Robert H. Hagen, Deborah R. Smith, and Steven W. Rissing.
Genetic relatedness among co-foundresses of two desert ants, Veromessor pergandei and Acromyrmex versicolor (Hymenoptera: Formicidae).
Psyche 95(3-4):191-201, 1988.

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GENETIC RELATEDNESS AMONG CO-FOUNDRESSES OF TWO DESERT ANTS, VEROMESSOR PERGANDEI AND ACROMYRMEX VERSZCOLOR
(HY MENOPTERA: FORMICIDAE)
BY ROBERT H. HAGEN', DEBORAH R. SMITH^,
AND STEVEN W. RISSING~
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 estab- lishment 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 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 electro- phoretic analysis of intra-group relatedness among co-foundresses of Veromessor pergandei 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 pedi- gree 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 deter- mining behavior patterns and thus can be treated as selectively neutral within the context of social evolution (Pamilo 1984). 1. Department of Entomology, Michigan State University, East Lansing, Michigan 48824.
2. Insect Division, Museum of Zoology, University of Michigan, Ann Arbor, Michi- gan 48109.
3. Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501. To whom correspondence should be addressed. Manuscript received by the editor September 20, 1988.



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192 Psyche [vo~. 95
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 -80å¡ and stored until electrophoresed.
Electrophoretic methods. We prepared frozen ants for electro- phoresis by grinding them individually in an extraction buffer at 4OC. We removed the gasters of V. pergandei queens before grind- ing 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 electro- phoretic 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 vol- tages 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 4O C.
To visualize the allozymes we used enzyme-specific stains (Harris and Hopkinson 1978, Richardson et al. 1986)? mixed 1: 1 with 1.5% agar solution and poured onto the plates. When sufficient stain intensity was reached, we rinsed off the agar layer and soaked the



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19883 Hugen, Smith, & Rksing - 7bo desert ants 193 plate in tap water to remove unred dye. Precipitated dye remains in the cellulose acetate layer, so stained plates were pix- served directly or photocopied, Genetic interpretations of variation in resulting bands was based on known enzyme quaternary structure (Hank and Hopkkison 1978, Richardson et al. 19861, supplemented by comparison of haploid males where possible. We resdvd allozym products of 18 psumptive genetic lwi from V. pergatidei queens, 4 of which were polymorphic with 2 akles each: Est-1 and fib2 (general esterase; beta-naphthyl acetate as substrate), Mdh-I (malate dehydrogenase, EC 1. I.l.3i'J and Pgm (pho~p~og~ucomutase, EC 2.7.5. I), Optimal separation for the V. pergandei esterase allozymes was given by buffer 1 (25 rnin at 250 V); for Mdh-1 by buffer C (30 min at 250 V); and for Pgm by buffer I (25 min at 250 V). An additional po~ymorphism for Idh-1 &xi- trate dehydrogenase, EC 1.1- I .&!) was present in one small sample (buffer I, 20 min at 250 V). Banding patterns and allele designations for the esteras loci are shown in Figure 1; esterase genotypes could not be scored from all individuals,
Figure I. Zymogrm of Werase loci i~ V. pergandei Allelta for &I-1 an designated "C* (cathodal] and
(anodal); the enzyme behave as a monomer. Est-1 CC ad 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 10muA~C~andIane9kT/C~.For E+21ana2-4,7, loand 11 mnF/F'an&hml,7,9d 12are*FjSu.



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194 Psyche [vo~. 95
An anamalous Gpi (glucose phosphate isomerase, EC 5.3.1.9) and Ao (aldehyde oxidase, EC 1.2.3.1) banding pattern was present in low frequency among V. pergandei queens (buffer I, 25 min at 250 V). Since we were unable to verify Mendelian inheritance of the variant pattern, which could not be interpreted easily in terms of known enzyme quaternary structure, we have omitted these loci from the analysis. The monomorphic loci resolved from I? pergan- dei were (enzyme trivial name, E.C. code and best running buffer are listed after each): Aat-1 (aspartate aminotransferase, EC 2.6.1.1; I); Ac (aconitase, EC 4.2.1.3; C); Dia (diaphorase, EC 1.6.*.*; D); G3p (glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; A); G6pdh (glucose-6-phosphate dehydrogenase, EC 1.1.1.49; A) Hk (hexose kinase, EC 2.7.1.1; I) Lap (leucine aminopeptidase, EC 3.4.11.1; I); Ldh (lactate dehydrogenase, EC 1.1.1.27; D); Mdh-2 (C); Pep (pep- tidase, phenylalanyl-prolyl substrate; C); Pgd (6-phosphogluconate dehydrogenase, EC 1.1.1.44; D).
We resolved allozyme products of 30 presumptive genetic loci from A. versicolor queens. Only Pgm was polymorphic with 2 alleles designated F (fast migrating) and S (slow). Best resolution was given by buffer D, run for 30 min at 250 V. The monomorphic loci were (enzyme trivial name and EC code [if not listed above] and best buffer are listed after each): Aat-1 and Aat-2 (D); Ac (D); Ada (adenosine deaminase, EC 3.5.4.4; D); Ak (Adenylate kinase, EC 2.7.1 -20; A); Ald (aldolase, EC 4.1 -2.13; D); Ao (D); Apk (arginine phosphokinase, EC 2.7.3.3; D); Dia (I); Est (D); Fum (fumarase, EC 4.2.1 -2; D); Gapdh (glycgraldehyde phosphate dehydrogenase, EC 1.2.1.12; C); Gldh (glucose dehydrogenase, EC 1.1.1.47; A); Gpi (C); G3p (A); G6pdh (B); Hbdh (hydroxybutyrate dehydrogenase, EC 1.1.1.30; D); Hk (C); Idh-1 and Idh-2 (C); Lip (C); Ldh (B); Mdh-1 and Mdh-2 (C); Me (malic enzyme, EC 1.1.1.40; A); Pep (glycyl-leucyl substrate; C); Pgd (A); Sod (superoxide dismutase, EC 1.15.1.1 ; D); Sordh (sorbitol dehydrogenase, EC 1.1.1.14; I). Statistical analyses. The within group relatedness for foundress associations was calculated from the relationship: where Fst and Fit are Wright's inbreeding coefficients (Hamilton 1972, Pamilo 1984, McCauley et al. 1988). The Fst and Fit were estimated for each polymorphic locus using Long's (1986) proce-



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196 Psyche [vo~. 95
dure, which is corrected for sample size bias. Since all of the poly- morphic loci detected in this study had 2 alleles, the method is essentially identical to that of Pamilo (1984, Crozier et al. 1984, Pamilo and Rosengren 1984), when groups are weighted by the number of individuals. Weighting of groups by size appears prefer- able in the case of V. pergandei and A. versicolor foundress associa- tions, which varied from 2 to 15 queens in these samples. 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 pres- ent 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 foun-
dress 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 related- ness 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. versico-
lor 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 and McCracken 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.



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19881 Hagen, Smith, & Rissing - Two desert ants 197 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. LOCUS r S.E. Fst Fit N
V. pergandei Est-1 0.043 0.106 0.0208 -0.0308 24 Est-2 -0.009 0.093
-0.0049 0.0527 31
Mdh-1 -0.1 17 0.043 -0.0554
-0.0561 31
pgm
0.174 0.158
0.1107 0.2685 31
Mean 0.033 0.0 176 0.0638
A. versicolor Pmn -0.125 0.028 -0.0632 0.0077 26 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, 1983; Wade 1985). Similar random association of genotypes occurs in polygy- nous 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, espe- cially social ones (social structure lowering effective population size and providing a nest structure that buffers environmental variabil- ity) (reviewed in Graur 1985, Sheppard and Heydon 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



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198 Psyche [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. Foundress
Pgm Genotype
Group FF FS S S
Total:
Solitary:
adjacent colonies (Pollock and Rissing 1985). Under the latter sys- tem, 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 (Myrmeco- cystus 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



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200 Psyche [vo~. 95
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