Mineralized tissues were abundant in the spotted ratfish, but they had lower tissue mineral density (TMD) compared to the little skate and the small-spotted catshark. (A) Photograph of the ratfish Hydrolagus colliei illustrating the anterior region that was micro-CT imaged. (B-G) Micro-CT 3D renderings of the anterior region of ratfish (B) showed mineralized tissues in neural arches, basidorsals, and centra in segmented vertebrae (C,D), in the ceratohyal and other skeletal elements in the pharyngeal skeleton (E), and in the fused vertebral synarcual (F,G). (H) Comparative quantitative analyses showed that TMD of the synarcual, neural arch, and centra in ratfish were significantly lower than TMDs of same regions in little skate and catshark. Abbreviations: A, anterior; bb, basibranchial; bd, basidorsal; bh, basihyal; c, centrum cb, ceratobranchial; ch, ceratohyal; D, dorsal; hbs, hypobranchials; L, left; MK, Meckel’s; na, neural arch; P, posterior; R, right; snf, spinal nerve foramina; V, ventral. * indicates statistically significant comparison (p<0.05). Scale bars: as indicated.

Ratfish lack neural arch bone-like tissue. (A-C) Micro-CT 3D renderings showed a compact mineralization pattern of bone-like tissue in neural arches of segmented vertebrae in little skate (A) and catshark (B), and an irregular pattern of mineralized tissues in ratfish (C). (D-F) Micro-CT 2D virtual slices showing that the bone-like tissue in little skate and catshark was extensively mineralized and surrounded a neural arch core (D,E), whereas irregular mineralized tissues in ratfish neural arches were restricted to medial and lateral periphery (F). (G-L) Section histology showed Alizarin red staining of continuous mineralization and birefringent collagen fibers in neural arch bone-like tissues in little skate (G,J) and catshark (H,K), but not in ratfish (I,L). Picrosirius red staining also revealed some birefringent fibers (likely reticular) in muscles on the lateral sides of neural arches in little skate, catshark, and ratfish (J-L). Irregular mineralized tissue in the ratfish neural arch was discretely organized, Alizarin red-positive, and Col2-positive mineralized cartilage (I,L). Other regions of ratfish were Alcian blue-positive and Col2-positive unmineralized cartilage (I,L). The neural arch core in little skate and catshark consisted of mineralized cartilage (MC) and mostly unmineralized cartilage, respectively (J,K). Abbreviations: 2D, two-dimensional; 3D, three-dimensional; Ab, Alcian blue; Ar, Alizarin red; BLT, bone-like tissue; c, cartilage; Col2, collagen type 2; D, dorsal; IF, immunofluorescence; L, lateral; M, medial; MC, mineralized cartilage; PSR, picrosirius red; V, ventral. Scale bars: as indicated.

Little skate, catshark, and ratfish all showed trabecular mineralization but without the bone-like cap zone in catshark and ratfish. (A-D) Micro-CT renderings of the little skate hyomandibula and precaudal vertebra (A,B), catshark caudal vertebra (C), and ratfish ceratohyal (D). (E-H) Tesseral and non-tesseral mineralization patterns in little skate, catshark, and ratfish. The little skate hyomandibula displayed a superficial polygonal tesseral pattern (E’) and an underlying trabecular tesseral pattern (E”), while regions of the little skate neural spine only displayed a trabecular tesseral pattern (F). The catshark haemal arch (G), and ratfish ceratohyal (H) displayed a non-tesseral trabecular pattern. Regions for hi-mag views in panels E’-H are indicated in corresponding panels of A-D. (I-T) Histological and immunofluorescent features of tesseral or non-tesseral mineralized tissues in little skate (I,J,M,N,Q,R), catshark (K,O,S), and ratfish (L,P,T). Alizarin red staining, Safranin O staining, and Col2 immunofluorescence showed that little skate, catshark, and ratfish had the body zone of mineralized cartilage (I-T). The bone-like tesseral cap zone consisting of elongate lacunae in matrix that is mineralized (I,J), Safranin O-negative (M,N), Col2-negative (Q,R), and contains birefringent collagen fibers (Q,R) was present only in polygonal tesserae and trabecular tesserae in little skate. Abbreviations: Ab, Alcian blue; Ar, Alizarin red; Bz, body zone; c, cartilage; Col2, collagen type 2; Cz, cap zone; D, dorsal; IF, immunofluorescence; L, lateral; M, medial; PSR, picrosirius red; NA, neural arch; NS, neural spine; V, ventral. Scale bars: as indicated.

Micro-CT segmentation clearly correlated histological features of mineralized tissues with their mineralization patterns. (A-J) Trichrome’s acid fucshin marked the larger and smaller cap zones of little skate polygonal and trabecular tesserae, respectively (A,F). 3D renderings of polygonal tesserae showed that the cap zone (Cz, segmented in red) was superficial and reflected the polygonal pattern (B-D). The smaller cap zone in trabecular tesserae was not laterally extensive enough to display a superficial polygonal pattern like that of polygonal tesserae (B-D,G-I). Spokes in polygonal and trabecular tesserae did not stain with Trichrome (* in A,F). 3D renderings showed that spokes (*, segmented in blue) in the deeper body zones displayed a radiating pattern like the trabecular patterns in little skate polygonal and trabecular tesserae (A-C,E,F-J). (K-O) Trichrome staining demonstrated that trabecular mineralized tissue in ratfish lacked a cap zone but had other features, such as a body zone and intertesseral fibres (K). Segmentation demonstrated that spokes in ratfish were patchy and did not display a radiating trabecular pattern (K-O). Abbreviations: Bz, body zone; Cz, cap zone; ITF, intertesseral fibres. Scale bars: as indicated.

Ratfish centra had the same features as areolar mineralized tissues in little skate and catshark. (A-C) Micro-CT renderings demonstrated that centra in little skate and catshark had a compact mineralization and a gross biconcave morphology (A,B), while centra in ratfish also displayed a compact mineralization pattern but were organized in multiple rings (C). (D-O) Consistent with areolar mineralized tissue in little skate (D,G) and catshark (E,H), Alcian blue/Alizarin section histology (Ab/Ar) showed that mineralized centra in ratfish consisted of a mineralized areolar centra layer with elongate lacunae in concentric lamellae (F,I). Regions for hi-mag views in panels G-O are indicated in corresponding panels of D-F; regions for hi-mag inserts in panels G-I are indicated in those panels. Areolar mineralized tissue in little skate lacked Safranin O staining and Col2 immunofluorescence (J,M), while catshark areolar mineralized tissue demonstrated weak Safranin O staining and Col2 immunofluorescence (K,N). Safranin O staining and Col2 immunofluorescence highlighted cartilaginous matrix of areolar mineralized tissue in ratfish (L,O). Little skate also had mineralization in both outer and inner centra layers (G), while catshark also had an outer mineralized layer (H), and these additional centra layers demonstrated weak Safranin O staining and Col2 immunofluorescence (J,M,K,N). Abbreviations: A, anterior; Ab, Alcian blue; Ar, Alizarin red; c, cartilage; Col2, collagen type 2; D, dorsal; IF, immunofluorescence; IL, inner layer; L, lateral; M, medial, OL, outer layer; P, posterior; V, ventral. Scale bars: as indicated; inserts in G-I are 20 µm.

Morphological and histological features of centra in little skate embryos and juveniles suggested that the adult ratfish demonstrated paedomorphic centra. (A-D) Desktop micro-CT renderings showed centrum and other mineralized structures in vertebral segments (dashed lines) of little skate embryos and juveniles (A-C) and adult ratfish (D). (E-H) Synchrotron radiation micro-CT renderings of centra in little skate embryos and juveniles (E-G) and adult ratfish (H) showed that centra in the ratfish were slightly constricted like those of little skate embryos (red arrows). (I-K) Alcian blue/Alizarin red section histology showed that areolar mineralized tissue constituted the only centra layer in little skate embryos (I,J), whereas an outer mineralized centra layer was present in 6.5 cm DW juveniles (K). (L) Tissue mineral density (TMD) of centra in ratfish was similar to those of stage 33 embryos and 6.5 cm DW juveniles of little skate. Abbreviation: A, anterior; Ab, Alcian blue; Ar, Alizarin red; bd, basidorsal; C, centrum; HA, haemal arch; L, lateral; M, medial; NA, neural arch; OL, outer layer; P, posterior.* indicates statistically significant comparison (p<0.05). Scale bars: as indicated.

Character states of mineralized tissues in various chondrichthyan clades suggest shared and derived features. The absence of neural arch bone-like tissue (BLT) in holocephalans and ancestral crown chondrichthyans (from fossils) would further support the conclusion that neural arch bone-like tissue is a synapomorphic (shared derived) character of elasmobranchs. Polygonal (PT) and trabecular (TT) tesseral mineralization patterns appear to be absent in extant holocephalans and stem chondrichthyans, but their presence in some elasmobranchs and some fossil holocephalans suggest that these tesseral mineralizations are synapomorphic characters of crown chondrichthyans. Non-tesseral trabecular mineralization (NTT) is a symplesiomorphic (shared ancestral) character of stem and crown chondrichthyans, because it is pervasive in ancestral and extant chondrichthyans. The histological cap zone in some elasmobranchs, along with the inference that polygonal tesserae in fossil holocephalans means that they had cap zones, suggests that the cap zone is also a synapomorphic character of crown chondrichthyans. The presence of a histological body zone in either tesseral or non-tesseral mineralized tissues of stem and crown chondrichthyans suggests that the body zone is a symplesiomorphic character of all chondrichthyans. Areolar mineralized tissue is present in elasmobranchs and holocephalans, but its absence in stem chondrichthyans suggests that it is a synapomorphic character of crown chondrichthyans.