The complex Robertsonian system of Dichroplus pratensis (Melanoplinae, Acrididae). I. Geographic distribution of fusion polymorphisms

1 Universidad Nacional de Misiones, Departamento de Genética, Facultad de Ciencias Exactas, Quimicas y Naturales, Félix de Azara 1552, 6°Piso, 3300 Posadas, Misiones, Argentina; 2 Biological Sciences Center, Dept of Genetics and Cell Biology, 1!,l,5 Gartner Ave, St Paul, MN, USA; 3 Dept Ciencias Biolôgicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Laboratorio de Genética, 1428 Buenos Aires, Argentina


INTRODUCTION
The role of chromosomal change in speciation has been extensively discussed (White, 1978a(White, ,b, 1982John, 1981;Mayr, 1982;Patton and Sherwood, 1983;Reig, 1984;Lande, 1985;Baker and Bickham, 1986;King, 1987;Sites and Moritz, 1987). Related species frequently have distinct karyotypes often assumed to be a consequence of a causal relationship between chromosomal rearrangements and speciation (White, 1978a). Karyotypic divergence may also be a by-product of speciation. This discussion is of interest since chromosomal models of speciation have been proposed (King, 1987;Sites and Moritz, 1987). Evidence for a role of chromosome change in speciation is far from clear, usually indirect and the critical properties of rearrangements have sometimes been overlooked or assumed without reliable data (John, 1981).
Chromosome polymorphisms and polytypisms allow the analysis of these issues.
The neotropical genus Dichroplus is interesting because of its inter-and intraspecific chromosomal variability. Of 40 known species, 33 have been studied chromosomally and centric fusion is a major source of differentiation (Mesa et al, 1982). Some cases of intraspecific Robertsonian variation have been reported and in this respect Hewitt (1979) and John (1983) mention D pratensis Bruner, originally studied by Mesa (1956) andSdez (1956). The cytogenetics of this species became very confused because of its morphologic similarity to D obscurus which has an entirely different karyotype and geographic distribution (Bidau, 1984). The situation became clearer after Mesa's 1971 paper in which 2 fusion polymorphisms superimposed upon the standard karyotype were reported. Unfortunately, Mesa (1971) and Sdez and P6rez-Mosquera (1970) called the different morphs &dquo;cytological races&dquo;. This is an error which was carried over to John's (1983) paper.
The aim of our study was to establish whether the polymorphisms were present in other areas of the species distribution range or if they were limited to a hybrid zone between 2 authentic chromosomal races. The situation uncovered is more complex.
Here we report the existence of several races differing in type and frequency of Robertsonian translocations.

MATERIALS AND METHODS
Adult grasshoppers of both sexes were collected between 1982 and 1989 at the localities shown in table I and figure 1. Testes dissected out in the field were fixed in 3:1 alcohol-acetic acid directly or after 15' hypotonic treatment in 50% insect saline. Females were injected with 0.05% colchicine. Ovaries and gastric caeca were removed after 6-8 h and fixed after hypotonic treatment. Some males were colchicinised after biopsy for the removal of part of the testes. Cytological methods have already been described (Bidau, 1986). The standard karyotype was determined in males from Puerto Madryn and Gaiman (table I) through measurements of photographic enlargements and camera lucida drawings of Cmetaphases and late pachytene cells. The same procedure was followed for the identification of the different fusions. Banding methods did not prove useful for chromosome identification (see Results).

The standard karyotype
The standard chromosome complement is shared in principle by all sampled populations and it is unique within the genus (Mesa et al, 1982). A quantitative analysis was possible in 2 populations (Puerto Madryn and Gaiman) where the frequency of standards is high (table I). The karyotype comprises 19 ( d' ) and 20 (Q) telocentrics, 18 of which are autosomes (fig 2a). The latter include 6 large (L I -L G ) and 3 small (S 7 -S 9 ) chromosome pairs; the X is about the size of L 4 (fig 2; table II). Relative lengths based on measures of C-metaphases and late pachytene bivalents are given in table II. S 7 is the megameric bivalent and has a heterochromatic interstitial block (fig 3a,b). S s carries a proximal NOR associated with a C-positive block (Bidau, 1986) (fig 3a,b). The only L-chromosome identifiable by C-banding is L6 , polymorphic for a distal heterochromatic block (fig 3a).
Male meiosis is well characterised (Bidau, 1990). L-bivalents have a proximaldistal chiasma distribution while S-bivalents always form a single chiasma of variable localisation (Bidau, 1990 Levan et al, 1964). They will be termed metacentric in this paper. The most symmetric fusion is 3/4; the least symmetric, 1/6. All populations but one are polymorphic for up to 4 fusions (table I). Populations polymorphic for 3 fusions exist that share the 3/4 fusion but have the 1/6 and 2/5 fusions in one case (San Luis and La Pampa populations) The total number of males analysed is 900 and that of females 122. 'Localities 1-12 and 25-37 belong to Buenos Aires Province; 13-15, to Rio Negro Province; 16,17,to Chubut Province; 18-20 to San Luis Province and 21-24, to La Pampa Province. b CD: Collection date. 'N: number of males ( d) and females (9) studied cytologically. CF: centric fusions found in each sample. In sample 13, fusion 3/5 appeared in one individual and its identification is doubtful. e K: Number of karyomorphs found in the sample (regardless of sex-chromosome differences). f St: Frequency of standard (all telocentric) individuals in the sample. 9 2n: Range of diploid numbers found in males and females jointly. and the 1/2 and 5/G fusions in the other (Monte Hermoso and Sierra de la Ventana populations). Monobrachial homologies are evident in these examples which explain the populations polymorphic for more than 3 fusions that occur within hybrid zones (Bidau, 1991;Tosto and Bidau, 1991).
Polymorphism for a fusion implies 3 karyotypes: standard (st), structural heterozygote (Het) and homozygote (Hom). Thus populations polymorphic for 2 or 3 simple fusions will potentially show 9 and 27 karyotypes respectively. For example, all 9 karyotypes were found in the El Condor population, polymorphic for 2/4 and 5/6. Considering the 7 fusions, 60 different karyotypes are expected in each sex within the species. Since monobrachial homologies occur, further karyotypes are expected in hybrid zones (see below and Bidau, 1991). (2 types of B chromosomes also produce karyotype variation (Bidau, 1986(Bidau, , 1987.) Meiotic behavior of Hets, Homs and hybrids has already been described. In Hets and Horns it is very regular as expected (despite low frequencies of nondisjunction in trivalents) (Bidau and Mirol, 1988;Bidau, 1990). Hybrids however, show a markedly abnormal meiotic behavior (Bidau, 1991). Nevertheless, a marked modification of the chiasma patterns occurs in all Hets and Homs: it mainly consists of a significant shift of chiasma positions to distal localisations in the chromosomes involved in the fusions, together with a reduction of cell mean chiasma frequency (Bidau, 1990). A more complex repatterning occurs in hybrids (Bidau and Fenocchio, in preparation).

Frequency and distribution of the fusions
The area sampled for fusions in Argentina, although large, represents but a limited picture of the potential situation in view of the wide distribution range of the species (Liebermann, 1963).
No strict geographic pattern emerges from the study of the karyotyped populations but some regularities are apparent. Fusions 3/4 and 1/6 are widespread in the northern sampled area while fusion 2/5, although associated with the former, has been found only in the westernmost part of the area. Futhermore, 2/5 is frequent at San Luis but decreases southwards, disappearing at Gral Acha (La Pampa) (table  IV). Fusion 1/2 seems limited to Sierra de la Ventana and Monte Hermoso. 5/6 occurs in the southern range of the species and 1/4 and 2/4 are limited to coastal populations of Rio Negro and Chubut provinces respectively. One individual from El C6ndor probably carried an eight fusion, 3/5. Within the same fusion system, frequencies of each fusion may vary widely as in the 2/5 case already mentioned. Nevertheless, all polymorphisms fit the Hardy-Weinberg equilibrium since no departures from the expected values were found (Bidau, 1984;Tosto and Bidau, 1991).

DISCUSSION
Chromosomal polymorphism is rather frequent in natural populations but its role in evolution is debatable. This has been thorougly studied in Drosophila (Brussard, 1984;Sperlich and Pfriem, 1986). When analysing the fate of chromosomal rearrangements in natural populations, their potential involvement in speciation is relevant (King, 1987;Sites and Moritz, 1987). Second, chromosome polymorphisms common within a species are frequently not of the type of rearrangement determining interspecific differences (Bidau, 1989). Third, the mechanical and genetic properties of chromosome rearrangements must be considered to establish their role in adaptive and/or speciation processes. Last, the distribution of polymorphisms may fit an ordered pattern such as the central-marginal model (Brussard, 1984), an unknown pattern or no pattern at all.
In the Acrididae, centric fusion has been a dominant form of change during the evolution of the group (John and Hewitt, 1968;John and Freeman, 1975;Hewitt, 1979;John, 1983). It is thus puzzling that very few cases of polymorphisms and polytypisms have been reported (Hewitt, 1979;John, 1983). Only 2 cases of single fusion polymorphism were previously analysed on a population basis (Hewitt and Schroeter, 1968;Bidau and Hasson, 1984;Colombo, 1987).
The fusions of D pratensis probably arose independently in different parts of the species range and spread subsequently (see below). Nevertheless, the possibilities that the same arose several times in different populations or else that two or more fusions appeared as a result of a single mutation, cannot be discarded. There is circumstancial evidence that the same rearrangement can occur repeatedly within a population (Sperlich and Pfriem, 1986) and that rapid multiple rearrangements do occur (King, 1982;Peters, 1982). Thus karyotypic orthoselection (White, 1978a) need not depend on a slow sequential process.
Centric fusion is the dominant form of chromosome variation in D pratensis (and within Diclaropdus) apart from B chromosomes (Bidau, 1986(Bidau, , 1987. It is thus possible that D pratensis chromosomes have a tendency to break non-randomly at the centromere, increasing the probability of fusions which could, depending on other factors (see below), become established as polymorphisms. Molecular mechanisms have been proposed to explain the high incidence of Robertsonian fusion in the mouse (Redi et al, 1986(Redi et al, , 1990Redi and Capanna, 1988) based on DNA sequence homology in pericentromeric areas of all chromosomes. These models could apply to D pratensis as well.
With one exception, all multiple polymorphisms of D pratensis fit the Hardy-Weinberg equilibrium (Tosto, 1989;Tosto and Bidau, 1991). Fixation of 3 fusions was only observed in one isolated populations (Bidau, 1989;Tosto and Bidau, 1991). Maintenance of such balanced polymorphisms is only possible if meiotic behaviour of trivalents in heterozygotes is regular (unless heterozygotes are positively heterotic despite loss of fertility due to meiotic misbehaviour). We have shown that all heterozygous fusions behave well at meiosis (Bidau and Mirol, 1988;Mirol and Bidau, 1991a) as demonstrated by their very low non-convergent orientation frequencies and production of abnormal sperm. However, aneuploidy and macrospermatid production increase with the number of heterozygous fusions (Bidau and Mirol, 1988) which could explain the higher frequencies of fusion metacentrics in populations with 3 fusions in order to minimise the frequencies of double and triple heterozygotes (Tosto, 1989;Tosto and Bidau, 1991). In comparable stable multiple polymorphisms such as those of the common shrew, heterozygotes do not have their fertility severely reduced (Searle, 1984(Searle, , 1988Garagna et al, 1989;Searle et al, 1990).
Stable meiotic behaviour is achieved by a repatterning of chiasma distribution of the fused chromosomes (Bidau, 1990;Mirol and Bidau, 1991b) which leads to the conclusion that fusions can affect intra-(and inter-) chromosomal recombination drastically. Thus they could serve to protect the integrity of coadapted supergenes and also allow for the maintenance of favourable linkage disequilibria. A rationale for the existence of the polymorphisms thus exists.
Each fusion system could become established because it protects a given set of coadapted supergenes adaptive to a given habitat (Bidau, 1989(Bidau, , 1990). Colonisation of a new environment may occur in the standard high recombination condition followed by adaptation and establishment of a particular fusion system (fig 7). This could explain the diversity of polymorphisms in relation to the wide ecological tolerance of the species. In this context it is worth recalling the &dquo;central-marginal&dquo; model. Although no clear pattern of distribution of polymorphisms emerges from the data (perhaps because only a part of the large distribution area of the species was sampled) 3 points are evident: 1), certain fusions occur only or are more frequent in certain areas; 2), frequency gradients for some fusions exist (ie 2/5); and 3), the less polymorphic populations are those from eastern central Patagonia (PM and G) which are ecologically marginal since their habitat conditions are certainly rougher and population densities lower than in the rest; they thus share properties of marginal populations (Brussard, 1984).
Frequently, chromosomal differences between species are not of types characterising their common polymorphisms (ie John and Weissman, 1977;John et al, 1983;Sperlich and Pfriem, 1986). This applies to D pratensis whose unique standard karyotype possibly derived through 2 tandem fusions from the basic Cryptossacci complement, but whose polymorphisms are essentially Robertsonian.
Centric fusions are candidates for the establisment of post-mating barriers (King, 1987;White, 1978) if conditions for the establishment of a balanced polymorphism are not met. Polymorphic fusions would thus have no impact on speciation. D pratensis however, presents a complex situation: since the same telocentrics are involved in different arm combinations, if populations polymorphic for metacentrics with monobrachial homologies intercross, some hybrids should form meiotic multivalents prone to non-convergent orientation and unbalanced segregation thus producing negative heterosis. The frequency of such hybrids will depend on the frequencies of monobrachially homologous metacentrics in each population. King (1987) contemplates this situation but considers that fixation is needed, which we believe is not so. In fact, in hybrid zones, the frequency of metacentrics could be boosted in order to avoid gene exchange between chromosomal races (Bidau, 1991;Tosto and Bidau, 1991). Furthermore, King (1987) says that through this interaction &dquo;neutral rearrangements have been transformed into negatively heterotic changes&dquo;. The rearrangements involved are not necessarily neutral and in D pratensis they certainly exert effects on recombination which may be adaptive since different supergenes may be involved (Bidau, 1990).
The interaction of polymorphic forms of D pratensis would produce a certain proportion of sub-fertile hybrids. This could be interpreted as an incipient postmating mechanism that could be enhanced in principle by the fixation of the fusions in the diverging populations. Hybrids zones such as predicted have been found and analyses of hybrid meiotic behaviour and frequency of metacentrics are currently being performed (Bidau, 1991;Tosto and Bidau, 1991).

ACKNOWLEDGMENTS
The senior author wishes to express his sincere gratitude to his sons, Claudio Jr and Pablo for help in the collection of grasshoppers. 1B4rs R Josens was very helpful during one of the field trips and Miss NI I<orenblum karyotyped some of the La Pampa specimens. Prof Dr J I-I Hunziker guided and stimulated the beginnings of the senior author's research on grasshopper cytogenetics. Special thanks to Miss M Gimenez for help in the preparation of this typescript. The work on D pratensis has been aided by CONICET grants to Dr IIunzil<er and UBA grants to CJB.