Updated life cycle of G. trihymene during vegetative growth
The life cycle during vegetative growth of G. trihymene is generalized in Figure 5, based on previous and current studies [21, 22]. The life cycle has multiple stages, as is typical in polyphenic ciliates. These life stages could be highly diverse and complex, depending on the total number of asymmetric divider morphotypes and food concentration. For simplification and clarity, most intermediate asymmetric dividers are not shown in Figure 5.
Some free-living ciliates, for example, Tetrahymena pyriformis, produce maximal progeny cells by shifting their physiological states during starvation [23]. Similarly, G. trihymene produces progeny cells by combining three reproductive modes: asymmetric division, reproductive cysts and equal fission. In addition, this is the first report of reproductive cysts in scuticociliates, though they are not uncommonly found in certain ciliate genera, like Colpoda and Tetrahymena[4]. If each morphotype of asymmetric dividers could be deemed as one life stage, which could probably be the case as many similar or continuous asymmetric divider morphotypes were repeatedly found in cultures with different "age" or media, then the updated life cycle of G. trihymene might rival most known life cycles of free-living ciliates in complexity (Figure 5). G. trihymene thus provides a special opportunity for studying ciliate polyphenism.
Although G. trihymene was first discovered early in 1966, it was believed to reproduce only by equal fission during vegetative growth [21, 22]. One reason for the persistence of this narrow view of G. trihymene reproduction is that, to date, few studies have been conducted on G. trihymene and they have mainly focused on morphology or systematics rather than reproduction dynamics [21, 22]. Secondly, some of the reproductive forms appear only under particular food conditions, for example, in the Hong Kong isolate, asymmetric dividers appeared on the 3rd or 4th day after inoculation, when bacterial supply was high and disappeared soon after the appearance of tomites. The disappearance of asymmetric dividers was probably associated with the transition from exponential culture growth to the stationary phase. Third, the relative immobility and irregular body shapes of most asymmetric dividers (Figures 1G, H; 2E, N), could cause them to be mistaken as cultural artifacts or debris. Lastly, some asymmetric dividers are easily mistaken as conjugating cells or equal binary dividers, if observed on low magnifications (<100×) (Figure 2J). Thus, it is no wonder that these usually large, irregularly shaped asymmetric dividers were unreported until this study.
The class Oligohymenophorea, to which all scuticociliates and the well-known Tetrahymena and Paramecium belong, contains highly diverse species [24], but only a few model species, such as Tetrahymena thermophila and Paramecium tetraurelia, are under intensive biological study. Most members of Oligohymenophorea, especially the marine species, are limited to taxonomic and systematic studies or are undescribed [2, 25]. We predict that as life histories of more species are closely examined, much more diversity in reproductive strategies will be discovered among free-living protists.
Proposed ecological roles of various life cycle stages
The high feeding efficiency, slow movement and arrested cytokinesis observed in G. trihymene asymmetric dividers may be advantageous. Based on the results of our culturing experiments, we conclude that asymmetric dividers are innate physiological states of G. trihymene, which can be induced to occur in bacteria-sufficient media. Cells with asymmetric divisions may ingest more food than those without; most asymmetric dividers had many oral apparatuses with oral membranes beating quickly. They may be able to consume as many bacteria as several trophonts in the same period of time (Figure 2N, arrowheads). In addition, the relative immobility of these asymmetric dividers may minimize their energy consumption [26]. The arrested cytokinesis could also save energy for asymmetric dividers, compared with equal dividers.
We propose the following ecological scenario that comes about as G. trihymene with a capacity for asymmetric divisions explores its surrounding environment. Suppose one G. trihymene trophont finds a food patch with plenty of bacteria, but also with many other bacteria-feeding protists. To avoid being a loser in this resource exploitation competition, for 2-3 days G. trihymene vigorously feeds on bacteria and divides equally. While plenty of bacteria remain, some trophonts asymmetrically divide, producing trophonts and more asymmetric dividers. When the food patch is nearly exhausted, most trophonts transform into tomites, and the asymmetric dividers instead of producing trophonts, produce tomites. After most of the bacteria are consumed, most tomites become resting cysts. Asymmetric dividers secrete a cyst wall and continue dividing inside, producing reproductive cysts, which ultimately become resting cysts. Some tomites transformed from trophonts or released by asymmetric dividers swim rapidly to seek more food patches, transforming back into trophonts when they find new food patches and repeating the above processes. The quickly dispersing tomites, the tolerating resting cysts, and the diverse reproductive strategy may enable G. trihymene to identify and dominate enough food patches and survive in the coastal water community.
Phylogenetic position of G. trihymene, and asymmetric division
G. trihymene groups with typical scuticociliates with high bootstrap support and posterior probability, though the precise relationships within the clades remain unresolved (Figure 4). In addition, G. trihymene has high SSU rDNA pair-wise identity with Anophryoides haemophila (96%), the scuticociliate causing the "Bumper car disease" of American lobsters and Miamiensis avidus (96%), a polyphenic, parasitic ciliate, which causes diseases in fish [27, 28]. Our result supports the monophyly of scuticociliatia, despite what was found in earlier studies utilizing a previously reported G. trihymene SSU rDNA sequence [GenBank Accession No.: AY169274] [29, 30], which we believe to be erroneous. AY169274 shares great similarity with SSU sequences of some flagellates, e.g. it has 96% identity with the 18S rDNA sequences of the nanoflagellate Spumella sp. GOT220 [GenBank Accession No.: EF027354]. In line with our interpretation, the most recent study on morphology and morphogenesis of G. trihymene (performed by the same group that submitted the previous Gt SSU rDNA sequence) showed that it is indeed a typical scuticociliate [22].
Asymmetric divisions, similar to those in G. trihymene, occur in certain apostome and many astome ciliates (see phylogenetic position in Figure 4), though the details of division had never been studied using continuous microscopy [5]. Such asymmetric dividers were called catenoid colonies in these host-dependent ciliates. Asymmetric dividers were so named in the present study to emphasize the difference between the two filial cells. As in the asymmetric division of G. trihymene in Figure 2A, long cell chains in the parasitic and commensal astome and apsotome ciliates are formed by repeated incomplete divisions without separation of the resulting filial products, after which some subcells are fully or partially pinched off. These subcells require subsequent metamorphosis to regain the form typical of the normal trophont stage of the life cycle [3, 5].
The results of the phylogenetic analysis suggest that complex life cycles including asymmetric division are either 1) an ancestral feature of these three groups that has been modified, lost, or not yet discovered in other free-living species, or 2) a convergent trait that has arisen multiple times independently in these closely related taxa.
Asymmetric division: one clue to multicellularity?
The colonial flagellate hypothesis, claiming that flagellated protists living as colonies evolved into the first animals, has inspired extensive productive exploration on the origin of multicellularity [31–34]. The asymmetric division of G. trihymene serves as an alternative mechanism through which ciliates may have led to a multicellular form: a multicellular form could arise by a ciliate with one macronucleus and one micronucleus subdividing itself as a result of growth followed by arrested cytokinesis. It should be noted, however, that such asymmetric division does not result in different developmental fates akin to truly multicellular ciliate species, such as Zoothamnium alternans[35, 36].
As is shown in this study, asymmetric dividers produce new asymmetric dividers and trophonts by successive asymmetric divisions, in favorable conditions, and the more available food, the longer the asymmetric divisions persisted (Figure 3, filled bars). If asymmetric dividers lived in consistently bacteria-rich environments for a long time, they might retain the multicellular form, but lose the ability to produce trophonts or tomites. Bacteria-rich environments were common in the ancient ocean, which had very different chemistry from that of today's [37, 38]. Thus, it is possible that some multicellular organisms, which have not yet been discovered or have since gone extinct, originated from certain asymmetric dividers of ciliates.