Supplementary Material Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

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Supplementary Material

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Racquel A. Singh¹, Vittorio Boscaro¹, Erick R. James¹, Anna Karnkowska¹, Martin Kolisko¹, Gregory S. Gavelis¹,Noriko Okamoto¹, Javier del Campo¹, Rebecca Fiorito¹, Elisabeth Hehenberger¹, Nicholas A. T. Irwin¹, Varsha Mathur¹, Rudolf H. Scheffrahn², Patrick J.

Keeling^1,*

1 Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada

2 Fort Lauderdale Research & Education Center, University of Florida, Davie, Florida, USA

*Corresponding author; pkeeling@mail.ubc.ca

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Supplementary Figure Legends

Supplementary Figure S1. Bayesian phylogeny of SSU rRNA sequences of the class Cristamonadea (1527 bp) including the sequence from M. yanesha. The tree is rooted with sequences from the closest known relatives to the Cristamonadea, members of the class Tritrichomonadea. Sequences obtained in this study are in bold type. Taxon names include GenBank accession numbers and the name of the termite host. Numerical values at nodes indicate Bayesian posterior probability (where greater than 0.90) and % ML bootstrap support (out of 1,000 replicates, where in agreement with the Bayesian topology and greater than 60%).

Sequences assigned to or likely belonging to the joeniid genera Joenia and Joenina are denoted by a thick vertical bar.Tree figures were generated using the FigTree software v1.4.4

(http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Illustrator (https://adobe.com/products/illustrator).

Supplementary Figure S2. Bayesian phylogeny of SSU rRNA sequences of the class

Cristamonadea (1526 bp) including the sequence from Joenoides intermedia. The tree is rooted with sequences from the closest known relatives to the Cristamonadea, members of the class Tritrichomonadea. Sequences obtained in this study are in bold type. Taxon names include GenBank accession numbers and the name of the termite host. Numerical values at nodes indicate Bayesian posterior probability (where greater than 0.90) and % ML bootstrap support (out of 1,000 replicates, where in agreement with the Bayesian topology and greater than 60%).

Sequences assigned to or likely belonging to the joeniid genera Joenia and Joenina are denoted by a thick vertical bar.Tree figures were generated using the FigTree software v1.4.4

(http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Illustrator (https://adobe.com/products/illustrator).

Supplementary Video S1. Differential interference contrast (DIC) microscopy video clips of new Devescovina, Macrotrichomonas, and Runanympha specimens. Symbionts were observed with an Axioplan 2 DIC microscope (Zeiss) and multiple cells of each symbiont species were filmed with a 3CCD HD video camera XL H1S (Canon).

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0.05

GQ254637 Simplicimonas similis (Uroplatus lineatus)

AB326380 Deltotrichonympha operculata (Mastotermes darwiniensis) FJ986219 Coronympha valentinia (Incisitermes snyderi)

FN377762 Devescovina sp. (Cryptotermes longicollis)

FJ986222 Coronympha mackinnonia (Incisitermes schwarzi)

DQ855404 Stephanonympha nelumbium (Cryptotermes domesticus) MT975291 Devescovina aymara (Rugitermes sp.)

AB458862 Macrotrichomonas sp. (Glyptotermes satsumensis)

AF052702 unidentified symbiont (Porotermes adamsoni)

AB032225 unidentified symbiont (Neotermes koshunensis)

AY063294 Calonympha grassii (Cryptotermes brevis)

AY063293 Metacoronympha senta (Incisitermes snyderi)

GQ254638 Simplicimonas moskowitzi (Chamaeleo cristatus)

FN377804 unidentified symbiont (Neotermes jouteli)

AB032224 unidentified symbiont (Neotermes koshunenis)

MT975292 Macrotrichomonas ashaninka (Comatermes perfectus)

HQ215840 unidentified symbiont (Neotermes castaneus)

FN377765 Devescovina sp. (Cryptotermes dudleyi)

AB458860 Foaina nana (Cryptotermes domesticus)

AB032214 unidentfied symbiont (Cryptotermes domesticus) DQ855405 Caduceia versatilis (Cryptotermes cavifrons)

AF215858 unidentified symbiont (Kalotermes flavicollis)

DQ855403 Stephanonympha nelumbium (Cryptotermes domesticus) MT975295 Runanympha illapa (Comatermes perfectus)

JX679411 Coronympha clevelandii (Incisitermes immigrans)

AB326382 Joenia sp. (Kalotermes flavicollis)

KJ493791 Macrotrichomonoides restis (Neotermes jouteli)

FJ986220 Coronympha sutherlandia (Incisitermes banksi)

FN377759 Devescovina lepida (Neotermes castaneus)

FN377802 unidentified symbiont (Cryptotermes havilandi)

HQ215836 Calonympha chia (Neotermes castaneus)

HQ215837 Snyderella kirbyi (Calcaritermes nigriceps) FN377801 unidentified symbiont (Cryptotermes havilandi)

AB458861 Gigantomonas herculea (Hodotermes mossambicus) U17506 Metadevescovina polyspira (Pterotermes occidentis)

MT975296 Runanympha pacha (Comatermes perfectus)

AB458855 Joenina pulchella (Porotermes adamsoni)

FN377766 Devescovina sp. (Cryptotermes havilandi)

AB032223 unidentified symbiont (Neotermes koshunensis) AF052696 unidentified symbiont (Cryptotermes dudleyi)

AJ583378 Deltotrichonympha nana (Mastotermes darwiniensis) AB458857 Stephanonympha sp. (Cryptotermes cavifrons)

AB032222 unidentified symbiont (Neotermes koshunensis) FM160645 Metadevescovina modica (Incisitermes marginipennis)

MT975290 Devescovina sapara (Rugitermes laticollis) FN377761 Devescovina arta (Neotermes castaneus)

KJ831625 Kofoidia loriculata (Paraneotermes simplicornis) KJ493793 Metadevescovina sp. (Neotermes jouteli)

AB458863 Macrotrichomonas sp. (Glyptotermes fuscus) KJ493792 Metadevescovina sp. (Neotermes jouteli)

AY055799 Tritrichomonas foetus (Bos taurus)

AB458859 Devescovina sp. (Glyptotermes fuscus)

X87132 Metadevescovina extranea (Mastotermes darwiniensis)

AB032220 unidentified symbiont (Glyptotermes fuscus)

FN377803 unidentified symbiont (Neotermes castaneus)

AJ583377 Mixotricha paradoxa (Mastotermes darwiniensis) AB458858 Devescovina sp. (Neotermes koshunensis) FM956077 Metadevescovina modica (Incisitermes marginipennis)

KJ493790 Macrotrichomonas lightii (Paraneotermes simplicicornis)

AY055803 Tritrichomonas nonconforma (Anolis bartschi) AJ132467 Koruga bonita (Mastotermes darwiniensis)

AY055802 Tritrichomonas augusta (Lacerta vivipara)

KJ831624 Kofoidia loriculata (Paraneotermes simplicornis)

MT975293 Macrotrichomonas secoya (Calcaritermes temnocephalus) U17504 Coronympha octonaria (Incisitermes snyderi)

FN377763 Devescovina sp. (Cryptotermes secundus)

MT975294 Macrotrichomonas yanesha (Calcaritermes rioensis)

HQ215838 Snyderella swezyae (Cryptotermes cylindroceps) FJ986221 Coronympha koidzumia (Incisitermes milleri)

AB032221 unidentified symbiont (Neotermes koshunensis)

AB458854 Joenia annectens (Kalotermes flavicollis)

HQ593144 Snyderella yamini (Calcaritermes nearcticus) AB183885 unidentified symbiont (Incisitermes minor)

0.98/66

1.0/100 1.0/99 1.0/99

1.0/100 1.0/100

1.0/76

1.0/98

1.0/94 1.0/99

1.0/100

1.0/97 1.0/100

1.0/83

1.0/99 1.0/100

1.0/97

1.0/100

1.0/71 1.0/97

0.98/73 1.0/86

0.93/95

1.0/100 1.0/96

1.0/100

0.98/63

1.0/98

1.0/85

1.0/99

1.0/100

1.0/95 1.0/82 1.0/80

1.0/73

1.0/79

1.0/64

1.0/100

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1.0/100 0.97/77

1.0/100

AB183884 unidentified symbiont (Incisitermes minor)

AY063290 Snyderella tabogae (Cryptotermes cavifrons)

CRISTAMONADEA TRITRICHOMONADEA (outgroup)

Devescovina

Macrotrichomonas Runanympha

Supplementary Figure S1.

Joenia

Joenina

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CRISTAMONADEA TRITRICHOMONADEA (outgroup)

Devescovina

Macrotrichomonas Runanympha

Joenia

0.06

MT975296 Runanympha pacha (Comatermes perfectus)

GQ254637 Simplicimonas similis (Uroplatus lineatus)

AB032224 unidentified symbiont (Neotermes koshunensis) KJ493793 Metadevescovina sp. (Neotermes jouteli)

KJ831624 Kofoidia loriculata (Paraneotermes simplicicornis)

AB032214 unidentified symbiont (Cryptotermes domesticus) AB032225 unidentified symbiont (Neotermes koshunensis)

GQ254638 Simplicimonas moskowitzi (Chamaeleo cristatus)

MT975295 Runanympha illapa (Comatermes perfectus)

AB032220 unidentified symbiont (Glyptotermes fuscus)

U17504 Coronympha octonaria (Incisitermes snyderi) KJ493791 Macrotrichomonoides restis (Neotermes jouteli)

KJ831625 Kofoidia loriculata (Paraneotermes simplicicornis)

AF052702 unidentified symbiont (Porotermes adamsoni) AB458854 Joenia annectens (Kalotermes flavicollis)

AJ132467 Koruga bonita (Mastotermes darwiniensis) FN377766 Devescovina sp. (Cryptotermes havilandi)

FN377761 Devescovina arta (Neotermes castaneus)

FJ986222 Coronympha mackinnonia (Incisitermes schwarzi) JX679411 Coronympha clevelandii (Incisitermes immigrans) KJ493792 Metadevescovina sp. (Neotermes jouteli)

FN377765 Devescovina sp. (Cryptotermes dudleyi)

AY063290 Snyderella tabogae (Cryptotermes cavifrons) AB458860 Foaina nana (Cryptotermes domesticus)

HQ215837 Snyderella kirbyi (Calcaritermes nigriceps)

AY055802 Tritrichomonas augusta (Lacerta vivipara)

MT975292 Macrotrichomonas ashaninka (Comatermes perfectus) AB458858 Devescovina sp. (Neotermes koshunensis)

AB458856 Joenoides intermedia (Hodotermes mossambicus) DQ855405 Caduceia versatilis (Cryptotermes cavifrons)

X87132 Metadevescovina extranea (Mastotermes darwiniensis)

DQ855403 Stephanonympha nelumbium (Cryptotermes domesticus) FJ986219 Coronympha valentinia (Incisitermes snyderi)

AB032222 unidentified symbiont (Neotermes koshunensis) HQ215836 Calonympha chia (Neotermes castaneus) MT975291 Devescovina aymara (Rugitermes sp.)

AB458861 Gigantomonas herculea (Hodotermes mossambicus) FN377763 Devescovina sp. (Cryptotermes secundus)

MT975293 Macrotrichomonas secoya (Calcaritermes temnocephalus) AB032221 unidentified symbiont (Neotermes koshunensis)

FN377801 unidentified symbiont (Cryptotermes havilandi) FN377804 unidentified symbiont (Neotermes jouteli)

FJ986221 Coronympha koidzumia (Incisitermes milleri)

MT975290 Devescovina sapara (Rugitermes laticollis)

KJ493790 Macrotrichomonas lightii (Paraneotermes simplicicornis)

AB458857 Stephanonympha sp. (Cryptotermes cavifrons) AB183885 unidentified symbiont (Incisitermes minor)

FM160645 Metadevescovina modica (Incisitermes marginipennis)

AF052696 unidentified symbiont (Cryptotermes dudleyi)

FJ986220 Coronympha sutherlandia (Incisitermes banksi)

AY063294 Calonympha grassii (Cryptotermes brevis)

HQ593144 Snyderella yamini (Calcaritermes nearcticus) FN377762 Devescovina sp. (Cryptotermes longicollis)

AB458862 Macrotrichomonas sp. (Glyptotermes satsumensis)

AY055799 Tritrichomonas foetus (Bos taurus)

FN377759 Devescovina lepida (Neotermes castaneus) FM956077 Metadevescovina modica (Incisitermes marginipennis)

AB458855 Joenina pulchella (Porotermes adamsoni)

AB458863 Macrotrichomonas sp. (Glyptotermes fuscus) AB183884 unidentified symbiont (Incisitermes minor)

FN377803 unidentified symbiont (Neotermes castaneus)

AJ583377 Mixotricha paradoxa (Mastotermes darwiniensis)

HQ215838 Snyderella swezyae (Cryptotermes cylindroceps)

DQ855404 Stephanonympha nelumbium (Cryptotermes domesticus) AB458859 Devescovina sp. (Glyptotermes fuscus)

AY055803 Tritrichomonas nonconforma (Anolis bartschi)

AB032223 unidentified symbiont (Neotermes koshunensis) AJ583378 Deltotrichonympha nana (Mastotermes darwiniensis) U17506 Metadevescovina polyspira (Pterotermes occidentis)

HQ215840 unidentified symbiont (Neotermes castaneus)

AY063293 Metacoronympha senta (Incisitermes snyderi)

FN377802 unidentified symbiont (Cryptotermes havilandi)

AB326380 Deltotrichonympha operculata (Mastotermes darwiniensis) 1.0/97

1.0/83 1.0/82

1.0/100 1.0/97 1.0/100 1.0/71

0.93/95 1.0/100 1.0/97

1.0/97 1.0/100

1.0/92 1.0/99 1.0/85 1.0/99

1.0/100

1.0/76

1.0/89

0.97/64

1.0/99

1.0/100 1.0/63

1.0/97 1.0/100 1.0/96

0.99/69

1.0/100 1.0/89

0.98/76 1.0/73

1.0/100 1.0/100

1.0/100 1.0/100 1.0/100 1.0/95

0.99/70 1.0/100

1.0/100 1.0/95

1.0/98 1.0/100

Supplementary Figure S2.

Joenina

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Supplementary Tables

Supplementary Table S1. Mean pairwise identities of SSU rRNA sequences within new Cristamonadea species and across species. Mean pairwise identity values across species are calculated between each new cristamonad specimen and the sequence with which the species shares the highest mean pairwise identity (also listed in the table). Sequences that were obtained in this study are marked with an asterisk (*) whereas sequences collected from Genbank are presented with Genbank accession numbers.

Species Mean pairwise distance of sequences within

species

Mean pairwise distance to closest sequence in the tree

Closest sequence in the tree

Devescovina sapara 99.21 % 97.43% FN377761.1 –

Devescovina arta

Devescovina aymara 98.51% 94.21% FN377763.1 –

Devescovina sp.

Macrotrichomonas

ashaninka 97.62% 93.74% AB458862.1 –

Macrotrichomonas sp.

Macrotrichomonas

secoya 98.12% 95.77% AB458862.1 –

Macrotrichomonas sp.

Macrotrichomonas

yanesha 99.13% 89.96% *Macrotrichomonas

ashaninka sequence 14_50_38Mac17_32

Runanympha illapa 97.46% 95.59% *Runanympha pacha

sequence 19_10_43_17Placo_8

Runanympha pacha 96.82% 95.58% *Runanympha illapa

sequence 18_10_26_35Joe_37

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