Strange Little Flies in the Big City: Exotic Flower-Breeding Drosophilidae (Diptera) in Urban Los Angeles

Urban landscapes are commonly considered too mundane and corrupted to be biotically interesting. Recent insect surveys employing 29 Malaise traps throughout Los Angeles, California, however, have uncovered breeding populations of two unexpected species of one of the most studied and familiar groups of organisms, Drosophila “fruit” flies. Unlike most introduced species of drosophilids, which breed in fresh or decaying fruits, these are specialized flower-breeders. A common species in the survey was Drosophila (Drosophila) gentica Wheeler and Takada, previously collected only once, in El Salvador. It belongs to the flavopilosa species group, all species of which have been known until now from central Chile, Argentina and Uruguay, to Veracruz, Mexico and the Caribbean, breeding in flowers of Cestrum (“jessamine”) and Sessea (Solanaceae). The Los Angeles populations are probably breeding in a native and/or introduced Cestrum; in addition, populations in San Luis Obispo County were visiting ornamental Cestrum. Drosophila gentica occurs as far north as San Francisco, where it was found breeding in Cestrum aurantiacum. D. gentica is redescribed and figured in detail for diagnostic and identification purposes. Specimens from Jamaica previously identified as D. gentica are a distinct species but are not formally described in lieu of complete male specimens. Rare in the Malaise traps was Drosophila (Sophophora) flavohirta Malloch, a common species in Australia on the blossoms of native Myrtaceae, found on introduced Eucalyptus in South Africa and both Eucalyptus and Syzygium in Madagascar; adults feed on myrtaceous pollen and nectar, larvae breed in the flowers. It is also redescribed in detail, including its unusual egg. This is the first New World report of this species; DNA sequences confirm it is a morphologically highly aberrant member of the D. melanogaster species group. This study reveals how intensive field sampling can uncover remarkable biodiversity in even the most urbanized areas.

Introduction drosophilid introductions have had unexpected consequences. The most dramatic such consequence concerns the spotted-winged fruit fly, Drosophila suzukii (Matsumura). For many years this fly was a well-known major pest of cultivated fruit crops in its native Japan [27]. It appears to have been introduced into California in approximately 2008 and within three years spread throughout the southern half of North America up to Ontario [28], as well as into southern Europe [29]. It is a serious agricultural pest in its introduced range, particularly of small, soft berries and drupes [30], and it is even common in hardwood forests of the eastern U.S. (D. Grimaldi, pers. observ.). It is now also established in southern Brazil [31]. Given the temperate climate of Japan, establishment of D. suzukii in North America and Europe is not surprising, although the speed of its spread was remarkable.
Another important drosophilid invasion was by Zaprionus indianus Gupta, native to Africa, the Middle East, and India, where it is a highly polyphagous breeder in fermenting fruits. It was first detected in the New World in São Paulo, Brazil in 1999 [32] and had spread to Panama by 2003 and Florida by 2005 [33]. Now it is a significant agricultural pest in Brazil, particularly on figs (Ficus sp.), and it even appears to be adversely affecting populations there of native and other introduced frugivorous drosophilids [34]. Specimens are occasionally found as far north as New York City, although it is unknown if the species is breeding there since the species seems limited to tropical and subtropical climates.
One of the main features of Drosophila suzukii and Zaprionus indianus, indeed of all major invasive drosophilids-like Drosophila melanogaster Meigen, simulans Sturtevant, busckii Coquillett, repleta Woollaston, hydei Sturtevant, and funebris (Fabricius)-is their polyphagy and fecundity. Each of them can breed quickly on a wide array of fresh, damaged, or decaying fruits and organic substrates (e.g., [10]). As invasives, Drosophila gentica and D. flavohirta are unique in their host specialization and apparent low fecundity, laying a single large, very mature egg at a time, a common feature of anthophagous Drosophilidae. Drosophila gentica, in fact, was the second most abundant species in our Malaise trap samples from Los Angeles, after the common, cosmopolitan invasive Drosophila simulans. Drosophila flavohirta in South Africa actually appears to be a competitor with Apis mellifera Linn., the presence of fly larvae in the flowers making them unattractive to bees and negatively impacting eucalypt honey production [25,[35][36][37].

Methods, Materials, Results
Specimens of both Drosophila species were originally collected as part of the BioSCAN project; D. gentica was subsequently found also on host plants. BioSCAN is an innovative project based at the Natural History Museum of Los Angeles County designed to survey urban biodiversity using a collecting method, Malaise traps, usually deployed in more natural habitats, while also engaging the general public in the sampling program. At each of 29 sites across the City of Los Angeles, California, a Malaise trap and weather station were set up (Fig 1). Almost all sites are backyards of private citizens who volunteered to host traps for the study; additionally, one is a school, one is the Natural History Museum of Los Angeles County (LACM) "Nature Garden", and one is a community food garden. Express, written permission for the set up and maintenance of the traps by the landowners is on file at the Natural History Museum of Los Angeles County. No endangered or threatened species were involved. Geographic coordinates are given in the Material Examined section of the species treatments, summarized in the map in Fig 1. Malaise traps are tent-like structures made of a very fine-screened fabric, into which flying insects are intercepted and collected (Fig 1B). The traps can be set up for more than a year and emptied on a regular basis; they often collect insect species rarely captured using hand netting or other traditional methods. The diversity of focal groups of insects in the BioSCAN Malaise traps is being analyzed in their association with various indices of urbanization and land use to assess the effects of the city on the insect fauna. Additionally, besides surveying focal taxa, samples are screened for unusual taxa such as the Drosophila reported herein. Our new Drosophila records from the Malaise traps were collected and identified in the first three months of sampling of the BioSCAN project, during the winter when insect populations and diversity are lowest. Therefore, further unrecorded species are expected in summer samples.
All measured specimens were point mounted. In contrast to older specimens, new specimens were preserved in 70% ethanol treated with the solvent hexamethyldisilixane (HMDS), which preserves the color of specimens with more fidelity, also making them more fully distended and cleaner. Older museum specimens dried directly after capture or straight from ethanol are slightly greasy and appear slightly darker. Measurements of point-mounted specimens were generally made at 60-100X using a Nikon SMZ 1500 stereoscope with a Nikon DSRi1 digital camera and NIS Elements software; error range is approximately ± 0.01 mm (including the variation due to positioning the specimen). Standard measurements and ratios were made as given in [38]. A total of 43 measurements were made for each of five specimens from LA, five paratypes of D. gentica from El Salvador, and the four specimens of "gentica-like" from Jamaica (in [13]). All six specimens of D. flavohirta from LA were measured, and compared with measurements of five randomly sampled specimens collected 28 December, 2010 by S. McEvey in Stroud, New South Wales, Australia, off of Syzygium flowers (32.4079°S, 151.9672°E). Variation in measurements and ratios between samples of native and introduced specimens are given in the redescriptions only where significant (p<0.05) as based on a pairwise ANOVA for equal or unequal sample sizes. Male and female terminalia were dissected in representative specimens from all localities. Terminalia were macerated in warm 10% KOH, rinsed in water and 70% ethanol, dissected in glycerine using fine tungsten needles, and mounted on microscope slides in glycerine jelly for observation at 100-400X.
One In order to genetically determine the species identity of D. flavohirta in California, DNA was compared with specimens collected by S.M. in Stroud, Australia (locality data given above). DNA was extracted from a female flavohirta from sample #15778, and a female from Stroud, Australia, using standard ethanol extraction from which COI partial coding sequences were obtained and compared. Universal primers 5'-CCTACAGGAATTAAAATTTTTAGA-3' and 5'-TCCAATGCACTAATCTGCCATAT-3' were used to amplify a ca. 600-bp region of the COI CDS [39]. PCR conditions were as follows: denaturation at 94°C for 30 sec., annealing at 55°C for 30 sec., and elongation at 72°C for 30 sec for 35 cycles. Sequence alignments were performed using MUSCLE 3.8 [40].
Alignment of the LA and Stroud, Australia sequences showed 98.2% identity. This level of divergence within the amplified region is typical of intraspecific divergence within various species of the D. melanogaster species group. For example, a standard nucleotide BLAST of the D. simulans sequence, homologous to that amplified from the flavohirta samples, revealed nucleotide identities ranging from 98.0-100% amongst populations of simulans (the homologous region was obtained from D. simulans isolate RU259 complete mitochondrial genome, accession number AF200849.1). Additional collections and analyses are needed to comprehensively determine divergence of D. flavohirta populations and the placement of flavohirta within Sophophora [41][42][43], as discussed below with some new molecular data (S1 Text).
Repositories for the specimens are the Los Angeles County Museum, California Department of Food Agriculture (Sacramento, California), The Australian Museum (Sydney), and American Museum of Natural History (New York).

Systematics
The Drosophila (Drosophila) flavopilosa Species Group The flavopilosa group is very distinctive, morphologically and ecologically. All 17 described species are light-bodied flies with faint or no markings (Fig 2; S1 Fig), which contrasts in the female with her remarkable oviscapt of large, heavily sclerotized, black teeth ( Fig 2E; S2 Fig). The teeth curve slightly outward, and in some species each tooth is situated on a peduncle; those on opposing valves interdigitate when the valves are pressed together. This structure suggests that the oviscapt may function like a surgical retractor: when the valves separate the teeth grab and separate the flower tissue, allowing an egg to be inserted. Like many species of flower-breeding drosophilids, flies in the flavopilosa group lay a single mature egg at one time, rather than a clutch typical of saprophagous species. The eggs either have a pair of very short, stubby subapical filaments, or lack them entirely, again typical of flower-breeders. The larva hatches quickly after oviposition and begins feeding on flower tissue. According to Brncic [12], mature larvae feed on pollen. There is very little information on whether these flies are detrimental to Cestrum flowers (e.g., cause premature flower abscission) or may even be beneficial, as pollinators or biocontrol agents of their weedy, toxic plant hosts. Though planted as ornamentals in California, at least in South Africa (and probably other regions) Cestrum is considered to be a weedy invasive [44]. Santos and Vilela [24] mentioned that flavopilosa-group flies can be "excluded" as pollinators of Cestrum since the "adult flies are larger than the diameter of the tiny tubular flowers and therefore unable to enter the corollas." Observations of D. gentica by one of us (B.B.) in Los Osos, San Luis Obispo County, CA revealed that the flies readily passed in and out of the corollas of a Cestrum diurnum x nocturnum hybrid ("Orange Peel") ( Fig 1C-1E).
Molecular phylogenetic studies indicate that the flavopilosa group is most closely related to another Neotropical species group of Drosophila, the annulimana group [45][46][47], near the base of the "virilis-repleta" radiation of Drosophila. Species of the annulimana group are not light bodied, and they have a typical Drosophila oviscapt with a marginal row of small pegs.
Drosophila (Drosophila) gentica Wheeler and Takada.    Measurements (see description in S1 Text) further indicate that the CA flies have a slightly narrower front (frons), slightly longer posterior dorsocentral seta, and differences in two wing indices. It is unlikely that these differences amount to species differentiation, though molecular comparisons would be very useful when fresh material is available from Central America.
COMMENTS: In ( [13]: p. 407) it was mentioned that there "seem to be differences in the copulatory apparatus [between the El Salvador and Jamaica specimens], but there have been too few males to settle this point." Diagrammatic as the figures are in [13] (reprinted here, S2F-S2G Figs), the Jamaica specimens indeed appear to be a distinct species, confirmed by the diagnostic female and external differences with mainland gentica described above. Unfortunately, the genitalic slide preparations for the only known male specimens from Jamaica are missing, and so this species remains undescribed.
The Drosophila (Sophophora) melanogaster Species Group Genetic evidence places D. flavohirta within the melanogaster species group, although, as described below, this species is morphologically anomalous for the group and even the subgenus. The melanogaster species group is the largest of nine named species groups within Sophophora, with more than 180 species [48]. Species groups, including the melanogaster group, are typically divided into subgroups. There have been numerous studies on the systematics and relationships of the melanogaster group, and three prior molecular phylogenetic studies have included D. flavohirta [42,43,49].
The study by Da Lage et al. [42] was based on a 1485-bp coding sequence of the nuclear Amyrel gene (GenBank sequence AY733051; geographic origin not indicated); that of Russo et al. [49] was based on regions of six nuclear genes, including the same Amyrel sequence posted in GenBank; some species were not fully sequenced; Barmina and Kopp [43] sequenced 5-14 nuclear and mitochondrial loci. In [42], relationships of D. flavohirta varied depending on the type of analysis (parsimony and Bayesian). In both types of analyses, as well as in the study by Barmina and Kopp ([43]: see their S1 Text), D. flavohirta is closely related to the melanogaster subgroup, the latter a well-supported, intensively-studied lineage of nine species that includes such familiar species as D. melanogaster Meigen, D. simulans Sturtevant, and D. sechellia Tsacas and Bächli. The consensus parsimony tree in [42] was the following: elegans subgroup (takahashii s.g. (flavohirta (ficusphila Kikkawa and Peng + melanogaster s.g.))); the Bayesian tree consisted of the following grouping: elegans s.g. (ficusphila + takahashii s.g.) (flavohirta + melanogaster s.g.). In both hypotheses the support values (Bremer, bootstraps, posterior probability) for relationships of flavohirta and ficusphila were very low.
In [49], the maximum-likelihood tree consisted of the following: elegans s.g. (ficusphila (melanogaster s.g. (eugracilis Bock and Wheeler (takahashii s.g. (flavohirta + levii Tsacas))))). Drosophila levii (put in the takahashii s.g. by [42]) is from New Caledonia, and was placed on morphological grounds with ficusphila into a separate small group [50]; these authors [50] did not study flavohirta. We have expanded the molecular data set to more confidently place D. flavohirta with a Bayesian phylogenetic analysis (see S1 Text).
Overall, molecular phylogenetic evidence consistently places D. flavohirta within the melanogaster group, and two published studies, plus our new molecular analysis (S1 Text), converge on a close relationship of this species to the melanogaster subgroup, despite a morphology that is highly divergent with this clade. Another biologically interesting aspect of this grouping is that D. flavohirta is the only melanogaster-group species in Australia that is strictly anthophilous, not at all attracted to fruit baits. The five species in the southeast Asian elegans subgroup, also in the melanogaster species group, breed in various large, tubular flowers, like Ipomoea (Convolvulaceae) and Brugmansia (Solanaceae) [51]; interestingly, at least some of these species can be bred on standard Drosophila cornmeal lab medium, whereas most anthophagous species cannot. Drosophila ficusphila, another putative close relative of D. flavohirta from some analyses, is attracted to fruit baits (as the name reflects, it has a predilection for figs); all of these species are typical melanogaster-group species that possess male protarsomere sex combs and an oviscapt with sclerotized pegs.
Drosophila (Sophophora) flavohirta Malloch. DIAGNOSIS: Body light yellow with very pale setae; eyes iridescent [color varying with preservation, see below], micropubescent; face slightly raised but not carinate; cheek relatively deep; arista with short braches, 1 ventral and 2-3 dorsal branches, no micropubescence; anterior reclinate orbital seta lateral to proclinate, relatively large, 0.7X length of proclinate; two pairs of dorsocentral setae, no prescutellars; male without thick, black sex-comb setae on protarsus; oviscapt developed, but with only fine apical setae (no pegs); spermathecal capsule short, heavily sclerotized, with well-developed introvert. Male terminalia with ventral lobe of epandrium and surstylus long and pendulous; epandrium without microtrichia, not connected to cercus; surstylus 2-segmented, with 2 rows of prensisetae, rows separated by suture; hypandrium with COMMENTS: A short redescription of the species [41] was based on specimens from New South Wales, Australian Capital Territory, Queensland, and Northern Territory, Australia, most details of which are consistent with our redescription. Bock [41] did not mention the unique female terminalia, but described and figured the male genitalia, even commenting [41] that "On structure of male genitalia this species is clearly closely allied to the melanogaster species-group. It differs from other species of the group in its highly unusual coloration and in the absence of a sex-comb in the male" (pg. 19). Indeed, the protarsomere setae in males and females are essentially identical and only slightly thicker than remaining setae on the legs ( Fig  3H; S6D and S6E Figs); this species lacks the comb of thickened, blunt, sclerotized setae distinctive to almost all other melanogaster group species contra [43] (their Table S1). There is a host of additional features in flavohirta that differ from the melanogaster group, in fact probably all species in Sophophora. These include the following (S6-S8 Figs): • Arista with a few, short branches (2-3 dorsal, 1 ventral); vs. generally >3 dorsal and 2 ventral ones.
• Face raised, broad, and flat, with antennae lying in shallow scrobe-like structures that are slightly recessed; vs. face with a narrow, short carina.
• Face with one large pair of vibrissal setae; vs. generally (but not always) two pairs of equal or nearly equal size in Sophophora; • Oviscapt uniquely devoid of sclerotized pegs, each valve only with row of 6 fine, short, marginal setae and one very long, subapical seta; vs. sclerotized pegs of varying sizes surrounding most of oviscapt margin.
• Surstylus (male genitalia) pendulous and two-segmented, with prensisetae separated into two rows; vs. surstylus a one-segmented lobe with a single row of prensisetae.
• Ejaculatory apodeme apparently absent; vs. present. This is a significant and potentially important character.
• Egg highly distinctive in flavohirta (Fig 3I and 3J; S7 Fig), indeed so far as known unique in Sophophora for the subapical pair of filaments reduced to short, stubby lobes; vs. subapical filaments varying from long and filamentous to flattened and paddle-shaped.
Our comparisons of the male and female genitalia of specimens from Los Angeles and from Stroud, NSW, Australia revealed them to be identical (S6-S8 Figs) ., Fig 3) by the following features: • Hypandrium much broader and shorter, L/W = 0.6, with aedeagal apodeme extending well beyond anterior margin of hypandrium (0.55 the apodeme length); vs. hypandrial L/ W = 1.05 in our specimens, with only 0.34X the aedeagal apodeme length exposed.
• Paraphysis bilobed, but with inner lobe longer; vs. outer lobe longer in our specimens.
• Surstylus one-segmented, short and straight; vs. two-segmented, with suture between the two rows of prensisetae; pendulous and inwardly curved in our specimens.
• Surstylus with ventral row having 9 long prensisetae; vs. 5 short ones in our specimens.
• Epandrial lobe shorter and broader; vs. longer and slender.
In lieu of dissecting the holotype of flavohirta, we conclude that the differences in genitalic figures between material we examined and what Bock [41] presented are probably due to the very diagrammatic nature of Bock's renderings, not differences among species. Despite this, his placement of the species into the melanogaster group was accurate.
There are striking similarities between D. flavohirta and the rarely collected Australasian genus Baeodrosophila Wheeler and Takada, and it is likely that some of these features might be convergence based on anthophilic habits (Baeodrosophila visit and probably breed in the inflorescences of Pandanus "screw pines" [Pandanaceae: Monocotyledonae]). The similarities (and differences) are the few, short aristal branches (though lacking micropubescence in flavohirta) that is found on the main branch of the arista in Baeodrosophila; anterior reclinate seta lateral to the proclinate (though the former seta is relatively large in flavohirta); postpronotum with two large setae; a distinctive aedeagus with a conical shape and granular surface; digitiform paraphysis (though bilobed in flavohirta), simple hypandrium with a pair of paramedian setae on the posterior margin (though these are very short and fine in flavohirta, quite long in Baeodrosophila); the aedeagal apodeme long and rod-like, and-significantly-the absence of an ejaculatory apodeme. Drosophila flavohirta differs from Baeodrosophila by being slightly larger, without brownish or infuscate integument; having eyes that are micropubescent and with greenish-purple iridescence; face raised and flat instead of carinate; the oviscapt lacking pegs and not apically slender; the spermathecal capsule heavily sclerotized and with an introvert; and various features of the male terminalia: ventral lobe of epandrium long, pendulous; surstylus pendulous, 2-segmented.

Discussion: Hosts and Invasiveness
Flower-breeding Drosophilidae are very rarely collected in natural areas by sweeping, in Malaise traps, or by other general methods used for collecting insects; they are virtually never captured using fruit baits (D. Grimaldi, S. McEvey, pers. observations). For example, among the thousands of drosophilids amassed during the Zurqui All Diptera Biodiversity Inventory project in Costa Rica (B. Brown, Principal Investigator), collected using all general and many specialized field methods (including Malaise traps), there are only eight flavopilosa-group specimens. Flies in the flavopilosa group readily avoid detection even though they occur in abundance at flowers of Cestrum spp. during appropriate seasons. That substantial numbers of Drosophila gentica have been captured in Malaise traps from various spots in Los Angeles, and the species has been found in Los Osos and San Francisco, indicates that the populations are significant in size and well established. Several females from the Malaise samples contained mature eggs, confirming that these are breeding populations, which can be quite large on very local scales. Brncic [12], for example, calculated the density of adult Drosophila flavopilosa Frey in an area of Cestrum parqui scrub in central Chile, based on larval infestation rates of hundreds of flowers. Allowing for 90% pre-adult mortality, Brncic estimated the density of adults exceeded 30,000 flies per 100 m 2 .
So far as known, all authoritative host records of the flavopilosa group indicate that species are restricted to flowers of Cestrum (Solanaceae) (references above), a Neotropical and warm temperate genus of some 175 species of trees and shrubs [52]. Santos and Vilela [24] reared two flavopilosa-group species from Sessea brasiliensis in São Paulo state, southern Brazil, the only host record from this genus of 18 species. Brncic [12] mentioned (pg. 361) that "in Central and tropical America, it seems that other members of the flavopilosa group may utilize other flowers," for which he cited [53]. Unfortunately, several of the drosophilid identifications in [53] are known to be incorrect (e.g., the common, cosmopolitan species Drosophila immigrans Sturtevant is described as D. flexipilosa Pipkin, 1964); thus, identifications in that paper are suspect. Indeed, there are no flavopilosa group flies in Pipkin's material in the U.S. National Museum of Natural History (D. Grimaldi pers. observ.). In a large survey of flower-breeding Drosophilidae from southern and eastern Brazil [54], flowers were collected from 125 plant species in 47 families. No Cestrum or Sessea were collected, though flowers of other Solanaceae were, including Brugmansia, Brunfelsia, Datura, Petunia, Solanum, and Streptosolen. In that study there were found 28 species of Drosophilidae breeding in 56 of the flower species and 18 of the families, but no specimens of the flavopilosa group. It is quite likely that these flies are indeed restricted to breeding in Cestrum and Sessea. The studies conducted and reviewed by [12] and by [24] indicate that some species of these flies breed in multiple species of Cestrum, others appear to be strictly monophagous. As noted, we found large numbers of D. gentica in the flowers of a Cestrum hybrid in Los Osos, CA (breeding them from the flowers was not attempted), and this fly was actually breeding in Cestrum aurantiacum in San Francisco. Cestrum aurantiacum is introduced into California, its native distribution being Mexico, Costa Rica, Guatemala, and Venezuela. More extensive field collections are needed to clarify the host and geographic range of D. gentica in California.
Host specialization of flavopilosa-group flies on Cestrum reflects the chemistry of this plant genus. The flowers, commonly called "sweet jessamine", are highly fragrant, and the plants are notoriously toxic, particularly the reproductive structures. Ingestion of foliage, fruits, and flowers can be lethal to humans and grazing livestock [55][56][57]. Among the toxic types of compounds in Cestrum are glycosides and alkaloids, the former of which are implicated in acute hypercalcemia, calcinosis, and strynchine-like poisoning in ungulates [58][59][60][61]. Brncic [12] mentioned that "filtered homogenates" of Cestrum tissues kill the larvae of highly polyphagous, common species like Drosophila melanogaster, simulans, immigrans, hydei and funebris.
Cestrum parqui L'Herr, the most ubiquitous species of the genus in California, was introduced from southern South America into California, and is reported to occur in Los Angeles and Santa Barbara counties, as well as several counties around and including San Francisco [62]. In the Jepson Herbarium at Univ. California, Berkeley, there are specimens collected in 1893 from Napa City (Napa Co.), 1933 in Amador Co., and 1961 in Santa Barbara Co. Univ. California [63]. Interestingly, there is no mention of C. parqui in the California state flora [64], nor in the first edition of the Jepson Manual, although the second edition [65] indicates it is a "waif/garden escape" species. In Los Angeles Cestrum parqui is not widely planted but is a weedy species that readily disperses (P. Rundel, UCLA Bot. Garden, pers. comm. to LT May 2014). The genus Cestrum is not mentioned at all in [66]. Cestrum parqui has also been introduced toTexas [67].
Purportedly, C. parqui flowers in these North American locales from July through December. The fly specimens in this study were recovered from traps within this time frame, specifically 14 August to 9 November, 2013. Interestingly, several C. parqui plants were in flower at the University of California, Davis Arboretum in July and August, 2014, but Drosophila gentica was not found on the plants. In Chile, Cestrum parqui supports dense populations of Drosophila flavopilosa, a fly that occurs throughout southern South America [12,13]. In addition, there are two native species of Cestrum in North America: Cestrum nocturnum L., native to the southern U.S. (California, Texas, Louisiana, Georgia, Florida), and which is planted as an ornamental in California, and Cestrum diurnum L. in Texas, Florida, and Puerto Rico [67]. The pharmacology of these species has been well investigated.
Our discovery of D. gentica in California indicates that the ecological niche modeling by [47] may need to be re-assessed. Using four flavopilosa-group species that had the best geographic sampling, and 19 environmental variables (analyzed using MAXENT), they determined that the fly distributions appear to be more limited by abiotic factors like temperature and rainfall than by the distribution of various Cestrum species. Unfortunately, sampling of flavopilosa-group flies is poor, both for hosts as well as geographically. Indeed, there are no published records of these flies for the entire Brazilian Amazon basin nor for the Orinoco Basin, including all of Venezuela and the Guianas, where Cestrum is known to also be diverse.
Drosophila flavohirta appears to be restricted to flowers of the large family Myrtaceae, which comprises more than 130 genera that are widely distributed in tropical and warm-temperate regions. To date, flies have been collected in large numbers only on flowers of Eucalyptus [25,26,36,41] and Syzygium [26], and most recently found on Callistemon pallidus in Australia, all genera with massed, nectariferous flowers. The genus Eucalyptus contains c. 700 species, all native to Australia and nearby islands, various species of which have been introduced around the world, principally as a source of timber or shade trees since some species grow extremely tall and the resin (or "gum", source of their popular name "gum trees") makes them more resistant to insect damage [68]. Syzygium is a much larger and more widely distributed genus (c. 1200 species, with a center of diversity in southeast Asia); it too has been naturalized around the world, but not as extensively as Eucalyptus. Natural distribution of Callistemon s.s. includes the entire eastern margin of Australia and portions of southern Australia; this genus is closely related to Melaleuca [69].
Eucalyptus was introduced into California during the Gold Rush era of the 1850's, and now there are some 250 species cultivated in the state [63,68], with Eucalyptus globulus (blue gum) the most common species. Syzygium (as Eugenia) also occurs in California, but is much more limited in distribution and species within the state (some 44 species). Unfortunately, the flowers of Eucalyptus and Eugenia were not sampled during our study, only the flowers of the myrtaceous genus Melaleuca. Also, since the BioSCAN project is restricted to the Los Angeles area, we did not examine Malaise samples from other regions of California. Eventually, sampling should be made directly from myrtaceous flowers througout the state, especially the San Francisco area (an epicenter of California eucalypts), to determine how far D. flavohirta has spread. Given the diversity of Myrtaceae, especially in the Australasian Region, and knowing that Drosophila flavohirta is not restricted to Eucalyptus, it is possible that there are closely related species as yet uncollected in that region.
Several questions thus arise: When was D. flavohirta introduced into California? How far has it spread? Was Drosophila gentica introduced into California, or is it native? Does it (and perhaps other flavopilosa-group species) occur in other areas in the U.S. outside of California where Cestrum occurs? Native U.S. occurrence of D. gentica is highly doubtful, since it is extremely unlikely that such abundant flies escaped the detection of several large, very active Drosophila research groups in the region. In 1928 the famed "fly group" of Thomas Hunt Morgan moved to the California Institute of Technology in Pasadena [70], less than 20 km from most of our collection sites. This group included Alfred Sturtevant, author of the first treatment of North American Drosophila [71]. The other group was at the University of Texas, Austin, active from the 1940's to 1970's and where Marshall Wheeler was the systematist [13]. Drosophila flavohirta is much rarer in Malaise traps than D. gentica, somewhat expected since it congregates around arboreal blossoms and is rarely found near the ground. Drosophila flavohirta might also be rarely encountered if these are the early days of its establishment in California.  (Fig 1a-1f, 1h-1j), and of Drosophila "near gentica" from Jamaica (Fig 1g: see text).