The Chromosomes of three species of the Nasonia complex ( Hymenoptera , Pteromalidae )

The karyotypes of three sibling species, Nasonia vitripennis (WALKER, 1836), N. longicornis DARLING, 1990 and N, giraulti Darling, 1990 were examined, the latter two species for the first time. All species have chromosome numbers of n=5 and 2n=10, their chromosomes being metacentric. The distribution of constitutive heterochromatin also appears to be similar in the genus Nasonia Ashmead, 1904. However, statistical karyotypic differences between studied species were revealed using chromosome morpho­ metries. These data are consistent with the order of divergence in the Nasonia complex found on the basis of molecular studies.

markers is available (Saul, 1990).First data on its chromosome number, n=5, were obtained by Gershenzon ( 1946) and later confirmed by other studies (Pennypacker, 1958;G ershenzon, 1968;W hiting, 1968;N ur et a l, 1988).The only detailed karyotypic description of N. vitripennis was made by Reed (1993) who provided data on chromosome morphometries as well as on C-and Ag-NOR-bandings in this species.
The genus Nasonia A shmead, 1904 was considered to be monotypic for many years.Sur prisingly enough, two new sibling species, N. longicornis DARLING, 1990 and N. giraulti DARLING, 1990 were found in this genus in North America about ten years ago (DARLING & W erren, 1990).An extensive study of this complex revealed a number of morphological characters which could be used as distinction features for these taxa.In addition, many other characters could be used for separating these species with 0.95-0.999%certainty.Furthermore, N. vitripennis, N. longicornis and N. giraulti substantially differ in their courtship behaviour (van den A ssem & W erren, 1994).Chromosome number and other karyotypic features of the Pteromalidae appear to be highly conservative.Specifically, eight studied species of the family have n= 5 (Gokhman & Quicke, 1995;Gokhman, in press).Except for N. vitripennis, neither morphometric studies of karyotypes nor differential chromosome staining in these species, however, were performed to date.Moreover, n=6 was found in a local population of N. vitripennis from California (Goodpasture, 1974).Even among laboratory stocks of this species normally having n=5, some populations carrying a particular B chromosome were revealed (Nur et al., 1988;W erren, 1991).The present paper deals with the results of a karyological study of N. vitri pennis, N. longicornis and N. giraulti using routine and differential (C-) chromosome staining.

Material and methods
Chromosomes of all three Nasonia species were studied in wasps from laboratory cultures maintained at the Institute of Evolutionary and Ecological Sciences, University of Leiden, namely: Lab II, a laboratory stock of Nasonia vitripennis originally collected in Leiden more than 20 years ago; strain IV7 R2 o f N. longicornis derived from the strain IV7 (nest UT007) which had been collected in Utah, USA, in July 1988; and strain R16A o f N. giraulti derived from a particular strain (nest VA002) which had been collected in Virginia, USA, in Septem ber 1988.Air-drying chromosome preparations were made from cerebral ganglia of male and female prepupae, according to the routine procedure described bylMAI et al. (1988).Differen tial chromosome staining (C-banding) was performed according to the method developed by Sumner (1972) and modified by Gokhman (1997).Chromosomes were studied under mi croscopes MBR-15 and Zeiss NU-2.For making chromosome measurements, ten diploid metaphase plates for each species were scanned using static TV camera equipped with the image analysis program ImageExpert version 1.00.Scanned images were measured using Adobe Photoshop version 3.0.5.Statistical data analysis was performed with the help of STA-TISTICA version 4.3.The t test for independent samples was used as a statistical criterion.

Results
Numbers of studied individuals and metaphase plates for each species are presented in Table 1.Relative lengths and centromeric indices of all chromosomes of the three species are shown in Table 2. Chromosomes of N. vitripennis, N. longicornis and N. giraulti are very similar, especially for their centromeric indices.Due to this similarity, a unified karyotypic description is given for all species, with interspecific differences being added.The same chromosome numbers, n=5 and 2n=10, were found in N. vitripennis, N. longicornis and N. giraulti, all chromosomes being obviously metacentric and gradually decreasing in size (Figs.1-3).The first chromosome pair is slightly larger than the others.Chromosome 2 is significantly shorter in N. vitripennis than in N. giraulti or N. longicornis.Similarly, chromosome 3 is longer in the latter species than in N. giraulti.
The distribution of the constitutive heterochromatin also appeared quite similar in the three Nasonia species.All chromosomes have characteristic large segments of centromeric heterochromatin.Moreover, the shorter arm of the chromosome 3 is also heterochromatic, at least in most of the metaphase plates (Figs.4-6).

Discussion
All karyotypes of the Nasonia complex are symmetrical and very much similar to each other.
However, they differ from chromosome sets of some other Pteromalidae also having n=5.
Unfortunately, these differences can usually be revealed only after making detailed chro mosome measurements.For example, one of the two species belonging to the Anisopteromalus calandrae (HOWARD, 1881) species complex also has n=5 (G o k h m a n et ah, 1998), but its haploid karyotype is more asymmetrical, with two smaller chromosomes being visibly shorter than the third one.Moreover, the smallest chromosome of the latter species is submetacentric, whereas the others are clearly metacentric.
Gokhman, V. E. & Westendorff, M.: Chromosomes of three species Beitr.Ent. 50 (2000) 1 197 Our data on relative chromosome lengths in N. vitripennis are very close to those calculated by Reed (1993), namely: 25.10,21.36,19.74,17.89 and 15.91 (transformed from absolute chro mosome lengths).The latter author was also able to demonstrate the presence of pericentromeric heterochromatin in all chromosomes of this wasp species.
Evidence obtained from the study of an rDNA internal transcribed spacer (ITS2) (Campbell et al., 1993) and mitochondrial 16S DNA of the Nasonia complex (Reed & WERREN, in lift.)suggests that the time of divergence between N. vitripennis and the two remaining species is approximately 200,000 years, and that between N. longicornis and N. giraulti is about 100,000 years.Our data are consistent with the order of divergence in the Nasonia complex found on the basis of molecular studies.Specifically, a comparison of relative lengths of the chromo some 2 demonstrates that N. vitripennis is likely to stem out first from the common lineage which split later into N. longicornis and N. giraulti.The significant difference on the relative lengths of chromosome 3 between the two latter species may reflect an autapomorphous chromosomal rearrangement occurred in one of these taxa.
Tab. 1: Number o f studied individuals of the Nasonia complex and their metaphase plates.