Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid-shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear-like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100-200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.

The heterotrophic flagellate Percolomonas cosmopolitus (Heterolobosea) is often observed in saline habitats worldwide, from coastal waters to saturated brines. However, only two cultures assigned to this morphospecies have been examined using molecular methods, and their 18S rRNA gene sequences are extremely different. Further the salinity tolerances of individual strains are unknown. Thus, our knowledge on the autecology and diversity in this morphospecies is deficient. Here, we report 18S rRNA gene data on seven strains similar to P . cosmopolitus from seven geographically remote locations (New Zealand, Kenya, Korea, Poland, Russia, Spain, and the USA) with sample salinities ranging from 4‰ to 280‰, and compare morphology and salinity tolerance of the nine available strains. Percolomonas cosmopolitus -like strains show few-to-no consistent morphological differences, and form six clades separated by often extremely large 18S rRNA gene divergences (up to 42.4%). Some strains grow best at salinities from 75 to 125‰ and represent halophiles. All but one of these belong to two geographically heterogeneous clusters that form a robust monophyletic group in phylogenetic trees; this likely represents an ecologically specialized subclade of halophiles. Our results suggest that P . cosmopolitus is a cluster of several cryptic species (at least), which are unlikely to be distinguished by geography. Interestingly, the 9 Percolomonas strains formed a clade in 18S rRNA gene phylogenies, unlike most previous analyses based on two sequences.

21The heterotrophic flagellate Percolomonas cosmopolitus (Heterolobosea) is often observed 22 in saline habitats worldwide that range from coastal waters to saturated brines. However, only two 23 cultures assigned to this morphospecies have been examined using molecular methods, and their 24 18S rRNA gene sequences are extremely different. Further the salinity tolerances of individual 25 strains are unknown. Thus, our knowledge on the autecology and diversity in this morphospecies is 26 deficient. Here, we report 18S rRNA gene data on seven strains similar to P. cosmopolitus from 27 seven geographically remote locations (New Zealand, Kenya, Korea, Poland, Russia, Spain, and the 28 USA) with sample salinities ranging from 4‰ to 280‰, and compare morphology and salinity 29 tolerance of the nine available strains. Percolomonas cosmopolitus-like strains show few-to-no 30 consistent morphological differences, and form six clades separated by often extremely large 18S 31 rDNA divergences (up to 42.4%). Some strains grew best at salinities from 75 to 125‰ and 32 represent halophiles. All but one of these belonged to two geographically heterogeneous clusters 33 that formed a robust monophyletic group in phylogenetic trees; this likely represents an ecologically 34 specialized subclade of halophiles. Our results suggest that P. cosmopolitus is a cluster of several 35 cryptic species (at least), which are unlikely to be distinguished by geography. Interestingly, the 9 36 Percolomonas strains formed a clade in 18S rDNA phylogenies, unlike most previous analyses 37 based on two sequences. 38 39 40 3 41 Introduction 42 The modern classification of eukaryotic microbes is usually based on molecular sequence 43 information combined with morphological characters obtained using diverse imaging techniques, 44 often including electron microscopy. Many taxonomists have a fundamental question whether small 45 morphological differences among the observed organisms represent multiple species or one variable 46 species [1-3]. Conversely, in protistology, many species (and genera) that were originally proposed 47 based on light microscopy alone have proved to encompass considerable genetic diversity. Also, 48 many morphospecies of protists are cosmopolitan and/or are found across a very wide range of 49 habitats, raising the possibility of recognizing species that are divided more by geography or 50 ecology than morphology [4]. Bickford et al. [1] regarded 'cryptic species' as cases where two or 51more species are distinguished that were previously assigned to a single morphologically defined 52 species (i.e. morphospecies). The concept of cryptic (usually genetically different) species has been 53 examined in a diverse range of protists, e.g. [4, 5, 6]. This concept is widely accepted due to an 54 inconsistency between morphospecies and their DNA sequencing data. Obviously, the estimated 55 number of protist species will vary tremendously depending on the species concept employed [7, 8]. 56The species Percolomonas cosmopolitus (former...