Descriptions of two new species of the genus Colocasiomyia (Diptera, Drosophilidae) breeding on Rhaphidophora host plants in Yunnan, China

Abstract The genus Colocasiomyia de Meijere (Diptera, Drosophilidae) is known to include 30 described and nearly 60 undescribed species classified into six species groups. Among these, the C. gigantea group of seven known species (two Southeast Asian and five Chinese) proved to be peculiar for its specificity on monsteroid (subfamily Monsteroideae, family Araceae) host plants. In this paper, two new species, C. todai Jiao & Gao, sp. nov. and C. liae Jiao & Gao, sp. nov., are described as members of the C. gigantea group with specimens collected from inflorescences of the monsteroid host species Rhaphidophora peepla (Roxb.) Schott and R. crassicaulis Engl. & Krause, respectively, in Yunnan, China. The two new species are delimitated, in comparison with all known species, based on not only morphological but also DNA barcode (partial sequence of the mitochondrial COI, i.e., cytochrome c oxydase subunit I, gene) data. A revised key to all the nine species of the C. gigantea species group is provided.


Introduction
The genus Colocasiomyia de Meijere, 1914 is among a few well known anthophilic genera in the family Drosophilidae (Brncic 1983;Grimaldi et al. 2003;Fu et al. 2016). Species in this genus (30 described and nearly 60 undescribed ones) are all discovered from tropical and subtropical regions of the Old World, and taxonomically classified into six species groups: i.e., the C. crassipes group of two (2 described + 0 undescribed) species associated with hosts from the family Magnoliaceae, the C. zeylanica group of six (2 + 4) species associated with hosts from the family Arecaceae, three species groups [C. toshiokai group of six (6 + 0) species, C. baechlii group of thirty (2 + 28) species, and C. cristata group of thirty three (11 + 22) species] associated with aroid hosts from the subfamily Aroideae, and the C. gigantea group of seven known species associated with aroid hosts from the subfamily Monsteroideae (e.g., Sultana et al. 2006;Fartyal et al. 2013;Li et al. 2014;Shi et al. 2019). The C. gigantea group was erected by Fartyal et al. (2013) for three species: C. gigantea (Okada) using Epipremnum pinnatum in Java, Indonesia and Solomon Is., C. rhaphidophorae Gao & Toda using Rhaphidophora hookeri in Yunnan, southwestern China, and C. scindapsae Fartyal & Toda using Scindapsus coriaceus in Sabah, Malaysia. Li et al. (2014) subsequently described four additional species, i.e., C. longifilamentata Li & Gao, C. longivalva Li & Gao, C. hailini Li & Gao and C. yini Li & Gao from western Yunnan with specimens collected from inflorescences of Rhaphidophora decursiva (Roxb.) Schott. Here we add two new, Chinese species, i.e., C. todai Jiao & Gao, sp. nov. and C. liae Jiao & Gao, sp. nov., to the C. gigantea group, with specimens collected from inflorescences of Rhaphidophora peepla (Roxb.) Schott and R. crassicaulis Engl. & Krause, respectively, in Yunnan (Fig. 1).

Specimens and morphological observation
Colocasiomyia specimens were collected in western (Baoshan) and southern (Lüchun) Yunnan using an insect net (for adults) or by dissecting host inflorescences or infructescences (for eggs or dormant larvae within egg capsules on host infructescences), and immediately preserved in 70% (for morphological observation) or 100% (for DNA sequencing) ethanol (Table 1).
We observed external morphology and detailed structures of dissected organs following the methods in Fartyal et al. (2013) and Li et al. (2014). For species illustration, we used a DinoLite Digital Eyepiece Camera to photo graph the entire body, the wing, foreleg, and male/female genitalia for representative specimens. We followed McAlpine (1981) for the morphological terminology and Zhang and Toda (1992) for the definitions of measurements and indices. The type specimens are deposited in Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China (KIZ).

Species delimitation
Adult Colocasiomyia specimens, either newly obtained or collected previously, were first identified and sorted into species of the C. gigantea group in light of morphology. DNA barcodes (i.e., the 658-bp barcoding region of the mitochondrial cytochrome c oxydase I, COI, gene) were determined for representing specimens (adults, eggs, or larvae) of each morpho-species (Table 1). We followed the methods of Li et al. (2014) for DNA extraction, PCR amplification and DNA sequencing, using Folmer et al.'s (1994) primer pair LCO1490 (5'-GGTCA ACAAA TCATA AAGAT ATTGG -3') and HCO2198 (5'-TAAAC TTCAG GGTGA CCAAA AAATC A -3'). Sequences obtained were edited in the SeqMan module of the DNAStar package version 7.1.0 (DNAStar Inc., Madison, WI). The newly determined sequences were then aligned with 45 previously determined barcodes of the C. gigantea group (Table 1) using the software MEGA7 (Kumar et al. 2016). Neighbor-joining (NJ) trees were constructed in MEGA7 with the sequence alignment based on p-distances and also Kimura 2-parameter (K2P) divergences for comparison, with node supports (bootstrap percentages) calculated with 1000 replicates. The intra-and interspecific p-and K2P-distances were calculated for all the species in MEGA7, and then the barcoding "gap" (Meyer and Paulay 2005) was evaluated with intra-and interspecific p-distances, following the methods in Meier et al. (2006Meier et al. ( , 2008.

DNA barcoding
A total of 60 adult specimens of the C. gigantea group were morphologically sorted into five species, including three known (C. longifilamentata, C. longivalva, and C. yini) and two new ones (C. todai sp. nov. and C. liae sp. nov.) (Table 1). COI barcodes were determined for all 60 adults and 15 immature specimens (12 of C. hailini, 3 of C. longifilamentata) ( Table 1). The alignment of the 75 newly determined barcodes (GenBank accession numbers: MT916851-MT916925) and the 45 previously determined ones spans 658 (494 conserved, 164 variable including 154 parsimonyinformative) nucleotide sites. The two NJ trees based on p-and K2P-distances showed the identical topology, but slightly differed in terms of BP: the tree based on p-distances yielded overall higher BPs ( Fig. 2; but the K2P-distance tree not shown), confirming Srivathsan and Meier's (2012) Figure 2. Unrooted neighbor-joining tree of the C. gigantea species group built based on p-distances between COI sequences. Label of each operational taxonomic unit (OTU) is given in the form of "voucher number-GenBank accession number". Numbers beside nodes are bootstrap percentages (shown when ≥ 50; BP based on p-distance/BP based on K2P-distance). observation that p-distance performs better than K2P-distance in NJ-tree construction for DNA barcoding. The sequence clusters corresponding to the morpho-species are all compact and supported with high BPs (≥ 80 in both of the p-and K2P-distance methods). The grouping of two small-bodied species, C. hailini and C. yini, was strongly supported (BP = 99 in both methods). In addition, the sister-relationship between C. rhaphidophorae and C. longifilamentata was strongly supported (BP = 99 and 96, respectively). Table 2 shows the intra-and interspecific p-distances in the C. gigantea group. The observed maximal intraspecific distance (0.0130 in C. longifilamentata) was the same as the minimal interspecific one (0.0130 between C. longifilamentata and C. rhaphidophorae). There was a gap (size = 0.0117 p-distance) between the overall mean intraspecific (0.0013) and the minimal interspecific p-distances, while by deleting the 5% largest intraspecific and the 5% smallest interspecific distances, a wider gap (size = 0.0491 p-distance) was observed.
Female terminalia (Fig. 4F): Tergite VII mid-dorsally not constricted; VIII pubescent nearly entirely, with 3 setae in a vertical row on discolored, posteroventral portion. Oviscapt with distal, narrow elongation in addition to proximal, broad portion; proximal portion with large patch of dense, distinct warts; distal portion apically more or less truncated, with ca 3−4, 7−8 and 6 trichoid ovisensilla per side on basal 2/5 of dorsal margin, entire ventral margin, and at apex, respectively, but lacking any peg-like ovisensillum.  Remarks. Li et al. (2014) described C. longifilamentata, C. hailini, C. yini and C. longivalva with specimens collected exclusively from inflorescences of R. decursiva at Baihualing, Baoshan, Western Yunnan, but mentioned that very few adults of the last two species, especially C. longivalva, were collected from inflorescences of R. decursiva. Our subsequent field work there has revealed that C. longifilamentata and C. hailini, rarely together with C. yini, share inflorescences/infructescences of R. decursiva as their breeding resources but that C. longivalva does not breed on this plant at all (data not shown). Recently, we have found that C. longivalva, together with C. todai sp. nov., use R. peepla as a host plant: adults of both species were abundantly collected from inflorescences of this plant (Table 1); and a large number of Colocasiomyia 1 st -instar larvae were found overwintering within egg capsules between growing pistils of infructescences, and they were later identified as C. longivalva or C. todai sp. nov. by DNA barcoding or by examining the morphology of adults obtained from rearing the 1 st -instars (data not shown). Diagnosis. This species closely resembles C. rhaphidophorae in external morphology and structure of male and female genitalia, but can be distinguished from the latter by epandrial ventral lobe rod-like, distally slightly broadened in lateral view, apically inlaid with a relatively long, claw-like peg (Fig. 5A, B); aedeagus broader in distal half in ventral view (Fig. 5D); surstylus apically expanded, with 1 dorsosubapical, 2 ventrosubapical, minute sensilla in addition to three small setulae at apex (Fig. 5A); distal, narrow elongation of oviscapt somewhat sinuate in lateral view (Fig. 5F).
Female terminalia (Fig. 5F): Tergite VII mid-dorsally not constricted; VIII pubescent nearly entirely, with 3 setae in a vertical row on discolored, posteroventral portion. Oviscapt with distal, narrow elongation in addition to proximal, broad portion; proximal portion with large patch of dense, distinct warts; distal, narrow portion, with ca 2, 8 and 5 trichoid ovisensilla per side on basal 1/3 of dorsal margin, entire ventral margin, and at apex, respectively, and a tiny, peg-like ovisensillum near subapical, dorsal margin.