Chromosomal relationships of Simulium armoricanum and its undescribed sister species in the Simulium vernum species group (Diptera: Simuliidae) Author Adler, Peter H. Author Belqat, Boutaïna Author González, Josefina Garrido Author Pérez, Alexandre Justo Author Seitz, Gunther text Zootaxa 2016 4137 2 211 222 journal article 10.11646/zootaxa.4137.2.3 7d88638e-5a4f-48f8-a2eb-8375b402c1f6 1175-5326 267215 85DEC9CE-5DF9-4E56-94FB-83BDDB982941 Simulium armoricanum The banding sequences of all 56 chromosome preparations ( n = 16 males , 40 females ) of S. armoricanum from Sites 1–9 ( Table 1 ) were analyzed completely. Chromosomal complement. The nucleolar organizer was in the standard location in the base of IS (section 19B; Figs. 2 , 3 ), a chromocenter was absent, homologues were tightly paired, and B chromosomes and heterobands were not found. Simulium armoricanum was removed from the standard sequence for the S. vernum group by the following 11 fixed inversions and 1 nearly fixed inversion: IL-2 , IL-8 , IL-9 (frequency = 0.98; Fig. 3 ), IIL-8 + 4 additional but unnumbered inversions ( Fig. 4 ), IIIP-1 , IIIS-8 , IIIS-9 ( Fig. 5 ), and IIIL-1cy , ca . All but IL-2 , IL-8 , IIIP-1 , and IIIL-1cy , ca are unique to S. armoricanum . The IL arm carried two new inversions: a large, central inversion, IL-8 , which reversed the polarity of the Z marker, and the nearly fixed IL-9, on top of the widely shared IL-2 inversion. A portion of section 41 in most larvae (though not all; Fig. 3 B) was puffed as a white, diffusely banded area ( Fig. 3 A), a trait found in numerous species in the S. vernum group ( Brockhouse 1985 , fig. 5; Hunter & Connolly 1986 ). The IIL arm was complexly rearranged, and five inversions were required to reassemble the bands into the standard sequence ( Fig. 4 ). Various breakpoint scenarios are possible for four of the inversions; hence, we do not number those inversions. Chromosome III included the pericentric inversion IIIP-1 ( Fig. 5 ). The centromeric region of chromosome III was unique among members of the S. vernum group, consisting of a well-defined centromere band within a slightly expanded area, bounded on either side by a diffuse area of heterochromatin delimited distally by a thin, darkly staining band ( Fig. 5 ). IIIS had two unique overlapping inversions, with one coincident breakpoint ( Fig. 5 ). Two sequences were found in the IIIL arm ( Table 2 ), relative to the standard sequence of Brockhouse (1985) : IIIL-1cy,ca (= p sequence) and IIIL-2,(1’)cy,ca (= v sequence) of Hunter (1987) . Establishing the fixed IIIL sequence for S. armoricanum is complicated by the uncertainty of whether IIIL-1cy,ca and IIIL-1’cy,ca are different inversions, as suggested by Hunter (1987) ; the similarity of terminal bands in the inversions renders the decision problematic. The most parsimonious explanation is that IIIL-1cy,ca and IIIL-1’cy,ca are the same inversion, and that it is fixed in S. armoricanum , rendering IIIL- 2cy,ca ( Fig. 5 ) polymorphic. FIGURE 2. IS arm; N.O. = nucleolar organizer. A , Simulium cryophilum (female larva from Vindrarp, Sweden), showing standard sequence, with breakpoints indicated (arrows) for IS-1am,2am. Alphabetical reassembly of fragments identified by the letters a–h will produce the inverted sequence for IS-1am,2am. B , Simulium armoricanum (male larva, Site 9) double heterozygote for IS-1am,2am. FIGURE 3. IL arm of Simulium armoricanum , with fixed inversions IL-2 and IL-8 and the nearly fixed IL-9. A , male larva (Site 9), showing puff in section 41; CI = centromere, Z = Z band marker. B , male larva (Site 3), showing details of the distal third of the arm and absence of puffing in section 41. FIGURE 4. IIL arm of Simulium armoricanum (male larva, Site 3) fixed for inversion IIL-8 and 4 other inversions with breaks indicated by arrows. The standard sequence can be obtained by alphabetically ordering the small letters a–n, corresponding with each fragment. CII = centromere, j = jagged marker, Pb = parabalbiani, pf = puffing band. FIGURE 5. IIIS arm of Simulium armoricanum (female larva, Site 2) fixed for inversions IIIS-8 , IIIS-9 , IIIP-1 , and with the proximal portion of IIIL-2cy,ca. CIII = centromere, bl = blister marker, bm = basal marker, ca = capsule. TABLE 2. Distribution of the IIIL-2cy,ca polymorphism in larvae of Simulium armoricanum ; site numbers correspond to locations in Table 1.

Site	Gender	pp1	pv	vv
1	Male Female	0 0	0 2	0 4
2	Male Female	1 1	1 5	2 13
3	Male Female	0 0	0 0	2 0
5	Male Female	0 0	0 0	1 1
6	Male Female	0 0	0 1	0 3
7	Male Female	0 0	1 1	0 0
8	Male Female	2 1	2 3	2 4
9	Male Female	0 0	1 0	1 1

1 p = p sequence of Hunter (1987) = IIIL-1cy,ca; v = v sequence of Hunter (1987) = IIIL-2,(1’)cy,ca. IIIL-1cy , ca = IIIL- 1’cy , ca is regarded here as the fixed sequence, rendering IIIL-2cy,ca polymorphic. Sex chromosomes. Inversions IS-1am and IS-2am were universally linked to one another, never having been found independently. In the heterozygous condition, they formed a characteristic, complex loop ( Fig. 2 ). At Site 2, three of four males were IS-1am,2am heterozygotes and one was an inverted homozygote, whereas the 19 females were predominantly (68.4%) standard homozygotes ( Table 3 ). Although we acknowledge that sample sizes are small, a test for independence of IS-1am,2am and sex is only marginally significant at England Site 2 (G-test: G = 3.822, p = 0.05, df = 1), and not significant at Spain Site 8 (G = 0.020, p> 0.05, df = 1). Given the marginal significance of sex linkage in England , we refrain from recognizing IS-1am,2am as Y linked and tentatively regard all populations as having undifferentiated sex chromosomes (X0Y0). We suspect that IS-1am,2am is equivalent to the complex Y chromosome mentioned by Bass & Brockhouse (1990) in their sample of unstated provenance from England . If so, different populations in England might represent a spectrum of Y linkage for IS-1am,2am. Simulium armoricanum would be the only member of the group, among sufficiently analyzed species, with cytologically differentiated sex chromosomes based on the IS arm. TABLE 3. Distribution of linked chromosomal inversions IS-1am,2am with regard to gender in larvae of Simulium armoricanum ; site numbers correspond to locations in Table 1.

Site	Gender	ss 1	si	ii
1	Male Female	0 5	0 1	0 0
2	Male Female	0 13	3 5	1 1
3	Male Female	0 0	2 0	0 0
5	Male Female	0 0	1 0	0 1
6	Male Female	0 1	0 3	0 0
7	Male Female	0 1	0 0	1 0
8	Male Female	0 1	4 4	2 3
9	Male Female	0 0	1 0	1 1

1 s = standard banding sequence, i = inverted banding sequence for IS-1am,2am. Autosomal polymorphisms. In addition to IS-1am,2am and IIIL-2cy,ca, the latter with an overall frequency of 0.76 for the inverted constituent ( Table 2 ), we found IL-9 heterozygously in 2 females , one at Site 6 and one at Site 9. The same female at Site 9 also had a small inversion in IIIL (sections 85c–86a inclusive on the map of Brockhouse 1985 ). Taxonomic status. Other than the texture of its silken cocoon, S. armoricanum is morphologically no more differentiated than any of the other chromosomally studied species ( Bass 1998 ), but it is the most chromosomally differentiated of the 14 fully resolved nominal European members of the S. vernum group in terms of the number of fixed inversions (11) removed from the standard. No ecological or morphological evidence previously had suggested that S. armoricanum is a complex of species; its ecological requirements and morphology, as far as they are known, are tightly defined ( Bass 1998 ). Until evidence to the contrary comes to light, we regard S. armoricanum as a single cytological entity over a significant portion of its geographic range spanning more than 1200 linear km from southern England to Portugal , with the type locality encompassed by this range. Some hedging is necessary, however, because the characterization by Bass & Brockhouse (1990) of S. armoricanum from an unspecified location in England suggests that perhaps a different species is involved; their analysis indicates that IIIS is standard, rather than fixed for two overlapping inversions, which, however, produce a sequence superficially similar to the standard sequence. Bionomics. Larvae were collected from cool streams 2 m or less in width, with closed or open canopies. In England , the streams had open canopies and were in the heart of the moorland. In Spain , the streams were 10.9–11.6 ºC, with pH of 5.9–7.1 and conductivity of 5.9–78.5 µS/cm. The streams (≤ 0.5 m wide) in Portugal had open canopies characteristic of the high granitic mountains, with substrates of sand and stone and, at Site 6, some macrophytes. Larvae of S. armoricanum were collected in England with those of the S. cryophilum complex, S. intermedium Roubaud , and the S. tuberosum (Lundström) complex; in Portugal with S. aureum Fries sensu stricto— recorded and confirmed chromosomally ( 1 female , classic sequence, with no polymorphisms) for the first time from Portugal (Site 3)—the S. cryophilum complex, S. vernum Macquart sensu stricto , and S. intermedium ; and in Spain with the S. cryophilum complex, S. vernum sensu stricto , and the S. ornatum Meigen group.