New Results

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

Denis V. Tikhonenkov, Soo Hwan Jhin, Yana Eglit, Kai Miller, Andrey Plotnikov, Alastair G.B. Simpson, Jong Soo Park
doi: https://doi.org/10.1101/612531

Abstract

The heterotrophic flagellate Percolomonas cosmopolitus (Heterolobosea) is often observed in saline habitats worldwide that range 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 rDNA divergences (up to 42.4%). Some strains grew best at salinities from 75 to 125‰ and represent halophiles. All but one of these belonged to two geographically heterogeneous clusters that formed 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 rDNA phylogenies, unlike most previous analyses based on two sequences.

  • Cryptic species
  • Heterolobosea
  • Molecular Phylogeny
  • Morphospecies
  • Percolomonas
  • Protist
  • Protozoa

Introduction

The modern classification of eukaryotic microbes is usually based on molecular sequence information combined with morphological characters obtained using diverse imaging techniques, often including electron microscopy. Many taxonomists have a fundamental question whether small morphological differences among the observed organisms represent multiple species or one variable species [1–3]. Conversely, in protistology, many species (and genera) that were originally proposed based on light microscopy alone have proved to encompass considerable genetic diversity. Also, many morphospecies of protists are cosmopolitan and/or are found across a very wide range of habitats, raising the possibility of recognizing species that are divided more by geography or ecology than morphology [4]. Bickford et al. [1] regarded ‘cryptic species’ as cases where two or more species are distinguished that were previously assigned to a single morphologically defined species (i.e. morphospecies). The concept of cryptic (usually genetically different) species has been examined in a diverse range of protists, e.g. [4, 5, 6]. This concept is widely accepted due to an inconsistency between morphospecies and their DNA sequencing data. Obviously, the estimated number of protist species will vary tremendously depending on the species concept employed [7, 8].

The species Percolomonas cosmopolitus (formerly Tetramitus cosmopolitus Ruinen [9]) is a heteroloboseid flagellate with one long and three shorter flagella at the head of a ventral feeding groove, and no known amoeba stage in its lifecycle. Cell size is given as 6–12 μm long and 3–9 μm wide in the seminal modern accounts [10, 11]; the largest cells reported by Ruinen [9] are somewhat longer. This species is a particularly interesting protist for taxonomists and ecologists for several reasons: Firstly, it is possible that P. cosmopolitus sensu lato may be a remarkably broad assemblage of cryptic species. To date, two strains identified as P. cosmopolitus have been studied using molecular methods, but their 18S rRNA gene sequences share remarkably low sequence identity (58.4%). Secondly, P. cosmopolitus has been reported in samples from an extremely wide range of saline habitats, from coastal waters to saturated brines, throughout the world [9–12]. It is unknown whether there are ecologically or geographically defined subtypes. Thirdly, in addition to the genetic divergence between them (see above) P. cosmopolitus strains are usually not inferred to be sisters in phylogenetic trees, forming instead a paraphyletic group with respect to the pseudociliate Stephanopogon, which has many more flagella and a completely different feeding system [13–21]. Consequently, Percolomonas cosmopolitus and similar organisms (e.g. [22]) are interesting candidates to study the interplay between species distinctions, autecology and evolutionary history among protists.

Here, we examined seven Percolomonas cosmopolitus-like organisms, which were isolated from samples with a salinity range from 4‰ to 280‰, from seven different countries (New Zealand, Kenya, Korea, Poland, Russia, Spain, and the USA). The nine Percolomonas strains examined (including the two P. cosmopolitus strains previously sequenced) were morphologically similar to each other, but their 18S rRNA gene sequences showed considerable genetic divergence, and they formed six genetically distinct clusters. The strains isolated from high salinity samples preferred to grow in artificial media with salinity higher than seawater (75‰ or above), indicating ecological specialization. Percolomonas formed a monophyletic group in the 18S rRNA gene phylogeny.

Materials and methods

Isolation and cultivation

Seven monoprotistan strains were isolated from brackish to high salinity (4–280‰) water/sediment interface samples collected from New Zealand, Kenya, Korea, Poland, Russia, Spain, and the USA between 2008 and 2015 (Table 1). Isolates from higher salinity waters were LRS, SD2A, XLG1-P, S4, and P5-P; isolates from lower salinity waters were LO, and HLM-6. Strain HLM-6 was examined morphologically by A.P. Mylnikov [22] under the name Percolomonas lacustris. Each monoprotistan culture was established by single-cell isolation (from raw samples or crude cultures) or by serial dilution. High salinity media (~100‰ salinity) was made by dilution of Medium V (300‰; 272g NaCl, 7.6g KCl, 17.8g MgCl2, 1.8g MgSO4·7H2O, 1.3g CaCl2 l−1 water, see [23]) with sterile distilled water, whereas normal salinity media (~35‰ salinity) used autoclaved seawater. Luria-Bertani Broth (final concentration of 0.5%, Difco) plus autoclaved barley grains were added into each media to grow indigenous prokaryotes in the culture. For maintenance of each culture, 0.1 ml of inoculum was added into 5 ml of 100‰ salinity liquid media (isolates LRS, SD2A, XLG1-P, S4, and P5-P) or 35‰ salinity liquid media (isolates LO and HLM-6 plus the already available strains ATCC 50343 and White Sea = ‘WS’). Strain HLM-6 was also maintained in marine Schmalz-Pratt’s medium (NaCl–28.15 g l−1, KCl–0.67 g l−1, MgCl2·6H2O–5.51 g l−1, MgSO4·7H2O–6.92 g l−1, CaCl2·H2O–1.45 g l−1, KNO3–0.1 g l−1, K2HPO4·3H2O–0.01 g l−1 with a final salinity of 35‰) with addition of Pseudomonas fluorescens bacteria as food. All cultures were incubated at 25 °C and subcultured every four weeks.

View this table:
Table 1. Characterizations of Percolomonas strains; sizes and salinity range supporting growth.

Light microscopy

Live flagellates mounted on glass slides were observed with phase contrast microscopy or differential interference microscopy using a Leica DM5500B microscope equipped with a DFC550 digital camera (Leica, Wetzlar, Germany) or Carl Zeiss AxioScope A.1 microscope equipped with a AVT HORN MC-1009/S analog video camera. To observe the number and shape of the flagella, cultures (1 ml) were centrifuged at ×2,000 g for 10 min, then 900 µL of the supernatant was discarded, and the remaining volumes (i.e. 100 µL) were fixed by addition of 50 µL of 25% v/v glutaraldehyde (electron microscopy grade). The sizes of the live cells (i.e. 20 cells per culture) were measured from digital images.

Scanning electron microscopy

Cultures (1 ml) were centrifuged at ×1,200 g for 10 min, then 900 µL of the supernatant was discarded, and the remaining volumes (i.e. 100 µL) were fixed by adding 50 µL of 25% v/v glutaraldehyde (electron microscopy grade). Fixed cells were allowed to settle (40 min) on glass coverslips coated with 1% poly-L-lysine. Cells were rinsed with sterile media, and then dehydrated with a graded ethanol series (30–100%). The glass coverslips were then critical-point dried. Fixed cells were coated with gold/platinum using an ion sputter system. Specimens were examined with a SU8220 field emission scanning electron microscope (Hitachi, Tokyo, Japan) or JSM-6510LV scanning electron microscope (JEOL Ltd., Tokyo, Japan).

Molecular sequencing and phylogenetic analysis

Nucleic acids from the seven new isolates were extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) or MasterPure Complete DNA and RNA Purification Kit (Epicentre, Madison, USA), as described in the supplied protocols.

For all strains except LRS and HLM-6, the 18S rRNA gene sequences were obtained by PCR amplification using a combination of the eukaryote primers EukA 5′-AACCTGGTTGATCCTGCCAGT-3′ and EukB 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ [24]. The 20-µL PCR reactions included 1.5 µL each of 10-µM stocks of the primers, 2 µL of a 0.25-mM dNTP-mix, 0.8 µL of 50 mM MgCl2, 0.2 µL of 5 U/µL Taq DNA polymerase (Solgent, Daegeon, Republic of Korea), and 1–3 µL of DNA template. The cycling conditions were as follows: an initial denaturing step at 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 1 min of annealing at 55 °C, and extension at 72 °C for 2 min, with a final extension step for 10 min at 72 °C. Amplicons were cloned into a pGEM-T Easy vector, at least five positive clones per sample were partially sequenced, and a positive clone was completely sequenced using various sequencing primers. For strain XLG1-P, the cycling conditions were slightly different: 35 cycles of 20 s at 94 °C, 1 min of annealing at 55 °C, and 3 min of extension at 72 °C. Strain LRS was amplified using the different eukaryote primers 82F (5’-GAAACTGCGAATGGCTC-3’) and 1498R (5’-CACCTACGGAAACCTTGTTA-3’). The optimized PCR condition was 2 min at 96 °C (an initial denaturation), followed by 35 cycles of 30 s at 96 °C, 1 min at 60 °C, 2 min at 72 °C, with a final extension for 10 min at 72 °C. The PCR products for strains XLG1-P and LRS were directly sequenced by Sanger dideoxy sequencing without cloning.

For Strain HLM-6, the primers used were PF1 5′-GCGCTACCTGGTTGATCCTGCC-3′ and FAD4 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ [25, 26]. The 25-µL PCR reaction included 0.5 µL each of 10-µM primer stocks, 1 µL of DNA template, 10.5 µL PCR-grade water and 12.5 µL EconoTaq® PLUS Green 2x Master Mix (Lucigen, Middleton, USA). The cycling conditions were 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 50 °C, and 1.5 min at 72 °C for 1.5 min, with a final extension of 5 min at 72 °C. Amplicons were cloned using Strata Clone PCR Cloning Kit (Agilent, Santa Clara, USA). The 18S rRNA gene sequences from the isolates have been deposited in GenBank under the accession numbers XXXXXXX.

The 18S rRNA gene sequences from 62 representative heterolobosean species, plus 16 other Discoba species selected as outgroups, were used for phylogenetic analysis (the seed alignment originated from Jhin and Park [16]). The dataset was aligned and masked by eye, with 1,033 unambiguously aligned sites retained for analysis. The alignment is available on request. Phylogenetic trees were inferred by Maximum Likelihood (ML) and Bayesian analyses. The GTR + gamma + I model of sequence evolution was selected for the dataset using MrModeltest 2.2 [27] and was used for both analyses. The ML tree was estimated using RAxML-VI-HPC v.7 [28] with the GTRGAMMAI model setting, 500 random starting taxon addition sequences, and statistical support assessed using bootstrapping with 10,000 replicates. The Bayesian analysis was conducted in MrBAYES 3.2 [29] with two independent runs, each with four chains running for 2 × 107 generations with the default heating parameter (0.1) and sampling frequency (0.01). A burn-in of 30% was used, by which point convergence had been achieved (the average standard deviation of split frequencies for the last 75% of generations was < 0.05).

Salinity ranges for growth

To estimate the salinity ranges supporting growth of the seven new isolates and two previously available isolates, we performed an experiment using media with 3‰ to 300‰ salinity, made from artificial seawater stock (Medium V; see above) as reported by Park and Simpson [18, 30]. In brief, the medium was supplemented with heat-killed Enterobacter aerogenes at an initial density of 3.46 × 107 cells per ml (20 µL) at 7- to 14-day intervals to support the growth of the protists. All treatments were performed in duplicate. Medium V (0.96 ml) with a range of salinities (3‰–300‰) were inoculated with 20 µL of actively growing stock culture (100‰ or 35 ‰ salinity media with autoclaved barley grain) and incubated in the dark at 25 °C for at least 49 days. We confirmed the salinity range supporting growth by transferring a sample of the isolate into fresh media with the same salinity (0.96 ml of media; inoculum size 20 µL), and re-examining the culture for actively moving cells at 7- to 14-day intervals over a period of 35 days.

Results

General morphology

Live cells were usually ovoid-shaped or spindle-shaped with average lengths and widths ranging from 4.7–9.7 µm and 2.2–4.3 µm (Fig 1 and Table 1). The ratio of length and width of the cells was between 1.8 and 3.3 on average (Table 1). The biggest cells (length: 9.7 µm, width: 4.3 µm on average) were from strain LO isolated from Lake Turkana, Kenya, whereas the smallest cells were from strain ATCC 50343 (length: 4.7 µm, width: 2.6 µm on average), but the sizes of different strains represented an overlapping continuum. Cells had four flagella inserted in the sub-anterior part of the cell, at the head of the ventral cytostomal groove (Fig 2). Three flagella were shorter, and one flagellum was longer. The three short flagella were similar in length, similar to that of the cytostomal groove (typically ~4 µm long). The long flagellum averaged 14.7–19.1 µm in length, depending on the strain, which was 1.7–3.1 times longer than the length of the cell body (Table 1). Most cells had an acroneme at the tip of the long flagellum (Fig 2), however, this seems not to be a permanent feature. All species showed jerking motility and sometimes rotated in a counterclockwise direction. The right side of the cytostomal groove was curved, whereas the left side was roughly linear (Fig 2). No amoeboid form was observed during the cultivation of the organisms, and a cyst form was observed occasionally in all cultures except for strain ATCC 50343. We did not observe discrete features that distinguished isolates from each other under a light microscope or a scanning electron microscope.

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

(A) New strain LRS isolated from a solar saltern at the Ebre River Delta, Catalonia, Spain (280‰ salinity). (B) New strain SD2A isolated from Chula Vista solar saltern, USA (200‰ salinity). (C) New strain XLG1-P isolated from Lake Grassmere, New Zealand (196‰ salinity). (D) New strain S4 isolated from a Korean solar saltern (180‰ salinity). (E) New strain P5-P isolated from Wielizka salt mine, Poland (73‰ salinity). (F) Strain HLM-6 isolated from Novoe Lake, Orenburg oblast, Russia (36‰ salinity); this strain was the type for Percolomonas lacustris as described by A.P. Mylnikov [22]. (G) “Percolomonas cosmopolitus” ATCC 50343. (H) “Percolomonas cosmopolitus” strain WS (White Sea), originally reported by Nikolaev et al. [17]. (I) New strain LO isolated from Lake Turkana, Kenya (4‰ salinity). 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 2.
Fig 2. 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. 2 is the same as that in Fig. 1 and Table 1. All scale bars represent 5 μm.

Molecular phylogeny of 18S rRNA gene sequences

The 18S rRNA gene sequences from the seven unstudied Percolomonas strains were closest by BLASTN search to Percolomonas cosmopolitus strain WS (AF 519443), but with a low identity of 74% to 83%. As in previous studies [14–16, 19, 31, 32], Heterolobosea formed a strong monophyletic group in phylogenetic trees of 18S rRNA gene sequences (Fig 3). The seven new Percolomonas strains and two previously identified sequences branched with the pseudociliate taxon Stephanopogonidae with strong support (100% ML; PP 1; Fig 3) forming the clade Percolatea. Interestingly, Percolomonadidae, including the seven new sequences, formed a monophyletic group, with moderate bootstrap support (ML: 76%) and posterior probability 1. Within Percolomonadidae, ATCC 50343 (USA) and P5-P (Poland) were distinct from each other and the remaining seven strains, which formed a maximally supported clade. This subdivided further into (i) WS (Russia), (ii) HLM-6 (Russia), and (iii) a maximally supported ‘putative halophile clade’ of 5 strains in 2 clusters, ‘A’ and ‘B’, for a grand total of 6 distinct clusters or single-strain lineages. The 18S rDNA sequences differences between these 6 clusters range from 15.1 to 42.4%. Group A of the putative halophile clade was composed of strains LRS (Spain), XLG1-P (New Zealand), and S4 (Korea) and formed a maximally supported clade (Fig 3), with low genetic divergence between the strain (98% to 99% identities). All members were isolated from hypersaline waters of 180 to 280‰ salinity (Table 1). Group B consisted of strains LO (Kenya) and SD2A (USA) which showed 98% sequence identity, though formed a weakly supported clade (62% ML; PP 0.75). Strain SD2A was isolated at 200‰ salinity, while strain LO was isolated from a low salinity (4‰) sample, though it cannot grow at this salinity (see below and Table 1).

Fig 3.
Fig 3. 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 together represent the putative halophilic Percolomonas clade.

Salinity tolerance of Percolomonas strains

The Percolomonas strains were isolated from a variety of habitats with 4‰–280‰ salinity. Salinity ranges supporting growth of all nine sequenced Percolomonas isolates were determined as described earlier [16, 18, 20, 21]. Percolomonas strain LRS isolated from 280‰ salinity showed the broadest salinity range for growth (15‰–200‰ salinity, Table 1). Strain ATCC 50343 grew in the narrowest salinity range (30‰–75‰ salinity, Table 1). Percolomonas strain LO isolated from a source salinity of 4‰ grew only at 30‰ to 100‰ salinity. This suggests that the strain existed as an alternative life-cycle form (e.g. cyst) in the original sample. The five Percolomonas strains (i.e. LRS, SD2A, XLG1-P, S4, and P5-P) isolated from hypersaline habitats with >70‰ source salinity could grow best at 75‰ to 125‰ salinity (Table 1). In contrast, the four Percolomonas strains (i.e. ATCC 50343, White Sea, HML-6 and LO) isolated from non-hypersaline habitats with < 40‰ source salinity appeared to grow best at 30‰ to 50‰ salinity (Table 1).

Discussion

Morphology of Percolomonas isolates

In general, the isolates studied here are morphologically indistinguishable by light and scanning electron microscopy from the seminal modern accounts of Percolomonas cosmopolitus [10, 11]. They all have four flagella, specifically three short flagella (~4 μm long) and one long flagellum (12–20 μm long) directed along a ventral groove. The size of the cell body of P. cosmopolitus is 6–12 μm in length, 3–9 μm in width [10], overlapping with all of our isolates. The curved right side of the cytostomal groove differs from the left side with a near-linear shape. All strains move with a jerky gliding motion. Thus, it is appears that all new isolates may be assigned to the morphospecies P. cosmopolitus. The nominal Percolomonas species most similar to P. cosmopolitus include P. denhami, with three flagella, two of which are long [33] and P. similis with two flagella, one short, and the other long [34]. There are no molecular data for either of these species. The third similar species is Percolomonas lacustris, which was introduced based on the description of strain HLM-6. This was distinguished morphologically from P. cosmopolitus primarily by having a small posterior bulbous projection and an acroneme on one short flagellum [22]. However, the bulbous projection appears by light microscopy just as a pointed tip, which was a feature of the original description of P. cosmopolitus [9], and was seen intermittently in several of our isolates (see Fig 1). We confirmed that one short flagellum bearing a proportionately long acroneme can be a feature of strain HLM-6 (compare our Fig 2f to Fig 2g in [22]), but note that other cells of the strain appear to lack it (Fig 2f in [22]), and that (shorter) acronemes can be observed on the short flagella of several of the strains studied here (our Fig 2). Thus, it seems that the morphological distinction between P. lacustris and other P. cosmopolitus-like strains is subtle at best.

Genetic structure in Percolomonas

The Percolomonas complex includes 6 distinct clades that are separated by genetic distances of 15.1% or more. Considering the similar morphology of P. cosmopolitus-like strains (see above), it is reasonable to regard the Percolomonas complex as a grouping of several (at least) cryptic (or sibling) species. Among protists, it has long been recognized that numerous cryptic species can exist within a single morphospecies [4, 8, 35–38]. Fenchel [36] suggested that the cryptic protists are regarded as allopatric speciation, and are distributed in limited geographic regions. In our case, however, no geographic clustering of Percolomonas strains was observed in the two clades represented by multiple isolates, although the numbers of sampling sites and sequencing data are limited. Three Percolomonas strains in ‘Group A’ form a robust clade with low genetic divergences (98% to 99% identities) and are widely dispersed geographically (Korea, New Zealand, and Spain). In addition, two Percolomonas strains in ‘Group B’ (with 98% identity) are from Africa and North America. This pattern is more consistent with extremely widespread dispersal, as inferred for other very small protists (e.g. [39]), and we speculate that environmental selection to different habitats may determine which groups appear where (see below).

Halophilicity in Percolomonas

Ruinen [9] reported that P. cosmopolitus was detected at salinities ranging from 30‰ to saturated brines. In our study, the nine examined Percolomonas strains derived from a wide range of salinities, up to near-saturation. In general, the halophilicity of these strains was related to the salt concentration of their original habitats. Five Percolomonas strains (i.e. LRS, SD2A, XLG1-P, S4, and P5-P) were isolated from various habitats with a salinity range of 73‰ to 280‰. These all grew best at 75‰–125‰ by our qualitative estimation and most could still grow at 175‰ or even 200‰ on our experimental media. This clearly makes them halophiles according to the definition by Oren [40], in which a halophile could grow at 50‰ or higher and tolerated at 100‰ salinity. None were obligate halophiles, however, since all could also grow at 30‰ salinity. By contrast, three Percolomonas strains (i.e. HLM-6, ATCC 50343, and WS) isolated from non-hypersaline habitats (20‰ to 36‰ salinity) grew optimally at 30‰ or 50‰, and failed to grow above 100‰ salinity. Interestingly, Percolmonas strain LO grew optimally at 50‰ salinity, and grew up to 100‰ salinity, although it was isolated from a Lake Turkana, Kenya, with a very low salinity of 4‰. Phylogenetically, LO belongs within the putative ‘halophile clade’, and it is possible that it descended from a more halophilic ancestor (see below). Interestingly, the Lake Turkana sample was also the origin for Pharyngomonas turkanensis, a heterolobosean amoeba that grows best at 15–30‰ salinity, but is inferred to have descended from halophilic Pharyngomonas ancestor [41].

The taxon Heterolobosea includes a substantial proportion of the known halophilic or halotolerant eukaryotes [18–21, 41–44], and is interesting for examining the evolution of halophiles [16, 42, 43]. Recently, Kirby et al. [43] and Jhin and Park [16] suggested that the Tulamoebidae clade (sensu lato) in Heterolobosea were an example of a radiation of morphospecies that stemmed from a common halophilic ancestor. This clade included Pleurostomum flabellatum, Tulamoeba peronaphora, Tulamoeba bucina, and Aurem hypersalina, all with optimal salinities for growth of at least 150‰ [16, 20, 21, 43]. It is possible that the Percolomonas clade consisting of ‘Group A’ and ‘Group B’ may also represent a radiation of halophiles (albeit one of cryptic species within a morphospecies). All of the cultivated Percolomonas in this clade are halophiles, with one borderline case (LO; see above). However, this possibility of an exclusively/predominantly halophile clade could be a biased sampling artefact, and should be tested through additional isolations and study of related strains. This would also be useful to understand the nature of the closest relative of the halophile strain P5-P, which is phylogenetically isolated from others at present.

Is Percolomonas monophyletic?

For a long time only two 18S rRNA gene sequences of P. cosmopolitus were available, and these were included in many phylogenetic analyses of Heterolobosea [13–21, 45]. Most of these phylogenies showed the two sequences forming a paraphyletic group, with one more closely related to Stephanopogon [45]. This inference may have been affected by the small number of sequences of Percolomonas available. In the present study, with seven additional and different 18S rRNA gene sequences, we instead inferred a (moderately supported) Percolomonas clade. Future research will address the cause of this difference, and test the relationships amongst Percolomonas and Stephanopogon using other markers.

Conclusions

On the basis of light and scanning electron microscopic observations, all Percolomonas strains studied here are morphologically very similar, in spite of the huge genetic diversity they encompass. Percolomonas strains form at least 6 genetically distinct clades in the molecular phylogenetic trees, and could be considered to represent at least as many cryptic species. The speciation of Percolomonas strains could be partially related to salinity preference, rather than spatial distribution. The clusters ‘Group A’ and ‘Group B’, which are specifically related, may collectively represent a halophilic clade.

Author contributions

Conceptualization: Jong Soo Park, Alastair G.B. Simpson

Investigation: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin, Yana Eglit, Kai Miller, Andrey Plotnikov

Formal analysis: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin

Visualization: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin

Supervision: Jong Soo Park, Alastair G.B. Simpson

Funding acquisition: Jong Soo Park, Denis V. Tikhonenkov, Alastair G.B. Simpson

Writing – original draft preparation: Jong Soo Park, Denis V. Tikhonenkov, Alastair G.B. Simpson

Writing – review and editing: all authors.

Financial Disclosure Statement

This research was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF), a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Education (2016R1A6A1A05011910, 2017K2A9A1A06049946, and 2019R1A2C2002379) and the Ministry of Environment (MOE) of the Republic of Korea (NIBR201801208), respectively, to J.S.P., a Russian Foundation for Basic Research grant (No 18-504-51028) to D.V.T., and a Natural Sciences and Engineering Research Council of Canada Discovery Grant (298366-2014) to A.G.B.S.

Acknowledgements

We thank Dr. Alexander Mylnikov for help with SEM preparations and Dr. Jan Janouškovec for help with SSU rDNA sequencing of strain HLM-6. We thank Dr. Noèlia Carrasco for providing access to the Delta de l’Ebre sampling location, and Golara Sharafi for isolating and obtaining DNA from strain LRS. This research was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF), a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Education (2016R1A6A1A05011910, 2017K2A9A1A06049946, and 2019R1A2C2002379) and the Ministry of Environment (MOE) of the Republic of Korea (NIBR201801208), respectively, to J.S.P., a Russian Foundation for Basic Research grant (No 18-504-51028) to D.V.T., and a Natural Sciences and Engineering Research Council of Canada Discovery Grant (298366-2014) to A.G.B.S.

References

  1. 1.↵
    Bickford D, Lohman DJ, Sodhi NS., Ng PKL, Meier R, Winker K, et al. Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 2007; 22:148–155.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.
    Ristau K, Steinfartz S, Traunspurger W. First evidence of cryptic species diversity and significant population structure in a widespread freshwater nematode morphospecies (Tobrilus gracilis). Mol Ecol. 2013; 22:4562–4575.
    OpenUrl
  3. 3.↵
    Trewick SA. Sympatric cryptic species in New Zealand Onychophora. Biol J Linnean Soc. 1998; 63:307–329.
    OpenUrl
  4. 4.↵
    Scheckenbach F, Wylezich C, Mylnikov AP, Weitere M, Arndt H. Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates. Appl Environ Microbiol. 2006; 72:6638–6643.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Finlay BJ. Global dispersal of free-living microbial eukaryote species. Science. 2002; 296:1061–1063.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Škaloud P, Rindi F. Ecological differentiation of cryptic species within an asexual protist morphospecies: a case study of filamentous green alga Klebsormidium (Streptophyta). J Eukaryot Microbiol. 2013; 60:350–362.
    OpenUrlCrossRefPubMed
  7. 7.↵
    Foissner W. Protist diversity: estimates of the near-imponderable. Protist. 1999; 150:363–368.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    Finlay BJ, Fenchel T. Divergent perspectives on protist species richness. Protist. 1999; 150:229–233.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Ruinen J. Notizen über Salzflagellaten. II. Über die Verbreitung der Salzflagellaten. Arch Protistenkd. 1938; 90:210–258.
    OpenUrl
  10. 10.↵
    Fenchel T. Patterson DJ. Percolomonas cosmopolitus (Ruinen) n. gen., a new type of filter feeding flagellate from marine plankton. J Mar Biol Assoc UK. 1986; 66:465–482.
    OpenUrl
  11. 11.↵
    Larsen J, Patterson DJ. Some flagellates (Protista) from tropical marine sediments. J Nat History. 1990; 24:801–937.
    OpenUrl
  12. 12.↵
    Post FJ, Borowitzka LJ, Borowitzka MA, Mackay B, Moulton T. The protozoa of a Western Australian hypersaline lagoon. Hydrobiologia. 1983; 105:95–113.
    OpenUrlCrossRef
  13. 13.↵
    Cavalier-Smith T, Nikolaev S. The zooflagellates Stephanopogon and Percolomonas are a clade (Class Percolatea: Phylum Percolozoa). J Eukaryot Microbiol. 2008; 55:501–509.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    Hanousková P, Táborský P, Čepička I. Dactylomonas gen. nov., a novel lineage of heterolobosean flagellates with unique ultrastructure, closely related to the amoeba Selenaion koniopes Park, De Jonckheere & Simpson, 2012. J Eukaryot Microbiol. 2019; 66:120–139.
    OpenUrl
  15. 15.
    Harding T, Brown MW, Plotnikov A, Selivanova E, Park JS, Gunderson JH, et al. Amoeba stages in the deepest branching heteroloboseans, including Pharyngomonas: Evolutionary and systematic implications. Protist. 2013; 164:272–286.
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    Jhin SH, Park JS. A new halophilic heterolobosean flagellate, Aurem hypersalina gen. n. et sp. n., closely related to the Pleurostomum-Tulamoeba clade: Implications for adaptive radiation of halophilic eukaryotes. J Eukaryot Microbiol. 2019; 66:221–231.
    OpenUrl
  17. 17.↵
    Nikolaev SI, Mylnikov AP, Berney C, Fahrni J, Pawlowski J, Aleshin VV, et al. Molecular phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: Evolutionary implications. J Eukaryot Microbiol. 2004; 51:575–581.
    OpenUrlCrossRefPubMed
  18. 18.↵
    Park JS, Simpson AGB. Characterization of Pharyngomonas kirbyi (= “Macropharyngomonas halophila” nomen nudum), a very deep-branching, obligately halophilic heterolobosean flagellate. Protist. 2011; 162:691–709.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    Park JS, Simpson AGB. Diversity of heterotrophic protists from extremely hypersaline habitats. Protist. 2015; 166:422–437.
    OpenUrlCrossRefPubMed
  20. 20.↵
    Park JS, Simpson AGB, Lee WJ, Cho BC. Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938). Protist. 2007; 158:397–413.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    Park JS, Simpson AGB, Brown S, Cho BC. Ultrastructure and molecular phylogeny of two heterolobosean amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., isolated from an extremely hypersaline habitat. Protist. 2009; 160:265–283.
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    Mylnikov AP. A new flagellate Percolomonas lacustris sp. n. (Excavata, Percolozoa) from an inland saline lake (Southeastern European Russia). Biol Bull. 2016; 43:587–594.
    OpenUrl
  23. 23.↵
    Park JS. Effects of different ion compositions on growth of obligately halophilic protozoan Halocafeteria seosinensis. Extremophiles. 2012; 16:161–164.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA coding regions. Gene. 1988; 71:491–499.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Keeling PJ. Molecular phylogenetic position of Trichomitopsis termopsidis (Parabasalia) and evidence for the Trichomitopsiinae. Eur J Protistol. 2002; 38:279–286.
    OpenUrlCrossRef
  26. 26.↵
    Deane JA, Hill DRA, Brett SJ, McFadden GI. Hanusia phi gen. et. sp. nov. (Cryptophyceae): Characterization of ‘Cryptomonas sp. Φ’’. Eur J Phycol. 1998; 33:149–154.
    OpenUrlCrossRef
  27. 27.↵
    Nylander JAA. MrModeltest. Version 2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, Sweden. 2004.
  28. 28.↵
    Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006; 22:2688–2690.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBAYES 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012; 61:539–542.
    OpenUrl
  30. 30.↵
    Park JS, Simpson AGB. Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms, and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles. Environ Microbiol. 2010; 12:1173–1284.
    OpenUrlCrossRefPubMed
  31. 31.↵
    Pánek T, Silberman JD, Yubuki N, Leander BS, Čepička I. Diversity, evolution and molecular systematics of the Psalteriomonadidae, the main lineage of anaerobic/microaerophilic heteroloboseans (Excavata: Discoba). Protist. 2012; 163:807–831.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    Pánek T, Simpson AGB, Hampl V, Čepička I. Creneis carolina gen. et sp. nov. (Heterolobosea), a novel marine anaerobic protist with strikingly derived morphology and life cycle. Protist. 2014; 165:542–567.
    OpenUrl
  33. 33.↵
    Tong SM. Heterotrophic flagellates from the water column in Shark Bay, Western Australia. Mar. Biol. 1997; 128:517–536.
    OpenUrl
  34. 34.↵
    Lee WJ, Brandt SM, Vørs N, Patterson DJ. Darwin’s heterotrophic flagellates. Ophelia. 2003; 57:63–98.
    OpenUrl
  35. 35.↵
    Beszteri B, Ács É, Medlin LK. Ribosomal DNA sequence variation among sympatric strains of the Cyclotella menghiniana complex (Bacillariophyceae) reveals cryptic diversity. Protist. 2005; 156:317–333.
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    Fenchel T. Cosmopolitan microbes and their ‘cryptic’ species. Aquat Microb Ecol. 2005; 41:49–54.
    OpenUrlCrossRefWeb of Science
  37. 37.
    Koch TA, Ekelund F. Strains of the heterotrophic flagellate Bodo designis from different environments vary considerably with respect to salinity preference and SSU rRNA gene composition. Protist. 2005; 156:97–112.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Von der Heyden S, Chao EE, Vickerman K, Cavalier-Smith T. Ribosomal RNA phylogeny of Bodonid and Diplonemid flagellates and the evolution of Euglenozoa. J Eukaryot Microbiol. 2004; 51:402–416.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Stoupin D, Kiss AK, Arndt H, Shatilovich AV, Gilichinsky DA, Nitsche F. Cryptic diversity within the choanoflagellate morphospecies complex Codosiga botrytis – Phylogeny and morphology of ancient and modern isolates. Europ J Protistol. 2012; 48:263–273.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Oren A. Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Syst. 2008; 4:2.
    OpenUrlCrossRefPubMed
  41. 41.↵
    Park JS, Simpson AGB. Characterization of a deep-branching heterolobosean, Pharyngomonas turkanaensis n. sp., isolated from a non-hypersaline habitat, and ultrastructural comparison of cysts and amoebae among Pharyngomonas strains. J Eukaryot Microbiol. 2016; 63:100–111.
    OpenUrl
  42. 42.↵
    Harding T, Simpson AGB. Recent advances in halophilic protozoa research. J Eukaryot Microbiol. 2018; 65:556–570.
    OpenUrl
  43. 43.↵
    Kirby WA, Tikhonenkov DV, Mylnikov AP, Janouškovec J, Lax G, Simpson AGB. Characterization of Tulamoeba bucina n. sp., an extremely halotolerant amoeboflagellate heterolobosean belonging to the Tulamoeba-Pleurostomum clade (Tulamoebidae n. fam.). J Eukaryot Microbiol. 2015; 62:227–238.
    OpenUrl
  44. 44.↵
    Park JS, De Jonckheere JF, Simpson AGB. Characterization of Selenaion koniopes n. gen., n. sp., an amoeba that represents a new major lineage within Heterolobosea, isolated from the Wieliczka salt mine. J Eukaryot Microbiol. 2012; 59:601–613.
    OpenUrlCrossRefPubMed
  45. 45.↵
    Pánek T, Čepička I. Diversity of Heterolobosea. In: Caliskan M, editor. Diversity of Microorganisms. Rijeka: Intech; 2012. pp. 3–26.
Back to top
Posted April 17, 2019.
Download PDF
Email
Ecological and evolutionary patterns in the enigmatic protist genus Percolomonas (Heterolobosea; Discoba) from diverse habitats
Denis V. Tikhonenkov, Soo Hwan Jhin, Yana Eglit, Kai Miller, Andrey Plotnikov, Alastair G.B. Simpson, Jong Soo Park
bioRxiv 612531; doi: https://doi.org/10.1101/612531
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area