1614 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 polymor- phic in all taxa studied. Patterns of variation for the PGI gene in these northern taxa suggest that the balancing selec- tion 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. Can. J. Zool. 83: 1614–1623 (2005) doi: 10.1139/Z05-159 © 2005 NRC Canada Wheat et al. 1615 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 differ- ences 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 tem- perature 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 per- formance 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,