Forever Love: The Hitherto Earliest Record of Copulating Insects from the Middle Jurassic of China

Background Mating behaviors have been widely studied for extant insects. However, cases of mating individuals are particularly rare in the fossil record of insects, and most of them involved preservation in amber while only in rare cases found in compression fossils. This considerably limits our knowledge of mating position and genitalia orientation during the Mesozoic, and hinders our understanding of the evolution of mating behaviors in this major component of modern ecosystems. Principal Finding Here we report a pair of copulating froghoppers, Anthoscytina perpetua sp. nov., referable to the Procercopidae, from the Middle Jurassic of northeastern China. They exhibit belly-to-belly mating position as preserved, with male's aedeagus inserting into the female's bursa copulatrix. Abdominal segments 8 to 9 of male are disarticulated suggesting these segments were twisted and flexed during mating. Due to potential taphonomic effect, we cannot rule out that they might have taken side-by-side position, as in extant froghoppers. Genitalia of male and female, based on paratypes, show symmetric structures. Conclusions/Significance Our findings, consistent with those of extant froghoppers, indicate froghoppers' genitalic symmetry and mating position have remained static for over 165 million years.


Introduction
Mating behaviors for extant insects have been studied and documented, for example, for froghoppers [1], scorpionflies [2] and planthoppers [3]. However, fossil records of unequivocal insect mating are fairly sparse. Boucot and Poinar [4] listed 33 instances of fossilized mating insects, such as fireflies, mosquitoes, planthoppers, leafhoppers, water striders, bees and ants, 27 of which are preserved in amber, others on compression fossils. The hitherto oldest example of copulation in fossil insects is a pair of chironomids (Diptera) discovered in Early Cretaceous amber from Lebanon [4,5].
Herein, we report a pair of well-preserved copulating froghoppers, Anthoscytina perpetua sp. nov., referable to the Procercopidae, from Jiulongshan Formation at the Daohugou Village in northeastern China. Procercopidae is an extinct family in the superfamily of froghoppers, Cercopoidea Leach, 1815. Froghoppers get their name because the adults hop around on plants and shrubs like tiny frogs. The nymphs of froghoppers are called spittlebugs because they cover themselves with foaming spittle, composed of tiny air bubbles trapped in secretions from their Malpighian tubules, which provides protection from predation, parasitism and desiccation [6]. This discovery of the earliest record of copulating insects hitherto sheds light on the evolution of mating behavior in this group of insects.

Materials
We examined more than 1200 specimens from the locality of Daohugou, Inner Mongolia, China. All fossil materials studied are housed in the fossil insect collection of the

Methods
The specimens were examined by a LEICA MZ12.5 dissecting microscope and illustrated with the aid of a drawing tube attachment. Fossil photographs were taken by Nikon Digital Camera DXM1200C.
We follow the traditional terminologies of Cercopoidea [12] and Nel et al. [13]. Venation abbreviations used in the text and Figures

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under the Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in Zoobank, the online registration system for the ICZN. The Zoobank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ''Http://zoobank.org''. The ISID for this publication is: urn: lsid: zoobank.org: pub: AF664AE6-A000-45B9-B9BC-6759CD0F63EE. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS.  2B) and paratypes listed in the referred material (Fig. 3). ScA terminating at running to costal margin, ScP arched and fusing with R at basal 1/7 of wing length. R branching into RA and RP at basal 2/5 of wing length. RA simple, a sinuous crossvein ir between RA and RP at basal 8/9 of wing length. RP with 1-2 branches at basal 9/10 of wing length.

Discussion
The superfamily of froghoppers Cercopoidea Leach, 1815 comprises approximately 3000 described species distributed in five extant families. Following Wang et al. [19], a tentative reconstruction of the phylogenetic relationships within the Cercopoidea is proposed in Fig. 5. Although fossil records of Cercopoidea are lacking during the Late Cretaceous, Wang et al. have hypothesized that extant families, with the earliest fossil records from Paleocene, evolved from Procercopidae [19].
On the mating pair, the male's aedeagus is inserted inside the bursa copulatrix of the female. The shaft of aedeagus has external  sclerotization from base to turning point, with an apical spine and a pin-like phallotrema (Figs. 1C, D). Abdominal segments 8 and 9 of the male are disarticulated (Figs. 1A, C, 2E-G), suggesting that these segments might have been twisted and flexed during mating. The female's valvulae are not visible on the fossils, but anal tube is distinctly elongate and apical margin concave. As shown by representative paratypes in Figs. 4A-G, male and female genitalia are symmetrical. The copulating pair exhibit belly-to-belly mating position as preserved. However, due to the potential taphonomic effect, we cannot rule out that they might have taken a side-by-side position when alive, as do extant froghopper taxa [1,6,20].
Mating positions and the evolution of asymmetric insect genitalia have been reviewed by Huber et al [21] and further reported by Huber [22]. Huber et al. proposed that the femaleabove mating position is plesiomorphic for Neoptera. The next process involves a change of positions to male-above, belly-tobelly, or side-by-side, in which the male's genitalia actually contact the female from below as in a female-above position. These positions presumably allow a better control for the males during mating. In most of these positions, both females and males have symmetric genitalia, except for the cases that the side of approach becomes fixed (''one-sided position''), genitalia become asymmetric. For end-to-end and belly-to-belly positions, insects usually rotate the abdomen or the genitalia by 180 degree [21]. Genital asymmetry is a recurring phenomenon in insect morphology and current data suggest that it has arisen multiple times independently in several neopteran orders [22].  number of specimens clearly underscores the rarity of fossils preserving behaviors.
As already argued by Alexander [26] and supported by Huber et al. [21], all evidence suggests that a female-above position is plesiomorphic for Neoptera. It is reported that mating position of extant Cicadomorpha is side-by-side, but the tips of male and female abdomens are connected the same as in the female-above position [20,21,27,28]. Twisting of the abdomen or genitalia is involved in the side-by-side and the belly-to-belly positions. The fossil pair of copulating A. perpetua sp. nov. show belly to belly (or side by side) position, which is consistent with the mating position of extant froghoppers (Fig. 6A). The fossil also shows that the male abdominal segments 8 to 9 disarticulated from segment 7, suggesting that during mating, the male's abdomen was twisted and flexed after the 8 th segment, as depicted in a 3-D reconstruction Fig. 1B.

Conclusions
In summary, our finding of the hitherto earliest record of copulating froghoppers, consistent with those of extant froghoppers, indicate froghoppers' genitalic symmetry and mating position have remained static for 165 million years. The evidence also confirms that symmetric genitalia are plesiomorphic for the taxon and by the Middle Jurassic, froghoppers have adopted the belly-tobelly (or side-by-side) position, which was proposed by Huber et al. [21] as the next step in the process of position changes from the basal female-above.