81 NORTHWESTERN NATURALIST 94:81–84 SPRING 2013 MOLECULAR IDENTIFICATION OF A HITCHHIKING FROG ERICA MSHERMAN AND ADAM DLEACHE´ Key words: biogeography, Cuba, Cuban The specimen arrived in a shipment of stereo Treefrog, DNA barcoding, introduced species, equipment from Amazon.com on 19 February molecular data, Osteopilus septentrionalis, phylo- 2011. The specimen (Fig. 1) has been deposited genetic analysis, phylogeography, Washington at the Burke Museum of Natural History and Culture (UWBM 3483). Tissue samples from the The introduction and expansion of non-native frog were removed from the 2nd tarsal of the species into an ecosystem can be detrimental left foot, in addition to 1 mm2 of skin from the and result in the decline of native species lower abdomen. We extracted DNA using a abundance and the possible extinction of native Qiagen DNEasy Blood and Tissue kit, using the species (Dorcas and others 2012). Most com- spin-column animal-tissue protocol. DNA sam- monly, the mode of introduction is unintention- ples were diluted to a concentration of 10ng/ml al and human driven. Many intentional intro- prior to PCR amplification. ductions historically have been motivated by We targeted the mitochondrial DNA 16S individuals or groups who believe that the rRNA gene (16S), because this gene has been newly introduced species will be in some way widely used in amphibian systematics and is beneficial to humans in its new location currently the most useful marker for molecular (Pimentel and others 2005). Conversely, unin- taxonomic identification and DNA barcoding in tentional introductions are most often a bypro- frogs (Vences and others 2005). We amplified duct of human movements, and are thus and sequenced a 550-bp region of the 16S gene unbound to human motivations. Introduced using standard amphibian primers 16SA-L and species can have negative impacts on native 16SB-H (Vences and others 2005). The 20 ml PCR populations, including population declines reactions included 13 mlofdH2O, 0.2 mlofTaq through niche displacement, interspecific com- polymerase (1 unit), 0.2 ml of 25 mM MgCl2, petition for food and habitat, direct predation, 2.0 mlof103 PCR buffer, 0.5 ml of each 16S and competitive exclusion (Suarez and others primer (20 mM), 0.8 ml of 10 mM dNTP, and 1.0 ml 2005). of DNA. The PCR amplification program In this study, we used forensic molecular consisted of an initial denaturation step at techniques to identity a stowaway frog that was 2 min at 946C. This was followed by 29 cycles shipped to Kirkland, Washington, in an Ama- of denaturation (30 s at 946C), annealing (30 s at zon.com package. The frog was deceased and of 486C) and extension (30 s at 726C), and a final unknown origin, despite our best attempts to extension of 5 min at 726C. PCR products were locate the original shipping location from visualized using a 1% agarose gel stained with Amazon and its subsidiary shipping companies. EtBr. A sample of Lithobates pipiens (Northern It was also severely desiccated, which made it Leopard Frog) was used as a positive control. difficult to identify to species using morpholog- The PCR products were purified using ExoSAP- ical characteristics alone (Fig. 1). IT (USB). We sequenced using dye-labeled 82 NORTHWESTERN NATURALIST 94(1) aligned using Muscle v3.6 (Edgar 2004). The nucleotide substitution model was selected using JModelTest v0.1 (Posada 2008). Phyloge- netic relationships were inferred using maxi- mum likelihood and Bayesian inference. Maxi- mum likelihood analyses were conducted using RAxML-VI-HPC v7.0.4 (Stamatakis 2006). The RAxML analyses used the GTRGAMMA model of nucleotide substitution. Support values were estimated from 1000 non-parametric bootstrap replicates. We conducted Bayesian phylogenetic analyses using parallel MrBayes v3.1.2 (Ron- FIGURE 1. Photograph of the desiccated Osteopilus quist and Huelsenbeck 2003). We ran 2 separate septentrionalis (UWBM 3483) shipped in an Amazon.- analyses with different starting seeds for 2 com package to Kirkland, Washington. (Photo credit: million generations using 4 heated Markov Duncan Reid). chains (using default heating values). We assessed convergence by inspecting the cumu- dideoxy terminator cycle sequencing with Big- lative posterior probabilities of clades using the Dye v3.1 (Applied Biosystems), and products online program Are We There Yet? (AWTY; were cleaned using hydrated sephadex placed Nylander and others 2008). Posterior probabil- in a Milipore plate. Sequencing (both directions) ity values were obtained by summarizing the was performed on an ABI 3730 automated DNA posterior distribution of trees (post burn-in) sequencer. We aligned and edited DNA se- with a 50% majority-rule consensus tree. quences using Sequencher v4.2. The DNA The 16S data from the unknown sample sequence data are deposited on Genbank included 440 base pairs. The top hits from the (Accession #KC170728). nucleotide similarity search match with frogs of We conducted a nucleotide BLAST search the Lophiohylini, and the unknown sequences (BLASTn) of the unknown frog 16S gene on shared 100% coverage and 99% sequence Genbank. We downloaded a phylogenetically- similarity (E-value 5 0.0) with Osteopilus septen- informative cluster of DNA sequences using the trionalis (Cuban Treefrog). The single specimen PhyLoTA Browser (rel. 1.5). The sequences were of O. septentrionalis in Genbank with 16S data is FIGURE 2. A phylogeny of 16S mitochondrial DNA shows that specimen UWBM 3484 is an Osteopilus septentrionalis. SPRING 2013 GENERAL NOTES 83 from Guantanamo Bay, Cuba (Accession Num- warming scenarios (Ro¨dder and Weinsheimer ber: AY843712.1). The Cuba specimen differs 2009). Across its introduced range, O. septen- from UWBM 3483 by 3 nucleotide substitutions trionalis consumes a wide variety of inverte- (2 C-T transitions and 1 A-C transversion). brate and vertebrate prey including beetles, A phylogenetically-informative sequence roaches, isopods, and lepidopterans; other cluster for the Lophiohylini, which contains frogs including the Green Treefrog (Hyla the genus Osteopilus, was downloaded from cinerea), Squirrel Treefrog (Hyla squirella), East- PhyLoTA for phylogenetic analyses. The data ern Narrowmouth Toad (Gastrophryne caroli- matrix contained 16 species and 572 aligned nensis), Southern Leopard Frog (Lithobates nucleotide positions. The GTR+I+C nucleotide sphenocephalus), and Southern Toad (Anaxyrus substation model was selected by JModelTest terrestris); and lizards including the Brown under all criteria (Akaike Information Criterion, Anole (Anolis sagrei) and Common House Bayesian Information Criterion, or dynamical Gecko (Hemidactylus mabouia) (Bartareau and likelihood ration tests), and this model was used Meshaka 2007; Meshaka 2011). According to in the Bayesian phylogenetic analyses. The tree Austin (1973), O. septentrionalis has had a was rooted using Phyllodytes luteolus (Yellow negative effect on native frog populations. Heart-tongued Frog) based on the results Although here we report a failed cross- presented in Faivovich and others (2005). continental accidental introduction, O. septen- The phylogenetic analyses of the Lophiohy- trionalis is an opportunistic settler, and the lini provided strong support for the placement expansion of this species into new regions could of unknown sample UWBM 3483 (Fig. 2). be further facilitated by an increase in suitable Phylogenetic analyses using maximum likeli- habitats as a result of climate change (Ro¨dder hood and Bayesian analyses both support a and Weinsheimer 2009). This event also illus- clade containing O. septentrionalis and UWBM trates that the Pacific Northwest is not neces- 3483 (bootstrap support 5 100%; posterior sarily immune to this type of accidental intro- probability 5 1.0; Fig. 2). Monophyly of the duction by non-native species that are genus Osteopilus is supported by a 93% boot- successful at colonizing and surviving in ap- strap value and a 0.99 posterior probability, propriate habitats. although the relationships among species are not fully resolved (Fig. 2). Acknowledgements.—We thank K Fries for donating the frog to the Burke Museum of Natural History and Osteopilus septentrionalis is a neotropical and Culture. mostly arboreal frog in the family Hylidae that has become widely distributed in the south- LITERATURE CITED eastern United States. The native range of the species is Cuba, the Isle of Youth, the Cayman AUSTIN DF. 1973. Range expansion of the Cuban Islands, and the Bahamas (McGarrity and Treefrog in Florida. Florida Naturalist 46:28. Johnson 2009), but O. septentrionalis is now BARBOUR T. 1931. Another introduced frog in North America. Copeia 1931:140. considered an invasive species in the southern BARTAREAU TM, MESHAKA WE JR. 2007. Osteopilus continental US. The 1st introduction of O. septentrionalis (Cuban Treefrog). Diet. Herpetolog- septentrionalis to the US is speculated to have ical Review 38:324–325. occurred in Key West, Monroe County, Florida DORCAS ME, WILLSON JD, REED RN, SNOW RW, in 1931 (Barbour 1931), and introductions of O. ROCHFORD MR, MILLER MA, MESHAKA WE JR, septentrionalis are recorded throughout the state ANDREADIS PT, MAXXOTTI FJ, ROMAGOSSA CM, of Florida (Meshaka 2011), as well as in Georgia HART KM. 2012. Severe mammal declines coincide (Jensen and others 2008), Virginia (Mitchell and with proliferation of invasive Burmese Pythons in Reay 1999), Maryland (Meshaka 1996), and Everglades National Park. Proceedings of the National Academy of Sciences 109:2418–2422. Colorado (Livo