Hyaloklossia spp

Tokiwa, Toshihiro, Chou, Shyun, Tochigi, Yuki, Katayama, Kentaro & Duszynski, Donald W., 2021, Hyaloklossia Labb´e, 1896 (Alveolata: Apicomplexa) in frogs: Description of a new species and proposing a new subfamily to accommodate these enigmatic parasites, International Journal for Parasitology: Parasites and Wildlife 15, pp. 199-207 : 204

3.2. Sequence analyses of Hyaloklossia spp

3.2.1. Sequence similarity

The partial 18S gene sequence (1449 bp) of H. kasumiensis was 99.7% identical (1445/1449 bp) with that of H. lieberkuehni (accession no. AF298623) reported from the edible frog in the Czech Republic ( Modrý et al., 2001) but similarity with other genera in the Sarcocystidae was less than 97.5%.

The partial 28S sequence (1422 bp) of H. kasumiensis revealed 98.6% identity (955/969 bp) with that of H. lieberkuehni (accession no. AF513499), also from the edible frog in the Czech Republic ( Modrý et al., 2001). However, H. kasumiensis displayed similarity values <90.8% identity with other genera in the Sarcocystidae .

A partial cox1 sequence (910 bp) also was identified from H. kasumiensis and it had 87.9%–87.3% identity with the sequence from Cystoisospora spp. (nos. MF774038, MF774037, KT184385, LC377843, KU184384). The cox1 sequence of H. lieberkuehni was not reported in the INSD, and therefore, was unavailable for comparison.

Accumulated nucleotide substitutions in the 18S and 28S genes between H. kasumiensis from P. p. porosus in Japan and H. lieberkuehni from the edible frog in the Czech Republic were 0.23% and 1.76%, respectively ( Table 4). Some pairs of congeners among the other sarcocystids showed up to 1.20% divergence in the 18S sequence and 3.35% divergence in the 28S sequence. However, several pairs showed divergence values lower than or equal to the divergences observed between H. kasumiensis and H. lieberkuehni . In particular, the inter-specific divergence for both 18S and 28S genes between our species and H. lieberkuehni was higher or equivalent to that observed for Heydornia heydorni vs. Heydornia triffittae (18S: 0%, 28S: 0.35%) and Besnoitia bennetti vs. Besnoitia tarandi (18S: 0.23%, 28S: 0.35%) ( Table 4). Furthermore, the inter-genus divergence between Toxoplasma gondii vs.

nc = not calculated.

Neospora caninum (18S: 0.23%), N. caninum vs. Hammondia hammondi (18S: 0.23%, 28S: 1.41%), and N. caninum vs. H. heydorni (18S: 0.23%) was lower than that observed between H. kasumiensis and H. lieberkuehni .