OVERVIEW OF PhD RESEARCH
Insects have developed several physiological and morphological strategies to survive
extreme seasonal conditions. The ability to predict seasonal changes based on environmental cues (photoperiod), is one of the most crucial and interesting strategy of insects. Among different adaptive traits, photoperiodic diapause (or the delay in development in response to regularly and recurring periods of adverse environmental conditions) is particularly important as it affects the organism at physiological, morphological and behavioral levels.
Lycaeides diapause as eggs in their developmental process. Lycaeides idas, which are found farther north exhibit obligate diapause, whereas Lycaeides melissa exhibit facultative diapause but with generally fewer generations per year in the north than south. By taking advantage of this extensively studied model system, my proposed research has two broad objectives:
Objective 1: Identifying causes and propensity of diapause in facultatively diapausing L.melissa populations
Objective 2: Document if recent introgression has provided the genetic variation necessary for the southern-most population of the butterfly L.idas to evolve facultative diapause.
Here is a brief overview of the above mentioned objectives:
Objective 1: Identifying causes and propensity of diapause in facultatively diapausing L.melissa populations
Phenotypic plasticity occurs when the phenotype expressed by a given genotype depends on the environment. Previous studies on the heritability of plasticity have tried to understand the response of plastic traits to selection and it is still unclear to what extent natural selection makes plastic responses in different environments adaptive. General conclusions about a plastic trait being adaptive are made by measuring correlations between the plasticity of a trait and fitness averages across environmental gradients. However, it is still unclear how multiple environmental gradients can add complexity to the adaptivity of plastic traits. Also, it has become important to understand the evolution of plasticity in genetically structured populations to differentiate intra and inter-population genetic variation underlying plasticity. Understanding these areas will further help quantify the amount and distribution of genetic variation for plasticity and the extent to which plasticity responds to natural selection.
For this objective, I will focus on the following two studies:
Study 1: Identify the proximate causes of diapause initiation in a facultatively diapausing population
The circadian clock genes have been studied extensively in insects. However, the gene networks underlying seasonal rhythms for photoperiodism have not been studied so well. Past research has extensively used circadian rhythm genes as candidate loci for understanding the genetic basis of diapause switches. However, it should be recognized that the effect of circadian genes on photoperiod cannot be always concluded as a causal effect. It is possible that circadian genes may affect the expression of other genes which in turn affect seasonal rhythms. This provides an important avenue to not be restricted by circadian clock genes as candidate loci and use genomic data to isolate additional regions of the genome responsible for diapause.
The main cue for seasonal photoperidoic switch is day length. Short days give the signal to insects to go into diapause whereas long days do not trigger the seasonal diapause switch. Long day-length (LD) and short day-length (SD) trigger different physiological, cellular and behavioral responses in all insects and are the major cue for seasonal change.
Study 2: Identify potential differences in the popensity to enter diapause in facultatively diapausing L. melissa populations at different elevation and latitude gradients
Although there are several studies which have focused on evolutionary adaptation across the geographic ranges of species, there have been fewer studies that have compared local adaptation across elevation and latitudinal ranges. Comparison of populations at different elevations is interesting because environmental cues such as temperature, day-length and heat intensity differ at different levels along an elevation gradient. L. melissa populations occur along elevation and latitudinal gradients. I have observed that low elevation population come out early in May and have multiple generations. However, a high elevation population has a single generation and comes out in late June or July. Both these populations make an ideal representation of genetically structured populations and therefore are a good experimental model for understanding the evolution of diapause. The geographical range of L. melissa extends through different latitudes and this can help me compare populations along various latitudinal gradients.
Objective 2: Document if recent introgression has provided the genetic variation necessary for the southern-most population of the butterfly L.idas to evolve facultative diapause.
Lycaeides are geographically widespread and occupy a variety of different habitats. Along these lines, there is evidence that individual populations have adapted to their local conditions. Geographically widespread hybridization has been documented between Lycaeides idas and Lycaeides melissa in the Rocky mountains, which has resulted in a series of admixed populations that are L.idas-like in terms of their overall genome composition, host plant use and phenology (i.e., these populations exhibit obligate diapause and have a single brood per year). These admixed populations are relatively old (admixture occured about 14,000 ybp) and are composed entirely of hybrid individuals. Herein we refer to these populations as L.idas to distinguish this older admixture event from more recent hybridization,which is relevant for this study.
By taking advantage of this extensively studied model system for hybridization, the second part of my dissertation will help understand if adaptive introgression has led to recent adaptive
change in Lycaeides species for diapause trait by focusing on the following two studies:
Study 1: Use admixture mapping to identify quantitative trait loci (QTL) for obligate versus facultative diapause in an active hybrid zone between L.idas and L.melissa
For this objective, I will focus on a narrow hybrid zone between L.idas and L.melissa that occurs near Dubois, WY (Study site 1 in figure below). Hybridization is ongoing in this area and individuals with a mixture of genome compositions and traits (including variation in diapause) can be found here. Segregating variation in the hybrid zone will be used to identify alleles associated with species differences in diapause (that is, diapause QTL).
Study 2: Quantify genomic patterns of introgression in the single L.idas population that exhibits facultative diapause to test for recent introgression of L.melissa diapause QTL alleles
For this objective, I will analyze a L.idas population located in the Wyoming Range (Periodic Springs, Study site 2 in figure below). Unlike all other L.idas populations we have encountered, the flight season of this population indicates that it has evolved facultative diapause (this is also the southern-most L.idas population that we have identified and it is near the southern terminus of this species’ suspected range). L.melissa can be found a few kilometers from this site where they feed on introduced alfalfa.
This summer, my focus is on Objective1-Study 1. The following page descibes the objectives for this study, methods we will use and the expected outcomes.
OBJECTIVES
The objectives of this study are the following:
Quantify the effect of short day length on the proportions of females laying diapausing eggs
Measure temporal changes and differences in gene expression for butterflies that will lay diapausing vs. non-diapausing eggs
METHODS
I will use a single population of L. melissa (BST in Logan, UT), capture females from the wild and rear their eggs in the laboratory. These eggs will be subjected to long day and short day treatments to measure the differences in their propensity to enter diapause. Next, I will generate gene expression data from these two treatments to quantify differences in patterns of gene expression over the course of development in these two different treatments.
TESTS FOR PHENOTYPE MEASUREMENTS:
The following tests can be performed to check for diapause phenotype:
Record the number of eggs that hatch, die and enter diapause.
Take 10 day and 15 day old larvae and flash freeze them for future gene expression sequencing.
Record adult size and weight of adults.
Simulate winter conditions by keeping some eggs from each population in the freezer at 2-4℃ and constant humidity and darkness.
(Optional) Check the ovaries of diapausing and non-diapausing females to record the differences in ovarian structure.
(Optional) Record the eclosion rhythms i.e. record time of eclosions under different treatments.
Expected results
I expect females reared under the SD treatment to lay eggs that enter diapause and females reared under the LD treatment to lay eggs that do not enter diapause.
I expect numerous genes to exhibit expression differences between the two treatments, and that this will include some known circadian genes and additional co-expressed genes associated with diapause.
I expect differences in gene expression at different stages of development of larvae.