The genus Scaptodrosophila Duda part II: the coracina species group from East Asia, with morphological and molecular evidence (Diptera, Drosophilidae)

Abstract Eight new species of the Scaptodrosophila coracina species group are described from China: S. fuscilimba sp. n., S. fusciventricula sp. n., S. helvpecta sp. n., S. longispinata sp. n., S. nigrolimbata sp. n., S. trivittata sp. n., S. ventriobscurata sp. n., and S. zebrina sp. n. One known species S. coracina (Kikkawa & Peng) is redescribed. A key to all the examined species in the coracina group is provided. Species delimitations have been improved by integrating the DNA sequences with morphological information. The intra- and interspecific pairwise p-distances (proportional distance) are summarized. Some nucleotide sites with fixed status in the alignment of the COI sequences (662 nucleotide sites in length) are used as “pure” molecular diagnostic characters to delineate species in the coracina group.

In the present study, eight new species discovered from China are described, and one known species is redescribed. DNA barcoding was conducted to evaluate morphological delimitation for the coracina group, and for this, a total of 35 COI (mitochondrial cytochrome c oxidase subunit I) gene sequences of the above-mentioned nine species are determined (Table 1).

Specimens
The coracina group flies were collected by net sweeping from tussocks and tree trunks near streams in forests. All the examined specimens were preserved in 75% ethanol.

Species identification
The specimens were first identified as of the coracina group in light of morphology referring to Bock's (1982) and van Klinken's (1997) diagnoses. Then, they were examined for morphometric characters and detailed structures of terminalia, and sorted into putative species. For each of these putative species, representative specimens suitable for DNA sequencing were selected, considering also the numbers, geographical origins, and genders of available specimens. The methods of Liu et al. (2017) on morphology and molecular phylogenetics were followed.
All the sequences determined in this study were submitted to BOLD (The Barcode of Life Data system) and GenBank (Table 1). A total of 35 COI sequences of the coracina group were examined and aligned in MEGA 7.0 (Kumar et al. 2016). Then, calculation of the inter-and intraspecific genetic distances, construction of the neighbor-joining (NJ) tree and the character-based species delimitation were performed in MEGA 7.0. In this paper, four species described in Liu et al. (2017), S. maculata (GenBank accession number for COI sequence: KR070820), S. melanogaster (KR070823), S. nigricostata (KR070829), and S. obscurata (KR070838) were used as outgroups.

Description of species
An Mshot Camera was used to microphotograph specimens, photograph illustration and line drawings were processed with Adobe Photoshop 7.0 and Easy PaintTool SAI Ver.1.0.0. Zhang and Toda (1992) and Chen and Toda (2001) are followed for the definitions of measurements, indices and abbreviations. The type specimens were deposited in Department of Entomology, South China Agricultural University, Guangzhou, China (SCAU).

Results
The alignment of the 35 COI sequences spanned 662 nucleotide sites in length, with 195 variable sites, among which 188 were parsimony informative. The inter-and intraspecific p-distances in the coracina group are shown in Table 2. In most cases, the intraspecific p-distances in the coracina group were range from 0 to 2.9%, while the largest intraspecific p-distance in the coracina group was found in S. coracina (= 5.6 %). The smallest interspecific p-distance was found between S. fuscilimba sp. n. and S. helvpecta sp. n. (= 4.7 %).
The NJ tree was shown in Fig. 1. In this tree, each morphologically recognized species was strongly supported [bootstraps percentage (BP) = 100, and them formed a monophyletic group with respect to the outgroups (BP = 69)]. Fig. 2 shows nucleotides at the sites of "pure" diagnostics for each species of the coracina group in this study. At least one diagnostic site was recognized for each species.

Scaptodrosophila coracina species group
Scaptodrosophila coracina species group Mather 1955: 550;Bock 1982: 70;van Klinken 1997: 424. Diagnosis (modified from Bock 1982 andvan Klinken 1997). Body yellowish brown to black; arista with three or four dorsal and two ventral branches in addition to terminal bifurcation; facial carina narrow and flat; prescutellar setae usually large, as long as anterior dorsocentral setae; hypandrium usually with a pair of very large paramedian setae.
Description. Male and female: Head (Figs 3-7A, B): Eyes red to brownish red. Ocellar triangle yellowish brown to brown, mostly with three pairs of setae above ocellar setae. Frons narrower than 1/2 width of head, with a few minute setulae medially. Anterior reclinate orbital setae usually outside and close to proclinate orbital setae; posterior reclinate orbital seta larger than others. Face usually yellowish brown to black. Clypeus mostly yellowish brown to brown. Palpus usually yellowish brown. Vibrissa prominent; subvibrissal setae small. Gena and postgena narrow.
Thorax (Figs 3-7C-F): Mesonotum yellowish brown to black, sometimes with longitudinal stripe(s). Postpronotal lobe mostly yellowish to dark brown, with three long setae, and a few of shorter setae. Acrostichal setulae mostly in ca. eight to ten regular rows. Pleura mostly yellowish brown to dark brown. One small proepisternal seta. Katepisternum medially with three large setae and some small ones. Scutellum yellowish brown to black. Wing hyaline, sometimes infuscate. Basal medial-cubital crossvein absent. R 4+5 nearly parallel with M 1 distally. Halter mostly white to yellowish. Legs mostly yellowish brown to black.
In the following individual species descriptions, only characters that depart from the above description are provided for brevity.
Abdomen (  Diagnosis. This species is similar to S. fuscilimba sp. n. in the pattern on the abdomen tergites and the shape of male terminalia, but can be distinguished from the latter by having the tergite VI yellowish brown with dark brown caudal bands (Fig. 5C, E); paramere small, finger like in lateral view (Fig. 11D); aedeagus inverted triangle shaped and round dorsally in lateral view (Fig. 11D).
Abdomen (Fig. 5D, F): Tergites II to V yellow with dark brown caudal bands, the caudal band on tergite II interrupted medially; tergite VI dark brown.
Abdomen (Fig. 6C, E): Tergites II to VI yellow with dark brown caudal bands, the caudal band on tergites II to V interrupted medially.
Abdomen (Fig. 6D, F): Tergites II to IV yellow with dark brown caudal bands, the caudal bands narrowed dorsomedially; tergites V to VI yellow.
Measurements Etymology. A combination of the Latin words tri and vittatus, referring to the three caudal bands on the tergites II to IV.
Abdomen (Fig. 7D, F): Tergites II to VI yellow with black caudal bands, the caudal bands on tergites interrupted medially.