Sea anemones through X-rays : visualization of two species of Diadumene (Cnidaria, Actiniaria) using micro-CT

Abstract

Diadumenidae is a monogeneric family comprised of 10 valid species in the genus Diadumene. Although these species are distributed in all but the Southern Ocean, Diadumene lineata is a cosmopolitan species and one of the most widely distributed marine organisms. Recent phylogenetic analyses with multiple species have recovered Diadumenidae as a monophyletic group with high support. Although generic placement is straightforward, species-level identifications within Diadumene are difficult because species are defined by a mosaic of characters that vary in degree rather than kind. Two species of the genus have been recorded in the southwestern Atlantic: D. paranaensis along the south coast of Brazil and D. lineata along the southeast and northeast coasts of Brazil as well as in Argentina. Here we record D. leucolena for the first time in the southern hemisphere and describe D. manezinha, sp. nov., raising to four the number of species of Diadumene for Brazil and to 11 worldwide. We incorporated microcomputed tomography (micro-CT) into the description of both species in the first application of the method for Actiniaria. We evaluate the utility of micro-CT imaging and its potential to generate fast, low-cost, and high-resolution datasets despite the anemone's low-density soft tissue. We present a protocol for sample handling, chemical staining, and scanning parameters that resulted in satisfactory imaging of the two specimens examined. We also analyze advantages and limitations of using micro-CT over traditional techniques in the study of sea anemones.

Supplemental Material for 'Psittacosaurus amitabha, a new species of ceratopsian dinosaur from the Ondai Sayr locality, central Mongolia. (American Museum novitates, no. 3932)'

“…The concatenated dataset (12S, 16S, CO3, 18S, 28S) had a total of 213 taxa and 8292 sites. Trends in marker variability for both broad and focused datasets followed those previously reported for Actiniaria (e.g., Daly et al, , 2010Gusmão and Daly, 2010;Rodríguez et al, 2012Rodríguez et al, , 2014Lauretta et al, 2014;Gusmão et al, 2018). All phylogenetic reconstructions (MP, ML) using the concatenated data for the broad ordinal dataset agreed basically with the topology depicted in figure 9: two major clades corresponding to the two actiniarian suborders and five superfamilies were recovered, including superfamily Edwardsioidea/family Edwardsiidae with high bootstrap support (98% ML/100% MP).…”

“…Similarly, though the general shape and morphology of retractors were easily established in the 2D scan images, it was not possible to determine the number and ramification of muscle processes in the retractor or parietal muscles. Although our results somewhat restrict the utility of micro-CT scanning for the study of edwardsiids as suggested by Gusmão et al (2018), at least under the current resolution, we agree with Daly (2002) that a detailed exploration and description of morphological characters will likely help us resolve relationships among edwardsiid genera. As suggested by Daly (2002), this means that when possible, morphological features should be quantified and qualified in detail (e.g., muscle morphology, number and distribution of tentacles per cycle, number and distribution of microcnemes), and character complexes (e.g., presence and distribution of microcnemes on distal column) should be broken down with its constituents evaluated separately for phylogenetic informative potential.…”

“…Mean and standard deviation are given to provide information about variability in capsule size, though these are not statistically significant (see Sanamyan and Sanamyan, 2013). We follow a nematocyst terminology that combines the classification of Weill (1934) and Carlgren (1940), thus differentiating "basitrichs" from "b-mastigophores" with that of Schmidt (1969Schmidt ( , 1972Schmidt ( , 1974, which captures the underlying variation seen in "rhabdoids" (see Gusmão et al, 2018, for more details). We include photographs of each type of nematocyst for reliable comparison across terminologies and taxa (see Fautin, 1988).…”

“…Because only one specimen of S. shrimp was available for study, it could not be fixed in osmium tetroxide for CT-scanning as it would render the specimen unavailable for morphological and histological slides. For details on the staining protocol used here see Gusmão et al (2018). Once stained and washed, the specimen was transferred to and scanned on a 50 ml polyethylene tube filled with 100% ethanol on a phoenix v|tome|x s240 GE (General Electric, Fairfield, CT) at 60 kV and 70 μA with a molybdenum target at the Microscopy and Imaging Facility (MIF) at AMNH.…”

Species Delimitation in Sea Anemones (Anthozoa: Actiniaria): From Traditional Taxonomy to Integrative Approaches

“…adopting a burrowing habitat); therefore, they have seemingly been developed and lost several times throughout history (Rodríguez et al, 2012;Rodríguez et al, 2014). Some of these evolutionary changes could be even microanatomical and, based on recent observations, much more complex than have been appreciated up until now (Daly et al, 2017;Gusmão et al, 2018).…”