﻿More than 80 years without new taxa: analysis of morphological variation among members of Mexican Aeneolamia Fennah (Hemiptera, Cercopidae) support a new species in the genus

﻿Abstract The genus Aeneolamia includes eight described species and 32 subspecies widely distributed in America. In Mexico, two species (A.contigua and A.albofasciata) and one subspecies (A.contiguacampecheana) are recognized. In a recent study of Cercopidae in Mexico, a new species of Aeneolamia was noted from Oaxaca, Mexico based on body color and the ornamentation patterns of tegmen, without a formal taxonomic description. To test the hypothesis of an extant new taxon within the genus a comprehensive analysis of intraspecific morphological variation from 46 morphological features was performed, four related to tegmen color patterns in both sexes, six to male genitalia, and 36 continuous characters measured in specimens of both sexes of Mexican Aeneolamia from several geographical localities using traditional univariate, multivariate morphometric, and geometric morphometric methods. This is the first time that this approach has been used in Cercopidae. Aeneolamiadanpecki Castro, Armendáriz & Utrera, sp. nov. from Oaxaca showed pronounced morphological differences in tegmen coloration patterns, the shape of different elements of the male genitalia, and body measurements compared to the other Mexican members of Aeneolamia; therefore, it is described as a new species.

In the compilation of Carvalho and Webb (2005), the six species considered by Fennah were reported together with two additional species, A. albofasciata (Lallemand, 1939) and A. sanguiniplaga (Lallemand, 1938), and more than 30 subspecies. Years later, a new species from Brazil, A. bucca Paladini & Cavichioli, 2013 was proposed within the genus (Paladini and Cavichioli 2013). However, it was later assigned by the same authors to the genus Gervasiella , based on a cladistic analysis of morphological characters . Currently, Aeneolamia includes eight species and 32 described subspecies widely distributed in Brazil, Colombia, Costa Rica, Guatemala, Guyana, Honduras, Mexico, Panama, Venezuela, and Trinidad and Tobago (Carvalho and Webb 2005;Armendáriz-Toledano et al. 2022). In a cladistics framework based on morphological characters, Aeneolamia is supported as a sister clade of Isozulia Fennah, 1953, and together as the sister group of Prosapia Fennah, 1949, within the tribe Tomaspidini . The most recent molecular phylogenetic analysis of Ischnorhininae supports Aeneolamia and Isozulia as sister genera; however, its position within Tomaspidini was separated from Prosapia and associated with Ferorhinella Carvalho & Webb, 2004, Aracamunia Fennah, 1968, and Tropidorhinella Schmidt, 1910(Paladini et al. 2018. Since the description of A. albofasciata Lallemand, 1939; A. flavilatera belenensis Guagliumi, 1956;and A. flavilatera guarici Guagliumi, 1956 no new species or subspecies have been added to the genus Aeneolamia. In Mexico, Aeneolamia is represented by two species, A. albofasciata (Lallemand, 1939) and A. contigua (Walker, 1851). Both Mexican species of Aeneolamia are polyphagous on Poaceae and inhabit almost most regions from Mexican Republic (Martin et al. 1995;López-Collado and Pérez-Aguilar 2012), where they are reported as important damaging pests in sugar cane areas (De la Cruz-Llanas et al. 2005; López-Collado and Pérez-Aguilar 2012; Morales-Pérez et al. 2014;García-González et al. 2017) and grasses (Oomen 1975;Martin et al. 1995;De la Cruz-Zapata et al. 2016). In A. contigua, three subspecies have been recognized, from southwestern Mexico: A. contigua campecheana Fennah, 1951 from Haltunchen, Campeche;A. contigua postica (Walker, 1858) from around Orizaba volcano, Veracruz; and A. contigua sanctaerosae (Fennah 1953) from Santa Rosa, Yucatan. These subspecies were proposed based on differences in coloration patterns of body and tegmina, without conspicuous differences in male genitalia morphology. In Arméndariz-Toledano et al. (2022), the type specimens of A. contigua, A. contigua postica, and A. contigua sanctaerosae were compared, leading to the conclusion that these subspecies corresponded only to variations of A. contigua in agreement with Clark et al. (1976). In a recent study of the taxonomy and diversity of Cercopidae in Mexico and based on body color and the ornamentation patterns of the tegmina, a new species of Aeneolamia was observed from the mountains and central valleys of Oaxaca State. This undescribed taxon was provisionally named Aeneolamia aff. albofasciata (handwritten label: "Aeneolamia aff. albofasciata nueva especie", UCV, deposited in CEAM) for its morphological similarities to A. albofasciata (Armendáriz-Toledano et al. 2022). Members of Aeneolamia display intra-and interspecific variation in tegmina color, both within and among localities, placing great importance on male genitalia characters as reliable species identifiers, because they are conservative within the species (Fennah 1949;Paladini and Cavichioli 2013). Thus, we tested the hypothesis that A. aff. albofasciata is a new taxon within the genus by analysis of the morphological variation of Mexican Aeneolamia species using traditional univariate and multivariate morphometrics of 46 discrete and continuous features of external morphology, tegmina color pattern, and male genitalia on 628 specimens from 59 localities representative of their entire distribution. In addition, we looked for new discrete characters, as well as assessed their usefulness in the identification of these taxa. Furthermore, we performed a geometric morphometric analysis to evaluate whether the variation in the shape of the aedeagus spine allows delimitation of these taxa. This is the first time that this approach has been used to support and define the taxonomic status of a new taxon of Cercopidae. Based on our results, we describe A. danpecki sp. nov. and provide a complete dichotomous key to the Mexican species of Aeneolamia, replacing the partial key of Armendáriz-Toledano et al. (2022).

Materials and methods
A total of 628 Aeneolamia adults from 59 Mexican localities corresponding to 260 females and 368 males were reviewed. From the total sample, 64 specimens (43 ♀, 21 ♂) correspond to A. danpecki sp. nov., 496 to A. albofasciata (178 ♀, 318 ♂), and 68 to A. contigua (39 ♀, 29 ♂). For the third species, we included specimens collected around the respective type localities of the previously recognized subspecies A. contigua campecheana, A. contigua postica, and A. contigua sanctaerosae because the type localities were not geographically detailed in the original descriptions or the habitat of the subspecies in the locality had disappeared (Table 1). The specimens reviewed were loaned by the following institutions: Taxonomic identifications of the species were based on male genitalia. In addition, we included two specimens identified as A. albofasciata (= A. albofasciata occidentalis) from CEAM (Table 1) and determined by W. E. Clark in 1975, an authority on the identification of Aeneolamia species. Aeneolamia danpecki sp. nov. was recognized by a dark brown to light brown tegmen, with two incomplete and barely visible transverse bands, one of them oblique on the basal third and another straight on the distal third, or only the basal band visible, or both absent. Males and females were recognized by their genitalia.

Discrete morphological characters.
Because of high polymorphism in color patterns of wings recorded in some species of Auchenorrhyncha families, particularly in cercopids, and due to the male genitalia traits providing robust evidence to support A. danpecki sp. nov. , a comparison of the variations of tegmina color patterns and male genitalia morphology was performed among Aeneolamia species. Tegmen color patterns were analyzed in the entire sample (n = 628), and male genitalia features from ten specimens of A. danpecki sp. nov., eleven specimens of A. albofasciata, and seven specimens of A. contigua. These characters are as follows: (1) The same color as tegmen (Fig. 1K), (2) an incomplete thin transversal line (Fig. 1L), (3) a complete broad transversal yellowish or white line (Fig. 1M), (4) a complete broad transversal orange or red line (Fig. 1N).

Continuous quantitative morphological characters
Because the Aeneolamia species display apparent differences in body size, 90 adults from 31 Mexican localities were compared using measurements of the head, mouthparts, pronotum, tegmina, and legs (Fig. 3). Using these features, a comparison of the , 42) ratio between body length with wings and length without wings (RBW l ), 43) postclypeus radio in lateral view (width/length) (RPC l ), 44) eye ratio in lateral view (width/length) (RE l ), 45) head ratio in lateral view (width/length) (HRAL l ), 46) and ratio between the length of body with wings and forewing length in lateral view (BLW l ).

Data analyses
The frequency of character states for each feature was calculated for each taxon in the contingency tables (Tables 2, 3). To evaluate if the differences in frequency among character states are associated with different taxa, both Chi-square Test and the contingency coefficient were performed (e.g., Zar 2010). The normality of the distribution for the quantitative continuous features was independently tested by Shapiro and Wilkinson's test; these features were log-transformed to meet the criteria of normality. Basic descriptive statistics were calculated (mean and standard deviation) and the variation of each character was compared among species and between sexes. To determine whether each characteristic differed between sexes and putative species, we performed a two-way analysis of variance (ANOVA) with sex and species as factors, and multiple comparisons with a Tukey test (Zar 2010), but we only provide values that were significantly different at the 5% level (Tables 4, 5, 6).

Multivariate analyses
To explore if the variation of morphological characteristics together segregates the specimens of A. danpecki sp. nov. in a discrete group within multidimensional spaces, a series of ordination analyses were performed. A principal coordinate analysis (PCoA) was performed from a Gower pairwise matrix among 28 male specimens using the ten discrete (male tegmina color pattern and male genitalia) and 36 continuous features. Also, three principal components analyses (PCAs) were performed to explore the geographical patterns of morphological variation among specimens using pairwise covariance matrices of 36 continuous characters. Additionally, we include canonical variate analyses (CVAs) to determine to what extent these features explained the possible taxonomic segregation based on the 90 specimens in males, females, and both sexes together. Multivariate analyses were performed considering each specimen as an operational taxonomic unit (OTU). Lastly, we looked for multivariate statistical differences among taxonomic groups of Aeneolamia recovered in the ordination analyses, with an analysis of similarities (ANOSIM) and the respective pairwise Hotelling's T non-parametric tests among groups representing putative species. Groups recovered in the multivariate space were confirmed by the comparative morphological analysis of male genitalia.

Geometric morphometry of aedeagus
From the male genitalia images that show the aedeagus intact, shape variation in patterns of aedeagus spines were quantified among A. danpecki sp. nov. (n = 4), A. albofasciata (n = 6), and A. contigua (n = 7) specimens using potential homologous landmarks (lm) and semi-landmarks (sml) (Bookstein 1991;Zelditch et al. 2004). The aedeagus shape was defined by two type I lm, and 16 sml. Semi-landmarks were defined using digital curves of equidistant points on photographs of aedeagus spines in lateral view with TPS tpsDig 1.40 software (Rohlf 2004). Semi-landmarks were specific sites located along the digital curvatures representing the outline of the aedeagus spine. Form configurations were digitalized as two-dimensional coordinates with tpsDig 1.40 software (Rohlf 2004). To remove scale effects, position, and orientation from configurations, and obtain shape coordinates, a generalized Procrustes analysis (Zelditch et al. 2004) was performed with the CoordGen6 program of IMP (Sheets 2003). The tangential variation of curvatures of shape coordinates was minimized using the minimum Procrustes distance criterion (Pérez et al. 2006). The highest proportion of shape variation in the data set was quantified by means of a relative warps analysis from adjusted coordinates (Zelditch et al. 2004). Shape variation was analyzed with the first three RWs and shape changes were visualized with Thin-Plate Spline technique by means of deformation grids.

Geographical records
To illustrate the geographic distribution of Aeneolamia spp., the records of the analyzed specimens were projected onto a map of Mexican biogeographical provinces (Morrone et al. 2017).

Results
In total, 46 morphological characters were evaluated: four discrete characters focused on tegmen color patterns in both sexes, six discrete characters on male genitalia, and 36 continuous characters were measured in specimens of both sexes: Six continuous quantitative morphological were reported by Rodríguez et al. (2002Rodríguez et al. ( , 2003, and 30 new ones are proposed in this study.

Discrete morphological characters
All tegmina and male genitalia features showed differences in character state frequencies among A. danpecki sp. nov., A. albofasciata, and A. contigua (Tables 2, 3). Two traits of the tegmen and four from the male genitalia exhibit exclusive character states for species: CAT, CDT, SGP, SEGP, SSP, and PRE.
Aeneolamia contigua: light brown (30%) to dark brown (70%) tegmen (Fig. 1C), with an orange or red line in the internal clavus (100%) (Fig. 1F), the anterior third of tegmen with a conspicuous broad transversal orange or red line (100%) (Fig. 1J), and the distal third with a complete broad transversal orange or red line (100%) (Fig. 1N) Table 3. Frequencies of multi-state or binary characters used to compare the variation male genitalia among Mexican Aeneolamia species. Abbreviations: EAE elevation of the anal tube sclerites SGP shape of subgenital plates SEGP shape of internal edge of subgenital plate apex ASP paramere, shape of primary apical spine SSP parameter, shape of secondary subapical spine PRE tip of aedeagus spines.

Continuous quantitative morphological characters
Combining morphometric data of both sexes, Aeneolamia danpecki is smaller than both A. albofasciata and A. contigua in most features analyzed except SW d , and HRAL l (  6); multiple comparisons support that these measurements were lower in A. danpecki than in A. albofasciata or A. contigua (Table 4). Two-way ANOVA also supported significant statistical differences between sexes in more than 20 features (Tables 5, 6). The interaction of "species" and "sex" factors was considered only to evaluate which features differed between sexes within each species (Tables 5,6 (Table 5).

Multivariate analysis
The first two principal coordinates of PCoAs of continuous and discrete features of males explained 65% of variations (PCo1 = 39.99%, PCo2 = 15.01%) (Fig. 4A). Scatterplots of these principal coordinates (PCo1 vs. PCo2) showed that the specimens of A. danpecki and the other two Aeneolamia species fell into discrete phenotypic groups in the multivariate space analysis (Fig. 4A). The PCAs corresponding to the 36 characters combining both sexes (PCA ♂♀ ), males alone (PCA ♂ ), and females alone (PCA ♀ ) explained more than 80% of the total variation in the first two principal components: PCA ♂♀ = 99% (PC1 ♂♀ = 62.0%, PC2 ♂♀ = 37.0%); PCA ♀ = 88.5% (PC1 ♀ = 72.5%, PC2 ♀ = 16.0%); PCA ♂ = 98% (PC1 ♂ = 68.0%, PC2 ♂ = 30.0%). In the corresponding three-dimensional scatter plots, the specimens fell into three clusters in multivariate space corresponding to A. albofasciata, A. contigua, and A. danpecki (data not shown). The CVAs using 36 linear measurements explained more than 90% of the total variation in the first two canonical vectors in the three analyses performed; both sexes (CVA ♂♀ ): CV1 ♂♀ = 64%, CV2 ♂♀ = 33%; males alone (CVA ♂ ): CV1 ♂ = 81%, CV2 ♂ = 18%; and females alone (CVA ♀ ): CV1 ♀ = 86% CV2 ♀ = 13%. The scatter plot between these variables showed that the specimens in well-differentiated phenotypic clusters correspond to A. albofasciata, A. contigua, and A. danpecki (Fig. 4B). Separate analyses by sex (CVA ♂ , CVA ♀ ) displayed the clearest segregations of operational taxonomic units in well-defined discrete clusters (Fig. 4C, D); in the multivariate space, the OTUs corresponding to A. danpecki Table 4. Measurements of morphological characteristics of three Mexican Aeneolamia spp. as mean ± standard deviation (mm); * Features that display statistically significant differences among species supported by two way ANOVA; in these cases mean values with the same letter were not significantly different at the 5% level by the Tukey test. Abbreviations: HWE d Head width with eyes HL d head length in dorsal view, PW d pronotum width in dorsal view PL d pronotum length in dorsal view SW d scutellum width in dorsal view SL d scutellum length in dorsal view PcL v postclypeus length in ventral view PCW v postclypeus width in ventral view Al v anteclypeus length in ventral view AW v anteclypeus width in ventral view SL v stylet length in ventral view SW v stylet width in ventral view PcW v posterior coxa width in ventral view PcL v posterior coxa length in ventral view BLW v body length without wings in ventral view PCL l postclypeus length in lateral view PCW l postclypeus width in lateral view EL l eye length in lateral view Ew l eye width in lateral view HL l head length in lateral view HW l head width in lateral view LLMP l length of lateral margin pronotum in lateral view BL l body length including wings in lateral view LAW l length of the anterior wing in lateral view WLH d width-length radio of head in dorsal view WLP d width-length ratio of pronotum in dorsal view WLS d width-lengh radio of scutellum in dorsal view WLC v width-length radio of clypeus PR v postclypeus ratio in ventral view (width/length) SR v stylet ratio in ventral view (width/length) RCR v coxa ratio (width/length) RBW l ratio between body length with wings and length without wings RPC l postclypeus radio in lateral view (width/length) REl eye ratio in lateral view (width/length) HRAL l head ratio in lateral view (width/length) BLW l ratio between the length of body with wings and forewing length in lateral view.

Geometric morphometry of aedeagus
The superimposition of 15 aedeagus spine configurations of Aeneolamia members (A. albofasciata, n = 5; A. contigua, n = 6; A. danpecki, n = 4) showed that shape variation is found on both proximal and medial regions (Fig. 5). The first three Rws explained 96.8% of total variation (Rw1 = 79.8%; Rw2 = 13.6%; Rw3 = 3.4%). The respective two-dimensional Figure 5. Scatter plots among the first three relative warps with its respective deformation grids ± 1.5 SD, corresponding to shape analysis of aedeagus spine (sp) of Mexican Aeneolamia spp. A Rw1 vs. Rw2 B Rw1 vs. Rw3 C position of landmarks (S1 and S2) and semi-landmarks (1-14) on aedeagus spine of A. contigua D deformation grids ± 1.5 SD.   scatterplot of these RWs displays three discrete groups corresponding to the three species (Fig. 5A, B). The deformations in the components Rw1, Rw2, and Rw3 were related to the curvature degree of proximal, medial, and distal areas of the spine, respectively (Fig. 5C, D). Specimens of A. danpecki showed a conspicuously curved proximal region upwardly bent to form an almost 90° angle, as was described in the character PRE. Because our analyses support qualitative and quantitative discrete phenotypic variation among Aeneolamia species (two tegmina features characters and five genitalia ones) and the most pronounced morphological differences compared to the previously recognized species A. albofasciata and A. contigua, the specimens of the new species are grouped into a new taxon, Aeneolamia danpecki Castro, Armendáriz, Utrera, sp. nov., described below.  (Fig. 6).
Etymology. The epithet is a noun in the nominative singular standing in apposition to the genus Aeneolamia, in honor of Dr. Daniel C. Peck for his contributions to the knowledge of Cercopidae and his friendship with UC-V.
Diagnosis. Aeneolamia danpecki Castro, Armendáriz, Utrera, sp. nov. is assigned to the genus Aeneolamia by virtue of its tubular aedeagus with a single pair of slender spines attached anteriorly near the middle of the shaft. It can be distinguished from the other known Mexican species of Aeneolamia by the following combination of characters: tegmen dark brown to black, with two incomplete and barely visible transverse bands, one oblique band on the basal third, and another straight band on the distal  (Figs 6, 7); the apex of subgenital plates acute with an acuminate pointed lobe and straight lateral edges (Fig. 2I), the primary apical spine of parameres long and thin spine with a continuous curvature that is not angulated (Fig. 2F) and secondary subapical spine of parameres with two rounded acute lobes similar in size and shape (Fig. 2L); aedeagus spines slightly sinuous conspicuously curved upward and touching the superior margin of phallobase, tips bent to form an almost 90° angle (Figs 2O, 8A, B). Description. Male measurements. Lateral view length (N = 15) 7.45 ± 0.51 mm; width of head in dorsal view (N = 15) 1.75 ± 0.12 mm.
Head. Dorsal view (Fig. 6A, D): black with brown setae; eyes black (discolored in figures); vertex black with median carina that originates in posterior margin of head and extends to tylus, a small depression between eye and median carina elongated and black, without setae, ocelli as close to each other as width of an ocellus; tylus quadrangular and black, with median carina. Ventral view (Fig. 6B, E): postclypeus black, inflated, with median carina black; anteclypeus black; basal segment of rostrum light brown in middle with black sides, distal segment black, reaching mesocoxae; antennae with scape and pedicel black to light brown, basal body of flagellum light brown, setae on pedicel scarce, flagellum brown, basal body of flagellum subcylindrical, smaller than pedicel and with arista. Lateral view (Fig. 6C, G): postclypeus black, convex, lateral grooves slightly marked.
Thorax. Dorsal view (Fig. 6A, F): pronotum black with brown setae, punctate, hexagonal shape without carina, anterior zone with irregular depressed areas, one on each side, anterior margin straight, lateral anterior margin straight, lateral posterior margin slightly grooved, posterior margin grooved. Scutellum black, apex light brown in some specimens. Ventral view (Fig. 6B, E): with hind wing transparent light brown, venation brown-reddish, setae on both faces light brown; prosternum black to light brown, mesosternum black to light brown, metasternum light brown to reddish; fore legs dark brown, and meddle legs dark brown, with trochanters dark brown to light brown; hind legs with coxae, trochanters, femurs light brown with reddish tints or reddish, tibiae and tarsi dark brown to black; tibiae with two lateral spines and an apical crown with two rows of spines, basal spine small, distal spine 2 × longer than basal one, basal spine same size as apical crown spines; basitarsus with two rows of spines covered with scarce setae. Lateral view (Fig. 6C, I): pronotum not curved; tegmen dark brown to black, with two incomplete and barely visible transverse bands, one oblique band on basal third and another straight band on distal third or only basal band visible or both absent, the junction between Cu and R brown.
Abdomen. Ventral view (Fig. 6B, H): black, except posterior and lateral edges of each sternite reddish, last sternite reddish and subgenital plates black or dark brown.
Genitalia. Pygofer in lateral view (Figs 2C, 9A): lateral digital process, superior and inferior margins subequal in length, at the level of the inferior margin of the anal tube with the apex directed forward to the anal tube; subgenital plates in ventral view (Figs 2I,9B) with lateral edge straight, interior margins parallel, not touching distally, wide along almost entire length, but not truncated apex, with shape acute, and tip acuminate with small hook. Paramere in lateral view (Fig. 9A, C): resting on subgenital  plates, basal two-thirds broad and last third curved and tapered at tip to form a long hook, with two dorsal processes, one rounded mesal process with setae, another small process where the primary apical spine like-hook and the lateral secondary subapical spine converge, the primary apical spine long and slender with a continuous nonangulated curvature, sharp point and sclerotized; the lateral secondary subapical spine with two rounded lobes similar in size and shape, superior lobe sclerotized; inferior margin straight, distally curved to form a long spine like-hook. Aedeagus in anterodorsal view (Fig. 2R): bottle-shape with a thin apex, two thin, sinuous spines touching phallobase, tips as small hooks and hugging phallobase. Aedeagus in lateral view (Figs 2C,O,8A,B,9A): tubular, wide at base, abruptly narrowed where two lateral spines join shaft, lateral slightly sinuous spines touching superior margin of phallobase, and tips bent to form an almost 90° angle, apex acute, gonopore apical.
Female measurements. Lateral view length (N = 15) 8.39 ± 0.07 mm; width of head in dorsal view (N = 15) 2.06 ± 0.01 mm. Same characteristics as the male, except larger and posterior and lateral edges of each sternite light brown or reddish ( Fig. 7A-K).
Remarks. Aeneolamia danpecki has black or dark brown subgenital plates with an acute end. In the type of material, San Martín Lachila is a municipality and not part of the Municipality of Zimatlán. Aeneolamia danpecki was recognized as distinct for the first time as "Aeneolamia aff. albofasciata (Lallemand, 1939)" by López-Posadas (2021: 63).
Key to species and subspecies of Aeneolamia Fennah, 1949from Mexico (based on Armendáriz-Toledano et al. 2022 1 Apex of subgenital plates obliquely truncate (Fig. 2K)  Tegmen light brown to dark brown, with two orange transverse lines (Fig. 1J,  N), with orange lines on claval edges V-shaped (Fig. 1F)  Tegmen dark brown to black, with one or two yellowish or white transverse lines (Fig. 1I, M), sometimes accompanied by lines on claval edges V-shaped (Fig.  1E), the secondary subapical spine of parameres with two acute lobes, the dorsal one conspicuously bigger than the ventral (Fig. 2M) (Fig. 10).

Discrete morphological characters
The evaluation of ten discrete characters of male tegmen and genitalia indicates that six of them (CAT, CDT, SGP, SEGP, SSP, and PRE) are useful to differentiate A. danpecki, and both sets of features together can differentiate this species from A. albofasciata and A. contigua as well as being diagnostic characters for A. danpecki (Tables 2, 3). The shape of subgenital plate apex, the shape of subapical spine of paramere, and the shape of the aedeagus spines of A. danpecki show unique character states (Figs 2I, L, O, 8A, B), and nothing similar was documented in the entire series of A. albofasciata and A. contigua examined. Additionally, the occurrence of diagnostic traits on the tegmen of both sexes allows reliable differentiation of both males and females of A. danpecki (Fig. 1A, D, G, H, K) from other species of the Aeneolamia (Fig. 1B, C, E, F, I, J, M, N). Regarding the tegmen, polymorphism is a common phenomenon in members of Cercopidae, with certain spittlebug species showing large variability in tegminal coloration patterns. Phenotypic variation in tegmen color among specimens within or among populations has been attributed to genetic causes (Aquino-Borges et al. 2020), resulting from differences in mating behavior, in attraction cues, or in geographic barriers (Hutchinson 1963;Farish and Scudder 1967). These factors have promoted highly diverse polymorphisms with dozens of morphotypes recognized throughout the species distribution in extreme cases (Farish 1972) and in others, only a few variants within and between localities (e.g., Thompson and Carvalho 2016;Aquino-Borges et al. 2020). Members of the genus Aeneolamia are not exempt from this pattern, in which considerable color variation in tegmen has also been recognized in some species, within and among different spatially separated localities (Fennah 1949). For this reason, in a taxonomic sense, traits related to coloration patterns have been given less weight in defining taxa in this group than other body features (Paladini andCavichioli 2013, 2015). As in other Cercopidae and Aeneolamia species, our analysis of discrete features in A. danpecki, A. albofasciata, and A. contigua allow us to recognize polymorphisms in the coloration patterns of the tegmen, reflected in that the species each presented traits with different character states (TC, CIE, CAT, CDT), and some of them were shared among the species (TC, CIE) (Table 2). However, the comparison of their frequencies supported the fact that the species have exclusive character states in two features, which constitute diagnostic features; in addition, they have different combinations of character states which together allow their recognition, at least in Mexico. The importance of the genital characters in Cercopidae studies was recognized by Fennah (1968) who stated that female and male genitalia characters can be used for grouping species. In comparison with the tegmen features of color, those characters of male genitalia have been shown to be conserved and therefore reliable for species identification and delimitation (Paladini andCavichioli 2013, 2015; this study). In species with polymorphic tegminal color patterns, the specimens' series display consistent discrete morphological features in different elements of male genitalia (Paladini and Cavichioli 2013;Paladini et al. 2018;Aquino-Borges et al. 2020). According to this pattern, our results show that elements of male genitalia easily discriminate males among the Aeneolamia analyzed (Figs 2, 8); despite the tegmen polymorphism found among them (Fig. 1), diagnostic characters of the genital plates, parameres, and aedeagus were found to be the same as in other Aeneolamia species (Paladini and Cavichioli 2013).

Continuous quantitative morphological characters
The statistical analysis of morphological variation of Mexican members of Aeneolamia supports the earlier suggestion that specimens identified previously as A. aff. albofasciata in Armendáriz-Toledano et al. (2022) represent a new species, described here as Aeneolamia danpecki Castro, Armendáriz, Utrera, sp. nov. Morphological differences in male genitalia ( Fig. 2F-T) also support the species separation. Aeneolamia danpecki exhibited smaller mean measurements than both A. albofasciata and A. contigua in the 36 features analyzed. Among these features Al v , BLW v , LAW I , PCW v , PW d , SW d , and SL v displayed the most pronounced differences (Table 4).

Multivariate analysis
From quantitative continuous and discrete characters (PCoA) of males and quantitative features of both sexes (PCA), permitted the recovery of discrete groups corresponding to the two previously recognized species, A. albofasciata and A. contigua, and the new species A. danpecki (Figs 4,5), supporting that these cercopid species have strongly differentiated phenotypes. The robustness of the taxon clusters was demonstrated when the data set of characteristics was divided by sex, with A. danpecki being the most distant taxon in multivariate space and therefore morphologically distinct from A. albofasciata and A. contigua. In other hemipterans, quantitative measures and multivariate analysis have been used extensively to identify and delimit morphological variation within and between species (e.g., Blackman 1987;Gorla et al. 1993;Margaritopoulos et al. 2000;Jayasekera et al. 2010). This is the first time it has been utilized in Cercopidae. An outstanding result was that the multivariate analyses corresponding to each sex alone (CVA♂, CVA♀) displayed the clearest segregation of species (Fig. 4A, B). In the combined CVA of both sexes, one A. contigua female was grouped with A. danpecki and a male was grouped with A. albofasciata (Fig. 4B). This pattern can be explained by the sexual dimorphism of the three species studied. As in other cercopids, their females are usually equal to or somewhat larger than males of the same species (Paladini 2011), so most of the features measured in Mexican Aeneolamia females were larger than those of males. Aeneolamia albofasciata was the species with least pronounced sexual dimorphism in size, while A. contigua displays a greater difference between males and females (Fig. 4B). In other cercopids different sexually dimorphic traits have been recognized, such as ornamentation patterns in the tegmen (Peck et al. 2004;Paladini and Carvalho 2008), the profile of the anteclypeus (Hamilton 1977;Liang 2020), the form of anteclypeus in ventral view (Liang 2020), the tibial glands in male adults (Liang 2003(Liang , 2020, and an elongated basal body of the antenna in males of some genera of Ischnorhinini (Fennah 1968;Carvalho and Sakakibara 1989). Also, in some species, size dimorphism goes in the other direction, where males are smaller than females (Peck et al. 2002a(Peck et al. , 2004Rodríguez et al. 2002Rodríguez et al. , 2003.

Geometric morphometry of the aedeagus
Morphometric analyses have been poorly explored in Cercopidae and quantitative analyses of the shape of genital structures have not been performed previously in the family. However, in studies of other Cicadomorpha and other Cercopoidea, morphometric analyses have been used to recognize and delimit new species. In the genus Cycloscytina Martynov, 1926, shape analysis of the wing allowed to elucidate the species status of its members and support an extinct new species from the Triassic (Chen et al. 2022). In Philaenus, the species limits and distribution boundaries between Philaenus spumarius L., 1758 and Philaenus tesselatus Stål, 1864, were established based on a classical morphometric analysis of aedeagus (Seabra et al. 2019). Our results of geometric morphometric analysis indicate that the shape of the genital structures is quantitatively different among Mexican species of Aeneolamia. The lack of overlap in the shape configurations of the aedeagus spine confirms that this anatomical structure is a robust diagnostic character useful in their identification ( Fig. 5A-D).

Geographical records
In this study, it is evident that, at the biogeographical level, A. danpecki sp. nov. is in sympatry with A. contigua and A. albofasciata in the Sierra Madre del Sur. However, the records of Mexican Aeneolamia species (Table 1) and some authors (López-Collado and Pérez-Aguilar 2012) do not support that A. danpecki coexists in the same localities with another congeneric species (Fig. 10). In addition, the distribution of A. danpecki is narrower than those of A. contigua or A. albofasciata, having been reported only within the eastern portion of the eastern Sierra Madre del Sur province, which corresponds to the central valleys and mountains between Sola de Vega and the city of Oaxaca, Oaxaca State ( Fig. 10