- Email: [email protected]
Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago
Acta Tropica 199 (2019) 105124
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago
T
Rusdiyah Sudirman Made Alia,b, Isra Wahida, Jassada Saingamsookb, Atiporn Saeungb, ⁎ Anchalee Wannasanb, Catherine Waltonc, Ralph E. Harbachd, Pradya Somboonb, a
Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand c School of Earth and Environment, Faculty of Science and Engineering, University of Manchester, Manchester M13 9PT, UK d Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Indonesia ITS2 D3 COII Anopheles maculatus Genetics Taxonomy
This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2–2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.
1. Introduction The Indonesian Archipelago consists of over 13,000 islands lying between latitudes 6° N and 11° S and between longitudes 95° and 141° E, extending ∼1760 km north to south and 5120 km east to west. The major islands include Borneo (comprising Kalimantan of Indonesia, Brunei and Malaysia), Java, Sulawesi, Sumatra and New Guinea (comprising Western New Guinea of Indonesia and Papua New Guinea). Due to size, tropical climate and archipelagic geography, Indonesia is well known to have a high diversity of plants and animals, including mosquitoes (Myers et al., 2000). Anopheles mosquitoes have been extensively studied because of their roles in the transmission of pathogens that cause diseases in humans, especially malaria. In 2017, 261,617 malaria cases were reported across the archipelago (WHO, 2018). Plasmodium falciparum and P. vivax are the common human malarial parasites. Plasmodium knowlesi, a primate malarial parasite, also causes disease in humans on some islands (Elyazar et al., 2013). The Ministry of Health of Indonesia has implemented malaria control, aiming for
⁎
elimination by 2030 (Sitohang et al., 2018). Therefore, knowledge of malaria epidemiology and vector species is essential for the development of effective control strategies. However, knowledge of the mosquito fauna of Indonesia is far from complete. About 80 Anopheles species are recorded from Indonesia, 21 of which are regarded as primary and secondary vectors of human malarial parasites (Bonne-Wepster, 1953; Elyazar et al., 2013; O’Connor and Sopa, 1981; Sugiarto et al., 2017). Anopheles maculatus Theobald is regarded as one of the important vectors in Java and southern Sumatra, but it plays little or no role in Plasmodium transmission on other islands due to low vectorial capacity (Barcus et al., 2002; Elyazar et al., 2013). Whether differences in vectorial capacity are related to genetic distinction is not known. Other important malaria vector species in Indonesia include An. aconitus Dönitz, An. balabacensis Baisas, An. flavirostris (Ludlow), An. kochi Dönitz, An. leucosphyrus Dönitz, An. nigerrimus Giles, An. vagus Dönitz and members of the An. barbirostris, An. subpictus and An. sundaicus complexes. Like An. maculatus, their distributions and involvement in the transmission of malarial parasites varies across the
Corresponding author. E-mail address: [email protected] (P. Somboon).
https://doi.org/10.1016/j.actatropica.2019.105124 Received 7 May 2019; Received in revised form 1 August 2019; Accepted 2 August 2019 Available online 05 August 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Morphological features of adult mosquitoes were examined under a stereomicroscope. The morphological keys of Rattanarithikul and Green (1986); Rattanarithikul and Harbach (1990) and Rattanarithikul et al. (2006) were used to confirm the identification of species. The morphological terminology and abbreviations follow the Anatomical Glossary of the online Mosquito Taxonomic Inventory (http://mosquitotaxonomic-inventory.info/anatomical glossary-overview).
archipelago (Elyazar et al., 2013). The taxonomic status of An. maculatus across the Indonesian Archipelago is not clearly known and it has been speculated that it may consist of more than one species (Bangs et al., 2002; Elyazar et al., 2013; Rattanarithikul and Harbach, 1990). This is because previous reports regarding An. maculatus in Indonesia are based mainly on morphological identifications that do not unambiguously separate it from other members in the Maculatus Group, which consists of nine formally recognized species: An. dispar Rattanarithikul & Harbach, An. dravidicus Christophers, An. greeni Rattanarithikul & Harbach, An. maculatus, An. notanandai Rattanarithikul & Green, An. pseudowillmori (Theobald), An. rampae Harbach & Somboon, An. sawadwongporni Rattanarithikul & Green and An. willmori (James) (Harbach, 2019). Genetic markers in both mitochondrial DNA (mtDNA) (such as cytochrome c oxidase subunits I and II, COI and COII) and nuclear ribosomal DNA (rDNA) (such as the internal transcribed spacer 2, ITS2, and the D3 region of the 28S ribosomal gene) have been widely used to identify closely related and isomorphic species of mosquito complexes, including the Maculatus Group (Torres et al., 2000; Ma et al., 2006; Walton et al., 2007; Singh et al., 2012; Sum et al., 2014). Recent studies provide clear evidence for the existence of a new species within An. maculatus s.l. in the Indonesian Archipelago (Ali et al., 2019; Garjito et al., 2019). For this reason, we extended our studies to include phylogenetic analyses of DNA sequence data obtained from specimens of An. maculatus s.l. collected in Borneo (Kalimantan), Java, Sulawesi, Sumatra and the Sumba islands.
2.2. DNA extraction, amplification and sequencing Genomic DNA was extracted from fore- and midlegs of individual mosquitoes using the Pure Link™ Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, USA) according to manufacturer’s instructions. The specimens were retained for morphological examination. The ribosomal ITS2 and D3 regions and the mitochondrial COII gene were sequenced in the present study. The ITS2 region was amplified by PCR using the primers 5.8 F (5TGTGAACTGCAGGACACATG-3) and 28R (5-ATGCTTAAATTTAGGGG GTA-3) (Walton et al., 2007). The 28S-D3 domain of the 28S gene was amplified using the primers D3A (5-GACCCGTCTTGAAACACACGGA-3) and D3B (5-TCGGAAGGAACCAGCTACTA-3) (Schmidt et al., 2006). PCR reactions for both regions were carried out in a 20 μl volume containing 1 μl (5 ng) of DNA, 0.8 μM of each primer, 1.2 mM MgCl2, 1.6 mM dNTP mix and 0.08 U of Platinum®Taq DNA polymerase in 1x PCR buffer. Thermal cycling conditions were initial denaturation at 95 °C for 2 min, 35 cycles at 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The COII gene was amplified using the primers SCTL2-J-3037 (5ATGGCAGATTAGTGCAATGA-3) and TK-N-3785 (5-GTTTAAGAGACC AGTACTTG-3) (Liu and Beckenbach, 1992). PCR reactions were carried out, as described by Ali et al. (2019), in a 20 μL volume containing 0.4 U of Platinum®Taq DNA polymerase, 1X of PCR buffer, 1.5–3.0 mM of MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer and 1 μL (5 ng) of extracted DNA. The amplification profile comprised initial denaturation at 95 °C for 2 min, 35 cycles at 95 °C for 30 s, 45 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Amplified products were electrophoresed in 2% agarose gels and stained with ethidium bromide. PCR products were purified using the illustra™ ExoProStar™ 1-Step (GE Healthcare Life Sciences, UK) and sequenced using the BigDye® Terminator v3.1 cycle sequencing kit chemistry (First BASE, Malaysia).
2. Materials and methods 2.1. Mosquito collection and morphological identification Since very few An. maculatus s.l. adults in our study areas were collected from human bait, we captured adult mosquito specimens resting in cowsheds by aspirator at eight localities on five islands: 1) Sabang, Aceh Special Region, off the northern tip of Sumatra; 2) Hargowilis, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province (previously collected by Ali et al., 2019); 3) Hargotirto, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 4) Sumba Island, East Nusa Tenggara Province, Lesser Sunda Islands; 5) Pucak, South Sulawesi Province, Sulawesi; 6) Labuan, Central Sulawesi Province; 7) Wulai, West Sulawesi Province; and 8) North Kalimantan Province, Borneo (Fig. 1, Table 1). The mosquitoes were killed with chloroform vapor and preserved in vials with silica gel.
Fig. 1. Map of the Indonesian Archipelago and collecting sites: 1) Sabang, Aceh Special Region, off the northern tip of Sumatra; 2) Hargotirto, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 3) Hargowilis, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 4) Sumba Island, East Nusa Tenggara Province, Lesser Sunda Islands; 5) Pucak, South Sulawesi Province, Sulawesi; 6) Labuan, Central Sulawesi Province; 7) Wulai, West Sulawesi Province; and 8) North Kalimantan Province, Borneo. 2
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Table 1 Details of collections of An. maculatus s.l. made in the Indonesian Archipelago. No.
Province
Location
Coordinates
Altitude (m)
No. specimens collected
Date of collection
1 2 3 4
Sumatra Central Java Central Java East Nusa Tenggara
Sabang Hargotirto Hargowilis Sumba
5°49′59.7″N 95°19′01.6″E 7°47′37.9″S 110°05′40.8″E 7°49′43.2″S 110°06′32.1″E 9°26′52.7″S 119°15′40.2″E
107 388 199 468
5 6 7 8
South Sulawesi West Sulawesi Central Sulawesi North Kalimantan
Pucak Wulai Labuan SebatikTengah
5°09′29.2″S 119°42′01.3″E 1°02′42.9″S 119°31′27.5″E 0°38′28.9″S 119°51′08.5″E 4°09′20.4″N 117°47′04.9″E
113 42 109 53
19 10 8* 6 13 20 10 7 5
Aug 2017 Dec 2018 Jun 2017 Jul 2017 Nov 2018 Jan–May 2017 Feb–Mar 2018 Nov 2017 Dec 2018
* Previously collected by Ali et al. (2019).
Due to limited budget, two to five specimens of An. maculatus s.l. from each site were selected at random for sequencing: Sabang (n = 3), Hargowilis (3) and Hargotirto (4) of Central Java; Sumba Island (5); Pucak (2), Labuan (2) and Wulai (2) of Sulawesi; and Sebatik Tengah (4) of North Kalimantan. The specimen from North Kalimantan (Sebatik-3) was also sequenced. The sequences are deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accession numbers, for ITS2: MK504453–MK504475; for D3: MK518025–MK518047; for COII: MK236369–MK236371 and MK507483–MK507501.
2.3. Sequencing alignment and phylogenetic analysis The sequences obtained during this study were compared with those of the Salatiga Lab strain of An. maculatus s.l. (GenBank accession numbers for ITS2: MK659773, MK67565 and MK675654), wild-caught females from Hargowilis, Central Java Province (Java-1,3,5) previously described by Ali et al. (2019) (GenBank accession numbers for ITS2: MK204645–MK204647; COII: MK236369–MK236371) and other members of the Maculatus Group in GenBank using the Basic Local Alignment Search Tool (BLAST, available at http://blast.ncbi.nlm.nih. gov/Blast.cgi) under default parameters (max High-Scoring Segment Pairs 250, expect threshold 10, word size 28, optimized for highly similar hits, not specific to any organism). Anopheles karwari (James) was used as the outgroup taxon because it is not a member of the Maculatus Group but is classified along with An. maculatus s.l. as a member of the Neocellia Series. Consensus sequences of ITS2, D3 and COII were aligned using CLUSTALW under default parameters (Larkin et al., 2007), and ragged ends were trimmed using MEGA version 10 (Kumar et al., 2018). The most appropriate model of nucleotide substitution was determined for each dataset using jModelTest version 2.1.10 (Guindon and Gascuel, 2003; Darriba et al., 2012), which was GTR for ITS2 and D3 and GTR + I+G for COII (Tavare, 1986). The phylogenetic analyses were conducted using Maximum Likelihood (ML) in MEGA version 10 (Kumar et al., 2018) and Bayesian inference (BI) with MrBayes version 3.2.7 (Ronquist et al., 2012). For ML, robustness of the tree was tested with 1000 bootstrapped data set with bootstraps support values shown on each node. For BI, the Markov chain Monte Carlo (MCMC) simulation was run for one million generations (which resulted in an average standard deviation of split frequencies below 0.01), using three heated chains and one cold chain, and sampling every 100 generations with a burnin of 25%. Two independent analyses were conducted to confirm convergence. A 50% majority rule consensus tree was created from the remaining trees. The BI trees were generated using Figtree software version 1.4.4 (http:// tree.bio.ed.ac.uk/software/figtree/). Bayesian support values of 95% (0.95) and Maximum Likelihood Bootstrap values of 70% were taken as being highly supportive of a node (Hillis and Bull, 1993).
3.1. ITS2 The length of ITS2 ranged from 367 to 370 bp (supplementary Fig. S1). Sequences of An. maculatus s.s. from GenBank were identical to each other across a wide geographic range with the exception of the sequence from Malaysia (AY491974.1) that differed at two bases (19 and 205). This may indicate intraspecific variation but it is hard to exclude sequencing error as the explanation of deviation for this one sequence. The sequences of specimens from Hargotirto (JavaHT13–16), Sabang, Sumba and North Kalimantan were identical to most An. maculatus s.s. from mainland Asia. Their sequences were very distinct from those from Sulawesi, Hargowilis (Java-HW1,3,5) and the Salatiga Lab strain, which were almost identical, except for Sulawesi differing at one base (C↔T, position 336). The sequences from Sulawesi and Hargowilis differed from An. maculatus s.s. by 20 and 21 fixed sites, respectively. 3.2. D3 The length of D3 ranged from 304 to 312 bp (supplementary Fig. S2). The sequences of specimens from Hargotirto (Java-HT13–16), Sabang, Sumba and North Kalimantan were identical to An. maculatus s.s. from mainland Asia. Like the ITS2 sequences, their D3 sequences were very distinct from those from Sulawesi and Hargowilis (JavaHW1,3,5), differing by 12 and 11 fixed sites, respectively. Specimens from Sulawesi differed from those from Hargowilis at one base (C↔G, position 150).
3. Results 3.3. COII A total of 98 females of An. maculatus s.l. were collected from the eight localities (Fig. 1, Table 1). Morphologically, all females resembled An. maculatus s.s., in particular in having abdominal terga II–IV without scales; terga V and VI without scales or with sparse pale falcate scales on the posterior margins; terga VII and VIII largely or posteriorly covered with narrow pale spatulate scales; and the posterolateral corners of terga VI–VIII, and rarely tergum V, bearing patches of dark scales. The accessory sector pale spot on the wings is confined to vein R1 and is never extended onto the subcosta and costa, except one female from North Kalimantan Province (Sebatik-3), which is a character of the two Philippine species of the Maculatus Group, An. dispar and An. greeni.
The length of COII sequences of all specimens was 576 bp (supplementary Fig. S3). The COII sequences of An. maculatus s.s. from several countries in mainland Asia were identical, except one variable site at base 82 (A↔G). They were distinct from those from Hargotirto (JavaHT13–16), Sabang, Sumba and North Kalimantan by 7, 12, 8 and 9 fixed sites, respectively (supplementary Fig. S3). The COII sequences of specimens from Sumba and North Kalimantan were similar without any different fixed site, indicating that they are conspecific. Specimens from Hargowilis and Sulawesi differed from each other by 19 fixed sites; they differed from An. maculatus s.s. by 9 and 20 fixed sites, respectively. 3
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 2. Maximum Likelihood tree of ITS2 sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Bootstrap values are shown at each node.
Asia (BS = 100%, PP = 1.0). Clade II of An. maculatus included specimens from Hargowilis (Java-HW1,3,5), the Salatiga Lab strain and Sulawesi (BS = 99%, PP = 1.0). The clades consisting of other members of the Maculatus Group were clearly distinct. The only specimen from North Kalimantan (Sebatik-3) corresponds to the Philippine species An. greeni. The ML and BI analyses of the COII sequences obtained from the Indonesian specimens also yielded a similar tree topology to each other (Figs. 4 and 5). Unlike the ITS2 trees, however, within An. maculatus from Indonesia the tree consisted of five clades with highly supportive values particularly by BI, two comprised of specimens from Central Java (Clades I, IV), one of specimens from Sumba and North Kalimantan (Clade II), one of specimens from Sabang (Clade III), and one of specimens from Sulawesi (Clade V). Clade V, that comprised sequences of specimens from the three localities in Sulawesi, was genetically distinct from Clades I-IV that comprised the other Indonesian specimens (BS = 64%, PP = 1.0). Although the mosquitoes in Clades I-III
The ITS2, D3 and COII sequences of other members of the Maculatus Group were very distinct from those from the Indonesian Archipelago. Interestingly, however, the only specimen from North Kalimantan (Sebatik-3), which has an accessory sector pale spot extending onto the costa, had sequences corresponding to the Philippine An. greeni for all three loci (supplementary Figs. S1-S3).
3.4. Phylogenetic analyses The ML and BI analyses of the ITS2 sequences yielded a similar tree topology, showing only two distinct clades within the Indonesian specimens with strong support by BI and moderate support by ML (ML bootstrap, BS value = 55%, Bayesian Posterior Probability, PP, value = 0.98) (Figs. 2 and 3). The trees of D3 sequences are similar to the ITS2 trees (figure not shown). Clade I of An. maculatus consisted of specimens from Hargotirto (Java-HT13–16), Sabang, Sumba and North Kalimantan; this clade also included An. maculatus s.s. from mainland 4
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 3. Bayesian tree of ITS2 sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Posterior probabilities are shown at each node.
the Philippine islands of Palawan and Mindanao where An. dispar and An. greeni are present in sympatry (Rattanarithikul and Harbach, 1990; Manguin et al., 2008). It is possible that the other Philippine member of the Maculatus Group, An. dispar, might also occur in Borneo. Since both species are regarded as secondary vectors of malaria in the Philippines (Manguin et al., 2008), more sequencing of specimens from various localities in Borneo is needed to shed light on the distributions of these two species. Based on the rDNA ITS2 and D3 sequence data, the Indonesian An. maculatus s.l. can be divided into two main clades that correspond to at least two species. The first group, including mosquitoes from Hargotirto of Central Java, Sabang, Sumba and North Kalimantan, have ITS2 and D3 sequences that are identical to most of those of An. maculatus s.s. in mainland Southeast Asia (Clade I, Figs. 2 and 3). This same taxon, based on ITS2 sequences, has also been reported from South Sumatra, Purbalingga and Cilacap of west of Central Java, North Kalimantan, and Belu of East Nusa Tenggara (east of Sumba Island) by Garjito et al. (2019). Unfortunately, no COII sequences were available from this
belonged to the same clade for ITS2 and D3 as An. maculatus s.s. from mainland Asia, at COII they were separated from the clade consisting of An. maculatus s.s. from mainland Asia with highly supportive values by BI (PP = 1.0) (Fig. 5). Similarly, even though they belonged to the same ITS2/D3 clade, the mosquitoes from Sulawesi were clearly distinguished from those from Hargowilis (Java-HW1,3,5) in Central Java by COII falling into Clades IV and V, respectively. 4. Discussion Previous records and reports reveal that An. maculatus s.l. is widely distributed across the Indonesian Archipelago, but not beyond Sulawesi and Timor, or Weber’s Line, a biogeographical boundary lying approximately along the Australo-Papuan Shelf (Elyazar et al., 2013). Based on the morphological distinction and the rDNA and mtDNA sequences obtained in the current study, we report for the first time the occurrence of An. greeni outside the Philippines, i.e. in Northern Kalimantan of Borneo Island. This is likely due to the proximity of Borneo to 5
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 4. Maximum Likelihood tree of COII sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Bootstrap values are shown at each node.
divergence was likely shaped predominantly by the biogeographical history of this region. The Malay Peninsula, Borneo, Java and Sumatra, and their surrounding islands, comprise the Sundaic biogeographical region (or Sundaland), that has been periodically connected as a single larger, predominantly forested landmass when the Sunda Shelf was exposed periodically throughout the past 2.6 mya when sea levels were 50–200 m lower than today (Tougard, 2001). Allopatric speciation and the distribution of species of Anopheles mosquitoes, including An. maculatus s.l., in the Indonesian Archipelago has been attributed to isolation imposed by sea barriers (Morgan et al., 2013). Within the ITS2/D3 Clade I the specimens from different geographical regions i.e. mainland Southeast Asia, north Kalimantan/ Sumba, Java and Sabang formed distinct, moderately divergent (1.2–2.1%), COII clades, consistent with their formation by periodic allopatric fragmentation on different landmasses. Whilst the possibility that these mtDNA clades correspond to distinct species cannot be excluded, the lack of divergence at ITS2/D3 provides no support for this. In contrast, there was greater differentiation within the ITS2/D3 Clade II with An. maculatus s.l. from Sulawesi differing from specimens
study for comparison with our specimens. The second group detected here included mosquitoes from Hargowilis of Central Java, the Salatiga Lab strain and Sulawesi that were not closely related to An. maculatus s.s. Based on ITS2 sequence similarity, this second group is conspecific with a species of An. maculatus recently detected from Kulon Progo of Central Java by Garjito et al. (2019). Whilst the specimens from Hargowilis belong to this newly identified species, they are distinct from those from Hargotirto (JavaHT13–16) that are more closely related to An. maculatus s.s., supporting the presence of two species in Central Java. Both Hargotirto and Hargowilis villages are situated in Kulon Progo Regency, Yogyakarta Special Region, Central Java Province. The two villages are about 4.5 km apart and are surrounded by forest, palm gardens and hills. Hargowilis is located at a lower altitude (199 m), whereas Hargotirto is located at a higher altitude (388 m). Further sampling is needed to determine if the two forms occur sympatrically, if they occupy distinct ecological niches and if they differ in vectorial capacity. The taxonomic status of specimens with distinct mtDNA lineages within the above two species is more difficult to interpret but their 6
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 5. Bayesian tree of COII sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Posterior probabilities are shown for each node.
taxonomic status at this stage. The results of the current study may not represent all sibling species that may occur in the Indonesian Archipelago because few specimens were examined from each location and many islands remain to be surveyed. At present, nine species of the Maculatus Group are formally recognised (Harbach, 2004; Harbach, 2019). However, the results of previous studies (Ali et al., 2019; Garjito et al., 2019) and this current study clearly indicate that An. maculatus is a complex of sibling species, consisting of An. maculatus s.s. from mainland Asia and at least one novel (previously unrecognized) species in Hargowilis of Central Java Province and potentially a third species in Sulawesi with the taxonomic status of distinct mtDNA lineages in Central Java Province (Hargotirto), Sabang and Kalimantan/Sumba Island undetermined. Thus, more information based on morphological study (especially the immature stages), cytogenetics and cross-mating experiments, as well as further mtDNA sequencing from additional specimens from more locations that would enable species delimitation analysis to be conducted. These would help to clarify the nature and status of these forms.
collected in Hargowilis of Central Java by one nucleotide base in each of ITS2 and D3 sequences, and their COII sequences forming highly divergent clades, differing by 19 fixed sites (3.3%). In general, sequence divergence of > 2% at COI and COII genes corresponds to variation at the interspecific level in Anopheles mosquitoes (e.g. Wang et al., 2012, 2017). On this basis, An. maculatus s.l. in Sulawesi would be inferred to be another new species distinct from that found in Hargowlis, Central Java. Speciation may be more likely to occur by allopatric isolation on Sulawesi since it is separated from the Sundaic Region by a deeper sea delineated by Wallace’s Line that likely severely limits gene flow compared to among landmasses within Sundaland. However, high mtDNA divergence coupled with low ITS2/D3 divergence between Sulawesi and Hargowilis is consistent with mtDNA introgression occurring into the ITS2/D3 Clade II species from geographically localized populations in Central Java making inference of species delimitation from mtDNA data alone particularly problematic in this case. While significant differentiation at ITS2 and D3 is a clear indication of discrete species (Walton et al., 1999b) the opposite is not necessarily true as recently diverged species can show little or no divergence at these markers; e.g. An. gambiae and An. arabiensis (Paskewitz et al., 1993) and An. dirus Peyton & Harrison and An. scanloni Sallum & Peyton of the Dirus Complex (Walton et al., 1999a) for ITS2; and species S of An. fluviatilis James and An. harrisoni Harbach & Manguin of the Fluviatilis and Minimus Complexes, respectively, for D3 (Chen et al., 2006; Singh et al., 2006). Consequently, within both of the ITS2/ D3 Clades I and II it is not possible to draw further conclusions on
Declaration of Competing Interest The authors declare that we have no conflict of Interest. Acknowledgements We thank the Faculty of Medicine, Chiang Mai University for 7
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
providing a PhD scholarship to Rusdiyah Sudirman Made Ali. This research was supported mainly by the Faculty of Medicine (PAR-256105869) and partially by the Research Administration Office of Chiang Mai University and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2015M3A9B6073666).
pp. 327–355. O’Connor, C.T., Sopa, T., 1981. A checklist of the mosquitoes of Indonesia. A special publication of the U.S naval medical research unit No.2, Jakarta, Indonesia. NAMRU SP 45, 1–26. Paskewitz, S.M., Wesson, D.M., Collins, F.H., 1993. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex. Insect Mol. Biol. 2, 247–257. Rattanarithikul, R., Green, C.A., 1986. Formal recognition of the species of the Anopheles maculatusgroup (Diptera: Culicidae) occurring in Thailand, including the descriptions of two new species and a preliminary key to females. Mosq. Syst. 18, 246–278. Rattanarithikul, R., Harbach, R.E., 1990. Anopheles maculatus (Diptera: Culicidae) from the type locality of Hong Kong and two new species of the Maculatus complex from the Philippines. Mosq. Syst. 22, 160–183. Rattanarithikul, R., Harrison, B.A., Harbach, R.E., Panthusiri, P., Coleman, R.E., 2006. Illustrated keys to the mosquitoes of Thailand. IV. Anopheles. SE Asian J. Trop. Med. Public Health 37 (Suppl 2), 1–128. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., et al., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. Singh, O.P., Chandra, D., Nanda, N., Sharma, S.K., Pe, Than Htun, Adak, T., et al., 2006. On the conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C. J. Biosci. 31, 671–677. Singh, S., Prakash, A., Yadav, R.N.S., Mohapatra, P.K., Sarma, N.P., Sarma, D.K., et al., 2012. Anopheles (Cellia) maculatus group: its spatial distribution and molecular characterization of member species in north-east India. Acta Trop. 124, 62–70. Schmidt, S., Driver, F., Barro, P.D., 2006. The phylogenetic characteristics of three different 28S rRNA gene regions in Encarsia (Insecta, Hymenoptera, Aphelinidae). Org. Divers. Evol. 6, 127–139. Sitohang, V., Sariwat, E., Fajariyani, S.B., Hwang, D., Kurnia, B., Hapsari, R.K., et al., 2018. Malaria elimination in Indonesia: halfway there. Lancet Glob. Health 6, PE604–E606. Sugiarto, H., Kesumawati, U., Soviana, S., Hakim, L., 2017. Bionomics of Anopheles (Diptera: Culicidae) in a malaria endemic region of Sungai Nyamuk village, Sebatik Island–North Kalimantan, Indonesia. Acta Trop. 171, 30–36. Sum, J.S., Lee, W.C., Amir, A., Braima, K.A., Jeffery, J., Abdul-Aziz, N.M., et al., 2014. Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA. Parasites Vectors 7, 309. Tavare, S., 1986. In: In: Miura, Robert M. (Ed.), Some Probabilistic and Statistical Problems in the Analysis of DNA Sequences, Lectures on Mathematics in the Life Sciences, vol. 17. American Mathematical Society, Providence, Rhode Island, pp. 57–86. Torres, E.P., Foley, D.H., Saul, A., 2000. Ribosomal DNA sequence markers differentiate two species of the Anopheles maculatus (Diptera: Culicidae) Complex in the Philippines. J. Med. Entomol. 37, 933–937. Tougard, C., 2001. Biogeography and migration routes of large mammal faunas in SouthEast Asia during the Late Middle Pleistocene: focus on the fossil and extant faunas from Thailand. Palaeogeogr. Palaeoclimatol. Palaeoecol. 168, 337–358. Walton, C., Sharpe, R.G., Pritchard, S.J., Thelwell, N.J., Butlin, R.K., 1999a. Molecular identification of mosquito species. Biol. J. Linn. Soc. Lond. 68, 241–256. Walton, C., Handley, J.M., Kuvangkadilok, C., Collins, F.H., Harbach, R.E., Baimai, V., et al., 1999b. Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction. Med. Vet. Entomol. 13, 24–32. Walton, C., Somboon, P., O’Loughlin, S.M., Zhang, S., Harbach, R.E., Linton, Y.-M., et al., 2007. Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA. Infect. Genet. Evol. 7, 93–102. Wang, G., Li, C., Guo, X., Xing, D., Dong, Y., Wang, Z., et al., 2012. Identifying the main mosquito species in China based on DNA barcoding. PLoS One 7, e47051. Wang, G., Li, C., Zheng, W., Song, F., Guo, X., Wu, Z., et al., 2017. An evaluation of the suitability of COI and COII gene variation for reconstructing the phylogeny of, and identifying cryptic species in, anopheline mosquitoes (Diptera Culicidae). Mitochondrial DNA Part A 28 (5), 769–777. WHO, 2018. World Malaria Report 2018. accessed 4 July 2019. https://www.who.int/ malaria/publications/world-malaria-report-2018/report/en/.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.actatropica.2019. 105124. References Ali, R.S.M., Wahid, I., Saeung, A., Wannasan, A., Harbach, R.E., Somboon, P., 2019. Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia. Parasites Vectors 12, 107. Bangs, M.J., Soelarto, T., Barodji, Wicaksana, B.P., Boewono, D.T., 2002. Colonization of Anopheles maculatus from Central Java, Indonesia. J. Am. Mosq. Control Assoc. 18, 359–363. Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Marwoto, H., Bangs, M.J., et al., 2002. Epidemic malaria in the Menoreh hills of Central Java. Am. J. Trop. Med. Hyg. 66, 287–292. Bonne-Wepster, J., 1953. In: de Bussy, J.H. (Ed.), The Anopheline mosquitoes of the IndoAustralian Region, Amsterdam. Chen, B., Butlin, R.K., Pedro, P.M., Wang, X.Z., Harbach, R.E., 2006. Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia. Med. Vet. Entomol. 20, 33–43. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. Elyazar, I.R., Sinka, M.E., Gething, P.W., Tarmidzi, S.N., Bangs, M.J., Kusriastuti, R., et al., 2013. The distribution and bionomics of anopheles malaria vector mosquitoes in Indonesia. Adv. Parasitol. 83, 173–266. Garjito, T.A., Widiastuti, U., Mujiyono, M., Prihatin, M.T., Widiarti, W., Setyaningsih, R., et al., 2019. Genetic homogeneity of Anopheles maculatus in Indonesia and origin of a novel species present in Central Java. Parasites Vectors 12, 351. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Harbach, R.E., 2004. The classification of genus Anopheles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships. Bull. Entomol. Res. 94, 537–553. Harbach, R.E., 2019. Subgenus Cellia. Mosquito Taxonomic Inventory. accessed on 4 July 2019. http://mosquito-taxonomic-inventory.info/. Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. Larkin, M.A., Blackshields, G., Brown, N., Chenna, R., McGettigan, P.A., Lopez, R., et al., 2007. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Liu, H., Beckenbach, A.T., 1992. Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects. Mol. Phylogenet. Evol. 1, 41–52. Ma, Y., Li, S., Xu, J., 2006. Molecular identification and phylogeny of the Maculatus Group of Anopheles mosquitoes (Diptera: Culicidae) based on nuclear and mitochondrial DNA sequences. Acta Trop. 99, 272–280. Manguin, S., Garros, C., Dusfour, I., Harbach, R.E., Coosemans, M., 2008. Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review. Infect. Genet. Evol. 8, 489–503. Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A., Kent, J., 2000. Biodiversity hotspots for conservation priorities. Nature 403 (6772), 853–858. Morgan, K., Somboon, P., Walton, C., 2013. Understanding Anopheles diversity in Southeast Asia and its applications for malaria control. In: Manguin, S. (Ed.), Anopheles Mosquitoes — New Insights into Malaria Vectors. InTech, Rijeka, Croatia,
8
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago
T
Rusdiyah Sudirman Made Alia,b, Isra Wahida, Jassada Saingamsookb, Atiporn Saeungb, ⁎ Anchalee Wannasanb, Catherine Waltonc, Ralph E. Harbachd, Pradya Somboonb, a
Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand c School of Earth and Environment, Faculty of Science and Engineering, University of Manchester, Manchester M13 9PT, UK d Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Indonesia ITS2 D3 COII Anopheles maculatus Genetics Taxonomy
This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2–2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.
1. Introduction The Indonesian Archipelago consists of over 13,000 islands lying between latitudes 6° N and 11° S and between longitudes 95° and 141° E, extending ∼1760 km north to south and 5120 km east to west. The major islands include Borneo (comprising Kalimantan of Indonesia, Brunei and Malaysia), Java, Sulawesi, Sumatra and New Guinea (comprising Western New Guinea of Indonesia and Papua New Guinea). Due to size, tropical climate and archipelagic geography, Indonesia is well known to have a high diversity of plants and animals, including mosquitoes (Myers et al., 2000). Anopheles mosquitoes have been extensively studied because of their roles in the transmission of pathogens that cause diseases in humans, especially malaria. In 2017, 261,617 malaria cases were reported across the archipelago (WHO, 2018). Plasmodium falciparum and P. vivax are the common human malarial parasites. Plasmodium knowlesi, a primate malarial parasite, also causes disease in humans on some islands (Elyazar et al., 2013). The Ministry of Health of Indonesia has implemented malaria control, aiming for
⁎
elimination by 2030 (Sitohang et al., 2018). Therefore, knowledge of malaria epidemiology and vector species is essential for the development of effective control strategies. However, knowledge of the mosquito fauna of Indonesia is far from complete. About 80 Anopheles species are recorded from Indonesia, 21 of which are regarded as primary and secondary vectors of human malarial parasites (Bonne-Wepster, 1953; Elyazar et al., 2013; O’Connor and Sopa, 1981; Sugiarto et al., 2017). Anopheles maculatus Theobald is regarded as one of the important vectors in Java and southern Sumatra, but it plays little or no role in Plasmodium transmission on other islands due to low vectorial capacity (Barcus et al., 2002; Elyazar et al., 2013). Whether differences in vectorial capacity are related to genetic distinction is not known. Other important malaria vector species in Indonesia include An. aconitus Dönitz, An. balabacensis Baisas, An. flavirostris (Ludlow), An. kochi Dönitz, An. leucosphyrus Dönitz, An. nigerrimus Giles, An. vagus Dönitz and members of the An. barbirostris, An. subpictus and An. sundaicus complexes. Like An. maculatus, their distributions and involvement in the transmission of malarial parasites varies across the
Corresponding author. E-mail address: [email protected] (P. Somboon).
https://doi.org/10.1016/j.actatropica.2019.105124 Received 7 May 2019; Received in revised form 1 August 2019; Accepted 2 August 2019 Available online 05 August 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Morphological features of adult mosquitoes were examined under a stereomicroscope. The morphological keys of Rattanarithikul and Green (1986); Rattanarithikul and Harbach (1990) and Rattanarithikul et al. (2006) were used to confirm the identification of species. The morphological terminology and abbreviations follow the Anatomical Glossary of the online Mosquito Taxonomic Inventory (http://mosquitotaxonomic-inventory.info/anatomical glossary-overview).
archipelago (Elyazar et al., 2013). The taxonomic status of An. maculatus across the Indonesian Archipelago is not clearly known and it has been speculated that it may consist of more than one species (Bangs et al., 2002; Elyazar et al., 2013; Rattanarithikul and Harbach, 1990). This is because previous reports regarding An. maculatus in Indonesia are based mainly on morphological identifications that do not unambiguously separate it from other members in the Maculatus Group, which consists of nine formally recognized species: An. dispar Rattanarithikul & Harbach, An. dravidicus Christophers, An. greeni Rattanarithikul & Harbach, An. maculatus, An. notanandai Rattanarithikul & Green, An. pseudowillmori (Theobald), An. rampae Harbach & Somboon, An. sawadwongporni Rattanarithikul & Green and An. willmori (James) (Harbach, 2019). Genetic markers in both mitochondrial DNA (mtDNA) (such as cytochrome c oxidase subunits I and II, COI and COII) and nuclear ribosomal DNA (rDNA) (such as the internal transcribed spacer 2, ITS2, and the D3 region of the 28S ribosomal gene) have been widely used to identify closely related and isomorphic species of mosquito complexes, including the Maculatus Group (Torres et al., 2000; Ma et al., 2006; Walton et al., 2007; Singh et al., 2012; Sum et al., 2014). Recent studies provide clear evidence for the existence of a new species within An. maculatus s.l. in the Indonesian Archipelago (Ali et al., 2019; Garjito et al., 2019). For this reason, we extended our studies to include phylogenetic analyses of DNA sequence data obtained from specimens of An. maculatus s.l. collected in Borneo (Kalimantan), Java, Sulawesi, Sumatra and the Sumba islands.
2.2. DNA extraction, amplification and sequencing Genomic DNA was extracted from fore- and midlegs of individual mosquitoes using the Pure Link™ Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, USA) according to manufacturer’s instructions. The specimens were retained for morphological examination. The ribosomal ITS2 and D3 regions and the mitochondrial COII gene were sequenced in the present study. The ITS2 region was amplified by PCR using the primers 5.8 F (5TGTGAACTGCAGGACACATG-3) and 28R (5-ATGCTTAAATTTAGGGG GTA-3) (Walton et al., 2007). The 28S-D3 domain of the 28S gene was amplified using the primers D3A (5-GACCCGTCTTGAAACACACGGA-3) and D3B (5-TCGGAAGGAACCAGCTACTA-3) (Schmidt et al., 2006). PCR reactions for both regions were carried out in a 20 μl volume containing 1 μl (5 ng) of DNA, 0.8 μM of each primer, 1.2 mM MgCl2, 1.6 mM dNTP mix and 0.08 U of Platinum®Taq DNA polymerase in 1x PCR buffer. Thermal cycling conditions were initial denaturation at 95 °C for 2 min, 35 cycles at 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The COII gene was amplified using the primers SCTL2-J-3037 (5ATGGCAGATTAGTGCAATGA-3) and TK-N-3785 (5-GTTTAAGAGACC AGTACTTG-3) (Liu and Beckenbach, 1992). PCR reactions were carried out, as described by Ali et al. (2019), in a 20 μL volume containing 0.4 U of Platinum®Taq DNA polymerase, 1X of PCR buffer, 1.5–3.0 mM of MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer and 1 μL (5 ng) of extracted DNA. The amplification profile comprised initial denaturation at 95 °C for 2 min, 35 cycles at 95 °C for 30 s, 45 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Amplified products were electrophoresed in 2% agarose gels and stained with ethidium bromide. PCR products were purified using the illustra™ ExoProStar™ 1-Step (GE Healthcare Life Sciences, UK) and sequenced using the BigDye® Terminator v3.1 cycle sequencing kit chemistry (First BASE, Malaysia).
2. Materials and methods 2.1. Mosquito collection and morphological identification Since very few An. maculatus s.l. adults in our study areas were collected from human bait, we captured adult mosquito specimens resting in cowsheds by aspirator at eight localities on five islands: 1) Sabang, Aceh Special Region, off the northern tip of Sumatra; 2) Hargowilis, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province (previously collected by Ali et al., 2019); 3) Hargotirto, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 4) Sumba Island, East Nusa Tenggara Province, Lesser Sunda Islands; 5) Pucak, South Sulawesi Province, Sulawesi; 6) Labuan, Central Sulawesi Province; 7) Wulai, West Sulawesi Province; and 8) North Kalimantan Province, Borneo (Fig. 1, Table 1). The mosquitoes were killed with chloroform vapor and preserved in vials with silica gel.
Fig. 1. Map of the Indonesian Archipelago and collecting sites: 1) Sabang, Aceh Special Region, off the northern tip of Sumatra; 2) Hargotirto, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 3) Hargowilis, Kulon Progo Regency, Yogyakarta Special Region, Central Java Province; 4) Sumba Island, East Nusa Tenggara Province, Lesser Sunda Islands; 5) Pucak, South Sulawesi Province, Sulawesi; 6) Labuan, Central Sulawesi Province; 7) Wulai, West Sulawesi Province; and 8) North Kalimantan Province, Borneo. 2
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Table 1 Details of collections of An. maculatus s.l. made in the Indonesian Archipelago. No.
Province
Location
Coordinates
Altitude (m)
No. specimens collected
Date of collection
1 2 3 4
Sumatra Central Java Central Java East Nusa Tenggara
Sabang Hargotirto Hargowilis Sumba
5°49′59.7″N 95°19′01.6″E 7°47′37.9″S 110°05′40.8″E 7°49′43.2″S 110°06′32.1″E 9°26′52.7″S 119°15′40.2″E
107 388 199 468
5 6 7 8
South Sulawesi West Sulawesi Central Sulawesi North Kalimantan
Pucak Wulai Labuan SebatikTengah
5°09′29.2″S 119°42′01.3″E 1°02′42.9″S 119°31′27.5″E 0°38′28.9″S 119°51′08.5″E 4°09′20.4″N 117°47′04.9″E
113 42 109 53
19 10 8* 6 13 20 10 7 5
Aug 2017 Dec 2018 Jun 2017 Jul 2017 Nov 2018 Jan–May 2017 Feb–Mar 2018 Nov 2017 Dec 2018
* Previously collected by Ali et al. (2019).
Due to limited budget, two to five specimens of An. maculatus s.l. from each site were selected at random for sequencing: Sabang (n = 3), Hargowilis (3) and Hargotirto (4) of Central Java; Sumba Island (5); Pucak (2), Labuan (2) and Wulai (2) of Sulawesi; and Sebatik Tengah (4) of North Kalimantan. The specimen from North Kalimantan (Sebatik-3) was also sequenced. The sequences are deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accession numbers, for ITS2: MK504453–MK504475; for D3: MK518025–MK518047; for COII: MK236369–MK236371 and MK507483–MK507501.
2.3. Sequencing alignment and phylogenetic analysis The sequences obtained during this study were compared with those of the Salatiga Lab strain of An. maculatus s.l. (GenBank accession numbers for ITS2: MK659773, MK67565 and MK675654), wild-caught females from Hargowilis, Central Java Province (Java-1,3,5) previously described by Ali et al. (2019) (GenBank accession numbers for ITS2: MK204645–MK204647; COII: MK236369–MK236371) and other members of the Maculatus Group in GenBank using the Basic Local Alignment Search Tool (BLAST, available at http://blast.ncbi.nlm.nih. gov/Blast.cgi) under default parameters (max High-Scoring Segment Pairs 250, expect threshold 10, word size 28, optimized for highly similar hits, not specific to any organism). Anopheles karwari (James) was used as the outgroup taxon because it is not a member of the Maculatus Group but is classified along with An. maculatus s.l. as a member of the Neocellia Series. Consensus sequences of ITS2, D3 and COII were aligned using CLUSTALW under default parameters (Larkin et al., 2007), and ragged ends were trimmed using MEGA version 10 (Kumar et al., 2018). The most appropriate model of nucleotide substitution was determined for each dataset using jModelTest version 2.1.10 (Guindon and Gascuel, 2003; Darriba et al., 2012), which was GTR for ITS2 and D3 and GTR + I+G for COII (Tavare, 1986). The phylogenetic analyses were conducted using Maximum Likelihood (ML) in MEGA version 10 (Kumar et al., 2018) and Bayesian inference (BI) with MrBayes version 3.2.7 (Ronquist et al., 2012). For ML, robustness of the tree was tested with 1000 bootstrapped data set with bootstraps support values shown on each node. For BI, the Markov chain Monte Carlo (MCMC) simulation was run for one million generations (which resulted in an average standard deviation of split frequencies below 0.01), using three heated chains and one cold chain, and sampling every 100 generations with a burnin of 25%. Two independent analyses were conducted to confirm convergence. A 50% majority rule consensus tree was created from the remaining trees. The BI trees were generated using Figtree software version 1.4.4 (http:// tree.bio.ed.ac.uk/software/figtree/). Bayesian support values of 95% (0.95) and Maximum Likelihood Bootstrap values of 70% were taken as being highly supportive of a node (Hillis and Bull, 1993).
3.1. ITS2 The length of ITS2 ranged from 367 to 370 bp (supplementary Fig. S1). Sequences of An. maculatus s.s. from GenBank were identical to each other across a wide geographic range with the exception of the sequence from Malaysia (AY491974.1) that differed at two bases (19 and 205). This may indicate intraspecific variation but it is hard to exclude sequencing error as the explanation of deviation for this one sequence. The sequences of specimens from Hargotirto (JavaHT13–16), Sabang, Sumba and North Kalimantan were identical to most An. maculatus s.s. from mainland Asia. Their sequences were very distinct from those from Sulawesi, Hargowilis (Java-HW1,3,5) and the Salatiga Lab strain, which were almost identical, except for Sulawesi differing at one base (C↔T, position 336). The sequences from Sulawesi and Hargowilis differed from An. maculatus s.s. by 20 and 21 fixed sites, respectively. 3.2. D3 The length of D3 ranged from 304 to 312 bp (supplementary Fig. S2). The sequences of specimens from Hargotirto (Java-HT13–16), Sabang, Sumba and North Kalimantan were identical to An. maculatus s.s. from mainland Asia. Like the ITS2 sequences, their D3 sequences were very distinct from those from Sulawesi and Hargowilis (JavaHW1,3,5), differing by 12 and 11 fixed sites, respectively. Specimens from Sulawesi differed from those from Hargowilis at one base (C↔G, position 150).
3. Results 3.3. COII A total of 98 females of An. maculatus s.l. were collected from the eight localities (Fig. 1, Table 1). Morphologically, all females resembled An. maculatus s.s., in particular in having abdominal terga II–IV without scales; terga V and VI without scales or with sparse pale falcate scales on the posterior margins; terga VII and VIII largely or posteriorly covered with narrow pale spatulate scales; and the posterolateral corners of terga VI–VIII, and rarely tergum V, bearing patches of dark scales. The accessory sector pale spot on the wings is confined to vein R1 and is never extended onto the subcosta and costa, except one female from North Kalimantan Province (Sebatik-3), which is a character of the two Philippine species of the Maculatus Group, An. dispar and An. greeni.
The length of COII sequences of all specimens was 576 bp (supplementary Fig. S3). The COII sequences of An. maculatus s.s. from several countries in mainland Asia were identical, except one variable site at base 82 (A↔G). They were distinct from those from Hargotirto (JavaHT13–16), Sabang, Sumba and North Kalimantan by 7, 12, 8 and 9 fixed sites, respectively (supplementary Fig. S3). The COII sequences of specimens from Sumba and North Kalimantan were similar without any different fixed site, indicating that they are conspecific. Specimens from Hargowilis and Sulawesi differed from each other by 19 fixed sites; they differed from An. maculatus s.s. by 9 and 20 fixed sites, respectively. 3
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 2. Maximum Likelihood tree of ITS2 sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Bootstrap values are shown at each node.
Asia (BS = 100%, PP = 1.0). Clade II of An. maculatus included specimens from Hargowilis (Java-HW1,3,5), the Salatiga Lab strain and Sulawesi (BS = 99%, PP = 1.0). The clades consisting of other members of the Maculatus Group were clearly distinct. The only specimen from North Kalimantan (Sebatik-3) corresponds to the Philippine species An. greeni. The ML and BI analyses of the COII sequences obtained from the Indonesian specimens also yielded a similar tree topology to each other (Figs. 4 and 5). Unlike the ITS2 trees, however, within An. maculatus from Indonesia the tree consisted of five clades with highly supportive values particularly by BI, two comprised of specimens from Central Java (Clades I, IV), one of specimens from Sumba and North Kalimantan (Clade II), one of specimens from Sabang (Clade III), and one of specimens from Sulawesi (Clade V). Clade V, that comprised sequences of specimens from the three localities in Sulawesi, was genetically distinct from Clades I-IV that comprised the other Indonesian specimens (BS = 64%, PP = 1.0). Although the mosquitoes in Clades I-III
The ITS2, D3 and COII sequences of other members of the Maculatus Group were very distinct from those from the Indonesian Archipelago. Interestingly, however, the only specimen from North Kalimantan (Sebatik-3), which has an accessory sector pale spot extending onto the costa, had sequences corresponding to the Philippine An. greeni for all three loci (supplementary Figs. S1-S3).
3.4. Phylogenetic analyses The ML and BI analyses of the ITS2 sequences yielded a similar tree topology, showing only two distinct clades within the Indonesian specimens with strong support by BI and moderate support by ML (ML bootstrap, BS value = 55%, Bayesian Posterior Probability, PP, value = 0.98) (Figs. 2 and 3). The trees of D3 sequences are similar to the ITS2 trees (figure not shown). Clade I of An. maculatus consisted of specimens from Hargotirto (Java-HT13–16), Sabang, Sumba and North Kalimantan; this clade also included An. maculatus s.s. from mainland 4
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 3. Bayesian tree of ITS2 sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Posterior probabilities are shown at each node.
the Philippine islands of Palawan and Mindanao where An. dispar and An. greeni are present in sympatry (Rattanarithikul and Harbach, 1990; Manguin et al., 2008). It is possible that the other Philippine member of the Maculatus Group, An. dispar, might also occur in Borneo. Since both species are regarded as secondary vectors of malaria in the Philippines (Manguin et al., 2008), more sequencing of specimens from various localities in Borneo is needed to shed light on the distributions of these two species. Based on the rDNA ITS2 and D3 sequence data, the Indonesian An. maculatus s.l. can be divided into two main clades that correspond to at least two species. The first group, including mosquitoes from Hargotirto of Central Java, Sabang, Sumba and North Kalimantan, have ITS2 and D3 sequences that are identical to most of those of An. maculatus s.s. in mainland Southeast Asia (Clade I, Figs. 2 and 3). This same taxon, based on ITS2 sequences, has also been reported from South Sumatra, Purbalingga and Cilacap of west of Central Java, North Kalimantan, and Belu of East Nusa Tenggara (east of Sumba Island) by Garjito et al. (2019). Unfortunately, no COII sequences were available from this
belonged to the same clade for ITS2 and D3 as An. maculatus s.s. from mainland Asia, at COII they were separated from the clade consisting of An. maculatus s.s. from mainland Asia with highly supportive values by BI (PP = 1.0) (Fig. 5). Similarly, even though they belonged to the same ITS2/D3 clade, the mosquitoes from Sulawesi were clearly distinguished from those from Hargowilis (Java-HW1,3,5) in Central Java by COII falling into Clades IV and V, respectively. 4. Discussion Previous records and reports reveal that An. maculatus s.l. is widely distributed across the Indonesian Archipelago, but not beyond Sulawesi and Timor, or Weber’s Line, a biogeographical boundary lying approximately along the Australo-Papuan Shelf (Elyazar et al., 2013). Based on the morphological distinction and the rDNA and mtDNA sequences obtained in the current study, we report for the first time the occurrence of An. greeni outside the Philippines, i.e. in Northern Kalimantan of Borneo Island. This is likely due to the proximity of Borneo to 5
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 4. Maximum Likelihood tree of COII sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Bootstrap values are shown at each node.
divergence was likely shaped predominantly by the biogeographical history of this region. The Malay Peninsula, Borneo, Java and Sumatra, and their surrounding islands, comprise the Sundaic biogeographical region (or Sundaland), that has been periodically connected as a single larger, predominantly forested landmass when the Sunda Shelf was exposed periodically throughout the past 2.6 mya when sea levels were 50–200 m lower than today (Tougard, 2001). Allopatric speciation and the distribution of species of Anopheles mosquitoes, including An. maculatus s.l., in the Indonesian Archipelago has been attributed to isolation imposed by sea barriers (Morgan et al., 2013). Within the ITS2/D3 Clade I the specimens from different geographical regions i.e. mainland Southeast Asia, north Kalimantan/ Sumba, Java and Sabang formed distinct, moderately divergent (1.2–2.1%), COII clades, consistent with their formation by periodic allopatric fragmentation on different landmasses. Whilst the possibility that these mtDNA clades correspond to distinct species cannot be excluded, the lack of divergence at ITS2/D3 provides no support for this. In contrast, there was greater differentiation within the ITS2/D3 Clade II with An. maculatus s.l. from Sulawesi differing from specimens
study for comparison with our specimens. The second group detected here included mosquitoes from Hargowilis of Central Java, the Salatiga Lab strain and Sulawesi that were not closely related to An. maculatus s.s. Based on ITS2 sequence similarity, this second group is conspecific with a species of An. maculatus recently detected from Kulon Progo of Central Java by Garjito et al. (2019). Whilst the specimens from Hargowilis belong to this newly identified species, they are distinct from those from Hargotirto (JavaHT13–16) that are more closely related to An. maculatus s.s., supporting the presence of two species in Central Java. Both Hargotirto and Hargowilis villages are situated in Kulon Progo Regency, Yogyakarta Special Region, Central Java Province. The two villages are about 4.5 km apart and are surrounded by forest, palm gardens and hills. Hargowilis is located at a lower altitude (199 m), whereas Hargotirto is located at a higher altitude (388 m). Further sampling is needed to determine if the two forms occur sympatrically, if they occupy distinct ecological niches and if they differ in vectorial capacity. The taxonomic status of specimens with distinct mtDNA lineages within the above two species is more difficult to interpret but their 6
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
Fig. 5. Bayesian tree of COII sequences from specimens of An. maculatus s.l. collected in the Indonesian Archipelago, including the Java strain, An. maculatus s.s. from mainland Asia and GenBank sequences from members in the Maculatus Group, with An. karwari as the outgroup. Posterior probabilities are shown for each node.
taxonomic status at this stage. The results of the current study may not represent all sibling species that may occur in the Indonesian Archipelago because few specimens were examined from each location and many islands remain to be surveyed. At present, nine species of the Maculatus Group are formally recognised (Harbach, 2004; Harbach, 2019). However, the results of previous studies (Ali et al., 2019; Garjito et al., 2019) and this current study clearly indicate that An. maculatus is a complex of sibling species, consisting of An. maculatus s.s. from mainland Asia and at least one novel (previously unrecognized) species in Hargowilis of Central Java Province and potentially a third species in Sulawesi with the taxonomic status of distinct mtDNA lineages in Central Java Province (Hargotirto), Sabang and Kalimantan/Sumba Island undetermined. Thus, more information based on morphological study (especially the immature stages), cytogenetics and cross-mating experiments, as well as further mtDNA sequencing from additional specimens from more locations that would enable species delimitation analysis to be conducted. These would help to clarify the nature and status of these forms.
collected in Hargowilis of Central Java by one nucleotide base in each of ITS2 and D3 sequences, and their COII sequences forming highly divergent clades, differing by 19 fixed sites (3.3%). In general, sequence divergence of > 2% at COI and COII genes corresponds to variation at the interspecific level in Anopheles mosquitoes (e.g. Wang et al., 2012, 2017). On this basis, An. maculatus s.l. in Sulawesi would be inferred to be another new species distinct from that found in Hargowlis, Central Java. Speciation may be more likely to occur by allopatric isolation on Sulawesi since it is separated from the Sundaic Region by a deeper sea delineated by Wallace’s Line that likely severely limits gene flow compared to among landmasses within Sundaland. However, high mtDNA divergence coupled with low ITS2/D3 divergence between Sulawesi and Hargowilis is consistent with mtDNA introgression occurring into the ITS2/D3 Clade II species from geographically localized populations in Central Java making inference of species delimitation from mtDNA data alone particularly problematic in this case. While significant differentiation at ITS2 and D3 is a clear indication of discrete species (Walton et al., 1999b) the opposite is not necessarily true as recently diverged species can show little or no divergence at these markers; e.g. An. gambiae and An. arabiensis (Paskewitz et al., 1993) and An. dirus Peyton & Harrison and An. scanloni Sallum & Peyton of the Dirus Complex (Walton et al., 1999a) for ITS2; and species S of An. fluviatilis James and An. harrisoni Harbach & Manguin of the Fluviatilis and Minimus Complexes, respectively, for D3 (Chen et al., 2006; Singh et al., 2006). Consequently, within both of the ITS2/ D3 Clades I and II it is not possible to draw further conclusions on
Declaration of Competing Interest The authors declare that we have no conflict of Interest. Acknowledgements We thank the Faculty of Medicine, Chiang Mai University for 7
Acta Tropica 199 (2019) 105124
R.S.M. Ali, et al.
providing a PhD scholarship to Rusdiyah Sudirman Made Ali. This research was supported mainly by the Faculty of Medicine (PAR-256105869) and partially by the Research Administration Office of Chiang Mai University and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2015M3A9B6073666).
pp. 327–355. O’Connor, C.T., Sopa, T., 1981. A checklist of the mosquitoes of Indonesia. A special publication of the U.S naval medical research unit No.2, Jakarta, Indonesia. NAMRU SP 45, 1–26. Paskewitz, S.M., Wesson, D.M., Collins, F.H., 1993. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex. Insect Mol. Biol. 2, 247–257. Rattanarithikul, R., Green, C.A., 1986. Formal recognition of the species of the Anopheles maculatusgroup (Diptera: Culicidae) occurring in Thailand, including the descriptions of two new species and a preliminary key to females. Mosq. Syst. 18, 246–278. Rattanarithikul, R., Harbach, R.E., 1990. Anopheles maculatus (Diptera: Culicidae) from the type locality of Hong Kong and two new species of the Maculatus complex from the Philippines. Mosq. Syst. 22, 160–183. Rattanarithikul, R., Harrison, B.A., Harbach, R.E., Panthusiri, P., Coleman, R.E., 2006. Illustrated keys to the mosquitoes of Thailand. IV. Anopheles. SE Asian J. Trop. Med. Public Health 37 (Suppl 2), 1–128. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., et al., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. Singh, O.P., Chandra, D., Nanda, N., Sharma, S.K., Pe, Than Htun, Adak, T., et al., 2006. On the conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C. J. Biosci. 31, 671–677. Singh, S., Prakash, A., Yadav, R.N.S., Mohapatra, P.K., Sarma, N.P., Sarma, D.K., et al., 2012. Anopheles (Cellia) maculatus group: its spatial distribution and molecular characterization of member species in north-east India. Acta Trop. 124, 62–70. Schmidt, S., Driver, F., Barro, P.D., 2006. The phylogenetic characteristics of three different 28S rRNA gene regions in Encarsia (Insecta, Hymenoptera, Aphelinidae). Org. Divers. Evol. 6, 127–139. Sitohang, V., Sariwat, E., Fajariyani, S.B., Hwang, D., Kurnia, B., Hapsari, R.K., et al., 2018. Malaria elimination in Indonesia: halfway there. Lancet Glob. Health 6, PE604–E606. Sugiarto, H., Kesumawati, U., Soviana, S., Hakim, L., 2017. Bionomics of Anopheles (Diptera: Culicidae) in a malaria endemic region of Sungai Nyamuk village, Sebatik Island–North Kalimantan, Indonesia. Acta Trop. 171, 30–36. Sum, J.S., Lee, W.C., Amir, A., Braima, K.A., Jeffery, J., Abdul-Aziz, N.M., et al., 2014. Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA. Parasites Vectors 7, 309. Tavare, S., 1986. In: In: Miura, Robert M. (Ed.), Some Probabilistic and Statistical Problems in the Analysis of DNA Sequences, Lectures on Mathematics in the Life Sciences, vol. 17. American Mathematical Society, Providence, Rhode Island, pp. 57–86. Torres, E.P., Foley, D.H., Saul, A., 2000. Ribosomal DNA sequence markers differentiate two species of the Anopheles maculatus (Diptera: Culicidae) Complex in the Philippines. J. Med. Entomol. 37, 933–937. Tougard, C., 2001. Biogeography and migration routes of large mammal faunas in SouthEast Asia during the Late Middle Pleistocene: focus on the fossil and extant faunas from Thailand. Palaeogeogr. Palaeoclimatol. Palaeoecol. 168, 337–358. Walton, C., Sharpe, R.G., Pritchard, S.J., Thelwell, N.J., Butlin, R.K., 1999a. Molecular identification of mosquito species. Biol. J. Linn. Soc. Lond. 68, 241–256. Walton, C., Handley, J.M., Kuvangkadilok, C., Collins, F.H., Harbach, R.E., Baimai, V., et al., 1999b. Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction. Med. Vet. Entomol. 13, 24–32. Walton, C., Somboon, P., O’Loughlin, S.M., Zhang, S., Harbach, R.E., Linton, Y.-M., et al., 2007. Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA. Infect. Genet. Evol. 7, 93–102. Wang, G., Li, C., Guo, X., Xing, D., Dong, Y., Wang, Z., et al., 2012. Identifying the main mosquito species in China based on DNA barcoding. PLoS One 7, e47051. Wang, G., Li, C., Zheng, W., Song, F., Guo, X., Wu, Z., et al., 2017. An evaluation of the suitability of COI and COII gene variation for reconstructing the phylogeny of, and identifying cryptic species in, anopheline mosquitoes (Diptera Culicidae). Mitochondrial DNA Part A 28 (5), 769–777. WHO, 2018. World Malaria Report 2018. accessed 4 July 2019. https://www.who.int/ malaria/publications/world-malaria-report-2018/report/en/.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.actatropica.2019. 105124. References Ali, R.S.M., Wahid, I., Saeung, A., Wannasan, A., Harbach, R.E., Somboon, P., 2019. Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia. Parasites Vectors 12, 107. Bangs, M.J., Soelarto, T., Barodji, Wicaksana, B.P., Boewono, D.T., 2002. Colonization of Anopheles maculatus from Central Java, Indonesia. J. Am. Mosq. Control Assoc. 18, 359–363. Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Marwoto, H., Bangs, M.J., et al., 2002. Epidemic malaria in the Menoreh hills of Central Java. Am. J. Trop. Med. Hyg. 66, 287–292. Bonne-Wepster, J., 1953. In: de Bussy, J.H. (Ed.), The Anopheline mosquitoes of the IndoAustralian Region, Amsterdam. Chen, B., Butlin, R.K., Pedro, P.M., Wang, X.Z., Harbach, R.E., 2006. Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia. Med. Vet. Entomol. 20, 33–43. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. Elyazar, I.R., Sinka, M.E., Gething, P.W., Tarmidzi, S.N., Bangs, M.J., Kusriastuti, R., et al., 2013. The distribution and bionomics of anopheles malaria vector mosquitoes in Indonesia. Adv. Parasitol. 83, 173–266. Garjito, T.A., Widiastuti, U., Mujiyono, M., Prihatin, M.T., Widiarti, W., Setyaningsih, R., et al., 2019. Genetic homogeneity of Anopheles maculatus in Indonesia and origin of a novel species present in Central Java. Parasites Vectors 12, 351. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Harbach, R.E., 2004. The classification of genus Anopheles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships. Bull. Entomol. Res. 94, 537–553. Harbach, R.E., 2019. Subgenus Cellia. Mosquito Taxonomic Inventory. accessed on 4 July 2019. http://mosquito-taxonomic-inventory.info/. Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. Larkin, M.A., Blackshields, G., Brown, N., Chenna, R., McGettigan, P.A., Lopez, R., et al., 2007. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Liu, H., Beckenbach, A.T., 1992. Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects. Mol. Phylogenet. Evol. 1, 41–52. Ma, Y., Li, S., Xu, J., 2006. Molecular identification and phylogeny of the Maculatus Group of Anopheles mosquitoes (Diptera: Culicidae) based on nuclear and mitochondrial DNA sequences. Acta Trop. 99, 272–280. Manguin, S., Garros, C., Dusfour, I., Harbach, R.E., Coosemans, M., 2008. Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review. Infect. Genet. Evol. 8, 489–503. Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A., Kent, J., 2000. Biodiversity hotspots for conservation priorities. Nature 403 (6772), 853–858. Morgan, K., Somboon, P., Walton, C., 2013. Understanding Anopheles diversity in Southeast Asia and its applications for malaria control. In: Manguin, S. (Ed.), Anopheles Mosquitoes — New Insights into Malaria Vectors. InTech, Rijeka, Croatia,
8