Molecular Phylogenetics and Evolution 64 (2012) 381392

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Phycoerythrin evolution and diversication of spectral phenotype in marine

Synechococcus and related picocyanobacteria
R. Craig Everroad a,b,, A. Michelle Wood a,c
a

Institute for Ecology and Evolutionary Biology, formerly Center for Ecology and Evolutionary Biology, 5289 University of Oregon, Eugene, OR 97403, USA
RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
c
Atlantic Oceanographic and Meteorological Laboratory, National Oceanographic and Atmospheric Administration, Miami, FL 33149, USA
b

a r t i c l e

i n f o

Article history:
Received 5 January 2012
Revised 12 April 2012
Accepted 20 April 2012
Available online 3 May 2012
Keywords:
Adaptive evolution
Approximately unbiased test
Cyanobacteria
Horizontal gene transfer
Phycoerythrin
Phylogenetics

a b s t r a c t
In marine Synechococcus there is evidence for the adaptive evolution of spectrally distinct forms of the
major light harvesting pigment phycoerythrin (PE). Recent research has suggested that these spectral
forms of PE have a different evolutionary history than the core genome. However, a lack of explicit statistical testing of alternative hypotheses or for selection on these genes has made it difcult to evaluate
the evolutionary relationships between spectral forms of PE or the role horizontal gene transfer (HGT)
may have had in the adaptive phenotypic evolution of the pigment system in marine Synechococcus. In
this work, PE phylogenies of picocyanobacteria with known spectral phenotypes, including newly co-isolated strains of marine Synechococcus from the Gulf of Mexico, were constructed to explore the diversication of spectral phenotype and PE evolution in this group more completely. For the rst time,
statistical evaluation of competing evolutionary hypotheses and tests for positive selection on the PE
locus in picocyanobacteria were performed. Genes for PEs associated with specic PE spectral phenotypes
formed strongly supported monophyletic clades within the PE tree with positive directional selection
driving evolution towards higher phycourobilin (PUB) content. The presence of the PUB-lacking phenotype in PE-containing marine picocyanobacteria from cyanobacterial lineages identied as Cyanobium
is best explained by HGT into this group from marine Synechococcus. Taken together, these data provide
strong examples of adaptive evolution of a single phenotypic trait in bacteria via mutation, positive
directional selection and horizontal gene transfer.
2012 Elsevier Inc. All rights reserved.

1. Introduction
Marine Synechococcus are a globally important group of photosynthetic prokaryotes found in a wide range of habitats where the
quantity and quality of light available for photosynthesis can vary
dramatically over relatively short time-scales (Waterbury et al.,
1986; Li and Wood, 1988; Olson et al., 1990; Li, 1994; Partensky
et al., 1999; Crosbie et al., 2003; Scanlan et al., 2009). These photosynthetic organisms rely on light capture for growth, thus the
changing spectral quality of their environment represents a selective agent expected to promote evolution of phenotypes adapted to
different spectral environments (Wood, 1985; Stomp et al., 2004).
This is supported by data showing that the photosynthetic performance or growth of cultured strains is highest in light elds that
complement the spectral phenotype of the culture (Wood, 1985;

Abbreviations: AU, approximately unbiased; CA, chromatic adaptation; HGT,

horizontal gene transfer; ML, maximum likelihood; MP, maximum parsimony; PBS,
phycobilisome; PE, phycoerythrin; PEB, phycoerythrobilin; PUB, phycourobilin.
Corresponding author. Fax: +81 45 508 7363.
E-mail address: rcraig@riken.jp (R.C. Everroad).
1055-7903/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.04.013

Glover et al., 1987; Stomp et al., 2004, 2007). Additionally, eld

studies indicate that while different spectral phenotypes often
co-exist, there is an optical biogeography for marine Synechococcus
such that the most spectrally complementary pigment types predominate in natural waters of different colors (Olson et al., 1988,
1990; Wood et al., 1998; Coble et al., 2004; Katano and Nakano,
2006; Haverkamp et al., 2008).
The molecular mechanisms that underlie the spectral phenotype of a given cell are due to the presence of complex watersoluble macromolecular structures called phycobilisomes (PBS)
used for light harvesting (Grossman et al., 1993; Six et al., 2007).
The dominant phycobiliprotein pigments in the PBS confer any of
a wide range of colors on cells ranging from blue-green to orange,
pink and brick-red. Phycobiliproteins are pigments composed of
colorless apoproteins that bind several light-absorbing phycobilin
chromophores. Within a PBS, these phycobiliproteins can be found
as antennae (light harvesting) and core (energy transfer to photosynthetic reaction center II) pigments (for reviews on PBS structure
and energy transfer see Gantt, 1981; Glazer, 1989; MacColl, 1998).
Synechococcus is a form-genus with marine representatives
belonging to three genetically distinct subclusters collectively

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R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

known as cluster 5; these likely are not a monophyletic grouping

and each subcluster may represent a genus-level group (Herdman
et al., 2001; Scanlan et al., 2009). In two of these subclusters, 5.1
and 5.3, the antennae phycobiliprotein phycoerythrin (PE) is found
in two distinct forms that enhance light absorption in the green or
blue regions of the light spectrum (Six et al., 2007). Always present
are Class I PEs (PEI), which always contain the green-absorbing
chromophore phycoerythrobilin (PEB, Absmax  550 nm). Additionally Class II PEs (PEII) can be found; these always contain both PEB
and the blue/bluegreen absorbing chromophore phycourobilin
(PUB, Absmax  495 nm; Ong and Glazer, 1991; Six et al., 2007).
These PE apoproteins are coded for by either cpeBA which forms
the basis of PEI, or mpeBA which forms the basis of PEII (Ong and
Glazer, 1991; Wilbanks et al., 1991). When a strain synthesizes
both PEI and PEII, the PEI apoprotein may also bind the PUB chromophore, but PUB chromophores are unknown in the PEs of isolated marine Synechococcus that lack PEII (Ong and Glazer, 1991;
Six et al., 2007).
Phenotypically, the ratio of PUB and PEB chromophores in the
assembled PE molecule produce characteristic peaks in the
in vivo uorescence excitation and absorption spectra of whole
cells. Thus, the ExPUB/ExPEB ratio can be used as a quantitative
descriptor of spectral phenotype within marine Synechococcus
(Fig. 1). These phenotypes are classied into four categories based
on ExPUB/ExPEB ratios measured in whole cells or on pigment extracts, with higher ExPUB/ExPEB ratios indicating stronger absorption of blue light relative to green light (Wood, 1985; Wood
et al., 1998; Everroad and Wood, 2006; Six et al., 2007). These phenotypes are: PEB-only or PUB-lacking, where the PUB chromophore is absent; low-PUB (ExPUB/ExPEB < 0.6); mid-PUB (ExPUB/
ExPEB = 0.61.0) and high PUB (ExPUB/ExPEB > 1.0). Physiological
adapters that regulate PUB:PEB ratios by a process called type IV
chromatic adaptation (CA) may be thought as a fth spectral phenotype (Palenik, 2001; Everroad et al., 2006).
Apt and colleagues (1995) suggested that PBS and PE evolution
has proceeded towards increased absorption of shorter wavelengths, but this hypothesis has never been formally tested. An
evolutionary analysis by Zhao and Qin (2006) found elevated dN/
dS ratios and possible positive selection between different

40
M12.1

Relative Fluorescence

35

M16.3
M11.2

30
25
20
15
10
5
0
450

475

500

525

550

575

phycobiliprotein lineages; among other places, these elevated ratios were observed in the chromophore-binding regions of the phycobiliproteins examined. PE phenotypes do not map on
phylogenies based on housekeeping and/or ribosomal loci (Supplementary Fig. S1; Toledo et al., 1999; Fuller et al., 2003; Everroad,
2007; Six et al., 2007) or other molecular data (Wood and Townsend, 1990). Recent analyses of PE genes from many types of cyanobacteria, red algae and of the PBS gene cluster from 11 marine
Synechococcus genomes have revealed PE and PBS genes follow a
pattern of evolution incongruent with that of the core genome; this
pattern has been interpreted as due to unspecied horizontal gene
transfer (HGT) events (Everroad, 2007; Six et al., 2007). Based on
the incongruence between phycocyanin (PC; cpcBA-IGS) and 16S
rRNA gene trees for several lineages of freshwater picocyanobacteria, and the by-phenotype clustering of PE- and PC-rich isolates in
the cpcBA-IGS tree, Jasser et al. (2011) proposed HGT as a likely
mechanism explaining the distribution of the PE phenotype in
these groups.
Marine Synechococcus are the most widely recognized PE-rich
picophytoplankton in the marine environment, yet they are often
accompanied by representatives from other lineages of PE-containing picocyanobacteria. In particular, PE-rich strains closely related
to Cyanobium spp. are observed frequently in coastal and brackish
waters where the predominant wavelengths of available light are
generally longer than in the open ocean (Wingard et al., 2002;
Ernst et al., 2003; Chen et al., 2004; Haverkamp et al., 2009). Cyanobium is traditionally described as a fresh- and brackish-water
lineage lacking PE that in most cyanobacterial phylogenies is found
sister to the radiation that gives rise to both marine Synechococcus
cluster 5 and Prochlorococcus along with several somewhat-related
but unique lineages of picocyanobacteria (Urbach et al., 1998;
Herdman et al., 2001; Rippka et al., 2001; Robertson et al., 2001;
Ernst et al., 2003). In an earlier study, Everroad and Wood (2006)
noted that PE genes from Cyanobium-like isolates shared a common ancestry with PEs from marine Synechococcus 5.1, but could
not determine if PE genes in the Cyanobium-like isolates evolved
by direct descent from a common PE-containing ancestor or by
HGT. Similarly, Synechococcus 5.3 appears basal to the marine Synechococcus 5.1 and Prochlorococcus lineages but contains a PBS containing both PEI and PEII and a spectral phenotype most similar to
those of PUB-containing Synechococcus 5.1 strains (Six et al., 2007;
Dufresne et al., 2008).
The aim of the present research was to test hypotheses about
the evolution of spectral diversity within marine picocyanobacteria and the evolutionary relationships between Cyanobium and
Synechococcus subclusters 5.1 and 5.3. The spectral and genetic
diversity of several strains of marine Synechococcus were explored
as part of a more phylogenetically comprehensive group of taxa
than has been examined to date. The deduced phylogenetic relationships between these and other picocyanobacteria were used
to explicitly test the evolutionary patterns inferred for PE genes
associated with different spectral phenotypes and the whole cell
phylogenies derived from 16S rRNA gene sequences.

2. Materials and methods

Wavelength (nm)
Fig. 1. In vivo excitation spectra for PE uorescence emission at 588 nm for the
study strains M12.1, M16.3, and M11.2. Phycoerythrobilin (PEB) absorbs maximally
around 550 nm, while phycourobilin (PUB) absorbs maximally around 495 nm.
Characteristic peaks or shoulders in the spectra at these wavelengths are due to
these chromophores. Strain M12.1 represents the PUB-lacking phenotype, M16.3
represents the low-PUB phenotype, and M11.2 represents the high-PUB phenotype.
The low-PUB M16B.1 is omitted for clarity, but it possesses a near identical
excitation spectrum to M16.3. Likewise spectra for the chromatic adapters M11.1
and M16.17 are omitted. Spectra for these adapters under blue and white light
conditions can be found in Everroad et al. (2006).

2.1. Isolation, selection, and maintenance of strains used in this study

The four newly reported Gulf of Mexico strains, along with the
previously-described chromatic adapters M11.1 and M16.17
(Everroad et al., 2006) used in this study are a subset of a larger
culture isolation project. Isolation details have been previously described (Everroad et al., 2006). Briey, these strains were isolated
from water collected from a 10-L Niskin bottle on a conductivitytemperaturedepth (CTD) cast on February 9th, 2003 from a

R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

depth of 275 m at a site immediately above the Brine Pool, a methane seep located in the Gulf of Mexico offshore Louisiana, USA
(27430 N, 91150 W; MacDonald et al., 1990). This water was enriched with nutrients equivalent to f/50 Si culture medium (Guillard and Ryther, 1962), and once growth was visible, cells were
slowly transitioned into sterile seawater with f/2 Si nutrient levels. Clonal, but not axenic isolates were obtained from these
enrichments using the three successive pour plate steps as described in Brahamsha (1996). Based on microscopic observations
of morphology, size and cell division pattern, each clonal strain
was determined to match the description of the form genus Synechococcus (Herdman et al., 2001).
2.2. Determination of spectral phenotype
Spectral phenotype for each strain was determined using uorescence criteria described in Wood et al. (1998) and Everroad
and Wood (2006). Fluorescence excitation spectra were obtained
on an Aminco Bowman series 2 luminescence spectrometer with
the following settings: scan range 450580 nm, variable excitation
monochromator, emission monochromator at 588 nm, 4 nm bandpass for both monochromators and a scan speed of 4 nm s1. PE
excitation spectra were collected and the ratio of excitation at
495 nm to excitation at 550 nm was used to determine ExPUB/ExPEB
values for classication of strains as high, medium, or low PUB
strains, or as lacking PUB. All strains were screened for CA by comparing the ExPUB/ExPEB ratios of acclimated cultures grown in blue
and white light. For these experiments cultures were maintained in
exponential phase as described in Wood et al. (2005).
2.3. DNA isolation, amplication, cloning and sequencing
Sequencing for subsequent identity and phylogenetic analyses
were performed for two loci: the cotranscribed PE apoprotein
genes cpeBA (PEI) and mpeBA (PEII), and a portion of the 16S ribosomal RNA gene. DNA extraction, PCR and sequencing procedures
were largely as performed in Everroad and Wood (2006). For each
strain, culture media was harvested by centrifugation at 27,000g
for 15 min. Cell pellets were vortexed and genomic DNA was isolated to sterile water using the Chelex 100 method (de Lamballerie et al., 1992). All PCR amplications utilized reagent types and
concentrations as previously described (Everroad and Wood,
2006). A fragment of the 16S rRNA gene was amplied using the
cyanobacteria-specic primers CYA106F (Nbel et al., 1997) and
PLG2.3 (Miller and Castenholz, 2000).
For amplication and sequencing of the PE genes, the primer
pairs SynB1F/SynA3R and SynB3F/SynA2R were used with conditions as previously reported (Everroad and Wood, 2006). Both
cpeBA and mpeBA are amplied by these primers; consequently
PCR products were cloned using the pGEM-T vector (Promega,
Madison, WI). For each clone, the PE sequence was re-amplied
and subsequently sequenced in both directions with the original
and internal primers on a Beckman Coulter CEQ capillary sequencer using dye terminator chemistry at the Genomics Core Facility,
University of Oregon (Eugene, OR, USA).
2.4. Nucleotide sequence editing, alignment, and phylogenetic
reconstruction
Sequences were manually edited and assembled using the BioEdit Sequence Alignment Editor v. 5.0.9 (Hall, 1999). For each
strain, consensus PE sequences were created from the overlapping
fragments. Nucleotide alignments of the 16S rRNA gene and amino
acid alignments of the translated cpeBA and mpeBA nucleotide sequence data were obtained using CLUSTALX (Higgins and Sharp,
1988, 1989; Thompson et al., 1997). Default settings were used

383

for gap penalties. It is known that the a and b subunits of cpe

and mpe form distinct evolutionary groups in phycobiliprotein
phylogenies, but they are co-transcribed (i.e. cpeBA and mpeBA)
and possess similar divergence patterns. Previous analyses have
shown these subunits are suitable for analysis together (Newman
et al., 1994; Apt et al., 1995; Everroad and Wood, 2006; Everroad,
2007). To conrm this with the added sequence data, an incongruence-length difference test (Farris et al., 1995) was performed on
the translated amino acid PE data using the partition homogeneity
test in PAUP 4.0 b v1 (Sinauer Associates, Sunderland, MA, USA;
Swofford, 2003).
16S rRNA gene and PE alignments were made with 46 and 38
sequences, respectively. The ribosomal gene alignment was composed with genes from members of the marine and freshwater picocyanobacterial clade, namely Synechococcus (subclusters 5.1, 5.2
and 5.3), Prochlorococcus, Cyanobium and Cyanobium-like strains.
The freshwater Synechococcus PCC 7942 (cluster 1) and Synechococcus PCC 7002 (cluster 3) were used as the outgroup taxa for this
dataset. The PE alignment was composed of representatives from
all spectral phenotypes for CpeBA and MpeBA in Synechococcus
5.1, the mid-PUB Synechococcus 5.3, as well as CpeBA from nonpicocyanobacterial taxa, Prochlorococcus, and red algae
(Rhodophyta).
Hierarchical likelihood ratio tests were performed on the 16S
and PE data sets using the BIC criterion in jModelTest version
0.1.1 and ProtTest version 2.4, respectively (Posada, 2008; Abascal
et al., 2005). Phylogenetic analyses for both datasets were performed using maximum likelihood (ML), maximum parsimony
(MP) and Bayesian methods. Selected models with model parameters estimated were used in subsequent parametric phylogenetic
analyses. Gapped positions were excluded from all phylogenetic
analyses.
Maximum likelihood was implemented with PhyML online
(Guindon and Gascuel, 2003; Guindon et al., 2010) using the best
of SPR and NNI tree rearrangements of 5 random starting BIONJ
trees (Gascuel, 1997). ML analyses were bootstrap replicated 100
times. Maximum parsimony (MP) was performed in PAUP using
the heuristic search option and the tree-bisection-reconnection
branch-swapping algorithm. Starting trees were obtained by stepwise addition with 100 replications of random sequence addition.
MP trees were bootstrapped 100 times. Bayesian analyses were run
using MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001). For these
analyses, two runs of four chains (one cold) were run until the
deviation of split frequencies between the runs was <0.01, i.e. for
5  105 Metropolis-coupled Markov chain Monte Carlo generations. Trees were sampled every 100 generations. From these sample trees, 2000 were discarded as burn-in.
2.5. Tests of evolution
The AU test, or approximately unbiased test for tree selection
of Shimodaira (2002) was implemented using CONSEL version 0.1j
(Shimodaira and Hasegawa, 2001). To address specic evolutionary
hypotheses about the evolution of spectral phenotype for PE and
the relationship between Cyanobium and marine Synechococcus,
the best trees from the 16S and PE ML analyses were modied to
match alternative hypotheses in Treeview 1.6.6 (Page, 1996). These
constraint trees were analyzed locally using PhyML 3.0 with the
original sequence alignments and selected models. The output sitewise likelihood values were converted to PAUP format manually
for input into CONSEL.
To test for positive selection within CpeBA, a simpler 11 taxon
alignment was used (same taxa as shown in Fig. S1). The software
program CRANN version 1.04 was used to implement the relative
rate ratio test of Creevey and McInerney (2002, 2003) on this dataset. This test reconstructs ancestral sequences within the tree

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R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

using a maximum parsimony approach. To identify positive (directional and non-directional) selection, synonymous and non-synonymous substitutions are classied as invariant (change is retained
in all descendant taxa) or variant (position is subject to additional
changes) to create four classes: replacement invariant (RI), replacement variant (RV), silent invariant (SI) and silent variant (SV). By
comparing the ratios of RI:RV with SI:SV using the G-test, the null
hypothesis of neutral selection (RI:RV will be similar to SI:SV) can
be tested against the hypotheses of positive selection (RI:RV significantly different from SI:SV). Further, either directional or nondirectional positive selection can be identied if the rejection of
neutrality is due to a high number of RI or RV substitutions, respectively (Creevey and McInerney, 2002).
2.6. Sequence data
Sequences have been deposited in Genbank under the accession
numbers JN566227JN566230 for 16S rRNA gene sequences and
JN566231JN566237 for cpeBA and mpeBA. The 16S accession
numbers are also available in Fig. 2.
3. Results and discussion
3.1. Phenotypic characterization of new strains
The study strains from the Gulf of Mexico represent all but one
of the basic categories of spectral phenotype described in the introduction. Fluorescence excitation spectra for representative strains
are given in Fig. 1. Strain M12.1 possesses the PUB-lacking phenotype, strains M16.3 and M16B.1 possess the low-PUB phenotype,
strain M11.2 possesses the high-PUB phenotype, and the previously reported strains M11.1 and M16.17 possess the CA phenotype (Everroad et al., 2006).
3.2. Evolutionary relationships between Cyanobium and marine
Synechococcus
The inferred ML tree for the 16S rRNA gene is shown in Fig. 2.
The phylogenetic position of all the strains from the Gulf of Mexico
indicates they belong to marine Synechococcus cluster 5.1. Three
strains of Prochlorococcus as well as most previously described
clades of marine Synechococcus based on 16S rRNA gene sequence
data (clades IIX, XVI and clade X/Synechococcus subcluster 5.3;
Rocap et al., 2002; Fuller et al., 2003; Dufresne et al., 2008) were
included in the analysis. Clades with multiple representatives in
Fig. 2 were recovered. Strains M16B.1, M16.3 and M12.1 afliated
with clade II strain CC9605, indicating considerable phenotypic
and genetic diversity within this clade. Previous work has demonstrated variation of spectral phenotype and nitrogen utilization
capabilities within clade II (Fuller et al., 2003; Ahlgren and Rocap,
2006). M16.17, a member of clade XVI, afliated most closely with
the clade IX chromatic adapter RS9916. Strains M11.1 and M11.2
clustered together sister to clade III members WH 8102, WH
8103 and Max42.
This analysis also included several freshwater, brackish and
marine picocyanobacteria (including the previously reported PEcontaining Arabian Sea strains from Wingard et al., 2002 and
Everroad and Wood, 2006) provisionally belonging to the form
genus Cyanobium (Rippka et al., 2001; Crosbie et al., 2003; Ernst
et al., 2003). The Synechococcus 5.3 strains RCC307 and Minos11
were found deep in the tree, sister to the picocyanobacterial radiation, as discussed below.
The evolutionary relationships between genera within the picocyanobacterial lineage are generally agreed to follow a pattern
recovered in Fig. 2 (Everroad and Wood, 2006; Scanlan et al.,

2009). However, these relationships are not fully resolved, with

Synechococcus 5.1 sometimes reported as paraphyletic with respect
to Prochlorococcus (Urbach et al., 1998; Herdman et al., 2001).
Additionally, ambiguities exist within Synechococcus 5.1. Recent
phylogenies (including the one presented here in Fig. 2) place the
PE-lacking Synechococcus 5.2 strain WH 8101 within clade VIII of
Synechococcus 5.1 (Scanlan et al., 2009). Likewise, Synechococcus
5.1 clade X, consisting of strains RCC307 and Minos11, was recently described as sister to Synechococcus 5.1 and Prochlorococcus
and proposed to be renamed Synechococcus 5.3 (Dufresne et al.,
2008).
The remaining strains of picocyanobacteria not included among
the marine Synechococcus or Prochlorococcus lineages appear closely related to presently dened Cyanobium strains and together
form a monophyletic lineage of Cyanobium-like strains (sensu Ernst
et al., 2003) that includes isolates from fresh, brackish and marine
waters. However, this monophyletic group is not well-supported in
this work (Fig. 2) or elsewhere (e.g. Crosbie et al., 2003; Ernst et al.,
2003; Jasser et al., 2011). Several distinct lineages and naming systems exist for these Cyanobium-like organisms (Fig. 2), and the
relationships of these lineages to one another remain ambiguous
(Crosbie et al., 2003; Ernst et al., 2003). Although Cyanobium is
formally dened as lacking PE in Bergeys Manual of Systematic
Bacteriology (Rippka et al., 2001), many strains from these Cyanobium-like lineages have since been identied that possess PE, including the previously reported marine Cyanobium strains from the
Arabian Sea (Wingard et al., 2002; Everroad and Wood, 2006). Thus
like marine Synechococcus in subcluster 5.1, which are dened as
having PE (Herdman et al., 2001 but occasionally lack PE, strains
in the Cyanobium clade do not always conform to the pigment
complement expected from the formal description of the lineage,
i.e. sometimes containing PE even though the taxon is dened as
lacking PE. In other words, both Synechococcus 5.1 and Cyanobium
appear to include members both with and without a PE phenotype.
Based on the results presented in Fig. 2, it is clear that future efforts
should be made to revise the taxonomic status, formal descriptions, and naming conventions for the Synechococcus 5 cluster as
well as the Cyanobium and Cyanobium-like lineages of picocyanobacteria, as has been called for previously (Herdman et al., 2001;
Robertson et al., 2001; Dufresne et al., 2008).
Given the somewhat unclear relationships between the individual picocyanobacterial groups, and the dispersal of similar pigment
types throughout the entire radiation, a more robust analysis of the
16S rRNA gene tree was performed. The AU test was used to determine if an alternative phylogeny that placed either the PE-containing Cyanobium from the Arabian Sea or the mid-PUB Synechococcus
5.3 as sister to the remaining members of the Synechococcus 5.1
radiation could be rejected. The results, shown in Table 1, clearly
reject a phylogeny with these marine Cyanobium as members of
Synechococcus 5.1.
Conversely, a phylogeny that places the proposed Synechococcus
5.3 as a sister group to Synechococcus 5.1 clade cannot be rejected
as signicantly worse than the best tree, although it could be considered only marginally non-signicant (p = 0.063, SE 0.004;
Table 1). As discussed briey by Dufresne et al. (2008), phylogenomic analyses lacked sufcient taxon sampling for rm assignment of Synechococcus 5.3 to an existing clade (Six et al., 2007;
Dufresne et al., 2008), but other evidence from analyses of peroxiredoxins and glnB suggest that Synechococcus 5.3 is more similar
to Synechococcus 5.2 than 5.1, incongruent with a placement of
Synechococcus 5.3 as a member of the 5.1 clade (Scanlan et al.,
2009).
3.2.1. Evolution of picocyanobacterial PEs
The PE genes of the new PUB-lacking, low-PUB and high-PUB
Synechococcus 5.1 strains were sequenced to build a more complete

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385

M16B.1 (JN566230)
M16.3 (JN566229)
CC9605 (II; CP000110)
M12.1 (JN566228)
CC9902 (IV; CP000097)
75/-/0.88
WH 8103 (AF311293)
* Max42 (AY172805)
Clade III
WH 8102 (BX548020)
M11.2 (JN566227)
Synechococcus
M11.1 (DQ224204)
68/60/0.76
A CC9311 (CP000435)
5.1
B WH 8020 (AY172835) Clade I
Almo3 (AY172800)
100/87/0.99
Oli31 (AY172810 )
84/75/0.74
Eum14 (AY172804)
52/57/Clades V/VI/VII
WH 7805 (AAOK01000001)
69/63/0.8
WH 7803 (CT971583)
C
RS9916 (IX; AY172826)
-/62/D
M16.17 (XVI; DQ224203)
WH 8101 (VIII; AF001480)
77/95
CCMP1375 (AE017126)
1.0
A) 94/88/0.91
PAC1 (AF001471)
Prochlorococcus
B) 86/82/82/95
MIT 9313 (AF053399)
C) 71/60/0.90
0.96
D) 68/69/0.94
* P211 (AF098373)
Group I
MW 100C3 (AY151249)
ABRAXAS
(AF098372)
*
-/75/0.98
Antarctic
ACE (AF098370)
E) 73/51/0.83
78/87/F) 84/91/0.85
WH 5701 (AY172832)
Subalpine II /
G) 65/54/0.99
BO 8805 (AF317073) Synechococcus 5.2
H) 50/-/0.99
PCC 6307 (AF001477)
*
Cyanobium 1 (Group A)
PCC 7009 (AF216945)
ML/MP/PP
MW 33B4 (AY151237)
77/53
*
Group H
0.93
MW 25B5 (AY151233)
66/81
MW 4C3 (AY151238)
Group B (subalpine I)
BO 8807 (AF317074)
*
BS5 (AF330253)
Bornholm sea
Non-marine
E
G6.1 (DQ248005)
F
G11 (DQ248008)
picocyanobacteria
Arabian Sea
G
G4.1 (DQ248003)
Cyanobium
H
G10.1 (DQ248007)
PCC 7001 (AB015058)
Cyanobium 2
PS717 (AF216953) Group E (Lake Biwa)
RCC307 (NC_009482)
*
Synechococcus 5.3 (5.1 clade X)
Minos 11 / RCC61 (AY172807)
PCC 7942 (CP000100)
PCC 7002 (CP000951)
0.05

No PE (PC-rich)
PUB-lacking
Low PUB
Mid PUB
High PUB
Variable PUB/PEB
div-Chl a/b

71/-/53/57/85/60/0.93
71/-/0.89

Fig. 2. Maximum likelihood (ML) phylogram of the marine cyanobacterial clade (Prochlorococcus/marine Synechococcus/Cyanobium) inferred from a 1414-bp alignment of the
16S rRNA gene. Clade names for recovered clades are given. For Synechococcus 5.1; where only one representative of a clade is present, the clade number is included in
parentheses with the strain name. The freshwater Synechococcus PCC 7942 (cluster 1) and Synechococcus PCC 7002 (cluster 3) were used as outgroups. Support for each node
is shown with MP/ML bootstrap values >50 above the node. Bayesian posterior probabilities >0.7 are shown below the node. Asterisks indicate bootstrap support for the node
indicated at >90 for both ML and MP and a Bayesian posterior probability >0.97. Dashes indicate bootstrap or posterior probability support below 50 or 0.7, respectively.
Strain pigment information derived from Herdman et al. (2001), Rippka et al. (2001), Robertson et al. (2001), Crosbie et al. (2003), Ernst et al. (2003), Fuller et al. (2003),
Everroad et al. (2006), Everroad and Wood (2006), Six et al. (2007), and Haverkamp et al. (2008). The PUB content of the Antarctic strains is unreported.

PE phylogeny for marine Synechococcus. The partition homogeneity

test did not detect incongruence between the a- and b-subunits of
the PE genes, so they were analyzed together in subsequent phylogenetic analyses. The best-t model of evolution for the deduced
PE apoprotein sequence alignment conformed to the LG substitution model with gamma distribution and empirical amino acid frequencies (LG + G + F). This model was coded into MrBayes using a
manually checked template substitution matrix from the GARLI
project (Zwickl, 2006). The inferred ML tree, which is unrooted
(Fig. 3), shows very strong support for the existence of ve major
clades of PE, as reported previously (Everroad and Wood, 2006).
These include the PE genes of red algae, Prochlorococcus, cyanobacteria not from the picocyanobacterial radiation (i.e. other cyanobacteria), and the CpeBA and MpeBA clades from Synechococcus

subcluster 5.1. None of the deeper relationships between these

groups were well supported, although all methods suggest the
red algal CpeBA lineage is sister to the Synechococcus 5.1 CpeBA
clade. PE sequences from Prochlorococcus appear the most evolutionarily distant, which may in part be due to differential selection
on PE in these taxa (Ting et al., 2001). The placement of the primitive cyanobacteria Gloeobacter spp. PCC 7421 sister to the red algal
lineage is another interesting feature of the overall tree (Fig. 3).
Within the CpeBA lineage from marine Synechococcus and Cyanobium, there are well-supported subclades in which the sequences
from strains with xed PUB-lacking, low-PUB and high-PUB phenotypes occur together (Fig. 3). Specically, all CpeBA sequences
obtained from strains with a PUB-lacking phenotype, including
from both marine Synechococcus and Cyanobium, are found

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Table 1
Approximately unbiased likelihood tests of alternative tree topologies.
Rank

Tree topoogy

D lnLa

AUb

Phycoerythrin spectral clades

1
2
3
4

Best treec
MpeBA (ML)Hd
CpeBA (HM)(LN)
CpeBA ((HM)N)L

2.3
2.3
3.6
5.0

0.796
0.460
0.459
0.112

Phycoerythrin taxonomic clades

1
2
3
4

Best treee
PUB-lacking Cyanobium sister to no-PUB Synechococcus 5.1
PUB-lacking Synechococus 5.1 as sister clade to all other CpeBAs
PUB-lacking Cyanobium as sister clade to all other CpeBAs

6.3
6.3
10.5
10.7

0.875
0.18
0.085

RCC307 as sister to remaining CpeBAs

20.2

0.036

RCC307 as sister to remaining MpeBAs

31.3

0.001

16S rRNA locus

1
2
3

Best treeg
Synechococcus 5.3 sister to remaining Synechococcus 5.1
Arabian Sea Cyanobium sister to remaining Synechococcus 5.1

6.1
6.1
23.1

0.975
0.063

0.025f

0.005

Log likelihood difference.

p-Value for the approximately unbiased test.
Tree in Fig. 3 except RS9916 CpeBA is excluded. For CpeBA ((High Mid)Low)No; For MpeBA (High Mid)Low; no-PUB Cyanobium paraphyletc with no-PUB Synechococcus;
RCC307 in mid-PUB Synechococcus 5.1.
d
N, L, M and H refer to the No, Low, Mid and High clades of the PE tree tested. Details in main text.
e
Tree shown in Fig. 3.
f
Underlined values indicate topologies of interest that were rejected at a signicance level of 0.05.
g
Tree shown in Fig. 2. Arabian Sea Cyanobium sister to Cyanobium cluster 2, Synechococcus 5.3 basal to all non-outgroup taxa.
b

exclusively together as a monophyletic group sister to the remaining CpeBA sequences. Within this sister clade, all sequences from
strains with a low-PUB phenotype form a second monophyletic
group sister to the remaining mid, high, and variable-PUB sequences. For this latter grouping of CpeBA sequences from strains
with mid, high and variable-PUB (or CA) strains, two subclades
are observed. One clade, not well supported, places the mid-PUB
RCC307 sister to a group of chromatic adapters. The second clade
puts the origin of the branch leading to the chromatic adapter
RS9916 basal to a well-supported monophyletic group of xed
high-PUB strains. Consequently, this analysis makes the CA phenotype paraphyletic with respect to both a monophyletic high-PUB
cluster and the mid-PUB RCC307. However these placements are
not well supported in Fig. 3, and for RS9916 are incongruent with
the evolutionary relationships found in the MpeBA sequences.
Likewise, the placement of the RCC307 CpeBA and MpeBA sequences are not well supported; until further mid-PUB strain PE
sequences are available a true mid-PUB cluster cannot be identied. As RCC307 appears to be the only member of Synechococcus
5.3 examined that does not have the CA phenotype it may also
be simply that this particular strain has lost its ability to chromatically adapt (Six et al., 2007).
The pattern found in the MpeBA sequences is similar to that of
CpeBA, but with better resolution between high- and mid-PUB
clades. The xed low-PUB group is placed sister to two strongly
supported clades that contain either the mid-PUB RCC307 with
chromatic adapters or the xed high-PUB strains. The nearly complete congruence of the phylogenies for MpeBA and CpeBA in
strains that contain both is consistent with a hypothesis that the
two sets of genes represent paralogs evolving in tandem after a
gene duplication event that facilitated accommodation of the
PUB chromophore. Earlier work by Apt et al. (1995) emphasized
the role of gene duplication in the evolution of phycobiliproteins
and this appears to have been an important mechanism for acquisition of a PUB phenotype by marine Synechococcus.
The clustering of the Synechococcus PEs by spectral type and the
branching patterns of these clades suggest successive evolution towards higher PUB content, particularly because PE sequences from

strains with lower or PUB-lacking phenotypes share more basal

ancestors compared to strains with mid- or high PUB phenotypes.
To further explore this idea, two sets of tests were performed. The
AU test was used to test the evolutionary branching order of PE
spectral clades within the Synechococcus 5.1 CpeBA and MpeBA lineages as shown in Fig. 3, while the relative rate ratio test of Creevey
and McInerney (2002) was used to reconstruct ancestral sequences
and test for positive selection on a simpler 11 taxon CpeBA phylogeny containing all phenotypic groups.
3.2.2. Approximately unbiased tests
For the PE AU tests, using the nomenclature H = high-PUB,
M = mid-PUB, L = low-PUB and N = PUB-lacking, all possible evolutionary relationships of the H, M, L and N clades within CpeBA and
MpeBA were tested. These clades were dened as follows: For
MpeBA, the low-PUB group L included all sequences from lowPUB strains, the mid-PUB clade M included RCC307 and several
strains capable of chromatic adaptation, while the high-PUB clade
H contained all the high-PUB strains. For CpeBA test, the no- and
low-PUB groupings were clear (N, and L, respectively). Based on
the best tree however, assignment of high and mid-PUB clades was
somewhat less clear. The clade containing Synechococcus spp.
CC9311, CC9902 and WH 8020 and the true mid-PUB RCC307 are
referred to as M. When acclimated to white light, these CA strains
possess PEs with a mid-PUB phenotype and were historically considered mid-PUB strains (Six et al., 2007). Due to the questionable
placement of the chromatically adapting RS9916 within the H
clade in the CpeBA phylogeny as discussed above, a preliminary
AU test was performed to asses whether an alternative tree with
it as a member of the M clade with the remaining chromatic
adapters could be rejected. This test could not reject such a placement (p > 0.205), so RS9916 CpeBA was removed from the analysis.
This result, combined with the phenotype of RS9916 and the pattern of the MpeBA locus makes it seem possible that the placement
of RS9916 with the CpeBA H clade in the ML and Bayesian trees is
the result of some phylogenetic artifact in the analysis.
For PE spectral clades, these tests effectively reduced to separate four and three taxon questions. Trees could be considered

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R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

73/71/0.98
100/100/1.0
74/-/0.98

98/98/1.0

B
75/87/1.0 C

WH 8102 CpeBA
High PUB H
M11.2 CpeBA
CC9605 CpeBA

RS9916 CpeBA
CC9311 CpeBA
Variable PUB/PEB
WH 8020 CpeBA
CC9902 CpeBA

Synechococcus 5.1

RCC307 CpeBA (Syn 5.3; Mid PUB M)

A) 87/93/1.0
B) 59/56/0.9
C) 51/52/0.94
D) 99/92/1.0
E) 70/88/1.0
F) 92/77/0.99
G) 76/94/1.0
H) 71/84/1.0

90/87/1.0

98/100/1.0 D
E
F

ML/MP/PP
69/53/1.0

72/55/
0.91
97/100/1.0

M16B.1 CpeBA
M16.3 CpeBA

Low PUB L

WH 7803 CpeBA
WH 7805 CpeBA
M12.1 CpeBA
G5.1 CpeBA
G4.1 CpeBA
G11 CpeBA
99/100/1.0
67/56/0.75
100/100/1.0

PUB-lacking N

Arabian Sea (Cyanobium)

Porphyra yezoensis CpeBA
Porphyra purpurea CpeBA

Ceramium boydenii CpeBA

Corallina officinalis CpeBA

86/78/1.0

Rhodophyta

Gloeobacter PCC 7421 CpeBA

86/92/1.0

100/100/1.0

WH 8102 MpeBA
High PUB
M11.2 MpeBA
H
CC9605 MpeBA
RS9916 MpeBA

-/99/1.0

CC9902 MpeBA
WH 8020 MpeBA

CC9311 MpeBA
RCC307 MpeBA (Syn 5.3; M)
89/94/
1.0 94/-/- M16B.1 MpeBA
Low PUB
100/100/
M16.3 MpeBA
1.0
L
WH 7803 MpeB

100/100/1.0
100/100/1.0

-/81/
1.0
67/53/0.7
100/100/1.0

Variable
PUB/PEB

Synechococcus
5.1

Pseudanabaena PCC 7409 CpeBA

Fremyella diplosiphon CpeBA
Synechocystis PCC 9413 CpeBA

Prochlorococcus MIT 9313 CpeBA

Prochlorococcus CCMP 1375 CpeBA
Prochlorococcus PAC1 CpeBA

0.1
Fig. 3. Unrooted ML phylogram inferred from a 321 amino acid alignment of the a- and b-subunits of phycoerythrin (PE) I and PEII (CpeBA and MpeBA) for selected
cyanobacteria and red algae. ML/MP bootstrap >50 and Bayesian posterior probabilities >0.7 are given for each node. Study strains are in bold. When comparing the topology
of the CpeBA and CpeBA genes of marine Synechococcus, note that MpeBA is absent from strains with a phycourobilin (PUB)-lacking phenotype. Letters indicate nodes with
support values given to the left of the tree.

rooted since all Synechococcus/Cyanobium PEs share a most recent

common ancestor (Everroad and Wood, 2006). Thus the CpeBA test
included 14 alternative trees (rooted four-taxon problem), while
the MpeBA test included 2 alternative trees (rooted three-taxon
problem), for a total of 17 trees (16 alternatives plus the best tree).
Five additional tests were performed to assess the evolutionary
relationships between the PE-sequences from the Arabian Sea
Cyanobium, Synechococcus 5.3, and Synechococcus 5.1 strains. Specically, these tests aimed to demonstrate whether the genes for
PEs from the Cyanobium and Synechococcus 5.3 strain groups were
truly members of the same PE clade as the CpeBA and MpeBA

genes from all other PE-containing strains from the Synechococcus

5.1 group, and secondarily if the sequences from PUB-lacking
Cyanobium and Synechococcus strains in the 5.1 group could form
mutually monophyletic groupings (and a clearer demonstration
of HGT) as had been shown previously (Everroad, 2007). For these
tests trees were constructed by alternatively placing PEs from
Cyanobium as a sister group to PUB-lacking Synechococcus 5.1 or
as a sister clade to all marine Synechococcus CpeBA. Likewise, the
alternative placement of the CpeBA and MpeBA sequences from
strain RCC307 as a sister clade to the respective Synechococcus
5.1 PE clades was tested. Finally, the PUB-lacking Synechococcus

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R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

(A) MpeBA
High Mid Low

ML best tree

Mid

Low High

p = 0.46

(B) CpeBA
High Mid Low No High Mid Low

ML best tree

No

p = 0.459

High Mid

No Low

p = 0.112
Fig. 4. Schematic representation of possible phycoerythrin (PE) evolutionary
patterns for spectral phenotype. (A) The most likely tree for MpeBA, followed by
one alternative tree not rejectable using the approximately unbiased (AU) test. (B)
The most likely tree for CpeBA, followed by two non-rejectable trees. p-Values are
for the AU test. Small bars in alternative CpeBA phylogenies represent presumed
losses of phycourobilin (PUB) and MpeBA.

5.1 strains were placed sister to the PUB-lacking Cyanobium strains

and remaining Synechococcus 5.1 strains (making them the most
ancestral-like Synechococcus 5.1 CpeBA).
Selected results of the AU tests are shown in Table 1 and Fig. 4.
For MpeBA the best tree was (HM)L with the mid- and high-PUB
PEs nested to the exclusion of the low-PUB PEs (Fig. 3, Fig. 4A).
However, the AU test could not reject an alternative possibility,
namely (ML)H where the mid- and low-PUB PEs grouped together
to the exclusion of the high-PUB PEs (Fig. 4A, Table 1). This would
indicate successive adaptation to lower PUB content, counter to
what was observed in the most likely tree. In either case, evolution
appeared directional, but the direction is unknown based on these
results alone.
In the CpeBA phylogeny however, evidence of directional adaptation towards higher PUB content could be seen clearly. The best
CpeBA tree is ((HM)L)N, with high- and mid-PUB PEs nested together to the exclusion of low-PUB PEs (just as in the best MpeBA
tree), followed by PUB-lacking PEs sister to all PUB-containing PEs
(Fig. 4B). Like the best MpeBA tree, this evolutionary pattern suggests strongly that successive adaptation towards higher PUB content has occurred in PE. Alternative statistically supported
phylogenies are also consistent with evolution towards higher
PUB content and include (HM)(LN), with the high- and mid-PUB
PEs sister and sharing a common ancestor with a clade that contains sister low- and PUB-lacking PEs (Fig. 4B). The second alternative tree of ((HM)N)L again places high- and mid-PUB PEs as sister,
but here, the PUB-lacking PEs are found at the next deepest bifurcation, followed by a basal low-PUB PE clade. All 12 remaining
topologies, including three consistent with evolution toward lower
PUB content were rejected at a signicance level of 0.05 (Table 1).
Identical AU tests were performed that included RS9916 CpeBA as
a member of the H clade (as in the ML tree presented in Fig. 3);

these tests resulted in identical patterns of signicance for the

clades (not shown).
The two alternative CpeBA patterns of (HM)(LN) and ((HM)N)L
both retain high-PUB, mid-PUB and CA PEs together to the exclusion of the low- and PUB-lacking PEs, but must invoke a secondary
loss of PUB and MpeBA in the PUB-lacking clades (shown in
Fig. 4B), with the relationships between low- mid- and high-PUB
PEs still following directional evolution toward higher PUB content. Thus for both CpeBA and MpeBA, the best tree and all but
one non-rejectable topology agree on the direction of evolution.
Only the MpeBA phylogeny of (ML)H, shown in Fig. 4A, disagrees
with the other potentially acceptable trees but it is incongruent
with all CpeBA phylogenies that could not be rejected. Conversely
the best MpeBA phylogeny is congruent with all three possible
CpeBA phylogenies. The actual evolutionary history for these cotranscribed and co-assembled loci should match even if the actual
signal is somewhat ambiguous and, if this reasonable assumption
is made, then the best tree from MpeBA can be accepted as correct.
Given that the branching patterns are congruent between the MpeBA and CpeBA loci, the best tree fully supports the hypothesis that
evolution of the apoprotein genes for PEs associated with different
phycobilin composition and different spectral phenotypes at the
whole organism level proceeded towards higher PUB content and
adaptation of PE to bluer water. Whether or not the ancestral forms
of marine Synechococcus had the genetic architecture now associated with a PUB-lacking phenotype or rather contained the genes
for both PEI and PEII apoproteins remains an unanswered question.
If the latter, then the PUB-lacking strains present in the lineage today would have to have evolved by gene loss since they do not retain any residual sign of the apoprotiens for MpeBA, an apparent
requirement for PUB-containing phenotypes.
Next, the AU test was used to determine if PEs grouped by their
taxonomic afliation based on the 16S rRNA gene (Fig. 5). The test
could not reject a monophyletic Cyanobium clade sister to a monophyletic PUB-lacking Synechococcus 5.1 clade, nor could it reject a
tree that has PUB-lacking Synechococcus 5.1 sister to a CpeBA clade
that includes the remaining Synechococcus 5.1 and Cyanobium sequences (Fig. 5A and Table 1). These two possible topologies, like
the ML topology in Fig. 3, make Synechococcus 5.1 CpeBA ancestral
to Cyanobium CpeBA, incongruent with the relationship between
these groups observed in the 16S rRNA gene phylogeny (Fig. 2).
However, the results did reject both CpeBA and MpeBA from
RCC307 as sister to the respective Synechococcus 5.1 PE clades (Table 1, Fig. 5B). Likewise the hypothesis that CpeBA sequences from
a PE-containing Cyanobium are sister to the Synechococcus 5.1
CpeBA clade, consistent with the 16S rRNA gene phylogeny, and
a hypothesis that Cyanobium CpeBA is the ancestral form, can be
rejected.
3.2.3. Relative rates ratio test
To further examine the evolution of the picocyanobacterial PE
genes, a smaller 11-taxon tree with all phenotypes represented
was made and tested for evidence of positive selection using the
relative rates ratio test of Creevey and McInerney (2002; Fig. 6).
This method is more sensitive than traditional approaches such
as pairwise dN/dS ratios, which are calculated for the whole sequence and can often overlook positive selection at one or a few
positions. For all pairwise sequence comparisons in Fig. 6, the null
hypothesis of neutrality could be rejected using a codon-based Ztest, and all had dN/dS ratios <1, suggesting purifying selection
(not shown; Nei and Gojobori, 1986). Using the relative rates ratio
test, positive directional selection was detected on the branch leading to the ancestor of all Synechococcus 5.1 sequences ((HM)L)N
from the ancestor shared with the outgroup. Likewise, positive
directional selection was detected on the branch leading to the
(HM)L clade, to the (HM) clade and to both the H and M clades.

R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

389

(A) Non-rejected CpeBA phylogenies

High-PUB (Syn 5.1)
Mid-PUB (Syn 5.3)
Mid-PUB (Syn 5.1)
Low-PUB (Syn 5.1)
PUB-lacking (Syn 5.1)
PUB-lacking (Cbm)
PUB-lacking (Cbm)
ML best tree p = 0.875

High-PUB (Syn 5.1)

High-PUB (Syn 5.1)

Mid-PUB (Syn 5.3)

Mid-PUB (Syn 5.3)

Mid-PUB (Syn 5.1)

Mid-PUB (Syn 5.1)

Low-PUB (Syn 5.1)

Low-PUB (Syn 5.1)

PUB-lacking (Cbm)

PUB-lacking (Cbm)

PUB-lacking (Syn 5.1)

PUB-lacking (Syn 5.1)

PUB-lacking clades sister p = 0.18

Synechococcus 5.1 PUB-lacking sister to

remaining CpeBA sequences p = 0.085

(B) Rejected CpeBA phylogenies

High-PUB (Syn 5.1)

High-PUB (Syn 5.1)

Mid-PUB (Syn 5.1)

Mid-PUB (Syn 5.3)

Low-PUB (Syn 5.1)

Mid-PUB (Syn 5.1)

PUB-lacking (Syn 5.1)

Low-PUB (Syn 5.1)

PUB-lacking (Cbm)

PUB-lacking (Syn 5.1)

PUB-lacking (Cbm)

PUB-lacking (Cbm)

Mid-PUB (Syn 5.3)

Synechococcus 5.3 sister to remaining
CpeBA sequences p = 0.036

Cyanobium sister to remaining

CpeBA sequences p = 0.025

Fig. 5. Schematic representation of possible CpeBA evolutionary patterns in context of the 16S rRNA gene phylogeny of the strains from which the CpeBA sequences were
derived. (A) The most likely tree for CpeBA with Cyanobium (Cmb) shown twice to emphasize its paraphyly with respect to Synechococcus (top), followed by two non-rejected
phylogenies placing phycourobilin (PUB)-lacking CpeBA from Cyanobium as sister to PUB-lacking CpeBA from Synechococcus 5.1 (left) or PUB-lacking CpeBA from
Synechococcus 5.1 as sister to all other CpeBA sequences (right) using the approximately unbiased (AU) test. (B) Two rejected phylogenies that place CpeBA sequences from
either Synechococcus 5.3 (left) or Cyanobium (right) sister to all CpeBA sequences from Synechococcus 5.1. Syn 5.1 = Synechococcus 5.1, Syn 5.3 = Synechococcus, 5.3 (RCC307),
Cmb = Cyanobium (Arabian Sea strains). p-Values are for the AU test.

Conversely, positive selection was not detected on the branch leading from the ancestor of the (HM)L clade to the ancestor of the L
sequences, nor on the branch leading to the N sequences from the
ancestor of all the Synechococcus 5.1 CpeBAs (Fig 6).
3.3. Hypervariable region of CpeB
Everroad and Wood (2006) reported a hypervariable region of
approximately 35 amino acids long near the C-terminus of the
CpeB and MpeB sequences. In this previous study, they proposed
this region as a suitable taxonomic marker for PE, as well as for distinguishing CpeB from MpeB. However, this previous analysis did
not contain PUB-lacking PEs from Synechococcus 5.1. With the
addition of several new CpeB and MpeB sequences, including CpeB

from PUB-lacking Synechococcus, the present study reassessed this

hypervariable area. Supplementary Fig. S2 reveals this larger alignment and the utility of this region. As previously shown, it can differentiate between PEs from Prochlorococcus, Rhodophyta, nonpicocyanobacterial PEs, and CpeB and MpeB from Synechococcus
5.1. It can also distinguish low-PUB MpeB sequences from other
MpeB sequences, and it can differentiate between PUB-lacking,
high-PUB and other PUB-containing CpeB sequences. However, it
is not truly a taxonomic marker as previously proposed; it cannot
differentiate between PUB-lacking PEs from Cyanobium and Synechococcus 5.1, nor can it differentiate between PEs from Synechococcus 5.3 and 5.1. However, since this hypervariable region of the
gene was excluded from the phylogenetic analysis shown in Fig. 3,
the similarities that make it a poorer taxonomic marker than

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R.C. Everroad, A.M. Wood / Molecular Phylogenetics and Evolution 64 (2012) 381392

18:10
66:137

WH 8102
High-PUB H
CC9605

51:37
91:288

WH 8020
24:17
66:137
75:53
141:353

CC9311

Variable-PUB M

CC9902
M16.3

156:99
190:462

M16B.1

Low-PUB L

WH 7803
M12.1
PUB-lacking N
WH 7805
PCC 7421
Fig. 6. Relative rate ratio test for CpeBA. The number of replacement invariant and
replacement variant, silent invariant and silent variant substitutions (RI:RV and
SI:SV, respectively) are shown above the branches where positive directional
selection was detected (all branches at p < 0.001).

originally proposed do provide additional evidence that the PEs

from the Arabian Sea Cyanobium and Synechococcus 5.3 are evolutionarily truly Synechococcus 5.1 PEs.
3.4. Concluding remarks
These data give an interesting picture of the evolution of PE in
marine picocyanobacteria. As a group, these organisms differ from
many cyanobacteria and red algae in their widespread distribution
over a vast, uid environment in which the spectral composition of
the natural light eld varies dramatically. Three clades of marine
picocyanobacteria were detailed in this study: marine Synechococcus 5.1, marine Synechococcus 5.3, and Cyanobium (for CpeBA). All
three share the same unique ancestry of CpeBA and MpeBA apoprotein genes when considered in the context of other PE-containing cyanobacteria and red algae, indicating that the apoprotein of
their PEs evolved independently with respect to these other PEs.
The inferred phylogenies for both these genes clearly indicate that
PE has evolved directionally towards higher PUB content.
These PE results, in the context of the 16S rDNA gene tree, reveal that while both Synechococcus 5.3 and the Arabian Sea Cyanobium clearly possess Synechococcus 5.1-like PEs, these members of
Cyanobium are distinct from Synechococcus 5.1 based on their 16S
rRNA gene sequences. This strongly supported taxonomic separation between strains associated with the Cyanobium lineage and
the 5.1 lineage, combined with the data on the evolution of the
PE apoprotein genes from members of the PE-containing Cyanobium provides strong support for the acquisition of PE by these marine Cyanobium strains by HGT. Specically, the PEs from the marine
Cyanobium strains and Synechococcus 5.1 are mutually paraphyletic
in the best CpeBA PE tree (Fig. 3 and Fig. 5A) and PE from
Cyanobium is nested within Synechococcus 5.1 in the two alternative spectral phylogenies for CpeBA not rejected by the AU test
(Fig. 5A). If these results are combined with rejection of a tree with
Cyanobium CpeBA sister to all Synechococcus CpeBAs (Fig. 5B and
Table 1), the data strongly support a conclusion that the study
Cyanobium strains have acquired PE from marine Synechococcus
by HGT. This line of evidence is supported independently by the
shared identity of the length and sequences of the hypervariable

region in the C-terminus of CpeBA as discussed above. The study

of additional PE genes from other Cyanobium and Cyanobium-like
lineages will likely reveal more clearly the extent and nature of
such transfer events and the relationship between the distinct evolutionary lineages within the PE-containing picocyanobacteria.
The periodic selection model for bacterial populations predicts
that in clonal lineages, selective sweeps of strongly favored alleles
results in purges of less favored genotypes from the population
(Atwood et al., 1951; Cohan, 2002; Whitaker and Baneld, 2006).
For individual genes this results in an evolutionary pattern characterized by several niche-specic clusters of closely related sequences separated from each other by considerable evolutionary
distance (Whitaker and Baneld, 2006). Here it has been shown
clearly with strong statistical support that this model holds for
the evolution of PE genes in marine picocyanobacteria even though
they have a different evolutionary history than that of housekeeping loci like the 16S rRNA gene. For the groups studied here, the
phylogenetic history of PE apoprotein genes is the same for sequences that underlie any single spectral phenotype. Additionally,
the data show that gene duplication appears to have preceded evolution of mpeBA genes, and the data indicate that this event was
also required for inclusion of a PUB chromophore on any picocyanobacterial PE. More revealing in terms of evolutionary mechanism is the fact that the evolution of PE clades associated with
different phenotypes is ordered in a sequence that suggests an evolutionary adaptation from a pigment system adapted to use green
wavelengths that predominate in waters with high chlorophyll
content towards one with a greater ability to use the blue wavelengths that predominate in the waters of the open ocean. This evidence for molecular evolution in response to natural selection
involves an apparent progression from simpler to more complex
pigment systems after an episode of gene duplication. Together
the data presented here show marine picocyanobacteria to be an
exceptional model system for demonstrating the molecular mechanisms of adaptive phenotypic evolution.
Acknowledgments
The authors wish to thank Joe Thornton for numerous insightful
and helpful discussions. We also thank Scott Miller for critical comments and Kevin Emerson for technical assistance. This research was
funded by the Ofce of Naval Research (ONR Grant 0149910177 to
AMW), and NSF Grant OCE-0527139 (to AMW, CM Young, R Emlet,
and W Jaeckles). RCE acknowledges the support of a RIKEN FPR
fellowship. RCE was additionally supported by an NSF Integrated
Training Grant (IGERT) in Evolution, Development, and Genomics
(to the University of Oregon). Fieldwork in the Gulf of Mexico for
RCE was also supported by NSF Grant OCE-0118733 and NOAA/
NURP UNCW Grant NA96RU0260 (both to CM Young).
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ympev.2012.
04.013.
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