Fleas in the genus Ctenocephalides serve as biological vectors or intermediate hosts of microorganisms such as bacteria, rickettsia, protozoa and helminths. Ctenocephalides felis has a worldwide distribution, while C. orientis has long been considered as a subspecies of C. felis in Asia. To help the morphological recognition of these two species and further explore their differences, we used the geometric morphometric approach applied to the head. Both sexes were examined. Five anatomical landmarks of the head were used, and to capture the curvature of the front head, 10 semilandmarks were added. There was a consistent difference in species classification accuracy when considering landmarks only versus their combination with semilandmarks, suggesting the importance of the curve of the head as a taxonomic signal. Using or not the labels in the reclassification analyses, the head shape allowed by itself almost perfect recognition of the two species, in both sexes, even after adjustment for prior probabilities. The same approach disclosed a high level of sexual size and shape dimorphism in both species. The contribution of size variation to the discrimination by shape was much more important between sexes (from 27% to 45%) than between species (from 0.7% to 7.1%). Nevertheless, in our data, size never could represent a way to reliably recognise the sex of an individual, even less its species. Geographical variation in head shape could only be explored for the C. orientis sample. No significant correlation of morphometric variation with geography could be detected, which would be consistent with gene flow between Thai provinces. The geometric morphometric approach of the flea head, when it incorporates head curves, is a promising tool for rapid, economical, and accurate species and sex identification. It is, therefore, a useful tool for future epidemiological and demographic studies.

Fly identification is the primary step of analysis in forensic entomology. Although morphology and molecular techniques are considered satisfactory methods, some constraints may arise from a financial or even human point of view. Over the past decade, the geometric morphometric approach has been increasingly advocated for the classification and identification of arthropods. This study explored the method for species identification of 800 third-instar larvae of eight blow fly species of medical and forensic importance: Chrysomya chani Kurahashi, Chrysomya megacephala (Fabricius), Chrysomya (Ceylonomyia) nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya (Achoetandrus) rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia cuprina (Wiedemann), and Lucilia porphyrina (Walker). Based on the posterior spiracles geometry, the cross-validation revealed a relatively high percentage of correct classification in most species, ranking from 86% to 100%. The results of this study confirmed that the geometric morphometric (GM) analysis of posterior spiracles might be utilized as a larva identification tool. Therefore, this GM method represents one way of overcoming difficulties with the identification of blow fly larvae and can support further studies of these flies.

Ascoschoengastia indica is one of the dominant chigger species in Southeast Asia and a potential carrier of scrub typhus, due in part to its cosmopolitan nature. This study explored the possible biological significance of the observed dimorphism in the shape of its scutum sensilla. Sensilla are specialized structures that are generally adapted to perform specific functions related to sensory capabilities, so their shape and sizes are expected to vary between taxa. We describe morphological variation of the sensilla of A. indica in Thailand. The sensilla had either a round or an ovoid, club-shaped form, which was not dependent on the particularly locality or host. Ignoring the precise function of the sensilla and their morphological variation, our study attempted to answer the following single question: Do the distinct forms of the sensilla indicate possible heterogeneity of the A. indica species? The two forms, named S1 and S2, were compared by genetic and morphometric techniques. The genetic analysis was based on the COI sequences, while the morphometric comparison used the scutum, an organ shown to be of taxonomic value for chigger mites. Neither morphometric nor genetic data revealed any evidence of a speciation process underlying the morphological variation in sensillum types.

In vector control terms, insecticide resistance is the development of the capacity, of an insect population, to tolerate doses of an insecticide that are lethal to most individuals in a typical population of the same species. The genetic changes that determine resistance may have adaptive costs in the resistant phenotype or, conversely, may result in an adaptive advantage when compared to susceptible insects in the environment without insecticides. Triatoma infestans is one of the main vectors of Trypanosoma cruzi in the southern cone of South America. High insecticide resistance in T. infestans was detected in Argentina in Salta and Chaco provinces. The objective of this study was to determine the possible morphometric changes in wings, heads, and the antennal phenotype of deltamethrin-resistant T. infestans (RR) males and females compared to susceptible insects (SS), evaluating its implication in adaptive processes such as olfactory capacity, dispersion, and probability of colonizing new habitats, among others. Nine type I landmarks were marked on wings, 5 type II landmarks on heads, and 10 antennal sensilla were counted on 106 adults of both sexes (resistant and susceptible from first and second laboratory generations). Morphological divergence was observed between the two groups (RR and SS). The RR insects showed smaller sizes of wings and heads and shape compatible with lower dispersal potential and different active dispersal behaviors. Antennae also revealed sensory simplification in RR and divergence between RR and SS, although more marked in females. This study characterizes for the first time T. infestans RR and SS through wings, heads, and antennae. The results suggest a lower dispersive potential in resistant insects and the differences described lay the foundations for the identification of a resistance biomarker in triatomines.

Background: Triatoma dimidiata is a vector of the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. Phenotypic plasticity allows an organism to adjust its phenotype in response to stimuli or environmental conditions. Understanding the effect of T. cruzi on the phenotypic plasticity of its vectors, known as triatomines, has attracted great interest because of the implications of the parasite-triatomine interactions in the eco-epidemiology and transmission of the etiologic agent of Chagas disease. We investigated if the infection of the vector with T. cruzi may be associated with a change in the antennal phenotype of sylvatic, domestic, and laboratory-reared populations of T. dimidiata. Methods: The abundance of each type of sensillum (bristles, basiconic, thick- and thin-walled trichoid) on the antennae of T.cruzi-infected and non-infected T.dimidiata reared in the laboratory or collected in sylvatic and domestic ecotopes were measured under light microscopy and compared using Kruskal-Wallis non-parametric tests and permutational multivariate analysis of variance. Results: We found significant differences between sensilla patterns of infected and non-infected insects within sylvatic and domestic populations. Conversely, we found no significant differences between sensilla patterns of infected and non-infected insects within the laboratory-reared population. Besides, for sylvatic and domestic populations, sexual dimorphism tended to be increased in infected insects. Conclusion: The differences observed in infected insects could be linked to higher efficiency in the perception of odor molecules related to the search for distant mates and hosts and the flight dispersal in search of new habitats. In addition, these insects could have a positive effect on population dynamics and the transmission of T.cruzi.

Abstract Pintomyia (Pifanomyia) veintemillasi n. sp., is described as a new anthropophilic sand fly species based on morphological diagnostic characteristics, which allow differentiating it from the closely related species Pintomyia (Pifanomyia) nevesi and Pintomyia (Pifanomyia) maranonensis within the Evansi series. This species was collected in the Marimonos mountain range, at around 900 m altitude in the Municipality of Palos Blancos, Sud Yungas Province, Department of La Paz, Bolivia, and was misidentified in the past as Pi. (Pif.) nevesi.

All members of the ant genus Odontomachus Latreille, 1804 are venomous ants. Four species in this genus have been identified from Thailand: Odontomachus latidens Mayr, 1867; O. monticola Emery, 1892; O. rixosus Smith, 1757; and O. simillimus Smith, 1758. The three latter species are available and have been used for an outline morphometric study. They display similar morphology, which makes their distinction very difficult except for highly qualified individuals. A total of 80 worker specimens were studied, exploring the contour shapes of their head and pronotum as possible taxonomic characters. The size of each body part was estimated determining the contour perimeter, the values for which were largely overlapping between O. rixosus and O. simillimus; most O. monticola specimens exhibited a significantly larger size. In contrast to the size, each contour shape of the head or pronotum established O. rixosus as the most distinct species. An exploratory data analysis disclosed the higher taxonomic signal of the head contour relative to the pronotum one. The scores obtained for validated reclassification were much better for the head (99%) than for the pronotum (82%). This study supports outline morphometrics of the head as a promising approach to contribute to the morphological identification of ant species, at least for monomorphic workers.

Abstract Background: Triatoma dimidiata is a vector of the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. Phenotypic plasticity allows an organism to adjust its phenotype in response to stimuli or environmental conditions. Understanding the effect of T. cruzi on the phenotypic plasticity of its vectors, known as triatomines, has attracted great interest because of the implications of the parasite-triatomine interactions in the eco-epidemiology and transmission of the parasite. We investigated if the infection of the vector with T. cruzi may be associated with a change in the antennal phenotype of sylvatic, domestic, and laboratory-reared populations of T. dimidiata. Methods: The abundance of each type of sensillum (bristles, basiconic, thick- and thin-walled trichoid) on the antennae of T. cruzi-infected and non-infected T. dimidiatareared in the laboratory or collected in sylvatic and domestic ecotopes were measured under light microscopy and compared using Kruskal–Wallis non-parametric tests and Permutational Multivariate Analysis of Variance. Results: We found significant differences between sensilla patterns of infected and non-infected insects within sylvatic and domestic populations. Conversely, we found no significant differences between sensilla patterns of infected and non-infected insects within the laboratory-reared population. Besides, for sylvatic and domestic populations, sexual dimorphism tended to be increased in infected insects. Conclusion: The differences observed in infected insects could be linked to higher efficiency in the perception of odor molecules related to the search for distant mates and hosts and the flight dispersal in search of new habitats. In addition, these insects could have a positive effect on population dynamics and the transmission of T. cruzi.

An infestation of a Cimicidae (Hemiptera: Cimicidae) member, especially the bed bug, can cause economic loss and impact health. A cost-effective and user-friendly method for identifying the infesting species will help with the early detection and control of infestations. A linear morphometric method is often used, but it requires the examination of many characters and a highly preserved specimen. We conducted a comparative morphometric study of the effectiveness of Cimicidae classification using a single organ, the pronotum, through outline-based and linear morphometric methods. Bat (Stricticimex parvus), human (Cimex hemipterus), and bird (Paracimex sp.) ectoparasites were subject of the study. With both methods, the properties of size and shape were compared and used separately to classify the specimens. Classification analyses of the two methods provided similar results, but more informative variables of size and shape were obtained with the outline-based approach. Size, as analyzed with the outline-based method, could detect sexual dimorphism, and produced better reclassification. The shape variables obtained from the linear measurements were strongly influenced by size variation, much more than the ones obtained from coordinates describing the pronotum contours. Our data suggest that the outline-based approach provides better characterization variables, thus we recommend them for a wider use in other Cimicidae family members.

Background Most sand fly species are located in the Americas; some act as vectors of leishmaniasis and other human diseases. In Bolivia, about 25% of Neotropical species have been identified, and only a few have been implicated as vectors of cutaneous and visceral leishmaniasis. A new species of anthropophilic sand fly from the sub-Andean region of Alto Beni is described herein. Methods A large systematic entomological survey was carried out in a subtropical humid forest located in the Marimonos mountain range, at around 900 m altitude, in the municipality of Palos Blancos, Sud Yungas Province, Department of La Paz, Bolivia. Sand flies were captured over a period of 26 months between January 1982 and February 1984, at the ground and canopy level, using both CDC light traps and protected human bait. A total of 24,730 sand flies were collected on the ground, distributed in 16 species, and 3259 in the canopy, with eight species. One of these species was labeled as Pintomia ( Pifanomyia ) nevesi , although certain morphological features allowed us to doubt that it was that taxon. To define the identity of this sand fly, a re-evaluation (this work) was recently carried out through morphological analyses and measurements of the available specimens mounted on Euparal, previously labeled as Pi . ( Pif .) nevesi . Results Based on the morphological traits and measurements, the re-evaluated specimens were definitively identified as a new sand fly species, Pintomyia ( Pifanomyia ) veintemillasi , closely related to Pi . ( Pif .) nevesi and Pintomyia ( Pifanomyia ) maranonensis within the Evansi series. This new sand fly was the third most numerous anthropophilic species at the floor (6.2%) and the second most numerous anthropophilic at the canopy (35.1%). Conclusions A new anthropophilic sand fly species is described as Pi . ( Pif .) veintemillasi n. sp. This sand fly species was caught at about 900 m altitude in the Marimonos mountain range, a highly endemic area for cutaneous and mucosal leishmaniasis due to Leishmania ( Viannia ) braziliensis . Therefore, this species could be involved in the leishmaniasis transmission in the sub-Andean foothills of Alto Beni, Department of La Paz, Bolivia. Graphical Abstract

Chagas disease persists as one of the most important, and yet most neglected, diseases in the world, and several changes in its epidemiological aspects have been recorded since its discovery. Currently, some of the most relevant changes are related to: (i) the reduction in the incidence of the endemic due to the control of the most important vectors, Triatoma infestans and Rhodnius prolixus, in many countries; (ii) the migration of human populations spreading cases of the disease throughout the world, from endemic to non-endemic areas, transforming Chagas disease into a global threat; and (iii) new acute cases and deaths caused by oral transmission, especially in the north of Brazil. Despite the reduction in the number of cases, new challenges need to be responded to, including monitoring and control activities aiming to prevent house infestation by the secondary vectors from occurring. In 1979, Lent & Wygodzinsky(1) published the most complete review of the subfamily Triatominae, encompassing 111 recognised species in the taxon. Forty-two years later, 46 new species and one subspecies have been described or revalidated. Here we summarise the new species and contextualise them regarding their ecology, epidemiologic importance, and the obstacles they pose to the control of Chagas disease around the world.

Tsetse flies (Diptera: Glossinidae) are pupiparous insects that transmit African trypanosomes to humans and animals. Using three species of tsetse flies belonging to two subgenera, Nemorhina and Glossina, our data provide a first answer to the following question: do the juvenile stages of tsetse flies provide metric traits allowing reliable species and/or sex identification? We compared three species using their wings and their pupae: Glossina palpalis gambiensis, Glossina tachinoides and Glossina morsitans submorsitans. To separate size and shape information, 11 landmarks per wing were submitted to the Generalised Procrustes Analysis (GPA). The contour of the pupae was processed by elliptic Fourier analysis (EFA).A validated reclassification method was used to compare the taxonomic power of each organ, as well as its utility for sex recognition. The contour of the pupae did not provide a satisfactory species signal (75 %), relative to the one obtained from wing landmarks (99 %). However, the overall pupae size was much more satisfactory than the wings size (93 % versus 57 %). In spite of a lower taxonomic signal, the morphometric variation observed on pupae was in agreement with known phylogenetic relationships. Contrary to the wings, sexual dimorphism was much reduced in pupae and not strong enough to allow any reliable sex identification.

Accurate identification of mosquito species is critically important for monitoring and controlling the impact of human diseases they transmit. Here, we investigate four mosquito species: Aedes aegypti, Ae. albopictus, Ae. scutellaris and Verrallina dux that co-occur in tropical and subtropical regions, and whose morphological similarity challenges their accurate identification, a crucial requirement in entomological surveillance programs. Previous publications reveal a clear taxonomic signal embedded in wing cell landmark configuration, as well as in the external contour of the wings. We explored this signal for internal cells of the wings as well, to determine whether internal cells could uniformly provide the same taxonomic information. For each cell to be tentatively assigned to its respective species, i.e., to measure the amount of its taxonomic information, we used the shape of its contour, rather than its size. We show that (i) the taxonomic signal of wing shape is not uniformly spread among internal cells of the wing, and (ii) the amount of taxonomic information of a given cell depends on the species under comparison. This unequal taxonomic signal of internal cells is not related to size, nor to apparent shape complexity. The strong taxonomic signal of some cells ensures that even partly damaged wings can be used to improve species recognition.

Abstract Background: Triatoma dimidiata is a vector of the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. Phenotypic plasticity allows an organism to adjust its phenotype in response to stimuli or environmental conditions. Understanding the effect of T. cruzi on the phenotypic plasticity of its vectors, known as triatomines, has attracted great interest because of the implications of the parasite-triatomine interactions in the eco-epidemiology and transmission of the parasite. We investigated whether the infection of the vector with T. cruzi can change the antennal phenotype of sylvatic, domestic, and laboratory-reared populations of T. dimidiata. Methods: The abundance of each type of sensillum (bristles, basiconic, thick- and thin-walled trichoid) on the antennae of T. cruzi-infected and non-infected T. dimidiata reared in the laboratory or collected in sylvatic and domestic ecotopes were measured under light microscopy and compared using Kruskal–Wallis non-parametric tests and Permutational Multivariate Analysis of Variance. Results: We found significant differences between sensilla patterns of infected and non-infected insects within sylvatic and domestic populations. Conversely, we found no significant differences between sensilla patterns of infected and non-infected insects within the laboratory-reared population. Besides, our results show that the infection with T. cruzi affects the sexual dimorphism linked to antennal phenotype in sylvatic and domestic populations. Conclusion: These differences could be linked, for infected insects, to higher efficiency in the perception of odor molecules related to the search of distant mates and hosts and for flight dispersal in search of new habitats, and the possibility of a positive effect on population dynamics and on the vectorial transmission of T. cruzi.

BACKGROUND:Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS:Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES:To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS:We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS:Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

BACKGROUND:Japanese encephalitis is a severe disease of acute encephalitis, with children and the elderly primarily affected, and with mortality rates reaching over 25%. The virus is transmitted mainly by species of the Culex (Culex) vishnui subgroup, primarily the widely spread Cx. tritaeniorhynchus Giles. The latter is known as a highly migratory mosquito which moves with airflow over large distances. We explored the geometric variation of the wing venation among distant areas of its geographic distribution. Our working hypothesis was that shape variation across geography could reveal known past and present migratory routes.MATERIALS METHODS:We compared the wing venation geometry of 236 female Culex tritaeniorhynchus from different locations in the Madagascan (La Reunion), Oriental (Thailand, Vietnam) and Paleartic (Japan) regions. To ascertain the taxonomic signal of the wing venation we also used two species as relative outgroups, Cx. whitmorei and Cx. brevipalpis.RESULTS:In spite of an increasing morphometric variation as expected with larger geographic dispersion, our Cx. tritaeniorhynchus samples were clustered as a single species when considered relative to other Culex species. The relationships between geographic sites of Cx. tritaeniorhynchus globally conformed with an isolation by distance model. The shape homogeneity of our Palearctic samples (Japan) contrasted with some heterogeneity observed in the Oriental region (Thailand, Vietnam), and could be related to the different regimes of wind trajectories in these regions.CONCLUSION:The average shape variation of Culex tritaeniorhynchus disclosed a separation between Madagascan, Oriental and Palearctic regions in accordance with geography. The wing venation not only could reflect geography, it also contained a clear taxonomic signal separating three Culex species. Within Cx. tritaeniorhynchus, a contrasting pattern of shape variation between the Palearctic and the Oriental regions is tentatively explained by the influence of wind trajectories.

Tabanus spp., also known as horse flies (Diptera: Tabanidae), are important vectors of several animal pathogens. Adult females of Tabanus megalops and Tabanus striatus, which are members of the T. striatus complex, are morphologically similar and hence difficult to distinguish using morphological characteristics. In addition, molecular identification by DNA barcoding is also unable to distinguish these species. These two species can occur sympatrically with Tabanus rubidus, which is morphologically similar to T. megalops and T. striatus. Wing geometric morphometrics has been widely used in various insects to distinguish morphologically similar species. This study explored the effectiveness of landmark-based geometrics at distinguishing and identifying T. megalops, T. rubidus, and T. striatus in Thailand. Specimens were collected from different geographical regions of Thailand, and only unambiguously identified specimens were used for geometric morphometric analyses. Left wings of females of T. megalops (n = 160), T. rubidus (n = 165), and T. striatus (n = 85) were photographed, and 22 wing landmarks were used for the analysis. Wing shape was able to distinguish among species with high accuracy scores, ranging from 94.38% to 99.39%. We showed that morphologically very close species of Tabanus can be reliably distinguished by the geometry of their wing venation, and we showed how our experimental material could be used as a reference to tentatively identify new field collected specimens.

Background Fasciola hepatica and F. gigantica cause fascioliasis in both humans and livestock. Some adult specimens of Fasciola sp. referred to as “intermediate forms” based on their genetic traits, are also frequently reported. Simple morphological criteria are unreliable for their specific identification. In previous studies, promising phenotypic identification scores were obtained using morphometrics based on linear measurements (distances, angles, curves) between anatomical features. Such an approach is commonly termed “traditional” morphometrics, as opposed to “modern” morphometrics, which is based on the coordinates of anatomical points. Methods Here, we explored the possible improvements that modern methods of morphometrics, including landmark-based and outline-based approaches, could bring to solving the problem of the non-molecular identification of these parasites. F. gigantica and Fasciola intermediate forms suitable for morphometric characterization were selected from Thai strains following their molecular identification. Specimens of F. hepatica were obtained from the Liverpool School of Tropical Medicine (UK). Using these three taxa, we tested the taxonomic signal embedded in traditional linear measurements versus the coordinates of anatomical points (landmark- and outline-based approaches). Various statistical techniques of validated reclassification were used, based on either the shortest Mahalanobis distance, the maximum likelihood, or the artificial neural network method. Results Our results revealed that both traditional and modern morphometric approaches can help in the morphological identification of Fasciola sp. We showed that the accuracy of the traditional approach could be improved by selecting a subset of characters among the most contributive ones. The influence of size on discrimination by shape was much more important in traditional than in modern analyses. In our study, the modern approach provided different results according to the type of data: satisfactory when using pseudolandmarks (outlines), less satisfactory when using landmarks. The different reclassification methods provided approximately similar scores, with a special mention to the neural network, which allowed improvements in accuracy by combining data from both morphometric approaches. Conclusion We conclude that morphometrics, whether traditional or modern, represent a valuable tool to assist in Fasciola species recognition. The general level of accuracy is comparable among the various methods, but their demands on skills and time differ. Based on the outline method, our study could provide the first description of the shape differences between species, highlighting the more globular contours of the intermediate forms.

Background Chagas disease is a parasitic infection transmitted by “kissing bugs” (Hemiptera: Reduviidae: Triatominae) that has a huge economic impact in Latin American countries. The vector species with the upmost epidemiological importance in Ecuador are Rhodnius ecuadoriensis (Lent & Leon, 1958) and Triatoma dimidiata (Latreille, 1811). However, other species such as Panstrongylus howardi (Neiva, 1911) and Panstrongylus chinai (Del Ponte, 1929) act as secondary vectors due to their growing adaptation to domestic structures and their ability to transmit the parasite to humans. The latter two taxa are distributed in two different regions, they are allopatric and differ mainly by their general color. Their relative morphological similarity led some authors to suspect that P. chinai is a melanic form of P. howardi . Methods The present study explored this question using different approaches: antennal phenotype; geometric morphometrics of heads, wings and eggs; cytogenetics; molecular genetics; experimental crosses; and ecological niche modeling. Results The antennal morphology, geometric morphometrics of head and wing shape and cytogenetic analysis were unable to show distinct differences between the two taxa. However, geometric morphometrics of the eggs, molecular genetics, ecological niche modeling and experimental crosses including chromosomal analyses of the F1 hybrids, in addition to their coloration and current distribution support the hypothesis that P. chinai and P. howardi are separate species. Conclusions Based on the evidence provided here, P. howardi and P. chinai should not be synonymized. They represent two valid, closely related species.

Additional file 1: Table S1. Geographical location and altitude of studied communities in Loja and Manabí provinces, Ecuador.