Population Structure and Small Scale Thermal Tolerance of a Non-native Colonial Ascidian

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Marian Litvaitis UNH - Department of Natural Resources & the Environment Principal Investigator
Jennifer Dijkstra Wells National Estuarine Research Reserve Co-Principal Investigator

Students Involved:

Andrea Frey UNH - Department of Natural Resources & the Environment
Jessica Brandt UNH - Department of Biological Sciences
Nikita Wong UNH - Department of Biological Sciences
2012 Accomplishments

Evaluation of Population Structure and Thermal Tolerance of Non-native Tunicates Helps Forecast Changes in Their Abundance

Many non-native tunicate species are increasing in numbers along coasts due in part to warming ocean waters and their ability to tolerate a broad range of temperatures. Within invaded habitats, they can alter natural marine community dynamics and can negatively impact aquaculture. In 2012, using NHSG development funds, researchers studied the population structure and thermal tolerance in two non-native colonial tunicates: Botrylloides violaceus and B. schlosseri. They determined that B. violaceus, which is a more recent invader, exhibits slower growth and is more sensitive to changes in temperature than more established species like B. schlosseri, although temperature affects reproduction in both species. The results from this project will help marine park managers, researchers and the aquaculture industry to forecast the effects of predicted changes in climate on the abundance of coastal nuisance species.

Model Predicts Greater Numbers of Non-native Tunicates in Gulf of Maine Waters in Future
There is increasing interest in expanding aquaculture efforts for shellfish and seaweed species in the Gulf of Maine coastal waters. However, invasive tunicates often grow on aquaculture infrastructure and cultured species, causing crop loss and increased maintenance and processing costs. Populations of many non-native tunicates in the Gulf of Maine appear to be increasing, a trend that may be caused in part by warming ocean waters. In 2012, using NHSG development funds, researchers created a model of the predicted effects of warmer seawater temperatures on the populations of two non-native colonial tunicates in the Gulf of Maine — Botrylloides violaceus and B. schlosseri. The model results indicate that warming waters will result in greater numbers of both of these species, particularly in northern Gulf of Maine coastal waters. These findings will allow aquaculturists and resource managers to predict the spread of these tunicates for potential mitigation of their economic and ecological impacts.

Project overview
Non-native tunicates, sea-squirts or ascidians, are cosmopolitan invaders. Within their invaded habitats, they can alter natural marine community dynamics by out-competing native species for food and space. Tunicates also threaten aquaculture activities by increasing the weight of cultivation gear, causing work to be more demanding and thereby raising operating costs for shellfish producers and processors. Many non-native tunicates are increasing in numbers along coasts due in part to the warming of the oceans and the ability of these species to tolerate a broad thermal range. While some studies have related physiology to species distributions,  few have coupled physiological with biological processes to evaluate individual species response to climate warming. We empirically examined the influence of a range of temperatures on metabolic rate, growth and reproduction of two species of colonial ascidians, Botryllus schlosseri and Botrylloides violaceus collected from several sites along the coast of the Southwestern Gulf of Maine that experience varying thermal regimes. Understanding species’ physiological and biological response to climate warming is critical to evaluate the mechanisms that underlie their theoretical success. 


Project findings

Genetic structure of individual colonies of Diplosoma listerianum was high within and between sites while genetic structure of Botrylloides violaceus was similar in colonies collected within and between sites, suggesting an overall low genetic diversity. This is consistent with other studies on invasive species that show non-native species experience a genetic bottleneck in their non-native environment. Our controlled laboratory studies revealed that Botrylloides violaceus has slower growth and is more sensitive to changes in temperature than the more established ascidian species, Botryllus schlosseri. In addition, temperature affects reproduction in both Botryllid species.
Tunicates threaten marine biodiversity and aquaculture activities. We anticipate that results from this project will be used by marine park managers, researchers and the aquaculture industry to forecast the effects of predicted changes in climate on coastal nuisance species abundance.



Relating species distribution and their relative abundance patterns has been a central goal of marine ecology and ecological physiology (Valentine 1966, Somero 2005). Recently, the introduction of non-native species has highlighted the need to predict species distribution patterns as well as their success in the recipient communities. Invasive species often inhabit habitats that span considerable geographic distances (Harris & Tyrrell 2001, Grosholz 2002). However, their performance within the invaded habitats will depend on several factors including the physiological response of the invader to local environmental parameters (Somero 2005, Portner et al. 2007, Dijkstra et al. 2008, Dijkstra et al. 2009).
Habitat temperature is an important environmental factor for ectothermal organisms as it controls critical biological processes such as growth and reproduction (Brunetti et al. 1980, Minchin 1992, Westerman et al. in press). Recently, a number of studies linking thermal tolerance to species distribution have been published (Stillman & Somero 1996, 2000, Helmuth et al. 2002, Stenseng et al. 2005). However, most of these studies were performed on mobile invertebrates living in the intertidal zone (e.g., crabs, mussels, snails) and not on sessile animals nor in the Gulf of Maine (GOM; e.g., Stillman & Somero 1996, Stenseng et al. 2005). Few studies have examined physiological responses of invertebrates, specifically non-native tunicates (but see Dijkstra et al. 2008) in the Gulf of Maine, a region of significant temperature changes.
Non-native colonial tunicates (ascidians or sea-squirts) are among the latest arrivals in the Gulf of Maine. They have lecithotrophic larvae that spend less than 24 hours in the water column before settling on suitable substrate and metamorphosing into adult colonies, allowing them to build up local populations. Restricted larval development periods that enable a build up of local populations may enable tunicates to respond to selective environmental pressures and permit significant population differentiation over relatively small spatial scales. One study using Botrylloides violaceus linked mortality of transplanted colonies from ~100km to an anomalous upwelling event based on NOAA weather buoy data (Grosholz 2001). A recent study relating physiological responses of botryllids to various salinities and temperatures, suggests local adaptation of non-native tunicates is possible (Dijkstra et al., in prep.). However, thermal tolerance at small spatial scales has not been rigorously tested or been tested on other non-native ascidian species.
The proposed study will focus on establishing population structure and thermal tolerance of the compound ascidian Diplosoma listerianum in the Gulf of Maine. D. listerianum is endemic to Europe, but has a wide global distribution, occurring in the Caribban (Rocha et al. 2005), Pacific (Carman et al., in prep.), southern Brazil (Rocha et al. 2009), Gulf of Maine (Harris et al. 1998), South of Cape Cod (Osman & Whitlatch 1995), Mediterranean (Brunetti et al. 1988). In the Gulf of Maine, it was first observed in 1993 at the Isles of Shoals (Harris et al. 1998) and has since spread north to Portland, ME and south to Cape Cod (Dijkstra et al. 2007). At the Isles of Shoals, D. listerianum is very abundant and colonies appear healthy throughout the year (L. Harris, pers. comm.). In contrast, colonies of D. listerianum experience seasonal die-back at coastal sites (Dijkstra et al., unpub. data). Seasonal temperature regimes are larger at coastal sites than oceanic sites (Isles of Shoals). The more moderate seasonal temperature regime at the Isles of Shoals coupled with the length of time since establishment (1993) and short larval period may allow this species to respond to thermal selective pressures and result in local adaptation.
Objective of Research
Funds obtained through this grant will be used to investigate population structure and thermal tolerance in the non-native colonial tunicate, Diplosoma listerianum. We will collect 10 colonies of Diplosoma listerianum at each of 4 sites in the GOM (Salem, Mass., Isles of Shoals, Maine, Woods Hole, Mass. and Portsmouth Harbor, N.H.). Salem and Portsmouth Harbor are coastal sites that exhibit extreme ranges in temperature, while the Isles of Shoals is an offshore site with more moderate temperature ranges. D. listerianum is a compound ascidian belonging to the family Didemnidae. Individual colonies (10 samples/site) will be collected from 4 sites and stored in 95% ethanol. To evaluate within species population structure of Diplosoma listerianum in the Gulf of Maine, we will develop molecular markers for D. listerianum that will allow us to distinguish among populations of D. listerianum. We will use the cytochrome oxidase subunit I gene (CO-I) to distinguish among populations of D. listerianum in the Gulf of Maine. Stach and Turbeville (2002) suggest that CO-I evolves too rapidly in tunicates to be useful for the delineation of species boundaries. However, because of its accelerated rate of evolution, CO-I may be useful for intra-specific comparisons, especially in assessing conspecificity of D. listerianum from different locations.
Samples will be stored in 95% ethanol until DNA extraction. Genomic DNA will be extracted and about 650 base pairs of the CO-I gene will be amplified via PCR using available universal primers (Folmer et al. 1994). Gel-purified amplifications will be used as templates in cycle sequencing reactions and sequenced either at the Hubbard Genomics Center at UNH or sent to a commercial lab (Geneway, Hayward, Calif.), dependent on schedule availability at the HGC. For each sample, both strands will be sequenced. Sequences edited in FinchTV (Geospizia) will be aligned using CLUSTALX (Thompson et al. 1997). A Minimum Spanning Tree and Network will be calculated using Arlequin vers. 3.0 (Excoffier et al. 2005).
To examine thermal tolerance of this species, we will employ basic physiological measurements (heart function and growth). We will culture colonies of D. listerianum at four different temperatures (5 °C, 10 °C, 15 °C and 20 °C) for two weeks and record growth and heart rates. We have successfully used heart function and growth to assess osmotic and thermal tolerance of botryllid ascidians (Dijkstra et al. 2008, Dijkstra et al. 2009). These studies, along with our preliminary studies on heart function in D. listerianum, suggest it is a good indicator of thermal tolerance.


Relevance of Research

The establishments of non-native tunicates in the Gulf of Maine have resulted in substantial changes in species composition (Harris & Tyrrell 2001, Dijkstra 2007, Valentine et al. 2007) and changes in critical ecosystem processes e.g., maintenance of species diversity (Dijkstra & Harris in press). They have also been identified as a costly pest for aquaculture. Presently, our current knowledge of the basic biology and genetics of tunicates is insufficient to predict their distribution or determine population and spatial variability in their response to environmental variables e.g., temperature.
Understanding species response to environmental factors will enable coastal managers (e.g., friends of Casco Bay, Maine marine invasive species group) to forecast the distribution and success of Diplosoma listerianum at regional and local scales. Therefore, an early warning detection system can be set-up at sites that appear vulnerable to invasion. The early detection of an invading species can be applied immediately in establishing a more intensive program to detect any spread and determine what measures can be used to control spread. In addition, a graduate student who is examining population structure of another invasive tunicate species, Botrylloides violaceus, will be involved in this study. Specifically, she will carry out the genetic analysis and help with laboratory studies.
The information collected will be available through publication to a broad cross-section of stakeholders including regional marine invasive species groups e.g., Northeast Aquatic Invasive Species. These results will also serve as preliminary data to develop a full proposal for submission through N.H. Sea Grant and NSF to study physiological variation and population structure of D. listerianum across large spatial scales. In 2002, I submitted a Sea Grant Development proposal, and data obtained through it, allowed me to successfully obtain funding from NSF at $590,000. Hence, obtaining preliminary data through a Sea Grant Development fund is a crucial first step for successful competitions at funding agencies.  

Literature Cited

Brunetti R, Beghi L, Bressan M, Marin MG (1980) Combined effects of temperature and salinity on colonies of Botryllus schlosseri and Botrylloides leachi (Ascidiacea) from the Venetian Lagoon. Marine Ecology-Progress Series 2:303-314
Brunetti R, Bressan M, Marin MG, Libralato M (1988) On the ecology and biology of Diplosoma listerianum (Milne Edwards, 1841) (Ascidiacea, Didemnidae). . Vie et Milieu 38:123-131
Dijkstra J (2007) Climate change and invasive species interact to impact succession and diversity in Gulf of Maine marine fouling communities. Dissertation, University of New Hampshire
Dijkstra J, Dutton A, Westerman E, Harris L (2008) Heart rates reflect osmotic stress in two introduced colonial ascidians: Botryllus schlosseri and Botrylloides violaceus. Marine Biology 154:805-811
Dijkstra J, Harris LG, Westerman E (2007) The distribution and long-term temporal patterns of four invasive colonial ascidians in the Gulf of Maine. J Exp Mar Biol Ecol 342:61-68
Dijkstra JA, Harris LG (in press) Maintenance of diversity altered by a shift in dominant species: Implications for species coexistence. Marine Ecology Progress Series
Dijkstra JA, Westerman EL, Brooks C, Harris LG (2009) Linking physiology, biogeography and success of non-natives in subtidal communities in the Gulf of Maine Abstract: Sixth Conference on Marine Bioinvasions
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294-299
Grosholz ED (2001) Small spatial-scale differentiation among populations of an introduced colonial invertebrate. Oecologia 129:58-64
Grosholz ED (2002) Ecological consequences of coastal invasions. Trends In Ecology & Evolution 17:22-27
Harris LG, Tyrrell M, Chester CM (1998) Changing patterns for two sea stars in the Gulf of Maine, 1976-1996. In: Mooi R (ed) Proceedings of the ninth international echinoderm conference, San Francisco California, p 243-248
Harris LG, Tyrrell MC (2001) Changing community states in the gulf of maine: synergisms between invaders, overfishing and climate change. Biological Invasions 3:9-21
Helmuth B, Harley CDG, Halpin PM, O'Donnell M, Hofmann GE, Blanchette CA (2002) Climate change and latitudinal patterns of Intertidal thermal stress. Science 298:1015-1017
Minchin D (1992) Multiple species, mass spawning events in an Irish sea lough: The effects of temperatures on spawning and recruitment of invertebrates. Inv Repr Dev 22:229-238
Osman RW, Whitlatch RB (1995) Predation on early ontogenic life stages and its effect on recruitment into a marine epifaunal community. Marine Ecology-Progress Series 117:111-126
Portner HO, Peck L, Somero G (2007) Thermal limits and adaptation in marine Antarctic ectotherms: an integrative view. Philosophical Transactions of the Royal Society B-Biological Sciences 362:2233-2258
Rocha RM, Faria SB, Moreno TR (2005) Ascidians from Bocas del Toro, Panama. I. Biodiversity. Caribbean Journal of Science 41:600-612
Rocha RM, Kremer LP, Baptista MS, Metri R (2009) Bivalve cultures provide habitat for exotic tunicates in southern Brazil. Aquatic Invasions 4:195-205
Somero G (2005) Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Frontiers in Zoology 2:(http://www.frontiersinzoology.com/content/2-1/1
Stach T, Turbeville J (2002) Phylogeny of Tunicata inferred from molecular and morphological characters. Mol Phyl Evol 25:408-428
Stenseng E, Braby CE, Somero GN (2005) Evolutionary and acclimation-induced variation in the thermal limits of heart function in congeneric marine snails (genus Tegula): Implications for vertical zonation. Biological Bulletin 208:138-144
Stillman JH, Somero GN (1996) Adaptation to temperature stress and aerial exposure in congeneric species of intertidal porcelain crabs (genus Petrolisthes): Correlation of physiology, biochemistry and morphology with vertical distribution. Journal of Experimental Biology 199:1845-1855
Stillman JH, Somero GN (2000) A comparative analysis of the upper thermal tolerance limits of eastern Pacific porcelain crabs, genus Petrolisthes: Influences of latitude, vertical zonation, acclimation and phylogeny. Physiology and Biochemistry Zoologist 73:200-208
Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876-4882
Valentine JW (1966) Numerical analysis of marine molluscan ranges on extratropical northeastern pacific shelf. Limnol Oceanogr 11:198-211
Valentine PC, Carman MR, Blackwood DS, Heffron ER (2007) Ecological observations on the colonial ascidian Didemnum sp. in a New England tide pool habitat. J Exp Mar Biol Ecol 132:109-121
Westerman EL, Whitlatch RB, Dijkstra JA, Harris LG (in press) Variation in brooding period masks similarities in response to changing temperatures. Marine Ecology Progress Series