Temporal frequency stability and absence of effects on mating behaviour for an autosomal supernumerary segment in two natural populations of the grasshopper Eyprepocnemis plorans

M.D. Lopez-Leon, A. Martin-Alganza, M.C. Pardo, J. Cabrero, and J.P.M. Camacho

Abstract: Interannual evolution of a polymorphism for a supernumerary segment in the smallest autosome of the grasshopper Eyprepocnemis plorans has been analysed in two natural populations. The polymorphism seemed to be stable in both populations, despite its undertransmission through heterozygous females carrying B chromosomes. Analyses of the effects of the extra segment on mating behaviour failed to show differential mating success in any sex or consistent effects on mating pattern. These results are discussed in relation to the maintenance of this polymorphism in natural populations.

Key words: supernumerary segments, heterochromatin, repetitive DNA.

Resume : La prksence, d'une annke a l'autre, d'un polymorphisme pour un segment surnumkraire chez le plus petit autosome de la sauterelle Eyprepocnemis plorans a kt6 ktudike chez deux populations naturelles. Ce polymorphisme semblait stable chez les deux populations malgrk une transmission rkduite chez les femelles hktkrozygotes porteuses de chromosomes B. L'analyse des effets de ce segment supplkmentaire sur I'accouplement n'a montrk aucune diffkrence ni au niveau du succ6s dans I'accouplement chez l'un ou l'autre sexe, ni de f a ~ o n consistante au niveau du comportement d'accouplement. Une discussion de ces rksultats en relation avec le maintien de ce polymorphisme dans des populations naturelles est prksentke.

Mots cle's : segments surnumkraires, hktkrochromatine, ADN rkpktk.

[Traduit par la Rkdaction]

lntroduct ion

Supernumerary chromosome segments constitute one of the most frequent polymorphisms in natural populations of grasshoppers (Cabrero 1985). Much is known about their cytological properties such as, for instance, their hetero- geneity as deduced from the various types of response to C-banding (Camacho et al. 1984), and their effects on chiasma frequency (Schroeter and Hewitt 1974), chiasma distribu- tion on the carrier chromosome (John 1981; Camacho et al. 1984; Navas-Castillo et al. 1985, 1987; de la Torre et al. 1986), and the activity of nucleolus organizing regions

Corresponding Editor: C.B. Gillies. Received September 20, 1994. Accepted October 30, 1994.

M.D. L6pez-Lebn, A. Martin-Alganza, M.C. Pardo, J. Cabrero, and J.P.M. carnacho.' Departamento de Genktica, Facultad de Ciencias, Universidad de Granada, E- 1807 1 Granada, Spain.

' Author to whom all correspondence should be addressed.

(Cabrero et al. 1986). However, less is known about the reasons for their maintenance in natural populations, for example, the existence of accumulation mechanisms and their effects on the fitness of individuals carrying them. The few cases where transmission of supernumerary seg- ments has been analysed (Rhoades 1978; Ainsworth et al. 1983; Wilby and Parker 1988; Ruiz-Rejon et al. 1988; Lopez-Leon et al. 1992a) indicate that accumulation mech- anisms may be as common for them as they are for B chromosomes. The supernumerary segment on the small- est autosome (S,,) of Eyprepocnemis plorans is unusual, because it is undertransmitted through heterozygous females carrying B chromosomes (Lopez-Leon et al. 1991; 1994b). Thus, unless the supernumerary segment provides a bene- fit to the carriers, it will surely be eliminated from nat- ural populations. Thus, the analysis of the effects of this supernumerary segment on fitness is crucial for ascer- taining its biological role in natural populations. Complementary analyses of the evolution of polymorphism frequency in natural populations over several years must

Genome, 38: 320-324 (1995). Printed in Canada / ImprimC au Canada

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Lopez-Leon et al.

also be performed in order to predict any long term tendency to change. In this paper, frequency stability of the S , , polymorphism in E. plorans and its effects on mating behaviour are analysed in two natural populations over several years, in order to ascertain the evolutionary mech- anisms involved in its maintenance in the wild.

Materials and methods

Adult males and females of the grasshopper E. plorans were collected in two natural populations, one at Jete (J) and one at Salobrefia (S), in the province of Granada (Spain), in the years 1986 (J), 1990 (J and S), 1991 (J and S), and 1992 (J and S). Additionally, four (J) or five (S) samples of adult males were caught during the 1990 season at intervals of about 3 weeks to analyse intra-annual variation of the polymorphism. Because E. plorans specimens carry out a very prolonged copulation (about 24 h on average; L6pez- Le6n et al. 1994c), it is not very hard to collect a relatively high number of mating pairs in the field to analyse mating behaviour. J was sampled for mating pairs in 1986 and 1990, and S was sampled in 1990 and 1992. Each mating pair was kept in a tube and, once in the laboratory, the male and the female were fixed for cytological analysis. Nonmating individuals were simultaneously collected in the same populations in order to test differential mating success. Testes were fixed in acetic acid - ethanol 1:3, without any pretreatment. Females were injected with 0.05% colchicine in insect saline solution for 6 h prior to fixation of ovarioles in acetic acid - ethanol 1:3. Squash preparations of testis follicles and ovarioles were C-banded by the technique described in Camacho et al. (1984). To apply contingency X2 tests, classes were grouped when the minimum expected frequency in a cell in the contingency table was lower than 5.

Results

Eyprepocnemis plorans specimens possess 2n = 22 + XO &/XX 9 chromosomes. Three different karyotypes were observed with respect to the supernumerary segment on the S , , chromosome: normal homozygotes (NN) with both S , , chromosomes lacking the supernumerary segment, seg- mented homozygotes (SS) with both S,, chromosomes pos- sessing the supernumerary segment, and heterozygotes (NS).

Frequency stability of the polymorphism Comparisons of chromosome frequencies between males and females failed to show any significant difference related to sex in all samples collected at both populations (data not shown). Between-year comparisons also failed to show any significant temporal variation in chromosome fre-

2 quencies in J ( x ( ~ , = 6.64, p = 0.08) or S (xt2, = 0.65, p = 0.72) (Fig. 1). In consequence, the S , , polymorphism seemed to show interannual frequency stability, although frequency fluctuations were close to significant in J.

Mating behaviour The three main components of mating behaviour, differ- ential male mating success, differential female mating suc- cess, and pattern of mating were analysed. Mating success was tested by comparing karyotype frequencies between mating and nonmating individuals in the same population

Fig. 1. Interannual evolution of segmented S, , chromosome frequency (9,) in two natural populations of the grasshopper E. plorans. N = Number of individuals analysed.

YEAR

and year. Pattern of mating was tested by analysing the frequencies of the different copulating pairs established in each population.

As Table 1 shows, no differential male or female mating success seems to exist in relation to the supernumerary segment, and this result was consistent among the different populations and years analysed.

Mating pattern, however, showed different results among the four samples (Table 2). J males and females of 1986 were nonrandomly mated with respect to the supernumerary segment owing to a significant excess of NN X S- mating pairs (negative assortment), but the three remaining samples suggested random mating, although in 1990 J and 1992 S there were almost significant tendencies to positively assorted mating. Thus, the supernumerary segment did not seem to produce a clear effect on mating pattern in the wild that was consistent in both time and space.

Discussion

The data presently available indicate that the S,, super- numerary segment does not possess any cytological accu- mulation mechanisms but, on the contrary, is eliminated through heterozygous females possessing B chromosomes by means of meiotic drive against the segmented S,, chro- mosome (L6pez-Le6n et al. 1991; 1994b). Despite this elimination, the frequency of the supernumerary segment seems to remain stable in both populations analysed, although the total time period analysed (7 years for J and 3 years for S) was quite short and most likely does not provide an accurate perspective for long term evolution.

Nevertheless, unless the supernumerary segment exerts any beneficial effect on the individuals carrying it, it is clear that the undertransmission through B-carrying females should lead to its extinction from natural populations. Our

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Table 1. Analysis of mating success in males and females collected from two natural populations (one at Jete and one at Salobrefia) in 2 different years.

Karyomorph Mating success

Population Sex Year Type of individual NN NS SS Total q, X 2 df p

Jete 8 1986

1990

Q 1986

1990

Salobrefia 8 1990

1992

Q 1990

1992

Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total Mating Nonmating

Total

Table 2. Mating pattern in relation to the supernumerary segment.

Male karyotype Mating pattern

Population Year Female karyotype NN NS SS Total Pattern 1 P

Jete 1986 NN 26 20 2 48 Nonrandom, 5.42 0.02 NS 19 4 0 23 negative assortment

Total 45 24 2 71 1990 NN 50 7 0 57 Random, 3.79 0.052

NS 9 4 1 14 tendency to positive assortment SS 1 0 0 1

Total 60 11 1 72 Salobrefia 1990 NN 19 8 3 30 Random 0.10 0.76

NS 12 5 3 20 SS 1 0 1 2

Total 32 13 7 52 1992 NN 46 23 0 69 Random, 3.62 0.057

NS 26 24 2 52 tendency to positive assortment SS 3 3 0 6

Total 75 50 2 127

Note: NS and SS classes were pooled to perform the X2 test.

analysis of mating behaviour with mating pairs collected in chromosome of E. plorans (Ldpez-Ledn et al. 1992a) that the field has shown that the supernumerary segment seems is transmitted in a Mendelian ratio in both sexes (Ldpez- to be neutral with respect to mating success and mating Ledn et al. 1992b) and is mainly composed of a 180 base pattern. This same result was obtained for the B,-type pair (bp) DNA tandem repeat and ribosomal DNA (Ldpez-

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Leon et al. 1994a). The S,, supernumerary segment contains the same 180 bp DNA repeat (M.D. Lopez-Leon, J. Cabrero, and J.P.M. Camacho, unpublished data), so it seems clear that this DNA is neutral for mating behav- iour. After discovering a fitness reduction associated with the deletion of a satellite DNA array in the Responder locus of the segregation distorter system in Drosophila melanogaster (Wu et al. 1989), it became likely that some kinds of repetitive DNA might be beneficial for carrier fitness. The recent finding that the same deletion removes the Bari-1 transposon-like middle repetitive DNA, how- ever, challenges this suggestion, because while the Responder DNA is extremely variable, the Bari-1 hetero- chromatic array is very stable (Caizzi et al. 1992). Consequently, it is interesting to test the possibility that repetitive DNA is beneficial for individual fitness in systems other than the segregation distorter system; the super- numerary chromosome segments, which usually harbour this kind of DNA, constitute highly appropriate systems.

Although most supernumerary segments analysed up to the present time show accumulation mechanisms, which make them strong candidates to contain parasitic or selfish DNA (Rhoades 1978; Ainsworth et al. 1983; Wilby and Parker 1988; Lopez-Leon et al. 1992a), their effects on fitness have rarely been analysed. Tulipa australis is one of the few examples; in this plant species the coexistence of an accumulation mechanism and a beneficial effect on seed fertility doubled the frequency of a supernumerary segment in a single generation (Ruiz-Rejon et al. 1988). In Scilla auturnnalis, it was recently observed that plants that are heterozygous for a euchromatic supernumerary segment yield significantly heavier seeds than do plants lacking it (M. Jamilena, F. Martinez, M.A. Garrido-Ramos, C. Ruiz-Rejon, A.T. Romero, J.P.M. Camacho, J.S. Parker, and M. Ruiz-Rejon., submitted for publication). Our present analysis of mating pairs of E. plorans collected in the wild demonstrates that the supernumerary segment on the S , , chromosome does not influence mating success. However, since in E. plorans there is paternity displacement owing to a very strong second male sperm precedence (Lopez-Leon et al. 1993), mating patterns observed in nat- ural populations might not coincide with the effective paternity patterns, since different karyotypes may differ in their capability to achieve sperm precedence, thus pro- viding a second opportunity for sexual selection (see Smith 1984). Consequently, this fitness component should be investigated, together with survival and female fertility, in order to explain the biological significance of this polymorphism. This work is currently underway.

Acknowledgements I This study was partially supported by grants from the

Direccion General de Investigacion Cientifica y Tecnica (no. PB93-1108) and the Plan Andaluz de Investigacion, Grupo no. 3 122 (Spain).

References

Ainsworth, C.C., Parker, J.S., and Horton, D. 1983. Chromosome variation and evolution in Scilla auturn- nalis. Kew Chromosome Conf. 2: 261-268.

Cabrero, J. 1985. Estudios citogenkticos en saltamontes de la subfamilia Gomphocerinae: Heterocromatina, reordenaciones cromosomicas y actividad nucleolar. Tesis Doctoral, Universidad de Granada, Spain.

Cabrero, J., Navas-Castillo, J., and Camacho, J.P.M. 1986. Effects of supernumerary chromosome segments on the activity of nucleolar organizer regions in the grasshopper Chorthippus binotatus. Chromosoma, 93: 375-380.

Caizzi, R., Caggese, C., and Pimpinelli, S. 1992. Bari-1 a new transposon-like family in Drosophila rnelanogaster with a unique heterochromatic organi- zation. Genetics, 133: 335-345.

Camacho, J.P.M., Viseras, E., Navas-Castillo, J., and Cabrero, J. 1984. C-heterochromatin content of super- numerary chromosome segments of grasshoppers: detection of a euchromatic extra segment. Heredity, 53: 167-175.

de la Torre, J., Lopez-Fernandez, C., Nichols, R., and Gosalvez, J. 1986. Heterochromatin readjusting chi- asma distribution in two species of the genus Arcyptera: The effects among individuals and populations. Heredity, 56: 177-1 84.

John, B. 198 1. Heterochromatin variation in natural populations. Chromosomes Today, 7: 128- 137.

Lopez-Leon, M.D., Cabrero, J., and Camacho, J.P.M. 199 1. Meiotic drive against an autosomal super- numerary segment promoted by the presence of a B chromosome in females of the grasshopper Eyprepocnernis plorans. Chromosoma, 100: 282-287.

Lopez-Leon, M.D., Pardo, M.C., Cabrero, J., and Camacho, J.P.M. 1992a. Random mating and absence of sexual selection for B chromosomes in two natural populations of the grasshopper Eyprepocnernis plorans. Heredity, 69: 558-561.

Lopez-Leon, M.D., Cabrero, J., Camacho, J.P.M., Cano, M.I., and Santos, J.L. 1992b. A widespread B chro- mosome polymorphism maintained without apparent drive. Evolution, 46: 529-539.

Lopez-Leon, M.D., Cabrero, J., Pardo, M.C., Viseras, E., and Camacho, J.P.M. 1993. Paternity displacement in the grasshopper Eyprepocnernis plorans. Heredity, 71: 539-545.

Lopez-Leon, M.D., Neves, N., Schwarzacher, T., Heslop-Harrison, T.S., Hewitt, G.M., and Camacho, J.P.M. 1994a. Possible origin of a B chromosome deduced from its DNA composition using double FISH technique. Chromosome Res. 2: 87-92.

Lopez-Leon, M.D., Pardo, M.C., Cabrero, J., and Camacho, J.P.M. 1994b. Undertransmission of a supernumerary chromosome segment through heterozygous females possessing B chromosomes in the grasshopper Eyprepocnernis plorans. Genome, 37: 705-709.

Lopez-Leon, M.D., Pardo, M.C., Cabrero, J., and Camacho, J.P.M. 1994c. Dynamics of sperm storage in the grasshopper Eyprepocnernis plorans. Physiol. Entomol. 19: 46-50.

Navas-Castillo, J., Cabrero, J., and Camacho, J.P.M. 1985. Chiasma redistribution in bivalents carrying

Gen

ome

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

UN

IV C

HIC

AG

O o

n 11

/10/

14Fo

r pe

rson

al u

se o

nly.

Genome, Vol. 38, 1995

supernumerary chromosome segments in grass- hoppers. Heredity, 55: 245-248.

Navas-Castillo, J., Cabrero, J., Camacho, J.P.M. 1987. Chiasma redistribution in presence of supernumerary chromosome segments in grasshoppers: dependence on the size of the extra segment. Heredity, 58: 409-4 12.

Rhoades, M.M. 1978. Genetic effects of heterochromatin in maize. In Maize breeding and genetics. Edited by D.B. Walker. Wiley Interscience, New York. pp. 641-671.

Ruiz-Rejon, C., Lozano, R., Ortega-Nieto, F.J., and Ruiz-Rejon, M. 1988. B chromosomes and supernu- merary chromosome segments in Liliaceae: selfish or heterotic DNA? In Kew chromosome conference 111.

Edited by P.E. Brandam. H.M.S.O., London. pp. 141-149.

Schroeter, G., and Hewitt, G.M. 1974. The effects of supernumerary chromatin in three species of grass- hopper. Can. J. Genet. Cytol. 16: 285-296.

Smith, R.L. 1984. Sperm competition and the evolution of animal mating systems. Academic Press, New York.

Wilby, A.S., and Parker, J.S. 1988. The supernumerary segment system of Rumex acetosa. Heredity, 60: 109-1 17.

Wu, C.I., True, J.R., and Johnson, N. 1989. Fitness reduction associated with the deletion of a satellite DNA array. Nature (London), 341: 248-25 1.

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from

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IV C

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