In this study, we sequenced and analyzed the genomes of 40 strains, in addition to the already-reported two type strains, of two Crithidia species infecting bumblebees in Alaska and Central Europe and demonstrated that different strains of Crithidia bombi and C. expoeki vary considerably in terms of single nucleotide polymorphisms and gene copy number. Based on the genomic structure, phylogenetic analyses, and the pattern of copy number variation, we confirmed the status of C. expoeki as a separate species. The Alaskan populations appear to be clearly separated from those of Central Europe. This pattern fits a scenario of rapid host-parasite coevolution, where the selective advantage of a given parasite strain is only temporary. This study provides helpful insights into possible scenarios of selection and diversification of trypanosomatid parasites.IMPORTANCE A group of trypanosomatid flagellates includes several well-studied medically and economically important parasites of vertebrates and plants. Nevertheless, the vast majority of trypanosomatids infect only insects (mostly flies and true bugs) and, because of that, has attracted little research attention in the past. Of several hundred trypanosomatid species, only four can infect bees (honeybees and bumblebees). Because of such scarcity, these parasites are severely understudied. We analyzed whole-genome information for a total of 42 representatives of bee-infecting trypanosomatids collected in Central Europe and Alaska from a population genetics point of view. Our data shed light on the evolution, selection, and diversification in this important group of trypanosomatid parasites.

Faculty of Biology M 5 Lomonosov Moscow State University Moscow Russia

Genetic Diversity Centre ETH Zürich Zürich Switzerland

Institute of Integrative Biology ETH Zürich Zürich Switzerland

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czech Republic

Martsinovsky Institute of Medical Parasitology Tropical and Vector Borne Diseases Sechenov University Moscow Russia

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Maslov DA, Votýpka J, Yurchenko V, Lukeš J. 2013. Diversity and phylogeny of insect trypanosomatids: all that is hidden shall be revealed. Trends Parasitol 29:43–52. doi: 10.1016/j.pt.2012.11.001. PubMed DOI

Podlipaev SA. 2000. Insect trypanosomatids: the need to know more. Mem Inst Oswaldo Cruz 95:517–522. doi: 10.1590/s0074-02762000000400013. PubMed DOI

Kostygov AY, Butenko A, Yurchenko V. 2019. On monoxenous trypanosomatids from lesions of immunocompetent patients with suspected cutaneous leishmaniasis in Iran. Trop Med Int Health 24:127–128. doi: 10.1111/tmi.13168. PubMed DOI

Pacheco RS, Marzochi MC, Pires MQ, Brito CM, Madeira MD, Barbosa-Santos EG. 1998. Parasite genotypically related to a monoxenous trypanosomatid of dog’s flea causing opportunistic infection in an HIV positive patient. Mem Inst Oswaldo Cruz 93:531–537. doi: 10.1590/S0074-02761998000400021. PubMed DOI

Chicharro C, Alvar J. 2003. Lower trypanosomatids in HIV/AIDS patients. Ann Trop Med Parasitol 97(Suppl 1):75–78. doi: 10.1179/000349803225002552. PubMed DOI

Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V. 2018. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol 34:466–480. doi: 10.1016/j.pt.2018.03.002. PubMed DOI

Lukeš J, Skalický T, Týč J, Votýpka J, Yurchenko V. 2014. Evolution of parasitism in kinetoplastid flagellates. Mol Biochem Parasitol 195:115–122. doi: 10.1016/j.molbiopara.2014.05.007. PubMed DOI

Kostygov AY, Yurchenko V. 2017. Revised classification of the subfamily PubMed DOI

Schwarz RS, Bauchan GR, Murphy CA, Ravoet J, de Graaf DC, Evans JD. 2015. Characterization of two species of PubMed DOI

Ravoet J, Schwarz RS, Descamps T, Yanez O, Tozkar CO, Martin-Hernandez R, Bartolome C, De Smet L, Higes M, Wenseleers T, Schmid-Hempel R, Neumann P, Kadowaki T, Evans JD, de Graaf DC. 2015. Differential diagnosis of the honey bee trypanosomatids PubMed DOI

Schmid-Hempel R, Tognazzo M. 2010. Molecular divergence defines two distinct lineages of PubMed DOI

Graystock P, Goulson D, Hughes WO. 2014. The relationship between managed bees and the prevalence of parasites in bumblebees. PeerJ 2:e522. doi: 10.7717/peerj.522. PubMed DOI PMC

Durrer S, Schmid-Hempel P. 1994. Shared use of flowers leads to horizontal pathogen transmission. Proc R Soc Lond 258:299–302.

Brown MJF, Schmid-Hempel R, Schmid-Hempel P. 2003. Strong context-dependent virulence in a host-parasite system: reconciling genetic evidence with theory. J Anim Ecol 72:994–1002. doi: 10.1046/j.1365-2656.2003.00770.x. DOI

Otterstatter MC, Thomson JD. 2008. Does pathogen spillover from commercially reared bumble bees threaten wild pollinators? PLoS One 3:e2771. doi: 10.1371/journal.pone.0002771. PubMed DOI PMC

Schmid-Hempel R, Eckhardt M, Goulson D, Heinzmann D, Lange C, Plischuk S, Escudero LR, Salathe R, Scriven JJ, Schmid-Hempel P. 2014. The invasion of southern South America by imported bumblebees and associated parasites. J Anim Ecol 83:823–837. doi: 10.1111/1365-2656.12185. PubMed DOI

Whitehorn PR, Tinsley MC, Brown MJ, Darvill B, Goulson D. 2011. Genetic diversity, parasite prevalence and immunity in wild bumblebees. Proc R Soc B 278:1195–1202. doi: 10.1098/rspb.2010.1550. PubMed DOI PMC

Schmid-Hempel P, Puhr K, Kruger N, Reber C, Schmid-Hempel R. 1999. Dynamic and genetic consequences of variation in horizontal transmission for a microparasitic infection. Evolution 53:426–434. doi: 10.1111/j.1558-5646.1999.tb03778.x. PubMed DOI

Baer B, Schmid-Hempel P. 2003. Bumblebee workers from different sire groups vary in susceptibility to parasite infection. Ecol Lett 6:106–110. doi: 10.1046/j.1461-0248.2003.00411.x. DOI

Barribeau SM, Schmid-Hempel P. 2013. Qualitatively different immune response of the bumblebee host, PubMed DOI

Brunner FS, Schmid-Hempel P, Barribeau SM. 2013. Immune gene expression in PubMed DOI PMC

Barribeau SM, Sadd BM, Du Plessis L, Schmid-Hempel P. 2014. Gene expression differences underlying genotype-by-genotype specificity in a host-parasite system. Proc Natl Acad Sci U S A 111:3496–3501. doi: 10.1073/pnas.1318628111. PubMed DOI PMC

Schmid-Hempel R, Salathe R, Tognazzo M, Schmid-Hempel P. 2011. Genetic exchange and emergence of novel strains in directly transmitted trypanosomatids. Infect Genet Evol 11:564–571. doi: 10.1016/j.meegid.2011.01.002. PubMed DOI

Tognazzo M, Schmid-Hempel R, Schmid-Hempel P. 2012. Probing mixed-genotype infections II: high multiplicity in natural infections of the trypanosomatid, PubMed DOI PMC

Wilfert L, Gadau J, Baer B, Schmid-Hempel P. 2007. Natural variation in the genetic architecture of a host-parasite interaction in the bumblebee PubMed DOI

Ulrich Y, Schmid-Hempel P. 2012. Host modulation of parasite competition in multiple infections. Proc Biol Sci 279:2982–2989. doi: 10.1098/rspb.2012.0474. PubMed DOI PMC

Ulrich Y, Sadd BM, Schmid-Hempel P. 2011. Strain filtering and transmission of a mixed infection in a social insect. J Evol Biol 24:354–362. doi: 10.1111/j.1420-9101.2010.02172.x. PubMed DOI

Salathé RM, Schmid-Hempel P. 2011. The genotypic structure of a multi-host bumblebee parasite suggests a role for ecological niche overlap. PLoS One 6:e22054. doi: 10.1371/journal.pone.0022054. PubMed DOI PMC

Ruiz-Gonzalez MX, Bryden J, Moret Y, Reber-Funk C, Schmid-Hempel P, Brown MJ. 2012. Dynamic transmission, host quality, and population structure in a multihost parasite of bumblebees. Evolution 66:3053–3066. doi: 10.1111/j.1558-5646.2012.01655.x. PubMed DOI

Schmid-Hempel P, Aebi M, Barribeau S, Kitajima T, Du Plessis L, Schmid-Hempel R, Zoller S. 2018. The genomes of PubMed DOI PMC

Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V. 2019. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology 146:1–27. doi: 10.1017/S0031182018000951. PubMed DOI

Koch H, Woodward J, Langat MK, Brown MJ, Stevenson PC. Flagellum removal by a nectar metabolite inhibits infectivity of a bumblebee parasite. Curr Biol, in press. PubMed

Baer B, Schmid-Hempel P. 2001. Unexpected consequences of polyandry for parasitism and fitness in the bumblebee, PubMed DOI

Baer B, Schmid-Hempel P. 1999. Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee. Nature 397:151–154. doi: 10.1038/16451. DOI

Schmid-Hempel P. 2001. On the evolutionary ecology of host-parasite interactions: addressing the question with regard to bumblebees and their parasites. Naturwissenschaften 88:147–158. doi: 10.1007/s001140100222. PubMed DOI

Flegontov P, Butenko A, Firsov S, Kraeva N, Eliáš M, Field MC, Filatov D, Flegontova O, Gerasimov ES, Hlaváčová J, Ishemgulova A, Jackson AP, Kelly S, Kostygov A, Logacheva MD, Maslov DA, Opperdoes FR, O’Reilly A, Sádlová J, Ševčíková T, Venkatesh D, Vlček Č, Volf P, Votýpka J, Záhonová K, Yurchenko V, Lukeš J. 2016. Genome of PubMed DOI PMC

Weir W, Capewell P, Foth B, Clucas C, Pountain A, Steketee P, Veitch N, Koffi M, De Meeus T, Kabore J, Camara M, Cooper A, Tait A, Jamonneau V, Bucheton B, Berriman M, MacLeod A. 2016. Population genomics reveals the origin and asexual evolution of human infective trypanosomes. Elife 5:e11473. doi: 10.7554/eLife.11473. PubMed DOI PMC

Bussotti G, Gouzelou E, Côrtes Boité M, Kherachi I, Harrat Z, Eddaikra N, Mottram JC, Antoniou M, Christodoulou V, Bali A, Guerfali FZ, Laouini D, Mukhtar M, Dumetz F, Dujardin J-C, Smirlis D, Lechat P, Pescher P, El Hamouchi A, Lemrani M, Chicharro C, Llanes-Acevedo IP, Botana L, Cruz I, Moreno J, Jeddi F, Aoun K, Bouratbine A, Cupolillo E, Späth GF. 2018. PubMed DOI PMC

Reis-Cunha JL, Valdivia HO, Bartholomeu DC. 2018. Gene and chromosomal copy number variations as an adaptive mechanism towards a parasitic lifestyle in trypanosomatids. Curr Genomics 19:87–97. PubMed PMC

Yourth CP, Schmid-Hempel P. 2006. Serial passage of the parasite PubMed DOI PMC

Tibayrenc M, Ayala FJ. 2013. How clonal are PubMed DOI

Koffi M, De Meeus T, Sere M, Bucheton B, Simo G, Njiokou F, Salim B, Kabore J, MacLeod A, Camara M, Solano P, Belem AM, Jamonneau V. 2015. Population genetics and reproductive strategies of African trypanosomes: revisiting available published data. PLoS Negl Trop Dis 9:e0003985. doi: 10.1371/journal.pntd.0003985. PubMed DOI PMC

Fürst MA, McMahon DP, Osborne JL, Paxton RJ, Brown MJ. 2014. Disease associations between honeybees and bumblebees as a threat to wild pollinators. Nature 506:364–366. doi: 10.1038/nature12977. PubMed DOI PMC

Kraeva N, Butenko A, Hlaváčová J, Kostygov A, Myškova J, Grybchuk D, Leštinová T, Votýpka J, Volf P, Opperdoes F, Flegontov P, Lukeš J, Yurchenko V. 2015. PubMed DOI PMC

Votýpka J, Suková E, Kraeva N, Ishemgulova A, Duží I, Lukeš J, Yurchenko V. 2013. Diversity of trypanosomatids ( PubMed DOI

Grybchuk-Ieremenko A, Losev A, Kostygov AY, Lukeš J, Yurchenko V. 2014. High prevalence of trypanosome coinfections in freshwater fishes. Folia Parasitol 61:495–504. doi: 10.14411/fp.2014.064. PubMed DOI

Hines HM. 2008. Historical biogeography, divergence times, and diversification patterns of bumble bees (Hymenoptera: Apidae: PubMed DOI

Freel KC, Friedrich A, Hou J, Schacherer J. 2014. Population genomic analysis reveals highly conserved mitochondrial genomes in the yeast species PubMed DOI PMC

Ruan J, Cheng J, Zhang T, Jiang H. 2017. Mitochondrial genome evolution in the PubMed DOI PMC

Salathé R, Tognazzo M, Schmid-Hempel R, Schmid-Hempel P. 2012. Probing mixed-genotype infections. I. Extraction and cloning of infections from hosts of the trypanosomatid PubMed DOI PMC

Schmid-Hempel P, Schmid-Hempel R. 1993. Transmission of a pathogen in DOI

Votýpka J, d’Avila-Levy CM, Grellier P, Maslov DA, Lukeš J, Yurchenko V. 2015. New approaches to systematics of PubMed DOI

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data, http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing Study. 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Ramirez-Gonzalez RH, Bonnal R, Caccamo M, Maclean D. 2012. Bio-SAMtools: ruby bindings for SAMtools, a library for accessing BAM files containing high-throughput sequence alignments. Source Code Biol Med 7:6. doi: 10.1186/1751-0473-7-6. PubMed DOI PMC

Quinlan AR. 2014. BEDTools: the Swiss-Army tool for genome feature analysis. Curr Protoc Bioinformatics 47:11.12.1–11.12.34. doi: 10.1002/0471250953.bi1112s47. PubMed DOI PMC

Peterlongo P, Riou C, Drezen E, Lemaitre C. 2017. DiscoSnp++: DOI

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly PubMed DOI PMC

Klambauer G, Schwarzbauer K, Mayr A, Clevert DA, Mitterecker A, Bodenhofer U, Hochreiter S. 2012. cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate. Nucleic Acids Res 40:e69. doi: 10.1093/nar/gks003. PubMed DOI PMC

Opperdoes FR, Butenko A, Flegontov P, Yurchenko V, Lukeš J. 2016. Comparative metabolism of free-living PubMed DOI

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552. doi: 10.1093/oxfordjournals.molbev.a026334. PubMed DOI

Talavera G, Castresana J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56:564–577. doi: 10.1080/10635150701472164. PubMed DOI

Huerta-Cepas J, Serra F, Bork P. 2016. ETE3: reconstruction, analysis, and visualization of phylogenomic data. Mol Biol Evol 33:1635–1638. doi: 10.1093/molbev/msw046. PubMed DOI PMC

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. doi: 10.1093/sysbio/syq010. PubMed DOI

Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. doi: 10.1093/bioinformatics/btu033. PubMed DOI PMC

Gascuel O. 1997. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol 14:685–695. doi: 10.1093/oxfordjournals.molbev.a025808. PubMed DOI

Price MN, Dehal PS, Arkin AP. 2009. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26:1641–1650. doi: 10.1093/molbev/msp077. PubMed DOI PMC

Alexa A, Rahnenfuhrer J, Lengauer T. 2006. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics 22:1600–1607. doi: 10.1093/bioinformatics/btl140. PubMed DOI

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