III.

Systematics and Related Issues o Paraphyletic  includes only the

ancestors but not all descendants
A. Systematic and Phylogeny o Polyphyletic  all descendants but
 Taxonomy not all ancestors
 Monophyly, Paraphyly, Polyphyly  Dichotomous tree: common to all
B. Principles/Concepts and Approaches in Systematics phylogenetic tree but not always
 Concepts and Tools
o Phenetics *Monophyly is a synapomorphic character
o DNA sequence *Paraphyly is a symplesiomorphic character
o Molecular characters *Polyphyly – group which share analogous characters
o Metabolic characters
o Polyphasic systematics
 Character Evolution and Phylogeny
Reconstruction

THE HISTORY OF LIFE: Looking at the Patterns

Phylogeny: evolutionary history of a taxon

Ontogeny: life history; focuses on the individual
Taxon: a group of real organisms at any stage of the
classification

Phylogenetic tree/cladogram/phylogeny Phylogenetic pitchforks

 Tree structure that represents the evolutionary
relationship within a group
 Cladogram  hypothesis with evidence about
the actual evolutionary history of a group;
clade or “branch”
 Dendrogram  lengths in branches are
arbitrary; does not show how far the group was
modified from its ancestors; dendro or “tree”
 “Phylogenies”  postulated evolutionary
history; branch lengths indicate the amount of
character change; phylogram
Common Errors in Reading Trees
Reading Tress
 Used when there are few evidences due to lack
of knowledge
 Adaptive radiation or rapid speciation
o Members of a taxon differentially
adapt to different environmental
conditions; reproductive isolation
 Charles Darwin’s discovery of
finches and tortoises in the
Galapagos Island
 Aquarium fishes
 Reading trees, not ladders
 A clade only indicates the grouping, not
hierarchy (no superiority among groupings)
 Regardless of the position, clades are the same
if they point toward the same meaning

Clade
 A monophyletic group; includes all
descendants and ancestors
o Paraphyletic and polyphyletic are not a
clade
Using Trees for Classification
 Phylogenetic classification
 Naming of clades
Artificial vs Natural Classification
 Artificial  no concept of evolutionary
history; may use one or several characteristics;
somehow biased because it only uses
morphological characteristics
 Natural  concept of evolution; uses several
characteristics to arrive at a more natural
classification
Important Assumptions in Trees
 Changes in characteristics occurs in lineages
over time
o Apomorphy: derived character
 Synapomorphy: shared derived
character
o Plesiomorphy: ancestral character
 Symplesiomorphy: shared
ancestral character
 Autapomorphy: unique
character
 Any group of organisms is related by descent
from a common ancestor
 Bifurcating or branching pattern of lineage-
splitting
Reconstructed Trees: Parsimony
 Parsimony principle – choosing of the simplest
scientific explanation which fits a clade; more
suitable or efficient in constructing a clade
Using Trees
 To make predictions about fossils
o E.g. the ancestors of whales had
double pulley ankles (terrestrial mammal)
 To make predictions about poorly-studied
species
o E.g. paclitaxel – a drug used to treat
cancer; it stabilizes the microtubules;
extracted from Pacific Yew tree bark
 If the leaves are always taken,
the roots cannot absorb
nutrients; will eventually make
the plant die
o E.g. Taxus brevitolia was split into (1) T.
baceata and (2) T. sumatrana
 To learn about the evolution of diversity
o The beetle’s diet (diversification of
angiosperm)
HOMOLOGIES AND ANALOGIES
 Use of characters that are reliable indicators of
a common ancestry to build a tree
 Homologous characters  share a
common evolutionary history; may or may not
share similar function
 Analogous characters  share a common
function (e.g. bird’s wing and bat’s wing)
Genetic Bricolage
 Hit-or-miss recruitment and adaptation of
pre-existing genes or materials for new
functions
Homology: Orthology and Paralogy
 Orthology  descent from a common
ancestor by genome division (speciation)
o E.g. 98% of genes are shared by
humans and chimpanzees
 Paralogy  descent from a common
ancestor by duplication within a genome that
may or may not perform the same function
o Typically occurs within a single species
o E.g. human hemoglobin alpha is
paralogous to human hemoglobin beta
Convergent and Divergent Evolution
 Convergent  two distinct lineages evolve
into similar characteristics independently
o Both lineages may have faced similar
environmental challenges and selective
pressures
 Divergent  similar lineages which evolve
into two distinct lineages
 o Factors: adaptive radiation, availability
of different niches for colonization;
low competition rates
*One’s fitness is measured by two factors: (1) survive
the current environmental conditions (2) capacity to
contribute to the next generation
*HNS gene  histone-like nucleoid structure
What Happened When?
 Stratigraphy
o Provides a sequence of events in
which relative dates can be extrapolated
based on the layers of deposited
sediments
 Relative dating: no exact date;
only examines which one is
younger/older
o Limitation: uplifting of sediments
 Radiometric dating
o Relies on half-life decay of radioactive
elements to allow scientists to date
rocks and materials directly
o Radiocarbon dating: Potassium-40 and
Carbon-14 (5730 years)
 Dendrochronology
o Annual rings of trees
 Molecular rocks
Primary metabolism vs secondary metabolism
 Primary metabolism  processes done regardless of the
environment; for maintenance (e.g. ATP production)
 Secondary metabolism  introduced
Virus: protein & DNA which causes harm when inside the cell
because it can adhere/intact to other bacteria
Horizontal gene transfer: transfer of genes from nonrelated species
PHENETICS: Numerical Taxonomy
Phenetics: based on phenotype (observable characters in
both form and function)
Fundamental Positions of Numerical Taxonomy
 Having a greater content of classification
allows better classification
o It should have more characters on
which it is based
 A priori, every character is of equal weight in
creating taxa
o No bias as to which characters are
more important
 Overall similarity between any two entities is a
function of their individual similarities
 Distinct taxa can be recognized because
correlations of characters differ in the groups
being compared
 Phylogenetic inferences
o Can be made from the taxonomic
structures of a group and from
character correlation, given certain
assumptions about evolutionary
pathways and mechanisms
 Taxonomy is viewed and practiced as an
empirical science (body of knowledge based on
observations: the use of physical senses)
o Classifications are based on phenetic
similarity
Phenetic Methodology
 Starts with the collection of raw measurement
data on the chosen set of morphs called
OTUs or Operational Taxonomic Unit (has no
branding)
 Data may be transformed in some manner to
remove redundancy and to normalize the
values to a common scale
o Numerical (e.g. number of petals)
o Categorical (e.g. male or female)
 A measure of similarity or dissimilarity is
compared for each pair of OTUs (correlation
coefficient)
o Every assumption = equal (no
superiority between characters)
 A clustering method is used to group OTUs
that are most similar; the agglomerative
clustering algorithms involve joining the two
most similar OTUs into a new compound
OTU and recomputing the similarity measure
 o Repeated until they are merged into a
single branch
 Clustering diagram is then converted into a
classification by selecting a cut level for each
taxonomic rank and identifying the clusters
that are distinct at the cut level as the taxa of
that rank (there must be evidences that will
support the classification)
Advantages of Numerical Taxonomy
 Power to integrate data from a variety of
sources such as:
o Morphology, physiology,
chemistry, affinities between DNA
strands, amino acid, sequences of
proteins, etc.
 Automation of large portions of the
taxonomic processes leads to greater
efficiency (e.g. use of softwares)
 Since characters are reduced in number,
data gathered in numerical form can be
integrated with existing electronic data
processing systems for the creation of
descriptions or keys
 A quantitative method provides greater
discrimination along the spectrum of
taxonomic differences and are more
sensitive in delimiting taxa
 The creation of empirically data tables has
forced workers to use more and better
described characters (more characters =
better classification)
Problems of Estimating Phenetic Relationships
 Different life history stages and sexual
dimorphism (e.g. peacocks and peahens; fruit
flies)
 Use of different statistical tools
o Differences in estimates of
relationships produced by different
similarity coefficients
 Possible effects of parallelism and
convergence on taxonomic judgments based
on estimates of phenetic
Some Issues
 Retiring taxonomists are leaving numerous
‘orphan’ taxa behind
 Few students enter the field
 Environmental impact assessments require
accurate taxonomic information
o Change in land use for commerce
 Needs comprehensive
evaluation because it may
cause great impact on the
environment
 E.g. Kaliwa Dam and swamps
in Pampanga
 Conservation managers need precise species
data
o Tamaraw  endemic to Mindoro
Solution for the Issues of Phenetics: Another Method
 Molecular Systematics
o Speed up the process of describing
species and identifying specimens; use
of DNA sequences
 Linnaeus first used
morphological classification; a
very cumbersome work
 Consortium for the Barcode of Life (CBOL)
o An international initiative devoted to
developing DNA barcoding in
specimen identification and
determination of species boundaries
 CO1 gene or cytochrome c oxidase
I  used in identification
 Delimits the species (easy to
identify those who do not
belong to the gene pool)
Why DNA?
 More molecular characters available for
analysis than morphological ones
o DNA  great source of information;
not debatable
o Types of DNA:
 Introns – intervening
sequences
 Exons – coding regions
 Identity is easier to define (e.g. ATCG or
whether a flower color is pink or white)
 Organisms evolutionary history is
documented in its genome
o Recapitulation – ontogeny recapitulates
phylogeny; resembling successive
adult stages in the evolution of the
animal’s ancestors (phylogeny)
Taxonomic Issues Addressed by DNA
Nuclear Chloroplast Mitochondrion
 DNA helps in resolving species boundaries Big Small Medium
o Morphological differences between Genetic history ≠ Origin: Origin: Engulfed
species may be very subtle; difficult to species history Cyanobacteria bacteria
describe and applicable only to some Inheritance: Inheritance: Inheritance:
life history stages (amphibians) or to Biparental Generally maternal Generally maternal
only one gender Linear shape Circular shape Circular shape
 E.g. African vs Asian elephant
 DNA sequences are very rich source of Molecular Clocks help track evolutionary time
additional data (e.g. translation of proteins)  Molecular clock uses constant rates of
 DNA sequences help assign species names to evolution in same genes to estimate the
tissues absolute time of evolutionary change;
o Many biological tissues are routinely reference point (based on how the
collected but only a fraction can be environment selects it)
identified (i.e. adult morphs)  To extend molecular phylogenies beyond the
o Quarantine solutions for conservation fossil record, one must make an assumption
and food safety (quality control) about how change occurs over time

Methods in DNA-based Systematics (1) Species which

 Isolation of genetic material diverged recently
o Genomic, plasmid, nuclear, showed few nucleotide
mitochondrial, chloroplastid differences
(1) Genomic (2) Species which
 Focuses on the structure, function, diverged a long time
evolution, mapping, and editing of genomes ago have many
 Uses high throughput DNA sequencing and nucleotide differences
bioinformatics
 Chromosomal DNA; larger than plasmid
 Genes encoded are for vital metabolic Each Gene Mutates at a Different Rate
activities (e.g. respiration and cell division)  Genes coding for vital enzymes or structures
 Associated with histone proteins tend to be more conserved
 Can be transferred only after cell division  Slow rates of gene mutation: for older groups
(slow evolution)
(2) Plasmid  Fast rates of gene mutation: for relationships
 Naked DNA in closely related populations (fast evolution)
 Genes encoded are for secondary metabolism Neutral Theory constant molecular clock
(e.g. antibiotic resistance or any other gene
tolerance)  States that much evolutionary change in genes
and proteins has no effect on fitness and is
 Can be transferred through horizontal way of not influenced by natural selection
gene transfer between similar or different species
o Transfection – bacteria to another  Genes should not be selected for/against in
an environment
plasmid
o Transduction – transfer of viral or  Molecular changes in these genes & proteins
bacterial DNA from one cell to are regular like a clock
another
o Conjugation – exchange of genetic DNA deoxyribonucleic acid
material between bacteria by adhering  Degeneration
to it  Numerous combinations (64) but only 22
amino acids present
(3) Nuclear, Chloroplast, Mitochondrial Genomes o Has no effect on fitness (i.e. silent
 Endosymbiosis; own DNA or genetic material mutations)
  Structures:  Amplifies the genes; random targeting (smear)
o Primary  linear
o Secondary
o Tertiary
o Quaternary

*Codons may change but the charges still stay the

same (lock and key mechanism)

Gene Mutation Rate Problems

 If a gene is mutating very slowly, the level of
variation approaches the sequencing error
rate and inferences now become unreliable
o “error in facilities or actual change?”
 If a gene is mutating quickly, parallelisms
and reversals accumulate so fast that all
phylogenetic information is lost, thus no
inferences can be inferred

Tools:

(1) RFLP or Restriction Fragment Length Polymorphism

 Use of gel (agarose gel or polyacrylamide)
 DNA molecules have a negative charge; they
migrate to the positive region
 A qualitative method (no amplification)
o Uses length or banding as the basis

(3) Amplified Fragment Length Polymorphism

 Combination of both (1) and (2)
 More useful; targets the fragments, thus
similarities can be easily deciphered
 Final reference: banding patterns or
fingerprint patterns

(2) Random Amplified Polymorphic DNA

 Use of primers (4)
 Banding patterns – used as a clue for
identification
 Denaturing stage (splitting)
 Annealing stage
 Extending stage
Sequencing
 Sanger Method
o Denature DNA (from double-
stranded to single-stranded)
o Make multiple copies of a segment
o Attach a primer
 o Add four polymerase solution
 Modifications in the OH
(phosphate group is
terminated)
 OH: where phosphate
can attach
 If the nucleotide is modified,
other substances cannot attach
to it
*The shortest is recognized first since the shortest
one travels faster than the larger ones
Transcriptomes
DNA microarrays  arrays containing DNA (cDNAs which came
from mRNAs are hybridized); show different gene expression;
microarray chips contain fragments from genes in the group
analyzed
Steps
Luminescence vs Fluorescence
Fluorescence  needs another source of light
Luminescence  produces its own light
Omics Technology for Systematics
 Central Dogma – interconversion of
information
 Functional genomics – only genomes are
expressed
 Proteins
o (1) structural – determines form
o (2) enzyme – metabolism to
metabolites
 Genome – all of hereditary information
encoded in the DNA (or RNA)
 Transcriptome – set of all mRNAs (transcripts)
produced from a genome
o (1) complete set of transcripts of an
organism (taxonomic approach)
o (2) specific subset of transcripts
presents in a particular cell type or
under specific growth conditions
 It varies because it reflects genes that are
actively expressed at any given time
 (1) isolate mRNAs from cells at two stages
 (2) convert mRNAs to cDNAs (complementary
DNA) by reverse transcriptase
o Different language but information is
still the same
 (3) add the cDNAs to a microarray;
fluorescent cDNAs anneal to complementary
sequences on the microarray
o Single-stranded DNA will later on
bind to given spots (hybridization)
 (4) removal of unhybridized probe; each
fluorescent spot represents a gene expressed
in the cells
o High throughput
Transcriptomics
 Experiments performed under different
conditions
 Determines effect of conditions on expression
 Produces huge amount of data
 Lots of repeats required; expensive
DNA Microarray: Applications
 Allows simultaneous screening of thousands
of genes
 Genome-wide genotyping  “What are the
genes present in an individual?”
 Tissue-specific gene expression  “What are
the genes used to make proteins?”
 Mutational analysis  “Which genes have been
mutated?”
Adaptations to PCR
 Reverse transcriptase PCR (RT-PCR)
o Used to amplify RNA sequences
o First step uses reverse transcriptase to
convert RNA to DNA
 Quantitative PCR
o Shows quantitative differences in gene
levels (Are genes absent or present?)
o Amounts are converted to numbers in
terms of copy numbers
Q-PCR
 Hybridization
 Quenching molecule absorbs the signal of
light
 No attachment = no fluorescence
 Higher fluorescence, higher copies of targets
 Higher copy numbers, higher light intensity
 Probe is folded without target molecules
General Steps of PCR
(1) Denaturation
(2) Attaching of primers
(3) Elongation
Proteomes
 Set of all proteins under a set of condition
o (1) complete set of proteins for a
given organism
o (2) specific subset of proteins that are
present in a particular cell type under
specific growth conditions
 Varies because it reflects genes that have
become proteins
 Uses 2D electrophoresis and mass
spectrometry
 High-throughput but lesser than
transcriptomes
Gel Electrophoresis
 Electrophoresis separates molecules by size
 Limited resolution
 Proteins  polyacrylamide gel electrophoresis
Isoelectric focusing
 Across a pH gradient
 Proteins migrate to their isoelectric pH
 The quantity of R group (from amino acid)
determines the change of proteins
Two-dimensional Gel Electrophoresis
 Across size
 SDS-PAGE performed perpendicularly from
the original position
 Separates proteins according to pH and mass
Mass Spectrometry
 Uses the molecular weight of proteins as
database
 Protein sample is ionized and exposed to
electric field
 MALDI-TOF  estimates molecular weights
 Used to identify a few proteins within a
mixture
Proteomic Analysis by Mass Spectrometry
 Proteins separated by 2D electrophoresis
 Single proteins excited
o Digestion with trypsin give fragments
with unique set of sizes
 Sizes identified by mass spectrometry and
matched to database
 Allows identification of unknown proteins
Transcriptomics vs Proteomics
 Both are powerful tools
 Differs in practical application:
o Transcriptomics is robust, cost-
effective and user-friendly
o Proteomics is limited (problems with
purification)
Metabolomics
 Mass spectrometry can be used to measure
metabolic and other chemical compounds
 Bioinformatics  wet lab and hypothesis
questions
o Enable the discovery of new biological
insights
o Create a global perspective in which
unifying principles can be discerned
o Phylogeny reconstruction based on
character evolution

Polyphasic Approach to Systematics

 Includes phenetics, genotypic, etc.
 Incorporates coherent profile which will
contribute to the systematics of the taxon of
interest
1. Restriction fragment length
polymorphism (RFLP) is a technique that exploits
variations in homologous DNA sequences, known
as polymorphisms, in order to distinguish individuals,
populations, or species or to pinpoint the locations
of genes within a sequence. The term may refer to a
polymorphism itself, as detected through the differing
locations of restriction enzyme sites, or to a related
laboratory technique by which such differences can be
illustrated. In RFLP analysis, a DNA sample is
digested into fragments by one or more restriction
enzymes, and the resulting restriction fragments are then
separated by gel electrophoresis according to their
size.
Gel electrophoresis is a method for separation and
analysis of macromolecules
(DNA, RNA and proteins) and their fragments, based
on their size and charge. It is used in clinical
chemistry to separate proteins by charge or size (IEF
agarose, essentially size independent) and
in biochemistry and molecular biology to separate a
mixed population of DNA and RNA fragments by
length, to estimate the size of DNA and RNA
fragments or to separate proteins by charge.[1]
Nucleic acid molecules are separated by applying
an electric field to move the negatively charged
molecules through a matrix of agarose or other
substances. Shorter molecules move faster and
migrate farther than longer ones because shorter
molecules migrate more easily through the pores of
the gel. This phenomenon is called sieving.[2] Proteins
are separated by charge in agarose because the pores
of the gel are too large to sieve proteins. Gel
electrophoresis can also be used for separation
of nanoparticles.
2. RAPD 'Random Amplification
of Polymorphic DNA' is a type of PCR, but the
segments of DNA that are amplified are random. The
scientist performing RAPD creates several arbitrary,
short primers (8–12 nucleotides), then proceeds with
the PCR using a large template of genomic DNA,
hoping that fragments will amplify. By resolving the
resulting patterns, a semi-unique profile can be
gleaned from an RAPD reaction.
No knowledge of the DNA sequence of the targeted
genome is required, as the primers will bind
somewhere in the sequence, but it is not certain
exactly where. This makes the method popular for
comparing the DNA of biological systems that have
not had the attention of the scientific community, or
in a system in which relatively few DNA sequences
are compared (it is not suitable for forming a cDNA
databank). In recent years, RAPD has been used to
characterize, and trace, the phylogeny of diverse plant
and animal species.
 The DNA amplification technique of the
polymerase chain reaction (PCR) is a
laboratory method for creating multiple
copies of small segments of DNA
AFLP uses restriction enzymes to digest genomic
DNA, followed by ligation of adaptors to the sticky
ends of the restriction fragments. A subset of the
restriction fragments is then selected to be amplified.
This selection is achieved by
using primers complementary to the adaptor
sequence, the restriction site sequence and a few
nucleotides inside the restriction site fragments (as
described in detail below). The amplified fragments
are separated and visualized on denaturing on agarose
gel electrophoresis , either
through autoradiography or fluorescence methodologi
es, or via automated capillary sequencing instruments.
1. Digestion of total cellular DNA with one or
more restriction enzymes and ligation of
restriction half-site specific adaptors to all
restriction fragments.
2. Selective amplification of some of these
fragments with two PCR primers that have
corresponding adaptor and restriction site
specific sequences.
3. Electrophoretic separation of amplicons on a
gel matrix, followed by visualisation of the
band pattern.
MONOPHYLY
Seed plant phylogeny inferred from all three plant genomes:
Monophyly of extant gymnosperms and origin of Gnetales from
conifers
 Generating a new molecular data set of
mitochondrial small subunit rRNA sequences
helped in resolving the issue on the
gymnosperm monophyly
 Gnetales is placed within conifers; sister
group to the Pinaceae
 Gnetales is viewed as an extremely divergent
conifers, and there are many morphological
similarities between angiosperm and Gnetales
o Double fertilization and flower-like
reproductive structures
 Method:
o Extraction of total DNA (22) from 35
plants
o PCR
 The major phylogenetic conclusions: (i) Gnetales are
not the sister group to angiosperms among
extant seed plants; (ii) Gnetales are a
monophyletic group; (iii) extant gymnosperms
are also monophyletic; (iv) cycads are the basal
group of gymnosperms; (v) conifers and
Gnetales together comprise a monophyletic
group; and (vi) Gnetales are nested within a
paraphyletic conifers as sister group to the
Pinaceae
Monophyly of Lampreys and Hagfishes Supported by Nuclear
DNA-coded Genes
 18S and 28S rRNA support the monophyly of
lampreys and hagfishes
 Mitochondrial DNAs suggest the close
association of lampreys and Gnathostomes
 Used the method of cloning and sequencing
to clarify this issue
 By analyzing the maximum likelihood (ML)
inferred by the proteins, it was shown that
they prefer the monophyly of cyclostomes
 Method: Isolation and Sequencing of
Hagfish and Lamprey cDNAs
o Hagfish RNA  extracted from the
liver; lamprey  whole body
o Total RNAs were transcribed to
cDNAs by reverse transcriptase
o These cDNAs were used as template
for PCR amplification
 (1) Denaturing stage
 (2) Annealing stage
 (3) Extending stage
 Phylogenetic analyses carried out by two
enzymes: aldolase and enolase
 Missing one of the duplicated genes on one
lineage of cyclostomes might infer an
incorrect relationship between cyclostomes
and gnathostomes
 Lamprey enolase-1 is the homolog of
gnathostome enolases which are likely to have
diverged by two gene duplications in the early
evolution of gnathostome split
 Lampreys are considered closely related to
gnathostomes than hagfishes based only on
morphological features
PARAPHYLY
1. From the article of Cai et al., homoplasy was
mentioned. What is homoplasy and homoplastic
character as mentioned in the paper? Why is this not a
good character for phylogenetic studies?
2. What is the recommendation of Le et al to address
the monophyly of the genus Kachuga? What was their
reason of suggesting?
3. Xiao and Zu et al mention that Lasianthus clade is
highly supported and at the same time paraphyletic.
Reconcile the two seemingly contradicting statements.
Paraphyly of Chinese Amolops and phylogenetic position of the
rare Chinese frog, Amolops tormotus
 Method: Maximum Parsimony, Bayesian
Inference, and maximum likelihood methods
using two mitochondrial DNA fragments
o DNA extraction, amplification, and
sequencing
 Extraction of DNA from
muscle or liver tissue samples
 PCR amplification
 Purified PCR products were
sequenced from both
directions with an ABI
automated DNA sequencer
 Acquired sequences were
blasted against the GenBank
database to verify the
amplified sequences
 Phylogeny of Chinese Amolops
 o Fei et al. recognized five species
groups within Chinese Amolops based
on morphology
o A. tormotus group did not cluster
with other Amolops species but rather
belonged to the monophyletic genus
Odoronna
o Amolops is not monophyletic and
recognition of the A. tormotus group
is not justified
 Phylogenetic Status of A. tormotus
o It is orginially named as Rana tormotus
o Controversial since it is similar to A.
(H.) cavitympanum in having a deeply
sunken tympanum
o It was then transferred from the genus
Rana into the genus Amolops
o However, it was found that the
tadpole of A. tormotus has neither an
abdominal disk nor poison glands that
characterize Amolops
o Therefore, Li et al. suggested to
establish a new genus Warana for A.
tormotus
o It was not followed, however, since
the study supports the transfer of A.
tormotus into the genus Odoranna
o In this sense, genus Wurana becomes a
synonym of Odorrana
 Phylogenies provide a logical foundation for
establishing taxonomy
On the paraphyly of the genus Kachuga
 The relationships among the three giant
genera (Kachuga, Batagur, and Callagur) are
poorly known
 Methods: Taxonomic sampling and
Molecular data
o Combined approach of nuclear and
mitochondrial genes
o Mitochondrial DNA was included to
address the phylogenetic relationships
at the species level
o Three nuclear genes are included:
Cmos, Ragl, and Rag2
o Extraction of DNA from tissue or
blood samples
o PCR polymerase chain reaction
o PCR products were then visualized
using electrophoresis with the use of
agarose gel
o The resulting gel-purified product was
used as a template in reamplification
reactions
 Results show the monophyly of the clade
consisting of Batagur, Callagur, Hardella,
Kachuga, and Pangshura
 Hardella is sister to Batagur + Callagur +
Kachuga
 Genus Kachuga is polyphyletic to Callagur and
Batagur
 Lee et al. proposed that the five species of the
three genera Batagur, Callgur, Kachuga are
placed in the genus Batagur because it has the
page priority over Kachuga
o These species share a unique
character: presence of the coastal
fontanelles on the carapace of adult
males
Paraphyly and phylogenetic relarionships in Lasianthus inferred
from chloroplast rps16 data
 Based on the chloroplast rps16 data,
Lasianthus is paraphyletic because Saprosma
crassipes, a representative of the species with
two locules per ovary developing into two
pyrenes per drupe with a thin wall, and
Litosanthes biflora, the species in the monotypic
genus Litosanthes are nested with Lasianthus
clade
POLYPHYLY
1. Compare and contrast studies by Alejando et al and
Motley et al on the polyphyly of Bikkia. Include the
resolution proposed by the authors.
2. Pedostibes is polyphyletic by current subscription,
with SEA Pedostibes traced to closer to Phrynoidis. As
compared to Pedostibes. Why were SEA Pedostibes
not transferred to Phynoidis to resolve the polyphyly
of the group?
3. Why is it important to conduct phylogenetic studies
in the elucidaton of Cepaea classification? How did
other evidences compliment the phylogenetic results
in each proposed grouping of taxonomic changes
within Cepaea species?
Molecular phylogeny reveals the polyphyly of the snail genus
Cepaea (Gastropoda: Helicidae)
 Considered monophyletic based on
morphological characteristics
  Molecular phylogenetic analyses based on
mitochondrial and nuclear sequences of 20
genera of Helicidae suggest the polyphyly of
Cepaea
 C. vindobonensis is referred to Caucasotachea
and C. sylvatica to Macularia
 Cepaea, Macularia, and Caucaotachea have large
phylogenetic distances
Polyphyly of Asian Tree Toads, Genus Pedostibes Gunther,
1876, and the Description of a New Genus from Southeast
Asia
 The Asian tree toad genus Pedostibes exhibits a
disjunct distribution
 The Indian Pedostibes is embedded within a
primarily South Asian clade of toads
 Southeast Asian Pedostibes are nested within a
Southeast Asian clade which is the sister
lienage to the Southeast Asian river toad
genus Phrynoidis
 Indian and Southeast Asian Pedostibes are not
only allopatric and polyphyletic but they also
exhibit significant differences in morphology
and reproductive mode
o This indicates that the Southeast Asian
species are not congeneric with the
true Pedostibes of India
Polyphyly of Bikkia Based on Multi-locus Sequence Analysis with
emphasis on the endemic Bikkia philippinensis including its
conservation status
- Polyphyly of Bikkia
o The phylogenetic relationships within
Chicocceae are almost congruent to
previous study by Motley et al
o Bikkia is confirmed to be polyphyletic in
its current circumscription as the New
Caledonian species are found to be in a
clade with the monotypic Morierina while
other species found in another clade are
sisters to Badusa.
 The discrepancy between Motley
et al is that Morierina is placed
here as sister to the New
Caledonian Bikkia with strong
support
 The two clades of Bikkia also
gained more support in the
combined tree than Motley et
al’s.
o Chicocceae is defined by a combination
of two synapomorphic features: stamens
inserted at base of corolla, and spinulose
pollen
 However as stamen insertion at
base of corolla is also a
characteristic of the tribe
Hamelieae, only the spinulose
pollen should be retained as a
synapomorphic characteristic of
Chiococceae
- Phylogenetic position of Bikkia philippinensis
o Data trees show that Bikkia philippinensis is
nestled within the coastal Bikkia group,
which happen to be congruent with the
habitat and morphology of the Philippine
Bikkia species.
 Similar to other species in that
clade Bikkia philippinensis is found
to thrive in coastal environments,
having solitary white flowers and
infundibular corrolas
 Bikkia tetranda (type species of
the genus) is also found in that
clade
- Proposed taxonomic Solution
o As genus Bikkia was found to be
polyphyletic based on the three-gene
analysis and that of Motley et Al’s, the
coastal species should be retained as
Bikkia as it includes the type species
Bikkia tetrandra.
o The New Caledonian inland forest Bikkia
should be transferred to another name
instead, as the differ with the coastal
group in terms of flower morphology (sila
nalang mag-adjust kasi sila yung iba sa
standard)
- Conversation status of Bikkia philippinensis
o They used the IUCN Red List Categories
and Criteria in classifying the species
 Criterion E was not used as no
quantitative analysis was
performed on the taxon
o Species is restricted on Dako island
though, and magpupungko, Surigao del
Norte, Mindanao. It is ranked as Critically
Endangered as population size is
estimated to be fewer than 250 mature
individuals, with no subpopulation
containing more than 50 individuals.