MOLECULAR TAXANOMY

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2. Division of Entomology
FOH, SHALIMAR SKUAST-K
DOCTORAL CREDIT SEMINAR-II
(ENTO-692)
Speaker : WASIM YOUSUF MIR
Registration number : 2020-1029-D
Seminar title : Molecular Taxonomy – A Way Forward
Seminar incharge : Dr. Zakir Husain Khan
Dr. Barkat Hussain
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3. Augustin Pyramus de Candolle(1778-1841)
Image source: Wikipedia (2020)
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Greek word
“Taxis” – Arrangement
“Nomos” - Law
Taxonomy is the identification, description,
naming and classification of organisms

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Carolus Linnaeus 1707-1778
Image source: Website National museum, Stockholm

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The theory and practice of
classifying organisms
The science of the diversity of
organisms
(Ernest Mayr)

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Why ‘Taxonomy’?
Identified
Named
Classified
Applied
studies
Biological
studies

8. Basis of Taxonomy, conventionally!
Phenotype
Features
Shape
Size
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Limitations of Traditional Taxonomy
Variations in taxonomically
important traits
Inadequate morphology based
approach to species identification
Requires high level of expertise for
species identification

10. The new horizon of ‘Taxonomy’
Nucleic
acid
Protein
Molecular
Taxonomy
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 The classification of organisms on
the basis of the distribution and
composition of chemical substances
in them

12. Why “Molecular Taxonomy”??
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Can you tell these apart?

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Origin of
“Molecular Taxonomy”
“We establish that the mitochondrial gene
cytochrome c oxidase I (COI) can serve
as the core of a global bio-identification
system for animals”
(Proceedings of Royal Society of London, 2003)
Paul D N Hebert (Born: 1947)
Image source: Official Website, University of Guelph, Canada

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DNA barcoding first came to the attention
in 2003 when Paul Hebert’s research group
published a paper titled "Biological
identifications through DNA barcodes`

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19. Uses of DNA Barcoding
• Identification of biotypes/genetic groups.
• Quarantine stations.
• Conservation biology and biodiversity.
• Fragments of insects can be used.
• Biosecurity.
• Determination of food webs
• Molecular phylogeny.
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Tools used in molecular taxonomy
 DNA barcodes
A 648 base-pair region in the Mitochondrial cytochrome c
oxidase 1 gene (COI) highly effective in identifying birds,
butterflies, fish, Insects and many other animal groups.
o Bacteria: 16s ribosome
o Plants: Plastid DNA (matK gene)
o Animals: Mitochondrial DNA (COI gene)

21. DNA Barcode generated for the following
natural enemies and pests
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Parasitoids DNA barcode
Trichogramma achaeae TRINB001-10
T. brassicae, T. chilotreae TRINB002-10, TRINB003-10
T. Chilonis TRINB004-10
T. dendrolimi TRINB005-10
T. flandersi TRINB006-10
Predator DNA barcode
Chrysoperla zastrowi sillemi
CZASG001-10

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23. Different insect pests identified by DNA
barcoding
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29. CASE STUDIES
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Case study 1: DNA barcoding of flies commonly prevalent in
poultry farms of Bengaluru District
(Archana et al., 2016)
 Specimens of flies collected from 11 different poultry farms
 Larval form
 Adult form
 Larva reared to form adult
 Identification:
 Morphological
 Adult identification
• Length of fly
• Eyes
• Antennae
• Wing pattern
• Colour of flies
• Head pattern
• Thorax pattern
 Larval identification
• Length of larvae
• Integument
• Spines
• Cephalopharyngeal
skeleton
• Respiratory structures
such as anterior and
posterior spiracles.

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 Molecular identification
 Barcoding cytochrome c oxidase subunit I of mitochondrial
gene
 Amplified by universal primers LCO1490/HCO2198
 Primer details
LCO1490(F) 5’-GGTCAACAAATCATAAAGATATTGG-3
HCO2198 (R) 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’
Results:
 Morphological identification
• Musca domestica
• Chrysomya megacephala
• Hydrotaea capensis,
• Hydrotaea illucens
• Sarcophaga ruficornis
 Molecular (COI barcoding)
identification
• Musca domestica
• Chrysomya megacephala
• Hydrotaea capensis,
• Hydrotaea illucens
• Sarcophaga ruficornis
(Archana et al., 2016)

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Phylogenetic analysis of flies
(Archana et al., 2016)

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Case study 2: Identifying the larva of Syrphid fly Merodon avidus
(Diptera: Syrphidae)
Merodon specimen collected from Cerdap National Park, Eastern Serbia from the
bulbs of grass lily, Ornithogalum umbellatum
Total genomic DNA extracted from 50 mg of larval tissue
Sequences retrieved from GenBank:
 Merodon clavipes (HE653247)
 Merodon desuturinus (DQ387899)
 Merodon avidus (DQ845109)
 Merodon italicus (HE653253)
 Merodon loewi (DQ885923);
 Merodon nigritarsis (DQ386323);
 Merodon pruni (HE653260)
 Merodon rufus (KM507174)
 Merodon trebevicensis (DQ885919)
 Merodon velox (HE653265)
Specie recognised through mt DNA sequencing: Merodon avidus
(Andric et al., 2014)

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Morphological description of Merodon avidus larva:
Overall appearance:
• Length 8.6 mm and greatest width 3.1 mm
• “Short tailed” larva with external mouth-hooks and sclerotised mandibular
lobes. Uniformly beige to brown in colour.
• Sub cylindrical in cross-section, tapered anteriorly, with the anal segment
inclined dorsally.
• Integumental vestiture well-developed, with short, blunt and slightly
sclerotised spicules smaller on the ventral surface.
• Prolegs absent.
• Anal segment with two pairs of lappets
Larva in ventral view (mh-mouth hooks; prp- posterior respiratory process)

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Am- antenno maxillary organs; mh- mouth hooks; as- anterior spiracles; pps – primordia of pupal spiracles
Head
Primordia of pupal spiracles
Thorax
(Andric et al., 2014)

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Lappet Posterior respiratory process
(Andric et al., 2014)

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Case study 3 : Use of DNA barcodes to identify invasive
armyworm Spodoptera species in Florida
 Spodoptera species not reported in Florida:
 Spodoptera litura
 Spodoptera littoralis
 Collection of Spodoptera species present in area using pheromone traps
 DNA barcoding of S. litura and S. littoralis as collected from Taiwan and Portugal
respectively
 Comparison of DNA barcodes between the locally collected and exotic
specimens
 Discrimination of the different species obtained using morphological
characteristics
 Derivation of DNA barcoding data base to aid further studies and monitoring
(Nagoshi et al., 2011)

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Case study 4 : DNA barcoding of Indian ant species based on
cox1 gene
 Sixteen species of ants collected from Karnataka, India
 Specimens sequenced and barcoded for 658 bp region of mitochondrial
cytochrome c oxidase subunit 1 gene (cox1)
 Species collected
 Camponotus irritans
 C. parius
 C. compressus GR-17
 C. Compressus
 Anoplolepis gracilipes
 Oecophylla samaragdina
 Paratrechina longicornis
 Plagiolepis sp.
 Aphaenogaster beccarii
 Pheidologeton diversus
 Solenopsis geminata
 Monomorium scabriceps
 Myrmicaria brunnea
 Leptogenys chinensis
 Technomyrmex albipes
 Tapinoma melanocephalum
(Ojha et al.,2014)

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Cox1 sequences submitted to NCBI-GenBank
 Camponotus compressus: JN886027
 Solenopsis geminata : JN886028
 Myrmicaria brunnea : JN886029
 Leptogenys chinensis: JN886030
 Aphaenogaster beccarii: JN886031
 Camponotus parius : JN886032
 Camponotus irritans: JN886033
 Paratrechina longicornis: JN886034
 Oecophylla smaragdina: JN886035
 Tapinoma melanocephalum: JN886036
 Plagiolepis sp. : JN886037
 Tapinoma albipes: JN886038
 Camponotus compressus GR17: JN987857
 Monomorium scabriceps: JN987858
 Pheidologeton diversus: JN987859
 Aphaenogaster gracilipes: JN987860
(Ojha et al., 2014)

40. WORK DONE BY DIVISION OF ENTOMOLOGY
ON DNA BARCODING OF INSECTS
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41. Case study 5: Whitefly Species identified through Sequencing of
partial mtCOI genes Meniaz Nissar
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Sample name Collection site Field condition Host plant Species identified
M1 Budgam(Dulatapora) open Beans Bemisia tabaci
M2 Srinagar (Dal lake) Protected Gerbera Trialeurodes
vaporariorum
M3 Ganderbal (wakura) open Potato Bemisia tabaci
M4 Srinagar (wanganpora) open Beans Bemisia tabaci
M5 Srinagar (wanganpora) open squash Bemisia tabaci
M6 Budgam (bugam) open Brinjal Bemisia tabaci
M7 Ganderbal (badampora) open Beans Bemisia tabaci
M8 Budgam (Narkara) open Brinjal Bemisia tabaci
M9 Budgam (Narkara) open Cucumber Bemisia tabaci
M10 Srinagar (Shalimar) open Cucumber Bemisia tabaci
M11 Budgam(Dulatapora) open Potato Bemisia tabaci

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M12 Srinagar(wanganpora) Open Potato Bemisia tabaci
M13 Budgam (CITH) Protected Gerbera Trialeurodes vaporariorum
M14 Srinagar (foreshore) Protected Hibiscus Trialeurodes vaporariorum
M15 Budgam (chadoora) open Beans Bemisia tabaci
M16 Budgam (Dulatpora) open Brinjal Bemisia tabaci
M17 Ganderbal (Badampora) open Brinjal Bemisia tabaci
M18 Srinagar(foreshore) Protected Geranium Trialeurodes vaporariorum
M19 Srinagar (P.I section,tulip garden) Protected Gerbera Trialeurodes vaporariorum
M20 Srinagar ( Shalimar) open Potato Bemisia tabaci
M21 Budgam (Bugam) open Squash Bemisia tabaci
M22 Srinagar (Habakh) Protected Tomato Bemisia tabaci
M23 Srinagar (Alochibagh) Protected Gerbera Bemisia tabaci
M24 Srinagar (Habakh) Protected Brinjal Bemisia tabaci
CONTD.

43. Dendrogram of whitefly by MEGA Software
v 8.05 Meniaz Nissar
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44. Accession number of sequences published
in Gene bank Meniaz Nissar
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45. Case study 6:Molecular identification of Geometrid moths on BLAST
similarity bases in Kashmir valley Deelak Amin 2020
Species identification Gene
sequence
Identification
percentage
Accession no. New accession No.
allotted by NCBI
Alcis repandata COI 99.73 % KX071762.1 MN689335
Alcis vaeiegate COI 95.76% KX072097.1 MN689347
Alcis jubata COI 100 % MH522795.1 MN689352
Alcis perpicuata COI 96.03% MH522795.1 MN689360
Hypomecis infixaria COI 96.42 % KF394488.1 MN689349
Arichanna lapsariata COI 96.24 % KJ183242.1 MN689327
Antipercnia cordiforma COI 100 % KF522339.1 MN689351
Antipercnia
pseudoalbinigrata
COI 94.49 % KF522338.1 MN689324
Ascotis selenaria COI 100 % LC047740.1 MN689337
Ascotis imparata COI 96.18 % KX047239.1 MN689334
Lassaba contaminata COI 100 % KX951570.1 MN689342
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Chorodna baramulia COI 97.42 % KF394785.1 MN689336
Medasina albidaria COI 100 % JQ344892.1 MN689361
Psilalcis inceptaria COI 96.33 % KX951566.1 MN689343
Opisthograptis moelleri COI 100 % KF522465 MN689345
Ourapteryx pluristrigata COI 95.19 % KX863183.1 MN689331
Ourapteryx sambucaria COI 95.46 % KX043018.1 MN689338
Ourapteryx
caschmirensis
COI 96.46 % KX071652.1 MN689339
Ourapteryx sambucaria COI 100 % KX043018.1 MN689325
Cepphis advenaria COI 100 % KX071413.1 MN689358
Eutrapela clemataria COI 100 % MG357360.1 MN689356
Abraxas cashmiria sp. COI 96.07 % KX043487.1 MN689326
Biston betularia COI 100 % KT145262.1 MN689344
Biston suppressaria COI 100 % KF748229.1 MN689346
Thinopteryx crocoptera COI 100 % KF522515.1 MN689330
Lomographa tributaria COI 100 % KU381109.1 MN689354
CONTD.

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Odontopera bidentata COI 100 % KX788620.1 MN689357
Psyra bluethgeni COI 100 % AB265329.1 MN689362
Aspitates pseudogilvaria COI 96.07 % MH522797.1 MN689353
Tanaoctenia haliaria COI 100 % JN269164.1 MN689329
Xenoplia kashmirensis COI 94.08 % KX951577.1 MN689348
Heterothera quadrifulta COI 96.61 % HM876588.1 MN689355
Callipia vicinaria COI 100 % MF422047.1 MN689333
Photoscotosia miniosata COI 100 % KX862449.1 MN689332
Aplocera plagiata COI 100 % KX343550.1 MN689363
Pingasa ruginaria COI 100 % HM422803.1 MN689340
Pachyodes amplificata COI 100 % MG014818.1 MN689328
Chlorissa viridata COI 100 % HM391782.1 MN689359
Problepsis albidior COI 100 % MH197457.1 MN689341
CONTD.

48. Case study 7: Molecular identification of Giant yellow
aphids(GWA), Tuberolachnus salignus based on mtCOI region
Hussain et al.,2020
 The mtCOI region of the insect was amplified using universal primers
LCO1490: GGTCAACAAATCATCATAAAGATATTGG
HCO2198: TAAACTTCTGGATGTCCAAAAAATCA
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49. Phylogenetic tree showing genetic relatedness among population using
Maximum Parismony Method
Hussain et al.,2020
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50. Case study 8: Sequence of different RAPD-primers used for
molecular differentiation among different generations of ERM
selected through repeated applications of fenazaquin
Dr. Asma sherwani 2006
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Conclusion
 With the existence of millions of species and significant variations in their life-
stage, the correct identification becomes a challenge task for taxonomy.
 The new method called DNA barcoding, a tool of molecular taxonomy is used
to identify known and unknown species on the basis of the pattern of
nucleotide arrangement in a fragment of DNA from target species.
 DNA-based approach for species identification are technically simple, more
accurate and applicable to large-scale screening and provides efficient
platforms to identify insect species .
 DNA barcoding is inseparably linked with traditional taxonomy which
complements the taxonomic studies .

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Future prospects
 DNA barcoding can be successfully employed in monitoring exotic insect
pest invasions.
 The integration of morphological taxonomy with molecular taxonomy shall
ease the integrated pest management programmes at early stages and speed
the execution of plan.
 The development of DNA barcode library at regional level can help in
monitoring species decline and loss of biodiversity.

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