Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle... Show moreTubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle‐class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL‐F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL‐F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree‐based methods for nrITS in 67% of the tested tubers, 58% for trnL‐F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross‐border trade of Salep tubers. Show less
Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain... Show moreNext-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. BAPS allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. NEWHYBRIDS, a hybrid index and HIEST confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus. Show less
Ghahramanzadeh, R.; Esselink, G.; Kodde, L.P.; Duistermaat, H.; Valkenburg, J.L.C.H. van; Marashi, S.H.; ... ; Wiel, C.C.M. van de 2013
