Apostasiads, systematic anatomy, and the origins of Orchidaceae

Anatomical study of several species of the putatively primitive orchids Apostasia and Neuiviedia was based on specimens of leaves, stems and rootS. Research was carried out in the hope of providing objective information from anatomy towards unravelling the relationships of these plants, and especially towards answering the question of whether extant orchids evolved from these two genera, or from plants like them. The morphology of Apostasia and Neuwiedia has evoked the dogma of primitiveness because of the two or three anthers borne on separate filaments and the free style and stigma which characterize the flowerS. In most other orchids there is only one anther and filaments and styles are fused to form the column (gynostemium). The general anatomical structure of apostasiads shows features present in other orchids; none is restricted to Apostasia and Neuwiedia. Tracheary anatomy, however, shows vessels in roots, but not in any part of the shoot system. Vessel members are characterized by chiefly simple perforation plates in contrast with other orchids where scalariform perforation plates predominate in vessel members of the root. These phenomena, considered from the phylogenetic standpoint, would cast serious doubt on the possibility that plants with scalariform perforation plates, the ancestral, or primitive condition, could have arisen from plants with simple perforation plates, the derived, or advanced condition. On this basis the apostasiads could not have given rise to the di- and monandrous orchidS. Of the several suggested origins for orchids, Hypoxidaceae, or plants similar to them, whether in Asparagales, Liliales or Haemadorales of different authors, could have been the progenitors of orchids as a group, including the apostasiadS. Because of the unique combination of floral features in the apostasiads, their predominantly simple perforation plates, and their overall anatomical similarity to orchids in general, it would appear appropriate to consider them as a subfamily, Apostasioideae, of Orchidaceae sensu lato.     

Mycorrhizal diversity in Apostasia (Orchidaceae) indicates the origin and evolution of orchid mycorrhiza

We demonstrated that "orchid mycorrhiza," a specialized mycorrhizal type, appeared in the common ancestor of the largest plant family Orchidaceae and that the fungal partner shifted from Glomeromycota to a particular clade of Basidiomycota in association with this character evolution. Several unique mycorrhizal characteristics may have contributed to the diversification of the family. However, the origin of orchid mycorrhiza and the diversity of mycobionts across orchid lineages still remain obscure. In this study, we investigated the mycorrhizae of five Apostasia taxa, members of the earliest-diverging clade of Orchidaceae. The results of molecular identification using nrDNA ITS and LSU regions showed that Apostasia mycorrhizal fungi belong to families Botryobasidiaceae and Ceratobasidiaceae, which fall within the order Cantharellales of Basidiomycota. Most major clades in Orchidaceae also form mycorrhizae with members of Cantharellales, while the sister group and other closely related groups to Orchidaceae (i.e., Asparagales except for orchids and the "commelinid" families) ubiquitously form symbioses with Glomeromycota to form arbuscular mycorrhizae. This pattern of symbiosis indicates that a major shift in fungal partner occurred in the common ancestor of the Orchidaceae.     

Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.     

Seedlings of Neuwiedia (Orchidaceae subfamily Apostasioideae) have typical orchidaceous mycotrophic protocorms

Naturally occurring seedlings of Neuwiedia veratrifolia were found in three localities in Sabah, Borneo, Malaysia. Seedlings consisted of an irregular oblong protocorm and a terminal leafy rooted shoot. Protocorms contained mycotrophic tissue of the kind typical of orchid mycorrhiza (tolypophagy). This finding demonstrates an important synapomorphy between Neuwiedia and other orchids and strongly supports the monophyly of Orchidaceae in the broad sense, including apostasiod orchids.     

Evolution and biogeography of the slipper orchids: Eocene vicariance of the conduplicate genera in the Old and New World Tropics

Intercontinental disjunctions between tropical regions, which harbor two-thirds of the flowering plants, have drawn great interest from biologists and biogeographers. Most previous studies on these distribution patterns focused on woody plants, and paid little attention to herbs. The Orchidaceae is one of the largest families of angiosperms, with a herbaceous habit and a high species diversity in the Tropics. Here we investigate the evolutionary and biogeographical history of the slipper orchids, which represents a monophyletic subfamily (Cypripedioideae) of the orchid family and comprises five genera that are disjunctly distributed in tropical to temperate regions. A relatively well-resolved and highly supported phylogeny of slipper orchids was reconstructed based on sequence analyses of six maternally inherited chloroplast and two low-copy nuclear genes (LFY and ACO). We found that the genus Cypripedium with a wide distribution in the northern temperate and subtropical zones diverged first, followed by Selenipedium endemic to South America, and finally conduplicate-leaved genera in the Tropics. Mexipedium and Phragmipedium from the neotropics are most closely related, and form a clade sister to Paphiopedilum from tropical Asia. According to molecular clock estimates, the genus Selenipedium originated in Palaeocene, while the most recent common ancestor of conduplicate-leaved slipper orchids could be dated back to the Eocene. Ancestral area reconstruction indicates that vicariance is responsible for the disjunct distribution of conduplicate slipper orchids in palaeotropical and neotropical regions. Our study sheds some light on mechanisms underlying generic and species diversification in the orchid family and tropical disjunctions of herbaceous plant groups. In addition, we suggest that the biogeographical study should sample both regional endemics and their widespread relatives.     

Crassulacean Acid Metabolism and Epiphytism Linked to Adaptive Radiations in the Orchidaceae

Species of the large family Orchidaceae display a spectacular array of adaptations and rapid speciations that are linked to several innovative features, including specialized pollination syndromes, colonization of epiphytic habitats, and the presence of Crassulacean acid metabolism (CAM), a water-conserving photosynthetic pathway. To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, to climatic variables and the evolution of epiphytism in tropical regions. Altitude was the most important predictor of photosynthetic pathway when all environmental variables were taken into account, with CAM being most prevalent at low altitudes. By creating integrated orchid trees to reconstruct ancestral character states, we found that C₃ photosynthesis is the ancestral state and that CAM has evolved at least 10 independent times with several reversals. A large CAM radiation event within the Epidendroideae, the most species-rich epiphytic clade of any known plant group, is linked to a Tertiary species radiation that originated 65 million years ago. Our study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low-elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae.     

Seven New Complete Plastome Sequences Reveal Rampant Independent Loss of the ndh Gene Family across Orchids and Associated Instability of the Inverted Repeat/Small Single-Copy Region Boundaries

Earlier research has revealed that the ndh loci have been pseudogenized, truncated, or deleted from most orchid plastomes sequenced to date, including in all available plastomes of the two most species-rich subfamilies, Orchidoideae and Epidendroideae. This study sought to resolve deeper-level phylogenetic relationships among major orchid groups and to refine the history of gene loss in the ndh loci across orchids. The complete plastomes of seven orchids, Oncidium sphacelatum (Epidendroideae), Masdevallia coccinea (Epidendroideae), Sobralia callosa (Epidendroideae), Sobralia aff. bouchei (Epidendroideae), Elleanthus sodiroi (Epidendroideae), Paphiopedilum armeniacum (Cypripedioideae), and Phragmipedium longifolium (Cypripedioideae) were sequenced and analyzed in conjunction with all other available orchid and monocot plastomes. Most ndh loci were found to be pseudogenized or lost in Oncidium, Paphiopedilum and Phragmipedium, but surprisingly, all ndh loci were found to retain full, intact reading frames in Sobralia, Elleanthus and Masdevallia. Character mapping suggests that the ndh genes were present in the common ancestor of orchids but have experienced independent, significant losses at least eight times across four subfamilies. In addition, ndhF gene loss was correlated with shifts in the position of the junction of the inverted repeat (IR) and small single-copy (SSC) regions. The Orchidaceae have unprecedented levels of homoplasy in ndh gene presence/absence, which may be correlated in part with the unusual life history of orchids. These results also suggest that ndhF plays a role in IR/SSC junction stability.     

Genome size variation in Orchidaceae subfamily Apostasioideae: filling the phylogenetic gap

With more than 160‐fold variation, Orchidaceae are currently the most diverse angiosperm family with respect to the amount of nuclear DNA. This study provides first genome size estimates for approximately 50% of species currently recognized in subfamily Apostasioideae, which is sister to the other four orchid subfamilies. The estimated 1C‐values range from 0.38 pg in Apostasia nuda to 5.96 pg in Neuwiedia zollingeri var. javanica, a nearly 16‐fold range. The two genera show non‐overlapping genome sizes, with those in Apostasia being distinctly smaller than those in Neuwiedia. In fact, most Apostasia spp. are at the lower end of the range of orchid C‐values. Observed discontinuities in DNA amounts in genera most probably reflect interspecific variation in ploidy. In addition to ploidy heterogeneity in N. zollingeri var. javanica, intraspecific variation in genome size (up to 17.7%) was also detected in some species; this can be plausibly related to the incidence of different geographical variants or unrecognized taxonomic heterogeneity. The AT content varied from 62.6 to 66.0%, which is in the upper range recorded for angiosperms. The genome size data obtained in this study fill a major phylogenetic gap in Orchidaceae and show that (very) small genomes prevail in subfamily Apostasioideae. © 2013 The Linnean Society of London     

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

A THEORY ON THE EVOLUTION OF THE EXINE IN ORCHIDACEAE

The development of secondarily derived features in orchid pollen precludes the possibility of applying evolutionary trends hypothesized for dicot pollen to orchid pollen and perhaps even monocot pollen. There are three lines of pollen wall development, all secondarily derived from a possible tectate-perforate ancestral type, i.e., tectate-imperforate with incipient columellae (Cypripedioideae), intectate lacking a foot layer (Orchidoideae), and tectate-imperforate with globular masses of sporopollenin (Vandoid Epidendroideae).     

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.     

Origins and nature of vessels in monocotyledons: 8. Orchidaceae

Xylem of the orchids studied provided unusually favorable material to demonstrate how conductive tissue evolves in monocotyledons. In the end walls of tracheary elements of many Orchidaceae, remnants of pit membranes were observed with scanning electron microscopy and minimally destructive methods. The full range from tracheids to vessel elements, featuring many intermediate stages, was illustrated with SEM in hand sections of fixed roots, stems, and inflorescence axes of 13 species from four subfamilies. Pit membranes in end walls of tracheary elements are porose to reticulate in roots of all species, but nonporose in stems of Cypripedioideae and Vanilloideae and porose to reticulate in stems of Orchidoideae and Epidendroideae. The distribution pattern of pit membranes and pit membrane remnants in end walls of tracheary elements of orchids parallels the findings of others. The position of Cypripedioideae and Vanilloideae as outgroups to Orchidoideae and Epidendroideae, claimed by earlier authors, is supported by clades based on molecular studies and by our studies. Little hydrolysis of pit membranes in tracheary element end walls was observed in pseudobulbs or inflorescence axes of epidendroids. The pervasiveness of network-like pit membranes of various extents and patterns in end walls of tracheary elements in Orchidaceae calls into question the traditional definitions of tracheids and vessel elements, not merely in orchids, but in angiosperms at large. These two concepts, based on light microscope studies, are blurred in light of ultrastructural studies. More importantly, the intermediate expressions of pit membranes in tracheary element end walls of Orchidaceae and some other families of angiosperms are important as indicators of steps in evolution of conduction with respect to organs (more rapid flow in roots than in succulent storage structures) and habitat (less obstruction to flow correlated with a shift from terrestrial to epiphytic).     

Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae

DNA sequences of the plastid gene psaB were completed for 182 species of Orchidaceae (representing 150 different genera) and outgroup families in Asparagales. These data were analyzed using parsimony, and resulting trees were compared to a rbcL phylogeny of Orchidaceae for the same set of taxa after an additional 30 new rbcL sequences were added to a previously published matrix. The psaB tree topology is similar to the rbcL tree, although the psaB data contain less homoplasy and provide greater bootstrap support than rbcL alone. In combination, the two-gene tree recovers the five monophyletic subfamilial clades currently recognized in Orchidaceae, but fails to resolve the positions of Cypripedioideae and Vanilloideae. These new topologies help to clarify some of the anomalous results recovered when rbcL is analyzed alone. Both genes appear to be absent from the plastid genome of several achlorophyllous orchids, but are present in the form of presumably non-functional pseudogenes in Cyrtosia. This study is the first to document the utility of psaB sequences for phylogenetic studies of plants below the family level.     

Systematic implications of wood and bark anatomy in the Pacific Island genus Meryta (Araliaceae)

Wood anatomy was examined in 16 species of Meryta (a genus of c . 35 species) and bark anatomy was studied in 12 species. All but two of these taxa form an assemblage corresponding to the Northern Arc clade, one of two major groups identified by a recent molecular phylogenetic study. M. sinclairii and M. tenuifolia (corresponding to the New Zealand/Fiji clade) differ distinctly by having more numerous simple perforation plates, multiseriate rays with few marginal rows, and the absence of sclerified cells in collapsed secondary phloem, a bark feature that has not yet been found elsewhere in Araliaceae. The increase in abundance of simple perforation plates in the wood of these two species is not accompanied by a decrease in the number of bars on scalariform perforation plates. The wood structure of Meryta bears a strong resemblance to members of the Pacific Schefflera clade, sharing similar ranges of variation of several features. Bark characters, such as the diameter of the cortical secretory canals, the types of crystal in cortical cells, the types of axial parenchyma cell in collapsed secondary phloem, and the presence of sheath cells by phloem rays, appear to be of diagnostic value for some species of Meryta .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 363–379.     

广东惠东莲花山白盆珠兰科植物多样性分析

基于野外实地调查和文献记录,分析广东惠东莲花山白盆珠自然保护区的野生兰科植物物种多样性、属种地理分布特点及其种群结构。结果表明,白盆珠保护区野生兰科植物有40种,隶属于27属,包括地生兰26种、附生兰13种和腐生兰1种;以人为干扰较少的山林和溪谷沟边较为丰富。白盆珠保护区兰科植物属和种的地理成分均以热带亚洲分布成分为主,与邻近地区七目嶂保护区最为相似,两地的属种相似性系数分别达71.11%和61.11%。此外,调查中发现1个广东省兰科植物新记录种全唇盂兰Lecanorchis nigricans,及深圳拟兰Apostasia shenzhenica 1个新分布点。     

Rangewide analysis of fungal associations in the fully mycoheterotrophic Corallorhiza striata complex (Orchidaceae) reveals extreme specificity on ectomycorrhizal Tomentella (Thelephoraceae) across North America

Fully mycoheterotrophic plants offer a fascinating system for studying phylogenetic associations and dynamics of symbiotic specificity between hosts and parasites. These plants frequently parasitize mutualistic mycorrhizal symbioses between fungi and trees. Corallorhiza striata is a fully mycoheterotrophic, North American orchid distributed from Mexico to Canada, but the full extent of its fungal associations and specificity is unknown. Plastid DNA (orchids) and ITS (fungi) were sequenced for 107 individuals from 42 populations across North America to identify C. striata mycobionts and test hypotheses on fungal host specificity. Four largely allopatric orchid plastid clades were recovered, and all fungal sequences were most similar to ectomycorrhizal Tomentella (Thelephoraceae), nearly all to T. fuscocinerea. Orchid-fungal gene trees were incongruent but nonindependent; orchid clades associated with divergent sets of fungi, with a clade of Californian orchids subspecialized toward a narrow Tomentella fuscocinerea clade. Both geography and orchid clades were important determinants of fungal association, following a geographic mosaic model of specificity on Tomentella fungi. These findings corroborate patterns described in other fully mycoheterotrophic orchids and monotropes, represent one of the most extensive plant-fungal genetic investigations of fully mycoheterotrophic plants, and have conservation implications for the >400 plant species engaging in this trophic strategy worldwide.     

Earliest orchid macrofossils: Early Miocene Dendrobium and Earina (Orchidaceae: Epidendroideae) from New Zealand

Fossil leaves of two Early Miocene orchids (Dendrobium and Earina) are reported from New Zealand. The distinctive, raised tetra- to cyclocytic stomatal subsidiary cells of Earina and characteristic papilla-like absorbing glands and "ringed" guard cells of Dendrobium support the placement of the fossils into these genera. These therefore represent the first Orchidaceae macrofossils with cuticular preservation, the oldest records for subfamily Epidendroideae, as well as the first New Zealand and southern hemisphere fossil records for Orchidaceae. These taxa belong in basal clades to the Vandeae/Cymbideae or Epidendreae (Earina) and the Australasian clade of Dendrobium sensu lato. This phylogenetic placement demonstrates expansion of epiphytic orchids into Zealandia by the mid-Cenozoic and an important role for southern continents in the diversification of Orchidaceae.     

A phylogenetic study of pollinator conservatism among sexually deceptive orchids

Orchids of the genus Chiloglottis are pollinated through the sexual deception of male wasps mainly from the genus Neozeleboria (Tiphiidae: Thynninae). The orchids mimic both the appearance and sex pheromones of wingless female thynnines but provide no reward to the deceived males. Despite the asymmetry of this interaction, strong pollinator specificity is typical. Such plant-pollinator interactions would seem to be relatively flexible in the plant's adaptive response to variation in the local pollinator resource. However, we present DNA sequence data on both orchids and wasps that demonstrate a pattern of pollinator conservatism operating at a range of taxonomic levels. Sequence data from the wasps indicate 15 of 16 Chiloglottis pollinators are closely related members of one clade of Thynninae. A pattern of congruence between orchid and wasp phylogenies is also demonstrated below the generic level, such that related orchids tend to use related thynnine wasps as specific pollinators. Comparative physiological data on the wasp responses to the floral scents of two Chiloglottis species and one outgroup, Arthrochilus, indicate similar attractive volatile chemicals are used by related orchid taxa. By extension, we infer a similarity of sex pheromone signals among related thynnines. Thus, the conservative pattern of pollinator change in sexually deceptive orchids may reflect phylogenetic patterns in the sex pheromones of their pollinators.