Tests of conspecificity for allopatric vectors: Simulium nodosum and Simulium shirakii (Diptera: Simuliidae) in Asia

Allopatric populations present challenges for biologists working with vectors. We suggest that conspecificity can be concluded in these cases when data from four character sets—chromosomal, ecological, molecular, and morphological—express variation no greater between the allopatric populations than between corresponding sympatric populations. We use this approach to test the conspecificity of Simulium nodosum Puri on the mainland of Southeast Asia and Simulium shirakii Kono & Takahasi in Taiwan. The validity of these two putative species has long been disputed given that they are morphologically indistinguishable. The mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rRNA, and 16S rRNA genes and the nuclear-encoded 28S rRNA gene support the conspecific status of S. nodosum from Myanmar, Thailand, and Vietnam and S. shirakii from Taiwan; 0 to 0.19 % genetic differences between the two taxa suggest intraspecific polymorphism. The banding patterns of the polytene chromosomes of the insular Taiwanese population of S. shirakii and mainland populations of S. nodosum are congruent. The overlapping ranges of habitat characteristics and hosts of S. nodosum and S. shirakii corroborate the chromosomal, molecular, and morphological data. Four independent sources of evidence (chromosomes, DNA, ecology, and morphology) support the conspecificity of S. nodosum and S. shirakii. We, therefore, synonymize S. shirakii with S. nodosum. This study provides a guide for applying the procedure of testing conspecificity to other sets of allopatric vectors.


Background
Simuliid vectors of human and animal pathogens typically are complexes of evolutionarily and ecologically distinct cryptic species, for which accurate identification is an essential first step in the epidemiological understanding and control of arthropod-borne diseases [1][2][3][4]. Compounding the challenge of recognizing cryptic species is the difficulty of evaluating the species status of allopatric populations. The problem is particularly acute when populations are widely disjunct on the mainland, between mainlands, or between an island and the mainland [5].
Assigning the same or different names to allopatric populations has consequences for understanding vector potential and developing control strategies [6].
A classic example of the challenge of evaluating species status for allopatric populations involves Simulium nodosum Puri, distributed from India across southern China, Myanmar, Thailand, and Vietnam [7], and the morphologically similar Simulium shirakii Kono & Takahasi from Taiwan, more than 130 km from the Chinese mainland. Although S. shirakii has been treated for 80 years as a separate species, its species status has long been questioned because of morphological similarity with S. nodosum [8]. Simulium nodosum is mammalophilic, attacking humans and bovids [9][10][11]. It is also a vector of the agents of filariasis to ruminants [10].
Although allopatry confounds the evaluation of reproductive isolation, we suggest that allopatric populations, including putative species, can be considered conspecific when differences in their molecular sequences, chromosomal profiles, morphology, and ecology are within the range of variation for a geographically cohesive, panmictic set of populations. All four character sources require evaluation, given the existence of homosequential sibling species [12] and the failure of up to three of the character sets to discriminate reproductively isolated sympatric species [13].
We applied this test to two members of the Simulium nobile species group, S. nodosum and S. shirakii. The detailed morphological comparisons by Takaoka & Suzuki [8] between S. nodosum and S. shirakii originally suggested conspecificity and prompted the present study. We used a multi-locus phylogenetic analysis of one nuclear and three mitochondrial genes, a comparative analysis of the polytene chromosomes, and an evaluation of ecological data associated with our collections and in the literature [9,[14][15][16].

Methods
No national permissions were required for this study, which did not involve endangered or protected species. No specific permissions were required to access the study sites; the collections were made on public lands.
Larvae were collected by hand into ethanol from five sites in Myanmar, Taiwan, Thailand, and Vietnam (Table 1). Additional samples of larvae from Taiwan were collected into 1:3 acetic ethanol for chromosomal comparison with published information [15]. Habitat characteristics at each collection site were recorded, including altitude, canopy cover, and stream depth, temperature, and width. Species identifications were performed using illustrated taxonomic keys [8,14,[17][18][19].
Genomic DNA was extracted from each of five specimens per location, using the i-genomic CTB DNA Extraction Mini Kit (iNtRON Biotechnology Inc., Seongnam, South Korea). Amplifications of the mitochondriaencoded COI, 12S rRNA, and 16S rRNA, and nuclearencoded 28S rRNA genes were undertaken in a final volume of 50 μL containing 50-100 ng genomic DNA, 25 μL of ExPrime Taq Master Mix (GENETBIO Inc., Daejeon, South Korea), and 10 pmol of each forward and reverse primer. The primers used in this study were adopted from Folmer et al. [24] for COI, Kocher et al. [25] and Simon et al. [26] for 12S rRNA, Xiong & Kocher [20] for 16S rRNA, and Low et al. [27] for 28S rRNA.
We used the Feulgen technique and chromosomal slide-mounting procedures outlined by Adler et al. [32] to prepare the polytene chromosomes of 51 larvae from two sites (19 larvae from Guangfu, 32 larvae from Ruisui) in Taiwan (Table 1). Larval carcasses were deposited in the Clemson University Arthropod Collection. The chromosomes of all 51 larvae (25 females, 26 males) were compared band-for-band with the standard map for the subgenus Simulium [33,34] and with the chromosomes of larvae analyzed by Tangkawanit et al. [15] from nine sites in Thailand.
No phylogenetic tree from the concatenated dataset nor the single-locus analyses separated S. nodosum from S. shirakii. Both putative species were distributed randomly in the trees (Additional file 1: Figures S1-S4). The pairwise genetic distance analysis revealed intraspecific variation ranging from 0 to 0.23 % for S. nodosum from Vietnam, Myanmar, and Thailand; 0 to 0.08 % for S. shirakii from Taiwan; and 0 to 0.19 % for S. nodosum/S. shirakii ( Table 2). The interspecific variation between S. nodosum/S. shirakii and S. nobile, and between S. nodosum/S. shirakii and S. timorense, ranged from 5.10 to 5.45 % and 5.18 to 5.45 %, respectively. Thus, the level of polymorphism (0-0.19 %) between S. nodosum and S. shirakii was less than that between all pairs of mainland populations and within the Myanmar population (0.04-0.23 %) and far below that of S. nobile or S. timorense.
Chromosomal banding patterns of all Taiwanese larvae were four fixed inversions (IS-1, IIL-a, IIIL-b, IIIL-2) removed from the Simulium subgeneric standard sequence, and matched the fixed banding sequence of all 247 Thai larvae studied by Tangkawanit et al. [14]. Taiwanese and Thai populations had the nucleolar organizer in the end of IIS, undifferentiated sex chromosomes, and only two autosomal polymorphisms each, one of which (IIIL-1) was shared. The frequency of IIIL-1 was 0.99-nearly fixed-in Taiwan, and an average of 0.28 in Thai populations analyzed by Tangkawanit et al. [15]. The chromosome map of Tangkawanit et al. [15] for IIIL, however, shows the homozygous sequence for the IIIL-1 inversion rather than the claimed standard sequence; in addition, the distal breakpoint is shown as extended by one band beyond the actual breakpoint. IIL-1 was an infrequent inversion (average frequency = 0.04) in Thailand [15] and absent in Taiwan. IL-1 was a new, but rare (0.01) inversion, with breakpoints before the last bands in each of sections 39 and 40, in one male larva in Taiwan. Ectopic pairing of centromeres formed a loose pseudochromocenter in populations in Thailand [15] but was present in only about 1 % of nuclei per larva in Taiwan.
Habitat characteristics for our collection sites of S. nodosum and S. shirakii, such as stream width, overlapped broadly. Altitude, however, was considerably greater for our mainland populations (733-1439 m) than for our Taiwanese populations (15-70 m) ( Table 1).

Discussion
All four data sources-molecular, chromosomal, morphological, and ecological-independently support the conspecificity of S. nodosum and S. shirakii across a longitudinal range of 2365 km, providing a powerful consensus that S. shirakii and mainland populations in Myanmar, Thailand, and Vietnam are a single species. Our multi-locus analysis demonstrates that genetic differences within mainland populations of S. nodosum are greater than the differences between S. nodosum collectively and S. shirakii.
Simulium nodosum from Thailand and S. shirakii from Taiwan are identical in all details of their fixed chromosomal inversions and sex chromosomes, and share one of three autosomal inversions, the other two being rare. Inversion IIIL-1, which is found in about one-third of all homologues of Thai larvae and is nearly fixed in Taiwanese larvae, possibly expresses clinal variation, with lowest polymorphism in the Taiwan population, a common characteristic of insular and peripheral populations [32].
Habitat characteristics that typically differ between closely related species, especially stream size [35], are broadly overlapping among populations from Taiwan and the mainland, in agreement with records from the literature [14,15]. Known hosts, which can differ between closely related species [36], are consistent (bovids and humans) across the distribution [9,11,14]. Altitude, however, which can be associated with genetic isolation [37], differs between our populations in Taiwan (15-70 m) and on the mainland (733-1439 m). The altitudinal distinction, however, narrows or disappears when we draw from published information [14], suggesting that some of the discrepancy is attributable to sampling artifact. Elevation for S. nodosum in Thailand, for instance, ranged from 168 to 800 m [15]. The available ecological data suggest that a   broad range of habitat conditions is typical for S. nodosum and S. shirakii. Insular populations, in particular, are more likely to have a broader ecological niche, resulting in part from ecological release [38]. The molecular separation of S. nodosum and S. shirakii from S. nobile and S. timorense is in concordance with their morphological characters. The simple claws of the females and the number and form of the pupal gill filaments (three inflated horn-like tubes) separate S. nodosum/S. shirakii from other members of the S. nobile species group [8].