A cryptic radiation of Caribbean sea slugs revealed by integrative analysis: Cyerce 'antillensis' (Sacoglossa: Caliphyllidae) is six distinct species.

Integrative studies have revealed cryptic radiations in several Caribbean lineages of heterobranch sea slugs, raising questions about the evolutionary mechanisms that promote speciation within the tropical Western Atlantic. Cyerce Bergh, 1871 is a genus comprising 12 named species in the family Caliphyllidae that lack the photosynthetic ability of other sacoglossans but are noted for vibrant colours on the large cerata (dorsal leaf-like appendages) that characterize many species. Two species are widely reported from the Caribbean: Cyerce cristallina (Trinchese, 1881) and Cyerce antillensis Engel, 1927. Here, we present an integrative assessment of diversity in Caribbean Cyerce. Four methods of molecular species delimitation supported seven species in samples from the Caribbean and adjacent subtropical Western Atlantic. Six delimited species formed a monophyletic lineage in phylogenetic analyses but were > 9% divergent at the barcoding COI locus and could be differentiated using ecological, reproductive and/or morphological traits. We redescribe C. antillensis, a senior synonym for the poorly known Cyerce habanensis Ortea & Templado, 1988, and describe five new species. Evolutionary shifts in algal host use, penial armature and larval life history might have acted synergistically to promote the rapid divergence of endemic species with restricted distributions in this radiation, substantially increasing global diversity of the genus.

The polyketide to fatty acid transition in the evolution of animal lipid metabolism.

Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.

Animal FAS-like polyketide synthases produce diverse polypropionates.

Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.

Ecological speciation by sympatric host shifts in a clade of herbivorous sea slugs, with introgression and localized mitochondrial capture between species.

Host shifting in insect-plant systems was historically important to the development of ecological speciation theory, yet surprisingly few studies have examined whether host shifting drives diversification of marine herbivores. When small-bodied consumers feed and also mate on a preferred host, disruptive selection can split a population into host races despite gene flow. Support for host shifts is notably lacking for invertebrates associated with macroalgae, where the scale of dispersal by planktonic larvae often far exceeds the grain of host patchiness, and adults are typically less specialized than terrestrial herbivores. Here, we present a candidate example of ecological speciation in a clade of sea slugs that primarily consume green algae in the genus Caulerpa, including highly invasive species. Ancestral character state reconstructions supported 'sea grapes' (C. racemosa, C. lentillifera) as the ancestral host for a tropical radiation of 12 Elysia spp., with one shift onto alternative Caulerpa spp. in the Indo-Pacific. A Caribbean radiation of three species included symaptric host shifts to Rhipocephalus brevicaulis in the ancestor of E. pratensis Ortea & Espinosa, 1996, and to C. prolifera in E. hamanni Krug, Vendetti & Valdes 2016, plus a niche expansion to a range of Caulerpa spp. in E. subornata Verrill, 1901. All three species are broadly sympatric across the Caribbean but are host-partitioned at a fine grain, and distinct by morphology and at nuclear loci. However, non-recombining mtDNA revealed a history of gene flow between E. pratensis and E. subornata: COI haplotypes from E. subornata were 10.4% divergent from E. pratensis haplotypes from four sites, but closely related to all E. pratensis haplotypes sampled from six Bahamian islands, indicating historical introgression and localized "mitochondrial capture." Disruptive selective likely fueled divergence and adaptation to distinct host environments, indicating ecological speciation may be an under-appreciated driver of diversification for marine herbivores as well as epibionts and other resource specialists.

Phylogenomic resolution of the root of Panpulmonata, a hyperdiverse radiation of gastropods: new insight into the evolution of air breathing.

Transitions to terrestriality have been associated with major animal radiations including land snails and slugs in Stylommatophora (>20 000 described species), the most successful lineage of 'pulmonates' (a non-monophyletic assemblage of air-breathing gastropods). However, phylogenomic studies have failed to robustly resolve relationships among traditional pulmonates and affiliated marine lineages that comprise clade Panpulmonata (Mollusca, Gastropoda), especially two key taxa: Sacoglossa, a group including photosynthetic sea slugs, and Siphonarioidea, intertidal limpet-like snails with a non-contractile pneumostome (narrow opening to a vascularized pallial cavity). To clarify the evolutionary history of the panpulmonate radiation, we performed phylogenomic analyses on datasets of up to 1160 nuclear protein-coding genes for 110 gastropods, including 40 new transcriptomes for Sacoglossa and Siphonarioidea. All 18 analyses recovered Sacoglossa as the sister group to a clade we named Pneumopulmonata, within which Siphonarioidea was sister to the remaining lineages in most analyses. Comparative modelling indicated shifts to marginal habitat (estuarine, mangrove and intertidal zones) preceded and accelerated the evolution of a pneumostome, present in the pneumopulmonate ancestor along with a one-sided plicate gill. These findings highlight key intermediate stages in the evolution of air-breathing snails, supporting the hypothesis that adaptation to marginal zones played an important role in major sea-to-land transitions.

A molecular phylogeny of Thuridilla Bergh, 1872 sea slugs (Gastropoda, Sacoglossa) reveals a case of flamboyant and cryptic radiation in the marine realm.

The genus Thuridilla Bergh, 1872 comprises mostly tropical sap-sucking sea slugs species with flamboyantly coloured forms. However, the potential for cryptic or pseudocryptic species masked by convergent or polymorphic colour patterns has not been tested using molecular characters. In this study, we sampled 20 of the 23 recognized worldwide species and performed the most comprehensive molecular phylogenetic analysis of the genus to date using a multi-locus approach combining two mitochondrial (cytochrome c oxidase subunit I, 16S rRNA) and two nuclear (Histone H3, 28S rRNA) genes using maximum likelihood, maximum-parsimony and Bayesian criteria. Three molecular species delimitation methods (ABGD, GMYC, bPTP) and the morphology of radular teeth were additionally used to aid in species delimitation. Our analyses supported 35 species within Thuridilla, of which more than one-third (13) are part of a single radiation here named the Thuridilla gracilis (Risbec, 1928) species-complex. This complex includes T. gracilis, T. splendens (Baba, 1949), T. bayeri (Er. Marcus, 1965), and T. ratna (Er. Marcus, 1965), plus nine additional undescribed species. All 13 species are distinguishable by radular characters, external morphology and their DNA. The detection of this radiation led diversity of Thuridilla to be underestimated by about 25% and provides a new comparative system for studying the role of colour patterns in marine diversification.

Differential Tolerance and Seasonal Adaptation to Temperature and Salinity Stress at a Dynamic Range Boundary Between Estuarine Gastropods.

AbstractInsight into how coastal organisms will respond to changing temperature and salinity regimes may be derived from studies of adaptation to fluctuating estuarine environments, especially under stressful range-edge conditions. We characterized a dynamic range boundary between two estuarine sea slugs, Alderia modesta (distributed across the North Pacific and North Atlantic) and Alderia willowi, known from southern and central California. The species overlap from Bodega Bay to San Francisco Bay, where populations are dominated by A. modesta after winter rains but by A. willowi after peak summer temperatures. Laboratory assays confirmed superior tolerance to low salinity for the northern species, A. modesta: encapsulated embryos developed at 8 ppt, larvae survived at 4-6 ppt, and adults survived repeated exposure to 2 ppt, salinities that reduced development or survival for the same stages of A. willowi. Adults did not appreciably differ in their high-temperature threshold, however. Each species showed increased tolerance to either temperature or salinity stress at its range margin, indicating plasticity or local adaptation, but at the cost of reduced tolerance to the other stressor. At its northern limit, A. willowi became more tolerant of low salinity during the winter rainy season, but also less heat tolerant. Conversely, A. modesta became more heat resistant from spring to summer at its southern limit, but less tolerant of low salinity. Trade-offs in stress tolerance may generally constrain adaptation and limit biotic response to a rapidly changing environment, as well as differentiating species niches.

Animal biosynthesis of complex polyketides in a photosynthetic partnership.

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans.

Eggs sunny-side up: A new species of Olea, an unusual oophagous sea slug (Gastropoda: Heterobranchia: Sacoglossa), from the western Atlantic.

Nearly all of the recognized species of sea slugs in clade Sacoglossa (about 300 taxa) are herbivorous, using a uniseriate radula in suctorial feeding. The only exceptions are a pair of monotypic genera in the ceratiform family Limapontiidae: Olea Agersborg, 1923 from the northeastern Pacific, and Calliopaea d'Orbigny, 1837 from the northeastern Atlantic coast of Europe and the Mediterranean. Both genera feed on the eggs of other heterobranchs, notably cephalaspideans, and lack cerata on the anterior dorsum. Major differences are that C. bellula d'Orbigny, 1837 has more cerata than O. hansineensis Agersborg, 1923, a more typical radula with ascending and descending rows of fully-formed teeth, and a much longer penial stylet. Here, we describe a new egg-eating sacoglossan species from the subtropical Gulf of Mexico coast of Florida, U.S.A. Phylogenetic relationships inferred from analyses of a four-gene dataset including 219 sacoglossan species indicated the new taxon (O. hensoni n. sp.) belongs to Olea. The generic placement of the new species is also supported by its highly reduced radula and comparatively short penial stylet. A full description of the reproductive, digestive, and nervous systems is also provided. Finding a new Olea species in the warm waters of the western Atlantic was surprising, given the genus was previously known only from the cold northern Pacific, and suggests further diversity in oophagous sacoglossans may await discovery.

A biting commentary: Integrating tooth characters with molecular data doubles known species diversity in a lineage of sea slugs that consume "killer algae".

Predicting biotic resistance to highly invasive strains of "killer algae" (Caulerpa spp.) requires understanding the diversity and feeding preferences of native consumers, including sea slugs in family Oxynoidae. Past studies reported low algal host specificity for Oxynoe (6 spp.) and Lobiger (4 spp.), but these taxonomically challenging slugs may represent species complexes of unrecognized specialists that prefer different Caulerpa spp. Here, we assess global diversity of these genera by integrating gene sequences with morphological data from microscopic teeth and internal shells, the only hard parts in these soft-bodied invertebrates. Four delimitation methods applied to datasets comprising mtDNA and/or nuclear alleles yielded up to 16 species hypotheses for samples comprising five nominal taxa, including five highly divergent species in Lobiger and five in Oxynoe. Depending on the analysis, a further four to six species were recovered in the O. antillarum-viridis complex, a clade in which mitochondrial divergence was low and nuclear alleles were shared among lineages. Bayesian species delimitation using only morphological data supported most candidate species, however, and integrative analyses combining morphological and genetic data fully supported all complex members. Collectively, our findings double the recognized biodiversity in Oxynoidae, and illustrate the value of including data from traits that mediate fast-evolving ecological interactions during species delimitation. Preference for Caulerpa spp. and radular tooth characteristics covaried among newly delimited species, highlighting an unappreciated degree of host specialization and coevolution in these taxa that may help predict their role in containing outbreaks of invasive algae.

Molecular and morphological systematics of Elysia Risso, 1818 (Heterobranchia: Sacoglossa) from the Caribbean region.

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.

Reduced genetic diversity and increased reproductive isolation follow population-level loss of larval dispersal in a marine gastropod.

Population-level consequences of dispersal ability remain poorly understood, especially for marine animals in which dispersal is typically considered a species-level trait governed by oceanographic transport of microscopic larvae. Transitions from dispersive (planktotrophic) to nondispersive, aplanktonic larvae are predicted to reduce connectivity, genetic diversity within populations, and the spatial scale at which reproductive isolation evolves. However, larval dimorphism within a species is rare, precluding population-level tests. We show the sea slug Costasiella ocellifera expresses both larval morphs in Florida and the Caribbean, regions with divergent mitochondrial lineages. Planktotrophy predominated at 11 sites, 10 of which formed a highly connected and genetically diverse Caribbean metapopulation. Four populations expressed mainly aplanktonic development and had markedly reduced connectivity, and lower genetic diversity at one mitochondrial and six nuclear loci. Aplanktonic dams showed partial postzygotic isolation in most interpopulation crosses, regardless of genetic or geographic distance to the sire's source, suggesting that outbreeding depression affects fragmented populations. Dams from genetically isolated and neighboring populations also exhibited premating isolation, consistent with reinforcement contingent on historical interaction. By increasing self-recruitment and genetic drift, the loss of dispersal may thus initiate a feedback loop resulting in the evolution of reproductive isolation over small spatial scales in the sea.

Species Selection Favors Dispersive Life Histories in Sea Slugs, but Higher Per-Offspring Investment Drives Shifts to Short-Lived Larvae.

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.

Integrative species delimitation in photosynthetic sea slugs reveals twenty candidate species in three nominal taxa studied for drug discovery, plastid symbiosis or biological control.

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.

Phylogenomics supports Panpulmonata: opisthobranch paraphyly and key evolutionary steps in a major radiation of gastropod molluscs.

Pulmonates, with over 30,000 described species, represent the largest radiation of non-marine animals outside of Arthropoda. The pulmonate lung was a key evolutionary innovation enabling diversification of terrestrial and freshwater snails and slugs. However, recent studies drew conflicting conclusions about pulmonate monophyly, and support for a sister group is lacking, hindering our understanding of this major animal radiation. Analyses of mitochondrial protein-coding genes recovered a paraphyletic Pulmonata grading into a monophyletic Opisthobranchia, a traditional group of sea slugs long considered sister to pulmonates. Conversely, analyses of datsets dominated by nuclear rDNA indicated Opisthobranchia is paraphyletic with respect to Pulmonata. No study resolved the placement of two key taxa: Sacoglossa, an opisthobranch group including photosynthetic sea slugs, and Siphonarioidea, intertidal limpet-like snails traditionally in Pulmonata. To examine evolutionary relationships at the base of the pulmonate radiation, we performed a phylogenomic analysis of 102 nuclear protein-coding gene regions for 19 gastropods. Opisthobranchia was recovered as paraphyletic with respect to Panpulmonata, a clade in which Sacoglossa was sister to Pulmonata, with Siphonarioidea as the basal pulmonate lineage. Siphonarioideans share a similar gill structure with shelled sacoglossans but lack the contractile pneumostome of pulmonates, suggesting descent from an evolutionary intermediate that facilitated the pulmonate radiation into non-marine habitats.

Nitric oxide signaling differentially affects habitat choice by two larval morphs of the sea slug Alderia willowi: mechanistic insight into evolutionary transitions in dispersal strategies.

In many marine animals, adult habitat is selected by lecithotrophic (non-feeding) larvae with a limited lifespan. In generalist species, larvae may increasingly accept sub-optimal habitat over time as energy stores are depleted ('desperate larva' hypothesis). If the fitness cost of suboptimal habitat is too high, larvae of specialists may prolong the searching phase until they encounter a high-quality patch or die ('death before dishonor' hypothesis). In generalists, starvation is hypothesized to lead to a decline in inhibitory nitric oxide (NO) signaling, thereby triggering metamorphosis. Here, we document alternative functions for identified signaling pathways in larvae having 'desperate' versus 'death before dishonor' strategies in lecithotrophic clutches of a habitat specialist, the sea slug Alderia willowi. In an unusual dimorphism, each clutch of A. willowi hatches both non-selective larvae that settle soon after hatching and siblings that delay settlement in the absence of cues from the alga Vaucheria, the sole adult food. Pharmacological manipulation of NO signaling induced metamorphosis in non-selective but not selective stages. However, decreased NO signaling in selective larvae lowered the threshold for response to habitat cues, mimicking the effect of declining energy levels. Manipulation of cGMP or dopamine production induced metamorphosis in selective and non-selective larvae alike, highlighting a distinct role for the NO pathway in the two larval morphs. We propose a model in which NO production (1) links nitrogen metabolism with sensory receptor signaling, and (2) shifts from a regulatory role in 'desperate larva' strategies to a modulatory role in 'death before dishonor' strategies. This study provides new mechanistic insight into how the function of conserved signaling pathways may change in response to selection on larval habitat choice behaviors.

Poecilogony and population genetic structure in Elysia pusilla (Heterobranchia: Sacoglossa), and reproductive data for five sacoglossans that express dimorphisms in larval development.

Credible cases of poecilogony, the production of two distinct larval morphs within a species, are extremely rare in marine invertebrates, yet peculiarly common in a clade of herbivorous sea slugs, the Sacoglossa. Only five animal species have been reported to express dimorphic egg sizes that result in planktotrophic and lecithotrophic larvae: the spionid polychaete Streblospio benedicti and four sacoglossans distributed in temperate estuaries or the Caribbean. Here, we present developmental and genetic evidence for a fifth case of poecilogony via egg-size dimorphism in the Sacoglossa and the first example from the tropical Indo-Pacific. The sea slug Elysia pusilla produced both planktotrophic and lecithotrophic larvae in Guam and Japan. Levels of genetic divergence within populations were markedly low and rule out cryptic species. However, divergence among populations was exceptionally high (10-12% at the mitochondrial cytochrome c oxidase I locus), illustrating that extensive phylogeographic structure can persist in spite of the dispersal potential of planktotrophic larvae. We review reproductive, developmental, and ecological data for the five known cases of poecilogony in the Sacoglossa, including new data for Costasiella ocellifera from the Caribbean. We hypothesize that sacoglossans achieve lecithotrophy at smaller egg sizes than do related clades of marine heterobranchs, which may facilitate developmental plasticity that is otherwise vanishingly rare among animals. Insight into the environmental drivers and evolutionary results of shifts in larval type will continue to be gleaned from population-level studies of poecilogonous taxa like E. pusilla, and should inform life-history theory about the causes and consequences of alternative development modes in marine animals.

Seasonal polyphenism in larval type: rearing environment influences the development mode expressed by adults in the sea slug Alderia willowi.

Dimorphisms occur when alternative developmental pathways produce discrete phenotypes within a species, and may promote evolutionary novelty in morphology, life history, and behavior. Among marine invertebrates, intra-specific dimorphism in larval type (poecilogony) is notably rare, but should provide insight into the selective forces acting on larval strategies. Most established cases of poecilogony appear to be allelic polymorphisms, with local expression regulated by population-genetic processes. Here, we present evidence that dimorphic larval development in the sea slug Alderia willowi is a seasonal polyphenism; the type of larvae produced by an adult slug depends on the rearing environment in which that slug matured. In field surveys of 1996-1999 and 2007-2009, the population in Mission Bay, San Diego (California, USA) produced only short-lived lecithotrophic larvae in summer and early fall, but a varying proportion of slugs expressed planktotrophy in winter and spring. In laboratory experiments, slugs reared under summer conditions (high temperature, high salinity) produced the highest proportion of lecithotrophic offspring, whereas winter conditions (low temperature, low salinity) induced the lowest proportion of lecithotrophy. The shift to a nondispersive morph under summer conditions may be an adaptive response to historical closure of coastal wetlands during the dry season in southern California, which would inhibit dispersal by larvae of back-bay taxa. In most animal polyphenisms, a single larval type is produced and the rearing environment determines which adult phenotype develops. In contrast, alternative larval morphs are produced by A. willowi in response to seasonal cues experienced by the adult stage, varying the phenotype and dispersal potential of offspring. As the only known case of polyphenism in mode of larval development, A. willowi should become a model organism for mechanistic studies of dimorphism and the evolution of alternative life histories.

Functional chloroplasts in metazoan cells - a unique evolutionary strategy in animal life.

BACKGROUND: Among metazoans, retention of functional diet-derived chloroplasts (kleptoplasty) is known only from the sea slug taxon Sacoglossa (Gastropoda: Opisthobranchia). Intracellular maintenance of plastids in the slug's digestive epithelium has long attracted interest given its implications for understanding the evolution of endosymbiosis. However, photosynthetic ability varies widely among sacoglossans; some species have no plastid retention while others survive for months solely on photosynthesis. We present a molecular phylogenetic hypothesis for the Sacoglossa and a survey of kleptoplasty from representatives of all major clades. We sought to quantify variation in photosynthetic ability among lineages, identify phylogenetic origins of plastid retention, and assess whether kleptoplasty was a key character in the radiation of the Sacoglossa. RESULTS: Three levels of photosynthetic activity were detected: (1) no functional retention; (2) short-term retention lasting about one week; and (3) long-term retention for over a month. Phylogenetic analysis of one nuclear and two mitochondrial loci revealed reciprocal monophyly of the shelled Oxynoacea and shell-less Plakobranchacea, the latter comprising a monophyletic Plakobranchoidea and paraphyletic Limapontioidea. Only species in the Plakobranchoidea expressed short- or long-term kleptoplasty, most belonging to a speciose clade of slugs bearing parapodia (lateral flaps covering the dorsum). Bayesian ancestral character state reconstructions indicated that functional short-term retention arose once in the last common ancestor of Plakobranchoidea, and independently evolved into long-term retention in four derived species. CONCLUSION: We propose a sequential progression from short- to long-term kleptoplasty, with different adaptations involved in each step. Short-term kleptoplasty likely arose as a deficiency in plastid digestion, yielding additional energy via the release of fixed carbon. Functional short-term retention was an apomorphy of the Plakobranchoidea, but the subsequent evolution of parapodia enabled slugs to protect kleptoplasts against high irradiance and further prolong plastid survival. We conclude that functional short-term retention was necessary but not sufficient for an adaptive radiation in the Plakobranchoidea, especially in the genus Elysia which comprises a third of all sacoglossan species. The adaptations necessary for long-term chloroplast survival arose independently in species feeding on different algal hosts, providing a valuable study system for examining the parallel evolution of this unique trophic strategy.