Crustaceans (Malacostraca and Thecostraca) from the International Minho River, Iberian Peninsula

Crustaceans (Arthropoda) are a diverse and abundant group with chitinous exoskeleton, living on coastal/estuarine environments, at community invertebrate fauna. A survey on the species of some crustacean groups found on the Minho River estuary (international section) in the Iberian Peninsula, is presented with specimens collected through glass eel fishing bycatch, grab sampler, trammel net, beam trawl and fyke net sampling methods. A total of 98 specimens were examined belonging to 7 orders, 21 genera and 23 species (17 new records from Minho River, including one for Portugal). Brief diagnosis, ecological notes, species distributions and figures are provided intending to present taxonomic support on future projects in this region.


Introduction
The subphylum crustacea Brünnich, 1772 is one of the most numerous and diverse groups with more than 60,000 known species [1], with significant importance on estuarine ecosystems for providing a major source in fish and bird diets [2][3][4][5]. Additionally, these marine and brackish arthropods play various ecological roles, like predation [2], parasitism [6] and mesograzing [7]. They are widely used commercially (e.g., Crangon and Palaemon spp.) [2], for the food industry, pharmacological products, aquaculture and indirectly with research involving biotechnology for the reuse of waste from aquaculture [8]. Although crustaceans are a well-studied group, taxonomic problems arise from the lack of knowledge about certain groups, especially species with high sexual dimorphism or differences in juvenile-adult morphology [2,9,10] compounded by information fragmentation. Estuarine ecosystems are of utmost biological importance, being productive and valuable for biodiversity maintenance [11]. Macroinvertebrate surveys on the Minho River estuary are still insufficient to access its biodiversity with few works on macrobenthic ecology or specimens collected through glass eel fishing bycatch [12][13][14][15] and a survey on crustaceans of the order Isopoda [16]. In this study we provide an overview of some groups of the Crustacean fauna (brief diagnosis, ecological notes, global and Portuguese distributions) collected on the Minho River estuary, with a total of 23 species, in which 17 are new records for the Minho River, including one new record for Portugal-Nebalia strausi Risso, 1826. In addition, an update to the list of isopods from Minho River is provided here.

Study Area
Located northwest of the Iberian Peninsula, the Minho River originates in the Meira mountains (Spain), with an extension of around 300 km [17]. The last section of 70 km represents the northwest Spanish/Portuguese border, which is part of the protected areas of the Natura 2000 network as a site of ecological importance and a special area of conservation, marked as an Important Bird Area [18,19]. The estuarine area has a total area of 23 km 2 and Hydrobiology 2022, 1 48 a length of approximately 40 km [13], characterized by a mesotidal partially mixed system tending towards a salt wedge during the high floods [20].

Sampling, Identification and Preservation of Specimens
Specimens examined were collected during previous sampling campaign (including ecological researches from [15,21] at the estuary of the international Minho River Hydrobiology 2021, 1, FOR PEER REVIEW 2 represents the northwest Spanish/Portuguese border, which is part of the protected areas of the Natura 2000 network as a site of ecological importance and a special area of conservation, marked as an Important Bird Area [18,19]. The estuarine area has a total area of 23 km 2 and a length of approximately 40 km [13], characterized by a mesotidal partially mixed system tending towards a salt wedge during the high floods [20].

DNA Extraction, Amplification and Sequencing
DNA samples were extracted using E.Z.N.A. Mollusc DNA Kit (Omega Bio-tek). Amplification of the mitochondrial gene cytochrome c oxidase I (COI-5P), was performed using the primer pairs LoboF1 and LoboR1 [34]. PCR (Polymerase Chain Reaction) reactions contained 21 µL of VWR company (Monroeville, PA, USA) Red Taq DNA Polymerase Master Mix, 1 µL of each primer (10 mM) and 2 µL of DNA template. The PCR cycling conditions used were: 1 min at 94 • C; 5 cycles of 30 s at 94 • C, 1.5 min at 45 • C, 1 min at 72 • C; 35 cycles of 30 s at 94 • C, 1.5 min at 52 • C, and 1 min at 72 • C; 5 min at 72 • C. PCR products were visualized in a 1% agarose gel and purified using ExoSap at 37 • C for 15 min, followed by 15 min at 80 • C. Bidirectional sequencing provided by service supplier (STAB Vida Ltd., Lisboa, Portugal). Obtained sequences were submitted to GenBank with the following accession numbers Ceratothoa aff. oestroides OK642789 and Cymodoce sp. OK646549.

Data Analysis
COI-5P sequences were edited and manually aligned using MEGA software (Kumar et al., 2018) and verified for the presence of stop codons, insertions or deletions. Homology searches were performed in BOLD database [35] and with BLASTn [36] in GenBank database [37]. Maximum Likelihood (ML) trees for the genus Cymodoce (dataset of 36 sequences (Table S1)) and Ceratothoa (dataset of 73 sequences (Table S1)) were constructed with MEGA software using HKY model with Gamma distribution with invariant sites (HKY + G + I), chosen as the best-fit model of nucleotide substitutions by MEGA software. Bootstraps were performed using 10,000 replicates. Mined Ceratothoa oestroides sequences GQ240268 to GQ240280, from GenBank, were trimmed on the final 18 base pairs due to the presence of a stop codon followed by a shift on the reading frame. On the mined sequences from Ceratothoa italica JN604342 to JN604371 the same pattern was observed, and the final 118 base pairs were also trimmed. As no trace files are available for these public sequences, it is not possible to confirm these as pseudogenes or just errors on sequence editing or during submission to GenBank. Intra and interspecific divergence were calculated with the Kimura-2-Parameter (K2P) model as the standard genetic distance used in DNA barcode studies.

Results
A total of 98 specimens were examined belonging to 23 species, six from the order Decapoda, four from Cumacea, three from Mysida, two from Leptostraca, one from Tanaidacea and one from Balanomorpha. An update to the list of isopods from the Minho River [16] was also performed with the addition of six new records.  Type material. Syntypes: Six specimens collected on Hollands Diep, Netherlands, deposited at Naturalis Biodiversity Center, catalogue number RMNH.CRUS.D.29725 [33].
Diagnosis: Rostrum slightly upcurved with seven to eight dorsal teeth and three to five ventral teeth, two of the dorsal teeth behind the orbit; lower half of the rostrum without red-pigmented spots; pereopod 2 carpus equal to or slightly longer than merus [2,22].
Global distribution: Northeast Atlantic from Germany to Morocco and Mediterranean Sea [22,25].
Diagnosis: Rostrum slightly upcurved with seven to eight dorsal teeth and three to five ventral teeth, two of the dorsal teeth behind the orbit; lower half of the rostrum without red-pigmented spots; pereopod 2 carpus equal to or slightly longer than merus [2,22].
Global distribution: Northeast Atlantic from Germany to Morocco and Mediterranean Sea [22,25].
Global distribution: Northeast Atlantic from the Netherlands to the Iberian Peninsula and Mediterranean [2]. Distribution in Portugal: Species recorded along the Portuguese west coast [94]. Ecological notes: Marine; depth range from 0.5 to 100 m; protandrous hermaphrodite [2]. Diagnosis: Male and female with teeth on dorsal midline of carapace; antennula with a single aesthtasc; antenna as long as the body size on male; telson with six perianal setae [27].
Global distribution: Atlantic Ocean from British isles to the Iberian Peninsula and Mediterranean [22,23].
Diagnosis: Female with two to six teeth on dorsal midline of carapace, smooth in male; antennula with a single aesthtasc; antenna as long as the body size on male; telson with two perianal setae [22,27].
Global distribution: Northeast Atlantic Ocean from Norway to the Iberian Peninsula and Mediterranean Sea [22]. Distribution in Portugal: Species recorded along the west coast [94,99,104]. Ecological notes: From coastal intertidal up to depths of 150 m, on fine sand [22].
Global distribution: Northeast Atlantic from Skagerak to Bay of Biscay [23]. Distribution in Portugal: Species recorded along the Portuguese southwest coast [99]. Ecological notes: On coarse sediments; depth range 0-30 m depth [23].

Diastylis cornuta
Global distribution: Northeast Atlantic from Norway to the Iberian Peninsula and Mediterranean [33].
Global distribution: Northeast Atlantic from Baltic Sea to Northwest Africa and Mediterranean Sea [22].
Distribution in Portugal: Common species along the Portuguese coast and estuaries [12,94,113].
Ecological notes: Benthic on coastal shallow to deep waters [30].
Genus Neomysis Czerniavsky, 1882 Neomysis integer (Leach, 1814) ( Figure 5B). Neomysis integer Tattersall  Diagnosis: Antennal scale tapering to a point, with setae all round; telson lateral sides converging gradually to a narrow almost pointed apex; uropods less than twice as long; endouropod with a comb of spines on the inner ventral side [22,30].
Global distribution: Northeast Atlantic [22]. Distribution in Portugal: Species recorded at Ria de Aveiro [118], Cávado [67] and Tejo [119] rivers. Diagnosis: Antennal scale tapering to a point, with setae all round; telson lateral sides converging gradually to a narrow almost pointed apex; uropods less than twice as long; endouropod with a comb of spines on the inner ventral side [22,30].
Order Tanaidacea  Diagnosis: Male with anterior part of the carapace abruptly narrower than posterior part; eyes prominent on both sexes; antenna 1 with three articles on female and five on male; uropod exopodite with two articles and endopodite with four [22].
Ecological notes: From coastal to brackish habitats, constructing tubes on algae and hydroids, or on muddy substrates [22,24]. Type material. Lectotype: One female from Bay of Naples, Italy deposited at Natural History Museum of London (NHMUK) [129].
Global distribution: Northeast Atlantic from northwest France and British isles to the Iberian Peninsula and Mediterranean Sea [29,131].
Distribution in Portugal: New record (this study). Ecological notes: On coarse sediments and rhodoliths-environments, at depths of 4-23 m [2,131].
Diagnosis: Antennal flagellum with thin long setae; supraorbital scale extending beyond the distal end of the eye; pigmented eye; pleonite 4 with acute denticles [29].
Global distribution: Only known from the west Iberian coast [94,132]. Distribution in Portugal: Species recorded along the Portuguese west coast with a southern limit at Aljezur [94].
Ecological notes: Collected at depths between 9 and 33 m, coarse sand bottoms with a high presence of red non-geniculate calcareous algae (Rhodoliths) [132].   Fig. 1C  Diagnosis: Antennal flagellum with thin long setae; supraorbital scale extending beyond the distal end of the eye; pigmented eye; pleonite 4 with acute denticles [29].
Global distribution: Only known from the west Iberian coast [94,132]. Distribution in Portugal: Species recorded along the Portuguese west coast with a southern limit at Aljezur [94].
Diagnosis: Shell with four wall plates, low conical shape, orifice diamond-shaped [22,133]. Global distribution: Native to New Zealand and introduced to northwest Europe, present from Britain to south Portugal [22]. Distribution in Portugal: Species recorded at Setúbal, Faro [136] and Madeira island [137].
Update to Isopods from the Minho River [14]: Diagnosis: Body oval in females, males smaller than females with eyes relatively large; lateral margins of both sexes fairly sparsely fringed with spines; pereopods 6 and 7 of male with the distal region of carpus extended as a lobe fringed with spines ( Figure 7B) [6].
Global distribution: Northeast Atlantic from Russia to France and northwest Atlantic from Greenland to Maine (USA) [6].
Distribution in Portugal: Species recorded along the Portuguese west coast [113,145]. Ecological notes: Common on sheltered shores, occurring often in estuaries usually beneath stones [6].      [157].
Diagnosis: Male cephalon with central concavity and an acute median projection [165]. Global distribution: North Atlantic from Greenland to the British Isles extending southwards to the Mediterranean [6].
Distribution in Portugal: Species recorded on the Mondego estuary [99] and in Sines [166].
Diagnosis: Body length slightly more than three times the width; antenna 2 flagellum longer than peduncle; antenna 1 reaching the distal end of article 3 of antenna 2 peduncle; pleotelson posterior border laterally rounded with a median obtuse tooth [6,165].
Global distribution: Northeast Atlantic from Norway to France [6]. Distribution in Portugal: Species recorded on Tagus estuary [169] and on Azores archipelago [170].
Global distribution: Northeast Atlantic from Norway to France [6]. Distribution in Portugal: Species recorded on Tagus estuary [169] and on Azores archipelago [170].
Ecological notes: Sublittoral on detached algae or fish waste, occasionally on intertidal but also on attached drift algae [6].
Description: Body: Juvenile specimens with uniform color varying bright red to pale greenish yellow ( Figure 9A); adult female with orange and yellow bands and molted white spots on pleotelson and most pereonites ( Figure 9A,B); head pereon and pleon with a smooth dorsal surface ( Figure 9A,B) and pereonite 1 with anterolateral angle extending to the anterior part of the eye on both juvenile and adult female ( Figure 9C).
Diagnosis: Antenna shorter than other Idotea species with flagellum shorter than peduncle; telson rounded or with an obtuse median point on adult specimens [6,165].
Description: Body: Juvenile specimens with uniform color varying bright red to pale greenish yellow ( Figure 9A); adult female with orange and yellow bands and molted white spots on pleotelson and most pereonites ( Figure 9A,B); head pereon and pleon with a smooth dorsal surface ( Figure 9A,B) and pereonite 1 with anterolateral angle extending to the anterior part of the eye on both juvenile and adult female ( Figure 9C).  Pleotelson: Telson rounded with slightly acute median point ( Figure 9D); pleotelson convex on side view ( Figure 9C).
Antennule: Juvenile pedunculum with four segments, first segment with an acute corner on inner margin ( Figure 9E); flagellum with 7 ( Figure 9E) and a large setae on the apex of last segment ( Figure 9F); adult female flagellum with 13 segments (Figure 10A,B).
Antenna: Juvenile pedunculum with four segments and flagellum with 10 ( Figure 10C); apex of flagellum with three large setae on the apex, posterior end of each segment with a tuff of setae ( Figure 10D); adult female pedunculum with four segments and flagellum with 14, posterior end of each segment with a tuff of setae ( Figure 10E).
Antennule: Juvenile pedunculum with four segments, first segment with an acute corner on inner margin ( Figure 9E); flagellum with 7 ( Figure 9E) and a large setae on the apex of last segment ( Figure 9F); adult female flagellum with 13 segments (Figure 10A,B).
Antenna: Juvenile pedunculum with four segments and flagellum with 10 ( Figure 10C); apex of flagellum with three large setae on the apex, posterior end of each segment with a tuff of setae ( Figure 10D); adult female pedunculum with four segments and flagellum with 14, posterior end of each segment with a tuff of setae ( Figure 10E).   Uropod: Juveniles uropods slightly smaller than telson; endopod with marked serrations on inner margin; exopod wither than endopod with subquadrate posterior margin and a slightly rounded inner tip ( Figure 11D). Female uropods slightly smaller than telson, endopod with crenulated posterior margin, exopod as juveniles with small crenulations on posterior margin ( Figure 10E).

Remarks:
The genus Cymodoce has six described species for European waters (Atlantic and Mediterranean) [175]. However, antennule, pleopod 1 and uropod shape on both juveniles and female do not seem to match any of the descriptions provided by Dumay [176][177][178][179]. The strong acute corner on the inner margin of antennule and pleopod 1 lack of setae and spines on the basis do not match any of the descriptions for females, also juvenile uropod shape has no match with Dumay's descriptions. As most detailed descriptions are for adult males and none male was examined the identification of these specimens is lacking on this aspect, nonetheless the possibility of this record being for a new species cannot be ruled out.

Genetic Analysis
Both specimens, Ceratothoa aff. oestroides and Cymodoce sp., have not nested on any clade with other publicly available sequences for their respective genera (Figures 12 and 13), nor matched any sequence available on BOLD or GenBank. Ceratothoa aff. oestroides grouped close to the north Pacific species Ceratothoa arimae (Nunomura, 2001), with a sequence divergence of 9.4%, however this branch bootstrap support was 56. In contrast our sequence did not form a group with other Ceratothoa oestroides sequences that nested within a clade with Ceratothoa italica Schiödte & Meinert, 1883 with an internal sequence divergence of 0.54% (Figure 12). Cymodoce sp. grouped closely with a clade formed by sequences of Cymodoce truncata Leach, 1814 and Cymodoce emarginata Leach, 1818 (sequence divergence of 17%). However, other sequences of Cymodoce truncata were placed on other branches ( Figure 13). Uropod: Juveniles uropods slightly smaller than telson; endopod with marked serrations on inner margin; exopod wither than endopod with subquadrate posterior margin and a slightly rounded inner tip ( Figure 11D). Female uropods slightly smaller than telson, endopod with crenulated posterior margin, exopod as juveniles with small crenulations on posterior margin ( Figure 10E).
Remarks: The genus Cymodoce has six described species for European waters (Atlantic and Mediterranean) [175]. However, antennule, pleopod 1 and uropod shape on both juveniles and female do not seem to match any of the descriptions provided by Dumay [176][177][178][179]. The strong acute corner on the inner margin of antennule and pleopod 1 lack of setae and spines on the basis do not match any of the descriptions for females, also juvenile uropod shape has no match with Dumay's descriptions. As most detailed descriptions are for adult males and none male was examined the identification of these specimens is lacking on this aspect, nonetheless the possibility of this record being for a new species cannot be ruled out.

Genetic Analysis
Both specimens, Ceratothoa aff. oestroides and Cymodoce sp., have not nested on any clade with other publicly available sequences for their respective genera (Figures 12 and 13), nor matched any sequence available on BOLD or GenBank. Ceratothoa aff. oestroides grouped close to the north Pacific species Ceratothoa arimae (Nunomura, 2001), with a sequence divergence of 9.4%, however this branch bootstrap support was 56. In contrast our sequence did not form a group with other Ceratothoa oestroides sequences that nested within a clade with Ceratothoa italica Schiödte & Meinert, 1883 with an internal sequence divergence of 0.54% (Figure 12). Cymodoce sp. grouped closely with a clade formed by sequences of Cymodoce truncata Leach, 1814 and Cymodoce emarginata Leach, 1818 (sequence divergence of 17%). However, other sequences of Cymodoce truncata were placed on other branches ( Figure 13).

Discussion
From the 23 species collected, six were previously recorded at the Minho River estuary: Procambarus clarkii (Girard, 1852), Carcinus maenas (Linnaeus, 1758), Atyaephyra desmarestii (Millet, 1831), Crangon crangon (Linnaeus, 1758), Gastrosaccus spinifer (Goës, 1864) and Heterotanais oerstedii (Krøyer, 1842) [12,13,15,49], with the remaining 17 species being new records (Table S2). Two of those species are exotic to the Minho River Procambarus clarkia, introduced in the 90s [49], and Austrominius modestus. Decapoda is the most representative group among crustaceans, with freshwater and marine species, while the remaining groups present only marine species on the saltmarsh and near the mouth of the river. Species from the orders Decapoda and Mysida are well documented on the estuary of the Minho River, with four of the six decapod species being already recorded in this area [12,13,49]. Species composition of the order Mysida is similar to assemblages sampled in Ria de Aveiro [113]. Groups like Cumacea, Tanaidacea and Leptostraca are still poorly known in this area.

Discussion
From the 23 species collected, six were previously recorded at the Minho River estuary: Procambarus clarkii (Girard, 1852), Carcinus maenas (Linnaeus, 1758), Atyaephyra desmarestii (Millet, 1831), Crangon crangon (Linnaeus, 1758), Gastrosaccus spinifer (Goës, 1864) and Heterotanais oerstedii (Krøyer, 1842) [12,13,15,49], with the remaining 17 species being new records (Table S2). Two of those species are exotic to the Minho River Procambarus clarkia, introduced in the 90s [49], and Austrominius modestus. Decapoda is the most representative group among crustaceans, with freshwater and marine species, while the remaining groups present only marine species on the saltmarsh and near the mouth of the river. Species from the orders Decapoda and Mysida are well documented on the estuary of the Minho River, with four of the six decapod species being already recorded in this area [12,13,49]. Species composition of the order Mysida is similar to assemblages sampled in Ria de Aveiro [113]. Groups like Cumacea, Tanaidacea and Leptostraca are still poorly known in this area.
All species are contained within their documented geographical distribution. Nebalia strausi Risso, 1826 was recorded for the first time on Portuguese waters and Austrominius modestus (Darwin, 1854) on the northern Portuguese coast expanding the known distribution of this species in Portugal. The first record of parasitism from the isopod Ceratothoa oestroides (Risso, 1826) on Alosa alosa (Linnaeus, 1758) is also presented, although confirma-tion on C. oestroides identification is still required as COI-5P sequence did not match any of the publicly available sequences for this same species. Although a possible misidentification on the sequences of C. oestroides provided by Mladineo et al., 2009 [180] cannot be ruled out as they nest within a clade formed by sequences of Ceratothoa italica Schiödte & Meinert, 1883 (internal divergence of 0.54%), provided by different groups of authors ( Figure 12). As for Cymodoce sp. identification was not possible due to the lack of descriptions and keys for juveniles, being morphologically different from female and male adults, and produced an unmatched COI-5P sequence, requiring a greater effort of adult sampling, identification and posterior DNA barcode sequencing in order to solve problems with juvenile identification or other problematic species in this genus.
Larval stages from Procambarus clarkii found in December are in concordance with the findings on Sousa et al., 2013 [49] which placed the presence of juveniles on the Minho River from June to December. The presence of ovigerous females and megalopes of Palaemon longirostris also coincides with its known breeding periods (April to August [2]), as well as for the presence of larvae and juveniles of Carcinus maenas (starting on March [25]).

Conclusions
This investigation represents the first taxonomic study of some crustacean groups from the international Minho River, especially some of the less known groups like Cumacea, Tanaidacea and Leptostraca contributing to the knowledge of the Portuguese and Iberian fauna. The species Ceratothoa aff. oestroides was found for the first time parasitizing Alosa alosa. Nebalia strausi had its first record on Portuguese waters. For the genus Cymodoce morphological and molecular approaches are recommended in order to solve its problematic species status and assign juveniles and females to the respective male descriptions as those are still lacking. Despite the number of sampling methodologies employed glass eel fishing bycatch seems to be the most efficient method for sampling marine adventitious crustacean fauna, with the possibility of the appearance of other marine species if the sampling efforts were greater and regular over time. To fully understand the composition and distribution of crustaceans on the estuary of the international Minho River, different sampling methods are required concerning different habitats and assemblages.