(updated June 2023)
The phylogenetic tree below shows relationship among 18S rRNA sequences for “almost complete” sequences (i.e., sequences that exceed 2000 bases in length in the DNA databases), from isolates classified as belonging to T4, as determined using the neighbor-joining. Attempts to examine the phylogenetic relationship within T4 by maximum-likelihood, maximum parsimony and Bayesian inference yield trees with similar shape, although relationships between isolates within a subtype shift, as does some of the branching order between subtypes. Further support for the hypothesis represented by the tree below must await additional information from other parts of the genome. Some additional support of the validity of subtypes has been obtained from analysis of the alleles within the DF3 region of the 18S rRNA gene sequence. Further consideration of species designations is given below.
FIGURE: Phylogenetic relationships among T4 subtypes. Tree rooted using relationships of T4 isolates with members of sequence types T3 or T11. The numbers for each subgroup listed on the left indicated the estimated totals of all sequences (partial or nearly complete) in the database that could be classified into a particular subtype as of January 2020. A list of the subtype classification for “almost complete” T4 sequences from the DNA databases will be posted soon. Note also that a small number of isolates represented in the figure above show some characteristics of chimeric sequences (either chimeric sequences between alleles within a subtype or chimeras between subtypes; chimeric isolates involving segments of T4 with segments from other sequence types have been removed). This further complicates the difficulty of defining monophyletic subgroups.
Note on new subtype T4H (see below) – this subtype, not shown in the tree above and first suggested in 2023, appears to be a sister group to the T4neff (or T4G) subtype.
We have broken the T4 sequence type into at least 8 sub-types which appear to represent significant phylogenetic subgroups within T4. The most frequent subtype (T4A) also shows evidence of having the most within subtype diversity of alleles within the DF3 region of the gene. However, sequence divergence between isolates within T4A is small and apparent sub-subgroups within T4A cannot be identified with high confidence (except within our own mind’s eye).
We hypothesize that the sub-types of T4 represent one of the the best guesses of species delimitation within Acanthamoeba. Certainly, sequence types could be considered to represent “species.” However, there is an additional level of variation below the level of sequence type. Given that the tree above is based on a single gene, we are hesitant to place final weight on the results here. However, we are working on finalizing information from seven other genes for a sample of isolates spread over the sub-types which should provide insight into whether these can be considered to represent “species”.
REPRESENTATIVE ISOLATES FOR SUBTYPES
The T4 subtypes can be associated with representative standard ATCC strains and corresponding GenBank accession numbers
T4A – A. castellanii [ATCC 30011] (U07413)
T4B – A. castellanii Ma [ATCC 50370] (U07414)
T4C – A. sp. Fernandez [ATCC 50369] (U07409)
T4D – A. rhysodes Singh [ATCC 30973] (AY351644)
T4E – A. polyphaga Page-23 [ATCC 30871] (AF019061)
Examination of partial sequence of the 18S rRNA gene suggests that there may be substantial variation within the T4E subtype that has yet to be recognized. This may result in further subtypes broken out from within this subtype.
T4F – A. triangularis SH621 [ATCC 50254] (AF346662)
T4-Neff or T4G – A. castellanii Neff [ATCC 30010] (U07416) [note that the Neff strain has been shown to share mitochondrial 16S-like rRNA sequence with the homologous gene from A. terricola (ATCC 30134; Genbank AF479561). However, information for the nuclear 18S rRNA gene is lacking in the DNA databases].
ARE SUBTYPES EQUIVALENT TO SPECIES ?
Note that in our list of representative isolates, several subtypes are defined by the same species name (castellanii). Thus, despite our reservations about naming “species”, several comments on the occurrence of important standard strains within this phylogenetic setting are appropriate.
1 – The type strain for Acanthamoeba (ATCC 30011), representing the original isolate made by Castellani, occurs in subtpe T4A, the most abundant genetic form within Acanthamoeba. This strain is also considered the type strain of Acanthamoeba castellanii. If subtypes correspond to species, this implies that any isolate outside of subtype T4A cannot be a member of A. castellanii.
2 – Point 1 has important implications concerning the Neff strain, which does not represent the most frequent subtype (T4A), but rather a less frequent type designated here T4Neff, which is significantly differentiated from T4A. The Neff strain is classified morphologically as a member of A. castellanii, but genetically is not grouped with the subtype T4A. Use of the term “A. castellanii” in reference to the Neff strain would be incorrect. In the future is should be referred to as A. sp. Neff until an appropriate name can be assigned.
3 – The species name Acanthamoeba polyphaga is very problematic. Although we use A. polyphaga Page-23 as a representative of subtype T4E, the species name appears in several other subtypes (and outside of sequence type T4 as well). Examination of species descriptions, and associated standard strains is underway to determine priority for the name if there is a reconsideration of species nomenclature in Acanthamoeba.
ALLELIC VARIATION WITHIN SUBTYPES
Variation within subtype classification is also augmented by consideration of allelic variation within the DF3 region of the 18S rRNA gene. Detailed information on allelic variants within subtypes is given elsewhere in this site. Specific allele types appear to be primarily (or in most cases exclusively) restricted into single subtypes.