Materials and methods
Habitat preferencenext section
Individuals of both C. alfacariensis and C. hyale were sampled for an allozyme study in France and Italy in the summer of 1992. Species determination was based on the studies of Berger and Fontaine (1947), Jarvis (1955), and Dutreix (1980). Habitat preference between C. alfacariensis and C. hyale was studied by means of field observations in the French Alps where both species occur sympatrically.
A total of 920 adult C. alfacariensis and 1155 adult C. hyale were observed from the 14th of August until the 8th of September, 1974 by HD. The location and geographical position of all sites is presented in Table 1. During the field observations the following environmental variables were noted at each locality: 1. Aspect, 2. Vegetation (cultivated – uncultivated), 3. Humidity (dry – irrigated), 4. Altitude 5. Date, 6. Weather (warm – cold), in addition to the frequency of occurrence of adults of C. alfacariensis and C. hyale.
The relative abundances of both species were regressed onto the first axis of a principal components analysis (PCA based on the environmental variables and using Statistica for Windows, version 5, StatSoft Inc.) to see if both species varied significantly (positively or negatively) in their response to this synthetic environmental gradient. The relative abundance here is the absolute abundance of species A at a given site divided by the absolute abundances of both species at the same site. We use relative abundance here because of the high variance in the absolute abundance among sites.
For the allozyme electrophoresis analyses, populations of both Colias species were sampled in the French Alps and other localities, including the Provence, Northern France and Italy (Table 1). After capture the butterflies were stored in liquid nitrogen. The head and thorax were subsequently homogenised in the laboratory in 180 ml of a 0.1M Tris-HCl buffer (pH 8.0) and the extracts were centrifuged for 6 minutes at 9000 x g. The supernatant from the extract was then frozen until used for electrophoresis.
Two gel matrices were used, namely cellulose acetate and starch gels. The starch gels were prepared according to Harris and Hopkinson (1977) and the exact preparation of buffers and gels follows Raijmann et al. (1997). Two buffer solutions were used in the starch gels, namely a Tris-Citric acid buffer (TC 8.0) and a Tris-Malic acid buffer (TM 7.4). Agar overlays were applied to all gel slices.
The following loci (locus abbreviations in parentheses) gave consistently good results and were used for the analyses: Aldolase (ald), Creatine Kinase 1 (ck1) and 2 (ck2), Glutamate-oxaloacetate transaminase 1 (got1) and 2 (got2), Glycerol-3-phosphate dehydrogenase (gpd), Hexokinase (hk), Isocitrate dehydrogenase 1 (idh1) and 2 (idh2), Malate dehydrogenase 1 (mdh1) and 2 (mdh2), Malic enzyme (me), Mannose phosphate isomerase (mpi), Phosphogluconate dehydrogenase (6pgdh), Glucose phosphate isomerase (pgi), and Phosphoglucomutase (pgm). The loci ck1, ck2, gpd, got2, and pgm gave the best resolution with TM; got1, hk, mdh1, mdh2, me, mpi, idh1, idh2, and pgi gave the best resolution with TC; 6pgdh gave the best resolution with a Tris-Glycine buffer (pH 8.5) on cellulose acetate. Locus number 1 refers to the least anodally migrating locus. Allelic designations use ‘a’ as the least anodally migrating allele, then ‘b’ and so forth.
Individual genotypes were used as input data for BIOSYS-1 (Swofford and Selander, 1981). The allelic frequencies and mean heterozygosity were derived directly from the BIOSYS output. Variation in heterozygosity between C. alfacariensis and C. hyale was tested for significance with a one-way Anova (using Statistica for Windows, version 5, StatSoft Inc.).
The inbreeding coefficients (FST FIT , and FIS ; Wright, 1951) were calculated according to Weir and Cockerham’s (1984) F, f, and Theta respectively. FST values were tested for departure from zero by the method of Workman and Niswander (1970), and inbreeding coefficients (FIS ) were tested by the method of Li and Horvitz (1953).
The amount of gene flow (Ne M) was estimated by the F statistic (FST ) method of Weir and Cockerham (1984), which uses Theta as an estimate of FST . Theta is assumed to be a better estimate of FST than Wright (1978) estimate and does not make assumptions about number of populations, sample sizes, or heterozygote frequencies so that it is more appropriate for small data sets (Weir and Cockerham, 1984). FST values were calculated using the computer programme THETA (Ellis, 1994), which uses the individual genotypes as input. FST is a useful estimator of gene flow according to the equation Ne M ≈ _([1/ FST ]-1) (Wright, 1951). Since there is an inverse relationship between FST and Ne M we consider FST values that do not differ significantly from zero to indicate unlimited gene flow, i.e., a panmictic populations structure.