The main aim of this work was to learn more about the paralarval phase of loliginid squids, probably the most difficult phase to study, which are of high interest for stock predictions, growth models or as a model for ontogenetic patterns. Two main difficulties are constraining this field, namely the scarcity of squid paralarvae in the plankton and the lack of reliable morphological characters to accurately identify the paralarvae. Previous studies showed an unexpected diversity of loliginid paralarvae in the plankton of NW Iberian Peninsula, traditionally considered to be exclusively inhabited by Loligo vulgaris. Accordingly, the hidden diversity of loliginid paralarvae was unraveled to establish baseline studies for future species-specific management strategies. Visual identification of early life stages in squids can be problematic because ethanol-preserved paralarvae might lose the chromatophore patterns that help to identify each species. Therefore, molecular approaches are the only reliable tools to identify different species, when morphological characters are lost or not yet developed. For this study, the mitochondrial cytochrome c oxidase subunit 1 (COI), also known as the barcoding gene, was used to unequivocally identify the different loliginid paralarvae. In Galician waters, three different species were identified: Loligo vulgaris (n=99), Alloteuthis media (n=118) and Alloteuthis subulata (n=15) showing a different spatial distribution pattern. While in the south-west of Galicia the most abundant species was A. media, in the north is A. subulata. Loligo vulgaris maintained its abundance similar in both locations. These differences between Alloteuthis species, might result from interspecific competitive interactions, in order to minimize resource competition by shifting the habitat, both horizontally and vertically.
Understanding the demographic structure of these three loliginid species along the Galician coast, involves estimating growth patterns for each species. Consequently, statoliths were extracted and growth increments were counted. Statistical analyses showed significant differences on dorsal mantle length (DML) at age between gender, being L. vulgaris larger at the same age than A. media and A. subulata. Additionally, instantaneous relative growth rates were higher in spring than in autumn for all species. The paralarvae of L. vulgaris and A. media reflected two well differentiated hatching peaks, one in late spring-early summer and another peak in late autumn. Meanwhile, the hatching peak for A. subulata occurred in spring, before than the other two loliginids, and a second peak that was almost negligible owed to the lack of paralarvae.
The following step was focused on L. vulgaris due to its high market value and importance for local fisheries along the Western Iberian Peninsula waters (WIP). The next biological question was to elucidate the diet of L. vulgaris paralarvae in the wild applying next generation sequencing (NGS). The digestive system of 31 paralarvae was dissected and a 300bp section of COI gene was sequenced with the MiSeq platform. In total, 45 taxa were identified belonging to 34 species: 20 species of crustaceans, 3 of fish, 4 of Cnidaria, 2 of Echinodermata and 5 of molluscs including cephalopods. This trophic study revealed that the European common squid, L. vulgaris, is a generalist predator during their paralarval phase.
Finally, the genetic diversity and population connectivity of L. vulgaris was studied in detail along the WIP. Two molecular approaches were used for this study: 1) COI data and double digest restriction-site associated DNA sequencing (ddRADseq). The results obtained revealed 79 haplotypes out of 160 COI sequences, while the SNP data set included 86,431 loci after filtering for 38 individuals with 86,319 neutral loci. Both techniques showed high genetic diversity and homogeneity with high gene flow, revealing a single population along the WIP, which can be managed as a single unit
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