Convergent Evolution of Placental Systems and the Independent Origin of Genomic Imprinting in Viviparous Fish
Abstract
The placenta is a complex organ that establishes a connection and facilitates physiological interactions between the mother and fetus. Placentation has independently evolved multiple times throughout the animal kingdom, yet the mechanisms underlying its evolution remain poorly understood. The fish family Poeciliidae comprises approximately 270 species exhibiting diverse reproductive modes, ranging from lecithotrophy (yolk provisioning) to matrotrophy (maternal provisioning), with varying levels of post-fertilization maternal provisioning. The viviparous matrotrophic species have evolved a structure with functional similarity to the mammalian placenta. This diversity provides a unique opportunity to investigate the evolution of placentation among closely related species. The blackstripe livebearer, Poeciliopsis prolifica, is a viviparous fish species with a placenta in the family Poeciliidae that exhibits a high level of post-fertilization maternal investment. In this dissertation, we investigated the molecular mechanisms underlying placental evolution in P. prolifica. The P. prolifica genome was sequenced using the PacBio long-read sequencing and 10× Genomics linked-read sequencing. A high-quality P. prolifica reference genome was generated with 674 Mbp assembly in 504 contigs with excellent continuity and completeness. A total of 27,227 protein-coding genes were annotated from the merged data sets based on bioinformatic predictions, RNA sequencing and homology evidence. Phylogenetic analyses revealed that Poeciliopsis prolifica diverged from the guppy (Poecilia reticulata) approximately 19 million years ago. Gene family analysis identified the rapid expansion of families with T-cell activation and other immune-related functions in P. prolifica, which may be functionally related to the evolution of placenta. Our research provides the necessary resources and the genomic toolkit for investigating the genetic underpinnings of placentation. Genomic imprinting is an epigenetic phenomenon in which only one allele, inherited from either the mother or the father, is expressed. It has only been found in angiosperms and placental mammals, both of which exhibit an active paternal genome during maternal offspring provisioning. It is hypothesized that imprinting modulates conflict between maternal control of resource allocation and paternal control of resource acquisition. If true, then imprinting should evolve in any organism that evolves a placenta. To investigate potential genomic imprinting in a placental fish, reciprocal crosses between inbred lines are required to track the parental origin of alleles and estimate allele-specific expression. Using transcriptomes from reciprocal crosses between two inbred strains, 84 protein-coding genes were identified with robust evidence of imprinting in P. prolifica placenta. These include a pair of imprinted genes with opposing effects on glucose metabolism that are imprinted in a fashion that potentially resolves parental conflict, and two genes that exhibit the same parent-of-origin expression biases as those observed in placental mammals. We also found that the convergent evolution of the placenta between the placental mammals and fish in Poeciliidae involves the recruitment of the dpp4-associated gene network, which is associated with gut nutrient transport, to perform a similar function in the placenta. Our results reveal convergent evolution of the placenta between placental mammals and fish and provide the first evidence of genomic imprinting in a non-mammalian vertebrate. Genomic imprinting is regulated by epigenetic marks such as DNA methylation and histone modification. A canonical feature of the imprinted gene regulated by DNA methylation is that the imprinting control region exhibits around 50% methylation rate, as one paternal allele is fully methylated and the other is unmethylated. To further validate the imprinted genes identified in our study, DNA methylation profiles were quantified in placenta samples from two inbred strains and their reciprocal crosses using Illumina EM-seq, as well as whole-body samples from P. prolifica and its closely related non-placental species P. infans, and PacBio HiFi sequencing. Comparative analyses revealed similar genome-wide methylation landscapes in the two species, characterized by a bimodal distribution of promoter methylation, with substantial gene promoters exhibiting either low or high methylation and high levels of gene-body methylation. Candidate imprinted genes exhibited diverse methylation patterns, suggesting that genomic imprinting may be regulated by multiple epigenetic mechanisms beyond DNA methylation alone. Notably, by integrating Illumina and PacBio HiFi sequencing data, we identified the allele-specific methylation pattern at the promoter of dpp4, providing strong epigenetic evidence supporting its imprinted expression. Together, our studies integrated transcriptomic and epigenomic sequencing to investigate the existence of genomic imprinting in a placental fish of the family Poeciliidae. By combining allele-specific gene expression analyses with DNA methylation profiling, we identified imprinted genes that exhibit both the parent-of-origin effect and its corresponding allele-specific methylation patterns. We further demonstrated that there is a convergent placental evolution between the placental mammals and fish in Poeciliidae through the co-option of gut genes for placental functions. Taken together, our findings provide the first demonstration of genomic imprinting in a non-mammalian vertebrate and support the potential role of imprinting in resolving parental conflict.