Memórias do Instituto Oswaldo Cruz

AnalyzingFor ambiguities Review in trypanosomatids Only taxonomy by barcoding

Journal: Memórias do Instituto Oswaldo Cruz

Manuscript ID MIOC-2019-0504.R1

Manuscript Type: Original Article

Date Submitted by the n/a Author:

Complete List of Authors: Boucinha, Carolina; Instituto Oswaldo Cruz Caetano, Amanda; Instituto Oswaldo Cruz Santos, Helena; Instituto Oswaldo Cruz Healers, Raphael; University Catholique of Louvain, de Duve Institute Vikkula, Miikka; University Catholique of Louvain, de Duve Institute Branquinha, Marta; Instituto de Microbiologia Paulo de Góes - Universidade Federal do Rio de Janeiro, Microbiologia Geral Santos, André; Universidade Federal do Rio de Janeiro, Grellier, Philippe; Muséum National d'Histoire Naturelle, RDDM Morelli, Karina; Universidade do Estado do Rio de Janeiro, Ecologia d'Avila-Levy, Claudia; IOC,

barcode, barcoding, Kinetoplastea, taxonomy, Trypanosomatida, Keyword: Trypanosomatidae

Theme: Taxonomy, Phylogeny, Parasitology

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1 2 3 4 5 6 7 8 Analyzing ambiguities in trypanosomatids taxonomy by barcoding Commented [CL1]: Running title deleted, as requested. 9 10 11 Carolina Boucinha1, Amanda C. Rodrigues1, Helena L. C. Santos1, Raphael Healers2, Miikka 12 Vikkula2, Marta Helena Branquinha3, André Luis Souza dos Santos3, Philippe Grellier4, 13 1,5 1,2* 14 Karina Alessandra Morelli , Claudia Masini d'Avila-Levy 15 16 1 Coleção de Protistas, Laboratório de Estudos Integrados em Protozoologia, Instituto 17 Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. 2 de Duve Institute, University 18 19 of Louvain, Brussels, Belgium. 3 Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de 20 Janeiro, Brazil. 4 Unité Molécules de Communication et Adaptation des Microorganisme 21 For Review Only (UMR 7245 CNRS MCAM), Muséum National d'Histoire Naturelle, Paris, France. 5 Instituto 22 23 de Biologia Roberto Alcântara Gomes, Departamento de Ecologia, UERJ, Rio de Janeiro, 24 Brazil. 25 26 27 * Corresponding author: https://orcid.org/0000-0001-8042-4695 (d'Avila-Levy CM):: 28 Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21040-360, 29 Rio de Janeiro, Brazil. Tel.: +5521 2562-1014; Fax: +5521 2590-3495; email: 30 [email protected] 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Abstract 9 10 BACKGROUND: Biodiversity screens and phylogenetic studies are dependent on 11 reliable DNA sequences in public databases. Biological collections possess vouchered 12 specimens with a traceable history. Therefore, DNA sequencing of samples available at 13 14 institutional collections can greatly contribute to taxonomy, and studies on evolution and 15 biodiversity. METHODS: We sequenced part of the glycosomal glyceraldehyde phosphate 16 dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid 17 cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE: The main 18 19 objective of this work was to use phylogenetic inferences, using the obtained DNA sequences 20 and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees 21 For Review Only that can simplify new isolates screenings. FINDINGS: A DNA sequence is provided for the 22 23 first time for several isolates, the phylogenic analysis allowed the classification or 24 reclassification of several specimens, identification of candidates for new genera and species, 25 as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS: This survey 26 27 aimed at presenting a list of validated species and their associated DNA sequences combined 28 with a short historical overview of each isolate, which can support taxonomic and biodiversity 29 research and promote culture collections. 30 31 32 Keywords: barcode, barcoding, Kinetoplastea, taxonomy, Trypanosomatida, 33 Trypanosomatidae, voucher 34 35 36 Sponsorships: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), 37 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos 38 Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) 39 40 41 42 43 44 45 46 47 Introduction 48 49 The class Kinetoplastea is a noted group of protists that has one or two flagella, 50 emerging from a flagellar pocket and that may exist in nature either as free-living species or 51 as parasites of vertebrates, invertebrates or plants. The class is defined by the presence of a 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 characteristic structure, the kinetoplast, a highly condensed DNA from a single mitochondrion 9 (1) 10 that ramifies throughout the cell body. 11 The family Trypanosomatidae encompasses members, which are the causative agents 12 of severe human diseases that are mainly transmitted by an insect vector, such as Chagas 13 14 disease (caused by Trypanosoma cruzi), sleeping sickness (caused by Trypanosoma brucei 15 sensu lato) and the various forms of cutaneous and visceral leishmaniasis (caused 16 by Leishmania spp.). (2) In addition, there are currently 18 formally described genera that are 17 generically known as insect trypanosomatids, because they are thought to have the life cycle 18 19 restricted to an insect host, namely: Angomonas, Blastocrithidia, Blechomonas, Borovskyia, 20 Crithidia, Herpetomonas, Jaenimonas, Kentomonas, Lafontella, Leptomonas, Lotmaria, 21 For Review Only Novymonas, Paratrypanomsoma, Rhynchoidomonas, Sergeia, Strigomonas, Wallacemonas 22 (3) (4-8) 23 and Zelonia. (reviewed by and new genera described in ) Finally, there is one genus, 24 Phytomonas, which alternates its life cycle between a plant and an insect. (9, 10) 25 In a historical perspective, trypanosomatids taxonomy and diversity have been 26 27 completely revolutionized by the so-called “molecular era”. For instance, from 1966 to 1990, 28 only one new genus has been described,(1, 11) after the 90’s, 14 new genera were described, 29 with a clear increase of genera description in the last years. (4-8, 12) This augment in the number 30 of described taxa is not a simple consequence of more field expeditions and sample collection, 31 32 it also reflects taxonomic revisions and proposals of new taxa to better reflect the phylogeny 33 of previously described trypanosomatid isolates, whose true diversity has been hidden by 34 traditional taxonomy approaches.(3, 8, 13) 35 36 It is clear nowadays that trypanosomatid taxonomy, previously strongly based on 37 morphotypes, host specificity or site/mode of host colonization, must also be supported by 38 phylogenetic inferences. To this end, glycosomal glyceraldehyde-3-phosphate dehydrogenase 39 40 (gGAPDH) and the V7/V8 variable region of the 18S small subunit ribosomal RNA (SSU) 41 have been widely used and have been advocated as barcodes for trypanosomatid taxonomy, 42 while the spliced leader (SL) RNA and the internal transcribed spacer (ITS) region of the 43 ribosomal RNA are suitable to analyze strain or isolate diversity.(3, 8, 14) The use of these 44 45 markers allows the identification of species through comparison with sequences available in 46 public databases. However, it is not uncommon that the public databases lack one or more of 47 the aforementioned molecular markers, or even worse, the associated data of the sequenced 48 49 organism is imprecise, incomplete, outdated or even incorrect. Therefore, culture collections 50 with vouchered species and associated information are of outmost importance to pave the road 51 for trypanosomatid taxonomy.(15) 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 The Fiocruz Protist Culture Collection (Fiocruz-COLPROT, www.colprot.fiocruz.br) 9 10 has more than 400 live specimens of protists from several families, most of them 11 representatives of the Kinetoplastea class, mainly members of the Trypanosomatidae family. 12 This collection is constantly enriched with deposits from various collaborators, as well as field 13 14 expeditions performed by its research team. To ensure reliable management of a culture 15 collection is extremely important because it greatly increases the chances of sample 16 availability and preservation for future generations.(15) 17 Here, we sequenced gGAPDH and SSU (region V7/V8) to provide an unequivocal 18 19 molecular signature to 102 trypanosomatid specimens that compose part of the COLPROT 20 catalogue. In addition, a detailed revision of the archived information regarding sample 21 For Review Only precedency, host, place and year of isolation and any relevant associated information was 22 23 detailed, revised and made public. Our data led to the reclassification of several specimens 24 based on recent taxonomic revisions and phylogenetic inferences, the identification of 25 candidates to new genus and species, as well as the confirmation of the molecular identity of 26 27 several deposits. Overall, this “molecular certification” offers to the scientific community a 28 broader view of the family Trypanosomatidae. 29 30 Materials and Methods 31 32 Chemicals 33 Media constituents, reagents used in electrophoresis, buffer components, agarose and 34 all other reagents are of analytical grade or superior. The following kits were purchased from 35 36 Promega: Wizard® Genomic DNA Purification kit and Wizard® SV Gel and PCR Clean-Up 37 System. GelRed™ fluorescent DNA stain was obtained from Biotium. The BigDye® 38 Terminator v3.1 Cycle Sequencing was obtained from Life Technologies. Fetal bovine serum 39 40 (FBS) was purchased from Cultilab and heat inactivated. 41 42 Trypanosomatid cultures 43 All organisms examined in this study are cryopreserved at Fiocruz Protist Culture 44 45 Collection (COLPROT) (http://colprot.fiocruz.br). The host, geographical origin, isolator, 46 depositor and year of isolation are summarized in Table S1. The initial taxonomic 47 identification used was the one provided by the original depositor, which are cataloged in the 48 49 COLPROT records. Ampoules with the specimens were defrosted, and all its content was 50 grown in biphasic medium NNN/LIT (Novy-MacNeal-Nicolle/Liver Infusion Tryptose) 51 supplemented with 10% FBS. When necessary, cells were subcultured in LIT to reach 52 53 logarithmic growth phase. 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 DNA extraction, PCR amplification and DNA sequence generation 11 Total DNA from at least 5 × 106 cells was extracted from cultured trypanosomatids at 12 mid-log growing phase using the Wizard® Genomic DNA Purification kit according the 13 14 manufacturer's protocol. The V7/V8 SSU rRNA (V7V8 SSU rRNA F 5'-CAC CCG CGG 15 TAA TTC CAG C-3' and V7V8 SSU rRNA R 5'-CTG AGA CTG TAA CCT CAA-3') and 16 the gGAPDH (gGAPDH F 5′- GGB CGC ATG GTS TTC CAG-3′ and gGAPDH R 5′- CCC 17 CAC TCG TTR TCR TAC C -3′) genes were PCR-amplified using previously described PCR 18 19 conditions with small modifications, when required.(12) Briefly, both genes were PCR- 20 amplified to approximately 800 bp fragments with the following program: V7V8 SSU rRNA 21 For Review Only - 94 °C, 300 s followed by 35 cycles of 30 s at 94 °C, 120 s at 52 to 60 °C, 120 s at 72 °C and 22 23 a final extension period of 72 °C for 600 s; gGAPDH - 94 °C, 180 s followed by 30 cycles of 24 60 s at 94 °C, 120 s at 52 to 60 °C, 120 s at 72 °C and a final extension period of 72 °C for 25 600 s. The PCRs were subjected to reagents and temperature adjustments to amplify the region 26 27 of each of the molecular markers used for each species analyzed. The amplified DNA 28 segments were visualized by staining with GelRed after electrophoresis separation in 1.5% 29 agarose gel in a horizontal vessel, prepared at TBE solution (1.1 M Tris, 0.9 M Borate and 25 30 mM ethylenediamine tetra-acetic acid, pH 8.3) for 50 min at 90 V. PCR reaction products 31 32 were purified by Wizard SV Gel and PCR Clean-Up System kit, and sequenced in both 33 directions using the BigDye Terminator v3.1 cycle sequencing kit and loaded in the ABI 3730 34 Sequencing Platform at Fundação Oswaldo Cruz. Sequences were analyzed and edited using 35 36 the SeqMan software (DNASTAR software package, DNASTAR Inc., Madison, WI, USA). 37 38 Molecular identification and phylogenetic analysis 39 40 The generated sequences were compared with available trypanosomatid sequences in 41 Genbank by using BLASTn search. For phylogenetic analyses using Maximum Likelihood 42 (ML) or Bayesian (BI) inferences, the DNA sequences obtained in this study (Table S1) or 43 retrieved from Genbank (Table S2) were aligned using multiple sequence alignment with high 44 45 accuracy and high throughput (MAFFT) online server.(16) The alignment was automatically 46 trimmed using METAPIGA v2.0.(17) Three alignments were created: (i) V7/V8 SSU rRNA 47 sequences, (ii) gGAPDH sequences and (iii) concatenated sequences. The final dataset, which 48 49 compiles sequences generated in this study and sequences retrieved from GenBank, contained 50 134 specimens and 410 nucleotide positions (V7/V8), 159 specimens and 720 nucleotide 51 positions (gGAPDH) and 161 taxa and 1130 nucleotides (concatenated analysis). The 52 53 evolutionary model (GTR+I+G – General Time Reversible with Invariant and Gamma 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 distribution) was selected using Akaike criterion in METAPIGA v2.0. ML phylogenetic 9 10 inference was performed in METAPIGA v2.0 with the selected model and 1,000 bootstrap 11 replicates. Other parameters were used as default states. We also inferred Bayesian analysis 12 (BI) to concatenated sequences tree (18S SSU and gGAPDH) using MrBayes v3.2.(18) For 13 14 this, we used GTR+I+G substitution model with 100.000 generations with trees sampled every 15 10.000 generations using chains, and 25% of the early sample trees were discarded. 16 17 Historical research of deposits of Protist Collection 18 19 For each COLPROT deposit used in this work, a historical survey was done through 20 revising the Collection database, research on abstracts of conferences and, whenever possible 21 For Review Only or necessary, interview with the depositors. Data from this research fueled the internal 22 23 database SICol (http://sicol.fiocruz.br/), and are available in Tables S1 and S3, the updated 24 curated species are listed in COLPROT on-line catalogue (http://colprot.fiocruz.br). 25 Results and Discussion 26 27 The phylogenetic analyses based on the concatenated gGAPDH and SSU rRNA 28 sequences or on the independent gene sequences (data not shown) using either ML or BI 29 analysis resulted in similar topologies (Fig. 1). A total of 102 trypanosomatids cultures were 30 sequenced for gGAPDH and/or for V7/V8 (Table S1). Through the combination of Blastn 31 32 searches, pair-to-pair distance and phylogenetic analyses, a reference sequence for each taxon 33 was selected (Table S2) and included in Table S1 together with the identity percentage to the 34 DNA sequences generated herein. The sequenced specimens comprehended after taxonomy 35 36 revision and update a total of ten genera (Angomonas, Crithidia, Herpetomonas, Kentomonas, 37 Lafontella, Leptomonas, Phytomonas, Strigomonas, Wallacemonas and Zelonia) distributed 38 in 38 species (Table S1). Four distinct outcomes arose from the present data: (i) isolate 39 40 certified, i.e., the DNA sequencing of the available isolate matched the sequences available in 41 Genbank and the available literature (Table S1), (ii) isolate classification, i.e., the isolate was 42 not assigned to species yet, (iii) isolate re-classification, i.e., the isolate was previously 43 assigned to a species before the “molecular era” and it was reassigned to another taxon to 44 45 better reflect the phylogeny, and (iv) new typing unit, when the DNA sequencing and the 46 phylogenetic analysis support for a new genus/species proposition (Table S1). Due to 47 extensive notes of data, we comment below only the taxa that deserves highlight or the 48 49 description of their taxonomic historical assembly. All information about the hosts of 50 COLPROT deposits can be found in Table S3, the deposits are mainly from Brazil and are 51 distributed in four insect orders, while Hemiptera and Diptera are the most abundant. 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 1. Angomonas spp. 9 (12) 10 The genus Angomonas was validated by Teixeira and colleagues to allocate 11 trypanosomatids harboring endosymbionts and presenting the choanomastigote form, but not 12 related to the genus Crithidia. 13 14 1.1. Angomonas deanei 15 The material type for this species is the TCC 036E culture deposited in the 16 Trypanosomatid Culture Collection of the University of Sao Paulo (TCC-USP), replacing the 17 culture of the American Type Culture Collection (ATCC) 30255 (unavailable)(12). TCC 036E 18 19 and COLPROT044 are duplicates of Angomonas deanei, which were described originally as 20 Crithidia deanei.(19) In 2015, Dr. E. Camargo donated TCC 036E to COLPROT (698), and 21 For Review Only DNA sequencing confirmed the expected molecular identity. The new deposit of old 22 23 preserved samples from different sources are considered a proof of good practices in Culture 24 Collections throughout the world.(15) 25 The isolate COLPROT212 was received in 1996 directly from the isolator, P.M. Faria- 26 (20) 27 e-Silva, as Herpetomonas roitmani, which was originally described as Crithidia 28 roitmani.(21) This strain was later classified as A. deanei(12) COLPROT212 and TCC 080E 29 represent duplicates of this isolate. 30 COLPROT226 was deposited in 1994 by J. E. Fiorini. The trypanosomatid was 31 32 isolated in city of Alfenas/MG/BR and the source is questionable, since it is stated that it was 33 isolated from a blackberry in the original records, but the cited reference refers to an isolate 34 from Cucurbita morchata, a pumpkin typical for the Americas.(22) Irrespective to which 35 36 information corresponds to the actual source of the isolate, both represent fruits, and as such, 37 this is the first report of isolation of Angomonas from a plant and adds more data to the 38 discussion on host specificity in trypanosomatids. However, caution must be taken from any 39 40 assumption since this represents an old specimen with doubts in its records. 41 1.2. Angomonas desouzai 42 Type material: Culture ATCC 50305, for which TCC 079E is the duplicate 43 hapantotype deposited at TCC-USP(12). COLPROT109 is a duplicate of TCC 079E. The strain 44 45 was isolated by Faria-e-Silva (1986) and later described as Crithidia desouzai.(21) This strain 46 was later moved to the Angomonas genus,(12) which contains an endosymbiont and forms a 47 strict clade with Strigomonas. An isolate of this species preserved at University of Ostrava 48 49 was deposited at COLPROT in 2015 (COLPROT708) and revealed low genetic divergence 50 for gGAPDH (Table S1). Also, during field expeditions done by Dr. Jan Votýpka (Charles 51 University, Prague), a strain was isolated from a fly (Brachycera) in 2015, in Angra dos 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Reis/RJ/Brazil (COLPROT704). These deposits had their taxonomic identification confirmed 9 10 through the use of the molecular markers in this work. 11 The deposits of the species Angomonas ambiguus (COLPROT702) presented very low 12 sequence divergence from Genbank reference strains (Table S1) and had therefore the 13 14 taxonomic status confirmed (Fig. 1). 15 16 2. Crithidia spp. 17 Classically, this genus would be easily distinguishable from another insect 18 19 trypanosomatids through morphology. The choanomastigote morphotype is advocated to be 20 easily recognizable in light microscopy, even for non-specialized observer. However, 21 For Review Only phylogenetic studies using SSU rRNA and gGAPDH clearly indicate that this genus is 22 23 polyphyletic and together with the genus Leptomonas need a revision and reclassification, as 24 clearly demonstrated in Fig. 1.(12, 14, 23-25) 25 26 27 2.1. Crithdia acantocephali and Crithidia flexonema 28 The type strain for Crithidia flexonema was deposited at ATCC (50211) by LS 29 Diamond in 1960. The trypanosomatid was isolated in Ames, IA, USA, from the host Gerris 30 remiges,(26) which was later renamed to Aquarius remigis (Table S3).(27) COLPROT016 is a 31 32 duplicate. Up to now, there was no reference sequence in Genbank for this species. 33 Another isolate without reference sequence in Genbank is Crithidia acantocephali,(28) 34 which was isolated from Acanthocephala femorata (Table S3), and deposited and preserved 35 36 in ATCC, for which COLPROT042 is a duplicate. This, together with a previously 37 unidentified deposit (COLPROT254) isolated in Brazil from Zelus sp., present high sequence 38 identity to COLPROT016 (Table S1) and formed a monophyletic group, as seen in Fig. 1. 39 40 Therefore, we suggest a synonymization to the first described species, which is C. flexonema. 41 Although the described year of isolation argues in favor of C. acantocephali, we took into 42 consideration the year of publication (1960 for C. flexonema and 1961 for C. acantocephali). 43 It is also interesting to note that the species display a high geographic dispersion and are 44 45 capable of colonizing very distinct hosts. 46 47 2.2. Crithidia bombi, Crithidia expoeki, Lotmaria passim and Crithidia mellificae 48 49 Trypanosomatids infecting honeybees have been poorly studied with molecular 50 methods up to early 2000s. After the description of Crithidia mellificae,(29) it took forty years 51 until molecular data for honeybee trypanosomatids became available. C. mellificae is a 52 53 trypanosomatid parasite of Apis mellifera that was first described in Australian bees in 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 1967.(29) Schmid-Hempel & Tognazzo(24) restarted the discussion on trypanosomatid 9 10 occurrence in honeybees and presented for the first time the molecular identity of the 11 associated trypanosomatids. 12 The species C. mellificae represents the oldest described trypanosomatid from A. 13 14 mellifera, and it has two available cultures deposited in ATCC (30254 and 30862). However, 15 the lack of associated molecular data of these taxa together with the recent molecular 16 screening of honeybee’s colonies resulted in assumptions that require considerations. In a 17 wide honeybee microbiome screen, a trypanosomatid was repeatedly identified. The parasite 18 19 was isolated, cultured and sequenced. Considering its choanomastigote shape and its host, it 20 was assumed that it was a new isolate of C. mellificae and a molecular identity was assigned. 21 For Review Only This isolate was afterwards named C. mellificae strain SF, and the whole genome was 22 (30) (31) 23 sequenced. Later, Cepero research group , while analyzing honeybee colonies in Spain, 24 compared the available gGAPDH sequences of C. mellificae isolates to the ones generated 25 under their analysis, including C. mellificae ATCC 30254. It is clear that confusion was 26 27 generated due to the deposit in Genbank of DNA sequences assumed to belong to C. mellificae 28 without a molecular signature for the reference strain. This issue was clarified by Cepero(31) 29 and Schwarz(32) research groups, who independently and almost simultaneously sequenced 30 the available presumed C. mellificae strains, as well as the old C. mellificae available ATCC 31 32 strains. Both authors identified considerable differences between the earlier described taxa 33 and the more recent description of a trypanosomatid from honeybees. Schwarz and colleagues 34 proposed to erect the genus Lotmaria(32) to accommodate the more recently described isolates 35 36 from honeybees. COLPROT035 is a duplicate of ATCC 30254, and the gGAPDH and V7/V8 37 sequences confirm its identity (Table S1). 38 Crithidia bombi was originally described morphologically(33) and was later described 39 40 based on classical molecular markers. This species has been described in bumble bees, has a 41 worldwide distribution and is a very important pathogen of this insect.(24) In fact, C. bombi, 42 C. mellificae and Crithidia expoeki are important bees pathogens with impact on the insects’ 43 fitness and survival. They represent a group of well-studied trypanosomatids due to the 44 45 negative impact in honeybee populations, and in the economy. Clearly, the population decline 46 causes serious consequences not only on honey production, but on the overall pollination 47 process, with ecological and economic impacts. The neotype of C. bombi is represented by 48 49 BJ08.085 strain, isolated in Movelier, district of Delémont, Switzerland. COLPROT683, 50 COLPROT684 and COLPROT678 represent distinct isolates of this species.(24) C. expoeki 51 was isolated in the same host set, however, it presented a clear genetic divergence from C. 52 53 bombi. The distinct clones of these three species can be differentiated based on ITS and 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 cytochrome b sequences, while the species can be differentiated based on gGAPDH and SSU 9 (24) 10 rRNA sequences. The hapantotype of C. expoeki is the isolate BJ08.175, recovered from 11 Bombus lucorum in Röschenz, Switzerland, COLPROT679 is a duplicate, and 12 COLPROT685-687 are isolates of the same species (Table S1). 13 14 15 2.3. Crithidia fasciculata 16 The first technical name given to a mosquito flagellate was Crithidia fasciculata 17 Léger.(34) Léger described the species as small flagellates, shaped like a barely corn truncate 18 19 at the anterior end, which contains a funnel-like depression.(35) Afterwards, the morphology 20 proved to produce difficulties to assign new isolates to this species, and Wallace(35) proposed 21 For Review Only the first taxonomic revision of monoxenic trypanosomatids. In this sense, the formerly 22 (36) 23 described Herpetomonas culicidrum was synonymized to C. fasciculata. This isolate is 24 deposited at ATCC (11745), and COPROT048 is a duplicate. It was isolated from Anopheles 25 quadrimaculatus (Table S3). This is the oldest preserved C. fasciculata strain with viable 26 27 available cultures. 28 However, the strain Cf:C1, which has a poor traceable history, has been considered a 29 reference sequence, and it has a publicly available genome. The strain was obtained from Dr. 30 Larry Simpson, University of California, Los Angeles, USA. The oldest reference refers to 31 32 this strain as “The cells represent a clone of a C. fasciculata culture originally obtained 33 from Dr. Stuart Krassner. This clonal cell line has been growing in our laboratory for 3 years 34 in Brain-Heart Infusion Medium”.(37) Curiously, two extensive revisions of available 35 36 trypanosomatid isolates failed to describe any C. fasciculata isolated by S. Kassner.(1, 11) 37 Therefore, although rich in genomic information and being considered in the literature as a 38 reference strain, the host, place of isolation and any other biological aspect regarding its 39 40 occurrence in nature are unknown. For barcoding/taxonomy purposes, we compared the 41 sequences generated for all C. fasciculata isolates with the available genome from Cf:C1 42 clone, as well as to the coding sequence of gGAPDH for the COLPROT048 (ATCC 11745) 43 strain.(38) COLPROT048, the oldest preserved culture, and COLPROT050, the second oldest 44 45 culture, have low sequence divergence of V7/V8 and gGAPDH markers to the Cf:C1 strain. 46 In addition, as previously published, Crithidia luciliae (COLPROT053) should be 47 synonymized to C. fasciculata (Fig. 1 and Table S1).(39) Notwithstanding, Crithidia 48 49 guilhermei (COLPROT051)(39) and Crithidia ricardoi (COLPROT184)(40) also present very 50 low sequence divergence and should all be considered isolates of a single species (Fig. 1 and 51 Table S1). Furthermore, COLPROT213, isolated from a plant, and COLPROT606, isolated 52 53 from a human patient, were also identified as isolates of C. fasciculata (Fig. 1 and Table S1). 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 The molecular revision of these isolates shows for the first time that C. fasciculata is 9 10 widespread, geographically and in host occurrence. It is reasonable to assume that further 11 screening of either old samples or new isolates will reveal further the distribution of this 12 species, particularly in the view that it can be found in plants, mammalian hosts (41) and in a 13 14 wide range of insects of the order Diptera (Table S3). 15 16 2.4. Crithidia thermophila 17 The thermal resistance of monoxenous trypanosomatids has for a long time attracted 18 19 the attention of researchers. As initially presumed, these trypanosomatids are unable to survive 20 in a mammalian host, and as such, it was somewhat surprising that they can grow well in 21 For Review Only temperatures that are typical for blood-warm hosts. In this sense, Roitman(42) described 22 23 thermo-resistant species of Crithidia, and indeed, used this criterion to describe Crithidia 24 lucilae thermophila. 25 The strain originally deposited as Crithidia hutneri was isolated and described by Isaac 26 27 Roitman (1975) from the host Cosmoclopius (Table S3) in Brazil and it was deposited as 28 COLPROT018, for which ATCC has a duplicate ATCC 30818.(42) Recently, we revised the 29 taxonomy of several Crithidia isolates and proposed to join them to Crithidia thermophila, 30 which has the type strain the COLPROT054:(43) C. hutneri (ATCC 30818, COLPROT018), 31 32 C. lucilae thermophila (ATCC 30817, COLPROT054) and Crithidia confusa (ATCC PRA- 33 346, COLPROT676).(43) In addition, as part of the curation process, we decided to acquire the 34 old preserved strains from ATCC to exclude sample mishandling, such as mixtures, 35 36 mislabelling, etc. The "freshly" acquired strains from ATCC received new COLPROT 37 numbers (C. hutneri - COLPROT688 and C. lucilae thermophila - COLPROT689). gGAPDH 38 and V7/V8 sequencing confirmed the previous finding, being possible to observe a 39 40 monophyletic clade in Figure 1 and by the high similarity in the BLASTn (Table S1). 41 Furthermore, a recently isolated strain from COLPROT field expeditions (COLPROT703) 42 was also assigned to this species, as well as an old preserved sample awaiting species 43 designation (COLPROT056),(43) which was isolated by Carvalho and Deane(19) after the 44 45 screening of 1000 Zelus leucogrammus for the presence of trypanosomatids. Several isolates 46 were obtained and subsequently characterized. COLPROT056 is one of these isolates, which 47 has been previously identified only up to the genus level.(44) 48 49 50 2.5. Crithidia abscondita,Crithidia brachylflageli, Crithidia brevicula, Crithidia insperata, 51 Crithidia otongatchiensis and Crithidia permixta 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 The deposits of the species Crithidia abscondita (COLPROT677), Crithidia 9 10 brachylflageli (COLPROT669), Crithidia brevicula (COLPROT110 and COLPROT111), 11 Crithidia insperata (COLPROT670), Crithidia otongatchiensis (COLPROT628 and 12 COLPROT673) and Crithidia permixta (COLPROT671) showed very low sequence 13 14 divergence from the Genbank reference strains using the Blastn tool and through the support 15 of the topology observed by the phylogenetic analyzes inferred by ML and BI. In this sense, 16 these deposits had the taxonomic status confirmed (Fig. 1 and Table S1). 17 18 19 3. Herpetomonas spp. 20 The genus Herpetomonas harbors monoxenic trypanosomatids that present 21 For Review Only promastigote forms and another form, unique to the group, opisthomastigotes (rarely seen in 22 23 insects and even in culture). However, promastigote forms can be observed in other genera, 24 such as Leptomonas and Phytomonas. This generated ambiguities in the taxonomic 25 classification of the genus Herpetomonas, mainly at the time when only classical taxonomy 26 (45) 27 was performed. This prompted, Borghesan and collaborators to propose a complete 28 revision of the genus Herpetomonas, since phylogenetic analyzes revealed a polyphyletic 29 group.(13, 45) 30 31 32 3.1. Herpetomonas costoris and Herpetomonas mirabilis 33 The hapantotype of H. costoris is culture ATCC 30262, of which culture TCC 019E 34 deposited at the TCC-USP(13), and COLPROT022 are duplicates. A similar situation of H. 35 36 mirabilis occurred with the species Leptomonas costoris. This species was first described as 37 belonging to the genus Leptomonas by Wallace (1965),(46) isolated from Gerris comatus 38 (Hemiptera) in the United States. Borghesan(13) reallocated the species in the genus 39 40 Herpetomonas based on sequence analysis of gGAPDH, SSU rRNA and ITS. Our results for 41 the gGAPDH gene corroborate this result and the COLPROT022 deposit was renamed as H. 42 costoris (Fig. 1 and Table S1). 43 The hapantotype for H. mirabilis is a culture deposited as ATCC 30263, of which TCC 44 45 301E deposited at the TCC-USP(13), and COLPROT024 are duplicates. This species was first 46 described by Roubaud(47), isolated from a blowfly in Congo. In 1926, Wenyon reported 47 trypanosomatids on blowflies from India, Italy and Sudan and described it as a new species 48 49 of Herpetomonas. However, Wallace and Todd(48) observed large flagellates in blowflies from 50 Guatemala, naming them as Leptomonas mirabilis. The work of Borghesan(13) has shown that 51 this flagellate is a species of Herpetomonas and prioritize the description proposed by 52 (49) 53 Wenyon , and the species was validated as H. mirabilis. Analysis of the gGAPDH and SSU 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 rRNA genes corroborate the results obtained by Borghesan and the L. mirabilis deposit 9 10 (COLPROT024) was also renamed to H. mirabilis (Fig. 1 and Table S1). 11 12 3.2. Herpetomonas elegans and Herpetomonas modestus 13 14 The type material of Herpetomonas elegans is culture TCC 1733 and of Herpetomonas 15 modestus is TCC 1444.(13) H. elegans has three isolates obtained from Ornidia obesa, a 16 syrphid. Here, we assign COLPROT211, a strain isolated from a Diptera and previously 17 identified only at the family level, to this species (Fig. 1 and Table S1). Up to the moment, all 18 19 isolates of this species are found in the family Syrphidae, which may indicate a host-specific 20 interaction. Unequivocally, the sampling must increase to warrant further conclusions. On the 21 For Review Only other hand, COLPROT707 was identified as H. modestus (Fig. 1 and Table S1) and it was 22 23 isolated from a Muscidae, while the isolates previously described were all from the family 24 Calliphoridae (Table S3). 25 26 27 3.3. Herpetomonas isaaci 28 The type material of Herpetomonas isaaci is culture TCC 266E and was described by 29 Borghesan,(13) a total of eighteen isolates were described, all of them from Diptera, mainly 30 from the family Calliphoridae. During field expeditions done by COLPROT research team, 31 32 we found two infected Chrysomya megacephala (Table S3) with this tripanosomatid (Fig. 1 33 and Table S1). The activities were part of a teaching practical course performed in FIOCRUZ, 34 Manguinhos campus (COLPROT710 and 711). In addition, we also isolated this species from 35 36 feces of pigs in a peri-urban area of Rio de Janeiro (COLPROT682). The sampling, initially 37 aimed at isolating Trichomonas and Blastocystis, revealed the presence of kinetoplastid 38 flagellates, which may be a consequence of the high Diptera abundance. However, it is worth 39 40 mentioning that kinetoplastids have been found consistently in feces from different animals, 41 even in fresh released stools.(50) 42 3.4. Herpetomonas muscarum (51) 43 The isolates COLPROT013 and COLPROT229 refer to field isolation of 44 45 trypanosomatids from tomatoes and their natural predators (Table S3). To our knowledge, this 46 is the first time that Herpetomonas muscarum (51) have been described in both the tomato fruit 47 and its associated predator. The data reinforce the questioning of trypanosomatids’ host 48 49 specificity.(52) To sum up, COLPROT185 was isolated from a small rodent in Rio de Janeiro, 50 Brazil. Although Dr R. P. Brazil and co-workers suspected from the beginning that the isolate 51 was more related to Herpetomonas than to Leishmania, skepticism prevented proper taxa 52 (53) 53 description (RP Brazil, personal communication). Finally, a previously unidentified strain 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 (COLPROT081)(54) isolated from a common weed in Brazil (previously assigned to 9 10 Phytomonas sp.), COLPROT076 (previously described as Phytomonas davidi) and 11 COLPROT033 (previously described as Herpetomonas megaseliae) presented very low 12 sequence divergence to the type strain (COLPROT020) (Table S1). Thus, our results support 13 (13) 14 the observations from Borghesan and colleagues for the synonymization of P. davidi and 15 H. megaseliae to H. muscarum. All the strains analyzed presented very close sequences and 16 are represented in the tree by COLPROT020, the type strain (Fig. 1). 17 It is worth noting that the historical description of trypanosomatids, its preservation 18 19 and taxonomic updates are full of controversies. Despite the classical and joyful disagreement 20 among research groups, there is also a lack of trustable re-traceability of strains. In this sense, 21 For Review Only Leptomonas pessoai description is full of uncertainty, the deposit COLPROT276 had few 22 (55) 23 records, but the work of Galvão that described L. pessoai was cited in the COLPROT 24 records. By this information and by the DNA sequencing analysis, this available isolate, if 25 actually related to the original description of Galvão, should be synonymized to H. muscarum, 26 (11) 27 while the taxonomic validation of L. pessoai still waits confirmation. 28 3.5. Herpetomonas samueli 29 The hapantotype culture is ATCC 30971 of which culture TCC 003E, deposited at the 30 TCC-USP, and COLPROT074 are duplicates. According solely to Leptomonas lactosovorans 31 32 Genbank sequences, this species should be considered a sister taxon of Herpetomonas 33 samueli.(13) L. lactosovorans (COLPROT023) is a duplicate of ATCC 30970, the known 34 culture of the isolate described by Manaia.(56) DNA sequencing allowed the comparison 35 36 between sequences in Genbank by using the BLASTn tool. This search returned that L. 37 lactosovorans (COLPROT023) showed hight identity with the H. samueli TCC 003E isolate 38 for the gGAPDH target (Table S1). The phylogenetic analyzes inferred by the ML and BI 39 40 methods of this marker showed very robust topology values for this monophyletic clade that 41 still includes the Leptomonas wallacei (COLPROT194) species (Fig. 1).(57) This last one, was 42 isolated from Oncopeltus fasciatus raised in an insect laboratory colony (Table S3). 43 44 45 3.6. Herpetomonas samuelpessoai (19) 46 Herpetomonas anglusteri, which is also available at ATCC (50304), was deposited at 47 Coleção de Tripanossomatídeos (CT-IOC) in 1992 directly by Maurilio Soares 48 49 (COLPROT059). It was isolated from a flesh fly of medical importance, either in forensic 50 entomology context or as a myiasis producing agent (Table S3).(58) According to the 51 divergence profile acceptable for this species paratypes,(13) we propose that this isolate 52 (19) 53 COLPROT067 should also be listed as a paratype of the Herpetomonas samuelpessoai , 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 also available at TCC (005E) and ATCC (30252). Borghesan and colleagues(13) suggested that 9 10 H. anglusteri, deposited at TCC-USP, should be closely related to C. fasciculata (according 11 to unpublished data from the research group). 12 13 14 4. Kentomonas sp. 15 4.1. Kentomonas sorsogonicus 16 This new genus was described by Votýpka and collaborators,(59) from a sample isolated 17 from Sarcophaga sp. (Diptera), collected in the province of Sorsogon (Philippines). The 18 19 flagellate was described as Kentomonas and only one species was identified: Kentomonas 20 sorsogonicus. COLPROT received a deposit of this species (COLPROT696) by Dr. 21 For Review Only Vyacheslav Yurchenko in 2015. This deposit presents a sequence identical to that deposited 22 23 in GB (KM242072) by the analysis of the gene gGAPDH gene, which allowed the vouchering 24 of the new species to the institutional collection (Fig 1 and Table S1). 25 26 27 5. Lafontella sp. 28 5.1. Lafontella mariadeanei 29 This new erected genus was proposed to accommodate the old species Herpetomonas 30 mariadeanei (TCC 004E) (5, 60). In the extensive revision of the Herpetomonas genus proposed 31 32 by Borghesan,(13) this species did not fit in the strongly supported clade composed of 33 herpetomonads. In 2016, Yurchencko and colleagues proposed the new genus Lafontella to 34 accommodate this species. Our data also support this reclassification and adds COLPROT062 35 36 as a type culture also available at COLPROT (Fig. 1 and Table S1). 37 38 6. Leptomonas sp. 39 40 The genus Leptomonas was designed based on morphological and host occurrence 41 diagnosis, as all ancient described taxa in trypanosomatids. Except for a few examples, such 42 as the phytomonads, which present a strong monophyletic clade. The analysis of single or 43 multiple genes reveals a divergence that more likely reflects the limitations and resolution- 44 45 power of the available methods for species cataloguing, than the actual phylogenetic 46 divergence that can impact on evolutionary theories. The genus Leptomonas is clearly a 47 phylogenetically artificial taxon with dubious types species, however, it is preferable to 48 49 preserve this name rather the promoting a massive renaming of the numerous species placed 50 in this genus over a long time.(61) If one analyzes strictly gGAPDH and V7/V8 sequences, there 51 are several species that overlap each other and some that better fit within the genus Crithidia, 52 53 as well as the other way around. 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 6.1. Leptomonas acus, Leptomonas spiculata and Leptomonas tarcoles 11 COLPROT672, deposited by Dr. V Y Yurchencko as an isolate of Leptomonas 12 tarcoles, shares identical gGAPDH and V7/V8 sequences to Genbank deposits of L. tarcoles 13 14 (EF546787 and EF546786, respectively), which is expected (Table S1). However, it is not 15 possible to differentiate it from L. acus and L. spiculata solely based on gGAPDH and V7/V8 16 (data not shown).(23, 61) These targets have not shown resolution enough. Although ecological 17 and biological data support differences between these isolates, there is strong phylogenetic 18 19 evidence supporting a careful revision of the species currently allocated in this genus. 20 Additional loci should be formally incorporated into the identification of these complex of 21 For Review Only cryptic species, since gGAPDH and V7/V8 genes do not have resolution enough. 22 23 24 6.2. Leptomonas jaderae 25 COLPROT674, Leptomonas jaderae (34EC), was isolated from Jadera obscura 26 27 (Rhopalidae) from Costa Rica (Table S3), deposited by V. Yurchenko at COLPROT in 2015 28 and was also deposited at the ATCC by the same research group. The sequencing of gGAPDH 29 and V7/V8 confirmed the identity of the isolate deposited by V. Yurchenko (Table S1).(62) 30 6.3. Leptomonas podlipaevi 31 32 COLPROT344 was isolated from Leptocoris triviattus by Manaia in Campinas, São 33 Paulo State, Brazil (Table S3), and directly deposited in CT-IOC in 1999, as an isolate of 34 Leptomonas sp.(63) It showed low genetic divergence when compared with Leptomonas 35 36 podlipaevi 59LI isolate for gGAPDH sequence (EU076604) and V7/V8 (EU079124) (Fig. 1 37 and Table S1).(62) Therefore, we assigned it as an isolate of L. podlipaevi. 38 39 40 6.4. Leptomonas pyrrhocoris 41 Leptomonas pyrrhocoris is a cosmopolitan species that mainly has the family 42 Pyrrhocoridae as host(64) (Table S3). COLPROT026 is a duplicate of ATCC 30974, which is 43 the oldest isolate available in culture and represents the species designation. COLPROT026, 44 45 COLPROT572 and ‘type sequences analyses’ from Genbank JN036651 (gGAPDH) and 46 JN036653 (V7/V8) for L. pyrrhocoris(64) possess no nucleotide variation. COLPROT706, 47 which has been recently isolated from Dysdercus ruficollis (Table S3), shows little or no 48 49 genetic divergence for the gGAPDH and V7/V8 markers, respectively (Table S1). 50 Collectively, this confirms the widespread distribution of this species around the world.(64) 51 52 53 6.5. Leptomonas seymouri 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Leptomonas seymouri,(65) the COLPROT277 deposit which is a duplicate of ATCC 9 10 30220, was isolated in 1959 from Dysdercus suturellus (Hemiptera: Pyrrhocoridae) in Florida, 11 USA (Table S3).(65) The molecular identity of this deposit was confirmed when compared to 12 the sequence of gGAPDH (KP717896) deposited in Genbank (Fig. 1 and Table S1). In 13 14 addition, another strain isolated from Dysdercus ruficollis, collected in Rio Claro (Brazil) 15 (Table S3) and deposited by Dr. Jan Votýpka of Charles University (Prague) (COLPROT705), 16 was also attributed to this taxon (Fig. 1 and Table S1). 17 18 19 7. Phytomonas spp. 20 The type species of the genus Phytomonas is Phytomonas françai. This genus 21 For Review Only comprises plant trypanosomatids with promastigote forms. This first isolate received the name 22 (66) 23 of P. françai, in honor of Carlos França. The name Phytomonas gained acceptance as the 24 generic name for all trypanosomatids of plants, which led to the precipitated allocation of 25 Herpetomonas and Crithidia species isolated from plants in the Phytomonas genus. Despite 26 27 this, the genus is phylogenetically defined as a monophyletic cluster according to SSU rRNA 28 and gGAPDH genes, and the isolates that are divergent accommodate through molecular 29 phylogeny in the genera Herpetomonas, Crithidia and Leptomonas.(10) 30 The trypanosomatid P. françai was isolated in 1927 by Aragão from Maniot palmata. 31 32 The currently available culture was isolated by Vainstein & Roitman(67) in Brazil (1986) from 33 Maniot esculenta, which was sent for analysis by Empresa Capixaba de Pesquisa 34 Agropecuária (EMCAPA), state of Espírito Santo State, Brazil, and is deposited as TCC064E. 35 36 The original isolate described by Aragão(66) is no longer available. COLPROT011 has the 37 records to be a duplicate of TCC064E, however, its molecular characterization did not validate 38 it as such. COLPROT011 shared identical, gGAPDH and V7/V8 sequences with 39 40 COLPROT082, isolated from Euphorbia pinea, which is a duplicate of TCC306E and 41 TCCEM1.Fr. These isolates are under Phytomonas phylogenetic lineage PhyD(10) 42 COLPROT082 was isolated from Euphorbia pinea and it was deposited as Phytomonas sp. 43 Sequencing of gGAPDH and V7/V8 reveals that it is an isolate of Phytomonas PhyD. The 44 45 culture was donated by Marta Teixeira in 1994. In the original article that describes the strain 46 isolation, Dollet and colleagues(68) described the successful cloning of three samples: TCC 47 051E, TCC 053E and TCC EM1.Fr. COLPROT082 shares identical sequences of gGAPDH 48 49 and V7/V8 with these samples, which are all isolates from Euphorbia pinea from France. In 50 addition, an isolate obtained from Euphorbia hirta from India also shares identical sequences. 51 These isolates present ITS sequence divergence.(10) COLPROT011 and COLPROT082 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 requires sequencing of additional loci in order to fully ascertain its position within 9 10 Phytomonas PhyD group. 11 12 7.1. Phytomonas serpens 13 14 The first description of isolates from tomato fruits and a phytophagic hemiptera, 15 Nezara viridula, was from Cape Peninsula, South Africa by Gibbs,(69) who designated the 16 isolate as Leptomonas serpens, which was not preserved. Therefore, considering the paradigm 17 host/morphological stages, Podlipaev renamed it to Phytomonas serpens.(70) Subsequently, all 18 19 promastigotes isolated from tomatoes were described as P. serpens. Therefore, it would be 20 expected as it occurred in several other genus or species group that a great heterogeneity 21 For Review Only should be encountered in the taxa. Interestingly, this is not the case and several isolates 22 (71, 72) 23 obtained by two distinct researchers in Brazil all fit within a closely related group. 24 Indeed, all phytomonad formed a consistent phylogenetically related group, as previously 25 described.(10) 26 27 COLPROT174, COLPROT186, COLPROT187, COLPROT188, COLPROT189 and 28 COLPROT675 compose the “T series isolates from tomatoes”.(72) The type isolate of this 29 species has not been defined, not even in the recent molecular revision of the genus.(10) To our 30 records, the work of Jankevicius and colleagues(72) is the oldest description of P. serpens 31 32 isolation that still has the preserved samples in culture. The work describes a series of isolates, 33 which were designated a number followed by the letter T. The most studied is 9T 34 (COLPROT189, of which COLPROT174 is a duplicate). As such, we suggest that this isolate 35 36 should constitute the type strain. In 2014, COLPROT received a strain, as part of a 37 collaboration work with Dr. Vyacheslav Yurchenko. This is also a duplicate from isolate 9T, 38 and the molecular identification confirmed the identity (Table S1). The 9T was isolated from 39 40 a tomato fruit in Rolandia, Paraná State, Brazil, the 10T isolate in Palmital, São Paulo State, 41 Brazil, while 15T derives from an experimental infection of the insect. Although we can find 42 citation to the 30T isolate in some old articles,(73) we could not track its origin exactly. 43 Brazil and colleagues have been reporting the isolation of trypanosomatids from 44 45 tomatoes and the feeding insect Phthia picta for many years, however without success in the 46 isolation and in long-term culture of the isolates. After they succeeded in establishing stable 47 cultures, the authors described the isolates as Trypanosomatidae sp. (COLPROT245 and 48 49 COLPROT247), due to the lack of an available culture for comparison and to the uncertainties 50 that morphological characterization plus host origin information already posed.(71) The present 51 data clearly assigns COLPROT245 and COLPROT247 as strains of P. serpens, since virtually 52 53 no sequence divergence was detected in both markers (Table S1). COLPROT080 was isolated 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 from the fruit bergamot (Citrus bergamia) by Conchon and colleagues(74) and deposited in 9 (10) 10 1992 in COLPROT by Marta Teixeira. This isolate had the ITS sequenced, which assigned 11 it to the P. serpens PhyA group. However, the authors did not generate V7/V8 or gGAPDH 12 sequences. Here, we provided these sequences, which reinforce its classification within PhyA, 13 (10) 14 and as such we propose it to be regarded as an isolate of P. serpens (Table S1). 15 COLPROT083 was deposited by Marta Teixeira in 1994. The phytomonad was 16 isolated from the latex of Jathropha macantha and was classified within PhyA group, which 17 contains the widely studied species P. serpens.(10) However, the authors claim that the strain 18 19 history is questionable; the strain is referenced to Burstein & Romero.(9, 75) Although Zanetti 20 and colleagues (10) decided not to make public a DNA sequence with a questionable history, 21 For Review Only here, we chose a different direction: make public all DNA sequences, and adding comments 22 23 to the vouchered sequence. Then, other researchers can compare with their own isolates, or 24 even the same vouchered species, which will greatly speed up taxonomic revisions. 25 The isolate COLPROT181 was deposited at COLPROT by Janckevicius in 1996. The 26 (76) 27 isolate was described as Herpetomonas macgheei. It was named in honor of Robert Barclay 28 McGhee, a major contributor to the knowledge on Phytomonas, and it represented the first 29 report of a flagellate infection on gramineous crops, since it was isolated from Leptoglossus 30 zonatus, which was feeding on Corn (Zea mays). Later, Borghesan(13) indicated that this 31 32 isolate should be reallocated to the Phytomonas genus, and recently, Zanetti and coleeagues(10) 33 grouped it in the PhyA lineage, which is represented by P. serpens. According to our data, 34 and the analysis of available sequences, we also strongly recommend it to be regarded as an 35 36 isolate of P. serpens. COLPROT161 is a duplicate of the original isolate obtained from 37 another researcher, A. Romeiro, which indicates that the available circulating cultures are 38 indeed the same isolate. COLPROT161 and COLPROT181 share 100% identity with TCC 39 40 297E. Our suggestion to include all these isolates as strains of P. serpens comes from other 41 strains grouping based in gGAPDH and V7/V8.(10, 12, 13, 43) However, it must be stressed that 42 other markers, or even whole genome sequencing, can reveal interesting strain differences and 43 intrinsic characteristics. 44 45 46 8. Strigomonas spp. 47 The genus Strigomonas, as Angomonas, was validated by Teixeira and colleagues(12) 48 49 to allocate trypanosomatids harboring endosymbionts and presenting the choanomastigote 50 form, but not related to the genus Crithidia. According to the authors, Strigomonas and 51 Angomonas can be distinguished by kinetoplast morphology, kDNA molecular characteristics 52 53 and phylogenetic analyses based on the SSU rRNA, ITS1 rDNA and gGAPDH genes. 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 8.1. Strigomonas culicis 9 10 The hapantotype is culture ATCC 30268, with duplicates at TCC 012E and 11 COLPROT041. This species was originally described as Trypanosoma (Herpetomonas) 12 culicis by Novy and colleagues(77), whom already questioned the uncertain boundaries of the 13 14 morphology-based taxonomy: “it is in order to emphasize, as Leger has done, the fragile 15 distinctions which exist between Crithidia, Herpetomonas, and Trypanosoma”.(77) The 16 cultures available nowadays refer to the isolate described by Wallace & Johnson,(78) as a new 17 genus, Blastocrithidia, isolated from Aedes vexans. It was recently reclassified as a member 18 19 of the Strigomonadinae subfamily under the genus Strigomonas.(12) The latter was raised to 20 accommodate trypanosomatids that contain an endosymbiotic bacterium, which is a trait of 21 For Review Only the group, as well as their monophyletic relatedness.(12) An aposymbiotic strain was generated 22 (79) 23 by prolonged antibiotic-treatment by Chang, and it is available at COLPROT034 and 24 ATCC . Intriguingly, Teixeira and colleagues(12) list ATCC 30257 as a duplicate of TCC012E, 25 however, according to our records and ATCC catalogue, ATCC 30257 refers to the 26 27 aposymbiotic strain (Table S1). 28 29 8.2. Strigomonas oncopelti 30 The hapantotype culture is ATCC 12982, of which culture COLPROT055 is a 31 32 duplicate, as well as TCC 043E. This specimen was originally described as Herpetomonas 33 oncopelti.(36) Later, the species was moved to the Crithidia genus,(11) subsequently assigned 34 to Strigomonas.(12) Although the host has been described as uncertain in ATCC files, the 35 36 original article unequivocally assigns the host as Oncopeltus fasciatus.(36) The nowadays 37 available cultures are originated from the ones kept by FG Wallace, which were subsequently 38 donated to KP Chang, who performed experiments to eliminate, by antibiotic treatment, the 39 (79) 40 endosymbiont (COLPROT019). 41 42 9. Wallacemonas sp. 43 The genus described as Wallaceina was created to harbor a species (Wallaceina 44 45 inconstans) that had choanomastigote and endomastigote forms and that was previously 46 described as Crithidia brevicula.(80) In 2014, Yurchenko and colleagues redefined the 47 Wallaceina genus based on the 18S, gGAPDH, and SL sequences. These markers showed that 48 49 the genus was polyphyletic: (81)four species (Wallaceina brevicula, Wallaceina inconstans, 50 Wallaceina vicina and Wallaceina podlipaevi) are indeed one single species that form a 51 distinct clade, and were then moved back to the genus Crithdia, as C. brevicula, the original 52 (25) 53 group name. In addition, the authors also proposed that the name Wallaceina should be 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 abolished and the three species that remained in the genus be relocated to a new genus named 9 (25) 10 Wallacemonas. This genus has as a type species Wallacemonas colossoma. 11 9.1. Wallacemonas collosoma 12 COLPROT073 was originally described as Leptomonas collosoma by Wallace and 13 (26) 14 colleagues, and later moved to the new erected genus Wallacemonas, which better suits the 15 molecular phylogeny.(25) COLPROT073 is a duplicate of ATCC 30261 and they represent the 16 type species. 17 10. Zelonia sp. 18 19 According to the need of taxonomic revision of the genus Leptomonas, recently, 20 Leptomonas costaricensis(82) was reassigned to a new genus, Zelonia, based on the SSU rRNA 21 For Review Only and gGAPDH barcode sequences. 22 23 24 10.1. Zelonia australiensis and Zelonia costaricensis 25 Espinosa and colleagues analyzed the genes SSU rRNA and gGAPDH and the results 26 27 showed that L. costaricensis is very distant from the L. pyrrhocoris and L. seymouri species 28 and therefore the new genus was created to accommodate this species: Zelonia 29 costaricensis.(6) The COLPROT has a deposit (COLPROT627), which was renamed to the 30 new genus. Another species was described in this genus, Zelonia australiensis.(83) This species 31 32 has been described to accommodate trypanosomatids isolated from females of Simulium 33 (Morops) dycei, (Diptera: Simuliidae) found in Australia, which form a taxon sister of the 34 relocated species Z. costaricensis.(83) 35 36 37 10.2. Zelonia dedonderi (Dedet, Geoffroy & Benichou 1986) Boucinha and d’Avila-Levy 38 n.comb. 39 (84) 40 Molecular and morphological data indicate that Herpetomonas dedonderi does not 41 belong to the genus Herpetomonas and does not fit into the genus Crithidia, although they 42 present the choanomastigote form in culture.(13, 45) The COLPROT has a deposit of H. 43 dedonderi (COLPROT060) isolated from Haemagogus janthinomys (Diptera) in 1991 in 44 45 French Guiana (Table S3). The analysis of the gGAPDH and SSUrRNA genes showed that 46 the genetic divergence between H. dedonderi species deposited in COLPROT and the recently 47 reclassified Z. costaricencis supports that H. dedonderi is more related to the genus Zelonia, 48 49 suggesting its elevation for this genus and the reclassification as Zelonia dedonderi, n. sp 50 (Fig.1 and Table S1). 51 52 53 10.3. Zelonia n. sp. 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 The COLPROT616 (CEU334) deposit was received in 2003 from Dr. Philippe Grellier 9 10 from the Nacional Museum of Natural History in Paris, France, a strain isolated from Rasahus 11 surinamensis (Reduvidae), collected in Guyana (Table S3). Analysis of the genes gGAPDH 12 and V7/V8 showed a divergence which suggests that this isolate can represent a new species 13 14 of the genus Zelonia in comparison with the old species of L. costarricensis (Fig. 1 and Table 15 S1). 16 17 11. New genera 18 19 Finally, there are three deposits, COLPROT100, COLPROT101 and COLPROT728, 20 which do not fit in any previously described clade. COLPROT100 and COLPROT101 were 21 For Review Only isolated from Psychodopygus ayrozai (Diptera), by Toby Barret in Brazil in 1993 (Table S3). 22 23 COLPROT728 from Oncopeltus fasciatus colony (Hemiptera) by Alexandre Romeiro in 24 1998. The colony was established by Dr. N. Ratcliffe, Swansea University-UK and further 25 maintained by Dr. Patricia Azambuja Penna, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. 26 27 This insect colony is still maintained, which prompted us to screen O. fasciatus insects. As 28 expected, the trypanosomatid infection was confirmed, and re-isolation of the trypanosomatid 29 revealed that the molecular identity of this new isolate (COLPROT751 and COLPROT755) 30 matched an old deposit done by Dr. A. Romeiro, which represented another isolate from the 31 32 same host (COLPROT728). Both isolates present sequence divergence to all known 33 trypanosomatids conceivable with the proposition of a new typing unit. 34 COLPROT100 and COLPROT101 showed closer identity with the species Sergeia 35 36 podlipaevi (85) for the SSU rRNA gene. For the gGAPDH gene, the species that came closest 37 was Leptomonas tenua (86) (Table S1). COLPROT728 showed similarity with the Leptomonas 38 jaculum species (87) for the SSU rRNA marker and similarity with the species Kentomonas 39 40 sorsogonicus for the gGAPDH gene (Table S1). 41 Although the species mentioned above have a closer genetic identity to the discussed 42 deposits, the percentages of divergency do not support their synonymy. Figure 1 shows the 43 well supported topology by the concatenated analysis of both genes inferred by ML and BI 44 45 and Table S1 the BLASTn values suggests that they belong to complete novel taxa that 46 deserve full description in upcoming studies, which will include morphological 47 characterization, host interaction, behavior in distinct growth conditions and whole genome 48 49 sequencing. 50 51 Conclusions 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Taxonomic changes and updates are followed by “non-taxonomists” researchers with 9 10 skepticism. There are long delays in changes of species names, reassemble, joining or 11 reclassification in publications. Here, we provided a list of validated species with traceable 12 histories and associated DNA sequences (using BLASTn tool) together the ML and BI 13 14 analyses. We thereby contribute to the overall taxonomy and biodiversity knowledge of 15 trypanosomatids and provide support for non-taxonomist researchers to clearly depict the 16 taxonomic and phylogenetic relatedness of each sample described. This allows to correlate 17 new isolates to the previously described ones easily and quickly. In addition, we carefully 18 19 update the taxonomic information in each Genbank submission, in accordance to what was 20 described in the present report. It is well known by the scientific community that old or 21 For Review Only inaccurate taxonomic description of DNA sequences in Genbank reduce the pace of 22 23 phylogenetic and taxonomic studies. 24 Taxonomic summary 25 Class Kinetoplastea Honigberg, 1963 26 27 Subclass Metakinetoplastina Vickerman, 2004 28 Order Trypanosomatida Kent, 1880 29 Family Trypanosomatidae Doflein, 1901 30 Subfamily Leishmaniinae Maslov et Lukeš in Jirků et al., 2012 31 32 Genus Zelonia Shaw, Camargo and Teixeira 2016 33 Zelonia dedonderi (Dedet, Geoffroy & Benichou 1986) Boucinha and d’Avila-Levy n.comb. 34 Synonyms: Herpetomonas dedonderi Dedet, Geoffroy & Benichou 1986 35 36 Type material: the culture IHAUGF/8 IICAYI13 deposited in the collection of the 37 Department of Medical Protozoology of the London School of Hygiene and Tropical 38 Medicine is the holotype, which COLPROT 060 is a duplicate. 39 40 Type host: Haemagogus janthinomys Dyar, 1921 (Diptera: Culicidae) 41 Type location: rain forest, area of FRG (52”35‘W/4”55“) near Montsinery, French Guiana 42 Diagnosis: DNA sequences from gGAPDH (DQ383650) and SSU rRNA (V7/V8) 43 (DQ383648) 44 45 Remarks: This isolate was originally described as Herpetomonas dedonderi by Dedet, 46 Geoffroy & Benichou 1986, and a duplicate of the type culture was deposited in COLPROT 47 by Erney Camargo in 1991. Teixeira(45) and Borghesan(13) suggested that this species does not 48 49 fit in the genus Herpetomonas. Phylogenetic analyses conducted in the present work indicated 50 the closer relationship with the genus Zelonia. 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Acknowledgments 9 10 We would like to thank all the members of our laboratories for helpful and stimulating 11 discussions. We acknowledge the use of research infrastructure of de Duve Institute that 12 received Dr. d’Avila-Levy for a sabbatical year, particularly Dr. Miikka Vikkula for 13 14 stimulating discussion. We are thankful to all researchers that contributed to enrich 15 COLPROT catalogue. We thank the Brazilian grant agencies Coordenação de 16 Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de 17 Desenvolvimento Científico e Tecnológico (MCT/CNPq), Fundação Carlos Chagas Filho de 18 19 Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for funding, as well as Fundação 20 Oswaldo Cruz (FIOCRUZ) for research infrastrucutre. 21 For Review Only 22 23 Authors’ contribution 24 Conceived and designed the experiments – CMdL and KAM; performed the 25 26 experiments – CB, ACR, KAM; analyzed the data – CB, HLCS, RH, MV, MHB, ALS, PG, 27 KAM, CMdL; writing (original draft preparation) – CB, KAM, CMdL; writing (review and 28 editing) – CB, HLCS, RH, MV, MHB, ALS, PG, KAM, CMdL; funding aquisition – MV, 29 30 MHB, ALS, PG, CMdL. All authors read and approved the final manuscript. 31 32 References 33 34 35 1. Wallace FG. The trypanosomatid parasites of insects and arachnids. Exp Parasitol. 1966; 18: 124-93. 36 2. Vickerman K. The evolutionary expansion of the trypanosomatid flagellates. Int J Parasitol. 37 1994; 24: 1317-31. 38 3. d'Avila-Levy CM, Boucinha C, Kostygov A, Santos HL, Morelli KA, Grybchuk-Ieremenko 39 A et al. Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era. Mem Inst Oswaldo Cruz. 2015; 110: 956-65. 40 4. Hamilton PT, Votypka J, Dostalova A, Yurchenko V, Bird NH, Lukes J et al. Infection 41 Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association. 42 MBio. 2015; 6: e01356-15. 43 5. Yurchenko V, Kostygov A, Havlova J, Grybchuk-Ieremenko A, Sevcikova T, Lukes J et al. Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n. 44 J Eukaryot Microbiol. 2016; 63: 198-209. 45 6. Espinosa OA, Serrano MG, Camargo EP, Teixeira MMG, Shaw JJ. An appraisal of the 46 taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and 47 Endotrypanum. Parasitology. 2018; 145: 430-42. 7. Kostygov AY, Dobakova E, Grybchuk-Ieremenko A, Vahala D, Maslov DA, Votypka J et 48 al. Novel Trypanosomatid-Bacterium Association: Evolution of Endosymbiosis in Action. MBio. 49 2016; 7: e01985. 50 8. Kostygov AY, Yurchenko V. Revised classification of the subfamily Leishmaniinae 51 (Trypanosomatidae). Folia Parasitologica. 2017; 64. 9. Camargo EP. Phytomonas and other trypanosomatid parasites of plants and fruit. Advances 52 in Parasitology: Elsevier, 1999; 29-112. 53 54 55 56 57 58 59 60

https://mc04.manuscriptcentral.com/mioc-scielo Memórias do Instituto Oswaldo Cruz Page 26 of 41

1 2 3 4 5 6 7 8 10. Zanetti A, Ferreira RC, Serrano MG, Takata CS, Campaner M, Attias M et al. Phytomonas 9 (Euglenozoa: Trypanosomatidae): Phylogenetic analyses support infrageneric lineages and a new 10 species transmitted to Solanaceae fruits by a pentatomid hemipteran. Eur J Protistol. 2016; 56: 232- 11 49. 11. Podlipaev S. Catalogue of world fauna of Trypanosomatidae (Protozoa). . Proc Zool Inst 12 Leningrad. 1990; 144: 1-178. 13 12. Teixeira MM, Borghesan TC, Ferreira RC, Santos MA, Takata CS, Campaner M et al. 14 Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring 15 bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts. Protist. 2011; 162: 503-24. 16 13. Borghesan TC, Ferreira RC, Takata CS, Campaner M, Borda CC, Paiva F et al. Molecular 17 phylogenetic redefinition of Herpetomonas (Kinetoplastea, Trypanosomatidae), a genus of insect 18 parasites associated with flies. Protist. 2013; 164: 129-52. 19 14. Votypka J, d'Avila-Levy CM, Grellier P, Maslov DA, Lukes J, Yurchenko V. New Approaches to Systematics of Trypanosomatidae: Criteria for Taxonomic (Re)description. Trends 20 Parasitol. 2015; 31: 460-9. 21 15. d'Avila-Levy CM, Yurchenko V,For Votypka J,Review Grellier P. Protist Collections: Only Essential for 22 Future Research. Trends Parasitol. 2016; 32: 840-2. 23 16. Katoh K, Kuma K, Toh H, Miyata T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005; 33: 511-8. 24 17. Helaers R, Milinkovitch MC. MetaPIGA v2.0: maximum likelihood large phylogeny 25 estimation using the metapopulation genetic algorithm and other stochastic heuristics. BMC 26 Bioinformatics. 2010; 11: 379. 27 18. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 28 2012; 61: 539-42. 29 19. Carvalho AL, Deane MP. Trypanosomatidae isolated from Zelus leucogrammus (Perty, 30 1834)(Hemiptera, Reduviidae), with a discussion on flagellates of insectivorous bugs. J Protozool. 31 1974; 21: 5-8. 20. Faria e Silva PM, Sole-Cava AM, Soares MJ, Motta MC, Fiorini JE, de Souza W. 32 Herpetomonas roitmani (Fiorini et al., 1989) n. comb.: a trypanosomatid with a bacterium-like 33 endosymbiont in the cytoplasm. J Protozool. 1991; 38: 489-94. 34 21. Fiorini JE, Silva PMF, Soares MJ, Brazil RP. Três novas especies de tripanosomatideos 35 isolados em Alfenas, Minas Gerais, Brasil. Mem Inst Oswaldo Cruz 1989; 84: 69-74. 22. Fiorini JE, Cucolichio G, Rezende D, Nascimento LC, Faria-e-Silva PM. Isolation and 36 cloning of a Trypanosomatid with choanomastigote form from Mulberry. Mem Inst Oswaldo Cruz. 37 1995; 90, suppl I: 247. 38 23. Yurchenko VY, Lukes J, Tesarova M, Jirku M, Maslov DA. Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus Crithidia. Protist. 2008; 39 159: 99-114. 40 24. Schmid-Hempel R, Tognazzo M. Molecular divergence defines two distinct lineages of 41 Crithidia bombi (Trypanosomatidae), parasites of bumblebees. J Eukaryot Microbiol. 2010; 57: 337- 42 45. 25. Kostygov AY, Grybchuk-Ieremenko A, Malysheva MN, Frolov AO, Yurchenko V. 43 Molecular revision of the genus Wallaceina. Protist. 2014; 165: 594-604. 44 26. Wallace FG, Clark TB, Dyer MI, Collins T. Two New Species of Flagellates Cultivated 45 from Insects of the Genus Gerris. J Protozool 1960; 7: 390-2. 46 27. Andersen NM. Phylogeny and taxonomy of water striders, genus Aquarius Schellenberg (Insecta, Hemiptera, Gerridae), with a new species from Australia. Steenstrupia. 1990; 16: 37-81. 47 28. Hanson WL, McGhee RB. The Biology and Morphology of Crithidia acanthocephali n. sp., 48 Leptomonas Zeptoglossi n. sp., and Blastocrithidia euschisti n. sp. . J Protozool 1961; 8: 200-4. 49 29. Langridge DF, McGhee RB. Crithidia mellificae n. sp. an acidophilic trypanosomatid of the 50 honey bee Apis mellifera. J Protozool. 1967; 14: 485-7. 30. Runckel C, DeRisi J, Flenniken ML. A draft genome of the honey bee trypanosomatid 51 parasite Crithidia mellificae. PLoS One. 2014; 9: e95057. 52 53 54 55 56 57 58 59 60

https://mc04.manuscriptcentral.com/mioc-scielo Page 27 of 41 Memórias do Instituto Oswaldo Cruz

1 2 3 4 5 6 7 8 31. Cepero A, Ravoet J, Gómez-Moracho T, Bernal JL, Del Nozal MJ, Bartolomé C et al. 9 Holistic screening of collapsing honey bee colonies in Spain: a case study. BMC Research Notes. 10 2014; 7: 649. 11 32. Schwarz RS, Bauchan GR, Murphy CA, Ravoet J, de Graaf DC, Evans JD. Characterization of Two Species of Trypanosomatidae from the Honey Bee Apis mellifera: Crithidia mellificae 12 Langridge and McGhee, and Lotmaria passim n. gen., n. sp. J Eukaryot Microbiol. 2015; 62: 567-83. 13 33. Lipa JJ, Triggiani O. Crithidia bombi sp n, a new flagellated parasite of a bumble bee 14 Bombus terrestris L (Hymenoptera, Apidae). . Acta Protozool. 1988; 27: 287-90. 15 34. Léger L. Sur un flagellé parasite de l'Anopheles maculipennis. Comp R Séances Soc Biol Ses Fil. 1902; 54: 354-6. 16 35. Wallace FG. Flagellate parasites of mosquitoes with special reference to Crithidia 17 fasciculata Leger, 1902. J Parasitol. 1943: 196-205. 18 36. Noguchi H, Tilden EB. Comparative Studies of Herpetomonads and Leishmanias : I. 19 Cultivation of Herpetomonads from Insects and Plants. J Exp Med. 1926; 44: 307-25. 37. Simpson AM, Simpson L. Isolation and characterization of kinetoplast DNA networks and 20 minicircles from Crithidia fasciculata. J Protozool. 1974; 21: 774-81. 21 38. Hannaert V, Opperdoes FR, MichelsFor PA. Comparison Review and evolutionary Onlyanalysis of the 22 glycosomal glyceraldehyde-3-phosphate dehydrogenase from different Kinetoplastida. J Mol Evol. 23 1998; 47: 728-38. 39. Soares MJ, Brazil RP, Tanuri A, Souza W. Some ultrastructural aspects of Crithidia 24 guilhermei n. sp. isolated from Phaenicia cuprina (Diptera: Calliphoridae). Can J Zool. 1986; 64: 25 2837-42. 26 40. Sibajev A, Pacheco RS, Soares MJ, Cupolillo E, Santos ALB, Momen H. Crithidia ricardoi 27 sp. n. a new species of trypanosomatidae isolated from Culex saltanensis Dyar, 1928 (Diptera: Culicidae). Mem Inst Oswaldo Cruz. 1993; 88: 541-5. 28 41. Maruyama SR, de Santana AKM, Takamiya NT, Takahashi TY, Rogerio LA, Oliveira CAB 29 et al. Non-Leishmania Parasite in Fatal Visceral Leishmaniasis-Like Disease, Brazil. Emerg Infect 30 Dis. 2019; 25: 2088-92. 31 42. Roitman I, Mundim MH, Azevedo HP, Kitajima EW. Growth of Crithidia at High Temperature: Crithidia hutneri sp. n. and Crithidia luciliae thermophila s. sp. n. Journal Protozool. 32 1977; 24: 553-6. 33 43. Ishemgulova A, Butenko A, Kortisova L, Boucinha C, Grybchuk-Ieremenko A, Morelli KA 34 et al. Molecular mechanisms of thermal resistance of the insect trypanosomatid Crithidia 35 thermophila. PLoS One. 2017; 12: e0174165. 44. Sá-Xavier C, Santos SM, Sousa MA. A new trypanosomatid belonging to the genus 36 Crithidia isolated from Zelus leucogrammus (Hemiptera: Reduviidae). Mem Inst Oswaldo Cruz. 37 1998; 93: 95. 38 45. Teixeira MM, Takata CS, Conchon I, Campaner M, Camargo EP. Ribosomal and kDNA markers distinguish two subgroups of Herpetomonas among old species and new trypanosomatids 39 isolated from flies. J Parasitol. 1997; 83: 58-65. 40 46. Wallace FG, Todd SR, Rogers W. Flagellate Parasites of Water Striders with a Description 41 of Leptomonas costoris, n. sp. J Protozool. 1965; 12: 390-3. 42 47. Roubaud E. Sur un nouveau flagellé parasite de líntestin des muscides au Congo français. CR Sc Soc Biol. 1908; 64: 1106-8. 43 48. Wallace FG, Todd SR. Leptomonas Mirabilis Roubaud 1908 in a Central American Blowfly. 44 J Protozool. 1964; 11: 502-5. 45 49. Wenyon CM. Protozoology. A Manual for Medical Men, Veterinarians and Zoologists. 46 1926; vol. I. Bailliére, Tindall & Cox, London. 50. Pereira SMS. Elaboração de protocolos para padronização e sistematização dos 47 procedimentos para recebimento de depósitos de novas amostras na coleção de Protozoários da 48 FIOCRUZ. Universidade Federal do Rio de Janeiro, 2019; 270. 49 51. Rogers WE, Wallace FG. Two new subspecies of Herpetomonas muscarum (Leidy, 1856) 50 Kent, 1880. J Protozool. 1971; 18: 645-9. 52. Podlipaev SA. Insect trypanosomatids: the need to know more. Mem Inst Oswaldo Cruz. 51 2000; 95: 517-22. 52 53 54 55 56 57 58 59 60

https://mc04.manuscriptcentral.com/mioc-scielo Memórias do Instituto Oswaldo Cruz Page 28 of 41

1 2 3 4 5 6 7 8 53. Seixas RLR, Ponte CS, Santos GPL, P. BR. Isolation of Leishmania sp. of Nectomys 9 squamypes (Rodentia: Cricetidae) from Catimbau Grande, municipality of Rio Bonito, Rio de 10 Janeiro State. . Mem Inst Oswaldo Cruz. 1994; 89: 79. 11 54. Attias M, De Souza W. Axenic Cultivation and Ultrastructural Study of a Phytomonas sp. Isolated from the Milkweed Plant Euphorbia hyssopifolia. J Protozool. 1986; 33: 84-7. 12 55. Galvão AB, Oliveira RL, Carvalho ALM, Veiga GP. Leptomonas pessoai sp. 13 n.(Trypanosomatidae, Kinetoplastida, Protozoa). Rev Goiana Med. 1970; 16. 14 56. Manaia AC, Souza MCM, Lustosa ES, Roitman I. Leptomonas lactosovorans n. sp., a 15 Lactose‐Utilizing Trypanosomatid: Description and Nutritional Requirements. J Protozool. 1981; 28: 124-6. 16 57. Romeiro A, Sole-Cava A, Sousa MA, de Souza W, Attias M. Ultrastructural and 17 biochemical characterization of promastigote and cystic forms of Leptomonas wallacei n. sp. isolated 18 from the intestine of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae). J Eukaryot 19 Microbiol. 2000; 47: 208-20. 58. Suwannayod S, Sanit S, Sukontason K, Sukontason KL. Parasarcophaga (Liopygia) 20 ruficornis (Diptera:Sarcophagidae): a flesh fly species of medical importance. Trop Biomed. 2013; 21 30: 174-80. For Review Only 22 59. Votýpka J, Kostygov AY, Kraeva N, Grybchuk-Ieremenko A, Tesařová M, Grybchuk D et 23 al. Kentomonas gen. n., a New Genus of Endosymbiont-containing Trypanosomatids of Strigomonadinae subfam. n. Protist. 2014; 165: 825-38. 24 60. Yoshida N, Freymüller E, Wallace FG. Herpetomonas mariadeanei sp. n. (Protozoa, 25 Trypanosomatidae) from Muscina stabulans Fallen, 1816 (Diptera, Muscidae). J Protozool. 1978; 26 25: 421–5. 27 61. Jirku M, Yurchenko VY, Lukes J, Maslov DA. New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae. J Eukaryot 28 Microbiol. 2012; 59: 537-47. 29 62. Yurchenko VY, Lukes J, Jirku M, Maslov DA. Selective recovery of the cultivation-prone 30 components from mixed trypanosomatid infections: a case of several novel species isolated from 31 Neotropical Heteroptera. Int J Syst Evol Microbiol. 2009; 59: 893-909. 63. Manaia AC, Cordeiro NS. Description and axenic cultivation of an insect trypanosomatid. . 32 Mem Inst Oswaldo Cruz. 1989; 84: 151. 33 64. Votypka J, Klepetkova H, Jirku M, Kment P, Lukes J. Phylogenetic relationships of 34 trypanosomatids parasitising true bugs (Insecta: Heteroptera) in sub-Saharan Africa. Int J Parasitol. 35 2012; 42: 489-500. 65. Wallace FG. Leptomonas seymouri sp. n. from the cotton stainer Dysdercus suturellus. J 36 Protozool. 1977; 24: 483-4. 37 66. Aragão H. Sur un flogelle du latex de Manihot palmata, Phytomonas francai n. sp. Compte 38 Rendu de la Societe de Biologie. 1927; 1077: 1080. 67. Vainstein MH, Roitman I. Cultivation of Phytomonas françai Associated with Poor 39 Development of Root System of Cassava. J Protozool. 1986; 33: 511-3. 40 68. Dollet M, Cambrony D, Gargani D. Culture axénique in vitro de Phytomonas 41 sp.(Trypanosomatidae) d'Euphorbe, transmis par Stenocephalus agilis Scop (Coreide). Comptes 42 Rendus des Séances de l'Académie des Sciences Série 3, Sciences de la Vie. 1982; 295: 547-50. 69. Gibbs AJ. Leptomonas serpens n. sp., parasitic in the digestive tract and salivary glands of 43 Nezara viridula (Pentatomidae) and in the sap of Solanum lycopersicum (tomato) and other plants. 44 Parasitology. 1957; 47: 297-303. 45 70. Podlipaev SA. Phytomonas elmassiani (Mastigophora: Trypanosomamonadida) from the 46 plant Cynanchum sibiricum (Asclepiadaceae) in Central Asia and Kazakhstan. Trudy Zoologicheskogo Instituta, Akademiya Nauk SSSR (Sistematika prosteĭshikh i ikh filogeneticheskie 47 svyazi s nizshimi ėukariotami). 1986; 144: 61-5. 48 71. Brazil RP, Fiorini JE, de Faria e Silva P. Phytomonas sp. a trypanosomatid parasite of 49 tomato, isolated from salivary glands of Phthia picta (Hemiptera: Coreidae) in Southeast Brazil. 50 Mem Inst Oswaldo Cruz. 1990; 85: 239-40. 72. Jankevicius JV, Jankevicius SI, Campaner M, Conchon I, Maeda LA, Teixeira MMG et al. 51 Life cycle and culturing of Phytomonas serpens (Gibbs), a trypanosomatid parasite of tomatoes. J 52 Protozool. 1989; 36: 265-71. 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 73. Sa-Carvalho D, Perez-Morga D, Traub-Cseko YM. Characterization of kinetoplast DNA 9 from Phytomonas serpens. J Eukaryot Microbiol. 1993; 40: 317-22. 10 74. Conchon I, Campaner M, Sbravat C, Camargo EP. Trypanosomatids, other than Phytomonas 11 spp., isolated and cultured from fruit. J Protozool. 1989; 36: 412-4. 75. Burstein ZA, Romero OR. Flagelados en el latex de la Jatropha macrantha (Huanarpo 12 Hembra). Arch Per Pat Clin. 1956; 10: 1-11. 13 76. Jankevicius SI, De Almeida IL, Jankevicius JV, Cavazzana JR M, Attias M, De Souza W. 14 Axenic cultivation of trypanosomatids found in corn (Zea mays) and in phytophagous hemipterans 15 (Leptoglossus zonatus Coreidae) and their experimental transmission. J Euk Microbiol. 1993; 40: 576-81. 16 77. Novy FG, MacNeal WJ, Torrey HN. The trypanosomes of mosquitoes and other insects. J 17 Infect Dis. 1907: 223-76. 18 78. Wallace FG, Johnson A. The infectivity of old cultured strains of mosquito flagellates. J 19 Insect Pathol. 1961; 3. 79. Chang KP. Ultrastructure of symbiotic bacteria in normal and antibiotic‐treated 20 Blastocrithidia culicis and Crithidia oncopelti. J Protozool. 1974; 21: 699-707. 21 80. Frolov A, Malysheva M. DescriptionFor of Crithidia Review allae sp. n. and Crithidia Only brevicula sp. n. 22 (Protozoa, Trypanosomatidae) from the predator bug Nabis brevis Scholtz (Hemiplera, Miridae). 23 Russ J Zool. 1989; 68: 5-10. 81. Yurchenko V, Votypka J, Tesarova M, Klepetkova H, Kraeva N, Jirku M et al. 24 Ultrastructure and molecular phylogeny of four new species of monoxenous trypanosomatids from 25 flies (Diptera: Brachycera) with redefinition of the genus Wallaceina. Folia Parasitol (Praha). 2014; 26 61: 97-112. 27 82. Yurchenko VY, Lukes J, Jirku M, Zeledon R, Maslov DA. Leptomonas costaricensis sp. n. (Kinetoplastea: Trypanosomatidae), a member of the novel phylogenetic group of insect 28 trypanosomatids closely related to the genus Leishmania. Parasitology. 2006; 133: 537-46. 29 83. Barratt J, Kaufer A, Peters B, Craig D, Lawrence A, Roberts T et al. Isolation of novel 30 trypanosomatid, Zelonia australiensis sp. nov.(Kinetoplastida: Trypanosomatidae) provides support 31 for a Gondwanan origin of dixenous parasitism in the Leishmaniinae. PLoS Negl Trop Dis. 2017; 11: e0005215. 32 84. Dedet JP, Geoffroy B, Benichou JC. Herpetomonas dedonderi N. Sp.(Sarcomastigophora, 33 Trypanosomatidae) from Haemagogus janthinomys Dyar, 1921 (Diptera, Culicidae). J Protozool. 34 1986; 33: 530-3. 35 85. Svobodova M, Zidkova L, Cepicka I, Obornik M, Lukes J, Votypka J. Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges 36 (Ceratopogonidae, Diptera). Int J Syst Evol Microbiol. 2007; 57: 423-32. 37 86. Votypka J, Sukova E, Kraeva N, Ishemgulova A, Duzi I, Lukes J et al. Diversity of 38 trypanosomatids (Kinetoplastea: Trypanosomatidae) parasitizing fleas (Insecta: Siphonaptera) and description of a new genus Blechomonas gen. n. Protist. 2013; 164: 763-81. 39 87. Kostygov A, Frolov AO. [Leptomonas jaculum (Leger, 1902) Woodcock 1914: a 40 leptomonas or a blastocrithidia?]. Parazitologiia. 2007; 41: 126-36. 41 42 43 44 45 TABLES AND FIGURES 46 47 48 Table S1. Voucher information from the trypanosomatids barcoded, cultures are available 49 upon request at www.colprot.fiocruz.br 50 51 52 Table S2. Genbank sequences used in taxonomic comparison to barcode the trypanosomatids. 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 Table S3. Trypanosomatids species grouped by host. 9 10 11 Figure Legends 12 Fig. 1. Molecular phylogenetic analysis by ML and BI inferences from concatenated data 13 set. Phylogenetic tree of isolates studied in this work and Genbank sequences inferred by ML 14 15 and BI analyses using V7/V8 SSU rRNA region + gGAPDH concatenated set, with General 16 Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate 17 differences among sites (6 categories (+G, parameter = 0.5618)). The rate variation model 18 19 allowed for some sites to be evolutionarily invariable ([+I], 22.26% sites). The tree is rooted 20 with Paratrypanosoma confusum. NumbersFor at nodesReview correspond respectively Only to BI and ML 21 support values. The dash indicates support values below 80% from bootstraps derived from 22 23 1000 replicates. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 1. Molecular phylogenetic analysis by ML and BI inferences from concatenated data set. Phylogenetic 46 tree of isolates studied in this work and Genbank sequences inferred by ML and BI analyses using V7/V8 47 SSU rRNA region + gGAPDH concatenated set, with General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (6 categories (+G, parameter = 48 0.5618)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 22.26% 49 sites). The tree is rooted with Paratrypanosoma confusum. Numbers at nodes correspond respectively to BI 50 and ML support values. The dash indicates support values below 80% from bootstraps derived from 1000 51 replicates. 52 53 54 55 56 57 58 59 60 https://mc04.manuscriptcentral.com/mioc-scielo Memórias do Instituto Oswaldo Cruz Page 32 of 41

1 2 Table S1. Summarized voucher information from the trypanosomatids barcoded, cultures available upon request at www.colprot.fiocruz.br

3 Original deposit information Isolation Genbank acession number Blast results Code in other Updated classification Relevant reference§ Outcome of the study 4 collections Deposited originally as Origin/ Date Host species Isolated by/ Province/ Country /Date* gGAPDH SSUrRNA gGAPDH SSUrRNA

5 Angomonas ambiguus Teixeira et al. 2011 HM593019 HM593015 6 COLPROT702 TCC1780 Angomonas ambiguus E. Camargo/ 2015 Chrysomya albiceps M.M.G. Teixeira/Campo Grande/BR/2011 KX901495 KX901566 Teixeira et al. 2011 Isolate certified 99.72% 100.00% Angomonas deanei Teixeira et al. 2011 HM593022 HM593011 7 CT-IOC044 COLPROT044 TCC036E Crithidia deanei E. Camargo/ 1991 Zelus leucogrammus A.L.M. Carvalho/ Goiania/BR/ 1973 KX901497 KX901625 Carvalho & Deane 1974 Isolate certified 99.72% 100.00% 8 ATCC30255 CT-IOC212 COLPROT212 Herpetomonas roitmani P.M. Faria-e-Silva/ 1996 Ornidia obesa P.M. Faria-e-Silva/Alfenas/BR/1986 KX901496 KX901595 Faria e Silva et al. 1991, Teixeira et al. 2011 Isolate certified 99.17% 100.00% 9 TCC080E COLPROT226 CT-IOC226 Trypanosomatidae sp. J.E. Fiorini/ 1994 Morus sp. J.E. Fiorini/Alfenas/BR/ 1994 KX901499 - Fiorini et al. 1995 Isolate classification 99.45% - 10 COLPROT698 TCC036E Angomonas deanei E. Camargo/ 2015 Zelus leucogrammus A.L.M. Carvalho/ Goiania/BR/ 1973 KX901498 - Carvalho & Deane 1974 Isolate certified 99.31% - Angomonas desouzai Teixeira et al. 2011 HM593020 HM593016 11 CT-IOC109 COLPROT109 TCC079E Crithidia desouzai E. Camargo/ 1995 Ornidia obesa P.M. Faria e Silva/Alfenas/ BR/ 1986 KX901492 KX901604 Fiorini et al. 1989, Teixeira et al. 2011 Isolate certified 100.00% 100.00% 12 ATCC50305 COLPROT704 Trypanosomatidae sp. J. Votypka/ 2015 Cyrtoneuropsis conspersa J. Votýpka/Angra dos Reis/BR/ 2015 KX901494 - COLPROT field expedition Isolate classification 99.71% - 13 CT-IOC109 COLPROT708 TCC079E Angomonas desouzai V. Yurchencko/ 2015 Ornidia obesa P.M. Faria e Silva/ Alfenas/ BR/ 1986 KX901493 - Fiorini et al. 1989, Teixeira et al. 2011 Isolate certified 99.00% - 14 ATCC50305 Crithidia abscondita For Review Only Yurchenko et al. 2009 EU076606 EU079126 ATCCPRA-262 15 COLPROT677 Crithidia abscondita V. Yurchenko/ 2014 Largus sp. V. Yurchenko/ Puerto Napo/ EC/2007 KX901538 KX901572 Yurchenko et al. 2009 Isolate certified 99.86% 99.84% 127AL 16 Crithidia bombi Lipa & Triggiani 1988 GU321192 GU321194 17 COLPROT678 AK08.475 Crithidia bombi R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Alaska/US/ 2008 KX901528 KX901571 Schmid-Hempel & Tognazzo 2010 Isolate certified 99.57% 99.68% 18 COLPROT683 AK08.053 Crithidia bombi R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Alaska/US/ 2008 KX901529 KX901570 Schmid-Hempel & Tognazzo 2010 Isolate certified 98.47% 98.08% 19 COLPROT684 BJ08.068 Crithidia bombi R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Movelier/ CH/2008 KX901527 KX901569 Schmid-Hempel & Tognazzo 2010 Isolate certified 100.00% 99.36% Crithidia brachyflagelli Jirku et al. 2012 JF717835 JF717840 340 VL COLPROT669 Crithidia brachyflagelli V. Yurchenko/ 2014 Prepos accinctus M. Jirku / Tarcoles/CR/ 2009 KX901553 KX901580 Jirku et al. 2012 Isolate certified 99.86% 99.84% 20 ATCCPRA-345 Crithidia brevicula Kotysgov et al. 2014 KJ443343 - 21 CT-IOC110 COLPROT110 Wallaceina brevicula E. Camargo/ 1995 Nabis brevis A.O. Frolov / Pskov region/ RU/ 1986 KX901539 - Frolov & Malysheva 1989 Isolate certified 99.45% - TCC093 22 CT-IOC111 COLPROT111 Wallaceina inconstans E. Camargo/ 1995 Calocoris sexguttatus S.A. Podlipev/ Pskov region/ RU/ 1986 KX901540 - Podlipaev et al. 1990 Isolate certified 99.31% - TCC091 23 Crithidia expoeki Schmid-Hempel & Tognazzo 2010 GU321193 GU321195 24 COLPROT679 BJ08.175 Crithidia expoeki R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Röschenz/CH/ 2008 KX901530 - Schmid-Hempel & Tognazzo 2010 Isolate certified 99.71% - 25 COLPROT685 AK08.287 Crithidia expoeki R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Alaska/US/ 2008 KX901531 - Schmid-Hempel & Tognazzo 2010 Isolate certified 99.84% - 26 COLPROT686 BJ08.083 Crithidia expoeki R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Röschenz/CH/ 2008 KX901532 KX901568 Schmid-Hempel & Tognazzo 2010 Isolate certified 99.71% 100.00% COLPROT687 AK08.231 Crithidia expoeki R. Schmid-Hempel/ 2015 Bombus lucorum R. Schmid-Hempel/ Alaska/US/ 2008 KX901533 KX901567 Schmid-Hempel & Tognazzo 2010 Isolate certified 99.85% 100.00% 27 Crithidia fasciculata Léger 1902 AF047493 - CT-IOC048 COLPROT048 Crithidia fasciculata Nerad/ 1992 Anopheles quadrimaculatus H. Noguchi & E.B. Tilden/New Jersey/US/ 1926 KX901558 KX901624 Wallace 1943 Isolate certified 99.86% - 28 ATCC11745 CT-IOC050 COLPROT050 Crithidia fasciculata Nerad/ 1992 Culex pipiens H.N. Guttman/ St. Paul/ US/ 1942 KX901559 KX901623 Wallace 1943 Isolate certified 99.29% - 29 ATCC12857 COLPROT051 CT-IOC051 Crithidia guilhermei J. Anguster/ 1993 Lucilia cuprina R.P. Brazil/Rio de Janeiro/BR/ 1984 KX901557 KX901622 Soares et al. 1986 Isolate re-classification 98.14% - CT-IOC053 30 COLPROT053 Crithidia luciliae L.M. Previato/ 1992 Lucilia sericata F.G. Wallace & T. Clark /Minneapolis/ US/ 1958 KX901560 KX901621 Wallace & Clark 1959, Ishemgulova et al. 2017 Isolate re-classification 99.43% - ATCC30258 CT-IOC184 COLPROT184 Crithidia ricardoi R.L. de Oliveira/ 1996 Culex saltanensis A. Sibajev/Rio de Janeiro/BR/ 1993 KX901556 - Sibajev et al. 1993 Isolate re-classification 99.57% - 31 ATCC50523 32 COLPROT213 CT-IOC213 Trypanosomatidae sp. A. Romero/ 1996 Chamaesyce thymifolia A. Romeiro/ BR/ 1994 KX901554 - no associated publication Isolate classification 99.59% - COLPROT606 - Crithidia sp. A.A. Brandão/ 2011 Homo sapiens E. Martinez/ Cusco/ PE/ 1994 KX901555 KX901584 no associated publication Isolate classification 99.72% - 33 Crithidia flexonema Wallace et al. 1960 KX901534 KX901634 CT-IOC016 COLPROT016 Crithidia flexonema ATCC/ 1994 Aquarius remigis F.G. Wallace/ Ames/US/ 1960 KX901534 KX901634 Wallace et al. 1960 Isolate certified - - 34 ATCC50211 CT-IOC042 COLPROT042 Crithidia acanthocephali E. Camargo/ 1991 Acanthocephala femorata R.B. Mcghee/Athens/US/ 1959 KX901535 KX901626 Hanson & Mcghee 1961 Isolate certified 99.86% 100.00% 35 ATCC30251 COLPROT254 CT-IOC254 Crithidia sp. - Zelus sp. - KX901536 KX901587 CT-IOC field expedition Isolate classification 99.83% 99.84% 36 Crithidia insperata Yurchenko et al. 2009 EU076605 EU079125 COLPROT670 ATCCPRA-263 Crithidia insperata V. Yurchenko/ 2014 Leptopetalops sp. V. Yurchenko/ EC/ 2009 KX901549 KX901579 Yurchenko et al. 2009 Isolate certified 99.43% 100.00% 37 Crithidia mellificae Langridge & McGhee 1967 KJ713332 KJ713366 CT-IOC035 COLPROT035 Crithidia mellificae ATCC/ 1994 Apis mellifera R.B. Mcghee/Athens/US/ 1974 KX901537 KX901627 Langridge & McGhee 1967 Isolate certified 99.86% 98.87% 38 ATCC30254 Crithidia permixta Yurchenko et al. 2009 EU076607 EU079127 39 ATCCPRA261 COLPROT671 Crithidia permixta V. Yurchenko/ 2014 Mirini sp. V. Yurchenko/ Casanga/EC/ 2007 KX901541 KX901578 Yurchenko et al. 2009 Isolate certified 98.86 100.00% 128SI 40 Crithidia thermophila Roitman et al. 1977, Ishemgulova et al. 2017 KY264932 KY264937 41 42 43 https://mc04.manuscriptcentral.com/mioc-scielo 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 41 Memórias do Instituto Oswaldo Cruz

1 CT-IOC018 COLPROT018 Crithidia hutneri ATCC/ 1994 Cosmoclopius sp. I. Roitman/ Mambaí/BR/ 1975 KX901548 KX901633 Roitman et al. 1977; Ishemgulova et al. 2017 Isolate certified 99.86% 100.00% 2 ATCC30818 CT-IOC054 Roitman et al. 1977; Carvalho & Deane 1974; Ishemgulova COLPROT054 Crithidia luciliae thermophila E. Camargo/ 1991 Zelus leucogrammus A.L.M. Carvalho/ BR/ 1973 KX901547 KX901620 Isolate certified 99.86% 100.00% 3 ATCC30817 et al. 2017 Sá-Xavier et al. 1998, ; Carvalho & Deane 1974; COLPROT056 CT-IOC056 Crithidia sp. M. Deane/ 1991 Zelus leucogrammus N. Thomas/ BR/ 1991 KX901546 KX901618 Isolate certified 99.86% 100.00% Ishemgulova et al. 2017 4 ATCCPRA346 COLPROT676 Crithidia confusa V. Yurchenko/ 2014 Largus maculatus M. Jirku/Lake Arenal/CR/ 2009 KX901543 KX901573 Jirku et al. 2012; Ishemgulova et al. 2017 Isolate certified 99.86% 99.84% 320AR 5 same as COLPROT688 Crithidia hutneri ATCC/ 2015 Cosmoclopius sp. I. Roitman/ Mambaí/ BR/1975 KX901545 - Roitman et al. 1977; Ishemgulova et al. 2017 Isolate certified 99.86% - COLPROT018 same as Roitman et al. 1977; Carvalho & Deane 1974; Ishemgulova 6 COLPROT689 Crithidia luciliae thermophila ATCC/ 2015 Zelus leucogrammus A.L.M. Carvalho/ BR/ 1973 KX901544 - Isolate certified 99.86% - COLPROT054 et al. 2017 7 COLPROT703 Trypanosomatidae sp. J. Votypka/ 2015 Cyrtoneuropsis conspersa J. Votypka/Rio de Janeiro/BR/ 2015 KX901542 - Ishemgulova et al. 2017 Isolate certified 100.00% - Crithidia otongatchiensis Yurchenko et al. 2014 KF482058 KC205989 8 COLPROT628 CEU119 Trypanosomatidae sp. P. Grellier/ 2013 Ploeogaster sp. D. Blanchet/ Maripasoula/GF/ 2004 KX901551 KX901581 no associated publication Isolate classification 99.54% 100,00% COLPROT673 ECU-08 Crithidia otongatchiensis V. Yurchenko/ 2014 Eristalis sp. V. Yurchenko/ Otongatchi/EC/ 2008 KX901550 KX901576 Yurchenko et al. 2014 Isolate certified 99.43% 100.00% 9 Herpetomonas costoris Borghesan et al. 2013 JQ359738 JQ359728 CT-IOC022 10 TCC019E COLPROT022 Leptomonas costoris ATCC/ 1994 Gerris comatus F.G. Wallace/Lake Minnetonka/ US/1965 KX901476 - Wallace et al. 1965, Borghesan et al. 2013 Isolate certified 99.86% - ATCC30210 11 (prev30262) Herpetomonas elegans Borghesan et al. 2013. JQ359735 JQ359725 12 COLPROT211 Trypanosomatidae sp. M. Ziccardi/ 1996 Diptera: Syrphidae M. Ziccardi/Rio de Janeiro/BR/1996 KX901470 KX901596 No associated publication Isolate classification 99.72% 99.52% 13 Herpetomonas isaaci Borghesan et al. 2013 JQ359745 JQ359720 14 COLPROT682 - Trypanosomatidae sp. H.L.C. Santos/ 2015 Feces from Sus domesticus C. Boucinha/São Gonçalo/BR/2015 KX901471 - COLPROT field expedition Isolate classification 98.96% - COLPROT710 - Trypanosomatidae sp. K.A. Morelli/ 2015ForChrysomya Review megacephala K.A. Morelli/Rio de Janeiro/BR/2015 OnlyKX901472 - COLPROT field expedition Isolate classification 99.31% - 15 COLPROT711 - Trypanosomatidae sp. K.A. Morelli/ 2015 Chrysomya megacephala K.A. Morelli/Rio de Janeiro/BR/2015 KX901473 KX901562 COLPROT field expedition Isolate classification 99.45% 100.00% 16 Herpetomonas mirabilis Borghesan et al. 2013 JQ359739 JQ359729 CT-IOC024 17 COLPROT024 TCC301E Leptomonas mirabilis ATCC/ 1994 Cynomyopsis cadaverina F.G. Wallace/ Chichicastenango/GT/ 1963 KX901474 KX901630 Wallace & Todd 1964, Borghesan et al. 2013 Isolate certified 100.00% 99.22% ATCC30263 18 Herpetomonas modestus Borghesan et al. 2013 JQ359736 JQ359726 COLPROT707 Trypanosomatidae sp. J. Votypka/ 2015 Cyrtoneuropsis conspersa J. Votypka/Angra dos Reis/BR/2015 KX901475 KX901563 COLPROT field expedition Isolate classification 98.75% 99.84% 19 Herpetomonas muscarum Borghesan et al. 2013, Wallace 1966 JQ359748 JQ359731 COLPROT013 CT-IOC013 Trypanosomatidae sp. R.P. Brazil/ 1992 Phthia picta R.P. Brazil/Alfenas/ BR/ 1990 KX901464 KX901635 Brazil et al. 1990 Isolate classification 100.00% 99.83% 20 CT-IOC020 COLPROT020 TCC001E Herpetomonas muscarum muscarum ATCC/ 1994 Musca domestica F.G. Wallace/ Minneapolis/ US/ 1968 KX901463 KX901632 Rogers & Wallace 1971, Borghesan et al. 2013 Isolate certified 100.00% 99.32% ATCC30260 21 CT-IOC033 COLPROT033 TCC002E Herpetomonas megaseliae ATCC/ 1994 Megaseliae scalaris J. Janovy/ Lincoln/US/ 1972 KX901468 KX901628 Daggett et al. 1972, Borghesan et al. 2013 Isolate certified 99.58% 99.49% 22 ATCC30209 CT-IOC076 23 COLPROT076 TCC048E Phytomonas davidi E. Camargo/ 1991 Euphorbia heterophyla R.B. Mcghee/ Okeechobe/US/ 1975 KX901466 KX901612 Mcghee & Postell 1976, Borghesan et al. 2013 Isolate certified 100.00% 99.66% ATCC30287 24 COLPROT081 CT-IOC081 Phytomonas sp. M. Attias/ 1992 Euphorbia hyssopifolia M. Attias /Rio de Janeiro/BR/1982-1984 KX901469 KX901609 Attias & De Souza 1986 Isolate classification 99.84% 99.66% COLPROT185 CT-IOC185 Herpetomonas sp. R.P. Brazil/ 1996 Nectomys squamipes R.P. Brazil/Rio de Janeiro/BR/1994 KX901467 KX901601 Seixas et al. 1994 Isolate classification 99.86% 99.83% 25 COLPROT229 CT-IOC229 Phytomonas serpens JE.. Fiorini/ 1996 Solanum lycopersicum J.E. Fiorini/Alfenas/BR/ 1986 KX901465 KX901590 Fiorini et al. 1986 Isolate re-classification 100.00% 99.66% Isolate re-classification, COLPROT276 CT-IOC276 Herpetomonas samuelpessoai M. Cavazzana/ 1997 - - KX901484 KX901586 Podlipaev 1990, Galvao et al. 1970# 100.00% 99.49% history uncertain 26 Herpetomonas samueli Borghesan et al. 2013 JQ359742 - CT-IOC023 COLPROT023 Leptomonas lactosovorans ATCC/ 1994 Zelurus martinsi A.C. Manaia/Goiania/BR/ 1977 KX901478 - Manaia et al. 1981 Isolate re-classification 100.00% - 27 ATCC30970 CT-IOC074 28 COLPROT074 TCC003E Leptomonas samueli LM Previato/ 1992 Zelus leucogrammus A.L.M. Carvalho/Goiania/ BR/ 1973 KX901477 - Carvalho & Deane 1974 Isolate certified 100.00% - ATCC30971 29 COLPROT194 CT-IOC194 Leptomonas wallacei A Romeiro/ 1998 Oncopeltus fasciatus A. Romero/Rio de Janeiro/BR /1988 KX901479 - Romeiro et al. 2000 Isolate re-classification 99.52% - Herpetomonas samuelpessoai Borghesan et al. 2013 JQ359743 JQ359716 CT-IOC059 30 COLPROT059 Herpetomonas anglusteri M.J. Soares/ 1992 Parasarcophaga (Liopygia) ruficornis J.E. Fiorini/Alfenas/BR/ 1986 KX901482 KX901617 Fiorini et al. 1989 Isolate re-classification 99.86% 99.52% ATCC50304 31 CT-IOC 067 TCC COLPROT067 Herpetomonas samuelpessoai J. Anguster/ 1991 Zelus leucogrammus A.B. Galvão/Goiania/BR/ 1970 KX901481 KX901614 Galvão et al. 1970# Roitman et al. 1976 Isolate certified 99.86% 99.52% 005E ATCC 30252 32 COLPROT223 CT-IOC223 Trypanosomatidae sp. J.E. Fiorini/ 1996 Musca sp. J.E. Fiorini/ Alfenas/BR/ 1996 KX901480 KX901593 No associated publication Isolate classification 98.90% 99.84% COLPROT227 CT-IOC227 Herpetomonas sp. J.E. Fiorini/ 1989 Phaenicia eximia J.E. Fiorini/ Alfenas/BR/ 1996 KX901483 KX901591 No associated publication Isolate classification 99.58% 99.52% 33 Kentomonas sorsogonicus Votýpka et al. 2014. KM242072 KM242075 34 COLPROT696 MF-08.01 Kentomonas sorsogonicus V. Yurchenko/ 2015 Sarcophaga sp. J. Votypka /Donson/PH/ 2014 KX901516 - Votýpka et al. 2014. Isolate certified 99.31% - Lafontella mariadeanei Yurchenko et al. 2016 JQ359741 JQ359714 35 CT-IOC062 COLPROT062 ATCC30708 Herpetomonas mariadeanei A. Romanha/ 1992 Muscina stabulans N. Yoshida/ São Paulo/BR/ 1974 KX901491 KX901615 Yoshida et al. 1978, Yurchenko et al. 2016 Isolate certified 99.58% 99.67% 36 TCC004E Leptomonas jaderae Yurchencko et al. 2009. EU076603 EU079123 ATCCPRA-264 37 COLPROT674 Leptomonas jaderae V. Yurchenko/ 2014 Jadera obscura J. Lukes/ Tarcoles/CR/ 2005 KX901521 KX901575 Yurchencko et al. 2009 Isolate certified 99.72% 100.00% 34EC 38 Leptomonas podlipaevi Yurchencko et al. 2006. EU076604 EU079124 COLPROT344 CT-IOC344 Leptomonas sp. A.C. Manaia/ 1999 Leptocoris trivittatus A.C. Manaia/ Campinas/BR/ 1989 KX901520 KX901585 Manaia & Cordeiro 1989 Isolate classification 99.72% 99.52% 39 Leptomonas pyrrhocoris Votypka et al. 2012 JN036651 JN036653 CT-IOC026 COLPROT026 Leptomonas pyrrhocoris ATCC/ 1994 Pyrrhocoris apterus FG Wallace/ Karlstein Castle, Prague/CZ/ 1978 KX901526 KX901629 Zotta 1912, 1921 Isolate certified 100.00% 100.00% 40 ATCC30974 41 COLPROT572 CT-IOC572 Leptomonas sp. B.N. dos Santos/ 2005 Dysdercus sp. B.N. dos Santos/Rio de Janeiro/BR/ 2005 KX901525 - CT-IOC field expedition Isolate classification 100.00% - 42 43 https://mc04.manuscriptcentral.com/mioc-scielo 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Memórias do Instituto Oswaldo Cruz Page 34 of 41

1 COLPROT706 - Trypanosomatidae sp. J. Votypka/ 2015 Dysdercus ruficollis J. Votypka/Rio Claro/BR/ 2015 KX901524 KX901564 COLPROT field expedition Isolate classification 99.72% 100.00% 2 Leptomonas seymouri Kraeva et al. 2015 KP717896 KP717894 CT-IOC277 COLPROT277 Leptomonas seymouri A. Romanha/ 1997 Dysdercus suturellus F.G. Wallace/ Desveaux/US/ 1959 KX901523 - Wallace 1977 Isolate certified 99.72% - 3 ATCC30220 COLPROT705 - Trypanosomatidae sp. J. Votypka/ 2015 Dysdercus ruficollis J. Votypka/Rio Claro/BR/ 2015 KX901522 KX901565 COLPROT field expedition Isolate classification 99.86% 100.00% Leptomonas tarcoles Yurchenko et al. 2008 EF546787 EF546786 4 ATCCPRA-241 COLPROT672 Leptomonas tarcoles V. Yurchenko/ 2014 Prepops sp. V. Yurchenko/ Tarcoles/CR/ 2008 KX901552 KX901577 Yurchenko et al. 2008 Isolate certified 100.00% 100.00% 47VL 5 Phytomonas sp. PhyB Frolov et al. 2016, Zanetti et al. 2016 KU659030 KU659042 CT-IOC079 COLPROT079 Phytomonas sp. M. Teixeira/ 1992 Allamanda cathartica P. Kastelein/ Suriname/1984 KX901502 KX901611 Kastelein & Parsadi 1988 Isolate classification 100.00% 99.82% 6 TCC083E Phytomonas sp. PhyD Aragão1927#, Zanetti et al. 2016 EU084895 AF016322 7 COLPROT011 CT-IOC011 Phytomonas françai M. Attias/ 1994 Manihot esculenta I. Roitman/ Espírito Santo/ BR/1986 KX901504 KX901636 Vainstein & Roitman 1986 Deposit questioning 100.00% 98.68% 8 COLPROT082 CT-IOC082 Phytomonas sp. M. Teixeira/ 1994 Euphorbia pinea M. Dollet/FR/1982 KX901503 KX901608 Dollet et al. 1982 Isolate classification 100.00% 97.75% 9 Phytomonas serpens Gibbs 1957, Jankevicius et al. 1989, Zanetti et al, 2016 KU659028 KU659040 Isolate certified, history COLPROT080 CT-IOC080 Phytomonas sp. M.M.G. Teixeira/ 1992 Citrus bergamia I. Conchon / PR/BR/1989 KX901505 KX901610 Conchon et al. 1989 99.30% 99.46% uncertain 10 Isolate certified, history COLPROT083 CT-IOC083 Phytomonas sp. M.M.G. Teixeira/ 1994 Jathropha macrantha Perú, 1956 KX901508 KX901607 Burstein & Romero 1956# 99.30% 99.64% uncertain 11 CT-IOC161 Herpetomonas macgheei >> Phytomonas COLPROT161 A. Romero/ 1996 Leptoglossus zonatu S.I. Jankevicius/ Londrina/BR/ 1993 KX901509 KX901603 Jankevicius et al. 1993 Isolate certified 99.31% 99.64% TCC297E macgheei 12 CT-IOC181 Herpetomonas macgheei >> Phytomonas COLPROT181 J.V. Jankevicius/ 1996 Leptoglossus zonatu S.I. Jankevicius/ Londrina/BR/ 1993 MN108259 - Jankevicius et al. 1993 Isolate certified 99.31% - TCC297E macgheei 13 COLPROT174 CT-IOC174 9T Phytomonas serpens J.V. Jankevicius/ 1996 Solanum lycopersicum J.V. Jankevicius/ Rolândia/BR/ 1989 KX901510 KX901602 Jankevicius et al. 1989 Isolate certified 100.00% 99.64% CT-IOC186 14 COLPROT186 Phytomonas serpens J.V. Jankevicius/ 1996 Solanum lycopersicum J.V. Jankevicius/ Londrina★/BR/ 1989 KX901511 KX901600 Jankevicius et al. 1989 Isolate certified 100.00% 99.82% 15T For Review Only 15 COLPROT187 CT-IOC187 30T Phytomonas sp. J.V. Jankevicius/ 1996 Solanum lycopersicum J.V. Jankevicius/ BR/1989 KX901512 KX901599 Jankevicius et al. 1989 Isolate certified 100.00% 99.28% 16 COLPROT188 CT-IOC188 10T Phytomonas sp. J.V. Jankevicius/ 1996 Solanum lycopersicum J.V. Jankevicius/ Palmital/BR/ 1989 KX901513 KX901598 Jankevicius et al. 1989 Isolate certified 100.00% 99.46% 17 COLPROT189 CT-IOC189 9T Phytomonas serpens J.V. Jankevicius/ 1996 Solanum lycopersicum J.V. Jankevicius/ Rolândia/BR/ 1989 KX901514 KX901597 Jankevicius et al. 1989 Isolate certified 100.00% 99.82% COLPROT245 CT-IOC245 Trypanosomatidae sp. J.E. Fiorini/ 1997 Phthia picta J.E. Fiorini/Alfenas/ BR/ 1997 KX901506 KX901589 Brazil et al. 1990 Isolate classification 100.00% 99.64% 18 COLPROT247 CT-IOC247 Trypanosomatidae sp. J.E. Fiorini/ 1997 Solanum lycopersicum J.E. Fiorini/Alfenas/ BR/ 1997 KX901507 KX901588 Brazil et al. 1990 Isolate classification 100.00% 99.64% 19 COLPROT675 9T Phytomonas serpens V. Yurchenko/ 2014 Solanum lycopersicum J.V. Jankevicius/ BR KX901515 KX901574 Jankevicius et al. 1989 Isolate certified 100.00% 99.28% Strigomonas culicis Teixeira et al. 2011 EU079137 HM593009 CT-IOC034 20 COLPROT034 (apo) Blastocrithidia culicis (apo) ATCC 1994 Aedes vexans K.P. Chang/ US / 1975 KX901487 - Chang 1974, Teixeira et al. 2011 Isolate certified 100.00% - ATCC30257 CT-IOC041 21 COLPROT041 TCC012E Blastocrithidia culicis E. Camargo/ 1991 Aedes vexans FG Wallace/Mound/US/ 1942 KX901488 - Wallace & Johnson 1961, Teixeira et al. 2011 Isolate certified 100.00% - ATCC30268 22 Strigomonas oncopelti Teixeira et al. 2011 EU079134 AF038025 CT-IOC019 23 COLPROT019 (apo) TCC045E Crithidia oncopelti (apo) ATCC/ 1994 Oncopeltus fasciatus K.P. Chang/ US/1975 KX901486 - Chang 1974, Teixeira et al. 2011 Isolate certified 100.00% - ATCC30264 24 CT- IOC 055 TCC COLPROT055 Crithidia oncopelti Nerad/ 1992 Oncopeltus fasciatus H. Noguchi/ Shandaken/US /1926 KX901485 KX901619 Noguchi & Tilden 1926, Teixeira et al. 2011 Isolate certified 100.00% 96.33% 25 043E ATCC 12982 Wallacemonas collosoma Kostygov et al. 2014 EU084898 JN582046 CT-IOC073 26 COLPROT073 Leptomonas collosoma E. Camargo/ 1991 Gerris dissortis F.G. Wallace/ Minneapolis/ US/1960 KX901489 KX901613 Wallace et al. 1960 Isolate certified 99.17% 100.00% ATCC30261 Zelonia costaricensis Espinosa et al. 2016 DQ383650 DQ383648 27 COLPROT627 CEU120 Leptomonas costaricensis P. Grellier/ 2013 Ploeogaster (Aricosus) sp. D. Blanchet/ Maripasoula/GF/ 2003 KX901518 KX901582 MNHN field expeditions Isolate certified 99.29% 99.84% 28 Zelonia dedonderi n. comb. Present report DQ383650 DQ383648 COLPROT060 CT-IOC060 Herpetomonas dedonderi E. Camargo/ 1991 Haemagogus janthinomys J.P. Dedet/ Montsinery/GF/ 1981-1983 KX901519 KX901616 Dedet et al. 1986 Isolate re-classification 95.45% 96.81% 29 Zelonia n. sp. DQ383650 DQ383648 COLPROT616 CEU334 Trypanosomatidae sp. P. Grellier/ 2013 Rasahus surinamensis D. Blanchet/ Kaw/GF/2003 KX901517 KX901583 MNHN field expeditions Isolate classification 95.87% 97.92% 30 Trypanosomatidae n. gen. n. sp. CT-IOC021 COLPROT021 Herpetomonas muscarum ingenoplastis ATCC/ 1994 Phormia regina F. G. Wallace /Minneapolis/ US/ 1968 KX901490 KX901631 Rogers & Wallace 1971 New typing unit ! ! 31 ATCC30259 Trypanosomatidae n. gen. n. sp. KF054086 DQ394362 32 COLPROT100 CT-IOC100 Trypanosomatidae sp. W. Degrave/ 1994 Psychodopygus ayrozai T. Barret/ Porto Urucu/ BR/ 1993 KX901500 KX901606 no associated publication New typing unit 89.20% 91.72%

33 COLPROT101 CT-IOC101 Trypanosomatidae sp. W. Degrave/ 1994 Psychodopygus ayrozai T. Barret/ Porto Urucu/ BR/ 1993 KX901501 KX901605 no associated publication New typing unit 89.06% 92.40% 34 Trypanosomatidae n. gen. n. sp. KM242072 EF184218 COLPROT728 CT-IOC288 Trypanosomatidae sp. A. Romeiro/ 1998 Oncopeltus fasciatus A. Romeiro/ Rio de Janeiro/ BR/ 1988 MN108258 MN121011 Romeiro et al. 2000 New typing unit 83.14% 89.77% 35 COLPROT751 LEIP 436 Trypanosomatidae sp. A. Veloso/ 2017 Oncopeltus fasciatus A. Veloso/ Rio de Janeiro/ BR/ 2016 - MN121009 no associated publication New typing unit - 95.28% 36 COLPROT755 LEIP 440 Trypanosomatidae sp. A. Veloso/ 2017 Oncopeltus fasciatus A. Veloso/ Rio de Janeiro/ BR/ 2016 - MN121010 no associated publication New typing unit - 95.24% ATCC - American Type Culture Collection 37 CEU - Collection d'Eucaryotes Unicellulaires du Muséum National d'Histoire naturelle (MNHN) 38 COLPROT - Protist Culture Collection from Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil CT-IOC: Coleção de Tripanossomatídeos do Instituto Oswaldo Cruz, Fundacao Oswaldo Cruz, Brazil 39 TCC: Trypanosomatid Culture collection (TCC-USP) of the University of São Paulo, São Paulo, Brazil. 40 § The reference cited either refers to the original description of the isolate, or to the diagnosis of the species based on DNA sequences, or taxonomic updates #Refrence refers to the origianl taxa description, however, the authors were unable to access it. All other historical references cited are available from the auhors upon request 41 42 43 https://mc04.manuscriptcentral.com/mioc-scielo 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 41 Memórias do Instituto Oswaldo Cruz

1 2 * The year of isolation and the responsible for isolation were considered the year of publication and the first author of the article that described the strain isolation, unless this information was readily made availabe by the depositor or in the publication. ★ Isolate 15T was isolated from artificially infected insects, it is not clearly specified in the article which isolate was used in the infection experiments, nor the place, we are assuming the first author institution as the place of isolation 3 ! The blast values were not significant to group these Trypanosomatids with any sequence deposited in Genbank 4 The Genebank sequences highlighted in blue indicate that our study provided for the first time a reference sequence for a type strain 5 6 7 8 9 10 11 12 13 14 For Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 https://mc04.manuscriptcentral.com/mioc-scielo 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Memórias do Instituto Oswaldo Cruz Page 36 of 41

1 Table S2. Genbank sequences used in taxonomic comparison to barcode the trypansomatids. Species/strain Reference for taxonomy 2 Genera Updated taxonomy Deposit name in genbank Previous nomenclature Reference for sequence deposit (A), species description (B) gGAPDH SSU code update 3 Angomonas Angomonas ambiguus TCC1780 - Angomonas ambiguus - Teixeira et al. 2011 (A+B) HM593019 HM593015 TCC036E Angomonas deanei Teixeira et al. 2011 Angomonas deanei Crithidia deanei Teixeira et al. 2011 (A), Carvalho & Deane 1974 (B) HM593022 HM593011 4 ATCC30255 TCC079E Angomonas desouzai Teixeira et al. 2011 Angomonas desouzai Crithidia desouzai Teixeira et al. 2011 (A), Fiorini et al. 1989 (B) HM593020 HM593016 5 ATCC50305 Blastocrithidia 6 Blastocrithidia largi - Blastocrithidia largi Maslov et al. 2010 (A+B) FJ968528 FJ968531 Blechomonas 7 Blechomonas danrayi B08 - Blechomonas danrayi - Votýpka et al. 2013 (A+B) MH064439 KF054136 Blechomonas luni B09 - Blechomonas luni - Votýpka et al. 2013 (A+B) MH064438 MH055750 8 Blechomonas maslovi B6 - Blechomonas maslovi - Votýpka et al. 2013 (A+B) KF054106 KF054125 Blechomonas campbelli B06 - Blechomonas campbelli - Votýpka et al. 2013 (A+B) KF054097 KF054133 9 Blechomonas keelingi B86 - Blechomonas keelingi - Votýpka et al. 2013 (A+B) KF054110 KF054132 Blechomonas pulexsimulantis ATCC 50186 Votýpka et al. 2013 Blechomonas pulexsimulantis Leptomonas pulexsimulantis Votýpka et al. 2013 (A), Beard et al. 1989 (B) KF054090 KF054128 Borovskyia 10 Borovskyia barvae Kostygov et al. 2017 Leptomonas barvae Leptomonas barvae Maslov et al. 2010 (A+B) FJ968529 FJ968532 Crithidia 11 Crithidia bombi BJ08.085 - Crithidia bombi - Schmid-Hampel & Tognazzo 2010 (A), Lipa & Triggiani 1988 (B) GU321192 GU321194 Crithidia expoeki BJ08.175 - Crithidia expoeki - Schmid-Hampel & Tognazzo 2010 (A+B) GU321193 GU321195 ATCC 30862 12 Crithidia mellificae - Crithidia mellificae - Schwarz et al. 2015 (A), Langridge & McGhee 1967 (B) KJ713357 KJ713379 ATCC 30254 COLPROT054 13 Crithidia termophila Ishemgulova et al. 2017 Crithidia luciliae termophila Crithidia luciliae termophila Ishemgulova et al. 2017 (A), Rotiman et al. 1977 (B) KY264932 KY264937 ATCC30817 14 Crithidia fasciculata ATCC11745 - Crithidia fasciculata - Hannaert et al. 1998 (A), Noguchi & Tilden 1926 (B) AF047493(A) - 127AL Crithidia abscondita - Crithidia abscondita - Yurchenko et al. 2009 (A+B) EU076606 EU079126 15 ATCCPRA-262 16 Crithidia permixta isolate 128SI - - - Yurchenko et al. 2009 (A+B) EU076607 EU079127 119YS Crithidia insperata - Crithidia insperata - Yurchenko et al. 2009 (A+B) EU076605 EU079125 17 ATCCPRA-263 Crithidia otongatchiensis ECU-08 - Crithidia otongatchiensis - Yurchenko et al. 2014 KF482058 KC205989 18 340VL Crithidia brachyflagelli - Crithidia brachyflagelli - Jirku et al. 2012 JF717835 JF717840 ATCCPRA-345 19 Proteomonas brevicula Crithidia brevicula isolate ZK Kostygov et al. 2014 Crithidia brevicula Kotysgov et al. 2014 (A), Podlipaev et al. 1990 (B) KJ443343 KJ443353 Wallaceina brevicula 20 Endotrypanum Endotrypanum schaudinni TCC224 For ReviewEndotrypanum schaudinni Only Espinosa et al. 2018 (A+B) KX790715 KX790771 21 Herpetomonas TCC001E Herpetomonas muscarum Borghesan et al. 2013 Herpetomonas muscarum - Borghesan et al. 2013 (A), Rogers & Wallace 1971 (B) JQ359748 JQ359731 22 ATCC30260 Herpetomonas isaaci TCC266E - Herpetomonas sp. - Borghesan et al. 2013 (A+B) JQ359745 JQ359720 23 Herpetomonas elegans TCC1733 - Herpetomonas sp. - Borghesan et al. 2013 (A+B) JQ359735 JQ359725 Herpetomonas modestus TCC1444 - Herpetomonas sp. - Borghesan et al. 2013 (A+B) JQ359736 JQ359726 TCC019E 24 Herpetomonas costoris Borghesan et al. 2013 Leptomonas costoris Leptomonas costoris Borghesan et al. 2013 (A), Wallace, Todd & Rogers 1965 (B) JQ359738 JQ359728 ATCC30262 TCC301E Herpetomonas mirabilis Borghesan et al. 2013 Leptomonas mirabilis Leptomonas mirabilis Borghesan et al. 2013 (A), Wallace & Todd 1964 (B) JQ359739 JQ359729 25 ATCC30263 TCC003E Herpetomonas samueli Borghesan et al. 2013 Leptomonas samueli Leptomonas samueli Borghesan et al. 2013 (A), Carvalho 1973 (B) JQ359742 JQ359722 26 ATCC30971 37EC Herpetomonas samueli this work Leptomonas cf. lactosovorans Leptomonas cf. lactosovorans Yurchenko et al. 2009 (A), Manaia et al. 1981(B) EU076602 EU079122 27 ATCC30970 TCC005E Herpetomonas samuelpessoai Borghesan et al. 2013 Herpetomonas pessoai - Borghesan et al. 2013 (A), Roitman et al. 1976 (B) JQ359743 JQ359716 28 ATCC30252 Jaenimonas 29 Jaenimonas drosophilae - - - - Votýpka and Hamilton 2015 (A+B) KP260535 KP260534 Kentomonas 30 Kentomonas sorsogonicus MF-08.01 - Kentomonas sorsogonicus - Votypka et al. 2014 (A+B) KM242072 KM242075 Lafontella 31 TCC004E Lafontella mariadeanei Yurchenko et al. 2016 Herpetomonas mariadeanei Herpetomonas mariadeanei Borghesan et al. 2013 (A), Yoshida, Freymüller & Wallace 1978 (B) JQ359741 JQ359714 ATCC30708 32 Leishmania Leishmania infantum CBT13 Voltypka et al. 2015 Leishmania chagasi Leishmania chagasi Marcili et al. 2014 (A), Voltypka et al. 2015(B) KF041811 KF041776 33 Leishmania enriettii TCC2114 Leishmania enriettii Espinosa et al. 2018 (A), Muniz & Medina 1948 (B) KX790725 KX790777 Leptomonas 34 34EC Leptomonas jaderae - Leptomonas jaderae - Yurchenko et al. 2009 (A+B) EU076603 EU079123 35 ATCCPRA-264 Leptomonas podilipaevi 59LI - - - Yurchenko et al. 2009 (A), Yurchenko et al. 2006 (B) EU076604 EU079124 Leptomonas pyrrhocoris strain H10 - Leptomonas pyrrhocoris - Votypka et al. 2014 (A), Zotta 1921 (B) JN036651 JN036653 36 Leptomonas seymouri isolate Ld_39 - Leptomonas seymouri - Kraeva et al. 2015 (A), Wallace et al. 1977 (B) KP717896 KP717894 Lotmaria 37 Lotmaria passim - Lotmaria passim Schwarz et al. 2015 (A+B) KJ713352 KM066239 Novymonas 38 Novymonas esmeraldas AK2016a - Tripanosomatidae sp. Kostygov et al. 2016 (A+B) KT944300 KT944308 Paraleishmania 39 Paraleishmania deanei TCC256 Kostygov et al. 2017 Leishmania deanei Leishmania deanei Espinosa et al. 2016 (A), Lainson & Shaw 1987 (B) KX790720 KX790775 Paratrypanosoma 40 Paratrypanosoma confusum CUL13 - Paratrypanosoma confusum Flegontov et al. 2013 (A+B) KF963539 KF963538 Phytomonas 41 Phytomonas sp PhyD TCC EM1 - - - Zanetti et al. 2016 (A), Dollet et al. 2001 (B) EU084895 AF016322 Phytomonas sp. group PhyB TCC082E - Phytomonas sp. Phytomonas sp. Zanetti et al. 2016 (A+B) KU659030 KU659042 Phytomonas serpens group PhyA TCC060E - Phytomonas serpens Phytomonas serpens Zanetti et al. 2016 (A), Gibbs 1957 (B) KU659028 KU659040 42 Phytomonas serpens group PhyA TCC297E Zanetti et al. 2016 Phytomonas sp. Herpetomonas macgheei Zanetti et al. 2016 (A), Janckevicius et al. 1993 (B) KU659036 KX219757 Phytomonas dolleti PhyE TCC215E - - - Zanetti et al. 2016 (A+B) KX219748 KX219754 43 Phytomonas sp PhyE TCC148E - - - Zanetti et al. 2016 (A+B) KU659031 KU659043 Phytomonas sp PhyC TCC299e - - - Zanetti et al. 2016 (A), Sanchez-Moreno et al. 1995 (B) KX219750 KX219758 44 Sergeia Sergeia podlipaevi CER3 - Sergeia podlipaevi Svobodova et al. 2007 (A+B) DQ394365 DQ394362 45 Strigomonas TCC045E Yurchenko et al. 2009 (A), Hollar, Lukes & Maslov 1998 (A'), Noguchi Strigomonas oncopelti Teixeira et al. 2011 Crithidia oncopelti Crithidia oncopelti EU079134 (A) AF038025 (A') 46 ATCC30264 & Tilden 1926 (B) TCC012E Blastocrithidia culicis (A) Yurchenko et al. 2009 (A), Teixeira et al. 2011 (A'), Wallace & Strigomonas culicis Teixeira et al. 2011 Blastocrithidia culicis EU079137 (A) HM593009 (A') ATCC30268 Strigomonas culicis (A') Johnson 1961 (B) 47 Trypanosoma Trypanosoma cruzi strainG Trypanosoma cruzi Cavazzana et al. 2010 (A), Chagas 1909 (B) GQ140351 AF239981 48 Wallacemonas Wallacemonas collosoma Yurchenko et al. 2009 Wallacemonas collosoma Leptomonas collosoma Yurchenko et al. 2009 (A), Wallace et al. 1960 (B) EU084898 JN582046 49 Zelonia 15EC Zelonia costaricensis Espinosa et al. 2016 Leptomonas costaricensis Leptomonas costaricensis Yurchenko et al. 2006 (A+B) DQ383650 DQ383648 50 TCC2165 51 Zelonia australiensis - Barrat et al. 2017 Zelonia australiensis - Barrat et al. 2017 (A+B) KY273493 KY273498 52 53 54 55 56 57 58 59 60

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1 2 Table S3. Trypanosomatids species list organized by host Host taxonomy in the original COLPROT Updated taxonomy / Order Family Country /Date COLPROT number Species name 3 records Synonimys 4 Acanthocephala femorata - USA COLPROT042 Crithidia flexonema Leptocoris trivittatus Boisea trivittata Brazil COLPROT344 Leptomonas podlipaevi 5 Leptoglossus zonatu - Brazil COLPROT161 Phytomonas serpens Coreidae Leptopetalops sp. - Ecuador COLPROT670 Crithidia insperata 6 - Brazil COLPROT013 Herpetomonas muscarum Phthia picta 7 - Brazil COLPROT245 Phytomonas serpens Calocoris sexguttatus Grypocoris sexguttatus Russia COLPROT111 Crithida brevicula Mirini sp. - Ecuador COLPROT671 Crithidia permixta 8 Miridae Prepops sp. - Costa Rica COLPROT672 Leptomonas tarcoles 9 Prepos accinctus - Costa Rica COLPROT669 Crithidia brachyflagelli 10 Cosmoclopius sp. - Brazil COLPROT688 Crithidia thermophila - French Guiana COLPROT627 Zelonia costaricensis Ploeogaster (Aricosus) sp. 11 - French Guiana COLPROT628 Crithidia otongatchiensis Rasahus surinamensis - French Guiana COLPROT616 Zelonia n. sp. 12 Zelurus martinsi - Brazil COLPROT023 Herpetomonas samueli - Brazil COLPROT698 Angomonas deanei 13 - Brazil COLPROT054 Crithidia thermophila Reduviidae 14 - Brazil COLPROT056 Crithidia thermophila Zelus leucogrammus - Brazil COLPROT689 Crithidia thermophila 15 Hemiptera - Brazil COLPROT074 Herpetomonas samueli - Brazil COLPROT067 Herpetomonas samuelpessoai 16 - Brazil COLPROT044 Angomonas deanei Zelus sp. - *untraceable COLPROT254 Crithidia flexonema 17 Cosmoclopius sp. - Brazil COLPROT018 Crithidia thermophila 18 Dysdercus suturellus - USA COLPROT277 Leptomonas seymouri Pyrrhocoris apterus - Czech republic COLPROT026 Leptomonas pyrrhocoris 19 Pyrrhocoridae - Brazil COLPROT706 Leptomonas pyrrhocoris Dysdercus ruficollis - Brazil COLPROT705 Leptomonas seymouri 20 Dysdercus sp. - Brazil COLPROT572 Leptomonas pyrrhocoris Gerris comatusFor Review- OnlyUSA COLPROT022 Herpetomonas costoris 21 Gerridae Gerris dissortis - USA COLPROT073 Wallacemonas collosoma 22 Rhopalidae Jadera obscura - Costa Rica COLPROT674 Leptomonas jaderae Largus maculatus - Costa Rica COLPROT676 Crithidia thermophila Largidae 23 Largus sp. - Ecuador COLPROT677 Crithidia abscondita Nabidae Nabis brevis - Russia COLPROT110 Crithidia brevicula 24 - Brazil COLPROT194 Herpetomonas samueli - USA COLPROT019 (apo) Strigomonas oncopelti (apo) 25 Lygaeidae Oncopeltus fasciatus - USA COLPROT055 Strigomonas oncopelti 26 - Brazil COLPROT728 Trypanosomatidae sp. - USA COLPROT034 Strigomonas culicis (apo) Aedes vexans 27 - USA COLPROT041 Strigomonas culicis 28 Anopheles quadrimaculatus - USA COLPROT048 Crithidia fasciculata Culicidae 29 Culex pipiens - USA COLPROT050 Crithidia fasciculata Culex saltanensis - Brazil COLPROT184 Crithidia fasciculata 30 Haemagogus janthinomys - French Guiana COLPROT060 Zelonia dedonderi Chrysomya albiceps - Brazil COLPROT702 Angomonas ambiguus 31 - Brazil COLPROT710 Herpetomonas isaaci Chrysomya megacephala 32 - Brazil COLPROT711 Herpetomonas isaaci Cynomyopsis cadaverina - Guatemala COLPROT024 Herpetomonas mirabilis Calliphoridae 33 Lucilia cuprina - Brazil COLPROT051 Crithidia fasciculata Lucilia sericata - USA COLPROT053 Crithidia fasciculata 34 Phaenicia eximia Lucilia eximia Brazil COLPROT227 Herpetomonas samuelpessoai Phormia regina - USA COLPROT021 Trypanosomatidae n. gen. n. sp. 35 Diptera - Brazil COLPROT704 Angomonas desouzai 36 Cyrtoneuropsis conspersa - Brazil COLPROT707 Herpetomonas modestus - Brazil COLPROT703 Crithidia thermophila Muscidae 37 Musca domestica - USA COLPROT020 Herpetomonas muscarum Musca sp. - Brazil COLPROT223 Herpetomonas samuelpessoai 38 Muscina stabulans - Brazil COLPROT062 Lafontella mariadeanei 39 *untraceable - Brazil COLPROT211 Herpetomonas elegans Eristalis sp. - Ecuador COLPROT673 Crithidia otongatchiensis 40 Syrphidae - Brazil COLPROT708 Angomonas desouzai Ornidia obesa - Brazil COLPROT212 Angomonas deanei 41 - Brazil COLPROT109 Angomonas desouzai Phoridae Megaseliae scalaris - USA COLPROT033 Herpetomonas muscarum 42 Parasarcophaga (Liopygia) ruficornis - Brazil COLPROT059 Herpetomonas samuelpessoai Sarcophagidae 43 Sarcophaga sp. - Philippines COLPROT696 Kentomonas sorsogonicus - Brazil COLPROT101 Trypanosomatidae sp. Psychodidae Psychodopygus ayrozai 44 - Brazil COLPROT100 Trypanosomatidae sp. Apis mellifera USA COLPROT035 Crithidia mellificae 45 - USA COLPROT678 Crithidia bombi 46 - USA COLPROT683 Crithidia bombi - Switzerland COLPROT684 Crithidia bombi Hymenoptera Apidae 47 Bombus lucorum - Switzerland COLPROT679 Crithidia expoeki 48 - USA COLPROT685 Crithidia expoeki - Switzerland COLPROT686 Crithidia expoeki 49 - USA COLPROT687 Crithidia expoeki Heteroptera Gerridae Aquarius remigis Gerris remigis USA COLPROT016 Crithidia flexonema 50 Gentianales Apocynaceae Allamanda cathartica - Suriname COLPROT079 Phytomonas sp. 51 Chamaesyce thymifolia - Brazil COLPROT213 Crithidia fasciculata Euphorbia heterophyla - USA COLPROT076 Herpetomonas muscarum 52 Euphorbia hyssopifolia - Brazil COLPROT081 Herpetomonas muscarum Malpighiales Euphorbiaceae Euphorbia pinea - France COLPROT082 Phytomonas sp. 53 Jathropha macrantha - Peru COLPROT083 Phytomonas serpens 54 Manihot esculenta - Brazil COLPROT011 Phytomonas sp. Sapindales Rutaceae Citrus bergamia - Brazil COLPROT080 Phytomonas serpens 55 Rosales Moraceae Morus sp. - Brazil COLPROT226 Angomonas deanei Brazil COLPROT229 Herpetomonas muscarum 56 Brazil COLPROT174 Phytomonas serpens 57 Brazil COLPROT186 Phytomonas serpens Lycopersicon esculentum Brazil COLPROT187 Phytomonas serpens Solanales Solanaceae Solanum lycopersicum 58 Lycopersicon lycopersicum Brazil COLPROT188 Phytomonas serpens Brazil COLPROT189 Phytomonas serpens 59 Brazil COLPROT247 Phytomonas serpens Brazil COLPROT675 Phytomonas serpens 60 Artiodactyla Suidae Feces from Sus domesticus - Brazil COLPROT682 Herpetomonas isaaci Primate Hominidae Homo sapiens - Peru COLPROT606 Crithidia fasciculata Rodentia Cricetidae Nectomys squamipes - Brazil COLPROT185 Herpetomonas muscarum

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