bs_bs_banner A revised evolutionary history of armadillos (Dasypus) in North America based on ancient mitochondrial DNA BETH SHAPIRO, RUSSELL W. GRAHAM AND BRANDON LETTS Shapiro, B. Graham, R. W. & Letts, B. 2015 (January): A revised evolutionary history of armadillos (Dasypus)in North America based on ancient mitochondrial DNA. Boreas, Vol. 44, pp. 14–23. 10.1111/bor.12094. ISSN 0300-9483. The large, beautiful armadillo, Dasypus bellus, first appeared in North America about 2.5 million years ago, and was extinct across its southeastern US range by 11 thousand years ago (ka). Within the last 150 years, the much smaller nine-banded armadillo, D. novemcinctus, has expanded rapidly out of Mexico and colonized much of the former range of the beautiful armadillo. The high degree of morphological similarity between these two species has led to speculation that they might be a single, highly adaptable species with phenotypical responses and geographical range fluctuations resulting from environmental changes. If this is correct, then the biology and tolerance limits for D. novemcinctus could be directly applied to the Pleistocene species, D. bellus. To investigate this, we isolated ancient mitochondrial DNA from late Pleistocene-age specimens of Dasypus from Missouri and Florida. We identified two genetically distinct mitochondrial lineages, which most likely correspond to D. bellus (Missouri) and D. novemcinctus (Florida). Surprisingly, both lineages were isolated from large specimens that were identified previously as D. bellus. Our results suggest that D. novemcinctus, which is currently classified as an invasive species, was already present in central Florida around 10 ka, significantly earlier than previously believed. Beth Shapiro ([email protected]), Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA; Russell W. Graham, Department of Geosciences, Pennsylvania State University, University Park, PA 16802, USA; Brandon Letts, Department of Biology, Pennsylvania State University, University Park, PA 16802, USA; received 27th February 2014, accepted 23rd June 2014. Xenarthra, an enigmatic superorder of mammals, first documented morphological differences in the appears in the fossil record in South America during the calcaneus. The early evolutionary history of D. bellus Palaeocene (Rose et al. 2005), around 60 million years remains unresolved. Blancan (Pliocene) forms are ago (Ma). The superorder quickly diversified into three smaller than those of the Late Pleistocene, but it orders: the Phyllophaga (sloths), Vermilingua (anteat- appears there is also a size reduction just before extinc- ers) and Cingulata (armadillos) (Gaudin & McDonald tion (Klippel & Parmalee 1984). 2008). Dasypodidae, the family that includes armadil- There are two different hypotheses proposed to los, along with two other cingulates, Glyptodontidae explain the origin of the modern nine-banded arma- and Pampatheridae, dispersed northward into North dillo, D. novemcinctus. One hypothesis suggests that the America around 3 Ma with the formation of the Pana- nine-banded armadillo is directly descended from the manian isthmus (Marshall et al. 1982). Glyptodonts and beautiful armadillo (McNab 1980; McBee & Baker pampatheres went extinct about 11 000 years ago (ka) 1982). This hypothesis is supported by the fact that the (Kurtén & Anderson 1980). body size of D. bellus decreased during the latest Pleis- Today, the Dasypodidae contains 21 extant species tocene (Klippel & Parmalee 1984). On the other hand, (Möller-Krull et al. 2007), seven of which are within the the same data (Klippel & Parmalee 1984) show that genus Dasypus, which probably diversified in South D. bellus was extremely variable in size, with small America around 7 Ma (Delsuc et al. 2003). One species, D. bellus overlapping in size with large D. novemcinctus Dasypus bellus (beautiful armadillo), occurred in the (e.g. band osteoderm widths varied from 13.2–6.8 mm USA from about 2.5 Ma (Voorhies 1987; Woodbourne in beautiful armadillos compared with 5.4–6.9 mm 2010) until about 11 ka when it went extinct (Schubert in nine-banded armadillos (Klippel & Parmalee 1984: & Graham 2000). It ranged across the southeastern p. 157, table 3)). Consequently, D. bellus and USA from New Mexico and Texas to Florida, and D. novemcinctus may not actually be different species, north to Nebraska (Voorhies 1987) and Iowa across to but rather a single, highly adaptable species that has West Virginia (Klippel & Parmalee 1984: p. 154, fig. 2). been through a series of phenotypic changes along with No records of the beautiful armadillo exist from South range expansions and contractions in response to envi- America (Schubert & Graham 2000). ronmental fluctuations. This evolutionary change may The beautiful armadillo was large (∼1.2 m long), have happened in Mexico or even further south with about twice the size of the modern nine-banded arma- the extinction of D. bellus. dillo, D. novemcinctus. Other than size, there appears to Today, D. novemcinctus is the most widespread be a high degree of morphological similarity between xenarthran, with a continuous range extending from the two species (Auffenberg 1957; Slaughter 1961; Patagonia in Argentina northward into Nebraska in the Voorhies 1987), although Jansinski & Wallace (2014) USA (McBee & Baker 1982). In fact, it is the only DOI 10.1111/bor.12094 © 2014 Collegium Boreas. Published by John Wiley & Sons Ltd BOREAS Evolutionary history of armadillos (Dasypus) in North America 15 xenarthran in the USA, where the US Department of and 10 thousand years old. We additionally attempted Fish and Wildlife Services consider it to be an invasive to extract DNA from four glyptodont and five (non-native) species (Simonson et al. 2004). It rapidly pampathere bones and teeth (Table 1) sampled from expanded its range out of Mexico through Texas the Florida Museum of Natural History. and into the mid-central southern states within the From each specimen, we performed DNA extraction last 150 years (Humphrey 1974). Independently, in the for poorly preserved bones as described previously middle 1800s, humans introduced populations of (Letts & Shapiro 2012). As with all ancient DNA D. novemcinctus into Florida. Likewise, this population research, we performed DNA extraction and PCR expanded its distribution into the southeastern USA set-up in a sterile, positive pressure clean laboratory, in (Humphrey 1974). In the last 15 years, D. novemcinctus this instance the ancient DNA laboratory at the Penn- has continued its range extension in to central Missouri sylvania State University, which is physically isolated and southern Illinois (Taulman & Robbins 1996). With from modern molecular biology research. Prior to this the projection for future climate warming (IPCC 2013), project, no molecular biology research was carried out expectations are that it will continue to expand north- on either living or extinct armadillos in either the ward (McBee & Baker 1982; Taulman & Robbins 1996; ancient DNA laboratory or the modern DNA labora- Fig. 1). tory in which downstream molecular biology work was The recent, rapid expansion of nine-banded armadil- performed. Workflow was always from the ancient los into North America is reflected in their present-day DNA laboratory to the modern DNA laboratory, and genetic diversity. Two divergent mitochondrial lineages full protective coverings were worn at all times. Nega- of nine-banded armadillos are known (Huchon et al. tive controls were used at all steps and PCR products 1999; Frutos & Van den Bussche 2002; Arteaga et al. were cloned to characterize DNA damage and identify 2012), but only one of these has been identified in the environmental contaminants. Prior to extraction, we USA (Huchon et al. 1999; Arteaga et al. 2012). In addi- removed subsamples from each specimen using a tion, in an analysis of diversity within the mitochondrial Dremel tool. We collected bone powder either by drill- control region, which is the most rapidly evolving ing directly into the interior of the bone with a drill tip, segment of the mitochondrial genome, only two very or by removing a larger piece of bone with a cutting closely related haplotypes were observed amongst a disk and reducing this piece to powder with a sample of 20 nine-banded armadillos from across their Mikrodismembrator (Braun). Of those that we were US range (Huchon et al. 1999). By contrast, a far greater able to powder (Lab ID is listed as n/a in Table 1) we diversity of mitochondrial haplotypes is found in South collected 200–400 mg of bone powder, from which we and Central America, where both lineages of nine- extracted DNA using the silica-binding method banded armadillos are found as far north as Mexico described by Rohland et al. (2010). In the final step of (Huchon et al. 1999; Frutos & Van den Bussche 2002; the extraction, we eluted the DNA in 50 μl of TE buffer Arteaga et al. 2012). and added Tween-20 to a concentration of 0.5%, as this Here, we investigated the evolutionary relationship has been shown to prevent DNA molecules from between the extinct D. bellus and extant D. binding to the walls of the tube in which the extract is novemcinctus using ancient mitochondrial DNA. We stored (Rohland & Hofreiter 2007). isolated genetic data from two North American indi- Initial results suggested that DNA was preserved in viduals dating to the Pleistocene, one from Florida and two of the 12 armadillo samples (see below). However, the other from Missouri, both identified as D. bellus.We these samples were heavily contaminated with human performed a phylogenetic analysis of these and previ- DNA, probably introduced during excavation, han- ously isolated mitochondrial DNA data from nine- dling and storage. We therefore performed an addi- banded armadillos and from the most closely related tional extraction from these two samples, this time living species to the nine-banded armadillo, the greater including a bleach step in which coarsely powdered long-nosed armadillo (D. kappleri). bone was immersed in 3% bleach for 15 min. This pro- tocol has been shown to effectively remove nearly all Material and methods exogenous DNA contamination (Kemp & Smith 2005).