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SHORT REPORT
Open Access
Microbial mats in the Turks and Caicos
Islands reveal diversity and evolution of
phototrophy in the Chloroflexota order
Aggregatilineales
Lewis M. Ward
1*
, Usha F. Lingappa
2
, John P. Grotzinger
2
and Woodward W. Fischer
2
Abstract
Genome-resolved metagenomic sequencing approaches have le
d to a substantial increase in the recognized diversity of
microorganisms; this included the discovery of novel metaboli
c pathways in previously recognized clades, and has enabled a
more accurate determination of the extant distribution of ke
y metabolisms and how they evolved over Earth history. Here,
we present metagenome-assembled genomes of members of the
Chloroflexota (formerly Chlo
roflexi or Green Nonsulfur
Bacteria) order Aggregatilineales (formerly SBR1031 or Therm
ofonsia) discovered from sequencing of thick and expansive
microbial mats present in an intertidal la
goon on Little Ambergris Cay in the Turks and Caicos Islands. These taxa included
multiple new lineages of Type 2 reaction center-conta
ining phototrophs that were not closely related to
previously described phototrophic Chloroflexota
revealing a rich and intricate history of horizontal gene
transfer and the evolution of phototrophy and other core metabolic pathways within this widespread phylum.
Background
Most of the known diversity of phototrophic Chloroflex-
ota (formerly Chloroflexi or Green Nonsulfur Bacteria)
was derived from isolation- and sequencing-based efforts
primarily on hot spring microbial mats (e.g. [
35, 42]).
However microbial communities with diverse members
of phototrophic Chloroflexota are commonly found in
other environments, including coastal marine environ-
ments and carbonate platforms (e.g. [
36]). While
cultivation-based efforts have yet to isolate phototrophic
Chloroflexota from outside of the Chloroflexia class in
pure culture [
35], genome-resolved metagenomic se-
quencing has made substantial progress in uncovering
novel diversity of phototrophic lineages that have
otherwise remained inaccessible and unknown (e.g. [
6,
19
, 42, 48]).
The Turks and Caicos Islands occur at the southern end
of the Bahamian archipelago. The Caicos platform (Fig.
1)
is a contiguous shallow (< 5 m), grainy carbonate platform
situated in the trade winds (mean wind velocity of 8 m/s
from the east), and has a dry climate with net evaporation
in excess of precipitation [
10]. We studied microbial mats
found throughout Little Ambergris Cay
a small ~ 6 km-
long uninhabited island near the southern margin of the
platform. Little Ambergris Cay contains a bedrock rim,
formed by amalgamated cemented beach ridges and fossil
eolian dunes, enclosing a tidal lagoon with a dozen large
cut channels that communicate between the lagoon with
well-mixed platformal waters. The typical diurnal tidal
range is ~ 0.3 m. Polygonal microbial mats occur within
the lagoon amongst sparse black mangroves. These mats
are inundated daily by high tides. The mats are dissected
into individual decimeter-sized heads that take on an
© The Author(s). 2020
Open Access
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) applies to the
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* Correspondence:
lewis_ward@fas.harvard.edu
1
Department of Earth & Planetary Sciences, Harvard University, Cambridge,
MA 02138, USA
Full list of author information is available at the end of the article
Envir
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https://doi.org/10.1186/s40793-020-00357-8
upward domed shape by mature polygons with 60° angles
created by episodic desiccation. On average twice a decade,
tropical storms transport ooid sediment forming across the
platform into the lagoon and onto the mats [
37]
creating a
mode of lamination in addition to that created by microbial
succession. Dominant mat building taxa are a thickly-
sheathed, heterocystous cyanobacterium of the genus
Scyto-
nema
and the thin-walled cyanobacterium,
Halomicronema
[
36]. The mats host a rich sulfur cycling community (Gomes
et al.: Microbial mats on Little Ambergris Cay, Turks and
Caicos Islands: taphonomy and the selective preservation of
biosignatures/submitted). Deeper layers of the mat contain
abundant purple/red- and green- colored microbes visible in
exposed cross sections of the mats (Fig.
1); these have been
confirmed by 16S rRNA gene amplicon sequencing to in-
clude diverse and abundant phototrophs in the Proteobac-
teria and Chloroflexota phyla [
36].
In order to better characterize the diversity and evolu-
tionary histories of phototrophic Chloroflexota, we recov-
ered five metagenome-assembled genomes (MAGs) from
genome-resolved metagenom
ic sequencing of microbial
mats from the Turks and Caicos Islands which include
novel phototrophic lineages of
Chloroflexota not closely re-
lated to previously described phototrophs.
Methods
Methods for metagenomic sequencing and genome bin-
ning followed those published previously [
45, 46] and
described briefly here. Samples of microbial mat were
collected using an ethanol-sterilized spatula (~ 0.25 cm
3
of material per sample). Immediately after sampling,
cells were lysed and DNA preserved with a Zymo Terra-
lyzer BashingBead Matrix and Xpedition Lysis Buffer.
Lysis was achieved by attaching tubes to the blade of a
cordless reciprocating saw (Black & Decker, Towson,
MD) and operating for 1 min. Following return to the
lab, bulk environmental DNA was extracted and purified
with a Zymo Soil/Fecal DNA extraction kit. Purified
DNA was submitted to SeqMatic LLC (Fremont, CA)
for library preparation and sequencing via Illumina
NextSeq.
Raw sequence reads from four samples were co-assembled
with MegaHit v. 1.02 [
22] and genome bins constructed
based on nucleotide compositi
on and differential coverage
using MetaBAT [
18], MaxBin [
50], and CONCOCT [
1]prior
to dereplication and refinement with DAS Tool [
32]topro-
duce the final bin set. Annotation was performed using
RAST [
2]. Genome completeness and redundancy/contam-
ination was estimated with CheckM [
28], and likelihood of
Fig. 1
Geological context of the microbial mats from which the genomes in this study were recovered.
a
location of the Turks and Caicos Islands.
b
location of Little Ambergris Cay.
c
microbial mats and mangroves in tidal flat.
d
cross section of microbial mat showing pigmented layers
containing phototrophic bacteria
Ward
et al. Environmental Microbiome
(2020) 15:9
Page 2 of 9
presence or absence of metabolic pathways was estimated
with MetaPOAP [
43]. Visualization of the presence of anno-
tated metabolic pathways
was done via KEGG-decoder [
13]
following annotation of proteins sequences by GhostKOALA
[
17]. Taxonomic assignments were verified with GTDB-Tk
[
8, 29].
Protein sequences used in analyses (see below) were
identified locally with the
tblastn
function of BLAST +
[
7], aligned with MUSCLE [
11], and manually curated in
Jalview [
49]. Positive BLAST hits were considered to be
full length (e.g. > 90% the shortest reference sequence
from an isolate genome) with
e
-values better than 1e
20
.
Phylogenetic trees were calculated using RAxML [
33]on
the Cipres science gateway [
25]. Transfer bootstrap sup-
port values were calculated by BOOSTER [
20], and trees
were visualized with the Interactive Tree of Life viewer
[
21]. Concatenated ribosomal protein alignments were
built following methods from Hug et al. [
16]. Evolution-
ary histories of vertical versus horizontal inheritance of
metabolic genes were inferred by comparison of the top-
ologies of organismal and metabolic protein phylogenies
[
9, 42, 47, 48].
Results
Illumina NextSeq sequencing of four samples from the
Turks and Caicos Islands produced a total of 243,782,
642 reads of 151 nucleotides. These were coassembled
into 3,464,316 contigs totaling 2,510,159,592 nucleotides.
Binning of this dataset produced five medium- to high-
quality genomes (according to accepted quality stan-
dards, [
5]) which could be taxonomically classified by
GTDB-Tk into the lineage of the Chloroflexota phylum
currently annotated as order SBR1031 (Table
1, Fig.
2).
Other genomes previously assigned to this clade in-
cluded those proposed as the class
Candidatus
Thermo-
fonsia [
42] along with an isolate for which the order
Aggregatilineales was recently proposed [
26]. Following
the isolation and characterization of
Aggregatilinea lenta
[
26], and the clustering of these organisms into an
order-level clade by GTDB-Tk, we propose the reassign-
ment of the organisms previously described as
Candi-
datus
Thermofonsia
into the order Aggregatilineales.
This order is primarily made up of nonphototrophic or-
ganisms but also contains some members with full suites
of phototrophy genes, which appear to have been de-
rived via horizontal gene transfer from members of the
Chloroflexia class of Chloroflexota [
42].
Discussion
The environmental range exhibited by known members
of the SBR1031/Aggregatilineales was previously some-
what limited. Previously recovered genomes of members
of this group were sourced from hot spring environ-
ments [
42, 46], with the single known isolate isolated
from subseafloor sediments [
26]. 16S rRNA sequences of
SBR1031 have been recovered from more diverse envi-
ronments including hot springs [
39, 40], contaminated
soils [
24], and wastewater [
3]. Recovery of MAGs be-
longing to this order from carbonate tidal flats therefore
expands the available genomic diversity and known
range of Aggregatilineales to environments that also
have an extensive geological record.
The SBR1031 MAGs reported here encoded similar sets
of functional genes to previously reported members of this
order (Fig.
3). Like previously described members of
SBR1031/Aggegatilineales (e.g. [
42]), these organisms en-
code aerobic respiration via an A-family heme copper O
2
reductase, and contain both a
bc
complex and an alterna-
tive complex III [
42]; based on these electron transport
chain complexes, it is likely that these organisms are at
least facultatively aerobic. All genomes described here (ex-
cept for TC_22, the least complete genome) also encode a
bd
oxidase (O
2
reductase) capable of functioning for O
2
detoxification or respiration at low O
2
concentrations
[
4]
a trait observed in both aerobic and anaerobic mem-
bers of the Anaerolineae class (e.g. [
14, 27, 38, 42]).
Of the five SBR1031/Aggregatilineales genomes re-
ported here, three encode partial or full components
necessary for phototrophic energy transduction via a
Type 2 reaction center. TC_71 and TC_152 encode
complete sets of marker genes for phototrophy, includ-
ing those encoding PufL and PufM subunits of the re-
action center and bacteriochlorophyll synthesis (e.g.
BchX, BchY, and BchZ). MetaPOAP False Positive esti-
mates for phototrophy in these organisms were low (<
0.04), suggesting that it is very unlikely that these genes
were recovered as a result of contamination in the
MAGs. The TC_22 genome encodes the BchXYZ com-
plex but did not recover genes for PufL or PufM; Meta-
POAP False Positive and False Negative estimates were
similarly low (~ 0.04) for this genome, and based on
this analysis it remains unclear whether or not this or-
ganism contains a complete set of genes for phototro-
phy. However, the gene cluster encoding BchX, BchY,
and BchZ in TC_22 is located on the end of a contig,
and the region of the chromosome syntenous to that
encoding PufL and PufM in other phototrophic Chloro-
flexota (e.g. 6.5 kb upstream of
bchX, bchY,
and
bchZ
in
TC_152) is missing in the TC_22 genome. Based on
this, it is possible that this organism hosts a complete
phototrophy pathway but that some genes simply were
not recovered in the MAG. Like previously described
phototrophs in SBR1031, these organisms do not en-
code a BchLNB complex or the capacity for carbon fix-
ation via either the 3-hydro
xypropionate bi-cycle or the
Calvin cycle [
42]. It is worth noting, however, that TC_
22 does encode a Form IV rubisco-like protein on a
small contig; however enzymes in this family are not
Ward
et al. Environmental Microbiome
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Table 1
SBR1031/Aggregatilineales MAGs and genome statistics described in this study
Bin
Id
Classification
Completeness Contamination Strain
heterogeneity
Genome
size (mb)
Coding
sequences
GC% Contigs Shortest
contig
(nt)
RNAs N50 Phototrophy
markers (of
5)
Phototrophy
False Positive
estimate
Phototrophy
False
Negative
estimate
NCBI
WGS ID
TC_
15
d__Bacteria;p__
Chloroflexota;c__
Anaerolineae;o__SBR1031;
f__UBA2029;g__;s__
88.07
2.94
0
5.45
5127
60.1 662
2501
43 10,
293
0
N/A
2.40x10
-5
JAADYV
TC_
152
d__Bacteria;p__
Chloroflexota;c__
Anaerolineae;o__SBR1031;
f__;g__;s__
92.66
2.75
0
3.86
3445
55.7 312
2554
40 17,
396
5
0.0372
N/A
JAADYW
TC_
195
d__Bacteria;p__
Chloroflexota;c__
Anaerolineae;o__SBR1031;
f__;g__;s__
95.41
0
0
4.06
4178
61.5 431
2518
44 11,
646
0
N/A
1.98x10
-7
JAADYX
TC_
22
d__Bacteria;p__
Chloroflexota;c__
Anaerolineae;o__SBR1031;
f__A4b;g__;s__
77.88
1.83
0
4.43
4167
61.7 746
2501
30 6808 3
0.0424
0.049
JAADYY
TC_
71
d__Bacteria;p__
Chloroflexota;c__
Anaerolineae;o__;f__;g__;
s__
87.61
1.33
33.33
4.06
4216
56.9 576
2481
44 8059 5
0.039
N/A
JAADYZ
Ward
et al. Environmental Microbiome
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Fig. 2
(See legend on next page.)
Ward
et al. Environmental Microbiome
(2020) 15:9
Page 5 of 9
(See figure on previous page.)
Fig. 2
a
Concatenated ribosomal protein phylogeny of the
Chloroflexota, focusing on order SBR1031 (Aggregat
ilineales). Organisms encoding phototrophy w
ith
a Type 2 reaction center highlighted in green (including TC_22, which did not recover reaction center genes but may be a phototroph, as discussed in the
text).
Genomes first described here noted with pink circles. As species names ar
e not available for MAGs of uncultured organisms, strains are labelled with M
AG IDs (this
study) or NCBI WGS database IDs (others) followed by taxonomy as derived from
GTDB-Tk. Clades not the focus of this study have been collapsed and labele
d
with GTDB-Tk taxonomy.
b
Tanglegram showing phylogenetic (in)congrue
nce between concatenated ribo
somal proteins (left) reflecting organismal relationships
with PufM (right) as a marker of the horizontal gene transfer of phototrophy
proteins. Dotted lines show topological congruence within some lineages
of
Chloroflexia (in black) and incongruence between
Roseiflexus, Roseilinea,
and phototrophic members of SBR1031 (in red). This is indicative of horizontal gene
transfer of phototrophy proteins from the
Roseiflexus
lineage to
Roseilinea
and SBR1031
Fig. 3
Heatmap of metabolic functions of Aggregatilineales genomes produced by KEGG-decoder. The color gradient reflects the fractional
abundance of genes associated with a pathway encoded by a particular genome (i.e. white encodes 0 genes, and darkest red encodes all genes
annotated as part of the pathway). Genome IDs as used in this study (TC##) or as WGS identifiers (all others). Note that the apparent presence of
Calvin cycle genes in TC_22 and other members of Aggregatilineales is due to the presence of a Form IV rubisco-like protein that does not
catalyze CO
2
fixation, as described in the text
Ward
et al. Environmental Microbiome
(2020) 15:9
Page 6 of 9
capable of catalyzing CO
2
fixation and instead are used
for a variety of other functions [
34] and this genome
does not encode phosphoribulose kinase.
Comparisons of organismal (based on concatenated
ribosomal proteins and other vertically inherited markers)
and phototrophy protein (e.g. PufL, PufM) phylogenies in-
dicated substantial incongruences between organismal
and phototrophy tree topologies. These relationships are
indicative of a history of horizontal gene transfer (Fig.
2b)
(e.g. [
30]). In particular, the reaction centers found in
members of SBR1031 branch with those of
Roseiflexus
ra-
ther than as a clade separate from those of Chloroflexia
(e.g.
Chloroflexus + Roseiflexus
), suggesting that horizontal
transfer of phototrophy proteins occurred from the
Rosei-
flexus
branch to members of SBR1031
a pattern previ-
ously recognized in other members of the SBR1031/
Aggegatilineales [
42]. Interestingly, like
Roseiflexus
and
some other phototrophic members of SBR1031/Aggegati-
lineales [
42], TC_71 and TC_152 encode fused
pufL/pufM
genes encoding the two subunits of the Type 2 reaction
center heterodimer. Together with reaction center protein
phylogenic relationships, this observation indicated that
the reaction centers of these organisms are more closely
related to those of the
Roseiflexus
lineage of Chloroflexia
than to the more closely related
Ca.
Roseilinea gracile,
which encodes unfused
pufL
and
pufM
genes. A corollary
of these observations of is that phototrophy in SBR1031
must postdate the acquisition and diversification of photo-
trophy in the Chloroflexia, events that have been esti-
mated to have occurred in the last ~ 1 billion years [
31].
Conclusions
While horizontal gene transfer can be confidently deter-
mined to have been responsible for the presence of photo-
trophy in SBR1031, it remains unclear to what extent
intra-order horizontal gene transfer played a role in the
extant distribution of phototrophy within the clade.
Phototrophic lineages of SBR1031 show a polyphyletic dis-
tribution, separated by many nonphototrophic lineages
(Fig.
2a). This distribution could be reasonably explained
by the presence of phototrophy in the last common ances-
tor of SBR1031 followed by extensive loss in most line-
ages; however differences in the topology of phototrophy
proteins and organismal phylogenies within SBR1031 may
indicate later acquisition followed by multiple instances of
horizontal gene transfer between members of the clade.
Discovery and study of additional phototrophic members
of SBR1031 will be valuable to confidently resolve phylo-
genetic relationships of SBR1031 phototrophy proteins to
assess organismal relationships in this clade.
The expanded environmental distribution and genetic
diversity of Chloroflexota phototrophs described here
further reinforces that genome-resolved metagenomic
sequencing can provide an effective avenue for
discovering novel microbial diversity, particularly of
hard-to-culture phototrophs (e.g. [
6, 15, 35, 41, 48]).
These data also reinforce hypotheses that horizontal
gene transfer has been a major mechanism behind the
extant distribution of anoxygenic phototrophy (e.g. [
12,
42
, 48]). Together with the mounting evidence that most
microbial lineages that have ever lived are now extinct
[
23], the continuing lack of discovery of donor lineages
for horizontal gene transfer of phototrophy leads toward
a consistent hypothesis: most phototrophic lineages that
have ever existed have gone extinct, but relatively fre-
quent horizontal gene transfer has allowed phototrophy
pathways to persist in new lineages (e.g. [
44]).
Acknowledgements
Not applicable.
Authors
contributions
UFL, JPG, and WWF performed field work and collected samples. UFL and
LMW processed and analyzed data. LMW wrote the manuscript with assistance
from UFL, JPG, and WWF. All authors read and approved the final manuscript.
Funding
This work was made possible with support from the Agouron Institute, NSF
IOS project # 1833247, and the Caltech Center for Environment-Microbe In-
teractions. LMW acknowledges support from an Agouron Institute Postdoc-
toral Fellowship and the Simons Foundation Collaboration on Marine Microbial
Ecology. UFL was supported by an NSF Graduate Research Fellowship.
Availability of data and materials
The datasets generated during and analysed during the current study are
available in the NCBI WGS repository under project ID PRJNA602167 with
Accession IDs of JAADYW-JAADYZ.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Earth & Planetary Sciences, Harvard University, Cambridge,
MA 02138, USA.
2
Division of Geological & Planetary Sciences, California
Institute of Technology, Pasadena, CA 91125, USA.
Received: 21 January 2020 Accepted: 19 March 2020
References
1. Alneberg J, Bjarnason BS, de Bruijn I, Schirmer M, Quick J, Ijaz UZ, Loman NJ,
Andersson AF, Quince C. CONCOCT: clustering contigs on coverage and
composition. arXiv preprint arXiv:1312.4038; 2013.
2. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K,
Gerdes S, Glass EM, Kubal M, Meyer F. The RAST server: rapid annotations
using subsystems technology. BMC Genomics. 2008;9(1):75.
3. Björnsson L, Hugenholtz P, Tyson GW, Blackall LL. Filamentous Chloroflexi
(green non-sulfur bacteria) are abundant in wastewater treatment processes
with biological nutrient removal. Microbiol. 2002;148(8):2309
18.
4. Borisov VB, Gennis RB, Hemp J, Verkhovsky MI. The cytochrome bd
respiratory oxygen reductases. Biochim Biophys Acta. 2011;1807:1398
413.
https://doi.org/10.1016/j.bbabio.2011.06.016
.
5. Bowers RM, Kyrpides NC, Stepanauskas R, Harmon-Smith M, Doud D, Reddy
TBK, Schulz F, Jarett J, Rivers AR, Eloe-Fadrosh EA, Tringe SG. Minimum
information about a single amplified genome (MISAG) and a metagenome-
Ward
et al. Environmental Microbiome
(2020) 15:9
Page 7 of 9
assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol. 2017;
35(8):725
31.
6. Bryant DA, et al. Candidatus Chloracidobacterium thermophilum: an aerobic
phototrophic acidobacterium. Science. 2007;317:523
6. https://doi.org/10.
1126/science.1143236
.
7. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K,
Madden TL. BLAST+: architecture and applications. Bmc Bioinform. 2009;10:
421.
https://doi.org/10.1186/1471-2105-10-421
.
8. Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify
genomes with the genome taxonomy database. Bioinformatics. 2020;36(6):
1925
7.
9. Doolittle RF. Of urfs and orfs: a primer on how to analyze derived amino
acid sequences. Mill Valley: California, University Science Books; 1986.
10. Dravis JJ, Wanless HR. Caicos platform models of Quaternary carbonate
deposition controlled by stronger easterly Trade Winds
application to
petroleum exploration. In: Proceedings Developing models and analogs for
isolated carbonate platform
Holocene and Pleistocene carbonates of
Caicos Platform, British West Indies, vol. 22; 2008. SEPM, SEPM Core
Workshop.
11. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and
high throughput. Nucleic Acids Res. 2004;32:1792
7. https://doi.org/10.1093/
nar/gkh340
.
12. Fischer WW, Hemp J, Johnson JE. Evolution of oxygenic photosynthesis.
Annu Rev Earth Planet Sci. 2016;44:647
83.
13. Graham ED, Heidelberg JF, Tully BJ. Potential for primary productivity in a
globally-distributed bacterial phototroph. ISME J. 2018;350:1
6.
14. Hemp J, Ward LM, Pace LA, Fischer WW. Draft genome sequence of
Levilinea saccharolytica KIBI-1, a member of the Chloroflexi class
Anaerolineae. Genome Announc. 2015;3:e01357
15. https://doi.org/10.1128/
genomeA.01357-15
.
15. Holland-Moritz H, Stuart J, Lewis LR, Miller S, Mack MC, McDaniel SF, Fierer
N. Novel bacterial lineages associated with boreal moss species. Environ
Microbiol. 2018;20(7):2625
38.
16. Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ, Castelle CJ,
Butterfield CN, Hernsdorf AW, Amano Y, Ise K, Suzuki Y. A new view of the
tree of life. Nat Microbiol. 2016;1(5):16048.
17. Kanehisa M, Sato Y, Morishima K. BlastKOALA and GhostKOALA: KEGG tools
for functional characterization of genome and metagenome sequences. J
Mol Biol. 2016;428:726
31.
18. Kang DD, Froula J, Egan R, Wang Z. MetaBAT, an efficient tool for accurately
reconstructing single genomes from complex microbial communities. PeerJ.
2015;3:e165.
19. Klatt CG, et al. Community ecology of hot spring cyanobacterial mats:
predominant populations and their functional potential. ISME J. 2011;5:
1262
78. https://doi.org/10.1038/ismej.2011.73
.
20. Lemoine F, Domelevo Entfellner JB, Wilkinson E, Correia D, Davila Felipe M,
De Oliveira T, Gascuel O. Renewing Felsenstein's phylogenetic bootstrap in
the era of big data. Nature. 2018;556(7702):452
6. https://doi.org/10.1038/
s41586-018-0043-0
.
21. Letunic I, Bork P. Interactive tree of life (iTOL) v3: an online tool for the
display and annotation of phylogenetic and other trees. Nucleic Acids Res.
2016;44(W1):W242
5. https://doi.org/10.1093/nar/gkw290
.
22. Li D, Liu CM, Luo R, Sadakane K, Lam TW. MEGAHIT: an ultra-fast single-
node solution for large and complex metagenomics assembly via succinct
de Bruijn graph. Bioinformatics. 2015;31(10):1674
6.
23. Louca S, Shih PM, Pennell MW, Fischer WW, Parfrey LW, Doebeli M. Bacterial
diversification through geological time. Nat Ecol Evol. 2018;2(9):1458.
24. Militon C, Boucher D, Vachelard C, Perchet G, Barra V, Troquet J,
Peyretaillade E, Peyret P. Bacterial community changes during
bioremediation of aliphatic hydrocarbon-contaminated soil. FEMS Microbiol
Ecol. 2010;74(3):669
81.
25. Miller, M. A., W. Pfeiffer and T. Schwartz (2010). Creating the CIPRES Science
Gateway for inference of large phylogenetic trees. 2010 Gateway
Computing Environments Workshop (GCE).
26. Nakahara N, Nobu MK, Takaki Y, Miyazaki M, Tasumi E, Sakai S, Ogawara M,
Yoshida N, Tamaki H, Yamanaka Y, Katayama A. Aggregatilinea lenta gen.
Nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated
from subseafloor sediment, and proposal of the new order
Aggregatilineales Ord. Nov. within the class Anaerolineae of the phylum
Chloroflexi. Int J Syst Evol Microbiol. 2019;69(4):1185
94.
27. Pace LA, Hemp J, Ward LM, Fischer WW. Draft genome of Thermanaerothrix
daxensis GNS-1, a thermophilic facultative anaerobe from the Chloroflexi
class Anaerolineae. Genome Announc. 2015;3:e01354
15. https://doi.org/10.
1128/genomeA.01354-15
.
28. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM:
assessing the quality of microbial genomes recovered from isolates, single
cells, and metagenomes. Genome Res. 2015;25(7):1043
55.
29. Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil PA,
Hugenholtz P. A standardized bacterial taxonomy based on genome
phylogeny substantially revises the tree of life. Nat Biotechnol. 2018;36(10):
996
1004..
30. Raymond J, Zhaxybayeva O, Gogarten JP, Gerdes SY, Blankenship RE. Whole-
genome analysis of photosynthetic prokaryotes. Science. 2002;298(5598):
1616
20.
31. Shih PM, Ward LM, Fischer WW. Evolution of the 3-hydroxypropionate
bicycle and recent transfer of anoxygenic photosynthesis into the
Chloroflexi. Proc Natl Acad Sci. 2017;114(40):10749
54.
32. Sieber CM, Probst AJ, Sharrar A, Thomas BC, Hess M, Tringe SG, Banfield JF.
Recovery of genomes from metagenomes via a dereplication, aggregation
and scoring strategy. Nat Microbiol. 2018;3(7):836
43.
33. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-
analysis of large phylogenies. Bioinformatics. 2014;30(9):1312
3. https://doi.
org/10.1093/bioinformatics/btu033
.
34. Tabita FR, Satagopan S, Hanson TE, Kreel NE, Scott SS. Distinct form I, II, III,
and IV Rubisco proteins from the three kingdoms of life provide clues
about Rubisco evolution and structure/function relationships. J Exp Bot.
2008;59(7):1515
24.
35. Thiel V, Tank M, Bryant DA. Diversity of chlorophototrophic bacteria
revealed in the omics era. Annu Rev Plant Biol. 2018;69:21
49.
36. Trembath-Reichert E, Ward LM, Slotznick SP, Bachtel SL, Kerans C, Grotzinger
JP, Fischer WW. Gene sequencing-based analysis of microbial-mat
morphotypes, Caicos platform, British West Indies. J Sediment Res. 2016;
86(6):629
36.
37. Trower EJ, O
Reilly SS, Gomes M, Cantine M, Stein N, Grotzinger H, Strauss
JV, Lamb MP, Grotzinger JP, Knoll AH, Fischer WW. Active ooid growth
driven by sediment transport in a high energy shoal, Little Ambergris Cay,
Turks and Caicos, British Overseas Territories. J Sedimentary Res. 2018;88:
1132
51.
38. Ward LM, Hemp J, Pace LA, Fischer WW. Draft genome sequence of
Leptolinea tardivitalis YMTK-2, a mesophilic anaerobe from the Chloroflexi
class Anaerolineae. Genome Announc. 2015;3:e01356
15. https://doi.org/10.
1128/genomeA.01356-15
.
39. Ward LM. Microbial evolution and the rise of oxygen: the roles of
contingency and context in shaping the biosphere through time (Doctoral
dissertation, California Institute of Technology); 2017.
40. Ward LM, Idei A, Terajima S, Kakegawa T, Fischer WW, McGlynn SE. Microbial
diversity and iron oxidation at Okuoku-hachikurou Onsen, a Japanese hot
spring analog of Precambrian iron formations. Geobiology. 2017;15(6):817
35.
41. Ward LM, McGlynn SE, Fischer WW. Draft genome sequence of
Chloracidobacterium sp. CP2_5A, a phototrophic member of the phylum
Acidobacteria recovered from a Japanese hot spring. Genome Announc.
2017;5(40):e00821
17.
42. Ward LM, Hemp J, Shih PM, McGlynn SE, Fischer WW. Evolution of
phototrophy in the Chloroflexi phylum driven by horizontal gene transfer.
Front Microbiol. 2018;9:260.
43. Ward LM, Shih PM, Fischer WW. MetaPOAP: presence or absence of
metabolic pathways in metagenome-assembled genomes. Bioinformatics.
2018;34(24):4284
6.
44. Ward LM, Shih PM. The evolution of carbon fixation pathways in response
to changes in oxygen concentration over geological time. Free Radic Biol
Med. 2019;140:188
99.
45. Ward LM, Shih PM, Hemp J, Kakegawa T, Fischer WW, McGlynn SE. Genomic
evidence for phototrophic oxidation of small alkanes in a member of the
Chloroflexi phylum. bioRxiv. 2019:531582.
46. Ward LM, Idei A, Nakagawa M, Ueno Y, Fischer WW, McGlynn SE.
Geochemical and metagenomic characterization of Jinata Onsen, a
Proterozoic-analog hot spring, reveals novel microbial diversity including
iron-tolerant phototrophs and thermophilic lithotrophs. Microbes Environ.
2019;34(3):278
92.
Ward
et al. Environmental Microbiome
(2020) 15:9
Page 8 of 9
47. Ward LM, Johnston D, Shih PM. Phanerozoic radiation of ammonia oxidizing
bacteria. bioRxiv. 2019:655399.
48. Ward LM, Cardona T, Holland-Moritz H. Evolutionary implications of
anoxygenic hototrophy in the bacterial phylum Candidatus Eremiobacterota
(WPS-2). Front Microbiol. 2019d;10:1658.
https://doi.org/10.3389/fmicb.2019.
01658
.
49. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview version
2-a multiple sequence alignment editor an
d analysis workbench. Bioinformatics.
2009;25(9):1189
91. https://doi.org/10.1093/
bioinformati
cs/btp033
.
50. Wu YW, Tang YH, Tringe SG, Simmons BA, Singer SW. MaxBin: an
automated binning method to recover individual genomes from
metagenomes using an expectation-maximization algorithm. Microbiome.
2014;2(1):26.
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