Karyotype evolution in Aeshna ( Aeshnidae, Odonata)

1994. Karyotype evolution in Hered- itcts 121: ISSN 1994 The haploid DNA content of Aeshnu confiisa (2n = 27, n = 13 + XO, male). A. bonuric,nsis (2n = 26, n = 12 + neo-XY, male) and A. cornigero plunultica (2n = 16, n = 7 + neo-XY, male) has been determined (2.16 i 0.16 pg, 1.81 k 0.17 pg, and 2.08 k 0.08 pg, respectively). Despike the differences in chromosome size end number, differences in DNA content between species are nok significanl. The karyotypic analysis of ,4e.shnu species leads to the conclusion that fusions between autosomes or autosome and thc scx chromo- some, are the only chromosome rearrangement that occurred during evolution. In the species here studied, fusions have taken place with a minimal loss of DNA; however, other species of the genus show important differences in genome size, which cannot only he justified by fusion events.

The comparison of DNA content of related species is of great value in any analysis of karyotype evolution. This is particularly true in holokinetic systems, in which the lack of a localized centromere rcstricts thc numbcr of karyotypes charactcristic possible to consider, and also because fusions and fragincntations are the principal chromosome rearrangcnicnts obscrvcd (SYHENGA 1972;WHITE 1973). In spite of this, DNA content has been seldom determined in insects with holokinetic chromosomcs ( HUGHES-SCHRADER and SCHRADER 1956;SCHRADER andHUGHES-SCHRADER 1956, 1958;SCHREIBER et al. 1972;MELLO et al. 1986;PAPESCHI 1988PAPESCHI , 1991 and are almost absent in Odonata ( C U M M I N G 1964;PETROV and ALJESHIN 1983;PETROV et al. 1984). In the genus Aeshna, twenty-five spccies have been cytogenetically analyzed up to date, and the diploid number varies in malcs bctwccn 16 (14 + nco-XY) and 27 (26 + XO).
In the present work DNA content has been determined in Aeslina crrnfusa (2n = 26 + XO, male), A. honariensis (2n = 24 + neo-XY) and A . Cytological preparations for DNA measurements of the three species were obtained by squashing a piece of testis in 60 YO acetic acid; the coverslip was then removed by the dry-ice method and slides were air-dried. DNA charges were estimated by using the Feulgen reaction in conjunction with scanning densitometry. Thc Feulgen reaction was carried out as follows: air-dried slides were rinsed 3 times for 10 min each in distilled water, immersed in 5 N HCI for 20 min at 25 & I T , washed 3 times for 10 min each in distilled water, stained with the Schiff reagent for 2 h, and washed 3 times 10 min each in SO, water. The optimal hydrolysis time was previously determined through the calculation of a hydrolysis curve. Although the material had different fixation times, a previous analysis has shown that differences in DNA measurements in the same species with different fixation times are statistically non-significant ( MOLA 1992).
Only spermatid nuclei which have just begun to elongate, were measured. The number of slides per individual (from 1 to 3) and the number of individuals per species varied according to the number of cells and individuals suitable for this study. Chicken crythrocytes were used as standard of reference for the dctection and correction of any differences in thc Feulgen reaction among the slides used; according to RASCH et al. (1971) the DNA content of chicken erythrocytes is 2.5 pg. Twenty spermatid nuclei and twenty chicken erythrocyte nuclei were measurcd in each slide, and slides were coded and randomized prior to scoring. The measurements were conducted with a cytospectrophotometer Zeiss MPC 64 at a wavelength of 570 nm, attached to a Kontron MOP-Videoplan computer, with the program APAMOS 99.

Chromosome complement
The chromosome complement and meiotic behaviour of the three specics havc been previously describcd in detail (MOLA 1992). The principal features of the karyotype of these species can be summarized as follows: Aeshna confusu (2n = 27, n = 13 + XO) has a larger bivalent and a very small one (m bivalent), while the other eleven bivalents decrease gradually in size. The X chromosomc is larger than the m bivalent and of similar sizc to the second smallest one (Fig. la).
In A . bonariensis (2n = 26, n = 12 + neo-XY), the autosomal bivalents decrease gradually in size, except for the very small nz bivalent; the neo-XY is the largest bivalent and is heteromorphic (Fig. Ib).
A. cornigeru plunultica (2n = 16, n = 7 + neo-XY) has a reduced chromosome number and larger chromosomes than the Cormer two species. The autosomal bivalents can be grouped in five largc and two small ones, while the sex chromosome bivalent is the smallest one and noticeably heteromorphic (Fig. lc).  1). The analysis of variance of the data shows that differences between the three species and between individuals within each species are non-significant (Table I); however, differences between slides within each individual are significant.

Discussion
The modal karyotype of Aeshna (2n = 26 + XO, male) is present in 72 YO of the species (Fig. 2). In most of them a larger autosomal pair and a noticeably smaller one (in pair) are readily distinguished; the X chromosome is small and of similar size to the smallest bivalent ( MAKALOVSKAJA 1940;OK-SALA 1943;CUMMING 1964;CRUDEN 1968;KI-AUTA 1969;HUNG 1971;KIAUTA 1971KIAUTA , 1973KIAUTA andKIAUTA 1980, 1982). Thc chromosome complement of A. confusa presents these characteristics. Considering this modal karyotype as the ancestral one, it can be observed that during karyotype evolution in the genus fusions have taken place, involving autosomes and/or the sex chromosome. No increase in diploid number has been reported until present. The chromosome complement of A . bonuriensis (2n = 26, n = 12 + neo-XY, male) would have originated through the fusion of the original X chromosome with the largest autosomal pair, giving rise to a neo-XY system. A quite similar situation has been described in A. grandis, a species in which the neo-XY is the largest pair, heteromorphic and, hence, easily identified (OKSALA 1943;KIAUTA 1969). Thc chromosome complement of A . cornigera planaltica (2n = 16, n = 7 + neo-XY, male), which is much more reduced, would have originated through six fusions: five between autosomes and one between the original X chromosornc and thc smallcst autosornal pair.  Fig. l(a-c). DNA content in Aeshnu confusa (n = 13 + XO), A . honariensis (n = 12 + neo-XY) and A . cornigera planaltica (n = 7 + neo-XY). In a, b, and c, one cell at diakinesis from each species is shown in order to compare sizc and iiumbcr of chromosomes; the sex univalent or sex pair is indicated. Bar = I0 jim.
Hcredirah 121 (1994)  The large size of the chromosomes of A . cornigera plmcrlticri, when compared with those of A . con?fusa and A . honariensis, suggests that all the fusions that gave rise to such a reduced chromosome complement were probably accompanied by a minimal loss of DNA. As in A . cornigera plunalticcr, OKSALA ( 1943) described in A . coerulea that the sex bivalent was the smallcst of thc complement and hetcromorphic. The neo-XY system is particularly frequent in Aeshna (28 'YO of the species) since in the order only 5.4 'YO of the species have the neo-system. In many species of Aeslinrr, the heterotnorphism of the sex bivalent is easily recognized, a fact that is also unusual in other genera of Odonata.
DNA content is not always correlated with chromosome number and size, and particularly in insecls with holokinctic chromosomes different situations have been cncountcrcd. Related species whose karyotypcs diffcr by one or more fusions or fragmentations, can show constancy in DNA content, as in Thynntu and Bancisci (Heteroptera) (SCHRAIER and HUGHES-SCHRADER 1956, 1958 or significant differences in genome size, as described in some species of Balo.rtomr1 (Heteroptera) (PAPESCHI 1988). On the other hand, there arc also examples of related species with the same diploid chromosome number but significant differences in DNA content, as in Triutoma and other species of Belostoma (Heteroptera) ( SCHREIBER et al. 1972;PAPESCHI 1991).
In the species of Aeshna here analyzed, no diffcr-  THOMAS 1992). This fact suggests that fusions could be associatcd with a noticeable loss of DNA, not necessarily involving loss of information. PETROV andALJESHIN (1983), andPETROV et al. (1984) cstimated the haploid genome size of clcvcn species of Odonata belonging to 6 families, by means of DNA reassociation kinetics. They obtained values ranging from 0.37 pg to 1.7 pg. According to thesc authors, the DNA content of A. c.ocwdLw (as A . .rquamata) (2n = 24, n = I I fneo-XY) and A . ,junceu (2n=26, n = 12 + nco-XY) is 1.6 pg and 1 .0 pg, respectively. As both spccies differ only in one autosomal fusion and the species with higher chromosome number has a lowcr DNA content, it is evident that the DNA differences are not associated with the fusion itself; instead, thcy have probably occurred indc-