Background
Deforestation is the permanent destruction of forests in order to harvest timber, develop farms and pasture, and build roads and urban areas. Many of the world’s most biologically diverse regions are now subject to the highest rates of deforestation and one of the most severely affected of these is the Amazon tropical rainforest. In Brazil over the past 50 years, deforestation in the Amazon has reached unprecedented levels, with estimated losses of between 4 and 29 thousand km
2 annually [
1]. During the same period, the human population in the region has increased from approximately 4 to 24 million [
2]. Additional effects of regional deforestation include a dramatic loss of endemic species [
3], creation of social conflict, contribution to global climate change, and elevated human vulnerability to socio-environmental conditions [
4]. It is also having a negative impact on public health [
5‐
7], and increases vector-borne diseases in the Amazon River basin [
8]. One of the most important public health impacts is the increased transmission of
Plasmodium species responsible for human malaria [
9‐
13].
The increased incidence of
Plasmodium infection in humans in Amazonian villages resulting from deforestation has been described as “Frontier Malaria”. This has been characterized as a temporal process comprised of epidemic, transition, and endemic phases [
14,
15]. The epidemic phase involves changes in the natural forest landscape, including the biotic and abiotic conditions of forest larval habitats, that can favour species that are actively involved in the transmission of the
Plasmodium to humans. Ongoing anthropogenic changes in the environment, which alter the abiotic characteristics and ecology of larval habitats [
16], lead to an increase in abundance of the most important vector species and, ultimately, higher biting rates and
Plasmodium infection in settler communities with poor housing conditions, lack of access to health services and low or no immunity to the pathogens [
17]. The transition phase occurs several years after the initial settlements and sees gradual declines of transmission as a result of improved housing, infrastructure and public health services. The final endemic phase is reached when transmission reaches low and stable levels, normally within 10 years of the initial settlement. A developed public health service can achieve effective diagnosis and treatment of
Plasmodium falciparum infection, due to symptoms occurring before gametocyte production.
Plasmodium vivax infection, however, is more difficult to diagnose and treat, as the pathogen can exist at much lower densities [
18] and persist as hypnozoites in the liver [
19]. The only licensed treatments of the liver stage of
P. vivax are primaquine and tafenoquine, but they are of limited use in some populations due to the adverse effect of acute haemolysis in people with G6PD deficiency [
20,
21].
Plasmodium falciparum transmission emerges in the early stages of settlement and is joined by
P. vivax in later stages, with the latter generally becoming more prevalent. An emerging public health consensus is that the dynamics of malaria in the Amazon River basin is unstable, with waves of disease emergence accompanied by explosive epidemics in many localities. These are usually associated with changes to natural environments and ecologies, waves of economic development, and migratory influxes between endemic and non-endemic areas. This dynamic process has challenged control programmes developed to mitigate the burden of malaria in the Amazon [
22].
There are several vectors of malaria in the Neotropics that vary in importance at the local, regional and continental scale [
23]. Although the highest rates of experimental
Plasmodium infection have been found in
Nyssorhynchus aquasalis and
Nyssorhynchus albitarsis [
24], with the latter presenting higher biting indices [
25],
Nyssorhynchus darlingi has the highest rates of natural infection and is seen as the most important malaria vector through much of South America [
25,
26] (herein
Nyssorhynchus is elevated from subgenus to genus rank, as proposed by Foster et al. [
27]). This species is the most anthropophilic, endophilic and opportunistic vector, and is relatively abundant in the more populated localities of the Amazon [
16]. It is also strongly associated with human environments in deforested areas in the Amazon [
28,
29].
A great variety of potential vectors have been recorded in the deforested areas of the Amazon [
30‐
32], but the frequent occurrence of morphologically indistinguishable sibling species, which form species complexes [
33], means that vectors are often misidentified or cryptic species boundaries go undetected [
34‐
36]. Some vectors, such as
Nyssorhynchus triannulatus and
Anopheles peryassui, inhabit the forest edge, away from domestic environments and conventional vector control activities, but may be associated with human activity such as deforestation [
37]. Failure to properly elucidate the diversity present in vector species complexes at locations where forest gives way to frontier settlements will likely obstruct the understanding of vector ecology and the
Plasmodium transmission cycle associated with deforestation in the Amazon and impede efforts to develop strategies to control frontier malaria [
10,
38]. It is, therefore, essential to develop and employ a range of tools to discover potential vectors in malaria endemic areas.
Molecular tools are commonly employed to identify known species and delimit new species [
39]. DNA barcoding is now seen as an important first step in biodiversity assessment and sorting specimens into tentative species, and
COI the marker of choice because of the availability of universal primers that amplify across a diverse range of species, and the relatively low intraspecific and high interspecific divergence that occurs at this locus. It is clear that barcoding approaches are helping to resolve morphologically indistinguishable species within Anophelinae [
31,
34,
40,
41].
Although barcoding studies frequently employ phylogenetic (and normally NJ) trees to delimit species, resolving intraspecific from interspecific phylogenetic structure is frequently subject to observer bias [
42,
43]. Approaches explicitly designed for empirically and objectively delimiting species boundaries have been developed, among which are clustering and tree-based approaches, which do not require a priori definitions of taxa [
44] or even a threshold for intraspecific diversity [
45]. The use of such approaches can therefore allow species richness and delimitation to be independently estimated and tested against a priori species morphological definition.
The current study aims to describe Anophelinae species diversity in rural settlements affected by frontier malaria in the Amazon River basin, employing clustering-based (Automatic Barcode Gap Discovery; [
44]) and tree-based (multi-rate Poisson tree processes; [
45]) species delimitation approaches. The ability to describe diversity and delimit species in this setting is fundamental to understanding the vectors driving malaria transmission and allows for the development of more effective vector control and management strategies in frontier settlements in the Amazon River basin. In the study we pay particular attention to vector complexes and the potential for these to harbour new morphologically similar and potentially medically important species.
Discussion
The pattern of deforestation and anthropogenic changes in natural environments, especially in the Amazon tropical rainforest, and the insidious poverty of settlers living in precarious conditions, have been associated with increases in the incidence of malaria [
62]. Compounding this problem in areas of the Amazon River basin with active malaria is the primary vector
Ny. darlingi, which is a generalist and opportunistic species that can blood-feed both indoors and outdoors complicating the dynamics of transmission [
63]. There are also other important vectors involved in malaria transmission in the region, depending on the local ecological conditions. Considering the urgent need for studies focusing on practical malaria that includes accurate identification of malaria vectors, this study demonstrates the capability of the barcode region of the
COI mitochondrial gene and commonly used species delineation approaches to delimit a diverse range of Anophelinae species found in rural settlements affected by frontier malaria in the Amazon River basin. After assigning collection specimens to 19 morphospecies, conservative estimates delimit between 23 and 27 potential species, up to 13 of which appear to be new species.
Several species are well resolved in our phylogenetic tree and consistently delineated as coherent groups across analyses. These include
Ny. darlingi,
Ny. rangeli and
An. intermedius.
Ny. darlingi is the primary vector of malaria in the Amazon River basin [
16]. It is a highly anthropophilic species and can vary from endophagy to exophagy, and endophily to exophily [
64]. As such, vector control can be extremely challenging and the presence of this species at six rural settlements in Acre and Amazonas is significant for malaria transmission and vector control. In addition,
Ny. rangeli has been found to be important in local malaria transmission in Colombia [
65] and its larval habitat is associated with human-modified environments [
66]. Its presence in rural settlements in Acre and Rondônia may indicate a potential role in frontier malaria at these localities.
Anopheles intermedius sequence data (a singleton from São Paulo state) was included in the analysis as a reference as it is a potential malaria vector in the region, with natural infection detected in Amapa [
67], Pará [
68], and French Guiana [
69]. However, this species was not detected at any of the study’s collection sites.
Although
Ny. braziliensis was recovered as a monophyletic species and singularly grouped in both species delineation approaches, the species appeared to be highly diverse and partitioned into multiple groups in the relaxed ABGD partition. Insufficient sampling meant that we could not attribute any significance to this structure. The local abundance of this species in the Amazon River basin varies considerably [
37,
67,
70] but it appears to be common in human-modified environments [
71]. The species is highly anthropophilic [
37] and it has been found naturally infected with
P.
malariae [
70],
P.
vivax [
25,
72] and
P.
falciparum [
25,
73]. Its presence in rural settlements may therefore have implications for frontier malaria transmission.
To date, there is little genetic data available for
An.
peryassui, whose range covers most of northern South America. There is some evidence to suggest that the species may play a role in malaria transmission, with natural infection by
P. falciparum and
P. vivax reported in the state of Amazonas [
74]. The current study included specimen reference data from Colombia in Gómez et al. [
31], the only other source of
An.
peryassui genetic data available in GenBank. Results of the analyses demonstrated that
An.
peryassui appears to exist as a species complex with at least two (and possibly three) lineages identified, with the first found in Rondônia and the second found in Acre, Amapa, Colombia and Para (with the Amapa haplotype possibly splitting into a third lineage). However, the systematics of this complex remains poorly understood, because of the lack of material from the type locality in Rio Bonito in the state of Minas Gerais.
The genetic diversity of
An. fluminensis has also been poorly explored. This lack of interest may be due to the understanding that
An. fluminensis is not an important malaria vector [
75], although specimens identified as
An. near
fluminensis have been incriminated as a malaria vector in the department of Junin, Peru [
76] and more recently the species was found naturally infected by
Plasmodium malariae in the Atlantic Forest of São Paulo, Brazil [
77]. A study of mosquito diversity in the Ecuadorian Amazon found some genetic support for
An. near
fluminensis, demonstrating a 2.75–3.77% difference between sequences from the province of Orellana in Ecuador and São Paulo state, Brazil. However, despite the identification of similar levels of diversity between the two regions in this study, this is considered to be intraspecific, according to the approaches used, and grouped with the topotype specimen (Accession number: MF381677) from Rio de Janeiro. However,
An. fluminensis was split into two sympatric lineages (with Ecuadorian specimens and sensu stricto specimens grouped together), which, given their distinct evolutionary histories, may be of varying epidemiological importance. Clearly,
An. fluminensis has, to date, been understudied and overlooked but, given the species diversity that has been identified in this study from specimens morphologically identified as
An. fluminensis and the limited sampling effort (only collected from Acre), a more thorough evaluation of lineage diversity and relationships across its considerable South American range is to be encouraged.
Like
An.
peryassui and
An. fluminensis, there are scant genetic data available for
An. costai. The species appears to be found mainly in forest habitat [
78] and is not believed to be an important malaria vector. However,
An. costai has been frequently misidentified as
An.
mediopunctatus [
79], which has been found naturally infected with
Plasmodium vivax in the state of Amazonas [
74] and it may yet be recognized as epidemiologically important. This study reveals that
An. costai exists as a diverse species complex (between 3 and 6 lineages), and several of its lineages have geographical distributions that span the Amazon River basin and beyond. More extensive sampling will be required to determine whether
An. costai s.s. lineage identified in the clustering-based delimitation occurs in the Amazon River basin, while the ecologies of each species complex member should be explored to determine their potential importance in rural settlements affected by frontier malaria.
Anopheles punctimacula ranges from Mexico to Argentina and the Caribbean and has previously been implicated as a malaria vector in Panama [
80] and Colombia [
81], although this occurred prior to
An.
malefactor and
An.
calderoni emerging from this taxon [
82,
83].
Anopheles punctimacula has previously been reported to exist as a species complex, with at least two lineages from Panama supported across multiple genes [
36]. The two lineages identified in this study appear to be distantly related to those identified in Loaiza et al. [
36], which included the sensu stricto form collected near the type locality in Colón, Panama (and consistently grouped with the Lineage A and B form denoted in that study). One of these lineages exists over quite a considerable range (~ 1000 km; Acre, Brazil—Putumayo, Colombia) and further studies of the phylogeny and ecology of these taxa necesitate employing a range of molecular and morphological markers and more comprehensive sampling in order to more clearly define evolutionary relationships, geographical boundaries, ecological niches and, ultimately, their role in malaria transmission in the region.
Anopheles malefactor, previously elevated from synonomy with
An. punctimacula [
82], collected from a rural settlement in Acre, was clearly distinct from the
An. malefactor from the Panama (type locality) and Colombia, and appears to be a new species and sister to a newly delineated
An. fluminensis G1 lineage.
The close relationships observed among the
Ny. konderi and
Ny. oswaldoi complexes indicate that these should be considered a unique species complex named Oswaldoi-Konderi [
34]. There are now considered to be five species in this Oswaldoi–Konderi Complex, which include
Ny. oswaldoi s.s.,
Ny. oswaldoi A,
Ny. oswaldoi B,
Ny.
konderi and
Nyssorhynchus nr.
konderi [
34,
61]. Although
Ny. konderi and
Ny.
oswaldoi are generally not considered vectors of human malaria, Quiñones et al. [
65] found
Ny. oswaldoi B (although denoted
Ny. oswaldoi in their study) infected with
P. vivax in the state of Putumayo, Colombia. The complex may therefore be of epidemiological importance. Although all five species could be effectively delineated in the current study, only four were identified from the collection sites in Acre, Amazonas and Rondônia.
Ny. oswaldoi s.s. was identified from a site south of Amazonas and (with the inclusion of other data from GenBank) it can also be found Espírito Santo state (type locality; Accession number: JF923721), Paraná, Rondônia and São Paulo. This range covers both the Paraná and Amazon river basins, which are connected by the Parapetí River alluvial fan and may allow fluvial species relatively uninhibited expansion between basins [
84]. Whereas
Ny. oswaldoi A appears to be found through much of the Amazon River basin (Amazonas, Acre, Pará, Rondônia),
Ny. oswaldoi B was not identified at the collection sites, although it has previously been identified from a site in the northern reaches of the Brazilian Amazon (Santana, Amapá; [
34]). A
Ny. oswaldoi specimen (Field Specimen ID: SP22_9) from Pariquera-Mirim, Pariquera-Açu, São Paulo was included in this study to aid in delineating geographic distribution. However, this specimen was consistently resolved from
Ny. oswaldoi s.s. and is likely a new species within the Oswaldoi–Konderi Complex. The
Ny. konderi specimens collected in this study were confined to sites in the state of Acre but this species has also been collected from sites in Amapá (previously denoted
An. konderi Amapa [
60]). Saraiva et al. [
34] also identified this species from Amazonas, Pará and Rondônia. With respect to
Ny. nr.
konderi, previous studies have found this species in Colombia, Ecuador, Peru [
61] and the Brazilian states of Amazonas and Rondônia [
34]. In this study, this species was collected from Amazonas but also from a large number of rural settlements in Acre.
Recent studies have confirmed
Ny. benarrochi as a species complex, with the identification of
Ny. benarrochi B in Colombia [
85] and Peru [
86]. Although
Ny. benarrochi is predominantly zoophilic [
87] and therefore not believed to be an important malaria vector, highly anthropophilic behaviour does occur in some areas and it may be play a role in malaria transmission in the Peruvian Amazon [
85,
88]. This study confirms the existence of
Ny. benarrochi B, using data from Conn et al. [
86], in rural settlements in the state of Acre. It also identifies at least one other
Ny.
benarrochi lineage from Acre and Rondônia (and perhaps representing two). Unfortunately, this lineage cannot be confirmed as
Ny. benarrochi s.s. as material from the type locality in the municipality of La Ceiba, Trujillo State, Venezuela is unavailable. It is, therefore, essential that collections be obtained from this type locality in order to undertake a meaningful systematic review of the
Ny.
benarrochi complex and determine whether species diversity within this complex is associated with variation in anthropophilia and vector competence.
Nyssorhynchus triannulatus is found throughout Central and South America, and has been incriminated in the transmission of human malaria in the states of Amapa [
67], Amazonas [
74] and Pará [
68], Brazil. Recent studies have shown that
Ny. triannulatus forms a species complex with two other recently identified species;
Ny. halophylus [
89] and
Ny. triannulatus C [
90].
Nyssorhynchus triannulatus sensu lato has been found to form paraphyletic (at a combined ITS2,
white and
COI gene tree; [
91]) and monophyletic sister [using RAPD markers; 92] relationships with an
Ny.
halophylus and
Ny. triannulatus C clade. Within
Ny.
triannulatus s.l. two further clades have been recovered with the
COI gene, one from Central America and northern Colombia and the other primarily from the Amazon river basin [
91], and several additional biologically meaningful subclades from within the Amazonian clade were also proposed, based on multiple gene (
COI,
white and ITS2) and haplotype network analysis. The haplotypes included in this study come from the Amazonian states of Acre and Amapá, as well as from the states of Minas Gerais to the east, Espírito Santo on the Atlantic coast, São Paulo in the south east of Brazil, and neighbouring Colombia. Despite a considerable geographic distribution and habitat range [
91,
92], results from the current analysis show that, although the
Ny. triannulatus s.l. clade is extremely diverse, relationships among its
COI haplotypes cannot be meaningfully partitioned into separate species i.e., all branch structure and pairwise distances within
Ny. triannulatus are considered intraspecific. It appears that relationships may be better explained by complex population histories [
92], where elevated levels of genetic diversity have been maintained perhaps by historical fragmentation, secondary contact and gene-flow followed by more recent divergence due to geographical isolation. Further phylogenetic and species delimitation analyses at multiple loci may reveal more compelling support for species designation within
Ny. triannulatus, as phylogenetic analysis of
COI frequently fails to resolve well supported species of Anophelinae mosquitoes, e.g.,
Ny. strodei and
Ny. albertoi. Conflict among gene trees due to incomplete lineage sorting and horizontal gene transfer (gene-flow) is pervasive in very closely related species, making molecular identification of such species difficult. High density codominant markers such as single nucleotide polymorphisms (SNPs) offer some opportunities to overcome such phylogenetic challenges [
93‐
96] and an exploration of the hierarchy of structure within
Ny. triannulatus (and
Ny. braziliensis) may be better achieved using such markers in combination with population genetic approaches [
97].
Several of the morphospecies collected in this study are highly diverse complexes, the diversity of which is supported by a range of ecological, morphological and genetic data. However, the use of species delimitation approaches with the
COI gene failed to detect several of these species boundaries. Currently, 10 species have been identified within the
Ny. albitarsis complex, five of which (
Ny. albitarsis,
Ny. albitarsis H,
An. deaneorum,
Ny. marajoara and
Ny. oryzalimnetes) were collected in this study. A range of morphological and molecular data has been employed to resolve and support species in this complex [
98‐
100]. Nevertheless, the close relationships observed among
Ny. albitarsis H,
Ny. deaneorum and
Ny. marajoara at the
COI gene in the present study were consistent with patterns of intraspecific variation when employing cluster- and tree-based species delimitation. It appears that species delimitation of this complex at the
COI gene will fail to detect important species boundaries supported by other data and further exploration and discovery of species diversity in the Albitarsis Complex will require a multilocus approach, possibly using loci such as ITS2 and the
white gene, which have been used to resolve many of the species within the complex [
101,
102].
Similar issues were encountered when dealing with the Nuneztovari Complex and Strodei Subgroup. The former is comprised of
Ny.
dunhami,
Ny. goeldii,
Ny.
nuneztovari [
103], supported by a range of morphological and molecular data [
104‐
106]. Its geographical distribution runs from the Isthmus of Panama to northern South America and
Ny. nuneztovari sensu lato is considered one of the most important malaria vectors in the region [
26]. A recent study by Scarpassa et al. [
40] found
Ny.
nuneztovari s.l. haplotypes were variously identified as
Ny. dunhami,
Ny.
goeldii,
Ny. nuneztovari and an additional unknown clade. They found that population genetic and phylogenetic analysis were in some agreement, although lineage delineation and relationships among
COI trees that differed in sampling effort were to some degree incongruent. Although the current study combined the sequences from Scarpassa et al. [
40] with those from specimens collected from the Brazilian Amazonian states of Acre, Amapá, Amazonas, Pará, Rondônia, it failed to delineate any species within the
Nyssorhynchus nuneztovari complex. The Strodei Subgroup is made up of
Ny. albertoi,
Ny. strodei [
107],
Ny. striatus [
108],
Ny. rondoni and finally
Ny. arthuri, which is comprised of four species A - D [
35]. Given the systematic complexity of this group and the recent emergence of many of its species, the importance of these species in malaria transmission is unknown. However,
Ny.
strodei has previously been found naturally infected with
P.
vivax in Ariquemes, Rondônia [
73], although this case may refer to
Ny. arthuri C [
35].
Nyssorhynchus albertoi and
Ny. strodei, despite having considerable support as separate species within the Strodei Subgroup [
107], are known to be unresolved with the
COI gene [
35] and, unsurprisingly, species delimitation in the current study using this same gene failed to identify the respective species. The other members of the subgroup included in the study were resolved and clearly delineated. As in Bourke et al. [
35],
Ny. arthuri A was not detected from the Amazon region and
Ny. arthuri C has so far only been identified from the state of Rondônia. Further analysis of this species range and habitat is required before its epidemiological importance in rural settlements can be determined.
The work presented here demonstrates a clear advantage to employing species delimitation approaches when the objective is to explore species diversity and discover new species in Anophelinae where cryptic species boundaries are common. Due to the relative speed at which
COI can be sequenced and analysed, the study demonstrates the power of single-locus species delimitation approaches to establish a baseline of species diversity in Anophelinae in remote and unexplored regions of the Amazon River basin. However, it must be clearly stated that single-locus data alone should only be used to provide a preliminary description of species boundaries (possible gene tree-species tree discordance) and the new species delimited in this study remain putative. Rather, it allows for the establishment of viable species hypotheses, to be tested against a range on independent data sources that may be, for example, morphological, molecular and/or ecological in nature. In particular, there have been few or no classical studies of Anophelinae taxonomy conducted in the Amazon River basin in recent years, and further studies of species diversity in Anophelinae are, therefore, encouraged to better explore morphological variation among these species. Future work on species exploration and discovery in the Amazon River basin should also employ a multilocus delimitation approach [
43,
109] to better enable the resolution of all recognized species within important complexes, such as Albitarsis and Nuneztovari. In addition, the study demonstrates that exceptional diversity detected in morphospecies can be consistent with a model of intraspecific diversity and is suggestive of a complex evolutionary history that may be better explored using high-density markers and population genetic approaches.
This study has been successful in revealing a large number of unknown Anophelinae species that are likely to be new to science and occur in areas with endemic malaria transmission. One of the great challenges for malaria control in the Amazon River basin is the transmission that occurs outside of rural dwellings and the detection of new species that belong to groups containing important vectors will, therefore, have an important impact on the development of effective vector management and control strategies in the region. The World Health Organization recommends that vector management and control interventions should take account of potential impacts on the environment and biodiversity and should be focused on avoiding unintended impacts on non-vector species. The findings from this study will assist in refining such strategies, help build capacity in public health entomology and provide an important contribution to effective malaria control in the region.