Acmaeidae is a family of Patellogastropoda (called “true limpets”) that represents one of the most primitive groups of the Gastropoda and contains approximately 350 species worldwide. They are found in lower intertidal to subtidal zones and constitute macrobenthic communities on coastal rocky shores, playing an important role in littoral ecosystems (Branch, 1985). This family is characterized by having a foliated shell with a fibrillar prismatic shell microstructure, one feather-shaped ctenidium, and a single pair of tricuspid radular teeth (Lindberg, 1998). Over the last century, external shell morphology has been the feature most widely used for both species-level taxonomy and investigating the internal phylogeny of the Patellogastropoda. However, morphological traits of limpet species are highly variable with different morphs from the ontogenic or ecophenotypic origin, resulting in substantial taxonomic confusion and disagreement on their phylogenetic relationships (Nakano and Ozawa, 2007). Soft body anatomy including radula characters has also been considered phylogenetically informative and widely used to investigate the relationships among the Patellogastropoda families in the past decades (Lindberg, 1998; Sasaki, 1998). Nevertheless, the taxonomic confusion associated with the absence of key characters for defining the family boundaries, and convergent evolution and ecophenotypic plasticity in their morphological features has resulted in different classification systems of the Patellogastropoda over the last decades (Sasaki et al., 2006; Nakano and Sasaki, 2011).

Against this background, phylogenetic relationships including the taxonomic status of the “Acmaeidae” with the Patellogastropoda have remained unresolved from previous studies that employed different sources of taxonomic information, such as shell characters, internal morphology, and molecular sequence data [Dall, 1876; Lindberg, 1998; Sasaki, 1998 (morphology); Harasewych and McArthur, 2000; Nakano and Ozawa, 2007 (molecular data)]. The Acmaeidae was traditionally divided into two subfamilies, Acameinae and Pectinodontinae, based on morphological perspectives (Lindberg, 1998), but these two subfamilies (sensu Lindberg, 1998) were not found as a monophyletic group by subsequent molecular analysis of three mitochondrial genes (mtDNA 12S rDNA, 16S rDNA, and cox1; Nakano and Ozawa, 2007). Moreover, despite this molecular study reporting that the Acmaeidae species (Acmaea mitra, Niveotectura pallida, and Erginus sybaritica) were grouped with Lottiidae species, their relationships were not concordant with other studies using the morphology or using different molecular markers, i.e., nuclear vs. mitochondrial DNA: The Acmaeidae species were included as a subgroup of Lottiidae based on their morphological similarity with lottiid species (e.g., shell microstructure, radula characters, and presence of a ctenidium) (Lindberg, 1998), but they were revived as a separate family (Nakano and Sasaki, 2011) from the Lottiidae. These phylogenetic uncertainties of the “Acmaeidae” necessitate further study using different molecular evidence to elucidate their relationships with other Patellogastropoda families.

During recent decades, comparative analysis of the complete mitochondrial genome information (i.e., nucleotide sequence and gene order data) has been widely used for investigating the phylogenetic relationships in many molluscan groups including Patellogastropoda groups where interrelationships were not fully resolved (Uribe et al., 2019; Feng et al., 2020; Xu et al., 2022). In this study, we aimed to (1) resolve phylogenetic uncertainty of the Acmaeidae and (2) extend our knowledge of mitochondrial genome diversity among major groups of the Patellogastropoda. For this, we determined two complete genome sequences of “Acmaeidae” (Acmaea mitra and Niveotectura pallida), the first representatives from the Acmaeidae, and one complete genome sequence of Lottiidae (Discurria insessa), combined them with the mitochondrial genome sequences of 20 other published limpet species plus 12 non-patellogastropod species (including 2 chiton species), and performed phylogenetic analysis based on nucleotide sequence information and gene order data.

The Acmaeidae is one of the most taxonomically debated Patellogastropoda families, particularly in terms of their taxonomic status and phylogenetic relationships with other groups. Nonetheless, to date there has been only minimal study of this group using molecular data (Nakano and Sasaki, 2011). The relationships inferred in many previous studies did not agree with each other, according to the data sources used in the analysis, such as morphology vs. molecular data, or even between molecular markers (i.e., nuclear vs. mitochondrial DNA). Members of the family Acmaeidae were originally assigned to many lottiid genera and species (e.g., Pilsbry, 1891). However, this scheme was substantially revised based on the shell structure (Lindberg, 1986). After this revision, the genus Acmaea was restricted to the type species Acmaea mitra, and the rest of the species formerly belonging to Acmaeidae were transferred to the Lottiidae (Lindberg, 1986). The mitochondrial genome phylogeny reconstructed in this study provides new and invaluable insights into the internal phylogeny of the Patellogastropoda. The monophyletic grouping of Acmaeidae + Pectinodontidae species contradicts the previous analysis of partial gene fragments of mtDNA (Nakano and Ozawa, 2007), in which the “Acmaeinae” was nested within the Lottiidae, whereas the “Pectinodontinae” was clustered with Lepetidae species. These inferred relationships provided a basis of taxonomic treatment in which the “Pectinodontinae” was elevated to the familial rank (Nakano and Ozawa, 2007). Nakano and Sasaki (2011) later corrected their earlier erroneous interpretation derived from sequence contamination of Acmaea mitra by placing it as a junior synonym of Lottiidae. The Pectinodontidae still retained its familial status thereafter, awaiting taxonomic clarification from further analysis. In this regard, our mitochondrial genome tree highlights that Acmaeidae and Pectinodontidae are the most closely related, and their close affinity lends strong support to the previous morphology-based classification in which the pectinodontid species were treated as a subfamily of the family Acmaeidae (Lindberg, 1998). Nevertheless, further extensive studies based on re-evaluation of their morphological characters including shell and soft body anatomy will be necessary to clarify the taxonomic status of the pectinodontid species as a subgroup or confirm them as a taxonomically valid, separate family from the Acmaeidae.

Historically, phylogenetic relationships of the Acmaeidae with other groups have not yet clearly defined (Nakano and Sasaki, 2011). Based on morphological similarity [e.g., shell microstructure, radula characters (Lindberg, 1998), and presence of a left ctenidium (Sasaki, 1998)] and molecular analysis [12S and 16S mtDNA sequence data (Nakano and Ozawa, 2004)], Acmaeidae was once considered more closely related to Lottiidae than to Patellidae, Nacellidae, and Lepetidae, and was included as a subgroup of Lottiidae. In contrast, later analysis of mtDNA cox1 and histone H3 sequence data revealed that they were distinct from Lottiidae and were subsequently revived as a separate family from Lottiidae (Nakano and Sasaki, 2011). Polyphyletic relationships between Acmaeidae and Lottiidae species discovered from this mitochondrial genome phylogeny provide strong taxonomic justification of their independent family status. Moreover, the clade I monophyly (Patellidae, Nacellidae, Pectinodontidae + Acmaeidae) that is separated from clade II (Eoacmaeidae + Lottiidae) indicates that Acmaeidae is a member of the superfamily Patelloidea along with other clade I family members, not belongs to the superfamily Lottioidea. Note that the time-calibrated phylogenetic tree ( Figure 3 ) indicates that the divergence of the Acmaeidae from the Pectinodontidae is estimated to begin in the Jurassic-Cretaceus boundary (132.62 MYA), being much more recent event than the clade I/clade II separation (in the middle Carboniferous; 337.20 MYA) and the Lottiidae/Eoacmaeidae splitting (in the Carboniferous-Permian boundary; 295.84 MYA).

In many metazoan groups including molluscan species, comparison of the gene arrangement of the complete mitochondrial genome has been widely used and sharing similar synteny pattern observed among certain taxonomic groups can often be interpreted as additional evidence of their phylogenetic affinity (Lee et al., 2019). Likewise, gene order comparison across the limpet species provides additional insights into the phylogenetic relationships and mitochondrial genome evolution among the family members of the Patellogastropoda. The gene arrangement patterns of the two Acmaeidae species (A. mitra and N. pallida) showed the same gene order. Excluding the tRNAs that are often highly mobile even between closely related species, the arrangement patterns of PCGs and rRNA genes of the clade I members are highly conserved showing very similar synteny blocks within each family member ( Figure 2 ). Notably, the three families Acmaeidae, Pectinodontidae, and Nacellidae display the identical gene arrangement in PCGs and rRNA genes, lending additional support to their phylogenetic relatedness. By contrast, the gene order of the Lottiidae species belonging to clade II is highly variable according to the genus and even between congeneric species (see Lottia species), as previously reported (Xu et al., 2022). Moreover, the gene order of Lottiidae species shows substantially different patterns from those of clade I members (see Figure 2 for more details). Inferred relationships among Patellogastropoda species from gene order analysis ( Supplementary Figure 3 ) agreed with tree topology from the sequence analysis ( Figure 1 and Supplementary Figures 1 , 2 ) in that Patellogastropoda species were subdivided into two clades (clade I and clade II) and inferred relationships among clade I family members. One exception is that the two Pectinodontidae species (B. lactea and B. nipponica) were not grouped as monophyletic in their gene order tree: the former was clustered with Nacellidae species, whereas the latter was grouped with Acmaeidae species ( Supplementary Figure 3 ). This discrepancy results from B. lactea species shows an intermediate pattern between the Nacellidae and Acmaeidae in some tRNA synteny blocks. Within clade II, most of branches leading to the Lottiidae species were unresolved and showed extremely long branches that are ascribed to their extensive gene rearrangement events between the Lottiidae species. In addition, it is also interesting to note that the Acmaeidae and one Pectinodontidae species (B. nipponica) retain the same consecutive tRNA block of “tRNA KARNI” (trnK, trnA, trnR, trnN, and trnI), which has long been considered an ancestral state across the major clades of the Gastropoda including other subclasses (Uribe et al., 2016b; Gaitán-Espitia et al., 2019) and outgroup (chiton) species (Irisarri et al., 2014). At present, we are unable to precisely address the evolutionary history of gene arrangement patterns across the patellogastropods. To this end, in-depth analysis of complete mitochondrial genome sequences based on a full breadth of taxon coverage from relatively unstudied and/or underrepresented taxa is required to fully understand the mitochondrial genome evolution and a more comprehensive phylogeny among the major groups of the Patellogastropoda.

The data presented in the study are deposited on GenBank (accession numbers: OQ133456, OQ133457 and OQ133458).

J-KP designed and led the study. EP, DL, HK, YK collected samples, conducted the laboratory work, and performed phylogenetic analysis of the sequence data. EP, TN, and J-KP wrote the manuscript. All authors contributed to the article and approved the submitted version.