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7 Limitations of DNA Barcoding Employing mtCOI

7 Limitations of DNA Barcoding Employing mtCOI

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K.B. Rebijith et al.




Planococcus kraunhiae (12)

Planococcus lilacinus (6)


Planococcus ficus (4)

JF965419_Planococcus minor


Planococcus citri (9)



Crisicoccus pini (2)



Atrococcus paludinus (3)


Pseudococcus calceolariae (7)


Dysmicoccus lepelleyi (7)


Pseudococcus cryptus (5)



Pseudococcus com stocki (9)

Paracoccus marginatus (7)


Balanococcus takahashii (2)



Pseudococcus jackbeardsleyi (8)


Pseudococcus viburni (3)




Crisicoccus matsumotoi (19)

Dysmicoccus brevipes (3)

Dysmicoccus neobrevipes (15)


Maconellicoccus hirsutus (4)


Palmicultor lumpurensis (3)


Ferrisia virgata (2)

Ferrisia malvastra (4)

HM474183_Heliococcus puerariae



Dysmicoccus wistariae (2)

Pseudococcus longispinus (12)


Phenacoccus aceris (11)


Heliococcus kurilensis (7)

HM474263_Phenacoccus avenae


Phenacoccus solani (9)


Fig. 5.3 NJ tree with bootstrap support (1000 replicates)

showing clusters of species for mtCOI sequences. Distinct

clades for 29 species of aphids can be seen in the figure, in

which four species, namely Planococcus ficus, Crisicoccus

matsumotoi, Phenacoccus solani and P. aceris showing

two distinct groups with >90 % bootstrap support. The

numbers indicated in brackets represent the individuals

analysed in the corresponding species

sible to estimate species boundaries which

would have been estimated from a broader

data set.

• Mutation rate: For the DNA barcode to be

used as standalone, there should be a consis-

tent mutation rate, such as the proposed 2–3 %

divergence to correlate with species limit on a

consistent basis. Speciation, uniquely driven

by changes in mtDNA or speciation event,

necessarily alters the mtDNA haplotypes.


Molecular Identification of Mealybugs


Fig. 5.4 Neighbour joining (NJ) showing intra-specific variation in the barcode region for four species of mealybugs,

namely Planococcus ficus (a), Phenacoccus solani (b), Crisicoccus matsumotoi (c) and P. aceris (d)

• Heteroplasmy: This refers to the classical

view of mitochondria functionally haploid

with multiple identical copies. However, single nucleotide differences are common in

some species and are also abundant in some,

especially at the restriction sites.

• Compounding genetic factors: Coinheritance

factors that bias single mitochondrial inheritance and most obvious are (a) mitochondrial

selection either on the barcoding gene itself or

on the other linked genes; (b) cytoplasmically

inherited bacteria like Wolbachia and some

Rickettsia which alter the inheritance factors

(Rubinoff et al. 2006).

• Identification depends on the intra- and interspecific genetic variations.

• Difficult to resolve, recently diverged species

that arose through hybridization.

• No single gene is conserved in all domains of

life and exhibit enough sequence divergence

for species discrimination.

There are about 52 million sequence records

available currently, and it is expected that the

barcoding initiative of the animal kingdom will

produce about 100 million sequences and will be

available through GenBank.

An international database called BOLD

(Barcode of Life Data system) organizes the

sequence data on species identification, which

was initially developed as an informatics work

bench for a single, high-volume DNA barcode

facility. Later, the same has been selected by

the Canadian Barcode of Life Network (www.

bolnet.ca) to barcode all the eukaryotic life of

Canada, and subsequently, it has been adopted

by major barcode communities like birds,

fishes, lepidoptera, etc. BOLD provides an integrated bioinformatics platform that supports all

phases of analytical pathway from specimen

collection to a tightly validated barcode library.

It also provides a vehicle for collaboration

across research communities by coupling flexible security and data entry features with web

based delivery. A copy of all sequences in

BOLD is also sent to NCBI, DDBJ, and EMBL

as soon as the results are ready for public


K.B. Rebijith et al.



Other Targets for Molecular

Identification of Insects


Ribosomal DNA

Ribosomes are the major components of cells

that are involved in translating the mRNA into

proteins. Ribosomes consist of both proteins

and RNAs. The ribosomal RNA (rRNA) regions

that are conserved and more variable regions

can serve as both slow and fast clocks in identifying and unravelling the molecular phylogeny.

In eukaryotes (including insects), the genes

encoding both 18S and 28S rRNA are clustered

as tandem repeats in the nucleolus; in most animals, there are 100–500 copies of rDNA in the

nuclear genome in tandemly repeated transcription units. The repeated transcription unit is

composed of a leader promoter region known as

external transcribed spacer region (ETS), 18S

rDNA coding region, internal transcribed spacer

region (ITS), 28S rDNA coding region and an

internal non-coding transcribed spacer region

(IGS). In addition to the above, R1 and R2 retro

transposable elements are found in specific

locations (Fig. 5.5).

Different portions of the repeated transcription units evolve at different rates in the nuclear

genome; a higher degree of polymorphism is

found in the non-coding segments (IGS, ITS,

ETS), and the most variable part of the repeated

unit is IGS, which contains reiterated sub-repeats

ranging from 50 to several hundred base pairs in

length. The coding regions of the repeated unit

change relatively less and can be used for systematic studies of higher taxa or for ancient lineages. Ribosomal RNA genes undergo concerted

evolution so that the sequence similarity of the

members of an RNA family is expected to be

greater within species than between species.



Fig. 5.5 Gene organization of ribosomal genes

In addition to the above reterotransposons, R1

and R2 have been in the 28S rRNA genes of most

insects, are associated with arthropods, and are

usually precisely located at the same nucleotide

position within the 28S rRNA gene. Most of the

R2 elements are located about 74 bp upstream

from the site of R1 insertions. R1 and R2 do not

have long terminal repeats and block the production of functional rRNA, since there are many

rRNA genes, and R2 are kept from invading by

miRNA/siRNA. Usually, R1 and R2 do not have

accumulated mutations that would make them



Satellite DNA

Satellite DNA may consist of a large fraction of

the total DNA in an insect. Microsatellites are

usually species specific, and evolve at very high

rates. Satellite DNA can also be used for species

identification and analysis of populations.


Nuclear Protein Coding Genes

A variety of protein coding loci have been used

in molecular systematics, and some of them are

listed below:

1. alpha amylase 2. acetyl choline esterase 3.

actin 4. alcohol dehydrogenase 5. arylphorin

6. cecropin 7. chorin 8. dopa carbaxylase 9.

elongation factor 1 alpha 10. esterase 11. glycerol 3 phosphate 12. glycerol 6 phosphate

dehydrogenase 13. guanylate cyclase 14. globin family genes 15. histones 1 and 4 16.

hunch back 17. kruppel 18. luciferase 19.

lysozyme intron 20. myosin alkali light

chain intron 21. nullo 22. opsin 23. period 24.




Molecular Identification of Mealybugs

phosphogluco isomerase 25. phosphoenol

pyruate carboxy kinase 26. prune 27. copper,

zinc superoxide dismutase 28. sodium channel

para locus I 29. snail 30. timeless 31. triosephosphate isomerase 32. vestigial 33. white

34. wingless 35. xanthine dehydrogenase 36.

yolk protein 1 and 2 37. zeste.



• May be heterozygous and present in low copy


• Many genes contain large introns that makes it

difficult to amplify more than one exon.

• Many single-copy loci are actually are present

in more than one copy.

• Pseudogenes may create problem if comparisons are made inadvertently.

Even with all the limitations, the molecular

identification of insects employing mtCOI is

gaining momentum, and as of now it can be an

effective adjunct tool for the integrated taxonomy. Many barcoding initiatives are beginning to

take shape, such as the recent initiative on

Barcoding of butterflies of India funded by the

Department of Biotechnology. With the increase

in international trade on agricultural produces

where the danger of introduction of invasive species looms large, DNA barcoding is going to play

a vital role in the quick identification of insectpests at the port of entry. As ambitiously envisaged, the development and deployment of the

hand-held sequencer, which is supported by the

global networked database, is going to revolutionize the way we identify insects that are

already described, along with the new ones.



1. The relationship of six mealybug species (Pl.

citri, Pl.ficus, P.ovae, Ps.longispinus, Ps.vibruni,

Ph.aceris) was studied using randomly amplified

polymorphic DNA-polymerase chain reaction

(RAPD-PCR) in Turkey. Cluster analyses of


RAPD data clearly separated the species into

two groups (Serce et al. 2007).

2. Seven species of mealybugs (Ps maritimus,

Ps.vibruni. Ps.longispinus, Ps.calceolariae,

Pl.ficus, Pl.citri, Ferrisia gilli Gullan) were

identified using a Multiplex PCR based on the

mitochondrial cytochrome oxidase subunit

gene (Daane et al. 2011).

3. There was a slight difference in morphological characters in the populations of

Planococcus ficus, indicating that there are

two different populations of the same species

in Tunisian vineyards. Likewise, in the molecular analyses, two separate clades were

revealed in the neighbour-joining phylogenetic tree, supporting the morphological studies and suggesting there are two distinct

populations of grape vine in Tunisia, which

might be two different biotypes (Mansour

et al. 2012).


Andersson SG, Kurland CG (1998) Reductive evolution

of resident genomes. Trends Microbiol 6:263–268

Asokan R, Rebijith KB, Singh SK, Sidhu AS, Siddharthan

S, Karanth PK, Ellango R, Ramamurthy VV (2011)

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Brunner PC, Chatzivassilious EK, Katis NI, Frey JE

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Downie DA, Gullan PJ (2004) Phylogenetic analysis of

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