Introduction
The field of population genetics came into being in the 1920s and 1930s thanks to the theoretical basis framed by R.A. Fisher, J.B.S. Haldane and S. Wright among many others. Their achievement was to integrate the principles of Mendelian genetics – which had been rediscovered at the turn of the century – with Darwinian natural selection. Fisher, Haldane and Wright showed that Darwinism and Mendelism were not just compatible but they worked hand in glove with each other; this finding played a key part in the formulation of the ‘New Synthesis’ and explains why population genetics came to occupy such a pivotal role in evolutionary theory (Barton, Reference Barton2022).
Sturtevant's (Reference Sturtevant1917) conclusion that chromosome inversions suppress recombination in Drosophila melanogaster in heterozygotic state was later corroborated by means of cytogenetical analysis (Sturtevant and Beadle, Reference Sturtevant and Beadle1936); from then on, the empirical study of population genetics became not one of genes, but one of chromosome polymorphisms (Kapun and Flatt, Reference Kapun and Flatt2019). At that time, molecular gene markers were still unavailable, but Drosophila specialists in those early times had plenty of cytological variation to work with. Drosophila pseudoobscura has five chromosome pairs, with >30 different arrangements on chromosome 3 alone, due to more than ten overlapping inversions (Fuller et al., Reference Fuller, Haynes, Richards and Schaeffer2017); then the number of possible karyotypes is somewhere close to 310 = 59.049, and this is an underestimation. Hence it is not a hyperbole to affirm that the fulcrum of population genetics – which, in time, is the hard core of the New Synthesis – was population cytogenetics. The existence of chromosome polymorphisms and the assignment of different fitness values to them became the touchstone of our current idea of biological evolution.
The adaptive importance of chromosome inversions in Drosophila consisted in the suppression of recombination in structural heterozygotes (Sturtevant and Beadle, Reference Sturtevant and Beadle1936). These rearrangements would capture and accumulate ‘coadapted gene complexes’, which involve epistasis (Dobzhansky, Reference Dobzhansky1950). Kirkpatrick and Barton (Reference Kirkpatrick and Barton2006) proposed instead ‘local adaptation’ as an alternative (see below). There is plenty of evidence for both scenarios, coming from steep frequency clines, correlations with environmental (climatic) factors, predictable seasonal changes in inversion frequencies, evidence of epistatic selection and so forth (see Kapun et al., Reference Kapun, Fabian, Goudet and Flatt2016 and references therein).
Grasshopper karyotypes, by contrast, are rather conservative, with a prevailing chromosome number of 22 + X0/XX in males and females, respectively (Hewitt, Reference Hewitt1979). Therefore, the study of inversion polymorphisms in this group was restricted to a few species (White, Reference White1973). The Australian grasshopper Moraba ( = Keyacris) scurra was thoroughly studied by White and co-workers in the 1950s and 1960s, and a great deal of effort was devoted to the assignment of adaptive value to these polymorphisms (White, Reference White1958, Reference White1973; Lewontin and White, Reference Lewontin and White1960; White and Andrew, Reference White and Andrew1960). Another inversion-bearing Australian grasshopper with consistent latitudinal variation is Caledia captiva (Groeters and Shaw, Reference Groeters and Shaw1992; Shaw and Groeters, Reference Shaw and Groeters1998; Shaw et al., Reference Shaw, Coates and Arnold2011). In South America, Vaio et al. (Reference Vaio, Goñi and Rey1979) and Goñi et al. (Reference Goñi, de Vaio, Beltrami, Leira, Crivel, Panzera, Castellanos and Basso1985) discovered several chromosome inversions in some Argentine populations of Trimerotropis pallidipennis, but the authors concluded that the inversions did not follow any environmental or geographic patterns). Later analysis (Confalonieri, Reference Confalonieri1988, Reference Confalonieri1994; Confalonieri and Colombo, Reference Confalonieri and Colombo1989; Colombo and Confalonieri, Reference Colombo and Confalonieri1996; see Guzmán et al., Reference Guzmán, Kemppainen, Monti, Castillo, Rodriguero, Sánchez-Restrepo, Cigliano and Confalonieri2022 and quotations therein) showed the geographical and environmental patterns of these inversions and their stability along space and time. In chromosome inversions, local adaptation favours reduced recombination between locally adapted loci (Charlesworth and Charlesworth, Reference Charlesworth and Charlesworth1979; Lenormand and Otto, Reference Lenormand and Otto2000). This is a well-studied mechanism for the evolution of chromosome inversions whereby they capture two or more locally adapted alleles; later on, this high fitness chromosome spreads in the population in which those alleles are beneficial (Kirkpatrick and Barton, Reference Kirkpatrick and Barton2006; Charlesworth and Barton, Reference Charlesworth and Barton2018). By reducing recombination, inversions preserve the divergence between populations in the presence of gene flow, setting the stage either for permanent polymorphism or potential speciation (Navarro and Barton, Reference Navarro and Barton2003; Kirkpatrick and Barton, Reference Kirkpatrick and Barton2006).
This was the picture for population cytogeneticists up to the 1970s–1980s. But, at a time when allozyme electrophoresis, RFLPs and microsatellite polymorphisms allowed to obtain several molecular alleles which were easy to analyse simultaneously, population cytogenetics fell out of fashion because it could not keep pace with the variability evidenced by molecular research (Kirkpatrick, Reference Kirkpatrick2010; Fuller et al., Reference Fuller, Koury, Phadnis and Schaeffer2019).
Centric fusion polymorphisms
Robertsonian translocations ( = centric fusions) owe their name to W.R.B. Robertson, who first discovered a V-shaped structure in a metaphase I of Locusta migratoria which was not present in all the individuals (Robertson, Reference Robertson1916). Polymorphic centric fusions are more frequent among mammals, the house mouse being the best studied case (Davisson and Akeson, Reference Davisson and Akeson1993; Castiglia and Capanna, Reference Castiglia and Capanna2000; Dumas and Britton-Davidian, Reference Dumas and Britton-Davidian2002; Merico et al., Reference Merico, Giménez, Vasco, Zuccotti, Searle, Hauffe and Garagna2013; Förster et al., Reference Förster, Jones, Jóhannesdóttir, Gabriel, Giménez, Panithanarak, Hauffe and Searle2016) followed by phyllotines (Lanzone et al., Reference Lanzone, Labaroni, Suárez, Rodríguez, Herrera and Bolzan2015), Holochilus (D'Elia et al., Reference D'Elía, Hanson, Mauldin, Teta and Pardiñas2015), the genus Ctenomys (Bidau et al., Reference Bidau, Martí and Giménez2003) and the common shrew Sorex araneus (Giagia-Athanasopoulou and Searle, Reference Giagia-Athanasopoulou and Searle2003; Basset et al., Reference Basset, Yannic, Hausser, Searle, Polly and Zima2019). They were also thoroughly studied in populations of some fishes (Farr-Cox et al., Reference Farr-Cox, Leonard and Wheeler1996; Ostberg et al., Reference Ostberg, Hauser, Pritchard, Garza and Naish2013; Wellband et al., Reference Wellband, Mérot, Linnansaari, Elliott, Curry and Bernatchez2019), and the gastropod Nucella lapillus (Pascoe, Reference Pascoe2006).
Among grasshoppers, McClung (Reference McClung1917) discovered a polymorphism for a centric fusion between the X chromosome and an autosome in Hesperotettix viridis; six more fusion polymorphic grasshopper species were discovered ever since, all of them from the New World (Hewitt and Schroeter, Reference Kapun, Fabian, Goudet and Flatt1968; Colombo, Reference Colombo1987, Reference Colombo1989, Reference Colombo1990, Reference Colombo2007, Reference Colombo2008; Bidau and Mirol, Reference Bidau and Mirol1988; Bidau, Reference Bidau1990; Remis, Reference Remis2008; Taffarel et al., Reference Taffarel, Bidau and Martí2015; Castillo et al., Reference Castillo, Taffarel, Maronna, Cigliano, Palacios-Gimenez, Cabral-de-Mello and Martí2018; see Colombo, Reference Colombo2013 for a review). Other species were omitted from this list because they are polytypic rather than polymorphic (i.e. the variation lies between, not within populations, and heterozygotes are indeed interracial hybrids), such as the remarkable wetas from New Zealand (McKean et al., Reference McKean, Trewick and Morgan-Richards2015).
Fusions are an altogether different matter. The reduction of recombination they trigger is due both to the loss of independent segregation and because of a more distal localisation of chiasmata both in heterozygotes and in homozygotes (Colombo, Reference Colombo1987, Reference Colombo1989, Reference Colombo1990; Bidau, Reference Bidau1990; Remis, Reference Remis1990; Castiglia and Capanna, Reference Castiglia and Capanna2000; Bidau et al., Reference Bidau, Gimenez, Palmer and Searle2001; Dumas and Britton-Davidian, Reference Dumas and Britton-Davidian2002). On the other hand, as mentioned above, inversions reduce recombination in heterozygotes alone. Because of this asymmetry, it is unclear whether the evolutionary forces that operate on locally adapted inversions can act on centric fusions in a comparable way (Guerrero and Kirkpatrick, Reference Guerrero and Kirkpatrick2014).
In our laboratory two acridid models have been worked out: both of them are semiaquatic leptysmine grasshoppers which are unusually polymorphic from the cytogenetic standpoint, namely Leptysma argentina and Cornops aquaticum.
Bidau and Hasson (Reference Bidau and Hasson1984) reported the survey of two contiguous populations, sited 400 m apart, of L. argentina, a grasshopper whose range is restricted to Central-North Eastern Argentina and all of Uruguay. It is unusually polymorphic for a grasshopper, with a centric fusion between chromosomes 3 and 6 of the basic complement (fusion 3/6) and a large supernumerary segment on the smallest member of the karyotype, namely chromosome 10. The number of possible karyotypes was then 32 = 9 (18 if we added the presence or absence of a B-chromosome).
Besides, C. aquaticum is an all-out Neotropical grasshopper, and it occurs between the south of Mexico (23°N) and the latitude of Buenos Aires and Punta del Este (34°S–35°S) (Adis et al., Reference Adis, Bustorf, Lhano, Amédégnato and Nunes2007). Prior to coming under our scrutiny, C. aquaticum was an already appealing semiaquatic leptysmine because it has a strict host ecological specificity (it feeds on, and lays eggs in, floating plants of the also Neotropical Pontederiaceae family, called ‘water-hyacinths’) exclusively (Adis and Junk, Reference Adis and Junk2003). Moreover, after its introduction elsewhere as an ornamental, the blue-flowered water-hyacinth Pontederia crassipes became a weed that infested freshwater environments in tropical, subtropical and even temperate regions all around the world (Center et al., Reference Center, Hill, Cordo, Julien, Van Driesche, Blossey and Hoddle2002; Coetzee et al., Reference Coetzee, Hill, Byrne and Bownes2011). That's why C. aquaticum has been considered as a possible biological control of the obnoxious water-hyacinth (Oberholzer and Hill, Reference Oberholzer and Hill2001) and a great deal of effort has been devoted to study its phenology and ecology.
Mesa (Reference Mesa1956) and Mesa et al. (Reference Mesa, Ferreira and Carbonell1982) reported the presence of three polymorphic centric fusions in C. aquaticum in Uruguay (about lat. 31°S). Rocha et al. (Reference Rocha, Souza and Moura2004) described its karyotype for several collection sites in the state of Pernambuco (Brazil), at lat 8°S. Cornops aquaticum bears an all-telocentric karyotype, with three large (L), five medium (M) and three small (S) pairs. The sex determination system is X0/XX and the X is M. However, the centric fusions were nowhere to be seen. In Colombo (Reference Colombo2007, Reference Colombo2008, Reference Colombo2009), Colombo and Remis (Reference Colombo and Remis2018), Colombo et al. (Reference Colombo, Zelarayán, Franceschini and Remis2021, Reference Colombo, Bressa and Remis2023) and Romero et al. (Reference Romero, Colombo and Remis2014, Reference Romero, Colombo and Remis2017) we found and described all three fusion polymorphisms forming clines in the lower course of the Paraná and Uruguay Rivers.
The purpose of the present contribution consists in reviewing the quest, in a material generally regarded as intractable, for a feat any population (cyto)geneticist would fancy about: to have plenty of heritable variation to work with. This was the basis for our endeavour, i.e. the assignment of differential adaptive values for the different chromosome morphs. We consider the results of this research programme on the (mostly) univoltine, more terrestrial, well-behaved L. argentina, with its synchronous generations, as a necessary step for the study of the unruly C. aquaticum, which lives on the floating mats of an aquatic plant. This quest may yield a twofold benefit, given that C. aquaticum, beyond its theoretical value, is poised for introduction in many alien environments where P. crassipes has made inroads. The knowledge of the genetic system of a biocontrol is central for its success, and the parallel clines for three centric fusions have probably much to say about it.
Results and discussion
Leptysma argentina
The aim of the present cytological research in L. argentina consisted in testing the operation of natural selection, chiefly on the centric fusion (F) and the large supernumerary segment (S) polymorphisms, and to a lesser extent on the B-chromosome. Having analysed 11 populations over a span of 7 degrees of latitude (between 27°S and 34°S) (table 1, fig. 1) a latitudinal cline for F and another for B (which forms a reverse slope) were detected. Data come mostly from already published material (Colombo, Reference Colombo1987, Reference Colombo1989, Reference Colombo1990, Reference Colombo1993, Reference Colombo1997, Reference Colombo2000; Norry and Colombo, Reference Norry and Colombo1999; Colombo et al., Reference Colombo, Pensel and Remis2001, Reference Colombo, Pensel and Remis2004); data from the Tucumán (T) population remained unpublished until now.
Leptysma argentina: geographic and climatic variables associated with the populations studied here
Lat, South latitude; MinT, mean annual minimum temperature; MaxT, mean annual maximum temperature (in °C); Monthly rainfall, average yearly rainfall (in mm); Etp/yr, mean yearly evapotranspiration (in mm/year). Frequencies of chromosome polymorphisms for F, S and B (see text) are also indicated. N = sample size. Abbreviations of grasshopper populations are given in fig. 1.
a Previously unpublished data.
Sampling area and Leptysma argentina populations included in this study: Tucumán (T), Santa Fe (SF), Los Loros (LL), Yarará Guazú (YG), El Palmar (EP), Zárate (ZA), Puerto Talavera (PT), Isla Talavera (IL), Río Luján (RL) and Pilar (P). Pie diagrams indicate the frequencies of the centric fusion 3/6 (F), the segment S and the B-chromosome (B) in dark grey. Data from T were unpublished so far.
Latitudinal clines are compatible with (but not conclusive of) the operation of natural selection (Endler, Reference Endler1977). Endler (Reference Endler1986) listed ten criteria to detect the operation of natural selection in the wild, as follows:
I) Correlations of the polymorphisms with environmental (climatic) variables;
II) Comparison between closely related species;
III) Comparison between unrelated species living in similar habitats;
IV) Deviation from formal null-models, such as Hardy–Weinberg or gametic phase (linkage) equilibria;
V) Long-term study of trait frequency distribution, such as (a) long-term stability or (b) regular directional change;
VI) Perturbation of natural populations such as: (a) predictable seasonal changes and (b) introduction into new equivalent and non-equivalent habitats;
VII) Genetic demography or cohort analysis;
VIII) Comparisons among age-classes or life-history classes such as (a) comparison of breeding and non-breeding adults or (b) comparison of juveniles and adults and (c) fitness component analysis such as mating ability, mating preference, fecundity, viability, age of reproduction, etc.
IX) Non-equilibrium predictions of changes in trait distributions; and
X) Equilibrium predictions about trait frequency distribution.
Criteria II, III, VII, IX and X would not (likely) apply for the purposes of the present research and they were not discussed.
As for (I), besides the latitudinal clinal variation, the statistical analysis showed that:
i) F correlates negatively with average and maximum temperature (P = 0.0001 and 0.0002), positively with latitude (P = 0.0005) and negatively with yearly evapotranspiration (Etp yr–1) (P = 0.0111) and rainfall (P = 0.0189). A stepwise regression analysis showed a positive regression on latitude (P = 0.00063) and a negative one on Etp yr–1 (P < 0.00181).
ii) B correlates negatively with latitude (P = 0.0001) and positively with average and maximum temperature (P = 0.0002 and P = 0.0076 respectively). As for the stepwise regression analysis, B displays an inverted pattern with respect to F, with a highly significant negative selection on latitude (P = 0.0063) and a positive significant one on average temperature (P 0.0142),
iii) All 11 populations considered, S showed a significant negative regression against rainfall (P = 0.0189) despite the absence of any obvious geographic pattern (table 1, fig. 1). However, the IT population is very much of an outlier. A small sample from an extremely small population, in an area of intense human perturbation, it was suppressed from a second statistical analysis. As supposed, negative correlation with rainfall became highly significant (P = 0.0009).
It may seem odd that some of the polymorphisms show better regressions on latitude than on the environmental variable they ought to be related with, presumably temperature (but also rainfall in this region of South America). However, this seems to be a frequent feature of chromosomal clines of other organisms such as Drosophila (see Kapun et al., Reference Kapun, Fabian, Goudet and Flatt2016 and references therein). Apparently this may indicate that the underlying factor of clinal variation is related with temperature, but not temperature as such (Krimbas, Reference Krimbas, Krimbas and Powell1992).
(II) Two areas were sampled with special intensity: the northeast of Buenos Aires province (RL, O, Z, IT, PT) (lat. 34°S) and a protected area 300 km further north (‘El Palmar’, LL, EP, YG) (lat. 31.8°S). The karyotypes always adjusted to the Hardy–Weinberg expectations everywhere, but preliminary analyses in the first area suggested that
(i) there was a non-significant excess for S heterozygotes (BS) at the onset of the breeding season (spring), and a non-significant defect at the end (summer); the X2 comparison between both samples was significant, although the frequency of S did not change.
(ii) By contrast, the F frequency showed a consistent increase between spring and summer samples everywhere. The following year, however, the population had the same F frequency as the precedent. The polymorphism was apparently stable, but the frequencies changed along the year. (i) and (ii) turned out to be the first evidences of seasonal selection for F and S (Endler's VI a) (Colombo, Reference Colombo1989).
This evidence of cyclical change was constant for over 20 years, between, 1983 and 2004 and was replicated elsewhere, i.e. in the ‘El Palmar’ area, which was geographically distant and with different karyotypic frequencies when compared to the previous one.
Gametic phase (linkage) equilibrium analyses were carried out between F and S, both in early and late spring in both populations, taking advantage of the fact that L. argentina has synchronous and non-overlapping generations (Aquino and Turk, Reference Aquino and Turk1997). In the El Palmar population, young individuals (spring sample) were in equilibrium; in aged ones (summer sample) gametic phase disequilibrium was significant instead, with a prevalence – according to the simulation – of FB (fused and without S) and US (unfused and with the segment) gametes (Colombo, Reference Colombo1993). The same result was replicated further south, with a much higher F frequency, only that linkage disequilibrium D was non-significant (Colombo, Reference Colombo2000). This is yet another evidence (Endler's IV), albeit indirect, of the operation of natural selection (Endler, Reference Endler1986). Therefore, gametic phase equilibrium is a null model this species does not fit into.
As generations were synchronous and non-overlapping in this species (Aquino and Turk, Reference Aquino and Turk1997), age-class comparisons in both areas were possible. We compared the frequencies of all nine karyotypes (for F and S) at spring (young males) and summer (aged males) in both sampling regions. In the young male sample from El Palmar, karyotypes for both polymorphisms distributed independently; in aged ones the FF individuals tended to be unsegmented (BB), and the segmented (SS) ones tended to be unfused (UU); furthermore, there was always a clear excess of double heterozygotes (Colombo, Reference Colombo1993). The same result was obtained further south, where F displays a much higher frequency in both places, only with a different frequency (Colombo, Reference Colombo1993; Colombo et al., Reference Colombo, Pensel and Remis2001). All individuals are already adult by the onset of October (spring), and at this latitude there is just one generation a year (personal observation); therefore, the change of frequency must be due to differential survival alone. According to Endler (Reference Endler1986), comparison between age classes, in this case juveniles and adults (Endler's VIIIb), is a fairly direct evidence of natural selection for both polymorphisms, and it may come to explain the gametic disequilibrium described above.
(IV) Coincidental correlations between environmental variables both between and within populations along the year: the evidence here is contradictory and spotty. F is inversely correlated with temperature. However, it always increases its frequency from spring to summer; F showed a positive regression on Tmax along a series of seven (not strictly consecutive) years; the regression on rainfall changes its sign in El Palmar with respect to Buenos Aires' NE. Segment S does not change its frequency along the season, it is just the heterozygotes BS who decrease among the aged males. No attempt to perform these studies on the B-chromosome was done.
(V) Selection component analyses (Endler's VIIIc) confirmed that the F dosage was correlated with adult male longevity (Norry and Colombo, Reference Norry and Colombo1999). The selective male longevity value of F is clearer since its frequency has always increased along the breeding season, i.e. from October to December (spring to summer) (Colombo, Reference Colombo1993; Colombo et al., Reference Colombo, Pensel and Remis2001).
Moreover, a unisexual approach revealed that the F carriers had an advantage in mating selection (Colombo et al., Reference Colombo, Pensel and Remis2001). Furthermore, F dosage also increased female mating success (Colombo et al., Reference Colombo, Pensel and Remis2004). No effect on either phenotype or mating success was detected for segment S.
The quest for the selection target
At the time these studies were undertaken for the first time, except for a few cases (White and Andrew, Reference White and Andrew1960; Butlin et al., Reference Butlin, Read and Day1982) chromosomal change was generally deemed to be neutral for the external phenotype (Lande, Reference Lande1979; John, Reference John, Sharma and Sharma1983) and it was believed that only the internal phenotype (i.e. meiosis) was affected by them (John, Reference John, Sharma and Sharma1983). The study of body size in all three karyotypes of F on most body size-related traits in two populations of L. argentina (Colombo, Reference Colombo1989, Reference Colombo1997) revealed that F carriers were significantly larger, and that there is a good correlation between its frequency, latitude and body size-related traits. The populations of ‘El Palmar’ (lat. 31.8°) had smaller individuals and a lower F frequency; those of Buenos Aires (lat. 34°C) have a larger body size, and a higher F frequency (Colombo, Reference Colombo1989, Reference Colombo1997). The karyotype–body size correlation was consistent and highly significant. Santa Fe individuals, one degree of latitude further north, have no F and are smaller than the others; finally, males from the norther monomorphic population of Tucumán (27°S) are tiny (data unpublished).
The selection component analyses mentioned above (Endler's VIIIc criteria) showed that F is positively selected for longevity from spring to summer, i.e. with increasing temperature; aged males are larger than young ones, the direct target of selection being thorax height (Norry and Colombo, Reference Norry and Colombo1999). Additionally, when mating choice was studied it was found that sexual male selection advantage is associated with a larger third femur length in males, and total body length (TL) as well as third femur length (FL) in females. However, these variables are not the actual selection targets (Colombo et al., Reference Colombo, Pensel and Remis2001, Reference Colombo, Pensel and Remis2004).
To summarise:
i) Along the latitudinal cline, F correlates negatively with temperature but positively with size.
ii) Within a population, at a given time, F correlates positively with body size.
iii) Within a population, F increases from spring to summer (i.e. its frequency correlates positively with temperature) and, as always, keeps correlating positively with body size.
This supposed fit to the ‘Bergmann rule’ would rather reflect the case of a change of voltinism along a latitudinal transect (Shelomi, Reference Shelomi2012). There is evidence of this in Aquino and Turk (Reference Aquino and Turk1997). Further analysis is needed to shed light on the other two phenomena.
Both longevity and mating choice would lead to an increase of F; however, along the more than 20 years of monitoring these frequencies did not modify.
No phenotypic effect could be attributed on a consistent basis to segment S, nor was it revealed to be associated to mating success. It is certainly correlated against rainfall but no geographic pattern was observed. The target of selection on S (detected through gametic phase disequilibrium and joint age class comparison with F) remains unidentified.
Effects of centric fusions on recombination
In addition to the effects of F on phenotype, there were more evident outcomes on cytological features that led to a significant reduction of recombination in F carriers, especially heterozygotes (Colombo, Reference Colombo1987, Reference Colombo1989, Reference Colombo1990):
1) Formation of a new linkage group from the two erstwhile independently segregating chromosome pairs.
2) Reduction of proximal (P) and interstitial (I) chiasma formation in the fusion trivalent, which elicits an abrupt decrease of chiasma frequency, and a strict distal (D) localisation in these elements (intrachromosomal effects) (P, I and D with respect to the centromere) (Colombo, Reference Colombo1990).
3) Decrease in the frequency of total (T), proximal (P) and interstitial (I) chiasmata, and a slight increase in the frequency of distal chiasma frequency (D) in the fusion bivalent (but not so marked as that in the fusion trivalent). The rise in D takes place at the expense of I (both are negatively correlated) and the decrease of T is explained by a reduction of P (both are positively correlated, Colombo, Reference Colombo1990).
4) These within-population results were mirrored by the comparison among populations that display different F frequencies (Colombo, Reference Colombo1989).
These results (Colombo, Reference Colombo1987, Reference Colombo1989, Reference Colombo1990) along with those of Bidau and Mirol (Reference Bidau and Mirol1988), Bidau (Reference Bidau1990), Remis (Reference Remis1990), Colombo (Reference Colombo2007, Reference Colombo2008), Taffarel et al. (Reference Taffarel, Bidau and Martí2015), Martí et al. (Reference Marti, Castillo, Marti and Taffarel2017) and Castillo et al. (Reference Castillo, Taffarel, Maronna, Cigliano, Palacios-Gimenez, Cabral-de-Mello and Martí2018) corroborated that this feature of Robertsonian polymorphisms is consistent among grasshoppers (Colombo, Reference Colombo2013) and beyond. Suppression of recombination in trivalents of heterozygotes and chiasma reduction in fusion bivalents of fusion homozygotes has been reported in house mice (Davisson and Akeson, Reference Davisson and Akeson1993; Bidau et al., Reference Bidau, Gimenez, Palmer and Searle2001; Dumas and Britton-Davidian, Reference Dumas and Britton-Davidian2002; Merico et al., Reference Merico, Giménez, Vasco, Zuccotti, Searle, Hauffe and Garagna2013; Förster et al., Reference Förster, Jones, Jóhannesdóttir, Gabriel, Giménez, Panithanarak, Hauffe and Searle2016), and plants (Rieseberg, Reference Rieseberg2001). Moreover, recombination is shifted towards distal parts of heterozygous metacentrics in the shrew S. araneus (Borodin et al., Reference Borodin, Karamysheva, Belonogova, Torgasheva, Rubtsov and Searle2008).
Comparison between a spontaneous and a stable polymorphic centric fusion
A spontaneous fusion between pairs 5 and 7 (fusion 5/7) was found in a double heterozygote male that also conveyed the 3-3/6-6 trivalent. The number and position of chiasmata in both trivalents were compared, the 5/7 trivalent did not show any change of proximal (to the centromere) chiasma number or position, whereas the effect of the 3/6 centric fusion was conspicuous (Colombo, Reference Colombo1987). The higher frequency of proximal chiasmata in 5/7 trivalent when compared with the 3/6 one was associated with an increased proportion of linear orientation, thus conducing to the formation of unbalanced gametes. As both trivalents occurred in the same individual, it can be assumed that the genetic background was the same.
Hence, it was concluded that the 3/6 fusion triggered a shift of chiasma position towards the distal extremes – not so the 5/7 – and this may have been crucial in the maintenance of the polymorphism. Furthermore, the reduction of recombination between the arms of the newly arisen metacentric and its acrocentric homologues would have entailed a genetic differentiation – above all close to the centromere – between them. Rieseberg and Burke (Reference Rieseberg and Burke2001) stated that rearrangements reduce recombination in the vicinity of the rupture points; in this case, this point is the centromere, where two linkage groups no longer segregate independently. This linkage disequilibrium (LD) may have encompassed genes for local adaptation that would explain the creation of a chromosomal cline such as that exposed here. Wellerenreuther et al. (Reference Wellerenreuther, Rosenquist, Jaksons and Larson2017) demonstrated the existence of such genes within the inversions of the dipteran Coelopa frigida, which shows clines for inversions in the North and Baltic Seas correlated with temperature.
Three parallel fusion clines: the case of C. aquaticum
Here we review the results of our previous work on newly collected populations of C. aquaticum on the abundant, southwardly flowing rivers Paraná and Uruguay (Colombo, Reference Colombo2007, Reference Colombo2008, Reference Colombo2009; Romero et al., Reference Romero, Colombo and Remis2014, Reference Romero, Colombo and Remis2017; Colombo and Remis, Reference Colombo and Remis2018; Colombo et al., Reference Colombo, Zelarayán, Franceschini and Remis2021, Reference Colombo, Bressa and Remis2023). Both large parallel rivers are born in the Brazilian rainforest and converge into the River Plate at latitude 34.5°S in the Paraná Delta, just north of Buenos Aires city. Seven populations from Argentina were sampled along the Paraná Basin since 2005, namely: Corrientes (LP, 27°S), Santa Fe (SF, 31.8°S), Rosario (RO, 33°S), San Nicolás (SN, 33.33°S), San Pedro (SP, 33.5°S), Zárate (ZA, 34°S) and Tigre (TI, 34.4°S). Three parallel fusion clines were found for fusions between L and M pairs, namely 2/5, 1/6 and 3/4. In fact, these fusions (the same ones reported by Mesa (Reference Mesa1956) and Mesa et al. (Reference Mesa, Ferreira and Carbonell1982)) were detected along the middle and lower course of the river; therefore, there are 33 = 27 possible karyotypes. Fusion frequencies increase southward and downstream and the clines are steep: at the southernmost point they arrive to a frequency of 0.85 (they do not reach fixation) (Colombo, Reference Colombo2008). At this point the Paraná River flows into the River Plate, where there are no water-hyacinths, and consequently no C. aquaticum. In table 2a the frequencies for each fusion are shown, along with environmental variables they were correlated with, such as maximal, minimal and average temperature and yearly rainfall. Furthermore, the average fusion frequency per individual (fpi) is displayed for each population.
Geographic and climatic variables associated with Cornops aquaticum populations from the Paraná (a) and Uruguay (b) River basins
Lat, South latitude; MinT, mean annual minimum temperature; MaxT, mean annual maximum temperature (in °C); AvgT, mean annual average temperature; Rainfall, average yearly rainfall (in mm); fusion 2/5, 1/6 and 3/4 frequencies are also indicated; fpi: average fusion number per population. Abbreviations of grasshopper populations are given in fig. 2.
a In these populations fpi could be scored, but they were not identified due to poor fixation.
b May also be regarded as part of the Uruguay Basin (see explanation in the text). All of the data were previously published (see quotations in the text).
Later, in co-operation with Dr M.I Remis and Lic. M.L. Romero, we set forth to sample six more populations on the parallel flowing Uruguay Basin, another oversized tributary of the River Plate, with an additional latitudinal cline, only steeper. This cline displays higher fusion frequencies when compared to the same latitude of the Paraná cline up to the GU population, which has an average number of fusions per individual (fpi) of 3.3 (table 2, fig. 2). For example, SN, on the Paraná basin, at the same latitude has an fpi of only 0.57.
Sampling area and Cornops aquaticum populations included in this study. Populations from the Paraná River Basin: Laguna Pampín (LP), Santa Fe (SF), Rosario (RO), San Nicolás (SN), San Pedro (SP), Villa Paranacito (VP), Zárate (ZA) and Tigre (TI). Populations from Uruguay River Basin: Monte Caseros (MC), Salto Grande (SG), Colón (CO), Concepción del Uruguay (CU) and Gualeguaychú (GU) Pie diagrams indicate the frequencies of the centric fusions 2/5, 1/6 and 3/4 in dark grey. All data were previously published elsewhere (see quotations in the text).
However, further downstream from GU, Villa Paranacito (VP) has an fpi of only 1.65. A possible explanation for this anomaly may be that the Paranacito River, sited at the deltaic inundation zone of both convergent basins, is one of the many sprawling branches of the much mightier Paraná (hence its name), and flows into the final course of the Uruguay River. It communicates both basins: the stream (and the floods) of the Paraná basin would drag the floating mats of P. crassipes in which C. aquaticum lives into the smaller Uruguay. We think therefore that the fact that VP fusion frequencies fit closer to the Paraná than to the Uruguay cline is not a shocking anomaly. In fact, if we include it among the Paraná basin populations (to which it may arguably belong), VP fits neatly into this cline on account of its latitude (table 2b).
Correlation with environmental variables
The latitudinal, parallel clines between all three fusions and both rivers set the stage for level I in Endler's scale (Reference Endler1986): correlation between the trait (fusions in this case) and environmental variables. First a correlation was performed between individual fusions (and fpi) and climatic variables. All three fusions and fpi were positively correlated with latitude (cf. .L. argentina) and highly and negatively so with maximum temperature (P = 0.0036 for 2/5, P = 0.0017 for 1/6 and P = 0.0037 for 3/4; P = 0.0004 for fpi) (Colombo and Remis, Reference Colombo and Remis2018). None of the other variables analysed displayed any significant association with the fusions. As for stepwise correlations, there were no changes: all three fusions and fpi showed a negative regression on maximum temperature (2/5: P = 0.0076; 1/6: P = 0.0026; 3/4: P = 0.0040 and fpi: P = 0.0002). None of the other variables were even allowed into the analysis.
Phenotypic effects of C. aquaticum's fusions
Romero et al. (Reference Romero, Colombo and Remis2014) found an association between the fused 1/6 chromosome dosage and body size, which was significant for tegmina length, the provenance of the sample being the deltaic ZA and SP totalling 32 males. Colombo and Remis (Reference Colombo and Remis2018) analysed a more robust sample of 272 individuals, and the joint analysis revealed that fusions 2/5 and 3/4 are significantly associated with larger body size-related traits such as femur and tegmen length, fusion 1/6 not showing any significant effect. Therefore, the average fusion number per individual (fpi), which can take any number between zero and 6, is positively correlated with tegmen and femur length; otherwise stated, the regression of body size on fusion number is significant (Colombo and Remis, Reference Colombo and Remis2018).
As fusion frequency increases southwards, and fusion number (0–6) is correlated with body size, it could be expected that body size correlates with latitude as well. Indeed, it's the way round: body size decreases with latitude and increases correspondingly with temperature and rainfall (Colombo and Remis, Reference Colombo and Remis2018). In this case, the within-population effect is unlinked with the difference between populations.
This finding contradicts Adis et al. (Reference Adis, Sperber, Brede, Capello, Franceschini, Hill, Lhano, Marques, Nunes and Polar2008), who studied morphometrical variables of C. aquaticum all along a latitudinal transect between Trinidad (10°N) in the Caribbean and Punta del Este (35°S) on the River Plate's mouth. The insects were larger as the sampling site approached the Equator in this continent-wide study. The contradiction may lay on the scale of the survey. At Argentina's latitude C. aquaticum is bivoltine, but further north the number of generations a year increases, forming (seemingly) a seesaw effect. We failed to notice this effect in L. argentina because it does not occur much further north than Tucumán (27°S), where it is bivoltine and tiny, and further south is univoltine, with plenty of time for development.
We observed that, in C. aquaticum, there is a correlation between fusion dosage and body size (although fusion 1/6 does not affect body size related traits in C. aquaticum, Romero et al., Reference Romero, Colombo and Remis2014; Colombo and Remis, Reference Colombo and Remis2018).
Needless to say that the correlation of body size on fusion dosage does not mean necessarily that fusions are causing it: it is just a correlation. Secondly: if we do accept that some fusion is causing an effect, it would be too much of a leap of faith to assume that it is its function. However, it must be noted that the association between karyotype and phenotype is consistent in both species. This effect may be the target of selection (Norry and Colombo, Reference Norry and Colombo1999) or not (Colombo et al., Reference Colombo, Pensel and Remis2001, Reference Colombo, Pensel and Remis2004).
Did we demonstrate selection in these grasshoppers?
In the case of L. argentina there are many evidences of natural selection acting on the F and S polymorphisms (no matter how indirect this evidence may be) especially when we focus on the centric fusion: Endler's I, IV, V, VI and VIII criteria are comfortably met. In C. aquaticum we have three centric fusions, forming steep parallel clines, starting at the same point and always with the same frequency until they don't reach fixation because even the Paraná River was too short for them. To be sure, we have the Uruguay River besides, with a comparable cline. Too much of a coincidence. In addition, all three fusions correlate negatively and highly significantly with maximum temperature (same as L. argentina). But not much else.
Colombo (Reference Colombo2007) reported the recombination effects of all three fusions, which were coincidental in both species, i.e. (1) formation of a new linkage group from the two erstwhile independently segregating chromosome pairs, and (2) increase of distal (D) chiasma localisation, to the expense of proximal (P) and interstitial (I) chiasma formation in the chromosomes involved in the fusion, leading to a reduction of recombination in the fusion bivalent and an almost suppression in the trivalent.
A constant feature of large centric fusion polymorphisms is that recombination between the resulting metacentric and their acrocentric homologues is greatly reduced in the resulting trivalent of heterozygotes. The reason for this is that unrestricted chiasma formation in trivalents would lead to proximal and interstitial (with respect to the centromere) chiasmata. This would entail a rise of linear orientation of the trivalent due to spatial reasons to the expense of alternate orientation that renders V-shaped trivalents and balanced gametes (Bidau and Mirol, Reference Bidau and Mirol1988; Colombo, Reference Colombo2009, Reference Colombo2013). The ensuing reduction of recombination between the fused metacentric and the acrocentrics may provoke a partial genetic differentiation between both, especially in the regions close to the centromere, thus capturing locally adapted genes.
This differentiation between the rearranged and the non-rearranged homologue chromosomes of structural heterozygotes was often invoked as an explanation for inversion polymorphisms in Drosophila (Kirkpatrick, Reference Kirkpatrick2010), which capture advantageous haplotypes in linkage disequilibrium (Kirkpatrick and Barton, Reference Kirkpatrick and Barton2006; Charlesworth and Barton, Reference Charlesworth and Barton2018). According to this hypothesis the inversion is favoured because it prevents the breakdown of linkage disequilibrium caused by migration.
The parallel with C. aquaticum suggests an origin, somewhere in the Paraná middle course, of the Rb fusions and a local adaptation to it, and a southward migration helped by the current that drives the water-hyacinth floating meadows downstream. Upstream migration would be difficult for obvious reasons, although not impossible for a good flyer as C. aquaticum (Colombo et al., Reference Colombo, Zelarayán, Franceschini and Remis2021).
The hypothesis of the reduced recombination is endorsed by the fact that the most downstream populations, with high fusion frequencies, are both the most genetically disparate and most genetically impoverished when a microsatellite study was performed (Romero et al., Reference Romero, Colombo and Remis2017). The other populations could not be distinguished by the model and were lumped together under a Bayesian approach (Romero et al., Reference Romero, Colombo and Remis2017). When it came to population genetics, there were just three populations: the deltaic ZA and TI, and all the other lumped together.
Being neutral, these microsatellite distributions could be taken as null hypotheses when it came to compare the morphological distribution; in this case, the morphological Bayesian approach singled out just the northern, distant and subtropical LP and SF, pooling together all the others, deltaic or not, chromosomally polymorphic or not. Therefore, the morphological pattern must reflect season length and water availability and does not seem to reflect either genetic or chromosomal patterns (Colombo et al., Reference Colombo, Zelarayán, Franceschini and Remis2021).
Conclusions
The evidences for natural selection operating on chromosome polymorphisms in wild populations of L. argentina are based on the rejection of several null hypotheses, such as random geographical and environmental distribution of the rearrangements, gametic phase equilibrium, unsteadiness of chromosome morphs along the years (or the seasons), non-significant differences between age classes and random mating (Endler, Reference Endler1986). Additional, more indirect (but concurrent) evidence for F are: consistent correlation with phenotypic variables and an inverted association with recombination, due to both the loss of independent segregation and of proximal chiasma reduction.
Cornops aquaticum is less docile. All of the evidence we got is circumstancial, such as geographical and environmental correlations, association with phenotypic variables and the well-known twofold reduction of recombination of Robertsonian rearrangements. What makes it special is the amount of centric fusions involved in the clines (second only to Dichroplus pratensis, Bidau and Martí, 2005) and the microsatellite study performed by us (Romero et al., Reference Romero, Colombo and Remis2017) which revealed that the most polymorphic populations (ZA and TI) had the least genetic variability and were genetically detached from the rest.
However, part of Endler's sixth criterion (Reference Endler1986) consists in the perturbation of natural populations by introduction into equivalent, or non-equivalent habitats: the reproduction of clinal patterns where the species did not use to live. There are many examples of it in the literature, to mention only two: D. melanogaster (Kapun et al., Reference Kapun, Fabian, Goudet and Flatt2016) and D. subobscura (Prevosti el al., Reference Prevosti, Serra, Segarra, Aguadé, Ribó and Monclús1990) whose latitudinal clines from European flies reproduced elsewhere upon spontaneous transplant, even in an inverted fashion for South American populations. If C. aquaticum is introduced in South-Africa as a plague control – at roughly the same latitude studied by us in South America, i.e. 27°S and 34°S – and the cline is spontaneously reproduced, that would be the real and ultimate test of adaptive Robertsonian clines among the Orthoptera.
One more segment yet
All of L. argentina's sampled populations are polymorphic for segment S; being heterochromatic, it should bear no genes. But it's always there. It shows highly significantly negative regression on rainfall, and heterozygotes are subject to longevity selection in interaction with F. In addition, heterozygote frequency for S decreases significantly along the breeding season (see above). We cannot even start to speculate about the causes, only notice that neutrality may be an inadequate explanation for these phenomena.
Colombo (Reference Colombo2005) reported the existence of a latitudinal cline for a massive SS polymorphism on pair 9 of C. aquaticum between 27ªS and 34ªS whose slope was contrary to that of the fusions. Furthermore, given that in our long and extensive survey of this species we frequently noticed the presence of other heteromorphic bivalents, we performed the time-honoured C-banding technique to understand this abundance of supernumerary heterochromatin as well (Colombo et al., Reference Colombo, Bressa and Remis2023). By doing this, we detected three more C-positive polymorphic supernumerary segments in two nearby populations (ZA and VP). Now we have seven chromosomal polymorphisms in C. aquaticum to make sense of: three centric fusions, plus four supernumerary segments – at the moment. It makes 37 = 2187 possible different karyotypes: not Drosophila, but not too bad for a grasshopper.
Acknowledgements
The author is indebted to Dr María Isabel Remis for kindly reading the manuscript and providing helpful suggestions.
Financial support
Financial support from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2018-02567) (Argentina) to Dr Mª Isabel Remis is gratefully acknowledged.
Competing interests
None.
