Effects of allogeneic contact on life-history traits of the colonial ascidian Botryllus schlosseri in Monterey Bay

The formation of chimeric colonies following allogeneic contact between benthic invertebrates may strongly affect colony fitness. Here we show that, in a field population of the colonial ascidian Botryllus schlosseri in Monterey Bay, California, more than 20% of all colonies occur in allogeneic contact with conspecifics. We experimentally assessed the effects of allogeneic contact on the following life-history traits under natural field conditions: growth, age and size at first reproduction, and egg production (fecundity). When compared with isolated colonies, and in some cohorts also with colonies that rejected allogeneic neighbors, colonies that fused with neighbors incurred reduced fitness in terms of most life-history traits measured. We propose that one of the benefits of precise allorecognition is that, in fused colonies, it limits the unit of selection to chimeric individuals composed of closely related kin.     

Tunic cell populations during fusion events in the colonial ascidian Didemnum vexillum (Tunicata)

We documented changes in the abundance and distribution patterns of tunic cells involved in the allorecognition response of the colonial aplousobranch Didemnum vexillum, whose zooids do not share a common vascular system. A histological examination of the fusion zone of isogeneic (CIAs) and allogeneic (CAAs) fused colony assays revealed that tunic cuticles were rapidly regenerated. The underlying tunic matrix fused readily in all assays and controls. We identified four different types of tunic cells. Phagocytic cells represented the most abundant cell type in allogeneic fusions, followed by morula cells. These cells were more abundant at the immediate fusion junction than at 120 μm or 240 μm from the junction, most likely because they mediate the allorecognition reaction. Elongated filopodial cells also were present, although only at very low abundances, and a layer of bladder cells was located immediately below the cuticle. Our results provide quantitative evidence for the involvement of tunic cells in the allorecognition response of a highly invasive ascidian.     

Not like Botryllus: indiscriminate post-metamorphic fusion in a compound ascidian

Fusion trials between metamorphs of the aplousobranch compound ascidian Diplosoma listerianum indicated that chimera formation was not dependent on relatedness. Similar, high rates of union were observed between full siblings, half siblings, unrelated individuals from the same population, and individuals from two geographically distant localities. This is in contrast to the well-studied ascidian genus Botryllus, in which a highly polymorphic allorecognition system governing the fusion–non-fusion reaction (colony specificity) largely limits fusion to close relatives. Fusion in Botryllus establishes a vascular chimera throughout which stem cells may circulate, promoting cell lineage competition between the fusion partners. The restriction of fusion to close kin in Botryllus is thought to reduce the inclusive fitness costs of competitive interactions between cell lineages within the chimera. In contrast to Botryllus, modules (zooids) of a D. listerianum colony are not interlinked by blood vessels, seemingly precluding the exchange of stem cells. The apparent absence of strict colony specificity in D. listerianum is thus in keeping with the predictions of the Botryllus model for the maintenance of allorecognition polymorphism. However, colony specificity has been reported in other species of aplousobranch ascidian that also lack a common vascular system. In these, the threat of migrating blood-borne stem cells cannot be responsible for the presence of colony specificity. One possibility, requiring experimental investigation, is that stem cells could perhaps migrate between zooids by another route, such as through the matrix of the colonial tunic. Even in the absence of stem cell exchange, cheating on the costs of colony maintenance and defence could also produce selective forces favouring colony specificity. In compound ascidians, this could involve unequal contribution to extrazooidal structures, principally the tunic and related tissues. This consideration seems potentially relevant to the lack of discrimination during fusion in D. listerianum, since extrazooidal somatic investment in this species appears minimal, severely limiting the scope for this other form of cheating. The various possible modes of exploitative interaction between fused colonies are not mutually exclusive, and offer fundamentally similar explanations for colony specificity. If none of them can be shown to occur in non-botryllid species possessing colony specificity, the generality of the Botryllus model may require re-evaluation.     

Formal Genetics of Ascidians

SYNOPSIS. Our knowledge of ascidian genetics is reviewed. Thepaper is primarily concerned with the author's past and currentwork on the colonial species Bolryllus schlosseri. Five Mendelianloci account for most of its impressive polychromatism. Breedingexperiments have substantiated the hypothesis of a single multialleliclocus for each of three enzymes (MDH, SOD, PGI) suggested byelectrophoretic patterns. The nuclei of three linkage groupshave been revealed. Self—fertilization entails a severeinbreeding depression. A specific self, nonself recognition,expressed by fusion or repulsion of contacting colonies, occursin this species also. At variance with Botryllus primigenus,fusible colonies of B. schlosseri are completely interfertile.This has allowed a more direct genetic analysis of the phenomenon,confirming the alleged control by a single multiallelic locus.In order to fuse, the confronted colonies must share at leastone allele. Young buds grafted in the tunic after removal ofall the zooids develop a new colony at the host's expense onlyif donor and host are fusible. This means that fusibility andhistocompatibility are strictly correlated. Chimerical colonies,obtained either in this way or following the resorption of oneof two fused colonies, are now being investigated for theirrecognition specificity and electrophoretic pattern. Preliminarydata indicate that both can be durably altered, suggesting thatthe allogeneic cell populations are persistent and renewing.     

Chimerism following fusion in a clonal ascidian (Urochordata)

Many marine invertebrates bud vegetatively to produce a modular colony of individuals derived from a single zygote. Fusion of different colonies to produce a genetically composite entity (a chimera) is known from experiments on sponges, hydroids, corals, bryozoans and ascidians – groups which together dominate sessile faunas on marine hard substrates. Random amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR) analysis was applied to individual modules (zooids) dissected from colonies of a colonial ascidian, Diplosoma listerianum (Milne Edwards), to investigate the presence and extent of chimerism. The technique revealed chimerism in wild material. In total, 288 colonies from eight different natural populations were analysed. Chimeric colonies were present in all populations, at frequencies of up to 61%, with up to six different genotypes present in some colonies. Zooids of different genetic origin often intermingled within a chimeric colony to produce a zooidal mosaic. Although fusion of colonies has been observed directly in the laboratory, an unknown proportion of the chimerism detected in wild populations might have arisen through somatic mutation. To assess this possibility, tissue of 12 clones in culture was sampled repeatedly over a period of 3 years and subjected to RAPD–PCR analysis. RAPD banding patterns were generally very stable; the changes noted mostly involved minor bands that would not, on their own, have been taken as evidence for chimerism under the conservative criteria adopted for the study of wild populations. It was concluded that a large proportion of natural chimerism is attributable to colony fusion.  © 2003 The Linnean Society of London. Biological Journal of the Linnean Society , 2003, 79 , 183–192.     

Predation Limits Spread of Didemnum vexillum into Natural Habitats from Refuges on Anthropogenic Structures

Non-indigenous species can dominate fouling assemblages on artificial structures in marine environments; however, the extent to which infected structures act as reservoirs for subsequent spread to natural habitats is poorly understood. Didemnum vexillum is one of few colonial ascidian species that is widely reported to be highly invasive in natural ecosystems, but which in New Zealand proliferates only on suspended structures. Experimental work revealed that D. vexillum established equally well on suspended artificial and natural substrata, and was able to overgrow suspended settlement plates that were completely covered in other cosmopolitan fouling species. Fragmentation led to a level of D. vexillum cover that was significantly greater than was achieved as a result of ambient larval recruitment. The species failed to establish following fragment transplants onto seabed cobbles and into beds of macroalgae. The establishment success of D. vexillum was greatest in summer compared with autumn, and on the underside of experimental settlement plates that were suspended off the seabed to avoid benthic predators. Where benthic predation pressure was reduced by caging, D. vexillum establishment success was broadly comparable to suspended treatments; by contrast, the species did not establish on the face-up aspect of uncaged plates. This study provides compelling evidence that benthic predation was a key mechanism that prevented D. vexillum’s establishment in the cobble habitats of the study region. The widespread occurrence of D. vexillum on suspended anthropogenic structures is consistent with evidence for other sessile invertebrates that such habitats provide a refuge from benthic predation. For invasive species generally, anthropogenic structures are likely to be most important as propagule reservoirs for spread to natural habitats in situations where predation and other mechanisms do not limit their subsequent proliferation.     

Random amplified polymorphic DNA (RAPD) analysis reveals extensive natural chimerism in a marine protochordate

Random amplified polymorphic DNA (RAPD) analysis was applied to individual modules (zooids) of a colonial ascidian to investigate the presence and extent of chimerism, the parabiotic association of different genetic entities. The technique proved to be rapid and efficient for distinguishing different genotypes present in a colony, and revealed genetic mosaicism in wild material, as well as in laboratory cultures following planned fusion. Approximately one-third of colonies in the natural population studied possessed multiple genotypes, presumably as the result of fusion of different colonies. Furthermore, individual zooids of different genetic origin often intermingled after colony fusion, spreading each genotype throughout a larger total area.     

Effect of colony size on the competitive outcome of encrusting colonial organisms

The relative importance of colony size ratio of interacting species was studied in Tomioka Bay, Japan. Six encrusting colonial species belonging to the following three different taxonomic groups were tested: Ascidia (three species), Bryozoa (two) and Porifera (one). Colonies of these organisms were grown in the community of sessile organisms developed on plastic panels. Logistic regression analysis was carried out to determine the effect of size ratio on the competitive outcome of interacting colonies. The results between all possible combinations among these six species did not show a significant size effect in competitive outcome (i.e. a larger colony size did not always prove important in the success of a competitive interaction with smaller colonies of other species). On the contrary, competitive success depends on the types of species interacting. Certain species such asDidemnum moseleyi (ascidian) andHaliclona sp. (sponge), in spite of being smaller in colony size, won in competitive interactions with larger colonies of other species such asDiplosoma mitsukurii (ascidian) andWatersipora subovoidea (bryozoan). These results contradict the one reported earlier: that the larger the colony size, the more chance the colony will have to win in competitive interactions.     

Intracolonial genetic variation in the scleractinian coral Seriatopora hystrix

In recent years, increasing numbers of studies revealed intraorganismal genetic variation, primarily in modular organisms like plants or colonial marine invertebrates. Two underlying mechanisms are distinguished: Mosaicism is caused by somatic mutation, whereas chimerism originates from allogeneic fusion. We investigated the occurrence of intracolonial genetic variation at microsatellite loci in five natural populations of the scleractinian coral Seriatopora hystrix on the Great Barrier Reef. This coral is a widely distributed, brooding species that is at present a target of intensive population genetic research on reproduction and dispersal patterns. From each of 155 S. hystrix colonies, either two or three samples were genotyped at five or six loci. Twenty-seven (~17%) genetically heterogeneous colonies were found. Statistical analyses indicated the occurrence of both mosaicism and chimerism. In most cases, intracolonial variation was found only at a single allele. Our analyses suggest that somatic mutations present a major source of genetic heterogeneity within a single colony. Moreover, we observed large, apparently stable chimeric colonies that harbored clearly distinct genotypes and contrast these findings with the patterns typically observed in laboratory-based experiments. We discuss the error that mosaicism and chimerism introduce into population genetic analyses.     

Contact reactions between young colonies of the coral Pocillopora damicornis

 Newly settled larvae (primary polyps) or young colonies of the coral Pocillopora damicornis were brought into contact at various periods after planulation to examine isogeneic and allogeneic responses. While young colonies derived from the same colony always fused, those derived from different colonies showed either fusion, nonfusion, or incompatible fusion. Tissues were continuous in incompatibly fused pairs, but a white zone, without zooxanthellae, was observed at the interface. The skeleton was also continuous but a groove with skeletal spines on both sides was observed under the white zone. Polyps originating near the white zone later disappeared or were partially resorbed. After 2–8 months, several incompatibly fused pairs became separated by a skeletal ridge, or by a narrow zone of skeleton without living tissue. Incompatible fusion appears to be a distinct histoincompatible response which later transforms into nonfusion. The period between planulation and initial contact of colonies did not affect the outcomes of the contact experiments. Accepted: 31 January 1996     

Chimerism and population dieback alter genetic inference related to invasion pathways and connectivity of biofouling populations on artificial substrata

Disentangling pathways by which nonindigenous species expand and spread regionally remains challenging. Molecular ecology tools are often employed to determine the origins and spread of introduced species, but the complexities of some organisms may be reducing the efficacy of these tools. Some colonial species exhibit complexities by way of chimerism and winter colony regression, which may alter the genetic diversity of populations and mask the connectivity occurring among them. This study uses nuclear microsatellite data and simple GIS‐based modeling to investigate the influence of chimerism and winter regression on the genetic diversity and patterns of genetic population connectivity among colonies of Didemnum vexillum on artificial substrates. Colonies sampled in summer were shown to form a metapopulation, with high levels of admixture, extreme outcrossing, and some substructure. These patterns were consistent within the subsampled winter colonies and with the inclusion of chimeric data. However, allelic richness and diversity were significantly different between winter and summer samples, altering interpretations relating to population connectivity and pelagic larval duration. This study demonstrates the importance of including seasonal sampling and imperative life history traits in genetic studies for clear interpretations and the successful management of introduced species.     

Natural dispersal mechanisms and dispersal potential of the invasive ascidian Didemnum vexillum

Over the past decade, several species of non-indigenous ascidians have had adverse effects on a range of coastal ecosystems, and associated industries like aquaculture. One such species, the colonial ascidian Didemnum vexillum, poses a threat to the highly-valued New Zealand green-lipped mussel industry, and there is interest in whether and to what extent its spread can be managed at a regional scale (<100 km). An important component in the decision-making process for managing human-mediated pathways of spread is an understanding of D. vexillum’s natural dispersal potential. Here we use a weight-of-evidence approach, combining laboratory and field studies, to assess the role of natural dispersal mechanisms in the spread of D. vexillum. Under laboratory conditions, >70 % of D. vexillum larvae remained viable and were able to settle and undergo metamorphosis successfully following an artificial delay of 2 h. Larval viability decreased with increasing delay duration, although 10 % of larvae remained viable following a 36 h delay. A field-based study documented larval dispersal from two discrete source populations, with recruitment consistently detected on settlement plates at 250 m from source populations at one experimental site. Exponential decay models used to predict maximum larval dispersal distances at this site indicated that dispersal greater than 250 m is theoretically possible (>1 km in some situations). That being so, we recognise that the successful establishment and persistence of populations will depend on a wide range of processes not taken into account here. Our findings are supported by surveillance of D. vexillum spread in the wider study region; there are a number of instances where the species established on artificial structures that were several kilometres from known source populations, at a time when intensive regional-scale management of anthropogenic vectors was underway. Collectively, our findings indicate that D. vexillum has the ability to spread further by natural dispersal than previously assumed; probably hundreds of metres to kilometres depending on the local hydrological conditions, which has important implications for the ongoing management of this pest species world-wide.     

Tissue compatibility between colonies and between newly settled larvae of Pocillopora damicornis

Grafting experiments with newly settled larvae and with adult colonies of Pocillopora damicornis were performed. When pairs of newly settled larvae released from different colonies were kept in contact, they fused to form an aggregated colony. Even newly settled larvae derived from colonies belonging to different color morphs fused with each other and no sign of allogeneic rejection was observed. However, when branches of adult colonies belonging to different color morphs were kept in contact, they did not fuse. Fusion was observed only when branches derived from the same colony were paired. The present results suggest that juvenile corals lack the functional histocompatibility system as shown by adult colonies.     

Development and application of tools for incursion response: Lessons learned from the management of the fouling pest Didemnum vexillum

In October 2001, an unidentified didemnid ascidian, was recorded for the first time in New Zealand, smothering wharf piles and moorings in a northern harbour. A heavily-fouled barge then translocated the ascidian to an international shipping port some 500 km south, near the heart of the New Zealand mussel industry. The species was subsequently identified as Didemnum vexillum, but its status as indigenous or non-indigenous was disputed. Nonetheless, its presence was regarded as a significant threat to the mussel industry because of its demonstrated invasiveness on artificial structures, and its ability to over-grow and smother mussels.From the barge's mooring area, D. vexillum subsequently spread to the seabed beneath, and to nearby vessels and artificial structures (i.e., barges, recreational vessels, moorings, salmon cages and wharf piles). Given the likelihood that infected vectors would spread the ascidian to mussel farms in the region, and in consideration of a benefit-cost analysis, an eradication program for D. vexillum was instigated. This paper provides a chronology of events surrounding the initial detection and spread of the ascidian, and describes the development of incursion response tools for the different substrata that were infected. The treatments included smothering soft-sediment habitats with uncontaminated dredge spoil, wrapping wharf piles with plastic, smothering rip-rap habitats using a geotextile fabric, and various other approaches based on water blasting, air drying or chlorine dosing. While many of the response methods were completely effective at eliminating D. vexillum from different substrata, the program overall failed to eradicate the organism from the region. The reasons for this failure are documented, and the important lessons learned are highlighted, as a contribution to the successful management of invasive species in the future.     

A discrete,stochastic model of colonial phytoplankton population size structure: Development and application to in situ imaging-in-flow cytometer observations of Dinobryon

The scientific community lacks models for the dynamic changes in population size structure that occur in colonial phytoplankton. This is surprising, as size is a key trait affecting many aspects of phytoplankton ecology, and colonial forms are very common. We aim to fill this gap with a new discrete, stochastic model of dynamic changes in phytoplankton colonies' population size structure. We use the colonial phytoplankton Dinobryon as a proof-of-concept organism. The model includes four stochastic functions—division, stomatocyst production, colony breakage, and colony loss—to determine Dinobryon population size structure and populations counts. Although the functions presented here are tailored to Dinobryon, the model is readily adaptable to represent other colonial taxa. We demonstrate how fitting our model to in situ observations of colony population size structure can provide a powerful approach to explore colony size dynamics. Here, we have (1) collected high-frequency in situ observations of Dinobryon in Lac (Lake) Montjoie (Quebec, Canada) in 2013 with a moored Imaging FlowCytobot (IFCB) and (2) fit the model to those observations with a genetic algorithm solver that extracts parameter estimates for each of the four stochastic functions. As an example of the power of this model-data integration, we also highlight ecological insights into Dinobryon colony size and stomatocyst production. The Dinobryon population was enriched in larger, flagellate-rich colonies near bloom initiation and shifted to smaller and emptier colonies toward bloom decline.     

Growing or reproducing in a temperate sea: optimization of resource allocation in a colonial ascidian

Relatively little is known about the life cycles of ascidians in temperate seas. Here, we investigated the biological cycle of the colonial ascidian Didemnum fulgens, a dominant species in some shallow localities of the NW Mediterranean Sea. Growth rates and frequencies of fission/fusion events were calculated over a period of 13 months, and the reproductive cycle determined after 32 months of observation. For analyses of reproduction, zooids were dissected in the laboratory and classified into five reproductive categories; these data were used to calculate a maturity index. For growth analyses, underwater photographs of marked colonies were used to estimate the surface area of D. fulgens colonies, calculate monthly growth rates, and document fusion and fission events. Clear seasonal patterns in reproduction and growth were observed, with distinct periods of investment into each function. Gonad maturation started in winter and larval release occurred in early summer, just before maximal sea temperatures were reached. After reproducing, colonies shrank and aestivated during the warmer summer months. Growth occurred during the cooler months, with maximal and minimal growth rates observed in winter and summer, respectively. Fusions and fissions occurred year‐round, although fissions were more frequent in fall (coincident with high growth rates) and fusions in spring (coincident with reproduction). These results add to the mounting evidence that ascidian life cycles in temperate seas are characterized by a trade‐off between investment in reproduction and growth, triggered by seasonal temperature shifts and constrained by resource availability during summer.     

Increased inter-colony fusion rates are associated with reduced COI haplotype diversity in an invasive colonial ascidian Didemnum vexillum

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species.     

Studies on Japanese botryllid ascidians. IV. A new species of the genus Botryllus with a unique colony shape, from the vicinity of Shimoda

The morphology and life history of a strange and unidentified botryllid ascidian were investigated. This ascidian was first collected from the stony shore of Ebisu Island in Shimoda, a city on Izu peninsula in central Japan. Unlike other botryllid ascidians, whose colonies are flat and smooth, this ascidian's colonies are rugged. In each colony, zooids are arranged into several oval systems, each of which has a thick part containing zooids and very thin parts that do not. The arrangement of ovary and testis in this species is the same as in other species of the genus Botryllus; the ovary is situated anterior to the testis. The embryo of this ascidian develops in the peribranchial cavity of its mother zooid without any brooding organs, as is the case with Botryllus scalaris and Botryllus puniceus. Meanwhile, the results of cut colony assay experiments did not show the existence of colony specificity in this ascidian. Even when two syngeneic colonies were brought into contact at their growing edges, none fused together. On the other hand, when two colonies were brought into contact with each other at their cut surfaces, they always fused into a single colony, regardless of their origin. Therefore, this species may be the only species that lacks colony specificity among the botryllids studied so far.     

Allogeneic interactions in Hydractinia: is the transitory chimera beneficial?

The colonial marine hydroid, Hydractinia, exhibits four possible outcomes to allogeneic contacts: passive rejection, aggressive rejection, stable fusion and transitory fusion. In the special case of transitory fusion, Hydractinia colonies undergo tissue fusion, followed by tissue death at the original contact area, and colony separation. This type of rejection is different in several aspects from the rejection process that accompanies incompatible encounters. It has been suggested that in transitory fusion, the colonies gain immediate benefits from fusion, mainly due to size increase, without succumbing to costs associated with fusion (germ line parasitism). We report a long-term observation of repeated fusion and separation cycles in clones featuring transitory fusion that revealed a slow-down of specific growth rates following fusion, and recovery in growth rates following separation. Very rapid transfer of stained material between partners in transitory chimeras provides suggestive evidence that protection against germ line parasitism is far from being guaranteed by separation. Our data cast doubt as to whether the benefits considered for transitory fusion are sustainable and support the already made suggestion that fusion with self, rather than fusion with kin, has been the major selective force governing the evolution of allorecognition in colonial invertebrates.