Physella acuta: atypical mitochondrial gene order among panpulmonates (Gastropoda)

Abstract

Mitochondrial (mt) sequences are frequently used for phylogenetic reconstruction and for identification of species of molluscs. This study expands the phylogenetic range of Hygrophila (Panpulmonata) for which such sequence data are available by characterizing the full mt genome of the invasive freshwater snail Physella acuta (Physidae). The mt genome sequences of two P. acuta isolates from Stubblefield Lake, New Mexico, USA, differed in length (14,490 vs 14,314 bp) and showed 11.49% sequence divergence, whereas ITS1 and ITS2 sequences from the nuclear genome differed by 1.75%. The mt gene order of P. acuta (cox1, P, nad6, nad5, nad1, D, F, cox2, Y, W, nad4L, C, Q, atp6, R, E, rrnS, M, T, cox3, I, nad2, K, V, rrnL, L1, A, cytb, G, H, L2, atp8, N, nad2, S1, S2, nad4) differs considerably from the relatively conserved gene order within Panpulmonata. Phylogenetic trees show that the 13 protein-encoding mt gene sequences (equivalent codons) of P. acuta group according to gastropod phylogeny, yet branch lengths and dN/dS ratios for P. acuta indicate elevated amino acid substitutions relative to other gastropods. This study indicates that mt sequences of P. acuta are phylogenetically informative despite a considerable intraspecific divergence and the atypical gene order in its mt genome.

Figure 1.

Phylogenetic placement of Physella acuta isolates A and B within Physidae. Experimentally derived sequences were incorporated into NCBI popset 164430598 (Wethington & Lydeard, 2007) COI sequences from snails of the family Physidae (boxed) to generate a ML tree; NJ and MP yielded the same results. Original identifiers of strains or isolates of P. acuta are indicated in brackets. The outgroup includes sequences from Lymnaeidae and Planorbidae. Isolates A and B, which coexist in Stubblefield Lake, NM (bolded), cluster with different clades of P. acuta. The tree has been simplified for clarity, bootstrap values are indicated from 1,000 replicates.

Figure 2.

The mitochondrial genomes of Physella acuta isolates A and B. The outer circle represents the positive strand, the inner circle the negative strand. Protein-encoding genes are darkened to distinguish from rRNA genes. Bars (with length in bp) indicate location of sequence overlap between protein-encoding genes. Note the size difference of the mt genomes of the two P. acuta isolates, especially the indel beginning in cox2 following the intergenic region upstream of trnY.

Figure 3.

Potential origin of replication (POR), location by GC skew analysis. GC skew [(G − C)/(G + C)] ratios plotted in a bar graph relative to a linear representation of the mt genome of P. acuta (isolate A shown). Positive values indicate greater G content and negative values indicate increased C content. The vertical dotted line indicates the predicted location of the POR; note the GC skew maximum at 0.162 that further supports this prediction. This high peak is the origin of the sequence interval (window size 2,500 nt) with the highest GC skew, transitioning from low GC skew upstream (Xia, 2012). Shading of protein-encoding and RNA genes as in Figure 2.

Figure 4.

Physella acuta isolates A and B: tRNA sequence and structure. Predicted secondary structures of the 22 tRNAs encoded in the mt genomes from P. acuta isolates A and B. Only three tRNA genes are identical between isolates A and B. Two graphical representations are shown for all tRNA genes that differ in sequence between isolates A and B. Typically such differences occurred in the loops, not the stems. Three letter codes identify the amino acid anticodon specificity. Irregular tRNAs are Gly, Ser (AGN) and Ser (UCN).

Figure 5.

Linear alignment of mt gene order of Physella acuta compared with other panpulmonate gastropods. Phylogenetic relationships shown are based on analysis of 18S, 28S, 16S and COI sequences (Jörger et al., 2010). Protein-encoding and rRNA genes are colour coded to emphasize patterns and gene rearrangements. Single letters designate tRNA genes. The genes encoded on the negative strand (underlined) are the same for all species shown. Bold lines under the alignment delineate clusters of genes with the same internal order for the majority of the panpulmonates. Note that gene rearrangements among the panpulmonates are modest and rarely affect protein-encoding gene orders, with the exception of P. acuta. Alignment is not to scale.

Figure 6.

Phylogenetic analysis of selected gastropods. Representative gastropods with fully characterized mt genomes were selected to compare protein-encoding genes with those of Physella acuta. Optimized alignments of amino acid sequences of all 13 protein-encoding genes were concatenated for ML analysis (1,000 bootstrap replicates). Longer branch lengths indicate increased mutation rates of amino acid sequences across the mt genome. Note the relatively long branch of the Physella clade.