Simulium tenebrosum

Adler, Peter H., Srisuka, Wichai, Low, Van Lun, Takaoka, Hiroyuki & Saeung, Atiporn, 2019, High-Elevation Chromosomal Diversity of Black Flies (Diptera: Simuliidae) in Thailand, Insect Systematics and Diversity 3 (2019), No. 1, pp. 1-10 : 3

Simulium tenebrosum

The banding patterns of all 107 chromosomally prepared larvae were analyzed completely, including two males infected with a microsporidium resembling Amblyospora varians (Léger) sensu Vávra and Undeen (1981) (Microsporidia: Amblyosporidae ) and

one female infected with an unidentified mermithid nematode ( Mermithidae ). Homologs were tightly paired (98–100%). All larvae had the standard chromosomal banding sequence for the Southeast Asian S. tuberosum lineage (sensu Adler et al. 2016c) and were also fixed for IIIL-8, IIIL-11, and IIIL-13 ( Fig. 3 ). All larvae additionally had either of two mutually exclusive fixed-inversion sequences in the IIIL arm: IIIL-64 or IIIL-96 ( Fig. 3 ). The complete absence of individuals heterozygous for either of these inversions indicated the presence of two separate breeding populations (i.e., species): 57.9% (35 females and 27 males) with IIIL-64 and 42.1% (24 females and 21 males) with IIIL-96 ( Table 2). A presorted subsample of 33 final-instar larvae yielded 82% correct assignment to one or the other chromosomal sequences, with IIIL-64 representing the largest larvae (94% correct, n = 18) and IIIL-96 the smallest (67% correct, n = 15). Larvae of intermediate size carried either IIIL-64 or IIIL-96, indicating considerable size overlap ; only the extreme sizes agreed reasonably well with the two chromosomal sequences.

A small subterminal inversion, IIIL -10 ( Fig. 3D ), was distributed nonrandomly among larvae ( Table 3). Most of the 27 males (90.9%) with the IIIL-64 sequence carried IIIL -10 heterozygously. The remaining 9.1% of males with IIIL-64, plus all males with IIIL-96 and all females, lacked the inversion. IIIL -10, therefore, marked a differentiated Y chromosome (Y 1) in males with IIIL-64 ; the remainder (9.1%) of the males with IIIL-64 were, thus, Y 0. IIIL- 64 females (n = 35) were either standard (X 0, 77.1%) for section 100 of IIIL or had heteroband 100hb1 (X 1, 22.9%). Two males were heterozygous for IIIL -10 and IIIL 100 hb1, but on different homologs, and were, therefore, X 1 Y 1. All individuals with IIIL-96 had cytologically undifferentiated sex chromosomes (X 0 Y 0) .

Autosomal polymorphisms were limited to one inversion (IS-33, Fig. 2A ), six different secondary nucleolar organizers ( Figs. 3C and 4 ), three of which were in a single female larva, and two different heterobands ( Fig. 3A, C, E ); all polymorphisms had frequencies less than or equal to 0.02 ( Table 2). Heteroband 100hb1, which was X-linked in individuals with IIIL-64, was expressed heterozygously as an autosomal polymorphism ( Fig. 3C and E ) in two female larvae with IIIL-96 at an overall frequency of 0.02.

We assigned informal handles to the two groups of S. tenebrosum : cytospecies A for individuals fixed for IIIL-64 and cytospecies B for individuals fixed for IIIL-96.

Figures

Fig. 3. Diagnostic sequences of chromosome IIIL for Simulium tenebrosum s. l. from Doi Inthanon,Thailand. (A, C, E) Simulium tenebrosum cytospecies A; female larvae. (B, D) Simulium tenebrosum cytospecies B. (A) Centromere and adjacent region of chromosome III; C, centromere; hb, heterozygous expression of heteroband 82; N.O., primary nucleolar organizer. (B) IIIL (female larva), showing fixed inversions IIIL-1, IIIL-8, IIIL-11, IIIL-13, IIIL-64 sequence with breakpoints indicated by arrows and brackets; C, centromere; cs, cup, and saucer marker. (C) IIIL end with heterozygous expression of secondary nucleolar organizer (2°N.O.96) and heteroband (hb); location of 2°N.O.94 indicated by arrow;inversion IIIL-96 indicated by bracket; IIIL-11 and IIIL-13 are present but not indicated. (D) IIIL end with heterozygous expression ofY-linked IIIL-10 (X0Y1) indicated by dashed bracket; inversion IIIL-64 indicated by bracket; IIIL-11 and IIIL-13 are present but not indicated. (E) IIIL, showing fixed inversions IIIL-1, IIIL-8, IIIL-11, IIIL-13, IIIL-96 sequence with breakpoints indicated by arrows and brackets; C, centromere; cs, cup, and saucer marker; N.O. primary nucleolar organizer.

Fig. 2. Species-specific chromosomal sequences of species from Doi Inthanon,Thailand. (A) IS base of Simulium tenebrosum cytospecies B (female larva), with bracket indicating limits of IS-33; C, centromere. (B) IIIL end of S. kiewmaepanense (male larva), showing IIIL-1 sequence (bracketed). (C) IIL distal half of S. undecimum (male larva) with bracket indicating limits of IIL-1 and enhanced, glassy band (+). (D) IIS of S. kiewmaepanense (male larva) showing IIS-1, IIS-2, IIS-3 sequence.The sequence involves two coincident breakpoints.Thus, for the hypothesis shown, inverting IIS-3 will create the e/g junction representing the IIS-1 break proximal to the centromere; when IIS-2 is then inverted, it creates the h/f junction representing the IIS-1 break distal to the centromere.The Simulium subgeneric standard sequence is, thus, obtained by alphabetically ordering the fragments indicated by the letters a–h; bu, bulge; C, centromere; RoB, ring of Balbiani.

Fig. 4. Expression and locations of secondary nucleolar organizer (2°N.O.) in Simulium tenebrosum cytospecies A from Doi Inthanon,Thailand; female larvae. (A) IIIS distal to the primary nucleolar organizer (N.O.); arrow indicates location of 2°N.O.; bl, blister; ca, capsule. (B) IIS from centromere (C) to ring of Balbiani (RoB); arrow indicates location of 2°N.O. (C) IS end with heterozygous expression of 2°N.O. (D) IL end; arrow indicates location of 2°N.O.; n, neck.