Supplementary Material 

New approaches to systematics of Trypanosomatidae: criteria for taxonomic (re)descriptionJan 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,

2 Biology Centre, Institute of Parasitology, Czech Academy of Sciences, 370 05 České Budějovice (Budweis), Czech Republic,

3 Coleção de Protozoários, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-360 Rio de Janeiro, Brazil,

4 UMR 7245 MCAM, Muséum National d'Histoire Naturelle, CNRS, Sorbonne Universités, 75005 Paris, France,

5 Department of Biology, University of California - Riverside, Riverside, 92521, USA,

6 Faculty of Sciences, University of South Bohemia, 370 05 České Budějovice (Budweis), Czech Republic,

7 Canadian Institute for Advanced Research, Toronto, ON M5G 1Z8, Canada,

8 Life Science Research Centre, University of Ostrava, 710 00 Ostrava, Czech Republic,

9 Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Corresponding author: Yurchenko, V. (vyacheslav.yurchenko@osu.cz)

1

Minimum information required for formal taxon (re)description

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].

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-

6]. The only exceptions allowed are the cases of integrated symbionts when species in question

cannot survive without its partner(s) [S7-8].

Below we summarize information that are recommended and should be provided for any

new trypanosomatid species or genus description or redescription.

General morphological description

This is probably the most problematic part of any species/genus (re)descriptions.

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

2

morphotypes observed in situ and in culture by light microscopy should be documented on

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].

Ultrastructure

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

(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.

The important morphometric parameter measured using TEM microphotographs is

kinetoplast width (thickness). In some cases they can be used as species-specific characters

[S17-19]. Our recent side-by-side comparison has revealed that different methods of fixation

(HPF versus chemical) can affect overall parameters of the subcellular structures. The

3

measurements of the kinetoplast, for example, can vary within approximately 20% of each other

(J.V., J.L., V.Y., unpublished results). Thus it is important to compare only numbers obtained

using the same techniques.

Taxonomic summary

This is a formal taxon (re)description governed by the International Code of Zoological

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

(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

(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

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).

Sequences

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

4

even invalid names. The only solution proposed by the National Center for Biotechnology

Information (NCBI) is to keep both names in the database.

Presence or absence of bacterial endosymbionts

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.

Culture

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

(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.

Symptoms, pathogenicity, and infection prevalence

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,

21-22].

Xenotype, culture, and DNA deposits

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).

Etymology and synonymy

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

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

Nomenclature.

Additional information to complement formal taxon (re)description

If available, the following information can supplement formal taxa (re)description:

a) Presence or absence of dsRNA viruses [S16, 23-24].

b) Biological characteristics, e.g. cultivation in different media, growth curves, temperature

resistance, pH dependence, etc. [S6].

c) Host (vector) specificity [S25].

d) Development in vivo and/or in vitro [S26-27].

e) Presence of other trypanosomatids in the host specimen (co-infections) [S14].

f) Sequences of the marker genes for additional environmental samples belonging to the

same taxon [S6].

Species in Trypanosomatidae

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-

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

6

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).

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.

7

Supplemental references

S1. Grybchuk-Ieremenko, A., Losev, A., Kostygov, A.Y., Lukeš, J., and Yurchenko, V. (2014)

High prevalence of trypanosome co-infections in freshwater fishes. Folia Parasitol 61,

495-504.

S2. Borghesan, T.C., Ferreira, R.C., Takata, C.S., Campaner, M., Borda, C.C., Paiva, F.,

Milder, R.V., Teixeira, M.M., and Camargo, E.P. (2013) Molecular phylogenetic

redefinition of Herpetomonas (Kinetoplastea, Trypanosomatidae), a genus of insect

parasites associated with flies. Protist 164, 129–152.

S3. Losev, A., Grybchuk-Ieremenko, A., Kostygov, A.Y., Lukes, J., and Yurchenko, V. (2015)

Host specificity, pathogenicity, and mixed infections of trypanoplasms from freshwater

fishes. Parasitol Res 114, 1071-1078.

S4. Yurchenko, V., Lukeš, J., Xu, X., and Maslov, D.A. (2006) An integrated morphological

and molecular approach to a new species description in the Trypanosomatidae: the case

of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera:

Rhopalidae). J Eukaryot Microbiol 53, 103–111.

S5. Votýpka, J., Kostygov, A.Y., Kraeva, N., Grybchuk-Ieremenko, A., Tesařová, M.,

Grybchuk, D., Lukeš, J., and Yurchenko, V. (2014) Kentomonas gen. n., a new genus of

endosymbiont-containing trypanosomatids of Strigomonadinae subfam. n. Protist 165,

825-838.

S6. Votýpka, J., Suková, E., Kraeva, N., Ishemgulova, A., Duží, I., Lukeš, J., and Yurchenko,

V. (2013) Diversity of trypanosomatids (Kinetoplastea: Trypanosomatidae) parasitizing

fleas (Insecta: Siphonaptera) and description of a new genus Blechomonas gen. n.

Protist 164, 763–781.

8

S7. Maslov, D.A., Yurchenko, V.Y., Jirků, M., and Lukeš, J. (2010) Two new species of

trypanosomatid parasites isolated from Heteroptera in Costa Rica. J Eukaryot Microbiol

57, 177–188.

S8. Suh, S.O., Maslov, D.A., Molestina, R.E., and Zhou, J.J. (2012) Microbotryozyma

collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria

oleosa (Miridae). Antonie Van Leeuwenhoek 102, 99-104.

S9. Kostygov, A.Y., Grybchuk-Ieremenko, A., Malysheva, M.N., Frolov, A.O., and Yurchenko,

V. (2014) Molecular revision of the genus Wallaceina. Protist 165, 594-604.

S10. Zídková, L., Čepička, I., Votýpka, J., and Svobodová, M. (2010) Herpetomonas trimorpha

sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of the biting midge Culicoides

truncorum (Ceratopogonidae, Diptera). Int J Syst Evol Microbiol 60, 2236–2246.

S11. Maslov, D.A., Votýpka, J., Yurchenko, V., and Lukeš, J. (2013) Diversity and phylogeny

of insect trypanosomatids: all that is hidden shall be revealed. Trends Parasitol 29, 43–

52.

S12. Wheeler, R.J., Gluenz, E., and Gull, K. (2013) The limits on trypanosomatid

morphological diversity. PLoS One 8, e79581.

S13. Teixeira, M.M., Borghesan, T.C., Ferreira, R.C., Santos, M.A., Takata, C.S., Campaner,

M., Nunes, V.L., Milder, R.V., de Souza, W., and Camargo, E.P. (2011) Phylogenetic

validation of the genera Angomonas and Strigomonas of trypanosomatids harboring

bacterial endosymbionts with the description of new species of trypanosomatids and of

proteobacterial symbionts. Protist 162, 503–524.

S14. Yurchenko, V., Lukeš, J., Jirků, M., and Maslov, D.A. (2009) Selective recovery of the

cultivation-prone components from mixed trypanosomatid infections: a case of several

novel species isolated from Neotropical Heteroptera. Int J Syst Evol Microbiol 59, 893–

909.

9

S15. Schwarz, R.S., Bauchan, G.R., Murphy, C.A., Ravoet, J., de Graaf, D.C., and Evans, J.D.

(2015) Characterization of two species of Trypanosomatidae from the honey bee Apis

mellifera: Crithidia mellificae Langridge and McGhee, and Lotmaria passim n. gen., n. sp.

J Eukaryot Microbiol.

S16. Yurchenko, V., Votýpka, J., Tesařová, M., Klepetková, H., Kraeva, N., Jirků, M., and

Lukeš, J. (2014) Ultrastructure and molecular phylogeny of four new species of

monoxenous trypanosomatids from flies (Diptera: Brachycera) with redefinition of the

genus Wallaceina. Folia Parasitol 61, 97-112.

S17. Lukeš, J. and Votýpka, J. (2000) Trypanosoma avium: novel features of the kinetoplast

structure. Exp Parasitol 96, 178-181.

S18. Yurchenko, V., Hobza, R., Benada, O., and Lukeš, J. (1999) Trypanosoma avium: large

minicircles in the kinetoplast DNA. Exp Parasitol 92, 215-218.

S19. Zídková, L., Čepička, I., Szabová, J., and Svobodová, M. (2012) Biodiversity of avian

trypanosomes. Infect Genet Evol 12, 102-112.

S20. Yurchenko, V., Lukeš, J., Tesařová, M., Jirků, M., and Maslov, D.A. (2008) Morphological

discordance of the new trypanosomatid species phylogenetically associated with the

genus Crithidia. Protist 159, 99–114.

S21. Sundar, S. and Chakravarty, J. (2012) Recent advances in the diagnosis and treatment

of kala-azar. Natl Med J India 25, 85-89.

S22. Neuber, H. (2008) Leishmaniasis. J Dtsch Dermatol Ges 6, 754-765.

S23. Weeks, R., Aline, R.F., Jr., Myler, P.J., and Stuart, K. (1992) LRV1 viral particles in

Leishmania guyanensis contain double-stranded or single-stranded RNA. J Virol 66,

1389-1393.

S24. Marche, S., Roth, C., Manohar, S.K., Dollet, M., and Baltz, T. (1993) RNA virus-like

particles in pathogenic plant trypanosomatids. Mol Biochem Parasitol 57, 261-267.

10

S25. Votýpka, J., Klepetková, H., Yurchenko, V.Y., Horák, A., Lukeš, J., and Maslov, D.A.

(2012) Cosmopolitan distribution of a trypanosomatid Leptomonas pyrrhocoris. Protist

163, 616–631.

S26. Frolov, A.O. (1987) [Life cycle of Blastocrithidia miridarum (Kinetoplastida,

Trypanosomatidae)]. Zoologichesky Zhurnal 66, 655-661.

S27. Frolov, A.O., Malysheva, M.N., and Podlipaev, S. (1997) [Unusual pattern of cyst

formation in Blastocrithidia sp. (Kinetoplastida, Trypanosomatidae), parasites of the

intestine of water striders (Hemiptera, Gerridae)]. Parazitologiia 31, 356-363.

S28. Hey, J. (2006) On the failure of modern species concepts. Trends Ecol Evol 21, 447-450.

S29. Boenigk, J., Ereshefsky, M., Hoef-Emden, K., Mallet, J., and Bass, D. (2012) Concepts in

protistology: species definitions and boundaries. Eur J Protistol 48, 96-102.

S30. Caron, D.A., Countway, P.D., Savai, P., Gast, R.J., Schnetzer, A., Moorthi, S.D., Dennett,

M.R., Moran, D.M., and Jones, A.C. (2009) Defining DNA-based operational taxonomic

units for microbial-eukaryote ecology. Appl Environ Microbiol 75, 5797-5808.

S31. Westenberger, S.J., Sturm, N.R., Yanega, D., Podlipaev, S.A., Zeledon, R., Campbell,

D.A., and Maslov, D.A. (2004) Trypanosomatid biodiversity in Costa Rica: genotyping of

parasites from Heteroptera using the spliced leader RNA gene. Parasitology 129, 537–

547.

11