Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): Two novel cyanobacterial constituents isolated from a tripartite lichen of headstones

Abstract

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen-fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.

Full text

BRASILONEMA LICHENOIDES SP. NOV. AND CHROOCOCCIDIOPSIS LICHENOIDES SP. NOV.
(CYANOBACTERIA): TWO NOVEL CYANOBACTERIAL CONSTITUENTS ISOLATED FROM
A TRIPARTITE LICHEN OF HEADSTONES
1
Chelsea D. Villanueva
Department of Biology, University of North Florida, Jacksonville, Florida 32224, USA
Petr Hasler, Petr Dvorak, Aloisie Poulıckova
Department of Botany, Faculty of Sciences, Palacky University Olomouc,

Slechtitelu 27, CZ-771 46 Olomouc, Czech Republic
and Dale A. Casamatta
2
Department of Biology, University of North Florida, Jacksonville, Florida 32224, USA
Cyanolichens are an assemblage of fungi and
cyanobacteria from diverse, cosmopolitan habitats.
Typically composed of a single species of
cyanobacterium, with or without another eukaryotic
alga, here we present two novel cyanobionts isolated
from an undescribed tripartite lichen. This
endolithic lichen was isolated from a granite
cemetery tombstone from Jacksonville, FL, and
contains two potentially nitrogen-fixing cyanobionts.
Employing a total evidence approach, we
characterized the cyanobionts using molecular (the
16S rDNA and ITS gene region), morphological,
and ecological data. Phylogenetic analyses revealed
two novel taxa: Brasilonema lichenoides and
Chroococcidiopsis lichenoides, both of which fell
within well-supported clades. To our knowledge,
this represents the first instance of a tripartite
lichen with two cyanobacterial and no eukaryotic
members. These types of lichens may well represent
an unexplored reservoir of cyanobacterial diversity.
The specific epithets are proposed under the
provisions of the International Code of
Nomenclature for algae, fungi, and plants.
Key index words: 16S rDNA gene; 16S-23S ITS; biodi-
versity; cyanolichen; taxonomy
Abbreviations: BLAST, basic local alignment search
tool; GPS, global positioning system; GTR, general
time reversible; ML, maximum likelihood; MP, max-
imum parsimony; OTU, operational taxonomic unit;
rDNA, ribosomal deoxyribonucleic acid
Cyanobacteria are a group of photooxygenic
prokaryotes, and among the most important primary
producers on Earth. Much of the basic biodiversity of
this group is poorly understood, and many habitats
have been only cursorily explored for their cyanobac-
terial members (Dvo
r
ak et al. 2015b). Ubiquitous in
nearly all known aquatic habitats, they are also com-
mon components of numerous terrestrial environ-
ments (e.g., Holland 1977, 
Reh
akov
a et al. 2007).
Frequently encountered in soils, cyanobacteria are
often present due to their capabilities of fixing atmo-
spheric nitrogen, resisting the harmful impacts of UV
light, and their ability to enter a quiescent state dur-
ing times of environmental stress (Adams 2000,
Flechtner et al. 2002). Cyanobacteria are often also
integral components of symbiotic relationships, such
as with mosses, angiosperms, and cycads (Rai 1990).
Lichens are composed of an algal or cyanobacterial
photobiont incorporated into the body of a fungi,
with distribution patterns often reflecting algal cli-
mate and substrate preference (Sanders 2001, Peksa
and

Skaloud 2011). A single fungal symbiont may
envelope and direct the growth of multiple photosyn-
thetic endosymbionts, while hosting bacterial sym-
bionts living on fungal surfaces and in intercellular
spaces (Grube and Berg 2009, Bjelland et al. 2011,
Muggia et al. 2013, Dal Grande et al. 2014). The fun-
gal host, or mycobiont, provides a stable habitat for
the photosynthetic member and in exchange, the
photobiont provides fixed carbon and, in the case of
cyanolichens, fixed atmospheric nitrogen (Sanders
2001). Recently, it has been noted that many com-
mon lichens also contain basidomycete yeasts,
although their role is currently under study (Spribille
et al. 2016). Cyanolichens, or lichens containing a
cyanobacterial member, are common components of
epilithic and epiphytic habitats throughout the world
(Rikkinen 2002). Epilithic lichen populations inhabit
stone surfaces, often directly on top of endolithic
populations living just within stone, but may host dis-
tinct microbial consortia (McNamara and Mitchell
2005, McNamara et al. 2006).
Cyanobacteria photobionts of lichen biocrusts are
often studied from a stone biodeterioration
1
Received 30 August 2017. Accepted 15 December 2017. First
Published Online 27 January 2018. Published Online 21 February
2018, Wiley Online Library (wileyonlinelibrary.com).
2
Author for correspondence: e-mail dcasamat@unf.edu.
Editorial Responsibility: T. Mock (Associate Editor)
J. Phycol. 54, 224–233 (2018)
©2018 Phycological Society of America
DOI: 10.1111/jpy.12621
224

perspective, less so as a source of cyanobacterial diver-
sity (Crispim and Gaylarde 2005). The cyanobacterial
member of a lichen is typically filamentous and hete-
rocyte forming (e.g., Nostoc,Calothrix), putatively con-
tributing carbon and nitrogenous compounds (Rai
and Bergman 2002). Other cyanolichens may be coc-
coid forms that fix nitrogen (e.g., Chroococcidiopsis,
Gloeocapsa), filamentous forms without nitrogen-fix-
ing capabilities (e.g., Oscillatoria,Pseudanabaena), or
involved in tripartite interactions with eukaryotic
algae (e.g., Trentepohlia; Henskens et al. 2012, P
erez-
Ortega et al. 2012). In this study, we describe two
novel cyanobacteria isolated from a tripartite lichen
containing no eukaryotic algal symbionts, a new spe-
cies of a filamentous, nitrogen-fixing cyanobacterium
(Brasilonema lichenoides), and a new coccoid species
(Chroococcidiopsis lichenoides) inhabiting granite head-
stones (Jacksonville Beach, FL, USA).
MATERIALS AND METHODS
Sampling site. Isolates were obtained from H. Warren
Smith cemetery (Jacksonville Beach, FL, USA). Six individual
headstones were sampled, which provided three to five epi-
lithic and five endolithic samples each (Fig. S1 in the Sup-
porting Information). Samples were teased from headstones
using a sterile scalpel and transported in 1.5 mL micro cen-
trifuge tubes using nondestructive, sterile tape sampling tech-
niques (Cutler et al. 2012). Irradiance was measured at each
collection site using a basic quantum meter (Apogee Instru-
ment Inc., Logan, UT, USA) and the age and type of stone,
the condition of the stone, the presence of effluents or nearby
plant growth, as well as the class and color of dominant lichen
growth forms, and any color change in growth after wetting
was recorded (Table S1 in the Supporting Information).
Culturing. Microthallus samples were used to inoculate
cultures to isolate photosymbionts (for additional images of
the lichen, including members of the consortium, see
Figs. S1 and S2 in the Supporting Information). Cyanobacte-
rial isolates were cultured in liquid Z8 medium (Staub 1961)
with the addition of 10 lL(19) fungicide (Amphotericin,
Cell Grow Virginia). Cultures were kept on a desktop, at
ambient conditions (23°C, ~12:12 h light:dark photoperiod).
In addition, cultures were grown on nitrogen-free Z8 medium
to test for potential nitrogen fixation. Microthalli were
homogenized using sterile cotton swabs and isolated on
Sabouraud medium (30 g L
1
) by directly placing the
homogenate onto plates. Thallus dissection was not possible
due to the small size.
Morphological assessment. Morphology of the strains was ana-
lyzed via light microscopy (Zeiss AxioImager, Thornwood, NY,
USA objectives EC Plan–Neofluar 409/1.3 N.A., oil immersion,
DIC; Plan–Apochromat 1009/1.4 N.A., oil immersion, DIC).
Images were taken with a high-resolution camera (AxioCam
HRc 13MPx). Pictures were processed using with Zeiss AxioVi-
sion software (version 4.9.1.). During morphological evaluation
of natural samples and strains, the following characters were
assessed: cell shape, cell dimensions, type of cell reproduction,
sheaths, and granulation of cells. Measurements were per-
formed on 100 cells of both natural and culture materials.
Molecular techniques. DNA from fungal and cyanobacterial
isolates was extracted with the PowerSoil
TM
DNA Kit from
0.25 g of culture samples (Mo Bio Laboratories Inc., Carls-
bad, CA, USA). DNA quality was checked on an ethidium
bromide stained 1.5% agarose gel.
PCR amplification of the partial 16S rDNA and the whole
16S–23S ITS was performed using primers forward 27F (50-A
GAGTTTGATCCTGGCTCAG-30) and reverse B23S (50-CTT
CGCCTCTGTGTGCCTAGG-30) previously described in Lane
(1991). To amplify the mycobiont component for identifica-
tion purposes, primers ITS1 (50-TCCGTAGGTGAACCTGCG
G-30) and ITS4 (50-TCCTCCGCTTATTGATATGC-30) were
used as described in White et al. (1990). The 50 lL PCR reac-
tion contained: 27 lL sterile water, 1 lL of each primer
(0.01 mM concentration), 20 lL PCR Master Mix (Promega,
Madison, WI, USA), and 1 lL template DNA (50 ng lL
1
)
and PCR amplification proceeded as detailed in Casamatta
et al. (2005). Amplified rDNA was cloned into pGEM
â
T Vec-
tor System I and JM-109 High Efficiency Competent Cells (Pro-
mega) and cultured using carbenicillin-infused LB media.
Plasmid DNA was purified from eight replicate transformed
competent cell colonies per isolate, using QIAprep
â
Spin Mini-
prep Kits (Qiagen, Hilden, Germany). Sequencing of cDNA
libraries from two operons of varying size was performed by
Eurofins Genomics (MWG Operon Inc., Louisville, KY, USA).
A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
was used to obtain closely related taxa. New 16S sequences
were combined with sequences from GenBank having ≥93%
sequence similarity via BLAST searches. For both phyloge-
netic trees, sequences were aligned together using the Clus-
talX web interface (Thompson et al. 1997) and manually
checked and edited using Maclade v.4.06 (Maddison and
Maddison 2000 Maddison, W. P., & Maddison, D. R. 1992.
MacClade ver. 3, Sinauer, Sunderland, MA.). The GTR+I+
gamma model was selected using MEGA7: Molecular Evolu-
tionary Genetics Analysis version 7.0 for bigger data sets
(Kumar et al. 2015). An unweighted maximum-parsimony
(MP) and maximum-likelihood (ML) analyses were carried
out using MEGA 7 (Kumar et al. 2015), and bootstrap sup-
port was obtained from 1,000 pseudoreplicate data sets.
The 16S-23S ITS region (~800 bp) was analyzed by determin-
ing secondary structure of the following conserved domains:
D1-D10helix, Box-B helix, and the V3 helix. Secondary struc-
tures of specific ITS motifs were predicted using comparative
analysis combined with confirmation in Mfold (Zuker 2003).
RESULTS
Morphological assessment. The new Brasilonema was
similar to other species, but with several distinctions.
First, this strain appeared brownish orange when
initially isolated, but subsequently lost in culture
(Fig. 1). Second, B. lichenoides cells were discoid and
lacked obvious inclusion bodies. Third, heterocytes
were rounded and never squared or rectangular.
Fourth, B. lichenoides exhibited rare heteropolar fila-
ments. Fifth, B. lichenoides possessed undulated tri-
chomes (Table S2 in the Supporting Information).
The new species of Chroococcidiopsis was morphologi-
cally similar to other taxa, but with the simultaneous
production of baeocytes (Fig. 2). Furthermore, strains
produced copious amounts of extracellular vesicles in
culture (Fig. 2).
Molecular assessment. For both new taxa, both ML
and MP trees were constructed, which yielded simi-
lar topologies. A total of 70 taxa (~1,400 bp) were
used in the construction of each tree, which was
subsequently winnowed down to a smaller set of
OTUs, including all available species sequences, for
assessment in the respective genera.
NOVEL LICHEN CYANOBIONTS 225

Brasilonema sp. nov. Both the winnowed ML and
MP trees yielded similar topologies and thus only
the ML tree is included employing all available Bra-
silonema strains plus 14 sister taxa (Fig. 3a). Brasilo-
nema is a well-supported, monophyletic clade and
the closest relative of our strain was B. roberti-lamii
(85% and 90% support, respectively), which was iso-
lated from central Mexico. The new taxon shared
between 98.5% and 98.7% sequence similarity to
the closest relatives, and 95.1% similarity with
B. bromeliae, the type (Table S3 in the Supporting
Information).
Chroococcidiopsis sp. nov. The new isolate fell
within a highly supported (91%) clade containing
other Chroococcidiopsis taxa (Fig. 3b). Specifically,
C. cubana SAG39.79, isolated from a soil sample in
Cuba, numerous C. thermalis (e.g., CCAP 1423/1
from Roman baths in the United Kingdom, SAG
42.79 from German soils), and assorted Chroococcid-
iopsis spp. (e.g., PCC8201 from mineral springs in
Cuba and CCMP2728 from Pennsylvania, USA) fell
within this clade. The closest relative to our strain
was C. thermalis PCC7203, isolated initially from soil
near Greifswald, Germany. The new taxon shared
FIG. 1. Photomicrographic plate of Brasilonema lichenoides sp. nov. (A) Natural sample of filaments ensheathed by brownish sheaths,
tolypotrichoid false branching at heterocytes (HTC), (B) freshly isolated strain exhibits a thick, colorless sheath, necridic cells (NC), in
contrast to most Brasilonema which display a purple color, (C) formation of false branching after trichome disintegration, and (D–F) hor-
mogonia formation in apical parts of filaments surrounded by widened and layered mucilaginous sheath (>3 weeks old), scale
bars =10 lm. [Color figure can be viewed at wileyonlinelibrary.com]
226 CHELSEA D. VILLANUEVA ET AL.

between 98.4 and 99.7 sequence similarity to the
closest relatives in the genus Chroococcidiopsis
(Table S4 in the Supporting Information).
ITS assessment.Brasilonema: Several sister taxa with
available ITS regions were selected to examine the
secondary folding structures of the D1-D10helix. The
new taxon, for example, was most similar to B. octoge-
narum, with an A-C side bulge off the initial stem loop
and shared the exact same terminal loop, with two
nucleotide changes midstem, constraining the side
bulge (Fig. S3, a and d in the Supporting Informa-
tion). The new taxon did not contain any tRNAs, sim-
ilar to other Brasilonema isolates, e.g., B. angustatum
HQ847566 and B. octagenarum HQ847562 (although
other Brasilonema isolates do have tRNAs). The D1-
D10length was the same as the other Brasilonema that
lacked tRNAs (67 bp), as were three other regions
(Table S5 in the Supporting Information). While the
size of the sequence for the B. angustatum with
the tRNAs was larger as it contained the sequence,
the new taxon was closer to B. octogenarum in terms of
overall length (139 vs. 138) compared to either B. an-
gustatum (126 and 129 bp, respectively).
Chroococcidiopsis: The new taxon was structurally
identical to the closely related C. thermalis
PCC7203, with the only difference being the pres-
ence of a UUU segment at the terminal tip in our
taxon and single nucleotide substitution (G to a
C) immediately above the second internal loop,
below the A- side bulge (Fig. S4, a and b in the
Supporting Information). A close relative, C. ther-
malis PCC7203 which formed a sister clade to the
majority of other Chroococcidiopsis, showed a similar
folding pattern, but with distinct differences in the
ITS region (Fig. S4c, Table S6 in the Supporting
Information). Another Chroococcidiopsis,Chroococcid-
iopsis sp. SAG2025, which fell within this clade, had
a very different structure (Fig. S4d). Two other
outgroup taxa from the sister clade (Fig. S4, e and
f) were markedly different in ITS structure, with
corresponding differences in overall conserved seg-
ment lengths (Table S6).
Description of new species.Brasilonema lichenoides Vil-
lanueva, P. Ha
sler & Casamatta sp. nov.
Description: Culture (Fig. 1): Colonies initially iso-
lated from lichens and consisting of interwoven fila-
ments that occasionally stood erect from the
substrate. Filaments straight or bent, typically nonta-
pering (very infrequently heteropolar toward the
end), false branched. Trichomes straight, bent, or
undulated inside the sheath, constricted at cross
walls. Sheaths brown-orange (fresh isolates) to color-
less (culture), distinct, firm, becoming thin and col-
orless or disappearing in culture, up to 2.5 lm
wide, seldom layered at the basal part near the false
branching. Cells flattened or barrel shaped, green
to blue-green with peripheral chromatoplasm and
central nucleoplasm, frequently granulated, nonvac-
uolated, 4.1 0.9 wide (avg. SD) 99.1 0.7 lm
long, apical cells rounded. Heterocytes common,
intercalary, occasionally flattened but typically
spherical or hemispherical, 8.4 1.2 wide 99.7 
0.9 lm long; akinetes not present. Reproduction by
hormogonia and fragmentation of trichomes using
help of necridic cells. Meristematic zones if present
usually short and located near the apical or basal
parts.
Holotype: Botany 24: Lichens and others, No. 9226
(as a dried sample), OLM (Regional Museum in
Olomouc, Czech Republic), illustration from culture
Figure 4.
Type strain: No. 168/2015, deposited at the cul-
ture Collection of Department of Botany, Palacky
University in Olomouc, Czech Republic.
Type locality: Granite tombstones from the H. War-
ren Smith Cemetery, Jacksonville Beach, Florida,
USA (GPS: 30.2890°N, 81.4071°W).
Genbank accession numbers: MF423720 (16S rRNA)
and MF423719 (ITS region)
Etymology: Name is based on habitat of isolation as
a cyanobiont of a tripartite lichen.
Habitat: Growing in consortia as a lichen on ceme-
tery tombstones.
Chroococcidiopsis lichenoides Villanueva, P. Ha
sler et
Casamatta sp. nov.
FIG. 2. Photomicrographic plate of Chroococcidiopsis lichenoides
sp. nov. Note the presence of both baeocytes and nanocytes. Cul-
tures also produced copious amounts of extracellular vesicles of
unknown function. Scale bars =10 lm. [Color figure can be
viewed at wileyonlinelibrary.com]
NOVEL LICHEN CYANOBIONTS 227

Description: Culture (Fig. 2): Thallus microscopic
to macroscopic, colonies usually spherical or
hemispherical, 31.4 4.2 (avg. SD) 926.1 
5.6 lm, aggregated into thin greenish layer.
Mucilaginous envelopes thin, firm, and colorless.
Cells variable in shape from almost spherical, oval
to irregular, 4.7 0.79 wide 93.3 0.5 lm long,
bright green or grey-green, densely, and irregularly
aggregated inside the colony into sarcinoid pack-
ages. Reproduction by growth and fragmentation
of colonies into subcolonies, by gelatinization and
splitting of envelopes and liberation of cells and
small oval or irregular baeocytes 3.1 0.62 9
2.2 0.3 lm.
Holotype: Botany 24: Lichens and others, No. 9227
(as a dried sample), OLM (Regional Museum in
Olomouc, Czech Republic), illustration from cul-
ture, Figure 5.
Type strain: No. 165/2015, deposited at the cul-
ture collection of Department of Botany, Palacky
University in Olomouc, Czech Republic.
Type locality: H. Warren Smith Cemetery, Jack-
sonville Beach, Florida, USA (GPS: 30°17020.2″N,
81°24024.9″W).
Genbank accession numbers: MF423482 (16S
rRNA) and MF423720 (ITS region)
Etymology: Name is based on habitat of isolation as
a cyanobiont of a tripartite lichen.
Habitat: Growing in consortia as a lichen on gran-
ite cemetery tombstones.
DISCUSSION
Most cyanobionts are capable of nitrogen fixation
(Rai and Bergman 2002). Cyanobionts may be fila-
mentous or unicellular, with the former employing
heterocytes and the later using micro-anaerobic
zones to fix atmospheric nitrogen. In the sampled
lichen, we note the presence of both forms, which
were each capable of growth on nitrogen-free med-
ium. However, it cannot absolutely determine if they
both fix nitrogen in situ, as has been noted elsewhere
(Rai 2002). Likewise, the potential role of associated
bacteria cannot be discount. It is important to note
that cyanobionts may exhibit phenotypic variability
between lichenized and free-living states (e.g., Casa-
matta et al. 2006), and was also noted in this study.
Brasilonema lichenoides sp. nov. is the first Brasilo-
nema species isolated from a lichen thallus. The type
species B. bromeliae is a member of the Scytonemat-
aceae isolated from subaerophytic habitats in tropical
and subtropical Brazil (Fiore et al. 2007). Several
aerophytic, epiphytic, and epilithic Brasilonema spe-
cies have been isolated from Hawaii, central Mexico,
and Brazil (Aguiar et al. 2008, Vaccarino and Johan-
sen 2012, Becerra-Absal
on et al. 2013, Rodarte et al.
FIG. 3. (A) Maximum-likelihood
(ML) tree of 16S rDNA gene
sequence data for Brasilonema
lichenoides.Numbersabovethe
nodes represent ML bootstrap
values, while numbers below are
from maximum parsimony. The
new taxon is in bold.
228 CHELSEA D. VILLANUEVA ET AL.

2014). Brasilonema lichenoides was most closely related
to B. roberti-lamii, and both strains were growing
epilithically in warm humid climates (Rodarte et al.
2014). One of the defining features of Brasilonema is
the presence of vacuole-like structures (actually free
spaces within protoplasts surrounded by thylakoids;
Fiore et al. 2007), but the new strain did not exhibit
such vacuolization in natural populations. Our strain
did exhibit both “C” and “J” shaped trichomes, similar
to our closest relative, B. roberti-lammi (Rodarte et al.
2014). While Brasilonema is described as nonattenu-
ated, our new taxon did exhibit rare heteropolarity.
Furthermore, we note that the images of B. angustatum
show a similar degree of filament heteropolarity and
J-shaped trichomes to the new taxon (Fig. 2, sensu
Vaccarino and Johansen 2012). The new strain repre-
sents a unique taxon based on ecology (photobiont),
morphology, and molecular (16S) data.
Chroococcidiopsis is a widely distributed genus of
cyanobacteria, often found in xeric habitats with high
UV light levels (Dor et al. 1991). They may be found
in freshwater, marine, and subaerial habitats (Cum-
bers and Rothschild 2014). Notoriously difficult to
phylogenetically elucidate based solely on morphologi-
cal characters (e.g., Norris and Castenholz 2006), this
genus traditionally belongs to subsection II (Pleuro-
capsales) of the cyanobacteria (Rippka et al. 2001, Wil-
motte and Herdman 2001), which includes
FIG. 3. (B) ML tree of 16S
rDNA gene sequence data for
Chroococcidiopsis lichenoides.Numbers
above the nodes represent ML
bootstrap values, while numbers
below are from maximum parsi
mony. The new taxon is in bold.
NOVEL LICHEN CYANOBIONTS 229

cyanobacteria which produce baeocytes. However,
recent work by Kom
arek et al. (2014) have transferred
this clade to the Chroococcidiopsidales ordo nov. due to
baeocyte formation coupled with cell division in three
planes. Chroococcidiopsis rarely show their typical mode
of reproduction while lichenized, even if they would
typically do so in a free living or cultured state (Friedel
and Budel 1996). The new strain clearly showed baeo-
cyte formation and division in three planes in culture
(Fig. 2). Furthermore, results of 16S rDNA sequence
data and ITS secondary folding patterns showed that
this strain fell within the “Chroococcidiopsis” sensu stricto
clade with high bootstrap support. However, the new
isolate did not match any morphological description
or ecology (e.g., tripartite lichens of headstones in
Florida) of any currently circumscribed species. B€udel
and Hennsen (1983) note similar strains isolated as
phycobionts from the lichen family Lichinaceae, but
their isolates were of different sizes, different baeocyte
arrangements, or different sheath production.
Chroococcidiopsis lichenoides forms individual spherical
colonies or clusters resembling sarcinoid packages.
Colonies fragment and continue their growth or pro-
duce small spherical, oval, or irregular baeocytes.
Shape, colony structure, and symbiotic mode of life
are specific for this species.
Species concepts within the cyanobacteria are sub-
ject to much debate (Dvo
r
ak et al. 2015b). Given the
dearth of morphological features from which to
choose, coupled with issues related to both phenotypic
plasticity and cryptic diversity, describing and elucidat-
ing cyanobacterial species may be problematic (Dvo
r
ak
et al. 2015a,b, Dvo
r
ak et al. 2017, Struneckyetal.
2017). The monophyletic species concept sensu Johan-
sen and Casamatta (2005) has been proposed as the
standard for cyanobacterial systematics (e.g., Kom
arek
2013), which requires the description of an autapo-
morphy to test phylogenetic hypotheses. The new
strain was only 98% similar to Chroococcidiopsis thermalis,
the closest relative based on 16S sequence data. It is
proposed that the morphological disjunction, unique
ecological setting, 16S sequence dissimilarity, and dif-
ference in ITS sequence justify the erection of a new
taxon.
All lichens contain at least a single photobiont, and
only ~10% of all lichens contain cyanobacteria as their
primary photobiont (Friedel and Budel 1996). The
majority of tripartite lichens (those with two photosyn-
thetic members) contain a single cyanobacterial pho-
tobiont and a green alga (Tschermak-Woess 1988). In
these cases, the cyanobacterial component is seques-
tered into a separate region of the thallus, the
FIG. 4. Designated holotype line drawing of Brasilonema lichenoides sp. nov. Scale bar =10 lm.
230 CHELSEA D. VILLANUEVA ET AL.

cephalodia. The novel cyanobionts were not separated
into cephalodia, but rather loosely organized into the
thallus of the endolithic lichen.
Headstones represent an interesting substrate to
explore algal diversity and colonization. The sam-
pled headstones were all exposed to the environ-
ment >50 years ago, and provide a stable (e.g., no
history of headstone cleaning or preservation) envi-
ronment for primary colonization. This is also an
intriguing habitat to examine patterns of long-dis-
tance cyanobacterial dispersal. Many lichens contain
photobionts with cosmopolitan distribution (Chua
et al. 2012), and some cyanolichen guilds share sim-
ilar cyanobionts (Rikkinen 2002). However, it
remains unclear if the new taxa have broad or lim-
ited distribution as more sampling is needed. Tomb-
stones are also interesting habitats to answer
questions relating to ecological succession and facili-
tation of microbial communities given their preva-
lence, cultural significance, and stable nature.
The lead author thanks Gordon Rakita for an introduction to
the exciting world of cemetery tombstone preservation. The
authors gratefully acknowledge financial support from the
Department of Biology (UNF), the Coastal Biology Flagship
program (UNF), and the Internal Grant Agency of Palacky
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Supporting Information
Additional Supporting Information may be
found in the online version of this article at the
publisher’s web site:
Figure S1. Close-up photograph of a represen-
tative headstone. (A) Arrow denotes tripartite
endolithic cyanolichen growth, protruding from
stone crevices. (B) A close-up of dried tripartite
endolithic lichen samples.
Figure S2. Photomicrographs of fresh material
from the tripartite lichen. (A) 4009image of
endolithic lichen sample with colonies of Chroococ-
cidiopsis (cc), filaments of Brasilonema (b), and
brown, pigmented fungi (f). (B) Close-up
(1,0009) of the members of the lichen after
being in liquid Z-8 medium for >30 d (note, the
Brasilonema filaments have lost their coloration
and the fungi appear associated with the fila-
ments and appear colorless at this point).
Figure S3. D-stem of the 16S-23S ITS region for
Brasilonema lichenoides and the closest taxa contain-
ing available ITS data. (A) B. lichenoides CDV
clone 2, (B) B. angustatum HA4787-MV1 B2/p1h,
(C) B. angustatum HA4787-MV1 B2/p1f, (D) B. oc-
tagenarum HA4786-MV1 B7A/p4.
Figure S4. D-stem of the 16S-23S ITS region for
Chroococcidiopsis lichenoides and taxa that are phylo-
genetically related. (A) C. epilithica, (B) C. ther-
malis PCC7203, (C) C. sp. SAG2025, (D) C. sp.
UFS-A4UI-NPMV4-B4 clone B4.
Table S1. Relevant physical data from cemetery
tombstone sample collections.
232 CHELSEA D. VILLANUEVA ET AL.

Table S2. Comparative morphology of Brasilo-
nema lichenoides and other members of the genus
Brasilonema.
Table S3. Similarity matrix based on the 16S
rRNA gene sequence data from Brasilonema liche-
noides sp. nov. and sister taxa.
Table S4. Similarity matrix based on the 16S
rRNA gene sequence data from Chroococcidiopsis
lichenoides and sister taxa.
Table S5. Comparing nucleotide lengths of
conserved ITS domains for Brasilonema lichenoides
sp. nov. and closest relatives that have available
ITS data.
Table S6. Comparing conserved ITS domains
for Chroococcidiopsis lichenoides sp. nov. and closest
relatives that have available ITS data.
NOVEL LICHEN CYANOBIONTS 233