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ssBioMed CentBMC Evolutionary Biology
Open AcceSoftware
PhyloSort: a user-friendly phylogenetic sorting tool and its
application to estimating the cyanobacterial contribution to the
nuclear genome of Chlamydomonas
Ahmed Moustafa1 and Debashish Bhattacharya*1,2
Address: 1University of Iowa, Interdisciplinary Program in Genetics, 456 Biology Building, Iowa City, Iowa 52242, USA and 2University of Iowa,
Department of Biological Sciences and the Roy J. Carver Center for Comparative Genomics, 446 Biology Building, Iowa City, Iowa 52242, USA
Email: Ahmed Moustafa - ahmed-moustafa@uiowa.edu; Debashish Bhattacharya* - debashi-bhattacharya@uiowa.edu
* Corresponding author
Abstract
Background: Phylogenomic pipelines generate a large collection of phylogenetic trees that
require manual inspection to answer questions about gene or genome evolution. A notable
application of phylogenomics is to photosynthetic organelle (plastid) endosymbiosis. In the case of
primary endosymbiosis, a heterotrophic protist engulfed a cyanobacterium, giving rise to the first
photosynthetic eukaryote. Plastid establishment precipitated extensive gene transfer from the
endosymbiont to the nuclear genome of the 'host'. Estimating the magnitude of this endosymbiotic
gene transfer (EGT) and determining the functions of the prokaryotic genes remain controversial
issues. We used phylogenomics to study EGT in the model green alga Chlamydomonas reinhardtii.
To facilitate this procedure, we developed PhyloSort to rapidly search large collection of trees for
monophyletic relationships. Here we present PhyloSort and its application to estimating EGT in
Chlamydomonas.
Results: PhyloSort is an open-source tool to sort phylogenetic trees by searching for user
specified subtrees that contain a monophyletic group of interest defined by operational taxonomic
units in a phylogenomic context. Using PhyloSort, we identified 897 Chlamydomonas genes of
putative cyanobacterial origin, of which 531 had bootstrap support values ≥ 50% for the grouping
of the algal and cyanobacterial homologs.
Conclusion: PhyloSort can be applied to quantify the number of genes that support different
evolutionary hypotheses such as a taxonomic classification or endosymbiotic or horizontal gene
transfer events. In our application, we demonstrate that cyanobacteria account for 3.5–6% of the
protein-coding genes in the nuclear genome of Chlamydomonas.
Background
Phylogenomics
Recent advances in sequencing technologies and reduc-
sequence tag (EST) data and facilitated a revolution in the
field of comparative genomics. The availability of this
massive amount of genome data has been both a boon
Published: 15 January 2008
BMC Evolutionary Biology 2008, 8:6 doi:10.1186/1471-2148-8-6
Received: 29 August 2007
Accepted: 15 January 2008
This article is available from: http://www.biomedcentral.com/1471-2148/8/6
© 2008 Moustafa and Bhattacharya; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 7
(page number not for citation purposes)
tions in the cost of sequencing have fostered an unprece-
dented explosion of complete genome and expressed
and a major challenge for biologists. Phylogenomics
offers an useful avenue for dealing with genome data,
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allowing researchers to investigate gene phylogeny in a
genome-wide context [1]. This approach can be used to
annotate genes of unknown function in newly sequenced
genomes or to identify phylogenetic markers to infer an
accurate genealogy of life, or to understand the role of
endosymbiotic and horizontal gene transfer in eukaryotic
evolution [2]. Existing phylogenomic pipelines (e.g., Phy-
loGenie [3] and PhyloGena [4]) generate a large collec-
tion, often hundreds or thousands, of phylogenetic trees
that require manual inspection to observe general pat-
terns of genome evolution or to address specific hypothe-
ses about gene phylogeny.
Endosymbiosis
The engulfment of a free-living photosynthetic cyanobac-
terium by a heterotrophic protist (primary endosymbio-
sis) introduced photosynthesis into the eukaryotic
domain. The primary endosymbiosis occurred about 1.6
billion years ago (BYA) [5] and was a turning point for
evolution of life on our planet allowing the later develop-
ment of multicellular plants and animals. The first algae
diversified over time into the three primary photosyn-
thetic lineages, the green (later including land plants),
red, and glaucophyte algae. These taxa are united in the
eukaryotic supergroup Plantae. Some time after establish-
ment of the endosymbiotic relationship there was selec-
tive pressure to reduce the endosymbiont genome by
outright gene loss or transfer of genes to the host nuclear
genome. This latter process is termed endosymbiotic gene
transfer (EGT). Determining the extent of 'primary' EGT in
Plantae and whether only genes involved in plastid func-
tion were retained or were augmented by many other
genes of non-organellar function remain controversial
issues. Previously, using the flowering plant Arabidopsis, it
was estimated that the cyanobacterial endosymbiont con-
tributed 18% of the total set of nuclear genes in this spe-
cies [6]. In another study, EGT was shown to have
contributed only about 4% of the nuclear genes in Arabi-
dopsis, and 12% to the reduced nuclear genome of the
extremophilic red alga Cyanidioschyzon merolae [7]. In a
more recent study using the free-living glaucophyte alga
Cyanophora paradoxa, we estimated that about 4% of the
nuclear genome in this taxon was of cyanobacterial prov-
enance. However this latter study was based on incom-
plete EST data and is a provisional result. There exists
therefore a need to apply modern methods to analyze the
complete genome sequence of a mesophilic, free-living,
unicellular alga to generate a robust estimate of primary
EGT in a relatively 'simple' ancestor of land plants [2].
Furthermore, once the Plantae split into its constituent
lineages, a red (and likely also a green) alga was captured
by the ancestor of the chromalveolate protists via second-
nucleus of the algal endosymbiont to that of the chroma-
lveolate host through 'secondary' EGT. The chromalveo-
late supergroup includes a broad swath of protist diversity
including both photosynthetic (stramenopile algae, hap-
tophytes, dinoflagellates) and plastid-lacking (oomycetes,
ciliates, telonemids) lineages. Presumably the latter group
have lost their plastid secondarily. Phylogenomic analysis
of nuclear genes in these algae is expected therefore to
return trees which show gene origin in Plantae from a
cyanobacterial ancestor, followed by their transfer to
chromalveolates via a red or green algal secondary endo-
symbiosis [9] and [10].
Given the need to better understand EGT in algae using a
phylogenomic approach, we developed PhyloSort to ana-
lyze topologies in a high-throughput fashion. This open-
source Java tool inspects the topology of phylogenetic
trees to address the most frequently asked question in the
field: does a specific gene support the monophyly of cer-
tain operational taxonomic units (OTUs; e.g., cyanobacte-
ria and Plantae)? Here, we provide an overview of
PhyloSort and its application to the complete set of pre-
dicted proteins in the green alga Chlamydomonas rein-
hardtii to estimate the contribution through EGT of
cyanobacteria to the nuclear genome of a green alga.
Implementation
PhyloSort can be used via a graphical user interface (GUI;
Figure 1) and a text mode command line interface. Input
phylogenetic trees are read and parsed from a source
folder, where trees are stored as one tree per file in Newick
format, which is supported and produced by many phyl-
ogenetic inference tools such as PHYLIP [11], PAUP* [12],
PhyML [13] and RAxML [14]. Of the input trees, those
that satisfy the search criteria are copied or moved to an
output folder.
To begin the analysis, the hypothesized monophyletic
taxa are selected from a pool of taxa. This pool of taxa can
be loaded as a simple list from a plain text file. Alterna-
tively, a tree can be loaded that acts as a taxonomy refer-
ence for organizing the taxa in a phylogenetic format
(Figure 2). Finally, if no list is loaded or no reference tree
exists, the program will unite all taxa in all trees into a sin-
gle non-redundant list of taxa.
In a typical phylogenomic analysis, homologs within and
among different genomes give rise to multiple sequence
alignments (using, for example, MUSCLE [15] or Clus-
talW [16]) that do not all necessarily share among them
the same set of taxa. Therefore, the number of taxa that
represent members of any monophyletic group (e.g.,
cyanobacteria and Plantae) may (and likely will) varyPage 2 of 7
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ary endosymbiosis [8]. This process necessitated the
movement of genes involved in plastid function from the
from tree to tree. This can be explained, for example, by
lineage-specific gene duplications or gene losses. For this
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reason, in PhyloSort, the hypothesized taxa are arranged
in groups such that for any number of groups to be mono-
phyletic each group has to be represented by at least one
of its constituent taxa. In addition, because the names of
the sequences used in reconstructing the trees would have
different formats from one project or research group to
another, "regular expressions" [17] are used to extract taxa
(or species names) from the sequence names.
In addition to the topological constraint of monophyly,
the PhyloSort search can be adjusted by setting a mini-
mum bootstrap support value associated with the mono-
phyletic clades. This allows the identification of trees with
significantly supported (therefore, more reliable) mono-
phyletic relationships. Minimum and maximum number
of taxa in a trees and average number of genes per taxon
can also be chosen to sample differing levels of gene fam-
ily complexity.
PhyloSort offers two search modes, exclusive and inclu-
sive. In the exclusive mode, the taxa that exist in the tree
and belong to the hypothesized monophyletic taxa have
to be located in a single monophyletic clade. In the inclu-
sive mode, any group of taxa forming a clade that matches
the search criteria qualifies the trees regardless of whether
other taxa belong to the hypothesized monophyletic taxa
exist elsewhere in the tree. Prior to the monophyly search,
each tree is searched for an outgroup and then rerooted on
that taxon.
A genome-wide analysis generally produces a significant
number of trees that share multiple genes due to multiple
gene copies and gene families. Accordingly, to provide an
to merge trees by identifying overlapping genes among
trees and placing the trees into 'tree clusters' representing
gene families. A minimum number of overlapping genes
can be set for merging trees into clusters.
Through the GUI interface, a taxonomy reference tree
(Figure 2) can be used to hierarchically collect taxa and
simplify the assignment of taxa into groups, and phyloge-
netic trees can be visually inspected using ATV [18]. In
addition to the GUI and command line interfaces, Phy-
loSort provides a set of reusable and extendable applica-
tion programming interfaces (APIs) that can be
incorporated into other applications that may utilize the
monophyly search or other utility components such as
Screenshot of the Reference Taxonomy tree in PhyloSort (Windows XP) that can be used to selec the groups of taxaFigure 2
Screenshot of the Reference Taxonomy tree in PhyloSort
(Windows XP) that can be used to select the groups of taxa.
Screenshot of the main window of PhyloSort (Windows XP)Figure 1
Screenshot of the main window of PhyloSort (Windows XP).Page 3 of 7
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estimate of the number of unique gene families, PhyloS-
ort has a simple clustering functionality that can be used
Newick format parsing and basic phylogenetic tree
manipulation.
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To determine whether a set of taxa is monophyletic, there
are two main steps. First, the lowest common ancestor
(LCA) is located. Second, the subtree rooted by the LCA is
verified to not contain outgroups. The following subsec-
tions summarize the approaches that have been imple-
mented to determine whether a tree contains a clade that
matches the monophyletic criterion and any additional
constraints in both search modes.
LCA Identification
To locate the LCA for a set of leaves (taxa), the path from
the root to each leaf in the set is determined. Next, all
paths are compared to find the longest shared segment
(i.e., number of shared consecutive nodes). Then, the LCA
is the furthest node from the root on the longest shared
segment (Figure 3).
Exclusive search
Under the exclusive mode, all taxa in the tree that belong
to the set of taxa being examined for monophyly are
located and the LCA for the entire set is determined and
the clade rooted by the LCA is tested for monophyly.
Inclusive search
Under the inclusive mode, each leaf that belongs to the
groups of taxa being examined for monophyly is used as
a starting point to traverse the tree in a tip to root direc-
tion. Each internal node is examined as described above
in the exclusive search to test whether it contains a quali-
fying monophyletic clade. If the clade rooted by the inter-
nal node has a taxon that does not belong to the
hypothesized monophyletic taxa, the clade is rejected, all
nodes belong to the clade are marked not to be revisited,
and the next leaf is examined. If a clade is not rejected but
it does not satisfy the monophyly constraint or any addi-
tional filtering criterion, the parent of the clade is located
and the clade rooted by the parent is examined. Other-
wise, if the clade satisfies all criteria, the search stops
returning the tree as a matching tree. This search strategy
was specifically designed to identify trees that contain par-
alogs, a subset of which may satisfy the monophyly crite-
rion and therefore should be considered.
Results
As an example of the application of PhyloSort to address
biological questions, we quantified primary (cyanobacte-
rial) EGT in the green alga Chlamydomonas reinhardtii. Of
the 15,143 predicted Chlamydomonas nuclear-encoded
proteins, we found 4,631 that had cyanobacterial
homologs with a BLAST e-value < 1e-5 (i.e., ~30% of the
total number of genes) for which 4,129 trees were
inferred. With no restriction on the bootstrap support
value (i.e., bootstrap support value ≥ 0%), there were 897
(~6% of the genes) trees that showed monophyly of
cyanobacteria and Plantae (and in many cases, chromal-
veolates). By enforcing the minimum bootstrap support
values at ≥ 50% or ≥ 75%, we found 531 (~3.5% of the
genes) and 406 trees, respectively, that satisfied the mono-
phyly constraint. Clustering the 50% bootstrap category
resulted into at least 267 unique gene families. Based on
the gene ontology analysis (see Methods), at least 44% of
these predicted cyanobacterial genes were identified as
encoding plastid-targeted proteins and at least 47% were
involved in metabolic processes.
Discussion
Of the trees that supported the monophyly of cyanobacte-
ria and photosynthetic eukaryotes (represented by Plantae
and chromalveolates), we present here as an example the
phylogenetic tree of the thylakoid lumenal 17.4 kDa pro-
tein (Figure 4). This plastid-targeted protein belongs to
the "pentapeptide repeat" family of proteins of which the
function has not yet been characterized in Plantae. How-
ever, it has been shown that the pentapeptide repeats are
required for a proper localization of heterocyst glycolipids
in cyanobacteria [19].
A graphical cladogram representation of the tree (((a, b), c), (d, e))Fi ure 3
A graphical cladogram representation of the tree
(((a, b), c), (d, e)). In the tree, x is root, a, b, c, d and e are
terminals (leaves), and y and z are internals. To determine
whether a, b and c are monophyletic, the following steps are
performed: I. The paths from x to a, b, and c are (x → y → z
→ a), (x → y → z → b), and (x → y → c) respectively. II. The
longest shared segment among the three paths is (x → y). III.
The LCA of a, b, and c is y. IV. The subtree rooted by y con-
tains only a, b, and c. V. a, b, and c are monophyletic in the Page 4 of 7
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clade rooted by y.
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The tree demonstrates the phylogeny predicted for plastid
primary and secondary endosymbiosis [2]. The nuclear-
encoded Plantae proteins are of cyanobacterial origin (pri-
mary EGT), whereas the chromalveolate sequences are
rooted within red algae (secondary EGT; 72% bootstrap
support value). Here the Plantae are represented by its
three member lineages, the glaucophyte Cyanophora, the
red alga Cyanidioschyzon, the green algae Ostreococcus,
Chlamydomonas, Volvox and Physcomitrella, and the plants
Arabidopsis and Oryza, whereas the chromalveolates are
represented by the diatoms Phaeodactylum and Thalassio-
sira, the haptophyte Emiliania, the cryptophyte Guillardia,
and the dinoflagellate Alexandrium). Previous analyses
suggest that the chromalveolate red algal secondary endo-
symbiosis occurred 'soon' (ca. 1.3 BYA [5]) after the
cyanobacterial capture. Many other Chlamydomonas pro-
teins display the same topology as shown in Figure 4 and
are available as individual trees in [Additional file 1].
Specifically, of the 531 genes that belong to the 50% boot-
strap category, about 51% of the genes were shared among
red and green algae and about 12% were shared among
glaucophytes, red and green algae. The unevenness of the
distribution of cyanobacterial genes among the Plantae
lineages can be explained by limited EST data that are
available from glaucophytes (the nuclear genome is cur-
rently being sequenced to completion [20]), the highly
reduced nature of the genome of the red alga Cyanidio-
schyzon (16.5 Mb [21]) which likely precipitated the loss
of many target genes, and the sampling bias towards the
green lineages due to the use of Chlamydomonas as the
query for the phylogenomic analysis. In summary, using
PhyloSort we were able to rapidly inspect thousands of
phylogenetic trees of differing complexities and number
of terminal taxa and demonstrate the contribution of
cyanobacterial primary EGT to Plantae nuclear genomes.
Phylogenetic tree of thylakoid lumenal 17.4 kDa proteinFigure 4
Phylogenetic tree of thylakoid lumenal 17.4 kDa protein. This is a maximum likelihood tree inferred using RAxML-VI-
HPC, v2.2.1 with the JTT evolutionary model and 100 bootstrap replicates (bootstrap values < 50% are omitted from the phy-
logeny). We used a random starting tree (one round of taxon addition) and the rapid hill-climbing algorithm (i.e., option -f d in
RAxML). The tree was drawn using Drawtree . Lineages are color-coded as follows: green → green algae and land plants, red Page 5 of 7
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→ red algae, brown → chromalveolates, blue → glaucophytes, and black → cyanobacteria.
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