Description and biology of two new species of Neotropical Liriomyza Mik (Diptera, Agromyzidae), mining leaves of Bocconia (Papaveraceae)

Abstract Liriomyza mystica Boucher & Nishida, sp. n., and Liriomyza prompta Boucher & Nishida, sp. n. are described from Costa Rica. Both species were reared from leaves of Bocconia frutescens L. (Papaveraceae). The latter species was also reared from B. arborea S. Watson. Larvae of L. mystica mine primary veins of large, relatively old, mature leaves, and L. prompta mine blades of small to large, mature leaves. These represent the first record of agromyzids feeding on Bocconia. Biological information is also given and illustrated.


introduction
Liriomyza Mik is the second largest genus of agromyzid flies (after Phytomyza Lioy) with approximately 390 described species worldwide. Due to its high diversity, small size and mostly uniform external characters, identification at the species level is sometimes difficult for this genus. Host plant association is often an important tool for species identification especially for host-specific species. Host plants are known for 177 species (45%) of Liriomyza (Benavent-Corai et al. 2005) with almost all species feeding as leaf miners, except a few species that develop in stems, flower buds or potato tubers (Parrella 1987). Most species are monophagous or oligophagous, although several of the most highly polyphagous agromyzid species belong to this genus. As many as 76 plant families have been recorded as hosts for Liriomyza species, which is the widest host range known among Agromyzidae (Spencer 1990). In the Neotropical region, Liriomyza is the dominant genus with 92 species described (L. avicenniae Martinez; L. pectinimentula Sasakawa and L. pervensis Zlobin added to species list in Martinez and Etienne 2002). Host plants are known for approximately one-third of these species (Benavent-Corai et al. 2005) with some considered to be of major economic importance due to the damage they cause to ornamental plants and cultivated crops (Spencer 1973) or for their highly polyphagous habits (Boucher 2010). Species of Papaveraceae have previously been recorded as host plants for six agromyzid species (Benavent-Corai et al. 2005): Phytomyza horticola Goureau (on Eschscholzia Cham., Glaucium Miller, Papaver L.) Liriomyza strigata (Meigen) (on Glaucium, Meconopsis Viguier and Papaver), Calycomyza jucundacea (Blanchard) (on Papaver), Liriomyza huidobrensis (Blanchard) (on Papaver), Liriomyza xanthocera (Czerny) (on Papaver), and Phytomyza parvicella (Coquillett) (on Papaver), but none were previously recorded from the genus Bocconia.
Bocconia frutescens L., commonly known as Tree poppy, Parrotweed, Plume poppy, Sea oxeye daisy or simply Bocconia is a large shrub to small tree native to tropical America occurring from Mexico to Argentina, and Bahamas (Wagner et al. 1999;Hammel et al. 2007). It is easily recognized by its relatively dry and soft trunk and deeply lobed leaves reaching 30 cm wide and 55 cm long or larger (90 cm) (Hammel et al. 2007, K. Nishida, pers. observation). The species was intentionally introduced as an ornamental plant in Hawai`i in the early 1900's (Wester 1992), but has now been listed as a noxious weed by the State of Hawai`i (Benitez and Saulibio 2007). In Costa Rica, the species has been recorded between 100 and 3300 m on both Atlantic and Pacific slopes (INBio 1997(INBio -2006Hammel et al. 2007), and is commonly found in middle to high elevation cloudforests, along road sides, river banks, open fields, and light gaps (K. Nishida, pers. observation). According to Hammel et al. (2007), a second species of Bocconia, B. arborea S. Watson occurs in Costa Rica-it has the same appearance as B. frutescens except for deeper lobed foliage with more whitish texture on the underside. It has a smaller distributional range, occurring from Mexico to Costa Rica, possibly to Panama at an elevation varying from 1100 to 1600 m.
Here we describe two new species of Liriomyza reared from Bocconia at various localities in Costa Rica.

Materials and methods
Leaves of Bocconia frutescens and B. arborea infested by the Liriomyza species have been collected at multiple localities in Costa Rica (Figs 1-3, Table 1). Most of the observation, collecting and rearing was conducted between April 2007 and July 2011. The leaves collected were taken back to a laboratory in San Isidro de Coronado (Table  1, site 15) for rearing. Approximate average temperature of the rearing chamber was 25 °C (day) and 18 °C (night). Infested parts of the leaf blade were separated from the primary vein in order to separate the two fly species, which are site-specific on the leaf. The leaf blade and the primary veins were placed either in large transparent plastic bags or translucent Tupperware for observation and rearing of the larvae, puparia and parasitoids. Most adults were obtained from rearing, except a few caught while mating on Bocconia frutescens at site 13. For some of the sites, only leaf mines were recorded via observations. Larvae, puparia, adults and parasitoids were preserved in 75% ethanol. Most adults were dried using HMDS (hexamethyldisilazane). Photographs of the life histories and live specimens of both adults and immature stages were taken with digital cameras (Nikon Coolpix 4500, 8700, and Canon PowerShot G7). The final digital images were processed using Adobe Photoshop CS4. Type specimens are deposited in the following collections (acronyms used in the text are in parentheses): Canadian National Collection of Insects, Arachnids & Nematodes, Ottawa, ON, Canada (CNC); Museo de Zoología, Universidad de Costa Rica, San José, Costa Rica (MZUCR); Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica (INBio); Lyman Entomological Museum, McGill University, Ste-Anne-de-Bellevue, QC, Canada (LEM); National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (NMNH).
Specimens of immature stages are deposited in CNC and MZUCR, and parasitoid wasps in MZUCR.

Results
Both species of Liriomyza were present at most of the field sites, sometimes containing larvae of the two species on the same leaf. A total of 127 adult specimens representing two new species of Liriomyza were obtained from nine localities in Costa Rica ( Table 1). Most of these specimens were reared from Bocconia frutescens except four specimens that were collected while mating under leaves of Bocconia frutescens, and   (Table 1). No adult specimens were successfully reared from some of the sites, but species identification was still possible with larvae and/or puparia obtained. Species description and details on biology follow. Diagnosis. This species can be distinguished from other Neotropical species of Liriomyza by its completely yellow head and anepisternum, mesonotum almost completely brown to margin of scutellum, usually 2 + 1 dc, legs completely yellow, calypter brown on apical half with margin and fringe brown, and by the shape of the male genitalia and the shape of the anterior and posterior larval spiracles.

Liriomyza mystica
Description. Frons width 0.25 mm; ratio of frons width to eye width 2.3; orbit 0.23 times width of frons at midpoint; frons slightly projecting above or in front of eye in profile (Fig. 8), forming a distinct ring (cheek) below eye; 2 reclinate ors and 2 inclinate ori (Fig. 9) (lower ori sometimes reduced or missing on one side); orbital setulae reclinate, varying in number from about 4-8; first flagellomere rounded, not enlarged in males, with slight apical pubescence; arista 0.30-0.40 mm, with short but dense pubescence; gena deep, slightly extended at rear (Fig. 8); gena height at midpoint: 0.44 times maximum eye height. Eye oblique, bare. Normally 2+1 dc (except 4 specimens with 2+0 dc and 3 specimens with 3+1 dc); acrostichals in about 4 irregular rows; prescutellar acrostichal bristles absent; 2 notopleural bristles; 1 strong postpronotal bristle with 1 or 2 small setulae; anepisternum with 1 strong bristle on posterior margin at midpoint, sometimes with a few extra setulae; katepisternum with one strong bristle on posterodorsal corner, on yellow ground. Fore and mid-tibia without lateral bristle. Wing length 1.50-1.95 mm in male and 1.85-2.20 mm in females; M 1+2 ending at wing tip; costa extending to M 1+2 ; last section of CuA 1 : 1.5-1.9 times length of penultimate. Cross-vein r-m located at midpoint of cell dm. Stridulatory mechanism apparently absent.
Colour. Head (including frons, orbit, face, antenna, palp) entirely bright yellow. Hind margin of eye black for a small section beyond vte; both vt on yellow ground. Occiput black. Eye sometimes with a slight bluish or greenish reflection (not as pronounced as in L. prompta, Fig. 29); mesonotum almost completely dark brown except for narrow yellow margin posteriorly (Fig. 10), prescutellar area and intra-alar area sometimes slightly paler brown resulting in a weakly defined banded pattern on thorax, most visible in teneral specimen (Fig. 11); scutellum completely yellow with small brown patches laterally. Basal scutellar bristles on brown ground (but at the limit of yellow). Postpronotum, notopleuron and anepisternum completely yellow (at most with a very small pale brown patch on one or two of the sclerites). Katepisternum mostly brown except for upper margin yellow. Calypter brown on apical half, margin and fringe also brown; halter completely white. Legs completely yellow. Abdominal tergites pale brown.
Host plant. Bocconia frutescens L. (Papaveraceae). Biology. The larvae feed on spongy parenchyma and other tissues of primary veins and petioles of large, relatively old, mature leaves. Most of the larvae were in leaves of >30 cm long, with >10 mm petiole width and >10 mm thickness (n=120). The larvae were more frequently found mining in the thicker part of the primary vein including the petiole (i.e. less frequently near the leaf apex). One larva was found mining inside of a 2.2 mm width primary vein near the leaf apex. A few larvae were mining thick secondary veins. The mining appears to occur longitudinally, mostly near the upper leaf surface; the larvae left some brown to reddish brown scars along the leaf blade where the vein and blade join (Figs 20, 21, 23). These scars were more easily seen with a strong transmitted light (Fig. 21). The mines (internal tunnels) can also be distinguished by narrow pale lines (Fig. 22). When infested veins were longitudinally dissected, usually one to a few white Liriomyza prompta larvae were observed mining singly and scattered (n=12 leaves) (Figs 23, 24). Some solitary parasitoid wasp pupae were also found among the spongy parenchyma (Fig. 50). The mature fly larvae exited from either the upper side or underside of the veins (n=12 holes) (Fig. 25), each larva making a small oval-shaped hole of 1.1-1.3 mm wide (n=12) (Fig. 26). The tissue around the old exit holes was brown to reddish brown (n=5). The newly emerged larvae wiggled around in rearing plastic bags/cases for a couple of hours to a few hours before settling (Fig. 27) and starting to form a puparium. The larvae readily pupated on  the plastic surfaces. In general, the puparia (Fig. 48) were more translucent (translucent pale brown) than those of L. prompta (translucent brown to dark brown) and the pupa inside was visible. Duration of the larval stage was not recorded. The larvae that exited from veins pupated between 26 to 29.vi.2010 and the adults emerged between 20 to 22.vii.2010, i.e. the pupal stage lasted approximately 25 days (n=29). A mating pair was observed on the underside of a leaf around 7:00 am (site 13). No oviposition behavior was observed for this species.
Comments. Liriomyza mystica is most similar to L. prompta described below and to the Neotropical species L. commelinae (Frost) and L. robustae Spencer, especially in the form of the phallus with paired tubules. But these two latter species differ from L. mystica in a number of characters, including their host plants (both known from plants in the family Commelinaceae); mesonotum with a distinctive black and yellow pattern; surstylus with a distinct spine; third antennal segment enlarged in males; shape of both anterior and posterior spiracles and pupation occurring inside the mine (Silva and Oliveira 1952, Spencer 1984, Valladares 1984. The anterior and posterior spiracles of L. mystica are most similar to those of L. caesalpiniae Valladares reared from a Caesalpiniaceae, Caesalpinia gilliesii Benth. (Valladares 1984: figs 14, 15).
Most adult specimens of L. mystica were reared from site 5, but it was also found at other sites, up to an elevation of 2765 m ( Table 1). Considering that L. mystica larvae feed inside primary veins and petiole of large, mature leaves, it made it difficult to establish Bocconia arborea as possible host due to the problems in studying large trees with large leaves. In sapling trees of ca. 1 m tall (n=2) at Santo Domingo de Heredia (site 9), no larvae or evidence of feeding was observed.
Etymology. The species name is derived from the Latin mysticus (secret, mystic), referring to the hidden and inconspicuous leaf mines in primary vein and petiole.  Diagnosis. This species can be distinguished from other Neotropical species of Liriomyza by its completely yellow head and anepisternum, mesonotum almost completely brown to margin of scutellum, usually 3 + 1 dc, legs completely yellow, calypter brown on apical half with margin and fringe brown, and by the shape of the male genitalia and the shape of the anterior and posterior larval spiracles.
Male genitalia. Similar to L. mystica, but tubules of distiphallus more sclerotized, slightly wider, parallel sided (not diverging in ventral view). Mesophallus widest apical section (Fig. 33a), more than twice as large as narrower basal tubular section (Fig.  33b). Mesophallus in lateral view with a prominent curve near midpoint. Surstylus absent. Ejaculatory apodeme (Figs 34, 35) slightly more sclerotized than in L. mystica, and with blade slightly less expanded.
Early stages. Larval length: 1.95-2.70 mm, slightly smaller than L. mystica larva. White to creamy white with an internal orange spot at head (Fig. 47). Anterior spiracles about 0.1 mm distance from each other. Similar to L. mystica: fan-shaped with 5 small openings. Posterior spiracles with apparently 3 bulbs, but two very small and 1 much longer, curving toward anal segment (Figs 36, 37). Cephalopharyngeal skeleton ( Fig. 39) more elongated with side arms narrower than in L. mystica. Puparium translucent brown to dark brown (Fig. 49).
Host plants. Bocconia frutescens L. and B. arborea S. Watson (Papaveraceae) Biology. At site 15 in late October 2012 a few males were perched on small young leaves at the apical shoot of a 2 m host tree in the morning between 6:00 and 7:00 am. The males were either sitting or flying and perching on apical young leaves. A mating couple was observed on the underside of host leaf at 7:00 am (site 13, Fig.  6). Oviposition was observed a few times by two females at site 2. At 3:00 pm (17. vi.2011), overcast with slight drizzle, the two females were walking on the upper side of leaves of a Bocconia frutescens sapling, ca. 50 cm tall. The females either oviposited in the leaf tissue at the edge of the leaf margin (n=2) or along narrow leaf veins on the upper side of the leaf blade (n=3) (Fig. 7). It was difficult to locate the eggs because of their small size and translucent colour (n=1). After oviposition, the leaf was collected and a small larva was found on 28.vi.2011. Also at site 5, three leaves were randomly collected on 24.vi.2010, and newly started mines were observed on 2-3.vii.2010 (n=6 mines). These observations suggest that the duration of the egg stage is approximately 10 days. The larvae mine mesophyll of the leaf blade either singly or gregariously (up  to 7 larvae) (n=70 mines) (Figs 40-45). Mines were found on very small mature leaves of ca. 1.5 cm (n=5, at site 13) to 90 cm long (n=10, at site 10). The mature mines with a single larva were narrow and more or less linear (Figs 20,40,41), and mines with multiple larvae were blotch-shaped (Figs 40, 42). These mines were conspicuous on the upper side of leaves. With regard to location of mines on leaves, no patterns were noticed; mines appear to occur on any part of the leaf blade -some mines were found near the primary vein, leaf apex, or anywhere in between (uncounted). Presence of the larva in the mine can be recognized by the orange spot of the larval anterior end (Figs 43-45, 47). The early part of the mine becomes brown and the frass is seen in dark green to black linear dots (Figs 42-44). The mature larvae each made an elliptical exit hole 1.1-1.3 mm wide on the upper side of the leaves, (n=7 holes) (Fig. 46). Under rearing conditions, from the time of recognizing very early mines, the larvae completed mining within 3 to 4 days, and on the 5th day the puparia were usually formed on the plastic surface of rearing bags or containers. Young pupae were observed within a day or two after forming of the puparium (n=16). The puparia (Fig. 48) were less translucent than those of L. mystica. The pupa becomes dark 2-3 days prior to adult emergence. A cohort of larvae from site 5 which pupated on 20-23.xii.2008 emerged as adults on 16-20.i.2009 (n=6, 1 ♂, 5 ♀), other data as follows: pupation 28.vi.2011, adult emergence 24.vii.2011 (a cohort, n=4, 2 ♂, 2 ♀, site 2); pupation 12-13.ii.2012, emergence 8.ii.2012 (n=2, site 12); pupation 20.iv.2012, emergence