Independent evolution of intermediate bill widths in a seabird clade.

Interspecific introgression can occur between species that evolve rapidly within an adaptive radiation. Pachyptila petrels differ in bill size and are characterised by incomplete reproductive isolation, leading to interspecific gene flow. Salvin's prion (Pachyptila salvini), whose bill width is intermediate between broad-billed (P. vittata) and Antarctic (P. desolata) prions, evolved through homoploid hybrid speciation. MacGillivray's prion (P. macgillivrayi), known from a single population on St Paul (Indian Ocean), has a bill width intermediate between salvini and vittata and could also be the product of interspecies introgression or hybrid speciation. Recently, another prion population phenotypically similar to macgillivrayi was discovered on Gough (Atlantic Ocean), where it breeds 3 months later than vittata. The similarity in bill width between the medium-billed birds on Gough and macgillivrayi suggest that they could be closely related. In this study, we used genetic and morphological data to infer the phylogenetic position and evolutionary history of P. macgillivrayi and the Gough medium-billed prion relative other Pachyptila taxa, to determine whether species with medium bill widths evolved through common ancestry or convergence. We found that Gough medium-billed prions belong to the same evolutionary lineage as macgillivrayi, representing a new population of MacGillivray's prion that originated through a colonisation event from St Paul. We show that macgillivrayi's medium bill width evolved through divergence (genetic drift) and independently from that of salvini, which evolved through hybridisation (gene flow). This represents the independent convergence towards a similarly medium-billed phenotype. The newly discovered MacGillivray's prion population on Gough is of utmost conservation relevance, as the relict macgillivrayi population in the Indian Ocean is very small.

Cooccurring Activities of Two Autotrophic Pathways in Symbionts of the Hydrothermal Vent Tubeworm Riftia pachyptila.

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.

Compound-specific stable isotope analyses in Falkland Islands seabirds reveal seasonal changes in trophic positions.

BACKGROUND: While nitrogen and carbon stable isotope values can reflect ecological segregation, prey choice and spatial distribution in seabirds, the interpretation of bulk stable isotope values is frequently hampered by a lack of isotopic baseline data. In this study, we used compound-specific isotope analyses of amino acids (CSIA-AA) to overcome this constraint and to study interspecific differences, seasonal and historical changes in trophic positions of five seabird species, three penguins and two petrels, from a sub-Antarctic seabird community. RESULTS: CSIA-AA allowed comparing trophic positions of seabirds with temperate and polar distributions. Gentoo and Magellanic penguins had the highest trophic positions during the breeding season (3.7 and 3.9), but decreased these (2.9 and 3.3) during the feed-up for moult. Intra-specific differences were also detected in Thin-billed prions, where carbon isotope values clearly separated individuals with polar and temperate distributions, both in the breeding and interbreeding periods. Thin-billed prions that foraged in polar waters had lower trophic positions (3.2) than conspecifics foraging in temperate waters (3.8). We further investigated historical changes by comparing museum samples with samples collected recently. Our pilot study suggests that Rockhopper penguins, Magellanic penguins and Thin-billed prions with temperate non-breeding distributions had retained their trophic levels over a 90-100 year period, while Gentoo penguins and Thin-billed prions with polar non-breeding distributions had decreased trophic levels compared to historical samples. In contrast, Wilson's storm-petrels had slightly increased trophic levels compared to samples taken in 1924-1930. CONCLUSIONS: We applied compound-specific stable isotope analyses across a range of contexts, from intra-specific comparisons between stages of the breeding cycle to inter-specific seabird community analysis that would not have been possible using bulk stable isotope analyses alone due to differences in isotopic baselines.

Hydrogen Does Not Appear To Be a Major Electron Donor for Symbiosis with the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila.

Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.

Year-round at-sea movements of fairy prions from southeastern Australia.

Effective conservation assessments require detailed information of species' ecological niches during the whole annual cycle. For seabirds, this implies investigating the at-sea distribution and foraging behaviour during both the breeding and non-breeding periods. However, until recently, collecting information about small species has been precluded by the excessive size of the required devices. This lack of knowledge is exacerbated in the case of polytypic genera with species sharing very similar appearance and behaviour, such as the super-abundant prions (Pachyptila spp.). The present study investigates the year-round at-sea distribution and foraging ecology of the fairy prion (Pachyptila turtur) in southeastern Australia. Miniaturized GPS loggers during the breeding season and geolocators (GLS) during the non-breeding period were used over 4 consecutive years (2017-2021), with results that highlight the importance of the continental shelf-edge waters for fairy prions throughout the year. In addition, contrary to previous assumptions, the GLS data revealed an unsuspected post-breeding migration to the waters south of Australia, during which individuals probably undergo a rapid moult of flight feathers. Understanding the at-sea distribution and ecology of prions during the whole annual cycle will be fundamental to their conservation as it can reveal species- or population-specific threats that have been overlooked because of their status as abundant species.

The complete and closed genome of the facultative generalist Candidatus Endoriftia persephone from deep-sea hydrothermal vents.

The mutualistic interactions between Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone (short Endoriftia) have been extensively researched. However, the closed Endoriftia genome is still lacking. Here, by employing single-molecule real-time sequencing we present the closed chromosomal sequence of Endoriftia. In contrast to theoretical predictions of enlarged and mobile genetic element-rich genomes related to facultative endosymbionts, the closed Endoriftia genome is streamlined with fewer than expected coding sequence regions, insertion-, prophage-sequences and transposase-coding sequences. Automated and manually curated functional analyses indicated that Endoriftia is more versatile regarding sulphur metabolism than previously reported. We identified the presence of two identical rRNA operons and two long CRISPR regions in the closed genome. Additionally, pangenome analyses revealed the presence of three types of secretion systems (II, IV and VI) in the different Endoriftia populations indicating lineage-specific adaptations. The in depth mobilome characterization identified the presence of shared genomic islands in the different Endoriftia drafts and in the closed genome, suggesting that the acquisition of foreign DNA predates the geographical dispersal of the different endosymbiont populations. Finally, we found no evidence of epigenetic regulation in Endoriftia, as revealed by gene screenings and absence of methylated modified base motifs in the genome. As a matter of fact, the restriction-modification system seems to be dysfunctional in Endoriftia, pointing to a higher importance of molecular memory-based immunity against phages via spacer incorporation into CRISPR system. The Endoriftia genome is the first closed tubeworm endosymbiont to date and will be valuable for future gene oriented and evolutionary comparative studies.

Discovery of a readily heterologously expressed Rubisco from the deep sea with potential for CO2 capture.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key CO2-fixing enzyme in photosynthesis, is notorious for its low carboxylation. We report a highly active and assembly-competent Form II Rubisco from the endosymbiont of a deep-sea tubeworm Riftia pachyptila (RPE Rubisco), which shows a 50.5% higher carboxylation efficiency than that of a high functioning Rubisco from Synechococcus sp. PCC7002 (7002 Rubisco). It is a simpler hexamer with three pairs of large subunit homodimers around a central threefold symmetry axis. Compared with 7002 Rubisco, it showed a 3.6-fold higher carbon capture efficiency in vivo using a designed CO2 capture model. The simple structure, high carboxylation efficiency, easy heterologous soluble expression/assembly make RPE Rubisco a ready-to-deploy enzyme for CO2 capture that does not require complex co-expression of chaperones. The chemosynthetic CO2 fixation machinery of chemolithoautotrophs, CO2-fixing endosymbionts, may be more efficient than previously realized with great potential for next-generation microbial CO2 sequestration platforms.

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis.

The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

Behavioural plasticity in the early breeding season of pelagic seabirds - a case study of thin-billed prions from two oceans.

BACKGROUND: In long-lived seabirds that migrate large distances independently of each other, the early part of the breeding season is crucially important for a successful reproductive attempt. During this phase, pair bonds are re-established and partners coordinate their breeding duties. We studied the early breeding season in Thin-billed prions Pachyptila belcheri breeding in the Atlantic Ocean (Falkland/Malvinas Islands) and Indian Ocean (Kerguelen). Despite overlap in the wintering areas, these two populations exhibit differences in their timing and direction of migration. We hypothesised that these differences would influence behaviour during the early breeding season. RESULTS: In line with our hypothesis, we found very strong differences in colony attendance patterns. Thin-billed prions of the Falkland population spent the late winter period over shelf waters close to the colony, first arrived back at the colony in September, and attended the nests interruptedly for one month, before departing on a pre-laying exodus. In contrast, Kerguelen birds remained in the non-breeding areas until mid-October and spent much less time attending the burrow before their pre-laying exodus. Despite this asynchronous arrival to the two colonies, the subsequent patterns resulted in remarkably synchronous incubation in both populations, with males taking on the first long incubation shift in late November, whereas females returned to sea soon after egg laying. During the pre-laying exodus and incubation, Thin-billed prions from the Falklands spread north over the Patagonian Shelf, while prions from Kerguelen travelled much further, reaching southern oceanic waters and moved at faster speeds (> 400 km per day). Although prions from Kerguelen moved much further, their isotopic niches were considerably narrower, suggesting a stronger dependence on Antarctic waters. CONCLUSIONS: The study thus suggests that Thin-billed prions show a high intraspecific plasticity in their use of either neritic or oceanic waters during the early breeding season. Breeding birds from the Falkland Islands can exploit an extensive shelf area, while Kerguelen birds have adapted to the need to forage in distant southern open waters. This difference in foraging ecology may thus have shaped the phenology of the early breeding phase.

A Novel ω3-Desaturase in the Deep Sea Giant Tubeworm Riftia pachyptila.

One paradox of the trophic biochemistry of the deep sea giant tubeworm Riftia pachyptila, endemic to hydrothermal vent sites and nourished by polyunsaturated fatty acid (PUFA) deficiency chemolitoautotrophic sulfide-oxidizing bacteria, is the source of their PUFAs. Biosynthesis of PUFA starts with two precursors C18:2n-6 and C18:3n-3, which cannot be biosynthesized by most animals due to lack of ω6- and ω3-desaturase; thus, C18:2n-6 and C18:3n-3 are generally essential fatty acids for animals. Here, we characterized a gene derived from the R. pachyptila located by hydrothermal vent, which encoded a novel ω3-desaturase (Rp3Fad). The gene was identified by searching the R. pachyptila transcriptome database using known ω3-desaturases, and its predicted protein showed 37-45% identical to ω3-desaturases of fungus and microalgae, and only 31% identitical to nematode Caenorhabditis elegans ω3-desaturase. Expression in yeast Saccharomyces cerevisiae showed that the Rp3Fad could desaturate C18:2n-6 and C18:3n-6 into C18:3n-3 and C18:4n-3, respectively, displaying a Δ15 activity similar to plant ω3-desaturase, but it showed no activity towards C20 n-6 PUFA substrates, differing from the well-characterized C. elegans ω3-desaturases. Δ5, Δ6, Δ8, and Δ12 activity were also tested, resulting in no corresponding production. The function of ω3-desaturase identified in R. pachyptila could produce C18:3n - 3 used in synthesis of n - 3 series PUFAs, suggesting an adaption to PUFA deficiency environment in deep sea hydrothermal vent.

Evolutionary factors affecting the cross-species utility of newly developed microsatellite markers in seabirds.

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio-temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next-generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub-Antarctic seabird, the thin-billed prion (Pachyptila belcheri), that we tested for cross-species amplification in other Pachyptila and related sub-Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific FST provided better approximations of mtDNA relationships among the studied species than those estimated using DC , even though FST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross-species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.

Tubeworm succession at hydrothermal vents: use of biogenic cues to reduce habitat selection error?

Species colonizing new deep-sea hydrothermal vents along the East Pacific Rise show a distinct successional sequence: pioneer assemblages dominated by the vestimentiferan tubeworm Tevnia jerichonana being subsequently invaded by another vestimentiferan Riftia pachyptila, and eventually the mussel Bathymodiolus thermophilus. Using a manipulative approach modified from shallow-water ecological studies, we test three alternative hypotheses to explain the initial colonization by T. jerichonana and its subsequent replacement by R. pachyptila. We show that R. pachyptila and another vestimentiferan, Oasisia alvinae, colonized new surfaces only if the surfaces also were colonized by T. jerichonana. This pattern does not appear to be due to restricted habitat tolerances or inferior dispersal capabilities of R. pachyptila and O. alvinae, and we argue the alternative explanation that T. jerichonana facilitates the settlement of the other two species and is eventually outcompeted by R. pachyptila. Unlike the classic model of community succession, in which facilitating species promote their own demise by modifying the environment to make it more hospitable for competitors, we suggest that T. jerichonana may produce a chemical substance that induces settlement of these competitors. This process of selecting habitat based on biogenic cues may be especially adaptive and widespread among later-successional species that occupy a physically variable and unpredictable environment. In these cases, the presence of weedy species implies some integrated period of environmental suitability, whereas an instantaneous assessment of physical habitat conditions, such as water temperature for vent tubeworms, provides a poorer predictor of long-term habitat suitability.