Mon Dec 19 14:50:05 2022

By Simon Penel & Laurent Duret

1. Introduction

All P. aurelia genomes sequenced to date have been subject to a recent WGD event (WGD1). The question is whether the WGD1s detected in these genomes all result from a single WGD event in the ancestor of the P. aurelia lineage (scenario 1), or from several independent WGD1 events (scenario 2).

Eric noticed that in most gene families, ‘WGD1’ ohnologs identified in Pson or Pjen appear to have a recent last common ancestor (in the Pson/Pjen ancestor). Similarly, many ‘WGD1’ ohnologs identified in Psex appear to originate in the PSEX terminal branch. These observations tend to support scenario 2.

It should be noted however that gene conversion can occur between ohnologs. Hence the last common ancestor of a pair of ohnologs in a given gene family does not correspond the WGD event itself, but to the point when they stopped recombining. Thus, last common ancestor of a pair of ohnologs corresponds to the Last Conversion Event (LCE) between the two ohnologs. The fact that many ohnologs have a recent LCE is therefore not necessarily in contradiction with scenario 1: it may simply reflect the fact that the rate of gene conversion has remained relatively high in some lineages.

To distinguish between the two scenarios, we systematically analyzed gene family phylogenies to investigate the distribution of LCEs of ohnologs resulting from the last WGD event.

Here, gene trees are build and reconciled with GeneRax, based on CDS alignment, and using a DNA model (and using the IQtree tree as starting tree).

2. Data

Gene families

We analyzed reconciled phylogenetic trees from PhyloParameciumDB, which includes 26 genomes (22 paramecia + 4 tetrahymena). The P. aurelia clade is divided in two subclades: S2J (Psex, Pson, Pjen), and BDNOPT (Pbia, Pded, Pdoc, Pnov, Poct, Ppen, Ppri, Ptet, Ptre):

Fig. 1 Fig. 1: species tree of genomes represented in PhyloParameciumDB. Speciation nodes are numbered.**

WGD1 ohnologs

Ohnologs have been identified by O. Arnaiz in 17 P. aurelia strains (15 species), based on similarity search and synteny conservation within each genome (using the protocol of Aury et al. 2006):

pbiaurelia_V1-4_annotation_v2.0.WGD.tree
pdecaurelia_223_annotation_v1.0.WGD.tree
pdodecaurelia_274_annotation_v1.0.WGD.tree
pjenningsi_M_annotation_v1.0.WGD.tree
pnovaurelia_TE_annotation_v1.0.WGD.tree
poctaurelia_138_annotation_v1.0.WGD.tree
poctaurelia_K8_annotation_v1.0.WGD.tree
ppentaurelia_87_annotation_v1.0.WGD.tree
pprimaurelia_AZ9-3_annotation_v1.0.WGD.tree
pquadecaurelia_NiA_annotation_v1.0.WGD.tree
primaurelia_Ir4-2_annotation_v1.WGD.tree
psexaurelia_AZ8-4_annotation_v2.0.WGD.tree
psonneborni_ATCC_30995_annotation_v1.0.WGD.tree
ptetraurelia_32_annotation_v1.0.WGD.tree
ptetraurelia_mac_51_annotation_v2.0.WGD.tree
ptetraurelia_mac_annotation_v1.WGD.tree
ptredecaurelia_209_annotation_v1.1.WGD.tree

It should be noted that ohnologs have been identified in each genome, without using information from the other species. There might be some errors or missing data in these datasets (e.g. some pairs of ohnologs may have been missed because of assembly errors). Thus, the comparison of different species can provide additional support on ohnology inferences.

[NB: among the 15339 pairs of WGD1 ohnologs detected in Pson, only 12569 pairs (82%) are classified in a same PhyloParameciumDB gene family. The missing data correspond to pairs of WGD1 ohnologs that do not match the clustering criteria that we use to build PhyloParameciumDB gene families (we impose that homologs align over at least 80% of their length, and hence we exclude truncated homologs). Similar proportion are observed in other species. In absence of gene phylogeny, all pairs of WGD1 ohnologs that are not classified in the same PhyloParameciumDB gene family were excluded of the analysis].

3. Results

3.1 Dating the origin of Pson ohnologs by analysis of tree topology

We analyzed all pairs of WGD1 ohnologs identified in Pson and classified in a same PhyloParameciumDB gene family (N=12569 pairs of WGD1 ohnologs).

To date the age of LCE of each pair of ohnolog, we first analyzed the topology of gene phylogenies. To explain the approach, we will consider a simplified species tree:

Fig. 2 Fig. 2: exemplar species tree. According to scenario 1, one single event (WGD1) occurred in the last common ancestor of the Aurelia clade (branch b7).

Let us consider a pair of ohnologs in a given species (say PsonA/PsonB in species P. sonneborni). We can identify in the gene tree the node corresponding to the last common ancestor of this pair of ohnologs. We refer to this node as the Last Conversion Event (LCE) between these two ohnologs.

Fig. 3 shows several examples of gene phylogenies to illustrate how LCEs can be dated (relative to speciation events). For a given pair of ohnologs, we looked in the GeneRax-reconciled gene tree for the node corresponding to its LCE (red star in Fig. 3). For each node in the tree, GeneRax indicates to which category it was assigned (duplication, horizontal transfer or speciation). If the node is a duplication or a transfer, it indicates on which branch of the species tree the event occurred. We thus retrieve this information for each LCE node.

Fig. 3 Fig. 3: Examples of possible tree topologies for different LCE events. The red star indicate the Last Conversion Event (LCE) for the PsonA/PsonB pair of ohnologs.

In principle, the LCE of a pair of ohnologs is expected to correspond to a duplication node, and this node should occur in a branch of the species tree on the path from the root to the leaf (Psonn). There are however a few pairs that do not fit with that prediction:

## Warning in (function (node, family = "", pair = "") : Unexpected case in
## get_class_age: LCE age = PMUL in family FAM004335 (pair PSONN_101.PE110/
## PSONN_109.PE98)
## Warning in (function (node, family = "", pair = "") : Unexpected case in
## get_class_age: LCE age = PTRED in family FAM002945 (pair PSONN_118.PE65/
## PSONN_107.PE61)
## Warning in (function (node, family = "", pair = "") : Unexpected case in
## get_class_age: LCE age = node_19 in family FAM007581 (pair PSONN_050.PE248/
## PSONN_058.PE91)
## Warning in (function (node, family = "", pair = "") : Unexpected case in
## get_class_age: LCE age = PTRED in family FAM016024 (pair PSONN_051.PE160/
## PSONN_041.PE169)
## Warning in (function (node, family = "", pair = "") : Unexpected case in
## get_class_age: LCE age = node_24 in family FAM004692 (pair PSONN_166.PE118/
## PSONN_162.PE104)

Among the 12569 pairs of WGD1 ohnologs analyzed, the distribution of estimated LCE age is given below:

ClassLCE Nb_Ohnolog_Pairs Percentage PcDuplication
Recent 12043 95.82% 96.89
Intermediate 366 2.91% 75.68
Aurelia 130 1.03% 41.54
Old 25 0.20% 48.00
NA’s 5 0.04% NaN

In summary, the LCE age distribution of Pson ohnologs is the following:

  • Recent LCE = in b10 or b13
  • Intermediate LCE = in b9
  • Aurelia LCE = in the last common ancestor au the Aurelia clade (b8)
  • Old LCE = predating the Aurelia divergence

Thus, the analysis of tree topologies confirms that a large majority of Pson ohnologs have a recent LCE, as already noticed by Eric. However, these analyses also suggest that a substantial fraction of them are more ancient, which is a priori not consistent with scenario 2. Moreover, a large fraction of the oldest LCE node are not inferred as being duplication by GeneRax (because of HT events in the gene tree).

3.2 Validation by analysis of ohnologs identified in other species

It should be noted that the previous estimates of LCE ages are only based on the tree topology, which might be subject to errors. Notably, because of the long-branch attraction artefact, fast-evolving sequences tend to be erroneously placed near the root of the tree. For instance, in Fig. 3F, the tree topology would suggest that the LCE occurred in b8. However, the position PsonB, alone in clade B suggests that it might be subject to a long-branch attraction artefact, and hence that the age of the LCE might be overestimated. Such artefacts probably explain why some Pson WGD1 ohnologs appear to predate the divergence between the aurelia clade and its outgroups (P. caudatum or P. Multimicronucleatum), which have not been subject to WGD1 nor WGD2.

To circumvent this issue, I then used information on ohnology relationships available from other species. A LCE inferred in branch b9 was considered as confirmed if Psex homologs are present both in clade A and in clade B (as in Fig. 3D), and that if these homologs have been classified as ohnologs by Oliver. If they are no ohnolog, or if Psex homologs are present only in one clade (as in Fig. 3E), then the dating of this pair is considered as ‘unsure’.

Similarly, LCE event predating the Aurelia clade are considered as confirmed if at least 2 species of the BDNOPT clade have pairs of ohnologs present both in clade A and in clade B.

The table below gives the final number of WGD1 pairs classified as recent (b10 or b13), intermediate (b9) or Aurelia (b8 or earlier). Cases for which the dating is unsure are indicated by a ‘?’:

AgeLCE Nb_Ohnolog_Pairs Percentage MeanNbWGD1Pairs_BDNOPT PcDuplication
Recent 12043 95.8% 0.00 96.89
Intermediate 222 1.8% 0.00 98.65
?Intermediate 144 1.1% 0.00 40.28
?Aurelia 94 0.7% 0.02 20.21
Aurelia 36 0.3% 7.97 97.22
?Old 18 0.1% 0.06 44.44
Old 7 0.1% 6.57 57.14
NA’s 5 0.0% NaN NaN

Thus, 36 pairs of Pson WGD1 ohnologs have a LCE that predates the divergence of the aurelia clade. These ‘aurelia’ LCEs are supported by ohnology data from species of the BDNOPT clade (on average 7.97 species with a pair of ohnologs in clades A and B). Most of these pairs (97.22%) are inferred by GeneRax to descend from a duplication node.

Besides, 222 pairs of Pson WGD1 ohnologs have a LCE that predates the divergence between PSONN and PSEX. These ‘Intermediate’ LCEs are supported by ohnology data from P. sexaurelia . Most of these pairs (98.65%) are inferred by GeneRax to descend from a duplication node.

3.3 Distribution of dS between Pson WGD1 ohnologs

Given that synonymous sites are under weak selection, dS is expected to be a good proxy of divergence time. According to scenario 1, WGD1 is more ancient than the radiation of the aurelia clade. Hence, in absence of gene conversion, the dS values between WGD1 ohnologs should be larger than the dS between orthologs of the S2J and BDNOPT clades. The distribution of dS between Pson (S2J) and P. biaurelia (BDNOPT) orthologs is shown below:

Fig. 5 Fig. 5: Distribution of dS between Pson and P. biaurelia orthologs.

The median dS between Pson and P. biaurelia is 0.8. Thus, at least 50% of WGD1 ohnologs for which the LCE is inferred to predate the divergence of the aurelia clade should have a dS > 0.8.

To check that, I looked at the distribution of dS (and dN) for pairs of Pson ohnologues of different age classes:

##    Min. 1st Qu.  Median    Mean 3rd Qu.    Max.    NA's 
##  0.0000  0.0889  0.1444  0.1671  0.2190  1.3981     469

As expected, the distribution of dS is shifted towards lower values for ohnologs with a very recent LCE, and the proportion of ohnologs with dS>0.8 tends to increase with the inferred LCE age. However, among the 36 ohnologs inferred to have an ‘Aurelia’ LCE, 85.3% have a dS<0.8, more than the 50% expected if all of them really had a LCE predating the aurelia radiation. This implies that at least 70.6% of these ‘Aurelia’ ohnologs have in fact been subject to gene conversion more recently.

One possible explanation for this discrepancy is that in the above analyses, we considered the tree topology inferred by GeneRax, based on the CDS alignment. When genes are strongly conserved, the alignment contains little phylogenetic signal. GeneRax searches for the tree topology that minimizes the number of gene duplications/losses. Hence, it tends to favor a topology supporting scenario 1 (i.e. one single WGD1).

To avoid this issue, we can focus on WGD1 ohnologs that are sufficiently divergent (dS> 0.4), to provide phylogenetic signal.

Thus, ohnologs that are predicted to be ‘Aurelia’ (based on topology) but for which dS< 0.4 are therefore hereafter considered as unsure (‘?Aurelia’).

AgeLCE Nb_Ohnolog_Pairs Percentage MeanNbWGD1Pairs_BDNOPT PcDuplication
Recent 12043 95.8% 0.00 96.89
Intermediate 222 1.8% 0.00 98.65
?Intermediate 144 1.1% 0.00 40.28
?Aurelia 114 0.9% 1.43 34.21
?Old 18 0.1% 0.06 44.44
Aurelia 16 0.1% 7.88 93.75
Old 7 0.1% 6.57 57.14
NA’s 5 0.0% NaN NaN

Thus, there are 16 pairs of Pson WGD1 ohnologs for which we have evidence that their LCE predates the divergence of the aurelia clade:

  • Supported by the topology of the GeneRax reconciled tree
  • Supported by ohnology data from species of the BDNOPT clade (on average 7.88 species with a pair of ohnologs in clades A and B)
  • dS>0.4

3.4 Gene conversion between WGD2 ohnologs??

The previous analyses clearly show that some WGD1 ohnologs identified in Pson have a LCE that predates the divergence of the aurelia clade. This is a priori not consistent with scenario 2. However, the previous analyses did not take into account the fact that the position of WGD1 ohnologs in the gene tree can be affected by gene conversion with WGD2 ohnologs, posterior to the WGD1 event.

Let us consider a pair of WGD1 ohnolog (say 2A/2B) belonging to a set of 4 ohnologs (1A, 1B, 2A, 2B) resulting from WGD2+WGD1. As shown in Fig. 4B, if one gene (say 2A) gets converted by one of its WGD2 ohnolog (say 1B), then the LCE of 2A/2B would in fact correspond to WGD2. Thus, at the first step of our protocol, based on the tree topology, this pair of WGD1 ohnolog would have been classified as ‘Aurelia’ (b8). But at the 2nd step, it would have been classified as ‘unsure’ because it is not validated by any pair of WGD1 ohnolog in the BDNOPT clade. However, if a second independent conversion event occurs between WGD2 ohnologs in the BDNOPT clade, then it is possible to obtain tree topologies with an ‘Aurelia’ LCE supported by pairs of WGD1 ohnologs in the BDNOPT clade (Fig. 4C). Given that the fraction of ‘Aurelia’ LCE among Pson WGD1 ohnologs is rather small (0.1%), even though this complex scenario seems a priori quite unlikely, it cannot be directly discarded.

Fig. 4 Fig. 4: Examples of possible tree topologies under scenario 2 with gene conversion between WGD2 ohnologs.

To avoid this potential issue, we focused on pairs of WGD1 honologs that do not have any WGD2 or WGD3 ohnolog in Pson. The rationale is that this subset should correspond mostly to genes that have lost their WGD3/WGD2 ohnologs prior the WGD1 event, and hence for which the gene tree topology cannot have been affected by gene conversion with WGD2 ohnologs after the WGD1 event.

Among the 16 pairs of WGD1 Pson honologs with an ‘Aurelia’ LCE, 10 (62.5%) do not have any WGD2 or WGD3 ohnolog (NB: this proportion is higher than for pairs of WGD1 Pson honologs with a ‘Recent’ LCE: 56.5%). Thus, most pairs of ‘Aurelia’ WGD1 Pson honologs are robust to this potential issue.

3.5. Summary of phylogenetic analyses

In summary, among the 12569 pairs of Psonn WGD1 ohnologs that we analyzed, 130 (1.03%) are dated on the aurelia branch, based on the topology of the reconciled gene tree. Among them, 36 (0.29%) are confirmed by at least 2 pairs of ohnologs from the BDNOPT clade. We then excluded pairs of Psonn ohnologs for which the synonymous divergence was lower than expected (dS<0.4), which we suspect that there was not enough phylogenetic signal (and hence GeneRax tends to put the duplication at the aurelia node). After this step, there remained 16 (0.13%) pairs of Psonn ohnologs in our list of candidates.

I looked at the original gene trees obtained by IQTREE (i.e. without reconciliation by GeneRax). In 5 cases, the gene tree clearly supports an ancient WGD1 (prior the aurelia divergence). In the other cases, the gene tree is not well resolved (it is compatible with the hypothesis of a single WGD1, but no enough phylogenetic signal in the alignment to reject the alternative model).

NB: trees and alignments are available here: http://pbil.univ-lyon1.fr/members/duret/misc/Fam_aurelia_WGD1_ohnologs/

The table below provides links to the phylogenetic trees of the 16 ‘Aurelia’ pairs of Pson WGD1 ohnologs. The total number of species for which pairs of WGD1 ohnologs have been identified in clade_A and clade_B is indicated in parenthesis. Members of such pairs are labeled with ‘>>>> <<<<’ in the gene phylogeny (by putting the mouse pointer over branches displayed in red in the tree, you can see the path linking two members of a given WGD1 pair).

For the first 5 pairs (out top candidates), there’s a link to a PDF of the corresponding gene tree, where the ohnolog pair from Psonn and from a species of the BDNOPT clade are highlighted in red.

GeneTree Scaffold_A Scaffold_B AID_A AID_B LCE_age HasWGD23paralog
FAM000316 7 15 PSONN_007.PE253 PSONN_015.PE191 LCE_Aurelia (FAM000316 14 pairs, dS=99.00) 1
FAM002449 29 21 PSONN_029.PE32 PSONN_021.PE344 LCE_Aurelia (FAM002449 13 pairs, dS=0.71) 0
FAM005810 113 103 PSONN_113.PE29 PSONN_103.PE10 LCE_Aurelia (FAM005810 12 pairs, dS=1.27) 0
FAM006172 73 63 PSONN_073.PE146 PSONN_063.PE52 LCE_Aurelia (FAM006172 4 pairs, dS=NA) 0
FAM013981 52 33 PSONN_052.PE272 PSONN_033.PE70 LCE_Aurelia (FAM013981 8 pairs, dS=1.45) 0
FAM000298 7 15 PSONN_007.PE271 PSONN_015.PE210 LCE_Aurelia (FAM000298 12 pairs, dS=0.41) 1
FAM001446 8 35 PSONN_008.PE493 PSONN_035.PE316 LCE_Aurelia (FAM001446 7 pairs, dS=0.43) 0
FAM006172 60 43 PSONN_060.PE90 PSONN_043.PE209 LCE_Aurelia (FAM006172 10 pairs, dS=1.21) 1
FAM007206 149 117 PSONN_149.PE81 PSONN_117.PE86 LCE_Aurelia (FAM007206 9 pairs, dS=0.52) 1
FAM007690 95 84 PSONN_095.PE50 PSONN_084.PE58 LCE_Aurelia (FAM007690 15 pairs, dS=0.53) 0
FAM008348 39 57 PSONN_039.PE268 PSONN_057.PE98 LCE_Aurelia (FAM008348 13 pairs, dS=0.69) 0
FAM009082 114 116 PSONN_114.PE172 PSONN_116.PE162 LCE_Aurelia (FAM009082 12 pairs, dS=NA) 1
FAM009510 72 97 PSONN_072.PE240 PSONN_097.PE191 LCE_Aurelia (FAM009510 11 pairs, dS=0.43) 0
FAM009747 75 61 PSONN_075.PE162 PSONN_061.PE154 LCE_Aurelia (FAM009747 12 pairs, dS=0.63) 0
FAM011843 56 27 PSONN_056.PE57 PSONN_027.PE159 LCE_Aurelia (FAM011843 14 pairs, dS=1.36) 1
FAM012692 10 13 PSONN_010.PE427 PSONN_013.PE411 LCE_Aurelia (FAM012692 15 pairs, dS=0.76) 0

3.6 Are ‘Aurelia’ Pson WGD1 honologs really located in WGD1 paralogons?

The ‘Aurelia’ WGD1 Pson honologs might correspond to errors of ohnology assignments (e.g. a WGD2 paralogon misclassifed as being a WGD1 paralogon). To check that, I show below the paralogons of the 16 ‘Aurelia’ WGD1 pairs that we identified [the total number of species for which pairs of WGD1 ohnologs have been identified in clade_A and clade_B, and the presence of WGD2 or WGD3 paralogs are indicated in parenthesis]: for each of these ‘Aurelia’ WGD1 pair, I select up to 15 pairs of WGD1 ohnologs upstream and downstream, from the same scaffold pair, and I draw all genes contained in this interval. The 5 best candidates are shown first.

3.6.1 Top 5 candidates

Plot of paralogons for the 5 best candidates:

[1] “# 1: FAM000316 scaffold 7 vs scaffold 15 LCE_Aurelia (FAM000316 14 pairs, dS=99.00) (this pair has WGD2 or WGD3 paralog)” [1] “# 2: FAM002449 scaffold 29 vs scaffold 21 LCE_Aurelia (FAM002449 13 pairs, dS=0.71) (No WGD2 or WGD3 paralog)” [1] “# 3: FAM005810 scaffold 113 vs scaffold 103 LCE_Aurelia (FAM005810 12 pairs, dS=1.27) (No WGD2 or WGD3 paralog)” [1] “# 4: FAM006172 scaffold 73 vs scaffold 63 LCE_Aurelia (FAM006172 4 pairs, dS=NA) (No WGD2 or WGD3 paralog)” [1] “# 5: FAM013981 scaffold 52 vs scaffold 33 LCE_Aurelia (FAM013981 8 pairs, dS=1.45) (No WGD2 or WGD3 paralog)”

Several of the candidate ‘Aurelia’ ohnologs are located at an extremity of the paralogon. It is possible that they have been mis-assigned to the WGD1 paralogon (i.e. they might result from an independent duplication event) => to be checked.

3.6.2 Other candidates

Plot of paralogons for the other candidates:

[1] “# 6: FAM000298 scaffold 7 vs scaffold 15 LCE_Aurelia (FAM000298 12 pairs, dS=0.41) (this pair has WGD2 or WGD3 paralog)” [1] “# 7: FAM001446 scaffold 8 vs scaffold 35 LCE_Aurelia (FAM001446 7 pairs, dS=0.43) (No WGD2 or WGD3 paralog)” [1] “# 8: FAM006172 scaffold 60 vs scaffold 43 LCE_Aurelia (FAM006172 10 pairs, dS=1.21) (this pair has WGD2 or WGD3 paralog)” [1] “# 9: FAM007206 scaffold 149 vs scaffold 117 LCE_Aurelia (FAM007206 9 pairs, dS=0.52) (this pair has WGD2 or WGD3 paralog)” [1] “# 10: FAM007690 scaffold 95 vs scaffold 84 LCE_Aurelia (FAM007690 15 pairs, dS=0.53) (No WGD2 or WGD3 paralog)” [1] “# 11: FAM008348 scaffold 39 vs scaffold 57 LCE_Aurelia (FAM008348 13 pairs, dS=0.69) (No WGD2 or WGD3 paralog)” [1] “# 12: FAM009082 scaffold 114 vs scaffold 116 LCE_Aurelia (FAM009082 12 pairs, dS=NA) (this pair has WGD2 or WGD3 paralog)” [1] “# 13: FAM009510 scaffold 72 vs scaffold 97 LCE_Aurelia (FAM009510 11 pairs, dS=0.43) (No WGD2 or WGD3 paralog)” [1] “# 14: FAM009747 scaffold 75 vs scaffold 61 LCE_Aurelia (FAM009747 12 pairs, dS=0.63) (No WGD2 or WGD3 paralog)” [1] “# 15: FAM011843 scaffold 56 vs scaffold 27 LCE_Aurelia (FAM011843 14 pairs, dS=1.36) (this pair has WGD2 or WGD3 paralog)” [1] “# 16: FAM012692 scaffold 10 vs scaffold 13 LCE_Aurelia (FAM012692 15 pairs, dS=0.76) (No WGD2 or WGD3 paralog)”

Again, several of the candidate ‘Aurelia’ ohnologs are located at an extremity of the paralogon. It is possible that they have been mis-assigned to the WGD1 paralogon (i.e. they might result from an independent duplication event) => to be checked.

4. Conclusion

Under scenario 2, all WGD1 ohnologs are expected to have a recent LCE, except in case of multiple conversion events with WGD2 ohnologs posterior to the WGD1 events (see 3.4), or in case of confusion in WGD assignment (see 3.5)

Here we find some pairs of WGD1 ohnologs in PSON for which there is a phylogenetic signal indicating that their LCE predates the divergence of the P. aurelia clade. If true, then this would argue in favor of scenario 1. However, given the limited number of cases, it is difficult to exclude that they might result from errors in WGD assignment.

5. Annex: corresponding list of PhyloParamecium gene families and ohnolog ACNUC IDs

Family List_pairsAB
FAM000316 PNOVA:PNOVA_0001.PE206:PNOVA_0003.PE69;PPENT:PPENT_004.PE260:PPENT_008.PE238;PPRIMAZ9:PPRIMAZ9_004.PE310:PPRIMAZ9_008.PE289;PBIAU:PBIAU_0004.PE257:PBIAU_0001.PE360;PDECA:PDECA_0080.PE100:PDECA_0078.PE84;PDODE:PDODE_0287.PE14:PDODE_0342.PE22;POCTA138:POCTA138_010.PE287:POCTA138_004.PE280;POCTAK8:POCTAK8_277078.PE219:POCTAK8_277030.PE236;PTETR51:PTETR51_6.PE188:PTETR51_3.PE283;PTETR32:PTETR32_003.PE382:PTETR32_008.PE286;PQUAD:PQUAD_0002.PE169:PQUAD_0020.PE61;PTRED:PTRED_129357.PE169:PTRED_129381.PE230;PJENN:PJENN_0230.PE22:PJENN_0288.PE22;PSONN:PSONN_015.PE191:PSONN_007.PE253;
FAM000298 PDODE:PDODE_0455.PE18:PDODE_0389.PE20;PDECA:PDECA_0078.PE103:PDECA_0080.PE116;POCTA138:POCTA138_004.PE262:POCTA138_010.PE269;POCTAK8:POCTAK8_277030.PE255:POCTAK8_277078.PE203;PTETR32:PTETR32_008.PE268:PTETR32_003.PE398;PTETR51:PTETR51_3.PE264:PTETR51_6.PE204;PBIAU:PBIAU_0001.PE342:PBIAU_0004.PE239;PPENT:PPENT_008.PE256:PPENT_004.PE243;PTRED:PTRED_129381.PE247:PTRED_129357.PE185;PNOVA:PNOVA_0003.PE85:PNOVA_0001.PE190;PJENN:PJENN_0288.PE41:PJENN_0230.PE42;PSONN:PSONN_007.PE271:PSONN_015.PE210;
FAM001446 PBIAU:PBIAU_0333.PE9:PBIAU_0354.PE14;PPENT:PPENT_001.PE670:PPENT_007.PE463;PQUAD:PQUAD_0005.PE306:PQUAD_0004.PE297;PTRED:PTRED_129389.PE34:PTRED_129329.PE44;PJENN:PJENN_0197.PE14:PJENN_0241.PE12;PSONN:PSONN_008.PE493:PSONN_035.PE316;PSEXA:PSEXA_008.PE45:PSEXA_001.PE696;
FAM012692 PJENN:PJENN_0002.PE272:PJENN_0048.PE86;PSONN:PSONN_013.PE411:PSONN_010.PE427;PSEXA:PSEXA_013.PE68:PSEXA_040.PE74;PDECA:PDECA_0077.PE111:PDECA_0016.PE193;PDODE:PDODE_0301.PE4:PDODE_0333.PE2;POCTA138:POCTA138_008.PE388:POCTA138_016.PE355;POCTAK8:POCTAK8_277002.PE81:POCTAK8_276987.PE78;PTETR32:PTETR32_012.PE78:PTETR32_016.PE76;PTETR51:PTETR51_7.PE79:PTETR51_11.PE77;PPRIMAZ9:PPRIMAZ9_012.PE383:PPRIMAZ9_002_2.PE372;PPENT:PPENT_010.PE368:PPENT_013.PE348;PQUAD:PQUAD_0242.PE35:PQUAD_0123.PE36;PTRED:PTRED_129371.PE337:PTRED_129379.PE318;PNOVA:PNOVA_0554.PE23:PNOVA_0553.PE21;PBIAU:PBIAU_0237.PE42:PBIAU_0179.PE30;
FAM002449 PJENN:PJENN_0032.PE155:PJENN_0154.PE68;PSONN:PSONN_021.PE344:PSONN_029.PE32;PNOVA:PNOVA_0026.PE116:PNOVA_0005.PE35;PQUAD:PQUAD_0019.PE12:PQUAD_0050.PE131;PTRED:PTRED_129395.PE232:PTRED_129361.PE300;PPENT:PPENT_060.PE17:PPENT_025.PE335;PPRIMAZ9:PPRIMAZ9_050.PE282:PPRIMAZ9_117.PE39;PBIAU:PBIAU_0015.PE273:PBIAU_0007.PE324;PDECA:PDECA_0462.PE11:PDECA_0134.PE68;PDODE:PDODE_0211.PE63:PDODE_0001.PE348;POCTA138:POCTA138_063.PE276:POCTA138_022.PE40;POCTAK8:POCTAK8_277053.PE18:POCTAK8_276713.PE326;PTETR51:PTETR51_42.PE278:PTETR51_14.PE49;
FAM008348 PSONN:PSONN_057.PE98:PSONN_039.PE268;PSEXA:PSEXA_045.PE226:PSEXA_034.PE241;PQUAD:PQUAD_0225.PE23:PQUAD_0304.PE29;PTRED:PTRED_129447.PE220:PTRED_129433.PE82;PNOVA:PNOVA_0234.PE28:PNOVA_0462.PE24;PPENT:PPENT_039.PE82:PPENT_031.PE258;PPRIMAZ9:PPRIMAZ9_035.PE240:PPRIMAZ9_024.PE269;PTETR32:PTETR32_039.PE87:PTETR32_035.PE272;PTETR51:PTETR51_45.PE240:PTETR51_33.PE262;POCTA138:POCTA138_065.PE234:POCTA138_029.PE91;POCTAK8:POCTAK8_277037.PE81:POCTAK8_277036.PE86;PDECA:PDECA_0135.PE90:PDECA_0121.PE93;PDODE:PDODE_0155.PE74:PDODE_0152.PE70;
FAM013981 POCTA138:POCTA138_039.PE250:POCTA138_067.PE53;PTETR32:PTETR32_004.PE69:PTETR32_055.PE51;PTETR51:PTETR51_29.PE68:PTETR51_41.PE48;PJENN:PJENN_0080.PE81:PJENN_0172.PE24;PSONN:PSONN_033.PE70:PSONN_052.PE272;PQUAD:PQUAD_0022.PE6:PQUAD_0024.PE214;PTRED:PTRED_129426.PE454:PTRED_129079.PE207;PBIAU:PBIAU_0648.PE1:PBIAU_0254.PE29;
FAM011843 PJENN:PJENN_0049.PE108:PJENN_0052.PE107;PSONN:PSONN_056.PE57:PSONN_027.PE159;PDODE:PDODE_0047.PE67:PDODE_0005.PE135;PDECA:PDECA_0079.PE66:PDECA_0075.PE53;PTETR51:PTETR51_102.PE130:PTETR51_40.PE144;PTETR32:PTETR32_106.PE67:PTETR32_047.PE157;POCTA138:POCTA138_133.PE127:POCTA138_055.PE142;POCTAK8:POCTAK8_276947.PE139:POCTAK8_276911.PE120;PPRIMAZ9:PPRIMAZ9_108.PE125:PPRIMAZ9_094.PE56;PPENT:PPENT_126.PE123:PPENT_092.PE57;PNOVA:PNOVA_0010.PE112:PNOVA_0011.PE90;PQUAD:PQUAD_0051.PE94:PQUAD_0065.PE63;PTRED:PTRED_129334.PE109:PTRED_129432.PE140;PBIAU:PBIAU_0145.PE55:PBIAU_0141.PE54;
FAM006172 PBIAU:PBIAU_0030.PE145:PBIAU_0163.PE27;PDECA:PDECA_0017.PE60:PDECA_0009.PE182;POCTAK8:POCTAK8_276662.PE61:POCTAK8_276675.PE78;PTETR51:PTETR51_72.PE147:PTETR51_54.PE182;PTETR32:PTETR32_097.PE59:PTETR32_049.PE87;POCTA138:POCTA138_042.PE66:POCTA138_058.PE183;PTRED:PTRED_129377.PE133:PTRED_129365.PE86;PPENT:PPENT_077.PE69:PPENT_064.PE177;PPRIMAZ9:PPRIMAZ9_081.PE63:PPRIMAZ9_060.PE76;PSONN:PSONN_043.PE209:PSONN_060.PE90;
FAM009510 PBIAU:PBIAU_0076.PE11:PBIAU_0381.PE21;PPENT:PPENT_050.PE206:PPENT_087.PE192;PQUAD:PQUAD_0145.PE11:PQUAD_0055.PE9;POCTA138:POCTA138_081.PE29:POCTA138_068.PE201;PTETR51:PTETR51_64.PE218:PTETR51_79.PE202;PTETR32:PTETR32_080.PE29:PTETR32_109.PE197;PDECA:PDECA_0084.PE141:PDECA_0097.PE28;PDODE:PDODE_0127.PE72:PDODE_0158.PE40;PNOVA:PNOVA_0979.PE3:PNOVA_0686.PE10;PSONN:PSONN_097.PE191:PSONN_072.PE240;PSEXA:PSEXA_063.PE200:PSEXA_085.PE29;
FAM006172 PTRED:PTRED_129439.PE47:PTRED_129418.PE129;POCTA138:POCTA138_069.PE44:POCTA138_040.PE144;PTETR32:PTETR32_086.PE46:PTETR32_048.PE129;PSONN:PSONN_063.PE52:PSONN_073.PE146;
FAM009747 PSEXA:PSEXA_072.PE110:PSEXA_069.PE90;PSONN:PSONN_075.PE162:PSONN_061.PE154;PDODE:PDODE_0073.PE80:PDODE_0004.PE186;POCTA138:POCTA138_026.PE86:POCTA138_088.PE151;POCTAK8:POCTAK8_277090.PE159:POCTAK8_276961.PE130;PTETR51:PTETR51_76.PE160:PTETR51_67.PE140;PTETR32:PTETR32_028.PE283:PTETR32_067.PE151;PQUAD:PQUAD_0109.PE46:PQUAD_0041.PE105;PTRED:PTRED_129423.PE248:PTRED_129449.PE137;PPENT:PPENT_076.PE133:PPENT_088.PE130;PPRIMAZ9:PPRIMAZ9_064.PE96:PPRIMAZ9_071.PE107;PBIAU:PBIAU_0213.PE35:PBIAU_0078.PE110;
FAM007690 PDODE:PDODE_0021.PE173:PDODE_0015.PE171;PDECA:PDECA_0018.PE63:PDECA_0296.PE13;POCTA138:POCTA138_075.PE155:POCTA138_082.PE175;POCTAK8:POCTAK8_276937.PE40:POCTAK8_276721.PE38;PTETR51:PTETR51_94.PE44:PTETR51_81.PE171;PTETR32:PTETR32_107.PE162:PTETR32_065.PE173;PBIAU:PBIAU_0206.PE42:PBIAU_0165.PE8;PNOVA:PNOVA_0059.PE45:PNOVA_0044.PE52;PQUAD:PQUAD_0097.PE95:PQUAD_0257.PE9;PTRED:PTRED_129089.PE226:PTRED_129127.PE45;PPENT:PPENT_121.PE44:PPENT_069.PE41;PPRIMAZ9:PPRIMAZ9_107.PE44:PPRIMAZ9_056.PE164;PSEXA:PSEXA_095.PE42:PSEXA_078.PE168;PJENN:PJENN_0117.PE74:PJENN_0076.PE73;PSONN:PSONN_095.PE50:PSONN_084.PE58;
FAM005810 PSONN:PSONN_113.PE29:PSONN_103.PE10;PSEXA:PSEXA_083.PE26:PSEXA_102.PE161;PBIAU:PBIAU_0318.PE20:PBIAU_0281.PE36;PNOVA:PNOVA_0065.PE62:PNOVA_0051.PE16;PQUAD:PQUAD_0007.PE27:PQUAD_0059.PE10;PTRED:PTRED_129098.PE25:PTRED_129394.PE174;PPENT:PPENT_097.PE152:PPENT_051.PE175;PPRIMAZ9:PPRIMAZ9_103.PE20:PPRIMAZ9_069.PE131;PDECA:PDECA_0060.PE24:PDECA_0058.PE16;POCTA138:POCTA138_120.PE171:POCTA138_107.PE184;POCTAK8:POCTAK8_277077.PE154:POCTAK8_276890.PE187;PTETR32:PTETR32_050.PE154:PTETR32_122.PE191;
FAM009082 PSONN:PSONN_116.PE162:PSONN_114.PE172;PBIAU:PBIAU_0067.PE128:PBIAU_0146.PE11;PPENT:PPENT_147.PE9:PPENT_144.PE143;PPRIMAZ9:PPRIMAZ9_146.PE7:PPRIMAZ9_073.PE14;PNOVA:PNOVA_0043.PE10:PNOVA_0006.PE150;PTRED:PTRED_129403.PE15:PTRED_129327.PE10;PDECA:PDECA_0094.PE8:PDECA_0034.PE10;PDODE:PDODE_0242.PE52:PDODE_0077.PE13;POCTA138:POCTA138_141.PE144:POCTA138_083.PE11;POCTAK8:POCTAK8_276956.PE122:POCTAK8_276949.PE152;PTETR32:PTETR32_146.PE8:PTETR32_126.PE155;PTETR51:PTETR51_155.PE8:PTETR51_107.PE156;
FAM007206 PSONN:PSONN_149.PE81:PSONN_117.PE86;PSEXA:PSEXA_117.PE75:PSEXA_118.PE76;PPENT:PPENT_108.PE82:PPENT_095.PE78;PPRIMAZ9:PPRIMAZ9_110.PE87:PPRIMAZ9_106.PE82;PBIAU:PBIAU_0058.PE71:PBIAU_0095.PE36;PDODE:PDODE_0304.PE31:PDODE_0328.PE29;POCTAK8:POCTAK8_276998.PE78:POCTAK8_277028.PE81;PTETR51:PTETR51_130.PE86:PTETR51_140.PE86;PTETR32:PTETR32_141.PE91:PTETR32_096.PE90;
LCE=node_10; left=node_10: right=node_10:
6302
LCE=node_10; left=node_10: right=X
3171
LCE=PSONN; left=X right=X
1871
LCE=node_9; left=node_9: right=node_9:
204
LCE=node_10; left=X right=node_10:
168
LCE=PSONN; left=node_10: right=X
75
LCE=node_10; left=node_10: right=node_9:
54
LCE=node_10; left=node_9: right=node_10:
51
LCE=node_8; left=node_8: right=node_8:
34
LCE=node_10; left=node_5: right=node_10:
27
LCE=PSONN; left=node_9: right=X
22
LCE=node_10; left=node_8: right=node_10:
18
LCE=node_10; left=node_3: right=node_10:
17
LCE=node_10; left=node_4: right=node_10:
17
LCE=node_8; left=X right=node_8:
17
LCE=PSONN; left=X right=node_10:
17
LCE=node_10; left=node_10: right=node_3:
15
LCE=node_9; left=node_9: right=X
15
LCE=node_10; left=node_10: right=node_5:
14
LCE=node_9; left=node_10: right=node_9:
14
LCE=node_9; left=node_10: right=X
14
LCE=PSONN; left=X right=node_9:
14
LCE=node_9; left=node_9: right=node_10:
12
LCE=PSONN; left=X right=node_8:
11
LCE=node_8; left=node_8: right=node_10:
10
LCE=node_8; left=node_8: right=node_9:
10
LCE=node_9; left=node_10: right=node_8:
9
LCE=node_10; left=node_6: right=node_10:
8
LCE=node_10; left=X right=node_9:
8
LCE=node_9; left=node_8: right=node_10:
8
LCE=node_9; left=X right=node_9:node_11:
8
LCE=node_10; left=node_10: right=node_4:
7
LCE=node_8; left=node_8: right=X
7
LCE= node_9; left=node_10: right=node_9:node_11:
7
LCE= node_9; left=node_9:node_11: right=node_10:
7
LCE=node_9; left=X right=node_8:
7
LCE=PSONN; left=node_8: right=X
7
LCE=node_10; left=node_10: right=node_8:
6
LCE=node_10; left=node_24: right=node_10:
6
LCE=node_10; left=node_5: right=X
6
LCE=node_9; left=X right=node_10:
6
LCE=node_10; left=node_10: right=node_24:
5
LCE=node_10; left=node_10: right=node_6:
5
LCE=node_8; left=node_9: right=node_11:
5
LCE=n ode_10; left=node_10:node_0: right=node_10:
4
LCE=node_10; left=node_9: right=X
4
LCE=node_5; left=node_5: right=node_5:
4
LCE=node_8; left=node_9: right=node_8:
4
LCE=node_9; left=node_10: right=node_11:
4
LCE=node_9; left=node_8: right=node_9:
4
LCE=PSONN; left=X right=node_3:
4
LCE=node_10; left=node_24: right=X
3
LCE=node_10; left=node_3: right=X
3
LCE=node_10; left=node_8: right=X
3
LCE=node_6; left=node_6: right=node_6:
3
LCE=node_8; left=node_10: right=node_8:
3
LCE=node_8; left=X right=node_9:node_11:
3
LCE=node_9; left=node_3: right=node_9:
3
LCE =node_9; left=node_9:node_11: right=node_9:
3
LCE=PSONN; left=node_3: right=X
3
LCE=PSONN; left=X right=node_9:node_11:
3
LCE=n ode_10; left=node_10: right=node_10:node_0:
2
LCE=no de_10; left=node_10: right=node_10:node_16:
2
LCE=node_10; left=node_10: right=node_24:node_0:node_3:
2
LCE=node_10 ; left=node_10: right=node_4:node_0:node_1:
2
LCE= node_10; left=node_10: right=node_4:node_5:
2
LCE=node_10; left=node_10: right=node_7:
2
LCE=n ode_10; left=node_10: right=node_9:node_11:
2
LCE= node_10; left=node_10:node_0: right=node_9:
2
LCE=node_10; left=node_11: right=node_10:
2
LCE=node_10; left=node_24:node_0:node_3: right=node_10:
2
LCE=n ode_10; left=node_24:node_0:node_3: right=X
2
LCE=node_10; left=node_4: right=X
2
LCE=node_10 ; left=node_4:node_0:node_1: right=node_10:
2
LCE=node_10; left=X right=node_4:
2
LCE=node_10; left=X right=node_8:
2
LCE=node_10; left=X right=node_9:node_11:
2
LCE=node_5; left=node_6: right=node_5:
2
LCE =node_8; left=node_11:node_9: right=node_9:
2
LCE=node_8; left=node_3: right=node_8:
2
LCE=node_8; left=node_8: right=node_5:
2
LCE= node_8; left=node_9: right=node_10:node_11:
2
LCE=node_8; left=node_9: right=node_5:
2
LCE=node_8; left=node_9:node_11: right=X
2
LCE=node_9; left=node_10: right=node_24:
2
LCE=node_9; left=node_3: right=X
2
LCE=node_9; left=node_5: right=node_9:
2
LCE=node_9; left=node_8: right=X
2
LCE=node_9; left=node_9: right=node_4:
2
LC E=node_9; left=node_9:node_0: right=node_9:
2
LCE=n ode_9; left=node_9:node_18:node_12: right=X
2
LCE=node_9; left=X right=node_9:
2
LCE=n ode_9; left=X right=node_9:node_18:node_12:
2
LCE=node_9; left=X right=X
2
LCE=PSONN; left=node_11: right=X
2
LCE=PSONN; left=node_24: right=X
2
LCE=PSONN; left=node_4: right=X
2
LCE=PSONN; left=node_5: right=X
2
LCE=PSONN; left=X right=node_0:
2
(Other)
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Mon Dec 19 14:51:21 2022