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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0
forBiggerDatasets
Sudhir Kumar
1,2,3
, Glen Stecher
1
, and Koichiro Tamura*
,4,5
1
Institute for Genomics and Evolutionary Medicine, Temple University
2
Department of Biology, Temple University
3
Center for Excellence in Genome Medicine and Research, King Abdulaziz University, Jeddah, Saudi Arabia
4
Research Center for Genomics and Bioinformatics, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
5
Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
*Corresponding author: E-mail: ktamura@tmu.ac.jp
Associate editor: Joel Dudley
Abstract
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many
sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized
for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of
thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a
new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available
in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows
application that can also be used on Mac OS X. The command line MEGA is available as native applications for
Windows, Linux, and Mac OS X. They are intended for use in high-throughput and scripted analysis. Both versions
are available from www.megasoftware.net free of charge.
Key words: gene families, timetree, software, evolution.
Molecular Evolutionary Genetics Analysis (MEGA) software is now
being applied to increasingly bigger datasets (Kumar et al. 1994;
Tamura et al. 2013). This necessitated technological advance-
ment of the computation core and the user interface of MEGA.
Researchers also need to conduct high-throughput and scripted
analyses on their operating system of choice, which requires
that MEGA be available in native cross-platform implementation.
We have advanced the MEGA software suite to address these
needs of researchers performing comparative analyses of DNA
and protein sequences of increasing larger datasets.
Addressing the Need to Analyze Bigger Datasets
Contemporary personal computers and workstations pack
much greater computing power and system memory than
ever before. It is now common to have many gigabytes of mem-
ory with a 64-bit architecture and an operating system to match.
To harness this power in evolutionary analyses, we have ad-
vanced the MEGA source code to fully utilize 64-bit computing
resources and memory in data handling, file processing, and
evolutionary analytics. MEGA’s internal data structures have
been upgraded, and the refactored source code has been tested
extensively using automated test harnesses.
We benchmarked 64-bit MEGA7 performance using 16S ribo-
somal RNA sequence alignments obtained from the SILVA
rRNA database project (Quast et al. 2013;Yilmaz et al. 2014)
with thousands of sites and increasingly greater number of se-
quences (as many as 10,000). Figure 1 shows that their
computational analysis requires large amounts of memory
and computing power. For the Neighbor-Joining (NJ) method
(Saitou and Nei 1987), memory usage increased at a polynomial
rate as the number of sequences was increased. The peak mem-
ory usage was 1.7 GB for the full dataset of 10,000 rRNA se-
quences (fig. 1B). For the Maximum Likelihood (ML) analyses,
memory usage increased linearly and the peak memory usage
was at 18.6 GB (fig. 1D).Thetimetocompletethecomputation
(fig. 1Aand C)showedapolynomialtrendforNJandalinear
trend for ML. ML required an order of magnitude greater time
and memory. We also benchmarked MEGA7 for datasets with
increasing number of sites. Computational time and peak mem-
ory showed a linear trend. In addition, we compared the mem-
ory and time needs for 32- and 64-bit versions (MEGA6and
MEGA7, respectively), and found no significant difference for NJ
and ML analyses. This is primarily because both MEGA6and
MEGA7 use 8-byte floating point data types. However, the 32-
bit MEGA6 could only carry out ML analysis for fewer than 3,000
sequences of the same length. Therefore, MEGA7isasignificant
upgrade that does not incur any discernible computational or
resource penalty.
Upgrading the Tree Explorer
The ability to construct a phylogenetic tree of >10,000 se-
quences required a major upgrade of the Tree Explorer as well,
because it needed to display very large trees. This was accom-
plished by replacing the native Windows scroll box with a
Brief communication
ßThe Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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Mol. Biol. Evol. doi:10.1093/molbev/msw054 Advance Access publication March 22, 2016 1
MBE Advance Access published April 13, 2016
at :: on April 19, 2016http://mbe.oxfordjournals.org/Downloaded from
custom virtual scroll box, which increased the number of taxa
that can be displayed in the Tree Explorer window from
4,000 in MEGA6 to greater than 100,000 sequences in
MEGA7. This is made possible by our new adaptive approach
to render the tree to ensure the best display quality and
exploration performance. To display a tree, we first evaluate
if the tree can be rendered as a device-dependent bitmap
(DDB), which depends on the power of the available graphics
processing unit. If successful, the tree image is stored in video
memory, which enhances performance. For example, in a
computer equipped with GeForce GT 640 graphics card,
Tree Explorer successfully rendered trees with more than
100,000 sequences and responded quickly to the user scroll-
ing and display changes. When a DDB is not possible to
generate, then Tree Explorer renders the tree as a device in-
dependent bitmap. Because of the extensive system memory
requirements, we automatically choose a pixel format that
maximizes the number of sequences displayed. Basically, the
pixel format dictates the number of colors used: 24 (2
24
colors), 18, 8, 4, or 1 bit (monochrome) per pixel. Memory
needs scale proportional to the number of bits used per pixel.
Cross-Platform MEGA-CC for High-Throughput and
Scripted Analyses
We have now refactored MEGA’s computation core (CC,
Kumar et al. 2012)sothatitcanbecompilednativelyfor
Linux, Windows, and Mac OS X systems in order to avoid the
need for emulation or virtualization. This required porting the
computation core source code to a cross-platform program-
ming language and replacing all the Microsoft Windows sys-
tem API calls. For instance, the App Linker system, which
integrates the MUSCLE (Edgar 2004) sequence alignment ap-
plication with MEGA, relied heavily on the Windows API for
inter-process communication and was refactored extensively.
In order to configure analyses in MEGA7-CC, we have chosen
to continue requiring an analysis options file (called .mao file)
that specifies all the input parameters to the command-line
driven MEGA-CC application; see figure 1 in Kumar et al. (2012).
To generate this control file, we provide native prototyper
applications (MEGA-PROTO) for Windows, Linux, and Mac OS
X. MEGA-PROTO obviates the need to learn a large number of
commands, and, thus, avoids a steep learning curve and po-
tential mistakes for inter-dependent options. It also enables us
to deliver exactly the same experience and options for those
who will use both GUI and CC versions of MEGA7.
Marking Gene Duplication Events in Gene Family Trees
We have added a new functionality in MEGA to mark tree
nodes where gene duplications are predicted to occur. This
system works with or without a species tree. If a species tree is
provided, then we mark gene duplications following Zmasek
and Eddy (2001) algorithm. This algorithm posits the smallest
number of gene duplications in the tree such that the min-
imum number of unobserved genes, due to losses or partial
sampling are invoked. When no species tree is provided, then
all internal nodes in the tree that contain one or more
FIG.1. Timeand memory requirements for phylogenetic analyses using the NJ method (A,B) and the ML analysis (C,D). For NJ analysis, we used the
Tamura–Nei (1993) model, uniform rates of evolution among sites, and pairwise deletion option to deal with the missing data. Time usage
increases polynomially with the number of sequences (third degree polynomial, R
2
¼1), as does the peak memory used (R
2
¼1) (A,B). The same
model and parameters were used for ML tree inference, where the time taken and the memory needs increased linearly with the number of
sequences. For ML analysis, the SPR (Subtree–Pruning–Regrafting) heuristic was used for tree searching and all 5,287 sites in the sequence
alignment were included. All the analyses were performed on a Dell Optiplex 9010 computer with an Intel Core-i7-3770 3.4 GHz processor, 20 GB
of RAM, NVidia GeForce GT 640 graphics card, and a 64-bit Windows 7 Enterprise operating system.
Kumar et al. .doi:10.1093/molbev/msw054 MBE
2
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common species in the two descendant clades are marked as
gene duplication events. This algorithm provides a minimum
number of duplication events, because many duplication
nodes will remain undetected when the gene sampling is
incomplete. Nevertheless, it is useful for cases where species
trees are not well established.
Realizing that the root of the gene family tree is not always
obvious, MEGA runs the above analysis by automatically root-
ing the tree on each branch and selecting a root such that the
number of gene duplications inferred is minimized. This is
done only when the user does not specify a root explicitly. A
Gene Duplication Wizard (fig. 2)walkstheuserthroughallthe
necessary steps for this analysis. Results are displayed in the
Tree Explorer (fig. 3) which marks gene duplications with blue
solid diamonds. When a species tree is provided, speciation
events are marked with open red diamonds. Results can also
be exported to Newick formatted text files where gene du-
plications and speciation events are labeled using comments
in square brackets. In the future, we plan to extend this sys-
tem with the capability to automatically retrieve species tree
from external databases, including the NCBI Taxonomy
(http://www.ncbi.nlm.nih.gov/guide/taxonomy/) and the
timetree of life (Hedges et al. 2015).
Timetree System Updates
We have now upgraded the Timetree Wizard (similar to
the wizard shown in fig. 2), which guides researchers
through a multi-step process of building a molecular phy-
logeny scaled to time using a sequence alignment and a
phylogenetic tree topology. This wizard accepts Newick
formatted tree files, assists users in defining the out-
group(s) on which the tree will be rooted, and allows users
to set divergence time calibration constraints. Setting
time constraints in order to calibrate the final timetree
is optional in the RelTime method (Tamura et al. 2012), so
MEGA7 does not require that calibration constraints be
available and it does not assume a molecular clock. If no
calibrations are used, MEGA7 will produce relative diver-
gence times for nodes, which are useful for determining
the ordering and spacing of divergence events in species
and gene family trees. However, users can obtain absolute
divergence time estimates for each node by providing
FIG.2. The Gene Duplication Wizard (A) to guide users through the process of searching gene duplication events in a gene family tree. In the first
step, the user loads a gene tree from a Newick formatted text file. Second, species associated with sequences are specified using a graphical
interface. In the third step, the user has the option to load a trusted species tree, in which case it will be possible to identify all duplication events in
the gene tree, from a Newick file. Fourth, the user has the option to specify the root of the gene tree in a graphical interface. If the user provides a
trusted species tree, then they must designate the root of that tree. Finally, the user launches the analysis and the results are displayed in the Tree
Explorer window (see fig. 3).
Molecular Evolutionary Genetics Analysis .doi:10.1093/molbev/msw054 MBE
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calibrations with minimum and/or maximum constraints
(Tamura et al. 2013). It is important to note that MEGA7
does not use calibrations that are present in the clade
containing the outgroup(s), because that would require
an assumption of equal rates of evolution between
the ingroup and outgroup sequences, which cannot be
tested. For this reason, timetrees displayed in the Tree
Explorer have the outgroup cluster compressed and
grayed out by default to promote correct scientific anal-
ysis and interpretation.
Data Coverage Display by Node
In the Tree Explorer, users will be able to display another set
of numbers at internal tree nodes that correspond to the
proportion of positions in the alignment where there is at
least one sequence with an unambiguous nucleotide or
amino acid in both the descendent lineages; see figure 5
in Filipski et al. (2014). This metric is referred to as mini-
mum data coverage and is useful in exposing nodes in the
tree that lack sufficient data to make reliable phylogenetic
inferences. For example, when the minimum data coverage
is zero for a node, then the time elapsed on the branch
connecting this node with its descendant node will always
be of zero, because zero substitutions will be mapped to
that branch (Filipski et al. 2014). This means that diver-
gence times for such nodes would be underestimated.
Such branches will also have very low statistical confidence
when inferring the phylogenetic tree. So, it is always good
to examine this metric for all nodes in the tree.
Conclusions
We have made many major upgrades to MEGA’s infrastructure
and added a number of new functionalities that will enable
researchers to conduct additional analyses with greater ease.
These upgrades make the seventh version of MEGA more ver-
satile than previous versions. For Microsoft Windows, the
64-bit MEGA is made available with Graphical User Interface
and as a command line program intended for use in high-
throughput and scripted analysis. Both versions are available
from www.megasoftware.net free of charge. The command
line version of MEGA7 is now available in native cross-platform
applications for Linux and Mac OS X also. The GUI version of
MEGA7 is also available for Mac OS X, where we provide an
installation that automatically configures the use of Wine for
compatibility with Mac OS X. Since Wine only supports 32-bit
software, we provide 32-bit MEGA7GUIforMacOSX.
However, Mac and Linux users can run the 64-bit Windows
version of MEGA7 GUI using virtual machine environments,
including VMWare, Parallels, or Crossover. Alternatively,
64-bit MEGA-CC along with MEGA-PROTO can be used as they
run natively on Windows, Mac OS X, and Linux.
Acknowledgments
We thank Charlotte Konikoff and Mike Suleski for extensively
testing MEGA7. Many other laboratory members and beta
testers provided invaluable feedback and bug reports. We
thank Julie Marin for help in assembling the rRNA data ana-
lyzed. This study was supported in part by research grants
from National Institutes of Health (HG002096-12 to S.K.) and
Japan Society for the Promotion of Science (JSPS) grants-in-
aid for scientific research (24370033) to K.T.
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