www.elsevier.com/locate/meegid

Infection, Genetics and Evolution 7 (2007) 93–102

Genetic diversity and molecular identification of mosquito species in

the Anopheles maculatus group using the ITS2 region of rDNA

C. Walton a,*, P. Somboon b, S.M. O’Loughlin a, S. Zhang c, R.E. Harbach d, Y.-M. Linton d,B. Chen c,e, K. Nolan f, S. Duong g, M.-Y. Fong h, I. Vythilingum i, Z.D. Mohammed j,

Ho Dinh Trung k, R.K. Butlin c,l

a Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UKb Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

c School of Biology, University of Leeds, Leeds LS2 9JT, UKd Department of Entomology, The Natural History Museum, London SW7 5BD, UK

e College of Plant Protection, Southwest Agricultural University, Chongqing 400716, PR Chinaf Department of Biological Sciences, University of Warwick, Warwick CV4 7AL, UK

g National Center for Malaria, Parasitology and Entomology, Ministry of Health, Phnom Penh, Cambodiah Department of Parasitology, University of Malaya, Kuala Lumpur, Malaysia

i Institute for Medical Research, Kuala Lumpur, Malaysiaj Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia

k Department of Entomology, National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnaml Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

Received 6 September 2004; received in revised form 11 May 2006; accepted 12 May 2006

Available online 19 June 2006

Abstract

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated

using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto

unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next

most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that

chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/

peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the

ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to

distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist

field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no

intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification

method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are

either connected by gene flow and/or share a recent common history.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Anopheles maculatus group; Malaria; Southeast Asia; ITS2; Genetic structure

1. Introduction

Anopheline mosquitoes occur typically as groups of closely

related species that cannot always be distinguished reliably

* Corresponding author. Tel.: +44 161 275 1533; fax: +44 161 275 3938.

E-mail address: cathy.walton@man.ac.uk (C. Walton).

1567-1348/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2006.05.001

using morphological characters. Members of species com-

plexes or groups can differ in biological attributes such as

anthropogenicity, exophagy/endophagy, exophily/endophily,

longevity and larval habitat preference. These characteristics

relate to the vectorial capacity of a species and the means by

which effective vector control can be implemented (Subbarao,

1998). Consequently, the development of reliable molecular

tools for species identification, and an understanding of

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–10294

intraspecific genetic diversity and population structure play

important roles in the development of vector control strategies

(Collins et al., 2000).

The Anopheles maculatus group is an assemblage of eight

recognised species in the Oriental Region (Harbach, 2004).

Members of the group occur from the Indian subcontinent

through Southeast Asia to Taiwan. Adults are difficult to

distinguish morphologically due to overlapping characters. The

presence of several species within the group was resolved

primarily with the use of cytological methods (Green and

Baimai, 1984; Green et al., 1985, 1992; Baimai et al., 1993).

The group was revised by Rattanarithikul and Green (1986) and

Rattanarithikul and Harbach (1991), who recognised eight

morphologically similar species. Table 1 shows how the species

designations relate to the cytological forms and their

geographic ranges. Chromosomal form K (Baimai, 1989;

Baimai et al., 1993) has not yet been formally recognised as a

species. Chromosomal forms B and E are currently regarded as

cytotypes of An. maculatus. As noted by Green et al. (1985),

these chromosomal forms either represent sibling species or

reflect geographic variation within An. maculatus. In general,

form B is found throughout northern Thailand but is replaced

by form E in southern Thailand and peninsular Malaysia (Green

et al., 1985). Cross-mating studies found no evidence of post-

mating reproductive incompatibility between the two cytotypes

(Baimai et al., 1984). The two chromosomal forms can be

distinguished using cuticular hydrocarbons (Kittayapong et al.,

1990), and using these markers it was inferred that the two

chromosomal forms are sympatric at some sites in peninsular

Malaysia (Kittayapong et al., 1993). This suggests that the

chromosomal forms correspond to separate species, and any

barriers to reproduction are likely to be pre-mating.

Members of the Maculatus Group are known to be involved

in malaria transmission, but the vectorial capacity of individual

species remains unclear from previous studies due to the

difficulty in species identification using morphology alone.

This uncertainty is aggravated by the observation that the

ability of a species to transmit malaria can vary depending upon

local factors such as environmental conditions and population

size. For example, although An. willmori has been recorded as a

major vector in Nepal (Pradhan et al., 1970), it has never been

Table 1

The chromosomal forms of the An. maculatus group with their corresponding form

Formal species name Chromosomal form

An. maculatus (Theobald) B, E, Fa, K

An. sawadwongporni (Rattanarithikul and Green) A

An. pseudowillmori (Theobald) I

An. dravidicus (Christophers) C

An. notanandai (Rattanarithikul and Green) G

An. willmori (James) H

An. dispar (Rattanarithikul and Harbach) J

An. greeni (Rattanarithikul and Harbach) D

Information is taken from Rattanarithikul and Green (1986), Green et al. (1992) aa Form F has now been excluded.

implicated as such in Thailand where it is apparently rare. An.

maculatus is widespread but is only considered to be a major

vector in southern Thailand and peninsular Malaysia (Hodgkin,

1956; Rahman et al., 1993). Since the presence of malaria

transmitted by An. maculatus correlates with the presence of

form E, it is possible that only this chromosomal form is able to

transmit malaria to any significant extent (Rongnoparut et al.,

1996; Kittayapong et al., 1993; Upatham et al., 1988). An.

maculatus is considered to be a principal vector in Java (Barcus

et al., 2002), but its cytotype and specific identity remain

undetermined. Anopheles pseudowillmori (Green et al., 1991)

and An. maculatus and An. sawadwongporni (Rattanarithikul

et al., 1996; Somboon et al., 1998), have been found infected

with malaria parasites in Thailand.

As part of a large-scale study carried out in northwestern

Thailand to understand the ecology and biting behaviour of all

potential malaria vector species in relation to land cover and

land use change, it was necessary to be able to distinguish

species of the Maculatus Group. This excludes An. dispar and

An. greeni which are confined to the Philippines (Rattanar-

ithikul and Harbach, 1991), and which can readily be identified

using the ITS2-RFLP assay of Torres et al. (2000). The other six

species of the An. maculatus group potentially occur in

northwestern Thailand, although only An. maculatus and An.

sawadwongporni are considered to be widespread (Green et al.,

1991). Two molecular methods have been developed to

distinguish some members of the An. maculatus group in

China (Ma et al., 2002; Li et al., 2003), but these were

unavailable at the start of this study and to our knowledge they

have not been tested for use in Thailand. The number of

specimens to be screened in such studies is often large.

Cytological methods of identification are not suitable as they

are stage-specific, time-consuming and laborious to perform.

PCR-based methods of identification are preferable as they are

relatively quick, straightforward and reliable. Regions of the

ribosomal DNA (rDNA) are often the markers of choice in

Anopheles for this purpose as there are often fixed differences

even between closely related species (Collins and Paskewitz,

1996; Walton et al., 1999a).

The aim of this work was two-fold: (1) to explore the genetic

diversity of the An. maculatus group and (2) to develop a PCR-

al names and reported distributions

Distribution

Bangladesh, Cambodia, China, India, Indonesia, Laos, Malaysia,

Myanmar, Nepal, Pakistan, Sri Lanka, Taiwan, Thailand, Vietnam

Cambodia, China, Myanmar, Thailand, Vietnam

China (Yunnan), India (Punjab, Assam, Kasauli), Nepal, Thailand

(northwest), Vietnam (Tonkin)

India (Nilgiri Hills), Myanmar (Kale Valley), Thailand (northern)

Thailand (Kanchanaburi, Nakhon Phanom, Phetchaburi)

India (Punjab, Almora Kumao, Kasauli, Kalpa, Assam), Nepal,

Pakistan (Kashmir), Thailand (Chiang Mai)

Philippines

Philippines

nd Baimai et al. (1993).

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–102 95

based identification method to reliably distinguish species of

the group in northwestern Thailand, which could be used in a

large-scale epidemiological and ecological study. The marker

used for these purposes was the second internal transcribed

spacer (ITS2) that separates the 5.8S and 28S rDNA subunits.

The diversity of the An. maculatus group was investigated, not

only within the study foci in northwestern Thailand, but also in

the rest of Thailand and other Southeast Asian countries. This

enabled us to evaluate the geographic extent over which the

identification method is potentially applicable since this is

dependent upon the distribution of genetic diversity of ITS2

within and between species.

2. Materials and methods

2.1. Mosquito collection and morphological and

chromosomal identification

Adult mosquitoes were collected using animal and human

baits at sites 1 and 2 in northwestern Thailand (Fig. 1, Table 2),

and progeny broods were raised from some females. Larvae

were collected from the edges of running streams and reared to

adulthood. Collections of larvae and adults were made at the

mountain site 3 (Doi Inthanon) in an attempt to collect the

higher altitude species An. willmori, but only An. maculatus and

An. pseudowillmori were found. Specimens were identified to

species based on adult and egg morphology (Rattanarithikul

and Green, 1986) (keys are unavailable for immature stages of

the An. maculatus group) and/or metaphase karyotypes (Baimai

et al., 1993). Mosquitoes that could be reliably identified to

species (some of which were from progeny broods and some of

which were field-caught mosquitoes) were designated as

reference specimens. Some siblings of the progeny broods

were retained as vouchers in The Natural History Museum,

London. In collections from sites 4 to 8, made during routine

Fig. 1. Outline map of Thailand and part of Southeast Asia showing the 33 mosquito

the epidemiological and entomological study area in northwestern Thailand.

collections in our ecological and epidemiological study, the

mosquitoes were identified to the group level only and

subsequently sequenced or tested using the identification assay

developed herein. Mosquitoes from other localities in Thailand,

Cambodia, mainland China, Malaysia, Taiwan and Vietnam

were also sequenced to examine geographical diversity.

2.2. DNA extraction, amplification and sequencing of ITS2

DNA was extracted from whole individual mosquitoes using

a salting-out protocol (Sunnucks and Hales, 1996). One

microlitre of DNA (equivalent to 1/800 of a mosquito) was used

in each 50 ml PCR reaction. The rDNA ITS2 was amplified

using primers 5.8F (50-TGTGAACTGCAGGACACATG-30)and 28R (50-ATGCTTAAATTTAGGGGGTA-30) (Collins and

Paskewitz, 1996). The concentrations of the reactants were:

0.2 mM of each primer, 200 mM dNTP, 2.5 mM MgCl2, 20 mM

(NH4)2SO4, 75 mM Tris–HCl (pH 8.8) and 0.01% (w/v)

‘Tween’. One unit of Thermoprime Plus DNA Polymerase

(ABgene, Epsom, UK) was used per reaction. The samples

were heated at 94 8C for 5 min before 35 cycles of amplification

at 94 8C for 1 min, 61 8C for 30 s and 72 8C for 30 s followed by

a final extension step of 5 min. The amplification products were

purified on columns and sequenced using the PCR primers and

fluorescent chemistry (Applied Biosystems, Warrington, UK).

Sequences were aligned and checked manually in SeqEdit

(version 1.0.3) (Applied Biosystems, Warrington, UK). Most of

the sequencing was done in both directions, including that for

all reference specimens, all specimens of An. pseudowillmori,

chromosomal form K and Anopheles dravidicus from Thailand,

and at least two specimens from each sampling site. The

remainder were confirmed to be one of the established

sequences by comparison of the ITS2 sequence generated by

sequencing in a single direction, but if there was any ambiguity

they were then sequenced in both directions.

collection sites listed in Table 2. The box indicates the approximate coverage of

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–10296

Table 2

Number of specimens of each species sequenced for ITS2 from each site

Site no. Date (month/year) Collection sites (country, province, village/town) Species

MAC SAW PSEU DRAV K

1 November 2000 Thailand, Mae Hong Son, Ban Mae Top Nua 10 6 3 3 –

2 November 2000 Thailand, Lampang, Ban Den Udom – 8 – – –

3 April 2001 Thailand, Chiang Mai, Doi Inthanon 6 – – – –

4 June 2001–December 2002 Thailand, Mae Hong Son, Ban Nong Khao Klang 2 – – – –

5 June 2001–December 2002 Thailand, Mae Hong Son, Ban Huai Pong Khan Nai – 2 – – –

6 June 2001–December 2002 Thailand, Mae Hong Son, Huai Chang Kham 3 8 – 1 –

7 June 2001–December 2002 Thailand, Chiang Mai, Ban Huai Ngu 2 1 – – –

8 June 2001–December 2002 Thailand, Lamphun, Ban Pang – 8 – – –

9 October 1996 Thailand, Loei, Ban Pa Kow Lam 4 3 – – –

10 October 1996 Thailand, Sakhon Nakhon, Ban Kok Klang 1 – – – –

11 October 1996–December 2004 Thailand, Ubon Ratchathani, Na Chaluai and Kang

Ka Lao National Park

– – – – 3

12 July 2001 Thailand, Kanchanaburi, Ban Phu Toei 3 – – – –

13 July 2001 Thailand, Prachaup Khiri Khan, Huey Rae 1 1 – – –

14 July 2001 Thailand, Prachaup Khiri Khan, Palau-U 3 2 – –

15 July 2001 Thailand, Chumphon, Ban Noi Chok Kwa 5 – – –

16 July 2001 Thailand, Ranong, Ban Hing Chang 6 – – – –

17 July 2001 Thailand, Song Khla, Pedang Besar 5 – – – –

18 August 2003 Malaysia, Terengganu, Kampungs Jenagor, Basong,

Payah Kayu and Dura

11 – – – –

19 August 2003 Malaysia, Pahang, Sungai Beruas 4 – – – –

20 August 2003 Malaysia, Johor, Kota Tinggi 3 – – – –

21 August 2001 China, Guangxi, Pubei, Chengguan 13 – – – –

22 August 2001 China, Guangdong, Huidong, Daling 14 – – – –

23 September 2000 China, Taiwan, Taitung 8 – – – –

24 June 2001 China, Hainan, Changjiang, Shilu – 1 – – –

25 June 2003 Vietnam, Lang Son, Trang Dinh, Chi Minh 1 – 1 – –

26 July 2000 Vietnam, Ninh Binh, Cucphuong National Forest 5 – – – –

27 May 2004 Vietnam, Nghe An, Thanh Chuong, Thanh Lam 1 – – – –

28 2003 Vietnam, Quang Binh, Le Thuy, Ngan Thuy 3 – – 3

29 June 2004 Vietnam, Quang Ninh, Ba Che, Thanh Lam 5 – – – –

30 October 2003 Cambodia, Preah Vihear, Ror Vieng, Romeny – – – – 3

31 June 2003 Cambodia, Ratanakiri, Ochum, Chaongchan 6 1 – – –

32 October 2003 Cambodia, Pailin, Pang Rolim – 1 – – –

33 June 2003 Cambodia, Pursat, Dey Kra Hom – 3 – – –

Total per species 122 48 4 4 9

The morphologically identified specimens came from sites 1 and 2 and cytologically identified specimens of form K were collected at site 11. Other specimens were

identified from field collections as belonging to the An. maculatus group and identified to species based on ITS2 sequences. Collection sites were at or near the

localities indicated and their approximate locations are shown in Fig. 1. MAC, An. maculatus; SAW, An. sawadwongporni; PSEU, An. pseudowillmori; DRAV, An.

dravidicus; K, form K (see text).

2.3. Sequence alignment and phylogenetic analysis

DNA sequences were aligned using Clustal W version 1.7

(Thompson et al., 1994). Phylogenetic relationships were

inferred using maximum-likelihood (ML), maximum parsi-

mony and neighbour-joining methods in PHYLIP version 3.5c

Table 3

Primers used in the multiplex PCR and expected sizes of the fragments amplified

Primer Sequence Lengt

5.8F 50-ATCACTCGGCTCGTGGATCG-30 20

MAC 50-GACGGTCAGTCTGGTAAAGT-30 20

PSEU 50-GCCCCCGGGTGTCAAACAG-30 19

SAW 50-ACGGTCCCGCATCAGGTGC-30 19

K 50-TTCATCGCTCGCCCTTACAA-30 20

DRAV 50-GCCTACTTTGAGCGAGACCA-30 20

(Felsenstein, 1989). Kimura two-parameter distances with a

transition/transversion ratio of two were used for tree

construction with the neighbour-joining method. The model

for the ML method used one category of substitution rates,

empirical base-frequencies and an expected transition/transver-

sion ratio of 2. The global search option was also used in ML

from each species

h (bp) Species Fragment size (bp)

Universal forward

An. maculatus 180

An. pseudowillmori 203

An. sawadwongporni 242

Form K 301

An. dravidicus 477

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–102 97

tree construction to further explore alternative tree topologies.

Bootstrap supports were based on 1000 re-sampled datasets

using SEQBOOT in PHYLIP. Trees were visualised using

TREEVIEW (Page, 2001).

2.4. PCR conditions for the identification method

Reactants: 0.2 mM of primers 5.8F, MAC, DRAV, K and

0.1 mM of primers SAW and PSEU (primers defined in

Table 3); 2.5 mM MgCl2; 200 mM dNTP; 20 mM (NH4)2SO4,

75 mM Tris–HCl (pH 8.8) and 0.01% (w/v) ‘Tween’; 0.5 units

of Thermoprime Plus DNA Polymerase (ABgene, Epsom, UK);

and 1 ml of genomic DNA sample in a total reaction volume of

25 ml. The samples were heated at 94 8C for 5 min before 35

cycles of amplification at 94 8C for 1 min, 61 8C for 30 s and

72 8C for 30 s followed by a final extension step of 5 min.

3. Results

Adult and larval mosquitoes from sites 1 to 3 in northwestern

Thailand (Fig. 1, Table 2) were identified morphologically to

obtain a set of reference specimens for the study area. Four

species of the An. maculatus group were identified: An.

maculatus, An. sawadwongporni, An. dravidicus and An.

pseudowillmori. Despite numerous entomological surveys, An.

notanandai has not been recorded from this region of Thailand,

so it is possible that it does not occur there. Although An. willmori

has been recorded in northwestern Thailand, it is associated with

higher altitudes and is therefore unlikely to occur in the majority

of entomological and epidemiological surveys.

The reference specimens from sites 1 to 3 (16 An. maculatus,

14 An. sawadwongporni, 3 An. pseudowillmori and 3 An.

dravidicus (Table 2, Fig. 1)) were sequenced for ITS2. The

sequences for the four species are quite distinct from each other

and no intraspecific variation was found (Fig. 2). To further

establish that only these species were present at the field sites in

northwestern Thailand, the ITS2 sequences of 27 additional

specimens from sites 4 to 8 (Table 2, Fig. 1) were also obtained.

Each was found to have a sequence identical to one of the four

reference sequences.

To assess the level of geographical variation, another 146

mosquitoes, collected from other areas within Thailand and

from other countries in Southeast Asia, were sequenced for

ITS2 (Table 2). Despite the wide geographical range covered,

no intraspecific variation was found. In the case of An.

maculatus, this involved specimens from northern and southern

Thailand, Cambodia, China (Guangxi, Guangdong and

Taiwan), Malaysia and Vietnam, and for An. sawadwongporni

specimens were from mainland China, Cambodia, Vietnam and

northern and southern Thailand. However, three specimens that

were identified as chromosomal form K from eastern Thailand

(site 11, Table 2 and Fig. 1) had a quite distinct ITS2 sequence

(sequence K in Fig. 2) differing by 14 base substitutions and the

length of an indel from the next most closely related sequence,

of An. sawadwongporni (Fig. 2). This sequence was also

obtained from three specimens from Cambodia (site 30) and

three from Vietnam (site 28).

Comparisons were made between the species-specific ITS2

sequence found in this study and ITS2 sequences of the same

species from mainland China and Malaysia that were available

from GenBank. Several bases (�20) from the beginning and

end of the database sequences were excluded in these

comparisons as the multiple differences observed in these

regions, in comparisons with our sequences, are most likely due

to sequencing errors in the database sequences (as the region

near to the primer can be difficult to read), or possibly due to the

inclusion of primer sequences with the submitted sequence. On

this basis, ITS2 sequences from An. maculatus (AF261950),

An. sawadwongporni (AF512551) and An. dravidicus

(AF261951) from mainland China were identical to those

found in our study. The ITS2 sequence of An. pseudowillmori

(AF512550), however, differed at seven sites from the sequence

of Thai specimens. A second (partial) sequence from An.

pseudowillmori (AF261952) was more similar to the sequence

of Thai specimens although it still differed by at least one base.

(This sequence was not included in the phylogenetic analysis

because it was incomplete.) The two database sequences of An.

maculatus from Malaysia (AF500072 and AF500073) both

differed at three bases (32, 150 and 240, Fig. 2) from the

sequence of this species that we obtained from Thai, Chinese,

Malaysian and Cambodian mosquitoes. AF500072 (MAC

Malay1) differed at another two bases and AF500073 (MAC

Malay2) differed at another six bases from our sequence of An.

maculatus.

A molecular phylogeny (Fig. 3) was constructed using all the

unique ITS2 sequences of members of the An. maculatus group

available from this study and from GenBank, using the

alignment in Fig. 2. The gene tree in Fig. 3 was constructed

using maximum-likelihood and is unrooted because an

outgroup with easily aligned ITS2 is not available. ML and

maximum parsimony methods of tree construction resulted in

the same tree topology. However, the relative positions of An.

willmori, An. dravidicus, An. dispar and An. greeni were altered

when the neighbour-joining method was used, although the

latter two species still clustered with An. maculatus. There is

low bootstrap support (�56%) for the deeper branching events.

Nevertheless, Fig. 3 illustrates clearly that the specimens of

chromosomal form K are most closely related to An.

sawadwongporni, yet show a level of sequence divergence

comparable to that between other species of the group. These

sequences have been deposited in GenBank (accession numbers

DQ518615–DQ518629).

The identification method is based on the principle of allele-

specific amplification in which Thermus aquaticus (Taq) DNA

polymerase is unable to extend primers that are mismatched to

their template DNA (Ugozzoli and Wallace, 1991; Scott et al.,

1993). The alignment of the sequences from each species

(Fig. 2) was used to design species-specific amplification

primers with a reverse orientation (Table 3, Fig. 2). A single

universal primer (5.8F) binds to the 5.8S gene in all species in

the forward orientation (Collins and Paskewitz, 1996). The

reaction conditions were optimised with respect to annealing

temperature, magnesium concentration, primer concentration

and polymerase concentration to ensure that each species-

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–10298

Fig. 2. Alignment of ITS2 sequences of members of the An. maculatus group collected and sequenced in this study: An. maculatus (MAC), An. sawadwongporni

(SAW), An. pseudowillmori (PSEU), An. dravidicus (DRAV) and chromosomal form K (K); together with differing sequences obtained from GenBank: An.

pseudowillmori (PSEU China; accession number: AF512550) and An. willmori from China (WILL; AF512552), An. dispar (DISPAR; AF234778) and An. greeni

(GREENI; AF234779) from the Philippines; and two sequences of An. maculatus, one from Jeram Kedah, Negeri Sembilan, central peninsular Malaysia (MAC

Malay1; AF500072) and the other from Johore in southern peninsular Malaysia (MAC Malay2; AF500073). Dots indicate identity with the reference sequence from

An. maculatus and a dash denotes a deletion with respect to the other sequences. Boxes indicate the binding sites of the species-specific primers (Table 2).

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–102 99

Fig. 3. Unrooted phylogenetic tree constructed using maximum-likelihood,

showing bootstrap values �50%. (Asterisk (*) indicates a partial sequence of

An. pseudowillmori from China, AF261952 only differs by a single base

insertion from sequences of Thai and Vietnamese specimens.)

specific primer (in combination with the universal primer) only

amplifies DNA from the corresponding species and there is no

cross-amplification with any of the other primers. The

optimised reaction conditions for the identification method

are given in Section 2. Inclusion of all the primers in a single

PCR reaction with DNA from one of the five species generates a

PCR product of a diagnostic length (Table 3) that can be

detected by agarose gel electrophoresis (Fig. 4).

Since large number of mosquitoes need to be identified for

meaningful epidemiological and ecological field studies, we

sought to develop a quick, reliable method of processing

mosquitoes. The use of a crude extraction method (see Section

2), rather than a complex DNA extraction method that yields

Fig. 4. A 1.5% agarose gel showing the amplification products generated from

the multiplex PCR using DNA isolated from individual mosquitoes of known

species. Lane 1: 20 bp marker; lanes 2 and 15: 100 bp marker; lanes 3 and 4: An.

maculatus; lanes 5 and 6: An. pseudowillmori; lanes 7 and 8: An. sawadwong-

porni; lanes 9 and 10: chromosomal form K; lanes 11 and 12: An. dravidicus;

lane 13: no DNA control; lane 14: 50 bp marker.

DNA of high quality, was therefore investigated. Ten-fold serial

dilutions of crude DNA samples (prepared from heads) were

used in the multiplex PCR. Amplification was successful across

four orders of magnitude of sample concentration, indicating

that this simple method of DNA extraction is extremely robust.

Extracted samples have been used successfully for up to 2

weeks after preparation when stored frozen. The DNA is

unlikely to be suitable for long-term storage but the method

does enable the identification procedure to be repeated if

necessary. Legs or heads can be used for DNA extraction

enabling the remainder of the mosquito to be used for other

assays, preserved for long-term storage for other studies, or

retained as a voucher. Routinely, whole mosquitoes are boiled

for 15 min in 200 ml of water and 1 ml used in the above PCR

identification method.

4. Discussion

4.1. Species diversity

The sequencing survey and phylogenetic analysis (Fig. 3)

indicate greater species diversity in the An. maculatus group

than has been recognised formally up to now. In the general

context of low intraspecific variation, the high degree of

differentiation of the specimens of chromosomal form K from

the other species (3.7% divergence from the most closely

related species, An. sawadwongporni; Fig. 3) is strong evidence

that these belong to another species. Divergence among

members of mosquito species complexes varies but can be

substantial (e.g. �10–18% at ITS2 among members of the An.

annularis group, unpublished data). However, divergence at

ITS2 can be much lower than this even between well-

recognised species; for example, it is only 0.6% between An.

dirus and An. baimaii (Walton et al., 1999b), 1.0% between An.

maculipennis and An. daciae (Nicolescu et al., 2004), and 0.4–

1.6% among members of the An. gambiae complex (Paskewitz

et al., 1993). The species status of this chromosomal and

genetic form needs to be confirmed by observing sympatry

without heterozygotes using chromosomal or other markers.

Although it is possible that form K corresponds to An.

notanandai, it is unlikely since An. notanandai corresponds to

chromosomal form G and has previously only been reported

from eastern Thailand (Table 1, Rattanarithikul and Green,

1986). According to R. Rattanarithikul (personal communica-

tion to REH), form K is morphologically similar to, but

distinguishable from, An. notanadai, providing additional

evidence that form K represents another species of the An.

maculatus group.

There are two sequences for An. pseudowillmori from

Yunnan Province, China in the database. One sequence

(AF261952) appears to be the same as the Thai and Vietnamese

sequences generated in this study (see below). The other

sequence (AF512550) exhibits seven differences from the

sequences of Thai An. pseudowillmori even after the ends are

trimmed. It is therefore possible that these sequences

correspond to two different, yet very closely related species.

However, since it is unknown whether the Chinese specimens

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–102100

were collected from the same or different places within Yunnan,

they may merely represent geographically isolated and diffe-

rentiated populations of An. pseudowillmori.

The taxonomic status of chromosomal forms B and E of An.

maculatus remains unclear, although the apparent difference in

vectorial capacity between them makes this distinction

particularly worthwhile to clarify. Previous collection records

with associated chromosomal data indicate the northernmost

collection of chromosomal form E is from Phato, Rangong (at

latitudes of 098460N and less) (Green et al., 1985), but

according to Rongnoparut et al. (1999) this form occurs as far

north as �128N. The majority of our specimens are therefore

expected to be form B, but those from sites 13, 14, 15, 16 and 17

in peninsular Thailand, and especially those from sites 18, 19

and 20 in Malaysia, are most likely to be chromosomal form E.

Despite the likelihood that our specimens include both

chromosomal forms, only a single ITS2 sequence was detected.

The two GenBank sequences of Malaysian An. maculatus

differ by several bases from the sequences that we obtained from

specimens of An. maculatus collected in Thailand, China,

Cambodia, Vietnam, Malaysia and Vietnam. This difference

could correspond to the two chromosomal forms if the Malaysian

mosquitoes from the database are all form E and our mosquitoes

are all chromosomal form B, although, as argued above, this

seems unlikely. Rather than indicating any form of intraspecific

variation or the presence of cryptic species, the differences in the

Malaysian sequences may be due to sequencing errors. This is

supported by the fact that the sequencing we report here was

carried out independently in two laboratories without any

conflict. Furthermore, errors in GenBank sequences have been

noted previously (Linton et al., 2002). It is clearly important to

gather more sequence data (from more variable loci), ideally

coupled with chromosomal data and epidemiological data, from

An. maculatus in Malaysia.

4.2. Phylogenetic relationships

Fig. 3 shows clearly that the putative species corresponding to

chromosomal form K is the sister of An. sawadwongporni.

Ongoing studies have shown that the adults of both taxa have

overlapping morphological characters, but their eggs are clearly

distinct. Moreover, crossing experiments revealed post-zygotic

isolation between chromosomal form K and both An. maculatus

(form B) and An. sawadwongporni (P. Somboon, unpublished

data). It can also be seen that the two genetic forms of An.

pseudowillmori are substantially divergent from other members

of the An. maculatus group. However, the low bootstrap support

(�56%) for the deeper branching events in Fig. 3, illustrates that

the rapidly evolving ITS2 locus is unable to resolve the deeper

relationships. A full understanding of the phylogenetic relation-

ships within the group will require the analysis of other loci, such

as more conserved regions of the rDNA or mtDNA genes.

4.3. Intraspecific diversity

Despite considerable geographic sampling, no intraspecific

variation was found in ITS2 in the 187 specimens that we

sequenced. (This assumes that we have correctly interpreted the

level of divergence observed between putative species K and

other members of the group as representing interspecific

divergence rather than intraspecific variation.) The only

indication of intraspecific variation came from comparisons

made with sequences obtained from sequence databases. The

difference of a one base insertion in An. pseudowillmori from

China (AF261952) relative to the sequences of Thai specimens

could be due to sequencing error since the other three species in

Thailand and China (An. sawadwongporni, An. maculatus and

An. dravidicus) were identical to each other (once starting and

ending sequences are edited from the Chinese sequences). Given

that we found no variation in An. maculatus from Thailand,

China, Cambodia, Vietnam and Malaysia, the variation observed

between the sequences of the two Malaysian specimens is

unexpectedly high. It would be helpful if this locus could be

sequenced from more individuals from countries neighbouring

Thailand, particularly China and Malaysia, to establish the extent

of any intraspecific variation within members of the group.

The lack of intraspecific variation that is generally observed

over a large geographic area could imply sufficient gene flow to

allow homogenisation of sequences of the rDNA genes by

concerted evolution (Elder and Turner, 1995). Alternatively, it

could reflect a demographic history of these populations in

which they have been derived sufficiently recently to have

prevented diversification from the ancestral sequence. The very

low level of population structure detected in An. maculatus in

Thailand using microsatellites (Rongnoparut et al., 1999) is

consistent with both of these hypotheses.

4.4. Applicability of the identification method

When the molecular identification method was applied to

240 adult mosquitoes of the An. maculatus group from our field

study in northwestern Thailand, it was able to amplify>94% of

the specimens, with the unidentified specimens being attributed

to degraded DNA due to poor preservation. An. maculatus and

An. sawadwongporni are the most abundant species (51.7% and

42.1% of the total, respectively), correlating with their

widespread distribution indicated in Table 2. An. dravidicus

and An. pseudowillmori were rarely encountered (2.1% and

4.2% of the total, respectively), but this could, in part, be due to

differences in feeding preferences as all specimens were

captured on human bait. Chromosomal form K was never

encountered, which together with its incidence at sites 11, 28

and 30, suggests that it has an eastern distribution in mainland

Southeast Asia.

The general lack of intraspecific variation in ITS2 sequence

makes the identification method likely to be very useful over a

large geographic area—apparently at least in Malaysia, most of

Thailand, parts of China, and most probably Cambodia,

Vietnam and Taiwan. However, the method will not distinguish

the two sequences of ITS2 obtained from An. pseudowillmori in

China. The inclusion of chromosomal form K in the

identification method will help to extend its usefulness to

eastern Thailand, and to Vietnam and Cambodia where this

form also occurs. In some areas it will be necessary to adapt the

C. Walton et al. / Infection, Genetics and Evolution 7 (2007) 93–102 101

method to include the identification of additional species, for

example, An. willmori in high altitude areas and An. notanandai

in west-central Thailand. The sequence alignment (Fig. 2)

illustrates that there appears to be ample variability between

species, and the region is sufficiently lengthy to enable primers

to be designed for additional species. Before the method is

deployed in new areas, it is clearly advisable to assess which

species are present and the extent of intraspecific variation by

sequencing the ITS2 region of specimens from the area.

In conclusion, the identification method presented here is

likely to work over a large geographic area with scope to

modify it to include additional species. Furthermore, it is very

robust to the use of a simple and rapid DNA extraction method

and to the concentration of DNA used. For this reason, and the

fact that the method requires only a single step, a PCR reaction,

before running the samples on an agarose gel, the method is an

eminently practical tool for large-scale field-based studies

where reliable species identification is important.

Acknowledgements

We thank the many people who contributed to the mosquito

collections for this study, including: Dr. Samsak Prajakwong,

former director of the Office of Vector-Borne Disease Control

No. 2 in Chiang Mai, Thailand, and his staff, particularly Mr.

Raksakul Kantawong, for their assistance in collection and

identification of mosquito specimens in Thailand; Conor Cahill

and Mark Isenstadt for assistance with collections in north-

western Thailand; Dr. Hwa-Jen Teng, Division of Vector-borne

Infectious Diseases, Taiwan, and Prof. Masahiro Takagi and Dr.

Yoshio Tsuda, Institute of Tropical Medicine, Nagasaki

University, Japan, for organizing the collection of specimens

in Taiwan; Dr. Ngyuen Duc Manh and staff of the National

Institute of Malariology, Parasitology and Entomology, Hanoi,

Vietnam, for collecting and providing specimens from

Vietnam; Dr. Tho Sochanta and other staff of the National

Center for Malaria, Parasitology and Entomology, Cambodia,

for their assistance in the collection and identification of

mosquitoes in Cambodia. This work was part of the

RISKMODEL and MALVECASIA projects funded by the

European Union, grant numbers QLK2-CT-2000-01787 and

ERBIC 18CT970211, respectively.

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