5.a. Palaeoecology

The Ediacaran microfossils occur in various facies in shallow to offshore platform and slope settings, which represent holomarine environments (Grey, Reference Grey2005; Jiang et al. Reference Jiang, Shi, Zhang, Wang and Xiao2011; Moczydłowska & Nagovitsin, Reference Moczydłowska and Nagovitsin2012; Anderson et al. Reference Anderson, Macdonald, Jones, McMahon and Briggs2017), and many are cosmopolitan. Such distribution is typical of extant phytoplankton (algae and bacteria) that may be passively dispersed globally by ocean gyres and currents over a short time of a few thousand years (Reynolds, Reference Reynolds2006). Wide geographic distribution is also known among zooplankton (Lipps, Reference Lipps1993; Garrison, Reference Garrison1999) but their cyst morphology is dissimilar to the studied microfossils (compare Porter, Reference Porter, Xiao and Kaufman2006; Bosak et al. Reference Bosak, Macdonald, Lahr and Matys2011; Morais et al. Reference Morais, Fairchild, Lahr, Rudnitzki, Schopf, Garcia, Kudryavtsev and Romero2017). Studied microfossil species are cosmopolitan (with the exception of T. spinosa so far as is known) and facies-independent which is consistent with the phytoplankton (see also Grey, Reference Grey2005; Moczydłowska, Reference Moczydłowska2005; Liu & Moczydłowska, Reference Liu and Moczydłowska2019).

5.b. Wall biochemical properties

Various species of Appendisphaera, Urasphaera and Tanarium, including those studied herein, have been previously extracted from the sedimentary rocks and preserved as 3D and robust vesicles comprising organic matter with biochemical decay resistance, a property evidenced by organic matter survival through hundreds of millions of years of geologic history, and its negative reaction to HF acid upon extraction (Moczydłowska et al. Reference Moczydłowska, Vidal and Rudavskaya1993; Grey, Reference Grey2005; Moczydłowska, Reference Moczydłowska2005, Reference Agić, Moczydłowska and Canfield2016; Vorobeva et al. Reference Vorobeva, Sergeev and Knoll2009; Moczydłowska & Nagovitsin, Reference Moczydłowska and Nagovitsin2012). Species of Tianzhushania and Mengeosphaera have not yet been extracted from the sedimentary rocks as organically preserved microfossils, but their 3D preservation shown by circular outlines of vesicles that survived the early diagenesis and permineralization by silica and phosphate without collapse as observed in thin-sections (Liu et al. Reference Liu, Xiao, Yin, Chen, Zhou and Li2014; Xiao et al. Reference Xiao, Zhou, Liu, Wang and Yuan2014; Shang et al. Reference Shang, Moczydłowska, Liu and Liu2018; Liu & Moczydłowska, Reference Liu and Moczydłowska2019) indicates mechanical and chemical resistance. Although the fossil biopolymers are usually transformed to more recalcitrant components during diagenesis and their chemical composition may not be original, even fossil molecules (biomarkers) and traces of biopolymers could be detected in primary composition without full fossil structures in favourable conditions (Briggs & Summons, Reference Briggs and Summons2014). Specific conditions, such as aluminosilicate and kaolinite mineral coating, may stabilize organic matter and facilitate preservation of organic fossils (Anderson et al. Reference Anderson, Tosca, Cinque, Frogley, Lekkas, Akey, Hughes, Bergmann, Knoll and Briggs2020). These previously studied (Moczydłowska et al. Reference Moczydłowska, Vidal and Rudavskaya1993; Grey, Reference Grey2005; Moczydłowska, Reference Moczydłowska2005, Reference Agić, Moczydłowska and Canfield2016; Moczydłowska & Nagovitsin, Reference Moczydłowska and Nagovitsin2012; Liu et al. Reference Liu, Xiao, Yin, Chen, Zhou and Li2014; Xiao et al. Reference Xiao, Zhou, Liu, Wang and Yuan2014; Shang et al. Reference Shang, Moczydłowska, Liu and Liu2018, Reference Shang, Liu and Moczydłowska2019; Liu & Moczydłowska, Reference Liu and Moczydłowska2019) and present microfossils derive from sediments that have undergone mild diagenesis and low thermal maturity, suggesting limited potential for organic biopolymer change over time. Consequently, the original organism biopolymers likely had similar decay resistance to those presently comprising the microfossils. Very few microfossil taxa have been studied as to their geochemical composition but, in general, phytoplankton microfossils are decay-resistant due to their cyst wall properties and therefore are abundantly preserved (Evitt, Reference Evitt1985; Colbath & Grenfell, Reference Colbath and Grenfell1995; Kokinos et al. Reference Kokinos, Eglinton, Goni Mam Boon, Martoglio and Anderson1998; Arouri et al. Reference Arouri, Greenwood and Walter2000; Marshall et al. Reference Marshall, Javaux, Knoll and Walter2005; Briggs & Summons, Reference Briggs and Summons2014). Ediacaran Tanarium conoideum examined by micro-Fourier transform infrared (FTIR) spectroscopy showed spectra that are consistent with those obtained from algaenans isolated from extant chlorophyte and eustigmatophyte microalgae (Marshall et al. Reference Marshall, Javaux, Knoll and Walter2005). The studied Tanarium species preserved not only resistant cyst wall but also internal cells.

The wall resistance properties of the studied microfossils are known among the algaenan, mannan, sporopollenin, cellulose, cutan and chitin groups of biopolymers and among these, the first three groups are known in algal cysts (Atkinson et al. Reference Atkinson, Gunning and John1972; Evitt, Reference Evitt1985; Derenne et al. Reference Derenne, Largeau, Berkalo, Rousseau, Wilhelm and Hatcher1992 a, b, Reference Derenne, Largeau and Berkalo1996; Gelin et al. Reference Gelin, Volkman, Largeau, Derenne, Sinninghe Damsté and De Leeuw1999; Allard & Templier, Reference Allard and Templier2000; Hagen et al. Reference Hagen, Siegmund and Braune2002; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006; De Leeuw & Largeau, Reference De Leeuw, Largeau, Engel and Macko2006; De Leeuw et al. Reference De Leeuw, Largeau, Versteegh and Van Bergen2006). Sporopollenin, cellulose and cutan are synthesized by plants (Evitt, Reference Evitt1985; Buchanan et al. Reference Buchanan, Gruissem and Jones2000), which originated from algae that acquired chloroplasts from their ancestral cyanobacteria (Delwiche, Reference Delwiche1999; Raven et al. Reference Raven, Evert and Eichhorn2005; Keeling, Reference Keeling2010; Adl et al. Reference Adl, Bass, Lane, Lukes, Schoch, Smirnov, Agatha, Berney, Brown, Burki, Cárdenas, Cepicka, Chistyakova, del Campo, Dunthorn, Edvardsen, Eglit, Guillou, Hampl, Heiss, Hoppenrath, James, Karnkowska, Karpov, Kim, Kolisko, Kudryavtsev, Lahr, Lara, Le Gall, Lynn, Mann, Massana, Mitchell, Morrow, Soo Park, Pawlowski, Powell, Richter, Rueckert, Shadwick, Shimano, Spiegel, Torruella, Youssef, Zlatogursky and Zhang2019), and cellulose is synthesized by certain cyanobacteria (Römling & Galperin, Reference Römling and Galperin2015). All these biopolymers are produced by photosynthesizing organisms. Chitin is polymerized by rhizaria, fungi, protistan holozoans and animals (Webster & Weber, Reference Webster and Weber2007; Gupta, Reference Gupta2011; Taylor et al. Reference Taylor, Krings and Taylor2015; Torruella et al. Reference Torruella, de Mendoza, Grau-Bové, Antó, Chaplin, del Camplo, Eme, Pérez-Cordón, Whipps, Nichols, Paley, Roger, Sitjà-Bobadilla, Donachie and Ruiz-Trillo2015; Loron et al. Reference Loron, Francois, Rainbird, Turner, Borensztajn and Javaux2019). The resistant compounds were recognized as chitin in fungal microfossils at c. 1.0–0.9 Ga (Loron et al. Reference Loron, Francois, Rainbird, Turner, Borensztajn and Javaux2019), but these microfossils have no morphologic comparison to those studied here. Chitin commonly occurs in animal integuments, but chitin is also found in the egg cysts of only a few known taxa of derived phyla among the invertebrates (nematods, tardigrades, and arthropods including crustaceans and insects; Scholtz & Wolff, Reference Scholtz, Wolff, Minelli, Boxhall and Fusco2013). The fossil record indicates that these invertebrate groups evolved in the Cambrian and insects in the Devonian (Maas & Waloszek, Reference Maas and Waloszek2001; Engel & Grimaldi, Reference Engel and Grimaldi2004; Erwin & Valentine, Reference Erwin and Valentine2013). Based on molecular clock analysis, the origin of crown group animals is suggested to occur in the Tonian–Cryogenian interval at c. 833–650 Ma, yet a precise timeline of animal evolution cannot be currently obtained (dos Reis et al. Reference Dos Reis, Thawornwattana, Angelis, Telford, Donoghue and Yang2015). This estimate is approximate and the minimum age is not much older than the fossil records for the emergence of major animal phyla in the terminal Ediacaran and Cambrian (Sperling et al. Reference Sperling, Pisani, Peterson, Vickers-Rich and Komarower2007; Budd, Reference Budd2008). The above-considered phyla likely evolved in this transitional interval. Conversely, the Ordovician metazoan egg cases of ‘chitinozoans’ are not made of chitin (Jacob et al. Reference Jacob, Paris, Monod, Miller, Tang, George and Beny2007). Thus, among photosynthesizing clades in the Ediacaran time (Moczydłowska, Reference Moczydłowska2008b, Reference Agić, Moczydłowska and Canfield2016; Butterfield, Reference Butterfield2015), cyanobacteria and algae are the most likely to have produced resistant cyst walls. Although filamentous cyanobacteria produce heterocysts and akinetes with thick walls that are preservable and resistant, they lack ornamentation. The algal cysts are the best candidates because their morphology is characteristic and recognizable among the microfossils studied.

The biochemical synthesis pathway of decay-resistant, refractory biopolymers in the algal cyst wall is thought to be a shared ancestral (symplesiomorphic) character of phylogenetic lineages of basal chlorophytes and derived streptophytes leading to plants (Raven et al. Reference Raven, Evert and Eichhorn2005; Falkowski & Raven, Reference Falkowski and Raven2007; O’Kelly, Reference O’Kelly, Falkowski and Knoll2007; Baldauf, Reference Baldauf2008; Turmel et al. Reference Turmel, Brouard, Gognon, Otis and Lemieux2008; Burki et al. Reference Burki, Roger, Brown and Simpson2020). Cellulose, the most abundant biopolymer on Earth, is exceptionally resistant and is produced by plant cellulose synthase complexes; these enzyme complexes have a cyanobacterial origin and have been genetically inherited from cyanobacterial ancestors that became chloroplasts in algae and then in plants (Römling & Galperin, Reference Römling and Galperin2015). Enzymes that bind specific compounds during bacterial photosynthesis are present in all photosynthesizing organisms (David & Alm, Reference David and Alm2011); these organisms include some bacteria, algae and embryophytes (Raven et al. Reference Raven, Evert and Eichhorn2005). Among green algae, the phenotypic cyst characters are expressed in various clades of chlorophytes and streptophytes (the group Chloroplastida; Adl et al. Reference Adl, Bass, Lane, Lukes, Schoch, Smirnov, Agatha, Berney, Brown, Burki, Cárdenas, Cepicka, Chistyakova, del Campo, Dunthorn, Edvardsen, Eglit, Guillou, Hampl, Heiss, Hoppenrath, James, Karnkowska, Karpov, Kim, Kolisko, Kudryavtsev, Lahr, Lara, Le Gall, Lynn, Mann, Massana, Mitchell, Morrow, Soo Park, Pawlowski, Powell, Richter, Rueckert, Shadwick, Shimano, Spiegel, Torruella, Youssef, Zlatogursky and Zhang2019; Burki et al. Reference Burki, Roger, Brown and Simpson2020) and the endosymbiotically derived green lineage of dinoflagellates among alveolates (Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989; Keeling, Reference Keeling2004; Raven et al. Reference Raven, Evert and Eichhorn2005; Falkowski & Raven, Reference Falkowski and Raven2007; Fehling et al. Reference Fehling, Stoecker, Baldauf, Falkowski and Knoll2007; O’Kelly, Reference O’Kelly, Falkowski and Knoll2007; Graham et al. Reference Graham, Graham and Wilcox2009; Leliaert et al. Reference Leliaert, Smith, Moreau, Herron, Verbruggen, Delwiche and De Clerck2012). In the Chloroplastida that have primary plastid originated directly from cyanobacterium, this common origin implies a strong morphological/cell biological synapomorphy (Burki et al. Reference Burki, Roger, Brown and Simpson2020). Alongside biochemical synthesis and certain enzymes acquired from early photosynthesizing ancestors, the genetic toolkits for reproduction and zygotic cyst formation were conceivably inherited and shared within the ‘green’ lineages of algae (including photosynthesizing dinoflagellates).

5.c. Cell division: palintomy

Palintomic cell division, the process during which a parental cell or zygote undergoes a rapid sequence of repeated divisions that result in decreased size of cells (Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989, p. 778), has been observed in all microfossils studied here and some previously interpreted as animal embryos or holozoans (Xiao & Knoll, Reference Xiao and Knoll2000; Huldtgren et al. Reference Huldtgren, Cunningham, Yin, Stampanoni, Marone, Donoghue and Bengtson2011; but see Butterfield, Reference Butterfield2011; Donoghue et al. Reference Donoghue, Cunningham, Dong, Bengtson and Wanninger2015; Cunningham et al. Reference Cunningham, Vargas, Yin, Bengtson and Donoghue2017; Section 6.c). Palintomy in reproductive stage is common to unicellular green algae, protistan holozoans and metazoans but only at the early developmental stages up to 16-cell metazoan embryos (Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989, pp. 610, 632; Mathews, Reference Mathews1986, pp. 24–5, 30–1; Gilbert & Raunio, Reference Gilbert and Raunio1997; Jurd, Reference Jurd2004; Lee, Reference Lee2008, pp. 192, 214, 217; Nielsen, Reference Nielsen2012; Leadbeater, Reference Leadbeater2015, p. 61). Looking at the pattern of cell divisions at the early stages alone, the microfossils cannot be discriminated between these clades. At the later ontogenetic stages with multiple cells that are identical and randomly clustered, microfossils may be considered among protistan holozoans and algae (Butterfield, Reference Butterfield2011; Huldtgren et al. Reference Huldtgren, Cunningham, Yin, Stampanoni, Marone, Donoghue and Bengtson2011). The decisive features in favour of either of these two are the cyst morphology (see Section 5.d) and, if possible to detect, the biochemistry or cyst wall properties.

The palintomy that is observed in Appendisphaera grandis, Mengeosphaera bellula and Tianzhushania spinosa is evident in cysts of the same size that contain one or four to multiple, and in Tianzhushania thousands of, identical cells (Figs 2a–c and 3a, b, e, f). Palintomic division is observed during sporogenesis in algae as well as in protistan holozoans and metazoans (Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Jurd, Reference Jurd2004; Butterfield, Reference Butterfield2011; Huldtgren et al. Reference Huldtgren, Cunningham, Yin, Stampanoni, Marone, Donoghue and Bengtson2011). However, in algae and protistan holozoans, this process leads to formation of morphologically identical offspring cells (which may be very numerous; Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Lee et al. Reference Lee, Leedale and Bradbury2000; Graham et al. Reference Graham, Graham and Wilcox2009; Leadbeater, Reference Leadbeater2015). In metazoans, palintomic division occurs only at the morula and early blastula stages, and thereafter the cells are programmed to follow routes to specification (Kessel & Shih, Reference Kessel and Shih1974; Gilbert, Reference Gilbert2010; Nielsen, Reference Nielsen2012; Shilo, Reference Shilo2014). In progressing cell divisions, the cells are differentiated in shape and size, oriented into poles, asymmetrically segregated and aligned and grow into tissues. In animal embryos, cell differentiation, their orientation and polarization begin from the stage of 16 cells and after the third round of cell divisions (Anderson, Reference Anderson1973; Kessel & Shih, Reference Kessel and Shih1974; Mathews, Reference Mathews1986; Gilbert & Raunio, Reference Gilbert and Raunio1997; Jurd, Reference Jurd2004; Gilbert, Reference Gilbert2010; Nielsen, Reference Nielsen2012; Shilo, Reference Shilo2014; Gross et al. Reference Gross, Treffkorn, Mayer and Wanninger2015). Even in tardigrades, which may not have obvious blastocoel and in which the sterroblastula is a ball of cells, the cells are differentiated in shape (Gross et al. Reference Gross, Treffkorn, Mayer and Wanninger2015; Levin et al. Reference Levin, Anavy, Alison, Cole, Winter, Mostov, Khair, Senderovich, Kovalev, Silver, Feder, Fernandez-Valverde, Nakanishi, Simmons, Simakov, Larsson, Liu, Jerafi-Vider, Yaniv, Ryan, Martindale, Rink, Arendt, Degnan, Degnan, Hashimshony and Yanai2016). The animal fertilization membrane or egg cyst is discarded and the embryo grows from a blastocyst to a gastrula stage and then into a larva (Mathews, Reference Mathews1986; Gilbert & Raunio, Reference Gilbert and Raunio1997; Jurd, Reference Jurd2004; Gilbert, Reference Gilbert2010). This developmental pattern is the current embryology dogma without exception in extant animals of any phylogenetic position, from sponges and cnidarians to higher phyla (Fig. 5 further below). None of the microfossils studied here or those previously inferred to be animal embryos (Tianzhushania and its putative developmental stages Megasphaera, Parapandorina and Megaclonophycus; Xiao & Knoll, Reference Xiao and Knoll2000; C Yin et al. Reference Yin, Bengtson and Yue2004; L Yin et al. Reference Yin, Zhu, Knoll, Yuan, Zhang and Hu2007, Reference Yin, Zhou and Yuan2008; Chen et al. Reference Chen, Xiao, Pang, Zhou and Yuan2014) have ever shown the presence of a blastocoel or gastrocoel or cell differentiation, which are animal embryonic characters, even in the thousands of cells stages (Hagadorn et al. Reference Hagadorn, Xiao, Donoghue, Bengtson, Gostling, Pawlowska, Raff, Raff, Turner, Yin, Zhou, Yuan, McFeely, Stampanoni and Nealson2006; Huldtgren et al. Reference Huldtgren, Cunningham, Yin, Stampanoni, Marone, Donoghue and Bengtson2011; Chen et al. Reference Chen, Xiao, Pang, Zhou and Yuan2014; Donoghue et al. Reference Donoghue, Cunningham, Dong, Bengtson and Wanninger2015; Cunningham et al. Reference Cunningham, Vargas, Yin, Bengtson and Donoghue2017). Cell differentiation claimed to occur in Megaclonophycus-like microfossils (Chen et al. Reference Chen, Xiao, Pang, Zhou and Yuan2014) is not substantiated and this record is alternatively interpreted (Tang, Reference Tang2016; see Section 6.c). New specimens of A. grandis, M. bellula and T. spinosa (Figs 2 and 3) preserved in the late developmental stages provide the decisive evidence to dismiss the animal embryo interpretation for Tianzhushania (Xiao & Knoll, Reference Xiao and Knoll2000; Yin et al. Reference Yin, Bengtson and Yue2004) and Appendisphaera (Yin et al. Reference Yin, Zhu, Knoll, Yuan, Zhang and Hu2007). Mengeosphaera is not different in this respect.

Palintomic cell division was inferred in peanut-shaped microfossils that were attributed to the protistan holozoan Tianzhushania life cycle (Huldtgren et al. Reference Huldtgren, Cunningham, Yin, Stampanoni, Marone, Donoghue and Bengtson2011; Donoghue et al. Reference Donoghue, Cunningham, Dong, Bengtson and Wanninger2015; Cunningham et al. Reference Cunningham, Vargas, Yin, Bengtson and Donoghue2017) and resulted in forming thousands of tightly arranged cells. However, these cells are not identical and are aligned into layers in the peripheral portion of the microfossil. This appears not to be strictly palintomic division, or the layers were formed after the palintomy ceased. In any case, the cell differentiation and layering in peanut-shaped microfossil is very different from that of Tianzhushania spinosa, and does not belong to the same life cycle (see section 6.c).

5.d. Cyst morphology

Cyst morphology, alongside pattern of cell division and cyst wall biochemistry, is a characteristic phenotypic feature. Morphologically complex cysts as observed in the studied microfossils are well-known among green microalgae (Bold & Wynne, Reference Bold and Wynne1985, pp. 93, 143, 167; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995, pp. 352, 471; Hagen et al. Reference Hagen, Siegmund and Braune2002; Raven et al. Reference Raven, Evert and Eichhorn2005, pp. 296, 327, 337; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006; Lee, Reference Lee2008, pp. 152, 192–3; Graham et al. Reference Graham, Graham and Wilcox2009, pp. 434–5; Van Westen, Reference Van Westen2015; Figs 4 and 5) but are not produced by protistan holozoans (Leadbeater, Reference Leadbeater2015, pp. 47, 49, 61; Torruella et al. Reference Torruella, de Mendoza, Grau-Bové, Antó, Chaplin, del Camplo, Eme, Pérez-Cordón, Whipps, Nichols, Paley, Roger, Sitjà-Bobadilla, Donachie and Ruiz-Trillo2015; Adl et al. Reference Adl, Bass, Lane, Lukes, Schoch, Smirnov, Agatha, Berney, Brown, Burki, Cárdenas, Cepicka, Chistyakova, del Campo, Dunthorn, Edvardsen, Eglit, Guillou, Hampl, Heiss, Hoppenrath, James, Karnkowska, Karpov, Kim, Kolisko, Kudryavtsev, Lahr, Lara, Le Gall, Lynn, Mann, Massana, Mitchell, Morrow, Soo Park, Pawlowski, Powell, Richter, Rueckert, Shadwick, Shimano, Spiegel, Torruella, Youssef, Zlatogursky and Zhang2019) or the lower animal phyla known today and inferred to exist in the Ediacaran (sponge, cnidarians, placozoans, lophotrochozoans; Sperling & Vinther, Reference Sperling and Vinther2010; Zhuravlev et al. Reference Zhuravlev, Liñán, Gámez Vintaned, Debrenne and Fedorov2012; Wood et al. Reference Wood, Liu, Bowyer, Wilby, Dunn, Kenchington, Hoyal Cuthill, Michell and Penny2019; Fig. 5). The higher animal phyla such as tardigrades and arthropods that may form spinose cysts (Sanoamuang et al. Reference Sanoamuang, Saengphan and Murugan2002; Rabet, Reference Rabet2010; Scholtz & Wolff, Reference Scholtz, Wolff, Minelli, Boxhall and Fusco2013) had not yet evolved at the time, if relying on the fossil record (Budd & Jensen, Reference Budd and Jensen2000; Kouchinsky et al. Reference Kouchinsky, Bengtson, Runnegar, Skovsted, Steiner and Vendrasco2012) and in agreement with some molecular clock estimates (Erwin et al. Reference Erwin, Laflamme, Tweedt, Sperling, Pisani and Peterson2011). The earliest tardigrade fossils are middle Cambrian (Müller et al. Reference Müller, Walossek and Zakharov1995; Gross et al. Reference Gross, Treffkorn, Mayer and Wanninger2015) and arthropods are known from the early Cambrian (Fortunian Stage trace fossils, and Stage 3 body fossils; Erwin & Valentine, Reference Erwin and Valentine2013), thus not supporting comparisons of their cysts’ morphology with Ediacaran microfossils. Based on the new specimens and evaluating their features at the late reproductive stages, and as Tianzhushania spinosa has never before been observed at such multicellular stage, we further explore the possible affinities of cyst-like vesicles bearing processes and membranes.

Fig. 4. Extant green algal (desmidiacean) zygotic cysts that are morphological counterparts to the studied microfossils. (a) Staurastrum borgeanum. (b) Staurodesmus dejectus. (c) Micrasterias papillifera. (a, b) Specimens from ponds in the Netherlands. (c) Algaebase, Galway, National University of Ireland, Online Collection (Guiry & Guiry, Reference Guiry and Guiry2019), http://algaebase.org. All are transmitted-light micrographs.

Fig. 5. Schematic comparative morphology of studied microfossils, reproductive cysts with offspring cells in Chloroplastida (green algae), and embryology of Holozoa, including eggs, developing embryos and diapause cysts. (a–e) Microfossils with processes- and external membranes-bearing (m) cyst-like vesicles containing endocyst (en) inside vesicle cavity and internal spheroidal cells of equal sizes and tightly clustered, numbering from four (Fig. 2a) to numerous to hundreds (T. spinosa) seen in vesicle sections. (f–j) Examples of reproductive cysts in the group Chloroplastida, showing morphologic pattern of overall shape and characteristic processes, external membranes (m), rod-like elements supporting membrane (r), excystment structure (ex) and endocysts (en), and containing palintomically dividing offspring cells (in green). (k–w) Embryos, diapause cysts and eggs of representative organisms from the Supergroup Holozoa, including protistan (unicellular) and metazoan (multicellular) holozoans. (k) Codosiga botrytis, stalked (s) cell with flagellum (f) and collar (c) and cyst (cy) that contains dividing cells and releases many small flagellated cells (after Leadbeater, Reference Leadbeater2015). (l–w) Metazoan holozoans; micromeres (mm) marked in red colour, macromeres (mc) in orange colour, blastcoel (b). Details in the Supplementary Material available online at https://doi.org/10.1017/S0016756820001405.

Among the body plan and described morphological features, the studied microfossil taxa show excystment structures (pylome in Appendisphaera and median split in others), which are characteristic of reproductive cysts in extant algae (Evitt, Reference Evitt1985; Dale, Reference Dale2001; Head et al. Reference Head, Lewis and De Vernal2006; Moczydłowska, Reference Agić, Moczydłowska and Canfield2016). These structures differ from structures in some heterotrophic protist cysts, such as in amoebae and ciliates, which are morphologically predetermined to be openings with collars, necks or rims, or open by lysis of the wall (Tappan, Reference Tappan and Lipps1993; Porter, Reference Porter, Xiao and Kaufman2006; Bosak et al. Reference Bosak, Macdonald, Lahr and Matys2011; Morais et al. Reference Morais, Fairchild, Lahr, Rudnitzki, Schopf, Garcia, Kudryavtsev and Romero2017), in addition to dissimilar shape of cyst. Diapause egg cysts in extant animals are opened by enzymatic autolysis of the cyst wall and are discarded without any morphologically defined opening structure (Gilbert & Raunio, Reference Gilbert and Raunio1997; Jurd, Reference Jurd2004). For our interpretation, however, we analysed not one, the excystment, but a combined set of features to recognize the possible phylogenetic relationships between microfossils and extant biota. The described individual features (shape of processes, their sizes and distribution) in various combinations in ornamented vesicles with occasionally preserved openings and with resistant walls, as well as newly observed reproductive cells and their spatial arrangements, as observed in Appendisphaera, Urasphaera, Mengeosphaera and Tanarium, are representative for their affinities and are found in extant microalgae. The set of morphological features expressed in studied taxa are consistent with the overall morphology of reproductive cysts (Bold & Wynne, Reference Bold and Wynne1985, pp. 93, 143, 167; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995, pp. 352, 364, 471; Hagen et al. Reference Hagen, Siegmund and Braune2002; Raven et al. Reference Raven, Evert and Eichhorn2005, pp. 331, 337; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006; Graham et al. Reference Graham, Graham and Wilcox2009, pp. 414, 434–5, 464; Van Westen, Reference Van Westen2015; Guiry & Guiry, Reference Guiry and Guiry2019; see summary by Moczydłowska, Reference Agić, Moczydłowska and Canfield2016; Figs 4 and 5).

A morphological element that is peculiar to Tianzhushania – the external multilayered or multilamellar membrane surrounding the cyst (Fig. 3e) – has not been considered as a possible indicative feature, neither in the animal nor protistan holozoan interpretations. Such morphology is unknown in egg-cases, diapause cysts or any reproductive stages in animals or protistan holozoans (Gilbert & Raunio, Reference Gilbert and Raunio1997; Lee et al. Reference Lee, Leedale and Bradbury2000; Jurd, Reference Jurd2004; Gilbert, Reference Gilbert2010; Leadbeater, Reference Leadbeater2015; Adl et al. Reference Adl, Bass, Lane, Lukes, Schoch, Smirnov, Agatha, Berney, Brown, Burki, Cárdenas, Cepicka, Chistyakova, del Campo, Dunthorn, Edvardsen, Eglit, Guillou, Hampl, Heiss, Hoppenrath, James, Karnkowska, Karpov, Kim, Kolisko, Kudryavtsev, Lahr, Lara, Le Gall, Lynn, Mann, Massana, Mitchell, Morrow, Soo Park, Pawlowski, Powell, Richter, Rueckert, Shadwick, Shimano, Spiegel, Torruella, Youssef, Zlatogursky and Zhang2019). In extant algae, the external membrane is a common feature of the cyst in the initial process of its formation, during which the wall is secreted inside the membrane as the primary and secondary wall and thus may be multilayered and have a complex ultrastructure (Schlösser, Reference Schlösser, Irving and John1984; Bold & Wynne, Reference Bold and Wynne1985; Kokinos & Anderson, Reference Kokinos and Anderson1995; Dale, Reference Dale2001; Hagen et al. Reference Hagen, Siegmund and Braune2002; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006). The external membrane is present in several resistant organic-walled microfossils, which have been identified as possible algal cysts and of much older ages, at the time when animals had certainly not yet appeared (i.e. in the Mesoproterozoic; see dos Reis et al. Reference Dos Reis, Thawornwattana, Angelis, Telford, Donoghue and Yang2015). A few heterotrophic protists (amoebozoans and cercozoans) known in the Tonian (Porter, Reference Porter, Xiao and Kaufman2006) do not possess any membranes. Mesoproterozoic Shuiyousphaeridium and Gigantosphaeridium (Agić et al. Reference Agić, Moczydłowska and Yin2015, Reference Agić, Moczydłowska and Yin2017) and the Tonian Trachyhystrichosphaera and Cymatiosphaeroides (Vidal & Ford, Reference Vidal and Ford1985; Butterfield et al. Reference Butterfield, Knoll and Sweet1994) are examples of microfossils with external membranes. Cymatiosphaeroides has a single to multilaminated membranous envelope supported by processes and vesicle diameter ranging from 30 to 350 μm (Butterfield et al. Reference Butterfield, Knoll and Sweet1994, fig. 15c), and a body plan like that of Tianzhushania. These features are phenotypically consistent with some green algal cysts (Fig. 5). Some Mesoproterozoic taxa have been interpreted as stem-group Chloroplastida (Moczydłowska et al. Reference Moczydłowska, Landing, Zang and Palacios2011; Agić et al. Reference Agić, Moczydłowska and Yin2015), and Cymatiosphaeroides as chlorophycean alga (Moczydłowska, Reference Agić, Moczydłowska and Canfield2016).

5.e. Reproductive cycle

In the studied species, the cysts with one zygotic to four and multiple dividing cells show a morphological pattern that is characteristic of green algal cysts containing dividing offspring cells (spores) (Schlösser, Reference Schlösser, Irving and John1984; Bold & Wynne, Reference Bold and Wynne1985, pp. 129, 133, 141; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995, pp. 352, 364, 366, 471; Yamamoto et al. Reference Yamamoto, Nozaki and Miyzawa2003; Raven et al. Reference Raven, Evert and Eichhorn2005, pp. 331, 327, 337; Lee, Reference Lee2008, p. 217; Graham et al. Reference Graham, Graham and Wilcox2009, pp. 414, 434, 464; Van Westen & Coesel, Reference Van Westen and Coesel2014). The offspring cells (aplanospores, autospores, spores, swarmers) may be numerous (32–64 to hundreds; above citations) within algal cysts and are not limited to any strict number. The lack of a cavity in the multiple-celled clusters is dissimilar to animal embryos, which would have a blasto- or gastrocoel in multiple-celled developing stages as well as differentiated cells arranged into layers and poles (Kessel & Shih, Reference Kessel and Shih1974; Mathews, Reference Mathews1986; Jurd, Reference Jurd2004; Gilbert, Reference Gilbert2010; Nielsen, Reference Nielsen2012; Fig. 5). The absence of these diagnostic animal embryonic characters indicates that the studied microfossils are not of animal origin. In particular, this interpretation opposes the placement of Appendisphaera tenuis among animal diapause egg cysts (Yin et al. Reference Yin, Zhu, Knoll, Yuan, Zhang and Hu2007).

The new record of multiple identical cells of various numbers within specimens of the same species documents the ontogenetic stages as is seen in unicellular green algal maturing cysts (Bold & Wynne, Reference Bold and Wynne1985; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Hagen et al. Reference Hagen, Siegmund and Braune2002; Raven et al. Reference Raven, Evert and Eichhorn2005; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006; Lee, Reference Lee2008). The extant unicellular green algae show, in general, a similarly simple mode of reproduction forming multiple equal offspring cells tightly clustered inside the cyst, and this pattern might have been followed from the ‘generalist ancestor’ because immediate living relatives are hitherto unknown (Torruella et al. Reference Torruella, de Mendoza, Grau-Bové, Antó, Chaplin, del Camplo, Eme, Pérez-Cordón, Whipps, Nichols, Paley, Roger, Sitjà-Bobadilla, Donachie and Ruiz-Trillo2015).

In the cyst of Tianzhushania spinosa, the offspring cells were formed in the membranous, smooth-walled endocyst within its cavity. The offspring cells were likely released through the rupture in the cyst wall and endocyst and when freed they began to grow to vegetative cells. This life cycle included vegetative cells which are unrepresented as fossils, and only one kind of reproductive stage of which is the cyst represented by T. spinosa. This is because thousands of offspring cells indicating the late developmental stage were produced directly in the T. spinosa cyst. This evidence does not support the previous interpretations on the presence of several intervening developmental stages with different morphology in the life cycle of Tianzhushania (see Section 6.c). The T. spinosa cyst was likely zygotic and formed around the two fused (mating) vegetative cells of opposite orientation, e.g. + – strains, as it is known in algae (Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995, p. 352; Raven et al. Reference Raven, Evert and Eichhorn2005, p. 331; Lee, Reference Lee2008, p. 192; Graham et al. Reference Graham, Graham and Wilcox2009, p. 375). The diploidal zygote first divided meiotically to return to haploidal cells characteristic of algae, and then mitotically and palintomically in a series of divisions producing offspring cells (spores). After the cyst matured and contained the critical mass of cells, which reached the minimum viable size, they were released and the life cycle was closed. Based on the present record, new observations and the evaluation of previous interpretations of Tianzhushania as metazoan or protistan holozoan (see Section 6.c), we propose algal affinity for this taxon.

5.f. Comparisons to modern algae

The cyst morphology and reproductive cycle in various lineages of extant algae, which are well-recognized and accepted knowledge substantiated by new case studies (Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Hagen et al. Reference Hagen, Siegmund and Braune2002; Yamamoto et al. Reference Yamamoto, Nozaki and Miyzawa2003; Raven et al. Reference Raven, Evert and Eichhorn2005; Damiani et al. Reference Damiani, Leonardi, Pieroni and Cáceres2006; Lee, Reference Lee2008; Graham et al. Reference Graham, Graham and Wilcox2009; Van Westen & Coesel, Reference Van Westen and Coesel2014; Van Westen Reference Van Westen2015; see the summary in Moczydłowska, Reference Agić, Moczydłowska and Canfield2016; Fig. 5), provide striking phenotypic analogues to the microfossils studied. These analogues are found in the group Chloroplastida, in its basal division Chlorophyta and derived Streptophyta (according to classification by Adl et al. Reference Adl, Bass, Lane, Lukes, Schoch, Smirnov, Agatha, Berney, Brown, Burki, Cárdenas, Cepicka, Chistyakova, del Campo, Dunthorn, Edvardsen, Eglit, Guillou, Hampl, Heiss, Hoppenrath, James, Karnkowska, Karpov, Kim, Kolisko, Kudryavtsev, Lahr, Lara, Le Gall, Lynn, Mann, Massana, Mitchell, Morrow, Soo Park, Pawlowski, Powell, Richter, Rueckert, Shadwick, Shimano, Spiegel, Torruella, Youssef, Zlatogursky and Zhang2019; Burki et al. Reference Burki, Roger, Brown and Simpson2020). Modern morphological counterparts are observed in cysts of numerous marine species of basal chlorophytes, such as Chlamydomonas and Golenkinia (Bold & Wynne, Reference Bold and Wynne1985; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Raven et al. Reference Raven, Evert and Eichhorn2005; Falkowski & Raven, Reference Falkowski and Raven2007; Fehling et al. Reference Fehling, Stoecker, Baldauf, Falkowski and Knoll2007; O’Kelly, Reference O’Kelly, Falkowski and Knoll2007; Guiry, Reference Guiry2013) and in derived lineages of some freshwater streptophytes, such as Closterium, Cosmarium, Staurastrum, Staurodesmus and Micrasterias (Bold & Wynne, Reference Bold and Wynne1985; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Lee, Reference Lee2008; Graham et al. Reference Graham, Graham and Wilcox2009; Van Westen & Coesel, Reference Van Westen and Coesel2014; Van Westen, Reference Van Westen2015; Figs 4 and 5). For example, the zygotic cyst of Chlamydomonas, which is thick-walled, resistant and ornamented by spines, contains a single large cell, the zygote, which after multiple divisions produces numerous, small spheroidal offspring cells (Schlösser, Reference Schlösser, Irving and John1984; Bold & Wynne, Reference Bold and Wynne1985; Van den Hoek et al. Reference Van den Hoek, Mann and Jahns1995; Raven et al. Reference Raven, Evert and Eichhorn2005; Lee, Reference Lee2008). If fossilized, Chlamydomonas would show a morphological pattern similar to some of the studied microfossils.

Further morphological analogues, including three genera of extant desmidiacean algae in the streptophytes, exemplify the zygotic cyst morphology of acanthomorphic vesicles with conical simple or divided process tips and a wall composed of refractory biopolymers (Figs 4a–c and 5). The cyst encloses spheroidal dividing cells (spores), initially two as found in Staurodesmus (Fig. 4b), to multiple, as illustrated in Staurastrum and Micrasterias (Fig. 4a, c). At the early developmental stage, the zygote forms a single internal body, and the cyst still contains chlorophyll (Fig. 4a, b) and is metabolically active, not only in the process of zygotic subdivision. The mature cyst is devoid of chlorophyll (Fig. 4c). Despite being freshwater representatives, these algae cannot be excluded from comparisons since algae either originated from marine ancestors (Falkowski & Raven, Reference Falkowski and Raven2007; Fehling et al. Reference Fehling, Stoecker, Baldauf, Falkowski and Knoll2007; Hackett et al. Reference Hackett, Yoon, Butterfield, Sanderson, Bhattacharya, Falkowski and Knoll2007; Knoll et al. Reference Knoll, Summons, Waldbauer, Zumberge, Falkowski and Knoll2007) or in low-salinity habitats (Sánchez-Baracaldo et al. Reference Sánchez-Baracaldo, Raven, Pisani and Knoll2017). However, the latter habitat is not reconciled with the fossil record and is exemplified by the habitats of modern taxa. These habitats are ephemeral, contrary to the expected robustness of the system to sustain the evolving biota. It is also well-known that even the same genus, such as prasinophycean extant algae and fossil Cymatiosphaera, may occupy marine, brackish and freshwater environments preserving the same morphology (Tappan, Reference Tappan1980; Dotzel et al. Reference Dotzel, Taylor and Krings2007).

The combined features of resistant organic-walled vesicles with surface ornamentation in the form of processes and/or membranes, an excystment opening, an internal membranous endocyst containing a single to multiple cells, and the evidence of multiple palintomically dividing cells with wall furrows that are preserved inside the vesicle cavity are consistent with inferring that the studied microfossils represent zygotic reproductive cysts of green algae. The earlier interpretation of Appendisphaera and Tanarium as representing algal zygotic cysts (Moczydłowska, Reference Moczydłowska2005, Reference Agić, Moczydłowska and Canfield2016; Moczydłowska et al. Reference Moczydłowska, Landing, Zang and Palacios2011) or conventionally existing among phytoplankton (Grey, Reference Grey2005) and made prior to observing internal cells is now reinforced by evidence of reproductive stages with cell division diagnostic of algae.

5.g. Comparisons to microfossils of other ages

Ediacaran cysts are, in all the features analysed here, similar to many microfossil species of various ages from the Mesoproterozoic to Palaeozoic, which were inferred to be algal in affinity based on comparative morphology with extant taxa (Tappan, Reference Tappan1980; Zang & Walter, Reference Zang and Walter1989; Moczydłowska, Reference Moczydłowska1991, Reference Moczydłowska2005, Reference Moczydłowska2010, Reference Agić, Moczydłowska and Canfield2016; Moczydłowska et al. Reference Moczydłowska, Vidal and Rudavskaya1993, Reference Moczydłowska, Landing, Zang and Palacios2011; Colbath & Grenfell, Reference Colbath and Grenfell1995; Arouri et al. Reference Arouri, Greenwood and Walter2000; Grey, Reference Grey2005; Willman & Moczydłowska, Reference Willman and Moczydłowska2007; Lamb et al. Reference Lamb, Awramik, Chapman and Zhu2009; Moczydłowska & Willman, Reference Moczydłowska and Willman2009; Agić et al. Reference Agić, Moczydłowska and Yin2015, Reference Agić, Moczydłowska and Canfield2016, Reference Agić, Moczydłowska and Yin2017; Miao et al. Reference Miao, Moczydłowska, Zhu and Zhu2019; Shang et al. Reference Shang, Liu, Moczydłowska and Yang2020). This similarity between fossil cysts and extant algal cysts is argued not to be coincidental or the result of convergent morphology in polyphyletic organisms, but the expression of phylogenetic relationships within some green algal lineages from their early ancestors to those living today. As mentioned above, the extant algae inherited and share their mode of reproduction with related and early diverging lineages, along with the biosynthesis of polymers that comprise resistant cyst walls and some enzymes metabolically vital for photosynthesis (Raven et al. Reference Raven, Evert and Eichhorn2005; David & Alm, Reference David and Alm2011). The sexual reproduction and biosynthesis of refractory polymers in the walls of protective cysts harbouring zygote and dividing cells acquired in the deep phylogenetic history by algae in marine environments was an evolutionary success due to certain directly and endosymbiotically inherited features (Margulis et al. Reference Margulis, Corliss, Melkonian and Chapman1989; Woese et al. Reference Woese, Kandler and Wheelis1990; Falkowski & Raven, Reference Falkowski and Raven2007; O’Kelly, Reference O’Kelly, Falkowski and Knoll2007; Torruella et al. Reference Torruella, de Mendoza, Grau-Bové, Antó, Chaplin, del Camplo, Eme, Pérez-Cordón, Whipps, Nichols, Paley, Roger, Sitjà-Bobadilla, Donachie and Ruiz-Trillo2015; Burki et al. Reference Burki, Roger, Brown and Simpson2020), as were some biochemical processes and the ability to form zygotic cysts. These best-fitted characters in reproductive cysts might have been selected for and shared in photosynthetic algae that may be traced back by microfossils to c. 1.7 Ga (Moczydłowska et al. Reference Moczydłowska, Landing, Zang and Palacios2011; Agić et al. Reference Agić, Moczydłowska and Yin2015, Reference Agić, Moczydłowska and Yin2017; Moczydłowska, Reference Agić, Moczydłowska and Canfield2016; Miao et al. Reference Miao, Moczydłowska, Zhu and Zhu2019). The evolutionary history of green algae, the chlorophytes, if accepting the affinity of such microfossils, may be inferred from the fossil record in marine environments beginning prior to c. 1.7 Ga and that of phylogenetically more distant streptophytes adapted to freshwater environments prior to c. 1.0 Ga (Raven et al. Reference Raven, Evert and Eichhorn2005; Knauth & Kennedy, Reference Knauth and Kennedy2009). Microfossil algal affinities (Moczydłowska, Reference Agić, Moczydłowska and Canfield2016; Loron & Moczydłowska, Reference Loron and Moczydłowska2018) are disputed (Javaux & Knoll, Reference Javaux and Knoll2017; Del Cortana et al. Reference Del Cortana, Jackson, Bucchini, Van Bel, D’hondt, Škaloud, Delwiche, Knoll, Raven, Verbruggen, Vandepoele, De Cleck and Leliaert2020), so microfossil taxa may be selectively used to calibrate molecular clock estimates, resulting in younger estimates for the divergence of Chlorophyta, e.g. c. 1 Ga (Del Cortana et al. Reference Del Cortana, Jackson, Bucchini, Van Bel, D’hondt, Škaloud, Delwiche, Knoll, Raven, Verbruggen, Vandepoele, De Cleck and Leliaert2020). This estimate has not considered more recent records (Agić et al. Reference Agić, Moczydłowska and Yin2015, Reference Agić, Moczydłowska and Yin2017; Miao et al. Reference Miao, Moczydłowska, Zhu and Zhu2019) for fossil calibration, or dealt only with the multicellular algae (Tang et al. Reference Tang, Pang, Yuan and Xiao2020). The wide timespans for divergence of Archaeoplastida at c. 1.6–1.1 Ga and the symbiotic origin of the plastid at c. 1.7–1.1 Ga (Betts et al. Reference Betts, Puttick, Clark, Williams and Donoghue2018) may account for uncertainties in the methods used for estimates and are not precise (see also dos Reis et al. Reference Dos Reis, Thawornwattana, Angelis, Telford, Donoghue and Yang2015). Mis-calibration of the molecular clock estimates is affected by the difficulty in interpreting fossils with no extant exemplars and the reconstruction of ancestral characters (Leliaert et al. Reference Leliaert, Smith, Moreau, Herron, Verbruggen, Delwiche and De Clerck2012).

The c. 1.7 Ga record of green algae, if further confirmed, broadly coincides with that of red algae at c. 1.6 Ga, which however diverged earlier according to the phylogenomics of green plants (Corlett et al. Reference Corlett2019). Taking fossil record biases into account, both the red and green algae are still very ancient organisms. The red algae, the rhodophytes, represented by the putative florideophycean and bangiophycean classes, are recognized in the fossil record in the 1.0 Ga Hunting Formation, Canada (Butterfield et al. Reference Butterfield, Knoll and Sweet1990; Butterfield, Reference Butterfield2000; see Gibson et al. Reference Gibson, Shih, Cumming, Fischer, Crockford, Hodgskiss, Wörndle, Creaser, Rainbird, Skulski and Halverson2018 for age 1.0 Ga), and the 1.6 Ga Lower Vindhyan Supergroup in India and are classed among crown-group eukaryotes (Bengtson et al. Reference Bengtson, Sallsted, Belivanova and Whitehouse2017; but cf. Betts et al. Reference Betts, Puttick, Clark, Williams and Donoghue2018, who suggests they belong to total group Archaeoplastida instead, and concerning age see also Ray, Reference Ray2006). Multicellular red, brown and/or green algae with parenchymatous thalli are also well-represented in the Ediacaran Doushantuo Formation (Steiner, Reference Steiner1994; Xue et al. Reference Xue, Tang, Yu and Zhou1995; Xiao et al. Reference Xiao, Zhang and Knoll1998, Reference Xiao, Knoll, Zhang and Hua1999, Reference Xiao2004; Xiao, Reference Xiao2002; Yuan et al. Reference Yuan, Xiao, Yin, Knoll, Zhou and Mu2002, Reference Yuan, Chen, Xiao, Zhou and Hua2011; Yin et al. Reference Yin, Zhu, Tafforeau, Chen, Liu and Li2013).