Why all those spines?: Anachronistic defences in the Didiereoideae against now extinct lemurs

Abstract

Plants evolve physical defences, such as spines, against browsing herbivores. However, in some cases, these defences may be anachronistic because the principal consumers of protected parts of the plant are extinct. In such cases, there may be few extant species consuming heavily defended resources. Here we examine the spiny defences of Madagascar's endemic Didiereoideae, and ask whether they may be anachronistic. To accomplish this aim, we reviewed the literature to determine which species consume these plants today, and then used stable isotope biogeochemistry to determine who may have exploited Didiereoideae in the recent past. There are four major groups of browsers that are now extinct in Madagascar: giant lemurs, elephant birds (Aepyornis and Mullerornis: Aepyornithidae), pygmy hippopotamuses (Hippopotamus) and giant tortoises (Aldabrachelys: Testudinidae). Each group was evaluated for isotopic evidence of didiereoid plant consumption. Given the structure of members of this plant clade (especially Alluaudia), we predicted that lemurs would be their most important consumers. Three extant lemur species consume Didiereoideae. Several of the extinct lemurs, particularly Hadropithecus stenognathus, may have relied heavily on these spiny plants. None of the non-lemur megafaunal browsers (elephant birds, hippopotamuses and giant tortoises) were important consumers of Didiereoideae.

Full text

1Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 1 of 7
Why all those spines? Anachronistic defences in
the Didiereoideae against now extinct lemurs
Plants evolve physical defences, such as spines, against browsing herbivores. However, in some cases, these
defences may be anachronistic because the principal consumers of protected parts of the plant are extinct.
In such cases, there may be few extant species consuming heavily defended resources. Here we examine
the spiny defences of Madagascar’s endemic Didiereoideae, and ask whether they may be anachronistic.
To accomplish this aim, we reviewed the literature to determine which species consume these plants today,
and then used stable isotope biogeochemistry to determine who may have exploited Didiereoideae in the
recent past. There are four major groups of browsers that are now extinct in Madagascar: giant lemurs,
elephant birds (Aepyornis and Mullerornis: Aepyornithidae), pygmy hippopotamuses (Hippopotamus) and
giant tortoises (Aldabrachelys: Testudinidae). Each group was evaluated for isotopic evidence of didiereoid
plant consumption. Given the structure of members of this plant clade (especially Alluaudia), we predicted
that lemurs would be their most important consumers. Three extant lemur species consume Didiereoideae.
Several of the extinct lemurs, particularly Hadropithecus stenognathus, may have relied heavily on these
spiny plants. None of the non-lemur megafaunal browsers (elephant birds, hippopotamuses and giant
tortoises) were important consumers of Didiereoideae.
Motivation
Madagascar is renowned for its wealth of endemic flora and fauna. In particular, the arid south and southwest
is famous for its ‘spiny forests’ full of spiny bushes and trees belonging to the Apocynaceae (e.g. Pachypodium
lamerei), Euphorbiaceae (e.g. Euphorbia stenoclada), Fabaceae (e.g. Acacia bellula), Salvadoraceae (e.g. Azima
tetracantha) and Didiereoideae,1 an endemic subfamily of the Didiereaceae.2,3 Indeed, species from the latter
subfamily are limited almost entirely to the Spiny Thicket and Succulent Woodland ecoregions in southern and
southwestern Madagascar, which are characterised by hot temperatures and brief rainy seasons.4
The 12 species of the Didiereoideae belong to four genera: Alluaudia, Alluaudiopsis, Decarya and Didierea. All
members of this subfamily possess sharp, thick spines along their axes which protect their leaves5,6; however,
none of the closely related Didiereaceae from mainland Africa (Calyptrotheca, Ceraria, Portulacaria) possesses
spines.1 Experimental research on plant taxa in mainland Africa has demonstrated that the spines reduce foliage
loss to browsing ungulates.7,8 This protection suggests that the common ancestor of the Madagascan forms
was subjected to intense leaf predation shortly after its arrival. Arakaki and colleagues9 reported a diversification
estimate for Madagascan Didiereoideae of 17 million years ago (mya) based on molecular data. These data imply
an earlier date for the dispersal of the basal didiereoid from continental Africa to Madagascar. According to these
authors, Alluaudia itself began diversifying only 11 mya. Ocampo and Columbus10 suppor t a slightly more recent
radiation of Madagascan didiereoids, with the divergence of the Madagascan lineage from the closest continental
African relative at around 15 mya.
Spines on these tall, emergent plants may be defences against leaf predation by climbing animals such as
lemurs.6 Spines of most Alluaudia spp., for example, are found at heights above the ground (5–9 m) that were
likely prohibitive for terrestrial browsers such as tortoises, hippopotamuses and elephant birds. Whereas it is
conceivable that these taxa browsed juvenile forms or the lower portions of adult plants, widescale herbivory by
tortoises or hippopotamuses seems unlikely. Furthermore, although the ‘wiry’ qualities of Alluaudia humbertii and
Decarya madagascariensis may have provided some defence against elephant bird herbivory,11 their spines are
relatively ineffective against birds and, presumably, other animals with hard beaks that protect their mouths, such
as tortoises.12 Another reason to suspect that the spines on the Didiereoideae evolved to protect leaves against
climbing animals and not against other major groups of herbivores is the timing of arrival of major herbivore groups
to Madagascar. Both hippopotamuses and testudines arrived relatively recently13 – likely after the appearance of
spines and diversification of Madagascan didiereoids. Only lemurs and elephant birds would have been present
when the ancestral didiereoid arrived. If Bond and Silander11 are correct in characterising elephant birds as poorly
suited to exploit the leaves of the Didiereoideae, then lemurs become the most plausible contenders. Additionally,
if few extant lemurs exploit these plants, then the giant extinct lemurs may be implicated.
Ideally, testing the hypothesis that spines served to defend the leaves of the Didiereoideae against giant lemurs
requires more than compiling evidence that certain giant lemurs likely consumed these plants. We would like to
know the degree to which the spines acted as a deterrent to overconsumption of small and vulnerable young leaves
by giant lemurs. The latter question is challenging, at best, within the context of palaeobiology. Palaeontological
evidence is often indirect, and arguments may depend on unspoken assumptions. Thus, it is important to make
explicit the questions that can be addressed with the tools we have at our disposal. How, using those tools, can
plant anachronisms in Madagascar be discerned?
As evolutionary biologists we can ascertain, first, whether or not the Didiereoideae are native or endemic to
Madagascar (i.e. not recently introduced). Secondly, we can establish whether the presumed anachronistic spines
are derived. Thirdly, we can determine whether or not the hypothesised consumers (lemurs) were present when
AUTHORS:
Brooke E. Crowley1,2
Laurie R. Godfrey3
AFFILIATIONS:
1Department of Geology,
University of Cincinnati,
Cincinnati, OH, USA
2Department of Anthropology,
University of Cincinnati,
Cincinnati, OH, USA
3Department of Anthropology,
University of Massachusetts,
Amherst, MA, USA
CORRESPONDENCE TO:
Brooke Crowley
EMAIL:
brooke.crowley@uc.edu
POSTAL ADDRESS:
Department of Geology,
University of Cincinnati,
500 Geology Physics Building,
345 Clifton Court, Cincinnati, OH
45221, USA
DATES:
Received: 24 June 2012
Revised: 16 Aug. 2012
Accepted: 25 Aug. 2012
KEYWORDS:
Madagascar; lemur;
crassulacean acid metabolism;
δ13C; δ15N
HOW TO CITE:
Crowley BE, Godfrey LR. Why
all those spines? Anachronistic
defences in the Didiereoideae
against now extinct lemurs. S
Afr J Sci. 2013;109(1/2), Art.
#1346, 7 pages. http://dx.doi.
org/10.1590/sajs.2013/1346
© 2013. The Authors.
Published under a Creative
Commons Attribution Licence.

2Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 2 of 7
these features likely originated. We can probe whether spines can be
understood outside the context of the proposed plant–animal interactions
and we can examine palaeodistribution data to test the plausibility of the
proposed interactions. Finally, we can explore whether spines serve any
apparent purpose today, or were likely used in the past in a manner that
no longer holds.
Our inferences in this paper are based explicitly on the combination of
a literature review and stable isotope biogeochemistry that addresses
these issues. We ask the following specific questions:
1. To what extent do modern lemurs feed in southern and southwestern
Madagascar on C3, C4 and crassulacean acid metabolism (CAM)-
based plants? How much do they feed on Didiereoideae?
2. Can we distinguish Didiereoideae from other CAM plants using
stable isotopes? Such discrimination is needed if we are to
use stable isotope data to successfully test the hypothesis that
Didiereoideae spines are anachronistic.
3. To what extent were lemurs feeding on CAM plants in the past?
4. Do stable isotopes suggest that any of the extinct non-lemur
herbivores were major consumers of Didiereoideae?
Background on stable isotope biogeochemistry
Stable isotopes can be used to reconstruct the diets of living and extinct
animals. The relative proportion of heavy and light isotopes (e.g. 13C/12C
or 15N/14N) in a substance is reported using a standardised ’δ’ notation
(e.g. δ13C, δ15N). These values are measured as parts per thousand (‰)
higher or lower than an international standard.
Carbon isotope (δ13C) values can, in some cases, be used to
distinguish plants that fix carbon via C3 photosynthesis (most trees
and herbs), C4 photosynthesis (many grasses) and CAM (stem and
leaf succulents).14 Many succulents have the ability to switch between
full CAM photosynthesis and C3 photosynthesis, which can result in
highly variable δ13C values.14 However, in arid environments, such as
those in southwestern Madagascar, carbon fixation is strongly biased
towards CAM photosynthesis.15 Nitrogen isotope (δ15N) values in
plants are affected by environmental conditions, plant physiology,
nutrient availability and microbial associations.14,16 Nitrogen isotope
values clearly distinguish plants growing in different habitats. Plants
from moist, cool localities have lower δ15N values than plants from dry,
warm localities.16,17 Coastal localities can evince exceptionally high δ15N
values.18 Most plants obtain their nitrogen directly from soil nitrate and
ammonium, and their δ15N values are greater than that of air (~0‰).
Plants with symbiotic nitrogen-fixing bacteria can have δ15N values close
to 0‰.16,18 No consistent differences in δ15N have been reported among
the three photosynthetic groups, but CAM plants can have significantly
higher δ15N values than sympatric C3 or C4 plants.18,19 Differences in
plant physiology or differential use of water sources may result in δ15N
differences between the Didiereoideae and non-spiny leaf succulents.20
Isotopic patterns in plants are reflected in animal consumers with some
isotopic enrichment. Carbon and nitrogen isotope values in herbivore
bone collagen tend to be, respectively, ca. 5‰ and 3‰ higher than
those in plants.21 Once we have accounted for isotopic enrichment
between collagen and diet, we may be able to use carbon isotope values
in consumer tissues to estimate the relative ingestion of C3, C4 and CAM
plants. As with plants, nitrogen isotope values in animals can be used
to distinguish habitat types.17,22 Within a particular habitat, δ15N values
increase with increasing consumption of animal matter.21,22 However,
because most of the lemur species included in our analyses are, or
were, predominantly herbivorous, we do not believe our results are
confounded by the effects of faunivory on nitrogen isotope values.23-32
The two possible exceptions are Archaeolemur majori (whose diet
likely included some animal matter) and Daubentonia robusta. Extant D.
madagascariensis consumes more animal matter than any of the other
lemurs included in our study33 and it is likely that the extinct D. robusta,
which lived in the southwest, would have had a similar diet.34 If this
species consumed insects that in turn fed on CAM resources, elevated
δ15N isotope values might falsely suggest CAM consumption, when in
fact they really reflect trophic omnivory. We therefore omitted D. robusta
from our analyses.
Methods
To explore the extent to which modern lemurs feed on C3, C4 or
CAM plants, we conducted a thorough review of the literature. We
examined 74 manuscripts, books and book chapters that discuss the
feeding behaviour of living lemurs in southwestern Madagascar. All
sources included in our survey are listed in Supplementary table 1 (see
supplementary material online), and all documented observations of
feeding on CAM are provided in Supplementary table 2. We used the
website www.tropicos.org and Petitjean and colleagues35 to identify
scientific names and families for recorded food species. Succulence was
assessed using species-specific isotopic or anatomical data whenever
possible.36-41 If no data were available for particular species, we used
published information at the generic or familial level.42,43
To determine if the spiny Didiereoideae can be isotopically distinguished
from other plants, we compared δ13C and δ15N values from leaves
that we collected in the spiny forest at Beza Mahafaly Special Reserve
(BMSR) in south-central Madagascar (taxa provided in Table 1). We
included previously published isotope values for C3 and CAM plants17
and new isotope values for C4 plants. All plant specimens were collected
between 2006 and 2009. We sampled Alluaudia procera, which is the
only member of the Didiereoidea that occurs in abundance at the reserve.
Alluaudia is both the most speciose and the most widespread didiereoid
genus. Additionally, although carbon isotope values have been measured
for a number of spiny CAM species,10,37,41 nitrogen isotope data have
been published only for Alluaudia procera.17 We used an analysis of
variance (ANOVA) with Tukey’s tests of honestly significant differences
(HSD) and Student’s t-tests to test the significance of isotopic differences
between C3, C4 and CAM plants, and between Alluaudia and other CAM
plants. All statistical tests were performed using JMP (version 7.0).
Significance was set at α = 0.05. We assessed the assumptions of
normality and homoscedasticity of variance for all analyses. We tested
for homogeneity of variances using Levene tests.
To address the extent to which lemurs and non-primate herbivores fed on
CAM plants in the past, we used δ13C and δ15N values from bone collagen.
We analysed 72 bones of extant and extinct lemurs as well as extinct giant
tortoises and pygmy hippopotamuses from subfossil sites in the Spiny
Thicket and Succulent Woodland ecoregions (coastal and inland). Collagen
was prepared following previously published methods.44 Samples were
analysed at the Stable Isotope Laboratory at the University of California,
Santa Cruz. We verified collagen preservation using collagen yield, atomic
C:N ratios, and carbon and nitrogen isotope values. We added these data
to our existing database of previously published isotope data.45-48 Raw
isotope data for all individuals are presented in Supplementary table 3.
Carbon isotope values for subfossil individuals were corrected to account
for δ13C shifts in atmospheric CO2 following the industrial revolution (The
Suess Effect).45 Carbon isotope values for individuals younger than 150
years BP were corrected using an age-dependent correction of -0.004‰
per year between 1860 and 1965 AD and -0.02‰ per year between 1965
and 2005 (modern). All individuals older than 150 years were corrected by
-1.2‰. In order to avoid sampling bias, we used nonparametric Wilcoxon
signed ranks tests to compare mean δ13C values for subfossil extant and
extinct lemur species.
We calculated mean %CAM consumption using mixing models in
ISSOERROR version 1.04.49 We used mean δ13C values for C3 and CAM
plants from the spiny forest at BMSR as end members, correcting for
the +5‰ difference in δ13C values between collagen and plants. We
did not include C4 plants in these models because they are relatively rare
in southern Madagascar and no modern lemurs are known to consume
them. If δ15N values do, indeed, differentiate spiny Didiereoideae from
sympatric non-spiny CAM plants, then we may be able to use mean
δ15N values in addition to δ13C values to distinguish consumption of
Didiereoideae. We corrected plant values by +3‰ to account for
the difference in δ15N values between collagen and plants.21 We also

3Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 3 of 7
corrected for the small 1.6‰ difference in δ15N between coastal and
inland animals (Supplementary table 4).
Results
What do modern lemurs eat in southwestern Madagascar?
Six lemur species have been observed to feed on endemic and introduced
CAM plants in southern and southwestern Madagascar: Eulemur rufifrons
(the red-fronted brown lemur), Lemur catta (the ring-tailed lemur), Lepilemur
leucopus (the white-footed sportive lemur), L. petteri (Jean-Jaques Petter’s
sportive lemur), Microcebus griseorufus (the reddish-grey mouse lemur)
and Propithecus verreauxi (Verreaux’s sifaka) (Supplementary table 2).
Historically, behavioural studies have tended to concentrate on individuals
living in gallery forest habitats. However, recent research has documented
significant CAM consumption by individuals living in dry or spiny forest at
Berenty, Beza Mahafaly, Cap Sainte Marie and Tsimanampetsotsa.24,28,32,50
Loudon et al.50 and Gould et al.32 estimate 15% CAM consumption for
L. catta living in spiny forest at Tsimanampetsotsa and Berenty Reserve,
respectively. Consumption of CAM resources by L. catta at Cap Sainte
Marie can be >75% during some months.28 Although the vast majority
of the CAM plants consumed by members of the latter population are
introduced, including Opuntia (prickly pears), native CAM species such as
Aloe and Kalanchoe can each comprise >10% of the diet of L. catta during
some months of the year.
Only Lemur catta, Lepilemur leucopus and Propithecus verreauxi have
been observed to consume Didiereoideae (Table 2). All but one of these
published observations have involved Alluaudia spp. The exception was
Didierea trolli which is consumed by L. catta.27 Some modern lemurs
have been reported to feed heavily on Alluaudia. For example, during
certain months at Cap Sainte Marie nearly 14% of the diet of L. catta is
Alluaudia procera.28 Lepilemur leucopus was observed to rely entirely on
leaves and flowers of Alluaudia spp. during the dry season at Berenty.24,25
Table 2: Observations of lemurs feeding on Didiereoideae taxa in southwestern Madagascar
Lemur species Genus and species Parts consumed Locality Source on feeding Source on CAM
Lemur catta Alluaudia dumosa Leaves, fruit, flowers Berenty Gallery Forest, Cap
Sainte Marie 28, 52 37, 41
Lemur catta Alluaudia humbertii Mature leaves Berenty Gallery Forest 27, 52 10, 37, 41
Lemur catta Alluaudia procera Young leaves, mature
leaves, flowers Berenty Gallery Forest
and Spiny Forest; Cap
Sainte Marie
27, 28, 32, 52, 53 17, 37, 41
Lemur catta Didierea trollii Young leaves, flowers Berenty Gallery Forest 27, 52 10, 37, 41
Lepilemur leucopus Alluaudia ascendens Buds, leaves, flowers Berenty Dry Forest 2, 24, 25 10, 37, 41
Lepilemur leucopus Alluaudia procera Buds, leaves, flowers Berenty Dry Forest 2, 24, 25 17, 37, 41
Propithecus verreauxi Alluaudia ascendens Flowers Hazofotsy 54 10, 37, 41
Propithecus verreauxi Alluaudia procera Flowers Hazofotsy 54 17, 37, 41
Table 1: Plant taxa included in this study
Family Genus Species NPhotosynthetic
pathway
Spiny?
Apocynaceae Landolphia sp. 2 C3No
Apocynaceae Pentopetia androsaemifolia 1 C3No
Burseraceae Commiphora sp. 11 C3No
Celastraceae Reissantia angustipetala 1 C3No
Combretaceae Grewia grevei 5 C3No
Combretaceae Terminalia spp. 9 C3No
Euphorbiaceae Croton geayi 4 C3No
Meliaceae Cedrelopsis grevei 5 C3No
Phyllanthaceae Phyllanthus decaryanus 5 C3No
Rhamnaceae Gouania glandulosa 2 C3No
Salvadoraceae Salvadora angustifolia 3 C3No
Mimosaceae Dichrostachys humbertii 6 C3Yes
Asclepiadaceae Cynanchum mahafalense 1 CAM No
Cucurbitaceae Seyrigia sp. 2 CAM No
Cucurbitaceae Xerosicyos perrieri 9 CAM No
Euphorbiaceae Euphorbia tirucalli 21 CAM No
Didieraceae Alluaudia procera 17 CAM Yes
Cyperaceae Cyperus sp. 1 C4No
Poaceae Panicum spp. 7 C4No

4Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 4 of 7
On the other hand, Lepilemur living in the gallery forest at BMSR does
not consume any Didiereoideae but relies to some degree on non-spiny
Euphorbia tirucalli.51 These differences underscore the potential site
specificity of variation in feeding observations.
Do Alluaudia differ isotopically from sympatric CAM plants?
Carbon isotope values differ significantly for CAM, C3 and C4 plants from
BMSR (Figure 1; F2,111=714.9, p<0.0001). Post-hoc HSD tests indicate
that all three are distinct. Carbon isotopes cannot distinguish Alluaudia
procera from sympatric CAM plants (p>0.05). However, nitrogen isotope
values do clearly separate these two plant groups (t=5.38, df=40,
p<0.0001). Alluaudia has distinctly elevated δ15N values (Figure 1).
Figure 1: Box-and-whisker plots of (a) δ13C values for C3, CAM and C4
plants and (b) δ15N values for spiny Didiereoideae and non-
spiny CAM from the spiny forest at Beza Mahafaly Special
Reserve, Madagascar.
To what extent were lemurs feeding on CAM plants in the past?
We found no differences in mean δ13C values between subfossil
extant and extinct lemur species (Wilcoxon signed ranks, S =17, z
=0, p=1.0), although our subfossil sample showed greater variance.
Mixing models based on δ13C values suggest that subfossil individuals
belonging to each of the three extant species consumed mostly C3
resources (Table 3). CAM consumption was negligible for subfossil
Lepilemur, but modest CAM consumption is indicated for subfossil
Lemur catta (8.5%) and Propithecus verreauxi (5%). These values
are slightly higher than CAM consumption estimates for P. verreauxi
and L. catta living today in gallery forest,27 but they are not as high as
values for L. catta in dry forest at coastal localities in the south.28,32,50
Importantly, substantial CAM consumption by modern lemurs, even at
coastal localities, is a seasonal phenomenon.28 Because isotope values
in bone collagen integrate several years of dietary input,21 modest %CAM
estimates for subfossil individuals may reflect seasonal fluxes in CAM
consumption. Among the extinct taxa living in the south and southwest,
Megaladapis edwardsi, M. madagascariensis, Pachylemur insignis and
Palaeopropithecus ingens show no evidence of CAM consumption
(Table 3; Supplementary table 3). In contrast, our data indicate modest
CAM consumption by Archaeolemur majori (5%) and significant CAM
consumption by Mesopropithecus globiceps (25%) and Hadropithecus
stenognathus (92%). In summary, while it is evident that not all southern
lemurs consume CAM plants today, and it is unlikely that all consumed
them in the past, some CAM consumption can be documented in a wide
variety of lemur species.
Table 3: Descriptive statistics including number of specimens analysed,
and mean δ13C and δ15N values ±1σ for each species
Carbon Nitrogen
Genus and
species NMean
δ13C ±1σ a%CAMbNMean
δ15N±1σd
Lemur catta 25 -20.5±1.4 8.5 25 10.6±2.0
Lepilemur
leucopus 8 -21.9±1.7 0 8 9.5±0.7
Propithecus
verreauxi 29 -20.9±1.0 5 29 9.6±2.1
Archaeolemur
majori 23 -20.9±1.4 5 19 11.3±1.9
Hadropithecus
stenognathus 9-10.8±1.5 92 7 13.8±3.2
Megaladapis
edwardsi 8 -22.0±0.5 0 5 11.5±2.6
Megaladapis
madagascariensis 14 -21.7±1.2 0 12 11.3±1.4
Mesopropithecus
globiceps 4-18.6±2.9 25 3 13.1±1.8
Pachylemur
insignis 18 -22.2±1.4 0 16 11.4±1.9
Palaeopropithecus
ingens 30 -21.7±0.7 0 27 13.5±2.0
Aepyornis spp. 60 -24.4±0.9c 11c
Aldabrachelys
spp. 19 -20.6±3.3 8 16 10.3±1.4
Hippopotamus
lemerlei 14 -21.1±2.0 3 14 9.0±1.8
%CAM values were calculated using ISSOERROR version 1.04.49
aCollagen δ13C values have been corrected to account for δ13C shifts in atmospheric CO2
following the industrial revolution.
bMean %CAM consumption was estimated using δ13C values from C3 and CAM plants from
the spiny forest at Beza Mahafaly Special Reserve (-21.5‰ and -9.9‰, respectively). Plant
δ13C values were corrected by +5‰ to account for the isotopic difference between collagen
and plants.21
cδ13C values and %CAM estimates are from the organic portion of eggshell.54 Carbon isotope
values were corrected for the isotopic difference between eggshell and plants.57 We corrected
carbon isotope data for eggshells to account for atmospheric changes in δ13C (-1.2‰).
dPlant δ15N values were corrected by +3‰ to account for the isotopic difference between
collagen and plants.21
Because isotope values in plants are reflected in their animal consumers,
we may be able to use differences in δ15N between Alluaudia and
sympatric CAM plants to identify lemurs that consumed Didiereoideae
in the past. Among those species identified as CAM consumers by
their δ13C values, differing δ15N values suggest varying degrees of
Alluaudia consumption (Figure 2). Nitrogen isotope values indicate that
Didiereoideae were not a dominant element of L. catta or P. verreauxi
diets. Among the extinct taxa, A. majori may have consumed small
amounts of Didiereoideae. However, M. globiceps may have consumed
substantial amounts of Didiereoideae, and H. stenognathus, which is
characterised by exceptionally high δ13C and δ15N values, may have
relied heavily on Didiereoideae (Figure 2).

5Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 5 of 7
Figure 2: Mean δ13C and δ15N values ±1σ for lemur species that likely
consumed varying degrees of non-spiny and spiny CAM plants.
Bubbles represent mean carbon and nitrogen isotope values
±1σ for C3, C4, non-spiny CAM plants and Alluaudia procera.
Subfossil carbon isotope values for subfossils were corrected
for the post-industrial shift in atmospheric δ13C values.
Carbon and nitrogen isotope values in plants were corrected
by +3‰ and +5‰ to account for the isotopic difference
between collagen and plants. Finally, nitrogen isotope values in
plants were shifted +1.6‰ to account for the mean isotopic
difference between inland Beza Mahafaly Special Reserve and
coastal localities (Supplementary table 3) in Madagascar.
The living lemur most reliant on Didiereoideae is likely Lemur catta. This
species may have consumed more Didiereoideae in the past than it
currently does in moist gallery forests.23,45 Goodman and colleagues55
noted the distributional overlap of L. catta and the Didiereoideae. They
suggested that this lemur species may have evolved in dry forests and
subsequently moved into moister riparian forest, where didiereoid taxa
do not exist. In fact, even today, in some arid habitats where L. catta still
thrives and CAM resources abound (e.g. Tsimanampetsotsa, Cap Sainte
Marie), these lemurs consume substantial amounts of Didiereoideae and
other CAM plants.28,50 More research is needed to document the degree
to which L. catta exploits Didiereoideae as opposed to other CAM plants.
The fact that both Alluaudia and Hadropithecus have extreme δ13C and
δ15N values is striking. The geographic overlap of the Didiereoideae and
Hadropithecus stenognathus is also remarkable (Figure 3). With the
exception of Ampasambazimba in Central Madagascar, all subfossil
localities yielding Hadropithecus fall within the modern distributional
range of the Didiereoideae. Compellingly, δ13C values for the two H.
stenognathus individuals sampled from Ampasambazimba suggest
a pure C3-based, rather than a CAM-based, diet (δ13C <-22‰).
This geographic overlap combined with the match for both δ13C and
δ15N between Alluaudia and Hadropithecus, strongly suggests that
Didiereoideae was a staple in the diet of Hadropithecus in the Spiny
Thicket and Succulent Woodland ecoregions of Madagascar.
Our subfossil isotope data do not support the notion that Lepilemur
consumed large quantities of Alluaudia in the past. This finding might be
considered curious, because Lepilemur is the only living lemur that has
been reported to consume large quantities of Alluaudia today. The diet
of Lepilemur has been studied in detail only at two localities in southern
and southwestern Madagascar: the spiny forest at Berenty Private
Reserve where Alluaudia exists, and the gallery forest at BMSR, where
didiereoid taxa do not exist. Alluaudia spp. may comprise close to 100%
of this species’ diet at Berenty Private Reserve at least during the dry
season.2,24,25 Yet δ13C values for Lepilemur from multiple subfossil sites
in the southwest indicate negligible CAM consumption in the past (Table
3; Supplementary table 3).
Source: adapted from www.tropicos.org.
Figure 3: Map of Madagascar including localities where Hadropithecus
stenognathus remains have been found. The distribution of the
Didiereoideae is shaded in grey.
Because our subfossil Lepilemur specimens come from several,
geographically widespread, localities (two inland, one coastal), it is
unlikely that this result reflects sampling bias. Instead it would appear
that modern individuals might have recently shifted their diet at Berenty
to include a resource that was inconsistently exploited (if at all) in the
past. Recent transitions in diet or habitat may be widespread among
modern lemurs living in southwestern Madagascar.45 Isotope values
for subfossil Lepilemur do not differ significantly from those for extinct
Archaeolemur majori, Megaladapis edwardsi, M. madagascariensis,
Palaeopropithecus ingens or Pachylemur insignis. Four of these (all
except Archaeolemur) have a 0% CAM signal, as does subfossil
Lepilemur. Of these four, the two Megaladapis species have dental
topography much like Lepilemur,23 as well as relatively small infraorbital
foramina29 and dental microwear30,56 that suggest dominant foliage
consumption.
Did now extinct non-lemur herbivores consume Didiereoideae?
Estimated CAM consumption for extinct hippopotamuses, tortoises
and elephant birds is minor compared to that for Mesopropithecus and
Hadropithecus. Mixing models suggest that, on average, Hippopotamus
spp. and the giant tortoise Aldabrachelys spp. consumed only 3% and
8% CAM, respectively (Table 3). Clarke et al.57 used δ13C values in
Aepyornis eggshells to estimate that elephant birds consumed ca. 11%
CAM. Nitrogen isotope values are similar in subfossil Hippopotamus,
Aldabrachelys, Lemur catta and Propithecus verreauxi (Table 3;
Supplementary table 3), indicating nominal consumption of Didiereoidea
for these species. Nitrogen isotope values do not exist for Aepyornis.
However, their δ13C values suggest that elephant birds did not exploit
large amounts of Didiereoideae.

6Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 6 of 7
Conclusions
Stable isotope data do not support significant CAM consumption by
non-climbing extinct herbivores such as elephant birds, giant tortoises
or pygmy hippopotamuses, but they do support significant CAM
consumption in several extinct lemur lineages. It seems likely that
spines evolved in the ancestral didiereoid as a defence against lemur
folivory. At the very least, as didiereoids diversified to include relatively
large spiny trees in southern and western Madagascar, they must have
been exploited by climbing herbivores of some kind. Because so many
herbivores in the south and southwest have become extinct, one might
hypothesise that the spines on these plants are today anachronistic. The
unusual isotopic signal of these plants allows us to test the plausibility
of this hypothesis, and to offer new insights into likely past consumers.
Our data support the conclusions that the herbivores exploiting the
leaves of Alluaudia were largely climbing lemurs, and that the loss of
giant climbing lemurs has rendered the spines of didiereoid plants,
such as Alluaudia, increasingly anachronistic. With the exceptions of
Lepilemur and Lemur catta in some locations, lemur species today
consume little CAM. However, carbon isotope values indicate that both
extant and now-extinct lemurs may have consumed more CAM plants
in the past, including didiereoid taxa such as Alluaudia. In particular,
Lemur catta, Mesopropithecus globiceps, and especially Hadropithecus
stenognathus, may have relied heavily on Didiereoideae in the recent
past. If indeed the dominant consumers of Alluaudia leaves are now
extinct, these plants may no longer require formidable defence.
Acknowledgements
We thank Dyke Andreasen, Lalao Andriamahefarivo, Anne Axel,
Zachary Rogers, Wendy Applequist, the Madagascar Institut pour la
Conservation des Ecosystèmes Tropicaux, the Missouri Botanical
Garden and the BMSR staff for technical and logistical assistance; the
Université d’Antananarivo Ecole Supériure des Sciences Agronomiques
Département des Eaux et Forêts for permission to collect plants at
BMSR; Margaret Schoeninger and Leanne Nash for providing Lepilemur
isotope data from BMSR; and Stephen Nash/Conservation International
for permission to reproduce illustrations of lemurs. Subfossils were
sampled under collaborative agreements between LRG, D.A. Burney,
W.L. Jungers and the Département de Paléontologie et d’Anthropologie
Biologique, Université d’Antananarivo. Funding was provided by a
Guggenheim fellowship to L.R.G. and the UC Lab Fee Research Program
(09-LR-07-115818-DOMIN to BEC).
Authors’ contributions
Both authors participated in the planning and design of the research.
L.R.G. conceived of the project and B.E.C. conducted analyses. Both
authors wrote the manuscript.
References
1. Applequist WL, Wallace RS. Expanded circumscription of Didiereaceae and
its division into three subfamilies. Adansonia. 2003;25(1):13–16. Available
from: http://www.mnhn.fr/publication/adanson/a03n11a12.pdf
2. Charles-Dominique P, Hladik CM. Le Lepilemur du sud de Madagascar:
Écologie, alimentation et vie sociale. Rev Ecol (Terre Vie). 1971;25(1):3–66.
3. Fenn MD. The spiny forest ecoregion. In: Goodman SM, Benstead JP, editors.
The natural history of Madagascar. Chicago: University of Chicago Press;
2003. p. 1525–1530.
4. Burgess ND, D’Amico Hales J, Underwood EC, Dinerstein E, Olson D, Itoua
I, et al. Terrestrial ecoregions of Africa and Madagascar: a conservation
assessment. Washington, DC: Island Press 2004.
5. Applequist WL, Wallace RS. Phylogeny of the Madagascan endemic family
Didiereaceae. Plant Syst Evol. 2000;221:157–166.
6. Grubb PJ. Interpreting some outstanding features of the flora and vegetation
of Madagascar. Perspect Plant Ecol. 2003;6(1,2):125–146.
7. Cooper SM, Owen-Smith N. Effects of plant spinescence on large mammalian
herbivores. Oecologia. 1986;68(3):446–455.
8. Milewski AV, Young TP, Madden D. Thorns as induced defenses: Experimental
evidence. Oecologia. 1991;86(1):70–75.
9. Arakaki M, Christin P-A, Nyffeler R, et al. Contemporaneous and recent
radiations of the world’s major succulent plant lineages. Proc Natl Acad Sci
USA. 2011;108(20):8379–8384.
10. Ocampo G, Columbus JT. Molecular phylogenetics of suborder Cactineae
(Caryophyllales), including insights into photosynthetic diversification and
historical biogeography. Am J Bot. 2010;97(11):1827–1847.
11. Bond WJ, Silander JA. Springs and wire plants; anachronistic defences
against Madagascar’s extinct elephant birds. Proc R Soc Lond B.
2007;274(1621):1985–1992. http://dx.doi.org/1910.1098/rspb.2007.0414
12. Bond WJ, Lee WG, Craine JM. Plant structural defences against browsing
birds: A legacy of New Zealand’s extinct moas. Oikos. 2004;104:500–508.
13. Samonds KE, Godfrey LR, Ali JR, Vences M, Sutherland MR, Irwin MT. Spatial
and temporal arrival patterns of Madagascar’s vertebrate fauna explained
by distance, ocean currents, and ancestor type. Proc Natl Acad Sci USA.
2012;109:5352–5357.
14. Marshall JD, Brooks JR, Lajtha K. Sources of variation in the stable isotopic
composition of plants. In: Michener R, Lajtha, K, editors. Stable isotopes
in ecology and environmental science. 2nd ed. Malden, MA: Blackwell
Publishing; 2007. p. 22–60.
15. Kluge M, Razanoelisoa B, Brulfert J. Implications of genotypic diversity and
phenotypic plasticity in the ecophysiological success of CAM plants, examined
by studies on the vegetation of Madagascar. Plant Biol. 2001;3(3):214–222.
16. Handley LL, Austin AT, Robinson D, Serimgeour CM, Raven JA, Heaton THE,
et al. The 15N natural abundance of (δ15N) of ecosystem samples reflects
measures of water availability. Aust J Plant Physiol. 1999;26:185–199.
17. Crowley BE, Thorén S, Rasoazanabary E, Vogel ER, Barrett MA, Zohdy S, et al.
Explaining geographical variation in the isotope composition of mouse lemurs
(Microcebus). J Biogeogr. 2011;38:2106–2121.
18. Muzuka ANN. Isotopic compositions of tropical East African flora and their
potential as source indicators of organic matter in coastal marine sediments.
J Afr Earth Sci. 1999;28(3):757–766.
19. Codron J, Codron D, Lee-Thorp JA, Sponheimer M, Bond WJ, De Ruiter D,
et al. Taxonomic, anatomical, and spatio-temporal variations in the stable
carbon and nitrogen isotopic compositions of plants from an African savanna.
J Archaeol Sci. 2005;32:1757–1772.
20. Evans RD, Ehleringer JR. Water and nitrogen dynamics in an arid woodland.
Oecologia. 1994;99(3/4):233–242.
21. Crowley BE. Stable isotope techniques and applications for primatologists. Int
J Primatol. 2012;33:673–701.
22. Sealy JC, Van der Merwe NJ, Lee-Thorp JA, Lanham JL. Nitrogen isotopic
ecology in southern Africa: Implications for environmental and dietary tracing.
Geochim Cosmochim Acta. 1987;51(10):2707–2717.
23. Godfrey LR, Winchester JM, King SJ, Boyer DM, Jernvall J. Dental topography
indicates ecological contraction of lemur communities. Am J Phys Anthropol.
2012;148(2):215–227.
24. Hladik CM, Charles-Dominique P. The behaviour and ecology of the sportive
lemur (Lepilemur mustelinus) in relation to its dietary peculiarities. In: Martin
RD, Doyle GA, AC W, editors. Prosimian biology. London: Duckwor th; 1974.
p. 23–37.
25. Russell RJ. The behavior, ecology and environmental physiology of a noctur nal
primate, Lepilemur mustelinus (Strepsirhini, Lemuriformes, Lepilemuridae).
Durham, NC: Duke University; 1977.
26. Richard AF. Variability in feeding behavior of a Malagasy prosimian,
Propithecus verreauxi: Lemuriformes. In: Montgomery G, editor. The ecology
of arboreal folivores. Washington, DC: Smithsonian Institution Press; 1978.
p. 519–533.
27. Simmen B, Hladik A, Ramasiarisoa P. Food intake and dietary overlap in native
Lemur catta and Propithecus verreauxi and introduced Eulemur fulvus at
Berenty, southern Madagascar. Int J Primatol. 2003;24(5):949–968.
28. Kelley EA. Lemur catta in the region of Cap Sainte-Marie, Madagascar:
Introduced cacti, xerophytic Didiereaceae-Euphorbia bush, and tombs
[unpublished dissertation]. Saint Louis (MO): Washington University; 2011.

7Volume 109 | Number 1/2
January/February 2013
South African Journal of Science
http://www.sajs.co.za
Research Article Didiereoideae consumption by now extinct lemurs
Page 7 of 7
29. Muchlinski MN, Godfrey LR, Muldoon KM, Tongasoa L. Evidence for
dietary niche separation based on infraorbital foramen size variation
among subfossil lemurs. Folia Primatol. 2011;81:330–345. http://dx.doi.
org/310.1159/000323277
30. Godfrey LR, Semprebon GM, Jungers WL, Sutherland MR, Simons EL,
Solounias N. Dental use wear in extinct lemurs: Evidence of diet and niche
differentiation. J Hum Evol. 2004;47(3):145–169.
31. Sauther ML, Sussman RW, Gould L. The socioecology of the ringtailed lemur:
Thirty-five years of research. Evol Anthropol. 1999;8:120–132.
32. Gould L, Constabel P, Mellway R, Rambeloarivony H. Condensed tannin intake
in spiny-forest-dwelling Lemur catta at Berenty Reserve, Madagascar, during
reproductive periods. Folia Primatol. 2009;80:240–263.
33. Sterling EJ, Dierenfeld EC, Ashbourne CJ, Feistner ATC. Dietary intake,
food composition and nutrient intake in wild and captive populations of
Daubentonia madagascariensis. Folia Primatol. 1994;62(1–3):115–124.
34. Simons EL. The giant aye-aye Daubentonia robusta. Folia Primatol.
1994;62(1–3):14–21.
35. Petitjean A, Petijean M, Rakotovao P. Les plantes utiles de Madagascar, tome
1: Répertoires des noms vernaculaires, tome 2: inventaire. Antananarivo:
Bibliotheque de l›Academie Malgache; 1992.
36. De Luca P, Alfani A, De Santo VA. CAM, transpiration, and adaptive
mechanisms to xeric environments in the succulent Cucurbitaceae. Botanical
Gazette. 1977;138(4):474–478.
37. Winter K. δ13C values of some succulent plants from Madagascar. Oecologia.
1979;40(1):103–112.
38. Winter K, Wallace BJ, Stocker GC, Roksandic Z. Crassulacean acid
metabolism in Australian vascular epiphytes and some related speces.
Oecologia. 1983;57(1/2):129–141.
39. Winter K, Aranda J, Holtum JAM. Carbon isotope composition and water-
use efficiency in plants with crassulacean acid metabolism. Funct Plant Biol.
2005;32(5):381–388.
40. Martin CE, Loeschen VS, Coke LB. Crassulacean acid metabolism in
selected terrestrial succulents in southeastern Jamaica, including two
species in the Commelinaceae. Oecologia. 1990;84(1):99–102. http://dx.doi.
org/110.1007/BF00665601
41. Kluge M, Brulfert J, Rauh W, Ravelomanana D, Zielger H. Ecophysiological
studies on the vegetation of Madagascar: A δ13C and δD survey for
incidence of crassulacean acid meteabolism (CAM) among orchids from
montane forests and succulents from the xerophytic thorn-bush. Isotopes,
Environment and Health Studies. 1995;31(2):191–210.
42. Silvera K, Neubig KM, Whitten WM, Williams NH, Winter K, Cushman JC.
Evolution along the crassulacean acid metabolism continuum. Funct Plant
Biol. 2010;37(11):995–1010.
43. Sayed OH. Crassulacean acid metabolism 1975-2000, a check list.
Photosynthetica. 2001;39(3):339–352.
44. Crowley BE, Godfrey LR, Irwin MT. A glance to the past: Subfossils, stable
isotopes, seed dispersal, and lemur species loss in southern Madagascar. Am
J Primatol. 2011;73(1):25–37.
45. Crowley BE, Godfrey LR, Guilderson TP, Zermeño P, Koch PL, Dominy NJ.
Extinction and ecological retreat in a community of primates. Proc R Soc Lond
B. 2012;279:3597–3605.
46. Burney DA, Burney LP, Godfrey LR, Jungers WL, Goodman SM, Wright HT, et
al. A chronology for late prehistoric Madagascar. J Hum Evol. 2004;47:25–63.
47. Burney DA. Rates, patterns, and processes of landscape transformation and
extinction in Madagascar. In: MacPhee R, ed. Extinction in near time. New
York: Kluwer/Plenum; 1999. p. 145–164.
48. Burleigh R, Arnold EN. Age and dietary differences of recently extinct Indian
Ocean tortoises (Geochelone s. lat.) revealed by carbon isotope analysis.
Proc R Soc B. 1986;227:137–144.
49. Phillips DL, Gregg JW. Uncertainty in source partitioning using stable
isotopes. Oecologia. 2001;127:171–179.
50. Loudon JE, Whitelaw DC, Sponheimer M, Sauther ML, Cuozzo FP. Lemurs
eating isotopes: A stable isotope analysis of ring-tailed lemurs (Lemur catta)
and their menu at the Beza Mahafaly Special Reserve. Am J Phys Anthropol.
2008;135(Suppl 46):142.
51. Nash LT. Vertical clingers and sleepers: Seasonal influences on the activities
and substrate use of Lepilemur leucopus at Beza Mahafaly Special Reserve,
Madagascar. Folia Primatol. 1998;69(Suppl 1):204–217.
52. Simmen B, Sauther ML, Soma T, Rasamimanana H, Sussman RW, Jolly A, et
al. Plant species fed on by Lemur catta in gallery forests of the southern domain
of Madagascar. In: Jolly A, Koyama N, Rasamimanana H, Sussman RW, eds.
Ringtailed lemur biology. New York: Springer, 2006; p. 55–68. Available from:
http://rd.springer.com/chapter/10.1007/978-0-387-34126-2_5
53. Gould L, Power ML, Ellwanger N, Rambeloarivony H. Feeding behavior and
nutrient intake in spiny forest-dwelling ring-tailed lemurs (Lemur catta) during
early gestation and early mid-lactation periods: Compensating in a harsh
environment. Am J Phys Anthropol. 2011;145:469–479.
54. Richard A. The feeding behavior of Propithecus verreauxi. In: Clutton-Brock
TH, editor. Primate ecology: Studies of feeding and ranging behaviour in
lemurs, monkeys and apes. New York: Academic Press; 1977. p. 71–96.
55. Goodman SM, Rakotoarisoa SV, Wilmé L. The distribution and biogeography
of the ringtailed lemur (Lemur catta) in Madagascar. In: Jolly A , Sussman RW,
Koyama N, Rasamimanana H, editors. Ringtailed lemur biology. New York:
Springer; 2006. p. 3–15.
56. Scott JR, Godfrey LR, Jungers WL, Scott RS, Simons EL, Teaford MF, et al.
Dental microwear texture analysis of two families of subfossil lemurs from
Madagascar. J Hum Evol. 2009;54(4):405–416.
57. Clarke SJ, Miller GH, Fogel ML, Chivas AR, Murray-Wallace CV. The amino
acid and stable isotope biogeochemistry of elephant bird (Aepyornis)
eggshells from southern Madagascar. Quaternary Sci Rev. 2006;25(17–
18):2343–2356.