I have identified fungus isolates from the fruit surface by SSU +ITS+ LSU regions, such as :
1) Fusarium kyushuense , 2) Fusarium proliferatum ;3) Aspergillus aculeatus ;4) Aspergillus europaeus ; 5) Aspergillus flavus, etc
now i want to construct a phylogenetic tree,
1-so how many reference sequences (From NCBI) should use for an individual fungus isolate? and those references isolate should be from fruit source (isolated from fruit host)?
2-If found many references isolate sequence so what's the criteria for selection and use in the phylogeny?
3-If can not found some reference sequence for a fungus isolate from fruit source then can use from other sources such as soil, etc?
I may suggest you this crash course: https://viralzone.expasy.org/e_learning/phylogenetics/content.html
Citing the crash-course: "What to choose for the Outgroup? There are two requirements for the outgroup:
- It should absolutely not belong to the group under study, otherwise the tree's topology will be hopelessly wrong
- It should not be too distantly related either, because it must be aligned with the other sequences. If it is too distantly related, the alignment quality may suffer.
In conclusion, a good outgroup would be a member of a sister clade. For example to produce a phylogeny of FMDV, one would choose another Picornavirus. But the sister clade of the group under study may not be known, and it may be safer in this case to choose a more distant relative."
*My direct answer for you would be that you only need one, chosen correctly
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