Genetic Relatedness among Co-foundresses of Two Desert Ants, Veromessor Pergandei and a Cromyrmex Versicolor (hymenoptera: Formicidae)

Cooperative colony foundation occurs in some social hymenop-tera. Polistine wasp foundress associations are usually composed of close relatives (reviewed in Gamboa et al. 1986, Michener and Smith 1987) suggesting kin selection may play an important role in establishment of such groups. Cooperative colony foundation, however, may be advantageous even if cofoundresses are not related (Lin and Michener 1972, Pollock and Rissing 1988a). Indeed, several behav-ioral (reviewed in Rissing and Pollock 1988) and one electrophoretic (Ross and Fletcher 1985) study suggest ant foundress associations form without respect to relatedness. Here we report on an electro-phoretic analysis of intra-group relatedness among co-foundresses of Veromessorpergandei and Acromyrmex versicolor, two common desert ants with cooperative colony foundation (Pollock and Riss-ing 1985, Rissing and Pollock 1986, Rissing et al. 1986). Ideally, relatedness should be measured directly through pedigree analysis of interacting individuals (Hamilton 1972). Since this is impractical for most natural populations of social insects, the alternative is indirect estimation using neutral genetic markers (Pamilo and Crozier 1982, Pamilo 1984). We used polymorphic allozyme loci, detected by protein electrophoresis, for this purpose (Richardson et al. 1986). Allozyme loci offer the advantage that homozygous and heterozygous individuals are readily distinguisha-ble; in addition, these loci are not likely involved directly in determining behavior patterns and thus can be treated as selectively neutral within the context of social evolution (Pamilo 1984).

Cooperative colony foundation occurs in some social hymenoptera.Polistine wasp foundress associations are usually composed of close relatives (reviewed in Gamboa et al. 1986, Michener andSmith 1987) suggesting kin selection may play an important role in establishment of such groups.Cooperative colony foundation, however, may be advantageous even if cofoundresses are not related (Lin andMichener 1972, Pollock andRissing 1988a).Indeed, several behavioral (reviewed in Kissing and Pollock 1988) and one electrophoretic (Ross and Fletcher 1985) study suggest ant foundress associations form without respect to relatedness.Here we report on an electrophoretic analysis of intra-group relatedness among co-foundresses of Veromessor pergandei and Acromyrmex versicolor, two common desert ants with cooperative colony foundation (Pollock and Rissing 1985, Rissing and Pollock 1986, Rissing et al. 1986).
Ideally, relatedness should be measured directly through pedigree analysis of interacting individuals (Hamilton 1972).Since this is impractical for most natural populations of social insects, the alternative is indirect estimation using neutral genetic markers (Pamilo andCrozier 1982, Pamilo 1984).We used polymorphic allozyme loci, detected by protein electrophoresis, for this purpose (Richardson et al. 1986).Allozyme loci offer the advantage that homozygous and heterozygous individuals are readily distinguishable; in addition, these loci are not likely involved directly in determining behavior patterns and thus can be treated as selectively neutral within the context of social evolution (Pamilo 1984).

MATERIALS AND METHODS
Foundress associations of V. pergandei were collected from two sites, "Main" and "Granite" (2 km apart) immediately south of the southeast corner of South Mountain Park, Phoenix, AZ during February-March 1988.Foundress associations of A. versicolor were collected from a site in North Scottsdale, AZ (described in Rissing et al. 1986) in September 1987.In each case, existence of a single characteristic mound of freshly excavated soil indicated a single foundress association.Live co-foundresses were air expressed immediately to Michigan State University where they were frozen at -80Â° and stored until electrophoresed.
Electrophoretic methods.We prepared frozen ants for electrophoresis by grinding them individually in an extraction buffer at 4OC.We removed the gasters of V. pergandei queens before grinding in a pH 7.09 0.1 M tris buffer (with 40 mg EDTA, 20 mg NAD, 10 mg NADP and 250 pl beta-mercaptoethanol per 100 ml: Buffer 1) or in an unbuffered detergent solution (with 100 pl Triton-X, 10 mg NADP and 100 pl beta-mercaptoethanol per 100 ml: Buffer 2).Buffer 2 gave superior results for esterases but was no better, and in some cases worse, than Buffer 1 for other enzymes.We ground whole A. versicolor in buffer 1.For each ant we adjusted the amount of buffer from 10 to 100 pl to give an approximately equal ratio of buffer to ant tissue.
We applied extracts from 12 ants (ca. 1 pl from each) to thin-layer cellulose acetate plates (Titan 111: Helena Laboratories, Beaumont, TX).Plates were soaked for at least 30 min in a running buffer before sample application; we used the same buffer for the electrophoretic run.We used cellulose acetate running buffers "A", "B", "C", "D", and "I" of Richardson et. al. (1986); no single buffer gave good resolution for all enzymes tested.Run durations ranged 15-35 minutes?under constant voltage (200-300 V); durations and voltages were adjusted to optimize separation for each enzyme that showed clear activity.Combinations of running buffer, voltage and time giving best results are noted below.All electrophoresis was done at 4 O C.
Standard errors (S.E.) for the relatedness estimates were obtained by a jacknife procedure over groups (Sokal and Rohlf 1981, Pamilo 1984, Crozier et al. 1984).Simulation studies have shown that S.E.estimated by this method tend to be overly conservative and can be unreliable when allele frequencies are highly unequal (Crozier et al. 1984, Wilkinson McCracken 1985).Because of this, use of these S.E. in formal statistical hypothesis testing is not justified with the present data.In the case of V. pergandei foundress associations, a more robust estimate of r is possible by combining estimates across the 4 informative loci (Wilkinson and McCracken 1985).For this pooled estimate we used the weighted means of Fsi and Fit across loci (Long 1986).
Verornessor pergandei.Relatedness within V. pergandei foundress associations does not differ from 0 (Table 1; mean estimate across loci = 0.033, Table 2).Allele frequencies of V. pergandei are similar between subsites (Table 1); therefore, we treat them as a single population.Pooling subsites would inflate estimated relatedness if subsites differed.Pgm and Mdh-1 allele frequencies are highly unequal, which limits their usefulness as genetic markers for relatedness; they are most informative in combination with Est-1 and Est-2 alleles.None of the loci appear linked.
Acromyrmex versicolor.Genetic relatedness among A. versicolor co-foundresses is no greater than that expected from randomly associating queens (Table 2, 3).Negative value of the estimate (-0.125) is likely a statistical artifact resulting from unequal Pgm allele frequencies and relatively small numbers of queens in each foundress association (mean = 3.8) (Crozier et al. 1984, Wilkinson andMcCracken 1985) rather than an indication that queens avoid kin (Hamilton 1972).The small standard error associated with the estimate (0.028) indicates little variation in relatedness among groups, consistent with purely random mixing of genotypes.Foundress associations of V. pergandei and A. versicolor are not composed of close kin.Veromessor pergandei and A. versicolor foundresses do not deviate from random assortment of genotypes, precluding the operation of kin selection (Wilson 1977(Wilson , 1983;;Wade 1985).Similar random association of genotypes occurs in polygynous Solenopsis invicta colonies (Ross and Fletcher 1985), which are likely founded cooperatively.No other cooperatively founding ant species have demonstrated behavioral evidence of preferential association among relatives (Rissing and Pollock 1988), suggesting that results from the three species now studied electrophoretically are likely to be general.The genetic basis for cooperative behavior among co-founding queens, therefore, cannot be described as a direct consequence of kin selection.
Genetic diversity of many Hymenoptera is lower than found in other insect orders owing either to haplodiploid sex determination (increasing selective pressure on deleterious alleles exposed in haploid males or decreasing effective population size) or behaviorall environmental peculiarities characteristic of many species, especially social ones (social structure lowering effective population size and providing a nest structure that buffers environmental variability) (reviewed in Graur 1985, Sheppard andHeydon 1986).The decreased variability of A. versicolor relative to V. pergandei may reflect differences in the mating systems of the two: while all A. versicolor colonies in an area release alates on a single day (Wheeler 19 17, Rissing et al. 1986), V. pergandei colonies release alates over a three month period with little coordination of reproduction among  adjacent colonies (Pollock and Rissing 1985).Under the latter system, small numbers of reproductives (especially males) released per day enhance sampling error associated with the distribution of genes with colonies, thus enhancing genetic variance within a population.Starting colonies of four North American ant species (Myrmecocystus mimicus, Solenopsis invicta, V. pergandei and A. versicolor) have clumped starting nests, yet adult colonies of these species are highly territorial, leading to strong intraspecific competition among starting colonies in the form of brood raiding (reviewed in Pollock Figure I. Zymogrm of Werase loci i~ V. pergandei Allelta for &I-1 a n designated "C* (cathodal] and (anodal); the enzyme behave as a monomer.Est-1 CC a d CA gtnotypa are distinguished by the relative intensity of each band, since an artifact band cornigrates wi?h the A aIimymc mi-2 hm allelm T" (fast) and uS*(s!ow) and hhaves as a dimer, migin; *-I--= amdaL For fit-], lanes, 2,5, &and 11 arc"A/Ag;lanes 1,3,4,7,8md 1 0 m u A ~C ~a n d I a n e 9 k T / C ~. F o r E+21ana2-4,7, loand 11 m n F / F ' a n & h m l , 7 , 9 d 12are*FjSu.

[VOI. 95 Table 3 .
Pgm genotype frequencies of A. versicolor queens from foundress associations.Groups are listed from largest to smallest in size; the frequencies among 14 solitary queens are given for comparison.

Table 2 .
Relatedness and F-statistics for V. pergandei and A. versicolor foundress associations.The S.E. is standard error of relatedness, r, based on jacknifing over N groups of foundresses or nestmates.The mean r for V. pergandei foundresses is calculated from the weighted mean Fsi and Fit across loci.