A new species group in the genus Dichaetophora, with descriptions of six new species from the Oriental region (Diptera, Drosophilidae)

Abstract The genus Dichaetophora Duda comprises 61 described species classified into four species groups: agbo, tenuicauda, acutissima and sinensis. This genus is distributed exclusively in the Old World, and is rich in species in the tropical and subtropical areas of the Oriental, Australasian, and Afrotropical regions. In this paper, a new species group, the trilobita group, is established for six new species discovered from the Oriental region. The delimitation of these species is firstly performed in light of morphology and further with the aid of DNA sequences of the mitochondrial COI and COII (cytochrome c oxydase, subunits I and II, respectively) genes, considering also their respective geographical origins. Then, the new species (trilobita Yang & Gao, sp. n., heterochroma Yang & Gao, sp. n., flatosternata Yang & Gao, sp. n., borneoensis Yang & Gao, sp. n., javaensis Yang & Gao, sp. n., and sumatraensis Yang & Gao, sp. n.) are described, and a key, based on not only morphological but also molecular information, is provided.


Introduction
The genus Dichaetophora is widely distributed in the Old World, especially its tropical and subtropical regions. This genus was originally established by Duda (1940) as a subgenus in the genus Drosophila Fallén, for the Seychellean species, Drosophila aberrans Lamb. Burla (1954) supplemented three African new species (including an informally named one) into Dichaetophora and revised its diagnostic characters. Since then, the species composition of this subgenus [genus since Grimaldi's (1990) upgrading] had been altered time and again in relation to the genus Nesiodrosophila Wheeler & Takada (see Hu and Toda 2002). Hu and Toda (2002) examined the relationships among the genera Dichaetophora, Nesiodrosophila, the Lordiphosa tenuicauda species group and some presumably related genera by a cladistic analysis of morphological characters. As a result, the revised and enlarged genus Dichaetophora was proposed and subdivided into three species groups, i.e., the agbo, acutissima and tenuicauda groups. Hu and Toda (2005) established the forth (sinensis) species group for four species newly described from China, raising the number of known Dichaetophora species to 61. In the present study, a new species group is established for six new species of Dichaetophora recently discovered from the Oriental region, the trilobita group. The species delimitation is based on not only morphological but also geographical and DNA sequence data. A key to the six species is provided.

Specimens
A summary of the specimens employed in the present study is shown in Table 1. The flies were mostly captured by net sweeping on herbs growing along watersides in open forests or at forest edges. Specimens were preserved in either 70% (after fixing with Kahle's solution for morphological observation) or 100% ethanol (for DNA sequencing).

Species delimitation
The specimens were first identified as of Dichaetophora in light of morphology referring to Hu and Toda's (2005) diagnosis of this genus. Then, they were examined for external morphology, morphometric characters and detailed structures of some dissected organs by the same methods as in Li et al. (2014), and sorted into morpho-species. For each of these morpho-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 a hind-leg (usually the right one) or small piece(s) of abdominal tissue picked from the dissection cut of terminalia, using the TIANamp® Genomic DNA Kit. DNA sequences of the 658-bp barcoding region of the mitochondrial COI (cytochrome c oxydase subunit I) gene were then amplified and sequenced with the Folmer primers (Folmer et al. 1994; Table 2), using the same PCR cycle program as in Li et al. (2014). In addition, we determined the DNA sequences of the whole 688-bp region of the mitochondrial COII (cytochrome c oxidase subunit II) gene, using the primer pair designed by Simon et al. (1994;Table 2), with the same PCR cycle program used in Gao et al. (2007). The sequences obtained were edited in the SeqMan module of the DNAStar package, version 7.1.0 (DNAStar, Inc., Madison, WI), and aligned in MEGA7 (Kumar et al. 2016). We performed a tree-based DNA barcoding with the COI and COII sequences, respectively, with Bayesian trees constructed using MrBayes 3.1 (Ronquist and Huelsenbeck 2003). For this, the sequence alignment of each gene was partitioned into two subsets (codon positions 1 plus 2, and codon position 3), with choice of substitution models justified via model testing (Srivathsana and Meier 2012) in MEGA7 using the Bayesian Information Criterion. In Bayesian inference, sampling frequency was set as every 1000 generations, and numbers of chains = 4. Two analyses were run simultaneously till the average deviation of split frequencies fell well below 0.01. Therefore, in all analyses, full runs of 5,000,000 generations were performed. In each analysis, 1000 early-phase samples were discarded as burn-in for each run, yielding a total of 8,002 trees to construct a 50% majority consensus tree with nodes characterized by posterior probability (PP). We then summarized the information of intra-and interspecific p-distances calculated without data partitioning. The morpho-species were then reconsidered by integrating information from the morphology, the geographical distribution ( Fig. 1) and DNA barcodes.

Descriptions
In species illustration, a DinoLite® Digital Eyepiece Camera was used to microphotograph some organs for representative specimens. McAlpine (1981) was followed for the morphological terminology, and Zhang and Toda (1992) for the definitions of measurements and indices. The examined specimens are deposited in the following institutes:

Species delimitation
The specimens examined were first sorted into four morpho-species (Table 1). We got 23 COI and 26 COII sequences. The GenBank accession numbers are KY809802− KY809824 for the COI, and KY809825−KY809850 for the COII sequences. Table 3 shows the result of model selection. The COI and COII Bayesian trees (both unrooted) are shown in Fig. 2. Each of the morpho-species sp.K1 [from Peninsular Malaysia and Borneo (Sarawak and Sabah)], sp.K2 (Sabah) and sp.K2-like (Xishuangbanna, Yunnan, southwestern China) was strongly suggested to be monophyletic. Sp.K2 and sp.K2-like formed a well-supported clade (PPs = 1.00 in both of the COI and COII trees, respectively). Sp.K1, which is sympatric with sp.K2 in Sabah, formed a clade independent from the clade of sp.K2+sp.K2-like. Thus, these three morpho-species, spp.K1, K2 and K2-like, were recognized as independent species, i.e., trilobita sp. n., heterochroma sp. n. and flatosternata sp. n., respectively. Specimens of the morpho-species sp.K3 clustered into three more or less diverged, allopatric lineages each endemic to Borneo (Sabah), West Java, or Sumatra (Jambi) in the COII tree (PP = 1.00 for each lineage). While the former two lineages were recovered in the COI tree (PPs = 0.99 and 1.00, respectively), the last one was not supported in this tree. Table 4 shows the summary of intra-and interspecific p-distances for the six putative species. The interspecific mean p-distances for COI sequences among these three lineages of sp.K3 varied from 0.0349 (Java vs. Sumatra) to 0.0751 (Borneo vs. Java), coinciding with the smallest interspecifc distance variability of 5.9±4.1% (uncorrected divergence) for COI sequences in Diptera (Meier et al. 2008) and being larger than their intraspecific mean distances ranging from 0.0045 (Java) to 0.0185 (Sumatra). However, these lineages are morphologically very similar, differing from each other in so few morphological characters that it is hard to distinguish between them (see descriptions). On the other hand, comparison of the COI and COII nucleotide sequences among these lineages has revealed that there are fixed, lineagespecific nucleotides at more than one sites, where nucleotides remain unchanged in the other three species (Table 5). Such sites can therefore be used as pure molecular diagnostic characters (Sarkar et al. 2002, DeSalle et al., 2005 for respective lineages. Taking into account their geographically isolated situations as well, we regard these lineages as three independent, cryptic species, i.e., borneoensis sp. n., javaensis sp. n. and sumatraensis sp. n.

Taxonomy
In the following descriptions of the new species group and new species, and also the key to species, some figures in Hu and Toda (2005) are referred to, with their original numbers given in double quotation marks.
The six new species to be described here certainly belong to the genus Dichaetophora, according to its diagnosis revised by Hu and Toda (2002): cibarium only slightly protruded at anterolateral corners; oviscapt with apical ovisensillum robust and largest, distinguishable from the others; basal lobe of palpus without setulae; hypopharyngeal apodeme expanded anteriorly; labellum with less than six pseudotracheae; ocellar setae outside triangle made by ocelli. Within Dichaetophora, they should be related to the  sinensis group, sharing some characters regarded by Hu and Toda (2005) as diagnostic for the latter group: very large ocellar triangle (" Fig. 1"); large number (≥ 40 per side) of medial sensilla on cibarium (" Fig. 6"); ventral surface of prementum forming discrete bump (Fig 4E, J, O, T, Y, D'; "Figs 8-11A"). However, they lack some other diagnostic characters of the sinensis group: foreleg tibia with stout apical seta distinctly thicker than preapical dorsal seta (" Fig. 2"); aedeagus apically with membranous, trumpet-like Table 4. Summary of intra-and interspecific p-distances. Values of p-distance below diagonal, values of standard error above diagonal.  dilation ("Figs 8F,. And, three of them, trilobita sp. n., heterochroma sp. n. and flatosternata sp. n., share a particular character, i.e., 4 pseudotracheae varying in thickness (" Fig. 4"), with the agbo species group (Hu and Toda 2005). Furthermore, all the six new species possess some characters specific to themselves: there are two or three prominent setae on the anteromedial portion of cercus (Figs 5-10B); the cercus is strongly sclerotized along the anterior to caudoventral margin, which seems to be homologous with the strong sclerotization of the caudoventral portion of cercus seen in three species of the sinensis group ("Figs 9-11B"), but the sclerotized plates of cerci are fused with each other caudoventrally and to epandrium anteroventrally (Figs 5-10A,B). Based on these morphological characteristics, we establish a new species group, the trilobita species group, in Dichaetophora, for the six new species.
Common characters. Head (Fig. 4): Eye red, with dense interfacetal setulae; longest axis nearly orthogonal to body axis. Frons, face, gena, occiput, postgena and clypeus glossy black; facial carina blackish brown. Ocellar very large, nearly rectangular, anteriorly reaching to ptilinal fissure; frontal vitta narrow, without interfrontal setulae. Pedicel grayish brown; arista with 6−7 dorsal and 2−3 ventral branches in addition to terminal fork. Subvibrissal seta not differentiated, as small as other orals. Palpus slender, apically with one prominent ventral and one subprominent dorsal setae. Cibarium not thickened on anterior margin, with four anterior sensilla arranged square; dorsal wall pear-shaped, anteriorly somewhat dilated in dorsal view and strongly convex in lateral view; anterior portion of hypopharynx shorter than posterior tubular portion. Prementum with 5-6 (one proximal, one central, 2-3 lateral, and one distal longest) pairs of setae. Labellum with four pseudotracheae per side.
Legs (Fig. 3A, E, H, K, N, Q): Preapical dorsal setae present on all tibiae; foreleg apical seta as thick as preapical dorsal one. Foreleg first tarsomere slightly shorter than total length of four succeeding tarsomeres; all tarsi narrowing distally, with small, apical claws.

Key to the species
In the following key, not only morphological characters but also the selected pure diagnostic nucleotide sites of COI and COII sequences (Table 5) are used to identify the three cryptic species, borneoensis sp. n., javaensis sp. n. and sumatraensis sp. n.

Description of new species
The characters described above for the genus, the species group, and the key are not referred to in the following descriptions. Diagnosis. Postocellar setae absent; wing without distinct cloud (Fig. 3C); sclerotized, caudoventral bridge of cerci with small, narrow, apically pointed, median process and a pair of lateral, broad, apically rounded lobes (Fig. 5A); aedeagus apically curved, hook-like, and finely wrinkled all over, subapically with numerous, coarse serrations, submedially not swollen dorsally (Fig. 5G); spermatheca without distinct introvert (Fig. 5K).
Wings (Fig. 3C): Veins pale brown to brown. Halter pale gray; stem darker. Legs (Fig. 3A) pale grayish yellow; mid-and hindleg femora distally, foreleg tibia and tarsi proximally to medially darker. Apical setae present on all tibiae; hindleg apical seta short, stout. Mid-leg first tarsomere longer than total length of four succeeding tarsomeres; hindleg first tarsomere as long as total length of four succeeding tarsomeres.
Description Wings (Fig. 3G): Veins pale brown to dark brown, but pale within central pale patch. Halter pale gray; stem darker.
Legs (Fig. 3E) pale grayish yellow; foreleg coxa, tibia and tarsus, except for fifth tarsomere, dark brown. Mid-leg first tarsomere longer than total length of four succeeding tarsomeres; hindleg first tarsomere as long as total length of four succeeding tarsomeres.
Description  Wings (Fig. 3J): Veins pale brown to dark brown, but pale within central pale patch. Halter pale gray; stem darker.
Legs (Fig. 3H) pale grayish yellow; foreleg tibia and tarsus, mid-leg femur and distal portion of hindleg femur darker. Hindleg tibia lacking apical seta. Mid-leg first tarsomere longer than total length of four succeeding tarsomeres; hindleg first tarsomere as long as total length of four succeeding tarsomeres.
Legs (Fig. 3K): All femora blackish brown; foreleg coxa, tibia and tarsus (except 5 th tarsomere) grayish brown; rest grayish yellow. Apical setae present on foreleg and mid-leg tibiae. Mid-leg first tarsomere as long as total length of four succeeding tarsomeres; hindleg first tarsomere longer than total length of four succeeding tarsomeres.
Wings (Fig. 3P) slightly fuscous; veins yellowish brown to brown. Halter and stem gray. Legs (Fig. 3N): All femora blackish brown; foreleg coxa, tibia and tarsus (except 5 th tarsomere) grayish brown; rest grayish yellow. Apical setae present on foreleg and midleg tibiae. Mid-leg first tarsomere as long as total length of four succeeding tarsomeres; hindleg first tarsomere longer than total length of four succeeding tarsomeres.
Remarks. The last three species somewhat resemble trilobita sp. n. in having the following morphological characters: wing without distinct, dark cloud; surstylus with medial patch of pubescence on outer surface; sclerotized, caudoventral bridge of cerci with narrow, median process; and oviscapt valve dorsomedially narrowly extended. However, the three species are very hard to distinguish from each other because of their least morphological differentiation. To overcome this difficulty, we employed 19 nucleotide sites of COI and COII genes as molecular diagnostic characters to identify these cryptic species (Table 5). Nucleotide substitutions at two of these sites are nonsynonymous, i.e., causing changes of amino acids, and specific to borneoensis sp. n. (Table 5), thus providing more reliable (less changeable) characters for this species.