The female penis acts as an intromittent organ and anchors the female to the male

Sex-specific elaborations are common in animals and have attracted the attention of many biologists, including Darwin []. It is accepted that sexual selection promotes the evolution of sex-specific elaborations. Due to the faster replenishment rate of gametes, males generally have higher potential reproductive and optimal mating rates than females. Therefore, sexual selection acts strongly on males [], leading to the rapid evolution and diversification of male genitalia []. Male genitalia are sometimes used as devices for coercive holding of females as a result of sexual conflict over mating []. In contrast, female genitalia are usually simple. Here we report the reversal of intromittent organs in the insect genus Neotrogla (Psocodea: Prionoglarididae) from Brazilian caves. Females have a highly elaborate, penis-like structure, the gynosome, while males lack an intromittent organ. The gynosome has species-specific elaborations, such as numerous spines that fit species-specific pouches in the simple male genital chamber. During prolonged copulation (∼40–70 hr), a large and potentially nutritious ejaculate is transferred from the male via the gynosome. The correlated genital evolution in Neotrogla is probably driven by reversed sexual selection with females competing for seminal gifts. Nothing similar is known among sex-role reversed animals.

Results and Discussion

The gynosomal structures are species specific. The distal sclerotized part is strongly curved in N. curvata ( Figures 2 A–2D), but is straight or only slightly curved in other species ( Figures 3 and S2 ). The membranous region of N. curvata has a smooth dorsal lobe (yellow) and five areas bearing sclerotized spines: a dorsal (red), a pair of lateral (green), and a pair of ventrolateral (purple) spiny areas ( Figure 2 ). In N. aurora and N. brasiliensis, the dorsal and lateral spiny areas are present ( Figure S2 ), but the dorsal lobe and ventrolateral spiny areas are absent. The gynosome of N. truncata lacks all elaborations, but its membranous part is densely covered by tiny bristled spines ( Figure 3 ).

3 Eberhard W.G. Sexual Selection and Animal Genitalia. 5 Lange R.

Reinhardt K.

Michiels N.K.

Anthes N. Functions, diversity, and evolution of traumatic mating. 9 Kamimura Y. Correlated evolutionary changes in Drosophila female genitalia reduce the possible infection risk caused by male copulatory wounding. 10 Bonduriansky R. The evolution of male mate choice in insects: a synthesis of ideas and evidence. 11 Eberhard W.G. Rapid divergent evolution of genitalia. 12 Chapman T.

Arnqvist G.

Bangham J.

Rowe L. Sexual conflict. 13 Hotzy C.

Polak M.

Rönn J.L.

Arnqvist G. Phenotypic engineering unveils the function of genital morphology. 5 Lange R.

Reinhardt K.

Michiels N.K.

Anthes N. Functions, diversity, and evolution of traumatic mating. 9 Kamimura Y. Correlated evolutionary changes in Drosophila female genitalia reduce the possible infection risk caused by male copulatory wounding. Spiny genitalia are present in many male animals []. These may be used as stimulatory devices [] or may result from sexual conflict [], in addition to having an anchoring function to grasp and hold mates. Species-specific membranous pouches in female genitalia are reported in some insects to accommodate the male genitalia [] and thereby reduce the cost of mating imposed by the corresponding male genital spines. The relative function and pattern of elaboration of male and female genitalia in Neotrogla are completely reversed relative to that generally observed.

14 Klimov P.B.

Sidorchuk E.A. An enigmatic lineage of mites from Baltic amber shows a unique, possibly female-controlled, mating. 15 Nyholm T. Über Bau und Funktion der Kopulationsorgane bei den Cyphones (Col. Helodidae). Studien über die Familie Helodidae X. 16 Williams W.G. Sex and Evolution. 17 Müller G.B.

Wagner G.P. Innovation. In certain astigmatan mites [] and scirtid beetles [], male genitalia are reduced and females possess an elongated intromittent tube or an eversible genital duct, respectively. Although these organs are used to obtain sperm or spermatophores, no anchoring mechanism has been observed. Female seahorses have an intromittent ovipositor to deposit eggs in the male brood pouch [], but this is not a penis. The evolution of a female penis is likely to be strongly constrained because, in internally fertilizing animals, the ancestral condition is exclusively that of an inseminating male requiring an intromittent organ and a receiving female, so that integrated modifications in male and female structures and behaviors are required []. Therefore very few animals have reversed intromittent copulatory organs.

18 Gwynne D.T. The secondary copulatory organ in female ground weta (Hemiandrus pallitarsis, Orthoptera: Anostostomatidae): a sexually selected device in females?. It is known that reversed sexual selection has sometimes caused the evolution of masculine characteristics or of secondary genitalia in females. For example, female ground weta (Orthoptera: Anostostomatidae: Hemiandrus pallitarsis) benefit from seminal gifts and compete intensely for them [], using an elbow-shaped structure located in the middle of the abdomen to obtain them from males. An anchoring intromittent organ, such as the gynosome, might be even more effective at taking up seminal gifts from males. Nevertheless, reversed intromittent organs were previously unknown among animals with reversed sexual selection.

19 Wearing-Wilde J. The sclerotized spermatophore of the barklouse Lepinotus patruelis. 19 Wearing-Wilde J. The sclerotized spermatophore of the barklouse Lepinotus patruelis. 20 Wearing-Wilde J. Mate choice and competition in the barklouse Lepinotus patruelis (Psocoptera: Trogiidae): The effect of diet quality and sex ratio. 6 Lienhard C.

Do Carmo T.O.

Ferreira R.L. A new genus of Sensitibillini from Brazilian caves (Psocodea: ‘Psocoptera’: Prionoglarididae). 7 Lienhard C.

Ferreira R.L. A new species of Neotrogla from Brazilian caves (Psocodea: ‘Psocoptera’: Prionoglarididae). 21 Gwynne D.T. Sexual conflict over nuptial gifts in insects. 6 Lienhard C.

Do Carmo T.O.

Ferreira R.L. A new genus of Sensitibillini from Brazilian caves (Psocodea: ‘Psocoptera’: Prionoglarididae). Insects related to Neotrogla suggest the potential of nuptial gifts favoring the evolution and diversification of the gynosome. In Lepinotus patruelis (Trogiidae), the direction of sexual selection is reversed (without reversal of the intromittent organ). Males of this species transmit specialized spermatophores (seminal capsules formed during copulation within the female’s spermatheca), which are produced by the enlarged seminal duct []. Males are choosier about mates than females, indicating the costs of spermatophore production, while females compete for the nutritious seminal gift []. In Neotrogla, similarly shaped spermatophores ( Figures 3 F, S3 C, and S3D) and an enlarged seminal duct producing voluminous spermatophore material ( Figures 2 B, S3 A, and S3B) are present, suggesting that Neotrogla males also donate a nutritious seminal gift to females. All known Neotrogla species inhabit extremely dry oligotrophic caves and feed on bat guano and bat carcasses, which are relatively scarce resources []. Under such circumstances, nutritious seminal gifts cause a strong selection pressure for increased female mating rate []. During their life, Neotrogla females may acquire several spermatophores (up to 11 have been observed in N. brasiliensis) ( Figures 3 F and S3 C) []; they are evidently polyandrous. We also observed that females consumed the contents of the spermatophores after their first mating before producing mature eggs (n = 5; Figure S3 D), suggesting that the contents of the spermatophores are probably used for nutrition as well as fertilization.

19 Wearing-Wilde J. The sclerotized spermatophore of the barklouse Lepinotus patruelis. 22 Klier E. Zur Konstruktionsmorphologie des männlichen Geschlechtsapparates der Psocopteren. 8 Lienhard C. Psocoptères euro-méditerranéens. 22 Klier E. Zur Konstruktionsmorphologie des männlichen Geschlechtsapparates der Psocopteren. 8 Lienhard C. Psocoptères euro-méditerranéens. 19 Wearing-Wilde J. The sclerotized spermatophore of the barklouse Lepinotus patruelis. This interpretation may explain the following unique characteristics of the female internal genitalia and of the coupling behavior of Neotrogla. The spermathecal duct of Neotrogla is divided by a spermathecal plate, such that the female can simultaneously maintain two filled spermatophores ( Figures 3 F and S3 C), something unknown in related taxa []. The duration of copulation in N. curvata is exceptionally long (52.5 ± 11.2 hr, mean ± SD, range 41–73 hr, n = 12; Table S2 ) in comparison with related taxa: approximately 40 min in Prionoglaris stygia [], approximately 2 hr in Trogium pulsatorium [], and a maximum of 4 hr in the genus Lepinotus []. In Neotrogla, females have structures that can coercively hold males. The very long copulation, as well as polyandry, is probably controlled by females to obtain more seminal gifts from males.