Molecular phylogenetics (/məˈlɛkjʊlər ˌfaɪloʊdʒəˈnɛtɪks, mɒ-, moʊ-/[1][2]) is the

branch of phylogeny that analyzes genetic, hereditary molecular differences,

predominantly in DNA sequences, to gain information on an organism's
evolutionary relationships. From these analyses, it is possible to determine the
processes by which diversity among species has been achieved. The result of a
molecular phylogenetic analysis is expressed in a phylogenetic tree. Molecular
phylogenetics is one aspect of molecular systematics, a broader term that also
includes the use of molecular data in taxonomy and biogeography.[3][4][5]
Molecular phylogenetics and molecular evolution correlate. Molecular evolution is
the process of selective changes (mutations) at a molecular level (genes, proteins,
etc.) throughout various branches in the tree of life (evolution). Molecular
phylogenetics makes inferences of the evolutionary relationships that arise due to
molecular evolution and results in the construction of a phylogenetic tree.[6]
History
Further information: History of molecular evolution
The theoretical frameworks for molecular systematics were laid in the 1960s in the
works of Emile Zuckerkandl, Emanuel Margoliash, Linus Pauling, and Walter M.
Fitch.[7] Applications of molecular systematics were pioneered by Charles G.
Sibley (birds), Herbert C. Dessauer (herpetology), and Morris Goodman (primates),
followed by Allan C. Wilson, Robert K. Selander, and John C. Avise (who studied
various groups). Work with protein electrophoresis began around 1956. Although
the results were not quantitative and did not initially improve on morphological
classification, they provided tantalizing hints that long-held notions of the
classifications of birds, for example, needed substantial revision. In the period of
1974–1986, DNA–DNA hybridization was the dominant technique used to measure
genetic difference.[8]
Theoretical background
Early attempts at molecular systematics were also termed chemotaxonomy and
made use of proteins, enzymes, carbohydrates, and other molecules that were
separated and characterized using techniques such as chromatography. These
have been replaced in recent times largely by DNA sequencing, which produces the
exact sequences of nucleotides or bases in either DNA or RNA segments extracted
using different techniques. In general, these are considered superior for
evolutionary studies, since the actions of evolution are ultimately reflected in the
genetic sequences. At present, it is still a long and expensive process to sequence
the entire DNA of an organism (its genome). However, it is quite feasible to
determine the sequence of a defined area of a particular chromosome. Typical
molecular systematic analyses require the sequencing of around 1000 base pairs.
At any location within such a sequence, the bases found in a given position may
vary between organisms. The particular sequence found in a given organism is
referred to as its haplotype. In principle, since there are four base types, with 1000
base pairs, we could have 41000 distinct haplotypes. However, for organisms within
a particular species or in a group of related species, it has been found empirically
that only a minority of sites show any variation at all, and most of the variations
that are found are correlated, so that the number of distinct haplotypes that are
found is relatively small.[9]

In a phylogenetic tree,
numerous groupings (clades) exist. A clade may be defined as a group of
organisms having a common ancestor throughout evolution. This figure illustrates
how a clade in a phylogenetic tree may be expressed.
In a molecular systematic analysis, the haplotypes are determined for a defined
area of genetic material; a substantial sample of individuals of the target species or
other taxon is used; however, many current studies are based on single
individuals. Haplotypes of individuals of closely related, yet different, taxa are also
determined. Finally, haplotypes from a smaller number of individuals from a
definitely different taxon are determined: these are referred to as an outgroup. The
base sequences for the haplotypes are then compared. In the simplest case, the
difference between two haplotypes is assessed by counting the number of
locations where they have different bases: this is referred to as the number
of substitutions (other kinds of differences between haplotypes can also occur, for
example, the insertion of a section of nucleic acid in one haplotype that is not
present in another). The difference between organisms is usually re-expressed as
a percentage divergence, by dividing the number of substitutions by the number of
base pairs analysed: the hope is that this measure will be independent of the
location and length of the section of DNA that is sequenced.
An older and superseded approach was to determine the divergences between
the genotypes of individuals by DNA–DNA hybridization. The advantage claimed for
using hybridization rather than gene sequencing was that it was based on the
entire genotype, rather than on particular sections of DNA. Modern sequence
comparison techniques overcome this objection by the use of multiple sequences.
Once the divergences between all pairs of samples have been determined, the
resulting triangular matrix of differences is submitted to some form of
statistical cluster analysis, and the resulting dendrogram is examined in order to
see whether the samples cluster in the way that would be expected from current
ideas about the taxonomy of the group. Any group of haplotypes that are all more
similar to one another than any of them is to any other haplotype may be said to
constitute a clade, which may be visually represented as the figure displayed on
the right demonstrates. Statistical techniques such
as bootstrapping and jackknifing help in providing reliability estimates for the
positions of haplotypes within the evolutionary trees.
Techniques and applications
Every living organism contains deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), and proteins. In general, closely related organisms have a high degree of
similarity in the molecular structure of these substances, while the molecules of
organisms distantly related often show a pattern of dissimilarity. Conserved
sequences, such as mitochondrial DNA, are expected to accumulate mutations
over time, and assuming a constant rate of mutation, provide a molecular clock for
dating divergence. Molecular phylogeny uses such data to build a "relationship
tree" that shows the probable evolution of various organisms. With the invention
of Sanger sequencing in 1977, it became possible to isolate and identify these
molecular structures.[10][11] High-throughput sequencing may also be used to obtain
the transcriptome of an organism, allowing inference of phylogenetic relationships
using transcriptomic data.
The most common approach is the comparison of homologous sequences for
genes using sequence alignment techniques to identify similarity. Another
application of molecular phylogeny is in DNA barcoding, wherein the species of an
individual organism is identified using small sections of mitochondrial
DNA or chloroplast DNA. Another application of the techniques that make this
possible can be seen in the very limited field of human genetics, such as the ever-
more-popular use of genetic testing to determine a child's paternity, as well as the
emergence of a new branch of criminal forensics focused on evidence known
as genetic fingerprinting.
Molecular phylogenetic analysis
There are several methods available for performing a molecular phylogenetic
analysis. One method, including a comprehensive step-by-step protocol on
constructing a phylogenetic tree, including DNA/Amino Acid contiguous sequence
assembly, multiple sequence alignment, model-test (testing best-fitting substitution
models), and phylogeny reconstruction using Maximum Likelihood and Bayesian
Inference, is available at Nature Protocol.[12]
Another molecular phylogenetic analysis technique has been described by Pevsner
and shall be summarized in the sentences to follow (Pevsner, 2015). A phylogenetic
analysis typically consists of five major steps. The first stage comprises sequence
acquisition. The following step consists of performing a multiple sequence
alignment, which is the fundamental basis of constructing a phylogenetic tree. The
third stage includes different models of DNA and amino acid substitution. Several
models of substitution exist. A few examples include Hamming distance, the Jukes
and Cantor one-parameter model, and the Kimura two-parameter model
(see Models of DNA evolution). The fourth stage consists of various methods of
tree building, including distance-based and character-based methods. The
normalized Hamming distance and the Jukes-Cantor correction formulas provide
the degree of divergence and the probability that a nucleotide changes to another,
respectively. Common tree-building methods include unweighted pair group
method using arithmetic mean (UPGMA) and Neighbor joining, which are distance-
based methods, Maximum parsimony, which is a character-based method,
and Maximum likelihood estimation and Bayesian inference, which are character-
based/model-based methods. UPGMA is a simple method; however, it is less
accurate than the neighbor-joining approach. Finally, the last step comprises
evaluating the trees. This assessment of accuracy is composed of consistency,
efficiency, and robustness.[13]

Five Stages of Molecular Phylogenetic Analysis

MEGA (molecular evolutionary genetics analysis) is an analysis software that is
user-friendly and free to download and use. This software is capable of analyzing
both distance-based and character-based tree methodologies. MEGA also contains
several options one may choose to utilize, such as heuristic approaches and
bootstrapping. Bootstrapping is an approach that is commonly used to measure
the robustness of topology in a phylogenetic tree, which demonstrates the
percentage each clade is supported after numerous replicates. In general, a value
greater than 70% is considered significant. The flow chart displayed on the right
visually demonstrates the order of the five stages of Pevsner's molecular
phylogenetic analysis technique that have been described.[13]
Limitations
Molecular systematics is an essentially cladistic approach: it assumes that
classification must correspond to phylogenetic descent, and that all valid taxa must
be monophyletic. This is a limitation when attempting to determine the optimal
tree(s), which often involves bisecting and reconnecting portions of the
phylogenetic tree(s).
The recent discovery of extensive horizontal gene transfer among organisms
provides a significant complication to molecular systematics, indicating that
different genes within the same organism can have different phylogenies. HGTs
can be detected and excluded using a number of phylogenetic methods
(see Inferring horizontal gene transfer § Explicit phylogenetic methods).
In addition, molecular phylogenies are sensitive to the assumptions and models
that go into making them. Firstly, sequences must be aligned; then, issues such
as long-branch attraction, saturation, and taxon sampling problems must be
addressed. This means that strikingly different results can be obtained by applying
different models to the same dataset.[14][15] The tree-building method also brings
with it specific assumptions about tree topology, evolution speeds, and sampling.
The simplistic UPGMA assumes a rooted tree and a uniform molecular clock, both
of which can be incorrect.[13]
The low resolution power of single genes have been overcome using multigene
phylogenies, though the issue of HGT still merits careful algorithmic design.[16]