JOURNAL OF QUATERNARY SCIENCE (2018) 33(8) 958–968 ISSN 0267-8179. DOI: 10.1002/jqs.3073 Nitrogen isotope (d15N) patterns for amino acids in lemur bones are inconsistent with aridity driving megafaunal extinction in south-western Madagascar SEAN W. HIXON,1* EMMA A. ELLIOTT SMITH,2 BROOKE E. CROWLEY,3,4 GEORGE H. PERRY,1,5 JEANNOT RANDRIANASY,6 JEAN FREDDY RANAIVOARISOA,6 DOUGLAS J. KENNETT1,7 and SETH D. NEWSOME2 1Department of Anthropology, Pennsylvania State University, State College, PA, USA 2Department of Biology, University of New Mexico, Albuquerque, NM, USA 3Department of Geology, University of Cincinnati, Cincinnati, OH, USA 4Department of Anthropology, University of Cincinnati, Cincinnati, OH, USA 5Department of Biology, Pennsylvania State University, State College, PA, USA 6Department of Biological Anthropology and Sustainable Development, University of Antananarivo, Madagascar 7Institutes for Energy and the Environment, Pennsylvania State University, State College, PA, USA Received 21 February 2018; Revised 25 September 2018; Accepted 3 October 2018 ABSTRACT: Most endemic species with body masses >10 kg on Madagascar went extinct within the past 1000 years. The extent to which human predation, anthropogenic landscape transformation and aridification may separately or together explain this extinction pattern remains controversial. We present nitrogen isotope (d15N) values of individual amino acids preserved in bones from now-extinct Pachylemur insignis and extant Propithecus verreauxi from two subfossil sites in south-western Madagascar: Tsirave and Taolambiby. The amino acid-specific approach allows us to identify environmental signals that are otherwise difficult to recognize in bulk collagen d15N values. Specifically, we use the d15N values of source amino acids (phenylalanine and lysine) as a proxy for habitat aridity between ca. 4000 years ago and present and the spacing of d15N values between trophic and source amino acids to quantify trophic levels for these two lemur species. Despite paleoenvironmental evidence for lowering water tables and the expansion of relatively arid savanna between 4000 and 1000 years ago, our isotope data suggest that these lemurs did not live in increasingly arid habitats and did not change their trophic level. Together, our results support the hypothesis that aridity alone did not play a major role in late Holocene megafaunal extinctions in south-western Madagascar. # 2018 John Wiley & Sons, Ltd. KEYWORDS: glutamic acid; habitat baseline; habitat modification; lysine; phenylalanine; trophic level. Introduction Humans and their associated domesticated animals (e.g. dogs, cattle, goats and sheep) spread across the island during Controversy exists regarding the drivers of past and ongoing the past 1000 years (Verin and Battistini, 1971; Dewar, 1984; extinctions of Madagascar’s endemic fauna. Some scenarios Wright et al., 1993). Landscape modification, disease and direct attribute species loss to human activities such as hunting, interspecific interactions associated with this spread may have landscape burning and the introduction of invasive species to contributed to the decline of endemic taxa (Battistini, 1971; the island (Dewar, 1984; Virah-Sawmy et al., 2016), but MacPhee, 1997; Godfrey and Jungers, 2003; Burney et al., climate change in the form of aridification may have also 2004; Crowley, 2010; Burns et al., 2016; Crowley et al., 2017). played a significant role in this process (Mahe and Sourdat, Additionally, aridification may have contributed to extirpations 1972; Burney et al., 2004; Virah-Sawmy et al., 2010). South- (Battistini, 1971; Mahe and Sourdat, 1972; Burney et al., 2004). western Madagascar is an ideal region to study late Holocene A paleohydrological record from Lac Ihotry, south-western ecological change, because this region has a high diversity of Madagascar (Figs 1 and 2; Supporting Information, Fig. S1) endemic species and some of the earliest evidence for reveals that the arrival of both humans and bovids coincided humans on the island (Perez et al., 2003; Burney et al., 2004; with a drying trend that included a lowering of the water table Douglass and Zinke, 2015). Early human colonists, who were and salinization of coastal pans and wetlands between roughly present in south-western Madagascar by at least 2300 calen- 2250 and 700 cal a BP (Vallet-Coulomb et al., 2006). dar years before present (cal a BP, MacPhee and Burney, Palynological changes in the sediment record of the saline 1991; Perez et al., 2005), encountered a diverse endemic coastal basin of Ambolisatra, south-western Madagascar (Fig. 1), megafauna that included pygmy hippos, elephant birds, and a also suggest aridification during the past several thousand years wide array of giant lemur species (Burney et al., 2004; (Burney, 1993; Virah-Sawmy et al., 2016) as do contemporane- Crowley, 2010). Most of these species went extinct after ous declines in the abundance of aquatic birds at coastal 1000 cal a BP (Crowley, 2010), and, despite oral histories subfossil sites such as Belo-sur-Mer (Goodman and Rakotozafy, and European accounts that describe relatively recent sight- 1997). These data provide support for the possibility that the ings of now-extinct megafauna (e.g. Burney, 1998; De combination of human activities and climate change triggered Flacourt, 2007), the most recent credible radiocarbon dates species declines (Burney et al., 2004; Clarke et al., 2006; Virah- > for endemic animals with body mass 10 kg are ca. 500 cal Sawmy et al., 2010). a BP (Simons, 1997; Muldoon et al., 2009). Although a variety of environmental factors influence the d15 Ã nitrogen isotope ( N) values of soil and overlying terrestrial Correspondence: Sean W. Hixon, as above. plants, moisture availability is one of the most important E-mail: [email protected] # 2018 John Wiley & Sons, Ltd. MEGAFAUNAL EXTINCTION IN SOUTH-WESTERN MADAGASCAR 959 habitats during the Holocene. Rather, the authors hypothe- sized that agropastoralism probably put a greater strain on endemic fauna compared with aridification. However, the possibility that past aridification acted in concert with negative interactions between introduced and endemic spe- cies to drive megafaunal extinctions could not be excluded. Interpretation of bulk collagen d15N values is complicated by the fact that these values are influenced by both trophic level (TL) and the aridity-dependent isotopic baseline (d15N values in primary producers in a given habitat). Animals have higher d15N values than their diet due to physiological processes that discriminate against 14N; d15N offsets between consumer bulk collagen and diet D15 ‰ ( Nconsumer-diet) are 2–5 (Vanderklift and Ponsard, 2003). Even when the TL of an organism can be assumed with reasonable accuracy, there exists uncertainty regarding D15 the magnitude of Nconsumer-diet, because this value can vary significantly among taxa and is affected by diet quality as well as physiological condition (Vanderklift and Ponsard, 2003; Herrera et al., 2006; Miron et al., 2006; Ramı´rez and Hobson, 2006). Omnivorous lemurs have a greater potential for variable TL than obligate grazers or folivores. Crowley et al. (2017) made reasonable assumptions regarding the TL of various terrestrial vertebrates in Madagascar, and differ- ences in diet can explain much of the variability in measured d15N values among taxa. Nevertheless, there remains the possibility that shifts in either TL or baseline d15N values d15 ( Nbaseline) over time could have contributed to the observed changes in bulk collagen d15N values, which complicates interpretation of the data. Figure 1. Map of south-western Madagascar (Laborde projection). The search for a climate-related signal can be expanded by Sites that produced bones analyzed in this study (Tsirave and measuring d15N values of individual amino acids in bone Taolambiby/Beza Mahafaly) are marked with squares. Other relevant collagen. There are significant differences in D15N sites (black circles) include important paleoecological sites such as consumer-diet Belo-sur-Mer (Goodman and Rakotozafy, 1997), Lac Ihotry (Vallet- values among individual amino acids in terrestrial organisms Coulomb et al., 2006) and Ambolisatra (Burney, 1993; Virah-Sawmy (Edgar Hare et al., 1991; Chikaraishi et al., 2011). For et al., 2016). example, glutamic acid (Glu) has consistently elevated d15N in consumer tissues relative to those in diet, and it is (Austin and Vitousek, 1998; Schulze et al., 1998; Handley consequently considered a trophic amino acid (AA, Chikar- et al., 1999; Amundson et al.,2003;Craineet al., 2009). aishi et al., 2011). Conversely, phenylalanine (Phe) in Preferential loss of 14N from the inorganic soil nitrogen consumers is isotopically similar to that in primary producers, pool due to nitrification, denitrification and ammonia and is consequently considered a source AA (Chikaraishi 15 d15 volatilization results in N-enriched soil nitrogen under et al., 2011). Thus, NPhe values in any consumer will d15 arid conditions (Austin and Vitousek, 1998). Spatial or resemble the NPhe at the base of the food web in its habitat d15 d15 temporal patterns in the N values of primary producers ( Nbaseline), and the TL of an organism can be approximated cascade up the food chains. Thus, animals that live in based partly on the offset in d15N values between trophic AA relatively arid habitats tend to have high d15N values (typically glutamic acid) and source AA (typically phenylala- relative to those at similar trophic levels that live