Description of a new species of Dacus from Sri Lanka, and new country distribution records (Diptera, Tephritidae, Dacinae)

Abstract A fruit fly survey in the Sinharaja and Knuckles National Parks in Sri Lanka (2016), using traps baited with the male lures methyl eugenol, cue-lure, and zingerone, yielded 21 species of Dacini fruit flies. Of these, three species, viz. Bactroceraamarambalensis Drew, B.dongnaiae Drew & Romig, and B.rubigina (Wang & Zhao), are new country occurrence records, and Dacus (Mellesis) ancoralis Leblanc & Doorenweerd, sp. n. is described as a new species. The Sri Lankan Dacini fruit fly fauna is now comprised of 39 species.


Introduction
Dacine fruit flies are a large group of Old World tropical Diptera, with 932 described species, of which 83 are pests of fruit and fleshy vegetables (Vargas et al. 2015. Previous intensive surveys were carried out in Sri Lanka between 1993 and 1996 and resulted in a greatly improved understanding of the island's diversity of fruit flies, including the description of eleven new species (Tsuruta et al. 1997, Tsuruta and White 2001. This increased the number of known species for Sri Lanka to 35 Romig 2013, 2016). We report here the results of new surveys carried out twenty years after the last one, which include three new country occurrence records, and we describe one new species of Dacus.

Materials and methods
We built traps from modified urine sample cups (described in Leblanc et al. 2015a) and deployed them along walking trails for 2-5 days in August 2015, to sample fruit flies in the Sinharaja National Park (40 sites) and the Knuckles National Park (31 sites). At each site, we maintained three traps, separately baited with the fruit fly lures methyl eugenol, cue-lure and zingerone. We used a 1×1 cm strip of dichlorvos insecticide to kill the flies entering traps. Collected specimens were preserved in 95% ethanol and later stored in a -20 °C freezer for long-term conservation, or double-mounted using 10 mm minuten pins. Prior to final mounting, we soaked minuten-pinned specimens for 3-12 hours in ethyl ether, to preserve the coloration. We identified flies using available keys White 2001, Drew andRomig 2016). We photographed the newly described species using a Nikon D7100 camera attached to an Olympus SZX10 microscope, with Helicon Focus pro v6.7.1 software used to stack photos taken at different focal planes. We took measurements using an ocular grid mounted on an Olympus SZ30 dissecting microscope. Morphological terms used in the description generally follow White (1999) and generic assignment for each species follows the checklist published by Doorenweerd et al. (2018). We extracted DNA and sequenced the mitochondrial COI-3P and COI-5P region from selected specimens to help confirm species identity. The entirety of the mitochondrial gene COI was sequenced using two PCR primers L1-DCHIM (5'-TCGCCTAAACTTCAGCCATT-3') and PAT-K508 (5'-TCCAATGCAC and two additional internal primers (HCO-2198, 5'-TAAACTTCAGGGTGACCAAAAAAT-CA-3' and HCO-2198RC 5'-TGATTTTTTGGTCACCCTGAAGTTTA-3') resulting in a 1,535 base-pair (bp) long fragment. We sequenced up to six additional genes (two fragments of CAD, Wingless, White-eye, PGD, EF1-alpha and Period) for selected representatives of all species, including the one described here, and already available from a published dataset (San . Pairwise genetic distance (p-distance) between specimens was calculated in Geneious R10.2.3. We performed maximum likelihood analysis of 1535 base-pairs of COI (3P + 5P region) of the newly described species and several closely related congeners using RaxML v8.2.11 (Stamatakis 2014).
We repeated the best-scoring tree search 20 times and employed 1,000 multiparametric bootstrap searches with automatic halting following the extended majority rule criterion, halting the bootstraps after 200 searches, to estimate branch support. All specimen taxonomy and collecting data, as well as DNA sequences have been added to BOLD (dx.doi.org/10.5883/DS-DACANC) and the sequences have been deposited in Genbank (COI-5P: MH272136-MH272144 and COI-3P: MH272145-MH272155).

Results
We collected a total of 3,498 specimens representing 21 species; 14 species in Sinharaja N.P. and 15 in Knuckles N.P. (Table 1). The majority (76.0 %) belong to seven pest species, dominated (64.5%) by Bactrocera kandiensis Drew and Hancock. We treat the previously described Bactrocera invadens Drew, Tsuruta and White (Drew et al. 2005) as a junior synonym of B. dorsalis (see Schutze et al. 2015). Three species represent new country records: B. rubigina (Wang and Zhao), B. dongnaiae Drew and Romig, and B. amarambalensis Drew, and one new species, Dacus ancoralis Leblanc & Doorenweerd, is described below, increasing the number of species known to occur in Sri Lanka to 39 (Table 1).
One species not attracted to the traditional male lures, B. syzygii White & Tsuruta, was captured in zingerone-baited traps, as well as cue-lure responding B. bipustulata (Bezzi), B. perigrapha White & Tsuruta, B. rubigina (Wang & Zhao), and Dacus discophorus (Hering). The new species was also collected at zingerone. The specimen identified as B. amarambalensis Drew was collected in a cue-lure trap, whereas the two other known specimens (holotype and paratype) were collected at methyl eugenol. This may represent lure contamination or a superficially very similar species that shares the markings on postpronotal lobes and other distinctive features of the described species.
In addition to fruit flies, lacewings (Neuroptera: Chrysopidae) were collected in the methyl eugenol traps (133 specimens in Sinharaja N.P. and 55 in Knuckles N.P.). All specimens are consistent with Ankylopteryx anomala, a widely distributed species previously recorded from Sri Lanka (Breitkreuz et al. 2015), and for which attraction to methyl eugenol is well documented (Leblanc et al. 2015b). Differential diagnosis. Dacus ancoralis is similar to other Asian species of Dacus with a red-brown scutum lacking the yellow medial and lateral vittae and with a costal  Molecular diagnostics. Figure 3 shows the maximum likelihood tree based on combined COI-5P and COI-3P regions (1535 base-pairs [bp]) for Dacus ancoralis and the closest congeners in our dataset. In the COI-3P fragment (836 bp), the minimum p-distance to Dacus vijaysegarani is 1.38%, and in the COI-5p DNA barcode fragment (658 bp) it is 2.43%. Because there is only one specimen of D. ancoralis we cannot test for reciprocal monophyly. The overall next closest relative in our dataset is D. siamensis, at a minimum p-distance of 8.61% in COI-3P, 8.81% in COI-5P.

Dacus (Mellesis) ancoralis
Description of adult. Head ( Figure 1A). Vertical length 2.00 mm. Frons, of even width, length 1.06 times as long as broad; red-brown with fuscous around orbital setae and on anteromedial hump; latter covered by short red-brown hairs; orbital setae dark fuscous: one pair of superior and two pairs of inferior fronto-orbital setae present; lunule fulvous. Ocellar triangle dark fuscous. Vertex fuscous. Face fulvous with medium sized oval black spots in each antennal furrow, a fuscous band along lower margin between spots and a dark fuscous triangular marking below antennal sockets; length 0.55 mm. Genae red-brown, with fuscous subocular spot; dark fuscous seta present. Occiput fulvous and yellow along eye margins; occipital row with two parallel rows of adjacent setae (with 11 and 17 setae). Antennae with segments 1 (scape) and 2 (pedicel) fulvous and segment 3 (first flagellomere) fuscous; a strong red-brown dorsal seta on segment 2; arista black (fulvous basally); length of segments: 0.83 mm; 0.70 mm; 1.23 mm.
Thorax ( Figure 1B, E). Scutum red-brown with a broad light fuscous lanceolate pattern on its posterior third, anteriorly prolonged into three very narrow lines reaching anterior margin, light fuscous narrow outer bands parallel to lanceolate pattern and reaching notopleural suture. Pleural areas red-brown except a broad vertical dark fuscous band in front of mesopleural stripe, a large dark fuscous spot occupying central portion of katepisternum, and a dark fuscous spot on katepimeron above hind coxa. Yellow markings as follows: notopleura (notopleural callus); narrow parallel-sided mesopleural (anepisternal) stripe, reaching midway between anterior margin of notopleura and anterior notopleural seta dorsally, continuing to katepisternum as a transverse spot and to scutum as moderately broad yellow markings along anterior margin of notopleural suture; lower 25% of anatergite (remainder dark fulvous); anterior 70 % of katatergite (remainder black). Postpronotal lobes dark fulvous. Medial and lateral postsutural vittae absent. Postnotum red-brown with two broad longitudinal fuscous bands. Scutellum yellow except for narrow black basal band. Setae (number of pairs): 1 scutellar; prescutellar absent; 1 intraalar; 1 posterior supraalar; 1 anterior supraalar; 1 mesopleural; 2 notopleural; 4 scapular; all setae well developed and black.
Wings ( Figure 1D). Length 7.00 mm; basal costal (bc) and costal (c) cells fuscous and covered with microtrichia; remainder of wings colorless except dark fuscous subcostal cell, broad dark fuscous costal band overlapping R 4+5 and of uniform width, not reaching vein M; anal streak absent; supernumerary lobe weakly developed.
Abdomen ( Figure 1C, E, F). Elongate, clavate and petiolate; terga tightly joined but with medial protuberances; pecten of cilia present on tergum III; posterior lobe of surstylus short; abdominal sternum V with a slight concavity on posterior margin. Tergum I and sterna I and II longer than wide. Tergum I orange-brown with apical third yellow and a median light fuscous band on apical half of red-brown portion. Tergum II orange-brown with medial dark fuscous narrow band and two short basal bands, lateral to medial band, forming an anchor-shaped pattern, and broad fuscous markings on lateral margins. Tergum III orange-brown with dark fuscous as along base and extended to whole lateral margins and into a triangular medial band. Tergum IV orange-brown with dark fuscous medial basal triangular marking, narrowly along base of tergum and broadly along entire lateral margins. Tergum V orangebrown with dark fuscous medial basal triangular marking, and large lateral bands covering basal half of tergum and reaching lateral margins. A pair of basally fuscous and apically orange-brown ceromata (shining spots) on tergum V. Abdominal sterna dark except pale sternite II.
Etymology. The name ancoralis is a noun in apposition that refers to the anchorshaped fuscous pattern on abdominal tergum II in the holotype ( Figure 1F).
Notes. Although Dacus ancoralis is genetically closely related to D. vijaysegarani and there is only one specimen, they do not appear to be sympatric, with D. vijaysegarani only known from Malaysia, Thailand and Vietnam, and with the clear differences in coloration of all body parts we are confident in describing it as a new species. The holotype of Dacus ancoralis was referred to as "ms7321 Dacus (Mellesis) sp-78", sister to D. vijaysegarani, in the seven-gene phylogeny presented in San . It keys to couplet 37 (p 467) in the Keys to the Fruit Flies of South-East Asia (Drew and Romig 2016), where it can be added as a unique combination of having dark fuscous postpronotal lobes and a red-brown scutum. Dacus ancoralis was collected in a trap with zingerone lure. A number of other species of Dacus were found to be drawn to zingerone in recent years , but because there is only one specimen known we cannot yet confirm it as a zingeroneattracted species. This species is assigned to subgenus Mellesis, as defined by Drew and Romig (2013) based on the petiolate abdomen with tergum I longer than wide and sternum V weakly concave apically, the presence of anterior supraalar setae and absence of prescutellar setae, the combined length of antennal segment greater than vertical length of face, and the absence of anal streak on wing. Its nearest relatives all belong to subgenus Mellesis (Figure 3).