The spread of the introduced ascidians Ciona robusta Hoshino & Tokioka, 1967 and Rhodosoma turcicum (Savigny, 1816) in the southwestern Atlantic Ocean and Coastal Research

New records of the introduced solitary ascidians, Ciona robusta and Rhodosoma turcicum, have been added to the southwestern Atlantic Ocean, from Cabo Frio to Ilha Grande Bay, Brazil, in surveys conducted since 2009. Both species occurred on natural and artificial substrates, in predator-protected habitats, and regions close to harbors or other maritime activities. The distribution of Ciona robusta was related to water temperature, influenced by upwelling intensity and temperatures below 25oC, while R. turcicum was not sensitive to the range of recorded water temperatures. The occurrence of both species on sites close to maritime terminals suggests vessels as potential vectors.


INTRODUCTION
Marine bioinvasions have been recorded globally and have increased in recent decades in terms of the numbers of recorded species and numbers of scientific papers published (Seebens et al., 2013;Dias et al., 2019). Invasive species (IS) and nonindigenous species (NIS) have been identified as being responsible for reducing diversity in invaded regions, leading to local extinctions, and causing impacts on economic activities (Creed et al., 2017;O'Brien et al., 2017;Blackburn et al., 2019). Academic discussions concerning those issues are continually increasing (Junqueira, 2013;Fowler et al., 2020). Awareness of the importance of ascidians as invasive species is growing worldwide (Carlton and Eldredge, 2009;Zhan et al., 2015;Colarusso et al., 2016).
Eutrophication (Marins et al., 2010;Crooks et al., 2011), aquaculture facilities (McKindsey et al., 2007;, and harbor and shipping operations (Darbyson et al., 2009;Grey, 2010) have been indicated as important influences in increasing their spread. However, NIS introduction and spread are also subjected to limiting factors. Predation by fish (Marins et al., 2009;Dumont et al., 2011;Dias et al., 2013;Roth et al., 2017) and competition with the local fauna (Paetzold et al., 2012) are two biological factors that can regulate invasiveness, while environmental factors (such as temperature) can act as selective forces that prevent the establishment and persistence of invasive species. Therefore, environmental and biological factors will delimit species niches and are determinants for successful bioinvasions (Granot et al., 2017).
Ascidians can be found in many different habitats, and the photonegative behavior of their larvae Barboza and Skinner favors their occurrence in crevices or under rocks, more protected from predation and sedimentation (Millar, 1971;Lambert, 2005). Communities that settle on artificial substrates have often been found to be different from those encountered on natural surfaces (Perkol-Finkel et al., 2006). Artificial substrates are common in ports and marinas and serve as gateways for species introductions (Lambert, 2005;Ignacio et al., 2010). Surveys of invasive species must, therefore, use specific methodologies for data collection (Kakkonen, 2019).
We describe here the current distribution of Ciona robusta and Rhodosoma turcicum in coastal areas of Rio de Janeiro (~250 km in total), highlighting the importance of sea temperature on their distributions. We also discuss the current knowledge on possible transport vectors and their spreading along the southeast Brazilian coast.

sTUDy aRea
The present study was conducted within the coastal transition zone between Tropical and Warm Temperate regions (Spalding et al., 2007) in Rio de Janeiro State ( Figure 1A and 1B). The area has intense maritime activities like shipping, harbor, oil industry  Barboza and Skinner and other nautical activities, such as tourism, fishing, and coastal development (Silva et al., 2018;INEA, 2015;Ignacio et al., 2010). The climate is considered tropical humid, with a rainy season from October to April, and a dry season from May to September. Despite that general pattern, rainfall can vary greatly along the coast, mainly due to the proximity of the Serra do Mar Mountains, with average annual rainfall rates varying from 800 to 2,100mm (INEA 2015;Coe et al., 2007). There is a seasonal upwelling, which is stronger close to Cabo Frio, becoming weaker close to Ilha Grande Bay (Valentin, 2001;Creed et al., 2007).

WaTeR MONITORINgsea sURfaCe TeMpeRaTURes
Sea surface temperatures (SST) were measured using IButton (R) sensors programmed to record every hour at Cabo Frio and Ilha Grande Bay (at two sites in the latter: Dois Rios and Ponta Leste). The sensors were accommodated inside falcon tubes (to protect them from direct contact with the seawater) and placed at depths of 1-2 m, depending on the site. The shallow positions of the sensors ensured that no effects of water stratification were measured. SST data were aggregated monthly, and mean, and standard deviations were calculated. SST measurements were initiated in 2012 and extended until January 2014 in Cabo Frio and December 2019 in Ilha Grande Bay. Additionally, we extracted monthly SST data from satellite imagery during the period between January 2009 and December 2019 (data provided by NCEP, OISST Version 2, available from https://iridl.ldeo.columbia.edu/maproom/Global/Ocean_Temp), from the Brazilian Navy Oceanographic Buoys Program (PIRATA), and from the National Oceanographic Data Center (BNDO, 2020) for Cabo Frio, covering 2009Frio, covering -2010Frio, covering , 2012Frio, covering -2013Frio, covering , and 2016Frio, covering -2018 saMplINg We employed two sampling strategies: on artificial experimental structures (Marins et al., 2009;Kremer and Rocha, 2011;Skinner et al., 2013) and by active SCUBA or free diving searches. Our approach was similar to Kakkonen et al. (2019), which emphasize habitats favoring ascidian recruitment.

aRTIfICIal sTRUCTURes
Granite plates (21 x 12cm) and sandwich black polyethylene plates (15×15cm), sensu Kremer and Rocha (2011) were used to collect ascidians within protected cages, needed to prevent fish predation on ascidian recruits (Marins et al., 2009;Skinner et al., 2013). The experimental units were immersed 1 m below the surface at 11 sites positioned in the study areas: Cabo Frio (at Itajuru Inlet, Forno Harbor, and Cabo Frio Island); Guanabara Bay (Urca and Praia Vermelha); Sepetiba Bay (Junqueira and Guaíba Island); and Ilha Grande Bay (Piraquara de Fora, Praia do Anil, Ponta Leste, Abraão, and Dois Rios). Polyethylene plates were installed only at sites off Ilha Grande (Table 1).

sCUba aND fRee DIvINg
Exploratory scuba diving was undertaken for periods of 50 minutes each, actively searching for solitary ascidians on potentially predator-protected substrates such as holes, caves, or under boulders (Rocha, 1995). Those searches were performed at 26 sites at Ilha Grande Bay and two sites at Cabo Frio (Table 1). Free diving was used at some sites with submerged artificial structures; no scuba diving was undertaken in Guanabara Bay or Sepetiba Bay due to low water visibility.

DaTa aNalysIs
Mean monthly SST was calculated from daily data for Cabo Frio and Ilha Grande Bay collected by Ibutton® sensors and buoys. Satellite SST was retrieved to gain a wide temporal and regional perspective. Mean SST values (< 25.0 o C and > 25.1 o C) were used in combination with records of Ciona robusta and Rhodosoma turcicum in the selected temperature ranges to perform a square Fisher analysis (Underwood 1997) testing the hypothesis that temperature influences Ciona robusta distribution, but not Rhodosoma turcicum occurrences. The SST value of 25 o C was chosen as the upper physiological limit of survival for C. robusta (Caputi et al., 2015;Rocha et al., 2017;Kim et al., 2019). The physiological temperature limits for R. turcicum are unknown, but due to its tropical distribution the species may thrive in water temperatures higher than 22 o C, as suggested by Shenkar and Loya (2009).

WaTeR TeMpeRaTURe MONITORINg
SST data from Ibutton® sensors, buoys, and satellites indicated large SST variations between seasons, years, and monitoring methods ( Figure 2). Satellite Barboza and Skinner   Combining the SST data from all sources, we estimated the mean monthly SST values for 99 months, of which the means of 53 months (54%) were lower than or equal to 25°C, while those of 43 months (46%) were higher than or equal to 25.1°C. Those data were used to test the influence of temperature on the presence of the two species.

speCIes OCCURReNCes
Ciona robusta Hoshino and Tokioka, 1967 Ciona individuals found along the coast of Rio de Janeiro agreed with the description of C. robusta by Hoshino and Tokioka (1967), as well as the revisions conducted by Sato et al. (2012) and Brunetti et al. (2015). The tunic of C. robusta is cartilaginous, yellowish, with the siphons having almost equal sizes at the anterior end of the body. The tunic has many tubercular protuberances, although they are sometimes restricted to the siphon or anterior regions. The body is covered by six strong longitudinal bands, from the posterior end of the body up to the siphons and red spots were observed at the margin between adjacent siphon lobes. The circular muscles of the siphons are Barboza and Skinner not as strong as the longitudinal muscles, and do not form bands.
We analyzed the material collected between 2008 and 2013 from all regions of Rio de Janeiro State (Figure 3), and those deposited at the Zoological Collection of the Universidade do Estado do Rio de Janeiro (CZFFP), as listed in Table 2.
Ciona robusta was recorded on artificial plates in all surveyed regions (Figure 3). Ilha Grande Bay was the only site with occurrences on natural substrates, specifically at Jorge Grego Island (depth 18m), where eight individuals were found under large boulders, which are habitats resembling those simulated by experimental cages.
The lengths of the 33 individuals collected ranged from 40 to 150 mm, with the largest specimens being those collected on natural substrates at Jorge Grego Island, at depths of 18m. The maximum number of individuals collected was 8, at Jorge Grego Island (natural substrate, August/2013), and six at Guaíba Island (artificial substrate, May/2009) (Table 3).
Rhodosoma turcicum (Savigny, 1816) The material analyzed comprised specimens collected between 2009 and 2019 at Cabo Frio and Ilha Grande Bay (Figure 3). Most individuals were encountered within protected cages, while those found on    Table 4. We have identified 114 individuals obtained on 30 sampling dates. The maximum numbers of individuals collected were 13 (July/2011) and 11 (October/2010 and June/2014) found at Forno Harbor, in the Cabo Frio Region, all on artificial substrates (Table 5). Most individuals were collected during 2010 (n=31) and 2011 (n=32), the most intensively studied years at Cabo Frio.
The entire specimens examined (with tunic) varied from 9 to 41 mm in length and 3 to 23 mm in width. Their tunics were thick with epibionts and had greenish colorations that were lost after fixation. They were attached to the substrate by the right sides of their bodies. We found small papillae on the tunics, mainly close to the anterior region of the body. A lid in that region can be displaced by internal muscles to cover both oral and atrial siphons. It is easier to spot individuals in the field when the lid is open. Dissected individuals showed short and simple oral tentacles. Muscles were visible close to the siphons and extended onto the hinge of the lid. The dorsal tubercles were horseshoe-shaped. The pharynx was plain, without folds, and had straight stigmata and papillae sustaining the vessels. Our specimens conformed well with the descriptions provided by Van Name (1945), Kott (1985Kott ( , 2005, and Rocha et al. (2012a, b).

CURReNCes
Ciona robusta was recorded 15 times within the temporal window considered. Among those records, 86.7% occurred with SST between 21-25°C, and 53.3% occurred with SST between 23.1 to 25.0°C. Only two records (13.3%) occurred with SST higher than 25.1°C. The Fisher exact test was significant (p=0.023; 1 d.f.), indicating this environmental variable as a driver of the presence of C. robusta.

DIsCUssION
We detected a wider distribution range of Ciona robusta than previously recorded for the studied area (Millar, 1958;Costa, 1969;Marins et al., 2009). The only previously collected specimens that we have Barboza and Skinner Marins et al. (2009) but considering the confirmation of recent specimens in Rio de Janeiro as C. robusta, and the widespread occurrence of that species in the Southern Hemisphere, we believe that earlier records in Brazil belongs to this species and not to C. intestinalis, and should be corrected (Sato et al., 2012;Brunetti et al., 2015). Ciona robusta was detected at Ilha Grande Bay during an early phase of our monitoring, which appears to be related to intermediate water temperatures. This is because we have not recorded C. robusta at Ilha Grande Bay since 2013, when higher than normal SSTs (sometimes reaching 33°C) have occurred (Skinner, 2018a, b). The temperature tolerance range of C. robusta was experimentally tested by Kim et al. (2019) and showed that egg development and larval settlement is greater at temperatures between 16 and 20°C. Caputi et al. (2019) indicated that C. robusta density is associated with SST, with low densities from May to September, the time period with the highest SSTs (24.3 to 27.5 o C). Rocha et al. (2017) reported that a temperature range of 20 to 25 o C is best for egg, larval, and adult development and recruitment. Our data, in a multiyear comparison, indicate predominance of C. robusta records during periods with mean SST lower than 25.0 o C, corroborating the findings of Caputi et al. (2015).
Previous records of C. robusta in Brazil were made from Cabo Frio (RJ) to São Paulo (Millar, 1958;Costa, 1969;Marins et al., 2009;Rocha and Bonnet, 2009;Dias et al., 2013), covering the Warm Temperate Southwestern Atlantic (WTSA) Marine Ecoregion (Spalding et al., 2007;Bouchemousse et al., 2016), influenced by upwelling events, mainly during spring and summer months. SST values in the Cabo Frio region may drop below 20 o C Batista et al., 2017;Boltovskoy and Valentin, 2018). Extreme high SST, however, can prevent reproduction or even kill adults growing in shallow waters in the region (Caputi et al., 2015;Rocha et al., 2017), which would contribute to the irregular historical records of the species along the coast of Rio de Janeiro. Those records, and successful introductions of C. robusta along the southeastern coast of Brazil, could be related to El Niño/La Niña events that increase or reduce SSTs in the Atlantic Ocean. The , 2010 seasons, when some of our records were made, were characterized by strong to neutral La Niña seasons with negative average anomalies (NOAA, 2019). Data from sensors and buoys confirmed lower SSTs during those years. Records of C. robusta at Ilha Grande Bay ceased after the strong El Niño events of 2014-2015-2016, when SST reached 33 o C (at depths down to 10m) during summer months. There is no evidence of the presence of C. robusta at Cabo Frio in summer 2016 when SST presumably reached favorable conditions for the species (17 to 22 o C). Shenkar et al. (2018) argues that C. robusta is acclimated to both temperate and tropical areas, matching the environmental conditions of the southwestern Brazilian Coast. Bouchemousse et al. (2016) published the global distribution of C. robusta, which included the South Brazilian coast (after introductions in the mid-20 th century). According to the SST patterns recorded along the coast of Rio de Janeiro State, the most suitable region for the continuous occupation of C. robusta is the region near Cabo Frio, where SST usually range from 12.5 to 29.0 o C Batista et al., 2017).
Ciona robusta is an invasive species, often fouling substrates in aquaculture farms (Clancey and Hinton, 2003;Daley and Scavia, 2008;Ramsay et al., 2008;Fofonoff et al., 2019;Global Invasive Species Database, 2019;Kim et al., 2019), and their establishment in bivalve farms along the southern Brazilian coast is a threat to that industry. The socking or pearl net methods (Baylon, 1990) usually employed by farmers help to prevent predation events but could favor C. robusta recruitment, growth, and spread. Some fouling prevention procedures can also deter C. robusta development, but certain suspended structures can serve as refuge for the species.
Rhodosoma turcicum was first recorded at Cabo Frio from 2009 to 2012 (Skinner et al., 2013), at Sepetiba Bay in 2012, and at Ilha Grande Bay from 2012 to 2019. There are other reports about this species on northeast Brazil, at Bahia and Ceará, but those records remain unpublished on thesis and reports. The species was recorded on both artificial and natural substrates at Ilha Grande and Cabo Frio. The first records of R. turcicum at Ilha Grande Bay were on eastern sites (2012); records from the most western site refer to 2018 and 2019 samples. Thus, our records and those from Granthom- Costa et al. (2016) indicate that this species is spreading in Rio de Janeiro Barboza and Skinner State, mainly at Cabo Frio and Ilha Grande Bay. The alternative hypothesis of variable sampling effort is not supported because even at sites where sampling was performed along an extensive period, from early 2012 to 2019, records of this species were scarce. SST lower than 25 o C and up to 31 o C do not appear to be limiting for R. turcicum, as our surveys both at the warmest (Piraquara de Fora) and at the coldest sites (Arraial do Cabo Bay) recorded the species. Based on the global distribution of R. turcicum, its temperature range is from 20 to 30 o C, with most records within the 25 -30 o C range (OBIS, 2019). Shenkar and Loya (2009) suggested 22 o C as the lower limit for this species. It is a tropical species recorded from the Caribbean, Mediterranean, and the Red Sea, and is tolerant to high temperatures.
Some aspects are relevant to the detection of C. robusta and R. turcicum. The tunics of both species are reasonably soft as compared to other solitary ascidians recorded on natural or artificial substrates in Rio de Janeiro State, such as Phallusia nigra Savigny, 1816, Styela plicata (Lesueur, 1823, Herdmania pallida (Heller, 1878), and Microcosmus exasperatus Heller, 1878 (Marins et al., 2010;Granthom-Costa et al., 2016;Skinner et al., 2016). Those differences in tunic thicknesses and chemical/physical defenses will be determinant for ascidian palatability to predators such as fish (Monniot et al., 1991;Tarallo et al., 2016). The recently described Pyura beta Skinner, Rocha & Counts (2019), recognized as introduced into the SW Atlantic, also have a hard tunic that reduces predation. The effects of predator control on Ciona spp. (Marins et al., 2009;Dumont et al., 2011) and other ascidian species are frequently cited in the literature (Epelbaum et al., 2009;Freestone et al., 2011).
Another common and important factor potentially affecting species introduction and spreading is the proximity of harbors and marinas. The region from Cabo Frio to São Sebastião in the SW Atlantic experiences intense marine ship traffic, potentially connecting those coastal areas to several tropical and subtropical biogeographical realms throughout the world (Seebens et al., 2013;Brasil, 2019;Sardain et al., 2019). Transoceanic cruise ships, passenger vessels, and many fishing boats and yachts cross those waters daily, and could locally spread introduced species (Zabin et al., 2014;Skinner et al., 2016;Kauano et al., 2017).
It will be necessary to establish continuous monitoring and experimental procedures considering predator exclusion near Cabo Frio to test the hypothesis that the continuous records of C. robusta there, in contrast to the discontinuous sightings at Ilha Grande and other regions, can be attributed to the lower SST related to upwelling. It would also represent a good opportunity to test the effects of upwelling strength and other climatic and oceanographic variables such as wind intensity and direction on C. robusta establishment. aCKNOWleDgMeNTs Authors would like to thank Dr. Helena Passeri Lavrado (UFRJ) for the suggestions on statistical analysis and the two referees who suggested many improvements in this manuscript. This work was supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro -FAPERJ (Proc. #E-26/11.454/2011). The authors are grateful for the scholarship awarded to DFB by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001, the logistic support provided by CEADS-UERJ.