Introduction
Razor clams are ecologically and economically important marine bivalves (Saeedi and Costello, Reference Saeedi and Costello2019a), inhabiting sand and muddy bottoms within the lower intertidal and subtidal areas. Razor clams have been part of the pre-glaciation malacofauna of the mid-North Atlantic nine-island Azores archipelago (36–40°N, 24–31°W). Ensis minor (Chenu, 1843) was present on Pleistocene deposits in Santa Maria Island (Ávila et al., Reference Ávila, Amen, Azevedo, Cachão and García-Talavera2002), although, possibly due to a severe drop in sea surface temperature (Ávila et al., Reference Ávila, Madeira, Mendes, Rebelo, Medeiros, Gomes, Garcia-Talavera, Silva, Silva, Cachão, Hillaire-Marcel and de Frias Martins2008), disappeared from the archipelago with other littoral bivalves living in fine sand. Solen marginatus Pulteney, 1799 was referenced for the shores of São Miguel by Drouet (Reference Drouët1861: 47). Solen vagina Linnaeus, 1758 was also indicated to the Azores (0 to −18 m) by Jeffreys (Reference Jeffreys1881: 929), but it has been considered a dubious record (Ávila et al., Reference Ávila, Azevedo, Gonçalves, Fontes and Cardigos2000). After the submission of this manuscript, Álvaro et al. (Reference Álvaro, Sinigaglia, Madeira, Hipólito, Melo, Arruda, Fernandes, Baptista and Ávila2024) reported the presence of S. marginatus from beach observations at one site in Praia da Vitória, Terceira Island (Figure 1). Based on 11 individuals and opportunistic observations down to 2.5 m, the species was validated through molecular tools, advocating the existence of a new population (Álvaro et al., Reference Álvaro, Sinigaglia, Madeira, Hipólito, Melo, Arruda, Fernandes, Baptista and Ávila2024), however, no phylogenetic analysis or information on species density and dispersion were provided.
Observations of Solen marginatus in Praia da Vitória bay, Terceira Island, Azores; (a) Circles indicate presence, (A, G–K), squares indicate absence, (B–F) with letters representing different sampling sites of S. marginatus in August 2023, triangle indicates the single sighting location of Álvaro et al. (Reference Álvaro, Sinigaglia, Madeira, Hipólito, Melo, Arruda, Fernandes, Baptista and Ávila2024); (b) largest and a smaller specimen collected at Site A; (c) size distribution at Site A (n = 118); (d) dorsal and (e) ventral view of shells: aam, anterior adductor muscle; avp, antero-ventral mantle projection; ant, anterior; dor, dorsal; lig, ligament; LV, left valve; mf, marginal furrow, pam, posterior adductor muscle; pl, pallial line; pos, posterior; pvp, postero-ventral mantle projection; ps, pallial sinus; rct, right cardinal tooth; lct, left cardinal tooth; RV, right valve.
As part of the ecological studies undertaken onboard ‘OCEANUS II’ around the Archipelago of the Azores, S. marginatus was identified on the 17 August 2023 in several locations in the bay of Praia da Vitória, Terceira Island. The species was verified by classic and molecular taxonomy using cytochrome oxidase I (COI) mitochondrial gene to improve the accuracy of species-level identification and allow to perform intraspecies diversity analysis in an attempt to understand level genetic divergence and population source (Hebert et al., Reference Hebert, Cywinska, Ball and deWaard2003; Fernández-Tajes and Méndez, Reference Fernández-Tajes and Méndez2007; Cunha et al., Reference Cunha, Tenorio, Afonso, Castilho and Zardoya2008; Leray and Knowlton, Reference Leray and Knowlton2015). In support of conservation measures, the species' habitat range and population biology were further addressed for the bay of Praia da Vitória, by analysing dispersion and densities through visual surveys and size ranges, providing the first ecological insights on habitat and phylogeny of the new population of grooved razor clam in the Azores.
Material and methods
Razor clams were observed and collected on several locations in the north part of the bay of Praia da Vitória, Terceira Island on the 17 August 2023. Samples were kept in 96% ethanol for molecular studies and −20°C for subsequent analysis at the Atlantic Naturalist Collection in Horta, Faial Island, Azores. Identification was conducted by morphology-based taxonomy and molecular analysis. For the molecular identification 20 razor clam individuals from Praia da Vitória, Terceira Island, together with an additional sample of five specimens obtained in Faro (south Portugal) were used to establish a control group. A fragment of the foot was dissected and preserved in 96% ethanol until DNA processing. DNA extractions were conducted on 10–20 mg of foot tissue using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The COI amplification of the ‘Folmer’ barcode region at the 5′ start of the cytochrome c oxidase 1 gene (CO1), was done with LCO 1490 (5′-GGTCAACAAATCATAAAGATATTG-3′) and HCO 2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) universal primers (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994). Amplifications consisted of 25 μl mix containing 1 μl (10–100 ng) of genomic DNA, 5 mM GoTaq buffer (5×), 0.2 mM dNTP (Promega, Madison, WI, USA), 1.5 mM MgCl2, 0.2 mM of each primer, and 1 U of GoTaq DNA polymerase (Promega). The PCR cycling profile for COI amplification included an initial denaturation step at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, extension at 72°C for 40 s, and a final extension at 72°C for 5 min. The PCR products were purified through ethanol/sodium acetate precipitation Field (Green and Sambrook, Reference Green and Sambrook2016) and subsequently sequenced using the corresponding PCR primers. Sequencing was performed on an Applied Biosystems 3130xl Genetic Analyser, utilising Sanger technology and the BigDye® Terminator v3.1 kit. The generated COI gene sequences were assembled and trimmed using the Geneious Prime (version 2020.0.3, Biomatters, New Zealand, https://www.geneious.com). The assembled sequences were BLASTn-searched in the National Centre for Biotechnology (NCBI) and compared with closely related sequences. Sequences were aligned using MUSCLE in Geneious Prime. Overall mean diversity was computed with MEGA v 11.0.13. The maximum likelihood (ML) analysis was conducted in RAxMLGUI v 2.0.10 (Tamura et al., Reference Tamura, Stecher and Kumar2021) with 100 bootstrap replicates and K81uf + gamma model. As an outgroup, we used a COI sequence from Ensis ensis (ACCN: HF970367).
To address habitat range, a visual assessment was conducted on the seafloor at 10 locations throughout the bay of Praia da Vitória (Figure 1). The presence of S. marginatus was verified using specific bioturbation burrows with an eight or keyhole shape. With a low diversity of sand bivalves, the identification was straightforward, however, caution was taken by excluding old or unclear lebensspurren (e.g. Pereyra et al., Reference Pereyra, Bel Haouz and Lagnaoui2023). Quadrats of 0.5 × 0.5 m were used three times in each location to assess densities per m2 at the various locations throughout the bay density was estimated in more detail using visual line transects conducted by two scuba divers at Site A, within the area of occurrence of the species at 8.6 m depth (Site A). Four non-overlapping random line transects of 20 square metres were conducted at this site, totalling 80 sqm.
Population biology was assessed through size distribution by collecting 118 specimens at site A with a ‘salting method’ using a saline solution and measuring shell length from posterior to the anterior furthest distance using a vernier calliper (0.1 cm). Size distribution was evaluated with a Kolmogorov–Smirnov test (P < 0.05).
Results
Morphological analysis
The species was identified from its equivalve shell, elongated and rather broad shape–shell ratio 1:6.1, with razor-like dorsal and ventral margins straight and parallel (Figure 1; Fischer et al., Reference Fischer, Bauchot and Schneider1987; Carpenter and De Angelis, Reference Carpenter and De Angelis2016). The shells are anterior obliquely truncated and longer at the ventral edge. There is only one cardinal tooth on each valve (with no lateral teeth). Anterior adductor muscle scar is nearly as long as the ligament. The distinct marginal furrow bordering the anterior margin of valves is very conspicuous and distinct from other species (Figure 1d; Dautzenberg, Reference Dautzenberg1897; Luczak and Dewarumez, Reference Luczak and Dewarumez1992). The outer colour of the shell ranges from yellowish to pale brownish growth zones.
Phylogenetic analysis
The COI gene sequence generated from the Solen samples from Azores and Faro shared 100% similarities with S. marginatus from Ria de Aveiro, Portugal (ACCN: MK779736) and Asturias, Spain (ACCN: KJ818881). Furthermore, the Azorean samples shared 99% similarity with samples from Asturias, Spain (ACCN: KJ818881, KJ818886, KJ818884, KJ818877, KJ818890, KJ818889, KJ818882, KJ818879) and Ria de Aveiro, Portugal (ACCN: MK779734). Within the S. marginatus the lowest shared similarity (90%) was with a sample from Italy (ACCN: MN630857). The overall mean diversity of the S. marginatus clade (including S. marginatus and the Azorean samples) was 1%. Phylogenetic relationships of the samples from Azores with other closely related Solen species, as inferred by the COI gene sequences using ML analysis, showed a formation of a distinct S. marginatus clade (Figure 2).
Phylogenetic relationships of the samples from the Azores with other closely related Solen species, as inferred by the COI gene sequences using ML analysis. Numbers above branches are >50% bootstrap values.
Habitat range and density
The habitat range of Solen marginatus in Praia da Vitória, Terceira Island from the preliminary visual census is presented in Figure 1a. The species was present on the sites encircled (average densities given from the quadrats) as follows: A (8,6 m depth; 12.4 ind/m2), I (9.6 m; 9.3 ind/m2), K (8.9 m; 10.7 ind/m2); with some of the sites of occurrence hosting densities below 1 ind/m2: G (7.1 m depth), H (7.5 m), and J (14.5 m). No burrows were observed on locations B (7.2 m depth), C (20 m), D (14.2 m), E (10 m), and F (4.6 m).
The distribution seems confined to the northern part of the bay (Figure 1). Presence was noted northward of a theoretical line between the green lighthouse (right peer as you enter the bay), and the peer south of Praia Grande (next to location F, Figure 1). Next to Prainha they were mainly observed on depths of 8 m and deeper (Figure 1, sample location K), although generally spread, with greater incidence at the bottom of the slope. At this site, only at specific locations S. marginatus was found up to 2.5 m depths. Densities estimated by visual transects at Site A were 12.69 individuals/m2 (STDEV = 2.6; n = 1015 per 80 sqm). Shell length of S. marginatus at Site A averaged 11.3 cm (STDEV = 1.3), ranging between 8.79 and 15.4 cm. Size distribution is presented in Figure 1c. Kolmogorov–Smirnov test revealed a non-normal distribution (D = 0.345), with a leptokurtic positive skewness (excess kurtosis = 1.2585) around size classes between 10 and 11.99 cm length, consisting of 60% of the population. The preliminary weight measurements averaged 19.4 g (Stdev = 6.9) per specimen.
Discussion
Solen marginatus is widespread along the European continent, east of the Azores, from the Baltic Sea to the Mediterranean, and from the Black Sea along the African coast to down to Senegal (Saeedi and Costello, Reference Saeedi and Costello2019b). The mid-North Atlantic Azorean malacofauna has affinities with the Mediterranean, Portugal, and Madeira Island (Ávila et al., Reference Ávila, Azevedo, Gonçalves, Fontes and Cardigos2000), so the occurrence of this population is not unexpected. It is noticeable however, that such a dense aggregation of a species with high commercial value remained unreported until present. Solen marginatus has not been reported on other islands since the original report by Drouet (Reference Drouët1861), and was not included in the recent check-list for the Azores (Ávila et al., Reference Ávila, Azevedo, Gonçalves, Fontes and Cardigos1998; Borges et al., Reference Borges, Bried, Costa, Cunha, Gabriela, Gonçalves, Martins, Melo, Parente, Raposeiro, Rodrigues, Santos, Silva, Vieira, Vieira, Mendonça and Boieiro2010). Our findings on size distribution and preliminary habitat range of the community demonstrate that this population is well established and reaching high densities in the northern area of Praia da Vitória Bay, with no individuals observed in the south of the Bay. The settling location is one of the less hydrodynamic areas of the bay, including the recreational dock and larger sandy beaches. Considering the densities revealed and the historical presence of the solenoids in the region, the grooved razor clam may expand to other locations in the archipelago.
The dominance of reproductive sizes also indicates a well-established population, as S. marginatus reaches maturity between the first and the second year (ca. 4.7 cm length; Maia, Reference Maia2006, in Ria de Aveiro, Portugal). Having 60% of the population with a medium age of 4 years old shows potential for reproduction and growth. While settlement occurred more than 10 years ago, judging from the existence of individuals above 14 cm length (Maia, Reference Maia2006), a settlement period of over 20–25 years is expected, as it is very unlikely that only 10 years of recruitment as proposed by Álvaro et al. (Reference Álvaro, Sinigaglia, Madeira, Hipólito, Melo, Arruda, Fernandes, Baptista and Ávila2024) would result in such high densities, as here reported over a considerably large area. Genetically it is a recent settlement as supported by the shallow phylogenetic divergence with other populations. The origin of this population remains undetermined, with greater genetic affinity to the Ria de Aveiro (North of Portugal) and the Asturias (North of Spain) populations. Our findings on the source populations, agree with the hypothesis of Álvaro et al. (Reference Álvaro, Sinigaglia, Madeira, Hipólito, Melo, Arruda, Fernandes, Baptista and Ávila2024), that suggested an anthropogenic origin, likely from Portugal or Spain, due to the 8–9 day settlement period of the razor clam larvae (Da Costa et al., Reference Da Costa, Nóvoa, Ojea and Martínez-Patiño2012). Intentional or non-intentional anthropogenic sources remain possible, as well as natural dispersion. The regular arrival of boats, coupled with the availability of live specimens for purchase in local marketplaces for use as human consumption or fish bait, will more likely function as introduction vectors once the distance to the Northeast Atlantic coastal areas (at least 1800 km) makes natural dispersion more complex.
Future works should cover the entire sand bank habitats on the island as well as other potential habitats in the archipelago. The use of different genetic markers may prove useful in the search for genetic divergence and understanding the population origin. Future population studies should comprise reproductive histological analysis, toxicological and finer distribution assessments if fisheries exploitation is to be considered. These would support establishing no-take areas, closure dates, minimum sizes, or total allowable catches. Future extractive techniques may include dredging, which places this newly described and confined population and the overall habitat under imminent threat requiring urgent conservation.
Data
Data is kept at Atlantic Naturalist collection in Horta, Faial and can be made available under request.
Acknowledgements
This study was financed by the regional ONG Atlantic Naturalist Association and the Portuguese national funds from FCT – Foundation for Science and Technology through projects UIDB/04326/2020 (DOI:10.54499/UIDB/04326/2020), UIDP/04326/2020 (DOI:10.54499/UIDP/04326/2020) and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020). The authors wish to acknowledge Maria, Margarida, and Cristina Machado from GEO – Grupo de Estudos Oceânicos for field work assistance, Carlos Afonso from CCMAR/Universidade do Algarve and Mafalda Albuquerque Naturalist – Science and Tourism.
Author Contributions
A. M., D. M. and J. N. G.-P., species discovery, field work, and paper writing, G. D. data analysis and paper writing, R. C. and L. C. molecular analysis and paper writing.
Financial Support
This study was financed by the regional ONG Atlantic Naturalist Association and the Portuguese national funds from FCT – Foundation for Science and Technology through projects UIDB/04326/2020 (DOI:10.54499/UIDB/04326/2020), UIDP/04326/2020 (DOI:10.54499/UIDP/04326/2020) and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020).
Competing interests
All authors have no conflict of interest.
Ethical Standards
The present survey complied with all ethical standards.
