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Red Algal Phylogenomics Provides a Robust


Framework for Inferring Evolution of Key
Metabolic Pathways
December 2, 2016 Tree of Life

Citation
Qiu H, Yoon HS, Bhattacharya D. Red Algal Phylogenomics Provides a Robust Framework for
Tweet
Inferring Evolution of Key Metabolic Pathways. PLOS Currents Tree of Life. 2016 Dec 2 . Edition 1.
doi: 10.1371/currents.tol.7b037376e6d84a1be34af756a4d90846.

Authors
Huan Qiu

Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, New Jersey, United States
of America.

Hwan Su Yoon

Department of Biological Sciences, Sungkyunkwan University, Suwon, South Korea.

Debashish Bhattacharya

Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, New Jersey, United States
of America.

Abstract
Red algae comprise an anciently diverged, species-rich phylum with morphologies that span unicells to large
seaweeds. Here, leveraging a rich red algal genome and transcriptome dataset, we used 298 single-copy
orthologous nuclear genes from 15 red algal species to erect a robust multi-gene phylogeny of Rhodophyta.
This tree places red seaweeds (Bangiophyceae and Florideophyceae) at the base of the mesophilic red algae
with the remaining non-seaweed mesophilic lineages forming a well-supported sister group. The early
divergence of seaweeds contrasts with the evolution of multicellular land plants and brown algae that are
nested among multiple, unicellular or filamentous sister lineages. Using this novel perspective on red algal
evolution, we studied the evolution of the pathways for isoprenoid biosynthesis. This analysis revealed losses
of the mevalonate pathway on at least three separate occasions in lineages that contain Cyanidioschyzon,
Porphyridium, and Chondrus. Our results establish a framework for in-depth studies of the origin and evolution
of genes and metabolic pathways in Rhodophyta.

Funding Statement
HQ would like to thank the National Science Foundation of the United States (https://www.nsf.gov/) for funding
and support (EF-1416785). HSY and DB acknowledge the Marine Biotechnology Program (PJT200620) that is

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funded by Ministry of Oceans and Fisheries of Korea (http://www.mof.go.kr/). HSY wishes to thank the National
Research Foundation of Korea (http://www.nrf.re.kr/) for funding and support (MEST: 2014R1A2A2A01003588).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.

Introduction
Red algae (Rhodophyta) form a monophyletic lineage containing ~7,000 described species1 that exhibit a wide
variety of morphological and ultra-structural forms and have complex reproductive strategies. The
Cyanidiophytina (e.g., Galdieria and Cyanidioschyzon) include extremophiles that thrive in volcanic areas
surrounding hot springs. In contrast, their mesophilic sisters (Rhodophytina) are globally distributed from
freshwater environments to open oceans and deep oceans (>200 m) to the intertidal zone. Despite a highly
reduced core gene inventory that resulted from an ancient phase of genome reduction2, red algae represent
one of the few eukaryotic lineages that have evolved complex multicellularity3, typified by red seaweeds such
as Porphyra and Gracilaria. Red seaweeds account for ~95% of known red algal taxa and are important
sources of agricultural (e.g., nori) and industrial products (e.g., agar and carrageenan).

Studies of red algal systematics have largely relied on a handful of plastid and nuclear genes4,5,6,7,8 and
focused on a broad diversity of lineages within the Florideophyceae9,10. One of the major findings of these
analyses is the separation of Cyanidiophytina from the Rhodophytina4,8. Whereas Cyanidiophytina contain only
two known families (Cyanidiaceae and Galdieraceae), Rhodophytina encompass six classes: Bangiophyceae,
Florideophyceae, Compsopogonophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae4.
Excluding the well-supported monophyly of Bangiophyceae and Florideophyceae (hereafter, collectively
referred to as red seaweeds), relationships among the remaining classes remain controversial4,5,6,7,8.

In this study, we applied phylogenomics to a rich genomic dataset to erect a robust red algal tree of life. The
dataset encompassed 298 orthologous nuclear-encoded genes from all major red algal lineages. In contrast to
previous phylogenies built using smaller datasets4,5,6,7,8, our results support a fundamental, ancient split
between red seaweeds and non-seaweed lineages among mesophiles. We discuss the implication of this new
perspective on red algal phylogeny to understanding the evolution of multicellularity in red algae, and
demonstrate the utility of this phylogenetic framework to infer the evolution of the mevalonate (MVA) pathway of
isoprenoid biosynthesis in Rhodophyta.

Methods
Construction of single-copy orthologous gene alignments

We created a local database that includes protein sequences (translated from EST or predicted from genome
sequences) from 15 red algal taxa2,11,12,13,14,15,16 (Fig. 1A) and 3 green algae17,18,19 (Table 1, Appendix 1). This
database, after removing short sequences with length <100 amino acids, was used in a self-query using
BLASTp (e-value cutoff = 1e-5). The BLASTp search output was used as input for OrthoMCL20 with parameters
(evalueExponentCutoff = -10, percentMatchCutoff = 40, inflation = 1.5) to construct orthologous gene families.
Among these families, we searched for single-copy orthologous genes with one gene copy per species
(allowing missing data in up to three red algae and in no more than one green alga). For each orthologous
gene family, the corresponding sequences were retrieved and aligned using MUSCLE (version 3.8.31) under
the default settings21. The alignments were then trimmed using TrimAl (version 1.4)22 in automated mode
(-automated) and then polished with T-COFFEE (version 9.03)23 to removed poorly aligned residues
(conservation score 5) among the aligned blocks. A total of 298 single-gene alignments (length >150 amino
acids and with 15 sequences) were retained for downstream analysis.

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Construction of the multi-protein phylogeny

The 298 single-copy gene alignments were concatenated into a super-protein alignment. A phylogenetic tree
was inferred using Phylobayes (version 3.3)24 under the CAT model25. This is a mixture model that takes into
consideration site-specific evolutionary properties (such as rate and profile) within the alignment25. The CAT
model generally fits data significantly better than one-matrix models such as LG and WAG. We set up two
chains that ran in parallel and assessed convergence periodically using bpcomp and tracecomp functions.
Convergence assessments were done based on sampled trees (taking one from every 10 trees) following
burnin equal to 20% of the entire length of the chain. The two chains were stopped when they converged to an
acceptable level that allows good qualitative measurement of the posterior consensus. According to the user
instructions (www.phylobayes.org/), an acceptable run corresponds to a maximum discrepancy across all
bipartitions (maxdiff <0.3) when monitored with the bpcomp function, and statistical discrepancies <0.3 and
effective sizes >50 for all parameters when monitored with the tracecomp function.

Construction of coalescence model-based species trees

We built a coalescence model-based red algal phylogeny with 100 replicates following Seos method26. For
each replicate, we randomly sampled 298 genes with replacement. For each sampled alignment, a pseudo-
alignment was generated by random sampling of amino acid site from the original alignment with replacement.
Only one green algal sequence (as outgroup) was retained with the priority given to Chlamydomonas
reinhardtii, Chlorella variabilis, and Micromonas RCC299 in order. A ML tree was built for each pseudo-
alignment using IQtree (version 0.9.6)27 under the best-fit amino acid evolutionary model selected on the fly (-m
TEST). The resulting 298 ML trees, rooted with outgroup sequences, were then used for maximum pseudo-
likelihood tree construction using MP-EST (version 1.4) under the default settings28. This procedure was
repeated 100 times and the resulting 100 maximum pseudo-likelihood trees were summarized under majority
rule using the consense function in Phylip (http://evolution.genetics.washington.edu/phylip.html).

Phylogenetic analyses of mevalonate pathway genes

Galdieria sulphuraria proteins in the MVA pathway (module identifier: M00095) and the methylerythritol
phosphate (MEP) pathway (module identifier: M00096) were retrieved from the KEGG database29 and used as
queries against NCBI (nr) using BLASTp (e-value cutoff = 1e-5) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
representative sequences (e.g., from Metazoa and land plants) were retrieved from Genbank. Local BLASTp
searches (e-value cutoff = 1e-5) were done against our red algal database aforementioned followed by retrieval
of the significant hits. Galdieria phlegrea sequences were retrieved from the previous study30. Each G.
sulphuraria query, together with the homologs (from Genbank and our local database), were aligned using
MUSCLE (version 3.8.31)21 under the default settings. The alignment was trimmed using trimAl (version 1.4)22
in the automated mode (-automated). ML trees were built using IQtree (version 0.9.6)27 under the best amino
acid evolutionary model selected using (-m TEST) with branch support values estimated using 1,500 ultrafast
bootstrap replicates (-bb 1500). The resulting trees were manually inspected. Distantly related paralogs (if any)
were removed manually and the trees were rebuilt following the procedure described above.

Validation of gene losses in red algae

We searched for the G. sulphuraria MVA and MEP proteins in a red algal nucleotide database (genome and
transcriptome) using tBLASTn (e-value cutoff = 1e-5). The homologous protein sequences translated from the
hit nucleotide sequences were collected using an in-house script. For each query sequence, the translated
proteins corresponding to the three top bit-score hits and the three top-identity (query-hit identity) hits were
incorporated into the single-gene ML tree building procedure described above. Distantly related homologs were
manually identified and removed. Red algal sequences that were monophyletic with G. sulphuraria were
considered to be orthologs.

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Results and Discussion


Red algal tree of life

We constructed single-gene alignments for a total of 298 one-to-one orthologous genes (98,494 amino acid
positions in total) that are conserved in 15 red algal and 3 green algal taxa (see Methods). Analysis of the
concatenated super-protein alignment under the CAT model led to a highly supported phylogenetic tree that
received 1.00 posterior probability for all interior nodes (Fig. 1A). This tree confirmed the early split between
Cyanidiophytina and Rhodophytina4,8 and monophyletic relationship between Bangiophyceae and
Florideophyceae4,8. The relationships within Florideophyceae are consistent with previous analyses10,31 with
Hildenbrandiophycidae (Hildenbrandia) in the basal position. Nemaliophycidae (Palmaria) is sister to the
monophyletic group containing Corallinophycidae (Calliarthron) and Rhodymeniophycidae (Chondrus)10,31. The
remaining non-seaweed mesophilic lineages formed a robust monophyletic group, with Stylonematophyceae in
the basal position. Compsopogonophyceae formed a sister group to the monophyletic Porphyridiophyceae and
Rhodellophyceae.

Concatenation-based analysis has previously been shown in some instances to result in inflated statistical
support for incorrect topologies32 due to heterogeneity across genes and gene-specific evolution, such as gene
duplication33. To minimize this problem, we used a tree summarization approach that does not rely on the
concatenation of multiple single-gene alignments. This method takes a population of single-gene trees as input
and estimates the species tree using a coalescence model28. This analysis led to the same tree topology (Fig.
1A) to the concatenation-based analysis with high bootstrap support for the monophyletic group comprising red
seaweeds (bootstrap support = 100%) and non-seaweed mesophilic red algae (bootstrap support = 90%). The
relationships among non-seaweed red algal lineages are however weakly supported (bootstrap support =
49-51%). Taken together, our phylogenomic analyses strongly support a separation between seaweeds and
non-seaweed lineages at the base of mesophilic red algae (Fig. 1A).

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Fig. 1: Red algal phylogenomics


(A) A phylogenetic tree inferred from a concatenated 298-protein alignment. The outgroup species are not shown.
Statistical supports (separated by a back slash) for each branch are derived from the super-protein analysis
(posterior probability) and from the coalescence model-based analysis (bootstrap support). (B) Schematic
representation of the positions of red seaweeds and land plants (thick branches) in red algae and Viridiplantae,
respectively. The phylogenies are derived from this study (panel I), Scott et al (Ref. 6, panel II) and Leliaert et al.
(Ref. 35, panel III). The arrows indicate genome reduction (GR). Bangiophyceae (Bangio.), Compsopogonophyceae
(Compsopogo.), Cyanidiophyceae (Cyanidio.), Florideophyceae (Florideo.), Porphyridiophyceae (Porphyridio.),
Stylonematophyceae (Stylonemato.), Coleochaetophyceae (Coleochaeto.), Chlorokybophyceae (Chlorokybo.),
Klebsormidiophyceae (Klebsormidio.), Mesostigmatophyceae (Mesostigmato.), Zygnematophyceae (Zygnemato.).

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The early divergence of red seaweeds within mesophilic red algae is consistent with the antiquity of the
Bangiophyceae. A putative bangiophyte (Bangiomorpha pubescens) has been found in rocks dated at ca. 1.2
billion years old34. This result suggests the existence of distinct fates for the two lineages that split from the
common ancestor of mesophilic red algae. One lineage remained unicellular with the development of filaments
in some species (e.g., Rhodochaete and Purpureofilum), whereas the other developed complex filamentous
plant bodies leading to red seaweeds with bi- and tri-phasic life cycles. The basal position of red seaweeds
among mesophiles (Fig. 1B, scenario I) contrasts with previous analyses4,5,6,8,10,34 (Fig. 1B, scenario II) and to
the highly derived position of land plants in Viridiplantae35. Land plants evolved within streptophyte green algae
that have simpler morphological forms (Fig. 1B, scenario III)35. Likewise, kelps (phaeophytes) are also nested
among multiple unicellular stramenopile lineages36. Among mesophilic red algae, red seaweeds appear to
have recovered from the extensive genome reduction they shared with the red algal ancestor2 and that was
further exacerbated in Cyanidiophytina due to their extremophilic lifestyles30. The early (>1 billion year old)
emergence of the multicellular lineage is all the more remarkable when placed in context to the early evolution
of red algae. Alternatively, the lack of simpler lineages (in terms of morphology) in the red seaweed clade may
suggest their high rate of extinction or the existence of yet unknown species that remain to be discovered in
this clade. An early emergence of a peculiar type of multicellularity (green seaweed Palmophyllophyceae) was
also discovered recently in the basal position of Chlorophyta37.

Parallel losses of MVA pathway

To demonstrate the usefulness of this novel perspective on red algal phylogeny, we used the reference tree to
elucidate the evolution of the isopentenyl pyrophosphate (IPP) biosynthetic pathway. IPP is the building block of
isoprenoids that comprises a large diversity of lipids found in all three domains of life. In photosynthetic
eukaryotes, two independent pathways exist to produce IPP, the cytosolic and peroxisome localized MVA
pathway and the plastid MEP pathway38. Whereas the MEP pathway is conserved across many species, the
MVA pathway has been lost in green algae (Chlorophyta)38 and in some red algal lineages such as C.
merolae16 and P. purpureum12. Our analysis of red algal sequence data (see Methods) showed that the MEP
pathway is present in all examined lineages. The minor gene losses that were found are most likely to be
explained by missing data commonly associated with transcriptome datasets (Fig. 3, Appendix 2). In contrast,
the MVA pathway is largely absent (3rd to 6th enzymes in the pathway, Fig. 2A) in most red algal lineages
except the Stylonematophyceae (Rhodosorus marinus and Purpureofilum apyrenoidigerum) and G.
sulphuraria. Presence of the MVA pathway in G. sulphuraria39 and Cyanidium caldarium40 is supported with
genetic and biochemical evidence39,40. This result suggests that loss of MVA pathway is more widespread than
previously thought. The red algal origin of the MVA genes in Stylonematophyceae is supported with
phylogenetic data (see Methods). For example, in the phylogeny of HMG-CoA reductase (HMGR, Fig. 2B), R.
marinus and P. apyrenoidigerum form a monophyletic group with and Galdieria species, whereas no other red
algae were present in this clade. A similar pattern is found for other MVA pathway genes that were lost in most
red algal species (Fig. 4, Appendix 3).

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Fig. 2: MVA pathway in red algae


(A) The distribution of MVA pathway genes across red algal species. Black and open circles denote the presence
and absence of the genes, respectively. For each gene, the gray boxes indicate gene presence for the
corresponding classes. Arrows indicate genome reduction. Red vertical bars indicate gene losses. ACAT
(acetyl-CoA acetyltransferase), HMGS (hydroxymethylglutaryl-CoA synthase), HMGR (3-hydroxy-
3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), PMK (phosphomevalonate kinase), MVD (mevalonate
decarboxylase), IDI (isopentenyl-diphosphate delta-isomerase). (B) A ML tree of HMGR. The taxa in red color: red
algae, green: Viridiplantae, orange: chromalveolates, brown: Opisthokonta.

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Absence of the MVA pathway in all five sampled red seaweeds suggests it was most likely lost in their common
ancestor. BLASTp searches (e-value cutoff = 10) against nucleotide databases (expressed sequence tag and
transcriptome shotgun assembly) in NCBI did not return any significant hits to MVA pathway genes from
Bangiophyceae and Florideophyceae. In addition, their losses in C. merolae, P. purpureum, and C. crispus that
have both transcriptome and genome data available are well supported. Given the red algal phylogeny (Fig.
1A), these losses were unambiguously resulted from three parallel events (Fig. 2A). Under this scenario, the
MVA pathway survived the ancient phases of genome reduction (arrows, Fig. 2A) and underwent gene loss
more recently after the split of the seaweed and non-seaweed lineages. MVA pathway loss in C. merolae likely
resulted from an additional phase of genome reduction specific to this lineage30 (Fig. 2A). The selective forces
that led to the retention or loss of the MVA pathway across the mesophilic red algal lineages are presently
unknown. Nonetheless, MVA pathway loss suggests that IPP biosynthesis is dependent on the plastid MEP
pathway and requires transporters for the export of IPP from the plastid to the cytosol38. The MVA pathway was
also lost in Chlorophyta (including most unicellular green algae)38 and G. sulphuraria is physiologically distinct
from mesophilic species. For this reason, the discovery of possible MVA pathway-containing and -absent
lineages among mesophilic red algae provides an algal model for studying the evolution of isoprenoid
biosynthesis and intracellular trafficking among compartments.

Conclusion
Our phylogenomic analyses resulted in a well-supported red algal phylogeny that provides new insights into the
evolution of red seaweeds. Our results will allow more accurate reconstruction of evolutionary events (e.g.,
gene family evolution2 and molecular calibration10) and provide a framework to map the distribution of red algal
functions and traits. Further efforts are needed to substantiate the relationships among non-seaweed
mesophilic red algae with high quality genome data from these taxa41.

Data Availability
The multi-protein alignment is available for download (ID: 20087) from TreeBASE (https://treebase.org).

Competing Interests
The authors have declared that no competing interests exist.

Corresponding Author
Huan Qiu, Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, NJ
08901, USA.

E-mail: huan.qiu.bio@gmail.com

Appendix 1

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Table 1
Algal genome and transcriptome data used for the phylogenomic analysis

Classification Species Source Data type MMETSP ID


Seaweed Hildenbrandia rubra Ref. 2 Transcriptome
Seaweed Palmaria palmata Ref. 2 Transcriptome
Seaweed Calliarthron tuberculosum Ref. 14 Partial genome
Seaweed Chondrus crispus Ref. 13 Whole genome
Seaweed Porphyra umbilicalis Ref. 14 Transcriptome
Mesophiles Purpureofilum apyrenoidigerum Ref. 2 Transcriptome
Mesophiles Rhodochaete pulchella Ref. 2 Transcriptome
Mesophiles Rhodosorus marinus Ref. 11 Transcriptome MMETSP0315
Mesophiles Rhodella maculata Ref. 11 Transcriptome MMETSP0167
Mesophiles Compsopogon coeruleus Ref. 11 Transcriptome MMETSP0312
Mesophiles Erythrolobus australicus Ref. 11 Transcriptome MMETSP1353
Mesophiles Timspurckia oligopyrenoides Ref. 11 Transcriptome MMETSP1172
Mesophiles Porphyridium purpureum Ref. 12 Transcriptome
Extremophiles Galdieria sulphuraria Ref. 15 Transcriptome
Extremophiles Cyanidioschyzon merolae Ref. 16 Whole genome
Green algae Chlorella variabilis Ref. 17 Whole genome
Green algae Chlamydomonas reinhardtii Ref. 18 Whole genome
Green algae Micromonas pusilla Ref. 19 Whole genome

Appendix 2

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Fig. 3: Distribution of the MEP pathway across red algal lineages


Black and open circles denote the presence and absence of the genes, respectively. For each gene, the gray boxes
indicate the gene presence for the corresponding classes. DXS (1-deoxy-d-xylulose 5-phosphate synthase), DXR
(1-deoxy-d-xylulose 5-phosphate reductoisomerase), MCT (2-C-methyl-d-erythritol 4-phosphate
cytidylyltransferase), CMK (C-methyl-d-erythritol kinase), MDS (2-C-methyl-d-erythritol 2,4-cyclodiphosphate
synthase), HDS (4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase), HDR (4-hydroxy-3-methylbut-2-en-1-yl
diphosphate reductase), IDI (isopentenyl-diphosphate isomerase).

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Appendix 3

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Fig. 4: ML trees for six MVA pathway genes


The taxa in red color: red algae, green: Viridiplantae, orange: chromalveolates, brown: Opisthokonta.

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Acknowledgements
The authors would like to thank the editor and reviewer for their valuable comments and suggestions to
improve the manuscript.

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