Cryptic speciation in a model invertebrate chordate

Abstract

We applied independent species concepts to clarify the phylogeographic structure of the ascidian Ciona intestinalis, a powerful model system in chordate biology and for comparative genomic studies. Intensive research with this marine invertebrate is based on the assumption that natural populations globally belong to a single species. Therefore, understanding the true taxonomic classification may have implications for experimental design and data management. Phylogenies inferred from mitochondrial and nuclear DNA markers accredit the existence of two cryptic species: C. intestinalis sp. A, genetically homogeneous, distributed in the Mediterranean, northeast Atlantic, and Pacific, and C. intestinalis sp. B, geographically structured and encountered in the North Atlantic. Species-level divergence is further entailed by cross-breeding estimates. C. intestinalis A and B from allopatric populations cross-fertilize, but hybrids remain infertile because of defective gametogenesis. Although anatomy illustrates an overall interspecific similarity lacking in diagnostic features, we provide consistent tools for in-field and in-laboratory species discrimination. Finding of two cryptic taxa in C. intestinalis raises interest in a new tunicate genome as a gateway to studies in speciation and ecological adaptation of chordates.

Fig. 1.

Sexual interactions and Bayesian clustering. (A) Heterotypic and homotypic crosses between allopatric and sympatric individuals of C. intestinalis A and B. Gamete compatibility is represented as key developmental steps from fertilization to sexual maturation. Each stage is expressed as percentage of the previous one (y axis). (B) Absence of natural hybridization between C. intestinalis A and B is uncovered by Bayesian-based clustering analysis of 12 microsatellite loci. Data were normalized. Error bars indicate standard deviations.

Fig. 2.

Genetic divergence. (A) A single maximum parsimony tree (1,148 steps of length, confidence interval = 0.96) inferred from 2,578-bp concatenated nuclear (Hox5, Gsx, Hox13, and ITS-2) and mitochondrial (COX1) sequences is shown (192 parsimony informative sites, 7.44%). Maximum likelihood/maximum parsimony/neighbor joining bootstrap support (1,000 iterations) and Bayesian posterior probability values are shown, respectively, on the left and right of each node. Numbers after locality abbreviations indicate distinct haplotypes. (B) Microsatellite genotype-based factorial correspondence analysis in three-dimensional representation. Color codes: black, FuI; light gray, PlyMBA; dark gray, Fis; white, PlyS. (C) Median joining haplotype network based on 515-bp COI alignment. Haplotypes are shaded according to minimum–maximum latitudes. I, PlyMBA; II, FuI, FuE, CdS, VC, Ve, Ta, LT, Al, and Br-1; III, HMB; IV, JyB-2; V, OnM; VI, JyB-1; VII, Fis-2; VIII, PlyS-2; IX, BH; X, PlyS-1 and Br-2; XI, Fis-1; XII, Ed.

Fig. 3.

Geographical distribution of C. intestinalis sp. A (pink) and sp. B (pale blue) and corresponding patterns of spermiduct pigmentation. See Morphology for detailed information on pattern distribution. Empty ovals indicate uncharacterized records of C. intestinalis populations. Arrowheads point to the ellipsoidal papillae at the end of the spermiduct.