The Solanaceae family is one of the major groups of angiosperms with ≈2500 species and 100 genera (Filipowicz and Renner, 2012). The family contains species with agricultural and economical importance worldwide such as Solanum tuberosum, Solanum melongena, Solanum lycopersicum, Nicotiana tabacum, and Capsicum annuum, as well as numerous toxic or poisonous species. Puerto Rico is considered a biodiversity hotspot due to its tropical location and the high concentration of endemic species that have been threatened by habitat loss (Helmer et al., 2002; Myers et al., 2000). According to Axelrod (2011), in Puerto Rico, there are 46 species of Solanaceae from 14 genera. Of these 46 species, Bunfelsia densifolia, Bunfelsia lactea, Bunfelsia portoricensis, Goetzea elegans, Solanum ensifolium, and Solanum woodburyi are endemic. Some of these species are endangered or rare, with only a few studies conducted on them.
Filipowicz and Renner (2012) included the endemic and nonendemic Brunfelsia species of Puerto Rico in their systematic study. They hypothesized that these species reached the island 3 to 5 million years ago, and their seeds were dispersed by birds from South America. In research by Muscarella et al. (2014), the Puerto Rican endemic Solanaceae species G. elegans and S. ensifolium were included as part of the trees of Puerto Rico that were evaluated. They studied 89% of Puerto Rico’s trees to analyze the community phylogeny based on regions with different climate areas.
In Puerto Rico, there are two endemic and endangered Solanaceae species: G. elegans and S. ensifolium. In 1985, G. elegans was listed by the U.S. Fish and Wildlife Service under the Endangered Species Act. At that time, there were fewer than 50 individuals in three known sites (U.S. Fish and Wildlife Service, 1985). More recently, this species has increased in number of individuals and has been reported to be present in 10 localities, but it still is considered an endangered species (Vargas, 2013). Comparatively, the species distribution and status of S. ensifolium is less known, as it has not been evaluated since 1992. In 1992, 150 individuals were found in the locality of Las Tetas de Cayey (Vargas, 2015). It was hypothesized that the species could be present in other localities, but that has not been confirmed. Unfortunately, during this study, S. ensifolium was not found in Las Tetas de Cayey or at other localities where it was thought to be, such as in Florida, Puerto Rico. Its presence at other localities is currently unknown. Due to Hurricane María in 2017, there is a high possibility that S. ensifolium might be even more critically endangered than in 1992, and in the worst-case scenario, it has gone extinct in the wild. More studies and conservation efforts are critically needed to determine the current status of S. ensifolium.
In addition, studies of rare species such as the endemic S. woodburyi are also needed, especially using molecular tools. This species has not been classified as endangered, but only a few individuals are known to be present in specific localities, such as the Sierra de Luquillo (Axelrod, 2011). Our study is the first to include S. woodburyi in a molecular study. Previous research on this species has focused on the description of the species in a synopsis of the endemic species of the West Indies (Howard, 1966; Knapp, 2009). Of the 46 Solanaceae species found in Puerto Rico many, such as Jaltomata antillana and Solanum polygamum, are not well studied with no molecular data available in public databases such as GeneBank. Therefore, we apply molecular techniques, specifically a DNA barcoding approach, to better understand the species relationships and delimitations for the taxa of Solanaceae in Puerto Rico.
DNA barcoding was proposed as an easy and cost-effective technique for the identification of species. It consists of the amplification of a universal, robust, and standard region. The first region proposed for this analysis was the mitochondrial gene cytochrome c oxidase I (COI) for animals (Hebert et al., 2003). It has been successfully used in studies of taxonomy, population genetics, forensics in wildlife crimes, and conservation and in species identification in cases of fish mislabeling, among others (Cywinska et al., 2006; Di Pinto et al., 2015; Hebert et al., 2004; Rolo et al., 2013). In plants, the use of the COI region is not recommended for DNA barcoding studies due to its low evolutionary rate (Kress et al., 2005). DNA barcoding for plants has been more challenging than for animals, and more than one barcode region is needed for species identification. Currently, the most accepted and widely used regions in DNA barcoding studies in plants are rbcL, matK, psbA-trnH, and ITS (Bolson et al., 2015; CBOL Plant Working Group, 2009; Kress, 2017; Li et al., 2014). Different studies have shown successful identification of species using a combination of these barcode markers, but some studies have also found limited ability to discriminate species within groups consisting of recently diverged species (Collins and Cruickshank, 2013; Kress, 2017; Kress et al., 2009; Muscarella et al., 2014; Spooner, 2009).
In this study, the barcode regions used were two chloroplast regions and a nuclear region, matK, psbA-trnH, and ITS, respectively. The matK region is 1500 bp long and encodes for maturase-like polypeptide, which is believed to be involved in Group II intron splicing (Selvaraj et al., 2008). It has an evolutionary rate suitable for distinguishing taxa at high taxonomic levels such as order, family, and, in some cases, at the genus levels (Yu et al., 2011). For a coding region, matK has higher variability compared with rbcL, but it has not been successfully amplified in all plants (such as gymnosperms). However, it has worked well for angiosperms (CBOL Plant Working Group, 2009; Dunning and Savolainen, 2010; González et al., 2009; Hollingsworth et al., 2011; Kress and Erickson, 2007). Because matK cannot distinguish at lower taxonomic levels, it is usually combined with other regions such as psbA-trnH, which has a higher substitution rate (Hollingsworth et al., 2011). The psbA-trnH region is an intergenic spacer of ≈400 to 500 bp in length. Because of its variability, it has been demonstrated to discriminate successfully at the species level when combined with other barcodes (Bolson et al., 2015; Kress and Erickson, 2007; Kress et al., 2009; Štorchová and Olson, 2007).
For discrimination at the species level using DNA barcoding, the nuclear internal transcribed spacer (ITS) region been successfully used to complement chloroplast barcodes (Kress et al., 2005; Li et al., 2011). The internal transcribed spacers (ITS1 and ITS2) are between the nuclear ribosomal 18S-5.8S-26S genes, and altogether the marker consists of less than 700 bp (Feliner and Rosselló, 2007). Although ITS has helped discriminate at the species level, there are two main concerns when using it in DNA barcoding studies: the possibility of amplifying the ITS region from fungal contaminants and the amplification of divergent paralogous copies (Hollingsworth, 2011).
Our overall objectives in this study were 1) to understand the feasibility of using DNA barcoding for identification of Solanaceae species found in Puerto Rico and 2) to study the phylogenetic relationships among the endemic and nonendemic Solanaceae species of Puerto Rico.
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Interspecific and intraspecific pairwise distance for the MatK DNA barcode.
Interspecific and intraspecific pairwise distance for the ITS DNA barcode.
Interspecific and intraspecific pairwise distance for the psbA-trnH DNA barcode.