Developmental tuning of mineralization drives morphological diversity of gill cover bones in and their relatives

Eli G. Cytrynbaum, Clayton M. Small, Ronald Y. Kwon, Boaz Hung, Danny Kent, Yi-lin Yan, Matthew L. Knope, Ruth A. Bremiller, Thomas Desvignes, Charles B. Kimmel

Supplementary Information

Supplementary Figure Legends

Figure S1. Nonmineralized, “extended osteoid” tissue is a prominent feature of gill cover bones. A. A simplified cartoon of three major gill cover bones, drawn from the “fork- bearing” maculosus. The opercle (OP) in red, the subopercle (SOP) in purple, and the interopercle (IOP) in blue are drawn to emphasize mineralized (dark) and osteoid (light) regions. B-C. Actual images of an O. maculosus IOP and SOP, respectively, with mineralized bone stained pink by Alizarin Red and extended osteoid shown by the transparent, unstained regions. (See Fig. 1 of the main text for an Alizarin Red-stained OP image of this .)

Figure S2. Extended osteoid also exists in eelpouts (Zoarcidae). Adult Ophthalmolycus amberensis (A.) and Lycenchelys tristichodon (B.) OPs stained with Alizarin Red, showing lack of stain in the region of extended osteoid. Cross sections of Ralis-Watkins-stained O. amberensis (C.-C.') and L. tristichodon (D.-D.') gill covers, with the mineralized portions of the OP and SOP in red and the extended osteoid in blue, showing a lack of mineralization in the extended osteoid region. bsr = branchiostegal ray. E. In O. amberensis, extended osteoid and the resulting "fork" morphology appear after an established "fan" shape, relatively late in OP development. TL = total length.

Figure S3. Cells in extended osteoid regions express sp7, a transcriptional marker for osteoblasts. A. A juvenile Oligocottus maculosus OP and SOP. Deep purple indicates in situ hybridization signal for sp7, expressing most strongly along the growing edges of the extended osteoid on the posterior ventral edges of the OP and SOP, but also to a lesser extent in non-extended osteoid regions. B. Same image for a juvenile globiceps.

Figure S4. OP morphology is strikingly variable across the Cottoidea radiation. Note the variation in this phylogenetically diverse panel of Alizarin Red-stained adult OPs and the clear “growth bands” continuous between extended osteoid and mineralized tissues in E. bison and C. analis. Not only do the bones differ with respect to extended osteoid, but they are highly variable regarding general 2-D shape, thinness, presence of “trabeculae” (e.g. I. cavifrons and E. bison) and presence of ridges (e.g. D. setiger).

Figure S5. The major axis of OP shape variation in cottoids and several outgroups is strongly associated with the proportion of OP area that is extended osteoid. A. A standardized major axis regression (SMA) model fit to the data shows the negative relationship between proportion of extended osteoid and PC1 score. The solid line was drawn using slope and intercept estimates from the model, whereas the dashed lines reflect 95% confidence intervals for these estimates. Families are colored according to the scheme from Fig 3 and Fig 5. B. The same SMA regression approach, but applied to phylogenetically independent contrasts (PIC) to account for phylogenetic structure in the dataset.

Supplementary File Legends

Supplementary File 1. A .xlsx file containing species information and specimen sources, for each species used in the study. OCA = Oregon Coast Aquarium, OS = Oregon State University, UW = University of Washington Burke Museum, VA = Vancouver Aquarium.

Supplementary File 2. A .fasta file containing an alignment of assembled sp7 DNA sequences used to design probes for in situ hybridization. Included are protein-coding sequences from Cottus perplexus, Clinocottus globiceps, and Oligocottus maculosus. 䄀 伀倀 䐀

䄀 倀 嘀 匀伀倀

䤀伀倀 䜀 椀 氀 氀 䌀 漀 瘀 攀 爀 ㄀洀洀

䈀 䌀

Fig S1 A O. amberensis B L. tristichodon

2 mm 2 mm

C O. amberensis C'

OP

C'

OP osteoid SOP

SOP

Skin 500 µm 200 µm D L. tristichodon D' Gills

Skin OP D'

SOP OP osteoid

bsr SOP 500 µm 200 µm

E O. amberensis

2 mm

12.0 cm TL 16.2 cm TL 27.6 cm TL Fig S2 䄀 伀氀椀最漀挀漀琀琀甀猀 洀愀挀甀氀漀猀甀猀䈀 䌀氀椀渀漀挀漀琀琀甀猀 最氀漀戀椀挀攀瀀猀

伀倀 伀倀

攀搀最攀 漀昀 伀倀 漀猀琀攀漀椀搀 攀搀最攀 漀昀 伀倀 漀猀琀攀漀椀搀

Fig S3 匀伀倀 ⸀㔀 洀洀 匀伀倀 ㄀ 洀洀 䠀攀洀椀氀攀瀀椀搀漀琀甀猀 最椀氀戀攀爀琀椀 䤀挀攀氀椀渀甀猀 挀愀瘀椀昀爀漀渀猀 䈀氀攀瀀猀椀愀猀 挀椀爀爀栀漀猀甀猀

⸀㔀 洀洀 ⸀㜀㔀 洀洀 ⸀㔀 洀洀

䔀渀漀瀀栀爀礀猀 戀椀猀漀渀 䌀栀椀琀漀渀漀琀甀猀 瀀甀最攀琀攀渀猀椀猀 䌀氀椀渀漀挀漀琀琀甀猀 愀渀愀氀椀猀

⸀㔀 洀洀 ㈀⸀㔀 洀洀 ⸀㜀㔀 洀洀

䐀愀猀礀挀漀琀琀甀猀 猀攀琀椀最攀爀 䌀氀椀渀漀挀漀琀琀甀猀 愀挀甀琀椀挀攀瀀猀 䐀

䄀 倀

㈀⸀㔀 洀洀 ⸀㔀 洀洀 嘀Fig S4 Fig S5 File S1

Genus Species Short ID Common Name Source Suborder Cottoidei? Family Artedius harringtoni Ahar scalyhead sculpin OS 11055 Yes Psychrolutidae Artedius fenestralis Afen padded sculpin author-collected Yes Psychrolutidae Artedius corallinus Acor coralline sculpin author-collected Yes Psychrolutidae Aspidophoroides monopterygius Amon alligatorfish OS 8551 Yes Agonidae Blepsias cirrhosus Bcir silverspotted sculpin OS 551 Yes Agonidae Chitonotus pugetensis Cpug roughback sculpin OS 5269 Yes Psychrolutidae Clinocottus embryum Cemb calico sculpin OS 3357; 5782 Yes Psychrolutidae Cacu sharpnose sculpin VA Yes Psychrolutidae Clinocottus analis Cana woolly sculpin author-collected Yes Psychrolutidae Clinocottus globiceps Cglo mosshead sculpin author-collected Yes Psychrolutidae Cottus rhotheus Crho torrent sculpin OS 5269 Yes Cottus gulosus Cgul riffle sculpin author-collected Yes Cottidae Cottus asper Casp prickly sculpin author-collected Yes Cottidae Cottus perplexus Cper reticulate sculpin author-collected Yes Cottidae Dasycottus setiger Dset spinyhead sculpin OS 15251 Yes Psychrolutidae Enophrys bison Ebis buffalo sculpin OS 5945 Yes Psychrolutidae Enophrys diceraus Edic antlered sculpin OS 11133 Yes Psychrolutidae Enophrys taurina Etau bull sculpin author-collected Yes Psychrolutidae Gasterosteus aculeatus Gacu threespine stickleback Kimmel et al. 2017 No (outgroup) Gasterosteidae Hemilepidotus hemilepidotus Hhem red Irish lord OS 3248; VA Yes Agonidae Hemilepidotus gilberti Hgil Gilbert's Irish lord author-collected Yes Agonidae Hexagrammos decagrammus Hdec kelp greenling Kimmel et al. 2017 No (outgroup) Hexagrammidae Icelinus borealis Ibor northern sculpin UW 0151700 Yes Psychrolutidae Icelinus burchami Ibur dusky sculpin author-collected Yes Psychrolutidae Icelinus cavifrons Icav pit-head sculpin author-collected Yes Psychrolutidae Jordania zonope Jzon longfin sculpin UW 018012 Yes Jordaniidae Leptocottus armatus Larm Pacific staghorn sculpin author-collected Yes Cottidae Liparis gibbus Lgib variegated snailfish UW 119087 Yes Liparidae Liparis florae Lflo tidepool snailfish Kimmel et al. 2017 Yes Liparidae Lycenchelys tristichodon Ltri eelpout author-collected No (outgroup) Zoarcidae Micropterus dolomieu Mdol smallmouth bass Kimmel et al. 2017 No (outgroup) Centrarchidae Micropterus salmoides Msal largemouth bass Kimmel et al. 2017 No (outgroup) Centrarchidae Myoxocephalus polyacanthocephalus Mpol great sculpin OS 7011; VA Yes Psychrolutidae Nautichthys oculofasciatus Nocu sailfin sculpin OS 3197 Yes Agonidae Oligocottus maculosus Omac author-collected Yes Psychrolutidae Oligocottus snyderi Osny fluffy sculpin author-collected Yes Psychrolutidae Ophiodon elongatus Oelo lingcod Kimmel et al. 2017 No (outgroup) Hexagrammidae Ophthalmolycus amberensis Oamb eelpout author-collected No (outgroup) Zoarcidae Pholis ornata Porn saddleback gunnel Kimmel et al. 2017 No (outgroup) Pholidae Pholis gunnellus Pgun rock gunnel Kimmel et al. 2017 No (outgroup) Pholidae Porocottus camtschaticus Pcam NA UW 042695 Yes Psychrolutidae Pungitius pungitius Ppun ninespine stickleback Kimmel et al. 2017 No (outgroup) Gasterosteidae Radulinus asprellus Rasp slim sculpin author-collected Yes Psychrolutidae Rhamphocottus richardsonii Rric grunt sculpin OCA Yes Rhamphocottidae Scorpaenichthys marmoratus Smar cabezon author-collected Yes Scorpaenichthyidae Sebastes maliger Smal quillback rockfish Kimmel et al. 2017 No (outgroup) Sebastidae Sebastes auriculatus Saur brown rockfish Kimmel et al. 2017 No (outgroup) Sebastidae File S2

>Cper_sp7_CDS ATGGCCGCATCTATTCTGGAGGTAGGGAATGTAATTGAAGACGCACGCTATGGCTCCAGT CCTCTGGCTATGTTAACGGCTACCTGTAACAAGTTCGGCAGCACAAGCCCCATCAGGGAT TCGGCTACACCCGGTAAGATTGGCAGCGCTATTCCAGTAAAGAAGCTCTACACCATGACC TCTGACCTTCAGACGGCGAAGAACGGACGGACCGCAGACGGCAGTGGCCTGGCAGACTCC TACACTGGCTCCTTCACCACAGCTGGAGGAGGAGGAGGTGGCGGGCTGCTTACCCCCAAT GGAAGCCCCCCTCCTTCCGCCGGAGGCTACACTACAGAATATAACCCTTTCTCCCACTCC TTCCAAACCTCCATCACCCAGGACCCATCTCTTTTAGTGTCCAAGGGCCACGCGACAGCT GACTGCCTCACCAGTGTCTACACCTCGCTGGATATGACGCACCCTTATGGCTCCTGGTAT AAAGCCGGTATCCACCCTGGCATTACTGCTGCTCCGGCTAATGCCACGTCCTCCTGGTGG GATGTCCACCCCAACTCCAACTGGCTGTCCTCAACCCAGCCCCAGGTGGACGGAGGTCTC CAGGCCTCCCTGCAGCCTGTGGCACAACAGGCCTCCCTGAGCCCACAGTTGCCCAGTTAC AACACCGACTTTACATCACTCAACCCGGCTTCTTACCCGTCTGTGGGGCTGGGCTCCTCC TCACACCTCCTCCAGCCCTCTCAGCACATGCTGCCCCAGGACATGTACAAGCCCAAGCCT GTGCCAAGTGCAGGGATGATTGAGAGTCCCATGGGCCTAAAGCCTGCTCGGGGATCAGGG GGCTACAGCGGAGGGGGCGCGCCCACCAGGTCCTCATGTGACTGCCCCAACTGCCAGGAG CTGGAGAGGCTGGGAGCCTCAGCTGCATCCCTGAGGAAGAAGCCAGTCCACAGCTGCCAC ATCCCAGGCTGTGGGAAAGTCTACGGCAAGGCCTCCCACCTCAAGGCCCACCTGCGCTGG CACACTGGCGAGCGGCCCTTTGTTTGCAACTGGCTGTTCTGCGGGAAGCGTTTCACCCGC TCGGACGAACTGGAGAGGCACGTGCGCACCCACACGCGGGAGAAGAAGTTCACCTGTCTA CAGTGCAACAAGCGTTTCACACGCAGCGACCACCTCTCCAAGCACCAGAAGACCCATGCA GAGTCTGCAATGCAGGGTAAAGCGGTAGCGGTGGAGGGAGAGACAGATCCTCGGAGTGAG GAGACCACGGAGCTCAACGCCAGCGCGGTGCCCACCAATCCTGTCACCGACCAAATCACC AACGGAGATGAGAAGACGGGCACACCGAATGGAGTGGAGAACAGCAGTGGACTGTTGGAG ATCTGA >Cglo_sp7_CDS ATGGCCGCATCTATTCTGGAGGTAGGGAATGTAATTGAAGACGCACGCTATGGCTCCAGT CCTCTGGCTATGTTAACTGCTACCTGTAACAAGTTTGGCAGCACAAGCCCCATCAGGGAT TCGGCTACACCCGGTAAGATTGGCAGCGCTATTCCAGTAAAGAAGCTCTACACCATGACC TCTGACCTTCAGGCAGCGAAGAACGGACGGACCGCAGACGGCAGTGGCCTGGCAGACTCC TACACTGGCTCCTTCACCACAGCTGGAGGAGGAGGAGGTGGCGGGCTGCTTACCCCCAAT GGAAGCCCCCCTCCTTCGGCCGGAGGCTACACTACAGAGTATAACCCTTTCTCCCACTCC TTCCAAACCTCCATCTCCCAGGACCCGTCTCTTTTAGTGTCCAAAGGCCACGCTACAGCT GACTGCCTCACCAGTGTCTACACCTCGCTGGATATGACGCACCCTTATGGCTCCTGGTAT AAAGCCGGTATCCACCCTGGCATTACTGCCGCTCCGGCGAATGCCACGTCATCCTGGTGG GATGTCCACCCCAACTCCAACTGGCTGTCATCAACCCAGCCCCAGG---ACGGAGGTCTC CAAGCCTCCCTGCAGCCTGTGGCACAACAGGCCTCCCTGAGCCCACAGTTGCCCAGTTAC AACACCGACTTTACACCACTCAACCCGGCTTCTTACCCTTCCGTGGGGCTGGGCTCCTCC TCACACCTCCTCCAGCCCTCTCAGCACATGCTGCCCCAGGACATGTACAAGCCCAAGCCT GTGCCAAGTGCAGGGATGATTGAGAGTCCCATGGGCCTAAAGCCCGCTCGGGGATCAGGG GGCTACAGCGGAGGGGGCGCGCCCACCAGGTCCTCATGTGACTGCCCCAACTGCCAGGAG CTGGAGAGGCTGGGGGCCTCGGCTGCATCCCTGAGGAAGAAGCCAGTCCACAGCTGCCAC ATCCCAGGCTGTGGGAAAGTCTACGGCAAGGCCTCCCACCTCAAAGCCCACCTGCGCTGG CACACTGGCGAGCGGCCCTTTGTTTGCAACTGGCTGTTCTGCGGGAAGCGTTTCACCCGC TCGGACGAACTGGAGAGGCACGTGCGCACCCACACGCGGGAGAAGAAGTTCACCTGTCTA CTGTGCAACAAGCGTTTCACACGCAGCGACCACCTCTCCAAGCACCAGAAAACCCATGCA GAGTCCGCAATGCAGGGCAAAGCTGTAGCTGTGGAGGGAGAGACAGATCCTCGCAGTGAG GAGACCACAGAGCTCAACGCCAGCGCTGTGCCCACCAATCCTGTCGCTGACCAAATCACC AACGGAGATGAGAAGACCGGCACACCTAATGGAGTGGAGAACAGCAGTGGACTGTTGGAG ATCTGA >Omac_sp7_CDS ATGGCCGCATCTATTCTGGAGG------AAGACGCACGCTATGGCTCCAGT CCTCTGGCTATGTTAACTGCTACCTGTAACAAGTTCGGCAGCACAAGCCCCATCAGGGAT TCGGCTACACCCGGTAAGATTGGCAGCGCTATTCCAGTAAAGAAGCTCTACAACATGACC TCTGACCTTCAGGCAGCGAAGAACGGACGGACCGCAGACGGCAGCGGCCTGGCAGACTCC TACACTGGCTCCTTCACCACAGCTGGAGGAGGAGGAGGTGGCGGGCTGCTTACCCCCAAT GGAAGCCCCCCTCCTTCGGCCGGAGGCTACACTACAGAGTATAACCCTTTCTCCCACTCC TTCCAAACCTCCATCTCCCAGGACCCGTCTCTTTTAGTGTCCAAAGGCCACGCTACGGCC GACTGCCTCACCAGTGTCTACACCTCGCTGGATATGACGCACCCTTATGGCTCCTGGTAT AAAGCCGGTATCCACCCTGGCATTACTGCCGCTCCGGCGAATGCCACGTCCTCCTGGTGG GATGTCCACCCCAACTCCAACTGGCTGTCATCAACCCAGCCCCAGG---ACGGAGGTCTC CAAGCCTCCCTGCAGCCCGTGGCGCAACAGGCCTCCCTGAGCCCACAGTTGCCCAGTTAC AACACCGACTTTACACCACTCAACCCGGCTTCTTACCCTTCCGTGGGGCTGGGCTCCTCC TCACATCTCCTCCAGCCCTCTCAGCACATGCTGCCCCAGGACATGTACAAGCCCAAGCCT GTGCCAAGTGCGGGGATGATTGAGAGTCCCATGGGCCTAAAGCCCGCTCGGGGATCAGGG GGCTACAACGGAGGGGGCGCGCCCACCAGGTCCTCTTGTGACTGCCCCAACTGCCAGGAG CTGGAGAGGCTGGGGGCCTCGGCTGCATCCCTGAGGAAGAAGCCAGTCCACAGCTGCCAC ATCCCGGGGTGTGGGAAAGTCTACGGCAAGGCCTCCCACCTCAAAGCCCACCTGCGCTGG CACACCGGCGAGCGGCCCTTTGTTTGCAACTGGCTGTTCTGCGGGAAGCGTTTCACCCGC TCGGACGAACTGGAGAGGCACGTGCGCACCCACACGCGGGAGAAGAAGTTCACCTGTCTA CTGTGCAACAAGCGTTTCACGCGCAGCGACCACCTCTCCAAGCACCAGAAAACCCATGCG GAGTCCGCAATGCAGGGCAAAGCTGTAGCTGTGGAGGGAGAGACAGATCCTCGCAGCGAG GAGACCACAGAGCTCAACGCCAGCGCTGTGCCCGCCAATCCTGTCGCTGACCAAAGCACC AACGGAGATGAGAAGACCGGCACACCTAATGGAGTGGAGAACAGCAGTGGACTGTTGGAG ATCTGA