Cretaceous amber fossils highlight the evolutionary history and morphological conservatism of land snails

Other than hard bones and shells, it is rare for soft tissues to fossilize, but occasionally they are well-preserved in amber. Here, we focus on both modern and fossilized species of the land snail superfamily Cyclophoroidea. Phylogenetic relationships within the Cyclophoroidea were previously studied using extant species, but timing of divergence within the group remains unclear. In addition, it is difficult to observe morphological traits such as the chitinous operculum and periostracum of fossil snails due to their poor preservation potential. Here we describe nine species including a new genus and five new species of well-preserved fossil cyclophoroideans from the mid-Cretaceous Burmese amber. These fossils include not only the shell, but also the chitinous operculum and periostracum, soft body, and excrements. We present the first estimation of divergence time among cyclophoroidean families using fossil records and molecular data, suggesting extreme morphological conservatism of the Cyclophoroidea for nearly 100 million years.


Supplementary figures
Fig. S1. Maximum clade credibility trees generated with the BEAST2 analysis from the combined sequences (COI, 16S, and H3 genes) using a COI molecular clock rate.

Supplementary tables
Supplementary tables in this manuscript longer than one page, hence we provide tables as another word file. Table captions are here:   Table S1. Mesozoic to early Cenozoic fossil records of the Cyclophoridae, Diplommatinidae, and Pupinidae.  Excrements around the aperture elliptical. Apex blunt, and embryonic slightly spherelike shape with smooth and round whorls.

Remarks:
The morphological characters of the peristome and small number of whorls suggest that this specimen is very likely to be a juvenile. Although it is difficult to identify the species, we believe this specimen belongs to Cyclophoridae due to a lamellar and thin operculum. This specimen has a grobosely conical shell with a smooth surface, similar to the extinct genus Palaeocyclophorus Wenz, 1923 and the extant genus Leptopoma L. Pferffer, 1847. In Leptopoma, however, the shell is considerably thick and somewhat smooth. Therefore, this specimen is probably related to Palaeocyclophorus, but we do not identify any genus or species.
Hence, we assign these five species to Palaeocyclophorus. In addition, According to the fossil records of pupinid land snails 7-23 , the five genera have been reported from the Santonian of the Upper Cretaceous to the Lower Oligocene 7 . In these species, Kallomastoma aberrans, Ventriculus dolium and Cyclomastoma pachygaster resemble S. electrothauma sp. nov. and have a pupiniform shell profile and a thickened peristome. However, in K. aberrans, the shell is much bigger (three times in length and width of S. electrothauma sp. nov.) and rounder shell profile than that of S. electrothauma sp. nov., and the shape of the aperture is clearly different from that of S. electrothauma sp. nov. Kallomastoma aberrans has a deformed drop-shape relative to the former and a circular shape relative to S. electrothauma sp. nov. Ventriculus dolium and C. pachygaster have a small shell but a much rounder shell profile. They can be easily discriminated from S. electrothauma sp. nov.

Specifics of DNA extraction, PCR, alignment parameters, and model selection
We extracted DNA and performed PCR-based sequencing of a Pupinella rufa individual according to a previous study 24 . Alignment of the COI and H3 sequences was straightforward and required no gaps; 16S sequences were aligned using MUSCLE 25 ; GBLOCKS v0.91b 26 was used to select regions in the aligned sequences that were confidently aligned for analysis (Table S5). For divergence time estimation, Kakusan4-4.0.2011.05.28 27 was used to select the appropriate models for sequence evolution. Fig. S1. Maximum clade credibility trees generated with the BEAST2 analysis from the combined sequences (COI, 16S, and H3 genes) using a COI molecular clock rate. The outgroups (Conus and Pomacea) are not shown. For convenience, we assign numbers to the major nodes (Table S2)