Phylogenetic character reconstruction at the species level is useful to test for trait changes associated with speciation events . The incompatibility of rearranged chromosomes could be an important factor leading to reproductive isolation and speciation, in particular where such changes are linked to the sex chromosomes. The genus Cicindela is very species rich, which is usually ascribed to ecological shifts in habitat associations [34, 35] and a tendency for the isolation of local populations [36, 37]. However, karyotypic features have not previously been considered to affect species diversification in this group. Karyotype analyses in Cicindela are technically challenging, due to the small size of chromosomes and the absence of the polytene chromosomes common in Diptera, plus the difficulties of obtaining meiotic cells (where multiple sex chromosomes can be accurately identified) except during a short season of the year. Yet, our sampling was sufficiently dense for a comparative analysis. We found that the multiple sex chromosomes provide an evolutionarily dynamic system affected by repeated gains and losses of X chromosomes, and repeated shifts in the localization of the rDNA loci between autosomes and heterosomes, with potential consequences for speciation.
Mechanistically, these shifts in X chromosome numbers do not appear to involve reciprocal rearrangements in the autosomes, as the latter remain invariable, arguing against a mechanism for the origination and loss of X chromosomes through fusions with autosomes. The independence of autosome and heterosome numbers has previously been established in lineages of Cicindela in the Palearctic, India and Australia, although in those cases changes affected mainly the autosome numbers while heterosomes were invariable for the modal X1X2X3Y system [38, 39]. The absence of autosomal-heterosomal fusions and fissions was also supported by the fact that we did not observe any physical associations of a chiasmatic type between autosomes and heterosomes during meiosis which would be required for such rearrangements. Instead, in all cases studied here the multiple X chromosomes and the Y were connected at the telomeric ends during pairing in male meiosis and were visible as a conspicuous multivalent where (Fig. 1), as had already been described for the Palearctic species C. hybrida .
The lack of chiasmatic associations between heterosomes and autosomes also argues against a role of fusions and fissions to be responsible for the positional changes of the rDNA clusters. The number and position of rDNA loci can readily be altered by Robertsonian changes (fissions or fusions) via terminal nucleolus organizer regions (NOR) usually containing the rDNA clusters. Two chromosomes each carrying a terminal NOR may either fuse to produce an interstitial NOR, or a single chromosome with an interstitial NOR may undergo fissions resulting in two chromosomes with terminal NORs. However, except perhaps for the case of C. marginata with six rDNA loci, we did not see the expected changes in number and position of rDNA loci, as changes in chromosome number and rDNA loci were independent. Therefore neither fissions nor fusions at NORs are supported, unless one invokes additional rearrangements such as pericentric inversion to accommodate the predominantly mediocentric chromosomes in Cicindela. Furthermore, considering the achiasmatic nature of heterosomes, Robertsonian rearrangements cannot explain the changes that occur simultaneously in autosomes and heterosomes.
An alternative scenario are non-reciprocal translocations affecting the rDNA clusters which could lead to changes in position (translocation) or numbers of rDNA sites (transposition retaining a copy at the origin). These changes can be facilitated by the presence of transposable elements, as e.g. in the Type I and II ribosomal gene insertions in Drosophila melanogaster, Bombyx mori , Apis mellifera and other Hymenoptera . Translocations between chromosomes bearing rDNA loci and those lacking them have been invoked to explain the rearrangements observed in Paeonia  and Allium , and also may occur in the ground beetle genus Zabrus which presents the highest variability in the number of rDNA sites (2–12) found so far in insects . However, these rearrangements occur among autosomes only, not affecting the sex chromosomes. The peculiar situation in C. marginata, where translocation of rDNA copies between chromosomes and even within a chromosome may be responsible for the unusual number of rDNA loci, might be a good model for elucidating the cytogenetic basis for the changes in number of X chromosomes and gene content.
Whatever the mechanisms that produce the change of number and position of rDNA clusters, they seem to be affected by various constraints to the rearrangements of karyotypes. Careful inspection of the chromosome preparations suggested that the rDNA loci were not always in the same pair of autosomes, and may be localized on different homeologous pairs, even in closely related species. Therefore, numerous additional chromosomal rearrangements between autosomes and heterosomes may be present which are not detected here when scoring chromosome numbers and rDNA autosomal-heterosomal localization only. Yet, the overall gestalt of the karyotype was maintained despite these apparent changes in gene content. Nine pairs of autosomes were found in all species, showing very similar sizes and phenotypes. This degree of conservation appears to be specific to the multiple-X karyotype, as such constraints on chromosome morphology are not seen in the ancestral simple-X system of distantly related cicindelids which differ greatly in chromosome number and overall morphology . Similarly, there are apparent constraints to the number and gene content of heterosomes. For example, the X1X2Y and X1X2 X3 X4Y karyotypes represented derived states which were confined to a single species (or perhaps small clades, had denser taxon sampling been available), indicating that deviations from the modal X1X2X3Y system are evolutionarily short-lived and unstable. Finally, the chromosomal positions of rDNA clusters also appeared to be constrained in the multiple-X system. The autosomal number of clusters was two (one pair of allelic copies), but never exceeded this number, and when more than two rDNA copies were found in Cicindela they always appeared on the heterosomes, most commonly on the X only, and in some species a further rDNA copy on the Y chromosome. This is in contrast to the single-X chromosome systems in the basal groups of Cicindelidae which exhibit between four and eight (two to four pairs) autosomal rDNA clusters . The nature of these constraints on the karyotype remains unknown but they may be linked to the evolutionary stability of this multiple-X system .
Despite the morphological conservation of the multiple-X system, the frequent movements of genes between autosomal to heterosomal positions expose the affected loci to greatly altered dynamics of gene evolution and recombination. As there is no cross-over in the male heterosomes in cicindelids, rates of homologous recombination in the sex chromosomes are reduced by half for the X chromosomes (recombination only in females) and to virtually zero for the Y (resulting in their inevitable degradation; ). The hemizygous nature of the X will greatly increase selection on recessive mutations, altering the rate and kind of mutational changes. This would cause the rearranged genes to diverge quickly, even if rearranged gene regions are duplicated. In the case of the rDNA clusters, this could reduce the rate of homogenization of copies in different parts of the genome. For example, an analysis of sequence variation in the ITS1 region of the rDNA cluster in C. dorsalis, a species shown here to exhibit rDNA copies on a pair of autosomes plus a single copy on the X, exhibited greatly divergent ITS types, possibly corresponding to heterosomal and autosomal copies . This supports the idea that the translocation to the sex chromosomes results in changes of evolutionary dynamics.
These kinds of chromosomal rearrangements might also have an effect on speciation. Translocations of genes between autosomes and sex chromosomes will greatly change the possibilities for gene flow, and changes in the number of X chromosomes may alter the production of balanced gametes. Incompatibility of gametes with different numbers of X chromosomes, or indeed changed position of rDNA clusters, could lead to incorrect separation of chromosomes during anaphase I in a hybrid, and produce a number of unbalanced gametes resulting in reproductive disadvantage. In the case of changes in X chromosome numbers this effect could be exacerbated by altering the sex determination control and the gene regulation associated with changes in heterosome number. As pointed out in the recent literature [1, 6, 7, 47], it is not likely that these 'underdominant' variants become established in a population. This has provided a strong argument against the stasipatric model of speciation  which suggests that chromosomal rearrangements cause reproductive isolation due to hybrid dysfunction. However, when involving sex chromosomal unidirectional rearrangements as those in Cicindela, this model may still be valid. Depending on the precise genotypes participating in a mating, the combination of certain gametes could lead to a significant proportion of inviable (e.g., no rDNA clusters, unbalanced number of X chromosomes) zygotes, selecting against heterozygotes and maintenance of polymorphisms in a population. These effects may be exacerbated by the reduction of gene flow from suppressed recombination and extending the effect of linked isolation genes, a mechanism proposed as the main driver for the fixation of novel karyotypes under more recent models [8, 48]. As gene flow is more restricted between sex chromosomes than autosomes, sex linked genes are particularly efficient to produce such postzygotic barriers , and hence rearrangements involving sex chromosomal portions of the genome may be a particularly effective isolating mechanism.
Therefore, the high level of apparent chromosome rearrangements and the deposition of a substantial portion of the genome in the low-recombining sex may promote speciation in Cicindela. With some 1,000 species world-wide, this is one of the largest genera of insects. In particular, the observation of evolutionarily short-lived X1X2Y and X1X2X3X4Y lineages suggests that these chromosomal changes could initiate reproductive isolation. If these rearrangements are frequent relative to other (ecological or geographical) processes influencing speciation rates, the cytogenetic parameters could drive speciation and possibly be responsible for the great species richness in Cicindela. In addition, the population structure of Cicindela is also favoring the fixation of chromosomal mutations locally, as most species are early succession specialists frequently occurring in isolated habitat patches where a dynamic system of colonization and extinction may enhance the separation of local genetic entities. In support of this possibility, we found two Iberian species, C. flexuosa and C. littoralis, where in each case a single population was fixed for an rDNA localization different from all other populations , indicating that local genetic races arise frequently.