Dimorphic male scutal patterns and upper-eye facets of Simulium mirum n. sp. (Diptera: Simuliidae) from Malaysia
© Takaoka et al. 2016
Received: 13 November 2015
Accepted: 18 February 2016
Published: 9 March 2016
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.
The black flies (Diptera: Simuliidae) in Sabah and Sarawak, Malaysia, in the northern part of Borneo, were recently reviewed [1–3]. 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 [3–7]. 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 , has been treated as Simulium sp. (sensu Smart & Clifford, 1969) . This species is herein described as new.
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 . 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 (iNtRON 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.  for COI, Simon et al.  for COII, Kocher et al.  and Simon et al.  for 12S rRNA, and Xiong & Kocher  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 ChromasPro Version 1.7.6 (Technelysium Pty Ltd., Australia). All sequences were preliminarily aligned using CLUSTAL X and edited using BioEdit 126.96.36.199 . 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  implemented in PAUP 4.0b10 . 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 . 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 two-parameter model of substitution (K2P distance) evolution model. Maximum likelihood (ML) analysis was performed with GTR substitution model using PhyML 3.0 . 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 . Specimens used for the description are listed in Type Material.
Ranges of intra- and inter-specific pairwise genetic distances based on concatenated gene sequences of COI (630 bp), COII (614 bp), 12S rRNA (434 bp) and 16S rRNA (508 bp) genes
1. S. mirum n. sp.
2. S. crassimanum
3. S. laterale
4. S. maklarini
5. S. nigripilosum
Description of new species
Simulium mirum Takaoka, Sofian-Azirun & Low
Simulium (Simulium) laterale (not Edwards): Smart & Clifford , pages 22–26 (Female,
male, pupa and larva)
Simulium (Simulium) sp.: Takaoka et al. , page 28.
Female (n = 10)
Male (n = 8 for form A, n = 7 for form B)
Pupa (n = 25)
Mature larva (n = 7)
HOLOTYPE: Female, reared from a pupa collected from a small stream (width 0.2 m, depth 2–3 cm, water temperature 16.4 °C, shaded, altitude 1728 m, 06°01.397’N, 116°32.611’E), slow flowing in a natural forest, Mt. Kinabalu, Sabah, 18-VI-2014, by M. Sofian-Azirun, Z. Ya’cob, C.D. Chen & K.W. Lau. PARATYPES: Three males (form A), one male (form B), all reared from pupae, and one pupal exuviae, and seven mature larvae, same data as holotype; one female, one male (form A), both reared from pupae, and one pharate female, collected from a small stream (width 3.5 m, depth10-12 cm, water temperature 17.5 °C, shaded, altitude 1591 m, 06°00.665’N, 116°32.399’E), moderate to fast flowing in a natural forest, Mt. Kinabalu, Sabah, 18-VI-2014, by M. Sofian-Azirun, Z. Ya’cob, C.D. Chen & K.W. Lau; one male (form A), reared from pupa collected from a small stream (width 6.0 m, depth 0.5 m, water temperature 17.0 °C, partially shaded, altitude 1714 m, 06°01.337’N, 116°36.420’E), moderate to fast flowing in a natural forest, Mesilau, Kundasang, Sabah, 18-VI-2014, by M. Sofian-Azirun, Z. Ya’cob, C.D. Chen & K.W. Lau; eight females, three males (form A), six males (form B), two intersexes (one with female head, though frons much narrower, female abdomen, male wings, male legs, and thorax of intermediate sex with indefinite scutal color pattern; the other with female head, though frons much narrower, female wings, male legs, male abdomen except female terminalia with abnormal structures between ovipositor valves and paraprocts, and thorax of intermediate sex with indefinite scutal color pattern), all reared from pupae collected from a main channel of a stream (width 3.0-4.0 m, stream bed rocky, water temperature 23.0 °C, partially shaded), moderately flowing in a pasture, Bakalalan, Sarawak, Malaysia (altitude 928 m, 03°50.624’N, 115°36.312’E, 21-VIII-2008), by H. Takaoka.
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.
The pupae of this species were collected attached to plastic sheets and dead leaves of trees in the water. Associated species were S. (N.) borneoense Takaoka, S. (S.) beludense Takaoka, S. (S.) laterale and S. (S.) keningauense Takaoka.
Borneo (Sabah, Sarawak and Kalimantan).
The species name mirum refers to the extraordinary finding that this new species has dimorphic males. The Latin word ‘mirus’ means ‘extraordinary’.
To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:27FDFE51-721B-488C-BAAF-010822CDD626. The LSID for the new name Simulium mirum is urn:lsid:zoobank.org:act:E8B9D049-317B-4F8F-9235-53222BC3A172.
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 [24–26]. 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 ) 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 . 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 ).
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, Ya’cob & Sofian-Azirun by the length ratio of the second antennal segment against the first (1.15–1.19) (c.f., 0.95–0.98 in S. cheedhangi). The larvae of five other related species are unknown.
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.
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.
We are grateful to Prof. P.H. Adler, Clemson University, Clemson, USA, for his kindness in reading the current manuscript and providing valuable comments. We express our thankfulness to The Board of Trustees of Sabah Parks for providing the permit to conduct research and collect specimens in Sabah Parks. Thanks are due to Mr. J. Guntabid (Park Manager), Mr. S. Dunsul (Research Collaborator), Mr. Martin (Park Ranger), Mr. H. Minel (Park Guide), the Kinabalu Parks; Mr. M. Rasul and Mr. N.A. Jamil, University of Malaya; Mr. K. Fernandez, Mrs. M. Mahali, and Mr. E. Silin, Universiti Malaysia Sabah, for their assistance in the field. Our sincere appreciation goes to Dr. C. Leh (Sarawak Museum) and Prof. Emeritus I. Miyagi (Ryukyu University), for their kind invitation and arrangement to conduct the surveys in Bakalalan. This work was supported by the research grants from University of Malaya (RP003A/13SUS & CG072/2013) and Universiti Malaysia Sabah (SBK-0107-STWN-2013).
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