RESEARCH ARTICLE
Phylogenetic Analysis of Anopheles (Cellia) subpictus Grassi UsingrDNA-ITS2 Sequence
Jainder S. Chhilar • Sudarshan Chaudhry
Received: 6 February 2011 / Revised: 22 March 2012 / Accepted: 27 March 2012 / Published online: 11 April 2012
� Zoological Society, Kolkata, India 2012
Abstract Anopheles subpictus Grassi is one of the most
abundant malaria vector mosquitos in Indian subcontinent
especially in post monsoon months. This taxon has been
speculated to be composed of four sibling species recog-
nised using morphological and cytogenetic parameters
provisionally named A, B, C and D. One of the sibling
species ‘B’ has well documented status as a vector of
malaria parasite in the Oriental region. Molecular phylo-
genetic analysis of this mosquito was done to discern sib-
ling/cryptic species using internal transcribed spacer 2
region of the nuclear ribosomal DNA (rDNA-ITS2). An.
culicifacies sibling species A, a member of Myzomia ser-
ies, was used as an out-group to root the trees. In the
present analysis rDNA-ITS2 region was PCR amplified,
sequenced and the sequences obtained were then subjected
to phylogenetic analysis with the sequences already present
in the sequence database GenBank. Multiple sequence
alignment was performed using ClustalX and further
manually annotated in MEGA 4. Initial analysis suggested
extreme 30 end sequence divergence within different pop-
ulations. Phylogenetic analysis of this spacer was done
using maximum parsimony, maximum likelihood, and
distance matrix—neighbour joining methods performed
with bootstrapped dataset that were executed in DNA-
PARS, DNAML, DNADIST and neighbour programs in
Phylip software package respectively. Phylogenetic trees
produced were similar in topology but differed in bootstrap
support. Results support the division of this taxon into at
least two major clades differing in their sequence compo-
sition and product size that are recommended to be
renamed as An. subpictus inland form and An. subpictus
coastal form. Differences include insertions/deletions,
transitions and transversions; especially one large 30 end
deletion event. The sibling species status of An. subpictus
has been analysed critically.
Keywords Anopheles subpictus � rDNA-ITS2 �Phylogeny � Sibling species
Introduction
Mosquitoes belonging to the family Culicidae, subfamily
Anophelinae act as vectors of various pathogens including
malarial parasite. For more than a century, mosquito tax-
onomists and systematists have concentrated their efforts on
the biology, identification and classification of members of
the family Culicidae attempting to determine their diversity,
vectorial status and phylogeny (Coluzzi 1970; Kitzmiller
1976; Harbach 1994; Harbach and Kitching 1998, 2005;
Sallum et al. 2002). Presently, there are 420 morphologically
distinguishable anopheline species in the world of which 70
are considered to be vectors of malaria. Out of these 420, 56
are prevalent in Indian subcontinent including 13 malaria
vectors (Knight and Stone 1977; Rao 1984; Nagpal and
Sharma 1995). Anopheles (Cellia) subpictus Grassi 1899 is
the most abundant anopheline in most parts of the Indian
subcontinent and South-east Asia (Rao 1984; Chandra et al.
2010). Its role as a vector of malaria, filariasis, and West Nile
virus has been well documented (Panicker et al. 1981; Ku-
lkarni 1983; Banerjee et al. 1991; Amerasinghe et al. 1992;
Abhyawardana et al. 1996; Sahu 1998; Amerasinghe and
J. S. Chhilar (&)
Department of Zoology, Government PG College, Gohana,
Sonipat 131301, Haryana, India
e-mail: [email protected]
S. Chaudhry
Mosquito Cytogenetic Unit, Department of Zoology, Panjab
University, Chandigarh 160014, India
123
Proc Zool Soc (Jan-June 2012) 65(1):1–10
DOI 10.1007/s12595-012-0021-8
TH
EZ
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OLOGICAL SOC
IET
YKO LK ATA
Amerasinghe 1999; Hubalek and Halouzka 1999; WHO
1999; Chatterjee and Chandra 2000; Chandra et al. 2010).
Presently An. subpictus is a member of the Pyretophorus
series of subgenus Cellia having a sister group relationship
with An. indefinitus (Anthony et al. 1999).
To solve the problem sibling species have been created
especially in vector species discrimination, various tools
and techniques were successfully used including morphol-
ogy, polytene chromosome comparative cytogenetics, and
molecular level tools viz. enzyme electrophoresis, cuticular
hydrocarbons and DNA based markers resulting in identi-
fication of more than 30 different sibling species complexes
in The genus Anopheles (Coluzzi 1970; Green et al. 1985;
Pape 1992; Subbarao and Sharma 1997; Xu et al. 1998;
Marrelli et al. 1999; Huong et al. 2001; Linton et al. 2001;
Manonmani et al. 2001; Goswami et al. 2005; Ma et al.
2006; Foley et al. 1998, 2007). Ribosomal DNA (rDNA)
has been extensively used in the past for identification and
discrimination of sibling species in anophelines especially
the internal transcribed spacer 2 (ITS2) region (Fritz et al.
1994; Collins and Paskewitz 1996; Favia et al. 2001; Chen
et al. 2003). Also the problems and complexities in analysis
of cryptic taxa at molecular level, the systematics and
classification of anophelines, application of various
molecular phylogenetic approaches and troubleshooting
during analysis have been thoroughly reviewed (Nei 1996;
Harbach and Kitching 1998, 2005; Besansky 1999; Krzy-
winski et al. 2001; Sanderson and Bradley Shaffer 2002;
Krzywinski and Besansky 2003; Philippe et al. 2005).
In this context, based on the presence of inversion
genotypes two sibling species provisionally named A and B
were reported within An. subpictus (Suguna 1982; Reuben
and Suguna 1983). Further in 1994, Suguna and co-workers
presented morphological evidences in support of the pres-
ence of two additional species C and D raising the number of
sibling species in this taxon to four. Reports of presence of
morphological and cytogenetic polymorphism in An. sub-
pictus (Kirti and Kaur 2004; Chhilar and Chaudhry 2005)
made it necessary to have a molecular viewpoint on its
sibling species status. Recently, Chhilar (2009) and Suren-
dran et al. (2010) have reported that published morpholog-
ical characters are not enough to identify some members of
the An. subpictus complex hence the present study based on
the sequence characteristics of rDNA-ITS2 has implications
on the sibling species within the An. subpictus complex.
Materials and Methods
Collection of Mosquitoes and Identification
Wild populations of An. subpictus constituted the material
for the present study. The larvae and adults were collected
from the States of Haryana and Punjab (North-western
India) (Table 1). The larvae, in various stages of devel-
opment were segregated, put in separate enamel bowls and
fed on a protein rich diet of finely powdered dog biscuits
and yeast tablets in the ratio of 6:4 (Chaudhry et al. 2005).
For the preliminary sorting the dichotomous keys of Wattal
and Kalra (1967), Nagpal and Sharma (1995) and a rapid
field key prepared by the present authors for all the species
prevalent in the Chandigarh region (unpublished) were
followed. The final confirmation was done from the stan-
dard banding pattern of the larval salivary polytene
X-chromosome from fourth instar larvae (Chaudhry et al.
2005).
DNA Extraction and PCR Amplification
DNA was extracted from more than ten individual adult
specimens from each population by following the standard
phenol–chloroform extraction method (Sambrook et al.
1989). The wings and head of individual mosquito females
were mounted on the slides for population identification
and differentiation and the rest of the body parts were used
for DNA extraction. Primers were designed for rDNA-ITS2
spacer region using primer3 web interface software from
rDNA-ITS2 sequence (accession number AY049004)
(Table 2). These primers aligned to sites in the conserved
5.8S (ITS2FP) and 28S (ITS2RP) regions flanking the
ITS2. The working PCR reaction mixture had 19 PCR
reaction buffer containing 1.5 mM MgCl2; 0.2 mM dNTPs
and 2.5 lM of each primer. To this 1 ll of sample DNA
(diluted 50 times) and 1 ll of Taq polymerase enzyme (3
units per reaction) were also added. Thermocycler was
programmed for the PCR with the following reaction
conditions: step 1: initial denaturation at 94 �C for 4 min,
step 2: denaturation at 94 �C for 1 min, step 3: annealing at
50 �C for 1 min, step 4: amplification at 72 �C for 1 min,
step 5: repeat step 2–4 for 30 cycles, step 6: final extension
at 72 �C for 5 min. The PCR amplified products were
resolved on 2 % Agarose gels using 0.59 TBE buffer.
Table 1 Collection details of An. subpictus populations
S. no. Place of collection Longitude/latitude Date of collection Life stage of mosquito
1 Bank Colony, Rohtak Road, Bhiwani (HR) 28.46 N 76.18 E 10.10.03 Adults, larvae, pupae
2 Gohana Road at Purkhas Road diversion, Sonipat (HR) 29.00 N 70.00 E 21.09.03 Larvae
3 Kishan Pura, Patiala Road, Sangrur (PB) 30.12 N 75.53 E 2.11.03 Adults, larvae, pupae
2 Proc Zool Soc (Jan-June 2012) 65(1):1–10
123
Standard 100 bp DNA ladder was used for checking the
amplified product size. All the chemicals were purchased
from Bangalore Genei P Ltd, Bangalore, India. PCR
products were commercially sequenced from genoMbio
Technologies Pvt. Ltd., Pune, India. The PCR end product
size varied from 667 to 680 bp while the ITS2 spacer size
varied from 558 to 559 bp. Sequences were annotated
using ChromasPro version 1.41 and submitted using stand
alone sequence preparation tool ‘Sequin’ to the GenBank
database. The sequences submitted to GenBank database
were assigned accession numbers EF1601868–EF1601870.
Apart from the populations sequenced during the present
study from North-west India other sequences of rDNA-
ITS2 available at GenBank database having the acces-
sion numbers AY049004.1, AF406615.1, AF406614.1,
AF406613.1, and AF406616.2 were retrieved and used for
phylogenetic analysis (Table 3). rDNA-ITS2 sequence
having accession no AY702488.1 of An. culicifacies sibling
species A, a member of Myzomia series in subgenus Cellia,
was used as out-group to root the phylogenetic trees.
Phylogenetic Analysis
Multiple sequence alignment (MSA) was performed using
ClustalX and then it was manually edited in MEGA version
4 (Tamura et al. 2007). The MSA was obtained in a *.phy
format file to perform phylogenetic analysis with the
Phylip software package (Felsenstein 2004). For a thor-
ough Phylogenentic analysis both the distance and char-
acter based approaches—neighbour joining (NJ) analysis,
maximum-parsimony (MP) analysis, and maximum-likeli-
hood (ML) analysis respectively were preformed and
Kimura two-parameter distances were calculated using
Total substitution and transition/transversion ratio (Nei
1996; Sanderson and Bradley Shaffer 2002; Philippe et al.
2005). The bootstrapping was carried out prior to analysis
with 1000, 100, and 10000 replicates for parsimony, ML,
and NJ approach respectively. The protocols and steps
were followed from Aiyar (2000) for ClustalX and Retief
(2000) for Phylip.
Results
The MSA of annotated rDNA-ITS2 sequences of An.
subpictus populations with out-group An. culicifacies
resulted in a total of 662 alignment sites out of which the
base sequence of only 134 sites was constant (Fig. 1). In
other regions as many as 21 insertions/deletions (indels)
varying in length requiring gaps in alignment could be
identified. When compared carefully, it was found that ten
of these indels were due to sequence divergence between
the coastal populations (A, B and E) and inland populations
(F, G, H, C and D) (see Table 3 for details). Out of the ten
Table 2 rDNA-ITS2 forward and reverse primers used in the present study
Primer Sequence Properties
ITS2 FP 50-GTGAACTGCAGGACACATGAA-30 Length: 21 bases,
Tm: 59.74 �C,
GC%: 47.62
ITS2 RP 50-TGCTTAAATTTAGGGGGTAGTCA-30 Length: 23 bases,
Tm: 59.10 �C,
GC%: 39.13
Table 3 Details of ITS2 sequence composition in An. subpictus
Species/population Population code Accession no. ITS2 length GC% content
AP ITS2 AP ITS2
Anopheles subpictus/Sri Lanka coastal region A Gb/AY049004.1 618 477 53.23 55.04
Anopheles subpictus/Sri Lanka inland B Gb/AF406615.1 662 474 54.68 56.54
Anopheles subpictus/Sri Lanka inland 2 C Gb/AF406614.1 755 564 53.24 54.78
Anopheles subpictus/Sri Lanka inland 3 D Gb/AF406613.1 754 564 53.05 54.78
Anopheles subpictus/Sri Lanka coastal region 2 E Gb/AF406616.2 668 521 53.44 53.84
Anopheles subpictus/India Sonipat F Gb/EF601868 670 558 55.82 56.81
Anopheles subpictus/India Bhiwani G Gb/EF601869 682 559 55.27 56.17
Anopheles subpictus/India Sangrur H Gb/EF601870 667 558 55.47 57.52
AP amplified product, length in base pair
Proc Zool Soc (Jan-June 2012) 65(1):1–10 3
123
Fig. 1 MSA of rDNA-ITS2 spacer sequences of An. subpictus using ClustalX software in Phylip (*.phy) format. Indels in alignment are denoted
by dash (–)
4 Proc Zool Soc (Jan-June 2012) 65(1):1–10
123
indels, seven were found in the 50 half of ITS2 sequences
under study. Further, one large 30 terminal indel of 121
bases between ‘541 base and 662 base’ was found in
populations A, B, and E (the coastal populations).
Phylogenetic Distance
Kimura’s two-parameter distances were calculated using
total substitutions i.e. transitions ? transversions (d =
s ? v) and transition/transversion ratio (R = s/v) using
MEGA 4 software (Table 4).
Kimura Two-Parameter Distance Using d = s ? v
As a result of the comparisons of sequences it was noticed
that the average genetic distance was 0.454 among the
members of all the populations (referred hereafter as taxa)
when all gaps were considered as complete deletions,
whereas it was 0.453 when all gaps were considered as
pair-wise deletions. The maximum and minimum distance
between different taxa was found between populations A
and E (0.000) and B and D (0.253) when all gaps were
considered as complete deletion whereas, on the basis of all
the gaps considered as pair-wise deletion, the minimum
and maximum distances were present between population
A and E (0.000) and D and E (0.301) respectively.
Kimura Two-Parameter Distance Using R (Transition/
Transversion Ratio)
In all the populations under study, the average transition/
transversion ratio was 1.246 when all gaps were considered
as complete deletions, whereas it was 1.168 when all gaps
were considered as pair-wise deletions. The minimum and
maximum ratios were observed between population G and
D (0.480) and G, A, and E (1.312) when all gaps were
considered as complete deletion, whereas with all gaps
being considered as pair-wise deletion it was between
population C and D (0.330) and G and B (1.310).
Phylogenetic Relationships
The MSA file in Phylip format called outfile was manually
edited using a standard text editor and ‘–’ or ‘.’ were
replaced by ‘?’ characters so that they are counted as a
single gap. In all the analysis no weights were used and the
gaps were excluded. The bootstrapped data was used for
analysis using DNAML, DNAPARS, and DNADIST (ML,
MP, and NJ) that resulted in consensus trees with similar
topology but varying in bootstrap support. Most of the
phylogenetically critical nodes were significantly supported
by bootstrap values of [90 % (Table 5; Figs. 2, 3, 4).
Table 4 Nucleotide P-distance matrix between rDNA-ITS2 sequen-
ces of An. subpictus populations and An. culicifacies (out-group)
using Kimura two-parameter method with d: transitions ? transver-
sions (lower-left diagonal), with R = s/v (transition/transversions)
(upper-right diagonal)
Population A B C D E F G H
A 0.000 1.182 0.897 0.884 ? 1.056 1.312 1.189
B 0.030 0.000 0.976 0.960 1.182 1.017 1.350 1.066
C 0.234 0.244 0.000 0.500 0.897 1.098 0.564 0.899
D 0.243 0.253 0.028 0.000 0.884 0.766 0.480 0.852
E 0.000 0.030 0.234 0.243 0.000 1.056 1.312 1.189
F 0.207 0.192 0.079 0.084 0.207 0.000 0.604 1.132
G 0.226 0.217 0.095 0.097 0.226 0.057 0.000 0.983
H 0.251 0.225 0.105 0.102 0.251 0.045 0.095 0.000
Table 5 Bootstrap value variation
S. no Taxon/clade MP ML NJ
1 (F ? H) 939 86 8153
2 (C ? D) 981 100 9853
3 (A ? E) 999 100 9975
4 ((F ? H) ? G) 460 – –
5 ((A ? E) ? B) 1000 86 9780
6 ((F ? H) ? (C ? D)) – 48 6830
7 ((F ? H) ? G) ? (C ? D)) 1000 – –
8 ((F ? H) ? (D ? C) ? G) – 91 9299
Fig. 2 Phylogenetic tree generated by MP An. culicifacies was used
as out-group to root the tree. Numbers on branches are bootstrap
values using 1,000 replicates. Taxa are abbreviated as in Table 3
Fig. 3 Phylogenetic tree generated by ML An. culicifacies was used
as out-group to root the tree. Numbers on branches are bootstrap
values using 100 replicates. Taxa are abbreviated as in Table 3
Proc Zool Soc (Jan-June 2012) 65(1):1–10 5
123
Populations (A ? E), (D ? C), and (F ? H) clustered
together in different clades while population B clustered
with clade (A ? E) as a basal group. Population G was
found to be basal to either (F ? H) or (C ? D) clade.
Population G clustered paraphyletically in inland popula-
tions clades either as ((F ? H) ? G) in MP tree where it
was supported by a bootstrap value of 460 only, whereas it
clustered as ((F ? H) ? (D ? C) ? G) in ML and NJ
trees with significant bootstrap values of 91 and 9,299
(Table 5; Figs. 2, 3, 4).
Discussion
Variability in Length and Sequence Composition
The sequence analysis revealed two types of rDNA-ITS2
spacers in this species—one consisting of 558–564 bp
(populations F, G, H, C, and D) while the other consisting of
474–521 bp (populations A, B, and E) (Table 3). When the
length of ITS2 was reviewed within the subgenus Anoph-
eles, Maculipennis complex has an average length of
305 bp (Porter and Collins 1991) while Quadrimaculatus
complex has 305–310 bp (Cornel et al. 1996), An. petrag-
nani has 302 bp and An. claviger has 341 bp (Kampen et al.
2003). Similarly, within the subgenus Nyssorhynchus, An.
nuneztovari has a range of 363–369 bp (Fritz et al. 1994).
To the contrary the members of the subgenus Cellia have
longer ITS2 spacers for example: An. gambiae has 426 bp
(Paskewitz et al. 1993), Dirus complex has 710–716 bp,
while Punctulatus group has 549–563 bp (Beebe et al.
2000c), an exception being the Minimus group members
having ITS2 region as small as 227 bp in An. varuna and
375 bp in An. minimus C (Phuc et al. 2003). Beebe and
Cooper (2000) reported in their study on the Punctulatus
group that the 50 end sequence was more conserved than the
30 sequence, a condition which is similar to the one found in
the present populations of An. subpictus. Interestingly 30
end of the ITS2 region has been contended to be useful in
attaining a stable secondary structure (Joseph et al. 1999;
Schultz et al. 2005; Dassanayake et al. 2008).
The comparative base composition of the ITS2
sequences reveals that purines are more than pyrimidines in
all the populations (Table 6). The average base composi-
tion for An. subpictus was found to be 21.6 % of A, 22.6 %
of T, 29.5 % of G and 26.2 % of C. The MSA revealed
genomic divergence in the spacer length and base com-
position which manifested as Transitions, Transversions
and indels in the alignments. In comparison to the ampli-
fied products that included 5.8s and 28s rDNA regions
(which were annotated) the ITS2 spacer was found to be
GC rich (Tables 3, 6). When compared to other anopheline
species, the average GC content of 56.68 % is very close to
the An. gambiae complex in the same Pyretophorus series
that is having 55 % of the total GC content. However,
some of the members of subgenus Cellia have longer ITS2
spacers leading to higher GC content. For example, An.
dirus and An. punctulatus have as much as 61–71 % where
the increase is attributed to evolutionary drift and natural
selection for greater fitness and survival of the species
against the natural mutational load. There is a general
observation that, within the species complexes, the extent
of sequence variations among the members is not consis-
tent (Beebe et al. 1999, 2000a, b, c; Marinucci et al. 1999;
Beebe and Cooper 2000).
Table 6 Comparative base composition of ITS2 sequences in populations of An. subpictus
Population
code
Nucleotide frequency
of base G
Nucleotide frequency
of base C
Nucleotide frequency
of base A
Nucleotide frequency
of base T
A 30.5 24.6 20.6 24.4
B 31.4 25.1 19.6 23.8
C 28.7 26.1 22.5 22.7
D 28.0 26.8 22.3 22.9
E 29.4 24.4 21.3 24.8
F 30.3 26.5 21.7 21.5
G 29.5 26.7 21.3 22.5
H 30.5 27.1 21.0 21.5
Fig. 4 Phylogenetic tree generated by NJ with Kimura two-param-
eter distances An. culicifacies was used as out-group to root the tree.
Numbers on branches are bootstrap values using 1,000 replicates.
Taxa are abbreviated as in Table 3
6 Proc Zool Soc (Jan-June 2012) 65(1):1–10
123
Phylogenetic Distance and Relationships
The analysis of Kimura two-parameter and other genetic
distances revealed that the minimum distance was
encountered between members of the inland populations of
An. subpictus and the maximum distance is between
An. subpictus coastal populations and An. culicifacies
(out-group) using both total substitution and transition/
transversion ratio approach. The same is reflected in the
phylogenetic trees derived using bootstrapped datasets
Fig. 5 ITS2 consensus
sequence of An. subpictusinland type (population C, D, F,
G, and H)
Fig. 6 ITS2 consensus
sequence of An. subpictuscoastal type (A, B, and E
populations)
Fig. 7 MSA of consensus sequences for inland type A and coastal type B (showing the large 30 indel)
Proc Zool Soc (Jan-June 2012) 65(1):1–10 7
123
(Figs. 2, 3, 4). All the three phylogenetic trees derived
from rDNA-ITS2 sequence dataset support the presence of
sibling species in An. subpictus complex as most of the
important branches have [70 % bootstrap support. When a
condensed tree for 75 % bootstrap value is considered all
the branches collapse and the populations clearly form the
two basal clades representing the inland and coastal pop-
ulations. Similarly, the inner nodes for ancestral taxonomic
units of inland populations and coastal populations are
significantly supported by bootstrap values in trees
obtained in all the phylogenetic approaches. As the prob-
ability of obtaining the correct tree topology is above 95 %
in a 500 bp length sequence using any of the MP, ML or
NJ-based approach (Nei 1996), it can be inferred that the
tree topology obtained using different approaches in the
present study is also correct.
Finally, it can be concluded that population B from Sri
Lanka is actually a member of provisionally named An.
subpictus coastal form, while the population from Bhiwani
(G) India is basal to all the inland populations representing
the inland type form. In relevance to the results discussed
in the foregoing text consensus sequences were generated
for both the inland and coastal form (for various popula-
tions as detailed in Table 3; Figs. 5, 6) of this species and
aligned using ClustalX (Fig. 7) as a ready reference stan-
dard for future studies. It is recommended that the two
sibling species A and B be renamed as inland and coastal
forms. Though, there is very little support for sibling spe-
cies C and D in the present molecular study still further
inter-laboratory joint research effort to describe proper
taxonomic status of these forms using multiple parameters
from whole of its geographical and vectorial range are
required before deciding about the status of sibling species
C and D.
Acknowledgments The authors are thankful to Chairperson,
Department of Zoology, Panjab University, Chandigarh for providing
the laboratory facilities under its special assistance programme (SAP)
and Centre of Advance Studies (CAS) of University Grants Com-
mission, New Delhi. The first author is grateful to the Council of
Scientific and Industrial Research, New Delhi for providing research
grant vide F. no. 9/135(441)/2K2/EMR-I. The authors are highly
grateful to the reviewers for their critical suggestions.
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