Insights into the fauna associated with Egeria densa at the upper section of the international Minho River estuary (NW Iberian Peninsula) 3 decades after its establishment

The macrophyte Egeria densa Planchon, 1849 is a freshwater plant native to the subtropical regions of South America. Fast vegetative reproduction and efficient dispersal allow this species to form extensive beds that produce high levels of oxygen in freshwater ecosystems, generating microhabitats that act as refuge and nursery for an array of organisms, increasing sedimentation and light availability. Despite its undisputable ecological role, it is considered invasive and is present on all continents except Antarctica with the first records in Europe occurring at the 20th century. At the international Minho River (NW Iberian Peninsula), the observation of its presence was noted in the 1990s and is now an established population in this ecosystem. This study is the first descriptive-taxonomical assessment of the associated fauna with the exotic macrophyte E. densa, using both morphological and molecular approaches, three decades after its establishment in the international Minho River. Results indicate the presence of a faunal assemblage, composed a total of 20 identified species, including platyhelminths, hydrozoans, bryozoans, molluscs (Gastropoda), annelids (Oligochaeta and Hirudinea), crustaceans (Ostracoda, Copepoda, Branchiopoda and Amphipoda), aquatic mites and insects. Paludicella aff. articulata (Ehrenberg, 1831), Girardia sinensis Chen & Wang, 2015, Lebertia insignis Neuman, 1880 and Ceriodaphnia rigaudi Richard 1894 are recorded for the first time in Portugal.


Introduction
The species Egeria densa Planchon, 1849 is a freshwater perennial plant native to the subtropical regions of South America, which forms dense mats that cover extensive areas. Considered invasive is now present on all continents except Antarctica (Yarrow et al. 2009). Development of E. densa beds lowers water velocity causing sedimentation of suspended particles, increasing light availability (Yarrow et al. 2009). Thus this aquatic plant plays an important role on the structure and function of freshwater environments, generating microhabitats used as refuge and nursery for zooplankton, macroinvertebrates and fish (Pelicice and Agostinho 2006;Yarrow et al. 2009). Furthermore E. densa promotes phytoplankton biomass decline (Mazzeo et al. 2003), stimulates an increase in epiphytic macroinvertebrate biomass which subsequently increases carnivorous fish density (Diehl and Kornijów 1998). The first records in Europe occurred at 20th century (Yarrow et al. 2009), and the first observation at the international Minho River (NW Iberian Peninsula) occurred during the 90ʼs (Antunes, pers. comm.). Since then E. densa developed populations along this freshwater system, generating dense mats on otherwise sandy intertidal environments, forming a microhabitat distinct from its native counterparts that cover mainly the subtidal section. A first attempt at a descriptive-taxonomical assessment of the fauna associated with the exotic macrophyte E. densa at the International Minho River, using both morphological and molecular approaches, provided 12 new records for the study area, including the exotic species Girardia sinensis Chen & Wang, 2015, Menetus dilatatus (Gould 1841) and Helobdella europaea (Kutschera 1985).

Study area
The estuarine area of the international Minho River, located at the northwest Spanish/Portuguese border (NW Iberian Peninsula), has a length of approximately 40 km (Sousa et al. 2008a), with a mesotidal partially mixed system tending towards a salt wedge estuary during the high floods (Sousa et al. 2005). Sampling was performed at the upper section of the estuary, on the Portuguese margin in front of Morraceira Island (Vila Nova de Cerveira), approximately 19 km from the river's mouth (Fig. 1a). This section of the river is a predominantly freshwater system throughout most of the year, characterized by saline intrusions during the summer season, especially during the dryer months (Pereira et al. 2022) and its margins are covered by monospecific dense mats of Egeria densa (Fig. 1b,c) that overlays the supratidal area, extending to the upper subtidal.

Specimen sampling, identification and preservation
Samplings were performed in the upper section of the international Minho River estuary ( Fig. 1), near Morraceira Island, Vila Nova de Cerveira, Portugal. Aerial parts of E. densa were sampled monthly from October 2021 to March 2022, in an intertidal zone, with each sample being collected manually in a area delimited by a square box of  ,1 m 2 and bagged for posterior analysis. Species identification was performed using specialized literature (Brinkhurst 1971;Brinkhurst and Jamieson 1971;Lincoln 1979;Cornelius 1995;Alonso 1996;Tachet et al. 2000;Wood 2015;Govedich and Moser 2015;Rogers 2019;Horne et al. 2019;Lee and Lee 2019;Noreña et al. 2019;Conesa-García 2021). All specimens were photographed with a Leica EZ4W stereomicroscope, a Nikon Digital Sight D5-L1 camera using a Nikon SMZ800 stereomicroscope and a Nikon ECLIPSE 50i microscope. Specimens were preserved in 70% ethanol and deposited at the Natural History Museum of the Iberian Peninsula (NatMIP -"Museu de História Natural da Península Ibérica"), sited at Aquamuseu do Rio Minho, Vila Nova de Cerveira, North Portugal.

Genetic analysis
Genomic DNA extraction was performed using a using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-tek), following manufacturer's instructions. Amplification of the COI-5P region was carried out using the primers LoboF1 (5'-KBTCHACAAAYCAYAARGAYATHGG-3') and LoboR1 (5'-TAAACYTCWGGRTGWCCRAARA-AYCA-3') (Lobo et al. 2013), and a pre-made PCR mix (VWR International, LLC, Pennsylvania, USA). PCR reactions were comprised of 1× PCR buffer, 1.5 mM of MgCl2, 0.2 mM of the dNTP mixture, 1 U of DNA Taq polymerase, plus 0.5 µM of each primer, ca. 30-50 ng of genomic DNA, and sterile MilliQ-grade water to make up a total volume of 25 µL. The reactions were run on a Hybaid PxE Thermocycler (Thermo Electron Corporation, Milford, Massachusetts), following the PCR conditions in Lobo et al. (2013). PCR products were electrophoresed through a 1% agarose 1× Tris-acetate-EDTA buffer (TAE) gel stained with GreenSafe Premium (Nzytech, Lisbon, Portugal). Positive PCR products were purified using the ExoFast method, in which an enzymatic cleanup that eliminates unincorporated primers and dNTPs is performed with Exonuclease I (Escherichia coli) and FastAP Thermosensitive (SAP), and then sequenced directly. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems kit (Applied Biosystems, Carlsbad, California), and were run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Stabvida, Oeiras, Portugal). DNA sequence homology searches and species level identifications were performed using BOLD (Hebert and Ratnasingham 2007) and with BLAST (Altschul et al. 1990) in GenBank databases (Benson 2004). COI sequences were edited and manually aligned using MEGA X (Kumar et al. 2018) and checked for insertions, deletions and stop codons. For phylogenetic reconstruction, COI-5P sequences were retrieved from public databases as follows: sequences for the species in analysis, sequences from the respective genus and sequences from a species of the same family used as outgroup. For the species Bothrioneurum vejdovskyanum, Cypridopsis vidua and Menetus dilatatus, which had no available sequences for congeners, only sequences belonging to members of the same family were retrieved. Specimens of Paludicella aff. articulata and Embolocephalus sp., had no match in BOLD or GenBank. Thus, phylogenetic reconstruction for P. aff. articulata was carried out using sequences belonging the order Ctenostomida and Cyclostomatida for outgroup. For Embolocephalus sp. other sequences from the genus Embolocephalus, as well as from the subfamily Tubificinae were included with Naidinae used as outgroup. Phylogenetic relationships were reconstructed through Maximum-Likelihood, using the substitution model GTR + I + G, as chosen by JModelTest (Posada 2008). MOTUʼs were delimited using the species delineation tool Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012). Intra-and inter-specific distances were calculated on MEGA X using the K2P substitution model.

Morphological analysis
A total of 3370 specimens were examined, representing 20 species belonging to 6 phyla, 16 families and 16 genera. Among these 20 species identified using morphological characters, 4 could only be identified to the family level only (Table 1).

Integrated taxonomy (morphology and genetic analysis)
A total of 3370 specimens were examined, representing 20 species belonging to 6 phyla, 16 families and 20 genera. Twelve species constituted new records for the International Minho River (SI). Four species had species level misidentifications, using morphological characters, which could be resolved through the integrative analysis of morphological and genetic features. Another 4 species could only be assertively identified after genetic analysis ( Table 1) lineages with 4-5% genetic distance, two of which occur sympatrically in the study area (Fig. 5b). Embolocephalus sp. (identified morphologically as Embolocephalus cf. velutinus) is revealed to be a sister taxon of E. velutinus (15% genetic distance) forming a distinct clade within the genus Embolocephalus (Fig. 5c). Exotic species, H. europaea, M. dilatatus and G. sinensis present shared haplotypes spanning multiple continents (Figs. 2a and 3b and c). COI amplification of the hydrozoan Sertularia cupressina failed, revealing a need of specific primers for this group. Physella acuta amplification has shown the presence of chimerical DNA between this gastropod and the epizoic oligochaete Chaetogaster limnaei, which was posteriorly confirmed inside P. acuta mantle cavity. Ecological notes Freshwater leach, reaching up to 20 mm (Kutschera 1985); predator (Kutschera 1987); sampled in salinities ranging from 0.06 to 0.05, more abundant in October when water temperatures reached 17.75 °C.
Remarks Kutschera (2004), suggested that this species is exotic to Europe based on how the species COI sequences clustered closely to Helobdella triseralis (Blanchard, 1849) from South America, instead of its European counterparts; haplotypes shared between Europe, Oceania, Asia and North America is indicative of a generalized colonization event originating from a similar founding population (Fig. 3a). Helobdella europaea (Kutschera 1985) (Fig. 6a).
Material examined 28 specimens collected from October to December 2021, on E. densa leaves; 1 specimen with COI sequence, GenBank accession number: OP912901.

Distribution in Portugal
International Minho River (this study) and Ferreira River (Valongo, Porto) (Ferreira et al. 2022). Ecological notes Common on stagnant waters rich in organic matter specially among macrophytes (Dumnicka 2007); sampled in salinities ranging from 0.06 to 0.04, water temperatures ranging from 17.75 to 7.69 °C.
Remarks Species level identification based on molecular data; detected the presence of two sympatric lineages with 5% genetic distance in the study area (Fig. 5b).
Remarks This specimen requires further examination to clarify its identification. Nonetheless this is the first record of the genus Chaetogaster for the study area. Remarks Genetic analysis shows this specimen as a sister taxon to Embolocephalus velutinus with a 15% distance between the two species, thus forming a distinct clade within the genus Embolocephalus, indicating the need for a revision of this genus in western Europe (Fig. 5c).
Geographical distribution Species recorded in Europe, Africa, North America and Asia (Brinkhurst and Jamieson 1971; Jongwoo 2011). Ecological notes Sampled in salinities ranging from 0.06 to 0.04, water temperatures ranging from 17.75 to 7.69 °C; found on pools of streams (Sabatino et al. 2010).
Remarks Species level identification based on molecular data; although the specimen formed a MOTU within the 2% threshold with DNA barcodes of Lebertia insignis from Norway and Montenegro, it forms a clearly distinct lineage (Fig. 2b)

Distribution in Portugal
International Minho River (this study).
Geographical distribution Commonly distributed in tropical and subtropical regions, more rarely in temperate regions (Alonso 1996). Macrocyclops sp. (Fig. 7d) Material examined 1 specimen collected January 2022, on E. densa leaves.
Ecological notes Predator (Abdullahi 1992); sampled in salinity of 0.06, water temperature 9.62 °C. Material examined 6 specimens, collected from January to February 2022, on E. densa leaves.

Distribution in Portugal
International Minho River (this study) and Coimbra (Frey 1971), however details for this second record are lacking.
Ecological notes Maximum body size up to 3.3 mm (Alonso 1996); filter feeder (Alonso 1996); sampled in salinities ranging from 0.06 to 0.04, water temperatures ranging from 9.62 to 9.45 °C.

Distribution in Portugal
International Minho River (this study), species recorded between Bragança and Chaves, in River Divor, on Arraiolos (Cobo et al. 2001) and at the Azores archipelago (Murray et al. 2004). Remarks Species level identification based on molecular data; obtained DNA sequence formed a singleton MOTU, nested within group 1 of Cypridopsis vidua complex (Fig. 4b), comprised of several MOTUʼs. This unusual complex is further described in Cywinska and Hebert (2002 Psectrocladius limbatellus (Holmgren 1869) (Fig. 9a).
Remarks Phylogenetic reconstruction through COI sequences did not reveal any reliable placing or grouping within Ctenostomatida (Fig. 4a). Remarks Species level identification based on molecular data; genetic analysis revealed the presence of 5 different MOTUʼs; the sequence obtained nested within a MOTU comprised of exclusively of European sequences (Fig. 5a); minimum and maximum K2P distances between lineages are 14 and 22% respectively.
Geographical distribution Europe and Asia, from Iberian Peninsula to Japan (Boudot and Salamun 2015).
Material examined 20 colonies, collected from September 2021 to February 2022, on E. densa leaves; 1 specimen with COI sequence, GenBank accession number OP913190. Ecological notes Sampled in salinities ranging from 0.06 to 0.04, water temperatures ranging from 17.75 to 7.69 °C.

Distribution in Portugal
Remarks Shared haplotypes between Europe, Asia and North and Central America is suggests a generalized colonization event originating from a same founding population (Fig. 3b).
Material examined 326 specimens collected from October 2021 to March 2022 on E. densa leaves.
Remarks As this species first description is based on specimens collected in France (Draparnaud 1805), its introduction in Europe probably occurred before the 19th century, with the first record from the Iberian Peninsula dating from 1845 (García-Berthou et al. 2007) and from 1872 at Macaronesian archipelagos (Taylor 2003).
Material examined 4 specimens, size 1.5 mm, collected November 2021 and February 2022 on E. densa leaves.
Remarks Pfenninger et al. (2003) suggested the existence of four cryptic species of Ancylus fluviatilis, three of which occur in Portugal (one at the north region, one at the south and another occurring along the coastal line). (Holyoak et al. 2019) (Bodt et al. 2020).
Species level identification of Oligochaeta remains rather challenging for non-specialists (Table 1), even with highly detailed taxonomic guides, all specimens collected required further morphological and genetic analysis. The specimens identified as E. cf. velutinus have shown an incongruent identification with known COI-5P sequences for this species (15% genetic distance) which indicates the need for a revision of this genus in western Europe (Fig. 5c). Phylogenetic analysis of (B) veydovskyanum revealed a possible species complex forming three marked monophyletic clades with 4-5% genetic distance, with two of those lineages occurring sympatrically in the study area (Fig. 5b).
In turn the sequence of P. aff. articulata, did not retrieve significant percentage of similarity to any other sequence available on BOLD and GenBank. A placement within the order Ctenostomatida was attempted, however COI phylogenetic signal did not produce significant results, with low bootstrap support (Fig. 4a) and erratic topologies, which can be explained by an incipient state of Ctenostomida DNA reference libraries.
Species identification is becoming a highly dynamic process with the addition of DNA barcoding methodologies, forcing the re-evaluation of identifications based on morphological characters in a constant feedback loop. The absence of dichotomous keys for recently introduced exotic species (such as G. sinensis, M. dilatatus and H. europaea) in identification guides clearly hampered our study, as did the lack of historic data regarding macroinvertebrate assemblages before and after the introduction of E. densa in the study area. Furthermore, without the performed genetic analysis, misidentifications (M. dilatatus identified as Gyraulus parvus, G. sinensis identified as Schmidtea sp. and H. europaea identified as Helobdella stagnalis) and incomplete identifications (Table 1), could had further propagate the error on to Girardia sinensis Chen & Wang, 2015 (Fig. 10d).
Type material Specimens deposited at Institute of Zoology, Chinese Academy of Sciences, Beijing, China (Chen et al. 2015).
Material examined 521 specimens collected from October 2021 to March 2022 on E. densa leaves; 1 specimen with COI sequence, GenBank accession number: OQ185384.
Geographical distribution Scattered records worldwide.

Distribution in Portugal
International Minho River (this study).
Ecological notes Maximum body size up to 15.2 mm (Chen et al. 2015); sampled in salinities ranging from 0.06 to 0.04, water temperatures ranging from 17.75 to 7.69 °C.
Remarks Species level identification based on molecular data; shared haplotypes between Europe, Oceania, Asia, North Africa, North and Central America suggests a generalized colonization event originating from small founding populations (Fig. 3c). Considering that the genus Girardia is known for its distribution from North to South America only (Sluys et al. 2005), and based on the phylogenetic placement as a sister taxa to the North American Girardia tigrina and Girardia dorotocephala, G. sinensis is most likely of North American ancestry.

Declarations
Competing interests The authors have no competing interests to declare that are relevant to the content of this article.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. other efforts developed in the study area. This highlights the necessity of well-prepared DNA reference libraries that can provide points of comparison between species identifications, reduce errors of interpretation and incomplete information provided by most identification guides. Comparing our faunal composition, associated with E. densa, with results obtained by Pedroza-Ramos et al. (2016) on Lake Tota, Colombia, reveals a similar composition at the family level and overlapping genera such as Helobdella, Eurycercus, Cypridopsis, Macrocyclops, Ischnura, Girardia and Physella. Since most studies related to E. densa associated fauna have been performed in freshwater environments (Collier et al. 1999;Pedroza-Ramos et al. 2016), this study is one of the first attempts to characterize taxonomically its interactions on estuarine habitats, and so it is interesting that we found such similarities, especially regarding the American ancestry of some of this genera.

Conclusions
This study represents the first taxonomic characterization of the fauna associated with the macrophyte Egeria densa at the upper section of the international Minho River. Twenty species were recorded, 12 of which constituted new occurrences in the study area, and 4 were exotic species, with the first records for Portugal of the species Paludicella aff. articulata (Ehrenberg, 1831), Girardia sinensis Chen & Wang, 2015, Lebertia insignis Neuman, 1880 and Ceriodaphnia rigaudi Richard 1894. Local fauna catalogues remain largely unfinished, or outdated, and rarely account for the dynamic shifts of macroinvertebrate assemblages related to established bioengineer exotic species, such as E. densa. However, it is important to know this associated biodiversity for a better cause-effect assessment of species introductions and understand its impact on local ecosystems in order to optimise environmental management.