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I am performing an AMOVA analysis via Arlequin.

At this point, I have:

  1. collected sequences of mtDNA from the cox1 gene,
  2. aligned the different genes using clustalX
  3. converted the files into .arp format.

I then altered the structure of my populations in Arlequin so that all the different populations are in one group. When I tried to run AMOVA I get the error message

"Ill-Defined genetic structure".

Any clues as to what I have to adjust?

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If one group is one population this will not work with Arlequin AMOVA. There must be more than one population (I might have misread the question however).

There could several reasons for this output. The most likely is there are insufficient SNPs within the cox1 locus for MANOVA to identify a population structure with sufficient statistical robustness. What it requires are a minimum of 3 independent alleles (SNPs), really 3 genotypes. If the SNPs are linked on the same bit of DNA that doesn't count - thats just one genotype. What I suspect is happening is there is a dearth (not enough) genetic information within cox1 to perform MANOVA.

There could be other explanations, but this is the first issue to address. This isn't just Arlequin it is standard theory for any allele frequency based calculations and why this is important is more complicated to explain.


Please note, it is the run-up to Christmas .. okay put Christmas on hold. To respond to the comments ...

Firstly, if you hold the data set it is very easy to identify the number of genotypes. There are past posts on this herein. I am not inclined to describe alternative approaches to an algorithm I've still not yet published :-) ... however it is really easy and needs an empirical test. It is good to assess number of genotypes per se Arlequin will do this analysis ... finding that within the output I dunno.

Extending loci is always good and for mtDNA I would not recommend with 16S nor cox2. rRNA should be avoided in allele frequency calculations, its okay for taxonomic assignment (bacteria rather than mtDNA). cox2 is a small locus. I would recommend for mtDNA

  • cobB, formerly called cytB
  • intergenic region of mtDNA

cobB is a good target, it has a lot more selection on it for within species analysis than cox1. The intergenic region is rarely used, its a good locus because of the higher rates of mutation therein.

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    $\begingroup$ Would you suggest using a different genetic marker for this type of analyses? I do not have access to SNPs so the next best thing would be other mtDNA genes like COX2 and rRNA. $\endgroup$
    – Nickmofoe
    Commented Dec 19, 2022 at 14:01
  • $\begingroup$ @Nickmofoe answered ... Merry Christmas BTW. $\endgroup$
    – M__
    Commented Dec 20, 2022 at 16:22
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    $\begingroup$ Alright so: The COX1 locus ended up not being the issue. I made some corrections to the flat files and they seemed to run with no issues. I got the results and everything seems to check out. I concluded that the issue was not the locus itself but rather the incredibly complicated .arp format. Thanks for the advice though! Much appreciated and merry Christmas!!! $\endgroup$
    – Nickmofoe
    Commented Dec 22, 2022 at 9:23
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    $\begingroup$ Merry Christmas to you @nickmofoe! The "complicated format" was why Arlequin was never popular, because everybody felt it was unnecessarily complicated. This why your original question on PGDSpider was interesting because it as overdue requirement to make Arlequin accessible. $\endgroup$
    – M__
    Commented Dec 22, 2022 at 13:34

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