Triatoma yelapensis sp. nov. (Hemiptera: Reduviidae) from Mexico, with a Key of Triatoma Species Recorded in Mexico

Simple Summary Triatominae (Hemiptera, Reduviidae) bugs are considered species of medical relevance because they are vectors of Chagas disease. In Mexico, most species of the Triatoma genus are distributed in the lowlands of the west of Mexico, including the Mexican Pacific coast. Here, we describe Triatoma yelapensis sp. nov. from the Pacific coast of Jalisco (Mexico). We provide statistical support for the morphological distinctiveness of the new species and an updated key of the genus Triatoma for species recorded in Mexico. Abstract Thirty-four species of Triatominae (Hemiptera, Reduviidae) are recorded in Mexico, Triatoma Laporte, 1832 the most speciose genus in this country. Here, we describe Triatoma yelapensis sp. nov. from the Pacific coast of Jalisco (Mexico). The most similar species to T. yelapensis sp. nov. is T. recurva (Stål, 1868), but they differ in head longitude, the proportion of labial segments, coloration pattern of corium and connexivum, spiracles location, and male genitalia. To provide statistical support for the morphological distinctiveness of the new species, we performed a geometric morphometric analysis of T. yelapensis sp. nov., T. dimidiata s.s. (Latreille, 1811), T. gerstaeckeri (Stål, 1859), and T. recurva (Stål, 1868), considering head morphology. We also provide an updated key of the genus Triatoma for species recorded in Mexico.

In 2006, the Institute of Epidemiological Diagnosis and Reference (InDRE) processed several specimens of triatomines from Jalisco, whose morphological characters were different from any other known species of Triatoma. Considering these specimens, here we describe a new species of the genus Triatoma Laporte, 1832. This new species seems closely related to the T. phyllosoma species group, particularly resembling T. recurva (Stål, 1868). We provide statistical support for the morphological distinctiveness of the new species using geometric morphometry. Finally, we provide an updated key of species of the genus Triatoma recorded in Mexico.

Materials and Methods
The Secretary of Health of the State of Jalisco sent eight Triatoma sp. specimens to the InDRE Entomology Laboratory. These specimens were collected manually, from 2006 to 2017, after performing entomological surveillance in a human settlement called "Selva El Tuito". Specimens were collected from Yelapa (20.4875 N, −104.5536 W) and Pizota (20. disease is mainly attributed to some members of the Triatoma phyllosoma species group, which currently includes 17 species [18,19]. Most species of the T. phyllosoma species group are distributed in the lowlands of the west of Mexico, including the Mexican Pacific coast. In this area, Jalisco stands out among other states because of its large richness of species of the T. phyllosoma species group, high rates of natural infection of T. cruzi in these triatomines and prevalence of Chagas disease in humans [20,21]. Seven species of the T. phyllosoma species group are recorded in Jalisco: T. brailovskyi Martínez, Carcavallo and Peláez, 1984, T. dimidiata, T. mazzottii Usinger 1941, T. longipennis Usinger 1939, T. pallidipennis (Stål, 1872), T. phyllosoma (Burmeister, 1835), and T. picturata Usinger, 1939 [18-20, 22-26]. However, records of T. mazzottii in Jalisco are controversial [19,22].
In 2006, the Institute of Epidemiological Diagnosis and Reference (InDRE) processed several specimens of triatomines from Jalisco, whose morphological characters were different from any other known species of Triatoma. Considering these specimens, here we describe a new species of the genus Triatoma Laporte, 1832. This new species seems closely related to the T. phyllosoma species group, particularly resembling T. recurva (Stål, 1868). We provide statistical support for the morphological distinctiveness of the new species using geometric morphometry. Finally, we provide an updated key of species of the genus Triatoma recorded in Mexico.

Materials and Methods
The Secretary of Health of the State of Jalisco sent eight Triatoma sp. specimens to the InDRE Entomology Laboratory. These specimens were collected manually, from 2006 to 2017, after performing entomological surveillance in a human settlement called "Selva El Tuito". Specimens were collected from Yelapa (20.  We use the following terminology: general morphology of Lent and Wygodzinsky [7] and Weirauch [28], antennal sensilla of May-Concha et al. [29], and genitalia of Lent and [13]. Habitus images were obtained using a Nikon D100 camera, with a 55 mm macro lens. Detailed observations and measurements of the head (1.6×), pronotum (1×), wings (1×), legs (1×), and male genitalia (6.3×) were made using a Zeiss Discovery V8 stereoscope and an Axion Vision V 4.8.2.0 camera. For male genitalia drawings, we used a Zeiss Stemi V6 stereoscope with a light camera at 5× magnification. Drawings were edited using Adobe Illustrator v.24.3 and Photoshop ® software.
To provide statistical support for the morphological distinctiveness of the new species, we performed a geometric morphometric analysis of T. yelapensis sp. nov., T. dimidiata s.s., T. gerstaeckeri (Stål, 1859), and T. recurva, considering head morphology [30]. The last three species were chosen for this analysis because they morphologically resemble T. yelapensis sp. nov. more than other species of the T. phyllosoma complex. Additionally T. dimidiata s.s., T. gerstaeckeri, and T. recurva come, respectively, from the three main clades of the T. phyllosoma species group [20]. We chose 12 landmarks of the dorsal view of the head (Figure 2A) for the following specimens: T. yelapensis sp. nov. (3 males, 5 females), T. recurva (3 males from Chihuahua; 6 males, 8 females from Sinaloa), T. dimidiata s.s. (10 males, 16 females from Chiapas; 5 males from Yucatán), and T. gerstaeckeri (2 males, 5 females from Tamaulipas; 1 male from San Luis Potosi; 1 male from Puebla; 1 male, 2 females from Hidalgo; 2 males from Coahuila). Landmarks were located in homologue structures of previously referenced landmarks for triatomines [4,13], which are easily detectable among specimens. We selected 12 landmarks: 4 of type I and 8 of type II. Landmarks were captured using the XYOM-CLIC V 99 software [31]. Males and females were analyzed together instead of each genus on its own because of the low number of specimens available for some examined species. Anatomical landmarks were processed using a generalized procrustes analysis (GPA). This analysis orthogonally aligns and projects residual Procrustes onto a flat plane tangent to the consensus of the aligned objects [31]. Residual Procrustes of the 69 studied specimens were retrieved from the superimposition of Procrustes and used as input for the analysis of principal components (ACP). The first six principal components were used as final conformation variables for canonical variate (CVA) analysis. ANOVA statistics were used to compare the variance between group means for centroid size (CS). Analyses were performed using the XYOM V2 online software available at https://xyom.io (accessed on 14 May 2021).

Coloration
General coloration dark brown for the holotype, to black. Head uniformly black, except for a reddish-brown narrow area from maxillary plate to clypeus ( Figure 2B); reddish-brown narrow area is more evident in some specimens than in others. Antenna color: pedicellus dark brown in the apex, slightly lighter basally; basiflagellomere uniformly dark brown; distiflagellomere light brown in the apex and darker basally ( Figure 2C). Neck dark brown to black, with a yellowish spot laterally. Labium dark reddish brown; connections between labial segments light yellow ( Figure 2D). Pronotum uniformly dark brown ( Figure 3A). Scutellum dark brown; central depression dark reddish brown; posterior process dark brown. Hemelytra with corium yellow; large dark central spot no surpassing R and R + M veins and extending approximately to the basal area of corium ( Figure 3A); apex dark brown; costal margin basally dark brown; clavus basally black and apically smoked black; membrane smoky brown, lighter than dark brown portion of corium ( Figure 3A); a cloudy spot dark brown between cubitus and postocubitus veins. Legs uniformly dark. Connexivum dark brown; subtriangular yellow-orange spot on dorso-ventral external margin, wide in posterior half, reaching the connexival suture (Figures 3 and 4). Sternites uniformly dark brown ( Figure 4). Spiracles yellowish, surrounded by a yellow spot on the tegument (Figure 4).
Head. Cylindrical, narrow, rugose transversally on dorsum and irregular laterally, slightly rugose below, 2.5 times as long as wide across eyes (1:0.40); anteocular region 3 times as long as postocular (1:0.3). Eyes in lateral view reaching ventral but not dorsal margin of head. Ratio between width of eye and synthlipsis 1:1.59. Ocellar lens small (0.23 mm) located at U-shaped tubercle with subparallel sides. Antenniferous tubercles situated in the middle of the anteocular region, not surpassing the mandibular plate. First antennal segment not surpassing the apex of clypeus. Labium slender; first visible segment extending but not surpassing level of the base of antenniferous tubercle; second visible segment extending to the level of the yellow spot on neck, attaining level of base of head. Ratio of labial segments 1:1.73:0.45. Clypeus long and narrow apically. Maxillary plate not attaining apex of clypeus, slightly sharped apically ( Figure 2B). Anterolateral projections large, subconical, rounded. Head longer than pronotum (1:0.77).
Pronotum. Constricted at the level of transversal sulcus. Disc of anterior lobe granulose with 1 + 1 discal tubercles and 1 + 1 lateral tubercles, smaller than half of length of discal tubercles, but still perceptible. Posterior lobe heavily wrinkled; submedian carinae on posterior half of lobe; humeral angles rounded. Scutellum triangular heavily rugose with central portion depressed; apical process elongate sub-cylindrical and apically rounded. Mesosternum with a knob transverse T-shaped on median portion ( Figure 4C). Hemelytra surpassing urotergite VI, not attaining VII margin in either sex, leaving entire connexivum and lateral portions of urotergites exposed ( Figure 3A). Spiracles on abdominal sternites, adjacent, but not touching connexival suture ( Figure 4B).
Legs. Slender and sub-cylindrical; fore femora with a row of five denticles in small elevations on anterolateral portion; fore and mid femora with one pair of small denticles subapically under surface. Males with small fossula spongiosa on fore tibia; legs of females without fossula spongiosa. Abdomen one-third as wide as total length of body, convex slightly flattened ventrally in the middle ( Figure 3A).
Vestiture. Setae decumbent, sparse, inconspicuous (0.1-0.2 mm). Head with setae very sparse and decumbent. Pedicellus and basiflagellomere of antenna with short and decumbent setae; distiflagellomere with bristles erected and numerous sub-erect sensilla trichoidea. Second labial segment with few inconspicuous setae; apex of third labial segment with conspicuous setae. Pronotum and scutellum glabrous. Mesosternum and metasternum with scattered and very sparse short setae, decumbent. Hemelytra glabrous. Lateral margin of connexivum with dark short and thick setae; female with abundant setae on seventh segment. Pilosity of venter short and decumbent. Table 1. Triatoma yelapensis character measures for females, males, the holotype and T. recurva (mm). ARL-length of anteocular region; bfla-length of basiflagellomere (the fourth was missing in all specimens); HL-length of head (excluding neck); lb1-lb3-length of first to third labial segments; Max = maximum value; Min = minimum value; PedL-length of pedicellus; PL-length of pronotum; PRL-length of postocular region; WE-width of eye; WH-width of head; TL-total length of the body (mm); SD = standard deviation; Sy-length of synthlipsis; WPPL-width of posterior pronotal lobe.

T. yelapensis T. recurva
Female ( Male genitalia (Figures 5 and 6). Pygophore globose, rugose transversally in the posterior third on venter, marginate distally; general color piceo; in lateral view longer than width (1:0.7); transverse bridge of pygophore sclerotized and large. Median process of pygophore triangular, short, not depressed at baseline, with long setae. Parameres elongated, cylindrical, subapically curved; rugose in the externo-apical portion; setae very scarce on both surfaces ventral and dorsal. Articulatory apparatus long. Basal plate extension rectangular, shorter than basal plate (1:0.83), strongly curvated in lateral view; arms elongated, divergent but united by a somewhat narrow and slightly elongated basal bridge. Gonopore process cylindrical, hollow, elongated, occupying the entire medial extension of basal plate, lateral margin sclerotized. Phallosoma support with two elongated lateral arms, joined at basal portion and separated at apex. Medial basal sclerite of phallosoma, large, rounded at apex, strongly sclerotized. Dorsal phallotecal sclerite ovoid, laminar plate with parallel sides, basally opening, less sclerotized on the top. Endosomal process medium to small size, discoid-shaped, sclerotized, and denticulate in the anterior half.

Etymology
The specific epithet derives from the type locality, Yelapa, and the Latin adjectival suffix "-ensis", meaning "originating in" or "pertaining to".

Host-Parasite Data
Only one specimen of Triatoma yelapensis sp. nov. was examined for Trypanosoma cruzi infection using the PCR technique and the following primers: Fw: GCAGTCGGCK-GATCGTTTTCG; Rv: TTCAGRGTTGTTTGGTGTCCAGTG. The evaluated sample was positive for T. cruzi infection (Figure 7).

Etymology
The specific epithet derives from the type locality, Yelapa, and the Latin adjectival suffix "-ensis", meaning "originating in" or "pertaining to".

Host-Parasite Data
Only one specimen of Triatoma yelapensis sp. nov. was examined for Trypanosoma cruzi infection using the PCR technique and the following primers: Fw: GCAGTCGGCK-GATCGTTTTCG; Rv: TTCAGRGTTGTTTGGTGTCCAGTG. The evaluated sample was positive for T. cruzi infection (Figure 7).

Species Group Assignment
The combination of characters suggested by Rengifo-Correa et al. [20] to diagnose the Triatoma phyllosoma species group was observed in T. yelapensis sp. nov. The following character combination is shared between T. yelapensis sp. nov. and the Triatoma phyllosoma species group members: head slightly longer than pronotum; location of annteniferous tubercles in the middle of the anteocular region, not surpassing the mandibular plate; genae tapering apically, not surpassing apex of clypeus; first and third rostral segments much shorter than second; third rostral segment shorter than first; the shape of humeral angles rounded; the shape of scutellar process elongated sub-cylindrically and apically rounded; legs dark, slender, and cylindrical; spongy fossula present in fore tibiae of males, absent in females; abdomen wide with urotergites exposed. Considering this information, we propose T. yelapensis sp. nov. as a member of the T. phyllosoma species group. Further phylogenetic analyses are needed to corroborate this hypothesis of the relationship.

Morphometry
Geometric morphometry. According to PCA and CVA analyses of factorial maps of

Species Group Assignment
The combination of characters suggested by Rengifo-Correa et al. [20] to diagnose the Triatoma phyllosoma species group was observed in T. yelapensis sp. nov. The following character combination is shared between T. yelapensis sp. nov. and the Triatoma phyllosoma species group members: head slightly longer than pronotum; location of annteniferous tubercles in the middle of the anteocular region, not surpassing the mandibular plate; genae tapering apically, not surpassing apex of clypeus; first and third rostral segments much shorter than second; third rostral segment shorter than first; the shape of humeral angles rounded; the shape of scutellar process elongated sub-cylindrically and apically rounded; legs dark, slender, and cylindrical; spongy fossula present in fore tibiae of males, absent in females; abdomen wide with urotergites exposed. Considering this information, we propose T. yelapensis sp. nov. as a member of the T. phyllosoma species group. Further phylogenetic analyses are needed to corroborate this hypothesis of the relationship.

Morphometry
Geometric morphometry. According to PCA and CVA analyses of factorial maps of the head shape for T. yelapensis sp. nov., T. dimidiata s.s., T. recurva, and T. gerstaeckeri, T. yelapensis sp. nov constitutes an independent group that differs from other analyzed species. Total variation in the explained conformation was 98.1%. In the discriminant space, the first canonical variable represented 93.2% of the total conformation variation between groups (Figure 8). In this axis, T. yelapensis sp. nov. and T. recurva constitute fully isolated groups, whereas T. gerstaeckeri and T. dimidiata cluster together. The second canonical variable represented 4.9% of the total variation in the conformation, exhibiting low discrimination between analyzed species. Correspondence of each specimen to its own group was 92.8%, according to the K-means algorithm; however, 5/69 individuals of T. dimidiata and T. gerstaeckeri were discrepant with the initial plan of the group. groups, whereas T. gerstaeckeri and T. dimidiata cluster together. The second canonical variable represented 4.9% of the total variation in the conformation, exhibiting low discrimination between analyzed species. Correspondence of each specimen to its own group was 92.8%, according to the K-means algorithm; however, 5/69 individuals of T. dimidiata and T. gerstaeckeri were discrepant with the initial plan of the group. Linear morphometry. Univariate comparison of 14 morphological characters between T. yelapensis and T. recurva shows significant differences in 5 of them. These characters were length of anteocular region (ARL: P<0.000), length of head excluding neck (HL: P < 0.000), length of first and second labial segments (Llb1: p < 0.03; Llb2: P < 0.001), and length of postocular region (PRL: P < 0.001) ( Table 1). [7], Alejandre-Aguilar et al. [32], and Rengifo-Correa et al. [20]) 1. Pronotum with humeral angles acute.
Head and thorax with abundant pilosity; first antennal segment attaining or surpassing the level of apex of clypeus; spongy fossulae absent in all tibiae of both sexes.

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Head and thorax appearing glabrous dorsally; first antennal segment rarely attaining but not projecting beyond the level of apex of clypeus; males with spongy fossulae present on fore or fore and mid tibiae; females lack spongy fossulae. 9 4. Pronotum black; connexivum largely dark brown, with small yellow or orange-red spot.

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Pronotum with posterior lobe extensively orange-yellow; connexivum largely orange-yellow, with a small anterolateral dark brown spot.
T. gerstaeckeri (part) -Dark markings of connexival segments smaller than orange-yellow markings. 12 12. Clavus completely black; venter uniformly black, except yellow or orange-yellow spot continuous from the external margin of connexivum to connexival suture, wider on the posterior portion of each segment and occupying almost 70% of each segment; corium glabrous, yellow with one central and basal black spot.
T. yelapensis sp. nov -Clavus black basally, yellow to orange, yellow apically; venter uniformly light yellow, except dark pygophore, if extensively dark brown to black, then coloration of margin of venter adjacent to connexival suture yellow to orange yellow. 13 13. Venter largely light yellow, except dark pygophore; connections between each segment of the labium with a light yellow annulus, conspicuously contrasting with surrounding tegument.
T. huehuetenanguensis -Venter largely piceous or dark brown, except yellow to orange yellow area adjacent to connexival suture; connections between each segment of the labium concolorous with labium, or at most slightly lighter than surrounding tegument. 14 14. Pronotum with anterior lobe with 1+1 discal tubercles, and 1+1 round, smaller lateral tubercles; membrane of hemelytra almost as pale as corium; pygophore almost round, as long as wide; postocular portion of head with sides subparallel.. . . . . . . ..

T. mopan
-Pronotum with anterior lobe without distinctive tubercles; membrane of hemelytra distinctly darker than corium, rarely almost as pale as corium; pygophore ovoid, longer than wide; postocular portion of head rounded laterally.
T. dimidiata 27. Size 23 mm or more; postocular portion of head with sides subparallel; pronotum with discal and lateral tubercles; fore femora relatively slender, from eight to nine times as long as it is wide; base of hemelytra conspicuously light-colored.
T. gerstaeckeri -Size 22 mm or less; postocular portion of head distinctly rounded laterally; pronotum without lateral tubercles; fore femora relatively stout, about six times as long as wide; base of hemelytra only slightly lighter than remainder.

T. indictiva
28. Base of clypeus strongly swollen, conspicuously convex in lateral view; under surface of head sinuate in side view; venter evenly rounded.

T. incrassata
-Base of clypeus less swollen, flat above, its upper surface only very slightly convex in lateral view; under surface of head almost straight in lateral view; venter slightly flattened longitudinally along middle.

Discussion and Conclusions
Thirty-four species of Triatominae have been recorded in Mexico, of which nineteen consistently invade human houses; the remaining species are found only occasionally in association with humans, preferring sylvatic haenbitats [18]. Triatoma yelapensis sp. nov. increases the number of triatomines recorded for Mexico to 35. This species has shown potential as a T. cruzi vector because it was naturally infected with this parasite and it was collected regularly, from 2006 to 2015, in the peridomicile of a human settlement in Jalisco, Mexico. Triatoma yelapensis sp. nov. seems closely related to species of the T. phyllosoma species group. Hybridization is common between species of this species group [19], but T. yelapensis sp. nov. does not resemble any hybrid or valid species already described. Triatoma yelapensis sp. nov. differs from other species of Triatoma in external morphology-such as head morphology, corium and connexivum coloration pattern-and male genitalia.
Triatoma yelapensis sp. nov. shares several morphological characters with the T. phyllosoma species group members, particularly T. recurva. For T. recurva, Lent and Wygodzinsky [7] observed specimens "with orange-yellow outer margin dorsal and ventrally wider toward posterior portion of each segment; light color in some cases slightly extended mesad along intersegmental connexival sutures". These characters are slightly similar to those exhibited by T. yelapensis sp. nov. However, the light portion of the connexivum of T. yelapensis sp. nov. is wider than it is for T. recurva, particularly toward the posterior portion of each segment, and is in a sub-triangular shape. The corium in T. yelapensis sp.nov. is yellow with a large dark central spot, whereas the corium of T. recurva is uniformly dark brown.
Head morphometry is also useful to characterize T. yelapensis sp. nov. conformation variables; the geometric morphometry of the head of triatomines is frequently used to provide statistical support for differences observed between similar species [33]. Here, we were able to detect differences between conformation variables of the head of T. yelapensis sp. nov., T. dimidiata s.s., T. gerstaeckeri, and T. recurva. Additionally, we also found differences between the linear morphometry of the head of T. yelapensis sp. nov. and T. recurva. These differences were found in the length of the first and second labial segments, the length of anteocular and postocular regions, and the length of the head, excluding the neck. Linear morphometry of the head permitted us to see differences between very close species in the T. phyllosoma species group, such as those between T. mopan vs. T. dimidiata [4]. Lent and Wygodzinsky [7] and Zhao et al. [13] suggested that endosomal sclerites are potentially useful for species-level diagnosis. For instance, male genitalia was important in distinguishing T. bassolsae in Alejandre-Asguilar et al.'s 1999 study of T. phyllosoma (Alejandre-Aguilar et al., 1999). The size and shape of the endosomal process and the position of the denticles allow T. yelapensis sp. nov. to be distinguished from other species of the T. phyllosoma species group. The endosomal process of T. yelapensis sp. nov. is a medium size (0.45 mm) and discoid-shaped, with denticles in the anterior half ( Figures 5 and 6), whereas the endosomal processes of T. gerstaeckeri [34] and T. recurva are bigger than the endosomal process of T. yelapensis sp. nov., and denticles are numerous on all external margin ( Figure 5); Triatoma dimidiata s.l has a small endosomal process (0.33-0.37 mm) and denticles are scarce or absent [35]. Phallosoma support is similar in most species analyzed, i.e., separated at the apex, except for T. dimidiata s.s., which may also share similarities. The dorsal phallotecal sclerite is similar between all species, with a general oval shape with small variations, mainly in size [34,35].