Journal of Plant Research

, Volume 132, Issue 3, pp 383–394 | Cite as

Germination niches and seed persistence of tropical epiphytic orchids in an urban landscape

  • Muhammad IzuddinEmail author
  • Tim Wing Yam
  • Edward L. Webb
Regular Paper


Urbanisation has contributed to significant biodiversity loss, yet, urban areas can facilitate biodiversity conservation. For instance, there is evidence of urban trees supporting natural establishments of orchids, the most species-rich plant family on Earth. However, the germination niches—which include both suitable biophysical conditions and orchid mycorrhizal fungus/fungi (OMF)—are not sufficiently known for most species, especially tropical epiphytic orchids. The fate of their dispersed seeds is poorly understood as well. We conducted fungal baiting and seed sowing experiments, next-generation sequencing, generalised linear models, and seed viability tests to detect and identify potential OMF, investigate biophysical factors that influenced OMF availability and orchid germination, and assess seed longevity. Ceratobasidiaceae- and Serendipitaceae-associated OMF were successfully detected in three of four orchid species. In general, orchid species and humus presence had significant effects on OMF availability. Orchid species and temperature were predictive of germination. Post-experiment viability tests revealed that one orchid species, Grammatophyllum speciosum Blume, may produce long-lived seeds. The results suggest that urban trees can support OMF and orchid germination, but both processes are limited by biophysical factors. This study also indicates the possibility of seed persistence among epiphytic species. As orchid germination niches are complex and tend to be unique to individual species, we do not encourage generalisations. In contrast, species-specific information can help formulate useful recommendations towards conservation.


Niche requirements Orchid mycorrhizal fungus Seed viability Ex situ conservation Singapore Urban ecology 


With ongoing rapid urban expansion and more forecasted worldwide this century, biodiversity loss continues to escalate (McKinney 2002; Seto et al. 2011). This loss is further compounded by recent projections of urban development in biodiversity hotspots and near protected areas (Güneralp and Seto 2013). Urbanisation contributes to habitat fragmentation, degradation, and loss, which in turn may lead to orchid population isolation and species extinction (Gardner et al. 2009; Ricketts 2001). And yet, although urbanisation has reduced biodiversity considerably, appropriate strategies in urban areas can contribute to biodiversity conservation (Fischer et al. 2016; Goddard et al. 2010; Wang et al. 2017). With urban areas becoming more prevalent, the need to assess the capacity of these spaces for biodiversity conservation is a high conservation priority.

Vascular epiphytes represent a large proportion of plant diversity that has undergone dramatic population declines in recent years due to land use changes (Sodhi et al. 2008; Turner et al. 1994; Zotz 2016). Among these plants, orchids have the distinction of being the most diverse plant family as well as the most vulnerable to environmental changes because of the family’s extreme dependence on specific biotic agents (mycorrhizal fungi, pollinators) closely tied to the life cycle (Dearnaley et al. 2012; Dixon et al. 2007; Gravendeel et al. 2004). Nevertheless, previous findings have shown that epiphytes, including orchids, are able to colonise urban trees (Bhatt et al. 2015; Izuddin and Webb 2015; Ranta 2008; Wee 1978). Moreover, some orchids are amenable to ex situ translocation efforts in urban habitats (Izuddin et al. 2018; Scade et al. 2006; Yam et al. 2011) but a more mechanistic understanding of the underlying factors determining germination and establishment success is required to address potential species loss and guide conservation and management strategies.

The species-specific germination niche is a combination of compatible orchid mycorrhizal fungus/fungi (OMF) and suitable biophysical conditions; for most orchid species and in particular tropical epiphytic orchids, however, their niche has not been evaluated (Bonnardeaux et al. 2007; Brundrett et al. 2003; Dearnaley et al. 2012; Jacquemyn et al. 2015; McCormick et al. 2012; Rasmussen et al. 2015; Zi et al. 2014). The OMF plays a vital role in determining orchid establishment as well as survival and growth because the minute orchid seeds lack appreciable food reserves (e.g., endosperm), and are therefore fully reliant on symbiotic interactions with compatible OMF to gain water, carbon, and other essential nutrients (Dearnaley 2007; Rasmussen and Rasmussen 2009; Smith and Read 2008; Yoder et al. 2000). However, different fungal taxa may vary in nutrient uptake efficiency and capacity to stimulate orchid germination (Brundrett 2007; Jacquemyn et al. 2015; van der Heijden et al. 2003; Zi et al. 2014). Furthermore, OMF are not necessarily widespread, but instead, may be limited by biophysical variables (Jacquemyn et al. 2014, 2015). Thus, detecting and identifying orchid-specific OMF as well as determining their availability and the associated biophysical factors can benefit future ex situ efforts (e.g., direct seeding, fungal inoculation of microsites).

In addition to OMF availability, variation in germination patterns in natural populations suggests that biophysical conditions may limit orchid establishment (Kartzinel et al. 2013; Rasmussen et al. 2015). Biophysical factors such as humus presence, water availability, and microsite temperature can induce germination as well as stimulate protocorm and seedling growth (Diez 2007; McCormick et al. 2012; Zettler et al. 2011). Influential biophysical factors may differ from one orchid species to another (Kartzinel et al. 2013; Rasmussen et al. 2015; Zi et al. 2014). Hence without the ideal conditions, OMF availability alone may not be sufficient to support germination. The complexity of germination niches can become even greater when biophysical factors that directly influence OMF presence are considered as well (Rasmussen et al. 2015). An ideal establishment site is therefore a microsite that supports biophysical conditions that simultaneously promote seed germination and OMF occupancy. Fundamentally, the identification of biophysical factors that favour seed germination could facilitate future intervention actions, resulting in strategies that are tailored to specific orchid species.

In addition to species-specific niche, seed longevity may influence the likelihood of successful establishment (Leck et al. 1989; Whigham et al. 2006). Seed longevity of epiphytic orchids remains largely unstudied, and has often been assumed to be short-term, i.e., orchid seeds degrade rapidly when dispersed to unsuitable microsites (Kartzinel et al. 2013; Rasmussen 1995). However, previous studies have presented clues indicating the robustness of orchid seeds. For instance, orchid seeds are known to remain viable for decades when stored at low temperature and humidity (Batty et al. 2001; Ramsey and Dixon 2003; Seaton et al. 2018). They are also highly resistant to chemical surface treatments (e.g., sodium hypochlorite, sulphuric acid) that are frequently applied to sterilise seeds (Batty et al. 2001; Hicks 1999). Accordingly, knowledge on the persistence of orchid seeds may improve seed-based conservation efforts, especially for endangered orchid species.

In this study, we asked whether urban trees support germination niches for native epiphytic orchids, i.e., are OMF and biophysical conditions that are suitable for native epiphytic orchids present on urban trees? Here, we use the city-state of Singapore as a model system. The dynamic nature of Singapore’s urban-green matrix makes the country an ideal study model for developing cities in relation to biodiversity conservation in the urban landscape. This context also provides an ideal opportunity to evaluate the feasibility of ex situ orchid conservation in urban environments. We inferred the availability of OMF—associated with four native epiphytic orchids—on urban trees using a novel orchid mycorrhizal fungal baiting technique. Potential OMF was identified using DNA barcodes and Illumina® MiSeq technology. We also identified biophysical factors that influenced OMF availability and germination success. The latter was achieved using a modified seed sowing experiment. We then assessed species-specific seed longevity using post-experimental, non-germinated seeds, i.e., do orchid seeds exhibit persistence? As orchids have been observed on modified landscapes, we expected OMF to be present on urban trees. We also hypothesised OMF availability and species-specific orchid germination to be related to specific biophysical factors. Since orchid seeds are very small and nutrient limited, we suspected that seed persistence is unlikely, i.e., seed degradation would be expected.

Materials and methods

Study area and species

Singapore is one of the most urbanised countries in the world, with more than 99% of its original forest cover transformed (Corlett 1992; Sodhi et al. 2004). To improve vegetation cover, the city-state has implemented multiple programmes and strategies that are devoted to urban greening (Ministry of National Development 2008; Tan et al. 2013; Yuen 1996).

Dendrobium crumenatum Sw., Bulbophyllum vaginatum (Lindl.) Rchb. f., Cymbidium finlaysonianum Lindl., and Grammatophyllum speciosum Blume are epiphytic, photosynthetic orchids native to Southeast Asia, typically distributed across Thailand, Peninsular Malaysia, Singapore, Borneo, and Indonesia (Yam et al. 2011). The common D. crumenatum grows in clusters of multiple, pendulous stems with deciduous and lanceolate leaves. Bulbophyllum vaginatum is an endangered species with a creeping, freely-branching growth habit. Individuals have rhizomes that bear numerous pseudobulbs, each with one short-stalked leaf. Cymbidium finlaysonianum is a critically endangered species that forms a basket of lengthy, leathery leaves enabling it to trap leaf litter from the canopy. The largest orchid species in the world, G. speciosum, is presumed to be nationally extinct. It has long, fleshy stems that harbour stalkless leaves that are 31–60 cm in length.

These species were selected mainly for two reasons: (1) dissimilar conservation statuses and (2) readily available seeds. As part of an orchid conservation programme, the seeds of these species were previously collected and stored (Yam et al. 2011). Mature, nearly dehisced seed capsules of D. crumenatum and B. vaginatum were acquired from natural populations found on urban trees whereas mature capsules of C. finlaysonianum and G. speciosum were collected from protected reserves and hand-pollinated nursery plants. About 8–10 capsules were collected per species. All capsules were surface-sterilised using 20% Clorox® and ethyl alcohol before seed removal. The seeds of each species were then pooled, dried in a lab drying oven for 24 h at 23 °C, and stored at −20 °C for 1–3 months prior to the experiments. To assess seed viability, isolated seeds were soaked in a 1% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) for 48 h at 30 °C in darkness (Samala et al. 2014; Van Waes and Debergh 1986). The pH was adjusted to 7 with 1 M NaOH. Seeds were then washed with sterilised Milli-Q® water (Merck & Co., Inc., Kenilworth, New Jersey, USA) to remove excess stain and observed under a stereo microscope. Red-stained embryos were classified as viable (Lauzer et al. 1994).

Sampling and experimental sites

A list of all roads planted with the roadside tree Albizia saman (Jacq.) Merr. were compiled with the help of Singapore’s National Parks Board. This species originates from South America and was selected chiefly for two reasons: (1) it is the most widely planted tree in the country (Tan et al. 2009) and (2) its distinct traits such as flaky bark and leaf-devoid inner branches allow it to be favourable host trees for epiphyte colonisation (Wee 1978). Fifteen roads were randomly selected as study sites, five in each of three “habitat-types” defined by the dominant surrounding land use type (Izuddin and Webb 2015): secondary forest (surrounded by lowland tropical forest, young secondary forest patches, or tree-dominated urban parks), grassland (surrounded by expanses of manicured turf grass, possibly fringed with shrubs or trees), and urban (surrounded by man-made structures) (Yee et al. 2011). The dominant land use was defined as > 50% of the area within an octagon of ~200 m diameter (drawn in Google™ Earth around the selected road; Fig. 1). Three individual trees were randomly selected at each road and the geographic coordinates of each tree were recorded using a Garmin® GPSMAP 60CSx GPS receiver (Garmin International, Inc., Olathe, KS, USA). Subsequently, three microsites—stem, fork, and branch—were randomly selected per tree (i.e., total microsites = 135). All trees were not colonised by any orchid.
Fig. 1

Map of the bark sampling and seed packet deployment locations in Singapore. The insets are examples of sites that were categorised based on dominant surrounding land use, i.e., “habitat-type”, from top left (clockwise): urban, grassland, and secondary forest. All octagons were 200 m in diameter (drawn to scale)

Availability of orchid mycorrhizal fungi on urban trees

We implemented an ex situ fungal baiting technique modified after Brundrett et al. (2003) for terrestrial orchids to allow simultaneous baiting and detection of OMF associated with various epiphytic orchid species under controlled conditions. Bark samples (~25 mm2) were acquired from all selected microsites using sterilised scalpel. They were then packaged in sterile plastic storage bags and consequently placed individually in 60 mm Petri dishes (germination plates; three replicates per microsite). Each bark sample was saturated with sterilised Milli-Q® water and left overnight for absorption before draining the excess water the following day. Approximately 150–200 seeds of D. crumenatum, B. vaginatum, C. finlaysonianum, and G. speciosum were sprinkled on to the bark sample with one species per corner, avoiding any clumping of seeds.

All germination plates were placed in a growth chamber (Sanyo MLR-351H, Sanyo Electric Co., Ltd, Osaka, Japan) under the following conditions: temperature 24/29 °C (day/night), relative ambient humidity 90%, photoperiod 12 h, and fluorescent light: 0–40 μmol m−2 s−1 (Panasonic Corporation, Osaka, Japan). All plates were checked every 3–4 days to ensure that they remained moist but not saturated; sterilised Milli-Q® water was added when required. There were two controls (five plates per control): (1) bark samples without orchid seeds and (2) orchid seeds on sterile filter paper (substitute for sterilised bark); as complete bark sterilisation (i.e., free of all fungi) was difficult to achieve, the latter was considered instead. Each plate was inspected under a stereo microscope once monthly for 12 months (January 2016–January 2017). Germination indicated OMF presence and was recorded when the enlarged embryo had emerged from the testa (Whigham et al. 2006; Fig. 2). We also checked for other signs of germination, particularly changes in colour (mainly green, which tend to signify the capacity for photosynthesis) as well as shape (possible indication of shoot/root development) (Islam et al. 2015; Kartzinel et al. 2013; Samala et al. 2014; Tawaro et al. 2008). The seed viability test was conducted on subsets of post-experiment, non-germinated seeds. For each species, ca. 150–200 seeds with visible embryo were randomly selected from various germination plates and carefully removed with a pair of precision tweezers. Only qualitative comparison was done as the sample size for each species was limited.
Fig. 2

Seed sowing and orchid mycorrhizal fungus (OMF) baiting experiments—a seed packet with iButton® hygrochron temperature/humidity logger, b germination initiation of a C. finlaysonianum seed on sponge, c mass development of D. crumenatum protocorms on bark, and d swollen embryo of a G. speciosum seed on bark

Several biophysical variables were recorded for sampling each tree: host tree DBH (diameter at breast height; measured with a diameter tape, at 1.3 m above the base), microsite location (stem/fork/branch), and substrate (presence/absence of humus and moss). Climate data—mean rainfall, mean ambient temperature, and mean wind speed—were 5-year daily averages, calculated from compiled weather records provided by Meteorological Service Singapore (2017). Using Google™ Earth, we also measured the distance of host trees from nearest forest vegetation and nearest distance between trees located on the same road (i.e., distance between host trees). Due to the limited timeframe of the study, bark chemistry (e.g., hydrocarbon content, pH) was not analysed in this study.

Biophysical factors influencing ex situ germination

A modified seed sowing technique was developed to allow simultaneous seed germination of different orchids enclosed within a single seed packet, while preventing undesirable seed clumping. This modification was based on existing seed sowing methods for single epiphytic orchid species (Cruz-Higareda et al. 2015; Kartzinel et al. 2013; Rasmussen and Whigham 1993). We cut polyurethane sponges into 40 × 40 × 2 mm sizes (germination sponges). The 2 mm thickness was sufficient to retain seeds while avoiding water retention and hence moisture bias. To sterilise the sponges, we soaked them in a 5% solution of decon® 90 (Decon Laboratories Limited, East Sussex, UK) for 2 h, thoroughly rinsed with sterilised Milli-Q® water, autoclaved, and dried in a fume hood. Approximately 250–400 seeds of each orchid species were uniformly sprinkled over each corner with one species per corner, avoiding mixing (for illustrations, see Cruz-Higareda et al. 2015).

To affix the germination sponges to the tree, each sponge was directly placed on the bark, covered with a sterilised 70 × 120 mm nylon mesh with 50 μm diameter holes, and secured using stainless steel tacks and staples (Fig. 2a). This pore size allowed entry of fungal hyphae, yet prevented seed loss (Rasmussen and Whigham 1993; Wang et al. 2011). In May 2016, a total of 135 seed packets were established on 135 microsites—stem, fork, or branch—among 45 trees (i.e., 9 seed packets per study site). During the experimental period, one study site was destroyed for urban development, reducing the final sample size to 126 seed packets among 42 trees (i.e., 14 sites; Fig. 1).

The following biophysical variables were recorded at each tree: height of seed packet from ground (measured using a laser distance meter), host tree DBH (measured with a diameter tape, at 1.3 m above the base), and canopy openness. Percentage canopy openness was calculated from digital hemispherical photographs (taken at 1.5 m above ground) using Gap Light Analyzer (GLA) version 2.0 (Frazer et al. 1999). We logged ambient temperature and relative humidity of each microsite by continuously running the iButton® DS1923 temperature/humidity loggers (Maxim Integrated™, San Jose, CA, USA). Daily rainfall data were acquired from weather records provided by Meteorological Service Singapore (2017). We also noted microsite location (stem/fork/branch), substrate (presence/absence of humus and moss), directional exposure (aspect), and direct cover [presence/absence of epiphyte(s) directly above seed packet, e.g., Davallia denticulata (Burm.) Mett.].

After 10 months, seed packets were removed and each sponge was immediately placed in a Petri dish. Dislodgement of seed packets—possibly due to tree pruning, torrential rain, and/or damage by urban birds—were also noted. Germination sponges were examined under a stereo microscope for germination, indicated by the emergence of a swollen embryo from the ruptured testa (Whigham et al. 2006; Fig. 2). We also inspected for other signs of germination, mainly changes in colour (primarily green, which tend to signify the capacity for photosynthesis) as well as shape (possible indication of leaf or root primordia) (Islam et al. 2015; Kartzinel et al. 2013; Samala et al. 2014; Tawaro et al. 2008). We then conducted seed viability tests on subsets of post-experiment, non-germinated seeds. For each species, ca. 150–200 seeds with visible embryo were randomly selected from various seed packets and carefully removed with a pair of precision tweezers. Only qualitative comparison was done as the sample size for each species was limited.

Statistical analyses

To assess the effects of biophysical factors on OMF presence and orchid seed germination rates, we used binomially-distributed generalised linear models (GLMs). Both models were conducted at two levels: overall (i.e., all species) and species-specific. When overdispersion was evident, the model was fitted with quasi-binomial error structure. The explanatory variables for the models were species and all recorded biophysical variables. Multicollinearity was assessed and highly collinear variables were removed from the models. Akaike’s information criterion (AIC; Akaike 1974) was used for step-wise simplification and model evaluation. Model deviance was estimated as goodness-of-fit. Spatial autocorrelation was examined using variograms and correlograms (Legendre and Fortin 1989). All analyses were conducted using R version 3.4.2 (R Development Core Team 2015), including the packages “spatial” (Venables and Ripley 2002) and “car” (Fox and Weisberg 2011).

Identification of potential protocorm-associated fungus

After 1 year, all germinated seeds of each species—retrieved from the ex situ fungal baiting experiment—were pooled for identification of OMF via DNA barcodes and next-generation sequencing (see Appendix S1). Due to their minute size and delicate nature, seed-pooling was necessary to yield sufficient fungal DNA (Kartzinel et al. 2013). Hence, OMF-related sequences may represent either orchid-specific OMF or an asymbiotic fungal taxa found on the germination plates. Because all germinated seeds were pooled, we could not compare OMF across habitat-types or microsites.


The orchid mycorrhizal fungal baiting method successfully detected OMF presence in three of four epiphytic species. In total, 117 (87%) plates contained germinated D. crumenatum seeds, 79 (59%) contained germinated B. vaginatum seed, and 27 (20%) contained germinated G. speciosum seed (Fig. 3). Germination was not observed in any of the control plates. The overall GLMs revealed that variation in OMF presence was associated with orchid species (Table 1). Species-wise, microsite location—specifically branch—corresponded to presence of D. crumenatum-associated OMF. Both overall and species-specific GLMs also indicated the positive relationship between presence of humus and OMF presence. Distance of individual trees from nearest forest vegetation negatively influenced presence of B. vaginatum-associated OMF, and conversely, positively influenced presence of G. speciosum-associated OMF. For D. crumenatum, OMF presence showed a positive association with distance between individual trees. As the fungal baiting experiment did not yield any germinated C. finlaysonianum seeds, we could not determine the biophysical variables that influence the presence of C. finlaysonianum-associated OMF. No spatial autocorrelation was detected in any dataset.
Fig. 3

Percentage germination per orchid species across all germination sponges (seed sowing experiment) and germination plates (orchid mycorrhizal fungal baiting experiment)

Table 1

Results of generalised linear models (GLMs) illustrating the biophysical variables that were influential predictors of orchid mycorrhizal fungus (OMF) presence

Biophysical variables



P value



< 0.001

 Presence of humus



< 0.001

Dendrobium crumenatum Sw.a


< 0.05




< 0.05

 Presence of humus



< 0.001

 Distance between host trees



< 0.05

Bulbophyllum vaginatum (Lindl.) Rchb. f.b

 Presence of humus



< 0.05

 Distance of host tree from nearest forest vegetation

− 0.001


< 0.05

Grammatophyllum speciosum Blumea


 Presence of humus



< 0.01

 Distance of host tree from nearest forest vegetation



< 0.05

Since the fungal baiting experiment did not yield any germinated C. finlaysonianum seeds, we could not determine the biophysical variables that influence the presence of C. finlaysonianum-associated OMF. All models were fitted with a binomial or quasi-binomial error structure

aBinomial or bquasi-binomial error structure

Recovery rate of seed packets was moderate with 73 of 126 (58%) packets retrieved. Fifty-one (74%) packets contained germinated D. crumenatum seeds, 53 (73%) contained germinated B. vaginatum seeds, 36 (51%) contained germinated C. finlaysonianum seeds, and 17 (25%) contained germinated G. speciosum seeds (Fig. 3). In general, orchid species and temperature were predictive of germination (Table 2). Similarly, temperature negatively influenced germination of C. finlaysonianum seeds. Germination of D. crumenatum seeds was significantly related to humus presence and host tree diameter. For G. speciosum, germination was positively associated with average daily rainfall. No biophysical variable was influential in determining germination of B. vaginatum.
Table 2

Results of generalised linear models (GLMs) illustrating the biophysical variables that were influential predictors of orchid germination

Biophysical variables



P value




< 0.001


− 0.664


< 0.01

Dendrobium crumenatum Sw.b





< 0.05

 Presence of humus



< 0.001

Cymbidium finlaysonianum Lindl.b



− 1.171


< 0.05

Grammatophyllum speciosum Blumeb


 Average daily rainfall



< 0.001

No predictor was influential in determining germination of B. vaginatum. All models were fitted with a binomial or quasi-binomial error structure

aBinomial or bquasi-binomial error structure

All seeds had high pre-experiment seed viability: D. crumenatum 88.3%, B. vaginatum 72.7%, C. finlaysonianum 87.7%, and G. speciosum 80.7% (Table 3). Seed viability of non-germinated D. crumenatum seeds decreased to 47.0% and 34.2% under controlled (1-year period; orchid mycorrhizal fungal baiting experiment) and variable environmental conditions (10-month period; seed sowing experiment) respectively. Seed viability after the same time periods and conditions were 50.7% and 49.4% for B. vaginatum, 46.5% and 55.2% for C. finlaysonianum, and 63.8% and 65.7% for G. speciosum.
Table 3

Pre- and post-experimental seed viability of orchid seeds


% of seeds stained



Fungal baiting

Seed sowing

Dendrobium crumenatum Sw.a




Bulbophyllum vaginatum (Lindl.) Rchb. f.b




Cymbidium finlaysonianum Lindl.c




Grammatophyllum speciosum Blumed




Statuses of orchid species: acommon, bendangered, ccritically endangered, dextinct

Overall, 478 047 reads were obtained and assigned to three pooled protocorm samples. Clustering quality-filtered sequences to OTUs yielded 108 OTUs (334 unique sequences), of which three were assigned to putative OMF-OTUs in two families (see Table S1). Multiple fungal taxa, including OMF, saprotrophs (e.g., Hypocreales, Capnodiales), and parasites (e.g., Pleosporales) were detected and identified. The OMF-orchid associations for germination were as follows: D. crumenatum with Ceratobasidium sp. (Ceratobasidiaceae), B. vaginatum with Sebacinales, and G. speciosum with Sebacinaceae (see Table S1). Since the orchid mycorrhizal fungal baiting experiment did not yield any germinated C. finlaysonianum seeds, the associated OMF could not be identified.


In this study, we investigated whether urban trees support germination niches for native epiphytic orchids. We found that orchid mycorrhizal fungi are present on urban trees, but they were limited by various biophysical conditions. Likewise, seeds germinated on microsites of urban trees, but germination was constrained by biophysical factors. We also found evidence of seed persistence among epiphytic orchid species. Overall, our study builds upon previous studies indirectly indicating that urban trees can support unassisted orchid germination and establishment (Bhatt et al. 2015; Izuddin and Webb 2015; Ranta 2008; Wee 1978).

Orchid mycorrhizal fungi on urban trees

We found fungal taxa that are commonly associated with epiphytic and terrestrial orchids (Jacquemyn et al. 2017). Each study species was associated with a unique fungal taxon. These characteristic affiliations may differentiate their germination niches, leading to possible co-occurrence of orchid species (Jacquemyn et al. 2012; Těšitelová et al. 2012). Furthermore, knowing the identity of available OMF can help discern the trophic strategy of compatible fungus and thus the principal source of organic carbon for germinated seeds (Rasmussen et al. 2015). We also detected common non-mycorrhizal taxa such as Capnodiales and Pleosporales. These saprotrophic fungi may indirectly enhance nutrient access to orchids via substrate decomposition (Herrera et al. 2010). While we did not quantify the fungal density/potential (abundance and vitality) of each taxon, symbiont density/potential can influence a microsite’s capacity to support seed germination (McCormick et al. 2012). Our identification results were also based on germinated seeds retrieved from the ex situ fungal baiting experiment only and thus may be limited. Therefore, although challenging, we encourage future studies to attempt isolation of OMF directly from bark substrates and orchids at higher growth stages to improve morphological and molecular identification as well as affirm OMF presence. Moreover, the controlled nature of our orchid mycorrhizal fungal baiting method may increase the likelihood of successful fungal isolation.

Based on the orchid mycorrhizal fungal baiting experiment, the germination patterns indicate the variation in availability of species-specific OMF among microsites, most likely delimited by a single or multiple biophysical factor(s). Our results highlight the central role of humus in determining OMF presence. Humus usually accumulates in cracks and crevices of microsites, most notably on the tree fork (Böhnert et al. 2016; Yam et al. 2011). These pockets of organic substrate are not only vital sources of nutrients (e.g., carbon, phosphorus) for mycelial development, but of water as well (Yam et al. 2011). Improved substrate moisture can promote mycelial survival and growth, which in turn can benefit developing orchid seeds by augmenting their capacity for moisture imbibition (Mújica et al. 2018; Osono et al. 2003; Yoder et al. 2000).

The orchid mycorrhizal fungal baiting experiment indicates distance-dependent relationships between OMF availability, host trees, and secondary forest vegetation. The OMF associated with D. crumenatum (Ceratobasidium sp.) was the most prevalent orchid mycorrhizal fungal taxon and occurred at sites whereby host trees were considerably spaced out, implying that dispersal and subsequent establishment of this taxon is largely unhindered in the urban landscape. This OMF was also primarily present on branches; given that tree branches are typically exposed and structurally angled (cf. vertical stem and fork), it is possible that this part of the tree has the highest capacity for inoculum/spore interception (Chapela and Boddy 1988). For B. vaginatum, the associated OMF was detected on microsites located near secondary forest patches, indicating that nearby vegetative patches may serve as sources of orchid mycorrhizal fungal spore/inoculum. Peay et al. (2012) demonstrated that availability of fungal spores in a non-host vegetation (i.e., vegetation devoid of a specific fungus) decreases with increasing spatial distance from host vegetation. However, this trend may not always hold true as the opposite pattern was observed for G. speciosum-associated OMF. In general, due to the complex dynamics of air currents and passive nature of diaspore movement, diaspore dispersal and deposition remains broadly unpredictable (Peay and Bruns 2014). Insects, birds, and mammals may potentially spread fungal inoculum from one tree to another as well (Malloch and Blackwell 1992).

Germination on urban trees

As expected, germination and biophysical influencers varied across orchid species. We found that increasing microsite temperature negatively affects germination. High temperatures directly promote undesirable water loss in seeds and indirectly in substrates, which in turn hinder mycelial development (Yoder et al. 2000). This was particularly evident for the germination of C. finlaysonianum seeds. We detected germinated C. finlaysonianum seeds in seed packets but not on germination plates. It is possible that temperature fluctuations function as biological cues that release seed dormancy or enhance seed germination (Rasmussen et al. 2015; Zettler et al. 2001).

Dendrobium crumenatum’s germination tended to occur on larger trees, perhaps the result of greater humus content resulting from greater surface area of larger trees. The larger surface area also permits greater fungal diaspore interception, increasing the probability of OMF establishment over time (Izuddin and Webb 2015). The association between D. crumenatum and humus suggests that this species’ germination may be more dependent on mineral nutrients (e.g., nitrogen, phosphorus) than other study species (Zotz and Asshoff 2010; Zotz et al. 2011). As mentioned, pockets of humus are also important water sources and hence may facilitate germination by preventing seed desiccation and promoting water imbibition (Scott and Carey 2002; Yoder et al. 2000).

Similarly, water availability is a critical factor in determining the germination success of G. speciosum. Microsites experiencing higher levels of rainfall can engender sustained moisture in substrate, encouraging both seed and protocorm development (Rasmussen et al. 2015). Seed development of this species may require a higher demand for moisture as compared to other study species.

Seed persistence

After being subjected to varying field conditions, more than 80% of G. speciosum seeds retrieved remained viable. This result suggests that this species may produce long-lived seeds, and thus, has the potential to persist for years on exposed bark. Although persistent seeds are typically associated with terrestrial orchid species (Bakker et al. 1996; Baskin and Baskin 1998), given the right conditions, it is possible that seeds of epiphytic orchids can remain viable within bark layers or crevices of host trees over a period of time as well. The possible advantage of persistent seeds is that there is less temporal urgency for germination as the chances of encounter with a suitable OMF will increase over time, especially if there are expanding patches of compatible fungus nearby (Whigham et al. 2006). Since seed longevity of orchids—notably epiphytic species—has often been overlooked, we encourage future investigations into this biological aspect that may have valuable conservation implications.


Knowledge on the identity and availability of relevant OMF, as well as biophysical influencers, is imperative for future plans of establishing orchids, especially endangered species, in natural or urban landscapes (Batty et al. 2002). This is the first orchid mycorrhizal fungal baiting experiment that utilises bark samples from urban trees to detect the OMF of multiple orchid species. Our results suggest that mycorrhizal fungi must be taken into account for orchid conservation programmes to be successful on a long-term basis for tropical epiphytes, not just temperate terrestrials. We also paired the lab-based method with a field-based seed sowing method to achieve a comprehensive understanding of species-specific germination niches [i.e., suitable OMF and (micro-)environmental conditions]. Indeed, abiotic factors—though often overlooked—are equally crucial due to their contrasting influence in determining carbon and fungi sources as well as orchid germination and growth (Dearnaley et al. 2012; Izuddin et al. 2018; Rasmussen et al. 2015). We also found evidence of seed persistence among epiphytic species. This finding may shed new light on future seed-based research and intervention attempts such as long-term seed sowing experiments (e.g., > 2 years) and direct seeding.

Overall, our results indicate that the requirements for OMF presence and orchid germination, when collectively contemplated, differ among orchid species. This implies that germination niches tend to be unique for each orchid species, making any generalisation unadvisable. Nevertheless, such species-specific information can be useful in formulating hypotheses for subsequent species. More studies on epiphytic orchid germination are required, particularly investigations in the canopy region, which are relatively scarce. We also encourage prospective studies to isolate and identify fungi directly from protocorms or small seedlings to affirm species-specific association. Ultimately, pertinent insights on germination niches gained can guide future in situ and ex situ conservation efforts such as fungal inoculation of microsite followed by seeding or transplantation of propagated orchids. As orchids are a major component of the epiphytic ecosystem and tropical biodiversity in general, future research should continue to evaluate how both forest and non-forest habitats can contribute to orchid conservation.



The authors thank Peter Ang, Maria Lee, Amrita Srivathsan, Muhammad Noh Al, and Maryam Nadheera for their valuable assistance. The molecular work and computational resources were supported by SEABIG (Grants R-154-000-648-646 and R-154-000-648-733).

Supplementary material

10265_2019_1110_MOESM1_ESM.pdf (49 kb)
Supplementary material 1 (PDF 49 kb)


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Copyright information

© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • Muhammad Izuddin
    • 1
    Email author
  • Tim Wing Yam
    • 2
  • Edward L. Webb
    • 1
  1. 1.Department of Biological SciencesNational University of SingaporeSingaporeSingapore
  2. 2.Singapore Botanic GardensSingaporeSingapore

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