Abad‑Franch et al. Parasites Vectors (2021) 14:195 https://doi.org/10.1186/s13071‑021‑04647‑z Parasites & Vectors RESEARCH Open Access 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 diferentiation. 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 camoufage coloration) and a shift from palm‑crown to vertebrate‑nest microhabitats (yielding smaller bodies and shorter and stouter heads). These fndings shed new light on the origins of phenotypic diversity in triatomines, warn against *Correspondence: [email protected]; [email protected] 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 © The Author(s) 2021, corrected publication 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Keywords: Triatominae, Rhodnius, Chagas disease, Systematics, Morphometrics, Genetics Introduction ecuadoriensis populations spanning most of the geo- Triatomine bugs transmit Trypanosoma cruzi among the graphic/ecological range of the species (Fig. 1). Rho- mammalian hosts they associate with in shared micro- dnius ecuadoriensis is a major vector of T. cruzi in habitats [1, 2]. Bugs that occur in human-made habitats western Ecuador and northwestern Peru [24–26]. In may transmit the parasite to people, fueling the spread of Ecuador, northern wild populations are primarily asso- Chagas disease [1, 3]. It has been proposed that occupa- ciated with the endemic Phytelephas aequatorialis palm tion of new microhabitats may trigger selection for dis- in both Andean wet forests and lowland dry forests; in tinct phenotypic variants in these blood-sucking bugs [4]. the dry inter-Andean valleys of southwestern Ecuador, Over the last few decades, molecular studies have iden- where native palms are rare or absent, wild R. ecuado- tifed examples of phenotypic convergence or divergence riensis seem to primarily associate with arboreal squir- at several systematic levels [5–9]. Perhaps most remark- rel nests [1, 2, 27–33]. Te natural habitats of Peruvian ably, molecular phylogenetic analyses suggest that none populations remain unclear, with a few records suggest- of the three genera to which the main Chagas disease ing association with vertebrate nests/refuges in hollow vectors belong (Triatoma, Panstrongylus and Rhodnius), trees and perhaps cacti [2, 26, 27, 34]. In addition, some which are all defned after morphological characters [1], R. ecuadoriensis populations have adapted to live in is monophyletic [8–14]. A few named species have been and around houses in coastal Ecuador and, especially, shown to be phenotypic variants of another species [8]; in the dry valleys of southwestern Ecuador and north- for example, Triatoma melanosoma is now regarded western Peru—where the bugs contribute to endemic as a T. infestans chromatic variant [15, 16]. Conversely, Chagas disease [24–26, 35–39]. In coastal and in south- some named species have been shown to include several western Ecuador, bugs from wild and human-made cryptic taxa [8]; for example, Rhodnius robustus (s.l.) is habitats have overlapping phenotypes [40] and identi- composed of at least fve distinct lineages, two of which cal or nearly identical mitochondrial cytb haplotypes have been formally described as R. montenegrensis and R. [41]; this, together with frequent, rapid reinfestation marabaensis [8, 17–21]. Further examples of phenotype/ of insecticide-treated dwellings [36, 42] and prelimi- genotype mismatch are reviewed in [8]. nary microsatellite data [41, 43], indicates that wild and Te sometimes striking variation of triatomine-bug non-wild R. ecuadoriensis populations are locally highly phenotypes has been attributed to a propensity of mor- interconnected. Our comparative phenotypic and phological characters to change in response to chang- genetic analyses cover most of the known geographic ing habitat features [2, 4, 22]. Tus, within-species and ecological diversity in R. ecuadoriensis; they reveal divergence may be driven by habitat shifts (e.g., wild that phenotypic divergence and convergence can both to domestic) involving subsets of genetically homoge- occur within a single nominal triatomine-bug species, neous populations [4], and the use of similar habitats and suggest that microhabitat adaptations likely play a by genetically distinct populations may result in con- crucial role in this phenomenon. vergence or in the retention of ancestral phenotypes [2, 4, 23]. When using morphological characters only, Methods therefore, taxonomists are in peril of describing spu- Origins of bugs and qualitative phenotype assessment rious species or overlooking cryptic taxa [8]. Despite We compared R. ecuadoriensis type specimens from La the practical importance of accurate taxonomic judg- Toma, Catamayo, Loja Province, Ecuador (Laboratório ment when the organisms of interest transmit a Nacional e Internacional de Referência em Taxonomia life-threatening parasite, the degree, direction and de Triatomíneos [LNIRTT], Fiocruz, Brazil; [44]) and underlying causes of phenotypic change and diversity canonical descriptions of the species [1, 45] with feld- in triatomines remain obscure. collected Ecuadorian bugs, including (i) northern bugs In this paper, we combine a detailed pheno- from Ph. aequatorialis palms of Santo Domingo de los typic characterization, qualitative and quantitative, Tsáchilas Province (Andean wet forests; “Tsáchilas” here- with mitochondrial cytochrome b gene (cytb) and after) and Manabí Province (lowland dry forests), and (ii) nuclear ribosomal second internal transcribed spacer primarily nest-dwelling southern-Andean bugs caught in/ (ITS2) DNA sequence analyses to study Rhodnius around houses in El Oro (Puyango river basin) and Loja Abad‑Franch et al. Parasites Vectors (2021) 14:195 Page 3 of 21 Fig. 1 Sampling of Rhodnius ecuadoriensis populations. The map shows the approximate