18S Rdna Sequence-Structure Phylogeny of the Trypanosomatida

Home , Blastocrithidia, Crithidia, Euglenozoa, Kinetoplastida, Leishmania, Leptomonas

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1 18S rDNA Sequence-Structure Phylogeny of the

2 Trypanosomatida (Kinetoplastea, Euglenozoa) with

3 Special Reference on Trypanosoma

5 Alyssa R. Borges1,§, Markus Engstler1 & Matthias Wolf2,§*

7 1Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Am

8 Hubland, 97074 Würzburg, Germany

10 2Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, 97074

11 Würzburg, Germany

13 § These authors contributed equally to this work

14 * Corresponding author: [email protected] (M. Wolf)

16 Abstract

17 Background: Parasites of the order Trypanosomatida are known due to their medical

18 relevance. Trypanosomes cause African sleeping sickness and Chagas disease in South

19 America, and Leishmania ROSS, 1903 species mutilate and kill hundreds of thousands of

20 people each year. However, human pathogens are very few when compared to the great

21 diversity of trypanosomatids. Despite the progresses made in the past decades on

22 understanding the evolution of this group of organisms, there are still many open questions

23 which require robust phylogenetic markers to increase the resolution of trees.

24 Methods: Using two known 18S rDNA template structures (from Trypanosoma cruzi

25 CHAGAS, 1909 and Trypanosoma brucei PLIMMER & BRADFORD, 1899), individual 18S rDNA

26 secondary structures were predicted by homology modeling. Sequences and their secondary

27 structures, automatically encoded by a 12-letter alphabet (each nucleotide with its three

28 structural states, paired left, paired right, unpaired), were simultaneously aligned. Sequence-

29 structure trees were generated by neighbor joining and/or maximum likelihood.

30 Results: With a few exceptions, all nodes within a sequence-structure maximum likelihood

31 tree of 43 representative 18S rDNA sequence-structure pairs are robustly supported (bootstrap

32 support >75). Even a quick and easy sequence-structure neighbor-joining analysis yields

33 accurate results and enables reconstruction and discussion of the big picture for all 240 18S

34 rDNA sequence-structure pairs of trypanosomatids that are currently available.

35 Conclusions: We reconstructed the phylogeny of a comprehensive sampling of trypanosomes

36 evaluated in the context of trypanosomatid diversity, demonstrating that the simultaneous use

37 of 18S rDNA sequence and secondary structure data can reconstruct robust phylogenetic

38 trees.

40 Keywords: Evolution, SSU, Phylogeny, RNA, Secondary Structure, Sequence-Structure,

41 Trypanosoma.

43 Background

44 Kinetoplastids (Kinetoplastea HONIGBERG, 1963) are a remarkable group of unicellular

45 organisms. They include free-living and parasite protists of invertebrates, vertebrates, and

46 plants [1–3]. Among them, we find the obligatory parasites of the order Trypanosomatida

47 KENT, 1880, emend VICKERMAN in Moreira et al. 2004 [3], including the human pathogens T.

48 brucei, which causes African sleeping sickness, T. cruzi, the causative agent of Chagas

49 disease in South America, and Leishmania species which infect and harm hundreds of 2

50 thousands of people each year [2,4,5]. African trypanosomes are likely the most well-known

51 trypanosomatids. Due to their dixenic life cycle and the extracellular lifestyle in the vertebrate

52 blood, they have evolved interesting, and sometimes unusual, mechanisms to deal with such

53 different environments and challenges imposed by the immune system. Thus, it does not come

54 as a surprise that major discoveries in cell biology have been made in trypanosomes, such as

55 antigenic variation [6], glycolysis compartmented in unique organelles [7], GPI-anchoring of

56 membrane proteins [8], and unprecedented nucleotide modifications [9].

57 For harboring such a diverse group of organisms, it is unsurprising that the evolution of

58 parasitism inside Kinetoplastea has been intriguing scientists for decades. Given that each

59 parasitic group has closely affiliated free-living relatives and reversion to a free-living state

60 did not occur, it is probable that at least four independent adoptions of obligate parasitism or

61 commensalism have occurred [2,5]. Currently, the earliest diverging lineage inside

62 Trypanosomatida is the genus Paratrypanosoma VOTYPKA & LUKES, 2013, represented by

63 one species found in mosquitoes, Paratrypanosoma confusum VOTYPKA & LUKES, 2013

64 [10,11]. The vast majority of trypanosomatids are monoxenic parasites of insects with few

65 dixenic genera due to the capacity of infect vertebrates, such as Leishmania and Trypanosoma

66 GRUBY, 1843 [2]. Thus, the most likely origin of Leishmania and Trypanosoma is from within

67 monoxenic trypanosomatids, implicating that their origins were no earlier than 370 million

68 years ago, when the invasion of land by vertebrates occurred [5,12,13]. The transmission of

69 an insect-living trypanosomatid into a warm-blooded host has most likely occurred many

70 times with rare successful cases [5,10,13]. So far, only Trypanosoma and Leishmania have

71 left surviving descendants and only trypanosomes have adopted an extracellular lifestyle in

72 vertebrates.

73 Once having passed the vertebrate colonization bottleneck, Trypanosoma radiation and

74 adaptation to diverse vertebrate species became an unprecedented evolutionary success story.

75 Today, these parasites prosper in essentially all vertebrate Classes, from fish to birds [13–15]. 3

76 The 18S rDNA marker has been extensively used to analyze the phylogenetic relationship

77 inside this group [5,12,16–19]. The incorporation of glycosomal glyceraldehyde phosphate

78 dehydrogenase (gGAPDH) to the analysis allowed the generation of more resolved trees,

79 consolidating, for example, the long-lasting question about the monophyly inside

80 Trypanosoma [13,15,17–21].

81 The advent of modern sequencing technologies has greatly advanced our understanding of

82 trypanosomatid phylogeny, with more new genera described in the last decade than within the

83 past century [2,3,5]. These days, trypanosomatid phylogeny has sufficiently advanced to

84 provide a solid framework for comparative studies, with genomic data available for more than

85 just the medically relevant kinetoplastids. Interestingly, the basic layout of trypanosomatid

86 genomes appears to be strikingly similar, with high overall synteny, within and between

87 monoxenic and dixenic species [2]. The constantly growing genome data might become a

88 powerful tool for evolutionary inference. In the future, trypanosomatids will be studied not

89 only as infective agents of devastating neglected tropical diseases, or powerful genetic and

90 cellular model systems, but also to unravel basic principles of the evolution of unicellular

91 eukaryotes. What we know today is just the tip of an iceberg. The origin of Trypanosoma, for

92 example, remains enigmatic. Further, the presence of prokaryotic endosymbionts and viruses

93 in trypanosomatids, or the full biodiversity and ecological role of insect trypanosomatids

94 remain superficially explored [2,22].

95 Here, we present the first large scale study in which trypanosomatid RNA secondary structure

96 is used as an additional source of phylogenetic information. We use 18S rRNA sequence-

97 structure data simultaneously in inferring alignments and trees. This approach was recently

98 reviewed and shown to increase robustness and accuracy of reconstructed phylogenies

99 [23,24]. So far, all conclusions have been made with multigene trees. The most important

100 result of our study is that there are only a few places in our robustly supported trees where

101 branching does not match with multigene phylogenomic trees. It is these discrepancies with 4

102 which we focus our discussion, as they are potentially critical branches that are ambiguous

103 and require more attention.

104

105 Methods

106 Taxon Sampling, Secondary Structure Prediction, Sequence–Structure Alignment, and

107 Phylogenetic Tree Reconstruction

108 We have used all 240 SSU 18S ribosomal RNA gene sequences from Trypanosomatida

109 available at NCBI (GenBank) with a sequence length >1500 nucleotides and with a full

110 taxonomic lineage down to a complete species name. Secondary structures of the 18S rDNA

111 sequences were obtained by homology modeling [25] using the ITS2 database [26] as well as

112 T. cruzi (AF245382) and T. brucei (M12676) as templates (Fig S1). The two template-

113 secondary structures (without pseudoknots) were obtained from the Comparative RNA Web

114 (CRW) site [27]. For sequence-structure alignments, the four nucleotides multiplied by three

115 states (unpaired, paired left and paired right) are encoded by a 12-letter alphabet [24]. Using a

116 specific 12×12 sequence-structure scoring matrix [28], global multiple sequence-structure

117 alignments were automatically generated in ClustalW2 1.83 [29] as implemented in 4SALE

118 1.7.1 [28,30]. Based on Keller et al. [23], using 12-letter encoded sequences, sequence-

119 structure neighbor-joining (NJ) trees were determined using ProfDistS [31]. Further, using 12-

120 letter encoded sequences, a sequence-structure maximum likelihood (ML) tree [32] - for only

121 a representative subset of 43 sequence-structure pairs - was calculated using phangorn [33] as

122 implemented in the statistical framework R [34]. The R script is available from the 4SALE

123 homepage at http://4sale.bioapps.biozentrum.uni-wuerzburg.de [24]. Bootstrap support for all

124 sequence-structure trees was estimated based on 100 pseudo-replicates. Trees were rooted

125 with non-Trypanosoma sequences from Trypanosomatida.

126

127 Results and discussion

128 From the analysis of 240 18S rDNA sequence-structure pairs (Fig. 1) and selection of 43

129 different species (Fig. 2), we obtained trees supported by high bootstraps values (> 75) on

130 sister groups displaying the following Trypanosoma clades: the Trypanosoma pestanai

131 BETTENCOURT & FRANCA, 1905 clade, represented in our tree by this species found in the

132 Eurasian badger [13]; the T. brucei clade, consisting of trypanosome species naturally

133 transmitted by tsetse flies, such as Trypanosoma vivax ZIEMANN, 1905, Trypanosoma

134 congolense BRODEN, 1904, Trypanosoma godfrey MCNAMARA ET AL., 1994, Trypanosoma

135 simiae BRUCE ET AL., 1913, Trypanosoma equiperdum DOFLEIN, 1901, Trypanosoma evansi

136 STEEL, 1885, and T. brucei [13,17,19,21]; the T. cruzi clade, comprising mammalian

137 trypanosomes with worldwide distribution, such as T. cruzi, Trypanosoma rangeli TEJERA,

138 1920, and Trypanosoma wauwau TEIXEIRA & CAMARGO, 2016, endemic of Latin America,

139 Trypanosoma conorhini (DONOVAN, 1909) SHORTT & SWAMINATH, 1928 found in Europe,

140 South America and Africa, and Trypanosoma dionisii BETTENCOURT & FRANCA, 1905

141 distributted in Latin America, Africa, Asia and Europe [19–21,35]; the Trypanosoma lewisi

142 (KENT, 1880) LAVERAN & MESNIL, 1901 clade, including the rodent parasites Trypanosoma

143 microti LAVERAN & PETTIT, 1910, Trypanosoma grosi LAVERAN & PETTIT, 1909 and T. lewisi

144 [36]; the Crocodilian clade, harboring Trypanosoma grayi NOVY, 1906 from Africa and

145 Trypanosoma ralphi TEIXEIRA & CAMARGO, 2013 from South America [15,37]; the Avian

146 clade, with Trypanosoma corvi STEPHENS & CHRISTOPHERS, 1908, Trypanosoma avium

147 DANILEWSKY, 1885 and Trypanosoma thomasbancrofti SLAPETA, 2016 [38]; the Trypanosoma

148 theileri LAVERAN, 1902 clade, with T. theileri, a worldwide distributted cattle parasite, and

149 the subclade representant Trypanosoma cyclops WEINMAN, 1972 [13,21]; and the Aquatic

150 clade, harboring trypanosomes from fish, anurans and platypus [14,39,40]. Interestingly, the

151 lizard/snake clade is also represented in our tree with Trypanosoma varani WENYON, 1908, a

152 snake trypanosome, branching together with the mammal parasite Trypanosoma freitasi REGO 6

153 ET AL., 1957. The branching of marsupial and rodent trypanosomes inside this clade has been

154 previously observed [36,41]. Thus, our analysis corroborates the existence of the lizard-

155 snake/marsupial-rodent clade composed by trypanosomes transmitted by sandflies [36].

156 The phylogenetic analyses using sequence-structure data of 18S rDNA (Fig. 2) supports the

157 monophyly of Trypanosoma as previously observed in trees constructed with partial/

158 complete sequences of 18S rDNA and/or gGAPDH sequences [15,17–19]. Intriguingly, in the

159 tree obtained using a greater number of sequences (Fig. 1) Strigomonas culicis (WALLACE &

160 JOHNSON, 1961) TEIXEIRA & CAMARGO, 2011 (U05679-1 and HQ659564-1) appear as a basal

161 group of African trypanosomes (Fig. 1). To date, studies on Trypanosomatida showed a basal

162 position of Trypanosoma in relation to Strigomonas LWOFF & LWOFF, 1931 [2,22,42]. Of

163 notice, S. culicis is a monoxenic parasite of the order Diptera [22], the same order of the well-

164 known vector of T. brucei clade trypanosomes, the tsetse fly. Although it may be tempting to

165 speculate the derivation of the tsetse lifestyle of African trypanosomes from Strigomonas, we

166 cannot exclude the possibility of poor sequence assembly, which could interfere with the

167 topology observed. Finally, one sequence of the bat parasite T. dionisii clustered within the T.

168 brucei clade (Fig. 1). This species is distributed worldwide, with its origin in Africa, and

169 presents a high phyletic diversity [35]. However, its branching inside T. cruzi clade is strongly

170 supported [13,17–19,21].

171 The first branching of Trypanosoma (Fig. 2) forms two major groups: one lineage composed

172 by T. brucei and T. pestanai clades, and another with trypanosomes from Terrestial (T. cruzi,

173 T. lewisi, T. theileri, snake-lizard/marsupial-rodent, avian and crocodilian clades) and Aquatic

174 lineages. Thus, our tree corroborates the hypothesis of the independent evolutionary history of

175 both human pathogens, T. brucei and T. cruzi [13]. The topology of our tree shows the

176 Aquatic clade as a solid lineage, in accordance with previous observations [15,18,19,21].

177 However, the origin of this clade is still under debate. Many studies using different DNA

178 markers, such as long (> 1.4 kb) 18S rDNA sequences, v7v8 hypervariable region of 18S 7

179 rDNA and/or partial sequences of gGAPDH, showed either an early division between Aquatic

180 and Terrestrial lineages as a single event [15,18,19,21] or in subsequent events with

181 amphibian trypanosomes and Trypanosoma therezieni BRYGOO, 1963 at the basis of

182 Trypanosoma [17,43,44]. Interestingly, our tree suggests a later evolution of the Aquatic clade

183 from Terrestrial trypanosomes (Fig. 2), which agrees with the insect-first hypothesis [13,36].

184 This hypothesis assumes that trypanosomes were originated from a monogenetic insect

185 parasite that adapted to live inside terrestrial vertebrates and later spread to leeches and other

186 aquatic animals, most likely through amphibians [13].

187 Trypanosomes of the T. brucei clade are virtually restricted to Africa, having an exception in

188 T. vivax [45,46]. The early divergence of T. vivax inside the T. brucei clade (Fig. 1, 2) is in

189 accordance to previous results showing a higher evolutionary rate of this species among the

190 Salivarian trypanosomes [19,21,47]. It is interesting to consider that a previous analysis of

191 18S rDNA sequences revealed that members of the T. brucei clade show an evolutionary rate

192 higher than other trypanosomes [47]. However, this high divergence has proven not to alter

193 the topology of sequence-based trees [17]. Inside this clade, a low bootstrap value (ML = 53)

194 is observed in the differentiation between T. brucei and T. evansi (Fig. 2), suggesting an

195 unresolved positioning. In fact, the relationship between these species is controversial, with

196 results supporting either T. evansi as a subspecies of T. brucei or showing great diversity

197 between both depending on the T. evansi strains [48,49].

198 Regarding the other major group of our analysis (Terrestrial/Aquatic lineages), the first

199 branching inside this group suggests the differentiation of the snake-lizard/marsupial-rodent

200 clade as a basal group of other trypanosomes (Fig. 2). However, other studies have suggested

201 avian trypanosomes as a basal group among terrestrial lineages [13,17]. This can be

202 associated with the low bootstrap values of our tree in either three events: the snake-

203 lizard/marsupial-rodent clade (ML = 54) differentiation, the divergence of crocodilian

204 trypanosomes (ML = 55), and the internal branch of avian trypanosomes (ML = 61), which

205 will be further explored in our discussion.

206 The T. cruzi and T. lewisi clades appear as sister groups in our analysis (Fig. 2), as has been

207 previously demonstrated [17–19,21]. The T. cruzi clade can be subdivided into three

208 subclades: Schyzotrypanum, T. wauwau (and other Neotropical bat trypanosomes,

209 Trypanosoma noyesi BOTERO & COOPER, 2016, and Trypanosoma livingstonei TEIXEIRA &

210 CAMARGO, 2013) and T. rangeli/T. conorhini [21,35]. The specific sequences of

211 Trypanosoma minasense CHAGAS, 1908 and Trypanosoma leeuwenhoeki SHAW, 1969

212 grouped with T. rangeli in our tree were previously considered synonyms of this species by

213 18S rDNA sequence analysis, explaining their positioning [13,50]. Regarding the T. lewisi

214 clade, our results suggest the existence of two subclades inside the group (ML = 100), one

215 harboring T. microti, and the other with T. lewisi and T. grosi. This finding is in accordance

216 with a recent analysis of long fragments of 18S rDNA which demonstrated this subdivision

217 despite the similarities in the v7v8 hypervariable region [51].

218 Concerning avian trypanosomes, our tree reflects previous findings in both the divergence

219 between T. avium and T. corvi and the highly supported (ML = 100) proximity between T.

220 avium and T. thomasbancrofti [38,50]. A low bootstrap value (ML = 61) is observed in the

221 divergence of T. corvi and T. avium/ T. thomasbancrofti. It is interesting to note that we

222 currently have two topologies known in literature, with the possibility of paraphyly

223 demonstrated by analysis of long sequences of 18S rDNA [19,38]. This, however, was not

224 observed in trees constructed with v7v8 hypervariable region of 18S rDNA, gGAPDH

225 sequences, or concatenated trees using both [15,37,52]. Thus, our tree indicates the need for a

226 better resolution on avian trypanosome positioning. Considering that we used only three

227 species of avian and two species of crocodilian trypanosomes in our reconstruction, our

228 approach represents an interesting method to be applied in further studies.

229 In our analysis we see the crocodilian/alligator trypanosomes (T. grayi and T. ralphi)

230 branching together with a high support value (ML = 100) (Fig. 2). Although T. grayi is found

231 in Africa and T. ralphi in South America [15,37,52], in a tree with distant external groups like

232 ours, this topology is expected due to their proximity inside the Crocodilian clade [15,37].

233 Our tree reflects the proximity of the crocodilian trypanosomes with T. cruzi clade, as

234 previously observed through full genome analysis [53]. Interestingly, crocodilian

235 trypanosomes, such as T. grayi and Trypanosoma kaiowa TEIXEIRA & CAMARGO, 2019 are

236 tsetse-transmitted species that are not restricted to the sub-Saharan belt [15,37,53], suggesting

237 higher adaptive plasticity of crocodilian trypanosomes.

238 The trypanosomes of the Aquatic lineage branched together (Fig. 2). The subgroups observed

239 are anuran trypanosomes (Trypanosoma rotatorium (MAYER, 1843) LAVERAN, 1901,

240 Trypanosoma mega DUTTON & TODD, 1903, Trypanosoma fallisi MARTIN & DESSER, 1990,

241 Trypanosoma ranarum (LANKESTER, 1871) DANILEWSKY, 1885, and Trypanosoma

242 neveulemairei BRUMPT, 1928) and fish trypanosomes (Trypanosoma siniperca CHANG, 1964,

243 Trypanosoma ophiocephali CHEN, 1964, Trypanosoma cobitis MITROPHANOW, 1884,

244 Trypanosoma granulosum LAVERAN & MESNIL, 1902, Trypanosoma pleuronectidium

245 ROBERTSON, 1906) along with the platypus parasite Trypanosoma binneyi MACKERRAS, 1959,

246 which is in accordance to the literature [14,15,39,40]. Interestingly, the anuran parasite,

247 Trypanosoma chattoni MATHIS & LEGER, 1911, appears in our analysis more related to fish

248 and platypus trypanosomes than to the anuran clade. This positioning of T. chattoni was

249 shown in a previous study using complete 18S rDNA sequences and non-trypanosomes as the

250 outgroup [54]. However, recent trees using complete 18S rDNA sequences and concatenated

251 analysis of v7v8 hypervariable region and gGAPDH rooted by other trypanosomes sustained

252 a monophyletic anuran clade [39,40]. Thus, T. chattoni positioning in our tree can be related

253 to the use of non-trypanosomes as the outgroup.

254 10

255

256 Conclusions

257 18S rDNA is one of the most used markers in inferring phylogenies at higher taxonomic

258 levels. However, this molecule has often been claimed to be inadequate for reconstructing

259 phylogenetic relationships at lower taxonomic levels, in particular, because of its conservative

260 rate of evolution. Thus, the use of the v7v8 hypervariable region has become popular inside

261 the trypanosome community as a barcode suitable to describe new species along with

262 gGAPDH [13]. However, some questions regarding the positioning of the groups still need to

263 be answered. To this, evaluating bigger sequences and using different genetic markers can

264 bring more information to the analysis improving resolution of the trees [13]. In this work, we

265 demonstrate that the simultaneous use of 18S rDNA sequence and secondary structure data

266 (i.e., the consideration of the individual secondary structures of the rRNA genes) could indeed

267 provide important information for reconstructing more robust phylogenetic trees. Our

268 topology highlights the need for further exploration of some groups, such as the avian and

269 snake-lizard/marsupial-rodent clades, which are less explored in trypanosome phylogenies.

270 Therefore, this study provides an additional fundament for upcoming phylogenetic

271 reconstructions and/or barcoding approaches which can be used not only in trypanosomatids.

272

273 Declarations

274 Ethics approval and consent to participate

275 Not applicable

276

277 Consent for publication

278 Not applicable

279

280 Availability of data and materials

281 The sequence data analysed in this paper is publicly available in NCBI (GenBank) and their

282 respective accession numbers are included in the trees. Data supporting the conclusions of this

283 article are included within the article. Data and materials are available upon reasonable

284 request to the corresponding author.

285

286 Competing interests

287 The authors declare that they have no competing interests.

288

289 Funding

290 Ph.D. scholarship was granted to Alyssa Rossi Borges by Brazilian agency CAPES (program:

291 CAPES/DAAD - Call No. 22/2018; process 88881.199683/2018-01). ME is supported by

292 DFGgrants EN-305.

293

294 Authors' contributions

295 MW conceived the study and performed the bioinformatic analysis. ARB analyzed the data

296 and majorly contributed to the manuscript writing. ME and MW assisted with data analysis

297 and manuscript writing. All authors read and approved the final manuscript.

298

299 Acknowledgments

300 We thank Julius Lukeš (Institute of Parasitology, Biology Centre CAS, Czech Republic) for

301 fruitful discussions and a pre-review of our manuscript, Jaime Lisack for proof-reading, and

302 Elisabeth Meyer-Natus for obtaining the electron microscopy image used in the graphical

303 abstract.

304

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459

460

461 Figure legends:

462

463 Fig. 1: 18S rDNA sequence-structure neighbor-joining (NJ) tree obtained by ProfDistS

464 [31]. All 240 18S rDNA sequences from Trypanosomatida (Kinetoplastea, Euglenozoa)

465 available at NCBI (GenBank) with a sequence length >1500 nucleotides and with a full

466 taxonomic lineage down to a complete species name have been used for the analysis. For tree

467 reconstruction the global multiple sequence-structure alignment (.xfasta format) as derived by

468 4SALE [28,30] was automatically encoded by a 12-letter alphabet [24]. GenBank accession

469 numbers accompany each taxon name. Key taxa are off and on marked in gray and

470 additionally named alongside the tree. Non-monophyletic taxa are indicated by quotation

471 marks. Singletons are highlighted red and polyphyletic taxa are highlighted blue. The scale

472 bar indicates evolutionary distances. The tree is rooted at non-Trypanosoma sequences.

473

474 Fig. 2: 18S rDNA sequence-structure maximum likelihood (ML) tree, representative

475 subset of 43 sequences from Fig. 1, obtained by phangorn as implemented in R [33].

476 Bootstrap values from 100 pseudo-replicates, mapped at the appropriate internodes, are from

477 maximum likelihood- (ML) and neighbor-joining- (NJ, obtained by ProfDistS, Wolf et al.

478 [31]) analyses. For NJ tree reconstruction the global multiple sequence-structure alignment

479 (.xfasta format) as derived by 4SALE [28,30] was automatically encoded by a 12-letter

480 alphabet [24]. For ML tree reconstruction the “one letter encoded” fasta format (12-letter

481 alphabet) as derived by 4SALE [28,30] was used. GenBank identifiers accompany each taxon

482 name. The scale bar indicates evolutionary distances. Highly supported branches are indicated

483 by thicker lines. The tree is rooted at Crithridia mellificae (KM980182) and Leishmania

484 amazonensis (JX030087). Clades discussed in the text are highlighted.

485

486 Supplementary Material 17

487

488 Fig. S1: Trypanosoma brucei (M12676). Secondary structure: small subunit ribosomal RNA

489 taken from the Comparative RNA Web (CRW) site [27].

490

AF359495_1_Trypanosoma_cruzi AF359496_2_Trypanosoma_cruzi AF359480_1_Trypanosoma_cruzi AF359472_1_Trypanosoma_cruzi AF359492_1_Trypanosoma_cruzi AF359491_1_Trypanosoma_cruzi AF359478_1_Trypanosoma_cruzi AF359490_1_Trypanosoma_cruzi AF359489_1_Trypanosoma_cruzi AF359477_1_Trypanosoma_cruzi AF359471_1_Trypanosoma_cruzi AF359468_1_Trypanosoma_cruzi AF359465_1_Trypanosoma_cruzi AF359462_1_Trypanosoma_cruzi AF359461_1_Trypanosoma_cruzi AF288661_1_Trypanosoma_cruzi AJ620544_1_Trypanosoma_cruzi AF292942_1_Trypanosoma_cruzi NJ AJ009148_1_Trypanosoma_cruzi AY785566_1_Trypanosoma_cruzi AY785565_1_Trypanosoma_cruzi AY785582_1_Trypanosoma_cruzi AY785581_1_Trypanosoma_cruzi AY785572_1_Trypanosoma_cruzi 0.02 AY785574_1_Trypanosoma_cruzi AY785573_1_Trypanosoma_cruzi AY785571_1_Trypanosoma_cruzi

AF359479_1_Trypanosoma_cruzi AF359493_1_Trypanosoma_cruzi AF359494_1_Trypanosoma_cruzi AF359463_1_Trypanosoma_cruzi AF359464_1_Trypanosoma_cruzi_ AF303659_1_Trypanosoma_cruzi AF359466_1_Trypanosoma_cruzi AY785580_1_Trypanosoma_cruzi AF359467_1_Trypanosoma_cruzi AF359469_1_Trypanosoma_cruzi KX007998_1_Trypanosoma_cruzi AF359474_1_Trypanosoma_cruzi AF359484_1_Trypanosoma_cruzi AF359483_1_Trypanosoma_cruzi AJ009147_1_Trypanosoma_cruzi AF359473_1_Trypanosoma_cruzi AF359481_1_Trypanosoma_cruzi AY785583_1_Trypanosoma_cruzi AF359482_1_Trypanosoma_cruzi AF359470_1_Trypanosoma_cruzi AY785564_1_Trypanosoma_cruzi AF359475_1_Trypanosoma_cruzi AF359486_1_Trypanosoma_cruzi AY785576_1_Trypanosoma_cruzi AF359485_1_Trypanosoma_cruzi AF359476_1_Trypanosoma_cruzi AY785584_1_Trypanosoma_cruzi AF359487_1_Trypanosoma_cruzi AF359488_1_Trypanosoma_cruzi AY785575_1_Trypanosoma_cruzi AF288660_1_Trypanosoma_cruzi AF228685_1_Trypanosoma_cruzi AY785568_1_Trypanosoma_cruzi AF303660_1_Trypanosoma_cruzi AY785577_1_Trypanosoma_cruzi AY785570_1_Trypanosoma_cruzi AY785578_1_Trypanosoma_cruzi AY785563_1_Trypanosoma_cruzi AY785579_1_Trypanosoma_cruzi AJ009149_1_Trypanosoma_cruzi AY785569_1_Trypanosoma_cruzi AJ009141_1_Trypanosoma_brucei_gambiense AY785585_1_Trypanosoma_cruzi AJ009154_1_Trypanosoma_evansi cruzi KX007996_1_Trypanosoma_brucei_gambiense „brucei/evansi/equiperdum“ AY785562_1_Trypanosoma_cruzi LC326397_1_Trypanosoma_dionisii AY785561_1_Trypanosoma_cruzi AJ009153_1_Trypanosoma_equiperdum AY912268_1_Trypanosoma_evansi AY785586_1_Trypanosoma_cruzi AY912269_1_Trypanosoma_evansi AY785567_1_Trypanosoma_cruzi AJ009142_1_Trypanosoma_brucei_rhodesiense AJ009150_1_Trypanosoma_cruzi_marinkellei KX007997_1_Trypanosoma_brucei_rhodesiense AF301912_1_Trypanosoma_cruzi KY114576_1_Trypanosoma_evansi KY114578_1_Trypanosoma_evansi AJ009152_1_Trypanosoma_dionisii KT023565_1_Trypanosoma_evansi KY114580_1_Trypanosoma_evansi AJ009151_1_Trypanosoma_dionisii KY114575_1_Trypanosoma_evansi AJ012411_1_Trypanosoma_conorhini congolense XR_003828665_1_Trypanosoma_conorhini KY114577_1_Trypanosoma_evansi KY114579_1_Trypanosoma_evansi AJ009166_1_Trypanosoma_vespertilionis AJ009144_1_Trypanosoma_congolense AJ009145_1_Trypanosoma_congolense AJ012415_1_Trypanosoma_rangeli AJ009146_1_Trypanosoma_congolense AJ012414_1_Trypanosoma_rangeli KP307024_1_Trypanosoma_congolense KP307025_1_Trypanosoma_congolense AJ012413_1_Trypanosoma_minasense dionisii KP307026_1_Trypanosoma_congolense AJ012417_1_Trypanosoma_rangeli AJ009155_1_Trypanosoma_godfreyi AF065157_1_Trypanosoma_rangeli conorhini Trypanosoma AJ009162_1_Trypanosoma_simiae KP307021_1_Trypanosoma_simiae AJ012416_1_Trypanosoma_rangeli AJ404608_1_Trypanosoma_simiae AJ012412_1_Trypanosoma_leeuwenhoeki „rangeli“ simiae KP307022_1_Trypanosoma_simiae AJ009160_1_Trypanosoma_rangeli EU477537_1_Trypanosoma_vivax HQ659564_1_Strigomonas_culicis Strigomonas KT030821_1_Trypanosoma_wauwau U05679_1_Strigomonas_culicis KT030810_1_Trypanosoma_wauwau XR_001548753_1_Leptomonas_pyrrhocoris Leptomonas KT030809_1_Trypanosoma_wauwau KM980182_1_Crithidia_melliﬁcae KM980183_1_Crithidia_melliﬁcae KR653211_1_Trypanosoma_wauwau wauwau KM980184_1_Crithidia_bombi KR653210_1_Trypanosoma_wauwau KM980185_1_Crithidia_bombi KT030813_1_Trypanosoma_wauwau KY264937_1_Crithidia_luciliae_thermophila corvi KM980186_1_Crithidia_expoeki KT030835_1_Trypanosoma_wauwau KM980187_1_Crithidia_expoeki KT030830_1_Trypanosoma_wauwau JN624299_1_Crithidia_dedva KT030823_1_Trypanosoma_wauwau GQ920678_1_Leishmania_aethiopica XR_002460808_1_Leishmania_major AY461665_2_Trypanosoma_corvi Crithidia XR_002460809_1_Leishmania_major JN006854_1_Trypanosoma_corvi outgroup: XR_002460810_1_Leishmania_major XR_002460811_1_Leishmania_major KT728396_1_Trypanosoma_thomasbancrofti thomasbancrofti Trypanosomatida XR_002460812_1_Leishmania_major XR_002460813_1_Leishmania_major KT728395_1_Trypanosoma_thomasbancrofti (non-Trypanosoma ) JX030087_1_Leishmania_amazonensis KT728394_1_Trypanosoma_thomasbancrofti JX030088_1_Leishmania_amazonensis JX030083_1_Leishmania_amazonensis major KT728393_1_Trypanosoma_thomasbancrofti JX030084_1_Leishmania_amazonensis KT728392_1_Trypanosoma_thomasbancrofti JX030085_1_Leishmania_amazonensis JX030086_1_Leishmania_amazonensis KT728391_1_Trypanosoma_thomasbancrofti JX030191_1_Leishmania_braziliensis KT728390_1_Trypanosoma_thomasbancrofti JX030192_1_Leishmania_braziliensis avium JX030188_1_Leishmania_braziliensis KT728389_1_Trypanosoma_thomasbancrofti JX030189_1_Leishmania_braziliensis JX030190_1_Leishmania_braziliensis KT728388_1_Trypanosoma_thomasbancrofti lewisi JX030135_1_Leishmania_braziliensis KT728387_1_Trypanosoma_thomasbancrofti JX030136_1_Leishmania_braziliensis JX030137_1_Leishmania_braziliensis amazonensis KT728386_1_Trypanosoma_thomasbancrofti JX030138_1_Leishmania_braziliensis JX030139_1_Leishmania_braziliensis KT728385_1_Trypanosoma_thomasbancrofti JX030140_1_Leishmania_braziliensis JX030187_1_Leishmania_braziliensis KT728384_1_Trypanosoma_thomasbancrofti JN003595_1_Leishmania_panamensis KT728383_1_Trypanosoma_thomasbancrofti KX138599_1_Blastocrithidia_triatomae Leishmania KX138600_1_Blastocrithidia_miridarum JN624300_2_Herpetomonas_nabiculae KT728382_1_Trypanosoma_thomasbancrofti HG425174_1_Herpetomonas_ztiplika KT223609_1_Phytomonas_nordicus KT728381_1_Trypanosoma_thomasbancrofti JN582046_1_Leptomonas_collosoma JN582047_1_Leptomonas_rigidus KT728380_1_Trypanosoma_thomasbancrofti JN582048_1_Leptomonas_rigidus JN582049_1_Leptomonas_rigidus MF401951_1_Trypanosoma_freitasi braziliensis KT728379_1_Trypanosoma_thomasbancrofti AJ005279_1_Trypanosoma_varani AJ009159_1_Trypanosoma_pestanai AJ009143_1_Trypanosoma_cobitis KT728378_1_Trypanosoma_thomasbancrofti AJ620551_1_Trypanosoma_granulosum DQ494415_1_Trypanosoma_siniperca EU185634_1_Trypanosoma_ophiocephali AJ132351_1_Trypanosoma_binneyi KT728377_1_Trypanosoma_thomasbancrofti DQ016613_1_Trypanosoma_pleuronectidium DQ016617_1_Trypanosoma_pleuronectidium DQ016618_1_Trypanosoma_pleuronectidium DQ016616_1_Trypanosoma_murmanensis AJ009157_1_Trypanosoma_mega AJ009161_1_Trypanosoma_rotatorium AF119806_1_Trypanosoma_fallisi KT728376_1_Trypanosoma_thomasbancrofti AF119808_1_Trypanosoma_mega KT728375_1_Trypanosoma_thomasbancroftiKT728401_1_Trypanosoma_avium KT728374_1_Trypanosoma_thomasbancroftiKT728400_1_Trypanosoma_avium KT728373_1_Trypanosoma_thomasbancroftiKT728399_1_Trypanosoma_avium KT728398_1_Trypanosoma_avium KT728397_1_Trypanosoma_avium KT728402_1_Trypanosoma_avium Blastocrithidia AF416563_2_Trypanosoma_avium AY099320_2_Trypanosoma_avium AY099319_3_Trypanosoma_avium Herpetomonas AF416559_1_Trypanosoma_avium AJ009140_1_Trypanosoma_avium FJ694763_1_Trypanosoma_grosi AJ009158_1_Trypanosoma_microti Phytomonas GU252213_1_Trypanosoma_lewisi GU252211_1_Trypanosoma_lewisi GU252210_1_Trypanosoma_lewisi AB242273_1_Trypanosoma_lewisi AJ009156_1_Trypanosoma_lewisiAJ005278_1_Trypanosoma_grayi Leptomonas

KF546526_1_Trypanosoma_grayi

KF546521_1_Trypanosoma_ralphi

AJ009164_1_Trypanosoma_theileri AJ009163_1_Trypanosoma_theileri

AJ131958_1_Trypanosoma_cyclops

AF119807_1_Trypanosoma_chattoni grayi AF119810_1_Trypanosoma_ranarum theileri AF119809_1_Trypanosoma_neveulemairei pleuronectidium bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.235945; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

K 980182M Crithidia melliﬁcae outgroup J 030087X Leishmania amazonensis A 009159J Trypanosoma pestanai T. pestanai clade 79/73 E 477537U Trypanosoma vivax 100/100 65/92 A 009144J Trypanosoma congolense 100/100 A 009155J Trypanosoma godfreyi clade 70/78 A 009162J Trypanosoma simiae 100/100 A 009153J Trypanosoma equiperdum A 009154J Trypanosoma evansi 53/100 A 009141J Trypanosoma brucei brucei T. 100/100 A 005279J Trypanosoma varani 100/100 Snake-lizard/marsupial-rodent clade M 401951F Trypanosoma freitasi A 119807F Trypanosoma chattoni 77/71 88/- A 132351J Trypanosoma binneyi D 016613Q Trypanosoma pleuronectidium 80/94 A 620551J Trypanosoma granulosum Fish/platypus 100/ 99/100 100 A 009143J Trypanosoma cobitis trypanosomes 100/100 E 185634U Trypanosoma ophiocephali 54/96 100/100 D 494415Q Trypanosoma siniperca A 009161J Trypanosoma rotatorium A 009157J Trypanosoma mega

77/- 84/100 Aquatic clade A 119806F Trypanosoma fallisi 80/97 A 119810F Trypanosoma ranarum 81/79 96/98 A 119809F Trypanosoma neveulemairei

Anuran clade 95/92 A 119808F Trypanosoma mega 100/100 A 009163J Trypanosoma theileri 99/100 T. theileri clade A 131958J Trypanosoma cyclops 100/100 K 546526F Trypanosoma grayi Crocodilian clade K 546521F Trypanosoma ralphi 61/80 J 006854N Trypanosoma corvi 83/69 A 009140J Trypanosoma avium Avian clade 100/100 K 728373T Trypanosoma thomasbancrofti 100/100 A 009158J Trypanosoma microti 55/52 A 009156J Trypanosoma lewisi T. lewisi clade 100/100 F 694763J Trypanosoma grosi 99/100 A 012413J Trypanosoma minasense 100/100 69/53 A 009160J Trypanosoma rangeli A 012412J Trypanosoma leeuwenhoeki T. rangeli/T. conorhini 64/- A 012411J Trypanosoma conorhini clade 79/- A 009166J Trypanosoma vespertilionis 84/98 78/94 A 301912F Trypanosoma cruzi Schyzotrypanum

100/100 A 009151J Trypanosoma dionisii cruzi T. 0.1 KT030823 Trypanosoma wauwau T. wauwau

ML/NJ bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.235945; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Secondary Structure: small subunit ribosomal RNA C A C 5' - AUCAUUGGGUGC A A CCGUGUGGCGGC C G GA C G a CUUUUGUGCCGA U U U A U U G C CCCUCGGCCCCA G C U A G C U G AUUUAUUUAUCA A C A U G G G G U U A A U AUUUACGUGCCU C G G A G A U C C U G C U A U AUUCUAUCACCC ACGGAC CCAAGCUG CAG AGGUGCC CCUCCGGCGGGGA UU U C A A C U A A G A C A G U UUC CGCACCAA CG A GCG CCGGUUCCCUCU U A A U G GUCGGU G UUUGG CUGCGG GGCCCCU UCCCCAACGGUGG U G A UUUGAGGUUCUU U G U U U A U U GAG CUGGUGCG CG GCA G G C A G C C C G A C U A U CCGGGGUUUUUU U A A U U A G A A G A G U A A U ACGGGAAUAUCC A U U G C UG U C U C G U U A A U UCAGCAC G U G - 3' U A U U U U C U GAACUGUGGGCCACGUAGUUUUGUGCCGUG G U A U C G G C C U U C G C A G A G U G C CCAGUCCCGUCCACCUCGGACGUGUUUUGA C A G C G A U G G U C G A U CCCACGCCCUCGUGGCCCGUGAACACACUC G U A U A U G A G U C G A U A C A C U C G AGAUACAAGAAACACGGGAGCGGUUCCUCC C G C C G UC G C A U G C U G U A G UCACUUUCACGCAUGUCAUGCAUGCGAGGG U A G G UG C U A U A G G U U A U A G U GGCGUCCGUGAAUUUUACUGUGACCAAAAA G U U U C U A A A G C G U U AGUGCGACCAAAGCAGUCUGCCGACUUGAA C A C U C A U G U G G U A A UUACAAAGCAUGGGAUAACGAAGCAUCAGC G C G C G U C G A C G U U U G C U CCUGGGGCCACCGUUUCGGCUUUUGUUGGU G C A U G U G C G G G UUUAGAAGUCCUUGGGAGAUUAUGGGGCCG C G G A G C G C A A U U C U C CGUGCCUUGGGUCGGUGUUUCGUGUCUCAU G C A C U A A C A G G U A U UUUUGUGGCGCGCACAUUCGGCUCUUCGUG A G C a C C G C A U A U C A U A C U U AUGUUUUUUUACAUUCAUUGCGACGCGCGG U G A C A U A G C C A A U A U U A G G G C CUUCCAGGAAUGAAGGA G G A A U C G A A G U A U A A A G U G U C C C A G C U U U C G U G U U C A G C A A U C U U A C G C G U A G C G U A C A G C A G A A U A A G C U U G A U C G A G G U C G G G C U A C GACACC U G C C G C C A C G U A C G C A U A A U U C A UUC A C U G C C G A GUC GGU A C U A U G G C G U U U A G G C G G A U C A A A G C AC G G C G G A G G C G C A A G C A G U C A C A C U A U G C U G A A A U G C U A G C G A C G C C C A U G G C CC A G U AA C A A G U G A U A A C C U G G C G C G G A C U U U GGG GUUC G A U U A A A G C C G A A G C C G A A C A C A A C G A C U U U U A G CUU GAGU A G C A U U U C A G A G G A U A UC G U A A A U G A C A A G A U G A A C C A A C G G C G A G C C A G G A A A A A A G A C G U A U U U UGACG AAUGGCA AC U C A G G A G G C U C C U C U A G A G A G A U U C C C U A U CA UGCCAUUUGU GU U G C G A U A G U A A U G U A C A C C C G U U A U U G U G C C A U U A G A C C A G G G U A C U A G C GCAUGGUUG UUUCA G A U C U G A A U U A G A C A U G G A A U A A C AUAGAGCCGACCGUGC A C G C A G C C A U G C C A G C G GC G U A C C A G C C G A A U 5' U G G G G A U C G C C A G U U A U U A A C C G C G G A CGAUG GGCA G A UAGCUGUAGG U G G U A U U G U C A G C U G A C G A U A CCUGCAGCUG A A C U A UGUCG GCC AU G A U U A A A C U A G A A A A U A A U A G A A C U A U U C U C G C C A A U G G A C G U A A U A C G UU C G A C G C G G A G C A G U G C A U A C G A U G U U A G C G A A U U A C AG C U A N C G G U G A G C U G U A U C U U U C U U 3' A A C C A A G A G C A C U A GC AGA CUGC G C C A C G A U A U G C G A U CU G G G UCUGC GCAG A U G A G U A G G GA A A U U A G A U C U G U U A C C A U A C A G C G G C A G G U G G U G C G G C G C G C A U U A G G C A U G A U G A U U G C G A C C U A C G U C C U G C G A U U A G A G C A C U C G A A Trypanosoma brucei G A A A C G G G A C C C (M12676) U C C G G C A C A U U A C A A C A G A C G 1.cellular organisms 2.Eukaryota 3.Euglenozoa U G U A U G G G C G C U G U C G 4.Kinetoplastida 5.Trypanosomatidae C C C G A C G U G G G A C C G 6.Trypanosoma 7.Trypanozoon U U G C G C G U G U U G U C July 2003 A C C U UGCGG U A C U C U A U CCGCA U C A A U A U A U U G A U U GCUCGGCG ACUG ACUUGGC G U A U A A U C AGCCGAGC CAUG GCCAAGC C A A A U A A U A A G C C G C U A G A C U C U C G U G A A C A A C C U U A G C G C G C U G U C A A A G U U A C C U U U U U U C A C U G G C A A A G A U G A A U U U U G U A U G U G C U A A C A C A C C A G G G G G C A G C A G G U G C C G C U C C U U C U U C G A G C G C G G U U A C A U G C C G A A G C A U U G C C U G U C A Citation and related information available at http://www.rna.icmb.utexas.edu