science.sciencemag.org/content/368/6498/eaaz5667/suppl/DC1

Supplementary Materials for

Exploring whole-genome duplicate gene retention with complex genetic interaction

analysis

Elena Kuzmin, Benjamin VanderSluis, Alex N. Nguyen Ba, Wen Wang, Elizabeth N. Koch, Matej Usaj, Anton Khmelinskii, Mojca Mattiazzi Usaj, Jolanda van Leeuwen, Oren Kraus, Amy Tresenrider,

Michael Pryszlak, Ming-Che Hu, Brenda Varriano, Michael Costanzo, Michael Knop, Alan Moses, Chad L. Myers*, Brenda J. Andrews*, Charles Boone*

*Corresponding author. Email: charlie.boone@utoronto.ca (C.B.); brenda.andrews@utoronto.ca (B.J.A.); chadm@umn.edu(C.L.M.)

Published 26 June 2020, Science 368, eaaz5667 (2020) DOI: 10.1126/science.aaz5667

This PDF file includes: Materials and Methods Figs. S1 to S9 Captions for tables S1 to S13 References

Other supplementary material for this manuscript includes: MDAR Reproducibility Checklist (PDF)Tables S1 to S13 (Excel format)

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Materials and Methods General information about the SGA dataset

In this study, 240 double mutants and 480 corresponding single mutant control ‘query’ strains were screened for genetic interactions against a diagnostic set of ~1,200 mutant ‘array’ strains. Every double mutant query strain was screened alongside its two single mutant control strains, in two independent replicates, for a total of 1,440 screens. The scoring of 537,911 double and 256,861 triple mutants identified 4,650 negative and 2,547 positive digenic interactions and 2,466 negative and 2,091 positive trigenic interactions. The raw genetic interaction data is available in Table S1. The final trigenic interaction scores adjusted for digenic interactions are available in Table S2, where we used an established interaction magnitude cut-off for digenic interactions (|𝜀 | > 0.08, p < 0.05) and trigenic interactions (|𝜏 | > 0.08, p < 0.05). The dataset can be browsed interactively at http://boonelab.ccbr.utoronto.ca/paralogs/. Tables S1 to S13 were also deposited in the DRYAD Digital Repository (https://doi.org/10.5061/dryad.g79cnp5m9). MATLAB routines that produce SGA digenic and trigenic interaction scores are available at https://doi.org/10.5281/zenodo.3665423. SGA query strain construction Strain maintenance

All query strains were maintained on YEPD media (1% yeast extract, 2% peptone, 2% glucose, 0.012% adenine) supplemented with 100 μg/mL nourseothricin (Werner Bioagents) and array strains on YEPD supplemented with 200 μg/mL geneticin (Agri-Bio). Array strains deleted for non-essential genes were obtained from the yeast deletion collection (71) and the available collection of temperature-sensitive alleles of essential genes (72). Query strain construction

We constructed and successfully screened 240 double mutant query yeast strains, each deleted for a pair of nonessential WGD paralog genes (Table S1-S4). These pairs included 171 paralogs from a list of 261 that were previously identified to be non-interacting as well as 17 interacting paralogs (34) out of 143 interacting pairs that were viable and thus amenable for screening, chosen based on the original list of WGD paralogs (6) as well as additional 52 out of 83 which were unique to the updated WGD paralog list (12). Using a starting strain from our lab collection of SGA background strains harbouring paralog 1 deletion marked by natMX4, we generated a heterozygous diploid strain by mating it with a wild type SGA background strain, Y7091, and deleted the second paralog replacing it with K. lactis URA3 reporter. Thus, the 240 strains represent mutants that were successfully isolated by tetrad dissection analysis and passed our internal quality control steps during screening. Paralogs that were essential as single mutants and for which there was no starting strain, or which would have been too sick to screen (double mutant fitness < 0.7) based on our global network analysis at the time of starting this project (32), in linkage with SGA markers, and those that our tetrad analysis identified to be synthetic lethal, non-sporulating, exhibited poor spore viability, or abnormal marker segregation pattern in tetrad analysis were excluded. Digenic and trigenic SGA screens that failed QC and did not show gene linkage with query genes and the gene deletion mutations of interest of their respective query strains were not confirmed by PCR were excluded from the analysis. Single mutant control strains (480 in total) deleted for one member of a paralog pair were constructed, such that each strain carried the deletion of one member of a paralog pair with the relevant control marker

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inserted at the benign HO locus. To construct single mutant controls, strains with the following genotype were used: 1) SN deletion strain MATα par1Δ::natMX4 can1Δ::STE2pr-Sp_his5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 LYS2+; or 2) newly constructed MATα par2Δ::KlURA3 can1Δ::STE2pr-Sp_his5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 LYS2+. They were transformed with either a KlURA3 or natMX4 PCR product containing 55 bp of homology flanking the HO ORF. Correct gene disruption was confirmed by colony PCR. p5749 plasmid was used to PCR amplify K.lactis URA3. It belongs to pFA6a series (73). The sequence of KlURA3 can be found using GenBank accession number D00431. This plasmid is ampicillin resistant and was cultured in 2YT + 120 μg/ml ampicillin. Primers that were used for deleting YFG and marking it with KlURA3 (5’ à 3’): (fwd) 55 b of homology specific to 5’ of YFG followed by cggagacaatcatatgggag, (rvs) 55 b of homology specific to 3’ of YFG followed by tctggaggaagtttgagagg. Primers that were used to delete HO and mark it with KlURA3 (5’ à 3’): (fwd) CATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAAATGcggagacaatcatatgggag (rvs) TTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTTAtctggaggaagtttgagagg p4339 plasmid was used to mark gene deletions with natMX4 (74). This plasmid is ampicillin resistant and was cultured in 2YT + 120 μg/ml ampicillin. Primers that were used to delete YFG and mark it with natMX4 (5’ à 3’): (fwd) 55 b of homology specific to 5’ of YFG followed by acatggaggcccagaatacc (rvs) 55 b of homology specific to 3’ of YFG followed by cagtatagcgaccagcattc. Primers that were used to delete HO and mark it with natMX4 (5’ à 3’): (fwd) CATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAAATGacatggaggcccagaatacc (rvs) TTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTTAcagtatagcgaccagcattc

Query strain ploidy was verified using Fluorescence-Activated Cell Sorting (FACS). Briefly, strains were cultured in YEPD overnight then diluted to OD600 = 0.1 in fresh YEPD and grown 5-6 hr, 30°C, 200 rpm. The cell suspension was centrifuged, 1,000 x g, 3 min and resuspended in cold 1 ml 70% ethanol, then incubated overnight at 4°C with rotation. The cells were washed in 1 ml sterile water and 500 μl were discarded. The remaining 500 μl were pelleted and resuspended in 200 μl of 50 mM Tris-Cl pH 8.0 + 0.2 mg/ml RNase A, then incubated 3 hr, 37°C. The cell suspension was centrifuged, 1000 x g, 3 min and resuspended in 200 μl of 50 mM Tris-Cl pH 7.5 + 2 mg/ml proteinase K, incubated 30-60 min, 50°C. The cell suspension was centrifuged, 1,000 x g, 3 min and resuspended in 0.5 ml FACS buffer. Then, on average 14 μl were transferred to a round bottom 96-shallow well plate with the addition of 200 μl SYBRgreen (1:5,000, 50 mM Tris-Cl pH 7.5). A benchtop flow cytometer running the GUAVA program was used to obtain a FACS profile, which was analyzed using FLOWJO. Another quality control step consisted of verifying the mating type of the strain by performing crosses with Y12978 (MATa lys1Δ) and Y12979 (MATα lys1Δ) and testing for complementation and growth on SD minimal media. Genotypes of all query strains, as well as other strains and plasmids that were used in this study are in Table S4. Synthetic genetic array analysis for trigenic and digenic interactions Query fitness estimation

A high-density array was assembled to estimate query strain fitness. The strains on the array were arranged such that those carrying mutations in genes that are on the same chromosome were maximally separated. The gaps on the array (including the border rows and columns) were filled with either Y13096 (MATα ura3Δ::natMX4 hoΔ::KlURA3 can1Δ::STE2pr-

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Sp_his5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 LYS2+) or Y14412 (MATα his3Δ1::natMX4 hoΔ::KlURA3 can1Δ::STE2pr-Sp_his5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 LYS2+), which enabled the arrays to be crossed to two different control strains, each carrying a marked deletion in a benign locus, (his3 and ura3): DMA1 (MATa his3Δ1::kanMX4 leu2Δ0 ura3Δ0 met15Δ0) and Y14420 (MATa his3Δ1 leu2Δ0 ura3Δ0::kanMX4 met15Δ0). This minimized the amount of missing data caused by genes of interest being linked genetically to HIS3 or URA3 genomic loci. Mutants with both query genes residing in HIS3 or URA3 linkage groups were assigned a fitness value of NaN. Once the arrays were crossed to the aforementioned strains, they were subjected to SGA, as described below, and scored for colony size in order to estimate fitness (Fig. S1, Table S5). The quantitative scoring method employed for single and double mutant fitness estimation was described previously (75), with the exception that bootstrapped means, instead of medians, across replicates were used in variance estimation and final fitness values. Each high-density array was screened in triplicate for a total of 6 replicates. Since the arrays were in 1536-colony format, there are 4 technical replicates of mutants on the array resulting in a total of between 12 and 24 colony measurements for each fitness estimate. To control for differences associated with the choice of the control strain (his3Δ1 or ura3Δ0), we fit a first-order polynomial, which was then applied to adjust the values for all ura3Δ0::kanMX4 strains. After the adjustment, double mutant fitness estimates from the two assays were averaged together, and the resulting standard can be found in Table S5. Fitness estimates were compared to another SGA dataset (30) and any mutant with |difference| > 0.2 was set to NaN. During the genetic interaction scoring process, all NaN fitness estimates were assigned a value of 1.0, as described previously (75). Array fitness estimation

The mean single mutant fitness estimates of each array strain were obtained from a previous study (37). The variance of array single mutant fitness estimates were used to calculate the interaction p-values and were derived from screening the wild-type control query strain (Y13096) against the diagnostic array (n = 91). Query pair interaction estimation

The array used to quantify query fitness included both double mutant queries and their respective pairs of single mutant controls. We therefore used these measurements to calculate genetic interactions between paralog sisters, providing a higher quality estimate for these focused interactions than was obtained in our comprehensive genetic interaction network (30). As in that network, the expected double mutant fitness is defined as the product of the two relevant single mutants, and the interaction (epsilon) is defined as the difference (observed – expected). To establish significance for these interactions, we assume these residuals are normally distributed under the null model (no interaction), and derive a p-value from the observed epsilon and its expected variance of the corresponding mutant. These interaction scores are available in Table S5.

𝑑𝑚𝑓!"#!$%!& = 𝑠𝑚𝑓' × 𝑠𝑚𝑓( 𝜀 = 𝑑𝑚𝑓)*+!,-!& − 𝑑𝑚𝑓!"#!$%!&

𝜎.&/0_!"#!$%!& = ,𝜎.+/0' × 𝜎.+/0(- + ,𝜎.+/0' × 𝑠𝑚𝑓(- + ,𝜎.+/0( × 𝑠𝑚𝑓'-

𝑝 = 𝑛𝑜𝑟𝑚𝑐𝑑𝑓 4−|𝜀|

𝜎&/0_!"#!$%!&6

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Trigenic synthetic genetic array analysis technique (𝜏-SGA) Trigenic Synthetic Genetic Array was conducted as previously described (37, 76, 77, 78).

Briefly, lawns of query mutant strains were grown at 26°C for 2 days and then pinned onto fresh YEPD plates. The diagnostic mutant array was then pinned on top of the query strain. Diagnostic array was composed of a total of ~1,200 mutants which provided a representative view of the global digenic interaction network as described previously (37, 79). The mated mix was incubated at room temperature for a day and then diploids were selected by pinning the resulting MATa/α diploid zygotes to YEPD + G418/clonNAT and incubating at 26°C for 2 days. The resulting natR/Ura+/kanR MATa/α diploids were sporulated by pinning onto enriched sporulation agar plates and incubating at 22°C for 7 days. To select for MATa meiotic haploid progeny, spores were pinned onto SD – His/Arg/Lys + canavanine/thialysine (50 μg/ml of each analog) and incubated at 26°C, 2 days. Following haploid selection, the resulting colonies were pinned onto SDMSG – His/Arg/Lys/Ura + canavanine/thialysine/G418 (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.1% monosodium glutamic acid, 0.2% amino acid supplement, 2% agar, 2% glucose, 50 μg/mL of each analog, for antibiotic concentration see ‘Strain Maintenance’ section), after which Ura+/kanR/natR MATa meiotic haploid progeny were selected by pinning the double/triple mutant haploid mix onto SDMSG – His/Arg/Lys/Ura + canavanine/thialysine/G418/clonNAT to select for final triple mutants. The incubation temperature for all the selection steps was 26°C, except for sporulation, which was conducted at 22°C. Every double mutant query strain was screened alongside its two single mutant control strains in two independent replicates. Because trigenic interactions were derived by profile subtraction, single mutant control queries and their corresponding double mutant control query were screened in the same batch to minimize the compounding of systematic effects. Pilot screens (Fig. S4, Table S3) were conducted in a similar manner except all strains were screened against the genome-wide array of non-essential (NES) gene deletion mutants (32) and the array of temperature sensitive alleles of essential (ES) genes (72). Quantifying trigenic interactions Quantitative fitness-based model of genetic interactions

Digenic and trigenic interactions were scored as previously described (37, 75):

(𝑑𝑖𝑔𝑒𝑛𝑖𝑐)𝜀3,5 = 𝑓35 − ,𝑓3𝑓5- (𝑡𝑟𝑖𝑔𝑒𝑛𝑖𝑐)𝜏3,5,6 =𝑓356 − 𝑓3𝑓5𝑓6 − 𝜀5,6𝑓3 − 𝜀3,6𝑓5 − 𝜀3,5𝑓6

where ε is the digenic interaction score, τ is the final adjusted trigenic interaction score, ƒ is

fitness and i, j, and k are individual mutations. The final adjusted trigenic interaction profile consists only of trigenic interaction scores that

have been corrected for all digenic interactions. All unthresholded digenic interactions are used to compute trigenic interactions. The interactions are available in Table S1&2. Pilot screens in which 11 double mutant query strains and their corresponding single mutant control strains were crossed to the genome-wide array of non-essential (NES) gene deletion mutants (71) and the array of temperature sensitive alleles of essential (ES) genes (72) were scored with a random set of 1,211 and 2,494 additional queries, respectively, to achieve numerical stability. The interactions from pilot screens are available in Table S3. MATLAB routines that score initial colony images to produce SGA digenic interaction scores, as well as those that apply this model to produce trigenic interaction scores are available at https://doi.org/10.5281/zenodo.3665423.

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Trigenic interaction confirmations were previously conducted using tetrad analysis and random spore analysis (37). Briefly, 15 interactions that overlapped a previous study (80) and were re-confirmed in (37) showed a significant trigenic score in 9 cases resulting in an estimated recall of ~60%, consistent with another high-throughput SGA study (32). The difference could be attributed to the reproducibility or magnitude of the genetic interaction in addition to the genotype of the background strain used in the two studies. Screening the cln1Δ cln2Δ query mutant strain against the diagnostic array identified 73 trigenic interactions at an intermediate cut-off, τ ≤ -0.08, p < 0.05. An arbitrary set was selected for confirmations by random spore analysis showing that 26 of 34 trigenic interactions were confirmed, resulting in an estimated precision of 76%, and therefore, a false discovery rate of 24%. All triple mutant screens and the corresponding double mutant screens reported in this study have been conducted in two replicates to obtain a sufficient level of precision for the detection of trigenic interactions, as previously described (37). In order to ascertain the reproducibility of trigenic interaction screens, we screened 9 query sets (one double mutant with two single mutant control query strains) in 4 replicates, for a total of 108 screens. Each screen was crossed to our diagnostic array of ~1,200 mutants (themselves each arranged in 4 adjacent biological replicates). The following paralog pairs comprised the query sets: GPB1-GPB2, DPB3-DLS1, ARE1-ARE2, VPS64-FAR10, YGR283C-YMR310C, SBE2-SBE22, BCH2-CHS6, MLP1-MLP2 and SKI7-HBS1. Each query set was scored twice using two independent replicates to obtain independent sets of trigenic interactions (A and B) with technical characteristics similar to the dataset as a whole. To estimate the precision and recall characteristics in the absence of a gold-standard for trigenic interactions, we used the following formula AB = ( A × P ) × R, where A is the total number of interactions seen in the first experiment, AB is the number of interactions observed in both A and B experiments, P is precision, and R is recall. A×P is, therefore, the expected number of true positives among A, and R is the fraction of these that we would expect to be captured again in an independent experiment. Fig. S2D shows precision as a function of recall using this formula for nine jackknife samples, each leaving out interactions from one query set. Classifying trigenic interactions into novel vs. modified

Trigenic interactions were classified into novel and modified as previously described (37) (Fig. 2, S3A, Table S1&2). Briefly, our trigenic interaction scoring model allows for a trigenic interaction involving two genes connected by a digenic interaction, given that the triple mutant exhibits a significant deviation from the expected fitness of the double mutant when combined with the third mutation. Thus, “modified” trigenic interactions are observed when the third mutation exacerbates or alleviates a previously known digenic interaction leading to a more extreme phenotype than expected, and thus can be said to modify an existing interaction. On the other hand, “novel” trigenic interactions occur when none of the pairwise genetic combinations within the triplet overlaps with a previously known digenic interaction. In these cases, we identified a novel functional connection for genes that were not previously observed to interact digenically. In practice, a trigenic interaction (τijk) between a double mutant query (Qij) and an array (Ak) is called novel if there is no significant interaction between either single mutant control query (Qi or Qj) and the array (Ak), and also no interaction between query gene pair itself. Digenic interactions between Qi-Ak or Qj-Ak were measured using our single mutant control queries. Query pair interactions (Qi-Qj) were measured using the single and double mutant fitness standard (Table S5) by applying the multiplicative model to derive the genetic interaction

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between the two query genes. Each query mutant fitness score has an associated standard deviation, and these were combined to calculate the expected variance of the double mutant fitness under the product. As residuals (epsilon scores) are approximately normally distributed under the null model of no interaction, this expected variance can be used to calculate a p-value. If any such digenic interaction exists, either positive or negative, the trigenic interaction is called modified. We classified trigenic interactions into 2,560 modified (1,236 negative, 1,324 positive) and 1,033 novel (621 negative, 412 positive) at the following thresholds for digenic interactions (p < 0.05, |e| > 0.08) and trigenic interactions (p < 0.05, |t| > 0.08). Trigenic interactions that have no overlapping digenic interaction but stem from double mutant queries for which double mutant fitness estimates have not been generated in our standard due to quality control and have corresponding NaN values have been withheld from the novel class. Comparison of trigenic interactions with external functional standards

We compared the functional information reflected by digenic and trigenic interactions by assessing their ability to predict other functional relationships (Fig. S3C-F). Specifically, we evaluated the precision (relative to background) of each class of interaction against a collection of pairwise functional gold-standards. Protein-protein interactions were taken from the union of five high-throughput studies (81-85). Localization data were taken from (86), and two genes were considered colocalized, if they shared one or more cellular compartment annotations above the recommended thresholds (cytoplasm excluded) (Table S14). Co-expression values were taken from an integration of high throughput expression studies (87), genes were considered a true positive for co-expression, if their score fell in the top 3 percent, and a true negative, if their score fell in the bottom 50 percent. Genes were considered co-annotated, if they shared one or more annotations to functional neighborhoods as defined previously (30). Each of these standards captures pairs of genes, and thus they do not map directly to trigenic interactions. We chose to try and capture cases where either query paralog had a functional relationship to the array gene (OR). However, we also wanted to account for standards which have true negatives (e.g. co-expression) or are incomplete. To do this we used the following truth table where 1 denotes a True Positive, -1 denotes a True Negative, and 0 denotes an unsure relationship or a missing value. Gold-standard input

Q1A 1 1 1 0 0 0 -1 -1 -1 Q2A 1 0 -1 1 0 -1 1 0 -1

‘OR’ standard output

Q1Q2A 1 1 1 1 0 0 1 0 -1

Precision for combination of interaction-class and standard was then calculated normally:

𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 =𝑇𝑃

𝑇𝑃 + 𝐹𝑃 Digenic interactions (e < -0.08, p < 0.05) and trigenic interactions (t < -0.08, p < 0.05) were used for this analysis. For all digenic interactions, annotations for the query gene and the identified interacting genes were considered; HO is a benign control locus that houses the marker, and it is excluded from the analysis.

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Correlation of trigenic interaction fraction with physiological and evolutionary features The magnitude of the genetic interaction between paralogs was quantified in this study

(Table S5). Genetic interaction degree asymmetry was derived from this study where the paralogs were deemed asymmetric, if the ratio of their interactions exceeded 5 as previously described (33) and in cases where one of the degrees was zero, it was set to 1 if the high degree paralog met the degree threshold at the stringent score cut-off of 𝜀 ≤ -0.12 to provide confidence of the resulting asymmetry (Table S10).

Sequence divergence rate (Table S10) was calculated in the following manner: previously, we have shown that we can computationally predict large conserved protein segments, which are very likely to be functional domains of proteins and are efficient filters for studying disordered protein regions (59). These large conserved protein segments can be predicted in species prior to the whole-genome duplication and analyzed for changes in rates of evolution post-genome duplication. Focusing only on these domains (which we concatenated to prevent mispredictions of a single domain into multiple smaller domains), PAML was used (88) to estimate the number of amino acid substitutions per site for both post-WGD clade and the pre-WGD clade (PAML program: AAML, clock=0, cleandata=0, fix_omega=0, ncatG=8, WAG rate matrix). The same pre-WGD and post-WGD species that were used for a previous study (12) were also used for the current analysis. The rates of substitutions are normalized against a pruned species tree to account for differing species composition between clades and overall different rates of evolution, and we then obtain a fold-change in substitutions per site post-WGD. The raw difference between the fold-change between the two paralogous clades is used as our measure of sequence evolution asymmetry. Formally, a species tree is calculated on the whole dataset to obtain an expected rate of evolution (αpre-WGD, αpost-WGD) and the rates of evolution are estimated for a particular paralog pair (βpre-WGD, βpost-WGD_par1, βpost-WGD_par2). Because some proteins evolve faster than others, the calculated rates of evolution are scaled such that:

𝛼#,!789: = 𝜆𝛽#,!789:

The final score for the sequence divergence rate is given by

𝑆𝑒𝑞𝑢𝑒𝑛𝑐𝑒𝑑𝑖𝑣𝑒𝑟𝑔𝑒𝑛𝑐𝑒𝑟𝑎𝑡𝑒 = abs 4𝜆𝛽#)+%789:_#;,<

𝛼#,!789:−𝜆𝛽#)+%789:_#;,.

𝛼#,!789:6

Paralog pairs that exhibited genetic interaction asymmetry were tested for induction

during developmental programs: meiosis, filamentous growth and glucose starvation (Table S11). A previously published meiotic mRNA-seq and ribosome profiling dataset was used to find genes induced in meiosis (51). First, the average of all 8 meiotic time points was taken for both mRNA-seq and ribosome profiling datasets. Using the average meiotic expression, the fold-change between either vegetative growth or starvation (MATa/a cells in sporulation media for 4.5 hours) was calculated. A gene was then considered to be expressed in meiosis, if the fold-change from vegetative growth to meiosis and starvation to meiosis was greater than 4. The same cutoffs were applied to both mRNA-seq and ribosome profiling. A gene was considered to be required during filamentous growth, if its deletion mutant exhibited an invasion defect and met a previously established cut-off of ‘invasion score’ ≤ -0.7 (52). Gene expression was considered to be induced in response to glucose starvation, if the maximum transcript levels, expressed as log2 ratios of the zero timepoint, exceeded 2 as reported in a previous study (53).

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Dosage selection was estimated as previously described (33, 38) using global digenic profile correlation similarity from a previously published study (30) (Table S10). Analysis was restricted to paralogs (WGD) with at least 6 trigenic or digenic interactions in one of the screens.

The list of sparsely connected paralogs based on the global digenic network analysis is available in Table S6. Family size was determined by sequence homology and further refined by literature curation (Table S9).

The list of ohnologs and homeologs was obtained from a previous study as reported in their data associated with Fig. 1C (50). In our data (Table S8) ohnologs consisted of 22 (37%) paralog pairs with a low trigenic interaction fraction and 13 (63%) paralog pairs with a high trigenic interaction fraction, whereas homeologs consisted of 16 (36%) paralog pairs with a low trigenic interaction fraction and 9 (64%) paralog pairs with a high trigenic interaction fraction, resulting in a statistically insignificant difference in their proportions (Fisher’s exact test, p = 1).

Functional annotation of digenic and trigenic interactions using SAFE

SAFE (Functional annotations based on the Spatial Analysis of Functional Enrichment) of the global genetic interaction profile similarity network was used to annotate gene function (30, 72) (Fig. 5A). The trigenic interactions of paralogs with a high trigenic interaction fraction at the intermediate threshold (t< -0.08, p < 0.05) were taken as inputs to SAFE analysis. An enrichment score was computed for each gene on the global similarity network from theCellMap.org (89) based on the overlap of its direct neighborhood with a specified set of trigenic interactions. The hypergeometric test was used to assess the significance of enrichment (Fig. 5B&C, S5I). Trigenic interactions predict novel paralog function (ECM13-YJR115W) (Fig. S5A-F) Benomyl sensitivity assays

Yeast strains (WT: Y13096, ecm13Δ: TM427, yjr115wΔ: TM1130, ecm13Δ yjr115wΔ: TM3405, mad3Δ: SN1472) were grown overnight at 30°C in YPD, serially diluted, and spotted onto YPD plates containing 15 µg/ml benomyl or DMSO only (Fig. S5C). Plates were incubated at 30°C for 2 days. The mad3Δ mutant has a known fitness defect on media containing benomyl (90). Latrunculin B sensitivity assays

Yeast strains (WT: Y13096, ecm13Δ: TM427, yjr115wΔ: TM1130, ecm13Δ yjr115wΔ: TM3405, bni1Δ: SN570) were grown overnight at 30°C in YPD, diluted to OD600 of 0.1, and grown in 200 ul YPD containing 10 µM lantranculin B or DMSO only (Fig. S5D). Growth was monitored for 24 hr in a TECAN by measuring the OD600. The bni1Δ mutant has a known fitness defect on media containing latrunculin B. Spindle morphology

Yeast strains lacking ECM13, YJR115W, or ECM13-YJR115W and expressing Tub1-GFP (p5575) were grown overnight to saturation then sub-cultured the next morning and grown to mid-log phase at 30°C in synthetic dextrose media lacking uracil (SD-Ura) to maintain plasmid selection (Fig. S5E). Cells were deposited onto an agar slide and imaged using a spinning-disc confocal microscope (WaveFX, Quorum Technologies) connected to a DMI 6000B fluorescence microscope (Leica Microsystems) controlled by Volocity software (PerkinElmer), and equipped with an ImagEM charge-coupled device camera (Hamamatsu C9100-13, Hamamatsu Photonics)

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and 63x/NA1.4 Oil HCX PL APO objective. Imaging was done at room temperature. Cells with normal or abnormal spindle morphology were counted using ImageJ Cell Counter plugin (91). On average 63 cells were imaged per field of view for a total of three samplings in two replicates. GFP strain construction and microscopy

N-terminal tagging of Ecm13 and Yjr115w was performed by conventional PCR targeting (92) using plasmid pMaM173 (pYM-N-sfGFPΔC-SceIsite-CYC1term-URA3-TEF1pr-SceIsite-sfGFP (93)) as template. The resulting strains, Y14798 and Y14801, expressing sfGFP-tagged fusion proteins of Ecm13 and Yjr115w, respectively, from the TEF1 promoter, were validated by colony PCR to verify correct cassette integration and immunoblotting with anti-GFP antibodies (Abcam, #ab6556). Yeast strains Y14798 and Y14801 were grown to mid-log phase at room temperature in synthetic complete media supplemented with 2% glucose. Live cells were stained in Hoechst 33258 (Thermo Fisher Scientific) staining solution (1:100 dilution of 1 mg/ml dye in fresh media) for 15 min, rinsed 3 times, transferred to a Concanavalin A coated 384-well PerkinElmer CellCarrier Ultra imaging plate, and centrifuged for 45 s at 500 rpm before imaging. Micrographs were obtained at room temperature on the Opera Phenix (PerkinElmer) automated spinning disk confocal microscope with a 60x water-immersion objective. Hoechst 33258 was excited using a 405 nm laser, and emission collected through a 435-480 nm filter. GFP was excited using a 488 nm laser and emission collected through a 500-550 nm filter. The two channels were imaged sequentially. Z-stacks of 5 optical sections with 1 µm spacing were acquired. Micrographs were processed using ImageJ (Fig. S5F) (91). Trigenic interactions predict novel paralog function (STB2-STB6) (Fig. S5G-J) Bap2-GFP localization

Yeast strains (WT: Y13096, stb6Δ: TM481, stb2Δ: TM2239, stb6Δ stb2Δ: TM3367) carrying Bap2-GFP were grown overnight to saturation in YEPD then sub-cultured (1:50) the next morning and grown for 1 hour in YEPD at 30°C in the presence of 8 µM FM4-64 dye for 1 hour at room temperature to allow complete internalization of the dye to the vacuolar membrane, washed twice with water, resuspended in SDminimal containing histidine, leucine, methionine, uracil and grown for 3 hours in YEPD at 30°C. Cells were deposited onto an agar slide and imaged using a spinning-disc confocal microscope as described above (Fig. S5J).

CellProfiler (v2.2.0rc3) was used to measure the sub cellular localization of Bap2-GFP to the vacuole and cell periphery in WT, stb6Δ, stb2Δ and stb6Δ stb2Δ strains (Fig. 5J) (94). The illumination of images was corrected using a median filter with a dilation radius of 5. Then Otsu thresholding was applied to the GFP channel to identify cell objects, and then 3 pixels were subtracted from the boundaries of these objects to identify the cell periphery. The RobustBackground adaptive thresholding method was used on the RFP channel based on the signal from the FM4-64 dye to identify the vacuolar regions in the cell objects. Following these steps, the intensity of the GFP channel in these regions was measured. The median GFP intensity was calculated in the vacuolar regions compared to the vacuolar size, and the ratio of GFP intensity in the cell periphery region compared to the entire cell. The stb6Δ stb2Δ double mutant showed a prominent mislocalization of Bap2 into the vacuoles consistent with these paralogs playing a role in the MTC pathway.

11

Estimating constraints on paralog evolution We sought a metric that measured differences in which columns were constrained in the

paralogs after the whole-genome duplication, but that was insensitive to the overall average rate of evolution of any paralog (Fig. 6, S6). To do so, rates of evolution were obtained for specific columns in multiple sequence alignments using the discrete gamma model of protein evolution as implemented in PAML (88). These rates were computed for the pre-WGD sequences (12), and for each paralog separately. Pearson correlation coefficients were calculated between the rates of the pre-WGD clade to each paralog (pre-WGD & Paralog 1 and pre-WGD and Paralog 2), and between the two paralogs (Paralog 1 and Paralog 2) and classified paralogs into those with high and low correlation of position specific evolutionary rate patterns (Table S12). If both paralogs share constraints on specific residues of domains during evolution and evolve similarly after the WGD (they contain residues that are strongly entangled), then they would be expected to be more similar to each other than the pre-WGD species. If paralogs do not share constraints on specific residues of domains during evolution and evolve differently after the WGD, then they would be less similar to each other than to the pre-WGD species. Therefore, pairs with a high correlation of position specific evolutionary rate patterns were designated as those pairs for which the correlation of rates between extant sisters was greater than or equal to both correlations between each sister and the pre-WGD ancestor. Although this metric should be insensitive to overall changes in the rate of evolution of any of the two paralogs, we find that this distance deviation and the rate asymmetry are still significantly correlated, implying that changes in protein function both change which columns are constrained, but also changes the overall rate of evolution of proteins. Our analysis was restricted to protein domains with position-specific conservation in the pre-WGD clade, and those are typically unlikely to be intrinsically disordered and by definition have position-specific conservation. If we looked at the whole protein, then the analysis could be masked by intrinsic disorder, but since our analysis requires a faithful alignment, and disordered regions do not align well, we had to resort to doing the analysis on domains, which are mostly structured (Fig. 6, S6).

WGD paralogs were defined to be synthetic lethal (SL), if the genetic interaction score between them was e ≤ -0.35 as reported in a previous study (30) or, if they were shown to be synthetic lethal by tetrad analysis in this study (Table S13). Mitochondrial carrier protein family genetic interaction network. Negative digenic and trigenic interactions from this study that meet the intermediate cut-off (e or t < -0.08, p < 0.05) were combined with literature-curated genetic interactions, as reported by BioGrid (95) and included negative genetic interactions, phenotypic enhancement, dosage rescue and phenotypic suppression to construct the network (Fig. S8). Simulating paralog divergence. The computational framework was based on the Duplication-Degeneration-Complementation model with elements of the Escape from Adaptive Conflict model to explain evolutionary outcomes stemming from gene duplication (Fig. 7A-D, S9A-D). The Duplication-Degeneration-Complementation model proposes that the accumulation of deleterious mutations in regulatory regions can increase the probability of the retention of duplicated genes and that duplicate gene preservation can occur by partitioning ancestral functions (39). The Escape from Adaptive Conflict model proposes that ‘gene sharing’ precedes gene duplication, whereby the ancestral gene is involved in more than one process or carries out more than one function and thus it is ‘shared’ between processes. After duplication, each

12

daughter gene can carry out one of these functions by optimizing its multiple domains such that each gene would adapt in gene expression or function (96). In our model, each gene was created with a fixed length drawn randomly from a uniform distribution [25, 100]; length represented the number of positions subject to a degenerative mutation. Hypothetical functions were then assigned to contiguous regions of each “gene,” and these regions were allowed to overlap in “sequence” space (i.e. multiple functions could be affected by a single degenerative mutation). We note, however, that because each position was treated as statistically independent of all others in terms of degenerative mutations, the fact that they were spatially contiguous in our model does not affect the results of the simulations (this model accommodates real scenarios in which a single molecular function maps to non-contiguous regions of a gene’s sequence). The number of functions for each gene and the length of each functional region were drawn randomly from uniform distributions ([3, 15], [3, 20], respectively). The position of each functional segment was likewise randomly selected. Once the gene was created, it was duplicated and subjected to random degenerative mutations at a constant rate.

A mutation anywhere within a functional region was considered to disable that function, and as functions could overlap, a single mutation could disable multiple functions simultaneously. Mutations to a gene that disabled a function that could be carried out by the sister (buffered) were allowed to persist. Mutations which disabled a function sustained by only one copy were discarded as evolutionary dead ends, which reflects an assumption that all functions are essential and must be maintained by at least one of the sister paralogs. A paralog pair was deemed to have reached steady-state when no more divergent mutations could occur while maintaining viability.

The evolution of 500,000 paralog pairs was simulated and results were binned according to each pair’s initial structural entanglement. Here, structural entanglement is defined as the fraction of a gene’s mutable positions that are responsible for two or more functions. The probability of reversion to singleton was estimated by counting the number of simulated pairs with all functions assigned to a single sister, relative to the total number of simulated pairs (Fig. 7B). Bias in sister functions was calculated as the number of functions carried out by one sister (greater of the two) divided by the gene’s original number of functions (Fig. 7C). Retained functional overlap was calculated as the fraction of sequence positions carrying out one or more functions in both sisters simultaneously after steady-state had been reached (Fig. 7D).

Establishment and acceleration of asymmetric functional divergence can be understood by considering which functional regions would be susceptible to a degenerative mutation, and which would be protected by a consequential decrease in fitness. At the beginning of each simulation, there is an equal probability that any given function will survive in each sister. As a mutation arises in one paralog, natural selection “protects” the corresponding sequence in the sister from mutation, in the sense that lineages with these secondary random mutations die out and are not observed. However, if multiple functional regions overlap, this “protection” can extend to shared regions of the second function, and thus, there is a slightly higher probability that a degenerative mutation will appear in the same sister that lost the first function (33).

Single mutant fitness (this study)

Sing

le m

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Single mutant fitness Double mutant fitness

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Fig. S1.

A B

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Fig. S1. Quantification of fitness of query strains using high-density arrays and interaction score replicate correlations. (A) Histogram of single mutant fitness estimates of all single mutant control query strains used in this study, n = 472. (B) Histogram of double mutant fitness of all double mutant query strains used in this study, n = 201. (C) Scatter plot comparing the single mutant fitness derived from this study and another study (30), n = 426, r = 0.51, p = 3x10-30. (D) Scatter plot comparing double mutant fitness derived from this study and another study (30), n = 139, r = 0.72, p = 2x10-23. r denotes Pearson correlation coefficient.

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Fig. S2.

Fig. S2. Reproducibility of digenic and trigenic interactions. (A) Correlation of digenic interaction scores, (B) raw trigenic interaction scores and (C) adjusted trigenic interaction scores from two independent replicates. Significant interactions scores (p < 0.05) are shown; the dashed lines represent intermediate, (τ or ε) > 0.08, p < 0.05 (blue) and stringent, (τ or ε) > 0.12, p < 0.05 (red), cut-offs. (D) Estimates of precision and recall were obtained by analyzing 9 double mutant and their corresponding single mutant control queries, screened in 4 replicates, for a total of 108 screens. Trigenic interaction scores from two replicate screens of each query were compared against two independent replicate screens (τ ≤ -0.08 & p < 0.05). Grey lines depict precision as a function of recall for jackknife samples, red dotted line denotes the median, black dotted line is displayed as a reference using the estimate of recall derived from CLN1-CLN2 validation experiments (see Supplementa-ry Methods for details).

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Data density

-1 -0.5 0.5 10-1.5 -1.5 -1 -0.5 0.5 10-1.5 -1.5 -1 -0.5 0.5 10-1.5 -1.5SGA score (ε), Replicate 1

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Fig. S3.

Pos. digenic interactionsNeg. digenic interactionsC D

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Fig. S3. Characterization of trigenic interactions of paralogs. (A) Pie chart depicting the total number of trigenic interactions of different classes for paralogs which are characterized by low (left) and high (right) trigenic interaction fraction. Negative ((τ or ε) < -0.08, p < 0.05) genetic interactions are depicted as blue edges, positive ((τ or ε) > 0.08, p < 0.05) as yellow. A trigenic interaction between a double mutant query and the array strain is called novel (bright blue/orange) if there is no significant digenic interaction between either single mutant control query and the array strain or between the query gene pair. Trigenic interactions that overlap with one or more negative or positive digenic interactions are called modified, and are further classified by the type of the digenic interaction. Of our 240 double mutant query strains (P1-P2), 38 show a negative and 43 show a positive digenic interaction between query gene pair (P1-P2) (|score| > 0.08, p < 0.05), thus all trigenic interactions of these queries are modified. Interactions may further be classified by digenic interactions (if any) between a single mutant query control strain and the array strain (P1 and/or P2-A negative, P1 and/or P2-A positive). Modified trigenic interactions that 1) overlap digenic interactions of the same sign are in medium blue/orange, 2) overlap digenic interactions of the opposite sign are in light blue/orange and 3) overlap a mix of positive and negative digenic interactions (mixed) are depicted in grey. (B) Negative genetic interaction degree ((τ or ε) < -0.08, p < 0.05) in singletons compared to all duplicates and duplicates with low (<0.4) and high (>0.4) trigenic interaction fraction. Single and double mutants are denoted by ∆ or ∆∆, respectively. Singleton degree was calculated using data set from (37). Bars represent means, errorbars are SEM. Statistical significance was assessed using Wilcoxon rank sum test. Overlap with common functional standards for (C) Negative digenic interactions, (D) Positive digenic interactions, (E) Negative trigenic interactions, (F) Positive trigenic interactions. All genetic interactions meet the intermediate cut-off, (τ or ε) > 0.08, p < 0.05. The standards are the following: merged protein-protein interaction (PPI) standard, co-annotation to GO biological process, co-expression, co-localization. The dashed line denotes background expectation.

Pos. modified trigenicpos. and neg. digenic

Pos. modified trigenicneg. digenic

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GPB2-GPB1 ARE1-ARE2MLP1-MLP2 CIK1-VIK1SBE22-SBE2

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Fig. S4.

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Genome-wide Diagnostic

Fig. S4. Comparison between genome-wide and diagnostic array screens. (A) Genetic interaction fraction for 11 pilot paralog pairs screened against genome-wide array of nonessential gene deletion mutants and temperature sensetive alleles of essential genes and the diagnostic array. Genetic interaction fraction was similar between array types, negative genetic interactions were used at the intermediate cut-off, τ or ε < -0.08, p < 0.05. (B) Frequency of negative genetic interactions, τ or ε < -0.08, p < 0.05, within and across biological processes for screens conducted against genome-wide array of non-essential gene deletion mutants and temperature sensetive alleles of essential genes and the diagnostic array. The fraction of screened query-array combinations that exhibit negative interactions was measured for 14 broadly defined gene sets (32). Significance was assessed with a hypergeometric test; p < 0.05.

CIK1

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CFig. S5.

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tcp1-2tcp1-1act1-4act1-136act1-101chs6∆

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Fig. S5. Functional characterization of poorly known paralogs. (A) ECM13-YJR115W represents a poorly characterized functionally redundant paralog pair possibly involved in tubulin assembly, consistent with the fraction of their trigenic interactions being 0.77 and very few paralog specific interactions. Summarized GO term enrichment is consistent with a role in tubulin assembly. (B) The similarity of the unadjusted trigenic profile of ECM13-YJR115W to the genetic interaction profiles of genes on the global similarity network. Genes with profile similarity > 0.2 are listed. Unadjusted trigenic profile refers to raw trigenic interaction scores before digenic interactions were subtracted (see Supplementary Methods). (C) Growth response to benomyl for the wild-type, ecm13Δ, yjr115wΔ and ecm13Δ yjr115wΔ mutants, showing tubulin cytoskeleton defect in the double mutant and a slight defect in the ecm13Δ single mutant. YEPD, yeast extract, peptone, dextrose. (D) Growth response to lantranculin B for the wild-type, ecm13Δ, yjr115wΔ and ecm13Δ yjr115wΔ mutants showing no actin cytoskeleton defect. (E) Delayed polymerization/failed nucleation of the spindle was scored for the wild-type, ecm13Δ, yjr115wΔ and ecm13Δ yjr115wΔ mutants. Data are shown as mean, SEM. Representative brightfield and Tub1-GFP fluorescent micrographs are displayed for the wild-type and ecm13Δ yjr115wΔ double mutant. (F) Ecm13 and Yjr115w were N-terminally tagged with sfGFP and overexpressed from the TEF1 promoter using seamless tagging cassettes. Nuclei were visualized using Hoechst stain.Scale bar is 5 µm. (G) STB6-STB2 represents a poorly characterized functionally redundant paralog pair possibly involved in the MTC pathway, consistent with the fraction of their trigenic interactions being 0.95 and very few paralog specific interactions. (H) The similarity of the unadjusted trigenic profile of STB2-STB6 to the genetic interaction profiles of genes on the global similarity network. Genes with profile similarity > 0.2 are listed. Unadjusted trigenic profile refers to raw trigenic interaction scores before digenic interactions were subtracted (see Supplementary Methods). (I) SAFE (70) analysis was used to visualize regions of the global digenic interaction profile similarity network (30) that were enriched for genes in the trigenic interaction profiles of the following paralog pairs STB2-STB6. (J) Bap2-GFP localization was monitored in wild-type, stb6Δ, stb2Δ and stb6Δ stb2Δ mutants. The vacuole is stained with a fluorescent dye FM4-64. Data are shown as mean, SEM and a two-tailed ttest was used to asseess statistical significance.

Dwild typebni1∆ecm13∆ yjr115w∆

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Unadj. Trigenic Interaction Profile Correlation

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p = 8 x 10-55

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Fig. S6A

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Fig. S6. Analysis of correlated evolutionary sequence changes. Multiple sequence alignments for (A) Mrs3, Mrs4, and pre-WGD proteins (B) Ski7, Hbs1 and pre-WGD proteins were visualized using Jalview. The position specific evolutionary rates are reflected in the blue to white colouring from low to high rates, respectively, and the line graph.

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

Tbla2/1-271Ndai2/1-275Scer2/1-272Skud2/1-272Kafr2/1-272Knag2/1-272Vpol2/1-272Ncas2/1-272Suva2/1-272Smik2/1-272Cgla2/1-273

E V E D Y E S M P S T A P L S S Q L MA G A F A G I M E HM VM F P V D S L K T R I Q S T Q L K N I I H T Q G Y L A PW KG VQ A I L V G A G P A H A I Y F A T Y E A C K S R L I K E - ND T G Y H - - - P F K I A L CG A T A T T V S D F L F N P F D T V K Q R L Q L - - - N T K T I Y Q N EG L A A F Y Y S Y P T T I AM D I P F A A F N F V I Y E S T T K F F N P T N S Y N P F I H C L CG G I S G A T C A A I T T P L D C I K T I L Q V RG S E T L G SG Q L K K A S T MT E A A K A I Y S V R GW KG F V RG MK P R V I A NM P A T A I SW T A Y E C A K H F LE D T D Y E A L P S H A P L A H Q L MA G A F A G I M E H S I M F P I D A L K T R I Q S S Q I S K I S T A E G S F A L W KG VQ S V I L G A G P A H A V Y F A T Y E F WK S Y L I K D - E D L E T H Q - - P L K T A F S G A MA T V A S D A L MN P F D T I K Q R MQ L L K M K S K S I Y Q N E G I S A F Y Y S Y P T T I AM N I P F A A F N F M I Y E S A S K F F N P T H V Y N P L I H C L CG G I S G T I C A A I T T P L D C I K T V L Q V RG S K S V S ME I F K N A N T F K K A A N A I YQ V H GW KG FW RG L K P R I I A NM P A T A I SW T A Y E C A K H F LD L P D Y E A L P T H A P L Y H Q L I A G A F A G I M E H S VM F P I D A L K T R I Q S S Q I S H I S T S E G T L A L W KG VQ S V I L G A G P A H A V Y F G T Y E F C K K N L I D S - S D T Q T HH - - P F K T A I SG A C A T T A S D A L MN P F D T I K Q R I Q L - - - N T K Q I Y Q S EG L A A F Y Y S Y P T T L VM N I P F A A F N F V I Y E S S T K F L N P S N E Y N P L I H C L CG S I SG S T C A A I T T P L D C I K T V L Q I R G S Q T V S L E I MR K A D T F S K A A S A I Y Q V YG WK G F WR GW K P R I V A N M P A T A I SW T A Y E C A K H F LD L P D Y E A L P T H A P L Y H Q L I A G A F A G I M E H S VM F P I D A L K T R I Q S S Q I S H I S T S E G T L A L W KG VQ S V I L G A G P A H A V Y F G T Y E F C K K N L I D S - ND T Q T HH - - P F K T A I SG A C A T T A S D A L MN P F D T I K Q R I Q L - - - N T K Q I Y Q S EG L A A F Y Y S Y P T T L VM N V P F A A F N F V I Y E S S T K F L N P S N E Y N P L I H C L CG S I SG S T C A A I T T P L D C I K T V L Q I R G S Q T V S L E I M K K A D T F S K A A T A I YQ V Y GW KG F S RG WK P R I V A N M P A T A I SW T A Y E C A K H F LDN I N Y E S L P E D S S L Y A Q L L A G A F A G I M E H S VM F P I D A L K T R I Q A S Q I S K I S A S E G S F A L W KG VQ S V I L G A G P A H A V Y F G T Y E F C K A H L I E K - D K L H T HQ - - P V K T A I SG AM A T I A S D A L L N P F D T I K Q R MQ L - - - AM K S I Y K N EG F I A F Y Y S Y P A T I A MN I P F T A L N F V V Y E S S I K L F N P T E S Y N P L I H C L SG G I S G A L A A A T T T P L D V I K T T L Q V RG S E K V Q L Q V L R K A D T F N K A A V A I Y K I YG WK G F L K G L K P R V I A S I P A T A I SW T S Y E C A K H F LL D L D Y E S M P S N S P L S H Q L L A G A F A G I M E H S VM F P I D A L K T R I Q S Q N I S K I S T L E G S T T L WK G V Q S V I L G AG P A H A V Y F G T Y E F C K S R L I D E - Q DM H T HQ - - P I K T A I SG A C A T V A S D A L MN P F D T L K Q R VQ L - - - S A G E MY R T E G I S A F Y Y S Y P T T I AM N I P F T A L N F V I Y E S S T K I L N P T GG Y N P L V H C L CG G I S G T L C A A I T T P L D V I K T T L Q V RG S D R V S L E I F R Q A D T F S K A A R A I F K V H G Y KG FW RG L Q P R I V A T M P A T A I SW T A Y E C A K H F LE E I D Y E A L P E N A S L P S Q L L A G A F A G I M E H L VM F P I D A L K T R V Q S K E I S K I T T T E G S MA L W KG VQ SM I L G A G P A H A V Y F G T Y E L M K A R L I T P - E DM H T HQ - - P L K T A I SG A T A T I A A D A L MN P F D T I K Q R MQ L - - - S T K N I Y K K E G L R A F Y Y S Y P T T I AM N I P F V S L N F V I Y E S S T K I F N P S NN Y N P L I H C I C GG L S G A T C A A L T T P L D C I K T V L Q V RG S E S V S L D I M K K A D T F T K A A K A I Y Q V HG WG G F L R G L K P R V V A N M P A T A I SW T S Y E C A K H F LA D V D Y E A L P A H A P L S H Q L L A G A F A G I M E H S TM F P I D A L K T R I Q S K Q I S K I S T ME G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E F T K A H L I P D - S Q R E T HQ - - P I K V A V S G A T A T V A S D F F MN P F D T I K Q R MQ I - - - S A K K I Y N L EG L S A F Y Y S Y P T T I AM N I P F A A F N F M I Y E S A S K F F N P L HH Y N P L I H C L CG G I S G A I A A A V T T P L D C I K T V I Q I R G S S V V S L E V M K K A N T F K K A T S A I L MV YG WK G F WR G L Q P R I L A NM P A T A I SW T A Y E C A K H F LD S P D Y E A L P S H A P L Y H Q L I A G A F A G I M E H S VM F P I D A L K T R I Q S S Q I S H I S T S E G T MA L W KG VQ S V I L G A G P A H A V Y F GM Y E F C K K N L I D P - ND T Q T HH - - P F K T A I SG A C A T T A S D A L MN P F D T I K Q R I Q L - - - N T K Q I Y Q S EG L A A F Y Y S Y P T T L VM N I P F A A F N F V I Y E S S T K F L N P S N E Y N P L I H C L CG S I SG S T C A A I T T P L D C I K T V L Q I R G S Q T V S L E L MR K A D T F S K A A G A I Y Q V YG WK G F WR GW K P R I V A N M P A T A I SW T A Y E C A K H F LD L P D Y E A L P T H A P L Y Y Q L I A G A F A G I M E H S VM F P I D A L K T R I Q S S Q I S H I S T S E G T L A L W KG VQ S V I L G A G P A H A V Y F G T Y E F C K K N L I D S - ND T Q S HH - - P F K T A I SG A C A T T A S D A L MN P F D T I K Q R I Q L - - - N T R Q I Y Q S EG L A A F Y Y S Y P T T L VM N I P F A A F N F V I Y E S S T K F L N P S N E Y N P L I H C L CG S I SG S T C A A I T T P L D C I K T V L Q I R G S Q T V S L E I MR K A D T F S K A A S A I Y Q V YG WR G F S R GW K P R I V A N M P A T A I SW T A Y E C A K H F LD V I D Y E A L P DH A P L A H Q L MA G A F A G I A E H S V I F P L D A L K T R L Q A R Q L S S I S AQ EG SM V L WK G V Q S V L L G AG P A H A V Y F A T Y E M V K S F L I D E A T S T S K Y H - - F F K T A F S G A T A T I A A D A L MN P F D V I K Q R I Q L - - - N A K R I Y S K E G F Q A F Y S S Y P T T L A I N I P F A A F N F G I Y D T A T R Y F N P S G V Y N P F I H C L CG G I S G A A C AG L T T P L D C I K T A L Q V RG S E K V S M E V F KQ A D T F K K A T R A I Y Q V YG WR G F WS G V K P R I L A NM P A T A I SW T A Y E F A K H F L

Rate s

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

Scer1/1-272Suva1/1-272Skud1/1-272Ndai1/1-272Kafr1/1-272Tbla1/1-274Smik1/1-272Knag1/1-272Ncas1/1-272Cgla1/1-272Tpha1/1-272Vpol1/1-272

E E I D Y E A L P S H A P L H S Q L L A G A F A G I M E H S L M F P I D A L K T R V Q A S Q I S K I S T ME G S MA L W KG VQ S V I L G A G P A H A V Y F G T Y E F C K A R L I S P - E DMQ T HQ - - PM K T A L SG T I A T I A A D A L MN P F D T V K Q R L Q L - - - D T K Q I Y Q N EG F A A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A S K F F N PQ N S Y N P L I H C L CG G I S G A T C A A L T T P L D C I K T V L Q V RG S E T V S I E I M K D A N T F G R A S R A I L E V H GW KG FW RG L K P R I V A N I P A T A I SW T A Y E C A K H F LE E I D Y E A L P S H A P L Y S Q L L A G A F A G I M E H S L M F P I D A L K T R V Q A S Q I S K I S T I E G S MA L W KG VQ S V ML G A G P A H A V Y F A T Y E F C K A R L I S P - E DMQ T HQ - - PM K T A L SG T V A T I A A D A L MN P F D T V K Q R L Q L - - - D T K Q I Y Q H EG F A A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A S K Y F N P Q N S Y N P L V H C L CG G L SG A T C A A L T T P L D C I K T V L Q V RG S E T V S I G I M K D A N T F G R A S R A I L E V H GW KG FW RG L K P R I V A N I P A T A I SW T A Y E C A K H F LE E I D Y E A L P S H A P V H SQ L L A G A F A G I M E H S L M F P I D A L K T R V Q A S Q I S K I S T ME G S MA L W RG VQ S V I L G A G P A H A V Y F A T Y E F C K A R L I S P - E DMQ T HQ - - PM K T A L SG T I A T I A A D A L MN P F D T V K Q R L Q L - - - D T K H I Y Q N EG F A A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A S K F F N PQ N S Y N P L I H C L CG G I S G A T C A A L T T P L D C I K T V L Q V RG S E T V S I G I M RDA D T F G R A S R A I L E V H GW KG FW RG L K P R I V A N I P A T A I SW T A Y E C A K H F LE E I D Y E A L P S N A P L T H Q L L A G A F A G I M E H S VM F P I D A L K T R I Q S SQ L S K I S S A E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E Y A K S H L I D E - K D I Q T HQ - - P L K T A L SG T C A T I A A D A L MN P F D T I K Q R MQ L - - - N S K Q I Y K N EG F S A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A S K F F N P V N T Y N P L I H C L CG G L SG A T C A A I T T P L D C V K T V L Q V RG S E T V S L D V MK Q A D T F K K A A S A I L E V H GW KG FW RG L K P R V I A NM P A T A I SW T A Y E C A K H F LG E I D Y E A L P S S S P L S H Q L L A G A F A G I M E H S V L F P V D A I K T R I Q C L Q L S R I S A L EG S L A L WK G V Q S V I L G AG P A H A V Y F A T Y E F T K S H L I R P - E D I Q T HQ - - P F K T A I SG A T A T I M A D A L MN P F D T I K Q R MQ L - - - K S K S I Y Q K EG L K A F Y Y S Y P T T L L M N I P F A A CN F T I Y E S A T K Y L N P S D T Y N P F V H C T AG G I S G A A C A A L T T P L D C I K T V L Q T RG S K D I S S D I M R R A D T F I K A CDA I Y S T L G WK G F WR G L K P R V I A NM P A T A I SW T A Y E C A K H F LE E I D Y E S L P I N T P L A S Q L F A G A F A G V ME H T VM F P I D V L K T R I Q S T Q L T K I T T N EG F K S L WK G L S S V L L G AG P A H A V Y F A T Y E F T K S K L MT E - N A Y S S P R WN P L K I A L SG A S A T I L S D A L L N P F D T V K Q R MQ I - - - S T K L I Y Q K EG L R A F Y Y S Y P T T L AM N I P F V S L N F V I Y E T S T A F L N P S N K Y N P Y I H C L CG G I S G A T C A A L T T P L D C I K T V L Q V RG S N N I S E P I L K N A D T F A K A S R A I Y K L NG Y R G F L K G L K P R V I A NM P A T A I SW T A Y E C A K H F FE E I D Y E A L P S H A P L R S Q L L A G A F A G I M E H S L M F P I D A L K T R V Q A AQ I S K I S S V E G S MA L W KG VQ S V I L G A G P A H A I Y F A T Y E F C K A R L I S P - E DMQ T HQ - - P I K T A L SG T I A T I A A D A L MN P F D T I K Q R L Q L - - - D T K Q I Y Q N EG F A A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A S K F F N PQ N S Y N P L I H C L CG G I S G A T C A A L T T P L D C I K T V L Q V RG S E T V S I G I M RDA N T F G R A S R A I L E V H GW KG FW RG L K P R I V A N I P A T A I SW T A Y E C A K H F LE E I D Y E A L P S T A P L R H Q L MA G A F A G I M E H S V L F P I D A L K T RM Q S AQ I T R I S T A E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E WA K T S L I N P - E D I Q T I Q - - P L R V A A S G A L A T I A A D A L MN P F D T I K Q R I Q L - - - K A S R I Y KG EG L S A F Y T S Y P T T L AM N I P F A A F N F M I Y D T T T K V L N P T N T Y N P F V H C F CG G L SG A L C A A I T T P L D C I K T V L Q V RG S D S V S T D I L K R A D T F N K A A R A I F Q L Y GW KG F L RG L N P R V I S F I P A T A I SW T S Y E M A K H F LE E I D Y E A L P D S A P L S H Q L L A G A F A G I M E H S VM F P I D A L K T R I Q S SQ MA K I S T A E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E Y T K K Y L I D E - K D MQ T H Q - - P L K T A L S G T V A T I A A D A L MN P F D T L K Q R MQ L - - - N T K Q I Y K N EG F S A F Y Y S Y P T T L AM N I P F A A F N F M I Y E S A T K F F N P T ND Y N P L V H C L SG G L SG A T C A A I T T P L D C I K T V L Q V RG S E S V S L Q V M K E A N T F Q K A T K A I Y Q V HG A K G F WR G L Q P R V F A NM P A T A I AW T A Y E C A K H F LE E I D Y E A L P P C A P L HH Q L L A G A F A G I M E H S V L F P V D A I K T R I Q S K Q I S K I T T A EG S L A L WK G V Q S V I L G AG P A H A V Y F A T Y E F S K S K L I D P - Q D MH T H Q - - P I K T A I S GM A A T T V A D A L MN P F D V I K Q R MQ L - - - N T K N I Y H K EG F A A F Y Y S Y P T T L VM N I P F A A F N F A I Y E S A T K F MN P S N E Y N P F I H C I SG G L SG A T C A A I T T P L D C I K T V L Q V RG S E T V S N E I MK Q A N T F Q R A A S A I Y K I HG WK G F L R G L K P R V I A NM P A T A I SW T S Y E C A K H F LE E I D Y E A L P D S A S L S S Q L MA G A F A G I M E H F VM F P F D A L K T R I Q S K Q I S K I T T T E G S L A L W KG VQ SM I L G A G P A H A V Y F S T Y E Y M K K T L I DQ - K DMQ T HQ - - P L K T A L SG A T A T I A S D A L MN P F D T I K Q R MQ L - - - S T K N I Y H K EG L R A F Y Y S Y P T T I AM N I P F V S L N F V I Y E S S T K L F N P T N E Y N P L V H C L CG G L SG A T C A A I T T P L D C I K T V L Q V RG S K S V S L E V M K K A N T F R K A A D A I Y H V H GW KG F L R G I K P R I I A N V P A T A I SW T A Y E C A K H F LI P I E Y E S MP N E S P L H Y QM V A G A F A G I M E H S VM F P V D T I K T K I Q A G S L Y N V I K L E G A S S L WK G I Q P I L L G AG P A H A V Y F G A Y E Y L K T V L I D E - ND T S K Y H - - P L K V A L SG F V A T V A S D A V MT P I D T I K Q R MQ L - - - E T K S I S K N EG L K A F F Y S Y P T T V AM D V P F S I L N F V I Y D S SM Q F F N P S H I Y N P Y I H C G C G A L S G G I A A I V T T P L D C I K T V L Q V RG S K K I S MQ A F K E A D S F S K A A K A I Y T T Y GW T G F F R G L R P R V V A N V P A T A I SW S S Y E L A K H L L

Rate s

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

Klac/1-275Zrou/1-272Lwal/1-272Lthe/1-272Ecym/1-272Tdel/1-272Egos/1-272Lklu/1-272

Q E I D Y E A L P D T A P L S Y Q L I A G A F A G I M E H S I M F P I D A L K T RM Q A Q Q I S R I S S T EG S L A L WR G V Q S MV MG AG P A H A V Y F A T Y E F C K EQ L I D A - K D F N T HQ - - P L K T A V S G V A A T V A A D A L MN P F D T I K Q R L Q L Q S K S A F N I Y K N EG PM A F F Y S Y P T T L AM N I P F A A L N F V I Y E S S T K F F N P T N A Y N PW I H C L CG G I A G A T C A A V T T P L D C I K T V L Q I R G S D T V H V E S F K T A N T F K K A A Q A I WQ S YG WK G F WR G L Q P R V I S N I P A T A I SW T S Y E F A K H L LE E I D Y E S L P P D A P L Y S Q L MA G A F A G I M E H S VM F P I D A L K T R I Q S A Q I S K I S T ME G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E F T K SQ L I D R - R D Y Q T HQ - - P L K T A L SG T A A T V A A D F L MN P F D T I K Q R MQ L - - - N A K G I Y Q K EG L A A F Y Y S Y P T T I VM N I P F A A MN F V I Y E S S T K I F N P S N G Y N P L V H C L CG G I S G A A C A A I T T P L D C I K T V L Q V RG S E S V S H E V L R K A D T F T K A T K A I Y Q L RG L K G F L R G L K P R I I A NM P A T A I SW T A Y E C A K H F LE E I D Y E S L P T N A P L T H Q L A A G A F A G I M E H S I M F P I D A I K T RM Q A Q Q I A R I S T T E G S MA L W KG VQ S V I L G A G P A H A V Y F A T Y E MC K S Y L I D P - Q D F Q T H Q - - P L K T A A S G I A A T V A A D L L MN P F D T I K Q R MQ L - - - R A S R I Y R N E G L A A F F Y S Y P T T I AM N I P F A A F N F A I Y E S A T K F F N P E N T Y N P L I H C L CG G I S G A T C A A I T T P L D C I K T V L Q V RG S E S V V D P L F R Q A D T F S R A A S A I S K V Y GW SG FW RG L K P R I I S N M P A T A I SW T A Y E C A K H T LE E I D Y E A L P S S A P L T H Q L A A G A F A G I M E H S I M F P I D A I K T RM Q A Q Q I A R I S T T E G S MA L W KG VQ S V I L G A G P A H A V Y F A T Y E MC KG Y L I D P - Q D F Q T H Q - - P L K T A A S G V A A T I A A D ML MN P F D T I K Q R MQ L - - - R A S R I Y R N E G L A A F F Y S Y P T T I AM N I P F A A F N F V I Y E S S T K L MN P NN S Y N P L I H C L CG G L SG A T C A A I T T P L D C I K T V L Q I R G S E S V V H P L F R S A D T F S K A A S A I F K I YG WS G F WR G L K P R I I S N M P A T A I SW T A Y E C A K H F LQ E I D Y E S L P E S A P L G Y Q L T A G A F A G I M E H S I M F P I D A I K T R I Q A A Y I A K I S T T E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E V C K F N L I N A - E D MQ T H Q - - P L K T A L S G T A A T I A A D A L MN P F D T I K Q R L Q L - - - H A L R I Y Q N EG Y A A F F Y S Y P T T I AM N I P F A A L N F V I Y E S S I K F V N P S N S Y S P W I H C L CG G I S G A T C A A I T T P L D C V K T V L Q V RG S D T V Q S Q I F R R A D T F K K A A S A I Y Q T YG WK G F WR G L K P R V V S N M P A T A I SW T T Y E F A K H F LG E I D Y E S L P A N A P L A S Q L MA G A F A G I M E H S VM F P I D A L K T R I Q S S Q I S K I S T A E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E Y T K S Q L I D P - Q D YQ T H Q - - P L K T A L S G T A A T I A A D A L MN P F D T I K Q R MQ L - - - S A K Q I Y Q K EG I M A F Y Y S Y P T T I AM N I P F A A F N F V I Y E S S T K V F N P S ND Y N P L I H C L CG G I S G A T C A A V T T P L D C I K T V L Q V RG S E T V S L P I F R N A D T F S K A T K A V Y K I H GW NG FW RG L K P R V I A NM P A T A I SW T A Y E C A K H F FP E L D Y E A L P E N A P L V Y Q L A A G A F A G I M E H S I M F P I D A I K T RM Q A AQ I A K I S T T E G S L A L W KG VQ S V V L G A G P A H A V Y F A T Y E MC K S R L I D P - E D R Q T HQ - - P L K T A L SG T L A T V A A D A L MN P F D T I K Q R L Q L - - - H A V R MY Q R E G I A A F F Y S Y P T T I AM N I P F A A L N F V I Y E S S T K I F N P S NN Y N P W I H C L CG G I S G A T C A A I T T P L D C V K T V L Q I R G A D S VQ SQ L F K E A D T F R K A A S A I H K T YG WS G F F R G L K P R I I S N M P A T A I SW T S Y E F A K H L LE E I D Y E S L P A N A P L S H Q L A A G A F A G I M E H S I M F P I D A I K T RM Q T Q T I A R I S T T E G S L A L W KG VQ S V I L G A G P A H A V Y F A T Y E MC KG Y L I D P - Q D YQ T H Q - - P L K T A L S G T A A T V A A D A L MN P F D T I K Q R MQ L - - - Q A S N I Y RQ EG L A A F F Y S Y P T T I AM N I P F A A F N F V I Y E S S T K L L N P S NN Y N P L I H C MC G G I A G A A C A A V T T P L D C I K T V L Q V RG S E T V V D P L F K Q A D T F R K A A R A I YQ V Y G S KG FW RG L K P R I I S N M P A T A I SW T A Y E C A K H F L

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10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460

Scer2/1-401Kafr2/1-361Tbla2/1-382Tpha2/1-379Skud2/1-401Ndai2/1-392Knag2/1-346Vpol2/1-378Ncas2/1-375Suva2/1-401Smik2/1-400Cgla2/1-390

P L N L T C L F L G D T NA G K S T L L G H L L Y D L N E I S MS SM R E L Q K K S S N - - - - - - - - - - L D P S S S N S F K V I L DN T K T E R E - - - NG F S MF K K V I Q V E N - - - - - D L L - P P S S T L T L I D T PG S I K Y F N K - - - - E T L N S I L T F D P E V Y V L V I D CN Y D S WE K - - S L D G P NN Q I Y E I L K V I S Y L N K N - - S A C K K H L I I L L N K A D L I SW DK H R L EM I Q S E L N Y V L K E N F Q W - - - T D - A E F Q F I P C S G L L G S N L N K T F V G T I L Q S S V L Q P - - - - - - - - I A E I N Y V S L K V L I N SG Y I Q S GQ T I E I H T Q Y E D F H Y Y G I V S R MK N S - - - KQ I I S V G L N P D I L E V L V K I H N - - T E D F T K KQ F H I R KG D I I I H S R K T N T L Q T HA L S D - - P V D L G S E L L L Y HN L - - - - T H N A V K L V K I L G T ND I S - - - - - - - - - - - - I N P NQ S L I V E V E I I E P D F - A L N V I - - - D S K - - Y I T NN I V L T S I D H K V I A V GN P R L N V I M CG H S K S G K S T I L G R I L HD L R C I S I E K I R D L K R Q I E K - - - - - - - - R D P L A D S N L Y L SW I T N T S DD E R R - - - L S K T L H V H K - - - - K - - - - - E F N I S D K H T F R MV D I P S D R K F S S - - - - - S V I P Y L - - WN S D V A I L V I D C S I DG F E I G F S L DG Q T L E - - - - - - - - H A KM I K - S E P H I K N V I I L M N K MD T I DW Y E T R Y I E I K N E L I A F - L L N I G F - - - QG - DQ L SW I P CD G T T G HG I T QQ L V L Q I S Q T E L S K Q - - - - - - - - - - - - - - - L I YG N I L S G S I Q PG E T I T I Y P S R Q S V - - - - T I S K I M S L - - - K E E R R I S T VG E S V V L K I L N G D K - L N G - - - - - - - V I VG D Y - - - - - - V S G V L K MF DN L - - D L A V G S RM VM I K G F - - - - - - - - - - - - - - - - - - - - - - - - - - F D R K V K I L K K V D K F T F E V E L I D K T H C S MP F F P E E DD K - - - L D L NN V I L L Q NN R I MG S AI K S I N I L I MG D T G S G K S T L MG R L I Q D F N L L D Y D T I R R I K WD S E K - - - - - - - - - - - H L K S T N Y L A WL V D K S K N E R Q - - - SG S S L F P H T I E I H K R N L F L DN E - L N S T A Y N I N E I PG N K S Y V S - - - - - K A R TW V - - F N S N L I V I C I D CN I E S F E K G F S L DG Q T RD - - - - - - - - H I L L T K - - T MG I K Y L I F AM NK MD T V EW D E V R FM S I K N E L T V F - L K D I Q F - - - K H V E N V S W I P I SG R T G E G V HNV F I F Q I L K R KM H E S - - - - - - - - - - D K HA T L I T G K V I NG T I Q P G E N I T V Y P S K Q S L - - - - I V D R I S S T - - - - - - L P I A L E S D S V T L K V L NA D - - Y Q S - - - - - - - I S I G N I - - - - - - A T S V K T L Y MN E - - H L Q VG D L MT V Y S G F - - - - S R YG V K I T E I L S - - - - - - - N D I I P V N K S H I G S D E I A T F S L D V I E P D H - D I P I L I H L A N N - - P MA L N E V I F R R QG A F V A I GK P H C T F V V L G H V D AG K S T L MG R L L Y D I G A V D S N L I R K L K R E S E S - - - - - - - - - - - I G K G S F H L A WV MDQ T N E E R A - - - RG V T V S I C T - - - - S - - - - - D F E - T D K S R F T I V D A PG H RD F V P - - - - - S A I S G I - - SQ A D V A I I T I D C G T DA F E SG F N L D GQ T K E - - - - - - - - H S L L A K - - S L G V K R L I V A MN K L D S V D WF EG R F NQ I Q S E L K I F - F D D I G F - - - K E - E Q L DW V P V S G L T G EG V H K I F L F S I L E V S E S N K - - - - - - - - - - - H E T G V I SG R V E S G T I Q PG E T I T I Y P S E Q S V - - - - I V D K I T V G - - - DG KQ K I A I K ND F V S I R I R N A F - - I E D - - - - - - - I Q P G D L - - - - - - C A S V L T F K L D R - - P V L PG T S FM MF RG V - - - - C E Q P A R I G K L I S S V D K K D P T K I L K K K I R H L A T NQ G A I V E I L L T E K K R - W I P L L S Y NDN K - - - - H L G R I V L R K D G R T I G A GP L N S T C L F F G V T T S G K T T L L G H L L Y E L N E I S I S S I R E L Q K K V N S - - - - - - - - - - L S L P A S NH F K I I L DN T K L E R E - - - NG F S M C R K I I Q I E N - - - - - D L L - P P S S S L T L I D T P G N I K Y F E K - - - - E T I N S I L T F N P D I F T L V I D CD Y D S WE K - - S L D G L NN Q I Y E V L R I I S Y L N E N - - S A Y K K Q L I V L L N K A D L I SW DK Q R L E M I Q S E L N Y L L T E T F Q W - - - K N - T Q F Q F I P C S G I L G S N L NNA F I G I I L Q N P I L Q P - - - - - - - - T A E HNC V S L K V L I K SG Y I Q S GQ T I E I H T H Y E E V CH Y G I I T KM T K P - - - K L T L P I G V H S D I L E V F V K I H S - - T E E L T K KQ A H I R K ND L V I S S R K A S I S Q T H L L N D - - P V N L G S E L I L Y HD L - - - - MC K T V K L V K I L G T NA T S - - - - - - - - - - - - I I S NQ S I I V E V E I T E P N F - A L N V I - - - N S E - - Y I T N Y I V L T T T DH K V V A V GN P F L N V I L L G H S K S G K S T I L G R L L K D L K L L S I E E I R S T K F Q L E R - - - - - - - - QQ E K N P N S L Y L A K I G E T K L S K D K - - - H Y - - - - - - - - - - - R - - - - - Q I K - H E ND T F N I F D I P I F R Q S R K NNN F Q S L I A T I - - NQ C S V A I L T I D CN T DQ F E S N F N I DG E L I Q - - - - - - - - L I Y L L K F S A R K L N K I V I L L N KM D S I DWY HDR F I E I K T E L S L F - F K D L N L CDD P D - N E I V W I P T SG L Y GQ G I V D R F I F H I E N A S K I N S R S E K L K - - - - - Q D E C F V T G H V H S G S I Q I G E S MT I F P S K I S V - - - - T I E S I T C L D T L T RQ S K I A V A DD Y V I L R V S K L F D F Q ND - - - - - - - V N VG D F - - - - - - A T S I T I F P N I E K E R L A I G S S F V L I R G N GG E N S T Y K V R V S K I L S L S S N T Y - - - - - - - - - - - - - Q N T T I G F D V E L - E S E S - P I P M I S V K D NG S D F T C F N E F I L EQ N E R I YG L GV P A T N V V V L G H T A AG K T T L L G R L L Q D L HA V G I E D V RD A K R Q C E R - - - - - - - - - R K ME D T N A Y L VW L V E QGG D S G S L Y HH A V T - - - - - - - - - - - - - - - - - - - Y R DH S Y N F T V A P S R G ND T Q - - - - - R L V S E I - - P HM DM A I L V V D C S T D G F E S G F N L T G H T I E - - - - - - - - H A L L A K - - Y SG V K K L I V AM NK L D T V DW Y V E R Y R E V E A Q L R L F - L T E I G Y - - - S D - DQ V CW V P CD S T S G A T V A H R L L L C V S Q V Q E - - - - - - - - - - - - - - G K Q L C V EG WV GQG H V A R G E T L V S V T ND S L F - - - - T V A R L Q V R - - - Q S DA A I A L PG Q Y V K L Q L K V T RG G - - - - - - - - - - V S V N E Y - - - - - - L T C A Q L L E V F K - C D I R VG ME L K L L R E F - - - - Q R V T V R V C E I - - - - - - - - - - - - - - - - - - - - - - S D RG F I R C E V T DD S G - G V T V F R R L E N R - - - - - - - - F Y V T C E G R T V G V CK P H C T F V V L G H V D AG K S T L MG R L L Y D I G A V D S N H I R K L K R E S E R - - - - - - - - - - - I G K G S F H L A WV MDQ T T E E R E - - - RG V T V S I C T - - - - S - - - - - D F E - T D K I R F T V V D A P G H RD F V P - - - - - N A I A G I - - SQ A D V A V L T I D C G T DA F E HG F N L D GQ T K E - - - - - - - - H A L L A R - - S I G V T H V V V A MN KM D S V E WD K S R FM D I K S E L S I F - F E D I G F - - - K E - T Q I T WV P C SG L S G E G V H K T S Y F - L L E V I S S N K - - - - - - - - - - - H E E A V V S G R V E S G S I Q PG E T I T F Y P S E Q S V - - - - L V D K I I AG - - - S S Q I P A A F K ND F I T L K L R Q A H - - A E D - - - - - - - I EG G D L - - - - - - A A S V L T F N L E R - - P M L PG T S MMM F R G V - - - - C E Q P AM V N K L V S L V D K HD P S K I I K K K V RH L G S NQ S A I V E I E L T E K K R - W I P L L S F K Q N R - - - - H L G R V V L R K DG K T I G AGK P Y L N F VM L G N E S A G K S T I I G R L L E D S G L V R I D E I R S V K K E L E K - - - - - - - - - S K L N A EM L Y L S K L M E K K M S S T F - - - - - - - - - - - - - - - - - - - - - - S L D - K N V S E F S A F D I PG D L K H L S - - - - - S S I K A I - - RQ C T T A I L T I D CN T DA F E S A F NMG S A T I Q - - - - - - - - H I Y L C K - - Q A N I DN I I I MM NK MD T I DW DQ G R Y F Q I K N E L Q S F - L S R L G F - - - K K - E Q F TW I P S S G L Y GQ G I V H S F F F S L T K K P R P A K - - - - L D V E D K R G D V Y T L MG E V L SG S I Q I G E SM T I Y P S K Q S V - - - - T V E K I S K V - - - N T E K S I A I E MDQ V I L T V S N L Y N - D K D - - - - - - - I R I S D V - - - - - - A A S I F I F E T N K K P S I G I G S R G S L Y RD G - - - - A V I P V K I K N L T S T Q E S A E T S D Q S Q S K - - - M P S N CN V N I E C - - - E T E R - P V V L L DH K NG K - - - - K C G N L V L Y H E E T I V A T GP L N L T C L F L G D T S S G K S T L V G H I L Y E L D E I S MA S I R D L K K S V D N - - - - - - - - - - F D A T T S NH F K I I V DN T K T E R E - - - SG F S MF K K T I Q ME N - - - - - S L L - P S S S S L T L I D T P G N S E F F N K - - - - E T I N S I L T F N S D V F T L V I D CN Y D S WE K - - S L D S P T N K I Y E I L R I I S Y L NM N - - S T Y K K Q L I V L L N K A D L I SW DK L R L E M I Q S E L Q Y ML T E T F QW - - - E T - T Q L Q F I P C S G L SG S N L N G A F T G I V L HN P MH P P - - - - - - - - S A E S N Y I S L K V F I K SG Y I Q S GQ T I E I H T P Y E E S S Y Y G I I T KM I K P - - - K L T S S V G V H S D I L E I H V K I H H - - T N E F T K K Q I H I H K ND L I I S P R K A N T L Q T C L F S N - - P I V V D S E L V L Y HD L - - - - I Y T T I K L MK I V G T N A S S - - - - - - - - - - - - I N P NQ S L I I E V E I M A P D F - A L N V I - - - S S K - - Y V T ND V V L T S T DH K I V A V GP L D L T C L L L G D A N SG K S T L L G H L L Y E L N E L S I S SM R E L Q K K A S N - - - - - - - - - - L D P L L S NH F K I I L DN T K I E R E - - - NG F S MF K K MV K I K N - - - - - N F L - - P S S S L T L I D T P G N S E Y F N K - - - - E T I N S I L T F N P D V F A L V I D CN Y E F WE K - - S L D G P NN R I Y E I L R V I S Y L YQ K - - S T R K K H L I V L L N K A D L I SW DK Q R L E I I Q S E L N Y L L T E H F Q W - - - K A - A Q F Q F I P C S G L L G S N L NN S F I G I I L Q S P V T Q P - - - - - - - - S A E A NC L S L K V L I K SG Y I Q S GQ T I E I H T Q F KG L Q Y Y G I V T E MR K A - - - K Q I L A V G V N S D I L E V L V K I H N - - T E D FM K N R T L I R K G D SM I L S R R A NN L Q T HM L N D - - R I D L G S E L I L Y HN L - - - - T Y S V A K L L K I F G T ND T S - - - - - - - - - - - - I G P YQ S L M V E V E I V E P N F - D L T V I - - - D S K - - Y V T DN I V L T S I D H K V I A V GA T S F Q V PM F G L P G S G K S T I L GQ L S F H L G L T T R E D I R V I K T DM E R MH F R R I D K L D T R L L T T S P F CW V V D E T T E E R Q - - - F G H S NN I K P - - - - L - - - - - H I K - Y ND T D I I I N E V P G S F D L C S - - - - - T I E T K R - - Y V G N S V I I V V S A E L G D Y E A N F D M K K R L I E - - - - - - - - K L I Y C N - - G V G V R R I L T I I N KM D L I DWD MDR Y T V MK H E L E L I - Y QQ VG I - - - D I - L K CD F I G T S A I T G E A L T ND L V N SQ EQ T L L D S E K L I A I Q T G N K H E QQ L S V P F Y I E R G L I L KG Q K L K I N R T G L E V - - - - V V K S I K E S - - L R K K V N V A V V GQ S V D L L F E P S E A I T S S - - - - - - - - N I N N I - - - - - - L T S C WS L K N C S - - KQ L D F N N I L I F C - - - - - - - - - - - - VG R F L N I C I S N V EQ T E NN E K - - - L NN F Q Y I F C R A C L K S S E N - M L A F E PG Y E S A - - - - - - K S F V L F F E N R A I G F G

Rate s

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460

Scer1/1-379Suva1/1-379Ndai1/1-379Kafr1/1-381Tbla1/1-379Smik1/1-379Knag1/1-380Ncas1/1-379Skud1/1-379Cgla1/1-379

L P H L S F V V L G H V D AG K S T L MG R L L Y D L N I V N Q S Q L R K L Q R E S E T - - - - - - - - - - - MG K S S F K F AW I M DQ T N E E R E - - - RG V T V S I C T - - - - S - - - - - H F S - T H R A N F T I V D A P G H RD F V P - - - - - N A I MG I - - SQ A DMA I L C V D C S T N A F E S G F D L DG Q T K E - - - - - - - - HM L L A S - - S L G I H N L I I AM NK MDNV DW SQ Q R F E E I K S K L L P Y - L V D I G F - - - F E - D N I NW V P I S G F SG EG V Y K I F L F S V L E I I P S K K - - - - - - - - - T S ND L A L V S G K L E SG S I Q P G E S L T I Y P S E Q S C - - - - I V D K I Q V G - - - SQ Q T D V A I KG D F V T L K L R K A Y - - P E D - - - - - - - I Q N G D L - - - - - - A A S V T T F D MN R - - P L L P G T P F I L F I G V - - - - K E Q P A R I K R L I S F I D K G N - - T A S K K K I R H L G S KQ RA F V E I E L I E V K R - W I P L L T A H E N D - - - - R L G R V V L R K DG R T I A AGL P H L S F V V L G H V D AG K S T L MG R L L Y D L K I V N Q V Q L R K L Q K E S E T - - - - - - - - - - - L G K A S F K F AW I M DQ T N E E R E - - - RG V T V S I C T - - - - S - - - - - H F S - T E K A N F T I V D A PG H RD F V P - - - - - N A I MG I - - SQ A DMA I L C V D CN T NA F E KG F D L D GQ T K E - - - - - - - - HM L L A S - - S L G I Q N L I I AM NK MD S V N WS QQ R F E E I K S K L L P Y - L V D I G F - - - SG - D N I SW V P I S G F SG EG V H K I F L F S I L E I I P L K K - - - - - - - - - T NN E L A L V SG K L E S G S I Q PG E S L T I Y P S E Q S C - - - - I V D K I Q V G - - - SQ Q T D V A I KG D F V T L R L R K A Y - - P E D - - - - - - - I Q D G D L - - - - - - A A S V T T F E MN R - - P L L P G T P F I L F I G V - - - - K E Q P A K I K R L I S L I D K DN - - N V I K K K V RH L G S KQ RA L V E I E L I E V K R - W I P L L T A S E N D - - - - R L S R V V L R K DG R T I A AGK P H L S F V V L G H V D AG K S T L MG R L L Y D I G A V N S N Q I R K L K K E S E Q - - - - - - - - - - - I G K G S F H L A WV MDQ T T E E R E - - - RG V T V S I C T - - - - S - - - - - D F E - T E K A N F T I V D A PG H RD F V P - - - - - N A I A G V - - SQ A D V A V L S I D C G T DA F E SG F N L D GQ T K E - - - - - - - - H T L L A K - - S MD V NNV I V AM NK MD S V N WS L G R Y L E I Q G K L S H Y - F E E V G F - - - S E - DQ I K WV P C SG F S G E G V Y K I F L F S I L E V I P S K K - - - - - - - - - - - N E E A I I T G K L G S G S I Q PG E T I T L Y P S E Q S V - - - - V V D K I L MG - - - K E Q V P I A I KG D F V T L K L R HA H - - P E D - - - - - - - I QGG D L - - - - - - G A S V L T F K MD R - - P L L P G T S F ML F R G V - - - - C E Q P A R V S K L V S T V D K RD P S K I I K K K I R H L S S N Q A A I I E V E L T E R R R - W I P ML T F N E N K - - - - H L G R V V L R K DG R T I A AGK P Q L S F V V L G H V D AG K S T L MG R L L Y D I G A V D T K HM R K L K K E S E S - - - - - - - - - - - I G K G S F H L A WV MDQ T T E E R E - - - RG V T V S I C T - - - - S - - - - - H F E - T E K A K F T I V D A P G H RD F V P - - - - - N A I A G V - - SQ A D I A V L T I D C G I G A F E SG F S L D GQ T K E - - - - - - - - H T L L A R - - S MD I S N I L V VM NK L D S V QW S E E R F N E I K T K L S D F L L ND V G F - - - K K - E Q I S WV P C SG F S G E G V Y K I F L F S V ME V I T T K K - - - - - - - - - - - D D E C F I S G R V E S G T I Q PG E S I T I F P S E Q S V - - - - L V D K I L L N - - V N S H L N V A S K G D F V T L K L R N S H - - P A D - - - - - - - I E N G D L - - - - - - C A S V L T F QM S R - - P L L P G T P L ML F R G V - - - - C E H P A R I N K L I S V L D K ND P T K V L K K K V K H I S S N Q V A I V E V E L T E R K R - WL PM L T F D E N K - - - - H L G R I I L R K D G R T I A T GK P H C S F V V L G H V D AG K S T L MG R L L Y D I G A V D NQ L I R K L K K E S E S - - - - - - - - - - - I G K G S F H L A WV MDQ T K E E R E - - - RG V T V S I C T - - - - S - - - - - D F E - T D K V K F T I V D A P G H RD F V P - - - - - N A I Q G I - - SQ A D VG V L S I D C G T DA F E SG F N L D GQ T K E - - - - - - - - H A L L A R - - S MG V H Y L I I VM N K MD S V N WS E E R F Q K V K A D L S V F - F D E I G F - - - K E - NQ I E WV P V S G L SG AG V Y K I F L F A I T D V T T S K K - - - - - - - - - - - AG E A I I T G R V E A G S I Q PG E T I T I Y P S E Q S V - - - - L V D K I T SG - - - N E D V P I A I K ND F V T L K L R HA F - - P E D - - - - - - - I Q A G D L - - - - - - G A Y V L T F K L G R - - P I L PG T P VM L F KG V - - - - R E Q P A R I N K L V S L V D K HD V E K I I K K K V RH L S S GQ A A I V E V E L V E K K R - W I P I L T F QQ NK - - - - H L G R V I L R K D G K T I A A GL P H L S F V V L G H V D AG K S T L MG R L L Y D L K I V N Q S Q L R K L Q K E S E T - - - - - - - - - - - MG K S S F K F AW I M DQ T H E E R E - - - RG V T V S I C T - - - - S - - - - - H F S - T Q R A S F T I V D A P G H RD F V P - - - - - N A I MG I - - SQ A DMA I L C V D C S A N A F E S G F D L DG Q T K E - - - - - - - - HM L L A S - - S F G I H N L I I AM NK MDNV DW SQ Q R F E E I K S K L L P Y - L V D I G F - - - C E - D N I SW V P I S G F SG EG V Y K I F L F S V L E I I P L K K - - - - - - - - - T S N E L A L I SG K L E S G S I Q PG E S L T I Y P S E Q S C - - - - I V D K I Q V G - - - SQ Q T D V A I KG D F V T L K L R K A Y - - P E D - - - - - - - I Q N G D L - - - - - - A A S V T T F E MN R - - P L L P G T P F I L F I G V - - - - K E Q P A R I K R L I S L I D K DN - - N T I K K K V RH L G S KQ RA L V E I E L I E V K R - W I P L L T A S E N D - - - - R L G R A V L R K DG R T I A AGK P H L S F V V L G H V D SG K S T L MG R V L Y D VG V V S QQQ L H K L Q R E S E K - - - - - - - - - - - I G K S S F H L AW VM DQ T S E E R E - - - RG V T V S I C T - - - - S - - - - - E F E - T K GG A F T I V D A P G H RD F V P - - - - - N T I G G V - - YQ S D V A L L T I D CN V NG F E SG F D L D GQ T K E - - - - - - - - H T L L A R - - S MD I R T V V V A MN KM D T V N WS Q K R F E E I R G K L T P Y - F R E I G F - - - Q D - DQ I K WV P L SG MT G E G V H K I F L F S V M E S D P S S K - - - - - - - - - - K ND E A I I T G K L E SG S I Q P G E T I N I Y P S E Q S A - - - - L V D K I M F G - - - K DD K P V A V K G E F V S L R L RQ A F - - P K E - - - - - - - I E N G D I - - - - - - A A A V L T F K MN R - - P L L P G T S V ML F H G V - - - - Y E Q P A R I S K L I A L L D K A D P T K V Q K R K V K H L P S N Q I A V V E V E L T D K R Q - WL P V L P F D K N K - - - - H L S R I I L R K D G R S I A T GK P H L S F V V L G H V D AG K S T L MG R L L Y D I G A V D T N H I R K L K K E S E R - - - - - - - - - - - I G K G S F A L A WV MDQ T T E E R E - - - RG V T V S I C T - - - - S - - - - - D F D - T P K A N F T I V D A PG H RD F V P - - - - - N A I A G V - - SQ A D V A V L S I D C G T DA F E SG F N L D GQ T K E - - - - - - - - H T L L A K - - S MD V N N I V V A MN KM D S V N WS Q E R F MD I K Y K L S A F - F E E V G F - - - H E - DQ I K WV P V S G F S GQG V F K I F L F S I L E V I P S K K - - - - - - - - - - - N E E A I I SG K L G S G S I Q PG E T I T V Y P S E Q S C - - - - V V D K I L K G - - - K N Q V G I A I K G D F V T L K L R HA H - - A E D - - - - - - - I QGG D I - - - - - - A A S V L T F K MD R - - P L L P G T S F ML F R G V - - - - C E Q P A R I S K L V S T V D K HN P E K I L K K K I R H L G S NQ A A I V E I E L T E R K R - W I PM L T F K E N R - - - - H L G R V V L R K DG R T I A AGL P H L S F V V L G H V D AG K S T L MG R L L Y D L K I V N Q S Q L R K L Q R E S E T - - - - - - - - - - - MG K S S F K F AW I M DQ T S E E R E - - - RG V T V S I C T - - - - S - - - - - H F S - T E R A N F T I V D A P G H RD F V P - - - - - N A I MG I - - SQ A DMA I L C V D C S I N A F E S G F D L DG Q T K E - - - - - - - - HM L L A S - - S L G I H N L I I AM NK MDNV NW SQ Q R F E E I K S K L L P Y - L I D I G F - - - C K - D N I CW V P I S G F SG EG V H K I F L F S V L E I I P L K K - - - - - - - - - T N S E L A L I S G K L E SG S I Q P G E S L T I Y P S E Q S C - - - - I V D K I Q V G - - - SQ Q T G V A I KG D F V T L K L R K A Y - - P E D - - - - - - - I Q N G D L - - - - - - A A S V T T F E MN R - - P L L P G S P F I L F I G I - - - - K E Q P A R I K K L I S L I D K S S - - N V L K K K V RH L G S KQ RA F V E I E L I E V K R - W I P L L T A S E N D - - - - R L G R V V L R K DG R T I A AGK P H V S F V V L G H V D AG K S T L MG R V L Q D VG A V D K T Y I R K L K K E S E N - - - - - - - - - - - I G K G S F H L A WV MDQ T K E E R E - - - RG V T V S I C T - - - - S - - - - - D F D - T P NA N F T I V D A P G H RD F V P - - - - - N A I A G V - - SQ A D V A I L S I D C G I N A F E S G F N L DG Q T K E - - - - - - - - H A L L A K - - S MD I K R V I V AM NK MD T V QW S H E R Y E D I K Q K L V K F - L Y D I G F - - - T D - NQ L L W V P C SG F S G E G V Y K I F I F S I L E S S A S N K - - - - - - - - - - - N E E A I I SG K V E S G T I Q PG E T L T I Y P S E Q S V - - - - T V D K I I MG - - - K E Q V P I A V K G E F V T I K L R HA H - - P E D - - - - - - - I QGG D I - - - - - - A A S V L T F K MD R - - P L L P G T P F ML F R G V - - - - C E Q P A R I S K L I S L V D K DD F E T V I K K K V RH L S S H Q A A I V E I E L T E K R R - W I P L L T F S Q N E - - - - H I G R I V C R K DG R T I A T G

Rate s

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460

Lwal/1-379Egos/1-379Lthe/1-379Tdel/1-379Klac/1-379Zrou/1-379Lklu/1-379

K P H L S F V V L G H V D AG K S T L MG R L L F D V G A V DN K L I R K L K R E S E L - - - - - - - - - - - AG K S S F H L AW VM DQ T S E E R A - - - RG V T V D I C T - - - - S - - - - - D F K - T S K A T F T I V D A PG H RD F V P - - - - - N A I A G V - - SQ V D V A V L S I D C S T D A F E S G F N L DG Q T K E - - - - - - - - H T L L A R - - S L G V R H I I V AM NK MD S V D WY EG R F E D I K F E L R N F - F E D I G I - - - K E - E Q L SW V P C SG L S G E G V Y E T F V F S VM E V S P G N K - - - - - - - - - - - A N E A M I F G R V E S G H I Q SG E T I T I Y P S E Q S V - - - - L V DQ I S SG - - - N DH T P V A V K G D F V S L K L R NA F - - Y E D - - - - - - - I Q S G D I - - - - - - A A I V L T F K L D R - - P L L P G T S F M F F R G S - - - - C E Q P A R V K R L V S T V D K S N S E K I L K K K V K H L G S N Q A A I V E I E L T E K K R - R V P ML P F D E N K - - - - H L A R I V L R K E G R T I A A GK P H MS F V V L G H V D AG K S T L MG R L L Y D V G A V D T K L I RQ L K R E S E L - - - - - - - - - - - AG KG S F H L AW VM DQ T N E E R A - - - RG V T V D I C T - - - - S - - - - - E F E - T A K S T F T V I D A P G H RD F V P - - - - - N A V T G V - - N L A D V A I V T I D C A T D A F E S G F N L DG Q T R E - - - - - - - - H I I L A R - - S L G V K H I I L A MN KM D T V E WH EG R F K A I R L E L L S F - L E D I G F - - - K E - P Q T SW V P C SG L T G E G V YQ K F L F S L L D T H S S G K - - - - - - - - - - - N N E V Y V S G K V L GG S V Q P G E T I T I Y P S E Q T V - - - - V V D N I Y V G - - - D K R V G A A V Q ND F V T L K L K NA N - - F E D - - - - - - - I KG G D L - - - - - - A A I V L T F K V D K - - P V L PG A S F I L F R GG - - - - AQ C S A R I K S L HQ A V D KQ D P S K V L K K K V R H I G S NQ S A I V T I E L T D RG L - KM P L L T F D Q N K - - - - R F G R I L L R R D G K T I A A GK P H L S F V V L G H V D AG K S T L MG R L L Y D V G A V DN K L I R K L K K E S E M - - - - - - - - - - - I G K S S F H L AW VM DQ T S E E R N - - - RG V T V D I C T - - - - S - - - - - D F K - T K DA T F T I V D A PG H RD F V P - - - - - N A I A G V - - SQ V D V A V L S I D C S T D A F E S G F N L DG Q T K E - - - - - - - - H T L L A R - - S L G V R H I V V A MN KM D S V D WY EG R F E D I K F E L R N F - F E D I G I - - - K D - DQ L SW V P C SG L T G E G V YQ K F V F S I L E V S P G S K - - - - - - - - - - - A N E A V V S G R V E S G H I QGG E T I T V F P S E Q S V - - - - L V DQ I L T G - - - N E Q A P V A I K G D F V S L K L R NA F - - Y DD - - - - - - - I QGG D L - - - - - - A A V V L T F N L D R - - P L L P G T S F I L F R G S - - - - C E Q P A R V K K L V S V V D K S D P T K I L K K K V RH L G S KQ A A I I E I E L V E K K R - R V P ML T F G E N K - - - - H L G R I V L R K E G K T I A A GK P H C S F V V L G H V D AG K S T L MG R L L Y D I G A V D I S H I R K L K K E S E R - - - - - - - - - - - I G K G S F H L A WV MDQ T A E E R E - - - RG V T V S I C T - - - - S - - - - - D F E - T N A A R F T I V D A PG H RD F V P - - - - - S A I A G I - - SQ A D V A L L S I D CG T D A F E S G F N L DG Q T K E - - - - - - - - H T L L A R - - S MG V S R I V V A MN KM D T A D X X X X X X X X X X X X X X X X - X X X X X X - - - X X - X A I P C V P C SG L S G E G V Y K K F L F S I L E V I P S S K - - - - - - - - - - - N E E A I I T G K I E S G S I Q PG E S I T I Y P S GQG V - - - - L V D K I Q T G - - - N D K S R I A V K G D F V T L K L R HA Y - - P E D - - - - - - - I EG G D L - - - - - - G A S V L T F N ME R - - P L L P G T P F I L F R G V - - - - CQ Q P A R I S K L E C L V D K S D P S K V L K K K V K H L G S N K A A L V E V E L T E R K R - W I P T L K F S Q N K - - - - H L G R V L F R K D G R T I A A GK P H L N F V V L G H V D AG K S T L MG R L L Y D V G A V D T K L I R K L Q K E S E M - - - - - - - - - - - I G K S S F H L AW VM DQ T S E E R N - - - RG V T V D I C T - - - - S - - - - - D F A - T T K S S F T I V D A P G H RD F V P - - - - - N A I V G I - - SQ A D V A V L S V D CG T D A F E S G F N L DG Q T K E - - - - - - - - H A L L A R - - S L G I K H I V V A MN KM D S V S WY EG R F ND I R S E L A V F - F E E I G F - - - KG - ND V SW V P C SG L S G E G I F K T F L F N I L D V T P T S K - - - - - - - - - - - N T S A I I SG K V E S G T I Q PG E T I T I Y P S E Q S C - - - - V V D S I L C G - - - N D S V D I A L H G D F V Q L K L HNA F - - P E D - - - - - - - I QGG D L - - - - - - A S I V L T F R L D R - - P L L P G T S L ML F R G A - - - - T E Q P C R I K K L C C T V D K S N P S K V L K K K V K H L G SQ Q A A I V E I E L V E K K R - R I P M L T F E Q N K - - - - K L G R V V L R K EG R T I G A AK P H C S F V V I G H V D SG K S T L MG R V L Y D L G V V D I S H L R K L K R E S E I - - - - - - - - - - - VG K S S F H L AW VM DQ T P E E R E - - - RG V T V S V C T - - - - N - - - - - D F E - T P S T R F T I V D A P G H RD F V P - - - - - N A I A G I - - S E A D A A V L S I D C G T DA F E SG F N L D GQ T K E - - - - - - - - H T L L A R - - S L G VG H I I V AM NK MD T V DW YQ E R F E Q I R R E L S S F - F E T I G Y - - - R P - E Q I S W I P C SG L T G A N V V K R F L F S I T E V I S V N K - - - - - - - - - - - N E E V V V S G K V E S G S I Q PG E T L N I F P S E Q S V - - - - V V N R I T I D - - - N N E V P V A T KG D F A V L R L R NA F - - A E I - - - - - - - I E A G D L - - - - - - A A S V L T F QM S R - - P L L P G T P F M F F K G V - - - - N E Q P A R V S K L N S I I D K Q D P S K I I K K K V K H L G S N Q A A I F E I E L V E K E R - W I P F L T S T Q N R - - - - RM S R I V MR K E G R T I A A GK P H L N F V V L G H V D AG K S T L MG R L L Y D V G A V N Y K L I R K L K K E S E Q - - - - - - - - - - - AG KG S F H L AW VM DQ T S E E R D - - - RG V T V D I C T - - - - S - - - - - D F E - T D R A T F T I I D A PG H RD F V P - - - - - N A I T G I - - SQ A D A A V L T I D C C V DA F E SG F S L D GQ T K E - - - - - - - - H T L L A R - - S L G A R H I V V A MN KM DH E GW Y P T R F F D I K WE L E S F - F K D I G I - - - K K - E Q V SW V T C S G L SG EG V Y N I F L F S I L D V S P T S K - - - - - - - - - - - N N E V I V S G K V E A G S I Q PG E T I T I Y P S E Q S V - - - - L V D S I L SG - - - N D R V K I G V AG D F VM L K L R E A Y - - Y E D - - - - - - - I Q S G D L - - - - - - A T T V L T F K L D R - - P L L P G T S F ML F R GG - - - - C E Q P A R I K K L V S I V C K K D P K K I L K K K V K H L G S D Q A A I V E I E L I E K K R - R I P I L T I E K S K - - - - H L G R I V L R K E G R T V A A G

Rates

Mitochondrial inner membrane

Intermembrane space

N C

Fig. S7A

Fig. S7. MRS3-MRS4 structural and functional entanglement . (A) A simplified illustration of the mitochondrial carrier family of proteins to which MRS3-MRS4 belong. The primary structure consists of three repeats denoted in light purple, green and pink colours, whereby each repeat is 100 amino acids in length and is composed of two hydrophobic membrane spanning α-helices (H1&H2, H3&H4 and H5&H6) as well as a characteristic amino acid sequence motif PX[D/E]XX-[K/R]X[K/R] (20–30 residues) [D/E]GXXXX[W/Y/F][K/R]G that forms a loop connecting each pair of membrane-spanning domains within the mitochondrial matrix (61). (B) An illustrative hypothetical example of in silico modeling results, which would be consistent with the evolutionary steady-state of MRS3-MRS4, capturing the paralog pair’s retained functional redundancy and divergence.

Mitochondrialmatrix

H1 H2 H3 H4 H5 H6

B

Randommutations

Functional divergence & retained redundancy

Evolutionary steady state

Paralog 2Paralog 1Immediately upon duplication

Transport Specialty 1Transport Specialty 2

Core Channel Functionality

GGC1

SAL1

YIA6

AAC1

MRS3

UGO1

YMR166C

MRS4

PIC2

RIM2

YHM2

YMC1

LEU5 CTP1ODC2

ODC1YMC2

YEA6

AAC3

YDL119C

MTM1

PET8

MIR1

PET9

SFC1 ORT1

FLX1

Negative digenic ( ), trigenic ( ) interaction (this study)Negative genetic interaction (BioGrid)Phenotypic enhancement (BioGrid)Dosage rescue (BioGrid)Phenotypic suppression (BioGrid)

Fig. S8.

Fig. S8. Mitochondrial carrier protein family genetic interaction network. Genetic interactions among members of the mitochondrial carrier family, as defined previously (62) is depicted. Whole genome duplicate pairs screened in this study: YMC1-YMC2, ODC1-ODC2, YEA6-YIA6, MRS3-MRS4 are highlighted in blue, red, green and yellow colours, respectively. Genetic interactions from this study that meet the intermediate cut-off, (τ or ε) > 0.08, p < 0.05 (dark blue) were combined with literature-curated genetic interactions, as reported by BioGrid (95): negative genetic interactions (light blue), phenotypic enhancement (orange), dosage rescue (black) and phenotypic suppression (grey).

0 10 20 30 40 50 60 70 80 90 1000

0.2

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Entanglement (%)

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Fig. S9. In silico modeling of paralog divergence . Paralogs were generated to represent a range of overlapping functions at the onset of their evolutionary trajectories and the propensities to revert to (A) singleton state, (B) evolve asymmetrically (C) evolve asymmetrically (null model) and (D) retain overlap at the evolutionary steady-state are depicted. X-axis represents bins of initial functional overlap in sequence space at the start of the simulations. That is, the number of positions responsible for two or more functions divided by the total number of positions in the ‘gene’, rounded down to the nearest decile.

A

D

Fig. S9.

B

C

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rate

Fu

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13

Supplementary Tables: Table S1. Raw genetic interaction dataset. This file contains digenic interaction scores as well as raw and adjusted trigenic interaction scores in a tab-delimited format with 12 columns: 1) Query Strain ID, 2) Query allele name, 3) Array strain ID, 4) Array allele name, 5) Combined mutant type, 6) Raw genetic interaction score (epsilon), 7) Adjusted trigenic interaction score (tau), 8) p-value, 9) Query fitness, 10) Array single mutant fitness, 11) Combined mutant fitness relative to wild-type, 12) Combined mutant fitness standard deviation. Column descriptions:

1. Query Strain ID 2. Query Allele name 3. Array Strain ID 4. Array Allele name 5. Combined mutant type

a. ‘digenic’ for double mutants resulting from a cross between a single mutant control query and a single mutant array strain

b. ‘trigenic’ for triple mutants resulting from a cross between a double mutant query and a single mutant array strain

6. Raw genetic interaction score (epsilon) 7. Final genetic interaction score (trigenic tau / digenic epsilon). Trigenic scores are

adjusted according to the τ-SGA model. Digenic scores receive no further adjustment and the epsilon value is repeated.

8. Interaction p value 9. Query fitness

a. single mutant fitness for single mutant queries b. double mutant fitness for double mutant queries

10. Array single mutant fitness 11. Combined mutant fitness.

a. Double mutant fitness for the resulting combined digenic mutants b. Triple mutant fitness for the resulting combined trigenic mutants

12. Combined mutant fitness standard deviation Table S2. Digenic and adjusted trigenic interaction dataset. This file contains digenic and trigenic interaction scores at an established interaction magnitude cut-off for digenic interactions (p < 0.05, |e| > 0.08) and trigenic interactions (p < 0.05, |t| > 0.08 in a tab-delimited format with 8 columns: 1) Query Strain ID, 2) Query allele name, 3) Array strain ID, 4) Array allele name, 5) Combined mutant type, 6) Final genetic interaction score (tau), 7) p-value, 8) Interaction type. Column descriptions:

1. Query Strain ID 2. Query Allele name 3. Array Strain ID 4. Array Allele name 5. Combined mutant type

14

a. ‘digenic’ for double mutants resulting from a cross between a single mutant control query and a single mutant array strain

b. ‘trigenic’ for triple mutants resulting from a cross between a double mutant query and a single mutant array strain

6. Final genetic interaction score (trigenic tau / digenic epsilon). Trigenic scores are adjusted according to the τ-SGA model and account for any digenic interactions. Digenic scores receive no further adjustment and the epsilon value is repeated from Table S1.

7. Interaction p value 8. Interaction types:

a. Digenic is digenic b. Novel is novel trigenic c. Unclassified apparently novel but with unknown query-query interaction score

thus cannot be distinguished from modified and novel d. Modified Q-, Modified Q-A-, Modified Q-A+, Modified Q-A+-, Modified A-,

Modified A+ or Modified A-+ are modified trigenic interactions are further broken down by where the overlapping digenic interaction is found: Q- for a negative interaction between query genes, A for a negative interaction between one or both of the query genes and the array gene, A+ for a positive interaction between one or both of the query genes and the array gene, A+- if both query genes have a digenic interaction with the array but of opposing signs)

Table S3. Raw genetic interaction dataset from pilot screens. This file contains digenic interaction scores as well as raw and adjusted trigenic interaction scores in a tab-delimited format with 12 columns: 1) Query Strain ID, 2) Query allele name, 3) Array strain ID, 4) Array allele name, 5) Combined mutant type, 6) Raw genetic interaction score (epsilon), 7) Adjusted trigenic interaction score (tau), 8) p-value, 9) Query fitness, 10) Array single mutant fitness, 11) Combined mutant fitness relative to wild-type, 12) Combined mutant fitness standard deviation. Column descriptions:

1. Query Strain ID 2. Query Allele name 3. Array Strain ID 4. Array Allele name 5. Combined mutant type

a. ‘digenic’ for double mutants resulting from a cross between a single mutant control query and a single mutant array strain

b. ‘trigenic’ for triple mutants resulting from a cross between a double mutant query and a single mutant array strain

6. Raw genetic interaction score (epsilon) 7. Final genetic interaction score (trigenic tau / digenic epsilon). Trigenic scores are

adjusted according to the τ-SGA model. Digenic scores receive no further adjustment and the epsilon value is repeated.

8. Interaction p value 9. Query fitness

a. single mutant fitness for single mutant queries b. double mutant fitness for double mutant queries

15

10. Array single mutant fitness 11. Combined mutant fitness.

a. Double mutant fitness for the resulting combined digenic mutants b. Triple mutant fitness for the resulting combined trigenic mutants

12. Combined mutant fitness standard deviation Table S4. Query strains and plasmids list. This file contains the complete list of yeast strains and plasmids that were used in this study. The ‘Strain Pairing List’ tab lists the double mutants and their corresponding single mutant control strains that were used to generate trigenic interaction scores. The ‘Genotypes’ tab lists the complete genotype of each strain including control strains that were used to derive the fitness standard. The ‘Plasmids’ tab lists the plasmids that were used in this study. Table S5. Fitness standard for single and double mutant query strains and query-query interactions. This file contains the fitness standard for single and double mutant query strains as well as the genetic interaction between the query genes. The ‘Fitness standard’ tab lists the fitness values for single and double mutants. The ‘Query interactions’ tab lists the interactions between the query genes for each double mutant query strain. Table S6. List of WGD paralogs that are sparsely connected on the global digenic interaction network. The list was obtained from (30). Table S7. Trigenic interaction fraction of paralogs. This file contains the fraction of trigenic interactions for all paralogs. Table S8. Classification of paralogs into ohnologs and homeologs. This file contains the list of paralogs classified into ohnologs and homeologs based on their evolutionary origins as reported in a previous study (50). Table S9. Family size of WGD paralogs. This file contains the list of paralogs and their associated gene family size. Table S10. Evolutionary and physiological properties. This file contains ‘Features’ tab with the evolutionary and physiological features that correlate with trigenic interaction fraction, digenic interaction degree asymmetry, rate of sequence divergence and digenic global profile similarity. It also contains the ‘Seq div rate’ with the terms that were used to calculate the sequence divergence rate described in the ‘Correlation with physiological and evolutionary features’ section above. Table S11. Paralog pairs exhibiting asymmetric digenic interactions. Paralogs in which the low degree sister is induced in a developmental program such as meiosis, filamentous growth and glucose starvation is denoted by ‘1’, otherwise it is ‘0’.

16

Table S12. Correlations of position specific evolutionary rate patterns for WGD paralog pairs. Cor12 represents the correlation of evolutionary sequence changes between the two sister clades. Cor1p represents the correlation of evolutionary sequence changes between sister 1 clade and pre-WGD clade. Cor2p represents the correlation of evolutionary sequence changes between sister 2 clade and pre-WGD clade. Table S13. List of synthetic lethal WGD paralogs. ORFs and gene names for synthetic lethal WGD paralogs are provided along with the average digenic interaction score (30) and results of tetrad analysis performed in this study.

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