Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Comparative maxicircle analysis in Trypanosoma species from the LSRM clade highlights patterns in an underexplored lineage

  • Fanny Rusman,

    Roles Data curation, Formal analysis, Methodology, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Valeria Aramayo,

    Roles Formal analysis

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Noelia Floridia-Yapur,

    Roles Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Anahí Guadalupe Díaz,

    Roles Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Tatiana Ponce,

    Roles Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Soledad Hodi,

    Roles Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Juan José Aguirre,

    Roles Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Gonzalo Greif,

    Roles Methodology

    Affiliation Laboratorio de Interacciones Hospedero-Patógeno - UBM, Institut Pasteur de Montevideo, Montevideo, Uruguay,

    ⨯
  • Luisa Berná,

    Roles Methodology

    Affiliation Unidad de Bioinformática, Institut Pasteur de Montevideo, Montevideo, Uruguay

    ⨯
  • Carlos Robello,

    Roles Methodology

    Affiliation Laboratorio de Interacciones Hospedero-Patógeno - UBM, Institut Pasteur de Montevideo, Montevideo, Uruguay,

    ⨯
  • Patricio Diosque,

    Roles Supervision, Writing – review & editing

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯
  • Nicolás Tomasini

    Roles Conceptualization, Funding acquisition, Resources, Writing – review & editing

    nicolas.tomasini@conicet.gov.ar

    Affiliation Instituto de Patología Experimental - Dr. Miguel Ángel Basombrío IPE-CONICET, Salta, Argentina

    ⨯

Abstract

Trypanosoma lainsoni, Trypanosoma platydactyli, and Trypanosoma scelopori are kinetoplastid flagellates belonging to the LSRM clade of the genus Trypanosoma. These parasites have a unique mitochondrial DNA structure, the kinetoplast, comprising catenated maxicircles and minicircles. However, genetic information on the kinetoplasts of these species remains unknown. In this study, we assembled and analyzed maxicircles from different isolates of T. lainsoni, T. platydactyli, and T. scelopori to address the current gap in genetic information about the LSRM clade and explore their phylogenetic relationships within the Trypanosoma genus. The maxicircle of T. lainsoni isolate Le29, including the coding and divergent regions, was de novo assembled combining Illumina and Oxford Nanopore Technologies, revealing a length of 49,306 bp. Additional isolates of T. lainsoni (Ca37 and Ca47), as well as T. platydactyli and T. scelopori, were sequenced with Illumina, yielding complete coding regions and partial divergent regions for all. As in other trypanosomes, coding regions exhibited conserved synteny in gene order and RNA editing patterns. We found that mRNA editing by U-insertion was higher in T. lainsoni than in T. cruzi, suggesting a partial loss of editing in the latter. Phylogenetic analyses based on the coding region positioned the LSRM clade alongside the Aquatic clade as one of the most ancestral groups within non-salivarian trypanosomes and supported the ancestral position of the Trypanosoma brucei clade, contrasting with previous reports. Finally, analysis of the maxicircle divergent region suggests a gradual transition from long to short repeat structures in non-salivarian trypanosomes. This study provides the first characterization of the T. lainsoni maxicircle and related LSRM clade species. These findings provide new insights into the ancestral relationships within the group, the evolution of the divergent region of the maxicircles and propose RNA editing has been evolving within the genus.

Citation: Rusman F, Aramayo V, Floridia-Yapur N, Díaz AG, Ponce T, Hodi S, et al. (2025) Comparative maxicircle analysis in Trypanosoma species from the LSRM clade highlights patterns in an underexplored lineage. PLoS One 20(9): e0332749. https://doi.org/10.1371/journal.pone.0332749

Editor: Vyacheslav Yurchenko, University of Ostrava: Ostravska univerzita, CZECHIA

Received: March 28, 2025; Accepted: September 3, 2025; Published: September 22, 2025

Copyright: © 2025 Rusman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data for this study are publicly available from the NCBI’s GenBank (https://www.ncbi.nlm.nih.gov/genbank) repository (PP751618) and BioProject (https://www.ncbi.nlm.nih.gov/bioproject) repository (PRJNA1106468).

Funding: This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, https://www.conicet.gov.ar) and the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, https://www.argentina.gob.ar/ciencia/agencia), award number: PICT2019-02855 to NT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Trypanosomes constitute a diverse group of protozoan parasites found within the genus Trypanosoma belonging to the higher class Kinetoplastida [1,2]. Trypanosomes conform a monophyletic clade composed by species able to infect all classes of vertebrate hosts, exhibiting various morphological forms throughout their life cycle, and being transmitted by blood-sucking invertebrates [3,4]. Some studies have explored the intricate relationships among members of the Trypanosoma genus [5,6]. The most taxon-rich phylogenetic trees were constructed based on alignments of 18S rDNA and gGAPDH genes and have shed light on certain relationships within the genus, delineating almost ten well-supported clades within it [7–10]. Despite these advances, controversies persist regarding the evolutionary relationships among major clades, particularly the placement of the Aquatic clade and its relation to terrestrial lineages. The Aquatic clade includes Trypanosoma species that infect aquatic vertebrates, such as fish, amphibians, turtles, and platypus. These species are typically transmitted by leeches and sand flies [7–10]. Some studies suggest the Aquatic clade occupies a basal position within the genus, supported by specific phylogenetic reconstructions [11–14]. However, this interpretation is controversial as other analyses using alternative methods suggest its position is not consistently resolved across datasets [10]. Similarly, the placement of the T. brucei clade remains a subject of debate. While some phylogenetic studies position it as an ancestral lineage within the genus [10], contrasting evidence supports its classification as a more derived group, reflecting specific evolutionary adaptations [4,12,14–16]. Furthermore, the Trypanosoma pestanai and LSRM (lizard-snake-rodent-marsupial) clades illustrate some of the ongoing challenges in resolving phylogenetic relationships, as their precise evolutionary positions relative to other terrestrial lineages remain controversial [10,17,18]. Despite these efforts, a clear understanding of the evolutionary relationships between different clades remains elusive, highlighting the need for alternative genetic markers, to clarify these complex phylogenetic patterns. However, by correlating the hosts of trypanosomatids with phylogenetic trees, significant associations were uncovered between clades and specific hosts [11].

Among the main clades of trypanosomes, members of the LSRM clade are distinguished by their close association with their hosts [11]. This clade includes species such as Trypanosoma platydactyli that has been identified in lizards, such as geckoes (Tarentola mauritanica) in Italy and France [19] and Trypanosoma scelopori that was originally described from the North American western fence lizard (Sceloporus occidentalis) [20]. It is important to note that these clade-host associations are not exclusive and may change as new studies emerge, as demonstrated by the discovery of Trypanosoma lainsoni, which was first isolated from a rodent (Mesomys hispidus) in the Amazon Forest (Amazonas state, Brazil) [21]. Its geographical range was later extended to the Argentine Chaco (Parque Nacional Copo), and its host record also expanded, as it was isolated for the first time from a feline (Leopardus geoffroyi) [18]. Recently, the list of non-volant small mammals infected by T. lainsoni in Brazil (Minas Gerais state, Brazil) was expanded [22]. While various characteristics of T. lainsoni have been described, critical aspects such as its vector(s), life cycle, pathogenicity, and potential infection of other hosts or reservoirs remain elusive.

Trypanosomes, like other kinetoplastids, exhibit distinctive features such as a unique mitochondrial DNA structure known as the kinetoplast (kDNA). The kDNA is composed of two classes of circular DNA molecules that are concatenated: minicircles and maxicircles [23–26]. Minicircles exhibit sizes ranging from 0.33 to 10 kb depending on species and are found in tens of thousands of copies per network [23–25,27,28]. Meanwhile, maxicircles which are equivalent to the mitochondrial genome of other eukaryotes exhibit species-specific sizes ranging from 20 to 64 kb and are found in dozens of copies per network [26,29–33]. Maxicircles are characterized by a coding region (CR) that encodes components of the respiratory chain, and a divergent region (DR) composed of a short repetitive region and a long repetitive region, both of unknown functions [6,29–32,34–36]. This region is challenging to sequence and assemble due to its repetitive nature and length variability across different species [6,29,37]. Although comparative studies have identified common patterns in the organization of the DR, its functional role remains largely unknown [34]. Whereas the DR has been proposed as a regulator of gene expression, or a potential replication origin, topoisomerase II binding sites, and transcripts of varying lengths within this genomic region suggests a more intricate functional role than previously hypothesized [38]. Despite these findings, the evolution of the maxicircle remains elusive. The role of the DR and its repetitive nature are not fully elucidated, and most CR genes require extensive RNA editing for functionality. The origin and diversification of this process within Trypanosoma remain largely unexplored, as do the evolutionary pressures shaping the DR. Clarifying these aspects is key to understanding the evolution of mitochondrial features in the genus.

The CR of the maxicircle is composed of several genes, most of them are typical protein-coding genes [30,39,40]. Of the eighteen protein-coding genes in the maxicircles, twelve are encoded as cryptogenes and lack functional open reading frames [41]. These transcripts become functional only after post-transcriptional processing—specifically, RNA editing—which involves the insertion and deletion of uridine residues [41–43]. The process is guided by gRNAs mostly encoded by the minicircles [44–47].

Here we provide the first characterization of maxicircle sequences within the LSRM clade. Using a hybrid sequencing approach, we successfully assembled and annotated the maxicircle of T. lainsoni. In addition, we retrieved raw sequencing data from public repositories and performed de novo assembly and annotation of the maxicircles of T. platydactyli and T. scelopori, which had not been previously analyzed despite the availability of their genomic datasets [48]. Phylogenetic analyses placed the LSRM clade as one of the most ancestral lineages among non-salivarian trypanosomes, confirming its basal position alongside the Aquatic clade. These findings also support the ancestral position of the T. brucei clade and provide new insights into evolutionary dynamics of maxicircles and RNA editing within the Trypanosoma genus.

Materials and methods

Trypanosoma lainsoni isolates

For this study, three isolates of T. lainsoni were analyzed, obtained from different hosts, all sourced from Parque Nacional Copo, Argentina. The three isolates were previously identified as T. lainsoni in a prior study through the amplification of 18S and gGAPDH genes [18]. Isolate Le29 was collected from Leopardus geoffroyi, while isolates Ca37 and Ca47 were obtained from Calomys sp. The isolates were cultivated in a biphasic medium, comprising a solid phase (4% agar supplemented with rabbit blood) and liver infusion-tryptose (LIT) medium enriched with 20% fetal bovine serum, hemin (20 µg/mL), penicillin (100 IU), and streptomycin (100 µg/mL). Cultures were maintained under agitation at 25°C. Parasites in the exponential growth phase were harvested through centrifugation (800 xg, 10 min, 4°C) for subsequent DNA extraction. Genomic DNA was extracted using a commercial kit (Quick-DNA MiniPrep, Zymo) following the manufacturer instructions. DNA quality and quantity were assessed by measuring the absorbance ratio at 260/280 nm and the absorbance at 260 nm using Nanodrop (Thermo Fisher Scientific, USA). Genomic DNA integrity was confirmed through electrophoresis on a 0.8% agarose gel.

Deep sequencing

The whole genome libraries of T. lainsoni isolates Le29, Ca37 and Ca47 were sequenced on an Illumina NovaSeq (Illumina, San Diego, USA) with a 150 bp paired-end read approach performed by a sequencing service (Novogene, USA), obtaining a total of 41 million paired-end reads for Le29, 35 million for Ca37, and 43 million for Ca47. Additionally, a whole genome library was prepared for Le29 with the SQK-NBD112–24 Kit (Oxford Nanopore Technologies, UK) according to the manufacturer instructions and sequenced using a MinION Mk1C platform (Oxford Nanopore Technologies, UK). The library was run for 48 hours in a R10.4 FlowCell (FLO-MIN112, Oxford Nanopore Technologies, UK) starting from 1 μg of total genomic DNA, yielding a total of 45,397 reads. Basecalling of ONT reads was performed using Guppy v6.1.5 (Oxford Nanopore Technologies, UK) in high-accuracy mode, executed through MinKNOW v22.05.8.

Preprocessing reads

The analyses were carried out on Galaxy (https://nanopore.usegalaxy.eu/). The first step involved quality control for both ONT and Illumina reads using different strategies. Long reads (ONT) were assessed using NanoStat [49], while Illumina reads were evaluated using FastQC [50] and Falco [51]. Illumina reads for T. lainsoni isolates, T. platydactyli (SRR24223604), and T. scelopori (SRR24223650) were trimmed and quality-filtered using Trimmomatic (version 0.38.01) [52] with the following parameters: LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:20 MINLEN: 50.

Maxicircle assembly and gene annotation

A de novo hybrid assembly was generated using MaSuRCA (v4.1.4) [53], combining ONT long reads and Illumina paired-end short reads from the Le29 isolate, with the specific aim of recovering the mitochondrial maxicircle. To identify the maxicircle within the assembly, BLASTn searches were performed using known maxicircle sequences (S1 Table) as queries. This search yielded a single contig with high similarity to reference maxicircles, hereafter referred to as the maxicircle-like contig. This contig was manually reordered to place the 12S rDNA gene at the starting coordinate, in accordance with the orientation commonly used for maxicircle sequence reports. The reordered sequence was then aligned with representative maxicircle sequences (S1 Table) using MEGA7.0 [54] to assess gene collinearity and support subsequent annotation and comparative analyses. Subsequently, a two-step polishing strategy was applied. First, ONT reads were mapped with minimap2 (v2.28) [55] to the reordered contig, which served as the reference for Medaka (v1.7.2) (https://github.com/nanoporetech/medaka) polishing using the appropriate model for R10.4.1 chemistry and high-accuracy basecalling. Second, quality-trimmed Illumina reads were mapped with BWA-MEM2 (v2.2.1) [56] to the Medaka-polished contig, which served as the reference for Pilon (v1.20.1) [57] to correct any residual base-level errors. The final maxicircle sequence obtained after this step was used in all downstream analyses. To validate the integrity and continuity of the conserved region (CR) obtained from the hybrid assembly, we also performed an independent de novo assembly using NOVOPlasty (see below). This independent reconstruction served as confirmation of the structure recovered by the hybrid strategy. Finally, to assess sequencing depth and assembly accuracy, ONT and quality-filtered Illumina reads were independently mapped to the polished maxicircle sequence using minimap2 (v2.28) and BWA-MEM2 (v2.2.1), respectively, using default parameters. The resulting BAM files were sorted with Samtools sort (v2.0.7) [58] and duplicate reads were marked using Picard MarkDuplicates (v3.1.1.0). Per-base depth and overall coverage were calculated with Samtools (v1.21) [58]. Assembly consensus quality (QV) was estimated from mismatch and indel statistics obtained with Qualimap BamQC (v2.3.0) [59] for the entire maxicircle, and in particular for the coding region, and coverage profiles were visualized with Matplotlib [60].

Annotation of maxicircle genes was performed manually based on multiple sequence alignments (MSA) generated with MEGA7.0. Our assembled maxicircle sequences were aligned to annotated maxicircle references from T. cruzi- Esmeraldo (DQ343646.1), T. marinkellei (KC427240.1), T. cruzi- Sylvio (FJ20396.1), T. equiperdum (EU185800.1), T. vivax (KM386508.1), T. musculi (KT368148.1), T. lewisi (OM000219.1), T. copemani (MG948557.1), and L. tarentolae (NC000894.1). Gene boundaries (start and stop codons) for non-edited genes were determined based on conserved synteny and ORFs that translated into coherent protein sequences.

For extensively edited genes, inferred mature mRNA sequences were used to define gene limits and validate annotation. The RNA editing patterns of T. lainsoni maxicircle genes were inferred by manually reconstructing edited mRNA sequences for each gene, using the editing profiles of orthologous genes in T. cruzi as reference. Uridine insertions and deletions were introduced into unedited sequences to generate functional open reading frames, following the approach described previously in [40,61–63]. The total number of U-indels per gene was recorded to estimate the extent of editing. GC content was calculated separately for each gene and used only as a complementary metric to describe GC variation across genes, since pan-edited genes have been associated with elevated GC content in previous studies [30,36,64], without being used to infer editing. The GC percentage and the GC skew were analyzed at a 50 bp window and visually represented with the annotated maxicircle genome using pyCirclize (https://github.com/moshi4/pyCirclize). Additionally, AT percentage and AT skew were calculated for the complete maxicircle sequences of Le29 and T. cruzi-Dm28c using a 100 bp sliding window, to address the existence of an AT-rich region. The Dm28c sequence was selected for comparison because it represents a fully assembled maxicircle, including both the conserved and divergent regions, and has been previously reported to contain an AT-rich region [29].

A dot plot graph illustrating the self-alignment of the T. lainsoni maxicircle sequence was generated using YASS webserver (https://bioinfo.univ-lille.fr/yass/yass.php) with default parameters. Additionally, dot plot graphs were created to compare the T. lainsoni maxicircle CR sequence with that of five other Trypanosomatidae species.

Assembly of the maxicircle coding region of Trypanosoma lainsoni isolates Ca37 and Ca47, Trypanosoma platydactyli and Trypanosoma scelopori

For T. lainsoni isolates Ca37 and Ca47, T. platydactyli (NCBI-SRA: SRR24223604) and T. scelopori (NCBI-SRA: SRR24223650) [48] the respective coding regions of the maxicircle were assembled using NOVOPlasty (version 4.3.1) [65]. The parameters employed were as follows: the trimmed paired-end reads (R1 and R2), generated by Trimmomatic, were used as input for the assembly. The seed sequence used for assembly was obtained from the T. cruzi (Sylvio) maxicircle sequence (NCBI code: FJ203996.1), utilizing the non-edited gene cytochrome oxidase subunit 1 (COI) as the initial point for assembly. Other parameters were configured as follows: The Illumina platform was specified with a read length of 150 bases and a total insert size of 300 bases for paired-end reads. The assembly parameters were designated for mitochondrial assembly, anticipating a genome size range of 12,000–52,000 bases. It is worth noting that the program reconstructs the complete CR along with DR sequences flanking it. However, due to the highly repetitive nature of the DR, these sequences are collapsed by the program, preventing the resolution of its full extent.

To assess structural conservation, dot plot graphs using YASS with default parameters were generated to compare the CR of T. lainsoni (Le29, obtained from the hybrid assembly) with those obtained for Ca47, Ca37, T. platydactyli and T. scelopori. Additionally, self-alignments of Le29 and Ca47 (NOVOPlasty obtained contig) were performed to explore internal repetition patterns within the DR, particularly at its junction with the CR.

Divergent region analyses

Dot plot graphs were generated using YASS webserver with default parameters to compare the complete T. lainsoni maxicircle sequence, including its divergent region, with those of T. cruzi (MW421590.1), T. musculi (OM000218.1), T. brucei brucei (M94286.1), T. lewisi (OM000219.1), and T. vivax (MT090068.1). Outputs from the YASS webserver were used to identify palindromic sequences within the DR of T. lainsoni. Upon completing the search, palindromic sequences were extracted from the YASS alignment files. Subsequently, the nucleotide sequences were aligned using MEGA7.0.

Phylogenetic analysis

The maxicircle coding regions of LSRM clade species were compared with the available trypanosome maxicircles sequences in Nucleotide (NCBI) (S1 Table). The entire CR of the maxicircles were aligned and trimmed using MEGA7.0. Phylogenetic analysis was performed by the maximum likelihood (ML) method, using MEGA7.0. The best substitution model was selected using the AIC criterion. The ML tree was generated with 1,000 bootstrap replicates. In addition, the tree was edited using FigTree (version 1.4.4) (http://tree.bio.ed.ac.uk/software/figtree/).

Results

Assembly and annotation of kDNA maxicircles in the LSRM clade

Genomic DNA from T. lainsoni Le29 isolate was sequenced using both the Illumina and ONT platforms to obtain the maxicircle sequence. A de novo hybrid assembly was generated with MaSuRCA using the complete set of reads from both platforms. The maxicircle was identified by performing BLASTn searches against the hybrid MaSuRCA assembly contigs, using available maxicircle sequences of other Trypanosoma species as queries (Supplementary File 3). This approach allowed the detection of a 49,255 bp maxicircle-like contig containing the complete coding region flanked by the divergent region. The maxicircle-like contig was manually reordered at the 12S gene. The sequence was then polished in two steps: first with Medaka using ONT long reads, and subsequently with Pilon using quality-filtered Illumina short reads. The final curated T. lainsoni maxicircle sequence was 49,306 bp in length, comprising a 15,340 bp coding region and a 33,966 bp divergent region (Fig 1). Notably, the T. lainsoni maxicircle does not present an AT-rich region (S1 Fig). To further evaluate the structure of the CR, an independent de novo assembly was carried out using NOVOPlasty with Illumina reads. The resulting contig corresponded to the expected organization of the CR and showed no inconsistencies with the curated maxicircle sequence. Sequencing coverage across the ~ 49 kb maxicircle was evaluated independently for both platforms after polishing. Illumina reads provided high-quality coverage across the whole molecule (Fig 1B). ONT reads, while fewer in number, included long reads that spanned the entire structure and supported the continuity of both conserved and divergent regions. Summary statistics on read quality (S2 Fig and S3 Fig) and length distribution for both platforms are provided in S2 Table, and S3 Table. For Illumina reads mapped to the complete maxicircle, the mean coverage was ~ 472.9× with a consensus quality value (QV) of ~27.2 (~99.81% accuracy), while ONT reads achieved a mean coverage of ~10.48 × . For the coding region, Illumina reads yielded a mean coverage of ~418.6 × , supporting both the structural and nucleotide-level fidelity of the assembly.

thumbnail
Download:
Fig 1. Trypanosoma lainsoni (Le29) maxicircle schematic representation.

(A) Annotated genes are displayed in the outer circle in light orange (outer + strand and inner – strand) and the divergent region in orange. The GC skew with a window size of 50 bp is illustrated in violet. The GC content (also with a window size of 50 bp) is illustrated in light blue. (B) Depth of coverage was calculated for each position along the maxicircle using long-read (Nanopore; upper panel) and short-read (Illumina; lower panel) sequencing data. Nanopore reads were aligned using minimap2 and Illumina reads using BWA-MEM2. The x-axis represents the nucleotide positions along the maxicircle (in base pairs) and the y-axis indicates the number of reads mapped to each position (read depth) calculated using Samtools and plotted with Matplotlib. Each panel shows the coverage profile corresponding to the sequencing platform.

https://doi.org/10.1371/journal.pone.0332749.g001

The nucleotide sequences and gene arrangement within the CR of the maxicircle were highly conserved across trypanosomatids, while allowed for some species-specific variations. This conservation provided a framework for the annotation of 20 genes in the kDNA maxicircles from the three species (Table 1), including the start and end positions of these genes and their length comparison with T. cruzi. Additionally, the maxicircles from two isolates of T. lainsoni (Ca37 and Ca47), as well as from T. platydactyli and T. scelopori, were assembled (S1 File) and annotated. Furthermore, CR regions of these isolates were analyzed and presented in S4 Fig. These analyses revealed a high degree of sequence conservation across the CR of the LSRM clade, with remarkable preservation in gene order and overall size. However, specific differences were observed. For instance, MURF5 showed notable variation in length, being 321 bp in T. lainsoni, 252 bp in T. scelopori, and 213 bp in T. platydactyli, while significantly shorter in T. cruzi at 148 bp based on NCBI annotations. Notably, previous studies have reported a longer MURF5 sequence of 261–267 bp for T. cruzi [40]. Similarly, in 8 out of the 9 extensively edited genes, the length is shorter in T. lainsoni compared to T. cruzi, primarily due to a lower thymine content in T. lainsoni. These findings are summarized in Table 1.

thumbnail
Download:
Table 1. Comparison of gene positions and lengths among LSRM clade species and T. cruzi.

https://doi.org/10.1371/journal.pone.0332749.t001

The overall architecture of the T. lainsoni maxicircle, including annotated genes of the CR, DR, GC skew and content across the whole maxicircle is illustrated in Fig 1.

Eighteen protein-coding genes were identified and classified based on the extent of RNA editing required to produce functional transcripts (Table 1). To complement this annotation, GC skew analysis revealed a strong G-rich bias, particularly in genes subjected to extensive RNA editing (S5 Fig). The corresponding inferred edited mRNAs are available in S2 File. Finally, the number of insertions required to obtain the open reading frame was analyzed, revealing that in seven out of nine extensively edited genes, T. lainsoni requires a higher number of uridine insertions compared to T. cruzi (Fig 2A). A specific example is shown for the gene ND9 in Fig 2B, which illustrates the increased editing complexity in T. lainsoni.

thumbnail
Download:
Fig 2. Differences in inferred insertion events between Trypanosoma lainsoni and Trypanosoma cruzi.

(A) Bar plot showing the difference in the number of insertion events inferred from multiple sequence alignments between T. lainsoni Le29 and T. cruzi Sylvio X10 across nine mitochondrial maxicircle genes. Insertions were estimated as alignment positions where the edited transcript contains a nucleotide aligned to a gap in the genomic sequence. Values represent Δ insertions (T. lainsoni – T. cruzi). A value > 0 indicates more U-insertions in T. lainsoni than in T. cruzi. (B) Schematic representation of inferred U-insertion sites for the ND9 gene, based on the alignment between T. cruzi Sylvio X10 and T. lainsoni Le29. The fully edited mRNA sequence of T. cruzi Sylvio X10 is shown in blue (top). Asterisks indicate positions with U-insertions in the edited transcript relative to the genomic sequences: light purple for T. cruzi Sylvio X10 and orange for T. lainsoni Le29. The yellow box highlights a region that was not evaluated due to sequence differences between T. cruzi and T. lainsoni.

https://doi.org/10.1371/journal.pone.0332749.g002

Ancestral position of T. brucei, LSRM and aquatic clades within Trypanosoma

The phylogenetic analysis of the CR revealed a robust clustering of trypanosome species into eight distinct clades within the genus Trypanosoma. These clades include the basal T. brucei clade, which predominantly comprises trypanosomes infecting African mammals; the Aquatic clade, the LSRM clade; the Crocodilian clade, including trypanosomes infecting crocodilians and alligators; the T. theileri clade, with species infecting marsupials and placental mammals; the T. pestanai clade, represented by T. copemani, a trypanosome infecting Australian marsupials, and T. caninum, which infects Brazilian dogs; the T. lewisi clade, predominantly found in rodents; and the T. cruzi clade, which includes trypanosomes infecting a variety of mammals with a global distribution.

Within the phylogenetic tree, T. brucei clade emerges as the basal lineage, diverging first among the Trypanosoma species, indicating its ancestral position. Following this, two main lineages branch out: one comprising the aquatic trypanosomes, and the other including various terrestrial clades. The LSRM clade is positioned as one of the most ancestral groups alongside the aquatic clade, supported by a bootstrap value of 100%. Furthermore, isolates Le29, Ca37, and Ca47 formed a well-defined clade, indicating their close phylogenetic relationship (Fig 3).

thumbnail
Download:
Fig 3. Phylogenetic analysis of the Trypanosomatidae species using the whole maxicircle coding regions.

Phylogenetic tree is performed based on Maximum likelihood method based on the General Time Reversible (GTR) substitution model with discrete Gamma distribution (+G) and Invariant Sites (+ I) parameters with 1,000 bootstrap replicates with the respective bootstrap confidences indicated at branch points. The sequences used in the analysis and their corresponding GenBank accession numbers are as follows: T. cruzi Esmeraldo (DQ343646.1), T. cruzi CL Brener (DQ343645.1), T. cruzi Sylvio (FJ203996.1), T. marinkellei (KC427240.1), T. conorhini (MKKU01000412.1), T. lewisi (OM000219.1), T. musculi (KT368148.1), T. copemani (MG948557.1), T. grayi (OM049542.1), T. congolense (GCA_003013265.1), T. brucei brucei (MK584625.1), Leishmania tarentolae (NC000894.1), T. rangeli (KJ803830.1), T. theileri (GCF_002087225.1), T. brucei rhodesiense (OM049543.1), T. mega (GCA_030849715.1), T. boissoni (GCA_030849725.1), T. melophagium (GCA_022059095.1), T. equiperdum (CZPT020000280.1), T. caninum (GCA_036321205.1).

https://doi.org/10.1371/journal.pone.0332749.g003

LSRM clade sheds light on divergent region evolution

Upon sequence analysis of the maxicircle DR, various tandem repeats were identified. Dot plots of maxicircles for six different trypanosomatid species, including T. lainsoni, are shown in Fig 4. The plots show variants of DR organization, highlighting the unique features across species. Architectural features of the DR sequence revealed several common traits: repetitive elements were consistently arranged in a head-to-tail manner, and within DR sequences, a short repeat element (also known as P5 or DRI) and a long repeat (also named P12 or DRII) could be identified. These regions consist of repeated sequences characterized by distinct differences in repeat lengths, structures, and repetition patterns. The short repeat constitutes a tandem repeat with a large period but a limited number of repetitions. However, as shown in Fig 4A, T. lainsoni lacks a clearly differentiated short repeat region, unlike T. cruzi or T. musculi, or T. lewisi (Fig 4B, C, E).

thumbnail
Download:
Fig 4. Dot plot of six whole maxicircle sequences.

Dot plots were generated using YASS of self-self sequence of the six species analyzed. (A) T. lainsoni, (B) T. cruzi-Dm28c, (C) T. musculi, (D) T. brucei brucei, (E) T. lewisi, (F) T. vivax. The color-coded regions on the right represent key structural features of the maxicircle: blue represents the conserved region (CR). Orange and purple correspond to the divergent region, which includes the short repeats and long repeats, respectively.

https://doi.org/10.1371/journal.pone.0332749.g004

In contrast, a long repeat comprises highly repetitive units with a small period and is organized into repeat arrays of variable length. This organization was also observed in assemblies generated independently with NOVOPlasty for Le29 and Ca47 (S6 Fig), confirming the presence of tandem arrays with short repeat units in multiple T. lainsoni isolates. The YASS dot plot analysis of T. platydactyli and T. scelopori reveals an apparent progressive reduction in sequence identity within the long repeat array in relation to T. lainsoni (Fig 5). In T. platydactyli, an emerging short repeat is observed with some regions that maintain some degree of sequence identity with elements of the long repeat. Instead, in T. scelopori, a clearly differentiated short repetitive region becomes evident. Considering the basal position of the LSRM in relation to other terrestrial trypanosomes in the phylogenetic tree (Fig 3) with a short repeat region, these findings indicate that the short repetitive region originated from a unit of the long repeat array through the gradual loss of sequence identity and the posterior period increase.

thumbnail
Download:
Fig 5. Dot plots of the CR-DR boundary in maxicircles from the LSRM clade.

Dot plots were generated using YASS to compare the self-self sequences of the analyzed LSRM species, allowing the visualization of both conserved and divergent regions. (A) T. lainsoni (Le29), (B) T. platydactyli, (C) T. scelopori. The color-coded regions on the right represent key structural features of the maxicircle: blue represents the conserved region (CR). In A, a magnified view of the 5’ start of the divergent region is shown to highlight the internal repeat structure. The sparser pattern in this plot reflects the zoomed-in scale and the complex organization of repeats. Orange and purple correspond to the divergent region, which includes the short repeats and long repeats, respectively.

https://doi.org/10.1371/journal.pone.0332749.g005

The arrays in the long repeat region are frequently interspersed with repeat units of a different type, known as I-elements. These elements were originally described as conserved palindromic sequences in the variable region of the T. cruzi maxicircle [36], and later formalized as “I-elements” in comparative studies of kinetoplastid, where they mark the boundaries of repeat arrays within the divergent region. These I-elements influence the nature of large repeats; however, in some species, this element is absent (Fig 4D) [34]. For T. lainsoni the repetitive element I, had a length of about 390 bp that remarks the arrays borders (S7 Fig A). The cross-structure (S7 Fig B) present within the conserved element is due to a match to the reverse complement that forms an imperfect palindrome. Three types of palindromes were detected, differing in length, location, and number of occurrences relative to element I (S7 Fig B). Palindrome type I (53 bp) was located at the first occurrence of element I in the divergent region. Palindrome type II (39 bp) was found in the second repetition of element I. Finally, palindrome type III (65 bp) was observed in the remaining repetitions (S7 Fig C). Despite their varying lengths and sequences, palindromes share a 39 bp core that contains a highly conserved A5C element (S7 Fig D).

Discussion

Here, we successfully assembled the T. lainsoni Le29 maxicircle through a de novo hybrid assembly combining long-read ONT and short-read Illumina sequences. Additionally, partial maxicircle sequences were assembled for two other T. lainsoni isolates, as well as for T. platydactyli, and T. scelopori. To the best of our knowledge, these represent the first descriptions of maxicircle sequences for these species and within the LSRM clade. We also analyzed intraspecific diversity and phylogenetic relationships with other trypanosomes, providing insights into the evolutionary relationships among clades within the genus Trypanosoma and the evolution of the divergent region of maxicircles. In addition, these sequences will help to better describe T. lainsoni distribution and host range by designing PCR specific primers to directly detect it on samples without requiring sequencing such as previous molecular methods that identified this parasite.

The estimated size of the maxicircle is 49 kb in the Le29 isolate, comprising two distinct regions: a CR of 15 kb and a DR of 33 kb (Fig 1). The maxicircle assembled with MaSuRCA for Le29 encompassed an almost complete sequence and is close to sizes reported for maxicircles of other trypanosomatid species (∼20–64 kb) [26], in particular those reported for some Trypanosoma cruzi strains [27,29]. The robustness of the final maxicircle sequence was supported by sequential polishing with ONT and Illumina reads, which corrected residual errors and reinforced confidence in the structural and nucleotide-level accuracy of the assembly. This is further substantiated by the high mean coverage (~472.9×) and a consensus quality value (QV) of ~27.2 (~99.81% accuracy) from read-to-assembly alignments, indicating both depth and quality sufficient to ensure reliable reconstruction. In addition, to further support the structural accuracy of the conserved region, a separate de novo assembly was performed using NOVOPlasty, confirming consistency in gene order within this region (Supplementary File 1). The primary size variation between the T. lainsoni maxicircle described in this study and those of other kinetoplastids can be attributed to differences within the DR.

Notably, the size of the CR and gene order is similar to those reported for T. brucei, T. cruzi and T. lewisi, which are approximately 15 kb in length [27,29,30,33,37]. Interestingly, extensively edited genes ATPase6, ND9, ND8, ND7, COIII, CR3, CR4, ND3 and RPS12 showed variations in size when compared to genes reported for T. cruzi (Table 1). It is predicted that these size discrepancies will be corrected through the insertion and deletion of uridine residues in the primary transcripts via the process of RNA editing. However, it is notable that in 8 of the 9 extensively edited genes, the size is smaller in T. lainsoni than in T. cruzi. These size differences are primarily due to a lower T content in T. lainsoni. In addition, more U-insertions were predicted for seven out of nine extensively edited genes in T. lainsoni than in T. cruzi to obtain the complete open reading frame. As T. lainsoni is positioned more basally than T. cruzi in the trypanosome phylogenetic tree, this finding supports the hypothesis that RNA editing is being partially lost during the evolution of trypanosomes, likely through a mechanism distinct from the retroposition of already edited mRNAs [66,67]. In relation to these findings, a study by [64] showed that the maxicircles of salivarian trypanosomes have high GC content and that pan-edited genes are shorter, thus requiring greater RNA editing than non-salivarian trypanosomes. However, while [64] associates these patterns with the recent evolutionary divergence in Salivarian and proposed that mRNA editing is not always “on the way out”, our results suggests a contrasting perspective: mRNA editing may be undergoing a partial reduction during the evolution of Trypanosoma.

In this regard, the CR sequence has proven to be a valuable taxonomic marker for phylogenetic analyses [68,69], allowing us to better resolve the evolutionary relationships between the LSRM, T. brucei clade and other trypanosomes. Our analysis built a robust phylogeny with high bootstrap support, effectively resolving major clades within Trypanosoma. Notably, the phylogenetic analyses positioned the T. brucei clade as basal and the LSRM clade alongside the Aquatic clade as one of the most ancestral groups within non-salivarian trypanosomes (Fig 3). This finding highlights the value of using maxicircle CR sequences to clarify evolutionary relationships that remain ambiguous with other currently used markers, offering a faster mutation rate and providing a greater number of phylogenetically informative sites [64,70]. While our findings contribute to a more comprehensive understanding of Trypanosoma clades relationships, further research including more species is needed to finally confirm relationships among clades.

Lastly, the discussion surrounding the DR of the kinetoplast maxicircle has long been debated. Initially characterized as a variable and non-coding region, the structural diversity observed in the DR across different species added complexity to its nature. The distinct differences in DR structures among various species have led to questions about its potential functional significance. Our analyses uncovered a 33,966 bp repetitive region comprising the DR of the T. lainsoni maxicircle, which lacks an AT-rich region or a clearly differentiated short repeat region (S1 FigA), as reported in other trypanosomes such as T. cruzi [29]. Instead, T. lainsoni appears to have one of the elements from the long repeat array with regions where it has lost sequence identity with other repeats. Notably, this architecture was consistently recovered not only in the hybrid assembly, but also in the ONT long reads themselves and in independent reconstructions using NOVOPlasty for both Le29 and Ca47, supporting the biological reality and conservation of this structure across isolates (Supplementary File 1, and S6 Fig). This loss of sequence identity is more evident in T. platydactyli, showing the emergence of a short repetitive region and, ultimately, a well-differentiated short repetitive region in T. scelopori. In addition, the short repeat region in T. scelopori has short and long period repeats (only long period repeats are observed in T. cruzi and others), which suggest an increase in the repetition period, possibly by reparation based on homology or slippage during replication. These results suggest a potential mechanism for the origin of the short repetitive region from the long repeat arrays in trypanosomes. A recent study by Gerasimov et al. [34] proposed that the divergent region of the maxicircle is organized into two distinct compartments, P5 and P12, composed of repeats of different lengths and periodicities. This model, developed primarily from analyses of Leishmania and a subset of Trypanosoma cruzi strains, provides a useful conceptual framework for exploring repeat architecture in the DR. In our study, the distinction between “short” and “long” repeats observed across several Trypanosoma species is broadly compatible with this compartmentalization.

Although the mechanisms underlying the short repetitive region changes remain unknown, it has been suggested that repetitive region sequence arrays may contain binding sites for specific transcription factors, DNA maintenance proteins or may serve as sites for initiating maxicircle replication and transcription [30]. Moreover, an intriguing conserved sequence element of ~390 bp is present in the divergent region of the T. lainsoni with imperfect palindromes (type I, II and III) containing a core palindromic sequence of 39 bp within the element. Palindrome structures have previously been identified in the maxicircles of other trypanosomatids [29–31,36]. These sequences have been reported in a wide range of genomes [71], most in mitochondrial DNA [72,73], as well as in chloroplasts genomes [74]. In eukaryotes, palindromic sequences have been associated with a diversity of functions. They have been linked to genetic instability, genome stability, and replication processes [75]. In addition, palindromes containing an A5C-element have been proposed as potential recognition sites for the binding of transcription factors or initiation of transcription [31], the exact biological function of these elements has yet to be fully explored.

In conclusion, this study presents the first description of the kDNA maxicircles in the LSRM clade. Our findings revealed that the coding region of the T. lainsoni maxicircle retains a conserved gene order analogous to that of other closely related trypanosomatids, with an anticipated RNA editing pattern closely resembling that of T. cruzi. However, T. cruzi requires relatively few U-insertions, supporting the idea that RNA editing may be undergoing a gradual reduction in non-salivarian Trypanosoma lineages. This study also supports the T. brucei clade as the most ancestral and shows that LSRM and aquatic trypanosomes are ancestral to non-salivarian (terrestrial) trypanosomes. Furthermore, the structure of the DR in LSRM species suggests a possible evolutionary trajectory leading to the emergence of short repeat elements. Finally, we described the presence of a palindrome containing an A5C within the T. lainsoni DR, suggesting its functional conservation across trypanosomes.

Supporting information

S1 Fig. AT skew graphs for the whole maxicircle sequence in T. lainsoni (A) and T. cruzi (B).

AT-rich region in T. cruzi Dm28c is highlighted in yellow. The window size was 100 bp.

https://doi.org/10.1371/journal.pone.0332749.s001

(TIF)

S2 Fig. Per-base Phred quality scores of Illumina reads mapped to the polished T. lainsoni maxicircle.

Quality scores were calculated using Falco, based on Illumina reads previously mapped to the polished maxicircle assembly. Both forward (left) and reverse (right) reads display consistently high Phred scores across most base positions, with values exceeding Q30.

https://doi.org/10.1371/journal.pone.0332749.s002

(TIF)

S3 Fig. Quality and length distribution of ONT reads mapped to the T. lainsoni maxicircle.

Summary plot generated with NanoPlot showing the relationship between read quality and read length for the Oxford Nanopore reads that mapped to the T. lainsoni maxicircle.

https://doi.org/10.1371/journal.pone.0332749.s003

(TIF)

S4 Fig. Dot plot comparisons of the maxicircle coding regions obtained using NOVOPlasty of various Trypanosoma species generated with YASS.

The plots illustrate the sequence similarity within the LSRM clade, comparing T. lainsoni (Le29) with (A) T. platydactyli, (B) T. scelopori, (C) T. lainsoni (Ca37), and (D) T. lainsoni (Ca47). The axes represent sequence positions, highlighting coding regions (green lines) and regions with potential inversions or deletions (red lines). Continuous diagonal lines indicate high sequence similarity without interruptions, whereas breaks or disruptions suggest sequence variation, such as insertions, deletions, or rearrangements.

https://doi.org/10.1371/journal.pone.0332749.s004

(TIF)

S5 Fig. GC percentage graphs for the coding regions of the maxicircle in T. cruzi (A) and T. lainsoni (B).

The x-axis represents the nucleotide position along the maxicircle coding region, and the y-axis represents the percentage of GC content. The window size used was 25 bp. Regions with GC content values exceeding the 40% threshold are indicative of RNA editing. Gray arrows: Ribosomal RNA genes. Magenta arrows: extensively edited genes. Light purple arrows: minor edited genes. Purple arrows: non-edited genes.

https://doi.org/10.1371/journal.pone.0332749.s005

(TIF)

S6 Fig. Dotplot comparison of maxicircle assemblies from T. lainsoni Le29 and Ca47 generated with NOVOPlasty.

YASS alignment between the assembled contigs shows complete conservation of the coding region and a sharp divergence at the transition to the divergent region (DR). The diagonal line confirms high collinearity and sequence identity across the coding block in both isolates, while the alignment break marks the onset of the DR, consistent with the expected maxicircle architecture.

https://doi.org/10.1371/journal.pone.0332749.s006

(TIF)

S7 Fig. Divergent region of T. lainsoni maxicircle.

(A) Dot plot analysis generated with YASS comparing the sequence of T. lainsoni maxicircle to itself, illustrating three palindromic structures: blue arrow for palindrome I, violet arrow for palindrome II, and magenta arrow for palindrome III. (B) Detailed view of the divergent region in the dot plot. (C) Presentation of palindrome sequences, their respective lengths, and their positions in the highlighted example in (B). (D) Core palindromic sequence of 39 bp within the conserved element of the divergent region.

https://doi.org/10.1371/journal.pone.0332749.s007

(TIF)

S1 Table. The maxicircle coding region sequences analyzed in this study.

https://doi.org/10.1371/journal.pone.0332749.s008

(PDF)

S2 Table. ONT reads summary statistics.

https://doi.org/10.1371/journal.pone.0332749.s009

(PDF)

S3 Table. Illumina reads summary statistics.

https://doi.org/10.1371/journal.pone.0332749.s010

(PDF)

S1 File. Le29, Ca37, Ca47, T. platydactyli and T. scelopori maxicircle sequences.

https://doi.org/10.1371/journal.pone.0332749.s011

(ZIP)

S2 File. Edited mRNAs.

https://doi.org/10.1371/journal.pone.0332749.s012

(ZIP)

S3 File. BLASTn results.

https://doi.org/10.1371/journal.pone.0332749.s013

(XLSX)

References

  1. 1. Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, et al. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol. 2021;11(3):200407. pmid:33715388
  2. 2. Yurchenko V, Butenko A, Kostygov AY. Genomics of Trypanosomatidae: Where We Stand and What Needs to Be Done?. Pathogens. 2021;10(9):1124. pmid:34578156
  3. 3. Kaufer A, Ellis J, Stark D, Barratt J. The evolution of trypanosomatid taxonomy. Parasit Vectors. 2017;10(1):287. pmid:28595622
  4. 4. Hamilton PB, Stevens JR, Gaunt MW, Gidley J, Gibson WC. Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA. Int J Parasitol. 2004;34(12):1393–404. pmid:15542100
  5. 5. Kostygov AY, Albanaz ATS, Butenko A, Gerasimov ES, Lukeš J, Yurchenko V. Phylogenetic framework to explore trait evolution in Trypanosomatidae. Trends Parasitol. 2024;40(2):96–9. pmid:38065790
  6. 6. Kaufer A, Stark D, Ellis J. Evolutionary Insight into the Trypanosomatidae Using Alignment-Free Phylogenomics of the Kinetoplast. Pathogens. 2019;8(3):157. pmid:31540520
  7. 7. Hamilton PB, Stevens JR. Classification and phylogeny of Trypanosoma cruzi. American Trypanosomiasis Chagas Disease. Elsevier. 2017. p. 321–44.
  8. 8. Fraga J, Fernández-Calienes A, Montalvo AM, Maes I, Deborggraeve S, Büscher P, et al. Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene. Infect Genet Evol. 2016;43:165–72. pmid:27180897
  9. 9. Stevens JR. Kinetoplastid phylogenetics, with special reference to the evolution of parasitic trypanosomes. Parasite. 2008;15(3):226–32. pmid:18814685
  10. 10. Borges AR, Engstler M, Wolf M. 18S rRNA gene sequence-structure phylogeny of the Trypanosomatida (Kinetoplastea, Euglenozoa) with special reference to Trypanosoma. Eur J Protistol. 2021;81:125824. pmid:34352687
  11. 11. Hamilton PB, Gibson WC, Stevens JR. Patterns of co-evolution between trypanosomes and their hosts deduced from ribosomal RNA and protein-coding gene phylogenies. Mol Phylogenet Evol. 2007;44(1):15–25. pmid:17513135
  12. 12. Acosta I da CL, da Costa AP, Nunes PH, Gondim MFN, Gatti A, Rossi JL Jr, et al. Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp. nov., from Atlantic Rainforest of southeastern Brazil. Parasit Vectors. 2013;6:349. pmid:24330660
  13. 13. Lima L, Espinosa-Álvarez O, Hamilton PB, Neves L, Takata CSA, Campaner M, et al. Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade. Parasit Vectors. 2013;6(1):221. pmid:23915781
  14. 14. Fermino BR, Viola LB, Paiva F, Garcia HA, de Paula CD, Botero-Arias R, et al. The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene. Parasit Vectors. 2013;6(1):313. pmid:24499634
  15. 15. McInnes LM, Gillett A, Ryan UM, Austen J, Campbell RSF, Hanger J, et al. Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala ( Phascolarctos cinereus). Parasitology. 2009;136(8):875–85. pmid:19570316
  16. 16. Cavazzana M Jr, Marcili A, Lima L, da Silva FM, Junqueira ACV, Veludo HH, et al. Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats. Int J Parasitol. 2010;40(3):345–55. pmid:19766649
  17. 17. Koual R, Buysse M, Grillet J, Binetruy F, Ouass S, Sprong H, et al. Phylogenetic evidence for a clade of tick-associated trypanosomes. Parasit Vectors. 2023;16(1):3. pmid:36604731
  18. 18. Díaz AG, Ragone PG, Rusman F, Floridia-Yapur N, Barquez RM, Díaz MM, et al. A Novel Genotype and First Record of Trypanosoma lainsoni in Argentina. Pathogens. 2020;9(9):731. pmid:32899895
  19. 19. Gramiccia M, Gradoni L, Maroli M. Caractérisation enzymatique deTrypanosoma platydactyliCatoulllard, 1909 isolé deSergentomyia minuta minutaRondani, 1843 en Italie. Ann Parasitol Hum Comp. 1989;64(2):154–6.
  20. 20. Telford SR Jr. A review of the trypanosomes from lizards of the family Iguanidae (sensu lato), including the descriptions of five new species, and an evaluation of the effect of host difference upon taxonomic characters of saurian trypanosomes. Syst Parasitol. 1996;34(3):215–37.
  21. 21. Naiff RD, Barrett TV. Trypanosoma (Megatrypanum) lainsoni n. sp. from Mesomys hispidus (Rodentia: Echimyidae) in Brazil: trypomastigotes described from experimentally infected laboratory mice. Parasite. 2013;20:51. pmid:24309069
  22. 22. de Oliveira MM, Ferrando CPR, Gómez-Hernández C, de Oliveira KR, Araújo IAC, Ribeiro PVA, et al. Prevalence of Trypanosoma lainsoni and its effects of parasitism on the health of non-volant small mammals from the Brazilian Cerrado. Parasitol Res. 2023;122(7):1509–18. pmid:37129625
  23. 23. Lukes J, Guilbride DL, Votýpka J, Zíková A, Benne R, Englund PT. Kinetoplast DNA network: evolution of an improbable structure. Eukaryot Cell. 2002;1(4):495–502. pmid:12455998
  24. 24. Michieletto D. Kinetoplast DNA: a polymer physicist’s topological Olympic dream. Nucleic Acids Res. 2025;53(2):gkae1206. pmid:39676656
  25. 25. Diggines B, Whittle S, Yadav I, Holmes EP, Rollins DE, Catley TE, et al. Multiscale topological analysis of kinetoplast DNA via high-resolution AFM. Phys Chem Chem Phys. 2024;26(40):25798–807. pmid:39354753
  26. 26. Herreros-Cabello A, Callejas-Hernández F, Fresno M, Gironès N. Mitochondrial DNA Structure in Trypanosoma cruzi. Pathogens. 2025;14(1):73. pmid:39861034
  27. 27. Callejas-Hernández F, Herreros-Cabello A, Del Moral-Salmoral J, Fresno M, Gironès N. The Complete Mitochondrial DNA of Trypanosoma cruzi: Maxicircles and Minicircles. Front Cell Infect Microbiol. 2021;11:672448. pmid:34268138
  28. 28. Li S-J, Zhang X, Lukeš J, Li B-Q, Wang J-F, Qu L-H, et al. Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi. Nucleic Acids Res. 2020;48(17):9747–61. pmid:32853372
  29. 29. Berná L, Greif G, Pita S, Faral-Tello P, Díaz-Viraqué F, Souza RDCMD, et al. Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex. PLoS Negl Trop Dis. 2021;15(8):e0009719. pmid:34437557
  30. 30. Lin R-H, Lai D-H, Zheng L-L, Wu J, Lukeš J, Hide G, et al. Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen. Parasit Vectors. 2015;8:665. pmid:26715306
  31. 31. Wang J-F, Lin R-H, Zhang X, Hide G, Lun Z-R, Lai D-H. Novel insertions in the mitochondrial maxicircle of Trypanosoma musculi, a mouse trypanosome. Parasitology. 2022;149(12):1546–55. pmid:35924587
  32. 32. Gerasimov ES, Afonin DA, Korzhavina OA, Lukeš J, Low R, Hall N, et al. Mitochondrial RNA editing in Trypanoplasma borreli: New tools, new revelations. Comput Struct Biotechnol J. 2022;20:6388–402. pmid:36420151
  33. 33. DeCuir J, Tu W, Dumonteil E, Herrera C. Sequence of Trypanosoma cruzi reference strain SC43 nuclear genome and kinetoplast maxicircle confirms a strong genetic structure among closely related parasite discrete typing units. Genome. 2021;64(5):525–31. pmid:33086026
  34. 34. Gerasimov ES, Zamyatnina KA, Matveeva NS, Rudenskaya YA, Kraeva N, Kolesnikov AA, et al. Common Structural Patterns in the Maxicircle Divergent Region of Trypanosomatidae. Pathogens. 2020;9(2):100. pmid:32033466
  35. 35. Flegontov PN, Guo Q, Ren L, Strelkova MV, Kolesnikov AA. Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri. Mol Genet Genomics. 2006;276(4):322–33. pmid:16909285
  36. 36. Westenberger SJ, Cerqueira GC, El-Sayed NM, Zingales B, Campbell DA, Sturm NR. Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region. BMC Genomics. 2006;7:60. pmid:16553959
  37. 37. Hoyos Sanchez MC, Ospina Zapata HS, Suarez BD, Ospina C, Barbosa HJ, Carranza Martinez JC, et al. A phased genome assembly of a Colombian Trypanosoma cruzi TcI strain and the evolution of gene families. Sci Rep. 2024;14(1):2054. pmid:38267502
  38. 38. Myler PJ, Glick D, Feagin JE, Morales TH, Stuart KD. Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites. Nucleic Acids Res. 1993;21(3):687–94. pmid:8382804
  39. 39. Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019;146(1):1–27. pmid:29898792
  40. 40. Ruvalcaba-Trejo LI, Sturm NR. The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer. BMC Genomics. 2011;12:58. pmid:21261994
  41. 41. Aphasizheva I, Alfonzo J, Carnes J, Cestari I, Cruz-Reyes J, Göringer HU, et al. Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes. Trends Parasitol. 2020;36(4):337–55. pmid:32191849
  42. 42. Read LK, Lukeš J, Hashimi H. Trypanosome RNA editing: the complexity of getting U in and taking U out. Wiley Interdiscip Rev RNA. 2016;7(1):33–51. pmid:26522170
  43. 43. Simpson L, Sbicego S, Aphasizhev R. Uridine insertion/deletion RNA editing in trypanosome mitochondria: a complex business. RNA. 2003;9(3):265–76. pmid:12591999
  44. 44. Hajduk S, Ochsenreiter T. RNA editing in kinetoplastids. RNA Biol. 2010;7(2):229–36. pmid:20220308
  45. 45. Blum B, Bakalara N, Simpson L. A model for RNA editing in kinetoplastid mitochondria: “guide” RNA molecules transcribed from maxicircle DNA provide the edited information. Cell. 1990;60(2):189–98. pmid:1688737
  46. 46. Benne R, Van den Burg J, Brakenhoff JP, Sloof P, Van Boom JH, Tromp MC. Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell. 1986;46(6):819–26. pmid:3019552
  47. 47. Aphasizhev R, Aphasizheva I. Mitochondrial RNA editing in trypanosomes: small RNAs in control. Biochimie. 2014;100:125–31. pmid:24440637
  48. 48. Albanaz ATS, Carrington M, Frolov AO, Ganyukova AI, Gerasimov ES, Kostygov AY, et al. Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae. BMC Genomics. 2023;24(1):471. pmid:37605127
  49. 49. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics. 2018;34(15):2666–9. pmid:29547981
  50. 50. Brown J, Pirrung M, McCue LA. FQC Dashboard: integrates FastQC results into a web-based, interactive, and extensible FASTQ quality control tool. Bioinformatics. 2017;33(19):3137–9. pmid:28605449
  51. 51. de Sena Brandine G, Smith AD. Falco: high-speed FastQC emulation for quality control of sequencing data. F1000Res. 2019;8:1874. pmid:33552473
  52. 52. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. pmid:24695404
  53. 53. Zimin AV, Marçais G, Puiu D, Roberts M, Salzberg SL, Yorke JA. The MaSuRCA genome assembler. Bioinformatics. 2013;29(21):2669–77. pmid:23990416
  54. 54. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4. pmid:27004904
  55. 55. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094–100. pmid:29750242
  56. 56. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. 2013.
  57. 57. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963. pmid:25409509
  58. 58. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10(2):giab008. pmid:33590861
  59. 59. Okonechnikov K, Conesa A, García-Alcalde F. Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2016;32(2):292–4. pmid:26428292
  60. 60. Hunter JD. Matplotlib: A 2D Graphics Environment. Comput Sci Eng. 2007;9(3):90–5.
  61. 61. Rusman F, Floridia-Yapur N, Tomasini N, Diosque P. Guide RNA Repertoires in the Main Lineages of Trypanosoma cruzi: High Diversity and Variable Redundancy Among Strains. Front Cell Infect Microbiol. 2021;11:663416. pmid:34136416
  62. 62. Thomas S, Martinez LLIT, Westenberger SJ, Sturm NR. A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing. BMC Genomics. 2007;8:133. pmid:17524149
  63. 63. Greif G, Rodriguez M, Reyna-Bello A, Robello C, Alvarez-Valin F. Kinetoplast adaptations in American strains from Trypanosoma vivax. Mutat Res. 2015;773:69–82. pmid:25847423
  64. 64. Kay C, Williams TA, Gibson W. Mitochondrial DNAs provide insight into trypanosome phylogeny and molecular evolution. BMC Evol Biol. 2020;20(1):161. pmid:33297939
  65. 65. Dierckxsens N, Mardulyn P, Smits G. Unraveling heteroplasmy patterns with NOVOPlasty. NAR Genom Bioinform. 2019;2(1):lqz011. pmid:33575563
  66. 66. Benne R. RNA editing in trypanosomes. Eur J Biochem. 1994;221(1):9–23. pmid:7513284
  67. 67. Simpson L, Thiemann OH, Savill NJ, Alfonzo JD, Maslov DA. Evolution of RNA editing in trypanosome mitochondria. Proc Natl Acad Sci U S A. 2000;97(13):6986–93. pmid:10860961
  68. 68. Kaufer A, Barratt J, Stark D, Ellis J. The complete coding region of the maxicircle as a superior phylogenetic marker for exploring evolutionary relationships between members of the Leishmaniinae. Infect Genet Evol. 2019;70:90–100. pmid:30738194
  69. 69. Kaufer A, Stark D, Ellis J. A review of the systematics, species identification and diagnostics of the Trypanosomatidae using the maxicircle kinetoplast DNA: from past to present. Int J Parasitol. 2020;50(6–7):449–60. pmid:32333942
  70. 70. Solana JC, Chicharro C, García E, Aguado B, Moreno J, Requena JM. Assembly of a Large Collection of Maxicircle Sequences and Their Usefulness for Leishmania Taxonomy and Strain Typing. Genes (Basel). 2022;13(6):1070. pmid:35741832
  71. 71. Lavi B, Levy Karin E, Pupko T, Hazkani-Covo E. The Prevalence and Evolutionary Conservation of Inverted Repeats in Proteobacteria. Genome Biol Evol. 2018;10(3):918–27. pmid:29608719
  72. 72. Medina R, Franco MEE, Bartel LC, Martinez Alcántara V, Saparrat MCN, Balatti PA. Fungal Mitogenomes: Relevant Features to Planning Plant Disease Management. Front Microbiol. 2020;11:978. pmid:32547508
  73. 73. Arunkumar KP, Nagaraju J. Unusually long palindromes are abundant in mitochondrial control regions of insects and nematodes. PLoS One. 2006;1(1):e110. pmid:17205114
  74. 74. Brázda V, Lýsek J, Bartas M, Fojta M. Complex Analyses of Short Inverted Repeats in All Sequenced Chloroplast DNAs. Biomed Res Int. 2018;2018:1097018. pmid:30140690
  75. 75. Wadkins RM. Targeting DNA secondary structures. Curr Med Chem. 2000;7(1):1–15. pmid:10637354