A new species, Dactylosoma piperis n. sp. (Apicomplexa, Dactylosomatidae), from the pepper frog Leptodactylus labyrinthicus (Anura, Leptodactylidae) from Mato Grosso State, Brazil.

The Dactylosomatidae Jakowska and Negrelli, 1955 are one of four families belonging to adeleorinid coccidia and comprise the genera Babesiosoma Jakowska and Nigrelli, 1956 and Dactylosoma Labbé, 1894. These blood protozoa occur in peripheral blood of lower vertebrates, and are commonly reported parasitising amphibians. The present study describes Dactylosoma piperis n. sp. from the pepper frog Leptodactylus labyrinthicus (Spix, 1824) (Anura: Leptodactylidae), collected in 2018 at the municipality of Araguaiana, Mato Grosso State, Brazil, based on morphology of intra-erythrocytic trophozoite, primary and secondary merogonic stages and a molecular analysis (partial 18S rDNA). Dactylosoma piperis n. sp. forms a well-supported clade with other Dactylosomatidae. This is the first molecular characterization of a species of Dactylosoma from a Brazilian anuran.

According to Barta [1,3], members of this family have undergone numerous reclassifications and systematic revisions, since the description of the first species, Dactylosoma ranarum (Kruse, 1890). Furthermore, there is a lack of information on the biology of this group of parasites, with the life cycles of only two species elucidated to date, namely Babesiosoma stableri Schmittner and McGhee, 1961 and Babesiosoma mariae (Hoare, 1930) [4,6,64]. Although leeches are considered to be the vectors of these parasites, in a recent study, possible developmental stages of Dactylosoma kermiti Netherlands, Cook and Smit, 2020 were observed in the gut and haemocoel of mosquitoes that had fed on infected hosts [64].
Species of Dactylosoma are characterised by merogonic development within the peripheral blood of their vertebrate hosts. During primary merogony, a large multinucleate meront is formed producing up to 16 merozoites. These merozoites then separate either repeating primary merogony or initiating secondary merogony. In secondary merogony, meronts produce up to eight merozoites that either repeat secondary merogony or mature into gamonts [65].
Currently there are six recognised species of Dactylosoma known globally. Two of these are described from fish hosts, and the remaining four species from anuran hosts. Namely D. ranarum described from the European frog Pelophylax kl. esculentus (Linnaeus, 1758); Dactylosoma sylvatica Fanthan, Porter and Richardson, 1942 reported in Lithobates sylvatica (LeConte, 1825) from Quebec, Canada; Dactylosoma taiwanensis Manwell, 1964 described infecting Fejervarya limnocharis Gravenhorst, 1829 collected in Taiwan; and D. kermiti described infecting the anurans, Ptychadena anchietae Bocage, 1868 and Sclerophrys gutturalis Power, 1927 from South Africa. Moreover, to date only two recognised species of Dactylosoma have been molecularly characterised, D. ranarum and D. kermiti, and one unidentified species of Dactylosoma from Belgium [65].
In Brazil, only two studies have reported on species of Dactylosoma from anuran hosts. Durham [23] briefly reported on two haemogregarine species infecting toads from Para State, the first species an unidentified haemogregarine possessing similar characteristics to Hemolivia stellata Petit, Landau, Baccam and Lainson, 1990 and the second a haemogregarine conforming morphologically to a species of Dactylosoma. The second was a study by Da Costa and Pereira [21] who screened a total of 100 frogs and toads captured and examined during 1964-1971 from Rio de Janeiro State, Brazil. Parasites from different groups were identified, including species of Hepatozoon and Dactylosoma. According to Da Costa and Pereira [21], a species of Dactylosoma and Hepatozoon leptodactyli were observed parasitising Leptodactylus latrans (Steffen, 1815) (syn. L. ocellatus). These authors suggested that although the dactylosomatid parasite observed resembles D. ranarum, more data are needed before final conclusions can be made. To date, there are no formal species descriptions of dactylosomatid species from Brazil.
Due to the limited data of anuran haemogregarine parasites from Brazil, the aim of this study was to characterise and describe a new species of Dactylosoma using morphological and molecular methods.

Ethics
All applicable international, national, and institutional guidelines for the ethical handling of animals were followed (IBAMA license 60640-1; CEUA-UNESP 1061).

Anuran collection
In August of 2018, a female adult of Leptodactylus labyrinthicus, with 105.56 mm snout-vent length and weight of 98 g, was collected at the municipality of Araguaiana, Mato Grosso State, Brazil (14°35 0 47 00 S; 51°43 0 9.59 00 W) (FAPESP 2018/09623-4; FAPESP 2018/00754-9). The animal was physically restrained and blood was collected by puncture of the cervical paravertebral sinus using sterile and disposable syringes and needles [81]. During the containment, the sex (male/female) and age of the specimen were estimated. No ectoparasites were observed on the animal.
After the blood collection, three thin blood smears were made on glass slides and the remaining blood sample was stored in EDTA tubes and frozen at À10°C for further molecular analysis.

Morphological and morphometric analysis
The blood smears were fixed with absolute methanol and stained with 10% Giemsa Methylene Blue Eosin Merck Ò diluted in distilled water (pH 7.0 for 50 min), according to Eisen and Schall [26], at the Parasitology division from UNESP, Botucatu. For morphological analysis of the intraerythrocytic parasite stages, digital images were captured and measured using a compound microscope at 1000Â magnification with the Leica software application suite LAS V3.8 (Leica Microsystems). Measurements are in micrometres (lm) comprising the parasite's length and width, with mean and standard deviation (means ± standard deviation) given. Parasitaemia was calculated per 100 erythrocytes, with~10 4 erythrocytes examined per blood smear following Cook et al. [16].

Molecular analysis
DNA was extracted from whole blood samples following the blood protocol of the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Partial 18S rRNA gene fragments (600 bp) were amplified using the primers HepF300/Hep900 [79]. PCR amplification reactions were carried out in a final volume of 25 lL, containing 1 lL each of 10 pmol primers, 12.5 lL of Master Mix MyFi TM Mix Bioline Ò and 5 lL of extracted DNA, with nuclease-free water accounting for the remaining volume; following the conditions of O'Dwyer et al. [69]. PCR amplification was performed on a Peltier 200 Thermocycler (MJ Research, Watertown, MA, USA), with initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 45 s, 50°C for 60 s and 72°C for 60 s, followed by a final extension at 72°C for 7 min.
PCR products were subjected to electrophoresis at 80 V in a 1.5% agarose gel, stained with Gel Red, and observed using ultraviolet transilluminator. The products of interest were purified by adding 2 lL of ExoSAP-IT Ò (Affymetrix, Santa Clara, CA, USA) to 5 lL of PCR product according to the manufacturer's recommendations. Amplicons were then sequenced using PCR primers on a 3500 Genetic Analyzer capillary sequencer (Applied Biosystems) and after using a BigDye Terminator Cycle Sequencing Ready Reaction Kit v.3.1 (Applied Biosystems), according to the manufacturer's recommendations.
The sequence chromatograms obtained (forward and reverse sequences) were assembled and edited using BioEdit v.7.0.9 [34] to obtain a partial 18S rDNA consensus sequence. Sequences from the haemogregarine group were aligned using For the BI analysis, the Markov Chain Monte Carlo (MCMC) algorithm was run for 1 million generations, sampling every 100 generations. The first 25% of the trees were discarded as "burn-in". The Tracer tool was used to assess convergence and the "burn-in" period [71]. For the ML analysis, nodal support was assessed using 1000 rapid bootstrap replicates [72].
Site of infection: Peripheral blood erythrocytes. Parasitaemia: 0.2%. Etymology: The host species L. labyrinthicus is commonly referred to in Brazil as the pepper frog. Therefore, the species epithet is derived from the Latin word piperis meaning pepper (noun in apposition).
Material deposited: Hapantotype, two blood smears from L. labyrinthicus deposited in the collection of the National Gene sequence: 18S rRNA gene sequence deposited in GenBank under accession number MW264134.
Note: The authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of the International Code of Zoological Nomenclature [41]. Description ( Fig. 1; Table 1): The developmental stages of the unidentified species of Dactylosoma observed were trophozoites, early stage meronts, meronts and merozoites from the primary merogony. For secondary merogony, it was possible to identify early stage meronts, meronts and merozoites. In addition, the early stage meronts and the mature meronts varied in morphology including the typical hand-like (dactylate shape), the quadrangular, the fan-like and circular shapes. Typically, primary merogony of species of Dactylosoma produces up to 16 merozoites and secondary merogony up to eight merozoites. However, in the present study, during primary merogony, meronts were observed producing up to ten chromatin divisions of the nuclei and during secondary merogony, meronts were observed producing up to eight chromatin divisions (Fig. 1).

Primary merogony
Trophozoite (Fig. 1A): Elongated, tapering towards one end and larger and rounded at opposite end, measuring 7.4 lm ± 1.3 lm long, 3.75 lm ± 1.5 lm wide, and with area of 19.31 lm 2 ± 0.4 lm 2 ; cytoplasmic vacuoles observed mainly in tapering end; nuclei placed at the rounded end, although chromatin division is not clearly defined; cytoplasm staining whitish-purple (n = 5).

Secondary merogony
Young secondary meront (Figs. 1G-1H): Elongated with one end tapered and the other rounded. Rounded end containing two to three nuclei, with dense and circular chromatin staining in deep magenta peripherally distributed, with or without cytoplasmic vacuole, 6.1 lm ± 1.2 lm length, 4.15 lm ± 0.9 lm width and 28.02 lm 2 ± 0.2 lm 2 in area (n = 3).

Differential diagnosis
Dactylosoma piperis n. sp. is characterised by its elongated and unique trophozoites, with one side rounded and the other tapered; the morphological variation of early stage meronts to mature meronts ranging between dactylate, fan-like, quadrangular and circular shapes, and the number of merozoites produced in primary merogony (up to 10) and secondary merogony (up to eight). This species can be distinguished from all currently recognised species of Dactylosoma from anuran hosts, namely D. kermiti, D. ranarum, D. sylvatica, and D. taiwanensis based on several developmental characteristics, such as the number of nuclear chromatin divisions present in primary and secondary merogony, unique trophozoite morphology and developmental stage morphometrics.
In comparison, D. piperis n. sp. differs from D. ranarum (the first described species in the Dactylosomatidae), in the number of chromatin divisions of up to six nuclei during secondary merogony and trophozoite morphology being slender and smaller with both ends rounded. Nevertheless, certain characteristics observed, such as meronts with merozoites arranged in fan-like fashion or quadrangular mass, and the two types of schizogony (primary and secondary), are typical of dactylosomatid parasites. The first type producing larger meronts with nuclei located peripherally and vacuoles present during merozoite formation, and the second type producing smaller meronts, with chromatin division of nuclei more condensed and staining dark-purple with fewer merozoites produced. For D. kermiti, primary merogony is characterised by up to 14 chromatin divisions and second merogony by up to six chromatin divisions, as compared to D. piperis n. sp., with up to 10 and up to 8 chromatin divisions observed in primary and secondary merogony, respectively. In addition, the trophozoites of D. kermiti are smaller and slender, and elongated to oval in shape with vacuoles present, measuring 6.7 lm ± 2.2 lm long and 3.5 lm ± 1.2 lm wide, which differs from the trophozoite size and shape of D. piperis n. sp. Also, the morphometric values of primary meronts and merozoites, and secondary meronts and merozoites from this study were larger as compared to D. kermiti.
In comparison, the developmental stages between D. sylvatica and D. piperis n. sp. differ in morphology and size, with trophozoites of D. sylvatica measuring lager (7.0-8.5 lm Â 6.3-7.6 lm), with an amoeboid shape, circular nuclei and alveolar cytoplasm without inclusions. Furthermore, meronts of D. sylvatica produce only up to eight merozoites, in the first and the second merogony, as compared to up to ten in the first merogony and eight in the second merogony of D. piperis n. sp.
With regard to D. taiwanensis and D. piperis n. sp., both species present similar trophozoite morphology, with trophozoites of D. taiwanensis measuring 3.9 lm wide and 7.3 lm long; distinguished morphology can be observed, with elongate or ovoid vacuolated form with equal rounded ends, compared to elongated with one end rounded and the other tapered from D. piperis n. sp. From secondary merogony, mature fan-like, quadrilateral-shape and hand-like meronts were observed with between four and eight nuclei, differing from the  The phylogenetic tree comprised sequences of adeleorinid apicomplexan parasites (Haemogregarinidae, Hepatozoidae, Karyolysidae, and Dactylosomatidae) available from GenBank ( Table 2). The BI and ML phylogenetic analysis had similar topologies, showing species of Haemogregarina forming a monophyly sister to a large clade consisting of isolates from species of Hepatozoon, Hemolivia, and Karyolysus. All species of Dactylosoma clustered together as a sister group to the Haemogregarinidae clade (Fig. 2).

Discussion
Amphibians are experiencing large-scale declines in species diversity. According to the IUCN Global Amphibian Assessment over the past decade, a third of the estimated amphibian species have declined. The major contributors to amphibian's species declines are environmental changes, fragmentation, and loss of habitat [29,30]. In addition, this group of vertebrates has a great diversity of parasites, ranging from helminths, bacteria and fungi to haemoparasites, such as trypanosomatids and haemogregarines [2,27,39,54,67,68]. Moreover, one disease has recently caught the attention of the scientific community: the amphibian chytridiomycosis panzootic is considered the most impactful example of disease spread and demonstrates its role in the decline of amphibian biodiversity worldwide [74]. However, although parasites usually have a negative connotation, they play a fundamental role in biology, ecology, evolution and population dynamics [39].
Costa and Bérnils [19] reported that Brazil has the greatest biodiversity of amphibians in the world, with more than 1,080 described species. Yet, studies on amphibian parasites from Brazil are scarce especially with regards to protozoan haemoparasites, such as the haemogregarines [21,23]. Therefore, there is a lack of data on the diversity, life cycles and possible vectors of protozoan haemoparasites of Brazilian anurans, highlighting the importance of screening these diverse hosts in Brazil [2,31].
The L. labyrinthicus was infected by a species of Dactylosoma. This anuran is widely distributed throughout South America [29,36,37]. In Brazil, L. labyrinthicus occurs mainly near wetlands and has been recorded in open habitats throughout the Cerrado, Caatinga regions, and in central Amazonia [11,38,52,57] It is a large frog from the Leptodactylus group [35] and opportunist predator feeding on invertebrate and vertebrate animals (amphibians, amphisbaenas, lizards, snakes, and small rodent species) [10,28,78,81]. In the IUCN Red list, this species is classified as LC -Least Concern [35].
In the present study from the blood smears of L. labyrinthicus, a new species of Dactylosoma, Dactylosoma piperis n. sp. is described, with parasitaemia of 0.2%. In a recent study, Netherlands et al. [65] described D. kermiti infecting anurans in South Africa, with parasitaemia varying between host species and individuals. In the host Ptychadena anchietae (Bocage, 1868), parasitaemia varied from 2% to 5.7%, and in the host Sclerophrys gutturalis (Power, 1927), parasitaemia averaged 0.2%, similar to the current study's findings.
Species of Dactylosoma have a wide distribution, infecting a variety of hosts [9,53,60]. These findings support the hypothesis of parasite distribution proposed by Metcalf [62], suggesting that parasite distribution could be explained by a Gondwana land link, so the same species could be reported in different hosts from distant geographic regions; however, according to Manwell [60], this theory was never accepted. However, the geographical locations of the six valid species do not include the regions of Central-and South America. Therefore, it is unlikely that D. piperis n. sp., is a previously described species from a different continent, with different biomes, ecosystems and also different vertebrate hosts and possible vectors. All these data support the description of D. piperis n. sp. as a new species with the aid of morphological and molecular analysis.
With regard to the molecular analysis, the phylogenetic relationships between different haemogregarines (Karyolysidae, Haemogregarinidae, Hepatozoidae, and Dactylosomatidae) and the isolate from the present study showed the forming of several well-supported clades. Species of Hepatozoon were polyphyletic, with species isolated from large mammals forming a well-supported clade sister to the Karyolysidae clade comprising species of Karyolysus, with species of Hepatozoon isolated from amphibians, reptiles and rodents forming a well-supported clade sister to the Karyolysidae clade comprising species of Hemolivia. The Haemogregarinidae clade formed a sister clade to the large monophyly comprising the Hepatozoidae and Karyolysidae clades. The Dactylosomatidae clade was found to be a well-supported monophyletic group sister to the Haemogregarinidae clade, these findings are similar to those reported by Netherlands et al. [65] In addition, the Dactylosomatidae clade formed a polytomy with Babesiosoma stableri (HQ224961); Dactylosoma sp.
Moreover, in an attempt to resolve the polyphyletic genus Hepatozoon, the genus Bartazoon Karadjian, Chavatte and Landau, 2015, was proposed to replace the Hepatozoon genus from species transmitted exclusively by haematophagous insects, with the aim of resolving the polyphyletic placement of the genus Hepatozoon [44]. However, Maia et al. [58] considered the idea premature, since some issues within Hepatozoon polyphyly still remain unsolved even with the use of the proposed genus Bartazoon.
Furthermore, Léveillé et al [55], shows the problematic designation of the genus Bartazoon based on the congenic and phylogenetic relationship of Hepatozoon griseisciuri and the type species of the genus Hepatozoon as described by Miller [63]. Léveillé et al. [55] reporting complete molecular data on nuclear 18S rDNA and the mitochondrial genome from Hepatozoon spp., showed significant pairwise differences observed between 18S rDNA and mitochondrial genome sequences; the sequences observed in their study support the idea of superiority of COI sequences on nuclear genes to describe species, and the mitochondrial genomes sequenced to date display staggering diversity. The adeleorinid coccidian will require additional sequence data from mitochondrial genomes to better understand the taxonomy and phylogenetic classifications, and the authors suggested that the genus Hepatozoon is likely distributed into multiple genera that have yet to be defined.
Notwithstanding, until the scientific community has complete knowledge about the transmission structure and life cycle of haemogregarines, as well as the real phylogenetic and genomic diversity, the addition of a new genus to the group is precipitated. Thus, the phylogenetic analysis from this study was based on Léveillé et al. [55] and Maia et al. [58], within the old classification of Hepatozoon spp., covering different groups of animals and transmission pathways, considered valid so far.
The importance of using techniques to correctly identify and describe a new species is emphasized in this manuscript. However, regarding the molecular technique, future studies should include using variable markers, such as a mitochondrial Table 3. The shaded matrix (upper) shows the percentage of similarity (%) of the nucleotide sequences and the non-shaded matrix (lower) shows the p-distance (pair-wise distance) between the Dactylosoma sequences in anurans available at GenBank (452 nt). gene, to increase the phylogenetic resolution and systematic position on dactylosomatid parasites. Furthermore, studies including a great variety of Brazilian anuran species from different localities should be done with the aim of increasing the biodiversity and prevalence knowledge of dactylosomatid species. Also, studies focusing on life-cycle experimental work, testing possible vectors in the transmission D. piperis n. sp., should be attempted to gain a better understanding of the ecology of this parasite. This study provides the first report with molecular characterisation of a species of Dactylosoma parasitising Brazilian anurans.