Patrick J. Krug

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

BackgroundAmong 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 slugs 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.ResultsThree 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.ConclusionWe 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.

Waterborne and Surface-Associated Carbohydrates as Settlement Cues for Larvae of the Specialist Marine Herbivore Alderia modesta

Larvae of the specialist marine herbivore Alderia modesta (Opisthobranchia: Ascoglossa) metamorphose in response to a chemical settlement cue from the alga Vaucheria longicaulis, the obligate adult prey. Bioactivity coeluted with both high and low molecular weight carbohydrates in solution, and with insoluble high molecular weight carbohydrates associated with the algal cell wall. Larvae metamorphosed in response to water conditioned by V. longicaulis, as well as to frozen and homogenized algal tissue. The inducer was efficiently extracted from the algae with boiling water, but after all soluble activity was extracted, residual tissue still induced larval settlement. Ethanol precipitation of a boiled-water extract followed by gel filtration chromatography showed that the precipitate contained carbohydrates of >100,000 Da molecular weight, while the supernatant contained only low molecular weight carbohydrates (<2,000 Da); in both cases all activity was associated with the carbohydrate peak. An aqueous-insoluble 4% NaOH extract was chromatographed in 7 M urea to yield a bioactive high molecular weight carbohydrate peak. Activity was not affected by proteinase K or mild acid hydrolysis, but was significantly decreased by periodate treatment. The results indicate that larvae of A. modesta metamorphose in response to both water-soluble and surface-associated carbohydrates of V. longicaulis, and that the soluble cue exists as both high and low molecular weight isoforms.

Not My “Type”: Larval Dispersal Dimorphisms and Bet-Hedging in Opisthobranch Life Histories

When conditions fluctuate unpredictably, selection may favor bet-hedging strategies that vary offspring characteristics to avoid reproductive wipe-outs in bad seasons. For many marine gastropods, the dispersal potential of offspring reflects both maternal effects (egg size, egg mass properties) and larval traits (development rate, habitat choice). I present data for eight sea slugs in the genus Elysia (Opisthobranchia: Sacoglossa), highlighting potentially adaptive variation in traits like offspring size, timing of metamorphosis, hatching behavior, and settlement response. Elysia zuleicae produced both planktotrophic and lecithotrophic larvae, a true case of poecilogony. Both intracapsular and post-hatching metamorphosis occurred among clutches of “Boselia” marcusi, E. cornigera, and E. crispata, a dispersal dimorphism often misinterpreted as poecilogony. Egg masses of E. tuca hatched for up to 16 days but larvae settled only on the adult host alga Halimeda, whereas most larvae of E. papillosa spontaneously metamorphosed 5–7 days after hatching. Investment in extra-capsular yolk may allow mothers to increase larval size relative to egg size and vary offspring size within and among clutches. Flexible strategies of larval dispersal and offspring provisioning in Elysia spp. may represent adaptations to the patchy habitat of these specialized herbivores, highlighting the evolutionary importance of variation in a range of life-history traits.

EVOLUTION OF POECILOGONY FROM PLANKTOTROPHY: CRYPTIC SPECIATION, PHYLOGEOGRAPHY, AND LARVAL DEVELOPMENT IN THE GASTROPOD GENUS ALDERIA

Abstract Poecilogony, a rare phenomenon in marine invertebrates, occurs when alternative larval morphs differing in dispersal potential or trophic mode are produced from a single genome. Because both poecilogony and cryptic species are prevalent among sea slugs in the suborder Sacoglossa (Gastropoda: Opisthobranchia), molecular data are needed to confirm cases of variable development and to place them in a phylogenetic context. The nominal species Alderia modesta produces long-lived, feeding larvae throughout the North Atlantic and Pacific, but in California can also produce short-lived larvae that metamorphose without feeding. We collected morphological, developmental, and molecular data for Alderia from 17 sites spanning the eastern and western Pacific and North Atlantic. Estuaries south of Bodega Harbor, California, contained a cryptic species (hereafter Alderia sp.) with variable development, sister to the strictly planktotrophic A. modesta. The smaller Alderia sp. seasonally toggled between planktotrophy and lecithotrophy, with some individuals differing in development but sharing mitochondrial DNA haplotypes. The sibling species overlapped in Tomales Bay, California, but showed no evidence of hybridization; laboratory mating trials suggest postzygotic isolation has arisen. Intra- and interspecific divergence times were estimated using a molecular clock calibrated with geminate sacoglossans. Speciation occurred about 4.1 million years ago during a major marine radiation in the eastern Pacific, when large inland embayments in California may have isolated ancestral populations. Atlantic and Pacific A. modesta diverged about 1.7 million years ago, suggesting trans-Arctic gene flow was interrupted by Pleistocene glaciation. Both Alderia species showed evidence of late Pleistocene population expansion, but the southern Alderia sp. likely experienced a more pronounced bottleneck. Reduced body size may have incurred selection against obligate planktotrophy in Alderia sp. by limiting fecundity in the face of high larval mortality rates in warm months. Alternatively, poecilogony may be an adaptive response to seasonal opening of estuaries, facilitating dispersal by long-lived larvae. An improved understanding of the forces controlling seasonal shifts in development in Alderia sp. may yield insight into the evolutionary forces promoting transitions to nonfeeding larvae.

Patterns of Speciation in Marine Gastropods: A Review of the Phylogenetic Evidence for Localized Radiations in the Sea*

Abstract: Modern speciation theory is heavily influenced by Mayrs postulate that prolonged geographical isolation is necessary for differentiated populations to evolve reproductive isolation. Present-day distributions of sister species are consistent with allopatric or peripatric speciation in many terrestrial and freshwater animal groups. However, the oceans present few obstacles to dispersal for marine taxa with planktonic larvae, and sister species are not often split at biogeographical breakpoints in the sea. Theory predicts that disruptive selection on habitat choice or resource use can split a population into divergent ecotypes without physical separation, yet sympatric speciation is still often viewed as improbable. Here, I review phylogenetic evidence from diverse marine gastropods to test Mayrs prediction that recently diverged sister species should not be sympatric over most of their ranges. In contrast to expectations, young sister species are often broadly sympatric in many gastropod groups, suggesting that classical models of allopatric divergence are insufficient to explain marine speciation. I discuss four mechanisms that may contribute to this deviation from predicted biogeographical patterns: transient allopatry along continuous coastlines, rapid evolution of gamete recognition proteins, shifts to non-planktonic development, and ecological divergence. The available evidence argues that patterns of marine speciation depend on complex interactions between geography, life history, and ecology, often resulting in local radiations within a basin or endemic to an island group. Whether selection acts on ecotypes in sympatry or on populations during secondary contact, ecological factors may promote speciation in the sea at smaller spatial scales than expected. I highlight areas for future study to improve our understanding of the forces generating marine biodiversity, and why the geography of speciation may be fundamentally different for shallow-water animals.

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.

Poecilogony and larval ecology in the gastropod genus Alderia

The gastropod genus Alderia (Allman, 1845) (Opisthobranchia: Sacoglossa) contains a rare case of poecilogony, or variable larval development within a species. This paper reviews the alternative larval morphs and dispersal strategies expressed by Alderia spp., and presents new data on larval ecology, environmentally induced changes in development, and rates of metamorphosis for larvae differing in age and life history. Recent morphological and molecular analyses revealed a cryptic poecilogonous species in the previously monotypic genus Alderia. The newly described Alderia willowi Krug et al., 2007 occurs south of Bodega Harbor, California, and was the subject of all prior studies by Krug and co-workers. Unlike its strictly planktotrophic congener Alderia modesta (Lovén, 1844), A. willowi produces either small feeding larvae that have a 30-day pre-competent period or large larvae that need not feed to metamorphose. Individuals can vary the developmental mode of their offspring, with a variable proportion switching from lecithotrophy (prevalent in summer) to planktotrophy in winter or spring; a similar shift is induced in some adults upon transfer to the laboratory. In a second dispersal polymorphism, a variable percentage of lecithotrophic larvae undergo spontaneous metamorphosis within 2 days of hatching, while their siblings disperse until induced to settle by carbohydrate cues from the host algae Vaucheria spp. The percentage of spontaneous metamorphosis is uncorrelated with fecundity and is generally between 15–30%, a possible product of stabilizing selection on this bet-hedging dispersal strategy. Despite their different ages, competent larval morphs produced by alternative developmental pathways are similar in size, swimming behavior, and responses to dissolved settlement cues. However, competent planktotrophic larvae and older lecithotrophic larvae initiated metamorphosis faster after settlement than newly hatched lecithotrophic larvae, suggesting a link between planktonic period and habitat choice. Although rare, poecilogonous species like A. willowi offer special insights into the evolutionary causes and ecological consequences of alternative life histories.

THE SCENT OF DANGER: TETRODOTOXIN (TTX) AS AN OLFACTORY CUE OF PREDATION RISK

Larvae of the California newt (Taricha torosa) exhibit striking predator-avoidance behavior, escaping to refuges in response to a chemical cue from cannibalistic adults. In laboratory flow-tank experiments, stream water collected near free-ranging adults induced hiding responses in 100% of the larvae tested. Solutions prepared by bathing adults (in field and laboratory) also evoked strong hiding behaviors. Insensitive to adult feeding status (fed or starved), and clearly not an excretory product, the chemical cue was released from adult skin (i.e., in swabs of adult backs, sides, and bellies). Tetrodotoxin (TTX) was found in skin swabs of adults and in bathwater at 1 × 10−7 mol/L using reversed-phase high-pressure liquid chromatography (HPLC). Concentrations of 1 × 10−7 to 1 × 10−9 mol/L TTX standard, and equivalent dilutions of bathwater, triggered hiding behaviors in larvae, with no subsequent sublethal toxicity. The presence of TTX-sensitive cells within larval olfactory epithelium was confirmed by beh...

Developmental Dimorphism: Consequences for Larval Behavior and Dispersal Potential in a Marine Gastropod

Specific effects of alternative developmental programs on swimming and settlement behavior for marine larvae have not been identified experimentally. A major impediment to this research has been the rarity of species with variable development. Here, we compared traits related to movement and habitat selection for different ontogenetic stages of long-lived, feeding larvae (planktotrophic) and short-lived, nonfeeding larvae (lecithotrophic) of the herbivorous gastropod Alderia modesta. Newly hatched planktotrophic larvae swam in meandering paths with equal rates of upward and downward movement. As planktotrophic larvae developed towards competence (physiological ability to metamorphose), their swimming paths became straighter, faster, and increasingly directed towards the bottom, traits shared by newly hatched lecithotrophic larvae. Despite differing in developmental history, competent planktotrophic (32-d-old) and lecithotrophic larvae (competent upon hatching) exhibited qualitatively similar swimming behaviors and substrate specificity. However, lecithotrophic larvae moved downward at twice the speed of competent planktotrophic larvae, potentially producing a 5-fold higher rate of contact with the bottom in natural flows. Competent larvae swam downwards rather than passively sinking, even though sinking rates were faster than swimming speeds; active swimming may allow larvae to keep the velum extended, permitting rapid response to chemical settlement cues and promoting successful habitat colonization. Differences between larvae of the two development modes may reflect fine-tuning by selection of traits important for dispersal and settlement into patchy adult habitats.

Endogenous signaling pathways and chemical communication between sperm and egg.

SUMMARY Red abalone (Haliotis rufescens) sperm detect a waterborne chemical cue released by conspecific eggs, and change their swimming behavior to increase the likelihood of fertilization success. Previously, we isolated the natural sperm attractant by bioassay-guided fractionation and high-performance liquid chromatography, and chemically identified it as the free-amino acid l-tryptophan (l-Trp). In the present study, levels of this ecologically meaningful compound were quantified in various abalone tissues, and in freshly spawned eggs. Tryptophan was the least abundant of 19 dissolved free amino acids (DFAAs) in ovary, testis, foot muscle, gill, stomach and hemolymph. As a proportion of the DFAA pool, however, Trp concentrations were significantly elevated in eggs (three- to seven-times higher) relative to all other sampled tissues. Natural rates of Trp release from eggs also were measured and correlated with fertility. Fertilization success peaked during an initial 30 min period (post-spawn), but decreased to nil over the next 50 min. Closely paralleling these events, Trp accumulated in seawater around freshly spawned eggs for the first 45 min (post-spawn) before decaying rapidly from solution. Older eggs stopped releasing Trp approximately when they became infertile, revealing a critical link between gamete physiology and chemical signaling. This apparent negative feedback loop did not arise from tryptophan oxidation, uptake by bacteria in seawater, or a degrading enzyme released by eggs. As a metabolic precursor critical to development of the larval nervous system, Trp could be an honest indicator of egg fitness for prospective sperm suitors. Our results suggest that endogenous signaling pathways have been co-opted for external communication between gametes, as an adaptation to increase reproductive success by promoting sperm navigation towards fertile eggs.