DNA barcoding can highlight taxa in which conventional taxonomy underestimates species richness, identifying mitochondrial lineages that may correspond to unrecognized species. However, key assumptions of barcoding remain untested for many groups of soft-bodied marine invertebrates with poorly resolved taxonomy. Here, we applied an integrative approach for species delimitation to herbivorous sea slugs in clade Sacoglossa, in which unrecognized diversity may complicate studies of drug discovery, plastid endosymbiosis, and biological control. Using the mitochondrial barcoding COI gene and the nuclear histone 3 gene, we tested the hypothesis that three widely distributed “species” each comprised a complex of independently evolving lineages. Morphological and reproductive characters were then used to evaluate whether each lineage was distinguishable as a candidate species. The “circumtropical” Elysia ornata comprised a Caribbean species and four Indo-Pacific candidate species that are potential sources of kahalalides, anti-cancer compounds. The “monotypic” and highly photosynthetic Plakobranchus ocellatus, used for over 60 years to study chloroplast symbiosis, comprised 10 candidate species. Finally, six candidate species were distinguished in the Elysia tomentosa complex, including potential biological control agents for invasive green algae (Caulerpa spp.). We show that a candidate species approach developed for vertebrates effectively categorizes cryptic diversity in marine invertebrates, and that integrating threshold COI distances with non-molecular character data can delimit species even when common assumptions of DNA barcoding are violated.

For 40 years, paleontological studies of marine gastropods have suggested that species selection favors lineages with short-lived (lecithotrophic) larvae, which are less dispersive than long-lived (planktotrophic) larvae. Although lecithotrophs appeared to speciate more often and accumulate over time in some groups, lecithotrophy also increased extinction rates, and tests for state-dependent diversification were never performed. Molecular phylogenies of diverse groups instead suggested lecithotrophs accumulate without diversifying due to frequent, unidirectional character change. Although lecithotrophy has repeatedly originated in most phyla, no adult trait has been correlated with shifts in larval type. Thus, both the evolutionary origins of lecithotrophy and its consequences for patterns of species richness remain poorly understood. Here, we test hypothesized links between development mode and evolutionary rates using likelihood-based methods and a phylogeny of 202 species of gastropod molluscs in Sacoglossa, a clade of herbivorous sea slugs. Evolutionary quantitative genetics modeling and stochastic character mapping supported 27 origins of lecithotrophy. Tests for correlated evolution revealed lecithotrophy evolved more often in lineages investing in extra-embryonic yolk, the first adult trait associated with shifts in development mode across a group. However, contrary to predictions from paleontological studies, species selection actually favored planktotrophy; most extant lecithotrophs originated through recent character change, and did not subsequently diversify. Increased offspring provisioning in planktotrophs thus favored shifts to short-lived larvae, which led to short-lived lineages over macroevolutionary time scales. These findings challenge long-standing assumptions about the effects of alternative life histories in the sea. Species selection can explain the long-term persistence of planktotrophy, the ancestral state in most clades, despite frequent transitions to lecithotrophy.

The Caribbean is a biodiversity hotspot for photosynthetic sea slugs, with about 27 described species in the genus Elysia Risso, 1818. However, many species are poorly known or have complex taxonomic histories, complicating assessments of regional biodiversity and impeding studies of plastid symbiosis, speciation, and larval biology. Using an integrative approach, we address the taxonomy and systematics of Caribbean elysiids by performing robust tests of existing species hypotheses, and describe six new species. Species delimitation included DNA barcoding of up to 189 nominal conspecific specimens; nuclear gene sequences were then used to confirm that divergent lineages were genetically distinct candidate species. New synonymies and species descriptions are based on external anatomy, penial and radular morphology, developmental characters, and host ecology of all species described from the region, plus a critical review of the literature. We synonymized three species (Elysia annedupontae Ortea, Espinosa & Caballer in Ortea, Caballer, Moro & Espinosa, 2005, Elysia clarki Pierce et al. 2006, and Elysia leeanneae Caballer, Ortea & Espinosa in Ortea, Espinosa, Buske & Caballer, 2013), transfered one species from Bosellia (Elysia marcusi), and described six new species (Elysia pawliki n. sp., Elysia zemi n. sp., Elysia christinae n. sp., Elysia hamanni n. sp., Elysia taino n. sp., and Elysia buonoi n. sp.). We resurrected the name Elysia velutinus Pruvot-Fol, 1947, a senior synonym of Elysia tuca Ev. Marcus & Er. Marcus, 1967. Based on a four-gene phylogeny of 76 Elysia spp., we identified shifts in host use and penial armature that may explain patterns of endemic diversification in Elysia, invoking both ecological and non-ecological mechanisms. Non-monophyly of stylet-bearing species rejects previous attempts to classify species based on presence of a stylet (i.e., the genus Checholysia Ortea, Caballer, Moro & Espinosa, 2005). Our findings show how integrative approaches can resolve the taxonomic status of problematic species (e.g., Elysia papillosa Verrill, 1901) for soft-bodied marine taxa.

Large-scale analysis of the fossil record requires aggregation of palaeontological data from individual fossil localities. Prior to computers, these synoptic datasets were compiled by hand, a laborious undertaking that took years of effort and forced palaeontologists to make difficult choices about what types of data to tabulate. The advent of desktop computers ushered in palaeontology's first digital revolution-online literature-based databases, such as the Paleobiology Database (PBDB). However, the published literature represents only a small proportion of the palaeontological data housed in museum collections. Although this issue has long been appreciated, the magnitude, and thus potential significance, of these so-called 'dark data' has been difficult to determine. Here, in the early phases of a second digital revolution in palaeontology--the digitization of museum collections-we provide an estimate of the magnitude of palaeontology's dark data. Digitization of our nine institutions' holdings of Cenozoic marine invertebrate collections from California, Oregon and Washington in the USA reveals that they represent 23 times the number of unique localities than are currently available in the PBDB. These data, and the vast quantity of similarly untapped dark data in other museum collections, will, when digitally mobilized, enhance palaeontologists' ability to make inferences about the patterns and processes of past evolutionary and ecological changes.

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