Remarkably preserved cysts of the extinct synurophyte, Mallomonas ampla, uncovered from a 48 Ma freshwater Eocene lake

Chrysophyte algae produce a siliceous stage in their life cycle, through either asexual or sexual reproduction, known as a cyst. Cysts form in response to shifts in environmental conditions, population density, or predation pressure, and upon germination provide a seed source for future populations. Cysts are morphologically distinct for each species, and since their remains become part of the sediment or fossil record cysts are valuable tools in ecological and paleolimnological investigations. However, their value as biological indicators is limited because the vast majority of cyst morphotypes have not been linked to specific vegetative species. In the current work, an exquisitely preserved and morphologically complex cyst type is described from a 48 million year old early Eocene fossil site. This finding is remarkable since many of the cysts were still associated with components of the living vegetative cells that produced them, enabling the morphotype to be immediately linked to the synurophyte, Mallomonas ampla. Fusion of identifiable components of the living cell post cyst formation is unknown in modern investigations. The identification of the cyst structure for M. ampla could be valuable in determining cyst morphotypes for other species in the lineage.


Site Description
Details of the Giraffe Pipe fossil locality are given in previous works e.g. [26][27][28] and briefly summarized here. The Giraffe Pipe locality is a kimberlite diatreme that was emplaced into the Slave Craton in the Northwest Territories of Canada (64°44′N, 109°45′W) approximately 48 million years ago during the Eocene. The diatreme crater filled with water, becoming a maar lake, and subsequently infilled over thousands of years with a sequence of lacustrine and eventually paludal sediments. The sediment strata were later capped by Neogene glacial deposits, entombing them within the crater. A 163 m long drilled core, collared at a 47° angle, was uncovered from the Giraffe maar in 1999 by BHP Billiton Inc. A 113.1 m portion of the core contains well preserved stratified organic sediment, including 68.3 m of lacustrine mudstones, overlain with 44.8 m of peaty and terrestrial remains. An air-fall tephra bed located near the end of the aquatic phase indicates that the lake sediments were all deposited during the Eocene 28 .
This region of the Arctic was warm and wet compared to today, with reconstructed mean annual temperature and mean annual precipitation values 17 °C higher and 4 times greater, respectively, than present, and the region supported a warm mixed forest 28 . The entire lacustrine phase of the core contains an extensive array of microfossils which reflect a lake teaming with organisms, including chrysophytes, synurophytes, diatoms, euglyphids, heliozoans and sponges 26,[29][30][31][32][33] . Shifts in the complement of organisms indicate changing lake conditions, including between alkaline and acidic periods, and shallow versus deep phases 32 . The presence of synurophytes, diatoms and sponge lineages found today largely in tropical regions reflect the warm climate of the region 26 . In addition, the remains of palm trees indicate ice-free conditions and winter temperatures above freezing 34 . The current hypothesis is that post phreatomagmatic kimberlite emplacement, a waterbody formed within the crater, varying in depth and physical attributes over time, slowly infilling, and eventually transitioning to a terrestrial environment.

Materials and Methods
Samples for this study were obtained from seven sections of a drilled core taken from the Giraffe Pipe locality. Samples from this core are identified using a three-part number 29 . The first number represents the core box in which the sample is stored. Core boxes are numbered sequentially starting with box 1 (closest to the land surface), and increasing with depth in the core. The larger the number, the deeper the section is within the core. Box 11 represents the top and end of the lacustrine phase within the sequence. Each box contains three 1.5 m core lengths, stored in channels 1, 2 and 3. The second number represents the channel. The third number is the measurement in cm down from the top of a core length within the channel. For example, sample 19-1-100 represents a sample taken from 100 cm down along the core length positioned in channel 1 from box 19. This study includes samples from 17-1-15, 17-2-40, 17-2-94, 17-2-138, 19-1-100, 19-2-98 and 19-2-100.
From 50-100 mg of mudstone was retrieved from each section and oxidized using 30% H 2 O 2 under low heat for a minimum of an hour, and longer if rock fragments remained mostly intact 23,29 . Samples were rinsed with distilled water a minimum of five times with centrifugation, and the final slurries stored in glass vials at 4 °C. For analysis with scanning electron microscopy (SEM), an aliquot of each slurry was air dried onto a piece of heavy duty aluminum foil, trimmed, and attached to an aluminum stub with apiezon wax. Samples were coated with a mixture of gold and palladium for 2 min with a Polaron Model E sputter coater and observed with a Leo (Zeiss) 982 FESEM, or a FEI Nova NanoSEM 450, field emission scanning electron microscope 23,29 .
Details of the cyst are as described by the guidelines prepared by the International Statospore Working Group 35 . Measurements of the diameters of cysts, pores, collars and spines were made directly from SEM micrographs. Measurements of the pore and collar were made on specimens where these structures were facing straight up. Specimens were not tilted. Spine lengths were estimated from ones along the midsection positioned parallel with the stub surface. (2020) 10:5204 | https://doi.org/10.1038/s41598-020-61993-1 www.nature.com/scientificreports www.nature.com/scientificreports/

Results
Large concentrations of an unidentified cyst were uncovered in seven samples from the Giraffe Pipe core, with especially abundant numbers of specimens found in samples from stratum 19-1-100. Many of the cysts from 19-1-100 were unique in that they still contained attached scales, allowing identification as Mallomonas ampla Siver & Lott. (Fig. 1a-c). Scales of M. ampla are oval with a perforated base plate, posterior rim, V-rib and a shallow dome structure (Fig. 1a-c). Scales are large and can range upwards in length to 7 µm 23 . The posterior rim, arms of the V-rib, and anterior submarginal ribs are all thinly constructed. The posterior rim is especially shallow and typically extends slightly further along one margin. Base plate pores cover the entire scale, are more or less evenly spaced, and form distinctive concentric rows in the posterior flange. The base of the V-rib can be strongly hooded, but is thin, and the V-rib arms are continuous with the anterior submarginal ribs. On scales with broader domes, the anterior submarginal ribs are often extended into short wing-like structures. The dome is shallow, usually broadly oval, and set back from the anterior margin. The shield and dome are covered with numerous, small, evenly spaced papillae. The posterior flange may possess scattered papillae, but this portion of the scale generally lacks numerous papillae. Cyst description. Cysts are large, spherical with a mean diameter of 19.9 ± 1.3 µm, covered with spines, and with a wide flattened collar that is surrounded by a ring of spines (Figs. [2][3][4]. With the exception of the spines, the cyst wall is smooth. The majority of spines range in length from 0.5-0.9 µm, have a wide base, decrease in diameter with distance from the cyst wall, and terminate with a splayed apex consisting of a series of finger-like projections that are more or less parallel with the cyst wall ( Fig. 3a,e,f). On most spines the splayed apices consist of five to ten projections, each of which may further bifurcate. Although spines are solid, there is a depression on the apical end within the center of the terminal projections. Spines are widely spaced and occur singularly, or often in groups of two and rarely three or four. Spines in the anterior hemisphere in the vicinity of the collar are often longer, more slender, with bifurcate apices; these spines have a mean length of 1 µm and lack heavily splayed tips (arrows on Fig. 3a,e). The pore is small, circular, with slightly conical sides and a mean diameter of 1.3 ± 0.1 µm (Fig. 4a-f). The collar is wide with a mean diameter of 4.5 ± 0.3 µm, circular, apically flattened and fused with the cyst wall ( Fig. 4a-f). The outer margin of the collar is slightly thickened and often lined with a series of siliceous nodules of twisted projections (Fig. 4a-f). The collar is, in turn, surrounded by a ring of long slender spines that project slightly away from the collar; most of these spines have bifurcate or trifurcate apices.

Scales of Mallomonas ampla
Variation in cyst characters. Although the diameter of cysts ranged from 17-23 µm (n = 34), 29 were between 19-21 µm (Fig. 5a). The diameter of the collar (Fig. 5b) and pore (Fig. 5c) were also relatively stable ranging from 4-5 µm and 1-1.5 µm, respectively. A few cysts had spines with highly splayed and elongated finger projections that often bend downward, sometimes resting on the cyst wall (Fig. 3f). On most cysts, the bases of the spine forming the ring that surrounds the collar were closely spaced and often fused together (Fig. 4a-c,e). Sometimes the spines become fused over half their length, forming what could be considered as a secondary collar (Fig. 4a). On the other end of the spectrum, on a few specimens the spines were fewer and more widely spaced (e.g. Fig. 4d), or not even juxtaposed to the collar itself (e.g. Fig. 4f).
Additional observations. Scales from the forming vegetative cell, and in their original orientation, were fused in place onto most cysts. Although ribbed bristles with the unique hooked foot characteristic of M. ampla were abundant in the rock samples, only broken pieces were found on cysts (Fig. 3d). There were also a number of cysts www.nature.com/scientificreports www.nature.com/scientificreports/ in cleaned preparations that appeared to be at least partially surrounded by an additional cyst (Fig. 6a-d). That is, a cyst within a cyst. The inner cyst on most, if not all, of these specimens had attached scales, and in all cases both cyst morphotypes represented those of M. ampla. On some of the specimens, the outer cyst appeared to encircle more than half of the outer circumference of the inner cyst (e.g. Fig. 6a).
Museum specimens. The type specimen for Mallomonas ampla is from section GP 17-2-94 of the Giraffe Pipe core and deposited at the Canadian Museum of nature (CANA 85744). As a result of the current project, material from the GP 19-1-100 stratum and two permanent glass slide mounts were also deposited at the Canadian Museum of nature (CANA 128453). Each slide has numerous specimens of the cyst for M. ampla. They also contain specimens of the rare diatom genus Ambistria which is detailed in a separate publication 31 .

Discussion
Of the vast number of cyst morphotypes described from studies around the world, only 5% or fewer are linked to specific species 6 . This fact severely limits the usefulness of cysts in ecological, biogeographic and paleolimnological investigations. The limited number of cysts that have been successfully linked to specific vegetative states were done so through direct observation of living organisms in field collections, or induced from cultures in laboratory www.nature.com/scientificreports www.nature.com/scientificreports/ experiments 6 . Cysts uncovered from sediments are no longer associated with the living cells that produced them, making it impossible to assign them directly to specific organisms 8 . No cyst morphotypes described from sediment samples have ever been found still associated with siliceous scales from the cell covering. This fact is what makes the findings reported here for the Giraffe locality truly remarkable.
All samples from the Giraffe locality containing cysts of Mallomonas ampla also had isolated scales of this species, but only specimens from the 19-1-100 stratum still had attached scales allowing for a positive identification. This is extraordinary since specimens were initially treated with oxidizing acids in order to remove them from the rock matrix. Even after treatment with acids, scales remained fused to the cysts in their original orientations. www.nature.com/scientificreports www.nature.com/scientificreports/ This indicates that not only did the cells forming cysts remain intact upon death, but that the scales became fused together and with the cyst wall during the fossilization process. This phenomenon is unique for fossil remains from the Giraffe locality, but it is not unique for M. ampla. Cysts of other species with intact scales have also been uncovered from the Giraffe locality, indicating that the conditions in the sediments and overlying water column of this maar lake that resulted in the cells remaining intact were unusual, possibly highly anoxic and undisburbed. Scales still attached to the cysts of another species, Mallomonas aperturae Siver, were so well preserved that the original arrangement of the scales on the cell covering could be critically examined 36 . The same situation is now documented for M. ampla from a different section of the extensive core, and in this case with a significantly greater number of specimens.
The specimens of cysts partially surrounded by a second cyst are curious and perplexing. The most logical explanation is that during the fossilization process the inner cysts became compressed against the outer cyst, breaking through the outer cyst, and ending up within the rock matrix appearing to be within the outer cyst. However, in some cases the outer cyst appears to encircle more than half of the diameter of the inner cyst, which could not be explained by this compression hypothesis. In addition, the inner cysts were never found surrounded by ones other than those representing M. ampla, despite the presence of many other cyst morphotypes in the sample rock. A second explanation is that after formation, what would be the outer cyst "germinated" internally forming a cell with scales, which, in turn, produced the inner cyst. This phenomenon has never been observed and seems unlikely, yet could explain those specimens where more than half of the circumference of the inner cyst is surrounded by the outer cyst. It is also interesting as remarkable that it is to have uncovered cysts with scales still attached in their original overlapping patterns, it is important to note that these "triple" fossil specimens depicting an inner cyst-attached scale coat-outer cyst also remain attached and fused to each other. Does this indicate that the three units were indeed formed together, supporting the internal germination idea? None of these specimens were found attached to a third outermost cyst which would seem likely under the compression hypothesis.
In a recent literature survey that included 1409 records of modern cysts (chrysophytes and synurophytes), Siver 37 reported a range in diameter from 2-30 µm, with the majority of cyst types between 5-10 µm. This matches nicely cyst diameter estimates of ca. 3-35 μm given by previous researchers 8,13,38 . In addition, the range in diameter for cyst specimens formed by the same species was remarkably small with 85% of the species forming cysts with a range in diameter of approximately 3 µm or less 37 . This was especially true for taxa with scale forming cell coverings. Some of the variation in cyst diameter is likely due to differences in the sizes of vegetative cells at the time of formation 6 . Compared to the results reported by Siver 37 , the cyst formed by Mallomonas ampla is on Figure 6. Scanning electron micrographs of Mallomonas ampla cysts from the Giraffe Pipe locality that appear to be contained within slightly larger, or "parent, " cysts. Inner cysts in (a,b,d) have attached scales, while no scales are observed on the cyst in (c). The broken outer cyst in (a) appears to encircle more than 50% of the circumference of the inner cyst, and the one in (c) at least 50% of the inner cyst diameter. Scale bars = 5 µm.
www.nature.com/scientificreports www.nature.com/scientificreports/ the larger end of the spectrum, and the range in diameter is consistent with the results reported in that study. In addition, the diameters of the collar and of the pore were both remarkably consistent, yielding a mean ratio of 3.5 ± 0.5. The ratio of cyst diameter to pore diameter was large with a mean of 16 ± 0.8. The small size of the pore relative to the cyst is remarkable since when the cyst germinates, the newly formed (and naked) cell must squeeze out of the pore.
Compared to the structure of the collar and pore, there was a wide range of variation in the character of the spines, both within and between specimens. Most of the variation was in the length of the spine and the degree to which the apex was splayed into the finger projections. Shorter spines with more complex apices are the most common morphology and these spines typically cover the majority of the cyst wall. Longer and more slender spines with less splayed apices were restricted to the anterior hemisphere and usually near the collar complex. A hypothesis explaining the variability in spine morphology is that spine length and the degree of lateral splaying of the spine apex for chrysophytes with a siliceous scale covering is dependent on the distance from the developing cyst to the confining cell covering. Formation of spines begins from the cyst wall and progresses outwards. As spines lengthen in an organism with a confining outer scale covering, they come up against the scale coat causing the silica deposition process to bend laterally, resulting in the splayed structure of the apex. Specimens with scales still intact showing splayed apices in contact with the scales clearly supports this hypothesis. Under this hypothesis, the shorter the distance from the cyst wall to the scale covering, the smaller would be the length of the spine. The majority of synurophytes have oval or obovate-shaped cells 1,25,39 . Most cysts are spherical and when they form the anterior hemisphere usually faces the anterior end of the cell 1 . This means that if the cell has an oval shape, that the distance between the developing cyst and scale covering would be greater extending from the anterior hemisphere, and less between the sides and posterior portions of the cyst. Thus, there would be more space for development of longer spines and more complex and projecting collars on the anterior hemisphere, yielding longer spines with no or less splayed apices.
Although the structures of cysts are unknown for many species bearing scale coverings, there is support for the confinement hypothesis resulting in splayed spine apices. At least four other species of Mallomonas are known to produce cysts with various types of splayed apices. These include Mallomonas muskokana (Nicholls) Siver & Wolfe, M. acaroides var. acaroides Perty emend. Ivanov, M. pseudocoronata Prescott, and M. crassisquama (Asmund) Fott. Siver 40 reported an almost identical spine structure for M. muskokana as reported here for M. ampla. Kristiansen 25 noted the spines of M. acaroides var. acaroides to be "irregularly curved with blunt disc-like tips, which are surrounded by a whorl of short, thin twisted projections". This describes spines referred to herein as those with splayed apices. Kristiansen 25 further notes that the cyst formed by M. crassisquama has spines similar to those of M. acaroides var. acaroides. Spines of M. pseudocoronata are described as slightly curved with flat splayed tips 25,41 . Interestingly, a similar type of spine spaying is found on the ends of linking spines of some species of the diatom genus Aulacoseira (e.g. A. crassipunctata and A. lirata 42 ). In this case, developing spines become splayed laterally into free spaces as they come in contact with confining silica structures on adjoining valves.
Knowing the morphological structure of M. ampla cysts could prove useful in discovering the cyst morphotypes for the two closely related species in this lineage, M. multisetigera and M. neoampla. Collections containing these species, or surface sediments from waterbodies with active populations, could be examined for cysts with similar spine and collar characters as those for M. ampla. Although matches made using this technique would only provide indirect evidence, it would nonetheless be worthwhile evidence.
In summary, the numerous specimens of M. ampla cysts with attached scales from the cell covering in the Giraffe Pipe core are remarkable and further emphasize the uniqueness of this fossil locality. The morphology of the cyst, especially the complex collar, is unique among known cyst types, and the splayed nature of the spine tips is likely a function of forming within a space confined by the scales of the cell covering.

Data availability
All data generated for this study is included in this publication. Raw material from stratum 19-1-100 of the Giraffe Pipe core and permanent prepared glass slides with numerous specimens have been deposited at the Canadian Museum of Nature and can be borrowed on request.