Multilocus genetic data diversification patterns in Laevilitorininae

The high-latitude littorinids include three intertidal and subtidal genera: Laevilitorina Pfeffer 1886, Pellilitorina Pfeffer 1886 and Laevilacunaria Powell 1951 (Reid 1989). The genus Laevilacunaria has a restricted distribution in the Southern Ocean (SO), mainly in some sub-Antarctic islands (South Georgia, Crozet and Kerguelen) and the Antarctic Peninsula. In contrast, Laevilitorina is one of the most widely distributed marine gastropod genera in high latitudes of the Southern Hemisphere and the SO. An important trait of both genera is that they have a life cycle without free-living pelagic larvae. Therefore, these genera represent a very interesting biogeographical model, since one presents a wider distribution and a greater number of species than the other despite having similar developmental modes and ecologies. This research aims to establish the origin and diversification of Laevilitorina and Laevilacunaria in the SO, in addition to addressing the taxonomy and systematics of these genera. The phylogenetic analysis shows that the genera Laevilacunaria and Laevilitorina are paraphyletic. In this work we propose a reclassification of Laevilitorininae into four distinct genera based on molecular and morphological differences. Furthermore, our results suggest that the evolutionary origin and diversification of Laevilitorininae in the SO shows that the ancestor of the Laevilitorininae subfamily is Gondwananic, whose first diversification coincides with the final phase of Gondwana fragmentation. The early diversification of this group would have been influenced by vicariance processes where four major clades would be formed. Finally, more recently long-distance dispersal events with establishment played a key role in the current geographic distribution of Laevilacunaria and Laevilitorina. These events, probably facilitated by rafting through floating macroalgae, allowed Laevilacunaria and Laevilitorina species to cross oceanographic barriers and colonize new areas.

Methods
DNA extractions were performed from portions of the animal's foot, or from the whole animal in case of very small individuals, using the DNeasy Blood& Tissue Kit (Qiagen), with a modified protocol for small amounts of tissue (Maturana et al., 2021). Four molecular markers were used for this study: genes previously utilized in littorinid phylogenies (Reid et al., 2012; Saha et al., 2022; Williams et al., 2003). These genes include the mitochondrial cytochrome c oxidase subunit I (COI; 620 bp), 12S ribosomal RNA (12S rRNA; 320 bp), 16S ribosomal RNA (16S rRNA; 480 bp) and the nuclear gene 28S ribosomal RNA (28S rRNA; 1300 bp). Amplification of the different markers were done following Williams et al., (2003). Primer details and annealing temperature (Tm) for corresponding genes are described in supplementary (Table S1). PCRs were performed in 25 μl reaction volume containing 2 μL template DNA (10–20 ng), 0.8 mM of each primer, 12,5 μL of Phusion Hot Start II High-Fidelity PCR MasterMix (Thermo Fisher Scientific, Vilnius, Lithuania) and sterilized Milli-Q water. The reaction conditions included an initial denaturation step of 3 min at 98◦C, followed by 34 cycles of 10 s at 98 ◦C, 30 s at a gene-specific annealing temperature (Table S1), 15s at 72 ◦C, and a final extension of 10 min at 72 ◦C. PCR amplicons were purified and sequenced in both directions at Macrogen (Korea). Forward and reverse sequences were manually examined using Phred scores to ensure all sequenced bases matched and were of good quality. Contigs were assembled using Geneious 10.2.2 (Kearse et al., 2012) and independently aligned using Muscle (Edgar, 2004) with standard settings. Conserved sites, variable sites and parsimony-informative sites for each region were estimated using MEGA 5 software.