Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.     

Molecular evidence for host–parasite co-speciation between lizards and Schellackia parasites

Current and past parasite transmission may depend on the overlap of host distributions, potentially affecting parasite specificity and co-evolutionary processes. Nonetheless, parasite diversification may take place in sympatry when parasites are transmitted by vectors with low mobility. Here, we test the co-speciation hypothesis between lizard final hosts of the Family Lacertidae, and blood parasites of the genus Schellackia, which are potentially transmitted by haematophagous mites. The effects of current distributional overlap of host species on parasite specificity are also investigated. We sampled 27 localities on the Iberian Peninsula and three in northern Africa, and collected blood samples from 981 individual lizards of seven genera and 18 species. The overall prevalence of infection by parasites of the genus Schellackia was ～35%. We detected 16 Schellackia haplotypes of the 18S rRNA gene, revealing that the genus Schellackia is more diverse than previously thought. Phylogenetic analyses showed that Schellackia haplotypes grouped into two main monophyletic clades, the first including those detected in host species endemic to the Mediterranean region and the second those detected in host genera Acanthodactylus, Zootoca and Takydromus. All but one of the Schellackia haplotypes exhibited a high degree of host specificity at the generic level and 78.5% of them exclusively infected single host species. Some host species within the genera Podarcis (six species) and Iberolacerta (two species) were infected by three non-specific haplotypes of Schellackia, suggesting that host switching might have positively influenced past diversification of the genus. However, the results supported the idea that current host switching is rare because there existed a significant positive correlation between the number of exclusive parasite haplotypes and the number of host species with current sympatric distribution. This result, together with significant support for host–parasite molecular co-speciation, suggests that parasites of the genus Schellackia co-evolved with their lizard hosts.     

Compilation of morphological and molecular data,a necessity for taxonomy: The case of Hormogaster abbatissae sp. n. (Annelida,Clitellata, Hormogastridae)

Conflict among data sources can be frequent in evolutionary biology, especially in cases where one character set poses limitations to resolution. Earthworm taxonomy, for example, remains a challenge because of the limited number of morphological characters taxonomically valuable. An explanation to this may be morphological convergence due to adaptation to a homogeneous habitat, resulting in high degrees of homoplasy. This sometimes impedes clear morphological diagnosis of species. Combination of morphology with molecular techniques has recently aided taxonomy in many groups difficult to delimit morphologically. Here we apply an integrative approach by combining morphological and molecular data, including also some ecological features, to describe a new earthworm species in the family Hormogastridae, Hormogaster abbatissae sp. n., collected in Sant Joan de les Abadesses (Girona, Spain). Its anatomical and morphological characters are discussed in relation to the most similar Hormogastridae species, which are not the closest species in a phylogenetic analysis of molecular data. Species delimitation using the GMYC method and genetic divergences with the closest species are also considered. The information supplied by the morphological and molecular sources is contradictory, and thus we discuss issues with species delimitation in other similar situations. Decisions should be based on a profound knowledge of the morphology of the studied group but results from molecular analyses should also be considered.     

The Taxonomy and Phylogeny of Echinometra (Camarodonta: Echinometridae) from the Red Sea and Western Indian Ocean

The number of valid species in the genus Echinometra (Echinodermata, Echinoidea) and their associated identification keys have been debated in the scientific literature for more than 180 years. As the phylogeny and dispersal patterns of these species have been widely used as a prominent model for marine speciation, new insights into their taxonomy have the potential to deepen our understanding of marine speciation processes. In this study we examine Echinometra taxonomy, combining morphology and molecular tools. We present the taxonomy and phylogeny of Red Sea and Western Indian Ocean Echinometra. The currently available morphological keys were found to be limited in their ability to delineate all species within this genus. Nonetheless, morphological similarities between the Red Sea and Western Indian Ocean populations were high, and delimited them from the other species. These latter populations together formed a monophyletic clade, genetically distant from any of the other Echinometra species by more than 3%. Combining both traditional taxonomy and molecular evidence, we found that these populations were neither Echinometra mathaei nor E. oblonga, as previously considered. The morphological discrepancies of these populations, and their genetic divergence from the other Echinometra species, suggest that they should be considered as a new Echinometra species.     

Tree-based delimitation of morphologically ambiguous taxa: a study of the lizard malaria parasites on the Caribbean island of Hispaniola

Malaria parasites in the genus Plasmodium have been classified primarily on the basis of differences in morphology. These single-celled organisms often lack distinguishing morphological features, and this can encumber both species delimitation and identification. Six saurian malaria parasites have been described from the Caribbean island of Hispaniola. All six infect lizards in the genus Anolis, but only two of these parasites can be distinguished using morphology. The remaining four species overlap in morphology and geography, and cannot be consistently identified using traditional methods. We compared a morphological approach with a molecular phylogenetic approach for assessing the taxonomy of these parasites. We surveyed for blood parasites from 677 Anolis lizards, representing 26 Anolis spp. from a total of 52 sites across Hispaniola. Fifty-five of these lizards were infected with Plasmodium spp., representing several new host records, but only 24 of these infections could be matched to previously described species using traditional morphological criteria. We then estimated the phylogeny of these parasites using both mitochondrial (cytb and coxI) and nuclear (EF2) genes, and included carefully selected GenBank sequences to confirm identities for certain species. Our molecular results unambiguously corroborated our morphology-based species identifications for only the two species previously judged to be morphologically distinctive. The remaining infections fell into two well-supported and reciprocally monophyletic clades, which contained the morphological variation previously reported for all four of the morphologically ambiguous species. One of these clades was identified as Plasmodium floridense and the other as Plasmodium fairchildi hispaniolae. We elevate the latter to Plasmodium hispaniolae comb. nov. because it is polyphyletic with the mainland species Plasmodium fairchildifairchildi and we contribute additional morphological and molecular characters for future species delimitation. Our phylogenetic hypotheses indicate that two currently recognised taxa, Plasmodium minasense anolisi and Plasmodium tropiduri caribbense, are not valid on Hispaniola. These results illustrate that molecular data can improve taxonomic hypotheses in Plasmodium when reliable morphological characters are lacking.     

Molecular characterization of haemococcidia genus Schellackia (Apicomplexa) reveals the polyphyletic origin of the family Lankesterellidae

The current taxonomy on the haemococcidia establishes that the two genera of protozoan parasites that integrate the family Lankesterellidae are Lankesterella and Schellackia. However, the phylogeny of these genera, as well as the other coccidia, remains unresolved. In this sense, the use of type and described species is essential for the resolution of systematic conflicts. In this study, we molecularly characterize the type species of the genus Schellackia, that is, S. bolivari from Europe and also a described species of the same genus from Asia. At the same time, we contribute with the molecular characterization of another species of the genus Lankesterella. All this put together supports the polyphyly of the family Lankesterellidae. Therefore, we propose the resurrection of the zoological family, Schellackiidae Grassé 1953 , to include species within the genus Schellackia.     

Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens that alternate between Salicaceae and other plant hosts. Species delineation and identification are difficult within this group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is not always reliable since different Melampsora rust species can overlap on one host but specialize on a different one. To date, two different species recognition methods are used to recognize and define species within the Melampsora genus: (i) morphological species recognition, which is based solely on morphological criteria; and (ii) ecological species recognition, which combines morphological criteria with host range to recognize and define species. In order to clarify species recognition within the Melampsora genus, we applied phylogenetic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15 Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance between phylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogenetic species. All 12 lineages were concordant with host specialization, but only three belonged to strictly defined morphological species. The estimation of the species tree obtained with Bayesian concordance analysis highlighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial host plants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evolution, whereas telial host specificity seems to have evolved more recently. The morphological characters initially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary and genetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificity must be considered an important criterion for delineating and describing species within the genus Melampsora as previously suggested by ecological species recognition.     

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

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

Challenges in the delimitation of morphologically similar species: a case study of <Emphasis Type="Italic">Tuber brumale</Emphasis> agg. (Ascomycota,Pezizales)

Tuber brumale (winter truffle) is one of the most controversial true truffles, not only in regard to its ecological and economical role but also its taxonomy. Multilocus phylogenetic analyses have revealed that specimens identified earlier as T. brumale belong to two species. These species were deemed cryptic right away, because preliminary morphological measurements did not show any phenotypical differences. In this study, we measured the morphology of 119 T. brumale agg. specimens, identified by DNA-based phylogenetic tools. We found several continuous morphological characters which show strong statistical differences between the two species, albeit not without overlap. Using a combination of these characters, we show that efficient separation of the two species is possible. We describe T. cryptobrumale sp. nov. and present the environmental demands and the potential area reconstruction of both species. We argue that non-representative sampling is a major culprit in most failures to detect both the existence of morphologically similar species and their morphological differences. Our findings illustrate the benefits of integrative taxonomy: the use of a combination of molecular, morphological and ecological tools.     

Extended phylogeny of the Craspedida (Choanomonada)

Currently choanoflagellates are classified into two distinct orders: loricate Acanthoecida and non-loricate Craspedida. The morphologically based taxonomy of the order Craspedida is in need of a revision due to its controversial, paraphyletic and inconsistent systematics and nomenclature. In this study, we add molecular data (SSU and parts of the LSU rDNA) of six new Craspedida species isolated from saline, brackish and freshwater habitats to the existing knowledge. Four of these six organisms could be described as new species: Paramonosiga thecata, “Salpingoeca” euryoecia, “Salpingoeca” ventriosa, “Sphaeroeca” leprechaunica, whereas two are assigned to previous morphologically described species: “Salpingoeca” fusiformis Saville Kent, 1880 and “Salpingoeca” longipes Saville Kent, 1880. Paramonosiga is established as a new genus of the Craspedida based on its phylogenetic position. Extending the dataset by six additional sequences shows that the craspedid taxonomy is still unsolved as the type specimen Salpingoeca gracilis has not yet been sequenced and hence a clear assignment to the genus Salpingoeca is not possible. Trying to assign morphological and ecological data to phylogenetic clades is not successful. We give an improved/emended morphological diagnosis for the two redescribed species and add molecular data for all six species, shedding light on their phylogenetic position.     

New species of avian Hepatozoon (Apicomplexa: Haemogregarinidae) and a re-description of Hepatozoon neophrontis (Todd & Wohlbach, 1912) Wenyon, 1926

Seven new species of avian Hepatozoon, H. lanis, H. malacotinus, H. numidis, H. pittae, H. estrildus, H. sylvae and H. zosteropis, respectively, are described from the Laniinae, Malaconotinae, Numidinae, Pittidae, Poephilinae, Sylviinae and Zosteropidae. Hepatozoon adiei Hoare, 1924 is synonymised with Hepatozoon neophrontis (Todd &; Wolbach, 1912) Wenyon, 1926 from the Accipitridae and H. neophrontis re-described. Four species of Hepatozoon described by de Beaurepaire Aragão from Brazil are reviewed and Hepatozoon tanagrae (de Beaurepaire Aragão, 1911) Hoare, 1924 is synonymised with H. rhamphocoeli (de Beaurepaire Aragão, 1911) Hoare 1924 and H. brachyspizae (de Beaurepaire Aragão) Hoare, 1924 with H. paroariae (de Beaurepaire Aragão, 1911) Hoare, 1924. Illustrations and measurements for Hepatozoon albatrossi Peirce &; Prince, 1980, H. atticorae (de Beaurepaire Aragão, 1911) Hoare, 1924 and H. parus Bennett &; Peirce, 1989 are also presented to complete the review of the known avian species. The value of some potential morphological characteristics for distinguishing species of Hepatozoon is discussed.     

Thecamoeba cosmophorea n. sp. (Amoebozoa,Discosea, Thecamoebida) — An example of sibling species within the genus Thecamoeba

A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.     

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80–18.0%), while 38 animals (47.5%; 95% CI: 36.8–58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was ‘poor’ (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.