A DNA Metabarcoding Study of a Primate Dietary Diversity and Plasticity across Its Entire Fragmented Range

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.     

Non-invasive conservation genetics of the critically endangered golden-crowned sifaka (<Emphasis Type="Italic">Propithecus tattersalli</Emphasis>): high diversity and significant genetic differentiation over a small range

The genetic structure of the golden-crowned sifaka (Propithecus tattersalli) was evaluated in three forest types in the Daraina region between the Loky and Manambato Rivers in northeastern Madagascar. This critically endangered lemur species, only found within this restricted region (~1,300 km²), comprises populations which survive in forest fragments of varying size. While this remote region is already affected by human deforestation, the scheduled paving of the national road can potentially bring significant changes in the near future. In 2006, sifakas feces from 82 putative individuals were collected immediately after defecation. DNA samples were genotyped at 13 microsatellite loci to characterize patterns of genetic diversity within and among three sets of forest fragments representing different ecological types. The recent 2CTAB/PCI extraction method and the ‘comparative’ genotype validation approach allowed us to significantly improve the efficiency of amplification procedures. We found that golden-crowned sifaka genetic diversity was surprisingly high despite its narrow distribution and the current level of habitat fragmentation. We used both spatial and non-spatial Bayesian clustering methods to determine if forest edges correlated with cryptic genetic clusters and found that it was overall the case. However, the results also indicate that migrations are possible between two of the three studied forest complexes despite the presence of the national road. This is probably due to remaining riparian and small forest patches between these two forest complexes. The third forest complex is more differentiated although some migrations/connections may still exist through non-sampled fragments. This strongly suggests that it is necessary to maintain corridors and small patches, and also to explore the genetic diversity of the fragments outside the protected area.     

Spatial variation in density and total size estimates in fragmented primate populations: the golden‐crowned sifaka (Propithecus tattersalli)

The golden‐crowned sifaka (Propithecus tattersalli) is an endangered lemur species found only in the Daraina region, a very restricted area in north‐eastern Madagascar. Its forest habitat is highly fragmented and expected to suffer from significant changes in the near future. The species is poorly known and only one census study, carried out in 2000, has ever been published. It is thus crucial to update the conservation status of the golden‐crowned sifaka before major anthropogenic environmental changes take place. Using the line‐transect approach, we estimated the species density in the main forest fragments located in both the peripheral and central parts of the distribution range, including both protected and unprotected areas. In parallel, we tried to determine whether an edge effect could be detected by comparing densities at different distances from the forest edges. We found important variation of sifaka densities among forest fragments. The total species abundance is thus difficult to determine, but we estimated that it is likely to be over 18,000, two to three times higher than previously thought. However, our data also suggested that most P. tattersalli live in forests located in the central part of the distribution range and that the estimated densities in the central part were high (>80 individuals/km²). Two forest fragments, found to host a large part of the total population, are currently outside the managed area and their incorporation to the managed area is strongly recommended. Lastly, as expected for a folivorous and not heavily hunted species, our results are consistent with the hypothesis that this species does not experience a clear edge effect, at least during the first half of the dry season. This could be due to a high resiliency to habitat fragmentation or to the fact that fragmentation has been going on for some time. Am. J. Primatol. 72:72–80, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of 20 microsatellites developed from <Emphasis Type="Italic">Propithecus deckeni coronatus</Emphasis> with cross-amplification in <Emphasis Type="Italic">Propithecus deckeni deckeni</Emphasis>

The crowned sifaka (Propithecus deckeni coronatus) is found between the Mahavavy the Betsiboka Rivers in either Anjahamena or Antrema Classified Forest. The crowned sifaka is endangered due to the loss and fragmentation of its natural habitat by deforestation. Twenty polymorphic nuclear microsatellite loci were isolated from genomic DNA derived from a P. d. coronatus from Anjahamena, Madagascar. Population genetic parameters were estimated on 10 individuals sampled from Anjahamena, Madagascar, to determine marker utility and preliminary baseline values to study the species’ genetic diversity and phylogeography. In a cross-amplification test, each marker in the suite was informative in P. d. deckeni.     

Characterization of 20 microsatellite marker loci in Coquerel's sifaka (Propithecus coquereli)

The Coquerel's sifaka (Propithecus coquereli) is one of the species of Propithecus, living in the dry forest of northwest Madagascar. This species is endangered due to the loss and fragmentation of its natural habitat, consequential of deforestation. Twenty nuclear microsatellite loci were isolated from genomic DNA derived from a free ranging Coquerel's sifaka from the Mariarano Classified Forest, Madagascar. Population genetic parameters were estimated as baseline values from samples collected in that forest.     

Characterization of 13 microsatellite marker loci in Verreaux's sifaka (Propithecus verreauxi)

The Verreaux's sifaka (Propithecus verreauxi) is one of the species of Propithecus, living in the dry forest of southwest Madagascar. This species is endangered due to the loss and fragmentation of its natural habitat, a consequence of deforestation. Thirteen novel nuclear microsatellite loci were isolated and characterized in three populations of Verreaux's sifaka. The marker suite proved informative with an average of 8.9 alleles per locus and observed heterozygosity across the three populations of 0.675.     

Relative growth of the limbs and trunk in sifakas: Heterochronic,ecological, and functional considerations

Limb, trunk, and body weight measurements were obtained for growth series of Milne-Edwards's diademed sifaka, Propithecus diadema edwardsi, and the golden-crowned sifaka, Propithecus tattersalli. Similar measures were obtained also for primarily adults of two subspecies of the western sifaka: Propithecus verreauxi coquereli, Coquerel's sifaka, and Propithecus verreauxi verreauxi, Verreaux's sifaka. Ontogenetic series for the larger-bodied P. d. edwardsi and the smaller-bodied P. tattersalli were compared to evaluate whether species-level differences in body proportions result from the differential extension of common patterns of relative growth. In bivariate plots, both subspecies of P. verreauxi were included to examine whether these taxa also lie along a growth trajectory common to all sifakas. Analyses of the data indicate that postcranial proportions for sifakas are ontogenetically scaled, much as demonstrated previously with cranial dimensions for all three species (Ravosa, 1992). As such, P. d. edwardsi apparently develops larger overall size primarily by growing at a faster rate, but not for a longer duration of time, than P. tattersalli and P. verreauxi; this is similar to results based on cranial data. A consideration of Malagasy lemur ecology suggests that regional differences in forage quality and resource availability have strongly influenced the evolutionary development of body-size variation in sifakas. On one hand, the rainforest environment of P. d. edwardsi imposes greater selective pressures for larger body size than the dry-forest environment of P. tattersalli and P. v. coquereli, or the semi-arid climate of P. v. verreauxi. On the other hand, as progressively smaller-bodied adult sifakas are located in the east, west, and northwest, this apparently supports suggestions that adult body size is set by dry-season constraints on food quality and distribution (i. e., smaller taxa are located in more seasonal habitats such as the west and northeast). Moreover, the fact that body-size differentiation occurs primarily via differences in growth rate is also due apparently to differences in resource seasonality (and juvenile mortality risk in turn) between the eastern rainforest and the more temperate northeast and west. Most scaling coefficients for both arm and leg growth range from slight negative allometry to slight positive allometry. Given the low intermembral index for sifakas, which is also an adaptation for propulsive hindlimb-dominated jumping, this suggests that differences in adult limb proportions are largely set prenatally rather than being achieved via higher rates of postnatal hindlimb growth. Our analyses further indicate that the larger-bodied P. d. edwardsi has a higher adult intermembral index than P. tattersalli and P. verreauxi, thus supporting the allometric argument regarding the interspecific scaling of limb proportions in arboreal primates which employ vertical postures (Cartmill, 1974,1985; Jungers, 1978, 1985). Lastly, additional analyses indicate that P. d. edwardsi exhibits significant sexual dimorphism where adult females are larger than adult males in about one-third of all adult comparisons, whereas P. tattersalli exhibits significant sexual dimorphism in about one-fifth of all adult comparisons. Among western sifakas, adult P. v. coquereli exhibit significant sex dimorphism in about one-third of all comparisons, whereas adult P. v. uerreauxi show no significant differences between the sexes. Given that all taxa are ontogenetically scaled, this suggests that sexual dimorphism develops via such processes as well. Interestingly, our data indicate that sex dimorphism is allometric, with larger-bodied taxa like P. d. edwardsi being more dimorphic.     

Characterization of 21 microsatellite marker loci in the silky sifaka (Propithecus candidus)

The silky sifaka, Propithecus candidus, considered one of the rarest and most endangered primates in the world, exists in only a few fragmented forests in northeastern Madagascar. This species faces increasing pressures as a direct result from loss of habitat in the form of tavy (slash and burn agriculture), illegal logging and mining along with hunting for subsistence, even within protected areas. We report a marker suite of 21 loci developed from genomic DNA from a silky sifaka collected in 2003 from Anjanaharibe-Sud Special Reserve. Polymorphism of each locus evaluated in 18 individuals pooled from Marojejy National Park and Anjanaharibe-Sud Special Reserve. The number of observed alleles per locus ranges from 2 to 7. The observed and expected heterozygosity were 0.389–0.889 and 0.322–0.789, respectively. The information revealed in this study will provide useful tools for further study of the social structure and population dynamics of the silky sifaka to facilitate conservation management in the imminent future.     

Craniodental Characters in the Taxonomy of Propithecus

The indriid genus Propithecus comprises the sifakas, medium-sized lemurs endemic to the forests of Madagascar. Traditionally, scientists divided the genus into only 2 or 3 species —Propithecus diadema, P. verreauxi, and, since 1988, P. tattersalli— with 4 or 5 subspecies in each of the first 2 taxa, but recent authors have suggested that many more distinct species should be recognized. We draw from quantitative and qualitative studies of craniodental traits to evaluate further the phenetic distinctiveness and taxonomic status of each named form of Propithecus. We recognize 9–10 species in the genus. The 4 or 5 species of the Propithecus diadema group —P. diadema, P. candidus, P. perrieri, P. edwardsi, and perhaps P. holomelas, if distinct— share several derived features, including large average body size and a mandible specialized for rotational chewing, and clearly comprise a closely related complex. The 5 species of the Propithecus verreauxi group —P. verreauxi, P. coquereli, P. deckenii, P. coronatus, P. tattersalli— are each highly distinctive morphologically and likely do not comprise a monophyletic group. In particular, we point out the highly distinctive cranial features of Propithecus coronatus, which researchers have traditionally largely overlooked.     

Infant development and parental care in two species of sifakas

This paper reports the results of a three-month field study on parental care and infant development in the diademed sifaka (Propithecus diadema edwardsi) in the primary rain-forest of south-eastern Madagascar. They are compared with a three-and-a-half-month study of captive white sifaka (Propithecus verreauxi coquereli) in the Duke University Primate Centre. Records were taken by means of focal animal and instantaneous sampling. In both species the mother was the primary carrier and caretaker; theP. verreauxi father carried the infant significantly more than did any animal other than the mother in theP. diadema group. The infantP. verreauxi spent less time off the mother than didP. diadema from week 4 through week 10. It is concluded thatP. verreauxi shows more non-maternal care thanP. diadema and also develops at a slower rate. The difference in the two species' habitats is discussed as a possible cause.     

Climate change and human colonization triggered habitat loss and fragmentation in Madagascar

The relative effect of past climate fluctuations and anthropogenic activities on current biome distribution is subject to increasing attention, notably in biodiversity hot spots. In Madagascar, where humans arrived in the last ~4 to 5,000 years, the exact causes of the demise of large vertebrates that cohabited with humans are yet unclear. The prevailing narrative holds that Madagascar was covered with forest before human arrival and that the expansion of grasslands was the result of human‐driven deforestation. However, recent studies have shown that vegetation and fauna structure substantially fluctuated during the Holocene. Here, we study the Holocene history of habitat fragmentation in the north of Madagascar using a population genetics approach. To do so, we infer the demographic history of two northern Madagascar neighbouring, congeneric and critically endangered forest dwelling lemur species—Propithecus tattersalli and Propithecus perrieri—using population genetic analyses. Our results highlight the necessity to consider population structure and changes in connectivity in demographic history inferences. We show that both species underwent demographic fluctuations which most likely occurred after the mid‐Holocene transition. While mid‐Holocene climate change probably triggered major demographic changes in the two lemur species range and connectivity, human settlements that expanded over the last four millennia in northern Madagascar likely played a role in the loss and fragmentation of the forest cover.     

Berenty 2006: Census of <Emphasis Type="Italic">Propithecus verreauxi</Emphasis> and Possible Evidence of Population Stress

We provide a survey of a folivorous lemur, Propithecus verreauxi (sifaka), in the Berenty Reserve, southern Madagascar. Higher densities of folivores in small patches occur in either high-quality food areas or in less disturbed refugia. The skewness of sex ratio, which in lemurs is often male-biased, can be critically exacerbated in population stress. We predicted that sifaka would show higher densities in areas where protein-rich food is abundant (prediction 1a) and in refugium areas (prediction 1b). Owing to increased competition by brown and ring-tailed lemurs and decreased food production by tamarind trees, we expected an extremely male-biased sex ratio (prediction 2). In November–December 2006, we counted and sexed 206 adult/subadult sifaka (49 groups) during daily walks in different forest zones (Ankoba secondary forest, to the north, Malaza gallery/scrub areas, and spiny forest, to the south). Sifaka may have decreased in the gallery forest to concentrate in Ankoba (in a sort of out-of-Malaza). The area contains protein-rich food (prediction 1a confirmed). Sifaka are proportionally more concentrated in the spiny area than in the degraded scrub forest (prediction 1b confirmed). The sex ratio is extremely male biased, possibly due to either high sifaka density, in Ankoba, or food availability reduction, in Malaza (prediction 2 confirmed). The sifaka population seems to be under stress: researchers need to resume demographic studies, interrupted in Berenty in the mid-1980s, to preserve in situ a species that is difficult to protect ex situ.     

Landscape genetics of an endangered lemur (Propithecus tattersalli) within its entire fragmented range

Habitat fragmentation may strongly reduce individuals’ dispersal among resource patches and hence influence population distribution and persistence. We studied the impact of landscape heterogeneity on the dispersal of the golden‐crowned sifaka (Propithecus tattersalli), an endangered social lemur species living in a restricted and highly fragmented landscape. We combined spatial analysis and population genetics methods to describe population units and identify the environmental factors which best predict the rates and patterns of genetic differentiation within and between populations. We used non‐invasive methods to genotype 230 individuals at 13 microsatellites in all the main forest fragments of its entire distribution area. Our analyses suggest that the Manankolana River and geographical distance are the primary structuring factors, while a national road crossing the region does not seem to impede gene flow. Altogether, our results are in agreement with a limited influence of forest habitat connectivity on gene flow patterns (except for North of the species’ range), suggesting that dispersal is still possible today among most forest patches for this species. Within forest patches, we find that dispersal is mainly among neighbouring social groups, hence confirming previous behavioural observations.     

Characterization of twenty-one microsatellites developed from <Emphasis Type="Italic">Propithecus diadema</Emphasis>

Diademed sifaka (Propithecus diadema) distributed throughout Madagascar’s eastern rain forests from the Mangoro and Onive Rivers north to the Mananara River. Twenty-one polymorphic nuclear microsatellite loci were isolated from genomic DNA derived from a P. diadema, from Mantadia National Park. Population genetic parameters were estimated on 10 individuals each, sampled from Mantadia National Park and Maromizaha Unprotected Forest. Allele sizes ranged from 4 to 14 and observed heterozygosities ranged from 0.200 to 0.900 per locus. This marker suite will be informative in further population genetic studies.     

Characterization of 21 microsatellite marker loci in the Milne-Edwards’ Sifaka (Propithecus edwardsi)

Milne-Edwards’ Sifaka (Propithecus edwardsi) is a dark furred lemur with white flanks living in the southern region of the eastern rainforest belt of Madagascar. This species is endangered due to habitat destruction primarily from deforestation. Twenty-one polymorphic nuclear microsatellite loci were isolated from a genomic DNA derived from an individual from Ranomafana National Park, Madagascar. Population genetic parameters were estimated on 10 individuals each, from Andringitra National Park and the Vatoharana Forest in Ranomafana National Park.     

Evidence of Invasive Felis silvestris Predation on Propithecus verreauxi at Beza Mahafaly Special Reserve, Madagascar

Increasing evidence supports the idea that endemic avian and mammalian predators have profoundly impacted primate populations in Madagascar (Goodman, S. M. Predation on lemurs. In S. M. Goodman, & J. P. Benstead (Eds.), The natural history of Madagascar (pp. 1221–1228). Chicago: University of Chicago Press, (2003).). The role in regulating lemur populations of the 3 introduced mammalian carnivorans —small Indian civets (Viverricula indica, Desmarest 1804), domestic dogs (Canis lupus familiaris, Linnaeus 1758), and invasive wildcats (Felis silvestris, Schreber 1775)— is less clear, but recent evidence suggests that the latter 2 are becoming important predators of diurnal lemurs. We report evidence for invasive wildcat predation on sifaka (Propithecus verreauxi verreauxi) in Parcel 1 at Beza Mahafaly Special Reserve, Madagascar, including skeletal remains of apparent Propithecus sifaka victims, observations of wildcat predatory behavior, and behavioral responses of the lemurs in the presence of wildcats.     

Interspecific Responses of Ringtailed Lemurs to Playback of Antipredator Alarm Calls Given by Verreaux's Sifakas

The ringtailed lemur, Lemur catta, and Verreaux's sifaka, Propithecus verreauxi verreauxi, are diurnal prosimians living sympatrically in Madagascar. Species-specific alarm calls emitted by each of these two species in response to aerial and terrestrial predators differ acoustically. Behavioural responses of ringtailed lemurs evoked by playbacks of conspecific alarm calls differ when the vocalizations were produced in response to aerial predators as opposed to terrestrial predators. We conducted playback experiments on two populations of ringtailed lemurs, using two types of sifaka alarm calls. One population consisted of free-ranging groups which lived sympatrically with sifakas, the other was a colony group which had no contact with sifakas. The results illustrate that the former group of lemurs can perceive what type of predators the sifaka calls refer to, whereas the latter group was not able to recognize the difference in the calls.     

Characterization of 21 microsatellite marker loci in the ring-tailed lemur (<Emphasis Type="Italic">Lemur catta</Emphasis>)

Ring-tailed lemur (Lemur catta) is the only species in the Genus Lemur, distributed in the deciduous and spiny forests of southwestern Madagascar. This species is listed as endangered due to the loss and fragmentation of its natural habitat, a consequence of deforestation. Twenty-one nuclear microsatellite loci were isolated from a genomic DNA derived from a free-ranging ring-tailed lemur population from the Tsimanampetsotsa National Park, Madagascar. We report various parameter estimates and measures to establish the utility of this marker suite as screened among individuals this single forest fragment.     

Characterization of 21 microsatellites developed from Propithecus deckeni deckeni

Decken's sifaka (Propithecus deckeni deckeni) is found between the Mahavavy and Manambolo Rivers, in western Madagascar. Twenty‐one polymorphic nuclear microsatellite loci were isolated from genomic DNA derived from a P. d. deckeni, from Tsingy de Bemaraha National Park. Population genetic parameters were estimated on 10 individuals each, sampled from Tsingy de Bemaraha National Park and Tsiombikibo Classified Forests. Significant null alleles were detected in seven loci which were dropped before population genetic parameters were re‐estimated and compared to the full marker suite.