# The vertebrate small leucine-rich proteoglycans: amplification of a clustered gene family and evolution of their transcriptional profile in jawed vertebrates

Nathan Gil, Nicolas Leurs, Camille Martinand-Mari, Mélanie Debiais-Thibaud

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Nathan Gil, Nicolas Leurs, Camille Martinand-Mari, Mélanie Debiais-Thibaud. The vertebrate small leucine-rich proteoglycans: amplification of a clustered gene family and evolution of their transcriptional profile in jawed vertebrates. G3, 2025, 10.1093/g3journal/jkaf003/7945327. hal-04873230

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# The vertebrate small leucine-rich proteoglycans: amplification of a clustered gene family and evolution of their transcriptional profile in jawed vertebrates

Nathan Gil, Nicolas Leurs, Camille Martinand-Mari*, and Mélanie Debiais-Thibaud*

Institut des Sciences de l'Evolution de Montpellier, Université de Montpellier, CNRS, IRD, 34090 Montpellier, France.

* Corresponding authors:

camille.martinand-mari@umontpellier.fr

melanie.debiais-thibaud@umontpellier.fr

## Running head

Evolution of vertebrate SLRPs

## Abstract

Small Leucine-Rich Proteoglycans (SLRPs) are a major family of vertebrate proteoglycans. In bony vertebrates, SLRPs have a variety of functions from structural to signaling and are found in extracellular matrices, notably in skeletal tissues. However, there is little or no data on the diversity, function and expression patterns of SLRPs in cartilaginous fishes, which hinders our understanding of how these genes evolved with the diversification of vertebrates, in particular regarding the early events of whole genome duplications that shaped gnathostome and cyclostome genomes. We used a selection of chromosome-level assemblies of cartilaginous fish and other vertebrate genomes for phylogeny and synteny reconstructions, allowing better resolution and understanding of the evolution of this gene family in vertebrates. Novel SLRP members were uncovered together with specific loss events in different lineages. Our reconstructions support that the canonical SLRPs have originated from different series of tandem duplications that preceded the extant vertebrate last common ancestor, one of them even preceding the extant chordate last common ancestor. They then further expanded with additional tandem and whole-genome duplications during the diversification of extant vertebrates. Finally, we characterized the expression of several SLRP members in the small-spotted catshark *Scyliorhinus canicula* and from this, inferred conserved and derived SLRP expression in several skeletal and connective tissues in jawed vertebrates.
1 **Keywords:** Genomic evolution; evolution of gene expression; gnathostomes; cartilaginous fishes; small-
2 spotted catshark; SLRP; skeleton evolution

3

4 Introduction

A diversity of connective tissues has emerged with the evolution of vertebrates, including their skeletal tissues (Root et al. 2021, 2022). The macromolecular content of their extracellular matrices (ECM) consists firstly of collagen fibers (e.g. Collagen type I in bone and Collagen type II in cartilage), together with other types of proteins, and lipids. Among non-collagenous proteins, proteoglycans are particularly abundant in the highly hydrated ECMs (proteoglycan content in cartilage: 5-7% w/w; Hardingham 2006). Within proteoglycans, the largest known vertebrate family is the Small Leucine-Rich Proteoglycans (SLRPs). In this family, the protein moiety is relatively small (36-42 kDa) and has a distinctive leucine-rich repeat (LRR) domain (Nikitovic et al. 2012; Iozzo & Schaefer 2015). They can be bound to any of the three glycosaminoglycans (Zappia et al. 2020): heparin/heparan sulfate (HS), chondroitin/dermatan sulfate (C/DS) and keratan sulfate (KS). SLRPs play critical roles in the structure and assembly of various ECMs and hence in the development, structure and homeostasis of connective tissues (Hardingham 2006; Nikitovic et al. 2012; Park et al. 2008; Boskey 2010). For instance, they are known to interact with several types of collagen fibers by regulating their assembly via their protein moiety, while their glycosaminoglycans control the correct spacing between fibers. SLRPs can also regulate apatite formation in mineralized tissues, which are vertebrate innovations, and interact with skeletal growth factors such as TGFs and BMPs (Nikitovic et al. 2012; Iozzo & Schaefer 2015; Boskey 2010; Schaefer & Iozzo 2008).

In bony vertebrates, twenty-three SLRP paralogs have been identified so far and most can be found along four different chromosomes (Iozzo & Schaefer 2015; Park et al. 2008; Schaefer & Iozzo 2008; Costa et al. 2018). They are classically divided into five classes based on protein sequence similarity and gene chromosomal localization (Schaefer & Iozzo 2008). Class I contains asporin (Aspn), biglycan (Bgn), decorin (Dcn), and four “ECM proteins” (Ecm2, Ecm2L, EcmX and EcmXL). Class II includes fibromodulin (Fmod), keratocan (Kera), lumican (Lum), lumican-like (LumL), osteomodulin (or osteoadherin, Omd) and prolongin (Prelp). Class III consists of epiphycan (Epyc), opticin (Optc) and osteoglycin (or mimecan, Ogn). Class IV encloses chondroadherin (Chad), chondroadherin-like (ChadL), nyctalopin (Nyx) and tsukushin (Tsku). Class V encompasses podocan (Podn) and podocan-like (PodnL). Nephrocan (Npc) does not belong to any of these classes for structural reasons, despite its classification into SLRPs (Iozzo & Schaefer 2015; Schaefer & Iozzo 2008; Costa et al. 2018; Mochida et al. 2006). The sixteen SLRPs from classes I to III are classified as ‘canonical’ defined by the presence of an
extended repeat (called ear repeat) in the LRR C-terminal capping motif (LRRCE) of their protein moieties (Park et al. 2008). Despite the expectations of an already diverse canonical SLRP repertoire prior to vertebrate evolution, only few sequences have been identified in the vertebrate sister group (two *Ciona* SLRPs with LRRCEs, Park et al. 2008). The history of the SLRP family amplification in vertebrates is therefore still uncharacterized, in particular regarding the two rounds of whole genome duplications (2R event; Ohno 1970) that shaped the evolution of jawed vertebrate genomes, while cyclostome genome evolution shared the first (1R event) and then underwent additional polyploidization events (Marlétaz et al. 2024; Nakatani et al. 2021).

To assess the evolution of the SLRP gene family in vertebrates we took advantage of high-quality genomic data in the cartilaginous fish lineage (in particular with the small-spotted catshark *Scyliorhinus canicula*) and the cyclostome lineage (including the lampreys *Petromyzon marinus* and *Lethenteron reissneri* and the hagfish *Myxine glutinosa*) together with the cephalochordates *Branchiostoma lanceolatum* and *B. floridae*. We identified the outcome of the two rounds of whole genome duplications (1R and 2R events in gnathostomes; 1R and following events in cyclostomes) on clustered and non-clustered SLRPs. From these, we inferred a hypothetical ancestral state prior to the 1R event and lineage-specific gene losses. Taking advantage of the small-spotted catshark being an amenable organism in the laboratory and with extensive transcriptomic data, we thoroughly characterized SLRP gene expression patterns in this species with a special focus on the processes of differentiation of connective and skeletal tissues. From this, we could infer some ancestral features of SLRP expression in jawed vertebrates, but also some derived features of these genes in either the bony or cartilaginous fish lineages.

## 21 Materials and Methods

## 22 Protein sequence sampling

SLRP protein sequences were recovered from public databases (NCBI, Genbank) via BLASTP or TBLASTN (Altschul et al. 1990) with default parameters of each source database program) on nine sarcopterygians, seven actinopterygians and four chondrichthyans (the details of the source database for each sequence are given in Supplemental Table S1). The mouse *Mus musculus* and the zebrafish *Danio rerio* sequences were further used to screen the locally assembled transcriptome of the thornback ray *Raja clavata* (Debiais-Thibaud et al. 2019). Reciprocal BLASTs were performed to restrain the recovered sequences to actual SLRPs. Seven teleostean and two sarcopterygian sequences for EcmX were recovered via BLAST of the *D. rerio* sequence, to strengthen the resolution in teleosts. *Petromyzon marinus* SLRP sequences (Park et al. 2008; Ota et al. 2013) were used to screen the *P. marinus* genome
1 (assembly kPetMar1.pri) for additional sequences. These recovered *P. marinus* SLRP sequences were
2 used to screen the *Lethenteron reissneri* and the *Myxine glutinosa* genomes in NCBI (respectively
3 assembly ASM1570882v1 and UKY_Mglu_1.0). The 'SLRP' sequences of any *Branchiostoma* species
4 were obtained with BLASTP against the Protein sequence database in NCBI filtered for *Branchiostoma*
5 annotation. The Lingo gene family (including Lingo 1, 2, 3 and 4 in jawed vertebrates) were used as an
6 outgroup for the SLRP phylogeny as they contain Leucine Rich Repeat domains shared with SLRP
7 proteins. Lingo protein sequences were obtained using BLASTP of the Lingo1, Lingo2, Lingo3 and
8 Lingo4 annotated mouse sequences on the *Branchiostoma* and vertebrate species. All sequences used in
9 this study are listed with accession numbers in Supplemental Table S1.

10

11 SLRP phylogeny

12 The 549 protein sequences from vertebrate and *Branchiostoma* species were aligned with MAFFT v7.453
13 using the E-INS-i strategy (--genafpair --maxiterate 1000) recommended for sequences with multiple
14 conserved domains and long gaps (Katoh & Standley 2016). The resulting alignment was then filtered to
15 exclude sites containing gaps in more than 95% of the sequences. The final alignment used for subsequent
16 phylogenetic reconstruction included 1439 amino acids and is available in the Supplemental File S1. The
17 phylogeny was then inferred by Maximum Likelihood using IQ-Tree v2.1.3 (Nguyen et al. 2015) under
18 the best-fitting model of amino-acid sequence evolution (JTT+R7) as selected using ModelFinder
19 (Kalyaanamoorthy et al. 2017) based on the corrected Akaike Information Criterion (AICc). Statistical
20 node support was estimated by performing 1000 ultrafast bootstrap replicates (below named 'UFBoot')
21 (Hoang et al. 2018) and SH-like approximate likelihood ratio tests based on 1000 replicates (SH-aLRT;
22 Guindon et al. 2010). Branch support (SH-aLRT/UFBoot values) is indicated on the tree files. The full
23 phylogeny is available as a treefile in Supplemental File S2.

24

25 SLRP synteny

26 SLRP synteny was explored in seven reference genomes available on NCBI: mouse *M. musculus*
27 (GCF_000001635.27), small-spotted catshark *S. canicula* (GCF_902713615.1), zebrafish *D. rerio*
28 (GCF_000002035.6), elephant shark *Callorhinchus milii* (GCF_000165045.1), sea lamprey *P. marinus*
29 (GCF_010993605.1), Far Eastern brook lamprey *Lethenteron reissneri* (GCF_015708825.1) and hagfish
30 *Myxine glutinosa* (GCF_040869285.1). In addition, the reedfish *Erpetoichthys calabaricus* genome
31 (GCF_900747795.1) was consulted through the UCSC genome browser. Data from *D. rerio* and *C. milii*
32 are shown only when they differ from *E. calabaricus* and *S. canicula*, respectively. *E. calabaricus* was
1 chosen over *D. rerio* as a non-teleost actinopterygian devoid of the 3R (teleost whole-genome duplication event) duplicates.

3

4 SLRP protein domain screening

5 SLRP protein sequences were screened using LRR finder (Bej et al. 2014), EML (cell compartment: 6 extracellular) (Kumar et al. 2019) and Sulfinator (Monigatti et al. 2002) that predict, respectively, 7 putative positions for Leucine-Rich Repeats (LRRs), glycosaminoglycan and N-glycosylation sites, and 8 tyrosine sulfation sites. Additionally, the proteins were screened for two LRRCE motifs: (I) the standard 9 one from (Park et al. 2008) typical of canonical SLRPs; and (ii) a version relaxed in the C-terminal 10 region, using the ScanProsite tool (de Castro et al. 2006). The model LRRCE motif was (*PROSITE* 11 syntax): 12

12

13

Standard LRRCE Motif

14 [LIV]-X(2)-[LVIYFMA]-X-[LIFM]-X(2)-[NH]-X-[ILVF]-X(2)-[VIMFLY]-X(4)-[FIMLV]-C-X(7,20)- 15 [LYIMV]-X(2)-[ILVTMF]-X-[LVMI]-X(2)-N-X-[IVLMAFT]-X(8,9)-[FYMPVAIS]-X-C 16

16

17

LRRCE Motif, Relaxed at C-terminal

18 [LIV]-X(2)-[LVIYFMA]-X-[LIFM]-X(2)-[NH]-X-[ILVF]-X(2)-[VIMFLY]-X(4)-[FIMLV]-C-X(7,20)- 19 [LYIMV]-X(2)-[ILVTMF]-X-[LVMI]-X(2)-N-X-[IVLMAFT]-X(8,12)-C 20

20

21 RT-qPCR

22 Total RNAs from 22 anterior vertebrae (AV) of *S. canicula* embryos from 5 to 8 cm Total Length (TL) 23 were isolated with ReliaPrep RNA tissue Miniprep system (Promega) and used for cDNA preparation 24 performed by Superscript II reverse transcription (Invitrogen) with an oligoT primer. Each cDNA was 25 run in triplicate on a 384-well plate for each primer pair by using thermal cycling parameters: 95°C for 26 2 min, 95°C for 10 s, 68°C for 10 s, 72°C for 10 s (45 cycles), and an additional step 72°C for 10 min 27 performed on a LightCycler 480 with the SensiFAST SYBR No-ROX kit (Meridian Bioscience) (qPHD 28 UM2/GenomiX Platform, Montpellier – France). Forward and reverse primers were defined using 29 Primer3Input version 4.1.0 (see Supplemental Table S2). Results were normalized with the expression 30 of three reference genes *eefla*, *actin* and *gapdh* by geometric mean, and data were further analyzed with 31 the LightCycler 480 software 1.5.1. The reference point used was the highest value of ΔCp for a given

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1 gene: all expression values of any given gene are all above or equal to 1-fold. Developmental trajectories

2 were plotted on R (v4.04) using the ggplot2 package.

3

4 **Embryo Collection**

5 Embryos of the small-spotted catshark originated from a Mediterranean population of adult females

6 housed at Observatoire Océanique de Banyuls, France. Embryos were raised in seawater tanks at 16–

7 18 °C and euthanized by overdose of tricaine (MS222, Sigma) at appropriate stages. Whole embryos

8 were fixed in paraformaldehyde 4% in phosphate buffered saline solution for 48h and then stocked in

9 ethanol 100% before the tissue was sampled for cryostat sectioning. Handling of small-spotted catshark
10 embryos followed all institutional, national, and international guidelines [European Communities
11 Council Directive of September 22, 2010 (2010/63/UE)]: no further approval by an ethics committee
12 was necessary as the biological material is embryonic and no live experimental procedures were carried
13 out.

14

15 **In situ mRNA hybridization**

16 *In situ* hybridizations were performed on 14 µm thick cryostat sections of samples cut transversely in the
17 body trunk of fixed 6,5 cm TL embryos, at the level of the pectoral fins. All subsequent procedures were
18 previously described (Leurs et al. 2021). Slides were scanned on a Hamamatsu NanoZoomer 2
19 (Montpellier RIO Imaging facility, INM Optique). Primers designed to generate the DNA matrix used
20 for RNA probe synthesis were defined using Primer3Input version 4.1.0 (see Supplemental Table S2).

21

22 **Results**

23 A conserved SLRP repertoire in vertebrates that diversified in early chordates/early vertebrates

24 The complete SLRP phylogeny (Figure 1 and Supplemental Figure S1) was rooted using the Lingo
25 protein family as an outgroup, which shares a leucine-rich repeat region with SLRPs but are
26 transmembrane proteins (Homma et al. 2009): vertebrate Lingo paralogs grouped with a sister gene
27 annotated as ‘carboxypeptidase N’ in *Branchiostoma floridae*, making this outgroup a chordate-rooted
28 outgroup. This root branched to a first bifurcation separating a chordata Chad clade (non-canonical SLRP,
29 supported by 96.7/99 SH-aLRT/UFBoot values) from the rest of the SLRP proteins. In the chordata Chad
30 clade were three well-supported gnathostomata Chad orthology groups that we named: Chad1 for the
31 classically recognized Chondroadherin paralog, Chad2 for the sometimes identified Chondroadherin-like
32 paralog, and Chad3 for the latter, never identified before, paralog (all supported by 100/100 SH-
1 aLRT/UFBoot). Chad3 sequences were only found in chondrichthyans and in non-amniote
2 sarcopterygians (Supplemental Table S1) and a single lamprey sequence was 1:1 orthologous to Chad2.

3 These phylogenetic relationships could have suggested an origin of three gnathostome paralogs from the
4 two rounds of whole-genome duplication. However, *chad2* and *chad3* were found to be tandemly
5 arranged genes in the elephant shark *C. milii* and the small-spotted catshark (on loci
6 NW_006890314.1:268,311-282,341 and chr23: 22,986,334-23,090,131 respectively), invalidating this
7 hypothesis.

8 Sister to the chordata Chad clade, low support of branches resolved in a trifurcation joining the vertebrata
9 Tsku clade (100/100 SH-aLRT/UFBoot), the vertebrata Nyx clade (88.8/93), and the remaining SLRPs
10 that appear monophyletic with intermediate support values (90.4/95). The Nyx clade was made of two
11 gnathostomata paralogs (herein named Nyx1 and Nyx2, respectively 98.3/99 and 100/100) and two
12 lamprey sequences. The vertebrate Tsku clade included one copy for each species.

13 In the remaining part of the tree, the gnathostomata Npc clade (one gene for each species, 91.6/93) was
14 sister to a clade made of a multifurcation of five well supported groups of orthology: (i) the chordata
15 Podn clade (95/97) with two gnathostome paralogs herein named Podn1 and Podn2 (99.4/100 and
16 100/100); (ii) the chordata clade herein named Clade 2 (100/100); (iii) the vertebrata Clade 1 (94.6/94);
17 (iv) the vertebrata Clade 3 (99.8/100); (v) the vertebrata Clade 4 (100/100).

18 Amphioxus sequences robustly grouped with vertebrate sequences in the Podn, Clade 2, and Chad
19 chordata clades (Figure 1), placing the origin of each of these clades earlier than the divergence of the
20 cephalochordate lineage from the other chordate lineages. Although less robust in our results (90.4/95
21 SH-aLRT/UFBoot), the grouping of (chordate Clade 2; chordate Podn; vertebrate Clades 1, 3 and 4)
22 rather support the same age for the origin of the Tsku and Nyx clades, although only identified in
23 vertebrate species. In the elephant shark *C. milii* and the small-spotted catshark genomes, *rpc*, *nyx1*,
24 *podn1*, *podn2* and *tsku* were all located as single SLRPs on independent loci. However, in *Branchiostoma
25 lanceolatum*, the Podn- and Clade 2-associated genes are located 56kb apart on chromosome 5, opening
26 the hypothesis of an initial tandem duplication at the origin of the Podn and Clade 2 ancestral genes.

27 Canonical SLRP paralogs were amplified both with the gnathostome 2R events and local tandem
duplications
28

29 Clade 2 includes vertebrate and amphioxus sequences, but Clades 1, 3, and 4 only include vertebrate
30 sequences (Figure 1), placing the origin of these three later clades at an undetermined timing located
31 between the time of divergence of cephalochordate from other chordates and the time of
1 cyclostome/gnathostome divergence. In the following we focus on the gnathostome groups of orthology
2 and their relationship to cyclostome SLRPs (whose sequences we named SLRP 1 to 13, with numbers
3 randomly distributed) so as to better identify events of duplication associated to the 1R+2R events.

4 Within Clade 1, three gnathostome paralogs were identified as Ecm2, EcmX and Ecm2L (Figure 2) with
5 Ecm2L lost in most tetrapods and EcmX lost in chondrichthyans and birds. Ecm2 was the sister clade to
6 EcmX with the placement of cyclostome SLRP5 as sister group of Ecm2 (SH-aLRT/UFBoot: 97.7/92;
7 Figure 2). Within the EcmX clade, a teleost-only clade (99.7/100) branched out of all other bony fish
8 sequences, but low internal node support prevented from supporting either of two possible scenarios: this
9 clade was previously identified as EcmX and considered another Ecm2 related gnathostome paralog
10 (Costa et al. 2018) or it may be the product of the teleost-specific third whole genome duplication
11 identified as the 3R event (Jaillon et al. 2004; Kasahara et al. 2007).

12 Within Clade 2 (Figure 3) gnathostome Bgn and Aspn were sister clades and teleost 3R duplicates were
13 only conserved for Bgn (*bgna* and *bgnb*). Their cyclostome sister clade included: two paralogs in
14 lampreys (herein identified as SLRP2 and 3) and one myxine paralog (SLRP2/3). The gnathostome Dcn
15 clade (98.2/100) included one clade with osteichthyans and chondrichthyan sequences (Dcn1), and a
16 second clade with only chondrichthyan sequences (Dcn2), despite poor support for the Dcn1 clade (see
17 next section on synteny for a robust argument to support with group of orthology). The cyclostome
18 SLRP1 and SLRP4 clades were placed as the outgroup of all other, or as part of the (Dcn1, Dcn2, Aspn,
19 Bgn) clade respectively.

20 We displayed the phylogeny of Clade 3 by illustrating the two major clades that were identified into
21 Clade 3: the gnathostome Lum, Fmod and LumL groups and three cyclostome (lamprey) sequences
22 constituted Clade 3a (SLRP6, 7 and 8; Figure 4), sister to Clade 3b and made of the gnathostome Omd,
23 Kera and Prelp groups along with another three cyclostome sequences (SLRP9, 10 and 11; Figure 5). In
24 Clade 3a, teleost 3R paralogs were identified only in the Fmod clade, while LumL was identified only in
25 elephant shark, the coelacanth and actinopterygian species, so inferred to be secondarily lost in
26 elasmobranchs and in tetrapods. LumL was the only paralog for which a 1:1 orthology relationship with
27 the cyclostome SLRP7 was identified. Lamprey paralogs SLRP6 and SLRP8 could not be robustly
28 identified as sister to either of the gnathostome paralogs (Figure 4).

29 In Clade 3b, all gnathostome lineages displayed the presence of the Kera, Prelp and Omd paralogs, with
30 only one copy of each in teleost species, despite the 3R event (Figure 5). No 1:1 orthology relationship
31 could be identified between the gnathostome clades and the cyclostome SLRP 9, 10 or 11.
1 Within Clade 4, the gnathostome Ogn and Epyc clade were well supported, but the Optc group had lower
2 support (78.4/97) together with the sister relationship between Optc and Epyc (79.5/97; Figure 6). The
3 Optc paralog was lost in chondrichthyans. Teleost-specific duplication was partially conserved for ogn
4 (ogna and ognb) as previously shown (Costa et al. 2018). The cyclostome sequences branched outside
5 of the whole clade of gnathostome sequences, and as the hagfish sequence branched even outside of all
6 others, we decided to name it SLRP13, when the lamprey sequences were named SLRP12.

7 **Genomic organization of the vertebrate canonical SLRPs**

In gnathostome species, canonical SLRP genes were clustered along four conserved genomic loci (Figure 7, and Supplemental Table S1 for detailed genomic location). These gene clusters will be referred to as gene clusters A, B, C and D. Genes found on cluster A were *ecm2*, *aspn*, *omd* and *ogn*; on cluster B: *ecmX* (illustrated here with the reedfish *E. calabaricus*) and *bgn*; on cluster C: *ecm2L* (in the catshark and zebrafish), *dcn1*, *lum*, *kera*, and *epyc*; on cluster D: *fmod*, *dcn2* (only in cartilaginous fishes), *lumL* (in the reedfish and elephant shark), *prelp*, and *optc* (only found in bony fishes). For each cluster, at least one neighbor gene was conserved in synteny between the compared genomes, supporting orthology between compared loci (Figure 7). We oriented all clusters so that the first position along the gene cluster was occupied by Clade 1 genes (*ecm2*, *ecmX*, *ecm2L*; absent on cluster D) and/or so that the last position of the gene cluster was occupied by Clade 4 genes (*ogn*, *epyc*, *optc*) (absent on cluster B). Clade 2 and 3 genes showed less conserved positions on cluster D (Figure 7). Clade 2 genes (*aspn*, *bgn*, and *dcn1*) were positioned on the second locus in clusters A, B and C but *dcn2* was positioned between *fmod* and *lumL* on cluster D (Figure 7). The location of gnathostome *dcn1* genes on one orthologous locus along gene cluster C in all gnathostomes led us to support the monophyly of gnathostome Dcn1 clade despite its low support in the phylogeny (Figure 3). No Clade 3 gene was found on cluster B: one paralog was identified on cluster A (*omd*), two paralogs on cluster C (*lum* and *kera*) and a maximum of three paralogs on cluster D (*fmod*, *lumL* and *prelp*).

In the lamprey genomes, four SLRP gene tandems were identified (Figure 7). *SLRP5* shared synteny with *SLRP6* (Clade 1 and 3 genes, respectively), and *SLRP3* shared synteny with *SLRP12* (Clade 2 and 4 genes, respectively). *SLRP8*, *9* and *11* all belonged to Clade 3 and were all found on the same chromosome, but *SLRP11* was megabases away from the two other clustered sequences, making this shared synteny a potential result of secondary chromosomal rearrangements. In the brook lamprey, *LrSLRP1* was also found in the same chromosomal region, but again, more than 200kb away from its closest *LrSLRP8* gene. Finally, *SLRP7* was found with *SLRP10* (both Clade 3 genes). Shared synteny with a sequence coding for a member of the *Atp2b* gene family was verified on most of the loci where a
1 lamprey *SLRP* gene was identified (Figure 7), showing a greater number of homologous loci linking
canonical SLRPs with an ATP2B-related gene in cyclostomes (a minimum of 6) than in gnathostome (a
maximum of 4). No such clustering of SLRP genes could be identified in the hagfish genome (Figure 7)
4 as only *SLRP1*, *2/3* and *5* were found on the same chromosome but megabases away one from another.
5

6

Conservation of SLRP protein domains in vertebrates

We screened specific SLRP protein structural data, such as LRR number and length and putative N-glycosylation or glycosaminoglycan attachment sites (see Supplemental Table S3). We found a small degree of variation in the number of LRRs in the small-spotted catshark proteins which often lacked one to two LRRs as compared to mammal sequences (Matsushima et al. 2021). Within the small-spotted catshark canonical SLRPs, we found a standard LRRCE motif in Aspn, Ecm2, Dcn1, Dcn2, Lum, Ogn and Epyc. In the remaining sequences only a screening with the C-terminal unconstrained LRRCE yielded hits. In addition to the LRR and LRRCE motifs, protein alignment showed good conservation of the cysteine rich motifs on the N terminal capping region of all small-spotted catshark SLRPs (Low et al. 2021). Several small-spotted catshark SLRP sequences showed high similarity in terms of number and position of putative N-glycosylation and glycosaminoglycan attachment sites (e.g. Clade 3 SLRPs except Omd) compared to described bony fish orthologs. Twelve lamprey SLRPs belonged to the four clades where gnathostome canonical sequences were found. In both lampreys, ten sequences out of these twelve also displayed a standard LRRCE motif (SLRP1-3, 5-8, 10 and 12), while in the hagfish, five out of six canonical SLRP displayed a standard or relaxed LRRCE motif (SLRP1, 2/3, 5, 9 and 13). No LRRCE motif was identified in any of the *Branchiostoma* sequences but displayed 63 LRR domains.

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23

Tissue- and embryonic stage-specific expression of SLRP genes in the small-spotted catshark

Gene expression levels were extracted from transcriptomic data published in Mayeur et al. 2024. These transcriptomic data were acquired in a variety of small-spotted catshark embryonic stages and adult tissues and were compared through TPM values (a proxy for expression quantification in a given sample) and Z-score values (a proxy to quantify overexpression bias towards one given sample), both summarized in Table 1 (and see Supplemental Tables S4, S5). Some SLRP transcripts displayed very low TPM values across all sampled tissues (*dcn2*, *kera*, *chad3*, *rpc*, *nyx*, *podn1* and *podn2*, mean TPM < 15), while others reached high expression levels in some tissues (e.g. *chad1* in vertebrae > 2700 TPM). In most sampled tissues, *bgn* displayed high levels of expression (TPM > 50). Expression of many SLRPs was biased (Z-score > 1) towards endoskeletal tissues (the Meckel's cartilage, vertebrae and chondrocranium samples)

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and/or exoskeletal tissues (dental lamina), and some were exclusively enriched in the endoskeletal system: *chad1*, *chad2*, *epyc*, *fmod* and *lum*.

Several SLRPs from different clusters and different clades had expression biased towards the ampullae of Lorenzini (AOL) (*ecm2*, *bgn*, and *prelp*, Table 1) or the eye (*nyx* and *podn1*), making these sensory organs another important site of SLRP expression. Biased expression was also found for *ogn* in the esophagus (Supplemental Tables S4, S5). Compared to the other SLRPs, *tsku* displayed a more divergent expression pattern being the only one enriched in the liver, spiral intestine and the uterus but not in endoskeletal tissues (Supplemental Tables S4, S5). Finally, *ecm2L* was exclusively enriched in early embryonic stages. Most SLRPs also showed strong and/or biased expression in late embryonic stages (stages 30 and 31), up to exclusive expression of *dcn2*, *kera* and *omd* for these stages, when the skeleton is known to engage into cell differentiation (Table 1 and (Enault et al. 2016; Berio et al. 2021)). Based on parallel higher enrichment in the skeletal tissues and in late embryonic stages, we further controlled for SLRPs expression during skeletal development in later small-spotted catshark embryonic stages.

## Cell-specific expression of SLRPs in developing skeletal tissues of the small-spotted catshark

We tested the timing and location dynamics of genes expressed in endoskeletal tissues (vertebrae) first by relative qPCR measurement (Supplemental Figure S2) and then by *in situ* hybridization on embryonic tissues. The results of qPCR amplification showed that several SLRPs were lowly expressed or even could not be amplified in our samples of embryonic vertebrae: *kera*, *ecm2L*, *dcn2*, *chad3*, *podn1*, *podn2*, *ivan*, *nyx* and *tsku*. These genes were therefore not selected for further analysis by *in situ* hybridization. The relative qPCR data showed that most of the SLRPs expressed at that stage were downregulated over the course of tissue differentiation (Supplemental Figure S2) except for four of them (out of 18 genes): *omd*, *lum*, *fmod* and *podn1* (Supplemental Figure S2). In 5- to 8-cm TL small-spotted catshark embryos, skeletal tissues develop from poorly differentiated cell populations to a variety of differentiated tissues including: non-mineralized and mineralized cartilage and perichondrium, mineralized fibrous sheath of the notochord and non-mineralized notochord (Figure 8 a-c and (Enault et al. 2016; Berio et al. 2021)), skin denticles develop as the dermis and epidermis differentiate, muscle tissue differentiates (Figure 8c). To better identify the cell-specific gene expression patterns of SLRPs, we therefore used *in situ* hybridization on sections of 6.5cm TL embryos for a selected set of genes (based upon TPM values (mean endoskeletal tissue TPM > 30, Table 1) and positive results of qPCR amplification).
Expression patterns of other SLRPs followed two main patterns were compared to either *col2a1* or *col1a1* expression (Figure 8) that were assigned to either chondrocyte or perichondrial cells, respectively. The *aspn*, *chad1*, *chad2*, *epyc*, *omd*, *ogn* and *prelp* genes were expressed by chondrocytes (Figure 9, a-e and k-l), while *bgn*, *dcn1* and *lum* displayed perichondrial cell expression (Figure 9, m-o). Additional expression sites were observed: in chordocytes of the notochord for both *chad1* and *epyc* and in neural cells of the spinal cord for *omd*. In developing scales, ameloblastic expression was detected for *bgn*, *lum*, *omd* and *ogn* (Figure 9, j, p, r and t), while mesenchymal (odontoblastic) expression was observed for *dcn1* and *lum* (Figure 9, r and s). Additionally, expression was detected in the mesenchyme of scale roots for *aspn*, *chad2*, *lum* and *omd* (Figure 9, f, h, j and r). Expression in the dermal cells was detected for *bgn*, *dcn1*, *lum*, *omd* and *ogn* (Figure 9, j, p and r-t). Finally, muscle expression was detected for *dcn1*, *lum*, *omd* and *bgn* (Figure 9, j and r-t). No expression could be detected by *in situ* hybridization for *ecm2* and *fmod*.

## Discussion

In this study we recovered previously described gnathostome orthology groups, resolved the phylogenetic relationships among canonical SLRPs within each Clade 1, 2, 3 and 4, and identified several new gnathostome paralogs of the SLRP family: one canonical SLRP (Dcn2, only conserved in chondrichthyans); and three non-canonical SLRPs that we named Nyx2, Podn2 and Chad3.

By integrating amphioxus sequences in the phylogeny, we can infer that all non-canonical SLRP clades evolved before the divergence between vertebrates and cephalochordates, despite the fact that some of them were not conserved in extant cephalochordates (Figure 1). Only one amphioxus sequence showed orthology relationships with canonical SLRP sequences, all grouped into the chordate Clade 2, also supporting the evolution of Clade 2 in an early chordate ancestor. In the genome of the amphioxus *Branchiostoma lanceolatum*, the genes identified in the Podn and Clade 2 groups were genes arranged in tandem on the chromosome 5, supporting a single zone of tandem duplications that evolved into the Podn and Clade 1, 2, 3, 4 genes. In our phylogeny, cephalochordate/vertebrate orthology relationships could not be inferred for Clade 1, 3 and 4: these tandem duplications may therefore have occurred later, but still before the last common ancestor of extant cyclostome and gnathostome.

## Evolutionary scenario for canonical SLRPs expansion in vertebrates

Chondrichthyans and cyclostomes have proven highly valuable for inferring general and specific properties regarding the evolution of vertebrate gene families (Smith et al. 2018; Leurs et al. 2022; Suzuki et al. 2017; Debiais-Thibaud et al. 2019). Using chondrichthyan genome data, we show the clustering of
gnathostome canonical SLRPs along four different loci and the branching of paralogs in Clade 1-4
phylogenies are all congruent with the expected pattern for cluster multiplications by the 2R whole
genome duplications (Dehal & Boore 2005) following a ((cluster A, cluster B), (cluster C, cluster D))
relation (Figure 10). For instance, within Clade 2, *aspn* (cluster A) and *bgn* (cluster B) are more recent
paralogs, while *dcn1* (cluster C) and *dcn2* (cluster D) are more recent paralogs (Figure 3). This pattern is
also visible for Clades 1 and 4 if we consider gene loss for the sister genes to *ecm2L* and *ogn* respectively
(Figures 2 and 6). However, the observed topology for Clade 3 cannot be explained using only two rounds
of whole genome duplication and gene loss. To explain the several additional paralogs of Clade 3
observed in clusters C and D, we propose a most-parsimonious scenario where two events of tandem
duplication occurred, the first one before the first round of whole-genome duplication (R1 event) and the
other one before the split between gnathostomes and cyclostomes (Figure 10). Before the gnathostome
second round of whole genome duplication, one ancestral cluster with four SLRPs duplicated into
clusters A and B, and another ancestral cluster with six SLRP paralogs gave rise to clusters C and D
(Figure 10). Subsequent gene loss (and the 3R additional whole genome duplication in teleosts) would
explain the genomic organization of extant gnathostomes and the inferred phylogeny. A recent study
showed a clear case of clustered, tandemly duplicated genes diversifying through the 2R-event in the
zone neighboring SLRPs with similar ((A,B),(C,D)) relationship (Ocampo Daza et al. 2022)), making
the excellent conservation of the gnathostome SLRP loci a wider characteristic of a whole chromosomal
section.

In addition to gnathostome data, we identified twelve lamprey and seven hagfish sequences as
canonical SLRPs, both from their grouping with gnathostome canonical SLRPs into Clades 1-4, and
supported by the presence of a LRRCE motif. Their exact phylogenetic relationship to each of the
gnathostome paralogs are mostly strongly supported (Figures 2-6). Lamprey sequences were found as
smaller gene tandems, composed of mixes of genes belonging to Clades 1 and 3, or 2 and 4, or several
members of the Clade 3 with a member of Clade 2 (Figure 7). These characteristics are similar to
gnathostome clusters, and support a scenario where tandem duplications that gave rise to the four SLRP
clades predated the cyclostome/gnathostome divergence (Figure 10). The SLRP clusters identified in
extant species would then all derive from a single five-gene vertebrate ancestral cluster that underwent
the 1R event, and subsequently duplicated again through the parallel genome duplication histories of
gnathostomes and cyclostomes (Figure 10). The cyclostome group (SLRP2 and SLRP3) is sister to the
gnathostome (*Bgn* and *Aspn*), supporting their shared evolution from the ancestral A/B cluster. Similarly,
robust phylogenetic relationship between the cyclostome SLRP5 and the gnathostome Ecm2 (Figure 2)
suggests descendance from the ancestral A/B cluster. However, SLRP5 is found in shared synteny with
SLRP6 (Figure 7) which is phylogenetically related to the Lum and Fmod gnathostomes genes (Figure 4) which are descendants of the ancestral C/D cluster (Figure 4). This observation comes in contradiction to the proposed evolutionary scenario and may be the result of chromosomal rearrangements in cyclostomes where SLRP clusters appear much less well conserved than in gnathostomes, as also exemplified with the hagfish genomic data (Figure 7). The evolutionary scenario accounting for Clade 3 topology involves tandem duplications in addition to the gnathostome two rounds of duplications: one of these tandem duplications produces: (i) the ancestor for the Omd and the (Kera, Prelp) group (Clade 3b, Figure 5, duplicate 3b in Figure 10); (ii) the other copy (duplicate 3a in Figure 10) will undergo an additional tandem duplication to generate a series of three Clade 3 genes on the ancestral C/D cluster. The complete series of hypothetical genes duplication and losses are illustrated in Figure 10.

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## 12 Evolution of SLRP transcriptional profile in gnathostome skeletal tissues

Two main types of SLRP expression patterns can be observed in the small-spotted catshark embryonic endoskeletal tissues: perichondrium- or chondrocyte-associated expressions. For instance, *bgn*, *dcn1* and *lum* all show a perichondrium-associated expression pattern (Figure 9, Table 2) congruent with these genes being described to interact with collagen I in mice (Chen & Birk 2013). The chondrocyte expression of *chad1*, *chad2*, *epyc*, *omd*, *ogn* and *prelp* (Figure 9, Table 2) is a conserved feature between the small-spotted catshark and bony fishes (Shinomura & Kimata 1992; Funderburgh et al. 1997; Wilda et al. 2000; Grover & Roughley 2001; Tillgren et al. 2015). Most of bony fish SLRPs are also components of the bone matrix, associate with type I collagen fibers, and are expressed by osteoblasts (Bgn, Dcn1: (Kamiya et al. 2001); Aspn, Lum, Epyc, Ogn: (Khayal et al. 2018); Omd, (Sommarin et al. 1998); Prelp: (Li et al. 2016); Chad1: (Shen et al. 1998) despite Chad2 (previously named Chondroadherin Like) was detected expressed in cartilage but not bone cells (Tillgren et al. 2015). These observations suggest a large part of SLRPs (canonical and non-canonical) were involved in specific skeletal and connective tissues already in the last common ancestor of extant gnathostomes, in particular in cartilage. The single report of SLRP gene expression in cyclostomes was in the hagfish *Eptatretus burgeri* (Ota et al. 2013) where one Class I SLRP gene (MgSLRP4 is the most similar to this sequence) was found expressed in mesenchymal and cartilaginous cells. Comparable expression data in lamprey species, although challenging, would shed light on potentially vertebrate-wide shared function of SLRPs in either cartilaginous or mesenchymal/perichondrial tissues, resulting from the evolution of skeletal tissues in early vertebrates.

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Within gnathostomes, a major exception to this conservation status is *aspn*, that was shown to be expressed in the perichondrium but not in differentiated cartilage (Henry et al. 2001) and to interact with Collagen type I in mice (Kalamajski et al. 2009) (Table 2). In the small-spotted catshark however, *aspn* was only detected in chondrocytes and not in the perichondrium, suggesting divergent evolution of *aspn* expression patterns. In addition, the small-spotted catshark Omd protein sequence is one of the most divergent SLRPs when comparing it to its bony fish ortholog: it showed no glycosaminoglycan attachment sites but six N-glycosylation sites; there were no detected putative tyrosine sulfation sites while several are found in bony fishes; no acidic C-terminal region was found. Its expression was located in chondrocytes in the embryonic small-spotted catshark while it has been detected in mice osteoblasts (Ninomiya et al. 2007) and in rat fetal femur bone with a function in binding hydroxyapatite (Wendel et al. 1998). Since chondrichthyans lost the ability to make bone and have novel modes of mineralization (Seidel et al. 2016), evolution of the *aspn* and *omd* expression patterns and sequences might be linked to this phenotypic evolution. Conversely, the *bgn* and *lum* genes are expressed in mice chondrocytes and known to interact with Collagen II and Aggrecan (Chen & Birk 2013; Wilda et al. 2000) but they were not detectable in the cartilage in the small-spotted catshark (Figure 9). Functional studies on these four genes may shed light on protein evolution events that correlate with the lineage-specific evolution of skeleton in extant gnathostomes.

Expression in small-spotted catshark embryonic exoskeletal tissues in this study was also focused on dermal denticles that develop similarly to teeth (Debiais-Thibaud et al. 2015). The observed patterns in the small-spotted catshark can be ameloblast-associated (*bgn*, *ogn*, *omd*), odontoblast-associated (*dcn1*) or both (*lum*) (Figure 9, Table 2). In mice teeth, *bgn* and *omd* were detected in both ameloblasts and odontoblasts while *aspn*, *dcn1*, *lum*, *ogn* and *fmod* were only found in odontoblasts (Buchaille et al. 2000; Matsuura et al. 2001; Hou et al. 2012; Houari et al. 2014; Randilini et al. 2020). Therefore, we can speculate on a conserved function of *bgn* and *omd* in enamel/enameloid formation and of *dcn1* and *lum* in dentin formation. Odontoblasts, the dentine-producing cells, show more differences in SLRP expression between bony and cartilaginous fishes than ameloblasts, which is surprising as dentine is considered a stable tissue in vertebrates, while the evolution of extant forms of enamel and enameloid are considered more derived tissues (Kawasaki & Weiss 2008; Leurs et al. 2022). The odontoblasts are thought to be very similar to osteocytes in their differentiation and secretion pathways and again, the lack of *aspn* expression in small-spotted catshark odontoblasts might be another outcome of bone loss. To obtain a general trend in the evolution of exoskeleton cell types and tissues, a much denser characterization of genes expressed in teeth or scales in a variety of bony and cartilaginous fishes is necessary.
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In contrast to other SLRPs, *ecm2L* and *kera* expression is enriched in very early embryonic stages (Table 1). Protein motifs and expression of *kera* is quite similar to its ortholog in bony fishes, that has been shown to be expressed very early during chick embryogenesis with possible roles in neural-crest cells migration (Conrad & Conrad 2003). We show that *ecm2L* was lost in amniotes and that the small-spotted catshark ortholog is very peculiar since we found eighteen putative N-glycosylation sites before the LRRs. Together, these results suggest that Kera and Ecm2L may be important targets for future studies of SLRP involvement in the earliest step of skeletal development, through their potential role in neural crest cell migration.

## Specific SLRP evolutionary trends in cartilaginous species

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In our species sampling, *dcn2* was present only in chondrichthyans and inferred to be lost in all other gnathostome lineages. Screening of the protein motifs showed high similarity with its paralog Dcn1 which is itself very similar to its bony fish orthologs (Low et al. 2021; Kalamajski & Oldberg 2010), suggesting functional redundancy between these genes. However, *dcn2* expression enrichment in the small-spotted catshark kidney is not found for *dcn1*, which may be a functional reason for its maintenance in chondrichthyan genomes.

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An enrichment of expression for *ecm2*, *bgn*, *prelp* was detected in the ampullae of Lorenzini, which are sensory organs that can detect electromagnetic fields and temperature gradients, and are filled with a keratan sulfate rich gel. Previous studies on ampullae of Lorenzini did not identify the proteoglycans to which these keratan sulfate chains might be linked to (Zhang et al. 2018; Melrose 2019), but specifically *lum*, *ogn*, *prelp* have been reported to carry keratan sulfate in mammals (Iozzo & Schaefer 2015; Funderburgh et al. 1997; Hultgårdh-Nilsson et al. 2015) and are highly expressed in the ampullae of Lorenzini (TPM > 50, Table 1). Predicted N-glycosylation and glycosaminoglycan attachment sites in the small-spotted catshark SLRP sequences were on conserved positions, including those where keratan sulfate attachment was shown in mammals (Low et al. 2021) (Supplemental Table S3). Therefore, *lum*, *ogn* and *prelp* are excellent candidates for structural SLRPs involved in the secretion of the specialized gel of the ampullae of Lorenzini in chondrichthyan fishes. More surprising are *bgn* and *ecm2* expression in the ampullae of Lorenzini. The small-spotted catshark Ecm2 is divergent when compared to its bony fish ortholog: it is predicted to have five possibly sulfated tyrosines in the N-terminal region (none predicted in mice), four putative glycosaminoglycan attachment sites (only one in mice) and three N-glycosylation sites (none in mice), all possible sites for keratan sulfate attachment. This SLRP might carry keratan sulfate chains in cartilaginous fishes, but these results are highly speculative since Ecm
1 proteins have been very poorly studied in bony fishes, and our data are only predictions which are more

2 permissive than the actual observation of glycosaminoglycan attachment (Kalamajski & Oldberg 2010).

3

4 **Conclusion**

5 Here we characterized the repertoire of SLRPs in vertebrates by including cartilaginous fish, cyclostome

6 and cephalochordate sequence data. The relative genomic stability of clustered SLRP genes in

7 gnathostomes might be related to the observation of conserved transcriptional profiles suggesting high

8 developmental constraints on the SLRP proteins, notably in relation to skeletal development and

9 homeostasis. Our data support that several key features that evolved in early vertebrates (endo- and exo-

10 skeleton, sensory organs) depend on the expression of several members of the SLRP gene family. A

11 thorough characterization of glycosaminoglycan chain linkage of chondrichthyan and other vertebrate

12 SLRPs is now critical to better understand lineage-derived and ancestral features associated with the

13 function of these proteoglycans.

14

15 **Data Availability Statement**

16 The authors affirm that all data necessary for confirming the conclusions of the article are present

17 within the article, figures, and tables or through public databases as cited in Supplemental Table S1. All

18 supplemental material available at G3 online.

19 **Acknowledgments:** We acknowledge the MRI platform (ANR-10-INBS-04) for imaging support, the

20 GenSeq platform, Labex CEMEB (ANR-10-LABX-04-01) and NUMEV (ANR-10-LABX-0020) for

21 sequencing support and the RHEM facility (INCa_Inserm_DGOS_12553, FEDER-FSE 2014-2020

22 Languedoc Roussillon) for histology expertise. We thank Hélène Mayeur, Sylvie Mazan and

23 Nathanëlle Saclier for discussions on RNAseq data analysis and Frédéric Delsuc for his help and

24 expertise with phylogenetic reconstructions.

25 **Funding:** NL and NG were supported by the LabEx CEMEB, and ANR "Investissements d'avenir"

26 program (ANR-10-LABX-04-01).

27

28 **Conflict of Interest:** The authors declare that they have no known competing financial interests or

29 personal relationships that could have appeared to influence the work reported in this paper.
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29
<table><caption>**Table 1.** Selection of transcriptomic data (TPM values) to characterize SLRP gene expression profiles in adult skeletal tissues, sensory organs and embryonic stages of the small-spotted catshark, data extracted from Mayeur et al. 2024. Samples with both Z-score > 1 and TPM > 50 are bold and underlined. Samples with Z-score > 1 but TPM < 50 are bold. AOL: ampullae of Lorenzini; E-St-X: embryo stage x. See the complete tables with the 31 adult tissues/embryo stage TPM and Z-score values in Supplemental Tables S4 and S5.</caption><thead><tr><th></th><th></th><th>Meckel's cartilage</th><th>Vertebrae</th><th>Chondrocranium</th><th>Skin denticles</th><th>Dental lamina</th><th>Eye</th><th>AOL</th><th>E-St-12</th><th>E-St-22</th><th>E-St-24</th><th>E-St-26</th><th>E-St-30</th><th>E-St-31</th></tr></thead><tbody><tr><td rowspan="2">Clade 1</td><td>ecm2</td><td><strong>63,0</strong></td><td><strong>64,9</strong></td><td><strong>46,7</strong></td><td>18,7</td><td>32,6</td><td>24,4</td><td><strong>45,6</strong></td><td>7,5</td><td>10,0</td><td>13,4</td><td>13,2</td><td>26,4</td><td>19,9</td></tr><tr><td>ecm2L</td><td>1,0</td><td>0,9</td><td>1,3</td><td>0,9</td><td>1,3</td><td>1,3</td><td>1,0</td><td>1,0</td><td><strong>141,5</strong></td><td><strong>182,2</strong></td><td><strong>149,2</strong></td><td>4,3</td><td>1,8</td></tr><tr><td rowspan="5">Clade 2</td><td>aspn</td><td><strong>129,2</strong></td><td><strong>130,9</strong></td><td>54,2</td><td>30,0</td><td><strong>130,6</strong></td><td>33,5</td><td>26,5</td><td>1,2</td><td>1,5</td><td>2,6</td><td>2,4</td><td>33,0</td><td>61,6</td></tr><tr><td>bgn</td><td>388,0</td><td><strong>711,6</strong></td><td><strong>550,2</strong></td><td>208,0</td><td>170,1</td><td>284,1</td><td><strong>1048,8</strong></td><td>1,0</td><td>38,6</td><td>119,7</td><td>63,1</td><td>134,3</td><td>121,1</td></tr><tr><td>dcn1</td><td><strong>93,0</strong></td><td><strong>244,5</strong></td><td>29,9</td><td>22,9</td><td>33,0</td><td>4,1</td><td>7,4</td><td>3,5</td><td>2,2</td><td>6,2</td><td>8,6</td><td>50,6</td><td><strong>72,7</strong></td></tr><tr><td>dcn2</td><td>2,1</td><td>2,5</td><td>1,0</td><td>0,5</td><td>0,5</td><td>4,5</td><td>0,5</td><td>0,2</td><td>0,5</td><td>0,5</td><td>0,9</td><td><strong>5,9</strong></td><td><strong>11,0</strong></td></tr><tr><td>fmod</td><td><strong>144,7</strong></td><td><strong>305,2</strong></td><td>56,1</td><td>6,4</td><td>7,6</td><td>7,1</td><td>6,5</td><td>6,0</td><td>6,3</td><td>12,1</td><td>9,9</td><td>16,8</td><td>8,8</td></tr><tr><td rowspan="4">Clade 3</td><td>kera</td><td>4,5</td><td>4,3</td><td>3,6</td><td>4,3</td><td>5,2</td><td>4,8</td><td>3,7</td><td>2,9</td><td>5,3</td><td>9,0</td><td>15,6</td><td><strong>48,7</strong></td><td><strong>31,1</strong></td></tr><tr><td>lum</td><td><strong>688,7</strong></td><td><strong>825,9</strong></td><td>259,9</td><td>93,6</td><td>211,3</td><td>51,2</td><td>268,6</td><td>1,0</td><td>16,1</td><td>8,6</td><td>24,4</td><td>119,1</td><td>189,0</td></tr><tr><td>omd</td><td>33,4</td><td>31,1</td><td>26,3</td><td>21,6</td><td>32,9</td><td>17,8</td><td>22,8</td><td>14,8</td><td>13,8</td><td>17,3</td><td>16,2</td><td><strong>93,3</strong></td><td><strong>133,3</strong></td></tr><tr><td>prelp</td><td><strong>118,5</strong></td><td><strong>222,6</strong></td><td><strong>124,0</strong></td><td>59,6</td><td>48,5</td><td>71,6</td><td><strong>103,8</strong></td><td>9,5</td><td>17,1</td><td>18,9</td><td>19,5</td><td>51,3</td><td>44,7</td></tr><tr><td rowspan="2">Clade 4</td><td>epyc</td><td>199,1</td><td><strong>1939,3</strong></td><td>46,1</td><td>1,9</td><td>2,6</td><td>9,0</td><td>2,9</td><td>1,3</td><td>42,6</td><td>39,4</td><td>49,2</td><td>43,5</td><td>16,1</td></tr><tr><td>ogn</td><td><strong>152,1</strong></td><td><strong>246,9</strong></td><td><strong>136,9</strong></td><td>58,7</td><td>39,5</td><td>55,4</td><td>70,8</td><td>1,3</td><td>1,6</td><td>2,8</td><td>2,7</td><td>91,8</td><td><strong>129,3</strong></td></tr><tr><td rowspan="8">Non-canonical</td><td>chad1</td><td><strong>1325,9</strong></td><td><strong>2715,1</strong></td><td><strong>704,3</strong></td><td>12,0</td><td>4,0</td><td>57,7</td><td>9,8</td><td>0,5</td><td>0,4</td><td>0,9</td><td>3,2</td><td>37,9</td><td>21,3</td></tr><tr><td>chad2</td><td><strong>234,7</strong></td><td><strong>152,7</strong></td><td><strong>90,1</strong></td><td>0,5</td><td>1,8</td><td>9,5</td><td>3,3</td><td>1,2</td><td>3,9</td><td>4,2</td><td>6,9</td><td>17,4</td><td>10,0</td></tr><tr><td>chad3</td><td>9,5</td><td>7,4</td><td>15,2</td><td>9,9</td><td>6,6</td><td>13,9</td><td>11,0</td><td>1,8</td><td>6,1</td><td>9,4</td><td>8,7</td><td>10,6</td><td>11,5</td></tr><tr><td>rpc</td><td>0,1</td><td>0,1</td><td>0,1</td><td>0,0</td><td>0,0</td><td>0,0</td><td>0,1</td><td>0,1</td><td><strong>0,9</strong></td><td><strong>1,1</strong></td><td><strong>1,7</strong></td><td>0,1</td><td>0,1</td></tr><tr><td>nyx</td><td>2,7</td><td>2,7</td><td>2,6</td><td>2,0</td><td>2,8</td><td><strong>5,7</strong></td><td>2,2</td><td>1,3</td><td>2,1</td><td><strong>3,1</strong></td><td>2,2</td><td><strong>3,3</strong></td><td>2,6</td></tr><tr><td>podn1</td><td><strong>9,5</strong></td><td><strong>13,6</strong></td><td><strong>17,4</strong></td><td>1,1</td><td>1,7</td><td><strong>9,0</strong></td><td>8,2</td><td>0,3</td><td>0,7</td><td>1,7</td><td>3,4</td><td><strong>10,3</strong></td><td><strong>8,9</strong></td></tr><tr><td>podn2</td><td><strong>40,7</strong></td><td><strong>32,0</strong></td><td><strong>24,3</strong></td><td>14,0</td><td>16,2</td><td>18,0</td><td>10,1</td><td>9,6</td><td>11,1</td><td>13,9</td><td>11,8</td><td>12,3</td><td>10,3</td></tr><tr><td>tsku</td><td>21,6</td><td>15,0</td><td>18,6</td><td>33,4</td><td><strong>50,3</strong></td><td>36,1</td><td>11,9</td><td>22,1</td><td>23,0</td><td>15,3</td><td>26,0</td><td>23,0</td><td>20,2</td></tr></tbody></table>
<table><caption>**Table 2.** Compared expression patterns of *SLRP* genes in mineralized tissues; osteich: osteichhyan species (see main text for references) and chondr: chondrichthyan species as exemplified by the small-spotted catshark (this study); *na*: non-applicable; empty boxes for missing data.</caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">chondrocytes</th><th colspan="2">perichondrium</th><th colspan="2">osteoblasts</th><th colspan="2">odontoblasts</th><th colspan="2">ameloblasts</th></tr><tr><th>chondr</th><th>osteich</th><th>chondr</th><th>osteich</th><th>chondr</th><th>osteich</th><th>chondr</th><th>osteich</th><th>chondr</th><th>osteich</th></tr></thead><tbody><tr><td rowspan="3">Clade 2</td><td>aspn</td><td>+</td><td>-</td><td>-</td><td>+</td><td>na</td><td>+</td><td>-</td><td>+</td><td>-</td><td>3</td></tr><tr><td>bgn</td><td>-</td><td>+</td><td>+</td><td>+</td><td>na</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>dcn1</td><td>-</td><td>+</td><td>+</td><td>+</td><td>na</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-4</td></tr><tr><td rowspan="3">Clade 3</td><td>lum</td><td>-</td><td>+</td><td>+</td><td>+</td><td>na</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>omd</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>prelp</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="3">Clade 4</td><td>epyc</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ogn</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>chad1</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>+</td><td>-</td><td>-</td><td>-</td><td>6</td></tr><tr><td></td><td>chad2</td><td>+</td><td>+</td><td>-</td><td>-</td><td>na</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

**Figure 1:** Phylogenetic relationships of SLRP sequences in chordates obtained by maximum likelihood (best fit model JTT+R7; 549 sequences, 1439 amino-acid positions; full non-collapsed phylogeny given in Supplemental File S2 and Supplemental Figure S1; non-collapsed Clades 1, 2, 3, and 4 in the following Figure 2, 3, 4, 5 respectively) and rooted by the vertebrate Lingo1/2/3/4 sequences and their closest amphioxus sequence. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both > 95 are shown as black dots, or as open circles nodes if values are < 95 and > 85, SH-aLRT/UFBoot values for internal nodes are shown on the branches. The four clades of canonical SLRPs are collapsed and shown in color, non-canonical SLRPs are highlighted in grey.

**Figure 2:** Detail of internal nodes within the vertebrate Clade 1 as defined in Figure 1. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both > 95 are shown as black dots, or as open circles nodes if values are < 95 and > 85, SH-aLRT/UFBoot values for internal nodes are shown on the branches. Dotted box locates teleost-specific duplicates.
**Figure 3:** Detail of internal nodes within the vertebrate Clade 2 as defined in Figure 1. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both> 95 are shown as black dots, or as open circles nodes if values are < 95 and >85, SH-aLRT/UFBoot values for internal nodes are shown on the branches. Dotted boxes locate teleost-specific duplicates.

**Figure 4:** Detail of internal nodes within the vertebrate Clade 3 as defined in Figure 1: Clade 3a. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both> 95 are shown as black dots, SH-aLRT/UFBoot values for internal nodes are shown on the branches. Dotted boxes locate teleost-specific duplicates.

**Figure 5:** Detail of internal nodes within the vertebrate Clade 3 as defined in Figure 1: Clade 3b. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both> 95 are shown as black dots, SH-aLRT/UFBoot values for internal nodes are shown on the branches.

**Figure 6:** Detail of internal nodes within the vertebrate Clade 4 as defined in Figure 1. Nodes of highlighted clades supported by ultrafast bootstrap and SH-aLRT values both> 95 are shown as black dots, or as open circles nodes if values are < 95 and >85, SH-aLRT/UFBoot values for internal nodes are shown on the branches. Dotted boxes locate teleost-specific duplicates.

**Figure 7:** Genomic organization of canonical SLRP gene clusters in selected vertebrate genomes. Distance between genes is indicated but not represented to scale. Gene names are similar to Figure 1 (for gene accession numbers, see Supplemental Table S1).

**Figure 8:** Histology and gene expression in anterior cross sections of 6.5 cm TL *S. canicula* embryo. (a-c): HES staining, (d-f): *collal* *in situ* hybridization; (g-i): *col2al* *in situ* hybridization. Close-up on vertebral tissues (b, e, h) and skin layers (c, f, i). Legends: d: dermis; e: epidermis; m: mesenchyme of a scale; mu: muscle; nt: notochord; sc: spinal cord. Black arrowheads indicate expression in the perichondrium; white arrowheads indicate expression in chondrocytes. Dotted lines mark separations between tissues. Scale bars are in µm and given at the top of each column.
1

2 **Figure 9:** mRNA *in situ* hybridizations of selected SLRPs in vertebral tissues (a-e and k-o) and skin

3 layers (f-j and p-t) in 6.5 cm TL *S. canicula* embryo. Legends: a: ameloblast; b: scale base and legends

4 as in Figure 8. Scale bars are in µm and given on the top panel of each column.

5

6 **Figure 10:** Hypothesized scenario for the amplification of the canonical SLRP gene family in chordates

7 with regard to the R1 (genome duplication in a common ancestor to cyclostomes and gnathostomes) and

8 successive genome multiplications in either the gnathostome (R2) or the cyclostome lineage. Only

9 selected sea lamprey SLRP loci are shown (see Main text for details). Color code similar to Figure 1.

10 White rectangle indicates hypothesized gene loss. Loci not to scale.

11
<figure><img src="image_2.png" /><figcaption>Figure 1
353x559 mm (DPI)</figcaption></figure>

1

2

3
<figure><img src="image_3.png" /><figcaption>Figure 2
432x559 mm (DPI)</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3
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<figure><img src="image_5.png" /><figcaption>Figure 4
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<figure><img src="image_6.png" /><figcaption>Figure 5
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VERTEBRATA
Clade4

<figure><img src="image_7.png" /><figcaption>Figure 6
396x559 mm (DPI)</figcaption></figure>
## Gnathostomes

### Cluster A

<figure><img src="image_8.png" /><figcaption>reedfish</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>mouse</figcaption></figure>

<figure><img src="image_10.png" /></figure>

### Cluster C

<figure><img src="image_11.png" /><figcaption>reedfish</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>mouse</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>zebrafish</figcaption></figure>

<figure><img src="image_14.png" /></figure>

### Cluster B

<figure><img src="image_15.png" /><figcaption>reedfish</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>mouse</figcaption></figure>

<figure><img src="image_17.png" /></figure>

### Cluster D

<figure><img src="image_18.png" /><figcaption>reedfish</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>mouse</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>elephant shark</figcaption></figure>

<figure><img src="image_21.png" /></figure>

## Cyclostomes

<figure><img src="image_22.png" /><figcaption>sea lamprey</figcaption></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>brook lamprey</figcaption></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /><figcaption>Figure 7
437x559 mm (DPI)</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>Figure 8
347x347 mm (DPI)</figcaption></figure>

1
2
3
<figure><img src="image_37.png" /><figcaption>Figure 9
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1
2
3
<figure><img src="image_38.png" /><figcaption>Figure 10
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1
2
3
<figure><img src="image_39.png" /></figure>

Graphical Abstract
559x367 mm (DPI)