Ryan D. Phillips

Reconnecting plants and pollinators: challenges in the restoration of pollination mutualisms

Ecological restoration of plant-pollinator interactions has received surprisingly little attention, despite animal-mediated pollination underpinning reproduction of the majority of higher plants. Here, we offer a conceptual and practical framework for the ecological restoration of pollination mutualisms. Through the use of targeted restoration plantings to attract and sustain pollinators and increased knowledge of the ecological requirements of pollinators, we propose that pollination could be successfully restored in degraded ecosystems. The challenge for pollination biologists is to integrate their findings with those of plant restoration ecologists to ensure sustainable pollination in restored ecosystems.

Implications of pollination by food and sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae)

Caladenia is very unusual in that it contains species that attract pollinators by two different strategies, food and sexual deception. Among the sexually deceptive species, baiting for pollinators has shown that within populations orchid species are typically pollinated by a single species of thynnine wasp. However, some wasp species can be pollinators of more than one species of orchid usually when their ranges do not overlap. There is a trend for closely related orchids to exploit wasps from the same genus, with different lineages of orchids often pollinated by different genera. Very little is known about pollination of food-deceptive Caladenia species, although it is evident they attract a suite of generalist food-seeking insects. Food-deceptive species have a higher pollination rate than do sexually deceptive species. Studies of population genetics and pollen movements are few, although they suggest a pattern of fine-scale genetic structuring within populations, owing to predominantly restricted seed dispersal and low genetic differentiation among populations as a consequence of rare long-distance seed-dispersal events. Both evolutionary and ecological research of Caladenia will greatly benefit from a better understanding of the insect species involved in pollination, their ecological requirements and the ecological and genetic consequences of food and sexual deception.

Low population genetic differentiation in the Orchidaceae: implications for the diversification of the family

A leading hypothesis for the immense diversity of the Orchidaceae is that skewed mating success and small, disjunct populations lead to strong genetic drift and switches between adaptive peaks. This mechanism is only possible under conditions of low gene flow that lead to high genetic differentiation among populations. We tested whether orchids typically exhibit high levels of population genetic differentiation by conducting a meta‐analysis to compare mean levels of population genetic differentiation (FST) between orchids and other diverse families and between rare and common orchids. Compared with other families, the Orchidaceae is typically characterized by relatively low genetic differentiation among populations (mean FST = 0.146) at allozyme loci. Rare terrestrial orchids showed higher population genetic differentiation than common orchids, although this value was still lower than the mean for most plant families. All lines of evidence suggest that orchids are typically characterized by low levels of population genetic differentiation, even in species with naturally disjunct populations. As such, we found no strong evidence that genetic drift in isolated populations has played a major role in the diversification of the Orchidaceae. Further research into the diversification of the family needs to unravel the relative roles of biotic and environmental selective pressures in the speciation of orchids.

Caught in the act: Pollination of sexually deceptive trap-flowers by fungus gnats in Pterostylis (Orchidaceae)

BACKGROUND AND AIMS Pterostylis is an Australasian terrestrial orchid genus of more than 400 species, most of which use a motile, touch-sensitive labellum to trap dipteran pollinators. Despite studies dating back to 1872, the mechanism of pollinator attraction has remained elusive. This study tested whether the fungus gnat-pollinated Pterostylis sanguinea secures pollination by sexual deception. METHODS The literature was used to establish criteria for confirming sexual deception as a pollination strategy. Observations and video recordings allowed quantification of each step of the pollination process. Each floral visitor was sexed and DNA barcoding was used to evaluate the degree of pollinator specificity. Following observations that attraction to the flowers is by chemical cues, experimental dissection of flowers was used to determine the source of the sexual attractant and the effect of labellum orientation on sexual attraction. Fruit set was quantified for 19 populations to test for a relationship with plant density and population size. KEY RESULTS A single species of male gnat (Mycetophilidae) visited and pollinated the rewardless flowers. The gnats often showed probing copulatory behaviour on the labellum, leading to its triggering and the temporary entrapment of the gnat in the flower. Pollen deposition and removal occurred as the gnat escaped from the flower via the reproductive structures. The labellum was the sole source of the chemical attractant. Gnats always alighted on the labellum facing upwards, but when it was rotated 180 ° they attempted copulation less frequently. Pollination rate showed no relationship with orchid population size or plant density. CONCLUSIONS This study confirms for the first time that highly specific pollination by fungus gnats is achieved by sexual deception in Pterostylis. It is predicted that sexual deception will be widespread in the genus, although the diversity of floral forms suggests that other mechanisms may also operate.

Pollination ecology and the possible impacts of environmental change in the Southwest Australian Biodiversity Hotspot

The Southwest Australian Biodiversity Hotspot contains an exceptionally diverse flora on an ancient, low-relief but edaphically diverse landscape. Since European colonization, the primary threat to the flora has been habitat clearance, though climate change is an impending threat. Here, we review (i) the ecology of nectarivores and biotic pollination systems in the region, (ii) the evidence that trends in pollination strategies are a consequence of characteristics of the landscape, and (iii) based on these discussions, provide predictions to be tested on the impacts of environmental change on pollination systems. The flora of southwestern Australia has an exceptionally high level of vertebrate pollination, providing the advantage of highly mobile, generalist pollinators. Nectarivorous invertebrates are primarily generalist foragers, though an increasing number of colletid bees are being recognized as being specialized at the level of plant family or more rarely genus. While generalist pollination strategies dominate among insect-pollinated plants, there are some cases of extreme specialization, most notably the multiple evolutions of sexual deception in the Orchidaceae. Preliminary data suggest that bird pollination confers an advantage of greater pollen movement and may represent a mechanism for minimizing inbreeding in naturally fragmented populations. The effects of future environmental change are predicted to result from a combination of the resilience of pollination guilds and changes in their foraging and dispersal behaviour.

The discovery of 2-hydroxymethyl-3-(3-methylbutyl)-5-methylpyrazine: a semiochemical in orchid pollination.

Drakaea livida (Orchidaceae) is pollinated by sexual deception of the wasp Zaspilothynnus nigripes (Thynnidae). It is shown that the orchid emits the same compound, 2-hydroxymethyl-3-(3-methylbutyl)-5-methylpyrazine, that females emit when calling for mates. This novel pyrazine was isolated and identified by GC-EAD and GC-MS and confirmed by synthesis. This compound may represent the first known case of pyrazines as sex pheromones in Hymenoptera.

Convergent specialization - the sharing of pollinators by sympatric genera of sexually deceptive orchids

Summary 1. Pollinator sharing can offer powerful insights into the floral traits associated with the evolution of a pollination system and the consequences of floral differences for pollinator behaviour. Here, we investigate the first known case of pollinator sharing between two sexually deceptive plant genera. Floral manipulations were used to test the importance of floral traits for pollinator behaviour and pollination efficiency. We also explored the ecological differences enabling species co-occurrence. 2. Drakaea livida and Caladenia pectinata (Orchidaceae) exhibit dramatic differences in floral display and the insectiform appearance of the labellum, yet both are pollinated by sexually attracted males of the thynnine wasp Zaspilothynnis nigripes. Because of the prevalence of cryptic species in some genera of thynnine wasps, we confirmed pollinator sharing by a mark–recapture study and sequencing of the mtDNA CO1 region. 3. Floral dissections revealed that semiochemicals used to attract the pollinator are released from the labellum in D. livida and sepaline clubs in C. pectinata. Drakaea livida was more efficient at converting pollinator attraction into potential pollen deposition leading to higher fruit set. Floral manipulations showed that pollinator contact with the labellum increases when it is the point of semiochemical release. However, sexual attraction to the labellum remained infrequent in C. pectinata in all experimental treatments. 4. While their distribution and climatic range show extensive overlap, the differences in edaphic requirements of the two orchid species suggest that they rarely co-occur. Therefore, the potential cost of sharing the same pollinator species is not realized. 5. Synthesis. This case of pollinator sharing confirms that morphological traits do not place a strong constraint on the evolution of sexual deception. However, interspecific differences in floral traits have important consequences for converting attraction into pollination, suggesting that selection can act to increase efficiency at multiple steps of the pollination process. This system provides a novel opportunity to elucidate the chemical, visual and morphological adaptations underpinning the evolution of sexual mimicry.

Congruent species delineation of Tulasnella using multiple loci and methods

Understanding evolutionary and ecological processes requires accurate delimitation of species. Species are most commonly defined under the general lineage concept (GLC), where they are considered to be segments of diverging population-level lineages (de Queiroz, 2007). Within the umbrella of the GLC, various species concepts have been developed, including themorphological species concept (MSC), the biological species concept (BSC) and the phylogenetic species concept (PSC). The BSC is characterized by species representing populations that potentially can interbreed. The PSC includes the genealogical concordance phylogenetic species recognition concept, which uses phylogenetic concordance of multiple unlinked genes to identify evolutionary independence of lineages (Taylor et al., 2000). PSC is applied frequently in taxonomic groups where it is difficult to quantify morphological variation or performmating studies. One such group is Tulasnella, a group of fungi where morphological identification of species is problematic. Tulasnella includes putatively saprotrophic species on decayed wood (Roberts, 1999; Cruz et al., 2011). Some species are also encountered as ectomycorrhizas (Tedersoo et al., 2010) or orchid mycorrhizal symbionts (Dearnaley et al., 2012). However, many questions remain about species delimitation in this group. We therefore need a robust multilocus method for species delimitation to establish a framework for studying the evolution, ecology and physiology of orchid–fungus relationships. It is now recognized that the most effective approach to species delineation is the integration of multiple datasets and analytical methods (Sites &Marshall, 2004; Leach e & Fujita, 2010; Yang & Rannala, 2010; Barrett & Freudenstein, 2011). Here we use evidence from six nuclear loci, twomitochondrial loci, orchid-host association and geographical location of samples in a multifaceted approach to delineate species of Tulasnella associated with the Australian orchid genera Chiloglottis, Drakaea, Paracaleana and Arthrochilus. Specifically, we employ gene tree construction methods to resolve fungal species boundaries; use coalescent species tree construction methods (using the programs *BEAST and BPP) to test for host and geographic association; and apply population genetic assignment methods to test for admixture between populations. Finally, germination data are used to explore the correlation between physiological traits and phylogenetic boundaries among Tulasnella. In light of the outcomes we evaluate the implications of multigene approaches for fungal species delimitation in this Tulasnella group.

Biogeography of Caladenia (Orchidaceae), with special reference to the South-west Australian Floristic Region

Caladenia contains 376 species and subspecies, of which almost all are endemic to temperate and southern semiarid Australia. Eleven species occur in New Zealand, 10 of which are endemic, and one species is widely distributed in eastern Australia and the western Pacific. Only three species occur in both south-western and south-eastern Australia. At subgeneric level, Drakonorchis is endemic to the South-west Australian Floristic Region (SWAFR), Stegostyla to eastern Australia and New Zealand, whereas three subgenera, Calonema, Phlebochilus and Elevatae occur on both sides of the Nullarbor Plain. Subgenus Caladenia is primarily eastern Australian but also extends to the western Pacific. The largest subgenera (Calonema and Phlebochilus) have radiated extensively, with Calonema exhibiting a greater concentration of species in more mesic parts of the SWAFR than Phlebochilus. Within the SWAFR, the major biogeographic division within Caladenia follows the 600-mm isohyet. Within rainfall zones, biogeographic districts for Caladenia correlate with a combination of underlying geology and surface soils. Areas of high endemism contain diverse edaphic environments. Climatic and edaphic requirements are likely to be key drivers of rarity in Caladenia, although these parameters may be acting in concert with mycorrhizal and pollinator specificity.

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

BACKGROUND AND AIMS Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale. METHODS Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia. KEY RESULTS With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent. CONCLUSIONS The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.