(Propithecus Tattersalli) Within Its Entire Fragmented Range

Molecular Ecology (2010) doi: 10.1111/j.1365-294X.2010.04581.x

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

ERWAN QUE´ ME´ RE´ ,*† BRIGITTE CROUAU-ROY,*† CLE´ MENT RABARIVOLA,§ EDWARD E. LOUIS JR– and LOUNE` SCHIKHI†‡ †CNRS UMR EDB, Toulouse, France, *Universite´ Paul Sabatier UMR 5174 EDB, 118 route de Narbone Toulouse, France, ‡Instituto Gulbenkian de Cieˆncia, Rua da Quinta Grande, no. 6, 2780-156 Oeiras, Portugal, §Universite´ de Mahanjanga, Faculte´ des Sciences, Campus Universitaire Ambondrona BP 652 401 Mahajanga, Madagascar, –Center for Conservation and Research, Henry Doorly Zoo, 3701 South 10th Street, Omaha, NE 68107, USA

Abstract 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.

Keywords: causal modelling, dispersal, habitat fragmentation, lemur, primate, Propithecus tattersalli, spatial autocorrelation Received 10 April 2009; revision received 19 January 2010; accepted 28 January 2010

ham et al. 2002; Frankham 2005; Olivieri et al. 2008; Craul Introduction et al. 2009). It is believed that, in the medium or long Habitat loss and fragmentation are among the greatest term, it can lead to an increase in the level of inbreeding, threats to biodiversity worldwide (Myers et al. 2000; the erosion of species’ evolutionary potential, and an Ganzhorn et al. 2003), particularly so for tropical ecosys- increase in the risk of extinction (Saccheri et al. 1998; tems (Sala et al. 2000; Foley et al. 2005; Laurance & Peres Keller & Waller 2002; Johansson et al. 2007). Local extinc- 2006). They reduce habitat area, quality and connectivity tions of small fragmented populations are relatively for many wild species with potentially dramatic conse- common (Fahrig & Merriam 1994; Kindlmann & Burel quences for population viability (Andre´n 1994; Fahrig 2008). Consequently, persistence of species strongly 2003; Walker et al. 2008). In particular, it can lead to a depends on the ability of dispersing individuals to move rapid decline of population size and genetic variation across heterogeneous landscapes to re-colonize empty within habitat fragments (Crnokrak & Roff 1999; Frank- patches and maintain the functional connections among remaining populations. One aim of conservation is to Correspondence: E. Que´me´re´, Fax: (+33) 561 557 327; identify the most suitable areas in the matrix (i.e. puta- E-mail: [email protected] and L. Chikhi, Fax: (+33) 561 557 327, tive corridors) to restore and maintain habitat connectiv- E-mail: [email protected] ity among populations (Taylor & Fahrig 2006; Cushman

2010 Blackwell Publishing Ltd 2 E. QUE´ ME´ RE´ ET AL. et al. 2009). Consideration of the matrix heterogeneity and particularly in the evergreen forests along the eastern and the level of permeability of its components to dis- coast and the deciduous forests of western Madagascar persal movements are thus key issues for the manage- (Green & Sussman 1990; Smith 1997; Harper et al. 2007). ment of endangered species with patchy distributions Most previous studies have focused on the impact of hab- (Taylor et al. 2006; Lindenmayer et al. 2008). itat fragmentation on species richness (Ganzhorn et al. The effects of environmental features on individual 2003; Scott et al. 2006; Dunham et al. 2008; Allnutt et al. dispersal are difficult to assess using mark-recapture or 2008) or on the distribution of species among and within radio-tracking approaches because a relatively large patches (Lehman et al. 2006; Irwin 2007; Que´me´re´ et al. number of individuals have to be captured and moni- 2010). However, for Malagasy species, the genetic conse- tored (Vignieri 2005; Cushman 2006; Broquet et al. 2006). quences of habitat fragmentation have been poorly stud- Furthermore, these direct methods are time consuming, ied (but see Olivieri et al. 2008; Radespiel et al. 2008a,b often invasive and thus not generally suitable for the Craul et al. 2009 for recent exceptions). The Daraina study of threatened species. Genetic data, which can be region, where P. tattersalli lives, is expected to suffer from obtained using non-invasive sampling methods, offer an significant disturbances in the near future and was iden- attractive alternative approach to infer patterns of dis- tified as a high-priority area for biodiversity conservation persal. Genetic methods can highlight the natural and (Ganzhorn et al. 1999; ZICOMA, 1999). While we have anthropogenic factors shaping migration patterns shown that the population densities were higher than between habitat patches (Holderegger & Wagner 2008; previously thought (Que´me´re´ et al. 2010), recent political Balkenhol et al. 2009). The genetic study of functional instability has led to a renewed level of hunting and habitat connectivity (i.e. the ‘degree to which the land- poaching. Thus, to facilitate conservation in this species, scape impedes or facilitates movements among resources studies focused on identifying the natural and anthropo- patches’, Taylor et al. 2006) has received growing interest genic factors influencing patterns of dispersal and levels principally because of the improvement of methodologi- of genetic diversity are needed. cal and technical tools for assessing habitat heterogeneity To characterize the P. tattersalli spatial genetic struc- (e.g. high-resolution satellite images, Geographic Infor- ture, we first combined field observations and remote mation Systems). This has favoured the emergence of sensing data within a Geographic Information System ‘Landscape genetics’ (Manel et al. 2003), a new interdisci- (GIS) to provide an a priori definition of the landscape plinary research area combining population genetics and heterogeneity in Daraina area. Specifically, we assessed landscape ecology for quantifying the effect of landscape (i) the extent of forest habitat and structural connectiv- composition, configuration and matrix quality on spatial ity and (ii) environmental features such as rivers, roads patterns in neutral and adaptive genetic variation (Storfer or riparian corridors that may impede or facilitate indi- et al. 2007; Holdegger & Wagner 2008). For instance, sev- vidual dispersal. To obtain the genetic data we used a eral recent landscape genetic studies used previously non-invasive sampling strategy (Taberlet et al. 1997; existing data with new statistical tools to uncover the Kohn et al. 1999) to genotype 230 individuals from the spatial genetic discontinuities corresponding to anthro- main forest patches of the golden-crowed sifaka range pogenic barriers such as roads or canals (Epps et al. 2005; using 13 microsatellites. We first assessed the level of Coulon et al. 2006; Perez-Barberia et al. 2008; Radespiel, genetic diversity across all individuals sampled and et al. 2008b). Others characterized environmental fea- then within each of the main inhabited forest patches. tures that facilitate migration such as riparian areas in To build a species-specific model of habitat suitability jumping mice (Vignieri 2005), high-slope areas in bighorn (i.e. a posteriori definition of the landscape reflecting sheep (Epps et al. 2007) or eucalyptus forests in small the golden-crowned sifaka behavioural response in marsupials (Banks et al. 2005). terms of distribution and dispersal), we employed two In this study, we investigated the impact of forest frag- individual-based approaches: (i) the commonly used mentation on patterns of genetic differentiation in the Bayesian clustering method STRUCTURE (Pritchard et al. golden-crowned sifaka (Propithecus tattersalli). This rela- 2000; Falush et al. 2003), which identifies panmictic sub- tively large, social species of lemur is found only in the population units (i.e. clusters) delineated by discrete Daraina region of north-eastern Madagascar (Fig. 1) and boundaries and (ii) a causal modelling approach based has one of the most limited ranges of any lemur on resemblance matrices (Legendre 1993; Cushman et (2450 km2) (Mittermeier et al. 2006). The island of Mada- al. 2006) which evaluates the ability of alternative gascar is among the most important biodiversity hotspot hypotheses to identify a landscape model (i.e. a combi- and conservation priority areas in the world (Myers et al. nation of environmental factors) that predicts rates and 2000; Ganzhorn et al. 2003). However, deforestation and patterns of gene flow. We compare the results from the habitat fragmentation have increased precipitously dur- two approaches and discuss their relevance for P. tat- ing the 20th century, primarily due to human pressure tersalli conservation strategies.

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 3

Fig. 1 Sampling distribution of golden- crowned sifakas. Distribution range of Propithecus tattersalli in the Daraina area. The green colour represents the forest fragments where we detected P. tattersalli individuals. Red circles correspond to the location of the sampled social groups. The commune chieftowns are represented by black squares. The national road is represented by a brown line and the main rivers by blue lines. The correspondence between circled letters and forest fragment complex name is indicated in Table 1.

Fanamby, in order to minimize direct anthropogenic Methods pressures such as intensive wood and mining exploita- tion. In practice, it appears that golden-crowned sifaka Study area and landscape structure characterization populations are present both within and outside the To capture the landscape heterogeneity in the Daraina SFUM (Vargas et al. 2002; Que´me´re´ et al. 2010). Finally, region and differentiate forest and non-forest vegetation it is important to note that the Daraina region, delin- areas, we conducted a landscape classification using a eated by the Loky and the Manambato rivers, is crossed LandSat Thematic Mapper imagery acquired in 2002 by a third large river (the Manankolana River) flowing and the maximum likelihood supervised method, south to northeast and by the unpaved national road implemented in the software ENVI 4.1. The accuracy of RN5A running southeast to northwest (Fig. 1). the classification was checked in the field and using SPOT imagery. The forest habitat in the Daraina region Sampling strategy and DNA analysis is highly fragmented, covering about 17% of the total area (44,000 ha) and with only 6% of the patches larger We conducted a non-invasive hierarchical sampling than 1000 ha (n = 9) and 20% larger than 100 ha design including social groups from across the species (n = 28) (Fig. 1). Forest patches (mainly lowland dry range. Faecal samples were collected in all the main for- forests) are surrounded by a heterogeneous matrix of est patches within the golden-crowned sifaka’s distribu- riparian gallery forests, agricultural areas, zebu cattle tion (Fig. 1). Within the larger patches, we also grazing pastures, human settlements and by large collected samples from several valleys, and within grasslands and dry scrubs. Some of the forest patches these valleys several neighbouring social groups were were included in 2005 in the Station Forestie`re a` Usages sampled to capture genetic variation at multiple spatial Multiples—’Multiple Usage Forest Station’ (SFUM) scales. We collected faecal pellets for genetic analyses Loky-Manambato managed by the Malagasy NGO during two field missions in 2006 and 2008. The data

2010 Blackwell Publishing Ltd 4 E. QUE´ ME´ RE´ ET AL. from 292 putative individuals belonging to 107 social for the first level analysis) using the admixture model groups are analysed here. All the samples were and assuming correlated allele frequencies (Pritchard et obtained immediately after defecation as described in al. 2000; Falush et al. 2003). We used a uniform prior for Que´me´re´ et al. (2009), and their geographical coordi- alpha, the parameter representing the degree of admix- nates were assigned at the faeces collection point. DNA ture, with a maximum of 10.0 and set Alphapropsd (the extraction was performed following the 2CTAB ⁄ PCI standard deviation of the proposal distribution) to 0.05. protocol adapted from Vallet et al. (2008). The extracted Lambda, the parameter representing the amount of cor- DNA was amplified using a set of 13 microsatellite loci relation in the parental allele frequencies, was set at 1.0 isolated for Propithecus tattersalli (8 loci) and for the and the prior Fst was set to the default values with a closely related species P. coquereli (5 loci) (see Que´me´re´ mean of 0.01 and a standard deviation of 0.05. For each et al. 2009 for details of protocols). In order to ensure run, we checked the stabilization of the likelihood. To the reliability of genotypes, we performed a sequential determine the optimal number of clusters, we compared replicate-based approach as described in Frantz et al. the log-likelihood of the data given the number of clus- (2003). This was found to be more efficient for P. tatters- ters (Ln P(X|K)), evaluated the individual membership alli than the commonly used multi-tube approach (Tab- coefficient (Q) (Pritchard et al. 2007) and computed the erlet et al. 1996) because the same reliable consensus standardized second order rate DK of change of Ln genotypes were reached using a smaller mean number P(X|K) (Evanno et al. 2005), for different values of K. of replicates (Que´me´re´ et al. 2009). To quantify the The DK method is known to detect only the uppermost reliability of our genotyping, we calculated the mean level of structure (Evanno et al. 2005). We thus repeated quality index (QI) across samples and loci following the the process in a hierarchical way until no substructure rules defined in Miquel et al. (2006). Using the software could be uncovered and by assigning at each hierarchi- CERVUS 3.03 (Kalinowski et al. 2007), we computed the cal level, the individuals to the group where more than probability of identity (pID) (i.e. the overall probability 90% of their genome is assigned (i.e. membership pro- that two individuals drawn at random from a given portion Q > 0.9). Since the golden-crowed sifaka indi- population share identical genotypes at all typed loci) viduals within social groups are expected to be closely and identity assuming sibling (pIDsib). related (Meyers 1993), the ‘optimal’ value of K might be reflecting family-induced structure rather than the overall geographical population structure as it has been Genetic diversity and structure analysis recently noticed in gorillas (Bergl & Vigilant 2007) or in The level of genetic variation for the whole data set and salmon and simulated data (Anderson & Dunham within each of the nine studied forest patches was mea- 2008). We, therefore, treated the clustering analyses as a sured as the mean number of alleles per locus (MNA), data-exploration tool as was suggested by Chikhi and the allelic richness (AR), the observed heterozygosity Bruford (2005) or Anderson and Dunham (2008). Since

(Ho) and the expected heterozygosity (He) (Nei 1978). In the assumption of correlated allele frequencies may also order to investigate the level of genetic structure among lead to an overestimation of K (Pritchard et al. 2007), forest patches, we estimated the pairwise Fst between we repeated the analyses using the independent allele all populations (Wright 1978) following Weir & Cocker- frequency model with lambda set to 1.0. We report only ham (1984). Departure from HWE was calculated with the results from runs assuming correlated allele fre-

Fis (Wright 1978). We tested the signiﬁcance of Fis and quencies, since the outcomes of our analyses were not

Fst values using permutations. We performed all analy- affected by this change in model choice. To discuss the ses using the GENETIX software (Belkhir et al. 1996–2004), relationship between genetic discontinuities and envi- except for Allelic Richness (AR), which was computed ronmental features, we computed the mean member- using FSTAT (Goudet 2001). ship coefficients of each social group for each cluster and plotted the results on the map of the Daraina region. These coefficients were assigned to the group Identification of population units and structuring sampling locations, and we used the inverse distance environmental factors weighted (IDW) interpolation function implemented in STRUCTURE analysis. To detect discontinuities in genetic ArcGis 9.2 to predict the overall pattern of cluster mem- variation and define genetically distinct units, we first bership through the species range. used the commonly used individual-based clustering Bayesian methods implemented in the STRUCTURE 2.2 CAUSAL MODELLING analysis. To identify the environ- (Pritchard et al. 2000; Falush et al. 2003). We performed mental factors influencing P. tattersalli dispersal across 10 runs of 500,000 iterations with a burn-in period of landscape, we employed a causal modelling on resem- 100,000 for each K value (with K ranging from 1 to 12 blance approach (Legendre 1993; Cushman et al. 2006).

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 5

This factorial, multi-model approach aims to combine radius of 200 m. (i.e. resolution of the friction maps). least cost path modelling and partial Mantel tests to We used this value because (i) it corresponds to the identify the landscape organizational models (i.e. combi- estimated radius of sifaka social groups’ territory nation of environmental factors) which appear to drive (Meyers 1993) and (ii) it allows us to consider small the patterns of genetic relatedness among individuals elements like riparian forests that are likely to favour (Cushman et al. 2006). Contrary to clustering methods, sifakas dispersal (Adriaensen et al. 2003; Broquet et al. this approach needs to define a priori the environmental 2006). The three isolation-by-canopy resistance surfaces factors potentially driving genetic structure. We detail [C1, C2 and C3] corresponded to a low ⁄ medium ⁄ high now the different steps of this approach. level of selectivity in which the most opened areas are two ⁄ five ⁄ ten times, respectively, more difficult to cross First step—identification of potential drivers of genetic than the most forested areas (Fig. S1, Supporting structure. By combining field observations, remote sens- Information). We then used these resistance surfaces to ing data and GIS tools, we identified four potential estimate cost distances between individuals and to pro- drivers of P. tattersalli genetic structure in Daraina area. duce three pairwise matrices using the PATHMATRIX tool These are the geographical distance [Factor D], the (Ray 2005). Cost distances are the cumulative costs national road (as a barrier) [Factor R], the Manankolana corresponding to the least-cost route between pairs of River (as a barrier) [Factor M], and the forest habitat individual locations. structural connectivity (for simplicity we refer to it as ‘canopy’) [Factor C]. We did not test the effect of Third step—identification and test of organizational slope ⁄ altitude since P. tattersalli range mostly corre- models. We considered 15 possible organizational mod- sponds to lowland landscapes. Human density was also els corresponding to all the patterns of causality among not considered since it is relatively low in the Daraina the four factors described above (Table 2c and Fig. S2, area and a strong fady (i.e. local taboo) limited the direct Supporting Information). To assess the support of each pressures on the golden-crowned sifaka (poaching and of these models, we followed the procedure detailed in hunting mentioned above appear to have been caused Cushman et al. (2006). Each of the 15 models is related to by non-local people settling in the region during a a set of hypotheses corresponding to statistical relation- ‘gold rush’ in the 1990s and during the recent political ships between genetic structure and putative structuring instability). environmental factors (Table 2a and c). Support for a model can be simply computed as the number or the Second step—computation of genetic and resistance distance proportion of statistical predictions that are fulfilled. A matrices. Genetic dissimilarity [G] among individuals model is fully supported only if the entire set of hypoth- was estimated by computing the matrix of Rousset eses is verified. To evaluate the relationship between the

(2000)’s genetic distance ar between pairs of individuals genetic structure and each of the potential factors, we using the program SPAGeDI (Hardy & Vekemans used simple (Mantel 1967) and partial (Smouse et al. 2002). The isolation-by-distance hypothesis [D] was 1986) Mantel tests within a causal modelling framework modelled as a single hypothesis with genetic dissimilar- (Legendre 1993). We first computed the Mantel correla- ity predicted to increase linearly with Euclidian dis- tion and associated Monte Carlo P value for each simple tances. A matrix of Euclidian distances was thus and partial Mantel correlation between genetic-distance computed between pairs of individual locations. The matrix and the matrices corresponding to the different two Isolation-by-barrier hypotheses (Isolation-by-road factors or set of factors (Table 2b). We then compared [R] or Manankolana River [M]) were represented as cat- the significant and non-significant (NS) tests to the egorical model matrices (Legendre & Legendre, 1998) expectations under the 15 models (Table 2c) to identify with 0s and 1s for pairs of individuals on the same or the organizational model with the greatest support (i.e. different side of the barrier, respectively. To model the most consistent with the observed pattern of genetic Isolation-by-canopy hypothesis [C], we computed three data). For each model, we computed a support rate cor- resistance surfaces representing three level of selectivity responding to the ratio of the number of non-rejected for the forest connectivity. We first carried out a grad- hypotheses to the total number of tested hypotheses. The ual forest numeric model (FNM) (Coulon et al. 2004) confidence intervals of the support rates were obtained from the landscape classification (see above): the using a resampling procedure (Manly 1997) in which we smoothing function implemented in the software ENVI randomly selected 75% of the 230 individuals without 4.1 was used to assign a value between 1 and 10 to each replacement (100 iterations). We recomputed the support pixel corresponding to 30 · 30 m2 squares indicating rates for each of the 15 models using the 100 data sets the amount of open area surrounding the pixel within a repeating the procedure detailed above.

2010 Blackwell Publishing Ltd 6 E. QUE´ ME´ RE´ ET AL.

ily surprising given that all samples were immediately Spatial genotypic structure at the patch scale collected after defecation. All 13 loci used were poly- Isolation-by-Euclidian distance appears to be an impor- morphic with an average of 7.7 alleles (SD = 1.98) per tant factor structuring genetic diversity in P. tattersalli locus across all samples. The pID (Evett & Weir 1998) ) populations (see the ‘Results’ section below). In order to was 8.82 · 10 14 and the pIDsib (Evett & Weir 1998) ) investigate the spatial scale of variation of the dispersal was 7.98 · 10 6 for a 13-locus genotype, allowing ample process, we performed a spatial autocorrelation analysis power to discern individuals. Among the 250 samples, in GENALEX 6.2 (Peakall & Smouse 2006) for individuals 20 were discarded as they corresponded to the same belonging to the same forest patch hence assuming the multi-locus genotype as another individual from the Euclidian distance and the rate of dispersal as the only same social group at all 13 loci. We note here that we factors influencing genetic structure. Data from all expected this to happen since defecation of the mem- patches were combined using the ‘Multiple Pops’ option. bers of a sifaka group often occurs over a relatively The spatial autocorrelation analysis assesses the genetic short period of time and often under the same tree. similarity between pairs of individuals at different geo- Based on our 2006 experience, our sampling strategy graphical distance classes. These classes were defined so during the 2008 fieldwork season was hence done so as as to (i) represent sensible biological classes (see below), to ensure that all individuals were sampled in a group, (ii) be of similar length (in meters), and (iii) have a mini- accepting the risk of sampling the same individual mum number of within class comparisons. The first dis- more than once. The remaining 230 individuals were tance class was defined to only include comparisons thus considered for the analyses. Altogether this corre- among individuals from the same social group (i.e. the sponded to a total of 2960 genotypes (i.e. 30 missing ‘within groups’ class). The following distance classes cor- genotypes 1%). responded to increments of 500 m, up to 5000 m, and Genetic diversity of golden-crowned sifakas across had between 58 and 264 pairwise comparisons. The auto- all nine forest patches, as measured by He had a mean correlation patterns may result from processes other than value of 0.66, ranging between 0.57 (Antsiasia) and dispersal, such as mutation, which is known to be 0.73 (Ampondrabe). The average AR was 3.83 ranging affected by sampling scheme (Guillot et al. 2009). Since from 3.00 (Antsiasia) to 4.36 (Bekaraoka) (Table 1). we collected the samples at random across the forest Average Ho values were slightly higher than He in patches (Fig. 1), we assume that our sampling scheme most of the sampling sites resulting in mostly negative ) does not reflect species density and is not informative Fis values ranging from 0.18 to 0.01 with a significant about the process. Thus, the patterns of autocorrelation departure from HWE for three sites (Bobankora, Am- should reflect the sifaka dispersal and their social and pondrabe and Antsaharaingy). In these three cases, reproductive structure. Since behavioural data indicated significant heterozygosity excess was observed for only that P. tattersalli dispersal is biased toward neighbouring one to four loci which appear to drive the overall social groups (Meyers 1993), we should obtain a signifi- result. We found that the genetic diversity levels cant positive autocorrelation in the first distance classes within all forest patches were also relatively high and and the correlogram should flatten out at the scale where that this AR was slightly correlated with patch surface dispersal is not connecting social groups (Aars et al. areas but not significantly (Pearson correlation, P 2006). The significance of r values was tested by doing value = 0.06). Levels of differentiation, as measured by

1000 random permutations of the data to compute the pairwise Fst, were signiﬁcant between most of the sites 95% conﬁdence interval about the null hypothesis of no (values ranging between 0.03 and 0.30—Table S1, Sup- spatial substructure (r = 0) compared with the bootstrap porting Information) except between the neighbouring

95% conﬁdence intervals around each estimate of r Bemokoty and Tsarahitsaka forest patches (Fst = 0.01).

(Peakall et al. 2003). The highest Fst values were found for Antsiasia (aver-

age pairwise Fst = 0.2) and Antsaharaingy (0.16) Results located in the far northern and south-eastern edges of the distribution area, respectively (Patches I and Genetic diversity and patterns of differentiation among A—Fig. 1). By excluding these two isolated patches, patches which were responsible for the largest Fst values, we observed that the level of differentiation among We obtained multi-locus genotypes from 250 out of the patches located on the same side of the Manankolana

292 extracted samples (78%). Overall the faecal sam- River was much more limited (Fst values between 0.01 ples were of good quality, with a mean QI per sample and 0.07) in comparison with the values obtained of 0.81 (SD = 0.16) and the mean QI per locus of 0.82 among patches located on different sides (Fst ranged (SD = 0.04). This relatively high quality is not necessar- between 0.09 and 0.16).

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 7

Table 1 Summary statistics for the nine forest patches populations

Forest massif [ID] Sample size MNA AR Ho He n.b. Fis

ANTSAHARAINGY [A] 22 4.62 3.29 (0.75) 0.69 (0.19) 0.58 (0.15) )0.18*** AMPONDRABE [B] 16 4.77 4.19 (0.86) 0.79 (0.12) 0.73 (0.09) )0.08* AMPOETANY [C] 37 5.23 3.71 (0.51) 0.68 (0.10) 0.65 (0.09) )0.05 BEMOKOTY [D] 25 5.08 3.99 (0.77) 0.69 (0.13) 0.67 (0.10) )0.03 TSARAHITSAKA [E] 20 4.92 3.88 (0.84) 0.69 (0.10) 0.66 (0.10) )0.04 BAA [F] 18 5.15 4.05 (0.87) 0.69 (0.15) 0.69 (0.09) )0.01 BEKARAOKA [G] 49 6 4.36 (0.73) 0.72 (0.12) 0.72 (0.09) 0.01 BOBANKORA [H] 31 5.54 4.03 (0.87) 0.70 (0.13) 0.66 (0.12) )0.07** ANTSIASIA [I] 6 3 3.00 (0.71) 0.60 (0.22) 0.57 (0.19) )0.06 Average 25 4.92 3.83 (0.77) 0.69 (0.14) 0.66 (0.11) )0.03

ID, forest massif reference in Fig. 1; MNA, mean number of alleles; Ho, observed heterozygosity; He, n.b., unbiased expected heterozygosity; AR, allelic richness; Standard deviations are in brackets. *P < 0.05; ** P < 0.01; ***P < 0.001.

strongly suggests that one of the main factors structur- Identification of population units and environmental ing present-day genetic diversity is the Manankolana factors River and not the road. STRUCTURE analysis. The STRUCTURE analysis showed that the likelihood of the data increased quickly up to K =3 Causal modelling analysis. The results of the simple and then reached an asymptote (data not shown). For Mantel tests for the seven independent factors in K > 3, the results were not consistent among runs and Fig. 3 show (i) that all correlations are significant, but the uncovered genetics units corresponded to related also that (ii) all correlation values are relatively similar individuals belonging to the same or neighbouring (between 0.25 and 0.33), with only one exception, social groups. It thus appeared that K = 3 captured the namely the road which exhibits a much lower value major genetic structure in our sample while producing (r = 0.05). The two highest values appear to be those a very high average value for Q (0.92 ± 0.11). The three corresponding to the Manakolana River [M] and clusters, KA, KB and KC, corresponded to three regions Euclidean distance [D] hypotheses, followed by the with limited overlap, and KA was mostly composed of different ‘canopy’ models. For each of the 15 hypothet- individuals from east of the Manankolana River ical organizational models, Table 2c represents the (Fig. 2). Using the method of Evanno et al. (2005), the expected results of the causal modelling analysis in results were at first slightly different as the highest DK terms of partial Mantel tests. For instance, model 1 value was obtained for K = 2 which corresponded, with [D + C + M] assumes that in Propithecus tattersalli gene very few exceptions, to individuals sampled on the flow is influenced by all factors except the road. If this Eastern (i.e. KA) or Western (i.e. KB and KC) sides of model were correct, we would expect the first partial the Manankolana River (data not shown). When we Mantel test corresponding to the correlation between repeated the DK analysis within these two main clus- road and pattern of gene flows controlling for distance ters, as suggested by Evanno et al. (2005), we found that (RG.D) to be NS because the road factor is absent of the western river side group could be further divided the model. We observed that this is indeed the case into two subgroups with one genetically distinct group (Table 2b, r = )0.06, P = 1.00). In order to make this corresponding to most of the individuals from Antsaha- Table 2 easier to read, we highlighted the cases where raingy in the extreme north of the distribution area. We the P-value of the partial Mantel tests matched with found no substructure on east of the Manankolana the expectation of the model (i.e. when the hypothesis River (Fig. 2). When we interpolated the KA cluster is thus consistent and hence potentially supported). membership coefficients obtained with STRUCTURE on the The global support rates of the models (i.e. the ratio of map of the Daraina region (Fig. 2), we observed that the number of non-rejected hypotheses to the total the main genetic discontinuity overlapped with the geo- number of tested hypotheses) vary between 0.14 graphic location of the river, even though the analysis (±0.06) for model 15 (only one prediction was correct) did not use any prior geographical information. In con- and 0.83 (±0.04) for model 1 (10 predictions out of 12) trast, the genetic clusters did not coincide with the (Table 2c). None of the 15 models is fully supported, national road bisecting the area, although it is parallel but seven models (models 1–7) seem to provide to the river for about one third of its length. This relative good predictions in terms of partial Mantel

2010 Blackwell Publishing Ltd 8 E. QUE´ ME´ RE´ ET AL.

Legend Indian Ocean Interpolation (IDW) of structure assignments (cluster KA)

0.00 – 0.20 0.20 – 0.40 0.40 – 0.60 0.60 – 0.80 0.80 – 1.00 Ampiskina

Maromokotra Rivière Manankolana

NR 5A

Nosibe

Daraina

Fig. 2 Membership of social groups genetic clusters. The social groups are represented by pie charts at the location where they were sampled (n = 105). The size of the pie charts is proportional to the number of sampled individuals in the social groups (between one and five individuals). The colours indicate the average membership coefficients for each social group to each of the three clusters uncovered by STRUCTURE (blue, red and yellow colour for cluster KA, KB and KC, respectively). The red gradient illustrates the interpolated (IDW) membership coefficients of the cluster 1 (see text for details). tests (support > 0.6), three of which have support CG.D, NS). This suggests that the effect of canopy in values ‡0.8 (models 1–3). Among the six best- model 1 is caused by a correlation between canopy and supported models, five appear to have the geographi- geographic distance. In others words, model 1 should be cal distance factor [D], three the Manankolana [M], or canopy [C] factor.

Manankolana river [M] *** Identification of the best organizational model. To identify Euclidian distance [D] *** the best model, we considered not only the global Canopy [C1] support of each model but also analysed in detail the out- *** Canopy [C2] put of associated predictions. Indeed, the different factors *** are not independent and a partial Mantel test may reveal Canopy [C4] *** a significant correlation not because of the direct effect of Canopy [C3] *** a factor but rather through a correlation with another fac- National road [R] *** tor. For instance, we can see a significant canopy effect 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.5 partialling out the river (Hypothesis CG.M, P Mantel r value < 0.01). However, we also observed that there is no Fig. 3 Results of simple Mantel tests. *= P < 0.05, **P < 0.001, effect of canopy controlling for distance (Hypothesis ***P < 0.001.

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 9

Table 2 Evaluation of the 15 organizational models

F = Forest; C = Canopy; M = Manankolana River; R = Road; D = Euclidian distance; G = Genetic distance. In Table 2a, we represented the set of diagnostic resistance hypotheses (partial mantel tests) related to each model. A period separates the main matrices on the left from the covariate matrix on the right that are partialled out in the partial Mantel test. For example, RG.D is a partial Mantel test between the road (R) and the genetic distance (G) matrices with the geographical distance matrix (D) partialled out. For each model, the expectations for the Mantel tests were indicated in Table 2c. S and NS mean that the corresponding partial Mantel tests are expected to be signiﬁcant or NS, respectively for this model. The grey boxes are a visual help to identify tests where the P value (Table 2b) matched the expected result of the test (see text for a simple example). Boxes where a particular test is meaningless for a speciﬁc model were represented in black. The support rates corresponding to the ratio of total hypotheses minus the rejected hypotheses to the total number of tested hypotheses, and their standard deviation values (SD) are indicated for each model.

modified to exclude canopy, which would reduce to Model 3 (D + M) thus appears to explain best most sig- model 3. Similarly, model 2 predicts that the pattern of nificant and NS correlations while keeping the number of gene flow is only a function of distance. However, we factors to a limited level (two factors instead of three found a significant correlation between the river and the for model 1). Additionally to the tests performed on pattern of gene flow while controlling the effect of dis- the whole data set and to control for the Manankolana tance (Hypothesis MG.D, S). This suggests that the river River effect, we also performed causal modelling analy- should be added to this model, producing again model 3. ses on each side of the river. For both sides, the Isola-

2010 Blackwell Publishing Ltd 10 E. QUE´ ME´ RE´ ET AL.

Table 3 Organizational models for the two sides of the river

See Table 2 for legend.

tion-By-Distance model was the only fully supported Scale of isolation-by-distance model, hence confirming our previous results (data not shown). We also modelled the Isolation-by-canopy Significant and positive autocorrelation values were hypothesis as a new categorical model matrix [C4] with obtained in the first four distance classes (Fig. 4), corre- the values of 1 and 0 corresponding to pairs of individ- sponding to distances less than 1500 m. The highest uals located within the same patch, or in different value was observed in the first distance class corre- patches, respectively. Interestingly, we observed, in this sponding to comparisons between individuals from the case, that the model D + C (Distance and Canopy) was same social groups (r = 0.244, P value < 0.001). The the best supported model for individuals located on the autocorrelogram then flatten out and the autocorrelation west side of the river whereas the model D was still coefficient stabilised around 0.08 in the following three the best model for the east side (Table 3). When we distance classes before reaching values around zero excluded individuals from Antsaharaingy, the model D between 1500 and 2000 m. Then, the values remained was again the best supported for the west side (data slightly negative but were not significant up to 5000 m, not shown). as expected when there is isolation by distance.

0.35 Fig. 4 Spatial autocorrelation analysis.

) 0.3 r r Correlogram plots of the spatial genetic 0.25 U autocorrelation coefﬁcient r as a func- 0.2 L tion of distance for individuals within 0.15 0.1 forest fragments. Upper (U) and lower 0.05 (L) bounds for the 95% conﬁdence 0 interval for the null hypothesis of no –0.05 spatial structure (r = 0) and the upper Genetic correlation ( –0.1 Ur and lower Lr 95% error bars about r –0.15 50 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 as determined by bootstrap resampling are shown. Distance class (m)

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 11

Discussion tern is the result of significant human presence as many villages and rice fields are located near the river. A high-genetic diversity within a small distribution range? Another plausible explanation to this pattern is related to the sifakas dispersal behaviour. Indeed, behavioural The level of genetic diversity observed for the golden- studies by Meyers (1993) have shown that most of the crowned sifaka (MNA = 7.7 and He = 0.74 across loci dispersal events between groups appear to take place and for all samples) appeared higher than we expected during the mating season, which occurs in the middle for a species living in one of the smallest range and of the rainy season when the river is full and therefore most fragmented habitats of any lemur. Comparing this represents a significant barrier to dispersal. This is sup- estimate with those obtained from closely related spe- ported by the fact that rivers are increasingly recogni- cies remains problematic as population genetic studies sed as barriers to gene flow in many primate species, on other sifakas are still limited and often carried out both outside (Eriksson et al. 2004; Goossens et al. 2005) using a restricted number of individuals or locations and in Madagascar (Mittermeier et al. 2008) and that due to the difficulty in capturing sifakas. Only one this role has also been confirmed across other taxa in study conducted on a large number of Propithecus Madagascar (Wilme´ et al. 2006; Yoder & Nowak, 2006). verreauxi (>200 individuals) from the Beza Mahafaly This is particularly clear in the Microcebus and Lepilemur Special Reserve protected area (Lawler et al. 2001; Law- genera where many new species have been shown to ler et al. 2003) could be used as a comparison. Although have very small ranges whose limits are rivers (Rade- this species is known to have a wider range and a less spiel et al. 2008a, b; Louis et al. 2006; Mittermeier et al. disturbed habitat than Propithecus tattersalli, the authors 2008). Also, the geographical range of P. tattersalli is found similar values of genetic diversity (MNA = 9.29 nearly entirely delimited by two rivers, the Manambato and He = 0.74). Furthermore, the level of genetic and the Loky. The only exception is a small population diversity found in this study are higher than those north-west of the Loky (Fig. 1), in the most upstream observed for three Microcebus and one Lepilemur species region of this river. (L. edwardsi), even though these species are expected to have larger effective population sizes (Olivieri et al. The role of isolation by distance 2008; Craul et al. 2009). For L edwardsi, the low diversity observed (MNA £ 4 across 14 loci and He = 0.55) seems One of the main results provided by the causal model- to be due to the fact that the species is currently heavily ling approach is that the geographical distance (together hunted and undergoing a significant reduction in popu- with the Manankolana River) appeared to be the main lation size (Craul et al. 2009). The comparisons with the population-structuring factor in P. tattersalli. This sug- Microcebus should be taken cautiously because of ascer- gests that P. tattersalli effective migration events are tainment bias introduced by the choice of the microsat- mostly between neighbouring groups, and was con- ellite loci. Loci used in Olivieri et al. 2008 were firmed by the spatial autocorrelation analysis. This anal- originally developed for Microcebus murinus and were ysis showed that the highest autocorrelation values thus expected to produce an underestimation of genetic were found at the smallest spatial scale, within groups diversity (Chikhi 2008). Altogether we would like to (Fig. 4), suggesting that most individuals within social emphasize that the comparison of levels of genetic groups are closely related. This result is probably due diversity across species remains a tricky issue, as it to the fact that sifaka social groups are likely including heavily depends on how loci were isolated and how the lineages of philopatric related females and young males sampling was performed. born in the group who have not dispersed yet (Meyers 1993). We also observed a significant spatial autocorre- The role of the Manankolana River as a major gene lation among pairs of individuals in the first four dis- flow barrier tance classes (i.e. at distances <1500 m). These four classes included 87% of the distances between individ- One of the main results of our study was that we uals and the nearest groups hence suggesting that (i) clearly identified the Manankolana River as a major our sampling scheme was appropriate to assess fine- geographical feature shaping genetic differentiation in scale structure and (ii) most dispersal events within for- P. tattersalli and as possibly the main barrier to dis- est patches occur among neighbouring groups. This persal in the Daraina region. This result was surprising result is again in agreement with the behavioural stud- at first given that the river is dry most of the year, and ies on P. tattersalli (Meyers 1993) and also on P. ver- particularly during our fieldwork, and hence should reauxi (Richard et al. 1993). These authors observed, in a not be more difficult to cross than many other open 5-year census period, that all Verreaux’s sifakas males areas. One possible interpretation could be that this pat- migrated to groups no more than two home ranges

2010 Blackwell Publishing Ltd 12 E. QUE´ ME´ RE´ ET AL. away from their natal group. As suggested by Richard expect to find a limited level of genetic differentiation (1985) and Richard et al. (1993), these patterns of adja- between forest patches, even if migration had stopped cent transfers cause some neighbouring groups to have at the present time. Indeed, the genetic structure can related males, thus resulting in a ‘neighbourhood-like’ have a substantial time lag in its response to changes in social organization. gene flow (Wright 1978; Waples 1998). This possible time lag is difficult to assess since it may be influenced by many factors such as the nature of the genetic mark- Impact of forest habitat fragmentation on P. tattersalli ers employed, the population size and the level of sub- dispersal structure before fragmentation, and mating patterns The clustering and causal modelling approaches (Balkenhol et al. 2009). Although aerial and satellite brought us complementary and interesting results about images suggest that forest patch boundaries in the how the golden-crowned sifakas respond to current Daraina region have been relatively stable in the last habitat fragmentation at both landscape and patch 50 years (Jimenez & Vargas, 2000), we currently have scales. At the landscape scale (i.e. entire species range), no data before this date. We thus cannot determine the results of the causal modelling approach suggest whether the habitat was continuous before the 1950s. that canopy connectivity is not a major population The current patterns of genetic differentiation could structuring factor. This is in agreement with the cluster- thus potentially be the result of very recent fragmenta- ing analysis since two of the three uncovered genetic tion, say during the first half of the 20th century. Under units included several forest patches, suggesting that the second scenario, the forest fragmentation could be effective dispersal is actually occurring between at least significantly more ancient, perhaps a few centuries or some patches. A relatively large network of riparian millennia old. In this scenario, the golden-crowned gallery forests has been maintained and we suggest that sifaka may have ‘adapted’ to the fragmented landscape, they could have contributed to preserving dispersal and genetic differentiation observed today would corre- between most of the forest patches. However, when we spond to the actual migration events between present- explored the results at the smaller, regional scale, the day populations and fragments. This second scenario is situation appeared to be more complex. Indeed, the supported by the fact that we observed sifakas moving STRUCTURE results showed that habitat fragmentation and on the ground on several occasions (EQ pers. obs.). hence connectivity had a strong impact on dispersal, Jimenez & Vargas (2000) also observed P. tattersalli indi- since we found a significant genetic discontinuity in the viduals crossing more than 200 m of grasslands to reach very north of the Daraina area, identifying Antsahara- the closest forest. They also showed that golden- ingy as a cluster (KC). This highlights one limitation of crowned sifakas were able to survive in riparian forests the causal modelling approach that can only identify and forest fragments smaller than 10 ha, which were general processes working at the entire sampling scale sometimes located more than 2 km away from the clos- (i.e. the landscape scale). Here, habitat fragmentation est large forest. Additionally, they noted that straight appears to be important in only one region, but this line distances better explained the presence of P. tatters- effect is averaged out with other regions where frag- alli in forest fragments than the presence or absence of ments are still connected, leading to a NS effect when corridors among forest patches. the whole region is considered. Interestingly, we observed that the model implementing Isolation-by-can- On the use of causal and least-cost modelling opy hypothesis obtained the highest support when we restricted the causal modelling approach to individuals Here, we presented a practical case in which causal located on the west side of the Manankolana River and modelling was used to identify key features influencing used a categorical model (C4) to represent the Isolation- patterns of genetic differentiation. As expected we by-canopy hypothesis. found that the use of simple Mantel tests to identify Altogether (if we exclude the isolated Antsaharaingy structuring factors (see Epps 2007; Coulon 2004) could forest), our results are in agreement with a weak influ- produce spurious correlations (Cushman et al. 2006). ence of forest habitat structural connectivity on sifaka The reason for this is that different factors may be cor- dispersal, not because habitat fragmentation is not a related among themselves, and a significant test may potential threat for this species but because in most not be due to a particular factor but to another one to regions, forest fragments are still close enough from which it is correlated. The causal modelling approach, each other or still connected by corridors. Two radically by explicitly stating different partial tests and compar- different historic scenarios of forest fragmentation may ing the support for competing hypotheses, appeared explain our results. First, if the forest fragmentation able to identify at least some of these spurious correla- process in the Daraina area was recent then we would tions. Used together with STRUCTURE, it allowed us to

2010 Blackwell Publishing Ltd LANDSCAPE GENETICS OF GOLDEN-CROWNED SIFAKAS 13 identify important factors and importantly some limita- Acknowledgements tions. First, the analysis is necessarily dependent on the Financial support for this study was provided by CNRS and factors identified a priori as potentially important. This the French ministry of Research core funding to UMR5174 means that some ecological and behavioural knowledge CNRS Universite´ Paul Sabatier. EQ was funded by a MNRT of the species may be required on habitat and corridor (Ministe`re de l’Education Nationale, de la Recherche et de la preferences to define least-cost paths. Second, poten- Technologie) PhD grant. The field work was possible thanks tially important factors in one sub-region may not be to the support of the following associations and NGOs: detected as such when the analysis is performed across IDEA WILD, CEPA (Conservation des Espe`ces et Populations Animales, notably J.-M. Lernould) and FANAMBY (in partic- the whole region, as we saw above. The joint use of ular S. Rajaobelina, S. Wohlhauser and McG. Ranaivo Arivel- clustering methods may thus be necessary, particularly o). We warmly thank the Daraina local communities for as they do not require much prior information. welcoming us, Julie Champeau, Aubin Besolo, Emmanuel Another point to keep in mind in our case is that Rasolondraibe, all guides and research assistants for their the configuration of the forest habitat in the Daraina precious help on the field. We thank CAFF ⁄ CORE, the region may have played a role in the results. Indeed, ‘Direction geńe´rale des Eaux et Foreˆt’ for giving us permis- the surface area of several forest patches was relatively sion to conduct the fieldwork in Madagascar and for the authorizations to export the samples. We also thank A. Cout- large compared to the distances between patches and inho for his continuous support and for facilitating visits to the total area of the distribution range. The distance between the IGC and EDB. We also thank Kyle Dexter, between some individuals belonging to the same patch Simon Blanchet and four anonymous referees for useful and could thus be higher than the distance between indi- constructive comments which helped us improve the manu- viduals belonging to different patches. This could script significantly. The lab work was funded by European explain why it was difficult to demonstrate an effect Commission (research contract QLRI-CT-2002-01325 INPRI- of connectivity. Indeed, we found a high correlation MAT), the Center of Conservation and Research (CCR), Omaha’s Henry Doorly Zoo (Nebraska, USA), where the mi- between the geographical distance and the least-cost crosatellites were isolated and the Institut Francais de la Bio- distances that were maximizing the amount of forest diversite´, Programme Biodiversite´ de l’Oceán Indien. LC was areas in a particular path. This was observed for all also partly funded by the FCT grant PTDC ⁄ BIA- the levels of selectivity used for the canopy factor. BDE ⁄ 71299 ⁄ 2006. Thus, we suspect that least-cost modelling analyses might work better when the geographical scale of the study is significantly greater than the dispersal ability References of the species of interest (Vignieri 2005; Epps et al. Aars J, Dallas JF, Piertney SB et al. 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