Florent Martos

Two Asian orchids, the bamboo orchid ( Arundina graminifolia ) and the Philippine ground orchid ( Spathoglottis plicata ), are expanding into South and Central America, the Pacific, and Africa. Here, we tested whether seed traits and low in vitro selectivity toward mycorrhizal fungi may contribute to their successful spread outside the native range. We examined seed size and reserves, asymbiotic and symbiotic germination with orchid mycorrhizal fungi from different geographic regions. Both species have relatively large seeds, with A. graminifolia having some of the largest among orchids. Seeds contain lipids, proteins, and low amounts of soluble saccharides and starch. Despite these reserves, both orchids are initially mycoheterotrophic and require an external carbon source for early development. In asymbiotic culture, both species showed high germination on sucrose‐containing media, with maximum germination exceeding 95%. In symbiotic culture, both species formed protocorms with Tulasnellaceae isolates from different continents, including isolates obtained from locations separated by more than 9000 km. This capacity was broader in S. plicata . Our results suggest that the success of S. plicata outside its native range may be facilitated by broad compatibility with Tulasnellaceae, some of which have a global distribution, and partly by self‐pollination. In A. graminifolia , relatively large seeds may provide greater internal reserves for early development, but this species appears more specific toward certain Tulasnellaceae lineages and remains pollinator‐dependent. Together with the ability to colonize disturbed habitats, these traits may help explain the successful spread of both orchids outside their native range.

Les plantes n'exploitent pas seules les nutriments du sol, mais dépendent de champignons avec lesquels elles forment des symbioses mycorhiziennes dans leurs racines. C'est en particulier vrai pour les 25 000 espèces d'orchidées actuelles qui dépendent toutes de champignons mycorhiziens pour accomplir leur cycle de vie. Elles produisent des graines microscopiques qui n'ont pas les ressources nutritives pour germer, mais qui dépendent de la présence de partenaires adéquats pour nourrir l'embryon (hétérotrophie) jusqu'à l'apparition des feuilles (autotrophie). Les mycorhiziens restent présents dans les racines des adultes où ils contribuent à la nutrition, ce qui permet d'étudier plus facilement la diversité des symbiotes à l'aide des outils génétiques. Conscients des biais des études en faveur des régions tempérées, nous avons étudié la diversité des mycorhiziens d'orchidées tropicales à La Réunion. Nous avons montré que (1) les orchidées tropicales ont des partenaires semblables aux orchidées tempérées et méditerranéennes (Sebacinales, Ceratobasidiaceae et surtout Tulasnellaceae), et que ces taxons de champignons sont largement représentés dans différents biomes et dans différentes plantes hôtes. Nous avons aussi démontré pour la première fois que (2) les orchidées épiphytes (dont les associations étaient peu connues) ont des cortèges mycorhiziens différents de ceux des orchidées terrestres dans les communautés tropicales. De plus, en développant une approche à l'échelle de réseaux d'interactions (78 espèces de La Réunion), nous avons montré que (3) les espèces tropicales ont tendance à être généralistes et que (4) le réseau mycorhizien des orchidées montre des propriétés semblables à celles des réseaux d'interactions mutualistes (nestedness et asymétrie d'interaction), alors que la nature mutualiste de cette symbiose mycorhiziennes fait débat. Dans un second volet de la thèse, nous avons étudié les partenaires des orchidées non chlorophylliennes (mycohétérotrophes) tropicales. Nous avons montré que (5) les espèces tropicales peuvent s'associer à des champignons saprophytes qui les nourrissent en carbone issu de la décomposition de la litière dans les forêts tropicales humides et que (6) les modèles tropicaux (en n'étant pas spécifiques) remettent en question les idées reçues sur la mycohétérotrophie des plantes. Nous avons confirmé que (7) la mycohétérotrophie dérive d'un régime nutritionnel intermédiaire (mixotrophie) mis en place dans des lignées chlorophylliennes. Dans un dernier volet de la thèse, nous avons posé la question du déterminisme phylogénétique des associations orchidées-champignons. En analysant la force du signal dans les phylogénies des deux partenaires, nous avons vérifié que (8) les associations mycorhiziennes sont peu conservées à l'échelle supra-générique dans la phylogénie des orchidées, et qu'elles (9) peuvent être maintenues à une échelle plus récente (cas de certains clades d'angraecoïdes). Ces résultats soulignent l'empreinte relative des processus écologiques et évolutifs sur les patrons d'associations actuels, et remettent en question l'idée qu'un processus de coévolution pourrait guider le système.

Hiptage (Malpighiaceae) is a genus almost exclusively native to Asia, with a single species, Hiptage benghalensis, being a noxious invasive plant in several areas of the world and particularly threatening the native flora of the Mascarene Islands. Whereas 17 Hiptage species have been reported from Vietnam, there is currently no available genetic information that could be used to confirm the species diversity in the country. Through a study combining phylogenetic and morphological analyses of newly-collected samples and herbarium specimens, we were able to accurately identify five Hiptage species in Vietnam. One of these, Hiptage aptera, represents a species new to science. Specimens of the new species show white to slightly pink petals, erect to forward-curved petals, eight tiny calyx glands and wingless fruits, a unique feature within this genus. Our results strongly support Vietnam as one of the diversity centres of the Hiptage genus with 18 species out of the 48 species. Although we could not determine the source of introduction of Hiptage in the Mascarenes Archipelago, our findings highlight the genetic similarity of all Hiptage benghalensis samples collected in its secondary distribution areas, suggesting a probable single introduction. This is consistent with historical reports dating the plant’s introduction to the Mascarenes to the 18^th century.

In the tropics, one successful tool for orchid scientific studies and conservation is the creation of shade houses for ex situ cultivation of regional species. Here, we provide the first comparison of floral scent chemistry in shade houses and natural populations of orchids in central Africa. We investigate floral scent variations between two shade houses and one natural site in a sphingophilous epiphytic orchid, Cyrtorchis letouzeyi, in Cameroon. Samples were collected in and ex situ through the dynamic headspace method and analyzed using gas chromatography coupled with mass spectrometry. Both composition and relative emission rate were analyzed with temperature and humidity levels recorded at the time of sampling. The floral scent was dominated by both aromatics and isoprenoids and derivatives, including methyl benzoate, linalool, and pyranoid and furanoid forms of linalool oxide. Variations in temperature and humidity had no significant impact on the floral scent composition but on relative emission rates. Samples under shade house cultivation produced the same compounds as those in the natural environment. Quantitatively, differences were recorded in the proportions of volatile organic compounds (VOCs) produced, but the main compound remained unchanged in both environments. Dissimilarities were driven by the production of linalool and its derivatives. Based on these findings, we suggest that shade houses are a suitable tool to characterize the qualitative floral scent diversity of tropical epiphytic orchids and can therefore serve to complement in situ sampling.

Summary: While orchids germinate thanks to carbon from their symbiotic fungi, variable carbon exchanges exist between adult orchids and their mycorrhizal fungi. Although some truly autotrophic orchids reward their fungi with carbon at adulthood, some species remain achlorophyllous and fully dependent on fungal carbon (mycoheterotrophy). Others are photosynthetic but also import fungal carbon: The so‐called mixotrophic (MX) orchids rely on fungi of diverse taxonomy and ecology. Here, we classify MX nutrition of orchids into three types. Type I mixotrophy associates with diverse Asco‐ and Basidiomycota that are either saprotrophic or ectomycorrhizal, entailing enrichment of the orchids in ² H, ¹³ C, and ¹⁵ N. The two other types associate with rhizoctonias, a polyphyletic assemblage of Basidiomycotas that is ancestrally mycorrhizal in orchids. Type II mixotrophy associates with rhizoctonias that secondarily evolved into saprotrophic or ectomycorrhizal ecology, and thus enrich the orchid in ² H, ¹³ C, and ¹⁵ N. Type III mixotrophy, which remains debated, associates with rhizoctonias that have retained their ancestral lifestyle, that is saprotrophic and/or endophytic in nonorchids, and only entail orchid enrichment in ² H and ¹⁵ N. Based on a case study of achlorophyllous variants in Mediterranean Ophrys and on published data, we discuss the distinct nature and research perspectives of type III mixotrophy.

Specialised pollination has often been proposed as a major driver of the unrivalled diversification of angiosperms. With its unparalleled variation in flower depth, ranging from spurless to 40 cm spurred flowers pollinated by hawkmoths, angraecoid orchids (Angraecinae) provide unique opportunities to reveal the impact of floral specialisation on diversification rates in the Afrotropics and Neotropics. We compiled floral characters for 327 angraecoid species, and assigned these species to newly formalised floral syndrome categories calibrated against pollination case studies. We then estimated ancestral character states and state-dependent speciation rates using the latest time-calibrated phylogeny of angraecoids. We found that white flowers consistent with micro-sphingophily (2 cm≥ spurs <8.7 cm), macro-sphingophily (8.7 cm≥ spurs <18.6 cm) and mega-sphingophily (spurs ≥18.6 cm) evolved repeatedly in angraecoids, and are ancestral in some non-sphingophilous clades. Reversals to non-sphingophily and pigmented flowers suggest higher evolutionary lability of these traits than what is traditionally thought. Increasing floral specialisation with long spurs does not seem to enhance angraecoid orchids’ speciation rates, since micro-sphingophily appears to be associated with the highest speciation rates. An abundance of short-proboscid hawkmoths in Madagascar, as an ecological opportunity, may have accelerated the speciation of micro-sphingophilous taxa.

Societal Impact Statement: Vanilla is one of the most valuable spices in the world. In Madagascar and La Réunion, the world's leading producers, vanilla is of great economic and cultural importance. Like all orchids, vanilla plants associate with mycorrhizal fungi in their roots forming mutualistic associations that allow them to grow and thrive. Understanding the diversity of mycorrhizal fungi adapted to vanilla cultivation, particularly in the Indian Ocean islands where they have never previously been studied, is becoming a necessity for maintaining vanilla crops in these regions in the face of climate change and the emergence of new pathogens.Summary: The vanilla orchid (Vanilla spp.) is one of the most valuable cultivated plants worldwide. As with all orchids, vanillas form mycorrhizal associations with fungi in their roots, but their fungal partners have not been investigated outside their native geographic range in Central America. We investigated the whole fungal and mycorrhizal associations in cultivated vanilla (Vanilla planifolia) by sequencing the fungal ITS‐2 marker in the terrestrial and aerial roots using a metabarcoding approach. We selected plants cultivated in three conditions (i.e., cultivation under shade house, in openfield, or in the understory) in one locality of La Réunion island (Indian Ocean) and tested for a possible effect of cultivation practices on fungal communities. Cultivated vanillas in La Réunion mainly associate with Tulasnellaceae (75 OTUs) and Ceratobasidiaceae (8 OTUs). Among the seven most abundant Tulasnellaceae, six are similar to fungi detected in the roots of cultivated vanillas in Central America or in the roots of native orchids in La Réunion. Cultivation practices impacted both total fungal and mycorrhizal community compositions with no clear effect on fungal richness. Notably, Tulasnellaceae and Ceratobasidiaceae were scarce in aerial roots, except in the traditional cultivation in the forest understory. These results shed light on the geographical origins of mycorrhizal fungi of cultivated vanillas in La Réunion and show that they form a pool of both locally and globally distributed fungal partners. These mycorrhizal communities vary according to cultivation practices, and their potential roles in plant nutrition and resistance against pathogens await further attention.

Aim: Diversification of obligate parasites is often closely associated with historical shifts among host species. This phenomenon is well‐documented in animal parasites but still poorly understood for parasitic plants. We studied the associations between host shifts, biogeography and diversification in Cytinus, a genus of endophytic holoparasitic plants which show a remarkable disjunct distribution.Location: Mediterranean Basin, southern Africa, Madagascar.Taxon: Cytinus.Methods: We sampled numerous populations in South Africa and the western Mediterranean Basin, as well as a few populations in Madagascar where the genus is rare. We amplified two nuclear (ITS and SSU) and two mitochondrial (matR and nad1) loci, to infer its evolutionary history using maximum likelihood and time‐calibrated Bayesian analyses, to elucidate the factors responsible for its current distribution and to assess whether host switching is a main driver of speciation.Results: All genes recover lineages corresponding to the three geographic areas of Cytinus and sublineages associated with specific host plants, although they disagree on their deep‐level relationships. Ribosomal nuclear genes support an initial split of the Malagasy lineage and an Afrotropical origin of Cytinus before colonization of warm temperate latitudes. Mitochondrial genes support an initial split of the Mediterranean and Afro‐Malagasy lineages and a later split between the South African and Malagasy clades. Introgressive hybridization is the most likely explanation for this discrepancy, while horizontal gene transfers from hosts is less likely. We found strong support for cryptic host‐specific diversity, with five and eight host races in the Mediterranean and South Africa respectively, and a strong phylogenetic signal in the host–parasite associations suggestive of parasite diversification via phylogenetic host tracking.Main Conclusions: These dated phylogenies indicate that radiation through long‐distance dispersal events and adaptation to new hosts best explain the disjunct distribution of Cytinus. We emphasize the importance of host specificity and host‐switching in the diversification and biogeography of plant endoparasites.