting the formation of at least two paralogs in the U. florida-subflor- idana complex and U. praetervisa (originally labeled U. parafloridana), also underlined by the relative position of U. wasmuthii (see Fig. 16 for comparison). The second cluster on the upper right might reflect an additional, recent duplication event.

ITS was identical to that of two other accessions originally ITS-based topology, makes the distinction of U. pacificana labeled U. pacificana (KU352651; Mark et al. 2016a) and based on medullary chemistry questionable. RPB1 data U. glabrescens (AB051639; Ohmura 2002), and the three did not resolve this problem. Although two accessions accessions differed from those ofU. glabescens s.str. only labeled U. pacificana (KU352077, KU352094) formed in one position (File S1). Mark et al. (2016a) gave the a grade basal to U. glabrescens, another accession labe- chemistry of their U. pacificana clade as baeomycesic and led U. cf. fulvoreagens (KU352096), presumably with squamatic acid, but it is unclear whether this refers to the norstictic and salazinic acid, also clustered within that actual specimens or deduced from the published chem- grade (Fig. S5). IGS data, also with two accessions for istry for U. pacificana. The fact that Saag et al. (2011) U. pacificana, did not separate the latter from U. gla- identified the corresponding accession (JN086329) orig- brescens (Fig. S3). inally as U. substerilis suggests that it contains norstictic and salazinic acid, and the same applies to the accession Usnea glabrata aggregate. Usnea glabrata is another labeled U. glabrescens (AB051639) by Ohmura (2002). shrubby, sorediate species, characterized by swollen Mark et al. (2016a) suggested that the baeomycesic and branches, large and excavate soralia lacking isidio- squamatic acid chemotype may occur variably in the morphs, a lax medulla, and usually with protocetraric (and U. glabrescens-fulvoreagens complex, which, given the fumarprotocetraric) acid, although a salazinic-norstictic 340 Plant and Fungal Systematics 65(2): 303–357, 2020

acid chemotype is also known (Clerc 2007; Randlane substitutions: one from Taiwan (FJ494923), one from Japan et al. 2009). Eight ITS accessions have been deposited (AB051049), two from China (MT259216, MT259217), under this name (one as U. aff. glabrata). Two of them and two from Brazil (MF669900, MF669899). The four represented U. esperantiana (see above), whereas the Southeast Asian sequences were deposited under the name remaining six clustered with an unidentified sequence Usnea aciculifera Vain. and the Japanese sample was in a supported clade (78%) sister to U. durietzii (Fig. 7, published under this name (Ohmura 2002), whereas the Fig. S1). These seven accessions formed two lineages: other three sequences have apparently not been published. a singleton sequence from Switzerland on a rather long Clerc (2016) discussed U. aciculifera in connection with branch and a cluster of five sequences from the USA, U. perhispidella, maintaining U. aciculifera as a different Spain and Great Britain on a well-supported branch; the species, with aggregated soralia and short isidiomorphs seventh sequence clustering at the base also belonged rarely forming isidiofibrils. Assuming that the identifica- here but was very short (see above). Assuming that these tions of the Asian specimens are correct, which appears accessions represented two species, it remains unclear likely (Ohmura 2001, 2002, 2012), the ITS data supported which corresponded to U. glabrata s.str., as the latter was the separation of U. aciculifera from U. perhispidella described from Switzerland and so both lineages may and also indicate that both species have a wide, possibly correspond to this taxon. With nuLSU, the two lineages pantropical distribution. In that case, the two Brazilian formed a single, supported and homogeneous cluster. Data specimens should correspond to the aciculifera and not available for the RPB1 corresponded to the second lineage, the perhispidella morphology. forming a fully supported clade on a long branch (Fig. S5). Three further specimens from Brazil (MF669904, MF669879, MF669902) included in the Usnea perhispi- Usnea himalayana-nidifica aggregate. This small, della clade by Gerlach et al. (2019a) did not cluster with strongly supported clade (99%) contained nine acces- the above two groups, but from two separate, small clades sions. Two unidentified accessions from New Zealand phylogenetically more similar to two specimens from (Buckley et al. 2014) formed a fully supported subclade Peru (JQ837305) and Japan (AB051056) identified as on a long branch. A second, strongly supported subclade U. dasaea (Ohmura 2002; Truong et al. 2013a), but appar- (99%) consisted of two unpublished sequences identified ently not representing that species (see above), and a large as U. himalayana from Taiwan, deposited by Y.-M. Shen cluster of unidentified specimens from New Zealand for et al. in 2008. A third, supported subclade (74%) contained which no voucher information was available (Buckley three lineages: two originally unidentified sequences from et al. 2014). The two small U. perhispidella s.lat. clades Taiwan labeled U. pseudogatae in the corresponding were here labeled U. aff.perhispidella 1 (MF669904) and paper (Shen et al. 2012), one sequence from Malaysia U. aff. perhispidella 2 (MF669879 / MF669902). labeled U. himalayana (Ohmura 2002), and two unpub- A specimen identified as Usnea perhispidella from lished sequences from Taiwan identified as U. nidifica, Peru (JQ837290; Truong et al. 2013a) clustered with also deposited by Y.-M. Shen et al. in 2008. Given that a specimen deposited as U. dasaea, but published under U. himalayana was described from India (Babington the name U. perhispidella from Brazil (MF669883; Ger- 1852) and the specimens from tropical southeast Asia lach et al. 2019a). This strongly supported small clade was formed two separate lineages, it is possible that neither found to be more distantly related to the U. perhispidella of these actually represents that species. / U. aciculifera cluster (separated by the U. subpectinata complex, see below) were and is here labeled U. aff. Usnea perhispidella-aciculifera-subpectinata aggre- perhispidella 3. gate. Ten sequences bear the name Usnea perhispidella, The Usnea subpectinata complex formed an unsup- a species described from Kenya (Steiner 1897). This taxon ported clade of accessions originally deposited under is characterized by a subpendulous thallus with a pale base the names U. cornuta and U. aff. cornuta 3 (Gerlach and minute, plain, densely arranged soralia combined with et al. 2019a; see above). Usnea subpectinata, described numerous isidiomorphs and isidiofibrils, and the stictic from Scotland, was considered a synonym of U. cor- acid complex chemistry (Clerc 2016). Nine Brazilian sam- nuta (Clerc 2004, 2006). The clade labeled here with ples identified with this name formed a monophyletic, this name contained mostly accessions from Central and supported clade in the analysis by Gerlach et al. (2019a), South America, but also one from Europe (France). It including also MCM7 and RPB1. however, the internal formed a monophyletic, but unsupported clade in the ITS, structure suggested the presence of more than one species including three supported subclades (Fig. 7, Fig. S1). The (Gerlach et al. 2019a: fig. 2). first of these, here labeled U. aff. subpectinata, could be In our full ITS analysis, several additional specimens considered a separate species based on the number of con- clustered with these samples phylogenetically, forming sistent substitutions (File S1). The other two subclades, four groups. Three samples from Brazil (MF669898, although strongly supported, differed from each other at MF669903, MF669905) and one unpublished sequence the 98.5% threshold, but not from the other samples in of unknown origin, but likely from Southeast Asia this clade. The RPB1 recovered the entire clade (except (HQ671307), formed a cluster here interpreted as U. aff. subpectinata) as well-supported (87%) with only U. perhispidella s.str. Closely related to this cluster was little internal structure and uncorrelated with the subclades a strongly supported clade containing six terminals and in the ITS (Fig. S5). With MCM7, two larger, strongly differing from theU. perhispidella cluster in five consistent supported, but entirely separate clades, were found, plus R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 341 one smaller, supported clade, a potential indication of the A total of 34 ITS sequences have been deposited under presence of three paralogs (Fig. S7). the name Usnea rubicunda, originating from North and South America, Europe, and Asia (Ohmura 2002, 2008; Usnea pygmoidea-orientalis aggregate. This unsup- Kelly et al. 2011; Saag et al. 2011; Schoch et al. 2012; ported clade contained eight accessions, all from Asia: two Truong et al. 2013a; Millanes et al. 2014; Gerlach et al. from Japan identified as U. pygmoidea (Ohmura 2002), 2017, 2019a). Most of these clustered in the same part four from Taiwan identified as U. orientalis (Shen et al. of the tree, but did not form a coherent entity (Fig. 7, 2012), and two unidentified and unpublished sequences Table S1, Fig. S1). One supported clade (83%) contained from China forming a fully supported subclade. Neither 13 samples from North and South America, Macaronesia, U. pygmoidea nor U. orientalis were resolved as mono- the Mediterranean, and Asia, including one sequence from phyletic. The two taxa form shrubby to subpendent thalli Brazil (MF669912) identified as U. aff. rubicunda (Ger- and U. orientalis produces apothecia, whereas U. pyg- lach et al. 2019a) and one unpublished sequence from the moidea forms soralia. U. pygmoidea is considered to USA deposited by Z. R. Caven et al. in 2016, identified as include two chemical races, one corresponding to the U. pennsylvanica, presumably a synonym of U. rubicunda chemistry of U. orientalis in producing salazinic acid as (Clerc 2007). A second, unsupported clade contained 18 major compound (Ohmura 2001). This suggests a complex sequences, one of them (JQ837307) identified asU. erina- situation of a species pair aggregate. cea, apparently a fertile specimen not related to the latter species. A third, internally structured clade was supported Usnea rubicunda-rubrotincta aggregate. Usnea rubi- (83%) and contained mostly unidentified sequences from cunda is one of the most commonly collected species New Zealand (Buckley et al. 2014), one sequence identi- of Usnea, with a presumably cosmopolitan distribution fied as U. rubicunda from New Zealand (Millanes et al. (Fig. 26), characterized by a reddish cortical pigment, 2014), and three sequences labeled U. rubrotincta from small soralia producing isidiomorphs, and a stictic acid Asia (Ohmura 2002; Ohmura & Clerc 2019). chemistry (Swinscow & Krog 1979, 1988; Mies 1989; The first clade contained at least three supported line- Marcano et al. 1996; Elix & McCarthy 1998, 2008; Ste- ages: one with five terminals including the singleU. penn- vens 1999; Aptroot & Seaward 1999; Ohmura 2002, 2008; sylvanica sample from North and South America and Calvelo & Liberatore 2002; Spielmann 2006; Galloway the Mediterranean; a second lineage with three terminals 2007; Clerc 2007; Mercado-Díaz 2009; Randlane et al. from Asia, and a third lineage with five terminals from 2009; Schumm & Aptroot 2010; Singh & Sinha 2010; North and South America and Asia. The second clade Kelly et al. 2011; Saag et al. 2011; Truong et al. 2011, contained mostly a consistent haplotype, present in South 2013a; Millanes et al. 2014; Aptroot 2016; Herrera-Cam- America and Europe, and a few deviating singletons from pos 2016; Sipman & Aguirre-C. 2016; Truong & Clerc South America, Galapagos, and Macaronesia. The third 2016; Ohmura & Kashiwadani 2018; Rodríguez-Flakus clade contained up to four units, all from Asia and/or et al. 2016; Galinato et al. 2017; Gerlach et al. 2017, New Zealand, three of them remaining unnamed and the 2019a; Bungartz et al. 2018; Esslinger 2019; Lücking fourth, a grade with four sequences, bearing the labels et al. 2020c). U. rubicunda and U. rubrotincta. Given that the type

Figure 26. World distribution of Usnea rubicunda s.lat. according to the Global Biodiversity Information Facility, GBIF [https://www.gbif.org/ species/2606096]. 342 Plant and Fungal Systematics 65(2): 303–357, 2020

Figure 27. Pairwise identity matrix based on the ITS for the Usnea rubicunda aggregate. Dark grey = identity below 98.5%; the blue vs. beige areas indicate separate species, whereas the differently shaded beige areas indicate insufficiently separated lineages.

of U. rubicunda is from Great Britain (Stirton 1881), from U. rubicunda. However, all sequenced specimens we considered the cluster including all specimens from identified as U. rubicunda in that study corresponded Great Britain and several from South America as U. rubi- to the lineages in the first clade, thus not representing cunda s.str., further encompassing the somewhat deviating U. rubicunda s.str. Usnea rubrotincta itself formed a core haplotypes from Galapagos and parts of South America clade separate from U. rubicunda in another section of (Fig. 27). The Asian-New Zealand clade was not well sep- the tree (Fig. 7, Table S1, Fig. S1) with three sequences arated from the U. rubicunda cluster in the identity matrix from Japan (Ohmura 2002, 2008) and two unpublished (Fig. 27) and may be recognized at the subspecies level. sequences from Taiwan deposited by Y.-M. Shen et al. in In contrast, the first clade contained four well sep- 2012. Three further sequences identified asU. rubrotincta arated entities in topology, support and in the identity from Japan (Ohmura 2002; Ohmura & Clerc 2019) and matrix (Fig. 27). These should be considered separate, South Korea (deposited by K.-M. Lim et al. in 2005, but likely cryptic species. For the second clade, the name apparently not used in a published study), did not cluster U. pennsylvanica appears to be available. For the third with the other sequences, but fell into the third clade in clade from Asia, the name U. pseudorubicunda Asahina the U. rubicunda assemblage (see above). Ohmura (2008) would potentially apply (Ohmura 2001), whereas for the recovered all of these in a single clade, likely because of fourth clade, including specimens from South America, the limited taxon sampling and outgroup selection. the Mediterranean, and Asia (Japan), the situation is more Thus, while there are two clearly separate entities complicated. This entire assembly requires detailed study, corresponding to Usnea rubrotincta, it remains unclear including potential phenotypic differences and the proper how the name Usnea rubrotincta is to be applied, given assessment of type material. Some additional sequences that specimens identified with that name, all from Asia, originally deposited under the name Usnea rubicunda formed two distantly related clades in different portions were found in other clades, here labeled U. aff.moreliana of the tree and that the type material is from Madeira. and U. aff. rubrotincta. Clerc (2007) listed Usnea rubrotincta (described from Usnea subcornuta aggregate. This is another shrubby, Madeira) and U. rubescens (described from Australia) as sorediate species, characterized by a subcortical, orange- synonyms of U. rubicunda. Ohmura (2008) performed red pigment, large soralia with isidiomorphs, and a stic- a detailed morphological, chemical and phylogenetic tic-norstictic acid chemistry (Randlane et al. 2009; Truong analysis, resulting in a clear separation of U. rubrotincta & Clerc 2016). Four ITS accessions were available under R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 343 this name, forming three lineages in two different places of the NYBG C. V. Starr Virtual Herbarium at http:// of the tree (Fig. 7, Fig. S1). One sequence from Ecuador sweetgum.nybg.org/science/vh) and two from Europe (Truong et al. 2013a) fell close to several unidentified (Spain; Araujo 2016), and a second subclade with two accessions from New Zealand, whereas the other three accessions from Brazil (Gerlach et al. 2019a). The first sequences formed two lineages in close proximity, one subclade was here interpreted as U. subscabrosa s.str., singleton from Ecuador (Truong et al. 2013a) and two whereas the Brazilian subclade represents a separate spe- from Europe (Saag et al. 2011, Truong et al. 2013a). Given cies (U. aff. subscabrosa). The two subclades differed that U. subcornuta was described from Madeira (Stirton in 12 consistent substitutions and one indel (File S1), 1881), the European clade of two samples likely corre- resulting in 97.8% similarity. MCM7 data were availa- sponds to this species. The related singleton sequence ble for the two Brazilian samples, forming two separate from Ecuador (JQ837327) contained some odd base calls, singletons on long branches (Fig. S7), another example so its status requires further examination. of a potential paralog situation in this marker. Data from the nuLSU also placed Usnea subcornuta sequences as singletons in three separate lineages (Fig. S2), ITS-based DNA barcoding in Usnea: current versus whereas with MCM7, one Ecuadorian and one European updated metadata sequence clustered in a strongly supported clade, albeit on long individual branches (Fig. S7). The first Ecuadorian Comparison of updated identifications of sequence labels sample was found to be unrelated to U. subcornuta s.str., with the submitted identifications resulted in seven sce- whereas the second sample from Ecuador appeared to narios: (1) the submitted name matched the name in represent a distinct, yet closely related lineage. a phylogenetic species concept (exact match); (2) the submitted name matched an established synonym of the Usnea subflammeaaggregate . Usnea subflammea was updated name (exact match, but use of a synonym; e.g., recently described from the Azores and the Canary Usnea filipendula vs. U. dasopoga; see Arcadia 2013); Islands (Clerc 2006), characterized as being similar (3) the submitted identification was unnamed, but had to U. flammea, but with a (sub-)pendent growth habit, a specific label denoting species-level precision (precise, numerous surface tubercles and absence of lobaric acid but unnamed; e.g., Usnea sp. NW-2007-1 in Wirtz et al. (stictic acid only). Four accessions have been deposited 2008 or U. aff. cornuta 3 in Gerlach et al. 2019a); (4) under this name, two from Brazil and two from the the submitted name matched the correct species com- Canary Islands (Gerlach et al. 2019a). In the study by plex, but did not identify the precise subclade (impre- Gerlach et al. (2019a), one of the sequences from the cise; e.g., U. baileyi from Hawaii in Ohmura 2002 vs. Canary Islands (238ES = 238TEN) was labeled U. geis- U. baileyi from Cameroon in Orock et al. 2012); (5) the leriana, and all four fell in the same area of the species submitted name matched the corresponding species phe- tree together with a sequence identified as U. flammea. notypically, but corresponded to a separate, sometimes The latter was here revealed to form part of U. flam- unrelated clade, not the species in a strict sense (mislead- mea s.str. (Fig. 7, Fig. S1), whereas the two accessions ing; e.g., U. cornuta from Ecuador and Peru in Truong from the Canary Islands formed two separate lineages et al. 2013a); (6) the submitted name did not have a spe- in proximity to each other, but not a single clade. The cies-level identification, e.g., Usnea sp. (unresolved); two accessions from Brazil fell in a separate area of and (7) the submitted name identified a species different the ITS tree, there clustering with weak support (70%). from the actual species represented by the underlying RPB1 also separated the four accessions into three clades sequence data (incorrect; e.g., U. malmei from Taiwan (Fig. S5), similar to MCM7 (Fig. S7), supporting the in Greenwood et al. 2016). interpretation of the sample 238ES (= 238TEN) as rep- For DNA barcoding to work well, one would expect resenting U. geisleriana and the Brazilian lineage as most accessions to belong to categories 1–4. However, in a different taxon resembling U. subflammea phenotyp- our assessment of original submission labels, only about ically (U. aff. subflammea). half of the sequences provided an exact match, 47% cor- responding to the current name and 2% to an established Usnea subscabrosa aggregate. Usnea subscabrosa was synonym (Fig. 28). Furthermore, 5% provided a precise, described from Portugal (Motyka 1938). It is a shrubby yet unnamed match and 12% corresponded to species to (sub-)pendent species characterized mainly by a thick, complexes (imprecise). A total of 35%, i.e., one third of hard and vitreous cortex, small soralia and by the pres- all accessions, when appearing as best hits in BLAST ence of protocetraric acid in the medulla (Truong et al. searches, would give a misleading (9%) or incorrect (7%) 2013b). A second chemotype with thamnolic acid occurs identification or would be unresolved (19%). In terms of in the Azores (Clerc 2006) and in the Iberian Peninsula identification success, using a scoring system of catego- (Araujo 2016), but sequences for this chemotype are not ries (1–3) = 3, (4) = 2, (5, 6) = 1 and (7) = 0, this would yet available. give a weighted average of 70.6% for query sequences In our ITS-based tree, the five available sequences having a high-scoring match to a given reference sequence labeled U. subscabrosa formed an unsupported clade (Table 1). with two strongly to fully supported subclades (Fig. 7; If all accessions would be updated with the adjusted Fig. S1): one with one accession from North America identifications based on our analysis, the categories would (apparently unpublished; origin confirmed by consultation change as follows: exact matches = 58%, precise, but 344 Plant and Fungal Systematics 65(2): 303–357, 2020

threshold of 98.5% (Irinyi et al. 2015; Jeewon & Hyde 2016; Kõljalg et al. 2019; Nilsson et al. 2019) would underestimate species richness in this group and thus our assessments using identity matrices based on this value may be too conservative. Since metabarcoding pipelines often use a lower threshold of 97% (Majaneva et al. 2015; Sinha et al. 2017; Anslan et al. 2018), we also computed the number of species hypotheses in Usnea s.lat. using this value, resulting in 56 taxa. This was only 26.5% of our likely estimate of 211 species and a gross underestimation of the real species richness in the genus. If we assume that in the present data set, the number of previously recognized species more or less doubled based on phylogenetic resolution combined with available data on phenotype, the currently recognized number of nearly 450 taxa according to our checklist may actually correspond to up to 900 taxa, making Usnea an ultradi- verse genus even when Dolichousnea and Eumitria are treated separately (Lücking et al. 2017a).

Figure 28. Proportions of categories of inferred matches and mismatches Distribution patterns in Usnea: taxonomic versus based on submitted sequence labels compared to updated species iden- phylogenetic inventories tifications. Based on our assessment of taxonomic inventories of as yet unnamed matches based on names with qualifi- the genus Usnea over the past 50 years (Table S2), ers (e.g., Usnea baileyi 2, U. aff. cornuta 1) = 31%, and we assigned each species to one of the following dis- precise, but as yet unnamed matches (e.g., Usnea sp. 1 tribution patterns: (1) subcosmopolitan, (2) Pantropics, Wirtz) = 11%. This would increase identification success (3) amphi-Pacific, (4) amphi-Atlantic, (5) Gondwana to a weighted average of 99.0%. Since third-party annota- (= amphi-Atlantic but tropical only, i.e. American and tions to update sequence ID labels in primary repositories African tropics), (6) Afro-Eurasia, (7) Paleotropics, (8) such as GenBank are not currently possible, we provide Northern Hemisphere, (9) Southern Hemisphere, (10) here an updated ITS alignment for the genus Usnea using Americas, (11), North and Central America, (12), Central the adjusted identification labels, which can be used in and South America, (13) Russia-Asia, (14) Asia-Oceania, local BLAST approaches (File S8; Usnea Local BLAST (15) North America, (16) Central America (incl. Mexico), Release 1.0). We also submitted the data to the curated (17) South America, (18) Europe, (19) Africa, (20) Asia, UNITE database (Nilsson et al. 2018, 2019; Kõljalg et al. (21) Oceania, and (22) Antarctica. These were further 2019). In addition, we provide an updated voucher table classified into wide (1–5), contiguous (6–10), neighboring (Table S1). (11–14), and single regions (15–22). Based on taxonomic inventories using pheno- ITS-based DNA barcoding in Usnea: inferred species type-based identifications, a large proportion of the 438 richness species (23%) were inferred to have wide distribution including across discontiguous areas, 8% a distribution Based on the original identifications of the ITS acces- in broad, contiguous areas consisting of more than two sions for Usnea s.lat., the 1,751 sequences nominally regions, 7% in two neighboring regions, and the major- represented 143 taxa. Using our revised assessment, this ity (62%) in a single region (Fig. 29). However, when number increased to at least 211 taxa, with 294 individual species-level clades were considered, amounting to 286 lineages that might in part represent additional taxa based entities in our ITS-based tree, distribution patterns were on the detailed elaborations above (Table S1). Usnea substantially much narrower with only 5% having a wide s.lat. sequences in the curated UNITE database (Nils- distribution, 12% a distribution in broad, contiguous areas, son et al. 2018, 2019; Kõljalg et al. 2019) were resolved 1% in two neighboring regions, and 82% in a single region as 148 species hypotheses at the 98.5% threshold, 227 (Fig. 29). at the 99% threshold, and 322 at the 99.5% threshold When looking at individual distribution types, most [https://unite.ut.ee/search.php#fndtn-panel1; Taxon name types corresponding to wide distribution patterns substan- = Usnea (Genus)]. These numbers are just slightly above tially decreased when taking into account phylogenetically our assessment of taxa vs. lineages, suggesting that in defined species, with the exception of amphi-Atlantic Usnea s.lat., the 99% threshold denotes a conservative (Fig. 30). In contrast, several contiguous distribution types approach to species delimitation and the 99.5% threshold increased in proportion, namely Afro-Eurasia, Northern a progressive approach, the reality oscillating somewhere Hemisphere, and Southern Hemisphere (Fig. 30). All in between. Given these results, the often-used default neighboring distribution types also decreased, whereas R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 345

ITS-based barcoding in Usnea compared to other lichenized genera

ITS-based barcoding as a means to delimit and recognize species has been amply used in other large genera of lichenized fungi, generally with good success. Notably, ITS provides generally higher levels of resolution and support in basidiolichens, including in the backbone, as exemplified by the generaCora Fr. and Sulzbacheromyces B.P. Hodk. & Lücking, often to the point that unambigu- ous alignment of the ITS within a genus becomes difficult (Lücking et al. 2017b; Coca et al. 2018). Among ascoli- chens, sizable monophyletic genera with a large amount of ITS data include Cladia Nyl., Cladonia P. Browne, Melanelia Ess., Parmelia Ach., Parmotrema A. Massal., Peltigera Willd., Pseudocyphellaria Vain., Ramalina Ach., and Sticta (Schreb.) Ach., and Strigula Fr. Studies testing the utility of DNA barcoding markers often focus on the barcoding gap, with good success in genera of Parmeliaceae, such as Melanelia and relatives, Parmelia, and Parmotrema (Leavitt et al. 2014, 2016; Divakar et al. 2016; Del-Prado et al. 2019), but also in the genus Cladia (Parnmen et al. 2013). In other lineages, such as Cladonia, assessing the barcoding gap rendered ITS suboptimal compared to other markers (Kelly et al. Figure 29. Proportion of principal distribution patterns when species 2011; Pino-Bodas et al. 2013; Kanz et al. 2015; Marthin- are delimited by phenotype (traditional taxonomic inventories) vs. de- sen et al. 2019). The barcoding gap approach may suffer limitation by molecular data (DNA barcoding). from sampling bias and also may not reflect accurate phy- logenetic relationships, as it is based on pairwise compar- single region distribution types mostly increased, with the isons of sequence similarity (Lücking et al. 2020a, b), and exception of Central America (incl. Mexico) and Africa it may be biased when underlying species concepts to not (Fig. 30). While some of this is due to taxonomic and reflect phylogenetically delimited lineages (Marthinsen geographic sampling bias, it became obvious that wide, et al. 2019). The computation of barcoding gaps should discontiguous distribution patterns inferred through tradi- therefore always be based on phylogenetically delimited tional species concepts are much less frequent than pre- lineages and not on phenotypically identified specimens. viously presumed, whereas wide distributions following When being used in broad, multiple alignment-based contiguous areas are more frequent when species concepts context, as here in the genus Usnea, ITS generally works are adjusted based on molecular data. Also, regional ende- well, although in recently evolving species complexes it mism is more frequent. may pose problems of resolution. Examples include the recently emended Peltigeraceae (Kraichak et al. 2018; Lücking 2019), for instance the genera Peltigera, Pseu- docyphellaria and Sticta (Moncada et al. 2014; Magain & Sérusiaux 2015; Lücking et al. 2017c; Magain et al. 2018: Ranft et al. 2018; Simon et al. 2018). Microlichens in the genus Strigula (Dothideomycetes) exhibit high lev- els of resolution with ITS and good congruence with other markers (Jiang et al. 2016, 2017a, b; Ford et al. 2019; Woo et al. 2020). In contrast to some other fungal lineages (Lücking et al. 2020a), genome-wide analyses have also shown that intragenomic variation in the ITS is generally low, both in ascolichens (e.g., Rhizoplaca; Bradshaw et al. 2020) and basidiolichens (e.g., Cora; Lücking et al. 2014). Likewise, using a cloning approach, Saag et al. (2014) screened for potential ITS paralogs in Vulpicida and did not find evidence for paralogous copies of this marker. Thus, the main issue with ITS, at least in lichenized lineages, appears to be the occasional lack of resolution. Candidate Figure 30. Proportion of individual distribution patterns when species are delimited by phenotype (traditional taxonomic inventories) vs. de- cases, such as the Usnea antarctica / U. aurantiacoatra limitation by molecular data (DNA barcoding). species pair (Grewe et al. 2018; Lagostina et al. 2018), 346 Plant and Fungal Systematics 65(2): 303–357, 2020

are not only found in other groups of Usnea (U. florida demonstrating the ITS to be quite uniform in this clade, vs. U. subfloridana, U. lambii vs. U. perpusilla), but mor- regardless of the study source. This underlines the repro- phologically discrete taxa not resolved by ITS are also ducibility of this approach, regardless of the study and its known from other macrolichens, including Sticta fulig- objectives. On the other hand, analysis of highly resolving inosa (Dicks.) Ach. vs. S. limbata (Sm.) Ach. or S. filix microsatellite and RADseq markers showed that ITS was (Sw.) Nyl. vs. S. lacera (Hook. f. & Taylor) Müll. Arg. not able to properly resolve the two species present in (Magain & Sérusiaux 2015; Ranft et al. 2018; Moncada this complex. et al. 2020b). Besides resource-intensive methods such While lack of resolution appears to be an issue with as microsatellite markers and RADseq, IGS appears to be ITS in recently evolving species complexes in the genus a promising secondary barcoding marker in Usnea, but has Usnea, we did not find any evidence for gene duplica- also proven useful in other lichenized and non-lichenized tion (paralogs) or hybridization causing artifactual clades, fungal lineages, such as Thamnolia Ach. ex Schaer. and thus confirming aformentioned studies in Parmeliaceae Schizophyllum Fr. (James et al. 2001; Onuţ‐Brännström (Vulpicida) or Lecanoraceae (Rhizoplaca). Comparison et al. 2017). with other markers demonstrated that particularly IGS and RPB1 are useful to complement ITS-based phylogenies. Conclusions IGS shows better resolution and support at species level, whereas RPB1 has less terminal resolution and delineates The fungal ITS barcoding marker has been used for about larger species complexes. Both IGS and RPB1 also pro- two decades to assess species delimitation and recog- vide better backbone resolution and support than ITS. nition in the genus Usnea. As in other lichenized and IGS is therefore a promising secondary barcoding marker non-lichenized fungi, ITS has contributed substantially to in Usnea, a notion matching aformentioned studies in unravel and remedy artificial taxonomic concepts and has other Parmeliaceae. The nuLSU marker appears to be of helped to reshape taxonomy in the genus Usnea. However, limited use in Usnea, providing neither good resolution the large amount of available data showed that ITS-based nor good backbone support. The other three commonly DNA barcoding in this genus also has its limits, both employed protein-coding markers, TUB2, RPB2, and practically and conceptually. MCM7, showed variable evidence of aberrant topologies In practical terms, while sequence quality was overall here interpreted as paralog, particularly in the MCM7, excellent and we found only few low-quality accessions, and these markers should be used with great care, espe- information on voucher specimens and overall identifica- cially when it comes to multimarker coalescence species tions were inadequate in many cases. Particularly these delimitation approaches. two issues greatly challenge successful ITS-based barcod- Further challenges include difficult morphospecies ing in Usnea, as more often than not, the taxon identifica- that do not form coherent clades in ITS or other markers, tions on BLAST hits will not indicate the correct species. suggesting various levels of cryptic speciation. The most This can be largely mitigated by updating and correcting notorious example is the Usnea cornuta complex discussed voucher information. However, since this is not possible in detail above. In these cases, multimarker approaches directly in primary repositories such as GenBank, we have using ITS, IGS and RPB1 will help to assess supported here provided an updated alignment that can be used in lineages, but it cannot always be expected that these line- local BLAST applications or through the curated UNITE ages will be readily separable using phenotypic characters. ITS database. For future voucher information in Usnea Overall, we conclude that ITS is a good first approx- (and corresponding cases), we strongly recommend listing imation to assess species delimitation and recognition in key characters such as medullary chemistry in voucher the genus Usnea, as long as data are carefully analyzed tables, as has been done in a number of exemplar studies. and reference sequences are critically assessed and not Another practical challenge to successful DNA bar- taken at face value. In difficult groups, we recommend coding is taxonomic and geographic coverage. Currently, sequencing IGS as a secondary barcode marker, which only around 30% of the nearly 450 currently accepted spe- together with ITS should result in reliable species assess- cies in Usnea s.lat. have been sequenced, and there is both ments in most cases. However, additional studies using taxonomic and geographic bias. Thus, while Dolichousnea RADseq should be executed to assess the performance of and subgenus Neuropogon are well-represented among the the ITS-IGS barcoding approach in species complexes. data, sampling for Eumitria and Usnea s.str. remains mod- In the near future, attempts should be made to close tax- erate to poor and biased towards certain lineages. Geo- onomic and geographic gaps, in particular in Eumitria graphically, Antarctica and Europe are well-represented, and Usnea s.str. and in the highly diverse areas of North followed by South America, whereas North America, America, Africa, Asia, and Oceania. Africa, Asia and Oceania are undersampled. A peculiar situation arises with New Zealand, which is represented Acknowledgements by a large amount of ITS accessions from across both major islands, but most of them left unidentified. First and foremost, we take this opportunity to thank our es- As outlined above, the species pair Usnea antarctica teemed colleague, mentor and friend Philippe Clerc, for his vs. U. aurantiacoatra is one example of an extremely invaluable and continuous contributions to the taxonomy and well-sampled clade. Numerous ITS sequences were gen- systematics of the genus Usnea during the last 36 years. The erated both from taxonomic and from ecological studies, present study, which we dedicate to Philippe on the occasion R. Lücking et al.: Two decades of DNA barcoding in the genus Usnea 347 of his well-deserved retirement, reflects his influence and his File S5. Global RPB1 alignment for the genus Usnea based on GenBank work on many levels, including transferring his knowledge to accessions, using original identifications. Download file numerous colleagues and students. We also thank Nora Wirtz File S6. Global RPB2 alignment for the genus Usnea based on GenBank for providing voucher data on samples of Neuropogon s.lat. accessions, using original identifications. Download file from her studies. Yoshihito Ohmura placed the photograph of . Global MCM7 alignment for the genus Usnea based on Gen- Dolichousnea longissima at our disposal. Andreas Beck helped File S7 Bank accessions, using original identifications. Download file with information about annotations of some type specimens housed in M and James Lendemer shared some insight into File S8. Global ITS alignment for the genus Usnea based on GenBank the taxonomy of the Usnea strigosa complex. Henrik Nilsson accessions, using updated identifications (Usnea Local BLAST Release assisted with integrating the data set into UNITE. We thank 1.0). Download file Steve Leavitt and Yoshihito Ohmura for valuable comments that helped to improve the manuscript. References

Abbott, R. J., Barton, N. H. & Good, J. M. 2016. Genomics of hy- Supplementary electronic material bridization and its evolutionary consequences. Molecular Ecology 25: 2325‒2332. Figure S1. Global ITS-based, best-scoring maximum likelihood tree Acharius, E. 1803. Methodus Qua Omnes Detectos Lichenes Secundum for the genus Usnea based on GenBank accessions, using original iden- Organa Carpomorpha ad Genera, Species et Varietates Redigere tifications. Download file atque Observationibus Illustrare Tentavit Erik Acharius. Stockholm. Figure S2. Global nuLSU-based, best-scoring maximum likelihood Agassiz, L. 1850. Lake Superior: Tts Physical Character, Vegetation and tree for the genus Usnea based on GenBank accessions, using original Animals. With a narrative of the Tour by J. Elliot Cabot. Boston. identifications. Download file Ahti, T., Mayrhofer, H., Schultz, M., Tehler, A. & Fryday, A. M. 2016. Figure S3. Global IGS-based, best-scoring maximum likelihood tree First supplement to the lichen checklist of South Africa. Bothalia for the genus Usnea based on GenBank accessions, using original iden- 46: 1–8. tifications. Download file Ait Hammou, M., Miara, M. D., Rebbas, K., Slimani, A, Ravera, S. & Hamer El Ain, A. S. 2014. Mise à jour de l’inventaire des lichens Figure S4. Global TUB2-based, best-scoring maximum likelihood tree d’Algérie. Revue Ecologie-Environment 10: 75–103. for the genus Usnea based on GenBank accessions, using original iden- tifications. Download file Andreev, M., Kotlov, Y. & Makarova, I. 1996. Checklist of lichens and lichenicolous fungi of the Russian Arctic. The Bryologist 99: Figure S5. Global RPB1-based, best-scoring maximum likelihood tree 137–169. for the genus Usnea based on GenBank accessions, using original iden- tifications. Download file Anslan, S., Nilsson, R. H., Wurzbacher, C., Baldrian, P., Tedersoo, L. & Bahram, M. 2018. Great differences in performance and outcome Figure S6. Global RPB2-based, best-scoring maximum likelihood tree of high-throughput sequencing data analysis platforms for fungal for the genus Usnea based on GenBank accessions, using original iden- metabarcoding. MycoKeys 39: 29–40. tifications. Download file Aptroot, A. 2002. New and interesting lichens and lichenicolous fungi Figure S7. Global MCM7-based, best-scoring maximum likelihood in Brazil. Fungal Diversity 9: 15–45. tree for the genus Usnea based on GenBank accessions, using original Aptroot, A. 2016. Preliminary checklist of the lichens of Madagascar, identifications. Download file with two new thelotremoid Graphidaceae and 131 new records. Table S1. Updated, complete voucher table for ITS accessions of the Willdenowia 46: 349–365. genus Usnea, including original and updated identifications and voucher Aptroot, A. & Feijen, F. J. 2002. Annotated checklist of the lichens information. Additional recent accessions for RBP1 and MCM7 cor- and lichenicolous fungi of Bhutan. Fungal Diversity 11: 21–48. responding to the studies by Araujo (2016) and Gerlach et al. (2017, 2019a, 2020) are also given. Download file Aptroot, A. & Iqbal, S. H. 2010. Annotated checklist of the lichens of Pakistan, with reports of new records. Herzogia 25: 211–229. . Preliminary working checklist of regional and country/ter- Table S2 Aptroot, A. & Seaward, M. R. 1999. Annotated checklist of Hongkong ritory reports of species of Usnea, along with an assessment of cur- lichens. Tropical Bryology 17: 57–102. rently establish taxonomic synonyms. Selected infraspecific names are included, simplified by combining the species and infraspecies epithets Aptroot, A. & Sparrius, L. B. 2006. Additions to the lichen flora of with a hyphen. TYPE indicates records corresponding to type citations Vietnam, with an annotated checklist and bibliography. The Bryo- (country of origin), including for taxonomic synonyms. Some reports logist 109: 358–371. by Dodge from Africa (1956, 1957) have not been verified as they are Araujo, E. 2016. Sistemática Integrada del Género Usnea Dill. ex Adans. not treated in the various papers by Swinscow & Krog (1974, 1975, (Parmeliaceae) en la Península Ibérica. Doctoral Thesis, Univer- 1976a, b, 1978, 1979, 1988), and they have not been counted towards sidad Complutense de Madrid, Spain. the total; potentially they may represent up to 27 additional species, but most of the other names established by Dodge (1956, 1957) have been Arcadia, L. 2013. Usnea dasopoga, a name to be reinstated for U. fili- subsumed under synonymy of other species (Swinscow & Krog 1974, pendula, and its orthography. Taxon 62: 604–605. 1975, 1976a, b, 1978, 1979, 1988). Two unpublished names [ined.] have Armaleo, D. & Clerc, P. 1991. Lichen chimeras: DNA analysis suggests also not been included in the total count, leaving 447 accepted names that one fungus forms two morphotypes. Experimental Mycology (428 species and 19 infraspecies). Download file 15: 1–10. File S1. Global ITS alignment for the genus Usnea based on GenBank Articus, K. 2004. Neuropogon and the phylogeny of Usnea sl (Parme- accessions, using original identifications. Download file liaceae, Lichenized Ascomycetes). Taxon 53: 925–934. File S2. Global nuLSU alignment for the genus Usnea based on Gen- Articus, K., Mattsson, J. E., Tibell, L., Grube, M. & Wedin, M. 2002. Bank accessions, using original identifications. Download file Ribosomal DNA and β-tubulin data do not support the separation of the lichens Usnea florida and U. subfloridana as distinct species. File S3. Global IGS alignment for the genus Usnea based on GenBank Mycological Research 106: 412–418. accessions, using original identifications. Download file Arup, U., Ekman, S., Grube, M., Mattsson, J.-E. & Wedin, M. 2007. The File S4. Global TUB2 alignment for the genus Usnea based on GenBank sister group relation of Parmeliaceae (Lecanorales, Ascomycota). accessions, using original identifications. Download file Mycologia 99: 42–49. 348 Plant and Fungal Systematics 65(2): 303–357, 2020

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