Ecological and evolutionary patterns in the enigmatic protist genus Percolomonas (Heterolobosea; Discoba) from diverse habitats

Abstract

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.

Fig 1. Morphometric data of Percolomonas strains analyzed in this study (n = 50).

L:W: ratio of length and width of the cells, LF:L: ratio of long flagellum and length of the cells. Note that whiskers above and below the box plot (interquartile range) indicate the 90th and 10th percentiles. The individual marks (i.e. closed circles) and lines within the boxes represent each outlier outside the whiskers and the medians, respectively.

Fig 2. General light micrographs of Percolomonas cosmopolitus-like strains (ordered by source salinity as in Table 1).

(A) New strain LRS. (B) New strain SD2A. (C) New strain XLG1-P. (D) New strain S4. (E) New strain P5-P. (F) Strain HLM-6 (the type for Percolomonas lacustris as described by A.P. Mylnikov [38]). (G) “Percolomonas cosmopolitus” ATCC 50343. (H) “Percolomonas cosmopolitus” strain WS, originally reported by Nikolaev et al. [28]. (I) New strain LO. Note that the right cell image in each panel represents a fixed cell. Groups (A) and (B) indicate halophile clades ‘A’ and ‘B’ in Fig 3. All scale bars represent 5 μm.

Fig 3. Scanning electron micrographs of Percolomonas cosmopolitus-like strains.

(A) Strain LRS (B) Strain SD2A. (C) Strain XLG1-P. (D) Strain S4. (E) Strain P5-P. (F) Strain HLM-6 = Percolomonas lacustris. (G) ATCC 50343. (H) Strain WS. (I) Strain LO. The order of strains in Fig 3 is the same as that in Fig 2 and Table 1. All scale bars represent 5 μm.

Fig 4. Maximum likelihood phylogenetic tree of 18S rRNA gene sequences from Percolomonas strains, other representative heterolobosean species, and outgroups (i.e. Euglenozoa, Jakobida, and Tsukubamonas globosa).

Bootstrap support values (> 60%) are shown at the nodes. Solid circles represent a Bayesian posterior probability of 1 (posterior probability < 0.95 not shown). Note that ‘Group A’ and ‘Group B’ in Percolomonadidae (marked in red) together represent the putative halophilic Percolomonas clade.