Terrestrial orchid conservation in the age of extinction

Background

Conservation through reserves alone is now considered unlikely to achieve protection of plant species necessary to mitigate direct losses of habitat and the pervasive impact of global climate change. Assisted translocation/migration represent new challenges in the face of climate change; species, particularly orchids, will need artificial assistance to migrate from hostile environments, across ecological barriers (alienated lands such as farmlands and built infrastructure) to new climatically buffered sites. The technology and science to underpin assisted migration concepts are in their infancy for plants in general, and orchids, with their high degree of rarity, represent a particularly challenging group for which these principles need to be developed. It is likely that orchids, more than any other plant family, will be in the front-line of species to suffer large-scale extinction events as a result of climate change.

Scope

The South West Australian Floristic Region (SWAFR) is the only global biodiversity hotspot in Australia and represents an ideal test-bed for development of orchid conservation principles. Orchids comprise 6 % of all threatened vascular plants in the SWAFR, with 76 out of the 407 species known for the region having a high level of conservation risk. The situation in the SWAFR is a portent of the global crisis in terrestrial orchid conservation, and it is a region where innovative conservation solutions will be required if the impending wave of extinction is to be averted. Major threatening processes are varied, and include land clearance, salinity, burning, weed encroachment, disease and pests. This is compounded by highly specialized pollinators (locally endemic native invertebrates) and, in the most threatened groups such as hammer orchids (Drakaea) and spider orchids (Caladenia), high levels of mycorrhizal specialization. Management and development of effective conservation strategies for SWAFR orchids require a wide range of integrated scientific approaches to mitigate impacts that directly influence ecological traits critical for survival.

Conclusions

In response to threats to orchid species, integrated conservation approaches have been adopted (including ex situ and translocation principles) in the SWAFR with the result that a significant, multidisciplinary approach is under development to facilitate conservation of some of the most threatened taxa and build expertise to carry out assisted migration to new sites. Here the past two decades of orchid conservation research in the SWAFR and the role of research-based approaches for managing effective orchid conservation in a global biodiversity hotspot are reviewed.Key words: Orchids, pollination, mycorrhiza, integrated conservation, terrestrial, threats, ex situ conservation, in situ conservation     

Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media

Background and Aims

Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed.

Methods

Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken.

Key Results

It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media–mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility.

Conclusions

The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.     

A New Orchid Genus,Danxiaorchis, and Phylogenetic Analysis of the Tribe Calypsoeae

Background

Orchids have numerous species, and their speciation rates are presumed to be exceptionally high, suggesting that orchids are continuously and actively evolving. The wide diversity of orchids has attracted the interest of evolutionary biologists. In this study, a new orchid was discovered on Danxia Mountain in Guangdong, China. However, the phylogenetic clarification of this new orchid requires further molecular, morphological, and phytogeographic analyses.

Methodology/Principal Findings

A new orchid possesses a labellum with a large Y-shaped callus and two sacs at the base, and cylindrical, fleshy seeds, which make it distinct from all known orchid genera. Phylogenetic methods were applied to a matrix of morphological and molecular characters based on the fragments of the nuclear internal transcribed spacer, chloroplast matK, and rbcL genes of Orchidaceae (74 genera) and Calypsoeae (13 genera). The strict consensus Bayesian inference phylogram strongly supports the division of the Calypsoeae alliance (not including Dactylostalix and Ephippianthus) into seven clades with 11 genera. The sequence data of each species and the morphological characters of each genus were combined into a single dataset. The inferred Bayesian phylogram supports the division of the 13 genera of Calypsoeae into four clades with 13 subclades (genera). Based on the results of our phylogenetic analyses, Calypsoeae, under which the new orchid is classified, represents an independent lineage in the Epidendroideae subfamily.

Conclusions

Analyses of the combined datasets using Bayesian methods revealed strong evidence that Calypsoeae is a monophyletic tribe consisting of eight well-supported clades with 13 subclades (genera), which are all in agreement with the phytogeography of Calypsoeae. The Danxia orchid represents an independent lineage under the tribe Calypsoeae of the subfamily Epidendroideae. This lineage should be treated as a new genus, which we have named Danxiaorchis, that is parallel to Yoania. Both genera are placed under the subtribe Yoaniinae.     

Reproductive isolation and pollination success of rewarding Galearis diantha and non-rewarding Ponerorchis chusua (Orchidaceae)

Background and Aims

Increasing evidence challenges the conventional perception that orchids are the most distinct example of floral diversification due to floral or prezygotic isolation. Regarding the relationship between co-flowering plants, rewarding and non-rewarding orchids in particular, few studies have investigated whether non-rewarding plants affect the pollination success of rewarding plants. Here, floral isolation and mutual effects between the rewarding orchid Galearis diantha and the non-rewarding orchid Ponerorchis chusua were investigated.

Methods

Flowering phenological traits were monitored by noting the opening and wilting dates of the chosen individual plants. The pollinator pool and pollinator behaviour were assessed from field observations. Key morphological traits of the flowers and pollinators were measured directly in the field. Pollinator limitation and interspecific compatibility were evaluated by hand-pollination experiments. Fruit set was surveyed in monospecific and heterospecific plots.

Key Results

The species had overlapping peak flowering periods. Pollinators of both species displayed a certain degree of constancy in visiting each species, but they also visited other flowers before landing on the focal orchids. A substantial difference in spur size between the species resulted in the deposition of pollen on different regions of the body of the shared pollinator. Hand-pollination experiments revealed that fruit set was strongly pollinator-limited in both species. No significant difference in fruit set was found between monospecific plots and heterospecific plots.

Conclusions

A combination of mechanical isolation and incomplete ethological isolation eliminates the possibility of pollen transfer between the species. These results do not support either the facilitation or competition hypothesis regarding the effect of nearby rewarding flowers on non-rewarding plants. The absence of a significant effect of non-rewarding P. chusua on rewarding G. diantha can be ascribed to low levels of overlap between the pollinator pools of two species.     

Why are orchid flowers so diverse? Reduction of evolutionary constraints by paralogues of class B floral homeotic genes

Background

The nearly 30 000 species of orchids produce flowers of unprecedented diversity. However, whether specific genetic mechanisms contributed to this diversity is a neglected topic and remains speculative. We recently published a theory, the ‘orchid code’, maintaining that the identity of the different perianth organs is specified by the combinatorial interaction of four DEF-like MADS-box genes with other floral homeotic genes.

Scope

Here the developmental and evolutionary implications of our theory are explored. Specifically, it is shown that all frequent floral terata, including all peloric types, can be explained by monogenic gain- or-loss-of-function mutants, changing either expression of a DEF-like or CYC-like gene. Supposed dominance or recessiveness of mutant alleles is correlated with the frequency of terata in both cultivation and nature. Our findings suggest that changes in DEF- and CYC-like genes not only underlie terata but also the natural diversity of orchid species. We argue, however, that true changes in organ identity are rare events in the evolution of orchid flowers, even though we review some likely cases.

Conclusions

The four DEF paralogues shaped floral diversity in orchids in a dramatic way by modularizing the floral perianth based on a complex series of sub- and neo-functionalization events. These genes may have eliminated constraints, so that different kinds of perianth organs could then evolve individually and thus often in dramatically different ways in response to selection by pollinators or by genetic drift. We therefore argue that floral diversity in orchids may be the result of an unprecedented developmental genetic predisposition that originated early in orchid evolution.Key words: Orchidaceae, orchid evolution, evo-devo; perianth, class B genes, DEFICIENS, subfunctionalization, neofunctionalization, gene duplication, peloria, modularization     

Oeceoclades maculata, an alien tropical orchid in a Caribbean rainforest

Background and Aims

Undisturbed forest habitat can be relatively impenetrable to invasive, non-native species. Orchids are not commonly regarded as invasive, but some species have become invasive and these generally depend on habitat disturbance. One of the most aggressive orchids is Oeceoclades maculata, a terrestrial species with remarkable ecological amplitude. Originally from tropical Africa, it is now widespread in the neotropics. By associating its local distribution with land-use history and habitat characteristics, it was determined whether O. maculata is dependent on habitat disturbance. It was also investigated whether this exotic orchid occupies the same habitat space as two sympatric native species.

Methods

Six 10 m × 500 m transects were censused in June 2007 on the 16-ha Luquillo Forest Dynamics Plot, located in the Luquillo Mountains, Puerto Rico. The plot had been mapped for historical land use, topography and soil type.

Key Results

Oeceoclades maculata was the most abundant of three orchid species surveyed and was found in all four historical cover classes. In cover class 3 (50–80 % forest cover in 1936), 192 of 343 plants were found at a density of 0·48 plants per 5 × 5 m subplot. Over 93 % of the 1200 subplots surveyed were composed of Zarzal or Cristal soil types, and O. maculata was nearly evenly distributed in both. The orchid was most common on relatively flat terrain. The distribution and abundance of two sympatric orchid species were negatively associated with that of the invasive species.

Conclusions

Oeceoclades maculata does penetrate ‘old growth’ forest but is most abundant in areas with moderate levels of past disturbance. Soil type makes little difference, but slope of terrain can be important. The negative association between O. maculata and native species may reflect differences in habitat requirements or a negative interaction perhaps at the mycorrhizal level.Key words: Oeceoclades maculata, Wullschlaegelia calcarata, Prescottia stachyodes, Orchidaceae, land-use history, tropical forest disturbance, terrestrial orchids, invasive species, Luquillo Experimental Forest, Puerto Rico, forest recovery, Caribbean     

Variation in nutrient-acquisition patterns by mycorrhizal fungi of rare and common orchids explains diversification in a global biodiversity hotspot

Background and Aims

Many terrestrial orchids have an obligate requirement for mycorrhizal associations to provide nutritional support from germination to establishment. This study will investigate the ability of orchid mycorrhizal fungi (OMF) to utilize a variety of nutrient sources in the nutrient-impoverished (low organic) soils of the Southwest Australian Floristic Region (SWAFR) in order to effectively compete, survive and sustain the orchid host.

Methods

Mycorrhizal fungi representing key OMF genera were isolated from three common and widespread species: Pterostylis recurva, Caladenia flava and Diuris corymbosa, and one rare and restricted species: Drakaea elastica. The accessibility of specific nutrients was assessed by comparing growth including dry biomass of OMF in vitro on basal CN MMN liquid media.

Key Results

Each of the OMF accessed and effectively utilized a wide variety of nutrient compounds, including carbon (C) sources, inorganic and organic nitrogen (N) and inorganic and organic phosphorus (P). The nutrient compounds utilized varied between the genera of OMF, most notably sources of N.

Conclusions

These results suggest that OMF can differentiate between niches (micro-niche specialization) in a constrained, highly resource-limited environment such as the SWAFR. Phosphorus is the most limited macronutrient in SWAFR soils and the ability to access phytate by OMF indicates a characterizing functional capacity of OMF from the SWAFR. Furthermore, compared with OMF isolated from the rare D. elastica, OMF associating with the common P. recurva produced far greater biomass over a wider variety of nutritional sources. This suggests a broader tolerance for habitat variation providing more opportunities for the common orchid for recruitment and establishment at a site.     

Armament imbalances: match and mismatch in plant-pollinator traits of highly specialized long-spurred orchids

Background

Some species of long-spurred orchids achieve pollination by a close association with long-tongued hawkmoths. Among them, several Habenaria species present specialized mechanisms, where pollination success depends on the attachment of pollinaria onto the heads of hawkmoths with very long proboscises. However, in the Neotropical region such moths are less abundant than their shorter-tongued relatives and are also prone to population fluctuations. Both factors may give rise to differences in pollinator-mediated selection on floral traits through time and space.

Methodology/Principal Findings

We characterized hawkmoth assemblages and estimated phenotypic selection gradients on orchid spur lengths in populations of three South American Habenaria species. We examined the match between hawkmoth proboscis and flower spur lengths to determine whether pollinators may act as selective agents on flower morphology. We found significant directional selection on spur length only in Habenaria gourlieana, where most pollinators had proboscises longer than the mean of orchid spur length.

Conclusions/Significance

Phenotypic selection is dependent on the mutual match between pollinator and flower morphologies. However, our findings indicate that pollinator-mediated selection may vary through time and space according to local variations in pollinator assemblages.     

On the roles of colour and scent in a specialized floral mimicry system

Background and Aims

Sexually deceptive orchids achieve cross-pollination by mimicking the mating signals of female insects, generally hymenopterans. This pollination mechanism is often highly specific as it is based primarily on the mimicry of mating signals, especially the female sex pheromones of the targeted pollinator. Like many deceptive orchids, the Mediterranean species Ophrys arachnitiformis shows high levels of floral trait variation, especially in the colour of the perianth, which is either green or white/pinkinsh within populations. The adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in sexually deceptive orchids remain obscure.

Methods

The relative importance of floral scent versus perianth colour in pollinator attraction in this orchid pollinator mimicry system was evaluated by performing floral scent analyses by gas chromatography-mass spectrometry (GC-MS) and behavioural bioassays with the pollinators under natural conditions were performed.

Key Results

The relative and absolute amounts of behaviourally active compounds are identical in the two colour morphs of O. arachnitiformis. Neither presence/absence nor the colour of the perianth (green versus white) influence attractiveness of the flowers to Colletes cunicularius males, the main pollinator of O. arachnitiformis.

Conclusion

Chemical signals alone can mediate the interactions in highly specialized mimicry systems. Floral colour polymorphism in O. arachnitiformis is not subjected to selection imposed by C. cunicularius males, and an interplay between different non-adaptive processes may be responsible for the maintenance of floral colour polymorphism both within and among populations.     

Variation in Mycorrhizal Associations with Tulasnelloid Fungi among Populations of Five Dactylorhiza Species

Background

Orchid species rely on mycorrhizal symbioses with fungi to complete their life cycle. Although there is mounting evidence that orchids can associate with several fungi from different clades or families, less is known about the actual geographic distribution of these fungi and how they are distributed across different orchid species within a genus.

Methodology/Principal Findings

We investigated among-population variation in mycorrhizal associations in five species of the genus Dactylorhiza (D. fuchsii, D. incarnata, D. maculata, D. majalis and D. praetermissa) using culture-independent detection and identification techniques enabling simultaneous detection of multiple fungi in a single individual. Mycorrhizal specificity, determined as the number of fungal operational taxonomic units (OTUs), and phylogenetic diversity of fungi were compared between species, whereas discriminant analysis was used to compare mycorrhizal spectra across populations and species. Based on a 95% cut-off value in internal transcribed spacer (ITS) sequence similarity, a total of ten OTUs was identified belonging to three different clades within the Tulasnellaceae. Most OTUs were found in two or more Dactylorhiza species, and some of them were common and widespread, occurring in more than 50% of all sampled populations. Each orchid species associated with at least five different OTUs, whereas most individuals also associated with two or more fungal OTUs at the same time. Phylogenetic diversity, corrected for species richness, was not significantly different between species, confirming the generality of the observed orchid mycorrhizal associations.

Conclusions/Significance

We found that the investigated species of the genus Dactylorhiza associated with a wide range of fungal OTUs from the Tulasnellaceae, some of which were widespread and common. These findings challenge the idea that orchid rarity is related to mycorrhizal specificity and fungal distribution.     

Spatial patterns of photosynthesis in thin- and thick-leaved epiphytic orchids: unravelling C3–CAM plasticity in an organ-compartmented way

Background and Aims

A positive correlation between tissue thickness and crassulacean acid metabolism (CAM) expression has been frequently suggested. Therefore, this study addressed the question of whether water availability modulates photosynthetic plasticity in different organs of two epiphytic orchids with distinct leaf thickness.

Methods

Tissue morphology and photosynthetic mode (C₃ and/or CAM) were examined in leaves, pseudobulbs and roots of a thick-leaved (Cattleya walkeriana) and a thin-leaved (Oncidium ‘Aloha’) epiphytic orchid. Morphological features were studied comparing the drought-induced physiological responses observed in each organ after 30 d of either drought or well-watered treatments.

Key Results

Cattleya walkeriana, which is considered a constitutive CAM orchid, displayed a clear drought-induced up-regulation of CAM in its thick leaves but not in its non-leaf organs (pseudobulbs and roots). The set of morphological traits of Cattleya leaves suggested the drought-inducible CAM up-regulation as a possible mechanism of increasing water-use efficiency and carbon economy. Conversely, although belonging to an orchid genus classically considered as performing C₃ photosynthesis, Oncidium ‘Aloha’ under drought seemed to express facultative CAM in its roots and pseudobulbs but not in its leaves, indicating that such photosynthetic responses might compensate for the lack of capacity to perform CAM in its thin leaves. Morphological features of Oncidium leaves also indicated lower efficiency in preventing water and CO₂ losses, while aerenchyma ducts connecting pseudobulbs and leaves suggested a compartmentalized mechanism of nighttime carboxylation via phosphoenolpyruvate carboxylase (PEPC) (pseudobulbs) and daytime carboxylation via Rubisco (leaves) in drought-exposed Oncidium plants.

Conclusions

Water availability modulated CAM expression in an organ-compartmented manner in both orchids studied. As distinct regions of the same orchid could perform different photosynthetic pathways and variable degrees of CAM expression depending on the water availability, more attention should be addressed to this in future studies concerning the abundance of CAM plants.     

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

Background and Aims

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids.

Methods

Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network.

Key Results

Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine.

Conclusions

Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.     

Differences in pollen viability in relation to different deceptive pollination strategies in Mediterranean orchids

Background and Aims

To date, current research involving pollen viability has been evaluated in a relatively low number of orchid species. In the present study, we focused on five related Mediterranean orchid genera (Anacamptis, Orchis, Dactylorhiza, Ophrys and Serapias) that are characterized by different types of deceptive pollination.

Methods

The in vitro germination ability of increasingly aged pollinaria of eight food-, seven sexually and two shelter-deceptive species was evaluated. Pollination experiments on two food-, one sexually and one shelter-deceptive species were also performed and the percentage of embryonate seeds derived from the increasingly aged pollinaria was checked.

Key Results

All of the examined species showed long-term viabilities (=50 % pollen tube growth) that ranged from 8 to 35 d. Species with the same deceptive pollination strategies exhibited the same pollen viability trends. Interestingly, pollen viabilities of species groups with different deception types have shown significant differences, with sexually and shelter- deceptive species exhibiting a shorter life span than food-deceptive species.

Conclusions

This study confirms the prolonged germination and fertilization capacities of orchid pollinaria, and to our knowledge is the first report demonstrating a clear relationship between pollen viability and pollination system. It is proposed that this relationship is attributed to the different types of reproductive barriers, pre- or post-zygotic, that characterixe Ophrys and Serapias and the food-deceptive species, respectively.     

Desiccation tolerance,longevity and seed-siring ability of entomophilous pollen from UK native orchid species

Background and Aims

Pollinator-limited seed-set in some terrestrial orchids is compensated for by the presence of long-lived flowers. This study tests the hypothesis that pollen from these insect-pollinated orchids should be desiccation tolerant and relatively long lived using four closely related UK terrestrial species; Anacamptis morio, Dactylorhiza fuchsii, D. maculata and Orchis mascula.

Methods

Pollen from the four species was harvested from inflorescences and germinated in vitro, both immediately and also after drying to simulate interflower transit. Their tolerance to desiccation and short-term survival was additionally assessed after 3 d equilibration at a range of relative humidities (RHs), and related to constructed sorption isotherms (RH vs. moisture content, MC). Ageing of D. fuchsii pollen was further tested over 2 months against temperature and RH, and the resultant survival curves were subjected to probit analysis, and the distribution of pollen death in time (σ) was determined. The viability and siring ability, following artificial pollinations, were determined in D. fuchsii pollen following storage for 6 years at –20 °C.

Key Results

The pollen from all four species exhibited systematic increases in germinability and desiccation tolerance as anthesis approached, and pollen from open flowers generally retained high germinability. Short-term storage revealed sensitivity to low RH, whilst optimum survival occurred at comparable RHs in all species. Similarly, estimated pollen life spans (σ) at differing temperatures were longest under the dry conditions. Despite a reduction in germination and seeds per capsule, long-term storage of D. fuchsii pollen did not impact on subsequent seed germination in vitro.

Conclusions

Substantial pollen desiccation tolerance and life span of the four entomophilous orchids reflects a resilient survival strategy in response to unpredictable pollinator visitation, and presents an alternative approach to germplasm conservation.     

Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella

Background

Mycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species’ association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment.

Results

Using next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance.

Conclusions

G. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1422-7) contains supplementary material, which is available to authorized users.     

On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae)

Background and Aims

Most molecular phylogenetic studies of Orchidaceae have relied heavily on DNA sequences from the plastid genome. Nuclear and mitochondrial loci have only been superficially examined for their systematic value. Since 40% of the genera within Vanilloideae are achlorophyllous mycoheterotrophs, this is an ideal group of orchids in which to evaluate non-plastid gene sequences.

Methods

Phylogenetic reconstructions for Vanilloideae were produced using independent and combined data from the nuclear 18S, 5·8S and 26S rDNA genes and the mitochondrial atpA gene and nad1b-c intron.

Key Results

These new data indicate placements for genera such as Lecanorchis and Galeola, for which plastid gene sequences have been mostly unavailable. Nuclear and mitochondrial parsimony jackknife trees are congruent with each other and previously published trees based solely on plastid data. Because of high rates of sequence divergence among vanilloid orchids, even the short 5·8S rDNA gene provides impressive levels of resolution and support.

Conclusions

Orchid systematists are encouraged to sequence nuclear and mitochondrial gene regions along with the growing number of plastid loci available.Key words: 26S rDNA, 18S rDNA, 5·8S rDNA, atpA, nad1, orchids, plastid, Vanilla, vanilloid orchids, Vanilloideae     

Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.     

Morphological Variation of the Scorpionfly Panorpa obtusa Cheng (Mecoptera: Panorpidae) with a New Synonym

Background

The overabundance of synonyms is an unavoidable by-product of taxonomic practice in insects. How to reduce or even eliminate synonymy has long been a great challenge for insect taxonomists. The scorpionflies Panorpa obtusa Cheng, 1949 and Panorpa leei Cheng, 1949 (Insecta: Mecoptera: Panorpidae) were originally described from Taibaishan in the Qinling Mountains with identical collection data and both are based on a single gender, the former on a male and the latter on two females. However, whether P. leei is conspecific with P. obtusa or a good species remains an unsolved problem.

Results

On the basis of intensive morphological comparison of 93 males and 53 females of scorpionflies collected from the type locality using light and scanning electron microscopy, we found P. obtusa has considerable morphological variation (especially the wing markings and genitalia in both male and female), and Panorpa leei is totally comprised of one of the morphs of P. obtusa.

Conclusions

In combination with identical type localities and overlapping morphological variation, P. leei Cheng is proposed as a junior subjective synonym of P. obtusa Cheng. To avoid synonyms, taxonomists should pay more attention to individual variation and base decisions on a series of specimens to describe new species.     

Collection and trade of wild-harvested orchids in Nepal

Background

Wild orchids are illegally harvested and traded in Nepal for use in local traditional medicine, horticulture, and international trade. This study aims to: 1) identify the diversity of species of wild orchids in trade in Nepal; 2) study the chain of commercialization from collector to client and/or export; 3) map traditional knowledge and medicinal use of orchids; and 4) integrate the collected data to propose a more sustainable approach to orchid conservation in Nepal.

Methods

Trade, species diversity, and traditional use of wild-harvested orchids were documented during field surveys of markets and through interviews. Trade volumes and approximate income were estimated based on surveys and current market prices. Orchid material samples were identified to species level using a combination of morphology and DNA barcoding.

Results

Orchid trade is a long tradition, and illegal export to China, India and Hong Kong is rife. Estimates show that 9.4 tons of wild orchids were illegally traded from the study sites during 2008/2009. A total of 60 species of wild orchids were reported to be used in traditional medicinal practices to cure at least 38 different ailments, including energizers, aphrodisiacs and treatments of burnt skin, fractured or dislocated bones, headaches, fever and wounds. DNA barcoding successfully identified orchid material to species level that remained sterile after culturing.

Conclusions

Collection of wild orchids was found to be widespread in Nepal, but illegal trade is threatening many species in the wild. Establishment of small-scale sustainable orchid breeding enterprises could be a valuable alternative for the production of medicinal orchids for local communities. Critically endangered species should be placed on CITES Appendix I to provide extra protection to those species. DNA barcoding is an effective method for species identification and monitoring of illegal cross-border trade.