Heredity 83 (1999) 428±434 Received 5 November 1998, accepted 21 June 1999

The B chromosome polymorphism of the grasshopper plorans in North Africa. I. B variants and frequency

M. BAKKALI, J. CABRERO, M. D. LOÂ PEZ-LEOÂ N, F. PERFECTTI & J. P. M. CAMACHO* Departamento de GeneÂtica, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain

Polymorphism for B chromosomes has been detected in all nine populations of the grasshopper ssp. plorans sampled in Morocco. The most frequent B chromosome in all

populations showed a C-banding pattern and size similar to those of the B1 variant found in the Iberian Peninsula. In addition, other B chromosome variants (B1iso1,B1iso2,B1d1,B1di1,B3and B1dd1) were discovered in these populations, although at a very low frequency. No signi®cant di€erences in B chromosome frequency were found either in the nine populations or, for some of them, in up to three consecutive years. These results are discussed in the light of current hypotheses on the evolution of this B chromosome polymorphism in the Iberian Peninsula.

Keywords: B chromosomes, Eyprepocnemis plorans ssp. plorans, grasshopper, .

Introduction chromosome variants have been described in natural Spanish populations of this grasshopper , B chromosomes, also called accessory or supernumerary according to size, morphology and C-banding pattern chromosomes, are large pieces of dispensable indepen- (Henriques-Gil et al., 1984; Henriques-Gil & Arana, dently segregating DNA (Camacho et al., 1997a,b). 1990; Lo pez-Leo n et al., 1993); however, only ®ve Reported in more than 1300 plant species and nearly 500 variants have had success in establishing polymorphisms species (Jones & Puertas, 1993), variation in the (B1,B2,B5,B6and B24), the remaining types being found number and morphology of B chromosomes is a at extremely low frequency (Henriques-Gil & Arana, frequent chromosomal polymorphism found in natural 1990; Lo pez-Leo n et al., 1993). New B variants appear populations of many eukaryote organisms. B chromo- frequently (Lo pez-Leo n et al., 1993) and the replace- somes show extensive variation in size and morphology, ment of a B variant by another one (Henriques-Gil & and most of them are heterochromatic with a variety of Arana, 1990) can be caused by a detectable meiotic drive di€erent responses to C-banding. Because of their favouring the substituting variant (Zurita et al., 1998). harmful e€ects on carriers, most of them are considered Evidence for the monophyletic origin of most B chro- genome parasites (OÈ stergren, 1945; MuÈ ntzing, 1963; mosome variants, including the predominant types, has Nur, 1966, 1969; Puertas et al., 1985) that maintain been provided by C-banding (Henriques-Gil et al., 1984; themselves by accumulation mechanisms (Jones, 1985, Lo pez-Leo n et al., 1993) and ¯uorescent in situ hybrid- 1991). ization (Cabrero et al., 1999). B chromosomes are especially frequent in Orthoptera, The B chromosome polymorphism of E. plorans has where they sometimes constitute apparently stable provided evidence for the long-term evolution of these polymorphisms (Hewitt, 1979). The grasshopper Eypre- genome parasites as a result of an `arms race' between pocnemis plorans ssp. plorans shows a highly remarkable them and the A genome. Thus B chromosomes, starting B chromosome polymorphism where the great majority as parasites (possessing drive and damaging host of populations harbour B chromosomes (Camacho et al., ®tness), can be neutralized because of the evolution of 1997a,b). This subspecies inhabits the Mediterranean drive-suppressor genes in the A genome and the parallel region, the Caucasus, Turkey, Turkmenistan, Iran and evolution both of less damaging B chromosome variants south-western Arabia (Dirsh, 1958). More than 40 B and of more tolerant A genomes (Camacho et al., 1997a,b). This explains the existence of polymorphisms for B chromosome variants lacking drive (Lo pez-Leo n *Correspondence. E-mail: [email protected] et al., 1992a) and apparent e€ects on host ®tness

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(Lo pez-Leo n et al., 1992b; Martõ n-Alganza et al., 1997). Materials and methods These neutralized B chromosome variants, however, are condemned to random extinction and the B chromo- Specimens of the grasshopper E. plorans ssp. plorans some polymorphism would disappear unless replaced by were collected at nine natural populations from north- newly arising sel®sh B chromosome variants (Camacho western Morocco during September in 1995, 1996 and et al., 1997b). Evidence for such a regeneration of the B 1997. The geographical location and the number of chromosome polymorphism has also been found in specimens caught in each of these nine populations are E. plorans (Zurita et al., 1998). shown in Fig. 1 and Table 1. The populations sampled The geographical distribution of di€erent B chro- in 1995 were Smir (between Ceuta and Tetouan), Frain mosome variants of this species has been extensively and SO.DE.A (close to Ksar-el-Kebir), and Mechra studied in Spain and the presence of B chromosomes (close to Mechra-bel-Ksiri). In 1996 we collected grass- has been reported in almost all natural populations hoppers at Ain l'abid and Tatouft (near Ksar-el-Kebir), analysed (Camacho et al., 1980; Henriques-Gil et al., in addition to taking new samples of the same popula- 1984; Henriques-Gil & Arana, 1990), the only non-B tions sampled in 1995. In 1997, we captured new populations being located in an inland region of the specimens from the six populations sampled in previous Segura River basin (Cabrero et al., 1997). The single years and collected grasshoppers in three new sites: North African sample hitherto analysed (Melilla) Larache, Asilah and Rabat. showed the presence of a B chromosome very similar Testes were ®xed in 1:3 acetic acid-ethanol without to those found in the Iberian Peninsula (Henriques-Gil any pretreatment. Females were injected with 0.1 mL of & Arana, 1990). 0.05% colchicine in saline solution 6 h before The present paper seeks to provide a more complete ®xation of the ovaries. Fixed gonadal tissues were stored picture of B chromosome distribution in natural Afri- at 4°C for at least one month before analysis. can populations of E. plorans. We report the presence B chromosome presence was studied by squashing of B chromosomes in all nine populations sampled testis follicles and ovarioles in acetic orcein. For from Morocco, and the existence of several low- characterization on the basis of morphology and frequency B chromosome variants. We also perform C-banding patterns, gonadal tissues of B-chromosome- an analysis of B chromosome frequency variation in carrying individuals were C-banded as described by time and space. Camacho et al. (1991). The B chromosome structure was

Fig. 1 Geographical location of the nine populations of the grasshopper Eyprepocnemis plorans sampled in Morocco from 1995 to 1997. e, Cities; d, Populations (1, Smir; 2, Asilah; 3, Larache; 4, Ain l'abid; 5, Tatouft; 6, Frain; 7, SO.DE.A; 8, Mechra; 9, Rabat). Principal rivers are also indicated.

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Table 1 Spatial and temporal distribution of B chromosome frequencies in specimens of the grasshopper Eyprepocnemis plorans ssp. plorans caught at nine Moroccan localities

Comparison of B1 freq. between years Total B1 Total B 2 Population Year 0B 1B 2B 3B Total B1 freq. B freq. v d.f. P prevalence prevalence Smir 95 8 3 0 0 11 0.273 0.273 27.273 27.273 96 18 5 0 0 23 0.217 0.217 21.739 21.739 97 21 10 2à 1§ 34 0.382 0.500 32.353 38.235 Total 47 18 2 1 68 0.309 0.368 2.365 4 0.779 27.941 30.882 Asilah 97 7 1 0 0 8 0.125 0.125 Ð Ð Ð 12.500 12.500 Larache 97 26 13 1 0 40 0.375 0.375 Ð Ð Ð 35.000 35.000 Ain l'abid 96 15 9 1 0 25 0.440 0.440 40.000 40.000 97 38 9± 0 0 47 0.170 0.192 17.021 19.149 Total 53 18 1 0 72 0.264 0.278 5.519 2 0.055 25.000 26.389 Tatouft 96 13 2 4 0 19 0.526 0.526 31.579 31.579 97 26 12 2 0 40 0.400 0.400 35.000 35.000 Total 39 14 6 0 59 0.441 0.441 5.346 2 0.080 33.898 33.898 Frain 95 13 5 2 0 20 0.450 0.450 35.000 35.000 96 17 2 1 0 20 0.200 0.200 15.000 15.000 97 28 10 0 0 38 0.237 0.263 23.684 26.316 Total 58 17 3 0 78 0.282 0.295 5.494 4 0.213 24.359 25.641 SO.DE.A 95 25 8àà 1 0 34 0.265 0.294 23.529 26.471 96 49 14 1 0 64 0.250 0.250 23.438 23.438 97 27 14§§ 0 0 41 0.268 0.342 26.829 34.146 Total 101 36 2 0 139 0.259 0.288 5.519 4 0.055 24.460 27.338 Mechra 95 34 9 1 0 44 0.250 0.250 22.727 22.727 96 54 25±± 5 1ààà 85 0.400 0.447 34.118 36.471 97 14 5 2 0 21 0.429 0.429 33.333 33.333 Total 102 39 8 1 150 0.360 0.387 2.795 4 0.596 30.667 32.000 Rabat 97 11 3§§§ 2 0 16 0.375 0.438 Ð Ð Ð 25.000 31.250

One individual carried one B1iso1. àOne individual carried one B1iso1 and one B1iso2. §This individual carried two B1 and one B1iso2. ±One individual carried one B1iso2. One individual carried one B1iso2. ààOne individual carried one B1d.d1. §§Three individuals carried one B1d.d1. ±±One individual carried one B1iso2. One individual carried one B1d1 and one B1d.et1. àààThis individual carried two B1 and one B1iso1. §§§One individual carried one B3. examined in male pachytene±diplotene cells, because of Our study of gonadal tissues of E. plorans ssp. plorans the less condensed state of B-chromatin in these meiotic specimens revealed the presence of B chromosomes in all stages. nine Moroccan populations sampled. Only two individ- uals (Mechra-1996 and Smir-1997) were found bearing Results three B chromosomes, this being the highest number of B chromosomes found in the same individual (Table 1). The standard chromosome complement of males and The most frequent B chromosome in all nine popula- females of the grasshopper E. plorans is composed of 23 tions was an acrocentric chromosome, about half the (22 + X0) and 24 (22 + XX) telocentric chromosomes, size of the X chromosome, with two proximal C-bands respectively. of similar sizes. Fluorescent in situ hybridization

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Parker et al., 1991). In the case of the grasshopper E. plorans, Camacho et al. (1997b) reported nonsignif- icant temporal variation in B chromosome frequency at two Spanish localities (Jete and SalobrenÄ a), sharply contrasting with another Spanish population (Torrox)

where rapid changes in B24 frequency were demonstrat- Fig. 2 C-banded B chromosome variants found in the nine ed (Zurita et al., 1998). The nonsigni®cant interannual Moroccan Eyprepocnemis plorans ssp. plorans natural popu- changes of the B chromosome frequency observed in the lations sampled. (a) The most frequent B chromosome found six Moroccan populations of E. plorans sampled at least in all the nine populations studied; (b) found in one individual twice (Smir, Ain l'abid, Tatouft, Frain, SO.DE.A and in Mechra 1996; (c) found in one individual in SO.DE.A 1995 Mechra) corroborate that the time needed to record and in three from the same population in 1997; (d) present in signi®cant changes in B chromosome frequency depends one individual in Mechra 1996; (e) observed in one individual on the B variant, the population, and the evolutionary in Rabat 1997; (f) described as B and B by Henriques-Gil 12 1iso status of the polymorphism at the time of sampling. et al. (1984) and Lo pez-Leo n et al. (1993), respectively. One The proportion of individuals carrying B chromosomes individual carrying this B variant was found in Mechra 1996 and two in Smir 1997; (g) one individual carried this B variant was similar in the nine Moroccan populations studied. in Mechra 1996, four in Smir 1997, one in Ain l'abid 1997, and However, Spanish populations of this species show another one in Frain 1997. di€erent local B chromosome frequency (Henriques-Gil et al., 1984; Henriques-Gil & Arana, 1990; Camacho et al., analysis shows that this B chromosome is made up of 1997b). The low spatial di€erentiation in Morocco could the same two DNA types (rDNA and a 180-bp repeat be explained by a rapid invasion of the North African DNA) present in B chromosomes from the Iberian populations by the B chromosome polymorphism, to- Peninsula (Cabrero et al., 1999). All these data suggest gether with high gene ¯ow between the nine populations that the predominant B chromosome in Morocco is studied. This scenario is supported by the ¯ying capacity similar (in terms of size, morphology, C-banding pattern of this grasshopper species and the absence of major and DNA content) to the predominant one in the geographical barriers between the nine localities. The Iberian Peninsula, which was named B1 by Henriques- presence of some B variants at very low frequency in some Gil et al. (1982) (Fig. 2). Temporal analysis of B1 populations, and their absence in others, does not frequency variation in Moroccan populations revealed diminish the importance of gene ¯ow as a possible cause no signi®cant di€erence between years of collection in of the spatial stability of B chromosome frequency any of the six populations where this comparison could through the Moroccan populations analysed, because be made (Table 1). Likewise, a comparison of the these are, presumably, short-lived variants usually with frequency of individuals with di€erent numbers of B1 low transmission eciency (Lo pez-Leo n et al., 1993). chromosomes in the nine populations also failed to Thus, it is unlikely that they will migrate to other 2 detect signi®cant di€erences (v16 ˆ 18.292; P ˆ 0.309). populations before disappearing. It is remarkable that The mean prevalence (percentage of individuals carrying the only two B variants detected in more than one at least one B chromosome) was 28.32% ‹ 2.26, and population (B1iso1 and B1iso2) are those produced by a there were no signi®cant di€erences between popula- recurrent phenomenon (centromere misdivision); they are 2 tions (v8 ˆ 3.983; P ˆ 0.861) or years within populations also frequently found in Spanish populations at a very low (P ranging from 0.093 to 1). frequency because of their low transmission ratio (Lo pez-

In addition to the principal type (B1), we found six other Leo n et al., 1993). B1iso1 has been described by Henriques- B chromosome variants (Fig. 2), for which the structure Gil et al. (1984) as B12 and as B1iso by Lo pez-Leo n et al. and possible mode of origin are summarized in Fig. 3. (1993), and according to the code proposed by Lo pez-

Leo n et al. (1993) B1iso2 can be considered to be a Bmini. The highly dynamic character of this B chromosome Discussion polymorphism (Camacho et al., 1997b) is supported by With the exception of the grasshoppers Atractomorpha the presence of other B variants detected in some of the bedeli and Acrida lata inhabiting sites heavily disturbed nine Moroccan populations studied. This dynamism of by human activity (Sannomiya & Kayano, 1968), and B chromosomes is an important characteristic for the the grasshopper Myrmeleotettix maculatus, which shows maintenance of the polymorphism, because it means a cline movement in East Anglia (Shaw, 1983), the that a new B variant with drive can replace the principal frequencies of most plant and animal B chromosomes neutralized B chromosome, thereby regenerating and that have been studied over several years have remained prolonging the polymorphism life cycle (Zurita et al., stable (Jones & Rees, 1982; Cano & Santos, 1989; 1998).

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Fig. 3 Hypothetical origin of the six minority B variants arising from the

most widespread variant, B1. (a) Cen- tromere misdivision and subsequent chromatid nondisjunction; (b) deletion of part of the proximal dark C-band and part of the distal light region; (c) deletion of part of the proximal dark C-band; (d) extra-radial transposition; (e) deletion of part of the distal light region; (f) dupli- cation of a dark C-band and deletion of part of the distal light region.

In the chive Allium schoenoprasum, Bougourd & et al., 1993). Lo pez-Leo n et al. (1994) proposed the X Parker (1979) traced with high precision the sites where chromosome as the ancestor of the B chromosomes of B chromosomes probably originated and their mode of the Jete and SalobrenÄ a Spanish populations. Moreover, spreading. Despite the extensive study of many Iberian FISH studies show that B chromosomes from Moroccan E. plorans populations, such results have not yet been and Spanish E. plorans populations have a common achieved in this grasshopper, partly because of almost origin because both are made up of the same DNA universal B chromosome presence in Spanish popula- sequences (Cabrero et al., 1999). All these data lead us to tions, and partly because of the scarcity of information deduce the possible intercontinental migration of these B from African populations. chromosomes and to discard the third hypothesis. Henriques-Gil et al. (1984) postulated two possibilities The common origin of B chromosomes from Spain for the origin of the B chromosomes of E. plorans: (i) B and Morocco, with B1 as the original B in both chromosomes originated prior to the colonization of the countries, their similar response to C-banding, their Iberian Peninsula by this grasshopper (in this case the similar DNA content (Cabrero et al., 1999) and the North African populations should have B chromosomes) existence of di€erent B variants in both Spain and or (ii) B chromosomes originated in a Spanish popula- Morocco, indicate that the near-neutral model devel- tion and spread throughout Spain. We can add another oped for Spanish populations (Camacho et al., 1997a,b) hypothesis: (iii) B chromosomes of E. plorans originated might be useful to apply to the polymorphism in independently in Spain and Morocco. Our study shows Morocco. An inland region in the Iberian Peninsula that B chromosomes are present in all the Moroccan lacking B chromosomes has been interpreted as evidence populations studied, and the fact that the principal B of relict populations preceding B origin, as they are chromosome found in all the studied populations is a isolated by geographical barriers from the B-carrying very similar B variant (B1) indicates that we can consider ones (Cabrero et al., 1997). This indicates that B this B variant as the original B chromosome in Moroc- chromosomes most likely originated after the species can populations. The observed north±south continuity colonized the Iberian Peninsula, and this would run of B chromosome presence can be interpreted as the counter to hypothesis (i). The demonstration of several result of a migration of B1-carrying individuals through Spanish populations where B1 has been substituted by the studied populations. B1 is also considered the other B variants (B2 in Granada province, B5 in original B chromosome variant in Spanish natural Fuengirola; see Henriques-Gil & Arana, 1990), and the populations (Henriques-Gil et al., 1984; Lo pez-Leo n recent polymorphism regeneration shown in Torrox (see

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Zurita et al., 1998), indicate that the polymorphism is CANO, M. I. ANDSANTOS , J. L. 1989. Cytological basis of the B presumably old in Spain, so that it has reached the near- chromosome accumulation mechanism in the grasshopper neutral stage in many populations and the regeneration Heteracris litoralis (Ramb). Heredity, 62, 91±95. , V. M. 1958. Revision of the genus Eyprepocnemis FIEBER, stage in a few localities. In Morocco, the polymorphism DIRSH 1853 (Orthoptera: Acridoidea). Proc. Roy. Ent. Soc. Lond. B, shows lower B frequencies than in Spain, only the 27, 33±45. original B (B ) constituting a polymorphism, with no 1 HENRIQUES-GILHENRIQUES-GIL.,., N. ANDARANA , P. 1990. Origin and substitution observed regeneration. The fact that B1 shows drive in of B chromosomes in the grasshopper Eyprepocnemis some populations (Bakkali et al. unpubl. data) suggests plorans. Evolution, 44, 747±753. that the B polymorphism is still in the sel®sh stage (the HENRIQUES-GILHENRIQUES-GIL..N. N.,SANTOS, J. L.AND GIRALDEZ , R. 1982. beginning) and thus it is still young in Morocco. All B-chromosome polymorphism and interchromosomal chi- these facts suggest that the B polymorphism most likely asma interference in Eyprepocnemis plorans (; arose in the Iberian Peninsula after the colonization of Orthoptera). Chromosoma, 85, 349±359. HENRIQUES-GILHENRIQUES-GIL..N. N.,SANTOS, J. L.AND ARANA , P. 1984. Evolution this species, and B1 has recently colonized the north of Africa where the polymorphism is in the sel®sh starting of a complex B-chromosome polymorphism in the grass- hopper Eyprepocnemis plorans. Chromosoma, 89, 290±293. stage of the near-neutral cycle. HEWITT, G. M. 1979. Grasshoppers and crickets. In: John, B. (ed.) Animal Cytogenetics, vol. 3, Insecta 1, Orthoptera. Acknowledgements GebruÈ der Borntraeger, Berlin. JONES, R. N. 1985. Are B chromosomes sel®sh? In: Cavalier- M. Bakkali wishes to thank the Agencia EspanÄ ola de Smith, T. (ed.) The Evolution of Genome Size, pp. 397±425. Cooperacio n Internacional for funding a studentship, Wiley, London. and Mr S. Bakkali for his help in capturing specimens. JONES, R. N. 1991. B Chromosome drive. Am. Nat., 137, 430±442. This study was partially supported by grants from the JONES, R. N.AND PUERTAS , M. J. 1993. The B chromosomes of Spanish Direccio n General de EnsenÄ anza Superior (no. Rye (Secale cereale L.). In: Dhir, K. K. and Sareen, T. S. PB96±1433) and Plan Andaluz de Investigacio n, Grupo (eds) Frontiers in Plant Science Research,pp.81±112. no. CVI-165. Bhagwati Enterprises, Delhi, India. JONES, R. N.AND REES , H. 1982. B Chromosomes. Academic Press, New York. References LOLOPEZ PEZ--LEONLEO N, M. D., PARDO, M. C., CABRERO, J.AND CAMACHO , J. P. M. 1992a. Random mating and absence of sexual selection BOUGOURD, S. M.AND PARKER , J. S. 1979. The B chromosome for B chromosomes in two natural populations of the system of Allium schoenoprasum. II. 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