Siliceous spicules in a vauxiid sponge (Demospongia) from the Kaili Biota(Cambrian Stage 5), Guizhou, South China

Fossils of the sponge Angulosuspongia sinensis from calcareous mudstones of the middle and upper part of the Kaili Formation (Cambrian Stage 5) in the Jianhe area of Guizhou province, South China, exhibit an apparently reticulate pattern, characteristic of the Vauxiidae. Energy Dispersive X-Ray Spectrometry (EDS) and Raman spectroscopy analysis indicate the presence of silica in the skeletal elements of these fossils, suggesting that this taxon possessed a skeleton comprised of spicules. This is the first confirmation of siliceous skeletal elements in fossils of the family Vauxiidae, and it lends support to the hypothesis that some early demosponges possessed biomineralized siliceous skeletons, which were subsequently lost and replaced by spongin later in the evolutionary history of this lineage. The new materials provide critical insight into the phylogeny and evolution of biomineralization in the Demosopongiae.

The earliest stages of animal evolution remain highly unclear 1 . Recent studies of molecular genetics suggest that sponges lay near the base of the animal phylogenetic tree [2][3][4] . Whether the sponges are monophyletic or polyphyletic is subject to interpretation 2 . Based on aspects of cellular structure and chemistry, some workers have favored the possibility that sponges have multiple origins from two or three different single-celled ancestors 5,6 . Sponges certainly have a deep evolutionary history 7 . Articulated body fossils and isolated spicules have been reported from the Ediacaran [8][9][10][11][12] and the earliest Cambrian [13][14][15][16] . Biomarker evidence suggests that sponges evolved even earlier, perhaps as early as the Cryogenian Period 17 . The majority of Proterozoic fossils interpreted as sponges, however, have been questioned 18 , owing to the presumed non-spicular nature of some 19 , which would have resulted in a poor fossil record of the group 20 . Thus, while fossils of early aspiculate sponges are unusual, they are essential in unraveling sponge origins and in understanding demosponge phylogeny and the history of spicule evolution.
To date, the best-known early aspiculate poriferans are those assigned to the family Vauxiidae Walcott, 1920, which is known primarily from the Cambrian. The Vauxidae are characterized an apparently reticulate, aspiculate fibrous skeleton 21 . The skeletal composition of the Vauxidae has been reinterpreted a number of times [21][22][23][24][25][26] , but recently, Ehrlich et al. 27 confirmed chitin in the skeleton of Vauxia gracilenta, and concluded that it was a "keratose" demosponge rather than mineralized spicules, and proposed that the Vauxiidae were likely to be the most basal definitive demosponge group known. This contrasts with a recent hypothesis that some aspiculate sponge skeletons were derived evolutionarily through demineralization of siliceous spicules and loss of spicules may have happened at least twice in the Demospongiae 20 . Botting et al. 28 identified spicules within skeletal strands of Vauxia bellula from the Cambrian Burgess Shale. On the basis of this evidence, Botting et al. 29 suggested that diactines were primitive for some Keratosa, and that spicules predate the appearance of the demosponge crown group and were subsequently lost in Myxosongiae and Keratosa.
This study seeks to test the loss of spicules hypothesis in order to shed light on the major pathways in the skeletal evolution of Demosopongiae and the skeletal composition of the Vauxiidae. For this reason we have studied a new genus and species, Angulosuspongia sinensis 30 25 . The Jianhe material illustrates apparent spicular structures, composed of silica and preserved in relief, which were previously unknown in the Vauxiidae. Although spicules were previously described in Vauxia bellula 28 the original mineralogy of its spicules is unkown. The Jianhe specimens add an interesting dimension to the evolutionary history of the Demospongea, as they clearly indicate that some early Paleozoic ancestors of nonbiomineralizing 'Keratosa' possessed siliceous spicules. The new material lends support to the hypothesis that some early demosponges possessed biomineralized siliceous skeletons, and later in their evolutionary history, silica was replaced by spongin.

Results
Skeletal composition. Taphonomic characteristics of the vauxiid sponge Angulosuspongia sinensis from the Kaili Biota suggest a rather rigid skeletal network, in contrast to flexible fibers comprised of spongin or chitin. Skeletons of Jianhe materials, although compacted in mudstone, are consistently preserved three-dimensionally, with robust rays extending beyond the margin of the sponge body (Figs 1 and 3a). This manner of preservation indicates that the skeletal elements were capable of resisting flattening during sediment compaction. Energy-dispersive X-ray spectroscopy (EDS) and elemental mapping were used to determine the composition of skeletal elements. The analyses reveal O and Si to be major components in both the sponge body and rock matrix, but the sponge spicules have far less Al, K and Fe than rock matrix, and the sponge body is enriched in C relative to the matrix ( Fig. 4; Table 1). It is worth noting that skeletons were more silicon-rich and with less carbon than the inner of polygonal openings produced by fused spicules (Fig. 4a,b,d,e). Raman spectroscopy analysis (Fig. 5) indicates that the vauxiid sponge skeletons of the Kaili Biota are composed of kerogen and silica and demonstrates that the kerogen is composed of geochemically moderately altered amorphous carbonaceous matter (interlinked polycyclic aromatic hydrocarbons) like that of other Burgess Shale-type fossils of Cambrian age 31,32 .
Confirmation of the presence of SiO 2 in the spicular skeleton provides strong support for the interpretation that this species possessed siliceous spicules. It is unlikely that silica is a secondary diagenetic product in specimens of vauxiid sponges of the Kaili Biota, as co-occurring, biomineralized brachiopods do not show evidence of mineral replacement (Fig. 4j-o). As is typical of the Burgess Shale-type preservation, high-fidelity preservation of labile soft tissues in fossils of the Kaili biota resulted primarily from conservation of primary organic remains,   Fig. 1b; (b) close-up view of the rectangular area in Fig. 1g. Crosses denote EDS points, crosses 1-3 in Fig. 2a,b mark sponge spicules, crosses 4-6 in Fig. 2a and 4-5 in Fig. 2b mark the inner of polygonal openings produced by fused spicules, and crosses 7-9 in Fig. 2a and 6-8 in Fig. 2b mark the surrounding matrix. All scale bars equal 0.5 mm. All specimens were photographed dry in reflected light.

Discussion
Prior to the description of the Jianhe materials, the family Vauxiidae was monogeneric, embracing only Vauxia. In Vauxia and putatively related sponges, three interpretations of skeletal composition have emerged.
Finks 24 regarded Vauxia as a specialized offshoot of the hexactinellid Protospongiidae. Implicit in this classification is a skeletal composition of opaline silica. Walcott 22 reported that in all specimens he examined from the Burgess Shale of British Columbia, Canada, the original siliceous matter of the spicules was removed and replaced by pyrite or a black carbonaceous material, or a combination of the two. (2) Keratose spongin fiber hypothesis. Rigby 25 reassigned the family Vauxiidae to the lithistid Demospongea on the basis of skeletal symmetry. He stated that the symmetry of Vauxia is more similar to the symmetry of the Demospongea than the Hexactinellida. Later, Rigby 26 concluded that the vauxiid skeleton was probably comprised of spongin fibers rather than biomineralized spicules, and Rigby and Collins 21 characterized the skeleton as having a double-layer construction of apparently fused keratose fibers. According to Ehrlich et al. 35 spongin in poriferans results from a hierarchical, multilevel organization of collagen microfibrils. The Maldonado 20 hypothesized that spongin skeletons evolved at least twice in the Demospongiae. Spicules, which he considered to have been siliceous, were, in his view, lost and replaced by spongin fibers at least once in the Chondrosida-Verongida lineage, and also in the Haplosclerida -Dictyoceratida -Dendroceratida lineage. The skeletal composition of the Jianhe specimens are intriguingly consistent with Walcott's 22 interpretation that Vauxia from the Burgess Shale originally possessed siliceous spicules that were later replaced, and offer support for Maldonado's 20 hypothesis that spongin fibers replaced siliceous spicules in some demosponge lineages. These findings support the view that spicules were present among skeletal strands of Vauxia bellula 28   originally siliceous composition. This new information does not entirely refute Rigby's 26 interpretation of the Vauxiidae as non-biomineralized, as siliceous spicules appear to have been present in some species, but not others. The simultaneous occurrence of spicules and spongin fibers in vauxiid sponges may also support Botting's hypothesis of a single origin of spicules prior to the appearance of crown-group Silicea, and the subsequent loss of spicules in early 'keratosan' sponges 28,29 rather than the traditional and molecular-based views of demosponge phylogeny 36 .

Methods
Sixty-two specimens of vauxiid sponge remains were examined in this study. They are housed at the Guizhou Research Center for Paleontology, Guizhou University, Guiyang, China (GRCP, GU). Some specimens (Figs 1  and 2) were imaged using a Canon EOS Rebel T3i Digital SLR camera with MP-E 65 mm macro lens. Others (Figs 3 and 4) were imaged at finer scale using a LEO1530VP Scanning Electron Microscope (SEM) equipped with an Energy-dispersive X-ray Spectrometer (EDS), located in the State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences. One sample, GM-16-1192 was gold-coated, whereas GTBM9-4598a imaged under the SEM were left uncoated. Raman spectroscopy analyses for skeletal composition of vauxiid sponges from the Kaili Biota were performed on an Invia model Raman spectrograph of the Renishaw company. In this instrument, two laser devices with wavelengths of 514 nm and 785 nm excite monochromatic light. Experiment conditions: optical laser wavelengh was 514 nm, scanned area was 100 to 2000 cm −1 , time of exposure was 10 s.