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Spatial distribution and genetic structure of loliginid paralarvae along the Galician coast (NW Spain)
Fisheries Research 222 (2020) 105406
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Spatial distribution and genetic structure of loliginid paralarvae along the Galician coast (NW Spain)
T
Elsa García-Mayorala,b,*, Álvaro Rouraa, Andrea Ramiloa, Ángel F. Gonzáleza a b
Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain Campus Do Mar, Universidad de Vigo, 36310, Vigo, Spain
ARTICLE INFO
ABSTRACT
Handled by: Chennai Guest Editor
Squids belonging to the cephalopod family Loliginidae have an important economic value for the small-scale fisheries in Galicia and recreative fishing. Early life stages of loliginids are studied along the Iberian coast to forecast their population abundance based on correct species identification, but visual identification of the paralarvae is not possible for this family. Molecular analyses using cytochrome oxidase subunit I (COI) have shown that the three most common species of loliginids in the Galician coast are Alloteuthis media, Loligo vulgaris and Alloteuthis subulata. This study compared the abundance and spatial distribution of the three loliginid species between the northern and southern waters of Galicia. Results showed that A. media is the most abundant in the south and A. subulata in the north, while L. vulgaris maintains similar percentage of frequency. Genetically, L. vulgaris and A. media have complex haplotype networks and high diversity indices, while A. subulata has a reduced haplotype network and low diversity indices. The genetic variability observed in COI data showed a consistent genetic flow between north and south Galicia and no population structuring was observed along the Galician coast with this mitochondrial marker. Fine scale studies with nuclear and mitochondrial markers are needed to have a better understanding of the population structure of this important resource.
Keywords: Loliginidae Cephalopod paralarvae Genetic diversity Population structure NE Atlantic
1. Introduction The cephalopods belonging to the family Loliginidae have an important commercial value in the north-eastern Atlantic and Mediterranean, especially in the coastal Iberian Peninsula and in the Mediterranean countries (Simón et al., 1996). In this area, the loliginid family is represented by four species: Loligo vulgaris, L. forbesii, Alloteuthis media and A. subulata (Anderson et al., 2008; Guerra and Rocha, 1994; Laptikhovsky et al., 2019). In the western Mediterranean and the western coast of the Iberian Peninsula, the European squid, L. vulgaris, is caught as by catch by bottom and pelagic trawling and also by smallscale hand-jig artisanal fisheries, while A. media is caught as bycatch by bottom trawl fisheries and beach seine. On the other hand, A. subulata is considered as a secondary target species in Spain, Portugal and Italy and has low commercial value due to its small size being captured as bycatch in trawl fisheries. Specific statistics on catches for each species are impossible to know with accuracy, because most of the landings of L. vulgaris are mixed with L. forbesi under the name L. vulgaris or Loligo spp. (Jereb et al., 2015; Roper et al., 2010; Simón et al., 1996). The two Loligo species used to have overlapping distribution ranges, being L. forbesi more
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abundant in the northern area (Northern and Celtic Sea) and L. vulgaris in the south (Iberian Peninsula). During the 1990s the presence of L. forbesi declined dramatically in the west coast off the Iberian Peninsula increasing in Scottish waters (Chen et al., 2006). The other loliginid genus, smaller in size, has also been wrongly identified and used to be reported as Alloteuthis spp. or Loligo spp. (Jereb et al., 2015). In Galicia (NW Spain), the loliginids L. vulgaris and both Alloteuthis species are captured by small scale fisheries as it is hand-jigging and boliche (boat seine) located close to the coast (Jereb et al., 2015; Tasende et al., 2005; Simón et al., 1996). The landings of L. vulgaris in the last ten years were 3472 t, going from 338 t to 87 t during 2007–2016, while a total of 172 t of Alloteuthis spp. were caught in the same period. Captures of L. vulgaris in Galicia decreased to 75 t while Alloteuthis spp. increased to 8 t in 2018 (https://www.pescadegalicia. gal). Loliginids have a pelagic larval stage that spend two to three months as zooplankton, known as paralarvae. During this dispersal stage, they remain over the continental shelf by means of controlling their vertical position (Roura et al., 2016, 2019). Previous studies made in the west coast of Iberian Peninsula (NE Atlantic) classified the loliginid paralarvae at family level (González et al., 2005; Moreno et al., 2009; Rocha
Corresponding author at: Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain. E-mail address: [email protected] (E. García-Mayoral).
https://doi.org/10.1016/j.fishres.2019.105406 Received 9 July 2019; Received in revised form 24 September 2019; Accepted 2 October 2019 0165-7836/ © 2019 Elsevier B.V. All rights reserved.
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and Guerra, 1999; Roura et al., 2013) because the chromatophore pattern, a species-specific character present is fresh specimens (Sweeney et al., 1992), practically disappears in ethanol-preserved specimens. However, current molecular techniques amplifying the cytochrome oxidase I (COI), showed that the loliginid paralarval assemblage of the western Iberian Peninsula and north of Africa is dominated by Alloteuthis media and A. subulata, followed by Loligo vulgaris (OlmosPérez et al., 2018; Roura et al., 2019). The abundance of paralarvae is a potential tool that can be used to make stock predictions (González et al., 2010). However, proper identification of loliginid paralarvae is essential for a correct management of this resource in order to estimate biomass and quota uptake (Ward, 2000). Loliginid paralarval abundance is related positively with upwelling intensity in the southern waters of Galicia with a peak in their abundance in the water column at night (González et al., 2005; Otero et al., 2009; Roura et al., 2016; Olmos-Pérez et al., 2018). The only data gathered from the north revealed the presence of loliginid paralarvae on the shelf (Rocha, 1999), but only 15 individuals were collected and visually identified based on chromatophore pattern. Recent studies have shown that the different loliginid species occupy different niches in the horizontal and vertical axis of the water column. Alloteuthis media is found closer to the shore than A. subulata, while L. vulgaris overlaps the distribution of these two species. In the water column, A. subulata is found in deeper strata than A. media (Olmos-Pérez et al., 2018). Despite these spatial differences that imply different dispersal capabilities, the genetic flux of the loliginid assemblage is enough to keep high nucleotide and haplotype diversity indices for A. media and L. vulgaris but low in A. subulata along the Canary Current upwelling system (CanC), both in south Galicia (Olmos-Pérez, 2018) and between the northern and southern limits of the CanC (> 2000 km apart, Roura et al., 2019). Given that the northern part of Galicia is not under the influence of the CanC, it could represent an oceanographic barrier for genetic flux between the Western and Northern Iberian Peninsula squid populations. Accordingly, the aims of this study are three: 1) genetically identify the loliginid paralarvae present along the Galician coast, 2) recognise their distribution patterns and 3) evaluate if there exists a genetic barrier between the northern and southern waters of Galicia that may affect population structure and diversity indices.
Ons islands (Fig. 1D). Zooplankton samples were caught with a Multinet Hydrobios Mammoth net of 250 μm mesh size by filtering about 200 m3 at seven different strata: 105–85, 85–55, 55–35, 35–20, 20–10. 10–5 and 5 to 0 m. The samples were fixed on board in 96% alcohol and stored at −20 °C. Afterwards, loliginid paralarvae were separated from the zooplankton samples in the laboratory and prepared for molecular analyses. 2.3. Mitochondrial DNA sequencing Loliginid paralarvae from north and south Galicia were preserved in 96% ethanol for molecular analysis. Individual mantle from each loliginid paralarvae was dissected and genomic DNA purifications were performed employing QIAamp DNA Micro Kit (QIAGEN) according to the manufacturer’s protocol for isolation of genomic DNA from tissues. DNA quality and quantity were checked in a spectrophotometer Nanodrop® ND-2000 (Thermo Scientific). To identify the loliginid species, a fragment of cytochrome c oxidase subunit I (COI) was amplified using the universal primers HCO2198 and LCO1490 (Folmer et al., 1994). PCR reactions were performed in a total volume of 25 μl containing 1 μl of genomic DNA, PCR buffer at 1x concentration, 0.3 μM primers, 0.2 mM nucleotides and 0.025 U. μl −1 DreamTaq DNA Polymerase (Thermo Scientific). The PCR assays were carried out in a Tgradient thermocycler (Biometra), under the following reaction conditions: 3 min at 95 °C, 40 cycles of 30 s at 95 °C, 45 s at 48 °C and 1 min at 72 °C, followed by 7 min at 72 °C. A negative control (no DNA) was included in all PCR amplifications. PCR products were separated on a 2% agarose gel in Tris acetate EDTA buffer, stained with Red Safe and scanned in a GelDoc XR documentation system (Bio-Rad Laboratories). The amplified fragments were cleaned for sequencing using ExoSAP-IT™ PCR Product Cleanup (Applied Biosystems) for 15 min at 37 °C, followed by inactivation for 15 min at 80 °C. Sequencing was performed in StabVida (Portugal) and the chromatograms were analysed using ChromasPro 2.1.8. (Technelysium Pty Ltd). All generated sequences were searched for species identity using BLAST (Basic Local Alignment Search Tool) through web servers of the National Center for Biotechnology Information (NCBI). 2.4. Population genetic analysis
2. Material and methods
To evaluate the genetic diversity of the different loliginid species, the software DnaSP v. 6 (Librado and Rozas, 2009) was used to calculate the number of haplotypes (H), the haplotype diversity (h), nucleotide diversity (π), number of segregating sites (S), and the average number of nucleotide differences (k). The neutrality tests of Tajima’s D (Tajima, 1984) and Fu’s FS (Fu, 1997) were tested with Arlequin v3.5.2 (Excoffier et al., 2005). In addition, to examine population differentiation, pair-wise Fst values were computed counting the haplotype frequencies for each species using Arlequin v3.5.2. A final alignment with 195 sequences of 623bp was used to build the median joining haplotype networks using Nexus software (www.fluxus-engineering. com). Additionally, in order to get better results, three haplotype sequences of A. subulata from the Ría de Vigo (Olmos-Pérez et al., 2018) obtained in BOLD (MF157701, MF157702, MF157703) were assembled manually. Furthermore, for this study sequences of loliginid paralarvae from the CanC (Roura et al., 2019) were downloaded from the online project CEPAR in BOLD.
2.1. Study area The NE Atlantic coast constitutes one of the four eastern boundary upwelling systems of the world, known as the Canary Current upwelling system (CanC; Arístegui et al., 2009). The western Iberian Peninsula is at the northern end of this upwelling system, characterised by seasonal upwelling, between May-June to September-October. During this season north-eastern winds force the Eastern North-Atlantic Central Water (ENACW), a cold and nutrient-rich current, to the surface sustaining an important fishery industry along the coasts of Portugal and Galicia (NW of Iberian Peninsula). Galicia is composed by coastal embayment called Rías where the upwelling is intensified (Tenore et al., 2008, 1995). 2.2. Sampling Overall, 278 zooplankton samples were collected in the north of Galicia (Fig. 1A) during June 2017 on board of R/V Sarmiento de Gamboa with a bongo net of 500 μm mesh size, doing double oblique transects at 2.5 knots. Zooplankton samples (n = 338) were obtained in southern Galicia (Fig. 2B) on board of R/V Mytilus from June to November 2017. Two types of surveys were carried out in the area: six surveys were undertaken at night in the Ría de Vigo (Fig. 1C) and four 24 h surveys were carried out along six transects surrounding Cíes and
3. Results 3.1. Genetic identification and diversity A total of 207 loliginid paralarvae were collected from north (N = 54) and south Galicia (N = 153). The identification of 198 of 207 paralarvae revealed three species of loliginids (Table 1). From 144 2
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Fig. 1. Map locating the sampling sites. A) Samplings from the north, with depths ranging from 50 to 500 m and B) the south of Galicia, showing in detail the transects carried out in C) the Ría de Vigo and D) around the Cíes and Ons Islands, with depths ranging from 10 to 105 m.
individuals sequenced in southern Galician waters, 79 were classified as Alloteuthis media (56%), followed by 55 Loligo vulgaris (39%) and ten A. subulata (5%). Out of 54 specimens sequenced in northern Galicia, 40 were classified as A. subulata (74%), eight as L. vulgaris (15%) and six as A. media (11%). The COI sequences analysed revealed a total of 78 haplotypes: 35 from L. vulgaris, whose populations from north and south of Galicia shared three haplotypes; 34 from A. media, with three haplotypes shared between both localities; and nine from A. subulata sharing two haplotypes. The highest haplotype and nucleotide diversity was observed in L. vulgaris and the lowest in A. subulata (Table 1). Overall, species from the south have a higher haplotype diversity than those from the north, despite the southern samples were collected in an area much smaller than that of the north. In contrast, nucleotide diversity was higher in the north for A. subulata and L. vulgaris, but lower for A. media (Table 1).
Conversely, A. subulata had a reduced haplotype network with one main haplotype and four branches emerging from it, where two haplotypes from the northern coast are separated by 11 and 13 mutations. Finally, L. vulgaris presents a complex haplotype network with a high number of mutations and four main haplotypes (H1, H3, H5 and H15, accounting for 8; 5; 10 and 6 individuals out of 63). Three of these four main haplotypes are shared between northern and southern populations, except for haplotype H5, which was only present in the southern region. 3.3. Spatial distribution and abundance The spatial distribution of loliginid paralarvae revealed differences in the species composition between northern and southern Galician waters. A. media is the most abundant species in the south-western, while A. subulata is the dominant species in the north. Horizontal distributions in both locations showed that A. media is found closer to the coast than A. subulata, which are found in offshore waters (Fig. 3). A similar pattern was observed in the north, with a gradient from the border of the continental shelf (200 m depth) dominated by A. subulata, followed by L. vulgaris and A. media in shallower samples (Fig. 3A, B). Furthermore, A. subulata paralarvae were vertically distributed from 10 to 55 m in southern waters. Meanwhile, A. media and L.vulgaris overlap their distribution from the surface down to 55 m (Fig. 4B).
3.2. Population structure Pairwise comparison showed non-significant values (Fst, p > 0.05) for the three species between north and southern squid populations, thus indicating a constant genetic flux between them. Moreover, detection of negative significant departures from the null hypothesis of neutrality using Tajima´s D were detected in A. media, A. subulata and L. vulgaris (except for the northern specimens, Table 1). On the other hand, results of the Fu’s Fs test for A. subulata cannot rejected the null hypothesis of neutral evolution but significant negative values were obtained for A. media and L. vulgaris. Alloteuthis media had a star-shaped haplotype network with two main haplotypes (H2 and H7 accounting for 30 and 11 individuals respectively out of 85, Suppl. Table 1) that are shared by the populations from north and south Galicia and numerous single haplotypes (Fig. 2).
4. Discussion This study analysed the taxonomic composition and abundance of cephalopod paralarvae belonging to the family Loliginidae along the Galician coast, a seasonal upwelling region. Results obtained showed that A. media prevails in the south coast of 3
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Fig. 2. Haplotype networks for each species. In black the paralarvae belonging to the south and in light grey individuals from the northern waters of Galicia. Haplotype number is represented in black and the position of the different mutation within the aligned sequences are shown in red.
Galicia, while in the northern coast is A. subulata. The study done by Olmos-Pérez et al. (2018) between 2012–2014 in the southern waters of Galicia also agreed with our research, which also found that A. media was the most common loliginid paralarvae (57.5%), with L. vulgaris abundance in an increasing tendency during the last years with A. subulata in an opposite variation trend (Fig. 5). At a wider scale, the loliginid paralarvae collected at the limits of the CanC in 2009 (from north-western Iberian Peninsula and southwest Morocco) showed that A. media (85%) was the most abundant, followed by A. subulata (14%) and L. vulgaris (1%). Altogether, these data suggest that L. vulgaris is increasing its relative abundance and the opposite is occurring with A. subulata, maybe related with the decreasing intensity of the upwelling along the Iberian coast and the increasing in thermal stratification (Bode et al., 2009; García Reyes et al., 2015): However, more data are needed to evaluate in detail this hypothesis.
4.1. Population structure The genetic diversity indices calculated for L. vulgaris and A. media indicate high haplotype (h > 0.7013) and nucleotide diversity (π > 0.00356), taking into account the values calculated by GoodallCopestake et al. (2012). Alloteuthis subulata showed low genetic differentiation but numerous segregating sites. Similar results were obtained in Olmos-Pérez et al. (2018), where L. vulgaris had lower values than those obtained in this study but higher in absolute terms, whereas A. media had a medium-high (h = 0.680, π = 0.0019) genetic variation. Loligo vulgaris showed a much more complex network with higher number of mutations than the network obtained by Olmos-Pérez et al. (2018). Given that the samples were collected in the same area as Olmos-Pérez et al. (2018), but three years later, the differences could be attributed due to the total number of L. vulgaris caught in the different years. In south of Galicia 29 paralarvae were caught by Olmos-Pérez
Table 1 Molecular diversity indices and population demographic statistics for each loliginid species. H, number of haplotypes; h, haplotype diversity; π, nucleotide diversity; S, number of segregating sites; k, average number of nucleotide differences; D, Tajima’s D (Tajima, 1984); Fs, Fu’s Fs (Fu, 1997). Species
Location
n
H
h ( ± standard deviation)
π
S
K
D
Fs
A. media A. media A. media A. subulata A. subulata A. subulata L. vulgaris L. vulgaris L. vulgaris
North South All North South All North South All
6 79 85 40 10 50 8 55 63
4 33 34 7 4 9 6 32 35
0.800 0.865 0.857 0.433 0.644 0.473 0.929 0.949 0.948
0.00193 0.00275 0.00269 0.00242 0.00171 0.00228 0.01181 0.00982 0.01008
3 25 26 21 4 22 13 42 44
1.20 1.71 1.67 1.51 1.07 1.42 7.36 6.12 6.28
−0.447ns −2.079** −2.113** −2.311** −0.943ns −2.344** 1.390ns −1.327ns −1.325ns
−1.454* −27.709** −27.883** −0.911ns −0.742ns −2.532ns 0.3049ns −16.887** −18.709**
± ± ± ± ± ± ± ± ±
0.172 0.032 0.033 0.095 0.152 0.084 0.084 0.018 0.015
4
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Fig. 3. Maps showing the relative abundances of loliginid paralarvae in the sampling area. A) Map showing the north and B) the south Galician coast.
and for this study 55 individuals were obtained. The haplotype networks of Alloteuthis media and A. subulata are quite similar to the networks obtained by Olmos-Pérez et al. (2018) in the south of Galicia and Roura et al. (2019) throughout the CanC (from NW Iberian Peninsula to SW Morocco). It is remarkable that similar haplotype networks were obtained with specimens collected more than 2000 km apart, thus showing that the dispersal of these paralarvae and adult migrations
effectively mixes the population along the CanC. Comparing the haplotypes obtained in this study with those from different studies carried in the same area four and eight years ago (Olmos-Pérez et al., 2018 and Roura et al., 2019, respectively), it is interesting to note that A. media specimens have two haplotypes in common (H1 and H2) in the different studies, while the paralarvae from CanC shared seven unique haplotypes with the south and three from the
Fig. 4. A) Horizontal distribution of paralarvae in the north, where samples were collected with bongo and B) vertical distribution through the water column in the southern coast, where samples were collected with a multinet. 5
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Fig. 5. Relative abundance of the 3 species through the different sampling years. Asterisk shows samples collected in other studies: NW Iberian Peninsula in 2009 (Roura et al., 2019), and between 2012 and 2014 in the Ría de Vigo (Olmos-Pérez et al., 2018). Samples collected in 2017 belong to this study.
north of Galicia (Suppl. Table 1). This indicates a clear connection of the species along the Atlantic coast. Similarly, A. subulata main haplotype is the same in the different years and locations. On the other hand, L. vulgaris individuals from the south collected in this study shared five haplotypes with specimens sampled in 2013–2014 in the same area (Olmos-Pérez et al., 2018), while individuals sampled in the north only shared two haplotypes with those sampled by Olmos-Pérez et al. (2018). In terms of neutrality tests, A. media obtained significant negative Tajima’s D and Fu’s Fs for the southern population revealing an excess of low frequency polymorphisms (rare alleles) relative to expectation, suggesting population expansion after a recent bottleneck (GoodallCopestake et al., 2012). Nevertheless, A. subulata presented significant negative values for Tajima’s D in the northern population that evidence that COI has evolved under non-random processes, possibly affected by a recent selective sweep, which is shown by the absence of numerous haplotypes. This low number of haplotypes has been previously observed in the Eastern Atlantic (Olmos-Pérez et al., 2018; Roura et al., 2019 and Suppl. Table 1). For L. vulgaris the neutrality tests were not significant except for the southern population that can result from demographic events (Sotelo et al., 2009). Because of the change in the orientation of the coast of Cape Finisterre, it has been mentioned as a biogeographical boundary for gene flow between north and south of Galician waters (López-Jamar et al., 1992; López et al., 2015). However, genetic analyses carried in this study using COI data showed a clear homogeneity along the Galician coast for the three species meaning that there is an effective gene flow between the two areas. This lack of genetic structure was seen in other molluscs such as Mytilus galloprovincialis (Diz and Presa, 2009) and Donax trunculus with microsatellites (Nantón et al., 2017). Also, the velvet swimming crab Necora puber and the seahorse Hippocampus guttulatus showed lack of structure along the Galician coast (López et al., 2015; Sotelo et al., 2009). Nonetheless, some caution needs to be taken when interpreting this dataset owed to two factors: the differences in sample size for each species that may affect the resolution of genetic indices and that this study has been conducted with just one mitochondrial marker, which gives a broad idea of the population structure along the Iberian Coast, but more markers would be necessary to assess finer scale population structure.
coast. Loliginid paralarvae are suggested to display a coastal strategy (Roura et al., 2016, 2019), being retained between the coast and the limit of the continental shelf due to the interaction of their vertical behaviour and coastal currents, thus reducing offshore transport and increasing alongshore dispersal. The distribution pattern found in this work, with all the loliginid paralarvae collected within the limits of the continental shelf (0–200 m depth), supports the coastal strategy suggested for this family of cephalopod paralarvae, which are evenly distributed in the coastal area with some intraspecific differences. The fact that paralarvae occur close to the coast could suggest that adults migrate to the coast for spawning. Research done in the Mediterranean Sea (Cabanellas-Reboredo et al., 2012) inferred that L. vulgaris hatchlings occur close to the coast and later migrate to deeper waters over the continental shelf, such as L. gahi in Falkland Islands or L. vulgaris reynaudii in South Africa (Rodhouse et al., 1992). Other studies suggested that L. vulgaris occurs between shore and 100 m isobaths, whereas Alloteuthis spp. adults are found over the shelf and upper slope until the 350 m (Anderson et al., 2008; Moreno et al., 2007). Other research of the loliginid species Doryteuthis plei and D. sanpaulensis (Araujo and Gasalla, 2018) found that although they are coastal, their abundance is influenced by environmental factors such as the upwelling and sea surface temperature. Additionally, there were differences in the horizontal distribution where D. plei was found in the southernmost area and D. sanpaulensis in the northernmost off the South Brazil Bight. Moreover, Meath et al. (2019) found that D. pealeii and D. plei which, are two species that co-occur in the Gulf of Mexico, presented different migration movements, while Doryteuthis pealeii moves through southward waters, D. plei moves to northward. This behavior has been observed in other marine species such as the fish from the genus Etheostoma (Hlohowskyj and White, 1983). This behavioural adaptation permits to each species to have the same prey as food resource, but the intense intraspecific competition is reduced by habitat shift (Mendelson, 2006). The data reported by Olmos-Pérez et al. (2018) states that the spatial distribution of Alloteuthis species is quite different, both horizontally and vertically, with A. media found through all the water column and A. subulata appearing from 10 to 55 m in the south and along the continental platform in the north. At a wider scale, A. media is more abundant in the south and A. subulata in the north of Galicia. These differences could be due to competitive interactions, and Alloteuthis spp. could be minimizing the resource competition by habitat shift, with subtle changes in their vertical position that affect their horizontal distribution.
4.2. Spatio-temporal variability In this study, we observed differences on the spatial distribution of the three Loliginidae paralarvae found in the north-western Spanish 6
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An annotated and illustrated catalogue of species known to date. Volume 2. Myopsid and Oegopsid Squids Vol. 2 FAO Species Catalogue for Fishery Purposes No. 4. Roura, Á., Amor, M., González, Á.F., Guerra, Á., Barton, E.D., Strugnell, J.M., 2019. Oceanographic processes shape genetic signatures of planktonic cephalopod paralarvae in two upwelling regions. Progr. Oceanogr. 170, 11–27. https://doi.org/10. 1016/j.pocean.2018.10.005. Roura, Á., Álvarez-Salgado, X.A., González, Á.F., Gregori, M., Rosón, G., Otero, J., Guerra, Á., 2016. Life strategies of cephalopod paralarvae in a coastal upwelling system (NW Iberian Peninsula): insights from zooplankton community and spatio-temporal analyses. Fish. Oceanogr. 25, 241–258. https://doi.org/10.1111/fog.12151. Roura, Á., Roura, Á., Álvarez-Salgado, X.A., González, Á.F., Gregori, M., Rosón, G., Guerra, Á, Álvarez-Salgado, X.A., González, Á.F., Gregori, M., Rosón, G., Guerra, Á, 2013. Short-term meso-scale variability of mesozooplankton communities in a coastal upwelling system (NW Spain). Progr. Oceanogr. 109, 18–32. https://doi.org/10. 1016/j.pocean.2012.09.003. Simón, F., Rocha, F., Guerra, A., 1996. The small-scale squid hand-jig fishery off the north-western Iberian Peninsula: application of a model based on a short survey of fishery statistics. Fish. Res. 25, 253–263. Sotelo, G., Posada, D., Morán, P., 2009. Low-mitochondrial diversity and lack of structure in the velvet swimming crab Necora puber along the Galician coast. Mar. Biol. 156, 1039–1048. https://doi.org/10.1007/s00227-009-1148-7. Sweeney, M.J., Roper, C.F.E., Mangold, K.M., Clark, M.R., Boletzky, S.V., 1992. “Larval” and juvenile cephalopods: a manual for their identification. Smithson. Contrib. Zool. 1–182. https://doi.org/10.5479/si.00810282.513. Tajima, F., 1984. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 3, 607–612. Tasende M.G., Quintero F., Arnáiz R., Bañón R., Campelos J.M., Lamas F., Gan-cedo A., Rodríguez M.E. and Ribó J., La pesquería de calamar y puntilla con boliche en las Rías Baixas gallegas 1999-2003. Los Recursos Marinos de Galicia. Xunta de Galicia, Serie técnica, nº 3, Santiago de Compostela, 2005, 108 pp.
Elsa García-Mayoral: Investigation, Conceptualization, Formal analysis, Resources, Writing - original draft. Álvaro Roura: Supervision, Conceptualization, Funding acquisition, Methodology, Writing - review & editing. Andrea Ramilo: Methodology, Validation, Data curation, Writing - review & editing. Ángel F. González: Supervision, Project administration, Funding acquisition, Conceptualization, Methodology, Resources, Writing - review & editing. Acknowledgements This work was supported by the Project CALECO (CTM2015-69519R) funded by the Spanish Ministry of Economy and Competitiveness. We thank the crew and technicians of R/V Mytilus (IIM, CSIC Vigo) and R/V Sarmiento de Gamboa (CSIC) for their assistance. We are also grateful to Lara García, Sara Mohamed, Miguel Gil, Alexandra Castro, Silvia Blanco, Nicolás Robineau, Javier Tamame, Fernando Alonso and the group of Fisheries Ecology for their valuable assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fishres.2019.105406. References Anderson, F.E., Pilsits, A., Clutts, S., Laptikhovsky, V., Bello, G., Balguerías, E., Lipinski, M., Nigmatulin, C., Pereira, J.M.F., Piatkowski, U., Robin, J.P., Salman, A., Tasende, M.G., 2008. Systematics of Alloteuthis(Cephalopoda:Loliginidae) based on molecular and morphometric data. J. Exp. Mar. Bio. Ecol. 364, 99–109. https://doi.org/10. 1016/j.jembe.2008.07.026. Araujo, C.C., Gasalla, M.A., 2018. Distribution patterns of loliginid squid paralarvae in relation to the oceanographic features off the South Brazil Bight (22°–25°S). Fish. Oceanogr. 27. https://doi.org/10.1111/fog.12238. Arístegui, J., Barton, E.D., Álvarez-Salgado, X.A., Santos, A.M.P., Figueiras, F.G., Kifani, S., Hernández-León, S., Mason, E., Machú, E., Demarcq, H., 2009. Sub-regional ecosystem variability in the Canary Current upwelling. Progr. Oceanogr. 83, 33–48. https://doi.org/10.1016/j.pocean.2009.07.031. Bode, A., Álvarez-Ossorio, M.T., Cabanas, J.M., Miranda, A., Varela, M., 2009. Recent trends in plankton and upwelling intensity off Galicia (NW Spain). Prog. Oceanogr. 83 (1–4), 342–350. https://doi.org/10.1016/j.pocean.2009.07.025. Cabanellas-Reboredo, M., Alós, J., Palmer, M., March, D., O’Dor, R., 2012. Movement patterns of the European squid Loligo vulgaris during the inshore spawning season. Mar. Ecol. Prog. Ser. 466, 133–144. https://doi.org/10.3354/meps09925. Chen, C.S., Pierce, G.J., Wang, J., Robin, J.P., Poulard, J.C., Pereira, J., Zuur, A.F., Boyle, P.R., Bailey, N., Beare, D.J., Jereb, P., Ragonese, S., Mannini, A., Orsi-Relini, L., 2006. The apparent disappearance of Loligo forbesii from the south of its range in the 1990s: trends in loligo spp. abundance in the northeast Atlantic and possible environmental influences. Fisheries Research. https://doi.org/10.1016/j.fishres.2005.12.002. Diz, A.P., Presa, P., 2009. The genetic diversity pattern of Mytilus galloprovincialis in Galician Rías (NW Iberian estuaries). Aquaculture 287, 278–285. https://doi.org/10. 1016/j.aquaculture.2008.10.029. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1, 47–50. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Fu, Y.X., 1997. Statistical Tests of Neutrality of Mutations Against Population Growth. Genet. Soc. Am. 147, 915–925. García-Reyes, M., Sydeman, W.J., Schoeman, D.S., Rykaczewski, R.R., Black a, B., Smit, A.J., Bograd, S.J., 2015. Under pressure: climate change, upwelling, and eastern boundary upwelling ecosystems. Front. Mar. Sci. 2, 1–10. https://doi.org/10.3389/ fmars.2015.00109. González, Á.F., Otero, J., Guerra, A., Prego, R., Rocha, F.J., Dale, A.W., 2005. Distribution of common octopus and common squid paralarvae in a wind-driven upwelling area (Ria of Vigo, north-western Spain). J. Plankton Res. 27, 271–277. https://doi.org/10. 1093/plankt/fbi001. González, Á.F., Otero, J., Pierce, G.J., Guerra, Á., 2010. Age, growth, and mortality of Loligo vulgariswild paralarvae: implications for understanding of the life cycle and longevity. ICES J. Mar. Sci. 67, 1119–1127. https://doi.org/10.1093/icesjms/fsq014. Goodall-Copestake, W.P., Tarling, G.A., Murphy, E.J., 2012. On the comparison of population-level estimates of haplotype and nucleotide diversity: a case study using the gene cox1 in animals. Heredity (Edinb). 109, 50–56. https://doi.org/10.1038/hdy. 2012.12. Guerra, Á., Rocha, F., 1994. The life history of Loligo vulgaris and Loligo forbesi (Cephalopoda: Loliginidae) in Galician waters (NW Spain). Fish. Res. https://doi.org/
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Tenore, K.R., López-Jamar, E., Santiago, G., Gonzalez, G., Rey, J., Cal, R.M., Hanson, R.B., 2008. Upwelling and outwelling effects on the benthic regime of the continental shelf off Galicia, NW Spain. J. Mar. Res. 50, 465–488. https://doi.org/10.1357/ 002224092784797584. Ward, R.D., 2000. Genetics in fisheries management. Hydrobiologia. https://doi.org/10. 1023/A:1003928327503.
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Spatial distribution and genetic structure of loliginid paralarvae along the Galician coast (NW Spain)
T
Elsa García-Mayorala,b,*, Álvaro Rouraa, Andrea Ramiloa, Ángel F. Gonzáleza a b
Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain Campus Do Mar, Universidad de Vigo, 36310, Vigo, Spain
ARTICLE INFO
ABSTRACT
Handled by: Chennai Guest Editor
Squids belonging to the cephalopod family Loliginidae have an important economic value for the small-scale fisheries in Galicia and recreative fishing. Early life stages of loliginids are studied along the Iberian coast to forecast their population abundance based on correct species identification, but visual identification of the paralarvae is not possible for this family. Molecular analyses using cytochrome oxidase subunit I (COI) have shown that the three most common species of loliginids in the Galician coast are Alloteuthis media, Loligo vulgaris and Alloteuthis subulata. This study compared the abundance and spatial distribution of the three loliginid species between the northern and southern waters of Galicia. Results showed that A. media is the most abundant in the south and A. subulata in the north, while L. vulgaris maintains similar percentage of frequency. Genetically, L. vulgaris and A. media have complex haplotype networks and high diversity indices, while A. subulata has a reduced haplotype network and low diversity indices. The genetic variability observed in COI data showed a consistent genetic flow between north and south Galicia and no population structuring was observed along the Galician coast with this mitochondrial marker. Fine scale studies with nuclear and mitochondrial markers are needed to have a better understanding of the population structure of this important resource.
Keywords: Loliginidae Cephalopod paralarvae Genetic diversity Population structure NE Atlantic
1. Introduction The cephalopods belonging to the family Loliginidae have an important commercial value in the north-eastern Atlantic and Mediterranean, especially in the coastal Iberian Peninsula and in the Mediterranean countries (Simón et al., 1996). In this area, the loliginid family is represented by four species: Loligo vulgaris, L. forbesii, Alloteuthis media and A. subulata (Anderson et al., 2008; Guerra and Rocha, 1994; Laptikhovsky et al., 2019). In the western Mediterranean and the western coast of the Iberian Peninsula, the European squid, L. vulgaris, is caught as by catch by bottom and pelagic trawling and also by smallscale hand-jig artisanal fisheries, while A. media is caught as bycatch by bottom trawl fisheries and beach seine. On the other hand, A. subulata is considered as a secondary target species in Spain, Portugal and Italy and has low commercial value due to its small size being captured as bycatch in trawl fisheries. Specific statistics on catches for each species are impossible to know with accuracy, because most of the landings of L. vulgaris are mixed with L. forbesi under the name L. vulgaris or Loligo spp. (Jereb et al., 2015; Roper et al., 2010; Simón et al., 1996). The two Loligo species used to have overlapping distribution ranges, being L. forbesi more
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abundant in the northern area (Northern and Celtic Sea) and L. vulgaris in the south (Iberian Peninsula). During the 1990s the presence of L. forbesi declined dramatically in the west coast off the Iberian Peninsula increasing in Scottish waters (Chen et al., 2006). The other loliginid genus, smaller in size, has also been wrongly identified and used to be reported as Alloteuthis spp. or Loligo spp. (Jereb et al., 2015). In Galicia (NW Spain), the loliginids L. vulgaris and both Alloteuthis species are captured by small scale fisheries as it is hand-jigging and boliche (boat seine) located close to the coast (Jereb et al., 2015; Tasende et al., 2005; Simón et al., 1996). The landings of L. vulgaris in the last ten years were 3472 t, going from 338 t to 87 t during 2007–2016, while a total of 172 t of Alloteuthis spp. were caught in the same period. Captures of L. vulgaris in Galicia decreased to 75 t while Alloteuthis spp. increased to 8 t in 2018 (https://www.pescadegalicia. gal). Loliginids have a pelagic larval stage that spend two to three months as zooplankton, known as paralarvae. During this dispersal stage, they remain over the continental shelf by means of controlling their vertical position (Roura et al., 2016, 2019). Previous studies made in the west coast of Iberian Peninsula (NE Atlantic) classified the loliginid paralarvae at family level (González et al., 2005; Moreno et al., 2009; Rocha
Corresponding author at: Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain. E-mail address: [email protected] (E. García-Mayoral).
https://doi.org/10.1016/j.fishres.2019.105406 Received 9 July 2019; Received in revised form 24 September 2019; Accepted 2 October 2019 0165-7836/ © 2019 Elsevier B.V. All rights reserved.
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and Guerra, 1999; Roura et al., 2013) because the chromatophore pattern, a species-specific character present is fresh specimens (Sweeney et al., 1992), practically disappears in ethanol-preserved specimens. However, current molecular techniques amplifying the cytochrome oxidase I (COI), showed that the loliginid paralarval assemblage of the western Iberian Peninsula and north of Africa is dominated by Alloteuthis media and A. subulata, followed by Loligo vulgaris (OlmosPérez et al., 2018; Roura et al., 2019). The abundance of paralarvae is a potential tool that can be used to make stock predictions (González et al., 2010). However, proper identification of loliginid paralarvae is essential for a correct management of this resource in order to estimate biomass and quota uptake (Ward, 2000). Loliginid paralarval abundance is related positively with upwelling intensity in the southern waters of Galicia with a peak in their abundance in the water column at night (González et al., 2005; Otero et al., 2009; Roura et al., 2016; Olmos-Pérez et al., 2018). The only data gathered from the north revealed the presence of loliginid paralarvae on the shelf (Rocha, 1999), but only 15 individuals were collected and visually identified based on chromatophore pattern. Recent studies have shown that the different loliginid species occupy different niches in the horizontal and vertical axis of the water column. Alloteuthis media is found closer to the shore than A. subulata, while L. vulgaris overlaps the distribution of these two species. In the water column, A. subulata is found in deeper strata than A. media (Olmos-Pérez et al., 2018). Despite these spatial differences that imply different dispersal capabilities, the genetic flux of the loliginid assemblage is enough to keep high nucleotide and haplotype diversity indices for A. media and L. vulgaris but low in A. subulata along the Canary Current upwelling system (CanC), both in south Galicia (Olmos-Pérez, 2018) and between the northern and southern limits of the CanC (> 2000 km apart, Roura et al., 2019). Given that the northern part of Galicia is not under the influence of the CanC, it could represent an oceanographic barrier for genetic flux between the Western and Northern Iberian Peninsula squid populations. Accordingly, the aims of this study are three: 1) genetically identify the loliginid paralarvae present along the Galician coast, 2) recognise their distribution patterns and 3) evaluate if there exists a genetic barrier between the northern and southern waters of Galicia that may affect population structure and diversity indices.
Ons islands (Fig. 1D). Zooplankton samples were caught with a Multinet Hydrobios Mammoth net of 250 μm mesh size by filtering about 200 m3 at seven different strata: 105–85, 85–55, 55–35, 35–20, 20–10. 10–5 and 5 to 0 m. The samples were fixed on board in 96% alcohol and stored at −20 °C. Afterwards, loliginid paralarvae were separated from the zooplankton samples in the laboratory and prepared for molecular analyses. 2.3. Mitochondrial DNA sequencing Loliginid paralarvae from north and south Galicia were preserved in 96% ethanol for molecular analysis. Individual mantle from each loliginid paralarvae was dissected and genomic DNA purifications were performed employing QIAamp DNA Micro Kit (QIAGEN) according to the manufacturer’s protocol for isolation of genomic DNA from tissues. DNA quality and quantity were checked in a spectrophotometer Nanodrop® ND-2000 (Thermo Scientific). To identify the loliginid species, a fragment of cytochrome c oxidase subunit I (COI) was amplified using the universal primers HCO2198 and LCO1490 (Folmer et al., 1994). PCR reactions were performed in a total volume of 25 μl containing 1 μl of genomic DNA, PCR buffer at 1x concentration, 0.3 μM primers, 0.2 mM nucleotides and 0.025 U. μl −1 DreamTaq DNA Polymerase (Thermo Scientific). The PCR assays were carried out in a Tgradient thermocycler (Biometra), under the following reaction conditions: 3 min at 95 °C, 40 cycles of 30 s at 95 °C, 45 s at 48 °C and 1 min at 72 °C, followed by 7 min at 72 °C. A negative control (no DNA) was included in all PCR amplifications. PCR products were separated on a 2% agarose gel in Tris acetate EDTA buffer, stained with Red Safe and scanned in a GelDoc XR documentation system (Bio-Rad Laboratories). The amplified fragments were cleaned for sequencing using ExoSAP-IT™ PCR Product Cleanup (Applied Biosystems) for 15 min at 37 °C, followed by inactivation for 15 min at 80 °C. Sequencing was performed in StabVida (Portugal) and the chromatograms were analysed using ChromasPro 2.1.8. (Technelysium Pty Ltd). All generated sequences were searched for species identity using BLAST (Basic Local Alignment Search Tool) through web servers of the National Center for Biotechnology Information (NCBI). 2.4. Population genetic analysis
2. Material and methods
To evaluate the genetic diversity of the different loliginid species, the software DnaSP v. 6 (Librado and Rozas, 2009) was used to calculate the number of haplotypes (H), the haplotype diversity (h), nucleotide diversity (π), number of segregating sites (S), and the average number of nucleotide differences (k). The neutrality tests of Tajima’s D (Tajima, 1984) and Fu’s FS (Fu, 1997) were tested with Arlequin v3.5.2 (Excoffier et al., 2005). In addition, to examine population differentiation, pair-wise Fst values were computed counting the haplotype frequencies for each species using Arlequin v3.5.2. A final alignment with 195 sequences of 623bp was used to build the median joining haplotype networks using Nexus software (www.fluxus-engineering. com). Additionally, in order to get better results, three haplotype sequences of A. subulata from the Ría de Vigo (Olmos-Pérez et al., 2018) obtained in BOLD (MF157701, MF157702, MF157703) were assembled manually. Furthermore, for this study sequences of loliginid paralarvae from the CanC (Roura et al., 2019) were downloaded from the online project CEPAR in BOLD.
2.1. Study area The NE Atlantic coast constitutes one of the four eastern boundary upwelling systems of the world, known as the Canary Current upwelling system (CanC; Arístegui et al., 2009). The western Iberian Peninsula is at the northern end of this upwelling system, characterised by seasonal upwelling, between May-June to September-October. During this season north-eastern winds force the Eastern North-Atlantic Central Water (ENACW), a cold and nutrient-rich current, to the surface sustaining an important fishery industry along the coasts of Portugal and Galicia (NW of Iberian Peninsula). Galicia is composed by coastal embayment called Rías where the upwelling is intensified (Tenore et al., 2008, 1995). 2.2. Sampling Overall, 278 zooplankton samples were collected in the north of Galicia (Fig. 1A) during June 2017 on board of R/V Sarmiento de Gamboa with a bongo net of 500 μm mesh size, doing double oblique transects at 2.5 knots. Zooplankton samples (n = 338) were obtained in southern Galicia (Fig. 2B) on board of R/V Mytilus from June to November 2017. Two types of surveys were carried out in the area: six surveys were undertaken at night in the Ría de Vigo (Fig. 1C) and four 24 h surveys were carried out along six transects surrounding Cíes and
3. Results 3.1. Genetic identification and diversity A total of 207 loliginid paralarvae were collected from north (N = 54) and south Galicia (N = 153). The identification of 198 of 207 paralarvae revealed three species of loliginids (Table 1). From 144 2
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Fig. 1. Map locating the sampling sites. A) Samplings from the north, with depths ranging from 50 to 500 m and B) the south of Galicia, showing in detail the transects carried out in C) the Ría de Vigo and D) around the Cíes and Ons Islands, with depths ranging from 10 to 105 m.
individuals sequenced in southern Galician waters, 79 were classified as Alloteuthis media (56%), followed by 55 Loligo vulgaris (39%) and ten A. subulata (5%). Out of 54 specimens sequenced in northern Galicia, 40 were classified as A. subulata (74%), eight as L. vulgaris (15%) and six as A. media (11%). The COI sequences analysed revealed a total of 78 haplotypes: 35 from L. vulgaris, whose populations from north and south of Galicia shared three haplotypes; 34 from A. media, with three haplotypes shared between both localities; and nine from A. subulata sharing two haplotypes. The highest haplotype and nucleotide diversity was observed in L. vulgaris and the lowest in A. subulata (Table 1). Overall, species from the south have a higher haplotype diversity than those from the north, despite the southern samples were collected in an area much smaller than that of the north. In contrast, nucleotide diversity was higher in the north for A. subulata and L. vulgaris, but lower for A. media (Table 1).
Conversely, A. subulata had a reduced haplotype network with one main haplotype and four branches emerging from it, where two haplotypes from the northern coast are separated by 11 and 13 mutations. Finally, L. vulgaris presents a complex haplotype network with a high number of mutations and four main haplotypes (H1, H3, H5 and H15, accounting for 8; 5; 10 and 6 individuals out of 63). Three of these four main haplotypes are shared between northern and southern populations, except for haplotype H5, which was only present in the southern region. 3.3. Spatial distribution and abundance The spatial distribution of loliginid paralarvae revealed differences in the species composition between northern and southern Galician waters. A. media is the most abundant species in the south-western, while A. subulata is the dominant species in the north. Horizontal distributions in both locations showed that A. media is found closer to the coast than A. subulata, which are found in offshore waters (Fig. 3). A similar pattern was observed in the north, with a gradient from the border of the continental shelf (200 m depth) dominated by A. subulata, followed by L. vulgaris and A. media in shallower samples (Fig. 3A, B). Furthermore, A. subulata paralarvae were vertically distributed from 10 to 55 m in southern waters. Meanwhile, A. media and L.vulgaris overlap their distribution from the surface down to 55 m (Fig. 4B).
3.2. Population structure Pairwise comparison showed non-significant values (Fst, p > 0.05) for the three species between north and southern squid populations, thus indicating a constant genetic flux between them. Moreover, detection of negative significant departures from the null hypothesis of neutrality using Tajima´s D were detected in A. media, A. subulata and L. vulgaris (except for the northern specimens, Table 1). On the other hand, results of the Fu’s Fs test for A. subulata cannot rejected the null hypothesis of neutral evolution but significant negative values were obtained for A. media and L. vulgaris. Alloteuthis media had a star-shaped haplotype network with two main haplotypes (H2 and H7 accounting for 30 and 11 individuals respectively out of 85, Suppl. Table 1) that are shared by the populations from north and south Galicia and numerous single haplotypes (Fig. 2).
4. Discussion This study analysed the taxonomic composition and abundance of cephalopod paralarvae belonging to the family Loliginidae along the Galician coast, a seasonal upwelling region. Results obtained showed that A. media prevails in the south coast of 3
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Fig. 2. Haplotype networks for each species. In black the paralarvae belonging to the south and in light grey individuals from the northern waters of Galicia. Haplotype number is represented in black and the position of the different mutation within the aligned sequences are shown in red.
Galicia, while in the northern coast is A. subulata. The study done by Olmos-Pérez et al. (2018) between 2012–2014 in the southern waters of Galicia also agreed with our research, which also found that A. media was the most common loliginid paralarvae (57.5%), with L. vulgaris abundance in an increasing tendency during the last years with A. subulata in an opposite variation trend (Fig. 5). At a wider scale, the loliginid paralarvae collected at the limits of the CanC in 2009 (from north-western Iberian Peninsula and southwest Morocco) showed that A. media (85%) was the most abundant, followed by A. subulata (14%) and L. vulgaris (1%). Altogether, these data suggest that L. vulgaris is increasing its relative abundance and the opposite is occurring with A. subulata, maybe related with the decreasing intensity of the upwelling along the Iberian coast and the increasing in thermal stratification (Bode et al., 2009; García Reyes et al., 2015): However, more data are needed to evaluate in detail this hypothesis.
4.1. Population structure The genetic diversity indices calculated for L. vulgaris and A. media indicate high haplotype (h > 0.7013) and nucleotide diversity (π > 0.00356), taking into account the values calculated by GoodallCopestake et al. (2012). Alloteuthis subulata showed low genetic differentiation but numerous segregating sites. Similar results were obtained in Olmos-Pérez et al. (2018), where L. vulgaris had lower values than those obtained in this study but higher in absolute terms, whereas A. media had a medium-high (h = 0.680, π = 0.0019) genetic variation. Loligo vulgaris showed a much more complex network with higher number of mutations than the network obtained by Olmos-Pérez et al. (2018). Given that the samples were collected in the same area as Olmos-Pérez et al. (2018), but three years later, the differences could be attributed due to the total number of L. vulgaris caught in the different years. In south of Galicia 29 paralarvae were caught by Olmos-Pérez
Table 1 Molecular diversity indices and population demographic statistics for each loliginid species. H, number of haplotypes; h, haplotype diversity; π, nucleotide diversity; S, number of segregating sites; k, average number of nucleotide differences; D, Tajima’s D (Tajima, 1984); Fs, Fu’s Fs (Fu, 1997). Species
Location
n
H
h ( ± standard deviation)
π
S
K
D
Fs
A. media A. media A. media A. subulata A. subulata A. subulata L. vulgaris L. vulgaris L. vulgaris
North South All North South All North South All
6 79 85 40 10 50 8 55 63
4 33 34 7 4 9 6 32 35
0.800 0.865 0.857 0.433 0.644 0.473 0.929 0.949 0.948
0.00193 0.00275 0.00269 0.00242 0.00171 0.00228 0.01181 0.00982 0.01008
3 25 26 21 4 22 13 42 44
1.20 1.71 1.67 1.51 1.07 1.42 7.36 6.12 6.28
−0.447ns −2.079** −2.113** −2.311** −0.943ns −2.344** 1.390ns −1.327ns −1.325ns
−1.454* −27.709** −27.883** −0.911ns −0.742ns −2.532ns 0.3049ns −16.887** −18.709**
± ± ± ± ± ± ± ± ±
0.172 0.032 0.033 0.095 0.152 0.084 0.084 0.018 0.015
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Fig. 3. Maps showing the relative abundances of loliginid paralarvae in the sampling area. A) Map showing the north and B) the south Galician coast.
and for this study 55 individuals were obtained. The haplotype networks of Alloteuthis media and A. subulata are quite similar to the networks obtained by Olmos-Pérez et al. (2018) in the south of Galicia and Roura et al. (2019) throughout the CanC (from NW Iberian Peninsula to SW Morocco). It is remarkable that similar haplotype networks were obtained with specimens collected more than 2000 km apart, thus showing that the dispersal of these paralarvae and adult migrations
effectively mixes the population along the CanC. Comparing the haplotypes obtained in this study with those from different studies carried in the same area four and eight years ago (Olmos-Pérez et al., 2018 and Roura et al., 2019, respectively), it is interesting to note that A. media specimens have two haplotypes in common (H1 and H2) in the different studies, while the paralarvae from CanC shared seven unique haplotypes with the south and three from the
Fig. 4. A) Horizontal distribution of paralarvae in the north, where samples were collected with bongo and B) vertical distribution through the water column in the southern coast, where samples were collected with a multinet. 5
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Fig. 5. Relative abundance of the 3 species through the different sampling years. Asterisk shows samples collected in other studies: NW Iberian Peninsula in 2009 (Roura et al., 2019), and between 2012 and 2014 in the Ría de Vigo (Olmos-Pérez et al., 2018). Samples collected in 2017 belong to this study.
north of Galicia (Suppl. Table 1). This indicates a clear connection of the species along the Atlantic coast. Similarly, A. subulata main haplotype is the same in the different years and locations. On the other hand, L. vulgaris individuals from the south collected in this study shared five haplotypes with specimens sampled in 2013–2014 in the same area (Olmos-Pérez et al., 2018), while individuals sampled in the north only shared two haplotypes with those sampled by Olmos-Pérez et al. (2018). In terms of neutrality tests, A. media obtained significant negative Tajima’s D and Fu’s Fs for the southern population revealing an excess of low frequency polymorphisms (rare alleles) relative to expectation, suggesting population expansion after a recent bottleneck (GoodallCopestake et al., 2012). Nevertheless, A. subulata presented significant negative values for Tajima’s D in the northern population that evidence that COI has evolved under non-random processes, possibly affected by a recent selective sweep, which is shown by the absence of numerous haplotypes. This low number of haplotypes has been previously observed in the Eastern Atlantic (Olmos-Pérez et al., 2018; Roura et al., 2019 and Suppl. Table 1). For L. vulgaris the neutrality tests were not significant except for the southern population that can result from demographic events (Sotelo et al., 2009). Because of the change in the orientation of the coast of Cape Finisterre, it has been mentioned as a biogeographical boundary for gene flow between north and south of Galician waters (López-Jamar et al., 1992; López et al., 2015). However, genetic analyses carried in this study using COI data showed a clear homogeneity along the Galician coast for the three species meaning that there is an effective gene flow between the two areas. This lack of genetic structure was seen in other molluscs such as Mytilus galloprovincialis (Diz and Presa, 2009) and Donax trunculus with microsatellites (Nantón et al., 2017). Also, the velvet swimming crab Necora puber and the seahorse Hippocampus guttulatus showed lack of structure along the Galician coast (López et al., 2015; Sotelo et al., 2009). Nonetheless, some caution needs to be taken when interpreting this dataset owed to two factors: the differences in sample size for each species that may affect the resolution of genetic indices and that this study has been conducted with just one mitochondrial marker, which gives a broad idea of the population structure along the Iberian Coast, but more markers would be necessary to assess finer scale population structure.
coast. Loliginid paralarvae are suggested to display a coastal strategy (Roura et al., 2016, 2019), being retained between the coast and the limit of the continental shelf due to the interaction of their vertical behaviour and coastal currents, thus reducing offshore transport and increasing alongshore dispersal. The distribution pattern found in this work, with all the loliginid paralarvae collected within the limits of the continental shelf (0–200 m depth), supports the coastal strategy suggested for this family of cephalopod paralarvae, which are evenly distributed in the coastal area with some intraspecific differences. The fact that paralarvae occur close to the coast could suggest that adults migrate to the coast for spawning. Research done in the Mediterranean Sea (Cabanellas-Reboredo et al., 2012) inferred that L. vulgaris hatchlings occur close to the coast and later migrate to deeper waters over the continental shelf, such as L. gahi in Falkland Islands or L. vulgaris reynaudii in South Africa (Rodhouse et al., 1992). Other studies suggested that L. vulgaris occurs between shore and 100 m isobaths, whereas Alloteuthis spp. adults are found over the shelf and upper slope until the 350 m (Anderson et al., 2008; Moreno et al., 2007). Other research of the loliginid species Doryteuthis plei and D. sanpaulensis (Araujo and Gasalla, 2018) found that although they are coastal, their abundance is influenced by environmental factors such as the upwelling and sea surface temperature. Additionally, there were differences in the horizontal distribution where D. plei was found in the southernmost area and D. sanpaulensis in the northernmost off the South Brazil Bight. Moreover, Meath et al. (2019) found that D. pealeii and D. plei which, are two species that co-occur in the Gulf of Mexico, presented different migration movements, while Doryteuthis pealeii moves through southward waters, D. plei moves to northward. This behavior has been observed in other marine species such as the fish from the genus Etheostoma (Hlohowskyj and White, 1983). This behavioural adaptation permits to each species to have the same prey as food resource, but the intense intraspecific competition is reduced by habitat shift (Mendelson, 2006). The data reported by Olmos-Pérez et al. (2018) states that the spatial distribution of Alloteuthis species is quite different, both horizontally and vertically, with A. media found through all the water column and A. subulata appearing from 10 to 55 m in the south and along the continental platform in the north. At a wider scale, A. media is more abundant in the south and A. subulata in the north of Galicia. These differences could be due to competitive interactions, and Alloteuthis spp. could be minimizing the resource competition by habitat shift, with subtle changes in their vertical position that affect their horizontal distribution.
4.2. Spatio-temporal variability In this study, we observed differences on the spatial distribution of the three Loliginidae paralarvae found in the north-western Spanish 6
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CRediT authorship contribution statement
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Elsa García-Mayoral: Investigation, Conceptualization, Formal analysis, Resources, Writing - original draft. Álvaro Roura: Supervision, Conceptualization, Funding acquisition, Methodology, Writing - review & editing. Andrea Ramilo: Methodology, Validation, Data curation, Writing - review & editing. Ángel F. González: Supervision, Project administration, Funding acquisition, Conceptualization, Methodology, Resources, Writing - review & editing. Acknowledgements This work was supported by the Project CALECO (CTM2015-69519R) funded by the Spanish Ministry of Economy and Competitiveness. We thank the crew and technicians of R/V Mytilus (IIM, CSIC Vigo) and R/V Sarmiento de Gamboa (CSIC) for their assistance. We are also grateful to Lara García, Sara Mohamed, Miguel Gil, Alexandra Castro, Silvia Blanco, Nicolás Robineau, Javier Tamame, Fernando Alonso and the group of Fisheries Ecology for their valuable assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fishres.2019.105406. References Anderson, F.E., Pilsits, A., Clutts, S., Laptikhovsky, V., Bello, G., Balguerías, E., Lipinski, M., Nigmatulin, C., Pereira, J.M.F., Piatkowski, U., Robin, J.P., Salman, A., Tasende, M.G., 2008. Systematics of Alloteuthis(Cephalopoda:Loliginidae) based on molecular and morphometric data. J. Exp. Mar. Bio. Ecol. 364, 99–109. https://doi.org/10. 1016/j.jembe.2008.07.026. Araujo, C.C., Gasalla, M.A., 2018. Distribution patterns of loliginid squid paralarvae in relation to the oceanographic features off the South Brazil Bight (22°–25°S). Fish. Oceanogr. 27. https://doi.org/10.1111/fog.12238. Arístegui, J., Barton, E.D., Álvarez-Salgado, X.A., Santos, A.M.P., Figueiras, F.G., Kifani, S., Hernández-León, S., Mason, E., Machú, E., Demarcq, H., 2009. Sub-regional ecosystem variability in the Canary Current upwelling. Progr. Oceanogr. 83, 33–48. https://doi.org/10.1016/j.pocean.2009.07.031. Bode, A., Álvarez-Ossorio, M.T., Cabanas, J.M., Miranda, A., Varela, M., 2009. Recent trends in plankton and upwelling intensity off Galicia (NW Spain). Prog. Oceanogr. 83 (1–4), 342–350. https://doi.org/10.1016/j.pocean.2009.07.025. Cabanellas-Reboredo, M., Alós, J., Palmer, M., March, D., O’Dor, R., 2012. Movement patterns of the European squid Loligo vulgaris during the inshore spawning season. Mar. Ecol. Prog. Ser. 466, 133–144. https://doi.org/10.3354/meps09925. Chen, C.S., Pierce, G.J., Wang, J., Robin, J.P., Poulard, J.C., Pereira, J., Zuur, A.F., Boyle, P.R., Bailey, N., Beare, D.J., Jereb, P., Ragonese, S., Mannini, A., Orsi-Relini, L., 2006. The apparent disappearance of Loligo forbesii from the south of its range in the 1990s: trends in loligo spp. abundance in the northeast Atlantic and possible environmental influences. Fisheries Research. https://doi.org/10.1016/j.fishres.2005.12.002. Diz, A.P., Presa, P., 2009. The genetic diversity pattern of Mytilus galloprovincialis in Galician Rías (NW Iberian estuaries). Aquaculture 287, 278–285. https://doi.org/10. 1016/j.aquaculture.2008.10.029. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1, 47–50. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Fu, Y.X., 1997. Statistical Tests of Neutrality of Mutations Against Population Growth. Genet. Soc. Am. 147, 915–925. García-Reyes, M., Sydeman, W.J., Schoeman, D.S., Rykaczewski, R.R., Black a, B., Smit, A.J., Bograd, S.J., 2015. Under pressure: climate change, upwelling, and eastern boundary upwelling ecosystems. Front. Mar. Sci. 2, 1–10. https://doi.org/10.3389/ fmars.2015.00109. González, Á.F., Otero, J., Guerra, A., Prego, R., Rocha, F.J., Dale, A.W., 2005. Distribution of common octopus and common squid paralarvae in a wind-driven upwelling area (Ria of Vigo, north-western Spain). J. Plankton Res. 27, 271–277. https://doi.org/10. 1093/plankt/fbi001. González, Á.F., Otero, J., Pierce, G.J., Guerra, Á., 2010. Age, growth, and mortality of Loligo vulgariswild paralarvae: implications for understanding of the life cycle and longevity. ICES J. Mar. Sci. 67, 1119–1127. https://doi.org/10.1093/icesjms/fsq014. Goodall-Copestake, W.P., Tarling, G.A., Murphy, E.J., 2012. On the comparison of population-level estimates of haplotype and nucleotide diversity: a case study using the gene cox1 in animals. Heredity (Edinb). 109, 50–56. https://doi.org/10.1038/hdy. 2012.12. Guerra, Á., Rocha, F., 1994. The life history of Loligo vulgaris and Loligo forbesi (Cephalopoda: Loliginidae) in Galician waters (NW Spain). Fish. Res. https://doi.org/
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