Nine cytological segregates in the Simulium damnosum complex (Diptera: Simuliidae).

974 NOTES Nine Cytological Segregates in the Simulium damnosum Complex (Diptera: Simuliidae) * by ROBERT W. DUNBAR, Associate Professor, Department ofZoology, University of Western Ontario, London, Ontario, Canada A preliminary examination of salivary gland chromosomes of Simulium damnosum sent from Uganda revealed 4 distinct cytological categories. Because of the relative closeness of the 3 localities from which the material came and the sympatry of 2 of the forms it was considered in a previous paper a that these categories represent sibling species, and that S. damnosum is a sibling species complex. Reported here are the results of a more detailed re- examination of the material on which the previous paper was based, and a similar examination of about two-thirds of the material received by the present author since 1964. Nine categories are recognized so far; the original 4 (with 2 minor name changes)-namely, Sebwe (formerly Sebwe B), Sanje (formerly Sebwe A), Nile, and Kagera-and additionally, Bandama, Nkusi, Ketaketa, Kulfo, and Kibwezi. It should be recognized that these are not legitimate species names but convenient designations lifted from accompanying collection labels. Cytological categorization of the 9 forms While writing the previous paper a the author realized that there could be errors in the details of each category, but considering the constancy of each category this was not thought to be serious. The re-examination of the original 4 categories has revealed several homologous inversions which were overlooked. However, finding these additional homologous differences has strengthened, not weakened, these categories; but relative positions in the phylogenetic scheme have shifted. Identification of a specimen depends on the identification of specific segments of the banding pattern. In a poor series of chromosome slides it is quite possible that known segments could be misread, or that other homo- zygously inverted segments could be overlooked and the error remain undetected until revealed by a better slide series. * This research was supported by grants from the National Research Council of Canada. a Dunbar, R. W. (1966) Nature (Lond.), 209, 597-599. The forms are described starting with Sanje, the arbitrarily chosen standard and a phylogenetically central form (Fig. 1), then the three phylogenetic lines radiating from it: line 1, Sebwe, Kibwezi; line 2, Nkusi; line 3, Ketaketa, Nile, Kulfo, Ban- dama, Kagera. Only interspecific inversion differ- ences from the previously described form are men- tioned; differences from species described previously are cumulative. The idiograms summarize the inver- sions present (Fig. 2). Collection numbers (A63/1, A67/7 etc.) refer to collections listed in the accom- panying table. For technical details the reader is referred to previous papers.a b The distribution of the forms in Central Africa is shown in Fig. 3. Sanje. Standard banding pattern. Two floating inversions; IIL-1 and IIIS-1. Centric fusion is pre- valent. IIL-1 has been found twice heterozygously with standard; it is homozygous in all other speci- mens (it was previously a reported erroneously that all homozygotes were standard). This form can be subdivided into 2 populations based on the presence or absence of IIIS-1, which is restricted to males, thereby providing a distinct Y chromosome. (a) Northern subpopulation. IIIS-1 absent (S/IIL-1 present). Distribution: Uganda and north- ern Tanzania, 136 specimens; A63/1, A63/2, A65/7, A65/8, A66/18, A66/19, A68/3, A68/5. (b) Southern subpopulation. IIIS-1 present in 26 out of 35 males, thus providing an alternate Y chromosome (S/IIL-1 is absent). Distribution: southern Tanzania, 64 specimens; A66/9, A66/10, A66/1 1. Sebwe. Differs from Sanje by IIIL-I. No floating inversions yet noted. Distribution: western Uganda, 51 specimens; A63/1, A63/2, A68/3, A68/7, A68/8. Kibwezi. Differs from Sebwe by an inversion com- plex in IIL tentatively identified as IIL-15.16. No floating inversions yet noted. Distribution: south- west Kenya, 23 specimens; A67/9. b Dunbar, R. W. (1967) Canad. J. Zool, 45, 377-396. 2350i NINE CYTOLOGICAL SEGREGATES IN SIMULIUM DAMNOSUM COMPLEX FIG. I PHYLOGENETIC RELATIONSHIPS AMONG THE 9 SIBLING SPECIES, WITH INTERSPECIFIC INVERSIONS SHOWN BETWEEN THE SIBLINGS Sebwe IIIL-I IIL-15 16 Kibwezi Nkusi II L-5 Sanje - Bandama Nkusi. Differs from Sanje by IlL-5. No floating inversions yet noted. Centric fusion is present. Distribution: Uganda, southwest Kenya, southern Tanzania, 73 specimens; A65/2 to A65/6, A65/13, A66/1, A66/2, A66/1 1, A66/12, A67/9. Ketaketa. Differs from Sanje by IS-I and IL-3. There are 4 floating inversions: IL-7, once hetero- zygously; IIL-I and IIL-l 1 are linked and were found homozygously 8 times, and 6 times hetero- zygously with IIL-4. The sample produced poor chromosome slides and the identifications of IIL-1 and IIL-4 are tentative. The heterozygote IIL-1i+ IIL-l1/IIL-4 is complex. Some centric fusion is found. Distribution: southern Tanzania, 14 spec- imens; A66/11, A66/12, A67/8. Nile. Differs from Ketaketa by IL-I and IIIL-2. (IS-i and IL-3 were previously overlooked a). There are 10 floating inversions, differences in the I s-I I L-3 IIL-6 III L-4 Nile IL-I III L- 2 Ketaketa II L- 4 kulfo IIIL-3 kagera proportions of which may be used to distinguish 3 subpopulations. (a) Jinja focus population. IS-2, IS-3 and IL-2 are found in from 40% to 60% of the chromo- somes. IS-4, IIS-1, IIL-2 and IIL-8 are in 0.5% of the chromosomes. IIL-3 is homozygous in all speci- mens but 3; of these heterozygotes, 2 were with standard and the third was with IIL-8. Distribution: Uganda, 154 specimens; A64/1, A65/1, A65/3 to A65/5, A65/13 to A65/16, A66/4. (b) Abu Hamad population. IIS-1 was found heterozygously once; all specimens were homo- zygous for IIL-3. There is very little chromosome polymorphism in this population. Distribution: Sudan, 14 specimens; A67/4. (c) West Africa population. IS-3, IIL-2, IIL-8, IIIL-6 and IIIL-7 are all found in about 30% to 40% of all chromosomes. (IS-2 and IIL-3 are absent 975 FIG. 2 IDIOGRAMS OF THE 9 SIBLING SPECIES a I. I! Sebwe 2 A,I. E 11I 3 . 72 SIDne 6 3g a, 13 _J kulfo Nkusi KRitaketa 5 IW&1 E1 Bandama N 5] 4 db p 3 kagera T 43 73 b 72 _ 100 42 %TCL a The inversions and chromosomal features are plotted according to their percentage total complement length (%TCL). Brackets indicate inversions: heavier ones to the left, interspecific (fixed) inversions; lighter ones to the right, intraspecific (floating) inversions; detached brackets to the right, only inversions restricted to Y chromosomes. C = centromere; N = nucleolus. Descrip- tive names: b = blister; db = double bubble; R = Ring of Balbiani; p = para-Balbiani; Ex = expanded region associated with a centromere. 1s I kibwnzi EI 1! I a NINE CYTOLOGICAL SEGREGATES IN SIMULIUM DAMNOSUM COMPLEX FIG. 3 DISTRIBUTION OF CYTOLOGICAL COLLECTIONS while IIL-2 and IIL-8 have increased considerably compared with Jinja.) Distribution: Mali, Upper Volta, Ivory Coast, 34 specimens; A66/5 to A66/7, A66/15, A66/16, A67/2. Kulfo. Differs from Ketaketa by IIL-4. There are 11 floating inversions involved in about 40 hetero- zygous configurations in 12 specimens; IS-6, IS-7, IS-8, IL-5, IL-8, IL-9, IIS-2, IIS-3, IIL-12, IIL-13 and IIIL-8. (IIL-5 found in both sexes here is re- stricted to males in Kagera.) Distribution: Ethiopia, 12 specimens; A67/1. Bandama. Differs from Kulfo by IIL-6 and IIIL-4. There are 4 floating inversions: IS-5 found once; IL-6 and IIL-7, each twice heterozygously and 21 times homozygously; IIIL-5 heterozygously 11 times and homozygously 4 times. Distribution: Ivory Coast, 23 specimens; A66/5. Kagera. Differs from Kulfo by IIIL-3 (IIL-4 and IIIL-3 were previously overlooked a). The only 2 floating inversions, IL-4 and IL-5, are restricted to, and in, males, thus providing a distinct Y chromo- some. Distribution: Uganda, 48 specimens; A63/3, A64/2. Discussion There is a rather interesting division of the phylo- genetic chart based on chromosomal polymorphism. The 4 forms of the left-hand limb of Fig. 1 have almost no chromosomal polymorphism; only Sanje has any-S/IIL-1 twice in 200 specimens and S/IIIS-I only in males of a subpopulation (Fig. 2). Of those forms in the right-hand limb, all but one have con- siderable chromosomal polymorphism. In Kagera only the males showed heterozygous loops-for IL-4 and IL-5. This division is also characterized by the presence or absence of the 2 interspecific inver- sions between Sanje and Ketaketa-IS-1 and IL-3. Before a cytological category can be designated as a species it is necessary to show that the cytological category indeed repesents a distinct biological entity. The simplest proof would be hybridization experi- 977 NOTES CYTOLOGICAL COLLECTION DATA Author's Cytological form and no. of collection Location and date specimensb number a specimens Sebwe river, Toro (Ruwenzori), Uganda; 31 Jan. 1963 As for A6311; 10 March 1963 Kagera river, near Kikagati, Kyansore Island, Ankole, Uganda; 10 March 1963 Nile river, Kalagala Falls, 14 mi (22 km) downstream from Jinja, Uganda; 28 March 1964 As for A63/3; 11 April 1964 As for A64/1; 8 April 1965 Nkusi river, road bridge, Uganda; 7 May 1965 Sonso river, mid-dosing point in high forest, Budongo Forest, Uganda; 1 June 1965 Malaba river, on Kenya-Uganda border near Toro, Uganda; 12 June 1965 Aswa river, on Atiok-Palabek road, Uganda; 23 June 1965 Nkusi river, on Hoima-Fort Portal road between Budongo and Ruwenzori areas, Uganda; 25 June 1965 Sebwe river, from rocks at bridge, Uganda; 14 July 1965 Sebwe river, from vegetation at bridge, Uganda; 14 July 1965 Sebwe river, from vegetation 3/4 mi (1.2 km) upstream from bridge, Uganda; 14 July 1985 Kibaa river (small warm tributary of Murchison Park Nile), Uganda; 25 Aug. 1965 Murchison Park Nile, near mouth of Kibaa river, Uganda; 27 Aug. 1965 Just above Murchison Falls on Nile, Uganda; 28 Aug. 1965 Jinja Nile, Kangulumira, Uganda; 10 Dec. 1965 Wei or Nagak river on Uleppi-Oflrka road, Uganda; 16 Jan. 1966 Mpanga river, near Rwengo, Toro, Uganda; 10 Feb. 1966 Nile river, Kalagala Falls, Uganda; 21 Jan. 1966 Bandama river, Gauthier plantation, 4050'W, 5050'N, Ivory Coast; 14 April 1966 Bandama river, Ferk4-Korhago road, 5020'W, 9°3D'N, Ivory Coast; 14 April 1956 Samandeni, Black Volta river, 4030'W, 11°30'N, Upper Volta; 22 April 1966 Sanje river (water temp., 15.5°C), Tanzania; 24 June 1966 Kisawasawa river (water temp., 19°C), Tanzania; 24 June 1966 Mselezi (water temp., 23°C), Tanzania; 23 June 1966 Ketaketa, Luhumbero river (water temp., 21.5°C), Tanzania; 23 June 1965 Keralie river at M6n6 near Tansilla, Cercle of Nouna, 4011'W, 12035'N, Upper Volta; 15 Nov. 1966 Yame river at Bandiagara, Cercle of Bandiagara, 3036'W, 14021'N, Mali; 30 Sept. 1966 Simulium damnosum rearing tank, eggs from Lukungwi, Tanzania; 29 Nov. 1966 Lukungwi, just below crossing-place, Tanzania; 1 Dec. 1966 8 Sebwe; 11 Sanje; ?6 23 Sebwe; 25 Sanje 4 Kagera; ?2 27 Nile; ?3 44 Kagera; ?3 20 Nile; ?14 20 Nkusi; ?14 1 Nile; 16 Nkusi; ?5 2 Nile; 3 Nkusi; ?15 23 Nile; 2 Nkusi; ?10 21 Nkusi; ?7 34 Sanje 32 Sanje; ?2 4 Sanje 3 Nile; 2 Nkusi; ?6 9 Nile; ?6 35 Nile; ?19 17 Nile; ?10 10 Nkusi; ?5 1 Nkusi; ?1 27 Nile; ?9 23 Bandama; 9 Nile; ?3 11 Nile 7 Nile 29 Sanja; ?2 31 Sanje; ?1 34 Nkusi; I Sanje; I Ketaketa; ?6 9 Ketaketa; 19 Nkusi; ?14 4 Nile 2 Nile 9 Sanje; ?5 15 Sanje 978 A63/1 A6312 A63/3 A64/1 A64/2 A65/1 A65/2 A65/3 A65/4 A65/5 A65/6 A65/7 A65/8 A65/1 0 A65/13 A65/14 A65/15 A65/16 A66/1 A66/2 A66/4 A66/5 A66/6 A66/7 A66/9 A66/10 A66/11 A66/12 A66/15 A66/16 A66/18 A66119 NINE CYTOLOGICAL SEGREGATES IN SIMULIUM DAMNOSUM COMPLEX 979 CYTOLOGICAL COLLECTION DATA (continued) Author s Cytological form and no. ofcollection Location and date specim.ensb number se A67/1 Kulfo river at 4400 ft (1340 m), Arba Minch, Gamu-Gofa Province, Ethio- pia; 25 Feb. 1967 17 Kulfo; ?12 A67/2 Senegal river at Kayes, Cercie of Kayes, 11°26'W, 14°26'N, Mali; 12 Feb. 1967 1 Nile A67/4 Abu Hamad area, Main Nile, 33.5°E, 19°N, Sudan; 26 February 1967 14 Nile; ?2 A67/8 Ketaketa, Luhumbero river, Tanzania; 29 Aug. 1967 . 4 Ketaketa A67/9 Kibwezi river, tributary of Athi river, Kenya; 28 June 1967 23 Kibwezi; I Nkusi; ?4 A68/3 Nyamagasani river at old Nyaberongo bridge, 3600 ft (1090 m), Uganda; 7 Jan. 1968 10 Sebwe; I Sanje A68/5 Nyamagasani river at Katwe bridge, 1530 ft (466 m), Uganda; 4 Jan. 1968 9 Sanje; incomplete A68/7 Bwamba: Rivigo river at Bwsaru, 3100 ft (945 m), Uganda; 6 Jan. 1968 8 Sebwe; ?2 A68/8 Bwamba: Ndugutu river at Nyahuka, 2700 ft (820 m), Uganda; 6 Jan. 1968 2 Sebwe; ?1 a Collectors are as follows: A63/1 to A66/4, and A68/3 to A68/8: Angus McCrae, Uganda. A66/5 to A66/7, A66/15, A66/16, and A67/2: Bernard Philippon, Upper Volta. A66/9 to A66/12, and A67/8: W. HaUserman, Tanzania. A66/18 and A166/19: John N. Raybould, Tanzania. A67/1: Mrs Mary Lou Schmidt, Ethiopia. A67/4: 0. M. Abdelnur, Sudan. A67/9: R. B. Highton, Kenya. b Number of unidentifiable specimens preceded by a query, thus: " ?6". ments. But the laboratory rearing of blackflies at a level sufficient to allow the study of the genetics of a species is still an elusive goal. Alternatively, sympatric populations of phylogenetically contiguous forms which fail to show the critical heterozygous inversion configuration between two alternate homozygous sequences could be considered discreet species. By this criterion Sebwe, Sanje, Nkusi and Ketaketa represent species (see the table; A63/1, A63/2, A66/1 1, A66/12, A68/3). There is some doubt about the status of the other forms on purely cytological grounds but reasonable cases can be made that they, too, could represent species.c These arguments are based on various combinations of the following criteria: (1) the proximity ofknown ranges, (2) homo- zygous inversion differences, (3) different spectra of c Dunbar, R. W. (1959) Canad. J. Zool., 37, 495-525. d Basrur, P. K. (1959) Canad. J. Zool., 37, 527-570. e Basrur, P. K. (1962) Canad. J. Zool., 40, 1019-1033. f Basrur, V. R. & Rothfels, K. H. (1959) Canad. J. Zool., 37, 571-589. floating inversions, (4) different XY mechanisms, (5) micromorphological features, and (6) the observa- tion that the cytological categories in S. damnosum are as distinct as any observed in other groups of simuliids.a-J However, the possibility that 2 or more phylogenetically contiguous forms, where the evid- ence is not conclusive, may be widely divergent populations within the same species must not be entirely rejected until proof to the contrary is presented. ** * The author acknowledges with thanks the help of those who collected the material which was the basis fOr this report, and the assistance of Miss A. Carlson and Mrs M. Clark for the preparation of the chromosome slides. g Landau, R. (1962) Canad. J. Zool., 40, 921-939. h Ottonen, P. 0. (1966) Canad. J. ZooL, 44, 677-701. i Pasternak, J. (1964) Canad. J. Zool., 42, 135-158. J Rothfels, K. H. & Freeman, M. (1966) Canad. J. ZooL, 44, 937-945. 12