incorrectly mutation-annotated branch on phylogeny

[akifoss]

Hello,

We recently used TreeTime to infer ancestral sequences for a set of Influenza samples and then visualized the phylogenetic tree with the branches being annotated by the inferred mutations.
In this phylogeny we observed an incorrectly mutation-annotated branch, which doesn't seem to come from an upstream step (see image).

On the very first branch to the left the mutation "T216A" is displayed in the picture. On the branch highlighted by the first red circle one can however observe the reverse mutation "A216T". This is correct for the 3 samples at the very top, but incorrect for the 2 highlighted with the second red circle. As of now we have no possible explanation for this and hope that we can receive some tips on how to solve this.

General Information:
The GISAID IDs used are:
EPI_ISL_391019, EPI_ISL_392654, EPI_ISL_4005759, EPI_ISL_9140500, EPI_ISL_417210, EPI_ISL_406339, EPI_ISL_404460, EPI_ISL_8767089, EPI_ISL_12109635, EPI_ISL_13531231, EPI_ISL_18044584, EPI_ISL_18008523, EPI_ISL_17832068, EPI_ISL_17392718, EPI_ISL_17392712, EPI_ISL_18110329, EPI_ISL_18008533, EPI_ISL_17625845, EPI_ISL_15928563, EPI_ISL_16714267, EPI_ISL_16681646, EPI_ISL_17543516, EPI_ISL_16968010, EPI_ISL_16465046, EPI_ISL_16043978, EPI_ISL_16003478, EPI_ISL_17102775, EPI_ISL_17072386, EPI_ISL_17465826, EPI_ISL_17352967, EPI_ISL_18900944, EPI_ISL_18900945, EPI_ISL_18900946, EPI_ISL_18900947, EPI_ISL_18900948, EPI_ISL_18900949, EPI_ISL_18900950, EPI_ISL_18900951, EPI_ISL_18900952, EPI_ISL_18900953, EPI_ISL_18900954, EPI_ISL_18900955, EPI_ISL_18900956, EPI_ISL_18900957, EPI_ISL_18900958, EPI_ISL_18900959, EPI_ISL_18900960, EPI_ISL_18900961, EPI_ISL_18900962, EPI_ISL_18900963, EPI_ISL_18900964, EPI_ISL_18900965, EPI_ISL_18900966, EPI_ISL_18900967, EPI_ISL_18900968, EPI_ISL_18900969, EPI_ISL_18900970, EPI_ISL_18900971, EPI_ISL_18900972, EPI_ISL_18900973, EPI_ISL_18900974, EPI_ISL_18900975, EPI_ISL_18900976, EPI_ISL_18900977, EPI_ISL_18900978, EPI_ISL_18900979, EPI_ISL_18900980, EPI_ISL_18900981

Upstream Steps:
iqtree==2.2.2.7 (phylogenetic inference)
mafft==7.520 (MSA)

Any help/tips are appreciated - thanks!

[@DimitriTernovoj watching too]

[rneher]

that is very strange indeed. Would you mind sharing tree and alignment (via email for example). I am a registered GISAID user, so this should be ok and would save me the the trouble of retracing your steps.