Functional similarity and molecular divergence of a novel reproductive transcriptome in two male‐pregnant Syngnathus pipefish species

Abstract

Evolutionary studies have revealed that reproductive proteins in animals and plants often evolve more rapidly than the genome-wide average. The causes of this pattern, which may include relaxed purifying selection, sexual selection, sexual conflict, pathogen resistance, reinforcement, or gene duplication, remain elusive. Investigative expansions to additional taxa and reproductive tissues have the potential to shed new light on this unresolved problem. Here, we embark on such an expansion, in a comparison of the brood-pouch transcriptome between two male-pregnant species of the pipefish genus Syngnathus. Male brooding tissues in syngnathid fishes represent a novel, nonurogenital reproductive trait, heretofore mostly uncharacterized from a molecular perspective. We leveraged next-generation sequencing (Roche 454 pyrosequencing) to compare transcript abundance in the male brooding tissues of pregnant with nonpregnant samples from Gulf (S. scovelli) and dusky (S. floridae) pipefish. A core set of protein-coding genes, including multiple members of astacin metalloprotease and c-type lectin gene families, is consistent between species in both the direction and magnitude of expression bias. As predicted, coding DNA sequence analysis of these putative “male pregnancy proteins” suggests rapid evolution relative to nondifferentially expressed genes and reflects signatures of adaptation similar in magnitude to those reported from Drosophila male accessory gland proteins. Although the precise drivers of male pregnancy protein divergence remain unknown, we argue that the male pregnancy transcriptome in syngnathid fishes, a clade diverse with respect to brooding morphology and mating system, represents a unique and promising object of study for understanding the perplexing evolutionary nature of reproductive molecules.

The genome of the Gulf pipefish enables understanding of evolutionary innovations

“…As documented previously in S. scovelli and S. floridae [52], two similar astacin-like metalloproteases demonstrated strikingly opposite patterns of gene expression: one markedly pregnancy-enriched and the other highly pregnancy-depressed (Table 2, Table 3, Fig. 7b, c).…”

“…Previous work identified members of the astacin-like metalloprotease gene family as candidates for playing a functional role in male pregnancy [18, 52]. We confirmed extreme transcriptional differences for two of these patristacins between brood pouch tissue of pregnant and non-pregnant males (see “Differential expression analysis” section) and set out to characterize the distribution of this gene family in the Gulf pipefish and other teleost genomes.…”

“…Prior to our characterization of the Gulf pipefish genome, however, with the exception of a few transcriptomic resources [51, 52, 54], virtually no information existed for how key developmental genes and their modification might be responsible for derived syngnathid phenotypes. Now, with the availability of the genome of Syngnathus scovelli , and likely other related genomes soon to follow, we expect researchers interested in the developmental genetic underpinnings of novel vertebrate morphologies to make the critical experimental connections between genomic differences in syngnathids and their functional consequences.…”

“…Consistent with this functional diversity, our genome-based analysis of male pregnancy in S. scovelli revealed a transcriptionally rich brood pouch in which over 73% of annotated genes were expressed robustly and over 1000 were differentially expressed as a consequence of pregnancy (Additional file 2: SH2). Previous studies, based on de novo transcriptome assemblies, characterized pregnancy-specific gene expression in pipefish species of Syngnathus [52] and in the seahorse Hippocampus abdominalis [54], but lack of a reference genome in those surveys limited insights into the transcriptional breadth of the pouch and single gene resolution for transcript abundance measurements. Our differential expression analysis comparing early-stage pregnant to non-pregnant male pouch tissue echoes many of the patterns described in the comprehensive seahorse study [54], including evidence for positive regulation of developmental processes, lipid transport, homeostasis, and the immune system during pregnancy.…”

The evolution and physiology of male pregnancy in syngnathid fishes

“…Molecular tools, as well as enabling phylogenetic and conservation genetic studies of syngnathids, offer an unprecedented opportunity to resolve the genetic basis of male pregnancy (Mobley, Small & Jones, ). For example, transcriptomic and other gene‐expression studies of the brood pouch during pregnancy have identified genes putatively involved in a variety of pouch functions (Melamed et al ., ; Harlin‐Cognato et al ., ; Small et al ., ). Given that the physiology of the pouch in some species appears to vary throughout pregnancy, an approach which has proved particularly powerful is to use RNA sequencing (RNA‐seq) at a number of reproductive stages to compare gene expression at a fine scale as embryos develop (Whittington et al ., ).…”