Hypothesis:
If an anadromous population of stickleback spends most of its life in a marine environment, then the pelvic enhancer for its Pitx1 gene will be present, resulting in the appearance of pelvic spines.
Rationale:
The stickleback from Deep Creek lake are anadromous, meaning that they spend parts of their lives in both freshwater and marine environments. However, the majority of their life is spent in oceanic conditions, only moving to fresh water temporarily to breed once in their life cycle. As a result, the environmental pressures of marine life are very prevalent – the most significant of these is predation by other carnivorous fish. The general stickleback population is known for the presence of bodily armor, and when its main predator’s method of attack involves biting, then having as much armor as possible is favorable for survival, including in the pelvic region. In the long run, then, those with a vulnerable pelvic area would face much lower chances of survival. Those with more armor are able to survive and reproduce, passing on their genes. While the Pitx1 gene, which controls the presence of the pelvis, is present in all stickleback, be it marine or freshwater, a specific enhancer (a section of DNA that allows for expression in specific areas) must be present in order for the gene to be expressed in the pelvic region. Thus, the environmental pressures of the marine environment should favor the presence of this enhancer, as it would make for a more survivable experience. Due to the various marine environmental pressures acted upon the Deep Creek stickleback, pelvic spines are favored and will be observed in the population. For this reason, the pelvic spine enhancer will be present in the Pitx1 gene of the PCR amplified DNA samples of both Deep Creek stickleback and the marine control. As the enhancer is present in both samples, the gel electrophoresis will not reveal a significant difference in the lengths of the genes.
Results :
The gel electrophoresis revealed that PCR amplified samples of Deep Creek and the marine control were the same length, supporting the hypothesis that the pelvic enhancer was present in both (Figure 1). Despite these findings it is critical to note this presence was also detected in the negative control (water), indicating the results had been contaminated (Figure 1).
Discussions and future directions:
Pelvic armor is a favorable trait in a marine environment on account of the protection they provide from carnivorous fish. Positioned perpendicularly on the fish, the spines make it difficult for marine predators to swallow them and the plates on their back are bite resistant. Conversely, when the Deep Creek Stickleback move to the freshwater area to breed the armor is to their detriment, because of the fluctuation in environmental pressures. The once useful plates now act as target on their backs, making it easier for birds to hunt them. Dragonfly larvae, a major contributor to freshwater predation, use the pelvic spines to catch and eat the stickleback. Considering that these anadromous stickleback were acted on by the various pressures that encompass life in a marine environment, it was logical to hypothesize the appearance of pelvic spines due to their advantageousness.
After running PCR and completing gel electrophoresis, the gels showed that the DNA samples of the marine control and the Deep Creek stickleback were the same length. These results indicate that both genes had a functioning enhancer, supporting our initial hypothesis. As predicted, due to the environmental pressures acted on the Deep Creek Stickleback a functioning enhancer was present in the gene. The enhancer’s presence and functionality determined that the Deep Creek Stickleback sample had developed armor, because of the extensive time spent in a marine environment. The biological significance of the results is that the findings highlight the convergent evolution that has occurred in this population of stickleback. In order for the Deep Creek stickleback to adapt among the pressures of their environment they developed the armor that would allow them to thrive. In light of the results of the study it can be determined that armor is a favorable trait among anadromous stickleback as well as marine, because of the extensive time spent under the pressures of a marine environment.
It cannot be ignored that in running the experiment, the enhancer’s presence was also found in the negative control (water), which can only be explained by the occurrence of contamination (Figure 1). This presence in the negative control also indicates that contamination could have occurred at any point in the experiment. We cannot be sure which samples were contaminated and which were not therefore, the experiment should be rerun in order to ensure ultimate scientific accuracy and collect results that are devoid of contamination. The contamination could be due to a number of factors, the most probable of which is human error. It can be inferred that human error was the main source because humans also have an enhancer on the Pitx1 gene and cross contamination could have occured at any point in running the experiment.
The hypothesis is not merely supported by the results of the experiment but also by a variety of similar studies. One study remarks the adaptation of anadromous stickleback by examining their morphological armor changes over a few years, “A combination of traditional and geometric morphometric methods were used to study variation over multiple years in an anadromous population that breeds in Rabbit Slough, Cook Inlet, Alaska. Major armor anomalies were extremely rare but their occurrence at measurable frequencies suggests that significant standing variation for armor phenotypes exists in anadromous populations (Aguirre et al. 2008).” This study, much like our experimental results, found that the anadromous Rabbit Slough-Cook Inlet population of stickleback (minus a few outliers as described in the abstract) also exhibited pelvic spines. Another study remarks that it may predation may not be the main environmental pressure that “Substantial pelvic reduction in threespine stickleback sampled from 179 lakes around Cook Inlet, Alaska is strongly associated both with an absence of predatory fishes and a low calcium concentration (Bell et al. 1993).” The results of this study prompt questions for future research including, Is it low calcification or predation that affects stickleback armor morphology? Testing the effects of both isolated variables individually and examining the pelvic spine structure in stickleback would be a very interesting new direction for a future study. Additionally, an avenue of study that could provide more insight into the evolution or devolution of pelvic enhancer could be, are predators affected by fluctuating calcium concentration making them less likely to prey on the stickleback resulting in morphological changes in the pelvic spines? As a future study, this would also make an interesting new direction as it focuses on the effects of the environmental pressures on predators as well.
Literature Cited:
Windsor E. Aguirre, Kaitlyn E. Ellis, Mary Kusenda, Michael A. Bell. 2008. Phenotypic variation and sexual dimorphism in anadromous threespine stickleback: implications for postglacial adaptive radiation. Biological Journal of the Linnean Society, Volume 95: 465–478.
Michael A. Bell, Guillermo Ortí, Jeffrey A. Walker, Jeffrey P. Koenings. 1993. Evolution of pelvic reduction in threespine stickleback fish: a test of competing hypotheses. Wiley Online Library, Volume 47: 906-914.