The genus Scaptodrosophila Duda part I: the brunnea species group from the Oriental Region, with morphological and molecular evidence (Diptera, Drosophilidae)

Abstract Seven new species of the Scaptodrosophila brunnea species group are described from east Asia: S. maculata sp. n., S. melanogaster sp. n., S. nigricostata sp. n., S. nigripecta sp. n., S. obscurata sp. n., S. protenipenis sp. n. and S. rhina sp. n. Three known species, S. parabrunnea (Tsacas & Chassagnard), S. pressobrunnea (Tsacas & Chassagnard) and S. scutellimargo (Duda) are redescribed. A key to all the examined species in the brunnea 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 (664 nucleotide sites in length) are used as “pure” molecular diagnostic characters to delineate species in the brunnea group.


Introduction
To date, a total of 280 species (Bächli 2016) has been described in the genus Scaptodrosophila Duda, 1923 from around the world: four species from the Nearctic region, two species from the Neotropical region, ten species from the Palearctic region, 32 species from the Afrotropical region, 79 species from the Oriental region and 167 species from the Australasian region (Brake and Bächli 2008;Bächli 2016). So far, 12 species groups (Bächli 2016) have been established in Scaptodrosophila: the albifrontata group (Wheeler and Takada 1966), the aterrima group (Tsacas et al. 1988), the barkeri group , the brunnea group (Tsacas and Chassagnard 1976), the brunneipennis group , the bryani group (Throckmorton 1962), the coracina group (Mather 1955), the inornata group , the latifasciaeformis group (Burla 1954), the rufifrons group (Papp et al. 1999), the saba group (Burla 1954) and the victoria group (Wheeler 1949).
In the present study, seven new species from East Asian are described, and three known species are redescribed. DNA barcoding was conducted to evaluate morphological delimitation for the brunnea group, and for this, a total of 44 COI (mitochondrial cytochrome c oxidase I) gene sequences of the above-mentioned ten species mentioned above are determined (Table 1).

Specimens
The brunnea 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. In the species descriptions, an asterisk * denotes a new record.

Species identification
The specimens were first identified as of the brunnea group in light of morphology referring to Bock (1982) diagnosis of it. 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. For each of the selected specimens, the total DNA was extracted from the abdominal tissue of samples after the dissection of the genitalia, using the TIAN-GEN™ DNA extraction kit following the recommended protocol. The PCR/sequencing primer pair was either that designed by He et al. (2009, 5'-CGCCT AAACT TCAGC CACTT -3'), or that by Folmer et al. (1994, 5'-GGTCAA CAAAT CATAA AGATA TTGG -3', 5'-TAAAC TTCAG GGTGA CCAAA AAATC A-3'). The COI fragments were amplified using the cycle protocol as in Zhao et al. (2009).
All sequences generated determined in this study were submitted to BOLD (The Barcode of Life Data system) and GenBank (Table 1). A total of 44 COI sequences of the brunnea group were examined and aligned in MEGA 7.0 (Kumar et al. 2016). Then the inter-and intraspecific genetic distances were calculated for the species of the brunnea group using the p-distance model in MEGA 7.0. A NJ (Neighbor-joining) tree was constructed in MEGA 7.0 with p-distances.
In addition, we also conducted a character-based species delimitation. In the sequence alignment, sites being fixed within the focal species but differing from the remaining species were manually selected as diagnostic sites (i.e. "pure" diagnostics; Sarkar et al. 2002, Desalle et al. 2005 for each species. In this analysis, S. latifasciaeformis Duda, 1940 (Gen-Bank accession number: GU597448) and S. dorsocentralis Okada, 1965 (GU597447), S. puncticeps Okada, 1956 (KJ841770, KJ841771) were used as the outgroups.

Description of species
A Mshot Camera was used to microphotograph all the photographs, illustrations and line drawings were processed with the software Adobe Photoshop 7.0 and Easy Paint Tool 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 44 COI sequences spanned 664 nucleotide sites in length, with 202 variable sites, among which 177 were parsimony informative. The inter-and intraspecific p-distances between species of the brunnea group are given in Table 2. In most cases, the intraspecific p-distances in the brunnea group were less than 1%, while the largest intraspecific p-distance in the brunnea group was found in S. melanogaster sp. n. (= 2.7%). The interspecific p-distance ranged from 3.3% to 13.0%, while the smallest interspecific one was found between S. maculata sp. n. and S. parabrunnea sp. n.
The NJ tree was shown in Fig. 1. In this tree, each morphologically recognized species was strongly supported [bootstraps percentage (BP) = 99 or 100, apart from S. parabrunnea with single specimen), and they formed a monophyletic group with respect to the outgroups (BP = 56). Fig. 2 shows nucleotides at the sites where "pure" diagnostics for any species of the brunnea group in this study. Except S. maculata sp. n., at least one diagnostic site was recognized for each species. For example, the site 124 is diagnostic for S. rhina sp. n.: this site has a fixed nucleotide status of C (Cytosine) in this species, but T (Thymidine) in the other species.
Description. Male and female: Head (Figs 3-7A, E): eyes red to brownish red. Ocellar triangle yellowish brown to brown, mostly with 3 pairs of setae above ocellar setae. Frons nearly 1/3 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 brown. Clypeus mostly yellowish brown to brown. Palpus usually yellowish brown. Vibrissa prominent; subvibrissal setae small. Gena and postgena narrow. Thorax (Figs 3-7B, C, F, G): mesonotum yellowish brown to brown, usually with longitudinal stripe(s). Postpronotal lobe mostly yellowish, with 2-3 long setae and a few of shorter setae. Acrostichal setulae mostly in ca. 8-10 irregular rows. Prescutellar setae usually weak. Pleura mostly brown to dark brown. One small proepisternal seta. Katepisternal with three large setae and some small medially. Scutellum yellowish brown to brown, dark around basal scutellar setae, paler at tip. Wing hyaline, sometimes infuscate. Basal medial-cubital crossvein absent. R 4+5 nearly parallel with M 1 distally. Halter mostly white. Legs mostly yellowish brown.
In the following individual species descriptions, only characters that depart from the above universal characters are provided for brevity. Thorax (Fig. 3B, C): mesonotum yellowish brown, with a brown longitudinal stripe medially. Acrostichal setulae in ca. 8-10 irregular rows. Scutellum yellow, dark brown near basal scutellar setae, pale at tip. Pleura brownish.
Abdomen (Fig. 4H): tergites II to V yellow with dark brown caudal bands, the caudal bands on tergites II and III narrowed medially; tergite VI dark brown.
Measurements Etymology. From the Latin word "maculatus" (= spotted), referring to the mesonotum with dark patch.
Distribution. China (Yunnan). Diagnosis. This species is similar to S. rhina sp. n. in the male terminalia, but can be distinguished from the latter by having the paramere expanded and not divided distally in lateral view (Fig. 12D), the aedeagus distally protruded ventrally in lateral view (Fig. 12D), the mesonotum yellowish brown, with four brown longitudinal stripes sublaterally (Fig. 5B); see under that species.
Abdomen (Fig. 5D): tergites II to V brown with dark brown caudal bands, the caudal band on tergite II interrupted medially; tergite VI brown.
Distribution. China (Yunnan). Diagnosis. This species is similar to S. nigripecta sp. n. in the shape of the paramere and the pattern on the mesonotum (Fig. 13C, D), but can be distinguished from the latter by having the mesonotum mostly yellow (Fig. 5G), and the aedeagus slender and rod-like (Fig. 13C, D); see under that species.
Abdomen (Fig. 5H): all tergites yellowish brown with dark brown caudal bands, the caudal bands on tergites II and III narrowed medially.
Measurements   , alt. 760 m, 22.iv.2007, 9.v.2012 Diagnosis. This species is similar to S. protenipenis sp. n. in the aedeagus with pubescence (Fig. 14D), but can be distinguished from the latter by having the paramere apically divided into two triangular lobes in lateral view (Fig. 14D); aedeagus with a cluster of pubescence on small apical part in lateral view (Fig. 14D); see under that species.
Abdomen (Fig. 6D): all tergites brownish with dark brown caudal bands, the caudal bands on tergites II and III narrowed medially.
Measurements Etymology. A combination of the Latin words: "niger" + "pectus", referring to the black thorax.
Distribution. China (Yunnan). Diagnosis. This species differs from the other known species of this group in having the paramere with a hook-shaped projection basoventrally (Fig. 15D), and the aedeagus apically acute in lateral view (Fig. 15D).
Measurements Etymology. From the Latin word "obscurata" (= dark), referring to the thorax dark.
Abdomen (Fig. 7D): tergites II to V brownish with dark brown caudal bands, the caudal bands on tergite II narrowed dorsomedially; tergite VI brownish.
Distribution. China (Yunnan).  Diagnosis. This species differs from the other species of the brunnea group in the mesonotum being yellowish brown, lacking a longitudinal stripe (Fig. 7F), the pleura being yellowish brown (Fig. 7G), and the paramere distally divided in lateral view (Fig. 17D).
Abdomen (Fig. 7H): tergites II to V yellow, with dark brown caudal bands on tergites III to V, the caudal bands on tergite III and IV interrupted medially; tergite VI dark brown to black.
Measurements Etymology. From the Greek words: "rhnios", referring to the facial carina large and prominent.

Discussion
The specimens identified as S. pressobrunnea and S. scutellimargo putatively in this study mostly match the original descriptions, especially in the male terminalia described and illustrated by Tsacas and Chassagnard (1976) and Duda (1924), respectively, while differences are found in the color patterns on mesonotum (lacking longitudinal stripes) in Tsacas and Chassagnard (1976) and Duda (1924). Actually, color patterns of mesonotum can varied intraspecifically in the family Drosophilidae. Similar cases had been reported in Leucophenga piscifoliacea Huang &Chen, 2013 andL. rectifoliacea Huang &Chen, 2013. Thus, specimens of S. pressobrunnea and S. scutellimargo putative in this study were recognized as the known species. The integration of morphological and DNA-based approaches has revealed an effective way to improve accuracy for species identification (Dayrat 2005;Lumleyand Sperling 2010;Padial and De La Riva 2010). In the present study, we try to use the molecular data to text the putative, morpho-species. Each of the new species S. melanogaster sp. n., S. nigricostata sp. n., S. nigripecta sp. n., S. obscurata sp. n., S. protenipenis sp. n. and S. rhina sp. n. is supported as monophyletic in the NJ tree, and their maximum intraspecific distances are lower than the minimum interspecific distances. In addition, "simple pure characters" are all successfully found in these putative species. Thus, the validity of these seven new species described in the present study was confirmed by the DNA data and morphological research.
It is noteworthy that no "simple pure character" is found for S. maculata sp. n. in the character-based analyses, and the smallest interspecific distance in the brunnea group is detected between S. maculata sp. n. and S. parabrunnea (3.3%), which is above the 3% (or 2%) sequence divergence threshold (Hebert et al. 2003a(Hebert et al. , b, 2004. Actually, the "simple pure character" are not a perfect fix in some case, especially for species in the brunnea group with extremely similar COI haplotypes, or in cases hybridization and introgression will influence the success of mitochondrial identification methods, which had been observed in turtles of the genus Graptemys (Reid et al. 2011), as species often lacked identifying characters simply because of the lack of available variation in COI. Although S. maculata sp. n. is morphologically similar to S. parabrunnea, they can be distinguished easily by the shape of facial carina (Figs 3A, 4E), paramere and aedeagus (Figs 8D, 11D). In the phylogenetic analyses, the NJ tree recovered them as distinct clades (Fig. 1). Therefore, S. maculata sp. n. putative was designated as new species. Dayrat (2005) has previously proposed the use of different sources of evidence in taxonomic practice (i.e. geography, ecology, reproductive isolation, phylogeography, comparative morphology, population genetics, development, behavior), which is now called 'integrative taxonomy'. In fact, wide overlap between intra-and interspecific distances (0-15.5%) has been repeatedly observed in Diptera (Meier et al. 2006), indicating the necessity of using additional marker(s), and incorporating other sources of information (e.g., geographical and ecological) in species discrimination in this order (Huang et al. 2013).