Molothrus ater (Brown-headed Cowbird) Lays Egg in Artificial Nest in Highlands County, Florida Katherine K. Thorington1,2,*, Reed Bowman1, and Robert Fleischer3 Abstract - On April 28, 2001, we found an unidentified egg in an artificial nest in Highlands County, FL. The artificial nests used in this study contained one Coturnix japonica (quail) egg and one tethered clay sham egg. The unidentified egg was added to the nest between days 12 and 18 of exposure. The quail egg was unmarked, but the sham was covered in small beak markings suggesting that a bird had manipulated it. Molothrus ater (Brown-headed Cowbirds) and M. bonariensis (Shiny Cowbirds) occur in the area, but Brown-headeds are far more common; however, we never observed either species near this particular artificial nest. An mtDNA sequence (300 base pairs of cytochrome b) amplified from this egg was compared to DNA sequences from GenBank, and we found a nearly perfect sequence match with the Brownheaded Cowbird. Parasitism by these birds in unattended and artificial nests is unusual, especially when host activity is not mimicked by researchers. This is the first record of Brownheaded Cowbird parasitism in Highlands County. Molothrus ater Boddaert (Brown-headed Cowbirds, hereafter cowbirds) have substantially expanded their range with anthropogenic changes to North American forest structure (e.g. Cruz et al. 1998, Johnsgard 1997). They reached Florida in the early 1950s and spread south through the peninsula. The current breeding population occurs in the panhandle and along the west coast of the peninsula, although there are isolated inland breeding records including those from De Soto, Glades, and Polk counties (Cruz et al. 2000). Cowbirds are brood parasites and have been documented laying their eggs in the nests of over 220 bird species (Friedmann et al. 1977, Jobin and Picman 1994), although open-cup nesting passerines are their preferred hosts (Friedmann et al. 1977). Parasitism occurs more often in disturbed or fragmented habitats than in continuous tracts of similar cover types (Chace et al. 2005, Hersek et al. 2002, Wilcove 1985). Cowbird reproductive success depends on the females’ ability to find an acceptable host nest, the frequency with which the host species rejects foreign eggs, and the timing of parasitism in relation to host-laying. Female cowbirds watch potential hosts for cues to nest location and stage (Clotfelter 1998, Hauber 2001). Cowbird parasitism of unattended nests is rarely observed, although it does occur and may be more prevalent in areas lacking perching sites from which female cowbirds can watch host activity (Freeman et al. 1990; Thompson and Gottfried 1976, 1981). Experimental field studies using artificial bird nests have been employed to test a variety of nest-predation hypotheses (Major and Kendal 1996, Robinson et al. 2005). Many of these studies have been conducted in areas where cowbird parasitism is common, but parasitism of artificial nests is rarely observed, probably because these nests lack appropriate cues (i.e., parental activity). In Canada, Jobin and Picman (1994) described one instance of a cowbird laying an egg in an unattended artificial nest during a study of marsh-nesting birds. In studies using real, rather than artificial nests, some parasitism was observed (4–7% of all nests) when host activity was simulated by adding a real egg to nests each day (Lowther 1979; Thompson and Gottfried 1976, 1981). In contrast, no incidents of parasitism were recorded when experimental nests were placed in the study area with a complete “clutch” (Thompson and Gottfried 1976, 1981). Further, in a similar study, in which ceramic artificial eggs were added to artificial nests, no parasitism occurred (Yahner and DeLong 1992). Despite infrequent observations, cowbirds do make 2007 Notes 559 560 Southeastern Naturalist Vol. 6, No. 3 poor host-nest choices (Friedman et al. 1977) where the probability of their eggs hatching is low. For example, Freeman et al. (1990) studied 1325 Agelaius phoeniceus L. (Red-winged Blackbird) nest records from four breeding seasons in eastern Washington and found that 28 of 130 parasitized nests were parasitized after young had fledged or been depredated. Thus, in both artificial and natural nest situations, Brown-headed Cowbirds do occasionally make bad laying decisions. From April to June of 2001, we conducted an experimental nest study to examine variation in nest predation rates across a housing density gradient in a residential suburb of Lake Placid, Fl (Highlands County). At this site, native xeric oak scrub (Myers 1990) occurred in a variety of patch sizes in a heterogeneous mosaic of cleared lots and residential homes (see Thorington and Bowman [2003] and Bowman and Woolfenden [2001] for a detailed description of the study area). For the purposes of this study, we grouped house density as low (< 20 houses/40 ha), medium (21–39 house/40 ha), or high (> 40 houses/40 ha). We placed 120 artificial nests (40 per Figure 1. All three eggs in the artificial nest (canary nest basket) in Highlands County, FL. The cowbird egg is the small egg at the bottom left, the quail egg is at the top, and the clay sham is at the bottom right. 2007 Notes 561 density treatment) in patches of native scrub habitat. Each nest contained one Coturnix japonica Temminck and Schlegel (Coturnix Quail) egg and one tethered clay sham egg. On April 28, 2001 at 9:00 am, we checked a nest located in the medium housing density area and noted that the quail egg and clay sham were still present, but that a third egg was present (Fig. 1). The quail egg was unmarked, but the clay sham had several shallow beak marks, suggesting it had been pecked. The new egg was much smaller than either the quail egg or the clay sham. It was similar in appearance to a cowbird egg, but differed in that the spotting appeared dense, the base color was dark, and the egg was relatively narrow (21.8 x 14.6 mm; Fig. 2). However, cowbird eggs are highly variable in color, pattern, and size (Baicich and Harrison 1997, Bent 1958). The nest had originally been deployed on April 10, and thus was exposed to potential predation or parasitism for 18 days. The nest was checked on day 6 and 12, at which time the contents were undisturbed, and no additional eggs were observed. The parasitized nest was one of only 6 nests on the medium-density transect that had not been depredated by day 12. By day 18, however, 2 had been depredated, the above nest had been parasitized, and the remaining 3 appeared undisturbed. The parasitized nest was located in a Lyonia spp. shrub covered in Vitas spp. and Smilax spp. vines. The nest was placed 1.2 m above the ground and was hidden by Vitas and Smilax leaves. Although we did not specifically quantify nest concealment, this nest was more exposed than some, but not as exposed as others that were placed near trails or other habitat disturbances. The shrub in which this artificial nest was placed was in unmowed vegetation approximately 15 m from a mowed road berm. Figure 2. Close-up of the cowbird egg. Scale is in centimeters and millimeters. 562 Southeastern Naturalist Vol. 6, No. 3 Mitochondrial DNA was extracted from the egg using standard phenol-chloroform extraction protocols followed by centrifugal dialysis (Fleischer et al. 2000). A 300-base pair sequence of the Cytochrome-b gene was amplified from the purified DNA by PCR using primers Cytb1, Cytb2, Cytb-wow, and Cytb2rc (Dumbacher et al. 2003). Cleaned products were sequenced using standard ABI protocols and run on an ABI 3100 automated capillary sequencer. Sequences were aligned using Sequencher 4.1 (Lifecodes), and used in a blast search of GenBank. Other than a single ambiguity in the unknown egg sequence (0.0% divergence), all bases matched Brown-headed Cowbird cytochrome-b sequence AF290172. The other likely brood parasite in Florida is M. bonariensis Gmelin (Shiny Cowbird), which has spread into southern Florida from South America and the West Indies (Cruz et al. 2000); however, 7 of 300 base pairs (2.3% divergence) differed between the Cytochrome-b sequences of the unidentified egg and that of Shiny Cowbirds. Based on the appearance of the egg in the nest, its physical description and measurements, and its cytochrome-b sequence, we conclude this was a rare record of a Brown-headed Cowbird laying in an artificial nest (see Jobin and Picman 1994, Lowther 1979, Thompson and Gottfried 1981, and Yahner and DeLong 1992 for other records). This parasitism event was unusual in that we did not mimic host activities, we added all eggs on the first day, and we limited nest checks to minimize attraction of potential predators to the nest. These activities appear to have influenced parasitism of experimental nests in previous instances where it has been noted. Finally, this is the first breeding record of a Brown-headed Cowbird in Highlands County, FL. As such, it confirms the continued spread of this brood parasite throughout peninsular Florida. Ecological consequences may include reduced breeding success of resident passerines due to the lack of nest defense strategies (but see Fleischer and Woolfenden 2004). Acknowledgments . We thank Liza Merly, Craig Carter, and Arthur Fleischer for assistance in the field. Previous versions of the manuscript were improved by comments from Richard W. Thorington, Jr., Jesse Barber, and Peter D. Weigl. Archbold Biological Station supported K. Thorington with an undergraduate internship. Research in our suburban study area was supported by Archbold Biological Station and by NSF grant IBN-00777469 to R. Bowman. Lab expenses were provided by the Smithsonian Institution. Literature Cited Baicich, P.J., and C.J.O. Harrison. 1997. A Guide to the Nests, Eggs, and Nestlings of North American Birds. 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