BUSCADOR RECOLECTA

(a) Syntenic analysis of sea urchin LVA and LPI shows remarkable microsynteny conservation (i.e., linear relationships between chromosome pairs). Sea urchin LVA2 corresponds to SPU6 and SPU18, indicating LVA2 was fused from 2 ancestral chromosomes (b). Similarly, sea urchin LPI2 also corresponds to SPU6 and SPU18 (c), suggesting that this fusion event is a common trait in the Lytechnus genus. Furthermore, LVA1 corresponds to SPU8 and SPU19 (b), and LPI5 corresponds to SPU13 and SPU19 (c), indicating additional lineage-specific fusion events in sea urchin LVA and LPI. The data underlying this figure can be found in S1 Data., Peer reviewed

A stepwise process of sea urchin chromosomal evolution. We divided the process into 4 time points: t0, t1, t2, and ts (bottom right panel). At “t0,” individual chromosomes have not fused. At “t1,” 2 chromosomes are fused by either end-end translocation or centric insertion. At “t2,” intra-chromosomal translocations occur, although long stretches of chromosomal regions are still maintained. At “ts,” extensive intra-chromosomal rearrangements have occurred, and the fused chromosome becomes scrambled (fusion-with-mixing). We deduced 5 major fusion events that occurred during sea urchin chromosomal evolution, as follows. (1) Echinoderm EALGs E and B2⊗C2 fused and mixed to become sea urchin SALG E⊗(B2⊗C2) (t0 to ts in green). (2) EALGs B3 and J1 fused via centric insertion, followed by translocation to become SALG J1↘B3(t0 to t2 in maroon). (3) A Lytechinus-specific fusion event resulted from end-end fusion of SALGs G and D without obvious translocation (t0 to t1 in gray). (4) An LVA-specific fusion event involved Lytechinus LALGs F and J1⊗B3 without obvious translocation (t0 to t1 in Navajo white). (5) An LPI-specific fusion resulted from end-end fusion of Lytechinus LALGs F1 and C1, followed by an intrachromosomal translocation event (t0 to t2 in blue). Box sizes do not reflect the actual sizes of chromosomes., Peer reviewed

(a) Reconstruction of deuterostome ALGs E, B2, C2, and Q based on pairwise comparisons. The sea star POC2 and POC9 correspond to sea urchin SPU1 (b). POC2 corresponds to a single hemichordate chromosome PFL6, and POC9 corresponds to PFL18 and PFL23 (c). These 3 PFL chromosomes (PFL6, PFL18, and PFL23) also correspond to SPU1 (d). This observation suggests that the chromosomes in the sea star (POC2, POC9, and POC20) correspond to those in the LCA of the 2 echinoderm species, while SPU1 resulted from fusion of the 2 echinoderm ancestral chromosomes (echinoderm ALGs E and B2⊗C2). To infer the ambulacrarian ancestral condition, the amphioxus BFL genome was compared to the ambulacrarian genomes. POC2 and PFL6 correspond to a single amphioxus chromosome BFL5, supporting the conclusion that echinoderm ALG E has a deeper root in the ambulacrarian LCA and deuterostome LCA (ambulacraria/deuterostome ALG E). On the other hand, POC9 and both PFL18 and PFL23 correspond to 2 amphioxus chromosomes, BFL16 and BFL3 (e–g). Based on this observation, it may be inferred that POC9 could represent the ambulacraria ancestral chromosome (ambulacraria ALG B2⊗C2), and hemichordate PFL18 and PFL23 resulted from a split of ambulacraria ALG B2⊗C2. Notably, in addition to POC9, amphioxus BFL3 also corresponds to POC20 (e). POC20 shows one-to-one correspondence with SPU21 and PFL22 (b–d), suggesting that an ancestral chromosome was present at least in the LCA of ambulacrarians (ambulacraria ALG Q) and remained intact in the echinoderm lineage (echinoderm ALG Q). To infer the deuterostome ancestral condition and the evolutionary history of BFL3, the scallop PYE genome was compared to those of the deuterostome genomes (h–k). The observation that BFL16 corresponds to a single PYE chromosome (PYE1) supports the idea that the deuterostome LCA possessed this chromosome (deuterostome ALG B2). Additionally, BFL3 corresponds to PYE17 and PYE2. PYE2 also corresponds to BFL13 and 2 one-to-one corresponding chromosomes in ambulacrarian species (POC20/SPU21/PFL22 and POC3/SPU9/PFL15). Therefore, the deuterostome LCA likely possessed ALGs C2 and Q. In the lineage leading to ambulacrarians, deuterostome ALGs B2 and C2 fused and became ambulacraria ALG B2⊗C2. Furthermore, BFL3 also corresponds to 2 vertebrate chromosomes [19,20], so the chordate LCA likely inherited deuterostome ALGs C2 and Q, and these 2 chromosomes then fused specifically in amphioxus to become BFL3. The data underlying this figure can be found in S1 Data., Peer reviewed

(a) Reconstruction of deuterostome ALGs O2, B3, and J1 based on pairwise comparisons. The sea star POC6 corresponds to sea urchin SPU20 and SPU8 (b) and hemichordate PFL2 and PFL11 (c); SPU20 and SPU8 also correspond to these 2 PFL chromosomes (d), indicating that these 2 chromosomes were present at least in the ambulacrarian and echinoderm LCAs, and POC6 resulted from fusion of the 2 ancestral chromosomes (ALGs O2 and B3). Intriguingly, in addition to POC6, SPU8 also corresponds to POC14, while POC14 corresponds to a single hemichordate chromosome PFL17. Consistently, SPU8 corresponds to PFL11 and PFL17 (d), indicating that a single chromosome corresponding to POC14/PFL17 is an ancestral trait (ALG J1), while SPU8 resulted from chromosomal fusion (ALGs B3 and J1). Therefore, it can be inferred that the LCAs of ambulacrarians and echinoderms possessed these 3 ALGs (O2, B3, and J1), which remained as individual chromosomes in hemichordates but underwent different fusion events in different echinoderm lineages. Fusion of ALGs O2 and B3 led to sea star POC6, while fusion of ALGs B3 and J1 resulted in sea urchin SPU8. Consistent with this hypothesis, 3 distinct amphioxus chromosomes BFL19, BFL18, and BFL17 correspond to POC6 and POC14 (e); SPU20 and SPU8 (f); and PFL2, PFL11, and PFL17 (g). This correspondence supports the idea that the presence of the 3 ALGs can be traced back to the LCA of deuterostomes and remained in the chordate LCA. This conclusion is further reinforced by the observation that the scallop genome contains 3 distinct chromosomes (PYE3, PYE19, and PYE18) corresponding to POC6 and POC14 (h); SPU20 and SPU8 (i); PFL2, PFL11, and PFL17 (j); and BFL19, BFL18, and BFL17 (k). Additionally, the 3 amphioxus chromosomes BFL19, BFL18, and BFL17 have been shown to correspond to 3 distinct vertebrate chromosomes [19,20], supporting the conclusion that the chordate LCA possessed these 3 chromosomes. The data underlying this figure can be found in S1 Data., Peer reviewed

(a) Reconstruction of deuterostome ALGs R and B1 based on pairwise comparisons. Using the same logic as described for S6 Fig, sea star POC12 and POC18 appear to correspond to sea urchin SPU3 and SPU17, respectively (b), supporting the conclusion that their LCA possessed these 2 chromosomes (echinoderm ALGs R and B1). Comparison between hemichordate PFL and echinoderm species revealed that POC12/SPU3 and POC18/SPU17 correspond to a single hemichordate chromosome PFL9 (c and d). This observation suggests a fusion event occurred in the ambulacraria ancestor leading to PFL9 or a split event leading to POC12/SPU3 and POC18/SPU17. Using amphioxus BFL as an outgroup, the analysis showed that POC18/SPU17 corresponds to BFL10 (e and f), while amphioxus orthologs of POC12/SPU3 genes spread in the genome and no single BFL chromosome could be assigned to POC12/SPU3. Another outgroup scallop PYE was then used, revealing that POC12/SPU3 and POC18/SPU17 respectively correspond to PYE13 and PYE12 (h and i). Based on these comparisons, 3 major inferences can be made: (1) both deuterostome and ambulacraria ancestors possessed the 2 distinct chromosomes (deuterostome/ambulacraria ALGs R and B1); (2) at least in the LCA of hemichordates PFL and SCA, ALGs R and B1 were fused, leading to PFL9/SCA5; (3) in amphioxus, orthologous genes of deuterostome ALG R were dispersed to other chromosomes. Notably, in addition to POC12/SPU3, PYE13 also corresponds to POC22/SPU15, explaining the comparability between PYE13 and the hemichordate PFL1 and amphioxus BFL4 (j and k) and suggesting a fusion event led to PYE13. Consistent with this idea, the hemichordate PFL9 (fused from ALGs R and B1) corresponds to BFL10 (ALG B1) (g). It has been proposed that all chromosomes of vertebrates correspond to amphioxus chromosomes [19,20], suggesting that one ancestral chromosome (ALG R) spread to other chromosomes in the LCA of chordates. The scallop chromosome name was labeled and sorted according to chromosome size. Here, PYE12 is chromosome number 13 and PYE13 is chromosome number 12 in the previous study [25]. The data underlying this figure can be found in S1 Data., Peer reviewed

(a) Reconstruction of deuterostome ALGs J2, C1, A2, A1, I, and O1 based on pairwise comparisons among amphioxus BFL, hemichordate PFL, sea urchin SPU, sea star POC, and scallop PYE. First, the comparison of POC with SPU showed that POC16, POC8, POC21, POC1, POC11, and POC22 have one-to-one correspondence with SPU10, SPU13, SPU2, SPU5, SPU11, and SPU15, respectively (b), suggesting that these 6 chromosomes were already present in their LCA (echinoderm ALGs J2, C1, A2, A1, I, and O1). These 6 chromosomes also have one-to-one correspondence with hemichordate PFL5, PFL21, PFL3, PFL14, PFL20, and PFL1 (c and d), indicating that the existence of these 6 chromosomes could be traced further back to the ambulacrarian LCA (ambulacraria ALGs J2, C1, A2, A1, I, and O1). Comparisons with the amphioxus BFL genome showed that both POC16/SPU10/PFL5 and POC8/SPU13/PFL21 correspond to a single amphioxus chromosome BFL2 (e–g). Similarly, POC21/SPU2/PFL3 and POC1/SPU5/PFL14 correspond to amphioxus BFL1; POC11/SPU11/PFL20 and POC22/SPU15/PFL1 correspond to amphioxus BFL4. To infer the deuterostome ancestral condition, scallop PYE was used as an outgroup. This analysis showed that the 6 ambulacraria chromosomes correspond to 6 distinct PYE chromosomes (PYE4, PYE9, PYE16, PYE5, PYE11, and PYE13, see h–j), supporting the conclusion that these 6 chromosomes are ancient and were present in the deuterostome LCA (deuterostome ALGs J2, C1, A2, A1, I, and O1). Accordingly, the 3 amphioxus chromosomes (BFL 2, BFL1, and BFL4) correspond to the aforementioned 6 PYE chromosomes (k). Therefore, the amphioxus BFL2, BFL1, and BFL4 were formed from respective fusion events between deuterostome ALGs J2 and C1, ALGs A2 and A1, and ALGs I and O1. These 3 fusion events are likely amphioxus-specific because the 6 deuterostome ALGs correspond to 6 vertebrate ALGs [19,20], which support the notion that these 6 chromosomes remained intact in the LCA of chordates (chordate ALGs J2, C1, A2, A1, I, and O1). The data underlying this figure can be found in S1 Data., Peer reviewed

(a) A Hi-C contact map of P. flava genome assembly based on the HiC-pro pipeline [91]. Note that the 3′ end of PFL3-1 interacts with the 5′ end of PFL3-2, and the 5′ end of PFL3-1 interacts with the 3′ end of PFL3-3 (green arrows). The boxed area is magnified to show the chromosomal interactions around the PFL chromosome 23. (b) The 3′ end (right side) of PFL23-1 interacts with the 3′ end of PFL23-2 (blue arrow), suggesting that the 2 scaffolds are closely linked at their 3′ ends. These 2 scaffolds also highly interact with several smaller scaffolds (blue arrowheads). Similarly, the 3′ end of PFL3-4 interacts with the 5′ end of PFL3-3 (green arrow). Based on the contact information, PFL3-1 to PFL3-4 were assembled in the order of PFL3-4, PFL3-3, PFL3-1, and PFL3-2. PFL3-2 and PFL3-3 also interact with several smaller scaffolds (green arrowheads). P. flava chromosome #3 (PFL3) was thus assembled by joining PFL3-1 to PFL3-4; PFL23 was assembled by joining PFL23-1 and PFL23-2. The data underlying this figure can be found in S5 Data., Peer reviewed

Cartoon representation of the dimers observed in the crystal structures of Rap3T, Rap105, RapJ (PDB 4GYO), RapH (PDB 3Q15), RapI (PDB 4I1A), and RapF (PDB 4I9E). All proteins show a similar dimerization mode interacting mainly with the TPR domains (orange-yellow) mainly through the C-terminal helix (in tones of red) with the 3HB domains (blue tones) facing outward from the dimer., pbio.3002744.s003.tiff, Peer reviewed

(a) RMSD calculation from the superimposition of Cα atoms of Rap3T structures in complex with peptides SRGHTS, RGHTS, RRGHTA, and RRGHTAS. RMSD were calculated from individual monomers (lower) and dimers (upper). Number of atoms used in RMSD calculation is showed between parenthesis. (b) Superposition of 4 Rap3T-Peptide complexes with different pheromone variants in monomeric state RRGHTAS (blue), RRGHTA (pink), RGHTS (light green), and SRGHTS (orange). (c) Superposition of dimers for the 4 Rap3T complexes with peptides RRGHTAS (blue), RRGHTA (pink), RGHTS (light green), and SRGHTS (orange)., pbio.3002744.s004.tiff, Peer reviewed

pbio.3002744.s005.tiff, Peer reviewed