The genitals of rhinoceros beetles: a general overview of the endophallus in the tribe Agaocephalini (Coleoptera: Scarabaeidae: Dynastinae) - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice PeerJ . 2026 Apr 15;14:e21077. doi: 10.7717/peerj.21077 Search in PMC Search in PubMed View in NLM Catalog Add to search The genitals of rhinoceros beetles: a general overview of the endophallus in the tribe Agaocephalini (Coleoptera: Scarabaeidae: Dynastinae) Wonseok Choi Wonseok Choi 1 The Diversity of Small Worlds, Seoul, Republic of South Korea Find articles by Wonseok Choi 1, ✉ , Adrian Troya Adrian Troya 2 Departamento de Biología, Escuela Politécnica Nacional, Quito, Ecuador Find articles by Adrian Troya 2 Editor: Viktor Brygadyrenko Author information Article notes Copyright and License information 1 The Diversity of Small Worlds, Seoul, Republic of South Korea 2 Departamento de Biología, Escuela Politécnica Nacional, Quito, Ecuador ✉ Corresponding author. Received 2025 Nov 7; Accepted 2026 Feb 26; Collection date 2026. ©2026 Choi and Troya This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. PMC Copyright notice PMCID: PMC13091580 PMID: 42004705 Abstract The endophallus is the intromittent, membranous structure inside the aedeagus. It provides unique information for distinguishing taxa: species and genera. In addition to taxonomy, this organ is also useful to get insights about the evolution of the studied taxa, thus informing their classification. Despite the extensive reports on the structure and function of insect genitalia, the endophallus has received less attention than the endophallites. Several genera in the Agaocephalini rhinoceros beetles can be classified in three distinct groups based on the morphology of their endophalli. By examining the genitals of about 60% of the currently recognized species in this morphologically heterogeneous tribe, we provide for the first time a general overview of this structure. Furthermore, based on existing differences between genera, we discuss about the potential implications for the internal tribal systematics, as currently accepted. Novel information about endophalli structures will shed light on the missing links to other dynastine tribes. Keywords: Aedeagus, Genitalia, Intromittent organ, Raspulae, Systematics Introduction The genital structures have been widely used in taxonomy and systematics in Coleoptera ( Blaisdell, 1939 ; Miller, 2001 ; Medina, Molano & Scholtz, 2013 ; Schilthuizen et al., 2016 ), and their relatively rapid divergence is especially convenient in distinguishing closely related species ( Eberhard, 2010 ; Moctezuma & Halffter, 2021a ; Nolasco-Soto et al., 2023 ; Moctezuma et al., 2025 ), but also for studying the classification of beetles above the species level in some families ( Tschinkel & Doyen, 1980 ; Calder, 1990 ; Medina, Molano & Scholtz, 2013 ; Moctezuma & Halffter, 2020 ; Moctezuma & Halffter, 2021b ). The taxonomic utility of the multicomponent genital system, which has its basis on the comparison of intra-population versus inter-population variants ( Cohn, 1994 ), has led to numerous discoveries driving the progress of morphological research in beetles. Because of this, the genital structures are extensively reported in the literature. The scarab beetles (Scarabaeidae) show some of the most extreme cases of inter- and intrapopulation morphological disparities, usually polyphenic or allometric in nature ( Moczek, 2002 ; Rowland, Qualls & Beaudoin-Ollivier, 2005 ; Warren et al., 2014 ), most cases of which are well-exemplified in the head armature of the long-horned beetles (Dynastinae). This phenotypic plasticity usually obscures taxonomic differentiation ( Moczek, 2002 ). Besides the examination of external body structures, and in the absence of alternative sources of evidence, for example, DNA, ecology, behavior, the morphology of the genitalia, also called terminalia (see, for example, Cristóvão & Vaz-de Mello, 2021 ) can be of valuable support to the taxonomist. Most species of dynastines display species-specific genital structures which are informative for their diagnoses ( Ratcliffe, 2003 ). The morphological features of the male’s genitalia, or aedeagus, like the parameres, which are involved in tactile stimulations ( Düngelhoef & Schmitt, 2010 ), the phallobase, and the endophallus, which is a sperm-transferring device, are the main structures of interest. The external sclerotized structure of the aedeagus is composed of a bilobed tegmen, formed by two parameres ( Fig. 1A ), and a phallobase ( Fig. 1B ) ( Cristóvão & Vaz-de Mello, 2021 ; Scholtz, 1990 ), whereas the endophallus, which is a membranous sac encased inside the aedeagus, specifically within the median lobe, is composed of two sclerotized temones, a lobe (or lobes), and a number of spine-like endophallites ( Medina, Molano & Scholtz, 2013 ; Génier, 2019 ; Cristóvão & Vaz-de Mello, 2021 ). In an evolutionary context the complexity of these structures could be the result of cryptic female choice ( Roig-Alsina, 1993 ), acting as an important sexual selection mechanism ( Sloan & Simmons, 2019 ). Contrary to the well-studied, conspicuous sclerotized structures of the aedeagus, the endophallus has received less attention, likely due to the difficulty of dissecting and accessing this complex, fragile intromittent organ. Recently, Cristóvão & Vaz-de Mello (2021) provided a revised glossary of terms for the terminalia of Scarabaeoidea, including the structures of the endophallus, which was certainly needed. Figure 1. Main dynastine genital structures referred in this study. Open in a new tab (A) Aedeagus, dorsal view; (B) Aedeagus, lateral view; (C) endophallus; (D) seta-like type I raspulae; (E) digitiform type II raspulae; (F) endophalliculus. Scale bars. (C, E, F) one mm; (D) 100 µm. The genitalia of insects show a remarkable diversity due to selection mechanisms that have not been fully elucidated ( Hosken & Stockley, 2004 ; Hunt et al., 2009 ; Sasakawa, 2022 ). Nevertheless, due to its “rapid” evolution ( Hosken & Stockley, 2004 ), genital characters are sometimes omitted in phylogenetic analyses, because genital traits may not be informative. Some studies, however, are proving otherwise, see for example, Song & Bucheli (2010) , Zhou et al. (2020) , Fang et al. (2023) and Fernández-Campos et al. (2024) . The endophallus morphology is nowadays more understood than in the past and is increasingly used in taxonomic treatments of some Scarabaeidae groups. For example, in Scarabaeinae, see Moctezuma & Halffter (2021a) , Moctezuma & Halffter (2021b) , Nolasco-Soto et al. (2023) , and Moctezuma et al. (2025) . In Melolonthinae, the endophallus provides a large number of useful taxonomic characters ( Sanmartín & Martín-Piera, 2003 ), thus it has been employed for taxonomic treatments ( e.g. , Uliana & Gallerati, 2022 ). Endophallic structures are also important in the taxonomy of Orphninae as they can be a key character defining species and genera ( e.g. , Frolov & Akhmetova, 2016 ; Frolov & Akhmetova, 2020 ). Regarding dynastine genera, Morón (1995) reported on features of the endophallites of the Golofa Hope, 1837 of Mexico; Rowland (2003) suggested the morphology of the raspulae (a type of endophallite) may be a unique diagnostic trait in the genus Xylotrupes Hope, 1837; Neita-Moreno & Ratcliffe (2017) and Neita-Moreno (2021) provided details of endophallic traits in a review of Tomarus Erichson, 1847 from Argentina, Chile, and Uruguay, and of Cyclocephala Dejean, 1821 from Colombia, respectively. In the present contribution we provide the first comparative overview of the endophallus morphology of selected species representing all the component genera in the dynastine tribe Agaocephalini. This is a small group of about 60 species distributed in the Neotropics. This tribe is hard to diagnose because of the heterogeneity of external morphological characters of the body of its component genera ( Ratcliffe, 2003 ). Endrődi (1985) , for example, pointed out that Democrates Burmeister, 1847 and Colacus Ohaus, 1910 are closely related to the Cyclocephalini. More recently, Sobral & Grossi (2023) suggested to move those genera to the Pentodontini. Based on our observations, we here also comment on their suggested changes. Materials and Methods Material examined In order to compare homologous structures, besides the below selected Agaocephalini species, we also included members of the tribes Cyclocephalini, Dynastini, Oryctini, Pentodontini, and Phileurini (see Table 1 and the Supplementary File ). The specimens were identified by the first author using the taxonomic treatments of Endrödi (1970) , Endrodi (1985) , Ratcliffe (2003) , Ratcliffe, Cave & Mondaca (2021) and Sobral & Grossi (2023) , as well as by examining specimens in the following collections: WCPC, Wonseok Choi Personal Collection; NHM, Natural History Museum London; MNHN, Muséum National d’Histoire Naturelle. The material, examined specimens and preparations, is preserved in the WCPC collection and is freely available for research upon request. These specimens were legally purchased online or donated, and all of them had already been sacrificed at the time of acquisition. The corresponding bills can be obtained from the lead author upon request. Portions of this text were previously published as part of a preprint ( https://www.biorxiv.org/content/10.1101/2025.09.18.677223v1 ). Table 1. Material examined in this study. Tribe Species No. examined specimens Specimens’ origin Agaocephalini Aegopsis bolboceridus (Thomson) 3 Bra Aegopsis curvicornis Burmeister 3 Col, Ven Aegopsis diceratops Sobral & Grossi 1 Bra Aegopsis peruvianus Arrow 1 Bol Aegopsis vazdemelloi Sobral & Grossi 1 Bra Agaocephala bicuspis Erichson 1 Ven Agaocephala cornigera Serville 2 Bra Agaocephala margaridae Alvarenga 1 Bra Antodon goryi (Laporte) 1 Bra Brachysiderus quadrimaculatus Waterhouse 2 Per Colacus bicolor Ohaus 1 Arg Colacus moroni Neita-Moreno 1 Arg Democrates burmeisteri Reiche 2 Ecu Gnathogolofa bicolor (Ohaus) 2 Ecu Horridocalia delislei Endrödi 1 Ecu Lycomedes bubeniki Milani 2 Ecu Lycomedes buckleyi Waterhouse 1 Ecu Lycomedes burmeisteri Waterhouse 20 Ecu Lycomedes hirtipes Arrow 4 Col Lycomedes lydiae Arnaud 1 Col Lycomedes ohausi Arrow 3 Ecu, Per Lycomedes reichei Brême 6 Col Lycomedes salazari Pardo-Locarno, et al. 1 Col Lycomedes velutipes Arrow 4 Ecu Minisiderus benjamini (Abadie) 1 Bra Minisiderus goyanus (Ohaus) 3 Bra Minisiderus martinae (Abadie) 1 Bra Minisiderus matogrossensis (Ohaus) 1 Bra Minisiderus mielkeorum (Grossi & Grossi) 1 Bra Minisiderus minicola (Ohaus) 7 Bra Mitracephala humboldti Thomson 2 Per Spodistes batesi Arrow 1 Pan Spodistes hopei Arrow 3 Col, Pan Spodistes mniszechi (Thomson) 2 Mex Spodistes monzoni Warner 1 Mex Cyclocephalini Cyclocephala sexpunctata Laporte 1 Mex Dynastini Golofa porteri Hope 1 Ven Oryctini Megaceras morpheus Burmeister 1 Per Pentodontini Pentodon idiota (Herbst) 1 Ukr Phileurini Phileurus didymus (Linnaeus) 1 Per Total 93 Open in a new tab Notes. Abbreviations Arg Argentina Bol Bolivia Bra Brazil Col Colombia Ecu Ecuador Mex Mexico Pan Panama Per Peru Ukr Ukraine Ven Venezuela Specimens preparation General procedures and terminology followed Cristóvão & Vaz-de Mello (2021) . Selected specimens were softened by immersion in hot water for 10–20 min. The entire abdomen was removed, and its contents were softened, including the aedeagus, which was extracted as described in Ratcliffe, Cave & Mondaca (2021) . The removed tissues were immersed in 10% KOH solution at room temperature until connective tissues were digested, in average about 6 h. Older specimens or badly preserved needed longer digesting time, up to 24 h. After internal tissues were digested and softened, whole tissues including genitalia were transferred to distilled water. Membranous structures were inflated due to osmosis, and spontaneously, a part of the endophallus everted out of the phallobase. The endophallus was extracted by gently pulling out the temones and median lobe, then placed in alcohol. If the endophallus remained solid or was difficult to observe due to opaque fragments, it was reimmersed in a 10% KOH solution for 24 h. Each piece was washed in 70% ethyl alcohol and stored in glycerol for posterior examination. Terminology We divided the endophallus into the following three areas, so as to facilitate the recognition of specific structures: (1) A basal area where the temones are embedded; (2) a medial area where most endophallites and endophalliculi are placed; (3) an apical area bearing the lobes ( Medina, Molano & Scholtz, 2013 ; Fig. 1C ). The term ‘endophalliculus’ (singular) and ‘endophalliculi’ (plural), here first proposed, refers to the relatively small sacs usually covered with minute seta-like type I raspulae ( Fig. 1D ), which extend from the main endophallus body ( Fig. 1F ). This term is a neologism, and is composed of the words ‘endophallus’, and the masculine form of the Latin diminutive suffix ‘culus’, meaning ‘little’. The other narrow, long, and tube-like form of extended structures from the main body is referred to ‘lobe’, following the usage of Genier & Moretto (2017) . We adopted the term ‘raspula’ (singular) or ‘raspulae’ (plural), as in Cristóvão & Vaz-de Mello (2021) , with the following variation: Type I, which are densely grouped, seta-like or hair-shaped setae, typically placed in the medial area and around the endophalliculi ( Fig. 1D ); and Type II, which are spine-shaped or digitiform, and are usually placed on the endophalliculi and on the main endophallus body ( Fig. 1E ). The terms ‘plate-shaped,’ ‘knob-shaped,’ and ‘ring-like’ are used to describe a specific form of endophallites. When these were broad and flat, we referred to these as plate-shaped, which are usually placed on the endophalliculi and the main endophallus body. We observed knob-shaped endophallites, which are single and thick structures, placed at the connecting area between the main endophallus body and the endophalliculus, Ring-like endophallites are tiny, circular structures embedded in the endophalliculus. Finally, the term ‘columna’ (singular), or ‘columnae’ (plural), here first proposed, refers to the usually longest section of each temone, which is connected to its respective arm. Both the arm and the columna form a single structure. Here, the aim of naming a new term is to facilitate the distinction and description of the examined morphological feature. Specimens observation The prepared endophalli were mounted in 2.5% carboxymethyl cellulose (product # C0292; Samchun Co. Ltd.) solution on microscope slides. Some endophalli were hard to observe due to transparency, thus they were stained with 10% Nigrosin (product # 53075-1210; Junsei Co., Ltd.) aqueous solution. To examine the structures of inflated endophalli, 2.5% carboxymethyl cellulose solution was injected through the phallobase using a 15 g oral zonde needle. The inflated endophalli were photographed immediately and stored in glycerol. Imaging All specimens were examined with a Nikon SMZ645 stereoscope and Eclipse 50i microscope, then photographed with a Nikon D5200 coupled with a Tamron 90 mm Macro lens and Laowa 25 mm f/2.8 Ultra 2.5-5X lens mounted on a Wemacro macro rail. The resulting images were stacked using Zerene Stacker (Zerene Systems LLC) and retouched using Adobe Photoshop. All images by the first author. Results General overview of dynastine endophallus morphology The morphology of the endophallus in examined dynastines is overall similar with respect to the presence of two symmetric, elongated, and usually Y-shaped temones forming a ring-like structure ( Figs. 2A , 2D , 2E ), and a relatively long main sac bearing one or more endophalliculi ( Fig. 2A ). While the basal and apical area show little variation in shape, the medial area varies broadly in terms of the number and shape of lobes, endophalliculi, and endophallites. For example, Golofa porteri Hope, 1837 (Dynastini) shows four endophalliculi, three of which bear endophallites and two are distally connected by narrow lobes ( Fig. 2B ); Cyclocephala sexpunctata Laporte, 1840 (Cyclocephalini; Fig. 2C ) bears a single, uniformly sized endophalliculus and an elongated narrow lobe; Pentodon idiota (Herbst, 1789) (Pentodontini; Fig. 2D ) bears four lobes; Megaceras morpheus Burmeister, 1847 (Oryctini; Fig. 2E ) shows an enlarged endophalliculus; Phileurus didymus (Linnaeus, 1758) (Phileurini; Fig. 2F ) bears an enlarged endophalliculus extended from the medial area and numerous lobes. Figure 2. General view of the endophallus in some Dynastinae tribes. Open in a new tab (A) Lycomedes reichei Brême (Agaocephalini); (B) Golofa porteri Hope (Dynastini); (C) Cyclocephala sexpunctata Laporte (Cyclocephalini); (D) Pentodon idiota (Herbst) (Pentodontini); (E) Megaceras morpheus Burmeister (Oryctini); (F) Phileurus didymus (Linnaeus) (Phileurini). Black arrows point to the endophalliculi; red arrows point to lobes. Scale bars: one mm. Endophallites may be present or absent depending on each species, and their shape and number vary accordingly. For example, Cy. sexpunctata ( Fig. 2C ), Pe. idiota ( Fig. 2D ), Ph. didymus (Phileurini) ( Fig. 2F ) are not armed with prominent endophallites. Dense seta-like type I raspulae ( Fig. 1D , see also Fig. 5A in Binaghi, Dellacasa & Poggi, 1969 ), are present in Ph. didymus , and digitiform type II raspulae are present in Go. porteri ( Fig. 3A ). Additionally, endophallites are shaped as plates as in Go. porteri ( Fig. 3B ) and Me. morpheus ( Fig. 3C ), or knob-shaped as in several Agaocephlini species, including Mitracephala humboldti Thomson, 1859 and Agaocephala bicuspis Erichson, 1849, Ag. cornigera Le Peletier de Saint-Fargeau & Audinet-Serville, 1828, Ag. margaridae Alvarenga, 1958 ( Fig. 3D , Table 2 ). The apical area of all observed endophalli shows a similar structure with two lobes, and a short, narrow, tubular structure at the vertex of the main endophallus body ( Fig. 3E ). Figure 3. Close ups of some morphological features of the endophallus in Dynastinae. Open in a new tab (A) Digitiform type II raspulae ( Golofa porteri Hope); (B) plate-shaped endophallites (black arrows) ( G. porteri ); (C) plate-shaped endophallite ( Megaceras morpheus Burmeister); (D) knob-shaped endophallite of Mitacephala humboldti Thomson; (E) apex of endophallus bearing two lobes and a short tubular structure at the vertex (black arrows), ( Lycomedes burmeisteri Waterhouse). Scale bars: one mm. Table 2. Differences in certain endophallite features along all examined species. Tribe Species Group Hair-shaped type I raspulae Type II raspulae Plate-shaped endophallites Knob-shaped endophallites Agaocephalini Colacus bicolor Ohaus I 0 0 0 0 Colacus moroni Neita-Moreno 1 0 0 0 Democrates burmeisteri Reiche 1 0 0 0 Gnathogolofa bicolor (Ohaus) 1 0 0 0 Horridocalia delislei Endrödi II 0 0 0 0 Lycomedes bubeniki Milani 0 0 0 0 Lycomedes buckleyi Waterhouse 0 0 0 0 Lycomedes burmeisteri Waterhouse 0 0 0 0 Lycomedes hirtipes Arrow 0 0 0 0 Lycomedes lydiae Arnaud 0 0 0 0 Lycomedes ohausi Arrow 0 0 0 0 Lycomedes reichei Brême 0 0 0 0 Lycomedes salazari Pardo-Locarno, et al. 0 0 0 0 Lycomedes velutipes Arrow 0 0 0 0 Spodistes batesi Arrow 0 0 0 0 Spodistes hopei Arrow 0 0 0 0 Spodistes mniszechi (Thomson) 0 0 0 0 Spodistes monzoni Warner 0 0 0 0 Aegopsis bolboceridus (Thomson) III 0 0 0 0 Aegopsis curvicornis Burmeister 0 0 0 0 Aegopsis diceratops Sobral & Grossi 0 0 0 0 Aegopsis peruvianus Arrow 0 0 0 0 Aegopsis vazdemelloi Sobral & Grossi 0 0 0 0 Agaocephala bicuspis Erichson 0 0 0 1 Agaocephala cornigera Serville 0 0 0 1 Agaocephala margaridae Alvarenga 0 0 0 1 Minisiderus benjamini (Abadie) 0 0 0 0 Minisiderus goyanus (Ohaus) 0 0 0 0 Minisiderus martinae (Abadie) 0 0 0 0 Minisiderus matogrossensis (Ohaus) 0 0 0 0 Minisiderus mielkeorum (Grossi & Grossi) 0 0 0 0 Minisiderus minicola (Ohaus) 0 0 0 0 Mitracephala humboldti Thomson Undefined 0 0 0 1 Antodon goryi (Laporte) Undefined 0 1 0 0 Brachysiderus quadrimaculatus Waterhouse Undefined 0 0 0 0 Cyclocephalini Cyclocephala sexpunctata Laporte 0 0 0 0 Dynastini Golofa porteri Hope 0 1 1 0 Oryctini Megaceras morpheus Burmeister 0 0 1 0 Pentodontini Pentodon idiota (Herbst) 0 0 0 0 Phileurini Phileurus didymus (Linnaeus) 0 0 0 0 Open in a new tab Notes. 1= present; 0 = absent. Type II raspulae are only present in Go. porteri and Antodon goryi (Laporte, 1832). Of our set of examined material, Go. porteri is the only species among the Dynastinae showing two types of prominent endophallites: digitiform raspulae and plate-shaped endophallites ( Table 2 ). The endophallus of the Agaocephalini Basal area The temones vary in size and shape among genera. We will refer here only to the pair of temones whose arms are in opposed direction to the other endophallic structures because in most cases the other pair was broken in the dissection process. In Aegopsis curvicornis Burmeister, 1847 , the length of the proximal region, that is, the columna of each temone is about twice as long as its base ( Fig. 4A ), whereas in Ag. cornigera ( Fig. 4B ), Co. bicolor Ohaus, 1910 ( Fig. 4D ), Horridocalia delislei Endrödi, 1974 ( Fig. 4G ), Lycomedes reichei Brême, 1844 ( Fig. 4H ), and Spodistes batesi Arrow, 1902 ( Fig. 4K ), it is three times longer or more. The columnae of several Lycomedes Brême, 1844 species, including L. hirtipes Arrow, 1902 and L. reichei , show acute and well-defined vertices ( Fig. 4H ). On the contrary, other species in the same genus exhibit sub-triangular temones, which are less acute and have round vertices ( Fig. 5A ). In Minisiderus matogrossensis (Ohaus, 1930), the columna is about as long as wide ( Fig. 4I ). In Gnathogolofa bicolor (Ohaus, 1910), we could not compare said proportion because the columna is incomplete ( Fig. 4F ). The temonal arms in this species, as well as those of Co. moroni Neita-Moreno, 2015 ( Fig. 5B ), are peculiar because there is no distinction between a differentiated arm and the base of the columna. Here, the arm is as broad as the base of the columna and runs continuous with it, forming a bull’s-like horn structure ( Fig. 4F ). In Gn. bicolor the arm is directed lateriad, while in Co. moroni, it is strongly curved ( Fig. 5B ). Interestingly, the temones of Brachysiderus quadrimaculatus Waterhouse, 1881 are connected medially ( Fig. 4C ), this was observed only in this species. Figure 4. Endophallus of the Agaocephalini. Open in a new tab The complete organ is shown for each genus, except Antodon , which was damaged while dissecting it. (A) Aegopsis curvicornis Burmeister; (B) Agaocephala cornigera Serville; (C) Brachysiderus quadrimaculatus Waterhouse; (D) Colacus bicolor Ohaus; (E) Democrates burmeisteri Reiche; (F) Gnathogolofa bicolor (Ohaus), black arrow points to hair-shaped type I raspulae; (G) Horridocalia delislei Endrödi; (H) Lycomedes reichei Brême; (I) Minisiderus matogrossensis (Ohaus); (J) Mitracephala humboldti Thomson (black arrows points to endophallites at the connecting area between endophalliculus and the main endophallus body); (K) Spodistes batesi Arrow. Scale bars: one mm. Figure 5. Close-up of some morphological features of agaocephaline endophalli. Open in a new tab (A) Temones of Lycomedes burmeisteri Waterhouse; (B) temones of Colacus moroni Neita-Moreno; (C) hair-shaped type I raspulae at the endophallic medial area of Democrates burmeisteri Reiche; (D) seta-like type I raspulae in Lycomedes burmeisteri Waterhouse; (E) endophallus of Lycomedes velutipes Arrow (black arrows point at the endophalliculi); (F) endophallus of Antodon goryi (Laporte) (the black arrow is showing the club-shaped lobe, and the red arrows indicate spine-like type II raspulae); (G) endophallus of Aegopsis bolboceridus (Thomson); (H) ring-shaped endophallites of Mitracephala humboldti Thomson. Scale bars. (A, B, C, E, F, G) one mm; (D, H) 100 µm. Medial area Several endophalliculi and lobes are extended throughout the medial area. Lycomedes velutipes Arrow, 1902 has four endophalliculi ( Fig. 5E ), whereas the other species in that genus have three. Horridocalia delislei ( Fig. 4G ) and S. batesi ( Fig. 4K ) show two to three endophalliculi covered with seta-like type I raspulae, as well as the presence of one long lobe which is similar to that of Lycomedes. Democrates burmeisteri Reiche, 1852 ( Fig. 4E ) and Gn. bicolor ( Fig. 4F ) show a long distinctive band of hair-like type I raspulae ( Fig. 5C ), while L. bubeniki Milani, 2017, L. burmeisteri Waterhouse, 1879 , and L. ohausi Arrow, 1908 show seta-like type I raspulae ( Fig. 5D ). The raspulae at the basal area are more sparse and thicker than that at the surface of the medial area. Antodon goryi is unique among the Agaocephalini because it is the only species that has distinct type II raspulae, which are thick and spine-shaped ( Fig. 5F ). The endophallus of An. goryi has one endophalliculus. The endophalli of Ae. bolboceridus (Thomson, 1860) and Ae. curvicornis exhibit clear differences. The latter has one endophalliculus and one lobe ( Fig. 4A ), whereas Ae. bolboceridus has an enlarged medial area covered with dense type I raspulae, with the lobes extending to the apical area ( Fig. 5G ). The endophalliculus of Br. quadrimaculatus as well as that of all species in Lycomedes is similar in shape and length ( Figs. 4C , 4H ), but with distinct type I raspulae. Here, part of the endophalliculus is covered with a band of dense type I raspulae, and the rest is seta-like type I raspulae, a feature shared by all species in Lycomedes ( Fig. 5D ). The endophallic medial area of Agaocephala Le Peletier de Saint-Fargeau & Audinet-Serville, 1828 ( Fig. 4B ) and Mit. humboldti ( Fig. 4J ) has one endophalliculus; the connecting area between it and the main body is blocked by a single knob-shaped endophallite ( Figs. 4B , 4J ). Agaocephala cornigera has a patch of type I raspulae, which are similarly placed, this is, at the juxtaposition of the knob-shaped endophallite ( Figs. 4B , 4I ). However, Min. minicola (Ohaus, 1930) does not have endophallites except for minute surface-covering type I raspulae. Interestingly, we noted that the endophalli of some specimens of Mit. humboldti and Min. goyanus (Ohaus, 1930) have a small, transparentendophalliculus, where ring-shaped endophallites are embedded ( Fig. 5H ). Apical area All examined dynastine species exhibited a similar configuration of features in the apical area. Two lobes and a narrow tubular structure at the vertex are observed. These lobes vary from club-shaped, as in An. goryi ( Fig. 5F ), to long and twisted, as in Ae. curvicornis ( Fig. 4A ), to simple and long, as in the remaining species. Geographic correlation of endophallic structures Some endophallic traits correlate geographically, thus reflecting possible divergence among certain lineages. We noticed this in two species of the genus Aegopsis Burmeister, 1847 Ae. curvicornis and Ae. bolboceridus , whose distribution is clearly distinct. Aegopsis curvicornis is mostly Andean with its populations inhabiting mountainous regions of Trinidad & Tobago, Panama, Colombia and Ecuador ( Endrodi, 1985 ; Choi et al., 2023 ). The endophallus of this species has a narrow body, one endophalliculus, and multiple long lobes ( Fig. 4A ). On the other hand, Ae. bolboceridus , inhabits the Brazilian Cerrado and the southern region of the Atlantic Forest ( Endrodi, 1985 ; Choi et al., 2023 ). The endophallus of this species is mostly broad with narrow lobes ( Fig. 5G ). In contrast to their internal endophallus morphology, these two species are very similar externally, differing only in the number of tibial teeth, and in the form and size of their cephalic horns. All other species of Aegopsis that are distributed from Amazonian Peru to mid-southern Brazil, exhibited similar endophalli to that of Ae. bolboceridus . Preliminary grouping of the Agaocephalini Agaocephaline external morphology is diverse ( Fig. 6 ), several features, as the pronotal and cephalic horns, the elytral integument, the tarsal claws, are highly variable between genera. This obscures the internal classification of the tribe, However, based on patterns in gestalt, we here propose a new internal organization conformed by three groups of genera ( Fig. 7 ). Group I is formed by Colacus , Democrates and Gnathogolofa ( Figs. 6E – 6G , 7A – 7C ), their species are characterized by the following: absence of a pronotal horn, the head hasa tubercle on the frons, symmetric tarsal claws, broad and leaf-like mandibles, and the body integument has a general matte surface. The parameres of this group, as seen in dorsal view, are slender and elongated, with short apical setae ( Figs. 7A – 7C ). Group II is formed by Horridocalia Endrödi, 1974, Lycomedes and Spodistes Burmeister, 1847, the following characters define their species: prominent cephalic and pronotal horns ( Figs. 6H , 6I , 6L ), asymmetric claws, mandibles with two to three teeth, and the body has a tomentose surface ( Fig. 6L ). The parameres of the species in this group, as seen in dorsal view, are less elongated than those of group I, and are broadened distally, without apical setae ( Figs. 7D – 7F ). Group III is formed by Agaocephala , Aegopsis , and Minisiderus (Fig. G–J), the shared traits among species in these genera are not as evident as in the previous groups. For example, species in Aegopsis and Agaocephala bear two cephalic horns, while in Minisiderus the cephalic horn forms a single trunk proximally but diverges in two prominent teeth apically ( Fig. 6J ). Minisiderus bear a knob-like tubercle ( Fig. 6J ) on the pronotum, while in Aegopsis and Agaocephala this tubercle is protruding anteriad and is larger than that of Minisiderus ( Figs. 6A – 6B ). Exceptions to this are Ag. bicuspis , Ag. duponti Laporte, 1832, and Ag. inermicollis Arrow, 1914 where the pronotal tubercle is absent. The parameres in these genera, in dorsal view, are usually asymmetric and somewhat rectangular ( Figs. 7G – 7I ), except for those in Minisiderus which are broadened medially ( Fig. 7J ). The protarsal claws are symmetric in most species within this group, whereas only in Ag. cornigera , Ag. mannerheimi (Laporte, 1832) , and Ag. urus (Thomson, 1860) , these claws are asymmetric. Figure 6. A sample of the male taxonomic diversity of Agaocephalini rhinoceros beetles whose endophalli were examined in this study. Open in a new tab Each image represents the type species of its respective genus. (A) Aegopsis bolboceridus (Thomson); (B) Agaocephala cornigera Serville; (C) Antodon goryi (Laporte); (D) Brachysiderus quadrimaculatus Waterhouse; (E) Colacus bicolor Ohaus; (F) Democrates burmeisteri Reiche; (G) Gnathogolofa bicolor (Ohaus); (H) Horridocalia delislei Endrödi; (I) Lycomedes reichei Brême; (J) Minisiderus minicola (Ohaus); (K) Mitracephala humboldti Thomson; (L) Spodistes mniszechi (Thomson). Scale bars: five mm. Figure 7. Parameres (dorsal view) of the three here recognized agaocephaline groups. Open in a new tab Group I: (A) Colacus bicolor Ohaus; (B) Democrates burmeisteri Reiche; (C) Gnathogolofa bicolor (Ohaus). Group II: (D) Lycomedes burmeisteri Waterhouse; (E) Horridocalia delislei Endrödi; (F) Spodistes grandis Sternberg. Group III: (G) Agaocephala cornigera Serville. (H) Aegopsis curvicornis Burmeister. (I) Aegopsis bolboceridus (Thomson). (J) Minisiderus mielkeorum (Grossi & Grossi). Images of adults not to scale. Scale bars: one mm. The observed endophallic features also support our present proposal. For example, narrow and small lobes ( Figs. 4D – 4F ) are present only in Colacus , Democrates and Gnathogolofa Arrow, 1914, here classified under group I ( Table 2 ), whereas a broad and large endophalliculus ( Figs. 4G , 4H , 4K ) is present in Horridocalia Endrödi, 1974, Lycomedes Brême, 1844, and Spodistes Burmeister, 1847, here classified under group II ( Table 2 ). The genera in group III ( Table 2 ), on the other hand, show modestly extended endophalliculi as in Minisiderus ( Endrödi, 1970 ) ( Fig. 4I ), a minute endophalliculus connected to the main endophallus body, as in Agaocephala ( Fig. 4B ), or with both types of endophalliculi as in Aegopsis ( Fig. 4A ). In addition, long and often twisted, tube-like lobes are present in the three genera comprising group III ( Figs. 4A – 4B , 4I ). As of the remaining three genera, Antodon Brême, 1844, Brachysiderus Waterhouse, 1881, and Mitracephala Thomson, 1859, we could not find enough evidence, either from endophallic structures or from external morphological characters, to justify their placement within any of the aforementioned groups, and this is why we left them as “undefined” in Table 2 . The raspulae of An. goryi and Br. quadrimaculatus are differently shaped, with the first showing type II ( Fig. 5F ), while in the second the raspulae form a dense band of seta-like type I ( Fig. 4C ). Although Mit. humboldti ( Fig. 4J ) shares some endophallic traits with species in group III, it differs with all of them by a number of external body traits, most notably a single, thick cephalic horn and thickened protarsi, while all species of group III show two cephalic horns and thinner protarsi. Mitracephala humboldti has been collected in montane forests of the northern Ecuadorian Andes, Peru, and northern Yungas of Bolivia, whereas most species in group III have been recorded from the Brazilian Cerrado, except for Ae. curvicornis , Ag. bicuspis , and Ag. margaridae , which are known only from Andean or Amazonian sites in Venezuela, Colombia, and Northern Brazil. Discussion Heterogeneity of agaocephaline endophalli and its implications for the internal tribal classification Our observations underscore considerable morphological diversity of the endophallus in the examined agaocephaline species, particularly in the form and composition of sclerites. This heterogeneity contrasts with the more conservative, but diverse configurations reported in Scarabaeinae lineages. In Onthophagini, for example, the endophallic morphology is highly informative due to its low homoplasy, which means that endophallic characters likely correlate with the observed phylogenetic relationships among the genera and species in that tribe ( Tarasov & Solodovnikov, 2011 ; Tarasov & Génier, 2015 ; Moctezuma & Halffter, 2020 ; Moctezuma & Halffter, 2021b ). Comparable patterns of variability have been documented in Ateuchini ( Kohlmann & Solis, 2009 ; Moctezuma, Sánchez-Huerta & Halffter, 2018 ), Dichotomini ( Montoya-Molina & Vaz-de Mello, 2021 ; Arias-Buriticá & Vaz-de Mello, 2025 ; Moura et al., 2025 ), Sericini ( Ahrens, 2006 ), Phaneini ( Moctezuma & Halffter, 2021a ; Cupello, Ribeiro-Costa & Vaz-De-Mello, 2022 ), Trichiini ( Lis et al., 2008 ), and Deltochilini: Canthon Hoffmannsegg, 1817 ( Nolasco-Soto et al., 2023 ; Moctezuma et al., 2025 ) and Deltochilum Eschscholtz, 1822 ( González-Alvarado & Vaz-de Mello, 2014 ; González-Alvarado & Vaz-de Mello, 2021 ; Moctezuma et al., 2021 ). In this regard, Medina, Molano & Scholtz (2013) demonstrated that, despite the high variation of shared traits among several dung beetle taxa, the endophallus morphology is generally stable so as to characterize broader phylogenetic patterns across the subfamily. In contrast, at the intergeneric level, our observations reveal that Agaocephalini exhibits an even greater degree of variation reflected in the presence or absence of sclerites, as well as in their shape, than what has been published in Scarabaeinae. The most noticeable difference among the examined Agaocephalini species is the type and presence of endophallites. Hair-shaped type I raspulae at the medial area are found in Co. moroni ( Fig. 5B ), D. burmeisteri ( Fig. 5C ), and Gn. bicolor ( Fig. 4F ). Distinct type II raspulae are only found in An. goryi ( Fig. 5F ). Mitracephala humboldti and Ag. cornigera have knob-shaped endophallites or raspulae at the connecting area between the main body and an endophalliculus ( Figs. 4B and 4J ). The remaining species listed in Table 2 have only minute-sized, type I raspulae ( Figs. 1D , 4C , 4G – 4I ). The morphological characters reflecting this variation, however, are apparently not shared and derived along all the lineages that compose the tribe, as currently defined. This not only raises questions about the phylogenetic relationships among the currently known genera in the tribe, but also about the functional and evolutionary significance of endophallus architecture. Further evidence is needed, however, to confirm or reject our proposal depicting at least three agaocephaline groups. The observed differences in general endophallus morphology along our sample of agaocephaline species reinforce our idea of a paraphyletic tribe. The presence of specialized structures, such as type I and II raspulae, as well as various forms of endophalliculi, show a degree of divergence which we cannot explain from an evolutionary perspective based on the current sample size (93 individuals), even though these represent a relatively broad geographic range in South America. A future phylogenetic analysis using a scoring matrix including endophallic- and other external morphological traits, is needed to dig deeper into the relationships among genera. In addition, in order to elucidate whether some endophallic traits are the result of homoplasy or homology, evolutionary-developmental analyses are required. Morphological diversity and sexual selection The configuration of the observed endophallic traits among the examined lineages may be the result of selective pressures, perhaps linked to reproductive isolation or mating mechanisms like the structural complexity of endophallic architecture, which may have shaped the evolution of those lineages ( Rönn, Katvala & Arnqvist, 2007 ). For example, the length of the endophallus has been associated with that of the female ovipositor in some cerambycid beetles of the tribes Trachyderini and Torneutini ( Long & Tong, 2025 ). Therefore, the general reduction or complete absence of endophallites in other species, such as Min. minicola , a member of group III, may represent a secondary loss given that these structures are otherwise present in all of the remaining members of that group. In a number of insect species, females nonrandomly distribute sperm into multiple spermathecae during post-copulatory selection, a phenomenon known as cryptic female choice ( Birkhead, 1998 ; Hellriegel & Bernasconi, 2000 ; House et al., 2016 ). In order to succeed in sexual competition, male genitalia have evolved various mechanisms ( Hosken & Stockley, 2004 ). One of those mechanisms is sexual conflict using harmful male genitalia ( Rönn, Katvala & Arnqvist, 2007 ; Řezáč, 2009 ). For example, the genitalia of seed beetles ( Callosobruchus maculatus (Fabricius, 1775)) armed with spines that can hurt female reproductive tracts ( Cayetano et al., 2011 ). We observed similar spine-like endophallites in the endophalli of Go. porteri ( Fig. 3A ) and An. goryi ( Fig. 5F ). The exclusive occurrence of type II raspulae, in An. goryi may point to a lineage-specific innovation. In this study, the temones of all the Dynastinae examined were symmetric, except for that of D. burmeisteri . Although asymmetry in genital structures is a common trait among insects ( Schilthuizen, 2013 ), asymmetry of endophallic structures in Dynastinae has rarely been investigated ( Breeschoten, Clark & Schilthuizen, 2013 ). As far as we know, this is the first report of temonal asymmetry in an insect taxon. Temones may influence the directionality of the endophallus, possibly resulting in asymmetric sperm delivery. Further investigation is required to uncover the role of temones and the consequences of asymmetry in the context of female choice. Whether this is an isolated case being the product of, for example, a random mutation generating a malformation, or it is in fact a trait of D. burmeisteri , it remains to be tested because we only examined a single individual of this rarely found species. Limitations of the endophallus as a tool in agaocephaline taxonomy As noted before, the morphology of the examined endophalli allowed us to recognize three putative groups of genera. However, because the whole set of features may not be good synapomorphies, these may not be useful as a taxonomic tool for diagnosing the tribe, if its current taxonomic composition continues to hold after a proper phylogenetic study. This is the first comprehensive examination of endophallus structures of a dynastine tribe, however, without a broader understanding of endophallus morphology at the subfamily level, definitive conclusions about their taxonomic utility cannot yet be drawn. For example, the tribal placement of the three genera in group I remains uncertain. Endrödi (1970) noted that Colacus , Democrates , and Gnathogolofa might belong to Cyclocephalini, while Sobral & Grossi (2023) proposed transferring them to Pentodontini. Nevertheless, our current observations are insufficient to support either hypothesis, because the endophallus morphology of both tribes remains largely undocumented. Moreover, only 34 of the 57 known Agaocephalini species were examined in the current study. Broader sampling and comparative analyses are needed to critically evaluate the taxonomic utility of endophallus structures in the Dynastinae. Conclusion This is the first attempt to scrutinize the complex morphological architecture of the internal intromittent organ of all currently accepted agaocephaline genera. Although our sample size is still incomplete, we have shown that the observed characters are insufficient to formally propose a new classification for the tribe. Some features such as the number and shape of raspulae, endophalliculi, and lobes, proved useful for diagnosing some genus-groups we have here identified. The endophallic features described here can provide deeper support for the classification of evolutionarily related groups, ideally integrating data from a comprehensive scoring matrix of both internal and external structures, alongside DNA evidence. Expanding the taxonomic sampling and combining these datasets will bring a higher resolution of the evolutionary relationships among genera, potentially leading to a formal reclassification of the Agaocephalini, which we suspect may not represent a natural group. Supplemental Information Supplemental Information 1. Metadata of the material examined. Locality, including country and coordinates, collecting date, code, and collectors. peerj-14-21077-s001.xlsx (15.6KB, xlsx) DOI: 10.7717/peerj.21077/supp-1 Acknowledgments We thank Dr. Victor Moctezuma and two anonymous reviewers for their insightful comments, which led to improving the quality of this work. Funding Statement The authors received no funding for this work. Additional Information and Declarations Competing Interests The authors declare there are no competing interests. Author Contributions Wonseok Choi conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft. Adrian Troya conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft. Data Availability The following information was supplied regarding data availability: The data of specimens used in the study are available in the Supplemental File . All available photos of the examined endophalli are available at Zenodo: Choi, W., & Troya, A. (2025). Photographs of examined endophalli. Zenodo. https://doi.org/10.5281/zenodo.18028228 . References Ahrens (2006). Ahrens D. The phylogeny of Sericini and their position within the Scarabaeidae based on morphological characters (Coleoptera: Scarabaeidae) Systematic Entomology. 2006;31(1):113–144. doi: 10.1111/j.1365-3113.2005.00307.x. [ DOI ] [ Google Scholar ] Arias-Buriticá & Vaz-de Mello (2025). Arias-Buriticá JA, Vaz-de Mello FZ. Contributions to the taxonomy of Dichotomius Hope, 1838 (Scarabaeidae: Scarabaeinae: Dichotomiini): description of a new subgenus and redefinition of Dichotomius sensu stricto species groups. Revista Brasileira de Entomologia. 2025;69(1):e20240023. doi: 10.1590/1806-9665-rbent-2024-0023. 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[ DOI ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials Supplemental Information 1. Metadata of the material examined. Locality, including country and coordinates, collecting date, code, and collectors. peerj-14-21077-s001.xlsx (15.6KB, xlsx) DOI: 10.7717/peerj.21077/supp-1 Data Availability Statement The following information was supplied regarding data availability: The data of specimens used in the study are available in the Supplemental File . All available photos of the examined endophalli are available at Zenodo: Choi, W., & Troya, A. (2025). Photographs of examined endophalli. Zenodo. https://doi.org/10.5281/zenodo.18028228 . 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