DOI: 10.5061/dryad.t1g1jwtck
title: Multilocus genetic data diversification patterns in Laevilitorininae

description: 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.
description: 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 <em>c
 </em>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.
description: # Multilocus genetic data diversification patterns in Laevilitorininae
 [https://doi.org/10.5061/dryad.t1g1jwtck](https://doi.org/10.5061/dryad.t1g1jwtck) ## Description of the data and file structure This study examines the phylogeny, systematics, and biogeography of the subfamily Laevilitorininae. This research was focused on the extreme south of South America, including the Magellanic region and Cape Horn, also different Subantarctic Islands, West Antarctica and the South Island of New Zealand. Although this work was a collaborative effort involving five countries, Chile, France, Belgium, United Kingdom and New Zealand, it was led by researchers from a Global South country, with funding provided mostly by Chile. By analysing multi-locus genetic (12S, Cox1, 16S and 28S) data and radular morphology, our findings reveal that the genera Laevilacunaria and Laevilitorina are paraphyletic, indicating a more complex taxonomy and biogeography within the Laevilitorininae. The genus Laevilacunaria, previously underestimated in its diversity, encompasses at least two distinct clades. One clade includes both Antarctic species and a species present north of the Antarctic Polar Front, while the other is restricted to West Antarctica and South Georgia. ### Files and variables #### File: 12S\_dataset.fasta **Description:** Mitochondrial 12S gene data of the different species of Laevilitorininae. #### File: 16S\_dataset.fasta **Description:**  Mitochondrial 16S gene data of the different species of Laevilitorininae. #### File: Cox1\_dataset.fasta **Description:**  Mitochondrial Cox1 gene data of the different species of Laevilitorininae. #### File: 28S\_dataset.fasta **Description:**  Nuclear 28S gene data of the different species of Laevilitorininae. Localities code: Magellan Province= CH: Chabunco Beach, PO: Porvenir, TF: Tierra del Fuego, WI: Puerto Williams, HI: Horn Island; Flk/Mal: Falkland Is/Malvinas. West Antarctica=SG: South Georgia Island, SO: South Orkney, FB: Fildes Bay, EB: Esperanza Bay, CI: Cockburn Island, DI: Deception Island, YB: Yelcho Base, AV: Avian Island, Subantarctic Island, South Indian Ocean= CZ: Crozet Island, KER: Kerguelen Island ## Code/software Visual inspection of the data was performed in the paid software Geneious 11.1.5 with a personal R11 student license. To elucidate the phylogenetic relationships of *Laevilacunaria *species, 12S rRNA, 16S rRNA, cox1 and 28S rRNA sequences available from GenBank were aligned with our datasets (see Table S2) using MAFFT 6 [36] with default parameters [36] and then manually checked. Optimal nucleotide substitution models and partitioning scheme were selected for each marker separately using the corrected Akaike information criteria (AIC) in partitionfinder v2.1, allowing for cox1 codon partitioning [37] (Table S3). The datasets of the different loci were combined in sequencematrixv1.8 [38]. 36.        Katoh K., Rozewicki J., Yamada K.D. 2019 MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. *Brief Bioinform* **20**(4), 1160-1166. (doi:10.1093/bib/bbx108). 37.        Lanfear R., Frandsen P.B., Wright A.M., Senfeld T., Calcott B. 2017 PartitionFinder 2: New Methods for Selecting Partitioned Models of Evolution for Molecular and Morphological Phylogenetic Analyses. *Mol Biol Evol* **34**(3), 772-773. (doi:10.1093/molbev/msw260). 38.        Vaidya G., Lohman D.J., Meier R. 2011 SequenceMatrix: concatenation software for the fast assembly of multi-gene dataset with character set and codon information. *Cladistics* **27**, 171-180.