Unbiased analyses of ITS folding motifs in a taxonomically confusing lineage: Anagnostidinema visiae sp. nov. (CYANOBACTERIA)

Abstract

Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in nearly every known ecosystem. Recent investigations around the world have recovered vast amounts of novel biodiversity in seldom sampled habitats. One phylogenetically significant character, the secondary folding structures of the 16S-23S ITS rDNA region, has allowed an unprecedented capacity to erect new species. However, two questions arise: is this feature as informative as is proposed, and how do we best employ these features? Submerged sinkholes with oxygen-poor, sulfur-rich ground water in Lake Huron (USA) contain microbial mats dominated by both oxygenic and anoxygenic cyanobacteria. We sought to document some of this unique cyanobacterial diversity. Using culture-based investigations, we recovered 45 strains, of which 23 were analyzed employing 16S-23S rDNA sequences, ITS folding patterns, ecology, and morphology. With scant morphological discontinuities and nebulous 16S rDNA gene sequence divergence, ITS folding patterns were effective at articulating cryptic biodiversity. However, we would have missed these features had we not folded all the available motifs from the strains, including those with highly similar 16S rDNA gene sequences. If we had relied solely on morphological or 16S rDNA gene data, then we might well have missed the diversity of Anagnostidinema. Thus, in order to avoid conformation basis, which is potentially common when employing ITS structures, we advocate clustering strains based on ITS rDNA region patterns independently and comparing them back to 16S rDNA gene phylogenies. Using a total evidence approach, we erected a new taxon according to the International Code of Nomenclature for Algae, Fungi, and Plants: Anagnostidinema visiae.

Full text

Journal of Phycology. 2023; 00:1 –16. wileyonlinelibrary.com/journal/jpy
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© 2023 Phycological Society of A merica.
RESEARCH ARTICLE
Unbiased analyses of ITS folding motifs in a taxonomically
confusing lineage: Anagnostidinema visiae sp. nov.
(cyanobacteria)
Callahan A.McGovern1 | Alyson R.Norwich1 | Aimee L.Thomas1 |
Sarah E.Hamsher2,3 | Bopaiah A.Biddanda3 | Anthony D.Weinke3 |
Dale A.Casamatta1
Received: 17 June 2022
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Accepted: 24 March 2023
DO I: 1 0.1111 / jp y.1333 7
Abbreviations: BLAST, basic local alignment search tool; ICN, International Code of Nomenclature for Algae, Fungi, and Plants; ITS, internal transcribed
spacer; ML, maximum likelihood; MP, maximum parsimony.
1Departm ent of Biology, Universit y of
North Florida, Jacksonvil le, Florida, USA
2Department of Biology, Grand Valley
State University, Allendale, Michigan, USA
3Robert B. Annis Water Resources
Institute, Grand Valley State University,
Muskegon, Michigan, USA
Correspondence
Dale A. Casamatta, Department of
Biolog y, University of North Florida,
Jacksonv ille, Flori da, USA.
Email: dcasamat@unf.edu
Funding information
National Science Foundation, Grant/
Award Number: 2046958; NASA Michigan
Space Gra nt Consor tium, Grant/Award
Number: NNX15AJ20H
Editor: J.L. Collier
Abstract
Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in
nearly every known ecosystem. Recent investigations around the world have
recovered vast amounts of novel biodiversity in seldom sampled habitats.
One phylogenetically significant character, the secondary folding structures
of the 16S– 23S ITS rDNA region, has allowed an unprecedented capacity to
erect new species. However, two questions arise: Is this feature as informative
as is proposed, and how do we best employ these features? Submerged sink-
holes with oxygen- poor, sulfur- rich ground water in Lake Huron (USA) contain
microbial mats dominated by both oxygenic and anoxygenic cyanobacteria.
We sought to document some of this unique cyanobacterial diversity. Using
culture- based investigations, we recovered 45 strains, of which 23 were ana-
lyzed employing 16S– 23S rDNA sequences, ITS folding patterns, ecology,
and morphology. With scant morphological discontinuities and nebulous 16S
rDNA gene sequence divergence, ITS folding patterns were effective at ar-
ticulating cryptic biodiversity. However, we would have missed these features
had we not folded all the available motifs from the strains, including those with
highly similar 16S rDNA gene sequences. If we had relied solely on morpho-
logical or 16S rDNA gene data, then we might well have missed the diversity
of Anagnostidinema. Thus, in order to avoid conformation basis, which is
potentially common when employing ITS structures, we advocate clustering
strains based on ITS rDNA region patterns independently and comparing
them back to 16S rDNA gene phylogenies. Using a total evidence approach,
we erected a new taxon according to the International Code of Nomenclature
for Algae, Fungi, and Plants: Anagnostidinema visiae.
KEYWORDS
16S rDNA, ITS folding, microbial mats, secondary structure, systematics

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MCGOVERN et al.
INTRODUCTION
Cyanobacteria are photo- oxygenic bacteria that are
intimately associated with the major biogeochemical
cycles (Stal, 2007). Cyanobacteria are also among
the oldest lineages of bacteria with fossil records dat-
ing back over 3 billion years ago (BYA; Schopf,2006).
Profound ecosystem- level engineers, cyanobacteria
produced oxygen leading to the Great Oxidative Event
2.8 BYA (Dvořák et al.,2014).
The last decade has seen an explosion of newly
erected taxa based on molecular (16S rDNA gene
and 16S– 23S ITS rDNA region) data, coupled with
revisions and articulation of an accepted species
concept (Johansen & Casamatta,2005). This has al-
lowed the erection of many new genera with clear
morphological apomorphies (e.g., Nodosilinea) with
concurrent transferring of previously described species
(Perkerson et al.,2011). Strunecky et al.(2017 ) erected
Anagnostidinema and subsequently transferred some
taxa into this genus based on morphology. However,
this led to a potential issue: Are those transferred spe-
cies, previously erected based solely on morphology,
truly members of the new genus? Cyanobacteria are
renown for the difficulty in assessing taxonomy due to
issues arising from phenotypic plasticity, lack of mor-
phological variability, and cryptic diversity (Casamatta
et al.,2005). Thus, caution is warranted when assign-
ing new taxonomy to previously described taxa without
a total evidence approach (e.g., morphological, ITS,
and molecular data sets).
Extant microbial mats in Lake Huron's Middle Island
Sinkhole (MIS) are composed of cyanobacteria, diatoms,
and chemosynthetic sulfur- oxidizing bacteria that cover
the floor and walls of the submerged sinkhole (Biddanda
et al.,2 012; Nold, Pangborn, et al.,2010; Nold, Zajack,
& Biddanda,2010; Voorhies et al.,2012). Previous work
on the composition of the primary producer community
in the upper layers of the MIS mat ecosystem revealed
relatively depauperate cyanobacterial types correspond-
ing to the form- genera "Phormidium" and "Oscillatoria"
(e.g., Biddanda et al., 2015; Grim et al., 2021; Snider
et al., 2017 ). The prevailing consensus has been that
the sinkhole mat cyanobacteria are functionally versatile,
but with low genetic and phenotypic diversity (Biddanda
et al.,2012; Nold, Pangborn, et al.,2010; Nold, Zajack, &
Biddanda,2010; Voorhies et al.,2012). However, these
lineages (i.e., Phormidium and Oscillatoria) are pheno-
typically plastic, polyphyletic, and replete with cryptic
taxa— potentially masking considerable taxonomic di-
versity (Casamatta et al.,2012). Moreover, these earlier
studies of Lake Huron's sinkhole mat communities have
utilized next- generation sequencing technology (e.g.,
Kinsman- Costello et al., 2 017; Voorhies et al.,2012) or
clone libraries (e.g., Nold, Pangborn, et al.,2010; Nold,
Zajack, & Biddanda, 2010) to examine the mat com-
munity diversity, but none have isolated, cultured, and
characterized these communities morphologically and
molecularly.
We isolated and sequenced 23 unique strains from
these communities that fell within the recently erected
genus Anagnostidinema (Strunecky et al., 2017).
Anagnostidinema represents a genus whose members
are distinguished from Geitlerinema by a lack of capitate
apical cells, and recent phylogenetic transfers are based
mainly on morphology (Strunecky et al.,2017). Of the 23
strains employed in this study, three exhibited a morpho-
logical variation: the occasional presence of a thin sheath
in culture. In addition, those strains also clustered to-
gether in 16S rDNA gene phylogenies, but with only lim-
ited sequence divergence (>99% sequence similarity).
An additional character set, the folding patterns of the
three predominant structures in the cyanobacterial 16S–
23S ITS rDNA region (the V3, Box B, and D1– D1′ motifs)
revealed the same patterns. However, we only recov-
ered these clusters when examining a rather large set of
strains, including folding all available ITS rDNA regions.
Using a polyphasic approach and the blind ITS structure
clustering method, we propose the erection of a new
taxon of Anagnostidinema according to the International
Code of Nomenclature for Algae, Fungi, and Plants.
MATERIALS AND METHODS
Isolates
Microbial samples were collected from benthic com-
munities in Lake Huron in 2019 (Figure1). Samples
were kept under low- light conditions at ambient tem-
peratures in a cooler during transport to the laboratory.
Once in the laboratory, the samples were grown on Z- 8
medium (Rippka et al.,1979) on a desktop, at ambient
conditions (23°C, ~12:12 h light:dark photoperiod).
Morphological observation
Morphology of exponentially growing cultures of the 23
strains from which 16S rDNA gene sequenc e data was ob-
tained was analyzed via light microscopy (Nikon Eclipse
Ni with DIC). Images from exponential and stationary-
phase cultures were taken with a high- resolution cam-
era (Nikon digital sight DS- U3). Pictures were processed
using with Nikon NIS Elements version 4.51.00. During
morphological evaluation of strains, the following charac-
ters were assessed: cell shape, cell dimensions, type of
cell reproduction, sheaths, and granulation of cells.
Molecular techniques
Cells were suspended in DI H2O, vortexed, and
placed into −20°C for 30 min. The lysed cells were
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A. VISI AE ITS UTILITY
then centrifuged, and the supernatant containing DNA
was collected. Direct PCR amplification of the partial
16S rDNA gene and the whole 16S– 23S ITS rDNA
region was performed on isolated strains (Gaylarde
et al., 2004) using primers CYA8F and CYAB23R
(Neilan et al., 1997). The 50 μL PCR reaction con-
tained: 27 μL DNA containing supernatant, 0.5 μL of
each primer (0.01 mM concentration), and 22 μL PCR
Master Mix (Promega). PCR amplification proceeded
as detailed in Casamatta et al. (2005). The following
thermocycler parameters were used for amplification:
95°C for 5 min, followed by 35 cycles of 95°C for 1 min,
57°C for 45 s, 72°C for 4 min, and a final extension at
72°C for 10 min. PCR products were cleaned using
PureLink Quick PCR Purification Kit (Invitrogen) ac-
cording to manufacturer's protocols. Purified samples
were subsequently sequenced via Eurofins Genomics.
Phylogenetic analysis
A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.
cgi) was used to obtain closely related taxa. Our re-
covered 16S rDNA gene sequences were combined
with sequences from GenBank having ≥93% sequence
similarity via BLAST searches and additional cyano-
bacterial sequences from related genera. For the larger
16S rDNA gene tree, Gloeobacter violaceus PCC 7421
served as the outgroup, and Geitlerinema splendidum
PSE0519C for the focused tree. For all phylogenetic
trees, forward sequences were aligned using CLUSTAL
W Multiple Sequence Alignment Program (Thompson
et al., 1997). The maximum likelihood method and
Kimura 2- parameter model were selected using MEGA7:
Molecular Evolutionary Genetics Analysis version
7 (Kimura , 1980; Kumar et al.,2018). An unweighted
maximum parsimony (MP) and maximum likelihood
(ML) analyses were carried out using MEGAX (Kumar
et al.2018), and bootstrap support was obtained from
1000 pseudo- replicate data sets. The same parame-
ters were used in the construction of the homologous
16S– 23S ITS rDNA region (~500 bp) trees (FigureS1
in the Supporting Information). Using 16S gene data, a
Bayesian Inference Analysis was computed in MrBayes
3.2.4 (Ronquist & Huelsenbeck,2003) using two runs
of four Markov chains for 5 million generations, sam-
pling every 1000 generations. The initial 25% of gener-
ated trees were discarded as burn- in.
Homologous 16S– 23S ITS rDNA regions (~500 bp)
were further analyzed by determining the secondary
structure of the following conserved domains: D1- D1′
stem, Box- B helix and V3 region. Initially, all homologous
ITS rDNA structures were folded in Mfold (Zuker,2003)
with temperature set to default and draw mode at untangle
with loop fix. Next, all names/identifiers (with the excep-
tion of accession numbers) were removed. Gross second-
ary structures were visually assessed, clustered based on
features, then taxonomy re- assigned. Lastly, the cluster-
ing was compared to the 16S rDNA gene phylogeny.
RESULTS
Phylogenetic analyses
Anagnostidinema is a highly supported genus (1/99)
based on 16S rDNA gene phylogeny (Figure2). Our 23
sequenced strains fell within a cluster of other members
of Anagnostidinema (Figure3). Some of our new strains
(A. visiae LHM- M/L/U) fell within a modestly supported
FIGURE 1 Benthic microbial mat images from the Middle Island Sinkhole, Lake Huron. (a) A diver obser ves the purple cyanobacterial
mats covering karst boulders in the Sinkhole at ~15 m depth (Note: The diver and mat are in colder and denser groundwater, and the
sharp transition zone with the overlying warmer and lighter lake water is visible). (b) Diver view of purple cyanobacterial mats covering
the sinkhole bottom at ~23 m (Note: The finger like projection is due to the mat layer being buoyed up by gasses generated beneath the
mats in the sediments). Photo Credit: NOAA Thunder Bay National Marine Sanctuary, Alpena, Michigan.
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MCGOVERN et al.
cluster (0.905) and sister to the type, A. pseudacutissi-
mum CCALA 150 (Figure3). Since 20 of the 23 strains
were genetically identical, we used LHM- Y/X as repre-
sentatives of the sequenced strains in Figure3. All non- -
M/L/U strains fell within A. pseudacutissimum sensu
lato (LHM- Y/X), with strains identified as "Geitlerinema,"
"Leptolyngbya," etc., likely the result of misidentification
(Figure3).
ITS rDNA region analysis
We folded all ITS rDNA region motifs available via
GenBank and for all 23 of our strains (FigureS2 in the
Supporting Information).
Recovered ITS rDNA region motifs of all strains
of our newly identified Anagnostidinema (LHM-
M, LHM- U, LHM- L) were identical to each other for
FIGURE 2 Maximum Likelihood
tree using 16S rDNA gene sequences.
Node support values are: BPP, Bayesian
posterior probabilities (top); MLbs,
bootstrap values for ML (bottom left);
MPbs, bootstrap values for MP (bottom
right). Nodes with the highest support
values (BPP of 1.0 and MLbs and MPbs of
100%) marked with asterisks.
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A. VISI AE ITS UTILITY
all three motifs (FiguresS2– S4 in the Supporting
Information) and thus provide a distinction from all
other Anagnostidinema. For this study, all ITS rDNA
regions employed were homologous (e.g., operons
containing two tRNA's: tRNAIle and tRNAAla). All of our
23 strains contained both tRNAs; we observed only a
single, completely sequenced Anagnostidinema with
a single tRNA (“Geitlerinema” sp. LD24, accession
KT315932), which was not included in our analyses.
The D1– D1´ helices revealed similar patterns to the
16S rDNA gene sequence data, with the three strains
in question identical among themselves and all other
FIGURE 3 Maximum Likelihood tree using 16S rDNA gene sequences, with our new strains bolded. Node support values are (top and
bottom): BPP, Bayesian posterior probabilities and MLbs, bootstrap values for ML. Strains designated with quotation marks (“ ”) indicate
names taken from GenBank that likely do not reflect actual phylogenetic affiliation. Colored vertical lines correspond to Box B, V3, and
D1– D1'. Horizontal lines demarcate clusters of strains with all three identical ITS motifs.
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MCGOVERN et al.
A. pseudacutissimum sensu stricto (60 in total, with
representative forms in Figure4). The Box B region
showed similar patterns (60 in total, with represen-
tative forms in Figure5). It must be noted that while
some ITS rDNA gene motifs had identical topologies,
the actual sequence data (e.g., the nucleotides that
created the structures) were variable (Figure5). For
the V3 motif, not all the strains from GenBank used in
the other folding endeavors possessed this region (15
in total). Three of our strains (LHM- M, LHM- U, LHM-
L) possessed a 14 bp insert, altering the confirma-
tion of the structure (Table1, Figure6). The other 14
strains containing a V3 region did not have this 14 bp
insert. A phylogenetic tree using homologous, com-
plete ITS rDNA region data from our strains and those
available on GenBank revealed a similar topology to
trees generated using 16S rDNA gene sequence data
(FigureS5).
16S rDNA gene similarity matrix
The 16S rDNA gene similarity matrix revealed little
overall sequence divergence within or among strains
of Anagnostidinema (Table2). Our three strains were
99.99%– 100% similar to each other and >99% similar
to all other published strains. The remaining strains
of Anagnostidinema recovered were ca. 99% similar.
Although this is a remarkably high degree of similarity,
it must be noted that even A. amphibium HA4216- MV1
showed little sequence dissimilarity, but it fell far out-
side of our clade in the phylogenetic analyses.
Dissimilarity matrix
Calculated ITS rDNA region sequence p- distances
showed Anagnostidinema visiae strains were dissimilar
FIGURE 4 D1– D1′ stems from all available Anagnostidinema grouped by structural similarity.
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A. VISI AE ITS UTILITY
to the type, A. pseudacutissimum CCALA150 (4.0).
Within- species dissimilarity for the three strains of the
newly proposed Anagnostidinema was 0.00 (Table3).
Morphological observations
Our Anagnostidinema strains were similar to others in
the genus but with one potential distinction: the pres-
ence of slight sheaths in older, stationary- phase cul-
tures (Table4).
Anagnostidinema visiae C.A. McGovern & D.A.
Casamatta, sp. nov. (Figure7).
Description
Colonies spreading, olive green to blue green (gray
green in older cultures), penetrating the agar. Cultured
trichomes mainly straight, with occasional curves,
blue green to olive green. Sheathes occasionally
present depending on culture age. Trichomes mainly
growing in parallel, occasionally entangled, and
rarely coiled, forming thin mats. Slightly constricted
at cross- walls, 1.5– 2.2 μm wide, slightly attenuated
and bent at the ends. Cells olive green to blue green,
sometimes with granules at cross- walls, 2– 3 times
longer than wide (1.7– 3.5 μm long). Apical cells in
established cultures elongated, conical, sometimes
bent or hooked, non- calyptrate, necritic cells not
observed.
Holotype here designated
OL 100180, deposited in the culture collection of the
Palacký University in Olomouc as a preserved sample
of Anagnostidinema visiae LHM- M.
FIGURE 5 Box B helices from all available Anagnostidinema grouped by structural similarity.
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MCGOVERN et al.
TABLE 1 ITS rDNA region motif bp characteristics.
Sample Leader D1D1
Spacer + D 2 +
spacer D3 + spac er
tRNA Ile
gene
Spacer + V2 +
spacer
tRNA
ala gene Spacer Box- B + spacer
Box
AD4 + space r
V3 + ITS
end
Anagnostidinema
LHM- U
751 37 12 74 18 73 13 46 11 28 65
Anagnostidinema
LHM- L
751 37 12 74 18 73 13 46 11 28 65
Anagnostidinema
LHM- M
751 37 12 74 18 73 13 46 11 28 65
A. sp. (other 17 LHM
strains)a
751 38 12 74 18 73 13 46 11 28 51
A. sp. CHAB 751 38 12 74 18 73 13 46 11 28 51
A. carotinosum AICB 37 751 38 12 74 18 73 13 46 11 28 51
A. pseudacutissimum
CCALA 151
751 39 12 74 18 73 13 45 11 28 51
A. pseudacutissimum
JR16
751 38 12 74 18 73 13 46 11 28 51
A. pseudacutissimum
CCALA 150 TYPE
751 38 12 74 18 73 13 46 11 28 51
A. pseudacutissimum
M2
751 38 12 74 18 73 13 46 11 28 51
a All other 17 sequenced strains of Anagnostidinema LHM had identical ITS regions.
The bolded texts are our samples a nd the typ e of the spec ies.
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A. VISI AE ITS UTILITY
Etymology
The specific epithet (visiae) named in honor of Morgan
L. Vis, an American phycologist and mentor to two of
the authors.
Type locality
Benthic mat at 23 m depth from Lake Huron (GPS
45.19838° N, 83.32756° W), collected June 12, 2019, by
Phil Hartmeyer and Wayne Lusardi.
Molecular characterization
Nucleotide sequences of the 16S– 23S rDNA re-
gion transcript from strain Anagnostidinema vi-
siae LHM- M deposited in GenBank with accession
#ON258648.
Diagnosis
Differs from all other Anagnostidinema in the occa-
sional presence of a thin, clear sheath in culture, by
habitat (benthic, freshwater mats from sulfur- rich en-
vironments), and by a unique ITS rDNA region (14 bp
insert in V3 region).
DISCUSSION
Articulation of the biodiversity of cyanobacteria has
experienced a renaissance due to modern mo-
lecular methods (Dvořák et al., 2021; Johansen &
Casamatta,2005). Numerous new lineages (and spe-
cies) have been proposed from both previously, seldom
sampled and more traditional (e.g., planktonic) habitats
by employing the 16S rDNA gene (Zammit,2018). While
this approach has allowed an unprecedented explosion
in identifying new taxa, another trend has appeared:
FIGURE 6 V3 structures from all available Anagnostidinema grouped by structural similarity.
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MCGOVERN et al.
TABLE 2 16S rDNA region genetic similarity matrix among Anagnostidinema members.
12345678910 11 12 13 14
1Anagnostidinema
LHM- L
–
2Anagnostidinema
LHM- M
100 –
3Anagnostidinema
LHM- U
100 10 0 –
4G. carotinosum AICB37 99.99 100 100 –
5A. pseudacutissimum
CCALA 142
99.99 99.99 99.99 100 –
6A. amphibium
HA4 216MV1
99.98 99.98 99.98 99.97 99.97 –
7G. sp. LD24 99.99 100 100 99.99 100 99.98 –
8A. pseudacutissimum
CCALA 151
99.99 99.99 99.99 100 100 99.97 100 –
9A. pseudacutissimum
CCALA 150 TYPE
100 10 0 100 10 0 99.99 99.98 99.99 99.99 –
10 A. pseudacutissimum
M2
100 10 0 100 10 0 99.99 99.98 99.99 99.99 100 –
11 A. carotinosum MK80 99.97 99.97 99.97 99.97 99.97 99.98 99.97 99.97 99.97 99.96 –
12 G. sp. CHAB 100 10 0 100 99.99 99.99 99.98 99.99 99.99 100 10 0 99.97 –
13 A. LH M- X 99.99 99.99 99.99 100 100 99.97 100 100 99.99 99.99 99.97 99.99 –
14 A. L HM- Y 99.99 99.99 99.99 100 100 99.97 100 10 0 99.99 99.99 99.97 99.99 100 –
Note: Our new taxa are bolded.
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A. VISI AE ITS UTILITY
TABLE 3 ITS rDNA region genetic dissimilarity matrix among Anagnostidinema members.
Strain 12345678 9 10 11 12 13 14
1Anagnostidinema
LHM- U
–
2Anagnostidinema
LHM- M
0 –
3Anagnostidinema
LHM- L
0 0 –
4A. LH M- X 5.9 5.9 5.9 –
5A. L HM- Y 5.9 5.9 5.9 0 –
6A. sp. CHAB 4443.9 3.9 –
7A. pseudacutissimum
ladakh27
5.1 5 .1 5 .1 2.1 2.1 3.2 –
8A. pseudacutissimum
LD27
5.1 5 .1 5 .1 2.1 2.1 3.2 0 –
9A. sp. LD9 5.1 5 .1 5 .1 2.1 2.1 3.2 0 0 –
10 A. sp. Sai0 01 5.5 5.5 5.5 0.7 0.7 3.6 2.1 2.1 2 .1 –
11 A. carotinosum AICB 37 4.7 4.7 4.7 3.9 3.9 5 .1 3.6 3.6 3.6 3.2 –
12 A. pseudacutissimum
CCALA 151
5.5 5.5 5.5 0.7 0.7 3.6 2.1 2.1 2 .1 03.2 –
13 A. pseudacutissimum
JR16
6.3 6.3 6.3 2.5 2.5 5.1 1.7 1.7 1.7 2.5 3.9 2.5 –
14 A. pseudacutissimum
CCALA 150 TYPE
4443.2 3.2 4.3 1.7 1.7 1.7 3.2 1.7 3.2 2 .1 –
15 A. pseudacutissimum
M2
3.9 3.9 3.9 3.9 3.9 5.1 3.6 3.6 3.6 3.2 1.4 3.2 3.9 1.7
Note: Our new taxa are bolded.
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MCGOVERN et al.
TABLE 4 Morphological and ecological comparison of Anagnostidinema taxa.
Tax a Filaments Constrictions Granulation
Filament width
(μm) Sheath Habitat
A. pseudacutissimum
TYPE
Thin, straight or slightly bent Occasional and slight Many carotenoid granules
at poles
1.3– 2.2 Absent Freshwater, at littoral
of lakes, flooded
meadows, thermal
springs
A. splendidum Slightly bent and entangled,
sometimes coiled
Slight Translucent cyanophycin
granules at poles
2– 2.3 Absent Shallow stagnant
waters and wet
rocks
A. carotinosum Nearly straight None Several carotenoid
granules
1.8 – 2 Absent Freshwater, littoral
of lake
A. ex ile Thin, straight, slightly
attenuated at ends
Slight Fine granules at
cross- walls
2.5– 3 Absent Freshwater, peaty
waters
A. acutissimum Straight with slight attenuation
at ends
None or inconspicuous Prominent cyanophycin
granules
1.5– 2.5 Absent Freshwater, benthic
A. ionicum Straight and flexible None Fine cyanophycin granules 1.5 Absent Freshwater,
waterfalls, rice
fields
A. tenue Straight with attenuated ends None Refractive granules 1.2 – 1.5 Absent Freshwater, planktic,
benthic
Anagnostidinema LHM- L,
LHM- M, LHM- U
Mostly straight, slightly bent Slight Translucent granules at
poles
1.5 – 2. 2 Occasional, thin,
clear
Benthic mats, Lake
Huron
Note: The bolded texts are our samples and the type of the spec ies.
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13
A. VISI AE ITS UTILITY
taxonomic “noise.” By this, we mean that not all 16S
rDNA gene data is of comparable “quality,” and many
sequences on GenBank are only small fragments of
the 16S rDNA gene. Further, many times the accom-
panying taxonomic descriptors (e.g., "Oscillatoria") may
not actually represent the strain in question. Likewise,
the 16S rDNA gene lacks resolution at the species level
(Johansen & Casamatta, 2005; Komárek, 2016). To
create more discrimination for novel taxa, researchers
have turned to the folding motifs in the 16S– 23S ITS
rDNA region, in particular the D1– D1′, V3, and Box B
structures (Boyer et al.,2002; Cai et al.,2020; Iteman
et al., 2000). Numerous new taxa have been pro-
posed due to apomorphies or discontinuities in these
regions and, in some cases, concurrent transfer of
previously described species (e.g., Nodosilinea hunan-
ensis sensu, Cai et al.,2022; Oculatella coburnii sensu
Osorio- Santos et al.,2014; Koksharova & Wolk,2002).
Although an admirable endeavor, questions arise: How
variable are these structures themselves? What is the
background level of variability? What is the significant
variability of these structures? Can (should) we feel
emboldened to, carte blanche, transfer cyanobacterial
species based on morphological and/or ITS rDNA re-
gion structures without articulating this variability?
To examine these questions, we present our newly
described species: Anagnostidinema visiae. Although
p- distances >7.00 are considered good evidence that
two strains are separate species in lieu of any diag-
nostic features (Erwin & Thacker,2008; Osorio- Santos
et al.,2014), we note several lines of evidence to support
erecting a new taxon. First, morphologically our taxon
exhibited a potentially plastic feature: a thin, occasional
sheath in culture. Second, ecologically A . visiae origi-
nates from a unique habitat: benthic, sulfide- rich sink-
holes. Third, our new species contained a unique 14 bp
insert in the V3 motif of the ITS rDNA region. Likewise,
ITS rDNA region dissimilarity shows that A. visiae is
between 3.9% and 6.3% dissimilar from the other
identified taxa in the genus. Interestingly, this work
re- emphasizes a potentially confounding note when
constructing phylogenies using only the 16S rDNA
gene (Cai et al.,2022; Osorio- Santos et al.,2014). Our
strains, and indeed all strains in the genus for which we
had sequence data, shared remarkedly high 16S rDNA
gene similarity (99.9%– 100%, Table2). Thus, caution
when making taxonomic decisions is always warranted
as in this case the 16S rDNA gene is excellent for
placement into the genus, but rather poor at species
level discrimination.
We were able to isolate a sizable number of strains
(45) and sequenced the 16S– 23S rDNA region transcript
for 23 exemplars. In addition, we folded all available
ITS rDNA region motifs for our strains and all available
Anagnostidinema from GenBank. What we noted was
that the 16S rDNA gene tree topology matched very
well with ITS rDNA region folding but differed among
the three motifs. The Box B and D1– D1′ structures were
phylogenetically informative and matched clustering ev-
idenced by 16S sequences (Figure3). The V3 region
was highly conserved for all Anagnostidinema except
for our three strains, which showed a stark difference
in both bp length (a 14 bp insert) and confirmation
(Table3, Figure6). While an excellent character for our
taxon, and an excellent apomorphy, this does beg some
questions (Vaccarino & Johansen,2012).
First, are all ITS rDNA region motifs of equal phylo-
genetic weight? In our case, the V3 was different, but
does this hold for all cyanobacteria? What of the vari-
ability within a species versus a genus versus a family?
Does a nucleotide substitution in a loop region (e.g.,
which does not lead to a different structure), have equal
weight to an alteration in a stem region (e.g., with a con-
current compensatory change in the complementarily
bound nucleotide)? What of an insertion/deletion into a
stem that results in a conformation change?
FIGURE 7 Photomicrographs of Anagnostidinema visiae
strains. Slight granulation and constrictions at cross- walls present.
(a– d) Filaments with sheath formation. (e– h) Filaments without
sheath, mostly straight trichomes with slightly bent end cells. Scale
bar 10 μm in 1000× magnification.
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14
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MCGOVERN et al.
Second, this presents a cautionary tale about trans-
ferring taxa based solely on morphology. Our 16S rDNA
gene analyses, augmented with samples retrieved
from GenBank, did not recover a monophyletic, well-
supported phylogeny, but when coupled with ITS rDNA
region data did in fact resolve the relationships be-
tween the taxa in the genus. But what if a transfer was
initiated using just morphological character sets? What
if a morphological feature is plastic? What if it is only
inducible? In these cases, transfers based on classic
morphologies may be in error (MacKeigan et al.,2022).
Thus, we sought to address the nature of the vari-
ability of the ITS rDNA region motifs within strains of the
genus Anagnostidinema. We asked if these motifs jus-
tify the recent transfer of other taxa (e.g., Geitlerinema,
Limnothrix, Leptolyngbya, etc.) into this genus. Alas,
many of the ITS rDNA region transcripts are not avail-
able on GenBank. Furthermore, obtaining the original
types for genetic analyses, especially from taxa erected
long ago before sequencing was expected, is problem-
atic. Museum exsiccatae may be logistically difficult to
obtain, no longer available due to natural disasters or
loss from collections, and difficult to sequence (Dvořák
et al.,2020).
This also leads to a possible source of error: con-
firmation bias. We propose that all newly erected taxa
have all ITS rDNA region data present in the descrip-
tions. For example, if we had only folded a few ITS
rDNA region structures (based on the 16S rDNA gene
phylogenies), we might have missed the larger (more
granular) character set to resolve the relationships.
While we started with 20 strains, they were basically
identical (>99.9% sequence similarity) and had nearly
identical ITS rDNA region patterns for the three motifs
except for the unique V3 structure in the three strains
comprising the new species. This process makes it ap-
parent that we (cyanobacterial systematists) should be
wary of erecting new taxa with a paucity of strains and
ITS rDNA region structures. Thus, we propose some
potential suggestions for erecting new taxa:
1. When describing new taxa, make as certain as
possible to describe as much of the rDNA sequence
space as possible. This includes sequencing nu-
merous strains.
2. GenBank should not be used for identification with-
out morphological confirmation! By this we mean
that GenBank accession numbers and their corre-
sponding taxonomic names are replete with misi-
dentifications. Furthermore, these names may have
been accurate when deposited, but may have subse-
quently changed names.
3. We recommend sequence submitting authors to take
on the long- term responsibility of updating names
associated with their sequence data once new taxo-
nomic information becomes available. That over time
would improve GenBank records for all.
4. Very carefully edit phylogenetic trees. For example,
we have noted several instances of incorrect ac-
cession numbers and/or taxonomic identifiers from
the literature in published trees. As Alexander Pope
(1711) noted “to err is human; to forgive, divine.” We
cast no dispersions on anyone but note that this type
of error can have a domino effect.
5. When generating phylogenies and employing ITS
rDNA region structural motifs, it is imperative to fold
as many structures as possible. Although it is under-
standable why researchers do not necessarily fold all
available sequences, it may mean that we are mak-
ing less than ideal decisions about evolutionary re-
lationships. If the goal of systematists is articulating
evolution, then we must be certain to avoid the errors
of phenetics.
We note that it is possible, if not likely, that research-
ers have an inherent bias when employing ITS rDNA
region and 16S rDNA gene data (Lorenzi et al.,2019).
Anagnostidinema is an excellent test case showing
how this bias affects phylogenetic analyses for sev-
eral reasons. First, Anagnostidinema was recently
erected based on molecular data with several other
species (e.g., A. defelexum, A . epiphloeophyticum,
A. exile) transferred into it based solely on morphology
(Strunecky et al., 2017). Second, there exists much
overlap in species descriptions leading to little morpho-
logical realm space. The bias we are describing occurs
when researchers select which ITS rDNA region struc-
tures to fold and compare based on the trees gener-
ated via 16S rDNA gene data (Brooks et al.,2015). In
order to avoid this bias, we folded all Anagnostidinema
structures from all available strains then clustered them
based on shape without referencing the 16S rDNA
gene tree data. The clusters of like ITS rDNA region
structures were then compared to generated trees to
arrive at well- supported lineages. We note that this
approach provided a robust assessment of the phy-
logeny even though the 16S rDNA gene data set did
not resolve species level distinctions (e.g., there were
few clear demarcations). Thus, we use these recently
isolated and characterized strains to understand the
nature of “what are the pragmatic limitations of ITS
structures” and if we “can we wholesale transfer pre-
viously described botanical taxa” when erecting new
cyanobacterial genera.
AUTHOR CONTRIBUTIONS
Callahan A. McGovern: Conceptualization (equal);
formal analysis (equal); writing – original draft (equal);
writing – review and editing (equal). Aimee Lynn
Thomas: Conceptualization (equal); formal analy-
sis (equal); writing – original draft (equal); writing
– review and editing (equal). Alyson R Norwich:
Conceptualization (equal); formal analysis (equal);
writing – original draft (equal); writing – review and
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15
A. VISI AE ITS UTILITY
editing (equal). Sarah E Hamsher: Conceptualization
(equal); formal analysis (equal); funding acquisi-
tion (equal); writing – original draft (equal); writing
– review and editing (equal). Bopaiah A. Biddanda:
Conceptualization (equal); formal analysis (equal);
funding acquisition (equal); writing – original draft
(equal); writing – review and editing (equal). Anthony
D. Weinke: Formal analysis (equal); writing – origi-
nal draft (equal); writing – review and editing (equal).
Dale Casamatta: Conceptualization (equal); formal
analysis (equal); funding acquisition (equal); writing
– original draft (equal); writing – review and editing
(equal).
ACKNOWLEDGMENTS
The authors gratefully acknowledge funding from the
National Science Foundation grant (#2046958) to BAB,
DAC, and SEH and a NASA Michigan Space Grant
Consortium (NASA grant #NNX15AJ20H) to SEH. The
authors are grateful to two reviewers, one of which pro-
vided the excellent suggestion about updating names
on GenBank.
ORCID
Sarah E. Hamsher https://orcid.
org/0000-0002-5748-9770
Dale A. Casamatta https://orcid.
org/0000-0002-8056-0715
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SUPPORTING INFORMATION
Additional supporting information can be found online
in the Supporting Information section at the end of this
article.
Figure S1. Maximum Likelihood tree of 16S rDNA
sequences of all Anagnostidinema strains sequenced
in this study.
Figure S2. D1– D1¢ ITS rDNA region motif structures
for all Anagnostidinema strains sequenced in this study
(LHM strain designations).
Figure S3. Box B ITS rDNA region motif structures for
all Anagnostidinema strains sequenced in this study
(LHM strain designations).
Figure S4. V3 ITS rDNA region motif structures for all
Anagnostidinema strains sequenced in this study.
Figure S5. Maximum Likelihood tree of ITS rDNA
region sequences.
How to cite this article: McGovern, C. A.,
Norwich, A. R., Thomas, A. L., Hamsher, S. E.,
Biddanda, B. A., Weinke, A. D., & Casamatta, D. A.
(2023). Unbiased analyses of ITS folding motifs in
a taxonomically confusing lineage:
Anagnostidinema visiae sp. nov. (cyanobacteria).
Journal of Phycology, 00, 1–16. https://doi.
o r g /1 0.1111 / j py .13 3 3 7
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