Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida

Accepted Manuscript Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida Carolina Hernández-Lara, Alejandro Espinosa de los Monteros, Carlos Napoleón Ibarra-Cerdeña, Luis García-Feria, Diego Santiago-Alarcon PII: DOI: Reference:

S0020-7519(18)30236-4 https://doi.org/10.1016/j.ijpara.2018.10.002 PARA 4102

To appear in:

International Journal for Parasitology

Received Date: Revised Date: Accepted Date:

24 April 2018 27 September 2018 4 October 2018

Please cite this article as: Hernández-Lara, C., de los Monteros, A.E., Ibarra-Cerdeña, C.N., García-Feria, L., Santiago-Alarcon, D., Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida, International Journal for Parasitology (2018), doi: https://doi.org/10.1016/j.ijpara.2018.10.002

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Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida

Carolina Hernández-Laraa, Alejandro Espinosa de los Monterosb, Carlos Napoleón Ibarra-Cerdeñac, Luis García-Feriaa, and Diego Santiago-Alarcona,*

a

Red de Biología y Conservación de Vertebrados, Instituto de Ecología A.C. Carretera Antigua a

Coatepec 351, El Haya, C.P. 91070 Xalapa, Veracruz, México b

Biología Evolutiva, Instituto de Ecología A.C. Carretera Antigua a Coatepec 351, El Haya, C.P.

91070 Xalapa, Veracruz, México c

Departamento de Ecología Humana, Centro de Investigación y de Estudios Avanzados (Cinvestav)

Unidad Mérida. Antigua carretera a Progreso Km. 6, Cordemex, C.P. 97310, Mérida, Yucatán, México

*Corresponding author. Diego Santiago-Alarcon, Red de Biología y Conservación de Vertebrados, Instituto de Ecología A.C. Carretera Antigua a Coatepec 351, El Haya, C.P. 91070 Xalapa, Veracruz, México E-mail address: [email protected]

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Abstract The traditional classification of avian Haemosporida is based mainly on morphology and life history traits. Recently, molecular hypotheses have challenged the traditional classification, leading to contradictory opinions on whether morphology is phylogenetically informative. However, the morphology has never been used to reconstruct the relationships within the group. We inferred the phylogeny of avian Haemosporida from 133 morphological characters present in blood stages. We included all species with at least one mitochondrial gene characterized (n= 93). The morphological hypothesis was compared with the one retrieved from mitochondrial DNA (mtDNA) nucleotide sequences and a hypothesis that used a combination of morphological and molecular data (i.e., total evidence). In order to recover the evolutionary history and identify phylogenetically and taxonomically informative characters, they were mapped on the total evidence phylogeny. The morphological hypothesis presented more polytomies than the other two, especially within Haemoproteus. In the molecular hypothesis, the two Haemoproteus subgenera are paraphyletic, and some relationships within Parahaemoproteus were resolved. By combining the morphological and molecular data, we were able to resolve the majority of polytomies and posterior probabilities increased. We identified a unique combination of morphological traits, clearly differentiating avian Haemosporida genera, sub-genera of Leucocytozoon and Haemoproteus, and some Plasmodium sub-genera. Plasmodium had the highest number of synapomorphies. Furthermore, 86% of the species presented a unique combination of taxonomically informative characters. A limiting factor was the mismatch of traits characterized in species descriptions, leading to a morphological matrix with a considerable amount of missing data, particularly for the stages of early young and young gametocytes (67% of all missing data). Characters lacking information for the majority of species included the color of pigment granules, the cytoplasm appearance, and the presence and dimensions of vacuoles. According to our results, the combination of morphology and mtDNA proved to be a robust alternative to reconstruct the relationships among avian Haemosporida, obtaining a

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resolution and support similar to that obtained using full mitochondrial genome sequences for over 100 lineages.

Keywords: Avian Haemosporida phylogeny, Avian Haemosporida morphological phylogeny, Avian malaria, Plasmodium, Haemoproteus, Parahaemoproteus, Leucocytozoon, Akiba

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1. Introduction Avian Haemosporida belong to an Order of blood parasites composed of four families (Haemoproteidae, Plasmodiidae, Leucocytozoidae and Garniidae) and four genera (Haemoproteus, Plasmodium, Leucocytozoon and Fallisia; Valkiūnas, 2005), which are transmitted by bloodsucking dipterans from the families Ceratopogonidae, Hippoboscidae, Culicidae and Simuliidae (Valkiūnas, 2005; Santiago-Alarcon et al., 2012). The genus Haemoproteus is divided into two subgenera, Haemoproteus and Parahaemoproteus; Plasmodium is divided into five subgenera, Bennettinia, Novyella, Haemamoeba, Giovannolaia and Huffia; Leucocytozoon is divided into two subgenera, Leucocytozoon and Akiba; and Garniidae comprises one genus, Fallisia; to date only one species has been described for Leucocytozoon (Akiba) and for Fallisia (Plasmodioides; Valkiūnas, 2005). Some authors suggest that the subgenera Haemoproteus and Parahaemoproteus should be considered as two different genera because they have been recovered in separate clades in molecular phylogenies (Martinsen et al., 2008; Borner et al., 2016; Galen et al., 2018). Furthermore, the subgenus Haemoproteus includes parasites infecting non-passerine bird species from the Columbiformes (Santiago-Alarcon et al., 2010; Valkiūnas et al., 2010), Pelecaniformes (Levin et al., 2011; Bastien et al., 2014), and Charadriiformes (Levin et al., 2012), whereas Parahaemoproteus seems to infect birds across all of the avian phylogeny (Valkiūnas, 2005; Atkinson, 2008). Traditionally, avian haemosporidians have been classified based on their morphology during stages in blood cells, life-history traits, and host taxa (Valkiūnas, 2005; Martinsen et al., 2008). Of primary importance are morphological characteristics of several blood life stages: early young gametocytes, young gametocytes, growing gametocytes, fully grown micro and macrogametocytes, trophozoites, and meronts. It is therefore the combination of several characters, and not one or a few, that defines each morphospecies (Valkiūnas, 2005). In the traditional classification, Leucocytozoon is considered as the ancestor of a clade formed by Haemoproteus/Parahaemoproteus and Plasmodium (see Martinsen et al., 2008; Fig. 1D)

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Phylogenetic hypotheses prior to the molecular era relied heavily on the vertebrate host and the parasite’s life history as the major defining characteristics (Martinsen et al., 2008). In 1989, Barta performed a parsimonious phylogenetic analysis of the class Sporozoa, using 26 characters of the ultrastructural and developmental features, and included species of Haemoproteus, Leucocytozoon and Plasmodium. Species from the three genera were recovered in the same clade with an unresolved relationship among them. The incorporation of molecular phylogenetic methods has challenged the traditional classification of avian haemosporidians, and therefore the phylogenetic importance of morphological traits (e.g., Martinsen et al., 2006, 2007; Outlaw and Ricklefs, 2011). It is considered that the details on the three-dimensional features from parasites and their host cells are lost when looking through the microscope because only two dimensions can be appreciated (Martinsen et al., 2006), added to the fact that the process of blood smear preparation could deform cell and parasite structures (Martinsen et al., 2007). Despite this, the phylogenetic importance of morphological traits used in traditional classifications has not been formally tested. Early molecular phylogenies of avian Haemosporida were inferred using limited numbers of taxa and DNA from one gene (mtDNA cyt b; Bensch et al., 2000; Perkins and Schall, 2002). Using the apicomplexan Theileria annulata (Apicomplexan: Piroplasmida) as the outgroup, Perkins and Schall (2002) recovered Leucocytozoon at the base of haemosporidians and avian Haemoproteus and Plasmodium as sister taxa, both monophyletic. Martinsen et al. (2008) included more taxa and genes rooting the tree with Leucocytozoon, recovering the subgenera Haemoproteus and Parahaemoproteus in two different clades, suggesting that they should be considered different genera. Outlaw and Ricklefs (2011) proposed a phylogeny with a relaxed clock outgroup-free approach, where Plasmodium and Hepatocystis from mammals were sister to all other haemosporidians from birds and reptiles. Avian Plasmodium was recovered at the base of avian haemosporidians, followed by Parahaemoproteus, and Haemoproteus was sister to Leucocytozoon (Outlaw and Ricklefs, 2011). A recent study which included 26 genes from the three haemosporidian genomes (23 nuclear, two mitochondrial and one aplicoplast; Borner et al., 2016),

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was rooted on Leucocytozoon with Haemoproteus at the base of Parahaemoproteus and Plasmodium (Fig. 1E). The most comprehensive mitochondrial phylogeny included complete mitochondrial genomes of over 100 Haemosporida spp. from birds, reptiles and mammals, recovering Leucocytozoon at the base (Pacheco et al., 2017), supporting results from previous studies (Perkins and Schall, 2002; Borner et al., 2016). The latest Haemosporida phylogeny was inferred from DNA sequences of 21 nuclear genes, correcting for differences in base composition and using Theileria annulata as an outgroup (Galen et al., 2018). In this phylogeny, there are two clades of avian Haemosporida sharing the same common ancestor: one of them is formed by Plasmodium and Haemoproteus catharti, and the other by Leucocytozoon at the base and a clade formed by Parahaemoproteus and Haemoproteus (Galen et al., 2018; Fig. 1F). The first study comparing molecular phylogenies with species morphology found support for most of the morphologically identified species by genetic and phylogenetic species concepts (Martinsen et al., 2006). An early phylogenetic analysis using the ssrRNA gene revealed that avian Plasmodium subgenera do not correspond to their morphology-based classification (Kissinger et al., 2002). In contrast, a two-gene mitochondrial phylogeny (cyt b and coI) of avian Plasmodium found support for three (Haemamoeba, Huffia and Bennettinia) out of the five morphology-based subgenera (Martinsen et al., 2007). There has been a long debate about the use of morphology or molecules to infer relationships among organisms, but in the end both phenotype and genotype provide complementary information, and when used together, the resolution and support of the phylogeny tend to increase (Lee and Palci, 2015). Hence, more thorough investigations using morphological characters in a phylogenetic context are necessary in order to clarify relationships both at the genus and subgenus taxonomic levels of haemosporidians. Phylogenies within avian Haemosporida commonly use one to a few (usually no more than four) genes and a limited number of taxa. Some of these studies have placed the origin of the two main morphological characters that differentiate between families at the base of each family. These two characters are the presence of pigment granules and merogony in blood cells. Nevertheless, the

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morphology of avian Haemosporida has not previously been used to infer phylogenetic relationships of the group. Here we used, to our knowledge for the first time, the morphology of 133 characters from 93 species to infer the phylogeny of avian haemosporidians. In our analyses we included representatives from three genera (Leucocytozoon, Haemoproteus and Plasmodium) and eight stages of development that are present in the blood cells of the avian host. Then, we compared this morphological hypothesis with the one obtained from molecular characters (mtDNA) and another obtained from the total evidence dataset (i.e., morphology + mtDNA). We also identified the phylogenetically informative characters for each genus, subgenus, and for currently described species. Finally, we compared our results with a previous molecular hypothesis that used a multigene approach. We expected to recover a congruent topology between the morphological and molecular hypotheses, and a better resolved and supported topology from the total evidence hypothesis. Furthermore, we expected to identify new phylogenetically informative morphological characters.

2. Materials and methods 2.1. Data collection We conducted a search of the literature in order to identify all avian Haemosporida spp. which have had their mtDNA characterized. We considered all species which have had their complete mitochondrial genome characterized, as well as those having sequences from one or more of the following genes: cyt b, coI, or cytochrome oxidase subunit III (cox3; n= 93; see Supplementary Table S1).

2.1.1. Morphological data collection We used the species descriptions to obtain all the morphological characters that are used to identify avian Haemosporida spp., with a total of 133 morphological characters that are present in blood stages (Supplementary Table S2). These characters belong to eight stages of parasite

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development: 1) early young gametocytes (12 characters), 2) young gametocytes (13), 3) growing macrogametocytes (14), 4) growing microgametocytes (13), 5) fully grown macrogametocytes (27), 6) fully grown microgametocytes (26), 7) trophozoites (13), and 8) meronts (15). The first six stages of development correspond to species belonging to Leucocytozoon, Haemoproteus and Plasmodium, while stages 7 and 8 correspond only to species of Plasmodium. All morphological characters included are discrete and were coded with numbers from 0 to 8, and missing data was coded as a question mark (?). Non-applicable characters were coded as the tenth state (9) because when running the phylogenetic analyses, gaps and missing data are treated the same way. In our case, non-applicable characters indicated the absence of a developmental stage (e.g., trophozoites, meronts) or a structure (e.g., pigment granules, vacuoles) together with its features (e.g., size, color, shape, arrangement, location). Most of the characters included are present in more than one stage of development (e.g., shape of gametocyte, presence of vacuoles) but were treated as independent characters, since they usually vary from one stage of development to the other.

2.1.2. Molecular data collection We obtained cyt b sequences belonging to the selected morphospecies from the MalAvi database (http://mbio-serv2.mbioekol.lu.se/Malavi/, Bensch et al., 2009). Sequences from other mitochondrial genes (coI and cox3), as well as complete mitochondrial genomes, were obtained from GenBank (Benson et al., 2013) and EuPathDB (https://eupathdb.org/eupathdb/, Aurrecoechea et al., 2017) databases (see Supplementary Table S1 for references). In order to avoid including in our analysis sequences from misidentified species, we estimated genetic distances with a JukesCantor model in MEGA7 (Kumar et al., 2016) and used the consensus sequence of only those lineages that differed by less than 5% in their cyt b region (see Hellgren at al., 2007).

2.2. Phylogenetic analyses

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All phylogenetic hypotheses were estimated using Bayesian inference in MrBayes version 3.2.6 (Ronquist et al., 2012) on CIPRES Science Gateway (https://www.phylo.org/portal2/login!input.action, Miller et al., 2010). Analyses were performed with two runs of four chains each, a 25% burn-in, and saving the last 10,001 trees. The number of generations for each analysis varied in order to reach convergence (average standard deviation of split frequencies < 0.01): morphological 20 million, molecular 10 million, and total evidence 15 million generations. The quality of the analyses (effective sampling size, traces of the two runs, and burn-in) was examined in Tracer version 1.6.0 (http://beast.bio.ed.ac.uk/, Rambaut et al., 20032014). Majority rule consensus trees with posterior probabilities were visualized using FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/, Rambaut, 2006-2014). The morphological matrix comprised 93 morphospecies and 133 characters. For the morphological analysis we used an equal substitution rate model with gamma distribution and set the prior for the stationary state frequencies to infinity. In a preliminary molecular analysis using T. annulata as an outgroup, we recovered the genus Leucocytozoon at the base of avian Haemosporida. Theileria annulata was then eliminated from further analyses for being very divergent from haemosporidian species and Leucocytozoon was used to root the trees. Gaps were coded as (-) and missing data as (?). Mitochondrial DNA sequences were aligned with MUSCLE (Edgar, 2004) in Mesquite version 3.51 (http://mesquiteproject.org, Maddison and Maddison, 2017). The molecular matrix had the same 93 taxa and a length of 5,991 bp. We performed the molecular analyses with a general time reversible model with gamma distribution and a proportion of invariable sites (GTR+G+I). Priors for state frequencies (A= 0.33, C= 0.15, G= 0.15, and T= 0.38), substitution rates (AC= 0.068, AG=0.202, AT= 0.225, CG= 0.063, CT= 0.407, and GT= 0.035), gamma shape (0.74), and proportion of invariable sites (0.43) were specified in MrBayes as obtained from jModelTest v2 (Darriba et al., 2012). For the total evidence analysis, we concatenated the morphological and molecular matrices manually in one partitioned matrix with 93 taxa and 6124 characters. The first partition corresponds

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to morphological characters (i.e., standard data) from characters 1 to 133, and the second partition corresponds to mtDNA sequences (i.e., DNA data) from characters 134 to 6124. For the analysis, settings for each partition were left as previously described. To recover the evolutionary history of morphological characters present in blood stages of avian Haemosporida, we mapped the characters on the total evidence tree using WinClada (http://www.diversityoflife.org/winclada/, Nixon, 19992002). This analysis allowed us to identify those traits that are synapomorphies for specific taxa, from genera to morphospecies; also, to differentiate between unique origin characters (synapomorphies/autapomorphies) and characters shared among taxa due to convergent evolution (homoplasious). Data used for all analyses can be downloaded from doi:10.6084/m9.figshare.7137860.

3. Results 3.1. Morphological hypothesis Using 133 morphological characters of blood stages of avian Haemosporida, we recovered Leucocytozoon at the base of avian Haemosporida, having Akiba as its sister taxa (Fig. 2). Plasmodium was recovered as a well-supported (posterior probability  99%) monophyletic group, sister to Haemoproteus pallidulus, Haemoproteus multipigmentatus, and Haemoproteus paramultipigmentatus. The rest of Haemoproteus and Parahaemoproteus spp. formed a polytomy at the base of the Plasmodium clade. All species of subgenus Haemamoeba were recovered in a monophyletic clade sister to Plasmodium (Huffia) elongatum (Fig. 2). Species of subgenera Giovannolaia and Bennettinia were recovered within a clade that includes species of subgenus Novyella. The two species of Giovannolaia are in the same clade, sister to Plasmodium homopolare from subgenus Novyella. Plasmodium juxtanucleare, the only representative from subgenus Bennettinia, was grouped with Plasmodium (Novyella) lucens (Fig. 2).

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In the mtDNA hypothesis, Plasmodium, subgenus Leucocytozoon, Haemoproteus and Parahaemoproteus, are well-supported monophyletic groups (Fig. 3). The genera Leucocytozoon and Haemoproteus are polyphyletic. Placed at the base of avian Haemosporida is subgenus Leucocytozoon, however it is not grouped with subgenus Akiba which is the sister taxa of H. antigonis, a parasite infecting Gruiform birds. The subgenus Haemoproteus is at the base of Plasmodium, the cluster Akiba-H. antigonis, and Parahaemoproteus (Fig. 3). Within Plasmodium, only Giovannolaia was recovered as monophyletic and well supported. Species of Plasmodium are in two groups: one including all representatives from subgenera Haemamoeba and Giovannolaia, as well as P. (Huffia) elongatum and two species from subgenus Novyella; and the other including Plasmodium (Bennettinia) juxtanucleare and the majority of Novyella.

3.3. Total evidence hypothesis The three genera of avian Haemosporida were recovered as well supported monophyletic clades (Fig. 4). At the base of avian Haemosporida is Leucocytozoon with Akiba (Leucocytozoon caulleryi) as the sister taxon to the rest of them. Plasmodium is sister to the genus Haemoproteus. The monophyly of the sister subgenera Haemoproteus and Parahaemoproteus is supported. Parahaemoproteus shows internal structure, recovering four monophyletic clades within it. The five subgenera currently accepted within Plasmodium do not form monophyletic clades. Instead, two groups of Plasmodium can be recognized, one formed by Giovannolaia, Haemamoeba and Huffia and the other by Novyella and Bennettinia (Fig. 5). In the first group Giovannolaia is at the base of Haemamoeba and Huffia, in a well-supported clade. In the second group Bennettinia is within a group of Novyella spp. in a polytomy next to Parablennius rouxi and Plasmodium multivacuolaris.

3.4. Identification of phylogenetic and taxonomic informative characters Morphological characters and their states present in blood stages of avian Haemosporida were mapped on the total evidence tree, in order to recover their evolutionary history (Fig. 4,

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Supplementary Fig. S1). Subgenus Leucocytozoon presents six unique characters and two homoplasious characters, such as the presence of valutine granules in fully grown gametocytes as well as a cap-like shape of the nuclei of discoid host cells infected by fully grown gametocytes (Fig. 4, characters 4 and 5). The subgenus Akiba can be recognized by a combination of two autapomorphic and two homoplasious characters. Autapomorphies for Akiba are that fully grown gametocytes are closely appressed to the envelope of enucleated erythrocytes or adhere to some of its margin (Fig. 4, characters 1 to 3). The close relationship between Akiba and the other species of Leucocytozoon is supported exclusively by molecular characters. We could not identify morphological synapomorphies shared by these lineages. Of the three genera, Plasmodium is the one that has accumulated more unique characters (22 characters), all of them belonging to the stages of trophozoites and erythrocytic meronts (Fig. 4, characters 6 to 11). The character regarding the arrangement of pigment granules aggregated in loose clumps in trophozoites, is recovered at the base of Plasmodium; nonetheless, its usefulness for identifying the genus is questionable. This character has been lost during the evolutionary history of several species of Plasmodium. Within Plasmodium, the morphology of Bennettinia is clearly differentiated from the other subgenera by a combination of 26 characters (Fig. 5, characters 18 to 24), including that trophozoites adhere to the erythrocytes nucleus, as well as the presence of discoid and oval fully grown gametocytes. Species of Giovannolaia can be distinguished by a combination of four synapomorphic and two homoplasious characters, such as erythrocytic meronts that markedly or completely enclose the nuclei of erythrocytes or fully grown gametocytes that hypertrophy infected erythrocytes (Fig. 5, characters 1 to 4). Subgenera Haemamoeba and Huffia share five characters, three unique and two homoplasious, including the displacement of the erythrocyte nucleus by meronts towards one pole and fully grown microgametocytes that do not touch the envelope of enucleated erythrocytes (Fig. 5, characters 5 to 8). Subgenus Huffia presents eight homoplasious characters amongst which are the presence of a long thread or finger-like outgrowth that exceeds the length of the main body of trophozoites and fully grown

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macrogametocytes that are closely appressed to the envelope of erythrocytes but do not touch the nucleus all along its margin (Fig. 5, characters 9 to 16). Subgenera Novyella and Bennettinia share the position of the nucleus of macrogametocytes, which is close to the erythrocyte nucleus but not to the envelope. Species of subgenera Giovannolaia, Haemamoeba, and Huffia present small (< 5 µm) vacuoles in trophozoites. Haemoproteus and Parahaemoproteus share the presence of growing macrogametocytes that adhere to the envelope of erythrocytes but do not touch its nucleus and the presence of fully grown microgametocytes that slightly enclose the nucleus of erythrocytes with their ends (Fig. 4, characters 12 and 13). Species of the subgenus Haemoproteus share fully grown macrogametocytes that displace the nucleus of erythrocytes laterally and that present valutine granules (Fig. 3, character 14). Finally, all species of Parahaemoproteus share one unique character that is the presence of oval early young gametocytes (Fig. 4, character15). Eighty-six percent of the morphospecies (n= 80) had a unique combination of characters; the majority of those species belonging to Parahaemoproteus (n= 51), which is also the subgenus with more morphospecies (Supplementary Fig. S1). Nearly 26% of the morphospecies present at least one autapomorphy, being Parahaemoproteus and Plasmodium, the two clades with more species easily identifiable by autapomorphies, 17 and 7, respectively. Finally, 13 out of the 93 species do not have a unique combination of characters, being seven from Parahaemoproteus, four from Leucocytozoon and two from Plasmodium (Supplementary Fig. S1).

3.5. Missing data on species descriptions Species descriptions usually do not include a characterization of the same set of morphological traits. For example, the color of pigment granules in all stages of gametocytes is not described for the majority of species, going from 59% for fully grown macrogametocytes to 75% for early young gametocytes. Another example is the cytoplasm appearance of gametocytes (e.g., homogeneous, granular) that was only reported in 14% and 20% of the species for early young and

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young gametocytes, respectively. Other characters with considerable amounts of missing data include the presence and dimensions of vacuoles and the position of the gametocyte in relation to the nucleus and envelope of the erythrocyte (Table 1). Missing data from early young and young gametocytes represent over 67% of all missing data (Fig. 6A). Growing macrogametocytes was the better represented stage, with only 6.4% of missing data. Growing and fully grown macrogametocytes have similar values of missing data in comparison with microgametocytes, although the percentage of missing data in growing and fully grown microgametocytes is slightly higher. In the species descriptions, we found a very detailed characterization of macrogametocytes; however, when it comes to microgametocytes there were only a few sentences regarding sexual dimorphism and mentioning that they share the same characters as macrogametocytes, although this is not necessarily true. Our approach when coding microgametocytes was to visually check in the figures if they actually shared the same character states as macrogametocytes. If they did not present the same state, we coded the corresponding one when possible (i.e., character state clearly shown in the figures), otherwise the character was left as uncertain. In particular, characters regarding the nucleus of the gametocyte are distinct from the same characters in macrogametocytes. The main characteristics of sexual dimorphism are related to the staining of the cytoplasm and nucleus, as well as the size of the nucleus of the gametocyte (Valkiūnas, 2005). In macrogametocytes the nucleus is compact, well defined, and stains darker, while in microgametocytes the nucleus is larger, usually not clearly defined, and stains pale. The fact that the size of the nucleus of microgametocytes is different can have implications for some other characters such as the position of the nucleus of the gametocyte in relation to the nucleus and envelope of the erythrocyte, or the position of the nucleus in relation to the poles (i.e., central, subpolar, polar). The amount of missing data was not distributed equally among genera. Haemoproteus accounted for the 45.4% of missing data in all stages of gametocyte development (from early young to fully grown gametocytes), followed by Leucocytozoon with 41.1%, and finally Plasmodium with

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13.5%. It should be noted that Haemoproteus was the better represented genus with 58 species, followed by Plasmodium with 23, and finally Leucocytozoon with 12 species. Almost half of the species of Haemoproteus and Plasmodium did not have information for early young and young gametocytes, from 39% to 47% (Fig. 6B). It should be noted that for the analyses we considered only half of the original characters for these two stages because there was no information for most species.

4. Discussion 4.1. Comparison of phylogenetic hypotheses When comparing our three topologies, the morphological one presented more polytomies, especially within Haemoproteus. In the molecular hypothesis, Parahaemoproteus is clearly differentiated from Haemoproteus and relationships within Parahaemoproteus deep in the phylogeny are resolved; however, the relationships of most Parahaemoproteus spp. at the tips of the phylogeny are still unresolved. By combining the morphological and molecular data, we were able to resolve all the polytomies within Leucocytozoon, as well as the majority of polytomies among species of Parahaemoproteus. Furthermore, posterior probabilities increased for Leucocytozoon, Plasmodium, Haemoproteus and Parahaemoproteus, as well as within species and groups of species. According to our results, the combination of morphology and mtDNA proved to be the best alternative to reconstruct the relationships among avian Haemosporida, obtaining a resolution and support similar to that obtained using full mitochondrial genome sequences of over 100 lineages (Pacheco et al., 2017). The topology of our morphological hypothesis (Fig. 1A) presents similarities to the traditional classification of avian Haemosporida (Fig. 1D), recovering Leucocytozoon and Plasmodium in separate clades, with Leucocytozoon at the base of haemosporidians and Haemoproteus sister to Plasmodium at the tip of the phylogeny. However, in our hypothesis we did not recover species of Haemoproteus grouped within the same clade. One explanation for the

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Haemoproteus polytomy is that there are fewer characters describing this genus, added to the considerable amount of missing data (see below) in the first two stages of development (early young gametocytes and young gametocytes). Our morphological phylogeny supports the traditional idea of placing the gain of hemozoin pigment at the base of Haemoproteus and Plasmodium, and the gain of blood merogony in the ancestor of Plasmodium (Fig. 1A, D), being more parsimonious than the mtDNA and nuclear hypotheses (Fig. 1B, F, respectively). The topology of the mtDNA hypothesis (Fig. 1B) is similar to that of Borner et al. (2016, Fig. 1E), with the exception that we focused on avian haemosporidians and included subgenus Akiba in our analysis. Our mtDNA hypothesis differs from the mitochondrial genome (5125 bp) hypothesis of Pacheco et al. (2017; not shown) in the placement of Akiba, mainly due to the newly described species H. antigonis. We recovered Akiba as a derived lineage within the subgenus Haemoproteus as the sister species of H. antigonis, while Pacheco et al. (2017) recovered Akiba as sister to Parahaemoproteus. However, both subgenera Akiba and Parahaemoproteus present important differences in morphology, as well as in the blood cells they parasitize. The topology of the nuclear phylogeny of Galen et al. (2018; Fig. 1F) contrasts most with our mtDNA hypothesis. According to the nuclear hypothesis, Plasmodium and H. catharti share a common ancestor and they are the sister clade of the rest of avian Haemosporida. The total evidence hypothesis recovered Haemoproteus and Parahaemoproteus as sister taxa and as the most derived Haemosporida genus, and Leucocytozoon at the base of the tree (Fig. 1C). As mentioned, Pacheco et al. (2017) recovered Akiba as sister to Parahaemoproteus. Akiba and Parahaemoproteus are transmitted by the same family of vectors, biting midges (Diptera: Ceratopogonidae; Valkiūnas, 2005). It is possible that Akiba and Parahaemoproteus share a common ancestor that was transmitted by biting midges, which could explain the similarities in their mitochondrial genomes. Another possibility is that there has been hybridization between Akiba and Parahaemoproteus during the sexual stages occurring in the vector. Although the recombination of mtDNA in haemosporidians has not been reported, recent experiments indicate

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that there can be hybridization between species from the same genus that are transmitted by the same vector (Valkiūnas et al., 2008; Palinauskas et al., 2017). Our total evidence hypothesis is consistent with that of Borner et al. (2016) that uses genes from the three genomes of Haemosporida (Fig. 1E). Similarities between these two hypotheses suggest that the use of morphology, contrary to previous ideas, is in fact phylogenetically informative. In the case of the five subgenera of Plasmodium, our total evidence results suggest the monophyly of subgenus Giovannolaia (Fig. 5). It is worth noticing that in all the analyses, Bennettinia was recovered within the Novyella clade. The subgenus Bennettinia was established long after the rest of the Plasmodium subgenera (Valkiūnas, 1997), containing only one species, P. juxtanucleare. According to the description of Bennettinia, the main differences from the rest of Plasmodium subgenera are that erythrocytic meronts are smaller than the nucleus of the infected erythrocytes, the presence of roundish erythrocytic meronts, roundish gametocytes, and pedunculated oocysts (Valkiūnas, 1997). We identified a combination of 26 characters that differentiate the morphology of Bennettinia from the rest of Plasmodium (Fig. 5, characters18 to 24), of which only those referring to roundish gametocytes coincide with the species description (Valkiūnas, 1997). Other character states identified in our morphological analysis which are relevant for the classification of Bennettinia include the presence of fully grown gametocytes that are appressed to the envelope and partially touch the nucleus of erythrocytes, pigment granules aggregated in loose clumps in fully grown gametocytes, and trophozoites with randomly scattered pigment granules (see Fig. 5 for a description of all characters defining Bennettinia). According to morphology (Fig. 1A), the subgenus Akiba is closely related to the rest of Leucocytozoon spp. at the base of avian Haemosporida. However, the mtDNA (Fig. 1B) places Akiba as the sister taxa to H. antigonis, both being part of the Parahaemoproteus clade. When combining both datasets (Fig. 1C), it seems that differences in the morphology between Akiba and H. antigonis have more weight than the similarity among their mtDNA sequences, placing Akiba in the same position as in the morphological hypothesis, and H. antigonis within Haemoproteus.

17

One particular species caught our attention due to the change in its placement within the three phylogenetic hypotheses. Haemoproteus antigonis is a parasite of gruiform birds initially classified as Parahaemoproteus, due to its morphology and its host family (see Valkiūnas, 2005). However, a recent study characterizing a fragment of the cyt b gene recovered H. antigonis and closely related cyt b lineages in a novel clade, independent of the rest of avian Haemosporida (Bertram et al., 2017). In our morphological hypothesis, H. antigonis is recovered in a polytomy with other Haemoproteidae and with Plasmodium (Fig. 2), although it is at the base of subgenus Haemoproteus in the total evidence hypothesis (Fig. 4). The inclusion of L. (Akiba) caulleryi in our analysis allowed us to resolve the relationships of H. antigonis with the rest of avian Haemosporida based on mtDNA sequences, recovering these two species in a strongly supported clade (posterior probability  99%, Fig. 1B, 3).

4.3. Phylogenetic and taxonomic informative characters For Plasmodium, the presence of merogony in blood cells has traditionally been considered as the only characteristic life history trait; however, merogony in blood cells is much more than one trait. We could identify 28 morphological characters that are present during merogony in blood cells, of which 15 correspond to the stage of erythrocytic meronts and 13 to trophozoites. Mapping informative morphological characters on the total evidence phylogeny allowed us to identify morphological traits that are unique to specific clades. Plasmodium presented a combination of 22 morphological characters, 21 of those being synapomorphies and one homoplasy. For example, in meronts there is an absence of a long (> 2 μm) outgrowth in growing meronts, merozoites that are randomly arranged in fully grown meronts, and the absence of vacuoles. In trophozoites, main characters include the absence of a long outgrowth that exceeds the length of the main body of the trophozoite, trophozoites develop in mature erythrocytes, the presence of vacuoles, presence of roundish dark brown pigment granules aggregated in loose clumps, subpolar position in erythrocytes, and they do not adhere to the nucleus of erythrocyte. These traits that are shared by all 18

species of Plasmodium may have evolved prior to the speciation of the genus, which could explain why they are present in all the species. On the other hand, the origin of meronts that markedly or completely enclose the nucleus of erythrocyte, both synapomorphies of Giovannolaia, evolved more recently. Other more recently evolved characters within Plasmodium include the presence of meronts that hypertrophy erythrocytes and displace the nucleus of erythrocytes towards one pole, as well as fully grown microgametocytes that do not touch the envelope of enucleated erythrocytes, traits shared by species of Haemamoeba and Huffia. Autapomorphies are present in six species of Plasmodium (Novyella) included in this study, being the characters that evolved most recently. Plasmodium ashfordi presents growing and fully grown macrogametocytes that are appressed to the envelope and partially touch the nucleus of erythrocytes, as well as light brown pigment granules in erythrocytic meronts; in P. delichoni the outline of trophozoites is wavy or slightly amoeboid; in P. unalis the size of pigment granules in meronts is small or dust-like (<0.5 µm); in P. homonucleophilum the shape of pigment granules is oval or rod-like; in P. globularis trophozoites are in a median or submedian position; and in P. parahexamerium the outline of growing gametocytes and fully grown microgametocytes is slightly irregular, and the nucleus of fully grown macrogametocytes is placed in a terminal position. Synapomorphies for Leucocytozoon (Leucocytozoon) include the presence of roundish host cells with a cap-like nucleus that extends up to half of the circumference around fully grown gametocytes, as well as the presence of valutine granules in fully grown macrogametocytes. Autapomorphies for Leucocytozoon (Akiba) caulleryi are that fully grown macrogametocytes partially adhere or are closely appressed to the margins of enucleated host cells. Species from subgenus Parahaemoproteus share one synapomorphy, being the oval shape of early young gametocytes. There are 14 Parahaemoproteus and two Haemoproteus spp. with autapomorphies. Specific character combinations for species identified in this study are an important contribution to the taxonomy of avian Haemosporida. Nevertheless, further studies on

19

morphological characters are necessary to identify specific morphological traits for those species lacking them in this study. According to the relative ages of each genus, Leucocytozoon and Plasmodium have had more time to accumulate synapomorphies, which coincides with the number identified for these genera. On the contrary, Haemoproteus seems to be the most recent genus having no unique characters and only two homoplasies. Recent studies (Outlaw and Ricklefs, 2011; Galen et al., 2018) support the hypothesis that avian Plasmodium is not as recent as previously proposed.

4.4. Missing data on species descriptions We encountered a challenge when coding species because descriptions usually do not include the same information, leaving out valuable details that could be helpful to solve relationships among parasite species. Frequently, parasitaemia is very low, making it difficult to find enough parasites in order to make a complete characterization for each stage of development. In many cases, early stages of development are not found, which could explain the considerable lack of information on characters regarding early young and young gametocytes. Also, there is usually a poor characterization of microgametocytes, including only one sentence regarding sexual dimorphism. As mentioned in the results section, microgametocytes do not necessarily present the same character states as macrogametocytes, although including a detailed characterization of both could help resolve more relationships among Haemosporida. We suggest including the same set of characters to describe micro and macrogametocytes, as well as the other stages of development, even when it seems repetitive, as minor details may become relevant when inferring phylogenies and identifying species. The morphological characters identified in the current study can be a starting point for future species diagnosis (Supplementary Table S2). Thus, we encourage avian Haemosporida researchers to consistently provide information on the same set of characters when describing new species, and to clearly state and include in species descriptions and in the character matrix any novel character.

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4.5. Conclusions The identification of species of Haemosporida by observing blood smears under the microscope requires experience and it is time consuming. Nevertheless, our study provides support for the phylogenetic importance of morphological characters, and the need to use those in combination with molecular markers. We believe it is time to leave aside the limiting statement regarding the existence of few morphological characters, as our study shows there are at least 133 potential taxonomic characters available, and as more species are described, other characters may be added. Also, the assumption that similar character states observed under the microscope are the result of evolutionary convergence should be used with caution, since the phylogenetically informative characters we found clearly demonstrated the presence of synapomorphies in Leucocytozoon and Plasmodium. Moreover, microscopic examination of blood smears is cheaper than DNA sequencing, being an informative source of data when financial resources are limited. Also, microscopy allows information to be obtained which is unavailable via PCR, such as the presence of other blood parasites (e.g. Trypanosoma spp., microfilarids) as well as the estimation of hematological parameters (Valkiūnas et al., 2006; Santiago-Alarcon et al., 2012; Lüdtke et al., 2013).

Acknowledgements D.S.-A. was supported by Consejo Nacional de Ciencia y Tecnología, Mexico (CONACYT, ciencia básica 2011-01-168524; CONACYT, problemas nacionales 2015-01-1628), C.H.-L. was supported by a PhD degree grant (CONACYT, scholarship number 280401).

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References Atkinson, C.T. 2008. Haemoproteus. In: Atkinson, C.T., Thomas, N.J., Hunter, D.B. (Eds.), Parasitic diseases of wild birds. Wiley-Blackwell, USA, pp. 13-34. Aurrecoechea, C., Barreto, A., Basenko, E.Y., Brestelli, J., Brunk, B.P., Cade, S., Crouch, K., Doherty, R., Falke, D., Fischer, S., Gajria, B., Harb, O.S., Heiges, M., Hertz-Fowler, C., Hu, S., Iodice, J., Kissinger, J.C., Lawrence, C., Li, W., Pinney, D.F., Pulman, J.A., Roos, D.S., Shanmugasundram, A., Silva-Franco, F., Steinbiss, S., Stoeckert, C.J., Spruill, D., Wang, H., Warrenfeltz, S., Zheng, J., 2017. EuPathDB: the eukaryotic pathogen genomics database resource. Nucleic Acids Res 45, D581–D591. Barta, J.R., 1989. Phylogenetic analysis of the class Sporozoea (Phylum: Apicomplexa Levine, 1970): Evidence for the independent evolution of heteroxenous life cycles. J. Parasitol. 75, 195-206. Bastien, M., Jaeger, A., Le Corre, M., Tortosa, P., Lebarbenchon, C., 2014. Haemoproteus iwa in Great Frigatebirds (Fregata minor) in the Islands of the Western Indian Ocean. PLoS ONE 9, e97185. Bensch, S., Stjernman, M., Hasselquist, D., Östman, Ö., Hansson, B., Westerdahl, H., Pinheiro, T., 2000. Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proc. R. Soc. Lond. B 267, 1583-1589. Bensch, S., Hellgren, O., Pérez-Tris, J., 2009. MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Mol. Ecol. Resour. 9, 1353e1358. Benson, D.A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2013. GenBank. Nucleic Acids Res. 41, D36-42. Bertram, M.R., Hamer, S.A., Hartup, B.K., Snowden, K.F., Medeiros, M.C., Outlaw, D.C., Hamer, G.L., 2017. A novel Haemosporida clade at the rank of genus in North American cranes (Aves: Gruiformes). Mol. Phylogenet. Evol. 109, 73–79.

22

Borner, J., Pick, C., Thiede, J., Kolawole, O.M., Kingsley, M.T., Schulze, J., Cottontail, V.M., Wellinghausen, N., Schmidt-Chanasit, J., Bruchhaus, I., Burmester, T., 2016. Phylogeny of haemosporidian blood parasites revealed by a multi-gene approach. Mol. Phylogenet. Evol. 94, 221-231. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797. Galen, S.C., Borner, J., Martinsen, E.S., Schaer, J., Austin, C.C., West, C.J., Perkins, S.L., 2018. The Polyphyly of Plasmodium: Comprehensive Phylogenetic Analyses of the Malaria Parasites (Order Haemosporida) Reveal Widespread Taxonomic Conflict. R. Soc. Open Sci. 5, 171780. Hellgren, O., Križanauskiene, A., Valkiūnas, G., Bensch, S., 2007. Diversity and phylogeny of mitochondrial cytochrome b lineages from six morphospecies of avian Haemoproteus (Haemosporida: Haemoproteidae). J. Parasitol. 93, 889–896. Kissinger, J.C., Souza, P.C.A., Soares, C.O., Paul, R., Wahl, A.M., Rathore, D., McCutchan, T.F., Krettli, A.U., 2002. Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare. J. Parasitol. 88, 769-773. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33, 1870–1874. Lee, S.Y., Palci, A., 2015. Morphological Phylogenetics in the Genomic Age. Curr. Biol. 25, R922R929. Levin, I.I., Valkiūnas, G., Santiago-Alarcon, D., Cruz, L.L., Iezhova, T.A., O’Brien, S.L., Hailer, F., Dearborn, D., Schreiber, E.A., Fleischer, R.C., Ricklefs, R.E., Parker, P.G., 2011. Hippoboscid-transmitted Haemoproteus parasites (Haemosporida) infect Galapagos Pelecaniform birds: Evidence from molecular and morphological studies, with a description

23

of Haemoproteus iwa. Int. J. Parasitol. 41, 1019-1027. Levin, I.I., Valkiūnas, G., Iezhova, T.A., O'Brien, S.L., Parker, P.G., 2012. Novel Haemoproteus species (Haemosporida: Haemoproteidae) from the Swallow-Tailed Gull (Lariidae), with remarks on the host range of hippoboscid-transmitted avian hemoproteids. J. Parasitol. 98, 847-854. Lüdtke, B., Moser, I., Santiago-Alarcon, D., Fischer, M., Kalko, E.K.V., Schaefer, M., SuarezRubio, M., Tschapka, M., Renner, S.C., 2013. Associations of forest type, parasitism and body condition of two European passerines, Fringilla coelebs and Sylvia atricapilla. PLoS ONE 8, e81395. Martinsen, E.S., Paperna, I., Shall, J.J., 2006. Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts. Parasitology 133, 279-288. Martinsen, E.S., Waite, J.L., Schall, J.J., 2007. Morphologically defined subgenera of Plasmodium from avian hosts: test of monophyly by phylogenetic analysis of two mitochondrial genes. Parasitology 134, 483-490. Martinsen, E.S., Perkins, S.L., Schall, J.J., 2008. A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): Evolution of life-history traits and host switches. Mol. Phylogenet. Evol. 47, 261-273. Outlaw, D.C., Ricklefs, R.C., 2011. Rerooting the evolutionary tree of malaria parasites. Proc. Natl. Acad. Sci. 108, 13183-13187. Pacheco, M.A., Matta, N.E., Valkiūnas, G., Parker, P.G., Mello, B., Stanley, C.E., Jr., Lentino, M., Garcia-Amado, M.A., Cranfield, M., Pond, S.L.K., Escalante, A.A., 2017. Mode and rate of evolution of haemosporidian mitochondrial genomes: timing the radiation of avian parasites. Mol. Biol. Evol., doi 10.1093/molbev/msx285. Palinauskas, V., Bernotienė, R., Žiegytė, R., Bensch, S., Valkiūnas, G., 2017. Experimental evidence for hybridization of closely related lineages in Plasmodium relictum. Mol. Biochem. Parasitol. 217, 1-6.

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Perkins, S.L., Schall, J.J., 2002. A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences. J. Parasitol. 88, 972-978. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MRBAYES 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539542. Santiago-Alarcon, D., Outlaw, D.C., Ricklefs, R.E., Parker, P.G., 2010. High lineage diversity of Haemosporidian parasites in New World doves: multiple colonization of the Galapagos Islands. Int. J. Parasitol. 40, 463-470. Santiago-Alarcon, D., Ricklefs, R.E., Parker, P.G., 2012. Parasitism in the endemic Galápagos Dove (Zenaida galapagoensis) and its relation to host genetic diversity and immune response. In: Paul, E. (Ed.), Emerging avian disease: Studies in Avian Biology (vol. 42). University of California Press, Berkeley, pp. 31–42. Valkiūnas, G., 1997. Bird Haemosporida. Vilnius: Institute of Ecology. Acta Zool. Lit. 3–5, 1-607. Valkiūnas, G. 2005. Avian malaria parasites and other Haemosporidia. CRC Press, Boca Raton. Valkiūnas, G., Bensch, S., Iezhova, T.A., Križanauskienė, A., Hellgren, O., Bolshakov, C.V., 2006. Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential. J. Parasitol. 92, 418422. Valkiūnas, G., Iezhova, T.A., Križanauskienė, A., Palinauskas, V., Bensch, S., 2008. In vitro hybridization of Haemoproteus spp.: an experimental approach for direct investigation of reproductive isolation of parasites. J. Parasitol. 94, 1385-1394. Valkiūnas, G., Santiago-Alarcon, D., Levin, I., Iezhova, T.A., Parker, P.G., 2010. Haemoproteus multipigmentatus sp. nov. (Haemosporida, Haemoproteidae) from the endemic Galapagos dove Zenaida galapagoensis, with remarks on the parasite distribution, vectors, and molecular diagnostics. J. Parasitol. 96, 783-792.

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Legends to figures

Fig. 1. Comparison of avian Haemosporida topologies from six different phylogenetic hypotheses. From this study: (A) morphological traits; (B) mitochondrial DNA (cyt b, coI, cox3, complete mtDNA); and (C) total evidence (morphological traits + mtDNA). From previous studies: (D) morphology and life history traits (modified from Martinsen et al., 2008); (E) nuclear (23 genes), mitochondrial (cyt b and coI), and apicoplast (clpc) DNA (modified from Borner et al., 2016); and (F) nuclear DNA (21 genes) correcting for base composition differences (modified from Galen et al., 2018). Leu, Leucocytozoon; Aki, Akiba; Hae, Haemoproteus; Par, Parahaemoproteus; and Pla, Plasmodium; in (B) and (F) H., Haemoproteus.

Fig. 2. Bayesian morphological phylogenetic hypothesis of avian Haemosporida. Circles on nodes indicate posterior probability: ≥ 99%, black; ≥ 90%, grey; and ≥ 85%, white. Branch color indicates the genus: black, Leucocytozoon; dark grey, Haemoproteus; and light grey, Plasmodium.

Fig. 3. Bayesian mitochondrial DNA phylogenetic hypothesis of avian Haemosporida. Circles on nodes indicate posterior probability: ≥ 99%, black; ≥ 90%, grey; and ≥ 85%, white. Branch color indicates the genus: black, Leucocytozoon; dark grey, Haemoproteus; and light grey, Plasmodium.

Fig. 4. Bayesian total evidence phylogenetic hypothesis of avian Haemosporida. Diamonds on branches indicate the number of unique (black) and homoplasious (white) morphological characters defining each clade. Letters H and S in parentheses indicate whether a character is homoplasious or synapomorphic, respectively. Numbered morphological character figures: 1, presence of large (12.5 μm) Vc in the cytoplasm of GMi (H); 2, FgMa adhere to some of the margin of enucleated Er (S); 3, FgGa are closely appressed to the envelope of enucleated Er (S); 4, valutine granules in FgGa (S, H), and roundish host cell of FgGa (S); 5, cap-like nucleus of roundish host cell of FgGa

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(S), nucleus of roundish host cell extends up to half of the circumference around FgGa (S); 6, absence of a long outgrowth that exceeds the length of the main body of Tr (S), subpolar position of Tr (S), Tr do not adhere to ErN (S) and do not or slightly displace ErN (S); 7, in Tr presence of Vc (S) and roundish (S) dark brown (S) Pg (S) aggregated in loose clumps (H), Tr develop in mature Er (S); 8, absence of a long (> 2 μm) outgrowth in growing Me (S), merozoites randomly arranged in fully grown Me (S), Me develop in mature Er (S) and may adhere (S) to ErN; 9, Me may not adhere (S) to ErN and do not encircle ErN (S); 10, absence of Vc in Me (S), roundish (S), small (< 0.5 μm, S) Pg randomly scattered in the cytoplasm of Me (S); 11, influence of Me on Er (S) and ErN (S) not evident or slightly evident; 12, GMa adhere to the envelope but not to ErN (H); 13, FgMi slightly enclose ErN (H); 14, valutine granules in FgMa (H), FgMa displace ErN laterally (H); 15, oval early young gametocytes (S). Vc, vacuole; FgMa, fully grown macrogametocyte; FgMi, fully grown microgametocyte; FgGa, fully grown gametocyte; GMi, growing microgametocyte; GMa, growing macrogametocyte; Er, erythrocyte; ErN, erythrocyte nucleus; Tr, trophozoite; Pg, pigment granules; and Me, erythrocytic meront. All character figures were modified from Valkiūnas (2005). See Fig. 5 for details on Plasmodium subgenera.

Fig. 5. Bayesian total evidence phylogenetic hypothesis of avian Plasmodium. Subgenera are in bold letters. On terminal branches, black diamonds indicate the number of autapomorphic morphological characters. Letters H and S in parentheses indicate whether a character is homoplasious or synapomorphic, respectively. Numbered morphological character figures: 1, FgGa markedly enclose ErN (H) or 2, they encircle ErN completely and occupy all available cytoplasmic space (H); 3, Me markedly (S) 4, or completely (S) enclose ErN; 5, Er hypertrophied by FgGa (H); 6, Er hypertrophied by Me (S); 7, FgMi do not touch the envelope of enucleated Er (S); 8, Me displace ErN towards one pole of Er (S); 9, FgMa closely appressed to the envelope of Er but do not touch the nucleus along all of its margin (H); 10, nucleus of FgMa close to ErN (H) or 11, close both to ErN and envelope (H); 12, long outgrowth that exceeds the length of the main body of Tr

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(H); 13, merozoites arranged in rows (H) 14, as a fan (H) or 15, as a rosette (H) in FgMe; 16, FgMe occupy less than half of Er cytoplasmic space (H); 17, nucleus of FgMa close to ErN (H); 18, Tr adhere to ErN (H), small (<1 µm) Vc in Tr (H); 19, GMa slightly elongated (H) with Pg aggregated in loose clumps (H); 20, discoid FgMa (H) with Pg aggregated in loose clumps (H); 21, oval FgMa (H) and ErN rotated to the normal axis by FgMa (H); 22, Er hypertrophied by FgMa (H); 23, discoid FgMi with Pg aggregated in loose clumps (H), nucleus of FgMi is not close to ErN or the envelope (H), ErN rotated to the normal axis by FgMi (H); 24, oval FgMi (H), nucleus of FgMi is located close to ErN (H), ErN displaced laterally by FgMi (H). Characters not shown for Bennettinia: discoid GMa (H), discoid or slightly elongated GMi (H), Pg aggregated in loose clumps in GMi (H), FgMa appressed to the envelope of Er and partially touching ErN (H), slightly elongated FgGa (H), ErN rotated to the normal axis by FgMa (H). FgGa, fully grown gametocyte; ErN, erythrocyte nucleus; Me, meront; Er, erythrocyte; FgMi, fully grown microgametocyte; FgMa, fully grown microgametocyte; FgMe, fully grown meront; Tr, trophozoite; Vc, vacuole; GMa, growing macrogametocyte; Pg, pigment granules; GMi, growing microgametocyte. All character figures were modified from Valkiūnas (2005).

Fig. 6. Missing data on avian Haemosporida species descriptions per stage of gametocyte development. (A) Percentage of missing data in the morphological matrix by genus and stage of development in relation to the total amount of missing data. (B) Percentage of missing data by genus in each developmental stage of gametocytes. Bars and triangles: Haemoproteus, black; Leucocytozoon, dark grey; and Plasmodium, light grey. EYG, early young gametocytes; YG, young gametocytes; GMa, growing macrogametocytes; GMi, growing microgametocytes; FgMa, fully grown macrogametocytes; FgMi, fully grown microgametocytes.

Supplementary Fig. S1. Evolutionary scenario for the blood stages of avian Haemosporida morphology inferred by direct optimization of characters on the total evidence hypothesis. Black

28

circles on branches indicate synapomorphies or autapomorphies; white circles indicate homoplasious characters. Numbers above circles indicate the number of the character in the morphological matrix; and numbers below circles indicate the character state (see Supplementary Table S2).

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Table 1. Morphological characters with missing data (%) in avian Haemosporida spp. descriptions by stage of development.

Character Color of pigment granules

EYG 75

YG 70

GMa 61

GMi 60

FgMa 59

FgMi 60

Cytoplasm appearance (e.g., homogeneous, granular)

86

80

17

25

16

22

Presence/absence of vacuoles

81

73 13

12

11

Dimensions of vacuoles (e.g., small- <5 µm, large- 1 to 2.5 µm)

75

Position of gametocyte in relation to the nucleus and envelope of infected erythrocytes

49

37

Influence on the nucleus of infected erythrocytes (e.g., absent, displaced laterally, rotated to the normal axis)

46

38

Outline of gametocyte

47

37

Shape of gametocyte

48

35

Shape of pigment granules

47

37

Peculiarities of location of pigment granules in the cytoplasm (e.g., randomly scattered, aggregated in loose clumps)

47

35

Position of gametocyte (e.g., central, subpolar, polar)

45

38

Dimensions of pigment granules (e.g., 44 small or dust-like- <0.5 µm, medium- 0.5 to 1.0 µm)

35

Presence/absence of pigment granules

25

Presence/absence of valutine granules

42

Me

12

14

20

EYG, early young gametocytes; YG, young gametocytes; GMa, growing macrogametocytes; GMi, growing microgametocytes; FgMa, fully grown macrogametocytes; FgMi, fully grown microgametocytes; Me, erythrocytic meronts.

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Figure 1

A

D

Leu Aki Hae/Par Pla

Leu Hae/Par Pla

B

E

Leu Hae Pla Par Aki H. antigonis

Leu Hae

Hemozoin pigment:

gain

Par

loss

Pla

F

C

Leu Aki

Pla Hae

Pla H. catharti Leu Par Hae

Blood merogony:

gain

Par

Figure 2 L. caulleryi L. californicus L. fringillinarum L. toddi L. lovati L. macleani L. quynzae L. schoutedeni L. dubreuili L. majoris L. danilewskyi L. pterotenuis H. ilanpapernai H. minutus H. minchini H. antigonis H. columbae H. concavocentralis H. danilewskyii H. homobelopolskyi H. macrovacuolatus H. palloris H. pastoris H. picae H. sanguinis H. tartakovskyi H. belopolskyi H. parabelopolskyi H. homohandai H. jenniae H. lanii H. ptilotis H. magnus H. noctuae H. attenuatus H. coatneyi H. erythrogravidus H. motacillae H. vireonis H. micronuclearis H. multivolutinus H. valkiunasi H. iwa H. sacharovi H. syrnii H. hirundinis H. majoris H. passeris H. witti H. bukaka H. nucleocondensus H. payevskyi H. enucleator H. nucleofascialis H. vacuolatus H. pallidus H. paranucleophilum H. cyanomitrae H. homopalloris H. coraciae H. zosteropis H. balmorali H. fringillae H. gavrilovi H. manwelli H. homovelans H. turtur H. pallidulus H. multipigmentatus H. paramultipigmentatus P. rouxi P. vaughani P. elongatum P. relictum P. lutzi P. cathemerium P. gallinaceum P. matutinum P. tejerai P. homopolare P. circumflexum P. homocircumflexum P. multivacuolaris P. juxtanucleare P. lucens P. unalis P. ashfordi P. homonucleophilum P. globularis P. parahexamerium P. megaglobularis P. delichoni P. nucleophilum

Figure 3

L_toddi L_lovati L_macleani L_pterotenuis L_dubreuili L_californicus L_majoris L_fringillinarum L_schoutedeni L_danilewskyi L_quynzae H_iwa H_jenniae H_columbae H_multipigmentatus H_multivolutinus H_paramultipigmentatus P_elongatum P_tejerai P_lutzi P_matutinum P_gallinaceum P_circumflexum P_homocircumflexum P_lucens P_cathemerium P_megaglobularis P_relictum P_nucleophilum P_ashfordi P_delichoni P_juxtanucleare P_homopolare P_rouxi P_globularis P_multivacuolaris P_parahexamerium P_homonucleophilum P_unalis P_vaughani H_antigonis L_caulleryi H_homohandai H_enucleator H_ilanpapernai H_macrovacuolatus H_noctuae H_sacharovi H_syrnii H_turtur H_valkiunasi H_homovelans H_picae H_witti H_danilewskyii H_sanguinis H_zosteropis H_concavocentralis H_vacuolatus H_minutus H_pallidulus H_pallidus H_homopalloris H_palloris H_majoris H_vireonis H_paranucleophilus H_bukaka H_ptilotis H_motacillae H_passeris H_micronuclearis H_homobelopolskyi H_nucleofascialis H_coatneyi H_erythrogravidus H_cyanomitrae H_tartakovskyi H_minchini H_pastoris H_attenuatus H_balmorali H_gavrilovi H_manwelli H_belopolskyi H_nucleocondensus H_parabelopolskyi H_payevskyi H_fringillae H_coraciae H_hirundinis H_lanii H_magnus

10.0 10.0

L. toddi L_toddi L. lovati L_lovati L. macleani L_macleani L. pterotenuis L_pterotenuis L. dubreuili L_dubreuili L. californicus L_californicus L_majoris L. majoris L_fringillinarum L. fringillinarum L. schoutedeni L_schoutedeni L_danilewskyi L. danilewskyi L. quynzae L_quynzae H. H_iwa iwa H. H_jenniae jenniae H. H_columbae columbae H. H_multipigmentatus multipigmentatus H. H_multivolutinus multivolutinus H. H_paramultipigmentatus paramultipigmentatus P. P_elongatum elongatum P. P_tejerai tejerai P. P_lutzi lutzi P. P_matutinum matutinum P. P_gallinaceum gallinaceum P. P_circumflexum circumflexum P. P_homocircumflexum homocircumflexum P. P_lucens lucens P. P_cathemerium cathemerium P. P_megaglobularis megaglobularis P. P_relictum relictum P. P_nucleophilum nucleophilum P. P_ashfordi ashfordi P. P_delichoni delichoni P. P_juxtanucleare juxtanucleare P_homopolare P. homopolare P. P_rouxi rouxi P. P_globularis globularis P. P_multivacuolaris multivacuolaris P. P_parahexamerium parahexamerium P_homonucleophilum P. homonucleophilum P. P_unalis unalis P. P_vaughani vaughani H. H_antigonis antigonis L_caulleryi L. caulleryi H_homohandai H. homohandai H. H_enucleator enucleator H. H_ilanpapernai ilanpapernai H. H_macrovacuolatus macrovacuolatus H. H_noctuae noctuae H_sacharovi H. sacharovi H. H_syrnii syrnii H. H_turtur turtur H. H_valkiunasi valkiunasi H. H_homovelans homovelans H_picae H. picae H. H_witti witti H. H_danilewskyii danilewskyii H. H_sanguinis sanguinis H. H_zosteropis zosteropis H. H_concavocentralis concavocentralis H. H_vacuolatus vacuolatus H. H_minutus minutus H. H_pallidulus pallidulus H. H_pallidus pallidus H. H_homopalloris homopalloris H. H_palloris palloris H. H_majoris majoris H. H_vireonis vireonis H. H_paranucleophilus paranucleophilum H_bukaka H. bukaka H. H_ptilotis ptilotis H. H_motacillae motacillae H. H_passeris passeris H. H_micronuclearis micronuclearis H_homobelopolskyi H. homobelopolskyi H. H_nucleofascialis nucleofascialis H. H_coatneyi coatneyi H. H_erythrogravidus erythrogravidus H. H_cyanomitrae cyanomitrae H_tartakovskyi H. tartakovskyi H. H_minchini minchini H. H_pastoris pastoris H. H_attenuatus attenuatus H. H_balmorali balmorali H_gavrilovi H. gavrilovi H. H_manwelli manwelli H. H_belopolskyi belopolskyi H. H_nucleocondensus nucleocondensus H. H_parabelopolskyi parabelopolskyi H. H_payevskyi payevskyi H. H_fringillae fringillae H. H_coraciae coraciae H. H_hirundinis hirundinis H. H_lanii lanii H_magnus H. magnus

FAMILY LEUCOCYTOZOIDAE

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30 Family 4 Leucocytozoidae 737-850.fm Page 845 Tuesday, September 21, 2004 2:31 PM

FAMILY LEUCOCYTOZOIDAE

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30 Family 4 Leucocytozoidae 737-850.fm Page 781 Tuesday, September 21, 2004 2:11 PM

FAMILY LEUCOCYTOZOIDAE

28 Family 2 Plasmodiidae

589-730.fm Page 700 Tuesday, September 21, 2004 10:51 AM

05 Life Cycle

17-45.fm Page 30 Monday, September 20, 2004 7:02 PM

Figure 285 Gametocytes of Leucocytozoon lovati from the blood of Lagopus lagopus: 1–6 – macrogametocytes; 7–9 – microgametocytes.

Figure 4

781

L_caulleryi

ge 722 Tuesday, September 21, 2004 10:51 AM 700

L_pterotenuis SYSTEMATIC SECTION

L_toddi GENERAL SECTION L_lovati L_macleani SYSTEMATIC SECTION L_fringillinarum 2 2 L_caulleryi L.L_caulleryi L_californicus L_pterotenuis L_pterotenuis L. L_dubreuili L_toddi L.L_toddi L_majoris 28 Family 2 Plasmodiidae 589-730.fm Page 639 Tuesday, September 21, 2004 10:51 AM L_lovati L. L_lovati L_schoutedeni L_macleani L. L_macleani FAMILY PLASMODIIDAE 700 SECTION 639 4 1–4 5L_danilewskyi Figure 339 Gametocytes ofSYSTEMATIC Leucocytozoon caulleryi from the blood of Gallus gallus: L_fringillinarum – young; 5–7, 9, 10 – macrogametocytes; 8 – macro- and microgametocyte in the same host L_fringillinarum L. L_quynzae cell; 11–13 – microgametocytes. Explanations are given in the text. megalomerogony of L. caulleryi. Meronts are about 4 to 11 m in diameter at this time. 6 2 L_californicus L.L_californicus 1 P_circumflexum Infected cells start to rupture, and growing megalomeronts are released from the host cells. L_dubreuili On the ninth day after infection, extracellular meronts are common in numerous organs and L_dubreuili L. P_homocircumflexum 2 megalomerogony of L. caulleryi. Meronts are about 4 to 11 m in diameter at this time. tissues. It is interesting to note that they were recorded not only in the visceral organs but FAMILY HAEMOPROTEIDAE 509 L_majoris L. L_majoris P_relictum also in the eyes and sciatic nerves (Chew, 1968). Megalomeronts rapidly increase in size Figure of Leucocytozoon caulleryi from blood offrom Gallus Infected cells339 start Gametocytes to rupture, and growing megalomeronts arethe released thegallus: host cells. L_schoutedeni – young; 5–7, 9, 10 – macrogametocytes; 8 – are macroand microgametocyte in theand same host defined capsular-like thick wall. As the megalomeront develops,Entebbe, Uganda, 4 L_schoutedeni L. On the1–4 ninth day after infection, extracellular meronts common in numerous organs T y pand e are menclosed a t e r i abyl. a well Hapantotype (No. 28183, Motacilla flava, 30.12.1971, P_cathemerium cell; 11–13 – microgametocytes. Explanations areatgiven in the text. cytomeres appear of (Morii Leucocytozoon et al., 1987). Parasites are located solely caulleryi (Fig. 338, 3, 4) and oftissues. L. caulleryi. Meronts areto about to 11they m in diameter this time. It isblood interesting note4 that were recorded not only in theofvisceral organsfringillinarum but 39 Gametocytes the of Gallus gallus: L_danilewskyi N. Okia) and parahapantotypes (No. 25240, 29.10.1971, other data are as megalomerogony forin thefrom hapantotype; Figure 302 Gametocytes Leucocytozoon from the blood of Fringilla coelebs: L.L_danilewskyi clusters containing from several to approximately 20 megalomeronts (Fig. 338, 5). Mature Infected cells start to rupture, and growing released1–6 from–the host cells.P_gallinaceum macrogametocytes; 7–9 – microgametocytes. Family 2 Plasmodiidae 589-730.fm Page Tuesday, September 21, 2004 10:51 AMH.E. McClure; No. 12568 also in the eyes and megalomeronts sciatic nerves are (Chew, Megalomeronts rapidly increase in size No. 40420, M. 722 alba, 20.12.1969, Rajastham, India, Dendronanthus 626 usually SYSTEMATIC SECTION1968). L_quynzae megalomeronts are located extracellularly. They vary from 100 to 200 m in diamL. L_quynzae On the ninth day after infection, extracellular meronts are common in numerous organs and 2 P_elongatum and are enclosed by a well capsular-like thick wall. the megalomeront develops, Bangphra, H.E.largest McClure) are deposited oung; 5–7,indicus, 9, 10 macrogametocytes; 830ofin theIRCAH. –oftypetheir macroand microgametocyte inAsbut4solely the host ofdefined L. caulleryi. areorgans about to 11 msame in diameter at this time. eter, 20.10.1966, and often – reach 300 m in Tailand, diameter. The megalomeronts can reach 500 m in tissues. It is interesting to megalomerogony note that they were recorded not only inMeronts the visceral P_circumflexum SECTION P.P_circumflexum cytomeres appear al., 1987). areGENERAL located 338,from 3, 4)the and in cells. E t ydiameter m o l o g(Omar, y. The specific name derived fromdepends theSYSTEMATIC generic host, Motacilla. 1968). The size of is megalomeronts on thename peculiarities Infected cells(Morii start toetMegalomeronts rupture, andParasites growing megalomeronts are(Fig. released SECTION also in the eyes and sciatic nerves (Chew, 1968). rapidlyfrom increase in sizeP_lutzi Parasitemia markedly increases fromhost the 5th to 12th day after inoculation of sporoFigure 339 Gametocytes of Leucocytozoon caulleryi the blood of Gallus gallus: clusters containing from several to approximately 20 megalomeronts (Fig. 338, 5). Mature location. Solely developing megalomeronts are usually larger than the parasites developing P_homocircumflexum the capsular-like ninth day infection, extracellular common in numerous and are enclosed a well On defined wall. As the megalomeront develops, P.P_homocircumflexum 6 of– Haemoproteus 8afterthickextracellularly. 7 5–7, 13 – microgametocytes. Explanations are given in– bymacrogametocytes; the text. zoites, meronts and thenare rapidly decreases (Fig. organs 301). Aand low parasitemia was recorded in birds, P_matutinum in clusters (Kitaoka al., 1972). It vertebrate should be1–4 noted megalomeronts in 10 megalomeronts locatedare Theywere usually varyinfrom 100 in to the 200 m in diamTableet113 List of hoststhat motacillae. cytomeres appear (Morii ettissues. al., 1987). located (Fig. 338, 3,recorded 4) and and young; developing 9, 8solely – they macroin period the ofsame It are isParasites interesting to note that notmicrogametocyte only organs but(the P_relictum which were infected once, up to avisceral 10-month period observation). P.P_relictum 1host P_tejerai and reach diameter. The largest megalomeronts canrapidly reach 500 m inin size clusters containing eter, from several to the approximately 20 in megalomeronts (Fig. 338, 5). Mature alsooften in eyes300 and m sciatic nerves (Chew, 1968). Megalomeronts Macrogametocytes (Fig. 302, increase 1–6; Table 153) develop in roundish host cells; P_cathemerium Anthus campestris megalomeronts are diameter located extracellularly. TheyThe usually vary from 100 to 200 mdepends in diamcell;Dendronanthus 11–13 –indicus microgametocytes. Explanations are given in the text. P.P_cathemerium (Omar, 1968). size of megalomeronts on the peculiarities of their P_lucens and are enclosed by a well defined capsular-like thick wall. As the megalomeront develops, cytoplasm frequently contains small vacuoles; valutin granule are usually present; gametoeter, and often reachlocation. 300 m in diameter. The largest megalomeronts can reach 500 m in A. hodgsoni Motacilla alba Solely developing megalomeronts areParasites usually larger than the parasites developing P_gallinaceum cytomeres appear (Morii et al., 1987). are located solely (Fig.oval 338,form; 3, 4) the andparasite in P.P_gallinaceum cytes are roundish or of slightly nucleus is of variable form and diameter (Omar, 1968). The size of megalomeronts depends on the peculiarities of theirP_megaglobularis A. trivialis in clusters (Kitaoka et al., 1972). It should be noted that megalomeronts (Fig. developing inMature containing from several to the approximately 20 megalomeronts 338, 5). 8 P_elongatum location. Solely developingclusters megalomeronts are usually larger than parasites developing position; the nucleolus is prominent and well seen; the nucleus of host cell is pushed aside, P.P_elongatum P_nucleophilum located They usually from 100 to 20027 m in clusters (Kitaoka et al.,megalomeronts 1972). It should are be noted thatextracellularly. megalomeronts developing Family 1 Haemoproteidae 251-473.fm 3311–5), Monday, September 20, 2004 7:12 PMP_lutzi deformed, and in liesvary peripherally usually asina diammore or less evident cap (Fig.Page 302, 3 P.P_lutzi 2 P_ashfordi eter, and reach 300in m in diameter diameter. The largest megalomeronts 500extends m in less than 1/2 of the circumference of merogonyM aof about 4 to 11often m at time. sometimes band-like (Fig.this 302, can 6), itreach usually i n dL. i a g ncaulleryi. o s t i c c h a r a c t e r s.Meronts A parasite of speciesare of the Passeriformes whose P_matutinum megalomerogony of L. caulleryi. Meronts 4 to 11 m in diameter at this time. diameter (Omar, 1968). Theare size ofabout megalomeronts depends on can the peculiarities of of their P.P_matutinum gametocyte but sometimes extend up to 1/2 the circumference; the cytoplasm of host P_delichoni FAMILY HAEMOPROTEIDAE gametocytes grow around the nucleus of infected erythrocytes but do not encircle it com-21 location.1Solely developing megalomeronts are usually larger than the parasites developing P_tejerai cells is largely replaced by gametocytes, and is sometimes even invisible (Fig. 302, 5, 6) P.P_tejerai331 P_juxtanucleare Gametocytes adhere and to the nucleus and envelope ofmegalomeronts thestart erythrocytes. Dumbbell- inand d cells startpletely. to rupture, growing are released from the host cells. Infected cells to rupture, growing released from host cells. clusters (Kitaoka et al.,megalomeronts 1972). It should be frequently noted thatare in the but more ismegalomeronts present arounddeveloping the gametocytes as a more or less evident and pale P_lucens P.P_lucens shaped gametocytes are present, and they predominate in growing macrogametocytes. P_multivacuolaris margin of variable form (Fig. 302, 1–4). 9 11 10 On ninthin day after infection, extracellular are P_rouxi common in numerous organs and P_megaglobularis Gametocytesinfection, with a highly ameboid outline do notthe predominate growing macrogametocytes. P.P_megaglobularis Microgametocytes (Fig. 302, 7–9).and The general configuration and other features are ninth day after extracellular meronts are common inmeronts numerous organs oooooo P_nucleophilum as for macrogametocytes with the usual sexual dimorphic characters. P.P_nucleophilum P_homopolare tissues. It is interesting to note that they were recorded not only in the visceral organs but Relapses are well evident and synchronized with the breeding period of birds. P_ashfordi P.P_ashfordi It is interesting to note thatalso they were recorded not only in the visceral organs but increase1 in size P_vaughani in the eyes and sciatic nerves (Chew, 1968).D eMegalomeronts rapidly P_delichoni P.P_delichoni v e l o p m e nP_unalis t i n v e c t o r was investigated by Khan and Fallis (1970a) at a tem26 P_juxtanucleare perature of 21°C.P_homonucleophilum Exflagellation was observed in the midgut of simuliid flies 2 to 5 min after P. P_juxtanucleare the eyes and sciatic nervesand (Chew, 1968). Megalomeronts rapidly increase in size are enclosedFigure by a well339 defined capsular-likeof thick wall. As the megalomeront develops, Gametocytes Leucocytozoon caulleryi from ingestion of gametocytes. Ookinetes are present in the midgut betweenthe 12 and blood 108 h after of Gallus gallus: P_multivacuolaris P.P_multivacuolaris P_globularis cytomeres appear (Morii et al., 1987). Parasites are located solely (Fig. 338, 3, 4) and in P_rouxi – young; 5–7,the 9, megalomeront 10 – macrogametocytes; 8 – macro- and microgametocyte P. inP_rouxi the same host enclosed by a well defined capsular-like1–4 thick wall. As develops, P_parahexamerium P_homopolare P.P_homopolare H_antigonis clusters containing from several to approximately 20 megalomeronts (Fig.given 338, 5). cell; 11–13 – microgametocytes. Explanations in Mature the text. 14andare P_vaughani P.P_vaughani res appear (Morii et al., 1987). Parasites are located solely (Fig. 338, 3, 4) in H_iwa megalomeronts are located extracellularly. They usually vary from 100 to 200 m in diamP_unalis P.P_unalis H_jenniae Figure 86 Gametocytes of HaemoP_homonucleophilum containing from several toeter, approximately 5). Mature P.P_homonucleophilum and often reach 20 300 megalomeronts m in diameter. The (Fig. largest 338, megalomeronts can reach 500 m in H_multivolutinus proteus antigonis from the blood of P_globularis P. Figure 263 Plasmodium nucleophilum from the blood of Serinus canaria: Grus canadensis P_globularis (2, 3, 6) and H_paramultipigmentatus diameter (Omar, 1968). The size of megalomeronts depends on the peculiarities of their 1, 2 – trophozoites; 3–10 – erythrocytic meronts; 11–17 – macrogametocytes; 18–20 – microP_parahexamerium meronts are located extracellularly. They usually vary from 100 to 200H_columbae m in diammelanogaster (1, 4, 5): P.P_parahexamerium megalomerogony of L. caulleryi. Meronts are about 4 to 11 m in Lissotis diameter at this time. gametocytes. 1, 3–5 – macrogametocytes; 2, 6 – H_antigonis H. H_antigonis location. Solely developing megalomeronts are usually larger than the parasites developing H_multipigmentatus microgametocytes (modified from H_iwa d often reach 300 m in diameter. The largest megalomeronts can reach 500 m in H_iwa H. 2 H_picae cells start to rupture, and growing megalomeronts are released the host cells. Bennettfrom et al., 1975a). cycle of merogony is closeInfected to 24 h. et The majority of meronts rupture around the midday or in clusters (Kitaoka al., 1972). It should be noted that megalomeronts developing in H_jenniae FAMILY HAEMOPROTEIDAE H.H_jenniae 571 during a chronic stage of H_noctuae afterward. A few parasites are present in the blood H_multivolutinus r (Omar, 1968). The size ofshortly megalomeronts depends on the peculiarities their infection. Relapses were recorded but are not well pronounced (Manwell, 1935a). H.H_multivolutinus On the ninth day after infection, extracellular meronts are common in numerous organs and Figure 240 Plasmodium circumflexum from the blood ofof Serinus canaria: H_homohandai Among the haemoproteids of birds belonging to the Gruiformes, H. antigonis is especially simiTrophozoites (Fig. 263, 1, 2) are seen in mature erythrocytes; the ‘ring’ stage 1–3 – trophozoites; 4 – acan fullybegrown trophozoite (top) and an earliest binuclear erythrocytic meront H_paramultipigmentatus H.aH_paramultipigmentatus H_ilanpapernai 12 circumflexumpresent 13 lar to H. 19, gallinulae. It can be distinguished from the latter species on the basis of smaller number of Figure 240 Plasmodium from but the blood of Serinus (bottom); 5–13in–the erythrocytic macrogametocytes; 20 – microgametocytes. the ‘rings’ arecanaria: uncommon; growinglarger trophozoites are variable form butparasites wellmeronts; 14–18 –developing n. Solely developing megalomeronts are usually than H_columbae tissues. It is interesting to note that they were recorded not only in the visceral organs but H_columbae H. 1–3 – trophozoites; 4 – a fully grownevident trophozoite (top) andoutgrowths an earliest binuclear erythrocytic meront earliest trophozoites can be seen pigment granules in its gametocytes. H_sacharovi ameboid are not characteristic; (bottom); 5–13 – erythrocytic meronts; 14–18 – macrogametocytes; 19, 20 – microgametocytes. H_multipigmentatus H.H_multipigmentatus anywhere in infected erythrocytes; as the parasite develops, trophozoites elongate and one H_syrnii 236 Plasmodium tejerai from the blood of Meleagris gallopavo: and sciatic nerves also in the eyes (Chew, 1968). Megalomeronts rapidly increase ers (Kitaoka et al., 1972).Figure It should be noted that megalomeronts developing in Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; H_picae two minute-size pigment granules trophozoites are now usually seen in H.H_picaein size 1–3 –ortrophozoites; 4–9 –dark erythrocytic meronts; appear, 10–15 and – macrogametocytes; 16–20 – microH_macrovacuolatus Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen gametocytes. H_noctuae H.H_noctuae H_valkiunasi earliest trophozoites which are roundish or oval, sometimes ofand irregular are shape, can be seen enclosed by a well defined capsular-like thick wall. As the megalomeront anywhere in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; H_homohandai 27 is Family 1 Haemoproteidae 251-473.fm Page 373 Monday, September 20, 2004 7:12 PM 23. Haemoproteus (Parahaemoproteus) beckeri Roudabush and develops, H.H_homohandai anywhere erythrocytes; the outline is even; Figure the ‘ring’ not characteristic; H_enucleator 12stage Plasmodium relictum from the of Passercanaria: hispaniolensis: 273 Plasmodium elongatum from the blood in ofinfected Serinus canaria: asblood the parasite develops, nucleus markedly increases in size, plentiful cytoplasm develops Figure 263 Plasmodium nucleophilum from the blood of Serinus H_ilanpapernai as the parasite develops, nucleus increases in plentiful cytoplasm develops D i s t rmarkedly i b u t i o n. This parasite been recorded in Venezuela so far. 2 – has trophozoites; 3–9 –only erythrocytic meronts; 10–14 – macrogametocytes; 15, 16 – microgametoH_ilanpapernai H. Coatney, 1935 rophozoites; 5–13 – erythrocytic meronts; 14–17 – macrogametocytes; 18–20 –1,size, microH_homovelans and one or several small roundish pigment granules appear (Fig. 240, 2, 3); fully grown cytomeres appear (Morii al., 1987). Parasites are located solely (Fig. 338, 3, 4) and in FAMILY and small roundish pigment appear (Fig. 240,HAEMOPROTEIDAE 2,meronts; 3);inNc fully grown 373 cytes; Me merozoite; – nucleolus; Ne – nucleus of erythrocyte; Npet – nucleus of18–20 parasite; Pg– – micro1,one2 or–several trophozoites; 3–10 erythrocytic 11–17 – ismacrogametocytes; Type m a2 t e r i agranules l. –Hapantotypes are– deposited CPGA; parahapantotype deposited in IRCAH. H_sacharovi cytes. H.H_sacharovi trophozoites areCPG variable be seen anywhere in infected erythrocytes but H_turtur pigmentin granule. trophozoites are variable inItform, they can beoriginal seen anywhere infected was noted in the description that parterythrocytes of parahapantotypes was deposed in the (Well- in form, they can 15 thebuttype material gametocytes. come H_syrnii H.H_syrnii Museumfrom of Medical Sciences, London). However,containing is absent from in the CPG. several to clusters approximately 20 megalomeronts 5). Mature H_minutus Haemoproteus beckeri Roudabush and Coatney 1935: 1, Fig. 1, 2.(Fig. 338, Figure 181 Gametocytes of Haemoproteus motacillae the blood of Motacilla flava: H_macrovacuolatus E m o l o g y. This species is named in honour of Venezuelan parasitologist Dr. Enrique Tejera. H.H_macrovacuolatus 1, 2and – young; – macrogametocytes; 9 t–ymicrogametocytes. series. one to three weeks) then3–7 turns into a chronic8,stage which lasts for merozoites several formed in metacryptozoites are able to infect the cells of the erythrocyticH_pallidulus H_valkiunasi H. H_valkiunasi megalomeronts are located vary from 100 to 200 m in diamT y p e They v e r t e b r a usually t e h o s t. Toxostoma rufum (L.) (Passeriformes). One part of merozoites developed in metacryptozoites induces the extracellularly. next generations of H_concavocentralis . Erythrocytic meronts are always rare in the peripheral blood, and gametocytes M a i n d i a g n o s t i c c h a r a c t e r s. Fully grown trophozoites and young erythrocytic metacryptozoites and phanerozoites, another part invades the erythrocytes,H_palloris giving d d i t i o n a l v e r t e b r a t e h o s t s. Dumetella carolinensis and MimusH_enucleator polyglottos (PasseriH. H_enucleator minate. The majority of erythrocyticcycle merontsof concentrate in the haemopoietic merogony is close toorgans. 24 agamic h. The of while meronts middayAor meronts possess a large (frequently greatermajority than 2 µm in diameter) vacuole, rupture and pigmentaround the rise to eter, stages and gametocytes, which simultaneously appear in the blood. At this formes). H_homovelans and often reach 300 m in diameter. The largest megalomeronts can reach 500 m in H_homovelans H. ythrocytic merogony is clearly synchronized. A cycle of merogony is equal to 24 h. H_danilewskyii shortly afterward. A few parasites are present in the blood during a chronic stage Tof y p e l o c a l i t y. Peru, Nebraska, USA. H_turtur H.H_turtur jority of meronts rupture in the morning. The number of gametocytes decreases as H_sanguinis D i s t r i b u t i o n. This species has been recordedon only inthe the Nearctic so far.H_minutus diameter (Omar, 1968). The size of megalomeronts depends peculiarities of their infection. Relapses were recorded but are not well pronounced (Manwell, 1935a). H.H_minutus mber of blood passages increases, and the laboratory strains can finally lose the H_homopalloris T y p e m a t e r i a l. Hapantotype (No. 45242, Toxostoma rufum, September 1934, Peru, Nebraska, H_pallidulus to produce gametocytes. H.H_pallidulus Trophozoites (Fig. 263, 1, 2)location. are seen inSolely mature erythrocytes; the megalomeronts ‘ring’ stage can USA, be G.R.are H_zosteropis Coatney) is deposited in IRCAH. than the parasites developing usually larger developing H_concavocentralis ophozoites (Fig. 273, 1–4) are seen in all types of erythrocytes but more frequently H. E t y m o l o g y. This haemoproteid is named in honour of Dr. Elery R. Becker, theH_concavocentralis friend and teacher H_pallidus present but the ‘rings’ are uncommon; growing trophozoites are variable in form but well 1 d in young erythrocytes including erythroblasts; earliest trophozoites are roundish H_palloris of the authors of the specific name. H_palloris in H.developing H_vacuolatus in clusters (Kitaoka et al., 1972). It should be noted that megalomeronts frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite evident ameboid outgrowths are not characteristic; earliest trophozoites can be seen H_danilewskyii H.H_danilewskyii H_witti ps, trophozoites take an irregular form, ameboid outgrowths appear and are now H_sanguinis M a i n d i a g n o s t i c c h a r a c t e r s. A parasite of species of the Passeriformes whose H.H_sanguinis anywhere in infected erythrocytes; as the parasite develops, trophozoites elongate and one H_majoris gametocytes grow along the nucleus of infected erythrocytes; they H_homopalloris slightly enclose the H. H_homopalloris H_nucleofascialis or two minute-size dark pigment granules appear, and trophozoites are now usually seennucleus in with their ends but never encircle it completely. MediumH_zosteropis and fully grown H.H_zosteropis H_fringillae gametocytes are closely appressed to the nucleus and envelope of erythrocytes. H_pallidus H. H_pallidus H_hirundinis Dumbbell-shaped gametocytes are absent or they represent less than 10% of the total H_vacuolatus H_vacuolatus H. Figure 214 Gametocytes of Haemoproteus columbae from the blood of Columba livia: H_nucleocondensus number of growing gametocytes. Fully grown gametocytes do not fillH_witti the erythrocytes up 1–5 – macrogametocytes; 6–8 – microgametocytes (modified from Valkiunas and Iezhova, 1990b). H. H_witti H_payevskyi to their poles. Pigment granules are of large (1.0 to 1.5 m) and medium (0.5 to 1.0 m) H_majoris H.H_majoris H_pastoris size, rod-like or oval, about ten per gametocyte on average. Infected erythrocytes are roundish, usually median or submedian in position and possesses a clump of chromatin; H_nucleofascialis H. H_nucleofascialis Figure 107 Gametocytes of Haemoproteus picae from the blood of Pica H_minchini pica: hypertrophied in length in comparison to uninfected ones. pigment granules are roundish, of medium (0.5 to 1.0 µm) and large (1.0 to 1.5 µm) size, – H_fringillae H. 1–3 – young; 4–8 – macrogametocytes (modified from Valkiunas and Iezhova, 1992b). H_fringillae H_ptilotis frequently aggregated into large compact masses12 (Fig. 214, 2, 5), randomly scattered Figure Plasmodium relictum from the blood of Passer hispaniolensis: H_hirundinis H.H_hirundinis H_homobelopolskyi throughout the cytoplasm. 2 1, 2The–general trophozoites; 3–9 – erythrocytic meronts; 10–14 – macrogametocytes; 15, 16 – microgameto-H_nucleocondensus H.H_nucleocondensus Microgametocytes (Fig. 214, 6–8). configuration is as for macrogametoH_attenuatus H_payevskyi cytes with the usual sexual dimorphic characters; pigment granules tend to aggregate into H.H_payevskyi H_balmorali cytes; Me merozoite; Nc and – nucleolus; Ne – nucleus of erythrocyte; Np – nucleus of parasite; Pg –H_pastoris large (over 1 µm in diameter) compact masses and, as a– result, the granules are larger H.H_pastoris H_belopolskyi their number approximately half as pigment many as in macrogametocytes; other characters are as H_minchini granule. H.H_minchini H_parabelopolskyi macrogametocytes. m the blood of forSerinus canaria: H_ptilotis H.H_ptilotis H_lanii The aggregation of pigment into large compact masses in gametocytes is a distinctive H_homobelopolskyi H. H_homobelopolskyi character of H. columbae. This aggregation takes place during the final stages of deH_magnus c meronts; 14–17 – macrogametocytes; 18–20 – microH_attenuatus velopment in the blood. It has not always been recorded in naturally infected birds H.H_attenuatus H_vireonis investigated only once (Roudabush and Coatney, 1935; Ahmed and Mohammed, 1978b; H_balmorali H.H_balmorali H_motacillae – merozoites formed in metacryptozoites are able to infect the cells of the erythrocytic series.H_belopolskyi Valkiunas and Iezhova, 1990b). H.H_belopolskyi H_coatneyi The dynamics of parasitemia, relapses, and some peculiarities of immunity were H.H_parabelopolskyi One part of merozoites developed in metacryptozoites induces the next generations ofH_parabelopolskyi investigated by Ahmed and Mohammed (1978a) in experimentally infected Columba livia. H_erythrogravidus oo H.H_lanii H_micronuclearis 3 part invades the erythrocytes, givingH_lanii metacryptozoites and phanerozoites, while another H_magnus H.H_magnus H_tartakovskyi H_vireonis H.H_vireonis H_bukaka to agamic stages gametocytes, which simultaneously appear in the blood. At thisH_motacillae n turns into a chronic stagerisewhich lasts for and several H.H_motacillae H_gavrilovi H_coatneyi H.H_coatneyi H_manwelli ways rare in the peripheral blood, and gametocytes H_erythrogravidus H.H_erythrogravidus H_coraciae 4 H_micronuclearis H.H_micronuclearis H_cyanomitrae Figure 108 Microgametocytes of Haemoproteus picae from the blood of Pica pica (modified H_tartakovskyi cytic meronts concentrate in the haemopoietic organs. H.H_tartakovskyi – H_paranucleophilus from Valkiunas and Iezhova, 1992b). H_bukaka H.H_bukaka H_passeris H_gavrilovi H.H_gavrilovi y synchronized. A cycle of merogonydevelops, is equal to 24 h. gametocytes adhere to the nucleus of erythrocytes and extend longitudinally along H_manwelli H.H_manwelli 4 the nucleus, frequently taking a crescent-like shape (Fig. 107, 2, 3). H_coraciae H.H_coraciae he morning. The number of gametocytes decreases as H_cyanomitrae H.H_cyanomitrae H_paranucleophilus paranucleophilum H.H_paranucleophilus ases, and the laboratory strains can finally lose the 10.0 H_passeris H.H_passeris

1

30

2

3

Family 2 Plasmodiidae 589-730.fm Page 700 Tuesday, September 21, 2004 10:51 AM Figure 339 Gametocytes of Leucocytozoon caulleryi from the28 blood of Gallus gallus: 1–4 – young; 5–7, 9, 10 – macrogametocytes; 8 – macro- and microgametocyte in the same host cell; 11–13 – microgametocytes. Explanations are given in the text.

27 Family 1 Haemoproteidae 474-587.fm Page 509 Monday, September 20, 2004 7:16 PM

Figure 339 Gametocytes of Leucocytozoon caulleryi from the blood of Gallus gallus: 1–4 – young; 5–7, 9, 10 – macrogametocytes; 8 – macro- and microgametocyte in the same host cell; 11–13 – microgametocytes. Explanations are given in the text.

28 Family 2 Plasmodiidae

FAMILY PLASMODIIDAE

28 Family 2 Plasmodiidae

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05 Life Cycle

17-45.fm Page 30 Monday, September 20, 2004 7:02 PM 639

Development in vertebrate host The exoerythrocytic merogony is only fragmentary investigated in naturally infected birds. Two types of meronts are recorded, the meronts in the liver and the megalomeronts in the kidneys. Merogony was not observed in the other organs. M a i n d i a g n o s t i c c h a r a c t e r s. A parasite of species of the Galliformes whose gametocytes develop in roundish and fusiform host cells. The nucleus of fusiform host cell is of cap-like form or almond-shaped or resembles the nucleus of uninfected erythrocyte; the nucleus extends less than 1/3 of the circumference of the gametocyte. Currently, this parasite can be identified with confidence only in birds of the family Tetraonidae. Bonasa umbellus Canachites canadensis Centrocercus urophasianus Dendragapus canadensis

Table 148

D. obscurus Lagopus lagopus L. leucurus L. mutus

Lyrurus tetrix Tetrao urogallus Tetrastes bonasia Tympanuchus phasianellus

List of vertebrate hosts of Leucocytozoon lovati.

755

FAMILY LEUCOCYTOZOIDAE

27 Family 1 Haemoproteidae 474-587.fm Page 571 Monday, September 20, 2004 7:16 PM

30 Family 4 Leucocytozoidae 737-850.fm Page 755 Tuesday, September 21, 2004 2:11 PM

Development in vertebrate host The exoerythrocytic merogony is only fragmentary investigated in naturally infected birds. Two types of meronts are recorded, the meronts in the liver and the megalomeronts in the kidneys. Merogony was not observed in the other organs. M a i n d i a g n o s t i c c h a r a c t e r s. A parasite of species of the Galliformes whose gametocytes develop in roundish and fusiform host cells. The nucleus of fusiform host cell is of cap-like form or almond-shaped or resembles the nucleus of uninfected erythrocyte; the nucleus extends less than 1/3 of the circumference of the gametocyte. Currently, this parasite can be identified with confidence only in birds of the family Tetraonidae. –

Bonasa umbellus Canachites canadensis Centrocercus urophasianus Dendragapus canadensis

Table 148

D. obscurus Lagopus lagopus L. leucurus L. mutus

Lyrurus tetrix Tetrao urogallus Tetrastes bonasia Tympanuchus phasianellus

List of vertebrate hosts of Leucocytozoon lovati.

755

AMILY LEUCOCYTOZOIDAE

30 Family 4 Leucocytozoidae 737-850.fm Page 755 Tuesday, September 21, 2004 2:11 PM

e seen in all types of erythrocytes but more frequently 10.0 10.0 ding erythroblasts; earliest trophozoites are roundish the ‘ring’ stage was seen occasionally; as the parasite ular form, ameboid outgrowths appear and are now

FAMILY PLASMODIIDAE

28 Family 2 Plasmodiidae

639

589-730.fm Page 603 Tuesday, September 21, 2004 10:51 AM

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603

L_danilewskyi L_quynzae L_schoutedeni L_californicus L_dubreuili Figure 5 L_majoris L_fringillinarum L_pterotenuis 1 2 3 4 L_toddi L_lovati L_macleani L_caulleryi P. circumflexum P_circumflexum 4 2 P. P_homocircumflexum homocircumflexum Giovannolaia P. P_relictum relictum 1 Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; P. P_cathemerium cathemerium earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; gallinaceum which are roundish or oval, sometimes of irregularP. shape, can be seen P_gallinaceum 5 6 7asanywhere the parasite 8 develops, nucleus earliest markedlytrophozoites increases in size, plentiful cytoplasm develops Haemamoeba anywhere in infected erythrocytes; outline is even; the ‘ring’ stage is not characteristic; and one or several small roundish pigment (Fig. 240, the 2, 3); fully grown 3 granules 2 appear P. tejerai P_tejerai thecan parasite develops, markedly increases trophozoites are variable in form,asthey be seen anywherenucleus in infected erythrocytes but in 1size, plentiful cytoplasm develops and one or several small roundish pigment granules appear (Fig. 240,P. 2, 3);matutinum fully grown P_matutinum trophozoites are variable in form, they can be seen anywhere in infected erythrocytes but P. P_lutzi lutzi (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; 8 Trophozoites P. elongatum Huffia P_elongatum earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen anywhere in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; 722 SYSTEMATIC SECTION P. lucens P_lucens as the parasite develops, nucleus markedly increases in size, plentiful cytoplasm develops 722 SYSTEMATIC SECTION and one or several P. small megaglobularis roundish pigment granules appear (Fig. 240, 2, 3); fully grown 4 P_megaglobularis trophozoites are variable in form, they can be seen anywhere in infected erythrocytes but 17 P. P_nucleophilum nucleophilum 6); fully grown meronts are roundish, oval, or irregular in form, they occupy more than half 3 P. P_ashfordi ashfordi of the cytoplasmic space in the infected erythrocytes; mature meronts contain 6 to 24 (on average 16) merozoites; pigment granules are roundish or oval, usually of small size 2 P. P_delichoni delichoni (<0.5 m), brown, and clumped into a spot; meronts markedly deform the infected Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; erythrocytes, they markedly displace their nuclei and can even enucleate the host cells (Fig. P. homopolare P_homopolare earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen 226, 7); as a rule, the1fully grown meronts are less than 10 m and are usually about 7 SYSTEMATIC SECTION Novyella anywhere in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; P. P_vaughani vaughani as the parasite develops, nucleus markedly increases in size, plentiful cytoplasm develops and one or several small roundish pigment granules appear 3); fully grown 1 (Fig. 240, 2,P. unalis P_unalis trophozoites are variable in form, they can be seen anywhere in infected erythrocytes but 9 10 11 12 1 P. P_homonucleophilum homonucleuphilum nuclei decrease in size as the parasite matures; fully grown meronts are roundish, gular, of oval or fan-like form with nuclei arranged usually as rosettes or fans; 1 P. globularis P_globularis meronts contain four merozoites; one round globule, which is colourless or of 4 quoise colour and is located close to the pigment granules, is frequently seen P. P_parahexamerium parahexamerium 1, 7, 9, 11–14); this globule is sometimes seen even in segmenters (Fig. 261, 13, P. P_rouxi rouxi ment granules are of variable size but never exceed 0.5 m in diameter, are not s (usually one to three), black or dark brown, clumped into a spot; two 4 P. P_multivacuolaris multivacuolaris -size pigment granules are most frequently seen; meronts can be seen anywhere in 13 14 15 16 erythrocytes but more frequently are seen in a polar or subpolar position in the 26 P. P_juxtanucleare juxtanucleare Bennettinia H_antigonis (within one to three weeks) and then turns into a chronic stage which lasts for several months. Erythrocytic meronts are always rare in the peripheral blood, and gametocytes H_iwa predominate. The majority of erythrocytic meronts concentrate in the haemopoietic organs. Erythrocytic merogony is clearly synchronized. A cycle of merogony is equal to 24 h. H_jenniae The majority of meronts rupture in the morning. The number of gametocytes decreases as the number of blood passages increases, and the laboratory strains can finally lose the H_multivolutinus ability to produce gametocytes. 18 20 21 23 24 22 H_paramultipigmentatus Trophozoites (Fig. 273, 1–4) are seen in all types of erythrocytes but more19 frequently recorded in young erythrocytes including erythroblasts; earliest trophozoites are roundish H_columbae or oval, frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite develops, trophozoites take an irregular form, ameboid outgrowths appear and are now H_multipigmentatus H_picae 5.0 H_noctuae Figure 273 Plasmodium elongatum from the blood of Serinus canaria: 1– 4 – trophozoites; 5–13 – erythrocytic meronts; 14–17 – macrogametocytes; 18–20 – microH_homohandai gametocytes. Figure 273 Plasmodium elongatum from the blood of Serinus canaria: Figure 273 Plasmodium elongatum from the blood of Serinus canaria: H_ilanpapernai 1–– 4macrogametocytes; – one trophozoites; 5–13 –and erythrocytic meronts; 14–17 – macrogametocytes; 18–20 – micro1– 4 – trophozoites; 5–13 – erythrocytic meronts; 14–17(within 18–20 –then microto three weeks) turns into a chronic stage which lasts for several Trophozoites 270, 1, 2) seenand in mature and polychromatic erythrocytes; the Trophozoites (Fig. 270, 1,(Fig. 2)H_sacharovi are seen in are mature polychromatic Trophozoites (Fig. 270, 1, 2) are seen inerythrocytes; mature and the polychromatic erythrocytes; the gametocytes. gametocytes. earliest trophozoites are of variable even orinirregular in outline, possess negligible months. Erythrocytic meronts are always rare in the peripheral blood, and gametocytes Trophozoites (Fig. 270,earliest 1, 2) are seen in mature polychromatic erythrocytes; the trophozoites are of and variable even form, orare irregular outline, earliest form, trophozoites of variable form, possess even or negligible irregular in outline, possess negligible cytoplasm (Fig. 270,pronounced 1);inclearly pronounced ameboid outgrowths arefully not seen; grown predominate. The majority of erythrocytic meronts concentrate in the haemopoieticearliest organs.trophozoites are of variable even or irregular outline, possess negligible cytoplasmform, (Fig. 270, 1); clearly are not seen; grownfully are cytoplasm (Fig.ameboid 270, 1); outgrowths clearly pronounced ameboid outgrowths not seen; fully grown H_syrnii trophozoites areoutgrowths roundish, oval, or ofseen; irregular form, usually adhere to the nuclei of infected pronounced ameboid areare not fully Erythrocytic merogony is clearly synchronized. A cycle of merogony is equalcytoplasm to 24 h. (Fig. 270, 1); clearly trophozoites are roundish, oval, or of irregular form, usually adhere to the nuclei infected trophozoites roundish, oval,grown or of irregular form,ofusually adhere to the nuclei of infected (within oneoftowhich three lasts weeks) then turns a chronic stage which lasts erythrocytes, possess one or two minute dark-brown pigment granules (Fig. 270, 2); a small trophozoites areseveral roundish, oval, or of irregular form, adhere topossess the nuclei of infected (within one to three weeks) and then turns into a chronic stage forinand several The majority meronts rupture the morning. Theinto number of gametocytes decreases as for erythrocytes, possess one usually or two minute dark-brown pigment granules (Fig. 270, 2); a smallgranules (Fig. 270, 2); a small erythrocytes, one or two minute dark-brown pigment H_macrovacuolatus vacuole is sometimes present in the cytoplasm, but the typical ‘ring’ stage is not months. meronts are always in the strains peripheral blood, and erythrocytes, possess one or vacuole two minute dark-brownpresent pigment (Fig. 270, 2);the a small is sometimes ingranules theis cytoplasm, but typical ‘ring’ stagebut is the not typical ‘ring’ stage is not vacuole sometimes present in the cytoplasm, numberErythrocytic of blood, blood passages increases, and therare laboratory can finally lose thegametocytes months. Erythrocytic meronts are always rare in thethe peripheral and gametocytes characteristic; infection of the same erythrocyte with several parasites is common during Figure 273 Plasmodium elongatum from blood of majority Serinus canaria: H_valkiunasi characteristic; infection ofbut thecharacteristic; same erythrocyte withstage several parasites is common during parasites is common during infection of theissame with several vacuole is sometimes in the cytoplasm, the typical ‘ring’ not erythrocyte predominate. The of erythrocytic meronts concentrate in the haemopoietic organs. present ability to the produce gametocytes. predominate. The majority concentrate in the haemopoietic organs. 1–of 4 erythrocytic – trophozoites;meronts 5–13 – erythrocytic meronts; 14–17 – macrogametocytes; 18–20 – microcharacteristic; infection of the same erythrocyte with several parasites is common during Trophozoites (Fig. 273, 1–4) are seen in all types of erythrocytes but more frequently Erythrocytic merogony is clearly synchronized. A cycle of merogony is equal to 24 h. ium elongatum from themerogony blood of Serinus canaria: gametocytes. Erythrocytic is clearly synchronized. A cycle of merogony is equal to 24 h. H_enucleator recorded in young including erythroblasts; trophozoites are roundishdecreases as The majority of erythrocytes meronts decreases rupture in as the morning. earliest The number of gametocytes 5–13 – erythrocytic – in macrogametocytes; 18–20 – microThe majority ofmeronts; meronts14–17 rupture the morning. The number of gametocytes or oval, frequently possess ainvacuole; thepolychromatic ‘ring’ stage was seen occasionally; as thecan parasite Trophozoites (Fig. 270, 1,of2) blood are seenpassages mature and erythrocytes; the strains H_homovelans the number increases, and the laboratory finally lose the the number of blood passages increases, earliest and the laboratory strains canchronic finally lose the trophozoites areturns of variable even irregular in outline, possess negligible appear and are now develops, trophozoites take an or irregular form, ameboid outgrowths (within one to three weeks) and then into aform, stage which lasts for several produce gametocytes. cytoplasm (Fig.ability 270, 1);to clearly pronounced ameboid outgrowths are not seen; fully grown H_turtur ability to produce gametocytes. months. Erythrocytic meronts are always rare in the peripheral blood, and gametocytes Trophozoites (Fig. 273, are seentoin types of erythrocytes but more frequently trophozoites are roundish, oval, or of irregular form,1–4) usually adhere theall nuclei of infected (Fig. 273, 1–4) arestage seenmajority in all types of but more frequently e weeks)Trophozoites and then turns into a chronic which lasts forerythrocytes several predominate. The of erythrocytic meronts concentrate the haemopoietic H_minutus erythrocytes, possess one or two minute dark-brownin pigment granules (Fig.organs. 270, 2); a small recorded in young erythrocytes including erythroblasts; earliest trophozoites are roundish recorded in young erythrocytes including erythroblasts; earliest trophozoites are roundish clearly Acytoplasm, cycle of merogony is equal to stage 24 h. is not c meronts are always rare in theErythrocytic peripheral merogony blood, and gametocytes vacuole is is sometimes present in thepossess but the the typical ‘ring’ or oval,synchronized. frequently a vacuole; ‘ring’ stage was seen occasionally; as the parasite H_pallidulus characteristic; infection of the same erythrocyte with several parasites is common during or oval, frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite The majority of meronts rupture in the morning. The number of gametocytes decreases as ajority of erythrocytic meronts concentrate in the haemopoietic organs. develops, trophozoites take an irregular form, ameboid outgrowths appear and are now the number ofofblood passages increases, laboratory strains can finally lose the develops, trophozoites take an irregular form, ameboid outgrowths and are now erogony is clearly synchronized. A cycle merogony is equal to 24 and h. theappear H_concavocentralis to produce gametocytes. decreases as onts rupture in the morning. ability The number of gametocytes H_palloris Trophozoites (Fig.can 273,finally 1–4) are seenthe in all types of erythrocytes but more frequently d passages increases, and the laboratory strains lose recorded in young erythrocytes including erythroblasts; earliest trophozoites are roundish ametocytes. H_danilewskyii oval, frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite Fig. 273, 1–4) are seen in all or types of erythrocytes but more frequently H_sanguinis develops, trophozoites take an irregular form, ameboid outgrowths appear and are now rythrocytes including erythroblasts; earliest trophozoites are roundish H_homopalloris ossess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite tes take an irregular form, ameboid outgrowths appear and are now H_zosteropis Trophozoites (Fig. 270, 1, 2) are seen in mature and polychromatic erythrocytes; the earliest trophozoites are of variable form, even or irregular in outline, possess negligible H_pallidus cytoplasm (Fig. 270, 1); clearly pronounced ameboid outgrowths are not seen; fully grown trophozoites are roundish, oval, or of irregular form, usually adhere to the nuclei of infected H_vacuolatus erythrocytes, possess one or two minute dark-brown pigment granules (Fig. 270, 2); a small vacuole is sometimes present in the cytoplasm, but the typical ‘ring’ stage is not H_witti characteristic; infection of the same erythrocyte with several parasites is common during H_majoris H_nucleofascialis H_fringillae H_hirundinis H_nucleocondensus H_payevskyi H_pastoris 28 Family 2 Plasmodiidae

589-730.fm Page 639 Tuesday, September 21, 2004 10:51 AM

FAMILY PLASMODIIDAE

a lood 13 – m half the b 1– than (on from nts; 1 more mero erium upy to 24 them rocytic y occ ontain 6 all size m ca th , the m ry e rm odiu nts c of s infected lasm 4 – 10 – r in fo re mero sually e 6 P s; ig. u gula tu a l, zoite a re 22 rm th lls (F m v r irre Figu – tropho al, o rocytes; ish or o dly defo e host ce ut 7 to h, ov e o d th 1 – 3 tocytes. roun ts mark ucleate th sually ab undis d ery e ro te re c a n re n gam are u ules e infe nts a mero ven e mero ace in th ent gran a spot; nd can e µm and m sp rown ia 10 to lly g ucle smic than s; pig d in 6); fu cytopla erozoite clumpe ce their n are less e of th ge 16) m wn, and dly displa meronts e n avera m), bro ey mark lly grow µ (< 0.5 cytes, th le, the fu ro eryth ); as a ru 7 226,

mily 2 Plasmodiidae

639

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SYSTEMATIC SECTION

603 SYSTEMATIC SECTION

Figure 240 Plasmodium circumflexum from the of blood of Serinus canaria: Figure 240 Plasmodium circumflexum from the blood Serinus canaria: 1–3 – trophozoites; 4 – blood a fully of grown trophozoite (top) and an earliest binuclear erythrocytic meront Figure 240 Plasmodium circumflexum the Serinus canaria: 1–3 – trophozoites; 4 from – a fully grown trophozoite (top) and an earliest binuclear erythrocytic meront (bottom); 5–13 – erythrocytic meronts; 14–18binuclear – macrogametocytes; 19, 20 – microgametocytes. 1–3 – trophozoites; 4(bottom); – a fully grown trophozoite (top) and an earliest erythrocytic meront 5–13 –Figure erythrocytic 14–18 –circumflexum macrogametocytes; 20blood – microgametocytes. 240 meronts; Plasmodium from19, the of Serinus canaria: (bottom); 5–13 – erythrocytic meronts; 14–18 – macrogametocytes; 19, 20 – microgametocytes.

4–1 : aria cytes; 1 to s can rinu crogame of Se

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1–3 – trophozoites; 4 – a fully grown trophozoite (top) and an earliest binuclear erythrocytic meront (bottom); 5–13 – erythrocytic meronts; 14–18 – macrogametocytes; 19, 20 – microgametocytes. Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; Trophozoites (Fig. 240, 1–4) are seen in polychromatic and mature erythrocytes; earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen earliest trophozoites which are roundish or oval, sometimes of irregular shape, can be seen anywhere in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; anywhere in infected erythrocytes; the outline is even; the ‘ring’ stage is not characteristic; as the parasite develops, nucleus markedly increases in size, plentiful cytoplasm develops as the parasite markedlypigment increases in size,appear plentiful and onedevelops, or severalnucleus small roundish granules (Fig.cytoplasm 240, 2, 3);develops fully grown and onetrophozoites or several small roundish pigment appear (Fig. 240, 2, 3); fully grown but are variable in form, theygranules can be seen anywhere in infected erythrocytes trophozoites are variable in form, they can be seen anywhere in infected erythrocytes but Figure 240 Plasmodium circumflexum from the blood of Serinus canaria: 1–3 – trophozoites; 4 – a fully grown trophozoite (top) and an earliest binuclear erythrocytic meront (bottom); 5–13 – erythrocytic meronts; 14–18 – macrogametocytes; 19, 20 – microgametocytes.

m 6–

(within one to three weeks) and then turns into a chronic stage which lasts f months. Erythrocytic meronts are always rare in the peripheral blood, and ga predominate. The majority of erythrocytic meronts concentrate in the haemopoieti Erythrocytic merogony is clearly synchronized. A cycle of merogony is equ Figure 273 Plasmodium elongatum from the blood of Serinus canaria: The majority of meronts rupture in the morning. The number of gametocytes de 1– 4 – trophozoites; 5–13 – erythrocytic meronts; 14–17 – macrogametocytes; 18–20 – microthe number of blood passages increases, and the laboratory strains can finall gametocytes. ability to produce gametocytes. Trophozoites (Fig. 273, 1–4) are seen in all types of erythrocytes but more f (within one to three weeks) and then turns into a chronicrecorded stage which lasts erythrocytes for several including erythroblasts; earliest trophozoites are in young months. Erythrocytic meronts are always rare in the peripheral blood, and gametocytes or oval, frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as th predominate. The majority of erythrocytic meronts concentrate in the haemopoietic develops, trophozoitesorgans. take an irregular form, ameboid outgrowths appear and Erythrocytic merogony is clearly synchronized. A cycle of merogony is equal to 24 h. The majority of meronts rupture in the morning. The number of gametocytes decreases as the number of blood passages increases, and the laboratory strains can finally lose the ability to produce gametocytes. Trophozoites (Fig. 273, 1–4) are seen in all types of erythrocytes but more frequently recorded in young erythrocytes including erythroblasts; earliest trophozoites are roundish or oval, frequently possess a vacuole; the ‘ring’ stage was seen occasionally; as the parasite develops, trophozoites take an irregular form, ameboid outgrowths appear and are nowicro589-730.fm Page 722 Tuesday, September 21, 2004 10:51 AM 28 Family 2 Plasmodiidae

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28 Family 2 Plasmodiidae

Figure 226 Plasmodium cathemerium from the blood of Serinus canaria: Family 2 Plasmodiidae 589-730.fm Page 722 Tuesday, September 21, 2004 10:51 AM 1–3 – trophozoites; 4–10 – erythrocytic 28 meronts; 11–13 – macrogametocytes; 14–16 – microgametocytes. 722

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Figure 273 Plasmodium elongatum from the blood of Serinus canaria: 1– 4 – trophozoites; 5–13 – erythrocytic meronts; 14–17 – macrogametocytes; 18–2 gametocytes. 28 Family 2 Plasmodiidae

716

SYSTEMATIC SECTION

61 Plasmodium rouxi from the blood of722 Serinus canaria and Oriolus oriolus: phozoites; 4–14 – erythrocytic meronts; 15–19 – macrogametocytes; 20 – microgameto716

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SYSTEMATIC SECTION 28 Family 2 Plasmodiidae 589-730.fm Page 716 Tuesday, 21, 2004 10:5121, AM2004 10:51 AM 28 Family 2 Plasmodiidae 589-730.fm PageSeptember 716 Tuesday, September 28 Family 2 Plasmodiidae 589-730.fm Page 716 Tuesday, September 21, 2004 10:51 AM

Figure 240 Plasmodium circumflexum from the blood of Serinus716 canaria: 716 716 1–3 – trophozoites; 4 – a fully grown trophozoite (top) and an earliest binuclear erythrocytic meront (bottom); 5–13 – erythrocytic meronts; 14–18 – macrogametocytes; 19, 20 – microgametocytes.

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28 Family 2 Plasmodiidae

SYSTEMATIC SECTION

dae

SYSTEMATIC SECTION

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28 Family 2 Plasmodiidae

28 Family 2 Plasmodiidae

722

590

Figure 223 Main morphological peculiarities of the structure of the erythrocytic stages of malaria parasites of the subgenus Haemamoeba, which are used for identification of the species: Ne – nucleus of erythrocyte; Np – nucleus of parasite; Pg – pigment granule; Rb – residual body; Vc – vacuole. Explanations are given in the text.

1. Subgenus HAEMAMOEBA Grassi and Feletti, 1890

Haemamoeba Grassi and Feletti, 1890b: 6 (pro gen., partim).

T y p e s p e c i e s. Plasmodium relictum (Grassi and Feletti, 1891), according to subsequent designation (Corradetti et al., 1963a).

Erythrocytic meronts contain plentiful cytoplasm. The size of fully grown erythrocytic meronts exceeds that of the nuclei of infected erythrocytes. Fully grown gametocytes are roundish, oval or of irregular form, and their size markedly exceeds that of the nuclei of infected erythrocytes. Exoerythrocytic merogony takes place in cells of the reticuloendothelial system. Pedunculated oocysts are absent.

KEY TO THE SPECIES 1 (8). Pigment granules in gametocytes are roundish or oval in form (Fig. 223, 4). Rod-like pigment granules (Fig. 223, 3) are absent. Large (>1 µm in diameter) vacuoles (Fig. 223, 5) are absent in erythrocytic meronts. 2 (9).

28 Family 2 Plasmodiidae

28 Family 2 Plasmodiidae 589-730.fm Page 603 Tuesday, September 21, 2004 10:51 AM 28 Family 2 Plasmodiidae 589-730.fm Page 626 Tuesday, September 21, 2004 10:51 AM

FAMILY PLASMODIIDAE 626

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to SYSTEMATIC SECTION

SYSTEMATICSYSTEMATIC SECTION SECTION SYSTEMATIC SECTION

589-730.fm Page 716 Tuesday, September 21, 2004 10:51 AM 28 Family 2 Plasmodiidae

589-730.fm Page 716 Tuesday, September 21, 2004 10:51 AM

SYSTEMATIC SECTION

716 SYSTEMATIC SECTION Figure 236cathemerium Plasmodium tejerai fromof theSerinus blood canaria: of Meleagris gallopavo: Figure 226 Plasmodium from the blood 1–3 4–10 – trophozoites; 4–9 meronts; – erythrocytic 10–15 – macrogametocytes; 1–3 – trophozoites; – erythrocytic 11–13 meronts; – macrogametocytes; 14–16 – micro- 16–20 – microgametocytes. gametocytes.

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28 Family 2 Plasmodiidae

PL ILY FAM

i s t r i b are u t iroundish, o n. This oval, parasite been recorded Venezuela far. half 6); fully grownDmeronts or has irregular in form,only theyinoccupy moresothan T y pspace e m a in t e the r i a infected l. Hapantotypes are deposited CPGA; parahapantotype deposited in IRCAH. of the cytoplasmic erythrocytes; matureinmeronts contain 6 to 24is(on It was noted pigment in the original description that part of deposed average 16) merozoites; granules are roundish or parahapantotypes oval, usually ofwas small sizein the CPG (Wellcome Museum of Medical Sciences, London). However, the type material is absent in the CPG. (<0.5 µm), brown, and clumped into a spot; meronts markedly deform the infected E t y m o l o g y. This species is named in honour of Venezuelan parasitologist Dr. Enrique Tejera. erythrocytes, theycanaria: markedly displace their nuclei and can even enucleate the host cells (Fig. Figure 273 Plasmodium elongatum from the blood of Serinus 226, 14–17 7); as a –rule, the fully grown meronts are less than 10 µm and are usually about 7 to 1– 4 – trophozoites; 5–13 – erythrocytic meronts; macrogametocytes; M a i n d i a g n o s t i18–20 c c h a–r microa c t e r s. Fully grown trophozoites and young erythrocytic gametocytes. meronts possess a large (frequently greater than 2 µm in diameter) vacuole, and pigment

µ

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28 Family 2 Plasmodiidae

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270 Plasmodium from blood of Gallus gallus: Figure 270 Figure Plasmodium juxtanuclearejuxtanucleare from the blood of the Gallus gallus:

Figure 270 Plasmodium juxtanucleare from the blood of Gallus gallus: Figure 270 Plasmodium juxtanucleare from blood of Gallus 1, 2the – trophozoites; 3–9gallus: – erythrocytic meronts;macrogametocytes; 10–14 – macrogametocytes; 15, 16 – micro1, 2 – trophozoites; 3–9 – erythrocytic meronts; 10–14 16 – –micro1, 2 – trophozoites; 3–9 –– erythrocytic meronts;15, 10–14 macrogametocytes; 15, 16 – microgametocytes. 1, 2 – trophozoites; 3–9 – erythrocytic 10–14 – macrogametocytes; 15, 16 – microgametocytes.meronts; gametocytes. gametocytes.

SYSTEMATI

722

28 Family 2 Plasmodiidae

Figure 270 Plasmodium juxtanucleare from the blood of Gallus gallus: 1, 2 – trophozoites; 3–9 of– Gallus erythrocytic meronts; 10–14 – macrogametocytes; 15, 16 – microFigure 270 Plasmodium juxtanucleare from the blood gallus: gametocytes. 1, 2 – trophozoites; 3–9 – erythrocytic meronts; 10–14 – macrogametocytes; 15, 16 – microgametocytes.

Trophozoites (Fig. 270, 1, 2) are seen in mature and polychromatic erythrocytes; the earliest trophozoites are of variable form, even or irregular in outline, possess negligible cytoplasm (Fig. 270, 1); clearly pronounced ameboid outgrowths are not seen; fully grown trophozoites are roundish, oval, or of irregular form, usually adhere to the nuclei of infected erythrocytes, possess one or two minute dark-brown pigment granules (Fig. 270, 2); a small vacuole is sometimes present in the cytoplasm, but the typical ‘ring’ stage is not characteristic; infection of the same erythrocyte with several parasites is common during

Figure 270 Plasmodium juxtanucleare from the blood of Gallus gallus: 1, 2 – trophozoites; 3–9 – erythrocytic meronts; 10–14 – macrogametocytes; 15, 16 – microgametocytes.

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0 Plasmodium juxtanucleare from the blood of Ga phozoites; 3–9 – erythrocytic meronts; 10–14 – es.

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30 Family 4 Leucocytozoidae 737-850.fm Page 845 Tuesday, September 21, 2004 2:31 PM

FAMILY LEUCOCYTOZOIDAE

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27 Family 1 Haemoproteidae 251-473.fm Page 331 Monday, September 20, 2004 7:12 PM

Leu

FAMILY HAEMOPROTEIDAE

331

Figure 302 Gametocytes of Leucocytozoon fringillinarum from the blood of Fringilla coelebs: 1–6 – macrogametocytes; 7–9 – microgametocytes.

Aki

Parasitemia markedly increases from the 5th to 12th day after inoculation of sporozoites, and then rapidly decreases (Fig. 301). A low parasitemia was recorded in birds, which were infected once, up to a 10-month period (the period of observation). Macrogametocytes (Fig. 302, 1–6; Table 153) develop in roundish host cells; cytoplasm frequently contains small vacuoles; valutin granule are usually present; gametoFigure 86 Gametocytes of Haemocytes are roundish or of slightly oval form; the parasite nucleus is of variable form and proteus antigonis from the blood of Grus canadensis (2, 3, 6) and position; the nucleolus is prominent and well seen; the nucleus of host cell is pushed aside, Lissotis melanogaster (1, 4, 5): deformed, and lies peripherally usually as a more or less evident cap (Fig. 302, 1–5), 1, 3–5 – macrogametocytes; 2, 6 – sometimes band-like (Fig. 302, 6), it usually extends less than 1/2 of the circumference of microgametocytes (modified from gametocyte but sometimes can extend up to 1/2 of the circumference; the cytoplasm of host Bennett et al., 1975a). cells is largely replaced by gametocytes, and is sometimes even invisible (Fig. 302, 5, 6) but more frequently is present around the gametocytes as a more or less evident pale Amongand the haemoproteids of birds belonging to the Gruiformes, H. antigonis is especially simimargin of variable form (Fig. 302, 1–4). lar to H. gallinulae. It can be distinguished from the latter species on the basis of a smaller number of Microgametocytes (Fig. 302, 7–9). The general configuration andpigment other features granules inare its gametocytes. as for macrogametocytes with the usual sexual dimorphic characters. Relapses are well evident and synchronized with the breeding period of birds.

Hae

Par

Figure 339 Gametocytes of Leucocytozoon caulleryi from the blood of Gallus gallus: 1–4 – young; 5–7, 9, 10 – macrogametocytes; 8 – macro- and microgametocyte in the same host Haemoproteus D e v e l o p m e n t i n v e c t o r was investigated by Khan and Fallis23. (1970a) at a tem- (Parahaemoproteus) beckeri Roudabush and cell; 11–13 – microgametocytes. Explanations given in the peratureare of 21°C. Exflagellation wastext. observed in the midgut of simuliid flies Coatney, 2 to 5 min 1935 after

Pla

ingestion of gametocytes. Ookinetes are present in the midgut between 12 and 108 h after Haemoproteus beckeri Roudabush and Coatney 1935: 1, Fig. 1, 2.

Parasite figures modified from Valkiūnas (2005) T y p eat v e r this t e b r a t e time. h o s t. Toxostoma rufum (L.) (Passeriformes). megalomerogony of L. caulleryi. Meronts are about 4 to 11 m in diameter A d d i t i o n a l v e r t e b r a t e h o s t s. Dumetella carolinensis and Mimus polyglottos (PasseriInfected cells start to rupture, and growing megalomeronts are released fromformes). the host cells. T y p e l o c a l i t y. Peru, Nebraska, USA. D i s t r i b u t i o n. This species has been recorded only in the Nearctic so far. On the ninth day after infection, extracellular meronts are common in numerous organs and T y p e m a t e r i a l. Hapantotype (No. 45242, Toxostoma rufum, September 1934, Peru, Nebraska, USA, G.R. Coatney) is deposited in IRCAH. tissues. It is interesting to note that they were recorded not only in the visceral but is named in honour of Dr. Elery R. Becker, the friend and teacher E t y m o l organs o g y. This haemoproteid of the authors of the specific name. also in the eyes and sciatic nerves (Chew, 1968). Megalomeronts rapidly increase in size M a i n d i a g n o s t i c c h a r a c t e r s. A parasite of species of the Passeriformes whose and are enclosed by a well defined capsular-like As the megalomeront develops,caulleryi Figurethick 339 wall. Gametocytes of Leucocytozoon from they theslightly blood gametocytes grow along the nucleus of infected erythrocytes; enclose theof Gallus gallus: nucleus3, with4) theirand ends but it completely. Medium and fully grown cytomeres appear (Morii et al., 1987). Parasites are located 338, innever encircle 1–4 – young; 5–7,solely 9, 10(Fig. – macrogametocytes; andof microgametocyte in the same host gametocytes are closely appressed8to – the macronucleus and envelope erythrocytes. Dumbbell-shaped gametocytes are absent or they represent less than 10% of the total clusters containing from several to approximately 20 megalomeronts (Fig. 338, 5). Mature Fully grown cell; 11–13 – microgametocytes. Explanations given indothe text. number of growing gametocytes. are gametocytes not fill the erythrocytes up to their poles. Pigment granules are of large (1.0 to 1.5 m) and medium (0.5 to 1.0 m) megalomeronts are located extracellularly. They usually vary from 100 to 200 m in diamsize, rod-like or oval, about ten per gametocyte on average. Infected erythrocytes are hypertrophied in lengthm in comparison eter, and often reach 300 m in diameter. The largest megalomeronts can reach 500 in to uninfected ones. diameter (Omar, 1968). The size of megalomeronts depends on the peculiarities of their megalomerogony of L. caulleryi. Meronts are about 4 to 11 m in diameter at this time. location. Solely developing megalomeronts are usually larger than the parasites developing Infected cellsthat start to rupture, and growing in clusters (Kitaoka et al., 1972). It should be noted megalomeronts developing in megalomeronts are released from the host cells.

On the ninth day after infection, extracellular meronts are common in numerous organs and tissues. It is interesting to note that they were recorded not only in the visceral organs but also in the eyes and sciatic nerves (Chew, 1968). Megalomeronts rapidly increase in size and are enclosed by a well defined capsular-like thick wall. As the megalomeront develops, cytomeres appear (Morii et al., 1987). Parasites are located solely (Fig. 338, 3, 4) and in clusters containing from several to approximately 20 megalomeronts (Fig. 338, 5). Mature megalomeronts are located extracellularly. They usually vary from 100 to 200 m in diameter, and often reach 300 m in diameter. The largest megalomeronts can reach 500 m in diameter (Omar, 1968). The size of megalomeronts depends on the peculiarities of their location. Solely developing megalomeronts are usually larger than the parasites developing in clusters (Kitaoka et al., 1972). It should be noted that megalomeronts developing in

Highlights -

Over 200 morphological character states are phylogenetically informative. mtDNA and morphology combined retrieve a phylogeny similar to the one using the three Haemosporida genomes. Relationships were better resolved and supported using mtDNA and morphology combined. Subgenera Haemoproteus and Parahaemoproteus were paraphyletic. Unique combinations of character states can taxonomically determine species of all genera except Parahaemoproteus.

31