Aspects of the Growth and Reproductive Ecology of the Introduced Ascidian Didemnum Perlucidum (Monniot, 1983) in Western Australia

Aquatic Invasions (2015) Volume 10, Issue 3: 265–274 doi: http://dx.doi.org/10.3391/ai.2015.10.3.02 Open Access

Research Article

Aspects of the growth and reproductive ecology of the introduced ascidian Didemnum perlucidum (Monniot, 1983) in Western Australia

1 2 1 1 Julieta Muñoz *, Mike Page , Justin I. McDonald and Samantha D. Bridgwood 1Marine Biosecurity Research and Monitoring Group, Department of Fisheries Western Australia, Government of Western Australia, PO Box 20, North Beach, Western Australia 6920, Australia 2National Institute of Water and Atmospheric Research Ltd, P.O. Box 893, Nelson 7010, New Zealand E-mail: [email protected] (JM), [email protected] (MP), [email protected] (JIM), [email protected] (SDB) *Corresponding author

Received: 12 June 2014 / Accepted: 13 March 2015 / Published online: 20 April 2015 Handling editor: Elizabeth Cook

Abstract

In 2010, the colonial ascidian Didemnum perlucidum (Monniot, 1983) was first recorded in Western Australia and is now widespread across the state. This species is classified as a target biosecurity species by the Department of Fisheries, Government of Western Australia. However, limited information on the biology of this ascidian is available for Australian waters. The aim of this study was to gather baseline information on the temporal variation in growth, survival, and reproduction of D. perlucidum. The mean average colony area was significantly higher in summer (29.7 cm2 ) decreasing towards winter (3.3 cm2 ). Colonies produced larvae year round with maximum production (1109.0 larvae cm-2) and highest recruitment (0.90 settlers cm-2) in summer. Larva density was positively correlated to salinity (r = 0.965) and chlorophyll a concentration (r = 0.958) in the water column. From a management point of view, the decrease in colony size, larvae production, and recruitment of D. perlucidum during winter could offer the best opportunity for the control or eradication of this ascidian at specific locations in the state. Key words: sea squirt, invasive, reproduction, marine pest, growth, temporal patterns

Introduction facilities has been linked to a reduction in growth and increased mortality of cultivated shellfish Ascidians are a key ecological group because of (Rocha et al. 2009) and finfish (Tan et al. 2002). their ability to thrive in eutrophic environments, Despite several recorded negative impacts, the high capacity to colonise and overgrow natural costs associated with ascidian biofouling have and artificial substrates (such as floating pontoons, yet to be fully quantified. pilings, buoys, and vessel hulls) in protected Ascidians are sessile marine filter feeders with harbours, and high invasive potential (Shenkar a short-lived, non-feeding, motile larva (Berrill and Swalla 2011). Introductions of non-native 1951; Millar 1971). Most ascidians are hermaphro- colonial ascidians to natural and artificial substrates dites, live as solitary individuals or form colonies, in tropical and temperate environments are now and often display complex life history cycles. commonplace (Minchin and Sides 2006; Valentine These cycles include phases of sexual and asexual et al. 2007; Lambert 2009; Lengyel et al. 2009). reproduction and growth phenomena such as Once introduced, ascidians have the potential to fission, fusion, and re-juvenescence as well as negatively affect the biodiversity and ecological periods of recession and senescence (Turon and function of the recipient location (Dijkstra et al. Becerro 1992). Reproduction and growth are affected 2007; Lengyel et al. 2009; Morris and Carman 2012) by environmental factors (e.g. temperature and and may cause negative economic impacts. For food availability); for example, populations from example, heavy ascidian fouling in aquaculture temperate locations often show large variation

265 J. Muñoz et al. between summer and winter months (Ribes et al. winter of ~1 to 25 cm2 (Bridgwood et al. 2014). 1998). Some colonial ascidians concentrate growth The aim of the present study was to gather and reproductive activity to the winter/autumn baseline information on the temporal variation in seasons (Turon 1988; Turon and Becerro 1992; colony growth, survival, and reproductive Dijkstra et al. 2007; Ritzmann et al. 2009) while characteristics of Didemnum perlucidum in others, such as Didemnum vexillum Kott, 2002 Western Australian waters. (Fletcher et al. 2013) and Didemnum perlucidum Monniot, 1983 (Kremer et al. 2010; Smale and Methodology Childs 2011), show highest levels of reproduction and growth during summer months. It remains Study site unclear, however, whether these seasonal patterns This research was carried out within the are consistent among different geographic locations boundaries of the Hillarys Boat Harbour, Western and species. Australia (31°49′30.70″S, 115°44′07.71″E). An Didemnum perlucidum is a tropical colonial area of 90 m2 was selected and monitored over a ascidian (Didemnidae) that has been introduced period of one year from June 2012 to June 2013 to many locations worldwide (Culbertson and (Figure 1). Harper 2000; Coles et al. 2002; Golbuu et al. 2005; Kremer 2008; Kremer et al. 2010). In Australia, Water quality despite extensive surveys of ascidian fauna being undertaken around the country by Kott (1985, Water quality was measured monthly from June 1990 a, b, 1992 a, b, 1997, 2001, 2005) and by 2012 to June 2013 at a depth of 50 cm (n=39). McDonald (2005 a, b), this species had not been Water temperature, salinity, pH, and dissolved reported in Australian waters. In 2010, D. perlu- oxygen were measured with a YSI® multiparameter cidum was recorded for the first time in Australia sensor (YSI Pro Plus®, Perth Scientific, Perth, from the Swan River Estuary, Perth, Western Australia) while water transparency was measured Australia (WA) (Smale and Childs 2011). Later with a Secchi disc of 20 cm diameter. in 2011, an unidentified “sponge look alike” Chlorophyll a and pheophytin a analyses were organism was recorded heavily fouling a mussel performed on filtered seawater samples (5 L) and farm in the Cockburn Sound in WA and analysed with standard spectrophotometric methods subsequently identified as D. perlucidum. (Strickland and Parsons 1972) at the Marine and The presence of D. perlucidum in WA is Freshwater Research Laboratories (MAFRL), regarded as a significant threat to the shellfish Murdoch University, Western Australia. Chlorophyll aquaculture industry because of its ability to a and pheophytin a concentration was then used quickly smother mussels and oysters in aquaculture as an indirect measurement of food availability facilities as well as in natural habitats (Lambert (plankton) in the water column. 2009; Kremer 2010, 2011). Local anecdotal information suggests this ascidian can increase Specimen collection, preservation and identification the mortality of farmed mussels and oysters possibly by outcompeting native fouling populations Four different types of samples were used in this (Glenn Dibbin pers. comm.). study: 1) marked colonies for determination of Despite multiple records of D. perlucidum colony size, 2) samples collected for the introductions worldwide, most of the information reattachment experiment, 3) samples collected for gathered has related to its distribution, taxonomy, reproductive analysis and colony thickness and morphological description (Monniot 1983; determination, and 4) new colonies settled on Monniot et al. 1991; Dias et al. 2008; Kremer settlement plates. In all the cases, the identity of 2010, 2011; Smale and Childs 2011; Bridgwood the samples suspected of being D. perlucidum et al. 2014) while few studies have addressed its was confirmed using molecular methods. This biology and ecology. In Western Australia, D. entailed collecting a subsample (~1 cm) perlucidum is well established in marinas such a preserved in absolute ethanol. All samples were Hillarys Boat Harbour, where it is often found barcoded by amplifying the cytochrome oxidase colonising floating pontoons and pylons from the subunit I (COI) mitochondrial region using the water line to a depth of 4 m. Preliminary COI primers following the methods developed observations have shown that in summer, colonies by Stefaniak et al. (2009). The criteria for positive formed extensive mats covering areas of ~100 to molecular identification was established when 900 cm2, contracting to a much smaller size in sequences had a 99–100% match with a sequence

266 Growth and reproductive ecology of Didemnum perlucidum in Western Australia

Figure 1. Map showing the relative location of Hillarys Boat Harbour, Western Australia. The study site within the harbour is indicated by a red rectangle, the location of settlement arrays is indicated with orange dots.

previously identified as D. perlucidum (Genbank The thickness was measured to determine if accession JQ731735, Swan River, WA, identified this variable could be an indicator of growth for by Gretchen Lambert, University of Washington). a colony. As colonies used for growth were permanently attached to the substrate, this Colony area variable was measured on colonies collected for reproductive analysis (Refer to Asexual and sexual To evaluate changes in the area of D. perlucidum, reproduction section below). Colony thickness colonies were permanently marked (n = 780) and was measured by taking three replicate observed over one year (June 2012–June 2013). measurements from the centre of the colony with Each marked colony was photographed on a a digital calliper straight after collection and monthly basis within a 10 cm × 10 cm quadrat. prior to preservation. Selected photographs were standardised to a 700 × 700 pixel size, calibrated using a linear scale Fragment reattachment of 10 cm and analysed using the software ImageJ® (National Institute of Health, USA). The A field experiment was undertaken to determine area of each D. perlucidum colony was calculated if D. perlucidum fragments could survive and by using the freehand measuring tool to trace the reattach to another substratum after becoming perimeter of the colony. The total area occupied detached from the parent colony. To test this, by D. perlucidum per quadrat was the sum of all fragments (approx. 3 cm length) from ten colony areas measured in a photograph. colonies were placed into individual cages and

267 J. Muñoz et al. suspended vertically 20 cm above the seabed by Larval density and recruitment a sub-surface float. The cages were constructed from plastic “gutter guard” mesh made of high To determine whether larvae were settling and density polyethylene plastic with a 5 mm mesh developing into new colonies, a settlement array size. After ten days, the cages were retrieved and was deployed at 1 m depth at two locations in the transferred to the laboratory. The total number of study area (Figure 1). Each array consisted of an attached and unattached fragments was quantified aluminium frame with 4 arms each with 4 poly- by observing whether the colony fragments moved vinyl chloride plates (10 cm × 10 cm) resulting or floated from the cage when washed with in a total of 16 plates per array. Each season seawater. If the fragment moved or floated it was (winter, spring, summer and autumn) the arrays considered unattached. This experiment was were soaked for one month and then retrieved to repeated on a seasonal basis (autumn, winter, collect the fouled plates. Collected plates were spring, summer; n = 40). kept cool in seawater and transported to the laboratory where they were observed under a dissecting microscope to determine the presence Asexual and sexual reproduction of newly settled colonies suspected of being D. perlucidum. These colonies were removed and Samples of D. perlucidum colonies were collected processed for molecular identification as previously seasonally and dissected to determine: 1) the described. abundance of eggs and larvae; 2) the presence of testes, ova and vegetative buds in the zooids; and 3) the maturity index of the colony. The latter was Statistical analysis used as an indicative measure of reproductive Data was tested for normality and homogeneity output and recruitment as suggested by Fletcher of variances to determine the most suitable et al. (2013). analyses. SPSS V16 (IBM, USA) was used to Fifteen D. perlucidum colonies were collected perform the following analyses. Colony area was each season for one year: winter (June 2012– analysed using repeated measures ANOVA at 0.05 August 2012), spring (September 2012–November level to determine differences in average colony 2012), summer (December 2012–February 2013) area across months. The Greenhouse-Geisser and autumn (March 2013–May 2013). After sphericity correction test was used to test for collection, a subsample of each colony was relaxed violations in sphericity in the data prior to running in seawater with menthol crystals then preserved the repeated measures ANOVA. Difference in in 4% formalin-seawater for morphological colony thickness between months was tested using 2 analysis. A 0.25 mm section was removed from a one way ANOVA. Tukey HSD a posteriori test each colony sample and the total number of eggs was subsequently performed to determine specific and larvae counted. Additionally 10 zooids from differences in colony size and thickness between each of the 15 colonies (150 zooids per season; n months. = 600) were dissected to quantify the presence or The effect of seasons on larval densities in the absence of visible ova and testes (sexual colonies and on the number of new colonies on reproduction) and vegetative buds (asexual the settlement plates was determined using two reproduction) in each zooid. independent ANOVA for each variable. Specific The maturity index (MI) of the colonies was effects were tested using Tukey HDS a posteriori calculated for each season using the equation of test at the 0.05 level. Fletcher et al. (2013): To test the effect of season on the presence of eggs, testes, and vegetative buds a MANOVA MI = ΣnF/N (Pillai’s trace) analysis was conducted. Specific effects on each were tested using Tukey HDS a Where n is the number of colonies in stage F, F posteriori test at the 0.05 level. The actual P is the reproductive stage score (1 to 5), and N is values obtained for all the tests are presented in the total number of colonies examined. Each the results section. colony was scored as follows: 1) absence of visible A Pearson’s correlation was used to determine gonads; 2) presence of eggs in the abdomens; 3) if any correlation existed between water quality presence of coiled testes; 4) presence of eggs in variables (temperature, salinity, pH, transparency, the matrix; and 5) presence of mature larvae dissolved oxygen, chlorophyll a and pheophytin (visible tail and ocelus) in matrix. a) and colony size and larvae density in the colony.

268 Growth and reproductive ecology of Didemnum perlucidum in Western Australia

Figure 2. Water quality recorded at the sampling site over a period of one year (June 2012 to June 2013, mean ± SE; n=39).

Results

Water quality Fragment reattachment The water quality in the study area varied across D. perlucidum fragments were able to survive months (Figure 2). Mean water temperature ranged when detached from the parent colonies. In from 17 °C in August to 24.3 °C in January/ autumn, spring, and summer, 100% of the colonies February, salinity ranged from 33.4 in August to were able to survive and attach to the plastic 36.4 in March, dissolved oxygen ranged from 4.5 mesh of the cages while in winter 90% of the mg L-1 in February to 6.9 mg L-1 in June while colonies survived and reattached. pH ranged from 7.2 to 8.18. Chlorophyll a (Chl a) ranged from 0.09 to 2.4 µg L-1 while phaeophytin Asexual and sexual reproduction a ranged from 0.1 to 1.13 µg L-1 and water transparency ranged from 2.9 to 5 m. D. perlucidum reproduced sexually as indicated by the presence of ova and testes, and asexually via vegetative buds throughout the year. Results Colony area from the MANOVA analysis demonstrated a The area of D. perlucidum colonies varied significant (P = 0.001) effect of season on these significantly (P = 0.001) between months (Figure variables. 3). During colder months (Jul-Sep, ~17 oC), Dissected zooids contained ova, testes, and colonies underwent retraction and fragmentation vegetative buds arising from the oesophageal region and were significantly smaller than the colonies of the zooid. The density of ova, testes and buds observed in the warmer months (Jan-Feb, ~24 oC). varied seasonally. Zooids had a significantly greater In September 2012, the area covered by the number of testes in summer (7.5 testes zooid-1) colonies was the smallest measured (3.2 cm2) compared to winter (3.3 testes zooid-1; P = 0.003) increasing to an average area of 29.7 cm2 by and the largest number of ova (5.2 ova zooid-1; P March 2013. = 0.002) and vegetative buds (2.5 buds zooid-1; P Colony thickness varied across months (Figure = 0.011) in autumn (Figure 4). 3). Colonies were thinnest (1.2 mm) in November The maturity index (MI) of the colonies becoming significantly (P = 0.021) thicker towards observed in spring, summer and autumn was 1.0 the summer months in January (1.7 mm). with a slight decrease to 0.93 in winter.

269 J. Muñoz et al.

Figure 3. Mean colony size (n=780) and thickness (n=60) of Didemnum perlucidum over a period of one year (June 2012 to June 2013) at Hillarys Boat Harbour, Western Australia; bars indicate the SE of the mean.

Figure 4. Abundance of ova, testes and vegetative buds in Didemnum perlucidum zooids over a period of four seasons (2012–2013) (n=600; mean ± SE). Different letters indicate significant differences between seasons.

Figure 5. Larval development of Didemnum perlucidum. A. Cross section showing matrix with eggs and larvae; B. Morulla stage, C. Gastrula stage, D. Formation of tail with well-defined miomers. E-F. Fully developed larva. Photomicrographs by Department of Fisheries WA.

270 Growth and reproductive ecology of Didemnum perlucidum in Western Australia

Table 1. Temporal variation in larval density in Didemnum perlucidum colonies and density of settlers on settlement plates. Different letters indicate significant differences between seasons at 0.05 level.

Seasonal mean (# per cm-2) ANOVA

Winter Spring Summer Autumn df MSS F P Larvae in colony (n=60) 300a 460a 1109.0c 953.3d 3,52 201.0 3.11 0.034 Settlers on plates (n=64) 0.00a 0.00a 0.90b 0.10c 3,45 184.3 65.2 0.001

Table 2. Pearson’s correlation coefficient (r) between water autumn and then decreased towards winter. This quality parameters, colony size and larval density, * denotes a pattern is typical of many ascidians (Becerro and correlation significant at 0.05 level. Turon 1992) including D. perlucidum from South Larval density Colony size America (Kremer et al. 2010). While this study reports on the temporal variation of D. perlucidum Temperature 0.938 0.889 at a single location, these results likely are Salinity 0.965* 0.978* representative observations of other temperate pH -0.052 0.114 populations in the state. Dissolved Oxygen -0.222 -0.104 Water temperature is one of the main factors Transparency -0.842 -0.724 correlated to growth and reproduction of ascidians Chlorophyll a 0.958* 0.892 (Hirose et al. 2007). We observed a peak in growth Pheophytin a 0.766 0.689 and reproduction in the warmer seasons of summer and autumn. It was also during this time that we measured the highest concentrations of Larval density and recruitment Chl a, which was positively correlated to higher The dissection of the colonies revealed the larval densities. In temperate environments, food presence of different stages of eggs and larvae in availability is highest during the summer due to the same colony, from eggs at morulla and an increase in phytoplankton growth, increased gastrula stages to unhatched and fully developed temperature and higher irradiance (Coma et al. larvae (Figure 5). 2000; Petersen 2006). The sex allocation theory There was a significant (P = 0.03) difference suggests that sexual reproduction is associated in density of larvae in the colonies between seasons, with periods of higher food availability and water increasing from winter (300 larvae cm-2) to a temperature (Newlon et al. 2003). For example, maximum density in summer (1109 larvae cm-2) it has been observed that feeding levels affect the (Table 1). Larval density was positively correlated reproductive efforts of the colonial ascidian to salinity and Chl a concentration in the seawater Botryllus schlosseri (Grosberg 1988). Lambert (Table 2). Colony size was also positively correlated (2005) also found that the reproductive season in to salinity (Table 2). ascidians usually coincides with the period when Recruitment was observed in summer and autumn phytoplankton productivity is highest. Similarly, when larvae settled on the arrays and developed in our study, D. perlucidum’s sexual and asexual into new colonies (settlers). In summer, up to reproduction peaked in summer when water 0.90 colonies cm-2 were observed after only a one temperature and Chl a concentration were highest. month soaking period. However, no recruitment The higher larval density and colony size also was observed in winter and spring (Table 1). coincided with the highest salinity at the study Initial weekly inspections on the plates revealed suite, which occurred in summer. that many colonies were present on the settlement Colonies decreased in size and fragmented during plates a few days after deployment but one month the cool winter season, which also coincided later, when the settlement arrays were retrieved with the period of lowest Chl a concentration; from the water, the density of new settlers had suggesting a reduction in food availability. Colony decreased. fragmentation caused by zooid regression and the subsequent decrease in colony size is common in Discussion ascidians, especially in didemnids (Ritzmann et al. 2009; Page et al. 2011). This is thought to be Didemnum perlucidum colonies from Hillarys a rejuvenation process that extends the lifespan Boat Harbour, WA, reached peak colony area, of the zooids, and the survival of the colony larval density, and recruitment in summer and (Turon and Becerro 1992). For D. perlucidum

271 J. Muñoz et al. colonies in WA, winter may represent a resting D. perlucidum fragments were shown to survive season for growth and reproduction when resources and capable of quickly reattaching and growing are scarce and energy budgets allow for only on a new artificial substrate. It is well known colony maintenance until the next active-growth that fragments of colonial ascidians are capable season. Salinity was also at lowest in winter and of reattachment and further that enlargement of a colonial ascidians are often negatively affected colony is hastened by asexual budding (Stoner by low salinity (Lambert 2005; Fletcher et al. 1989; Fletcher and Forrest 2011). This suggests that 2013). Salinity variation may affect reproduction D. perlucidum colony growth through vegetative in ascidians, such as recruitment (Fletcher et al. budding may be an important process in the 2013) and metamorphosis (Vázquez and Young colonization of new substrates, and explains the 2000), and may be also influence the reproductive presence of vegetative buds year round. The patterns of D. perlucidum. In our study, new capacity for survival and reattachment may also settlers were not observed on settlement plates in be influenced by environmental factors such as winter, consistent the low larval density in the water temperature. In our study we observed that colonies. It is possible that larval production or reattachment of D. perlucidum is slightly decreased survival during this period was so low that the in winter (90% reattachment). Carman et al. (2014) presence of new settlers was not detected on our found similar results for D. vexillum, where plates. Fletcher et al. (2013) found similar results fragment reattachment success declined with during for Didemnum vexillum, where low recruitment periods of low water temperature. was explained as lack of detection capability Knowledge of the temporal and spatial rather than absence of new settlers. patterns of growth and reproduction of marine As was observed for D. perlucidum in Brazil fouling organisms is of particular importance in (Kremer et al. 2010), larvae in different develop- formulating effective management strategies. mental stages were observed year round. The This paper provides baseline information on the presence of zooids with ova and testes year round biology and ecology of D. perlucidum in Western would explain the continuous presence of fully Australia. From a management perspective it is developed larvae in the colonies (indicated by possible that this species may be overlooked if the high MI). Continuous asexual reproduction also sampling occurs only in winter when colonies was observed. While larvae production favours undergo a significant reduction in colony size the formation of new colonies, vegetative budding and fragmentation. The reduction in colony size provides the means for colony growth and expansion and recruitment during the colder months also even when resources are being allocated to sexual indicates that winter could be the most appropriate reproduction. However, the D. perlucidum life cycle period to control this species if management is and the environmental factors affecting it remain being considered. However, additional information poorly understood require further investigation. on the biology of this species and other Our study showed that there were considerably management strategies such as control of source more zooids containing testes than ova. Similar population, larval dispersal, and ongoing human results were observed by Dias et al. (2008) who mediated introduction need to be considered to found that D. perlucidum zooids had fewer ova avoid reinfection events. when under the effect of competition. In hermaphroditic organisms, an emphasis on male reproduction is commonly observed in nutrient Acknowledgements depleted situations and when organisms are The authors would like to thank C. StRoas, Management Hillarys found in high densities; both leading to greater Boat Harbour for providing access to the sampling site and their competition for space and resources. This can be continuous support to this study. Thanks to S. Fotedar and J. Dias explained in terms of the greater energy investment for their help with the molecular analysis of the samples, to R. required for production of female gametes than Duggan, C. Wellington, and M. Hewitt for their invaluable support with the field component of this research; and to M. is required for the production of male gametes Hourston for his help with the analysis of the data. Special thanks (Newlon et al. 2003; Dias et al. 2008). Western to G. Muniz Dias for his helpful comments and to two anonymous Australian waters are known to be oligotrophic reviewers for their suggestions which greatly improved this in nature which can lead to competition for food manuscript. (Smale and Childs 2011). While competition for food may be affecting D. perlucidum colonies and favouring male gamete production, this question needs further work to confirm the mechanism.

272 Growth and reproductive ecology of Didemnum perlucidum in Western Australia

References Kott P (1990a) The Australian Ascidiacea Part 2, Aplouso- branchia (1). Memoirs of the Queensland Museum 29: 1–226 Becerro MA, Turon X (1992) Reproductive cycles of the Kott P (1990b) The Australian Ascidiacea, Phlebobranchia and ascidians Microcosmus sabatieri and Halocynthia papillosa Stolidobranchia, Supplement. Memoirs of the Queensland in the northwestern Mediterranean. Marine Ecology 13: 363– Museum 29: 267–298 383, http://dx.doi.org/10.1111/j.1439-0485.1992.tb00360.x Kott P (1992a) The Australian Ascidiacea Part 3, Aplousobran- Berrill NJ (1951) Regeneration and budding in tunicates. chia (2). Memoirs of the Queensland Museum 32: 375–620 Biological Reviews 26: 456–475, http://dx.doi.org/10.1111/ Kott P (1992b) The Australian Ascidiacea, Supplement 2. j.1469-185X.1951.tb01207.x Memoirs of the Queensland Museum 32: 621–655 Bridgwood S, Muñoz J, McDonald JI (2014) Catch me if you can! Kott P (1997) Tunicates (Sub-Phylum Tunicata) In: Shepherd SA, The story of a colonial ascidian’s takeover bid in Western Davies S (eds), Marine Invertebrates of Southern Australia, Australia. BioInvasions Records 4: 217–223, http://dx.doi.org/ Part III. South Australian Research and Development 10.3391/bir.2014.3.4.02 Institute, South Australia, pp 1092–1255 Carman MR, Grunden DW, Ewart D (2014) Coldwater Kott P (2001) The Australian Ascidiacea Part 4, Aplousobranchia reattachment of colonial tunicate Didemnum vexillum (3), Didemnidae. Memoirs of the Queensland Museum 47: 1– fragment to natural (eelgrass) and artificial (plastic) 407 substrates in New Egland. Aquatic Invasions 9: 105–110, Kott P (2005) New and little-known species of Didemnidae http://dx.doi.org/10.3391/ai.2014.9.1.09 (Ascidiacea, Tunicata) from Australia (Part 3). Journal of Coles SL, DeFelice RC, Eldredge LG (2002) Nonindigenous Natural History 39: 2409–2479, http://dx.doi.org/10.1080/002 marine species in Kane'ohe Bay, Oahu, Hawaii. Bishop 22930500087077 Museum technical report: http://hbs.bishopmuseum.org/pdf/ Kremer LP (2008) Potencial invasor de Didemnum perlucidum kbay-report.pdf (Tunicata, Ascidiacea) em um ambiente de cultivo de Coma R, Ribes M, Gili JM, Zabala M (2000) Seasonality in mexilhoes [Invasive potential of Didemnum perlucidum in an coastal benthic ecosystems. Tree 15: 448–453, http://dx.doi. oyster farm environment]. MSc Dissertation. Universidad org/10.1016/S0169-5347(00)01970-4 Federal do Paraná, Brazil Culbertson J, Harper D (2000) Settlement of a colonial ascidian Kremer LP, Rocha RM, Roper JJ (2010) An experimental test of on an artificial reef in the Gulf of Mexico. Minerals colonization ability in the potentially invasive Didemnum Management Service, Gulf of Mexico Fish and Fisheries, perlucidum (Tunicata, Ascidiacea). Biological Invasions 12: Bringing Together New and Recent Research. New Orleans, 1581–1590, http://dx.doi.org/10.1007/s10530-009-9571-8 Louisiana Kremer LP, Rocha RM (2011) The role of Didemnum perlucidum Dias G, Martin Delboni CG, Lembo Duarte LF (2008) Effects of F. Monniot, 1983 (Tunicata, Ascidiacea) in a marine fouling competition on sexual and clonal reproduction of a ascidian: community. Aquatic Invasions 6: 441–449, http://dx.doi.org/10. the importance of competitor identity. Marine Ecology 3391/ai.2011.6.4.09 Progress Series 362: 149–156, http://dx.doi.org/10.3354/meps Lambert G (2005) Ecology and natural history of the proto- 07447 chordates. Canadian Journal of Zoology 83: 34–50, Dijkstra J, Sherman H, Harris LG (2007) The role of colonial http://dx.doi.org/10.1139/z04-156 ascidians in altering biodiversity in marine fouling Lambert G (2009) Adventures of a sea squirt sleuth: unravelling communities. Journal of Experimental Marine Biology and the identity of Didemnum vexillum, a global ascidian invader. Ecology 342: 169–171, http://dx.doi.org/10.1016/j.jembe.2006. Aquatic Invasions 4: 5–28, http://dx.doi.org/10.3391/ai.2009.4.1.2 10.035 Lengyel NL, Collie JS, Valentine PC (2009) The invasive Fletcher LM, Forrest BM (2011) Inducing spawning and culture colonial ascidian Didemnum vexillum on Georges Bank – techniques for the invasive ascidian Didemnum vexillum ecological effects and genetic identification. Aquatic (Kott, 2002). Aquatic Invasions 6: 457–464, http://dx.doi.org/ Invasions 4: 143–152, http://dx.doi.org/10.3391/ai.2009.4.1.15 10.3391/ai.2011.6.4.11 McDonald JI (2005a) Solitary Ascidiacea from shallow waters of Fletcher LM, Forrest BM, Atalah J, Bell JJ (2013) Reproductive the Recherche Archipelago, Western Australia. In: Wells F, seasonality of the invasive ascidian Didemnum vexillum in Walker D, Kendrick G (eds), The Marine Flora and Fauna of New Zealand and implications for shellfish aquaculture. Esperance, Western Australia. Perth, Western Australia Aquaculture Environment Interactions 3: 197–211, Museum, pp 451–461 http://dx.doi.org/10.3354/aei00063 McDonald JI, Fromont J, Kendrick G (2005b) Sponge and Golbuu Y, Bauman A, Kuartei J, Victor S (2005) The state of ascidian communities of the Recherche Archipelago. Final coral reef ecosystem of Palau. In: Waddell J (ed), The state of Report to CSIRO Strategic Research Fund for the Marine coral reef ecosystems of the United States and Pacific freely Environment. Unpublished Report associated states. NOAA Technical Memorandum NOS Millar RH (1971) The biology of ascidians. Advances in Marine NCCOS 11, NOAA/NCCOS Center for Coastal Monitoring Biology 9: 1–100, http://dx.doi.org/10.1016/S0065-2881(08)60341-7 and Assesment’s Biogeography. Team Silver Spring. Minchin D, Sides E (2006) Appearance of a cryptogenic ascidian, Maryland, pp 488–507 a Didemnum sp. fouling marina pontoons and leisure craft in Grosberg RK (1988) Life history variations within a population of Ireland. Aquatic Invasions 1: 143–147, http://dx.doi.org/10.33 the colonial ascidian Botryllus schlosseri. I. The genetic and 91/ai.2006.1.3.8 environmental controls of seasonal variation. Evolution 42: Monniot F (1983) Ascidies littorales de Guadeloupe I. Didem- 900–920, http://dx.doi.org/10.2307/2408907 nidae. Bulletin du Museum National d'Histoire Naturelle 4a Hirose E, Kojima A, Nogami J, Teruya K (2007) Seasonality of ser. 5: 5–49 sexual reproduction in three photosymbiotic Trididemnum Monniot C, Monniot F, Laboute P (1991) Coral Reef Ascidians of species (Didemnidae: Ascidiacea: Tunicata) in a subtropical New Caledonia. Orstrom Editions. Paris, 247 pp sea grass bed. Journal of the Marine Biological Association Morris Jr JA, Carman M (2012) Fragment reattachment, repro- of the United Kingdom 87: 979–982, http://dx.doi.org/10.1017/ ductive status, and health indicators of the invasive colonial S0025315407057219 tunicate Didemnum vexillum with implications for dispersal. Kott P (1985) The Australian Ascidiacea Part 1, Phlebobranchia Biological Invasions 14: 2133–2140, http://dx.doi.org/10.1007/ and Stolidobranchia. Memoirs of the Queensland Museum 23: s10530-012-0219-8 1–440

273 J. Muñoz et al.

Newlon AW, Yund PO, Stewart-Savage J (2003) Phenotypic Stefaniak L, Lambert G, Gittenberger A, Zhang H, Lin S, plasticity of reproductive effort in a colonial ascidian, Whitlatch RB (2009) Genetic conspecificity of the worldwide Botryllus schlosseri. Journal of Experimental Zoology Part populations of Didemnum vexillum Kott, 2002. Aquatic A: Comparative Experimental Biology 297(2): 180–188, Invasions 4: 29–44, http://dx.doi.org/10.3391/ai.2009.4.1.3 http://dx.doi.org/10.1002/jez.a.10244 Stoner DS (1989) Fragmentation: a mechanism for the stimulation Page M, Morrisey D, Handley S, Middleton C (2011) Biology and of genet growth rates in an encrusting colonial ascidian. ecology of the introduced ascidian Eudistoma elongatum, Bulletin of Marine Sciences 45(2): 277–287 and trials of potential methods for its control. Journal of Strickland JDH, Parsons TR (1972) A Practical Handbook of Aquatic Invasions 6: 515–517, http://dx.doi.org/10.3391/ai.20 Seawater Analysis. Ottawa: Fisheries Research Board of 11.6.4.17 Canada, Bulletin No. 167, 293 pp Petersen JK (2006) Ascidian suspension feeding. Journal of Tan CK, Nowak BF, Hodson SL (2002) Biofouling as a reservoir Experimental Marine Biology and Ecology 342: 127–137, of Neoparamoeba pemaquidensis, the causative agent of http://dx.doi.org/10.1016/j.jembe.2006.10.023 amoebic gill disease in Atlantic salmon. Aquaculture 210: Ribes M, Coma R, Gili JM (1998) Seasonal variation of in situ 49–58, http://dx.doi.org/10.1016/S0044-8486(01)00858-4 feeding rates by the temperate ascidian Halocynthia Turon X (1988) The ascidians of Tossa de Mar (NE of Spain). 11. papillosa. Marine Ecology Progress Series 175: 201–213, Biological cycles of the colonial species. Cahiers de http://dx.doi.org/10.3354/meps175201 Biologie Marine 29: 407–418 Ritzmann NF, da Rocha RM, Roper JJ (2009) Sexual and asexual Turon X, Becerro MA (1992) Growth and survival of several reproduction in Didemnum rodriguesi (Ascidiacea, Didem- ascidian species from the north-western Mediterranean. nidae). Iheringia, Série Zoologia 99: 106–110, http://dx.doi. Marine Ecology Progress Series 82: 235–247, http://dx.doi.org/ org/10.1590/S0073-47212009000100015 10.3354/meps082235 Rocha RM, Kremer LP, Baptista MS, Metri R (2009) Bivalve Vázquez E, Young CM (2000) Effects of low salinity on cultures provide habitat for exotic ascidians in southern metamorphosis in estuarine colonial ascidians. Invertebrate Brazil. Aquatic Invasions 4: 195–205, http://dx.doi.org/10.3391/ Biology 119: 433–444, http://dx.doi.org/10.1111/j.1744-7410.20 ai.2009.4.1.20 00.tb00113.x Shenkar N, Swalla BJ (2011) Global diversity of Ascidiacea. Valentine PC, Carman MR, Blackwood DS, Heffron EJ (2007) PLoS ONE (6): e20657, http://dx.doi.org/10.1371/journal.pone. Ecological observations on the colonial ascidian Didemnum 0020657 Smale D, Childs S (2011) The occurrence of a widespread marine sp. in a New England tide pool habitat. Journal of invader, Didemnum perlucidum (Tunicata, Ascidiacea) in Experimental Marine Biology and Ecology 342: 109–121, http://dx.doi.org/10.1016/j.jembe.2006.10.021 Western Australia. Biological Invasions 14: 1325–1330, http://dx.doi.org/10.1007/s10530-011-0167-8

274