This is a more specific version of the slide-set "Major Characteristics Used in Microbial Classification". A presentation I could not deliver for some reasons yet turned out to be pretty nice. I hope to deliver it some day, but for the time being I am making it public. I hope it would be of some use. :)

1. Introduction To Modern
Biosystematics For Fungal
Classification
Mrinal Vashisth

2. Biosystematics
• The science of systematic identification,
classification, and nomenclature
• The methods of biosystematics helps in affective
organization of the huge biodiversity
• Also reveals relationships and evolutionary trends
amongst organisms
• It effectively deals with the “The species problem”

3. Ernst Mayr
(1904 – 2004)
The Species Problem
• The classical definition of species: A group of
interbreeding and fertile organism producing
individuals constitute species
• Cannot be applied to hybrid species, species
without sexual reproduction and even extinct
species
• The problem: assumptions behind the
Morphospecies concept can’t be fully applied
while there is a lack of data in case of
Phylogenetic concept
• The solution: a consensus based on
Morphospecies and Phylogenetic concepts
for the definition of species

4. Classical Methods
• Earlier methods- Classical methods –
Morphospecies concept: Classification
based on all observable characters i.e.
Phenetics
Alpha taxonomy (W.B. Turill in papers
from 1935-37) is the term which refers
to the discipline of finding, describing,
and naming taxa.
Beta taxonomy (E. Mayr, 1968) it is the
sorting of taxonomy in even higher
categories of hierarchy.
Numerical taxonomy (Sokal and Sneath,
1963) As more and more features were
being included, algorithms were devised
to take the computational load.
Numerical taxonomy makes use of Keys
(set of alternate characters for
Identification)
• Rarely used now
From top to bottom the specificity
of characters increase

5. Evolution
• Evolution is a population phenomenon characterized by a
changes in the average characteristics.
• It may or may not benefit the survival of a species.
• Evolution can be Adaptive or Neutral (i.e. on genes which do
not contribute to reproductive success)
• Evolution has two parts:
- Microevolution: The process
Whole organism traits are a product of natural selection
while DNA sequences are shaped by drift
- Macroevolution: The history
Natural selection is driven by variation in reproductive
success

6. Basis of Evolution
• Variations are the basis for evolution
• The origin of these variations can be boiled down to
either genetic or epigenetic
• Genetic variations can have phenomena such as
mutations, mendalion lottery, founder effects and
genetic bottlenecks, reproductive success variation
(genetic drift etc.)
• Epigenetic: variations due to covalent modifications
to the DNA or proteins mantaining the DNA, miRNA
levels, sRNA, prion based, structural inheritance,
nucleosome positioning etc.

7. • Sources of diversity in
microbes are:
(A.) Quick generation time
resulting in faster mutation
rate
(B.) Horizontal Gene Transfer
(HGT) via Transformation,
Transduction and Conjugation
(C.) Large population size and
low extinction rates.

8. Take away points from the concept of
Evolution
• Evolution works on the genes in hand and not on
a blank slate.
• Neutral evolution is a source of regularity and
thus proves to be a central tool in tree
construction e.g. highly conserved regions.
• Mutations are the ultimate origin of all genotypic
variations… recombination also have a huge
impact on variation.
• Studying macroevolution leads to the realization that we
are indeed- a community of genomes and that there is –
one tree of life.

9. Modern Methods
• The diversity that we see today is all
by common ancestry. All gene pools
share a modified form of common gene
pool that existed billions of years ago
• Modern biosystematics = phylogeny +
population genetics
• Émile Zuckerkandl, Linus Pauling,
Walter Fitch, Emanuel Margoliash laid
out the theoratical framework for
Molecular phylogenetics.

10. Basics
• Earlier chemotaxonomy that made use of proteins, enzymes etc.
and techniques such as chromatography
• Most of these – replaced by sequence analysis – better – evolution
is ultimately reflected in the DNA sequence
BASIS
• Typically a 1000 bp long sequence (haplotype) can do for molecular
systematic analysis
- In a particular genus there are species differing in only a few sites
(e.g. Chimps and Humans are 98.5 % identical, in chosen
haplotypes; determined for a specific area of genetic material)
• Haplotypes from individuals in same species (in group) can be
compared to those of other species/ genus (out group)
• The assessment is usually in the form of “number of differences” as
percentage divergence
• Through dividing the number of substitutions by the number of
base pairs analysed: then it is assumed that this measure will be
independent of the location and length of the section of DNA that is
sequenced

11. Criteria for selecting a molecule for
classification purposes
• Modern methods are essentially molecular methods.
So selection of ideal molecules is crucial.
• Some important criteria are:
1.) It should be present in all members of the target
group.
2.) The molecule must contain regions of conserved
sequences for comparison purposes.
e.g. The differences in Hb sequences can be of great
help in distinguishing one specie from other.
3.) The changes in sequence data should have slow rate
of mutation for proper measurement

13. Polyphasic/Modern Taxonomy

14. Phenotypic methods
• The are all those that do not include the DNA/RNA sequencing or
their typing.
• (1.) Classical methods: These include colony/ cell characteristics
(colour, shape, pigmentation, production of slime etc.)
-> Use of Microscopes, pH meters, Chromatography etc. can be used to
assess these.
(2.) Numerical taxonomy: Analysis of huge data by means of
algorithms.
-> The only difference is that each character/key is given a weightage
according to their importance.
(3.) Cell wall composition: While the mode of cross-linkage can vary
within a species and also between strains, the amino acid
composition is common to all species within a genus
-> e.g. In archaea, pseudomurein is present where N-acetyl muramic
acid is replaced by N-acetyl talosuronic acid.

15. (4.) Fatty acid analyses:
-> The total cellular fatty acids are extracted, esterified and the methyl
ester content is analyzed by gas chromatography.
(5.) Other diagnostic methods:
-> Whole cell protein analyses with the help of SDS-PAGE can help in
comparison between related strains as different proteins undergo
changes at different rates
-> Polyamines present in cytoplasm that help in mantaining DNA
stability and osmolarity are also important classification characters
(6.) Advanced spectroscopy and spectrometric methods:
-> UV Resonance Raman Spectroscopy (UVRR) can give information
about Gram type of bacteria as well as Guanine+Cytosine content

16. (1.) DNA-DNA hybridization: (1961, McCarthy, Bolton)
-> Based on entire genotypes than particular haplotypes
-> This method is an indirect measurement of sequence similarity
between genomes in the form of divergence matrices which
are obtained from annealing labelled radioactive DNA to that of
non-radioactive DNA from unknown sources… giving measures
of incorporated DNA
-> After determining the divergences, we get triangular matrices
of the samples which are fed to statistical cluster analysis
-> These clusters are what we call dendrograms, which can be
used to verify the hypothesis or simply get new insights
-> Group of similar haplotypes form a clade
-> Statistical techniques such as bootstrapping and jackknifing
provide reliability estimates for positions within the constructed
tree
Genotypic Methods

17. (2.) RFLP:
-> The most sensitive technique as it directly
makes use of DNA variations
-> RFLP (Restriction Fragment Length
Polymorphism) – involves isolation of
genome – digestion and observation of gel
electrophoresis band pattern
-> Each fragment represents a genetic allele
-> Ribotyping takes this a step further by
labelling the genes for 16S, 23S, 5S rRNA,
tRNA etc. and
analysing for presence of
bands by autoradiography
• The general drawback of most
of these modern techniques is
that there is just not enough
information for comparison.
Result from a Ribotyping experiment

19. 3.) Sequence analysis:
• Comparative sequencing of the ribosomal DNA ITS2 region forms
the gold standard for fungal classification.
• What is ITS?
ITS is the spacer DNA situated between the small subunit rRNA and
large subunit rRNA genes in the chromosome of the corresponding
transcribed region
• The 5S sequence provides information suitable for Order level
• The 18S rDNA has been more extensively used for filamentous fungi
• The ITS (Internal Transcribed Spacer) region is suitable to reveal close
relationships such as Species
The Gold Standard

20. • Requirements: A Single Isolated Fungal
Colony, Ethanol Extract, DNA Extract/ FTA
Micro Elute Card
• Extract genomic DNA from cells of this
colony
• PCR amplification of the 350 base pair
long ITS2 target DNA region

21. • Cycle sequencing for
fluorescent labeling of DNA
molecules
• Sequence visualization on an
automated sequencer
• Analysis of data against a
proprietary database
0.2 % error rate

22. Limitations of molecular
systematics
• Since molecular systematics is cladistic, it presumes a
phylogenetic descent, and that all valid taxa be
monophyletic.
• Molecular phylogenies are sensitive to the assumptions
and models for their synthesis.
- Long-branch attraction, saturation, and taxon sampling
problems
- This implies different outcomes by using different
datasets even if dataset remains the same
• Due to Horizontal gene transfer different genes within
the same organism can have different phylogenies.

23. Images taken from: Hibbett, David S Taylor, John W, Fungal systematics:
is a new age of enlightenment at hand?, Nature Reviews Microbiology, 2013, 11 (12), 129-
133, http://dx.doi.org/10.1038/nrmicro2963

24. Conclusion
• The field of Taxonomy has evolved a lot from when it
once started.
• The challenges that Classical taxonomy faced were
overcame by the use of Modern Taxonomical methods
• Classification is an art, which evolves concordantly with
information and technology in hand. It gets a lot of
benefit from multiple perspectives.
• Modern methods in a combination with Classical methods
form the basis of Modern Taxonomy or Biosystematics
“phylogenetic information can be used to compare
fungal diversity profile across habitats”

25. THANKYOU
FOR YOUR
PRECIOUS
TIME!
Image Credits: study.com, NPTEL, Wikimedia Commons,
Universe-review.ca, N and other CC0 image sources,
otherwise cited immediately with the image

26. References
• Sarethy IP, Pan S, Danquah MK, Modern Taxonomy for Microbial
Diversity, InTech, http://dx.doi.org/10.5772/57407
• Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J. Polyphasic
taxonomy, a consensus approach to bacterial systematics,
Microbiological Reviews 1996; 60 (2): 407-438
• Das S, Dash HR, Mangwani N, Chakraborty J, Kumari S, Understanding
molecular identification and polyphasic taxonomic approaches for
genetic relatedness and phylogenetic relationships of microorganisms,
LEnME, Dept. of Life Science, NIT, Rourkela, Odisha, India
• Hibbett DS, Taylor JW , Fungal systematics: is a new age of
enlightenment at hand?, Nat. Rev. Microbiol., 2013, 11(2), 129-133,
http://dx.doi.org/10.1038/nrmicro2963
• Bruns RD, White TJ, Taylor JW, Fungal molecular systematics, Annu.
Rev. Ecol. Syst.1991, 22, 525-64
• Singh AK, Molecular Taxonomy: Use of Modern Methods in The
Identification of A Species, ISJ, 2012, 2 (1), 143-147