A comprehensive comparison of four species of Onchidiidae provides insights on the morphological and molecular adaptations of invertebrates from shallow seas to wetlands.

The Onchidiidae family is ideal for studying the evolution of marine invertebrate species from sea to wetland environments. However, comparative studies of Onchidiidae species are rare. A total of 40 samples were collected from four species (10 specimens per onchidiid), and their histological and molecular differences were systematically evaluated to elucidate the morphological foundations underlying the adaptations of these species. A histological analysis was performed to compare the structures of respiratory organs (gill, lung sac, dorsal skin) among onchidiids, and transcriptome sequencing of four representative onchidiids was performed to investigate the molecular mechanisms associated with their respective habitats. Twenty-six SNP markers of Onchidium reevesii revealed some DNA polymorphisms determining visible traits. Non-muscle myosin heavy chain II (NMHC II) and myosin heavy chain (MyHC), which play essential roles in amphibian developmental processes, were found to be differentially expressed in different onchidiids and tissues. The species with higher terrestrial ability and increased integrated expression of Os-MHC (NMHC II gene) and the MyHC gene, illustrating that the expression levels of these genes were associated with the evolutionary degree. This study provides a comprehensive analysis of the adaptions of a diverse and widespread group of invertebrates, the Onchidiidae. Some onchidiids can breathe well through gills and skin when under seawater, and some can breathe well through lung sacs and skin when in wetlands. A histological comparison of respiratory organs and the relative expression levels of two genes provided insights into the adaptions of onchidiids that allowed their transition from shallow seas to wetlands. This work provides a valuable reference and might encourage further study

PCR primers used in gene cloning.

<p>PCR primers used in gene cloning.</p

Expression levels of the <i>NMHC II</i> gene in different tissues from four representative Onchidiidae species.

<p>S = <i>Onchidium reevesii</i>; M = <i>Platevindex mortoni</i>; R = <i>Paraoncidium reevesii</i>; V = <i>Peronia verruculata</i> (little expression in the tested tissues).</p

Primer sequences and characterization of 26 SNPs in <i>Onchidium reevesii</i>.

<p>Primer sequences and characterization of 26 SNPs in <i>Onchidium reevesii</i>.</p

Annotated unigenes based on gene ontology.

<p>Annotated unigenes based on gene ontology.</p

Sequence information of four Onchidiidae species.

<p>Sequence information of four Onchidiidae species.</p

Habitats of the four studied species from the family Onchidiidae.

<p>The illustration was created using Photoshop. <i>Onchidium reevesii</i> primarily occupies wetlands, <i>Platevindex mortoni</i> can survive well in both water and wetlands, and <i>Paraoncidium reevesi</i>i and <i>Peronia verruculata</i> predominantly dwell in water. Note: The photograph outlined in red shows the dendritic gills in the dorsal skin of <i>Peronia verruculata</i>.</p

Light microscopy of the dorsal skin of four species in the Onchidiidae family.

<p>(<b>A-D</b>) An overview of the dorsal skin of (<b>A</b>) <i>Onchidium reevesii</i> (×40), (<b>B</b>) <i>Paraoncidium reevesii</i> (×40), (<b>C</b>) <i>Platevindex mortoni</i> (×40) and (<b>D</b>) <i>Peronia verruculata</i> (×40). (<b>E-H</b>) Dermis layer of (<b>E</b>) <i>O</i>. <i>reevesii</i> (×40), (<b>F</b>) <i>P</i>. <i>reevesii</i> (×40), (<b>G</b>) <i>P</i>. <i>mortoni</i> (×40) and (<b>H</b>) <i>P</i>. <i>verruculata</i> (×40). (<b>I-L</b>) Histological observation of glands in four species of Onchidiidae: (<b>I</b>) <i>O</i>. <i>reevesii</i> (×40), (<b>J</b>) <i>P</i>. <i>reevesii</i> (×40), (<b>K</b>) <i>P</i>. <i>mortoni</i> (×40), and (<b>L</b>) <i>P</i>. <i>verruculata</i> (×40). E, epidermis; D, dermis; SS, stratum spongiosum; SC, stratum compactum; CM, cuticular membrane; SCO, stratum corneum; SGR, stratum granulosum; SGE, stratum germinativum; MG, mucous gland; GG, granular gland; PC, pigment cell; MF, muscle fiber; BS, blood sinus; CP, calcium particle.</p

Phylogenetic analysis of 13 species.

<p>The names presented in non-black text highlight the main objects of this research study. The phylogenetic tree was inferred using Bayesian methods with MrBayes version 3.2.4. This tree was generated using 18S sequences.</p

Dorsal skin thicknesses of four species in the family Onchidiidae (Unit: μm).

<p>Dorsal skin thicknesses of four species in the family Onchidiidae (Unit: μm).</p