Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae

Abstract

Background
Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. It has been proposed that occupation of new microhabitats may trigger selection for distinct phenotypic variants in these blood-sucking bugs. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood.

Methods/results
We combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA sequence analyses to study Rhodnius ecuadoriensis populations from across the species’ range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs primarily from vertebrate-nest microhabitats (Ecuador/Peru) are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs from wet-forest palms (Ecuador) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs primarily from dry-forest palms (Ecuador) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently evolving lineages, with little within-lineage phylogeographic structuring or differentiation.

Conclusions
We report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (nest-dwelling southern-Andean vs palm-dwelling northern bugs; and palm-dwelling Andean vs lowland), and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs primarily from vertebrate-nest (but not palm) microhabitats. This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selection (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter and stouter heads). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model system for the study of phenotypic change under ecological pressure"Image missing" .

Full text

Abad‑Franchetal. Parasites Vectors (2021) 14:195
https://doi.org/10.1186/s13071‑021‑04647‑z
RESEARCH
Under pressure: phenotypic divergence
andconvergence associated withmicrohabitat
adaptations inTriatominae
Fernando Abad‑Franch1,2* , Fernando A. Monteiro3,4*, Márcio G. Pavan5, James S. Patterson2,
M. Dolores Bargues6, M. Ángeles Zuriaga6, Marcelo Aguilar7,8, Charles B. Beard4, Santiago Mas‑Coma6 and
Michael A. Miles2
Abstract
Background: Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse
ecotopes. It has been proposed that occupation of new microhabitats may trigger selection for distinct phenotypic
variants in these blood‑sucking bugs. Although understanding phenotypic variation is key to the study of adaptive
evolution and central to phenotype‑based taxonomy, the drivers of phenotypic change and diversity in triatomines
remain poorly understood.
Methods/results: We combined a detailed phenotypic appraisal (including morphology and morphometrics) with
mitochondrial cytb and nuclear ITS2 DNA sequence analyses to study Rhodnius ecuadoriensis populations from across
the species’ range. We found three major, naked‑eye phenotypic variants. Southern‑Andean bugs primarily from
vertebrate‑nest microhabitats (Ecuador/Peru) are typical, light‑colored, small bugs with short heads/wings. Northern‑
Andean bugs from wet‑forest palms (Ecuador) are dark, large bugs with long heads/wings. Finally, northern‑lowland
bugs primarily from dry‑forest palms (Ecuador) are light‑colored and medium‑sized. Wing and (size‑free) head shapes
are similar across Ecuadorian populations, regardless of habitat or phenotype, but distinct in Peruvian bugs. Bayesian
phylogenetic and multispecies‑coalescent DNA sequence analyses strongly suggest that Ecuadorian and Peruvian
populations are two independently evolving lineages, with little within‑lineage phylogeographic structuring or
differentiation.
Conclusions: We report sharp naked‑eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis
(nest‑dwelling southern‑Andean vs palm‑dwelling northern bugs; and palm‑dwelling Andean vs lowland), and sharp
naked‑eye phenotypic similarity of typical, yet genetically distinct, southern‑Andean bugs primarily from vertebrate‑
nest (but not palm) microhabitats. This remarkable phenotypic diversity within a single nominal species likely stems
from microhabitat adaptations possibly involving predator‑driven selection (yielding substrate‑matching camouflage
coloration) and a shift from palm‑crown to vertebrate‑nest microhabitats (yielding smaller bodies and shorter and
stouter heads). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against
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Open Access
Parasites & Vectors
*Correspondence: abadfr@yahoo.com; fam@ioc.fiocruz.br
1 Núcleo de Medicina Tropical, Faculdade de Medicina, Universidade de
Brasília, Brasília, Brazil
3 Laboratório de Epidemiologia e Sistemática Molecular, Instituto
Oswaldo Cruz‑Fiocruz, Rio de Janeiro, Brazil
Full list of author information is available at the end of the article
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
Introduction
Triatomine bugs transmit Trypanosoma cruzi among the
mammalian hosts they associate with in shared micro-
habitats [1, 2]. Bugs that occur in human-made habitats
may transmit the parasite to people, fueling the spread of
Chagas disease [1, 3]. It has been proposed that occupa-
tion of new microhabitats may trigger selection for dis-
tinct phenotypic variants in these blood-sucking bugs [4].
Over the last few decades, molecular studies have iden-
tified examples of phenotypic convergence or divergence
at several systematic levels [5–9]. Perhaps most remark-
ably, molecular phylogenetic analyses suggest that none
of the three genera to which the main Chagas disease
vectors belong (Triatoma, Panstrongylus and Rhodnius),
which are all defined after morphological characters [1],
is monophyletic [8–14]. A few named species have been
shown to be phenotypic variants of another species [8];
for example, Triatoma melanosoma is now regarded
as a T. infestans chromatic variant [15, 16]. Conversely,
some named species have been shown to include several
cryptic taxa [8]; for example, Rhodnius robustus (s.l.) is
composed of at least five distinct lineages, two of which
have been formally described as R. montenegrensis and R.
marabaensis [8, 17–21]. Further examples of phenotype/
genotype mismatch are reviewed in [8].
e sometimes striking variation of triatomine-bug
phenotypes has been attributed to a propensity of mor-
phological characters to change in response to chang-
ing habitat features [2, 4, 22]. us, within-species
divergence may be driven by habitat shifts (e.g., wild
to domestic) involving subsets of genetically homoge-
neous populations [4], and the use of similar habitats
by genetically distinct populations may result in con-
vergence or in the retention of ancestral phenotypes
[2, 4, 23]. When using morphological characters only,
therefore, taxonomists are in peril of describing spu-
rious species or overlooking cryptic taxa [8]. Despite
the practical importance of accurate taxonomic judg-
ment when the organisms of interest transmit a
life-threatening parasite, the degree, direction and
underlying causes of phenotypic change and diversity
in triatomines remain obscure.
In this paper, we combine a detailed pheno-
typic characterization, qualitative and quantitative,
with mitochondrial cytochrome b gene (cytb) and
nuclear ribosomal second internal transcribed spacer
(ITS2) DNA sequence analyses to study Rhodnius
ecuadoriensis populations spanning most of the geo-
graphic/ecological range of the species (Fig. 1). Rho-
dnius ecuadoriensis is a major vector of T. cruzi in
western Ecuador and northwestern Peru [24–26]. In
Ecuador, northern wild populations are primarily asso-
ciated with the endemic Phytelephas aequatorialis palm
in both Andean wet forests and lowland dry forests; in
the dry inter-Andean valleys of southwestern Ecuador,
where native palms are rare or absent, wild R. ecuado-
riensis seem to primarily associate with arboreal squir-
rel nests [1, 2, 27–33]. e natural habitats of Peruvian
populations remain unclear, with a few records suggest-
ing association with vertebrate nests/refuges in hollow
trees and perhaps cacti [2, 26, 27, 34]. In addition, some
R. ecuadoriensis populations have adapted to live in
and around houses in coastal Ecuador and, especially,
in the dry valleys of southwestern Ecuador and north-
western Peru—where the bugs contribute to endemic
Chagas disease [24–26, 35–39]. In coastal and in south-
western Ecuador, bugs from wild and human-made
habitats have overlapping phenotypes [40] and identi-
cal or nearly identical mitochondrial cytb haplotypes
[41]; this, together with frequent, rapid reinfestation
of insecticide-treated dwellings [36, 42] and prelimi-
nary microsatellite data [41, 43], indicates that wild and
non-wild R. ecuadoriensis populations are locally highly
interconnected. Our comparative phenotypic and
genetic analyses cover most of the known geographic
and ecological diversity in R. ecuadoriensis; they reveal
that phenotypic divergence and convergence can both
occur within a single nominal triatomine-bug species,
and suggest that microhabitat adaptations likely play a
crucial role in this phenomenon.
Methods
Origins ofbugs andqualitative phenotype assessment
We compared R. ecuadoriensis type specimens from La
Toma, Catamayo, Loja Province, Ecuador (Laboratório
Nacional e Internacional de Referência em Taxonomia
de Triatomíneos [LNIRTT], Fiocruz, Brazil; [44]) and
canonical descriptions of the species [1, 45] with field-
collected Ecuadorian bugs, including (i) northern bugs
from Ph. aequatorialis palms of Santo Domingo de los
Tsáchilas Province (Andean wet forests; “Tsáchilas” here-
after) and Manabí Province (lowland dry forests), and (ii)
primarily nest-dwelling southern-Andean bugs caught in/
around houses in El Oro (Puyango river basin) and Loja
excess reliance on phenotype‑based triatomine‑bug taxonomy, and confirm the Triatominae as an informative model
system for the study of phenotypic change under ecological pressure.
Keywords: Triatominae, Rhodnius, Chagas disease, Systematics, Morphometrics, Genetics
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
(Catamayo-Chira basin) provinces (Fig. 1). CAC Cuba
(University of Brasília, Brazil) supplied additional field-
caught southern-Andean Peruvian bugs from dwellings
of Suyo (department of Piura, Catamayo-Chira basin)
and Cascas (department of La Libertad, Chicama basin)
[39] (Fig. 1). Finally, bugs from two colonies founded
with material collected from, respectively, Ph. aequato-
rialis palms of Manabí and houses of northwestern Peru
(department of Cajamarca, Mashcon-Marañón basin)
were supplied by J Jurberg (LNIRTT) (see details in Addi-
tional file1: TableS1). Using these specimens, which we
note were fresh at the time of processing, we conducted a
detailed review of external morphological and chromatic
characters central to classical triatomine-bug taxonomy
[1, 45] and placed the results in the broader context of
what we know about the systematics, biogeography and
ecology of R. ecuadoriensis [1, 2, 8, 18, 25–36, 38–43]. In
particular, we emphasize that northern populations pri-
marily exploit palm-crown microhabitats just like most
Rhodnius species do [2, 29], whereas wild southern-
Andean populations are primarily associated with verte-
brate tree-nests in dry ecoregions where palms are either
Fig. 1 Sampling of Rhodnius ecuadoriensis populations. The map shows the approximate known distribution of R. ecuadoriensis in Ecuador,
including the western‑Andean wet premontane (or “cloud”) forests (green), the drier coastal lowlands (orange), the Andes–lowland transition and
the southern, dry inter‑Andean valleys associated with the Puyango (PB; blue) and Catamayo‑Chira basins (C-CB; red). The approximate geographic
location of each study population in Santo Domingo de los Tsáchilas, Manabí, El Oro, Loja and Peru is indicated by green, orange, blue, red and black
circles, respectively; just one specimen was available from both Suyo and Cajamarca. Most Peruvian material came from Cascas, in the middle‑upper
Chicama basin (ChiB), La Libertad. Putative barriers to past or current bug dispersal are indicated with gold‑colored double lines: the Huamaní range
(HR), which closes off the C‑CB to the south; the Sechura desert (SeD) on the northern Peruvian coastal plains; and the Huancabamba depression
(HD). The semiarid Santa river basin (SB) appears to mark the southern limit of R. ecuadoriensis’ range. Rhodnius ecuadoriensis also occurs along the
middle‑upper (inter‑Andean) stretches of the Marañón river valley (MV). See details in Additional file 1: Table S1 and main text
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
rare or absent [2, 8, 25–34]. Our sampling thus captures
this key ecological difference, and we will hereafter refer
to northern Tsáchilas and Manabí bugs as “primarily
palm-dwelling” and to southern-Andean El Oro, Loja and
Peru bugs as “primarily nest-dwelling” [2]. We note again
that a growing body of evidence suggests that wild and
non-wild R. ecuadoriensis populations are locally highly
cohesive [36, 40–43].
Traditional morphometrics—heads
We used 79 adult R. ecuadoriensis specimens for this part
of the study; 77 were fresh bugs and two were collection
bugs (see specimen details in Additional file1: TableS1).
We measured lateral- and dorsal-view, calibrated head
images (Fig. 2) and calculated descriptive statistics for
the whole sample and for ecological populations (pri-
marily palm-dwelling vs primarily nest-dwelling) and
geographic groups. To assess head-size variation, we esti-
mated population means (over all head measurements)
and likelihood-profile 95% confidence intervals (CIs)
by fitting Gaussian generalized linear models (identity
link-function, no intercept) using package lme4 1.1-21
[46] in R 3.6.3 [47]. For multivariate analyses, log-trans-
formed data were centered by row to remove isometric
size; the resulting “log-shape ratios” [48, 49] were used as
input for principal component analysis (PCA) on covari-
ances. e derived principal components (PCs or “shape
variables”) were submitted to canonical variate analysis
(CVA). We assessed the overall significance of multi-
variate CVA using Wilks’ λ statistic [50]. We computed
canonical vectors (CVs) and used the first two CVs to
plot the position of each specimen on the shape discri-
minant space; “convex hulls” enclosing all points within
each group were overlaid on the plots. Finally, a space of
size-free shape variables was constructed by explicitly
removing size (represented by PC1) from the measure-
ments; for this, residuals of linear regression of PC1 on
each measurement were used as new variables for size-
free CVA [51–53]. e derived CV1 and CV2 were plot-
ted as described above. Different parts of these analyses
were conducted using R 3.6.3 [47], JMP 9.0 (SAS Insti-
tute, Cary, NC, USA), and NTSYS 2.10y [54].
Geometric morphometrics—heads andforewings
Dorsal-head and forewing images of, respectively, 84
and 82 adult bugs (all fresh except for 1 collection bug;
see details in Additional file1: TableS1) were digitized
for a series of two-dimensional coordinates (Fig.2) using
tpsDig 1.18 [55]. Raw coordinates were subjected to the
Procrustes superimposition algorithm [56] and thin
plate spline (TPS) analysis using tpsRelw 1.18 [57]. We
used TPS to compute “partial warps” with affine (global
stretching) and non-affine (non-linear localized distor-
tions or “shape changes”) components. PCA of partial
warps yielded shape components (“relative warps”),
which were subjected to CVA as described above. We
also computed: (i) “centroid sizes” as overall measures of
head and forewing size; and (ii) Mahalanobis distances
between population pairs [22, 40, 49].
Molecular analyses
We used 72 R. ecuadoriensis fresh specimens for mito-
chondrial DNA analyses and a subset of 17 bugs for
nuclear DNA analyses (see details in Additional file 1:
TableS1); R. colombiensis, R. pallescens and R. pictipes
were used as outgroup taxa. We extracted DNA from bug
legs using DNeasy kits (Qiagen, Valencia, CA). A 663-bp
fragment of the mitochondrial cytochrome b gene (cytb)
and the complete nuclear ribosomal ITS2 (707–715 bp)
were amplified, purified and Sanger-sequenced as pre-
viously described [9, 11, 17]. We visually inspected the
chromatograms of forward and reverse DNA strands
with SeqMan Lasergene 7.0 (DNASTAR Inc., Madi-
son, WI, USA); in inspecting ITS2 chromatograms, we
particularly checked for the “double signal” typical of
paralogous pseudogene sequences [58, 59]. We aligned
consensus and outgroup sequences in MAFFT 7.0 [60],
using the L-INS-i algorithm and further manual fine-
tuning. We then computed descriptive statistics using
MEGA X [61].
Fig. 2 Measurements and landmarks used in the morphometric analyses. Head measurements were used for traditional morphometrics: A
Maximum width across the eyes, B postocular distance (posterior eye limit to head/neck limit), C length of antenniferous tubercle (anterior eye
limit to distal tip of tubercle), D anteocular distance (anterior eye limit to base of anteclypeus), E maximum diameter of the eye, F length of second
rostral segment, G length of third rostral segment. The yellow dotted line indicates head length, which we used, together with A, to compute head
length:width ratios. Green dots show the landmarks used for geometric morphometrics
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
e best-fitting model of base substitution for each
marker was selected using the Bayesian information
criterion (BIC) in bModelTest 1.2 [62]. We used the
*BEAST 0.15 package of the BEAST 2.6 platform [63,
64] to reconstruct Bayesian locus-specific phylogenetic
trees and multispecies-coalescent species trees [63], with
three independent runs (5 × 107 generations) for each
analysis. For locus-specific trees, we used the coalescent
model and sampled parameters every 10,000 genera-
tions; for species trees, we used the Yule model of specia-
tion and sampled parameters every 50,000 generations.
We evaluated (using Tracer 1.7 [65]) parameter conver-
gence and proper mixing by inspecting individual chains
and by checking that effective sample sizes (ESSs) were
sufficiently large; in our case, all ESSs were ≥ 104. As a
complement to phylogenetic analyses, we used pegas 0.14
[66] to build haplotype networks (infinite-sites model,
Hamming distances) for both loci.
It has been suggested, based on limited mitochondrial
DNA data, that R. ecuadoriensis may comprise two dis-
tinct lineages: one primarily from Ecuador (“group I”)
and the other primarily from Peru (“group II”) [6, 18]. We
set out to formally assess the data support for this “two-
lineage” hypothesis (H1), relative to the null hypothesis
of a single lineage (H0), by computing and comparing
the marginal likelihood (mL) and posterior probability of
each hypothesis [67, 68]. To do this, we first used our cytb
and ITS2 sequence data to estimate species trees under
both H1 (with each R. ecuadoriensis sequence assigned
to a pre-defined, geography-based group, either “Ecua-
dor” or “Peru”; Fig.1) and H0 (with all R. ecuadoriensis
sequences assigned to a single group). We then estimated
the mL of each species tree using two approaches: (i)
nested sampling [68, 69], as implemented in the NS 1.1
package [69] of BEAST 2.6 [63], with 5 × 106 Markov
chain Monte Carlo (MCMC) generations, 2 × 104 sub-
chain length and five active points; and (ii) path sampling
(also known as “thermodynamic integration”) [68, 70],
as implemented in the Model Selection 1.0.1 package of
BEAST 2.6 [64], with a pre-burn-in of 2 × 105 MCMC
iterations followed by 90 steps of 2 × 106 iterations (50%
burn-in) and each step repeated 500 times. Using the
hypothesis-specific log-mL values, we finally computed
log-Bayes factors (BF) as log-BF = log-mL(H1) − log-
mL(H0) [67]. For two hypotheses with equal prior prob-
abilities, Pr(H1) = Pr(H0) = 0.5, the Bayesian posterior
probability (BPP), given the data (D), of H1 is Pr(H1|D)
= BF/(1 + BF), and the posterior probability of H0 is
therefore Pr(H0|D) = 1 − Pr(H1|D). Kass and Raftery [67]
proposed a set of rules of thumb, derived from those first
suggested by Jeffreys [71], to grade the evidence in favor
of H1: the evidence is weak when 1 ≤ BF < 3; positive
when 3 ≤ BF < 20; strong when 20 ≤ BF < 150; and very
strong when BF ≥ 150. In a two-hypotheses context like
ours, this means that evidence in favor of H1 (or against
H0) would be deemed very strong only if examination of
the data changed the 1:1 prior odds (Pr(H1) = Pr(H0) =
0.5) to a posterior odds of at least 150:1, so that Pr(H1|D)
≥ 0.993 and Pr(H0|D) ≤ 0.007.
We note that the R. ecuadoriensis cytb sequences
studied by Villacis etal. [41, 72] were not publicly avail-
able at the time of writing this report, when the National
Center for Biotechnology Information (NCBI) GenBank
held five such sequences. Sequence AF045715 [73] is
only 399 bp, and sequences KC543508–KC543510 [74]
are identical to some of the haplotypes we found (details
below). Finally, KC543507 from a Manabí bug [74] differs
at three positions from two of our Manabí haplotypes;
one of those substitutions, however, yields a thymine
at a second codon position (position 233 in Additional
file2: Alignment S1) where cytosine is conserved across
all cytb sequences from Rhodnius species we have been
able to examine (e.g., see [8]). is singular non-synon-
ymous substitution (valine instead of alanine as in all
other Rhodnius except R. prolixus and some R. robus-
tus, which have threonine) suggests that KC543507 [74]
may contain base-call errors, and we therefore excluded
this particular sequence from further consideration. Our
analyses, in sum, are based on all reliable R. ecuadorien-
sis sequences of reasonable length (here, 663 bp) that, as
far as we know, are currently available for the two loci we
investigated.
Results
Qualitative phenotypic assessment
All southern-Andean, primarily nest-dwelling bugs from
El Oro, Loja and Peru had comparable, typical pheno-
types [1, 45], whereas northern-Andean specimens from
wet-forest palms (Tsáchilas) were very large and dark
bugs with long, slender heads; northern-lowland bugs
from dry-forest palms (Manabí) had intermediate phe-
notypes (Figs. 3, 4). Below and in Table1 we present
summary descriptions of these diverse putative R. ecua-
doriensis phenotypes (or “forms”); detailed descriptions
are provided in Additional file3: Text S1.
Typical forms: primarily nest‑dwelling southern‑Andean bugs
Primarily nest-dwelling bugs caught in/around houses
in southwestern Ecuador (El Oro, Puyango basin; and
Loja, Catamayo-Chira basin) were virtually identical to
the type material—very small triatomines, light brown-
yellowish with dark-brown stripes and irregular mark-
ings on the body and appendages [1, 45] (Fig.3). Peruvian
bugs were collected in/around houses of the dry middle-
upper Chicama basin (Cascas, La Libertad, approx. 350
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Page 6 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
km south of our fieldwork sites in Loja), except for one
specimen collected in Suyo, Piura, within the Catamayo-
Chira basin (Fig.1; Additional file1: TableS1). e over-
all aspect of Peruvian bugs largely matches that of type
material (Fig. 3). However, Chicama-basin bugs are
noticeably lighter-colored than southern-Andean Ecua-
dorian bugs; this is more evident on the legs, where the
dark mottled pattern is limited to small clusters of dots
and stripes on the basal and distal thirds of femora and
tibiae (Fig.3). e posterior lobe of the pronotum is also
lighter than in Ecuadorian material, and Chicama-basin
bugs are more slender than the typical specimens from
El Oro-Loja. Similar to type material, the heads of these
Peruvian specimens are noticeably short and stout (Fig.3;
see also Additional file3: Text S1).
Intermediate forms: primarily palm‑dwelling
northern‑lowland bugs
Phytelephas aequatorialis palms often harbor wild R.
ecuadoriensis populations in the central coastal province
of Manabí, Ecuador [2, 28, 29, 31]. e overall aspect
and coloration of these lowland, primarily palm-dwelling
bugs largely match those of typical R. ecuadoriensis, but
Fig. 3 Phenotypic diversity in Rhodnius ecuadoriensis. a–d Southern‑Andean populations (primarily from vertebrate tree‑nests but often found
in/around houses): Loja female, dorsal (a) and ventral (b) views; and Peru male, dorsal (c) and ventral (d) views; the arrows in d indicate the lighter
central area of femora (see text). e–h Northern populations (primarily from Phytelephas aequatorialis palms): northern‑lowland Manabí female, dorsal
view (e), northern‑Andean Tsáchilas male, dorsal view (f), northern‑Andean Tsáchilas female, dorsal view (g; the inset highlights the well‑developed
denticle in the distal tip of the fore femora), northern‑Andean Tsáchilas female, ventral view (h). Scale bar: approx. 5 mm
Fig. 4 Heads (lateral view) of southern‑Andean and
northern‑Andean Rhodnius ecuadoriensis: typical bugs primarily
from vertebrate tree‑nests (Loja) vs atypical bugs primarily from
Andean Phytelephas aequatorialis palms (Tsáchilas). Note the striking
divergence, which clearly falls within the range of what are normally
considered interspecies differences in the Triatominae [1]. Drawings
by FA‑F
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
Table 1 Summary of phenotypic and ecological variation in Rhodnius ecuadoriensis
MPP Median process of the pygophore of the external male genitalia
a Detailed descriptions are provided in Additional le3: Text S1
b See [1, 2, 24–43]
c Using the original description of the species [45] as the benchmark; see also [1]
Form Phenotypic traitsaGeography Primary (wild) habitatsbOther habitatsb
El Oro‑Loja‑Peru; typical phenotypecSimilar to species types; pale yellowish;
mottled pattern conspicuous; small
size (body length approx. 13–14 mm);
short and stout heads; MPP pointed
Southern‑Andean populations; inter‑
Andean valleys with dry forests in
southwestern Ecuador (El Oro and
Loja) and northwestern Peru (Pacific
basins and middle‑upper Marañón
basin)
Squirrel tree‑nests (Ecuadorian popula‑
tions); hollow trees with Didelphis
(Peruvian populations); likely also bird
and rodent nests on trees and cacti
Houses and peridomestic structures;
breeding colonies mainly associated
with hen nests, dovecotes and guinea‑
pig pens
Manabí; intermediate phenotype Light brown‑yellowish; mottled pat‑
tern conspicuous; intermediate size
(approx. 15–16 mm); heads longer
than in typical specimens; MPP
pointed
Northern‑lowland populations; dry for‑
ests of coastal (i.e. western) Ecuador
Phytelephas aequatorialis palms Squirrel, bird, rat or mouse nests; occa‑
sionally in man‑made habitats (mainly
peridomestic, with adult bugs often
found invading houses)
Tsáchilas; highly atypical phenotype Dark brown‑black with brown‑reddish
markings; mottled pattern incon‑
spicuous due to very dark background
color; large (approx. 17–18 mm); long
and narrow heads; MPP generally
truncated
Northern‑Andean populations; wet pre‑
montane (or “cloud”) forests (approx.
300–1800 m a.s.l.) along the Andes
foothills in western Ecuador
Phytelephas aequatorialis palms None known; adult (winged) bugs occa‑
sionally found invading houses
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
Manabí bugs have larger bodies and longer, more slender
heads (Fig.3; see Additional file3: Text S1).
Atypical forms: primarily palm‑dwelling northern‑Andean
bugs
In 1998, a male Rhodnius specimen was collected at light
near Alluriquín, Santo Domingo de los Tsáchilas, approx-
imately 900 m a.s.l. on the central-western Ecuadorian
Andes foothills (Fig.1). is wet premontane forest site is
within the range of R. ecuadoriensis, which is not shared
by any other known Rhodnius species [8, 18, 25, 75], but
the morphology and coloration of the specimen differed
strikingly from those of R. ecuadoriensis type material
(Table 1). Field surveys in Alluriquín yielded abundant
material from Ph. aequatorialis palms [28, 76]. ese
bugs are much larger and darker, and have much longer
heads, than typical R. ecuadoriensis (Table1; Figs.3, 4;
Additional file 3: Text S1); they are, however, smaller
than the closely related, light-colored R. pallescens and R.
colombiensis [1, 77] (Additional file4: Figure S1). Naked-
eye phenotype differences between northern-Andean
bugs and typical southern-Andean specimens are in the
range customarily associated with distinct species in the
Triatominae—and tend towards the “highly divergent”
extreme of that range if we consider closely related spe-
cies within the genus Rhodnius [1, 5, 8, 17–19] (Table1;
Figs.3, 4; see also Additional file 3: Text S1 and Addi-
tional file4: Figure S1).
Fig. 5 Traditional morphometrics of Rhodnius ecuadoriensis heads. Upper panels a–c: head size divergence among geographic (a) and ecological
(b) populations. Population codes: TS Tsáchilas (dark green), MN Manabí (orange), EO El Oro (blue), LJ Loja (dark red), PE Peru (black). The primary
habitat of each population is indicated in b: “Palms” for the the northern TS + MN populations (light green) and “Nests” for the southern‑Andean
EO + LJ + PE populations (bright red). Box plots (a, b) show medians (thick horizontal lines), quartiles (boxes) and values that fall within 1.5‑fold
the inter‑quartile range (whiskers); note a single small‑sized outlier (empty circle in a) in the MN population. Colored circles and error bars show
population means and 95% confidence intervals. c Relation between head length (yellow dotted line in Fig. 2) and width (measurement “A” in Fig. 2),
as the mean and range of raw length:width values; note the elongated heads of TS and MN bugs and the shorter, stouter heads of LJ, EO and PE
bugs. d, e Size and shape divergence among geographic populations. d Isometry‑free canonical discriminant analysis of linear head measurements;
the asterisk indicates the position of a specimen from Suyo (Catamayo‑Chira basin, Peru) in discriminant space. e Size‑free canonical discriminant
analysis on the residuals of linear regression of the first principal component on each head measurement. CV Canonical vector. See text for
methodological details
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
Traditional morphometrics—heads
Northern-Andean bugs from Tsáchilas palms clearly
had the largest heads in our sample; northern-lowland
bugs from Manabí palms were smaller than Tsáchilas
specimens but larger than southern-Andean bugs—
among which those from Loja had the smallest heads
and those from Peru were larger on average (Fig. 5a).
Overall, the heads of primarily palm-dwelling bugs were
larger (Fig.5b) and more elongated (Fig.5c) than those
of primarily nest-dwelling bugs. CVA confirmed among-
group differences (Wilks’ λ = 0.024; P < 0.0001), with a
negative correlation between CV1 scores and head size;
again, discrimination between primarily palm-dwelling
northern populations and primarily nest-dwelling south-
ern-Andean populations was complete (Fig. 5d). Some
overlapping occurred between El Oro and Loja, and a
single Peruvian specimen was firmly nested within the
Loja cluster (asterisk in Fig.5d). is specimen was col-
lected in Suyo, approximately 20–50 km from our field-
work sites in Loja and also within the Catamayo-Chira
basin (Fig.1). When size effects were explicitly removed
(see “Methods”), all Ecuadorian populations (plus the
Fig. 6 Geometric morphometrics of Rhodnius ecuadoriensis heads (a) and forewings (b): canonical discriminant analysis of relative warps. The
left‑hand‑side plots are factorial maps based on the two first CVs, with convex hulls enclosing individual bugs of each population. Grids show head
and forewing thin plate spline configurations at selected CV values (dotted arrows); head and forewing landmarks are shown (as light‑green dots)
for reference. Note the striking elongation of the head with increasing CV1 scores (in a) and the distinctive configuration of the forewing in Peruvian
bugs, with low scores on both axes (in b). The right‑hand‑side panels show the results of UPGMA (unweighted pair group method with arithmetic
means) cluster analyses based on Mahalanobis distances (scale bar: 1.0 unit). Centroid‑size comparisons are shown in Additional file 5: Figure S2
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Page 10 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
Suyo bug) were remarkably similar to one another, with
bugs from Peru appearing as the most distinct (Fig.5e).
Geometric morphometrics—heads andforewings
Geometric head-shape analyses (Wilks’ λ = 0.051; P
< 0.0001) confirmed the striking contrast between the
large, elongated heads of northern, primarily palm-dwell-
ing bugs (and, in particular, northern-Andean bugs from
Tsáchilas) and the small, short-and-stout heads of pri-
marily nest-dwelling southern-Andean bugs (Fig.6a; see
also Figs.4, 5c). Centroid-size comparisons (Additional
file5: Figure S2) closely mirrored the results of the head-
size analysis shown in Fig.5a. e patterns revealed by
CVA of forewing-shape components (Fig.6b; Wilks’ λ =
0.016; P < 0.0001) were comparable to those revealed by
size-free traditional head morphometrics (see Fig. 5e),
although the divergence of Peruvian bugs was clearer
against a backdrop of broad similarity among Ecuadorian
populations (Fig.6b). in plate splines and CV1 scores
suggested that most Tsáchilas, some Manabí, and a few
El Oro bugs had more elongated forewings, particu-
larly in comparison with Peruvian material; most bugs
from El Oro and Loja, as well as some Manabí speci-
mens, were somewhat intermediate (Fig. 6b). Forewing
centroid-size values were similar across primarily nest-
dwelling southern-Andean populations, much larger
in northern-Andean bugs, and again intermediate in
Fig. 7 Phylogenetic relations among the mitochondrial cytochrome b gene (cytb) and nuclear ribosomal second internal transcribed spacer (ITS2)
haplotypes in Rhodnius ecuadoriensis. Mitochondrial cytb haplotypes found in bugs with each ITS2 haplotype are shown in parentheses in the
ITS2 tree. Note the lack of differentiation among Ecuadorian populations. Note also: (i) that ITS2 haplotype H13 (pink font) was found in several
bugs from Manabí province and in one bug from Tsáchilas and (ii) that good‑quality ITS2 sequences could not be determined for bugs with cytb
haplotypes MN3 (Manabí, Ecuador) and PECJ (Cajamarca, Peru). Bayesian posterior probabilities > 0.95 are shown close to key nodes. Scale bars are
in substitutions per site. Outgroup taxa are in gray font
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
northern-lowland Manabí bugs (Additional file5: Figure
S2).
Molecular analyses 1—mitochondrial DNA
We found ten cytb haplotypes, some recovered from dif-
ferent collection sites, in our R. ecuadoriensis samples
(see Additional file 1: Table S1; Figs. 7, 8; and below).
All 663-bp sequences comprised an open reading frame
with no stop codons or any other signs of pseudogene
sequences. e R. ecuadoriensis cytb alignment had 42
variable sites (6.3%); 36 were in third codon positions,
five in first codon positions, and one was in a second
codon position. ere were two non-synonymous point
mutations in a single codon of the only haplotype (PE)
found in all 13 Peruvian bugs—with ACT (threonine)
instead of GTT (valine). One Manabí haplotype (MN5)
also had a first codon position non-silent substitution—
ATT (isoleucine) instead of CTT (leucine) (see Addi-
tional file2: Alignment S1; sequences were deposited in
GenBank under accession codes MT497021–MT497035
for R. ecuadoriensis and MT497036–MT497038 for out-
group taxa).
We isolated six cytb haplotypes (MN1–MN6) from
northern-lowland Manabí bugs (Additional file 1:
TableS1). MN1, MN3 and MN5 were found in one speci-
men each. MN2 was detected in bugs from two sites
(codes MN2A and MN2B); MN2 is identical to KC543509
from Santa Ana, Manabí [74]. MN4 was also found in
bugs from two distinct Manabí sites (MN4A and MN4B),
as well as in all nine southern-Andean bugs from El Oro
(MN4EO), including those collected in two different sites
and in different years; MN4 is identical to KC543510, also
from Santa Ana, Manabí [74]. MN6 was found in one bug
from Manabí and in the 20 northern-Andean specimens
from Tsáchilas palms (MN6TS). MN4 and MN6 differ by
a single, third codon position C/T transition. We isolated
three unique haplotypes (LJ1, LJ2 and LJ3) from the 15
southern-Andean Loja bugs (Additional file1: TableS1);
LJ2 is identical to KC543508 from Quilanga, Loja, in
the Catamayo-Chira basin [74]. Finally, 13 Peruvian
bugs from at least three dwellings of the middle-upper
Chicama basin also yielded a single, unique haplotype
(PECH). One specimen from the reference R. ecuadorien-
sis colony at LNIRTT, founded in 1979 with bugs from
Cajamarca, had the same haplotype (coded PECJ) (Addi-
tional file1: TableS1). Haplotype PE was the most dis-
tinct among all the R. ecuadoriensis cytb sequences we
studied; it was separated by 25 nucleotide substitutions
from the closest Ecuadorian haplotype (MN1; see Figs.7,
8; Additional file2: Alignment S1; and below).
Overall, uncorrected cytb nucleotide diversity was π
= 0.0179 ± 0.0029 standard error (SE) (SEs estimated
with 1000 bootstrap pseudo-replicates). Mean Kimura
2-parameter (K2p) distances were 0.0185 ± 0.0031 SE
for the 72-sequence cytb alignment and 0.0184 ± 0.0029
SE for the ten-haplotype dataset. K2p sequence diver-
gence was substantially lower among Ecuadorian hap-
lotypes (0.0015–0.01995) than between any of these
and PE (0.03904–0.04894). Mean K2p distance between
the primarily palm-dwelling northern-Andean (Tsáchi-
las) and northern-lowland (Manabí) populations was
low (0.0070 ± 0.0023 SE), and much lower than dis-
tances between these two populations and the primar-
ily nest-dwelling southern-Andean bugs (0.0210 for
Tsáchilas and 0.0222 for Manabí; both ± 0.0037 SE).
ese larger distances were driven by the clearly diver-
gent Peruvian haplotypes; when these were placed in
a separate group, K2p distances between Ecuadorian
populations were between 0.15 and 1.43%, whereas dis-
tances between Ecuadorian and Peruvian populations
were all > 4.07% (Table2).
e best-fitting model for the cytb alignment had three
substitution rates (AT = CG = GT; AG = CT; and AC)
with a Gamma shape parameter (+Γ) and a propor-
tion of invariable sites (+I). e cytb gene tree shows
Fig. 8 Networks of mitochondrial cytb and nuclear ITS2 haplotypes
in Rhodnius ecuadoriensis. Each circle represents a haplotype, with
circle size proportional to haplotype frequency. Mutational steps are
represented by black dots, with numbers given for the smaller‑sized
dots of the cytb network. Alternative connections between
haplotypes (represented by gray‑broken edges) were only inferred for
the ITS2 network. Color codes are as in Figs. 1, 5, 6, and 7
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Page 12 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
an unambiguous separation (BPP = 1.0) of the Peruvian
haplotype from a monophyletic (BPP = 1.0) Ecuado-
rian clade. No clear patterns of geographic or ecological
segregation are apparent within the Ecuadorian clade,
although the three LJ haplotypes unique to Loja bugs (see
Fig.7) cluster together with BPP ≈ 0.95. ese patterns
are also apparent in the cytb haplotype network shown in
Fig.8.
Molecular analyses 2—nuclear DNA
We identified 14 unique ITS2 haplotypes (GenBank
codes KT267937–KT267950) in our R. ecuadoriensis
sample, including three (H1–H3) from Peruvian bugs
carrying the PECH cytb haplotype and 11 from Ecuado-
rian bugs: two from northern-Andean bugs (Tsáchilas,
H12 and H14); three from northern-lowland Manabí
bugs (H9–H11); one (H13) from several Tsáchilas and
Table 2 Kimura two‑parameter distances between pairs of Rhodnius ecuadoriensis populations from across the species’ range
Calculations are based on 72 mitochondrial cytochrome b (cytb) DNA sequences and 17 nuclear ribosomal second internal transcribed spacer (ITS2) DNA sequences
from ve populations primarily associated with two distinct microhabitats: Phytelephas aequatorialis palms to the north (N) and vertebrate tree‑nests to the south (S);
see Fig.1, Table1 and Additional le1: TableS1.
K2p Kimura 2‑parameter, SE Standard error computed from 1000 bootstrap pseudo‑replicates
Marker Population 1 Primary habitat Population 2 Primary habitat K2p distance SE
Cytb Tsáchilas (N) Andean palms Manabí (N) Lowland palms 0.00697 0.00227
Tsáchilas (N) Andean palms El Oro (S) Vertebrate nests 0.00151 0.00149
Tsáchilas (N) Andean palms Loja (S) Vertebrate nests 0.01283 0.00419
Manabí (N) Lowland palms El Oro (S) Vertebrate nests 0.00566 0.00185
Manabí (N) Lowland palms Loja (S) Vertebrate nests 0.01426 0.00383
El Oro (S) Vertebrate nests Loja (S) Vertebrate nests 0.01129 0.00396
Tsáchilas (N) Andean palms Peru (S) Vertebrate nests 0.04235 0.00823
Manabí (N) Lowland palms Peru (S) Vertebrate nests 0.04127 0.00782
El Oro (S) Vertebrate nests Peru (S) Vertebrate nests 0.04072 0.00804
Loja (S) Vertebrate nests Peru (S) Vertebrate nests 0.04806 0.00868
ITS2 Tsáchilas (N) Andean palms Manabí (N) Lowland palms 0.00308 0.00126
Tsáchilas (N) Andean palms El Oro (S) Vertebrate nests 0.00331 0.00148
Tsáchilas (N) Andean palms Loja (S) Vertebrate nests 0.00378 0.00152
Manabí (N) Lowland palms El Oro (S) Vertebrate nests 0.00402 0.00153
Manabí (N) Lowland palms Loja (S) Vertebrate nests 0.00402 0.00127
El Oro (S) Vertebrate nests Loja (S) Vertebrate nests 0.00473 0.00168
Tsáchilas (N) Andean palms Peru (S) Vertebrate nests 0.00519 0.00192
Manabí (N) Lowland palms Peru (S) Vertebrate nests 0.00638 0.00214
El Oro (S) Vertebrate nests Peru (S) Vertebrate nests 0.00614 0.00220
Loja (S) Vertebrate nests Peru (S) Vertebrate nests 0.00613 0.00221
Table 3 Marginal likelihoods, Bayes factors and hypothesis testing: one versus two independently evolving lineages in Rhodnius
ecuadoriensis
Pr(H), Prior probability of each alternative hypothesis [here, both hypotheses are equally likely a priori: Pr(H0) = Pr(H1) = 0.5], Log-mL natural logarithm of the marginal
likelihood, SD standard deviation of the log‑mL, Log-BF natural logarithm of the Bayes factor (i.e. the dierence in log‑mL between H1 and H0), Pr(H|D) posterior
probability of each hypothesis, given the data [here, Pr(H0|D) ≈ 0 and Pr(H1|D) ≈ 1 for both analyses]
a Estimated under the assumption of equal prior probabilities, as Pr(H1|D) ≈ BF/(1 + BF), and Pr(H0|D) = 1 − Pr(H1|D)
b Or “thermodynamic integration”; note that, in the implementation we used, this method does not provide SD estimates for the log‑mLs
Analyses and hypotheses Pr(H) Log‑mL SD Log‑BF Pr(H|D)a
Nested sampling [69]
H0: one lineage 0.5 − 3944.09 6.05 24.22 0
H1: two lineages (“Ecuador” and “Peru”) 0.5 − 3919.87 5.59 0 1
Path samplingb [70]
H0: one lineage 0.5 − 3888.23 – 12.94 < 0.00001
H1: two lineages (“Ecuador” and “Peru”) 0.5 − 3875.29 – 0 > 0.99999
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
Manabí bugs; two from El Oro (H7, H8); and three
from Loja (H4–H6) (see Additional file 1: Table S1).
All these ITS2 sequences were overall similar to each
other (see Table 2; Additional file 6: Alignment S2);
there were no signs of pseudogene sequences in the
chromatograms. e 720-bp R. ecuadoriensis 14-hap-
lotype alignment (Additional file6: Alignment S2) had
34 variable sites (4.7%), of which 18 were mutations
(2.5%) and 16 were indels (2.2%). Within Ecuador, hap-
lotypes H4 (Loja) and H13 (Manabí and Tsáchilas) dif-
fered by a single-nucleotide indel. Using the pairwise
deletion option in MEGA X [61], we found an overall,
uncorrected nucleotide diversity π = 0.0052 ± 0.0014
SE; the values were π = 0.0046 ± 0.0014 SE for Ecuado-
rian sequences and π = 0.0047 ± 0.0021 SE for Peruvian
haplotypes, with a mean uncorrected between-group
distance of 0.0062 ± 0.0019 SE. e ITS2-based K2p
distance between the primarily palm-dwelling popu-
lations (Tsáchilas vs Manabí: 0.0031 ± 0.0012 SE) was
somewhat smaller than the distances between these and
the primarily nest-dwelling southern-Andean popula-
tions (0.0042 for Tsáchilas and 0.0049 for Manabí; both
±0.0014 SE). Table 2 shows K2p distances between
population pairs; while overall low, ITS2-based dis-
tances were consistently larger in the comparisons
involving Peruvian bugs.
For the ingroup + outgroup alignment (Additional
file 7: Alignment S3; outgroup sequences deposited in
GenBank under codes KT351069–KT351071), the small-
est BIC model of nucleotide substitution included four
rates (AC = GT; AG = CT; AT; and CG), a Gamma shape
parameter (+Γ), and a proportion of invariable sites (+I).
Phylogenetic analysis revealed no geographic or ecologi-
cal structuring among Ecuadorian bugs, but the three
ITS2 haplotypes from Peruvian specimens clustered in
a separate clade with BPP = 1.0 (Fig.7). is lent sup-
port to our cytb findings and, importantly, indicated that
the similarity of mitochondrial DNA sequences across
phenotypically and ecologically distinct Ecuadorian bugs
(including the highly divergent Tsáchilas specimens; see
also Fig.8) is not due to introgression [78].
Molecular analyses 3—species trees andBayesian
hypothesis testing
Table3 summarizes the results of our assessment of the
competing hypotheses about the number of independ-
ent lineages (one vs two) within R. ecuadoriensis. We
found that the “two-lineage” hypothesis, H1, has very
strong support from our DNA-sequence data, with log-
mL estimates consistently larger (by > 24 and > 12 units,
depending on the estimation procedure) than those of H0
(Table3). ese nested- and path-sampling BF estimates
correspond to BPP between 0.999998 and 1.0 in favor
of H1; conversely, then, we found that our data provide
virtually no support for the “single-lineage” hypothesis
(Table 3). Multispecies-coalescent analyses, therefore,
substantiated locus-specific and size-free morphometric
findings in that the best-supported species tree corre-
sponds to the “two-lineage” hypothesis. is tree (Fig.9)
shows a well-supported R. ecuadoriensis clade within
which our study specimens consistently segregate into
two closely related lineages: (i) the Ecuadorian lineage,
including typical southern-Andean bugs primarily from
vertebrate tree-nests, atypical northern-Andean bugs pri-
marily from wet-forest palms and intermediate northern-
lowland bugs primarily from dry-forest palms; and (ii)
the Peruvian lineage, including primarily nest-dwelling
southern-Andean bugs (with naked-eye phenotypes sim-
ilar to type material) from the Chicama and Mashcon-
Marañón basins (Fig.9; see also Figs.1, 3, 4, 5, and 6).
Discussion
In this report we describe a striking instance of phe-
notypic divergence and convergence within a single
nominal (i.e. named) species of Triatominae, Rhodnius
ecuadoriensis. We found: (i) sharp, naked-eye pheno-
typic divergence of genetically similar Ecuadorian bugs
(primarily nest-dwelling southern-Andean populations
vs primarily palm-dwelling northern populations—and,
within northern palm-dwelling bugs, Andean vs low-
land populations); and (ii) marked, naked-eye phenotypic
similarity, most likely due to convergence, of primarily
nest-dwelling southern-Andean populations (northwest-
ern Peru vs southwestern Ecuador) whose distinct DNA
Fig. 9 Multispecies coalescent analysis results: Rhodnius ecuadoriensis
species tree estimated using mitochondrial cytb and nuclear ITS2
sequences under the two‑lineage hypothesis (“Ecuador” and
“Peru”). Maximum clade credibility tree based on 3000 replicate
trees; Bayesian posterior probabilities for cladogenetic events are
given close to each node. Scale bar is in substitutions/site. Rhodnius
colombiensis, R. pallescens and R. pictipes were used as outgroup taxa
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Page 14 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
sequences and forewing (plus, to a lesser extent, head)
shapes strongly suggest incipient evolutionary divergence
(Fig.10; Table3; Additional file4: Figure S1). Below we
argue that local adaptation to distinct microhabitats is
probably the key driver underpinning this remarkable
example of phenotypic diversity within a single putative
species.
Triatomines are blood-sucking bugs that live in shel-
tered microhabitats with a more-or-less stable food
supply [1, 2, 27]. All Rhodnius species, for example, are
primarily arboreal; most are tightly associated with palm-
crown habitats, but some species and populations also
exploit vertebrate nests built on tree branches, inside
tree hollows, in bromeliads or on palm crowns [2, 27,
29]. Populations of a few Rhodnius species also occupy
human-made habitats and can transmit T. cruzi to peo-
ple and their domestic mammals [1, 3, 27]. Rhodnius
ecuadoriensis is one such species. In the wild, it seems
to be primarily associated with the endemic Phytelephas
aequatorialis palm of western Ecuador, but has also
Fig. 10 Divergence and convergence in Triatominae: genotypes, phenotypes and habitats of Rhodnius ecuadoriensis populations. The map
illustrates the approximate distribution of the two major R. ecuadoriensis lineages: the Ecuadorian lineage (orange; “Rhodnius ecuadoriensis I” of
[8, 18]) and the Peruvian lineage (yellow; “Rhodnius ecuadoriensis II” of [8, 18]). Question marks highlight uncertainties as to the species’ northern
and southern range limits. The reddish shade in Loja suggests possible, partial differentiation of local populations in the Catamayo‑Chira basin,
as indicated by the identification of three closely related cytb haplotypes (LJ1–LJ3) not shared with other populations (Figs. 7, 8) and by limited
microsatellite [41] and 2b‑RAD (restriction site‑associated DNA tag sequencing/genotyping based on type IIB restriction enzymes) genotyping data
[79]. Colored circles show the approximate geographic location (on the map) of cytb haplotypes and their correspondence with each phenotype
(on bug pictures); color codes are as in Figs. 1 and 5–8. Nuclear ITS2 haplotypes differ between Ecuadorian (H2–H14; orange box) and Peruvian bugs
(H1–H3; yellow box), with no clear geographic, ecological or phenotype‑related genetic structuring within Ecuador. The primary (natural) habitat of
each population is given in bold italics. Gray‑white arrows emphasize phenotypic divergence (D) or convergence (C) between populations
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Abad‑Franchetal. Parasites Vectors (2021) 14:195
been found in vertebrate nests; southern-Andean popu-
lations, in particular, occur in dry ecoregions in which
palms are rare or absent, and appear to have shifted to
squirrel, bird and opossum nests [1, 2, 26–34]. In addi-
tion, some R. ecuadoriensis populations can infest houses
and peridomestic structures—with a preference for hen
nests, guinea-pig pens and dovecotes [1, 24–27, 35, 36,
38, 39]. ese synanthropic populations are major local
vectors of human Chagas disease [1, 24–27, 36–39];
importantly, available phenotypic, genetic and behavioral
evidence suggests that they most likely represent subsets
of locally sympatric wild populations [36, 40–43]. At a
broader spatial scale, R. ecuadoriensis is the only named
Rhodnius species known to occur on the western side of
the Andes south of the Magdalena-Urabá moist forests
of northwestern Colombia; the Chocó rainforests along
the Colombian Pacific coast separate R. ecuadoriensis
from its sister-species clade, R. pallescens–R. colombi-
ensis [8, 18]. Within its range, R. ecuadoriensis occurs
in widely different ecoregions [8, 18, 25, 26]. In central-
western Ecuador, presence records range from Andean
wet premontane (or “cloud”) forests to semiarid parts
of the coastal lowlands [25]. In southwestern Ecuador
and northwestern Peru, the species occupies seasonally
dry inter-Andean valleys up to 2700 m a.s.l. and is often
found infesting houses [25, 26].
We reasoned that the broad ecological flexibility of R.
ecuadoriensis was likely to correlate with similarly broad
intraspecific variation, and set out to examine the signs
of diversification and adaptation in this locally important
vector species. To address both macro-scale diversity and
micro-scale adaptations, we analyzed mitochondrial and
nuclear DNA markers that have proven useful in similar
study systems [5–9, 11, 12, 15–17, 19, 72–74, 80–84] and
undertook a detailed qualitative/quantitative phenotypic
assessment including head and forewing morphometrics
[22, 40, 49, 51–53]; we then used rich, specimen-specific
ecological metadata (Additional file1: TableS1) to guide
the interpretation of results.
Macro‑scale diversity: lineages andshape patterns
Our results provide strong support to the view that R.
ecuadoriensis is composed of two major, independently
evolving lineages [8, 18] (Table3). e core Ecuadorian
lineage has been dubbed “R. ecuadoriensis group I” [18];
it occupies highly diverse ecoregions from the wet cen-
tral-western Ecuadorian Andes down to the drier val-
leys of the Catamayo-Chira basin—apparently always
north of the Sechura desert-Huamaní range (Figs.1, 10).
e Peruvian lineage, or “R. ecuadoriensis group II” [18],
occurs in the dry inter-Andean valleys of northwestern
Peru, from the Huancabamba depression down to (and
apparently excluding) the semiarid Santa river basin [8,
25, 26]; this distribution includes: (i) Pacific-slope valleys,
from the eastern edge of the Sechura desert to the Chi-
cama and perhaps Moche basins; and (ii) the Amazon-
slope upper Marañón valley (Table1; Figs. 1, 10). Cytb
divergence levels suggest [17, 83] that these two lineages
may have been evolving independently for 2.2–3.6 mil-
lion years, with a late Pliocene–early Pleistocene most
recent common ancestor. K2p distances (4.0–4.9%) are
larger than those separating R. prolixus from its sister
species, the partly sympatric R. robustus I (3.0–3.3%) [8,
17].
Although these differences are in the limit of what
Wiemers and Fiedler [85] consider “low” levels of mito-
chondrial DNA K2p sequence divergence between recip-
rocally monophyletic sister clades, our ITS2 (Fig.7) and
multispecies coalescent results (Table3; Fig.9) lend fur-
ther support to the hypothesis that Ecuadorian and Peru-
vian R. ecuadoriensis are independently evolving lineages
[8, 18, 67]. An allozyme electrophoresis study [86] includ-
ing R. ecuadoriensis colony bugs originally from Ecuador
(Manabí and El Oro) and Peru (Cajamarca; same colony
as PECJ) provides additional insight into divergence at
multiple nuclear loci; in particular, different alleles of
Mdh, Pep3 and Pep4, for which no heterozygotes were
detected, segregated “according to the geographical ori-
gin of the specimens from Ecuador and Peru” ([86], p.
303). A later study showed that the 45S rDNA gene clus-
ter occupies different chromosomal loci in Ecuadorian (X
and Y chromosomes; bugs from Manabí) and Peruvian
specimens (X chromosomes only; bugs from La Libertad)
[87]. Taken together, our DNA results and these inde-
pendent findings are strongly suggestive of a relatively
long history of independent evolution of Ecuadorian and
Peruvian R. ecuadoriensis lineages, likely involving ongo-
ing or very recent speciation. Our detailed appraisal of
phenotypes (Table1; Figs.3, 4; see also Additional file3:
Text S1) provides the basis for distinguishing bugs car-
rying Peruvian and Ecuadorian genotypes; we expect
professional taxonomists to examine our findings and,
if warranted, formally describe a new Rhodnius species
based on Peruvian material.
Our DNA-based results, in addition, broadly mirrored
those of forewing and (size-free) head shape analyses; as
previously suggested [51, 53], explicit size partitioning
was necessary to single out genetically distinct groups
after traditional morphometrics. We expected wing shape
to be relatively conserved because of the crucial role of
flight in adult-bug dispersal [1, 27] and the importance of
wing geometry for flight efficiency [88]. Forewing shape
differences reflected the relatively deep genetic diver-
gence of the Ecuadorian and Peruvian lineages (Figs.6b,
7, 8). On the other hand, the adaptive value of head-shape
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Page 16 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
variants within triatomine-bug species remains obscure.
Our results suggest that, when size effects are removed,
R. ecuadoriensis head shape may fairly mirror genetic
divergence (Figs.5e, 7, 8). In general, the elongated heads
of northern, primarily palm-dwelling populations (Tsáchi-
las and Manabí) sharply contrast with the shorter, stouter
heads of southern-Andean populations (Figs.3, 5c, 6a). As
discussed below, this may be related to a transition from
palm to nest microhabitats [2, 89].
Phenotypic variability atthemicro‑scale: microhabitat
adaptations
Elongated heads and medium-sized bodies (relative to
other triatomines) are characteristic of the genus Rho-
dnius, which mainly comprises palm-living species [1,
2, 29]. e few exceptions to this morphological “rule”
seem to correspond to nest-dwelling species [2]. e
Psammolestes, for example, are an atypical Rhodnius
sub-lineage [5, 6, 8–10, 13] with strikingly distinct phe-
notypes—very small bodies and very short-and-stout
heads [1, 2]. ese clearly derived traits are probably
related to the adaptation of the Psammolestes com-
mon ancestor to the enclosed vegetative nests of some
ovenbirds [2, 18, 22]. Among Rhodnius species, the
most similar in head shape and body size to the typical,
southern forms of R. ecuadoriensis is R. paraensis, which
has to date only been reported from the tree-hole nests
of arboreal Echimys spiny rats [1, 2, 27, 90]. Rhodnius
domesticus also has a relatively short and stout head for
the genus; it is too among the few Rhodnius species not
specializing in palm habitats—instead, it is associated
with the nests and shelters of Phillomys tree-rats and
Didelphis and Marmosa opossums in bromeliads and
hollow tree-trunks [1, 2, 27].
ese observations suggest that the reduced body size
and head dimensions of typical R. ecuadoriensis popula-
tions may be a consequence of their shifting from the
original palm-crown habitat to new, vertebrate-nest
microhabitats [2]. is shift likely occurred in the dry
Andean environments of southwestern Ecuador and
northwestern Peru that overall lack native palm popula-
tions [25, 26, 30, 32, 39]. In Loja, wild R. ecuadoriensis
often breed inside the nests of the tree-squirrel, Sciu-
rus stramineus/nebouxii [2, 30, 32, 33, 91]. Within nest
microhabitats, the close physical proximity between the
(virtually ectoparasitic) bugs and their hosts would relax
selection for long/narrow heads and mouthparts, which
may be required for biting free-ranging hosts more
safely (at a longer distance) and sucking their blood
faster (thanks to larger cibarial-pump muscles) [89, 92].
In addition, and as has been also postulated for domes-
tic triatomine populations [4], an overall more predict-
able food supply within a nest (or human dwelling), with
a higher likelihood of repeated smaller blood meals,
would relax the need for growing bigger bodies capable
of storing larger amounts of blood [89]. Finally, host-
mediated (passive) dispersal is probably more important
among nest-dwelling than among palm-dwelling bugs,
which might reduce the need for highly efficient flight,
hence relaxing selection for elongated wings [27, 88].
We note that in Manabí R. ecuadoriensis occurs both in
Ph. aequatorialis palms and in bird and mammal nests
built on palms, trees or bromeliads [2, 28, 29, 31, 93].
is might help explain the intermediate phenotypes,
including body size and head/forewing shape, of these
primarily palm-dwelling lowland populations (Figs.3, 5,
6; Additional file4: Figure S1).
We also found a striking variability of overall color
among R. ecuadoriensis populations. In particular, the
dark hue of Tsáchilas bugs differs markedly from the
typical brown-yellowish, straw-like color of the remain-
ing populations (Fig. 3). is straw-like coloration is
shared by R. pallescens and R. colombiensis [1, 77], sug-
gesting that it is plesiomorphic (Additional file4: Fig-
ure S1). In the fresh, field-caught bugs we studied, color
variation involved mainly pigmentation intensity rather
than discrete changes in the arrangement of markings.
For example, some typically colored bugs may have a
larger amount of irregular dark spots and markings,
or their dark markings may have larger surfaces. e
highly divergent Tsáchilas forms have large and abun-
dant black markings on a reddish-brown, generally very
dark background color. Although Tsáchilas and Manabí
populations share Ph. aequatorialis as their primary
ecotope, extensive field observations [28, 76] led us to
notice that palm-crown microhabitats are often quite
different in the wet Andes and the dry lowlands. In the
dry Manabí lowlands, dead palm fronds and fibers tend
to dry up, resulting in a straw-colored habitat substrate.
In contrast, dead palm fronds and fibers quickly decay
in the wet Andes foothills—where, in addition, large
amounts of epiphytes grow on the palms. As a result,
the palm-crown microhabitat of northern-Andean
Tsáchilas bugs has a dark, actually reddish-brown,
background color. Hence, the color of palm-dwell-
ing bugs from each area (Fig.3) closely matches their
palm-microhabitat background, suggesting camouflage
against the substrate [90, 94–96]. Also in line with this
“camouflage hypothesis”, southern R. ecuadoriensis
populations (Fig.3) associate with rodent and/or bird
nests made of light brown-yellowish materials—twigs,
dry grass/leaves and straw. We therefore suggest that
sight-guided predators likely provide the main selective
pressure underlying color variability in R. ecuadorien-
sis—and probably in other triatomines.
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Page 17 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
Caveats
e first, general caveat of this study is that the results
are based on a relatively limited (albeit overall well-
representative) sample of geographic–ecological pop-
ulations and on only two genetic loci (albeit two that
are informative for problems like the one we tackled).
Our interpretations of these results, therefore, are
best viewed as testable hypotheses to be addressed
by future research. is should ideally include: (i) the
study of natural populations along putative contact
zones between genetic and phenotypic variants (e.g.
on the Huamaní range or along the Andes foothills
from Tsáchilas down to El Oro; Fig. 1), as well as in
the Chocó wet forests of Ecuador and Colombia; (ii)
population genetics/genomics analyses [41, 43, 79]; (iii)
assessing the extent of range overlap and cross-fertility
between lineages and populations [97]; or (iv) a detailed
characterization of microhabitats, with an emphasis
on bug/background color matching [98]. Second, the
microhabitat associations we consider (Table1) refer
to the known primary habitats of natural wild popula-
tions [1, 2, 24–39]; we note, however, that the evidence
of a primary link with vertebrate nests is still weak for
wild Peruvian populations [2, 26, 27, 34]. Further, our
southern-Andean samples came from human-made,
not wild, microhabitats; as has been shown for south-
ern-Andean Ecuadorian populations, we assume that
the phenotypes [40] and genotypes [41] of these bugs
do not differ significantly (or indeed at all) from those
of their wild, nest-dwelling conspecifics (see also [36,
42]). is is also consistent with the patterns of genetic
and/or morphometric similarity of wild and non-wild
bugs described for northern-lowland R. ecuadoriensis
[40, 43] and for other triatomine-bug species that often
infest houses within their native ranges—including, for
example, R. prolixus [82], T. infestans [52, 99], T. brasil-
iensis [100, 101] or T. dimidiata [102]. Finally, a small
minority of the specimens we studied did not come
from field collections. Seven bugs were from laboratory
colonies (Additional file 1: Table S1), but, except for
one head-size outlier (Fig.5), we found no differences
between these bugs and their field-caught relatives.
Our sample also included two bugs from older collec-
tions (Additional file1: TableS1); because the color of
pinned bugs can change over time, we did not consider
these two specimens in our qualitative assessment of
phenotypes—for which we only used bugs that were
fresh at the time of appraisal.
Conclusions
Adaptation of an organism to its habitat becomes
particularly evident when a human observer can pre-
dict habitat traits from organism traits. Our findings
suggest that this is likely the case with R. ecuadorien-
sis populations. us, bug color predicts microhabitat
background color, suggesting an adaptive response to
selective pressure from sight-guided predators [90, 96].
e small body size and short/stout heads of southern-
Andean bugs predict [2, 89] that wild populations pref-
erentially exploit nest microhabitats—a proposition
for which there is some empirical evidence, including
abundant squirrel-nest populations [2, 30, 32, 33] and
a strong association of domestic bugs with hen nests
and guinea-pig pens [1, 26, 27, 35, 36, 39]. Importantly,
we have also shown that populations with extremely
divergent phenotypes can share their genetic back-
grounds, at least for the two loci we examined; also
importantly, our sequence data indicate that genetic
similarity among Ecuadorian bugs is not due to mito-
chondrial DNA introgression [78]. In addition, our data
reveal that southern-Andean R. ecuadoriensis popula-
tions with near-sibling naked-eye phenotypes belong
in two distinct evolutionary lineages—the Ecuadorian
“R. ecuadoriensis I” and the Peruvian “R. ecuadoriensis
II” [18]. is can have implications for taxonomy and,
hence, for the interpretation of taxonomy-dependent
research results. We note, for example, that the ref-
erence R. ecuadoriensis strain kept at the LNIRTT
(Fiocruz, Brazil) has the PE cytb haplotype, which is
nearly 5% divergent from the LJ haplotypes that geo-
graphically correspond to the species’ type material
from Catamayo, Loja [45]. In their classic revision,
Lent and Wygodzinsky [1] illustrate R. ecuadoriensis
with a Peruvian bug from Cajamarca (Fig.257 in [1]).
Peruvian- and Ecuadorian-lineage bugs are also clearly
divergent in forewing and (size-free) head shape, at sev-
eral allozyme loci [86] and cytogenetically [87].
In sum, our detailed appraisal of phenotypic and
genetic diversity in R. ecuadoriensis revealed phe-
notypic divergence within genetically homogeneous
populations and phenotypic convergence of genetically
distinct lineages likely on their way to speciation—if
not separate species already. Such remarkable, bidirec-
tional phenotypic change within a single nominal taxon
was apparently associated with adaptation to particular
microhabitats. ese findings shed new light on the ori-
gins of phenotypic diversity in the Triatominae, warn
against excess reliance on phenotype-based triatomine-
bug systematics, and confirm the Triatominae as an
informative model-system for the study of phenotypic
change under ecological pressure.
Abbreviations
BF: Bayes factor; BIC: Bayesian information criterion; BPP: Bayesian poste‑
rior probability; CV: Canonical variate; CVA: Canonical variate analysis; cytb:
Mitochondrial cytochrome b gene; EO: El Oro province, Ecuador; ESS: Effective
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Page 18 of 21
Abad‑Franchetal. Parasites Vectors (2021) 14:195
sample size; ITS2: Nuclear ribosomal second internal transcribed spacer; K2p:
Kimura two‑parameter genetic distance; LJ: Loja province, Ecuador; LNIRTT
: Laboratório Nacional e Internacional de Referência em Taxonomia de
Triatomíneos, Fiocruz, Brazil; MCMC: Markov chain Monte Carlo; mL: Marginal
likelihood; MN: Manabí province, Ecuador; PC: Principal component; PCA: Prin‑
cipal component analysis; PE: Peru; SE: Standard error; TPS: Thin plate spline;
TS: Santo Domingo de los Tsáchilas province, Ecuador; 2b‑RAD: Restriction
site‑associated DNA tag sequencing/genotyping based on type IIB restriction
enzymes.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13071‑ 021‑ 04647‑z.
Additional le1: TableS1. Populations, specimen details and haplotype
codes of 106 Rhodnius ecuadoriensis bugs used in morphometric and/or
molecular analyses. A summary table with the numbers of bugs used in
each analysis is also provided.
Additional le2: Alignment S1. Mitochondrial cytochrome b haplo‑
types in Rhodnius ecuadoriensis from Ecuador and Peru, plus outgroup
species (R. colombiensis, R. pallescens, R. pictipes).
Additional le3: Text S1. Detailed descriptions of the diverse Rhodnius
ecuadoriensis phenotypes.
Additional le4: Figure S1. Phenotype–microhabitat–phylogeny corre‑
spondences. Multispecies coalescent species tree (as in Fig. 9 of the main
text), with pictures (approximately to the same scale) of adult Rhodnius
ecuadoriensis and its closest relatives—R. colombiensis, R. pallescens and
R. pictipes. The distribution of phenotypes along the phylogeny suggests
that the common ancestor of the diverse R. ecuadoriensis forms was most
likely a relatively large, straw‑like‑colored bug. Similarly, the distribution of
primary microhabitats suggests that a shift of southern‑Andean popula‑
tions from palm crowns (green stars) to vertebrate nests (orange circles)
resulted in convergence towards the small‑size, short‑head/wing typical
R. ecuadoriensis phenotype; the combined star/circle symbol indicates
that northern‑lowland Manabí bugs are primarily palm‑dwelling but
may also exploit nest microhabitats. Rhodnius ecuadoriensis populations:
N-A Northern‑Andean (Tsáchilas), N-L northern‑lowland (Manabí), S-A
southern‑Andean (El Oro and Loja in Ecuador; La Libertad and Cajamarca
in Peru).
Additional le5: Figure S2. Centroid‑size comparisons. Population box‑
plots and Tukey‑Kramer (T‑K) tests for head and forewing centroid sizes
derived from geometric morphometrics.
Additional le6: Alignment S2. Fourteen nuclear ITS2 haplotypes found
in Rhodnius ecuadoriensis from Ecuador and Peru.
Additional le7: Alignment S3. Nuclear ITS2 haplotypes in Rhodnius
ecuadoriensis from Ecuador and Peru, plus outgroup species (R. colombien-
sis, R. pallescens and R. pictipes).
Acknowledgements
We thank CAC Cuba and J Jurberg for providing bugs, and the staff of the
Ecuadorian Malaria Control Service for field assistance. The findings and con‑
clusions in this report are those of the authors and do not necessarily repre‑
sent the official position of the US Centers for Disease Control and Prevention/
the Agency for Toxic Substances and Disease Registry.
Authors’ contributions
FA‑F, FAM, JSP and MAM conceived the study. FA‑F, MDB, MA and MAM raised
funds. FA‑F, MA and MAM administered different parts of the project. FA‑F,
FAM, MGP, JSP, MDB, MAZ, MA, CBB, SM‑C and MAM contributed to the devel‑
opment of research methods. FA‑F, FAM, MGP, JSP, MDB, MAZ and MA per‑
formed research. FA‑F, FAM, MGP, JSP, MDB and MAZ curated different datasets
and analyzed the data. MAM, MDB, SM‑C and CBB supervised research. FA‑F
and MAM drafted the first version of the manuscript; all authors contributed to
the interpretation of results and commented on manuscript drafts. All authors
read and approved the final manuscript.
Funding
UNICEF/UNDP/World Bank/WHO TDR (Grant 970195); Red de Investigación de
Centros de Enfermedades Tropicales—RICET, Ministerio de Salud y Consumo,
Madrid, Spain (Grant No. RD16/0027/0023 of the PN de I+D+I, ISCIII‑Subdi‑
rección General de Redes y Centros de Investigación Cooperativa RETICS);
and PROMETEO Program, Ayudas para Grupos de Investigación de Excelencia,
Generalitat Valenciana, Valencia, Spain (Grant No. 2016/099).
Availability of data and materials
Data supporting the conclusions of this article are included in the article and
its additional files. DNA sequences have been deposited in GenBank under
accession numbers MT497021–MT497038 for mitochondrial cytb haplotypes
and KT267937–KT267950 plus KT351069–K T351071 for nuclear rDNA ITS2
haplotypes.
Declarations
Ethics approval and consent to participate
Our study did not involve endangered or protected species; collection of
disease vectors did not require specific permits at the time this project was
carried out. Prior to field activities, we obtained oral informed consent from all
home‑ and landowners.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Núcleo de Medicina Tropical, Faculdade de Medicina, Universidade de Bra‑
sília, Brasília, Brazil. 2 Faculty of Infectious and Tropical Diseases, London School
of Hygiene and Tropical Medicine, London, UK. 3 Laboratório de Epidemiologia
e Sistemática Molecular, Instituto Oswaldo Cruz‑Fiocruz, Rio de Janeiro, Brazil.
4 Division of Vector‑Borne Diseases, Centers for Disease Control and Prevention,
Fort Collins, USA. 5 Laboratório de Mosquitos Transmissores de Hematozoários,
Instituto Oswaldo Cruz‑Fiocruz, Rio de Janeiro, Brazil. 6 Departamento de
Parasitología, Facultad de Farmacia, Universidad de Valencia, Valencia, Spain.
7 Facultad de Ciencias Médicas, Universidad Central del Ecuador, Quito, Ecua‑
dor. 8 Instituto Juan César García, Quito, Ecuador.
Received: 15 November 2020 Accepted: 16 February 2021
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