Les orchidées et leurs mycorhizes
M.-A. SELOSSE Muséum nat. d’Histoire naturelle, Paris Universités de Gdansk (Pologne) & Viçosa (Brésil) Les réseaux mycorhiziens
Mycohétérotrophie et réseaux
Mixotrophie et réseaux mycorhiziens
La difficile transition vers l’hétérotrophie
… due à un attachement fatal à la lumière ? MYCORRHIZAL SYMBIOSIS
… a symbiosis between two partners ECTO- MYCORRHIZAE
Asco- and Basidiomycetes on most trees in temperate regions ORCHID MYCORHIZAE
Intracellular pelotons formed within root cells, usually with « rhizoctonias"
(Beyrle et al. 1995) Orchid germination, usually involving rhizoctonias MYCORRHIZAL SYMBIOSIS
>85% of plants associate with soil fungi
Reciprocal nutrient exchanges + protection:
Sugars PLANT FUNGUS Water and N, P, K
Protection against soil stresses ITS BARCODING
genreGenus // ordreorder NiveauTaxonomic de résolution resolution taxonomique : : espèceSpecies / / variétésub-species genetSub-species (individus) / genet ITS
ITS 2 ITS 1 IGS 1 IGS 2 18 S 5.8 S 28 S 5 S 18 S
Ribosomal DNA sub-unit in fungi: circa 10 kpb environ 10 kpb Les réseaux mycorhiziens
Mycohétérotrophie et réseaux
Mixotrophie et réseaux mycorhiziens
La difficile transition vers l’hétérotrophie
… due à un attachement fatal à la lumière ? Mycoheterotrophs
Gentianaceae Ericaceae
Polygalaceae
Geosiridaceae Corsiaceae Lacandoniaceae Burmanniaceae Orchidaceae Petrosaviaceae Triuridaceae
(from Brundett, 2004) Mycoheterotrophs
Gentianaceae Ericaceae
Polygalaceae
Geosiridaceae Corsiaceae Lacandoniaceae Burmanniaceae Orchidaceae Petrosaviaceae Triuridaceae
(from Brundett, 2004) Neottia nidus-avis
Selosse et al., 2002, Mol. Ecol. 11, 1831 McKendrick et al., 2002, New Phytol. 145, 523 Selosse et al., 2002, New Phytol. 155, 183 ITS BARCODING
genreGenus // ordreorder NiveauTaxonomic de résolution resolution taxonomique : : espèceSpecies / / variétésub-species genetSub-species (individus) / genet ITS
ITS 2 ITS 1 IGS 1 IGS 2 18 S 5.8 S 28 S 5 S 18 S
Ribosomal DNA sub-unit in fungi: circa 10 kpb environ 10 kpb Neottia nidus-avis associated with Sebacinales… Neottia nidus-avis associated with Sebacinales… themselves ectomycorrhizal on nearby tree roots
Epipogium aphyllum
… with Inocybe species Roy et al., Annals Bot., 2009. Epipogium aphyllum Epipogium aphyllum … with Inocybe species Roy et al., Annals Bot., 2009. MYCOHETEROTROPHY
Exploiting the mycorrhizal network...
MYCOHETERO- GREEN TROPHIC PLANT TREE
FUNGUS Mycorrhizal Mycorrhizal association Carbon flow association
… as reflected in their spontaneous stable isotopes (13C and 15N) abundances Mycoheterotrophs
Gentianaceae Ericaceae
Polygalaceae
Geosiridaceae Corsiaceae Lacandoniaceae Burmanniaceae Orchidaceae Petrosaviaceae Triuridaceae
(from Brundett, 2004) Mycoheterotrophic Ericaceae: Hypopitys monotropa Tedersoo et al., 2007, Oecologia Autotrophs: Arctostaphylos uva-ursi, Picea abies, Melampyrum sylvaticum Ectomycorrhizal fungi, including Tricholoma myomyces, mycorrhizal on H. monotropa Wullschlaegelia calcrata Rhizomorphs (groups of hyphae) In 14 individuals from 4 sites: only saprotrophic fungi !
Marasmius Mycena
Other saprobes Gymnopus
All are white rot and litter decaying fungi No strict specificity at individual level d15N ‰
2
0 W. aphylla Green Leaf- plant decaying -2 fungi
-4
-6 -34 -32 -30 -28 -26 -24 -22
13 d C ‰ Martos et al., 2009 New Phytologist 184: 668-681 Les réseaux mycorhiziens
Mycohétérotrophie et réseaux
Mixotrophie et réseaux mycorhiziens
La difficile transition vers l’hétérotrophie
… due à un attachement fatal à la lumière ? Green orchids related to Epipactis helleborine mycoheterotrophs Epipactis helleborinedistans Epipactis distansatrorubens Epipactis atrorubensmicrophylla Epipactis microphyllapalustris EpipactisAphyllorchis palustriscaudata E.g. the Neottieae tribe, Aphyllorchis caudatamontana AphyllorchisLimodorum abortivum montana mycoheterotrophy LimodorumNeottia nidus abortivum-avis arose repeatedly, by Neottia ovatanidus-avis convergent evolution NeottiaCephalanthera ovata exigua Cephalanthera austinaeexigua among green species Cephalanthera longifoliaaustinae Cephalanthera damasoniumlongifolia Roy et al., BMC Biol. 2009 … that associate with Cephalanthera rubradamasonium CephalantheraThaia saprophytica rubra ectomycorrhizal fungi ThaiaPalmorchis saprophyticasp. Palmorchis Limodorum abortivum
Russula delica group Girlanda et al., Mol. Ecol., 2006 Ph. P. Pernot Cephalanthera damasonium & Cephalanthera longifolia Cephalanthera damasonium & Cephalanthera longifolia
C. longifolia C. damasonium Julou et al., 2005 Abadie et al., 2006
2 3 2 5 10 5 34 19 6 11
19
« Rhizoctonias » Ectomycorhizal fungi Saprophytes Plant endophytes or parasites Epipactis microphylla
Diverse ectomycorrhizal fungi, mainly truffles spp.
Pernot Selosse et al., Microb. Ph. P. Ph. P. Ecol., 2006 Epipactis microphylla associates with truffles
x 800 Microscopy and antibodies demon- strate that truffles are mycorrhizal (Selosse et al., 2006 x 1 700 Microbial. Ecol.) Tedersoo et al., 2007, Oecologia « Green » orchids: Listera ovata, Platanthera bifolia, Epipactis atrorubens MIXOTROPHY
Exploiting both the mycorrhizal network & photosynthesis...
MYCOHETERO- GREEN TROPHIC PLANT TREE
FUNGUS Mycorrhizal Mycorrhizal association Carbon flow association
… as reflected in their 13C &15N abundances Limodorum abortivum
A green orchid photosynthesizing below the compensation point
light dark 0
1
2
CO2 produced 3 mol.m -2.s-1
Girlanda et al., Mol. Ecol., 2006 In mixotrophic orchid species, achlophyllous variants, the albinos,
… survive as mycoheterotrophs!
Cephalanthera damasonium
(P. Pernot & F. Dusak) Epipactis purpurata
(A. Hasenfratz) A. Soulié
M. Roy
J.C. Claessens
S. Acker Gas exchange in C. damasonium
Net CO2 exchange
green
albino
PAR = photosynthetic active radiations Julou et al. 2005, New Phytol. IsotopesC. longifolia in (C.Abadie longifolia et al., 2006)
green albino green
Green autotrophic plants
Abadie et al. 2006, Botany Some green plants
- are mixotrophic and use mycorrhizal networks
- are predisposed to evolve mycohetero- trophy
- … with albinos as transitions?
Gebauer & Meyer, 2003 Bidartondo et al., 2005 Selosse & Roy, 2009 Some green plants
- are mixotrophic and use mycorrhizal networks
- are predisposed to evolve mycohetero- trophy
- … with albinos as transitions?
Selosse & Roy, 2009 Some green plants
- are mixotrophic and use mycorrhizal networks
- are predisposed to evolve mycohetero- trophy
- … with albinos as transitions?
Selosse & Roy, 2009 Les réseaux mycorhiziens
Mycohétérotrophie et réseaux
Mixotrophie et réseaux mycorhiziens
La difficile transition vers l’hétérotrophie
… due à un attachement fatal à la lumière ? Many mutation could abolish greenness, yet albinos remain very rare in mixotrophic orchid populations…
Why then?
Roy et al. 2013, Ecol. Monographs 83: 95–117 A 4–year monitoring of two Cephalanthera damasonium populations, with 20-70 albinos Dormancy…
Year n n+1 n+2
dormancy Dormancy: more frequent!
Year n n+1 n+2 % of dormant individuals in 2007
12 10 8 6 4 dormancy 2 0 n=110 n=850 n=35 n=46 Montferrier Boigneville
Albinos were 16.5x more often dormant than green individuals Shoot survival…
Number of shoots
Apparition Flowering time of shoots Shoot survival: lower!
Number of shoots
% shoots dried in late June 2007
60 50 40 30 20 10 0
51% albino shoots dry in June (vs 7% for green ones) > no seeds! Seed production and quality: lower…
Number of seeds % of seeds germinating in situ per fruits (x10) in seeds pockets (3 months)
100 100 *** 1400 *** 90 90 1200 80 80 70 70 1000 ns 60 60 800 50 50 600 40 40 30 30 400 20 20 200 10 10 0 0 0 n=7200nombre n=22800 de n=7200 %n=22800 fertilité %n=3960 germination% fertilité n=10800 % germination graines 3 x 3 less viable seeds in albinos! Comparison green ind. / albinos
Patterns of dormancy 6.8x more
Patterns of shoot survival 16.5x less
Seed production 9x less
= 103 x lower fitness
Roy et al. 2013, Ecol. Monographs 83: 95–117 Comparison green ind. / albinos
Patterns of dormancy 6.8x more
Patterns of shoot survival 16.5x less
Seed production 9x less
= 103 x lower fitness WHY DID ALBINOS FAIL THE TRANSITION ? Les réseaux mycorhiziens
Mycohétérotrophie et réseaux
Mixotrophie et réseaux mycorhiziens
La difficile transition vers l’hétérotrophie
… due à un attachement fatal à la lumière ? Allocation of mycoheterotrophic C versus photosynthetic C in mixotrophic orchids
based on δ13C
(and N content) Allocation of fungal versus photosynthetic C
In shoots Shoot C nutrition over the growth season in C. damasonium
-22 δ13C (‰) -24 Albinos (enriched in 13C)
-26
-28
C. vitalba -30 Autotrophs (depleted -32 R. peregrina in 13C)
-34 Growth Mar 23 Apr 12 May 02 May 22 Jun 11 Jul 01 03/26 04/27 05/11 05/24 06/26 season First leaves All leaves First Flowers Ripen opened flowers pollinated fruits Shoot C nutrition over the growth season in C. damasonium
-22 δ13C (‰) -24 Albinos
-26
Green individuals -28
C. vitalba -30 Autotrophs
-32 R. peregrina
-34 Growth Mar 23 Apr 12 May 02 May 22 Jun 11 Jul 01 03/26 04/27 05/11 05/24 06/26 season First leaves All leaves First Flowers Ripen opened flowers pollinated fruits A shift from 80% heterotrophic to 20% at fruiting!
-22 δ13C (‰) -24 Albinos
-26
Green individuals -28
C. vitalba -30 Autotrophs
-32 R. peregrina
-34 Growth Mar 23 Apr 12 May 02 May 22 Jun 11 Jul 01 03/26 04/27 05/11 05/24 06/26 season First leaves All leaves First Flowers Ripen opened flowers pollinated fruits A shift from 100% heterotrophic to 20% in E. helleborine
Albinos Hypopitys monotropa
C. mayalis
Green individuals Autotrophs
S. virgaurea
Growth June 1st June 23rd July15st July 28st season
Gonneau et al. 2014, J. Ecol., in press A decrease of mycorrhizal colonization at fruiting in C. damasonium
Percentage of root sections colonized albinos
*** green individuals
0%
No leaves First leaves First flowers Ripen fruits Roy et al. 2013, Ecol. Monographs Gaz exchange in C. damasonium
Net CO2 exchange
green
albino
Julou et al., 2005, New Phytol. Gaz exchange in C. damasonium
Net CO2 exchange
green
albino 3-4x lower basal metabolism
… a carbon limitation ? Julou et al., 2005, New Phytol. CONCLUSIONS
- Shoot autotrophy increases over the growth season.
- Albinos have lower basal metabolism, and especially lack photosynthetic and fungal C at fruiting. Allocation of fungal versus photosynthetic C
In shoots
In fruits Fruit C nutrition in Cephalanthera damasonium
-22 δ13C (‰) Albino fruits Albinos -24
-26 Green individuals
Green fruits -28 C. vitalba
-30
-32 R. peregrina
-34 Growth Mar 23 Apr 12 May 02 May 22 Jun 11 Jul 01 03/26 04/27 05/11 05/24 06/26 season First leaves All leaves First Flowers Ripen opened flowers pollinated fruits Fruit C nutrition in Epipactis helleborine
Albinos Flowers
C. mayalis
Green individuals Fruits
S. virgaurea
Growth June 1st June 23rd July15st July 28st season
Gonneau et al. 2014, J. Ecol., in press TESTING THE ROLE OF FUNGAL C
Limodorum abortivum, control vs. treated with iprodione
Bellino et al., 2014 Oecologia 175: 875-885 TESTING THE ROLE OF FUNGAL C
Limodorum abortivum, control vs. treated with iprodione twice, during and after anthesis
In situ root of L. abortivum with fungus at fruiting
100%
75%
50%
25% None!
Control Iprodione TESTING THE ROLE OF FUNGAL C
Limodorum abortivum, control vs. treated
Control Iprodione
Fruit length (mm) 25.7 2.3 a 26.7 3.4 a (n=42)
Fruit diam. (mm) 8.4 0.3 a 9.0 0.5 a (n=42)
Seed (mm2) 0.26 0.06 a 0.28 0.07 a (n=713)
Fruit δ13C (‰) -25.06 0.08 a -25.50 0.17 b (n=21)
No impact on seed set, full compensation by photosynthesis CONCLUSIONS
- Shoot autotrophy increases over the growth season.
- Albinos have lower basal metabolism, and especially lack photosynthetic and fungal C at fruiting.
- Fruits use photosynthates, even if fungal C may help (a bit). Allocation of fungal versus photosynthetic C
In shoots
In fruits
In underground parts and emerging shoots RHIZOME FEED ON FUNGAL C
a
Rhizomes Shoots Gonneau et al. 2015 RHIZOME FEED ON FUNGAL C
Rhizomes Shoots Gonneau et al. 2015 PHOTOSYNTHESIS
green leaf fruit
stem
rhizome
roots
MYCOHETEROTROPHY PHOTOSYNTHESIS PHOTOSYNTHESIS stem
green leaf fruit
stem
Fungal C
dormancy (& winter?) rhizome
roots
MYCOHETEROTROPHY FUNGUS PHOTOSYNTHESIS
green leaf fruit
stem
Fungal C
dormancy (& winter?) rhizome at shoot emergence
roots
MYCOHETEROTROPHY FUNGUS PHOTOSYNTHESIS
green leaf fruit
stem
Fungal C
dormancy (& winter?) rhizome at shoot emergence at fruiting (summer) roots
MYCOHETEROTROPHY FUNGUS PHOTOSYNTHESIS
green leaf fruit Plant C at fruiting (summer)
stem
Fungal C
dormancy (& winter?) rhizome at shoot emergence at fruiting (summer) roots
MYCOHETEROTROPHY FUNGUS PHOTOSYNTHESIS
green leaf fruit Plant C -> fitness by seeds stem
Fungal C
rhizome
-> fitness roots by survival
MYCOHETEROTROPHY FUNGUS CONCLUSIONS
- Fruits use mostly photosynthates
- Underground tissues use mostly fungal C
- The resulting optimization of the use of fungal and photosynthetic C is problematic for albino fruiting. CONCLUSIONS
- Fruits use mostly photosynthates
- Underground tissues use mostly fungal C
- The resulting optimization of the use of fungal and photosynthetic C is problematic for albino fruiting.
… due to this fatal addiction to light of mixotrophic plants, you cannot lose photosynthesis in one step! CONCLUSIONS
- Fruits use mostly photosynthates
- Underground tissues use mostly fungal C
- The resulting optimization of the use of fungal and photosynthetic C is likely fatal to albino seed production.
The evolutionary emergence of pure C sinks in mycorrhizal networks is a complex process,
… and mixotrophy is metastable in evolution
My partner network…
Mélanie Roy, Cédric Gonneau
Soc. Française d’Orchidophilie: J.-P. Amardeihl, C. Blanchon, F. Dusak, G. Joseph, J. Koenig, P. Pernot, C. & J. Péternel, D. Prat, G. Scappaticci, R. Souche, J. Villeret Univ. Salerno: A. Bellino, A. Alfani, D. Baldantoni Univ. Ceske-Budejovice: J. Jersakova, T. Malinova, T. Hajcek Univ. Turin: P. Bonfante, A. Faccio, M. Girlanda, S. Perotto Univ. Tartu: U. Koljalg, L. Tedersoo, U. Püttsepp, M. Öpik Acad. Science of Russia: M. Logacheva Univ. Pointe-à-Pitre: A. Ba, M. Dulorme, A. Rousteau Univ. Basel: P.-E. Courty, T. Boller, F. Walder, A. Wiemken
… all papers online on ISYEB website