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Cartilaginous Fishes Provide Insights into the Origin,Diversification, and Sexually Dimorphic Expression ofVertebrate Estrogen Receptor Genes

Grant L. Filowitz,†,1 Rajendhran Rajakumar,†,1 Katherine L. O’Shaughnessy,†,1 and Martin J. Cohn*,1,2

1Department of Molecular Genetics and Microbiology, UF Genetics Institute, University of Florida, Gainesville, FL2Department of Biology, University of Florida, Gainesville, FL†These authors contributed equally to this work.

*Corresponding author: E-mail: [email protected].

Associate editor: John Parsch

Abstract

Vertebrate estrogen receptors (ERs) perform numerous cell signaling and transcriptional regulatory functions. ERA (Esr1)and ERb (Esr2) likely evolved from an ancestral receptor that duplicated and diverged at the protein and cis-regulatorylevels, but the evolutionary history of ERs, including the timing of proposed duplications, remains unresolved. Here wereport on identification of two distinct ERs in cartilaginous fishes and demonstrate their orthology to ERa and ERb.Phylogenetic analyses place the ERa/ERb duplication near the base of crown gnathostomes (jawed vertebrates). We findthat ERa and ERb from little skate (Leucoraja erinacea) and mammals share key subtype-specific residues, indicatingconserved protein evolution. In contrast, jawless fishes have multiple non-orthologous Esr genes that arose by parallelduplications. Esr1 and Esr2 are expressed in subtype-specific and sexually dimorphic patterns in skate embryos, suggest-ing that ERs might have functioned in sexually dimorphic development before the divergence of cartilaginous andbony fishes.

Key words: estrogen receptor, chondrichthyan, vertebrate evolution, sex steroid evolution, sexual dimorphism.

Estrogen receptors (ERs) regulate an array of physiological anddevelopmental processes in male and female vertebrates.Most gnathostomes (jawed vertebrates) possess two distinctERs, ERa and ERb, which are encoded by the Esr1 and Esr2genes, respectively (Thornton 2001; Katsu et al. 2008; Katsu,Kohno, et al. 2010; Katsu, Taniguchi, et al. 2010). Ligand bind-ing activates ERs either by triggering translocation to the nu-cleus, where ERs bind estrogen response elements (EREs) toregulate transcription, or by initiating nongenomic signalingat extranuclear sites, such as the plasma membrane(Bjornstrom and Sjoberg 2005; Levin and Hammes 2016).The evolutionary origin of ERa and ERb is not well resolved,and previous phylogenetic studies have led to debate over therelationships of ERs in cyclostomes (lampreys and hagfishes,the extant jawless fishes) and gnathostomes. Over the lasttwo decades, four alternative models have been proposed forthe evolution of ERs in vertebrates. The first model (fig. 1A)suggests that cyclostomes possess a pro-ortholog of the gna-thostome ERA/ERb (Thornton 2001). The second model(fig. 1B) places cyclostome ER at the base of the gnathostomeERA clade, implying that the ERA/ERb duplication occurred atthe base of vertebrates, that cyclostomes have a bona fideERA, and that ERb was subsequently lost (Baker andChandsawangbhuwana 2008). The third model (fig. 1C) pla-ces one cyclostome ER at the base of the gnathostome ERbclade, whereas the other cyclostome ER falls out as a sister tothe ERA/ERb clade (Baker et al. 2014; Nishimiya et al. 2017).The fourth model (fig. 1D), similar to the trees published by

Thornton (2001), places cyclostome ERs outside of thegnathostome ERA/ERb clades (Katsu et al. 2016).

The lack of resolution between the jawed and jawless ver-tebrate Esr sequences has been confounded, in part, by apaucity of data from chondrichthyans (cartilaginous fishes,including sharks, skates, rays, chimaeras, and their relatives),which occupy a critical phylogenetic position. In an effort toresolve the evolutionary history of vertebrate ER genes, weenriched taxonomic sampling of chondrichthyans and per-formed molecular phylogenetic analyses, protein homologymodeling, and in situ hybridization studies. Here we report onthe identification of true orthologs of osteichthyan ERa andERb in elasmobranchs and in a holocephalan, indicating thatERa and ERb arose prior to the divergence of chon-drichthyans and osteichthyans.

ResultsWe identified, cloned, and sequenced two potential ER ortho-logs in the little skate Leucoraja erinacea (Chondrichthyes:Elasmobranchii: Batoidea; see supplementary materials andmethods, Supplementary Material online). Based on initialprotein motif prediction, both ER orthologs possessed thecanonical ER motifs, including the ligand-independent trans-activation domain (AF-1), a DNA-binding domain (DBD),a hinge region, and a ligand-binding domain (LBD/AF-2)(supplementary fig. S1, Supplementary Material online).Furthermore, we found high percent identity and sequenceconservation of these critical motifs between the L. erincacea

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Mol. Biol. Evol. doi:10.1093/molbev/msy165 Advance Access publication September 10, 2018 1

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ERs and the ERs of other vertebrates (supplementary fig. S1Aand B, Supplementary Material online), suggesting orthologyto ERA and ERb.

To test the hypothesis that the two putative ER sequen-ces from L. erinacea are orthologs of vertebrate ERa andERb, we performed phylogenetic reconstructions using ERaand ERb sequences from osteichthyans, putative ERsequences from elephant shark (Callorhinchus milii;Chondrichthyes: Holocephali), whale shark (Rhincodon typus;Chondrichthyes: Elasmobranchii: Selachii), and catshark(Scyliorhinus torazame; Chondrichthyes: Elasmobranchii:Selachii), and all available ER sequences from cyclostomes

and cephalochordates (Venkatesh et al. 2014; Read et al.2017; Katsu et al. 2016) (supplementary table S1,Supplementary Material online). The steroid receptor (SR)of Amphioxus floridae served as an outgroup (supplementarytable S1, Supplementary Material online). Bayesian phyloge-netic analysis using full-length amino acid sequences (seesupplementary materials and methods, SupplementaryMaterial online) placed a single skate ER within each of thetwo gnathostome ER clades (supplementary fig. S2A,Supplementary Material online). The tree topology was sim-ilar to the third model (fig. 1C), with some cyclostomesequences falling within the respective ERb clade, whereas

FIG. 1. Evolution of vertebrate ERs. Gnathostome ERa clade is highlighted in pink, ERb clade in blue, and lamprey/hagfish (cyclostome) species ingreen. Trees from previous studies of ER evolution show contrasting topologies and different patterns of gene duplication. (A) Model 1 fromThornton (2001). (B) Model 2 from Baker and Chandsawangbhuwana (2008). (C) Model 3 from Baker et al. (2014) and Nishimiya et al. (2017). (D)Model 4 from Katsu et al. (2016). (E) Nucleotide tree generated from the new data reported in this study (full version shown in supplementary fig.S4, Supplementary Material online). All node values are posterior probabilities. Shaded boxes show the positions of chondrichthyan species.

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the remaining sequences grouped independently of the ERa/b clades. Though this topology was generally well supportedby posterior probabilities (major clades�0.73), long brancheswithin cyclostomes suggested long-branch attraction (LBA)bias. Analysis of the alignment revealed cyclostome-specificinsertion/deletions. To test for LBA, we removed suspectsequences in different combinations and re-evaluated treetopology. These subsequent analyses yielded different treetopologies depending on which sequence(s) were eliminated(supplementary fig. S2B–E, Supplementary Material online),suggesting that our initial tree (and those of other studies)was likely affected by LBA artifacts.

To limit the influence of LBA, we next restricted our anal-ysis specifically to the LBD of the receptors, which is highlyconserved across species but, unlike the DBD, retains subtype-specific residues at key positions that may provide adequateinformative characters for phylogenetic resolution (see sup-plementary materials and methods, Supplementary Materialonline). The LBD-specific analysis resulted in a tree topologyin which all cyclostome sequences formed clades indepen-dent of gnathostome ERa/b (supplementary fig. S3,Supplementary Material online; major node support�0.89), and identified skate sequences as ERa and ERb (sup-plementary fig. S3, Supplementary Material online,highlighted taxa). Although promising, limiting the data setto only the LBD resulted in some taxa (e.g., cyclostomes,amphibians) having identical sequences, and, as a conse-quence, these were unresolvable. Therefore, we analyzedthe corresponding nucleotide sequences of the LBD (supple-mentary table S1 and supplementary materials and methods,Supplementary Material online). In contrast to the protein-level analysis, this reconstruction had increased resolution,particularly with respect to the cyclostomes (fig. 1E; supple-mentary fig. S4, Supplementary Material online). To confirmthat LBA was not affecting these results, we systematicallyremoved hagfish and lamprey sequences and evaluated thedata set under equivalent parameters. In all cases, the treetopology was unchanged and support remained high at ma-jor nodes (compare supplementary fig. S5A–C with supple-mentary fig. S4, Supplementary Material online). Takentogether, these results show that chondrichthyans have twogenes that encode ERs, and these are orthologs of Esr1/ERAand Esr2/ERb, consistent with Thornton’s original hypothesis(fig. 1A and E; Thornton 2001).

In order to infer ligand-binding properties of the skatereceptors, we used protein homology modeling of the LBDto estimate ER structure and function in L. erinacea (supple-mentary materials and methods, Supplementary Material on-line). Our results show the secondary and tertiary structuresof the LBD of both skate and human ERA and ERb are strik-ingly similar, consisting of 12 antiparallel a-helices (supple-mentary fig. S6A and B, Supplementary Material online). As inhumans, 11 of these helices fold into a 3-layered “wedge-shaped” molecular scaffold that maintains a ligand-bindingcavity (Brzozowski et al. 1997; Ascenzi et al. 2006). Theremaining secondary structural elements are a small two-stranded antiparallel b-sheet and a final A-helix, which arelocated at the ligand-binding portion of the molecule and

flank the main three-layered motif (Brzozowski et al. 1997;Ascenzi et al. 2006). The accuracy of these models was sup-ported by global model quality estimation (GMQE) scores of0.95 and 0.87, respectively (see supplementary materials andmethods, Supplementary Material online). In addition to thestrong structural conservation of the skate ER orthologs, theyalso possess the amino acid residues in the ligand-bindingpocket that are known to facilitate binding of estradiol andother receptor agonists (pink boxes in supplementary fig. S6Aand B, Supplementary Material online; see also Kuiper et al.1997). Finally, the LBD of human ERA and ERb possessesamino acid substitutions that alter the conformation of theirligand-binding pocket and configure subtype-specific bindingof certain molecules (Paech et al. 1997; Barkhem et al. 1998;Paige et al. 1999). These substitutions were also identified inthe skate predictive model (black boxes in supplementary fig.S6A and B, Supplementary Material online). Therefore, theconformation of the skate and human ERA and ERb LBD arehighly similar, and the predicted skate structures possess vitalsubtype-specific residues, suggesting that functional diver-gence of the LBD in these two receptors is conserved in skates.

To determine whether the predicted structures of skateERA and ERb are conserved in other chondrichthyan lineages,we performed homology modeling of holocephalan andshark ER proteins. Like the skate proteins, ERa and ERbfrom C. milii (elephant shark) shared strong structural con-servation with the human orthologs (GMQE¼ 0.87 and 0.82,respectively; supplementary figs. S7A and B, SupplementaryMaterial online), as did ERb from Rhincodon typus (whaleshark; GMQE¼ 0.78; supplementary fig. S7C,Supplementary Material online). Interestingly, an aminoacid substitution within the LBD of ERb was found in allchondricthyans but not in humans (supplementary figs. S6and S7, Supplementary Material online, ERb, alignment posi-tion 76). This amino acid change, M76L, also has been de-scribed in lungfish and some amphibians and it does notconfer any appreciable differences in ERb-specific activity(Katsu et al. 2008; Katsu, Taniguchi, et al. 2010). These resultssuggest that the features of skate ERa and ERb proteins arebroadly applicable to chondrichthyans.

Although our LBD homology modeling suggests that skateERs can bind estrogenic ligands, we also examined the AF-1N-terminal domain, which is associated with transcriptionalactivity in response to ligand-dependent and ligand-independent activation of the ERs (Pettersson et al. 2000;Metivier et al. 2001; Zwart et al. 2010; Arao et al. 2012). Thepresence of a predicted A-helical domain in ERA, but not ERb,is associated with the different transactivation potentials ofthe receptors in several vertebrates (Metivier et al. 2000, 2001;Zwart et al. 2010; Fuchs et al. 2013). Hydrophobicity clusteranalysis of the AF-1 region in skates revealed an A-helix pre-sent in a similar position within the skate ERA subtype only(supplementary fig. S8A, Supplementary Material online;boxed). Thus, skate ERA possesses an important secondarystructure that has been characterized as a key difference as-sociated with functional divergence of the two ER subtypes.

To predict whether skate ERA and ERb are capable of DNAinteraction, we analyzed the DBD of both receptors.

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Alignment of this highly conserved region of ERA and ERbshowed that the “P” and “D” boxes of the DBD, the twostructural elements that confer DNA binding capabilities(Schwabe et al. 1990, 1993), are fully conserved in skates (sup-plementary fig. S8B, Supplementary Material online). Indeed,this conservation extended to all of the ER orthologs that weexamined, with the notable exceptions of arctic lamprey andsea lamprey ER2 (discussed below). Collectively, these struc-tural models suggest that skate ERA and ERb: 1) are capable ofbinding estrogenic ligands in a subtype-specific manner; 2)possess transcriptional responses that are subtype-specific;and 3) have the potential to bind ERE sequences.

We next investigated whether Esr1 and Esr2 have subtype-specific expression patterns during skate embryonic develop-ment. In situ hybridization analysis of embryos at stage 26showed that Esr1 and Esr2 are expressed in the developinghead, gill arches and early pectoral and pelvic fin buds; how-ever, no expression was detected in the flank region betweenthe emerging paired fins (fig. 2A and B). At stage 30, expres-sion of both genes persisted in the cranial region (fig. 2C andD), but they showed divergent expression patterns elsewhere.Specifically, Esr1 was detected throughout the fins buds,whereas Esr2 showed restriction to the posterior region ofthe pectoral fin and the anterior and posterior regions of thepelvic fin (compare fig. 2C and D). Furthermore, Esr1 wasexpressed throughout the gill arches at stage 30, whereasEsr2 showed stronger expression in posterior arches (fig. 2Cand D; supplementary fig. S9, Supplementary Material online).We also detected expression of both receptors in the lateralline, the superficial chain of mechanosensory organs (fig. 2Cand D). At stage 31, Esr1 began to show more intense stainingin the posterior gill arches and Esr2 expression was even moreposteriorly restricted (fig. 2E and F; supplementary fig. S9,Supplementary Material online). Thus, Esr1 and Esr2 showsubtle differences in their temporal dynamics of expression.

We previously demonstrated that androgen receptor (AR)is expressed in developing pelvic fins of skates, and that ARsignaling regulates sexually dimorphic development of clas-pers, which are the copulatory organs on male pelvic fins(O’Shaughnessy et al. 2015). Furthermore, in tetrapod limbs,sexually dimorphic development of the digits is regulated byboth AR and ER signaling (Zheng and Cohn 2011). We there-fore investigated whether ER subtypes show sexually dimor-phic expression patterns in skate fin buds. At early stages ofpelvic fin development (stages 28 and 29), Esr1 and Esr2 areexpressed throughout the fin bud mesenchyme and in thecloaca (supplementary fig. S10A–D, Supplementary Materialonline). At stage 30, when sexual differentiation of the pelvicfins begins (O’Shaughnessy et al. 2015), Esr1 and Esr2 begin toshow enhanced staining in the clasper-forming region of malepelvic fins (fig. 2G and H). In female pelvic fins, Esr1 and Esr2expression patterns are similar to males, but the Esr1 domainappeared broader than Esr2 (fig. 2G and H). By stage 32, Esr1and Esr2 showed strong expression anteriorly and posteriorly(in the clasper bud) in male pelvic fins. In contrast, Esr1showed little staining in the female pelvic fin at stage 32,but Esr2 was strong anteriorly and weaker posteriorly (fig. 2Iand J). Taken together, these results show that skate Esr1 andEsr2 have subtype-specific and sexually dimorphic expressionpatterns during embryonic development.

DiscussionOur results show that chondrichthyans have bona fide ortho-logs of Esr1 and Esr2, the genes that encode ERA and ERb.Comparisons of the predicted ERA and ERb structures tothose of other gnathostomes showed conservation of resi-dues that characterize the A and b subtypes, which are nec-essary for ligand binding. Furthermore, the presence of an a-helical domain in the AF-1 region of the ERA ortholog, but

FIG. 2. Expression of Esr1 and Esr2 in Leucoraja erinacea embryos. Whole mount in situ hybridizations; anterior is to left in (A)–(F) and top in (G)–(J). (A) Stage 26 embryo showing expression of Esr1. Arrowheads point to expression domains in early fin buds and head. (B) Stage 26 embryoshowing Esr2 expression in domains similar to Esr1 (compare with A). Stage 30 embryos showing sustained expression of Esr1 (C) and Esr2 (D) inpaired fins, and in the lateral line sensory system. Note that Esr2 expression domains are more restricted than at stage 26. Stage 31 embryos showingexpression of Esr1 (E) and Esr2 (F) in fins, gill arches, and the lateral line. Pelvic fins of male (right) and female (left) skates at stages 30 (G, H) and 32 (I,J) showing expression of Esr1 (G, I) and Esr2 (H, J). pec, pectoral fins; pel, pelvic fins; h, head; lat, lateral line; g, gill arches.


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not ERb, is consistent with the subtype-specific secondarystructures that are found in teleosts and humans (Metivieret al. 2000, 2001). Collectively, these data suggest that Esr1 andEsr2 arose by duplication of the ancestral Esr1/2 gene beforethe chondrichthyan and osteichthyan lineages diverged, andthey had evolved subtype-specific characteristics (includingsequence, protein structure, and spatiotemporal expressiondynamics) associated with development of sexually dimor-phic morphology.

Based on phylogenetic evidence, Thornton (2001) sug-gested that the ancestral Esr gene duplicated in gnathostomesto produce Esr1 and Esr2. At the time, only a single lampreyER had been identified, and the conclusion that the ER du-plication occurred in gnathostomes was consistent with aWGD (1R) before the vertebrate radiation, followed by agnathostome-specific WGD (2R). As additional ERs wereidentified in cyclostomes, the resolution of their phylogeneticpositions was confounded by a deficiency in sampling and bythe LBA artifacts that we described above. Our phylogeneticreconstruction, which includes the newly cloned L. erinaceaEsr1/ERA and Esr2/ERb, supports the existence of two sepa-rate paralogy groups of vertebrate ER genes, gnathostomeEsr1 and Esr2, and cyclostome genes that we designate Esrc

and Esrd. This new proposed naming scheme is intended toresolve the dissonance in the literature, as these cyclostomesequences have been called various other names based onconflicting phylogenetic placements (e.g., ERa and ERb, ER1and ER2, and Esr1a and Esr1b in Baker et al. 2014; Katsu et al.2016; Nishimiya et al. 2017, respectively). Thus, the resultspresented here validate the hypothesis that Esr1 and Esr2arose by a gnathostome-specific duplication of an ancestralEsr gene, as originally proposed by Thornton (2001).

Our tree places Esrd from arctic lamprey and sea lampreyas an outgroup to Esr1, Esr2, EsrÇ, and the other Esrd (fig. 1E).Although this could imply that Esrd is a pro-ortholog of thegnathostome and cyclostome ERs, an alternate explanation isthat this positioning reflects the degeneration or functionaldivergence of Esrd (Katsu et al. 2016). Arctic lamprey Esrd wasshown to be incapable of binding estrogen-related ligands,which is primarily thought to be due to an insertion of 4amino acids, in addition to other destabilizing forces on theligand binding pocket (Katsu, et al. 2016) (refer to supple-mentary fig. S6C, Supplementary Material online).Presumably, sea lamprey Esrd groups with the arctic lampreyEsrd, because they share this insertion in their LBD, as well asunique mutations within their DBD including the D-box

FIG. 3. The results presented here show that chondrichthyans have two ER subtypes, ERa and ERb, encoded by the Esr1 and Esr2 genes, respectively.The duplication that gave rise to ERa and ERb occurred near the base of gnathostomes, prior to the divergence of chondrichthyans andosteichthyans. Cyclostome ERs underwent parallel duplications and, because they lack strict orthology to gnathostome ERa and ERb, we proposethat they are named ERd/c. Black circles represent gene duplication; white circle indicates gene loss; gray circle indicates degeneration (loss ofligand and DNA binding activity; Katsu et al. 2016) It is noted the lamprey ERd represented here is either in the process of degrading, or has acquiredligand-independent activity.


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(supplementary fig. S8B, Supplementary Material online).Teleosts have two Esr2 genes (Esr2a and Esr2b; refer to sup-plementary table S1, Supplementary Material online) due tothe teleost-specific WGD (3R), and the single copy of Esr1suggests that its paralog was lost. A duplicated Esr1 has beenreported in sturgeon, which may be the result of anAcipenseriform-specific duplication (Katsu et al. 2008). Thus,several independent duplications of ERs have occurredthroughout vertebrates (fig. 3).

Although vertebrates shared a WGD (1R), a separate par-allel WGD (2R) in cyclostomes and gnathostomes (Mehtaet al. 2013) could have led to EsrÇ and Esrd in the formerand Esr1 and Esr2 in the latter. Alternatively, if vertebratesshared a 1R/2R WGD (Smith et al. 2013), then extensive in-dependent sequence evolution would be required for gna-thostome ERs to group separately from those of cyclostomes.A third possibility is that the two ER paralogs in gnathos-tomes resulted from a gnathostome-specific WGD (2R),whereas cyclostome ER paralogs arose by tandem orchromosomal-scale duplications. Support for each of thesethree scenarios can be found in other studies, and the timingof the WGD events in vertebrates remains a topic of debate(Smith and Keinath 2015; Smith et al. 2018). Hemoglobin andmyoglobin duplicated independently in cyclostomes and gna-thostomes (Schwarze et al. 2015), as did clade A fibrillar col-lagen genes (Zhang and Cohn 2006), which can explain thelack of obvious 1:1 orthology. Based on the recently assem-bled sea lamprey germline genome (and the associated scaf-folds), EsrÇ and Esrd are on different large scaffolds and,therefore, are not likely syntenic (Smith et al. 2018).Furthermore, the lack of related neighboring genes suggeststhat large chromosomal segment duplication is unlikely; incontrast, the Esr1 and Esr2 regions in jawed vertebrates docontain paralogous neighboring genes.

In addition to the localized expression of both ERs in skatepaired appendages (fins and male claspers), we also foundpolarized expression in the gill arches. The structure of thecartilaginous gill bars of sharks led Gegenbauer to posit thatpectoral fins may have evolved from posterior gill arches(Gegenbauer 1878). Although this hypothesis has receivedlittle support from the fossil record (Coates and Cohn1998), it has been suggested that molecular similarities be-tween chondrichthyan gill arches and fins, such as polarizedexpression of sonic hedgehog (Shh) and fibroblast growthfactors (Fgfs), may reflect shared ancestry of these structures(Gillis et al. 2009; Gillis and Hall 2016). Although our data doesnot necessarily support or refute Gegenbauer’s hypothesis, wefind it intriguing that Esr1 and Esr2 are expressed (i) in thedeveloping gill arches in spatial domains that are strikinglysimilar to Shh and its receptor Patched1 (Ptch1); (ii) during finbud initiation; (iii) throughout paired fin development; and(iv) within the developing claspers of male skates. We inter-pret these findings as evidence for deep conservation of theancient gene regulatory network (GRN) that governs devel-opment of appendages, including paired fins and gills (Shubinet al. 2009; Pieretti et al. 2015) rather than evidence for anactual embryonic morphological transformation of gills tofins. Moreover, a recent analysis of skate pelvic fin

development showed that another SR, the AR, controls tran-scription of Hand2, an upstream regulator of sonic hedgehog,to initiate sexually dimorphic fin (clasper) development inmales (O’Shaughnessy et al. 2015). This led to the proposalthat AR may have played a role in the evolution and devel-opment of vertebrate paired appendages through its cooper-ation with the appendage development GRN. Our new dataon Esr1 and Esr2 in skate fin development, together withprevious findings that AR and ER control sexually dimorphicdevelopment of tetrapod digits (Zheng and Cohn 2011), fur-ther support the hypothesis that sex steroids played a role inthe evolution of vertebrate appendages. We propose that theEsr1/2 duplication in gnathostomes allowed further modula-rization of sex hormone signaling and contributed to theevolution of sexually dimorphic development of vertebratemorphology.

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online.

AcknowledgmentsThe authors thank Scott Bennett and David Remsen of theMarine Biological Laboratory (Woods Hole, MA) for access toembryos, and members of our lab for stimulating discussionsand critical comments on this work. This project was sup-ported by a Howard Hughes Medical Institute Early CareerScientist award to M.J.C. K.L.O. was supported in part by aUniversity of Florida Alumni Fellowship. R.R. was supportedby an Natural Sciences and Engineering Research Council ofCanada postdoctoral fellowship.

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1

Supplementary Materials

Title: Cartilaginous fishes provide insights into the origin, diversification, and

sexually dimorphic expression of vertebrate estrogen receptor genes Authors: Filowitz, Rajakumar, O’Shaughnessy, and Cohn Supplementary Figures S1-S10 Supplementary Table S1 Supplementary Materials and Methods

2

Supplementary Figures S1-S10

Figure S1. Structural domains of ERa and ERb. Each color block corresponds to a specific motif, as denoted in the human proteins. Light green represents the ligand-independent transactivation function (AF-1), teal is the DNA-binding domain (DBD), dark gray is the hinge region (H), dark blue is the ligand-binding domain (AF-2), and light gray is the C-terminus (C). (A) Human ERa and ERb. (B) Domains of ERa in lungfish, zebrafish, and the predicted skate ortholog. Denoted within each domain is the percent identity to the human sequence. (C) Domains of ERb, with percent identity denoted within each functional domain. Note that, overall, the skate sequences display higher percent identity with human than do the zebrafish orthologs.

1 179 261 310 551 595

1 144 225 263 499 531

Human ERα

Human ERβ

ERα orthologs ERβ orthologs

A

B C

AF-1 DBD H AF-2 C

AF-1 DBD H AF-2 C

177 259 303 543 595

44% 100% 45% 68% 14%

Lungfish

1 168 249 327 566 604

40% 95% 19% 74%

Lungfish

1 143 224 273 513 562

AF-1 H AF-2 C

Zebrafish

19% 94% 27% 63% 6%

1 176 258 306 546 574

AF-1 DBD H AF-2 28%

Skate

1

47% 99% 33% 77%

1 154 235 313 561

27% 91% 11% 64% 15%

Zebrafish ERβ-1

1 165 246 324 561 583

33% 90% 23% 71% 26%

Skate

1

11%

598

1 160 241 296 548

15% 92% 16% 66% 12%

Zebrafish ERβ-2

592

3

Figure S2. Identification of long branch attraction (LBA) using full length amino acid sequences with Bayesian inference. Gnathostome ERa clade is highlighted in pink, ERb clade in blue, and lamprey/hagfish (cyclostome) species in green. (A) Tree includes all species in this study. Note that some lamprey ER sequences fall within the gnathostome ERa clade. All major nodes show high support. (B) Removal of the arctic lamprey ER2 sequence changed the tree topology, causing some cyclostome sequences to group with the gnathostome ERb clade. This topology was also well supported (posterior probabilities ³0.69). (C-E) To further test for LBA, we removed brook lamprey and sea lamprey ER sequences (C), removed all lamprey sequences, leaving only hagfish sequences (D), and used only arctic lamprey sequences (E). These iterations each resulted in different but well supported tree topologies, demonstrating that the ER dataset likely suffered from LBA artifacts. See Supplementary Table S1 for sequence information.

A

B C

D E

0.3

Goldfish_ERAlpha

Arowana_ERAlpha

AClawedFrog1L_ERAlpha

IndonesianCoelacanth_ERAlpha

WhaleShark_ERBeta

Lungfish_ERBeta

Perch_ERBeta1

AmphioxusFloridae_ER

Perch_ERAlpha

Salamander_ERAlpha

IndonesianCoelacanth_ERBeta

AmphioxusFloridae_SR

LittleSkate_ERAlpha

SpottedGrouper_ERBeta2Rockfish_ERBeta2

Trout_ERBeta2

Goldfish_ERBeta2

AmphioxusBranchiostoma_ER

Gar_ERAlpha

WrinkledFrog_ERAlpha

WhaleShark_ERAlpha

Sterlet_ERBeta

Hagfish_ER

Catfish_ERBeta2

Trout_ERAlpha

Zebrafish_ERBeta1

Loach_ERBeta2

Rat_ERAlpha

Zebrafish_ERBeta2

Guppy_ERAlpha

SpottedGrouper_ERAlpha

Dove_ERAlpha

LeopardGecko_ERAlpha

Eel_ERBeta1

AfricanCoelacanth_ERBeta

JapaneseStripedSnake_ERAlpha

Mouse_ERBeta

InshoreHagfish_ER1

Salmon_ERAlpha

ArcticLamprey_ER1

Salamander_ERBeta

SpinyDogfish_ERBetaElephantShark_ERBeta

InshoreHagfish_ER2

Newt_ERAlpha

Arowana_ERBeta2

Gar_ERBeta

Seahorse_ERBeta2

Eel_ERAlpha

Marmoset_ERAlpha

Pike_ERAlpha

LittleSkate_ERBetaLeopardGecko_ERBeta

Clownfish_ERBeta2

Salmon_ERBeta1

Lungfish_ERAlpha

SeaLamprey_ER

Axolotl_ERBeta

SailfinMolly_ERAlphaRainbowfish_ERAlpha

Axolotl_ERAlpha

Mouse_ERAlpha

ElephantShark_ERAlpha

Frog_ERBeta

Chicken_ERBetaAlligator_ERBeta

Crocodile_ERAlpha

Trout_ERBeta

CarolinaAnole_ERBeta

Ricefish_ERAlpha

SailfinMolly_ERBeta2

Frog_ERAlpha

MexicanTetra_ERBeta1

Clownfish_ERBeta1

AClawedFrog1S_ERAlpha

Salmon_ERBeta2

Goldfish_ERBeta1

CarolinaAnole_ERAlpha

PaintedTurtle_ERAlpha

Hummingbird_ERBeta

SpottedGrouper_ERBeta1

ArcticLamprey_ER2

Seahorse_ERAlpha

Seahorse_ERBeta1

RainbowTrout_ERAlpha

Eel_ERBeta2

PaintedTurtle_ERBeta

RainbowTrout_ERBeta2

Bichir_ERAlpha

MexicanTetra_ERAlpha

CloudyCatshark_ERBeta

Trout_ERBeta1

Sturgeon_ERBetaBichir_ERBeta

BrookLamprey_ER0.9715

1

1

0.902

11

1

1

1

1

0.5293

1

0.8685

1

1

1

1

0.9129

0.7856

1

1

0.7856

0.9975

1

1 1

0.5419

1

1

1

1

1

1

1

1

1

1

1

0.7638

1

1

1

1

0.7496

0.943

1

1

1

1

0.7094

1

1

1

1

0.9665

0.9966

1

0.9841

0.5611

1

1

1

1

0.9531

1

0.9858

0.9908

1

1

1

1

0.9799

1

0.964

0.7245

11

1

1

0.7312

0.9958

0.6072

1

1

1

0.933

1

1

0.5176

1

0.5

Salmon_ERAlpha

Zebrafish_ERBeta1

Mouse_ERAlpha

SailfinMolly_ERAlpha

Arowana_ERAlpha


Seahorse_ERAlpha

ArcticLamprey_ER2

Pike_ERAlpha

RainbowTrout_ERBeta2Seahorse_ERBeta2

Bichir_ERAlpha

Loach_ERBeta2


LittleSkate_ERBeta



SeaLamprey_ER

Rockfish_ERBeta2

InshoreHagfish_ER2


Sturgeon_ERBeta



Salmon_ERBeta2

InshoreHagfish_ER1

Salamander_ERBeta


Lungfish_ERAlphaNewt_ERAlpha



Salamander_ERAlpha



Axolotl_ERBeta


Clownfish_ERBeta2

Alligator_ERBeta


Trout_ERBeta2

Goldfish_ERBeta2


Guppy_ERAlpha


LittleSkate_ERAlpha

Clownfish_ERBeta1

Hummingbird_ERBeta

Rat_ERAlpha

LeopardGecko_ERBeta

Frog_ERBeta

Goldfish_ERBeta1

Crocodile_ERAlphaPaintedTurtle_ERAlpha

Marmoset_ERAlpha

Sterlet_ERBeta


Hagfish_ER

Eel_ERBeta1

Perch_ERAlpha

Dove_ERAlpha




Axolotl_ERAlpha

ElephantShark_ERBeta

Gar_ERAlpha

Salmon_ERBeta1

Eel_ERBeta2

Goldfish_ERAlpha

Frog_ERAlpha

Lungfish_ERBeta

Eel_ERAlpha

ArcticLamprey_ER1

Trout_ERBeta1

WhaleShark_ERAlpha

Seahorse_ERBeta1

Rainbowfish_ERAlpha

Catfish_ERBeta2


Arowana_ERBeta2

Gar_ERBetaBichir_ERBeta

Mouse_ERBeta

WhaleShark_ERBeta

Ricefish_ERAlpha


SpinyDogfish_ERBeta

Chicken_ERBeta


Trout_ERAlpha

Zebrafish_ERBeta2

Perch_ERBeta1

Trout_ERBeta

0.9976

1

0.5012

1

0.9359

1

1

1

1

1

0.5962

1

1

1

0.6698 1

1

1

1

1

1

0.5653

1

1

1

0.6508

1

1

1

0.9335

1

0.8919

1

1

0.6057

1

1

0.6758

0.9002

1

1

1

1

1

1

1

1

0.9857

1

1

1

1

1

1

0.8967

1

1

1

1

1

1

1

1

1

1

0.92641

1

0.9941

0.5571

0.7838

0.5594

1

1

0.6152

1

0.9192

1

1

0.9762

1

1

0.9347

1

0.9988

0.9917

1

0.5

Frog_ERBeta


Chicken_ERBeta


Seahorse_ERBeta2



Ricefish_ERAlpha

Eel_ERBeta1

Hummingbird_ERBeta

Crocodile_ERAlphaPaintedTurtle_ERAlpha

IndonesianCoelacanth_ERAlphaLittleSkate_ERAlpha


Salamander_ERBeta

SpinyDogfish_ERBeta



Arowana_ERBeta2

Gar_ERAlpha


Perch_ERAlpha

Goldfish_ERBeta2

Guppy_ERAlpha

WhaleShark_ERBeta

Salamander_ERAlpha

Loach_ERBeta2

Salmon_ERBeta2

Clownfish_ERBeta1

Alligator_ERBeta


WhaleShark_ERAlpha

Arowana_ERAlpha

Lungfish_ERAlpha

Dove_ERAlpha

Goldfish_ERBeta1Salmon_ERBeta1

Trout_ERBeta2


InshoreHagfish_ER2

Rockfish_ERBeta2


Trout_ERBeta1



Clownfish_ERBeta2

LittleSkate_ERBeta


Mouse_ERAlpha

Lungfish_ERBeta

Eel_ERAlpha

Zebrafish_ERBeta1




Zebrafish_ERBeta2

Axolotl_ERAlpha

Bichir_ERAlpha

Rat_ERAlpha

Sturgeon_ERBeta

LeopardGecko_ERBeta

Trout_ERAlpha


Gar_ERBeta

Seahorse_ERAlpha

Goldfish_ERAlphaSalmon_ERAlpha




Axolotl_ERBeta

Pike_ERAlpha

Mouse_ERBeta

InshoreHagfish_ER1

Rainbowfish_ERAlpha

Catfish_ERBeta2

Sterlet_ERBeta



Eel_ERBeta2

Perch_ERBeta1



Seahorse_ERBeta1

Frog_ERAlpha

Newt_ERAlpha

Marmoset_ERAlpha

Trout_ERBeta

Bichir_ERBeta

0.6232

0.9313

0.545

1

1

1

1

0.9242

0.9668

1

0.7962

10.5853

1

1

1

1

1

1

11

0.782

1

1

1

0.9858

11

0.9194

0.9976

1

1

1

1

1

1

1

1

0.9431

1

0.7014

1

1

1

1

1

1

0.9905

1

0.8934

0.7867

1

1

1

1

1

0.5948

1

1

1

1

1

1

1

0.7085

1

0.9668

1

0.955

1

0.7725

1

1

1

1

0.7441

1

1

1

1

1

1

1

1

0.5

WhaleShark_ERAlpha

Frog_ERAlpha

Marmoset_ERAlpha

Seahorse_ERAlpha

Rat_ERAlpha

Trout_ERAlpha

Eel_ERBeta1

ArcticLamprey_ER1

Guppy_ERAlpha

Hummingbird_ERBeta




Goldfish_ERBeta1Salmon_ERBeta1



Newt_ERAlpha

Salmon_ERAlpha


Trout_ERBeta1

Pike_ERAlpha

Eel_ERBeta2

Frog_ERBeta

Sturgeon_ERBeta


Seahorse_ERBeta1

Goldfish_ERBeta2

Bichir_ERBeta

Trout_ERBeta

Rainbowfish_ERAlpha


Gar_ERAlphaBichir_ERAlpha

LeopardGecko_ERBeta

Catfish_ERBeta2

Salamander_ERBeta



Mouse_ERBeta



Perch_ERBeta1



Rockfish_ERBeta2

Chicken_ERBeta

ArcticLamprey_ER2


Trout_ERBeta2

Alligator_ERBeta

WhaleShark_ERBeta

Arowana_ERAlpha

Zebrafish_ERBeta2



Ricefish_ERAlpha

Crocodile_ERAlpha

Loach_ERBeta2


Lungfish_ERBeta

Axolotl_ERBeta

Dove_ERAlpha

Lungfish_ERAlpha


Clownfish_ERBeta1

SpinyDogfish_ERBeta

Zebrafish_ERBeta1


Axolotl_ERAlphaSalamander_ERAlpha

Gar_ERBeta


LittleSkate_ERAlpha

Eel_ERAlpha


LittleSkate_ERBeta


Clownfish_ERBeta2


Seahorse_ERBeta2

Sterlet_ERBeta

Arowana_ERBeta2

Perch_ERAlpha

Mouse_ERAlpha

Goldfish_ERAlpha

Salmon_ERBeta2




1

1

1

1

1

0.872

1

1

0.6976

0.9829

1

1

1

1

1

1

1

0.5024

1

0.9963

1

0.8232

11

1

1

1

0.9341

1

1

0.6293

11

10.9293

1

0.9378

0.9841

10.7122

0.828

1

1

0.75

0.8537 1

1

11

0.9866

1

1

1

1

1

1

1

1

1

1

1

0.6061

0.6622

0.9988

1

1

1

1

1

1

1

1

1

1

1

0.6293

1

0.7085

0.989

0.9805

0.9951

0.9341

1

1

0.5

Goldfish_ERBeta2


Chicken_ERBeta

ArcticLamprey_ER1AmphioxusBranchiostoma_ER

Bichir_ERBeta


LittleSkate_ERBeta

Lungfish_ERAlphaNewt_ERAlpha


Salmon_ERBeta1

SeaLamprey_ER

WhaleShark_ERBeta


Hummingbird_ERBeta

Marmoset_ERAlpha

Salmon_ERBeta2

Dove_ERAlpha

Catfish_ERBeta2

InshoreHagfish_ER1


Frog_ERBeta

Mouse_ERBeta

WhaleShark_ERAlpha

Clownfish_ERBeta1

Axolotl_ERAlpha

Alligator_ERBeta

Seahorse_ERAlpha

Eel_ERAlphaMexicanTetra_ERAlpha

Rat_ERAlpha

Zebrafish_ERBeta1Goldfish_ERBeta1

Crocodile_ERAlpha

Salmon_ERAlpha


Arowana_ERBeta2

Seahorse_ERBeta2

Hagfish_ER


Frog_ERAlpha

Ricefish_ERAlpha

Trout_ERBeta2

Pike_ERAlpha

CloudyCatshark_ERBetaSpinyDogfish_ERBeta

Clownfish_ERBeta2

Perch_ERBeta1

Eel_ERBeta1


LittleSkate_ERAlpha


Trout_ERAlpha

Eel_ERBeta2


Trout_ERBeta

Rockfish_ERBeta2




Sturgeon_ERBeta

Perch_ERAlphaSpottedGrouper_ERAlpha

Axolotl_ERBeta


Goldfish_ERAlpha


Salamander_ERBeta


Lungfish_ERBeta



Arowana_ERAlphaBichir_ERAlpha

Sterlet_ERBeta

Zebrafish_ERBeta2


Gar_ERBeta

Salamander_ERAlpha

Trout_ERBeta1




InshoreHagfish_ER2

Seahorse_ERBeta1

Rainbowfish_ERAlpha

Gar_ERAlpha

LeopardGecko_ERBeta

Guppy_ERAlpha

Mouse_ERAlpha

Loach_ERBeta2


1

0.5746

1

1

1

1

1

1

1

1

1

1

0.999

1

0.8639

0.5716

1

0.8165

1

1

0.994

1

0.9839

0.873

1

1

0.76410.9748

1

1

1

1

1

0.88

1

1

1

1

1

0.9163

1

1

1

1

10.6976

0.9365

1

1

1

1

1

1

1

0.7651

1

0.881

1

0.9839

0.8377

1

1

0.874

1

0.9788

1

1

1

1

1

1

1

1

0.8518

1

1

1

0.9808

1

1

0.9567

1

0.997

1

1

0.6512

1

4

Figure S3. Full, uncollapsed amino acid tree using only the ligand-binding domain. All node values are posterior probabilities. Green branches denote cyclostome species; blue indicates gnathostome ERa; pink indicates gnathostome ERb. Shaded boxes show the positions of chondrichthyan species.

5

Figure S4. Full, uncollapsed nucleotide tree using only the ligand-binding domain. All node values are posterior probabilities. Green branches denote cyclostome species; blue indicates gnathostome Esr1; pink indicates gnathostome Esr2. Shaded boxes show the positions of chondrichthyan species.

6

Figure S5. Phylogenetic reconstructions using only the ligand-binding domains of ERs eliminate long branch attraction artifacts. Nucleotide trees were analyzed by Bayesian inference (see Supplementary Materials and Methods). Generation of well-supported topologies, regardless of inclusion or exclusion of cyclostome sequences in subsequent reconstruction runs, demonstrates robustness of trees. (A) Brook lamprey ER was excluded from the dataset. (B) Atlantic hagfish ER was excluded. (C) Arctic lamprey ER2 sequence was excluded. See Supplementary Table S1 for details of species and sequences.

A

B C

0.3

RaifishERA

SpGrouERBa

ClfishERBa

TurtleESRB

SeahorERBb

SeahorERBa

Human_ERSB

SterletERA

Perch_ERBa

LeGeckoERA

GuppfshERA

SterletERB

SfMollyERA

MarmoseERB

ArowanaERB

SalamanERB

HoMouseERA

RocfshERBb

SturgeoERBCatshrkERB

MexTetERBa

HoMouseERBPolecatERB

AngEelERBa

SturgeERA2

ICoelacERA

ALampreER2

CaAnoleERA

BichirEsr2

SeaLamprER

tropGarERB

Trout_EsrA

LiSkateERA

GallusERA

AlgatorERB

WhSharkERA

ICoelacERB

AFloridaER

RicefshERA

ABelcherER

LiSkateERB

IHagfshERB

LungfshERA

FirNewtERB

ChickenERB

AngEel_ERA

MexTetrERAArowanaERA

ACoelacERB

ZebfshERBa

WcFrog_ERA

ElSharkERA

LungfshERB

RicfshERBa

WriFrogERB

SfMollERBb

Horse_ESRA

ZebfshERBb

GoldfshERA

PolecatERA

LeGeckoERB

GldfshERBa

Sheep_EsrB

AFrogLESRA

AcoelacERA

Human_ESRA

AFrogLESRB

CatfishERA

SturgeERA1

TurtleEsrA

BichirEsr1

AngEelERBb

SpGroupERA

TroutEsRBa

TroutEsrBb

SpGrouERBb

Pike_EsrAl

tropGarERA

NoRat_EsrB

SLampreER2

JaSnakeERA

WhSharkERB

SpotGarERB

ALampreER1

WcFrog_ERB

FirNewtERA

ElSharkERB

SalmonEsrBb

SpotGarErA

SalamanERA

GldfshERBb

WriFrogERA

CatfshERBb

SpinDogERB

NoRat_EsrA

CaAnoleERB

SalmonEsrA

ZebrfshERA

AFloridaSR

CrocodiERA

Perch_EsrA

ClfishERBb

SalmonEsrBa

RocfshERBa

IHagfshERA

SeahorsERA

AHagfishER

MarmoseERA

1

1

0.5406

0.641

1

1

1

0.9975

1

1

1

1

0.9644

0.9217

0.8076

1

1

1

1

0.9745

0.9998

1

1

0.9923

0.6505

0.9999

0.993

0.8648

0.6866

1

0.6228

1

1

0.7816

0.9996

1

0.9399

1

1

0.51830.8889

1

0.9989

1

0.6085

1

1

0.7984

1

1

1

1

1

1

1

1

0.9949

0.9698

0.796

1

1

0.7191

1

0.9996

1

0.9871 1

0.9987

1

1

1

1

0.7654

1

0.5993

0.9997

1

1

1

1

11

0.9543

1

1

0.6419

1

0.998

0.8958

0.918

1

1

0.9933

0.9993

1

1

1

1

0.7632

0.9979

1

0.3

GldfshERBa

TurtleESRB

SalamanERB

JaSnakeERA

BLampreyER

RocfshERBa

Perch_EsrA

AFloridaER

Human_ERSB

CatshrkERB

SpGrouERBb

NoRat_EsrB

CatfshERBb

HoMouseERAPolecatERA

SalmonEsrBb

IHagfshERA

FirNewtERB

WcFrog_ERA

ICoelacERB

SterletERA

SterletERB

SpGroupERA

TurtleEsrAHuman_ESRA

SeahorERBb

MarmoseERB

RocfshERBb

ZebrfshERA

RicefshERA

SalmonEsrA

AFrogLESRB

AngEelERBa

BichirEsr2SpotGarERB

SturgeoERB

AFrogLESRA

WhSharkERB

AngEel_ERA

RaifishERA

ICoelacERA

ACoelacERB

SpotGarErA

Trout_EsrA

CaAnoleERB

WhSharkERA

GallusERA

FirNewtERA

ArowanaERA

SfMollERBb

SLampreER2

RicfshERBa

SpinDogERB

Perch_ERBa

MexTetERBa

SpGrouERBa

ArowanaERB

AFloridaSR

AngEelERBb

TroutEsRBa

SeaLamprER

ZebfshERBa

SeahorERBa

ZebfshERBb

SalmonEsrBa

LungfshERB

GoldfshERAMexTetrERA

CrocodiERA

tropGarERA

AcoelacERA

SfMollyERA

LungfshERA

PolecatERB

ClfishERBb

NoRat_EsrA

AlgatorERB

SturgeERA1

ABelcherER

CatfishERA

Horse_ESRA

WcFrog_ERB

CaAnoleERA

LiSkateERA

TroutEsrBb

SturgeERA2

ChickenERB

ALampreER2

ElSharkERB

MarmoseERA

ClfishERBa

SeahorsERA

ALampreER1

ElSharkERA

HoMouseERB

SalamanERA

GldfshERBb

WriFrogERA

LeGeckoERB

GuppfshERA

LeGeckoERA

BichirEsr1

Sheep_EsrB

LiSkateERB

Pike_EsrAl

WriFrogERB

tropGarERB

IHagfshERB

0.8719

1

1

11

0.9951

1

1

0.8927

0.9554

0.9982

0.9995

1

1

0.861

1

0.9823

0.9994

0.7239

0.9999

0.6773 1

0.9385

0.8158

1

1

0.964

0.5803

1 1

11

1

1

1

0.6258

1

1

1

1

1

1

1

1

0.9155

1

1

0.6267

1

0.5864

1

0.5464

0.9991

1

0.6778

0.9509

0.976

0.9965

1

1

1

1

0.7254

0.9929

1

1

0.9986

0.6873

1

1

0.511

1

0.9947

0.8381

0.7002

0.96660.9936

0.9946

0.9999

1

0.7489

1

0.6463

0.9999

0.8484

0.9992

1

0.9936

1

1

1

0.7826

1

0.9996

1

1

0.9453

1

1

0.9999

10.9994

0.3

AngEelERBb

MarmoseERB

LiSkateERB

LiSkateERA

SalamanERA

ElSharkERA

BLampreyERSLampreER2

GuppfshERAZebrfshERA

AFrogLESRB

AcoelacERA

RicfshERBa

WriFrogERA

SterletERA

SalmonEsrBb

CatshrkERB

FirNewtERB

GoldfshERA

NoRat_EsrA

CatfishERA

SpinDogERB

TurtleESRB

SalmonEsrA

SeaLamprER

ArowanaERA

GldfshERBb

WcFrog_ERA

SpotGarErA

MexTetERBa

ICoelacERB

Human_ESRA

GallusERALeGeckoERA

TurtleEsrA

RicefshERA

BichirEsr2

Horse_ESRA

Pike_EsrAl

SpGrouERBa

WcFrog_ERB

ClfishERBa

SturgeERA1

ICoelacERA

WhSharkERA

ACoelacERB

Perch_ERBa

GldfshERBaSalmonEsrBa

AFrogLESRA

SfMollyERARaifishERA

BichirEsr1

Human_ERSB

Perch_EsrA

HoMouseERB

CatfshERBb

Trout_EsrA

WriFrogERB

AngEelERBatropGarERB

PolecatERA

ZebfshERBa

SterletERB

CaAnoleERB

SturgeoERB

AFloridaSR

SeahorERBa

tropGarERA

SfMollERBb

IHagfshERA

JaSnakeERA

SpGrouERBb

RocfshERBa

IHagfshERB

SeahorERBb

NoRat_EsrB

Sheep_EsrB

TroutEsRBa

PolecatERB

TroutEsrBb

AHagfishER

SeahorsERASpGroupERA

MexTetrERA

AFloridaER

CrocodiERA

WhSharkERB

ZebfshERBb

ALampreER1

AngEel_ERA

LeGeckoERB

ElSharkERB

ABelcherER

MarmoseERA

ClfishERBb

HoMouseERA

SpotGarERB

RocfshERBb

ChickenERB

SalamanERB

LungfshERB ArowanaERB

FirNewtERA

SturgeERA2

CaAnoleERA

AlgatorERB

LungfshERA

0.9369

0.6711

0.9882

0.9923

1

1

1

1

0.9967

0.9232

0.9739

0.9983

0.9651

0.9437

1

1

0.6803

0.9749

0.9723

0.9699

0.9996

0.9651

0.9651

0.568

0.9965

0.7821

1

0.9548

11

1

1

11

0.9524

1

1

1

0.9926 1

0.9651

1

1

1

0.9999

0.7052

0.7225

1

0.9996

0.8652

1

1

0.9513

0.783

1

1

1

1

0.9669

0.7454

1

1

0.999

0.8835

0.9977

1

0.622

1

1

1

1

1

0.9995

0.9914

0.9884

1

0.9976

0.9904

0.623

0.6651

0.6111

1

1

0.992

0.9461

1

0.9918

1

0.8693

1

1

1

0.9993

0.9983

1

0.7288

1

1

1

0.6826

7

Figure S6. Homology modeling of skate and lamprey ER protein structures. ER structures were generated with SWISS-MODEL utilizing human ER crystal structures as templates. (A, B) Skate ERa and ERb are highly similar to their human orthologs. Residues necessary for ligand binding are conserved (pink boxes in alignments). Black boxes show conservation of subtype-specific residues in the ligand-binding domain. (C) Models of the arctic lamprey ER2 (left) using human ERb as the template. Our predicted structure of the LBD is similar to that described by Katsu et al., 2016. Specifically, the insertion (yellow box in alignment) constitutes a destabilizing looping of the alpha helix (yellow arrow) that renders this ER unresponsive to estrogen ligand (Katsu, et al. 2016). Sea lamprey ER2 contains a similar mutation within the LBD, which results in a similar destabilizing loop (yellow arrow in C) to that observed in arctic lamprey. This suggests that sea lamprey ER2 also has a loss of ligand-binding function, potentially mediated by accumulation of this unique protein sequence (yellow box in alignment; compare lampreys to human).

8

Figure S7. Homology modeling of holocephalan and shark ER protein structures. As described in Figure S6, ER models were generated with SWISS-MODEL utilizing human ER crystal structures as templates. (A, B) Elephant shark ERa and ERb, like the skate proteins, are highly similar to human ER orthologs. (C) The structure of whale shark ERb is also highly similar to the human ERb protein. Whale shark ERa was not estimated due to the availability of only a partial LBD sequence. In all models, the residues necessary for ligand binding are conserved and are highlighted in pink boxes. The black boxes show conservation of subtype-specific residues within the ligand-binding domain.

9

Figure S8. AF-1 N-terminal region and DNA binding domain. (A) Alignment of ERa and ERb AF-1 region in human, skate, and trout. Alignment is colored by hydrophobicity, with red residues being hydrophobic, blue hydrophilic, and purple intermediate. Previous work has identified a potential a-helix in ERa by hydrophobic cluster analysis (HCA) in human and trout, and showed that this region was important to ERa transcriptional activity (Metivier, et al. 2000; Petit, et al. 2000). We used HCA to corroborate those findings, and also identified a similar structure in skate (boxed). The predicted a-helix in skate has an iterative hydrophobic chain more similar to human than trout. Additionally, the secondary structure is in a closer relative position between skate and human (note gap in trout ERa). (B) DBD region of ERa and ERb in multiple vertebrates. The black rectangles indicate the P-box and D-box, the two regions that confer ER-specific DNA recognition and constitute the protein dimer interface. There is complete conservation of amino acids in the P-box and D-box of the gnathostome ERs.

10

Figure S9. Esr2 is expressed in the pharyngeal arches of skate embryos. Whole mount in situ hybridizations showing Esr2 mRNA localization (purple signal). The anterior-posterior axis of the embryos is indicated by A® P. Esr2 is expressed in the anterior and posterior regions of the developing gill arches, though staining appears to be stronger posteriorly.

11

Figure S10. Esr1 and Esr2 are expressed in skate pelvic fin buds. Whole mount in situ hybridizations showing Esr1 and Esr2 mRNA localization (purple). The cloacal opening is marked by (cl) at the anterior margin. (A, B) Esr1 and Esr2 are expressed broadly throughout the early pelvic fin bud mesenchyme at stage 28. (C, D) At stage 29, expression of both genes begins to show stronger expression in the posterior region of the pelvic fin buds (arrowheads). (E, F) Esr1 and Esr2 expression in the pelvic fins persists through stage 30, when sexual differentiation of the fins is initiated. Note dark staining in the regions where the male copulatory organs (claspers) begin to appear as posterior expansions of the pelvic fin buds (white arrows)

12

Supplementary Table S1. Complete list of sequences used in phylogenetic analyses

Common Name Scientific name NCBI annotation Proposed annotation NCBI accession code Chinese Amphioxus Branchiostoma belcheri ER ER AB510027 Florida Amphioxus Branchiostoma floridae ER ER EF554313 Arctic Lamprey Lethenteron camtschaticum ER1 Erγ AB626148 Inshore Hagfish Eptatretus burgeri ER1 Erγ KP987796.1 Sea Lamprey Petromyzon marinus ER1 Erγ AY028456 Atlantic Hagfish Myxine glutinosa ER Erδ EU439936 Arctic Lamprey Lethenteron camtschaticum ER2 Erδ AB626149 Inshore Hagfish Eptatretus burgeri ER2 Erδ KP987797.1 Northern Brook Lamprey

Ichthyomyzon fossor ER2 Erδ GBEL01000002

Sea Lamprey Petromyzon marinus ER2 Erδ APA19937.1 Whale Shark Rhincodon typus ER-like Esr1 XM_020532945.1 Atlantic Salmon Salmo salar Esr1 Esr1 NM_001123592.1 Axolotl Ambystoma mexicanum Esr1 Esr1 AB524912 Carolina Anole Anolis carolinensis Esr1 Esr1 NM_001290517 Channel Catfish Ictalurus punctatus Esr1 Esr1 NM_001200074.1 Crocodile Crocodylus niloticus Esr1 Esr1 AB209933 Dove Columba livia Esr1 Esr1 NM_001282825 Elephant Shark Callorhinchus milii Esr1 Esr1 XM_007894403.1 European Eel Anguilla anguilla Esr1 Esr1 LN879034.1 Goldfish Carassius auratus Esr1 Esr1 JX440380 Guppy Poecilia reticulata Esr1 Esr1 NM_001297487.1 Horse Equus ferus caballus Esr1 Esr1 NM_001081772 Human Homo sapiens Esr1 Esr1 NM_000125 Indonesian Coelacanth

Latimeria menadoensis Esr1 Esr1 HF562327

Japanese Puffer Takifugu rubripes Esr1 Esr1 XM_003971746.2 Japanese Striped Snake

Elaphe quadrivirgata Esr1 Esr1 AB548295

Killifish Fundulus heteroclitus Esr1 Esr1 AY571785.1 Leopard Gecko Eublepharis macularius Esr1 Esr1 AB240528 Little Skate Leucoraja erinacea Esr1 Esr1 ****** Lungfish Protopterus annectens Esr1 Esr1 AB435636 Marmoset Callithrix jacchus Esr1 Esr1 XM_008995270 Medaka Oryzias latipes Esr1 Esr1 XM_020714493.1 Mexican Tetra Astyanax mexicanus Esr1 Esr1 XM_007253897 Mouse Mus musculus Esr1 Esr1 NM_007956 Newt Cynops pyrrhogaster Esr1 Esr1 AB524908 Painted Turtle Chrysemys picta Esr1 Esr1 NM_001282246 Polecat Mustela putorius Esr1 Esr1 XM_004784330 Rainbowfish Melanotaenia fluviatilis Esr1 Esr1 GU319956.1 Rat Rattus norvegicus Esr1 Esr1 NM_012689

13

Salamander Hynobius tokyoensis Esr1 Esr1 AB524910 Silver Arowana Osteoglossum bicirrhosum Esr1 Esr1 LC057258.1 Spotted Gar Lepisosteus oculatus Esr1 Esr1 XM_006625845.2 Spotted Grouper Epinephelus coioides Esr1 Esr1 GU721076.1 Stickleback Gasterosteus aculeatus Esr1 Esr1 LC006094.1 Western Clawed Frog

Xenopus tropicalis Esr1 Esr1 AY310902

Trout Oncorhynchus mykiss Esr1b Esr1 NM_001124558.1 Sturgeon Acipenser schrenckii Esr1a Esr1a BAG82650.1 Sturgeon Acipenser schrenckii Esr1b Esr1b BAG82651.1 African Coelacanth Latimeria chalumnae Esr2 Esr2 XM_005986391 Alligator Alligator mississippiensis Esr2 Esr2 AB548298 Atlantic Salmon Salmo salar Esr2 Esr2 NM_001123577.1 Axolotl Ambystoma mexicanum Esr2 Esr2 AB524913 Carolina Anole Anolis carolinensis Esr2 Esr2 XM_008125840 Chicken Gallus gallus Esr2 Esr2 NM_204794 Cloudy Catshark Scyliorhinus torazame Esr2 Esr2 AB551715 Elephant Shark Callorhinchus milii Esr2 Esr2 XM_007910258.1 Firebelly Newt Cynops pyrrhogaster Esr2 Esr2 AB524909 Human Homo sapiens Esr2 Esr2 NM_001291723 Hummingbird Calypte anna Esr2 Esr2 XM_008501049 Indonesian Coelacanth

Latimeria menadoensis Esr2 Esr2 HF562328

Japanese Sturgeon Acipenser schrenckii Esr2 Esr2 AB435633.1 Leopard Gecko Eublepharis macularius Esr2 Esr2 AB240529 Little Skate Leucoraja erinacea Esr2 Esr2 ********* Lungfish Protopterus annectens Esr2 Esr2 AB435637 Mouse Mus musculus Esr2 Esr2 NM_207707 Painted Turtle Chrysemys picta Esr2 Esr2 XM_005285890 Rat Rattus norvegicus Esr2 Esr2 NM_012754 Salamander Hynobius tokyoensis Esr2 Esr2 AB524911 Senegal Bichir Polypterus senegalus Esr2 Esr2 LC057257 Silver Arowana Osteoglossum bicirrhosum Esr2 Esr2 LC057259.1 Spiny Dogfish Squalus acanthias Esr2 Esr2 AF147746 Spotted Gar Lepisosteus oculatus Esr2 Esr2 XM_006632189.2 Western Clawed Frog

Xenopus tropicalis Esr2 Esr2 NM_001040012

Whale Shark Rhincodon typus Esr2 Esr2 AB551716 Goldfish Carassius auratus Esr2 Esr2a AF061269.1 Catfish Ictalurus punctatus Esr2-1-like Esr2a XM_017456575 Seahorse Hippocampus comes Esr2-like Esr2a XM_019870757.1 Clownfish Amphiprion melanopus Esr2a Esr2a HM185180 Killifish Fundulus heteroclitus Esr2a Esr2a AY570922.1 Medaka Oryzias latipes Esr2a Esr2a NM_001104702.1 Perch Perca flavescens Esr2a Esr2a DQ984125.1

14

Rockfish Sebastes schlegelii Esr2a Esr2a FJ646610 Spotted Grouper Epinephelus coioides Esr2a Esr2a GU721077.1 Zebrafish Danio rerio Esr2a Esr2a NM_180966.2 Eel Anguilla anguilla Esr2b Esr2a LN879036.1 European Eel Anguilla anguilla Esr2b Esr2a LN879036.1 Sailfin Molly Poecilia latipinna Esr2-1 Esr2a KT022998.1 Rockfish Sebastes schlegelii Esr2a Esr2a FJ646610.3 Channel Catfish Ictalurus punctatus Esr2 Esr2b NM_001200083.1 Japanese Puffer Takifugu rubripes Esr2 Esr2b XM_003978635.2 Mexican Tetra Astyanax mexicanus Esr2-like Esr2b XM_007230932 Seahorse Hippocampus comes Esr2-like1 Esr2b XM_019877457.1 European Eel Anguilla anguilla Esr2a Esr2b LN879035.1 Arowana Osteoglossum bicirrhosum Esr2b Esr2b LC057259.1 Atlantic Salmon Salmo salar Esr2b Esr2b JF798871.1 Clownfish Amphiprion melanopus Esr2b Esr2b HM185178.1 Goldfish Carassius auratus Esr2b Esr2b AF177465.1 Killifish Fundulus heteroclitus Esr2b Esr2b AY570923.1 Medaka Oryzias latipes Esr2b Esr2b NM_001128512.1 Rainbow Trout Oncorhynchus mykiss Esr2b Esr2b NM_001124570.1 Rockfish Sebastes schlegelii Esr2b Esr2b HQ452829 Spotted Grouper Epinephelus coioides Esr2b Esr2b GU721078.1 Zebrafish Danio rerio Esr2b Esr2b NM_174862.3 Florida Amphioxus Branchiostoma floridae SR SR EU371729.1

15

Supplementary Materials and Methods Data Mining for Chondrichthyan ER Sequences

Initial searches for little skate (Leucoraja erinacea) ER sequences were performed on the

National Center for Biotechnology Information (NCBI) sequence databases using BLASTn

under the discontiguous megablast algorithm. West African lungfish (Protopterus annectens)

full-length nucleotide sequences for Esr1 (AB435636) and Esr2 (AB435637) were used as

queries to mine whole genome shotgun sequences of L. erinacea (taxid: 7782). Several hits for

potential orthologs of Esr1 (AESE012519307 and AESE010097393) and Esr2

(AESE011520100) were recovered, but these were either short fragments or had poor overall

query coverage. Therefore, these sequences were used as templates to clone Esr genes from

L. erinacea by polymerase chain reaction (PCR). Predicted mRNA sequences for elephant

shark (Callorhinchus milii) Esr1 (XM_007894403) and Esr2 (XM_007910258) and a partial

fragment for whale shark (Rhincodon typus) Esr1 (XM_020512638.1) were recovered from

NCBI RefSeq (see Supplementary Table S1).

Little Skate Husbandry and Tissue Collection

Leucoraja erinacea eggs were obtained from the Marine Biological Laboratory (Woods Hole,

MA). Eggs were maintained in Fluval marine salt adjusted to 32 ppt at ambient temperature in

tanks with both mechanical and biological filtration systems. For tissue collection in preparation

for RNA extraction, embryos were removed from their egg cases and staged (Maxwell, et al.

2008). Male and female animals of developmental stages 31 and 32 were euthanized in MS-

222 and the liver, gonads, and pelvic fins were dissected and preserved in RNAlater (Qiagen).

Cloning of Leucoraja erinacea ER orthologs

RNA was isolated using RNAeasy Mini Plus Kits (Qiagen) and quantified on a Nanodrop-1000

(Thermo Scientific). RNA integrity was evaluated by using a Bioanalyzer 2100 (Agilent

Technologies). For cDNA synthesis, only RNA with a 260/280 >1.9 and a RNA integrity number

(RIN) of >9.0 was used. For initial cloning experiments, 1 µg of RNA was used for cDNA

synthesis using the Maxima cDNA kit (Thermo Scientific). Two potential Esr fragments were

16

amplified by PCR. Amplicons were separated on a 1.2% gel stained with ethidium bromide, and

bands of ~500 bp were excised and purified using the Wizard SV Gel Clean-up System

(Promega). The fragments were then ligated into pGEM-T Easy Vectors (Promega) and

transformed into NEB Turbo Competent cells (New England Biolabs). Plasmids were harvested

using the Wizard Plus SV minipreparation system (Promega) and then sequenced in both

directions. Preliminary BLASTn searches showed that the two clones had high query coverage

for Esr1 and Esr2, respectively. Rapid amplification of cDNA ends (RACE)-PCR and mining of

the L. erinacea genome were used to recover full-length transcripts of the putative Esr1 and

Esr2 orthologs. To confirm the full-length sequences, RACE-PCR was carried out using the

SMARTer 5’/3’ system (Clontech) following the manufacturers protocol. Amplicons were ligated

into either StrataClone PCR Vectors (Agilent) or pGEM-T Easy Vectors (Promega), which were

introduced into NEB Turbo Competent cells by heat-shock. Plasmids were then isolated and

sequenced as described above.

Analyses of ER Structural Domains

For multi-species comparisons of ERa and ERb, skate Esr1 and Esr2 were translated and the

functional domains for human (AAD52984 and AAC05985), lungfish (BAG82655 and

BAG82656), and zebrafish (AAK16740 and AAK16742) were retrieved from NCBI. Sequences

were then aligned manually in Se-Al v2.0a11 (courtesy of Andrew Rambaut). Percent identity

(PID) was calculated relative to human sequences as follows: PID=100 (Identical

residues/average length of sequence).

Phylogenetic Analyses

Preliminary phylogenetic reconstruction of the Esr family was conducted using the skate

sequences reported here and the annotated Esr sequences from cyclostomes and

gnathostomes. Cephalochordate steroid receptor (SR) was used as an outgroup representative.

All sequences were imported into Se-Al v2.0a11, frame-corrected, translated, and trimmed at

the 5’ and 3’ untranslated region. Sequences were aligned using TranslatorX (Abascal, et al.

2010) and then imported into GENEIOUS (Kearse, et al. 2012) for manual adjustment to

compensate for large insertions/deletions. For analysis of the ligand-binding domain (LBD) after

discovery of long branch attraction (LBA; see below), the LBD was isolated from each species

17

and then manually examined and aligned using GENEIOUS. Phylogenetic reconstructions of

amino acid sequences were performed by Bayesian inference using MRBAYES 3.2.2

(Huelsenbeck and Ronquist 2001) via the Cipres Science Gateway (Miller 2010). We utilized

the Poisson distribution as an amino acid substitution model, with four estimated gamma

categories, and we performed each run for 5,000,000 generations with a burn-in of 25%. Run

convergence was confirmed when the average standard deviation of split frequencies was

£0.01. For phylogenetic analysis of nucleic acid sequences, we first used jModelTest 2.1.6

(Darriba, et al. 2012) to estimate an appropriate model of DNA substitution by AICc and BIC

criteria. In both scenarios, the GTR+G model was selected with four estimated gamma

categories, and we performed each run for 10,000,000 generations with a burn-in of 25%. As

with the amino acid analysis described above, nucleotide sequence run convergence was

confirmed when the average standard deviation of split frequencies was £0.01. All trees were

read and edited in FigTreev1.4 (courtesy of Andrew Rambaut).

Testing for Long Branch Attraction (LBA)

We tested for LBA as previously described (Siddall and Whiting 1999; Pol and Siddall 2001). In

short, potentially problematic sequences were identified and removed individually and in

alternative combinations to allow for reevaluation of tree topologies by equivalent phylogenetic

methods (described above). We considered an analysis to be affected by a LBA phenomenon

when removal of a sequence altered the tree topology.

Protein Homology Modeling

Homology modeling was done using the SWISS-MODEL server (Guex, et al. 2009; Biasini, et

al. 2014) utilizing human crystal structures as templates for ERa and ERb (RCSB Protein

Database ID 1gwr.2 (alpha) and 4j26.1 (beta). Human template structures were originally

obtained at resolution by X-ray crystallography (2.40 Å for ERa and 2.30 Å for ERb)(Warnmark,

et al. 2002; Fuchs, et al. 2013). Our alignments yielded 77% and 71% conservation of amino

acid residues between human and skate ERa and ERb, respectively. Model quality estimations

were evaluated using Global Model Quality Estimation and QMEAN scores (Benkert, et al.

2009), which were performed directly on the workspace. Both skate homology models received

strong quality scores, indicating high accuracy of the predicted structure from the input

18

sequence. ERa for elephant shark (Callorhinchus milii) and ERb for elephant shark and whale

shark (Rhincodon typus) were modeled using the same crystal structure templates as described

for skate. For lamprey homology modeling, the LBD of arctic lamprey (Lethenteron

camtschaticum, BAM48574.1) and sea lamprey (Petromyzon marinus, APA19937.1) were

estimated using human ERb as the template (RCSB Protein Database 4j26.1). Our estimated

structure for arctic lamprey was compared against previously published homology models for

this species (Katsu, et al. 2016).

Secondary Structure Prediction

Secondary structure of the AF-1 domain was estimated using hydrophobicity cluster analyses

(HCA)(Gaboriaud, et al. 1987), which was performed on the Mobyle server (Neron, et al. 2009).

To compare our data with previous analyses of the AF-1 functional core (Metivier, et al. 2000;

Metivier, et al. 2001), AF-1 domains of ERa and ERb in human and trout were analyzed in

addition to skate. Our analyses predicted an a-helix within the AF-1 region of ERa for all

species tested. For clarity of presentation, we generated an alignment of ERa and ERb (colored

by hydrophobicity) and boxed the predicted secondary structure.

In Situ Hybridization

RNA in situ hybridization was performed with digoxigenin-labeled probes for Esr1 and Esr2 in L.

erinacea embryos as previously described (Freitas and Cohn 2004; O'Shaughnessy, et al.

2015) with the following modifications: Proteinase K (PK) concentrations were 10 µg/ml for

embryos younger than stage 28, and 20 µg/ml for embryos at stage 29 and older; embryos

were not agitated during PK digestion and the reaction was stopped using 2 mg/ml glycine for

five minutes. The color reaction was completed in BM Purple (Roche) at 4°C overnight, and

embryos were dehydrated in a graded methanol series before imaging. Expression of each

gene was examined in a minimum of 3 males and 3 females at each stage.

19

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