TESIS DOCTORAL
Interacción entre orquídeas epífitas y hongos micorrízicos
en bosques tropicales del Sur de Ecuador
Paulo Ignacio Herrera Vargas
Directores:
María del Carmen Molina Marcos Méndez Juan Pablo Suárez
Programa de Doctorado en Ciencias Escuela Internacional de Doctorado
TESIS DOCTORAL
Interacción entre orquídeas epífitas y hongos micorrízicos
en bosques tropicales del Sur de Ecuador
Autor:
Paulo Ignacio Herrera Vargas
Directores:
María del Carmen Molina Marcos Méndez Juan Pablo Suárez
Programa de Doctorado en Ciencias Escuela Internacional de Doctorado
La Dra. María del Carmen Molina Cobos y el Dr. Marcos Méndez Iglesias, profesores titulares de Universidad del Departamento de Biología, Geología Física y Química Inorgánica de la Universidad Rey Juan Carlos, y el Dr. Juan Pablo Suárez, Docente titular del Departamento de Ciencias Biológicas de la Universidad Técnica Particular de Loja,
CERTIFICAN:
Que los trabajos de investigación desarrollados en la memoria de tesis doctoral: “Interacción entre orquídeas epifitas y hongos micorrízicos en bosques tropicales del Sur de Ecuador", han sido realizados bajo su supervisión y son aptos para ser presentados por el BQ. Paulo Ignacio Herrera Vargas ante el tribunal que en su día se consigne, para aspirar al Grado de Doctor en el Programa de Doctorado de Ciencias por la Universidad Rey Juan Carlos.
VºBº Director de Tesis VºBº Director y Tutor de Tesis
Dra. María del Carmen Molina Dr. Marcos Méndez
VºBº Director de Tesis
Índice
Resumen……….... 1
Introducción general…...………. 3
Objetivos………... 11
Capítulo 1……….. 25
Capítulo 2……….. 79
Capítulo 3……….. 131
Capítulo 4……….. 169
Discusión general………. 207
Resumen
Introducción- Las interacciones bióticas juegan un papel esencial en la formación, persistencia y estabilidad de las comunidades. Las interacciones simbióticas micorrízicas son un componente vital de los ecosistemas vegetales. En particular, el establecimiento y la supervivencia de las orquídeas depende, entre otros factores, de su interacción con hongos micorrízicos (o micobiontes) que son indispensables para la germinación, establecimiento y el desarrollo temprano de las plántulas y probablemente para el estado adulto. El estudio de las interacciones entre micobiontes y orquídeas está ganando mayor impulso en la investigación ecológica, pero está en un estado incipiente. Hay cuatro aspectos que requieren atención: (1) el estudio de las propiedades de las redes de interacciones orquídea-micobionte puede ayudar a entender la importancia de los procesos basados en nichos en el ensamblaje de las comunidades, que afectan la diversidad, la estabilidad y la dinámica coevolutiva entre especies simbióticas, (2) averiguar el grado de especialización orquídea-micobionte, lo cual implica conocer si los micobiontes están en todas partes o si están limitados a ciertos sitios (estructura espacial de esta interacción) y, además, si las diferentes especies de orquídeas comparten los micobiontes o dividen su nicho mediante su asociación a diferentes micobiontes locales, (3) comprobar si las interacciones micorrízicas muestran un conservadurismo filogenético y restricciones filogenéticas en los patrones de asociación o si la cantidad de interacciones puede estar limitada por la diversidad fúngica local, y (4) ahondar en la caracterización taxonómica y filogenética de los micobiontes y su carácter micorrízico, frente a otras posibilidades como el endofitismo o el parasitismo.
cambios en la interacción oquideas-micobiontes frente a factores bióticos y abióticos que pueden estar determinando la estructura de las comunidades de micorrizas de orquídeas.
Métodos- Se trabajó con orquídeas epífitas en bosques montanos del sur de Ecuador. Usando técnicas de código de barras molecular se identificaron OTUs (unidades taxonómicas operativas) de micobiontes a partir de las raíces de orquídeas y se elaboró una red de interacciones entre orquídeas y micobiontes. Además se estudió en más detalle la variación espacial en la diversidad de micobiontes en orquídeas comunes y se examinó la diversidad filogenética de los micobiontes y la existencia o no de conservadurismo filogenético en las interacciones con micobiontes de tres orquídeas que difieren en su proximidad evolutiva. Finalmente, se elaboraron hipótesis filogenéticas para uno de los grupos clave de micobiontes, las Serendipitaceae.
Introducción general
Las comunidades ecológicas son inmensamente complejas, pero también están muy organizadas (Fortuna et al. 2010). Se cree que todos los organismos que forman parte de las comunidades están inmersos en una o más interacciones bióticas con otros organismos (Bronstein et al. 2006), y de maneras muy diversas, por ejemplo, en interacciones de competencia, mutualistas, de predación, parasitismo, etc. (Boucher 1985). Estas interacciones bióticas desempeñan un papel esencial en la formación, persistencia y estabilidad de las comunidades (Fortuna et al. 2010). Por ejemplo, de las interacciones de polinización y de dispersión depende gran parte de la reproducción y reclutamiento exitoso de muchas especies de plantas (Jordano et al. 2009). Es por ello que el estudio de las redes de interacción se ha convertido en uno de los desafíos actuales de la investigación ecológica al abordar sistemas complejos y megadiversos. Comprender los procesos que estructuran estas redes ayudará a explicar cómo los conjuntos hiperdiversos de especies que interactúan coevolucionan y se mantienen en sus hábitats (Jordano et al. 2009).
La interacción entre orquídeas y micobiontes desde una perspectiva de las redes complejas
2001). De hecho, las interacciones simbióticas micorrízicas son un componente vital de los ecosistemas vegetales (Smith & Read 2008; Bonfante & Anca 2009), ya que influyen en su estructura, diversidad y productividad (van der Heijden et al. 1998). En particular, el establecimiento y la supervivencia de las orquídeas depende, entre otros factores, de su interacción con hongos micorrízicos (o micobiontes) particulares (Arditti & Ghani 2000). Estos micobiontes son indispensables para la germinación, establecimiento y el desarrollo temprano de las plántulas (Smith & Read 2008), pero la dependencia de las orquídeas con respecto a estos hongos eventualmente puede continuar hasta el estado adulto, favoreciendo la provisión de nutrientes minerales y la absorción de agua (Yoder et al. 2000; Rasmussen 2002). Actualmente se ha señalado también, que los micobiontes son importantes para la diversidad y distribución de las orquídeas (Otero & Flanagan 2006; Otero et al. 2007; McCormick & Jacquemyn 2014).
comunidades, que afectan la diversidad, la estabilidad y la dinámica coevolutiva entre especies simbióticas (Jacquemyn et al. 2011; Chagnon et al. 2012; Martos et al. 2012). Además, se puede abordar el efecto de las especies en la red de interacción mediante la evaluación (simulación) de la robustez de la red ante la pérdida de especies interactuantes (Bastolla et al. 2009; Memmott et al. 2004). Este enfoque puede ser útil para identificar los organismos que más influyen en la comunidad, las preferencias de asociación y su efecto en el ensamblaje de las comunidades.
El grado de especialismo en las interacciones entre orquídeas y sus micobiontes
una propiedad global de las especies (Fox & Morrow 1981; Herrera 2005). Las especies pueden por tanto diferir en su grado de especialismo local y general (Hughes 2000).
Una de las teorías más importantes que explican los procesos de ensamblaje de las comunidades es la teoría del nicho, que enfatiza la importancia de la diferenciación interespecífica y las respuestas no aleatorias de las especies a sus entornos (Giller 1984; Leibold 1995; Chase 2011). La diversidad y coexistencia de las especies dentro de una comunidad podría estar determinada por las diferencias en sus rasgos ecológicos (Leibold & McPeek 2006). Esta variación de rasgos entre especies puede deberse a la influencia de las adaptaciones convergentes de las especies a sus hábitats, o por un legado de sus ancestros (Prinzing et al. 2001). Este legado es llamado conservadurismo filogenético, que significa que las adaptaciones de la especie ancestral a un conjunto de condiciones ambientales tenderán a ser conservadas por las especies descendientes (en otras palabras, conservadurismo de nicho filogenético) (Wiens 2004). El conservadurismo de nicho filogenético es un factor clave en el ensamblaje de las comunidades (Wiens 2004; Wiens et al. 2010) y es relevante para una variedad de rasgos, incluidos los que participan en interacciones bióticas (Wiens et al. 2010; Fontaine & Thébault 2015). Por lo tanto, estudiar en qué medida la estructura filogenética de las comunidades es diferente de la aleatoria puede proporcionar información importante sobre cómo están estructuradas las comunidades (Webb et al. 2002).
micobiontes entre las especies de orquídeas puede ser un determinante clave de la segregación del nicho ecológico de orquídeas (Waterman et al. 2011; Těšitelová et al. 2013; Jacquemyn et al. 2014), y su comprensión es entonces necesaria para predecir la coexistencia de especies en los ecosistemas. Alternativamente, la cantidad de interacciones puede estar limitada por la diversidad fúngica local (Illyés et al. 2009) debido a las condiciones ambientales locales (Taylor & Bruns 1999; Jacquemyn et al. 2014, 2016), lo que lleva a la especialización ecológica. Lamentablemente aún se desconoce si las relaciones entre las orquídeas y el conservadurismo filogenético son consistentes en diferentes escalas taxonómicas y espaciales de las orquídeas, o incluso entre los grupos taxonómicos de micobiontes.
Diversidad filogenética de los hongos micobiontes de orquídeas epífitas
suelo y raíces de plantas (Seifert 2009; Vrålstad 2011) y recientemente se estableció como el “código de barras” oficial de los hongos (Schoch et al. 2012). Mediante el agrupamiento de estas secuencias por su similaridad se pueden delimitar Unidades Taxonómicas Operaciones (OTUs) (Lievens et al. 2010), que teóricamente podrían representar linajes evolutivos. Esta puede ser una buena herramienta para clarificar la delineación de especies (por ejemplo, usando el reconocimiento de especies filogenéticas de concordancia genealógica; Taylor et al. 2000) y la evolución de las micorrizas en orquídeas. Mientras tanto, la definición de OTUs puede inferir medidas útiles en el estudio de diversidad (Göker et al. 2009), en este caso de los hongos formadores de micorrizas orquideoides.
no discriminan entre biotrofos (endófitos, micorrízicos, epifitos, etc.). Sumado a esto, sabemos que estos hongos también pueden estar colonizando el velamen de las raíces orquideoides o el sustrato en el que se desarrollan las orquídeas (rizosfera, humus, corteza de árboles) (Herrera et al. 2010) y que no todos los hongos forman asociaciones mutualistas con plantas y podrían mostrar un estilo de vida sapróbico (Garnica et al. 2016).
A pesar de esto, las herramientas moleculares parecen ser la mejor opción para establecer la hipótesis filogenética de los hongos micobiontes porque su biodiversidad críptica no se resuelve mediante el análisis microscópico único de las características morfológicas (estadios sexuales) (Oberwinkler et al. 2013). La identificación de las especies de los hongos micorrízicos ofrecerá una oportunidad particular en el estudio de la diversidad de los hongos asociados a las orquídeas, su función y su evolución (Weiß et al. 2011, 2016).
Interacciones micorrizas de orquídeas en bosques tropicales
Objetivos
En vista de los vacíos de conocimiento general sobre las interacciones entre orquídeas y sus micobiontes, y el adecuado sistema de estudio que suponen las orquídeas epífitas de los bosques tropicales, el objetivo general de esta tesis es: mediante la delimitación de OTUs de los micobiontes, estudiar la estructura y composición de la comunidad de hongos micorrízicos asociados a orquídeas epifitas tropicales, para evaluar si existen cambios en la interacción oquideas-micobiontes frente a factores bióticos y abióticos que pueden estar determinando la estructura de las comunidades de micorrizas de orquídeas.
En el primer capítulo de la tesis, el objetivo fue evaluar la riqueza de los micobiontes de orquídea pertenecientes a Tulasnellaceae y el grado de especificidad o generalismo que se da en las interacciones entre orquídeas y Tulasnellaceae en bosques montanos en los Andes del Sur de Ecuador, desde una perspectiva de redes complejas.
En el segundo capítulo de la tesis se exploran la estructura espacial y filogenética de los hongos micorrízicos que interactúan con dos especies de orquídeas epífitas, Epidendrum marsupiale y Cyrtochilum pardinum, coexistentes en un bosque siempre verde montano alto del Sur de Ecuador.
En el tercer capítulo de la tesis se evalúa la hipótesis de una conservación filogenética de los micobiontes, mediante el estudio de los micobiontes de tres especies de orquídeas simpátricas que difieren en su grado de parentesco filogenético. Para ello, se estudió a Scaphyglottis punctulata y Stelis superbiens (tribu Epidendreae) y Cyrtochilum myanthum (tribu Cymbidieae) en un bosque tropical de montaña del sur de Ecuador.
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Capítulo 1
Generalism in the interaction of Tulasnellaceae
mycobionts with orchids characterizes a biodiversity
hotspot in the tropical Andes of Southern Ecuador
Paulo Herrera, Ingrid Kottke, M. Carmen Molina,
Marcos Méndez y Juan Pablo Suárez
Abstract
Biotic interactions play an important role in the assembly and stability of communities. All orchids depend on mycobionts for early establishment, but whether individual orchid species depend on a specific or broad spectrum of mycobionts is still a matter of debate. Tulasnellaceae (Basidiomycota) is the richest and most widespread mycobiont worldwide. We assessed Tulasnellaceae richness in epiphytic and terrestrial orchids in different habitats, and evaluated the degree of generalism in orchid-Tulasnellaceae interactions and the robustness of this mutualistic system to the extinction of mycobiont partners. We sampled 114 orchid individuals including all common and rare species in 56 plots of 1 m2 in 3 habitats: pristine forest, regenerating forest and a landslide site in a tropical montane rainforest in Southern Ecuador. We found 52 orchid and 29 Tulasnellaceae species. The composition of Tulasnellaceae OTUs was moderately to highly similar across habitats and between orchid growth forms. A significantly nested network architecture indicated the existence of a core of generalist Tulasnellaceae OTUs interacting with both rare and common orchids. Terrestrial and epiphytic orchids showed significant differences in robustness to the extinction of their Tulasnellaceae mycobionts. Thus, generalist mycobionts may be relevant for the preservation of hyperdiverse orchid communities in the tropics.
Introduction
Biotic interactions such as mutualism play an important role in the assembly, persistence, and stability of communities (Fortuna et al. 2010). Fungi interact with over 80% of land plants, forming a mutualistic interaction known as mycorrhizae (Smith & Read 2008). Mycorrhizal symbionts (mycobionts) are a vital component of plant ecosystems (Bonfante and Anca 2009), and they improve plant growth by promoting nutrient uptake especially when the availability of soil nutrients is low (van der Heijden et al. 2008; Yang et al. 2016). As orchids produce tiny seeds lacking stored nutrients, they require an obligate supplement of carbon and minerals supplied by mycobionts to establish protocorms in the wild (Rasmussen 1995).
Recently, much attention has been given to documenting the diversity of the mycobionts associated with orchids and their degree of specificity (McCormick et al. 2004; Jacquemyn et al. 2010; Kartzinel et al. 2013; Pellegrino et al. 2014). Among these fungi, members of Tulasnellaceae (Basidiomycota) are one of the richest and most widespread mycobionts forming mycorrhizae with a broad spectrum of photosynthetic Orchidaceae in temperate and tropical ecosystems (Suárez et al. 2006; Dearnaley 2007; Dearnaley et al. 2012; Martos et al. 2012; Jacquemyn et al. 2015a). As Tulasnellaceae mycobionts are widespread on rotten wood and humus-rich substrates (Roche et al. 2010; Cruz et al. 2011, 2014), they are available to associate with orchids in terrestrial and epiphytic habitats of the tropics.
studied in megadiverse tropical systems, a high degree of specialism of orchid-Tulasnellaceae was also found (Otero et al. 2004, 2007; Riofrío et al. 2007). In contrast, pioneer studies at the community level in biodiversity hotspots of tropical systems provide a different picture. For instance, Martos et al. (2012) found a broad sharing of mycobionts among highly diverse orchid species in rainforests and sub-alpine heathlands from La Reunion Island. Similarly, multiple epiphytic orchid species in Neotropical cloud forests were associated with a high number of Tulasnellaceae and Serendipitaceae (previously Sebacinales subgroup B) (Suárez et al. 2006, 2008, 2016; Kottke et al. 2013; Weiss et al. 2016).
architecture among the highly diverse orchids and 3 groups of diverse mycorrhizal fungi. However, they did not consider the effect of habitats, life forms or sites on the architecture of the fungi-orchids network based on Tulasnellaceae as mycobionts.
Orchidaceae are extraordinarily speciose and abundant, and form an essential part of the tropical mountain rainforest in the Southern Andes in Ecuador (Homeier & Werner 2007). This area is considered a hotspot of plant biodiversity (Barthlott et al. 2005; Brehm et al. 2008), but the habitat is constantly threatened by anthropogenic activities, making its preservation a priority in global conservation. Most of its orchid species are endemic and rare (Homeier & Werner 2007). Only 2 to 5 species are abundant (>2000 individuals in 0.1 ha), while most others are doubletons or singletons (Kottke et al. 2013). However, many species may still remain undescribed (Endara 2000; Winkler et al. 2009). This kind of asymmetric abundances is characteristic of extremely species-rich, mutualistically dependent communities (Medan et al. 2007). Orchid species richness in the tropical area may be supported by quite distinct habitats in the neighborhood such as pristine forests, young regenerating forests and frequent disturbance by landslides combined with a strong altitudinal gradient (Beck et al. 2008). However, we are lacking the corresponding information on orchid mycobionts.
robust against the loss of Tulasnellaceae mycobiont species? If so, does it differ between epiphytic and terrestrial orchids?
Materials and methods Study sites
Mycorrhizas of Orchidaceae were sampled at 4 sites which had different elevations and disturbance, from pristine forests to a landslide area. Sites 1 and 4 were located in pristine forests at 2000 m and 2170 m a.s.l., respectively. Site 3 was located in a 40-y-old regenerating forest at 2170 m a.s.l. close to Site 4, and Site 2 was located in an area at 1900 m a.s.l. where a human-caused landslide occurred 40 years ago (Supplementary Figs. S1, S2).
The 40-y-old regenerating forest is a product of anthropogenic deforestation (Weber et al. 2008) and is structurally characterized by a lower proportion of trees (> 10 cm diam at breast height (dbh)) and a greater proportion of bushes and tree ferns, thus appearing less homogeneous and more open than the primary forest surrounding it (Martinez et al. 2008). The human-caused landslide produced a large gap which has vegetation in a young succession stage and is characterized by the growth of pioneer species that are not found in the primary forest (Bussmann et al. 2008). Soil properties and fertility in landslide areas can vary considerably, providing less favorable conditions for plant growth than undisturbed soils (Bussmann et al. 2008). Ecological factors such as the amount of solar radiation, minimum and maximum temperatures at the forest ground and rainfall are higher in the regenerating forest and the landslide site than in the primary forest. In contrast, amount of litter fall, seed quantity and seed diversity are lower in these two habitats.
Mycorrhizae sampling, fungal DNA isolation, PCR, cloning and sequencing
Fungal phylogenetic analysis and OTU delimitation
Sequences were edited and assembled into a consensus sequence using Sequencher 4.6 (Gene Codes, Ann Arbor, USA). Homology searches for each nucleotide sequence were carried out with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in GenBank database (https://www.ncbi.nlm.nih.gov) using the option megablast (Altschul et al. 1997). Only sequences of our strains similar to Tulasnellaceae species were aligned using the G-INS-I option in MAFFT Ver. 6.602b (Katoh et al. 2005). Alignments were checked to eliminate chimeric sequences using the software Bellerophon (Huber et al. 2004) and also manually copying and comparing suspicious parts of sequences on BLAST (<200 bp by part).
A phylogenetic analysis was performed using the entire nrDNA ITS-5.8S region. Due to the known difficulties in aligning Tulasnellaceae sequences (Suárez et al. 2006; Cruz et al. 2014), we carried out a two-step procedure. Firstly, a neighbour-joining analysis was performed in PAUP* 4.0b10 (Swofford 2002) using the BioNJ modification with Kimura 2-distances of the sole 5.8S region (160 bp). The resulting tree displayed 4 prominent groups (Groups I–IV), and new alignments of the whole nrDNA ITS-5.8S region were performed for each group (Group I = 604 bp, group II = 611 bp, group III = 576 bp and group IV = 927 bp). Phylogenetic reconstructions were carried out using a maximum likelihood (ML) analysis for each group in MEGA5 (Tamura et al. 2011) under the general time reversible DNA substitution model with 1000 bootstrap replicates, eliminating gaps and missing data. Additionally, a maximum parsimony (MP) analysis was performed for each group in MEGA5, using the close-neighbour-interchange (CNI) on random trees method with 1000 bootstrap replicates. All trees were midpoint-rooted.
similarity and 0.5 of linkage fraction. For further analysis, we chose the 95% threshold, which was calibrated using molecular and morphological data of Tulasnellaceae by Cruz et al. (2011, 2014). These OTUs were mapped into the ML trees.
Morphological criteria and DNA barcoding for orchid identification
The orchids in bloom at the time of sampling were subjected to morphological identification referring to Dodson et al. (unpublished work), Dodson and Escobar (1993), Dodson (2001, 2002, 2003, 2004) and available web resources (e.g., http://www.pleurothallids.com).
(Iotti and Zambonelli 2006) and 1U Phusion High-Fidelity PCR Mastermix. A negative control included PCR mix without DNA template.
The 3' end of ycf1 plastid gene was amplified using the primers 3720F (5′
-TACGTATGTAATGAACGAATGG-3′) and 5500R (5′
-GCTGTTATTGGCATCAAACCAATAGCG-3′) (Neubig et al. 2009). PCR conditions were as follows: initial denaturation at 98 ºC for 3 min followed by 35 cycles of 98 ºC for 30 s, 58 ºC for 1 min, and 72 ºC for 3 min, ending with a final elongation step at 72 ºC for 3 min. PCR products were sequenced bi-directionally by ABI 3730xl in Macrogen using the same primers as for PCR amplification. Sequence chromatograms were edited using the software Sequencher 4.6.
BLAST search (Altschul et al. 1997) was used to find published sequences with a high similarity to the named orchid species. As most of our orchid species have not been sequenced before, naming was generally only successful at the genus level (Supplementary Table S1).
Estimating richness and evaluating similarity of Tulasnellaceae OTUs across orchid life forms and sites
The sampling effort needed to record 95% of the estimated proportion of OTUs was calculated according to Jiménez and Hortal (2003).
Similarity in the composition of Tulasnellaceae OTUs between epiphytic and terrestrial orchids and across sites was evaluated by Chao-Jaccard and Chao-Sørensen similarity indices based on incidence and abundance (Chao et al. 2005) using the program EstimateS Ver. 8.2.0. These indices account for shared, but unobserved species among samples.
Description of the mutualistic network
Qualitative interactions based on presence/absence data between orchid species and Tulasnellaceae OTUs were assembled in an ordered binary matrix (Fig. 1). Degree distributions, i.e., the number of links per Tulasnellaceae OTU and orchid species, were displayed and network connectance was calculated to further characterize the structure of the mutualistic network using R software Ver. 3.1.1 (R Core Team, Vienna, Austria) with the 'bipartite' package (function plotweb for visualization and function networklevel for connectance values) (Dormann et al. 2008). To determine if the structure of the orchid-Tulasnellaceae network was significantly nested, a formal nestedness index was calculated as NODF (nested overlap and decreasing fill), and its significance was tested against 2 null models with 1000 randomizations (ER model assigning interactions completely at random and CE model randomizing each species similar to the observed data matrix), implemented in ANINHADO Ver. 3.0 Bangu (Guimarães & Guimarães 2006; Almeida-Neto et al. 2008). Significant nestedness would indicate a non-random assemblage of the network.
annealing to find the partition with the largest modularity (Haug et al. 2013). The analysis was based on the interaction of each orchid individual with individual Tulasnellaceae OTUs. We used an iteration factor of 1.0, a cooling factor of 0.995 and 1000 randomizations. The significance of modularity was evaluated by calculating a Z score obtained from the modularity of the orchid-fungal network minus the mean modularity of the randomizations (M rand) divided by the standard deviation of the randomizations (sigma M rand) (Haug et al. 2013). The Z score was converted to a p-value using the free online calculator QuickCalcs (GraphPad Software; http://www.graphpad.com/quickcalcs/).
Evaluating the robustness of the mutualistic network to species loss
number of pollinators or, in our case, Tulasnellaceae fungi (minimum robustness to partner extinction).
Differences in the robustness of the network to the simulated Tulasnellaceae extinction scenarios were evaluated in R with a one-way general linear model (GLM) using the 300 iterations as replicates. The orchid life forms (epiphytic and terrestrial orchids) were considered factors in the GLM analyses. Significance of the differences in robustness was calculated by means of a posteriori Tukey's honesty significant difference test according to Santamaría et al. (2014).
Results
Fungi sampling
Phylogenetic analysis and OTU delimitation of Tulasnellaceae
The neighbour-joining analysis of the sole 5.8S region obtained 4 well-supported bootstrap groups (Groups I–IV). Group I had the highest number of sequences with a total of 78 sequences, while Groups II, III and IV had 56, 32 and 44 sequences, respectively. Alignments of the complete nrDNA ITS-5.8S region within these 4 groups resulted in well-resolved clades with high support values for ML and MP analyses (Supplementary Figs. S3–S6). When a fraction linkage of 0.5 was used, we obtained 29 OTUs based on a threshold of 95% sequence similarity and 33 OTUs were obtained at a threshold of 97%. The OTUs obtained at the 95% threshold are mapped in the 4 phylogenetic trees of Tulasnellaceae (Supplementary Figs. S3–S6). As the results of ML and MP analyses were consistent with the monophyly of most of the 29 delimited OTUs (Supplementary Figs. S3–S6), OTU delimitation was phylogenetically supported.
Orchid identity and occurrence
Fig. 1 – Binary matrix presenting the relationship between the studied Tulasnellaceae and orchids. The 29 Tulasnellaceae OTUs (rows) and 52 orchid species (columns) are ordered by decreasing number of interacting partners (bars and numbers to the left and below the matrix). The grayscale check marks the number of interactions: one (light grey); two (dark grey); three or more (black); no interaction (white).
superbiens and Stelis sp. 3, 11 and 30) were found in both epiphytic and terrestrial forest habitats (Supplementary Table S1).
Richness and similarity of Tulasnellaceae OTUs across life forms and sites
Table 1 – Similarity indices of Tulasnellaceae OTUs at 95% similarity of the epiphytic and terrestrial orchid communities and at the different sites located in the Reserva Biológica San Francisco, Southern Ecuador.
Incidence-based Abundance-based Comparison Chao-Jaccard Chao-Sørensen Chao-Jaccard Chao-Sørensen
Epiphytic versus terrestrial 0.74 0.85 0.74 0.85
Pristine versus landslide 0.5 0.66 0.5 0.67
Pristine versus regenerating 1 1 1 1
Landslide versusregenerating 0.33 0.49 0.33 0.49
Structure of the orchid-Tulasnellaceae network and stability against species loss
The bipartite network formed by the 29 Tulasnellaceae OTUs and the 52 orchid species (Supplementary Figs. S7, S8) had a connectance of 7.2% and was significantly nested (NODF = 16.2; p < 0.05 for both ER and CE null-models). NODF values were 18.2 for orchids and 9.9 for Tulasnellaceae. The number of singletons and doubletons was high for Tulasnellaceae and orchids (55% and 71%, respectively) (Fig. 1). The network had a few generalist fungi (OTUs T1, T9, T13, T16) frequently linked with a few common orchid species (S. superbiens, Stelis sp. 11, Pleurothallis sp. 25). OTU T1 was the most common phylogenetic species associated with 26 individuals and 19 orchid species, followed by 3 others frequently linked with OTUs T16, T13 and T9 (Fig. 1). Fungi and orchids with few links were mainly linked to partners with many links, with one exception (T21 linked to Maxillaria sp. 7). No significant modularity was found for the bipartite network (M = 0.6, p = 0.9).
Tulasnellaceae extinction showed R50 values between 0.32 and 1.00 (Fig. 3). One-way GLM showed a significant effect of orchid life form on robustness to Tulasnellaceae OTUs extinction (Fig. 3). In the random extinction sequence, terrestrial orchids were more robust than epiphytic orchids, while the opposite was found in the most- to least-connected sequence (Fig. 3).
Fig. 3 – Mean robustness (R50) to Tulasnellaceae extinction under scenarios of extinction at random, most to least connected and least to most connected. 300 interactions were used in all
cases. Robustness is indicated by circles for epiphytic orchids and squares for terrestrial orchids. Error bars indicate standard deviation. Chi-squared (χ2) and significance (P) of a
one-way general linear model (GLM) comparing differences in robustness between the groups of orchids to the extinction of Tulasnellaceae are shown at the top of each simulation
Discussion
We found that the interactions between orchids and Tulasnellaceae fungi in the tropical forest of Southern Ecuador were characterized by a high richness of Tulasnellaceae, shared between epiphytic and terrestrial orchids across sites (landslide, pristine and regenerating forest). The structure of this mutualistic network was nested, and its robustness differed only slightly between the orchid life-forms (epiphytic or terrestrial) of this network. Tulasnella richness in European temperate habitats has reached asymptotic values near 10–12 OTUs (Jacquemyn et al. 2011, 2012). Not surprisingly, this diversity is higher in tropical forests, as indicated by previous studies in La Reunion (58 OTUs) (Martos et al. 2012) and the preliminary results for our study area (33 OTUs at 3% sequence divergence in Kottke et al. 2013). Here, we obtained 29 Tulasnella OTUs, and the non-saturated accumulation of fungal OTUs suggests that more locally-distributed rare species may be present at our study sites.
Tulasnellaceae, other primer combination methods could possibly have detected additional species (Jacquemyn et al. 2011, 2012, 2015b; Waud et al. 2016). We suggest using multiple primer alternatives in future studies to detect a more complete diversity of Tulasnellaceae.
Tulasnellaceae communities in the study area were similar across habitats despite a high turnover of orchid species (Werner et al. 2005; Werner 2011). One may argue that pooling terrestrial orchids (from the humus layer and in mineral soil) in one group could potentially hide differences, but these two kinds of substrates commonly have very similar components in tropical forests. In fact, orchids at the landslide site and the terrestrial orchids in the pristine forest shared 7 of the 9 Tulasnellaceae OTUs, including the most frequent ones. These results were unexpected and contrast with the findings for La Reunion (Martos et al. 2012), where significant differences were found in mycobiont composition between epiphytic and terrestrial orchids. These contrasting findings may be due to several factors, such as climatic and edaphic niche differences (Beck & Richter 2008), the phylogenetic and biogeographic history of these regions, or simply to different sampling strategies (plots versus taxonomy-based sampling, and small areas versus large landscapes).
other non-tropical mycorrhizal interactions (Vályi et al. 2015). Specialization can sometimes be overestimated by low sampling effort, particularly in small ecological networks (Fründ et al. 2016). Although our network was undersampled and caution is needed in interpreting these results, we consider that the nested structure is reliable because this network feature has been found to be robust to undersampling (Nielsen & Bascompte 2007).
In the Tulasnellaceae-orchids network, robustness progressively decreased from the least- to most-linked extinction scenario to the random and the most- to least-linked extinction scenarios. This pattern has also been described in pollination networks (Memmott et al. 2004; Burgos et al. 2007; Santamaría et al. 2014). In our network, this reveals that generalist fungi and orchids play an important role in the stability of the mutualistic system. Terrestrial and epiphytic orchids showed similar robustness to the extinction of their mycobionts, and although differences were statistically significant, the effect was minor. This means that mycobionts are equally important for the persistence of both subsets of orchids. A broad generalization of interactions is important for network robustness (Pocock et al. 2012). Loss of generalist keystone species (most-connected species) may drive cascades of biodiversity loss. Furthermore, high specificity is unlikely in mutualistic interactions, as species that can associate with a greater variety of species have a wider range of ecological options for survival (Timms & Read 1999).
2010), and (2) obligate mutualisms are stabilized by the simultaneous association with facultative mutualists (Takimoto & Suzuki 2016). These conditions of Tulasnellaceae may support the development and coexistence of the high orchid richness in the tropical montane rainforest of Southern Ecuador.
Acknowledgments
This research was funded by the Deutsche Forschungsgemeinschaft (DFG RU 816). We thank Nature and Culture International (NCI) for providing research facilities at the Reserva Biológica San Francisco in Ecuador. We would also like to thank Alberto Mendoza for his help in orchid identification, Silvia Santamaría for sharing the R code for the robustness analysis and Lori De Hond for correcting the English text.
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