Age estimates of cheirogaleid divergences

The complete alignment consisting of five loci (cytb,cox2,adora3,fiba andvWF) contained six missing sequences: threecox2 sequences (Galago moholi,Nycticebus pygmaeus andOtolemur garnetti)plus threefibasequences(Galago moholi,Nycticebus coucang andLoris tardigradus).

Table 4.3: Nucleotide substitution models and substitution rates per locus. The best fit model according to AIC and the model used inBeastanalyses are given. The nucleotide substitution rates were calculated for the galago/loris split. For the cox2 locus the respective sequence data was not available and the galago/loris rate therefore estimated using rates from the human/chimpanzee split.

Model Rate

Furthermore, several sequences contained missing data in thefiba andvWF fragments, rang-ing from a few basepairs to around 250 bp. No indels were present in the adora3, cytb and cox2 fragments, but multiple indels characterized thefiba andvWF fragments. The complete data set contained 3712 bp of aligned sequence, of which 1888 characters were variable and 1522 were parsimony informative. When the nuclear loci were excluded, the mtDNA align-ment contained 1824 bp of aligned sequence, of which 996 characters were variable and 874 parsimony informative.

The cytb, fiba and vWF loci were each found to best fit a transversion model (TVM) according to AIC, but were analyzed under a general time reversible (GTR) model. The nuclear loci were best fit to a model with gamma distributed rate heterogeneity (Γ), whereas the cytb locus was best fit to a model with both gamma distributed rate heterogeneity (Γ) and a proportion of invariant sites (I). The adora3 locus was found to best fit a GTR + Γ model, whereas the cox2 locus was found to best fit a Hasegawa-Kishino-Yano (HKY) +Γ+ I model (Table 4.3). Substitution rates, calculated based on the dated galago/loris divergence, ranged from 0.002 for the cox2 locus to 0.0009 for thefiba and vWF loci. All rates are given in Table 4.3.

The estimation of a strepshirrine phylogeny was not the main aim of this study and for two of the three BEAST analyses the topology was fixed a priori according to analyses of 18 nuclear loci by Horvath et al. (2008). Instead, we focused on divergence date estimates within the Cheirogaleidae, but age estimates for deeper nodes are also given in Table 4.4 for comparative purposes.

Age estimates yielded by the ‘combined data’ and the ‘mtDNA’ analyses were very similar.

Only the 95% credibility interval for the root is larger in the ‘mtDNA’ than in the ‘combined data’ analysis, due to no calibration prior having been enforced on the root in this analy-sis. The ‘unconstrained topology’ analysis resulted in an altered topology, with Daubentonia madagascariensis placed basal to all other strepsirrhines andLepilemur ruficaudatus and Pro-pithecus tattersalli emerging as sister taxa. The age estimates for this analysis are slightly older among the deeper nodes. For the nodes of interest, within the Cheirogaleidae, the es-timates do not differ by more than 1 ma and the 95% credibility intervals largely overlap.

Results of the ‘combined data’ analysis are shown in Fig. 4.7 and Table 4.4 and only age estimates from this analysis will be discussed in detail. The first divergence within the genus Cheirogaleus is estimated at 15.5 mya and divergence within species at 6.2 mya forC. medius, 4.8 mya for C. crossleyi and 2.1 mya forC. major.

Figure 4.3: Map of Madagascar showing the sampling sites and the centers of endemism and retreat-dispersion watersheds according to Wilmé et al. (2006). The sampling sites are color-coded according to species as assessed in chapter 2 and 3: yellow=C.

medius, red=C. major and blue=C. crossleyi. The sampling sites in the southeast (Fort Dauphin region) are pooled according to species. For a detailed description of these sites refer to Hapke et al. (2005). Map modified after Wilmé et al. (2006).

Figure 4.4: Simplified phylogeny as shown in Fig. 4.1 superimposed onto a map of Madagascar showing the sampling sites and the centers of endemism and retreat-dispersion watersheds according to Wilmé et al. (2006). Clades representing the different species are color-coded: C. medius=yellow,C. major=red andC. crossleyi=blue and the clade number as given in Fig. 4.1 is denoted. The sampling sites in the southeast (Fort Dauphin region) are pooled according to geographic location; see Fig. 4.5. For a detailed description of these sites refer to Hapke et al. (2005). Map modified after Wilmé et al. (2006).

Figure 4.5: Map of Madagascar showing the 39 sampling sites and the eight biogeographic zones (N, E1, E2, W2, W1, NW, x/SA, CH) adapted from Martin (1972), Richard and Dewar (1991), Martin (1995), Ganzhorn et al. (2006). Limits of the biogeo-graphic zones, excluding the central highlands (CH), are defined through major rivers: Bemarivo, Mangoro, Tsiribihina, Betsiboka, Maevarano and Mahavavy du Nord rivers and a mountain chain: the Anosy hill chain. The limits of the bio-geographic zones towards the inland (CH) are defined mainly through elevation (800-1000m). The biogeographic zones are color-coded as indicated by the col-ored background of the label. The sampling sites are labeled by locality number as denoted in Table 4.1.

Figure4.6:Cladogramsofbiogeographiczones.A)SimplifiedphylogenyofCheirogaleusindividualsasshowninFig.4.1.Thebiogeographiczones,inwhichtheindividualsofeachcladearepresent,aregiveninparenthesestogetherwiththesamplingsitenumbers.Thebiogeographiczonesarecolor-coded(comparewithFig.4.5).Ancestraldistributions,asreconstructedbyDIVA,foreachinternalnodearedenotedbycoloredsquares.Asterisksdenoteancestraldistributionswithmorethanoneoptimalreconstruction.B)CladogramofbiogeographiczonesbasedonspeciessimilaritiesoflemurcommunitiesaccordingtoGanzhornetal.(2006).

Figure 4.7: Ultrametric tree with divergence age estimates resulting from the combined pos-terior distribution of the three replicates of the ‘combined data’Beast analysis.

The mean age estimate for each node is given in millions of years, with the re-spective 95% credibility intervals indicated by the blue bars. The means in bold indicate nodes used as calibrations. A geological time scale is given at the top:

Pa=Paleocene, Pl=Pliocene and Q=Quaternary. Full details of age estimates of all analyses are presented in Table 4.4. Cheirogaleus individuals are

as-Table4.4:Bayesiandivergencedateestimatesinmillionsofyears.Themeanand95%credibilityintervalsaregivenfortheestimatesofthree analysesofthisstudyandpublishedestimatesfor17nodesasdefinedinFigure4.2.Nodesusedascalibrationsaremarkedinbold. NodeThisstudyThisstudyThisstudyHorvath1 Yoder&Yang2 Kappeler3 Roos4 Poux5 Combined data MtDNAUnconstrained topology 18nuclear loci 2mtDNA loci 5loci: mtDNA+nDNA

cytbcytb3nuclear loci R)Root81.8886.2488.68n/an/an/an/an/a 73.26,90.8063.88,109.0767.35,112.76 C1)Lorisiformes39.2639.4339.2839.3840.039.1n/a46n/a 36.94,41.6437.04,41.836.93,41.6436.91,41.6438.1,41.938.0,41.537,60 C2)Homo/Pan6.656.676.71n/an/an/an/an/an/a 5.56,7.715.58,7.775.62,7.795-7asprior N1)Strepsirrhini54.1754.0962.0575.0472.968.5n/a6160.4 46.95,62.3244.61,65.5847.92,76.4466.85,84.4164.0,82.061.3,75.450,8051.6,69.6 N2)Lemuriformes48.4448.6n/a66.2267.162.0n/a5849.6 39.73,57.6537.99,59.7354.91,74.7456.8,77.257.9,73.047,7641.1,58.5 N3)Lemuriformes35.236.1437.6139.3346.742.3n/a43n/a w/oAyeAye28.75,41.9128.06,44.1628.93,46.0433.4,45.8436.9,57.535.4,49.535,56 N4)Cheirogaleidae*25.7926.1627.6323.0531.829.0n/an/an/a 20.88,30.9220.06,32.2521.18,34.3318.61,28.0823.4,41.622.7,35.9 N5)Mirza/Microcebus18.9519.220.1914.1124.219.924.2n/an/a 14.87,23.2714.29,24.0815.02,25.3110.83,17.9416.8,33.414.6,26.1n/a N6)Cheirogaleus15.4516.016.59n/an/an/an/an/an/a continuedonnextpage

NodeThisstudyThisstudyThisstudyHorvath1Yoder&Yang2Kappeler3Roos4Poux5 Combined data MtDNAUnconstrained topology 18nuclear loci 2mtDNA loci 5lociincl. mtDNA+nDNA

cytbcytb3nuclear loci 11.7,19.4411.74,20.5212.12,21.56 N7)C.major/C.crossleyi12.9312.813.87n/an/an/an/an/an/a 9.6,16.669.13,16.839.77,18.4 N8)Microcebus12.0212.2112.736.9612.08.912.5n/an/a 9.29,14.978.89,15.439.33,15.884.83,9.177.8,17.95.5,13.2 N9)Microcebus,cladeA10.039.8210.65n/a10.0n/a8.8n/an/a 7.57,12.577.05,12.77.76,13.546.3,15.2 N10)Microcebus,cladeB8.48.588.87n/a8.8n/a8.4n/an/a 5.77,11.175.74,11.66.01,12.05.3,13.6 N11)Cheirogaleusmedius6.246.586.65n/an/an/an/an/an/a 4.52,8.124.53,8.84.65,8.95 N12)Cheirogaleuscrossleyi4.754.895.05n/an/an/an/an/an/a 3.32,6.363.26,6.613.25,6.83 N13)Cheirogaleusmajor2.42.582.54n/an/an/an/an/an/a 1.53,3.371.54,3.691.53,3.61 N14)Mirza2.082.182.24n/an/an/a2.1n/an/a 1.25,3.011.22,3.181.28,3.34n/a 1 Horvathetal.(2008),2 YoderandYang(2004),3 Kappeleretal.(2005),4 Roosetal.(2004),5 Pouxetal.(2005) * PhanerandAllocebusmissing,sincePhaneristhemostbasallineagewithintheCheirogaleidaethisageisanunderestimate n/a=ageestimatenotavailable

4.4 Discussion