The genus Scaptodrosophila Duda (Diptera, Drosophilidae), part III: the riverata species group from China, with morphological and molecular evidence for five new species

Abstract A new species group, the riverata species group, is established within the genus Scaptodrosophila based on morphological and molecular evidence for five known and five new species from China: S. abdentatasp. nov., S. cederholmi (Okada, 1988), S. crocata (Bock, 1976), S. paraclubata (Sundaran & Gupta, 1991), S. platyrhinasp. nov., S. puncticeps (Okada, 1956), S. riverata (Singh & Gupta, 1977), S. serrateifoliaceasp. nov., S. sinuatasp. nov. and S. tanyrhinasp. nov. A key to this group is provided. Furthermore, 51 mtDNA COI sequences belonging to S. puncticeps, S. riverata and the five new species are used for verifying species boundaries defined by the morphological data.


Introduction
A total of 12 species groups have been erected within the genus Scaptodrosophila (Duda 1923): albifrontata group (Wheeler and Takada 1966); aterrima group (Tsacas et al. 1988); barkeri group ; brunnea group (Tsacas and Chassagnard 1976;Liu et al. 2017); brunneipennis group ; bryani group (Throckmorton 1962); coracina group (Mather 1955;Liu and Chen 2018); inornata group ; latifasciaeformis group (Burla 1954); rufifrons group (Papp et al. 1999); saba group (Burla 1954) and victoria group (Wheeler 1949). Together, these taxa include ca 300 species. Here we describe five new species from China that are morphologically similar to five known, yet unplaced, species: S. cederholmi (Okada, 1988) from Sri Lanka; S. crocata (Bock, 1976) from Australia; S. paraclubata (Sundaran & Gupta, 1991) from India; S. puncticeps (Okada, 1956) from China, Kuril Islands, Korea, Japan; and S. riverata (Singh & Gupta, 1977) from China, India, Myanmar. These ten species all have a yellowish-brown body; an arista with two dorsal and one ventral branch in addition to a terminal bifurcation; a large facial carina; and developed prescutellar setae. This morphological group is also supported by molecular data for the five new species and two of the five previously described taxa. The combined morphological and molecular evidence supports the establishment of a new species group, the riverata species group, based on five known and five new species from China. DNA barcoding technology was employed to investigate the relationship of the riverata species group. Based on the results of the phylogenetic reconstruction, 51 barcode sequences of the COI (mitochondrial cytochrome c oxidase subunit I) gene belonging to two known and five new species were used to evaluate these species boundaries.

Specimens
The riverata group species were collected by net sweeping from tussocks and tree trunks. All the examined specimens were preserved in 75% ethanol.

Species identification
Total DNA was extracted from the abdominal tissue of samples after the dissection of the genitalia, using the TIANGEN™ DNA extraction kit following the recommended protocol. The COI fragments were amplified using the cycle protocol as in Zhao et al. (2009). The PCR sequencing primer pair was 5′-CGCCTAAACTTCAGCCACTT-3′ (Wang et al. 2006) and 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al. 1994). All sequences generated in this study were supplied with BOLD Process ID and GenBank accession numbers (Table 1).
A total of 51 COI sequences of the riverata group were examined and aligned with MEGA 7.0 (Kumar et al. 2016). Intra-and interspecific genetic distances were calculated for species of the riverata group using the p-distance model (Nei and Kumar 2000). We also conducted character-based species barcoding where fixed sites of one species differed from those of the others; these were manually selected as diagnostic sites (i.e., "pure" diagnostics, Sarkar et al. 2002;Desalle et al. 2005). Three known species: S. melanogaster (GenBank accession number: KR070823), S. rhina (KR070845) and S. scutellimargo (KR070847), were used as outgroup taxa in the phylogenetic analyses. The alignment was subsequently employed to reconstruct a phylogenetic tree using the Neighbor-joining (NJ) method with p-distance model implemented in MEGA 7.0.26 (Kumar et al. 2016). Nodal support values (bootstrap percentages, BPs) were inferred by bootstrapping with 1000 replicates and other default settings.

Description of species
An Mshot Camera was used to photomicrograph all the examined species. All photographs, illustrations, and line drawings were processed with the software Adobe Photoshop 7.0 and Easy PaintTool SAI Ver.1.0.0. The morphological terminology follows McAlpine (1981) and the definitions of measurements, indices, and abbreviations follow Chen & Toda (2001). The type specimens were deposited in Department of Entomology, South China Agricultural University, Guangzhou, China (SCAU).

Results
The alignment of the 51 COI sequences spans 632 nucleotide sites, with 131 variable sites, 122 of which were parsimony informative. Intra-and interspecific p-distances were provided in Table 2. The results show that the largest intraspecific p-distances within the riverata species group was 0.032 detected in S. puncticeps, followed by 0.016 in S. platyrhina sp. nov. while the minimum interspecific variation was 0.014 detected between S. abdentata sp. nov. and S. tanyrhina sp. nov.
The NJ (Fig. 1) tree shows that this new group is monophyletic with respect to the outgroups. Figure 2 shows nucleotides representing "pure" diagnostic sites for all species of the riverata group; at least one diagnostic site was recognized for each species. For example, site 21 is diagnostic for S. serrateifoliacea sp. nov. with a fixed status of C (Cytosine), rather than T (Thymidine) in the other species.

Scaptodrosophila riverata species group
Diagnosis. Body mostly yellow to yellowish brown, without patches; arista with two dorsal branches and one ventral branch in addition to the terminal bifurcation; facial carina large; prescutellar setae usually large, as long as anterior dorsocentral setae; hypandrium usually with a pair of long paramedian setae. Description. Male and female: Head (Figs 3, 4, 5A, B, 6A): Eyes red. Ocellar triangle yellowish brown, with 4-6 setae above ocellar setae. Frons about 1/3 width of head, largely yellowish brown to brown, with a few minute setulae medially. Fronto-orbital pale yellowish brown; anterior reclinate orbital seta usually lateral to and about 1/3 length of proclinate orbital seta; posterior reclinate orbital seta larger than other two orbital setae. Pedicel yellowish brown to brown, with a few of fine setae; first flagellomere pale yellowish. Face yellowish brown. Clypeus mostly yellow. Vibrissa prominent; subvibrissal setae small. Gena and postgena narrow, yellowish brown. Palpus yellow to yellowish brown with several setae.  (Singh & Gupta, 1977) B, D, F S. puncticeps (Okada, 1956). Scale bars: 0.5 mm.
Abdomen (  peg-like prensisetae on caudal margin, and several setae on outer and inner surface. Hypandrium shallow, usually with a couple of paramedian setae distally. Cercus separated from epandrium, pubescent and setigerous. Paramere developed, with few sensillae. Gonopods (as dorsal extension of the hypandrium, see Ashley and Sinclair 2017) fused with each other, broadened to hood-shaped. Aedeagus bifid, glabrous.
In the following descriptions, only those characters differing from the above description were provided. Diagnosis. Surstylus bifid, the upper lobe with about 5 thin, peg-like prensisetae and 1 fine seta, the below one with 1 thin, peg-like prensiseta and 2 fine setae (Fig. 7A Male terminalia: Epandrium with ca 14 setae near posterior margin and ventral corner on each side (Fig. 7A, B). Hypandrium glabrous (Fig. 7C, D). Paramere slender and curved apically, with 3 sensillae subbasally (Fig. 7C, D).

Etymology.
A combination of the Latin words: "tanaos" + "rhinos", referring to the developed carina.