To effectively conserve biodiversity, we must identify existing species and recognize the forces that generate species richness. Rivers and mountains can separate terrestrial populations for millions of years, allowing them to split into distinct species; however, we have little idea how species form in the ocean where there are few such boundaries to movement. Speciation could follow if some individuals inherited a preference for a new habitat or food source, or if populations became genetically fragmented after a species evolved non-migrating larvae. To test these ideas, we will construct a family tree of herbivorous sea slugs from across the globe, inferring relatedness among species by phylogenetic analysis of their DNA. For each species we will determine the host seaweed used as both food and habitat, and the type of larvae produced (dispersing versus non-migratory). By establishing algal hosts, larval types, and relationships among species, we can determine which traits have contributed to speciation in this group. If new species form when slugs switch onto different host algae, this will overturn long-held assumptions about speciation in the sea. This study will also test whether non-migratory larvae evolve due to pressure on mothers to make bigger offspring, and whether such larvae in turn promote the formation of new species. The results will advance our understanding of why certain regions or groups of animals have more species than others, and lead to the description of many new sea slug species. Both aspects are critical to preserving the ocean's biological richness.
We used DNA sequences to infer the phylogeny, or "family tree", of a sea slug group called sacoglossans. These slugs are an exciting group with which to study important evolutionary processes, such as identifying traits that cause some groups to diversify into many species while other groups are limited to a few species. Sacoglossans are vegetarians that feed, mate and lay eggs on their food or "host" algae. Each species usually specializes on a single algal host. Most marine animals produce tiny, planktonic larval stages that can be either (a) tiny and long-lived, traveling great distances on ocean currents, or (b) larger, short-lived, and less prone to disperse. Following a change to non-dispersive larvae, populations should quickly adapt to local conditions and rarely migrate into new areas, increasing the odds that a population will evolve into its own species. Sacoglossans are unusual in that related species often differ in the type of larvae they produce, indicating larval type evolves rapidly in this group; in a few cases, members of the same species even produce different types of larvae, a very rare flexibility. The causes of evolutionary switches between larval types, and the consequences of such shifts over millions of years, are outstanding questions in evolutionary biology. Sacoglossans present opportunities to examine how changes in larval type or diet affect important processes like species formation in the sea. For instance, in some species, mother slugs put extra ribbons of colorful yolk into their egg masses, providing food for developing offspring (analogous to putting cupcakes in your child’s lunchbox). We found that mothers control the size of their offspring by the amount of yolk they invested rather than through the size of the eggs that they laid. Further, lineages with yolk ribbons were significantly more likely to switch from tiny larvae to large, non-dispersive larvae; maternal investment is thus a key trait influencing the evolution of larval type. As no previous trait was ever linked to changes in larval type, our work provides new insight into how larval and adult stages affect each other’s evolution. What are the consequences of switches in larval type? We found that species with larger, short-lived larvae in fact had less gene flow among Caribbean islands, confirming that larger larvae are less dispersive (a finding challenged by several recent studies). We also found that changes in larval type may contribute to speciation. In one species, slugs from populations with large larvae avoided mating with slugs from neighboring sites that made tiny larvae. Such mate discrimination can be the first step towards species formation. Across the whole family tree, however, lineages with non-dispersive larvae did not become more species-rich, but did accumulate changes in their DNA at a faster rate. Species formation appeared to be more strongly influenced by host use. We found that most host shifts were restricted to the most species-rich genus, Elysia. Slugs that begin feeding on a different host alga may thus evolve into a new species over time. Our results also have importance to applied problems including drug discovery and biological control of invasive species. For instance, one "species" called Elysia ornata is reported to occur throughout the tropics; in some places, E. ornata contains anti-cancer compounds called kahalalides that are currently in testing as potential new chemotherapeutic agents. Using a combination of DNA sequences, adult anatomy, and larval features, we showed that there are actually 5 distinct species masquerading under the name "ornata." Obviously, it is critical to know which of five possible species actually contains a potential cure for cancer; our work therefore showcases the importance of accurate taxonomy and species identification in poorly studied groups like sea slugs. We similarly studied a group of species called "E. tomentosa" and relatives, and found seven undetected species lumped under one name. Some species in this complex eat the toxic, highly invasive "killer alga" Caulerpa taxifolia, and may therefore be useful as biological control agents. Lastly, some sacoglossans have the remarkable ability to store functional chloroplasts (the part of plant and algal cells that perform photosynthesis) from their meals. Instead of being digested, the hijacked chloroplasts continue to pump out nutrients for the slug, in some species for many months. Species that can sustain chloroplasts are used to study the early stages of intracellular symbioses, which led to the evolution of chloroplasts themselves from bacterial ancestors over a billion years ago. Our studies revealed that one such "species", Plakobranchus occelatus, is actually at least 10 distinct species presently unrecognized by science. Studies of different species that use the same name can create confusion by reaching inconsistent or contradictory results; recognizing and describing the true species richness in a group is therefore critical for effective scientific study of the species involved.
