**5. Conclusions**

In this work, we confirmed that sampling in poorly studied regions, such as Central America, may reveal broad diversity of *Paramecium*, making it possible also to find new species. We found representatives of six *Paramecium* morphological species, and the collected strains belonged to di fferent groups within some of these species (as in the case of the *P. aurelia* species complex and *P. multimicronucleatum*). Numerous molecular phylogenetic data demonstrate that each *Paramecium* morphospecies includes a number of intraspecific groups, which in some cases are known to correspond to syngens [5,11]. Genomic analyses showed that the *P. aurelia* complex emerged as a result of three whole genome duplications followed by species radiation [59], and speciation has been actively going on in this group [60,61]. It is known that the survival rate of F2 progeny in crosses of strains belonging to the same species, for example, in *P. sexaurelia* [62], can vary significantly. Inability to form conjugating couples seems to be one of the most limiting components of the reproductive barrier between sibling species of the *P. aurelia* complex. If couples can be formed, the possibility that a small proportion of the progeny from interspecies crosses may survive cannot be completely ruled out. Comparative genomics can potentially elucidate if all sibling species of the *P. aurelia* complex are absolutely isolated from each other. Pairs of twin species, like *P*. *primaurelia* / *P. pentaurelia* and *P. biaurelia* / *P. quindecaurelia* n. sp. may serve as the best models to study genetic isolation and gene flow between the *P. aurelia* species.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/5/197/s1, Table S1: Morphometric characteristics of *P. quindecaurelia* n.sp. and *P. biaurelia.*, Table S2: COII gene sequence identity values for sister species *P. quindecaurelia* sp.n. and *P. biaurelia*.

**Author Contributions:** Conceptualization, A.P. and R.M.-E.; methodology, A.P.; formal analysis, A.P.; investigation, A.P. and R.M.-E.; resources, A.P. and R.M.-E.; data curation, A.P.; writing—original draft preparation, A.P.; writing—review and editing, R.M.-E.; visualization, A.P. and R.M.-E.; project administration, R.M.-E.; funding acquisition, R.M.-E. All authors have read and agreed to the published version of the manuscript.

**Funding:** The short stay of A. Potekhin at the Sciences Faculty, UNAM was funded by Programa de Estancias de Investigación en la UNAM (PREI-DGAPA, UNAM).

**Acknowledgments:** The authors are grateful to PREI-DGAPA-Universidad Nacional Autónoma de México for the gran<sup>t</sup> to AP. Biól. The authors would like to thank Margarita Reyes-Santos and Maksim Melekhin for their technical assistance, P. B. Erika S. Vásquez, Estefania Garay P., Natalia Lebedeva for their help with laboratory procedures and ciliate maintenance, and Irina Nekrasova for support in molecular analysis of the strains. Carlos Durán and Mireya Ramírez are granted for their help with samples collection, Jordan Salomon and Kyle M. Simpson (Texas A&M University, USA) for the language editing. Sébastien Santini (CNRS/AMU IGS UMR7256) is acknowledged for maintenance of the website Phylogeny.fr, and Anna Romanovich from the Centre of Core Facilities "Molecular and Cellular Technologies", St Petersburg State University, Russia, for fast and quality DNA sequencing.

**Conflicts of Interest:** The authors declare no conflict of interest.
