Relative to the majority of proteins encoded by animal and plant genomes, those responsible for reproductive processes change quickly over evolutionary time. More specifically, protein-coding genes expressed in reproductive tissues routinely show signatures of rapid evolution. This trend is currently thought to arise as a consequence of sexual selection after mating, which occurs when the gametes of one sex compete for access to the gametes of the other sex. In seahorses and pipefishes, males nurture developing offspring in a placenta-like reproductive tissue known as the brood pouch. There is great variation across this family of fishes in the strength of competition among females for access to this male reproductive resource. Using the latest in high-throughput DNA sequencing technologies, the investigators will test the prediction that male reproductive proteins evolve more rapidly in species subject to intense post-mating sexual selection compared to species with little post-mating sexual selection. The data will also shed light on evolutionary changes in patterns of gene expression specific to male reproductive roles and the male's brood pouch.

In addition to addressing the central hypothesis, this work will generate an unprecedented number of DNA sequence resources for seahorse and pipefish biology. This information will be useful in many basic and applied contexts, including the development of genetics tools for the conservation of seahorses and their relatives, many of which are imperiled due to over-harvesting and environmental change. The project will also stimulate collaboration with international scientists and public outreach through the the Dallas World Aquarium.

Project Report

Genes that encode proteins involved in reproductive processes evolve especially rapidly, but the fundamental reason for this pattern has perplexed biologists for the last few decades. One idea is that the production of offspring often involves conflicts at several levels, including conflicts among members of the same sex competing for a mate, or even conflicts between a parent and its developing offspring during pregnancy, especially if some of those offspring were fathered/mothered by less desirable mates. Because these particular conflicts are so intimately tied to reproductive success, the idea is that traits (or gene variants underlying them) which solve these problems for individuals should change rapidly in populations over time. We tested whether the existence of these types of conflicts (related to a process known as "sexual selection") are associated with the rapid evolution of genes expressed in a novel reproductive tissue, the brood pouch of male-pregnant syngnathid fishes (seahorses and pipefishes). Our approach was to use recent, very high-throughput DNA sequencing techniques (called "next-generation sequencing") to identify potential "male pregnancy genes" in eight syngnathid species, and measure whether these genes have evolved more rapidly in the species characterized by the strongest reproductive conflicts (i.e. those that are not monogamous). Specifically, we sequenced millions of messenger RNAs (transcripts encoded by the genome that are ultimately translated into functional proteins) from the brood pouch tissues of pregnant and non-pregnant male seahorses and pipefishes. We assembled these short sequences into full length RNA transcripts, then aligned the equivalent protein-coding stretches of these molecules from all species. Because we know the evolutionary relationships among our study species, from the sequence alignments we were able to infer how fast these proteins have evolved in specific lineages. Our primary finding of significance was that on average, the evolution of proteins expressed in the pregnant male brood pouch does appear to be faster in non-monogamous species, relative to those species that are monogamous. (Monogamous species are expected to endure the fewest reproductive conflicts.) Very promisingly, these results suggest that a positive relationship between the strength of sexual selection/conflict and rate of reproductive protein evolution exists, but further analysis of our data and, comparisons to other reprdoductive genes (such as those expressed in testes and ovaries), will strengthen our understanding of the system. Other scientific impacts from this project include the generation of thousands of gene sequences from the genomes of seahorses and pipefishes, which may be of use to researchers and managers interested in conservation plans for other syngnathid species, many of which face extinction. Also, virtually nothing was known about which genes are important for male pregnancy before this study. Our data allowed us to compare gene expression levels between pregnant and non-pregnant males, and therefore identify likely male pregnancy genes. Interestingly, many of our candidate male pregnancy genes involved in processes such as water and ion balance have counterparts that operate very similarly during mammalian pregnancy. Future work based on our data will more precisely define to what extent pregnancy types that evolved indepently are similar and how many of the same, ancient animal genetic pathways were recruited for pregnancy functions. Several broader impacts also resulted from the completion of this project. The involved research required assistance from both undergraduate and junior graduate students. Through their participation and interaction with the project's senior scientists, they received valuable training in molecular and evolutionary biology. Several students from diverse backgrounds were trained as a consequence of this project, promoting science-related careers to citizens from groups traditionally underrepresented in this respect. During the period of the project we also presented our basic findings and our general knowledge of syngnathid biology to young students (pre- and elementary school) and the public in general. Information about seahorses and pipefishes is always very popular, and an excellent medium for keeping life sciences education interesting. Lastly, this project required collaboration with researchers in Japan. One of the project scientists visited a Japanese research station for a week to collect data and discuss science with our collaborators. International science collaborations are extremely important, as they maintain ties across political borders, and foster a diverse but global culture of science.

National Science Foundation (NSF)
Division of Environmental Biology (DEB)
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George W. Gilchrist
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Texas A&M Research Foundation
College Station
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