Character states of the mineralized chondrichthyan endoskeleton

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 non-continuous pattern of mineralized tissues in neural arches of segmented vertebrae in ratfish (A), compared to the continuous mineralization pattern of compact, bone-like tissue in catshark (B) and little skate (C). (D-F) Micro-CT 2D virtual slices showed that mineralized tissues (arrows) in ratfish neural arches were restricted to medial and lateral peripheries (D), whereas the bone-like tissue in catshark and little skate extended much deeper in the neural arch (E,F). (G-I) Alizarin red staining of histological sections confirmed micro-CT representation of mineralization patterns in ratfish (G), catshark (H), and little skate (I) neural arches, and Alcian blue revealed cartilaginous regions. (J-L) Picrosirius red staining did not reveal birefringent collagen fibers in ratfish neural arches (J), but such staining was abundant in bone-like tissues in catshark (K) and little skate (L). Birefringent fibers (likely reticular) were present in muscles (vertical arrows) on the lateral sides of neural arches in all species. Col2 immunostaining confirmed Alcian blue staining, identifying cartilage regions in all species. The neural arch core in catshark (K) and little skate (L) consisted of mostly unmineralized cartilage and mineralized cartilage, respectively. 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.

Ratfish, catshark, and little skate all showed trabecular mineralization, but ratfish and catshark did not have a bone-like cap zone.

(A-H) Micro-CT renderings of the ratfish ceratohyal (A,E) and catshark caudal vertebra (B,F) demonstrated non-tesseral trabecular mineralization, while little skate hyomandibula had tesseral trabecular mineralization (C,G). Little skate precaudal vertebra had tesseral polygonal mineralization (D,H) overlying tesseral trabecular mineralization (H’). Regions for hi-mag views in panels E-H’ are indicated in corresponding panels of A-D. (I-P) Alcian blue/Alizarin red and Safranin O/fast green staining of histological sections demonstrated that the body zone of non-tesseral or tesseral trabecular mineralized tissues in ratfish (I,M), catshark (J,N), and little skate (K,L,O,P) was mineralized cartilage, while the cap zone of tesseral polygonal mineralization was bone-like (K,L,O,P). (Q-T) Col2 immunostaining of adjacent sections to panels I-P confirmed cartilage regions in all species, while picrosirius red staining confirmed the bone-like tesseral cap zone 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 and mineralization patterns.

(A-C) Trichrome staining showed that spokes in the body zone of ratfish non-tesseral trabeculae (A) or little skate trabecular (B) or polygonal (C) tesserae had low cellularity and were unstained, while intense acid fuchsin staining marked the smaller and larger cap zones of little skate trabecular (B) and polygonal (C) tesserae, respectively. (D-I) 3D renderings of synchrotron micro-CT data showed that hypermineralized spokes (*, segmented in blue) were in the body zone of ratfish non-tesseral trabeculae (D,G) or little skate trabecular (E,H) or polygonal (F,I) tesserae. Tesseral cap zones could also be segmented clearly (red) in little skate (E,F,H,I). (J-O) Segmented 3D renderings demonstrated that spokes in ratfish were patchy in deep (J) or superficial (M) views, while little skate spokes displayed a radiating pattern traversing most of the trabecular patterns in either trabecular (K,N) or polygonal (L,O) tesserae. The smaller cap zone in trabecular tesserae (N) was not laterally extensive enough to display a superficial polygonal pattern like that of polygonal tesserae (O). Abbreviations: Bz, body zone; Cz, cap zone. 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 ratfish (A), catshark (B), and little skate (C) all had a compact mineralization, but ratfish centra were organized in multiple rings and did not display the gross biconcave morphology seen in catshark or little skate. (D-O) Alcian blue/Alizarin red and Safranin O section histology and Col2 immunostaining demonstrated that mineralized centra in ratfish (D,G,J), catshark (E,H,K), and little skate (F,I,L) all comprised a middle layer with elongate lacunae in concentric lamellae, but in ratfish this was mineralized cartilage, while catshark and little skate had little to no Safranin O or Col2 staining, respectively, in this region. Catshark mineralized centra also comprised the outer layer, while little skate mineralized centra also comprised both inner and outer layers. These additional centra layers had rounded cells embedded in matrix of weak Safranin O staining and Col2 immunofluorescence. 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. 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 were shared between little skate embryos and juveniles and adult ratfish.

(A-D) Desktop micro-CT renderings showed centra and other mineralized structures in vertebral segments (dashed lines) of little skate embryos and juveniles (A-C) and adult ratfish (D). (E-H) Synchrotron 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 extant and fossil chondrichthyan species suggest shared and derived features.

Non-tesseral trabecular mineralization (NTT) is likely a symplesiomorphic (shared ancestral) character of stem and crown chondrichthyans, because it is pervasive in fossil (Coates et al., 2018; Maisey et al., 2021; Pears et al., 2020) and extant (Berio et al., 2021; Seidel et al., 2020) chondrichthyans. Polygonal (PT) and trabecular (TT) tesseral mineralization patterns appear to be absent in extant holocephalans and stem chondrichthyans, but their presence in some extant elasmobranchs (Atake et al., 2019; Berio et al., 2022; Berio et al., 2021; Seidel et al., 2016; Wilga and Ferry, 2015) and a fossil holocephalan (Pears et al., 2020) suggest that these tesseral mineralizations are synapomorphic (shared derived) characters of crown chondrichthyans, subsequently lost in extant holocephalans. Areolar mineralized tissue is present in extant elasmobranchs (Atake et al., 2019; Berio et al., 2021; Criswell et al., 2017; Eames et al., 2007; Porter et al., 2016; Seidel et al., 2020) and holocephalans, but its absence in stem chondrichthyans suggests that it is a synapomorphic character of crown chondrichthyans. The absence of neural arch bone-like tissue (BLT) in holocephalans further supports the conclusion that it is a synapomorphic character of elasmobranchs. 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. The histological cap zone in some elasmobranchs (Atake et al., 2019; Berio et al., 2021; Seidel et al., 2016; Wilga and Ferry, 2015), along with the inference that polygonal tesserae in a fossil holocephalan means that they had cap zones (Pears et al., 2020), suggests that the cap zone is also a synapomorphic character of crown chondrichthyans. 0, 1, andindicate negative, positive, and unknown observations, respectively, of characters for the listed species. References to data from the listed species are cited in the legend