Contributions to Zoology, 71 (4) (2002)Daniel F.R. Cleary; Henri Descimon; Steph B.J. Menken: Genetic and ecological differentiation between the butterfly sisterspecies Colias alfacariensis and Colias hyale

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Materials and methods

Habitat preference

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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 14^th of August until the 8^th 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.

Population analysis

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.

Table 1. Location and geographical position of all sampling sites. Allozyme indicates that the population sampled was used in allozyme electrophoretic analysis. Field indicates that the site was used in the habitat preference study. The two populations from northern France were collected by a colleague in the surroundings of Paris and Metz. Population refers to the populations used for electrophoresis. A – J: C. alfacariensis, K – M: C. hyale.
Population	Site	Location	Lat	Long	Allozyme	Field

A	Poggio del Commune	Italy	44 57.64	6 41.01	x
B+M	Val de Prés	French Alps	44 58.47	6 30.33	x	x
C+L	Le Monêtier les Bains	French Alps	44 53.52	6 37.02	x	x
D	Puy Saint Pierre	French Alps	44 53.52	6 37.02	x
E	Saint Zachery	French Alps	43 23.07	5 42.22	x
F	Pontèves	Provence	43 33.55	6 02.53	x
G	Artigues	Provence	43 36.05	5 47.54	x
H	Mimet	Provence	43 24.54	5 30.19	x
I	Seine River outside Paris	Provence			x
J+K	Surroundings of Metz	Northern France			x
	Gero	Northern France	44 59.03	6 28.56		x
	les Guibertes	French Alps	44 58.01	6 31.57		x
	le Cassset	French Alps	44 59.03	6 28.56		x
	Saint Chaffery	French Alps	44 55.32	6 36.11		x
	le Rosier	French Alps	44 56.02	6 40.58		x
	Villard Saint Pancrace	French Alps	44 52.09	6 38.05		x
	la Vachette	French Alps	44 54.58	6 39.55		x
	Laus de Cervières	French Alps	44 51.40	6 43.35		x
	Granon	French Alps	44 58.04	6 39.01		x
	Cervières	French Alps	44 51.56	6 43.21		x

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 (F_ST F_IT , and F_IS ; Wright, 1951) were calculated according to Weir and Cockerham’s (1984) F, f, and Theta respectively. F_ST values were tested for departure from zero by the method of Workman and Niswander (1970), and inbreeding coefficients (F_IS ) were tested by the method of Li and Horvitz (1953).

The amount of gene flow (N_e M) was estimated by the F statistic (F_ST ) method of Weir and Cockerham (1984), which uses Theta as an estimate of F_ST . Theta is assumed to be a better estimate of F_ST 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). F_ST values were calculated using the computer programme THETA (Ellis, 1994), which uses the individual genotypes as input. F_ST is a useful estimator of gene flow according to the equation N_e M ≈ _([1/ F_ST ]-1) (Wright, 1951). Since there is an inverse relationship between F_ST and N_e M we consider F_ST values that do not differ significantly from zero to indicate unlimited gene flow, i.e., a panmictic populations structure.