1. ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793
American International Journal of
Research in Formal, Applied
& Natural Sciences
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 1
AIJRFANS is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by
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Subspecies identification in aphids (Homoptera: Aphididae) by application
of partial sequence of cytochrome c oxidase subunit I (COI) gene: a view on
the potential of method
Nina V. Voronova
Zoology Department of Belarusian State University, Minsk, Belarus
I. INTRODUCTION
Today the species problem remains one of the most fundamental biological problems as well as one extremely
difficult to solve[1]
. Debates surrounding the concept of a species concept have been going on for decades[2–6]
.
Ignoring the philosophical aspect of this problem, the most important question of the discussion is – if the living
world is really discrete, on which level of the taxonomic system and by which criterion can we draw a line
between taxonomic units so that their distinction and individuality are not in doubt[7-9]
?
The species problem can be observed most clearly in evolutionarily young groups of animals where adaptive
radiation of low level taxa was particular wide. The origination of a generous amount of species, subspecies,
races and other forms in an evolutionarily short time have led to an abundance of difficult to distinguish taxa.
Aphids (Homoptera: Aphididae) is just such a group. There is a large number of closed related species and
subspecies and, perhaps, ecological races which only differ in their biology or host-adaptation among the
aphids, but the evolutionary significance of such features are not unquestionable. The major ecological and
morphological plasticity of aphids which had been repeatedly demonstrated in experiments[10-11]
introduces
additional complexity into construction of the phylogenetic system of this large group of animals. Nevertheless,
it is impossible to disregard the questions of the taxonomic status of the closed related forms, because such
forms of aphids may vary considerably in their harmfulness. Determination of the significance of each aphid
species and subspecies as a crop pest is the main factor that forces scientists to search for new methods to detect
morphologically similar groups. Another reason for researching the problem of the subspecies detection in
aphids is the importance of obtaining knowledge about every evolutionary event in the group, including (or most
importantly) the ones which belongs to the micro evolutionary level. Studying the micro evolutionary events
allows us both to discover their mechanisms and to identify the main evolutionary trends in a group of
phytophagous carrying such an importance for food production and plant ecology[12-13]
.
Cytochrome c oxidase subunit I gene (COI) is a mitochondrion gene of eukaryotes which was selected as the
most effective molecular marker for species identification[14]
. A 500–700-bp region at the 5' end of the COI gene
forms the primary barcode sequence for members of the animal kingdom[15-16]
. In our work we aimed to estimate
whether the usage of COI partial sequences as a single molecular marker allows manifesting of morphologically
indistinguishable subspecies in aphids.
II. MATERIALS AND METODS
Specimens and DNA extraction
Specimens of aphids were collected in 2008–2010 from Russia and Belarus. We analyzed COI sequences from
aphids of 17 species and subspecies belonging to 4 genera and 2 tribes within Aphididae (Table 1).
Abstract: The family Aphididae is one of the most important for crop production group of phytophagous. In
such groups, in which certain forms can noticeably vary in their host-specify and harmfulness, correct
species and subspecies identification has great significance. Aphids (Homoptera: Aphididae) is known their
high ecological plasticity and simultaneously this family includes a lot of closely related species,
morphologically similar subspecies and other forms which morphology-based identification cannot be
single-valued. Mitochondrial COI sequences are provided for not only species but subspecies identification
of aphids from Europe. Fragments about 540-bp were analyzed. Most of the studied subspecies (76.92
percent) had distinct COI sequences. The rate of nucleotide substitutions on 5'-end of the COI gene varied
from two to seven per length (0.37–1.30 %). Difference between subspecies reached the level typical for
closed related species, and showed no individual variability or geographic affinity. Based on these results,
we conclude that COI sequences can provide an effective tool for identifying aphid subspecies in such
applications as pest management, monitoring and plant quarantine.
Keywords: aphids • subspecies identification • DNA barcoding • COI • molecular taxonomy

2. Nina V. Voronova, American International Journal of Research in Formal, Applied & Natural Sciences, 6(1), March-May 2014, pp. 01-06
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 2
All aphid samples were stored in 75% ethanol for slide voucher specimens and 96% ethanol at –20 °C for DNA
extracting. Identification of each aphid was based on exterior morphology of slide-mounted specimens[17]
,
verified with slides of Zoological Institute Collection of the Russian Academy of Sciences and were preserved
in the Insect Collection of the Zoology Department of the Belorussian State University (Minsk, Belarus). Total
genomic DNA extraction was performed using Genomic DNA Purification Kit (Thermo Fisher Scientific,
Fermentas). Samples for extraction consisted of single or several individuals from the same colony.
Amplification and Sequencing of mitochondrial genome fragments
The target 708-bp fragment of COI was amplified by polymerase chain reaction (PCR) using universal primer
pair, LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-
TAAACTTCAGGGTGACCAAAAAATCA-3')[15,18]
. The reaction mixture contained 200 μmol of dNTP mix,
15 pmol of each primer, 2 mmol of MgCl2, PCR-Buffer, 1 U Pfu polymerase (Thermo Fisher Scientific,
Fermentas) and 0.5 μg of DNA. We used the following thermal cycle parameters for 25 µl amplification
reactions: initial denaturation for 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 90 s
at 72 °C and a subsequent final extension at 72 °C for 5 min.
Table 1. Aphid species and subspecies used in this study. Nomenclature according to Remaudiere 1997.
Species Locality Host plant Date
Amphorophora idaei Börn. Grodno region, Belarus Rubus idaeus L. 27/08/2010
Aphis fabae cirsiiacanthoidis (Scop.) Minsk region, Belarus Cirsium arvense (L.) Scop. 15/07/2008
Aphis fabae fabae Scop. Minsk region, Belarus Chenopodium album L. 05/08/2010
Aphis fabae philadelphi (Börn.) Minsk, Belarus Philadelphus coronarius L. 13/06/2008
Aphis fabae ssp. Minsk region, Belarus Chenopodium album L. 03/06/2008
Aphis ruborum Börn. Stolbtsy district, Minsk region, Belarus Rubus caesius L. 14/07/2010
Dysaphis aff. newskyi (Börn.) Turochaksky district, Altai, Russia Heracleum sp. 04/07/2010
Dysaphis newskyi ossiannilssoni
Stroyan
Turochaksky district, Altai, Russia Angelica sp. 21/07/2010
Macrosiphum cholodkovskyi Mordv. Lepel district, Vitebsk region, Belarus
Filipendula ulmaria (L.)
Maxim.
04/06/2010
Macrosiphum gei Koch Minsk region, Belarus Geum urbanum L. 05/06/2009
Macrosiphum gei Koch Minsk region, Belarus
Anthriscus sylvestris (L.)
Hoffm.
05/06/2009
Macrosiphum gei Koch Minsk region, Belarus Aegopodium podagraria L. 12/06/2009
Macrosiphum gei Koch Minsk region, Belarus Chaerophillum aromaticum L. 10/06/2009
Macrosiphum knautiae Holm. Minsk region, Belarus Knautia arvensis (L.) Coult. 11/08/2009
Macrosiphum knautiae Holm. Minsk region, Belarus Dipsacus fullonum L. 08/07/2009
Macrosiphum rosae (L.) Minsk, Belarus Rosa glauca Pourr. 26/05/2010
Myzus cerasi cerasi (F.) Stolbtsy, Minsk region, Belarus, Cerasus vulgaris Mill. 14/08/2010
Myzus cerasi pruniavium (Börn.) Nesvizh, Minsk region, Belarus Cerasus avium (L.) Moench 10/07/2010
PCR products were tested by electrophoresis on an agarose gel and, if a single band was observed, were purified
using a DNA Gel Extraction Kit (Thermo Fisher Scientific, Fermentas) and were sequenced in a forward
direction by the automated sequencer 3130 Genetic Analyzer (Applied Biosystems, USA) with BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) in the Institute of Bioorganic Chemistry
NASB, Belarus. The sequences were individually checked by eye in the software package BioEdit v 7.0.5.3 and
verified for protein coding frame-shifts to avoid pseudogenes. Sequences are deposited in GenBank (accession
numbers JF340096, JF340098, JF340100–JF340103, JF340105, JF340107, JF340108, JF340113, JF776568 and
JN004138).
Genetic Divergence and Phylogeny
The combined sequences were aligned by using ClustalW in the software package MEGA4[19]
. Genetic
distances between pairs of species were calculated using the Maximum Composite Likelihood method in
MEGA4[20]
. One hundred twenty-three COI sequences obtained from the GenBank NCBI and The Barcode of
Life Data System CBOL were used for construction of trees and phylogenetic inference (the accession numbers
for the downloaded sequences given in Table S of Supplementary Information to this article). Phylogenetic trees
were constructed by the Minimum Evolution method[21]
. Bootstrapping was conducted using 1000 replicates[22]
.
After construction of trees, if we found that the subspecies branches had formed clusters with high values on the
bootstrap test, all sequences were checked by eye to discover specific nucleotide substitutions.
III. RESULTS AND DISCUSSION
Sequence analysis
The COI sequences within six groups of aphids were compared. Groups of comparison had been formed: M.
cerasi subspecies, D. newskyi subspecies, A. fabae complex of species, four forms of M. gei, which differ in

3. Nina V. Voronova, American International Journal of Research in Formal, Applied & Natural Sciences, 6(1), March-May 2014, pp. 01-06
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 3
their host-plant specificity and may presumably belong to a different subspecies, and complexes of species close
to M. rosae and A. idaei Goot. We studied the region from 81 to 620 nucleotide of COI having performed
sequence alignment on the complete mitochondrion genome sequence of Acyrthosiphon pisum (Harr.)
[NC011594]. We also used the COI sequences obtained from GenBank (accession numbers and some voucher
specimens are given in Fig. 2 and Fig. 3) in order to compare the identity of nucleotide substitutions between the
COI genes of the same subspecies from samples obtained by other researchers in different regions and in some
cases on different continents.
In all cases some single nucleotide substitutions were found in the COI sequences obtained from different
subspecies of aphids (Fig. 1).
Most frequently, those were synonymous transitions. The ratio of A↔G and С↔Т transitions was almost equal,
while the number of sites bearing such replacements varies in different species. Namely, the COI sequences of
the M. cerasi subspecies differ in seven loci. In particular, there are five nucleotide substitutions between M.
cerasi cerasi and M. cerasi pruniavium each of which is a synonymous transition. Interestingly, all studied
sequences of M. cerasi, including those samples which had not been identified exactly as subspecies, strongly
divided into three groups. The first one, as expected, incorporated M. cerasi cerasi, the second one included M.
cerasi pruniavium.
We only found two nucleotide substitutions between D. aff. newskyi and D. newskyi ossiannilssoni, however
one of them was the transversion A↔T. The same level of difference was detected between the sequences of the
closely related species M. rosae and M. knautiae (two nucleotide transitions) as well as A. idaei and A. ruborum
(one nucleotide transition). After comparing the COI sequences of certain forms of M. gei, which had been
taken into the study because the question about the necessity to divide M. gei into subspecies according to their
host-adaptation is presently under consideration[23]
, we observed that M. gei from Ch. aromaticum differs to M.
gei from G. urbanum in four COI sites, where three of those four substitutions were transversions, and it differs
from other forms of the M. gei complex in three sites.
Fig. 1 COI sequences of Myzus cerasi, Dyzaphis newskyi subspecies, Aphis fabae, Aphis idaei, Macrosiphum
gei, and Macrosiphum rosae groups. Comparisons are made using DNA-barcodes by Foottit at al. (2008)
and Valenzuela et al. (2007)

4. Nina V. Voronova, American International Journal of Research in Formal, Applied & Natural Sciences, 6(1), March-May 2014, pp. 01-06
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 4
The A. fabae species complex turned out the most homogeneous on COI gene sequences. A. fabae fabae was
only differing from the other forms of the complex in four loci, two of which represented a different
transversion. The COI sequences of A. fabae cirsiiacanthoidis, A. fabae philadelphi and A. fabae solanella were
identical (Fig. 1).
To compare the data we obtained with the other investigators’ results we had additional COI sequences of the
Illinoia azalea subspecies from CBOL. Unfortunately, it was the only group of subspecies of aphids we found in
the databases of nucleotide sequences after extensive searching. Nevertheless, it is easy to observe that there are
six nucleotide substitutions between the analogical COI gene fragments of the I. azalea subspecies. This finding
is completely consistent with our findings given above on the nucleotide substitution rate between subspecies of
aphids.
Phylogenetic constructing
We could not always observe the separation of the subspecies to stable clusters when we constructed the trees
by using various phylogenetic methods such as minimum-evolution, maximum-parsimony, and neighbor-
joining. From all studied groups only M. cerasi subspecies divided into three clusters very reliably (Fig. 3),
which may be associated with a large number of nucleotide substitutions between subspecies’ sequences.
Genetic distance
Genetic distances between some unites within studied groups (dij) were lying in the range from 0.000 to 0.023,
and the mean value for all subspecies was 0.011 (Table 2).
To compare we calculated the genetic distances between certain species allocable to the same genus by using the
orthologous COI gene fragment. We found out that the genetic distances between closed related species are
equal to the ones between subspecies, for example the genetic distance between A. idaei and A. ruborum is
0.002 while it is 0.003 between M. rosae closely related species (Fig. 2).
Table 2. Pairwise genetic distances of COI gene between subspecies calculated with three different
models. a
Average, b
Minimum, and c
Maximum.
Model
Among all analyzed
subspecies
Between four
Myzus cerasi ssp.
Between four
Aphis fabae ssp.
Between four forms of
Macrosiphum gei
Between three
Illinoia azaliae ssp.
Avea
Minb
Maxc
Ave Min Max Ave Min Max Ave Min Max Ave Min Max
MCL 0.011 0.000 0.023 0.017 0.011 0.020 0.005 0.000 0.010 0.014 0.006 0.023 0.007 0.002 0.010
p–dist. 0.011 0.000 0.022 0.015 0.011 0.019 0.005 0.000 0.010 0.014 0.006 0.022 0.007 0.002 0.010
K2P 0.011 0.000 0.022 0.015 0.011 0.019 0.005 0.000 0.010 0.014 0.006 0.022 0.007 0.002 0.010
In this paper we leave aside the discussion of questions about the evolutionary history of the analyzed
subspecies as well as the validity of their existence, relying in this issue on the opinions of colleagues in
taxonomy[17,24]
. Our interest shall only be the feasibility of identification of closed aphid subspecies by COI
sequence.
It should be noted that nowadays the DNA barcoding technique is used more widely in purely taxonomic studies
of aphids[25-31]
. Nevertheless, the absoluteness of this approach is still being debated.
Particularly heated polemics arise when molecular taxonomy data conflicts with views which are accepted at
that time among taxonomists on the status of some forms or on the relationship between the groups[32]
. The
reason is because the question “on which taxonomic level relevant molecular signal debuts” is the great issue of
molecular taxonomy. When dealing with a very short partial sequence of DNA in comparison with the full
length genome (typically 500-1500 nucleotides in length that is about 6 per cent of mitochondrial genome and
about 2.15e–4 per cent of total size of aphid DNA[33]
) it is sometimes difficult to be sure that the analysis of this
very small portion of genome will allow one to accurately identify species or subspecies. Recent studies of the
Canadian and Korean researchers[34-35]
as well as our own have shown that COI allows detection of certain
species of aphids within a high level of statistical significance. And this study displays that COI haplotypes are
formed in aphids at a lower taxonomic level, namely at the level of subspecies. These haplotypes cannot be
qualified as a random (individual) variability because we found that, for example, the COI haplotype of M.
cerasi cerasi from Belorussia is completely identical to those from Canada [EU701789 и EU701784], and the
haplotype of Belorussian M. cerasi pruniavium is the same as M. cerasi from Australia [DQ499055] and Canada
[EU701786, EU701790]. Likewise, sympatric M. gei subspecies – the necessity of the description of which, as
mentioned above, is currently under discussion – living in the same habitats but on different host plants also
show differences in the COI sequence.
Only the A. fabae group introduces some uncertainty as to the reliability of our conclusions. The lack of
nucleotide substitutions between COI of A. fabae cirsiiacanthoidis, A. fabae philadelphi and A. fabae solanella
may have two possible explanations: (1) these subspecies had diverged more recently than others we compared
and the specific nucleotide substitutions were not formed yet or (2) the subspecies rank was improperly assigned
to these forms.

5. Nina V. Voronova, American International Journal of Research in Formal, Applied & Natural Sciences, 6(1), March-May 2014, pp. 01-06
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 5
Fig. 2 Minimum evolution identification trees of Myzus cerasi, Dyzaphis newskyi subspecies, Aphis fabae,
Aphis idaei, Macrosiphum gei, and Macrosiphum rosae group based on Maximum Composite Likelihood
genetic distances. Numbers on branches are bootstrap.
Despite the fact that the vast majority of found nucleotide substitutions is synonymous, the fact of their
existence is an important evolutionary evidence, because it is known that in genes which have a strong
functional or selective constraint[36-37]
, like COI has, even synonymous substitutions are often under the pressure
of purifying selection. In any case the patterns we observed allow us to expect that the usage of even a single
marker, COI, provides an important tool for the identification of not only species but subspecies in aphids, at
least when it comes to “good subspecies” (by analogy to “good species”[38]
).
IV. CONCLUSION
We compared the sequences from 81 to 620 nucleotide of the COI gene within six groups of aphids belonging to
low level taxa (subspecies and closed related species). COI allows identifying subspecies with the same
efficiency as separate species. Subspecies of aphids have their own haplotypes of COI, with no individual
variability or geographic affinity.
Acknowledgements
We are grateful to Dr A. V. Stekolshchikov of the Laboratory of Insect Taxonomy of the Zoological Institute of
the Russian Academy of Sciences, for kindly providing us some Disaphis’ samples. We are also thankful to Dr

6. Nina V. Voronova, American International Journal of Research in Formal, Applied & Natural Sciences, 6(1), March-May 2014, pp. 01-06
AIJRFANS 14-205; © 2014, AIJRFANS All Rights Reserved Page 6
A. N. Evtushenkov the Head of the Department of Molecular Biology of Belarusian State University for his help
in the technical support of our work. We thank Mr. J. Boleininger of the Materials Department of Imperial
College London, UK for his helpful suggestion in improving the manuscript.
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