Phylogenomics reveals Adeleorina are an ancient and distinct subgroup of Apicomplexa.

Apicomplexans are a diverse phylum of unicellular eukaryotes that share obligate relationships with terrestrial and aquatic animal hosts. Many well-studied apicomplexans are responsible for several deadly zoonotic and human diseases, most notably malaria caused by Plasmodium. Interest in the evolutionary origin of apicomplexans has also spurred recent work on other more deeply-branching lineages, especially gregarines and sister groups like squirmids and chrompodellids. But a full picture of apicomplexan evolution is still lacking several lineages, and one major, diverse lineage that is notably absent is the adeleorinids. Adeleorina apicomplexans comprises hundreds of described species that infect invertebrate and vertebrate hosts across the globe. Although historically considered coccidians, phylogenetic trees based on limited data have shown conflicting branch positions for this subgroup, leaving this question unresolved. Phylogenomic trees and large-scale analyses comparing cellular functions and metabolism between major subgroups of apicomplexans have not incorporated Adeleorina because only a handful of molecular markers and a couple organellar genomes are available, ultimately excluding this group from contributing to our understanding of apicomplexan evolution and biology. To address this gap, we have generated complete genomes from mitochondria and plastids, as well as multiple deep-coverage single-cell transcriptomes of nuclear genes from two Adeleorina species, Klossia helicina and Legerella nova, and inferred a 206-protein phylogenomic tree of Apicomplexa. We observed distinct structures reported in species descriptions as remnant host structures surrounding adeleorinid oocysts. Klossia helicina and L. nova branched, as expected, with monoxenous adeleorinids within the Adeleorina and their mitochondrial and plastid genomes exhibited similarity to published organellar adeleorinid genomes. We show with a phylogeneomic tree and subsequent phylogenomic analyses that Adeleorina are not closely related to any of the currently sampled apicomplexan subgroups, and instead fall as a sister to a large clade encompassing Coccidia, Protococcidia, Hematozoa, and Nephromycida, collectively. This resolves Adeleorina as a key independently-branching group, separate from coccidians, on the tree of Apicomplexa, which now has all known major lineages sampled.

Lizard host abundances and climatic factors explain phylogenetic diversity and prevalence of blood parasites on an oceanic island.

Host abundance might favour the maintenance of a high phylogenetic diversity of some parasites via rapid transmission rates. Blood parasites of insular lizards represent a good model to test this hypothesis because these parasites can be particularly prevalent in islands and host lizards highly abundant. We applied deep amplicon sequencing and analysed environmental predictors of blood parasite prevalence and phylogenetic diversity in the endemic lizard Gallotia galloti across 24 localities on Tenerife, an island in the Canary archipelago that has experienced increasing warming and drought in recent years. Parasite prevalence assessed by microscopy was over 94%, and a higher proportion of infected lizards was found in warmer and drier locations. A total of 33 different 18s rRNA parasite haplotypes were identified, and the phylogenetic analyses indicated that they belong to two genera of Adeleorina (Apicomplexa: Coccidia), with Karyolysus as the dominant genus. The most important predictor of between-locality variation in parasite phylogenetic diversity was the abundance of lizard hosts. We conclude that a combination of climatic and host demographic factors associated with an insular syndrome may be favouring a rapid transmission of blood parasites among lizards on Tenerife, which may favour the maintenance of a high phylogenetic diversity of parasites.

Genetic diversity, phylogenetic position, and co-phylogenetic relationships of Karyolysus, a common blood parasite of lizards in the western Mediterranean.

The genus Karyolysus was originally proposed to accommodate blood parasites of lacertid lizards in Western Europe. However, recent phylogenetic analyses suggested an inconclusive taxonomic position of these parasites of the order Adeleorina based on the available genetic information. Inconsistencies between molecular phylogeny, morphology, and/or life cycles can reflect lack of enough genetic information of the target group. We therefore surveyed 28 localities and collected blood samples from 828 lizards of 23 species including lacertids, skinks, and geckoes in the western Mediterranean, North Africa, and Macaronesia, where species of Karyolysus and other adeleorine parasites have been described. We combined molecular and microscopic methods to analyze the samples, including those from the host type species and the type locality of Karyolysus bicapsulatus. The phylogenetic relationship of these parasites was analyzed based on the 18S rRNA gene and the co-phylogenetic relationship with their vertebrate hosts was reconstructed. We molecularly detected adeleorine parasites in 37.9% of the blood samples and found 22 new parasite haplotypes. A phylogenetic reconstruction with 132 sequences indicated that 20 of the newly detected haplotypes clustered in a well-supported clade with another 18 sequences that included Karyolysus galloti and Karyolysus lacazei. Morphological evidence also supported that K. bicapsulatus clustered in this monophyletic clade. These results supported the taxonomic validity of the genus. In addition, we found some parasite haplotypes that infected different lizard host genera with ancient diverging histories, which suggested that Karyolysus is less host-specific than other blood parasites of lizards in the region. A co-phylogenetic analysis supported this interpretation because no significant co-speciation signal was shown between Karyolysus and lizard hosts.

The genetic and morphological diversity of Haemogregarina infecting turtles in Colombia: Are mitochondrial markers useful as barcodes for these parasites?

Adeleorinid parasites commonly infect turtles and tortoises in nature. Currently, our knowledge about such parasites is extremely poor. Their characterization is based on morphological and molecular approaches using the 18S rDNA molecular marker. However, there is a limitation with the 18S rDNA due to its slow rate of evolution. For that reason, the goals of this study were to 1) design primers for new molecular mitochondrial markers to improve the phylogenetic reconstructions of adeleorinid parasites and 2) to determine the morphological and genetic diversity of Haemogregarina infecting turtles and tortoises in Colombia. Turtles from 16 species representing six families were examined for the presence of haemoparasites. We analyzed 457 samples using PCR, and 203 of them were also analyzed by microscopy. Using a mitochondrial genome of Haemogregarina sequenced in this study, we designed primers to amplify fragments of the cytochrome oxidase I (coxI), cytochrome oxidase III (coxIII), and cytochrome b (cytb) mitochondrial markers in adeleorinid parasites. Lineages obtained from nuclear and mitochondrial molecular markers clustered according to the turtle lineages from which they were isolated. It is noteworthy that we found different evolutionary lineages within the same morphotype, which may indicate heteroplasmy and/or cryptic diversity in Haemogregarina. Due to this situation, we could not make a species delimitation, even when integrating the different lines of evidence we had in this study. However, the primers presented here are useful for diagnosis and, moreover, according to the available information, all three genes retain phylogenetic signals; thereby fragments amplified can be used in reconstructing evolutionary relationships. This effort contributes to the knowledge of the diversity of these parasites infecting continental turtles from Colombia.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships.

Haemogregarines (Adeleorina) have a high prevalence in turtles. Nevertheless, there is only one Hepatozoon species described that infects Testudines so far; it is Hepatozoon fitzsimonsi which infects the African tortoise Kinixys belliana. Colombia harbours a great diversity of chelonians; however, most of them are threatened. It is important to identify and characterize chelonian haemoparasite infections to improve the clinical assessments, treatments and the conservation and reintroduction programs of these animals. To evaluate such infections for the Colombian wood turtle Rhinoclemmys melanosterna, we analysed blood from 70 individuals. By using the morphological characteristics of blood stages as well as molecular information (18S rRNA sequences), here we report a new Hepatozoon species that represents the first report of a hepatozoid species infecting a semi-aquatic continental turtle in the world. Although the isolated lineage clusters within the phylogenetic clades that have morphological species of parasites already determined, their low nodal support makes their position within each group inconclusive. It is important to identify new molecular markers to improve parasite species identification. In-depth research on blood parasites infecting turtles is essential for increasing knowledge that could assess this potential unknown threat, to inform the conservation of turtles and for increasing the state of knowledge on parasites.

Multilocus sequencing of Hepatozoon cf. griseisciuri infections in Ontario eastern gray squirrels (Sciurus carolinensis) uncovers two genotypically distinct sympatric parasite species.

Intra-leukocytic gamonts consistent with the description of Hepatozoon griseisciuri Clark, 1958 are reported for the first time in Canadian eastern gray squirrels (Sciurus carolinensis Gmelin, 1788). Polymerase chain reaction (PCR) amplification and direct Sanger sequencing identified a pair of distinct genotypes at both a nuclear and mitochondrial locus; two 18S ribosomal RNA gene sequences (rDNA; genotype A and genotype B: 1816 base pairs (bp); 98.8% pairwise identity) and 2 distinct complete mitochondrial genome sequences (genotype A: 6311 bp; genotype B: 6114 bp; 89.1% pairwise identity) were obtained from 3 H. griseisciuri-infected squirrels sampled in Guelph, Ontario. The genetic content of both circular-mapping mitochondrial genomes was conventional for apicomplexan protists; each encoded for 3 protein-coding genes (cytochrome c oxidase subunit I (COI); cytochrome c oxidase subunit III (COIII); and cytochrome B (CytB)), 14 fragmented large subunit rDNA, 10 fragmented small subunit rDNA, and 8 unassigned rDNA. These genotypes, based on sequences obtained from a pair of loci from two parasite genomes, confirm the presence of at least two Hepatozoon species infecting Ontario eastern gray squirrels, one of which is likely to be conspecific with H. griseisciuri.

Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences.

Extrachromosomal genomes of the adeleorinid parasite Hepatozoon canis infecting an Israeli dog were investigated using next-generation and standard sequencing technologies. A complete apicoplast genome and several mitochondrion-associated sequences were generated. The apicoplast genome (31,869 bp) possessed two copies of both large subunit (23S) and small subunit (16S) ribosomal RNA genes (rDNA) within an inverted repeat region, as well as 22 protein-coding sequences, 25 transfer RNA genes (tDNA) and seven open reading frames of unknown function. Although circular-mapping, the apicoplast genome was physically linear according to next-generation data. Unlike other apicoplast genomes, genes encoding ribosomal protein S19 and tDNAs for alanine, aspartic acid, histidine, threonine and valine were not identified. No complete mitochondrial genome was recovered using next-generation data or directed PCR amplifications. Eight mitochondrion-associated (215-3523 bp) contigs assembled from next-generation data encoded a complete cytochrome c oxidase subunit I coding sequence, a complete cytochrome c oxidase subunit III coding sequence, two complete cytochrome B coding sequences, a non-coding, pseudogene for cytochrome B and multiple fragmented mitochondrial rDNA genes (SSUA, SSUB, SSUD, LSUC, LSUG, RNA6, RNA10, RNA14, RNA18). The paucity of NGS reads generating each of the mitochondrion-like sequences suggested that a complete mitochondrial genome at typically high copy number was absent in H. canis. In contrast, the complete nuclear rDNA unit sequence of H. canis (18S rDNA to 28S rDNA, 6977 bp) had >1000-fold next-generation coverage. Multiple divergent (from 93.6% to 99.9% pairwise identities) nuclear 18S rDNA contigs were generated (three types with 10 subtypes total). To our knowledge this is the first apicoplast genome sequenced from any adeleorinid coccidium and the first mitochondrion-associated sequences from this serious pathogen of wild and domestic canids. These newly generated sequences may provide useful genetic loci for high-resolution species-level genotyping that is currently impossible using existing nuclear rDNA targets.

Klossiella equi Infecting Kidneys of Ontario Horses: Life Cycle Features and Multilocus Sequence-Based Genotyping Confirm the Genus Klossiella Belongs In the Adeleorina (Apicomplexa: Coccidia).

Species in the genus Klossiella Smith and Johnson, 1902 are unique among the suborder Adeleorina because they are monoxenous in mammals exclusively, whereas all other reported members of the Adeleorina use invertebrates as definitive hosts. Unlike other coccidia, all members of the Adeleorina undergo syzygy, the association of microgamonts and macrogamonts before maturation to gametes and syngamy. After fertilization, many members of the Adeleorina produce thin-walled polysporocystic oocysts. Despite being biologically similar to other members of the Adeleorina, the phylogenetic placement of the genus Klossiella has been questioned based on its unique host affinity. In the present study, 2 cases of Klossiella equi were reported from the kidneys of horses in Ontario. Details of the life cycle as well as mitochondrial and nuclear 18S ribosomal DNA ( 18S rDNA) sequences were analyzed to provide both morphological and molecular evidence for the phylogenetic placement of K. equi. Initially, various stages of the life cycle were identified in histological slides prepared from the kidney tissue, and DNA was isolated from the infected tissue. Polymerase chain reaction and Sanger sequencing were used to generate a complete mitochondrial genome sequence (6,569 bp) and a partial 18S rDNA sequence (1,443 bp). The K. equi 18S rDNA sequence was aligned with various publicly available apicomplexan 18S rDNA sequences. This alignment was used to generate a phylogenetic tree based on Bayesian inference. Multiple K. equi stages were identified including meronts, microgamonts, and macrogamonts associating in syzygy as well as thin-walled oocysts in various stages of sporogonic development. The 18S rDNA sequence of K. equi positioned within the monophyletic Adeleorina clade. The mitochondrial genome of K. equi contained 3 coding sequences for cytochrome c oxidase I, cytochrome c oxidase III, and cytochrome b as well as various fragmented ribosomal sequences. These components were arranged in a unique order that has not been observed in other apicomplexan mitochondrial genomes sequenced to date. Overall, it was concluded that there were sufficient morphological and molecular data to confirm the placement of K. equi and the genus Klossiella among the Adeleorina. The biological and molecular data obtained from these cases may assist with future studies evaluating the prevalence and life history of this seemingly underreported parasite and better define the impact of K. equi on the health of domestic and wild equids.