Introduction To Modern Biosystematics For Fungal Classification

Mrinal Vashisth

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”

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

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

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 historyNatural selection is driven by variation in

reproductive success

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.

• 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.

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.

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 forMolecular phylogenetics.

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 sequenceBASIS• 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

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

Polyphasic/Modern Taxonomy

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.

(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

(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

-> A cut-off value of 70% similarity is considered to define a species

-> In Fungi: A sharp drop off in cross-hybridization defines species

Genotypic Methods

(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

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

• 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

• 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

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.

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

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”

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

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• 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

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