Improved basic cytogenetics challenges holocentricity of butterfly chromosomes

Abstract

Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be holokinetic. This particularity, observed in a number of other invertebrates, is associated with meiotic particularities known as "inverted meiosis", in which the first division is equational, i.e., centromere splitting-up and segregation of sister chromatids instead of that of homologous chromosomes. However, the accurate analysis of butterfly chromosomes is difficult. 1) Their size is very small, equivalent to a single band of a mammalian metaphase chromosome. 2) They lack satellite DNA/heterochromatin in putative centromere regions and therefore marked primary constrictions. Our improved conditions for chromosome preparations in six butterfly species belonging to the Nymphalidae and Pieridae families challenges the holocentricity of their chromosomes: in spite of the absence of primary constriction, sister chromatids are recurrently held together at definite positions during mitotic metaphase, which makes possible to establish karyotypes composed of acrocentric and sub-metacentric chromosomes. The total number of chromosomes per karyotype is roughly inversely proportional to that of non-acrocentric chromosomes, which suggests the occurrence of frequent Robertsonian-like fusions or fissions during evolution. Furthermore, the behaviour and morphological changes of chromosomes along the various phases of meiosis do not differ much from those of canonical meiosis. In particular at metaphase II, chromosomes clearly have two sister chromatids, which refutes that anaphase I was equational. Thus, we propose an alternative mechanism to holocentricity for explaining the large variations in chromosome numbers in butterflies: 1) in the ancestral karyotype, composed of about 60-62 acrocentric chromosomes, the centromeres, devoid of centromeric heterochromatin/satellite DNA, were located at contact with telomeric heterochromatin; 2) the instability of telomeric heterochromatin largely contributed to drive the multiple chromosome rearrangements, which occurred during butterfly evolution.