Individual differences in the ability to compete for mates can drive evolution through a process known as sexual selection. While examples of sexual selection acting on behavior or morphology are well known, much less established is the understanding of how sexual selection acts at the molecular level to affect individual reproductive success. In species where females mate with multiple males, sperm compete for access to a female?s eggs. Sperm competition can lead to selection on seminal fluid proteins (Sfps), a class of proteins transferred by a male during mating that aid in successful fertilization by that male while reducing success of subsequent matings by other males. Because of their link to sperm competitiveness, Sfps may evolve differently in species with polygamous mating systems (with high sperm competition) compared to monogamous species (with low sperm competition). This project investigates the relationship between mating behavior and selection on Sfps in New World blackbirds (genus Agelaius), a group whose diverse behavior enables the study of how sexual selection affects DNA sequence evolution. Genes coding for Sfps will be isolated to test the hypothesis that such genes evolve faster in polygamous than in monogamous species. These genes will be archived on the NIH sequence database GenBank, providing researchers with tools for future comparative studies. Ultimately, results will clarify how selection links traits across a wide range of biological scales, from the molecular to organismal level. Mentoring and outreach efforts are integral to this project. Undergraduate students will be introduced to the basic research process by learning fundamental lab techniques and writing original papers. Results will be shared at Caribbean field sites, where a primary challenge facing natural resources agencies is reconciling the need for preservation with those of a tourist-driven economy. Work will also be communicated broadly at the university, community and national levels.

Project Report

Males compete with each other to mate with females. The classic image of this competition has been one of a male winning a fight over another male to gain exclusive access to a female. However, it is now known that females of many species mate with multiple males during a single breeding season. As a result, competition between males does not end at mating, but instead extends to fertilization, because sperm from multiple males must compete within a female’s reproductive tract to reach her eggs. This post-mating process, known as sperm competition, is thought to be just as strong as the competition to secure a mate, yet its evolutionary role remains largely unknown. Just as weapons like antlers have evolved to help males compete to mate with females, certain proteins in the seminal fluid have evolved to help males compete to fertilize a female’s eggs. These seminal fluid proteins (Sfps) do so in a variety of ways, such as by disabling the sperm of other males or by altering the female’s physiology or behavior to the male’s advantage. Because Sfps act against the reproductive interests of females and of other males, they are thought to be under strong pressure to evolve rapidly. Indeed, Sfps studied in insects and mammals have been shown to be fast-evolving, reflecting the evolutionary tug-of-war in which they are locked. However, it is unclear whether this tug-of-war exists in all animal groups. Beyond insects and mammals, little is known about Sfp evolutionary patterns elsewhere. Do similar proteins exist across different groups? If so, do the proteins experience similar pressures to those observed in insects and mammals, or are these pressures present in certain groups and not in others? The funded work addressed these questions in songbirds, which are known for multiple mating but poorly studied at the molecular level. This project linked behavioral and molecular patterns of evolution by asking: Do closely related species show similar levels of sperm competition? Which proteins exist in songbird seminal fluid? And, do species with different levels of sperm competition exhibit different rates of protein evolution? Field, lab and computational approaches were used to address these questions in three species of Agelaius New World blackbirds, which were predicted to vary in intensity of sperm competition. To measure this intensity, blood samples were collected in wild populations of breeding birds across North America and the Caribbean. Paternity tests then determined the proportion of nestlings that were the products of extra-pair mating (i.e., sired by a male other than the one on whose territory they had hatched), which is a proxy for the prevalence of sperm competition. This work provided the first evidence of multiple mating in two of the species and found that, combined with the third species (whose extra-pair mating rate was known), all three species had about 20-30% of extra-pair young. Because of the similarity in levels of sperm competition across species, Sfps were thus not expected to evolve at different rates across species. However, the question remained open of whether, as in insects and mammals, songbird Sfps evolve faster than non-Sfps. Next, Sfps were identified, producing the first catalog of these proteins in songbirds. Because Agelaius blackbirds have few genetic resources, Sfps were isolated using field-collected RNA and protein samples. Samples were sequenced, matched against each other, and searched across databases to determine the original gene sequences in DNA and their corresponding functions. A total of 184 proteins were recovered, some playing explicit roles in fertilization but most associated with metabolism, with many previously reported in insects and mammals. Finally, from the Sfp catalog, six genes were selected to test for rapid evolution. Surprisingly, Sfp-coding genes were generally not found to evolve more rapidly than control genes. One reason for this result is that many Sfps, even in insects and mammals, are actually pressured to remained unchanged because of their importance in ensuring proper fertilization. An alternate reason is that bird genomes in particular are known to evolve more slowly than other animal groups. Nevertheless, the results contrast trends observed in other animals. Work has already begun to examine how consistently these findings hold across genes and species, with the aim of understanding how competition between males can help shape patterns of molecular evolution. Findings from this project were shared in multiple ways. During the funding period, more than 13 research presentations were given to diverse public audiences, including female middle-school students and local communities at field sites. Two minority undergraduate students were successfully trained in lab techniques. A collaboration with the USFWS was also established to assess population genetics of one of the blackbird species (currently endangered). Finally, molecular resources were generated that will be available to the research community. Together, these efforts ensured the findings have been communicated widely at the university, community and national levels.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Michelle M. Elekonich
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Duke University
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