Journal of Molecular Evolution

, Volume 86, Issue 9, pp 635–645 | Cite as

Positive Selection in the Evolution of Mammalian CRISPs

  • Alberto VicensEmail author
  • Claudia L. Treviño
Original Article


Cysteine-RIch Secretory Proteins (CRISPs) constitute a versatile family, with functions in reptilian venom and mammalian reproduction. Mammals generally express three CRISPs, four in mice, and all are highly expressed in male reproductive tissues, either testis or accessory organs. Because reproductive proteins often evolve adaptively in response to post-copulatory sexual selection, we hypothesized that mammalian CRISPs, with important roles in male reproduction, could have undergone positive selection promoting their divergence. We explored the molecular adaptation of mammalian CRISPs applying phylogenetic methods. Our analyses revealed the evidence of positive selection in all mammalian CRISPs. The intensity of positive selection was heterogeneous among CRISP members, being stronger in CRISP3 than in CRISP1 and CRISP2, and also across functional domains, having stronger impact on Pathogenesis-Related 1 (PR-1) in CRISP2 and on Ion Channel Regulator (ICR) in CRISP1 and CRISP3. In addition, we discovered a new CRISP in some rodent species, suggesting that the acquisition of new CRISP components could contribute to male reproductive success or to acquire new physiological roles. Signatures of positive selection were not focused on any particular mammalian group, suggesting that adaptive evolution is a recurrent pattern in mammalian CRISPs. Our findings support a model of CRISP family diversification driven by episodes of duplication and posterior neofunctionalization, and provide potential adaptive changes responsible for interspecific differences in CRISPs activity.


Mammalian CRISP Reproduction Positive selection Ion channel Fertility 



The authors would like to thank Jose Luis De la Vega, Paulina Torres, Francisco Herrera, and Shirley Ainsworth for technical assistance; Juan Manuel Hurtado, Roberto Rodríguez, David Santiago Castañeda, Omar Arriaga, and Arturo Ocádiz for computing services; and Miguel Arenas for the critical revision of this manuscript. Computational analyses were supported by the cluster of the Instituto de Biotecnología UNAM (


This work was supported by Dirección General de Asuntos de Personal Académico/Universidad Nacional Autónoma de México (DGAPA/UNAM) (Contract Grant Number IN203116 to C.T. and postdoctoral fellowship to A.V.), the Alexander von Humboldt Foundation (w/o Grant Number to C.T.), and Juan de la Cierva postdoctoral fellowship (IJCI-2016-29550) from Spanish Government to A.V.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

239_2018_9872_MOESM1_ESM.png (2.1 mb)
Supplementary Figure S1 Phylogenetic tree of mammalian CRISP1 inferred by ML. Terminal branches are presented with the accession sequence and species name. Taxonomic groups are indicated on right of tree. Node labels indicate the number of replicates of bootstrap support, based on 1000 iterations. The Eulipotyphla Sorex araneus was used to root the tree (PNG 2107 KB)
239_2018_9872_MOESM2_ESM.png (1.6 mb)
Supplementary Figure S2 Phylogenetic tree of mammalian CRISP2 inferred by ML. Terminal branches are presented with the accession sequence and species name. Taxonomic groups are indicated on right of tree. Node labels indicate the number of replicates of bootstrap support, based on 1000 iterations. The Eulipotyphla Sorex araneus was used to root the tree (PNG 1647 KB)
239_2018_9872_MOESM3_ESM.png (1.4 mb)
Supplementary Figure S3 Phylogenetic tree of mammalian CRISP3 inferred by ML. Terminal branches are presented with the accession sequence and species name. Taxonomic groups are indicated on right of the tree. Branches corresponding to rodent CRISP 3 duplications are colored in red. The rodent CRISP3 clade, in the box, was analyzed separately for positive selection. Node labels indicate the number of replicates of bootstrap support, based on 1000 iterations. The Afrotheria Echinops telfairi and Chrysochloris asiatica were used to root the tree (PNG 1473 KB)
239_2018_9872_MOESM4_ESM.png (2 mb)
Supplementary Figure S4 Histogram representing the site-specific ω estimates under neutral (below alignment) and selection (above alignment) branch-site models on one CRISP3 paralog of prairie vole (Microtus ochrogaster). The tested branch is indicated with a red box. Rodent CRISP3 phylogeny (on left) was used as background. The species names are indicated on terminal branches in species with a single CRISP3 or on internal branches in species with two CRISP3 paralogs, these indicated with bold labels. Significant PSSs are indicated with colored bars (yellow: BEB probability > 0.95; red: BEB probability > 0.99). Domain scheme of the CRISP3 protein is shown above the histogram (PNG 2079 KB)
239_2018_9872_MOESM5_ESM.fasta (28 kb)
Supplementary File S1 CRISP1 sequence alignment used for selection analysis (FASTA format) (FASTA 27 KB)
239_2018_9872_MOESM6_ESM.fasta (30 kb)
Supplementary File S2 CRISP2 sequence alignment used for selection analysis (FASTA format) (FASTA 30 KB)
239_2018_9872_MOESM7_ESM.fasta (23 kb)
Supplementary File S3 CRISP3 sequence alignment used for selection analysis (FASTA format) (FASTA 23 KB)
239_2018_9872_MOESM8_ESM.nwk (2 kb)
Supplementary File S4 CRISP1 phylogenetic reconstructed by Maximum Likelihood (NEWICK format) (NWK 1 KB)
239_2018_9872_MOESM9_ESM.nwk (2 kb)
Supplementary File S5 CRISP2 phylogenetic reconstructed by Maximum Likelihood (NEWICK format) (NWK 1 KB)
239_2018_9872_MOESM10_ESM.txt (2 kb)
Supplementary File S6 CRISP3 phylogenetic reconstructed by Maximum Likelihood (NEWICK format) (TXT 1 KB)
239_2018_9872_MOESM11_ESM.pdf (57 kb)
Supplementary Table S1 List of CRISP sequences retrieved for this study. Information about CRISP gene, accession number and organisms is indicated (PDF 57 KB)
239_2018_9872_MOESM12_ESM.pdf (28 kb)
Supplementary Table S2 Parameter estimates and likelihood scores of site-specific models in mammalian CRISP (PDF 27 KB)
239_2018_9872_MOESM13_ESM.pdf (28 kb)
Supplementary Table S3 Positive selection sites identified by the likelihood site models implemented in Datamonkey server (PDF 28 KB)


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Copyright information

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Authors and Affiliations

  1. 1.Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Department of Biochemistry, Genetics and Immunology, School of BiologyUniversity of VigoVigoSpain

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