Nassarius foveolatus (Gastropoda, Nassariidae), a new record of an exotic species in Brazil

Exotic species are those that occur in an area beyond their natural limit and they are considered invasive when they cause harm to the economy, environment, or human health. In coastal environments, ballast water and inlays on the hull and other parts of vessels are the main ways of introducing invasive aquatic alien species. Nassarius foveolatus (Dunker, 1847) is native from the Central and East Indian Ocean to the East China Sea. The first specimens (empty shells) of N. foveolatus were collected manually on November 11, 2017 on the Rocio footbridge, located in the Paranaguá Estuarine Complex, on the coast of the State of Paraná, southern Brazil. Posteriorly, live specimens were collected in other localities of this bay. It is already possible to infer that the specimens of N. foveolatus occur together with the native specimens of N. vibex (Say, 1822), having the same niche. As previously only N. vibex existed in that place, at least a displacement of this native species has been occurred. However, certainly future ecological studies may confirm this displacement and additional consequences to the local ecosystem, as nassariids can be predators and scavengers. Control procedures should be also greatly implemented. Key-Words. Invasive; Exotic; Mollusca; Nassarius foveolatus; Paraná coast.


INTRODUCTION
Exotic, non-native or introduced species are those that occur in an area beyond their natural limit and they are considered invasive when they cause harm to the economy, environment, or human health (Carlton, 1996). Bioinvasions, i.e., the introduction of alien species into different ecosystems, are the major causes of biodiversity loss in the world, causing damage to local species and to the functioning of ecosystems (Stachowicz et al., 1999;Silva & Barros, 2011).
In coastal environments, ballast water (with sediments) and inlays on the hull and other parts of vessels are the main ways of introducing invasive aquatic alien species (Ferreira et al., 2004). Ballast water introduces aquatic organisms that are harmful to the environmental balance (in-cluding bacteria and viruses), both in marine and freshwater ecosystems, degrading important commercial activities such as those associated with fishing (Souza et al., 2009).
Nassariidae is a family of almost exclusively marine detritivores snails inhabiting bottoms of unconsolidated substrates and, to a lesser extent, rocky shores in tropical waters, with greater abundance between 0 and 300 m deep (Nekhaev, 2014). This group belongs to the neogastropod superfamily Buccinoidea and consists of more than 400 species (Brown, 1982;Galindo et al., 2016) divided into 18 extant genera, of which Buccinanops d'Orbigny, 1841 and Nassarius Duméril, 1805, are the largest genera in the family currently represented in the Brazilian coast (Rios, 2009;Rosenberg, 2009). In Brazilian waters, nine species of Nassarius have been recorded . ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049ISNI: 0000-0004-0384-1825 The typical shells of the family Nassariidae are characterized as being small to medium, with short and curved siphonal canal, conical protoconch, and horny operculum (Rios, 2009). It presents a high shell spire, oval shape with or without shoulder in the opening. The surface of the shell may be smooth or may exhibit axial and spiral sculptures Rios, 2009). Most nassariids have a well-developed callus. It has a rich fossil record dated since the Late Cretaceous (Taylor et al., 1980;Van Dingenen et al., 2015;Galindo et al., 2016).
The genus Nassarius and its close relatives tend to be restricted to muddy environments, though a few species shelter among loose rocks and sand. Ecologically, most species of Nassarius are thought to be facultative scavengers inhabiting inter-to subtidal shallow marine environments. As scavengers, they are important in maintaining the balance of ecological systems, especially for the benthic community (Cernohorsky, 1984). Their taxonomy, especially the status of the alleged genera and subgenera, is still far from a resolution and the distribution of most species is poorly understood Galindo et al., 2016). Compared to other gastropods, Nassarius snails have a relatively long planktonic larval phase, leading to a high level of dispersal capacity by marine currents (Tallmark, 1980). They can also be transported by ballast water and oysters farming to other environments and, by surviving, consequently becoming exotic species (e.g., Carlton, 1992;Bachelet et al., 2004;Townsend et al., 2010;Fofonoff et al., 2018;Goka, 2019).
The aim of this study is to report the first occurrence of the exotic species N. foveolatus in Brazil, confirmed both by morphological and molecular approaches. A discussion on its possible via of transportation and environmental consequences is also included.

MATERIAL AND METHODS
The first specimens (empty shells) of an unknown Nassarius species were collected manually on November 11, 2017, on the Rocio footbridge (25°30.236′S 48°31.891′W), located in the Paranaguá Estuarine Complex, on the coast of the state of Paraná, southern Brazil ( Fig. 1). Subsequently, on December 17, 2017, live specimens were collected in the same locality. The material collected was deposited in the molluscan collections of the Museu de Zoologia da Universidade de São Paulo,   The morphological identification was done according to Cernohorsky (1984). To confirm the morphological identification, molecular identification was performed using the DNA Barcode method (Hebert et al., 2003). Firstly, the genomic DNA was extracted using the EZ-DNA kit (Biological Industries) from muscle. Then, a fragment of 650 bp of the cytochrome oxidase subunit 1 (COI) was sequenced. The amplification comprises a PCR with 25 ul final concentrations of 2.5x buffer, 3 mM of MgCl2, 0.4 uM of dNTP, 0.1 pmol of each HCO and LCO (Folmer et al., 1994), 0.1 U of Taq Polymerase and 50 ng of DNA template. PCR product was purified using PEG 8000 (Amresco Inc., Cleveland, OH, USA). The sequencing reactions were performed with BigDye® kit (Applied Biosystems) according the manufacturer protocol and was purified with Sephadex G-50 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The final product was sequenced in an ABI 3130 Genetic Analyzer (Applied Biosystems).
The identification was confirmed though Neighbor-Joining (NJ) and Bayesian (BI) trees. Sequences of Nassarius spp. accessed from GenBank (see species and accession number in Table 1) were used as reference sequences and Antillophos sp. (GU393391.1) was used as outgroup. Sequences were aligned using the Muscle Algorithm (Edgar, 2004) in Geneious 2019.1.1. The NJ was constructed in Geneious, using the Tamura-Nei evolution model (Tamura & Nei, 1993) and 10 thousand bootstraps. The BI tree was constructed in the Beast 1.8 (Drummond et al., 2012), with three independent runs of 100 million MCMC generations sampled each 10,000 trees, 10% of burn-in and substitution model indicated by jModeltest 2.1.10 (Darriba et al., 2012).

RESULTS AND DISCUSSION
As referred above, the specimens of N. foveolatus ( Fig. 2A -B) were initially collected in Rocio, Paranaguá, Paraná. This locality is adjacent to Dom Pedro II Port (Port of Paranaguá), one of the largest in Brazil, which suggests that the species may have been introduced through ballast water. On the site, there is a small patch of mangrove bordered by a tidal flat with marshes. During low tides the plain is exposed, keeping some pools of water. Inside and around these pools, active live specimens were found at densities of up to 11 individuals/m², in sympatry with N. vibex (Say, 1822) ( Fig. 2C-D). The native N. vibex can be easily distinguished by being smaller, with the peristome callus much more developed in the adult specimens, by the thicker outer lip, and by the spire sculpture of strong axial ribs.
For the Bay of Bengal, in Odisha state, India, Ghosh et al. (2017) found a density of up to 11.8 individuals/ m² of N. foveolatus, suggesting that the density of this species in the Paranaguá Estuarine Complex is already reaching the same levels as those observed in its natural environment. On June 5, 2019, living individuals of N. foveolatus were found in the Emboguaçu River, in the Lamin Island, in Europinha (Vista Bela), and also in Ponta da Pita, all localities in the Paranaguá Estuarine Complex (Fig. 1).
The genetic identification for N. foveolatus suggested both by NJ (Fig. 3A) and BI (Fig. 3B) trees is highly supported. Interestingly, a sequence deposited as N. hepaticus (Pulteney, 1799) (FJ660644.1) in GenBank was also assigned to this group. However, since the other sequences of this species were assigned in a distant clade with N. nodifer (Powys, 1835) (see Appendices session for the entire phylogeny), this sequence probably represents an error of identification (as both species have similar shells) or even introgression between N. hepaticus and N. foveolatus, process already proposed for other Nassarius species (for details, see Pu et al., 2017).
Despite the inference of ballast water as responsible for the artificial dispersal of N. foveolatus in Paranaguá Estuarine Complex, the confirmation of this via of dispersal must be verified by additional studies. These could serve as alert on the species worldwide. The event can be the tip of the iceberg, representing a wide range of invasive species in the region (e.g., Rocha & Kremer, 2005;Neves & Rocha, 2008;Lopes, 2009;Altvater & Coutinho, 2015).
Based only on the collection experience, it is already possible to infer that the specimens of N. foveolatus occur side by side with the native specimens of N. vibex, having the same niche. As previously only N. vibex existed in that place (Rios, 2009;Absher et al., 2015), at least a displacement of the native species has occurred. However, more ecological studies may certainly confirm this displacement and additional consequences in the local ecosystem, as nassariids can be predators and scavengers. Control procedures should be also greatly implemented.