A chromosomal analysis of three species of Timarcha (Coleoptera, Chrysomelidae, Chrysomelinae)

Abstract The karyotypes of three species of Timarcha Latreille, 1829 have been analysed. Timarcha (Metallotimarcha) metallica (Laicharting, 1781), has 18 + Xyp male meioformula and 2n = 38 chromosomes, similar to those found in the two species of subgenus Americanotimarcha Jolivet, 1948, in agreement with morphological and molecular phylogenetic grounds. Timarcha (Timarcha) carmelenae Petitpierre, 2013 displays 9 + Xyp and 2n = 20 chromosomes as in morphologically related Andalusian species, whereas Timarcha (Timarcha) parvicollis ssp. seidlitzi Kraatz, 1879 shows 11 + Xyp and 2n = 24 chromosomes, clearly differing from the previous species. These results are discussed in order to get an insight into the main trends of the chromosomal evolution in Timarcha.


Introduction
The highly speciose genus Timarcha Latreille, 1829 comprises more than three hundred described taxa, almost all from the Palaearctic (Gómez-Zurita 2008, Kippenberg 2010, Warchalowski 2010, and is relatively well-known from chromosomal standpoints because 42 taxa have been surveyed to date and their range of diploid numbers goes from 2n = 18 to 2n = 44 (Gómez-Zurita et al. 2004, Petitpierre 2011. Herein, we report the chromosome numbers, male sex-chromosome systems, and main features of their karyotypes of T. (Metallotimarcha) metallica (Laicharting, 1781), T. (Timarcha) carmelenae Petitpierre, 2013 and T. (Timarcha) parvicollis ssp. seidlitzi Kraatz, 1879 to enlarge the cytogenetic analysis of the genus and discuss the most relevant trends of its chromosomal evolution.

Material and methods
The three checked species and their geographical origins are given in Table 1. The chromosome analyses were only performed on male living individuals brought to our laboratory in Palma de Mallorca (Spain), where they were killed with ethyl acetate. The cytogenetic data were obtained by testis dissection of male adult specimens which were fixed in 45% acetic acid , later on teased into small pieces for five minutes, squashed under a coverslip, immediately frozen in liquid nitrogen to remove the coverslip, and finally treated using conventional Giemsa staining procedures. Most examined cells were at meiotic metaphase I, providing the male meioformulae, thus the number of autosomal bivalents plus the male sex-chromosome systems. Finally, we took micrographs by a ZEISS AXIOPHOT or a ZEISS AXIOSKOP photomicroscope, and subsequently enlarged them for printing.

Timarcha (Metallotimarcha) metallica (Laicharting, 1781)
Two males of this species have displayed 2n= 38 chromosomes and an 18 + Xy p male meioformula, with a "parachute" Xy p sex-chromosome system (Fig. 2). Its karyotype is composed of nine medium size and nine small autosome pairs plus a submetacentric X-chromosome of medium size and a tiny y-chromosome. Four of the medium size autosome pairs were acrocentrics and the remaining meta-or submetacentrics, and three of the small ones were acrocentrics and the other metacentrics, as shown by spermatogonial mitotic metaphases ( Fig. 1) and meiotic metaphases II (Fig. 3). Thus, the fundamental number (FN) of chromosomal arms is 50.  (4) and La Sagra (5) individuals, with 9 + Xy p meioformula, the Xy p are arrowed 6 T. parvicollis ssp. seidlitzi: meiotic metaphase I with 11 + Xy p meioformula, the Xy p is arrowed and two partly overlapped autosomal bivalents are arrowheaded. Bar: 5 µm.

Timarcha (Timarcha) carmelenae Petitpierre, 2013
One male individual from Sierra Seca and another from La Sagra provided meiotic metaphases I of 9 + Xy p , again with a "parachute" Xy p sex-chromosome system, that is 2n = 20(Xy p ) chromosomes, and showing two autosomal bivalents a bit larger than the others (Figs 4 and 5).

Discussion
The diploid number of 2n = 38 chromosomes shown in Timarcha (Metallotimarcha) metallica should correct a previous miscounting report of 2n = 20 chromosomes (Petitpierre 1982 (Jolivet and Petitpierre 1992). These high chromosome numbers are in agreement with the similar morphological traits, the male genitalia and the molecular phylogenetic resemblances between the subgenera Metallotimarcha Motschulsky, 1860 and Americanotimarcha (Jolivet 1948, Iablokoff-Khnzorian 1966, Gómez-Zurita et al. 2000, Gómez-Zurita et al. 2004, Jolivet et al. 2013. Although the species of both subgenera show some plesiomorphic features, such an incomplete fusion of elytra, weak sexual dimorphism, aedeagus with a long tegmen cap, and a basal position in the molecular phylogenetic tree, their high chromosome numbers can not be considered as an ancestral character. First, because 2n = 20(Xy p ) is assumed to be the plesiomorphic and most frequent karyotype condition for Coleoptera of the suborder Polyphaga (Smith andVirkki 1978, Angus et al. 2007). Besides, this is the most common karyotype in the genus Timarcha where more than a half of the 42 surveyed taxa show 2n = 20(Xy p ) (Petitpierre 2011). And third, the karyotypes of both T. metallica and T. intricata share a quite high number of acrocentric autosome pairs, seven and fourteen respectively, which is an indication of their derived origin by multiple centric fissions or chromosomal dissociations from meta-or submetacentric chromosomes. Therefore, we assume that a hypothetic karyotype of 2n = 20(Xy p ) chromosomes, mostly composed of metacentrics or submetacentrics, would have been the plesiomorphous state for the genus, from which all the taxa of the three present subgenera, Americanotimarcha, Metallotimarcha and Timarcha s.str. may have radiated.