Phylogenetic Analysis and DNA-based Species Confirmation in Anopheles (Nyssorhynchus) Peter G. Foster1*, Eduardo S. Bergo2, Brian P. Bourke3, Tatiane M. P. Oliveira3, Sandra S. Nagaki3, Denise C. Sant’Ana3, Maria Anice M. Sallum3 1 Department of Life Sciences, Natural History Museum, London, United Kingdom, 2 Superintendeˆncia de Controle de Endemias, Secretaria de Estado da Sau´de de Sa˜o Paulo, Araraquara, Sa˜o Paulo, Brazil, 3 Departamento de Epidemiologia, Faculdade de Sau´de Pu´blica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract Specimens of neotropical Anopheles (Nyssorhynchus) were collected and identified morphologically. We amplified three genes for phylogenetic analysis–the single copy nuclear white and CAD genes, and the COI barcode region. Since we had multiple specimens for most species we were able to test how well the single or combined genes were able to corroborate morphologically defined species by placing the species into exclusive groups. We found that single genes, including the COI barcode region, were poor at confirming species, but that the three genes combined were able to do so much better. This has implications for species identification, species delimitation, and species discovery, and we caution that single genes are not enough. Higher level groupings were partially resolved with some well-supported groupings, whereas others were found to be either polyphyletic or paraphyletic. There were examples of known groups, such as the Myzorhynchella Section, which were poorly supported with single genes but were well supported with combined genes. From this we can infer that more sequence data will be needed in order to show more higher-level groupings with good support. We got unambiguously good support (0.94–1.0 Bayesian posterior probability) from all DNA-based analyses for a grouping of An. dunhami with An. nuneztovari and An. goeldii, and because of this and because of morphological similarities we propose that An. dunhami be included in the Nuneztovari Complex. We obtained phylogenetic corroboration for new species which had been recognised by morphological differences; these will need to be formally described and named. Citation: Foster PG, Bergo ES, Bourke BP, Oliveira TMP, Nagaki SS, et al. (2013) Phylogenetic Analysis and DNA-based Species Confirmation in Anopheles (Nyssorhynchus). PLoS ONE 8(2): e54063. doi:10.1371/journal.pone.0054063 Editor: Luciano A. Moreira, Centro de Pesquisas Rene´ Rachou, Brazil Received August 20, 2012; Accepted December 6, 2012; Published February 4, 2013 Copyright: ß 2013 Foster et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation was financially supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP) (Grants no. 2011/20397-7; 2005/ 53973-0; http://www.fapesp.br/), and Conselho Nacional de Desenvolvimento Te´cnico e Cientı´fico (CNPq BPP no. 301666/2011-3 to MAMS; http://www.cnpq.br/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Nyssorhynchus, Anopheles darlingi is the primary vector in areas of the Amazon Region [9,10]. In addition, An. albimanus, species of Malaria is among the world’s most important infectious the An. albitarsis Complex, An. aquasalis, An. marajoara, and An. diseases. There were approximately 216 million cases of malaria nuneztovari are also dominant vectors of human Plasmodium [9]. reported in 2011 and an estimated 655,000 deaths in 2010 [1]. Other species of the Nyssorhynchus may be secondary, local vectors, The disease is a major obstacle to social and economic or were found naturally infected with Plasmodium sporozoites, for development in affected countries [2], and although considerable example, An. benarrochi [11], An. rangeli [12,13], An. oswaldoi [12,14], funding has helped decrease the incidence of malaria by an An. oswaldoi B [13], An. strodei [15], An. rondoni [16], An. trinkae [12], estimated 17% since 2000, the failure to maintain effective malaria and An. triannulatus [17]. control strategies can lead to resurgence in historically endemic The Anophelinae subfamily includes 465 formally recognized regions [1,3]. Climate change [4] and deforestation [5] are also species and 50 unnamed members of species complexes [7], which likely to play an important role in the appearance of malaria in are subdivided into three genera – Anopheles, Bironella, and Chagasia. non-endemic and newly inhabited regions, respectively. Effective Species of the genus Anopheles are subdivided into seven subgenera: approaches to species identification are essential in vector Anopheles, Baimaia, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus, and incrimination, the assessment of malaria risk and development of Stethomyia. Worldwide, the primary vectors of human malaria malaria control strategies [6]. The focus of such work centres on parasites belong to the subgenera Anopheles, Cellia and Nyssorhynchus the genus Anopheles, which contains all known vectors of malaria, [18]. Most phylogenetic and phylogeographic studies regarding with many forming cryptic species complexes [7]. Such species are Anophelinae involved medically important species or species morphologically indistinguishable and for many of the most groups. The most comprehensive phylogenetic study, in terms of important vectors of malaria a molecular approach is the only the number of species sampled, is that of Sallum et al. (2000) [19], effective tool for resolving species and species relationships [8]. which employed morphological characters. Results of phylogenetic There are nine dominant Anopheles vector species in the analysis, using either morphological characters [19] or sequences Americas, three species belong to the subgenus Anopheles and six of mitochondrial and nuclear genes [20] corroborated the to the Nyssorhynchus [9]. Among species of the subgenus PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e54063 Species Confirmation in Anopheles (Nyssorhynchus) monophyly of Anophelinae and of subgenera Cellia, Kerteszia and and poor phylogenetic information contained in the two genes Nyssorhynchus. employed in the analyses. The subgenus Nyssorhynchus has a Neotropical distribution, with Several genes have been found to be useful in Anopheles An. albimanus extending to the southern Neartic Region. The systematics. The 59 region of the COI gene has long been used Nyssorhynchus includes 39 formally named species [21] with a as a DNA barcode for species identification, and also for current listing of 44 [22], which are subdivided between the barcoding animal life [43,44]. The barcode has been shown to Albimanus Section (Faran 1980) [23], Argyritarsis Section be successful for mosquito identification in Canada [45], and to (Linthicum 1988) [24], and Myzorhynchella Section (Galva˜o both corroborate known lineages and define new lineages within 1941) [25]. In the Argyritarsis and Albimanus Sections there are the An. albitarsis Complex [37]. Besansky and Fahey [46] used the species that were not formally validated and therefore were nuclear white gene to confirm the monophyly of the subfamily designated by letters added to the name of the taxon morpholog- Anophelinae, and tribes Sabethini, Culicini and Aedini. More ically more similar. This is so in the case of An. nuneztovari which recently, arginine kinase, CAD, catalase, enolase, hunchback, and white includes chromosomal forms A, B, and C [26–28]. Using ITS2 nuclear protein-coding genes were used to assess genus level sequence data, Sierra et al. (2004) [29], showed that An. nuneztovari relationships within Culicidae and to infer divergence time for B and C are conspecific, and Calado et al. (2008) [30] demon- major lineages, strongly supporting the monophyly of Anophelinae strated that at least part of that which was identified as An. and the tribes Aedini and Sabethini [47]. A larger amount of nuneztovari A may include An. goeldii, and so this species was sequence data would allow broader and deeper phylogenetics, resurrected from the synonymy. The Strodei Complex was named such as for example the study by Regier and Zwick [48], who used by Faran (1980) [23] and includes An. strodei, An. benarrochi and An. 62 protein-coding nuclear genes, using only non-synonymous rondoni. Anopheles strodei contained five species in synonymy, which change, to recover a robust higher-level phylogeny of Arthropoda. were described by Unti [31,32] using characters either of the eggs Here we used the COI barcode region, and the single copy or larvae. Recently, Sallum et al. [33] resurrected An. albertoi and nuclear white and CAD protein-coding genes to confirm known An. arthuri from synonymy with An. strodei. The number of morphologically defined species, and to estimate phylogenetic recognized Nyssorhynchus species has increased over time, and it is relationships and assess species complexes within the subgenus expected that this trend will continue. It has been proposed that Nyssorhynchus. We found that species confirmation was poor using An. triannulatus [34], An. konderi [35,36], and An. oswaldoi [14,36] any of the three genes alone, but was good using concatenated may be more than one species