Deciphering fungal species complexes using

multigene sequence analysis

Gunjan Sharma
Regional Centre for Biotechnology
NCR Biotech Cluster, Faridabad
Email: gunjansharma128@gmail.com

Belle Damodara Shenoy

CSIR-National Institute of Oceanography
Regional Centre, Visakhapatnam
Email: belleshenoy@nio.org

Invited talk at Central University of Tamil Nadu on 8.3.2018

www.csir.res.in www.nio.org
 Contents
1. The Kingdom of Fungi
2. Taxonomy, classification, nomenclature, identification
3. Fungal species recognition criteria
4. Molecular phylogenetic analysis
5. Species recognition criteria in Colletotrichum
6. Colletotrichum gloeosporioides species complex
7. Colletotrichum siamense species complex
8. Artificial intelligence (AI) tools in future fungal taxonomy
Kingdom Fungi

The Tree of Life

http://tolweb.org/tree
 What is a fungus?

An eukaryotic, heterotrophic organism devoid

of chlorophyll that obtains its nutrients by
absorption, and reproduces by spores.
The Kingdom Fungi

Scazzocchio 2014
How many fungal species are there?
How many fungal species are there?
 Cataloging microbial diversity

• Microbes: Maintain the balance in ecosystems

• Diversity of microbes: Sustenance of life on earth

• Human intervention: Depletion of biodiversity

• Many hotspots: Losing their endemic biodiversity

• Microbes: Industrially important biomolecules

 Basics of taxonomy
• Also known as Biosystematics,
has three components

• Classification: arrangement of
organisms based on similarity

• Nomenclature: naming of the

organisms

• Identification: determining
whether an organism belongs to
the group under which it is
classified and named
 Basics of taxonomy
• Also known as Biosystematics,
has three components

• Classification: arrangement of
organisms based on similarity

• Nomenclature: naming of the

organisms

• Identification: determining
whether an organism belongs to
the group under which it is
classified and named
 How to recognize a fungal species?

• Species concept
- a description of the kind of entity that constitutes a species
- “Species are segments of evolutionary lineages that have evolved
independently from one another”

• Species recognition criteria

- practical standards for recognizing whether individuals should be
considered members of the same species

(Cai et al. 2011. Fungal Diversity 50: 121-133)

Species concept vs. Recognition Criteria

• Many so-called species concepts actually

correspond to species recognition criteria
• Biological species concept: intersterility
• Morphological species concept: morphological
divergence
• Ecological species concept: adaptation to a
particular ecological niche
• Phylogenetic species concept: nucleotide
divergence between monophyletic lineages
Morphological species recognition criterion

• The most commonly used species recognition criterion

• Many cryptic species have been recognized

http://directors-deviate.ga/trenpan/sex-drugs-and-recombination-the-wildlife-of-aspergillus174.php
 Genes as evolutionary chronometers
• Proteins and genes: Evolutionary chronometers which
measure the evolutionary change
• 18S/28S rRNA genes: Reliable in fungi at family level
• ITS: Reliable at Species level in many groups

• Such markers
• Present in all members of the target group
• Functionally homologous in the organisms
• Contain regions of conserved sequences for comparison
• The changes in sequence data must be at a slow enough
rate to permit measurement
Phylogenetic Species Recognition Criterion

• PSC – relies on phylogenetic

variable characters, usually
DNA sequences of selected
genes or genomes
• PSC – “defined” as the smallest
monophyletic clade of
organisms that share a derived
character state
 The limitation of the PSC

• Single gene analyses are dependent on the

genes having an evolutionary history that
reflects that of the entire fungus (genome)
• This is often not the case
• Gene tree vs. Species tree
Genealogical concordance phylogenetic
species recognition criterion (GCPSRC)

Use the phylogenetic concordance of multiple unlinked genes

to indicate a lack of genetic exchange and thus evolutionary
independent of lineages
 GCPSR in fungi

• Identified fungal species which

otherwise could not be
recognized due to lack of
distinguishing morphological
characters or incomplete
intersterility
• Immensely useful in fungi as it
is more finely discriminating
than the other criteria or
more convenient
Molecular phylogenetic analysis
 Phylogenetic tree

Terminal Nodes
Branches or (Leaves)
Lineages A Represent the
Taxa (genes,
populations,
B species, etc.)

D
ROOT
Internal Nodes E
(represent hypothetical
ancestors of the taxa)
 Phylogenetic tree

Terminal Nodes
Branches or (Leaves)
Lineages A Represent the
Taxa (genes,
populations,
B species, etc.)

D
ROOT
Internal Nodes E
(represent hypothetical
ancestors of the taxa)
Monophyly
How to construct a phylogenetic tree?

Assembling a dataset

Multiple sequence alignment

Find out the best tree/s

Bootstrapping

Data presentation
 Assembling a dataset
• Own sequences + homologous sequences from the
public domain

• Two basic kinds of search strategy

 Multiple sequence alignment

MEGA SEAVIEW MAFFT server

• For each column, every residue from every sequence is homologous

• Adjust ‘by eye’ to ‘minimize insertion/ deletion events
• DNA sequences for closely related sequences
• Amino acid sequences for distant relationships
• GARBAGE IN, GARBAGE OUT!
 Tree-building methods
• Distance-matrix methods (a.k.a. Clustering or
algorithmic methods)
• Neighbour-joining
• UPMGA
• Discrete data/ Character-based methods (a.k.a. Tree
searching methods)
• Parsimony
• Maximum likelihood
• Bayesian methods

PHYLIP MEGA PAUP MrBayes

Bootstrapping

• Bootstrapping
• take random subsamples,
• build trees from
subsamples,
• calculate the frequency of
occurrence different parts
of the tree
• Tests the phylogenetic
accuracy of the tree
• BS values of 70% or higher:
likely to indicate reliable
groupings
 Data presentation
• Branch lengths are drawn to scale
• Branch length = the amount of evolution estimated to have
occurred
• BS values should be displayed as percentages
• Only BS values of 50% or higher are reported
• You can choose to root the tree (to indicate the direction of
evolution)
• A cladogram displays branching pattern only
MEGA
YouTube videos on phylogenetic trees!

https://www.youtube.com/watch?v=dUB4xbLJ8L8
 Plant diseases
•Anthracnose •Canker •Leaf spots
•Fungus •Fungus / •Fungus /
Bacteria Bacteria

•Blight
•Fungus

•Scab •Rust •Wilt •Leaf curl

•Fungus •Fungus •Bacteria •Virus

•Smut •Powdery mildew •Root knot

•Fungus •Fungus •Nematode
The “Top 10” fungal pathogens
Ran Taxon
k
1 Magnaporthe oryzae
2 Botrytis cinerea
3 Puccinia spp.
4 Fusarium
graminearum
5 Fusarium oxysporum
6 Blumeria graminis
7 Mycosphaerella
graminicola
8 Colletotrichum spp.
9 Ustilago maydis
10 Melampspora lini
 Colletotrichum
• Economically important phytopathogens
• Distribution: Tropics

• Causes: Anthracnose
Anthracnose symptoms
 Colletotrichum: survival strategies

Pathogen

Epiphyte/
Endophyt
e
Saprobe
 Life cycle of Colletotrichum

Germination
Anthracnose of conidia

Ascosp Healthy Plant

ore Sexual Asexual
Glomerella Colletotrichum
Cylindrical
Ascus
spores

Early infection &

invasion of tissue
Perithicium Acervulus
 Species “concepts” in Colletotrichum
Ecological species “concept” (Host-association) >800 species
(Corda, 1831)

Morphological species “concept” (Phenotype) 40 species

(Sutton, 1922)

Phylogenetic species “concept” (Cladistics) 119 species (Cannon, 2012)

14
3
(20
DNA Barcoding
 Research gaps in Colletotrichum taxonomy
• Morphotaxonomic characters: Phylogenetically
uninformative
• Public databases: erroneous sequences (Cai et al. 2009

• Phylogenetic diversity: poorly understood

• Taxonomy: Poorly resolved

Colletotrichum genus:
9 major clades

Cannon et al., 2012;

Studies in Mycology, Vol. 73
Figure: Phylogenetic relationships
and species boundaries of C.
siamense s. lat. and related
species. Fifty percent majority
rule consensus tree from a
Bayesian analysis based on a six-
locus combined dataset (ApMat,
CAL, GAPDH, GS, ITS, TUB2).
Posterior probabilities (PP, > 0.95)
are displayed at the nodes.
Thickened branches indicate
branches also present in the ML
tree with > 50 % bootstrap
support values. Bars in the first
column at the right present the
results of the phylogenetic
analysis based on five-locus (CAL,
GAPDH, GS, ITS, TUB2) alignment,
respectively. The other three
columns present the results of
three coalescent-based species
delimitation methods (GMYC, PTP,
BPP). “A” and “B” represent the
two potential species inferred
from PTP analysis. Ex-type
cultures are emphasized in bold.
Stars indicate isolates included in
the mating test
A.I. tools in future fungal taxonomy

• Quantitative phenotypic classes

• Genome scale data
• Databases and tools

Scaria and Shenoy (2018, in preparation)

https://www.slideshare.net/rvosa/taxonomic-classification-of-digitized-specimens-using-machine-learning
 Acknowledgements

 Director, CSIR-NIO

Scientist In-charge, CSIR-NIO RC Visakhapatnam

Organizers

CSIR-IMTECH, Chandigarh
Biocontrol of pathogenic Colletotrichum