Legally protected plants are illegally traded through online sales platforms and orchids are a significant component of this wildlife trade. This study focused on salep, a compound product made from wild collected orchid tubers from several genera—including Anacamptis, Dactylorhiza, Himantoglossum, Ophrys, Orchis, Serapias—whose harvest endangers some of the species used, despite their collection and sale being restricted by national and international legislation. Using a custom designed web crawler in combination with DNA barcoding of a subset of products over 18 months 1942 items of salep were detected as sold at a total value of US$ 37,775, estimated to be equivalent to 90,000 to 180,000 wild orchids being destructively harvested. Wild harvested tubers traded at a value of $0.21 and equivalent cultivated orchids have a market price of $16–28; cultivation is currently no viable alternative to wild harvesting. Using a web crawler on open trade sites contributes to knowledge on illegal wildlife trade, which can be used to address illegal plant trade at the national and international level.

Botrylloides niger (class Ascidiacea) is an invasive marine filter-feeding invertebrate that is believed to originate from the West Atlantic region. This species of colonial tunicate has been observed in several locations along the coasts of Israel and around the Suez Canal, but it has not yet been reported on the coasts of the Northeastern Mediterranean Sea (NEMS), suggesting an ongoing Lessepsian migration. However, the extent of this invasion might be concealed by reports of other potentially misidentified species of Botrylloides, given that the strong morphological similarities within this genus renders taxonomical identification particularly challenging. In this study, we performed a phylogeographic and morphological analysis of B. niger in the NEMS. We collected 238 samples from 8 sampling stations covering 824 km of the coastlines of NEMS. We reported 14 different morphotypes, of which the orange-brown, orange, and brown-striped morphs were the most abundant. Using the mitochondrial cytochrome C oxidase I (COI) as a DNA barcode marker, we identified 4 haplotypes. The COI haplotypes clustered with the reference B. niger sequences from GenBank and differed significantly from the sister Botrylloides species. We confirmed our identification using three additional barcoding markers (Histone 3, 18S rRNA, and 28S rRNA), which all matched with over 99% similarity to reference sequences. In addition, we monitored a station for a year and conducted a temporal analysis of the collected colonies. The colonies were absent during the winter and spring, while new colonies were established in the summer and expanded during autumn. We performed demographic population analysis on our spatial data that identified a possible population subdivision at a sampling site, which might have been caused by local freshwater input. Herein, we present the first report on the presence of Botrylloides niger in the NEMS. This study represents a key step toward understanding the diversity and the propagation of this highly invasive species of colonial ascidians, both within the Mediterranean basin as well as globally.

Tunicates are marine filter-feeding invertebrates that can be found worldwide and which are the closest phylogenetic group to the vertebrates (Craniata). Of particular interest, colonial tunicates are the only known chordates that can undergo Whole-Body Regeneration (WBR) via vascular budding. In Botrylloides anceps, a fully functional adult regenerates from a fragment of the vascular system in around 2 weeks after amputation. In this chapter, we present protocols to collect B. anceps colonies, confirm their species, breed them in the lab, monitor WBR and perform histological staining on cryosections.

Members of the tunicates, a subphylum of marine filter-feeder chordates, inhabit all marine and oceanic habitats from the subtidal to the abyssal. Considered the closest relatives to the vertebrates, the tunicates are widely used as model organisms for evo-devo, allorecognition, senescence, and whole-body regeneration studies. However, species boundaries are poorly understood due to the high morphological and genetic plasticity that characterizes many tunicate taxa. Here, we present findings on seven tunicate species (Botrylloides israeliense, Botrylloides sp., Botryllus humilis, Botryllus schlosseri, Symplegma brakenhielmi, Polyclinum constellatum and Didemnum perlucidum) sampled from six Turkish sites at the North Eastern Mediterranean Sea and employed the mitochondrial barcoding marker (COI) for evaluating the relationships among geographically restricted and widely spread ascidian species. Species delimitation was conducted using sequences generated in the current study in addition to sequences obtained from GenBank. General morphological features and colors of colonies were recorded at sampling sites. Then, all Styelidae colonies were attached and cultured on slides in an aquaculture room, enabling the study of other features, such as zooid distributions and sizes, oral tentacle numbers, and life cycles, using stereo and light microscopes. The spicules of formalin-fixed Didemnum perlucidum samples were examined under a light microscope. Then, scientific names were assigned to all species based on the results of the species delimitation and on comparisons of the obtained COI sequences with GenBank sequences. A putative new Botrylloides species (Botrylloides sp.) from the Antalya region was revealed, with a 99% match with the COI gene of another specimen from Saudi Arabia; further waiting for detailed traditional taxonomy.