Evolutionary history of the vertebrate Piwi gene family

Abstract

PIWIs are regulatory proteins that belong to the Argonaute family. Piwis are primarily expressed in gonads and protect the germline against the mobilization and propagation of transposable elements (TEs) through transcriptional gene silencing. Vertebrate genomes encode up to four Piwi genes: Piwil1, Piwil2, Piwil3 and Piwil4, but their duplication history is unresolved. We leveraged phylogenetics, synteny and expression analyses to address this void. Our phylogenetic analysis suggests Piwil1 and Piwil2 were retained in all vertebrate members. Piwil4 was the result of Piwil1 duplication in the ancestor of gnathostomes, but was independently lost in ray-finned fishes and birds. Further, Piwil3 was derived from a tandem Piwil1 duplication in the common ancestor of marsupial and placental mammals, but was secondarily lost in Atlantogenata (Xenarthra and Afrotheria) and some rodents. The evolutionary rate of Piwil3 is considerably faster than any Piwi among all lineages, but an explanation is lacking. Our expression analyses suggest Piwi expression has mostly been constrained to gonads throughout vertebrate evolution. Vertebrate evolution is marked by two early rounds of whole genome duplication and many multigene families are linked to these events. However, our analyses suggest Piwi expansion was independent of whole genome duplications.

Keywords: Argonaute gene family; Gene duplication; Phylogenetics; RNAi; Selection; Synteny; Transcriptomics.

Figure 1. Phylogenetic reconstruction of the vertebrate Piwi family.

A phylogenetic tree constructed from a reduced dataset to summarize relationships among Piwi paralogs. The IQ-Tree2 model chosen was LG+F+I+G4 (LG model using empirical base Frequencies, a proportion of Invariable sites, and a discrete Gamma model with four rate categories). A tree constructed from all species used is presented in Fig. S2. Piwi paralogs are color coded on the tree and tip labels are color coded for major gnathostome groups. The displayed tree was the maximum likelihood tree constructed with IQ-Tree2. Numbers by nodes correspond to support values from the ultrafast bootstrap routine in IQ-Tree2.

Figure 2. Synteny comparisons of vertebrate Piwil1 and Piwil4.

Organization of genes up and downstream of (A) Piwil1 and (B) Piwil4. Distances are not drawn to scale. White boxes represent genes that are not homologous to any other genes in the synteny block. Boxes on top of the black line reflect genes in forward orientation relative to Piwi genes and boxes below the line are in the opposite orientation. An “X” indicates pseudogenization and empty space between Fut4 and Amotl1 in 2B indicates an absent Piwil4.

Figure 3. Evolutionary properties of Piwil3.

(A) Synteny around the Piwil3 gene. White boxes represent genes that are not homologous to any other genes in the synteny block, and empty space between Sgsm1 and Aym1 in the mouse locus indicates a missing Piwil3. (B) Evolutionary rate measured as d _N/d _S mapped to branches of a mammalian Piwil1 and Piwil3 phylogenetic tree calculated from a free-ratio model in Codeml. (C) Codon sites under selection estimated by Bayes Empirical Bayes (BEB) from Codeml model M2a. M2a sites were chosen over M8 because M2a is more conservative and sites under selection in M8 are also identified in M2a. (D) One-ratio d _N/d _S estimates among Piwis in major lineages calculated using model M0 in Codeml. (E) Pairwise d _N/d _S distances among Piwis and major lineages.

Figure 4. Piwi expression characteristics.

(A) Expression of Piwi genes from both somatic and gonadal tissue from representatives of vertebrate lineages. Expression values were normalized by taking the log₁₀ of transcripts per million (TPM) +1. (B) Bar plot of Piwi expression in just testes.

Figure 5. An evolutionary hypothesis describing the history of gene gain and loss among vertebrates.

The common ancestor of vertebrates had two Piwis (an ancestral Piwil1 and Piwil2). Piwil2 has not undergone any gain or loss during vertebrate evolution. Most likely, Piwil4 was derived from a tandem duplication of Piwil1 in the common ancestor of gnathostomes, but was independently lost in ray-finned fishes, birds, and monotreme mammals. Piwil3 was derived from a tandem duplication of Piwil1 in the common ancestor of therian mammals. Source credits: Lamprey/cyclostome, Gareth Monger, http://phylopic.org/image/f9313512-3ce4-4349-86bc-060d4faa013e/ (CC 3.0, http://creativecommons.org/licenses/by/3.0/); Lobe-finned fish, Maija Karala, http://phylopic.org/image/202c2ad3-48a7-471d-87ef-c6d8406640e8/ (CC NC 3.0, http://creativecommons.org/licenses/by-nc-sa/3.0/); Amphibian, Sarah Werning, http://phylopic.org/image/cd0f49a1-4adf-448e-859c-b703a73b9481/ (CC 3.0, http://creativecommons.org/licenses/by/3.0/) Monotreme, Sarah Werning, http://phylopic.org/image/cd0f49a1-4adf-448e-859c-b703a73b9481/ (CC 3.0, http://phylopic.org/image/b406c409-2735-4a3d-a7aa-8afe0b6e72dc/); Therian, Bogdan Bocianowski, http://phylopic.org/image/2d078b25-e6a0-4beb-a5d3-5d6f16be8ebf/) (CC 3.0, http://creativecommons.org/licenses/by/3.0/); Cartilaginous fish, Public Domain: http://phylopic.org/image/545d45f0-0dd1-4cfd-aad6-2b835223ea0d/; ray-finned fish, Public Domain: http://phylopic.org/image/6f4c653a-1da1-4e02-85ef-6344b2d8e02a/; Reptile, Public Domain: http://phylopic.org/image/dffda000-77cb-4251-b837-0cd2ab21ed5b/; Chicken, Public Domain: http://phylopic.org/image/aff847b0-ecbd-4d41-98ce-665921a6d96e/.