The expansion continues: Girardia arrives in Africa. First record of Girardia sinensis (Platyhelminthes, Tricladida, Continenticola, Dugesiidae) in Morocco Author Benítez-Álvarez, Lisandra Departament de Genètica, Microbiologia i Estadística and Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Avinguda Diagonal 643, 08028, Barcelona, Catalonia, Spain. Author Mateos, Eduardo Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals and Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona. Avinguda Diagonal 643, 08028, Barcelona, Catalonia, Spain. Author Ouanighi, Younes El 0000-0003-1572-766X LESCB URL-CNRST N ° 18, FS, Abdelmalek Essaadi University, Tetouan, Morocco. Avenue Sebta, Mhannech II. 93002 https: // orcid. org / 0000 - 0003 - 1572 - 766 X; https: // orcid. org / 0000 - 0003 - 3819 - 2717; https: // orcid. org / 0000 - 0003 - 2664 - 646 X Corresponding author. lbenitezalvarez 87 @ gmail. com; https: // orcid. org / 0000 - 0002 - 0473 - 4525 lbenitezalvarez87@gmail.com Author Bennas, Nard 0000-0003-1572-766X LESCB URL-CNRST N ° 18, FS, Abdelmalek Essaadi University, Tetouan, Morocco. Avenue Sebta, Mhannech II. 93002 https: // orcid. org / 0000 - 0003 - 1572 - 766 X; https: // orcid. org / 0000 - 0003 - 3819 - 2717; https: // orcid. org / 0000 - 0003 - 2664 - 646 X Corresponding author. lbenitezalvarez 87 @ gmail. com; https: // orcid. org / 0000 - 0002 - 0473 - 4525 lbenitezalvarez87@gmail.com Author Alami, Majida El 0000-0003-1572-766X LESCB URL-CNRST N ° 18, FS, Abdelmalek Essaadi University, Tetouan, Morocco. Avenue Sebta, Mhannech II. 93002 https: // orcid. org / 0000 - 0003 - 1572 - 766 X; https: // orcid. org / 0000 - 0003 - 3819 - 2717; https: // orcid. org / 0000 - 0003 - 2664 - 646 X Corresponding author. lbenitezalvarez 87 @ gmail. com; https: // orcid. org / 0000 - 0002 - 0473 - 4525 lbenitezalvarez87@gmail.com Author Riutort, Marta Departament de Genètica, Microbiologia i Estadística and Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Avinguda Diagonal 643, 08028, Barcelona, Catalonia, Spain. text Zootaxa 2022 2022-08-02 5169 5 497 500 journal article 111182 10.11646/zootaxa.5169.5.9 27d1c9ce-e9eb-44fe-b7d3-8c4b098d256e 1175-5326 6952773 19C1F59A-2CD2-4207-8F75-E04F4D8080DD Biological invasions are an important issue in the conservation of biodiversity. Special interest must deserve the freshwater environments, which are highly sensitive to change in the community composition ( Gallardo et al ., 2016 ; Havel, Kovalenko, Thomaz, Amalfitano, & Kats, 2015). The freshwater planarians are active members of aquatic ecosystems with important regulatory function as predators and used as biological indicators of water quality ( Knakievicz, 2014 ; Wu & Li, 2018 ). The genus Girardia Ball, 1974 is the sister group of a clade including Dugesia Girard, 1850 and Schmidtea Ball, 1974 ( Álvarez-Presas & Riutort, 2014 ). The three genera are the most studied groups in the Dugesiidae family. For Girardia a biogeographic hypothesis ( Ball, 1974 ) proposed that during the formation of the current continents, its lineage was restricted to the land masses which would give rise to South America resulting in the Americas as the area of origin for all present-day species. A recent molecular study has given support to that hypothesis (Benítez-Álvarez et al ., under review). However, in the 20’s of last century the presence of Girardia genus was reported in Germany ( Meinken, 1925 ), and by the end of the 60’s its presence had reached a great part of Western Europe ( Gourbault, 1969 ; Saló, Baguñà, & Romero, 1980). Nowadays, it is also present in Australia , Japan , and Hawaii (Sluys, Kawakatsu, & Ponce De León, 2005; Sluys, Kawakatsu, & Yamamoto, 2010). In addition, new reports of Girardia tigrina (Girard, 1850) in the Balcans region ( Ilić et al ., 2018 ) and East Europe ( Kanana & Riutort, 2019 ) have been published recently. Moreover, a new species, G. sinensis Chen & Wang, 2015 was described from a locality in China (Chen, Chen, Wu, & Wang, 2015), but molecular data analyses showed that the species have a North American origin, taking into account its close relationships with G. dorotocephala and G.tigrina ( Kanana & Riutort, 2019 ; Benítez-Álvarez et al ., 2022 ) and the biogeographical history of the genus, in consequence its presence in China will also be the result of an introduction. Here we report for the first time, the presence of Girardia sinensis in Morocco and consequently of Girardia genus in Africa. Since the specimens found were sexually immature, we identified them to species level using two molecular markers: a fragment of the mitochondrial gene Cytochrome Oxidase I (COI) and the nuclear gene Elongation Factor 1 alpha (EF1a). The specimens were collected in Laou river , Morocco , under a bridge on P4104 road, at 199 meters of altitude and the geographical coordinates: 35.26299 , - 5.26036 in April 2019 ( Figure 1 ). Three asexual individuals were identified as Girardia sp. by their external features (triangular shape of the head, dotted and light pigmentation) and were conserved in absolute ethanol . Because the individuals were very small ( 3 mm approximately), the entire animal was used for the DNA extraction with Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. DNA quantification, PCR amplification, purification, sequencing and contig obtaining were performed as described in Benítez-Álvarez et al ., (2022). Six new sequences were obtained, and 57 sequences of Girardia were downloaded from GenBank ( Figure 2 ). The downloaded sequences are representative of 11 species, including the North American species: G. tigrina , and G. dorotocephala Woodworth, 1897 , and G. sinensis described from China ; six South American species: G. schubarti Marcus, 1946 , G. biapertura Sluys, 1997 , G. multidiverticulata Souza, Morais, Cordeiro & Leal-Zanchet, 2015 , G. anderlani Kawakatsu & Hauser, 1983 , G. sanchezi Hyman, 1959 , G. clandistina Sluys & Benítez-Álvarez 2022 ( 2022 ) , and three non-identified individuals, one from Chile and two from Mexico . The final alignment had 1716 positions (837 for COI and 879 for EF1a). A phylogenetic tree was inferred with the Bayesian Inference method implemented in MrBayes v3.2.2 (Ronquist et al ., 2012) using a partitioned scheme by codon and the General Time Reversible model + Gamma Distribution + Invariable Sites (GTR + Г + I). FIGURE 1. Locality where the three samples were collected in Morocco river Laou The phylogenetic tree ( Figure 2 ) places the three individuals from Morocco in the G. sinensis clade, supporting its classification as belonging to this species. At the same time, we can confirm with a broader taxonomic sampling the assignment of Kanana & Riutort (2019) of samples from Ukraine (Gti_ukraine in the tree) to G. tigrina species. In summary, we report for the first time the occurrence of Girardia in the African continent, specifically of G. sinensis in the Laou river, Morocco . We used molecular data to perform the taxonomic identification of asexual individuals, validating the use of molecular techniques to species identification in this group. Our work supports previous results demonstrating the introduction of three species of Girardia in Europe, Asia and now, Africa. It is worrisome how the broadening of our samplings of freshwater planarians has taken us to uncover more and more introduced species all around the world. Taking into account the rapid colonization and the success of Girardia in Europe, its arrival to Africa deserves special attention for the potential damage that it could represent for African freshwater communities. Other American invaders as the Nearctic “water boatman” Trichocorixa verticalis verticalis (Fieber, 1851) , have been reported in Europe and Africa with high colonizing success ( Carbonell, Céspedes & Green, 2021 ; L’Mohdi et al ., 2010 ; Guareschi et al ., 2013 ; Taybi et al ., 2020 ). We want to make a call to the scientific community, about the importance of monitoring the introduced species in freshwater ecosystems, to detect any detrimental consequences to those delicate ecosystems, and to recall the usefulness of genetic data to rapidly identify and study the origin of the alien species.