A new red alga preserved with possible reproductive bodies from the 518‐million‐year‐old Qingjiang biota

Macroalgae have been a key ecological component of marine ecosystems since the Proterozoic period and are common fossil forms in Cambrian Burgess Shale‐type Lagerstätten. However, in most cases, it is difficult to place these early fossil algae into modern groups because little distinctive morphology is preserved. Here, we describe a new form of macroalgae, Qingjiangthallus cystocarpium gen. & sp. nov., from the Qingjiang biota of South China. The new taxon is represented by 546 specimens remarkably preserved with characteristics that allow a phylogenetic placement into crown groups of red algae. Centimeter‐sized thalli resemble members of the extant Rhodymeniophycidae (a subclass of the class Florideophyceae), and hence suggest a florideophycean affinity, which indicates that ahnfeltiophycidaen and rhodymeniophycidaen algae may have diverged at least 518 Ma, accordant with estimations of molecular studies. The presence of possible cystocarps on Qingjiangthallus thalli suggests that evolutionary innovation of a triphasic life cycle in red algae may have occurred no later than the Early Cambrian. The branching patterns and branch width of Qingjiangthallus are consistent with the coarsely dichotomously branched morphogroup, which was previously present in the Ediacaran, Ordovician, and afterward, but absent in the Cambrian.


Introduction
Red algae (phylum Rhodophyta) are a large and species-rich assemblage (Gawryluk et al., 2019) and morphologically more diverse than any other group of macroalgae (Woelkerling, 1990).They are unique among eukaryotes for having a triphasic life cycle (carposporophyte, gametophyte and tetrasporophyte) and the lack of both flagella and centrioles during their entire life (Gabrielson et al., 1990;Graham & Wilcox, 2000).As one of the ancient lineages of photosynthetic eukaryotes (Archaeplastida), they might have given rise to other algal groups through secondary endosymbiosis (Brawley et al., 2017).Red algae have a deep geological history, and their fossils of calcified forms are sparsely known in Paleozoic rocks (Brooke & Riding, 1998) and common in Mesozoic and Cenozoic carbonates (Aguirre et al., 2000;Bucur et al., 2020).Four related fossil records from the Precambrian (Butterfield, 2000;Xiao et al., 2014;Bengtson et al., 2017) were discovered in thin sections of silicified carbonates, phosphorites and phosphatized stromatolitic microbialites.However, noncalcified macroscopic red algae preserved as carbonaceous films have rarely been reported.
Here, we describe a new form of red algae occurring as carbonaceous compressions from the Qingjiang biota of South China.Specimens are abundant and well preserved.Thalli comprise flat and ribbon-shaped branches showing different branching patterns, some short branchlets, discoid or globose holdfasts and an unexpected occurrence of possible cystocarps (reproductive bodies).Considering the resemblances to extant Gracilariales and Gigartinales, two orders of extant red algae in the subclass Rhodymeniophycidae (phylum Rhodophyta, class Florideophyceae), we conservatively consider that the new taxon has an affinity with Florideophyceae.This new finding provides fossil evidence for the early divergence of the major florideophycean lineages within the Rhodophyta.
Province, South China (Figs. 1A, 1B).The middle member of the Shuijingtuo Formation is composed of a succession of laminated black siltstones interrupted by two intervals of calcareous claystones, each of which consists of couplets of laminated black claystone alternating with submillimeter-to centimeter-thick, light gray-colored claystones (Fig. 1C; Fu et al., 2019).For more details on stratigraphy and the age of fossiliferous layers refer to Fu et al. (2019).The present specimens are concentrated on the bedding surface of light gray-colored claystones, contrasting with their sparse occurrence in the laminated black claystone (Fig. 1C).

Optical and electron microscopic analyses
The fossil specimens were examined and photographed using a Zeiss Stemi 305 stereomicroscope and a Canon EOS 5DS digital camera.Some selected specimens were coated with a ca.10-nm conductive gold-palladium layer and further examined using a Thermo Fisher Helios G4 focused ion beam-scanning electron microscope (FIB-SEM) to reveal more morphological details.A cross-section was cut using FIB to examine the microstructure of cystocarps.Thin sections cut from rock samples perpendicular to the bedding surface were examined under a Nikon ECLIPSE LV100POL microscope equipped with a digital camera.Electron probe micro-analysis (EPMA) was carried out to analyze the elemental compositions of the fossils.

Data measurement and analysis
To assess the size of the cystocarps, the major and minor axes of 734 dark spots of 37 specimens were measured using Adobe Photoshop CS3.Bar charts were created in WPS 2019.The branch widths of 242 specimens were measured using Adobe Photoshop CS3 to fit the new taxon in the right morphogroup.Distribution intervals of the possible cystocarp size and branch width were defined by the mean value ±2 standard deviations.(Figs. 2,3,5,7) Holotype LELE1165 (a complete frond; Figs.2A, 2B).
Materials Apart from the holotype and paratypes, 542 additional specimens from the Qingjiang biota were examined in this study.
Repository Specimens are all deposited in the Shaanxi Key Laboratory of Early Life and Environments (LELE), Northwest University, Shaanxi, China.
Etymology The generic name is derived from the Qingjiang locality and the Latinized Greek thullos, twig, the vegetative tissue of some organisms.The specific epithet is from Latin, cystocarpium, referring to possible cystocarps on thalli.
Diagnosis Fronds usually have one or two branches growing from a discoid or globose holdfast.Thalli are centimeter scale and asymmetrically, dichotomously, or laterally branched.Branches are flat, ribbon-shaped, with sharp tips, and without stipe.Main branches are always curved, slightly wider than other branches, and sometimes bear short branchlets.Lateral branches grow upward, and are arranged regularly in the upper portion of the thallus.Elliptical to circular structures are densely distributed on the surface of thalli, hundreds of microns in diameter and usually show a moderate relief.Description Specimens are preserved as carbonaceous compressions (Fig. 2C).Fronds consist of branched or unbranched thalli with a holdfast (Figs.2A, 2B, 3A-3D).Thalli branch sparsely or profusely in three different patterns, that is, one to three times asymmetrically, one to two times dichotomously, and laterally branched patterns (Figs.3E-3G).Branching angles vary from 20°to 98°.Branches are flat, ribbonshaped, without stipe, usually curved slightly upward and sharpened at the tips (Fig. 2D).They vary from 1.225 to 3.953 mm in width (AVG = 2.589, SD = 0.682, n = 351) (Fig. 4A), and are up to 10.40 cm tall.For asymmetrically and laterally branched thalli, the main branches are always curved and slightly wider than other branches (Figs.2A, 3E, 3F).Some have one or more branchlets, which are very short, 1.484-3.681cm long and usually in the lower portion of thalli (Figs.2A, 3E).Lateral branches grow upward away from the insertion point, and are regularly arranged in the upper portion of thalli (Fig. 3E).For dichotomously branched thalli, all branches have almost the same width (Fig. 3G).Rarely, preserved holdfasts are discoid or globose in shape, 2.416-4.173mm in diameter, without rhizoids or any other appendage, and each bearing one or two branches (Figs.2E, 3A-3D).
Dense dark spots resembling cystocarps in modern red algae are present on the surface of the whole thallus and numerous on most branches.Their density increases toward the tip of each branch, where they closely contact each other
The present fossil thalli are asymmetrically, dichotomously, and laterally branched (Figs.3E-3G), and some have short branchlets (Figs.2A, 3E).Of the 182 fossil specimens preserved with branches, the proportions of asymmetrically, dichotomously, and laterally branched patterns are 60.5%, 19.2%, and 20.3%, respectively, and 12 specimens have short branchlets.Such branching patterns have not been seen in any macroalgal thallus previously described from the Precambrian and Cambrian strata.In extant macroalgae, dichotomously branched thalli are common in Chlorophyta, Rhodophyta, and Phaeophyceae, and asymmetrically branched thalli occur in Rhodophyta and Phaeophyceae (Tseng, 1983).But asymmetrically and laterally branched thalli with short branchlets are generally restricted to the Rhodophyta (Wang, 2018).For some taxa of Rhodophyta, their thalli show three or four different branching patterns, and always have short branchlets constricted at the base (Wang, 2018).Accordingly, branching patterns of the present fossil thalli are comparable with those of the Rhodophyta, such as Gracilaria Greville, 1830.
Additionally, dense dark spots have more significant phylogenetic indications.They are present on thalli but absent from the rock matrix, indicating that they are biologically associated with macroalgae rather than originating from other organisms as a result of geological processes.In most specimens, these dark spots are present within the margin of macroalgal branches.Only two of the 546 specimens (LELE0197, LELE0320) have dark spots partially beyond the branch margins, interpreted as threedimensional structures growing on the branch margins.Hence, these dark spots are more likely to be ornaments or integral parts (e.g., reproductive organs) of the present macroalgae, rather than exogenic organisms in origin.
Similar structures have been reported from the early Neoproterozoic Tawuia Hofmann from the Little Dal Group of Canada (Hofmann, 1985) and algal fossils from the lower Neoproterozoic Liulaobei (Steiner, 1994) and Shiwangzhuang (Tang et al., 2021) formations of North China, and these were interpreted as possible saprophytes (Hofmann, 1985), reproductive cells (Hofmann, 1985;Steiner, 1994), or epibionts (Tang et al., 2021), respectively.The thalli of Qingjiangthallus show no obvious indication of degradation before infestation by the saprophytes, and the increasing density of dark spots toward the tip of branches (Figs.2A,  3E) seems to be unrelated to a saprophytic relationship.The interpretation of epiphytic organisms on macroalgae is interesting and reasonable.In modern oceans, epiphytic organisms are common on the surface of macroalgae (Wu et al., 2014).However, for the present fossils, the dark spots are more likely to be ornaments or integral parts of macroalgal thalli based on the above discussion.Therefore, the dark spots on the present fossils are unlikely to be saprophytes or epibionts.Similar ornaments have not been observed on the thalli surface of extant macroalgae.Thus, we are apt to interpret these dark spots on Qingjiangthallus as reproductive organs.
Many extant macroalgae develop bisporangia, monosporangia, polysporangia, and tetrasporangia that release spores to complete asexual reproduction.These sporangia are also elliptical or circular in outline and scattered in macroalgal thalli (Lee, 2008).However, these sporangia are micron-scale and fully or almost fully embedded within macroalgal thalli (Lee, 2008).The dark spots on Qingjiangthallus are more consistent with fucalean conceptacles (ca.0.1-0.6 mm in diameter; Phaeophyceae) (Clayton et al., 1987), or florideophycean cystocarps (approximately up to 3 mm in diameter) and nemathecia (ca.1-3 mm in diameter) (Smith, 1964), which are all bulging superficially on some macroalgal thalli.Fucalean conceptacles are always embedded at the tips of the final dichotomoies (Smith, 1964), and they are impossible to extend beyond the branch margins when compressed.
Elliptical-circular nemathecia are produced on all thalli except the final dichotomies (Smith, 1964).In contrast, the dark spots on the present fossils closely resemble florideophycean cystocarps based on their morphology, dimension, and position.However, no cell, carpospore, or internal structure (such as carposporangia) is recognized in either the cross-section cut using FIB or in the traditional thin section (Figs.5D, 5F-5I).These diagnostic characteristics are probably lost in the geological process.

Affinities
The branched thalli, large size, and discoid or globose holdfasts of these macrofossils all suggest a multicellular and macroalgal interpretation, rather than a fungal or cyanobacterial affinity.Marine macroalgae occur in the phyla Charophyta, Chlorophyta, Phaeophyta, and Rhodophyta (Dawes, 2016).The present fossil thalli are obviously different from those of Charophyta (brittleworts or stoneworts) due to the absence of diagnostic features, for example, a node-internode structure, whorls of branchlets from the node, and rhizoids (Lee, 2008).A few marine species of Chlorophyta (green algae), which have dichotomously branching patterns, can be distinguished based on densely matted and crisped thalli, branches with intervals, rhizoids, or disk holdfasts with rhizoids (Smith, 1964;Tseng, 1983).Some members of the Phaeophyta also show asymmetrically and dichotomously branching patterns, such as chordariacean and dictyotacean algae.For these species of Phaeophyta, the asymmetrically branched thalli with short branchlets usually bear a central axis, and the dichotomously branched thalli have branches with midribs and/or rounded or obtuse apices (Tseng, 1983).But the present fossil thalli bear no central axis or midrib, and their branch apices are sharp (Fig. 2D).According to the gross morphology of thalli, Qingjiangthallus yields a number of features of the Rhodophyta (red algae), including asymmetrically, dichotomously and laterally branching patterns, ribbon-shaped branches with sharp apices and short branchlets, and the absence of central axis and midribs.Thus, we temporarily place Qingjiangthallus into the crown-group Rhodophyta (red algae).
Four Proterozoic fossils have been reported to be related to the Rhodophyta, that is, Rafatazmia chitrakootensis Bengtson and Ramathallus lobatus Sallstedt from the Chitrakoot Formation (ca.1600 Ma) (Bengtson et al., 2017), Bangiomorpha pubescens Butterfield from the Hunting Formation (ca.1050 Ma) (Butterfield, 2000;Gibson et al., 2018), and algal thalli from the Weng'an biota (600-582 Ma) (Xiao et al., 2014).Rafatazmia chitrakootensis and Ramathallus lobatus are phosphatized microfossils, which are characterized by nonbranching filamentous thalli, diffuse growth by septation and possible pit connections and pit plugs, and lobate thallus with pseudoparenchyma, "cell fountains" and apical growth, respectively (Bengtson et al., 2017).Bangiomorpha pubescens was identified as a bangiacean red alga based on diagnostic cell-division patterns (Butterfield, 2000).Algal thalli with conspicuous pseudoparenchyma, spatial cell differentiation, and specialized reproductive structures from the Weng'an biota are three-dimensionally preserved in phosphorites, their thalli with cellular structures are micrometer-to millimeter-scale, and most features were obtained from thin sections (Xiao et al., 2014).The unbranched filaments of R. chitrakootensis and B. pubescens are obviously different from the present macroalgae.Microstructures of the present macroalgae, such as cellular morphology, differentiation of cortex and medulla and internal structure of possible reproductive organs, are not preserved, and hence comparisons with R. lobatus and algal thalli from the Weng'an biota are unavailable.The presumed red alga Dalyia racemata Walcott from the Burgess Shale (Cambrian Series 3, Stage 5) has a central stem and several slender branched stems terminating with a whorl comprising five to seven short, thin branchlets (Briggs et al., 1994).Its gross morphology is markedly different from that of the present macroalgae.
In the new phylogeny, Florideophyceae is divided into five subclasses, namely, Ahnfeltiophycidae, Corallinophycidae, Hildenbrandiophycidae, Nemaliophycidae, and Rhodymeniophycidae (Saunders & Hommersand, 2004;Le Gall & Saunders, 2007).The present macroalgae may have a fundamentally triphasic life history based on the occurrence of possible cystocarps.If this presumption is true, their morphological characteristics and triphasic life history can be further comparable with extant Rhodymeniophycidae, perhaps related to Gracilariales or Gigartinales.However, as a result of metamorphism, many diagnostic characteristics of the present macroalgae are not preserved, and we cannot ensure that they are linked throughout by pit connections, and the reliability of the interpretation of dark spots as cystocarps.Collectively considering the above discussion and the early age, we conservatively speculate that Qingjiangthallus cystocarpium probably has an affinity with the Florideophyceae (Fig. 6).

Evolutionary significance
As one of the oldest groups of eukaryotic algae (Baweja et al., 2016), red algae originated at the end of the Paleoproterozoic (1693 Ma) (Yang et al., 2016) or early Mesoproterozoic (1500 Ma) (Yoon et al., 2004) or 1400-1300 Ma (Lim et al., 1986) according to the results of molecular clock analyses.So far, the oldest reliable fossil record of red algae is ca.1050 Ma Bangiomorpha pubescens assigned to Bangiophyceae (Butterfield, 2000;Gibson et al., 2018), suggesting major red algal radiation at the end of the Mesoproterozoic (Yoon et al., 2010;Strassert et al., 2021).In addition, biomarker evidence indicates that red algae may have become abundant and commenced ecological expansion during the Tonian (ca.820 Ma) (Love & Zumberge, 2021).Their evolution is in accordance with the view that eukaryotes were present but not significant in Mesoproterozoic ecosystems, showing a protracted evolution based on a study of molecular fossils (Duda et al., 2021).Compared with other algae, their additional carposporophyte and tetrasporophyte phases are thought to be evolutionary innovations that explain the success of red algae (Yang et al., 2016).Carposporophytes produce and release numerous diploid carpospores to enhance the reproductive efficiency of the group.Cystocarp is one type of carposporophyte growing in the female thallus of Florideophyceae.However, its fossils are extremely rare.Phosphatized algal thalli from the Weng'an biota have been reported to have reproductive structures similar to cystocarps (Xiao et al., 2014).Qingjiangthallus has numerous possible cystocarps on most branches of the fossil thalli.The possible cystocarps in Weng'an algal thalli and Qingjiangthallus together indicate that the evolutionary innovation of a triphasic life cycle in red algae may have occurred no later than the Early Cambrian.
As the most diverse red algal class, Florideophyceae and Bangiophyceae diverged from a common ancestor ca.943 (817-1049) Ma (Yang et al., 2016) or 800 Ma (Lim et al., 1986) based on relaxed molecular clock analysis.Saunders & Hommersand (2004) 6).Qingjiangthallus is interpreted to be possibly comparable with Rhodymeniophycidae, which is a latediverging group of Florideophyceae (Yang et al., 2016).If this interpretation is correct, it suggests that the split between Ahnfeltiophycidae and Rhodymeniophycidae occurred ca.518 million years before the present or earlier, consistent with estimations of molecular studies (Fig. 6).
To circumvent phylogenetic challenges, early Paleozoic noncalcified macroalgae were categorized into nine morphogroups (LoDuca et al., 2017).These principal morphogroups were adapted by Bykova et al. (2020) to categorize Precambrian macroalgae, and one more morphogroup was added to contain fossils that do not fit in any of the nine categories in LoDuca et al. ( 2017).Precambrian macroalgae have been reported worldwide, and their morphological and evolutionary patterns have been documented in rather broad terms.From the Paleoproterozoic to the Ediacaran, macroalgae showed progressive increases in morphospace range, morphological disparity, functionalform groups (FFGs), and the maximum dimension (LoDuca et al., 2017).Especially in the Ediacaran, they experienced significant taxonomic, morphological, and ecological diversification (Xiao & Dong, 2006;Bykova et al., 2020).In addition to delicately dichotomously and stoloniform branched morphogroups, two new branching patterns, coarsely dichotomously and simple monopodial branched morphogroups (Chen et al., 1994;Nagovitsin et al., 2015), also occurred in Ediacaran assemblages.However, Cambrian macroalgae showed a decline in taxonomic diversity, morphological disparity, and FFGs compared with Ediacaran macroalgae (Wu, 2011;Bykova et al., 2020), which was probably caused by their potential taphonomic biases (Xiao & Dong, 2006), a possible extinction event in the last ca.10 Ma to the Ediacaran-Cambrian boundary (Bykova et al., 2020), and rapid change in the marine ecological environment during the Cambrian Explosion (Erwin & Tweedt, 2012;Zhang & Shu, 2014, 2021).No new morphogroup emerged in the Cambrian.In contrast, two branching morphogroups, that is, coarsely dichotomously and simple monopodial branched morphogroups, disappeared in the Cambrian fossil record (LoDuca et al., 2017).
The coarsely dichotomously branched morphogroup consists of macroalgal fossils whose thalli comprise cylindrical or ribbon-shaped elements with anisotomous or isotomous bifurcations, and most or all branches are >2 mm in width (LoDuca et al., 2017).Based on previous fossil records, this morphogroup makes its first appearance (also a single occurrence) in the Khatyspyt assemblages of the Ediacaran, but is absent in the Cambrian.However, it becomes abundant in the Ordovician and afterward (Bykova et al., 2020).Qingjiangthallus has ribbon-shaped branches with anisotomous or isotomous bifurcations and most branches (84%) are greater than 2 mm in width (Fig. 4A), and hence fits well in the coarsely dichotomously branched morphogroup.In this regard, Qingjiangthallus fills the Cambrian gap in the fossil record of this particular morphogroup.

Conclusion
Qingjiangthallus, assigned to the crown-group Rhodophyta, is the first known noncalcified fossil of red algae preserved as carbonaceous compressions.Its comparability with Rhodymeniophycidae and early age indicate that Ahnfeltiophycidae and Rhodymeniophycidae may have diverged during or before the Early Cambrian.The presence of abundant possible cystocarps on thalli indicates that the evolutionary innovation of the triphasic life cycle in red algae may have evolved no later than the Early Cambrian.Morphologically, the occurrence of Qingjiangthallus fills the gap of the coarsely dichotomously branched morphogroup in the Cambrian fossil record, suggesting an evolutionary continuity since the Ediacaran.

Fig. 1 .
Fig. 1.Geological map and stratigraphic column of study area (derived from Fu et al., 2019).A, Lithofacies map of the Yangtze Platform during Cambrian Stage 3, with type locality of the Qingjiang biota.B, Geological map of Changyang area, showing the distribution of the Cambrian Shuijingtuo Formation and fossil location at the Jinyangkou Section.C, Composite stratigraphic column of Changyang area, showing fossil horizons from the middle member of Shuijingtuo Formation.

Fig. 4 .
Fig. 4. Statistic diagrams showing ranges of the branch width and diameters of possible cystocarps.A, Branch width.Branches greater than 2 mm in width account for 84%.B, Length of major axis of possible cystocarps in dense portions (n > 32 per square centimeter), sparse portions (n ≤ 32 per square centimeter) and entire thalli (all data).C, Length of minor axis of possible cystocarps.

Fig. 5 .
Fig. 5. Morphological details and internal structures of possible cystocarps.A, C, Thalli with dense possible cystocarps (LELE1115 and LELE1180).B, Magnified image of the framed area in (A), showing possible cystocarps with striae on surface (indicated by arrows).D, Scanning electron microscope (SEM) image of a cross-section of a possible cystocarp cut from specimen in (C) using focused ion beams (FIB) (position indicated by the short red line in C).E, Thallus preserved as a thick carbonaceous film (LELE1137).F, G, Optical images of the thin section cut along the red line in (E), showing a section of thallus.H, I, Magnified images of the framed area in (F) and (G) to observe the internal structure of the possible cystocarps.Neither cells nor other structures is recognized.Scale bars = 5 mm (A, E), 1 mm (B, C), 4 μm (D), 200 μm (F, G), 20 μm (H, I).