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1 advances.sciencemag.org/cgi/content/full/3/11/eaao4709/dc1 Supplementary Materials for Pushing the limits of photoreception in twilight conditions: The rod-like cone retina of the deep-sea pearlsides Fanny de Busserolles, Fabio Cortesi, Jon Vidar Helvik, Wayne I. L. Davies, Rachel M. Templin, Robert K. P. Sullivan, Craig T. Michell, Jessica K. Mountford, Shaun P. Collin, Xabier Irigoien, Stein Kaartvedt, Justin Marshall The PDF file includes: Published 8 November 2017, Sci. Adv. 3, eaao4709 (2017) DOI: /sciadv.aao4709 fig. S1. Gene coding region (CDS) inferred vertebrate opsin gene phylogeny. fig. S2. Reconstruction of amino acid changes at known key spectral tuning sites of pearlside opsins. fig. S3. Vertebrate Gα transducin gene phylogeny. fig. S4. Vertebrate arrestin gene phylogeny. fig. S5. rh2 opsin gene conversion phylogeny. fig. S6. Topographic distribution of rod-like cone photoreceptors in three individuals of M. muelleri. fig. S7. Topographic distribution of ganglion cells (excluding amacrine cells and glial cells) in three individuals of M. muelleri. fig. S8. Expression of rh2 and rh1 opsin genes from sectional RNA in situ hybridization analyses of the eye of M. muelleri. fig. S9. Location of the true rod photoreceptors and their distribution across the retina in M. muelleri. fig. S10. Manual approach to extract genes from back-mapped reads. table S1. Summary of transcriptomes, GenBank accession numbers, opsin mapping (including base pair coverage), and proportional opsin gene expression. table S2. Summary of transcriptomes, GenBank accession numbers, transducin mapping (including base pair coverage), and proportional transducin gene expression. table S3. Summary of transcriptomes, GenBank accession numbers, arrestin mapping (including base pair coverage), and proportional arrestin gene expression.

2 table S4. Single-gene GenBank accession numbers of gene sequences produced during this study. table S5. Summary of the stereology parameters used for the analysis of the rodlike cone photoreceptors and ganglion cell distribution along with the quantitative results obtained using the optical fractionator methods in six retinas of M. muelleri. Legends for movies S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/3/11/eaao4709/dc1) movie S1 (.mp4 format). 3D reconstruction of the two photoreceptor types in M. muelleri. movie S2 (.mp4 format). Close-up 3D reconstruction of the nucleus and synaptic terminal of the two photoreceptor types in M. muelleri.

3 Supplementary Materials fig. S1. Gene coding region (CDS) inferred vertebrate opsin gene phylogeny. The pearlside retinal transcriptomes (n = 5 per species) contained three opsin genes (one rod opsin and two cone

4 opsins) (darker coloured boxes). Note that the rh2 cone opsins cluster as sisters within pearlside species suggesting two separate duplication events. However, as shown in fig. S5, gene conversion affecting the rh2 CDS is the cause of this pattern and the duplication is therefore likely to have occurred ancestrally. Vertebrate ancient (va) opsin gene sequences from five fish and one reptile species were used as outgroups to reconstruct the phylogenetic relationship. Node numbers depict Bayesian posterior probabilities. GenBank Accession Numbers are depicted after the species names, pearlside specific accession numbers are given in table S4.

5 bovine aa # M. muelleri rh2-1 M - N G T E G D N F Y I P M S N A S G L V R S P Y E Y P Q Y Y L G E K W Q F Y L L A A Y M V F L I S M. muelleri rh2-2 M - N G T E G D N F Y I P M S N A S G L V R S P Y E Y P Q Y Y L G E K W Q F Y L L A A Y M V F L I S M. mucronatus rh2-1 M - N G T E G E N F Y I P M S N A S G L V R S P F E Y P Q Y Y L G E K W Q F Y L L A V Y M L F L I F M. mucronatus rh2-2 M - N G T E G D N F Y I P M S N A S G L V R S P F E Y P Q Y Y L G E K W Q F Y L L A F Y M L F L I F S. analis rh2 M E N G T E G K N F Y I P M N N R S G L V R S P Y E Y P Q Y Y L A D P I L Y K F Q A A Y M L F L M F M. muelleri rh1 M - N G T E G P Y F Y V P M S N A T G V V R S P Y E Y P Q Y Y L V N P V A F F V L G A Y M F F L I L M. mucronatus rh1 M - N G T E G P Y F Y I P M S N A T G V V R S P Y E Y P Q Y Y L V N P A A F F V L G A Y M F F L I L bovine aa # M. muelleri rh2-1 T G L P L N G L T L V V T A Q N K K L Q Q P L N F I L V N L A V A G L I M V C F G F T I T F V T A M M. muelleri rh2-2 T G L P L N G L T L V V T A Q N K K L Q Q P L N F I L V N L A V A G L I M V C F G F T I T F V T A M M. mucronatus rh2-1 T G L P L N G L T L I V T F Q N K K L Q Q P L N F I L V N L A V A G M I M V C F G F T I T F C T A M M. mucronatus rh2-2 T G L P L N G L T L I V T A Q N K K L Q Q P L N F I L V N L A I A G M I M V C F G F T I T F C T A M S. analis rh2 T G G P I N I L T L V V T A Q N K K L R Q P L N F I L V N L A L A G A I M V F G G F V I T F Y T S M M. muelleri rh1 T V F P I N F L T L Y V T I E H K K L R T A L N Y V L L N L A V A N L F M V T G G F T T T L Y S S M M. mucronatus rh1 T C F P I N F L T L Y V T I E H K K L R T A L N Y V L L N L A V A N L F M V V G G F T T T L Y T S M bovine aa # M. muelleri rh2-1 N G Y F V F G P M G C A I E G F M A T L G G Q I S L W S L V V L A V E R Y I V V C K P M G S F K F G M. muelleri rh2-2 N G Y F V F G P M G C A I E G F M A T L G G Q I S L W S L V V L A V E R Y I V V C K P M G S F K F G M. mucronatus rh2-1 S G Y F I F G A M G C A I E G F M A T L G G Q I S L W S L V V L A V E R Y I V V C K P M G S F K F A M. mucronatus rh2-2 S G Y F I F G P M G C A I E G F M A T L G G Q I S L W S L V V L A V E R Y I V V C K P M G S F K F A S. analis rh2 N G Y F L L G P T S C A V E G F M A T L G G Q I S L W S L V V L A V E R Y I V V C K P M G S F K F S M. muelleri rh1 H G Y F V L G R S G C I I E G F C A T H G G Q V A L W S L V V L A I E R Y L V V C K P I A N F R F G M. mucronatus rh1 A G Y F V L G R T G C I I E G F C A T H G G Q V A L W S L V V L A I E R Y L V V C K P I A N F R F G bovine aa # M. muelleri rh2-1 N S H A T I G V V F T W V M A I A C A A P P L F G W S R Y L P E G L Q C S C G P D Y Y T L N P K C N M. muelleri rh2-2 N S H A T I G V V F T W V M A V A C A A P P L F G W S R Y L P E G L Q C S C G P D Y Y T L N P K C N M. mucronatus rh2-1 N S H A T A G V V F T W I M A L A C A A P P L L G W S R Y L P E G L Q C S C G P D Y Y T L N P K C N M. mucronatus rh2-2 N S H A T A G V V F T W I M A M A C A A P P L L G W S R Y L P E G L Q C S C G P D Y Y T L N P K C N S. analis rh2 A T H A G I G C G I T W F M A M A C A A P P L V G W S R Y L P E G L Q C S C G P D Y Y T M A P G Y N M. muelleri rh1 E N H A I M G L V F S W V M A F S C S L P P L F G W S R Y I P E G M Q C S C G I D Y Y T R A E G F N M. mucronatus rh1 E N H A I M G L V F S W V M A S S C S V P P L F G W S R Y I P E G M Q C S C G I D Y Y T R A E G F N bovine aa # M. muelleri rh2-1 N E S Y V M Y M F S C H F M V P V C T I F F T Y G S L V L T V K A A A A S Q Q E S E S T Q K A E R E M. muelleri rh2-2 N E S Y V M Y M F S C H F M V P V C T I F F T Y G S L V L T V K A A A A S Q Q E S E S T Q K A E R E M. mucronatus rh2-1 N E S Y V M Y M F S C H F C V P L F T I F F T Y G S L V L T V K A A A A S Q Q E S E S T Q K A E R E M. mucronatus rh2-2 N E S Y V M Y M F S C H F C V P L F T I F F T Y G S L V L T V K A A A A S Q Q E S E S T Q K A E R E S. analis rh2 N E S Y V I Y M F T C H F C F P V F T I F F T Y G S L V L T V K A A A A Q Q Q E S E S T Q K A E R E M. muelleri rh1 N E S F V I Y M F V V H F T I P L T I I T F C Y G R L L C A V K E A A A A Q Q E S E T T Q R A E K E M. mucronatus rh1 N E S F V I Y M F I V H F T I P L L I I L F C Y G R L L C A V K E A A A A Q Q E S E T T Q R A E R E bovine aa # M. muelleri rh2-1 V T R M C V L M V C G F L M A W T P Y A S Y A A W I F V N K G V S F S P Q S M A I P A F F A K S A S M. muelleri rh2-2 V T R M C V L M V C G F L M A W T P Y A S Y A A W I F V N K G V S F S P Q S M A I P A F F A K S A S M. mucronatus rh2-1 V T R M C I L M V F G F L L S W L P Y A S Y A A F I F M N K G I A F T P Q S M A V P A F F A K S G A M. mucronatus rh2-2 V T R M C I L M V F G F L L S W L P Y A S Y A A F I F M N K G I A F T P Q S M A V P A F F A K S G A S. analis rh2 V T R M C V L M V L G F L T A W V P Y A S L A A W I F F N K G A A F S A V G M A V P A F F A K S A S M. muelleri rh1 V S R M V V L M V M S Y M V S W M P Y A A V A W Y I F C N Q G T E F G P L F M A V P A F F A K S S A M. mucronatus rh1 V S R M V I L M V M S Y M V S W S P Y A S V A W Y I F C N Q G T E F G P L F M A V P A F F A K S S A bovine aa # M. muelleri rh2-1 I V N P V I Y V V L N K Q F R N C M M A T L - - G M A P A G D D E S S S - - V S S S S K T E V S S V M. muelleri rh2-2 I V N P V I Y V V L N K Q F R N C M M A T L - - G M A P A G D D E S S S - - V S S S S K T E V S S V M. mucronatus rh2-1 I I N P M I Y V V M N K Q F R T C M L A T L - - G M A A A G D D E S S S - - V S S S S K T E V S S V M. mucronatus rh2-2 I I N P M I Y V V M N K Q F R T C M L A T L - - G M A A A G D D E S S S - - V S S S S K T E V S S V S. analis rh2 L T N P V I Y V L L N K Q F R N C M L T T I - - G M G G M V D D E S S A S G S S S S S K T E V S S V M. muelleri rh1 L Y N P V I Y I C M N K Q F R M C M I T T L F C G K N P F E D M E G D S - - T T S A S K T E A S S V M. mucronatus rh1 L Y N P V I Y I C M N K Q F R T C M I T T L F C G K N P F E D M E G D S - - T T S A S K T E A S S V bovine aa # muelleri rh S P A M. Key spectral tuning sites (20, 23 ) M. muelleri rh S P A RH2 amino acid changes within Maurolicus species M. mucronatus rh S P A RH2 amino acid change between Maurolicus species and S. analis at key spectral tuning sites M. mucronatus rh S P A Retinal binding pocket site in bovine rhodopsin S. analis rh S Q G Q122 in cones and deeps-sea fish rods M. muelleri rh1 S S S S T S S V G P A P189 in cones, I189 in rods M. mucronatus rh1 - S S S T S S V A P A Transmembrane region fig. S2. Reconstruction of amino acid changes at known key spectral tuning sites of pearlside opsins. rh2 paralogs in both species showed very few amino acid differences (marked in yellow). Accession number for S. analis is EF

6 fig. S3. Vertebrate Gα transducin gene phylogeny. The pearlside retinal transcriptomes (n = 5 M. mucronatus; n = 4 M. muelleri) contained three alpha transducin genes (one rod transducin and two cone transducin). Mammalian taste transducing alpha subunit (gnat3) was used as outgroup to reconstruct the phylogenetic relationship. Node numbers depict Bayesian posterior probabilities. GenBank Accession Numbers are depicted after the species names, pearlside specific accession numbers are given in table S4.

fig. S4. Vertebrate arrestin gene phylogeny. The pearlside retinal transcriptomes (n = 5 M. mucronatus; n = 4 M. muelleri) contained four arrestin genes (two rod arrestins and two cone arrestins).

7 fig. S4. Vertebrate arrestin gene phylogeny. The pearlside retinal transcriptomes (n = 5 M. mucronatus; n = 4 M. muelleri) contained four arrestin genes (two rod arrestins and two cone arrestins). The arrestin of the ascidian Ciona intestinalis (ciarr) was used as an outgroup sequence to reconstruct the phylogenetic relationship. Node numbers depict Bayesian posterior probabilities. GenBank accession numbers are depicted after the species names, pearlside specific Accession Numbers are given in table S4.

8 fig. S5. rh2 opsin gene conversion phylogeny. Using the information contained in the 3 - untranslated regions (3 -UTRs) and the last 50 bp of exon 5 revealed the ancestral origin of the duplicated pearlside rh2 genes, and shows that gene conversion affecting the gene coding sequences (CDS) within pearlside species may have maintained an almost identical function of these gene products (also see Fig. 3, and fig. S2).

fig. S6. Topographic distribution of rod-like cone photoreceptors in three individuals of M. muelleri. (A to C) individuals S2004, S2005, S3001, respectively (refer to table S5).

9 fig. S6. Topographic distribution of rod-like cone photoreceptors in three individuals of M. muelleri. (A to C) individuals S2004, S2005, S3001, respectively (refer to table S5). The black lines represent iso-density contours, and values are expressed in densities x 10 4 cells/mm 2. The arrows indicate the orientation of the retina. N = nasal, V = ventral. Scale bar = 1 mm. (D and E) wholemount view of the photoreceptors in a high-density (D) and low-density (E) area. Scale bar 10 µm.

10 fig. S7. Topographic distribution of ganglion cells (excluding amacrine cells and glial cells) in three individuals of M. muelleri. (A to C) individuals S2005, S2006, S3002, respectively (refer to table S5). The black lines represent iso-density contours, and values are expressed in densities x 10 3 cells/mm 2. The arrows indicate the orientation of the retina. N = nasal, V = ventral. Scale bars = 1 mm. (D and E) light micrographs of the Nissl-stained ganglion cells in a low-density (D) and highdensity (E) area. gc = ganglion cell, ac = amacrine cell, g = glial cell. Scale bars = 25 µm.

fig. S8. Expression of rh2 and rh1 opsin genes from sectional RNA in situ hybridization analyses of the eye of M. muelleri. (A to D) rh2 labeling and (E to H) rh1 labeling.

11 fig. S8. Expression of rh2 and rh1 opsin genes from sectional RNA in situ hybridization analyses of the eye of M. muelleri. (A to D) rh2 labeling and (E to H) rh1 labeling. (D and E) show the entire retina labelled with either rh2 (D) or rh1 (E). (A to C) higher magnification images of the black boxes in (D) from top to bottom, respectively. (F-H) higher magnification images of the black boxes in (E) from top to bottom, respectively. Note that while rh2 was labelled everywhere in the retina, rh1 labelled cells (arrowheads in F-H) were very sparse and nearly exclusively found in the retinal margins of the retina. Scale bars = 100 µm (A to C, F to H), 1 mm (D, E).

12 fig. S9. Location of the true rod photoreceptors and their distribution across the retina in M. muelleri. (A) Rod photoreceptors labeled with anti-rhodopsin antibody and (B) distribution of the true rods across the retina (B). Each dot on the map in (B) represent one labeled cell. The black arrows indicate the orientation of the retina. N = nasal, V = ventral. Scale bars (A) = 1 µm, (B) = 1 mm.

13 reference sequence allele 4 allele 3 allele 2 allele 1 fig. S10. Manual approach to extract genes from back-mapped reads. Using a heterozygous individual to map unassembled reads against a reference sequence reveals a polymorphic site with four distinct single polynucleotide polymorphism (SNP) combinations (alleles) i.e. two gene copies (copy 1 = alleles 1 and 3; copy two = alleles 2 and 4). The copy specific SNPs in combination with the paired-end information were then used to manually reconstruct sequences by moving from SNP to SNP along the reference sequence.

14 table S1. Summary of transcriptomes, GenBank accession numbers, opsin mapping (including base pair coverage), and proportional opsin gene expression. Sub-mapping refers to the re-mapping of rh2 specific reads against the 3 -UTR alone. rh1 = rod opsin, rh2 = rhodopsin-like 2. Reads refer to number of Paired End fragments. RNA sequencing Mapping Relative expression % Transcriptome Rod Cones rod vs cone cones Species SRA GenBank Acc. No. Tot. raw reads rh1 rh2-1 Sub- rh2-2 Mapping No. reads x Cov. No. reads x Cov. No. reads No. reads x Cov. Sub- Mapping No. reads rh1 vs rh2 rh2-1 vs rh2-2 SRX ,531,936 25,906 2,141 1,679, , ,204 1,039,146 68,051 46, SRX ,301,844 30,203 2,495 1,790, , ,412 1,117,667 73,251 60, Maurolicus muelleri (Norwegian Fjord) SRX ,332,288 23,925 1,978 1,829, , ,890 1,081,219 70,851 49, SRX ,544,348 22,186 1,833 1,634, , ,222 1,075,381 70,314 52, SRX ,078,936 14,338 1,185 1,714, , ,174 1,070,155 70,092 69, Mean 54,157,870 23,312 1,926 1,729, , ,380 1,076,714 70,512 55, Standard error 6,124,761 2, ,712 2,384 7,158 12, , SRX ,396,072 2, ,146 40,479 97, ,608 35,969 79, SRX ,499,520 1, ,425 9,243 34, ,889 8,072 29, Maurolicus mucronatus (Red Sea) SRX ,574,170 4, ,004,880 65, , ,288 62, , SRX ,036,026 1, ,127 24,708 91, ,565 21,167 67, SRX ,718,678 4, ,001,589 65, , ,289 59, , Mean 61,844,893 2, ,233 41, , ,928 37,345 79, Standard error SRA, short-read archive 9,136, ,419 11,112 21, ,076 10,537 15,

15 table S2. Summary of transcriptomes, GenBank accession numbers, transducin mapping (including base pair coverage), and proportional transducin gene expression. gnat1 = G protein subunit alpha transducin 1, gnat2 = G protein subunit alpha transducin 2. Reads refer to number of Paired End fragments. RNA sequencing Mapping Relative expression % Transcriptome Rod Cones rod vs cone cones Species SRA GenBank Acc. No. Tot. reads Filtered gnat1 gnat2-1 gnat2-2 N 0 reads x Cov. N 0 reads x Cov. N 0 reads x Cov. gnat1 vs gnat2 gnat2-1 vs gnat2-2 Maurolicus muelleri (Norwegian Fjord) SRX ,531,936 1, ,451 16,781 36,551 3, SRX ,301,844 1, ,756 19,090 44,018 3, SRX ,332,288 1, ,747 18,411 41,438 3, SRX ,544,348 1, ,728 21,878 42,270 3, Maurolicus mucronatus (Red Sea) Mean 60,177,604 1, ,171 19,040 41,069 3, Standard error 1,458, ,568 1,063 1, SRX ,396, ,533 8,736 12,730 1, SRX ,499, ,672 1,325 2, SRX ,574, ,230 15,375 23,277 1, SRX ,036, ,880 4,040 5, SRX ,718, ,743 14,745 22,143 1, Mean 61,844, ,812 8,844 13,139 1, Standard error 9,136, ,221 2,803 4, SRA, short-read archive

16 table S3. Summary of transcriptomes, GenBank accession numbers, arrestin mapping (including base pair coverage), and proportional arrestin gene expression. sag = s-antigen visual arrestin, arr3 = arrestin 3. Reads refer to number of Paired End fragments. RNA sequencing Mapping Relative expression % Transcriptome Rod Cones rod vs cone cones Species Maurolicus muelleri (Norwegian Fjord) SRA GenBank Acc. No. Tot. raw reads saga arr3a arr3b N 0 reads x Cov. N 0 reads x Cov. N 0 reads x Cov. saga vs arr3 arr3a vs arr3b SRX ,531, SRX ,301, SRX ,332, SRX ,544, Maurolicus mucronatus (Red Sea) Mean 60,177, Standard error 1,458, SRX ,396, SRX ,499, SRX ,574, SRX ,036, SRX ,718, Mean 61,844, Standard error 9,136, SRA, short-read archive

17 table S4. Single-gene GenBank accession numbers of gene sequences produced during this study. rh1 = rod opsin, rh2 = rhodopsin-like 2, gnat1 = G protein subunit alpha transducin 1, gnat2 = G protein subunit alpha transducin 2, sag = s-antigen visual arrestin, arr3 = arrestin 3. Species GenBank accession no. Photoreceptor Gene (length bp) Maurolicus muelleri MF rod rh1 (1077) MF cone rh2-1 (1365)* MF cone rh2-2 (1365)* MF rod gnat1 (1053) MF cone gnat2-1 (1053) MF cone gnat2-2 (1038) MF rod (outer segment) x saga (1176) MF rod (synapses) x sagb-v1 (isoform) (1176) MF rod (synapses) x sagb-v2 (isoform) (1194) MF rod (synapses) x sagb-v3 (isoform) (1161) MF cone arr3a (1095) MF cone arr3b (1092) Maurolicus mucronatus MF rod rh1 (1074) MF cone rh2-1 (1368)* MF cone rh2-2 (1380)* MF rod gnat1 (1053) MF cone gnat2-1 (1053) MF cone gnat2-2 (1041) MF rod (outer segment) x saga (1179) MF rod (synapses) x sagb (1176) MF cone arr3a (1104) MF cone arr3b (1095) x sag expression localization according to (16). * length includes CDS and 3 -UTR (all other genes only CDS).

18 table S5. Summary of the stereology parameters used for the analysis of the rod-like cone photoreceptors and ganglion cell distribution along with the quantitative results obtained using the optical fractionator methods in six retinas of M. muelleri. CE = Schaeffer coefficient of error, Ø = diameter, SRP = spatial resolving power, PR = photoreceptors, GC = ganglion cells. Indiv Lens Ø, mm Cell type Counting frame size (µm) Grid size (µm) CE Total cells (n) Peak density (cells/ mm 2 ) S2004L 2.1 PR 40 x x ,699, ,750 S2005L* 2.0 PR 40 x x ,576, ,000 S3001R 2.1 PR 40 x x ,610, ,875 S2005R* 2.0 GC 80 x x ,950 18,750 S2006R 1.9 GC 80 x x ,918 17,969 S3002R 2.2 GC 80 x x ,248 19,531 * Same individual with the two eyes analyzed (left eye for PR and right eye for GC). Supplementary movies movie S1. 3D reconstruction of the two photoreceptor types in M. muelleri. movie S2. Close-up 3D reconstruction of the nucleus and synaptic terminal of the two photoreceptor types in M. muelleri.

Visual pigments. Neuroscience, Biochemistry Dr. Mamoun Ahram Third year, 2015

Visual pigments. Neuroscience, Biochemistry Dr. Mamoun Ahram Third year, 2015 Visual pigments Neuroscience, Biochemistry Dr. Mamoun Ahram Third year, 2015 References Photoreceptors and visual pigments Webvision: The Organization of the Retina and Visual System (http://www.ncbi.nlm.nih.gov/books/nbk11522/#a127)