Chrysomelinae species (Coleoptera, Chrysomelidae) and new biological data from Rio de Janeiro, Brazil

Abstract Chrysomelinae is one of the largest subfamilies in Chrysomelidae, yet much basic information remains unknown for Neotropical species. The present study aims to compile the first regional list of Chrysomelinae for the State of Rio de Janeiro, Brazil, and assemble natural history traits obtained from our fieldwork from 2005 to 2010 in Serra dos Órgãos National Park, a mountainous area of Atlantic forest. The species list was compiled from data from field work, collections, and literature, and recorded a total of 100 species, belonging to 21 genera in one tribe (Chrysomelini) and three subtribes: Chrysolinina (91 species), Chrysomelina (eight species) and Entomoscelina (one species). Of these, 91 species are new records for the state. Serra dos Órgaõs National Park holds records of 43 species, with Platyphora being the most species-rich genus, and Solanaceae the most common host plant family. Some new records of reproductive mode (larviparous vs. oviparous) and larval behavior are also given. These Brazil Chrysomelinae species exhibited a clear seasonal pattern, with more species recorded in the hot and rainy season from October to January, and considerably fewer species from June to August, during the drier and colder months. The fraction of new records in comparison with published species and natural history information illustrates how little we know of Chrysomelinae in the state and in the country.


Introduction
Chrysomelinae is the fifth largest subfamily of Chrysomelidae, after Galerucinae, Eumolpinae, Cassidinae and Cryptocephalinae (Reid 2006), with 3,000 species and 132 genera (Daccordi 1994, 1996, Riley et al. 2002, but these numbers vary among authors (see Seeno andWilcox 1982, Reid 1995). Two tribes are generally recognized: Timarchini, which is monogeneric with Timarcha Latreille (ca. 100 species); and Chrysomelini containing the remainder (Seeno andWilcox 1982, Daccordi 1994). However, there are still many problems concerning Chrysomelinae taxonomy. Daccordi (1996) listed 38 genera for the Neotropical region, out of which 31 are exclusive to the area. Some of the main contributions for Neotropical Chrysomelinae taxonomy and cataloguing are those by Jan Bechyně (e.g. 1954Bechyně (e.g. , 1958Bechyně (e.g. , 1980, which include many species descriptions and some regional lists, and, more recently, a key to the genera in Costa Rica by Wills Flowers (2004), modified from Bechyně and Springlova de Bechyně (1965). Both larvae and adults normally feed on leaves of the same host plant species and species tend to be monophagous or to feed on a narrow group of related plant species (Jolivet 1988). The same author pointed out that host plants are known for nearly 40% of Chrysomelinae genera, and data are largely lacking for tropical species. In the Neotropical area, Chrysomelinae are frequently associated with Solanaceae, Asteraceae, Apocynaceae, and Zygophyllaceae (Jolivet and Hawkeswood 1995). However, knowledge of Chrysomelinae biology is rare in this region, remaining so two decades after being underlined by Jolivet (1997).
Except for some ecological studies and species records confined to entomological collections, no list of species exists for the subfamily in the State of Rio de Janeiro or even in Brazil. Since we have conducted extensive research in a protected area in the state and have accumulated considerable biological information on Chrysomelidae species, our aim here is to compile the first regional list of Chrysomelinae in Brazil, and to assemble natural history traits for the species found in Serra dos Órgãos National Park, State of Rio de Janeiro.

Materials and methods
For Chrysomelinae species list compilation for Brazil, four national collections were examined: Coleção entomológica do Laboratório de Ecologia de Insetos / Universidade Federal do Rio de Janeiro, Rio de Janeiro (CLEI); Museu Nacional / Universidade Federal do Rio de Janeiro, Rio de Janeiro (MNRJ), except species from Itatiaia; Coleção entomológica da Fundação Instituto Oswaldo Cruz, Rio de Janeiro (CEIOC); and Museu Paraense Emílio Goeldi, Belém (MPEG). The digital collection of the Museo del Instituto de Zoologia Agricola, Universidad Central de Venezuela (MIZA) was also consulted. Finally, the literature was searched for additional records. These records are indicated in Table 1. Location is given by the municipality within the State of Rio de Janeiro, which comprises 43,696 km² and represents less than 1% of the country's area. Taxonomy follows Daccordi (1994) and Seeno and Wilcox (1982).

Study Site
The park covers an area of 20,024 ha of well-preserved Atlantic Rain Forest (see Veloso et al. 1991 for more on local vegetation) and is located ca. 100 km from Rio de Janeiro, in a mountainous area ranging from 80 m to 2263 m elevation. The climate is tropical, with a colder drier season from May to August, and a rainy warmer period from November to February (Flinte et al. 2009b). Mean annual temperature is around 18 °C, maximum of 38 °C and minimum of 0 °C. Annual precipitation varies between 1250 and 1500 mm (Flinte et al. 2008).

Species study and collection
Species were sampled by a combination of manual collecting, sweep nets and malaise traps, during the conduction of other projects with Chrysomelidae in the park. When a species was initially found in the field, individuals in as many different developmental stages as possible were brought to the laboratory and reared in plastic containers for host plant confirmation and observations on behavior and biology. In an attempt to describe species seasonal distribution in the area, considering data on labels of specimens from all collections, we recorded the different months on which they were collected and summed the number of species per month (independent of year).

General patterns of richness and distribution
The Chrysomelinae Neotropical fauna is thought to comprise ca. 38 genera (Daccordi 1996) and 1,020 species (Blackwelder 1944), but these are outdated numbers and no such information could be found specifically for Brazil. One hundred species occurring in the State of Rio de Janeiro were recorded, belonging to 21 genera in one tribe (Chrysomelini) and three subtribes: Chrysolinina, Chrysomelina and Entomoscelina (Table 1). Chrysolinina was represented by 91 species, followed by Chrysomelina with eight and Entomoscelina with only one species (Table 1). According to Daccordi (1996), there are many endemic Chrysolinina and Chrysomelina taxa in the Neotropical region, where they reach their maximum diversity. Only nine species are from previously published sources, the other 91 species we found are new records for the state. The genus with most species records was Platyphora (n = 39) representing 42.4% of Chrysolinina found and 40% of total species records, followed by Stilodes (n = 13, 12.9% of all species recorded) and Deuterocampta (n = 11, 10.9%), genera restricted to the Neotropical region (Daccordi 1996). Indeed, Platyphora is the most species-rich genus in South America (Daccordi 1994), with approximately 500 species (Chaboo et al. 2014 and references therein).
The findings presented here also revealed a high diversity of species and genera, typical for the Atlantic rain forest, in comparison to other studies in South and Central America. Flowers (2004) documented 67 species in 11 genera for Costa Rica and, similar to our work, Platyphora and Stilodes were the most species-rich genera. During a six-year field study in a Mexican state, 47 species and eight genera were found; Leptinotarsa, Calligrapha and Zygogramma were the genera with most species records (Burgos-Solorio and Anaya-Rosales 2004). Chaboo and Flowers (2015) found 158 species and 18 genera for Peru, based on species catalogues.
Species were recorded from only 17 (18.5%) of the state's municipalities, and 62 species were known from only one location (Table 1). We found a similar pattern in an inventory of Cassidinae for the same state (Flinte et al. 2009a), with most records concentrated near the city of Rio de Janeiro and in large protected areas, such as Petrópolis and Teresópolis (Serra dos Órgãos National Park) and Itatiaia (Itatiaia National Park). The high number of single locality records is probably due more to sampling effort than to endemism, considering that species normally are not very abundant and are more easily collected manually than with traps.
Maternal care was not recorded for any species in this study, although subsocial behavior is known in the subfamily for several species, including some Doryphora and Prosicela species (Windsor et al. 2013, Chaboo et al. 2014. Other interesting behavioral defenses were recorded, including larval cycloalexy in Z. appendiculata (Fig. 1-Y3) and P. curticollis (Fig. 1-H3), a defensive behavior of gregarious circular formation at rest (Jolivet et al. 1990, Vanconcellos-Neto and Jolivet 1994, Dury et al. 2014. Additionally, we recorded larvae of P. fraterna (Fig. 1-N2) attaching trichomes from Solanum host plant leaves to hairs on their backs, a behavior already described in P. zonata (Bernardi and Scivittaro 1991), which may contribute to larval camouflage. Larval   Grissell et al. 1987;2 Flinte et al. 2015;3 Flinte et al. 2009b;4 Olckers 2000;5 Jolivet and Hawkeswood 1995. aggregations were observed in many species (Table 2), but also for adults of Z. appendiculata on young folded leaves in the field ( Fig. 1-Y4). Larval gregarious behavior may serve to reduce individual risk against small invertebrate parasitoids and predators, and promote defense against larger predators through the cumulative effect of individuals' toxins (Grégoire 1988). Thanatosis ("feigning death") was observed in adults of P. axillaris ( Fig. 1-E1), P. fervida ( Fig. 1-L1, L2), and P. fraterna ( Fig. 1-N1), and both in adults and larvae of Stilodes (Grammomades) impuncticollis ( Fig. 1-W1, W3). Seven oviparous and seven larviparous species were found, most being new records of reproductive biology (Table 2). Chrysomelinae is the subfamily of leaf beetles with the most diversity in reproductive biology, containing oviparous, ovoviviparous and viviparous species (Bontems 1988), sometimes in the same genus, and also different levels of social behavior (Chaboo et al. 2014). The last two types of development may be more costly to the mothers, but ensure a quicker development of the vulnerable larval stage, among other advantages, as proposed by Jolivet and Hawkeswood (1995) and Chaboo et al. (2014 and references therein), which is why it is sometimes considered to be a parental care preceding birth (Hinton 1981). Interestingly, viviparous species may result in solitary larvae, as in P. axillaris (Fig. 1-E2), or larval aggregations, as in P. jucunda (Fig. 1-P2). Oviparous species may also have solitary or gregarious larvae, as in P. dejeani (Fig. 1-I2) and Z. appendiculata (Fig. 1-Y2), respectively, although larval aggregations seem rarer in this type of reproduction. In their work on subsocial neotropical Doryphorini, Windsor et al. (2013) found, among Platyphora species, two with solitary larvae and nine which formed larval aggregations, but all eleven species were larviparous. We observed a single case of larval cannibalism in the viviparous P. curticollis during laboratory rearing ( Fig. 1-H2), a behavior already described for some Chrysomelinae genera (Wade 1994, Mafra-Neto and Jolivet 1996, Windsor et al. 2013) that grants nutritional benefits.
Except for the eggs of Z. appendiculata, which are laid in masses on the underside of its host plant leaves ( Fig. 1-Y5), no other eggs of oviparous Chrysomelinae were found in the field. This is probably because chrysomelids often lay their eggs in the soil or in secluded parts of plants (Selman 1994). All oviparous species reared in laboratory laid chorion-covered yellowish eggs on the bottom of the vials or on leaves, normally grouped in clutches ( Fig. 1-W2). In the field, the number of eggs of Z. appendiculata varied from 80 to 100 per group (90.4 ± 8.3 SD; n=7 egg masses), and larval aggregations comprised between 10 and 233 individuals per group (49.2 ± 38.7; n=50 groups). Larvae of different egg masses may cohabit the same aggregation of this species, since differently sized larvae were observed in the same aggregation. Platyphora fraterna larvae ( Fig. 1-N2) were grouped in aggregations of 24.9 ± 13.1 SD individuals (n=14 groups), with a minimum of seven and maximum of 44 larvae per group. No pupa has yet been found in the field, but in the laboratory, prepupae always buried themselves in earth layer at the bottom of the vials. While pupation in Chrysomelinae may be arboreal or underground (Takizawa 1976), it seems that underground pupation is most common in our taxa as indicated by laboratory rearing.
Only a few observations on natural enemies of Chrysomelinae were made. Phoretic wasps of Pteromalidae (Hymenoptera) were found on adults of Grammodesma rubroaenea ( Fig. 1-D1) and Deuterocampta sedula ( Fig. 1-B1). Pteromalidae are well known parasitoids of chrysomeline larvae (Cox 1994). On one occasion, a Podisus (Hemiptera) nymph was seen preying on a larva of Z. appendiculata ( Fig. 1-Y6). Many chrysomeline species presented unprotected larvae without any apparent behavioral defense, but several gain chemical defenses by the sequestration of host plants toxins or by synthesizing defensive compounds from plant precursors, especially in Platyphora (Pasteels et al. 2001, Termonia et al. 2002.

Conclusions
The high proportion of new host, biological data and occurrence records in Rio de Janeiro reflects the limited knowledge we have about this subfamily in this immediate area. In Brazil, the picture is not very different, as no inventory for the subfamily has been compiled and the relatively few published records come from ecological studies such as Medeiros and Vasconcellos-Neto (1994), Medeiros et al. (1996), Macedo et al. (1998), and Flinte et al. (2009b. However, Chrysomelinae is known to be very species-rich in Brazil, including known en-demic species, such as Elythrosphaera lahtivirtai (Macedo et al. 1998). Because of their high host specificity (Jolivet 1988, Jolivet andHawkeswood 1995) and low dispersal ability (Freijeiro and Baselga 2016) the chrysomelines are expected to have many narrowly distributed species, especially in mountainous areas, as has already been found for other tropical Chrysomelidae species (García-Robledo et al. 2016, Macedo et al. 2016. These traits then would make these Brazil species especially vulnerable to extinction as the mountains within the Atlantic forest biome are largely degraded and threatened (Martinelli 2007).