I am using a reciprocal transplant study to test for local adaptation in an insect that is dependent on rotting vegetation substrates for larval development.
Our study question: over a cline in environmental conditions that effect the quality of the vegetation substrate, can we detect local adaptation? Specifically, we can test for local adaptation with two responses, i.e. survival and development time, using two tests (Kawecki and Ebert 2006):
Local versus foreign, local individuals always do better on their own substrate versus foreign individuals.
Home verus away, individuals always do better on their own substrate than on other substrates.
These are standard tests for local adaptation with the first being seen as the strongest indicator.
We collected adults at four locations along the cline. Further, we collected the vegetation substrates at each location. Next we selected and paired males and females from each location separately. Then we collected the eggs masses from the resulting breeding efforts of all the adult pairs from each location separately, homogenized them (again by location) in a solution to separte the individual eggs. Next, we divided the eggs from each location into groups of 30 and put them onto each of the four location substrates to accomplish the reciprocal transplant. Finally, we repeated this last step 3 times to create three replicates.
By sampling the adults and substrates only once we attempted to control for temporal variation in environmental conditions.
My question, are there differences in opinions on whether this truly represents three 'replicates'?