Multicellular Eukaryotic fossils from the Mesoproterozoic Chuanlinggou Formation of North China.

All life found on Earth today is made up of cells, with the vast majority of organisms still being unicellular; it is generally presumed that the earliest forms of life would have been single-celled Prokaryotes (organisms with cells which lack internal divisions and organelles).  Multicellularity has arisen numerous times within both Prokaryotic and Eukaryotic groups, although complex multicellularity, with cells differentiated into specialist forms and organised communication between cells, has only arisen six or seven times, and only in Eukaryotes.

The earliest widely accepted multicellular Eukaryotic fossils, filaments and spherical groups of cells, appear around the Mesoproterozoic-Neoproterozoic boundary, while filamentous Prokaryotes are known from the Archaean. Early multicellular Eukaryotes include Bangiomorpha pubescens, a putative Red Alga from 1050 million-year-old deposits in the Canadian Arctic, Eosolena loculosa, a Eukaryote of uncertain affinities from 1030 million-year-old deposits in Siberia, Arctacellularia tetragonala, another species of uncertain affinities from 1000 million-year-old deposits in the Democratic Republic of Congo, Proterocladus antiquus, a possible Green Alga from 950 million-year-old deposits in North China, Archaeochaeta guncho, another species of uncertain affinities from 950 million-year-old deposits in northwestern Canada, and Ourasphaira giraldae, a possible Fungi from 890 million year old deposits in the Canadian Arctic. 

Some putative multicellular Eukaryotes have also been recorded from earlier in the Mesoproterozoic, including Eosolena minuta, from 1500 million-year-old deposits in northern Siberia, or the carbonacious impressions of the Gaoyuzhuang Formation in North China, which can reach tens of centimetres across, or the possible Eukaryotic microfossils from the 1600 million-year-old Tirohan Dolomite of central India. The oldest examples of the coilled microfossil Grypania are currently dated to about 2100 million years before the present (i.e. Late Palaeoproterozoic) although it is debated whether this is a Eukaryote or a Cyanobacterium. Of similar age are the pyritic macrostructures of the Francevillian Biota of Gabon, though there is some debate as to whether there are of biological origins at all.

In 1989, micropalaeontologist Yan Yuzhong published a description of a filamentous Eukaryotic fossil from the 1630 million-year-old Chuanlinggou Formation of North China in the Bulletin of the Tianjin Institute of Geology and Mineral Resources. At this time journals were only available in print, and the Bulletin, which was printed in Chinese, had almost no circulation outside of China. Furthermore, the quality of the images in Yan's paper were rather poor, leading to the publication being largely overlooked.

In a paper published in the journal Science Advances on 24 January 2024, Lanyun Miao and Zongjun Yin of the State Key Laboratory of Palaeobiology and Stratigraphy at the Nanjing Institute of Geology and Palaeontology, Andrew Knoll of the Department of Organismic and Evolutionary Biology at Harvard University, Yuangao Qu of the Institute of Deep-sea Science and Engineering of the Chinese Academy of Sciences, and Maoyan Zhu, again of the State Key Laboratory of Palaeobiology and Stratigraphy at the Nanjing Institute of Geology and Palaeontology, and of the College of Earth and Planetary Sciences of the University of the Chinese Academy of Sciences, re-examine the Chuanlinggou Formation fossils, and discuss the implications of these for the origin of multicellular Eukaryotic life.

Samples of grey shale were collected from the Wengjiazhuang Section of the Chuanlinggou Formation in Kuancheng County of Hebei Province, which has been dated to 1634.8 million years before the present (±6.9 million years) using uranium/lead ratios in zircons. Zircons are minerals formed by the crystallisation of cooling igneous (or in this case, impact) melts. When they form, they often contain trace amounts of uranium, which decays into (amongst other things) lead at a known rate. Since lead will not have been present in the original crystal, it is possible to calculate the age of a zircon crystal from the ratio between these elements. Microfossils were then extracted from these shales by acid maceration.

This technique recovered flattened greyish or pale brown filamentous fossils, which Miao et al. describe as Qingshania magnifica, the name used by Yan Yuzhong for his material. These are not the only filamentous fossils derived from the Chuanlinggou Formation shales, but are significantly larger than other forms, supporting the idea that they are Eukaryotic in origin, while the other forms are Prokaryotic, probably Cyanobacteria. The 278 individual specimens Miao et al. identified ranged from 20 to 194 μm in diameter, with a maximum length of 860 μm. The filaments were straight or curved, and made up of smooth-walled cells, more than 20 of which were present in the longest specimens. These cells are generally cylindrical in shape, with a cell length of 15 to 190 μm. Terminal cells, where preserved, are hemispherical. None of the specimens had any form of external sheath or holdfast.

Transmitted-light photomicrographs of Qingshania magnifica from the Chuanlinggou Formation. (A) to (D) and (K) Filaments with cells of varying length and width. (E) Four-celled filament with hemispherical terminal cell. (F) and (G) Filament with notably decreasing cell width toward one end. Note that (F) and (G) represent the same specimen; (F) lost the narrowest part of the filament as shown in (G). (H to J) Filaments displaying more uniformity of cell dimensions. (L) Two-celled filament with ovoid terminal cell. All specimens were handpicked from organic residues of acid maceration and photographed in wet mounts, except for (K), which was photographed from a permanent strew mount. Solid and empty gray triangles in (A), (C), and (K) indicate the longest and the shortest cells, respectively, within single filaments. tb, transverse band (interpreted as cross wall); tr, transverse ring (interpreted as partially preserved cross wall). Scale bars 50 μm (A) to (E), (I), (J), and (L) and 100 μm (F) to (H) and (K). Miao et al. (2024).

The specimens show considerable variation, with the largest being ten times as wide as the smallest, individual cells being cylindrical, barrel-shaped, or cup-shaped, and filaments being of even width or tapering towards one end. Despite this variation, Miao et al. treat them all as a single species, suggesting that the variations reflect s different growth or developmental stages within the population.

Micrographs of Qingshania magnifica from the Chuanlinggou Formation. (A) Transmitted-light photomicrograph of a five-celled filament with constant width and dark narrow transverse bands. (B) Scanning electron microscope image of (A) showing surface features and the preservation as a complete compression. Note the obliquely compressed cross wall of the right terminal cell showing smooth surface and no other particular features. (C) to (E) Magnifications of (B), showing smooth wall surface and the well-defined contact between adjoining cells manifested by a very shallow groove (marked by cyan arrowheads) along transverse bands. (C) and (E) represent dashed boxes in (A) and (B); (D) corresponds to the dashed box in (C). Scale bars, 50 μm (A) and (B), 10 μm (C), and 2 μm (D) and (E). Miao et al. (2024).

Some of the filaments have small, round-to-ovoid structures within some of their cells. These structures are faint, but always contained entirely within the cell, making it unlikely that they are separate structures superimposed upon the filaments. Inclusions within cells, from both Proterozoic and Phanerozoic settings, have variously been interpreted as endocysts, collapsed cytoplasm, or organelles. The fossils are interpreted as being compressed cell walls, which makes it likely that structures withing them would be endocysts. A variety of Eukaryotic and Prokaryotic groups produce endospores in response to worsening conditions (such as the end of a growing season), but these tend to have protective envelopes thicker than the outer cell wall, which is not the case with these structures. However, the structures are found only in larger cells, and are only slightly smaller than the smallest cells, which suggests that they may be some form of asexual reproductive spore; similar spores are produced by some extant filamentous Algae, such as Urospora wormskioldii.

Transmitted-light photomicrographs of Qingshania magnifica with a small round or ovoid inclusion from the Chuanlinggou Formation. (A), (C), and (D) Filaments with constant width. (B) and (E) Magnifications of dashed boxes in (A) and (C), respectively, showing details of round inclusions. (F) Filament of notably varying width. Note that the middle cell of the filament is cyathiform in shape. (G and H) Magnifications of dashed box in (F) and (D), respectively. All specimens were handpicked from organic residues of acid maceration and photographed in wet mounts. Scale bar, 50 μm (A), (C), (D), and (F). Miao et al. (2024).

Microscale Raman and Fourier transform infrared spectroscopic investigations of the composition of the filaments suggested that the cell walls of Qingshania magnifica were composed largely of aromatic compounds, with a lower proportion of aliphatic compounds, with the aliphatic compounds forming long chains with little branching. This is not sufficient to make any  assessment of the taxonomic status of Qingshania magnifica on its own, but is quite distinct from the composition of Cyanobacterial cells found in the same deposits.

The original specimens of Qingshania magnifica described by Yan in 1989 were identified from thin sections of yellowish-green shales, and had a maximum width of about 250 μm and were up to 6000 μm in length. Yan identified these as Green Algae, placing them in the modern family Ulotrichaceae. Miao et al.'s specimens are slightly smaller, but preserve more detail, allowing for a more detailed reconstruction.  They interpret Qingshania magnifica as a simple multicellular organism with large cells and a degree of morphological variation, with a life cycle that involved spores produced within cells, which then produced thin filaments, which grew into thicker filaments, which were capable of producing more spores.

A wide range of both Prokaryotic and Eukaryotic organisms produce filaments of cells today. Among Prokaryotes, these include at least eleven phyla of Bacteria and one of Archaeans. The most sophisticated filamentous Prokaryotes are Cyanobacteria, which produce a range of forms including straight, tapering, and branching filaments. However, no known Cyanobacterium, or other Prokaryote, living or fossil, closely resembles Qingshania magnifica. Filamentous Eukaryotes include Algae such as Archaeplastids (the group that includes both Red and Green Algae) and Ochrophytes (the group that includes Brown Algae, Golden Algae, and Diatoms), as well as filamentous Fungi and Oomycetes (Water Molds). The cells of Qingshania magnifica are completely surrounded by cell walls, which suggests that each cell acquires its own nutrition, by either photosynthesis or osmotrophy (absorbing nutrients from the environment). This is also quite different from the hyphal structure seen in Fungi and Oomycotes, even the septate forms. making it unlikely that Qingshania magnifica could be assigned to either of these groups. Furthermore, molecular clock estimates suggest that Fungi did not appear till about 1000 million years ago, and Oomycotes probably around the dawn of the Cambrian.

Based upon this analysis, Miao et al. conclude that Qingshania magnifica is most likely to have been a Eukaryotic Algae. This is consistent with molecular clock analyses, which suggest plastids (chloroplasts) were first acquired by unicellular Algae during the Palaeoproterozoic. The morphology of Qingshania magnifica is also consistent with younger fossils interpretted as Green Algae, as well as several modern members of that group. However, Miao et al. do no conclude there is sufficient evidence to confidently place Qingshania magnifica within the Green Algae, as originally proposed by Yan, instead concluding that it could be a Green Algae, a Red Algae, a stem group Archaeplastid, or even a member of an entirely extinct Eukaryotic group. Whichever of these is true, Qingshania magnifica provides strong support for a Late Palaeoproterozoic appearance of the crown group Eukaryotes, rather than a Late Mesoproterozoic one, which has sometimes been proposed. 

Overview of early evolution of the Eukarya along with fossil records. (A) Simplified Eukaryotic tree with divergence time estimates of major branches by molecular clock study. LECA, Last Eukaryotic Common Ancestor. Dashed grey lines represent hypothetical stem-group Eukaryotes, which are extinct. Abbreviation: Pha., Phanerozoic. (B) Representative fossil records of early Eukaryotes. The oldest unambiguous Eukaryotic fossils are unicellular forms, e.g., Tappania plana and Shuiyousphaeridium macroreticulatum from the approximately 1650 million-year-old Ruyang Group; Dictyosphaera macroreticulata, Germinosphaera alveolata, and Valeria lophostriata from the Changzhougou Formation and lowermost Chuanlinggou Formation of North China. The Qingshania magnifica represents the current oldest convincing multicellular Eukaryote from the approximately 1635 million-year-old upper Chuanlinggou Formation of North China. The oldest Red Alga is Bangiomorpha pubescens from the approximately 1050 million-year-old Hunting Formation, Canada. The oldest Green Alga is Proterocladus antiquus from the approximately 950 million-year-old Nanfen Formation of North China. The oldest putative Fungus is Ourasphaira giraldae from the approximately 890 million-year-old Grassy Bay Formation of Canada. The oldest Amoebozoans are vase-shaped microfossils, e.g., Cycliocyrillium torquata from the approximately 750 to 730 million-year-old Kwagunt Formation, part of the Chuar Group of Arizona. Scale bars, 500 μm for the oldest Green Alga and 50 μm for all other specimens. Miao et al. (2024).

See also...