Nicolas B David
email: nicolas.david@curie.fr

Charlotte A Martin

Marion Segalen

François Rosenfeld

François Schweisguth

Yohanns Bellaïche
email: yohanns.bellaiche@curie.fr

Drosophila Ric8 regulates Gαi cortical localisation to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localisation and activation of heterotrimeric G proteins play a crucial role during asymmetric cell division. The asymmetric division of the

Thus, Frizzled and heterotrimeric G protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.

In the dorsal thorax (notum) of the Drosophila pupa, around one hundred pI cells each divide asymmetrically with an antero-posterior planar polarity to produce a posterior cell, pIIa, and an anterior cell, pIIb, which will further divide to give rise to a mechanosensory organ [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF][START_REF] Gho | Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell[END_REF][START_REF] Fichelson | The glial cell undergoes apoptosis in the microchaete lineage of Drosophila[END_REF] . The antero-posterior planar polarity of the pI cell division is dependent on Frizzled (Fz) activity [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF] .

It is marked by the anterior asymmetric localisation of the cell fate determinants Numb and Neuralized [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF][START_REF] Bellaiche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF][START_REF] Roegiers | Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage[END_REF][START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF] . This anterior localisation of Numb depends on Bazooka (Baz) which localises at the posterior pI cell cortex, and on Pins and Gαi which accumulate at the anterior cortex [START_REF] Bellaiche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF][START_REF] Roegiers | Bazooka is required for localization of determinants and controlling proliferation in the sensory organ precursor cell lineage in Drosophila[END_REF][START_REF] Schaefer | Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system[END_REF] . Pins belongs to a family of guanine nucleotide dissociation inhibitors for Gα subunit [START_REF] Bernard | Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins[END_REF] and restricts the localisation of Baz to the posterior cortex of the dividing pI cell. Baz, in turn, promotes the asymmetric localisation of Numb [START_REF] Bellaiche | The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila[END_REF] . Analysis of Gαi null dividing pI cells reveals that Gαi is required for localisation of a functional Pins-YFP fusion protein and of Baz (Fig. 1a-f). Furthermore, the orientation of the mitotic spindle of the pI cell ensures that its division takes place along the antero-posterior axis and within the plane of the epithelium.

The antero-posterior orientation of the spindle depends on Fz activity [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF][START_REF] Bellaiche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF] , and Pins and Gαi have been proposed to participate in this process [START_REF] Schaefer | Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system[END_REF] . However, the mechanisms ensuring apicobasal orientation of the spindle have not been analysed.

Recently, Ric8, a GEF for Gαi and Gαo, has been characterised in C. elegans and in mammals [START_REF] Miller | RIC-8 (Synembryn): a novel conserved protein that is required for G(q)alpha signaling in the C. elegans nervous system[END_REF][START_REF] Tall | Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor[END_REF][START_REF] Afshar | RIC-8 Is Required for GPR-1/2-Dependent Galpha Function during Asymmetric Division of C. elegans Embryos[END_REF][START_REF] Couwenbergs | Control of Embryonic Spindle Positioning and Galpha Activity by C. elegans RIC-8[END_REF] . Here, we analysed the role of a Drosophila ric-8 homologue in pI cell polarity and spindle orientation. In doing so, we identified the first mechanism ensuring correct apicobasal orientation of the mitotic spindle during pI cell division.

While we identified putative ric8a and ric8b in the fly genome, ric8b is likely to be a pseudogene (Supplementary Information Fig. S1a,b). To study ric8a function, we used the expression of a ric8a dsRNA, which strongly reduced Ric8a protein level by RNAi (see Supplementary Information, Fig. S1a,d). We also used a severe hypomorph or null P element allele of ric8a, G0397 (see Supplementary Information Fig S1a,e for details). The ric8a G0397 insertion is lethal and this lethality was rescued by a Ric8a-YFP protein, which was uniformly distributed in the cytoplasm of both epithelial and pI cells during interphase and mitosis (see Supplementary Information Fig. S1c).

In ric8a-RNAi sensory organs, pIIa to pIIb cell fate transformations were observed (Fig. 1g), we therefore analysed whether these fate transformations might arise from a role of Ric8a in pI cell polarisation, by comparing the distribution of Numb, Baz and Pins in control and in ric8a-RNAi pI cells (similar results were obtained on a smaller number of dividing pI cells by analysing ric8a mutant somatic clones; see Supplementary Information, Fig. S2). While Numb formed an anterior crescent in control dividing pI cells (Fig. 1h), it failed to localise asymmetrically in 46% of ric8a-RNAi pI cells in prometaphase or metaphase (Fig. 1i,j). As observed for pins and Gαi 10, 15-17 , a telophase rescue mechanism operates in ric8a-RNAi cells as Numb formed a weak anterior crescent in 96% (n=25) of ric8a-RNAi pI cells in telophase.

The localisation of Baz, which was restricted to the posterior half of the cortex in control cells at metaphase (Fig. 1k), was affected in 63% of dividing ric8a-RNAi pI cells, leading to a circular localisation in 75% of the affected pI cells (Fig. 1l,m). Similarly, the localisation of Pins to the anterior cortex was lost in 88% of ric8a-RNAi pI cells in prometaphase or metaphase (Fig. 1n-p). These data demonstrate that ric8a is required to polarise dividing pI cells and may act upstream of, or in parallel to baz and pins.

Consistent with the involvement of ric8a in regulating the anterior accumulation of Pins, we found that, in ric8a-RNAi cells, the anterior accumulation of Gαi was weaker than in control cells and was even lost in 62% of cells (Fig. 2a-c). Strikingly, in these cells where Gαi anterior accumulation was lost, Gαi was absent from the lateral cortex (Fig. 2a-c). This phenotype is not a consequence of defective cell polarity, because Gαi is still present, although symmetric, at the cell cortex of dividing pins pI cells (Fig. 2l; n=28). Therefore ric8a not only affects pI cell polarity but also appears to be required for accumulation of Gαi at the cell cortex during division.

To test whether this unexpected function of ric8a was specific to the pI cell, we analysed Gαi localisation in epithelial cells. Gαi was detectable at the basolateral cortex of epithelial control cells and its staining was augmented during mitosis (Fig. 2d). But, as in the ric8a-RNAi pI cells, Gαi was lost from the cortex of both interphase and mitotic ric8a-RNAi epithelial cells (Fig. 2e). ric8a is thus required for the accumulation of Gαi at the cortex of both, pI and epithelial cells. Since mouse Ric8 is a GEF for both Gαi and Gαo 12 , we looked also at the localisation of Gαo in ric8a-RNAi cells. Gαo appeared uniformly distributed at the cortex of dividing pI and epithelial control cells, but unlike Gαi, the cortical localisation of Gαo was not affected in ric8a-RNAi cells (Fig. 2f-h and data not shown). Gβ13F was uniformly distributed at the cortex of control pI cells (Fig. 2i,k). Like the staining for Gαi, this staining was strongly reduced in ric8a-RNAi cells (Fig. 2j,k). Again, this phenotype was not due to defective cell polarity, because Gβ13F was still cortical in both pins and Gαi mutant pI cells (Fig. 2m,n). Gβ13F was also detectable at the cell cortex of both interphase and mitotic epithelial cells, and this staining was equally lost in ric8a-RNAi cells (Fig. 2i-k). These data demonstrate that ric8a is required for cortical accumulation of Gαi and Gβ13F.

The loss of cortical staining for Gαi and Gβ13F could result either from a reduction in the amount of these proteins or from their failure to localise at the plasma membrane. We therefore first quantified and compared the fluorescent signal for Gαi and Gβ13F between control cells and neighbouring ric8a mutant clones (Fig. 3a,b). We found only a slight reduction in signal intensity (on average 11% n=10 for Gαi and 19% n=10 for Gβ13F) which could not account for the drastic reduction of the cortical staining. Moreover, we compared the levels of Gαi and Gβ13F proteins in wild-type and ric8a second instar larval brains and could not detect any significant difference between the two strains (Fig. 3c). These results support the idea that Ric8a is required for the cortical localisation of Gαi and Gβ13F. To corroborate this idea, we overexpressed either Gαi or both Gβ13F and Gγ1 in ric8a mutant cells. While overexpressed Gαi or Gβ13F are cortical in wild-type cells (Fig. 3d,g), they are mainly cytoplasmic in ric8a mutant cells (Fig. 3e,h). Furthermore the overexpression of Gαi in ric8a mutant epithelial cells could not rescue Gβ13F cortical localisation (Fig. 3f) and the overexpression of both Gβ13F and Gγ1 in ric8a mutant epithelial cells did not lead to Gαi cortical localisation (Fig. 3i). Altogether these data demonstrate that Ric8a mildly regulates Gαi and Gβ13F stability but is mainly required for their localisation at the plasma membrane.

Palmitoylation of Gα and its association with Gβγ are both required to allow the Gαβγ trimer to reach the plasma membrane [START_REF] Michaelson | Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi[END_REF][START_REF] Takida | Heterotrimer formation, together with isoprenylation, is required for plasma membrane targeting of Gbetagamma[END_REF] . Drosophila Ric8a may be required for Gαi palmitoylation or for its association with Gβγ. In mammals, Ric-8 does not interact with Gβ [START_REF] Tall | Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor[END_REF] , so the effect on Gβ13F is likely to be indirect. Gβ13F remained cytoplasmic in ric8a,Gαi double mutant epithelial cells (Fig. 3j), excluding that Gβ13F was held in the cytoplasm by mislocalised Gαi in ric8a mutant cells. We therefore envisage that Ric8a affects other Gα subunits which are necessary for Gβ13F to reach its destination. This proposition is consistent with the demonstration that mammalian Ric8 is a GEF for Gαi and Gαo but can also interact with Gαq and Gα13 12 .

We then analysed the role of ric8a in mitotic spindle positioning. We expressed the microtubule-associated protein Tau-GFP under the control of a neuralized-GAL4 driver to follow the dynamics of the mitotic spindle in dividing pI cells [START_REF] Bellaiche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF][START_REF] Roegiers | Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage[END_REF][START_REF] Kaltschmidt | Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system[END_REF] . In wild-type cells, the spindle is oriented along the antero-posterior axis (Fig. 4a,d; mean αxy-wt=31°). This strict orientation is dependent on Fz signalling [START_REF] Gho | Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila[END_REF][START_REF] Bellaiche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF] (Fig. 4d). In ric8a mutant cells, the spindle is still oriented along the antero-posterior axis, and not randomised as in fz mutant cells (Fig. 4b,e; mean αxy-ric8a=14°; p wt/ric8a >0.5). This result led to us to reanalyse spindle orientation in Gαi and pins pI cells (see Ref. 8), and we found that Gαi and Pins are not required downstream of Fz for antero-posterior orientation of the mitotic spindle (Fig. 4e; mean αxy-pins=35° and αxy-Gαi=39°; p wt/pins >0.2 and p wt/Gαi >0.1).

The orientation of the spindle is also strictly controlled along the apico-basal axis, so that division takes place in the plane of the epithelium. We quantified this by measuring αz, the angle of the mitotic spindle relative to the plane of the epithelium. In wild-type cells, the spindle is almost, but not exactly, parallel to the plane of the epithelium, the posterior centrosome being always slightly more apical than the anterior one (Fig. 5a,h; mean αz-wt=17°; see also Ref. 2). In ric8a mutant pI cells, the spindle appeared more tilted along the apico-basal axis, with 25% of the cells displaying a tilt above 30° (mean αz-ric8a=23°), a situation never observed in wild-type cells (Fig. 5b,h; p wt/ric8a <0.05). This apico-basal phenotype was stronger in pins and Gαi mutant pI cells, the posterior centrosome being always largely more apical than the anterior one (Fig. 5c,d,i; mean αz-pins=33° and αz-Gαi=39°; p wt/mutant <0.001 for both). Thus Ric8a, Pins and Gαi are required in pI cells to maintain the spindle in the plane of the epithelium. Furthermore, pins,Gαi double mutant pI cells displayed a phenotype identical to the one observed in pins or Gαi single mutant pI cells (Fig. 5i; mean αz-pins,Gαi=34°; p Gαi/pins,Gαi >0.5 and p pins/pins,Gαi >0.3), demonstrating that Pins and Gαi act together in controlling apico-basal spindle orientation (we will refer to their activity as the Pins/Gαi signalling). We also analysed spindle orientation in Gγ1 mutant pI cells and found that the spindle is similarly tilted along the apico-basal axis (see Supplementary Information, Fig. S3d; mean αz-Gγ1=44°; p wt/Gγ1 <0.001). This can be a consequence of the absence of Gαi and Pins asymmetric localisation in the Gγ1 mutant (see Ref. 21 and data not shown).

However, two additional phenotypes were observed in the Gγ1 mutant: a mild effect on the antero-posterior orientation of the mitotic spindle and oscillatory movements of the mitotic spindle throughout division (see Supplementary Information, Fig. S3a-c). This latter phenotype is reminiscent of that observed in C. elegans Gβ knock-down [START_REF] Afshar | RIC-8 Is Required for GPR-1/2-Dependent Galpha Function during Asymmetric Division of C. elegans Embryos[END_REF][START_REF] Tsou | LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling[END_REF] which was interpreted as a result of Gα hyperactivation.

Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells (Fig. 5j). This demonstrates first that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and second that a pI specific activity tilts the spindle in absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI specific activity for two reasons. Firstly, Fz signalling is still active in the pins and Gαi mutants since the spindle was correctly oriented along the antero-posterior axis in these mutants. Secondly, Fz accumulates at the posterior apical cortex of pI cells [START_REF] Bellaiche | The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila[END_REF] and this accumulation of Fz is maintained in Gαi pI cells (Fig. 5f). We thus envisaged that, while orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. We analysed fz,pins double mutants to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium (Fig. 5e,k; mean αz-fz,pins=9°; p pins/fz,pins <0.001). In the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is thus Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells (Fig. 5k; p wt/fz,pins <0.001), suggesting that Fz may also tilt the spindle in wildtype cells along their apico-basal axis. To test this, we analysed spindle orientation in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells (Fig. 5k; mean αz-fz=11°; p wt/fz <0.03).

Altogether, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric8a/Pins/Gαidependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arise from this balance (Fig. 5m). Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex (Fig. 5l). The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (see Supplementary Information, Fig. S4).

In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR/Gα complex, the homologue of the Pins/Gαi complex [START_REF] Afshar | RIC-8 Is Required for GPR-1/2-Dependent Galpha Function during Asymmetric Division of C. elegans Embryos[END_REF][START_REF] Couwenbergs | Control of Embryonic Spindle Positioning and Galpha Activity by C. elegans RIC-8[END_REF] . Our data demonstrate that, in the dividing pI cell, Ric8a is required for asymmetric localisation of Pins, Baz and Numb and for mitotic spindle positioning. We propose that those activities of Ric8a depend on an unexpected function of Ric8a: localising Gαi and Gβ13F at the plasma membrane. Our study of ric8a also revealed that, in the pI cell, ric8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype observed in ric8a pI cells could be accounted for by some persistence of the Ric8a protein in somatic clones. Alternatively, an intriguing possibility is that ric8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling [START_REF] Katanaev | Trimeric G proteindependent frizzled signaling in Drosophila[END_REF] . ric8a loss of function would thereby affect both the Fz-and Gαi-dependent activities exerted on the spindle resulting in a milder apico-basal tilt.

Importantly, developmental processes ranging from gastrulation [START_REF] Gong | Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation[END_REF] , neural tube closure [START_REF] Sausedo | Role of nonrandomly oriented cell division in shaping and bending of the neural plate[END_REF] , neurogenesis [START_REF] Adams | Metaphase spindles rotate in the neuroepithelium of rat cerebral cortex[END_REF] and retina formation [START_REF] Das | In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina[END_REF] to asymmetric segregation of cell fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral…) and parallel to the plane of the epithelium. We show here that in dividing pI cells these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the anteroposterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz-and Gαi-dependent activities are likely to act through forces pulling on astral microtubules [START_REF] Cowan | Asymmetric Cell Division in C. elegans: Cortical Polarity and Spindle Positioning[END_REF][START_REF] Kusch | Spindle asymmetry: a compass for the cell[END_REF] . Fz and heterotrimeric G signallings are implicated in mitotic spindle positioning during both symmetric and asymmetric cell division [START_REF] Du | Mammalian Pins Is a Conformational Switch that Links NuMA to Heterotrimeric G Proteins[END_REF][START_REF] Adler | Asymmetric cell division: plane but not simple[END_REF][START_REF] Hampoelz | Heterotrimeric g proteins; new tricks for an old dog[END_REF] . The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to our understanding of the mechanisms controlling mitotic spindle positioning.

Materials and Methods

Flies

Live imaging was carried out on staged pupae of the following genotypes: w; neur PGal4 ,UAS-Tau-GFP/+ w; pins 62 , neur PGal4 ,UAS-Tau-GFP/pins 62 w; Gαi P8 , neur PGal4 ,UAS-Tau-GFP/Gαi P8 w; Gαi P8 , neur PGal4 ,UAS-Tau-GFP, pins 62 /Gαi P8 , pins 62 w; fz kd4a , neur PGal4 /fz K21 ,UAS-Tau-GFP w; fz kd4a , neur PGal4 , pins 62 /fz K21 ,UAS-Tau-GFP, pins 62 w,ric8a G0397 ,FRT19A/ hs-flp,tub-Gal80,FRT19A; neur PGal4 ,UAS-Tau-GFP/+ yw,ubx-flp;FRTG13Gγ1 N159 /FRTG13,tub-Gal80; neur PGal4 ,UAS-Tau-GFP/+ w; CG31363 G00147 /CG31363 G00147 w; CG31363 G00147 , pins 62 /CG31363 G00147 , pins 62 w; arm-fz-GFP ; Gαi P8 , neur PGal4 ,UAS-HB2-mRFP/Gαi P8 w; ubi-pins-YFP ; Gαi P8 , neur PGal4 ,UAS-HB2-mRFP/Gαi P8 w; ubi-pins-YFP ; neur PGal4 ,UAS-HB2-mRFP/+ w; ubi-ric8a-YFP ; neur PGal4 ,UAS-HB2-mRFP/+ w; ubi-YFP-ric8a ; neur PGal4 ,UAS-HB2-mRFP/+ w,baz XJ106 ,FRT19A/ hs-flp,tub-Gal80,FRT19A; neur PGal4 ,UAS-Tau-GFP/+ Fixed staged nota were dissected from pupae of the following genotypes: w; ap PGal4 /+;UAS-Ric8a RNAi27 /+ w;ap PGal4 /+ w,ric8a G0397 ,FRT19A/Arm-LacZ FRT19A; ey-flp w,ric8a G0397 ,FRT19A/Arm-LacZ FRT19A; Gαi P8 / Gαi P8 , ey-flp w; ubi-Pins-YFP; pins 62 w,ric8a G0397 ,FRT19A / hs-flp,tub-Gal80,FRT19A; scabrous PGal4 ,UAS-α-catenin-GFP/UAS-Gαi w,ric8a G0397 ,FRT19A / hs-flp,tub-Gal80,FRT19A; scabrous PGal4 ,UAS-α-catenin-GFP/UAS-Gβ13F, UAS-Gγ1.

w; scabrous PGal4 ,UAS-α-catenin-GFP/UAS-Gβ13F, UAS-Gγ1 w; scabrous PGal4 ,UAS-α-catenin-GFP/UAS-Gαi Larval brains were recovered from w; w,ric8a G0397 ,FRT19A/w,ric8a G0397 ,FRT19A or w,ric8a G0397 ,FRT19A/Y second instar larvae and tub-Gal;UAS-Ric8a RNAi27 . All alleles, P elements and transgenes are described in Flybase (http://flybase.bio.indiana.edu/). Gαi P8 is a viable null Gαi allele; pins 62 is viable null pins allele [START_REF] Le Borgne | Unequal segregation of Neuralized biases Notch activation during asymmetric cell division[END_REF] ; w;fz kd4a /fz K21 is a null transheterozygote; Gγ1 N159 is an amorph Gγ1 allele ; neur PGal4 4 ; baz XJ106 is an amorph baz allele; UAS-Histone2B-mRFP (Langevin et al., in press); UAS-Gαi, UAS-Gβ13F and UAS-Gγ1 21 ; CG31363 G00147 is a GFP protein trap P element inserted in CG31363 that labels the mitotic spindle (a gift from W. Chia). Pins-YFP, YFP-ric8a and ric8a-YFP cDNAs were cloned in a modified version of the P-Ubiquitin vector (details available on request). The EcoRI/NotI ric8 PCR fragment from nucleotide 494 to 1414 was used to generate a ric8a hairpin in pUAS vector as described in Ref. 34. Transgenic lines were generated by DNA injection into w or pins 62 strains using the ∆2.3 helper plasmid.

Functionality of the ubi-pins-YFP transgene was demonstrated by rescue of the formation of the Numb crescent in dividing pI cell of staged ubi-pins-YFP,pins 62 /pins 62 pupae.

Immunocytochemistry and GFP imaging.

Nota were dissected from staged pupae and fixed and stained as described in Ref. 4. Primary antibodies were rabbit anti-Gαi and rabbit anti-Gβ13F (a gift from J. Knoblich) diluted 1:1000; rabbit anti-Gαo (a gift from M. Semeriva) diluted 1:250; rabbit anti-Numb (a gift from Y.N. Jan) diluted 1:1000; rabbit anti-Pins (a gift from J. Knoblich, W. Chia and X.

Yang) diluted 1:1000; rabbit anti-Baz (a gift from A. Wodarz) diluted 1:4000; mouse anti-Fas3 (7G10, obtained from DSHB) diluted 1:100; mouse anti-β-Gal (Promega) diluted 1:500; guinea-pig anti-Scribble (a gift from D. Bilder) diluted 1:2000 and guinea pig anti-Senseless (a gift from H. Bellen) diluted 1:4000. References for primary antibodies are given in Supplementary Information Table S1. The Cy3-and Cy5-coupled secondary antibodies were from Jackson Laboratories and Alexa-488-coupled secondary antibodies were from Molecular Probes. Images were acquired on Leica SP2 confocal microscopes. Live imaging was carried out as described in Ref. 4 and images were acquired on Zeiss LSM510 Meta and Leica SP2 confocal microscopes. Images were processed and assembled using ImageJ, Adobe Photoshop and Adobe Indesign. Quantification of Gαi and Gβ13F staining intensities were performed using Metamorph. α xy and α z angles were measured at midpoint between spindle formation and anaphase (all presented data) and at the onset of anaphase (data not shown). Both analyses gave similar results.

Western blot

Larval brains were dissected in PBS and boiled in SDS loading buffer. Primary antibodies were rabbit anti-Gαi (a gift from J. Knoblich) diluted 1:100; rabbit anti-Gβ13F (as above) diluted 1:1000; rat-anti-α-catenin (a gift from H. Oda) diluted 1:2000, and rabbit affinity purified anti-Ric8a (1:1000) generated against both CKLPATPDELKQEER and CAMIDKDGRPQPLEH peptides of Drosophila Ric8a (Covalab). HRP-coupled secondary antibodies were from Jackson Laboratories. In vitro translation was carried out using the TNT Promega kit.

Figure legends

Figure 1 ric8a is required to polarise dividing pI cells.

To examine the role of ric8a during division of the pI cell, we used RNAi to reduce the level of Ric8a, as this strategy allowed us to analyse large numbers of dividing pI cells. We expressed ric8a-dsRNA under the apterous-GAL4 driver whose expression is restricted to the dorsal part of the wing disc, that gives rise to the notum among other tissues. We will refer to apterous-GAL4/+;ric8a-dsRNA as ric8a-RNAi and apterous-Gal4/+ as control.

Localisation of Numb (green, a-b') and Baz (green, c-d') in dividing wild-type (a and c) and Gαi (b and d) pI cells, identified by Senseless, a pI cell marker (red) at prometaphase (mitotic stage was determined by DAPI staining, blue). Localisation of Pins-YFP (green, e and f) in dividing wild-type (e) and Gαi (f) pI cells identified by Histone2B-mRFP (red) in living pupae. Whereas in wild-type pI cells in prometaphase or metaphase Numb (n=27) and Pins-YFP (n=28) formed an anterior crescent, in Gαi mutant pI cells in prometaphase or metaphase Numb crescents failed to form in 81% of the cells (n=27) and Pins-YFP was circular in 100% of the cells (n=24). Whereas Baz formed a posterior crescent in 87% of the wild-type pI cells in prometaphase or metaphase , Baz was circular in 58% of the Gαi mutant pI cells (n=12).

(g) Normal and transformed sensory organs in ric8a-RNAi pupae at 24 hours after pupae formation stained for Cut (blue), Su(H) (green) and HRP (red). The normal organ (left) is composed of 4 cells. The subepithelial Su(H) -Cut + shaft cell is located below the Su(H) + socket cell. The subepithelial HRP + Cut + sheath cell is located next to the neuron identified by HRP staining and its axon (not shown). The transformed sensory organ (right) is composed of 4 cells all of which are HRP + indicating that this pI cell divided to produce a pIIb and a pIIblike (pIIb*) daughter cells that divided to produce one neuron and one sheath cell, the neurons being identified by their axonal projections (not shown). The percentage of pIIa to pIIb cell fate transformation was 2.7% in ric8a-RNAi pupae (n=184 organs) while it was 2.9% (n=345 organs) and 0.7% (n=1066 organs) in pins and Gαi pupae, respectively.

Localisation of Numb (green, h-i'), Baz (green, k-l') and Pins (green, n-o') in dividing control (h, k, n) and ric8a-RNAi (i, l, o) pI cells, identified by Senseless, a pI cell marker (red) at prometaphase (DNA, blue). (j, m, p) Quantification of the ric8a-RNAi phenotype for Numb (j), Baz (m) and Pins (p) asymmetric localisation. The number of analysed cells is indicated above each column.

Anterior is to the left. Scale bar is 5µm. ,f,f',g,g') and Fas3 (red, f, f'', g, g'') in control (f-f'') and ric8a-RNAi (g-g'') dividing pI cells.

(i-j'') Localisation of Gβ13F (green, i, i', j, j') and Fas3 (red, i, i'', j, j'') in control (i-i'') and ric8a-RNAi (j-j'') dividing pI and epithelial (marked by an asterisk) cells.

(c, h, k) Quantification of the ric8a-RNAi phenotype for Gαi, Gαo and Gβ13F localisation in dividing pI cells.

(l) Gαi (green) asymmetric localisation is lost in dividing pins mutant pI cells, but Gαi is still located at the cell cortex (n=28). (m and n) The cortical localisation of Gβ13F (green) is not affected in pins (m, n=7) and Gαi (n, n=56) pI dividing cells.

Mitotic stage was determined by DAPI staining (white or not shown). pI cells were identified by Senseless (blue). Anterior is to the left. Scale bar is 5 µm. (g-h') Localisation of Gβ13F (green, g' and h') in wild-type (g') or ric8a (h') epithelial cells overexpressing Gβ13F and Gγ1 under the control of the scabrous PGal4 driver identified by expression of α-Catenin-GFP (red, g and h).

(i and i') Localisation of Gαi (green, i') in ric8a epithelial cells overexpressing Gβ13F and Gγ1 under the control of the scabrous PGal4 driver identified by expression of α-catenin-GFP (red, i).

(j and j') Localisation of Gβ13F (green, j') in control and ric8a,Gαi mutant epithelial cells.

Control epithelial cell were identified by nls-GFP expression (red, j ). ',b',e',f',h',i',j') or cells overexpressing Gαi or Gβ13F-Gγ1(d', g'). Scale bar is 5 µm. to the pIIa cell. This defect is not due to a loss of asymmetric localisation of cell fate determinants as it is rescued in fz,pins double mutant pIIb cells. Furthermore the pIIa and pIIb apical apex are almost equivalent in the fz,pins double mutant as expected from the planar orientation of the mitotic spindle in this mutant pupae. p wt/pins <0.001; p wt<fz,pins <0,001 and p pins/fz,pins <0,001. Note that the pIIa/pIIb overall size difference is not significantly different in wild-type versus pins (p>0,9), wild-type versus fz,pins (p>0,1) and fz,pins versus pins (p>0,1) pupae indicating that the difference in apical apex size observed in the different mutant background is not due to a difference in pIIa/pIIb size difference.

The dashed line indicates the border between control cells and mutant cells (a

(e-g) Maximal projection of two confocal sections acquired in the plane of the socket (e'-g') and in the plane of the neuron (e''-g'') of wild-type (e), pins (f) and fz,pins (g) sensory organs. At 24 hours after pupae formation the socket cell (green) expresses Su(H) and the neuron (red) is identified by HRP staining and its axonal projection. All sensory cells express the nuclear Cut marker (blue). Green (e''-g'') and red (e'-g') dashed lines outline the positions of the out of focus socket and neuron cells, respectively.

We analysed the relative position of the nucleus of the socket cell and the nucleus of the neuron to assess the consequences of apico-basal spindle orientation in the pI cell. This orientation correlates with the XY and XZ distances between the pI daughter cells. But, as the neuron is known to delaminate and thus move along the XZ axis during organ formation, we measured the XY distance between the nucleus of the socket cell and the nucleus of the neuron in differentiated organs. Consistent with the orientations observed in pI cells, the socket cell and the neuron tend to be more superposed in pins organs relative to wild-type organs whereas they tend to be more distant in fz,pins organs relative to wild-type. (h) Graph of the distribution of the XY distance between the nucleus of the socket cell and the nucleus of the neuron. p wt/pins <0,03; p wt<fz,pins <0,001 and p pins/fz,pins <0,001. We note that pins and fz may also affect pI daughter cells differentiation or morphogenesis per se and may therefore contribute to the observed phenotype. Scale bar is 5 µm.

  Drosophila sensory precursor cell (pI) is polarised along the antero-posterior axis by Frizzled signalling, and during this division, activation of Gαi depends on Partner of Inscuteable (Pins). We establish here that Ric8, which belongs to a family of guanine nucleotide exchange factors for Gαi, regulates the Gαi and Gβ13F subunits cortical localisation. Ric8, Gαi and Pins are not necessary to control the antero-posterior orientation of the mitotic spindle during pI cell division downstream of Frizzled signalling, but they are required to maintain the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis.

Figure 2

 2 Figure 2 ric8a is required for accumulation of Gαi and Gβ13F at the cell cortex.

Figure 3

 3 Figure 3 ric8a is required for cortical localisation of Gαi and Gβ13F.

  (c) Western blot using antibodies against Ric8a, Gαi, Gβ13F and α-catenin on extracts from wild-type and ric8a mutant larval brains. While Ric8a is undetectable in ric8a extracts, Gαi and Gβ13F levels are similar in wild-type and mutant brain extracts. α-catenin was used as loading control. The band corresponding to Gαi was identified by its absence from Gαi mutant brain extracts. (d-e') Localisation of Gαi (green, d' and e') in wild-type (d') or ric8a (e) epithelial cells overexpressing Gαi under the control of the scabrous PGal4 driver identified by expression of α-Catenin-GFP (red, d and e).(f and f') Localisation of Gβ13F (green, f') in ric8a epithelial cells overexpressing Gαi under the control of the scabrous PGal4 driver identified by expression of α-catenin-GFP (red, f).

Figure 4

 4 Figure 4 Antero-posterior orientation of the mitotic spindle is independent of Ric8a, Pins and

Figure 5

 5 Figure 5 Ric8a, Gαi and Pins are required to maintain the spindle in the plane of the

  (h) Cumulative plots of αz in wild-type (n=31) and ric8a (n=16) mutant pI cells.(i) Cumulative plots of αz in wild-type (n=31), pins (n=21), Gαi (n=26) and pins,Gαi (n=23) mutant pI cells.

Acknowledgments

We thank D. Bilder, H. Bellen, W. Chia, Y.N. Jan, J. Knoblich, F. Matsuzaki, H. Oda, M. Sémériva, D. Strutt, A. Wodarz, X. Yang, F. Yu, the Developmental Studies Hybridoma Bank and the Bloomington Stock Center for strains and antibodies. We also thank the members of the Curie Imaging facility for their help and advices with confocal microscopy. Y.B. thanks A. Morineau for encouragements and support. We thank M. Bornens, A. Echard, M. Morgan, J. Sillibourne and M. Théry for critical comments on the manuscript in preparation. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC 4726 and ARC 7744), the Fédération pour la Recherche Médicale, the CNRS, the Curie Institute, and grants from the Ministry of Research (ACI program grants). N.D. is supported by a postdoctoral fellowship from ARC.

(j) Cumulative plots of αz in wild-type and pins epithelial cells in which the spindle was labelled by expression of the protein trap CG31363 G00147 (n=20 for both).

(k) Cumulative plots of αz in wild-type (n=31), pins (n=21), fz (n=24) and fz,pins (n=40) mutant pI cells.

(l) Cumulative plots of αz in wild-type (n=31), fz (n=24) and baz (n=13) mutant pI cells. Note that Pins is asymmetrically localized in baz mutant pI cells [START_REF] Bellaiche | The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila[END_REF] .

(m) Model for planar orientation of the mitotic spindle in dividing pI cells. Schematic representation of spindle orientation in wild-type, pins or Gαi, fz and fz,pins mutant pI cells.

In wild-type, the orientation of the spindle results from the balance of two forces and is thus slightly tilted along the apico-basal axis. In the absence of either one force or the other, the spindle is oriented according to the remaining force. Interestingly, in absence of the two forces, orientation of the spindle is not randomised, suggesting that a default pathway, probably acting in all epithelial cells, maintains the spindle within the plane of the epithelium.

Supplementary Figure 1 (a) We identified two ric8 genes, ric8a (CG15797) and ric8b (CG15910), within a 6kb region of the Drosophila X chromosome based on sequence homology with the C. elegans gene ric-8. Drosophila ric8a encodes a 573 amino acid protein that has 35% sequence homology with mouse Ric-8A and 28% with C. elegans Ric-8. (b) ric8b is nearly identical to ric8a at the nucleotide level but it encodes a protein of only a 217 amino acid residues. Black boxes are regions 98% identical between Ric8a and Ric8b proteins. Since no EST has been found for ric8b, ric8b is likely to be a pseudogene. The localisation of the two epitopes against which Ric8a antibody was raised are also indicated.

(c, c') The ric8a G0397 allele is lethal at the second/third instar larval stage. This lethality was rescued by a Ric8a-YFP fusion protein (green), which was uniformly distributed in the cytoplasm of both interphase and mitotic epithelial and pI cells. pI cells in interphase (c) and mitosis (c') were identified by Histone2B-mRFP (red) expressed under the neuralized-GAL4 driver. Epithelial cells are indicated by asterisks in (c). time in minutes'seconds''. Scale bar is 5 µm.

(d) Western Blot using Ric8a and α-catenin antibodies on wild-type (lane 1) and tubulin-Gal4/UAS-dsRNA (lane 2) larval brain extracts. α-catenin was used as loading control. The Ric8a band is barely detectable in ric8a-RNAi extracts.

(e) The P element, G0397, is inserted in the open reading frame of the last exon of ric8a, and thus, can only lead to the production of the first 278 amino acid residues of the protein (a).

Western Blot using Ric8a antibodies on w;Ubi-Ric8a-YFP(lane 1), ric8a G0397 /Y;Ubi-Ric8a (lane 2) third instar larval brain extracts and a in vitro translated Ric8a N-terminal fragment (lane 3). Although it can be recognized by the antibody (lane 3), the Ric8a N-terminal fragment that could be expressed from ric8a G0387 insertional mutant is not detectable (lane 2). Therefore, this allele is likely to be either a severe hypomorph or a null allele Supplementary Figure 2 Localisation of Numb (green, a and b), Pins (green, c and d) and Gαi (green, e and f) in dividing control (a, c and e) and ric8a mutant (b, d and f) pI cells, identified by Senseless, a pI cell marker (red) at prometaphase (mitotic stage was determined by DAPI staining, not shown). ric8a mutant pI cells were identified by loss of β-Gal expression (blue, a-f). Whereas Numb (n=15), Pins (n=22) and Gαi (n=18) were asymmetrically localised in wild-type cells in prometaphase or metaphase, Numb (n=9), Pins (n=15) and Gαi (n=12) crescents failed to form in 67%, 100% and 100% of ric8a mutant pI cells in prometaphase or metaphase, respectively. Scale bar is 5 µm. Supplementary Figure 4 (a-c) Representative apical apex of the pIIa (indicated by two asterisks) and pIIb (indicated by one asterisk) cells marked by Scribble (red) and identified by Cut (green, not shown) in wildtype (a), pins (b) and fz,pins (c) mutant pupae. In wild-type and pins mutant pupae, pIIb was identified as the most anterior cell. In fz,pins double mutant pupae, pIIb was identified by its smaller apical apex.

Supplementary Figure 3

(d) Graph of the distribution of the ratio of the apical apex of the pIIb and pIIa cells in wildtype, pins and fz,pins pupae. The apical apex of the pIIa cell is larger than the one of the pIIb consistent with the tilt orientation of the mitotic spindle observed in wild-type cells. In pins mutant pI cell the pronounced tilted orientation of the mitotic spindle correlates with a more important delamination of the pIIb as judged from the reduced size of its apical apex relative


Julia Fischer
email: jfischer@dpz.eu

Conceptualization, Visualization James P Higham
email: jhigham@nyu.edu

Susan C Alberts

Louise Barrett

THORE Jacinta C Beehner

J Bergman

Alecia J Carter

Anthony Collins

Sarah Elton

Joe ¨l Fagot

Maria Joana Ferreira

D A Silva

Kurt Hammerschmidt

Peter Henzi

Clifford J Jolly

Sascha Knauf

Gisela H Kopp

Jeffrey Rogers

Christian Roos

Caroline Ross

Robert M Seyfarth

Joan Silk

Noah Snyder-Mackler

Veronika Staedele

Larissa Swedell

Michael L Wilson

Dietmar Zinner

Thore J Bergman

Maria Joana Ferreira Da Silva

Veronika Sta

Joe ¨l Fagot

Kurt Hammersch- Midt

THE NATURAL HISTORY OF MODEL ORGANISMS Insights into the evolution of social systems and species from baboon studies

Baboons, members of the genus Papio, comprise six closely related species distributed throughout sub-Saharan Africa and southwest Arabia. The species exhibit more ecological flexibility and a wider range of social systems than many other primates. This article summarizes our current knowledge of the natural history of baboons and highlights directions for future research. We suggest that baboons can serve as a valuable model for complex evolutionary processes, such as speciation and hybridization. The evolution of baboons has been heavily shaped by climatic changes and population expansion and fragmentation in the African savanna environment, similar to the processes that acted during human evolution. With accumulating long-term data, and new data from previously understudied species, baboons are ideally suited for investigating the links between sociality, health, longevity and reproductive success. To achieve these aims, we propose a closer integration of studies at the proximate level, including functional genomics, with behavioral and ecological studies.

Introduction

Humans have been captivated by baboons for thousands of years: from ancient Egypt, where the god of wisdom, Thoth, was depicted with a baboon head, to the mid-19 th century when Charles Darwin remarked, "He who understands baboon would do more towards metaphysics than Locke" [START_REF]Metaphysics on morals and speculations on expression[END_REF]. At the beginning of the 20 th century, the South African naturalist Eugene Marais provided one of the first detailed accounts of free-ranging baboons [START_REF] Marais | My Friends the Baboons[END_REF], and by the 1950s, baboons had become the subject of more systematic scientific enquiry, both in the field and in captivity. This was the decade that the American physical anthropologist Sherwood Washburn and his student Irven DeVore set out to investigate baboons in Kenya [START_REF] Vore | The social life of baboons[END_REF]. Washburn reasoned that these ground-living primates were a good model for early human adaptations because they evolved in African savannas alongside ancestral hominins. Meanwhile, increasing interest in the use of non-human primates as biomedical models for humans led to the funding in 1958 of a three-year proposal titled "Initiation and Support of Colony of Baboons" by the US National Institutes of Health, with the first group of baboons shipped to the United States from Kenya in 1960(Vande-Berg, 2009). Since then, research in captivity on baboons as a biomedical model has been complemented by extensive fieldwork on baboon populations across Africa. Knowledge of the links between health and fitness in baboons under natural circumstances, including natural levels of genotypic and phenotypic variation, appears critical to put results from captive studies into context. An understanding of the evolution and life history of these animals in the wild also allows the scientific community to assess the validity of results derived from captive populations.

While the earlier field studies set out to uncover a baboon archetype, subsequent research has revealed that there is no such thing as "the baboon". Indeed, many would argue that the value of this genus lies precisely in the substantial variation in the social systems, life histories and ecologies within and between the baboon species (see Box 1 for a glossary of specialist terms used in this article). Collectively, these characteristics make baboons an excellent model organism for investigating a range of fundamental biological processes, such as physiological and behavioral adaptation, hybridization and speciation with gene flow (Alfred and Baldwin, 2015). In this way, the baboon model constitutes an important complement to other mammalian model organisms, such as wild house mice(Mus musculus; Phifer-Rixey and Nachman, 2015) and deer mice (genus Peromyscus; [START_REF] Bedford | Peromyscus mice as a model for studying natural variation[END_REF].

Systematic classification and distribution

Within the primate order, all extant baboons belong to the genus Papio. The genus is part of the tribe Papionini, within the family Cercopithecidae. The fossil record and phylogeographic analyses indicate that baboons originated in southern Africa. Nuclear and mitochondrial estimates put the date of initial divergence of baboon lineages at 1.5-2.1 million years ago [START_REF] Newman | Mitochondrial phylogeny and systematics of baboons (Papio)[END_REF][START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF][START_REF] Zinner | Baboon phylogeny as inferred from complete mitochondrial genomes[END_REF]. At about the same time, during the Pleistocene epoch, baboons started to expand their range across sub-Saharan Africa into both northern and southern savannas.

Presently, six species are recognized: the chacma baboon (Papio ursinus), which is found in southern Africa; the yellow baboon (Papio cynocephalus), which inhabits large parts of eastern Africa; the Kinda baboon (Papio kindae), which is found in Zambia, eastern Angola, and southern DR Congo; the olive baboon (Papio anubis), whose distribution ranges from northern DR Congo to parts of Kenya and Tanzania across to Sierra Leone in the west and to Eritrea in the east; the Guinea baboon (Papio papio), which is found from Sierra Leone to Mauritania and Senegal; and the hamadryas baboon (Papio hamadryas), which inhabits parts of Eritrea, Ethiopia, Somalia and the south-western part of the Arabian peninsula (Figure 1). Hybrid zones are found where species' distributions come into contact.

While the systematic grouping into taxa within the genus Papio is well accepted on both phenotypic and genetic evidence, the taxonomic ranking is disputed. According to the biological species concept [START_REF] Mayr | Animal Species and Evolution[END_REF], all taxa would be considered one polytypic species (Papio hamadryas) because where they meet in the wild they interbreed freely, producing viable and fertile hybrid offspring. Given that the different taxa vary substantially in appearance, behavior, and the characteristics of their society, we follow the phylogenetic species concept [START_REF] Cracraft | Species concepts and speciation analysis[END_REF], and refer to the different taxa as "species". Box 1. Glossary.

Admixture: Genetic admixture refers to the exchange of genetic information among two populations or taxa that had been reproductively isolated and which genetically diverged (see introgressive hybridization).

Consortship: Consortships occur when females are sexually receptive and involve a male and female pair who associate in close proximity, often mating repeatedly. Typically, male-female consort pairs travel, feed and rest together. Consortships can last for hours or days. Genetic architecture: Refers to the underlying genetic basis of a phenotypic trait (morphological, physiological, behavioral) and the variation in the respective trait.

Ghost lineage: A term from paleontology and phylogenetics. It refers to a phylogenetic lineage that has no fossil record or living representatives, but is inferred to have existed, for example, by whole-genome analyses of related taxa.

Hybridization: The interbreeding between two differentiated populations, usually closely related species, resulting in the combination of genetic material from previously isolated gene pools.

Introgression or introgressive hybridization: Observed between species or between genetically well-separated populations. It refers to the movement of genes, or gene flow, from one species into the gene pool of another by the repeated backcrossing of interspecific hybrids with one of their parent species.

Life history: The life history of an organism is a characterization of its patterns of development, reproduction, aging and mortality. Key measures of primate life history include length of gestation, age at the first occurrence of menstruation, age at first reproduction, number of offspring per litter, number of births per year, interval between births and life span.

Mating system: The distribution of matings among sexually active individuals within a social unit. Primate species can be monogamous (mating occurs mostly between pair partners), polyandrous (one female mates with multiple males), polygynous (one male mates with multiple females), or polygynandrous (males and females have multiple mating partners).

Mitochondrial and nuclear lineages: Mitochondria, organelles of almost all eukaryotic cells, carry their own genome. In contrast to nuclear genomes, recombination of the mitochondrial genome is a rare event and, since mitochondria are almost exclusively inherited via the maternal lineage, nuclear and mitochondrial genetic lineages can experience independent evolutionary histories. This often results in discordant phylogenies when using sex chromosomes (gonosomal), non-sex chromosomes (autosomal) or mitochondrial markers. Even phylogenies based on different nuclear genes or parts of the nuclear genome can lead to some discordances. Nevertheless, one can use nuclear and mitochondrial lineages to infer different evolutionary events within the evolutionary history of a species. Phylogenetic species concept: This concept defines a species as an irreducible group or cluster whose members are descendants from a common ancestor and who all possess a combination of certain defining derived traits known as apomorphies. Such groups are monophyletic (contrasted with paraphyletic or polyphyletic groups). Reproductive isolation is not a precondition for the definition of species. Since monophyletic groups are often nested, ranking a particular group as a species can be problematic.

Social organization:

The number of individuals and the composition of a group, including when and where those individuals are distributed. Groups may for instance be stable or reveal a fission-fusion system where the group temporarily splits into smaller sub-groups. Baboon societies may be uni-level (individuals live in a stable group and generally roam together) or multi-level (groups consist of predictable sub-groups, which may in turn consist of smaller subgroups). An important aspect of the social organization is the dispersal behavior, that is, which sex typically remains in the group into which is was born (i.e. its natal group) and which sex leaves the natal group to breed. In most mammals, females stay (female philopatry) and males leave (male dispersal), which is considered the ancestral state. The timing and type of dispersal has important consequences for the genetic structure of groups.

Morphology

All baboon species are anatomically and morphologically well adapted to a quadrupedal terrestrial lifestyle [START_REF] Fleagle | Old World monkeys[END_REF]. They have doglike muzzles and males have large canine teeth. Depending on the species, body mass varies between 17 and 30 kg for adult males, and between 10 and 15 kg for females, resulting in a sexual dimorphism in mass ranging between 1.55 and 2.20 (Anandam et al., 2013;[START_REF] Fischer | Charting the neglected west: the social system of Guinea baboons[END_REF][START_REF] Swedell | African Papionins: Diversity of Social Organization and Ecological Flexibility[END_REF].

The species differ notably in their fur color, body size and sexually selected characteristics, such as the distinct capes which are most pronounced in Guinea and hamadryas baboons but also present in olive baboons. Females of all species develop sexual swellings of the anogenital region when they are fertile. These swelling change throughout the menstrual cycle, such that maximum swelling typically coincides with ovulation [START_REF] Higham | Baboon sexual swellings: information content of size and color[END_REF]. The size and shape of the swellings varies considerably among species (Figure 2; [START_REF] Petersdorf | Sexual selection in the Kinda baboon[END_REF].

Ecology

All baboon species are largely terrestrial during the day but retreat to sleeping trees or cliffs during the night. They exhibit great ecological flexibility, allowing them to occupy habitats including semi-deserts grasslands, woodland savannas, humid forests, and Afroalpine grasslands over 3,000 meters above sea level [START_REF] Chala | Species-specific effects of climate change on the distribution of suitable baboon habitats -Ecological niche modeling of current and Last Glacial Maximum conditions[END_REF][START_REF] Fuchs | Ecological niche modeling of the genus Papio[END_REF]. Baboons eat a broad range of foods, although their diet mainly consists of plants, including fruit, seeds, leaves, and roots. They also eat insects and other arthropods and, occasionally, kill small antelopes, hares, rodents, birds and smaller monkeys (Goffe and Fischer, 2016;[START_REF] Swedell | African Papionins: Diversity of Social Organization and Ecological Flexibility[END_REF].

Phylogeography

Phenotypic differences between species are well recognized [START_REF] Jolly | Species, subspecies, and baboon systematics[END_REF], and based on their molecular phylogeny, baboons are generally split in two major groups: north and south [START_REF] Dunn | Biogeographic variation in the baboon: dissecting the cline[END_REF][START_REF] Frost | Cranial allometry, Phylogeography, and systematics of large-bodied papionins (primates: cercopithecinae) inferred from geometric morphometric analysis of landmark data[END_REF]. However, genetic evidence reveals a complex evolutionary history of the genus Papio. Analysis of mitochondrial DNA yields a phylogeny that includes several major haplogroups or cladesgroups of individuals who belong to a specific mitochondrial lineage. These haplogroups reflect the geographic origin of the respective specimens better than their external phenotypes or taxonomic classification, making species appear to be paraphyletic and polyphyletic when mapped onto the mitochondrial phylogeny [START_REF] Zinner | Mitochondrial phylogeography of baboons (Papio spp. ) -Indication for introgressive hybridization?[END_REF][START_REF] Zinner | The strange blood: natural hybridization in primates[END_REF].

Comparisons of whole genome sequences confirm the six baboon species taxonomy and suggest that the initial evolutionary divergence separated a southern lineage that ultimately produced Kinda, chacma and yellow baboons, from a northern lineage that produced olive, hamadryas and Guinea baboons [START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF]. Ancient hybridization events appear to have affected the genetic makeup of all species. For instance, Guinea baboons most likely experienced genetic admixture with a "ghost lineage" that is probably extinct, or that is at least not represented in the sample of genomes analyzed to date [START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF].

Multiple episodes of admixture and introgression have been linked to climate change and range expansion [START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF][START_REF] Walker | Papio baboon species indicative alu elements[END_REF][START_REF] Wall | Genomewide ancestry and divergence patterns from low-coverage sequencing data reveal a complex history of admixture in wild baboons[END_REF][START_REF] Zinner | Baboon phylogeny as inferred from complete mitochondrial genomes[END_REF]. Similar evolutionary mechanisms, including gene transfer by introgressive hybridization, are now recognized to have influenced the evolution of Neanderthals, Denisovans and modern humans [START_REF] Green | A draft sequence of the neandertal genome[END_REF][START_REF] Pru ¨fer | A high-coverage Neandertal genome from Vindija Cave in Croatia[END_REF][START_REF] Pru ¨fer | The complete genome sequence of a Neanderthal from the Altai Mountains[END_REF][START_REF] Reich | Genetic history of an archaic hominin group from Denisova cave in Siberia[END_REF][START_REF] Ackermann | Hybridization in human evolution: insights from other organisms[END_REF]. However, the absence of genomic data from Social style: The degree of aggressiveness among group members in a species. In societies that exhibit steep dominance hierarchies ("high despotism"), aggression is extremely asymmetrical, while in tolerant species, aggression is mild and frequently bi-directional. A further important component is the degree of nepotism within the species, that is, how kin-biased affiliation is. The social style of a species is associated with variation in relationship quality, which in turn characterizes the social structure of a species. Social system: A primate species' social system encompasses its social organization, social style, mating patterns and parental care system [START_REF] Kappeler | A framework for studying social complexity[END_REF].

non-sapiens African hominins presently hinders our ability to ask questions about ancestral African hominin hybridization [START_REF] Scerri | Did our species evolve in subdivided populations across Africa, and why does it matter?[END_REF][START_REF] Stringer | The origin and evolution of Homo sapiens[END_REF]. Baboons allow us to study the impact of gene flow in an extant model.

Of particular interest for understanding baboon evolution is how changes in population density and spatial structure, such as the opening and closing of forest and other barriers, gave rise to different social systems [START_REF] Jolly | Philopatry at the frontier: a demographically-driven scenario for the evolution of multi-level societies in baboons (Papio)[END_REF]. The range expansion of the genus appears to be of particular relevance. Given a southern African origin, modern baboons experienced a tremendous expansion of their range, possibly linked to changes to the habitats, animal communities and climate that occurred during the Pleistocene and that gave baboons the chance to disperse into the savanna belt north of the tropical forest zone [START_REF] Dolotovskaya | Comparing mitogenomic timetrees for two african savannah primate genera (Chlorocebus and Papio)[END_REF][START_REF] Zinner | The strange blood: natural hybridization in primates[END_REF]. Like humans and other savanna species, baboons have thus been subject of recurrent range shifts, fragmentation, and isolation and reconnection of populations [START_REF] Zinner | The strange blood: natural hybridization in primates[END_REF] -dynamics that affected baboon genetic structure and speciation [START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF].

In summary, baboons can serve as a valuable model for evolutionary divergence and hybridization, triggered by climatic changes and the expansion and fragmentation of populations in the African savanna. Such analyses are also highly relevant for a better understanding of early hominins.

Variation in social organization and behavior

The six baboon species vary substantially in their social characteristics, including social organization, social style and mating patterns. Group size varies within and among species. In chacma, olive and yellow baboons, group size ranges from about a dozen up to roughly one hundred animals [START_REF] Markham | Optimal group size in a highly social mammal[END_REF][START_REF] Swedell | African Papionins: Diversity of Social Organization and Ecological Flexibility[END_REF], while hamadryas and Guinea baboons temporarily aggregate into groups of several hundreds of individuals [START_REF] Patzelt | Group composition of guinea baboons (Papio papio) at a water place suggests a fluid social organization[END_REF][START_REF] Swedell | Hamadryas Baboon (Papio hamadryas)[END_REF]. Sex ratios in baboons vary too; some groups are fairly balanced, while adult females in other groups can outnumber adult males by about 10 to 1 [START_REF] Swedell | African Papionins: Diversity of Social Organization and Ecological Flexibility[END_REF].

Chacma, olive, Kinda and yellow baboonsrecently dubbed "COKY" baboons [START_REF] Jolly | Philopatry at the frontier: a demographically-driven scenario for the evolution of multi-level societies in baboons (Papio)[END_REF] -live in multi-male-multi-female groups, in which related females constitute the stable core, while males leave the group they were born into and join another. Clear rank hierarchies among males and females can be discerned based on aggressive interactions, including threats, chases and physical aggression, as well as signals of submission. In females, related individuals (known as matrilines) typically occupy adjacent ranks. For female chacma, olive and yellow baboons, female kin constitute the most important social partners [START_REF] Silk | Female-male relationships influence the form of female-female relationships in olive baboons, Papio anubis[END_REF][START_REF] Silk | Strong and consistent social bonds enhance the longevity of female baboons[END_REF][START_REF] Silk | Social bonds of female baboons enhance infant survival[END_REF]. In Kinda baboons, however, males are the most significant grooming partners for females [START_REF] Petersdorf | Sexual selection in the Kinda baboon[END_REF].

Females of all COKY species interact and mate with several males in the group. High-ranking males generally experience higher mating success than lower-ranking ones, though this rank-related mating skew is more pronounced in chacma baboons than in olive or yellow baboons [START_REF] Bulger | Adult Male Savanna Baboon Mating Activity and Interactions with Infants[END_REF][START_REF] Henzi | The historical socioecology of savanna baboons (Papio hamadryas)[END_REF][START_REF] Sta ¨dele | Male-female relationships in olive baboons (Papio anubis): Parenting or mating effort?[END_REF]. During female receptive periods, males aggressively guard their female mating partner, resulting in sexual "consortships" [START_REF] Noe | Reproductive tactics of male savanna baboons[END_REF][START_REF] Smuts | Sex and Friendship in Baboons[END_REF]. Consorts may last from several hours up to several days. Consort success (and thus mating success) is often related to male dominance status [START_REF] Gesquiere | Life at the top: rank and stress in wild male baboons[END_REF]. In yellow and olive baboons, however, male coalitions may be able to take the female away from a dominant male [START_REF] Noe | Reproductive tactics of male savanna baboons[END_REF][START_REF] Smuts | Sex and Friendship in Baboons[END_REF].

Male competition and aggressiveness vary considerably between species. Infanticide is frequent in some populations of chacma baboons [START_REF] Palombit | Femalefemale competition for male 'friends' in wild chacma baboons (Papio cynocephalus ursinus)[END_REF], but rare in olive, yellow and hamadryas baboons [START_REF] Lemasson | Friendships between males and lactating females in a free-ranging group of olive baboons (Papio hamadryas anubis): evidence from playback experiments[END_REF][START_REF] Swedell | African Papionins: Diversity of Social Organization and Ecological Flexibility[END_REF]. Lactating females often form close ties to specific males, which are often the sires of their infants [START_REF] Huchard | More than friends? Behavioural and genetic aspects of heterosexual associations in wild chacma baboons[END_REF][START_REF] Moscovice | Hedging their bets? male and female chacma baboons form friendships based on likelihood of paternity[END_REF][START_REF] Nguyen | Friendships" between new mothers and adult males: adaptive benefits and determinants in wild baboons (Papio cynocephalus)[END_REF][START_REF] Sta ¨dele | Male-female relationships in olive baboons (Papio anubis): Parenting or mating effort?[END_REF]. These relationships appear to be an adaptation against infanticide by recent immigrant males [START_REF] Palombit | The adaptive value of 'friendships' to female baboons: experimental and observational evidence[END_REF] and harassment by other group members [START_REF] Buchan | True paternal care in a multi-male primate society[END_REF] as well as a form of paternal investment [START_REF] Buchan | True paternal care in a multi-male primate society[END_REF][START_REF] Huchard | Paternal effects on access to resources in a promiscuous primate society[END_REF]. Male alliances are absent in chacma baboons, while common in yellow and olive baboons [START_REF] Noe | Which adult male savanna baboons form coalitions?[END_REF]. These differences in male competitive regimes are reflected in their dispersal behavior: male chacma baboons in the Okavango delta, for instance, do not emigrate from their natal group until after they are fully grown [START_REF] Beehner | Testosterone related to age and life-history stages in male baboons and geladas[END_REF], while male olive and yellow baboons often emigrate during adolescence (Alberts and Altmann, 1995;[START_REF] Packer | Reproductive constraints on aggressive competition in female baboons[END_REF].

Over the past decade, studies of Kinda baboons have broadened our knowledge of morphological and behavioral variation within the genus. Kinda baboons are smaller in body size, have reduced sexual dimorphism in body and canine size, and have larger relative testis volume, compared to other baboon species [START_REF] Jolly | Philopatry at the frontier: a demographically-driven scenario for the evolution of multi-level societies in baboons (Papio)[END_REF]. Kinda females exhibit small sexual swellings (Figure 2) and give inconspicuous calls [START_REF] Petersdorf | Sexual selection in the Kinda baboon[END_REF]. Chacma females, in contrast, give loud copulation calls, which function to incite male-male competition (O' Connell and Cowlishaw, 1994).

Hamadryas baboons -in contrast to the COKY baboon species described above -live in a multi-level society with reproductive units, called "one male units" comprising one sexually active leader male, a variable number of females, and sometimes a follower male [START_REF] Kummer | Social Organization of Hamadryas Baboons[END_REF]. Associations between several one-male units constitute a clan (Abegglen, 1984); several clans and unaffiliated bachelor males form a band, the main ecological unit, and multiple bands coalesce at resources, especially sleeping sites, to form troops [START_REF] Schreier | The fourth level of social structure in a multi-level society: ecological and social functions of clans in hamadryas baboons[END_REF]. Recent behavioral and genetic studies of hamadryas baboons show that leader and follower males tend to be maternally related, in line with the fact that they disperse only rarely. Females within a unit are also more likely to be related than expected by chance [START_REF] Sta ¨dele | The ties that bind: maternal kin bias in a multilevel primate society despite natal dispersal by both sexes[END_REF].

Guinea baboons also live in a multi-level society. Several units comprising a primary male, 1-6 females, young, and occasional secondary males make up parties, and 2 to 3 parties constitute a gang within a larger community [START_REF] Fischer | Charting the neglected west: the social system of Guinea baboons[END_REF]. Male Guinea baboons maintain strong bonds and a high degree of spatial tolerance [START_REF] Fischer | Charting the neglected west: the social system of Guinea baboons[END_REF]. Some, but not all males with strong bonds are highly related, suggesting that the existence of kin in the group promotes male tolerance [START_REF] Patzelt | Male tolerance and male-male bonds in a multilevel primate society[END_REF]. In striking contrast to most other baboon species, aggression between males is so rare that it is not possible to discern a dominance hierarchy with certainty [START_REF] Dal Pesco | Greetings in male guinea baboons and the function of rituals in complex social groups[END_REF]. Males engage in extended ritualized greetings that apparently function to reinforce delineations between parties and to test bonds between males [START_REF] Dal Pesco | Greetings in male guinea baboons and the function of rituals in complex social groups[END_REF]. Females freely transfer between units, parties and gangs. Female tenure with a given male may vary between weeks and years (Goffe et al., 2016). Both Guinea and hamadryas baboons exhibit female-biased dispersal [START_REF] Kopp | Population genetic insights into the social organization of Guinea baboons (Papio papio): Evidence for female-biased dispersal[END_REF][START_REF] Sta ¨dele | Fine-scale genetic assessment of sex-specific dispersal patterns in a multilevel primate society[END_REF].

Note that many of the most significant differences in social behavior between species have been observed across different populations in multiple African sites, as well as in captivity. Thus, there is good evidence that the variation we describe here reflects true species differences and not just variation between populations. Yet, characterizing the variation within species in greater detail would be extremely valuable.

Despite the variation in social organization and aggressiveness between the different baboon species, there is very little variation in the vocal repertoires and call types within the genus [START_REF] Hammerschmidt | Baboon vocal repertoires and the evolution of primate vocal diversity[END_REF]. This suggests that the structure of vocal patterns is highly conserved. Because species vary in their aggressiveness and their propensity to affiliate, they also differ in the frequency with which they use signals that either relate to fighting ability or "benign intent", respectively (Faraut et al., 2019;[START_REF] Fischer | Charting the neglected west: the social system of Guinea baboons[END_REF].

Variation in social cognition

Variation in social organization and in the nature and extent of competition over resources between baboon species is thought to result in differential selective pressure on social cognition (Amici et al., 2008;Aureli et al., 2008). To date, most of the work on baboon social knowledge has been done on chacma baboons that exhibit steep dominance hierarchies (known as despotism). A suite of studies by the American primatologists Dorothy Cheney and Robert Seyfarth and colleagues revealed that chacma baboons have sophisticated social knowledge (reviewed in [START_REF] Cheney | Baboon Metaphysics: The Evolution of a Social Mind[END_REF]. For instance, the animals represent the nested hierarchical rank relationships of their group members [START_REF] Bergman | Hierarchical classification by rank and kinship in baboons[END_REF], track the consortship status of pairs in their group [START_REF] Crockford | Baboons eavesdrop to deduce mating opportunities[END_REF], and selective deploy aid to unrelated individuals that were former grooming partners [START_REF] Cheney | Contingent cooperation between wild female baboons[END_REF].

Field playback experiments revealed that baboon species respond differently to social information. While the territorial chacma baboons respond strongly to apparent intruders [START_REF] Kitchen | Male baboon responses to experimental manipulations of loud "wahoo calls": testing an honest signal of fighting ability[END_REF], the spatially tolerant Guinea baboons paid more attention to vocalizations from co-resident group members compared to neighbors or strangers [START_REF] Maciej | Social monitoring in a multilevel society: a playback study with male Guinea baboons[END_REF]. Similarly, chacma baboons respond strongly to simulated rank reversals [START_REF] Bergman | Hierarchical classification by rank and kinship in baboons[END_REF] or break-ups of existing consortships [START_REF] Crockford | Baboons eavesdrop to deduce mating opportunities[END_REF], while Guinea baboon males were more interested in social information consistent with current social association patterns (Faraut and Fischer, 2019). The somewhat surprising responses of the Guinea baboons may be a result of the high gregariousness of the species, where deviant interaction patterns may initially be classified as "social noise" (Faraut and Fischer, 2019). In summary, these findings suggest that the content of what is represented, namely the associations between different individuals or their group memberships, appears to be relatively similar across the two species, while the value of different types of social information may vary substantially in relation to the type of society.

Sociality, health, aging and fitness

Over the past decade, baboon research has provided ground-breaking insights into the relationships between social status, social relationships, health and fitness measures such as offspring survival and longevity. Data from two long-term studies of baboon behavior and life history suggest that sociality enhances the fitness of females. For example, infants born to yellow baboon females who are more socially integrated have higher survival than infants of less social mothers (Archie et al., 2014;[START_REF] Silk | Social bonds of female baboons enhance infant survival[END_REF]; similar patterns are also seen in chacma baboons [START_REF] Silk | Strong and consistent social bonds enhance the longevity of female baboons[END_REF]. As in many other primates, higher-ranking male baboons sire more offspring than other males (Altmann et al., 1996). Higherranking females have shorter periods before they resume menstrual cycling following birth [START_REF] Gesquiere | Interbirth intervals in wild baboons: environmental predictors and hormonal correlates[END_REF][START_REF] Johnson | Life history and the competitive environment: trajectories of growth, maturation, and reproductive output among chacma baboons[END_REF][START_REF] Packer | Reproductive constraints on aggressive competition in female baboons[END_REF][START_REF] Smuts | Reproduction in wild female olive baboons[END_REF][START_REF] Wasser | Population trend alters the effects of maternal dominance rank on lifetime reproductive success in yellow baboons (Papio cynocephalus)[END_REF], which may be linked to quicker restoration of positive energy balance [START_REF] Gesquiere | Interbirth intervals in wild baboons: environmental predictors and hormonal correlates[END_REF]). Consistent with this, feeding on crops in olive baboons [START_REF] Higham | Living on the edge: life-history of olive baboons at Gashaka-Gumti National Park, Nigeria[END_REF], or discarded human food for yellow baboons (Altmann et al., 1977), also leads to a quicker return to menstrual cycling and increases reproductive output.

A number of studies have investigated the proximate mediators of the relationship between behavior and fitness. In particular, many researchers have taken advantage of noninvasive ways to measure glucocorticoid hormones, a class of hormones known to mediate the energetic demands that accompany social and ecological challenges. Concentrations of glucocorticoid hormones increase during specific challenges that are known to threaten an individual's fitness. For example, lactating chacma females that were at risk for infanticide because a new male immigrated into the group exhibited elevated glucocorticoid hormones compared to female counterparts that were not at risk [START_REF] Beehner | The effect of new alpha males on female stress in free-ranging baboons[END_REF]. Additionally, loss of a close female relative increases glucocorticoid concentrations, and this increase may be responsible for initiating a compensatory broadening and strengthening of female grooming networks [START_REF] Engh | Female hierarchy instability, male immigration and infanticide increase glucocorticoid levels in female chacma baboons[END_REF].

Several studies have investigated the relationship between glucocorticoid concentrations, rank and social stability in male baboons. In a long term-study of yellow baboons, high-ranking males had lower glucocorticoid concentrations, regardless of hierarchy stability, while alpha males may experience higher concentrations than expected for their rank [START_REF] Gesquiere | Life at the top: rank and stress in wild male baboons[END_REF]. Nonetheless, high-ranking yellow baboon males get sick less often and heal from wounds faster, suggesting that these high-ranking males are in better health and do not suffer trade-offs from these extra demands (Archie et al., 2012). Higher-ranking chacma baboon males also had higher glucocorticoid concentrations [START_REF] Bergman | Correlates of stress in free-ranging male chacma baboons, Papio hamadryas ursinus[END_REF][START_REF] Kalbitzer | Social behavior and patterns of testosterone and glucocorticoid levels differ between male chacma and Guinea baboons[END_REF].

More recently, baboons were also established as a promising model for studying the impact of sexually transmitted diseases on mating behavior. Female olive baboons in a population infected with the bacterium Treponema pallidum, a substrain of which causes syphilis in humans, copulated less with males showing clinical signs of infection (Pacie ˆncia et al., 2019). These findings highlight how pathogens may impose important selective pressures in mate choice and ultimately social evolution.

Functional genomics

The addition of data on Kinda and Guinea baboons increases the value of the baboon as a model, as we begin to have data available for all baboon species. While one aim of future analyses will be to understand the sources of variation between species, documenting similarities is equally valuable. Technological developments in genomic sequencing [START_REF] Robinson | Analysis of 100 high-coverage genomes from a pedigreed captive baboon colony[END_REF][START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF], including from non-invasively collected samples such as feces [START_REF] Perry | Genomic-scale capture and sequencing of endogenous DNA from feces[END_REF][START_REF] Snyder-Mackler | Efficient Genome-Wide sequencing and Low-Coverage pedigree analysis from noninvasively collected samples[END_REF], have brought genomics to the forefront of baboon behavioral studies [START_REF] Tung | Evolutionary genetics in wild primates: combining genetic approaches with field studies of natural populations[END_REF]. Given the close phylogenetic relatedness of the six baboon species, variation in key aspects of social behavior, and the presence of hybrids displaying intermediate phenotypes within hybrid zones, investigation of causal pathways from genotype to phenotype seems particularly promising within the baboon model [START_REF] Bergey | Dopamine pathway is highly diverged in primate species that differ markedly in social behavior[END_REF][START_REF] Jolly | Cerebrospinal fluid monoaminergic metabolites in wild Papio anubis and P. hamadryas are concordant with Taxon-specific behavioral ontogeny[END_REF].

Formerly, research into primate behavioral genetics focused on identifying a few small specific functional polymorphisms in sequence or length, and on linking these to phenotypic variation (e.g., [START_REF] Kalbitzer | Insights into the genetic foundation of aggression in Papio and the evolution of two length-polymorphisms in the promoter regions of serotonin-related genes (5-HTTLPR and MAOALPR) in Papionini[END_REF]. However, such studies are likely to overestimate the effect of one single aspect of genetic variation. With genotyping of single nucleotide polymorphisms (SNPs), and whole-genome sequencing, primate field studies are beginning to explore the wider genomic architecture that underlies variation in social behavior [START_REF] Rogers | The behavioral genetics of nonhuman primates: status and prospects[END_REF]. As well as whole genome sequence data, researchers now have access to annotations for protein coding genes and transcriptomes [START_REF] Robinson | Analysis of 100 high-coverage genomes from a pedigreed captive baboon colony[END_REF][START_REF] Rogers | The comparative genomics and complex population history of Papio baboons[END_REF][START_REF] Vilgalys | Evolution of DNA methylation in Papio baboons[END_REF]. We therefore expect an exponential increase in the number and diversity of available genomes, which will facilitate the investigation of the basis of baboon adaptations and adaptive flexibility.

In conjunction with research on other model organisms, such as deer mice [START_REF] Bedford | Peromyscus mice as a model for studying natural variation[END_REF], such studies provide fundamental insights into the foundation of natural variation and adaptation in socially living mammals.

Baboons in the Anthropocene

Baboons allow us to study the effects of accelerating anthropogenic fragmentation, loss of natural habitats and climate change in a highly adaptable primate system. For example, baboons may rapidly change how long they allocate time and energy to different behaviors or where they range, in response to human-related activities and habitat changes [START_REF] Fehlmann | Extreme behavioural shifts by baboons exploiting risky, resource-rich, human-modified environments[END_REF][START_REF] Ferreira Da Silva | Disrupted dispersal and its genetic consequences: comparing protected and threatened baboon populations (Papio papio) in west Africa[END_REF]. Studies of individual baboon behavior can use sophisticated GPS tracking and non-invasive genetic tools to make broad-scale inferences about movements and processes at the population level [START_REF] Kopp | The influence of social systems on patterns of mitochondrial DNA variation in baboons[END_REF][START_REF] Strandburg-Peshkin | Shared decision-making drives collective movement in wild baboons[END_REF]. These inferences can then be applied to questions of how other large populations of mammals will respond to changes in their environment.

Baboons are not considered a global priority in conservation, with the exception of Guinea baboons which are categorized as Near Threatened by the IUCN [START_REF] Oates | Mating avoidance in female olive baboons (Papio anubis) infected by Treponema pallidum[END_REF]. However, populations geographically overlapping with human communities often damage crops and infrastructure and are persecuted as pests. In some locations, people consume substantial number of baboons and sell their meat in bushmeat markets (e.g., [START_REF] Minho ´s | DNA identification of primate bushmeat from urban markets in Guinea-Bissau and its implications for conservation[END_REF]. Humans and baboons often compete for space and hunting of specific individuals or even entire groups is increasingly frequent, leading to fragmented populations and local extinctions [START_REF] Ferreira Da Silva | Disrupted dispersal and its genetic consequences: comparing protected and threatened baboon populations (Papio papio) in west Africa[END_REF]. Non-monitored populations living outside protected areas may be at a higher risk of silently disappearing. The challenge is to assess the risk of different Box 2. Outstanding questions about the natural history of baboons.

. How have changes in population density and environmental conditions (e.g., opening and closing of forest and other barriers) affected dispersal and mating patterns, and ultimately given rise to different social systems?

. What is the genetic architecture of baboon social behavior (including social style, patterns of dispersal, and degree of reproductive skew according to social status)? How does that architecture promote or restrict evolutionary flexibility in social systems?

. Does the link between sociality and reproductive success vary among species or even local populations?

. Do the different species vary in the way they represent the social relationships around them and how they attend to social information?

. How responsive are baboons to changes in temperature patterns due to global warming, as well as to associated changes in aridity or habitat type? populations and develop appropriate conservation plans.

The long-term nature of many baboon field studies has provided great insight into how populations may rise and fall rapidly in response to changes in the environment. The Amboseli Baboon Project, for example, has been running continuously for 50 years, and has documented numerous periods of relative drought or rainfall abundance (Alberts et al., 2005;[START_REF] Gesquiere | Life at the top: rank and stress in wild male baboons[END_REF]. This variation in precipitation has been linked to variation in fecundity and survival [START_REF] Beehner | The ecology of conception and pregnancy failure in wild baboons[END_REF][START_REF] Lea | Developmental constraints in a wild primate[END_REF] and to subsequent changes in population structure (Altmann et al., 1985). Periods of environmental change, and consequent boom and bust cycles in populations, are driven by both natural phenomena, such as natural aging of woodland, and anthropogenic influences, such as overgrazing by pastoralists (Alberts and Altmann, 2011). Many long-term baboon field sites also carefully collect detailed data on temperature and rainfall, as well as food availability and diseases. They also monitor the habitats in addition to the baboon populations. The breadth and scope of such data ensure that the baboon represents an outstanding model of both individuallevel and population-level responses to environmental change.

Conclusion

Baboons constitute a fascinating and informative analog model for hominin evolution in savanna habitats, with their ongoing patterns of range expansions and contractions, and regular occurrences of hybridization where two species meet. Given the availability of long-term data and the variation in the types of societies baboons live in, they constitute an excellent test case to study the link between sociality, health, longevity and reproductive success, as well as the emergence and spread of diseases. Such studies are extremely important to put biomedical data from captive baboon studies into natural context. For future research, we propose an approach that integrates field observations and carefully designed field experiments with cutting-edge measures of genomic variation, gene expression, non-invasive endocrinology and immunology. The fact that baboons have been studied in a wide range of habitats at sites across Africa for several decades also make them an informative example to investigate how populations of large mammals respond to environmental diversity and change (see Box 2 for suggested future research questions).

Figure 1 .

 1 Figure 1. Distribution of the six Papio species. Species distributions are modified from Zinner et al. (2013). Male baboon drawings by Stephen Nash. Reprinted with permission from Fischer et al. (2017). DOI: https://doi.org/10.7554/eLife.50989.003

Figure 2 .

 2 Figure 2. Illustration of key traits across baboon species. (A) Phenotypic variation between species. Pictures show adult males and females. (B) Crania of male baboons. (C) Sexual swellings of female baboon during peak estrus. Species are grouped by social organization (uni-and multi-level) and dispersal behavior (male-or female-biased dispersal). Images from Alexis Amann, Andrea Cardini, Sarah Elton, Julia Fischer, Courtney Fitzpatrick, James Higham, Megan Petersdorf, Joan Silk and Larissa Swedell. DOI: https://doi.org/10.7554/eLife.50989.004

Fischer et al. eLife 2019;8:e50989. DOI: https://doi.org/10.7554/eLife.50989

Acknowledgements

This paper is dedicated to the memory of Dorothy L Cheney who loved baboons. We thank the funders of the Frontiers in Baboon Research Symposium, namely the Deutsche Forschungsgemeinschaft (FI 707/21-1), the Leibniz Science-Campus Primate Cognition and the University of Go ¨ttingen. We thank PJ Perry and the editors and reviewers, for valuable comments and suggestions.

Deutsche Forschungsgemeinschaft funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Data availability

This is a review article; there are no data sets associated with this article.

Additional files


Flore Nilly

Mélyssa Cambronel

Olfa Baccouri

Amine Boukerb

Sophie Rodrigues

Marc Feuilloley

Nathalie Connil

STRESS HORMONES INCREASE BIOFILM FORMATION OF ENTEROCOCCUS FAECALIS

come

STRESS HORMONES INCREASE BIOFILM FORMATION OF ENTEROCOCCUS FAECALIS

Flore Nilly, Mélyssa Cambronel, Olfa Baccouri, Amine Mohamed Boukerb, Sophie Rodrigues, Marc Feuilloley and Nathalie Connil Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM) EA 4312, Université de Rouen Normandie, Normandie Université, Évreux, France.

Objective:

Enterococcus faecalis is a Gram positive, commensal bacterium of the human gut. This bacterium has controversial status due to its emerging role in nosocomial infections, while some strains with beneficial effects are used as probiotics and starter cultures in dairy industry. All these bacteria can be found on skin or in gut where they are continually exposed to various eukaryotic molecules. In this context, the aim of our work was to evaluate the effect of stress hormones on some Enterococcus strains.

Methods:

Three E. faecalis strains were included in this study: E. faecalis MMH594 and E. faecalis V583, pathogenic strains of clinical origin, and E. faecalis OB15, a probiotic strain, previously isolated from tunisian Rigouta (Baccouri et al, 2019, Front Microbiol. doi: 10.3389/fmicb.2019.00881.) These bacteria were exposed to 1µM of epinephrine (Epi) or norepinephrine (NE), and their capacity to form biofilm were quantified by Confocal Laser Scanning Microscopy (CLSM) after staining with the SYTO-9 green fluorescent nucleic acid stain

Conclusions:

This study showed for the first time that stress hormones could increase biofilm formation in E. faecalis. Future experiments will aim to decipher the mechanisms involved and to identify an adrenergic putative sensor in E. faecalis. This may help to develop new strategies of antagonism to prevent the colonization by opportunistic pathogens.

Results:

Stress hormones (Epi and NE) were found to modulate the formation of biofilms (biomass, thickness) in E. faecalis. The major effect was observed for the E. faecalis OB15 probiotic strain, with significant increases of biomass (+25%) and thickness (+27%) with Epi.

  of E. faecalis biofilms treated by Epi Statistical analysis: Results were expressed as means ± standard error (SE) of three experiments done in triplicate. Analysis of statistical significance was performed with Student's t-test and GraphPad Prism8 (* : p < 0.05, ** : p < 0.01) Quantification by CLSM of E. faecalis biofilms treated by NE


Nathalie Connil
email: nathalie.connil@univ-rouen.fr

Olfa Baccouri

Mohamed Zommiti

Marc Feuilloley

Risk and safety of Enteroccocci in probiotic use

come