The distinctly orange color of A. quadriverberis sets these flagellates apart from other organisms living in the same benthic environment. To our knowledge, similar organisms have not been recorded previously [3, 9–12, 28]; however, the orange color of A. quadriverberis is most reminiscent of the anoxic euglenozoan Calkinsia aureus .
The presence of four recurrent flagella in A. quadriverberis is another distinctive feature. Most cercozoans possess two flagella, although Cholamonas cyrtodiopsidis also has four flagella that are inserted subapically [30, 31]. The flagella of C. cyrtodiopsidis form two symmetrical pairs comprising one long and one stubby flagellum [30, 31]. This flagellar organization differs from A. quadriverberis, which has two pairs of tightly bundled flagella originating from the same flagellar reservoir. Cholamonas cyrtodiopsidis was assigned to the Cercomonadida due to possession of a microbody and kinetid architecture that is similar to some species of Cercomonas [30, 31]. Although both A. quadriverberis and C. cyrtodiopsidis possess four flagella, this character state is unlikely to be synapomorphic for these species: A. quadriverberis inhabits marine sand, whereas C. cyrtodiopsidis inhabits the intestines of diopsid flies . Moreover, the distinctive features present in one species tend not to be shared by the other (e.g. the paranuclear bodies found in C. cyrtodiopsidis are not present in A. quadriverberis). Because the phylogenetic position of C. cyrtodiopsidis has not yet been evaluated with molecular phylogenetic data, our ability to infer the evolution of the tetraflagellated state within the Cercozoa is limited.
The flagella of A. quadriverberis are covered by hairs, and although this stands in contrast to the smooth flagella described in most other cercozoans, such as Cercomonas and Proleptomonas , the hairs could be homologous to those described in the predatory soil-dwelling flagellate Aurigamonas solis [16, 32]. The four flagella of A. quadriverberis were also recurrent and homodynamic during gliding motility, which is unlike the heterodynamic flagella of most other interstitial cercozoans (e.g. Cercomonas, Heteromita, Katabia, Proleptomonas, and Protaspis) [8, 31]. The gliding cells of A. quadriverberis were plastic and capable of slow changes in shape that was somewhat similar to that found in euglenids . This plasticity is probably generated by the row of microtubules locating underneath the cell membrane (Figure 3E).
The nucleus of A. quadriverberis is difficult to see in living cells, which is also unlike most other cercozoans (e.g. Aurigamonas, Cercomonas, Ebria, Euglypha, Heteromita, Protaspis, Thaumatomastix, and Thaumatomonas) [8, 10, 16, 23, 34]. The bloated shape of the cell and the dense distribution of minute orange muciferous bodies that subtend the entire surface of the cell obscured the nucleus. The ultrastructure of the nucleus is similar to that of other cercozoans (e.g. contained several nucleoli) [8, 16, 35–37]; however, A. quadriverberis lacked permanently condensed chromosomes like those found in Cryothecomonas, Ebria, and Protaspis [8, 16, 23, 35, 37, 38]. The shape of the nucleus in A. quadriverberis was indented at one side, a feature also noticed in the nucleus of Protaspis grandis , and had a prominent anterior projection oriented towards the flagellar pocket. An anterior projection was also observed in the nucleus of Cercomonas; in both genera, the anterior projection was associated with a broad striated band and the ventral (posterior) roots of the anterior and posterior flagella (VP) [31, 36]. However, the characteristic microtubular cone present in Cercomonas [31, 36] was not observed in A. quadriverberis.
The cytoplasm of A. quadriverberis contained lipid globules, Golgi bodies and muciferous bodies. The muciferous bodies were compartments organized in linear arrays and filled with an amorphous matrix that appeared bright orange under the light microscope. Extrusomes like these have also been reported in C. armigera as a minute peripheral concavities filled with a homogeneous matrix . Other types of extrusomes that have been found in different cercozoan species, such as trichocysts, microtoxicysts, kinetocysts and osmiophilic bodies, [8, 31, 36], were absent in A. quadriverberis. The lipid globules varied considerably in size and were most abundant in the posterior region of A. quadriverberis. These globules were reminiscent of those described in Protaspis . Although the mode of feeding in A. quadriverberis was not clearly observed, evidence of ingested bacteria was observed within its cytoplasm (Figure 4C).
The cytoplasm of A. quadriverberis was highly vacuolated and looked similar to the cytoplasm described in Cryothecomonas armigera and Protaspis grandis [8, 37]. The anterior part of the cell, however, contained black bodies similar to those that have been observed in other distantly related eukaryotes, such as some semi-anoxic euglenids and ciliates. Moreover, distinct mitochondria with tubular cristae, which are characteristic of other cercozoans, were not found in A. quadriverberis. Putative mitochondria were, however, observed around the cell periphery (Figure 4G), and the lack of cristae in these organelles reflects either degenerate mitochondria associated with a low-oxygen environment or fixation artifact . The size of the putative mitochondria ranged between 135–185 nm long, which is smaller than the mitochondria described in most cercozoans. For example, the mitochondria of Aurigamonas solis are about 630 nm , the mitochondria of Cercomonas are about 485 nm , the mitochondria of Cryothecomonas longipes are about 280 nm , and the mitochondria of P. grandis are about 500 nm . Although the implementation of fluorescent stains, like Mitotracker, could help establish the identity of these structures , this approach is limited by the scarcity of these organisms in natural environments and the unpredictability of finding them in our samples.
Putative primary endosymbionts
Several light orange bodies about 4–14 μm in diam. were distributed within the cell and were especially abundant towards the anterior end of the cell. Although the ultrastructure of these pigmented bodies is novel, the presence of thylakoid-like membranes and a central space containing a densely stained inclusion is consistent with three possible identities that differ by the degree of integration with the host cell: (1) the bodies are ingested (photosynthetic) prey cells that are in the earliest stages of being degraded, (2) the bodies are transient photosynthetic endosymbionts that are continuously replenished by kleptoplasty, or (3) the bodies are permanently integrated photosynthetic endosymbionts (i.e. plastids). The plausibility of each of these hypotheses is addressed below.
The orange color of these bodies is reminiscent of the plastids in some microalgae, such as dinoflagellates and diatoms that occupy the same habitats as A. quadriverberis. However, neither dinoflagellate theca nor diatom frustules were found associated with these bodies in any TEM sections, and the ultrastructure of the bodies was very different from the known ultrastructural diversity in the plastids of diatoms and dinoflagellates. Some cyanobacteria are known to have pale orange coloration that is similar to the orange bodies within A. quadriverberis . These orange bodies were surrounded by two tightly compressed inner membranes and sac-like vesicles. Whereas typical food bodies show degrees of being digested by cellular enzymes, nearly all of the pigmented bodies observed were completely intact in all of the cells we observed (n = 70), suggesting that they are constant fixtures of the host cell cytoplasm.
Primary endosymbiosis, involving a photosynthetic prokaryote within a eukaryotic cell, results in three surrounding membranes: two cyanobacterial inner membranes and a third, outer phagosomal membrane. Green algae/land plants, red algae, and glaucophytes possess primary plastids [43–45]. Two membranes surround the plastids of green algae and red algae, and the third outer phagosomal membrane is inferred to have been lost [43–46]. Secondary endosymbiosis occurs through the engulfment, integration and maintenance of either a green or red alga by a predatory eukaryote. This process produced the plastids of cryptomonads, haptophytes, stramenopiles, dinoflagellates, apicomplexans, and euglenids [43–45]. Two different lineages of cercozoans have independently acquired plastids through endosymbiosis: (1) chlorarachniophytes have secondary plastids derived from green algae [45, 47] and (2) Paulinella chromatophora has primary plastids derived from cyanobacterial prey [48–50].
Like in Paulinella and the cyanelles of glaucophytes, the ultrastructure of the pigmented bodies within A. quadriverberis is most consistent with the ultrastructure of free-living cyanobacteria, suggesting an independent primary endosymbiotic origin [44, 48–52]. For instance, TEM sections through the pigmented bodies demonstrated a mode of division that is similar to division described in the cyanelles of Cyanophora paradoxa  (Figure 5C). Moreover, the thylakoids in the endosymbionts of P. chromatophora, the cyanelles of glaucophytes, and coccoid photosynthetic cyanobacteria are unstacked and arranged concentrically around the periphery of the cell [48, 54, 55]. A similar arrangement was observed in the pigmented bodies of A. quadriverberis (Figure 5A–C), although the majority of the thylakoids projected inward towards the core of the body. The central area within the pigmented bodies of A. quadriverberis resembled the pyrenoids in the cyanelles of Glaucocystis nostochinearum .
The thylakoid-free core of the pigmented bodies also contained polygonal viral particles. TEM sections through these particles demonstrated complete tailed phages similar to those known to infect cyanobacteria [56–58] (Figure 5G). Viral particles similar to those described in the pigmented bodies of A. quadriverberis have also been described in the same region in the plastids of other eukaryotes, such as the "polyhedral bodies" in the primary endosymbionts of P. chromatophora , the cyanelles of the glaucophyte Gloeochaete wittrockiana , and the free-living photosynthetic cyanobacterium Nostoc punctiforme . Two other important characters that have been used to infer a cyanobacterial origin for primary plastids are: (1) the presence of phycobilisomes and (2) the presence of a peptidoglycan wall [48, 49, 51]. However, as previously mentioned, neither phycobilisomes nor a peptidoglycan layer was present in the orange bodies in A. quadriverberis.