Dimorphic male scutal patterns and upper-eye facets of Simulium mirum n. sp. (Diptera: Simuliidae) from Malaysia

A species of Simulium in the Simulium melanopus species-group of the subgenus Simulium (formerly misidentified as S. laterale Edwards from Sabah and Sarawak, Malaysia) is suspected to have dimorphic male scutal color patterns linked with different numbers of upper-eye facets. This study aimed to confirm whether or not these two forms of adult males represent a single species. DNA sequences generated from four genetic loci, the mitochondrial-encoded COI, COII, 12S rRNA and 16S rRNA genes, of both forms of Simulium sp. males were compared with each other and also with those of the females and larvae of the same species. Four other related Simulium spp. were also used for comparison. Both the concatenated dataset and single-locus phylogenetic analyses indicate that the two distinct morphological males of Simulium sp. are indeed conspecific, and represent, together with their associated females and larvae, a distinct species. Based on DNA analyses, Simulium sp. is proven to show dimorphism in males and is herein described as a new species, Simulium mirum Takaoka, Sofian-Azirun & Low. This is the first report of such a novel species among the family Simuliidae.


Background
The black flies (Diptera: Simuliidae) in Sabah and Sarawak, Malaysia, in the northern part of Borneo, were recently reviewed [1][2][3]. So far, 38 species have been recorded from Sabah and Sarawak, all of which are classified in three subgenera of the genus Simulium: 18 species in Gomphostilbia, three species in Nevermannia and 17 species in Simulium [3][4][5][6][7]. Biting habits of females and other biological aspects of these East Malaysian black fly species remain unknown, except for one female of S. nigripilosum Edwards, which was caught by a hand net while flying around a human along the trail to the peak of Mt. Kinabalu, 2 km up from the Carson Waterfall, on September 10, 2007.
In a revision of eight species of the Simulium melanopus species-group of the subgenus Simulium from Sabah, one species that was misidentified as Simulium laterale Edwards by Smart & Clifford [8], has been treated as Simulium sp. (sensu Smart & Clifford, 1969) [3]. This species is herein described as new.
This new species was suspected of being dimorphic in the males. Form A males have a white pruinose scutum with a medial vitta and lateral, round, nonpruinose spots ( Fig. 1b) and upper-eye facets in 12-14 vertical columns and 15 or 16 horizontal rows. Form B males have a white pruinose scutum with an inverted-T-shaped, large, medial, nonpruinose portion (Fig. 1c) and upper-eye facets in 16 or 17 vertical columns and 17 or 18 horizontal rows [3]. To test this hypothesis, we adopted four genetic markers most commonly used for black fly phylogeny and systematics [9][10][11]. Genetic evidence that supports the conspecificity of the two forms of the males of this new species is provided, and a description of Simulium sp. as a new species is given based on females, males, pupae and larvae.

Taxa used for DNA analyses
To determine whether form A and form B males of Simulium sp. comprised one species, males of both forms together with females (all reared from pupae) and larvae collected from Mt. Kinabalu, Sabah, and Bakalalan, Sarawak, Malaysia, were used for DNA analyses. Other related species used for comparison were S. crassimanum Edwards, S. laterale, S. nigripilosum and S. maklarini Takaoka, all Sabah members of the S. melanopus species-group [3]. For 16S rRNA, sequences of S. bishopi Takaoka & Davies (AB093105) from Peninsular Malaysia, S. dumogaense Takaoka & Roberts (AB093106) and S. tumpaense Takaoka & Roberts (AB093110) from Indonesia, S. melanopus Edwards (AB093108) and S. taalense Takaoka (AB093109) from the Philippines available from the NCBI GenBank database were also included in phylogenetic analyses.

DNA isolation, polymerase chain reaction (PCR) and DNA sequencing
The genomic DNA was isolated from each individual, using the i-genomic CTB DNA Extraction Mini Kit (iN-tRON Biotechnology Inc., Seongnam, South Korea). The DNA amplifications by PCR were conducted using an Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems Inc., Foster City, CA, USA). Amplifications of the mitochondrial-encoded COI, COII, 12S rRNA and 16S rRNA genes were performed in a reaction mixture containing 50-100 ng of genomic DNA, 25 μL of NEXpro e PCR 2x Master Mix (Genes Labs Inc., Gyeonggi-do, South Korea), and 10 pmol of each forward and reverse primer. The primers used in this study were adopted from Folmer et al. [12] for COI, Simon et al. [13] for COII, Kocher et al. [14] and Simon et al. [13] for 12S rRNA, and Xiong & Kocher [15] for 16S rRNA. The PCR products were sequenced in both directions using the Big Dye Terminator v3.1 kit and run on an ABI 3730XL Genetic Analyzer (Applied Biosystems Inc.).

DNA sequence analyses
Sequences were assembled and edited using Chroma-sPro Version 1.7.6 (Technelysium Pty Ltd., Australia). All sequences were preliminarily aligned using CLUS-TAL X and edited using BioEdit 7.0.9.0 [16]. The COI, COII, 12S rRNA and 16S rRNA sequences generated in this study were deposited in the NCBI GenBank database under accession numbers KT207386-KT207457.
Congruence between separate genes was tested using the partition homogeneity test [17] implemented in PAUP 4.0b10 [18]. The results showed no significant differences among separate gene regions (P = 0.459); hence, data were concatenated for further analyses. The aligned sequences of single genes and the concatenated dataset were subjected to Bayesian inference (BI) analysis using four chains of Markov chain Monte Carlo (MCMC) implemented in MrBayes 3.1.2 [19]. Neighbour-joining (NJ) and maximum parsimony (MP) analyses were performed using PAUP 4.0b10. The MP tree was constructed using the heuristic search option, 100 random sequences additions, tree bisection reconnection (TBR) branch swapping, and unordered and unweighted characters. The NJ tree was estimated using Kimura's twoparameter model of substitution (K2P distance) evolution model. Maximum likelihood (ML) analysis was performed with GTR substitution model using PhyML 3.0 [20]. In this study, S. (Simulium) nobile de Meijere and S. (Gomphostilbia) leparense Takaoka, Sofian-Azirun & Ya'cob were chosen as outgroups for the construction of phylogenetic trees. Uncorrected (p) pairwise genetic distances among species were estimated using PAUP 4.0B10.

Description of a new species
The methods of description and illustration, and terms for morphological features used here, follow those of Takaoka [21]. Specimens used for the description are listed in Type Material.

DNA analyses
The concatenated COI + COII + 12S rRNA + 16S rRNA phylogenetic tree revealed two main genetic clades. One well-supported clade comprised Simulium sp. from Sabah and Sarawak, with its respective sub-clades according to populations. The other well-supported clade comprised other members of the S. melanopus speciesgroup in Malaysia: S. crassimanum, S. laterale, S. nigripilosum and S. maklarini (Fig. 2).

Description of new species
Simulium mirum Takaoka Labrum 0.62 times as long as clypeus. Antenna composed of scape, pedicel and nine flagellomeres, brownish black except scape, pedicel and first flagellomere dark yellow when viewed ventrally, or scape, basal one-third of pedicel dark yellow, and apical two-thirds of pedicel and first flagellomere medium brown when viewed posterodorsally; flagellomeres 1-8 each with pit-like depressions of irregular shape, bearing numerous sensilla, near apical margin on outer and inner surface (though flagellomere 8 with such depression only on outer surface) (Fig. 3a). Maxillary palp composed of five segments, proportional lengths of third, fourth, and fifth segments 1.00: 1.09: 2.23; sensory vesicle ( Fig. 3b) Fig. 3f ) slightly depressed medially, covered with 18-31 long stout and short to medium-long fine hairs on each side. Ovipositor valves ( Fig. 3f, g) bent ventrally, making right angle to sternite 8, each tapered apically with transparent bare round apex, membranous except narrow area along inner margin well sclerotized, and covered with 39-46 short to long hairs; inner margins well sclerotized, moderately concave medially. Genital fork (Fig. 3h) of inverted-Y form; arms slender, each with strongly-sclerotized ridge directed dorsally. Paraproct in lateral view ( Fig. 3i) much produced ventrally, covered with seven or eight medium-long to long stout hairs and about 30-36 short fine hairs on lateral surface; paraproct anteromedially with thin elongate moderatelysclerotized plate having round apical tip and 14-16 short setae scattered on its surface (Fig. 3j). Cercus in lateral view ( Fig. 3i) short, with posterior margin nearly straight, 4.6 times as wide as its greatest length, and covered with numerous hairs. Spermatheca (Fig. 3k) globular, nearly as long as wide, well sclerotized except duct and area of junction with duct unsclerotized, without reticulate surface patterns; minute internal setae present.

Male (n = 8 for form A, n = 7 for form B)
Morphological characters are the same in form A and form B except the number of upper-eye facets and the scutal color patterns. Body length 2.5-3.5 mm. Head (Fig. 1b, Fig. 1b and Fig. 4b) [brilliantly iridescent with inverted-T-like median portion nonpruinose in form B ( Fig. 1c and Fig. 4c)], and moderately covered with dark short hairs and upright longer hairs on prescutellar area. Scutellum and postnotum as in female. Legs. Brownish black to black except fore coxa, basal two-thirds of mid basitarsus, little more than basal one-third of hind basitarsus (though base darkened) and basal one-third of second hind tarsomere yellow; brilliantly iridescent widely on outer surface of fore tibia and on posterolateral surface of mid tibia, and narrowly on posterior surface of base of hind tibia, when illuminated at certain angles. Fore basitarsus with thick dorsal hair crest and greatly dilated, 4.33 times as long as its greatest width. Hind basitarsus (Fig. 4d) greatly enlarged, gradually widened toward apical one-fourth, then slightly narrowed toward apex, 3.95 times as long as its greatest width, 0.93 and 1.07 times as wide as greatest widths of hind tibia and femur, respectively; calcipala nearly as long as its basal width, and 0.30 times as wide as greatest width of basitarsus; pedisulcus well developed. Wing. Length 2.0-2.4 mm; other features as in female except basal portion of radial vein and subcosta entirely bare. Abdomen. Basal scale dark brown with fringe of dark long hairs. Dorsal surface of abdomen dark brown to brownish black, moderately covered with dark hairs, with pair of brilliantly iridescent dorsolateral spots on segments 2 and 4-7, of which those on segment 2 widely connected medially to each other, and those on other segments narrowly connected anteromedially to each other; abdomen also with pair of shiny lateral portions on segments 8 and 9 when illuminated at certain angles. Genitalia. Coxites, styles and ventral plate in ventral view as in Fig. 4e. Coxite in ventrolateral view (Fig. 4f ) nearly quadrate, 0.75 times as long as wide. Style in ventrolateral view (Fig. 4g) elongate, 1.84 times length of coxite, 2.19 times as long as its greatest width, widened from base to basal one-fourth, narrowed to little less than basal three-fifths, then nearly parallel-sided toward round apex, and with stout subapical spine; style in medial view (Fig. 4h) flattened dorsoventrally, gradually narrowed toward apex, and without basal or subbasal protuberance. Ventral plate in ventral view (Fig. 4e) with body nearly rectangular having ventrally produced median process that is bare except parts of lateral and anterior surfaces densely covered with minute setae, furnished with several teeth in vertical row on each posterolateral margin; basal arms short, stout and divergent; ventral plate in lateral view ( Fig. 4i) with body and its ventrally produced process with rounded ventral apex, with dentate posterior margin, and densely covered with minute setae anteromedially and anterolaterally; ventral plate in caudal view (Fig. 4j) with body and ventrally produced median process slightly narrowed from base to basal one-fourth, widened to basal one-third, then tapered apically, bare, with several teeth in vertical row on each lateral margin. Median sclerite in lateral view (Fig. 4i) folded backward and then curved dorsally, and in posterior view (Fig. 4k) plate-like, somewhat pigmented except apicomedial portion unpigmented, nearly parallel-sided except basal portion tapered toward base. Paramere (Fig. 4l) wide basally and with several small parameral hooks apically. Aedeagal membrane (Fig. 4m) densely covered with minute setae, with short sclerotized dorsal plate (Fig. 4n). Ventral surface of tenth abdominal segment (Fig. 4o, p) without distinct hairs. Cercus (Fig. 4o, p) rounded posteriorly, with 11 or 12 distinct hairs.
Head. Integument yellowish, bare except area surrounding facial trichomes (Fig. 5a), and each lateral surface moderately covered with minute tubercles; frons with two unbranched short trichomes (Fig. 5b) on each side; face with one unbranched short trichome (Fig. 5c) on each side; frontal trichomes shorter than facial ones. Thorax. Integument yellowish, bare widely on dorsal and dorsolateral surfaces of anterior half, densely covered with round tubercles on ventrolateral surface on anterior half, and sparsely or moderately covered with cone-shaped smaller tubercles on dorsal surface of posterior half; thorax with two medium-long anterodorsal trichomes (Fig. 5d), two medium-long anterolateral trichomes (Fig. 5e), one medium-long mediolateral trichome (Fig. 5f ), and three ventrolateral trichomes (two medium-long, one short) (Fig. 5g), on each side; all unbranched. Gill (Fig. 5h) with six slender thread-like filaments in pairs arising from short common basal stalk; all pairs almost sessile; filaments decreasing in length and thickness from dorsal to ventral; upper filament of dorsal pair longest (1.4 mm long) and lower filament of ventral pair shortest (0.7 mm long); relative thickness of filaments from dorsal to ventral when basal portions were compared 1.00: 0.88: 0.81: 0.75: 0.69: 0.69; each filament light to medium brown, nearly parallel-sided from base to middle, then gradually tapered toward apex; upper filament of dorsal pair and lower filament of ventral pairs at angle of 90°or little more when basal portions viewed laterally; cuticular surface with marked annular ridges forming reticulate patterns (Fig. 5i), covered with minute tubercles, of which relatively larger ones on ridges and smaller ones on inter-ridge spaces.
Abdomen. Dorsally, segment 1 yellow, other segments unpigmented; segment 1 without minute tubercles (though submedial areas moderately covered with tubercles in two pupae) and with one unbranched short slender seta (Fig. 5j) on each side; segment 2 with one unbranched short slender seta and five minute setae, of which three are stout and pigmented, two are slender and unpigmented (Fig. 5k), on each side; segments 3 and 4 each with four unbranched hooked spines and one minute slender seta on each side; segment 8 with spinecombs; segments 6-9 each with comb-like groups of minute spines in transverse row on each side; segment 9 without terminal hooks. Ventrally, all segments unpigmented; segment 5 with pair of bifid hooklets submedially and few minute setae on each side; segments 6 and 7 each with pair of bifid inner and outer hooklets and few minute setae on each side; segments 4-8 each with comb-like groups of minute spines on each side. Cocoon (Fig. 5l, m). Shoe-shaped, with anterior collar of moderate height, thickly woven, ochreous, not extended ventrolaterally; individual threads invisible; 3.2-3.7 mm long by 1.2-1.5 mm wide.

Mature larva (n = 7)
Body length 4.8-5.2 mm. Body whitish gray with abdominal segments 6-9 faintly to moderately overlaid with ochreous to reddish brown pigments (and abdominal segments 1-5 faintly overlaid with reddish brown pigment in two larvae). Abdomen becoming slightly wider from segment 1 to segment 6, widest between segments 6 and 7. Cephalic apotome whitish-yellow on anterior half and yellow on posterior half except narrow portion along posterior margin somewhat darkened medially; head spots obscure but consisting of positive and negative spots: anteromedial and posteromedial spots and posterior spots of mediolateral spots faintly positive, and anterior spots of mediolateral spots and posterolateral spots faintly negative. Lateral surface of head capsule yellow except eye-spot region whitish, and area between eye-spot region and posterior margin somewhat darkened. Ventral surface of head capsule (Fig. 6a) yellow except elongate spot and posterior portion of each side of postgenal cleft darkened. Antenna composed of three segments and apical sensillum, slightly longer than stem of labral fan; proportional lengths of first, second, and third segments 1.00: 1.15-1.19: 0.65-0.73. Labral fan with 40 or 41 primary rays. Mandible (Fig. 6b) with mandibular serrations composed of two teeth; major and longer tooth at obtuse angle to mandible on apical side; comb-teeth decreasing in length from first to third. Hypostoma (Fig. 6c) with nine anterior teeth, median and corner teeth most prominent; lateral margins moderately serrate apically; six hypostomal bristles lying divergent posteriorly from lateral border on each side. Postgenal cleft (Fig. 6a

Depository of type-specimens
The holotype and paratypes of the new species are deposited in the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Ecological notes
The pupae of this species were collected attached to plastic sheets and dead leaves of trees in the water. Associated species were S.

Etymology
The species name mirum refers to the extraordinary finding that this new species has dimorphic males. The Latin word 'mirus' means 'extraordinary'.

ZooBank registration
To comply with the regulations set out in article 8.

Discussion
This new species is striking in that there are two forms of males, which are reared from pupae with apparently the same external morphology: form A with a scutal pattern as in Fig. 1b and Fig. 4b linked with a smaller number of upper-eye facets in 12-14 vertical columns and 15 or 16 horizontal rows, and form B with a scutal pattern as in Fig. 1c and Fig. 4c linked with a larger number of upper-eye facets in 16 or 17 vertical columns and 17 or 18 horizontal rows. Beside these two different characters, both forms of males have the same morphological characters including the genitalia. The pupae of these two forms of males are morphologically indistinguishable from each other, and usually coexist in the same habitats, strongly suggesting a single species with dimorphism in the male scutal color pattern linked with a different number of upper-eye facets. Our DNA analyses show that both forms of males are conspecific, although they can be genetically differentiated by locations, a common observation in black flies [22,23]. Further, our genetic data clearly show that S. mirum n. sp. is distinguished from four known members of the S. melanopus species-group.
In the family Simuliidae, genetically-inherited dimorphism in one sex, like the present new species, has not been known though sexual dimorphism is common. Simulium (Notolepria) gonzalezi Vargas & diaz Najera from Belize, Ecuador, Guatemala and Mexico was reported to show dimorphism in the males in that some males are dichoptic with a narrow frons and more elongated antennal segments similar to those of the female, whereas most males are holoptic [24][25][26]. Since dichoptic heads with eyes medially separated by a frons are a typical feminine expression (e.g., Fig. 1a), all or some of these males of S. (N.) gonzalezi with dichoptic heads might be sexual mosaics, a phenomenon (mainly caused by mermithid infections [27]) occasionally observed in various groups of black flies.
The cause of the dimorphism shown in the males of S. mirum n. sp. is unknown. Unlike the case of S. gonzalezi, there seems to be the least possibility, if any, that it has been caused by the intersexuality. It is because the heads of both form A and form B males are holoptic (a typical expression of the male heads, i.e., the left and right eyes medially contiguous, not separated by the frons) (Fig. 1b,  c), though the numbers of the enlarged upper-eye facets are different between form A and form B, thus having nothing to do with the expression of the feminine head morphology. In general, the abnormalities resulted from the intersexuality are mostly expressed longitudinally (e.g., the head feminine, the thorax and abdomen masculine, or vice versa), or asymmetrically in paired features (e.g., the left side of the body feminine, the right side masculine, or vice versa), or intermediate (e.g., two intersex specimens in the type-material).
In species of the S. melanopus species-group, the male scutal color pattern is usually monomorphic but rarely polymorphic, as shown in Figure 183A-E of S. dumogaense Takaoka & Roberts from Sulawesi [21]. However, unlike this new species, no linkage of these different scutal color patterns with the number of upper-eye facets or other characters has been reported.
Dimorphism in the males of S. mirum n. sp. may have originated before this new species had spread in Sabah and Sarawak because it was observed in at least two locations (Timpohon and Mesilau) in Sabah and three locations (Bakalalan, Bario and Pueh) in Sarawak (HT, unpublished data). Further studies are needed to determine the frequency of occurrence of each male form in each population and the underlying chromosomal and genetic mechanisms of this phenomenon.
By the unique scutal color pattern ( Fig. 1b and Fig. 4b), form A males of this new species are distinguished from six of eight species from Sabah and Sarawak, for which the males are known, i.e., S. crassimanum, S. lardizabalae Takaoka & Sofian-Azirun, S. laterale, and S. murudense Takaoka, Ya'cob & Sofian-Azirun, S. nigripilosum and S. timpohonense Takaoka & Sofian-Azirun. Form B males are similar to those of S. timpohonense in having the yellow fore coxa and ordinary scutal color pattern ( Fig. 1c and Fig. 4c), but are distinguished from the latter species by the entirely darkened hind tibia.
The female of S. mirum n. sp. is similar to those of S. maklarini and S. cheedhangi Takaoka, Ya'cob & Sofian-Azirun in having the yellow fore coxa, but is distinguished from them by the haired basal portion of the radial vein and entirely darkened hind tibia.
The pupa of this new species is similar to that of S. maklarini in having a similar arrangement of the gill filaments and similar shoe-shaped cocoon but it is distinguished by having the medial area surrounding the facial trichomes moderately covered with tubercles (Fig. 5a) and by sharply edged annular ridges of the gill filaments (Fig. 5i) (c.f., tubercles are absent on the face, and the annular ridges of the gill filaments are not sharply edged in S. maklarini [28]).
The mature larva of this new species is distinguished from S. laterale by the shorter body length 4.5-5.0 mm and posterior circlet with 84-90 rows of hooks (c.f., the body length 6-7 mm, and the posterior circlet with 115-170 rows of hooks in the latter species), and from S. cheedhangi Takaoka

Conclusions
Based on DNA analyses, Simulium sp. is proven to show dimorphism in the male scutal color patterns linked with different numbers of upper-eye facets. This species is described as a new species. This is the first report of such a novel species with dimorphic males among the family Simuliidae.

Ethics statement
All experiments were performed in accordance with relevant guidelines and regulations of the University of Malaya. The research protocols were regulated and approved by the University of Malaya. Prior to the commencement of the sample collections, permission was approved by The Board of Trustees of Sabah Parks, Malaysia (Reference Number: TS/PTD/5/4 Jld. 49:52). This study did not involve endangered or protected species.

Additional file
Additional file 1: Figure S1 Figure S3. Maximum likelihood phylogenetic tree of the Simulium melanopus species-group from East Malaysia based on 12S