Reference-Based RADseq Unravels the Evolutionary History of Polar Species in ‘the Crux Lichenogorum’ Genus Usnea (Parmeliaceae, Ascomycota)

Abstract

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.

Full text

Citation: Otero, A.; Barcenas-Peña,
A.; Lumbsch, H.T.; Grewe, F.
Reference-Based RADseq Unravels
the Evolutionary History of Polar
Species in ‘the Crux Lichenogorum’
Genus Usnea (Parmeliaceae,
Ascomycota). J. Fungi 2023,9, 99.
https://doi.org/10.3390/
jof9010099
Academic Editor: Silke Werth
Received: 6 December 2022
Revised: 3 January 2023
Accepted: 8 January 2023
Published: 11 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Fungi
Journal of
Article
Reference-Based RADseq Unravels the Evolutionary History
of Polar Species in ‘the Crux Lichenogorum’ Genus Usnea
(Parmeliaceae, Ascomycota)
Ana Otero *, Alejandrina Barcenas-Peña, H. Thorsten Lumbsch and Felix Grewe
The Grainger Bioinformatics Center & Negaunee Integrative Research Center, Science & Education,
The Field Museum, Chicago, IL 60605, USA
*Correspondence: aotero@fieldmuseum.org
Abstract:
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 delimi-
tation, 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 neuro-
pogonoid 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.
Keywords:
next-generation sequencing; lichen-forming fungi; phylogenomics; systematics; species
delimitation; species complex
1. Introduction
The kingdom Fungi represents one of the most diverse branches in the tree of life [
1
].
Naturalists have pointed mainly to the vast diversity of growth forms, reproductive strate-
gies, habitats, biotic associations, and lifestyles that fungi can show or be adapted to.
Unfortunately, all this complexity has often hindered understanding their place in the tree
of life and underestimated their species diversity [
2
]. The latest estimates indicated between
2.2 and 3.8 million species of fungi, from which only 148,000 (the majority in Ascomycota)
are currently accepted [
3
,
4
]. Thus, nearly 90% of fungi diversity remains unknown, versus
50% for better-studied groups, such as plants [
5
]. One of the major reasons for hidden
fungal diversity is poorly explored habitats, such as lichenicolous fungi [
4
]. Another reason
is the restudy of known taxa through cutting-edge molecular methods.
Lichenized fungi are spread along the Fungal Tree of Life with more than 16,000 species,
mostly belonging to Ascomycota [
6
]. The extraordinary nature of lichens as symbiotic
associations involving multiple kingdoms and complex hierarchies of living organisms
(e.g., primary and secondary fungi, algae/cyanobacteria, and microbiome elements) arises
as a major challenge for species delimitation [
7
]. Indeed, traditional phenotype-based
taxonomy (i.e., morphological and chemical traits) has led to a vast underestimation of
actual species diversity [6].
J. Fungi 2023,9, 99. https://doi.org/10.3390/jof9010099 https://www.mdpi.com/journal/jof

J. Fungi 2023,9, 99 2 of 20
The integration of molecular methods for species delimitation has revolutionized
lichen systematics [
8
–
10
]. From the early use of single barcoding loci, such as ITS, to the
Next-Generation Sequencing (NGS) tools, the systematics of lichenized fungi has benefited,
and a wide range of complex evolutionary processes hidden by morphology are now
coming to light [
11
]. This new knowledge through molecular studies contributes to an
increase in the number of described species. New lineages are inferred as a result of three
main processes: (1) homoplasy (i.e., similar traits have evolved independently and multiple
times along the phylogeny) is perhaps the most frequently inferred, and one of the major flaws
of a phenotypic-only-based taxonomy [
12
,
13
]; (2) so-called cryptic species (i.e., species with no
observable and apparent phenotypic differences) are being abundantly argued for many newly
described species [
14
–
16
]; (3) species with apparent phenotypic differences showed short
genetic distances, often interpreted as a result of recent diversification and incomplete lineage
sorting [
17
,
18
] or as different morphotypes of the same species [
19
–
21
]. This complex scenario
of lichen speciation and the exceptionally high infraspecific phenotypic plasticity reduce
the efficacy of some widely used barcode genes [
22
–
24
]; thus, genome-wide sequencing
approaches are required to build robust phylogenies [25,26].
Among the various NGS techniques developed over the past decades, restriction
site-associated sequencing (RADseq) is one of the most successful and cost-effective tools
for species delimitation. In RADseq, high-throughput sequencing generates data from
thousands of loci over the genome that elucidates complex speciation questions [
27
]. For
lichenized fungi, using reference-based RADseq (i.e., loci are mapped back to a reference
genome) allows for the exclusion of loci from other organisms occurring in these symbioses
so that robust phylogenomic trees can be generated among closely related species [
28
–
30
].
Nevertheless, the assembly of massive amounts of genomic loci with potentially conflicting
phylogenetic signals entails new challenges and trade-offs between the computational
capacity and the accuracy to integrate complex biological scenarios such as ancient intro-
gression, hybridization, or incomplete lineage sorting into the bioinformatic pipelines [
31
].
Fortunately, the rapid proliferation of workflows for NGS data that accomplish these possi-
ble biases is parallel to the popularization of these sequencing methods [
32
,
33
]. Ultimately,
incorporating high-throughput sequencing as a tool for species delimitation is helping to
assess the taxonomic value of phenotypical characters traditionally described, which may
have been considered taxonomically irrelevant under a plethora of morphological varieties
often found in lichens [6].
Neuropogonoid lichens comprise around 17 species of lichen-forming fungi belonging to
the most iconic beard-like macrolichen: the genus Usnea Dill. ex Adans., currently considered
among the ten most speciose genera (~350 spp, Parmeliaceae, Ascomycota; [
34
]). Neuro-
pogonoid species are distributed in polar regions and high-alpine regions. They are adapted
to harsh microclimates, including extreme temperature variation, high radiation and winds,
and scarce water availability [
35
]. Contrary to the most widely repeated pattern of higher
species richness towards the equator line [
36
], this group has its main center of diversity
in Antarctica and subantarctic regions [
35
]. Recently, these organisms were noted as global
warming bioindicators since their habitats are documented to be some of the most sensitive
to temperature increases [
37
–
41
]. Diagnostic characters for this group are the saxicolous
habit, strong black pigmentation of the thallus (often variegated) and apothecia discs, and
a relatively thick cortex [
42
]. Usnic acid is present in the cortex of all species, and varied
combinations of depsides and depsidones in the medulla are described to characterize dif-
ferent races within species, including an abundance of acid-deficient lineages [
35
]. These
markedly distinguishing morphological and chemical traits are often associated with the
adaptation to harsh environments [
42
] and have led lichenologists to consider this group
either as a subgenus [
35
,
43
–
46
], a section [
47
], or a separate genus [
48
–
55
]. Lastly, multi-gene
phylogenies revealed the group as polyphyletic within Usnea subgen. Usnea and the name
‘neuropogonoid’ were coined to refer to the Neuropogon core group [
56
]. The systematics
of Usnea at a specific level is considered exceptionally difficult, often known as ‘the crux
lichenogorum’ by modern lichenologists, and neuropogonoid lichens follow this pattern

J. Fungi 2023,9, 99 3 of 20
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. pal-
lidocarpa, 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
]. Still, questions remain for system-
atics and the spatio-temporal evolutionary framework that shaped the diversification and
distribution of neuropogonoid lichens.
In this study, we aim to keep filling the gap of fungal diversity by using cutting-edge
methods such as reference-based RAD sequencing to investigate the phylogenomic relation-
ships and species boundaries for the 17 species of neuropogonoid lichens in combination
with morphological and chemical characters to circumscribe these clades. To this end, we
considered the following specific objectives: (1) to reconstruct the backbone phylogeny for
neuropogonoid lichens; (2) to infer the evolutionary timeframe for the neuropogonoid clade;
and (3) to contribute to fungal taxonomy by addressing the taxonomic rearrangements
inferred from the phylogenetic findings.
2. Materials and Methods
2.1. Specimen Sampling
Taxon sampling of neuropogonoid lichens was based on the taxa validly described at
the species level in [
35
], which constitutes the most comprehensive monograph of the neu-
ropogonoid group to date. Thirteen of the fifteen species recognized by Walker [
35
] were
included in the present study (Table S1, in Supporting Information). The two species not
considered for the present study were: (1) U. durietzii Motyka, interpreted as intermediate
between the neuropogonoid group and sect. Usnea [
35
], which was subsequently shown to
be only distantly related to neuropogonoid lichens [
56
]; and (2) U. neuropogonoides Motyka,
described from few and immature specimens with unclear relationships to neuropogonoid
lichens [
35
]. Four additional species recently accepted were also included here (i.e., U. lam-
bii, U. messutiae, [
42
]; U. ushuaiensis, [
58
]; and U. pallidocarpa [
59
]). In total, the ingroup
comprises 17 currently accepted species. In addition, we propose below the acceptance of
five additional species. A total of 120 samples from neuropogonoid lichens were studied,
plus six samples of three other Usnea species used as the outgroup. Samples were obtained
from multiple field campaigns performed by Phylogenomic Initiative Centre at The Field
Museum as well as from herbaria collections, including The Field Museum (F), Herbarium
Senckenbergianum (FR), and external research collaborations (Table S1).
2.2. Morphological and Chemical Characterization
The morphological and chemical investigation was performed for the taxa belonging
to the four selected clades with a particular taxonomic interest: (1) Clade A: New Zealand
endemic species, (2) Clade B: U. rohmederi, (3) Clade C: U. perpusilla, and (4) Clade D:
U. trachycarpa complex (see Figure 1). Morphological features were examined using a stereo-
microscope Olympus SZX-ILLD100 (Olympus Co., Tokyo, Japan), focusing primarily on ten
morphological characters that were considered valuable for species delimitation by Walker
(1985, [
35
]): (1) habit, (2) holdfast branching, (3) branching pattern above the holdfast
(4) thallus pigmentation, (5) surface ornamentation of the thallus, (6) presence of papillae,
(7) presence of fibrils, (8) internal structure, (9) presence of apothecia, and (10) presence
of vegetative propagules (see Table S2). High-performance thin-layer chromatography
(HPTLC) was done for the same species of taxonomic interest. Accordingly, a total of
73 samples were soaked in acetone overnight and chromatographed using solvent system
C [
61
–
64
]. Major and minor substances were identified, and chemosyndromes for all species
were assigned based on the previous chemosyndrome characterization [65].

J. Fungi 2023,9, 99 4 of 20
J. Fungi 2023, 9, x FOR PEER REVIEW 4 of 21
presence of vegetative propagules (see Table S2). High-performance thin-layer
chromatography (HPTLC) was done for the same species of taxonomic interest.
Accordingly, a total of 73 samples were soaked in acetone overnight and
chromatographed using solvent system C [61–64]. Major and minor substances were
identified, and chemosyndromes for all species were assigned based on the previous
chemosyndrome characterization [65].
Figure 1. Phylogenetic reconstruction of neuropogonoid lichens based on concatenated DNA
sequences of 21,831 loci (m4 dataset) obtained from Bayesian inference (BI) through the ExaBayes
software. Values at nodes indicate posterior probability yielded by BI on the left and bootstrap
support (BS) from maximum-likelihood (ML) inference (RaxML-NG software) on the right.
Maximum support for both inferences (i.e., BI = 1; BS = 100) was obtained for those nodes with no
values. Dark circles on the nodes represent the crown of the monophyletic species considered.
Clades of taxonomic interest are marked with letters A-D. Chemical characters are shown for clades
A-D. Up to 12 chemical acids were found: Us, Usnic acid; Nor, norstictic acid; Sal, salazinic acid;
Csal, consalazinic acid; Pso, psoromic acid; Pro, protocetraric acid; Squ, squamatic acid; Gyr,
gyrophoric acid; Hyp, hypostrepsilic acid; Neu, neuropogolic acid; Pla, placodolic acid; Fat, fatty
acids. High concentration of the substance is indicated with black bands, low concentration or traces
are indicated in gray bands, and non-colored bands indicate the absence of the substance. Images
Figure 1.
Phylogenetic reconstruction of neuropogonoid lichens based on concatenated DNA se-
quences of 21,831 loci (m4 dataset) obtained from Bayesian inference (BI) through the ExaBayes
software. Values at nodes indicate posterior probability yielded by BI on the left and bootstrap sup-
port (BS) from maximum-likelihood (ML) inference (RaxML-NG software) on the right. Maximum
support for both inferences (i.e., BI = 1; BS = 100) was obtained for those nodes with no values. Dark
circles on the nodes represent the crown of the monophyletic species considered. Clades of taxonomic
interest are marked with letters A-D. Chemical characters are shown for clades A-D. Up to 12 chemical
acids were found: Us, Usnic acid; Nor, norstictic acid; Sal, salazinic acid; Csal, consalazinic acid; Pso,
psoromic acid; Pro, protocetraric acid; Squ, squamatic acid; Gyr, gyrophoric acid; Hyp, hypostrepsilic
acid; Neu, neuropogolic acid; Pla, placodolic acid; Fat, fatty acids. High concentration of the substance
is indicated with black bands, low concentration or traces are indicated in gray bands, and non-colored
bands indicate the absence of the substance. Images of the thallus, reproductive structures and inter-
nal structure are shown for representative samples from each species at Clades A-D: (
a
)U. sp New49;
(
b
)U. sp New35, thallus cross section; (
c
)U. sp New35, basis; (
d
)U. sp New35, apothecia; (
e
)U. pseu-
docapillaris 3760; (f)U. pseudocapillaris 3758, soredia; (g)U. pseudocapillaris 3760, thallus cross section;
(
h
)U. subcapillaris New 32; (
i
)U. subcapillaris N100; (
j
)U. subcapillaris New 32, thallus cross section;
(
k
)U. ciliata New 23, (
l
)U. ciliata New 6; (
m
)U. ciliata N27, thallus cross section; (
n
)U. rohmederi
221-3B; (o)U. rohmederi 218-1, thallus longitudinal section; (p)U. perpusilla 222-2; (q)U. perpusilla

J. Fungi 2023,9, 99 5 of 20
208-19, thallus longitudinal section (cortex is indicated in red, medulla in blue, axis in black);
(
r
)U. aymondiana 197-1; (
s
)U. aymondiana 196-6, thallus cross section; (
t
)U. fibriloides 197-3;
(
u
)U. fibriloides 197-3, thallus longitudinal section (cortex is indicated in red, medulla in blue, axis in
black); (
v
)U. trachycarpa 19072; (
w
)U. trachycarpa 19072, apothecia; (
x
)U. trachycarpa 18684, thallus
cross section; (
y
)U. sphacelata 220-3; (
z
)U. sphacelata 226-1, thallus longitudinal section; (
aa
)U. sphace-
lata 226-1; (
bb
)U. sphacelata N110, soredia; (
cc
)U. sphacelata 220-3, soredia; (
dd
)U. subantarctica 203-11;
(
ee
)U. subantarctica 203-11, thallus longitudinal section; (
ff
)U. subantarctica 203-11; (
gg
)U. subantarc-
tica 203-11, soredia; (
hh
)U. trachycarpoides 173-1; (
ii
)U. trachycarpoides 234-8; (
jj
)U. trachycarpoides
182-3, thallus longitudinal section; (
kk
)U. hyyppae 4070; (
ll
)U. hyyppae 4158; (
mm
)U. hyyppae 4158,
thallus cross section. All black scales indicate 2 mm except for those where specific number is shown.
More information about specimen vouchers is provided in Table S1 of the Supplementary Material.
2.3. DNA Extraction and RAD Library Preparation
DNA extraction and RAD libraries were conducted through the Next-Generation
Facility at the University of Wisconsin Biotechnology Center (UWBC). DNA extraction
was performed using the DNeasy mericon Food Kit (Qiagen, Hilden, Germany). Paired-
end RADseq libraries were prepared using restriction enzyme ApeKI following [
66
] and
sequenced on a Novaseq6000 Illumina Inc. (San Diego, CA, USA) at UWBC by setting one
million reads per sample. Processed data were returned in the FASTQ format, with Phred
quality scores for all bases.
2.4. RADseq Assembly
Forward reads of all samples were processed in ipyrad v.0.9.84 [
67
] using the High-
Performance Computing (HPC) cluster installed at The Grainger Bioinformatics Center
(The Field Museum). Ipyrad was run using the “reference” assembly method, which maps
RAD sequences to a reference genome to determine homology. The genome of the lichen-
fungal culture of U. hakonensis Asahina [
68
] was used as the reference. The mapping sorted
the metagenomic lichen sequences for reads derived from the lichen fungus. Unmapped
sequences that did not match the lichen-fungal reference were discarded (reference-based
method, [
67
]). Ploidy was set to one to reflect the haploid nature of the fungal thallus.
The sensitivity of the results regarding data matrix completeness and the number of loci
was assessed by testing three different values of minimum taxon coverage per locus (i.e.,
parameter ‘m’): (1) the default value, m = 4; (2) 25% of the total samples, m = 32; and
(3) 50% of the total samples, m = 65. Thus, a total of three assembled data sets were
produced and denoted as m4, m25, and m50, respectively.
2.5. Phylogenomic Analyses
The resulting matrix of concatenated loci for the three datasets was used as the input
to perform phylogenomic analyses. Maximum likelihood (ML) inference was performed
through RAxML-Next-Generation v.1.1. [
69
] by running the ‘all-in-one’ option (i.e., ML tree
search + bootstrapping including both Felsenstein Bootstrapping and Transfer Bootstrap
Expectation method) over 20 different starting trees, GTR + GAMMA model and using the
automatic bootstrap stopping parameter with the majority rule criterion (autoMRE, cutoff:
0.030000). Bootstrap supports are represented on the best-scored ML tree. This software
re-implements former algorithms already integrated in RAxML/ExaML and provides new
features that outperform the accuracy, flexibility, speed, and scalability making it more
suitable for large empirical datasets [
69
]. Additionally, in order to assess the performance of
this recently developed method, RAxML v8.2.12 [70] (rapid bootstrapping, GTRGAMMA
model, autoMRE) was also implemented for the three datasets. The matrix producing
the highest average bootstrap support was also analyzed using Bayesian inference (BI) in
ExaBayes 1.5.1 [
71
]. Bayesian inference was set for two runs, four coupled chains, and
one million generations with a sampling frequency of 500 and a burn-in proportion of
0.25. A coalescent-based method for phylogenomic inference was also implemented for
the three data matrices through SVDquartets [
72
] in PAUP [
73
]. Individuals were grouped

J. Fungi 2023,9, 99 6 of 20
according to current species circumscriptions, and all possible quartets were evaluated
with 100 bootstrap replicates.
Potential historical introgression between the early diverging lineages of U. patago-
nica and the clade of the neuropogonoid core was tested through four-taxon D-statistic
(ABBA-BABA) tests [
74
]. In a pectinate four-taxon tree [(((P1,P2),P3),P4)], where A and
B represent ancestral and derived alleles, respectively, the P3 taxon is expected to share
derived alleles with either of the two sister species, P1 (BABA) or P2 (ABBA) in the same
proportion. An excess of BABA or ABBA pattern is interpreted as evidence of ancient
admixture between lineages. Accordingly, using the loci matrix for m4 dataset as the input,
U. patagonica samples were set up as the P1 taxon. A selection of two samples (i.e., the
two with the highest number of loci in the m4 dataset) per species from clade A were set
up as the P2 taxon. Likewise, species of the neuropogonoid core were set to represent
the P3 taxon. Ultimately, samples from the outgroup were assigned to the P4 taxon. We
implemented D-statistic calculations using allele frequencies through the ipa.baba module
of ipyrad, specifically designed for RADseq data sets. Tests were run for all possible sample
combinations in a four-taxon tree (P1–P4). Significance was assessed through 1000 bootstrap
replicates for each test, and Z-score was calculated to measure the number of bootstrap
standard deviations in which the D-statistic deviates from zero. Significant patterns were
considered for a Z-score > 3, which correspond to a conservative cutoff
α
= 0.01 [
75
] after
the Benjamini–Hochberg correction [76].
2.6. Estimates of Divergence Times
A penalized likelihood method, implemented in TreePL [
77
], was used to estimate a
time-calibrated tree. TreePL is an upgraded version of r8s [
78
] that accounts for among-
branch rate heterogeneity by applying the so-called smoothing parameter [
79
]. This method
relies on branch-length estimates from prior ML or BI analyses and node age constraints
to estimate a different substitution rate for each branch using stochastic optimization and
hill-climbing gradient-based methods. The analysis is less time-consuming and is suitable
for dealing with large amounts of data with high percentages of missing data, such as
RADseq [
31
,
80
–
82
]. Taxon sampling for divergence time estimation was reduced to include
a single sample per species by choosing the samples with the highest number of loci retrieved
from the three datasets (Table S3). Then, the ipyrad assembly branch from the full matrix (m4)
was run from step 7, and RaxML analysis was performed on the reduced matrix (i.e., one tip
per species) following the same parameter settings as for the full matrix. Two calibration
points were set based on the highest posterior density intervals yielded by [
83
] for the genus
Usnea: (1) root age (minimum age = 17.1355; maximum age = 29.2712) and (2) crown node
of neuropogonoid lichens (ingroup) (minimum age = 4.5719; maximum age = 14.7433). A
first analysis using the “prime” option was run to select the optimal set of parameter values.
Accordingly, a second run was performed by setting the following optimal parameters:
gradient-based (opt) optimizer = 1; autodifferentiation-based (optad) = 2; autodifferentiation
cross-validation-based optimizers (optcvad) = 2. In this second run, random subsample
and replicate cross-validation (RSRCV) was set to identify the best value for the smoothing
parameter (lambda). The best chi-square value for the smoothing parameter (lambda = 1e-8)
was implemented for the third and final analysis. For the three runs, the thorough analysis
option, 200,000 penalized likelihood iterations, and 5000 cross-validation iterations were set.
To account for uncertainty in branch lengths due to variance in molecular substitution across
the RADseq loci, TreePL was run for each of the bootstrap trees obtained from the Maximum
likelihood analyses following [
27
]. The maximum clade credibility method implemented in
TreeAnnotator [84] was used to build the consensus-calibrated tree.
Ultimately, caution and sensitiveness are needed when interpreting divergence time
estimates from RAD-seq data. We here accounted for some of the main concerns argued [
85
]
by: (1) including a broad range of the loci rather than maximizing the number of individuals
sampled per locus (i.e., m4 dataset is used), (2) providing more than one calibration
point, and (3) accounting for the branch-length uncertainty due to variance in molecular

J. Fungi 2023,9, 99 7 of 20
substitution across the RADseq loci by running the TreePL on every bootstrap tree generated
in the ML reconstruction (see above).
3. Results
3.1. Morphological and Chemical Characterization
Morphological characterization was performed for the four clades of particular taxo-
nomical interest (Clades A–D; Figure 1, Table S2).
Clade A is characterized by a smooth, waxy surface of the thallus, a yellowish thal-
lus, blackened toward the tips, variegated in U. subcapillaris and violaceous grading in
U. ciliata that also has black-edge annulations. The habit and mode of the branching varied
from an erect, proliferating holdfast and moderately branched in U. ciliata to a pendu-
lous/subpendulous, delimited holdfast and extensively branched in U. pseudocapillaris
and U. subcapillaris (Figure 1c–k). Thick cortices distinguished the subclade of U. subcapil-
laris and U. ciliata from the clade of U. pseudocapillaris with thinner cortices. A wide axis
(more than half of the thallus width) was generally found in the taxa of clade A except in
U. ciliata with narrower axes. Placement and ornamentation of apothecia distinguished
U. ciliata (subterminal, short excipular rays) from U. subcapillaris (lateral, long excipular
rays). Soredia found in U. pseudocapillaris are plane, rounded to confluent or occasionally
minute and punctiform. Otherwise, among mostly apotheciate specimens, soredia are
also found in U. ciliata, which are plane, rounded, and black-spotted (Figure 1k–m). An
undescribed taxon is found as the fertile counterpart of U. pseudocapillaris (Figure 1a–d),
which showed some traits similar to those found in U. ciliata, such as erect, proliferating
holdfast, black-edge annulations, and terminal apothecia. However, the pigmentation
pattern and internal structure are similar to the sorediate sister clade U. pseudocapillaris.
Morphological characters also support the recognition of U. rohmederi at the specific
rank (currently in U. perpusilla) in agreement with the RADseq phylogenomic reconstruc-
tions (see below). Thus, U. rohmederi (clade B) is distinguished from U. perpusilla s.str. (clade
C) by a delimited holdfast, black variegated pigmentation toward the apices with frequent
long excipular rays in the apothecia that are often variegated. Meanwhile, U. perpusilla has
a richly proliferating holdfast, barely variegated pigmentation, and lacks excipular rays in
the apothecia (Figure 1n–q).
Morphological characterization of clade D resulted in the recognition of seven species,
including two species newly described below in congruence with the phylogenomic recon-
structions (Figure 1r–mm). Brownish to red apothecia discs distinguished the two early
diverging, newly described species (U. aymondiana and U. fibriloides) from the remaining
apotheciate taxa in clade D. In turn, U. fibriloides is distinguished from U. aymondiana by
the presence of numerous minute black fibrils along both thallus and the apothecial discs
(Figure 1t–u). Papillae in U. aymondiana are abundant but less dense, and fibrils are larger
than in U. fibriloides (Figure 1r–s). Besides, U. aymondiana has a narrower axis width than
U. fibriloides (Figure 1r–u). The clade of U. trachycarpa was confined to specimens from the
Kerguelen, where the type was described. Specimens from other regions were found to be-
long to other species. Usnea trachycarpa has a delimited holdfast that is moderately branched
above, with a yellowish to black tips pigmentation and numerous black pigmented papillae
and fibrils variable in length. This species is distinguished from other apotheciate-related
taxa by having a wide axis, compact medulla, cupular and flat apothecial discs, orange
and black pigmented, often ornamented with papillae and short fibrils (Figure 1w). Usnea
trachycarpoides and U. hyyppae showed similar traits as found in U. trachycarpa but differed
in having a sublax medulla, narrower axis, more flattened, orange and terminal apothecia,
and longer fibrils. Usnea trachycarpoides is variable in the density of papillae from absent to
abundant (Figure 1hh–jj, Table S2). Two species (U. sphacelata and U. subantarctica) were
found to be sorediate in clade D. Usnea sphacelata is distinguished from U. subantarctica by a
smooth thallus surface with spread papillae on main branches and moderately branching
above the holdfast, rare fibrils, and a thin cortex, with a sublax axis (Figure 1y–cc, Table S2).
Soredia in both species are globose and also excavate in U. sphacelata.

J. Fungi 2023,9, 99 8 of 20
A total of 11 medullary substances and five chemosyndromes already described for
Usnea [
65
] were identified through the HPTLC (Tables 1and S4): (1) the hypostrepsilic acid
chemosyndrome (constituted by the dibenzofurans isostrepsilic acid and hypostrepsilic
acid) and (2) the neuropogolic acid chemosyndrome (constituted by the aliphatic com-
pounds proto- and neuropogolic acids). The hypostrepsilic acid and neuropogolic acid
chemosyndromes were detected in low concentrations only for U. trachycarpa. Other identi-
fied chemosyndromes were (3) the fumarprotocetraric syndrome that is here represented
by the protocetraric acid detected in low concentrations for U. subcapillaris,U. ciliata, and
U. aymondiana, (4) the salazinic acid chemosyndrome (including salazinic/consalazinic
and norstictic acids) that was frequently present as major substance in clade A, and finally,
(5) the psoromic acid chemosyndrome (including psoromic and 2’-O-de-methylpsoromic
acids) that was sporadically present along the four clades as accessory substance except for
some lineages in clade A in which it constituted a major substance (Figure 1).
Table 1.
Chemosyndromes identified following [
65
] for each species of the four clades of taxonomical
interest (Clades A–D; Figure 1). + indicates chemosyndrome constantly or frequently present in
high concentration,
±
indicates chemosyndrome either sometimes present in high concentrations or
present in low concentration.
Phylogenetic
Clade Species Hypostrepsilic
Acid
Fumarprotocetraric
Acid
Psoromic
Acid
Salazinic
Acid
Neuropogolic
Acid
AU. ciliata ± ± +
AU. subcapillaris ± ± +
AUndescribed taxon + +
AU. pseudocapillaris ±+
BU. rohmederi
CU. perpusilla ±
DU. aymondiana ± ±
DU. fibriloides +
DU. sphacelata ±
DU. subantarctica +
DU. trachycarpa ± ± ± ±
DU. trachycarpoides +
DU. hyyppae ±
Within-species chemical variability was moderate (1-2 chemotypes) except for
U. ciliata, U. subcapillaris, and U. trachycarpa, which had up to six different chemotypes
(Table S4). Usnic acid was found in the cortex as the major substance in all species exam-
ined. Clade A, comprising endemic species from New Zealand (U. ciliata,U. subcapillaris,
U. pseudocapillaris), had similar chemical patterns in which the salazinic acid chemosyn-
drome was present in most chemotypes (Tables 1and S4). Usnea subcapillaris, the most
chemically diverse species of this clade, distinguished from the other species by the pres-
ence of squamatic acid as one of the major substances for some samples. Usnea rohmederi
(Clade B) and U. perpusilla (Clade C) usually lacked medullary substances, but some speci-
mens of U. perpusilla contained traces of psoromic acid (Tables 1and S4). High chemical
variability was observed for species of clade D, with U. trachycarpa as the most chemically
diverse (6 chemotypes). The two newly described species differ in their chemistry. While
U. aymondiana contains fatty acids, sparse psoromic acid, and traces of protocetraric acid,
U. fibriloides contains the salazinic acid chemosyndrome. Among the other apotheciate
species, U. trachycarpa showed the highest variability containing chemotypes assigned
up to four chemosyndromes. Usnea trachycarpoides contains the salazinic acid chemosyn-

J. Fungi 2023,9, 99 9 of 20
drome, whereas U. hyyppae only had norstictic acid occasionally in two samples and traces
of psoromic and protocetraric acids. Regarding the two sorediate species of this clade,
U. sphacelata was medullary deficient with only traces of psoromic acid in one of the
specimens, whereas U. subantarctica showed two chemotypes including salazinic acid
chemosyndrome.
3.2. Assembly of RAD Sequencing
Three matrices of 126 samples were obtained after the ipyrad assembly, filtering, and
processing of all reads sequenced. As a result, the total numbers of (1) loci filtered ranged
from 21,831 (m4) to 4504 (m50), (2) single nucleotide polymorphisms (snps) from 381,659
(m4) to 166,242 (m50), and (3) missing data percentage from 82.91% (m4) to 72.41% (m50)
(Table 2).
Table 2.
Main summary statistics yielded by the three datasets generated by ipyrad from the three
values of minimum taxon coverage tested. The three values aimed to represent (1) the default value,
m = 4; (2) 25% of the total samples, m = 32; and (3) 50% of the total samples, m = 65, denoted as m4,
m25, and m50, respectively. The main statistics provided are (1) number of loci filtered, (2) mean loci
per sample, (3) standard deviation (SD) of number of loci per sample, (4) minimum number of loci
per sample, (5) maximum number of loci per sample, (6) number of single nucleotide polymorphisms
(N snps), and (6) percentage of missing data in the loci matrix.
Dataset N Samples N Loci
Filtered
Mean Loci
per Sample
SD Loci
per Sample Min Loci Max Loci N snps % Missing
Data
m4 126 21,831 5917.376 3619.83 938 15,073 381,659 82.91
m25 126 8698 4705.896 2340.84 804 8182 271,485 76.22
m50 126 4504 3180.304 1141.65 706 4442 166,242 72.411
3.3. Phylogenomic Analyses and Divergence Times Estimation
The topologies inferred through RAxML-NG (ML) using the full matrix of concate-
nated RADseq loci were congruent among the three parameter settings tested, and only
minor differences in bootstrap supports (BS) were observed (Figure S1a). All species were
retrieved as monophyletic with maximum BS. Similarly, high BS was inferred for all phy-
logenetic relationships among neuropogonoid species except for U. patagonica-clade A with
moderate supports (BS = 65–84), U. trachycarpoides-U. hyyppae (BS = 44–65), and U. taylorii-U.
aurantiacoatra-U. antarctica with BS = 75 for m50 dataset (Figure S1a). The highest overall
BS average was yielded by m4 dataset that was therefore selected for Bayesian inference
(BI, Figure 1). In the BI analysis, maximum values of posterior probability (PP) confirmed
the monophyly of neuropogonoid species. Overall, PP values for all nodes ranged from
0.97 to 1, except for the clade of U. acromelana (PP = 0.83) (Figure 1). Topologies of ML
with RAxML-NG and BI were congruent, and only minor node support differences were
retrieved (Figure 1). Thus, U. patagonica is inferred as sister to clade A which comprised
three endemic species from Australasia: (1) U. pseudocapillaris, sister to a clade including
(2) U. ciliata and (3) U. subcapillaris. The neuropogonoid core is formed by an early diverg-
ing clade comprising U. lambii as sister to a clade including U. ushuaiensis and U. rohmederi
(clade B, Figure 1) and a major clade comprising three subclades: (1) U. perpusilla (clade
C) sister to U. messutiae-U. pallidocarpa; (2) U. acromelana,U. taylorii and U. aurantiacoatra-
U. antarctica; and (3) Clade D, including U. aymondiana and U. fibriloides as early diverg-
ing branches and a subclade formed by U. trachycarpa-U. sphacelata and U. subantarctica,
U. trachycarpoides-U. hyppae. Only one incongruence was found for the ML inference from
RAxML v8.2.12 that retrieved U. patagonica as sister to the Neuropogonoid core (Figure S1b).
This position contrasts with the topologies yielded by all the other phylogenomic ap-
proaches herein implemented where U. patagonica is sister to clade A (i.e., RAxML-NG
v.1.1., ExaBayes and SVDquartets).

J. Fungi 2023,9, 99 10 of 20
The coalescent-based analyses using the SVDquartets also resulted in overall high BS
values (Figures 2a and S2). Coalescent-based topologies agreed with the BI and RAxML-NG
retrieving U. patagonica as sister to clade A, however with lower BS values. (BS = 45–58;
Figures 2a and S2). Other differences in the coalescent-based topologies were a low-supported
sister relationship between U. subantarctica and U. hyyppae (BS = 77–84; Figures 2a and S2)
and a poorly supported sister relationship of U. taylorii and U. antarctica to U. aurantiacoatra
(BS = 48–54; Figures 2a and S2).
J. Fungi 2023, 9, x FOR PEER REVIEW 10 of 21
ranged from 0.97 to 1, except for the clade of U. acromelana (PP = 0.83) (Figure 1).
Topologies of ML with RAxML-NG and BI were congruent, and only minor node support
differences were retrieved (Figure 1). Thus, U. patagonica is inferred as sister to clade A
which comprised three endemic species from Australasia: (1) U. pseudocapillaris, sister to
a clade including (2) U. ciliata and (3) U. subcapillaris. The neuropogonoid core is formed
by an early diverging clade comprising U. lambii as sister to a clade including U.
ushuaiensis and U. rohmederi (clade B, Figure 1) and a major clade comprising three
subclades: (1) U. perpusilla (clade C) sister to U. messutiae-U. pallidocarpa; (2) U. acromelana,
U. taylorii and U. aurantiacoatra-U. antarctica; and (3) Clade D, including U. aymondiana and
U. fibriloides as early diverging branches and a subclade formed by U. trachycarpa-U.
sphacelata and U. subantarctica, U. trachycarpoides-U. hyppae. Only one incongruence was
found for the ML inference from RAxML v8.2.12 that retrieved U. patagonica as sister to
the Neuropogonoid core (Figure S1b). This position contrasts with the topologies yielded
by all the other phylogenomic approaches herein implemented where U. patagonica is
sister to clade A (i.e., RAxML-NG v.1.1., ExaBayes and SVDquartets).
The coalescent-based analyses using the SVDquartets also resulted in overall high BS
values (Figures 2a and S2). Coalescent-based topologies agreed with the BI and RAxML-
NG retrieving U. patagonica as sister to clade A, however with lower BS values. (BS = 45–
58; Figures 2a and S2). Other differences in the coalescent-based topologies were a low-
supported sister relationship between U. subantarctica and U. hyyppae (BS = 77–84; Figures
2a and S2) and a poorly supported sister relationship of U. taylorii and U. antarctica to U.
aurantiacoatra (BS = 48–54; Figures 2a and S2).
Figure 2. (a) SVDquartets species tree of neuropogonoid lichens for m4 dataset. BS are indicated for
each node.; (b) Time-calibrated phylogeny obtained from treePL by using maximum clade
credibility from bootstrap trees of the maximum-likelihood analysis of the m4 of reduced sampling
matrix. Bootstrap supports (BS) for all branches are 100 except for the subclade U. pallidocarpa–U.
messutiae with BS = 92. Mean ages in millions of years (myr) are indicated for each node. Node bars
in blue indicate the node age ranges taking into account the branch length variance along the
bootstrap trees. The international stratigraphic scale is included from 23 myr until the present.
The ML phylogenomic trees resulting from the reduced matrix (one sample per
species) yielded overall high BS support for all phylogenetic relationships, and the
topologies of the trees were congruent with coalescent-based trees, BI and RAxML-NG
tree for the relationship between U. patagonica and Clade A (Figure S3).
Figure 2.
(
a
) SVDquartets species tree of neuropogonoid lichens for m4 dataset. BS are indicated for
each node.; (
b
) Time-calibrated phylogeny obtained from treePL by using maximum clade credibility
from bootstrap trees of the maximum-likelihood analysis of the m4 of reduced sampling matrix.
Bootstrap supports (BS) for all branches are 100 except for the subclade U. pallidocarpa–U. messutiae
with BS = 92. Mean ages in millions of years (myr) are indicated for each node. Node bars in blue
indicate the node age ranges taking into account the branch length variance along the bootstrap trees.
The international stratigraphic scale is included from 23 myr until the present.
The ML phylogenomic trees resulting from the reduced matrix (one sample per species)
yielded overall high BS support for all phylogenetic relationships, and the topologies of
the trees were congruent with coalescent-based trees, BI and RAxML-NG tree for the
relationship between U. patagonica and Clade A (Figure S3).
Introgression analysis resulted in a total of 55,275 tests generated from all possible
sample combinations for a four-taxon tree [(((P1,P2),P3),P4)] (Figure S4). BABA pattern
indicating introgression between U. patagonica and the neuropogonoid core. It was the
most frequent in 97% of all the 1636 tests that resulted in significance with a Z-score
≥
3
(Table S5). An average of 2501.85 loci per test were included (Table S5).
The divergence time estimation (Figure 2b) indicated that most species (14 out of
21) originated either in the Pliocene-Pleistocene boundary (~3 myr) or early Pleistocene
(~1.5 myr) (Table 3). The earliest diverging species, U. patagonica was estimated to have
evolved during the late Miocene and Miocene-Pliocene boundary, around 7.3 myr ago
(4.91–8.54 Bootstrapped variance, BV).

J. Fungi 2023,9, 99 11 of 20
Table 3.
Node ages (myr) and Bootstrapped variance (BV) intervals inferred through TreePL for
Neuropogonoid species.
Species Node Age BV
U. acromelana 5.56 3.64–6.89
U. antarctica 1.8 0.49–3.09
U. aurantiacoatra 1.8 0.49–3.09
U. aymondiana 6.13 5.31–7.23
U. ciliata 2.75 1.14–5.91
U. fibriloides 5.09 3.62–6.58
U. hyyppae 1.36 0.43–2.27
U. lambii 5.75 2.66–7.72
U. messutiae 2.60 1–4.93
U. pallidocarpa 2.60 1–4.93
U. patagonica 7.3 4.91–8.54
U. perpusilla 5.45 2.79–7.34
U. pseudocapillaris 2.42 0.88–5.22
U. rohmederi 2.68 1.19–5.81
U. sphacelata 2.01 0.95–3.77
U. subantarctica 2.75 0.95–4.25
U. subcapillaris 2.75 1.14–5.91
U. taylorii 3.51 1.69–5.58
U. trachycarpa 2.01 0.95–3.77
U. trachycarpoides 1.36 0.43–2.27
U. ushuaiensis 2.68 1.19–5.81
3.4. Taxonomy
3.4.1. Usnea aymondiana A.Otero, Barcenas-Peña, Lumbsch & Grewe sp. nov. (Figure 1r,s)
MycoBank: MB846765
Diagnosis: Usnea aymondiana is distinguished from U. trachycarpa s.l. by having
brown to dark red apothecia discs and it is distinguished in turn from its closest relative,
U. fibriloides by having a more richly branched thallus, less dense papillae, larger fibrils in
both branches and apothecial discs and fatty acid compounds.
Type: Argentina, Santa Cruz Province, Mount Aymond, 52
◦
07
0
S, 69
◦
31
0
W, 12.2003, N.
Wirtz & M.I. Messuti 7-186-9, Herbarium code: C0172407F (BCRU—holotype; F—isotype).
Etymology: The epithet refers to the locality where the type was collected, Monte
Aymond in Santa Cruz Province, Argentina.
Description: Thallus approx. 2–4 cm tall, arising from a delimited unpigmented
holdfast. Erect thallus moderately branched with yellowish main branches that become
extensively ramified and black pigmented toward the tips. Side branches mostly variegated
with bands of black pigment towards the tips. Rugose thallus surface usually foveolate with
abundant yellow papillae in the main branches and short fibrils (c. 1 mm) variegated or
continuously black-pigmented on all branches. Cortex thick, medulla sublax, axis compact
less than half or half of the branch diameter. Soredia and isidiomorphs not seen. Apothecia
frequent, subterminal, with brownish-red discs, margin verrucose with short black fibrils
and frequent long yellow or black variegated excipular rays. Pycnidia not seen.
Chemistry: HPTLC: Usnic acid is always present, and fumarprotocetraric acid and
psoromic acid chemosyndromes are frequent or sometimes absent (Elix et al., 2007, [
65
]).
Chemotypes: (1) Major substance: usnic acid; minor substance: psoromic acid; (2): Major

J. Fungi 2023,9, 99 12 of 20
substance: usnic acid; minor substance:
±
protocetraric acid. Fatty acids found in both
chemotypes.
Distribution: Only known from a single locality in Mount Aymond at 200 m altitude,
close to Rio Gallegos (Santa Cruz province), at the border between Argentina and Chile.
Notes: The new species is distinguished from the other U. trachycarpa allies by having
brown to dark red apothecia discs and can be readily distinguished from U. fibriloides by
a more richly branched thallus, less dense papillae, the presence of larger fibrils in both
branches and apothecial discs and also chemical differences (see below).
Additional specimens examined: ARGENTINA. Santa Cruz Province, Mount Aymond,
52
◦
07
0
S, 69
◦
31
0
W, 12/.003, N. Wirtz & M.I. Messuti 196-6 (F). Santa Cruz Province, Mount
Aymond, 52◦070S, 69◦310W, 12.2003, N. Wirtz & M.I. Messuti 197-1 (F).
3.4.2. Usnea fibriloides A.Otero, Barcenas-Peña, Lumbsch & Grewe sp. nov. (Figure 1t,u)
MycoBank: MB846792
Diagnosis: Usnea fibriloides is distinguished from U. trachycarpa s.l. by having a barely
branched and unpigmented thallus covered by minute thick, black-pigmented fibrils and
densely minute fibrillated apothecial margins without excipular rays. Usnea fibriloides also
differs from its closest relative, U. aymondiana by the presence of norstictic and salazinic
chemosyndromes.
Type: Argentina, Santa Cruz Province, Mount Aymond, 52
◦
07
0
S, 69
◦
31
0
W, 12.2003, N.
Wirtz & M.I. Messuti, 197-3. Herbarium code: C0172408F (BCRU—holotype, F—isotype).
Etymology: The epithet refers to the presence of distinguishing minute black pig-
mented fibrils all along the thallus and at the margins of apothecial discs.
Description: Thallus approx. 3–4 cm tall, arising from a delimited unpigmented
holdfast. Erect thallus barely branched, unpigmented, and rarely ramified. The thallus
surface highly rugose and densely papillae, abundantly covered by minute thick, black-
pigmented fibrils (less than 1 mm). Cortex thick, medulla sublax, axis compact half of
the branch diameter. Soredia and isidiomorphs not seen. Apothecia frequent, terminal,
brownish-red disc, margin verrucose and abundant minute black fibrils, without excipular
rays. Pycnidia not seen.
Chemistry: HPTLC: Major substance: usnic acid; minor substances or traces: salazinic
acid, consalazinic acid, norstictic acid (Elix et al., 2007, [65]).
Distribution: Only known from a single locality in Mount Aymond at 200 m altitude,
close to Rio Gallegos (Santa Cruz province), at the border between Argentina and Chile.
Notes: The new species is distinguished from the other U. trachycarpa allies by having a
barely branched and unpigmented thallus covered by minute thick, black-pigmented fibrils
and densely minute fibrillated apothecial margins without excipular rays. Differences in
secondary metabolites are also found with its closest relative U. aymondiana (see above).
Additional specimens examined: ARGENTINA. Santa Cruz Province, Mount Aymond,
52◦070S, 69◦310W, 12.2003, N. Wirtz & M.I. Messuti 197-4 (F).
4. Discussion
The phylogenetic structure of neuropogonoid lichens has been debated in the lit-
erature [
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
]. 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. The reference-based RAD
sequencing method resulted in a robust phylogeny that unveils the evolutionary history
of this group of lichenized fungi (Figure 1). These phylogenomic results have enabled us
to resolve two species complexes: the U. perpusilla-complex and U. trachycarpa-complex.
Subsequently, five additional names are proposed for the neuropogonoid lichens, including

J. Fungi 2023,9, 99 13 of 20
three species names resurrected and two newly described species. Moreover, two species
are sequenced here for the first time: U. pseudocapillaris and U. taylorii, thus resulting in a
full taxon coverage of this group [34].
Neuropogonoid lichens were supported as a monophyletic group in agreement with
previous phylogenies [
34
,
42
,
56
]. All phylogenomic trees retrieved monophyly for all the
species within the neuropogonoid group and highly supported phylogenetic relationships
among lineages (Figure 1). Similarly, the coalescent-based approach resulted in a congruent
topology with maximum support for most phylogenetic relationships, only differing in
lower BS support for for three nodes: (1) early divergence of U. patagonica as sister to clade
A and shallow divergence within (2) the U. taylorii-U. antarctica-U. aurantiacoatra clade, and
(3) U. hyyppae-U. subantarctica. Incomplete lineage sorting (ILS) or ancient introgression
can be masked under high bootstrap supports when analyzing large-scale, concatenated
datasets. These effects can be driven by only a few loci [
87
–
89
]. Coalescent-based methods
are shown to integrate and overcome the conflicting signal derived from these processes. In
particular, SVDquartets is expected to be confidently applicable for empirical systems where
levels of gene flow were maintained time after the speciation [
90
]. Furthermore, methods
such as BABA-ABBA tests enable distinguishing between a more stochastic signal of ILS
from the directional asymmetry of shared ancestral loci in ancient introgression events
among closely related species [
74
,
89
,
91
]. Our introgression tests suggested a significative
signal for ancient introgression between U. patagonica and the core of neuropogonoid
lichens (Figure S4, Table S5) that was also reflected by the coalescent-based tree (Figure 2a)
and the incongruence retrieved between ExaBayes and RAxML-NG, versus RaxML v.8.
concatenated-based trees (Figure 1and Figure S1). Otherwise, further investigations on the
most recent divergences of U. taylorii-U. antarctica-U. aurantiacoatra clade and U. hyyppae-
U. subantarctica are needed to disentangle potential ILS or ongoing gene flow already
suggested by the moderate support of these nodes for coalescent-based reconstructions.
4.1. Contributions to the Systematics of Neuropogonoid Lichens
Circumscriptions of some of the major species of neuropogonoid lichens [
35
] mostly
agree with our phylogenetic reconstructions. Nevertheless, the information provided by
more than 20,000 loci yielded by the high performance of RAD sequencing allowed us to
disentangle some of the most complicated species aggregates [
58
]. Here, we discuss the
contributions to the systematics of neuropogonoid lichens by the RAD sequencing analysis.
4.2. The Usnea Perpusilla Complex
The Usnea perpusilla complex exhibits an array of variable morphological traits rep-
resented by both fertile and asexual taxa with a smooth, faveolate, waxy, epapillate and
extensively black-pigmented surface combined with a thin axis and a lax medulla [
35
,
58
].
Wirtz et al. (2008, [
58
]) addressed this complex through a cohesion approach of species
delimitation resulting in the recognition of five species: (1) U. lambii, (2) U. pallidocarpa,
(3) U. perpusilla, (4) U. messutiae, and (5) U. ushuaiensis. Nevertheless, limitations of the
data set prevented them from addressing the phylogenetic relationship among species.
Our study revealed the presence of a sixth lineage for which we propose to resurrect
the name U. rohmederi (I.M. Lamb) I.M. Lamb [
92
], previously regarded as a synonym of
U. perpusilla [
35
]. Our phylogenomic results suggest that the U. perpusilla complex is di-
vided into two independent lineages: (1) an early diverging clade formed by U. lambii sister
to a clade including U. ushuaiensis and U. rohmederi, and (2) a derived clade formed by
U. perpusilla sister to the clade of U. messutiae and U. pallidocarpa (Figure 1). Usnea perpusilla
and U. rohmederi overlap in their distributional ranges from Tierra del Fuego to higher
latitudes in Argentina and morphologically, U. rohmederi is distinguished by the presence of
a more delimited holdfast, black variegated pigmentation toward the apices and frequent
long, variegated excipular rays (Figure 1n–q, Table S2).

J. Fungi 2023,9, 99 14 of 20
4.3. The Usnea Trachycarpa Complex
The U. trachycarpa complex includes species exhibiting a wide range of fertile pheno-
types and closely related asexual lineages [56]. The abundant presence of fibrils, papillate
thallus, and rufous-brown apothecial discs with numerous excipular rays characterize this
group [
35
,
48
]. Chemically, various chemosyndromes of
β
-orcinol depsidones are found
as medullary substances [
35
]. The extensive phenotypical variation around the diagnostic
traits has led to the description of a plethora of varieties, subspecies, forms, and even
different species often associated with particular geographic distributions [
35
]. Some
authors regarded this variability as part of the intraspecific variation (e.g., [
35
,
48
]). Further-
more, the asexual species U. subantarctica has been considered as the sterile counterpart of
U. trachycarpa (e.g., [
35
]), which was subsequently supported based on the paraphyly of
U. trachycarpa (e.g., [
34
,
42
,
56
,
93
]). The combination of full taxon coverage of this species
complex and large-scale genomic data resulted in a revised species delimitation. We
here propose to accept seven distinct species: (1) U. aymondiana and (2) U. fibriloides, two
newly described species currently only known from Mt. Aymond (Patagonia, Argentina);
(3) U. hyppae Räsänen [
94
] and (4) U. trachycarpoides (Vain.) C.W. Dodge [
95
], two resur-
rected species names previously synonymized with U. trachycarpa [
35
]; and the tradition-
ally accepted species (5) U. trachycarpa, (6) U. subantarctica, and (7) U. sphacelata (Clade D,
Figure 1). The seven species have been congruently inferred as well supported mono-
phyletic lineages and are also supported by morphological and chemical characters
(Figure 1, Tables 1, S2 and S4). The two newly described species are inferred as early
diverging branches of this clade and are characterized by phenotypical differences, such as
darker brownish to red apothecia unlike more orange apothecia for the other apotheciate lin-
eages in this clade. Besides, U. fibriloides is readily distinguished by numerous particularly
short and thick fibrils all along the thallus and apothecia that give it a distinguishing aspect
(Figure 1t). Contrasting chemistry is found between the two new species with norstistic,
salazinic and consalazinic acids in U. fibriloides, whereas U. aymondiana only contained
psoromic acid and traces of protocetraric acid (Figure 1, Tables 1and S4). Usnea trachycar-
poides and U. hyyppae occur in southern Chile and formed a well-supported clade together
with the sorediate U. subantarctica. Maximum likelihood and Bayesian trees inferred a sister
relationship between the two apotheciate species although with moderate support under
ML (BS = 65; Figure 1), whereas coalescent-based methods revealed U. hyyppae as sister
to the sorediate U. subantarctica with higher support (BS = 77–83, Figures 2and S2). Our
results suggest that U. hyyppae is likely the fertile counterpart of U. subantarctica. However,
further studies including more thorough sampling in this area will be necessary to test our
hypothesis. Otherwise, RADseq phylogenies indicate that U. trachycarpa is restricted to the
Kerguelen Islands from where the type was described [
96
]. The bipolar sorediate species
U. sphacelata is strongly supported as sister species to U. trachycarpa, thus pointing to a new
case in which the asexual sister clade would show a much wider distribution range than
the fertile sister clade [60].
4.4. The New Zealand Clade
Around 28 species of Usnea are known to occur in New Zealand [
97
]. However, this
apparently low number of species is thought to be highly underestimated as a result of
the lack of regional studies and the complicated taxonomy of this group [
97
]. Our study
supports the presence of six species of neuropogonoid Usnea taxa in New Zealand [
35
]:
U. acromelana, U. ciliata,U. lambii,U. pseudocapillaris (sequenced here for the first time),
U. sphacelata and U. subcapillaris.Usnea antarctica has been recorded in New Zealand, but
our molecular study does not support the presence of this species in the country. Fur-
thermore, U. lambii, separated from U. sphacelata [
42
] is here recorded and sequenced in
New Zealand for the first time (Figure 1). Three species that appear to be endemic to
New Zealand (U. ciliata,U. pseudocapillaris, and U. subcapillaris) formed one of the early
diverging clades of neuropogonoid lichens (Clade A, Figure 1). Usnea ciliata and U. sub-
capillaris, two fertile species, have a sister group relationship and form a sister group

J. Fungi 2023,9, 99 15 of 20
to the sorediate U. pseudocapillaris, discarding U. subcapillaris as its species pair as sug-
gested by [
35
]. Interestingly, the fertile counterpart of U. pseudocapillaris (herein referred
as undescribed taxon) is inferred as the intermediate phenotype of U. ciliata and U. pseudo-
capillaris (Figure 1a–d, Tables 1, S2 and S4). Further investigations on this putative new
taxon are necessary before a formal description. For the three other species, independent
colonizations of New Zealand during the Pleistocene of asexual species are most likely
(see U. lambii,U. acromelana, and U. sphacelata, Figure 1). Thus, we here unveil multi-
ple and independent origins of neuropogonoid lichens from New Zealand in contrast to
previous hypotheses which suggested a unique monophyletic clade for all New Zealand
neuropogonoid taxa, questioning an amphi-Pacific disjunction within some species (e.g.,
U. acromelana; [34]).
4.5. A Spatio-Temporal Evolutionary Framework of Neuropogonoid Lichens
The Miocene and Pliocene have been the main periods for the divergence of major lin-
eages within parmelioid lichens (e.g., Flavoparmelia, [
98
]; Melanohalea, [
99
]; Montanelia, [
100
];
Xanthoparmelia, [
101
]) including the crown age of Usnea [
83
,
102
]. The progressive global
cooling since the Oligocene-Miocene boundary [
103
], together with the increase in the
aridity, promoted a transition to temperate forests and ultimately to more open habi-
tats [
104
,
105
] that seem to have triggered the diversity of Parmeliaceae [
101
]. Neuro-
pogonoid species are exceptionally well-adapted to the most extreme environment in polar
and alpine regions where harsh conditions on aridity, radiation exposure, and scarce water
availability reduce the number of competitors. Indeed, the estimated origin of neuro-
pogonoid lichens in the late Miocene (Figure 2b, Table 3) coincides with a time when the
two centers of diversity, such as alpine zones in South America and subantarctic regions
in the southern hemisphere (e.g., northern Andes, southern Chile, New Zealand) were
highly impacted by (1) tectonic movements (e.g., maximum height of Andes) and subse-
quent proliferation of arid exposed cold deserts and (2) by glaciation of west Antarctica
after the set of circumpolar current and subsequent triggering of tundra habitats [
106
,
107
].
Ultimately, the burst of diversification of neuropogonoid lichens took place around the
Pliocene-Pleistocene boundary (~3 myr) or early Pleistocene (~1.5 myr) (Figure 2b, Table 3).
In particular, our results shed light on the role of New Zealand in neuropogonoid lichen
diversification bringing new insights to the biogeography of this group. Early colonization
and diversification in New Zealand were inferred in the Miocene-Pliocene boundary (4.91–
8.53 myr) which led to an ancient Amphipacific-disjunction at species level as well as more
recent Pleistocene colonization events by sorediate species (Figures 1and 2). Long-distance
dispersal (LDD) is the most plausible hypotheses for both ancient and recent colonization
events since they postdate the split of the major landmasses. Indeed, this amphi-pacific
pattern mediated by LDD is being widely observed in the lichen flora of New Zealand [
108
]
where the climate became rapidly cool-temperate since the late Miocene and Pliocene being
more similar to that of central Chile by that time [109].
5. Conclusions
The reference-based RAD sequencing method has resulted in strongly supported phy-
logenetic hypotheses of neuropogonoid lichens showing its power to resolve phylogenetic
relationships in the genus Usnea, which is considered one of the most complicated groups of
lichenized fungi. Large-scale genomic data in combination with a first-time full taxon coverage
has led to: (1) the reconstruction of the backbone phylogeny for neuropogonoid lichens, (2) the
inference of the evolutionary timeframe, and (3) the contribution to our knowledge of species
diversity in the group. The massive genomic data obtained, coupled with morphological and
chemical examinations, resolved two species complexes (U. perpusilla complex, U. trachycarpa
complex) and circumscribed species endemic to New Zealand and the Kerguelen, respectively.
Two species are newly described here, and three species names have been resurrected. Increas-
ing aridity and global cooling during the late Miocene and the Pliocene-Pleistocene boundary
are suggested as major drivers of diversification in neuropogonoid lichens. While this study

J. Fungi 2023,9, 99 16 of 20
provided a robust phylogeny of neuropogonoid lichens, it raises new questions about the
extrinsic (e.g., historical contingency) and intrinsic factors (e.g., reproductive strategy) that
may have interplayed to shape the genomic diversity and distributional ranges within and
across both hemispheres of earth.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jof9010099/s1, Figure S1: (a) RAxML-NG trees for m4, m25,
and m50 settings of minimum taxon coverage. (b) RAxML trees for m4, m25, and m50 settings of
minimum taxon coverage. Bootstrap support values (BS) are indicated for all nodes.; Figure S2:
SVDquartets species tree of neuropogonoid lichens for m25 and m50 datasets; Figure S3: RAxML
trees of the reduced dataset (i.e., one specimen per species) for m4, m25 and m50 settings; Figure
S4: Tree topology and BABA-ABBA scheme used for D-stat tests performed in ipyrad. Table S1:
Data information and NCBI SRA accessions of all individuals sampled; Table S2: Morphological
characterization of the species included in the phylogenetic clades of taxonomic interest; Table S3:
Number of loci obtained per sample for the three datasets (i.e., m4, m25, and m50) tested for the
present study and number of raw reads obtained after demultiplexing step.; Table S4: Chemical
substances obtained through HPTLC analysis and chemotypes assigned for the species of taxonomic
interests (clades A–D). Table S5: Summary of D-statistic analysis performed for all tests resulted from
all possible sample combinations in a four-taxon tree [(((P1,P2),P3),P4)].
Author Contributions:
Conceptualization, H.T.L., F.G. and A.O.; methodology, all authors; soft-
ware, A.O.; validation, F.G. and H.T.L.; formal analysis, A.O. and A.B.-P.; investigation, all authors;
resources, F.G. and H.T.L.; data curation, A.O.; writing—original draft preparation, A.O.; writing—
review and editing, all authors; funding acquisition, F.G. All authors have read and agreed to the
published version of the manuscript.
Funding:
This research was funded by the Negaunee Foundation, the Grainger Foundation, the
Robert A. Pritzker Center for Meteoritics and Polar Studies and Tawani Foundation, and CRYPTO-
COVER (Spanish Ministry of Science CTM2015-64728-C2-1-R) headed by Leopold Garcia Sancho
supported fieldwork on Livingston Island of Felix Grewe.
Data Availability Statement:
The data that support the findings of this study are openly available
in the Short Read Archive (SRA), through the BioProject accession PRJNA902355 (https://www.
ncbi.nlm.nih.gov/bioproject/PRJNA902355 (accessed on 7 January 2023)). Accession numbers for
RADseq raw sequences are listed in Table S1.
Acknowledgments:
We thank those lichenologist colleagues who helped with material sampling,
particularly Bruce McCune from Oregon State University (Corvallis, OR, USA) and Damien Ertz from
Jardin Botanique du Meise (Belgium). Likewise, we acknowledge the valuable sampling material
provided by The Field Museum Herbarium (F) and Frankfurt Herbarium (FR) with particular thanks
to Robert Salm and Christian Printzen for their support. We thank Allision Knight and John Knight
in New Zealand, Matt von Konrat, Juan Larrain, and Todd Widhelm in Chile, and Ulrike Ruprecht,
Christian Printzen, Leopold Garcia Sancho, and Ulrik Søchtig in Antarctica for collection assistance.
We also thank the staff of the Spanish Antarctic Station Juan Carlos I. We gratefully acknowledge
the work and support from the genomic lab staff at the Field Museum, Isabel Distefano and Kevin
Feldheim, as well as Matt Von Konrat, the head of botanical collections, and Todd Widhelm, collection
manager, for their help with the herbarium samples and at the botany lab. We are thankful to Yukun
Sun for his support with the high-performance computing cluster at The Field Museum. We are also
grateful for the contributions made by the interns Humayra Munshi and Yejun Kim. We are also
highly grateful for the helpful methodological comments and advice made by our colleagues at the
Field Museum, Claudio Ametrano, Todd Widhelm, Matt Nelsen, and Lourdes Valdez, and Ekaphan
Kraichak as well. Finally, we deeply thank the comprehensive work and extensive collection legacy
made by Nora Wirtz through her PhD dissertation.
Conflicts of Interest:
The authors declare no conflict of interest. The founders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the
manuscript; or in the decision to publish the results.

J. Fungi 2023,9, 99 17 of 20
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