Over 150 independent evolutionary events of viviparity are found in vertebrates, with females representing the pregnant sex. The unique “male pregnancy” in the family Syngnathidae represents an exception (Figure 1). Members within this group (seahorses, pipefishes, and seadragons) are iconic teleost fish and amongst their most intriguing life history traits is their sex-role reversal and unusual mode of paternal care. The basal type of paternal care in this group is found (amongst others) in the seadragons, in which males incubate fertilized eggs attached to a brood patch under their tail, where eggs stick via a specialized integument. In contrast, some lineages, including the seahorses, evolved a form of more derived “male pregnancy”, in which females transfer unfertilized eggs to the males, which fertilize and incubate them in their protective enclosed brooding pouch. Here, the father nourishes the embryos and provides them with oxygen via a placenta-like structure – even after they have hatched – until the juveniles are released from the pouch as miniature seahorses (Figure 2). To date, what provided the evolutionary opportunity for this unique male pregnancy remains a riddle.

While pregnancy can provide many advantages to the developing offspring, it comes with immunological challenges for the pregnant parents to evolve tolerance towards the semi-allogenic embryos. As the immune system aims to recognize and reject non-self tissue efficiently, pregnancy must have coevolved with a fine-tuning of the immune system to achieve tolerance towards the embryo. To understand the immunological alterations seahorses might have gone through while evolving male pregnancy an important clue can be found in one of their exceptional trait: asplenia (i.e., the loss of spleen). The spleen is an important secondary lymphoid organ that performs mostly immunological functions in almost all jawed vertebrates (Figure. 2). Furthermore, seahorses have lost substantial parts of the adaptive immune system’s gene repertoire. These surprising immune modifications might thus also be a consequence of this immunological trade-off and be central for the evolution of the unique male pregnancy. Meanwhile, this also raises the question of how these fish might immunologically compensate the secondary loss of a vital immune-organ and why it was lost in the first place.

In our study, published in Nature Communications, we aimed to understand the genomic underpinnings of interspecific variation more thoroughly in the immune repertoire of syngnathids. We generated de novo high-quality, chromosome-level genome assemblies of two seahorse species and compared them to several other genomes of syngnathid fishes with simple and derived paternal care as well as other teleosts. We found that the gene families revealed signatures related to immune response pathways, such as the NOD-like receptor signaling pathway, allograft rejection, and antigen processing and presentation were significantly contracted or lost in seahorses. The contraction or loss of immune-related gene families must have significantly contributed to the modifications identified in the seahorse immune system and may be related to the unique evolution of “male pregnancy”.

We further focused on the key genes for spleen development to elucidate the genetic mechanism of splenic absence in seahorses. A large phylogenetic range (34 species) including mammals, birds, reptiles, amphibians, and additional fish species revealed a lineage specific mutation in the T-cell leukemia homeobox 1 (tlx1) gene in seahorses, where a hydrophilic threonine (T, Thr) has been replaced by the hydrophobic alanine (A, Ala). In contrast, more distantly related members with less complex brooding organs, such as the weedy seadragon (Syngnathoides biaculeatus) and the alligator pipefish (Syngnathoides biaculeatus) – where the eggs are typically simply attached to the ventral side of the males’ tail – have retained the ancestral Alanine in this position and possesses an intact spleen. Tlx1 has been reported to control splenic primordia cell fate specification and organ expansion in mice and zebrafish. It is the only known gene whose pseudofunctionalization can result in spleen loss without causing other developmental abnormalities. As mutations in exons of protein-coding genes can lead to substantial phenotypic changes, we speculated that mutations in this site may be casual for the asplenia in seahorses.

To confirm this hypothesis, we went on with CRISPR/Cas9-mediated genome editing to generate three zebrafish lineages: the full gene knockout line (tlx1^▲), the specific point-mutation line (tlx1^A208T) mimicking the seahorse missense mutation and the point mutation site adjacent to the seahorse-specific mutation (tlx1^A207T). We found that both the tlx1^▲ and tlx1^A208T mutation led to spleen loss in all examined zebrafish individuals, while the tlx1^A207T line had a normal spleen, similar to that in the wild-type zebrafish, which further strongly implies the species specificity and functional conservatism of this site.  

In female pregnancy, the successful implantation of a fertilized egg is the prerequisite for a successful pregnancy, and although it is recognized by maternal immune cells, the mother-fetus interface undergoes dynamic changes in the immune system to allow the fetus to grow and develop in utero. Our evidence suggests that many factors related to immune tolerance are involved in the male pregnancy process. In addition to the asplenia, we also found that many genes involved in antigen recognition and presentation, T/B lymphocyte development, and complement activation had a varying degree of lineage-specific loss or structural variation in seahorses or in other Syngnathidae. This result also confirms the significant changes in the immune system of Syngnathidae to achieve immune tolerance towards the offspring. In conclusion, we revealed that the seahorse-specific mutation in tlx1 likely contributes to asplenia, and we provide further genetic evidence that the modified immune system in this lineage is likely linked to the evolution of paternal pregnancy. Our results will pave the way for further studies on the evolutionary history of immune tolerance and reproduction.