A new species of the Neotropical genus Beebeomyia (Diptera: Richardiidae) with observations of its biology on Dieffenbachia oerstedii (Araceae)

A new species of the genus Beebeomyia Curran found in central Veracruz (Mexico) is described, including characters of the male and female terminalia, and a key to the identification of the known species is provided. This new species was found feeding within inflorescences of Dieffenbachia oerstedii Schott (Araceae). Larvae were found throughout the inflorescence, with a proportion nearly three times higher in the female (bottom) than the male (top) section. The life cycle from larval stage to adult emergence takes place in an average time of 42 days, while pupation occurs within the inflorescence. Observations of the oviposition behaviour suggest that after oviposition, the female marks the inflorescence with a deterrent pheromone to prevent further ovipositions by other conspecific females. http://zoobank.org/lsid:zoobank.org:pub:DC19F7E5-3AAF-4E5C-A35C-F4872C89D3B4


Introduction
The first species of the genus was described as Zeugma palposa (Cresson 1908) and, some time later, three other species from South America were described by Hennig (1937) under the genus Zeugma Cresson; however, this generic name was preoccupied (Westwood 1840). Near this time, Curran (1934) described the monotypic genus Beebeomyia versicolor from Guyana, being the type species by original designation. In the catalog of the Neotropical Richardiidae, Steyskal (1968) confirmed by typeexamination that B. versicolor is a junior synonym of Z. palposa, and therefore all species were transferred to Beebeomyia.
This genus is exclusive to the Neotropical region and comprises four valid species, B. palposa (Cresson) from Surinam, Guyana, Peru and Bolivia; B. senilis (Hennig) and B. calligastra (Hennig) from Peru; and B. flavimaculata (Hennig) from Brazil (Steyskal 1968). Currently, we know there are some additional undescribed species from Costa Rica (Hancock 2010). The group has never been revised comprehensively or using modern taxonomic methods, and the descriptions of all of species are based upon external morphology; hence, the features of the male and female genitalia are still unknown.
The biology of the Richardiidae is poorly known and it is assumed that most of the species are saprophagous, the larvae feed on rotting fruits or diseased coconut palms (Steyskal 1958). Some undescribed Beebeomyia species were reared from flowers and bracts of Anthurium (Araceae) and Heliconia species (Musaceae) (Hancock 2010). In this paper, we describe a new species of Beebeomyia found in a tropical rain forest of central Veracruz, Mexico, and we provide biological data on this phytophagous species, reared from inflorescences of Dieffenbachia oerstedii Schott (Araceae).

Study site
The study was carried out within the Biosphere Reserve of Los Tuxtlas, Veracruz, Mexico. This area is the easternmost extension of the Mexican Transvolcanic Belt and constitutes the northernmost limit of tropical rainforest distribution in the Americas (Dirzo and Miranda 1991). Topography is complex, with elevations from sea level to 1600 m altitude within a short distance (towards San Martin Volcano). Given its latitudinal position and location, adjacent to the Gulf of Mexico, the area experiences high precipitation of around 4700 mm per year, and a mean annual temperature of 25°C (Dirzo and García 1992;Dirzo et al. 1997). The predominant vegetation is a tropical lowland rain forest, with variations along the altitudinal range, including cloud forest and mixed forests at higher elevations (García-Aguirre et al. 2010). Floristic diversity in the area includes 950 known vascular plants, largely composed of plants of Neotropical origin, but higher elevation sites include combinations of Neotropical and Nearctic taxa (Ibarra et al. 1997). In the understory, aroids are an important component of the local vegetation. The Araceae family comprises 34 species in nine genera within the Biosphere Reserve of Los Tuxtlas (Acebey and Krömer 2008), and some species, such as Syngonium podophyllum Schott and Dieffenbachia oerstedii exhibit high population densities.

Dieffenbachia oerstedii Schott (Araceae)
In Mexico, D. oerstedii may be confused with D. wendlandii Schott and D. killipii Croat because they have similar blade shape (Croat 2004;Acebey and Krömer 2008). Herbarium material of D. oerstedii may also be confused with D. seguine (Jacq.) Schott, from the West Indies and the Amazon region. It is an herb usually less than 50 cm in height, (ranging from 30-75 cm), distributed from southern Mexico to Panama. In the Los Tuxtlas region (Veracruz, Mexico) it occurs from 100 to 1100 m above sea level (Acebey and Krömer 2008). Flowering occurs throughout the year; however, there are two main periods, one in April at the beginning of the dry season and a second peak during September, at the end of the rainy season; each individual presents one to three inflorescences, or, rarely, four (Croat 2004). The inflorescence, which consists of a spathe with male and female flowers, is dicogamous (protogynous), and is pollinated by beetles of the genera Cyclocephala and Erioscelis (Scarabaeidae) (Cuartas-Hernandez and Nuñez-Farfán 2006).

Biological samples
During the peak flowering season of Dieffenbachia oerstedii (September of 2013), infestation rates and feeding habits of a richardiid fly species that was found associated with the developing inflorescences were evaluated. In the field, we observed the female oviposition behaviour, and developing inflorescences were collected for further examination. Under laboratory conditions (temp 24°C ± 4; and RH 70% ± 10), we kept the inflorescences individually in plexiglass cages with permanent moisture at the base. Each inflorescence was measured for length and width, and later it was dissected lengthwise to locate any larvae and/or pupae, recording the particular sites where immature stages were found feeding. The pupae were kept in plastic rearing chambers until adult emergence. Adult specimens were preserved and dry-mounted; the abdomens of some specimens were also excised to study the terminalia of both sexes. The genitalia were processed in a 10% sodium hydroxide (NaOH) solution by heating for 10 minutes, subsequently washed with distilled water, placed in plastic microvials with glycerine and pinned under the rest of the specimen. Digital photographs for D. oerstedii were taken with a Nikon D5100 (18-55 VR, lenses king CU+1, +2, +4, 52 mm) camera, and the images of Beebeomyia specimens were taken with an Olympus C5050 camera, adapted to Olympus SZX7 and BX41 microscopes. The morphological terminology of flies used in this paper is based on Cumming and Wood (2009). The material examined was deposited in the following entomological collections: Instituto de Ecología AC (INECOL), Xalapa, México (IEXA); Colección Nacional de Insectos, Instituto de Biología -UNAM (CNIN); the Estación de Biología Tropical Los Tuxtlas -UNAM (EBLT); and the National Museum of Natural History (USNM).

Data analyses
To evaluate the infestation levels by immature stages found inside the inflorescences of D. oerstedii, we calculated the means and standard error (mean ± SE). Wilcoxon tests were used for comparing infestation levels by fly species, and between sexual sections of the inflorescences (Zar 2010). Statistical analyses were performed using Statistica software 7.1 (StatSoft, Inc. 2006).
Genus Beebeomyia Curran, 1934 Diagnosis This genus belongs to the subfamily Richardiinae and can be recognised by the following combination of characters: head little or no longer than high; frons twice as long as wide, ocellar triangle very long, the anterior ocellus near the middle of the frons, scarcely hollowed above; frons flat, level with eyes; basal antennal segment short, more or less hidden; one pair of frontal setae opposite the ocellar seta; postocellar setae extremely weak; outer vertical seta as strong as inner vertical seta. Thorax with middle portion of the propleura pubescent only; the following setae present: 1 postpronotal, 2 notopleural, 1 anepisternal, 0-1 presutural supra-alar; 1 postsutural supra-alar; 2 dorsocentral (except one pair in B. senilis); 1 postalar; 1 intra-postalar; acrostichal setae absent.
All femora moderately slender with spiny ventral setae on apical half, usually reduced or sometimes absent on the fore femur, always present on mid and hind legs; hind femur not more swollen than others; vein Sc ending near the apex of R 1 ; crossvein r-m slightly before or at middle of the discal cell; crossveins r-m and dmcu further apart than length of dm-cu; cell bcu rounded to apex; vein A 1 + CuA 2 reaching margin at least as a fold; R 2+3 straight, not undulate; male costa not expanded. First abdominal segment narrowed at base, the apical portion of abdomen oval.
Key to species of Beebeomyia from the Neotropical region

Diagnosis
Beebeomyia tuxtlaensis sp. nov. can be distinguished from all other species of Beebeomyia by the following combination of characters: mesonotum entirely reddish, postpronotal lobe and notopleuron covered by whitish or grayish pollinosity; two dorsocentral setae, with anterior pair smaller; presutural supra-alar seta present; wing with small apical spot, costal and subcostal cells dark brown; foreleg yellow, including coxa to tarsomeres; mid and hind leg black with yellow; male with medial and lateral surstyli elongate of similar length in lateral view; phallus coiled with strong setae along basal third; distiphallus broad, with two comb-like opposite basal rows of long yellow setae; female aculeus length 0.80-0.84 mm, nearly two times as long as wide; apex of aculeus triangular, nearly two times longer than wide.
Head ( Figure 1B). Height 1.06-1.22 mm, width in lateral view 0.88-1.04 mm. Frons shining reddish posterodorsally between vertex and ocellar triangle, matt anteroventrally from lunule almost to base of orbital seta. Face reddish yellow; first flagellomere reddish dorsally, somewhat darkened along apical half, reaching margin of clypeus; arista pubescent; occiput along posterior margin of eye with thin line of whitish pollinosity; gena narrow. A pair of long orbital seta located almost in line with ocellar setae; postocellar seta very small, inner and outer vertical setae present and similar sized; ventral margin of occiput, close to insertion of the head and thorax, with row of small black setae forming a fringe.
Foreleg entirely yellow including coxa, anteroventral margin of distal part of femur without short spines, or with 1-2 subapical spines, posteroventral margin with row of 6-8 long black setae. Mid leg with femur mostly black, yellow apically, with 4-5 short ventral spines in two rows, tibia black, and tarsus yellow. Hind femur black except base and apex yellow, anteroventral margin with 2-3 spines slightly reduced, and posteroventral margin with 4-5 spines; tibia black, and tarsus yellow.
Wing ( Figure 1D). Wing is 4.24-4.64 mm long, and 1.48-1.68 mm wide. Mostly hyaline except costal and subcostal cells entirely brown, a narrow apical dark spot, extended from apex of vein R 2+3 to apex of vein M; wing membrane evenly covered with microtrichia, except cell bcu completely bare, and cell bm basally bare on 3/4 of its length; base of vein R 4+5 with 2-3 small black setae; crossvein r-m located near mid-length of discal cell.
Abdomen. Shining black, with all tergites uniformly covered with black setulae, in addition to some noticeable transverse striations on tergites 3-5; syntergite 1 + 2 with 2 long black setae located on medial side, with a mid-transverse strip devoid of setulae.
Male genitalia (Figures 1G-I). Epandrium brown reddish; proctiger yellow; inner and outer surstyli approximately of same length in lateral view, inner surstylus with ventral triangular protuberance located just before mid-length, with short apical prensiseta; outer surstylus with mid-dorsal tooth-like projection, spoon-shaped apically; phallus long tangled, provided with numerous strong setae on basal third, the rest bare; distiphallus (or glans) broad, membranous, with two opposite basal comblike rows of long yellow setae; apical extreme membranous and translucent with two projections, an acute thorn weakly sclerotised, and other non-sclerotised bulbous projection.

Etymology
The specific epithet comes from the Mexican native word 'Los Tuxtlas', in addition to the Latin suffix ensis = coming from, in reference to the region of origin of the material examined.

Infestation rates and biology
Along with Beebeomyia tuxtlaensis, we also found another fly species of the family Drosophilidae, as yet unidentified, whose larvae were feeding simultaneously within the inflorescences of D. oerstedii. We assessed the infestation levels by the two species along sexual sections of the inflorescences. As a global infestation produced by larvae and pupae on 36 inflorescences, we found 1901 individuals of B. tuxtlaensis distributed on 20.5% and 79.5% of the male and female sections, respectively; while the drosophilid species accounted for 370 individuals distributed on 55.7% and 44.3% along the male and female sections, respectively.
Results of the distribution of B. tuxtlaensis among the inflorescences showed greater infestation levels in the female section (Wilcoxon test, Z = 3.81, N = 36 inflorescences, P = 0.000), while the drosophilid species did not show significant differences of infestation levels between sexual sections (Wilcoxon test, Z = 0.84, N = 36 inflorescences, P = 0.396).
In Beebeomyia tuxtlaensis, the average time of development for the larval stage was 27.7 days, whereas for pupal stage it was 14.3 days; therefore, the expended time from the collection of larvae to adult emergence was of 42 ± 1.02 days (mean ± SE, N = 42 individuals); however, for the drosophilid species, the expended time from the collection of larvae to adult emergence was of 16.47 ± 0.86 days (mean ± SE, N = 40 individuals).
In addition, two hymenopteran parasitoid species were associated with B. tuxtlaensis, one species of the family Eulophidae, represented by a gregarious parasitoid recovered from 13 pupae; and three pteromalid specimens (Pteromalidae sp. 1) recovered from the same number of pupae. The drosophilid species was also  The biology of the Richardiidae is poorly known. The majority of known larvae are saprophagous, found in decaying vegetable matter (Hancock 2010). Only a few species have been recorded as having phytophagous larvae, such as Sepsisoma erythrocephalum (Schiner) which damages the stems of grasses (Deeming 1985), and Melanoloma viatrix Hendel and M. canospila with larvae associated with damage of pineapple fruits in South America (Peñaranda and Ospina 1995;Hancock 2010). Larvae of an unidentified species of Melanoloma were also observed in inflorescences of Taccarum ulei Engl. & K. Krause (Araceae) in Brazil, feeding on the connectives of male florets and fruits (Maia et al. 2013). Seifert and Seifert (1976) reported an unidentified Beebeomyia species associated with Heliconia imbricata (Kuntze) Baker, and H. wagneriana Peterson (Musaceae) in Costa Rica. Some observations showed that flower parts, particularly the petals, and nectar seem to be the main food source; oviposition occurs on the rachis or inside the bract near the juncture of the rachis and bract. Larvae occasionally were found in H. latispatha Benth. and adults were observed copulating, as well as near H. imbricata and H. wagneriana. Other records include an unidentified Beebeomyia species as saprophagous, most living between buds and bracts of H. imbricata (Naeem 1990), and also in H. bihai in Venezuela (Frank and Barrera 2010). Richardiid flies have been recorded as visitors of Dieffenbachia nitidipetiolata (Garcia-Robledo et al. 2005), and several undescribed Beebeomyia species from Costa Rica have been reared from flowers and bracts of Anthurium (Araceae) and Heliconia (Musaceae) species (Hancock 2010).
The oviposition behaviour of Beebeomyia tuxtlaensis was recorded in its natural environment. Adult females were observed on developing inflorescences, when they were still closed; conversely, we did not observe any adult male either on inflorescences or elsewhere on the host plant, indicating that mating may occur on other plants. After a female lands on the inflorescence, it moves from top to bottom for inspection, and then begins to deposit individual eggs. This is done repeatedly, with dozens of eggs hatching throughout the inner edge of bracts ( Figures 3A-E).
Upon completion of the oviposition process, the female moves once again up and down the inflorescence, exposing her aculeus and dragging it along the entire length of the structure, which most likely indicates that a deterrent pheromone is left to prevent further oviposition by other conspecific females. The eggs were incubated at the inner edge of closed bracts and usually remained in the open bracts with numerous eggs along the border. The larvae and pupae were both found in the upper section of the male inflorescence, feeding on the rachis, causing its decay ( Figures 4A-C). Furthermore, larvae of Beebeomyia were also found feeding at the bottom in the female section, and pupae were found arranged in small groups housed into the bract ( Figures 5A-D).