Morphology and life cycle of a new species of Psilocladus Blanchard, 1846 (Coleoptera, Lampyridae, Psilocladinae), the first known bromeliad-inhabiting firefly

. Fireflies (Coleoptera: Lampyridae) are soft-bodied beetles usually associated with mesic and hydric habitats. As such, terrestrial firefly larvae are commonly found in marshy environments and stream banks, while aquatic larvae might dwell in ponds, streams, mangroves, and even brackish water. Larval biology is especially important in fireflies, as the majority of species are extremely semelparous – that is, adults rely on resources gathered during larval stages. Despite their crucial relevance in firefly biology, larvae of only near 1% firefly species have been studied, and the majority of species remain known only from adult stages. That is especially true in the Neotropical region, where they are most diverse. Here, we describe Psilocladus costae sp. nov. after the study of adults and immature stages, the latter reported for the first time for the monotypic subfamily Psilocladinae McDermott, 1964. Interestingly, adults were first obtained by rearing the larvae, the former usually fly fairly high ( ca. 10 m) and are therefore seldom collected at ground level by conventional methods ( e.g., active search, Malaise traps). The new species is found in the Brazilian Atlantic Rainforest, an unprecedented habit for fireflies.


INTRODUCTION
There are over 2,000 firefly species (Coleoptera: Lampyridae) world-wide (Branham, 2010). The bioluminescent behavior of this charismatic group draws attention of people all around the globe (Faust, 2017). Some species gather in numbers and have attracted large audiences in various continents (e.g., Jeong et al., 2005;Faust, 2010), while others have been model systems for scientific studies, biotechnological innovations (e.g., Chen et al., 2015), and/or pest control (e.g., Fu & Meyer-Rochow, 2013). Despite their charisma and economic value, knowledge about Neotropical fireflies is relatively incipient compared to other regions of the planet (e.g., Asia [Ballantyne et al., 2019]; North America [Faust, 2017]). Most Neotropical species are only known from century-old original taxonomic descriptions, which are virtually always restricted to adult males. Indeed, only near 1% of firefly species have been studied during their immature stages. However, immature stages are crucial parts of species' ecological niche, especially in fireflies, as most species do not feed as adults, and rely on resources gathered during the larval stage. Therefore, studies comprehensively addressing all life stages are sorely needed in the firefly literature.
In the Neotropical region, where fireflies are most diverse (Costa, 2000), the few species that have been studied have diverse life histories. Professor Cleide Costa and colleagues were the first to describe in detail firefly larvae in the Neotropical region, in a book that would set the standards for larval studies in beetles, the "Larvas de Coleoptera do Brasil" (Costa et al., 1988). Costa et al. (1988) compared the larval morphology of four firefly species, Lucio castelnaui Kirsch, Aspisoma sp., Cratomorphus sp. and Bicellonycha sp., all of which are terrestrial and found on the leaf litter and low vegetation. Later, three other species were found in similar habitats: Pyractonema nigripennis Solier (Archangelsky, 2010), Aspisoma lineatum (Viviani et al., 2012), and Photuris femoralis Curtis (Rosa, 2007;cf. Souto et al., 2019). Additionally, the larvae of Cratomorphus cossyphynus Perty (Campos et al., 2018), Aspisoma fenestrata Blanchard (Archangelsky, 2004), Aspisoma sp. (Viviani, 1989) and Aspisoma sticticum Gemminger (Matos, unpublished) are semi-aquatic (i.e. associated to water bodies, but lacking adaptations to breath under water) and live in ponds and marshes. Several genera endemic to the Neotropical region have no larval descriptions at all. Indeed, out of 36 genera occurring in South America, only 5 have any species where larvae are known, all in the subfamily Lampyrinae Rafinesque or Photurinae Lacordaire; larvae of Amydetinae Olivier and Psilocladinae McDermott remain unknown. Here, we describe Psilocladus costae sp. nov. after the study of adults and immature stages, the latter reported for the first time for the monotypic subfamily Psilocladinae sensu Martin et al. (2019).
The genus Psilocladus Blanchard, 1842 comprised 42 species distributed in the Neotropical region, of which 11 were recorded for Brazil (McDermott, 1966). Psilocladus costae sp. nov. is found in the Brazilian Atlantic Rainforest (South America), inhabiting bromeliads during the larval stage, an unprecedented habit for fireflies. This new species is named in honor of Professor Cleide Costa (MZSP), who fostered this study directly and indirectly, as she supervised S. Rosa, who tutored L. Silveira during his studies, and the latter mentored S. Vaz in her studies. Professor Cleide Costa continuously inspires us in the study of bioluminescent Coleoptera.

MATERIAL AND METHODS
We collected larvae and adults from May 2017 to October 2019, in a legal reserve at Campos do Jordão (São Paulo State, Brazil, Global Positioning System 22°40′51.3″S, 45°35′30.6″W) (Figs. 1A-D), which is close to Parque Estadual de Campos do Jordão, covering 503 ha of Atlantic Rainforest in different conservation levels. During the day, we collected the larvae in leaf sheaths of fallen epiphytic bromeliads Vriesea betuminosa Wawra (Bromeliaceae) following the protocol described in Biffi & Rosa (2019). During the night, we collected larvae and adults, both them localized outside bromeliads by their bioluminescence . In addition to these adults, we studied material available in the following institutions (curator in parenthesis): CLEI, Coleção entomológica do Laboratório de Ecologia de Insetos, Universidade Federal do Rio de Janeiro, Brazil (M. Macedo); Universidade Federal de Itajubá, Brazil (UNIFEI); MNHN, Muséum National d'Histoire Naturelle, France (A. Taghavian) and MZSP, Museu de Zoologia da Universidade de São Paulo, Brazil. In total, we examined 11 adult specimens of Psilocladus costae sp. nov. (two females and nine males), one pupa and 18 larvae. We dissected three males, one female and three larvae. Immatures are deposited at CLEI and UNIFEI.
We reared the larvae at Laboratório de Zoologia (UNIFEI), at ambient temperature of Itajubá municipality, Minas Gerais state, with climate normal temperatures of 15, 1°C-19,50°C in autumn-winter, and 20,5°C-22,2°C in spring-summer, which are approximately 5°C warmer than temperatures in Campos do Jordão in the same period (INMET, 2019). Larvae were individually kept in 80 ml capped plastic pots with sand in the bottom and 2-3 small pieces of bromeliad leaf above the sand. The substrate was kept very wet to mimic the environment, as much as the innermost leaf sheath of bromeliads where most larvae were found (Fig. 2C). Larvae were fed weekly with termite workers and scirtid (Coleoptera) larvae, the latter collected in the same bromeliads we found lampyrid larvae. Dead larvae that quickly decomposed were not preserved. The material examined included dead larvae in good condition, and larvae and pupae killed and fixed in boiling water. Immatures and adults were preserved in 70% ethanol following Costa et al. (1988) and 90% ethanol following Campos et al. (2018). Adults and larvae were left 24 hours in a KOH solution at 10% before examination, then dissected with scissors and fine-tipped tweezers. Photos were acquired by a multifocal photographic system LEICA M205C, using LEICA V4 application suite and a Canon Rebel T6. Drawings were made under a stereomicroscope coupled to a camera lucida. For morphological terminology, we followed Beutel (1995) and Branham (2010) for immature stages, and Silveira & Mermudes (2014) for adults.
For the sake of convenience, we compare the new species to the three other species of Psilocladus found in Southeastern Brazil (a region that comprises four states: Espírito Santo, Minas Gerais, Rio de Janeiro and São Paulo): P. grandis Olivier, 1888;P. pulcher Olivier, 1885;and P. sigillatus Olivier, 1907.
Male: Sternum VI with light organ as long as ¾ sternum length and as wide as ½ sternum width. Sternum VII with light organ as long as ⅔ sternum length and as wide as sternum width. Sternum VIII with posterior margin slightly emarginate; syntergite robust, slightly longer than sternum IX with anterior margin emarginate; phallus with dorsal and ventral plates; dorsal plate basally fused to parameres, slightly smaller than parameres, struts pro-jected toward phallobase; parameres symmetric, apically acute; phallobase symmetric, with posterior margin rounded and lateral margins emarginate.

Abdomen (Figs. 10, 11E-F):
Gradually narrowed posteriorly from segment III; terga I-VIII with well impressed sagittal ecdisial line, anterior angles rounded, posterior angles acute and strongly projected posteriad with stout long seta at tip; terga I-VI transversal, subequal in length;  Vaz, S. et al.: New bromeliad-inhabiting species of Psilocladus Pap. Avulsos Zool., 2020;v.60.special-issue: e202060(s.i.).24 10/15 tergum I about 4.3× wider than long, VI 3.7× wider than long; tergum VIII 1.5× wider than VII, with posterior margin broadly emarginate; tergum IX as long as wide, lateral margins subparallel, posterior angles weakly projected with tuft of foliaceus setae at tip, posterior margin slightly rounded. Sternites I-IX sclerotized, with two pairs of parasagittal very short stout setae (at midlength and posterior margin) and long stout seta at posterior angles; sternites VI-VIII with additional pair of stout seta near lateral margins; stout setae increasing in length from sternites I-IX; laterotergites I-VIII 0.6-0.8× as large as respective sternites, sclerotized, posterior corners projected, with 2 prominences; anterior prominence with biforous spiracle, posterior prominence with a stout long seta; sternite VIII with pair of light organ on anterior corners. Segment X (Figs. 10B-C) attached under segment IX, tergum broadly conate or fused to sternum IX; sternum glabrous, most part sclerotized, except by subapical membranous transverse area; pygopod present. Pupa (Fig. 13): Total length ca. 7.0 mm, adecticous, exarate, white. Pronotum semicircular, anterior and lateral margins densely setose, setae long and stout. Dorsal surface with short setae. Posterior margin with posterior angles rounded, projected posteriad, median region straight. Mesothorax with a pair of reniform spiracles on posterior half of pleurite. Metanotum 1.1× as long as mesonotum, with a pair of long setae on posterior third. Abdominal terga I-VIII transverse, with posterior angles acute, strongly projected posteriad; lateral margins densely setose; setae long stout; tergite IX subquadrate with posterior angles weakly projected, with tuft of stout setae. Pair of light organ on sterna VI and VII (Fig. 14B), inconspicuous in alcohol-preserved specimen. Abdominal spiracles located ventrally on posterior angles of pleurae on segments I-VIII. scribed. The larva of this species is more similar to those of Photurinae species: Photuris fulvipes Curtis (Rosa, 2007) and an unidentified species of Bicellonycha Motschulsky (Costa et al., 1988). Those species share the oblong body (with semioval pronotum, widest at metathorax and gradually decreasing in width from abdominal segment III) (Fig. 10A), head with frontal arms well impressed posteriorly and divergent anteriad (V-or U-shaped), clypeolabrum with a median tooth darkly sclerotized, sensorium on second antennomere flattened (Figs. 11A, 12A, E), pretarsus with one basal seta on each side, and presence of localized stout setae on thorax and abdomen (Fig. 11E). They differ in the density and distributional pattern of the stout setae on thoracic and abdominal segments. Stout setae are sparser in Psilocladus costae sp. nov. and in Photuris femoralis, but in P. costae sp. nov. they are shorter and absent on median part of thoracic and abdominal terga. Larvae of P. costae sp. nov. also differ from both P. femoralis and Bycellonycha sp. larvae by having mandible sparsely setose and abruptly curved on apical half (Fig. 12F), hypopharynx with single anterior lobe and posterior part elongate (Fig. 12D), vitreous spots on terga VII and VIII, and tergite IX somewhat quadrate (Fig. 11F) (in Photurinae larvae, mandible is densely setose and gradually curved, the hypopharynx is bilobed with posterior part short, vitreous spots are absent, and the tergite IX is semioval). The pupa of P. costae sp. nov. is also more similar to the pupae of the photurine species than to pupae of other Neotropical species belonging to the genera Aspisoma Laporte, Lucio Laporte, Cratomorphus Motschulsky and Pyractonema Olivier. Photurinae and P. costae sp. nov. pupae share the presence of dense, long and stout setae on pronotal margins and posterior angles of abdominal terga (Fig. 13). Psilocladus costae sp. nov. pupa differs by having photophores on abdominal segments VI and VII (Fig. 14B), whereas photurine pupae have only one pair of photophores on segment VIII.

Etymology:
The specific epithet is in honor to Professor Dr. Cleide Costa who dedicated many studies to immature beetles, especially Elateroidea. Singular genitive, feminine.

Field and laboratory observations:
We collected 49 larvae inside fallen epiphytic bromeliads. Few larvae were collected outside of bromeliads during the night when they were localized by their very bright bioluminescence (Fig. 14A). Those larvae were identified by comparison with the bromeliad larvae. Of the 61 fallen bromeliads investigated, 26 had larvae of Psilocladus costae sp. nov., the majority of which found in the innermost leaves of the bromeliad rosette. The number of larvae inside each bromeliad ranged from one to five, but in most of them (n = 16) we found only one larva. They were found inside bromeliads throughout the year (22 larvae from April to August and 21 larvae from September to March), whilst active glowing larvae outside of bromeliads were found in the beginning of the rainy season (six larvae in October and November). At night larvae were found by their intense glow near bromeliad tanks, above leaf blades (Fig. 14A) and on trunks and branches near bromeliads fixed on trees. Larvae emit green light, easily seen from above through the vitreous spots on tergum VIII when the larvae are stretched, as well as through  Vaz, S. et al.: New bromeliad-inhabiting species of Psilocladus Pap. Avulsos Zool., 2020;v.60.special-issue: e202060(s.i.).24 12/15   Vaz, S. et al.: New bromeliad-inhabiting species of Psilocladus Pap. Avulsos Zool., 2020;v.60.special-issue: e202060(s.i.).24 13/15 vitreous spots on tergum VII, that covers the VIII when larvae shrink. In the lab, larvae preyed on termites and scirtid larvae. Mortality was very high and only two firefly larvae molted into pupa and adult, therefore we could not determine the number of larval instars. Larval length ranged from 9 to 22 mm and the larvae which molted into pupa and adult were ca. 15 mm long, therefore we infer that mature larva may be 15-22 mm long. A few larvae possibly died due to acari infestation, but the causes of overall mortality were not known. Adults were observed on wet and dark nights in December and January. They flew at about 10 meters height whilst glowing green continuously, rarely below 5 meters, yet 10 specimens could be collected on the grass and on leaf blades of bromeliads fixed lower on the trees. The flying adults concentrated in areas with higher density of canopy bromeliads (Figs. 1B, C).

Bromeliad-inhabiting fireflies
Bromeliad tanks can be thought of as complex ecosystems which house several species, including beetles. Although the study of bromeliad-inhabiting beetles in South America dates back to 1900 (Ohaus, 1900), recently there has been renewed interest (e.g., Albertoni et al., 2016;Biffi & Rosa, 2019). Because bromeliads provide important environmental needs of firefly larvae, viz. food (gastropods and other soft-bodied invertebrates) and moisture (in the bromeliad tank), they are obviously well-suited spots for fireflies. Our study is the first detailed report of fireflies living in bromeliad tanks, as far as we are aware. Other studies in South America have indeed found firefly larvae in bromeliad tanks, but rather focusing on the plant's ecology, that is, not on the insects themselves, and the identity of the species were never addressed (e.g., Givnish et al., 1984;Aguilera-Arango et al., 2011;Rogy et al., 2019).
Fireflies are usually found in mesic and hydric environments, arguably because: (i) their soft carapaces make them relatively more subject to water loss, which is also seen in other soft-bodied beetles (e.g., Lycidae, see Bocak et al., 2008), in comparison with most other beetles; and (ii) they usually prey on gastropods, earthworms and other soft-bodied animals, also usually restricted to mesic and hydric environments. Bromeliads are well-suited spots for fireflies, damp and packed with soft-bodied invertebrates. Thus, future studies addressing the macroinvertebrate faunas in bromeliads are likely to find fireflies.
We found no obvious morphological adaptations to an aquatic lifestyle in Psilocladus costae sp. nov. However, it is reasonable to assume that P. costae sp. nov. is semi-aquatic, as larvae tolerate some level of water submersion, as the majority of them were found at basal older leaves, which are constantly submerged in water. Another evidence that larval P. costae sp. nov. withstands some time under water is that under lab conditions, it readily preyed on aquatic larvae of scirtid beetles, which were highly abundant in the bromeliad tank. It is yet un-known whether or not this firefly species feed on scirtid beetles under natural conditions. The presence of larvae outside bromeliads during night suggests that they migrate out of bromeliad through the water and possibly feed on preys that are attracted by their intense glow.