Background: Genetic systems involving multiple X chromosomes have arisen repeatedly in sexually reproducing animals. Tiger beetles (Cicindelidae) exhibit a phylogenetically ancient multiple-X system typically consisting of 2-4 X chromosomes and a single Y. Because recombination rates are suppressed in sex chromosomes, changes in their numbers and movement of genes between sex chromosomes and autosomes, could have important consequences for gene evolution and rates of speciation induced by these rearrangements. However, it remains unclear how frequent these rearrangements are and which genes are affected.

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Cicindela littoralis and Cicindela flexuosa were analysed at population level to determine the localization and activity of ribosomal genes. Fluorescence in situ hybridization (FISH), using a PCR-amplified 18S rDNA fragment as a probe, revealed the presence of polymorphism regarding the number of chromosomes with ribosomal genes as well as their localization within the genome. Nine populations of C. littoralis showed a consistent pattern of two loci located in an autosomal pair (active during spermatogenesis as shown by silver staining) and one locus located in one of the multiple X chromosomes (silent during spermatogenesis), whereas individuals from the population of Punta Entinas showed only signals in the autosomal pair, lacking the heterosomal locus. In C. flexuosa, two patterns were also observed. Nine populations showed two loci in an autosomal pair whereas individuals from the population of San Pedro del Pinatar showed the two loci located in the heterosomes (one of the multiple Xs and in the Y). The hypothesis that these two different populations may reflect a status of well-differentiated phylogenetic entities is not supported for C. littoralis after the phylogenetic analysis of a fragment of the cytochrome oxidase I gene.

In this work, the first cytogenetic data on Neotropical Collyrinae is provided, by way of their karyotypes, C-banding and ribosomal genes (rDNA) localization using fluorescence in situ hybridization (FISH). The two species analysed, Ctenostoma (Procephalus) ornatum ornatum (male) and Ctenostoma (Euctenostoma) rugosum (female) showed, respectively, a diploid number of 17 and 18 chromosomes. C. ornatum ornatum has a multiple sex chromosome system (n = 7 + X1X2Y), and mitotic and meiotic metaphase cells showed rDNA gene labelling in the smallest autosomal pair. In this species, no C-bands were obtained, while C. rugosum seems to exhibit centromeric and/or interstitial C-bands in almost all chromosomes. The observation of a multiple sex chromosome system in Ctenostomini ensured the appearance of this characteristic in the hypothetical ancestral of Collyrinae and Cicindelini. The subfamily Collyrinae is not uniform in what concerns diploid chromosome number and rDNA gene localization, because C. ornatum ornatum possesses a lower chromosome number and autosomal rDNA genes when compared with the other Collyrinae species studied (Neocollyris spp.). Independent events leading to the reduction in chromosome number might have taken place during the split of the Collyrinae into the tribes Ctenostomini and Collyrini.

-Karyological investigations were carried out on five species of tiger beetles from the Afrotropical Region belonging to the tribes Megacephalini and Cicindelini. The species have distinct karyotypes either in the number of autosomes and heterosomes or in size and shape of the chromosomes: Megacephala megacephala and Prothyma lepneurii have 2n = 24+X,X 2 Y/ 24+X 1 X 1 X 2 X 2 , Euryarthron festwum has 2n= 22+X ] X 2 Y/22+X 1 X 1 X 2 X 2 , Ropaloteres feisthameli has 2n = 20+X 1 X 2 X,X 4 Y/20+XiX 1 X 2 X 2 X 3 X 3 X 4 X 4 and Hipparid-ium intenuptum has 2« = 20+X ] X 2 X 3 Y/20+XjX 1 X 2 X2X3X 3 . The existence of 12 pairs of autosomes in tribe Cicindelini is reported for the first time, being found in the genus Prothyma, the most ancient genus of this tribe. This supports the hypothesis that the chromosomal evolution in the Cicindelidae involved a reduction in number of chromosomes in the derived groups since the same number of autosomes or even more were found to occur in the most primitive tribes Collyrini and Megacephalini. All species have multiple sex chromosomes, apparently a characteristic of this family. Ropaloteres feisthameli has a sex chromosome system with four X chromosomes, only described for Cicindela maroccana pseudomar-occana and Cylindera trisignata.

Mitotic and meiotic chromosomes of Megacephala brasiliensis, a representative of the tribe Megacephalini‐a basal group in the family Cicindelidae‐were studied using conventional Giemsa‐staining, C‐banding and fluorescence in‐situ hybridization (FISH) with 18S rDNA probe. The diploid chromosome number was 2n=12 and the karyotype contained five pairs of metacentric and submetacentric autosomes and one pair of sex chromosomes, XY and XX in male and female, respectively. The X chromosome was metacentric while the Y was subtelocentric. All autosomes and the X showed distinct, large C‐positive blocks around the centromere indicating extensive distribution of constitutive heterochromatin. The Y chromosome showed a conspicuous large C‐positive block in the subterminal region as well as a weak interstitial band. FISH with a rDNA probe gave consistently positive signals in the pericentromeric region of the fourth pair of chromosomes. Size polymorphism of this rDNA‐positive site was always observed. The karyotype of this beetle exhibits the smallest 2n known for any tiger beetle of the genus Megacephala and of other basal groups of the family though it shares single sex chromosome system with them. The extreme reduction in the autosome number appears to be the result of massive and combined rearrangenments of various types. The discovered case might parallel the famous “Muntjac scandal” in mammal cytogenetics.

Four Neotropical tiger beetle species, three from the genus Megacephala and one from the genus Oxycheila, currently assigned to the tribe Megacephalini were examined cytogenetically. All three Megacephala species showed simple sex chromosome systems of the X0/XX type but different numbers of autosomal pairs (15 in M. cruciata, 14 in M. sobrina and 12 in M. rutilans), while Oxycheila tristis was inferred to have a multiple sex chromosome system with four X chromosomes (2n = 24 + X 1 X 2 X 3 X 4 Y/X 1 X 1 X 2 X 2 X 3 X 3 X 4 X 4 ). Fluorescence in situ hybridization (FISH) using a PCR-amplified 18S rDNA fragment as a probe revealed the presence of rDNA clusters located exclusively on the autosomes in all the Megacephala species (five clusters in M. cruciata, eight in M. sobrina and three in M. rutilans), indicating variability in the number of clusters and the presence of structural polymorphisms. The same methodology showed that O. tristis had six rDNA clusters, apparently also located on the autosomes. Although our data also show cytogenetic variability within the genus Megacephala, our findings support the most accepted hypothesis for chromosome evolution in the family Cicindelidae. The description of multiple sex chromosomes in O. tristis along with phylogenetic analyses and larval morphological characters may be assumed as an additional evidence for the exclusion of the genus Oxycheila and related taxa from the tribe Megacephalini.

Two species of Odontocheila, O. confusa and O. nodicornis, from the Neotropical Region were studied regarding their karyotypes, localisation and activity of ribosomal genes and C-banding. The species, although belonging to the same genus, have quite distinct karyotypes. O. confusa has 10 pairs of autosomes and a single sex chromosome mechanism of the XY/XX type, thus a diploid value of 2n = 22 in males and females. One aneuploid male with a diploid number of 2n = 20 and one male with three B chromosomes were found in a total of eight males studied. O. nodicornis has 17 autosomal pairs and also a single chromosome system but of the X0/XX type, thus a diploid value of 2n = 35 in males and 2n = 36 in females. Fluorescence in situ hybridisation (FISH) revealed the presence of rDNA clusters in two autosomes in both species in mitotic and meiotic figures. Silver staining of male interphase nuclei confirmed the FISH results and showed that all rDNA genes were active. C-banding analysis revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes in the two species plus two pairs in O. nodicornis with terminal positive C-bands. These results are discussed from the cytogenetic and evolutionary point of view.