Architectural instability, inverted skews and mitochondrial phylogenomics of Isopoda: outgroup choice affects the long-branch attraction artefacts

Abstract

The majority strand of mitochondrial genomes of crustaceans usually exhibits negative GC skews. Most isopods exhibit an inversed strand asymmetry, believed to be a consequence of an inversion of the replication origin (ROI). Recently, we proposed that an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families resulted in a double-inverted skew (negative GC), and that taxa with homoplastic skews cluster together in phylogenetic analyses (long-branch attraction, LBA). Herein, we further explore these hypotheses, for which we sequenced the mitogenome of Asotana magnifica (Cymothoidae), and tested whether our conclusions were biased by poor taxon sampling and inclusion of outgroups. (1) The new mitogenome also exhibits a double-inverted skew, which supports the hypothesis of an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families. (2) It exhibits a unique gene order, which corroborates that isopods possess exceptionally destabilized mitogenomic architecture. (3) Improved taxonomic sampling failed to resolve skew-driven phylogenetic artefacts. (4) The use of a single outgroup exacerbated the LBA, whereas both the use of a large number of outgroups and complete exclusion of outgroups ameliorated it.

Evolutionary history of inversions in the direction of architecture-driven mutational pressures in crustacean mitochondrial genomes

“…We retrieved all 348 crustacean mitogenomes available in the curated RefSeq database (O'Leary et al 2016); added two Isopoda (Cymothoidae) species with double-inverted skews Cymothoa indica (MH396438) and Asotana magnifica (MK790137) (Zhang et al 2019b;Zou et al 2020); and a primitive arthropod Limulus polyphemus (Chelicerata) with a highly conserved ancestral gene order (Lavrov et al 2000) as outgroup. To check whether there may be ORIs in taxa not covered by this dataset, we extracted all 991 nominally crustacean mitogenomes from the GenBank, sorted them by GC skew magnitude, selected a cut-off value of GC>-0.2 (this step was conducted to identify taxa with positive and reduced skew magnitudes; as we visually assessed that a vast majority of taxa with conserved ancestral architecture have GC skews <-0.2), compared the taxa with the RefSeq dataset, and selected 16 mitogenomes for additional analyses.…”

“…Inversed skew as autapomorphic trait for all isopods apart from the putatively basal Asellota has been proposed almost a decade ago (Kilpert et al 2012), but recently we showed that Cymothoidae and Corallanidae underwent an additional ORI and exhibit double-inverted (D-I) skews (Zhang et al 2019b). Isopods generally have highly destabilized, hypervariable GOs, with almost all species sequenced so far exhibiting a unique GO (Zou et al 2020), and with a large number of genes exhibiting strand switches compared to the AAGO ( Figure 6A).…”

“…We also tried aligning the major NCRs of several isopod species, but failed to identify universally conserved segments, thus supporting the observation of (Pie et al 2008) that CRs in crustaceans tend to be too divergent to be comparable. However, as all Cymothoidae and Corallanidae species sequenced so far exhibit D-I skews (Zou et al 2020), an ORI event in their common ancestor remains the most parsimonious explanation, so we assume that the rearrangement event behind this ORI comprised only the CR. Sequencing of further mitogenomes from these two and other closely related lineages will be needed to infer the exact phylogenetic timing of this ORI event with confidence.…”

“…(Wetzer 2002;Tan et al 2015Tan et al , 2019Timm and Bracken-Grissom 2015;Cheng et al 2018). A majority of crustaceans exhibit negative GC skews for genes on the mitochondrial 'majority strand' (the strand encoding a majority of genes in the ancestral crustacean mitogenomic architecture is named so by convention), but several crustacean lineages, for example most isopods, exhibit inversed (positive) GC skews (Hassanin 2006;Kilpert and Podsiadlowski 2006;Kilpert et al 2012;Pons et al 2014;Yu et al 2018;Zhang et al 2019b;Zou et al 2020). As many studies that relied on mitochondrial data to infer the phylogeny of isopods ignored these compositional biases, this produced pervasive topological instability and multiple contradictory phylogenetic hypotheses (Wetzer 2002;Wilson 2009;Hata et al 2017;Lins et al 2017;Zou et al 2018;Zhang et al 2019b).…”

“…Despite this limitation, mitochondrial data, especially complete mitochondrial genomes, can be a suitable tool for inferring lower-level phylogenies and studying evolutionary patterns in crustacean taxa that did not undergo ORI events. While many crustacean lineages exhibit the highly conserved ancestral arthropod mitochondrial architecture (Boore et al 1998;Cheng et al 2018;Tan et al 2018), a number of lineages exhibit exceptionally high plasticity of mitochondrial architecture (Kilpert et al 2012;Tan et al 2019;Zou et al 2020). As this includes common CR duplication events and extremely low levels of conservedness of CR sequences (Pie 2008), no study has ever attempted to systematically infer the evolutionary history of ORI events in crustaceans.…”