Figure 1
Representatives of the four main groups of Usnea s.lat. ADolichousnea (D. longissima); B -Eumitria (E. baileyi); C -subgenus Neuropogon (U. sphacelata); D -Usnea s.str. (U. erinacea). Photographs by RL except (A), kindly provided by Y. Ohmura.
Source publication
We present an exhaustive analysis of the ITS barcoding marker in the genus Usnea s.lat., separated into Dolichousnea, Eumitria, and Usnea including the subgenus Neuropogon, analyzing 1,751 accessions. We found only a few low-quality accessions, whereas information on voucher specimens and accuracy and precision of identifications was of subpar qual...
Contexts in source publication
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... Protousnea (Motyka) Krog either recognizing a single genus Usnea s.lat., with several subgenera and sections (Motyka 1936(Motyka , 1938Ohmura 2002;Ohmura & Kanda 2004;Wirtz et al. 2006;Truong & Clerc 2013;Ohmura & Kashiwadani 2018;Temu et al. 2019) or separating Dolichousnea (Y. Ohmura) Articus, Eumitria Stirt., and Neuropogon Nees & Flot. (Fig. 1), as smaller genera from Usnea s.str. (Articus 2004). Given the underlying data, we follow Divakar et al. (2017) and Kraichak et al. (2017) to recognize Dolichousnea and Eumitria as separate genera, while retaining Neuropogon within Usnea at the level of subgenus. Besides considerations of evolutionary age, this reflects the fact that ...
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... 4). Particularly notable were four sequences of U. articulata (L.) Hoffm. (JN086277: 6; JN086278: 19; JN086279: 13; JN086280: 14) and two labeled U. dasaea Stirt. (JN086283: 16; JN086284: 15). In general, low quality did not affect the correct placement of the affected sequences, but often considerably reduced support for the containing clades (Fig. S1). In the case of the two sequences labeled U. dasaea from Portugal, the ambiguous base calls occurred principally in the second portion of the ITS2 and were associated with numerous odd base calls, resulting in an extremely long stem branch (see below). These were the only two instances where the underlying topology was considered ...
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... combination, e.g. in the ITS1 vs. ITS2. However, we could not detect issues with these eight sequences that would indicate them as potentially chimeric. Chimeric sequences composed of portions of two different taxa would be expected to fall on long, yet unsupported branches, which was not the case for any of these eight sequences (Table S1; Fig. S1). Rather, they appeared to represent variants comparable to those in other ...
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... was indicated for six samples from four localities, one from Ecuador, three from Argentina, and two from two different places in Antarctica. The same applied to haplotype 8. For six accessions (EF492146, EF492147, EF492153, EF492161, EF492213, EF492216), no haplotypes were indicated. These were given in the tree figure ( Wirtz et al. 2008: 478, fig. 1) as 'potential recombinants' (see above). Their geographic origin could be inferred by comparing the isolate numbers with those of haplotypes indicated in the table. Two further 'potential recombinants' given in the tree figure ( Wirtz et al. 2008: 478, fig. 1), with the isolate numbers 208-19 and 221-1, were apparently not included in ...
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... were indicated. These were given in the tree figure ( Wirtz et al. 2008: 478, fig. 1) as 'potential recombinants' (see above). Their geographic origin could be inferred by comparing the isolate numbers with those of haplotypes indicated in the table. Two further 'potential recombinants' given in the tree figure ( Wirtz et al. 2008: 478, fig. 1), with the isolate numbers 208-19 and 221-1, were apparently not included in the GenBank submissions. Upon request, we received the complete voucher table from the original author (N. Wirtz, pers. comm. July 2020), which enabled us to fill in most of the missing data, except geographic origin in 16 cases (Table ...
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... very positive aspect in some studies was that the secondary chemistry of the underlying vouchers was listed, either in the voucher table ( Truong et al. 2013a;Gerlach et al. 2017) or in other sources of information (e.g., Nadel 2016;Mark et al. 2016a: 510-511, fig. 1 fig. 3). Given that current taxonomy in Usnea for many taxa assumes chemical variability and so the applied name is not necessarily a reflection of the underlying chemistry, the inclusion of the chemotype in the voucher information is strongly recommended for phylogenetic and DNA barcoding ...
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... Walker (1985) under subgenus Neuropogon clustered in a sister clade, namely Usnea ciliata (Müll. Arg.) Vain. and U. subcapillaris (D.J. Galloway) F.J. Walker (see also Wirtz et al. 2006). This clade of 148 terminals was almost exclusively formed by samples from New Zealand, with the exception of one specimen from Antarctica and another from Chile (Fig. S1). Most of the samples were not identified ( Buckley et al. 2014), but the few identified specimens ( Rafat et al. 2015) include two names also associated with subgenus Neuropogon by Walker (1985), namely U. inermis Motyka and U. torulosa (Müll. Arg.) Zahlbr. Three further identifications in this clade, U. ciliifera Motyka, U. pusilla ...
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... ITS-based maximum likelihood tree of 1,751 ingroup sequences mirrored the topology obtained in other recent studies (Nadel 2016;Divakar et al. 2017;Kraichak et al. 2017), with Dolichousnea and Eumitria forming a monophyletic clade sister to Usnea including Neuropogon, and Neuropogon s.str. forming a monophyletic clade nested within Usnea s.str. (Fig. 7; Fig. S1). The sister group relationship of Dolichousnea and Eumitria was not supported, but each of the two genera was strongly supported on long stem branches (94-96%), matching other findings that recovered both genera as supported clades, but with varying positions as early diverging lineages within Usnea s.lat. (Ohmura 2002;Articus ...
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... the numerous samples from Argentina identified as U. acromelana ( Wirtz et al. 2006Wirtz et al. , 2012Seymour et al. 2007) may not belong to that species (see also below). On the other hand, given the confirmed wide distribution of U. sphacelata with sequence data from Antarctica, throughout South and North America and northern Europe (Table S1, Fig. S1), its occurrence in New Zealand appears ...
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... This strongly supported genus, which is formally accepted here following Divakar et al. (2017), formed four supported subclades based on the ITS ( Fig. 7; Fig. S1), corresponding to one as yet unnamed species composed of unpublished sequences from China, deposited by S. Guo et al. in 2019 andL. Han et al. in 2020, as well as E. firmula, E. pectinata s.lat., and E. baileyi s.lat. (Ohmura 2002;Nadel 2016;Jaouen et al. 2019;Temu et al. 2019). The E. pectinata clade contained one sample (MN080241) ...
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... Eumitria baileyi clade formed two rather well-supported (88% and 89%), reciprocally monophyletic subclades, indicating the presence of at least two species ( Fig. 7; Fig. S1). This was further supported by the identity matrix (JQ673442 excluded due to reduced length), which resulted in values below 98.5% for all cross-comparisons between the two clades ( Fig. 8). In addition, the early diverging singleton sequence (MW267138) in the first subclade was revealed as an additional candidate species based on the ...
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... within a single area, together with the notion that most of its geographic range has not yet been sampled for molecular data, indicates that E. baileyi represents a complex of several species. This is also underlined by the existence of unique phenotypes, such as an unusual specimen from the Colombian Amazon with a brightly orange inner axis (Fig. 1A). The latter may morphologically correspond to E. baileyi f. endocrocea Zahlbr. described from Taiwan (Zahlbruckner 1933: 60), although it is unclear whether the author referred to the medulla in the proper sense or the fistulate inner axis with 'Medulla ... crocea'. The secondary chemistry of E. baileyi s.lat. appears to be rather ...
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... clade exhibited internal structure, but no distinct division into reciprocally monophyletic clades. The backbone was structured into various singletons and several smaller, partly supported subclades, including one subclade with the three samples from Southeast Asia (Ohmura 2002), but without separation in the identity matrix at the 98.5% level (Fig. 10). However, three nested, well-supported subclades on longer branches appeared as candidate species in the identity matrix, including samples from São Tomé and Príncipe and/or Tanzania (Nadel 2016; Temu et al. 2019). The largely unresolved backbone had a variable chemistry including constictic, salazinic, protocetraric, and/or ...
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... inconsistent to assume that structured differences in the ITS, as observed in E. baileyi s.lat. and E. pectinata s.lat., may just be infraspecific variation in these cases. Dolichousnea. Dolichousnea is by far the best sampled group in Usnea s.lat., with all three currently recognized taxa represented by ample sequence data (Figs 2, 7; Table S1; Fig. S1). The three taxa were all recovered in 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 ...
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... 1978Truong et al. 2013a;Ohmura & Kashiwadani 2018), but one unpublished accession (MN006813), deposited by M. L. Hale and J. Taylor in 2019, appears to be from California, although the geographic origin was not precisely indicated. That sequence formed part of a small, strongly supported subclade also including sequences from Japan and Taiwan ( Fig. 7; Fig. S1). This taxon thus appears to represent an unresolved species complex, the various lineages rather well separated in the identity matrix (Fig. 11). Ohmura (2001Ohmura ( , 2012 reported three chemotypes for D. trichodeoides, including salazinic or fumarprotocetraric acids or atranorin as major compounds. For one sequenced accession ...
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... be from California, although the geographic origin was not precisely indicated. That sequence formed part of a small, strongly supported subclade also including sequences from Japan and Taiwan ( Fig. 7; Fig. S1). This taxon thus appears to represent an unresolved species complex, the various lineages rather well separated in the identity matrix (Fig. 11). Ohmura (2001Ohmura ( , 2012 reported three chemotypes for D. trichodeoides, including salazinic or fumarprotocetraric acids or atranorin as major compounds. For one sequenced accession 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%; ...
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... (2001Ohmura ( , 2012 reported three chemotypes for D. trichodeoides, including salazinic or fumarprotocetraric acids or atranorin as major compounds. For one sequenced accession Figure 10. Pairwise identity matrix based on the ITS for the Eumitria pectinata clade. ...
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... grey = identity below 98.5%; grey = identity exactly 98.5%; white, blue and beige = identity above 98.5%. Figure 11. Pairwise identity matrix based on the ITS for the Dolichousnea trichodeoides clade. ...
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... internal variation was also observed in the supported Dolichousnea longissima clade with samples from across the Northern Hemisphere (Figs 2, 7; Table S1; Fig. S1). The internal structure was largely reflected in a cascading backbone, without clearly discernable larger lineages. The corresponding identity matrix identified several subgroups at the 98.5% level, but pairwise comparison between samples across all subgroups also revealed partially high identity levels above 98.5% (Fig. 12). Thus, D. ...
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... (Figs 2, 7; Table S1; Fig. S1). The internal structure was largely reflected in a cascading backbone, without clearly discernable larger lineages. The corresponding identity matrix identified several subgroups at the 98.5% level, but pairwise comparison between samples across all subgroups also revealed partially high identity levels above 98.5% (Fig. 12). Thus, D. longissima apparently cannot currently be subdivided into more than one species based on ITS data, even if it exhibited considerable internal variation. Secondary chemistry may help to discern lineages, although it has not been reported for most of the sequenced specimens. The three most common chemotypes produce diffractaic, ...
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... the 48 samples with either ITS or IGS data, only ten had both markers. Based on these, the internal topology for D. longissima was largely congruent between both markers and did not exhibit supported conflict, although IGS provided a more resolved topology (Figs S1, S3). Due to a limited amount or lack of data, the other markers could not be compared with ITS. ...
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... formed separate singletons likely representing separate species. Of the remaining 148 sequences, only nine were identified as GenBank deposits, representing U. ciliata and U. subcapillaris Figure 12. Pairwise identity matrix based on the ITS for the Dolichousnea longissima clade. ...
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... this information, the entire clade can currently only be outlined provisionally (see Fig. 7; Fig. S1). Following three singletons as early diverging lineages, a paraphyletic grade of ten sequences (KM369335 through KM369241) included two subsequently identified as U. torulosa (Buckley et al. 2014;Rafat et al. 2015) and was here interpreted to represent that taxon. The grade contained an unsupported subclade of five sequences (KM369431 ...
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... as U. torulosa (Buckley et al. 2014), differed consistently in five substitutions (99% similarity) and was here considered a separate species (U. aff. torulosa), as it fell elsewhere. A similar situation was found with five specimens identified as U. inermis (Buckley et al. 2014;Rafat et al. 2015), which fell into two larger, unsupported clades ( Fig. 7; Fig. S1), the first considered U. inermis s.str. and the second U. aff. inermis. The first clade of 27 accessions (KM369276 through KM369332) was internally rather uniform, but contained several sequences with odd base calls (see above; Table S1). The second clade formed two subclades, each with one sequence subsequently identified as U. ...
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... 2012) formed a strongly supported clade, whereas a fourth accession fell separate (U. aff. subcapillaris). The same applied to U. ciliata ( Wirtz et al. 2006Wirtz et al. , 2012, where a single accession originally identified as U. cf. ciliata did not cluster with the remaining samples, which formed a paraphyletic grade basal to U. subcapillaris ( Fig. 7; Fig. S1), including also one originally unidentified sample ( Buckley et al. 2014). Four accessions (JX144646 through KM369371) formed a fully supported clade on a long branch; one was subsequently identified as U. articulata (Rafat et al. 2015) and was here labeled U. aff. articulata 4, although this lineage was obviously not related to U. ...
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... et al. 2015): U. ciliifera, U. pusilla, and U. xanthopoga. Twenty accessions (JX144648 through KM369173) formed an unsupported clade containing five terminals subsequently labeled U. ciliifera. These contained a well-unsupported subclade (88%) of eight accessions (KR091723 through KM369173), two with odd base calls (see above) on long branches ( Fig. 7; Fig. S1). This latter subclade had two consistent substitutions and one indel (99.4% similarity) and so the two groups were labeled U. ciliifera 1 and U. ciliifera 2. The Usnea pusilla clade (KM369188 through JX144650), including a single, subsequently identified accession ( Rafat et al. 2015), was well-supported and internally variable, ...
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... substitutions, and another of nine accessions (KM369409 through KM369408) with two consistent substitutions, all in the ITS1 (File S1). In addition to these subsequently identified or provisionally identified clades, the New Zealand clade of subgenus Neuropogon contained four separate lineages that remained unidentified ( Fig. 7; Table S1; Fig. ...
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... subgenus Neuropogon core clade. Although containing a much larger number of terminals (446), this clade was genetically more uniform compared to the New Zealand clade ( Fig. 7; File S1; Fig. 10). Species in this clade are generally characterized by annulate black pigmentation along the branches (Fig. 1C) and black apothecial discs (Walker 1985;Ohmura 2002;Ohmura & Kanda 2004;Articus 2004;Wirtz et al. 2006Wirtz et al. , 2008Wirtz et al. , 2012Seymour et al. 2007). Notably, this clade did not contain a single accession from New ...
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... subgenus Neuropogon core clade. Although containing a much larger number of terminals (446), this clade was genetically more uniform compared to the New Zealand clade ( Fig. 7; File S1; Fig. 10). Species in this clade are generally characterized by annulate black pigmentation along the branches (Fig. 1C) and black apothecial discs (Walker 1985;Ohmura 2002;Ohmura & Kanda 2004;Articus 2004;Wirtz et al. 2006Wirtz et al. , 2008Wirtz et al. , 2012Seymour et al. 2007). Notably, this clade did not contain a single accession from New Zealand and was largely restricted to Antarctica and South America, with the exception of the more widely ...
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... perpusilla and U. lambii were not resolved as reciprocally monophyletic clades in the ITS and instead formed several supported and unsupported subclades ( Fig. 7; Fig. S1). The same clades were also recovered in the IGS, although with higher support (Fig. S3). The two taxa are distinguished by their reproductive mode, U. perpusilla producing apothecia and U. lambii soredia. Wirtz et al. (2008) resolved U. perpusilla as paraphyletic relative to U. lambii, but this topology was likely caused by the ...
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... relative to U. lambii, but this topology was likely caused by the inclusion of RPB1, in addition to ITS and IGS. Our analysis showed that RPB1 behaved erratically for this complex and other species in the Neuropogon core clade, mixing accessions of various taxa in various shallow grades and clades, an indication of the formation of paralogs ( Fig. 13; Fig. S5). recovered U. perpusilla and U. lambii as reciprocally monophyletic, but this was an artifact of limited sampling, representing one subclade each of the complex assemblage evident when joining all available data. Based on both ITS and IGS data, the situation in U. perpusilla versus U. lambii appears similarly complex as in Letharia ...
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... messutiae formed two unsupported subclades in the ITS, associated with a strongly supported clade representing U. pallidocarpa ( Fig. 7; Table S1; Fig. S1), the latter including additional accessions from Wirtz et al. (2006). IGS data exhibited a partly conflicting topology. The second U. messutiae subclade was mirrored by a strongly supported subclade in the IGS, whereas the first subclade was not only dissected into a grade, a strongly supported subclade, and one singleton in the IGS, ...
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... were strongly supported each in monophyletic clades, U. sphacelata clustered in an unsupported clade, and U. trachycarpa and U. acromelana in paraphyletic grades, one basal to U. subantarctica, that relationship without support, and the other basal to the U. antarctica-aurantiacoatra complex, that relationship being strongly supported ( Fig. 7; Fig. S1). With IGS, U. patagonica and U. sphacelata formed supported clades, whereas U. trachycarpa was again rendered paraphyletic and the U. subantarctica clade was not supported; U. acromelana formed a strongly supported clade, but was nested within the U. antarctica-aurantiaca complex (Fig. S3). As mentioned, RPB1 exhibited an erratic ...
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... supported clade, but was nested within the U. antarctica-aurantiaca complex (Fig. S3). As mentioned, RPB1 exhibited an erratic topology for taxa in this and the previous group, joining U. sphacelata, U. subantarctica, and U. trachycarpa in a homogeneous grade also including isolated accessions of U. pallidocarpa, U. perpusilla, and U. ushuaiensis (Fig. 13). Another, unsupported shallow clade was composed of accessions of U. aurantiacoatra, U. lambii, U. perpusilla, U. sphacelata, and U. ushuaiensis. A third group formed a shallow grade including U. acromelana, the U. antarctica-aurantiacoatra complex, and a single accession of U. sphacelata. Only U. patagonia formed a fully supported ...
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... RPB1 Figure 13. Comparison of topologies in the Neuropogon core clade between the ITS, IGS and RPB1 (cladograms). ...
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... these, two taxa are common and presumably widespread, namely U. angulata (Fig. 14) and U. merrillii (Marcano et al. 1996;Herrera-Campos et al. 1998;Elix & McCarthy 1998Stevens 1999;Ohmura 2002;Galloway 2007;Lin 2007;Singh & Sinha 2010;Truong et al. 2013b;Herrera-Campos 2016;Sipman & Aguirre-C. 2016;Truong & Clerc 2016;Rodríguez-Flakus et al. 2016;Ohmura & Kashiwadani 2018;Bungartz et al. 2018;Ess- linger 2019;Lücking ...
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... & Sinha 2010;Truong et al. 2013b;Herrera-Campos 2016;Sipman & Aguirre-C. 2016;Truong & Clerc 2016;Rodríguez-Flakus et al. 2016;Ohmura & Kashiwadani 2018;Bungartz et al. 2018;Ess- linger 2019;Lücking 2020c), and hence their sparse representation by ITS sequence data was surprising. Usnea angulata, which is particularly common in the Americas (Fig. 14), formed an early diverging lineage in our phylogeny, although this topology was not supported ( Fig. 7; Table S1; Fig. S1). Usnea merrillii formed a separate lineage near U. clerciana Truong, but that relationship was also not supported. Another presumably widespread species is U. shimadae, reported from Mexico and Tai- wan (Clerc ...
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... al. 2016;Ohmura & Kashiwadani 2018;Bungartz et al. 2018;Ess- linger 2019;Lücking 2020c), and hence their sparse representation by ITS sequence data was surprising. Usnea angulata, which is particularly common in the Americas (Fig. 14), formed an early diverging lineage in our phylogeny, although this topology was not supported ( Fig. 7; Table S1; Fig. S1). Usnea merrillii formed a separate lineage near U. clerciana Truong, but that relationship was also not supported. Another presumably widespread species is U. shimadae, reported from Mexico and Tai- wan (Clerc 2007;Ohmura 2001Ohmura , 2012Shen et al. 2012;Herrera-Campos 2016). The only available sequence, that clustered with a ...
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... see below) from Brazil; U. cirrosa fell in the vicinity of U. cladocarpa and U. halei close to U. flammea Stirt., both relationships without support; U. meridionalis formed an unsupported clade with U. durietzii and U. glabrata; and U. subrubicunda was found, without support, basal to the U. barbata-dasopoga-wasmuthii clade ( Fig. 7; Table S1; Fig. ...
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... namely U. ghattensis, U. intumenscens, U. aff. sanguinea, and U. sinensis (Ohmura 2002;Lin 2007;Singh & Sinha 2010;Shen et al. 2012;Gerlach et al. 2017;Ohmura & Kashiwadani 2018). Usnea ghattensis fell close to U. articulata, while U. intumescens clustered with support with U. bismolliuscula and U. sinensis with U. nipparensis ( Fig. 7; Table S1; Fig. S1). The accession originally identified as U. aff. sanguinea (Nadel 2016) formed a singleton clade unrelated to U. erinacea with which it has been synonymized ( Clerc 2004Clerc , 2007Clerc , 2011a; see below). However, the voucher specimen, while lacking soredia, does not produce apothecia and has a deviating chemistry, so this ...
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... of the singletons. In RPB1, two taxa, U. aurantiaciparvula and U. ghattensis, also formed singletons, whereas two accessions of U. cirrosa formed a paraphyletic grade at the base of U. cornuta s.str. (Fig. S5). A total of 30 taxa were recovered as monophyletic and internally more or less homogeneous and could be considered well-defined species. (Fig. 7, Table S1, Fig. ...
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... supported clade were from Europe ( Articus et al. 2002;Kelly et al. 2011;Saag et al. 2011;Millanes et al. 2014;Araujo 2016). It is unclear whether this taxon has simply not been sequenced from other areas, but given the geographic coverage of ITS accessions for the genus and the documented main distribution of U. hirta in North America and Europe (Fig. 15), a possible explanation is that U. hirta is a holarctic taxon, absent from the tropics and the Southern Hemisphere, and has simply not yet been sequenced in its North American and temperate Asian ...
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... case of a 'snowballing effect' in DNA barcoding, where initially misidentified sequences caused subsequently matching depositions to perpetuate this error ( Gilks et al. 2002). The sequenced samples in this case appear to represent an Asian endemic, as suggested by their nested placement within a larger, unsupported clade of Asian lineages ( Fig. 7; Fig. ...
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... contrast, the following five Asian species were all represented by ITS accessions either from the type region or at least near the type region: U. masudana, described from Taiwan (Asahina 1970) and with sequences from that (Shen et al. 2012); U. nidifica, described from Oceania (Norfolk Island; Taylor 1847) and with two unpublished accessions from Taiwan; U. nipparensis, described from Japan (Asahina 1956), with accessions from Japan ( Ohmura 2002Ohmura , 2011) and unpublished sequences from China; U. pangiana, described from India (Stirton 1883), with sequences from Japan (Ohmura 2002) and China (unpubl.), and U. pseudogatae Asahina, described from Taiwan (Asahina 1970) and with sequences from the same area ( Shen et al. 2012). In the cases of U. masudana and U. pseudogatae, the sequences were only identified in the corresponding paper (Shen et al. 2012), but not in the original depositions (see above). ...
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... 2016;Mark et al. 2016a) and an apparently misidentified outlier from Ecuador ( Truong et al. 2013a); and U. wasmuthii Räsänen, described from Estonia (Räsänen 1931). The latter formed a more or less coherent clade of 34 accessions, including several from the type region, but was akin to a species complex, with considerable internal structure ( Fig. 7; Fig. ...
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... 2012;Mark et al. 2016a;Araujo 2016;Gerlach et al. 2017Gerlach et al. , 2019a. IGS data of four species rendered three of these monophyletic, although with internal structure, namely U. acanthella, U. cavernosa, and U. silesiaca. Usnea wasmuthii formed a paraphyletic grade and had one conflicting sequence (was-03) clustering with U. subfloridana ( Fig. 16; Fig. S3). Data of TUB2 were available for six taxa, with two singletons (U. dasaea, U. flavocardia), two monophyletic clades lacking support and with considerable internal variation (U. cavernosa, U. hirta), and two polyphyletic assemblages, suggesting the potential formation of paralogs (U. silesiaca, U. wasmuthii; Fig. 16; Fig. ...
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... U. subfloridana ( Fig. 16; Fig. S3). Data of TUB2 were available for six taxa, with two singletons (U. dasaea, U. flavocardia), two monophyletic clades lacking support and with considerable internal variation (U. cavernosa, U. hirta), and two polyphyletic assemblages, suggesting the potential formation of paralogs (U. silesiaca, U. wasmuthii; Fig. 16; Fig. ...
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... grade with internal variation (Fig. S5). The situation for RPB2 was different; of the three taxa with available data, only U. silesiaca was rendered monophyletic, although with internal variation (Fig. S6). Usnea cavernosa resulted polyphyletic, suggesting the formation of paralogs, and U. wasmuthii formed a grade plus one outlier (was-08; Fig. ...
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... were rendered monophyletic with the ITS, U. cladocarpa and U. dasaea, formed paraphyletic, heterogeneous grades with long internal branches. Finally, seven taxa appeared polyphyletic with MCM7, suggesting the formation of paralogs, including U. cavernosa, U. chilensis, U. erinacea, U. grandispora, U. silesiaca, U. subdasaea, and U. wasmuthii ( Fig. 16; Fig. ...
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... the accession from Cameroon was too short to be reliably assessed, missing the entire ITS1 portion (File S1). The ITS identity matrix indicated the largest clade from São Tomé and Príncipe to represent a different species, whereas the two smaller clades closer to U. articulata s.str. can be best interpreted as infraspecific lineages (Fig. 17). According to Nadel (2016), specimens in the large, separate clade differed from U. articulata s.str. in the pseudocyphellate cortex and the barbatic acid chemistry (protocetraric and fumarprotocetraric acid in U. articulata s.str.; Swinscow & Krog 1976a;Randlane et al. 2009) and may correspond to U. speciosa Motyka described from São ...
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... must be noted that, while the available sequence data originated from Europe and Africa, Usnea articulata is regarded as a cosmopolitan species ( Fig. 18; Swinscow & Krog 1976aElix & McCarthy 1998Stevens 1999;Clerc 2007Galloway 2007;Rand- lane et al. 2009;Schumm & Aptroot 2010;Gumboski & Eliasaro 2011;Kelly et al. 2011;Saag et al. 2011;Ohmura 2012;Orock et al. 2012;Schoch et al. 2012;Truong et al. 2013a, b;Millanes et al. 2014;Rafat et al. 2015;Nadel 2016;Galinato et al. 2017;Lücking et ...
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... barbata-intermedia-lapponica-substerilis aggregate. A large, unsupported cluster comprising 59 accessions from North America and Europe contained several identifications in largely unresolved patterns (Fig. 7, Table S1, Fig. S1), including U. arizonica (1), U. barbata (14), U. chaetophora (2), U. dasopoga (3), U. diplotypus (3), U. cf. glabrescens (1), U. intermedia (11), U. lapponica (17), U. rigida (1), and U. substerilis (5). Most of these are from the studies by Saag et al. (2011) andMark et al. (2016a). Usnea arizonica, corresponding to an apparently ...
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... species is distinguished from U. dasopoga mainly by the formation of annular cracks (Halonen et al. 1998;Randlane et al. 2009), but the accessions identified with that name fell into three different positions in the tree. Usnea chaetophora has also been considered a morphological variation of U. barbata or U. dasopoga and has been synonymized with the latter (Clerc 2011b, 2016). ...
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... ITS identity matrix separated Usnea diplotypus from the U. barbata-lapponica aggregate, but did not provide clear resolution at the species level for U. barbata-intermedia vs. U. lapponica-substerilis (Fig. 19). IGS data for this clade were largely congruent with ITS (Fig. S3). RPB1 did not resolve the barbata-intermedia and lapponica-substerilis subgroups, but place one sample in an entirely different, supported clade with U. florida/U. subfloridana (Fig. S5), an indication of a possible ...
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... brasiliensis-cornuta aggregate. The Usnea cornuta aggregate exhibited the most complex situation in the genus. No less than 100 accessions bear the names cornuta, aff. cornuta, or subcornuta, distributed in six, partly separate clades containing a total of 208 successions (Fig. 7, Fig. S1). Usnea cornuta s.lat. is defined as a shrubby, sorediate taxon with inflated branches constricted at their point of attachment, minute soralia producing isidiomorphs along the terminal branches, but the taxon was described as highly variable both morphologically and in the medullary chemistry (Clerc 2007;Randlane et al. 2009;Gerlach ...
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... 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%. 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 lineage 5, and the clade here defined as U. cornuta s.str. corresponded to most of their lineage ...
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... and subclades: U. subflammea, U. halei, U. flammea, the newly recognized U. tenuicorticata and U. trachyclada (Gerlach et al. 2020), U. cornuta s.str., U. aff. cornuta 1-10, U. cirrosa, U. cladocarpa, the newly recognized U. stipitata (Gerlach et al. 2020), and a large, homogeneous clade representing an unidentified species from New Zealand (Fig. 7, Table S1, Fig. ...
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... Andes), U. grandispora (Brazil), U. macaronesica (Azores), and U. subglabrata (Bolivia). Two newly recognized species were U. arianae from South America and Europe and U. rubropallens from Brazil ( Gerlach et al. 2020), corresponding to lineages 11 and 13 in Gerlach et al. (2019a). However, based on ITS data, U. arianae remained heterogeneous (Fig. 7, Fig. S1), as also shown in the ITS identity matrix (Fig. 20), whereas RPB1 resolved these samples in a single clade (Fig. S5). Also, the ITS revealed a subclade of two unidentified sequences from New Zealand ( Buckley et al. 2014) nested within U. arianae based on the long branch not representing that species in the strict sense. Thus, U. ...
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... ITS-based phylogeny divided Usnea ceratina into four clades, three smaller clades in a supported, monophyletic group and one large, separate clade (Fig. 7, Table S1, Fig. S1). The large clade, comprised of 14 specimens from Central Europe, Great Britain, and North America, was clearly distinct from the three smaller clades (Fig. 21). It had nine fully consistent and several partial substitutions compared to the smaller clades (File S1). Given that the lectotype of U. ceratina is from Poland ...
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... ITS-based phylogeny divided Usnea ceratina into four clades, three smaller clades in a supported, monophyletic group and one large, separate clade (Fig. 7, Table S1, Fig. S1). The large clade, comprised of 14 specimens from Central Europe, Great Britain, and North America, was clearly distinct from the three smaller clades (Fig. 21). It had nine fully consistent and several partial substitutions compared to the smaller clades (File S1). Given that the lectotype of U. ceratina is from Poland (Ohmura ...
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... ITS-based phylogeny divided Usnea ceratina into four clades, three smaller clades in a supported, monophyletic group and one large, separate clade (Fig. 7, Table S1, Fig. S1). The large clade, comprised of 14 specimens from Central Europe, Great Britain, and North America, was clearly distinct from the three smaller clades (Fig. 21). It had nine fully consistent and several partial substitutions compared to the smaller clades (File S1). Given that the lectotype of U. ceratina is from Poland (Ohmura 2001;Truong & Clerc 2012), this clade was here interpreted as U. ceratina s.str. This clade also included a fertile specimen from Spain identified as U. cristatula ...
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... clades were not clearly separated in our identity matrix and could conservatively be considered a single species, although up to three species could be distinguished if a more progressive concept was applied. The two Asian clades are well separated from each other in their identity values, but both overlap with the American-Mediterranean clade (Fig. 21). In any case, the members of this clade are not conspecific with U. ceratina s.str. and cannot be identified with that name. For the American-Mediterranean clade, three names are potentially available, U. calicornica Herre, U. subcomosa Vain., U. pachyclada Motyka, and U. solida Motyka (Herrera-Campos et al. 1998;Halonen et al. ...
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... The latter is characterized by alectorialic acid, although a smaller number of specimens (not sequenced) produce salazinic and barbatic acids (Clerc & Otte 2018). Usnea wasmuthii is characterized by salazinic and barbatic acids as major compounds (Ohmura 2001;Randlane et al. 2009;Clerc & Otte 2018). This taxon clustered in a subsequent clade (Fig. 7, Fig. S1) and hence the two identifications in the U. dasopoga aggregate apparently do not represent that species. The small clade including these accessions was here considered as U. aff. viktoriana, as the sequences in this portion all share a characteristic ITS sequence motif at the end of the ITS2 (File S1). The singleton sequence labeled ...
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... remaining accessions formed a supported clade (74%) with three clusters: a basal grade of four accessions identified as U. barbata and U. cf. cylindrica, a strongly supported subclade of six accessions identified as U. barbata, U. dasopoga, and U. substerilis, and a remaining shallow grade including the bulk of U. dasopoga identifications (Fig. 7, Fig. S1). The entire clade originated from the Northern Hemisphere, mostly from Europe (Table S1). Usnea cylindrica was only recently described from Sweden (Clerc 2011b), differing in branching pattern from U. dasopoga, and the only available sequence tentatively identified with that name by Mark et al. (2016a) is probably not that species, a ...
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... endochrysea-mutabilis-strigosa aggregate. This aggregate was strongly supported on a long branch in our ITS-based phylogeny (Fig. 7, Fig. S1). Few data were available from other markers, with U. endochrysea and U. mutabilis forming a monophyletic clade with MCM7 ( Fig. S7) and U. mutabilis a monophyletic clade in TUB2 (Fig. S4). With nuLSU, this clade was not recovered as monophyletic (Fig. ...
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... was described from Africa and used to name African and Australian samples corresponding to this morphology (Swinscow & Krog 1979Stevens 1999;Nadel 2016). Clerc (2004Clerc ( , 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. sanguinea represents a separate species. This interpretation is, however, tentative, as the single specimen from São Tomé and Principe differs in medullary chemistry from the type of U. sanguinea (barbatic vs. norstictic and acid) and was sterile, lacking both apothecia and soredia (Nadel. ...
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... our global phylogeny, sequences deposited under that name formed a monophyletic clade, but with clear internal structure and associated with numerous unidentified sequences from New Zealand (Fig. 7, Table S1, Fig. S1). The entire clade was strongly supported (90%) and contained five clusters: a fully supported subclade of 11 unidentified specimens from New Zealand ( Buckley et al. 2014), a more or less supported clade (70%) of another seven unidentified specimens from New Zealand from that same study, a further unidentified singleton from New ...
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... identified as U. subfloridana; a weakly supported clade (69%) with seven specimens of U. subfloridana from North America; another weakly supported clade (68%) with 14 accessions bearing either name from North America and Europe; and a large, rather homogeneous assembly of 50 samples also bearing both names from across the Northern Hemisphere (Fig. 7, Fig. S1). The latter included a low-quality sequence (AF117996) on a long branch, explaining the lack of support for this clade. These clades were separated by three other species, namely U. wasmuthii, U. fulvoreagens and U. glabrescens. The ITS identity matrix only supported the consideration of the strongly supported second clade as a ...
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... and the grade two potentially separate species. Usnea glabrescens formed a small grade including two samples originally identified as U. diplotypus and U. substerilis ( Saag et al. 2011), plus a larger, unsupported clade from across the Northern Hemisphere, including two specimens originally identified as U. pacificana and U. substerilis ( Fig. 7; Fig. ...
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... (and fumarprotocetraric) acid, although a salazinic-norstictic Figure 25. Circle phylogram of Usnea sect. Usnea based on MCM7, highlighting the formation of at least two paralogs in the U. florida-subfloridana 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 ...
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... chemotype is also known (Clerc 2007;Randlane et al. 2009). Eight ITS accessions have been deposited under this name (one as U. aff. glabrata). Two of them represented U. esperantiana (see above), whereas the remaining six clustered with an unidentified sequence in a supported clade (78%) sister to U. durietzii (Fig. 7, Fig. S1). These seven accessions formed two lineages: a singleton sequence from Switzerland on a rather long branch and a cluster of five sequences from the USA, Spain and Great Britain on a well-supported branch; the seventh sequence clustering at the base also belonged here but was very short (see above). Assuming that these accessions ...
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... above). Usnea subpectinata, described from Scotland, was considered a synonym of U. cornuta (Clerc 2004(Clerc , 2006). The clade labeled here with this name contained mostly accessions from Central and South America, but also one from Europe (France). It formed a monophyletic, but unsupported clade in the ITS, including three supported subclades (Fig. 7, Fig. S1). The first of these, here labeled U. aff. subpectinata, could be considered a separate species based on the number of consistent substitutions (File S1). The other two subclades, although strongly supported, differed from each other at the 98.5% threshold, but not from the other samples in this clade. The RPB1 recovered the entire ...
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... Usnea rubicunda, originating from North and South America, Europe, and Asia ( Ohmura 2002Ohmura , 2008Kelly et al. 2011;Saag et al. 2011;Schoch et al. 2012;Truong et al. 2013a;Millanes et al. 2014;Gerlach et al. 2017Gerlach et al. , 2019a). Most of these clustered in the same part of the tree, but did not form a coherent entity (Fig. 7, Table S1, Fig. S1). One supported clade (83%) contained 13 samples from North and South America, Macaronesia, the Mediterranean, and Asia, including one sequence from Brazil (MF669912) identified as U. aff. rubicunda (Ger- lach et al. 2019a) and one unpublished sequence from the USA deposited by Z. R. Caven et al. in 2016, identified as U. ...
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... analysis, resulting in a clear separation of U. rubrotincta from U. rubicunda. However, all sequenced specimens identified as U. rubicunda in that study corresponded to the lineages in the first clade, thus not representing U. rubicunda s.str. Usnea rubrotincta itself formed a core clade separate from U. rubicunda in another section of the tree (Fig. 7, Table S1, Fig. S1) with three sequences from Japan ( Ohmura 2002Ohmura , 2008) and two unpublished sequences from Taiwan deposited by Y.-M. Shen et al. in 2012. Three further sequences identified as U. rubrotincta from Japan (Ohmura 2002;Ohmura & Clerc 2019) and South Korea (deposited by K.-M. Lim et al. in 2005, but apparently not used in a ...
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... in a clear separation of U. rubrotincta from U. rubicunda. However, all sequenced specimens identified as U. rubicunda in that study corresponded to the lineages in the first clade, thus not representing U. rubicunda s.str. Usnea rubrotincta itself formed a core clade separate from U. rubicunda in another section of the tree (Fig. 7, Table S1, Fig. S1) with three sequences from Japan ( Ohmura 2002Ohmura , 2008) and two unpublished sequences from Taiwan deposited by Y.-M. Shen et al. in 2012. Three further sequences identified as U. rubrotincta from Japan (Ohmura 2002;Ohmura & Clerc 2019) and South Korea (deposited by K.-M. Lim et al. in 2005, but apparently not used in a published ...
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... subcornuta aggregate. This is another shrubby, sorediate species, characterized by a subcortical, orangered pigment, large soralia with isidiomorphs, and a stictic-norstictic acid chemistry ( Randlane et al. 2009;Truong & Clerc 2016). Four ITS accessions were available under this name, forming three lineages in two different places of the tree (Fig. 7, Fig. S1). One sequence from Ecuador ( Truong et al. 2013a) fell close to several unidentified accessions from New Zealand, whereas the other three sequences formed two lineages in close proximity, one singleton from Ecuador ( Truong et al. 2013a) and two from Europe ( Saag et al. 2011, Truong et al. 2013a). Given that U. subcornuta was ...
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... from the Canary Islands ( Gerlach et al. 2019a). In the study by Gerlach et al. (2019a), one of the sequences from the Canary Islands (238ES = 238TEN) was labeled U. geisleriana, and all four fell in the same area of the species tree together with a sequence identified as U. flammea. The latter was here revealed to form part of U. flammea s.str. (Fig. 7, Fig. S1), whereas the two accessions from the Canary Islands formed two separate lineages in proximity to each other, but not a single clade. The two accessions from Brazil fell in a separate area of the ITS tree, there clustering with weak support (70%). RPB1 also separated the four accessions into three clades (Fig. S5), similar to MCM7 ...
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... our ITS-based tree, the five available sequences labeled U. subscabrosa formed an unsupported clade with two strongly to fully supported subclades ( Fig. 7; Fig. S1): one with one accession from North America (apparently unpublished; origin confirmed by consultation of the NYBG C. V. Starr Virtual Herbarium at http:// sweetgum.nybg.org/science/vh) and two from Europe (Spain; Araujo 2016), and a second subclade with two accessions from Brazil ( Gerlach et al. 2019a). The first subclade was here ...
Citations
... CMA measurements developed by Clerc (1984Clerc ( , 1987 and based on Asahina's observations (Asahina 1956) added important characters to distinguish species (Clerc & Naciri 2021). Recently DNA sequencing opened a new area in the systematics of this genus (Articus et al. 2002;Ohmura 2002Ohmura , 2008Ohmura , 2020Wirtz et al. 2006;Kelly et al. 2011;, Mark et al. 2016Gerlach et al. 2019;Ohmura & Clerc 2019, 2023Lücking et al. 2020), bringing to light a high, and so far, hidden variability (Gerlach et al. 2019. Nowadays, the species number of this genus is truly uncertain. ...
Type material of twelve species of Usnea related to Asia and South Africa were studied and one species from North America is newly described. Holotype specimens of eight Chinese taxa described in 1975 were synonymized as follows: U. mengyangensis is lectotypified and synonymized with U. aciculifera. Usnea recurvata and U. subrectangulata are synonymized with U. baileyi. Usnea kirinensis is synonymized with U. barbata s.str. Usnea crassiuscula and U. yunnanensis (= U. australis J.D. Zhao et al., nom. illeg. non Fr.) are synonymized with U. bismolliuscula. Usnea iteratocarpa is synonymized with U. cristatula (new to Asia, China). Usnea decumbens is synonymized with U. intumescens (new to Asia, China). Usnea entoviolata and U. roseola are synonymized with U. fragilis (new to Asia, China, India, Japan). The lectotype of Usnea trichoideoides was found to belong to the Usnea pectinata aggr. and consequently U. montis-fuji was resurrected for U. trichodeoides sensu Ohmura. Usnea ceratina and U. trichodeoides are excluded from the Japan lichen flora. Usnea macaronesica a recently described Macaronesian and South American species is synonymized with the Asian taxa U. pycnoclada. Usnea chicitae is described as a new species endemic of the Southern Appalachian Mountains. It is characterized by stipitate efflorescent soralia, a tenuicorticata-type of CMA and the presence of salazinic and psoromic acid in the medulla.
... Usnea Adans. is a fruticose lichen genus of the family Parmeliaceae (Lecanoromycetes), comprising nearly 450 currently accepted species (Lücking et al. 2020), widely distributed in polar, temperate and tropical regions. The genus is readily distinguished by the shrubby to pendulous thallus, branches with an elastic central axis, and the presence of usnic acid in the cortex. ...
... To reconstruct the phylogeny, we used a total of 178 ITS sequences (see Supplementary Material File S2), 102 of which were obtained from GenBank, plus 76 newly generated sequences (54 from the Philippines (Table 1), 21 from Japan and Taiwan and one from Portugal) obtained for this study (Supplementary Material File S2). Almost all available sequences from Asia were added (Lücking et al. 2020), except those not identified or with dubious identification (U. nidifica_Li207c and U. nidifica_Li190c). Since we putatively found Usnea dasaea in the Philippine material, we also added a sequence for this species obtained from Portugal (type country). ...
... Since we putatively found Usnea dasaea in the Philippine material, we also added a sequence for this species obtained from Portugal (type country). Usnea longissima was chosen as outgroup following Lücking et al. (2020). ...
A first integrative survey of the genus Usnea in the southern Philippines, taking into account morphological, anatomical, chemical and molecular characters, resulted in the recognition of 20 taxa, including three species new to science: Usnea angulata Ach., U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr., U. brasiliensis (Zahlbr.) Motyka, U. confusa Asah., U. croceorubescens Stirt., U. dasaea Stirt., U. himalayana C. Bab., U. krogiana P. Clerc, U. longissima Ach., U. nidifica Taylor, U. norsticornuta A. Gerlach & P. Clerc sp. nov. (characterized by a moderately thick cortex and by the presence of norstictic acid), U. paleograndisora A. Gerlach & P. Clerc sp. nov. (characterized by an orange subcortical pigmentation in the medulla, with enlarging soralia and a moderately thick and shiny cortex), U. pectinata Taylor, U. pygmoidea (Asahina) Y. Ohmura, U. rubicunda Stirt., U. rubrotincta (Stirt.) Zahlbr., U. spinulifera (Vain.) Motyka, U. subscabrosa Motyka and U. yoshihitoi P. Clerc & A. Gerlach sp. nov. (characterized by a lax medulla with non-conglutinated hyphae). Usnea krogiana is a new record for Asia; Usnea brasiliensis , Usnea confusa and U. croceorubescens are new records for the Philippines. This is the first phylogenetic study to include DNA sequences of Usnea from the Philippines. Molecular data from the ITS rDNA (76 newly generated sequences) are presented for most taxa except for U. himalayana , U. longissima and U. subscabrosa . At least six further taxa remain unidentified, awaiting the collection of additional specimens.
... Hybridization has been detected in other genera in the Parmeliaceae, most recently in Xanthoparmelia (Keuler et al. 2022). Incongruent results obtained from the Mcm7 gene may also reflect gene duplication and paralog formation as shown in Usnea (Lücking et al. 2020). ...
In recent years, the genus Bryoria ( Parmeliaceae , Lecanoromycetes ) has been the subject of considerable phylogenetic scrutiny. Here we used information on six gene regions, three nuclear protein-coding markers ( Mcm 7, GAPDH and Tsr 1), two nuclear ribosomal markers (ITS and IGS) and a partial mitochondrial small subunit (mtSSU), to examine infrageneric relationships in the genus and to assess species delimitation in the Bryoria bicolor / B . tenuis group in section Divaricatae . For this purpose, phylogenetic analyses and several of the available algorithms for species delimitation (ASAP, GMYC single, GMYC multiple and bPTP) were employed. We also estimated divergence times for the genus using *BEAST. Our phylogenetic analyses based on the combined data set of six gene loci support the monophyly of sections Americanae , Divaricatae and Implexae , while section Bryoria is polyphyletic and groups in two clades. Species from Bryoria clade 1 are placed in an emended section Americanae . Our study reveals that section Divaricatae is young ( c . 5 My) and is undergoing diversification, especially in South-East Asia and western North America. Separate phylogenetic analyses of section Divaricatae using ITS produced a topology congruent with the current species concepts. However, the remaining gene regions produced poorly resolved phylogenetic trees and the different species delimitation methods also generated highly inconsistent results, congruent with other studies that highlight the difficulty of species delimitation in groups with recent and rapid radiation. Based on our results, we describe the new species B . ahtiana sp. nov., characterized by its bicolorous, caespitose, widely divergent thallus, conspicuously thickening main stems, well-developed secondary branches, and rather sparse third-order branchlets. Another new lineage, referred to here as B . tenuis s. lat., is restricted to western North America and may represent a new species recently diverged from B . tenuis s. str., though further work is needed.
... The conventional DNA barcode marker for Fungi is the internal transcribed spacer (ITS) [18]. Despite potential challenges arising for some groups when used as the only marker (e.g., intragenomic variation or lack of resolution for discrimination), the ITS marker remains a useful exploratory means to assess fungi species delimitation and identification [8,19]. For many species it may be necessary to include additional markers and interpret the phylogenetic results in an integrative context to obtain an accurate outline of their boundaries [20]. ...
DNA barcoding is a powerful method for the identification of lichenized fungi groups for which the diversity is already well-represented in nucleotide databases, and an accurate, robust taxonomy has been established. However, the effectiveness of DNA barcoding for identification is expected to be limited for understudied taxa or regions. One such region is Antarctica, where, despite the importance of lichens and lichenized fungi identification, their genetic diversity is far from characterized. The aim of this exploratory study was to survey the lichenized fungi diversity of King George Island using a fungal barcode marker as an initial identification tool. Samples were collected unrestricted to specific taxa in coastal areas near Admiralty Bay. Most samples were identified using the barcode marker and verified up to the species or genus level with a high degree of similarity. A posterior morphological evaluation focused on samples with novel barcodes allowed for the identification of unknown Austrolecia, Buellia, and Lecidea s.l. species. These results contribute to better represent the lichenized fungi diversity in understudied regions such as Antarctica by increasing the richness of the nucleotide databases. Furthermore, the approach used in this study is valuable for exploratory surveys in understudied regions to guide taxonomic efforts towards species recognition and discovery.
... They found that ITS was used in most studies of species pairs, but that it presents limited resolution for recently diverged lineages such as in the Usnea antarctica (sorediate, asexual) and U. aurantiacoatra (apotheciate, sexual) pair. Using ITS and RPB1 markers, the data were insufficient to delimit the two species, which clustered in one lineage (Seymour et al., 2007;Wirtz, Printzen & Lumbsch, 2008;Wirtz et al., 2012;Lücking et al., 2020). However, using RADseq or microsatellite markers, these species were found to be distinct Grewe et al., 2018). ...
Accurate species delimitations are fundamental to our understanding of the genetic diversity on Earth and a vital part in evolutionary and conservation biology research. In lichenized fungi, species pairs have the same morphology and chemistry. They only differ in how they reproduce with one species using sexual reproductive structures and the other using asexual propagules. To classify these as one species or two has been a point of contention, and conclusions based on Sanger sequencing, where sequence data are limited and species boundaries are usually not observed, have been refuted after analysis with genome-scale data such as restriction site-associated DNA sequencing that tends to find fixed genetic differences between the two morphs. Pseudocyphellaria glabra and P. homoeophylla have long been considered a species pair that differ in geographical ranges but co-occur in New Zealand. We used restriction site-associated DNA sequencing data and generated thousands of genetic loci across 53 individuals. The RADseq data provided high-resolution phylogenetic and population genomic information. A maximum-likelihood phylogenetic reconstruction recovered both species as separate lineages, whereas population genetics indicated some evidence for admixture among P. glabra and P. homoeophylla from New Zealand. It is not clear whether the latter is due to ancient polymorphism or recent gene flow. Our study represents another example of the usefulness of RADseq to test species boundaries that segregate closely related species in lichenized fungi.
... with several species delimitations [57]. Although phylogenetic studies based on a few loci have unveiled new clades of neuropogonoid lichens (e.g., U. lambii, U. messutiae, U. pallidocarpa, and U. ushuaiensis; [42,58,59]), these studies have not been able to disentangle phylogenetic relationships among these lineages and species boundaries of some groups remain unclear [34]. RADseq has already shed light on an example of the widely debated species-pairs for U. antarctica and U. aurantiacoatra [60]. ...
... The phylogenetic structure of neuropogonoid lichens has been debated in the literature [34]. For decades since the first phenotype-based reviews, lichenologists have discussed not only possible phylogenetic relationships among these lichens but also their evolutionary timeframe, including their biogeographical history [35,48,86]. ...
... For decades since the first phenotype-based reviews, lichenologists have discussed not only possible phylogenetic relationships among these lichens but also their evolutionary timeframe, including their biogeographical history [35,48,86]. Nevertheless, in most cases, either the lack of a complete sampling or the insufficient variability of genetic markers has masked a robust reconstruction of the evolutionary history [34]. For the first time, we present a complete phylogenomic representation of the neuropogonoid group using cutting-edge sequencing methods and NGS assembly tools. ...
Nearly 90% of fungal diversity, one of the most speciose branches in the tree of life, remains undescribed. Lichenized fungi as symbiotic associations are still a challenge for species delimitation, and current species diversity is vastly underestimated. The ongoing democratization of Next-Generation Sequencing is turning the tables. Particularly, reference-based RADseq allows for metagenomic filtering of the symbiont sequence and yields robust phylogenomic trees of closely related species. We implemented reference-based RADseq to disentangle the evolution of neuropogonoid lichens, which inhabit harsh environments and belong to Usnea (Parmeliaceae, Ascomycota), one of the most taxonomically intriguing genera within lichenized fungi. Full taxon coverage of neuropogonoid lichens was sampled for the first time, coupled with phenotype characterizations. More than 20,000 loci of 126 specimens were analyzed through concatenated and coalescent-based methods, including time calibrations. Our analysis addressed the major taxonomic discussions over recent decades. Subsequently, two species are newly described, namely U. aymondiana and U. fibriloides, and three species names are resurrected. The late Miocene and Pliocene-Pleistocene boundary is inferred as the timeframe for neuropogonoid lichen diversification. Ultimately, this study helped fill the gap of fungal diversity by setting a solid backbone phylogeny which raises new questions about which factors may trigger complex evolutionary scenarios.
... Sometimes voucher information is missing altogether, sometimes it is cited only in the corresponding paper, but not submitted to the sequence database. For instance, in the lichenized fungal genus Usnea, 30% of nearly 1,500 accessions of the fungal barcoding marker ITS had issues with voucher information or lacked such information all together, not only in the original submissions but also in the associated publications (Lücking et al. 2020). In diatoms, more than 30% of the sequences lack vouchers (pers. ...
The access to molecular collections worldwide greatly improves the quality of scientific research by making a growing number of data available for investigation. The efforts on digitization also aim at facilitating the exchange of material between institutions and researchers that must follow regulations in place and respect best practice. The handbook presented here proposes a workflow to follow to safely exchange materials, in accordance with international laws and legislations. We make numerous recommendations here to help the institutions and researchers to navigate the legal and administrative procedures, to manage molecular collections in the best way possible.
... Species such as U. chloreoides Motyka, U. duriuscula Motyka, U. gigas Motyka and U. himantodes Stirt. are putative synonyms of U. mexicana (Swinscow & Krog 1988;Herrera-Campos et al. 1998;Ohmura 2001;Truong et al. 2013b;Nadel 2016;Lücking et al. 2020). Furthermore, species such as U. africana Motyka, U. amaniensis Dodge, U. bakongoensis P. A. Duvign., U. fernandiae P. A. Duvign., U. gigas and U. savanarum P. A. Duvign. ...
... Until recently, U. pectinata s. lat. had been included in only a small number of molecular investigations (Ohmura 2002;Articus 2004;Truong et al. 2013a;Nadel 2016;Temu et al. 2019;Lücking et al. 2020). However, these studies have paid greater attention to the genetic variation rather than morphological and chemical variability. ...
This study investigated the molecular, chemical and morphological variation in the Usnea pectinata aggregate using 42 specimens, 22 from Tanzania and 20 from São Tomé and Príncipe. A total of 31 sequences (13 ITS, 13 nuLSU and 5 RPB 1) were generated. The results are presented in two phylogenies: first a three-markers ‘backbone’ phylogeny for the U. pectinata aggregate, where six distinct, strongly supported subclades indicate considerable genetic variation in the dataset; and second, an ITS phylogeny with 47 terminals along with a mapping of morphological and chemistry data. Several well-supported monophyletic clades were recovered in both phylogenies and these may well represent separate species in the complex referred to here as the U. pectinata aggregate. Three morphotypes characterized by axis pigmentation and four by branch shape were noted. Six chemotypes were observed.
... Usnea is one of the most speciose genera in the family Parmeliaceae, with estimates ranging from c. 350 taxa (Thell et al. 2012;Lücking et al. 2017) to over 400 (Lücking et al. 2020). This hyperdiverse genus has historically been difficult to delineate into species due to a high degree of variability and the lack of characters to draw from within the genus (Clerc 1998;Ohmura 2001). ...
... The similar appearance of the species that do occur in São Tomé surely led to these incorrect determinations. Originally, the specimens collected in 2012-2013 were determined to represent 11 species (Nadel 2016) and were later published in Lücking et al. (2020) based solely on molecular data, using the original names or slight variations of them, from the unpublished thesis of the first author. This study, for the first time, combines molecular, morphological and chemical data to publish species determinations from the São Tomé and Príncipe collection. ...
... Sequences were then manually inspected to determine correct assembly and direction. From the original 87 sequences (Nadel 2016;Lücking et al. 2020), 56 were selected for this study by choosing only specimens examined and removing sequences that were deemed of low quality based on ambiguous characters. These 56 sequences were combined with 82 sequences gathered from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm. ...
An investigation of the genus Usnea , in the biodiversity hotspot of the Republic of São Tomé and Príncipe in tropical West Africa, is presented here. Fifteen species, or species aggregates, were recorded for the islands: Usnea articulata aggr., Usnea baileyi (Stirt.) Zahlbr., Usnea beckeri P. Clerc & Nadel, Usnea bicolorata Motyka, Usnea erinacea aggr., Usnea exasperata (Müll. Arg.) Motyka, Usnea firmula (Stirt.) Motyka, Usnea krogiana P. Clerc, Usnea longiciliata P. Clerc & Nadel, Usnea nodulosa Swinscow & Krog, Usnea pectinata aggr., Usnea sorediosula Motyka, Usnea submollis J. Steiner, and two undetermined species. Two species of lichen are described as new to science: U. beckeri and U. longiciliata. Both species are characterized by a dense and brittle, dark green thallus, the presence of apothecia surrounded by long cilia-like fibrils, a lack of soredia, and the presence of two unknown substances; however, whereas U. beckeri has a pendant growth form and can reach 25 cm in length, U. longiciliata is differentiated by a shrubby growth form of less than 6 cm. These two species also have different ascospore dimensions. A molecular phylogenetic analysis is presented that lends support to their description as new species. Additionally, Usnea krogiana is noted as new to continental Africa and nine Usnea species or aggregates are noted as new to the Islands of São Tomé and Príncipe in the Gulf of Guinea.
... Observed differences in sequence patterns between lineages in cases not assessed with quantitative species delimitation methods were comparatively high, with percentage similarities ranged between 90.37% (Gyalideopsis vulgaris clade) and 99.19% (Echinoplaca marginata clade), but mostly below 98% (Table 2). These differences are substantial considering that the underlying marker (nuLSU) is relatively conserved and for the highly variable ITS, 99% is mostly regarded as a critical threshold when analysed in a phylogenetic framework (Lücking et al., 2020a(Lücking et al., , 2020b. Overall, 46 out of 51 tested taxa (90%) were either monophyletic or at least phylogenetically coherent, supporting the validity of current taxonomic concepts in terms of accuracy, whereas the remaining taxa (10%) represented polyphyletic entities. ...
We present the first broad molecular-phylogenetic revision of the lichenized family Gomphillaceae, based on 408 newly generated sequences of the mitochondrial SSU rDNA and nuclear LSU rDNA, representing 342 OTUs. The phylogenetic analysis of 20 out of the 28 currently accepted genera resulted in 48 clades. Twelve genera were resolved as monophyletic: Actinoplaca, Arthotheliopsis, Bullatina, Caleniopsis, Corticifraga, Gomphillus, Gyalectidium, Gyalidea, Jamesiella, Rolueckia, Rubrotricha, and Taitaia. Two genera resulted paraphyletic, namely Aulaxina (including Caleniopsis) and Asterothyrium (including Linhartia). Six genera were in part highly polyphyletic: Aderkomyces, Calenia, Echinoplaca, Gyalideopsis, Psorotheciopsis, and Tricharia. While ascoma morphology and anatomy has traditionally been considered as main character complex to distinguish genera, our study supported the notion that the characteristic asexual anamorph of Gomphillaceae, the so-called hyphophores, are diagnostic for most of the newly recognized clades. As a result, we recognize 26 new genus-level clades, three of which have names available (Microxyphiomyces, Psathyromyces, Spinomyces) and 23 that will require formal description as new genera. We also tested monophyly for 53 species-level names for which two or more specimens were sequenced: 27 were supported as monophyletic and representing a single species, 13 as monophyletic but with an internal topology suggesting cryptic speciation, four as paraphyletic, and nine as polyphyletic. These data suggest that species richness in the family is higher than indicated by the number of accepted names (currently 425); they also confirm that recently refined species concepts reflect species richness better than the broad concepts applied in Santesson's monograph. A divergence time analysis revealed that foliicolous Gomphillaceae diversified after the K–Pg-boundary and largely during the Miocene, a notion supported by limited data available for other common foliicolous lineages such as Chroodiscus (Graphidaceae), Pilocarpaceae, and Porinaceae. This contradicts recent studies suggesting that only macrofoliose Lecanoromycetes exhibit increased diversification rates in the Cenozoic.
