Food for Thought: Lactating Coquerel’s Sifaka (Propithecus Coquereli) Eat Foods High in Protein and Fiber During the Lean Season

Abstract

Infant-bearing, Coquerel’s sifaka ( Propithecus coquereli ) undergo gestation during a lean seasonal climate with weaning occurring during the abundant season. During this time, nutrient demand increases due to placental transport to the fetus and to the infant postpartum by milk. Females respond to this increased demand by ingesting larger food quantities, reducing expenditure, and/or using their nutrient stores. We collected foods (N=75) exploited by lactating females (N=10) in Ankarafantsika National Park, Madagascar to examine the nutritional landscape within which sifakas forage. We measured food nitrogen, neutral detergent fiber (NDF), acid detergent fiber (ADF), gross energy (GE) and ash to estimate crude protein (CP), available protein (AP), fiber, mineral content and metabolizable energy (ME). Two significant PCA (principal component analysis) axes corresponded to high protein and high fiber-low ME explaining 91.6% of the variance. Cluster 1 is categorized by foods that contained higher AP and cluster 2 is categorized by higher fiber foods. P. coquereli rely on a diverse range of foods inclusive of those with high AP and ME, but also high fiber foods with low ME. We hypothesize that the high fiber, low ME foods may be important for maintaining the gut microbiome.

Full text

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Food for Thought: Lactating Coquerel’s Sifaka (
Propithecus
Coquereli
) Eat Foods High in Protein and Fiber During the Lean
Season
Abigail C. Ross (  a.ross@rockvalleycollege.edu )
Rock Valley College
Michael L. Power
Smithsonian Institution
Research Article
Keywords: Fiber, lactation, lemurs, metabolizable energy, nutrient content, nutritional ecology, seasonality
Posted Date: January 21st, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1247752/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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Abstract
Infant-bearing, Coquerel’s sifaka (
Propithecus coquereli
) undergo gestation during a lean seasonal climate with weaning occurring
during the abundant season.During this time, nutrient demand increases due to placental transport to the fetus and to the infant
postpartum by milk.Females respond to this increased demand by ingesting larger food quantities, reducing expenditure, and/or using
their nutrient stores.We collected foods (N=75) exploited by lactating females (N=10) in Ankarafantsika National Park, Madagascar to
examine the nutritional landscape within which sifakas forage.We measured food nitrogen, neutral detergent ber (NDF), acid detergent
ber (ADF), gross energy (GE) and ash to estimate crude protein (CP), available protein (AP), ber, mineral content and metabolizable
energy (ME).Two signicant PCA (principal component analysis) axes corresponded to high protein and high ber-low ME explaining
91.6% of the variance.Cluster 1 is categorized by foods that contained higher AP and cluster 2 is categorized by higher ber foods.
P.
coquereli
rely on a diverse range of foods inclusive of those with high AP and ME, but also high ber foods with low ME. We hypothesize
that the high ber, low ME foods may be important for maintaining the gut microbiome.
Introduction
The nutrient content of foods eaten by wild primates is highly variable and resources are not interchangeable 1–3. The nutrient quantities
required for proper primate nutrition are contingent on body size, metabolism, digestive anatomy and physiology, sex, life history, and
habitat quality 4–9. Food selection indicates varying nutritional needs 10 by prioritizing nutrients to meet distinct nutritional goals within
environmental constraints 11. Assessing the nutrients and energy available from foods helps gauge these specic parameters within this
contextual framework. One effective way to examine these constraints is to measure the nutrient content of foods consumed by
individual animals to explore the nutritional options in their habitat.
The taxonomic Family Indriidae is composed of mostly folivorous-frugivorous primates endemic to Madagascar that have evolved an
extensive small intestine and enlarged hindgut to assist with nutrient extraction 5. The enlarged lower gut characteristic of hindgut
fermenters consists of the caecum, a portion of the large intestine, and colon 12 that stretches to 13—15 times the animal’s body length,
thereby requiring a 24—48 hour gut-passage time in
Propithecus
spp. 13,14. The lower gut serves as a fermentation chamber to aid in
ber digestion (Lambert 1998) with large populations of microbes housed in the caecum (Campbell et al. 1999). Microbes found in the
caecum and colon are capable of fermenting ber, in turn producing energy for indriids in the form of short-chain volatile fatty acids
(primarily acetate, butyrate and propionate), as well as amino acids, vitamins and a host of other bioactive molecules that may benet
the host 15.
Indriids are challenged with the unpredictability in abundance and distribution of food resources due to the extreme seasonality within
the region 16. Additionally, the majority of lemur species including indriids give birth during the dry, lean season when resources are of
lower quality (i.e., reduced protein and energy availability) and wean infants during the wet, abundant season when resources are higher
quality (i.e., greater protein and energy availability) 17,18.
P. coquereli
infants are born predominantly during the lean season from June—
August and weaned during the abundant season from January—February 19,20. This reproductive strategy intensies the already high
energetic demands on lactating females since infants are behaviorally and nutritionally dependent when resources are most seasonally
depletive. As an example of a related species, female Verreaux’s sifaka (
Propithecus verreauxi
) increase their overall food intake during
late lactation; including increased intakes of crude protein, fat, non-structural carbohydrates and energy relative to males 6. During
gestation, sex differences in macronutrient intakes and energy were not present (Koch et al. 2017). Even with a greater nutrient intake
during late lactation, lactating
P. verreauxi
lose 18% of their body weight throughout the dry season 21.
In the present study, we investigate the nutrient content of foods selected by lactating
P. coquereli
during the lean season. We assessed
protein, ber, energy, and minerals to explore the nutrients available to lactating females from which we characterize the nutritional
landscape in which sifakas forage and feed.
Methods
Study Site
This study was conducted in Ankarafantsika National Park (ANP), Madagascar. ANP is a dry deciduous forest with a pronounced lean
(dry) season from May to September 22 with the greater number of
P. coquereli
infants being born during this time; i.e., late May to

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August 19,23. Forested areas are experiencing anthropogenic disturbance from slash-and-burn agriculture, re, human trac, unregulated
presence and herding of domestic cattle, bushmeat hunting and hole digging for
Dioscorea maciba
tuber extraction 24–26, which
increases food scarcity during the lean season. Soils are either red, speckled, or white, with red soil containing the highest water content
and white sand the lowest 25. Many tree species grow in nutrient poor, acidic white sands and a thick layer of loose sand is present on
the soil surface because of sandstone erosion 27,28. Flora are speciose and the forest understory is moderately thick with sparse leaf
litter (Lourenço & Goodman, 2006).
Plant Collection
The collection of plants that were consumed by ten habituated
P. coquereli
lactating females occurred from June to December of 2010
and 2011 for 93 hours over 52 weeks (26 consecutive weeks/season). Plant parts identied included: leaves, fruits, owers, buds, and
bark. Samples were stored in manila envelopes until they were transported to a propane drying oven at the end of each focal follow.
Plant Processing and Preservation
Samples were dried on-site in a propane oven at a maximum of 50°C using a max/min digital thermometer (HBE International Inc.) until
a constant weight was reached for at least 48 hours 29. Samples were weighed daily to determine dry weights and not exposed to direct
sunlight to limit post-collection changes in nutrient composition. Samples were placed in 3M SCC Dri-Shield 2000 moisture barrier bags
with silica gel and stored in plastic containers in a concrete storage area.
Scientic name identications were conrmed by experts at Parc Botanique et Zoologique de Tsimbazaza, Antananarivo, Madagascar;
Missouri Botanical Gardens, Antananarivo, Madagascar; Université d'Antananarivo – Faculté des Sciences; and ANP. Voucher herbarium
specimens were sent to the Smithsonian National Zoological Park, Washington, D.C. and Missouri Botanical Garden, St. Louis, Mo.
Permissions were granted to export plant material including names from the Direction Generale des Forets, Direction de la Valorisation
des Ressources Naturelles, and Service de la Gestion Faune et Flore (N°128N_EV10/MG11).
Chemical Analyses and Calculations
Laboratory assays were conducted at the Nutrition Laboratory, Smithsonian National Zoo and Conservation Biology Institute. Dry food
samples were re-dried at 55° C for a minimum of 48 hours and ground to achieve a homogeneous subsample. Plant material was
ground using a Wiley mill or with a ceramic mortar and pestle depending on consistency and sample size and passed either through a
0.38 mm sieve (CHN procedure) or 0.86 mm sieve. Assays included: nitrogen (N) as an index for protein, neutral detergent ber (NDF),
acid detergent ber (ADF), gross energy (GE) (kcal/g), and ash as an index for total mineral content. N content were measured using a
combustion method (Dumas method) in a PerkinElmer 2400 Series II Analyzer (PerkinElmer, Waltham, MA). The ANKOM ber procedure
using an ANKOM Fiber 200 Analyzer or the Van Soest ber procedure 30 were used for neutral detergent (NDF) and acid detergent ber
(ADF determination). We did not assay ADL (acid detergent lignin) which would have represented the indigestible ber fraction and
acknowledge this may have affected our results and interpretation. GE of samples (kcal/g) was measured using adiabatic bomb
calorimetry to measure the heat from sample combustion. Pellets were formed from 0.25—0.75 g of sample and re-dried for one hour at
60°C. A Parr 1241 Adiabatic Calorimeter (Parr Instrument Company, Moline, IL) was used to measure GE. Samples were considered for
re-assay if duplicates varied by >0.2 kcal/g. Total mineral content was determined by ashing the samples in a mue furnace. Crucibles
were lled with 0.25—0.50 g of sample and heated for six hours at 450° C.
We estimated crude protein (CP) following Maynard and Loosli 31; available protein (AP) following 32 ; and metabolizable energy (ME)
using values for energy not available from NDF from Campbell, et al. 33 and Conklin-Brittain, et al. 34.

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The value of 2.17 kcal/g for energy not available from NDF was estimated using the value 61% NDF digestion factor 33 and accounting
for the energy lost to microbial metabolism estimated by as 1kcal/g of NDF 34. Thus, energy lost from NDF is estimated to be:
The mean value for NDF digestion was for captive foods33, and thus likely represents a maximum for wild foods, so our estimated non-
protein ME is likely an overestimate.
Statistical Analysis
A total of 139 plant samples were assayed, however, there were some duplicate samples of the same food type (e.g., fruit, leaf) and
plant species collected from different locations or times. Duplicate samples were averaged to produce macronutrient values for a unique
species-plant part except in the case of four species that displayed an apparent seasonal difference in nutrient composition (Table 1).
These eight samples were treated as different foods, based on the macronutrient composition. This resulted in 75 unique sifaka foods
for which we report data (Table 2). All nutrient results are reported on a dry matter basis to control for the effect of variable water
content. Values are reported as mean ± SEM and range. Pearson’s correlation was used to assess associations among nutrients
assayed. Data were analyzed using SPSS 20.0, IBM Corp, Armonk NY.
Table 1
Foods consumed by
Propithecus coquereli
with seasonal differences
Botanical Name Malagasy Vernacular
Name Plant Part Date CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
Abrahamia ditimena
DITIMENA Leaves July 7.9 5.2 45.0 36.6 4.8
Abrahamia ditimena
DITIMENA Leaves October 11.0 8.3 28.2 24.5 4.2
Seasonal change July vs.
October
39.2% 59.6% -37.3% -33.1% -12.5%
Dalbergia
bracteolata
VAHAFISAKA Leaves July 13.4 12.0 33.0 20.2 5.2
Dalbergia
bracteolata
VAHAFISAKA Leaves October 19.1 17.7 21.6 13.7 4.2
Seasonal change July vs.
October
42.5% 47.5% -34.6% -32.2% -19.2%
Dalbergia
trichophylla
MANARY Fruit September 10.4 7.8 40.3 31.2 3.5
Dalbergia
trichophylla
MANARY Fruit November 18.1 15.0 43.7 32.8 3.0
Seasonal change September vs.
November 74.0% 92.3% 8.4% 5.1% -14.3%
Grangeria porosa
MAEVALAFIKA Leaves June 12.0 10.4 42.1 26.6 3.9
Grangeria porosa
MAEVALAFIKA Leaves October 7.6 6.4 54.5 34.3 3.4
Seasonal change June vs.
October
-36.7% -38.5% 29.5% 29.0% -12.8%

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

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Table 2
Plants selected as food resources by lactating
Propithecus coquereli
during Madagascar’s lean (dry) season, including their respective
nutrient and energy values
+Botanical Name
(genus + specic
epithet)
Botanical
Family
+Malagasy
Vernacular Name
Plant
Part CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
ME
(kcal/g)
Abrahamia
ditimena
ANACARDIACEAE DITIMENA Leaves 11.0 8.3 28.2 24.5 4.2 3.8
Abrahamia
ditimena
ANACARDIACEAE DITIMENA Leaves 7.9 5.2 45.0 36.6 4.8 3.4
Abrahamia
ditimena
ANACARDIACEAE DITIMENA Bark 2.2 0.0 79.2 75.2 3.4 2.8
Abrahamia
ditimena
ANACARDIACEAE DITIMENA Fruit 4.5 n/a 14.8 8.2 2.5 n/a
Abrahamia
spp. ANACARDIACEAE MANGA Fruit 3.5 3.4 8.6 6.2 1.9 3.6
Abrahamia
spp. ANACARDIACEAE MANGA Leaves 9.9 8.4 50.4 39.8 4.8 n/a
Albizia boivinii
* or
Unidentied
FABACEAE KITSAKITSANALA Leaves 20.3 18.7 26.1 17.7 4.9 3.5
Albizia mainaea
FABACEAE ALIBIZAHA Leaves 16.9 15.8 40.2 15.0 4.6 3.8
Astrotricha
spp. MELIACEAE VALOMAMAY Fruit 8.6 7.7 19.3 14.6 5.2 5.0
Bathiorhamnus
spp. RHAMNACEAE KABIJALAHY Leaves 13.0 10.8 46.2 33.05 3.2 3.8
Bussea perrieri
FABACEAE MIMOZA Leaves 23.7 22.1 23.0 15.2 6.0 3.7
Capurodendron
perrieri
*
or
Asteropeia
amblyocarpa
*
or
Securinega
spp.*
SAPOTACEAE
or
ASTEROPEIACEAE
or
PHYLLANTHACEAE
HAZONJIA Leaves 10.4 8.4 45.9 34.7 5.8 3.6
Combretum
spp. COMBRETACEAE MANAKOBONGO Fruit 7.3 6.5 45.1 35.2 3.5 3.3
Commiphora
spp. BURSERACEAE MATAMBELONA Leaves 13.6 11.8 17.1 14.5 5.6 3.6
Commiphora
spp. BURSERACEAE MATAMBELONA Buds 7.8 7.0 24.2 19.2 4.2 n/a
Crateva excelsa
CAPPARIDACEAE PAMBA Flowers 15.1 13.4 26.8 17.3 6.9 3.3
Cynanchum
spp. ASCLEPIADACEAE RAHAMATSATSO Flowers 6.1 5.6 42.9 38.4 n/a 3.0
Dalbergia
bracteolata
FABACEAE VAHAFISAKA Leaves 13.4 12.0 33.0 20.2 5.2 3.0
AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; NPGE = non-protein gross energy; ME =
metabolizable energy; ash = total minerals.
+Botanical names and vernacular names have been provided by previous researchers, local guides, and
published sources.
*Annotated botanical names were initially unidentied specimens associated only with Malagasy vernacular names. Consultations
with Missouri Botanical Garden - Madagascar and Université d’Antananarivo - Faculté des Sciences resulted in translated
suggestions of possible species endemic to the Ankarafantsika National Park region and within the distribution range of
P. coquereli
and are not based on taxonomic identication of actual plant specimens.

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+Botanical Name
(genus + specic
epithet)
Botanical
Family
+Malagasy
Vernacular Name
Plant
Part CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
ME
(kcal/g)
Dalbergia
bracteolata
FABACEAE VAHAFISAKA Leaves 19.1 17.7 21.6 13.7 4.2 3.8
Dalbergia
trichophylla
FABACEAE MANARY Leaves 18.3 17.4 20.2 14.9 4.7 3.7
Dalbergia
trichophylla
FABACEAE MANARY Fruit 18.1 15.0 43.7 32.8 3.0 4.5
Dalbergia
trichophylla
FABACEAE MANARY Fruit 10.4 7.8 40.3 31.2 3.5 4.8
Dalbergia
trichophylla
FABACEAE MANARY Flowers 16.1 13.07 n/a 30.0 n/a n/a
Dichapetalum
spp. DICHAPETALACEAE FANTSIKATRA Flowers 12.8 11.7 n/a 30.1 3.4 n/a
Diospyros
spp.* or
Diospyros
tropophylla
*
or
Casearia
nigrescens
*
EBENACEAE
or
SALICACEAE
HAZOMAFANA Leaves 17.1 16.3 27.3 21.5 3.8 4.0
Entada
spp. FABACEAE ROIMENA Flowers 16.9 15.2 34.8 22.7 n/a n/a
Entada
spp. FABACEAE ROIMENA Fruit 23.9 22.0 18.4 18.3 n/a n/a
Eucalyptus
spp.*
or
Eucalyptus
camaldulensis
*
MYRTACEAE KINININA Leaves
& bark 3.0 0.0 81.8 72.7 2.7 2.6
Gambeya
boiviniana
*SAPOTACEAE VOATSIKIDY Leaves 22.2 20.9 44.8 28.3 7.2 3.3
Garcinia
verrucosa
CLUSIACEAE NATOVAVY Leaves 8.5 6.4 40.6 33.7 4.0 3.8
Garcinia
verrucosa
CLUSIACEAE NATOVAVY Fruit 6.2 5.7 17.7 12.0 3.6 4.0
Garcinia
verrucosa
CLUSIACEAE NATOVAVY Leaf
buds 9.4 8.4 31.7 18.8 4.4 4.4
Grangeria porosa
ROSACEAE MAEVALAFIKA Bark 5.9 1.4 79.6 68.5 19.4 1.9
Grangeria porosa
ROSACEAE MAEVALAFIKA Leaves 7.6 6.4 54.5 34.3 3.4 2.9
Grangeria porosa
ROSACEAE MAEVALAFIKA Leaves 12.0 10.4 42.1 26.6 3.9 3.5
AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; NPGE = non-protein gross energy; ME =
metabolizable energy; ash = total minerals.
+Botanical names and vernacular names have been provided by previous researchers, local guides, and
published sources.
*Annotated botanical names were initially unidentied specimens associated only with Malagasy vernacular names. Consultations
with Missouri Botanical Garden - Madagascar and Université d’Antananarivo - Faculté des Sciences resulted in translated
suggestions of possible species endemic to the Ankarafantsika National Park region and within the distribution range of
P. coquereli
and are not based on taxonomic identication of actual plant specimens.

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+Botanical Name
(genus + specic
epithet)
Botanical
Family
+Malagasy
Vernacular Name
Plant
Part CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
ME
(kcal/g)
Grangeria porosa
ROSACEAE MAEVALAFIKA Leaf
buds 9.0 7.3 37.2 25.2 9.1 3.5
Grangeria porosa
ROSACEAE MAEVALAFIKA Fruit 8.6 6.9 50.6 34.3 3.7 3.5
Grewia
ambongensis
TILIACEAE SELIVATO Fruit 18.2 17.0 29.1 22.7 6.5 4.2
Grewia
ambongensis
TILIACEAE SELIVATO Leaves 22.2 20.8 25.4 15.8 7.8 3.2
Grewia
spp. MALVACEAE SELIALA Fruit 5.5 3.5 74.4 57.7 3.3 2.7
Landolphia
gummifera
APOCYNACEAE PIRA Fruit 3.8 3.2 51.6 34.5 2.1 2.9
Macphersonia
gracilis
SAPINDACEAE MAROAMPOTOTRA Fruit 4.9 3.3 61.5 38.6 3.3 2.6
Malleastrum
gracile
MELIACEAE ANDRIAMANAMORA Leaves 19.3 17.6 50.2 38.5 5.2 2.8
Mammea
punctata
CLUSIACEAE TSIMATIMANOTA Leaves 8.2 6.6 49.1 38.4 3.5 3.7
Mammea
punctata
CLUSIACEAE TSIMATIMANOTA Fruit 3.4 2.9 22.4 9.9 2.3 4.0
Mascarenhasia
spp.* APOCYNACEAE GODROA Leaves 13.8 12.6 24.2 18.6 5.9 3.8
Mimusops
spp. SAPOTACEAE HAZOPIKA Fruit 2.9 2.1 64.2 41.0 2.2 3.0
Monanthotaxis
spp. ANNONACEAE FOTSIAVADIKA Leaves 16.5 15.0 28.3 19.3 3.9 n/a
Monanthotaxis
spp. ANNONACEAE FOTSIAVADIKA Buds 15.6 13.7 36.2 23.7 n/a 3.6
Noronhia
spp. OLEACEAE HAZOTSIFAKA Leaves 12.5 11.0 35.5 27.6 5.3 3.5
Noronhia
spp. OLEACEAE HAZOTSIFAKA Bark 8.9 0.0 71.2 62.7 11.2 2.1
Ochna ciliata
OCHNACEAE MORAMENA Leaves 17.4 14.6 32.5 22.9 3.9 3.6
Omphalea
oppositifolia
*EUPHORBIACEAE VOASALAY Flowers 11.0 10.1 46.2 36.7 3.9 3.4
Passiora
foetida
*PASSIFLORACEAE BONGAPISO Leaf
buds 12.5 11.1 44.1 22.8 8.1 3.0
Passiora
foetida
*PASSIFLORACEAE BONGAPISO Leaves 27.1 24.4 37.9 26.7 6.8 3.1
Passiora
foetida
*PASSIFLORACEAE BONGAPISO Fruit 14.3 13.2 35.8 15.3 5.4 3.1
AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; NPGE = non-protein gross energy; ME =
metabolizable energy; ash = total minerals.
+Botanical names and vernacular names have been provided by previous researchers, local guides, and
published sources.
*Annotated botanical names were initially unidentied specimens associated only with Malagasy vernacular names. Consultations
with Missouri Botanical Garden - Madagascar and Université d’Antananarivo - Faculté des Sciences resulted in translated
suggestions of possible species endemic to the Ankarafantsika National Park region and within the distribution range of
P. coquereli
and are not based on taxonomic identication of actual plant specimens.

Page 9/20
+Botanical Name
(genus + specic
epithet)
Botanical
Family
+Malagasy
Vernacular Name
Plant
Part CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
ME
(kcal/g)
Polyalthia
spp. ANNONACEAE AMBALAHY Leaves 14.8 11.9 48.1 28.2 4.2 3.4
Polyalthia
spp. ANNONACEAE AMBALAHY Flowers 19.7 19.1 22.4 25.5 4.7 4.1
Polycardia libera
CELASTRACEAE MAMOARAVINA Leaves 12.6 12.2 20.8 16.2 6.3 4.2
Polycardia libera
CELASTRACEAE MAMOARAVINA Flowers 5.3 5.0 69.4 16.9 n/a n/a
Poupartia
sylvatica*
or
Poupartia
spp.*
or
Sclerocarya
birrea*
ANACARDIACEAE SAKOALA Leaves 13.2 11.0 19.1 15.8 3.4 3.9
Poupartia
sylvatica
* or
Poupartia
spp.*
or
Sclerocarya
birrea*
ANACARDIACEAE SAKOALA Flowers 7.9 6.8 24.9 21.4 2.9 3.9
Rhopalocarpus
similis
RHOPALOCARPACEAE HAZONDRINGITRA Fruit 5.5 4.8 30.8 17.1 2.6 3.4
Sorindeia
madagascariensis
ANACARDIACEAE VOATSIRINDRANA Fruit 4.9 4.2 16.8 10.2 3.1 3.6
Sorindeia
madagascariensis
ANACARDIACEAE VOATSIRINDRANA Flowers 9.6 9.4 8.3 6.5 3.4 4.2
Strychnos
madagascariensis
LOGANIACEAE VAKAKOA Leaf
buds &
leaves
19.6 17.6 19.8 12.8 4.8 4.0
Strychnos
madagascariensis
LOGANIACEAE VAKAKOA Leaves 9.0 7.5 33.1 23.3 4.3 3.7
Tabernaemontana
coffeoides
or
Mimusops
spp.
APOCYNACEAE
or
SAPOTACEAE
HAZOPIKA Leaves 11.5 10.1 27.2 21.6 4.8 3.9
Tectonia grandis
VERBENACEAE KESIKA Fruit 6.2 4.7 78.5 64.6 4.1 2.7
Terminalia boivinii
COMBRETACEAE AMANINOMBY Leaves 8.2 7.5 52.9 43.7 3.3 2.9
AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; NPGE = non-protein gross energy; ME =
metabolizable energy; ash = total minerals.
+Botanical names and vernacular names have been provided by previous researchers, local guides, and
published sources.
*Annotated botanical names were initially unidentied specimens associated only with Malagasy vernacular names. Consultations
with Missouri Botanical Garden - Madagascar and Université d’Antananarivo - Faculté des Sciences resulted in translated
suggestions of possible species endemic to the Ankarafantsika National Park region and within the distribution range of
P. coquereli
and are not based on taxonomic identication of actual plant specimens.

Page 10/20
+Botanical Name
(genus + specic
epithet)
Botanical
Family
+Malagasy
Vernacular Name
Plant
Part CP
(%)
AP
(%)
NDF
(%) ADF
(%) Ash
(%)
ME
(kcal/g)
Treculia perrieri
MORACEAE TSITIPAHA Fruit 13.2 10.3 30.1 25.1 10.0 3.4
Trilepisium
madagascariense
MORACEAE KILILO Leaves 11.1 10.3 27.9 19.0 6.5 3.2
Unidentied UNIDENTIFIED UNKNOWN FALLEN
TREE Bark 2.5 0.6 81.2 65.1 n/a 2.4
Unidentied UNIDENTIFIED LIANA UNKNOWN LIANA Leaves 20.5 19.5 22.2 12.1 7.2 3.9
Unidentied UNIDENTIFIED LIANA UNKNOWN LIANA Leaves 16.6 15.5 30.8 25.7 10.0 3.7
AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; NPGE = non-protein gross energy; ME =
metabolizable energy; ash = total minerals.
+Botanical names and vernacular names have been provided by previous researchers, local guides, and
published sources.
*Annotated botanical names were initially unidentied specimens associated only with Malagasy vernacular names. Consultations
with Missouri Botanical Garden - Madagascar and Université d’Antananarivo - Faculté des Sciences resulted in translated
suggestions of possible species endemic to the Ankarafantsika National Park region and within the distribution range of
P. coquereli
and are not based on taxonomic identication of actual plant specimens.

Exploratory statistics were used to describe the variation in sifaka foods. Principal component analysis (PCA) was conducted on the
nutrient values to reduce the number of parameters (CP, AP, NDF, ADF, GE, ME, and ash). Only axes with an eigen-value greater than one
were considered signicant. The PCA was considered signicant if Bartlett’s Test for Sphericity was signicant and the Kaiser-Meyer-
Olkin measure of sampling adequacy was greater or equal to 0.5 35. The number of signicant axes from the PCA was used to set the
k
value for the
k
-means cluster analysis on the same parameter set.
Results
Nutrient values for the 75 unique plant foods are given in Table 2. The sifakas selected foods representing 48 unique plant taxa with a
wide range of nutrient content. AP, digestible protein not bound in ber, ranged from 0.0–24.4%, with a mean of 10.3 ± 0.7% and median
of 10.2%. NDF ranged from 8.3–81.8% with a mean of 38.2 ± 2.1% and median of 34.8%. ADF ranged from 6.2–75.2% with a mean of
27.8 ± 1.8% and median of 23.7%. ME ranged from 1.92 kcal/g to 4.96 kcal/g, with a mean of 3.49 ± 0.07 kcal/g and median of 3.56
kcal/g. Ash (total minerals) ranged from 1.85–19.37%, with a mean of 4.95 ± 0.32% and median of 4.22%. Four foods showed seasonal
differences in nutrient composition, with the highest percentage of change in the amount of protein in manary (
Dalbergia trichophylla
)
fruit from the end of the lean to the beginning of the wet season (Table 1).
Except for bark, plant part does not categorize sifaka foods by nutrient composition, as all plant part categories had examples of high
and low values for all nutrients. For example, the mean and range of NDF content of leaves (35.8%, 17.1 – 81.8%) was virtually the same
as the mean and range of NDF for fruit (37.7%, 8.6 – 78.5%). Although the mean value for available protein for leaves (12.7±0.9%) was
numerically higher than that for fruit (7.6±1.3%), the range again was essentially identical for the two plant parts (0 – 24.4% and 0 –
22.0%). Bark contained mostly ber, with essentially no available protein (Table 2).
The best t PCA model contained only ve of the seven parameters (CP, AP, NDF, ADF, and ME). The best model found two signicant
axes (eigen-values greater than one) that can be categorized as high protein and high ber. These two axes (protein factor and ber
factor) explained 91.6% of the variation in nutrient content between the foods. Bartlett's Test of Sphericity was signicant (Chi-square =
435. 6, df = 10, p<0.001) and the Kaiser-Meyer-Olkin measure of sampling adequacy was 0.659, suggesting that sampling is adequate.
Estimated ME was signicantly negatively correlated with the ber factor score from the PCA (r = -0.867, p<0.001; Figure 1) but was not
associated with the protein factor score. Ash was positively correlated with the protein factor score (r = 0.314, p = 0.012) but was not
correlated with the ber factor score.

Page 11/20
The cluster analysis had
k
set to 2 based on the number of signicant axes from the PCA. Cluster 1 foods (N=52) were higher in AP and
lower in ber (Table 3). The foods in cluster 2 (N=14) were higher in ber and lower in estimated ME (Table 3). Nine foods could not be
ascribed to a cluster because they were missing GE data, and thus an estimated ME could not be calculated. Figures 2 through 4 display
how the foods in the two clusters differ. Cluster 1 foods displayed a positive correlation between the protein and ber factor scores (r =
0.580, p<0.001, Figure 2) while cluster 2 foods showed no association (r = -0.136, p=0.642, Figure 2). Both cluster 1 and cluster 2 foods
had negative correlations between estimated ME and the ber factor score (r = -0. 728, p<0.001 and r = -0.855, p<0.001). Cluster 1 foods
had a tendency for estimated ME to be negatively associated with the protein factor score (r = -0.270, p=0.053), but there was no
association between estimated ME and the protein factor score for cluster 2 foods.
Table 3
Nutrients in wild plant foods consumed by
P. coquereli
compared to components of captive lemur diet supplements
Cluster Number Analysis Parameter CP
(%)
AP
(%)
NDF
(%)
ADF
(%)
Ash
(%)
GE
(kcal/g)
ME
(kcal/g)
1:
High AP/ Low Fiber
Mean 13.4 12.0 31.1 22.0 5.0 4.7 3.6
 N 52 52 52 52 50 52 52
 Std. Error of Mean 0.76 0.73 1.47 1.17 0.26 0.06 0.06
 Median 13.1 11.1 30.5 21.5 4.8 4.6 3.6
2:
High Fiber/
Low ME
Mean 5.6 3.3 66.4 52.7 5.0 4.4 2.5
 N 14 14 14 14 13 14 14
 Std. Error of Mean 0.65 0.74 3.43 4.07 1.35 0.08 0.13
 Median 5.7 3.3 67.7 50.9 3.4 4.4 2.5
Total Mean 11.8 10.1 38.6 28.5 5.0 4.6 3.4
 N 66 66 66 66 63 66 66
 Std. Error of Mean 0.73 0.74 2.25 1.99 0.34 0.05 0.08
 Median 11.0 10.1 35.6 24.8 4.3 4.6 3.4
Marion+Guaranteed Analysis ≥ 23%  ≥ 21% 13% -16% ≤ 7%  
Mazuri*Guaranteed Analysis‡≥ 23%   ≤ 14%‡≤ 9%  
CP= crude protein; AP = available protein; NDF = neutral detergent ber; ADF = acid detergent ber; ash = total minerals; GE = gross
energy; ME = metabolizable energy.
+Marion Zoological Inc., Plymouth, Minnesota, USA. SKU# LEL B25, Leaf Eater Foods Biscuit
*Mazuri Exotic Animal Nutrition, St. Louis, Missouri, USA. SKU# 0001472 and 0001448, Leaf-Eater Primate Diet, Biscuit and Mini-
Biscuit, respectively.
‡Note: Mazuri's guaranteed analysis for ber is measured as Crude Fiber (CF). NDF and ADF values are not publicly available.

Figure 3 displays how cluster 1 foods are lower in ADF (though there is overlap) and both higher and more variable in estimated ME.
Figure 4 displays the lower and less variable AP for cluster 2 foods. In addition, there is no relationship between AP and ADF for cluster 1
foods (Figure 4), but a signicant decline in AP with ADF for cluster 2 foods (r = -0.717, p=0.004). The ratio of AP to CP differed between
clusters 1 (0.88±.01) and 2 (0.53±0.1; p<0.001), indicating that a greater percentage of protein was bound to the ADF fraction for cluster
2 foods. Cluster 2 foods had a lower protein-to-ber ratio whether expressed as CP-to-NDF (0.48±0.04 versus 0.09±0.01, p<0.001) or CP-
to-ADF (0.70±0.05 versus 0.12±0.02, p<0.001).

Page 12/20
Cluster 2 foods were comprised of all 4 bark samples, 6 of 18 fruit samples, 4 of 29 leaf samples, but no buds or owers. There were 6
samples, 3 from cluster 1 and 3 from cluster 2, that overlap in the ber and protein factor space (Figure 2). The cluster 1 foods were
lower in NDF (44.3±0.7% versus 50.4±0.7%, p=0.004) with no overlap, but otherwise did not differ from the cluster 2 foods (Table 4).
Table 4
Foods from clusters 1 and 2 overlapping in the protein factor-ber factor space+
Botanical Name Plant Part CP
(%)
AP
(%)
NDF (%) ADF (%) Ash
(%)
ME (kcal/g)
Abrahamia ditimena
Leaves 7.9 5.2 45.0 36.6 4.8 3.4
Combretum
spp. Fruit 7.3 6.5 45.1 35.2 3.5 3.3
Cynanchum
spp. Flowers 6.1 5.6 42.9 38.4 n/a 3.0
Mean Cluster 1* 7.1
±0.54
5.8
±0.38
44.3
±0.71
36.7
±0.94
4.2
±0.69
3.2
±0.12
Grangeria porosa
Fruit 8.6 6.9 50.6 40.7 3.7 3.5
Landolphia gummifera
Fruit 3.8 3.2 51.6 34.5 2.1 2.9
Mammea punctata
Leaves 8.2 6.6 49.1 38.4 3.5 3.7
Mean Cluster 2* 6.8
±1.54
5.6
±1.18
50.4
±0.74
37.9
±1.78
3.5
±0.48
3.4
±0.22
+See Figure 2.
*Foods differed by cluster in neutral detergent ber (NDF) (F=35.769, df=1, p=0.004).
Discussion
We found that lactating
P. coquereli
exploited a nutritionally diverse set of foods that varied widely for all measured nutrients and
included many high ber foods. The PCA indicated that available protein, ber and metabolizable energy accounted for over 91% of the
variation among these foods. Our analysis revealed two potential categories of foods in our dataset, visually represented in Figure 2.
The relationship between ME and ADF (Figure 3) and AP and ADF (Figure 4) visually demonstrates the separation between the clusters
for ber. However, estimated ME and AP shows considerable overlap between the two clusters, suggesting that sifaka foods could be
described by a nutritional gradient. This approach is supported since some foods were moderate to higher in protein and metabolizable
energy while lower in ber, and other foods were lower in protein and metabolizable energy while higher in ber. The gradient approach
may better reect the continuous nature of nutrient values, particularly for foods on the cluster boundaries (Figures 1, 2, and 4). However,
the six foods overlapping in protein and ber factor space do differ in NDF (Table 4) and the two clusters vary in the proportion of ber
bound to ADF. Both these factors support the hypothesis that these foods cluster into at least two nutritionally distinct groups. We
propose that these two food types will have different physiological and metabolic effects, with cluster 1 foods contributing more to the
ingesting sifaka’s nutritional status directly while cluster 2 foods will affect nutritional status through effects on the sifaka gut
microbiome.
Protein and ber were the most consistently variable nutrients in the sifaka foods, which also varied considerably in the protein-to-ber
ratio. Primates are estimated to require a minimum of 14% protein per dry matter basis for reproduction, 7—11% for growth and
development 36, and 6.4—8% crude protein in their diet to satisfy maintenance nutritional requirements 37. The cluster 1 foods consumed
by lactating
P. coquereli
had a mean of 12.0% available protein, which exceeds minimum protein requirements for primate maintenance,
and growth and development, while nearly meeting the estimated reproductive nutritional requirements. Cluster 1 foods had a high ratio
of AP-to-CP, supporting the hypothesis that they are good protein sources.
Lactating
P. coquereli
appear to have a diet quite high in ber (means of 38.2% NDF, 27.8% ADF) with a relatively low protein-to-ber ratio
without experiencing adverse effects and routinely consumed high ber foods during the lean season (Table 3). Frequently consumed
foods of gestating ring-tailed lemurs (
Lemur catta
) during the dry season contained less than 21% ADF 3. During lactation, eight of ten

Page 13/20
of the most frequently consumed foods contained less than 30% ADF, while none of the foods contained over 50% ADF 3. The black-and-
white ruffed lemur (
Varecia variegata
), consumed fruits, leaves and owers with ADF content of approximately 30% 38. The average ADF
content of leaves eaten by the larger-bodied Indri (
Indri indri
) was 53%, and the fruit, leaves and owers consumed by diademed sifakas
(
Propithecus diadema
) averaged between 30 and 50% ADF 39. The ber levels for these larger lemur species are comparable to our
results for
P. coquereli
. Although, the sifakas did include many high protein/low ber foods in their diet, suggesting that exploiting
different foods has functionally distinctive physiological and metabolic consequences.
High ber food consumption may be a residual effect of lactating
P. coquereli
unselectively exploiting the foods available in the forest
during the lean season. We emphasize that this also has biological relevance, since it provides an assessment of seasonally available
nutrients consumed during the critical period of infant development. During the lean season in a dry deciduous forest the availability of
foods high in available protein and metabolizable energy may be insucient, thereby constraining females to select dicult to digest
resources to meet energy requirements. Perhaps the increased demand placed by lactation in conjunction with the food constraints of
the lean season force sifakas living in dry deciduous forest to ingest the high ber foods.
However,
Propithecus
spp. are hindgut fermenters 5 with highly specialized gut microbiomes that vary depending on seasonal fruit
availability 40. Dietary plant ber only become nutritious after its microbial conversion into vital nutrients like short-chain fatty acids 41,
facilitated by specic cellulose-degrading microbes present in the sifaka gut and an increased functional capacity for ber metabolism
42. The specialized morphology of hindgut fermenters (enlarged caecum and elongated colon) could enable the ecient digestion of
brous materials, increasing nutrient extraction from dicult to digest resources. Our ndings are consistent with previous studies that
have shown sifakas to be seasonally exible folivores, a novel dietary strategy that may mitigate potential energetic decits 43–45.
Recent evidence demonstrates sifakas possess molecular adaptions to folivory including rapidly evolving gene pathways that aid in
xenobiotic metabolism and nutrient absorption, which may assist in the detoxication of plant compounds while maximizing nutritional
gain from leaves 46. This capacity for augmented nutrient uptake 46 would be advantageous to foraging throughout periods of
pronounced seasonality in Madagascar 18,47.
Variation in dietary ber is a critical component to understanding gut microbiomes in folivores and has been shown to affect microbial
diversity in
P. coquereli
42. Sifaka gut microbiomes have been found to be signicantly richer and more diverse in comparison to
generalist and frugivorous lemurs 42. Less inter-individual variation in sifaka gut microbiomes is exhibited relative to frugivorous
V.
variegata
and generalist
L. catta
, suggesting that sifakas may be less exible in terms of their diet 42 and more susceptible to habitat
disturbance 48.
Captive
P. coquereli
provisioned with a more diverse diet that included local wild plant species had signicantly richer, more diverse gut
microbiomes in comparison to when their standard diet was supplemented with winged-sumac only 48. Signicantly higher
concentrations of short-chain fatty acids, including acetate and propionate, and moderately greater concentrations of butyrate were
present in
P. coquereli
colonic metabolomes when provisioned a more diverse diet 48. Additionally, the same study found that individuals
given the opportunity to forage more naturally in forested enclosures, even for limited durations, maintained greater gut microbiome
diversity relative to conspecics without forest access (Green et al., 2018). This supports that ber consumption can have a profound
inuence on gut microbiome structure and function. It is possible that a high-ber diet is a requirement for sifakas to maintain their
coevolved microbiota. We posit that many if not all the cluster 2 foods in our study may have a greater effect on the sifaka gut
microbiome than a direct nutritional effect on the host animal. In other words, cluster 2 foods may be important for maintaining gut
health by feeding the microbiome, while cluster 1 foods more directly affect the nutritional plane of the sifakas.
Sifakas are exceptionally dicult to maintain in captivity due to their specialized digestive anatomy and highly folivorous diet 49,50. Our
results suggest that incorporating high-ber foods (ADF greater than 30% or even 40%) into captive diets would better replicate foods
consumed in the wild. Table 3 highlights two leading commercial products for leaf-eating primates in various life cycle stages, health,
and seasonality versus our eld data collected on lactating sifakas during the lean season. The commercial supplements contain higher
concentrations of protein (CP) and lower concentrations of ber. Both Marion and Mazuri provide their products as supplements to
foraging and non-foraging fruit and vegetable produce diets. Because of this, percent nutritional values of the various nutrients do not
represent the overall lemurs’ diet, but only that of the commercial product itself. Similarly, food selection in the wild depends on
environmental factors and does not necessarily reect the ideal composition for the health of sifakas without food supply constraints as
in captivity. While we acknowledge the limitations of juxtaposing a partial wild diet to a partial captive diet, it is presented here to
highlight the importance of incorporating nutritional diversity in captive diet design based on wild plant foods acquired by lemurs. We

Page 14/20
suggest that incorporating foods like the cluster 2 foods in this study may be helpful for dietary management of captive sifakas,
possibly by improving gut health through effects on the microbiome.
Consistent with previous studies 11,29,51−53, our results conrm that botanical category (e.g., fruit versus leaf) is a poor means by which
to assess the nutritional contribution a food will make to animals that consume it. Fruit is often equated with high water and high non-
structural carbohydrate (sugar) content; however, wild fruits can be substantially different in nutrient prole from domesticated fruits,
and often are similar to leaves, buds, and owers, as seen in our study. The fruits in this study were not different from leaves in ber
content. The NDF content of fruit in our study ranged from 8.6–78.5% and the mean NDF for fruit (37.7%) was numerically higher than
the mean NDF for leaves (35.8%). Sifakas ingest high ber foods, whether those foods are classied as leaves, fruit, owers, or buds.
Our results also conrm that wild plant foods can vary seasonally in nutrient content, cautioning that the nutritional consequences of
consuming some foods can differ by time of year.
In summary, infant-bearing
P. coquereli’s
employ a mixed-diet strategy consuming foods with wide ranges in percent nutrient content to
compensate for nutrient deciencies in multiple plant parts and food availability. Food sources clustered into two categories: high in
protein and low-to-moderate in ber; or high in ber and low in metabolizable energy.
Declarations
Acknowledgements
We thank Madagascar National Parks, Ankarafantsika National Park and the Ministere de l’Environnement et des Forets for permission
to conduct our research. A colossal thank you to Ravalohery Fara Nomena and Njaka Frankin for your expertise and perseverance
collecting plants. This project would not have been possible without the both of you. We thank Benjamin Andriamihaja, MICET staff,
Rakotondradona Remi, Razaiarimanana Jacqueline, and Missouri Botanical Garden-–Madagascar for invaluable in-country assistance.
Thank you to Robert Lund, Armand Randrianasolo, Sylvie Andriambololonera, Harison Rabarison, Justin Rakotoroa, Jhoanny Rasojivola,
Parc Botanique et Zoologique de Tsimbazaza, Missouri Botanical Garden– St. Louis and Madagascar, and the Université d'Antananarivo
– Faculté des Sciences for plant identications. Thank you to Lalao Andriamahefarivo, Herisoa Manjakahery, and Faranirina
Lantoarisoa for your assistance with plant exports. We thank Michael Jakubasz and Michael Maslanka for your support that began in
Madagascar and continued in Washington, D.C. A gracious thank you to Christina Petzinger, Cari Lewis, Nicole Johnson, Jessica Cooper,
Katie Murtough and DaeKyu Lee for your diligent lab assistance. Thank you to Robert Lund, Shawn Lehman, Julie Teichroeb, Michael
Schillaci, Rebecca Stumpf, and Becky Raboy for your exceptional insights and feedback on this project. We thank the anonymous
reviewers from the
International Journal of Primatology
that commented on earlier versions of the manuscript.
Author Contributions
ACR designed the project and collected the data. ACR and MLP assayed the samples. MLP conducted statistical analyses. ACR and
MLP co-wrote the manuscript.
Statement of Ethics
This research complied with protocols approved by the University of Toronto Animal Care Committee (Protocol #: 2000), adhered to the
legal requirements of Madagascar and followed the American Society of Primatologists’ Principles for the Ethical Treatment of
Primates. Research permits were issued in Madagascar by the Ministry of Environment, Water and Forests (Permit #:
N°239/11/MEF/SG/DGF/DCB SAP/SCB). Export permits were issued in Madagascar by the Director of Natural Resources. Imports were
approved by the United States Department of Agriculture and U.S. Fish and Wildlife Service.
Funding Sources
Funding was provided by: Primate Conservation, Inc. Research Grant #920, American Society of Primatologists Conservation Committee
Small Grant, The Explorers Club Exploration Fund, and the Department of Anthropology/Department Graduate Fellowships and Awards
Committee Research Funds- University of Toronto.
Data Availability Statement
The data presented here are represented by Table 1. The full data set is available in ACR’s dissertation.

Page 15/20
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Figures
Figure 1
Relationship between metabolizable energy (ME) and ber by cluster in foods consumed by lactating
P. coquereli

Page 18/20
Figure 2
Relationship between protein and ber by cluster in foods consumed by lactating
P. coquereli

Page 19/20
Figure 3
Relationships between metabolizable energy (ME) and acid detergent ber (ADF) of foods consumed by lactating
P. coquereli
determined from PCA followed by cluster analysis

Page 20/20
Figure 4
Relationship between available protein (AP) and acid detergent ber (ADF) of foods consumed by lactating
P. coquereli
determined from
PCA followed by cluster analysis