DNA variation and symbiotic associations in phenotypically diverse sea urchin Strongylocentrotus intermedius

Abstract

Strongylocentrotus intermedius (A. Agassiz, 1863) is an economically important sea urchin inhabiting the northwest Pacific region of Asia. The northern Primorye (Sea of Japan) populations of S. intermedius consist of two sympatric morphological forms, "usual" (U) and "gray" (G). The two forms are significantly different in morphology and preferred bathymetric distribution, the G form prevailing in deeper-water settlements. We have analyzed the genetic composition of the S. intermedius forms using the nucleotide sequences of the mitochondrial gene encoding the cytochrome c oxidase subunit I and the nuclear gene encoding bindin to evaluate the possibility of cryptic species within S. intermedius. We have examined the presence of symbiont microorganisms by means of 16S rRNA sequences. The nucleotide sequence divergence between the morphological forms is low: 0.74% and 0.70% for cytochrome c oxidase subunit I and nuclear gene encoding bindin, respectively, which is significantly below average intrageneric sequence divergence among Strongylocentrotus species. We thus have found no genetic evidence of cryptic species within S. intermedius. Phylogenetic analysis shows that the bacteria symbionts of S. intermedius belong to the phylum Bacteroidetes, but the U and G forms predominantly harbor highly divergent bacterial lineages belonging to two different taxonomic classes, Flavobacteria and Sphingobacteria. We propose that the U and G forms of S. intermedius represent distinct ecomorphological adaptations to contrasting shallow- and deep-water marine environments and might be considered incipient species. We also propose that the symbiotic bacteria likely play an important role in the evolution of morphological divergence of S. intermedius.

Fig. 1.

The U (A) and G (B) morphological forms of Strongylocentrotus intermedius.

Fig. 2.

Neighbor-joining tree of the COI and BND sequences of Strongylocentrotus, based on Kimura 2-parameter distance. The numbers at the nodes are bootstrap percent probability values based on 10,000 replications. Some BND and COI sequences are obtained from GeneBank with their accession numbers: S. purpuratus, COI, NC_001453 (44) and BND, AF077309 (10); Hemicentrotus pulcherrimus, COI, AF525453 (11) and BND, AF077319 (10). The sequences of COI and BND in S. polyacanthus and S. pallidus are from Balakirev and Ayala (accession numbers EU700089-EU700091 and EU700092-EU700094). POL = S. polyacanthus; PAL = S. pallidus; PUR = S. purpuratus; PUL = Hemicentrotus pulcherrimus.

Fig. 3.

Neighbor-joining tree of the 16S rRNA sequences of Strongylocentrotus endosymbionts, based on Kimura 2-parameter distance. The Bacteroidetes sequences are from sea urchins inhabiting the northern Primorye coast region, Sea of Japan, Pacific Ocean: Zolotoi Cape (A), Olga Bay (B), and Povorotnyi Cape (C). The sequences are designated by the first letter of the corresponding locality, with second letter referring to the color variant, U or G, following by the clone number. The numbers at the nodes are bootstrap percent probability values based on 10,000 replications. The clones of S. nudus are in boldface. The clones ZG-7V-6 and ZG-7V-7 are representatives of the class Bacteroidetes (genus Prevotella) and used here as common references for all three localities. “FLAVO” and “SPHINGO” refer to the two bacterial classes Flavobacteria and Sphingobacteria, respectively. NUD = S. nudus.

Fig. 4.

First two principal coordinates from a principal coordinate analysis of the Bacteroidetes sequences obtained from three sea urchin settlements: Zolotoi Cape, Olga Bay, and Povorotnyi Cape. S. intermedius sea urchins are designated by the letters U and G, followed by numbers that refer to each particular individual. NUD refers to S. nudus. The numbers on the axes refer to the percent of the variation explained by each principal component (P1 and P2). Four sea urchin individuals (boxed), one in each of the Zolotoi Cape and Olga Bay localities, and two in the Povorotnyi Cape, are displaced from the expected position (see text).