Taxonomic Re-Evaluation of the Non-Native Cichlid in Portuguese Drainages
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Taxonomic re-evaluation of the non- native cichlid in Portuguese drainages João Carecho1, Flávia Baduy2, Pedro M. Guerreiro2, João L. Saraiva2, Filipee Ribeiro3, Ana Veríssimo44,5* 1. Instituto de Ciências Biomédicas Abel Salazar, Universidade do Poorrto, Porto, Portugal 2. CCMAR, Centre for Marine Sciences, Universidade do Algarve, 8005-139 Faro, Por- tugal 3. MARE – Marine and Environmental Sciences Centre, Faculty of Sciences, University of Lisbon, Lisbon, Portugal 4. CIBIO - Research Centre in Biodiversity and Genetic Resources, Caampus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal 5. Virginia Institute of Marine Science, College of William and Mary,, Route 1208, Greate Road, Gloucester Point VA 23062, USA * correspondence to [email protected] SUUMMARY A non-native cichlid fish firstly repo rted in Portugal in 1940 was originally identified as Cichlasoma facetum (Jenyns 1842) based on specimens reported from “Praia de Mira" (Vouga drainage, northwestern Portugal). Currently, the species is known only from three southern Portuguese river drainages, namely Sado, Arade and Gua- diana, and no other record has been made from Praia de Mira or the Vouga drainage since the original record. The genus Cichlasoma has since suffered major taxonomic revisions: C. facetum has been conssidered a species-complex and proposed as the new genus Australoheros, including many species. Given the currennt taxonomic re- arrangement of the C. facetum species group, we performed a taxonomic re- evaluation of species identity of this non-native cichlid in Portuguese drainages us- ing morphological and molecular analyses. Morphological data coollected on speci- mens sampled in the Sado river drainages confirmed the identification as Australo- heros facetus. Moreover, nucleotide sequences of the cytochrome b gene obtained from specimens from Sado, Arade and Guadiana showed the existence of a single haplotype across drainages, which was 100% identical to A. facetus specimens col- lected in native Argentinean waters (i.e. Uruguay River). The currrent non-native distribution range of the species in Portugal results from human-mediated introduc- tions across the southern drainages. Keywords: Non-native fish, Cichlids, Taxonomic identification, Morphology, Cytochrome b Citation: Carecho J, Baduy F, Guerreiro PM, Saraiva JL, Ribeiro F, Veríssimo A (2018) Taxonom- ic re-evaluation of the non-native cichlid in Portuguese drainages. Fishes in Mediterrane- an Environments 2018.001: 12p. https://doi.org/10.29094/FiSHMED.2018.001 1 Non-native cichlids in Portugal DOI: 10.29094/FiSHMED.2018.001 INTRODUCTION The Iberian NNFF include a single cichlid species, representing the oldest Neo- Non-native species, and particularly tropical ornamental fish brought alive to those that become invasive, are considered Europe, in 1889 (Říčan & Kullander, 2006). one of the major global threats to biodiversi- The first record of this cichlid in Portugal, ty, only surpassed by habitat loss and frag- originally identified as Cichlasoma facetum mentation (Mack et al. 2000; Clavero & (Jenyns 1842), refers to specimens deposited García-Berthou, 2005; Gallardo et al., 2016). in the Museum of Zoology of the University Recent estimates suggest that over 12 000 of Coimbra (Catalog no.s ZOO5459-5462; non-native species are currently present Figure 1). However, this record raises sev- within European Union borders, of which eral questions regarding the original cap- around 10–15% are invasive (Sundseth, ture location of the specimens, as it includes 2014). The economic consequences of biolog- four cichlid fish plus an incorrectly identi- ical invasions are remarkable and amount fied specimen of the marine fish genus to about €12 billion/year over the past 20 Balistes (Catalog no. ZOO6567), reported years; yet, this amount is expected to in- from Mira - Praia (Figure 1), on the Atlantic crease considering the increasing rates of coast. The presence of Balistes mixed in new introductions (Sundseth, 2014; Geno- with the cichlid fishes is confusing as it in- vesi et al., 2015). vokes a marine environment as the capture location, further suggested by the mention Among vertebrates, freshwater fish- of “Mira – Praia” (Mira Beach). However, es are the most widely introduced species the same record also mentions “Viveiros e globally and comprise the best-studied Valas” (i.e. hatchery and drainage chan- group regarding invasion impacts (Rahel, nels), alluding to the hatchery facilities at 2002; Leprieur et al., 2008; Strayer, 2010). Praia de Mira ("Posto Aquícola de Praia de Not surprisingly, non-native fishes are con- Mira"), a government facility from the Gen- sidered one of the major global causes of eral Forestry Directorate, that was closed decline in aquatic biodiversity (Cowx, 1998; down in 1980s. This hatchery facility was Helfman, 2007). This is of particular con- dedicated to acclimatize and promote the cern within the Mediterranean biodiversity introduction of “warm” water freshwater hotspot, where many regions show high lev- fishes (Bochechas, J. personal communica- els of aquatic endemism, namely freshwater tion). fishes (Myers et al., 2000; Smith & Darwall, 2006). In addition to the current ambiguous collection locality of the original record of C. The Iberian Peninsula (Portugal and facetum in Portugal, the genus Cichlasoma Spain) is a notable example of the current has since undergone major taxonomic revi- situation in Mediterranean freshwater eco- sions, and many of its species were left systems: non-native freshwater fish (NNFF) without a generic name until 2016 (Říčan et records are among the highest within the al., 2016). In particular, the Cichlasoma Mediterranean basin although the region facetum group was thoroughly revised and hosts a large number of endemic freshwater the new genus Australoheros was proposed fish species (Elvira & Almodóvar, 2001; Ri- including several species (Říčan & Kulland- beiro et al., 2009). A recent revision indicat- er, 2006, 2008; Říčan et al., 2011). Despite ed over 30 NNFF within Iberian drainages the recent taxonomic re-arrangement of C. (Marr et al., 2013), and this number has facetum, no study has critically evaluated since increased (e.g. Aparício, 2015; Ribeiro the species identity of the non-native cichlid et al., 2015). Indeed, estimates of one new species in Portuguese waters although species every two years have been made for many authors have assumed it corresponds Portuguese freshwater ecosystems alone to Australoheros facetus (e.g. Ribeiro et al., (Ribeiro et al., 2009; Ribeiro, F. unpublished 2007; Baduy et al., 2016). data). 2 Non-native cichlids in Portugal DOI: 10.29094/FiSHMED.2018.001 Given the ambiguity surrounding the guese drainages. To this end, we conducted first record of C. facetum in Portugal and morphological and molecular analyses on a the recent taxonomic re-arrangement of this sample of fish specimens collected in all ma- particular taxon, this study aims to re- jor Portuguese drainages where the species evaluate the species identification of the is currently known to occur. non-native cichlid fish present in Portu- Figure 1. Above, specimens associated with the original first record of Australohe- ros facetus (= Cichlasoma facetum) (Jenyns 1842), deposited in the Museum of Zo- ology of the University of Coimbra, together with a specimen of the genus Balistes (first from left), collected on October 13th 1940 by Helmut Helling at Mira – Praia, Viveiros (Portugal). Below, detail photographs of specimens ZOO5462 (letft) and ZOO5461 (right). 3 Non-native cichlids in Portugal DOI: 10.29094/FiSHMED.2018.001 METHODS Genomic DNA was extracted using the EasySpin® Genomic DNA Miniprep Tis- Specimens were sampled through sue Kit (Citomed, Lisbon, Portugal) follow- electric fishing in the Sado, Arade and Gua- ing the manufacturer’s instructions. A diana drainages (Figure 2), following proto- fragment of the cytochrome b gene (~1200 cols described in Ribeiro et al. (2007). Twen- bp) was amplified via the polymerase chain ty whole fish (standard length: 37 – 80 mm) reaction (PCR) with the primers GLuDG.L- from Ribeira de Corona, Sado drainage TGACTTGAARAACCAYCGTTG (Palumbi (38.0265°N, 8.4315°W), were collected and et al., 1991) and H15915- kept frozen until morphological analysis. AACTGCAGTCATCTCCGGGTTACAAGAC Morphological identification to the family-, (Irwin et al., 1991). Each sample was ampli- genus- and species-levels was based on ex- fied in 10 µl reactions including 5 µl of ternal morphological characters and taxo- MyTaq 2x Hot Start (BIOLINE), 0.4 µl of nomic descriptions and/or keys described in each primer (10 µM), 1 µl of gDNA and 3.2 Nelson et al. (2016) and in Říčan & µl of dH2O. The PCR temperature profile Kullander (2006, 2008), respectively. Total included an initial denaturation at 94°C for and standard length were measured to the 5 min, followed by 30 cycles with denatura- nearest millimeter, and meristics included tion at 94°C for 1 min, primer annealing at scale counts on the upper and lower lateral 48°C for 40s, and extension at 72°C for 1 lines, and on the first epaxial longitudinal min, with a final extension step at 72°C for series of scales (E1, sensu Figure 1 from 5 min. Successful amplification was con- López-Fernández & Taphorn 2004), as well firmed by 2% (w/v) agarose gel electrophore- as dorsal and anal fins pines and rays. All sis run at 300V in 0.5X TAE buffer. Ampli- ® meristic counts were made under a dissect- cons were cleaned with 1 µl of ExoSAP-IT ing scope. Fin clips were collected from a PCR clean-up Kit (GE Healthcare, Pisca- total of 41 specimens from the Sado, Arade taway, NJ, USA), and sent to Macrogen Eu- and Guadiana drainages (Table 1), and pre- rope (The Netherlands) for Sanger sequenc- served in 96% ethanol for molecular genetic ing of the forward and reverse strands. analysis. Figure 2. Adult fish captured in the Guadiana river basin with approx. 15 cm of total length. 4 Non-native cichlids in Portugal DOI: 10.29094/FiSHMED.2018.001 Table 1. Information on the samples collected for molecular genetic analyses.