First Molecular Detection of Polychromophilus Parasites in Brazilian Bat Species.

Blood parasites of the Haemosporida order, such as the Plasmodium spp. responsible for malaria, have become the focus of many studies in evolutionary biology. However, there is a lack of molecular investigation of haemosporidian parasites of wildlife, such as the genus Polychromophilus. Species of this neglected genus exclusively have been described in bats, mainly in Europe, Asia, and Africa, but little is known about its presence and genetic diversity on the American continent. Here, we investigated 406 bats from sites inserted in remnant fragments of the Atlantic Forest and Cerrado biomes and urbanized areas from southern Brazil for the presence of Polychromophilus species by PCR of the mitochondrial cytochrome b encoding gene. A total of 1.2% of bats was positive for Polychromophilus, providing the first molecular information of these parasites in Myotis riparius and Eptesicus diminutus, common vespertilionid bats widely distributed in different Brazilian biomes, and Myotis ruber, an endangered species. A Bayesian analysis was conducted to reconstruct the phylogenetic relationships between Polychromophilus recovered from Brazilian bats and those identified elsewhere. Sequences of Brazilian Polychromophilus lineages were placed with P. murinus and in a clade distinct from P. melanipherus, mainly restricted to bats in the family Vespertilionidae. However, the sequences were split into two minor clades, according to the genus of hosts, indicating that P. murinus and a distinct species may be circulating in Brazil. Morphological observations combined with additional molecular studies are needed to conclude and describe these Polychromophilus species.

1. Introduction

The phylum Apicomplexa forms one of the most diverse groups of unicellular protists with a wide environmental distribution. They are classified as mandatory intracellular parasites and they have mobile invasive stages. They are characterized by the presence of an evolutionarily unique structure called the apical complex, used to adhere and invade host cells. Many of the species that are part of this group are considered pathogens in humans and other vertebrates. All animal species are believed to host at least one species of apicomplexan parasites [1,2,3]. Apicomplexa are divided into two orders: Eucoccidiorida (coccidian parasites) and Haemosporida (haemosporidian parasites). Haemosporida are organized into four families: Garniidae, Haemoproteidae, Leucocytozoidae, and Plasmodiidae, which include malaria parasites that infect various vertebrates and invertebrate hosts [4].

The hosts of the order Chiroptera have the greatest diversity of haemosporidian parasites among mammals, including nine genera. In addition to the well-known genera (Plasmodium and Hepatocystis), seven genera exclusively infect chiropterans: Polychromophilus, Nycteria, Bioccala, Biguetiella, Dionisia, Johnsprentia, and Sprattiella [5,6], clearly highlighting this group of mammals as a vital tool in the taxonomic, systematic, and evolutionary study of haemosporidians in mammals. Although Bioccala was elevated to a genus in 1984 [7], many studies, as well as this work, still use it as a subgenus of Polychromophilus, since its species present similar morphological characteristics and its genetics have not been studied [8].

The genus Polychromophilus has been found in insectivorous bats in tropical and temperate regions [9,10,11,12]. Only five species of Polychromophilus are known. Although they can be distinguished by slight differences in ultrastructure, they are classified mainly based on the type of host [13]. Of the five species of Polychromophilus described, Polychromophilus (Polychromophilus) melanipherus and Polychromophilus (Bioccala) murinus are mainly linked to two bat families: Miniopteridae and Vespertilionidae, respectively [14]. However, occasionally, P. melanipherus has been reported in Hipposideridae and Vespertilionidae and P. murinus in Rhinolophidae, Hipposideridae, and Miniopteridae [6]. In addition, the species P. (P.) corradetti and P. (P.) adami have been described in bats from the African region: Miniopterus inflatus in Gabon and Miniopterus minor in the Republic of Congo [13].

Recent studies have demonstrated a greater concentration of molecular studies aimed at African and European bats, e.g., [8,15,16,17]. In contrast, our knowledge about haemosporidian parasites of Brazilian bats is still restricted to morphological investigations, such as the case of Polychromophilus (Bioccala) deanei found in Myotis nigricans (Vespertilionidae). Myotis nigricans is an evening bat from Brazil, and is the first chiropteran host in which this group of parasites was found in the New World [18,19]. Nevertheless, no molecular data is available for this parasite in Brazil, and the only sequence of Polychromophilus sp. of bats from the American continent is from Myotis nigricans, from the Vespertilionidae family, found in Panama [20].

2. Materials and Methods

2.1. Sampling

Brain tissue samples of bats with no identified species (n = 406) were acquired from the Parana State Reference Laboratory (LACEN) program for monitoring rabies virus circulation. They were collected between September 2019 and August 2020 in 67 different municipalities in the State of Paraná, most of them inserted in remnant fragments of Atlantic Forest and Cerrado biomes, as well as in urbanized areas (Figure 1).

Figure 1

Location of municipalities in the State of Paraná, Brazil, where bat samples were collected.

All tissue samples and bats were collected and handled under appropriate authorizations by the Brazilian government. The project was approved by the Ethics in Use of Animals Committee, CEUA/SESA, of the Centro de Produção e Pesquisa de Imunobiológicos—CPPI/PR (approval number 01/2019 and date of approval 3 March 2020).

2.2. Polychromophilus Detection

The extraction of total nucleic acid (DNA and RNA) from collected samples was performed using the BioGene Extraction kit (K204-4, Bioclin, Belo Horizonte, MG, Brazil), following the manufacturer’s instructions.

A fragment of ~1.1 kb (approximately 92% of the gene) from the mitochondrial cytochrome b gene (cytb) was amplified using a nested polymerase chain reaction (PCR), taking standard precautions to prevent cross-contamination of samples. The PCR reactions were conducted as previously described [21] using primers DW2 and DW4 and 5 uL of genomic DNA in the first reaction, and 1 uL aliquot of this product was used as a template for a nested reaction with primers DW1 and DW6.

PCR products were sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit in ABI PRISM® 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using nested PCR primers. The cytb sequences were obtained and aligned with the sequences available at the GenBank® database.

The phylogenetic relationship among reported parasites was inferred using partial cytb gene sequences (1116 bp). GenBank® accessions of the used sequences are shown in the phylogenetic trees. The phylogenetic reconstruction was performed using the Bayesian inference method implemented in MrBayes v3.2.0 [22]. Bayesian inference was executed with two Markov Chain Monte Carlo searches of 3 million generations, with each sampling 1 of 300 trees. After a burn-in of 25%, the remaining 15,002 trees were used to calculate the 50% majority-rule consensus tree. The phylogeny was visualized using FigTree version 1.4.0 [23].

2.3. Host Species Identification

The positive samples were processed using a PCR protocol that amplifies host DNA with primers L14841 and H15149 that were designed to amplify fragments with ~390 bp of the mitochondrial cytb gene from a wide range of animals, including mammals, birds, amphibians, reptiles, and fish [24]. Amplified fragments were sequenced directly using the corresponding flanking primers. Obtained sequences were compared to other sequences deposited in the GenBank® database (www.ncbi.nlm.nih.gov/blast/Blast.cgi accessed on 19 March 2021). The best close match (BCM) algorithm was used to identify the best barcode matches of a query, and the species name of that barcode was assigned to the query if the barcode was sufficiently similar [25]. Positive identification and host species assignment were made when sequences presented a match of >97%.

Alternatively, for some specimens, a fragment with ~650 bp from the mitochondrial cytochrome c oxidase (coi) gene was amplified by two methods: (i) using the primers VF1_t1 (5′-TGT AAA ACG ACG GCC AGT TCT CAA CCA ACC ACA AAG ACA TTG G-3′) [26] and VR1_t1 (5′-AGG AAA CAG CTA TGA CTA GAC TTC TGG GTG GCC AAA GAA TCA-3′) [27] with PCR conditions and cycling from Kumar et al. [28], and (ii) using the universal primers LCO 1490 and HCO 2198 [29] and PCR protocol based on Ruiz et al. [30].

3. Results

This study detected five samples that were positive for Polychromophilus sp. (sample IDs: 116, 198, 335, 650, and 69642), confirming the presence of parasites of this genus in Brazilian bats. The percentage of positives was 1.2% (5/406) of the number of samples analyzed. Accordingly, the sequences of cytb and coi genes from the positive host samples were from Myotis ruber (116), Myotis riparius (198, 335, and 69642), and Eptesicus diminutus (650), all bats belonging to the Vespertilionidae family, collected in four municipalities in the State of Paraná (Araucaria, Cruz Machado, Curitiba, and Pato Branco) (Figure 2). The two samples obtained in Curitiba city were probably from an urban area since Curitiba is the most populous municipality of Paraná state and the eighth in the country.

Figure 2

Distribution of the positive samples of Polychromophilus sp. isolates from Paraná state, Brazil.

The nucleic acid polymorphism in mitochondrial cytb sequences (1116 bp) of Polychromophilus sp. isolates from Brazil compared to the best match sequence from GenBank® (#LN483038 of Myotis nigricans from Panama with 595 bp) is shown in Table 1. Thirteen sites were polymorphic among Brazilian sequences (Table 1). The Panamanian sequence, the only available one obtained from bats from the American continent, showed two nucleic acid substitutions found only in this isolate (gray columns) (Table 1).

Table 1

Nucleic acid polymorphism in mitochondrial cytochrome b gene (cytb) sequences of Polychromophilus sp. isolates from Brazil (116, 198, 335, 650, and 69642) and Panama (MYOPA01).

Isolate	219	247	261	273	339	405	512	789	792	810	811	853	885	945	1086
116	C	T	A	T	T	T	T	T	C	C	T	C	A	T	A
198	C	T	A	T	G	T	T	T	T	C	T	C	A	T	G
335	C	T	A	T	G	T	T	T	T	C	T	C	A	T	G
650	T	T	C	A	A	C	T	C	T	T	C	T	T	T	A
69642	C	T	A	T	T	T	T	T	C	C	T	C	A	C	A
MYOPA01	C	C	A	T	T	T	G

MYOPA01 has 595 bp and thus, there was no overlap for the nucleotides from 789–1086 with the Brazilian sequences (1116 bp). Gray columns show two nucleic acid substitutions found only in this isolate.

The sequence obtained from bat 650 was the most divergent, with 98–99% of identity with the others (with 11 or 12 nucleic acid substitutions) (Table 2). The Panamanian sequence presented two to eight nucleic acid substitutions compared to Brazilian sequences (98–99% of identity) (Table 2).

Table 2

Similarity percentage between the mitochondrial cytochrome b gene (cytb) sequences of Polychromophilus sp. found in different bats from Brazil and Panama (MYOPA01).

	Bat Species	116	198	335	650	69642	MYOPA01
116	Myotis ruber	1116	99%	99%	99%	99%	99%
198	Myotis riparius	1113	1116	100%	99%	99%	99%
335	Myotis riparius	1113	1116	1116	99%	99%	99%
650	Eptesicus diminutus	1105	1105	1105	1116	98%	98%
69642	Myotis riparius	1115	1112	1112	1104	1116	99%
MYOPA01	Myotis nigricans	592	591	591	587	592	595

The phylogenetic tree in Figure 3 was generated with reference sequences found in the Genbank® database, covering different haemosporidian genera obtained from different hosts (Table A1, Appendix A). The Polychromophilus sequences found in this study and all sequences of the genus available in the Genbank® database (Table A2, Appendix A) were included. The clade of the genus Polychromophilus is shown in evidence, and the remaining haemosporidian from other genera were collapsed.

Figure 3

Bayesian phylogeny based on the mitochondrial cytochrome b gene (cytb) from Polychromophilus spp. of the sequences identified in the present study (1116 bp) and reference sequences listed in Table A1 and Table A2 in Appendix A. * Sequence HM055583 has also been reported in P. murinus from Eptesicus serotinus, Nyctalus noctule, and Myotis myotis (Table A2, Appendix A). Eimeria spp. were used as an external group. The support values of the nodes (in percentage) indicate posterior probabilities. The red branches highlight the haemosporidian sequences found in mammals. The yellow branches highlight the haemosporidian sequences found in birds. The green branches highlight the haemosporidian sequences found in reptiles. The sequences found in the present study are highlighted in bold. The remaining reference sequences are collapsed to highlight the branch of the Polychromophilus genus.

Table A1

Mitochondrial cytochrome b gene (cytb) sequences of the parasite used as references for phylogenetic analyses and their respective accession numbers in the Genbank® database.

GenBank® Accession Number	Parasite Species	Host
HQ173882	Eimeria magna	Rabbit
HQ173892	Eimeria vejdovskyi	Rabbit
AY099045	Haemoproteus majoris	Bird
HM222472	Haemoproteus sp.	Bird
KT367832, KT367833, KT367822, KT367828	Haemosporida sp.	Antelope
KT367830, KT367819, KT367837	Haemosporida sp.	Antelope
FJ168565	Hepatocystis sp.	Bat
JQ070951, JQ070956	Hepatocystis sp.	Monkey
AY099063	Leucocytozoon dubreuli	Bird
NC_012450, FJ168563	Leucocytozoon majoris	Bird
KF159690, KF159720, MK098843-MK098847	Nycteria sp.	Bat
GQ141581, GQ141585, KT367845, KM598212	Parahaemoproteus sp.	Bird
NC_012447, FJ168561	Parahaemoproteus vireonis	Bird
HM235081	Plasmodium adleri	Gorilla
AY099054, HQ712051	Plasmodium atheruri	Rodent
AY099055	Plasmodium azurophilum	Lizard
KP875474	Plasmodium billcollinsi	Chimpanzee
HM235065	Plasmodium blacklocki	Gorilla
KF159674	Plasmodium cyclopsi	Bat
AB444126	Plasmodium cynomolgi	Monkey
FJ895307	Plasmodium gaboni	Chimpanzee
AF069612	Plasmodium gallinaceum	Bird
AY099053	Plasmodium giganteum	Lizard
JF923751	Plasmodium gonderi	Mandrill
JQ345504	Plasmodium knowlesi	Human
HM000110	Plasmodium malariae	Chimpanzee
GU723548	Plasmodium ovale	Human
JF923762	Plasmodium praefalciparum	Monkey
KP875479	Plasmodium reichenowi	Chimpanzee
AY733090	Plasmodium relictum	Bird
HM222485	Plasmodium sp.	Bird
JF923753	Plasmodium sp.	Mandrill
KJ700853, KJ700854	Plasmodium vinckei	Rodent
KF591834	Plasmodium vivax	Human
KF159671	Plasmodium voltaicum	Bat
DQ414658	Plasmodium yoelii killicki	Rodent

Table A2

Genbank® accession numbers of Polychromophilus mitochondrial cytochrome b gene (cytb) sequences used as a reference for phylogenetic analyses and sequences found in this study.

GenBank Accession Number	Parasite Species	Host	Origin
KU318045	P. melanipherus	Anopheles marshallii	Gabon
HM055583	P. murinus	Myotis daubentonii	Switzerland
HM055583	P. murinus	Eptesicus serotinus	Switzerland
HM055583	P. murinus	Nyctalus noctula	Switzerland
HM055583	P. murinus	Myotis myotis	Switzerland
HM055584–HM055589	P. murinus	Myotis daubentonii	Switzerland
MW984521	Polychromophilus sp.	Eptesicus diminutus	Brazil (this study)
KT750375	Polychromophilus sp.	Miniopterus africanus	Kenya
MH744509–MH744511, MH744518, MH744521	P. melanipherus	Miniopterus gleni	Madagascar
MH744506, MH744519	P. melanipherus	Miniopterus griffithsi	Madagascar
MH744514–MH744516	P. melanipherus	Miniopterus griveaudi	Madagascar
MH744508, MH744522–MH744525	P. melanipherus	Miniopterus griveaudi	Madagascar
JQ995284–JQ995288	Polychromophilus sp.	Miniopterus inflatus	Gabon
MH744504, MH744505	P. melanipherus	Miniopterus mahafaliensis	Madagascar
MH744512, MH744526	P. melanipherus	Miniopterus manavi	Madagascar
KT750430	Polychromophilus sp.	Miniopterus minor	Tanzania
MK098848, MK098849	Polychromophilus sp.	Miniopterus minor	Gabon
MW007677	P. melanipherus	Miniopterus natalensis	South Africa
KT750376-KT750382, KT750401, KT750402	Polychromophilus sp.	Miniopterus natalensis	Kenya
KT750406, KT750408, KT750409	Polychromophilus sp.	Miniopterus natalensis	Kenya
MK088162–MK088168	P. melanipherus	Miniopterus orianae	Australia
KT750383-KT750386, KT750415, KT750418	Polychromophilus sp.	Miniopterus rufus	Kenya
JN990708–JN990711	P. melanipherus	Miniopterus schreibersii	Switzerland
KJ131270–KJ131277	P. melanipherus	Miniopterus schreibersii	Southern and Central Europe
MW007689	P. melanipherus	Miniopterus schreibersii	Spain
KT750389	Polychromophilus sp.	Miniopterus sp.	Tanzania
KT750387	Polychromophilus sp.	Miniopterus sp.	Kenya
KF159675, KF159681, KF159699	Polychromophilus sp.	Miniopterus villiersi	Guinea
JN990712, JN990713	P. murinus	Myotis daubentonii	Switzerland
MH744532–MH744536	P. murinus	Myotis goudoti	Madagascar
LN483038	Polychromophilus sp.	Myotis nigricans	Panamá
MW984519, MW984520, MW984522	Polychromophilus sp.	Myotis riparius	Brazil (this study)
MW984518	Polychromophilus sp.	Myotis ruber	Brazil (this study)
MT136168	P. murinus	Myotis siligorensis	Thailand
KF159700	Polychromophilus sp.	Neoromicia capensis	Guinea
MW007685	P. melanipherus	Nycteribia schmidlii	Spain
MW007680, MW007681	P. melanipherus	Nycteribia schmidlii	Hungary
MW007682	P. melanipherus	Nycteribia schmidlii	Italy
MW007671–MW007674, MW007676	P. melanipherus	Nycteribia schmidlii scotti	South Africa
KU182361–KU182367	P. melanipherus	Nycteribia schmidlii scotti	Gabon
MH744527	P. melanipherus	Nycteribia stylidiopsis	Madagascar
MH744520	P. melanipherus	Paratriaenops furculus	Madagascar
KU182368	P. melanipherus	Penicillidia fulvida	Gabon
MH744528–MH744531	P. melanipherus	Penicillidia leptothrinax	Madagascar
MH744537	P. murinus	Penicillidia sp.	Madagascar
KF159714	Polychromophilus sp.	Pipistrellus aff. grandidieri	Guinea
LN483036	P. murinus	Rhinolophus sp.	Bulgaria
MT750305–MT750309	Polychromophilus sp.	Scotophilus kuhlii	Thailand
MT136167	P. melanipherus	Taphozous melanopogon	Thailand

Phylogenetic analysis based on cytb did not produce conflict in any of the main nodes. All the main genera and subgenera were recovered and represented in the phylogenetic tree by separate monophyletic clades. The results show the existence of four clades within the Haemosporida order analyzed here. Phylogeny also showed Polychromophilus as a sister clade of a group that contains Plasmodium species of ungulates, but with a distant relationship between Plasmodium and Hepatocystis from other mammals, such as primates and rodents.

All Polychromophilus sequences from bats of different parts of the world were grouped into a monophyletic clade (posterior probability of 100) composed of four subclades, with all Polychromophilus found in Brazilian bats segregated in only one of them. The first distinct subclade comprised all sequences of P. melanipherus from Miniopterus bat hosts, and the second subclade exclusively included sequences of Polychromophilus from vespertilionids (including Brazilian ones), confirming a clear separation of parasites from miniopterid and vespertilionid hosts. The other subclade that was separated contained the Polychromophilus sequences from Scotophilus kuhlii from Thailand (MT750305-MT750309). Two samples of parasites of Pipistrellus aff. grandidieri and Laephotis capensis from Guinea (KF159700 and KF159714) formed a separate group.

The subclade of Polychromophilus from vespertilionids was divided into two branches: one contained sequences of P. murinus from bats in Europe (Switzerland, Bulgaria), Madagascar, and Thailand, and a sequence of Eptesicus diminutus (650) from Brazil, and the other clade with M. nigricans from Panama and all the other Brazilian sequences isolated from the Myotis species.

4. Discussion

Based on the results presented herein, although the total number of bat families tested is unknown, Polychromophilus infection in Brazilian bats appears to be limited to just one family (Vespertilionidae). This finding is in accordance with the only previous report of Polychromophilus from Brazil, described as P. deanei, found in Myotis nigricans, also a Vespertilionidae bat [18,19].

According to one study, Paraná state has poor fauna regarding the number of bat species, with only 53 species from five families recorded [31]. The Phyllostomidae family has the highest species richness (25; 47% of the total), followed by Molossidae (13; 24%), Vespertilionidae (12; 22%), Noctilionidae (2; 4%), and Emballonuridae (1; 2.5%) [31]. Miretzki also showed the occurrence of only 55% of the species of the Atlantic Forest biome and the relative predominance of vespertilionids and molossids over phyllostomids. Herein, we analyzed samples obtained from much of the state’s area, with great sampling opportunities for other families. However, we were unable to find Polychromophilus in bat species that were not vespertilionids, suggesting that this parasite may be restricted to this group of bats in Brazil.

Regarding the frequency, we found the lowest positivity rate reported to date, although the total number of samples analyzed herein is one of the highest among published studies (Table 3). This could be related to the sample type analyzed in this study. This was the first time that Polychromophilus DNA was obtained from brain tissue, probably from parasites in the blood vessels that irrigate the organ. Thus, the direct comparison of the prevalence data with published studies that used blood samples is impaired.

Table 3

Occurrence of Polychromophilus sp. in this study and previous studies worldwide.

Country or Continent	Analyzed Samples	Positive Samples (Positivity)	Positive Host Species	Reference
Africa 1	505	56 (11%)	Miniopterus africanus, M. fraterculus, M. minor, M. natalensis, M. rufus, Myotis tricolor	[17]
Australia 2	85	47 (55%)	Miniopterus orianae	[32]
Brazil 3	406	5 (1.2%)	Eptesicus diminutus, Myotis ruber, Myotis riparius	This study
Europe 4	310	231 (74.5%)	Miniopterus schreibersii	[33]
Gabon	164	5 (3%)	Miniopterus inflatus	[34]
Gabon	92	2 (2%)	Miniopterus minor	[35]
Guinea	274	5 (2%)	Miniopterus villiersi, Neoromicia capensis, Pipistrellus aff. grandidieri	[15]
Madagascar	947	130 (13.5%)	Paratriaenops furculus, Miniopterus aelleni, M. manavi, M. gleni, M. grifthsi, M. griveaudi, M. mahafaliensis, M. majori, M. sororculus, Myotis goudoti	[36]
Madagascar	222	27 (12.2%)	Miniopterus egeri, M. griveaudi, M. ambohitrensis, M. gleni, Scotophilus robustus, Myotis goudoti	[37]
Switzerland	207	70 (34%)	Myotis daubentonii, M. myotis, Nyctalus noctula, Eptesicus serotinus	[38]
Thailand	44	5 (11%)	Scotophilus kuhlii	[39]
Thailand	271	13 (4.8%)	Myotis siligorensis, Taphozous melanopogon	[40]

1 Kenya, Malawi, Mozambique, Tanzania, and Uganda. 2 Detection of haemosporidians was performed by microscopy in all samples (274), but the molecular analysis was performed on only part of them (85 samples). 3 Detection of Polychromophilus was performed in samples of brain tissue. 4 Croatia, Portugal, Spain, Switzerland, Italy, Slovakia, and France.

Three different Brazilian bats species were found to be positive for Polychromophilus sp.: two Myotis species (M. ruber and M. riparius) and one species from the Eptesicus genus (E. diminutus). There are reports of Myotis species infections in Africa (M. tricolor in Kenya and M. goudoti in Madagascar) [17,36,37], Europe (M. daubentonii and M. myotis in Switzerland) [38], and Asia (M. siligorensis in Thailand) [40]. However, the only record of Polychromophilus infection in Eptesicus comes from Europe (E. serotinus in Switzerland) [38].

Myotis riparius is present in Honduras, Uruguay, Bolivia, Argentina, Paraguay, Trinidad, and Brazil [41], including the state of Paraná [31,42,43]. Myotis ruber is an endangered species under the category of “vulnerable” according to the Brazilian Institute of Environment and Renewable Natural Resources—IBAMA [44], and under the category of “near threatened” at a global level according to IUCN [45]. It is distributed across Argentina, Uruguay, Paraguay [40,46,47,48], and southeastern Brazil, including Paraná [49].

It is important to note that in our molecular identification of the host species using cytb and sequence comparisons, Eptesicus furinalis was the species with the best close match with the sequence obtained from bat 650. However, the percentage of identity was low (89%) compared to sequences available in the GenBank® database, making it impossible to identify the species. Thus, alternatively, we used the coi gene and the BOLD database (https://www.boldsystems.org/ (accessed on 31 March 2021), finding 98% of identity with an Eptesicus diminutus sequence, a reliable value for the species identification using the BCM method. Eptesicus diminutus has a distribution in the north and east regions of Paraná state [31]. It is from the Vespertilionidae family, and it is absent from the GenBank® database, which explains the first finding. Thus, we considered specimen 650 to be Eptesicus diminutus.

Our phylogenetic analysis showed a strongly defined clade represented by Plasmodium infecting rodents and primate hosts, which also included Hepatocystis isolated from bats. Similar data were obtained by other authors [38,50]. Haemoproteus and Leucocytozoon species were grouped separately in individual clades, as previously shown [51,52].

Regarding Polychromophilus sequences, a similar topology in the phylogenetic tree was obtained by Chumnandee et al. [39], where they grouped into a monophyletic clade with a clear separation of parasites from miniopterid and vespertilionid hosts. Four Brazilian sequences (GenBank® MW984519, MW984520, MW984522 from Polychromophilus sp. isolated of Myotis riparius, and MW984518 from Polychromophilus sp. isolated of Myotis ruber) were positioned close to the sequence of Polychromophilus sp. of bats of the species Myotis nigricans, Vespertilionidae family, from the Latin American region (Panama) (GenBank® #LN483038) [20]. One Brazilian sequence (GenBank® #MW984521, from Polychromophilus isolated from Eptesicus diminutus) was grouped with all P. murinus sequences in a sister clade. The latter, likely P. murinus, presented 1% divergence in the cytb sequence compared to the other Brazilian or Panamanian sequences, and was obtained from a different genus of bats. Thus, the possibility of most Brazilian sequences being a different Polychromophilus species must be investigated.

The present study provides the first molecular description of Polychromophilus parasites in Myotis ruber, Myotis riparius, and Eptesicus diminutus from Brazil and confirms the presence of this parasite 50 years after its first and only report in Brazilian territory. Moreover, our results suggest the occurrence of two distinct Polychromophilus species infecting two different genera of hosts, improving the current knowledge on blood parasites infecting Brazilian bats. However, it is crucial to add additional molecular markers to the phylogenetic analysis for an in-depth investigation. A three-genome phylogenetic analysis for robust haemosporidian phylogenies has been recommended [53] and must be properly included as part of a follow-up paper. Moreover, additional studies including morphological observations of these parasites combined with molecular data are needed to resolve its taxonomy. Furthermore, due to the great Brazilian extensions and the immense diversity of species and biomes, new bat populations should be investigated to provide a complete portrait of the biology of host–parasite interactions.