<p>Supplementary Material New approaches to systematics of Trypanosomatidae: criteria for taxonomic (re)description Jan Votýpka1,2, Claudia M. d'Avila-Levy3, Philippe Grellier4, Dmitri A. Maslov5, Julius Lukeš2,6,7, Vyacheslav Yurchenko8,2,9 1 Department of Parasitology, Faculty of Science, Charles University, 128 44 Prague, Czech Republic, </p><p>2 Biology Centre, Institute of Parasitology, Czech Academy of Sciences, 370 05 České Budějovice (Budweis), Czech Republic, </p><p>3 Coleção de Protozoários, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-360 Rio de Janeiro, Brazil, </p><p>4 UMR 7245 MCAM, Muséum National d'Histoire Naturelle, CNRS, Sorbonne Universités, 75005 Paris, France, </p><p>5 Department of Biology, University of California - Riverside, Riverside, 92521, USA, </p><p>6 Faculty of Sciences, University of South Bohemia, 370 05 České Budějovice (Budweis), Czech Republic, </p><p>7 Canadian Institute for Advanced Research, Toronto, ON M5G 1Z8, Canada, </p><p>8 Life Science Research Centre, University of Ostrava, 710 00 Ostrava, Czech Republic, </p><p>9 Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA</p><p>Corresponding author: Yurchenko, V. ([email protected])</p><p>1 Minimum information required for formal taxon (re)description </p><p>While we firmly believe that molecular phylogeny should be a keystone of any modern taxonomical description or redescription, other canonical traits cannot be simply ignored. In fact, in some instances these old-fashion features are the only way to match new descriptions to the original ones that were often made in the era preceding the development of the contemporary molecular biology methods [S1-3]. </p><p>Very important note: we strongly recommended (re)describing and naming species or genera only if an isolate is available in (ideally axenic) trypanosomatid-monospecific culture [S4-</p><p>6]. The only exceptions allowed are the cases of integrated symbionts when species in question cannot survive without its partner(s) [S7-8]. </p><p>Below we summarize information that are recommended and should be provided for any new trypanosomatid species or genus description or redescription. </p><p>General morphological description </p><p>This is probably the most problematic part of any species/genus (re)descriptions. </p><p>Trypanosomatids, in particular, monoxenous groups are highly polymorphic and the most common situation is a continuous gradient of cells' morphotypes, shapes and sizes between two or more extremes [S2, 9-10]. In addition to this, different morphological types could occur in situ, where the morphology may additionally vary according to the tissue location in the host body, and in a culture, where the different morphology may be additionally affected by culture conditions. Such situations decrease the credit of morphological descriptions and morphological features constitute only supplementary data in many cases. Therefore, morphology cannot be considered as the only sign for the species determination and only rarely can be used for an assignment to previously described taxa. Despite all of the above mentioned objections, any new (re)description should give at least a few basic morphological characteristics. Major </p><p>2 morphotypes observed in situ and in culture by light microscopy should be documented on </p><p>Giemsa or similar way stained slides. The eight basic morphotypes of trypanosomatids that used to serve as genus and/or species-defining traits in the current taxonomy are: amastigotes, choanomastigotes, endomastigotes, epimastigotes, opisthomastigotes, opisthomorphs, promastigotes, and trypomastigotes [S11-12]. General morphological description should be complemented by DAPI (4',6-Diamidino-2-phenylindole) or another nucleic acid staining. In addition to direct visualization of DNA-containing structures (nucleus and kinetoplast), it may help to detect bacterial endosymbionts if present [S5, 13]. </p><p>Ultrastructure </p><p>Electron microscopy analysis is an integral part of any modern (re)description. It is customary to provide both Scanning (SEM) and Transmission (TEM) electron microscopy microphotographs, respectively. They serve different purposes. While SEM allows direct cell visualization </p><p>(confirming light microscopy observation), TEM enables a detailed view into the cell ultrastructure [S13-15]. The subcellular structures and organelles usually detectable by TEM are: nucleus, kinetoplast, flagellum, flagellar pocket, paraflagellar rod, glycosomes and acidocalcisomes, subpellicular microtubules, Golgi apparatus, etc. In particular cases TEM helps to confirm the presence of bacterial endosymbionts and viruses [S5, 13]. Recently, we have pioneered a new protocol for TEM in trypanosomatids [S16]. It includes a high-pressure freezing (HPF) step and was proved appropriate for the preservation of subtle or rarely detected ultrastructural traits. </p><p>The important morphometric parameter measured using TEM microphotographs is kinetoplast width (thickness). In some cases they can be used as species-specific characters </p><p>[S17-19]. Our recent side-by-side comparison has revealed that different methods of fixation </p><p>(HPF versus chemical) can affect overall parameters of the subcellular structures. The </p><p>3 measurements of the kinetoplast, for example, can vary within approximately 20% of each other </p><p>(J.V., J.L., V.Y., unpublished results). Thus it is important to compare only numbers obtained using the same techniques. </p><p>Taxonomic summary </p><p>This is a formal taxon (re)description governed by the International Code of Zoological </p><p>Nomenclature (http://iczn.org/code). Typically, it includes the following articles: 1) Taxon diagnosis based on morphological, including ultrastructural, characteristics. However, as discussed above, in many instances those traits do not differentiate the new or re-described taxon from the previously described ones. In such cases the usage of its phylogenetic position as a diagnosis is fully sufficient; 2) Type species (name-bearing type of a nominal genus) or type material. The authors should clearly identify a unique individual as a type specimen </p><p>(holotype and its paratype counterpart; and/or lectotype, neotype or syntype, if necessary) or a series of individuals (syntypes, hapantotypes) for protists with complex life-cycles; 3) Type host </p><p>(the host species with which the name-bearing type of a nominal species was associated) and site (tissue) of infection. If possible, we suggest providing a barcoding sequence, typically the mitochondrial cytochrome c oxidase subunit I (COI) of the host species to The Barcode of Life </p><p>Data Systems, BOLD (C.M.D.L. manuscript in preparation) as well as other “typing molecular markers” to GeneBank databases; 4) Type locality (the geographical place of capture, collection, or observation of the name-bearing type of a nominal species; ideally including GPS coordinates). </p><p>Sequences</p><p>It is strongly recommended to associate all the sequences determined with a newly described or re-described taxon. In some cases when new combinations are proposed or taxa are renamed, it may cause problems because previously determined sequences were associated with old or </p><p>4 even invalid names. The only solution proposed by the National Center for Biotechnology </p><p>Information (NCBI) is to keep both names in the database. </p><p>Presence or absence of bacterial endosymbionts</p><p>It must be explicitly stated whether bacterial endosymbionts were detected by Giemsa or DAPI staining or by TEM. For positive isolates, sequences of the 16S ribosomal RNA genes should be provided. </p><p>Culture </p><p>In most cases (axenic) trypanosomatid-monospecific cultures must be established [S5, 14]. Co- infections of hosts by several more or less related parasites are rather common in trypanosomatids [S1, 3, 20], thus analysis of the clonal lines becomes a mandatory part of any </p><p>(re)description of new species. It is advisable to analyze several independent clones and compare them side-by-side by at least one molecular marker. </p><p>Symptoms, pathogenicity, and infection prevalence</p><p>Particularly for medically and/or veterinary important species it is customary to characterize clinical outcomes of the infection including symptoms, seasonal dynamics, prevalence, etc. [S1, </p><p>21-22]. </p><p>Xenotype, culture, and DNA deposits</p><p>The following materials must be archived in Institutional collections or museums: xenotype of the type host, hapantotype (e.g. stained slide), axenic cultures of the primary isolate and clonal line(s), total DNA sample from the primary isolate and clonal line(s). </p><p>Etymology and synonymy</p><p>The etymology of the new taxa names or new combination must be elucidated. All previously used synonyms (each of two or more names of the same rank used to denote the same </p><p>5 taxonomic taxon), junior synonyms, objective synonyms, senior synonyms, or subjective synonyms should be noted. For more information refer to The International Code of Zoological </p><p>Nomenclature. </p><p>Additional information to complement formal taxon (re)description </p><p>If available, the following information can supplement formal taxa (re)description: </p><p> a) Presence or absence of dsRNA viruses [S16, 23-24].</p><p> b) Biological characteristics, e.g. cultivation in different media, growth curves, temperature </p><p> resistance, pH dependence, etc. [S6]. </p><p> c) Host (vector) specificity [S25]. </p><p> d) Development in vivo and/or in vitro [S26-27]. </p><p> e) Presence of other trypanosomatids in the host specimen (co-infections) [S14]. </p><p> f) Sequences of the marker genes for additional environmental samples belonging to the </p><p> same taxon [S6]. </p><p>Species in Trypanosomatidae</p><p>Criteria for detecting and describing species are disentangled from a basic common concept of species [S28]. This applies to all groups of protists, but it is particularly true for trypanosomatids, which were described mainly based on morphological characters that do not necessarily present evolutionary origin in common [S29]. There is no consensus on molecular markers that can be used for species and genera distinction [S30]. The DNA barcodes are promising in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment and species identification [S31]. However, lack of consensus on isolates, sub-</p><p>6 species, species and genera degree of identity together with the use of distinct markers for different set of species, still poses a challenge to this approach (see the main text). We are deliberately not proposing a fixed amount of sequence difference to delineate species within the family Trypanosomatidae. Rather, the emphasis for species delineation should be a combination of phylogenetic analysis of DNA sequences together with a set of phenotypic characters (for example, vector or host specificity, pathogenesis, and geographical distribution). </p><p>Supplemental Figure 1. Protist collections in the 21st century. In addition to the ‘classical’ smear or slide specimens, the deposit of type specimens must include as much of the complementary materials as possible. These can include (but are not limited to) DNA, sequences, photos, alive, cryopreserved or fixed specimens, electron microscopy or histology materials, ecological data, etc. The advantage is that under one inventory number, scientists can have an access to a large panel of information or materials related to a species. </p><p>7 8 Supplemental references</p><p>S1. Grybchuk-Ieremenko, A., Losev, A., Kostygov, A.Y., Lukeš, J., and Yurchenko, V. (2014) </p><p>High prevalence of trypanosome co-infections in freshwater fishes. 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