Revisiting the Taxonomy of Cylapocoris Carvalho, 1954 (Hemiptera: Miridae: Cylapinae) with Descriptions of Five New Species and Morphology-Based Phylogenetic Analysis of the Genus

Simple Summary Cylapocoris is a genus of the cylapine tribe Fulviini distributed in the Neotropics, with most species being recorded from Central America. By the descriptions of five new species and redescriptions of six species, we provide a robust amount of morphological data, including the novel study of female genitalia, offering an updated diagnosis and description of the genus. This paper also elucidates the phylogenetic position of the genus and the interrelationships of the species within it as well as confirming the monophyly of Cylapocoris. Abstract This paper provides descriptions of five new species of the Neotropical genus Cylapocoris Carvalho, 1954 (C. bimaculatus n. sp., C. brooksi n. sp., C. carvalhoi n. sp., C. scutellatus n. sp., and C. simplexoides n. sp.). Cylapocoris and Cylapocoroides Carvalho, 1989 are redescribed and rediagnosed. Illustrations of male genitalia, scanning electron micrographs of selected structures of certain taxa, and an identification key to species are provided. Female genitalia are described and illustrated for the first time for Cylapocoris in nine out of 19 known species. A cladistic analysis of the genus, based on 62 morphological characters, is presented as a contribution to the understanding of relationships within Cylapocoris and its relationships with other groups of Cylapinae. The analysis comprises 16 ingroup species and 15 outgroup taxa. Both equal and implied weighting parsimony analyses were used in the phylogenetic reconstruction. We confirm the monophyly of Cylapocoris and its sister-group relationship with Cylapocoroides. Additionally, we identify subgroupings within Cylapocoris. Intertribal relationships within Cylapinae are briefly discussed.


Introduction
Cylapinae Kirkaldy, 1903 [1] is a small subfamily within the highly diverse family Miridae Hahn, 1831 (Hemiptera: Heteroptera) currently including over 520 species [2][3][4][5][6][7][8][9]. Most cylapine genera and species are known from the tropics and subtropics. Cylapines are rare in collections owing to cryptic habitats, and many species are represented only by the holotype or a few specimens, with access to molecular data being extremely limited for most taxa. As a result, our understanding of their biology, taxonomy, morphology, and distribution as well as phylogenetic relationships remain poor. Currently, Cylapinae is classified into six tribes, including two large groupings, i.e., Fulviini Uhler, 1886 and Cylapini Kirkaldy, 1903 and four small tribes: Bothriomirini Kirkaldy, 1906, Rhinomirini Gorczyca, 2000, Psallopini Schuh, 1976, and Vanniini Gorczyca, 1997 [10]. However, the identity and placement of Bremer support values (BS) [43] were obtained in TNT for the unweighted analysis ( Figure 1). Symmetric resampling supports (SRS) [44] were also performed for the implied weights analysis in TNT. Unlike bootstrap and jackknife values, symmetric resampling values are not affected by character weight and transformation costs [40,44]. Statistics > 50% are given in Figure 2 within closed circles.

Phylogeny
The heuristic search with equal character weights of characters produced equally the six most parsimonious trees of 153 steps, with a consistency index CI = 53 and a retention index RI = 77. The strict consensus tree with Bremer support values for each branch is shown in Figure 1.
Each of the k = 3-10 IW analyses resulted in two trees. The tree topologies differed in terms of position of several ingroup taxa, but the major clades remained unaffected. Character optimizations are given for a consensus tree obtained from the implied weighting analysis with k-value = 3 ( Figure 2), which had the highest weighting impact.
The phylogenetic analyses using the EW and IW approaches yielded similar results in terms of establishing the phylogenetic relationships within the subfamily Cylapinae. The analyses identified two main clades within the subfamily: clade 1 which consists of Bothriomirini and Cylapini, and it received high nodal support in both analyses (BS 2, SRS 83%) and clade 4 which comprises Fulviini, Psallopini, and Rhinomirini. It also received high nodal support in both analyses (BS 8, SRS 100%). The relationships within clade 1 were consistent between the EW and IW topologies. The analyses revealed a sistergroup relationship of Bothriomirini to Cylapini, a moderately supported clade (clade 2) containing taxa of Cylapini (BS 2, SRS 74%), and a decisively supported lineage (BS 6, SRS 100%) including taxa of the Cylapus complex sensu Wolski [9].
Within the clade 4, the relationships were not fully resolved. Psallops Usinger, 1946, Rhinomiris Kirkaldy, 1902, Euchilofulvius tibialis Poppius, 1909, Fulvius pallens Gorczyca, 2002, and Peritropis saldaeformis Uhler, 1891 formed an unresolved polytomy with the remaining taxa in the EW analysis. However, the IW analysis recovered more resolved relationships compared to the EW analysis, although most of the clades lacked support or had only moderate support. There was a well-supported assemblage (BS 6, SRS 100%) composed of Comefulvius chingonus Carpintero, 1985, Incafulvius peruvianus Carvalho, 1976, Henryfulvius gracilis Wolski, 2015, and Xenocylapus tenuis Wolski, 2015 (clade 10, Xenocylapus complex) in both the EW and IW analyses. Within this grouping, the analysis showed a sister-group relationship of Incafulvius peruvianus to Henryfulvius gracilis + Xenocylapus tenuis (clade 11), but this relationship lacked statistical support. The latter lineage (Henryfulvius gracilis + Xenocylapus tenuis) was moderately supported in IW analysis (SRS 73%). Both the EW and IW analyses recovered a sistergroup relationship within a well-supported assemblage (BS 3, SRS 97%) containing Cylapocoroides and Cylapocoris (clade 13). Additionally, the monophyly of Cylapocoris was well supported (BS 3, SRS 95%). Within Cylapocoris, both the EW and IW analyses identified two subgroupings: simplex clade (node 15) and pilosus clade (node 22). In the IW analysis, the topology for Cylapocoris was resolved, but many of the subordinate clades within the genus lacked support or had weak to moderate support while the EW analysis revealed an unresolved polytomy of Cylapocoris bimaculatus, C. braylovskyi, C. costaricaensis, and C. pilosus with other taxa within the clade 23.
Node 8. This clade is supported by a single synapomorphy: scent gland evaporative area absent . The support values are insignificant.
Node 11. Incafulvius + Henryfulvius + Xenocylapus. This node is supported by one homoplasious feature: setae on hemelytron simple . The support values are insignificant.
Node 15. Cylapocoris simplex clade. This lineage is phylogenetically defined by one homoplasious character: sclerotized part of ductus seminis inside endosoma as long as wide (51-1). The support values are insignificant.
Node 17. This clade is supported by one homoplasious feature. Yellowish patch on apex of endocorium present . This clade is recovered as monophyletic by 50% SRS.
Node 19. C. sulinus + C. simplex + C. fulvus + C. simplexoides. This lineage is monophyletic with 70% SRS and is defined by one homoplasious character: portion between apex of clypeus and anterior margin of eye shorter than eye width in lateral view (10-1).
Node 20. C. simplex + C. fulvus + C. simplexoides. This clade is phylogenetically defined by two synapomorphies: pronotum less than two times longer than wide (28-1) and endosoma thin . This node is recovered as monophyletic by a high support value (85% SRS).
Node 23. This node is supported by one homoplasious feature: pale annulation on antennomere II apically present . The support values are insignificant.

Cylapocoris carvalhoi New Species
Description. COLORATION. Dorsum dark brown with black and dark yellow areas. Head. Dark brown, vertex with two large dark yellow tinges bordering margin of each eye; antenna dark brown with dark yellow and orange areas; antennomere I dark brown broadly tinged with dark yellow basally; antennomere II with apical one third with orange annulation; antennomeres III and IV dark brown, except for narrow yellow annulation of antennomere IV apically; labium dark brown. Thorax. Pronotum. Blackish; collar dark ochraceous. Mesoscutum and scutellum. Dark brown. Thoracic pleura. Blackish, metathoracic scent gland evaporative area contrastingly yellow. Hemelytron. Dark brown. Legs. Coxae dirty yellow; other segments missing in the examined specimens. Abdomen. Dark brown; ventrolateral portions of segments V-VIII and entire pygophore ochraceous. TEXTURE AND VESTITURE. Dorsum shining, covered with moderately dense and long, semirecumbent setae. Head. Somewhat rugose, covered with rather short, moderately dense setae, sparser on frons. Thorax. Pronotum. Punctures present, large and deep. Thoracic pleura. Covered with sparse, semi-recumbent setae. STRUCTURE. Head. Antennomere II weakly incrassate. Thorax. Pronotum. Calli flat, pit between them shallow and narrow. Abdomen. Genitalia. Sclerotized portion of ductus seminis inside endosoma caliciform, longer than wide ( Figure 10C,D); medial lobe small, sub-ovoid ( Figure 10C Etymology. Named after the late José Cândido de Melo Carvalho for his many outstanding contributions to miridology. Biology. Unknown. Distribution. Costa Rica (Puntarenas) ("2" in Figure 22). Remarks. C. carvalhoi is most similar to C. costaricaenis, C. cucculatus, C. funebris, and C. plectipennis in sharing the pronotum with deep and large punctures and hemelytron uniformly dark brown without any pale patches. It can, however, be distinguished by the shape of the endosomal sclerites and parameres.    Figure 11A,B); dorsal wall of bursa copulatrix with large, crescent-like sclerotization occupying anterior half of genital chamber; sclerotized rings paired, each situated posterolaterally, thick-rimmed, ovoid, large, anterior edge reaching middle of bursa copulatrix, fused with anterior sclerotization ( Figure 11B).
Redescription. Female. COLORATION. Dorsum yellow with large black and dark brown areas ( Figure 12B,E). Head. Vertex yellow with irregular tinge medially; frons with posterior and lateral portions yellow ( Figure 12B,E,F), remainder of head black ( Figure 12B,E,F); clypeus with yellowish tinge basolaterally ( Figure 12F); antennomeres I and II fuscous ( Figure 12E); antennomere II with narrow whitish annulation apically ( Figure 12E); antennomeres III and IV black ( Figure 12E); labium yellowish, weakly tinged with dark brown ( Figure 12D). Thorax. Pronotum. Collar dark yellowish black; calli, lateral margin, and humeral angle black, surface between posterior portions of calli sometimes with yellow spot; posterior lobe yellow ( Figure 12B,E). Mesoscutum and scutellum. Black ( Figure 12B,E). Thoracic pleura. Mostly black; propleuron with yellowish tinge ventrally; evaporative areas and peritreme yellow to dark yellow ( Figure 12A,D). Hemelytron. Clavus yellow with irregular brown pattern on basal two thirds, apical one third brown, claval vein pale yellow along entire length, row of punctures along claval vein brown, surface between row of punctures on clavus and claval suture yellow; endocorium yellow with irregular brown pattern from area near base to medial part, apex with broad, brown patch; exocorium almost entirely brown, narrowly yellow basally and apically, medial fracture pale yellow along entire length, row of punctures along medial fracture and R + M veins dark brown; cuneus dark brown except for small yellow spot apically; membrane fuscous, its minor cell with pale yellow venation, major cell venation fuscous from basal to subapical region, apical part yellow ( Figure 12B,E). Legs. Coxae yellow, sometimes weakly tinged with brown; femora yellow, with relatively large, brown tinges; tibiae yellow, base with narrow brown patch and with two broad, brown annulations: one situated near base and other near middle; tarsus yellow ( Figure 12A,D). TEX-TURE AND VESTITURE. Dorsum covered with moderately dense, semi-recumbent setae ( Figures 12A and 13A). Head. Smooth, covered with moderately dense, semi-recumbent setae ( Figure 13B,C,D); vertex with shining narrow and short transverse area on vertex, between eyes, and two long oblique area originating from posterior part of vertex and terminating at antennal insertion, clypeus, maxillary and mandibular plates and anterior part of buccula shiny, glabrous without microtrichia and setae ( Figure 13B-D); antennomere II with relatively long, semi-recumbent setae, basal half with sparse vestiture, apical half with relatively dense setae ( Figure 13A). Thorax. Pronotum. With dense and relatively shallow punctures. Legs. Covered with moderately dense, semi-recumbent setae. Abdomen. Covered with moderately dense reclining setae. STRUCTURE. Head. Antennomere II weakly incrassate. Thorax. Pronotum. Calli moderately upraised, pit between them relatively broad. Scutellum. Convex. Abdomen.
Remarks. C. salvadorensis is most similar to C. bimaculatus (see Remarks under C. bimaculatus for further details).
Remarks. C. salvadorensis is most similar to C. bimaculatus (see Remarks under C. bimaculatus for further details).  Cylapocoris scutellatus new species (Figures 15 and 16, Table 1)

Cylapocoris scutellatus New Species
Remarks. C. scutellatus is most similar to C. marmoreus in the small ovoid body, the convex scutellum, the hemelytron with mottled coloration, and by the medial lobe occupying most endosoma. It can, however, be distinguished by the coloration of scutellum and the shape of the male genitalia.
Female. Similar to male in coloration, texture, vestiture, and structure. Abdomen. Genitalia. Bursa copulatrix semiovoid, dorsal wall with single sclerotized ring, thickrimmed, semi-ellipsoid, large, occupying anterior half of genital chamber, its anterior margin strongly convex, posterior margin strongly concave ( Figure 14E Etymology. The specific epithet "scutellatus" is used to denote the dark scutellum with broad longitudinal dark yellow reddish framed stripe medially. Biology. Unknown. Distribution. Ecuador (Pichincha) ("13" in Figure 22). Remarks. C. scutellatus is most similar to C. marmoreus in the small ovoid body, the convex scutellum, the hemelytron with mottled coloration, and by the medial lobe occupying most endosoma. It can, however, be distinguished by the coloration of scutellum and the shape of the male genitalia. Female. Genitalia. Bursa copulatrix semiovoid, membranous without any sclerotizations or sclerotized rings ( Figure 19A,B); posterior wall of bursa copulatrix with interramal sclerite broad, subtriangular ( Figure 19C).  Description. COLORATION. Dorsum medium brown with small dirty yellowish areas ( Figure 17A). Head. Vertex, frons, ventral half of maxillary plate and buccula medium brown ( Figure 17A,B,D); clypeus, mandibular plate and dorsal half of maxillary plate yellow ( Figure 17D); antennomere I yellow with medium brown annulation near base and reddish tinge apically ( Figure 17B); antennomere II reddish with narrow dark brown annulation basally, broader whitish annulation near base and medium brown annulation bordering the whitish one ( Figure 17A,D); antennomeres III and IV missing in the examined specimen; labial segments I and II dirty yellow; segment III medium brown; segment IV dark brown ( Figure 17B). Thorax. Pronotum, mesoscutum, scutellum and hemelytron. Medium brown ( Figure 17A,B); hemelytron with small dirty yellow patch on posterior part of endocorium and inner angle of cuneus; venation surrounding minor cell and outer part of venation surrounding major cell dirty yellow, inner part of major cell venation medium brown ( Figure 17A). Thoracic pleura. Medium brown; ventral margin of prosternum dirty yellow tinged; metathoracic scent gland evaporative area yellow, except for narrowly brown posterior part ( Figure 17B). Legs. Coxae yellow; remaining leg segments missing in the examined specimen ( Figure 17B). TEXTURE AND VESTITURE. Dorsum matte, covered with moderately dense, relatively long semi-recumbent setae ( Figure 17A-C,E,F). Head. Weakly rugose, covered with moderately dense, semi-recumbent and erect setae sparser on frons ( Figure 17E,F); antennomere I basal half nearly glabrous, with several setae, apical half covered with relatively long, semi-recumbent setae ( Figure 17F); antennomere II covered with dense, relatively long, semi-recumbent and erect setae ( Figure 17A,C,E). Thorax. Pronotum with sparse and shallow punctation ( Figure 17A). Thoracic pleura. Covered with sparse, relatively long, semi-recumbent setae ( Figure 17B,C). STRUCTURE. Body elongate-oval ( Figure 17A). Head. Short, portion between apex and clypeus and outer anterior part of eye shorter than eye in lateral view ( Figure 17B,F); antennomere II cylindrical ( Figure 17A). Thorax. Scutellum weakly convex ( Figure 17B,C). Abdomen. Genitalia. Aedeagus. Endosoma thin and elongate ( Figure 18A-D); sclerotized part of ductus seminis inside endosoma (DSS) short, as long as wide, strongly incrassate, medial lobe (ML) occupying most of endosoma; basal sclerite (BS) present, short, subrectangular ( Figure 18B-D).
Measurements   established in the taxonomic studies [25,26]. The current analysis confirms this author's suggestion of a possible affinity between Cylapocoris and Cylapocoroides and provides compelling evidence for the sister-group relationship between these taxa, forming a monophyletic group with strong support values (97%). This conclusion is supported by multiple morphological characters. Additionally, Cylapocoris is confirmed as a monophyletic group with high support values (95%). Our analysis also supports the observation made by Wolski [25] that the genus Adcylapocoris Carvalho, 1989 should be considered a junior synonym of Cylapocoris, as we have found strong evidence by nesting C. castaneus deeply within Cylapocoris. Furthermore, our analysis reveals two main subgroupings within Cylapocoris: the simplex clade (node 15), with no statistical support and the pilosus clade (node 22), with weak nodal support. The assemblages observed within the simplex clade are statistically supported, although the strength of support varies from weak to moderate. The groupings identified within the pilosus clade lack statistical support. Additionally, within this clade, approximately half of the species exhibit an unresolved polytomy in the EW analysis (clade 23). These findings underscore the necessity for additional evaluation of the phylogenetic relationships within the genus, which could potentially be achieved by incorporating multiple datasets (see below).
Although not the primary focus of this study, our analysis reveals the presence of certain generic groups suggested in previous studies and provides a glimpse into the relationships at deeper nodes, i.e., the tribes of Cylapinae. The present study strongly supports the monophyly of the Neotropical group of genera belonging to the tribe Cylapini, the Cylapus complex (clade 3), as established by Wolski [9] in a morphology-based phylogenetic study. Moreover, our analysis provides robust statistical support (100%) for the grouping composed of Comefulvius, Incafulvius, Henryfulvius, and Xenocylapus (Xenocylapus complex, clade 10), which was previously identified by Chérot et al. [46] and Wolski [35] in their taxonomic studies. This assemblage is decisively supported by a number of character states.
Furthermore, our analysis reveals two major groupings within the subfamily. The first comprises Bothriomirini and Cylapini (clade 1), receiving strong statistical support. This phylogeny confirms the monophyly of Bothriomirini as showed in the analyses based solely on morphology [4,6,9,14] and combined, molecular and morphological datasets [5,14]. Our analyses align with previous morphology-based analyses [4,6,9,14] confirming the monophyly of the assemblage composed of Cylapini and Bothriomirini and establishing their sister-group relationship. These topologies deviated from the topology proposed by Namyatova and Cassis [5] in their total-evidence analysis, as they revealed an unresolved position of the Bothriomirini.
The second major grouping on our phylogenetic tree includes Psallopini, Rhinomirini, and Fulviini (clade 4), and is strongly supported (100% SRS). It is characterized by several distinctive character states. The presence of the grouping including Psallopini, Rhinomirini, and Fulviini is consistent with the results of previous phylogenetic analyses based solely on morphological data conducted by Namyatova and Cassis [6], Wolski [9], and Tyts et al. [14], as well as the total evidence analysis performed by Tyts et al. [14]. In all these analyses, the position of Psallops varied from being placed as a sister group to the remaining taxa within this clade (this analysis and [9]) to being nested more deeply within Fulviini [6,14]. This evidence supports the conclusion made by Wolski and Henry [66] that Psallopinae should be considered within the subfamily Cylapinae. On the other hand, Namyatova and Cassis [5] showed Psallops as a sister group to cylapines in the total evidence analysis. Our present analysis showed the clade including Rhinomirini and Fulviini which is in line with the results of the other morphology-based analyses [6,9] and contrasts with total-evidence approaches either suggesting the phylogenetic proximity of Rhinomirini with Cylapini and Vanniini [5] or that Rhinomirini is nested in a separate clade with Psallops [14]. Wolski [9] and Tyts et al. [14] suggested that this incongruence between the tree topologies obtained using different datasets may be caused by the widespread convergent evolution and many characters used in different morphological analyses may be affected by homoplasy, reducing their phylogenetic significance. Tyts et al. [14] showed that total-evidence analysis gives strong supports for generic complexes and suggested that molecular and morphological approaches may have a complementary role in creating more stable classification. Given the low supports for most of the internal nodes within Cylapocoris and most of the nodes within the clade Rhinomirini + Fulviini, it is likely that to receive the more stable phylogeny within the genus and at the deeper nodes, the inclusion of the combined datasets is required. Our study represents the most densely sampled comparative approach ever performed for the genus Cylapocoris. The robust morphological dataset here presented is important for future studies integrating multiple datasets aiming at resolving the phylogeny of the genus and intertribal relationships.

Conclusions
Our research presents a significant contribution to the understanding of taxonomy and phylogeny within of the genus Cylapocoris. By extensively analyzing morphological data, including the novel study of female genitalia, we were able to offer an updated diagnosis and description of the genus. Our study represents the first effort to test the monophyly of Cylapocoris and shed light on the relationships among its species, as well as determine the phylogenetic position of the genus. We confirm the monophyly of Cylapocoris and its sistergroup relationship with Cylapocoroides. Additionally, we identify subgroupings within Cylapocoris. We also support the monophyly of the Cylapus and Xenocylapus complexes. Our findings also highlight the need for integrating molecular and morphological approaches to achieve a more stable classification within the Cylapinae what was suggested by the previous authors. Further research utilizing combined datasets is necessary to obtain a more reliable phylogeny at both the genus and deeper node levels. Our study lays a strong foundation for future investigations into the phylogeny of the genus and intertribal relationships by offering comprehensive and reliable morphological information.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/insects14090721/s1, File S1: The morphological dataset used for the phylogenetic analysis.