Bracon zuleideae Perioto & Lara (Hymenoptera, Braconidae): seed predation potential, expansion of the geographic distribution and new data on host plant phenology

Here, by the first time, was quantified the predation of seeds of Protium ovatum Engl. (Burseraceae) by Bracon zuleideae Perioto & Lara (Hymenoptera, Braconidae), a phytophagous seed eater, at Parque Nacional Grande Sertão Veredas, located in the State of Minas Gerais, Brazil. B. zuleideae is now mentioned for the state of Minas Gerais, Brazil, which extends its geographical distribution about 900 km north of its type locality, its only previous record, and new data about host plant phenology are provided.

raconidae is the second most diverse family of Hymenoptera and includes about 19,500 species distributed by nearly 1,000 genera in 46 subfamilies and the estimated species richness in the world varies between 40-100 thousand species (Wharton 1993;Campos & Sharkey 2006;Shaw 2006;Yu et al. 2011). The vast majority of Braconidae act as parasitoids of other insects, especially holometabolous in egg-larva phase or larva and the parasitism of adults or nymphs of hemimetabolous insects occurs only in Aphidiinae and Euphorinae (Campos & Sharkey 2006).
Braconinae is one of the largest subfamilies of Braconidae with more than 200 genera and about 2,5 thousand species (Shaw & Huddleson 1991;Quicke 2015) that, in their vast majority, act as larval ectoparasitoids of Coleoptera, Lepidoptera, Diptera and Hymenoptera (Shaw & Huddleson 1991;Quicke 1997). This subfamily is dominated by the cosmopolitan and very common type genus Bracon Fabricius, 1804 composed by about 1,000 species described worldwide (Belshaw et al. 2001;Loni et al. 2016) which acts mainly as parasitoids of a wide range of lepidopterous, coleopterous and dipterous larvae (Campos & Sharkey 2006). However, among the Bracon species, three stand out because of their unusual food habit in association with plants of the family Burseraceae: the neotropical species B. phytophagous Quicke and Bracon zuleideae Perioto & Lara act as seed predators in plants of Protium tovarense Pittier and P. ovatum Engl. in Mart., respectively, and the oriental B. garugaphagae Ranjith & Quicke that act as entomophytophagous species (i.e. ones that display entomophagy and phytophagy sequentially) in leaf galls induced by the psyllid Phacopteron lentiginosum Buckton (Hemiptera: Psylloidea) on Garuga pinnata Roxb. (Flores et al. 2005;Perioto et al. 2011;Ranjith et al. 2016).
Protium ovatum is a native and endemic species from the Brazilian savannah and Amazon areas (Reflora 2019) popularly known as "breu-do-cerrado", "breu-arbustivo" and "almacega" is easily recognized in the field by the fact that its leaves, when crushed, exude a strong mango scent. P. ovatum is a shrub of 0.4-4.0 m high, with glabrous branches and few lenticels. Its leaves are oval, glabrous, composed, with 3-5 coriaceous leaflets; the species blooms from April to September when fruits can be observed (Lima & Pirani 2005) and is explored by agro-extractivists and its essential oils are used in the production of medicines and incenses (Souza 2016). P. ovatum seems to have promising potential for economic use since, in the literature, there are reports that Protium resins are widely used in traditional medicine for wound healing, with analgesic and antiseptic properties, as well as in the production of perfumes and cosmetics, among other uses (Rüdiger et al. 2007); in biological assays these resins showed potent antiinflammatory activity (Siani et al. 1999;Almeida et al. 2015) in addition to having compounds that can be used to treat liver disease (Oliveira et al. 2005).
The objective of this study was to evaluate the potential of predation of seeds of P. ovatum by the braconid wasp B. zuleideae, extend its geographic distribution and provide new data on host plant phenology.

MAtERIAL AND MEtHoDs
The studied specimens of B. zuleideae were obtained from fruits of P. ovatum (Figure 1) collected at random along a trail of about 350 m in length in area of Brazilian savannah vegetation (Cerrado strictu sensu) within the Parque Nacional Grande Sertão Veredas (15°10'31.7" S/45°43'14.9" W), located in the municipality of Chapada Gaúcha, Minas Gerais State, Brazil, in September 12, 2018.
A total of 153 almost ripe fruits were collected from about 30 plants. The fruits were maintained in plastic containers (10 cm diameter, 15 cm high) covered with synthetic gauze and sent to the Laboratório de Sistemática e Bioecologia de Predadores e Parasitoides of the Instituto Biológico, in Ribeirão Preto, São Paulo, Brazil.
In the laboratory the fruits were individualized in glass vials (2.4 cm diameter, 8.3 cm high) covered with plastic film and kept at ambient temperature and humidity and observed daily until the adult wasps emerged. The emerged insects were kept in a 15 mL plastic vial with 95% ethanol and later air dried, mounted on entomological pins and labeled. Thirty-six days after collection, fruits that did not display adult exit holes of P. ovatum were dissected to check the contents of their interior.
The specific identification of the studied specimens was made by the first author.
Images were taken using a digital camera Leica MC170 HD attached to a stereomicroscope Leica M205C APO and specimens illuminated with high diffuse dome illumination Leica LED5000 HDI. Focus stacking of images was done using Helicon Focus   The phenological behavior of the approximately 12,000 plant species that develop in the Cerrado is fragmented (Mendonça et al. 2008;Oliveira 2008), since only a very small fraction of this flora had such behavior studied. Knowledge of the phenology is fundamental to understand the plant community structure, function, and the quality and quantity of resources available for consumers organisms (Williams et al. 1999). The knowledge about the phenology of P. ovatum is inserted in this context: for the state of São Paulo, it is known that this plant is collected with flowers from April to September and with fruits in September (Lima & Pirani 2005). The fruits used in this study were collected almost ripe in September and, in the collection area, many of them were still at the beginning of development, indicating that the fruiting season of this widely distributed plant would extend even longer; such facts may indicate the existence of latitudedependent variations in the fruiting season.

Results and disCussion
Bracon zuleideae is the second obligately known phytophagous species of Braconinae and, except for the fact that only one larva was ever observed feeding within each seed of P. ovatum, nothing more is known about its biology. This behavior was also observed by Flores et al. (2005) for B. phytophagous, the first obligately known phytophagous species of Braconinae, in seeds of P. tovarense.
In this study were obtained 153 fruits and 190 seeds since each fruit of P. ovatum can harbor more than one seed. From the fruits emerged 30 adults of B. zuleideae (17♀, 13♂) and, within the dissected seeds were found the remains of ten larvae of that wasp, totaling 40 seeds predated, which resulted in a percentage fruit infestation of 21.1%. This value is lower than that found by Flores et al. (2005) (50-60%) for B. phytophagous in fruits of P. tovarense; however, it should be noted that the study of Flores et al. (opus cit.) lasted ten months.
In this study, the geographical distribution of B. zuleideae is extended by about 900 km north from the type locality and the species is now mentioned for the Minas Gerais State (Figure 3). P. ovatum is a plant widely distributed over vast areas of the Cerrado and Amazon biomes (Reflora 2019) and this fact allows us to infer that the geographical distribution of B. zuleideae can be much wider than currently known.