All non-indigenous species were introduced recently? The case study of Cassiopea (Cnidaria: Scyphozoa) in Brazilian waters andre’ c. morandini 1 , sergio n. stampar 2 , maximiliano m. maronna 1 and fa’ bio l. da silveira 1 1Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, n. 101, São Paulo, SP 05508-090, Brazil, 2Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, Av. Dom Antônio, n. 2100, Assis, SP 19806-900, Brazil Upside-down jellyfish (genus Cassiopea) can be found in tropical coastal waters worldwide. Until now reports of the genus from Brazilian waters have been scant. We report here medusae and scyphistomae collected from Cabo Frio, Rio de Janeiro state. Although we could not unambiguously identify the material using morphological criteria, genetic sequence data (COI) indicate that the Brazilian jellyfishes are genetically similar to those from Bermuda, Hawaii and Florida, which are related to specimens from the Red Sea (Cassiopea andromeda). We hypothesize that the presence of C. andromeda in Brazil is due to an invasion event, as the scyphistomae were found growing over the known invasive ascidian Styela plicata. Estimation of divergence time between Brazil (Cabo Frio) and Florida/Bermuda populations is that it occurred at the begin- ning of ship movement to South America. Keywords: Invasive species, marine, rhizostome, jellyfish, blooms Submitted 25 May 2015; accepted 24 February 2016; first published online 5 April 2016 I N T R O D U C T I O N Invasive alien species are the second greatest threat to global biodiversity loss (Sala et al., 1999). In the case of the marine environment, these invasive species may be escapees following intentional introduction, but are more usually the unintended consequence of global shipping and maritime transport (Carlton, 1987; Williams et al., 1988; Carlton & Geller, 1993; Sax et al., 2007; Rodrı́guez-Labajos et al., 2009; Briski et al., 2012; Guo et al., 2012). Although most alien taxa that are introduced into new environments following ballast-water containment or hull-fouling fail to establish viable populations in their new environment (Carlton, 1996), a sufficiently large number do to warrant international programmes of preven- tion and control. Although jellyfishes (Cnidaria: Medusozoa) have a number of biological features that allow them to be potentially invasive (Graham & Bayha, 2007; Bayha & Graham, 2014), there are relatively few species (five out of �201; Bayha & Graham, 2014) with proven reports of invasion worldwide, although with many records distributed in different ocean basins (Bayha & Graham, 2014). Part of the reason for this must reflect an incomplete understanding of the biology, taxonomy and distribution of most jellyfishes (Dawson, 2005a), many of which have a number of cryptic species (Dawson, 2003; Holland et al., 2004), which was estimated as 7% (Appeltans et al., 2012). In Brazilian waters (along 9230 km of coast) only one (Phyllorhiza punctata von Lendenfeld, 1884; Moreira, 1961; Haddad & Nogueira, 2006) of the 22 recorded species of scyphozoans (Morandini et al., 2005; Lopes et al., 2009) can be considered as exotic to date. The genus Cassiopea is widely distributed across the globe in shallow tropical waters (Kramp, 1961). Although a total of 10 species are considered valid (Kramp, 1961; Hummelinck, 1968; Thiel, 1975), a number of genetically distinct lineages have been identified (Holland et al., 2004). Species of the genus can be abundant in disturbed/eutrophicated environ- ments (Arai, 2001). Members of this genus have been consid- ered invasive in the Mediterranean (Galil et al., 1990; Çevik et al., 2006; Schembri et al., 2010). Holland et al. (2004) also mapped the distribution of some populations and attributed the ‘unusual’ pattern observed to maritime transport. The first report of the genus Cassiopea Péron & Lesueur, 1810 in Brazil was published in 2002 (Migotto et al., 2002), although a number of sightings and grey-literature accounts pre-date that, but unfortunately without any voucher specimens. Here we report specimens of Cassiopea from along the Brazilian coast using both morphological and molecular data and we compare our results with published information in order to try and determine their origin. The application of molecular (genetic) data to define and identify populations and species in marine research is now a well-recognized venture (Schander & Willassen, 2005; Radulovici et al., 2010; De Broyer et al., 2011). Together with approaches such as morphology and ecology, this information let us analyse historical aspects such as time of possible invasion based on the divergence of Brazilian jellyfishes compared with the available data in the literature (Holland et al., 2004). Corresponding author: A.C. Morandini Email: acmorand@ib.usp.br 321 Journal of the Marine Biological Association of the United Kingdom, 2017, 97(2), 321–328. # Marine Biological Association of the United Kingdom, 2016 doi:10.1017/S0025315416000400 https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. mailto:acmorand@ib.usp.br https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms M A T E R I A L S A N D M E T H O D S Field sampling Jellyfish specimens were sampled by hand at a small branch of the Itajuru Channel, a connection of the Araruama Lagoon to the open sea in Cabo Frio county, Rio de Janeiro state (22852′32′′S 42801′07′′W) (Figure 1) during the period November 2008 to October 2012 (weekly intervals from 2008–2009 and every 3 months from 2010–2012). Regular measurements of salinity, dissolved oxygen concentrations and temperature (recorded with an YSI 85 probe) indicate that salinity varied from 0–36, water temperature from 26.6–28.78C, and dissolved oxygen from 7.01–15.56 mg L21 in a period of 6 h (from high to low tide). Medusae were preserved either in ethanol 95 or 4% formal- dehyde solution in seawater. Two hundred freshly collected specimens were examined in the laboratory facilities to measure bell diameter, count number of rhopalia, lappets, mouth arms, define shape of the oral arm appendages and the organization of the canal system (with injection of dye). Morphological comparisons were conducted with descrip- tions in the literature as well as with specimens of Cassiopea frondosa (Pallas, 1774) and Cassiopea cf. xamachana Bigelow, 1892 that had been collected in February 2004 at Laguna Golf Club, Varadero (Cuba), and deposited at the Museu Nacional da Universidade Federal do Rio de Janeiro (MNRJ 5837 and 5838, respectively). Scyphistomae morphologically resembling Cassiopea scy- phistomae (Hofmann et al., 1978; Straehler-Pohl, 2009; Heins et al., 2015) were collected at the same place that medusae were found. The scyphistomae were found growing on a variety of substrata, including plastic, wood and rubber shoes as well as on the exotic ascidian Styela plicata (Lesueur, 1823). Ten scyphistomae (Figure 2) were measured in the laboratory following the parameters proposed by Straehler-Pohl et al. (2011). Voucher specimens of medusae and scyphistomae were deposited at the Museu de Zoologia da Universidade de São Paulo (MZUSP, 1944, 1945). Molecular protocols DNA was extracted from the oral arms removed from three specimens of medusae using an Agencourt DNAdvancew kit (#A48708); DNA extraction from scyphistomae was not suc- cessful. Partial gene sequences were amplified using PCR, Universal PCR primers for the cytochrome oxidase I gene (COI) were used to amplify �500 base pairs of this protein coding mitochondrial gene, using the original PCR program (forward LCO1490 and reverse HCO2198 primers; Folmer et al., 1994). The PCR products were purified with the Agencourt AMPurew kit (#A63881) and this amplified DNA sequences were prepared to sequencing using the Applied Biosystems BigDyew Terminator v3.1 kit (#4337455), with the same primers and temperature conditions from the PCR’s reactions. The sequencing procedure was carried out on a Hitachi ABI PRISMw3100 genetic analyser. DNA analysis Sequences were assembled and edited using Geneious TM 5.4.4 (Drummond et al., 2011), and new sequences were deposited in GenBank (KC464458-KC464459). COI sequences were compared to those of the other Cassiopea specimens available in GenBank (see table 1 in Holland et al., 2004): sequence alignment was made using the MUSCLE plugin in Geneious TM 5.4.4 (default parameters) (Edgar, 2004). Kimura’s two-parameter model of base substitution and p-distance was used to calculate genetic distances in MEGA 6.05 software (Tamura et al., 2011). The Maximum Likelihood analysis was conducted via PHyML 3.0.1 beta with a more inclusive model, the general time reversible model with gamma values estimated directly from data (GTR + GAMMA) (Guindon et al., 2010). The Maximum Parsimony analysis was conducted via MEGA 5.05 (Tamura Fig. 1. Map of Brazil, showing records of Cassiopea Péron & Lesueur, 1810 specimens. Squares refer only to polyp records (1–2), triangles refer to medusa photographic records (4–7), star refers to sampling site at Cabo Frio (3), numbers after each symbol refer to date of first sighting in each area: (1) Imbé (29858′26′′S 50808′18′′W; February 2005), Rio Grande do Sul state; (2) São Sebastião (23849′41′′S 45825′22′′W; March 1999), São Paulo state; (3) Cabo Frio (22852′32′′S 42801′07′′W; October 2008), Rio de Janeiro state; (4) Aracruz (19850′07′′S 40803′28′′W; August 2007), Espı́rito Santo state; (5) Salvador (12857′26′′S 38831′50′′W; February 2012), Bahia state; (6) Macau (5803′53′′S 36830′18′′W; February 2011), Rio Grande do Norte state; (7) Itarema (02852′48′′S 39854′15′′W; June 2012), Ceará state. Fig. 2. Cassiopea andromeda (Forskål, 1775) scyphistoma just after collection, note long stalk and planuloid budding at the base of calyx; polyp �5 mm long. 322 andre’ c. morandini et al. https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms et al., 2011): optimal tree was obtained by the search of optimal tree space using the Close-Neighbour-Interchange (CNI) algorithm (see more in Stampar et al., 2012). Support estimation was assessed with two non-parametric methods: bootstrap (500 pseudoreplicates) and SH-aLRT test (Anisimova et al., 2011) in PHyML 3.0.1 beta (Figure 3). R E S U L T S A N D D I S C U S S I O N The morphological and range meristic characters determined from the 200 specimens collected at Cabo Frio are shown in Table 1 in comparison with data from the other presently recognized species in the genus. It should be noted that all inspected specimens were males, suggesting that the popula- tion is perhaps maintained through asexual reproduction at the scyphistoma stage. The morphological and meristic data indicate that, with the possible exception of Cassiopea fron- dosa, it is not possible to distinguish between the different species (see Table 1) using the selected character set because of the high level of overlap between species (see Mayer, 1910; Kramp, 1961; Hummelinck, 1968; Thiel, 1975; Holland et al., 2004). The exception, Cassiopea frondosa, can be separated from the other recognized species by the number of rhopalia (12, although some variation is also found) and it is clear that the Brazilian material is not of this species. Collected scyphistomae were also measured (N ¼ 10): oral disc diameter 0.65–1.5 mm; total length 2–3.5 mm; stalk length 1.15–2.5 mm; calyx length 0.35–0.5 mm; hypostome length 0.2–0.7 mm; number of tentacles 27–36. These mea- surements are in accordance with the available data for the genus (Ludwig, 1969; Straehler-Pohl, 2009). The scyphistomae were kept in laboratory conditions, and produced ephyrae (by monodisc strobilation) similar to the young medusae found in the field. The COI data obtained from the three sequenced speci- mens were identical. The phylogenetic analysis (Figure 3) Fig. 3. Left panel – Cladogram representation for maximum likelihood tree (ML) of Cassiopea species with support values on branches (as in figure order: SH-aLRT/maximum parsimony’s tree bootstrap result/maximum likelihood bootstrap) ∗ ¼ less than 50; in cases where figure permits, ML bootstrap values are below their respective branch. Right panel: phylogram representation only for same result (ML phylogram). non-indigenous upside-down jellyfish in brazil 323 https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms including the new Brazilian specimens, and terminals (¼sequences) already presented in a previous study (Holland et al., 2004) clustered the Brazilian Cassiopea sequences group with the Cassiopea andromeda samples (from Florida, Bermuda and Hawaii) as defined by Holland et al. (2004). The molecular distance between the Brazilian material and Florida and Bermuda specimens is 0.54% based on Kimura 2-parameter model and 0.005 on p-distance (3 to 6 nucleotide variation). A comparison of the molecular diver- gence between the Cassiopea species considered by Holland et al. (2004) is shown in Table 2, and it reveals that intraspe- cific variation (from 0.6–10%) is much smaller than interspe- cific variation (11–26%). It is estimated that the C. andromeda specimens sampled here from Cabo Frio diverged from those in Florida and Bermuda �500 years ago, which is relatively soon after the ‘discovery’ of Brazil in 1500. In an alternative method (Dawson, 2005b) to estimate the divergence time between species the equation T ¼ d/2l (where d is the nucleo- tide distance between populations and the mutation rate l ¼ 4.87 × 1026) was applied. Based on this equation the Cabo Frio specimens diverged from Florida and Bermuda �554 years ago. Whilst early ships did not have the ballast capacity of modern vessels, and their ballast would invariably have been rocks or other heavy solid material, their hulls would have inevitably been subject to heavy fouling (see more in Carlton & Hodder, 1995). An introduction into Brazil via the fouling route is perhaps more likely than by ballast water – although the scyphistomae of Cassiopea can produce a number of planuloids asexually on a daily basis (Hofmann et al., 1978), which could persist within ballast tanks – because scyphistomae were observed on artificial sub- strates as well as on another invasive species, Styela plicata (Barros et al., 2008). Given that genetic evidence (same haplo- types) indicates that Styela plicata populations off Florida and Brazil are similar (Barros et al., 2008), it is possible that popu- lations of both species were introduced into Brazilian waters from Florida. Cassiopea andromeda clearly has a number of ecological characteristics that predispose it to invasion success, chiefly Table 1. Comparison of available characters from the literature of the 10 valid/nominal species of the genus Cassiopea Péron & Lesueur, 1810 (species list from Mayer, 1910; Kramp, 1961; Gershwin et al., 2010). n ¼ several appendages, not specified on the papers. For mouth arms we are considering number of branches, type of branching and general shape, according to the descriptions. Species Size cm Rhopalia Lappets Mouth arms Appendages Type locality C. andromeda (Forskål, 1775) 10–12 16? variable 4–6 branches n small, 5-more large/arm Red Sea C. depressa Haeckel (1880) 10–12 16? 144 6–8 branches n small Madagascar, Mozambique C. frondosa (Pallas, 1774) 12–26 12 60 30–40 small West Indies, Caribbean C. maremetens Gershwin, Zeidler & Davie (2011) 2–20 19 57 (3/rhopalia) 4–6 branches 1–2 central, 1 base arm, 1 tip arm Lake Magellan, Australia C. medusa Light (1914) 26 17 119 (7/rhopalia) n branches (3 main distal) n small/large Culion Bay, Philippines C. mertensi Brandt (1835) 10–12 16? 128 n branches n large Kosrae Island, Micronesia C. ndrosia Agassiz & Mayer (1899) 5 18–22 64 (4/rhopalia) ? n leaf Suva, Fiji C. ornata Haeckel (1880) 10–12 16? 80 (5/rhopalia) ? n small (only) Palau, Papua New Guinea C. vanderhorsti Stiasny (1922) 17 14–18 48–80 (3–5/ rhopalia) dichotomous n small/large Caracas Bay, Curaçao C. xamachana Bigelow (1892) 15 16? 80 (5/rhopalia) Triangular 10–15 alternate branches n large ribbon Kingston Harbor, Jamaica C. andromeda ( from Cabo Frio) 1–20 16–22 48–80 (3–5/ rhopalia) 3–10 (different sizes) n small/large Cabo Frio, Rio de Janeiro, Brazil Table 2. Estimates of average evolutionary divergence over sequence pairs within and between ‘species’ as defined by Holland et al. (2004). Analyses were conducted using the Kimura 2-parameter model. The rate variation among sites was modelled with a gamma distribution (shape parameter ¼ 1). All positions containing gaps and missing data were eliminated (total positions in the final dataset: 554). Evolutionary analyses were conducted in MEGA5 (Drummond et al., 2011). n/c ¼ not possible to estimate evolutionary distances. Figure 3 lists the individuals used in the comparison as well as their location. C. andromeda (%) C. frondosa (%) C. ornata (%) C. sp1 (%) C. sp2 (%) C. sp3 (%) C. andromeda 1.6 25 23 23 23 22 C. frondosa 1.3 26 25 24 22 C. ornata 1.8 11 22 23 C. sp1 n/c 20 23 C. sp2 10 21 C. sp3 0.6 324 andre’ c. morandini et al. https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms the ability of its scyphistomae to produce planuloids at a high rate (personal observation). Off Brazil, it is clearly tolerant to quite wide variations in salinity and temperature (salinity 0– 36, water temperatures up to �298C), and this allows it to be an opportunistic species (Fofonoff et al., 2003; Devin & Beisel, 2007; Zerebecki & Sorte, 2011). It is known that Cassiopea species can be tolerant to salinity fluctuations (Goldfarb, 1914). Several other reports of Cassiopea medusae exist from the coast of Brazil based on photographic records (Figures 1 & 4), as they do for scyphistomae (states of Rio Grande do Sul and São Paulo; Figure 1). Although we do not at this stage have genetic data to confirm their identity to species, it is clear that the genus Cassiopea is finding suit- able habitats along the Brazilian coast and that in the future they may be much more common. The detection of non-indigenous species in Brazilian waters is important (Rocha et al., 2013), because lists of such species shall serve as elements for conservation policies and priorities. Even as exhaustive biological surveys provide the best methods for detecting alien species, the appearance of unusual taxa in large numbers is often the first evidence of an invasion event. This is the case of the present species, with blooms of C. andromeda (Figure 5) in the Araruama Lagoon since 2008. But not all blooms of unusual species need be similarly interpreted owing to the lack of comprehen- sive species maps, the scarcity of long-term data on jellyfish blooms (Condon et al., 2012), and because jellyfish naturally exhibit episodic and seasonal interannual variability (Mills, 2001). We are aware that our genetic data only suggest the hypotheses of possible appearance of this species as following an invasive event in the Brazilian shoreline. Furthermore, whilst C. andromeda may have been resident in Brazilian waters for �500 years, only now are populations (in some areas) blooming. The reasons for this are not yet understood, neither are the impacts on local biological communities, but to date there appear to be no obvious economic or social conse- quences of the bloom. Regardless, further work on the species in Brazil is now needed to define a broad and complete picture on present and past evolution of the species C. andromeda. A C K N O W L E D G E M E N T S We are thankful to Solange Brisson and Sara Oliveira for the first observation and alerting us about the occurrence of the animals at Cabo Frio (RJ). Collections were in accordance with Brazilian regulations (SISBIO licence 15031-2 to ACM). We thank Drs Oberdan J. Pereira (UFES), Thelma L.P. Dias (UEPB), Claudio L.S. Sampaio (UFAL) and Helena Matthews-Cascon and Bruno B. Batista (UFC) for recognizing and informing about photographic records in other regions. We also thank Dr Fábio Di Dario (NUPEM, UFRJ) and Mr Enzo C. Morandini for helping in field samples. Different parts of this manuscript were presented as posters at three Fig. 4. Photographic records of Cassiopea Péron & Lesueur, 1810 specimens from Brazilian coast (A–F). (A) Itarema, Ceará state (photo Carlos H.P. Marques & Yara G. Oliveira); (B) Macau, Rio Grande do Norte state (photo Thelma L.P. Dias); (C) Salvador, Bahia state (photo Claudio L.S. Sampaio); (D) Aracruz, Espı́rito Santo state (photo Oberdan J. Pereira); (E–F) Cassiopea andromeda (Forskål, 1775) from Cabo Frio, Rio de Janeiro state (photos by André C. Morandini and Sergio N. Stampar). non-indigenous upside-down jellyfish in brazil 325 https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms conferences (II Congresso Brasileiro de Biologia Marinha, III International Jellyfish Blooms Symposium, II World Conference on Marine Biodiversity) and we thank all the audi- ences for further discussions. We are also indebted to Dr Mark J. Gibbons (UWC, South Africa) for corrections and com- ments on an earlier version of the text. 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(2011) Temperature tolerance and stress proteins as mechanisms of invasive species success. PLoS ONE 6, e14806. Correspondence should be addressed to: A.C. Morandini Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, n. 101, São Paulo, SP 05508-090, Brazil Email: acmorand@ib.usp.br 328 andre’ c. morandini et al. https://doi.org/10.1017/S0025315416000400 Downloaded from https://www.cambridge.org/core. UNESP, on 19 Jun 2019 at 16:40:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. mailto:acmorand@ib.usp.br https://doi.org/10.1017/S0025315416000400 https://www.cambridge.org/core https://www.cambridge.org/core/terms All non-indigenous species were introduced recently? The case study of Cassiopea (Cnidaria: Scyphozoa) in Brazilian waters INTRODUCTION MATERIALS AND METHODS Field sampling Molecular protocols DNA analysis RESULTS AND DISCUSSION ACKNOWLEDGEMENTS FINANCIAL SUPPORT REFERENCES