Evaluating the Evolutionary and Genetic Relationships of the Andean Orchids of Northwestern Ecuador

DNA barcoding is a molecular based technique used to separate and identify individual species. Here we establish a DNA Barcode library for the orchid flora of an Andean cloud forest in Northwestern Ecuador. The library contains 135 matK and 136 rbcL DNA Barcodes representing over 33 Orchidaceae genera. Sequence analysis shows percent species resolution was higher for matK (98.8%) than rbcL (70.24%), with a large portion of the unresolved species for the rbcL loci coming from taxonomically complex genera in the subtribe Pleurothallidinae. Neighbor Joining (NJ) trees revealed that the orchid flora of Siempre Verde is divided taxonomically into two large monophyletic clades at the sub family level; Orchidoideae and Epidendroideae. Sequences within Orchidoideae presented with high bootstrap support across all NJ trees (matK, rbcL and matK+rbcL), indicating species within the clade are well resolved. Resolution for sequences within sub family Epidendroideae varied depending on taxonomic clade and loci used. Overall the matK NJ tree outperformed the rbcL NJ tree by delivering monophyletic clades at the subfamily, tribe, and subtribe level with higher bootstrap values, separating a higher number of congeners, particularly those in taxonomically complex genera such as Pleurothallis, Stelis, and Lepanthes. Estimates of evolutionary divergence showed a very low level of intraspecific variation in DNA Barcodes of target cryptic species Oncidium heteranthum, acknowledging that floral traits in Oncidium are often highly plastic, and not indicative of species lines

Seed Cryopreservation and Germination of <i>Rhus glabra</i> and the Critically Endangered Species <i>Rhus michauxii</i>

Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations

Seed Cryopreservation and Germination of Rhus glabra and the Critically Endangered Species Rhus michauxii

Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations