Five hundred million years to mobility: directed locomotion and its ecological function in a turtle barnacle.

Movement is a fundamental characteristic of life, yet some invertebrate taxa, such as barnacles, permanently affix to a substratum as adults. Adult barnacles became 'sessile' over 500 Ma; however, we confirm that the epizoic sea turtle barnacle, Chelonibia testudinaria, has evolved the capacity for self-directed locomotion as adults. We also assess how these movements are affected by water currents and the distance between conspecifics. Finally, we microscopically examine the barnacle cement. Chelonibia testudinaria moved distances up to 78.6 mm yr-1 on loggerhead and green sea turtle hosts. Movements on live hosts and on acrylic panels occasionally involved abrupt course alterations of up to 90°. Our findings showed that barnacles tended to move directly against water flow and independent of nearby conspecifics. This suggests that these movements are not passively driven by external forces and instead are behaviourally directed. In addition, it indicates that these movements function primarily to facilitate feeding, not reproduction. While the mechanism enabling movement remained elusive, we observed that trails of cement bore signs of multi-layered, episodic secretion. We speculate that proximal causes of movement involve one or a combination of rapid shell growth, cement secretion coordinated with basal membrane lifting, and directed contraction of basal perimeter muscles.

A Global Synthesis of the Correspondence Between Epizoic Barnacles and Their Sea Turtle Hosts.

Barnacles that are obligate epizoites of sea turtles are not parasites in the traditional sense. However, they can impair their hosts in some instances, disqualifying the association as strictly commensal. Characterizing these interactions requires knowing which epibionts pair with which hosts, but records of barnacles from sea turtles are scattered and symbiont/host match-ups remain equivocal. The objective of this study was to collate global records on the occurrence of barnacles with sea turtles and describe each species pair quantitatively. Records reporting barnacles with sea turtles were searched spanning the last 167 years, including grey literature, and findings were enumerated for 30,580 individual turtles to evaluate prevalence. The data were summarized globally as well as subdivided across six geographic regions to assess constancy of the affiliations. Patterns of partnering were visualized by hierarchical clustering analysis of percent occurrence values for each barnacle/turtle pair and the relative selectivity of each symbiont and susceptibility of each host were evaluated. After adjusting for synonymies and taxonomic inaccuracies, the occurrence of 16 nominal species of barnacles was recorded from all 7 extant sea turtle species. Mostly, barnacles were not specific to single turtle species, partnering on average with three hosts each. Neither were barnacles entirely host-consistent among regions. Three barnacles were common to all sea turtles except leatherbacks. The most common, widespread, and least selective barnacle was Chelonibia testudinaria, the only symbiont of all turtles. Excluding single-record occurrences, the barnacle Stomatolepas transversa was the only single-host associate of any hard-shell sea turtle (the green sea turtle) and Platylepas coriacea and Stomatolepas dermochelys were exclusive associates of leatherback sea turtles. Green sea turtles were the most vulnerable to epibiosis, hosting 13 barnacle species and Kemp's ridley sea turtles were the least, hosting three. Geographically, there was an average of nine barnacle species per world region, with diversity highest in the Pacific Ocean (12 species) and lowest in the Mediterranean Sea (6 species). It is paradoxical that the flexibility of barnacles for multiple host species contrasts with their overall strict specificity for sea turtles, with each symbiont occupying a virtually unique suite of turtle hosts.

Spatial distribution of epibionts on olive ridley sea turtles at Playa Ostional, Costa Rica.

There is a wealth of published information on the epibiont communities of sea turtles, yet many of these studies have exclusively sampled epibionts found only on the carapace. Considering that epibionts may be found on almost all body-surfaces and that it is highly plausible to expect different regions of the body to host distinct epibiont taxa, there is a need for quantitative information on the spatial variation of epibiont communities on turtles. To achieve this, we measured how total epibiont abundance and biomass on olive ridley turtles Lepidochelys olivacea varies among four body-areas of the hosts (n = 30). We showed that epibiont loads on olive ridleys are higher, both in terms of number and biomass, on the skin than they are on the carapace or plastron. This contrasts with previous findings for other hard-shelled sea turtles, where epibionts are usually more abundant on the carapace or plastron. Moreover, the arguably most ubiquitous epibiont taxon for other hard-shelled sea turtles, the barnacle Chelonibia spp., only occurred in relatively low numbers on olive ridleys and the barnacles Stomatolepas elegans and Platylepas hexastylos are far more abundant. We postulate that these differences between the epibiont communities of different sea turtle taxa could indicate that the carapaces of olive ridley turtles provide a more challenging substratum for epibionts than do the hard shells of other sea turtles. In addition, we conclude that it is important to conduct full body surveys when attempting to produce a holistic qualitative or quantitative characterization of the epibiont communities of sea turtles.

Parallel Patterns of Host-Specific Morphology and Genetic Admixture in Sister Lineages of a Commensal Barnacle.

Symbiotic relationships are often species specific, allowing symbionts to adapt to their host environments. Host generalists, on the other hand, have to cope with diverse environments. One coping strategy is phenotypic plasticity, defined by the presence of host-specific phenotypes in the absence of genetic differentiation. Recent work indicates that such host-specific phenotypic plasticity is present in the West Pacific lineage of the commensal barnacle Chelonibia testudinaria (Linnaeus, 1758). We investigated genetic and morphological host-specific structure in the genetically distinct Atlantic sister lineage of C. testudinaria. We collected adult C. testudinaria from loggerhead sea turtles, horseshoe crabs, and blue crabs along the eastern U.S. coast between Delaware and Florida and in the Gulf of Mexico off Mississippi. We find that shell morphology, especially shell thickness, is host specific and comparable in similar host species between the Atlantic and West Pacific lineages. We did not detect significant genetic differentiation related to host species when analyzing data from 11 nuclear microsatellite loci and mitochondrial sequence data, which is comparable to findings for the Pacific lineage. The most parsimonious explanation for these parallel patterns between distinct lineages of C. testudinaria is that C. testudinaria maintained phenotypic plasticity since the lineages diverged 4-5 mya.

A family-level Tree of Life for bivalves based on a Sanger-sequencing approach.

The systematics of the molluscan class Bivalvia are explored using a 5-gene Sanger-based approach including the largest taxon sampling to date, encompassing 219 ingroup species spanning 93 (or 82%) of the 113 currently accepted bivalve families. This study was designed to populate the bivalve Tree of Life at the family level and to place many genera into a clear phylogenetic context, but also pointing to several major clades where taxonomic work is sorely needed. Despite not recovering monophyly of Bivalvia or Protobranchia-as in most previous Sanger-based approaches to bivalve phylogeny-our study provides increased resolution in many higher-level clades, and supports the monophyly of Autobranchia, Pteriomorphia, Heteroconchia, Palaeoheterodonta, Heterodonta, Archiheterodonta, Euheterodonta, Anomalodesmata, Imparidentia, and Neoheterodontei, in addition to many other lower clades. However, deep nodes within some of these clades, especially Pteriomorphia and Imparidentia, could not be resolved with confidence. In addition, many families are not supported, and several are supported as non-monophyletic, including Malletiidae, Nuculanidae, Yoldiidae, Malleidae, Pteriidae, Arcidae, Propeamussiidae, Iridinidae, Carditidae, Myochamidae, Lyonsiidae, Pandoridae, Montacutidae, Galeommatidae, Tellinidae, Semelidae, Psammobiidae, Donacidae, Mactridae, and Cyrenidae; Veneridae is paraphyletic with respect to Chamidae, although this result appears to be an artifact. The denser sampling however allowed testing specific placement of species, showing, for example, that the unusual Australian Plebidonax deltoides is not a member of Donacidae and instead nests within Psammobiidae, suggesting that major revision of Tellinoidea may be required. We also showed that Cleidothaerus is sister group to the cementing member of Myochamidae, suggesting that Cleidothaeridae may not be a valid family and that cementation in Cleidothaerus and Myochama may have had a single origin. These results highlight the need for an integrative approach including as many genera as possible, and that the monophyly and relationships of many families require detailed reassessment. NGS approaches may be able to resolve the most recalcitrant nodes in the near future.

Epibiotic Diatoms Are Universally Present on All Sea Turtle Species.

The macro-epibiotic communities of sea turtles have been subject to growing interest in recent years, yet their micro-epibiotic counterparts are almost entirely unknown. Here, we provide the first evidence that diatoms are epibionts for all seven extant species of sea turtle. Using Scanning Electron Microscopy, we inspected superficial carapace or skin samples from a single representative of each turtle species. We distinguished 18 diatom taxa from these seven individuals, with each sea turtle species hosting at least two diatom taxa. We recommend that future research is undertaken to confirm whether diatom communities vary between sea turtle species and whether these diatom taxa are facultative or obligate commensals.

Microsatellite loci discovery from next-generation sequencing data and loci characterization in the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758).

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

Into the deep: a phylogenetic approach to the bivalve subclass Protobranchia.

A molecular phylogeny of Protobranchia, the subclass of bivalve mollusks sister to the remaining Bivalvia, has long proven elusive, because many constituent lineages are deep-sea endemics, which creates methodological challenges for collecting and preserving genetic material. We obtained 74 representatives of all 12 extant protobranch families and investigated the internal phylogeny of this group using sequence data from five molecular loci (16S rRNA, 18S rRNA, 28S rRNA, cytochrome c oxidase subunit I, and histone H3). Model-based and dynamic homology parsimony approaches to phylogenetic reconstruction unanimously supported four major clades of Protobranchia, irrespective of treatment of hypervariable regions in the nuclear ribosomal genes 18S rRNA and 28S rRNA. These four clades correspond to the superfamilies Nuculoidea (excluding Sareptidae), Nuculanoidea (including Sareptidae), Solemyoidea, and Manzanelloidea. Salient aspects of the phylogeny include (1) support for the placement of the family Sareptidae with Nuculanoidea; (2) the non-monophyly of the order Solemyida (Solemyidae+Nucinellidae); (3) and the non-monophyly of most nuculoid and nuculanoid genera and families. In light of this first family-level phylogeny of Protobranchia, we present a revised classification of the group. Estimation of divergence times in concert with analyses of diversification rates demonstrate the signature of the end-Permian mass extinction in the phylogeny of extant protobranchs.

Introduction to the symposium--barnacle biology: essential aspects and contemporary approaches.

Barnacles have evolved a number of specialized features peculiar for crustaceans: they produce a calcified, external shell; they exhibit sexual strategies involving dioecy and androdioecy; and some have become internal parasites of other Crustacea. The thoroughly sessile habit of adults also belies the highly mobile and complex nature of their larval stages. Given these and other remarkable innovations in their natural history, it is perhaps not surprising that barnacles present a spectrum of opportunities for study. This symposium integrates research on barnacles in the areas of larval biology, biofouling, reproduction, biogeography, speciation, population genetics, ecological genomics, and phylogenetics. Pioneering comparisons are presented of metamorphosis among barnacles from three major lineages. Biofouling is investigated from the perspectives of biochemical and biomechanical mechanisms. Tradeoffs in reproductive specializations are scrutinized through theoretical modeling and empirical validation. Patterns of endemism and diversity are delineated in Australia and intricate species boundaries in the genus Chthamalus are elucidated for the Indo-Pacific. General methodological concerns with population expansion studies in crustaceans are highlighted using barnacle models. Data from the first, draft barnacle genome are employed to examine location-specific selection. Lastly, barnacle evolution is framed in a deep phylogenetic context and hypothetical origins of defined characters are outlined and tested.

Microbial biofilms facilitate adhesion in biofouling invertebrates.

Much interest has focused on the role of microbial layers--biofilms--in stimulating attachment of invertebrates and algae to submerged marine surfaces. We investigated the influence of biofilms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test surfaces that were either clean or coated with a natural biofilm. Measuring larval removal under precisely controlled flow forces, we found that biofilms significantly increased adhesion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attachment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the presence of a biofilm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research.

Bathymetric and geographic population structure in the pan-Atlantic deep-sea bivalve Deminucula atacellana (Schenck, 1939).

The deep-sea soft-sediment environment hosts a diverse and highly endemic fauna of uncertain origin. We know little about how this fauna evolved because geographic patterns of genetic variation, the essential information for inferring patterns of population differentiation and speciation are poorly understood. Using formalin-fixed specimens from archival collections, we quantify patterns of genetic variation in the protobranch bivalve Deminucula atacellana, a species widespread throughout the Atlantic Ocean at bathyal and abyssal depths. Samples were taken from 18 localities in the North American, West European and Argentine basins. A hypervariable region of mitochondrial 16S rDNA was amplified by polymerase chain reaction (PCR) and sequenced from 130 individuals revealing 21 haplotypes. Except for several important exceptions, haplotypes are unique to each basin. Overall gene diversity is high (h = 0.73) with pronounced population structure (Phi(ST) = 0.877) and highly significant geographic associations (P < 0.0001). Sequences cluster into four major clades corresponding to differences in geography and depth. Genetic divergence was much greater among populations at different depths within the same basin, than among those at similar depths but separated by thousands of kilometres. Isolation by distance probably explains much of the interbasin variation. Depth-related divergence may reflect historical patterns of colonization or strong environmental selective gradients. Broadly distributed deep-sea organisms can possess highly genetically divergent populations, despite the lack of any morphological divergence.

Multiple origins and incursions of the Atlantic barnacle Chthamalus proteus in the Pacific.

Chthamalus proteus, a barnacle native to the Caribbean and western Atlantic, was introduced to the Pacific within the last few decades. Using direct sequencing of mitochondrial DNA (COI), we characterized genetic variation in native and introduced populations and searched for genetic matches between regions to determine if there were multiple geographical sources and introduction points for this barnacle. In the native range, we found great genetic differences among populations (max. F(ST) = 0.613) encompassing four lineages: one endemic to Panama, one endemic to Brazil, and two occurring Caribbean-wide. All four lineages were represented in the Pacific, but not equally; the Brazilian lineage was most prevalent and the Panamanian least common. Twenty-one individuals spread among nearly every island from where the barnacle is known in the Pacific, exactly matched six haplotypes scattered among Curaçao, the Netherlands Antilles; St John, US Virgin Islands; Puerto Rico; and Brazil, confirming a multigeographical origin for the Pacific populations. Significant genetic differences were also found in introduced populations from the Hawaiian Islands (F(CT) = 0.043, P < 0.001), indicating introduction events have occurred at more than one locality. However, the sequence, timing and number of arrival events remains unknown. Possible reasons for limited transport of this barnacle through the Panama Canal are discussed. This and a preponderance of Brazilian-type individuals in the Pacific suggest an unexpected route of entry from around Cape Horn, South America. Unification in the Pacific of historically divergent lineages of this barnacle raises the possibility for selection of 'hybrids' with novel ecological adaptations in its new environment.

Protobranch bivalves.

The subclass Protobranchia comprises more than 600 species of bivalves that occur throughout the world ocean. Mostly deposit feeders in soft sediments, they are abundant in the deep sea. Apomorphies that unite them as a group include gill structure, hinge conformation, shell microstructure, larval development, foot morphology, respiratory pigments, trophic mode and digestion. They are relatively small and highly conserved in form, originating in the Cambrian era. They may represent an ancestral, derived or paraphylectic group of the Bivalvia. The protobranchs include two orders, the Nuculoida and Solemyoida, which previously were classified separately in the subclasses Paleotaxodonta and Cryptodonta, respectively. They are of ecological interest and have a unique functional morphology. They feed mostly under the surface of the sediment with highly modified labial palps, but the degree to which they are selective in diet remains difficult to determine. They are important bioturbators in many soft-sediment assemblages; their feeding and locomotion affects sediment structure and community development. Solemyoids are unusual in inhabiting reducing environments and hydrocarbon seeps and in deriving their nutrition from endosymbiotic chemosynthetic bacteria. A variety of species of protobranchs are found in oceanic trenches, near hydrothermal vents, and in submarine caves. Protobranchs produce a lecithotrophic larval stage, the pericalymma, making their development unique among bivalves. The pericalymma remains in the plankton for a short time and presumably has low dispersal ability. Recruitment may be intermittent. Growth is rapid in post-larvae but decreases with age, though rates may not necessarily be slow, especially in continental shelf species. Life spans are commonly 1 to 2 decades, but deep-sea representatives may grow more slowly and live longer. Bottom fish, seastars and gastropods are their major predators and a few parasites and commensals have been documented. The predominance of protobranchs in deep-sea sediments may be a result of deep-sea origin or displacement from shallow waters by lamellibranchs. Their ability to deposit-feed, digest food extracellularly, and develop by means of lecithotrophic larvae make them particularly well adapted to cold and oligotrophic habitats.