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ORIGINAL RESEARCH
published: 20 December 2021
doi: 10.3389/fmicb.2021.774839
Edited by:
Lucia Muggia,
University of Trieste, Italy
Reviewed by:
Gábor M. Kovács,
Eötvös Loránd University, Hungary
Julieta Orlando,
University of Chile, Chile
*Correspondence:
Jolanta Miadlikowska
jolantam@duke.edu
†These authors have contributed
equally to this work and share first
authorship
‡These authors share senior
authorship
Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants,
a section of the journal
Frontiers in Microbiology
Received: 13 September 2021
Accepted: 19 November 2021
Published: 20 December 2021
Citation:
Medeiros ID, Mazur E,
Miadlikowska J, Flakus A,
Rodriguez-Flakus P,
Pardo-De la Hoz CJ, Cie ´
slak E,
´
Sliwa L and Lutzoni F (2021) Turnover
of Lecanoroid Mycobionts and Their
Trebouxia Photobionts Along an
Elevation Gradient in Bolivia Highlights
the Role of Environment in Structuring
the Lichen Symbiosis.
Front. Microbiol. 12:774839.
doi: 10.3389/fmicb.2021.774839
Turnover of Lecanoroid Mycobionts
and Their Trebouxia Photobionts
Along an Elevation Gradient in Bolivia
Highlights the Role of Environment in
Structuring the Lichen Symbiosis
Ian D. Medeiros1†, Edyta Mazur2†, Jolanta Miadlikowska1*, Adam Flakus2,
Pamela Rodriguez-Flakus2, Carlos J. Pardo-De la Hoz1, El ˙
zbieta Cie´
slak2,
Lucyna ´
Sliwa2‡and François Lutzoni1‡
1Department of Biology, Duke University, Durham, NC, United States, 2W. Szafer Institute of Botany, Polish Academy
of Sciences (PAS), Kraków, Poland
Shifts in climate along elevation gradients structure mycobiont–photobiont associations
in lichens. We obtained mycobiont (lecanoroid Lecanoraceae) and photobiont (Trebouxia
alga) DNA sequences from 89 lichen thalli collected in Bolivia from a ca. 4,700
m elevation gradient encompassing diverse natural communities and environmental
conditions. The molecular dataset included six mycobiont loci (ITS, nrLSU, mtSSU,
RPB1,RPB2, and MCM7) and two photobiont loci (ITS, rbcL); we designed new
primers to amplify Lecanoraceae RPB1 and RPB2 with a nested PCR approach.
Mycobionts belonged to Lecanora s.lat., Bryonora,Myriolecis,Protoparmeliopsis, the
“Lecanora”polytropa group, and the “L.” saligna group. All of these clades except for
Lecanora s.lat. occurred only at high elevation. No single species of Lecanoraceae was
present along the entire elevation gradient, and individual clades were restricted to a
subset of the gradient. Most Lecanoraceae samples represent species which have not
previously been sequenced. Trebouxia clade C, which has not previously been recorded
in association with species of Lecanoraceae, predominates at low- to mid-elevation
sites. Photobionts from Trebouxia clade I occur at the upper extent of mid-elevation
forest and at some open, high-elevation sites, while Trebouxia clades A and S dominate
open habitats at high elevation. We did not find Trebouxia clade D. Several putative new
species were found in Trebouxia clades A, C, and I. These included one putative species
in clade A associated with Myriolecis species growing on limestone at high elevation
and a novel lineage sister to the rest of clade C associated with Lecanora on bark in
low-elevation grassland. Three different kinds of photobiont switching were observed,
with certain mycobiont species associating with Trebouxia from different major clades,
species within a major clade, or haplotypes within a species. Lecanoraceae mycobionts
and Trebouxia photobionts exhibit species turnover along the elevation gradient, but
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
with each partner having a different elevation threshold at which the community shifts
completely. A phylogenetically defined sampling of a single diverse family of lichen-
forming fungi may be sufficient to document regional patterns of Trebouxia diversity
and distribution.
Keywords: elevation gradients, systematics, symbiosis, Andes mountains, new PCR primer, Lecanoromycetes,
Trebouxiophyceae, lichen biogeography
INTRODUCTION
Alexander von Humboldt’s Essai sur La Géographie des Plantes
was revolutionary for biogeography (Schrodt et al., 2019),
but, with respect to lichens, Humboldt was limited by the
nascent state of lichenology in the early 19th century. Drawing
on observations from Europe and South America, Humboldt
described lichens as “independent of the influence of the
climates” (von Humboldt and Bonpland, 2009)—a statement
that, with a modern understanding of lichen systematics and
symbiosis, we now know is far from the truth. Climate does
matter for lichens. There is turnover in lichenized fungi along
elevation gradients, including those in the northern Andes (Wolf,
1993;Soto-Medina et al., 2019) that inspired Humboldt’s work.
Furthermore, the mycobiont–photobiont interaction that defines
the lichen symbiosis is affected by environmental conditions at
micro and macro scales (e.g., James and Henssen, 1976;Peksa
and Škaloud, 2011;Singh et al., 2017). What appears externally
as the same lichen may actually represent a mycobiont species
occurring with different photobiont species at different points in
its range (Werth and Sork, 2014;Dal Grande et al., 2018).
Lichen photobiont biodiversity and patterns in the
mycobiont–photobiont association have been most commonly
studied at a regional, intra-biome scale at which climate is largely
consistent [Hestmark et al., 2016;Jüriado et al., 2019;Yahr et al.,
2004; but see Lu et al. (2018) for an intra-biome study along
a climatic gradient] or at a global scale, at which climate may
vary widely but regional patterns may not be apparent (Muggia
et al., 2014;Nyati et al., 2014;Lutsak et al., 2016;Magain et al.,
2017, 2018;Vanˇ
curová et al., 2018;Chagnon et al., 2019). Less
commonly, regional photobiont turnover across adjacent biomes
has been studied along gradients of latitude (Werth and Sork,
2014) or altitude (Dal Grande et al., 2018). In most studies,
investigations of lichen photobionts have been structured around
a specific mycobiont taxon, whether that taxon is a species (e.g.,
Blaha et al., 2006;Werth and Sork, 2014), genus (e.g., Vargas
Castillo and Beck, 2012;Magain et al., 2017, 2018), or family
(e.g., Helms, 2003;Nyati et al., 2014).
Lecanoraceae is one of the three largest families of lichenized
fungi that associate with the green alga Trebouxia, the most
common genus of lichen photobionts (Miadlikowska et al.,
2006;Lücking et al., 2017;Muggia et al., 2018). The family is
cosmopolitan and occurs on a range of substrates, typically with
a crustose growth form (Brodo et al., 2001). Despite the family’s
global distribution and diversity of corticolous species, the
literature on the Lecanoraceae–Trebouxia association has mostly
dealt with mycobiont taxa that are saxicolous, phylogenetically
outside Lecanora s.str., and from Europe, North America, or
Antarctica (Blaha et al., 2006;Guzow-Krzemi´
nska, 2006;Pérez-
Ortega et al., 2012;Ruprecht et al., 2012, 2020;Leavitt et al.,
2015, 2016;Wagner et al., 2020). Photobiont studies that have
included corticolous species of Lecanoraceae have generally done
so in the context of sampling photobionts from across the
lichen community of a small geographic area in the northern
hemisphere (e.g., Singh et al., 2019).
Trebouxia is divided into five major clades: A, C, D, I, and
S (Muggia et al., 2020;Xu et al., 2020). Clade D was recently
identified and is known from Iceland, Svalbard, and Tierra del
Fuego (Xu et al., 2020). Clade C is largely tropical, with a few
temperate representatives. The remaining clades, A, I, and S, are
cosmopolitan, including Antarctica. At the level of these major
clades, lineages are distinguished by differences in pyrenoid
structure (Muggia et al., 2020). The species-level classification of
Trebouxia is still incompletely understood. Leavitt et al. (2015,
2016) and Muggia et al. (2020) implemented an alphanumeric
classification system for putative Trebouxia species based on a
so-called “barcode gap” species delimitation method (Puillandre
et al., 2012). Although some of the species recognized by Muggia
et al. (2020) correspond to named taxa, the majority have not
been formally described.
In this paper, we investigate mycobiont–photobiont
interactions in Lecanoraceae lichens from across an elevation
gradient in Bolivia. Recent studies on lichen photobionts in
Bolivia have uncovered putative new species of Trentepohlia and
Asterochloris (Kosecka et al., 2020, 2021a), but the diversity of
Trebouxia photobionts in Bolivia remains largely unexplored
outside of one global study on photobionts of Cetraria aculeata
(Lutsak et al., 2016). Our dataset includes corticolous and
saxicolous species from Lecanora and other genera in
Lecanoraceae and encompasses nearly 5,000 m of elevation
change in the Andes mountains, south of where Humboldt
developed his ideas on biogeography. We use this dataset to ask
three main questions: (1) How is the biodiversity of Bolivian
Lecanoraceae distributed, both in the phylogeny of this fungal
family and along the elevation gradient? (2) What major clades
and species of Trebouxia are associated with Lecanoraceae in
Bolivia? (3) How do climate, substrate, and mycobiont host
structure photobiont communities?
MATERIALS AND METHODS
Field Sampling
Specimens of Bolivian lichens suspected of belonging to
Lecanoraceae based on their morphology were collected from
2004 to 2019. Collection sites spanned over 4,700 m of
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
elevation and included diverse natural communities across
multiple biomes (Navarro and Maldonado, 2002;Navarro and
Ferreira, 2007). Habitats ranged from patches of trees in
Los Llanos de Moxos savanna below 200 m, to Yungas and
Tucumano-Boliviano montane forests at mid-elevation sites, to
dry, rocky grasslands of the high Andean Puna at about 4,000
m and subnival vegetation above 4,600 m (Figure 1). Voucher
specimens are stored in KRAM and LPB.
Morphological Studies
Morphology and anatomy were studied by standard techniques.
Cross sections of apothecia were mounted in water for
observation of apothecium anatomy or in ca. 25% KOH
for observation of ascospores. Crystals were observed under
polarized light in a compound fluorescence microscope (Nikon
Eclipse 80i) and their solubility was tested with KOH and
65% nitric acid. Lichen secondary metabolites were analyzed
by thin-layer chromatography (TLC) in solvents A, B’, and C
following Culberson and Kristinsson (1970) and Orange et al.
(2001). For saxicolous samples, rocks were tested for carbonate
minerals with 10% HCl.
DNA Extraction, PCR, and Sequencing
From a collection of approximately 550 specimens, 235
specimens representing diverse morphotypes and habitats were
selected for molecular study. Note that the genus Lecidella,
although part of Lecanoraceae, was not targeted in this
sampling focused on lecanoroid species. Material collected in
the years 2015–2018 was kept frozen at −20◦C prior to DNA
extraction, while collections from earlier years were stored under
standard herbarium conditions. Specimens collected in 2019
were extracted when fresh. Apothecia excised from a single
thallus—or soralia, if apothecia were absent—were cleaned in
sterile, distilled water on a microscope slide. Visible contaminants
(e.g., lichenicolous fungi) were removed with ultrathin tweezers
and a razor blade. DNA was isolated using either the QIAamp
DNA Investigator Kit or DNeasyTM Plant Mini Kit (Qiagen,
Germany), depending on the amount of lichen tissue available,
following the manufacturer’s instructions.
To conduct a multi-locus phylogenetic analysis of
Lecanoraceae, we amplified the mycobiont nuclear ribosomal
large subunit (nrLSU), internal transcribed spacer (ITS),
mitochondrial small subunit (mtSSU), RNA polymerase
II largest (RPB1) and second-largest (RPB2) subunits, and
minichromosome maintenance factor 7 (MCM7). The ribosomal
RNA (rRNA) loci ITS, mtSSU, and to some extent nrLSU
have been frequently sequenced for phylogenetic studies of
Lecanoraceae and represent most of the reference data for this
family on GenBank. The ITS region, including the ITS1 spacer,
5.8S rRNA, and ITS2 spacer, was amplified with primers ITS1F
(Gardes and Bruns, 1993) and either LR3 or ITS4 (Vilgalys and
Hester, 1990;White et al., 1990); nrLSU was amplified with
primers AL2R and LR6 (Vilgalys and Hester, 1990;Döring et al.,
2000); and mtSSU was amplified with primers MSU1 and MSU7
(Zhou and Stanosz, 2001). Thermal cycler conditions for the
three rRNA loci were 95◦C for 3 min, followed by 35 cycles of
95◦C for 40 s, 52◦C for 40 s, and 72◦C for 150 s, with a final
extension at 72◦C for 10 min.
Due to the very small amount of DNA present in our
extractions, the protein-coding loci RPB1,RPB2, and MCM7
were amplified with a nested PCR approach. For RPB1, the initial
amplification was performed with primers RPB1-Af and either
RPB1-Cr or RPB1-Dr (Stiller and Hall, 1997;Matheny et al.,
2002). In the first round of PCR, the thermal cycler conditions
were 94◦C for 10 min, followed by 25 cycles of 94◦C for 45 s,
50◦C for 50 s, and 72◦C for 100 s, with a final extension at 72◦C
for 10 min. The second round of PCR used forward and reverse
primers lecRPB1-F and lecRPB1-R (Table 1), which we designed
based on the Lecanoraceae RPB1 alignment from Zhao et al.
(2016). Suitable priming sites were identified by visual inspection
of alignments in Mesquite (Maddison and Maddison, 2018) and
potential primer sequences were evaluated using online tools1,2.
The thermal cycler conditions for the second round were 94◦C
for 5 min, followed by 35 cycles of 94◦C for 45 s, 55◦C for
60 s, and 72◦C for 90 s, with a final extension at 72◦C for
10 min. For RPB2, the first round of PCR used primers fRPB2-5F
and fRPB2-7cR (Liu et al., 1999), and thermal cycler conditions
were identical to the first-round program for RPB1. The second
round used primers lecRPB2-6F and fRPB2-7cR. The newly
designed forward primer lecRPB2-6F (Table 1) was modified
from bRPB2-6F (Matheny, 2005) using Lecanoraceae sequences
from Miadlikowska et al. (2014). Thermal cycler conditions for
this round were the same as for the first round, except that the
number of cycles was increased to 35 and the extension time was
reduced to 85 sec per cycle. For MCM7, the first round of PCR
used primers MCM7-709f and MCM7-1443r, while the second
round used MCM7-709f and MCM7-1348r (Schmitt et al., 2009).
The thermal cycler programs for both rounds followed Schmitt
et al. (2009), except that the number of cycles in the first round
was reduced to 25.
Sanger sequencing of PCR amplicons has been shown
to reliably recover the dominant photobiont in Trebouxia-
associated lichens even when other photobiont genotypes are
present at low levels (Paul et al., 2018). Photobiont ITS was
amplified with primers ITS1T and ITS4T (Kroken and Taylor,
2000) using the thermal cycler program described above for
the mycobiont rRNA loci. For a subset of specimens, we
also amplified a portion of the Rubisco large subunit (rbcL)
with primers a-ch-rbcL-203-5’-MPN and a-ch-rbcL-991-3’-MPN
(Nelsen et al., 2011). Thermal cycler conditions followed Nelsen
et al. (2011). Selection of specimens for rbcL sequencing was
based on the photobiont ITS results and focused on specimens
that were: (1) outliers in the elevation distribution or host breadth
of a photobiont clade, (2) photobionts that appeared to be
new species-level lineages, (3) species-level photobiont lineages
only found in a single specimen, or (4) previously recognized
Trebouxia lineages for which rbcL data were lacking.
1https://eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design/ (accessed June
24, 2021).
2https://www.thermofisher.com/us/en/home/brands/thermo-scientific/
molecular-biology/molecular-biology- learning-center/molecular-
biologyresource-library/thermo-scientific-web-tools/multiple-primer-analyzer.
html (accessed June 24, 2021).
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
FIGURE 1 | Habitats where Lecanoraceae lichens were collected. (A) La Paz, Bautista Saavedra, 14◦48’9”S, 69◦10’51”W, 4850 m, open high Andean vegetation
with siliceous rocks. (B) La Paz, Bautista Saavedra, 15◦15’0”S, 69◦2’50”W, 4549 m, open high Andean area with limestone rocks. (C) La Paz, Franz Tamayo,
14◦46’39”S, 69◦0’35”W, 2550 m, Yungas montane cloud forest. (D) Tarija, Aniceto Arce, 21◦41’36”S, 64◦29’33”W, 2195 m, Tucumano-Boliviano montane forest.
(E) Santa Cruz, Cordillera, 18◦27’29”S, 61◦23’1”W, 292 m, Chaqueño forest. (F) Beni, Yacuma, 14◦51’7”S, 66◦20’23”W, 175 m, Los Llanos de Moxos savanna. All
photographs by AF.
PCR amplicons were checked on 1% agarose gels to confirm
the presence of a single fragment size and cleaned following
an enzymatic cleanup protocol with exonuclease I and shrimp
alkaline phosphatase (ThermoFisher Scientific, Waltham, MA,
United States). Sanger sequencing was performed by Eurofins
Genomics (Louisville, KY, United States). Sequencing reactions
used PCR primers except in two cases. Primers mrSSU1
and mrSSU3R (Zoller et al., 1999) were used in place of
MSU1 and MSU7 to sequence the mtSSU amplicons. A newly
designed primer, lecRPB2-seq7R (Table 1) was used in place of
fRPB2-7cR for sequencing the RPB2 amplicons. Forward and
reverse sequence reads were assembled, trimmed, and checked
for base-calling errors in SeqMan Pro (DNASTAR, Madison,
WI, United States) or Geneious Prime 2021.0.33. When ITS
3https://www.geneious.com
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
TABLE 1 | Lecanoraceae-specific RPB1 and RPB2 primers designed in
the present study.
Locus Primer
name
Use Nucleotide sequence (5’–3’)
RPB1 lecRPB1-F PCR GAR ACN GTY TGY CAY AAY TGY GGC AAG
lecRPB1-R PCR C RAA YTC RTT NAC NAC RTG NGC NGG
RPB2 lecRPB2-
6F
PCR TGG GGN YTR GTM TGY CCD GC
lecRPB2-
seq7R
sequencing G RTT RTG RTC NGG RAA NGG
and partial nrLSU were sequenced as one amplicon, they
were split using the program ITSx (Bengtsson-Palme et al.,
2013). Sequences were checked for contamination or specimen
misidentification using two complementary methods: (1) BLAST
with the NCBI nucleotide database and (2) placement in a
phylogeny of Lecanoromycetes with T-BAS (Miadlikowska et al.,
2014;Carbone et al., 2017, 2019). GenBank numbers for all newly
obtained sequences are given in Supplementary Data Sheet 1.
Alignments and Phylogenetic Analyses
We compiled a reference dataset (Supplementary Data Sheet 2)
of Lecanoraceae and outgroups based on taxa included in
Miadlikowska et al. (2014) and Zhao et al. (2016). In
general, reference taxa were only included if multiple loci,
including protein-coding loci, were available. The families
Parmeliaceae (represented by one species each of Protoparmelia
and Letharia) and Gypsoplacaceae (represented by two species
of Gypsoplaca) were used as outgroups. Our Lecanoraceae
dataset included several species groups within Lecanora,
Palicella,Lecidella,Protoparmeliopsis,Rhizoplaca,Myriolecis, the
“Lecanora”polytropa group, and the “Lecanora”saligna group,
as well as Haematomma,Ramboldia, and Miriquidica. Reference
sequences were checked for specimen misidentifications or other
metadata errors using BLAST with the NCBI nucleotide database.
The mycobiont rRNA loci (mycobiont ITS, nrLSU, and
mtSSU) were initially aligned using the MAFFT online server4
with the G-INS-1 option (Katoh et al., 2019). The alignments
were corrected by eye in Mesquite (Maddison and Maddison,
2018) and introns and ambiguously aligned regions were
delimited manually and excluded from downstream analyses
(Lutzoni et al., 2000). Protein-coding loci were aligned by
translated amino acid in Mesquite. Introns were delimited
manually and excluded from downstream analyses.
Each single-locus alignment was used as input for a maximum
likelihood phylogenetic analysis in IQ-TREE version 2.1.2
(Nguyen et al., 2015;Chernomor et al., 2016) run on the
CIPRES server (Miller et al., 2010). We performed 5,000 ultrafast
bootstrap pseudoreplicates to calculate support for each single-
locus tree (Hoang et al., 2018). We inspected the resulting
trees for well-supported conflicts (≥95% ultrafast bootstrap;
Hoang et al., 2018) among the six mycobiont loci, which were
then concatenated to form a single dataset. The concatenated
mycobiont alignment was used as input for a partitioned
4https://mafft.cbrc.jp/alignment/server/
maximum likelihood analysis in IQ-TREE. We ran IQ-TREE with
the -p and -m MFP + MERGE options so that ModelFinder
(Kalyaanamoorthy et al., 2017) would both optimize the
partitioning scheme and find the best-fitting substitution model
for each partition. The input partitioning scheme divided the
concatenated alignment by locus and by codon position for
the protein-coding loci. The final partitioning schemes and
substitution models used in this analysis are provided in Table 2.
We performed 5,000 ultrafast bootstrap pseudoreplicates to
calculate bipartition support for the tree topology.
We performed two phylogenetic analyses with the photobiont
data. First, all sequences were included in a global ITS-rbcL
alignment for Trebouxia to assign each sample to one of the
major Trebouxia clades. We used the ITS and rbcL sequences
curated by Muggia et al. (2020) as a reference dataset, with
slight modifications. These sequences are listed in the file
“Supplementary Data 2” from Muggia et al. (2020). We removed
sequence L138 from the Trebouxia clade S ITS alignment
and sequences L1107, L1360, L1401, L1417, L1425, L1504,
AJ007387, AJ969549, AJ249482, and 9493 from the Trebouxia
clade A ITS alignment because they were missing a significant
portion of ITS1 or ITS2. None of these ITS sequences had a
corresponding rbcL sequence. L1107 and 9493 were the only
representatives of the putative species A37 and A49, respectively,
but preliminary analyses indicated that our new sequences
were not closely related to these taxa. We also added all
ITS and rbcL reference sequences for Trebouxia clade D from
“Supplementary Table 5” of Xu et al. (2020). Representatives
from three other genera of Trebouxiophyceae were used as
outgroup taxa: Asterochloris, GenBank accession JN573844;
Myrmecia, KM462861; and Vulcanochloris, KR952313. Only rbcL
was included for the outgroup taxa because ITS was not alignable
across the four genera.
ITS sequences were initially aligned with MAFFT using
the G-INS-1 option and regions that were not conserved
across Trebouxia were excluded by eye in Mesquite; rbcL
sequences were aligned by translated amino acid in Mesquite.
Phylogenies for each locus were inferred as described above
for the mycobiont loci. The two alignments were concatenated
after inspecting single-locus trees for well-supported conflicts.
We used the concatenated dataset as input for a maximum
likelihood phylogenetic analysis in IQ-TREE. Model selection,
tree inference, and bootstrapping parameters were the same as
described above for the concatenated mycobiont dataset, with the
initial partitioning scheme dividing the alignment by locus and
further splitting rbcL by codon position.
For the second set of analyses, we performed a separate
phylogenetic analysis for each major clade of Trebouxia
represented in our sampling. The genus-wide alignment allowed
unknown sequences to be assigned to one of the major clades, but
because variable regions of ITS1 and ITS2 had to be excluded,
relationships within these clades were often poorly resolved. For
each major clade, reference taxa and sample sequences were
extracted from the global ITS and rbcL alignments. The ITS
alignments were corrected by eye in Mesquite and ambiguous
regions were re-delimited. One rbcL sequence from each of the
other major clades of Trebouxia was retained for the outgroup.
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TABLE 2 | Statistics on the alignments for Lecanoraceae (mycobiont) after the removal of introns and ambiguously aligned regions.
Locus Length Invariable (%) PI (%) Missing taxa (%) Model/Partition
ITS 252 144(57)69(27)14(9)1
nrLSU 1024 701(68)214(21)35(23)1
mtSSU 789 531(67)172(22)29(19)2
RPB1 630 298(47)297(47)42(27)3:3:4
RPB2 744 329(44)367(49)56(36)3:2:4
MCM7 537 269(50)249(46)102(66)3:2:4
The full concatenated alignment included 154 taxa and 3,976 sites. PI, parsimony-informative sites. Substitution models were as follows: 1, TN + F + R4; 2, HKY + F + R3;
3, GTR + F + R3; 4, TIM2e + I + G4. Models for protein-coding loci are given as first:second:third codon positions. Loci and codon positions with the same substitution
model were fused into a single partition.
After checking for well-supported conflicts between the two loci
as described above, we concatenated the ITS and rbcL alignments
and performed a maximum-likelihood analysis in IQ-TREE.
Model selection, tree inference, and bootstrapping parameters
were the same as described above for the concatenated mycobiont
dataset. The initial partitioning scheme divided the alignment
by locus and further split the ITS by region (ITS1, 5.8S, and
ITS2) and rbcL by codon position. The final partitioning schemes
and substitution models used for these analyses are provided in
Table 3.
To explore intraspecific diversity in a subset of putative
Trebouxia species, we inferred haplotype networks in R version
3.6.1 (R Core Team, 2019) using the packages ape (Paradis and
Schliep, 2019) and pegas (Paradis, 2010). Sites with ambiguous
bases were excluded from the haplotype analysis.
Classification of Mycobionts and
Photobionts
Our preliminary delimitation of mycobiont species was based on
morphology, chemistry, and the inferred phylogeny. We used
the program bPTP (Zhang et al., 2013) to inform our species
delimitation, using the concatenated maximum likelihood
phylogeny as input and running the Markov chain Monte Carlo
(MCMC) analysis for 500,000 generations, sampling every 500
generations5. The first 25% of samples were discarded as burn-in.
Based on the ITS-rbcL phylogeny, photobiont sequences were
assigned to a clade and putative species based on the framework
from Muggia et al. (2020). Specimens that were phylogenetically
distinct from any previously delimited species were considered to
represent novel putative species.
Ecological Analyses
To better understand the ecological diversity across the elevation
gradient, we extracted 19 bioclimatic variables from the
WorldClim database (Fick and Hijmans, 2017) for each sampling
locality. Bioclimatic variables were sampled at a resolution of
10 arc-minutes using the R packages sp (Pebesma and Bivand,
2005) and raster (Hijmans, 2021). We used the hclust function to
perform a hierarchical clustering of the 19 bioclimatic variables,
which was visualized as a dendrogram. Vegetation types for each
site were classified with field locality descriptions and vegetation
maps from Navarro and Ferreira (2007).
5https://species.h-its.org/
We analyzed patterns of species richness (αdiversity) and
turnover (βdiversity) along the elevation gradient. From the
original data for each lichen specimen (Supplementary Data
Sheet 1), we prepared inferred presence/absence matrices for the
mycobiont and photobiont species. The elevation gradient was
split into 500 m bins starting at sea level and extending to 4,999 m.
For each Lecanoraceae or Trebouxia species, we noted the lowest
and highest elevations at which the species was found, and coded
the species as “present” for all bins included in that range. For
example, a Trebouxia species found at 2,400, 3,050, and 3,600 m
would be coded as present from 2,000 to 3,999 m even though it
was absent from the 2,500 to 2,999 m bin. The Sørensen index of
dissimilarity was calculated for all pairwise comparisons between
elevation bins using the vegdist function in the R package vegan
(Oksanen et al., 2020).
RESULTS
Sequenced Specimens
We obtained DNA sequence data from approximately half of
the 235 specimens for which extractions were performed. Most
of the specimens that failed to yield any sequence data were
those collected before 2015 and stored under standard herbarium
conditions. Of the specimens from which we obtained sequence
data, approximately ten were not Lecanoraceae and were
excluded from further analysis. A further ca. 20 specimens yielded
only mycobiont or only photobiont sequences; the sequences
from specimens without paired mycobiont–photobiont data
were not used for the present paper. Finally, five specimens
were excluded from the analysis because of well-supported
conflicts between loci or discordance between molecular and
morphological data. A total of 89 specimens with both mycobiont
and photobiont data were used for the remaining analyses. We
generated over 500 new DNA sequences from this group of
specimens: 83 mycobiont ITS, 68 nrLSU, 79 mtSSU, 70 RPB1, 64
RPB2,22MCM7, 89 photobiont ITS, and 27 rbcL.
Alignments
Summary statistics for the Lecanoraceae and Trebouxia
alignments are presented in Tables 2,3, respectively. For the
mycobiont, almost all of ITS1 and a large fraction of ITS2 could
not be aligned across Lecanoraceae and were excluded from the
phylogenetic analysis. The three protein-coding loci contained
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
TABLE 3 | Statistics on the alignments for Trebouxia (photobiont) after the removal of introns and ambiguously aligned regions.
Clade ITS rbcL
Length PI (%) Model/Partition PI (%) Model/Partition
A 516 150(29)1:2:1 83(11)2:2:3
C 514 129(25)4:5:4 121(15)5:5:6
I 505 101(20)7:5:7 86(11)5:5:8
S 552 123(22)9:5:9 39(5)5:5:3
PI, parsimony-informative sites. The rbcL alignment was 789 bases long for all clades and had no excluded positions. Substitution models were as follows: 1 = TVMe + R3;
2 = K2P + R2; 3 = TIM3 + F + R3; 4 = SYM + R3; 5 = K2P + I; 6 = TIM2 + F + R3; 7 = TPM2 + F + G4; 8 = TIM2 + F + I; 9 = TIM3e + G4. Models are given as ITS1:5.8S:ITS2
for the ITS and first:second:third codon positions for rbcL. ITS regions and codon positions with the same substitution model were fused into a single partition.
the greatest number of parsimony-informative sites in absolute
terms and relative to their length (Table 2). For the Trebouxia
sequences, most of ITS1 and ITS2 could be aligned within each
major clade. ITS provided most of the parsimony-informative
sites for all four Trebouxia single-clade alignments, while rbcL
varied from being nearly as informative as ITS in Trebouxia clade
C to being minimally informative in Trebouxia clade S (Table 3).
Complete alignments with excluded regions delimited are
available in the supplementary materials (Supplementary Data
Sheets 3–8).
Lecanoraceae and Trebouxia Phylogeny
and Biodiversity
Photobionts from members of the Lecanoraceae collected in
Bolivia represented approximately 21 putative species in four
of the five major clades within Trebouxia (Figure 2 and
Supplementary Figure 1). The majority of photobionts were
phylogenetically nested within previously delimited species,
but six lineages appear to represent new species-level taxa in
Trebouxia clades A, C, and I (Supplementary Figures 2–4).
All samples from Trebouxia clade S corresponded to previously
delimited species (Supplementary Figure 5). No samples were
from Trebouxia group D (Supplementary Figure 1).
The single-locus trees for the mycobiont ITS, nrLSU, and
mtSSU had some well-supported nodes near the tips, but
deeper relationships were for the most part poorly supported
(Supplementary Figures 6–8). RPB1,RPB2, and MCM7 were
better able to resolve deeper relationships in Lecanoraceae,
with RPB1 yielding the best-supported tree of any single
locus (Supplementary Figures 9–11). The maximum likelihood
tree from the concatenated six-locus dataset (Figure 3 and
Supplementary Figure 12) included strong support for many
putative genus- and subgenus-level clades and some relationships
among those clades, but the backbone was poorly supported
in several regions of the tree. For example, the relationships
among Haematomma,Miriquidica,Ramboldia, and the rest of
the family were not well supported, nor was the position of
Lecidella. However, none of our mycobiont specimens belonged
to these genera.
Sampled mycobionts belong to two well-supported clades
within Lecanoraceae (Figure 3). The first clade, which we call
Lecanora s.lat., includes the Lecanora subfusca group (Lecanora
clades I and II), L. subcarnea group (Lecanora clade III), a group
we call Lecanora clade IV, L. formosa and Palicella,L. cavicola,
and L. rupicola. None of the relationships among these clades
were well supported. Species of Lecanora s.lat. were found
predominantly on bark at low- to mid-elevation sites, but some
collections were from rock at high elevation (Figure 4). The high-
elevation, saxicolous specimens mostly belonged to L. rupicola,
L. cavicola, and L. formosa, but also included taxa from Lecanora
clades II and III (Figures 4,5A).
The second major clade, which we call the MPRPS clade for
lack of a formal name, included high-elevation collections from
Myriolecis,Protoparmeliopsis, the “Lecanora”polytropa group,
Bryonora, and the “Lecanora”saligna group. MPRPS also includes
Rhizoplaca, although none of our collections belong to that
genus (Figure 3). Myriolecis and Protoparmeliopsis form a well-
supported clade, and the “L.”saligna group is a well-supported
sister to the rest of the MPRPS clade, but the phylogenetic
placements of the “L.”polytropa group, Rhizoplaca, and Bryonora
are uncertain. Except for the single specimen of Bryonora and one
Myriolecis specimen that were found on moss and two specimens
of the “L.”saligna group that were lichenicolous lichens, species
in the MPRPS clade occurred on rock (Figure 4).
Based on morphology, chemistry, and the molecular
phylogeny, our sampling included approximately 27 species in
Lecanora s.lat. and approximately 10 species in the MPRPS clade
(Figure 4). The tree topology and species delimitation analysis
suggest that several of the morphospecies contain multiple
cryptic species. For example, there are three well-supported
lineages on long branches in the morphospecies Lecanora
ecoronata (Lecanora clade I), and the specimens identified as L.
caesiorubella were distributed across three lineages in Lecanora
clade III that did not form a monophyletic group (Figure 4).
Photobiont Ecology and Mycobiont
Associations
The distribution of Trebouxia species was structured by elevation,
substrate, and mycobiont identity (Figures 4,5). Species of
the MPRPS clade were always found with photobionts from
Trebouxia clades A and S. Myriolecis,Protoparmeliopsis, and the
single specimen of Bryonora were exclusively associated with
Trebouxia clade A, while the “Lecanora”polytropa and “L.”
saligna groups occurred only with Trebouxia clade S. All of these
mycobiont genera were restricted to high elevations (Figure 5A).
This pattern is also related to substrate chemistry: Saxicolous
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0.04
FIGURE 2 | Summary phylogeny showing relationships among major clades of Trebouxia based on maximum likelihood analysis of the ITS-rbcL dataset. Bold clade
names indicate clades represented in our Bolivian sampling. Bold branches indicate UFboot2 support ≥95. Scale represents substitutions per site. The color
scheme introduced here will be used for these Trebouxia clades throughout the rest of the paper. For the full tree with all tip names and bootstrap values, see
Supplementary Figure 1.
0.04
FIGURE 3 | Summary phylogeny showing relationships within Lecanoraceae based on maximum likelihood analysis of the six-locus dataset. The clade names
Lecanora I–IV, Lecanora s.lat., and MPRPS (Myriolecis,Protoparmeliopsis,Rhizoplaca, “L.” polytropa, “L.” saligna) will be used throughout the paper in the sense
shown here. Bold clade names and filled triangles indicate clades represented in our Bolivian sampling. Bold branches indicate UFboot2 support ≥95. Scale
represents substitutions per site. For the full tree with all tip names and bootstrap values, see Supplementary Figure 12.
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1000
2000
3000
4000
elevation (m) vegetation
Beni savanna
Chaceño−Amazon forest
Tucumano−Boliviano forest
Yungas forest
Humid Puna high−Andean
Humid Puna subnival
Other
substrate
bark
rock (siliceous)
moss
rock (calcareous)
lichens
Trebouxia
substrate
elevation
vegetation
mycobiont
FIGURE 4 | Photobiont association, substrate, elevation, and vegetation type in the context of the mycobiont phylogeny. The tree is from the same six-locus analysis
as Figure 3 and Supplementary Figure 12; reference taxa were removed in R with the drop.tip function in ape and data were added to the tree with the package
ggtree (Yu et al., 2017). Bold branches indicate clades with UFboot2 support ≥95 in Supplementary Figure 12. Lecanoraceae species highlighted in green were
found with photobionts from more than one major clade of Trebouxia. Lecanoraceae species highlighted in yellow were found with multiple Trebouxia species from
the same clade. Colors used for Trebouxia clades are the same as in Figure 2. Species-level relationships within Myriolecis are not well-resolved in this analysis.
species of Myriolecis and Protoparmeliopsis grew on calcareous
rock, while species of the “L.” polytropa group and the lichen
hosts of members of the “L.” saligna group occurred on siliceous
rock (Figure 4). All of these mycobiont genera were restricted to
high-elevation sites (Figure 5A).
Species of the Lecanora s.lat. clade were found in association
with Trebouxia clades A, C, I, and S. There was a pronounced
turnover in photobiont clade along the elevation gradient, with
Trebouxia clade C at low to mid-elevations (up to ca. 3,000 m),
clade I mostly at upper mid elevations (ca. 2,000–3,500 m), and
clade S at high elevations (above 3,500 m) (Figure 5A). Trebouxia
clade A was only rarely associated with Lecanora s.lat., always
above 2,000 m (Figure 5A).
Clades within Lecanora s.lat. occupied varying portions of
the total elevation gradient (Figure 5A). Lecanora clade I was
only absent from the highest elevations, while Lecanora clade
III was missing at low elevations; Lecanora clade IV occupied
intermediate elevations. Lecanora clade II was primarily at low
elevations, except for a single high elevation species. Lecanora
clades I–IV were each associated with photobionts from at least
two, and often three, Trebouxia clades. The same elevational
turnover of photobiont clades that is apparent for Lecanoraceae
as a whole also occurs within these smaller clades; see, for
example, Lecanora clades I and III (Figure 5A).
At the level of putative Trebouxia species, there is considerable
variation in the level of specificity exhibited toward habitat and
mycobiont (Figures 4,5B). Most Trebouxia species occupied a
500–1,000 m subset of the elevation gradient (Figure 5B). Some
taxa occurred only with a single mycobiont clade in that narrow
elevation range. For example, Trebouxia C13 was only found
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Mycobiont clade
Elevation (m)
Putative Trebouxia species
Elevation (m)
B
A
0
1000
2000
3000
4000
5000
Lecanora−II
Lecanora−IV
Lecanora−I
Lecanora−III
L. calvicola
Protoparmeliopsis
Bryonora
Myriolecis
“L.” polytropa group
L. rupicola
“L.” saligna group
L. formosa
0
1000
2000
3000
4000
5000
C36
C13
C35
C10
C34
C02
A19
I19
C09
I13 s.lat.
I20
I15
A05
I18
S10
A53
A aff. 07
A01
A04
S02
A13
I
II
III
IV
V
VI
VII
VIII
C09 haplotype network
Trebouxia
A
C
I
S
Substrate
bark
lichen
moss
rock (siliceous)
rock (calcareous)
FIGURE 5 | Elevation distribution of Lecanoraceae mycobionts and their Trebouxia photobionts. (A) Elevation distribution of the major mycobiont clades, showing
Trebouxia clades and substrates. Note that photobiont turnover across the elevation gradient is apparent for Lecanoraceae as a whole and within individual
mycobiont clades. There are relatively few samples from the 3,000 to 4,000 m range because specimens for molecular study were originally selected for a taxonomic
study of Lecanoraceae. Although we have many collections from this altitudinal range, they were not particularly diverse morphologically and therefore were not
sampled heavily in our sequencing. (B) Elevation distribution of putative Trebouxia species. A single photobiont species typically occupies 500–1,000 m of the
elevation gradient. Inset shows haplotype network for Trebouxia C09, which occupies 1,500 m of the gradient. The haplotypes clustered on the left occur across the
elevation range of this species, while those clustered on the right only occur at the bottom of the range.
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
below 500 m with species of Lecanora clade I, while Trebouxia
A53 only occurred with species of Myriolecis at about 4,500 m
(Figure 4). Other photobionts were generalists with respect to
mycobiont but were only found in a specific habitat. Trebouxia
A13, S02, and S10 occurred with mycobiont species from both
the Lecanora s.lat. and MPRPS clades, but nearly always above
4,000 m (Figure 4). On the other hand, some photobionts
exhibited substantial niche breadth for both mycobiont host
and elevation. Trebouxia C09, the most broadly distributed
photobiont species in our sampling, was associated with species
from Lecanora clades I–IV and occurred on both bark and rock
from 1,500 to 3,000 m (Figures 4,5B).
Numbers of mycobiont and photobiont species were tightly
linked across the elevation gradient, with the number of
mycobiont species nearly always greater; both Lecanoraceae
and Trebouxia had the greatest number of species at high
elevation (Figure 6A). Species turnover was consistently high
across the elevation gradient for both partners (Figure 6B).
Each partner had a mid- to mid-high-elevation threshold
across which no species were shared, but this threshold
differed between Lecanoraceae (2,500 m) and Trebouxia
(3,500 m) (Figure 6B).
Photobiont Switching
Three mycobiont species were associated with photobionts from
two or more Trebouxia clades (Figure 4). Lecanora sp. 1
(n= 4) occurred on sandstone in open areas between 2,700
and 3,300 m. Below 3,000 m, this species was found with
Trebouxia C09, while above 3,000 m it was found with Trebouxia
I15. Lecanora rupicola (n= 2) was found in association with
Trebouxia A13 and S02 at a single high-elevation site, where
it grew on siliceous rocks. Lecanora aff. farinacea (n= 4)
occurred with Trebouxia C09 at ca. 2,900 m and with Trebouxia
I18 and S02 at ca. 4,250 m. All four collections were from
siliceous rocks, either sandstone at the lower site or schist at
the upper sites.
A larger number of mycobiont species associated with
multiple photobionts within one of the major Trebouxia clades
(Figure 4). “Lecanora”intricata (n= 4) and L. cavicola (n= 3)
each associated with Trebouxia S02 and S10. At one site at
ca. 4,260 m, L. cavicola was found with both photobionts. All
specimens of these two species were collected from siliceous rocks
in open vegetation above 4,000 m. Protoparmeliopsis garovaglii
(n= 2), which was only collected at a single site at ca. 4,550 m, on
limestone, occurred with Trebouxia A01 and A aff. 07. Lecanora
caesiorubella 1 (n= 2) occurred with Trebouxia C34 at ca.
1,900 m and Trebouxia C02 at 2,250 m; both sites were in
Yungas forest. Lecanora ecoronata 1 (n= 9) was predominantly
associated with Trebouxia C13, but at the same savanna sites also
occurred with Trebouxia C35 and C36. Lecanora sp. 2 (n= 3)
occurred with Trebouxia C13 at the bottom of its range and
C10 at the top of its range. Lecanora flavidomarginata (n= 2)
occurred with Trebouxia I18 and I20, at ca. 4,000 m and ca. 3,400
m, respectively.
Haplotypes of Trebouxia C09 fell into two clusters
(Figure 5B). One cluster spanned the entire elevation range of
this putative species, while the other occurred only in the lower
portion of its range. Lecanora aff. stramineoalbida was associated
with both haplotype clusters.
DISCUSSION
Mycobiont Phylogeny
Phylogenetic studies of Lecanoraceae have long been bedeviled
by a poorly supported backbone (Grube et al., 2004;Pérez-
Ortega et al., 2010;Zhao et al., 2016;Yakovchenko et al.,
2019;Davydov et al., 2021). Most of these studies, including
recent publications, have used ITS and mtSSU to infer the
phylogeny of Lecanoraceae, but those loci are most useful for
resolving relationships within genera or species groups and
cannot resolve deeper relationships across the entire family.
Zhao et al. (2016) obtained better support with a six-locus
dataset, but their analysis excluded many morphologically
distinct species groups for which the protein-coding loci
were lacking. In the present study, we used the same six
loci as Zhao et al. (2016): ITS, nrLSU, mtSSU, RPB1,RPB2,
and MCM7. We expanded their taxon sampling, bringing
in additional clades that were missing from their six-locus
analysis: Bryonora,Lecanora IV, Lecanora cavicola,Lecanora
rupicola, and the “Lecanora” saligna group. Despite this expanded
sampling, we found that several regions of the phylogeny remain
poorly supported.
One particularly difficult problem in Lecanoraceae systematics
is the delimitation of Lecanora s.str. In the present paper, we
have sidestepped this issue by referring to a well-supported
Lecanora s.lat. and well-supported clades nested within it,
without making a taxonomic judgment about what should be
included in Lecanora s.str. If morphologically distinctive groups
such as Palicella,Pulvinora, and the Lecanora rupicola group
are to be excluded from Lecanora s.str., as various authors have
proposed (Grube et al., 2004;Rodriguez Flakus and Printzen,
2014;Davydov et al., 2021), Lecanora s.str. should ideally be
tied to a well-supported node within our Lecanora s.lat. Our
analysis recovered no such node deeper than the species groups
delimited in Figure 3. For example, the Lecanora subfusca group
as traditionally circumscribed (Lecanora clades I and II; Zhao
et al., 2016) was not well supported in our analysis (Figure 3
and Supplementary Figure 12). Further research, potentially
with genomic data, will be required to stabilize the genus-level
nomenclature of Lecanoraceae.
Based on our results and the existing literature, we
recommend sequencing at a minimum ITS, mtSSU, and RPB1 in
order to place a specimen within the phylogeny of Lecanoraceae.
The new primers and nested PCR protocols we describe in
this paper can be used to sequence RPB1 and RPB2 even from
extractions with a very small quantity of DNA. In this study,
we have substantially increased the number of RPB1,RPB2,
and MCM7 sequences available for Lecanoraceae, which should
facilitate the use of these loci in future phylogenetic studies of
this family. The inclusion of Bryonora in our multilocus dataset
illustrates how the protein-coding loci can clarify relationships in
Lecanoraceae. Bryonora was previously represented on GenBank
by a single ITS sequence from Grube et al. (2004), and its
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0
5
10
15
1000 2000 3000 4000 5000
elevation
alpha diversity (number of species)
0.00
0.25
0.50
0.75
1.00
1000 2000 3000 4000
elevation
beta diversity (pairwise Sørensen)
AB
mycobiont
photobiont
FIGURE 6 | Species richness and β-diversity of Lecanoraceae and Trebouxia along the elevation gradient. (A) Number of Lecanoraceae (solid line) and Trebouxia
(dashed line) species observed and inferred for 500 m elevation bins. (B) Pairwise comparisons of 500 m elevation bins by the Sørensen index of dissimilarity, where
0 means all species are shared and 1 means no species are shared. Note that each partner has a mid- to mid-high-elevation threshold not spanned by any species,
but this threshold is not the same for mycobiont and photobiont. Comparisons below 1,500 m (reduced line weight) may be unreliable because of the absence of
samples from the 500–999 m elevation band.
phylogenetic position was unknown. The phylogenetic analysis
of Grube et al. (2004) placed it outside Lecanoraceae, albeit
without support. Our results, which included ITS, nrLSU, mtSSU,
RPB2, and MCM7 sequences for a specimen of Bryonora,
strongly support Bryonora as a distinct genus belonging to
the MPRPS clade.
Although there have been morphological studies on
Lecanoraceae in Bolivia (´
Sliwa et al., 2013, 2014; to a minor
extent, Guderley, 1999), ours is the first study to generate
molecular data for Bolivian Lecanoraceae and one of the only
molecular phylogenetic studies of tropical Lecanoraceae (Kirika
et al., 2012;Papong et al., 2013). These data will be used in
forthcoming publications on the systematics of Lecanoraceae. As
a resource for the community, we have made our Lecanoraceae
and Trebouxia trees and alignments available on the T-BAS
online portal6to facilitate the phylogenetic placement of
specimens from these clades (Carbone et al., 2017, 2019).
Trebouxia Diversity and Ecology
We found Lecanoraceae mycobionts from Bolivia in association
with four of the five major clades of Trebouxia, with only
Trebouxia clade D absent from our sampling. Clades A, I, and
S have been recorded with Lecanoraceae in numerous previous
studies (Beck, 1999;Blaha et al., 2006;Guzow-Krzemi´
nska, 2006;
Hauck et al., 2007;Pérez-Ortega et al., 2012;Ruprecht et al.,
2012, 2020;Leavitt et al., 2015, 2016;Voytsekhovich and Beck,
2016;Singh et al., 2019;Muggia et al., 2020;Wagner et al.,
2020). Lecanoraceae species have not previously been reported
to associate with Trebouxia clade C. Our results show that
species of Lecanora s.lat.—specifically Lecanora clades I–IV—
frequently associate with Trebouxia clade C photobionts in low
to mid-elevation habitats (Figure 5A). That this association has
not been seen before is a result of the dearth of molecular
6https://decifr.hpc.ncsu.edu/index.php
phylogenetic studies on tropical Lecanora. Our results reinforce
the importance of Trebouxia clade C photobionts in tropical
lichens. The prevalence of Trebouxia clade C photobionts in
the tropics has been recognized for nearly two decades (Helms,
2003;Cordeiro et al., 2005), but it was not until recently
that the species richness of this clade was well understood
(Muggia et al., 2020).
Several Trebouxia ecological patterns seen in other regions
and with other mycobiont taxa are evident in our results. These
include the association of Trebouxia clade A with calcareous
rock (Helms, 2003) and open, intermittently humid Andean
vegetation (Vargas Castillo and Beck, 2012), as well as the
association of Trebouxia clade S with high elevation (Blaha
et al., 2006;Muggia et al., 2008). In an analysis of over 6,000
publicly available sequences of lichenized Trebouxia, originally
sampled from around the world and associated with diverse
mycobiont hosts, Nelsen et al. (2021) found that Trebouxia
clade I photobionts occupy a bioclimatic space intermediate
between clade C and clade S, while clade A—the most speciose
lineage in Trebouxia—overlaps with all three. Our results
confirm this pattern (Figures 5A,B) and show that it can be
observed on a regional scale within the symbionts of a single
mycobiont family.
Along an elevation gradient from ca. 700–2,100 m in Europe,
Dal Grande et al. (2018) found that Trebouxia photobionts of
the mycobiont genus Lasallia had larger altitudinal ranges at
higher elevations. We saw no evidence for this pattern across the
entire elevation range represented in our sampling (Figure 5B).
It is possible that greater inter-biome variation in climate and
vegetation along our nearly 5,000 m elevation gradient leads to
species turnover that overrides any such pattern.
Our photobiont data included putative new species of
Trebouxia in clades A, C, and I (Supplementary Figures 2–4). In
clade S, which is globally less diverse than the other three clades
(Muggia et al., 2020), all of our specimens belonged to previously
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delimited species (Supplementary Figure 5). Most of the putative
novel species were recovered from a single specimen (C34, C35,
C36, I19, and I20) (Figure 5B). The exception is A53, which
we found in 11 specimens of Myriolecis across four localities
in the humid puna subnival vegetation (Figures 4,5). A53 was
always found in lichen thalli growing on exposed, calcareous rock
(Figure 4).
Several Trebouxia species we found in our sampling were
previously known primarily, or exclusively, from east Africa (see
below). Shared Trebouxia genotypes between Bolivia and Kenya
have been previously reported (Lutsak et al., 2016), highlighting
that long-distance dispersal probably plays a major role in the
distribution of lichenized algae (Rolshausen et al., 2020).
Notes on Selected Trebouxia Species
A01 — We found this species associated with a single
thallus of Protoparmeliopsis garovaglli.Guzow-Krzemi´
nska
(2006) recorded this photobiont in association with P. muralis in
Europe, and Leavitt et al. (2015) found it associated with species
of Protoparmeliopsis,Lecanora,Rhizoplaca, and Xanthoparmelia.
A04 — We recovered Trebouxia A04 from a single specimen
of Bryonora. This is the first photobiont DNA sequence obtained
from a lichen thallus of this mycobiont genus.
A05 — This species was found in association with a species
of Lecanora clade IV from ca. 3,500 m. It has otherwise been
reported from various species of Parmeliaceae: Hypotrachyna and
Punctelia in Kenya (Muggia et al., 2020) and Oropogon in Central
and South America (Leavitt et al., 2015).
A13 — This species is globally distributed (De los Ríos et al.,
2002;Blaha et al., 2006;Guzow-Krzemi´
nska, 2006;Muggia et al.,
2008, 2014;Nyati et al., 2014;Leavitt et al., 2015;Voytsekhovich
and Beck, 2016).
A19 — We provide the first rbcL sequence from this previously
delimited species (Muggia et al., 2020).
A53 — This is the most common novel putative species we
recovered in our sampling (Figure 4). It was always found in
association with species of Myriolecis growing on calcareous rock.
C02 — We provide the first rbcL sequence from this previously
delimited species (Muggia et al., 2020).
C09 — This species was previously reported from multiple
genera of Parmeliaceae in Kenya and from Tephromela in Kenya,
Russia, and Peru (Muggia et al., 2014, 2020). In our sampling,
Trebouxia C09 was found in association with species of Lecanora
s.lat. It is clearly a generalist with respect to mycobiont host and
substrate, occurring with species of Lecanora clade IV on bark
at the lower end of its altitudinal range, then with species from
clades I, II, and III, on bark and on rock, at the upper end of its
range (Figures 4,5B).
C10 — This species was previously reported from multiple
genera of Parmeliaceae in Kenya and Parmotrema tinctorum in
Japan (Ohmura et al., 2006;Muggia et al., 2020). In our sampling,
Trebouxia C10 was only found in association with species of
Lecanora clades I and II (i.e., the Lecanora subfusca group)
(Figure 4).
C13 — This species is known from three specimens of
Parmotrema and Canoparmelia from Kenya (Muggia et al., 2020).
In our sampling, Trebouxia C13 was only found in association
with species of Lecanora clades I and II (i.e., the Lecanora
subfusca group) (Figure 4).
C34 — This novel putative species was also found by Kosecka
et al. (2021b). See discussion below.
C35 and C36 — These OTUs form a novel lineage sister to
the rest of Trebouxia clade C (Supplementary Figure 3). They
were both found in association with Lecanora ecoronata 1 in the
low-elevation savanna. The phylogenetic position of these species
is supported by both ITS and rbcL, but additional investigations
of the ultrastructure of these species should be conducted to
evaluate whether they would be better treated as a new major
clade (i.e., if they do not have a corticola-type pyrenoid).
I13 s.lat. — We found several specimens associated with
Trebouxia I13 but, despite having both ITS and rbcL data, the
clade is not well supported (Supplementary Figure 4). This
region of the Trebouxia clade I tree, which also includes I17, I18,
I19, and I20, appears to be primarily tropical (Muggia et al., 2020)
and requires further phylogenetic study.
I15 — We provide the first rbcL sequence from this previously
delimited species. Trebouxia I15 was reported from Punctelia and
Hypotrachyna in Kenya (Leavitt et al., 2015;Muggia et al., 2020).
I18 — This species was reported from Hypotrachyna in Peru
and Kenya and Tephromela in Peru (Muggia et al., 2014, 2020).
I19 and I20 — These novel putative species were also found by
Kosecka et al. (2021b). See discussion below.
S02 — This species is a generalist with respect to mycobiont
host, associating with species of multiple genera within both the
Lecanora s.lat. and MPRPS clades.
S10 — This OTU corresponds to the formally described
species Trebouxia simplex (Tschermak-Woess, 1978), the type
of which was originally isolated from Chaenotheca in Europe,
and has also been reported from Bryoria in North America and
Europe (Lindgren et al., 2014) and Lasallia in Europe (Sadowska-
De´
s et al., 2014). Our results confirm that S10/T. simplex is
a generalist with respect to mycobiont host, associating with
species from both the Lecanora s.lat and MPRPS clades.
Multiple Photobionts in a Single Thallus
Although intra-thalline genetic diversity has been documented
in lichenized Trebouxia (e.g., Mansournia et al., 2012;Dal
Grande et al., 2014), Sanger sequencing of PCR amplicons
has been shown to consistently identify the most abundant
photobiont genotype (Paul et al., 2018). Furthermore, when
multiple photobiont genotypes are present, they are generally
closely related, belonging to the same species (Mansournia et al.,
2012;Dal Grande et al., 2014), sister taxa (Casano et al., 2011), or
at least the same group within Trebouxia (Paul et al., 2018).
For one specimen (Flakus 29597), Sanger sequencing
recovered two different ITS sequences with the forward and
reverse primers, corresponding to the photobionts I18 and S02.
Repeated PCR and sequencing of this specimen generated the
same result: two clean chromatograms with different sequences.
The rbcL sequence obtained from this specimen was consistent
with I18, so we used that photobiont in the figures and
analyses, although S02 is also consistent with the ecology of this
specimen. A technique such as fluorescence in situ hybridization
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
(FISH) would be required to conclusively demonstrate that both
photobionts occur in the same lichen thallus.
Specimens for which the photobiont ITS PCR was successful
(as determined by a single clear band on an agarose gel) but
sequencing failed because the chromatograms were unusable
due to double peaks might plausibly represent cases where
multiple algal species were present in the thallus. Likewise,
cases where isolated double peaks occurred in otherwise high-
quality chromatograms may indicate the presence of multiple
algal haplotypes in a single thallus. Further investigation
with next-generation sequencing would be required to verify
these conjectures.
Photobiont Switching Within a
Mycobiont Species
Photobiont switching within a mycobiont species can occur at
multiple phylogenetic levels: between photobiont genera (Ertz
et al., 2018;Vanˇ
curová et al., 2018), between major clades within
a photobiont genus (Blaha et al., 2006), between photobiont
species within a subgeneric clade (Magain et al., 2017, 2018;
Dal Grande et al., 2018), or between haplotypes within a
photobiont species (Gasulla et al., 2020). We found no evidence
of switching at the genus level, reaffirming that Lecanoraceae
mycobionts are strict specialists on Trebouxia (Miadlikowska
et al., 2006). Conversely, we found evidence for all three of the
intrageneric types of switching, suggesting that the specificity
of these mycobionts for particular algal species may be lower
than commonly thought. It should be emphasized, however,
that our sampling included few thalli from any one mycobiont
species and is therefore not ideal for studying photobiont
interactions within a mycobiont species. The conclusions drawn
from the instances of switching we did observe should be
considered preliminary and should be tested with a more
focused sampling.
One of the mycobiont species we found with photobionts
from two different Trebouxia clades was Lecanora rupicola
(Figure 4). Blaha et al. (2006) sequenced the photobiont for
specimens of L. rupicola from across Europe. They found that
this species associated with diverse photobionts from three
of the main clades of Trebouxia, including in some cases
multiple photobiont species at a single site. The mycobiont was
not sequenced in that study, so we do not know what role
population structure or cryptic diversity may have played in the
observed patterns, although the authors noted that there was
no correlation between variation in secondary chemistry and
photobiont identity.
All of the mycobiont species associated with photobionts from
multiple Trebouxia clades occurred on siliceous rocks in open
areas at mid-high to high elevation (Figure 4). The same was true
for many of the mycobiont species that partnered with multiple
photobionts from a single Trebouxia clade (Figure 4). This is
consistent with previous work that has shown that mycobiont–
photobiont associations in lichens are more specialized in
warmer climates (Singh et al., 2017). One hypothesis to explain
this pattern is that there are simply more photobiont species,
and therefore greater opportunity for partner switching, at
high elevation (Figure 6A). However, many of the instances
of switching we observed were related to elevation change
(Figure 5), lending support to the alternate hypothesis that
mycobionts switch between locally-adapted photobionts along
their range (Muggia et al., 2014;Dal Grande et al., 2018).
Turnover of Mycobionts and Photobiont
Communities
Our results add to a growing body of literature showing that
elevation gradients have a substantial effect on the composition of
lichen photobiont communities (Dal Grande et al., 2018;Devkota
et al., 2019;Gasulla et al., 2020;Rolshausen et al., 2020;Wagner
et al., 2020). This literature is part of a broader realization that
microbial diversity is subject to some of the same biogeographic
influences as macroscopic organisms (e.g., Nottingham et al.,
2018; but see Bryant et al., 2008). For example, various taxa of
free-living cyanobacteria and green algae in the soil may vary
in their abundance along elevation gradients (ˇ
Reháková et al.,
2011;Stewart et al., 2021). The elevation difference between the
lowest and highest sites in our dataset—nearly 5,000 m, greater
than any other study of this type—presents a unique opportunity
to study mycobiont and photobiont turnover accompanying
the transition between geographically close but ecologically
distant biomes (Figure 7). In contrast to previous studies where
turnover of mycobiont and photobiont communities occurred
at different spatial scales (Dal Grande et al., 2018;Lu et al.,
2018;Gasulla et al., 2020;Rolshausen et al., 2020), we observed
turnover in Lecanoraceae communities and their associated
Trebouxia photobionts at roughly equivalent rates (Figures 5,6).
However, because there are no mycobiont species that occur
along the entire length of the gradient, we cannot tease apart the
impact of substrate, climate, and mycobiont host on photobiont
distribution to the degree possible in other studies (e.g., Dal
Grande et al., 2018). For example, species of Myriolecis are
almost always found on calcareous rock at high elevation with
Trebouxia clade A.
Our findings on species richness and β-diversity along the
elevation gradient are consistent with previously observed
patterns and also suggest phenomena which should be
investigated in a larger sampling replicated across multiple
transects. The finding that, within each elevation bin, Trebouxia
species were less diverse than Lecanoraceae (Figure 6A) recalls
the conventional wisdom that natural communities contain
more mycobiont species than photobiont species (e.g., Singh
et al., 2019;Wagner et al., 2020). The one exception at 3,500
m, where the number of mycobiont and photobiont species are
equal, corresponds to a poorly-sampled elevation band and may
be an artifact (Figure 5).
Our results for β-diversity suggest an interesting
phenomenon: Along an elevation gradient, species turnover
occurs for both partners in a mutualistic interaction, but the
elevation thresholds at which community composition shifts
most dramatically are different for the two partners (Figure 6B).
This pattern has not been seen in similar studies of smaller
elevation gradients and fewer mycobiont taxa (e.g., Dal Grande
et al., 2018). These thresholds may be equivalent to the “symbiont
turnover zones” described by Rolshausen et al. (2020), but while
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Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
1000
2000
3000
4000
elevation
elevation
vegetation
MAT (BIO1)
MAP (BIO12)
500
1000
1500
MAP (mm)
0.5
10
15
20
25
MAT (°C)
N
600 km
22qS
20qS
18qS
16qS
14qS
12qS
10qS
70qW 68qW66qW64qW62qW60qW 58qW
vegetation
Beni savanna
Chaceño−Amazon forest
Tucumano−Boliviano forest
Yungas forest
Humid Puna high-Andean
Humid Puna subnival
Other
FIGURE 7 | Hierarchical clustering dendrogram of 19 bioclimatic variables for the sampling localities. Sampling locations and vegetation types are displayed on the
inset map. MAT, mean annual temperature (WorldClim variable BIO1). MAP, mean annual precipitation (WorldClim variable BIO12).
those authors found a turnover zone for one partner, we show
turnover zones for both partners and, crucially, these are at
widely spaced elevations. No Lecanoraceae species are found
both above and below 2,500 m, while no Trebouxia species
are found on both sides of 3,500 m. The former elevation is
the threshold at which rock begins to appear as a substrate
for Lecanoraceae, and is also the transition point between
aseasonal Yungas forest and seasonal Tucumano-Boliviano
forest (Figures 4,7). The latter elevation corresponds to the
transition from Trebouxia clades C and I to Trebouxia clades A
and S (Figure 5B); this is also the threshold at which open, rocky
habitats fully replace forest vegetation (Figure 7). Perhaps not
coincidentally, 3,500 m is also very close to a similar threshold
for woody plant ranges in the Bolivian Andes (Tello et al., 2015).
These findings suggest two alternative hypotheses. One
possibility is that mycobiont and photobiont ranges are
responding to different biotic or abiotic factors (e.g., substrate
versus temperature; rainfall versus the presence of pathogens).
Another possibility is that both thresholds are a response to the
same factor, but the two partners are responding differently. Lu
et al. (2018) found that ranges of Peltigera species and Nostoc
phylogroups did not have the same response to climate shifts
along a latitudinal gradient, but their study was at an intra-biome
scale and lacked the sharp community discontinuities we see here.
While our sampling is not adequate to distinguish between these
hypotheses, it provides guidance as to an ideal region and study
design that would have the power to resolve this question.
Phylogenetically Versus Ecologically
Designed Sampling to Study Photobiont
Communities
Kosecka et al. (2021b) examined the community of Trebouxia
photobionts along a largely identical elevation gradient in Bolivia.
That study included a four- to five-fold larger sample size
from multiple families of Trebouxia-associated Lecanoromycetes,
including some Lecanoraceae, but only dealt with the photobiont
sequences from these specimens. The results of Kosecka et al.
(2021b) in terms of Trebouxia diversity and distribution are
largely consistent with our own findings, from the broad pattern
of turnover among major clades of Trebouxia along the elevation
gradient to many of the individual Trebouxia species found. Our
study recovered several novel lineages not found by Kosecka et al.
(2021b)—A53, C35, and C36; conversely, and commensurate
with their larger sample size, Kosecka et al. (2021b) found
several putative Trebouxia species which were missing from our
sampling. Nevertheless, the close correspondence between the
two studies suggests that dense sampling of a single mycobiont
family may be sufficient to reveal regional patterns of biodiversity
in lichenized Trebouxia. This stands in contrast to the situation
with cyanolichens, where the Nostoc community will look very
different if, for example, one samples from only Collemataceae,
Peltigeraceae, or Lobariaceae in the same geographic area
(Magain et al., 2018). Studies of Trebouxia photobionts from
a single mycobiont clade are the norm in the literature, but
it has not been clear whether their results can be generalized
to represent the entire Trebouxia community of a region. Our
results suggest that the answer may be yes, as long as the focal
mycobiont clade occurs in a sufficiently diverse range of micro-
and macrohabitats.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. New sequence data generated for this study are
available on GenBank with accession numbers OK665489–
OK665671, OL603980–OL604141, OL625025–OL625113, and
OL663852–OL663919. Other data are included in the online
Supplementary Material for this article.
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fmicb-12-774839 December 14, 2021 Time: 13:52 # 16
Medeiros et al. Lecanoraceae and Trebouxia in Bolivia
AUTHOR CONTRIBUTIONS
FL, JM, and L´
S contributed to the conception and design of
the study and supervised Ph.D. students. L´
S and JM secured
funding. AF and PR-F collected the specimens and ecological
data in the field. EM and L´
S studied specimen morphology and
delimited morphospecies. EM and EC prepared DNA extractions.
IM designed primers for PCR and sequencing and generated
the figures. EM, IM, and CP-DH collected the molecular data.
IM and EM curated, validated, and analyzed the data and wrote
the original draft of the manuscript. All authors participated in
review and editing of the final manuscript and approved the
submitted version.
FUNDING
Funding for this research was provided by the National
Science Centre, Poland, under a grant to L´
S (project
no. 2016/21/B/NZ8/02463). IM was supported by a
US National Science Foundation Graduate Research
Fellowship (DGE 1644868).
ACKNOWLEDGMENTS
AF and PR-F are greatly indebted to the staff of the
Herbario Nacional de Bolivia, Instituto de Ecología, Universidad
Mayor de San Andrés, La Paz, for their cooperation and
to the Servicio Nacional de Áreas Protegidas (SERNAP) and
all protected area staff for providing permits for scientific
studies, as well as assistance and logistical support during field
work. We thank the two reviewers for their helpful feedback
on the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.774839/full#supplementary-material
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