Nodulated tree legumes and their symbiotic Bradyrhizobium in African and South-American Tropical Rainforests

Y. Prin, B. Dreyfus, C. LeRoux, G. Bena, M. Diabaté, P. de Lajudie, A. Bâ, S. M. de Faria, A. Munive & A. Galiana

Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), Campus International de Baillarguet, 34398 Montpellier Cedex 5, France. [email protected] The Leguminosae family • 650 genera, 18,000 • Three sub-families: – Caesalpinioideae (about 26% nodulated) • 1,900 species (woody perennials, tropics) – Mimosoideae (about 95% nodulated) • 2,700 species (woody perennials, tropics) – Papilionoideae (about 97% nodulated) • 13,400 species (herbaceous to trees, temperate to tropical) A wide diversity of nodule types in tropical rainforest trees from West Africa (Guinea) and South America (French Guyana) Occcurence of nodulation in leguminous species of the primary rain forest in Guinea (West Africa)

Sub-family / Tribe /Species habit Location Nodulation New species New and height observed observed CAESALPINIOIDEAE CAESALPINIEAE Bussea occidentalis TŹ; 25 m Ziama - yes yes CChidlowiahidlowia san gsanguineauinea TT ;; 25 m m ZiamaZiama ++ yesŅ yesŅ Delonix regia T ; 12 m Nimba - no no Erythrophleum ivorense T ; 40 m Ziama + Ņ Ņ Erythrophleum suaveolens T ; 40 m Bˇ ro + Ņ ŅŹ Mezoneuron benthamianum S ; 5 m Diˇc kˇ - yes ŅŹ CASSIEAE Cassia aubrevillei TŹ; 15 m Nimba - yes no Cassia sieberiana TŹ; 15 m Bˇ ro - Ņ Ņ Chamaecrista mimosoides H ; 1.5 m Nimba + no Ņ Dialium aubrevillei T ; 30 m Ziama - yes Ņ Dialium dinklagei T ; 20 m Diˇc kˇ - ć Ņ Dialium guineense T ; 15 m Bˇ ro - no Ņ Dialium pobeguinii T ; 15 m Ziama - yes Ņ Distemonanthus T ; 35 m Ziama - Ņ yes benthamianus Duparquetia orchidacea SŹ; 8 m Nimba - Ņ ŅŹ Senna podocarpa S ; 5 m Nimba - Ņ no Senna siamea T ; 20 m Bˇ ro - no Ņ CERCIDEAE Bauhinia thonninguii T ; 9 m Pic de Fon - yes no Griffonia simplicifolia SŹ; 5 m NÕzˇ rˇk orˇ - Ņ yes DETARIEAE Afzelia africana T ; 30 m Bˇ ro - no no Afzelia bella var.gracilior T ; 35 m Ziama - yes Ņ Copaifera salikounda T ; 30 m Ziama - Ņ Ņ Daniellia ogea T ; 30 m Bˇ ro - ŅŹ yes Daniellia thurifera T ; 35 m Ziama - Ņ Ņ Detarium heudelotianum T ; 20 m - Ņ Ņ Detarium macrocarpum T ; 25 m Diˇc kˇ - Ņ Ņ Detarium senegalense T ; 25 m Bˇ ro - ŅŹ Ņ Guibourtia copallifera T ; 25 m Ziama - Ņ no Guibourtia ehie T ; 30 m Ziama - Ņ Ņ Guibourtia leonensis T ; 30 m Diˇc kˇ - Ņ Ņ Tessmannia baikiaeoides T ; 10 m Nimba - Ņ yes AMHERSTIEAE crassifolia T ; 20 m Bˇ ro - yes no Anthonotha fragrans T ; 30 m Ziama - no Ņ Anthonotha macrophylla T ; 12 m Diˇc kˇ - yes Ņ Cryptosepalum tetraphyllum T ; 25 m Ziama - Ņ Ņ Gilbertiodendron bilineatum T ; 15 m Ziama - Ņ Ņ Gilbertiodendron limba T ; 15 m Ziama - Ņ Ņ Nodulation of leguminous species from Guinea (continued)

Sub-family / Tribe /Species Plant habit Location Nodulation New New and height species genus observed observed MIMOSOIDEAE PARKIEAE Parkia bicolor T ; 30 m Nimba - yes no Parkia biglobosa T ; 15 m Bˇ ro - no Ņ Pentaclethra macrophylla T ; 25 m Ziama + Ņ Ņ MIMOSEAE Adenopodia scelerata V ; 30 m Nimba - yes yes Aubrevillea kerstingii T ; 30 m Ziama - Ņ Ņ AubrevilleaAubrevillea platycarpaplatycarpa TŹ ; 30 m ZiamaZiama ++ yesŅ yes Ņ Calpocalyx aubrevillei TŹ; 25 m Ziama + Ņ no Calpocalyx brevibracteatus T ; 25 m Ziama - Ņ Ņ Dichrostachys cinerea S ; 8 m Bˇ ro + no ŅŹ Entada africana TŹ; 12 m Bˇ ro + yes Ņ Entada gigas VŹ; 30 m Diˇc kˇ + ŅŹ Ņ Entada mannii S ; 15 m Ziama + Ņ Ņ Mimosa invisa S ; 0.5 m Diˇc kˇ + no Ņ Newtonia aubrevillei T ; 30 m Ziama - yes Ņ Newtonia duparquetiana TŹ; 25 m Nimba - Ņ Ņ Piptadeniastrum africanum TŹ; 40 m Ziama + no no TetrapleuraTetrapleura tetrapteratetraptera T ; 20 m ZiamaZiama + yeyess yesyes Xylia evansii T ; 25 m Diˇc kˇ + Ņ no INGEAE Albizia adianthifolia T ; 20 m Diˇc kˇ + no no Albizia altissima T ; 20 m Ziama + yes Ņ Albizia ferruginea T ; 20 m Bˇ ro + no Ņ Albizia glaberrima T ; 15 m Nimba + Ņ Ņ Albizia zygia T ; 20 m Ziama + Ņ Ņ Samanea dinklagei T ; 20 m Ziama + yes Ņ Nodulation of leguminous species from Guinea (continued) Sub-family / Tribe /Species Plant habit Location Nodulation New species New genus and height observed observed PAPILIONOIDEAE SWARTZIEAE Bobgunnia fistuloides T ; 15 m Diˇc kˇ - yes no SOPHOREAE AAmphimasmphimas pter ocapterocarpoidesrpoides T ; 25 m Z i ama + + ye s yes ye s yes Angylocalyx oligophyllus S ; 4 m Nimba - Ņ Ņ Baphia nitida T ; 10 m Nimba + no no Baphia capparidifolia T ; 8 m Diˇc kˇ + yes Ņ DALBERGIEAE Dalbergia afzeliana V ; 35 m Nimba - yes no Dalbergia bignonae V ; 25 m Bˇ ro + Ņ Ņ Dalbergia hostilis V ; 15 m Ziama + Ņ Ņ Dalbergia oblongifolia S ; V ; 10 m Diˇc kˇ - Ņ Ņ PterocarpusDalbergia saxatili erinaceuss S ; V ; 2 0 m N im ba - + Ņ Ņ PterocarpusPterocarpus erina mildbraediiceus TŹ; 15 m Bˇ ro ++ no Ņ Pterocarpus mildbraedii T ; 20 m Ziama + yes Ņ PterocarpusPterocarpus san santalinoidestalinoides TŹ; 15 m Ziama - - no Ņ MILLETTIEAE Dalbergiella welwitschii S ; V ; 20 m Nimba - yes no Leptoderris brachyptera S ; V ; 20 m Ziama + Ņ Ņ Leptoderris fasciculata S ; V ; 20 m Nimba + Ņ Ņ Lonchocarpus cyanescens S ; V ; 30 m Nimba - Ņ Ņ Millettia dinklagei S ; V ; 15 m Nimba + Ņ Ņ Millettia griffoniana T ; 15 m Nimba - Ņ Ņ Millettia lucens S ; V ; 15 m Bˇ ro - Ņ Ņ Millettia rhodantha T ; 12 m Ziama + Ņ Ņ Millettia zechiana S ; 10 m Ziama + Ņ Ņ Platysepalum hirsutum S ; V ; 20 m Nimba - Ņ yes Tephrosia flexuosa S ; 1.5 m Nimba - Ņ no Droogmansia scaettaiana H ; 2 m Nimba + yes no PHASEOLEAE Dioclea reflexa S ; V ; 15 m Nimba - no no Dolichos dinklagei S ; 1.5 m Nimba - yes Ņ Dolichos tonkouiensis SŹ; 1. 5 m Nimba + Ņ Ņ Erythrina milbraedii T ; 30 m Ziama + Ņ Ņ Erythrina senegalensis S ; 8 m Diˇc kˇ + no Ņ Mucuna flagellipes SŹ;V Ź;5 m Bˇ ro - yes Ņ Mucuna poggei S ; V ; 20 m Bˇ ro + no Ņ Physostigma venenosum S ; V ; 6 m Bˇ ro - yes yes AESCHYN OMENEAE Kotschya lutea S ; 2 m Nimba + yes no Kotschya ochreata S ; 2.5 m Nimba + Ņ Ņ Ormocarpum megalophyllum S ; 1. 5 m Nimba - Ņ Ņ CROTALARIEAE Crotalaria lathyroides S ; 2 m Bˇ ro + no Ņ Occurence of nodulation in leguminous tree species of the Amazonian bassin (French Guyana) Sub-family / Tribe /Species Nodulation New species New genus observed observed CAESALPINIOIDEAE CAESALPINIEAE Dimorphandra polyandra + yes no Tachigali melinonii + no Ņ Tachigali myrmecophila + yes Ņ CASSIEAE Chamaecrista diphylla + no no DETARIAE bifolium - yes no MIMOSOIDEAE MIMOSEAE Stryphnodendron + no no pulcherrimum INGEAE Hydrochorea corymbosa + no no Inga disticha + Ņ Ņ Inga sertulifera + yes Ņ Zygia latifolia + no Ņ Zygia cataracteae + Ņ Ņ PAPILIONOIDEAE SWARTZIEAE Bocoa prouacensis - no no Swartzia polyphylla + Ņ Ņ SOPHOREAE Diplotropis purpurea + no no Ormosia coutinhoi + Ņ Ņ SpirotropisSpirotropis lo ngilongifoliafolia ++ yyeses yesyes DALBERGIEAE Andira inermis + no no Dalbergia monetaria + Ņ Ņ Hymenolobium flavum + Ņ Ņ Machaerium lunatum + Ņ Ņ Paramachaerium + Ņ Ņ ormososioides Pterocarpus officinalis + Ņ Ņ Vatairea erythrocarpa - Ņ Ņ BRONGNIARTIEAE Poecilanthe hostmannii + no no PHASEOLEAE Clitoria fairchildiana + no no Erythrina amazonica + yes Ņ

Confirmation of nodulation status in nursery

Piptadeniastrum africanum Chidlowia sanguinea

Erythrophleum ivorense Tetrapleura tetraptera g Rickettsia rickettsii Neisseria meningitidis Methylobacterium extorquens Methylobacterium nodulans Azoarcus indigens Bradyrhizobium japonicum Alcaligenes eutrophus Bradyrhizobium sp Ralstonia taiwanensis Paracoccus denitrificans Burkholderia sp. STM678 Agrobacterium rhizogenes Burkholderia graminis Rhizobium leguminosarum Burkholderia brasilense Sinorhizobium meliloti Burkholderia sp. STM815 Mesorhizobium loti Burkholderia cepacia Azorhizobium caulinodans Bordetella pertusis Xanthobacter agilis Azospirillum brasilense Nitrosovibrio tenuis a Spirillum volutans b

d e

Phylogeny of 16S rDNA 0.01 changes Trifolium, Pisum, Vicia ... Trigonella, Lacking Inverted Melilotus, Repeat Clade Medicago...

Astragalus, Clianthus, Onobrychis ...

Robinia, Gliricidia...

Robinioids Lotus, Anthyllis, Sesbania... Phaseolus, Macrotyloma, Vigna... Glycine, Pueraria...

Milletia, Tephrosia... Millettioids Indigofera Mirbelioids Bossiaea, Daviesia...

Pterocarpus, Stylosanthes, Arachis...

Aeschynomene Dalbergia, Machaerium... Dalbergioids Chaetocalyx, Nissolia Papilionioids Zornia... Amorpha, Dalea...

Phylogeny of Leguminosae based on parsimony analyses of matK gene sequences, adapted from Wojciechowski et al. (2004). Genista, Lupinus, Cytisus Bradyrhizobium, Mesorhizobium, Rhizobium, Ochrobactrum Crotalaria Methylobacterium Sophora...

Genistoids Bowdichia, Diplotropis, Nodulated, by ? Hovea...

Vataira Uribea... Non-nodulated Papilionioids Cladrastris Cladastris Non-nodulated Myrospermum, Non-nodulated or no nodulation report Dipteryx...

Swartzia Ateleia... Nodulated, by ?

Albizia, Calliandra, Bradyrhizobium, Rhizobium, Sinorhizobium, Mesorhizobium, Acacia, Allorhizobium Neptunia, Leucaena, Prosopis...

Mimosoids Tetrapleura, Entada, Bradyrhizobium, nodulated by ?, or non-nodulated Xylia... Cercidium, Dinizia... Non-nodulated Senna Caesalpinioids crown clade Non-nodulated or Bradyrhizobium Chamaecrista, Caesalpinia... Gleditsia, Non-nodulated Ceratonia...

Berlinia, Prioria, Non-nodulated or no nodulation report Tessmannia, Cercis...

Polygala californica Quillaja saponaria Vauquelinia californica OUTGROUP Suriana maritima

Phylogeny of Leguminosae based on parsimony analyses of matK gene sequences, adapted from Wojciechowski et al. (2004). STM803 Andira inermis STM757 Dalbergia monetaria STM827 Dalbergia monetaria STM756 Dalbergia monetaria ITS phylogeny of Bradyrhizobium rDNA STM833 Dalbergia monetaria STM823 Dalbergia monetaria Group A STM835 Dalbergia monetaria STM938 Andira inermis STM826 Dalbergia monetaria STM845 Dalbergia monetaria Plant Genus species: Amazonian species STM834 Dalbergia monetaria STM838 Dalbergia monetaria STM788 Dalbergia monetaria STM723 Samanea dinklagei Plant Genus species: Guinean species STM741 Erythrophleum ivorensis STM724 Samanea dinklagei Group B STM929 Millettia zechiana STM930 Millettia zechiana STM928 Millettia zechiana STM924 Albizia zygia STM739 Albizia sassa Group C STM927 Millettia zechiana STM726 Albizia zygia Bradyrhizobium japonicum T Genomic species I Bradyrhizobium liaoningense T Genomic species III Bradyrhizobium sp Genomic species IV

Bradyrhizobium sp Genomic species V

STM732 Pterocarpus mildbraedii STM932 Piptadeniastrum africanum STM933 Piptadeniastrum africanum STM722 Pterocarpus mildbraedii STM922 Albizia ferruginea STM923 Albizia zygia STM926 Millettia zechiana STM916 Albizia adianthifolia Group D STM934 Pterocarpus mildbraedii STM931 Millettia rothonda STM725 Albizia ferruginea STM795 Millettia zechiana STM790 Millettia zechiana STM851 Millettia zechiana STM792 Piptadeniastrum africanum ORS170 Faidherbia albida ORS1812 Abrus pulchellus STM744 Erythrina amazonica STM745 Erythrina amazonica STM840 Indigofera sp Group E STM758 Andira inermis STM749 Indigofera sp STM783 Andira inermis STM750 Erythrina amazonica Bradyrhizobium elkaniiT Genomic species II Bradyrhizobium sp Genomic species X STM751 Erythrina amazonica STM786 Erythrina amazonica STM841 Erythrina amazonica Group F STM728 Chidlowia sanguinea STM794 Chidlowia sanguinea STM727 Chidlowia sanguinea Group G STM731 Chidlowia sanguinea Bradyrhizobium sp Genomic species XI STM759 Indigofera sp

STM257 Albizia ferruginea Bradyrhizobium sp Genomic species VII Bradyrhizobium sp Genomic species IX

Bradyrhizobium sp Genomic species VI

Bradyrhizobium sp Genomic species VIII Azorhizobium cluster Mesorhizobium cluster Inoculation trials in Republic of Guinea

- Inoculation in nursery (50 replicates) -Transplantation to the field after 3 months - Field growth response after 4 months Effect of rhizobial inoculation on growth of Albizia spp. and Samanea dinklagei (after 4 months)

Height

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A. altissima x STM916 A. ferruginea x STM922 A. adianthifolia x STM916 S. dinklagei x STM922 A. zigia x STM923 Effect of rhizobial inoculation on growth of Millettia spp. (after 4 months) Height

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M. Zechiana x M. rhodantha x M. zechiana x STM926 STM931 STM851 Effect of rhizobial inoculation on growth of Erythrophleum spp. (after 4 months) Height

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E. guineensis x E. ivorensis x STM934 STM934 CONCLUSION

• Tropical rainforests = huge reservoirs of symbioses • Predominance of the genus Bradyrhizobium • Need for collaborative research between plant and bacteria phylogenists • Large potential of applications for reforestation with endangered species - IRAG, BP 1523, Conakry, République de Guinée - Universita Autonoma del Puebla, Mexico - Embrapa/Agrobiologia, BR465, Seropedica, Brazil - LSTM, Montpellier, France