New patellogastropod mitogenomes help counteracting long-branch attraction in the deep phylogeny of gastropod mollusks
Graphical abstract
Introduction
One pervasive systematic error in phylogenetic reconstruction is the well-known long-branch attraction (LBA) artifact (Bergsten, 2005). Originally noted by Felsenstein (1978), the LBA artifact is defined as the clustering of fast-evolving taxa (with long branches) in the tree irrespective of their genuine phylogenetic position. LBA often deceives phylogenetic methods when the taxon sampling is poor or the outgroup is distant (Lartillot et al., 2007). Moreover, the LBA is particularly misleading because the inferred false sister relationships generally receive high statistical support (Felsenstein, 1978). Probabilistic methods such as maximum likelihood (ML) and Bayesian inference (BI) are affected by LBA due to model violations (Brinkmann et al., 2005, Lartillot et al., 2007, Roure and Philippe, 2011). Several approaches have been proposed to correct or at least alleviate LBA (Philippe et al., 2011, Philippe and Roure, 2011). Some methods focus on improving the taxon sampling by generally adding more taxa or by specifically including slowly-evolving taxa, which break up or substitute the long branches involved in the LBA, respectively (Brinkmann et al., 2005). Other approaches are aimed at reducing saturation (convergence problems) in the alignments by removing fast-evolving positions (Philippe, 2000). Finally, the most effective solutions to LBA directly target model misspecification problems. In particular, the devise of site-heterogeneous models of evolution such as the CAT-GTR model (Lartillot and Philippe, 2004) has been reported as particularly successful in mitigating artifacts caused by LBA and site-specific compositional heterogeneity (Lartillot et al., 2007). Despite these efforts, not many studies are able to unequivocally counteract LBA, which is still one of the primary causes of generating controversial phylogenetic results (Anderson and Swofford, 2004).
Gastropods are the most diverse lineage of extant mollusks comprising about 80% of the species of the phylum. With >70,000 living species, they are adapted to marine (any depth), brackish, freshwater, terrestrial and even arboreal habitats (Aktipis et al., 2008). They also show a rich fossil record and a wide variety of morphologies (Fryda et al., 2008). Therefore, they could constitute a sound model system for evolutionary biology studies, once a robust phylogeny for the group is available. Gastropods are currently divided into seven main lineages: Patellogastropoda, Cocculiniformia, Neomphalina, Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia, although differences arise regarding the taxonomic rank and phylogenetic relationships among these major groups. Recently, Bouchet et al. (2017) assigned the rank of subclasses to these groups, except for Cocculiniformia and Neomphalina, which were included in the subclass Neomphaloides. Among all living gastropod lineages, Patellogastropoda (true limpets) has been usually recognized as the earliest-branching group (Golikov and Starobogatov, 1975, Haszprunar, 1988, Lindberg, 1988). Cladistic phylogenies based on morphological characters recovered Patellogastropoda (Eogastropoda) as sister group of the remaining lineages (together forming the clade Orthogastropoda), and within the latter, Caenogastropoda sister group to Heterobranchia (forming the clade Apogastropoda) (Fig. 1a; Ponder and Lindberg, 1997). In contrast, the placement of Patellogastropoda varied in the early molecular phylogenetic analyses (e.g., Harasewych et al., 1997, Colgan et al., 2000, Colgan et al., 2003, McArthur and Harasewych, 2003). More recent phylogenomic studies based on large nuclear sequence datasets recovered the deep split between Patellogastropoda and Orthogastropoda (Fig. 1b; Kocot et al., 2011) or favored a sister group relationship of Patellogastropoda and Vetigastropoda, thus rejecting the monophyly of Orthogastropoda (Fig. 1c; e.g., Smith et al., 2011, Zapata et al., 2014). Furthermore, almost all phylogenies based on complete mitochondrial (mt) genomes consistently recovered Patellogastropoda sister to Heterobranchia (Fig. 1d, e, f; Grande et al., 2008, Arquez et al., 2014, Osca et al., 2014b, Uribe et al., 2016b), thus challenging the monophyly of both Orthogastropoda and Apogastropoda. Patellogastropoda and Heterobranchia exhibited very long branches in mt genome phylogenies and notably, Lottia digitalis was the only representative of Patellogastropoda, thus pointing to the possibility that this result is due to LBA artifacts. Altogether, the relative placement of Patellogastropoda within Gastropoda remains an unsettled phylogenetic question regardless of the type of data used for its inference (Aktipis et al., 2008).
Several studies found a positive significant correlation in mt genomes between gene order rearrangement rates and faster evolutionary rates, which ultimately lead to long branches (Xu et al., 2006). Compared to other gastropods, L. digitalis has a large mt genome (28 vs. 15 Kb) and displays a radically different mt gene order (Simison et al., 2006). Since Patellogastropoda are a diverse group encompassing up to eight families (Eoacmaeidae, Acamaeidae, Lepetidae, Lottiidae, Neolepetopsidae, Pectinodontidae. Patellidae and Nacellidae) (Nakano and Sasaki, 2011, Bouchet et al., 2017), it is possible that new mitogenomes from other lineages may have less rearranged gene orders, possibly leading to shorter branches more amenable for phylogenetic studies. Here, we sequenced the mt genomes of representatives of the other two more speciose and common families of patellogastropods, Nacellidae (Cellana radiata) and Patellidae (Patella ferruginea and Patella vulgata), as well as 5.8 Kb fragment of the mt genome of one Eoacmaeidae (Eoacmaea sp.). Our aims were: (1) to check mt gene arrangements across different lineages of Patellogastropoda; (2) to search for Patellogastropoda species with mt rates of evolution slower than those of Lottia; and (3) to try recovering the deep phylogeny of Gastropoda through probabilistic phylogenetic inference and methods specifically devised to mitigate LBA, including the use of complex mixture models, amino acid recoding, and removal of fast-evolving positions, as well as the exploration of the effect of using different outgroup taxa.
Section snippets
Samples and DNA extraction
Single specimens from each of the following species were collected with the corresponding permits and stored in 100% ethanol: C. radiata (Nacellidae) from south of Ambatomainty, Madagascar (MNHN-IM-2009-14093), P. vulgata (Patellidae) from Laredo, Cantabria, north of Spain (shell, MNCN 15.5/84.078; tissue, MNCN/DNA: 94620), P. ferruginea (Patellidae) from Chafarinas Islands, Southwestern Mediterranean Sea (shell, MNCN 15.95/84.068; tissue, MNCN/DNA: 94622), and Eoacmaea sp. (Eoacmaeidae) from
Results
The complete mt genome of C. radiata was 16,194 bp long (Table 1), as assembled starting from 111,217 reads, which rendered a mean sequencing depth of 1,036x. The mt genomes of P. vulgata and P. ferruginea were assembled almost completely to a total of 14,808 bp and 14,460 bp, respectively (Table 1). The number of analyzed reads, and the mean sequencing depths were 3,639,495 and 24,819x and 997,428, and 6,918x, respectively.
Discussion
Phylogenetic inference based on genomic sequence datasets and under probabilistic methods is currently the best strategy for reconstructing the Tree of Life (Vargas and Zardoya, 2014). This cumbersome task faces at least two main (interconnected) challenges: sparse taxon sampling and among-lineage rate heterogeneity. These two challenges perfectly apply to the reconstruction of the phylogeny of Gastropoda (Mollusca). Several gastropod lineages, such as Neomphalina, Cocculiniformia, and
Conclusions
The new mitogenomes of Patella, and particularly Cellana, have gene orders more similar to that inferred for the gastropod ancestor and, in agreement with their lower gene rearrangement rates, display shorter branches than Lottia in phylogenetic trees. The use of the new mt genomes (in replacement to Lottia) allows breaking the LBA between Patellogastropoda and Heterobranchia that has prevailed in previous studies, irrespective of other experimental conditions. Commonly used site-homogeneous
Acknowledgements
We thank two anonymous reviewers for insightful suggestions on previous version of the manuscript. We are grateful to Javier Guallart and Iván Acevedo for providing a fresh specimen of P. ferruginea and Marta Calvo for its dissection. We are indebted to Nicolas Puillandre and the Muséum National d’Histoire Naturelle of París, France for the loan of Cellana radiata material from the ATIMO VATAE expedition in Madagascar (PI: Philippe Bouchet; DOI 10.17600/10110040), as part of the Our Planet
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