Introduction
Phymactis papillosa (Lesson, 1830) is an abundant, widely distributed anemone of the Pacific coast of Central and South America (Chile, Peru, Mexico and Sea of Cortez) (Figure 1). This species is the most common sea anemone of the coastline in Chile, occurring in the intertidal and subtidal up to 16 m depth. It is most abundant in the inferior part of the intertidal, where it can occur at aggregations of one to more than 100 individuals. It can reach the mean high tide level (Haussermann, Reference Häussermann2004). The size of the individuals appears to be vertically distributed, the smallest at higher levels of the intertidal and the biggest specimens at its lower levels (Stotz, Reference Stotz1979).
Distribution of Phymactis papillosa (Source: www.gbif.org/pt/species/2256771).
Phymactis papillosa is a middle-sized anemone with a red, brown, green or blue tentacle and column colour with a pedal disc size up to 100 mm. The colour of the up to 400 tentacles is paler than the colour of the column, that is covered with distinctive non-adhesive vesicles. It has a variable number of acrorhagi (Haussermann, Reference Häussermann2004). It reproduces sexually and asexually by longitudinal fission (Uribe et al., Reference Uribe, Rubio, Carbajal and Berrú2013).
Phymactis papillosa was initially considered a synonymy of P. clematis (Drayton in Dana, 1846), which was thought to occur in the Pacific and Atlantic Ocean, off the coast of Argentina (Haussermann, Reference Häussermann2004). However, the morphological analysis made by Haussermann (Reference Häussermann2004) on the Pacific populations considered P. clematis as a synonymy of P. papillosa. More recently Gomes et al. (Reference Gomes, Schama and Solé-Cava2012) studied the Atlantic populations using morphological and genetic analysis. They used allozymes and sequenced a fragment of the nuclear internal transcribed spacer I, 5.8 rDNA and internal transcribed spacer II. Their genetic results were compatible using both kinds of genetic markers and supported the distinction between Pacific P. papillosa and the Atlantic populations. This study revealed that the latter population is a distinct species, belonging to a different genus (Bunodosoma).
In 2017, an unusual sea anemone was recorded in the region of Cascais, Portugal by the MARE–ISPA monitoring intertidal team. Since then, the abundance of this anemone has been increasing in this region, with green, red and brown individuals recorded. The increased abundance, with individuals of various size classes, and the occurrence of different morphotypes, suggest that this species reproduces locally and has established local populations. The spread of invasive, non-indigenous species has been recognized as a growing problem in marine habitats, with their early detection being one of the highest priorities (Xiong and Zhan Reference Xiong and Zhan2016), as invasive species can have multiple impacts such as displacement of native species, and changes in community structure and in food webs (Molnar et al., Reference Molnar, Gamboa, Revenga and Spalding2008).
In this paper we used genetic methods and external morphology analysis to identify this anemone.
Materials and methods
Two green sea anemones at 5-m distance from each other were collected at Bafureira beach, Cascais (38.6921°N 9.3659°W). Sea anemones were anaesthetized with menthol crystals, a pedal disc clip was conserved in 70% ethanol and individuals in a 4% formaldehyde in salt water. Total DNA was obtained using REDExtract-N-Amp kit (Sigma-Aldrich) from ~2 mg of ethanol conserved tissue, following the manufacturer's instructions. Total concentration of DNA was measured in Nanodrop. Amplifications were conducted in 20 μl total-reaction volume with 10 μl of REDExtract-N-amp PCR reaction mix (Sigma–Aldrich), 0.8 μl of each primer (10 μM), 4.4 μl of sigma-water, and 4 μl of template DNA. Ribosomal internal transcribed spacers I and II (ITS1-rDNA and ITS2-rDNA) were amplified using the primers 18SF (5′- TCA TTT AGA GGA AGT AAA AGT CG- 3′) and 28SR (5′- GTT AGT TTC TTT TCC TCC GCT T – 3′) (Lôbo-Hajdu et al., Reference Lôbo-Hajdu, Guimarães, Mendes, Lamarão, Vieiralves, Mansure and Albano2004). The amplicon is a single sequence of 689 bp, which includes a partial 18S ribosomal DNA sequence, complete ITS-I, 5.8S ribosomal DNA and ITS-II sequences and a partial 28S sequence. PCR thermal cycling protocol was 94°C for 2 min, 40 cycles of [94°C 30 s, 51.5°C 45 s, 72°C 1 min], 72°C 10 min. DNA purification with magnetic beads and forward and reverse sequencing (Sanger method) were performed in Stabvida (www.stabvida.com). Sequences were inspected and edited using CodonCode Aligner (CodonCode Corporation, www.codoncode.com). Edited sequences were compared with sequences from known specimens on the National Center for Biotechnology Information (NCBI) GenBank database using the Basic Local Alignment Search Tool (BLAST) on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Results
The sequences obtained from the two individuals were identical (GenBank accession numbers OM574908, OM574909). BLAST results showed 99.85% identity with P. papillosa with a high coverage (97%). This sequence corresponds to an individual collected in Coquimbo, Chile by Gomes et al. (Reference Gomes, Schama and Solé-Cava2012) (GenBank accession number JN118564.1). The second most identical species is Phymactys clematis with 99.19% identity but only 71% coverage (GenBank accession number KY789425.1) and the third Bunodossoma caissarum Corrêa in Belém, 1987, a species from Brazil, with 88.59% identity and 100% coverage (GenBank accession number JN118560.1).
The individuals collected in this study were both green (Figure 2). Their external morphology corresponds to the one described by Haussermann (Reference Häussermann2004) for P. papillosa (Table 1): the individuals had ~3 cm pedal disc diameter, the column was covered with rounded non-adhesive vesicles, the tentacles and oral disc, with a prominent mouth opening, were slightly paler than the column. The oral disc had numerous short tentacles in its margin, and acrorhagi were present in the fosse (Figure 2).
Left: Phymactis papillosa recorded at intertidal shore of Sintra, Portugal (date: 21 July 2021) (coordinates: 38°51′33.55″N 9°26′56.53″W); Right: Phymactis papillosa amplified photo to highlight column external morphology.
Description of Phymactis papillosa, after Häussermann (Reference Häussermann2004)
Discussion
Globally, invasive species have transformed marine habitats and have been described as the second leading cause of species extinction (Molnar et al., Reference Molnar, Gamboa, Revenga and Spalding2008; Bellard et al., Reference Bellard, Cassey and Blackburn2016). Habitat displacement of native species and changes in the structure of communities and in the food chain are among the impacts they can cause (Molnar et al., Reference Molnar, Gamboa, Revenga and Spalding2008). Although only a small fraction of introduced marine species manage to proliferate and invade new habitats, their impact can be quite detrimental (Mack et al., Reference Mack, Simberloff, Lonsdale, Evans, Clout and Bazzaz2000). Any knowledge regarding the invasive species is of extreme interest since it may be possible to predict their behaviour and expansion.
Many anemone species tolerate hard conditions and potentially can proliferate rapidly by asexual reproduction, making this group well-adapted for introduction. Although active dispersal by sea anemones is limited to crawling or floating of adults or short-range dispersal of gametes and larvae, passive transport by rafting or by human mechanisms enables long-distance (transoceanic and interoceanic) dispersal. There are several documented sea-anemone invasions associated with mariculture. Additionally, commercial shipping and cruise liners can also promote invasions by hull fouling, transport in ballast water, and attachment in sea-chests, which probably have been underestimated (Glon et al., Reference Glon, Daly, Carlton, Flenniken and Currimjee2020). Given the distance between the native distribution of P. papillosa and Europe, the passive dispersal of adult individuals attached to vessels seems the more probable vector although the exact vector and point of arrival of this species into Europe is not known.
After transport and establishment, an invasive species must be capable of spreading (Glon et al., Reference Glon, Daly, Carlton, Flenniken and Currimjee2020). The behaviour and interaction with native species are crucial at this point. In fact, agonistic behaviours with other anemones have already been reported for the species P. papillosa, namely with an invasive species Anemonia alicemartinae, revealing P. papillosa as a stronger competitor, even when it is at a disadvantage in number of individuals (Brante et al., Reference Brante, RIera and Riquelme2019). Furthermore, the different phenotypes that this species presents seem to be related to aggressiveness. Green anemones are generally more aggressive in interspecific interactions (Brante et al., Reference Brante, RIera and Riquelme2019). It is necessary to consider the aggressive behaviour characteristic of the species described in this study, since its interaction with native species can lead to disturbances in the ecosystem, such as changes in habitat occupation. The knowledge on this topic is extremely relevant since interactions of this anemone with native marine species in Europe are not described. Thus, further studies are needed to infer the effect of the rapid increase in the abundance of P. papillosa in the European marine ecosystems.
Data available from citizen science platforms (iNaturalist www.inaturalist.org/taxa/195551-Phymactis-papillosa and reflected in www.gbif.org/pt/species/2256771) suggest occurrences in Alentejo coast (south-west Portugal) and Mediterranean coast around Murcia (Spain) (although there is no scientific validation of the identity of the species for each record), but the exact distribution of Phymactis papillosa in European shores is yet to be understood.
In this study, DNA barcoding was used to identify a non-native sea anemone species. This method, widely used in other taxonomic groups, has a hindered application in Cnidaria, due to the difficulties in selecting the appropriate marker, as the conventional COI (cytochrome oxidase I) has a very slow rate of evolution in some groups (e.g. Hellberg, Reference Hellberg2006), but not in all (e.g. Ortman et al., Reference Ortman, Bucklin, Pagès and Youngbluth2010). In fact, Shearer & Coffroth (Reference Shearer and Coffroth2008) have shown that levels of intraspecific variability and interspecific divergence overlap in scleractinean corals, precluding the use of this molecular marker in barcoding. McFadden et al. (Reference McFadden, Benayahu, Pante, Thoma, Nevarez and France2011) found the same pattern in Octocorallia. The inclusion of additional molecular markers increased the success of identification of morphospecies, but it seems to have also a limited application (e.g. 28S ribosomal nuclear DNA – McFadden et al., Reference McFadden, Brown, Brayton, Hunt and van Ofwegen2014, Quattrini et al., Reference Quattrini, Wu, Soong, Jeng, Benayahu and McFadden2019; Internal transcribed spacer II – Oliverio et al., Reference Oliverio, Barco, Modica, Richter and Mariottini2008). Recently Gong et al. (Reference Gong, Ding, Wang, Jiang and Zu2018) recommended the use of nuclear DNA (ITS, 18S rDNA and 28S rDNA) for barcoding in Cnidaria. However, the success of this approach is dependent on complete and reliable DNA barcode reference libraries (Paz & Rinkevich, Reference Paz and Rinkevich2021). Phymactis papillosa, identified in this study by molecular barcoding, has only one available sequence on GenBank (ITS I and ITS-II), which stresses the need to compile DNA sequences from organisms identified by experts, to enable an effective use of DNA barcoding in this group.
Author contributions
AMP and OM collected the samples and designed this study; AMP and MMS performed the laboratory work; AMP conducted data analysis; all authors wrote the manuscript.
Financial support
This study had the support of national funds through Fundação para a Ciência e Tecnologia (FCT), under the project LA/P/0069/2020 granted to the Associate Laboratory ARNET.
Conflict of interest
The authors declare none.
Data availability
Genetic sequences are available in GenBank (accession numbers OM574908, OM574909). Voucher specimens used in this study are available upon request and stored at ISPA – Instituto Universitário collections.
