A Reconnaissance of Population Genetic Variation in Arctic and Subarctic Sulfur Butterflies (Colias Spp.; Lepidoptera, Pieridae)

Home , Colias christina, Colias meadii, Colias nastes, Colias palaeno

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A reconnaissance of population genetic variation in arctic and subarctic sulfur butterflies (Colias spp.; Lepidoptera, Pieridae)

Christopher W. Wheat, Ward B. Watt, and Christian L. Boutwell

Abstract: Genotype–phenotype–environment interactions in temperate-zone species of Colias Fabricius, 1807 have been well studied in evolutionary terms. Arctic and alpine habitats present a different range of ecological, especially thermal, conditions under which such work could be extended across species and higher clades. To this end, we survey variation in three genes that code for phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), and glucose-6-phosphate dehydrogenase (G6PD) in seven arctic and alpine Colias taxa (one only for G6PD). These genes are highly polymorphic in all taxa studied. Patterns of variation for the PGI gene in these northern taxa suggest that the balancing selection seen at this gene in temperate-zone taxa may extend throughout northern North America. Comparative study of these taxa may thus give insight into the mechanisms driving genetic differentiation among subspecies, species, and broader clades, supporting the study of both micro- and macro-evolutionary questions. Résumé : L’étude des interactions génotype–phénotype–environnement chez les papillons Colias Fabricius, 1807 de la région tempérée s’est faite dans une perspective évolutive. Les habitats arctiques et alpins offrent une gamme différente de conditions écologiques et, en particulier, thermiques dans lesquelles un tel travail peut s’étendre au niveau des espè- ces et des clades supérieurs. Dans ce but, nous avons étudié la variation de trois gènes — ceux de la phosphoglucose isomérase (PGI), de la phosphoglucomutase (PGM) et de la glucose-6-phosphate déshydrogénase (G6PD) — chez sept taxons de Colias arctiques et alpins (un seul taxon pour G6PD). Ces gènes sont fortement polymorphes chez tous les taxons étudiés. Les patrons de variation de gène PGI chez ces taxons nordiques laissent croire que la sélection d’équilibre qui affecte ce gène chez les taxons de la région tempérée peut s’étendre au travers de toute l’Amérique du Nord. L’étude comparée de ces taxons peut ouvrir des perspectives sur les mécanismes qui poussent à la différentiation génétique dans les sous-espèces, les espèces et les clades plus larges et conduire à l’étude de questions à la fois de micro- et de macro-évolution. [Traduit par la Rédaction] Wheat et al. 1623

Introduction Specifically, variation in the thermal ecology of Colias butterflies in northern climates may have important conse- Sulfur butterflies (genus Colias Fabricius, 1807) are con- quences for genetic variants in enzymes of central energy spicuous members of North American insect communities metabolism, and thus for their carriers’ flight performance from the shores of the Arctic Ocean to the grasslands of and resulting Darwinian fitness. Colias butterflies regulate Florida. Temperate-zone Colias butterflies are a model sys- their body temperature, Tb, by orienting perpendicular to tem for studying resource allocation (Boggs and Watt 1981; sunlight when cold and parallel to it (or seeking shade) Nielsen and Watt 1998), sexual selection (Sappington and when overheated (Watt 1968). They fly voluntarily at Tbsbe- Taylor 1990; Nielsen and Watt 2000), and how natural selec- tween 29 and 40 °C, and flight is maximized between 35 tion can act on identified genes (e.g., Watt 1977, 1983, 1992; and 38–39 °C (Watt 1968; Tsuji et al. 1986). Populations or Watt et al. 1983, 1985; Watt and Boggs 1987). However, species evolve differences in absorptivity for sunlight and in- most North American Colias butterflies occupy montane, al- sulation against convective heat loss, compensating for local pine, or arctic habitats (e.g., Layberry et al. 1998). These differences in habitat thermal variables, so that each popula- taxa and their habitats present a wide range of potential nat- tion has access to the thermal flight maximum, on which all ural selection for study (e.g., Roland 1982; Watt et al. 1996). aspects of adult fitness depend, in its habitat (Watt 1968;

Received 12 May 2005. Accepted 26 October 2005. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 16 December 2005. C.W. Wheat,1,2,3 W.B. Watt, and C.L. Boutwell.4 Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA, and Rocky Mountain Biological Laboratory, Crested Butte, CO 81224, USA. 1Corresponding author (e-mail: [email protected]). 2Present address (1 November 2005 to 1 February 2006): c/o Doug Crawford, University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, FL 33149, USA. 3Future address (after 1 February 2006): Helsinki University, Metapopulation Research Group, Department of Biological and Environmental Sciences, P.O. Box 65 (Viikinkaari 1), FIN-00014 University of Helsinki, Finland. 4Present address: Ph.D. Program in Virology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA.

Roland 1982; Kingsolver 1983a, 1983b). Nonetheless, ther- Fig. 1. Sampling localities of Colias spp. in the North American mal variability among habitats still yields different thermal arctic and subarctic. experiences for local Colias populations, leading to differences in population flight (Kingsolver and Watt 1983, 1984), and hence different balances of thermally driven selective North America pressure on metabolic processes (e.g., Watt 1992; Watt et al. 1996).

In this context, we have studied genetic variation in meta- C. hecla hecla, bolic enzymes of Colias butterflies that share the substrate, C. nastes glucose-6-phosphate (G6P). Phosphoglucose isomerase (PGI) isomerizes G6P to fructose-6-phosphate, which then pro- C. palaeno ceeds through glycolysis, the Krebs cycle, and oxidative phosphorylation to produce of ATP, the “fuel” of active flight muscle. Phosphoglucomutase (PGM) converts G6P to glucose-1-phosphate for trehalose production or glycogen C. c. kluanensis storage. Glucose-6-phosphate dehydrogenase (G6PD) begins the pentose shunt, producing five-carbon sugars, as well as NADPH for reductive biosynthesis. C. c. christina Most of our work so far has focused on the PGI gene, studying electromorph (EM) alleles which are numbered by increasing anodal mobility upwards from 1. Ten common EM genotypes in temperate-zone Colias eurytheme Boisduval, C. m. elis 1852 were isolated and their biochemical performances (i.e., kinetics and thermal stability) assessed under natural temperature conditions (Watt 1977, 1983). These functional stud- C. c. astraea ies led to successful predictions of which PGI genotypes should show the best flight performance through the daily thermal cycle within the thermoregulation limits of Colias butterflies (Watt et al. 1983). In turn, differential flight performance among PGI genotypes was predicted to yield genotypic differences in survivorship, male mating success, and female fecundity; experimental tests have confirmed these predictions against neutral alternatives in the wild, vantage in this polymorphism imply for the repeatability of showing strong selection on the PGI gene (e.g.Watt 1983, evolution? Why has natural selection been unable to find al- 1992; Watt et al. 1985, 1996). leles that produce maximally functional homozygotes at this gene? Studies of EM variation at PGM and G6PD genes reveal Studying PGI gene variation in diverse Colias butterflies positive correlations between heterozygosity and male repro- of different thermal niches, especially from arctic and sub- ductive success, although both genes are independent of the arctic regions that are ecological analogs of alpine areas, PGI gene and their variation acts through performance dif- may shed more light on these questions. Here, by exploring ferences other than flight (Carter and Watt 1988). These genetic variation among seven such taxa at the PGI and genes’ natural variants, especially because of their shared us- PGM (and in one case G6PD) genes, we lay the groundwork age of the G6P substrate, may help us explore the general for future northern studies of the evolutionary interaction of problem of genetic interaction, or “epistasis”, in the evolu- environmental and genetic variations. tion of metabolic organization (Carter and Watt 1988; Eanes 1999; Watt and Dean 2000). Methods We have begun to extend these studies to Colias meadii Edwards, 1871, a mid-latitude species inhabiting alpine and Freshly emerged Colias butterflies were collected in peak subalpine meadows of the central and southern Rocky flight conditions across northwestern North America in Mountains. Differential survivorship and male mating suc- 1996, as mapped in Fig. 1. Taxonomy follows Layberry et cess among its PGI genotypes were correctly predicted from al. (1998) (sample sizes, n, in Table 1 except for pooled their biochemical phenotypes (Watt et al. 1996), and as in samples indicated below): Colias christina christina Ed- C. eurytheme, PGI heterozygotes often have higher fitness in wards, 1863, 23–24 July, from the Racing River at mile 430, the wild than homozygotes. But, there are important differ- Alaska Highway, British Columbia (latitude 58°50′N); ences between PGI variants of these two species, whose Colias christina astraea Edwards, 1872, 30 July, from Pros- implications prompted the present study. Although PGI vari- pect Creek, 1 mi. (1 mi. = 1.609 344 km) south of Cadomin, ants of C. eurytheme and C. meadii appear similar on stan- Alberta (this and the previous “subspecies” are weakly dis- dard electrophoresis gels, high-resolution electrophoresis tinguished by clinal wing color variation; latitude 53°2′N); clearly differentiates them, revealing shape and charge dif- Colias christina kluanensis Ferris, 1981, 17–18 July, from ferences; moreover, these genotypes differ significantly in the Duke River, Alaska Highway, Kluane District, Yukon kinetics and thermal stability among taxa (Watt et al. 1996). Territory (latitude 61°37′N); Colias hecla Lefebvre, 1836, a What does this similarity among species of heterozygote ad- pooled sample from the north slope of the Brooks Range

Table 1. Electromorph allele frequencies of genes that code for phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), and glucose 6-phosphate dehydrogenase (G6PD) among arctic and subarctic Colias butterflies. Allele frequency Gene (n)p1p2p3p4p5p6 Colias eurytheme* PGI (100) 0 0.075 0.630 0.245 0.050 0 PGM (100) 0 0.145 0.680 0.175 0 0 G6PD (100) 0.670 0.310 0.020 0 0 0 Colias christina christina PGI (46) 0.022 0.239 0.446 0.196 0.054 0.044 PGM (46) 0 0 0.696 0.261 0.044 0 Colias christina astraea PGI (70) 0.007 0.236 0.429 0.229 0.100 0 PGM (70) 0 0.064 0.621 0.286 0.029 0 Colias christina kluanensis PGI (77) 0.046 0.351 0.169 0.435 00 PGM (77) 0 0 0.942 0.046 0.013 0 Colias hecla PGI (31) 0.016 0.177 0.290 0.403 0.097 0.016 PGM (24) 0.115 0.539 0.346 0 0 0 Colias meadii elis PGI (30) 0 0.183 0.417 0.350 0.050 0 PGM (8) 0.188 0.250 0.313 0.250 0 0 Colias meadii meadii* Low elevation PGI (165)† 0.024 0.521 0.418 0.036 0 0 PGM (84)‡ 0.190 0 0.375 0.429 0.006 High elevation PGI (84)† 0.018 0.625 0.327 0.030 0 0 PGM (44)‡ 0.181 0.011 0.420 0.386 0 0 Colias nastes PGI (40) 0.050 0.238 0.350 0.300 0.063 0 PGM (40) 0.025 0.475 0.400 0.075 0.025 0 G6PD (40) 0.725 0.275 0 0 0 0 Colias palaeno chippewa PGI (51) 0.353 0.471 0.137 0.039 0 0 PGM (51) 0 0.078 0.882 0.020 0.020 0 Note: Numbers of genotypes, n, are in parentheses and number of alleles is equal to 2n. Highest allele frequencies are shown in boldface type. *Samples of C. eurytheme and C. m. meadii (Mesa Seco) from Watt et al. (2003). †Pooled allele frequency data from 1977 and from 1978 showing the same trend (Watt 1983). ‡Allele frequency data for 1978 only. along the Dalton Highway (Franklin Bluffs, mi. 386, 9–10 Allozyme electrophoresis in 7% acrylamide gels followed July (n = 13), Ice Cut, mi. 325, 11 July (n = 6), Oil Spill Carter and Watt (1988). PGI, PGM, and G6PD allozymes Hill, mi. 320, 11 July (n = 12)), Alaska (average site latitude segregate in each case at single Mendelian genes in diverse 69°26′N) (these samples are from habitats that are very species complexes (Watt 1977; Carter and Watt 1988). close in space, with continuous habitat in between, and thus Allelic mobility classes were compared among species by likely represent a single, interbreeding population); Colias visual inspection of gels and comparison with internal pro- meadii elis Strecker, 1885, a pooled sample from Mt. tein standards. Sample size differences among genes were Spieker, British Columbia, on 27–8 July (n = 12), from due to failure of a few individuals to stain clearly for all Prospect Creek 1 mi. south of Cadomin, Alberta, on 30 July genes. While our interests were most sharply focused on the (n = 17), and from Wilcox Basin, Alberta, on 31 July (n =1; PGI gene, we also scored all individuals for the PGM gene average site latitude 53°2′N); Colias nastes aliaska Bang- since this can be done on the same gels (Carter and Watt Haas, 1927, a pooled sample from along the Dalton High- 1988); we also examined one northern sample for the G6PD way with the same dates as C. hecla for each locality gene for exploratory purposes. We did not employ multi-gel (Franklin Bluffs, n = 22; Oil Spill Hill, n = 18); Colias Ferguson plot analysis (Watt et al. 1996) in this work, rea- palaeno chippewa Edwards, 1872, 6 July, from Reed’s soning that clear distinctions among or within electromorph Creek, mi. 88 of the Steese Highway, Alaska (latitude allele classes will be forthcoming from planned sequence- 65°25′N). structure-function studies in any case (cf. Wheat et al. 2006). For comparison to these northern samples, we also present Descriptive population statistics and tests were calculated data (from other works, e.g., Watt et al. 1996, 2003) on using tools for population genetic analysis (TFPGA; Miller C. eurytheme (Tracy, California, 19 September 1985, lati- 2000). We tested for match to the Hardy–Weinberg distri- tude 37°45′N) and Colias meadii meadii Edwards, 1871 bution with a computed Monte Carlo approximation of (Mesa Seco, Colorado, both below and above treeline, lati- Haldane’s test (Miller 2000), using 10 batches per analysis, tude 38°N). In both taxa, strong balancing selection main- 1 000 permutations per batch, 10 000 total permutations. We tains PGI gene polymorphism, with up to 10-fold fitness also used Goldstein’s (1964) exact binomial “x*” test for this differences calculated in the case of C. eurytheme (summary purpose and for testing specific hypotheses about pairwise in Watt 2003). differences of allele or genotype frequencies. In cases of

Fig. 2. Electrophoretic mobility class comparisons among Colias christina kluanensis (lanes 1–3), Colias christina astraea (lanes 4–13), and Colias eurytheme (lanes 14–20). Lane 1 is a 2/2, lane 4 is a 3/3, and lane 20 is a 4/4 homozygote. PGI is a dimeric enzyme, so heterozygotes display three bands (two homodimers and one heterodimer). Compare lanes 3 and 6 for “the same” mobility-class genotype from different subspecies, and lanes 13 and 14 for where the 3/3 homodimer can be seen side by side between species. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

multiple simultaneous tests, we adjusted the minimum sig- the previously well-studied C. eurytheme and C. m. meadii nificance level by the Dunn–Šidák method (Sokal and Rohlf for comparison purposes (cf., e.g., Watt et al. 2003). The 1995): new level α′ =1–(1–α)1/k, where k is the number most common PGI EM allele differs among the species stud- of tests and α is 0.05. ied (Table 1), and indeed in C. p. chippewa the 1 EM allele To probe our genetic samples for indications of natural se- class becomes common. Usually, the 2nd and 3rd most com- lection, we calculated the Ewens–Watterson F statistic using mon EM alleles in each species show single charge changes, standard procedures (Manly 1985; Hartl and Clark 1989), in- positive or negative, from the most common allele. How- cluding a Fortran computer program modified from that of ever, in C. c. kluanensis, alleles 2 and 4, not adjacent charge Manly (1985). This is a common statistic for the testing of classes, are the most common. All genes studied in each spe- allozyme data for the action of natural selection on the cies were polymorphic and matched Hardy–Weinberg distri- standing genetic variation within a population (Hartl and bution expectations (Table 2). Clark 1989). Numerical calculations of the distribution of Northern taxa have much more even distributions of PGI 1000 replicate, neutrally expected values of the Ewens– allele frequencies than the widespread lowland and low- Watterson homozygosity F statistic were run for each sam- latitude C. eurytheme. For example, among northern taxa, ple with k representing the number of alleles and n repre- C. p. chippewa has the highest frequency of its common al- senting the number of gene copies (i.e., twice the sample lele, p2 = 0.471, yet this is much lower than p3 = 0.630 in size of diploid Colias butterflies) appropriate to each sam- C. eurytheme (x* = 2.58, P = 0.01, α = 0.05; cf. Table 1). ple. The resulting distribution of neutrally expected F, with The low-latitude but high-elevation C. m. meadii, above tree- µ σ its mean and standard deviation , was calculated for each line, has a higher frequency of its most common allele, p2 = sample and the rank of the sample’s actual F value (rank ta- 0.625, than that of C. p. chippewa, but below treeline it is bled as R in percent units) in the distribution determined. similar, p2 = 0.521 (cf. Watt et al. 2003). These are signifi- Ranks, R, observed for each sample thus are “P values”, cor- cantly (x* = 2.48, P = 0.013, α = 0.025) and not signifi- responding to those values’ positions in their cumulative cantly different (x* = 0.89, P = 0.58, α = 0.025), probability distributions. R values on the left tail of the F respectively. The northern taxa therefore also show more distribution show greater-than-expected evenness of allele even PGI genotype distributions, with only two samples hav- ≥ frequency and suggest balancing selection; ranks on the right ing any genotype frequency 0.3 (C. c. kluanensis:p2/4 = tail show less-than-expected evenness and suggest purifying 0.325; C. m. elis:p3/4 = 0.3; Table 2). selection against all but a “wild type” allele (Manly 1985; For the PGM gene, most taxa have high 3 allele frequen- Hartl and Clark 1989). cies, but in C. hecla and C. nastes 2 alleles are most com- Regressions were calculated by a standard algorithm mon. Colias christina kluanensis and C. p. chippewa have (Sokal and Rohlf 1995), and bootstrap and permutation pro- very high 3 allele frequencies, p3 = 0.942 and 0.882, respec- cedures were done to test significance and estimate confi- tively, and correspondingly low levels of heterozygosity (Ta- dence limits for the regressions, using Perm Reg version 2.0 ble 2). Otherwise, C. c. christina, C. c. astrea, and C. nastes (Watt et al. 2003). show PGM frequency distributions roughly similar to C. eurytheme or C. meadii, although C. nastes shifts towards Results a more common 2 allele. We obtained G6PD data only from C. nastes, finding two Allele and genotype frequency variation among species alleles. The two most common genotypes in C. nastes are PGI, PGM, and G6PD electromorph (EM) allele mobility very similar in frequency to those of C. eurytheme. classes were similar among all taxa compared. This similarity does not, however, imply identity or functional similarity among alleles and genotypes in question (Watt et al. 1996; Allele and genotype frequency variation among Wheat et al. 2006). Positions of these EM alleles with re- subspecies spect to standard proteins, ferritin and hemoglobin, are In cases of geographically separated “subspecies”, there are shown by Carter and Watt (1988). Figure 2 illustrates gel interesting patterns of allele and genotype frequency diver- patterns for PGI EM classes 1–5. gences (Table 1). For the purposes of comparative analysis of Table 1 presents summary genetic statistics for all our allelic diversity, we make the parsimonious assumption that northern samples, and also includes representative samples of alleles of similar mobility within and among subspecies are

Table 2. Genotype frequencies among nine North American Colias butterflies. Genotype Gene (n)* p1/1 p1/2 p1/3 p1/4 p1/5 p2/2 p2/3 p2/4 p2/5 Colias eurytheme PGI (100) — — — — — 0.010 0.100 0.040 0.010 PGM (100) — — — — — 0.030 0.170 0.060 — G6PD(100) 0.420 0.460 0.040 — — 0.080 — — — Colias christina christina PGI (46) — — 0.043 — — 0.065 0.217 0.065 0.043 PGM (46) — — — — — — — — — Colias christina astraea PGI (70) — 0.014 — — — 0.043 0.214 0.114 0.043 PGM (70) — — — — — — 0.071 0.057 — Colias christina kluanensis PGI (77) — 0.013 — 0.065 — 0.117 0.143 0.325 — PGM (77) — — — — — — — — — Colias hecla PGI (31) — 0.024 0.024 — 0.024 0.024 0.122 0.146 — PGM (24) 0.042 0.125 0.042 0.042 — 0.333 0.125 0.042 — Colias meadii elis PGI (30) — — — — — — 0.200 0.167 — PGM (8) — 0.250 0.125 ——0.125 ——— Colias meadii meadii Low elevation PGI(165) — 0.024 0.024 — — 0.267 0.467 0.018 — PGM(84) 0.048 — 0.155 0.119 0.012 — — — — High elevation PGI(84) — 0.024 0.012 — — 0.381 0.429 0.036 — PGM(44) 0.045 — 0.091 0.182 — — — 0.023 — Colias nastes PGI (40) — 0.025 — 0.075 — 0.050 0.250 0.100 — PGM (40) — 0.025 — 0.025 — 0.300 0.300 0.025 — G6PD (40) 0.525 0.400 — — — 0.075 — — — Colias palaeno chippewa PGI (51) 0.176 0.275 0.078 — — 0.275 0.078 0.039 — PGM (51) — — — — — — 0.137 — 0.020 Note: Frequencies greater than 0.1 are in boldface type. Tests for fit to Hardy–Weinberg distribution expectations were based on Goldstein’s (1964) Haldane’s test in TFPGA (Miller 2000) gave comparable results to the extent allowed by the small sample sizes. *Numbers of genotypes, n, are in parentheses. †Observed total heterozygosity. ‡Observed difference in total heterozygosity from Hardy–Weinberg expectations. §Probability of observing the same or greater difference by chance. roughly comparable, owing to their shared evolutionary his- P <1×10–6; vs. high-elevation C. m. meadii: x* = 5.9, tory. Recent sequencing of the PGI gene in C. meadii and P <1×10–6; for these three tests, α = 0.017). The result C. eurytheme showed only one fixed amino acid difference is that the EM 2 allele goes from most common in C. m. between them (Wheat et al. 2006), suggesting that the major- meadii to third most common in C. m. elis, with the EM ity of amino acid variation in Colias butterflies is maintained 4 allele class going from uncommon in C. m. meadii to within and among populations: second most common in C. m. elis (tests similarly sig- (a) Colias christina christina and C. c. astraea have similar nificant but not shown for brevity). PGI and PGM allele frequencies, while C. c. kluanensis Genotype frequency changes accompany these allele fre- differs from them. Colias christina kluanensis has much quency differences (Table 2), and indeed it is probable that genotypic changes drive allele frequency changes as they lower p3 (and higher p2 and p4) at the PGI gene than its clearly do in C. eurytheme and C. m. meadii (Watt 2003; Watt relatives (e.g., vs. C. c. christina:p3 = 0.169 vs. –6 et al. 2003). As discussed below, these changes accompany p3 = 0.446, x* = 4.7, P <1×10 ; vs. C. c. astraea: –6 shifts in organism–environment interactions, and thus may of- p3 = 0.169 vs. p3 = 0.429, x* = 4.9, P <1×10 ; for C. c. astraea vs. C. c. christina: x* = 0.26, P = 0.79; fer opportunity for study of the selective causes of phyletic di- α = 0.017). Colias christina kluanensis has much higher vergence in genetic variant frequency. However, it is also important to note that our extensive sampling among popula- p3 (and lower p4) at the PGM gene than its relatives tions and across years for C. eurytheme and C. m. meadii has (e.g., vs. C. c. christina:p3 = 0.942 vs. p3 = 0.696, –6 also revealed a remarkable stability of allozyme frequencies x* = 5.2, P <1×10 ; vs. C. c. astraea:p3 = 0.942 vs. –6 (Watt et al. 2003). This suggests that our population samples p3 = 0.621, x* = 6.7, P <1×10 ; for C. c. astraea vs. C. c. christina: x* = 1.16, P = 0.25; α = 0.017). here may be representative of many, if not all, populations (b) The identity of the most common PGI allele shifts within these taxa. dramatically between C. m. meadii and C. m. elis.In C. m. meadii populations above treeline, the most com- Ewens–Watterson tests mon EM 2 allele is higher in frequency than those in Table 3 presents results of Ewens–Watterson testing of populations below treeline (high p2 = 0.625 vs. low p2 = our allozyme samples for adherence to or deviation from the 0.521, x* = 2.20, P = 0.008; Table 1; Watt et al. 2003), expectations of a neutral allele frequency distribution (a esti- but in each case it is very much higher than in C. m. elis mate of total sample homozygosity in relation to number of (p2 = 0.183; vs. low-elevation C. m. meadii: x* = 4.8, alleles and sample size, derivable from the infinite alleles © 2005 NRC Canada Wheat et al. 1619

† ‡ § p2/6 p3/3 p3/4 p3/5 p3/6 p4/4 p4/5 p4/6 p5/5 p5/6 H Hexc P — 0.380 0.310 0.070 — 0.060 0.020 — — — 0.550 0.001 0.99 — 0.480 0.230 — — 0.030 — — — — 0.460 –0.026 0.28 — — — — — — — — — — 0.491 0.045 0.36 0.022 0.217 0.130 0.043 0.022 0.087 — 0.022 — 0.022 0.630 –0.070 0.25 — 0.478 0.370 0.065 — 0.065 0.022 — — — 0.457 0.011 0.89 — 0.171 0.171 0.129 — 0.086 — — 0.014 — 0.686 –0.013 0.78 — 0.429 0.271 0.043 — 0.114 0.014 — — — 0.457 –0.070 0.24 — 0.026 0.143 ——0.169 — — — — 0.675 0.018 0.73 — 0.883 0.091 0.026 — — — — — — 0.117 0.006 0.87 — 0.098 0.122 0.122 0.024 0.171 0.098 — — — 0.742 0.030 0.57 — 0.208 0.042 — — — — — — — 0.418 –0.003 0.94 — 0.133 0.300 0.067 — 0.100 0.033 — — — 0.767 0.099 0.25 — 0.125 0.250 ——0.125 — — — — 0.625 –0.117 0.45

— 0.145 0.055 — — — — — — — 0.588 0.036 0.35 — 0.143 0.310 ——0.214 — — — — 0.643 –0.029 0.64 — 0.095 0.024 — — — — — — — 0.524 0.023 0.67 — 0.205 0.341 ——0.114 — — — — 0.636 –0.006 0.94 — 0.075 0.225 0.075 — 0.100 — — 0.025 — 0.750 0.025 0.72 — 0.200 0.050 0.050 — 0.025 — — — — 0.475 –0.133 0.08 — — — — — — — — — — 0.400 0.001 0.99 — 0.039 0.039 — — — — — — — 0.510 –0.124 0.07 — 0.784 0.039 0.020 — — — — — — 0.216 0.001 0.99 x* test with k representing 22 tests, resulting in a corrected Dunn–Šidák significance level (α′) for multiple tests of 0.0023. Parallel testing by simulation of

neutral model (Manly 1985; Hartl and Clark 1989). This 6×10–5. This strongly suggests the action of balancing se- procedure is known to have low statistical power so that lection throughout the data set. very large deviations are needed to produce a “significant” For the PGM gene, which shows large heterozygote advan- result. For an example, our comparison species C. eurytheme tage in male mating success in C. eurytheme and relatives, but and C. meadii experience strong balancing selection on the not in flight performance or viability (Carter and Watt 1988), PGI gene. But, while their Ewens–Watterson F statistic for five of the seven northern samples have an F value in the left this gene is biased toward the left side of the neutral expec- half of their Ewens–Watterson distributions. However, the tation distribution in each sample, suggesting excessive probability of getting this result by chance is not significant evenness of allele frequency as produced by balancing selec- (0.55 + 0.52 = 0.281). tion (see caption to Table 3), the deviations are not significant as isolated samples, despite the strong (e.g., Watt 2003) Heterozygosity by latitude analysis selective maintenance of the polymorphism. Many animals and plants show decreases in genetic diver- Our one sample of the G6PD gene, from C. nastes,isnot sity with increasing latitude, usually attributed to the rapid significant but nonetheless is in the left 20% of its distribu- colonization of deglaciated habitats at the beginning of the tion. The G6PD gene in lowland Colias butterflies shows Holocene (Hewitt 2000). While we know neither the exact large male mating advantage to heterozygotes, although like location of these species during the Pleistocene nor the de- the PGM gene it shows no effects on flight or viability mographic changes they experienced, we can ask if those (Carter and Watt 1988). more northerly taxa exhibit such genetic diversity decreases with increasing latitude, i.e., do the PGI and PGM genes of Meta-analysis of Ewens–Watterson tests Colias butterflies show this effect? We find a nearly signifi- A pattern is apparent among Ewens–Watterson tests of the cant positive regression of PGI heterozygosity on latitude PGI gene: no sample ranks in the right half of its Ewens– (Fig. 3 and its caption). The one outlier, C. p. chippewa, also Watterson distribution. Given that the probability of any one shows the lowest P value for PGI heterozygosity deviation sample occurring in the left half by chance is 0.5, the proba- from Hardy–Weinberg expectations (P = 0.07, Table 2; if bility that all 10 samples do so is 0.510 = 0.00098, and for this taxon is removed, there is a significant positive regres- the seven northern samples alone it is 0.57 = 0.0078. Indeed, sion of PGI heterozygosity on latitude). Results from the none of the northern samples has a rank higher than 25% or PGM gene are different and more in line with results from 0.25, and the by-chance probability of that result is 0.257 = other organisms, showing a negative regression of hetero-

Table 3. Ewens–Watterson F statistic tests for deviation from neutrality of genetic variation in Colias butterflies. Number of Gene n alleles (k) F Mean (µ) σ* R† Colias eurytheme PGI 200 4 0.454 0.594 0.179 25.0 PGM 200 3 0.514 0.701 0.187 22.4 Colias colias christina PGI 92 6 0.265 0.417 0.138 10.2 PGM 92 3 0.554 0.667 0.182 36.5 Colias christina astraea PGI 140 5 0.302 0.508 0.166 7.1 PGM 140 3 0.473 0.587 0.182 33.5 Colias christina kluanensis PGI 154 4 0.343 0.597 0.181 5.7 PGM 154 3 0.889 0.685 0.187 80.2 Colias hecla PGI 62 6 0.288 0.388 0.128 22.3 PGM 26 3 0.423 0.585 0.151 15.3 Colias meadii meadii Low elevation PGI 330 4 0.448 0.641 0.193 18.2 High elevation PGI 168 4 0.499 0.603 0.183 37.2 Colias meadii elis PGI 60 4 0.332 0.541 0.161 5.6 PGM 16 4 0.258 0.417 0.105 1.0 Colias nastes PGI 80 5 0.275 0.475 0.151 4.4 Colias palaeno chippewa PGI 102 4 0.366 0.569 0.172 11.3 PGM 102 4 0.785 0.569 0.172 84.8 *Standard deviation of expected F distribution. †Rank (percentile) of sample F value in that distribution. zygosity on latitude. The slopes of PGI and PGM are signifi- both pervasive now and probably far older than in present- cantly different, as their slopes lie outside one another’s day species. The macroevolutionary implications of this are 95% confidence intervals (caption of Fig. 3). discussed below. As molecular, functional, and fitness-related studies of Discussion polymorphisms at the PGM and G6PD genes proceed further in C. eurytheme and C. meadii, our results argue for their ex- Evolutionary impacts of allozyme variation in northern tension to northern taxa as well. Modes of natural selection, Colias butterflies other than those on the PGI gene, may act on these metabol- Many population biologists assume that electrophoretic ically adjacent, but functionally distinct, genes in parallel variants of metabolic enzymes have no functional effect and among many Colias taxa. Testing this possibility may allow hence are neutral to natural selection. But, for the PGI gene extension of bioenergetics as a unifying context for the study in C. eurytheme and C. meadii, amino acid sequence vari- of molecular functional adaptations in living things at large ants, seen in molecular sequencing and electrophoresis, alter (e.g., Watt 1985; Watt and Dean 2000). protein structure (Wheat 2001; Wheat et al. 2006) and thus both kinetics and thermal stability; these large functional ef- Microhabitat differences, climatic history, and phyletic fects then successfully predict large differences in flight per- differentiation of PGI gene adaptations formance in the wild, hence genotypic differences in fitness The interaction of Colias butterflies with thermal ecologi- as large as 10-fold (e.g., Watt 2003). Molecular structural cal variables, a central feature of their niche structure, analysis therefore complements results on functional and fit- changes with both elevation and latitude (e.g., Watt 1968). ness differences among PGI variants, providing insight into In particular, greater variance of thermal microclimate above the potential causes of these differences in the evolution of than below treeline is associated with PGI allele and geno- protein structure. Thus, PGI genotypes cover a wide range of type frequency shifts, often despite large migrational ex- natural selection intensities, from strong balancing selection change, between steppe and tundra populations of C. meadii that maintains some variants to true neutrality of others (Kingsolver and Watt 1983, 1984; Watt et al. 2003). The (Watt 2003; Wheat et al. 2006). thermal niches of arctic and subarctic Colias butterflies may Our meta-analysis of Ewens–Watterson testing suggests differ from that of the lowland species complex even more that strong balancing selection on the PGI gene may occur in than Rocky Mountain C. meadii in some ways. Using thermal all the northern taxa studied here. Given the greater evenness ecological techniques honed in the study of temperate-zone of allele frequencies in these taxa than in C. eurytheme and Colias butterflies, we can investigate the interaction of genetic C. meadii, seen in Table 1 and reinforced by the Ewens– variants with the environmental variation to which arctic and Watterson results, selection on the PGI gene in northern taxa alpine Colias butterflies are subject. may be even stronger than in those already studied. If so In this connection, we note that arctic and alpine Colias (and much more field and laboratory studies will be needed butterflies have maintained genetic diversity at the PGI, to test this), strong selective maintenance of PGI variation is PGM, or G6PD genes right through the dramatic climate

Fig. 3. Regressions of sample heterozygosity on latitude for North different evolutionary histories. The isolation of populations American Colias butterflies: (a) PGI and (b) PGM. Because fre- resulting from refugial separation, or simply to the division of quency data are a priori not normally distributed, statistical valida- previous ranges, by glacial barriers, may lead to ecological, tion of slopes was done by bootstrapping (1000 iterations) to test hence both adaptive and phyletic, differentiation of those pop- representativeness of the primary slope for its data set, and by per- ulations (cf. Remington 1968; Schluter 2000; Hewitt 2001). mutation (1000 iterations) to test difference from true random The shift in PGI and PGM genotype frequency distributions slope, using the program Perm Reg (Watt et al. 2003). For PGI, of C. c. kluanensis, in open Artemisia-dominated steppe, ver- the primary slope is 0.0043, bootstrapped slope and 95% confi- sus C. c. christina and C. c. astraea in more mesic meadows dence interval are 0.0041 ± 0.0037, and randomly permuted slope at forest edges, may reflect changes in ecological interaction and 95% confidence interval are 0.0002 ± 0.0048. For PGM, pri- associated with such isolation. (Indeed, C. c. kluanensis mary slope is –0.0097, bootstrapped slope and 95% confidence in- shows greater hindwing absorptivity for sunlight, a trait terval are –0.0099 ± 0.0070, and randomly permuted slope and known to alter the thermal balance of Colias butterflies, than 95% confidence interval are –0.0004 ± 0.0093. its more southerly relatives (cf. Watt 1968; Roland 1982; ()a Kingsolver and Watt 1984; C.W. Wheat and W.B. Watt, un- 0.8 published data).) A similar habitat-associated shift in PGI

0.7 allele frequency between “subspecies” also occurs between C. m. meadii and C. m. elis. Thus, a diversity of intermedi- 0.6 ate states of adaptive change and of phyletic differentiation

0.5 may be found in northern populations of Colias butterflies.

0.4 Large-scale evolutionary implications 0.3 It now seems likely that all populations (let alone taxa) of 0.2 North American Colias butterflies harbor major PGI polymorphisms, and that strong selection maintaining this vari-

PGI Heterozygosity 0.1 ability may be ubiquitous. This supports the speculation that, 0 at least throughout North America and perhaps in the rest of 35 45 55 65 75 the genus’ global species distributions, PGI polymorphisms in Colias butterflies may be evolving by successive replace- ()b Latitude (°) ment of selectively maintained polymorphic alleles without 0.8 ever passing through monomorphic states. This underscores the question raised by Watt et al. (1996): what is it that con- 0.7 strains against the emergence of any allele in this gene, and 0.6 genus, which would co-maximize functional properties in homozygotes, and thus be able to replace species-specific ar- 0.5 rays of polymorphic genotypes with “optimized” homo- 0.4 zygosity for new “wild-type” alleles? Another way to ask these questions is why is selection on the PGI gene so recur- 0.3 rent among species and how old might this pattern of bal- 0.2 ancing selection be? The answers may lie anywhere between the molecular and the population levels of biological organi- PGM Heterozygosity 0.1 zation. In the mean time, the phenomena themselves under- 0 score a perspective on specific-gene molecular variation that 35 45 55 65 75 is new to many evolutionists: “molecules are more than markers” and molecular variants may often be effective Latitude (°) probes of organism–environment interactions that result in strong natural selection (Lenski 1994; Watt and Dean 2000). change events of the Pleistocene, during which much of The ability to study the interaction of adaptive innovation their current habitat was repeatedly covered in ice (e.g., with phyletic differentiation may allow the addressing of Pewe 1983). Many arctic and alpine species show decreased some noteworthy macroevolutionary questions (e.g., Eldredge genetic diversity with increased latitude as a result of the 1995). Molecular systematic analysis can be applied to genes migrations and population size fluctuations entailed by gla- independent of these polymorphic ones in Colias butterflies, cial episodes (Hewitt 2000). We did find a negative correla- leading to independently inferred estimates of phylogenetic tion between PGM heterozygosity and latitude. However, at relationships among taxa. Such phylogenetic reconstructions the PGI gene, we found a nearly significant positive correla- can then be used to “map” specialization of adaptive variabil- tion between heterozygosity and latitude, and this was sig- ity, allowing us to ask whether adaptive specialization pro- nificantly different from the PGM gene. motes species differentiation, or conversely the isolation These results are striking, given previous documentation associated with speciation “releases” organisms into the op- of selection at both these loci. While it is difficult at this portunity for emergence of novel adaptive innovations. A stage to infer what might be causing this difference among comparative system such as this one, extending across the species and between loci, it is clear that they have had very whole of northern North America, affords an outstanding

© 2005 NRC Canada 1622 Can. J. Zool. Vol. 83, 2005 opportunity to explore such questions, central to macro- Pieridae): resource and time budget analysis. Funct. Ecol. 12: evolutionary study, in a direct empirical fashion. 149–158. Nielsen, M.G., and Watt, W.B. 2000. Interference competition and sexual selection promote polymorphism in Colias (Lepidoptera; Acknowledgements Pieridae). Funct. Ecol. 14: 718–730. We thank Carol Boggs, Ken Phillip, Scott Armbruster, Pewe, T.L. 1983. The periglacial environment in North America during Wisconsin time. In Late-Quaternary environments of the Jeff Hunston of Yukon Heritage, the Yukon First Nations United States. Edited by S.C. Porter. University of Minnesota Council, and Norbert Kondla for permission to collect in Yu- Press, Minneapolis. pp. 157–189. kon, or assistance in collecting, or essential information. Remington, C.L. 1968. Suture-zones of hybrid interaction between Portions of this work were drawn from a thesis submitted by recently joined biotas. Evol. Biol. 2: 321–428. C.W.W. to Stanford University in partial fulfillment of re- Roland, J. 1982. Melanism and diel activity of alpine Colias quirements for the Ph.D. degree in Biological Sciences. This (Lepidoptera; Pieridae). Oecologia (Berl.), 53: 214–221. work was supported by grant ER61667 from the US Depart- Sappington, T.W., and Taylor, O.R. 1990. Disruptive sexual selec- ment of Energy to W.B.W., and grants DEB 91-19411 and tion in Colias eurytheme butterflies. Proc. Natl. Acad. Sci. IBN 01-17754 to W.B.W., and a graduate fellowship to U.S.A. 87: 6132–6135. C.W.W. from the US National Science Foundation. All find- Schluter, D. 2000. The ecology of adaptive radiation. Oxford Uni- ings are our own and represent no official policy of any versity Press, Oxford. agency or institution. Sokal, R.R., and Rohlf, F.J. 1995. Biometry. 3rd ed. W.H. 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