103 First Record of Invasive Burmese Python Oviposition and Brooding Inside an Anthropogenic Structure Emma B. Hanslowe1, Bryan G. Falk1, Michelle A.M. Collier1, Jillian M. Josimovich1, Thomas A. Rahill2, and Robert N. Reed1,* Abstract - We discovered an adult female Python bivittatus (Burmese Python) coiled around a clutch of 25 eggs in a cement culvert in Flamingo, FL, in Everglades National Park. To our knowledge, this is the first record of an invasive Burmese Python laying eggs and brooding inside an anthropogenic structure in Florida. A 92% hatch-success rate suggests that the cement culvert provided suitable conditions for oviposition, embryonic development, and hatching. Given the plenitude of such anthropogenic structures across the landscape, available sites for oviposition and brooding may not be limiting for the invasive Burmese Python population. Introduction Native to Southeast Asia, Python bivittatus Kuhl (Burmese Python) has been established in southern Florida for well over a decade (Meshaka et al. 2000, Willson et al. 2011), but only two Burmese Python nests in the invasive range in Florida have been reported in the literature. The first was in an overgrown debris pile comprised of vines, small forbs and shrubs, other vegetation, cut logs, PVC pipe, barbed wire, poster-board, planks, cardboard, pieces of a filing cabinet, and other materials (Snow et al. 2007). The second was in a canal bank in an area of loosely packed soil, dense roots, and trash (Snow et al. 2010). In their native range, Burmese Pythons have been documented nesting in open-ground sites, tree holes, termite mounds, and under fallen logs (Wall 1921). Here we describe the first known occurrence of Burmese Python oviposition and brooding inside an anthropogenic structure. Methods At 2311 h on 15 May 2015, we discovered an adult Burmese Python coiled around a clutch of eggs in a cement culvert in Flamingo, FL, in Everglades National Park (25°8'29.56''N, 80°55'34.98''W). We blocked off both culvert openings with plywood and cinder blocks to prevent escape of the adult or hatchlings. We monitored the clutch every Monday and Friday starting 29 May 2015 until eggs exhibited signs of hatching. We then checked the clutch nightly from 24 June 2015 until 02 July 2015. Clutch checks took place at approximately 2300 h, during which we removed the plywood and cinder blocks from the western opening of the culvert and used a flashlight to observe the adult female and the clutch. We recorded 1US Geological Survey, Fort Collins Science Center, 2150 Centre Avenue, Building C, Fort Collins, CO 80526. 2Swamp Apes, 16234 Livingston Avenue, Lutz, FL 33559. *Corresponding author - reedr@usgs.gov. Manuscript Editor: John Placyk Everglades Invasive Species 2016 Southeastern Naturalist 15(Special Issue 8):103–106 Southeastern Naturalist E.B. Hanslowe, B.G. Falk, M.A. McEachern, J.M. Josimovich, T.A. Rahill, and R.N. Reed 2016 104 Vol. 15, Special Issue 8 the status of the mother and clutch (e.g., mother coiled on eggs, mother moved off clutch), and then replaced the plywood and cinder blocks. Following hatching, we captured the adult and hatchling snakes. We humanely euthanized hatchlings via a CASH Special Captive .22 Caliber Bolt Stunner (Accles and Shelvoke Ltd; Sutton Coldfield, West Midlands, England), a method approved by the USGS Fort Collins Science Center Animal Care and Use Committee (FORT IACUC Approval 2015- 02), necropsied them to assess general health, and determined sex by measuring cloacal depth with a probe (Schaefer 1934). We disposed of the hatchlings in the field and retained the adult female alive for a dif ferent research project. The concrete culvert was 13.90 m long, 0.45 m wide, and 0.40 m in height, running east to west beneath a paved road. Inorganic limestone gravel lined the bottom of the culvert. The clutch was located 4.60 m from the eastern opening (Fig. 1). Adjacent habitat included landscaped, grassy vegetation with evenly dispersed Swietenia mahogani (L.) Jacq. (Florida Mahogany), asphalt parking lots, and mangrove-dominated coastal habitat bordering the brackish waters of Florida Bay (e.g., Meshaka et al. 2000). Roystonea regia (Kunth.) O.F. Cook (Royal Palm), Ficus aurea Nutt. (Strangler Fig ), and Bursera simaruba (L.) Sarg. (Gumbo Limbo) were also present at the site. Figure 1. A cement culvert (13.90 m long, 0.45 m wide, and 0.40 m in height) in Flamingo, FL, in Everglades National Park where an adult female Burmese Python was discovered coiled around a clutch of 25 eggs. Photo taken on 22 June 2015 after the female had moved away from the eggs. Southeastern Naturalist 105 E.B. Hanslowe, B.G. Falk, M.A. McEachern, J.M. Josimovich, T.A. Rahill, and R.N. Reed 2016 Vol. 15, Special Issue 8 Results The female was coiled around the eggs during all 17 of our observations until 19 June 2015 (12 days before hatching commenced) when she moved several meters towards the eastern opening of the culvert. After that date, we never observed the female coiled around her clutch. On 28 June 2015, several of the eggs had become concave and misshapen, and one had rolled away from the rest of the clutch. Hatching had commenced by 01 July 2015 (47 d after we discovered the clutch), when we observed 2 hatchlings in the culvert. We removed 16 live hatchlings from the culvert between 02 and 04 July 2015 and removed the adult female (SVL = 2.65 m; total length = 2.97 m; 9.19 kg) on 03 July. Total clutch size was 25; 23 eggs hatched successfully and 2 were nonviable (92% hatch-success rate). We were unable to account for 7 hatchlings; they may have escaped from the culvert despite containment efforts. Of the 16 captured hatchlings, 10 were males and 6 were females. Necropsies revealed that each hatchling had yolk in the caudal half of its body cavity and an empty gastrointestinal tract; all appeared to be in good health. Although we did not conduct a necropsy, the adult female also appeared to be in good health. Discussion To our knowledge, this is the first documented observation of a Burmese Python laying eggs and brooding inside an anthropogenic structure in Florida. A 92% hatch-success rate suggests that the cement culvert provided suitable conditions for oviposition, embryonic development, and hatching. Though the frequency with which Burmese Pythons use anthropogenic structures for reproduction is unknown, this observation is unlikely to be the only case because culverts and similar structures are common and widespread throughout Florida. Consequently, available sites for oviposition and brooding may not be limiting even in highly altered habitats. That a Burmese Python utilized an anthropogenic structure may also prove important for predicting and curtailing the species’ northward spread; culverts and other anthropogenic structures might offer suitable thermal refuges to Burmese Pythons during cold events. New information regarding the life history, biology, and behavior of invasive Burmese Pythons in southern Florida can better inform development of detection and control tools for use by resource managers. Based on our observation, efforts to prevent Burmese Pythons from using culverts and similar structures for reproduction may be warranted. Additionally, focusing search efforts near anthropogenic structures may be advantageous in spring when females are gravid (Krysko et al. 2008). The difficulty in detecting Burmese Pythons and the inaccessibility of much of the occupied habitat increases the value of individual natural-history observations such as those we report here. Acknowledgments Funding was provided by Everglades National Park, the US Geological Survey (USGS) Greater Everglades Priority Ecosystem Science Program, and the USGS Invasive Species Science Program. B. Smith, J. Brisbane, C. Pedrozo, S. Tillis, and J. Selden assisted with the Southeastern Naturalist E.B. Hanslowe, B.G. Falk, M.A. McEachern, J.M. Josimovich, T.A. Rahill, and R.N. Reed 2016 106 Vol. 15, Special Issue 8 snake captures, and S. McKnight, M. Conway, and C. Moriarty assisted with monitoring the clutch. We thank M. Cherkiss and 2 anonymous reviewers for providing helpful comments on this paper. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the United States Government. Literature Cited Krysko, K.L., J.C. Nifong, R.W. 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