Mitogenomes resolve relationships of two Atacama Desert land snails

A comparative mitochondrial-genome study produced the first reference mitogenomes for the Atacama Region land snails Bostryx erythrostoma and Plectostylus broderipii. The assemblies established their positions within the sampled Orthalicoidea, with the focal relationships recovered consistently by maximum-likelihood, Bayesian and amino-acid analyses.

Specimens of B. erythrostoma and P. broderipii were collected in northern Chile’s Atacama Region, at Copiapó–Barranquilla and Copiapó–Totoral, respectively, and preserved in 96% ethanol. Shell morphology and established identification criteria were used by Juan Francisco Araya and Ricardo Catalán-Garrido. DNA was extracted from foot tissue, and paired-end sequencing with 150-bp reads was performed on an Illumina NovaSeq 6000. The B. erythrostoma run generated 63.6 million spots and 19.1 Gb of sequence data, while the P. broderipii run generated 39.2 million spots and 11.8 Gb.

After adapter and low-quality sequence removal, researchers assembled the mitochondrial DNA with multiple workflows, including MEGAHIT, SPAdes and the mitochondrion-targeted GetOrganelle pipeline. One mitochondrial contig was recovered for each species. Read mapping with BWA-MEM showed 100% breadth of coverage for both assemblies, with mean depths of 1,202.9× for B. erythrostoma and 348.2× for P. broderipii. The P. broderipii assembly was treated as complete, whereas one localized region of the B. erythrostoma assembly had reduced coverage and complex soft-clipping. Independent assembly approaches did not resolve that region unequivocally from the short Illumina reads, so the researchers described the B. erythrostoma mitogenome as nearly complete.

The B. erythrostoma mitogenome is 15,525 base pairs long, while the P. broderipii mitogenome is 14,978 base pairs. Both have the standard metazoan complement of 37 genes: 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. A and T together account for 69.75% of the B. erythrostoma genome and 72.08% of the P. broderipii genome. Both species have the same strand distribution for protein-coding and transfer RNA genes, although the magnitudes of their A+T and G+C skews differ. MITOS2 annotations were manually curated and independently assessed with MitoFinder, while transfer RNAs were evaluated with ARAGORN and tRNAscan-SE. The two annotation approaches agreed on gene content, order and strand orientation, with differences confined to boundary refinements of 1–27 base pairs.

Much of the difference in total genome length was associated with non-coding DNA rather than major differences in coding-gene content. B. erythrostoma has 23 intergenic regions totaling 1,489 base pairs, or about 9.59% of its genome; its largest, at 959 base pairs, lies between trnV and trnP. P. broderipii has 21 intergenic regions totaling 949 base pairs, or about 6.34%, with its largest 370-base-pair spacer between trnK and cox1. The B. erythrostoma assembly also has an alternative cox1–rrnL–trnL1–trnV organization. The same arrangement was independently observed in a published Bostryx mitogenome, making a lineage-specific feature more likely than an isolated assembly error, although its distribution across the genus was not established by the limited comparison.

Most transfer RNAs adopted the canonical metazoan cloverleaf structure after manual refinement. A small number, particularly trnS2, retained reduced structural elements, a pattern also documented in animal mitochondrial genomes. In P. broderipii, nad3, cox3 and nad2 contain incomplete stop codons, consistent with post-transcriptional completion via polyadenylation. By contrast, no incomplete stop codons were identified in B. erythrostoma. The researchers cautioned that mitochondrial gene boundaries, especially near transfer RNA genes, remain partly tentative because current annotation methods cannot fully account for post-transcriptional RNA processing.

For the phylogenomic analysis, the researchers extracted and codon-aligned the 13 mitochondrial protein-coding genes, producing a nucleotide supermatrix of 11,829 aligned sites across 19 taxa. The dataset contained 17 ingroup taxa and two Systellommatophora outgroups, Imerinia grandidieri and Eleutherocaulis alte. Model and partition selection used the Bayesian Information Criterion, and the initial 39 codon-position partitions were optimized to 20 for the final maximum-likelihood analysis in IQ-TREE. Branch support was estimated with 1,000 ultrafast bootstrap and 1,000 SH-aLRT replicates.

The maximum-likelihood and Bayesian trees recovered the same focal relationships. B. erythrostoma was sister to a published Bostryx sequence, with an ultrafast bootstrap value of 100 and posterior probability of 1.00. The Rabdotus mooreanus–Naesiotus nux pair was also strongly supported and grouped with the two Bostryx sequences. P. broderipii was recovered as sister to the remaining sampled Orthalicoidea, with support values of 100 and 1.00 at these focal nodes. A separate amino-acid analysis also recovered the focal relationships.

Some deeper relationships among stylommatophoran superfamilies received lower support or varied slightly among inference methods. The authors noted that heterogeneous evolutionary rates and compositional variation among mitochondrial genomes from distantly related lineages can influence phylogenetic inference at deeper scales. Accordingly, the strongest conclusion concerns relationships among the sampled Orthalicoidea rather than a definitive resolution of the entire stylommatophoran tree. The new mitogenomes provide reference resources for comparative and phylogenetic studies of Bulimulidae and Bothriembryontidae, while the limited taxon sampling constrains broader conclusions about the genus and superfamily.

Subject of Research: Comparative mitogenomics and phylogeny of two Atacama Region Orthalicoidea land snails

Article Title: Mitochondrial genomes and phylogenomic analysis of Plectostylus broderipii and Bostryx erythrostoma ( Gastropoda , Stylommatophora , Orthalicoidea ) from the Atacama Desert, Chile

Article References: Contreras-Díaz, R., Barahona-Segovia, R. M., Catalán-Garrido, R., Araya, J. F., Arias-Aburto, M., & Pizarro-Gacitúa, C. (2026). Mitochondrial genomes and phylogenomic analysis of Plectostylus broderipii and Bostryx erythrostoma (Gastropoda, Stylommatophora, Orthalicoidea) from the Atacama Desert, Chile. ZooKeys, 1291, 1-18. https://doi.org/10.3897/zookeys.1291.197983

Image Credits: AI Generated

DOI: 10.3897/zookeys.1291.197983

Keywords: mitochondrial genomes, phylogenomics, Orthalicoidea, Bostryx erythrostoma, Plectostylus broderipii, land snails, Atacama Desert, Chile, next-generation sequencing, molecular systematics, Mitochondrial, genomes