  Citation: Hines, H.N.; McCarthy, P.J.; Esteban, G.F. A Case Building Ciliate in the Genus Pseudoblepharisma Found in Subtropical Fresh Water. Diversity 2022, 14, 174. https:// doi.org/10.3390/d14030174 Academic Editor: Michael Wink Received: 16 December 2021 Accepted: 24 February 2022 Published: 27 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). diversity Article A Case Building Ciliate in the Genus Pseudoblepharisma Found in Subtropical Fresh Water Hunter N. Hines 1, * , Peter J. McCarthy 1 and Genoveva F. Esteban 2, * 1 Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, FL 34946, USA; pmccart5@fau.edu 2 Department of Life and Environmental Sciences, Faculty of Science and Technology, Bournemouth University, Dorset BH12 5BB, UK * Correspondence: hunter.n.hines@gmail.com (H.N.H.); gesteban@bournemouth.ac.uk (G.F.E.) Abstract: The genus Pseudoblepharisma is currently comprised of only one species, P. tenue, and one variant, P. tenue var. viride, both described as free-living ciliates thriving in oxygen depleted freshwater habitats of Europe. Here we report on this genus being discovered from subtropical freshwaters of Florida, USA. The Florida strain diverges from the P. tenue by being much larger, as well as having a far higher density of intracellular symbiotic green algae, resembling P. tenue var. viride. Unlike its European counterparts, the North American strain was observed to build a lorica, likely useful for protection and feeding; this has not been previously described for this genus. In contrast to P. tenue, the Florida strain does not have endosymbiotic purple bacteria. Despite large distances between sampling zones, the species is a close match at a morphological level to Pseudoblepharisma tenue var. viride. We provide the first 18S rRNA gene sequence for this species, allowing future investigations into the biogeography of this genus. As intensive sampling efforts continue to increase, cryptic microbial species will continue to be recorded from diverse freshwater habitats at a global scale. Keywords: biogeography; ciliate; cryptic biodiversity; ecology; freshwater; microbiology; protist 1. Introduction The cryptic biodiversity of an environment is an important component of microbial food webs and the ecosystems they help to support [1,2]. Undersampling remains a persistent issue within microbial ecology [3,4], such that the true diversity of a particular site or region is difficult to quantify. This results in many species remaining cryptic, especially when present in low numbers; however, they will also remain cryptic, even when present in astronomically high numbers, if the habitat is not studied [5]. This is frequently the case considering the number of unstudied habitats worldwide. Many species described as restricted to one area often have their biogeographical distribution expanded when intensive sampling efforts are conducted [6] and may be very common locally despite being previously unrecorded. The genus Pseudoblepharisma comprises free-living freshwater ciliates originally docu- mented from ponds in Europe (Germany) [7]. Blepharisma tenuis was the name used in the original German description of this genus [7]; however, this was quickly acknowledged to be incorrect, and it was fully rectified [8]. The genus was further described from the same geographical area, with descriptions of Pseudoblepharisma tenue presented [9]. This original work recorded the basic morphology, and it was later recorded (also in Germany) by Kreutz and Foissner (2006) [10], including the presence of intracellular algae and pink bacteria which were recognized not to be ingested but likely symbionts [8]. A detailed description of the German strain of P. tenue has recently been published [11], in which the use of single-cell genomics has allowed the identification of the symbionts, as well the determination of the draft genome for the ciliate. Diversity 2022, 14, 174. https://doi.org/10.3390/d14030174 https://www.mdpi.com/journal/diversity