Abstract
Euglena gracilis is a major component of the aquatic ecosystem and together with closely related species, is ubiquitous worldwide. Euglenoids are an important group of protists, possessing a secondarily acquired plastid and are relatives to the Kinetoplastidae, which themselves have global impact as disease agents. To understand the biology of E. gracilis, as well as to provide further insight into the evolution and origins of the Kinetoplastidae, we embarked on sequencing the nuclear genome; the plastid and mitochondrial genomes are already in the public domain. Earlier studies suggested an extensive nuclear DNA content, with likely a high degree of repetitive sequence, together with significant extrachromosomal elements. To produce a list of coding sequences we have combined transcriptome data from both published and new sources, as well as embarked on de novo sequencing using a combination of 454, Illumina paired end libraries and long PacBio reads. Preliminary analysis suggests a surprisingly large genome approaching 2 Gbp, with a highly fragmented architecture and extensive repeat composition. Over 80% of the RNAseq reads from E. gracilis maps to the assembled genome sequence, which is comparable with the well assembled genomes of T. brucei and T. cruzi. In order to achieve this level of assembly we employed multiple informatics pipelines, which are discussed here. Finally, as a preliminary view of the genome architecture, we discuss the tubulin and calmodulin genes, which highlight potential novel splicing mechanisms.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, Mccourt RM, Mendoz L, Moestrup O, Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MFJR (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52:399–451
Adl SM, Simpson AG, Lane CE, Lukeš J, Bass D, Bowser SS, Brown MW, Burki F, Dunthorn M, Hampl V, Heiss A, Hoppenrath M, Lara E, Le Gall L, Lynn DH, McManus H, Mitchell EA, Mozley-Stanridge SE, Parfrey LW, Pawlowski J, Rueckert S, Shadwick RS, Schoch CL, Smirnov A, Spiegel FW (2012) The revised classification of eukaryotes. J Eukaryot Microbiol 59(5):429–493
Attardi G, Schatz G (1988) Biogenesis of mitochondria. Annu Rev Cell Biol 4:289–333
Aykut AO, Atilgan AR, Atilgan C (2013) Designing molecular dynamics simulations to shift populations of the conformational states of calmodulin. PLoS Comput Biol 9(12):e1003366. doi:10.1371/journalpcbi1003366
Bachvaroff TR, Sanchez Puerta MV, Delwiche CF (2005) Chlorophyll c-containing plastid relationships based on analyses of a multigene data set with all four chromalveolate lineages. Mol Biol Evol 22:1772–1782
Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, Lennard NJ, Caler E, Hamlin NE, Haas B, Böhme U, Hannick L, Aslett MA, Shallom J, Marcello L, Hou L, Wickstead B, Alsmark UC, Arrowsmith C, Atkin RJ, Barron AJ, Bringaud F, Brooks K, Carrington M, Cherevach I, Chillingworth TJ, Churcher C, Clark LN, Corton CH, Cronin A, Davies RM, Doggett J, Djikeng A, Feldblyum T, Field MC, Fraser A, Goodhead I, Hance Z, Harper D, Harris BR, Hauser H, Hostetler J, Ivens A, Jagels K, Johnson D, Johnson J, Jones K, Kerhornou AX, Koo H, Larke N, Landfear S, Larkin C, Leech V, Line A, Lord A, Macleod A, Mooney PJ, Moule S, Martin DM, Morgan GW, Mungall K, Norbertczak H, Ormond D, Pai G, Peacock CS, Peterson J, Quail MA, Rabbinowitsch E, Rajandream MA, Reitter C, Salzberg SL, Sanders M, Schobel S, Sharp S, Simmonds M, Simpson AJ, Tallon L, Turner CM, Tait A, Tivey AR, Van Aken S, Walker D, Wanless D, Wang S, White B, White O, Whitehead S, Woodward J, Wortman J, Adams MD, Embley TM, Gull K, Ullu E, Barry JD, Fairlamb AH, Opperdoes F, Barrell BG, Donelson JE, Hall N, Fraser CM, Melville SE, El-Sayed NM (2005) The genome of the African trypanosome Trypanosoma brucei. Science 309(5733):416–422
Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W (2011) Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27(4):578–579
Bolger AM, Lohse M, Usade B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120
Bolte K, Bullmann L, Hempel F, Bozarth A, Zauner S, Maier U (2009) Protein targeting into secondary plastids. J Eukaryot Microbiol 56(1):9–15
Buetow DE (1982) The biology of euglena, vol III. Academic, New York
Bumbulis MJ, Balog BM (2013) UV-C exposure induces an apoptosis-like process in Euglena gracilis. ISRN Cell Biol 2013(869216):6 pages
Burger G, Lang BF, Reith M, Gray MW (1996) Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes. Proc Natl Acad Sci U S A A93:2328–2332
Canaday J, Tessier L, Imbault HP, Paulus F (2001) Analysis of Euglena gracilis alpha-, beta-and gamma tubulin genes: introns and pre-mRNA maturation. Mol Genet Genomics 265:153–160. doi:10.1007/s004380000403
Cook JR (1972) Ultraviolet inactivation of Euglena chloroplasts. I. Effect of light intensity of culture. Biophys J 12:1467–1473
Cook JR (1981) Variation of DNA levels in Euglena related to pH of culture medium. J Protozool 28:148–150
Cook JR, Roxby R (1985) Physical properties of a plasmid-like DNA from Euglena gracilis. Biochim Biophys Acta 824(80):83
Daiker V, Lebert M, Richter P, Hader D (2010) Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis. Planta 231:1229–1236. doi:10.1007/s00425-010-1126-9
Davis EA, Epstein HT (1971) Some factors controlling step-wise variation of organelle number in Euglena gracilis. Exp Cell Res 65:273–280
Dobakova E, Flegontov P, Skalicky T, Lukes J (2015) Unexpectedly streamlined mitochondrial genome of the Euglenozoan Euglena gracilis. Genome Biol Evol 7(12):3358–3367. doi:10.1093/gbe/evv229
Dobell C (1932) Antony van Leeuwenhoek and his ‘little animals’: being some account of the father of protozoology and bacteriology and his multifarious discoveries in these disciplines. Constable, London, UK. Reprinted 1958 Russell and Russell, New York, New York, USA
Dolezel J, Bartos J, Voglmayr H, Greilhuber J (2003) Nuclear DNA and genome size of trout and human. Cytometry 51:127–128
Dooijes D, Chaves I, Kieft R, Dirks-Mulder A, Martin W, Borst P (2000) Base J originally found in Kinetoplastida is also a minor constituent of nuclear DNA of Euglena gracilis. Nucl Acids Res 28(16):3017–3021. doi:10.1093/nar/28163017
Dos Santos Ferreira V, Rocchetta I, Conforti V, Bench S, Feldman R, Levin MJ (2007) Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress. Gene 389:136–145
Ebel C, Frantz C, Paulus F, Imbault P (1999) Trans-splicing and cis-splicing in the colourless Euglenoid, Entosiphon sulcatum. Curr Genet 35:542–550
Ebenezer TE, O’Neill E, Zoltner M, Obado S, Hampl V, Ginger M, Jackson A, de Koning H, Lukes J, Dacks J, Lebert M, Carrington M, Kelly S, Field M et al (2017) Gene complement and expression in Euglena gracilis (in preparation)
El-Metwally S, Hamza T, Zakaria M, Helmy M (2013) Next-generation sequence assembly: four stages of data processing and computational challenges. PLoS Comput Biol 9(12):1–19
Epstein HT, Allaway E (1967) Properties of selectively starved Euglena. Biochim Biophys Acta 142:195–207
Flegontov P, Gray MW, Burger G, Lukes J (2011) Gene fragmentation: a key to mitochondrial genome evolution in Euglenozoa? Curr Genet 57:225–232. doi:10.1007/s00294-011-0340-8
Gibbs SP (1978) The chloroplasts of Euglena may have evolved from symbiotic green algae. Can J Bot 56:2883–2889
Gnerre S, MacCallum I, Przybylski D, Ribeiro F, Burton J, Walker B, Sharpe T, Hall G, Shea T, Sykes S, Berlin A, Aird D, Costello M, Daza R, Williams L, Nicol R, Gnirke A, Nusbaum C, Lander ES, Jaffe DB (2011) High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci USA 108(4):1513–1518
Gojdics M (1953) The Genus Euglena Madison. University of Wisconsin Press, Wisconsin
Goto K, Beneragama CK (2010) Circadian clocks and antiaging: do non-aging microalgae like Euglena reveal anything? Ageing Res Rev 9:91–100
Gray MW, Doolittle WF (1982) Has the endosymbiont hypothesis been proven? Microbiol Rev 46:1–42
Gull K (2001) Protist tubulins: new arrivals, evolutionary relationships and insights to cytoskeletal function. Curr Opin Microbiol 4:427–432
Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: quality assessment tool for genome assemblies. Bioinformatics 29(8):1072–1075. doi:10.1093/bioinformatics/btt086
Hader D-P, Hemmersbach R, Lebert M (2005a) Gravity and the behavior of unicellular organisms. Cambridge University Press, Cambridge
Hader D-P, Hemmersbach R, Lebert M (2005b) Gravity and the behavior of unicellular organisms. Development and cell biology series (no. 40). Cambridge University Press, Cambridge
Hallick RB, Hong L, Drager RG, Favreau MR, Monfort A, Orsat B, Spielmann A, Stutz E (1993) Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res 21:3537–3544
Hill HZ, Epstein HT, Schiff JA (1966) Studies of chloroplast development in Euglena. XIV. Sequential interactions of ultraviolet light and photoreactivating light in green colony formation. Biophys J 6:135–144
Hornett EA, Wheat CW (2012) Quantitative RNA-Seq analysis in non-model species: assessing transcriptome assemblies as a scaffold and the utility of evolutionary divergent genomic reference species. BMC Genomics 13:361. doi:10.1186/1471-2164-13-361
Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, Sisk E, Rajandream MA, Adlem E, Aert R, Anupama A, Apostolou Z, Attipoe P, Bason N, Bauser C, Beck A, Beverley SM, Bianchettin G, Borzym K, Bothe G, Bruschi CV, Collins M, Cadag E, Ciarloni L, Clayton C, Coulson RM, Cronin A, Cruz AK, Davies RM, De Gaudenzi J, Dobson DE, Duesterhoeft A, Fazelina G, Fosker N, Frasch AC, Fraser A, Fuchs M, Gabel C, Goble A, Goffeau A, Harris D, Hertz-Fowler C, Hilbert H, Horn D, Huang Y, Klages S, Knights A, Kube M, Larke N, Litvin L, Lord A, Louie T, Marra M, Masuy D, Matthews K, Michaeli S, Mottram JC, Müller-Auer S, Munden H, Nelson S, Norbertczak H, Oliver K, O'neil S, Pentony M, Pohl TM, Price C, Purnelle B, Quail MA, Rabbinowitsch E, Reinhardt R, Rieger M, Rinta J, Robben J, Robertson L, Ruiz JC, Rutter S, Saunders D, Schäfer M, Schein J, Schwartz DC, Seeger K, Seyler A, Sharp S, Shin H, Sivam D, Squares R, Squares S, Tosato V, Vogt C, Volckaert G, Wambutt R, Warren T, Wedler H, Woodward J, Zhou S, Zimmermann W, Smith DF, Blackwell JM, Stuart KD, Barrell B, Myler PJ (2005) The genome of the kinetoplastid parasite, Leishmania major. Science 309(5733):436–442
Jackson AP, Vaughan S, Gull K (2006) Evolution of tubulin gene arrays in Trypanosomatid parasites: genomic restructuring in Leishmania. BMC Genomics 7:261
Jackson AP, Sanders M, Berry A, McQuillan J, Aslett MA, Quail MA, Chukualim B, Capewell P, MacLeod A, Melville SE, Gibson W, Barry JD, Berriman M, Hertz-Fowler C (2010) The genome sequence of Trypanosoma brucei gambiense, causative agent of chronic human african trypanosomiasis. PLoS Negl Trop Dis 4(4):e658
Jackson AP, Otto TD, Aslett M, Armstrong SD, Bringaud F, Schlacht A, Hartley C, Sanders M, Wastling JM, Dacks JB, Acosta-Serrano A, Field MC, Ginger ML, Berriman M (2016) Kinetoplastid phylogenomics reveals the evolutionary innovations associated with the origins of parasitism. Curr Biol 26:161–172
Kawasaki H, Kretsinger RH (1995) Calcium-binding proteins 1: EF-hands. Protein Profile 2(4):297–490
Kim JT, Boo SM, Zakrys B (1998) Floristic and taxonomic accounts of the genus Euglena (Euglenophyceae) from Korean fresh waters. Algae 13(2):173–197
Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res 40:D1202–D1210
Leedale GF (1958a) Mitosis and chromosome numbers in the Euglenineae (Flagellata). Nature 181(4607):502–503
Leedale GF (1958b) Nuclear structure and mitosis in the Euglenineae. Arch Mikrobiol 32:32–64
Leedale GF (1968) The nucleus in Euglena. In: Buetow DE (ed) The Biology of Euglena. Academic, New York, pp 185–272
Leedale GF (1974) Preliminary observations on nuclear cytology and ultrastructure in carbon-starved streptomycin-bleached Euglena gracilis. Colloq Int CNRS 240:285–290
Lefort-Tran M, Bre MH, Pouphile M, Manigault P (1987) DNA flow cytometry of control euglena and cell cycle blockade of vitamin Bl2-starved. Cells Cytometry 8:46–54
Levasseur PJ, Meng Q, Bouck GB (1994) Tubulin genes in the algal protist Euglena gracilis. J Eukaryot Microbiol 41(5):468–477
Linton EW, Karnkowska-Ishikawa A, Kim JI, Shin W, Bennett MS, Kwiatowski J, Zakrys B, Triemer RE (2010a) Reconstructing euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen nov (Euglenophyta). Protist 161:603–619. doi:10.1016/jprotis201002002
Lonergan TA (1985) Regulation of cell shape in Euglena gracilis. IV. Localization of actin, myosin and calmodulin. J Cell Sci 77:197–208
Martin W, Herrmann RG (1998) Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol 118:9–17
Mazus B, Falchuk KH, Vallee BL (1984) Histone formation, gene expression, and zinc deficiency in Euglena gracilis. Biochemistry 23:42–47
McCormack E, Braam J (2003) Calmodulins and related potential calcium sensors of Arabidopsis. New Phytol 159:585–598
McDowall J (2016) “Calmodulin” InterPro Protein Archive. Accessed 19 May 2016
McFadden GI (2001) Primary and secondary endosymbiosis and the origin of plastids. J Phycol 37:951–959
McKean PG, Vaughan S, Gull K (2001) The extended tubulin superfamily. J Cell Sci 114:2723–2733
Milanowski R, Gumińska N, Karnkowska A, Ishikawa T, Zakryś B (2016) Intermediate introns in nuclear genes of euglenids—are they a distinct type? BMC Evol Biol 16:49. doi:10.1186/s12862-016-0620-5
Milanowski R, Karnkowska A, Ishikawa T, Zakryś B (2014) Distribution of conventional and nonconventional introns in tubulin (α and β) genes of Euglenids. Mol Biol Evol 31(3):584–593
Morton BR (1998) Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages. J Mol Evol 46:449–459
Morton BR (1999) Strand asymmetry and codon usage bias in the chloroplast genome of Euglena gracilis. Proc Natl Acad Sci U S A 96(9):5123–5128
Nakazawa M, Inui H, Yamaji R, Yamamoto T, Takenaka S, Ueda M, Nakano Y, Miyatake K (2000) The origin of pyruvate: NADP1 oxidoreductase in mitochondria of Euglena gracilis. FEBS Lett 479:155–156
Newton AC (2001) Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101:2353–2364
Newton AC (2003) Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J 370:361–371
Nikolenko SI, Korobeynikov AI, Alekseyev MA (2013) BayesHammer: Bayesian clustering for error correction in single-cell sequencing. BMC Genomics 14(1):S7
O’Donnell EHJ (1965) Nucleolus and chromosomes in Euglena gracilis. Cytologia 30(2):118–154
O’Neil ST, Emrich SJ (2013) Assessing De Novo transcriptome assembly metrics for consistency and utility. BMC Genomics 14:465
O’Neill EC, Trick M, Hill L, Rejzek M, Dusi RG, Hamilton CJ, Zimba PV, Henrissat B, Field RA (2015) The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry. Mol BioSyst 11:2808
Parra G, Bradnam K, Korf I (2007) CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23:1061–1067
Perez E, Lapaille M, Degand H, Cilibrasi L, Villavicencio-Queijeiro A, Morsomme P, González-Halphen D, Field MC, Remacle C, Baurain D, Cardol P (2014) The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae. Mitochondrion 19(Pt B):338–349. doi:10.1016/jmito201402001
Porterfield DM (1997) Orientation of motile unicellular algae to oxygen: oxytaxis in Euglena. Biol Bull 193:229–230
Rawson JRY (1975) The characterization of Euglena gracilis DNA by its reassociation kinetics. Biochim Biophys Acta 402:171–178
Richards OC (1967) Hybridization of Euglena gracilis chloroplast and nuclear DNA. Biochemistry 57:156–163
Rosati G, Verni F, Barsanti L, Passarelli V, Gualtieri P (1991) Ultrastructure of the apical zone of Euglena gracilis: photoreceptors and motor apparatus. Electron Microsc Rev 4:319–342
Roy J, Faktorovab D, Lukes J, Burger G (2007) Unusual mitochondrial genome structures throughout the Euglenozoa. Protist 158:385–396
Schantz M, Schantz R (1989) Sequence of a cDNA clone encoding β-tubnlin from Euglena gracilis. Nucleic Acids Res 17(16):6727
Schwartzbach SD, Schiff, JA (1983) Control of plastogenesis in Euglena. In: Shropshire W Jr, Mohor H (eds) Encyclopaedia of plant physiology, 6A. New series, Springer, Berlin, pp 312–335
Simpson JT, Durbin R (2011) Efficient de novo assembly of large genomes using compressed data structures. Genome Res 22:549–556
Souza RT, Lima FM, Barros RM, Cortez DR, Santos MF, Cordero EM, Ruiz JC, Goldenberg S, Teixeira MMG, da Silveira JF (2011) Genome size, karyotype polymorphism and chromosomal evolution in Trypanosoma cruzi. PLoS One 6(8):e23042. doi:10.1371/journalpone0023042
Spencer DF, Gray MW (2011) Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome. Mol Gen Genomics 285:19–31
Spencer DF, Gray MW (2012) Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome. Mol Genet Genomics 285:19–31
Stankiewicz AJ, Falchuk KH, Vallee BL (1983) Composition and structure of zinc-deficient Euglena gracilis chromatin. Biochemistry 22:5150–5156
Streb C, Richter P, Ntefidou M, Lebert M, Hader D-P (2002) Sensory transduction of gravitaxis in Euglena gracilis. J Plant Physiol 159:855–862
Thompson MD, Copertino DW, Thompson E, Favreau MR, Hallick RB (1995) Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena. Nucleic Acids Res 23(23):4745–4752
Toda H, Yazawa M, Yagi K (1992) Amino acid sequence of calmodulin from Euglena gracilis. Eur J Biochem 205:653–660
Tzagoloff A, Myers AM (1986) Genetics of mitochondrial biogenesis. Annu Rev Biochem 55:249–285
Vallee BL, Falchuk KH (1981) Zinc and gene expression. Philos Trans R Soc Lond B 1(294):185–196
Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM (2014) Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9(11):e112963
Wiegert KE, Bennett MS, Triemer RE (2012) Evolution of the chloroplast genome in photosynthetic euglenoids: a comparison of Eutreptia viridis and Euglena gracilis (Euglenophyta). Protist 163:832–843
Yoon HS, Hackett JD, Bhattacharya D (2002) A single origin of the peridinin- and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis. Proc Natl Acad Sci U S A 99:11724–11729
Zakrys B (1988) The nuclear DNA level as a potential taxonomic character in Euglena EHR (Euglenophyceae). Algol Stud 49:483–504
Zakrys B, Walne PL (1994) Floristic, taxonomic and phytogeographic studies of green Euglenophyta from the Southeastern United States, with emphasis on new and rare species. Algol Stud 72:71–114
Zakryś B (1986) Contribution to the monograph of Polish members of the genus Euglena Ehrenberg 1830. Nova Hedwig Beih 42:491–540
Zhang T, Zhang X, Hu S, Yu J (2011) An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform. Plant Methods 7:38
Acknowledgements
We are greatly indebted to the following for their contributions of data, advice and suggestions: Peter Myler (Seattle), Purificacion Gacia-Lopes and David Moreira (Orsay), Rob Field (Norwich) and Vladimir Hampl (Praha).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer International Publishing AG
About this chapter
Cite this chapter
Ebenezer, T.E., Carrington, M., Lebert, M., Kelly, S., Field, M.C. (2017). Euglena gracilis Genome and Transcriptome: Organelles, Nuclear Genome Assembly Strategies and Initial Features. In: Schwartzbach, S., Shigeoka, S. (eds) Euglena: Biochemistry, Cell and Molecular Biology. Advances in Experimental Medicine and Biology, vol 979. Springer, Cham. https://doi.org/10.1007/978-3-319-54910-1_7
Download citation
DOI: https://doi.org/10.1007/978-3-319-54910-1_7
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-54908-8
Online ISBN: 978-3-319-54910-1
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)