Amanita penetrans

Problems with deriving nrDNA gene sequences for Amanita penetrans.
Derivation of an nrITS sequence from Amanita penetrans by means of Sanger Process sequencing alone appears to be difficult.  Possibly, this is due to failure of the rDNA repeat to homogenize—as has been suggested for other species of Amanita (e.g., A. cornelihybrida).

In the case of the present species, the nrLSU sequence is also difficult to derive.  We have been able to obtain several fragments comprising up to the last 94 characters of ITS2 (with capability to differentiate the species moderately well) and 148 to 485 characters of the 5' end of nrLSU (including some hets).

We have been able to replicate derivation of portions (or nearly all) of a second, more conserved, region up to 858 characters long.  This region is largely covered by reads using the following primers (LR5, LR3R, and LR7).  We select the following sequence (with hets/SNPs as noted) to indicate the 5' end of the second region: 5'-GGGTSGTTTCGMCCAYGTTCACGCTTAGGATGTTGGYTTAATGGCATTAA.

As might be expected from the relatively high degree of conservation, the latter fragment is of limited use for identification purposes (e.g., by BLAST).  However, even in this region, the sequences may contain hets within the initial 82 characters; and the length of derived sequences have varied greatly—from 335 to 858 characters.

Distinguishing Amanita justicei and A. penetrans with apparently constant character differences in nrLSU gene

The nrITS sequences for these two species are difficult to derive and are confusingly similar and often contain a number of ambiguities.

We find that there are five features in the nrLSU gene that, together, distinguish the two taxa.  To find these taxa we created a 2,021 character alignment comprising all nrITS and nrLSU sequences obtained to date (9 Nov. 2019) for the either of the two species.  The number of such sequences is 18.

It is important to note that since deriving a sequence containing the entirety of nrITS has been possible for us only once in the case of the two species in question, no comparison could be made for the first 444 characters of the longest sequence in the alignment.  The nrLSU gene begins (with "TCTGAACTCAAATCA") at character 533 of the alignment.

The distinguishing differences between the two species and related positioning information in the alignment follow:

• char. 614:
  T (justicei), G (penetrans)
  
• char. 1191:
  T (justicei), A (penetrans)
  
• chars. 1270-1272:
  TTT (justicei), TT (penetrans)
  
• char. 1936
  C (justicei), T (penetrans)
  
• char. 1939
  A (justicei), T (penetrans)