Copyright 2004 by the Genetics Society of America DOI: 10.1534/genetics.103.026120 The Genetic Covariance Among Clinal Environments After Adaptation to an Environmental Gradient in Drosophila serrata Carla M. Sgro`*,1 and Mark W. Blows† *Centre for Environmental Stress and Adaptation Research, La Trobe University, Melbourne 3083, Victoria, Australia and †Department of Zoology and Entomology, University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia Manuscript received December 23, 2003 Accepted for publication April 15, 2004 ABSTRACT We examined the genetic basis of clinal adaptation by determining the evolutionary response of life- history traits to laboratory natural selection along a gradient of thermal stress in Drosophila serrata.A gradient of heat stress was created by exposing larvae to a heat stress of 36Њ for 4 hr for 0, 1, 2, 3, 4, or 5 days of larval development, with the remainder of development taking place at 25Њ. Replicated lines were exposed to each level of this stress every second generation for 30 generations. At the end of selection, we conducted a complete reciprocal transfer experiment where all populations were raised in all environments, to estimate the realized additive genetic covariance matrix among clinal environments in three life-history traits. Visualization of the genetic covariance functions of the life-history traits revealed that the genetic correlation between environments generally declined as environments became more different and even became negative between the most different environments in some cases. One exception to this general pattern was a life-history trait representing the classic trade-off between development time and body size, which responded to selection in a similar genetic fashion across all environments. Adaptation to clinal environments may involve a number of distinct genetic effects along the length of the cline, the complexity of which may not be fully revealed by focusing primarily on populations at the ends of the cline. ATITUDINAL clines are widespread and provide a stress. QTL studies are able to identify loci that contrib- L natural framework within which to examine the ute to major phenotypic differences. However, if clinal operation of natural selection (Endler 1977). Despite adaptation is controlled by a large number of genes of their prevalence, the genetic basis of adaptation re- small effect that respond to selection at various stages sulting in clinal variation is poorly understood (Barton along the cline and do not approach fixation (Barton 1999). Two main approaches have been applied to de- 1999), the genetic basis of clines may be difficult to termining the genetic basis of adaptation along clines. resolve using the cline-end-QTL approach in isolation. First, quantitative trait loci (QTL) studies have taken For example, Stratton (1998) found that QTL with advantage of the large difference in phenotype at the large effects on flowering time in Arabidopsis thaliana ends of clines that are crossed to generate mapping were insensitive to a resource gradient, and that most populations for linkage association scans. This approach of the genotype-environment interaction was likely to has been able to successfully identify loci contributing be caused by many genes of small effect. to the large phenotypic differences at the ends of clines A second, and complementary, genetic approach to for body size in Drosophila melanogaster (Gockel et al. the study of the genetic basis of clines that is designed 2002), some of which may be involved in replicate clines explicitly to examine the continuous (or otherwise) na- on different continents (Calboli et al. 2003). ture of the genetic basis of clinal adaptation involves A fundamental aspect of the biology of clines is that the determination of the pattern of genetic covariation clines in many traits are usually continuous in nature between multiple environments (Kirkpatrick et al. 1990; and may display either linear (e.g., James and Par- Gomulkiewicz and Kirkpatrick 1992; Cooper and tridge 1995; James et al. 1995; Hallas et al. 2002; Hoff- DeLacy 1994). Rather than attempting to identify single mann and Shirriffs 2002) or more complex (Azevedo loci, this approach endeavors to answer two different et al. 1996; Magiafoglou et al. 2002; Sgro` and Blows questions concerning the genetic basis of clinal adapta- 2003) associations with latitude or some environmental tion: (1) whether adaptation to environments adjacent along the cline involves more similar genetic responses than adaptation to very different environments such as those at the ends of clines and (2) whether the continu- 1Corresponding author: Centre for Environmental Stress and Adapta- tion Research, La Trobe University, Melbourne 3083, Victoria, Aus- ous reaction norms that describe the association be- tralia. E-mail: [email protected] tween the trait and the particular stress that may charac- Genetics 167: 1281–1291 ( July 2004) 1282 C. M. Sgro` and M. W. Blows terize a cline are constrained in their evolution by the development time and size (Hallas et al. 2002). Nonlin- available patterns of genetic covariance (Gomulkiewicz ear patterns for trait means are likely to be the result and Kirkpatrick 1992; Kingsolver et al. 2001). of adaptation to environmental factors that do not show Although latitudinal clines are widespread and exten- linear patterns along the latitudinal cline (Loeschcke sively studied, the environmental factors that have et al. 2000). Examination of climatic data for the east shaped the evolution of these clines remain largely un- coast of Australia obtained from the Bureau of Meteorol- known. A number of laboratory thermal experiments ogy indicated that environmental factors that have non- using D. melanogaster have provided some insight into linear patterns with latitude similar to those suggested the selective factors underlying latitudinal clines (Cav- for development time in D. serrata do exist: the number icchi et al. 1989; Partridge et al. 1994a,b), but have of days over 35Њ is one such factor. After performing a not specifically examined adaptation to environmental number of pilot studies, we decided to use exposure of gradients, since they have focused on two or three tem- larvae to a heat stress of 36Њ for 4 hr each day for the peratures at any one time. Yet environmental factors 1–5 days of larval development as our selective factor. along latitudinal clines are most often expressed as gra- This allowed us to create a gradient in environmental dients rather than in two or more spatially distinct zones stress that may have some relevance to that experienced (Endler 1977). In addition, stress resistance traits often by D. serrata along its latitudinal distribution from tropi- display continuous clinal variation (Hallas et al. 2002; cal to more temperate areas. Hoffmann et al. 2002), suggesting that environmental Experimental populations were initiated from the F7 factors along clines may vary in such a way as to form generation of the cross between two laboratory-adapted gradients of environmental stress. To understand the populations of D. serrata representing northern (tropi- role of environmental gradients in the formation of cal) and southern (temperate) areas of the distribution latitudinal clines, it will be necessary to experimentally of this species (Sgro` and Blows 2003). Both the north- evaluate how adaptation to environmental gradients oc- ern (Cooktown) and southern (Wollongong) popula- years as bottle 2ف curs. tions had been in the laboratory Here, we have created a gradient of increasing stress cultures at 25Њ and 12-hr light (L):12-hr dark (D) photo- to examine the genetic basis of adaptation to an environ- period, before the commencement of this study under mental gradient by populations of D. serrata. D. serrata identical culture conditions (three bottles per popula- flies per bottle). The two populations at the 300ف ,is a member of the melanogaster subgroup, in which tion genetic clines in body size, weight, and cold resistance cline ends were crossed to ensure that many of the (Hallas et al. 2002) and development time (Magiafog- alleles for determining trait differences along the spe- lou et al. 2002; Sgro` and Blows 2003) are exhibited by cies of the D. serrata cline were present within the base natural populations along the eastern coast of Australia. population. At the F7 generation of this cross, replicate Using a laboratory natural selection experiment, we ex- lines from this mass-bred population were set up and posed replicates of a single base population to an envi- placed in six environments to evolve in the laboratory ronmental gradient composed of six environments vary- natural selection experiment, three replicate lines per ing in the frequency of extreme larval temperature stress environment. Each replicate line consisted of two bot- for 30 generations. We measured the direct and corre- tles of 40 females and 40 males per bottle. The selection lated responses to selection of a number of life-history regime involved exposure of larvae to a heat stress of traits. Our experimental design consisted of a reciprocal 36Њ for 4 hr per day (and then returned to 25Њ) for 0, transplant experiment, in which all populations were 1, 2, 3, 4, or 5 days throughout larval development assessed for the life-history traits in all environments. (hereafter referred to as environments E1, E2, E3, E4, This experiment enabled us to extract the realized addi- E5, and E6, respectively), starting at the first instar, to tive genetic variance-covariance among environments generate a gradient of environmental stress. This was on the basis of the direct and correlated selection re- done by placing 6-day-old adults in fresh bottles and sponses of the replicate populations to determine the allowing them to lay for 24 hr at 25Њ and 12 hr L:12 hr pattern of genetic covariance among the clinal popula- D, after which time the adults were removed from the tions. bottles. The eggs were left to hatch for a further 24 hr at 25Њ and 12 hr L:12 hr D, and the bottles were then placed in their respective selection environments.