Integrative analyses of Nervilia (Orchidaceae) section Linervia reveal further undescribed cryptic diversity in Thailand

The delimitation of cryptic species is necessary to accurately classify and appropriately conserve biodiversity. Integrative analyses can be incisive in detecting and circumscribing cryptic diversity, especially in species complexes whose members are delineated by minor or overlapping morphological variation. We adopt an integrative approach to assess species relationships and resolve species boundaries in the taxonomically difficult Nervilia adolphi/punctata species alliance of N. sect. Linervia, an Old World complex of reduced, one-flowered terrestrial orchids that is both species-rich and poorly known in tropical and warm temperate Asia. We sampled 12 of the 27 known species of the alliance in Asia, including all four species reported from Thailand and a further 20 plants collected in that country that could not be satisfactorily identified using morphology alone. Phylogenetic analyses using one nuclear (ITS) and two plastid (matK and trnL-F) markers confirmed both N. sect. Linervia and the alliance itself as monophyletic, and corroborated 11 of the 12 sampled species; N. punctata proved polyphyletic, with the Thai samples referred to this Indonesian species falling sister to the Himalayan N. mackinnonii. The 20 unidentified Thai samples formed three distinct, strongly supported clades. STACEY, a Bayesian coalescence approach to species delimitation, resolved the same three clusters, but provided evidence suggesting that one comprised two distinct sub-clades. Building on this genetic evidence, we identify subtle morphological differences and invoke a diagnosable species concept to circumscribe three previously unrecognized cryptic species from Thailand. This objective approach to species delimitation validates ostensibly minor morphological differences as a basis for differentiating species within the alliance, paving the way for a global analysis of species boundaries throughout the genus as a whole.     

Cephalanthera exigua rediscovered: new insights in the taxonomy,habitat requirements and breeding system of a rare mycoheterotrophic orchid

Five mycoheterotrophic species of Cephalanthera have been proposed from tropical Asia. Until recently, all of them were only known from the type specimens, for which reason it has been difficult to judge if some of them were actually conspecific. The recent discovery of two large populations of C. exigua in Thailand made it possible to describe the range of variation of this species in much more detail, and we then found it to be well distinguished from the others. A phylogenetic analysis based on nuclear rDNA ITS sequences corroborated the systematic position of C. exigua at the generic level. Examination of the previously neglected type of C. pusilla from Myanmar lead to the conclusion that this taxon of disputed generic affinity likewise belongs to Cephalanthera, and that it is a clearly distinct species. Finally, we accept that also C. ericiflora from Laos as well as C. gracilis and C. calcarata from Yunnan are probably distinct species. Cephalanthera exigua grows in hill evergreen forest, but we found little floristic similarity between its two localities in Thailand (IS_S=22.5%). Ectomycorrhizal trees of the families Fagaceae and Ulmaceae are probably the ultimate carbon source for C. exigua at both sites, as the mycorrhizal partners of this orchid (basidiomycetes of the family Thelephoraceae) obtain their carbon nutrition through ectomycorrhizal association with forest trees. Pollination experiments demonstrated that the nectarless flowers of C. exigua are not spontaneously autogamous. Based on the pollination syndrome and previously published observations on other species of the genus, we suggest that C. exigua has an insect‐operated breeding system acting by deceit, and that the pollinators are probably solitary bees.     

Pollination biology of Luisia curtisii (Orchidaceae): indications of a deceptive system operated by beetles

A population of Luisia curtisii (Orchidaceae: Aeridinae) in northern Thailand was studied with regard to pollination biology. Although a high level of self-compatibility was demonstrated experimentally, the very low natural fruit set (1.4–1.9 %) clearly indicated that the species depends on external agents for pollination. Our observations suggest that L. curtisii is pollinated by beetles, as Lema unicolor (Chrysomelidae) and Clinteria ducalis (Scarabaeidae) were the only flower visitors observed to carry pollinaria of this species. The hypothesis of specialised cantharophily is further supported by 2-methylbutyric acid and caproic acid being striking components of the floral scent. Judging from the lack of nectar and the behaviour of visiting beetles, the pollination system seems to rely on food or brood site deception. Retention of the anther on the pollinarium for some time after pollinarium removal probably reduces the frequency of insect-mediated autogamy and geitonogamy in Luisia curtisii—a possibility that was supported by comparative data on (1) the anther retention time and inflorescence visitation time of Lema unicolor and (2) stigma and anther length in the orchid. Existing reports of specialised beetle pollination in orchids are reviewed, and we conclude that there is accumulating evidence that specialised cantharophily is more common in the Orchidaceae than previously assumed.     

Pollination-system diversity in <Emphasis Type="Italic">Epipactis</Emphasis> (Orchidaceae): new insights from studies of <Emphasis Type="Italic">E. flava</Emphasis> in Thailand

The rewarding orchid Epipactis flava was studied in NW Thailand. Its flowers were visited by a wide range of insects, most of which served as pollinators. The most frequent pollen bearers were (in decreasing order): the cricket Homoeoxipha lycoides, stingless bees of the Tetragonula testaceitarsis/hirashimai complex, hoverflies of subfam. Syrphinae, the wasp Polybioides gracilis and sweat bees of subfam. Halictinae. We found no evidence of a link between the rheophytic habit of E. flava and its pollinator fauna. Whereas most pollinators visited the flowers to feed on nectar, females of Episyrphus alternans (Syrphidae: Syrphinae) were observed to oviposit despite the absence of prey for their young. Hence, we suggest that dual pollination systems contribute to the opportunist strategy of E. flava, and we discuss, in a phylogenetic framework, how the strategy fits in with those previously reported for Epipactis sect. Arthrochilium. The elastic attachment of the epichile (a universal trait in sect. Arthrochilium) was found to promote outcrossing, and we hypothesize that loss of the elastic hinge has provided a key innovation facilitating recurrent evolution of obligate autogamy in sect. Epipactis (which is nested in sect. Arthrochilium).     

Orchids in the torrent: on the circumscription,conservation and rheophytic habit of Epipactis flava

The systematically difficult genus Epipactis (Orchidaceae) is often subdivided into two sections: Epipactis and Arthrochilium. Until now, the latter has attracted much less taxonomic attention than the former, but here we reassess the alleged distinction of two rare and nationally endangered taxa from tropical Asia (E. flava, E. atromarginata), based on morphological examination of live plants in situ in northern Thailand and of herbarium specimens from the entire range of the complex (Thailand, Laos and Vietnam). As the variation in vegetative and floral dimensions, flower colour and labellum morphology broadly overlaps between the two taxa, we merge them under the oldest valid name at species level, E. flava. Geomorphological features of our three study sites in Thailand and high shared occurrence of previously confirmed rheophytes among the accompanying species indicate that E. flava is itself rheophytic (a very rare feature in the Orchidaceae). Possible adaptations of E. flava to its rheophytic lifestyle are discussed. Among these, the formation of pure stands through dense clonal growth involving a creeping rhizome places E. flava as a ‘mat‐rooted rheophytic landplant’ (sensu van Steenis). Combining taxonomic, distributional and ecological data, we discuss the occurrence of this stream‐inhabiting Epipactis in a conservation context. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 358–370.     

Wildlife Trade and Human Health in Lao PDR: An Assessment of the Zoonotic Disease Risk in Markets

Although the majority of emerging infectious diseases can be linked to wildlife sources, most pathogen spillover events to people could likely be avoided if transmission was better understood and practices adjusted to mitigate risk. Wildlife trade can facilitate zoonotic disease transmission and represents a threat to human health and economies in Asia, highlighted by the 2003 SARS coronavirus outbreak, where a Chinese wildlife market facilitated pathogen transmission. Additionally, wildlife trade poses a serious threat to biodiversity. Therefore, the combined impacts of Asian wildlife trade, sometimes termed bush meat trade, on public health and biodiversity need assessing. From 2010 to 2013, observational data were collected in Lao PDR from markets selling wildlife, including information on volume, form, species and price of wildlife; market biosafety and visitor origin. The potential for traded wildlife to host zoonotic diseases that pose a serious threat to human health was then evaluated at seven markets identified as having high volumes of trade. At the seven markets, during 21 observational surveys, 1,937 alive or fresh dead mammals (approximately 1,009 kg) were observed for sale, including mammals from 12 taxonomic families previously documented to be capable of hosting 36 zoonotic pathogens. In these seven markets, the combination of high wildlife volumes, high risk taxa for zoonoses and poor biosafety increases the potential for pathogen presence and transmission. To examine the potential conservation impact of trade in markets, we assessed the status of 33,752 animals observed during 375 visits to 93 markets, under the Lao PDR Wildlife and Aquatic Law. We observed 6,452 animals listed by Lao PDR as near extinct or threatened with extinction. The combined risks of wildlife trade in Lao PDR to human health and biodiversity highlight the need for a multi-sector approach to effectively protect public health, economic interests and biodiversity.