A new ant-butterfly symbiosis in the forest canopy fills an evolutionary gap

Myrmecophilous butterflies can establish complex symbiotic relationships with ants. A caterpillar wandering among the brood of the aggressive ponerine ant Neoponera villosa was found inside the core of a nest built in the myrmecophytic bromeliad Aechmea bracteata. This is the first caterpillar found living inside a ponerine ant nest. Its DNA barcode was sequenced, and an integrative approach was used to identify it as Pseudonymphidia agave, a poorly known member of the subtribe Pachythonina in the riodinid tribe Nymphidiini. The cuticle of the tank-like caterpillar lacks projections or tubercles and is covered dorsally by specialized flat setae that form an armor of small plates. Ant-organs potentially related to caterpillar-ant signaling, such as perforated cupola organs and tentacle nectary organs, are present. These morphological traits, together with evidence of social integration (direct contact with host brood, protective morphology, slow movement, no host aggressiveness), suggest that P. agave is a symbiotic, social parasite of N. villosa, preying on its host brood. However, several knowledge gaps remain, including oviposition site, dependence on bromeliad association, steps to colony integration, and larval diet through development. Carnivory has been reported in all known members of the subtribe Pachythonina (caterpillars prey on honeydew-producing hemipterans) suggesting a shift to myrmecophagy inside the ant nests as a possible evolutionary transition.


Material and methods
The ant-plant symbiotic system. Neoponera villosa is widely distributed from Mexico to Argentina 55 . This is the largest Mexican ant; workers measure 1.2 to 1.3 cm 56 and have a powerful and painful sting. They are generalist predators and forage mainly in the canopy collecting liquid carbohydrate food sources [57][58][59] . Throughout its distribution range, this species nests opportunistically in pre-existing cavities in live or dead trees, hollow branches, or cacao pods 57,60-63 , among other plant cavities. However, in the southern part of the Yucatan Peninsula, Mexico, it preferentially uses the myrmecophytic tank-bromeliad Aechmea bracteata as a nest site and very few colonies are established on other plants 53,54,61,[64][65][66][67][68] . Founding queens show a clear spontaneous preference for A. bracteata material 64 and workers also significantly prefer this bromeliad over other species during nest relocation 54 .
The tightly intertwined leaves of A. bracteata collect rainwater and constitute true water reservoirs that are extremely attractive to a wide range of living organisms, from aquatic life forms to terrestrial invertebrates (especially ants) and even vertebrates 61,65,[69][70][71] . In the A. bracteata microcosm, a particularly diverse array of specialized and facultative myrmecophiles, mostly antagonists, establish complex trophic interactions with N. villosa 53 . To date, various invertebrates have been recorded in direct association with the brood of this species and can be considered as true myrmecophiles 53 . Three of them are brood parasitoids: an unidentified species of Kapala (Hymenoptera: Eucharitidae) 53 , an unidentified species of Blanchardiscus (Hymenoptera: Encyrtidae) 68 , and the hoverfly Hypselosyrphus trigonus (Diptera: Syrphidae) 67,68 . Two species (the pseudoscorpion, Chelodamus mexicolens, and an unidentified tenebrionid beetle possibly in the subfamily Alleculinae) are brood predators, while two mites (an unidentified species of the genus Oplitis and an unidentified galumnid species) are phoretic on the host adults or larvae and a third unidentified mite species (of the genus Cosmolaelaps) is cleptoparasite on the ant larvae. Finally, two other species (a staphylinid beetle of the genus Myrmigaster and a diapriid wasp of the genus Trichopria) have unclear relationships but have been found wandering on the cocoons with their antennae in direct contact with the host cocoon surface 53 . Sampling. Eighty-two colonies of N. villosa, all of them nesting in the core of a tank-bromeliad A. bracteata ( Fig. 1A,B) were collected between January 2016 and April 2018 in several sites in the southern part of the  Table S2). Sequences were then combined with a larger matrix of Riodinidae species for a total of 269 specimens comprising all previously sequenced Riodinidae 36,37,41 including eight nuclear genes as well as the mitochondrial barcode region as described in Seraphim et al. 37 : ArgKin, CAD, GAPDH, Ef1a, IDH, MDH, RpS5, wingless and COI. This expanded matrix was used to obtain a maximum likelihood tree using IQ-TREE software version 1.6.12 75 , with Curetis barsine (Lycaenidae: Curetinae) as an outgroup. The model of nucleotide substitution was estimated using the model selection implemented in IQ-TREE 76 . Support was estimated using 10,000 ultrafast boostraps 77 and the SH-aLRT test 78 . The resulting tree was compressed for clarity.

Results
A myrmecophilous caterpillar ( Fig. 1) was found among the ant brood in a Neoponera villosa colony collected on June 10, 2017 at Ejido Blasillo, Campeche, Mexico (18°7′13.6056″ N, 89°19′47.791″ W, 263 m asl). The caterpillar was isolated in a small humidified petri dish. After several days, the larva appeared to be in a poor condition and was preserved in alcohol. After this myrmecophilous larva was discovered, intensified collecting efforts uncovered 19 additional N. villosa colonies in the same locality 52 , but no additional riodinid larva.

Caterpillar identification.
We sequenced the COI DNA barcode of the caterpillar and included it with a dataset of other riodinid sequences prior to inferring a phylogenetic tree. This tree placed our caterpillar within the subtribe Pachythonina, sister to Pseudonymphidia agave from Mexico (C. Pozo et al., unpubl. data; specimens MAL-05053 and MAL-05054, Fig. 2 and Supplementary Table S2). Indeed, the caterpillar barcode was identical to the barcode of a Mexican P. agave adult (MAL-05054), from Calakmul, Campeche, collected in 1998. Pseudonymphidia agave was originally described as Lemonias agave Godman and Salvin, 1886. Two P. agave subspecies are currently recognized: the nominate subspecies and leucogonia (Stichel, 1911). Based on similarities in genital morphology, antennal length and wing pattern, Hall and Harvey 84 temporarily placed agave and leucogonia in the genus Pseudonymphidia. This genus forms a monophyletic clade along with Pachythone, Roeberella and Pixus in the phylogenetic hypothesis of Seraphim et al. 37 , who erected the subtribe Pachythonina to include several genera placed as incertae sedis in previous revisions of the family (see also 36 ). In two recent controversial studies, Zhang et al. 85,86 placed several species of the subtribe Pachythonina in the genus Pachythone, including the two currently valid species of Pseudonymphidia (P. agave and P. clearista) (see Figs. 9 and 32 in 85 and Fig. 30 in 86 ), but without considering the distribution nor the diversity or natural histories of the species of the whole genus, and without formally presenting the novel combinations or providing a reason for transferring the two species from the genus Pseudonymphidia. Here, we decided not to follow Zhang et al. 85,86 and use 'Pseudonymphidia agave' until new concrete integrative taxonomic evidence is presented. According to the phylogenetic tree obtained here (Fig. 2), the closest relative of the genus Pseudonymphidia is Archaeonympha drepana, a genus not included in the study of Seraphim et al. 37 that needs to be revised since its type has not yet been sequenced and Prevalence, natural history, and behavioral observations. Of the 82 N. villosa colonies collected between January 2016 and April 2018 around the southern part of the Yucatan Peninsula, only one colony was infested with a riodinid larva, even though 19 of the colonies were from the same locality (Ejido Blasillo) and its surroundings. The caterpillar of P. agave was wandering among the host cocoons and larvae in the presence of workers; no aggression was detected during a 3 h observation period. The host colony where the riodinid caterpillar was discovered nested in an Aechmea bracteata bromeliad established at a height of 4 m on a Haematoxylum campechianum (Fabaceae) tree, situated in a patch of deciduous forest. The colony was composed of 11 dealated queens, three gynes, 165 workers, 265 larvae, 173 pupae (in cocoons), and many eggs. Larvae and pupae were apparently not parasitized by endo-or ectoparasites. Apart from the riodinid larva, very few other myrmecophiles were found in the colony: only the cleptoparasitic mite Cosmolaelaps sp. (present on ant larvae, N = 5) and two species of staphylinid beetles (Myrmigaster sp. and Tyropsis sp., N = 2 and N = 1, respectively) were found in the nest chambers. Additionally, 17 adults of an unidentified species of Nitidulidae (Coleoptera), and 16 pseudoscorpions (Chelodamus mexicolens) were found in the nest refuse.
Morphology of the Pseudonymphidia agave caterpillar. Head capsule width: 2.06 mm, total length: 17.81 mm (N = 1). Head brown; body pale brown with mottled dark spots corresponding to the color of the microscopic setae present in each region (Fig. 3A). Body onisciform without projections or tubercles (small knoblike or rounded protuberances), similar in size to the host cocoons (Figs. 1C,3A). Head and appendages (legs and prolegs) concealed under the body, not visible dorsally (Fig. 3A,D,F). Anterior portion of the body wider than the posterior segments in dorsal view (Fig. 3A). Prothoracic shield bilobed (Fig. 3B). Tegument covered dorsally by arborescent flat setae that form an armor of small plates and perforated cupola organs (PCOs) (Fig. 3C,E,G). Downward pointing setae forming a ventral-lateral fringe (Fig. 3F), and elongated arborescent setae associated with the opening of tentacle nectary organs (TNOs) on A8 (Fig. 3H-J). Prothoracic spiracle located ventrally (Fig. 3D), abdominal spiracles aligned laterally, with exception of those on A2 and A8, which are located subdorsally; openings elevated with elliptical peritrema (Fig. 3I). The larval instar could not be determined with certainty, but from its size it corresponded clearly to an advanced instar.
Distribution records for Pseudonymphidia agave adults. Pseudonymphidia agave, the white-trailed metalmark or agave metalmark, is a rare species, recorded from a few localities. Based on museum specimens, P. agave has been recorded from as far north as San Luis Potosi in Mexico and as far south as Colombia (Fig. 4). Specimens have been collected between 0 and 1 600 m 87 . This species seems restricted to specific habitats in tropical forests; adult specimens have been collected in evergreen, semi-evergreen, deciduous, semideciduous, and cloud forests. Adults have been collected on rambutan flowers, Nephelium lappaceum (Sapindaceae), in a commercial plantation 87 , but caterpillar host plants are unknown and natural history information was not available prior to this study. Only 52 P. agave specimens from Mexico currently exist in museum collections (Supplementary Tables S3, S4). Regarding the museum specimens, flight activities have been recorded almost the whole year, with a significant peak in June and a smaller one in October ( Supplementary Fig. S1), a pattern previously observed for the Neotropical riodinids and other small butterflies whereby the greatest number of individuals has been recorded during the wettest season of the year 88 .

Discussion
The biodiversity of tropical forest canopies is a frontier of knowledge that can still reveal great surprises 89 . In these environments, ants are species rich and abundant, connecting species and trophic levels through antagonistic and mutualistic interactions (reviewed in 90 ). Our description of this specific type of biodiversity unveiled an unexpected symbiotic interaction between a butterfly caterpillar and an aggressive ant. Our report that Pseudonymphidia agave caterpillars are guests in the nests of Neoponera villosa is, to the best of our knowledge, the first case of a butterfly caterpillar in direct contact with the host brood inside a ponerine ant nest. Our finding is the first to provide information on the life cycle of a member of the genus Pseudonymphidia, of which the immature stages were previously unknown, and sheds light upon the possible evolutionary steps of social parasitism in riodinids. In recent years, much new information about the immature stages of Riodinidae has been discovered 11,91-96 . Nevertheless, immatures of some lineages remain completely unknown, principally in the Nymphidiini, suggesting that species are rare or that immature stages have not been found yet because they feed in the canopy. On the other hand, the presence of a P. agave larva among the members of a N. villosa colony is consistent with the idea that these taxa may exhibit specialized associations with ants, including long periods inside the ant nests 11,39,96 . Pseudonymphidia agave belongs to the recently erected subtribe Pachythonina (Nymphidiini), a clade diagnosed on the basis of genetic characters 37 . This group comprises rare and range-restricted taxa associated with tropical forests and is the latest subtribe of Nymphidiini whose natural history information has been published 42 . Species in the Pachythonina for which some life history information is known have armored carnivorous caterpillars specialized in preying on honeydew-producing hemipterans (e.g., Pachythone spp. 42,97 ) and are thought to carry out ant-mediated oviposition on harmful ant-plant symbioses, using specific ants as oviposition cues (e.g., Minstrellus grandis 44 ), or preying on ant brood (e.g., P. agave; this study). Although scattered, the available information indicates that Pachythonina species demonstrate morphological and behavioral traits allowing coexistence with extremely aggressive ants, including unusual associations with pseudomyrmecine and ponerine ants (Supplementary Table S1). In the miletine lycaenid Liphyra brassolis found in the nests of the arboreal formicine Oecophylla smaragdina, the tank-like morphology of the caterpillar is considered to be  98 . Although a commensal or scavenger lifestyle may be considered a preadaptation to stealing food stores or preying on the brood present in ant host nests 19 , the larvae of only a few species in various lepidopteran families (Tineidae, Psychidae, Pyralidae and Noctuidae) are known to eat on food stores or nest material or scavenge on dead organic matter 19,21 . Althought the larvae of some non-myrmecophilous riodinids are detritivores as is the case for Detritivora barnesi 39,99 , ant-associated riodinid caterpillars have never been considered as scavengers and the possibility that the larva of P. agave that we observed could eat stored food or wastes in N. villosa nests seems unlikely. Ponerine ants are a group of large aggressive ants with a powerful sting 3 . Most species are specialized or generalist predators, occupying a high trophic position 100,101 . The use of liquid foods on foliage, however, has been recorded for some species such as N. villosa that harvest liquid secretions from extrafloral nectaries, exudates  Table S1). According to DeVries 23 and Eastwood & Fraser 47 the evolution of symbiosis between caterpillars and ants is associated with ant genera that harvest liquid food on vegetation. The aggressiveness and conspicuous appearance of N. villosa ants associated with liquid feeding on plants may have generated an ecological opportunity for the evolution of exploitation by preadapted caterpillars 23,27,102,103 . This hypothesis can be corroborated both in ecological time 104 and evolutionary time if the specialization between P. agave caterpillars on N. villosa ants is confirmed. Detailed behavioral interactions between the P. agave caterpillar and N. villosa ants, could not be analyzed, and neither tentacle eversion nor liquid or volatile compounds released by the TNOs were observed. However, much evidence suggests that the nature of the relationship between caterpillars of this riodinid and N. villosa is not facultative or simply casual: (i) the larva was found among the brood in the core of the ant nest, (ii) it has a general protective morphology, with the head retracted or maintained under the shield-like, thick cuticle of the body much like the ant-brood feeding lycaenid L. brassolis 98 , (iii) it had a slow, gradual, slug-like movement behavior, and (iv) the ants did not behave aggressively. These traits indicate that the myrmecophile is well adapted to life inside the ant nest and are consistent with a symbiotic relationship with its host. Exchange of liquid food through regurgitation (stomodeal trophallaxis) is a highly evolved form of social food sharing, but it is infrequent in ponerine ants 58,105 . The first case of social parasitism in riodinids (exploitation of the colony resources through trophallaxis inside the ant host nest) has been recently reported in Aricoris arenarum (Riodininae: Lemoniadina) 11 , in nests of the formicine ant Camponotus punctulatus, and a similar "cuckoo" life-style could be possible for the P. agave caterpillar. However, stomodeal trophallaxis is not known to occur in N. villosa 58 ; instead, workers share liquid food by means of mandibular pseudo-trophallaxis: workers gather and transport liquid substances with surface tension to maintain a drop between the mandibles. In the nest, they offer the liquid to other workers who "spoon" some of the liquid 58 , but as the head of the P. agave larva is protected in a concealed, ventral position, and its rigid armoured cuticle does not allow the larva to bend its body easily, it is unlikely that the caterpillar's mouthparts could come in contact with the droplet of liquid that is occasionally held by the workers between their mandibles. Since the colony had no other source of food for the caterpillar aside from the ants themselves, and the general rigid morphology of the former likely prevents obtaining food by pseudo-trophallaxis, we further hypothesize that the caterpillar is most probably a brood predator (myrmecophagy), at least during the advanced larval instar as found in our study. Furthermore, in adults of P. agave, the wings present a greasy appearance, a trait that has been considered as a potential sign of carnivory 106,107 . Carnivory in riodinid caterpillars has been www.nature.com/scientificreports/ documented in five Nymphidiini lineages preying on ant-tended hemipterans on plants 42 , but myrmecophagy has only been recorded under artificial conditions 45,108 . The larva of P. agave differs from all other known riodinid caterpillars, though the general tank-like appearance is similar to other riodinids in the Lemoniadina (e.g., Menander spp. 39,109 ), and Pachythonina subtribes (e.g., Pachythone xanthe 42 ). Although phytophagous, Menander caterpillars are covered with a prominent carapace that flares outward to the substrate and covers the body and legs; they are thus heavily armored and possess a complete set of ant-organs 39,109 . In P. xanthe caterpillars, the prothoracic shield divided vertically into two movable plates, the absence of vibratory papillae and a carapace that protects the head and appendages are undoubtedly the most remarkable traits 42 . Some of these larval characters such as the body shape, the morphology of the lateral fringe setae, and the positioning of the spiracles, are shared with P. agave, suggesting that they may be morphological synapomorphies for the subtribe. In fact, these morphological characters led us to think that the larva potentially belonged to the subtribe Pachythonina before corroboration through molecular data.
Among myrmecophilous caterpillars, two strategies can be discerned: (i) free-living caterpillars that establish commensal or trophobiotic associations with ants on plants; and (ii) social parasite caterpillars that at some point of their development live within an ant nest 11 . This seems to be related to the progressive activation of antorgans and the production of putative chemical compounds that trigger ant adoption at a specific moment of the caterpillar development 110 . The life cycle of P. agave appears to conform to the latter strategy, but details of the adoption process by the ant colony are unknown. In summary, our data strongly suggest that P. agave is an obligate symbiont in N. villosa nests and that the advanced larval instar studied here is myrmecophagous. Whether females lay eggs directly on the leaves or inflorescences of the bromeliad is not known but seems possible. As suggested for obligate myrmecophilous lycaenids 111 , the paucity of records of this butterfly might be explained by its highly specialized life history, making the butterfly distribution dependent on the myrmecophytic association between N. villosa and the bromeliad A. bracteata. Such an assumption seems consistent with the known range of P. agave. The range of this species is much narrower than that of its ant host, N. villosa, which is consistent with what would be expected of an obligate and host-specific myrmecophilous butterfly, but it extends well beyond the Yucatan Peninsula and, in fact, overlaps perfectly the range of A. bracteata, from the 'Huasteca Potosina' area on the Mexican Gulf Coast and the southern part of Sinaloa on the Pacific Coast to northern Colombia 70 .