Phylogenetic analysis of host–symbiont specificity and codivergence in bioluminescent symbioses

Several groups of marine fishes and squids form mutualistic bioluminescent symbioses with luminous bacteria. The dependence of the animal on its symbiont for light production, the animal’s specialized anatomical adaptations for harboring bacteria and controlling light emission, and the host family bacterial species specificity characteristic of these associations suggest that bioluminescent symbioses are tightly coupled associations that might involve coevolutionary interactions. Consistent with this possibility, evidence of parallel cladogenesis has been reported for squid–bacterial associations. However, genetic adaptations in the bacteria necessary for and specific to symbiosis have not been identified, and unlike obligate endosymbiotic associations in which the bacteria are transferred vertically, bacterially bioluminescent hosts acquire their light-organ symbionts from the environment with each new host generation. These contrasting observations led us to test the hypotheses of species specificity and codivergence in bioluminescent symbioses, using an extensive sampling of naturally formed associations. Thirty-five species of fish in seven teleost families (Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae, Monocentridae, Acropomatidae, Leiognathidae) and their light-organ bacteria were examined. Phylogenetic analysis of a taxonomically broad sampling of associations was based on mitochondrial 16S rRNA and cytochrome oxidase I gene sequences for the fish and on recA, gyrB and luxA sequences for bacteria isolated from the light organs of these specimens. In a fine-scale test focused on Leiognathidae, phylogenetic analysis was based also on histone H3 subunit and 28S rRNA gene sequences for the fish and on gyrB, luxA, luxB, luxF and luxE sequences for the bacteria. Deep divergences were revealed among the fishes, and clear resolution was obtained between clades of the bacteria. In several associations, bacterial species identities contradicted strict host family bacterial species specificity. Furthermore, the fish and bacterial phylogenies exhibited no meaningful topological congruence; evolutionary divergence of host fishes was not matched by a similar pattern of diversification in the symbiotic bacteria. Re-analysis of data reported for squids and their luminous bacteria also revealed no convincing evidence of codivergence. These results refute the hypothesis of strict host family bacterial species specificity and the hypothesis of codivergence in bioluminescent symbioses. The Willi Hennig Society 2007. Members of several families of marine fishes and squids establish mutualistic associations with luminous bacteria, called bioluminescent symbioses (Herring and Morin, 1978; Hastings and Nealson, 1981; Dunlap and Kita-Tsukamoto, 2006). In these associations, the animal forms a complex of tissues called a light organ, in which typically a single species of light-producing bacteria is cultured extracellularly, along with accessory tissues that control, direct and diffuse the bacterial light from the animal’s body. The animal uses the bacterial light in various luminescence displays that are associated with sex-specific signaling, predator avoidance, locating or attracting prey, and schooling (Hastings, 1971; Morin et al., 1975; McFall-Ngai and Dunlap, 1983; McFall-Ngai and Morin, 1991; McFall-Ngai, 1991; Woodland et al., 2002; Sasaki et al., 2003; Jones and *Corresponding author: E-mail address: [email protected] The Willi Hennig Society 2007 Cladistics 10.1111/j.1096-0031.2007.00157.x Cladistics 23 (2007) 507–532 Nishiguchi, 2004; Sparks et al., 2005). In turn, the bacteria use nutrients obtained from the host to reproduce (e.g., Nealson, 1979; Graf and Ruby, 1998) and are disseminated from the animal’s light organ into the environment (Haygood et al., 1984; Nealson et al., 1984; Ruby and Asato, 1993). Along with the animal’s ecological dependence on bacterial light and its specialized anatomical adaptations for harboring bacteria and controlling light emission, several other features of bioluminescent symbioses suggest they are tightly coupled associations that might involve coevolutionary interactions (Nealson et al., 1981; Saffo, 2002). One such feature is their apparent specificity. Members of a given family of bacterially luminous fishes or squids are thought to consistently harbor the same species of luminous bacteria in their light organs (Reichelt et al., 1977; Ruby and Morin, 1978; Hastings and Nealson, 1981; Dunlap and KitaTsukamoto, 2006; Dunlap and Ast, 2005). This ‘‘host family bacterial species specificity’’ is believed to result from the host animal selecting its species of symbiotic bacteria and doing so with sufficient specificity that other kinds of bacteria are prevented from colonizing its light organ (Reichelt et al., 1977). Colonization efficiency of the ‘‘native’’ (i.e., specific) symbiont over other bacteria in experimental colonization studies (e.g., McFall-Ngai and Ruby, 1991; Fidopiastis et al., 1998) supports the concepts of species specificity and host selection, which presumably would have a genetic basis. A strict symbiont–host specificity, resulting from genetically based host selection of the symbiont, presumably could provide opportunities for coevolutionary events, although such reciprocal heritable changes (Page and Charleston, 1998) may be difficult to identify. Host– symbiont codivergence (i.e., cospeciation; Page and Charleston, 1998), a possible consequence of coevolution, however, may be easier to detect. Indeed, data indicating host–symbiont ‘‘parallel evolution’’ for the luminous bacterium Vibrio fischeri colonizing light organs of sepiolid squids have been reported (Nishiguchi et al., 1998). These data have been interpreted as evidence of coevolution in bioluminescent symbioses (Thompson, 2005), although genetic adaptations in the bacteria necessary for and specific to light-organ symbiosis have not been identified. In contrast to this view, other features of bioluminescent symbioses distinguish this class of symbiosis in fundamentalways frombacterial–animal endosymbioses. Endosymbioses are mutually obligate associations in which the intracellular symbiotic bacteria are maternally transferred and are generally assumed to involve coevolutionary interactions (e.g., Cary and Giovanonni, 1993; Baumann et al., 1995; Peek et al., 1998; Lo et al., 2003; Hosokawa et al., 2006). One major difference with endosymbioses is that in most bioluminescent associations, the luminous bacteria, which are extracellular, are not obligately dependent on the host for their reproduction; they colonize a variety of other marine habitats, including intestinal tracts, skin and body fluids of marine animals, sediment, and seawater, where they coexist and successfully compete with many other kinds of bacteria as members of commensal, saprophytic, pathogenic, and free-living bacterial communities. A second major difference is that light-organ symbiotic bacteria are acquired from the environment with each new generation of the host (Wei and Young, 1989; McFall-Ngai and Ruby, 1991; Wada et al., 1999); that is, they are acquired horizontally instead of being transferred vertically through the maternal inheritance mechanisms seen for obligate bacterial endosymbionts of terrestrial and marine invertebrates (e.g., Cary and Giovanonni, 1993; Baumann et al., 1995; Peek et al., 1998; Hosokawa et al., 2006). Furthermore, the substantial genomic diversity among strains of luminous bacteria populating individual light organs indicates that colonization of fish light organs is effected by multiple, genetically distinct strains, not by a single, specific strain type (Dunlap et al., 2004; Dunlap and Ast, 2005). A related difference is that the strict host– symbiont specificity expected for associations undergoing coevolutionary change may not consistently characterize light-organ symbioses; certain squids and fishes harbor two species of bacteria in their light organs (Fidopiastis et al., 1998; Nishiguchi, 2000; Kaeding et al., 2007). Because the bacteria are facultative, apparently opportunistic symbionts, selection for symbiosis-specific genetic changes in the bacteria seem less likely than they would be for obligate, maternally transferred endosymbionts. These different views led us to examine the issues of specificity and codivergence in bioluminescent symbioses. Our goals were to define the fidelity of host family bacterial species specificity in naturally occurring associations and test the extent of host–symbiont codivergence. For this work, we carried out an extensive sampling of bioluminescent associations, isolated and identified the light-organ bacteria specific to each host using phylogenetic criteria, and tested for host–symbiont phylogenetic congruence. We also reanalyzed data and results for bacteria symbiotic with sepiolid squids, using parsimony and cophylogeny mapping. Materials and methods Collection of fish specimens Fishes were collected from coastal and bentho-pelagic waters in various locations in Japan, Okinawa, Taiwan, and the Philippines (Fig. 1; Table 1). Collection efforts focused on local and regional fish markets where a variety of shallowand deep-dwelling fishes are landed. 508 P. V. Dunlap et al. / Cladistics 23 (2007) 507–532