See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/337959323

Phylogenomics — principles, opportunities and pitfalls of big‐data

phylogenetics

Article  in  Systematic Entomology · December 2019

DOI: 10.1111/syen.12406

CITATIONS READS

47 6,702

2 authors:

Andrew D. Young Jessica Gillung

University of Guelph McGill University
20 PUBLICATIONS   270 CITATIONS    44 PUBLICATIONS   353 CITATIONS   

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Jessica Gillung on 28 January 2020.

The user has requested enhancement of the downloaded file.

Systematic Entomology (2019), DOI: 10.1111/syen.12406
OPINION
Phylogenomics – principles, opportunities and pitfalls
of big-data phylogenetics
1
A N D R E W D . Y O U N G † and J E S S I C A P . G I L L U N G 2
1
California Department of Food and Agriculture, Plant Pest Diagnostics Center, Sacramento, CA, U.S.A. and 2 Department of Natural
Resource Sciences, McGill University, Sainte-Anne-de-Bellevue, Canada
Introduction
Phylogenetics is the science of reconstructing the evolution-
ary history of life on Earth. Traditionally, phylogenies were
constructed using morphological data only, but the introduc-
tion of Sanger sequencing and PCR in the late 1970s enabled
genetic information to be incorporated into phylogenetic
analyses. Early phylogenetic studies employing multilocus
analyses contributed greatly to our knowledge of phylogenetic
history and challenged some well-established views of the
relationships among many groups of plants and animals. Since
the publication of these pioneering studies, significant method-
ological advances in both sequencing and analytical techniques
have been made, and molecular phylogenies are now broadly
accepted to represent robust hypotheses of organismal relation-
ships. Next-generation sequencing techniques, developed in the
mid-2000s, revolutionized DNA sequencing and led to a dra-
matic reduction in sequencing cost per nucleotide and a sharp
increase in data generation speed. As a result, the generation
of unprecedented amounts of sequence data for both model
and nonmodel organisms has become affordable. This develop-
ment has transformed the field of molecular phylogenetics into
phylogenomics – where genome-scale data are obtained from
multiple samples at once at a much reduced cost (Mardis, 2011).
The phylogenomic pipeline can be very complex, present-
ing an overwhelming array of methodologies available for the
acquisition, manipulation, analysis and interpretation of massive
datasets. Researchers also have to overcome the challenges of
sequencing strategy design, identification of orthologous loci,
model selection and phylogeny estimation. This can be par-
ticularly daunting for researchers new to the field – both stu-
dents and established scientists – who wish to delve into novel
methods and data to reconstruct the evolution of their study
group. Here we present an entry-level overview of the theory
Correspondence: Jessica P. Gillung, Department of Natu-
ral Resource Sciences, McGill University, 21111 Lakeshore
Road, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada. E-mail:
jpg.bio@gmail.com
†Present address: School of Environmental Sciences, University of
Guelph, Guelph, Ontario, N1G 2W1, Canada.
© 2019 The Royal Entomological Society
and tools that are central to phylogenomics, with an emphasis
on the appropriate application of techniques useful for phylo-
genetic analysis of genomic data. We focus on the sequencing
technologies and statistical methods for phylogeny estimation,
and the software implementing these methods and their appli-
cation to large molecular datasets. We also discuss the tools
and tradeoffs for improving the accuracy of phylogenomic anal-
yses, including the biological and methodological sources of
systematic error in phylogeny estimation. Finally, we provide
a glossary of commonly encountered terms used in phyloge-
nomics that may be useful for those entering the field and hop-
ing to sort through the multitude of methods, analytical tools
and terminology inherent to this relatively new, but rapidly
advancing field.
What is phylogenomics?
The word ‘phylogenomics’ was first introduced in the con-
text of prediction of gene function for genome-scale data
(Eisen, 1998), and soon after in the context of phyloge-
netic inference (O’Brien & Stanyon, 1999). The discipline
of phylogenomics owes its existence to the advances made
in DNA sequencing technology over the past two decades
(Metzker, 2010). It comprises several areas of research at
the interface between molecular and evolutionary biology
and has two major goals: (i) to infer phylogenetic relation-
ships between taxa and gain insights into the mechanisms of
molecular evolution; and (ii) to use multispecies phylogenetic
comparisons to infer putative functions for DNA or protein
sequences.
Traditional Sanger sequencing studies include relatively few
loci and are therefore limited by stochastic or sampling error.
As there is a relatively small number of phylogenetically infor-
mative characters available in one or a few genes, this random
‘noise’ influences the inference of backbone nodes, potentially
leading to poorly resolved or poorly supported phylogenetic
trees. This problem can be addressed successfully by using much
larger amounts of sequence data. Modern phylogenomic analy-
ses, which take advantage of hundreds to thousands of loci from
across the genome, are, on average, orders of magnitude larger
1
2 A. D. Young and J. P. Gillung
than traditional Sanger sequencing datasets. The size of these
datasets therefore significantly reduces the impact of stochas-
tic error and data availability as a limiting factor, offering great
promise for resolving historically recalcitrant nodes in the tree
of life.
High-throughput sequencing technologies [also called
next-generation sequencing (NGS)] (Fig. 1) have yielded
genome-scale data in immense quantities. Next-generation
sequencing technologies differ fundamentally from the Sanger
method in that they allow for massively parallel DNA sequenc-
ing, providing extremely high throughput from multiple samples
simultaneously and at a much reduced cost (Mardis, 2011).
Millions to billions of DNA nucleotides can be sequenced in
parallel, yielding orders of magnitude more data and mini-
mizing the need for the fragment-cloning methods that are
used with Sanger sequencing (Fig. 1). Recent progress in
NGS technology and the rapid development of bioinformat-
ics tools now allow research groups of any size to generate
large amounts of genomic sequences for organisms of interest.
High-throughput sequencing can be used for whole-genome
sequencing (Lam, 2012), whole-transcriptome shotgun sequenc-
ing (also called RNA sequencing, RNA-seq, or transcriptomics;
Wang, 2009), whole-exome sequencing (Rabbani, 2014), and
reduced-representation genome sequencing (also called target
enrichment) (e.g., Faircloth, 2012; Lemmon, 2012).
Table 1 summarizes the most commonly used sequencing
technologies in phylogenomics. For more details on these
different technologies see the Beginner’s Handbook of Next
Generation Sequencing by Genohub (https://genohub.com/
next-generation-sequencing-handbook/) (see also Ambardar,
2016; Besser et al., 2018, and references therein). Choosing
the appropriate sequencing technology for a phylogenomic
study has important effects on downstream workflows, espe-
cially in terms of read length, as library preparation in some
phylogenomic techniques (e.g. ultraconserved elements and
anchored hybrid enrichment, discussed later) requires a read size
selection step.
A road map for the phylogenomic workflow
Strict experimental reproducibility is an integral – albeit
uncommon – aspect of biological sciences, mainly due to var-
ied technical challenges with implementation and curation of
experimental methods and procedures. Despite the importance
of phylogenetic analyses to most fields of biology, the repro-
ducibility of phylogenetic experiments can be very low, with
an estimated 60% of published phylogenetic analyses being
‘lost to science’ due to the unavailability of the underlying data
and methods (Magee, 2014). Published phylogenetic studies
can be difficult or impossible to replicate or expand upon, as
the utilized analytical software, software versions, software
parameters, dependencies and operating system versions can be
very challenging to uncover or recreate.
The promotion of open science and reproducible research can
create a more productive and responsible scientific culture in
phylogenomics, enabling researchers to build upon previous
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
studies and continuously address larger and more complex
questions. This philosophy encompasses the sharing of data and
code used to produce the analysis, as well as open archiving of all
raw data (Mork, 2015; Shade & Teal, 2015). Data provenance,
the recording of the input and transformation of information
used to generate a result, is a key issue in reproducibility. Several
recommendations and guidelines to promote the best practices
in reproducibility and data management in phylogenomics
and bioinformatics have been proposed (Cranston et al., 2014;
Magee, 2014; Debiasse & Ryan, 2019), and many tools for
ensuring provenance and curation of both data and methods have
been developed (e.g. Dunn, 2013; Oakley, 2014; Szitenberg,
2015).
To ensure the best practices in phylogenomics and bioin-
formatics, it is vital that reproducibility checkpoints are
enforced – places in a workflow devoted to scrutinizing its
integrity, so results are validated across multiple iterations to
ensure consistency of results.
Additionally, adopting an iterative, branching workflow to sys-
tematically explore the methodological space is crucial. Linear
methodology, with experimental and computational procedures
lined up one after the other, as presented in most published
studies, is rarely the reality of phylogenetic analysis. Instead,
estimating phylogenetic trees is more often than not a messy
enterprise, and a systematic exploration of the methods and data
is recommended in order to select the best tools and pipelines
to answer the question at hand. Finally, for good provenance of
experimental procedures and computational tools utilized in a
particular study, it is highly recommended that comprehensive
notes are kept throughout the process. In particular, keeping a
‘readme’ file at every step can be extremely helpful in keeping
track of the software versions used, parameter values utilized,
goals of each step and how they relate to the software utilized, as
well as indication of data format changes. All these can greatly
contribute to standardization and ease of downstream efforts
(Shade & Teal, 2015).
Phylogenomic data are a precious scientific resource: molec-
ular sequence alignments and phylogenies are expensive to
generate, difficult to replicate and have seemingly infinite poten-
tial for synthesis and reuse. For most phylogenomic analyses,
phylogeneticists are faced with a large combination of algo-
rithms, models and data manipulation techniques. To address
this issue, here we present a flowchart containing the major steps
and tools utilized in phylogenomics (Fig. 2). The flowchart is
not meant to be exhaustive, but merely a visualization of the
commonly utilized methodologies and pipelines in recent
phylogenomic studies.
Taxon sampling: a crucial first step in phylogenomics
Taxon sampling is of extreme importance for phylogenetic infer-
ence, and increased sampling of taxa – coupled with increased
sampling of loci – is commonly advocated as a solution to
resolving recalcitrant nodes of the tree of life. Ideally, sampling
 Understanding phylogenomics 3

Fig. 1. Comparison of Sanger sequencing (left) and Illumina next-generation sequencing (right). A flow cell is a glass slide with lanes, each lane being
a channel coated with two types of oligos, which are complementary to the adapters ligated in the DNA fragments. dsDNA, double-stranded DNA; F,
forward DNA strand; R, reverse DNA strand; ssDNA, single-stranded DNA. [Colour figure can be viewed at wileyonlinelibrary.com].

© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406

4 A. D. Young and J. P. Gillung

of both taxa and sequences should be increased at the same pace,

Indels, deletions
but the advances in high-throughput sequencing have caused

Read length,
up to 100 kb
increases in gene sampling to far outpace taxon sampling. As

Nanopore

portability
greater amounts of data are incorporated into phylogenetic stud-
Oxford

0.1–1

5–40
ies, new evidence and hypotheses regarding relationships among

SR
taxa can emerge, and placement of lineages within clades can
change dramatically. Taxon sampling can thus greatly influence

Speed, read length

hypotheses supported by phylogenetic inference (Rosenberg &
Kumar, 2003; Nabhan & Sarkar, 2012).
Biosciences

up to 60 kb Taxon selection meant to address a specific research question

0.5–10
Pacific

should take place early in a phylogenomic study. ‘Sufficient’

Indels
taxon sampling is always dependent on the questions being
SR
13
addressed. Ideally, in order to unravel the phylogeny of an
entire taxonomic unit, most, if not all, subordinate taxa in that
Speed, read length
unit should be sampled. Even though increasing the number
homopolymers

of taxa results in a more complex computational problem for

Ion Torrent

phylogenetic analysis, it has been demonstrated that denser

200–400

Indels,

taxon sampling improves phylogenetic accuracy (Heath et al.,

1–15

1–2
SR

2008).
Taxon sampling, however, can be greatly limited by the phy-
logenomic method of choice. Transcriptomics, for instance,
High throughput

requires specimens collected and stored directly into liquid

Substitutions

nitrogen or RNAlater, whereas other sequencing methods,

2000–6000
Table 1. Comparison of different next-generation sequencing (NGS) platforms. SR, single read; PE, paired-end read.

NovaSeq

including target enrichment, and shotgun and exome sequenc-

0.2–0.8
SR, PE

ing, will require molecular-grade specimens, preferably pre-

250

served in high-grade ethyl ethanol and stored in a laboratory

ultrafreezer. A notable exception to this is target enrichment of
ultraconserved elements (UCEs), a method that can successfully
Throughput, low

generate phylogenomic data from old, pinned insect museum

Substitutions
1000–1800

specimens (Blaimer, 2016).

error rate
SR, PE

Genome-scale projects may be particularly vulnerable to sys-

HiSeq

150

tematic error caused by nonproportional phylogenetic sampling.

0.2

As dataset size increases, so does the accumulation of non-

random systematic error and accompanying nonphylogenetic
Substitutions

Throughput

signal (Jeffroy, 2006). Bayesian analyses of macroevolutionary

NextSeq

10–120

patterns – including divergence-time estimation, ancestral state

SR, PE

reconstruction, and diversification rate estimation – assume pro-

150

0.8

portional sampling of lineages within a clade, and deviations

from it may potentially lead to biases (Stadler, 2009). However,
Substitutions

Read length

some implementations enable ‘corrections’ for uneven taxon

Illumina

sampling (e.g. revbayes implements corrections for birth-death

SR, PE
MiSeq

10–15

and various diversification rate models, except for fossilized

300

0.8

birth-death).
Before sequencing new specimens, it is also worth evaluat-
ing previously sequenced resources. The National Center for
Read accuracy

Biotechnology Information’s Sequence Read Archive (NCBI

Substitutions
Up to 1 kb

and length

SRA) contains user-uploaded raw sequence data and align-

c. 0.0005

0.001–1
Sanger

ment information from high-throughput sequencing projects

(Leinonen, 2011). Other resources include FlyBase (Thurmond,
SR

2019), a large database of Drosophila genes and genomes,

WormBase (https://www.wormbase.org), containing genomic
data of Caenorhabditis elegans and related nematodes, and the
Error rate (%)
range per run
Throughput

Read length

UCSC Genome Browser (Kent et al., 2002), a large repository

Advantages
Error type
Read type

of mostly vertebrate genomes. Utilizing sequences from these

databases can save money and/or increase taxon sampling in
(Gb)

ongoing phylogenomic projects.

© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406


Fig. 2. A generalized phylogenomic project workflow. WGSS,
whole-genome shotgun sequencing; AHE, anchored hybrid enrichment;
UCE, ultraconserved element; ML, maximum likelihood. [Colour
figure can be viewed at wileyonlinelibrary.com].
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 5
Acquisition of DNA sequences: from specimens
to plastic tubes
For a comprehensive overview of insect DNA methods,
see Moreau (2014), which offers a detailed description of
DNA extraction methods using either commercial kits or
phenol/chloroform protocols.
After DNA extraction, specimens should be deposited in
publicly accessible collections in association with their unique
identifier, and publications utilizing these data should always
include unique identifier, repository and specimen metadata
(including specimen collector, date and method of collection,
and geographic origin).
Vouchering specimens with unique identifiers (alphanumeric
database number) is crucial for all phylogenetic projects. There-
fore, nondestructive or partially destructive DNA extraction
methods should be used whenever possible, and in these cases,
the extracted specimen itself becomes the voucher. By con-
trast, when nondestructive DNA extraction is not possible,
such as in transcriptomic projects or small-bodied organisms,
a photographic voucher can be associated with the sequence
data. Moreover, when the destroyed specimen is part of a
sample of conspecifics (e.g. in communal or social insects),
another specimen from the same sample can serve as a voucher,
provided it is made clear that it is not the extracted speci-
men. Properly vouchering specimens used for DNA extraction
greatly increases reproducibility by alleviating issues related
to sample identity and unstable taxonomy (Pleijel et al., 2008;
Turney, 2015).
A vast array of techniques to generate phylogenomic
data
Although large phylogenomic datasets have become increas-
ingly more accessible and cost-efficient in recent years, it is now
widely accepted that simply increasing the amount of sequence
data will not unambiguously resolve some of the most diffi-
cult nodes in the tree of life, mainly due to systematic error
from nonphylogenetic signal or model inadequacy. Appropri-
ate locus selection is therefore crucial in phylogenomics, but
knowledge of the best molecular markers for resolving diffi-
cult branches at various evolutionary depths is still incipient.
Questions still remain about whether to use coding or noncod-
ing sequence data, conserved or highly variable loci, and long or
short alignments (Betancur-R. et al., 2014; Edwards et al., 2016;
Chen et al., 2017). Therefore, one of the most critical decisions
in a phylogenomic project is the sequencing method to be uti-
lized, a decision that must be made a priori as each method will
result in different types of genomic data sequenced. Different
methods have their own characteristics, advantages, and limita-
tions, including cost-effectiveness, ease of use, sample quality
required, and downstream data filtering and analysis workflow.
Phylogenomic sequencing methods (Table 2) can be broadly
subdivided into shotgun sequencing and target enrichment
sequencing. Shotgun sequencing is the process of sequencing
6 A. D. Young and J. P. Gillung

Table 2. Main characteristics of different phylogenomics sequencing methods. UCE, ultraconserved element; AHE, anchored hybrid enrichment;
nrDNA nuclear ribosomal DNA.

Whole-genome
UCEs AHE RNA-seq shotgun Genome skimming
Missing data Low Low Medium Very low if high Low
among species coverage
Type and quality High-grade, alcohol High-grade, alcohol Must be preserved High-grade, alcohol High-grade, alcohol
of specimens preserved best; preserved best; specifically for RNA preserved best; preserved best;
needed museum specimens museum specimens work in liquid museum specimens museum specimens
often acceptable sometimes acceptable nitrogen or RNAlater sometimes acceptable often acceptable

DNA template Low Medium High High Low

quality needed
Genome data Targeted nuclear loci; Targeted nuclear and Randomly sequenced Nearly complete Partial or complete
obtained partial or complete mitochondrial loci; loci, vast majority of genome; symbiont mitochondrial
mitochondrial partial or complete nuclear genome; genomes as by-catch. genome; nrDNA;
genomes as by-catch. mitochondrial symbiont loci as Coding and symbiont genomes as
Symbiont loci could genomes as by-catch. by-catch. Coding noncoding sequences by-catch. Coding and
be targeted in the Symbiont loci could sequences only noncoding sequences
probe set. Coding and be targeted in the
noncoding sequences probe set. Mostly
coding sequences

Taxonomic levels All levels from Mostly deep levels Deep levels All levels from All levels from
for phylogenetic shallow to deep shallow to deep shallow to deep
relationships
Relative cost Medium-low Medium High Medium-high, Low
depending on the
coverage and depth

from the entire fragmented genome at random, returning part or
all of the genome depending on the sequencing depth achieved,
whereas target enrichment uses bidirectional probes (analogous
to primers in Sanger sequencing) to recover only genomic
regions of interest. Popular methods of shotgun sequencing
include genome skimming, whole-genome shotgun sequencing
and transcriptome sequencing (i.e. RNA-seq). Popular methods
of target enrichment for phylogenetics include anchored hybrid
enrichment (AHE) (Lemmon, 2012) and UCEs (McCormack
et al., 2012; Faircloth et al., 2012) [see also Mandel (2014)
for an alternative method developed for plants in the family
Compositae]. These techniques are reviewed briefly in Table 2
and have been covered in more detail elsewhere (e.g. Lemmon
& Lemmon, 2013; McCormack, 2013; Wen et al., 2015; Zhang
et al., 2019).
Shotgun sequencing
Shotgun sequencing (Fig. 3) involves fragmenting template
DNA into short pieces, which are then randomly sequenced
to obtain reads. Next, various methods and software are used
to overlap different reads and assemble them into a longer
DNA sequence called a contig. RNA-seq can be considered a
special form of shotgun sequencing, where whole mRNA is
first extracted and reverse-transcribed into reverse-complement
DNA, which is then sequenced.
Sequencing depth, or the average number of times an individ-
ual base in the genome is sequenced, is a key concept in shotgun
sequencing. Because the genome is sequenced at random,
multiple-copy regions of the genome (i.e. mitochondrial, ribo-
somal, and plastid DNA) are sequenced more frequently than
single-copy regions. Therefore, when a genome is sequenced at
a relatively shallow depth, only fragments from multiple-copy
regions of the genome are sequenced in sufficient quantities to
be successfully recovered. Shallow-depth whole-genome shot-
gun sequencing is also called genome skimming (Straub et al.,
2012), a time and cost-efficient method of sequencing mostly
mitochondrial, ribosomal and plastid DNA. Conversely, when
near-complete genomes are desired from whole-genome shot-
gun sequencing, a much greater sequencing depth is required
in order to sequence sufficient numbers of fragments from
single-copy regions of the genome.
By contrast, in RNA-seq (or transcriptomics) the extracted
mRNA is used as a template to generate reverse-complement
DNA. This reverse-complement DNA is then sequenced, result-
ing in data generated from only the genomic regions undergo-
ing active transcription at the time of tissue preservation. This
method is therefore not only a genome-reduction strategy, but
also facilitates the comparison of transcription activity between
individual tissues, life stages, rearing conditions, etc. One of
the major drawbacks of transcriptomics is the high tissue qual-
ity required – specimens must be flash-frozen in liquid nitro-
gen or collected directly into RNAlater, thus precluding the

© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406

 Understanding phylogenomics 7

(A)

(B)

(C)

Fig. 3. Comparison of main shotgun sequencing methods used in phylogenomics. [Colour figure can be viewed at wileyonlinelibrary.com].

Fig. 4. Comparison of target enrichment sequencing methods commonly used in phylogenomics. [Colour figure can be viewed at
wileyonlinelibrary.com].

© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406

8 A. D. Young and J. P. Gillung
utilization of specimens already available in tissue collections
or museums.
Target enrichment sequencing
Targeted sequence capture, or target enrichment (Fig. 4), is
an umbrella term for multiple efficient, cost-effective methods
for generating phylogenomic datasets for nonmodel organisms.
These methods effectively reduce genomic DNA complexity
through the use of short (60–120 bp), single-stranded nucleotide
baits or probes that hybridize with template sequences, thus
enabling the recovery of particular sequences of interest with
high coverage. As a result, mostly genomic regions of interest
are recovered, although nontarget DNA (including mitochon-
drial and symbiont sequences) can be present in the result-
ing set of reads. Multiple samples can be multiplexed and
sequenced together, which enables the generation of DNA
sequence data for hundreds of loci from over 100 samples
simultaneously. There are two main methods of target enrich-
ment commonly used in animal phylogenomics: AHE (Lemmon
et al., 2012) and UCEs (McCormack et al., 2012). Both methods
are reduced-representation approaches that rely on the utiliza-
tion of a phylogenetically informative subset of the study organ-
isms’ genomes. Other target enrichment methods have been pro-
posed, with varied locus selection criteria, including Hyb-Seq
(Weitemier et al., 2014), Compositae COS loci (Mandel et al.,
2014) and RELEC (Karin, 2019), but AHE and UCEs have thus
far been the most commonly used methods in phylogenomic
studies of animals.
In these methods, probes hybridize in-solution with targeted
anchor sequences, which are subsequently enriched. Both the
genomic regions targeted by the probes and their flanking
regions are sequenced, such that both conserved and more
variable (and thus more phylogenetically informative) regions
are sequenced at once. These reduced-representation methods
enable researchers not only to capture the same sets of loci
across all taxa of interest, but also to exclude repetitive or
phylogenetically misleading regions of the genome, including
pseudogenes and paralogues. A great benefit of consistently
using the same sets of markers across studies is that it allows
for meta-analyses as more phylogenomic data accumulate over
time. By contrast, for RNA-seq datasets to be consistent, the
transcriptomes must be gathered from the same tissue types, and
it can be challenging to accurately assess orthologues and align
different isoforms during analysis.
Anchored hybrid enrichment sequencing targets mainly
protein-coding regions of the genome, meaning that enriched
loci comprise mostly coding, and in some cases intronic or
other genomic elements (e.g. untranslated region). This means
that phylogenetic data can be more easily coded and analysed
both as nucleotides and as amino acids. Candidate target loci
are identified using genomes, transcriptomes or raw genomic
reads from two or more reference species. Then, sequences for
the target loci for each reference species to be included in the
probe kit design are isolated, and an alignment for each locus is
generated. Probes are developed based on these alignments, for
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
which a substantial amount of quality control is applied such
that target loci are single copy and have the appropriate amount
of sequence variation to ensure both phylogenetic accuracy and
efficient enrichment (Lemmon, 2012). Full probe kits target
500–800 protein-coding loci on average, and marker genes
(also called traditional or legacy genes, e.g. COI) are often
included in the target pool of loci, which facilitates integration
with previous Sanger sequencing phylogenetic studies.
Ultraconserved elements, in turn, are highly conserved regions
of the genome shared among evolutionarily distant taxa. As
universal genetic markers for particular taxa of interest, UCE
data are useful for reconstructing the evolutionary history and
population-level relationships of many organisms. In short,
UCEs are identified by aligning several genomes to each other,
with subsequent detection and filtering of areas of very high
(95–100%) sequence conservation across all taxa of interest.
There are a number of different ways of identifying UCEs for
use as genetic markers and designing baits to target them, but
the most commonly used approach in animal phylogenetics
was described in detail in Faircloth (2017). Ultraconserved
elements have been shown to perform well when collecting data
from museum samples (Blaimer et al., 2016; McCormack et al.,
2016), which can greatly facilitate expansion of taxon sampling
as sequencing is no longer restricted to fresh specimens. Despite
older specimens producing fewer and shorter loci in general, it
is still possible to retrieve hundreds of markers from relatively
old specimens (McCormack et al., 2016).
A major advantage of UCEs over AHE data is phyluce, an
user-friendly and open-source software pipeline developed for
the processing and analysis of UCE data (Faircloth, 2016). phy-
luce contains several software packages and tutorials that are
extremely helpful and accessible, especially to beginners. These
resources, however, require some familiarity with working in
the command-line environment of a Unix or Unix-like system.
For a comprehensive and user-friendly guide to working on
the command line see the Happy Belly Bioinformatics tutori-
als (https://astrobiomike.github.io/unix/). For a comprehensive
and informative overview of the theory and practice of UCEs
for arthropod phylogenomics, see Zhang et al. (2019).
Building phylogenomic data: quality control
and assembly of raw reads
Performing quality-control on raw reads obtained from
high-throughput sequencing is a crucial, yet sometimes over-
looked step. Reads should ideally be inspected for sequence
quality, guanine-cytosine (GC) content, presence of adapter
sequences and read errors (i.e. base calling errors and small
insertions/deletions). Several programs have been proposed
for quality control of NGS data, including fastqc for Illu-
mina reads (Andrews, 2010), and ngsqc for reads from all
other platforms (Dai et al., 2010). These two resources offer
a means to detect and visualize potential errors, after which
a complementary software, such as trimmomatic (Bolger
et al., 2014), can be used to trim low-quality bases and remove
adapter contamination. While trimmomatic offers an all-in-one

solution to sequence quality control, being the most commonly
used in animal phylogenomics, other similar programs have
been proposed. cutadapt, for instance, is used only for read
trimming, but is especially useful for working with sequences
obtained from Applied Biosystems’ SOliD sequencer (Martin,
2011). Likewise, scythe is a Bayesian approach for removing
adapters from the 3′ end of sequences, where read quality is
often degraded (https://github.com/vsbuffalo/scythe). sickle
is a standalone program used only for read quality trimming
(Joshi & Fass, 2011). Finally, htstream consists of a com-
prehensive quality control pipeline that allows streaming from
application to application (https://github.com/ibest/HTStream).
The software can simultaneously handle both single-end and
paired-end reads and enables process parallelization.
Once quality control has been performed on raw reads, the
next step is the assembly of contigs. Sequence assembly refers
to aligning and merging small DNA fragments obtained from
a high-throughput sequencing platform in order to recon-
struct longer DNA sequences. Sequence assembly is necessary
because whole genomes cannot be sequenced in one go, but
rather small pieces of DNA of approximately 20 000–30 000
bases in length are sequenced at a time, depending on the
technology used. These short DNA pieces are called reads, and
these reads are then assembled into longer DNA sequences
called contigs.
There are two main techniques of genomic assembly: de
novo and reference-based. De novo assembly methods consist
of constructing, simplifying and resolving de Bruijn graphs to
extract contigs, and no reference genome is needed [see Com-
peau (2011) for a general introduction to de Bruijn graphs]. In
the case of reference-based methods, a previously assembled
genome is used as a reference to which sequenced reads are
independently aligned. Ultimately, almost every read is placed
at its most likely position, and in contrast to de novo assem-
bly, no synergies between reads occur. De novo assembly is
often the preferred method in phylogenomics, as it does not
require a fully assembled reference genome. A multitude of
methods for de novo assembly have been developed, and the
field is constantly evolving. Performance of different methods
depends greatly on data type and species to be assembled, and
each method has its own set of tradeoffs, including computer
memory needed and computational complexity exhibited. The
most commonly used programs for de novo assembly in phy-
logenomics include velvet (Zerbino & Birney, 2008), trinity
(Grabherr, 2011), soapdenovo (Li et al., 2010), spades (Banke-
vich, 2012) and abyss 2.0 (Jackman et al., 2017). For a compre-
hensive comparison of them, see Narzisi & Mishra (2011) and
Hölzer & Marz (2019). Finally, the software atram 2.0 (Allen
et al., 2015) enables easy manipulation of whole-genome,
genome-skimming and transcriptomic data. The program imple-
ments most of the aforementioned assemblers in a pipeline that
enables automation and parallelization of assembly tasks. In
addition, the program performs targeted assembly of contigs of
interest, which can greatly reduce the computational scale of
the assembly problem compared with full genome/transcriptome
de novo assembly, as well as avoiding errors introduced during
whole-genome/transcriptome assembly.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 9
As assembly is a major step in any phylogenomic analysis,
problems at this stage (incomplete assembly, assembly errors
and redundancy) can lead to difficulties in downstream work-
flows, including orthologue and paralogue identification, align-
ment and matrix construction. These problems increase the
amount of missing data in the final aligned matrix, ultimately
limiting the amount of useful data. For the best assembly,
high coverage, long read lengths, and good read quality are
all needed. However, current sequencing technologies do not
inherently possess all three; for instance, Illumina sequencing
results in good-quality reads, but short length, while pacbio
sequencing generates very long reads, but with low quality
(see Table 1).
Assessing common ancestry in molecular sequence
data: a case for orthology
Phylogenetic relationships should always be estimated based
on sequences that are related by orthology, i.e. whose common
ancestor diverged as a result of speciation (orthologues), rather
than a duplication event (paralogues) (Fitch, 1970). Genes
arising from duplication events complicate the inference of a
species tree based on concatenated gene alignments, because
the gene tree describing the relationships among paralogues may
differ from the species tree.
Orthology assessment has become a central problem for evo-
lutionary and molecular biologists. In phylogenetic inference, it
is assumed a priori that the loci used to infer evolutionary rela-
tionships are orthologues, and the violation of this assumption
can lead to phylogenetic error.
There are two main categories of orthology prediction:
(i) similarity or graph-based methods (Altenhoff & Dessi-
moz, 2009); and (ii) phylogeny-based methods (Gabaldón,
2008). Both methods can be computationally intensive, as
the exhaustive pairwise comparisons for blast-based meth-
ods and the tree inference for the phylogeny-based meth-
ods impose great computational burden. However, compara-
tive studies have shown that phylogeny-based approaches have
much higher specificity, particularly for detecting orthologues
among distantly related taxa (Chen et al., 2007; Altenhoff
& Dessimoz, 2009).
In similarity-based methods, loci are partitioned into groups
of orthologues based on the assumption that genes that share
similar sequences are orthologous. The precise definition of
a group of orthologous genes, however, depends on the par-
ticular method used, and several methods and algorithms to
cluster orthologous genes have been proposed, including blast
searches, Markov clustering, spectral clustering and hierarchical
orthology. Several similarity-based methods are commonly used
in phylogenetics and functional genomics, including orthomcl
(Li, 2003), cog (Tatusov et al., 2003), inparanoid (O’Brien,
2005), oma (Roth, 2008), proteinortho (Lechner et al.,
2011), eggnog (Powell et al., 2012), orthofinder (Emms &
Kelly, 2015), orthograph (Petersen et al., 2017) and orthodb
10 A. D. Young and J. P. Gillung
(Kriventseva et al., 2019). By contrast, phylogeny-based meth-
ods aim to identify orthologues by inferring the lowest common
ancestor relationships between genes, which is performed by
estimating a gene tree and identifying its speciation and duplica-
tion events using reconciliation with a species tree (Ullah et al.,
2015). Commonly used phylogeny-based methods of orthology
prediction in phylogenomics include loft (van der Heijden
et al., 2007), coco-cl (Jothi et al., 2006), orthologid (Chiu
et al., 2006) and phylotreepruner (Kocot et al., 2013). For
a comprehensive review and comparison of different orthology
prediction methods, see Salichos & Rokas (2011) and Trachana
et al. (2011).
Construction of a phylogenomic dataset: alignment
and concatenation
Following assembly and orthology prediction, the next step in a
phylogenomic workflow is the alignment of sequence data. For
phylogenomic-scale projects, two of the most commonly used
multiple-sequence alignment programs are clustal (Larkin
et al., 2007) and mafft (Katoh & Standley, 2013). Broadly,
both methods produce a pairwise distance matrix from input
sequences, construct an initial guide tree from the distance
matrix, and then align sequences based on the estimated tree.
The first alignment produced is then scored according to a
specified criterion, and used to produce a new guide tree, which
is in turn used to produce an updated alignment that is then
rescored, until a best-scoring final alignment is reached in an
iterative process.
mafft L-INS-i, E-INS-i and G-INS-i algorithms are most
appropriate for phylogenomic datasets as they were designed
to handle a large number of sequences at once. If sequences
have full length but low similarity, then the global option is
appropriate (G-INS-i). Alternatively, if sequences have one
similar core domain in between flanking unalignable regions,
then the local affine gap option works best (L-INS-i). Finally,
if sequence data are composed of multiple alignable domains
with varied length, then the local generalized affine gap scheme
is more appropriate (E-INS-i). For most phylogenomic datasets,
mafft E-INS-i algorithm is probably more appropriate, as it
makes the fewest assumptions about the data, and is better
at handling unalignable sections of data within a sequence.
However, several widely used alignment pipelines (including
phyluce) utilize the L-INS-i algorithm by default. For small to
medium-sized projects, both mafft and clustal have online
servers where sequence data can be uploaded and aligned
remotely.
Finally, individually aligned loci can be concatenated into a
supermatrix for use in concatenation-based analyses (see later).
Several software packages and scripts facilitate concatenation
of aligned loci, including scafos (Roure et al., 2007), phyluce
(Faircloth, 2016), and amas (Borowiec, 2016). These programs
generate a partition file delimiting the boundaries of individ-
ual loci within the supermatrix, which are then used for both
partitioning and model selection in downstream phylogenetic
analysis.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Describing the process of molecular evolution:
model selection and partitioning
Evolutionary models are at the centre of molecular sequence
data analyses. Parameter inference, whether performed within
a maximum likelihood (ML) or Bayesian inference (BI) frame-
work, relies on the explicit definition of the substitution process,
and different models and parameters account for different pro-
cesses of evolution.
Selecting the model that best describes the evolutionary
process that generated the data at hand is of utmost importance,
and failure to do so can lead to erroneous estimates of topology
and branch lengths.
Model selection is most commonly performed under a fre-
quentist framework, where the fit of the data to each substitu-
tion model (together with the substitution model parameters,
tree topology, and branch lengths) is assessed through itera-
tive optimizations of the likelihood function, where ML scores
of each model are compared through one of several possible
criteria. It is worth mentioning that the partition scheme is
part of the model, and so selecting a partition scheme is also
part of model selection. Some of the most widely used crite-
ria include the hierarchical and dynamic likelihood ratio tests
(hLRT and dLRT, respectively) (Posada & Crandall, 2001), the
Akaike information criterion (AIC; Akaike, 1974), the corrected
AIC (AICc; Sugiura, 1978), the Bayesian information crite-
rion (BIC) (Schwarz, 1978), and the decision-theory criterion
(DT) (Minin et al., 2003). Current widely used model selec-
tion programs available for phylogenomics are based on the fre-
quentist framework and employ several of the aforementioned
information criteria. Most commonly used programs in phyloge-
nomics include partitionfinder2 (Lanfear et al., 2016) and
modelfinder (Kalyaanamoorthy et al., 2017) (which is imple-
mented in the program iqtree; Nguyen et al., 2015). These
methods scale relatively well for phylogenomic-scale datasets
(using the rcluster algorithm in partitionfinder; Lanfear
et al., 2014) and require a concatenated dataset coupled with a
partition file for data partitioning as input.
Alternatively, under the Bayesian approach, model selection
can be performed using the ratio of the marginal likelihoods of
two models (i.e. Bayes factor; Goodman, 1999). The ratio of the
marginal likelihoods quantifies the strength of evidence for one
model being more appropriate to describe the data than the other.
Several methods that estimate the Bayes factors or the marginal
likelihood for model selection in phylogenetic analyses have
been proposed, with variable tradeoffs between computation
times and accuracy (Suchard, 2001; Huelsenbeck & Rannala,
2004; Lartillot & Philippe, 2006; Fan et al., 2010).
Protein-coding genes can be analysed as amino acids,
nucleotides or codons, and choosing which data type to analyse
in phylogenomics is a challenge that can significantly affect the
results. The statistical analysis of amino acid and nucleotide
data are fundamentally different (Huelsenbeck et al., 2008).
Model parameters of nucleotide models are estimated from
the data, while in amino acid models most of the parameters
are fixed to specific values estimated from a large number

of sequences and predetermined by the model (e.g. Dayhoff
et al., 1978; Jones et al., 1992; Le et al., 2012; etc.) (but see
+F variants of models implemented in partitionfinder2
and iqtree, where stationary frequencies are estimated from
the data). Although the fixed amino acid models reduce the
number of free parameters to be estimated, leading to less
computationally intensive analyses, it is possible that even
the best-fitting fixed amino acid model is not appropriate
for the data at hand. Several studies have shown that using
an inadequate model may result in erroneous estimates of
phylogeny (e.g. Buckley, 2002; Lemmon & Moriarty, 2004;
Cooper, 2014), and proper model selection is an integral
part of the phylogenetic reconstruction procedure, especially
when analysing amino acids. In the case of nucleotides, it
has been suggested that using the most parameter-rich model
(GTR + I + G) instead of conducting thorough model selec-
tion leads to phylogenies as accurate as those obtained when
model selection is performed. This implies that the stan-
dard practice of model selection before phylogeny inference
is unnecessary when a complex and parameter-rich model
is applied to nucleotide data (Abadi et al., 2019). However,
it is worth mentioning that combining a gamma distribution
(+G; Yang, 1993) with a proportion of invariant sites (+I; Fitch
& Margoliash, 1967) to account for among-site rate hetero-
geneity has been highly criticized, mainly because some of
the parameters of the +I and + G models cannot be optimized
independently of each other (Yang, 2006; Sullivan, 1999; May-
rose, 2005; Jia et al., 2014). Thus, it is more advisable to apply
a GTR + G mixture model for each partition, as opposed to a
GTR + I + G.
Estimating evolutionary history: phylogenetic trees
Model-based approaches have dominated phylogenetic studies
in recent decades, with ML and BI used most widely. Parsi-
mony analysis, by contrast, is not suitable for phylogenetic
analysis of molecular data, as it is prone to misinterpretation
of identical yet homoplastic character states as homologous,
resulting in incorrect grouping of taxa with increasing support
as additional data are included, a property referred to as incon-
sistency (Felsenstein, 1978). This problem is most prevalent
in the so-called ‘Felsenstein zone’, a region of theoretical tree
space in which divergent nonsister taxa with parallel charac-
ter state transformations are joined by long-branch attraction
(Huelsenbeck & Hillis, 1993). Moreover, parsimony methods
fail to make explicit an underlying model of evolution. For
an in-depth review of the strengths and weaknesses of par-
simony and model-based phylogenetic methods, see Yang &
Rannala (2012), and for an introduction to Bayesian statistics
in phylogenetics see Nascimento et al. (2017).
Broadly, model-based phylogenetic methods can be divided
into concatenation (or supermatrix) and coalescent-based
approaches. Concatenation-based methods infer phylogenies
from a single combined gene matrix and assume that all genes
have the same, or very similar, evolutionary histories (Huelsen-
beck et al., 1996). Even when the dataset is partitioned so that
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 11
different models of evolution are applied to individual loci, the
tree is linked across all partitions and a single species tree results
from the analysis. This assumption, however, may be incorrect,
as biological processes such as hybridization and incomplete
lineage sorting (ILS) can lead to discrepancy between gene trees
and the species tree (Degnan & Rosenberg, 2006; Edwards,
2009). To overcome this issue, coalescence-based approaches
have been proposed as an alternative, where gene trees are
allowed to vary across genes, and the assumption that all
genes have the same history is relaxed (Liu et al., 2015). Of
these approaches, the multispecies coalescent model (Rannala
& Yang, 2003) is the most popular for phylogenomic data
analysis.
Concatenation-based analyses
Commonly used ML software includes garli (Zwickl, 2006),
raxml (Stamatakis, 2014), iq-tree (Nguyen et al., 2015), and
examl (Kozlov et al., 2015). raxml and iqtree are both
relatively user-friendly, with well-annotated manuals available
on their respective webpages. iqtree, in particular, is extremely
well-documented and includes a variety of tutorials available on
the developer’s website. examl and garli, in turn, require more
familiarity with basic scripting and working in command-line
environments. examl is well suited to the analysis of extremely
large datasets, while garli is notable for including a wide range
of models of codon evolution.
The most well-known BI software includes mrbayes (Ron-
quist et al., 2012b), exabayes (Aberer et al., 2014), phy-
lobayes (Lartillot et al., 2013), revbayes (Höhna et al., 2016),
beast (Suchard et al., 2018), and beast2 (Bouckaert et al.,
2019). mrbayes is a well-established program, with very helpful
documentation and many online tutorials available. phylobayes
implements the CAT model, a site-heterogeneous mixture model
developed to account for site-specific features of protein evo-
lution (Le et al., 2008). revbayes, in turn, allows for greater
user control over individual parameters but may require more
significant time investment from new users, at least initially.
beast and beast2 are primarily used to co-estimate a species
tree and divergence times and are discussed in more detail sub-
sequently. Of all these programs, the most appropriate for large
phylogenomic datasets are revbayes and exabayes, which are
particularly suitable for mass parallelization and multithreading
on computer clusters.
Coalescent-based analyses
The most commonly used coalescent-based methods in
phylogenomics are called ‘shortcut’ or summary coalescence
approaches [e.g. stem (Kubatko et al., 2009), mdc (Than &
Nakhleh, 2009), steac (Liu, 2009), star (Liu, 2009), mp-est
(Liu, 2010), svdquartets (Chifman & Kubatko, 2014) and
astral (Zhang, 2018)], wherein gene trees and the species
tree are not co-estimated. In fact, these methods do not analyse
sequence data directly, and instead use individual gene trees
12 A. D. Young and J. P. Gillung
as inputs to calculate a single species tree while accounting
for variation across genealogies. Coalescence methods that
co-estimate gene trees and the species tree, such as *beast
(Heled & Drummond, 2010), are more desirable, but currently
are not computationally tractable for large datasets (see Liu
et al., 2015 for a comprehensive review). astral and svdquar-
tets, in particular, are fast and user-friendly, and have been
shown to be both statistically consistent under the multispecies
coalescent model and to perform well under high ILS conditions
(Chou et al., 2015).
However, these summary coalescent methods have been criti-
cized (Kubatko & Degnan, 2007), as they are unable to account
for causes of gene tree – species tree conflict other than ILS.
Of primary concern is recombination, as it violates one of the
primary assumptions of the multispecies coalescent model and
is likely to have occurred in long gene fragments. Conversely,
if analyses are limited to short loci where recombination is
unlikely, gene-tree estimation error can increase (Springer &
Gatesy, 2014). A possible workaround to this issue, known as
statistical binning, was proposed by Mirarab et al. (2014), where
genes with similar gene-tree topologies are grouped together
and analysed through concatenation, before using these concate-
nated supergene trees as inputs for coalescent analyses.
I have a phylogeny! What now?
Phylogenomic studies have greatly improved our understanding
of the tree of life and show remarkable promise for resolving
incongruences in previous analyses based on limited numbers of
loci. As previously mentioned, large phylogenomic datasets are
not as susceptible to stochastic (or sampling) error as analyses
of datasets with fewer characters per taxon, and generally result
in well-resolved and well-supported estimates of phylogeny.
However, phylogenomic analyses are vulnerable to systematic
error – biases caused by a failure of the method rather than a
lack of data (Kumar et al., 2012). In fact, adding more data
can exacerbate the effect of systematic error by increasing
support for erroneous relationships (Brown & Thomson, 2016).
Systematic error can arise as a result of improper modelling
of biological phenomena (e.g. heterotachy, compositional bias,
rate heterogeneity, ILS, horizontal gene transfer; Romiguier
et al., 2016) or methodological issues (e.g. poor model selec-
tion, biased taxon or gene sampling, poor orthology inference;
Hosner et al., 2016), both of which can lead to biased or
incorrect parameter estimates in phylogenomic analyses.
It is critical to examine phylogenomic analyses for evidence of
systematic error, even when the datasets are large and support
for phylogenetic relationships is high.
Poor model fit seems to be one of the major sources of sys-
tematic bias in phylogenomics. When the model fails to account
for important features of the data, phylogenetic inferences and
measures of confidence (i.e. branch support) can be inaccurate.
One way of incorporating model complexity and thus increas-
ing model fit to phylogenomic analysis is data partitioning (i.e.
when different models are applied to different portions of the
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
alignment). This means that only those sites that are assumed
to evolve under a single model of evolution are grouped during
analysis. Partitioning allows the incorporation of heterogeneity
in models of molecular evolution, freeing parameter values from
being joint estimates across all of the data in a particular dataset.
Model selection as traditionally performed in phylogenetics and
phylogenomics allows for the identification of the best-fitting
model among a pool of candidate models, which is then applied
to each data partition separately. However, this procedure only
enables the selection of an appropriate model when compared
with the other candidate models (i.e. relative model fit), but
it does not inform us whether the best-fitting model is indeed
appropriate for the data or not (i.e. absolute model fit). Absolute
model fit tests employing posterior predictive simulation (and
related approaches) have the potential to fill an important gap in
phylogenetic methodology by assessing a model’s absolute fit to
a given dataset (Bollback, 2002; Brown, 2014). Assessing the fit
of different models to empirical data in such a way that the data
can reject the fit of all models should be a fundamental step in the
phylogenomics pipeline. These methods are being implemented
in a growing number of phylogenetic software, making them
easier to apply as a routine step in phylogenetic analyses [e.g.
phylobayes (Lartillot et al., 2009) and revbayes (Höhna et al.,
2016)]. Posterior prediction allows for the removal of portions
of the data that cannot be adequately modelled, thus improving
overall accuracy of phylogenetic estimations (Doyle, 2015).
Evaluating the reliability of a given phylogenetic tree is just
as important as the phylogenetic estimate itself. Measures of
branch support indicate which parts of the tree have greater
credibility when interpreting the evolution of a group and can
pinpoint outstanding questions where data collection is needed
to resolve remaining uncertainties. Assessing the uncertainty
in individual clades on a tree has the benefit that it allows
the researcher to evaluate specific hypotheses of monophyly.
The reliability of a phylogenomic hypothesis can be assessed
using frequentist (i.e. ML) and Bayesian approaches. In the ML
framework, support values are estimated using nonparametric
bootstrapping (Efron, 1979; Felsenstein, 1985), a procedure
that involves the random resampling (with replacement) of
characters (i.e. columns in an alignment of DNA sequences)
from the original data to generate pseudo-replicate data matrices
identical in size to the original matrix. These pseudo-replicates
are then subjected to the same phylogenetic analyses as the
original dataset. Bootstrap support for a group of interest is
calculated as the proportion of times that the group is obtained in
the pseudo-replicates; for instance, if a group appeared in 95% of
the analyses of the bootstrapped data matrices, then its bootstrap
support value is c. 95%.
While posterior probabilities have a clear-cut interpretation,
representing the probability that the phylogenetic estimate is
true given the model, the priors and the data (Huelsenbeck
et al., 2002), bootstrap values are not as easily interpreted in
phylogenetics (Huelsenbeck & Rannala, 2004). Nonetheless,
most practising systematists erroneously interpret bootstrap
support as the probability that the clade is correct – even though
a high bootstrap proportion is probably associated with correct
clades more often than low bootstrap proportions. In fact, only

the Bayesian approach directly addresses the probability that a
clade is correct, conditional on the observations. In other words,
the posterior probability of a tree can be interpreted as the
probability that the tree is correct given the data and the model
(Huelsenbeck et al., 2001), but bootstrap values cannot.
Both posterior probabilities and bootstrap values are sensitive
to model misspecification and can be highly inflated when the
model is oversimplified (e.g. when an overly simplistic model
is utilized, or a concatenated alignment is not adequately
partitioned).
The sensitivity of posterior probabilities, however, seems to
be greater than the sensitivity of bootstrap values (Huelsen-
beck & Rannala, 2004; Lemmon & Moriarty, 2004; Brown &
Lemmon, 2007; Brown & Thomson, 2016). Bayesian support
values were shown to be consistently higher than bootstrap
values for the same clades both in simulations and in empirical
analyses (Erixon et al., 2003; Huelsenbeck & Rannala, 2004).
Posterior probabilities are more sensitive to underspecification
of the evolutionary model than to overspecification, meaning
that it is more detrimental to phylogeny estimation to use an
oversimplified model as opposed to an overly complex one
(Huelsenbeck & Rannala, 2004). Thus, appropriate model fit
is necessary for both reliable phylogeny estimation and inter-
pretation of results, especially in the presence of confounding
factors – the methodological and biological phenomena that
cause model violation. In the last few years, much effort has
been devoted to uncovering and understanding the effects
of confounding factors in phylogenomic reconstruction (see
Table 3). When the model fails to account for important fea-
tures of the data, inferences and measures of confidence can be
inaccurate, and as the complexity of datasets grows with size,
so does the potential for poor model fit to bias phylogenetic
inference.
Given the unreliability of branch support values under some
circumstances, it is desirable to perform additional analyses
to verify the robustness of phylogenetic estimates obtained
with either ML or BI. One such approach involves the cal-
culation of gene and site concordance factors, which allow
for the assessment of the fraction of loci and alignment sites
supporting a particular branch (Ané et al., 2007; Minh et al.,
2018). Importantly, this procedure highlights underlying dis-
agreement or discordance in phylogenomic datasets, serving
as a useful complement to bootstrap values and posterior
probabilities, especially for large phylogenomic datasets.
This method is implemented in the software iq-tree, and a
user-friendly tutorial is freely available from the developer’s
website. Another method to assess the statistical robustness
of phylogenomic estimates is four-cluster likelihood-mapping,
an approach that reveals the proportion of taxon quartets in a
data matrix that supports each of the three alternative topolo-
gies around a specific branch of interest (Strimmer & von
Haeseler, 1997). This approach allows for hypothesis-testing
of alternative relationships and the visualization of the phy-
logenetic information contained in a sequence alignment.
Four-cluster likelihood-mapping can also be easily carried out
in iq-tree.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 13
Beyond topologies: unravelling patterns
of biodiversity
For those interested in investigating additional questions besides
estimating the evolutionary relationships of taxa of interest,
there is a wide range of supplemental analyses available, includ-
ing divergence time estimation, ancestral state reconstruction,
patterns of diversification, biogeography, and others.
Divergence time estimation
Divergence time estimation is concerned with establishing the
timeline of evolution of a particular group, therefore allow-
ing cladogenesis (i.e. speciation) to be correlated with past
geological events, changes in climate, mass extinctions or the
diversification of other organisms. Divergence time estimation is
a complicated, but often essential, step for many phylogenomic
projects. Accurate estimates of species divergence times are vital
to understanding historical biogeography, estimating diversifi-
cation rates and identifying the causes of variation in rates of
molecular evolution.
Originally, phylogenies were dated by assuming a constant
molecular clock, with mutation or substitution rate estimated
with reference to the fossil record (Zuckerkandl & Pauling,
1962). Since then, new and more sophisticated methods have
been introduced, as it has become clear that the rate of molecu-
lar evolution varies significantly over time and among lineages.
Thus, it is now standard practice to accommodate such rate vari-
ation using relaxed-clock models (Drummond et al., 2006; Yang
& Rannala, 2006). There are two main alternative approaches to
estimating divergence times in phylogenomics: node-dating and
tip-dating methods. In node-dating approaches, the age and phy-
logenetic placement of fossil species are used to calibrate partic-
ular nodes in the phylogeny, and those calibration points are then
utilized to estimate the ages of the remaining nodes. In tip-dating
approaches (e.g. implementing the fossilized birth-death topol-
ogy and node-age prior; Heath et al., 2014), multiple fossils per
lineage or node can be utilized, and fossil placement is estimated
based on their ages, without the necessity for morphological data
or prior age densities on fossils. Total-evidence dating, proposed
by Ronquist et al. (2012a), is a special case of tip-dating where
fossils are included in the analysis along with the extant taxa,
so the phylogenetic tree, divergence times and fossil placement
are jointly estimated by combining morphological and molecu-
lar data. There are advantages and tradeoffs for both approaches,
but node-dating approaches have been recently criticized mostly
due to unpredictable and undesirable interactions among fossil
calibration densities (i.e. user-specified prior ages) (Brown &
Smith, 2018) [see Arcila et al. (2015), Matzke & Wright (2016)
and Smith et al. (2018a) for a review of the major concepts and
issues related to divergence dating in phylogenomics].
In node-dating approaches, estimating divergence times
requires a phylogenetic tree and associated branch length
information, and a list of fossil taxa of reliable age and phylo-
genetic placement to be used as calibration points. Depending
on the focal organism, there may be little information in the
14 A. D. Young and J. P. Gillung

Table 3. Major confounding factors in phylogenomic analyses and the strategies to mitigate them

Sources of systematic
error Strategies to overcome Main references

Compositional Remove the loci or taxa exhibiting extreme deviation in Jeffroy et al. (2006); Philippe et al. (2011); Duchêne
heterogeneity composition. Alternatively, explicitly modelling et al. (2017); Borowiec (2019)
compositional heterogeneity may be more satisfactory.
Missing data Design smaller (i.e. with fewer loci) but more complete Roure et al. (2013); Hosner et al. (2016); Kocot et al.
datasets, as opposed to larger (i.e. with more loci) but (2017); Smith et al. (2018b)
sparser datasets. Analyse subsamples of the data to see
if missing data cause conflict.
Branch-length Perform analysis with and without taxa and genes most Nosenko (2013); Struck (2014); Kück & Wägele
heterogeneity likely to be susceptible to long-branch attraction (e.g. (2015); Qu et al. (2017)
remove rapidly evolving sites or taxa).
Paralogy Filter and remove paralogous loci using an appropriate Struck (2013); Betancur-R (2014); Siu-Ting (2019)
method for orthology prediction. Carefully examine
gene topologies to detect paralogues.
Heterotachy Removal of most varied sites from alignment. Use Zhong (2011); Bouckaert & Lockhart (2015); Kocot
evolutionary models that explicitly model heterotachy et al. (2017); Crotty et al. (2019)
(e.g. ghost, implemented in iqtree).
Gene tree Use coalescence approaches. Perform statistical Betancur-R (2013); Copetti (2017); Richards (2018)
heterogeneity binning, tree concordance analysis.

fossil record, such that obtaining fossils with reliable age
and phylogenetic placement may be challenging. Therefore,
palaeontological literature should be carefully reviewed so
that fossil placement is critically evaluated. See Parham et al.
(2012) for a discussion on the best practices for fossil selection
for divergence time estimation studies. A valuable resource
for generating a list of potential fossil taxa is the Fossilworks
Paleobiology Database (https://fossilworks.org), a repository
of fossil species containing both their age and phylogenetic
placement.
Divergence time estimation in phylogenomics is generally
performed using BI and penalized likelihood methods. Com-
monly used Bayesian divergence time estimation software
includes mrbayes (Ronquist et al., 2012b; Zhang, 2016),
mcmctree (part of the paml package; Xu & Yang, 2013),
phylobayes (Lartillot et al., 2013), beast (Suchard et al.,
2018), and beast2 (Bouckaert et al., 2019). mrbayes, beast
and beast2 enable the co-estimation of the phylogeny and
divergence times and allow for the implementation of both
node-dating and tip-dating approaches, including total-evidence
dating (where morphological data can be used for fossil place-
ment). Comprehensive and user-friendly tutorials are available
for mrbayes (Zhang, 2017), mcmctree (https://github.com/
mariodosreis/divtime) and beast2 (Barido-Sottani et al., 2017).
Divergence time estimation using penalized-likelihood include
‘r8s’ (Sanderson, 2003), ‘treePL’ (Smith & O’Meara, 2012)
and the ‘chronos’ function of the R package ape (Paradis, 2004;
Paradis, 2013).
Ancestral state reconstruction
Ancestral state reconstruction is the extrapolation back in
time from observed characters of individuals or species to
their common ancestors. These methods enable the estima-
tion of the number of times a particular phenotype evolved,
the approximate timing of major evolutionary events, and
missing phenotypic characteristics of fossil species. Ancestral
state reconstruction can also help to contextualize correlated
shifts between phenotypic and ecological characters, as well as
perform phylogenetic prediction, in which phenotypic values
for unobserved or incompletely sampled taxa can be estimated.
Finally, ancestral state reconstruction can be used to recover
ancestral DNA sequences, the ancestral amino acid sequence of
a protein, the composition of a genome (including gene order
and copy number), as well as the geographic range of an ances-
tral population or species. However, as with other types of anal-
yses detailed here, it is important to carefully consider the scien-
tific question that is being asked and which analyses and meth-
ods are suitable to answering it. It is not advisable to tack on
analyses such as ancestral state reconstruction unless they con-
tribute to answering the question(s) the study seeks to address.
This is doubly true when said analyses are performed without
due diligence (e.g. using parsimony mapping when model-based
techniques are available, not performing model testing, etc.).
Ancestral state reconstruction in phylogenomics can be per-
formed in a ML or BI framework. Analyses using ML can be
performed in bayestraits (Pagel et al., 2004), the ‘ace’ func-
tion in the r package ape (Paradis, 2004), mesquite (Maddison
& Maddison, 2018), the ‘FastAnc’ function in the r package
phytools (Revell, 2012), and the ‘rayDISC’ function in the
r package corhmm (Beaulieu et al., 2013). Analysis under BI
can be performed in bayestraits, revbayes and mesquite.
These methods take a phylogenetic tree with branch length
information and a trait file containing the character states for
each taxon as input, and transition rates and ancestral states for
two or more characters are then calculated. mesquite and ‘ray-
DISC’ can only analyse discrete (i.e. categorical) characters and

© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406


implement only two to three models of trait evolution, while
bayestraits and revbayes enable much greater flexibility,
allowing the analysis of both discrete and continuously varying
traits, and implementing a variety of models of character evolu-
tion. In addition, these implementations can be used in a single
phylogenetic tree (so only uncertainty about model parameters
is explored), or it can be applied to a posterior sample of trees,
in which model parameters are jointly estimated and phyloge-
netic uncertainty is taken into account. Bayesian character state
estimation is preferred over ML approaches as it accounts for
uncertainty in both model parameters and phylogeny (Pagel,
2004; Vanderpoorten & Goffinet, 2006). For a review and com-
parison of the methods of character state reconstruction, see
Royer-Carenzi (2013), Joy et al. (2016) and Royer-Carenzi &
Didier (2016).
Diversification rate estimation
Rates of speciation and extinction vary greatly through time
and among lineages, leading to extensive heterogeneity in
species richness across the tree of life. By linking diversification
rate to traits of interest, evolutionary biologists can explore a
variety of hypotheses in a quantitative manner. For instance,
if a clade of interest possesses a synapomorphy (i.e. novel
evolutionary trait) that constitutes a putative key innovation,
diversification rate analysis can be used to determine whether
that clade actually diversified at a higher rate than related
lineages. Several approaches have been proposed to estimate
lineage-specific rates of diversification, which can be broadly
subdivided into two main categories: model-based approaches
(i.e. methods that utilize models that are parameterized with
speciation and extinction rates) and nonmodel-based approaches
(i.e. methods that simply rely on branch lengths and splitting
events) (Wiens, 2017).
Model-based approaches are dominant in phylogenomics.
For example, Bayesian analysis of macroevolutionary mixtures
(bamm; Rabosky, 2014) is widely used in empirical studies. One
of the major advantages of bamm over nonmodel methods is
that it can accommodate heterogeneity in the diversification rate
through time and among lineages in a clade. bamm simulates a
posterior distribution of macroevolutionary rate shift configura-
tions given a phylogeny of interest and then extracts marginal
rates of speciation and extinction for individual taxa based on
this distribution. However, studies have shown that bamm anal-
yses can result in problematic estimates of both diversifica-
tion rates and rate shifts (Moore et al., 2016; Meyer & Wiens,
2018). Estimation of diversification rates can also be carried
out in medusa (Alfaro, 2009), the r package rpanda (Morlon
et al., 2016) and revbayes, which offers a variety of different
hypothesis-testing options and diversification rate models in a
Bayesian framework. Additionally, the r package tess imple-
ments a variety of statistical approaches for inferring rates of
lineage diversification from empirical phylogenetic trees under
various stochastic branching process models (Höhna, 2013).
Some major features of tess include the ability to implement
various methods of incomplete taxon sampling, robust Bayesian
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 15
methods for assessing the relative fit of models of lineage diver-
sification, and phylogeny simulation for the generation of null
hypotheses (Höhna, 2015).
It has been shown that incomplete and biased taxon sampling
can cause unpredictable effects on inferences of diversification
rates, particularly in large empirical datasets (Höhna, 2014; Title
& Rabosky, 2017). For instance, Sun et al. (2019) found out
that, as compared with full empirical sampling, representative
and random sampling schemes either under- or overestimate
speciation rates at varying degrees, depending on the method
and sampling scheme utilized.
Caution in the inference and interpretation of diversification
estimates is necessary, especially when using very sparse or
incomplete taxonomic sampling – regardless of method utilized.
Because of this, it is also advisable to perform sensitivity
analyses to verify whether missing data might cause bias in the
empirical results.
Biogeography
Historical biogeography is concerned with understanding the
distributional patterns of organisms across the globe, as well as
the processes that have produced such distributions. In recent
years, biogeography has transitioned from parsimony-based
approaches (i.e. cladistic biogeography; Parenti, 2006) to a
statistical framework, where parametric models are used to
explore a much broader range of hypotheses as compared with
parsimony-only approaches (Ree & Sanmartín, 2009).
Since then, several biogeographic models have been proposed,
including statistical dispersal-vicariance analysis (S-DIVA;
Ronquist, 1997; Yu et al., 2010), dispersal – extinction –
cladogenesis (DEC; Ree & Smith, 2008), bayarea (Landis
et al., 2013) and Bayesian binary Markov chain Monte Carlo
(MCMC) analysis (BBM; Yu et al., 2015). In S-DIVA, ances-
tral distributions in a phylogeny are reconstructed based on a
three-dimensional cost matrix, in which extinctions and dis-
persals cost more than vicariance events. The biogeographic
histories with the lowest cost are selected, while accounting for
both phylogenetic and DIVA optimization uncertainty. In turn,
DEC assigns probabilities to various range-changing events,
and these event probabilities are then used to calculate the
likelihood of the observed geographic range at the tips of the
tree. Finally, the values of the parameters and ancestral states
that confer maximum probability given the data are selected
after ML optimization. BBM is primarily designed for recon-
structing ancestral distributions of nodes in a phylogenetic tree,
using simple probabilistic models and a fixed topology. Finally,
bayarea estimates biogeographic history by marginalizing
over all possible biogeographic scenarios given the current dis-
tributions of taxa, estimating model parameters using MCMC,
and comparing candidate biogeographic models using Bayes
factors.
These models, as well as their variations, are commonly
used in phylogenomics, and are implemented in a ML frame-
work in the program lagrange (Ree et al., 2005) and the r
16 A. D. Young and J. P. Gillung
package biogeobears (Matzke, 2013), and under BI in the pro-
grams bayarea (Landis, 2013) and rasp (Yu et al., 2015). bio-
geobears is particularly flexible, enabling both the probabilistic
inference of historical biogeography (i.e. inference of ances-
tral geographic ranges on a phylogeny) and the comparison
of different models of range evolution (i.e. model selection in
biogeography). In addition, biogeobears allows greater user
flexibility to relax parameter values and to introduce new
free parameters, including new cladogenic events such as the
founder-event speciation (+J).
The future of phylogenomics
The rate at which genomic data can be obtained has increased
exponentially in recent years, providing unprecedented opportu-
nities for the reconstruction of molecular phylogenies. In most
cases, the use of phylogenomic-scale data reduces the effects of
stochastic error in phylogenetic analysis. However, this influx
of data increases the potential for systematic error, and methods
and protocols for addressing this issue are still in development.
Current models of evolution may be overly simplistic in many
cases, and model misspecification, regarded as insignificant in
the Sanger era of phylogenetic reconstruction, may adversely
affect phylogenomic-scale analyses (Philippe et al., 2011). Sev-
eral approaches to alleviate the effects of systematic bias in
phylogenomics have been proposed, including tests of composi-
tional heterogeneity (Duchêne et al., 2017), posterior predictive
approaches to assess model fit (Bollback, 2002; Doyle et al.,
2015), and approaches to evaluate the sensitivity of results to
removal of sites or loci likely to introduce bias (Chen et al.,
2015; Goremykin et al., 2015) and to explore genealogical con-
cordance among different regions of the genome (Minh et al.,
2018).
Most, if not all, of these approaches should become a part of
the standard phylogenomics toolkit, and focus should be given
to interpreting, integrating and summarizing across different
datasets and tree-inference approaches for drawing major phy-
logenetic conclusions.
Coalescence-based phylogenetic analysis, which addresses
the issues of ILS, is currently gaining ground in phyloge-
netic circles, but there are valid criticisms of the summary
(or ‘shortcut’) coalescent approaches used most commonly
in phylogenomic-scale datasets (Kubatko & Degnan, 2007).
If future implementations of true coalescent analysis – with
co-estimation of phylogeny and coalescence time – become
viable, this could largely address some of the major sources
of systematic error. Until then, coalescent analysis is extremely
useful, mostly by showcasing gene tree conflict, but should prob-
ably be performed alongside traditional ML or BI analyses.
Unfortunately, current molecular phylogenies are often
entirely divorced from morphological analyses, making it
difficult to link current literature with both past phylogenetic
analyses and the natural history of organisms themselves.
In addition, as morphology is often our only link to extinct
species, morphological data should ideally be used to infer
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
fossil placement in a quantitative and comparative framework.
The analysis of combined morphological and molecular charac-
ters for both living and extinct taxa in a model-based framework
is an ideal approach for both phylogeny estimation and fossil
placement, which probably results in more robust divergence
time estimation and character state reconstruction.
Contrary to popular belief, novel phylogenomic studies can be
performed relatively inexpensively. Some early phylogenomic
studies performed somewhat simple data analyses, so down-
loading, combining and reanalysing published datasets using
several of the described techniques is a cost-effective way to
get acquainted with the field. By interrogating existing data
in more detail than in the original publication, novel insights
can be obtained (and published) without the need for expensive
sequencing. Furthermore, data scientists could develop new ana-
lytical tools or data-processing methods using existing datasets
to demonstrate their effectiveness and ability to analyse empiri-
cal data. By improving even a single step in the phylogenomics
pipeline (see Fig. 2), bioinformaticians can develop novel and
highly applicable tools for data processing.
Overall, the future of phylogenomic analysis is extremely
bright, with many independent avenues of exploration available.
Through awareness of the pitfalls of systematic error due to
either methodological or biological phenomena, increasingly
robust hypotheses of evolution can be proposed. Likewise, by
carefully considering taxon selection, experimental protocols
and data interpretation, advances in phylogenomic protocols can
greatly contribute to resolving the tree of life.
Glossary of the basic concepts in phylogenomics
Adapters. Short, synthetic, single-stranded DNA molecules
that are ligated to the ends of DNA fragments of interest.
These allow target DNA fragments to bind to a flow cell
for amplification in a NGS platform. Adapters are indexed or
‘barcoded’ with a short, unique sequence usually six to 10
bases long, which enables multiple samples to be sequenced
simultaneously (multiplexing). After sequencing, these indexes
are used to associate individual reads back to their correct
sample (demultiplexing).
Coalescence. A point in past evolutionary time where different
alleles originated from a single common ancestor gene variant.
Compositional heterogeneity. Variation in the equilibrium rate
of individual bases or amino acids between taxa. Not accounting
for compositional heterogeneity in phylogenetic analyses can
lead to tree estimation error.
Contig. A contiguous sequence of DNA constructed by merging
(or assembly) at least two individual DNA sequence reads. With
traditional Sanger sequencing, contigs are typically constructed
from a forward and reverse read of the same gene region,
whereas in NGS methods, contigs are typically assembled using
large numbers of partially overlapping reads in both the forward
and reverse directions.
De novo assembly. The processes of constructing contigs and
scaffolds without the use of a pre-existing reference genome
from a related organism. The methods used by de novo assembly

software are varied, but the most common type for short reads is
assembly by de Bruijn graphs.
Felsenstein zone. A region of theoretical tree space where
long branch attraction is especially pervasive. This leads to the
artificial grouping of two or more taxa that are situated in long
branches.
Gene tree. A phylogenetic tree based on data (or information)
from a single locus.
Gene tree estimation error. The inaccuracy in the topology of
individual gene trees. This can be caused by either systematic
error (e.g. model misspecification) or stochastic error (i.e. insuf-
ficient phylogenetic signal leading to inaccurate tree topologies).
Gene tree – gene tree conflict. The mismatch between individ-
ual gene tree topologies constructed from different loci from a
single organism. This phenomenon is most commonly caused
by incomplete lineage sorting, where only a random subset of
alleles for each gene within an organism is passed on during a
speciation event.
Heterotachy. Variation in the evolutionary rate of individual
genes, gene regions or taxa over time. Not accounting for
heterotachy in phylogenetic analysis can lead to tree estimation
error.
Horizontal gene transfer (HGT). The transfer of genetic
material from one organism to another that is not its offspring.
HGT causes unrelated organisms to share the same, or similar,
genetic material, thus misleading phylogeny estimation.
Incomplete lineage sorting (ILS). The random inheritance of
only some gene variants (or alleles) by a new species from a
founding population during speciation events. ILS can cause
mismatches between the species tree and individual gene trees.
Incongruence. Two or more phylogenetic trees are said to
be incongruent when they exhibit conflicting topologies or
branching orders and cannot be superimposed. This implies that
at least one node present in one tree is not found in the others,
where it is replaced by alternative groupings of taxa.
Kmer. A DNA sequence of length k, generally obtained from a
sequencing platform.
Long branch attraction (LBA). When two (or more) lineages
have much longer branches than the others, they tend to group
together irrespective of their true relationships (i.e. common
ancestry).
Missing data. Character information that is not sampled for
some taxa in a character matrix. In the context of phylogenomic
analyses, missing data occur as loci or portions of loci are not
sampled for particular taxa in an alignment.
Model of sequence evolution. A statistical description of the
process of substitution in nucleotide or amino acid sequences.
Complex models better approximate the evolutionary process
but at the expense of more parameters and computational time.
As more complex, and thus more parameter-rich, models require
more data to properly describe the process of evolution, they
have become more reliable with the advent of phylogenomic
datasets.
Multiplexing. A procedure that allows large numbers of DNA
libraries to be pooled and sequenced simultaneously during
a single run on a high-throughput instrument (e.g. Illumina).
Individual barcode sequences are added to each library so that
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 17
reads can later be identified and associated with their respective
species.
Nonparametric bootstrap. A procedure where replicate
bootstrap datasets are generated by randomly sampling (with
replacement) characters (i.e. columns) from a sequence matrix.
These replicates are then analysed phylogenetically under the
same parameters used for the original data, with bootstrap val-
ues corresponding to the proportion of trees/replicates in which
a group is recovered. Bootstrap values are thought to correspond
to assessments of confidence (or support) for each clade of an
observed tree, but they nonetheless have no straightforward
interpretation in a phylogenetic context.
Orthologue. A gene that is homologous to another gene in a
related species and arose through speciation. Orthologous genes
usually retain the same or similar functions in related organisms,
and therefore are under similar selection pressures.
Paralogue. A gene that is homologous to another gene in either
the same or a related species and arose through gene duplication
events. Paralogous genes often obtain new functions within a
genome, such that they may undergo entirely different selection
pressures when compared with the original gene from which
they descended.
Parsimony. A method to choose among possible phylogenetic
trees, which states that the phylogeny implying the simplest (i.e.
the least number of changes in character states) hypothesis is
the best.
Posterior probability. In Bayesian phylogenetics, the posterior
probability of a clade is the probability that the relationship is
correct, given the data and assuming that the model is correct.
The posterior probability of each clade is estimated based on
the frequency at which that clade is resolved among the whole
sample of posterior trees (i.e. across all possible phylogenetic
trees).
Read. A single-stranded DNA sequence that has been ‘read’ by
a DNA sequencer.
Reciprocal blast search. blasting (basic local alignment
search tool) is a procedure where a given locus is compared with
a predefined database of loci obtained from other organisms,
with the best (most likely) hit being returned. In phylogenetics,
reciprocal blast searches are generally used to assess gene
orthology. In a reciprocal blast search, the best hit is then
blasted against all loci from the original organism. If the best
hit from this reciprocal blast search is the same locus that was
used for the original query, then the two loci are likely to be
orthologous. If a different locus is returned as the best hit by a
significant margin, then the two loci are regarded as paralogous.
Reference-guided assembly. Also called referenced assembly,
this procedure aligns individual reads to a pre-existing reference
genome from a related organism to construct contigs and
scaffolds. The main limitation of reference-guided assemblies
is the reliance on existing annotated genomes from related taxa.
Depending on the study organism and the phylogenetic diversity
of taxa sampled, this technique may be more or less viable for
phylogenomic studies.
Saturation. An alignment is saturated when sequences have
undergone so many multiple substitutions that apparent
distances largely underestimate the real genetic distances.
18 A. D. Young and J. P. Gillung
Phylogenetic inference works best with datasets that are only
partially saturated. Due to their reduced number of possible
states (four bases), nucleotide sequences saturate more rapidly
than protein sequences (20 amino acids). In addition, third
codon positions saturate more rapidly than first and second
positions.
Scaffold. A portion of a genome reconstructed by merging
multiple contigs together, which are often separated by gaps of
known length. Ideally, complete scaffolds represent entire gene
regions, chromosomes, etc.
Sequencing depth. The number of times individual bases
are sequenced. To assemble contigs of single-copy loci in a
genome, greater sequencing depth is required, as multiple-copy
loci will be sequenced repeatedly before sufficient reads from
single-copy areas of the genome are obtained.
Species tree. A phylogenetic tree that shows the evolutionary
relationships between a group of species.
Stochastic error. The error in phylogenetic estimation caused
by the finite length of the sequences used in the reconstruction.
As the size of the sequences increases, the magnitude of the error
decreases. This is why phylogenomic datasets are thought to be
almost immune to stochastic error.
Systematic error (or bias). Inaccuracy in phylogenetic recon-
struction that is a result of the failure of the reconstruction
method to fully account for the properties of the data.
Tiling. In the context of NGS, tiling refers to an overlap
between short DNA sequences (e.g. probes/baits or reads).
Target enrichment probes are often designed in a tiled fashion in
order to maximize the chances of successfully hybridizing with
DNA fragments from across the locus of interest and in all target
organisms. Also, during contig assembly, many individual reads
are overlapped, or ‘tiled’, in order to build a consensus of reads
at each base position.
Acknowledgements
Thank you to Shaun Winterton for his support and encourage-
ment (i.e. productive nagging) during the preparation of this
manuscript. Thank you to Ziad Khouri for his valuable com-
ments and suggestions on an early version. Thank you to the
reviewers, Dr Steve Cameron and Dr Phil Ward, whose insight-
ful comments greatly improved the text. The authors declare no
conflict of interest. Work by ADY was conducted on the tradi-
tional territory of the Patwin and Nisenan peoples, while JPG’s
research was carried out on the traditional territory of Cayuga
and Onondaga Nations. The authors are grateful to have the
opportunity to work on their land.
References
Abadi, S., Azouri, D., Pupko, T. & Mayrose, I. (2019) Model selection
may not be a mandatory step for phylogeny reconstruction. Nature
Communications, 10, 934.
Aberer, A.J., Kobert, K. & Stamatakis, A. (2014) ExaBayes: massively
parallel Bayesian tree inference for the whole-genome era. Molecular
Biology and Evolution, 31, 2553–2556.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Akaike, H. (1974) A New Look at the Statistical Model Identification.
Selected Papers of Hirotugu Akaike (ed. by E. Parzen, K. Tanabe and
G. Kitagawa), pp. 215–222. Springer, New York, New York.
Alfaro, M.E., Santini, F., Brock, C. et al. (2009) Nine exceptional
radiations plus high turnover explain species diversity in jawed
vertebrates. Proceedings of the National Academy of Sciences, 106,
13410–13414.
Allen, J.M., Huang, D.I., Cronk, Q.C. & Johnson, K.P. (2015) aTRAM -
automated target restricted assembly method: a fast method for assem-
bling loci across divergent taxa from next-generation sequencing data.
BMC Bioinformatics, 16, 98.
Altenhoff, A.M. & Dessimoz, C. (2009) Phylogenetic and functional
assessment of Orthologs inference projects and methods. PLoS
Computational Biology, 5, e1000262.
Ambardar, S., Gupta, R., Trakroo, D., Lal, R. & Vakhlu, J. (2016) High
throughput sequencing: an overview of sequencing chemistry. Indian
Journal of Microbiology, 56, 394–404.
Andrews, S. (2010) FastQC: a quality control tool for high through-
put sequence data. URL http://www.bioinformatics.babraham.ac.uk/
projects/fastqc. [Accessed on 23 Nov 2019].
Ané, C., Larget, B., Baum, D.A., Smith, S.D. & Rokas, A. (2007)
Bayesian estimation of concordance among gene Trees. Molecular
Biology and Evolution, 24, 412–426.
Arcila, D., Alexander Pyron, R., Tyler, J.C., Ortí, G. & Betancur-R., R.
(2015) An evaluation of fossil tip-dating versus node-age calibrations
in tetraodontiform fishes (Teleostei: Percomorphaceae). Molecular
Phylogenetics and Evolution, 82, 131–145.
Bankevich, A., Nurk, S., Antipov, D. et al. (2012) SPAdes: a new
genome assembly algorithm and its applications to single-cell
sequencing. Journal of Computational Biology, 19, 455–477.
Barido-Sottani, J., Bošková, V., Plessis, L.D. et al. (2017) Taming the
BEAST – A community teaching material resource for BEAST 2.
Systematic Biology, 67, 170–174.
Beaulieu, J.M., O’Meara, B.C. & Donoghue, M.J. (2013) Identifying
hidden rate changes in the evolution of a binary morphological
character: the evolution of plant habit in Campanulid angiosperms.
Systematic Biology, 62, 725–737.
Besser, J., Carleton, H.A., Gerner-Smidt, P., Lindsey, R.L. & Trees, E.
(2018) Next-generation sequencing technologies and their application
to the study and control of bacterial infections. Clinical Microbiology
and Infection, 24, 335–341.
Betancur-R, R., Li, C., Munroe, T.A., Ballesteros, J.A. & Ortí, G. (2013)
Addressing gene tree discordance and non-stationarity to resolve a
multi-locus phylogeny of the flatfishes (Teleostei: Pleuronectiformes).
Systematic Biology, 62, 763–785.
Betancur-R, R., Naylor, G.J.P. & Ortí, G. (2014) Conserved genes,
sampling error, and Phylogenomic inference. Systematic Biology, 63,
257–262.
Blaimer, B.B., Lloyd, M.W., Guillory, W.X. & Brady, S.G. (2016)
Sequence capture and phylogenetic utility of genomic ultraconserved
elements obtained from pinned insect specimens. PLoS One, 11,
e0161531.
Bolger, A.M., Lohse, M. & Usadel, B. (2014) Trimmomatic: a flexible
trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120.
Bollback, J.P. (2002) Bayesian model adequacy and choice in phyloge-
netics. Molecular Biology and Evolution, 19, 1171–1180.
Borowiec, M.L. (2016) AMAS: a fast tool for alignment manipulation
and computing of summary statistics. PeerJ, 4, e1660.
Borowiec, M.L., Rabeling, C., Brady, S.G., Fisher, B.L., Schultz, T.R. &
Ward, P.S. (2019) Compositional heterogeneity and outgroup choice
influence the internal phylogeny of the ants. Molecular Phylogenetics
and Evolution, 134, 111–121.
Bouckaert, R., Lockhart, P. (2015) Capturing heterotachy through
multi-gamma site models. bioRxiv, 018101.

Bouckaert, R., Vaughan, T.G., Barido-Sottani, J. et al. (2019) BEAST
2.5: an advanced software platform for Bayesian evolutionary analy-
sis. PLoS Computational Biology, 15, e1006650.
Brown, J.M. (2014) Predictive approaches to assessing the fit of
evolutionary models. Systematic Biology, 63, 289–292.
Brown, J.M. & Lemmon, A.R. (2007) The importance of data parti-
tioning and the utility of Bayes factors in Bayesian Phylogenetics.
Systematic Biology, 56, 643–655.
Brown, J.M. & Thomson, R.C. (2016) Bayes factors unmask highly
variable information content, Bias, and extreme influence in Phyloge-
nomic analyses. Systematic Biology, 66, 517–530.
Brown, J.W. & Smith, S.A. (2018) The past sure is tense: on interpreting
phylogenetic divergence time estimates. Systematic Biology, 67,
340–353.
Buckley, T.R. (2002) Model misspecification and probabilistic tests of
topology: evidence from empirical data sets. Systematic Biology, 51,
509–523.
Chen, F., Mackey, A.J., Vermunt, J.K. & Roos, D.S. (2007) Assessing
performance of Orthology detection strategies applied to eukaryotic
genomes. PLoS One, 2, e383.
Chen, M.-Y., Liang, D. & Zhang, P. (2015) Selecting question-specific
genes to reduce incongruence in Phylogenomics: a case study
of jawed vertebrate backbone phylogeny. Systematic Biology, 64,
1104–1120.
Chen, M.-Y., Liang, D. & Zhang, P. (2017) Phylogenomic resolution of
the phylogeny of Laurasiatherian mammals: exploring phylogenetic
signals within coding and noncoding sequences. Genome Biology and
Evolution, 9, 1998–2012.
Chifman, J. & Kubatko, L. (2014) Quartet inference from SNP data
under the coalescent model. Bioinformatics, 30, 3317–3324.
Chiu, J.C., Lee, E.K., Egan, M.G., Sarkar, I.N., Coruzzi, G.M. &
DeSalle, R. (2006) OrthologID: automation of genome-scale ortholog
identification within a parsimony framework. Bioinformatics, 22,
699–707.
Chou, J., Gupta, A., Yaduvanshi, S., Davidson, R., Nute, M., Mirarab, S.
& Warnow, T. (2015) A comparative study of SVDquartets and other
coalescent-based species tree estimation methods. BMC Genomics,
16, S2.
Compeau, P.E., Pevzner, P.A. & Tesler, G. (2011) How to apply
de Bruijn graphs to genome assembly. Nature Biotechnology, 29,
987–991.
Cooper, E.D. (2014) Overly simplistic substitution models obscure
green plant phylogeny. Trends in Plant Science, 19, 576–582.
Copetti, D., Búrquez, A., Bustamante, E. et al. (2017) Extensive gene
tree discordance and hemiplasy shaped the genomes of north Ameri-
can columnar cacti. Proceedings of the National Academy of Sciences
of the USA, 114, 12003–12008.
Cranston, K., Harmon, L.J., O’Leary, M.A. & Lisle, C. (2014) Best
practices for data sharing in phylogenetic research. PLoS Currents,
6, ecurrents.tol.bf01eff4a6b60ca4825c69293dc59645.
Crotty, S.M., Minh, B.Q., Bean, N.G., Holland, B.R., Tuke, J., Jer-
miin, L.S., von Haeseler, A. (2019) GHOST: Recovering Historical
Signal from Heterotachously-evolved Sequence Alignments. bioRxiv,
174789.
Dai, M., Thompson, R.C., Maher, C. et al. (2010) NGSQC:
cross-platform quality analysis pipeline for deep sequencing data.
BMC Genomics, 11, S7.
Dayhoff, M.O., Schwartz, R. & Orcutt, B. (1978) A model of evolu-
tionary change in proteins. Atlas of Protein Sequence and Structure
(ed. by M.O. Dayhoff), pp. 345–352. National Biomedical Research
Foundation, Silver Spring, Maryland.
Debiasse, M.B. & Ryan, J.F. (2019) Phylotocol: promoting transparency
and overcoming Bias in Phylogenetics. Systematic Biology, 68,
672–678.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 19
Degnan, J.H. & Rosenberg, N.A. (2006) Discordance of species trees
with their most likely gene trees. PLoS Genetics, 2, e68.
Doyle, V.P., Young, R.E., Naylor, G.J. & Brown, J.M. (2015) Can we
identify genes with increased phylogenetic reliability? Systematic
Biology, 64, 824–837.
Drummond, A.J., Ho, S.Y.W., Phillips, M.J. & Rambaut, A. (2006)
Relaxed phylogenetics and dating with confidence. PLoS Biology, 4,
e88.
Duchêne, D.A., Duchêne, S. & Ho, S.Y.W. (2017) New statistical criteria
detect phylogenetic Bias caused by compositional heterogeneity.
Molecular Biology and Evolution, 34, 1529–1534.
Dunn, C.W., Howison, M. & Zapata, F. (2013) Agalma: an automated
phylogenomics workflow. BMC Bioinformatics, 14, 330.
Edwards, S.V. (2009) Is a new and general theory of molecular
systematics emerging? Evolution, 63, 1–19.
Edwards, S.V., Xi, Z., Janke, A. et al. (2016) Implementing and
testing the multispecies coalescent model: a valuable paradigm
for phylogenomics. Molecular Phylogenetics and Evolution, 94,
447–462.
Efron, B. (1979) Bootstrap methods: another look at the Jackknife. The
Annals of Statistics, 7, 1–26.
Eisen, J.A. (1998) Phylogenomics: improving functional predictions for
uncharacterized genes by evolutionary analysis. Genome Research, 8,
163–167.
Emms, D.M. & Kelly, S. (2015) OrthoFinder: solving fundamen-
tal biases in whole genome comparisons dramatically improves
orthogroup inference accuracy. Genome Biology, 16, 157.
Erixon, P., Svennblad, B., Britton, T. & Oxelman, B. (2003) Reliability
of Bayesian posterior probabilities and bootstrap frequencies in
Phylogenetics. Systematic Biology, 52, 665–673.
Faircloth, B.C. (2016) PHYLUCE is a software package for the analysis
of conserved genomic loci. Bioinformatics, 32, 786–788.
Faircloth, B.C., McCormack, J.E., Crawford, N.G., Harvey, M.G.,
Brumfield, R.T. & Glenn, T.C. (2012) Ultraconserved elements
anchor thousands of genetic markers spanning multiple evolutionary
timescales. Systematic Biology, 61, 717–726.
Fan, Y., Wu, R., Chen, M.-H., Kuo, L. & Lewis, P.O. (2010) Choosing
among partition models in Bayesian phylogenetics. Molecular Biol-
ogy and Evolution, 28, 523–532.
Felsenstein, J. (1978) Cases in which parsimony or compatibility
methods will be positively misleading. Systematic Zoology, 27,
401–410.
Felsenstein, J. (1985) Confidence limits on phylogenies: an approach
using the bootstrap. Evolution, 39, 783–791.
Fitch, W.M. (1970) Distinguishing homologous from analogous pro-
teins. Systematic Zoology, 19, 99–113.
Fitch, W.M. & Margoliash, E. (1967) A method for estimating the
number of invariant amino acid coding positions in a gene using
cytochrome c as a model case. Biochemical Genetics, 1, 65–71.
Gabaldón, T. (2008) Large-scale assignment of orthology: back to
phylogenetics? Genome Biology, 9, 235.
Goodman, S.N. (1999) Toward evidence-based medical statistics. 2: the
Bayes factor. Annals of Internal Medicine, 130, 1005–1013.
Goremykin, V.V., Nikiforova, S.V., Cavalieri, D., Pindo, M. & Lockhart,
P. (2015) The root of flowering plants and total evidence. Systematic
Biology, 64, 879–891.
Grabherr, M.G., Haas, B.J., Yassour, M. et al. (2011) Full-length
transcriptome assembly from RNA-Seq data without a reference
genome. Nature Biotechnology, 29, 644–652.
Heath, T.A., Hedtke, S.M. & Hillis, D.M. (2008) Taxon sampling and
the accuracy of phylogenetic analyses.
Heath, T.A., Huelsenbeck, J.P. & Stadler, T. (2014) The fossilized
birth–death process for coherent calibration of divergence-time
20 A. D. Young and J. P. Gillung
estimates. Proceedings of the National Academy of Sciences, 111,
E2957–E2966.
Heled, J. & Drummond, A.J. (2010) Bayesian inference of species
trees from multilocus data. Molecular Biology and Evolution, 27,
570–580.
Höhna, S. (2013) Fast simulation of reconstructed phylogenies under
global time-dependent birth–death processes. Bioinformatics, 29,
1367–1374.
Höhna, S. (2014) Likelihood inference of non-constant diversification
rates with incomplete taxon sampling. PLoS One, 9, e84184.
Höhna, S., Landis, M.J., Heath, T.A. et al. (2016) RevBayes: Bayesian
phylogenetic inference using graphical models and an interactive
model-specification language. Systematic Biology, 65, 726–736.
Höhna, S., May, M.R. & Moore, B.R. (2015) Phylogeny Simulation and
Diversification Rate Analysis with TESS, 98.
Hölzer, M. & Marz, M. (2019) De novo transcriptome assembly:
a comprehensive cross-species comparison of short-read RNA-Seq
assemblers. GigaScience, 8, giz039.
Hosner, P.A., Faircloth, B.C., Glenn, T.C., Braun, E.L. & Kimball,
R.T. (2016) Avoiding missing data biases in phylogenomic inference:
an empirical study in the Landfowl (Aves: Galliformes). Molecular
Biology and Evolution, 33, 1110–1125.
Huelsenbeck, J.P., Bull, J.J. & Cunningham, C.W. (1996) Combining
data in phylogenetic analysis. Trends in Ecology & Evolution, 11,
152–158.
Huelsenbeck, J.P. & Hillis, D.M. (1993) Success of phylogenetic
methods in the four-taxon case. Systematic Biology, 42, 247–264.
Huelsenbeck, J.P., Joyce, P., Lakner, C. & Ronquist, F. (2008) Bayesian
analysis of amino acid substitution models. Philosophical Transac-
tions of the Royal Society B: Biological Sciences, 363, 3941–3953.
Huelsenbeck, J.P., Larget, B., Miller, R.E. & Ronquist, F. (2002)
Potential applications and pitfalls of Bayesian inference of phylogeny.
Systematic Biology, 51, 673–688.
Huelsenbeck, J.P. & Rannala, B. (2004) Frequentist properties of
Bayesian posterior probabilities of phylogenetic trees under simple
and complex substitution models. Systematic Biology, 53, 904–913.
Huelsenbeck, J.P., Ronquist, F., Nielsen, R. & Bollback, J.P. (2001)
Bayesian inference of phylogeny and its impact on evolutionary
biology. Science, 294, 2310–2314.
Jackman, S.D., Vandervalk, B.P., Mohamadi, H. et al. (2017) ABySS
2.0: resource-efficient assembly of large genomes using a bloom filter.
Genome Research, 27, 768–777.
Jeffroy, O., Brinkmann, H., Delsuc, F. & Philippe, H. (2006) Phy-
logenomics: the beginning of incongruence? Trends in Genetics, 22,
225–231.
Jia, F., Lo, N. & Ho, S.Y.W. (2014) The impact of modelling rate
heterogeneity among sites on phylogenetic estimates of intraspecific
evolutionary rates and timescales. PLoS One, 9, e95722.
Jones, D.T., Taylor, W.R. & Thornton, J.M. (1992) The rapid generation
of mutation data matrices from protein sequences. Computer Appli-
cations in the Biosciences, 8, 275–282.
Joshi, N.A. & Fass, J.N. (2011) Sickle: A sliding-window, adaptive,
quality-based trimming tool for FastQ files. URL https://github.com/
najoshi/sickle. [Accessed on 23 Nov 2019].
Jothi, R., Zotenko, E., Tasneem, A. & Przytycka, T.M. (2006)
COCO-CL: hierarchical clustering of homology relations based
on evolutionary correlations. Bioinformatics, 22, 779–788.
Joy, J.B., Liang, R.H., McCloskey, R.M., Nguyen, T. & Poon, A.F.Y.
(2016) Ancestral reconstruction. PLoS Computational Biology, 12,
e1004763.
Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K., von Haeseler, A. &
Jermiin, L.S. (2017) ModelFinder: fast model selection for accurate
phylogenetic estimates. Nature Methods, 14, 587–589.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Karin, B.R., Gamble, T. & Jackman, T.R. (2019) Optimizing phyloge-
nomics with rapidly evolving long exons: comparison with anchored
hybrid enrichment and Ultraconserved elements. bioRxiv, 672238.
Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence align-
ment software version 7: improvements in performance and usability.
Molecular Biology and Evolution, 30, 772–780.
Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H.,
Zahler, A.M. & Haussler, D. (2002) The human genome browser at
UCSC. Genome Research, 12, 996–1006.
Kocot, K.M., Citarella, M.R., Moroz, L.L. & Halanych, K.M. (2013)
PhyloTreePruner: a phylogenetic tree-based approach for selection
of orthologous sequences for phylogenomics. Evolutionary Bioinfor-
matics, 9, 429–435.
Kocot, K.M., Struck, T.H., Merkel, J., Waits, D,S., Todt, C., Brannock,
P.M., Weese, D.A., Cannon, J.T., Moroz, L.L., Lieb, B., Halanych,
K.M. (2017) Phylogenomics of Lophotrochozoa with consideration
of systematic error. Systematic Biology, 66, 256–282, 2.
Kozlov, A.M., Aberer, A.J. & Stamatakis, A. (2015) ExaML version 3:
a tool for phylogenomic analyses on supercomputers. Bioinformatics,
31, 2577–2579.
Kriventseva, E.V., Kuznetsov, D., Tegenfeldt, F., Manni, M., Dias, R.,
Simão, F.A. & Zdobnov, E.M. (2019) OrthoDB v10: sampling the
diversity of animal, plant, fungal, protist, bacterial and viral genomes
for evolutionary and functional annotations of orthologs. Nucleic
Acids Research, 47, D807–D811.
Kubatko, L.S., Carstens, B.C. & Knowles, L.L. (2009) STEM: species
tree estimation using maximum likelihood for gene trees under
coalescence. Bioinformatics, 25, 971–973.
Kubatko, L.S. & Degnan, J.H. (2007) Inconsistency of phylogenetic
estimates from concatenated data under coalescence. Systematic
Biology, 56, 17–24.
Kück, P.J. & Wägele, W. (2015) Plesiomorphic character states cause
systematic errors in molecular phylogenetic analyses: a simulation
study. Cladistics, 32, 461–478.
Kumar, S., Filipski, A.J., Battistuzzi, F.U., Kosakovsky Pond, S.L. &
Tamura, K. (2012) Statistics and truth in phylogenomics. Molecular
Biology and Evolution, 29, 457–472.
Lam, H.Y., Clark, M.J., Chen, R. et al. (2012) Performance comparison
of whole-genome sequencing platforms. Nature Biotechnology, 30,
78–82.
Landis, M.J., Matzke, N.J., Moore, B.R. & Huelsenbeck, J.P. (2013)
Bayesian analysis of biogeography when the number of areas is large.
Systematic Biology, 62, 789–804.
Lanfear, R., Calcott, B., Kainer, D., Mayer, C. & Stamatakis, A. (2014)
Selecting optimal partitioning schemes for phylogenomic datasets.
BMC Evolutionary Biology, 14, 82.
Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T. & Calcott,
B. (2016) PartitionFinder 2: new methods for selecting partitioned
models of evolution for molecular and morphological phylogenetic
analyses. Molecular Biology and Evolution, 34, 772–773.
Larkin, M.A., Blackshields, G., Brown, N.P. et al. (2007) Clustal W and
Clustal X version 2.0. Bioinformatics, 23, 2947–2948.
Lartillot, N., Lepage, T. & Blanquart, S. (2009) PhyloBayes 3: a
Bayesian software package for phylogenetic reconstruction and
molecular dating. Bioinformatics, 25, 2286–2288.
Lartillot, N. & Philippe, H. (2006) Computing Bayes factors using
thermodynamic integration. Systematic Biology, 55, 195–207.
Lartillot, N., Rodrigue, N., Stubbs, D. & Richer, J. (2013) PhyloBayes
MPI: phylogenetic reconstruction with infinite mixtures of profiles in
a parallel environment. Systematic Biology, 62, 611–615.
Le, S.Q., Dang, C.C. & Gascuel, O. (2012) Modeling protein evolution
with several amino acid replacement matrices depending on site rates.
Molecular Biology and Evolution, 29, 2921–2936.

Le, S.Q., Gascuel, O. & Lartillot, N. (2008) Empirical profile mix-
ture models for phylogenetic reconstruction. Bioinformatics, 24,
2317–2323.
Lechner, M., Findeiss, S., Steiner, L., Marz, M., Stadler, P.F. &
Prohaska, S.J. (2011) Proteinortho: detection of (co-)orthologs in
large-scale analysis. BMC Bioinformatics, 12, 124.
Leinonen, R., Sugawara, H., Shumway, M. & International Nucleotide
Sequence Database Collaboration (eds) (2011) The sequence read
archive. Nucleic Acids Research, 39, D19–D21.
Lemmon, A.R., Emme, S.A. & Lemmon, E.M. (2012) Anchored
hybrid enrichment for massively high-throughput phylogenomics.
Systematic Biology, 61, 727–744.
Lemmon, A.R. & Moriarty, E.C. (2004) The importance of proper
model assumption in Bayesian Phylogenetics. Systematic Biology, 53,
278–298.
Lemmon, E.M. & Lemmon, A.R. (2013) High-throughput genomic
data in systematics and Phylogenetics. Annual Review of Ecology,
Evolution, and Systematics, 44, 99–121.
Li, L., Stoeckert, C.J. & Roos, D.S. (2003) OrthoMCL: identification
of ortholog groups for eukaryotic genomes. Genome Research, 13,
2178–2189.
Li, R., Zhu, H., Ruan, J. et al. (2010) De novo assembly of human
genomes with massively parallel short read sequencing. Genome
Research, 20, 265–272.
Liu, L., Wu, S. & Yu, L. (2015) Coalescent methods for estimating
species trees from phylogenomic data. Journal of Systematics and
Evolution, 53, 380–390.
Liu, L., Yu, L. & Edwards, S.V. (2010) A maximum pseudo-likelihood
approach for estimating species trees under the coalescent model.
BMC Evolutionary Biology, 10, 302.
Liu, L., Yu, L., Pearl, D.K. & Edwards, S.V. (2009) Estimating species
phylogenies using coalescence times among sequences. Systematic
Biology, 58, 468–477.
Maddison, W.P. & Maddison, D.R. (2018) Mesquite: a modular sys-
tem for evolutionary analysis. URL http://www.mesquiteproject.org.
[Accessed on 23 Nov 2019].
Magee, A.F., May, M.R. & Moore, B.R. (2014) The Dawn of open
access to phylogenetic data. PLoS One, 9, e110268.
Mandel, J.R., Dikow, R.B., Funk, V.A. et al. (2014) A target enrichment
method for gathering phylogenetic information from hundreds of loci:
an example from the compositae. Applications in Plant Sciences, 2,
1300085.
Mardis, E.R. (2011) A decade’s perspective on DNA sequencing
technology. Nature, 470, 198–203.
Martin, M. (2011) Cutadapt removes adapter sequences from
high-throughput sequencing reads. EMBnet Journal, 17, 10–12.
Matzke, N.J. (2013) Probabilistic historical biogeography: new models
for founder-event speciation, imperfect detection, and fossils allow
improved accuracy and model-testing. Frontiers of Biogeography, 5,
242–248.
Matzke, N.J. & Wright, A. (2016) Inferring node dates from tip dates
in fossil Canidae: the importance of tree priors. Biology Letters, 12,
20160328.
Mayrose, I., Friedman, N. & Pupko, T. (2005) A gamma mixture model
better accounts for among site rate heterogeneity. Bioinformatics, 21,
ii151–ii158.
McCormack, J.E., Faircloth, B.C., Crawford, N.G., Gowaty, P.A., Brum-
field, R.T. & Glenn, T.C. (2012) Ultraconserved elements are novel
phylogenomic markers that resolve placental mammal phylogeny
when combined with species-tree analysis. Genome Research, 22,
746–754.
McCormack, J.E., Hird, S.M., Zellmer, A.J., Carstens, B.C. & Brum-
field, R.T. (2013) Applications of next-generation sequencing to
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Understanding phylogenomics 21
phylogeography and phylogenetics. Molecular Phylogenetics and
Evolution, 66, 526–538.
McCormack, J.E., Tsai, W.L.E. & Faircloth, B.C. (2016) Sequence
capture of ultraconserved elements from bird museum specimens.
Molecular Ecology Resources, 16, 1189–1203.
Metzker, M.L. (2010) Sequencing technologies – the next generation.
Nature Reviews Genetics, 11, 31–46.
Meyer, A.L.S. & Wiens, J.J. (2018) Estimating diversification rates for
higher taxa: BAMM can give problematic estimates of rates and rate
shifts. Evolution, 72, 39–53.
Minh, B.Q., Hahn, M. & Lanfear, R. (2018) New methods to calculate
concordance factors for phylogenomic datasets. bioRxiv, 487801.
Minin, V., Abdo, Z., Joyce, P. & Sullivan, J. (2003) Performance-based
selection of likelihood models for phylogeny estimation. Systematic
Biology, 52, 674–683.
Mirarab, S., Bayzid, M.S., Boussau, B. & Warnow, T. (2014) Statistical
binning improves species tree estimation in the presence of gene tree
incongruence. Science, 346, 1250463.
Moore, B.R., Höhna, S., May, M.R., Rannala, B. & Huelsenbeck, J.P.
(2016) Critically evaluating the theory and performance of Bayesian
analysis of macroevolutionary mixtures. Proceedings of the National
Academy of Sciences, 113, 9569–9574.
Moreau, C.S. (2014) A practical guide to DNA extraction, PCR, and
gene-based DNA sequencing in insects. Halteres, 5, 32–42.
Mork, R., Martin, P. & Zhao, Z. (2015) Contemporary challenges for
data-intensive scientific workflow management systems. Proceedings
of the 10th Workshop on Workflows in Support of Large-Scale Science
- WORKS ’15 (ed. by J. Montagnat and I. Taylor), pp. 1–11. ACM
Press, Austin, Texas.
Morlon, H., Lewitus, E., Condamine, F.L., Manceau, M., Clavel, J.
& Drury, J. (2016) RPANDA: an R package for macroevolutionary
analyses on phylogenetic trees. Methods in Ecology and Evolution, 7,
589–597.
Nabhan, A.R. & Sarkar, I.N. (2012) The impact of taxon sampling
on phylogenetic inference: a review of two decades of controversy.
Briefings in Bioinformatics, 13, 122–134.
Narzisi, G. & Mishra, B. (2011) Comparing De novo genome assembly:
the long and short of it. PLoS One, 6, e19175.
Nascimento, F.F., dos Reis, M. & Yang, Z. (2017) A biologist’s guide
to Bayesian phylogenetic analysis. Nature Ecology and Evolution, 1,
1446–1454.
Nguyen, L.-T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015)
IQ-TREE: a fast and effective stochastic algorithm for estimating
maximum-likelihood phylogenies. Molecular Biology and Evolution,
32, 268–274.
Nosenko, T., Schreiber, F., Adamska, M. et al. (2013) Deep metazoan
phylogeny: when different genes tell different stories. Molecular
Phylogenetics and Evolution, 67, 223–233.
O’Brien, K.P., Remm, M. & Sonnhammer, E.L.L. (2005) Inparanoid:
a comprehensive database of eukaryotic orthologs. Nucleic Acids
Research, 33, D476–D480.
O’Brien, S.J. & Stanyon, R. (1999) Phylogenomics: ancestral primate
viewed. Nature, 402, 365–366.
Oakley, T.H., Alexandrou, M.A., Ngo, R., Pankey, M.S., Churchill,
C.K.C., Chen, W. & Lopker, K.B. (2014) Osiris: accessible and
reproducible phylogenetic and phylogenomic analyses within the
galaxy workflow management system. BMC Bioinformatics, 15, 230.
Pagel, M., Meade, A. & Barker, D. (2004) Bayesian estimation of
ancestral character states on phylogenies. Systematic Biology, 53,
673–684.
Paradis, E. (2013) Molecular dating of phylogenies by likelihood
methods: a comparison of models and a new information criterion.
Molecular Phylogenetics and Evolution, 67, 436–444.
22 A. D. Young and J. P. Gillung
Paradis, E., Claude, J. & Strimmer, K. (2004) APE: analyses of
phylogenetics and evolution in R language. Bioinformatics, 20,
289–290.
Parenti, L.R. (2006) Common cause and historical biogeography. Bio-
geography in a Changing World. Fifth Biennial Conference of the
Systematics Association (ed. by M.C. Ebach and R.S. Tangney), pp.
61–82. CRC Press, London.
Parham, J.F., Donoghue, P.C.J., Bell, C.J. et al. (2012) Best practices for
justifying fossil calibrations. Systematic Biology, 61, 346–359.
Petersen, M., Meusemann, K., Donath, A. et al. (2017) Orthograph: a
versatile tool for mapping coding nucleotide sequences to clusters of
orthologous genes. BMC Bioinformatics, 18, 111.
Philippe, H., Brinkmann, H., Lavrov, D.V., Littlewood, D.T.J., Manuel,
M., Wörheide, G., Baurain, D. (2011) Resolving difficult phyloge-
netic questions: why more sequences are not enough. PLoS Biology,
9, e1000602, 3.
Pleijel, F., Jondelius, U., Norlinder, E. et al. (2008) Phylogenies without
roots? A plea for the use of vouchers in molecular phylogenetic
studies. Molecular Phylogenetics and Evolution, 48, 369–371.
Posada, D. & Crandall, K.A. (2001) Selecting the best-fit model of
nucleotide substitution. Systematic Biology, 50, 580–601.
Powell, S., Szklarczyk, D., Trachana, K. et al. (2012) eggNOG v3.0:
orthologous groups covering 1133 organisms at 41 different taxo-
nomic ranges. Nucleic Acids Research, 40, D284–D289.
Qu, X.J., Jin, J.J., Chaw, S.M., Li, D.Z. & Yib, T.S. (2017) Multiple
measures could alleviate long-branch attraction in phylogenomic
reconstruction of Cupressoideae (Cupressaceae). Scientific Reports,
7, 41005.
Rabbani, B., Tekin, M. & Mahdieh, N. (2014) The promise of
whole-exome sequencing in medical genetics. Journal of Human
Genetics, 59, 5–15.
Rabosky, D.L. (2014) Automatic detection of key innovations, rate
shifts, and diversity-dependence on phylogenetic trees. PLoS One, 9,
e89543.
Rannala, B. & Yang, Z. (2003) Bayes estimation of species divergence
times and ancestral population sizes using DNA sequences from
multiple loci. Genetics, 164, 1645–1656.
Ree, R.H., Moore, B.R., Webb, C.O. & Donoghue, M.J. (2005) A
likelihood framework for inferring the evolution of geographic range
on phylogenetic trees. Evolution, 59, 2299–2311.
Ree, R.H. & Sanmartín, I. (2009) Prospects and challenges for para-
metric models in historical biogeographical inference. Journal of Bio-
geography, 36, 1211–1220.
Ree, R.H. & Smith, S.A. (2008) Maximum likelihood inference of geo-
graphic range evolution by dispersal, local extinction, and Cladogen-
esis. Systematic Biology, 57, 4–14.
Revell, L.J. (2012) Phytools: an R package for phylogenetic comparative
biology (and other things). Methods in Ecology and Evolution, 3,
217–223.
Richards, E.J., Brown, J.M., Barley, A.J., Chong, R.A. & Thomson, R.C.
(2018) Variation across mitochondrial gene Trees provides evidence
for systematic error: how much gene tree variation is biological?
Systematic Biology, 67, 847–860.
Romiguier, J., Cameron, S.A., Woodard, S.H., Fischman, B.J., Keller,
L. & Praz, C.J. (2016) Phylogenomics controlling for base composi-
tional Bias reveals a single origin of Eusociality in Corbiculate bees.
Molecular Biology and Evolution, 33, 670–678.
Ronquist, F. (1997) Dispersal–vicariance analysis: a new approach to
the quantification of historical biogeography. Systematic Biology, 46,
195–203.
Ronquist, F., Klopfstein, S., Vilhelmsen, L., Schulmeister, S., Murray,
D.L. & Rasnitsyn, A.P. (2012a) A total-evidence approach to dating
with fossils, applied to the early radiation of the hymenoptera.
Systematic Biology, 61, 973–999.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
Ronquist, F., Teslenko, M., Van Der Mark, P. et al. (2012b) MrBayes 3.2:
efficient Bayesian phylogenetic inference and model choice across a
large model space. Systematic Biology, 61, 539–542.
Rosenberg, M.S. & Kumar, S. (2003) Taxon sampling, bioinformatics,
and phylogenomics. Systematic Biology, 52, 119–124.
Roth, A.C.J., Gonnet, G.H. & Dessimoz, C. (2008) Algorithm of OMA
for large-scale orthology inference. BMC Bioinformatics, 9, 518.
Roure, B., Baurain, D. & Philippe, H. (2013) Impact of missing data
on phylogenies inferred from empirical phylogenomic data sets.
Molecular Biology and Evolution, 30, 197–214.
Roure, B., Rodriguez-Ezpeleta, N. & Philippe, H. (2007) SCaFoS:
a tool for selection, concatenation and fusion of sequences for
phylogenomics. BMC Evolutionary Biology, 7(Suppl 1), S2.
Royer-Carenzi, M. & Didier, G. (2016) A comparison of ancestral
state reconstruction methods for quantitative characters. Journal of
Theoretical Biology, 404, 126–142.
Royer-Carenzi, M., Pontarotti, P. & Didier, G. (2013) Choosing the
best ancestral character state reconstruction method. Mathematical
Biosciences, 242, 95–109.
Salichos, L. & Rokas, A. (2011) Evaluating ortholog prediction algo-
rithms in a yeast model clade. PLoS One, 6, e18755.
Sanderson, M.J. (2003) r8s: inferring absolute rates of molecular
evolution and divergence times in the absence of a molecular clock.
Bioinformatics, 19, 301–302.
Schwarz, G. (1978) Estimating the dimension of a model. The Annals of
Statistics, 6, 461–464.
Shade, A. & Teal, T.K. (2015) Computing workflows for biologists: a
roadmap. PLoS Biology, 13, e1002303.
Siu-Ting, K., Torres-Sánchez, M., San Mauro, D. et al. (2019) Inad-
vertent paralog inclusion drives artifactual topologies and timetree
estimates in Phylogenomics. Molecular Biology and Evolution, 36,
1344–1356.
Smith, B.T., Mauck III, W.M., Benz, B., Andersen, M.J. (2018b) Uneven
missing data skews phylogenomic relationships within the lories and
lorikeets. bioRxiv, 398297.
Smith, S.A., Brown, J.W. & Walker, J.F. (2018a) So many genes, so
little time: a practical approach to divergence-time estimation in the
genomic era. PLoS One, 13, e0197433.
Smith, S.A. & O’Meara, B.C. (2012) treePL: divergence time estimation
using penalized likelihood for large phylogenies. Bioinformatics, 28,
2689–2690.
Springer, M.S. & Gatesy, J. (2014) Land plant origins and coalescence
confusion. Trends in Plant Science, 19, 267–269.
Stadler, T. (2009) On incomplete sampling under birth–death models
and connections to the sampling-based coalescent. Journal of Theo-
retical Biology, 261, 58–66.
Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic
analysis and post-analysis of large phylogenies. Bioinformatics, 30,
1312–1313.
Straub, S.C.K., Parks, M., Weitemier, K., Fishbein, M., Cronn, R.C.
& Liston, A. (2012) Navigating the tip of the genomic iceberg:
next-generation sequencing for plant systematics. American Journal
of Botany, 99, 349–364.
Strimmer, K. & Haeseler, A.v. (1997) Likelihood-mapping: a simple
method to visualize phylogenetic content of a sequence alignment.
Proceedings of the National Academy of Sciences, 94, 6815–6819.
Struck, T.H. (2013) The impact of paralogy on phylogenomic stud-
ies – a case study on annelid relationships. PLoS One, 8, e62892.
Struck, T.H. (2014) TreSpEx-detection of misleading signal in phyloge-
netic reconstructions based on tree information. Evolutionary Bioin-
formatics Online, 10, 51–67.
Suchard, M.A., Lemey, P., Baele, G., Ayres, D.L., Drummond, A.J. &
Rambaut, A. (2018) Bayesian phylogenetic and phylodynamic data
integration using BEAST 1.10. Virus Evolution, 4, vey016.

Suchard, M.A., Weiss, R.E. & Sinsheimer, J.S. (2001) Bayesian selec-
tion of continuous-time Markov chain evolutionary models. Molecu-
lar Biology and Evolution, 18, 1001–1013.
Sugiura, N. (1978) Further analysts of the data by akaike’s information
criterion and the finite corrections: further analysts of the data by
akaike’s. Communications in Statistics, 7, 13–26.
Sullivan, J., Swofford, D.L. & Naylor, G.J.P. (1999) The effect of
taxon sampling on estimating rate heterogeneity parameters of
maximum-likelihood models. Molecular Biology and Evolution, 16,
1347–1347, 1356.
Sun, M., Folk, R.A., Gitzendanner, M.A., Guralnick, R.P., Soltis,
P.S., Chen, Z., Soltis, D.E. (2019) Estimating rates and patterns of
diversification with incomplete sampling: a case study in the rosids.
bioRxiv, 749325.
Szitenberg, A., John, M., Blaxter, M.L. & Lunt, D.H. (2015) Repro-
Phylo: an environment for reproducible Phylogenomics. PLoS Com-
putational Biology, 13, 11, 9, e1004447.
Tatusov, R.L., Fedorova, N.D., Jackson, J.D. et al. (2003) The COG
database: an updated version includes eukaryotes. BMC Bioinformat-
ics, 4, 41.
Than, C. & Nakhleh, L. (2009) Species tree inference by minimizing
deep coalescences. PLoS Computational Biology, 5, e1000501.
Thurmond, J., Goodman, J.L., Strelets, V.B. et al. (2019) FlyBase 2.0:
the next generation. Nucleic Acids Research, 47, D759–D765.
Title, P.O. & Rabosky, D.L. (2017) Do macrophylogenies yield stable
macroevolutionary inferences? An example from Squamate reptiles.
Systematic Biology, 66, 843–856.
Trachana, K., Larsson, T.A., Powell, S., Chen, W.-H., Doerks, T.,
Muller, J. & Bork, P. (2011) Orthology prediction methods: a quality
assessment using curated protein families. BioEssays, 33, 769–780.
Turney, S., Cameron, E.R., Cloutier, C.A. & Buddle, C.M. (2015)
Non-repeatable science: assessing the frequency of voucher specimen
deposition reveals that most arthropod research cannot be verified.
PeerJ, 3, e1168.
Ullah, I., Sjöstrand, J., Andersson, P., Sennblad, B. & Lagergren, J.
(2015) Integrating sequence evolution into probabilistic Orthology
analysis. Systematic Biology, 64, 969–982.
van der Heijden, R.T.J.M., Snel, B., van Noort, V. & Huynen, M.A.
(2007) Orthology prediction at scalable resolution by phylogenetic
tree analysis. BMC Bioinformatics, 8, 83.
Vanderpoorten, A. & Goffinet, B. (2006) Mapping uncertainty and phy-
logenetic uncertainty in ancestral character state reconstruction: an
example in the Moss genus Brachytheciastrum. Systematic Biology,
55, 957–971.
Wang, Z., Gerstein, M. & Snyder, M. (2009) RNA-Seq: a revolutionary
tool for transcriptomics. Nature Reviews Genetics, 10, 57–63.
Weitemier, K., Straub, S.C.K., Cronn, R.C., Fishbein, M., Schmickl,
R., McDonnell, A. & Liston, A. (2014) Hyb-Seq: combining target
enrichment and genome skimming for plant phylogenomics. Appli-
cations in Plant Science, 2, 1400042.
© 2019 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12406
View publication stats
Understanding phylogenomics 23
Wen, J., Egan, A.N., Dikow, R.B. & Zimmer, E.A. (2015) Utility of
transcriptome sequencing for phylogenetic inference and character
evolution. Next-generation Sequencing in Plant Systematics
Wiens, J.J. (2017) What explains patterns of biodiversity across the tree
of life? BioEssays, 39, 1600128.
Xu, B. & Yang, Z. (2013) PAMLX: a graphical user interface for PAML.
Molecular Biology and Evolution, 30, 2723–2724.
Yang, Z. (1993) Maximum-likelihood estimation of phylogeny from
DNA sequences when substitution rates differ over sites. Molecular
Biology and Evolution, 10, 1396–1401.
Yang, Z. (2006) Computational Molecular Evolution. Oxford University
Press, New York, New York.
Yang, Z. & Rannala, B. (2006) Bayesian estimation of species diver-
gence times under a molecular clock using multiple fossil cali-
brations with soft bounds. Molecular Biology and Evolution, 23,
212–226.
Yang, Z. & Rannala, B. (2012) Molecular phylogenetics: principles and
practice. Nature Reviews Genetics, 13, 303–314.
Yu, Y., Harris, A.J., Blair, C. & He, X. (2015) RASP (reconstruct
ancestral state in phylogenies): a tool for historical biogeography.
Molecular Phylogenetics and Evolution, 87, 46–49.
Yu, Y., Harris, A.J. & He, X. (2010) S-DIVA (statistical dispersal-
Vicariance analysis): a tool for inferring biogeographic histories.
Molecular Phylogenetics and Evolution, 56, 848–850.
Zerbino, D.R. & Birney, E. (2008) Velvet: algorithms for de novo
short read assembly using de Bruijn graphs. Genome Research, 18,
821–829.
Zhang, C. (2017) Molecular Clock Dating using MrBayes. arXiv,
1603.05707.
Zhang, C., Rabiee, M., Sayyari, E. & Mirarab, S. (2018) ASTRAL-III:
polynomial time species tree reconstruction from partially resolved
gene trees. BMC Bioinformatics, 19, 153.
Zhang, C., Stadler, T., Klopfstein, S., Heath, T.A. & Ronquist, F. (2016)
Total-evidence dating under the fossilized birth–death process. Sys-
tematic Biology, 65, 228–249.
Zhang, M.Y., Williams, J.L. & Lucky, A. (2019) Understanding UCEs: a
comprehensive primer on using ultraconserved elements for arthropod
phylogenomics. Insect Systematics and Diversity, 3, 1–12.
Zhong, B., Deusch, O., Goremykin, V.V. et al. (2011) Systematic error
in seed plant phylogenomics. Genome Biology and Evolution, 3,
1340–1348.
Zuckerkandl, E. & Pauling, L. (1962) Molecular disease, evolution and
genetic heterogeneity. Horizons in Biochemistry (ed. by M. Kasha and
B. Pullman), pp. 189–225. Academic Press, New York, New York.
Zwickl, D.J. (2006) GARLI: genetic algorithm for rapid likelihood
inference. PhD Dissertation, The University of Texas at Austin, Austin
Accepted 30 October 2019