One versus many independent assemblies of symbiotic nitrogen fixation in flowering plants

Abstract

Some species of legumes and nine other flowering plant families form symbioses with bacteria that fix atmospheric nitrogen within specialized plant structures called nodules. How and how often nodulation symbiosis originated has implications for engineering symbiotic nitrogen fixation in non-legume crops. The prevailing hypothesis of a single origin with massive parallel losses has been challenged in a phylogenomic study favoring 16 origins and 10 losses. Nodulation has been assembled once or many times from existing processes (e.g., mycorrhizal symbiosis) and therefore almost nothing about it is truly novel. Because any feature of nodulation can be explained either as divergence from a common origin or as convergence in unrelated taxa, tests are needed that can distinguish whether assembly of homologous components has occurred uniquely or convergently. Much needs to be learned about nodulation symbioses across the proposed independent origins, especially involving the master nodulation transcription factor, Nodule Inception (NIN).

Fig. 1. Multiple origins and losses of nodulation hypothesized by Kates et al. in the four orders of the NFNC.

a, b The two sister subfamilies of Fabaceae/Fabales, c Rosales, d Fagales, e Cucurbitales, with illustrations of floral and nodule diversity across nodulating lineages (note that nodule photos are not available for some taxa). Red numbered circles at nodes of phylogenetic trees indicate nodulation origins, and blue numbered X’s mark losses; numbers correspond to those in Kates et al.. Gain 3 in fig. 1 (a) is gray because Dussia has recently been identified as a non-nodulator. Photo credits: Lathyrus—Modified photo taken from Wikimedia Commons user Krzysztof Ziarnek (Kenraiz) from Openverse.org https://openverse.org/image/7758c4f4-c297-439b-bc26-bd569c062470?q=Lathyrus+japonicus&p = 20. CC BY-SA 3.0; Chorizema—Modified photo taken from Wikimedia Commons user jmcgross from Openverse.org https://openverse.org/image/74b0733e-8968-4a1f-9cfb-0d63ad654958?q=Chorizema&p = 3. CC BY 2.0; Baptisia—Modified photo taken from Flickr user scadwell from Openverse.org https://openverse.org/image/7b0c918e-f653-4380-bf8b-cec5a7268bfd?q=Baptisia&p = 10. CC BY-NC 2.0; Aeschynomene—Modified photo taken from Wikimedia Commons user Dinesh Valke (Thane, India) from Openverse.org https://openverse.org/image/d2183990-6a4e-4f80-acb8-7828ab4c8606?q=Aeschynomene&p = 20. CC BY-SA 2.0; Templetonia—Modified photo taken from Flickr user jeans_Photos from Openverse.org https://openverse.org/image/e38a4a70-191b-4797-80e2-430369178688. CC BY 2.0; Swartzia—Modified photo taken from Wikimedia Commons user Vojtěch Zavadil from Openverse.org https://openverse.org/image/4148c309-97d6-4397-8731-0d12db1af28d?q=Swartzia&p = 1. CC BY-SA 3.0; Mimosa—Modified photo taken from Flickr user João de Deus Medeiros from Openverse.org https://openverse.org/image/ad2bd259-bb73-4c53-b9f3-27afedec978e?q=Mimosa&p = 52. CC BY 2.0; Dimorphandra and Melanoxylon—Permission by Domingos Cardoso; Chamaecrista—Modified photo taken from Flickr user bob in swamp from Openverse.org https://openverse.org/image/89e3db92-edf3-4ef9-9cb4-afbfcff0ada0?q=Chamaecrista&p = 4. CC BY 2.0; Parasponia—Permission by Luuk Rutten; Ceanothus—Modified photo taken from Flickr user John Rusk from Openverse.org https://openverse.org/image/28ed838d-c3b0-4625-b219-05fec907bb10?q=Ceanothus&p = 2. CC BY 2.0; Discaria—Modified photo taken from Wikimedia Commons user Dick Culbert (Gibsons, B.C., Canada) from Openverse.org https://openverse.org/image/3e7ee66a-e7d4-403d-a23d-ee0e7b1771de?q=Discaria&p = 8. CC BY 2.0; Hippophae—Permission by Jan Thomas Johansson; Dryas—Modified photo taken from Wikimedia Commons user Krzysztof Ziarnek (Kenraiz) from Openverse.org https://openverse.org/image/2557c2d5-3a38-4324-a353-60faaa310c1d?q=Dryas+drummondii&p = 1. CC BY-SA 4.0; Alnus—Modified photo taken from Flickr user John Rusk from Openverse.org https://openverse.org/image/6ccfe034-cedf-4f24-8c22-d1130aa317f8?q=Alnus&p = 56. CC BY 2.0; Casuarina—Modified photo taken from Wikimedia Commons user Kevin Thiele (Perth, Australia) from Openverse.org https://openverse.org/image/ce9ef39d-4bb3-48ee-aa20-b70b252b7a89?q=Casuarina&p = 41. CC BY 2.0; Myrica—Modified photo taken from Wikimedia Commons user Hajotthu from Openverse.org https://openverse.org/image/75c68290-81d0-4942-85f4-a5d8ce5d0b6f?q=Myrica+gale&p = 11. CC BY-SA 3.0; Datisca—Modified photo taken from Wikimedia Commons user H. Zell from Openverse.org https://openverse.org/image/250d3f16-3764-4e40-9a50-dbde7a9c0730?q=Datisca&p=1. CC BY 3.0; Coriaria—Modified photo taken from Flickr user In Memoriam: Ecuador Megadiverso from Openverse.org https://openverse.org/image/e090111d-6a75-4a0d-98da-b3222d8a95db?q=Coriaria&p = 3. CC BY-NC-SA 2.0; Nodules: Lathyrus and Dimorphandra—Permission by Euan James; Chorizema and Templetonia—Permission by Julie Ardley; Baptisia—Permission by Jeff Doyle; Aeschynomene and Mimosa—Permission by Hukam Singh Gehlot; Swartzia—Permission by Domingos Cardoso; Chamaecrista—Permission by Eduardo Gross; Melanoxylon—Permission by Sergio de Faria; Parasponia—Permission by Luuk Rutten and Rene Geurts; Ceanothus—Permission by O’Dell et al.; Discaria and Coriaria—Permission by Luis G. Wall; Dryas—Permission by Dagmar Hann and Jessica Folgmann; Datisca, Casuarina, Alnus, Hippophae and Myrica—Permission by Nadia Binte Obaid and Katharina Pawlowski. Links to licenses for reuse restrictions: CC BY-SA 3.0: https://creativecommons.org/licenses/by-sa/3.0/. CC BY 2.0: https://creativecommons.org/licenses/by/2.0/. CC BY-NC 2.0: https://creativecommons.org/licenses/by-nc/2.0/. CC BY-SA 2.0: https://creativecommons.org/licenses/by-sa/2.0/. CC BY-SA 4.0: https://creativecommons.org/licenses/by-sa/4.0/. CC BY 3.0: https://creativecommons.org/licenses/by/3.0/. CC BY-NC-SA 2.0: https://creativecommons.org/licenses/by-nc-sa/2.0/.

Fig. 2. Stepwise recruitment of features of nodulation.

a The nodulation symbiosis is assembled by recruitment of modules from pre-existing complex functional traits (A–D; e.g., symbiosis signaling, lateral root development, etc.), each comprising several components (a₁, a₂, … d₃), beginning at t_o with the recruitment of component “a₂” of the “A” function for a new role (designated by italic font). Various other functions are recruited over a period of time (delta t) until at t_n a complete nodulation symbiosis evolves with the addition of component “d₂.” Functions a-d are shown as being recruited into a pre-nodulation trait with intermediate correlated states (ab and abc); gene or GRN duplication could produce components that could diverge from ancestral functions without such association, but this would not be “recruitment,” and would collapse delta t to 0. b Only the timeline of recruitment of a₂-d₂ differs between SNG and MUL, as illustrated for Medicago. For both SNG and MUL, t_o could be the same, but whereas under SNG the full nodulation symbiosis has evolved by the time of the most recent common ancestor (MRCA) of the NFNC (t_n >100 MYA), under MUL t_n does not occur until after the origin of legumes (~70 MYA) but before the origin of crown papilionoid legumes. For MUL, the precursor evolves prior to the NFNC MRCA, but all other components of the full RNS could evolve as quickly as in SNG, in this case, 60 and 70 MYA.

Fig. 3. Relationships between processes shared between nodulation and other functions under the two competing hypotheses for legumes (Medicago) and Betulaceae (Alnus).

a, b Single origin (SNG). a Nodulation originates in the NFNC ancestor (MRCA) by recruitment of modules from various processes (e.g., mycorrhizal signaling, lateral root development) as in Fig. 2a. Later, lineages leading to legumes and Betulaceae diverge, as shown by the slightly different colors (for convenience, the legume lineage is shown as retaining the ancestral darker coloration). Thus, recruited features have the potential to differentiate (e.g., specialize for nodulation vs. their previous role) prior to the divergence of legumes and Betulaceae. b Using recruitment of features from mycorrhizal signaling during the origin of nodulation in the NFNC MRCA as an example, there could be greater similarities between some features of mycorrhizal (brown box “M”) and nodulation symbiosis (green box “N”) in nodulating species of each lineage (many legumes; currently only Alnus in Betulaceae) than between either mycorrhizal or nodulation symbioses across species. c, d Multiple origins (MUL). c Divergence of taxa pre-dates independent origins of nodulation, therefore the individual processes have already diverged in the two taxa prior to their independent recruitment for nodulation, as shown by the slightly different colors. d Using recruitment of mycorrhizal signaling during the independent origin of nodulation in Medicago (legume) and Alnus (Betulaceae) as an example, divergence of the legume and Betulaceae lineages pre-dates convergent origins of nodulation, allowing time for details of mycorrhizal signaling to have diverged in the two lineages. This could be reflected in greater similarities between some features of nodulation and mycorrhizal symbiosis within each species than between either mycorrhizal or nodulation symbioses across species.

Fig. 4. Homologous and non-homologous gene recruitment inferred from TF binding sites.

a An ancestral gene, X_a (gray box, transcription start site shown by arrow), has a 5’ promoter region (narrow line) with two cis-regulatory elements (CREs) associated with two different functions (red and blue boxes). b In a descendant, the gene is recruited for nodulation by acquiring a NIN-binding CRE (green box) and is observed in nodulating species-1. c–e Nodulating species 2, 3, and 4 use gene X in nodulation symbiosis, also under the control of NIN; all three have the same conserved NIN-binding motif. However, only in gene X_n-2 is the NIN-binding motif in the same position relative to motifs for the original functions as in gene X_n-1. This can be taken as evidence for a single recruitment event (SNG) in the ancestor of the taxa with genes X_n-1 and X_n-2, but independent recruitment events in the taxa with genes X_n-3 and X_n-4 (MUL).