HOW THE DEVIL RAY GOT ITS HORNS

A Thesis submitted to the faculty of San Francisco State University / ^ I ( In partial fulfillment of Y i q ^ the requirements for the Degree

Master of Science

In

Biology: Marine Biology

by

John Daniel Swenson

San Francisco, California

June 2018 CERTIFICATION OF APPROVAL

I certify that I have read How the Devil Ray Got Its Horns by John Daniel Swenson and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Marine Biology at San Francisco State University.

Carmen Domingo, Ph.D. Professor

Pleuni Pennings, Ph.D. Assistant Professor How the Devil Ray Got Its Homs

John Daniel Swenson San Francisco, California 2018

A central goal of evolutionary-developmental biology is to elucidate the genetic underpinnings of body plan evolution. Batoids (skates and rays) are basal gnathostomes, known for their distinctive disc-like pectoral fins that develop via novel gene expression patterns at the anterior. Devil rays and their charismatic relatives of the family Myliobatidae are batoids with a particularly derived body plan, exhibiting kite-shaped pectoral fins and a unique set of fleshy appendages at the anterior of the body called, “cephalic lobes”. Cephalic lobes are used for feeding and are morphologically similar to the anterior pectoral fins of other batoid species. However, the process by which cephalic lobes develop is unknown and their presumed homology with the anterior pectoral fins of other batoids has not been demonstrated at a developmental or molecular level. We therefore examined the morphological and molecular development of the cownose ray, a myliobatid genus that is sister to the devil rays. We found that cephalic lobes unequivocally develop as anterior extensions of the pectoral fins. Using comparative transcriptomics, we found developmental gene expression profiles in cephalic lobes that are indicative of homology with the anterior pectoral fins of skates. Combined, our data provide strong evidence that cephalic lobes are specialized anterior pectoral fin domains rather than independent appendages.

I certify that the Abstract is a correct representation of the content of this thesis.

Chair, Thesis Committee Date PREFACE AND/OR ACKNOWLEDGEMENTS

This project has been the most challenging and rewarding undertaking of my life so far and it absolutely would not have been possible without the guidance, generosity, and support of a number of people.

First and foremost, I must thank my adviser, Karen Crow-Sanchez, for giving me freedom to pursue this project from the start, encouragement when it seemed impossible near the beginning, and guidance and mentorship every step of the way. Your impact on my life has been monumental: bringing me into the lab altered my course in life and helped me build momentum towards a career I’ve dreamt about for over a decade. Also, swimming in the bioluminescence at Catalina Island was truly a highlight of my life. For these reasons and more, I am tremendously grateful.

It is impossible to do any research without samples. Fortunately, we had some amazing collaborators without whom this project would have been impossible. Particularly, I am indebted to Bob Fisher and George Trice (Sr. and Jr.). Your patience, laid-back attitudes, and generosity combined to make the fuel that launched this project. I hope to continue working with you gentlemen for years to come.

I’d also like to thank Gregg Poulakis and Rachel Scharer (Case) for your hospitality and the opportunities you gave me to work with rare and beautiful animals; Barry, Mark, Steve, and Bo for your efforts and willingness to help when I was in Florida; Matt Kolmann and

Christine Bedore for teaching me how to navigate the world of elasmobranch scientists; Kristene Parsons for being the most inspirational person I know; Michael Izumiyama,

Kayla Hall, and Shannon Barry for being wonderful friends and for allowing me to bounce ideas off you and for helping me navigate the lab; Peter Hundt for your sarcasm and wisdom; Frank Cipriano for your guidance and for helping me refine my techniques in the lab (pipetting especially!); and Brandice Thompson for the financial and moral support during the first two difficult years.

I also must thank my family, whose unwavering support, encouragement, and advice have been essential through this whole process, particularly over the last tempestuous eight months.

And finally, thank-you to my committee, Carmen Domingo and Pleuni Pennings, for your advice, feedback, patience, and encouragement.

This project truly was a team effort and it would not have been possible without every person listed above.

v TABLE OF CONTENTS

LIST OF TABLES ...... VIII

LIST OF FIGURES ...... IX

INTRODUCTION ...... 1

METHODS ...... 5

S a m p l e c o l l e c t i o n ...... 5

D evelopmental s t a g in g ...... 6 RN A SEQUENCING OF DISTINCT PECTORAL FIN DOMAINS...... 6

M e r g in g r e a d s f r o m d o m a in s f o r interspecies comparisons ...... 8

A d d it io n a l R N A - s e q u e n c in g f o r d e n o v o transcriptome a s s e m b l y ...... 9

D a t a a n a l y s i s ...... 10 Data preprocessing...... 10 Transcriptome assembly and annotation...... 11 Alignment and additional filtering...... 12 Differential expression...... 12 Visualization...... 14

RESULTS ...... 15

D evelopmental s t a g in g ...... 15 Stage one...... 20 Stage two...... 20 Stage three...... 20 Stage four...... 21 Stage five...... 21 Stage six...... 22 Stage seven...... 22

P e c t o r a l f in f u s io n ...... 2 2

C e p h a l ic l o b e s d e v e l o p a s m o d if ie d a n t e r io r p e c t o r a l f i n s ...... 23

S h a r e d interspecific developmental m il e s t o n e s b e t w e e n b a t o id s ...... 24

Q u a l it y c o n t r o l a n d d o m a in - specificity o f R N A -S e q d a t a ...... 24

S h a r e d m o l e c u l a r m e c h a n is m s u n d e r l ie c e p h a l ic l o b e a n d p e c t o r a l f in development 25

M y l io b a t id c e p h a l ic l o b e s a r e h o m o l o g o u s t o t h e a n t e r io r p e c t o r a l f in s o f s k a t e s ...... 28

N o v e l g e n e e x p r e s s io n p a t t e r n s a r e a s s o c ia t e d w it h u n iq u e m y l io b a t id f e a t u r e s ...... 31

DISCUSSION ...... 33

D evelopmental v a r ia t io n in b a t o id s ...... 33 vi P u t a t iv e g e n e t ic underpinnings o f m y l io b a t id - s p e c if ic f e a t u r e s ...... 36 Dlx2 and Dlx4...... 36 Lhx9...... 37 D kkl...... 37 Alxl and omd...... 38

D e e p h o m o l o g y o f p a t t e r n in g m e c h a n is m s in b a t o id p e c t o r a l f i n s ...... 39 Hand2...... 39 Tbx3...... 40 A 1x4...... 41 A PUTATIVE ROLE FOR HOXA GENES IN THE EVOLUTION AND DEVELOPMENT OF CEPHALIC LOBES 42

CONCLUSION...... 44

REFERENCES...... 45

vii LIST OF TABLES

Table Page

1. Table 1: ...... 7 2. Table 2 : ...... 19 3. Table 3 : ...... 19 4. Table 4: ...... 27 5. Table 5: ...... 31 6. Table 6 :...... 32

viii LIST OF FIGURES

Figures Page

1. Figure 1...... 9 2. Figure 2 ...... 15 3. Figure 3 ...... 25 4. Figure 4 ...... 26 5. Figure 5 ...... 30 6. Figure 6 ...... 35 7. Figure 7 ...... 42 8. Supplementary Figure 1 ...... 55 9. Supplementary Figure 2 ...... 56

ix 1

Introduction The same mechanisms that drive body plan diversification underlie the evolution of biodiversity. In response to varying environmental pressures, organisms evolve body plans that are adapted to specific habitats and lifestyles. This continual adaptive interplay between organism and environment has led to the evolution of some truly spectacular forms that have fascinated people for centuries. Devil rays and their charismatic relatives of the family Myliobatidae (manta rays, eagle rays, cownose rays) are shaped like kites, with broad pectoral fins that are wider than they are long. Myliobatids are members of the batoid clade, a group which includes skates, stingrays, and guitarfish (Aschliman, 2014; Last et al., 2016). Batoids, colloquially called “flat sharks”, have a dorso-ventrally flattened body plan with distinctive broad pectoral fins that fuse to the rostrum. The large anterior half of the pectoral fin is a derived feature that arose in association with modifications to existing developmental-genetic programs (Nakamura et al., 2015). When these programs were interrupted by the addition of exogenous retinoic acid, the anterior of the fin was reduced and the skate pectoral fins resembled the pectoral fins of their shark relatives (Dahn et al., 2007). Most batoids use their large compressed pectoral fins to swim by perpetuating small waves along the fins (Rosenberger, 2001). Myliobatids, however, swim by oscillating their pectoral fins up and down as though they are flying underwater (Rosenberger, 2001). The capacity for underwater flight, otherwise called ‘oscillatory swimming,’ is derived from evolutionary modifications to the myliobatid pectoral fins. These modifications include a stiffening of some skeletal elements (Schaefer and Summers, 2005), a posterior shift in the distribution of fin rays (Hall et al., 2018), a lateral extension of the wing span resulting in a high aspect ratio (Fontanella et al., 2013), 2

and the evolution of a unique set of appendages called cephalic lobes at the anterior, which have assumed the role of feeding (Mulvany and Motta, 2013). Cephalic lobes, the horn-shaped structures that inspired the common name of the ‘devil’ ray, are a defining feature of the myliobatids. They are unique to this family of batoids and likely facilitated the restructuring of the pectoral fins from a broad ancestral disc shape (Aschliman et al., 2012) into the characteristic kite shape of present-day myliobatids (Mulvany and Motta, 2013). Cephalic lobes are modified anterior pectoral fins that perform a distinct function (feeding) (Mulvany and Motta, 2014; Sasko et al., 2006), have novel musculature (Gonzalez-Isais, 2003; Mulvany and Motta, 2013), and are positioned on the face, separate from the fins (Nelson, 2007). This morphological arrangement led Nelson (2007) to describe myliobatid genera Mobula (manta and devil rays) and Rhinoptera (cownose rays) as the only living vertebrates with three pairs of functional appendages. But are cephalic lobes truly independent appendages or are they specialized domains of the pectoral fin that have been optimized under distinct functional constraints? To address this question, we examined the morphological and molecular mechanisms driving development of the cephalic lobes. If cephalic lobes are truly independent, then we expect them to develop in a domain that is physically distinct from the pectoral fins and to express posterior patterning genes that are common to paired appendages. Alternatively, if cephalic lobes are specialized pectoral fin domains, then we expect their development to be physically tied to the pectoral fins and we hypothesize that they will exhibit similar gene expression patterns to the anterior pectoral fins of other batoids (with some degree of modification). The developmental mechanisms underlying body plan remodeling and appendage patterning are complex. Changes to regulatory networks that guide developmental gene expression profiles are often associated with the evolution of novel features and derived 3

body plans (Gillis et al., 2009; Schneider and Shubin, 2013; Shubin et al., 2009). One of the common ways in which modifications to regulatory networks effect morphological evolution is through spatiotemporal shifts in apical ectodermal ridge (AER) activity in developing appendages. The AER is composed of ectodermal cells that exchange signals with the underlying mesenchyme to drive cell proliferation and outgrowth while keeping cells exposed to these signals in an undifferentiated state (Berge et al., 2008). A heterochronic shift in AER signaling is thought to have contributed to the fm-to-limb transition (Freitas et al., 2007; Schneider and Shubin, 2013), while a novel AER domain in the anterior pectoral fin broadens the batoid pectoral fin into a disc (Nakamura et al., 2015). It is unknown whether AER signaling has been altered in the modified cephalic lobes and pectoral fins of myliobatids. The novel anterior AER in batoids was discovered by Nakamura et al. (2015) while examining development of the little skate, a member of the family Rajidae. This AER is established and maintained by a genetic regulatory network involving Hox genes (Barry and Crow, 2017; Nakamura et al., 2015). Hox genes are master regulatory genes that guide appendage patterning during development via ‘colinear’ expression, in which the anteriormost (3’) genes of the cluster are expressed first, and the posterior (5’) genes last in progressively restricted domains (Gaunt and Strachan, 1996; Izpisua-Belmonte et al., 1991; Mallo et al., 2010; Nelson et al., 1996; Zhu et al., 2017). Spatiotemporal shifts in Hox gene expression can give rise to morphological modifications, followed by novel refinements or adaptations to the modified trait. One mechanism by which this process occurs is through Hox-facilitated changes in the pattern of AER induction and maintenance (Nakamura et al., 2015; Zakany et al., 2007). Shifting Hox expression patterns may underlie shifts in AER expression by repressing or activating other Hox genes (Sheth et al., 2014). Accordingly, the anterior 4

AER in skates is associated with a novel expression domain of 3’ HoxA and HoxD genes (Nakamura et al., 2015), as well as HoxA 13 (Barry and Crow, 2017). This anterior domain may be unique to batoid pectoral fins, though limited taxonomic sampling has thus far hindered our capacity to generalize the gene expression patterns found in skates to other batoids. To shed light on the mechanisms underlying the evolution and development of batoid fins, we documented morphological development and sequenced RNA from the cephalic lobes and pectoral fins of cownose ray embryos (Rhinoptera bonasus). We then compared our data to previously published transcriptomic data from the little skate, Leucoraja erinacea (Nakamura et al., 2015). If cephalic lobes are derived from the anterior pectoral fins of ancestral batoids, as has been suggested by others (Mulvany and Motta 2013), we hypothesize that developing myliobatid cephalic lobes will exhibit gene expression profiles reminiscent of the anterior pectoral fins of skates, including genes involved in the anterior AER, like Wnt3 and 3’ Hox genes. However, if cephalic lobes represent independent appendages, we would expect posterior patterning mechanisms, such as 5’ HoxD genes, to be expressed in cephalic lobes. Testing for homology between myliobatid cephalic lobes and the anterior pectoral fins of skates requires samples of myliobatids undergoing early development, and these samples are very difficult to obtain. Myliobatids have notoriously long gestation times (>9 months) and give live birth to few pups (<5) (Fisher et al., 2013; Marshall and Bennett, 2010; Martin and Cailliet, 1988; Tagliafico et al., 2012; Wourms and Demski, 1993), leaving limited options for sampling. However, the cownose ray, Rhinoptera bonasus, is frequently caught as bycatch in commercial fisheries in Chesapeake Bay (Fisher et al., 2014). So, we worked with commercial fishermen, successfully sampled cownose ray embryos in early development, and characterized their developmental progression. To shed light on the genetic underpinnings of body plan remodeling in the Myliobatidae, we 5

preserved the RNA of the samples and leveraged a comparative transcriptomics approach. Combined, these approaches allowed us to address the following specific research questions: 1) Do cephalic lobes develop as independent appendages that are distinct from pectoral fins? 2) Do cephalic lobes share developmental gene expression patterns with (i.e. are they homologous to) the anterior pectoral fins of skates, which lack cephalic lobes? 3) Are there morphological and molecular features associated with cephalic lobes that are unique to myliobatids relative to batoids that lack cephalic lobes?

By examining the evolution and development of a unique feature in a basal gnathostome lineage, we expect to gain insight into the ways in which diverse body plans are generated during evolution. To our knowledge, this project is the first to describe the morphological or molecular development of any batoid outside the family Rajiformes.

Methods Sample collection It is extremely challenging to sample embryos of myliobatids at specific developmental stages, as there is no published staging scheme for a myliobatid taxon. However, Fisher et al. (2013, 2014) documented the annual cownose ray reproductive cycle in Chesapeake Bay, and this allowed us to estimate the timeline of embryonic development. We worked with commercial fishermen in July and August 2016 and collected ten embryos from gravid cownose rays that were caught as bycatch. After staging these embryos and identifying data gaps, we returned to Chesapeake Bay during an optimized time period in July and August 2017 and successfully collected 28 more developing cownose rays 6

spanning the key periods of pectoral fin development. Samples for RNA-sequeneing were preserved in RNALater immediately after euthanization, incubated at 4°C for 24-72 hours, and then kept frozen at -20°C until dissection and RNA extraction. Samples that were preserved specifically for staging were fixed in 4% paraformaldehyde for 48 hours before being transferred to methanol and stored at -80°C.

Developmental staging Embryos were staged in an RNase-free environment and grouped according to characters associated with pectoral fin and cephalic lobe development. To solidify our groupings, a numerical index was created representing the characters that were deemed to be most consistent and informative with respect to pectoral fin development (Table 1).

RNA sequencing of distinct pectoral fin domains RNA was extracted from eight distinct pectoral fin domains during stage three of cownose ray development (n=6) (Figure 1). The cephalic lobe was cut at the notch and the rest of the pectoral fin was cut into five equal-sized pieces, measured from the anterior-most point of the remaining fin (the notch) to the posterior-most point. Invariably, when measured in this way the cephalic lobe was the same size as the five other pectoral fin domains, resulting in six equal-sized tissues, measured from anterior to posterior.

RNA was extracted and isolated from each tissue sample using Trizol followed by column-based purification using the PureLink RNA mini kit (Invitrogen) and DNase I digestion. Purity was assessed using a NanoDrop and all 260/280 ratios measured between 2.0-2.5. When there was residual guanidine salt, as indicated by a 260/230 ratio < 1.5, then a SPRI bead purification was conducted and the eluate was reanalyzed to confirm purity. 1 1 Pectoral fin fusion Index Cephalic lobe (CL) fusion Index Notch position Index Posterior to 2nd None 0 None(CL hooked) 0 pharyngeal arch 0 None (CL partially Between 2nd and 4th To first gill arch 1 unfurled) 1 pharyngeal arch 1

To second gill arch 2 None (CL fully unfurled) 2 5th pharyngeal arch 2 ( Partial (cranciofacial Anterior to 5th tissue does not extend pharyngeal arch, but To third gill arch 3 beyond base of CL) 3 posterior to mouth 3 Partial (craniofacial tissue partially covers CL To fourth gill arch 4 distally) 4 Level with mouth 4 Complete (craniofacial tissue still To fifth gill arch 5 distinguishable) 5 Anterior to mouth 5

All gill arches plus Complete (craniofacial shoulder 6 tissue indistinguishable) 6 Table 1: Developmental index parameters. These parameters were added to the DW:BL ratio to create a staging ii development in myliobatids. 8

Sample aliquots were diluted and RNA integrity number (RIN) was evaluated using a BioAnalyzer RNA Pico chip. All samples exhibited a RIN > 8. Total RNA from each sample was quantified using a Qubit and 1 p.g of RNA from each sample was used for Poly(A) mRNA isolation using NEBNext oligo d(T) magnetic beads according to the manufacturer’s protocol. After mRNA isolation and fragmentation, cDNA libraries were constructed using the NEBNext Ultra Directional Library Prep Kit for Illumina and each sample was given a unique barcode. Double-sided size selection was performed using SPRI beads targeting 400 bp fragments. Fragment size, concentration, and library purity were verified using a High Sensitivity DNA chip on either a BioAnalyzer or Fragment Analyzer at the University of California, Berkeley Functional Genomics Lab.

In total, forty-eight libraries representing eight distinct fin domains with six biological replicates were normalized, pooled, and sequenced on an Illumina HiSeq 4000 set for 100 bp PE reads. A 1.5% PhiX spike-in was included. Sequencing was conducted at the Vincent Coates Genomics Laboratory at the University of California, Berkeley.

Merging reads from domains for interspecies comparisons Nakamura et al. (2015) divided the little skate pectoral fin into three equal-sized domains antero-posteriorly and cut from the distal edge in through the mesoderm to the proximal margin of the developing fin (T. Namakura pers. comm). Similarly, we divided the cownose ray pectoral fin (including cephalic lobe) into six equal-sized domains that included both ectoderm and mesoderm (Figure 1). When we combine reads from the two anterior-most domains, the two middle domains, and the two posterior-most domains, our data represent three evenly-sized pectoral fin domains that mirror those from Nakamura et al. (2015). Combining reads in this way allows us to broadly compare expression patterns between the two taxa to infer homology. Then, when we separate our reads into the specific domain from which they came, we can detect fine-scale gene expression patterns in the cownose ray. 9

Figure 1: Extraction domains for RNA- CL: Cephalic lobe ANT: Anterior pec fin sequencing. View is ventral side; anterior is at MID-ANT: Mid-anterior pec fin top. The left pectoral fin was cut into six evenly- MID: Mid pec fin sized pieces and separate libraries with distinct MID-POST: Mid-posteriorpec fin barcodes were prepared for each fin domain. In POST: Posterior pec fin PELVIC: Pelvic fin addition, RNA was extracted from the left pelvic fin and from the area actively undergoing fusion between the pectoral fin and gill arches (the CL + ANT (combined) ‘zipper’). The blue, yellow, and red lines delineate the medial boundary of tissue from > which RNA was extracted. Reads from the blue MID-POST (CL + ANT), yellow (MID-ANT + ANT), and MID-ANT + MID red (MID-POST + POST) were combined for (combined) POST DGE analysis, and then separated into the precise PELVIC domains from which the tissue came to

MID-POST ♦ POST investigate gene expression on a finer level. ; (combined)

When referencing analyses involving the combined domains in the cownose ray, we use the word “combined”, such as in the “combined anterior” (blue in Figure 1), “combined middle” (yellow in Figure 1), and “combined posterior” (red in Figure 1); when referencing an analysis with the reads separated, we refer to the specific domain from which the RNA was extracted (e.g. CL or ANT).

Additional RNA-sequencingfor de novo transcriptome assembly The cownose ray is a non-model organism, and the closest taxon for which a published genome exists is the elephant shark (Venkatesh et al., 2014), which diverged from the cownose ray and other elasmobranchs about 420 million years ago (Inoue et al., 2010). When sequence divergence is > 15%, de novo transcriptome assemblies are recommended over reference-based mapping approaches (Vijay et al., 2013). In anticipation of assembling a transcriptome de novo, we sequenced additional cephalic lobe, pectoral fin, and clasper domains on an Illumina MiSeq with longer reads (250 & 300 bp PE) to create a scaffold to which the shorter HiSeq reads could map 10

(Supplementary figure 1). For these samples, RNA was extracted from stage 1, stage 4, and stage 6 embryos (n=2 each) using an RNEasy micro kit, including an on-column DNase I digestion. BioAnalyzer traces showed that all samples had significant genomic DNA contamination, so all samples were subjected to a second DNase I digestion and re­ run on the BioAnalyzer to ensure DNase digestion was successful.

These samples were quantified using a Qubit, then 450 ng of RNA from each sample was used as input for mRNA isolation using magnetic d(T) beads. Samples were subsequently fragmented with heat in the presence of magnesium (Wery et al., 2013). cDNA libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and each sample was given a unique barcode. Samples were subjected to a double-sided size selection using SPRI beads targeting an average of 700 bp fragments. Successful library construction and size-selection was verified on a Fragment Analyzer (AATI) before samples were quantified by qPCR, diluted to 10 nM, and pooled. Over the course of five days, three aliquots from this pooled library were diluted to 4 nM and prepared for sequencing on an Illumina MiSeq at San Francisco State University following manufacturer protocols. Two of the runs were conducted using a 500 cycle v2 kit at 12 pM concentration, and one of the runs was conducted using a 600 cycle v3 kit at 20 pM concentration.

Data analysis Data preprocessing Adapters and low quality sequences from cownose ray data were trimmed from fastq files using the TrimGalore (Krueger, 2012) wrapper for Cutadapt (Martin, 2011) and FastQC (Andrews, 2010). Overlapping reads were combined with Flash (Magoc and Salzberg, 2011) prior to assembly and orphaned reads were retained. Data from sampled regions of three individuals were used in the assembly, including: 2 regions of a stage 1 embryo (broad anterior pectoral fin region and mid-anterior pectoral fin), 8 regions of a stage 3 embryo 11

(cephalic lobe, anterior pectoral fin, mid-anterior pectoral fin, mid-pectoral fin, mid­ posterior pectoral fin, posterior pectoral fin, zipper region, and pelvic fin), and the developing clasper tissue from an approximately stage 5 embryo.

Little skate (Ler) transcriptomic data come from Nakamura et. al.(2015). A counts table was generated according to the methods outlined therein and was shared with us by the authors.

Transcriptome assembly and annotation Abyss (Simpson et al., 2009) was used to assemble contiguous sequences (contigs) from the paired end (PE) and orphaned sequence data with 10 kmer sizes ranging from 25-70 nucleotides. Contigs of 500 nucleotides or greater were summarized according to similarity (e-value < le-160) with BLAST+ tools (Camacho et al., 2009). Annotation of contigs was conducted iteratively by best-reciprocal-blast using peptide, coding sequence, and non­ coding libraries from Danio rerio (GRCzlO) with BLAST+ tools and a minimum e-value threshold of 0.1. Specifically, each Rhinoptera bonasus (Rbo) contig was paired with D. rerio sequences and the lowest average e-value was chosen as the annotation. Therefore, more than one Rbo contig could be designated with the same D. rerio sequence, allowing for potential redundancy in the de novo Rbo transcriptome assembly. The longest open reading frames (ORFs) that were unannotated to D. rerio sequences were annotated by BLASTp using the RefSeq vertebrate protein database (downloaded 4-4-2017). Contig sequences without strong hits (e-value > le-10) were designated as potentially contaminated and removed if BLASTp of the ORFs strongly matched any RefSeq protozoa, viral, plant, plasmid, fungi, bacteria, or archaea sequences (downloaded 6-1- 2017). Remaining unannotated contigs were designated by their top forward BLAST hit in D. rerio, resulting in a total transcriptome size of 27,337 contigs. 12

Alignment and additional Jittering R base (Ihaka and Gentleman, 1996) and Bioconductor (Gentleman et al., 2004) software packages were used for alignment of quality filtered PE data and inference of differential expression. Strand-specific alignment was conducted using the seed-and-vote methodology for uniquely aligned reads, as implemented in the Rsubread package (Liao et al., 2013), to the annotated Rbo transcriptome. Rsamtools was used for file indexing and manipulation (Morgan et al., 2016). Due to potential biases unrelated to developmental gene expression differences, contig sequences annotated as ribosomal, mitochondrial, predicted, hypothetical, or uncharacterized were removed from consideration in differential expression. Moreover, only contigs that were represented by more than 100 reads across the full data set of 56 samples and more than 30 reads in at least one individual were considered reasonable to use for differential expression comparisons. Normalization of read count data was conducted with the trimmed mean of M-values (TMM) method (Robinson and Oshlack, 2010). EdgeR (Robinson et al., 2010) was used to conduct quasi­ likelihood F-tests between groups of samples and false discovery rate (Benjamini- Hochberg) was used to control for multiple testing.

Differential expression Differential expression scores were calculated on raw counts from stage 30 and 31 Ler and stage three Rbo samples using the R statistical computing environment (R Core Team, 2014) and limma-voom software (Law et al., 2014; Phipson et al., 2016; Ritchie et al., 2015). To identify the top DEGs in the anterior pectoral fin of each species, we filtered the top 40 DEGs (sorted by p-value) from the combined anterior relative to the combined middle and combined posterior pectoral fin in Rbo, and from the anterior relative to the middle and posterior in Ler. Each annotation was confirmed via a manual BLAST search. When multiple contigs were associated with the same gene and all of these contigs were differentially expressed (p < 0.05), the most significantly differentially 13

expressed contig was kept and the rest were removed from subsequent analyses. If multiple contigs were associated with the same gene and some were differentially expressed while others were not, then all contigs associated with that gene were removed from subsequent analyses. We also removed contigs for which no reliable annotation could be obtained. This resulted in retention of 20 of the top 40 differentially expressed transcripts.

To identify potential genes associated with cephalic lobe and/or notch development, we identified the top 50 DEGs in the Rbo combined anterior pectoral fin relative to the combined middle and queried the Ler transcriptome for these genes. If the gene was present in the Ler transcriptome and the annotation was correct (as determined by a manual BLAST search), then we visually examined the differential expression profile of the gene. We used intraspecific ratios of gene expression (Dunn et al., 2013) to identify genes that may be associated with phenotypic differences at the anterior pectoral fins of Rbo and Ler. Genes were pegged as potentially being associated with myliobatid-specific features if they were differentially expressed (p < 0.05) in the anterior combined domain of Rbo relative to the middle and not differentially expressed in the Ler anterior pectoral fin relative to the middle. We chose to use the middle domain for our comparison in both species due to pronounced morphological differences between the two species in the posterior (Hall et al., 2018), which could confound interpretation.

All cDNA libraries were analyzed prior to sequencing on a BioAnalyzer, as indicated above. Raw sequences were assessed with FastQC, and clean reads were visualized with an MDS plot in R. These QC analyses revealed that a handful of the libraries, which were prepared 1 -3 weeks after the other samples, appeared to exhibit significant batch effects. Some of these libraries exhibited BioAnalyzer traces with particularly low concentrations (<3 nM), while the FastQC analysis revealed that all of these samples were composed of 14

> 50% PCR duplicates, which was not the case in any of the other samples. Further, on the MDS plot, these samples grouped separately from the rest, which otherwise grouped together by fin domain.

To avoid the confounding influence of likely technical artifacts on measures of biological variance, we excluded these outlier samples from our DGE analyses, which still left us with at least four biological replicates for each condition in Rbo. However, while the statistics regarding raw numbers of DEGs presented here reflect this trimmed dataset, all specific genes that are highlighted in graphs and text exhibit the same statistically significant patterns in both the trimmed dataset and the dataset that includes all samples. In other words, the set of genes we inferred to be differentially expressed was consistent regardless of how the overall dataset was treated, though the specific numbers presented herein represent the trimmed dataset that was subjected to additional filtering.

Visualization Counts were normalized for sequencing depth and heat maps were created using the gplot package (Wames et al., 2009). Z-scores were calculated in R based on the average expression of each individual gene among fin domains. We normalized our data for visualization using counts per million (cpms), a simple ratio to account for sequencing depth across libraries and allow for between-library comparisons (Law et al., 2014). Heat maps of selected genes were superimposed on images of cownose ray embryos using Adobe Photoshop (e.g. Fig. 7).

Volcano plots were also created using the gplot package (Wames et al., 2009) after standardizing the data for interspecies comparisons using a ratio of expression levels (fold change) between fin domains within each species according to Dunn et al. (2013). 15

Results Developmental staging We’ve characterized seven distinct stages of development in the cownose ray, Rhinoptera bonasus (Rbo), spanning early development of the pectoral fins (Figure 2). Characters and measurements associated with pectoral fin development, including cephalic lobe morphology, extent of pectoral fin fusion to gill arches, and disc-width to body length ratios were assigned a value (Table 1). These values were ultimately combined into a single developmental index to distinguish among developmental stages (Table 2). The period of cownose ray development described herein corresponds to developmental stages 28-32 in the winter skate, Leucoraja ocellata (Maxwell et al., 2008), which have also been applied to the little skate (Barry and Crow, 2017; Nakamura et al., 2015, Table 3).

Figure 2: Stages of development in the cownose ray, Rhinoptera bonasus. Bars are 5mm. All pictures are of the ventral side unless indicated. Top of frame is anterior. CL = cephalic lobe, notch = notch, GA = gill arch, pelvic = pelvic fin, zip = zipper, GAE = gill arch ectoderm, clasp = clasper, CFT = craniofacial tissue.

Stage one: Cephalic lobe is curled posterior to the gill arches. Pectoral fin has only just begun to expand anteriorly. Pelvic fin is rounded, shaped like a tear drop. 16

notch

Pelvic

notch

Dorsal view

Stage two: The cephalic lobe remains mostly curled, though it is no longer flush with the pectoral fin. The pectoral fin has begun to expand anteriorly away from the body and overlaps no more than 4 gill arches. Pectoral fins are not yet fused to the axial skeleton. The pelvic fin is round, like a half circle. Stage three: The pectoral fin fuses to the body during this stage, visibly evident at the intersection of appendicular and axial tissue which we call the zipper (zip). The cephalic lobe is no longer curled towards the posterior, but is aligned with the developing primary cartilage of the pectoral fin. The cephalic lobe is positioned caudally and laterally relative to the mouth. In late stage 3 embryos, the dorsal notch has begun to expand and the cephalic lobe is slightly rotated and shifted ventrally. 17

Stage four: The entire pectoral fin is fused to the body, and the cephalic lobe has just begun settling into a ventral pocket of craniofacial tissue. As the notch fuses to the body, it expands ventrally, placing the cephalic lobe near the mouth. The claspers are becoming visible in males as indentations in the pelvic fin, and the gill arch ectoderm (GAE) is beginning to expand over the ventral pectoral fin. Stage five: The pocket of craniofacial tissue grows to partially cover the cephalic lobe, which still resembles a separate entity sandwiched between two sections of tissue outgrowth. The notch has expanded into a fold of tissue oriented in the dorsoventral direction. The claspers further differentiate from the pelvic fins. The GAE fuses to ventral ectodermal tissue on the pectoral fin, thereby completing the process of pectoral fin fusion. 18

Stage six: The craniofacial tissue covers the cephalic lobe, but is still distinguishable. The claspers begin to roll, appearing as distinct structures medial to the pelvic fins, which have assumed a squared morphology. Stage seven: The cephalic lobe and craniofacial tissue are fully fused and there is no evidence of fusion. The claspers are fully rolled. From stage 7 onwards, the developing cownose ray has the same outward morphology as a mature cownose ray. Pectoral Cephalic lobe Notch Gill arch Stage Replicates DW:BL ratio fin fusion fusion position ectoderm fusion T 1 5 0.4-0.6 0-1 0 0-1 0 0 2 5 0.6-0.7 1-3 0-1 1 0 3 3 6 0.7-0.9 4-6 2 1-2 0 8 4 8 0.7-0.9 6 3 3 1 1 5 7 0.9-1.2 6 4 4 1-2 1 6 3 1.2-1.4 6 5 4 2 1 7 4 1.2-1.7 6 6-7 4-5 2 1 Table 2: Range of developmental index scores for each stage of cownose ray (Rhinoptera bonasus) development.

Stage of development (Rbo) Stage of development (Ler) Shared milestones Anterior pectoral fin is hooked; 1 28-29 Pectoral fin unfused to body Anterior pectoral fin may or may not bi 2 30-31 Pectoral fin begins to fuse to body Anterior pectoral fin is uncurled; 3 31 Pectoral fin completes fusion to gill arc 4 32 Pectoral fin and rostrum fuse Fusion of pec fin to body is complete fr 5+ 32+ rostrum Table 3: Developmental milestones shared between developing pectoral fins in cownose ray and little skate and t\ each. Around stage five of cownose ray development (stage 32 of little skate), developmental processes in the pec difficult to draw parallels using these characters. 20

Stage one Pectoral fin development in Ler and Rbo proceeds with distal extension and anterior expansion that begins posterior to the gill arches. In Rbo, the anterior-most regions correspond to the developing cephalic lobes, which are medially curled into a hook-like morphology. By late stage one, a “notch” has formed which begins to distinguish the cephalic lobe from the rest of the pectoral fin. Over the course of stage one, the pectoral fins expand rostrally: by late stage one, the pectoral fin may extend rostrally as far as three gills arches and exhibit dorsal fusion over a single gill arch (dorsal fusion of the pectoral fin precedes ventral fusion). Pelvic fins are small and rounded, resembling a tear drop. Disc-width to body length ratios are < 0.6 and the total developmental index ranges from 0.4 (early stage one) to 2.6 (late stage one). Based on the morphology and position of the pectoral fin, stage one of Rbo development corresponds to stages 28 and 29 of Ler.

Stage two During stage two, the pectoral fins continue to expand rostrally and fuse to the body on the dorsal side, though the ventral side remains unfused. The cephalic lobes begin to unfurl and fin rays become visible, similar to the process that occurs at the anterior of Ler pectoral fins. The pelvic fins remain small and rounded. Disc-width to body length ratios range from 0.6-0.7, and the total developmental index ranges from 3.6-5.7. Based on the morphology and position of the pectoral fin, stage two of Rbo development corresponds to stage 30-31 of Ler.

Stage three Throughout stage three, the pectoral fin fuses, or zips, to the body on both the dorsal and ventral sides. Similar to stage 31 skates (Maxwell et al., 2008), the degree of pectoral fin fusion of stage three cownose rays is variable and may span anywhere between one and five gill arches. In stage three embryos, the cephalic lobes are fully unfurled, the medial margin of the cephalic lobe is aligned with the developing primary cartilage in the 21

pectoral fin and the notch is visibly distinct. By late stage three, the medial margin of the notch has begun to expand dorsoventrally, slightly rotating the cephalic lobe and positioning it ventrally. Pelvic fins remain small and rounded. Disc-width to body length ratios range from 0.7-0.9 and the total developmental index ranges from 8.7-10.9. Based on the morphology and position of the pectoral fins, stage three of Rbo development corresponds to stage 31 of Ler development.

Stage fo ur Stage four embryos begin to assume the form of adult cownose rays, though with some noteworthy differences. At stage four, pectoral fin fusion spans all five gill arches on both the dorsal and ventral sides. In addition, the notch has fused to the shoulder and zagged ventrally, leaving the cephalic lobe positioned just posterior to the mouth. A pocket of craniofacial tissue (CFT) has formed at the comer of the mouth in which the cephalic lobe sits, sandwiched between layers of dorsal and ventral CFT. The ventral CFT has an ectodermal component which extends to the pectoral fin ,where it flattens into a triangle­ shaped sheet that we call the gill arch ectoderm (GAE). The pelvic fin is beginning to transition from a round to a square shape and claspers are becoming visible in males as indentations in the pelvic fin. The DW:BL ratio (0.7-0.9) is not much different than stage three, but other index parameters clearly distinguish stage four embryos. The total developmental index spans a tight range of 13.7-13.9. Based on pectoral fin morphology and position, stage four of Rbo development corresponds to stages 32 and 33 of Ler.

Stage five At stage five, the base of the cephalic lobe fuses to the face in the CFT pocket that was established in stage four. Both the dorsal and ventral sides of the CFT begin to grow out and envelop the cephalic lobe. The GAE, though visibly distinct, has fully fused with ectoderm covering the ventral pectoral fin. Once GAE fusion is complete, the pectoral fin (besides the cephalic lobe) is fully fused to the body. The pelvic fin continues to square 22

off as the fin rays shift into a posterior orientation. Claspers appear as distinct offshoots of the pelvic fins. Cownose rays at stage five have begun to assume the wide kite shape characteristic of mature myliobatid rays, evident by a DW:BL ratio that approaches and surpasses 1.0 (0.9 - 1.2). The total developmental index spans 15.0-17.1.

Stage six By stage six, the CFT pocket has expanded to cover the entire cephalic lobe. Though the cephalic lobe superficially resembles the cephalic lobe of an adult cownose ray, it also appears to be composed of three layers (ventral craniofacial tissue, cephalic lobe, dorsal craniofacial tissue). These tissues are loosely connected distally and fused proximally. The pelvic fins are squared, with fin rays pointing towards the posterior, while claspers have curled and now resemble enclosed tubes. The DW:BL ratio of stage six embryos ranges from 1.2-1.4 and the total developmental index range from 18.2-18.4.

Stage seven During stage seven, the ventral and dorsal CFT and the cephalic lobe are fully fused. The fully fused cephalic lobe has shifted forward and now occupies a position anterior to the mouth, where it remains through ontogeny. With both the pectoral fin and cephalic lobe fully fused to the body, there is no outward evidence that fusion occurred. The DW:BL ratio has a wide range of 1.2-1.7 as growth slows along the antero-posterior axis and lateral pectoral fin outgrowth accelerates. The total developmental index of these samples ranges from 19.2-21.7, though we put little stock in the upper limit of that range, as the DW:BL ratio of cownose rays may continue to increase for up to ten months before parturition (Fisher et al., 2013).

Pectoral fin fusion The overall developmental progression of the cownose ray pectoral fin is similar to the process observed in skates. In Raja eglanteria (Luer et al., 2007), Leucoraja ocellata (Maxwell et al., 2008) and Rhinoptera bonasus, the expanded anterior pectoral fins first 23

grow away from the body before fusing to the gill arches in a caudal-rostral progression later in development. To our knowledge, this pattern of anterolateral outgrowth and subsequent fusion to the axial skeleton has not been observed in the developing appendages of any vertebrate besides batoids, suggesting that this developmental process is a batoid synapomorphy. Further, we observed that pectoral fin fusion occurs rapidly once it begins, with the process likely spanning no more than two weeks of the eleven- month cownose ray gestation period. Once cownose ray pectoral fins and gill arches have fused, the GAE expands from the gill arches to cover and fuse to the ventral pectoral fin. Following GAE fusion, the pectoral fin (excepting the cephalic lobe) is fully fused to the body.

Cephalic lobes develop as modified anterior pectoral fins The cephalic lobes of cownose rays do not develop as independent appendages; rather, it is more accurate to think of cephalic lobes as pectoral fin domains that are specialized for feeding, distinguished from the rest of the pectoral fin by a small notch of stunted tissue outgrowth and an altered pattern of fusion that places the cephalic lobe near the mouth.

Though clearly attached to the anterior pectoral fin during development, the cephalic lobe is distinguished by a small region of reduced tissue outgrowth, which we call the ‘notch’. Though most of the transitory morphologies observed during cownose ray cephalic lobe development are also present during development of skate pectoral fins (e.g. the anterior hook), the notch is a distinguishing feature of cephalic lobes. Therefore, we suggest that the presence of the notch is associated with cephalic lobe differentiation, as the notch delineates the posterior boundary of the cephalic lobe and neither the cephalic lobe nor the notch is present in developing skates.

Once the anterior of the cownose ray pectoral fin is fully fused to the gill arches, the notch fuses to the shoulder and bends ventrally. This process positions the unfused 24

cephalic lobe on the ventral side of the body next to the mouth. Simultaneously, a pocket of craniofacial tissue (CFT) develops next to the mouth and the cephalic lobe sits snugly in the middle of this pocket. Then, the CFT grows and envelops the cephalic lobe as the cephalic lobe continues expanding anteromedially. By stage seven, the CFT and cephalic lobe are completely fused and positioned anterior to the mouth, thereby completing the process of pectoral fin fusion. Though the process of pectoral fin fusion is likely shared among batoids, the ventral positioning of the anterior portion (which is the cephalic lobe in myliobatids) may be unique to myliobatids.

Shared interspecific developmental milestones between batoids One goal of this study is to assess developmental-genetic homology between the anterior pectoral fins of skates and the cephalic lobes of myliobatids. Because developmental- genetic programs can shift in space and time, it is imperative to ensure that the developmental stages being examined are as comparable as possible. The pectoral fins of the little skate and the cownose ray share several batoid-specific developmental milestones, which we used as a guide to determine comparability among developmental stages (Table 3). Chief among these are the angle and morphology of the hook (which is the cephalic lobe in cownose rays) at the anterior of the pectoral fin and the degree of pectoral fin fusion to the gill arches and rostrum. We found that stage 31 of Ler is comparable to stage three of Rbo. At this stage, several unique expression profiles are found in the anterior pectoral fin of both species; therefore, we chose to focus on this stage for our molecular analysis.

Quality control and domain-specificity of RNA-Seq data To assess the reliability and domain specificity of our RNA-Seq data, we looked for canonical fin patterning genes in the Rbo and Ler data separately. We found posterior HoxD expression to be confined to the posterior pectoral fin in each taxon (Figure 3). We also confirmed that expression of Tbx4 and Tbx5, genes which are restricted to 25

developing pelvic and pectoral fins/limbs, respectively (Gibson-Brown et al., 1998), exhibited expected expression profiles in Rbo (data not shown). Nakamura et. al. (2015) did not sequence a pelvic fin domain in Ler, but we were able to confirm expression of Tbx5 throughout the pectoral fin while Tbx4 was absent from their assembled transcriptome, as expected. These observations suggest that the RNA-Seq data from both species are reliable and domain-specific.

Shared molecular mechanisms underlie cephalic lobe and pectoral fin development To establish homology between the pectoral fins of the cownose ray and little skate, we combined reads from the two anterior-most, the two middle, and the two posterior-most domains of the cownose ray (Figure 1 red, yellow, green). When reads were combined in

Relative expression of paired fin patterning genes in little skate and cownose ray

Little skate Cownose ray Anterior I ill 1Anterior Middle Middle Posterior III! Posterior Q) ^ C\j £ % 2* £? £ S’ S' ^ iry Q £

Color K»y

*1 0 0.5 1 Column 2*Score Figure 3: Normalized expression of fin patterning genes in developing pectoral fins of the cownose ray (stage three) and little skate (stage 31). Counts were normalized to cpm (counts per million) to account for sequencing depth, and z-scores were calculated for each gene. Genes to the right of the black bar show expected expression patterns based on Nakamura et. al. (2015) and Barry & Crow (2017). Genes to the left of the black bar have expression patterns that have not been described in chondrichthyans and are unexpected given their expression patterns in other taxa. All are known fin patterning genes that are in the top 20 differentially expressed genes (sorted by p value) in the anterior of the little skate and anterior combined domain of the cownose ray. * gene is differentially expressed in the anterior relative to both the middle and posterior. 26

this way, our extraction domains mimicked those of Nakamura et. al. (2015), as the combined domains in Rbo each represented one third of the pectoral fin.

Under these conditions, we found far more differentially expressed genes (DEGs; p < 0.05) in the anterior Rbo domain than in Ler (anterior vs posterior DEGs: 88 Ler, 714 Rbo; anterior vs middle DEGs: 115 Ler, 644 Rbo). We suspect this has less to do with biology than technical differences in data generation and processing. Nakamura et al. (2015) used 2-3 replicates for each condition, while we had 4-6, which increases our power to detect differentially expressed genes in Rbo (Schurch et al., 2016). Additionally, the library preparation, sequencing, and bioinformatic analyses were conducted using different protocols and pipelines.

Such issues are common in studies comparing gene expression across species and studies, and can be overcome by comparing ratios (fold change) of expression among fin domains within each individual sample (Dunn et al., 2013). Accordingly, we examined differentially expressed genes in the combined anterior domain relative to the combined middle in Rbo and compared the logFC ratios and p values to the genes that are differentially expressed in the anterior domain relative to the middle in Ler (Figur

Figure 4: Differential expression Rbo Stage 3 Artenot vs Posterior Pectorai Fin ls t Stag* 31 A n w & vs Pectoral scores of fin patterning genes in cownose ray and little skate, showing similar patterns of expression in the anterior and posterior fin domains. Anterior is top, posterior is bottom. o Black dots: not significantly differentially expressed (p > 0.05), light blue dots: differentially expressed with small fold change (logFC) ratio (p < 0.05, logFC < 1.5), dark blue dots: differentially expressed with high logFC ratio (p < 0.05, logFC >1.5). 27

Of the top 20 DEGs in the Ler anterior pectoral fin relative to the posterior and middle at stages 30 and 31, over 60% of the DEGs were also differentially expressed (p < 0.05) in the stage three Rbo combined anterior domain (Table 4). Included in the top 20 anteriorly-biased DEGs of both species (sorted by p value) were known fin/limb patterning genes including HoxA2 and HoxA4 which have been linked to the expanded anterior domain of the batoid pectoral fin (Nakamura et al., 2015).

Anterior vs middle Anterior vs posterior Rbo Ler Rbo Ler dmrt3* bco2 A lxl bco2 slc6al5 dmrt3* Alx4* Pax9* Tbx3* otxl slc6al5 o txl NPTX1 Tbx2* NPTX1 Alx4* Runx3 Tbx3* npb HoxA2* Tbx2* Alx4* Lhx9 Tbx2* MCF2L2 HoxA2* dmrt3* HoxD3* Tbxl asip Tbx3* HoxB4* Alx4* Dlx4* Pax9* RUNX1* pou3f3 epb4H4a* Tbx2* epb4H4 Dlx4* HoxA4* HoxA2* Tbx3* Dlx2 Lhx2* CNTN1 mafb* A lxl M sxl* Runx3 FoxC2* NrCAM Fgf7 GABBR2 Tlx2 FAM69c Alkal2 SynDigl Lhx2* tnsflO EDAR Dlx2 tfpi* itgal Pax9* HoxA4* LAM Al fb ln l SCUBE3 SLIT3 HoxA4* SHOX LAM Al hs3stl EDAR myb tnfrsfl9* igf2 HoxD4

Table 4: Top 20 differentially expressed genes in the anterior pectoral fin of stage three cownose ray and stage 31 little skate relative to the middle and posterior. Bold text indicates that the gene is in the top 20 DEGs of both species. Asterisks next to skate genes indicate that the gene is also differentially expressed (p < 0.05) in the more extensive cownose ray dataset, even if the gene is not included in this table. 28

Other top 20 DEGs shared between both species, such as Dlx4, Tbx2, and Tbx3 were unexpected and, to our knowledge, have not been documented as enriched in the anterior domains of non-batoid appendages. On the other hand, Pax9 and A 1x4 are canonically expressed in anterior fins and limbs; as expected, we found these genes in the top 20 anterior DEGs of both batoid species examined. Interestingly, dmrt3, a gene that is mostly known for its role in sex determination and development of sexual organs (Kim et al., 2003) is also differentially expressed in the anterior pectoral fin of Ler and Rbo. These data suggest that development of the anterior pectoral fins of raj id skates and myliobatid rays occurs via homologous pathways, including some that are batoid- specific.

Myliobatid cephalic lobes are homologous to the anterior pectoral fins of skates To determine whether it is the cephalic lobe or anterior pectoral fin driving the gene expression profiles in the combined anterior pectoral fin domain of Rbo, we separated our reads into the specific domains from which they came. Heat maps revealed that a majority of the anteriorly-biased genes shared between Rbo and Ler are concentrated in the cephalic lobe (CL) in Rbo (Figure 5). We found that four of the genes highlighted above - Tbx2, Tbx3, Alx4 and dmrt3 - are even differentially expressed in the CL relative to the anterior pectoral fin (Table 5). Though it falls just short of statistical significance in the DGE analysis (p = 0.07), Dlx4 expression is also focused in the CL relative to the ANT. Thus, we found fin building and patterning genes involved in key regulatory networks in anterior Ler pectoral fins are highly enriched in the cephalic lobes and absent from the anterior pectoral fin in Rbo, suggesting that the cephalic lobes of Rbo are homologous with the anterior pectoral fin of Ler. 29

HoxA13, a gene associated with the anterior AER in skates (Barry and Crow, 2017), is also differentially expressed in the CL relative to the ANT, though its expression profile was masked in the combined analysis, likely because downregulation in the ANT domain diluted the higher expression levels in the CL when the reads were combined. Hand2 is also differentially expressed in the CL relative to the ANT, and its expression profile was similarly masked in the combined analysis. In both Ler and Rbo, HoxA13 and Hand2 are expressed in both the anterior and posterior pectoral fin domains, resulting in neither gene being differentially expressed in either domain relative to the other.

Thus, in the case of HoxA13 and Hand2, as well as Tbx2, Tbx3, dmrt3, and Dlx4, it is the cephalic lobe driving the expression patterns observed in the combined anterior pectoral fin domains of cownose rays. In contrast, the anterior Hox genes (HoxA2, HoxA4, and HoxA5) are differentially expressed in the ANT relative to CL, as is Pax9, implying that not all anterior pathways were inherited by the cephalic lobe in Rbo. Nevertheless, these data indicate that multiple batoid-specific expression patterns at the anterior of the pectoral fin are specifically expressed in the cephalic lobes, supporting the notion that myliobatid cephalic lobes and the anterior pectoral fins of skates are patterned by homologous molecular pathways. These findings also dispel the broad assumption that the anterior pectoral fins of skates and the anterior pectoral fins of myliobatids are directly homologous. Rather, the evolutionary relationship between these features is more complex, and the homologous regulatory networks that drive development of the anterior pectoral fins of skates appear to be split between the CL and ANT in myliobatids. 30

Color Key

-2-1012 Expression of key patterning genes (Rbo) Cofemrs Z-So&rg

Cephalic lobe Anterior Mid-Anterior Middle Mid-Posterior Posterior

Figure 5: Fine-scale gene expression of key fin-building and patterning genes in cownose ray pectoral fins (ventral side of embryo depicted). Genes that are differentially expressed in the combined anterior domain of cownose rays are concentrated in the cephalic lobe. Asterisks indicate that the gene is differentially expressed in the cephalic lobe relative to the anterior pectoral fin (p < 0.05). HoxA2, HoxA4, and HoxA5 are concentrated at the anterior pectoral fin in a region of reduced expression of HoxA13, which may downregulate these genes in the cephalic lobe. Hand2 is canonically known to be a posterior patterning gene, but we find high expression in the cephalic lobe as well, indicating that this gene may have been recruited to the cephalic lobe to set up and/or maintain antero-posterior patterning pathways. 31

Cephalic lobe vs. anterior pectoral fin Novel gene expression patterns are Gene logFC adj.P.Val associated with unique myiiobatid features Tbx2* 1.4 1.5E-07 Myliobatid cephalic lobes are distinguished Tbx3* 1.9 4.3E-08 from the rest of the pectoral fin by a notch, Dlx4 1.0 0.07 dmrt3* 3.1 8.0E-09 where tissue outgrowth appears to stall during A 1x4* 1.0 6.9E-04 development. To identify potential

Pax9* - 2.2 8.1E-09 mechanisms driving development and HoxA2 - 1.2 1.3E-04

HoxA4 - 1.2 1.4E-05 maintenance of the notch, we queried the Ler HoxA13* 2.2 0.01 transcriptome for the top 50 genes that were Hand2* 2.2 1.8E-04 Lhx9* 1.7 3.8E-04 differentially expressed in the combined omd -1.4 4.8E-04 anterior domain of Rbo pectoral fins relative Dlx2* 1.6 1.0E-03 to the combined middle domain, looking for A lx l 1.2 1.0E-05 genes that are represented in the Ler Table 5: Differential expression scores for cephalic lobe vs. anterior pectoral fin, transcriptome but not differentially expressed showing separation of anterior fin patterning mechanisms. Gray cells are at the anterior. differentially expressed in ANT, white cells are differentially expressed in CL. We were able to identify five genes that meet ♦statistically significant (p<0.05). these criteria: omd, Lhx9, Alxl, Dlx2, and Dkkl (Table 6). Each of these genes was differentially expressed in the combined anterior domain of Rbo relative to the combined middle domain (p < 0.05, logFC > 1.5), while none of these genes was differentially expressed under the same conditions in Ler at stage 31 (Supplementary Figure 2), nor were they differentially expressed in the anterior little skate pectoral fin at stages 29 and 30 relative to the posterior (data not shown). These genes, upregulated in the combined anterior pectoral fin of Rbo and not Ler, may be involved in regulating processes such as chondrogenesis, patterning, and initiation or maintenance of the notch (see discussion). m

Rbo Ler Gene Potential funct t i e LoeFC Fusion (esp. of cartilage), chondi A lxl 1.10E-06 2.3 0.99 -0.4 patterning (Qu et. al. 1999)

Neuron differentiation, apoptos dlx2 3.30E-06 2.4 0.99 0.2 (Bendall & Abate-Shen 2000, Ak Raja et. al. 2017)

Wnt/AER inhibitor, apoptosis (N dkkl 0.001 2.4 0.99 0.8 Murkopadhyay 2001)

Keep mesenchymal cells in prog Lhx9 0.002 1.7 0.99 -0.55 order regulatory complexes, AP/ (Tzchori et. al. 2009, Hobert & \A

Regulates diameter and shape o omd 0.003 1.4 0.03 -1.8 al. 2015)

Table 6: Fin building and patterning genes putatively implicated in development of myliobatid-specific featur differential expression in the combined anterior relative to the combined middle in Rbo and in the anterior rek Expression of these genes is upregulated at the anterior in cownose rays, but not in little skates, and are therefc involvement in the development of myliobatid-specific features at the anterior of the pectoral fin (e.g. cephalic compagibus laminum). 33

Discussion Based on multiple lines of evidence, including developmental observations and shared gene expression patterns, we conclude that cephalic lobes are modified anterior pectoral fins that share homology with the anterior pectoral fins of skates. That said, we also observed undocumented developmental processes that contribute to establishment of the myliobatid body plan and identified a handful of genes that may be associated with the evolution and development of cephalic lobes and other myliobatid-specific features at the anterior of the pectoral fin.

Developmental variation in batoids Cownose ray pectoral fins, like skate pectoral fins, develop via a multi-step process which includes anterolateral outgrowth at the anterior margin of the fin and subsequent fusion to the axial skeleton. Phenotypes with incomplete pectoral fin fusion have been observed in nature in various batoid clades, including the Rajidae (Prado et al., 2008), Torpedinidae (Reynaud et al., 2010), Dasyatidae (Prado et al., 2008; RAmiRez- HeRnAndez et al., 2011), Urotrygonidae (Mejia-Falla et al., 2011), Potamotrygonidae (Oldfield, 2007), Rhinobatidae (Bomatowski and Abilhoa, 2009), and Gymnuridae (Narvaez and Osaer, 2016). This deformity is so common among batoids that it has been termed the “batman” morphology (Oldfield, 2007). We have observed that anterior pectoral fin expansion occurs independently of fusion to the body in batoids and suggest that incomplete fusion of the batoid pectoral fin is associated with failure of the underlying developmental pathways driving the fusion process. Therefore, the batman morphology may occur when the molecular mechanisms driving outgrowth proceed normally and the mechanisms underlying fusion are interrupted.

Failure of the underlying genetic network may also explain developmental deformities in cephalic lobes, which also develop via distinct outgrowth and fusion processes. Ramirez- Amaro et al. (2013) documented a bat ray (Myliobatis californica) with a morphological 34

abnormality in the cephalic lobes: rather than exhibiting a single fused lobe, as is normal in bat rays, this particular individual had two separate cephalic lobes with a protuberant rostrum between them. Based on data from the cownose ray, we can infer that the deformity documented by Ramirez-Amaro et al. (2013) is associated with a failure of the left and right cephalic lobe to fuse to the rostrum in the middle.

With pectoral fins and cephalic lobes that grow outward and then fuse, small changes to the patterns of outgrowth, fusion, or both could result in vastly different morphologies. The notch, a localized area of reduced pectoral fin outgrowth, is present in cownose rays but not skates, and is likely associated with cephalic lobe differentiation among myliobatid taxa. We predict that the degree to which tissue outgrowth is reduced at the notch during development is correlated with the number of fin rays present in the shoulder of mature myliobatids. While mature cownose rays, devil rays, and eagle rays exhibit a total lack of skeletal elements in the shoulder, some bat rays of the genus Myliobatis), the putative ancestral myliobatid, display a radiation of stunted, but continuous, fin rays at the shoulder, between the cephalic lobe and pectoral fin (Figure 6) We hypothesize that the genes responsible for stunting tissue outgrowth in the notch are weakly expressed or deactivated early in these bat rays relative to other myliobatids.

The molecular machinery driving the fusion process likely varies among myliobatids. While the cownose ray cephalic lobe is positioned ventrally near the mouth during pectoral fin fusion, the single fused cephalic lobe of mature bat rays is found in the same plane as the pectoral fin, implying that the notch does not expand ventrally in bat rays during pectoral fin fusion. However, eagle rays (genus Aetobatus) and devil rays (genus Mobula) exhibit ventrally positioned cephalic lobes, indicating that ventral placement of the lobes appears to be consistent among most myliobatid genera. 35

Figure 6: Cephalic lobe position varies among myliobatid taxa and may be associated with the presence or absence of fin rays in the shoulder region, a) is the left side of the head of Myliobatis californica, b) is the ventral side of a cleared and stained M. californica specimen, c) is the left side of the head of Rhinoptera bonasus, d), is the ventral side of a cleared and stained R. bonasus specimen. The red line in (a) delineates the approximate boundary between the cephalic lobe (left) and pectoral fin (right) in M. californica. Notice that both the cephalic lobe and pectoral fin exist in the same plane, with no dorso-ventral break between them. This presumably permits the development of fin rays in the shoulder region [denoted by red brackets in (b)]. In contrast, the red arrows in (c) show a dorso-ventral shift in the cephalic lobe (below and left of red arrows) and pectoral fin (above and right of red arrows) of Rhinoptera bonasus. This shift may be associated with a lack of diagonal fin rays in the shoulder region [red brackets in (d)].

The notch expands ventrally during fusion, and it is also the feature that separates the cephalic lobe from the rest of the pectoral fin. Our observations suggest that the notch is an important distinctive feature of myliobatids. Slight modifications to the developmental-molecular machinery driving processes such as pectoral fin fusion and notch formation may have contributed to the morphological and ecological diversification of batoids, a group which comprises over half the diversity present in the chondrichthyan lineage (Kolmann et al., 2014). 36

Putative genetic underpinnings of myliobatid-specific features One of the greatest strengths of RNA-sequencing is its capacity to facilitate discovery, particularly in non-model taxa with few transcriptomic resources. Though functional studies are untenable in taxa like cownose rays that are not easily kept in captivity, we can putatively infer roles for genes with interesting expression patterns by examining their functions in model organisms.

Chief among the unique features that distinguish myliobatid pectoral fins from the pectoral fins of other species are the notch, an area of reduced tissue outgrowth between the cephalic lobe and the rest of the pectoral fin, and the compagibus laminum, a region of thickened fin rays at the anterior of the pectoral fin (Hall et al., 2018). Because these features are present in myliobatids and absent from skates, and due to the methods by which we parsed our RNA extractions (Figure 1), we expect to find genes associated with development of the notch and compagibus laminum enriched in the combined anterior of Rbo pectoral fins but not in the anterior of Ler pectoral fins. We also expect that when we split reads into their specific domains in Rbo, genes associated with the compagibus laminum will be enriched in the ANT relative to the CL. Using these criteria, we identified five genes that may be involved in the development of myliobatid-specific features, including the notch and compagibus laminum (Table 6). We speculate on potential roles for these genes below.

Dlx2 and Dlx4 We found a distinct expression pattern of distal-less (Dlx) genes that appears to be associated with development of anterior pectoral fin modifications, including cephalic lobes, in Rbo. Dlx genes are homeobox transcription factors which are commonly expressed in regions that give rise to derived structures in vertebrates (Bendall and Abate-Shen, 2000; Neidert et al., 2001). It has been suggested that different combinations of Dlx genes give rise to distinct morphological structures (Akimenko et al., 1994). In 37

Rbo, the domains of Dlx genes are concentrated in modified fin structures: Dlx2 and Dlx4 are differentially expressed in cephalic lobes (myliobatid synapomorphy), and anterior pectoral fins (batoid synapomorphy); they are also highly expressed in the claspers (chondrichthyan synapomorphy), while expression is downregulated in all other fin domains. These results are consistent with the hypothesis that specific combinations of Dlx genes may underlie the evolution and development of morphological modifications.

Lhx9 Lhx9, a member of the LIM homeobox transcription factor family, guides appendage patterning in all three dimensions: anteroposterior, proximodistal, and dorsoventral (Tzchori et al., 2009). LIM proteins, which possess two conserved protein-binding domains, have the ability to regulate gene expression by interacting with other proteins in a homomeric or heteromeric fashion (Hobert and Westphal, 2000). This may permit LIM genes to guide complex post-transcriptional regulatory processes at the anterior of Rbo pectoral fins and in the cephalic lobes. Lhx9 expression is two-fold higher in the CL relative to ANT (p < 0.001), and approximately two-fold higher in ANT relative to the rest of the pectoral fin domains. Thus, while Lhx9 is enriched in both the ANT and CL domains relative to the rest of the pectoral fin, our data suggest that Lkx9 is most likely to play a role in development of the cephalic lobe, at least during stage three of Rbo development.

Dkkl Wnt signaling is involved in AER formation and maintenance (Berge et al., 2008; Kawakami et al., 2001; Kengaku et al., 1998). One of the primary mechanisms driving cephalic lobe differentiation is initiation and maintenance of the notch, an area of reduced tissue outgrowth that delineates the boundary between the posterior cephalic lobe and the anterior pectoral fin. The notch persists even while the cephalic lobe and pectoral fin 38

continue to expand on either side of it, suggesting the existence of mechanisms that inhibit tissue outgrowth in the notch while outgrowth proceeds on either side of it.

Dkkl is a known inhibitor of Wnt signaling (Fedi et al., 1999; Mao et al., 2001; Niida et al., 2004), making it an intriguing candidate for repressing AER maintenance in the notch. In developing mouse limbs, Dkkl is expressed in areas undergoing programmed cell death in the anterior and posterior necrotic zones of developing limb buds (Mukhopadhyay et al., 2001). It is also expressed in the interdigital mesenchyme just prior to apopotosis (Mukhopadhyay et al., 2001). In Rbo, we find Dkkl to be most highly expressed in the cephalic lobe, with the ANT domain having the second highest expression levels. Though we can neither confirm localized expression of Dkkl in the notch, specifically, nor demonstrate its precise functional role, the expression pattern and known functions of this gene are concordant with our a priori expectations for potential notch genes: namely, high expression in at least one RNA extraction domain that includes the notch and a known role in suppressing AER function.

Alxl and omd Alxl and omd (osteomodulin/osteoadherin) are also differentially expressed in the combined anterior of cownose ray pectoral fins relative to the combined middle and do not show the same pattern in skates. Both of these genes are associated with chondrogenesis: Alxl, also known as Cartl, is associated with chondrogenic fusion in the nasal membrane of mice (Qu et al., 1999). It is also involved in anteroposterior (AP) patterning and is functionally redundant with Alx4 (Qu et al., 1999). In humans, omd regulates the diameter and shape of collagen fibrils, a primary component of cartilage (Tashima et al., 2015). With its expression being significantly higher in the ANT domain relative to the CL (p < 0.001), we suspect a putative role for omd in development of the compagibus laminum. Alxl may also be involved in development of these thick fin rays, perhaps by promoting chondrogenic fusion; however, due to its higher expression in the 39

CL relative to the ANT, we suspect this gene is more likely involved in AP patterning, as the thickened fin rays are specific to the ANT domain.

Though we have suggested putative roles for each of the five genes that are enriched at the anterior of myliobatid pectoral fins, functional studies are needed to confirm the validity of our suggestions. Unfortunately, functional studies are not feasible in live- bearing cownose rays. However, ectopic expression of these genes or gene editing using the CRISPR-Cas9 system in the little skate, an egg-laying species that is more amenable to experimental manipulation, may be possible.

Deep homology of patterning mechanisms in batoid pectoral fins In developing tetrapod limbs, the proximal limb bud expresses 3’ Hox genes and Fgf7, which together can induce expression of Fg/8 and induction of an AER (Zakany and Duboule, 2007). The 3’ Hox genes and Fgf7 are also involved in outgrowth and patterning in the expanded anterior pectoral fin domain of Ler (Nakamura et al., 2015). Thus, it was proposed that the same genetic network that patterns the proximal tetrapod limb bud may have been co-opted to the expanded anterior pectoral fins of skates, prompting formation of a novel anterior AER and broadening the batoid pectoral fin into a disc (Nakamura et al., 2015). Our data support this hypothesis and implicate Hand2, Alxl, Alx4, and TbxS as potential additions to this co-opted genetic network (Figure 7).

Hand2 Hand2 is canonically expressed at the posterior of developing fins and limbs where it interacts with HoxD13 to initiate expression of the morphogen shh (Galli et al., 2010; Osterwalder et al., 2014). Relative to mice, catsharks exhibit a more posteriorly-restricted expression domain of Hand2 with no expression at the anterior of the fin (Onimaru et al. 2015). In contrast to both tetrapods and catsharks, we find that Hand2 is enriched in both the anterior and posterior of batoid pectoral fins (Figure 5, Figure 7). 40

In tetrapods, an antagonistic relationship between Hand2 and Gli3 prepattems the anteroposterior axis of developing limbs, with Hand2 restricting Gli3 and Alx4 to the anterior of developing limbs (via Tbx3), while A 1x4 and Gli3 keep Hand2 confined to the posterior (Galli et al., 2010; Osterwalder et al., 2014; te Welscher et al., 2002). The pectoral fins of batoids appear to be patterned by a fundamentally different process. Hand2 is expressed in the cephalic lobe in a domain that overlaps that of Alx4 and Gli3. Gli3 expression also overlaps Hand2 in sharks and holocephalans, which led Onimaru et al. (2015) to conclude that the genetic antagonism between Gli3 and Hand2 is weak in chondrichthyans. Our data from batoids - the sister taxon of sharks - support this conclusion.

Without a mechanism restricting it to the posterior, Hand2 may have assumed a distinct role at the anterior of the batoid pectoral fin. Interestingly, in tetrapods, Hand2 is expressed in an overlapping proximal domain with Tbx3 (Osterwalder et al., 2014). Further, Hand2 is co-expressed in the flank with Tbx3 to prepattem and position the limb bud (Rallis et al., 2005). Misexpression of Tbx3 during limb bud initiation was found to recruit new genes from the flank into the developing limb bud (Rallis et al., 2005). It is conceivable that expression of Tbx3 in batoids, which is concentrated at the anterior of Ler and Rbo, was co-opted to the anterior pectoral fin, bringing with it a slew of genes from the flank, such as Hand2. This co-option may have led to distinct outgrowth and patterning systems at the anterior and posterior margins of batoid pectoral fins.

Tbx3 Tbx2 and Tbx3 are strongly upregulated in the combined anterior domains of cownose rays and little skates (Table 4, Figure 4); a similar pattern is observed in stage 30 catshark pectoral fins (Onimaru et al. 2015). In chicks and zebrafish, Tbx2 and Tbx3 are expressed in anterior and posterior stripes, the latter having higher levels of expression that persist longer relative to the former. Tbx3 is involved in Hand2-mediated posteriorizing 41

processes, including posterior repression of Gli3 (Osterwalder et al., 2014; Ruvinsky et al., 2000; Tumpel et al., 2002). As we have mentioned, the antagonism between Gli3 and Hand2 in tetrapods (Onimaru et al. 2015) does not occur in chondrichthyans, suggesting that Tbx3 (and likely Tbx2) acquired a distinct function in chondrichthyans.

Alx4 Alx4 is a transcription factor that is dependent on AER signaling and has been shown to perform a complimentary role to the posteriorizing morphogen sonic hedgehog (shh) in establishing the anterior-posterior axis in developing fins and limbs (Kuijper et al., 2005; Takahashi et al., 1998). In mice, Alx4 expression at the anterior is highest in the proximal domain (Onimaru et al. 2015) and in Rbo, Alx4 expression is highest in the cephalic lobe, an anterior region that begins developing with a hook-shaped morphology (Figure 7). Alx4 may be part of the ancestral genetic module that was co-opted to the anterior of batoid pectoral fins to establish the unique anterior AER in batoids, along with Wnt3, Hand2, Tbx3, and the 3’ Hox genes (Barry and Crow, 2017; Nakamura et al., 2015). Intriguingly, when A 1x4 is overexpressed, it has the ability to inhibit cell proliferation (Shi et al., 2017), implying that Alx4 could potentially contribute to formation of the notch in myliobatids. Of course, visualization and functional studies are necessary to determine the precise location and function of Alx4 in the expanded anterior pectoral fins of batoids. 42

a)

Catshark Cownose ray

b) I Alxl Alxl d)

m .. . l r

f F j Mouse Cownose ray

Figure 7: Proximal gene expression patterns in the catshark pectoral fin and mouse limb bud may have been co-opted to the anterior pectoral fin and cephalic lobe. Cownose ray images were created by superimposing heat maps on images of developing cownose ray pectoral fins (see Methods for more details). Darker colors indicate higher levels of expression, a) Alx4 is most highly expressed in the proximal margin of catshark pectoral fins (left) and in the cephalic lobe of cownose rays (right), b) Alxl was not examined in catshark, but in mouse expression is highest in proximal margins at anterior and posterior of developing limbs and, again, in cownose rays expression is highest in the cephalic lobe, c) and d) Hand2 and Tbx3 expression are highest in posterior limbs (green dots in lefthand images); however, expression is also present at the proximal anterior limb (white arrows). In the cownose ray, expression of both of these genes is highest in the cephalic lobe (though Hand2 also has a posterior expression domain). The catshark image in a) was modified from Onimaru et. al. (2016), the mouse image in b) was modified from Beaverdam and Meijlink (2001), and the mouse images in c) and d) were modified from Osterwalder et. al. (2012).

A potential rote for HoxA genes in the evolution and development o f cephalic lobes The role of HoxA genes in cephalic lobe evolution and development is likely to be significant. HoxA 13 is frequently associated with the evolution of fin and limb modifications and novel features and is also correlated with the anterior AER in the little skate (Archambeault et al., 2014; Barry and Crow, 2017; Fromental-Ramain et al., 1996; 43

O’Shaughnessy et al., 2015). We found HoxA13 upregulation (p<0.01, logFC > 2) in the CL relative to the ANT and MID-ANT domains of Rbo, once again providing evidence in support of cephalic lobe/anterior pectoral fin homology. This pattern may indicate that the anterior AER in skates has shifted forward in the myliobatids and is concentrated in the cephalic lobes.

Sheth et. al. (2014) demonstrated that HoxA13 can downregulate expression of 3’ Hox genes. Expression patterns in Rbo are consistent with a role for HoxA13 in downregulating the 3’ HoxA genes in the cephalic lobes (Figure 5). Contrastingly, in Ler the 3’ HoxA genes, including HoxA2, HoxA4, and HoxA5, are expressed in overlapping domains with HoxA13 at the anterior of the pectoral fin (Nakamura et al., 2015). Thus, expression patterns in Rbo are consistent with a role for HoxA 13 in downregulating other HoxA genes, but the same pattern is not present in Ler.

However, HoxA 13 exhibits substantial variation in its regulatory abilities. In tetrapod limbs, distal autopodial expression of HoxA 13 contributes to repression of HoxA 11 by acting on a novel enhancer in the HoxAll intron (Kherdjemil et al., 2016). The distal exclusion of HoxAll by HoxA 13 during late development is likely associated with the evolution of tetrapod limbs (Ahn and Ho, 2008; Sakamoto et al., 2009); however, during early tetrapod limb development, the two domains overlap (Metscher et al., 2005), implying that the regulatory capabilities of HoxA 13 are variable. It is thus conceivable that HoxA 13 could play distinct regulatory roles in the fins of developing skates and myliobatids.

Relative to HoxA13, AER-inducing 3’ HoxA genes (specifically, HoxA2, HoxA4, and HoxA5) are expressed in distinct domains during stage three of cownose ray development (Figure 5). Both the ANT and CL domain in cownose ray pectoral fins undergo continuous outgrowth during development, while the process is stifled in the notch 44

between the two domains. This suggests the existence of separate AERs in the cownose ray cephalic lobe and the anterior pectoral fin, which may be governed by distinct regulatory modules.

Though the precise roles of HoxA13 and the 3’ HoxA genes in the cownose ray pectoral fin cannot be confidently deduced without further studies into specific expression profiles (proximal vs. distal) and function, our data align with the conclusions of previous studies that have implicated roles for these genes in AER induction and the development of unique body plan modifications, including the anteriorly-expanded pectoral fins of skates.

Conclusion Our results demonstrate that the cephalic lobes of devil rays and their relatives are not independent appendages; rather, cephalic lobes are specialized domains of the anterior pectoral fin that exhibit distinct modifications that are associated with functional optimization, such as a notch and ventral diversion as the pectoral fin fuses. Anterior expansion and fusion of the pectoral fins to the body is likely a batoid synapomorphy; variations to this foundational process may be associated with the diversification of batoid lineages. For example, in the Myliobatidae, stunting tissue outgrowth at the notch and ventrally shifting the pattern of pectoral fin fusion at the anterior results in functionally-specialized cephalic lobes that are homologous to the anterior pectoral fins of skates. Thus, the cephalic lobes of devil rays and their relatives demonstrate how slight changes to developmental programs can lead to functional specialization and are likely associated with the remarkable taxonomic diversity observed among batoids. 45

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CL Cephalic lobe ANT: Anterior pec fin MID-ANT: Mid-anterior pectoral fin CLASP: Clasper

CL+ANT

MID-ANT

CLASP

Supplementary Figure 1: Additional RNA extraction domains for long-read sequencing. Extraction domains comprised stage 1 cephalic lobe and pectoral fin, stage 4 cephalic lobe (CL), and stage 6 clasper (CLASP). logFC 0

2

t ~log10(ad| Val) P 4

6

8 the little skate. Genes with a logFC value > 0 (top (top 0 > value logFC a with Genes skate. little the reveal distinct expression patterns in the combined combined the in patterns expression distinct reveal half of graph) have more expression in the middle middle inthe expression more have graph) of half (bottom 0 < value logFC a with genes while pectoral, anterior inthe expression more have graph) of half ratio (p < 0.05, logFC < 1.5); Dark blue dots: blue 1.5);Dark < logFC 0.05, < (p ratio anterior pectoral fins of the cownose ray relative to relative ray cownose the of fins pectoral anterior cownose ray-specific features. Black dots: not not dots: Black features. ray-specific cownose of maintenance and/or formation in role potential a indicating middle, combined the to relative fin pectoral anterior combined inthe expressed differentially highly are genes these however, ray, cownose in the middle; the to relative fin pectoral genes highlighted the skate, little the In fin. pectoral differentially expressed with high ratio (p < 0.05, 0.05, < (p ratio high with expressed differentially 0.05), > (p expressed differentially significantly anterior inthe expressed differentially not are scores (logFC) of fin-building and patterning genes genes patterning and fin-building of (logFC) scores 2: Figure Supplementary oF 1.5) > logFC small with expressed differentially dots: blue Light Differential expression expression Differential 56