Dietary isotopes of Madagascar's extinct megafauna reveal Holocene browsing and grazing guilds

Abstract

Megafauna play a disproportionate role in developing and maintaining their biomes, by regulating plant dispersal, community structure and nutrient cycling. Understanding the ecological roles of extinct megafaunal communities, for example through dietary reconstruction using isotope analysis, is necessary to determine pre-human states and set evidence-based restoration goals. We use δ¹³C and δ¹⁵N isotopic analyses to reconstruct Holocene feeding guilds in Madagascar's extinct megaherbivores, which included elephant birds, hippopotami and giant tortoises that occurred across multiple habitats and elevations. We compare isotopic data from seven taxa and two elephant bird eggshell morphotypes against contemporary regional floral baselines to infer dietary subsistence strategies. Most taxa show high consumption of C₃ and/or CAM plants, providing evidence of widespread browsing ecology. However, Aepyornis hildebrandti, an elephant bird restricted to the central highlands region, has isotope values with much higher δ¹³C values than other taxa. This species is interpreted as having obtained up to 48% of its diet from C₄ grasses. These findings provide new evidence for distinct browsing and grazing guilds in Madagascar's Holocene megaherbivore fauna, with implications for past regional distribution of ecosystems dominated by endemic C₄ grasses.

1. Introduction

Late Quaternary and older terrestrial ecosystems were typically dominated by megaherbivores, which shaped their environments through top–down interactions with plant communities and vegetation structure [1,2]. Megaherbivores impact diversity and structure of ecosystems by suppressing plant growth through physical disturbance and herbivory, influencing nutrient cycling within and between landscapes, and dispersing plant propagules [3,4]. However, megafauna have been disproportionately vulnerable to human-caused extinction, and many ecosystems now lack these keystone species [5]. Reconstructing the ecology of now-extinct megaherbivore guilds is essential to identify disrupted ecological processes and guide environmental management and restoration [1,6].

Late Quaternary Madagascar supported a diverse megafauna, including elephant birds, hippopotami and giant tortoises. Madagascar's megaherbivores became extinct in the late Holocene during a period of intensive anthropogenic forest clearance and conversion to open habitats around 1100–1000 BP [7–9]. Today 80% of Madagascar is covered by grassland [10,11], but the pre-disturbance distribution and extent of native grasslands remain uncertain [12,13]. Madagascar contains native grass lineages dating from the Miocene, and 40% of its grass species are unique; it contains among the world's highest grass diversity and endemism, with particularly diverse assemblages in the island's central highlands ecogeographical region [10,11]. However, there is limited evidence for regional existence of a late Quaternary vertebrate grazing guild, suggesting that endemic grasses may have been limited to small, low-density clearings [13–16].

Most trees, shrubs and herbs use a C₃ (Calvin) metabolic pathway for carbon fixation during photosynthesis, whereas most tropical grasses use a C₄ (Hatch-Slack) pathway [17]. Stable carbon isotope (δ¹³C) values in bones of animals that consumed these plants can indicate likely former presence of forests or grasslands, and isotope analysis is widely used for late Quaternary palaeoecological reconstruction [14]. Animals with pure C₃ diets have δ¹³C values below −21.5‰ and pure C₄ diets above −9‰. Reported δ¹³C values for Madagascar megaherbivores are interpreted as indicating forest environments [18–20]. However, some Madagascar megaherbivore subfossil sites are interpreted as open grassland biomes [21,22]. Using δ¹³C values to infer open-habitat grasses as dietary resources can be confused by plants using crassulacean acid metabolism photosynthesis (CAM plants); for example, the succulent plant Kalanchoë exhibits flexible CAM patterns across Madagascar, with δ¹³C values similar to C₃ plants in humid environments and to C₄ plants in dry environments [23]. The CAM-specialist extinct lemur Hadropithecus shows δ¹³C values of −24.2‰ in the mesic central highlands, and −9.6‰ in the arid southwest [14]. However, wetland C₄ plants (rushes, sedges) also occur in Madagascar, so comparison of δ¹⁵N values between co-occurring taxa can also be included in dietary assessments using δ¹³C data; plants in arid environments have higher δ¹⁵N values [24], enabling differentiation between wetland and dryland C₄ plants.

Research into Madagascar's extinct vertebrate ecology has mainly focused on giant lemurs, with studies of megaherbivores hindered by poorly resolved taxonomy [25]. However, recent taxonomic reassessments have clarified species diversity in elephant birds [26] and hippopotami [27], enabling the investigation of species-specific niches and landscape ecology. Here we investigate new and published dietary isotope data for all Madagascar hippopotamus and elephant bird species and for the regionally extinct giant tortoise Aldabrachelys across three distinct ecogeographical zones, to determine megaherbivore dietary niches and presence of natural open grassland habitats (e.g. savannahs, open wooded habitats) in Madagascar's late Quaternary ecosystems [25].

2. Material and methods

We assembled a database of 203 δ¹³C and 118 δ¹⁵N values for late Quaternary skeletal elements of all recognized Madagascar hippopotami (Hippopotamus lemerlei, n = 15; H. madagascariensis, n = 6), elephant birds (Aepyornis hildebrandti, n = 8; A. maximus, n = 3; Mullerornis modestus, n = 9; Vorombe titan, n = 11) and Aldabrachelys sp. (n = 19), and for both elephant bird eggshell morphotypes (‘thin eggshell’ representing M. modestus, n = 9; ‘thick eggshell’ representing Aepyornis or Vorombe, n = 93; [28]) (table 1). We include 243 published values (160 δ¹³C, 83 δ¹⁵N), and previously unreported data for 42 specimens from accelerator mass spectrometry (AMS) dating of bone collagen [9] performed at the Oxford Radiocarbon Accelerator Unit (ORAU). In total, 86 samples have associated radiocarbon dates, with just one predating the Holocene (NIUTSM 01539: 14 580 ± 460 BP). Collection localities cover three Madagascan ecoregions: southern arid spiny bush (n = 147, 14 localities), western succulent woodland (n = 25, six localities) and the central highlands (n = 21, four localities) (figure 1; electronic supplementary material, table S1). Ecoregions varied in megaherbivore composition; although Mullerornis occurred in highland and lower-elevation regions, A. hildebrandti and H. madagascariensis were largely/completely restricted to the central highlands, and most other species only occurred in southern and/or western ecoregions [25,30] (table 1). Aldabrachelys data are only available for the arid spiny bush, although specimens are also recorded from the central highlands [28] (table 1).

Table 1.

Mean isotope values and dietary proportion estimates for Madagascar's megaherbivores, inclusive of bone/eggshell correction and Suess correction.

taxon	no. specimens	δ13C (mean)	s.d.	est. diet proportion (mean) C3 plants	est. diet proportion (mean) CAM plants	s.e.
1. Arid spiny bush
Aepyornis maximus	2	−28.42	0.71	0.93	0.07	0.04
Mullerornis modestus (bone)	7	−26.44	0.81	0.79	0.21	0.02
Mullerornis modestus (eggshell)	9	−25.58	0.48	0.73	0.27	0.01
thick eggshell	93	−26.35	0.92	0.79	0.21	0.01
Hippopotamus lemerlei	10	−21.87	2.49	0.48	0.52	0.06
Hippopotamus sp.	18	−22.99	2.33	0.55	0.45	0.04
Aldabrachelys sp.	18	−25.18	2.75	0.71	0.29	0.05
2. Succulent woodland
Aepyornis maximus	1	−28.02	0.71a	0.9	0.1	0.05
Vorombe titan	11	−29.26	0.72	0.99	0.01	0.02
Hippopotamus lemerlei	4	−28.13	0.73	0.91	0.09	0.03
Hippopotamus madagascariensis	3	−20.17	1.56	0.36	0.64	0.06
Hippopotamus sp.	15	−26.07	3.71	0.77	0.23	0.07
Aldabrachelys sp.	1	−33.52	2.75a	1	0	0.19

taxon	no. specimens	δ13C (mean)	s.d.	est. diet proportion (mean) C3 plants	est. diet proportion (mean) CAM plants	s.e.
3. Central highlands
Aepyornis hildebrandti	8	−21.12	1.42	0.52	0.48	0.03
Mullerornis modestus (bone)	1	−28.22	0.81a	0.98	0.02	0.06
Hippopotamus madagascariensis	3	−28.27	3.25	0.99	0.01	0.12
Hippopotamus sp.	13	−27.83	4.29	0.96	0.04	0.08

^as.d. not available and arid spiny bush value used.

Figure 1.

Madagascar ecoregions, showing localities for specimens in this study. Adapted from [29].

We assessed dietary sources using mixing models in ISSOERROR v. 1.04 [31] to investigate dietary niche differentiation between taxa within ecoregions. We calculated proportionate consumption of C₃ versus CAM plants in the more arid southern and western ecoregions, and C₃ versus C₄ plants in the comparatively wet central highlands, which do not support significant CAM plant biomass and contain a regionally restricted endemic C₄ plant community [10,11,14,15]. We used δ¹³C isotope values for discrimination model end-members from ref. [32]: C₃ plants, arid spiny bush and succulent woodland (Beza Mahafaly), −29.4‰ (σ: 2.4, n = 240); central highlands (Tsinjoarivo): −28.5‰ (σ: 1.8, n = 49); CAM plants, arid spiny bush and succulent woodland (Beza Mahafaly): −15‰ (σ: 1.2, n = 67). C₄ grass end-member values used the global mean value of −13.1‰ [33], with substituted σ and sample size from central highlands C₃ plants. δ¹³C values used in fractionation were corrected to account for δ¹³C enrichment in bone (+5‰) [34] and eggshell (+2‰) collagen [35], and by +1.22‰ to account for δ¹³C shifts in atmospheric CO₂ (Suess effect; [36]).

3. Results

In arid spiny bush, elephant birds and giant tortoises show low δ¹³C values (species means: −25.18 to −28.42‰), with dietary fractionation indicating these taxa all consumed mainly C₃ plants and only limited amounts of CAM plants (mean estimated proportions of CAM consumption: 0.07–0.27). M. modestus (bone and thin eggshell) shows highest estimated CAM consumption (sample means: 0.21–0.27). Hippopotami (H. lemerlei and samples unidentified to species) show higher δ¹³C values (sample means: −21.87 to −22.99‰) and correspondingly much higher estimated proportions of CAM consumption, with C₃ and CAM plants both comprising about half of their diet (mean estimated proportions, C₃: 0.48–0.55, CAM: 0.45–0.52) (table 1 and figure 2; electronic supplementary material, file S1).

Figure 2.

Observed isotope ‰ for Madagascar's megaherbivores: (a) δ¹³C values. (b) δ¹³C and δ¹⁵N values. Key: green star, Aepyornis hildebrandti; pink triangle, A. maximus; orange square, Mullerornis modestus (bone); blue triangle: Vorombe titan; open square: thin eggshell (M. modestus); open triangle: thick eggshell (Aepyornis or Vorombe); blue diamond: Hippopotamus lemerlei; purple cross: H. madagascariensis; open diamond: Hippopotamus sp.; filled circle: Aldabrachelys sp.

In succulent woodland, elephant birds (A. maximus, V. titan), giant tortoises and H. lemerlei show low δ¹³C values (species means: −26.46 to −33.52‰) and very low estimated proportions of CAM consumption (0.01–0.09). By contrast, H. madagascariensis shows high δ¹³C values (species mean: −20.16‰) and much higher mean estimated CAM consumption (0.64).

In the central highlands, M. modestus and hippopotami (H. madagascariensis and samples unidentified to species) show high δ¹³C values (sample means: −28.22 to −28.83‰), and are estimated to have consumed almost entirely C₃ plants and minimal C₄ grasses (mean estimated proportions of C₄ consumption: 0.02–0.04). Conversely, A. hildebrandti shows high δ¹³C values (sample mean: −21.12‰) and much higher mean estimated C₄ consumption (0.48).

For hippopotami, H. madagascariensis had δ¹⁵N values of 3.0–5.2‰, and H. lemerlei of 7.0–13.3‰. The lowest hippopotamus δ¹⁵N value was from Antsirabe, central highlands (1.3‰) and the highest was from Beloha, arid spiny bush (13.3‰). For elephant birds, A. hildebrandti had δ¹⁵N values of 5.0–7.8‰, A. maximus of 11.8–15.3‰, M. modestus of 6.4–16.0‰, V. titan of 5.5–13.7‰ and ‘thick eggshell’ of 8.7−17.6‰. Aldrabrachelys in arid spiny bush had values of 8.4–13.3‰. Across ecoregions, non-overlapping δ¹⁵N ranges were observed for single measurements in A. maximus (dry deciduous forest, 11.8‰; arid spiny bush, 12.7–15.3‰) and M. modestus (central highlands, 6.4‰; arid spiny bush, 6.5–15.8‰).

4. Discussion

We present the first species-level dietary niche reconstruction for Madagascar's megaherbivores, revealing a range of δ¹³C and δ¹⁵N values and trophic ecologies across taxa and ecoregions. Our findings indicate the former existence of multiple herbivore guilds across Madagascar. These data support previous identification of a widespread browsing guild and provide the first direct evidence for a megaherbivore grazing guild in Madagascar's central highlands.

δ¹³C values in hippopotamus samples indicate broad trophic niches for both species, suggesting both browsing and grazing behaviours. This contrasts with the mainland African hippopotamus (H. amphibius), which is predominantly a terrestrial grazer [37]. Madagascar hippopotami were trophically closer to Africa's extant pygmy hippopotamus (Choeropsis liberiensis), which is comparable in size to Madagascar's extinct species, and browses on forest plants [38]. Interestingly, δ¹⁵N ratios indicate more aquatic feeding in H. madagascariensis than H. lemerlei. Aquatic habitats were available for H. madagascariensis in the central highlands [25]. This result contrasts with aquatic adaptations inferred from cranial morphology in H. lemerlei [39], but is consistent with behavioural ecology (emergence onto land for feeding) in the otherwise aquatic H. amphibius, suggesting a similar lifestyle for H. lemerlei. Aldabrachelys isotopes from arid spiny bush show a comparable δ¹³C/δ¹⁵N signal to H. lemerlei, indicating a similar browsing niche in this ecoregion.

CAM plants comprised a substantial proportion of the diets of one or both hippopotamus species in arid spiny bush and succulent woodland, but δ¹³C values are lower in the central highlands, suggesting higher reliance upon C₃ plants. CAM plants are relatively scarce in this region; however, they occur across numerous biomes and elevations in Madagascar, with a range of δ¹³C values (e.g. Kalanchoë, −11.4 to −27.3‰) [23]. CAM plant CO₂ is fixed by the C₃ pathway in humid environments such as the central highlands, producing δ¹³C values below −22‰ [23] and thus consistent with CAM consumption in this region as well. Hippopotami therefore probably consumed C₃ and CAM plants across Madagascar, matching the varying δ¹³C pattern in the CAM-specialist Hadropithecus across different ecoregions [14].

Elephant bird δ¹³C values from arid spiny bush and succulent woodland fall outside the range for C₄ consumption (open-habitat dryland grasses or wetland sedges and rushes). In arid spiny bush, δ¹³C values indicate that all elephant birds had predominantly C₃ diets, with some CAM consumption by M. modestus; higher δ¹⁵N values compared to sympatric hippopotami indicate that these plants were less likely to be from wetlands. Differences between sympatric elephant birds may indicate further species-specific dietary differences; for example, higher δ¹⁵N values (e.g. in A. maximus) are associated with frugivory or omnivory (including invertebrate or small vertebrate consumption) [32], which comprise extant ratite dietary strategies [40]. Eggshell and bone values also differ in M. modestus, possibly indicating seasonal reliance upon dietary resources during oogenesis, or that eggshell and bone fractionation rates may need separate assessment.

Our most striking result is that δ¹³C data for A. hildebrandti provide the first evidence for grazing ecology in elephant birds. Although unique within Madagascar's ratites, grazing is also the primary dietary strategy in greater rhea (Rhea americana) [41], and other large flightless birds (e.g. geese) also have important regulatory effects on island grasslands [42]. Our results thus identify A. hildebrandti as a likely top–down regulator of native grassland ecosystems in the central highlands [13,15,16]. δ¹³C values for this species indicate a mixed diet containing large quantities of C₄ plants (c. 48%), whereas co-occurring hippopotami consumed only tiny amounts of C₄ plants (1–4%). Although A. hildebrandti had higher mean δ¹⁵N values compared to sympatric hippopotami, this disparity is much lower than between species in other ecoregions. Indeed, lower δ¹³C values in CAM plants within mesic conditions [23] suggest that A. hildebrandti might not have consumed any forest plants and was exclusively an open-habitat forager, consuming a mixture of C₄ and CAM plants. High variability in CAM plant δ¹³C values complicates interpretation of results, but the likelihood of A. hildebrandti exhibiting grazing behaviour is supported by the non-matching regional δ¹³C signature of the CAM specialist Hadropithecus (mean: −24.2 δ¹³C‰) [14]. This hypothesis is consistent with the small olfactory bulb in skulls assigned to A. hildebrandti, comparable to the neuroanatomy of extant open-habitat palaeognaths [43]. However, the taxonomic identity of these crania is uncertain; they are not associated with diagnostic postcrania or locality data, and two separate skull morphotypes have been referred to A. hildebrandti [44–46].

δ¹³C data from skeletal collagen provide a comprehensive new understanding of Madagascar megaherbivore dietary ecology. Most available subfossils originate from southern Madagascar, and further research should investigate data across wider areas. For example, giant tortoises from the central highlands remain isotopically unstudied; these animals might also have been grazers, but their shell shape (associated with biomechanical advantage for grazing or browsing in extant species [47]) is poorly understood, making ecological inference difficult. However, whereas most modern-day open habitats on Madagascar are anthropogenic in origin, our results provide important evidence for former existence of native ecosystems dominated by endemic C₄ grasses. It is clear that Madagascar supported multiple megaherbivore trophic guilds with differing relationships to native vegetation, which must have played important roles in regulating diverse natural landscapes. Madagascar's ecosystems are now highly degraded, and protection and sustainable management of landscapes and ecosystem services represents a global priority for biodiversity conservation and human well-being [48,49]. Hypotheses of what constitutes a ‘natural’ Madagascar ecosystem must therefore consider the ecologies and regulatory roles of the island's now-extinct megafauna, to support evidence-based restoration of this ecologically complex island.

Data accessibility

All data are available in electronic supplementary material, file S1 [50].

Authors' contributions

J.P.H.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, validation, visualization, writing—original draft, writing—review and editing; S.T.T.: Funding acquisition, investigation, supervision, visualization, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Competing interests

The authors have no competing interests.

Funding

This study was supported by a NERC Radiocarbon Facility grant no. (NF/2015/1/4) to S.T.T./J.P.H., and Research England.