Absence of Hemolivia mauritanica (Apicomplexa: Haemogregarinidae) in natural populations of Testudo graeca in Morocco.

During spring 2018, we captured 101 spur-thighed tortoises, Testudo graeca, from seven localities in central Morocco. All tortoises were examined for the presence of blood parasites Hemolivia mauritanica and Hyalomma aegyptium ticks, the known vectors. We looked for H. mauritanica infection by examination of blood smears and by genetic analysis with PCR using extractions from both tortoises and ticks. On all tortoises collected, 71.29% were infested with at least one tick, with a mean infestation intensity of 7.12 ticks/tortoise and maximum of 15.55 ticks/tortoises in Had Draa locality (Essaouira region). Although the definitive host is present and abundant in all tortoise populations, all blood samples were Hemolivia-negative. Our results support and confirm the finding of studies previously conducted in other populations of Morocco and indicate that H. mauritanica has a narrower distribution range than its tick vector.

Morphometric and molecular characterisation of Hepatozoon bashtari n. sp. in painted saw-scaled viper, Echis coloratus (Ophidia, Viperidae).

Hepatozoon species are the most widely known haemogregarines infecting a wide range of vertebrates, although predominately snakes. Herein, Hepatozoon bashtari n. sp., originally infecting the painted saw-scaled viper, Echis coloratus, in Saudi Arabia is described using both morphological features and molecular data from 18S rDNA sequences. The overall prevalence of infection was 60% (9/15) with parasitaemia ranging from 52 to 60%. Gamonts were entirely intraerythrocytic and were observed to cause considerable hypertrophy within the host cell. The mean size of mature gamonts was 15.4 × 3.3 µm. Merogonic stages were confined to the lung endothelial cells with monomorphic meronts. The average size of mature meronts was 32 × 12 µm and they were estimated to produce 13-16 merozoites each. The phylogenetic tree generated from SSU rDNA sequences revealed that Hepatozoon bashtari sp. n. clusters with the vast majority of other Hepatozoon species infecting snakes, lizards and geckos in various regions of the world, which would appear to support the hypothesis of prey-predator transmission of the genus Hepatozoon. Through a combination of morphological comparison with closely related Hepatozoon spp. and 18S rRNA gene sequence analysis, it is possible to confirm Hepatozoon bashtari sp. n. as a new species.

Redescription of Hepatozoon ameivae (Carini and Rudolph, 1912) from the lizard Ameiva ameiva (Linnaeus, 1758).

Blood samples from 72 Ameiva ameiva lizards from Central Amazonian upland forests were collected, and thin smears of 40 (55.5%) animals were positive for gamonts of Hepatozoon with a mean level of intensity of infection of 14 parasites/2000 blood erythrocytes (0.73%). The gametocytes were found attached with host cells' nuclei, and their dimensions were 14.28 ± 1.05 µm in length and 4.50 ± 0.80 µm in width. Phylogenetic analyses of the 18S rRNA gene showed that the new sequences obtained from A. ameiva constitute a monophyletic sister clade to the Hepatozoon spp. from Brazilian snakes. Based on morphological features and new molecular data, we redescribe this hemogregarine as Hepatozoon ameivae. This study also provides the first molecular characterization of a Hepatozoon species from a Brazilian lizard.

Molecular characterisation and morphological description of two new species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina: Hepatozoidae) infecting leukocytes of African leopards Panthera pardus pardus (L.).

BACKGROUND: The African leopard Panthera pardus pardus (L.) is currently listed as a vulnerable species on the IUCN (International Union for the Conservation of Nature) red list of threatened species due to ongoing population declines. This implies that leopard-specific parasites are also vulnerable to extinction. Intracellular apicomplexan haemoparasites from the genus Hepatozoon Miller, 1908 have been widely reported from wild carnivores in Africa, including non-specific reports from leopards. This paper describes two new haemogregarines in captive and wild leopards from South Africa and provides a tabular summary of these species in relation to species of Hepatozoon reported from mammalian carnivores. METHODS: Blood was collected from nine captive and eight wild leopards at various localities throughout South Africa. Thin blood smears were Giemsa-stained and screened for intraleukocytic haemoparasites. Gamont stages were micrographed and morphometrically compared with existing literature pertaining to infections in felid hosts. Haemogregarine specific primer set 4558F and 2733R was used to target the 18S rRNA gene for molecular analysis. Resulting sequences were compared to each other and with other available representative mammalian carnivore Hepatozoon sequences from GenBank. RESULTS: Two species of Hepatozoon were found in captive and wild leopards. Of the 17 leopards screened, eight were infected with one or both morphologically and genetically distinct haemogregarines. When compared with other species of Hepatozoon reported from felids, the two species from this study were morphometrically and molecularly distinct. Species of Hepatozoon from this study were observed to exclusively parasitize a particular type of leukocyte, with Hepatozoon luiperdjie n. sp. infecting neutrophils and Hepatozoon ingwe n. sp. infecting lymphocytes. Phylogenetic analysis showed that these haemogregarines are genetically distinct, with Hepatozoon luiperdjie n. sp. and Hepatozoon ingwe n. sp. falling in well supported separate clades. CONCLUSIONS: To our knowledge, this is the first morphometric and molecular description of Hepatozoon in captive and wild African leopards in South Africa. This study highlights the value of using both morphometric and molecular characteristics when describing species of Hepatozoon from felid hosts.

Hepatozoon milleri sp. nov. (Adeleorina: Hepatozoidae) in Akodon montensis (Rodentia: Cricetidae: Sigmodontinae) from southeastern Brazil.

Members of the genus Hepatozoon (Miller, 1908) are blood parasites found in a wide range of host species, including wild rodents; however, information about the life cycle, distribution and Hepatozoon species diversity infecting these mammals are lacking. We studied the parasite stages and DNA sequences of Hepatozoon sp. of 11 naturally infected Akodon montensis. Thin blood smears, tissue samples and whole blood were obtained for morphology, morphometry and molecular analyses. Seven of the 11 rodents had gamonts on the blood smears. Biological and morphological features of the parasite such as tissue tropism, gamonts and meronts size and morphology, as well as the DNA sequence comparison and phylogenetic analysis, indicated that the Hepatozoon sp. detected in this study is distinct from those species previously reported in small rodents. Herein, we propose a new species, named Hepatozoon milleri sp. nov. This is the first description of a new Hepatozoon species from wild small rodents in Brazil, based on morphological and molecular characteristics.

Morphology and Phylogeny of Calyptospora paranaidji n. sp. (Eimeriorina: Calyptosporidae), an Apicomplexan Parasite of the Hepatic Tissue of Cichla piquiti Kullander & Ferreira, 2006, From a Reservoir in the Brazilian Amazon Region.

The coccidians of the family Calyptosporidae are parasites of the tissue and organs of fish and aquatic invertebrates, in particular in the tropical region. In contrast with other apicomplexans of the suborder Eimeriorina, the diversity and ecology of the species of the genus Calyptospora have been poorly investigated, resulting in a lacuna that restricts the understanding of the distribution and prevalence of this group of eukaryote microparasites in the Amazon region. In the present study, the integrated comparative analysis of morphological characteristics, histological and structural traits, and the sequences of a fragment of the 18S rRNA gene, provides support for the identification of a new species of Calyptospora, found parasitizing the hepatic tissue of the piscivorous blue peacock bass, Cichla piquiti, captured in the reservoir of the Estreito hydroelectric dam on the middle Tocantins River in northern Brazil. This new species was named Calyptospora paranaidji n. sp.

Two new species of Hepatozoon (Apicomplexa: Hepatozoidae) parasitising species of Philothamnus (Ophidia: Colubridae) from South Africa.

To date, only a few species of Hepatozoon Miller, 1908 have been described from amphibians and reptiles of South Africa, including two species from anuran hosts, three from saurians, one from chelonians, and two from ophidians. Hepatozoon bitis (Fantham, 1925) and Hepatozoon refringens (Sambon et Seligmann, 1907), parasitising Bitis arientans (Merrem) and Pseudoaspis cana (Linnaeus), respectively, were described in the early 1900s and since then there have been no further species of Hepatozoon described from snakes in South Africa. Blood smears, used in peripheral blood haemogregarine stage morphometrics, and whole blood used in molecular characterisation of haemogregarines were collected from the caudal vein of six snakes of three species, namely Philothamnus hoplogaster (Günther), Philothamnus semivariegatus (Smith) and Philothamnus natalensis natalensis (Smith). For comparison, a comprehensive table summarising available information on species of Hepatozoon from African snakes is presented. Haemogregarines found infecting the snakes from the present study were morphologically and molecularly different from any previously described from Africa and are thus here described as Hepatozoon angeladaviesae sp. n. and Hepatozoon cecilhoarei sp. n. Both haemogregarine species were observed to cause considerable dehaemoglobinisation of the host cell, in case of infection with H. angeladaviesae resulting in a characteristic peripheral undulation of the host cell membrane and karyorrhexis. To the authors' knowledge, these are the first haemogregarines parasitising snakes of the genus Philothamnus Smith described using both morphological and molecular characteristics in Africa.

A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adelerina) from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil.

Based on both unique morphological characteristics of the gamont, distinct changes caused to the host erythrocyte and analysis of partial 18S rRNA gene sequences, a new parasite of the genus Hepatozoon Miller, 1908 is described from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil. The new species, Hepatozoon musa n. sp., is characterized by large and curved mature gamonts (18.9 ± 0.9 µm in length and 3.8 ± 0.3 µm in width) that considerably engorge infected host erythrocytes and displace the nucleus laterally, which become longer and thinner. Phylogenetic estimates indicate the new species is more closely related to the recently described Hepatozoon cuestensis O'Dwyer, Moço, Paduan, Spenassatto, Silva & Ribolla, 2013, from Brazilian rattlesnakes. These recent findings highlight the need for further studies of Hepatozoon to better determine the biodiversity of this common but poorly-studied parasite group.

Hepatozoon silvestris sp. nov.: morphological and molecular characterization of a new species of Hepatozoon (Adeleorina: Hepatozoidae) from the European wild cat (Felis silvestris silvestris).

Based on morphological and genetic characteristics, we describe a new species of Hepatozoon in the European wild cat (Felis silvestris silvestris), herein named Hepatozoon silvestris sp. nov. The study also provides the first data on the occurrence of H. felis in this wild felid. Hepatozoon meronts were observed in multiple cross-sections of different organs of four (44%) cats. Additionally, extracellular forms, resembling mature gamonts of Hepatozoon, were found in the spleen and myocardium of two cats. Furthermore, tissues of six animals (67%) were positive by PCR. Hepatozoon felis was identified infecting one cat (11%), whereas the 18S rRNA sequences of the remaining five cats (56%) were identical, but distinct from the sequences of H. felis. Phylogenetic analyses revealed that those sequences form a highly supported clade distant from other Hepatozoon spp. Future studies should include domestic cats from the areas where the wild cats positive for H. silvestris sp. nov. were found, in order to investigate their potential role to serve as intermediate hosts of this newly described species. Identification of its definitive host(s) and experimental transmission studies are required for elucidating the full life cycle of this parasite and the possible alternative routes of its transmission.

Redescription, molecular characterisation and taxonomic re-evaluation of a unique African monitor lizard haemogregarine Karyolysus paradoxa (Dias, 1954) n. comb. (Karyolysidae).

BACKGROUND: Within the African monitor lizard family Varanidae, two haemogregarine genera have been reported. These comprise five species of Hepatozoon Miller, 1908 and a species of Haemogregarina Danilewsky, 1885. Even though other haemogregarine genera such as Hemolivia Petit, Landau, Baccam & Lainson, 1990 and Karyolysus Labbé, 1894 have been reported parasitising other lizard families, these have not been found infecting the Varanidae. The genus Karyolysus has to date been formally described and named only from lizards of the family Lacertidae and to the authors' knowledge, this includes only nine species. Molecular characterisation using fragments of the 18S gene has only recently been completed for but two of these species. To date, three Hepatozoon species are known from southern African varanids, one of these Hepatozoon paradoxa (Dias, 1954) shares morphological characteristics alike to species of the family Karyolysidae. Thus, this study aimed to morphologically redescribe and characterise H. paradoxa molecularly, so as to determine its taxonomic placement. METHODS: Specimens of Varanus albigularis albigularis Daudin, 1802 (Rock monitor) and Varanus niloticus (Linnaeus in Hasselquist, 1762) (Nile monitor) were collected from the Ndumo Game Reserve, South Africa. Upon capture animals were examined for haematophagous arthropods. Blood was collected, thin blood smears prepared, stained with Giemsa, screened and micrographs of parasites captured. Haemogregarine morphometric data were compared with the data for named haemogregarines of African varanids. Primer set HepF300 and HepR900 was employed to target a fragment of the 18S rRNA gene and resulting sequences compared with other known haemogregarine sequences selected from the GenBank database. RESULTS: Hepatozoon paradoxa was identified infecting two out of eight (25 %) V. a. albigularis and a single (100 %) V. niloticus examined. Phylogenetic analyses revealed that H. paradoxa clustered with the 'Karyolysus' clade, and not with those of reptilian Hepatozoon spp. CONCLUSIONS: In addition to this being the first morphological and molecular characterisation of a haemogregarine within the African Varanidae, it is the first report of a species of Karyolysus infecting the monitor lizard family. Furthermore, this constitutes now only the third described and named Karyolysus species for which there is a nucleotide sequence available.

Morphological and molecular characterisation of Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) from the blood of the Caspian freshwater turtle Mauremys caspica (Gmelin) (Geoemydidae) in Iran.

To date, a number of species of Haemogregarina have been described from different turtle hosts, mainly based on the morphology of the developmental stages detected in the host erythrocytes. The diversity and overlapping morphological features in the old and recent descriptions has led to considerable complications in the taxonomy of Haemogregarina spp. In this study, different stages of maturity and developing gamonts of a putative new species of Haemogregarina were detected in erythrocytes of the Caspian turtle Mauremys caspica (Gmelin) (Geoemydidae) originating from a southern province in Iran. Although some of the morphological characteristics were consistent with Haemogregarina stepanowi Danilewsky, 1885, some new observations were made, particularly in the gamont stage. The phylogenetic analysis based on 18S rDNA sequences revealed that the present isolate appears as basal to a large clade of Haemogregarina spp. with sequences available in the GenBank database. In accordance with the phylogenetic results, the present Iranian isolate showed a higher degree of interspecific divergence (up to 3.3%) compared to the data for the taxa available in the GenBank database. Thus, molecular data indicate that this isolate may represent a new species. However, further genetic analyses are needed as a complementary tool to the morphological characterisation in order to elucidate the phylogenetic relationships of Haemogregarina spp.

First report and description of a Cyrilia sp. (Apicomplexa: Haemogregarinidae) from a freshwater Cururu Stingray Potamotrygon cf. histrix (Elasmobranchii: Potamotrygonidae), from the Amazon Region, Brazil.

A haemogregarine is described in 12 cururu stingray (Potamotrygon cf. histrix), from Mariuá Archipelago, Negro River, in the Brazilian Amazon Basin. All animals, both male and female, were parasitized by the haemogregarine and parasitaemia varied between 0.8% and 10% of erythrocytes. The stages observed included trophozoites or merozoites, suspected meronts, and gamonts presumed to be of two types, macrogamonts and microgamonts. Most stages were observed inside mature erythrocytes, while others were extracellular. The stages observed were most similar to those characteristics of the genus Cyrilia, than to any other fish haemogregarine and may represent a new Cyrilia species.

First report of Hepatozoon (Apicomplexa: Adeleorina) from king ratsnakes (Elaphe carinata) in Shanghai, with description of a new species.

Hepatozoon species are the most common hemoparasites of snakes. In this study, Hepatozoon parasites were examined for the first time in king rat snakes (Elaphe carinata) from Shanghai, China. All 10 snakes were found to be infected with Hepatozoon gamonts. The gamonts were folded back in a hook-wise fashion for about 3 µm at one end. Parasitemia levels ranged from 4-43 infected erythrocytes per 1,000 examined. The gamonts changed the morphology of the parasitized erythrocytes. Although the gamonts showed some distinct variations in both the parasite and its nucleus, phylogenetic analysis indicated that all the E. carinata in this study formed a monophyletic group, and were distinct from all other published Hepatozoon species. A new species, Hepatozoon chinensis, was proposed based on the molecular and morphologic evidence.

Coccidial dispersion across New World marsupials: Klossiella tejerai Scorza, Torrealba & Dagert, 1957 (Apicomplexa: Adeleorina) from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) (Mammalia: Didelphimorphia).

Klossiella tejerai Scorza, Torrealba & Dagert, 1957 is a primitive coccidian parasite reported from the New World marsupials Didelphis marsupialis (Linnaeus) and Marmosa demerarae (Thomas). The current work describes K. tejerai from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) in Southeastern Brazil, evidencing the coccidial dispersion across opossums of the same family. The sporocysts recovered from urine samples were ellipsoidal, 20.4 × 12.7 µm, with sporocyst residuum composed of scattered spherules and c.13 sporozoites per sporocyst, with refractile bodies and nucleus. Macrogametes, microgametes, sporonts, sporoblasts/sporocysts were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. Didelphis marsupialis should not have transmitted K. tejerai to D. aurita because they are not sympatric; however M. demerarae is sympatric with D. marsupialis and D. aurita. Therefore, D. aurita becomes the third host species for K. tejerai in South America.

Haemogregarines from western Palaearctic freshwater turtles (genera Emys, Mauremys) are conspecific with Haemogregarina stepanowi Danilewsky, 1885.

The majority of Haemogregarina species have been based on the morphology of their erythrocytic stages and supposed strict host specificity. The quantity of species with a limited number of overlapping diagnostic traits has led to a considerable mess in haemogregarine taxonomy and significant synonymy. We analysed host specificity, intra- and interspecific variability, evolutionary relationships, and the distribution of the type species of the genus Haemogregarina--H. stepanowi. The morphology of blood stages and 18S rDNA sequences of this haemogregarine from four western Palaearctic hard-shelled freshwater turtles (Emys orbicularis, Mauremys caspica, Mauremys leprosa and Mauremys rivulata) were compared with Haemogregarina balli. Additional sequences of 18S rDNA of Haemogregarina-like isolates collected from three species of African hinged terrapins (genus Pelusios) were used to enlarge the dataset for phylogenetic analyses. Thirteen sequences (1085 bp) of Haemogregarina representing all four western Palaearctic turtle species were identical, corresponding to H. stepanowi, which is closely related to the Nearctic species H. balli. In our analyses, Haemogregarina spp. constituted a monophyletic clade sister to the genus Hepatozoon. Haemogregarina stepanowi possesses a wide distribution range from the Maghreb, through Europe, Turkey and the Middle East to Iran. We consider that the genus Haemogregarina has a low host specificity crossing the family level of its vertebrate hosts and that its distribution is likely to be linked to the vector and definitive host--the leech.

Experimental evidence against transmission of Hepatozoon canis by Ixodes ricinus.

Hepatozoon canis is among the most widespread tick-borne protozoa infecting domestic and wild carnivores. Its distribution is related to the occurrence of its major vector, the brown dog tick Rhipicephalus sanguineus. However, the role of Ixodes ricinus as a vector of H. canis has been hypothesized. In the present study, the development of H. canis was investigated in I. ricinus and R. sanguineus nymphs collected from a naturally infested dog. All I. ricinus ticks examined (n=133) were negative by cytological examination at days 20, 30, and 90 post collection, although H. canis DNA was detected in one nymph at day 20 and in 2 nymphs at day 30 post collection. On the other hand, H. canis sporogony was documented by cytology, and H. canis DNA was detected by PCR in R. sanguineus at day 30 post collection. These results indicate that H. canis sporogony does not occur in I. ricinus, but in R. sanguineus, suggesting that I. ricinus does not act as a vector of H. canis.

Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp. (Apicomplexa: Adele-orina: Hepatozoidae) from the crag lizard (Sauria: Cordylidae) Pseudocordylus langi from the North Eastern Drakensberg escarpment, Eastern Free State, South Africa.

Two new haemogregarine species, Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp., are described from the pe-ripheral blood of the high altitude crag lizard, Pseudocordylus langi, collected between October 2006 and April 2009 from the North Eastern Drakensberg, Eastern Free State. Hepatozoon langii n. sp. has maturing and mature gamonts that appear encapsulated and have narrow, curved tails. Their cytoplasm stains pinkish-purple with Giemsa, while their nuclei are pur-ple stained with stranded chromatin. Mature gamonts measure 19.1 ± 1.0 (15.4-28.1) µm long by 6.2 ± 1.1 (3.5-7.9) µm wide. Hepatozoon vacuolatus n. sp. gamonts are mostly broader at one pole than the other, have bluish-pink cytoplasm characterised by distinctive rounded and oval vacuoles, and demonstrate pink granules with Giemsa staining. Nuclei stain purple and are mainly coarsely granular. Mature gamonts measure 16.5 ± 1.0 (14.7 - 17.6) µm long by 5.9 ± 1.2 (4.0 - 7.7) µm wide. Both species parasitize erythroblasts, as well as erythrocytes and can dehaemoglobinize the cytoplasm of their host cells. Hepatozoon langii n. sp occurred in the absence of H. vacuolatus n. sp., but the latter haemogregarine always formed mixed infections with the former; no stages intermediate between the two haemogregarine types were observed.

Morphological characteristics of a new species of Haemogregarina Danilewsky, 1885 (Apicomplexa: Adeleorina) in naturally infected Acanthodactylus boskianus (Daudin) (Sauria: Lacertidae) in Egypt.

This paper describes the morphological characteristics of a new species of Haemogregarina Danilewsky, 1885, from naturally infected Acanthodactylus boskianus (Daudin) in Egypt, based on both blood and tissue stages. The prevalence of infection was found to be 66% (33/50). The blood stages were found to be confined to the erythrocytes and could be differentiated into small and large forms. The small form (trophozoites) measures 8 × 3.5 µm, whereas the large form (mature gamonts) measures 12 × 5 µm. The tissue stages were observed only in the liver. Meronts occurred within parasitophorous vacuoles in hepatocytes and ranged from mononucleate forms to mature forms that contained c.10-15 merozoites.

Haemogregarine specificity in two communities of Florida snakes, with descriptions of six new species of Hepatozoon (Apicomplexa: Hepatozoidae) and a possible species of Haemogregarina (Apicomplexa: Haemogregarinidae).

Five species of snakes in Florida, from Palm Beach County in the south and Alachua County 450 km to the north, occur in similar habitat but have distinctive Hepatozoon species characteristic of each host species. In Palm Beach County, Diadophis punctatus is host to Hepatozoon punctatus n. sp., Thamnophis sauritus sackenii to Hepatozoon sauritus n. sp., and Nerodia fasciata pictiventris to Hepatozoon pictiventris n. sp. In Alachua County, N. fasciata pictiventris is parasitized by Hepatozoon fasciatae n. sp., Seminatrix p. pygaea by Hepatozoon seminatrici n. sp., and Thamnophis s. sirtalis by Hepatozoon sirtalis n. sp. Each Hepatozoon sp. has distinctive gamonts and sporogonic characters and, in the 4 species where known, meronts. Nerodia floridana is host to Haemogregarina floridana n. sp. in both localities, with generic identification tentative, based upon presence of erythrocytic meronts. The presence of sporocysts in the proboscis of 31% of Aedes aegypti infected by H. pictiventris is the first report of infective stages of a reptilian Hepatozoon species within the mouthparts of a dipteran vector. This study suggests that in Florida, at least, the diversity of the Hepatozoon community not only equals but probably exceeds the diversity of the snake communities present, and that host specificity in nature may be much greater than that postulated from previous studies.

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics.

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.