Building the first SSU database of phagotrophic euglenids using single-cell approaches
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This document describes building the first SSU database of phagotrophic euglenids using single-cell approaches. 28 new SSU sequences were obtained from single phagotrophic euglenid cells using methods like multiple displacement amplification and PCR or shotgun genomics. These culture-independent techniques allowed both identification of cells through micrographs and sequencing of the SSU gene from individual cells, improving coverage of diversity in this poorly sampled group.
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Building the first SSU database of phagotrophic euglenids using single-cell approaches
- 1. Building the first SSU database of phagotrophic euglenids using single-cell approaches SSU Phylogeny RAxML GTR gamma 1000 bootstraps 957 sites Simpson Lab website: protists.biology.dal.ca Taxon sampling of phagotrophic euglenids is extremely limited for all molecular markers including the SSU gene, mainly because there are very few cultures available (see Table 1). The culture-independent approach demonstrated here enables collection of micrographic data (for identification) and SSU genes from a single cell, using either multiple-displacement amplification (MDA) and PCR, partial shotgun-genomics, or single-cell transcriptomics. The wide range of taxa successfully sampled with these methods shows that they can rapidly produce much-improved (and much-needed) coverage of phagotrophic euglenid diversity. Fig. 1: Examples of SSU-typed phagotrophic euglenid cells. Top row, left to right: Anisonema acinus (SDB4), Chasmostoma nieuportense (UB23), Urceolus sp. (UB25), Petalomonas planus (UB22). Bottom row, left to right: Ploeotia cf. vitrea (U4), Neometanema sp. (SDB8), Dinema sp. (UB26), Heteronema globuliferum (BP3). Colors and symbols correspond to Table 1. Scale bars are 20µm. Methods PHOTOTROPHS OSMOTROPHS SYMBIONTIDS OUTGROUP Table 1: Genera of phagotrophic euglenids ordered by availability of SSU data (outside this study). 2 = sequences from 2 species only, 1 = 1 species only, 0 = no sequences available. New data obtained in this study shown in colored symbols (correspond to Fig. 1). sediment sample extraction / analysis of SSU pick single cells photo- documentation transcript ome shotgun genome seq. SSU-PCR (via MDA) Gordon Lax & Alastair Simpson Dalhousie University Petalomonas Notosolenus Anisonema Peranema Ploeotia Heteronema Neometanema Dinema Keelungia Entosiphon Urceolus Jenningsia Sphenomonas Lentomonas Chasmostoma Calycimonas Scytomonas Dolium Dylakosoma 2 1 0 # SSU via PCR # shotgun genome data # transcriptomes 28 new SSU sequences +1 Entosiphon sulcatum (not shown) 0.1 Anisonema sp. W1 Lepocinclis ovum Ploeotia costata (brackish) Neobodo saliens Anisonema sp. SDB1 Bihospites bacati isolate 2 Parmidium circulare Trypanoplasma borreli Ploeotia corrugata UB21 Dinema platysomum U5 Notosolenus ostium U1 Neometanema sp. BP5 Neometanema exaratum U2 Anisonema sp. SMS2 Dinema litorale UBJ2 Peranema trichophorum CCAP1260/1B Petalomonas sphagnophila GU477296 Peranema trichophorum PtR Cryptobia helicis Ploeotia cf. vitrea U4 Cryptoglena pigra Monomorphina pyrum Keelungia pulex Heteronema scaphurum Peranema trichophorum Ploeotia costata AF525486 Trachelomonas abrupta Diplonema papillatum Petalomonas cantuscygni Colacium vesiculosum Anisonema acinus B1 Heteronema globuliferum BP3 Environmental sequence AY821956 Petalomonas cantuscygni CCAP 1259/1 Eutreptia viridis Urceolus sp. UB25 Bodo saltans Rapaza viridis Anisonema sp. SDB4 Distigma gracilis Euglena viridis Gyropaigne lefevrei Dimastigella trypaniformis Notosolenus urceolatus M49 Petalomonas planus UB22 Rhynchobodo sp. Phacus parvulus Anisonema sp. G1 Rhynchomonas nasuta Dinema sulcatum Calkinsia aureus Notosolenus ostium Peranema sp. AY048919 Environmental sequence GQ330643 Environmental sequence EF100248 Environmental sequence HM749952 Urceolus sp. UB24 Anisonema sp. PP1 Environmental sequence AY821958 Anisonema/Dinema sp. U3 Dinema/Heteronema sp. CD5 Neometanema sp. BP6 Neometanema sp. SDB8 Distigma curvatum Petalomonas sphagnophila GU477297 Ichthyobodo necator Neometanema larsenii SDB3 Neometanema parovale M51 Dinema sp. UB26 Environmental sequence AY821957 Petalomonas sphagnophila GU477295 Environmental sequence AB505539 Anisonema acinus Anisonema sp. SAL5 Dinema litorale UB10 Menoidium pellucidum Environmental sequence DQ310255 Heteronema sp. PP2 Rhabdomonas incurva Ploeotia costata (marine) Bihospites bacati isolate 1 97 100 53 100 100 52 100 100 100 57 100 53 100 53 95 54 100 78 91 67 100 100 82 60 100 100 100 50 100 50 100 80 100 100 100 100 100 100 100 100 89 52 93 51 97 59 86 100 92 100 100 100 100 51 99 100 53 100 100 95 98