Mark A. Clements

Quantifying Phytogeographical Regions of Australia Using Geospatial Turnover in Species Composition

The largest digitized dataset of land plant distributions in Australia assembled to date (750,741 georeferenced herbarium records; 6,043 species) was used to partition the Australian continent into phytogeographical regions. We used a set of six widely distributed vascular plant groups and three non-vascular plant groups which together occur in a variety of landscapes/habitats across Australia. Phytogeographical regions were identified using quantitative analyses of species turnover, the rate of change in species composition between sites, calculated as Simpsons beta. We propose six major phytogeographical regions for Australia: Northern, Northern Desert, Eremaean, Eastern Queensland, Euronotian and South-Western. Our new phytogeographical regions show a spatial agreement of 65% with respect to previously defined phytogeographical regions of Australia. We also confirm that these new regions are in general agreement with the biomes of Australia and other contemporary biogeographical classifications. To assess the meaningfulness of the proposed phytogeographical regions, we evaluated how they relate to broad scale environmental gradients. Physiographic factors such as geology do not have a strong correspondence with our proposed regions. Instead, we identified climate as the main environmental driver. The use of an unprecedentedly large dataset of multiple plant groups, coupled with an explicit quantitative analysis, makes this study novel and allows an improved historical bioregionalization scheme for Australian plants. Our analyses show that: (1) there is considerable overlap between our results and older biogeographic classifications; (2) phytogeographical regions based on species turnover can be a powerful tool to further partition the landscape into meaningful units; (3) further studies using phylogenetic turnover metrics are needed to test the taxonomic areas.

Codiversification of orchids (Pterostylidinae) and their associated mycorrhizal fungi

Fungal symbionts involved in mycorrhizal associations are known to vary considerably in both specificity and the level of benefits conferred on their plant hosts. For orchids, association with a suitable mycorrhizal fungus is vital for successfulgermination,growthandestablishment.UsinganevolutionarilydistinctgroupofAustralasianterrestrialorchids, the Pterostylidinae(Cranichiadeae: Orchidaceae), weassessedpotential codiversification andthelevel ofresponse between thisdiversehostgroup(~250species)andtheirassociatedfungalsymbionts.Allfungalisolatesrecovered(~200from41host species covering all major orchid clades) were identified as species of Ceratobasidium, which clustered into strongly supported groups using nuclear (ITS) and mitochondrial (ML 4-5) gene sequences. Three clades within the Pterostylidinae phylogeny showed associations with specific fungal clades. The results suggest the occurrence of local adaptation by the fungal symbionts to the orchid host, particularly in diverse and widespread host taxa. Results of cross-inoculation in vitro germination experiments revealed correlations between certain mycorrhizal fungal clades and particular orchid taxa, with germination generally being most effective when seeds were inoculated with fungal strains from the same clade as found naturallyassociatedwiththeorchidspecies.Wefoundonlygeneralcongruencebetweentheorchidandfungalphylogenies, suggesting that strict codivergerence between these orchids and their mycorrhizal associates has not occurred at the broad level of resolution studied.

Significant phorophyte (substrate) bias is not explained by fitness benefits in three epiphytic orchid species.

PREMISE Epiphytes rely on their phorophyte (host substrate) for support; epiphytic orchids also rely on mycorrhizal fungi for germination. Previous studies have proposed a degree of specificity in both interactions. Epiphytic orchids therefore provide an interesting system in which to examine multispecies interactions and the evolution of specialization. METHODS We examined the potential and actual distributions of three co-occurring, related epiphytic orchid species: Sarcochilus hillii, Plectorrhiza tridentata, and Sarcochilus parviflorus on phorophytes in Australias temperate dry rainforests. KEY RESULTS These three small epiphytic orchid species were all biased toward certain woody plant species, in particular, the tree Backhousia myrtifolia, though the extent of specificity varied. Biases toward the most common phorophyte species were not explained by increases in adult orchid fitness, nor did probability of flowering increase on B. myrtifolia. Indeed, individuals on this woody phorophyte tended to have fewer inflorescences than those on other woody phorophytes. Only S. hillii benefited from establishment on B. myrtifolia; it had more leaves on this phorophyte than on others. CONCLUSIONS In many cases what appear to be simple interactions between two species may be mediated by more complex symbioses. For this system, we propose that the cause for bias in orchid distribution occurs much earlier in an orchids life and may be due to a bias of their mycorrhizal fungi for the dominant orchid phorophytes.

Generalised pollination of Diuris alba (Orchidaceae) by small bees and wasps

Most Diuris species possess flowers of pea-like form and colour, and occur in association with flowering peas of the tribe Mirbeliae. Previous studies of the pollination of Diuris maculata sensu lato have found evidence for guild mimicry of pea flowers. The flowers of Diuris alba are also pea-like in form but not in colour, and this species is frequently found in habitats where peas are uncommon or absent. We investigated the pollination of Diuris alba, which we expected may have a distinct pollination system at Lake Munmorah, New South Wales. Many Diuris species lack floral rewards, but D. alba produced a small amount of nectar. Flower visitors, and hence putative pollinators, were mainly female Exoneura bees, but also the wasps Eurys pulcher and a Paralastor species. Reproductive success of D. alba, both in woodland containing abundant Dillwynia retorta and in heathland where this pea was absent, was higher than in the previously studied D. maculata s.l. We suggest that the pollination of D. alba is more generalised than that found in the legume guild mimic D. maculata s.l. Although its flowers may display structural similarity to pea flowers, other characteristics suggest that its pollination system has diverged from a presumed pea-mimicry ancestral condition.

The host bias of three epiphytic Aeridinae orchid species is reflected, but not explained, by mycorrhizal fungal associations

PREMISE OF THE STUDY The three co-occurring epiphytic orchid species, Sarcochilus hillii, Plectorrhiza tridentata, and Sarcochilus parviflorus vary in host specificity; all are found predominantly on the tree Backhousia myrtifolia but some also associate with a broad range of species. Despite this specialization, no fitness advantage has been detected for adult orchid plants growing on the preferred host. Therefore, we predicted that the host specialization of these orchid species is a consequence of a bias toward particular orchid mycorrhizal fungi, which are in turn biased toward particular woody plant species. METHODS To test this hypothesis, we sampled representatives of each orchid species on B. myrtifolia and other host species across sites. Rhizoctonia-like fungi were isolated from orchid roots and identified using molecular markers. KEY RESULTS Three groups of fungi were identified, and the orchid species varied in their specificity for these. All fungal groups were found on the host B. myrtifolia; yet at all sites, only one orchid species, S. hillii, associated with all three groups. CONCLUSIONS Our results demonstrate that these orchid species did vary in their mycorrhizal specificity; however, the distribution of their mycorrhizal associates did not directly explain their host associations. Rather, we propose that the mycorrhizal relationship of these orchid species is complex and have suggested future avenues of research.

Caladenia revisited: Results of molecular phylogenetic analyses of Caladeniinae plastid and nuclear loci

PREMISE OF THE STUDY The classification of the primarily Australasian group of orchids Caladenia and allied genera (Caladeniinae: Diurideae) containing 71 federally listed threatened species has proven controversial. Analyzing these species using genetic material will provide a sound basis for their classification and the capacity to ensure accurate conservation measures can be implemented. METHODS We present a multigene analysis based on nuclear ribosomal ITS and five plastid regions from 54 species representing all major taxonomic groups within Caladeniinae. KEY RESULTS In our combined analysis, apart from Leptoceras and Praecoxanthus, all Caladenia ingroup taxa form a strongly supported clade that is also supported by morphological synapomorphies (parallel leaf venation; leaf solitary, lanceolate, covered with glandular or eglandular trichomes). Characters and character states historically used to delimit taxa were revealed to be homoplasious and therefore do not support recognition of Arachnorchis, Cyanicula, Drakonorchis, Ericksonella, Jonesiopsis, Petalochilus, Pheladenia, and Stegostyla as previously proposed. Glossodia and Elythranthera are shown to be a specialist group embedded within Caladenia. CONCLUSIONS Based on our results, none of the current systems of classification of the subtribe is satisfactory. Instead our results point to Lindleys 1840 interpretation of Caladenia, but including Glossodia and Elythranthera, as being the most accurate reflection of the group. Accordingly, a renewed reclassification of Caladeniinae is proposed as well as several new combinations.

Phylogenetic relationships in Pterostylidinae (Cranichideae: Orchidaceae): combined evidence from nuclear ribomsomal and plastid DNA sequences

A study to evaluate the relationships in subtribe Pterostylidinae (Cranichideae: Orchidaceae) was undertaken using DNA sequences from the nuclear ribosomal ITS region (256 taxa) and plastid matK (subset of 37 taxa). Parsimony analysis of nuclear, plastid and combined datasets revealed that there is strong support for the monophyly of Pterostylidinae, and three major groups therein. Clades A–C contain nine, possibly 10, identifiable groups supported by morphological synapomorphies. Clade A comprises the following two major, strongly supported groups that correlate with morphological synapomorphies: (1) Speculantha (including Petrorchis) and (2) Linguella and Eremorchis, sister to an unresolved polytomy containing Taurantha, a paraphyletic Crangonorchis and polyphyletic Diplodium. There is no support for continued recognition of Taurantha, Crangonorchis, Linguella and Eremorchis, all of which are embedded within the broader, strongly supported, monophyletic Diplodium. Clade B represents true Pterostylis. Clade C contains the morphologically disparate Bunochilus, Hymenochilus, Oligochaetochilus, Pharochilum, Plumatichilos, Stamnorchis and Urochilus (including Ranorchis) in a partially resolved tree. There is strong molecular and morphological synapomorphic internal support for the recognition of these taxa as genera. Our results revealed that none of the presently proposed classification systems for Pterostylidinae truly accounts for the underlying phylogenetic signal. A streamlined classification system, therefore, seems warranted, although further research based on a larger plastid DNA dataset is required to elucidate relationships in Clade C.

Highly sensitive DNA fingerprinting of orchid pollinaria remnants using AFLP

Numerous Australian terrestrial orchid species in the genus Diuris may be pollinated by food source mimicry. In our field studies, direct observations of orchid–pollinator interactions were rare, but native bees were frequently captured carrying orchid pollinaria, or pollinaria remnants. Sometimes, pollinaria remnants were minimal and included only the viscidium, a sticky pad that was often highly persistent. Confirmation of such tissue as being of orchid source, and attributing them to a particular species can aid pollination studies. DNA-based methods that may identify more or less intact orchid pollinaria are available, but extremely small and degraded samples can pose technical challenges. We have developed an AFLP protocol for such difficult samples that offers some significant advantages over direct PCR-based analysis. We simulated AFLP profiling of very low-DNA samples using DNA template from serial dilutions. A DNA sample range from 6.4 picograms to at least as high as 100 nanograms (15 500-fold range) all yielded AFLP fingerprints. The practical application of this inherent sensitivity of AFLP is demonstrated by the identification of remnants of orchid pollinaria sampled from bees, presented here as a case study. It is expected that this approach will find many applications where sample DNA is limiting, or possibly where pollen of similar appearance may comprise species mixtures.

A molecular phylogenetic analysis of Diuris (Orchidaceae) based on AFLP and ITS reveals three major clades and a basal species.

Diuris is a terrestrial orchid genus of at least 61 and possibly more than 100 species, restricted to Australia except for one species endemic in Timor. Distinctive species groups have respective eastern and western centres of distribution. Although species affinities have been vaguely understood for many years, no formal infrageneric treatment has been undertaken as Diuris possesses few reliable morphological characters for a classification system. We have undertaken cladistic parsimony and Bayesian phylogenetic analyses of Diuris by using the ITS1–5.8S–ITS2 region of nuclear rDNA and morphological characters, with a subset of samples also studied by amplified fragment length polymorphism (AFLP) as an independent test of phylogenetic relationships. Four major clades with strong bootstrap support were resolved and are named here according to a recently published classification; D. sulphurea forms a lineage (subg. Paradiuris) of its own that is well supported as the sister to the rest of Diuris. Two other major eastern clades contained species related to D. maculata (subg. Xanthodiuris) and D. punctata (subg. Diuris), respectively. Although these latter two subgenera are genetically well resolved, there is minimal genetic variation at species level, consistent with recent, rapid speciation. A fourth clade (subg. Hesperodiuris) has a centre of distribution in Western Australia, and has more genetic and morphological variation than the eastern subgenera. Total evidence analysis provides support for the western clade being sister group to the two eastern subgenera Diuris and Xanthodiuris; however, this relationship was not resolved by molecular data. Hybridisation is known to be common among species within subgenera Diuris and Xanthodiuris. Instances of incongruence between different datasets were found suggestive of hybridisation events between species of different sections of Diuris.

Identification of the endangered Australian orchid Microtis angusii using an allele-specific PCR assay

The endangered orchid, Microtis angusii, was recently described from a single population consisting of approximately 100 plants. This species is morphologically very similar to close relatives, and taxonomic difficulties have hindered attempts to identify further populations for protection. Here we present a rapid, economical, PCR-based assay for the effective identification of this species based on rDNA sequence variation. Using two single nucleotide substitutions in the internal transcribed spacer (ITS) region of the ribosomal DNA that are diagnostic for M. angusii, we developed an allele-specific PCR that can be easily visualized on a standard agarose gel, avoiding the use of expensive restriction enzymes and DNA sequencing reagents and equipment. Using PCR primer pairs for both the M. angusii, and the alternate allele, we also detected an individual heterozygous for the two alleles, indicating a need for further detailed genetic study. We performed a ‘blind trial’ to confirm the utility of this assay. Microtis angusii samples were successfully discriminated from amongst several congeners, and a further, previously unknown, population of the species was identified.