how many species of woolly monkeys inhabit colombian forests?

American Journal of Primatology 72:1131–1140 (2010)

RESEARCH ARTICLE

How Many Species of Woolly Monkeys Inhabit Colombian Forests?

SERGIO BOTERO1�, LAURA Y. RENGIFO2, MARTA L. BUENO2, AND PABLO R. STEVENSON1

1Departamento de Ciencias Biologicas, Universidad de los Andes, Bogota, Colombia2Instituto de Genetica, Universidad Nacional de Colombia, Bogota, Colombia

There is a controversy regarding how many species the genus Lagothrix contains, since the Lagothrixlagothricha subspecies have been recently proposed to be actual species. Clarification of species statusis of particular importance in the case of L. l. lugens, because it is the most endangered and itsdistribution is restricted to the Colombian Andes, a highly deforested region. Using cytogenetic andmolecular markers, we obtained evidence indicating that the subspecies status is appropriate for thetwo taxa occurring in this country. We also report high levels of intraspecific variability in thekaryotype. We find evidence for a late Pleistocene separation of the subspecies, and we propose it isthe limited area of contact between the taxa that allowed for them to partially differentiate. Am. J.Primatol. 72:1131–1140, 2010. r 2010 Wiley-Liss, Inc.

Key words: Lagothrix lagothricha lugens; Pleistocene refugia; karyotype; cytogenetic; Neotropicalprimate conservation; Atelidae; Atelinae

INTRODUCTION

Woolly monkeys (Lagothrix, Atelidae) are largefrugivorous primates that are widely distributed overcentral and western Amazonia [1963]. Their size andhabits make them particularly sensitive to humandisturbance [Defler, 2004]. A recent review of theirIUCN classification now ranges from Vulnerable toCritically Endangered. IUCN has identified habitatdegradation as the main risk for populations in theAndean region, whereas hunting pressure is themost important factor in lowland Amazonia [Boubliet al., 2008; Cornejo et al., 2008; Palacios et al., 2008;Stevenson & Link, 2008; Stevenson et al., 2008].There is also disagreement as to how many speciesthe genus contains [Defler, 2004], which is of particularimportance given the possible effect of taxonomicposition in conservation policies [Morrison et al., 2009].

In a seminal revision of the genus [Fooden,1963], Lagothrix was described as containing twospecies, L. flavicauda and L. lagothricha, the lattercomposed of four subspecies L. l. lagothricha, L. l.lugens, L. l. poeppigii, and L. l. cana. This remainedthe accepted taxonomy until Groves [2001] elevatedthe status of all taxa in the genus, separatingOreonax flavicauda into another genus and propos-ing that the previous subspecies of L. lagothrichawould be considered as separate species. AlthoughGroves’ [2001] work was highly detailed and statis-tically well supported, it only relied on morphologicalcharacters to establish such separation. A recentarticle has questioned his proposition for O. flavi-cauda by using the same methodology, showing thatit resulted from an artifact of sampling [Matthews &

Rosenberger, 2008]. It is thus relevant to evaluatewhether the proposed species are indeed species. Toprevent ambiguity, we will use Fooden’s [1963]taxonomy for the rest of the article.

There are two reported subspecies of woollymonkeys in Colombia. L. l. lagothricha inhabitsthe south of the country in the Amazonas andthe southern Orinoquian region. L. l. lugens is thesubspecies having the smallest distribution and isrestricted to the eastern and central cordilleras inthe northern Andes and adjacent lowlands [Fooden,1963; Hernandez-Camacho & Cooper, 1976], seeFigure 1. If the taxon lugens is indeed a separatespecies, it is undoubtedly the most endangered oneowing to the elevated habitat transformation in theColombian Andes [Armenteras et al., 2003; Stevenson& Link, 2008]. L. l. lugens is classified by the IUCN asCritically Endangered [Stevenson & Link, 2008], andhas probably become extinct in Venezuela because norecent reports of its populations have been published;

Published online 2 September 2010 in Wiley Online Library (wileyonlinelibrary.com).

DOI 10.1002/ajp.20878

Received 14 December 2009; revised 25 July 2010; revisionaccepted 25 July 2010

Additional Supporting Information may be found in the onlineversion of this article.

Contract grant sponsor: Colciencias, the Universidad Nacionalde Colombia, and the Universidad de los Andes.

�Correspondence to: Sergio Botero, 1230 York Avenue, Box 18,New York, NY 10065. E-mail: [email protected]

Sergio Botero’s current address is Rockefeller University, 1230York Avenue, New York, NY 10065.

rr 2010 Wiley-Liss, Inc.

thus, its appropriate protection in Colombia is ofparticular importance.

Cytogenetic markers provide a valuable tool forspecies differentiation and have been used success-fully for this purpose in Neotropical primates [Defler& Bueno, 2007]. The L. lagothricha karyotype haslong been published and intraindividual variation

was reported in chromosome 7 [Egozcue & Perkins,1970]. Although karyotyping provides a way of deter-mining species boundaries by identifying possiblecytogenetic incompatibilities, its power is enhancedwhen used in conjunction with molecular markers:such a mixed approach should offer a way ofvalidating the results [Amaral et al., 2008]. To our

Fig. 1. Distribution map for the Colombian subspecies of Lagothrix. Known area of contact between the subspecies is circled. Thenorthern area of contact is hypothesized but has not been properly documented. Map is based on Defler [2004] and was plotted using theGMT software [Wessel & Smith, 1995].

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1132 / Botero et al.

knowledge, no study has been conducted to evaluatethe separation of Lagothrix taxa using this approach.

We devised this study to be a preliminary analysison the taxonomic status of the two Colombian taxaof woolly monkeys, by using both molecular andcytogenetic markers. Given the great discrepancybetween species concepts, which by nature is mainlyoperational [de Queiroz, 2007], we establish thefollowing measurable distinctions as requisites forthe taxa to be considered different species:

* The effective number of migrants per generation(estimated as Nm) between the taxa in questionshould be very close or equal to zero.

* Karyotypic differences, if present, should differ-entiate the taxa by showing a statistically signi-ficant segregation between them.

METHODS

Blood samples were obtained from 16 captiveLagothrix sp. monkeys kept in Colombian zoologicalinstitutions from June 2008 to August 2009. Samplingwas performed by each institution’s veterinarian incompliance with current Colombian laws and animalcare regulations, and adhered to the American Societyof Primatologist’s Principles for the Ethical Treat-ment of Nonhuman Primates. To the best of ourknowledge, the institutions in which the animals arekept at the present time are listed in Table I.

The geographic origin of the sampled animals wasuncertain, though none of the sampled individuals

was born in captivity. These animals have come to bein zoological institutions after having been seized bylocal authorities when they were being kept or sold aspets. The local custom for capturing woolly monkeysconsists of killing mothers in order to get thedependant infants; thus, it is sound to assume thatthey represent wild caught individuals, precluding thepossibility of being hybrid animals conceived incaptivity. We do not use the information on thelocation where the animals were seized as a proxy forprovenance of the sample, because the animals arelikely transported for long distances before being sold.This information is also not available in several cases.

We restricted our analysis to comparing taxaidentified by their phenotype, for which purpose wedetermined their phenotype according to Fooden’sdescription. The main difference between subspeciesis the dominant coat color: uniform brown forL. l. lagothricha and gray to black for L. l. lugens,although significant variation exists in each sub-species [Fooden, 1963]. Such separation fails to takeinto account the existence of intermediate pheno-types [Defler, 2004], but is the only alternative forour purposes. The inadvertent inclusion of hybridanimals in the study is unlikely, because the area ofcontact between the subspecies is small [Fooden,1963], though this possibility cannot be ruled outentirely. Given that the original division amongtaxa was based mainly on coat color [Fooden, 1963],we believe phenotype to be a good proxy forsubspecies determination. Nonetheless, our resultsshould be interpreted with caution until replicatedwith better sampling.

TABLE I. Karyotypes, GeneBank Accession Numbers, Institution Where the Individual is Kept and IdentificationNumber (for Fig. 3) for Individuals Included in the Study

Karyotype GeneBank accession number

ID/Institution Subspecies Chromosome 4 Chromosome 7 Chromosome 24 COII D-loop

S1 L. l. lagothricha V A N N N N GU212696 GU212679P1 L. l. lagothricha V A V V N N GU212698 GU212681P2 L. l. lagothricha V A N N N V GU212699 GU212682D1 L. l. lagothricha V A N N N N GU212700 GU212683M1 L. l. lagothricha N N N N N N GU212701 GU212684B1 L. l. lagothricha V A N V N V GU212697 GU212680S2 L. l. lugens A A V V N V GU212706 GU212689S3 L. l. lugens N N N N N V GU212704 GU212687D2 L. l. lugens A A V V N N GU212705 GU212688M2 L. l. lugens V V N N N V GU212702 GU212685M3 L. l. lugens A A N V V V GU212703 GU212686B2 L. l. lugens A A N N V V GU212708 GU212691V1 L. l. lugens N A N N V V GU212711 GU212694V2 L. l. lugens V A N V N V GU212710 GU212693V3 L. l. lugens V V N N N N GU212707 GU212690V4 L. l. lugens N N N V N N GU212709 GU212692

Karyotypes: N, normal; V, variant; A, alternative variant. ID/Institution: S, Fundacion Zoologico Santacruz, San Antonio del Tequendama,Cundianamarca; M, Zoologico Santa Fe, Medellin, Antioquia; B, Zoologico de Barranquilla, Barranquilla, Atlantico; V, Bioparque los Ocarros, Villavicencio,Meta; P, Zoologico Matecana, Pereira, Risaralda; D, Deceased.

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Colombian Woolly Monkeys’ Taxonomy / 1133

Karyotyping

Blood samples of approximately 3 ml wereadded to 0.1 ml Liquemin (Roche) and taken to thelaboratory in a temperature-isolated package. Wecultured about 200 ml of sample at 371C in 10% fetalbovine serum (GIBCO) with 10,000 U/ml penicillin(GIBCO) and 100 ml 0.4% Vicia faba lectin (purifiedby the GRIP, protein research group, chemistrydepartment, Universidad Nacional de Colombia).After 72 hr, we added 100 ml of 25 mg/ml colchicine(Winthrop) and standard metaphase fixation of cellswas performed after 20 min [Moorhead et al., 1960;Verna & Babu, 1989]. Chromosomes were analyzedwith G banding patterns as described previously[Sole & Woessner, 1992].

We visualized and photographed the karyotypeswith a Zeiss Axiophot (Zeiss) optical microscopeand analyzed them with LUCIA Karyo (LUCIA)software. After identifying each chromosome pair,we determined differences from the previouslydescribed karyotype [Stanyon et al., 2001] andcalculated their frequency. To determine whetherthe frequencies of the detected changes were con-sistent with a division between taxa, we performeda permutation test with 10,000 repetitions in thePermute software [Brookes et al., 1997].

Mitochondrial DNA Molecular Analyses

We extracted DNA from 200ml of the sample usingQIAamp DNA Blood Mini Kit (Qiagen), according tothe manufacturer’s protocol. Following extraction,the mitochondrial cytochrome oxidase II gene (COII)and the mitochondrial control region’s hypervariableregion (D-loop) were amplified by PCR. We checkedreactions on agarose gels at 1% and sequenced positivereactions through Macrogen Korea commercial ser-vice. At least three different sequencing reactions, eachfrom a different PCR reaction, were performed forboth primers for each gene to control for possiblepseudogene amplification.

Conditions for COII amplification were: 1Xreaction buffer, 2 mM magnesium chloride, 0.8 mMeach dNTP (Bioline), 0.35mM each primer, 0.05 u/mlTucanTaq Taq DNA polymerase (CorpoGen), and0.1mg/ml BSA (Promega), in a 50ml final volume.We used 3ml of DNA solution directly from theextraction; DNA concentration in the eluate wasnot quantified. The thermal profile included initialdenaturing at 941C for 10 min; followed by 27 cycles of941C for 45 sec; 521C for 25 sec; 721C for 25 sec; anda final extension step at 721C for 7 min, with L6955(50-AACCATTTCATAACTTTGTCAA-30) and H7766(50-CTCTTAATCTTTAACTTAAAAG-30) primers[Collins & Dubach, 2000]. Conditions for D-loop ampli-fication were: 1X reaction buffer, 2.5 mM magnesiumchloride, 0.8 mM each dNTP (Bioline), 0.5mM eachprimer, 0.05 u/ml TucanTaq Taq DNA polymerase(CorpoGen), and 0.1mg/ml BSA (Promega). The thermal

profile included initial denaturing at 941C for 10 min;followed by 27 cycles of 941C for 30 sec; 621C for 45 sec;721C for 45 sec; and a final extension at 721C for 7 min,using primers L15400 (50-TCCACCATTAGCACC-CAAAG-30) and H15940 (50-CCTGAAGTCGGAACCA-GATG-30) [Kocher et al., 1989]. We used the sameprimers for the PCR and sequencing reactions.

We checked protein translation for the COII geneto evaluate the possible presence of nuclear mitochon-drial DNAs (NUMTs); all observed mutations weresynonymous changes, suggesting that there were noNUMTs in the sequences, although the possibilitycannot be completely ruled out. It is not possible toperform this same approach for the D-loop region,because it does not code for a protein sequence. If theprimers used for D-loop amplification or COII as well,do anneal also in several NUMTs one would expect tosee a signal of this in the chromatogram owing to theprobabilistic nature of PCR. Given that the ratio ofnuclear genome to mitochondrial genomes per cell isin the order of 1/1,000 [Takamatsu et al., 2002], in theworst case scenario one would expect a similar amountof NUMTs and real mitochondrial sequences to beamplified. If the amplified NUMTs are very distant orinclude any insertions or deletions, many doublepeaks are to be expected, which was not observed.It is also possible that the primers anneal preferen-tially on a few or even only one NUMT which could berelatively conserved, and thus greatly confound theanalysis, but given the high number of haplotypesfound (15 in 16 samples; see results), the probability ofall the individuals being homozygous (because eachhaplotype should represent an allele under thisscenario) is too low for this to be expected. Thus,although we cannot rule out the possibility of NUMTsbeing amplified in our PCR amplifications, this is veryunlikely. Still, we caution the readers to take ourresults as preliminary until they are replicated.

We performed a multiple sequence align-ment using Bioedit’s [Hall, 1999] implementation ofClustalW [Larkin et al., 2007]. After alignment, wetrimmed sequence ends to obtain a homogeneousmatrix, obtaining a sequence length of 431 nucleo-tides for D-loop and 674 for COII. Because these areboth mitochondrial regions and no recombination isexpected, we concatenated both genes for subsequentanalyses while specifying data partitions wheneverdistinct models of evolution were applied. For phylo-genetic analyses, we used as out-groups the corres-ponding sequences of two other Neotropical primates;Ateles (GenBank FJ785422, complete mitochondrion,nucleotides 7006–7679 for COII, and GenBankAF213949 for D-loop), Cebus (GenBank AJ309866,complete mitochondrion, nucleotides 7016–7689 forCOII, and 15470–15893 for D-loop), and the distantlyrelated Homo (GenBank GU212695 for COII andGU212678 for D-loop; this study). Sequences havebeen deposited in GenBank under the accessionnumbers listed in Table I.

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We performed maximum parsimony (MP) andmaximum likelihood (ML) phylogenetic analysesusing PAUP�4.0b10 [Swofford, 2003], with thefollowing parameters: heuristic search with 1,000random addition sequence replications keeping tentrees per cycle for MP, heuristic search with 10random addition sequence replications keeping tentrees per cycle, using the K81uf1G model obtainedwith the AIC criterion in Modelest 3.7 [Posada &Crandall, 1998] for ML. Both analyses were alsobootstrapped with 1,000 repetitions to obtain nodesupport. For Bayesian phylogenetic reconstruction,we used an approach with seven Monte CarloMarkov chains (MCMC) with 10,000,000 steps each,a burn-in fraction of 0.5, a partition data functionwith the model GTR1I for the COII region andHKY1G for D-loop. Models were obtained with theAIC criterion in MrModeltest [Nylander, 2004], chainswapping, and all other parameters were set to defaultin MrBayes [Ronquist & Huelsenbeck, 2003]. Usingseven chains ensured that swapping between themwas high, effectively providing good sampling of thespace. We performed a S-H test [Shimodaira &Hasegawa, 1999] with a 1,000 replicates bootstrap tocompare the topologies of the trees obtained, asimplemented in PAUP� [Swofford, 2003]. We didnot perform further optimization of phylogeneticanalyses, because population genetic analyses aremore appropriate to evaluate the relationship of thesubspecies given the lack of reciprocal monophyly.

We used the DNAsp v5.00.07 software [Rozaset al., 2003], to get information about possibledemographic processes going on in the two subspe-cies and to estimate basic statistics about thesequences obtained. We used Hudson et al.’s equa-tions to calculate Fst as a proxy to calculate Nm[Hudson et al., 1992], Fs [Fu, 1997], and R2 tests[Ramos-Onsins & Rozas, 2002] to evaluate theassumption of constant population size, which isnecessary for the other analysis. Confidence intervalswere calculated using coalescent simulation with10,000 replicates for Fs and R2. Given the lowvariability observed in COII, we only used the D-loopdata to estimate values of Nm.

Additionally, we tested for an isolation withmigration model [Nielsen & Wakeley, 2001] andestimated divergence time, as implemented in the IMsoftware [Hey & Nielsen, 2007], with ten MCMCchains running for 100,000,000 steps, sampled every100 steps, with a burn-in of 500,000 steps, and ageometric heating scheme having parametersh1 5 0.8 and h2 5 0.9. Maximum values of 2 for them1 and m2 parameters, 2 for the t parameter, and1,000 for the q1 parameter were set after 13optimization runs, to achieve maximum resolutionat the parameters’ point of convergence for the timeto the most recent common ancestor (TMRCA)calculations. For the Nm calculations, we used onlythe D-loop data and we also performed an analysis in

which we used a value of 5 and 10 for the m1parameter (flux of genes into L. l. lagothricha fromL. l. lugens), with all other parameters as previouslydescribed. This was done because the m1 parameterdid not converge and we wanted to explore itsbehavior. Starting parameters were 15 for m1 andm2, 16 for t, and 70 for q1. To calculate the values ofthe TMRCA, we used the mutation rate of 0.0031substitutions per site per million years, as previouslyestimated for COII in New World monkeys [Hodgsonet al., 2009], and one order of magnitude higher(0.031 changes per site per Myr) for the D-loopregion, as this relationship has been shown to holdfor apes and other mammals [Ruvolo et al., 1993].These values were combined by multiplying eachmutation rate by the number of base pairs in eachregion, and adding these two values to create a‘‘mean’’ of the mutation expected in the concatenateof the COII and D-loop regions, which is1.54504� 10�5 mutations per concatenated sequenceper year. This is the value that is actually used forthe TMRCA calculations. Note that the mutationrate used does not affect the Nm calculations. Wechecked the probability and autocorrelation plots toevaluate parameter convergence.

For an additional estimation of TMRCA of thespecies, we use the BEAST v1.5.3 software [Drummond& Rambaut, 2007] with the relaxed lognormal clockmodel [Drummond et al., 2006], using Ateles as anout-group. We used the same mutation rate as in theIM analyses by implementing a prior with normaldistribution with mean 1.55� 10�5 and SD 0.1� 10�6

mutations per region per year. A partition of the datawas specified for this analysis in which the COII genefollowed a GTR model with Gamma and Invariantsites, with different rate of mutation for its third codonpositions, because all the variability observed waslocated there. The D-loop region followed a GTR modelwith Gamma and Invariant sites. A 1� 109 steps wereused for this analysis, using a 20% burn-in and allother parameters set to default. Results were visualizedwith the Tracer v1.4.1 software [Drummond &Rambaut, 2007].

RESULTS

Karyotypes showed variation in chromosomes 4,7, and 24 (Fig. 2). Chromosome 4 showed the mostvariability with three phenotypes: a conspicuous Gpositive terminal band on the short arm, a reducedterminal band, and the absence of this terminalband. All variants were observed in homozygous andheterozygous individuals, except for the normalband-diminished band combination that was notobserved. Chromosome 7 showed only one variantwhich was a diminished terminal G band on theshort arm. Chromosome 24 showed a pericentricinversion. Both variants in chromosomes 7 and 24were observed in homozygous and heterozygous

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individuals. Individual karyotypes are given inTable I. We evaluated the possibility of separatingtaxa by the frequency of their karyotypes througha permutation test with nonsignificant results(P 5 0.11), suggesting that there is indeed noseparation and supporting the subspecies ratherthan species status.

We found six haplotypes for L. l. lagothrichaand nine for L. l. lugens, but nucleotide diversitywas higher for L. l. lagothricha (Pi 5 0.02) than forL. l. lugens (Pi 5 0.01). COII showed 20 variable sitesof which 4 were singletons and the other 16 wereparsimony informative sites, whereas D-loop showed44 variable sites of which 3 were singletons and theremaining 41 were parsimony informative sites.Phylogenetic trees obtained by all three methods(MP, ML, and Bayesian inference) showed nosignificant differences (P40.05 in all possible com-binations, S-H test), although differences do occur inthe grouping of out-groups. In all cases, the treetopology showed no reciprocal monophyly betweentaxa. For simplicity, only the Bayesian inferencephylogram is shown in Figure 3, indicating nodesupport for nodes shared with the ML and MPanalyses. Note that node support is very low inthe basal nodes, thus, although it might seem as ifL. l. lagothricha is a more recent taxon, the obtainedtree does not support this conclusion.

There was no evidence of historic change inpopulation size indicated by the data in L. l. lagothricha(Fs 5 0.29, 95% CI: �1.22–5.14, P 5 0.34; R2 5 0.18,95% CI: 0.11–0.31, P 5 0.38) or L. l. lugens(Fs 5�0.992, 95% CI: �3.64–4.65, P 5 0.25; R2 5 0.17,

95% CI: 0.10–0.24, P 5 0.67). This also held forL. lagothricha as a species (Fs 5�2.43, 95% CI:�4.18–5.18, P 5 0.10; R2 5 0.18, 95% CI: 0.087–0.200,P 5 0.89). Although of no particular interest forthis study, the fact that population size can be takenas constant is a necessary starting point for meetingthe assumptions of the other methods used. Thisresult has no biological meaning, given the unknownorigin of sampled individuals and the very smallsample size used. Therefore, it should not be inter-preted to mean anything about the actual demo-graphics of the taxa, but simply as a requisite forother analysis.

Fig. 2. Karyotype of Lagothrix lagothricha, sensu Fooden [Fooden, 1963], and variation found in this study. Both L. l. lugens andL. l. lagothricha karyotypes showed all variants: N, normal; V, variant; A, alternative variant.

Fig. 3. Obtained Bayesian inference phylogram. Bootstrap MPand ML, and Bayesian analysis node supports are indicatedwhen they are available (MP|ML|Bayesian inference posteriorprobability� 100). ‘‘�’’ indicates the absence of the node inthe obtained tree. Out-groups have been excluded to allowmore detail in the phylogram. The repeated haplotype found inL. l. lugens is indicated in the figure.

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The estimated migration rate between popula-tions (Nm 5 0.68) reflects some level of isolation,because a value of 1 or higher would be expected ifthere was no distinction between taxa. As estimatedby the migration with isolation model, the effectivenumber of migrants per generation from L. l. lugensto L. l. lagothricha is 17.3 (95% CI: 0.5–17.1) and0.74 (95% CI: 0.19–10.1) in the opposite direction.Note that the estimation of Nm from L. l. lugens toL. l. lagothricha is outside the confidence interval.This occurs because the parameter tendency is torise up until reaching a plateau around a value of4.25, which is outside of the selected limit for theanalysis (we performed a test with the limit of theparameter being 5 and 10). Because a value higherthan 1 for the Nm estimate already means that thepopulations are not differentiated, we did not explorethe estimation under even higher values of theparameter m1. Directional Nm values are in agree-ment with the nondirectional calculation, but show ahigher level of gene flow. These later estimates are tobe taken as being more reliable, given the inclusionof directionality and the fact that they are based ona coalescent simulation which is robust for smallsample sizes [Harding, 1996]. Including more sam-ples would likely allow for a better estimation of themigration rate parameter.

Estimates of the TMRCA for each taxon arepresented in Table II. Effective sample sizes (ESS)for all the parameters estimated in BEAST wereabove 200, which is the rule of thumb given by thesoftware authors, and were above 750 for all TMRCAestimations. The ucld.stdev parameter was estimatedto be significantly higher than 1 (ucld.stdev 5 5.18,95% CI: 3.68–6.28), which indicates the data does notbehave in a clockwise manner and the choice ofthe relaxed lognormal clock model is adequate[Drummond & Rambaut, 2007]. ESS values wereabove 10,000 for all IM parameter calculations. Afteroptimization of the parameters, the probability andautocorrelation plots were checked and showed noabnormal patterns.

DISCUSSION

There is a remarkable level of variability in thespecies’ karyotype, but it is unlikely to interfere withreproduction as indicated by the presence of hetero-zygous individuals for all possible combinations,

except for the normal band-diminished band hetero-zygote in chromosome 4. It is likely that this missingcombination exists in nature and the limitedsampling size used did not allow for its detection.The lack of separation in the karyotype frequenciesbetween the two taxa is consistent with a scheme inwhich they interbreed. High levels of intraspecificchromosomal variability have been observed in theclosely related atelid genus Alouatta, suggesting thatthis is not an uncommon feature in the Atelidae[de Oliveira et al., 2002]. There are several hybridcaptive-born individuals in zoos around the world[Stevenson, 2010]. The existence of hybrids supportsthe absence of prezygotic reproductive isolationbetween these taxa, but an analysis of hybrid fertilitywould be necessary to confirm this.

Female dispersal represents the vast majority ofdispersal events in L. lagothricha and has beenhistorically considered to be the only dispersalmode for the species [Nishimura, 2003]. The use ofmitochondrial molecular markers is then of particu-lar relevance for this study, because the informationon gene flow obtained will be accurate. If femalesshowed complete philopatry, no gene flow could bedetected using this information [de Queiroz, 2007].There is some evidence for limited male dispersal[Di Fiore & Fleischer, 2005; Di Fiore et al., 2009], butit is certainly on a smaller scale and has not beenfully documented [Maldonado & Botero, 2009].If male dispersal were common, our results wouldbe a minimum estimation of the level of gene flow inthe species, but this is unlikely to be the case becausemale dispersal is in fact a rare observation.

If our assumption of no inclusion of NUMTS inthe dataset holds true, the lack of reciprocal mono-phyly observed in phylogenetic analyses could stemfrom both polymorphisms in the ancestral popula-tion, a true mixing of the taxa, or some level of both.There is still no definitive way of determining whichof these schemes is contained in the species tree;however, coalescent approaches allow for a distinc-tion between ancestral polymorphism and gene flowwhen two taxa are being analyzed [Hey & Nielsen,2004]. Estimating the level of migration (Nm)between taxa is a good proxy for supporting one ofthe two schemes, because a significant level ofmigration will indicate that the taxa are interbreed-ing [Joly et al., 2009]. Using a coalescent approach,we obtained different Nm values than those esti-mated by using a more classical approximation[Hudson et al., 1992]. However, in both cases, ourresults support the scheme in which L. l. lugens andL. l. lagothricha are interbreeding, although only to alimited extent because it seems that the gene flux isstronger from L. l. lugens to L. l. lagothricha than inthe opposite direction.

The small area of contact between the sub-species [Fooden, 1963] makes it unlikely for twodifferent individuals from this zone to have been

TABLE II. Estimates of TMRCA for Each Taxon

Analysis Taxon TMRCA

IM Lagothrixlagothricha

22,718, 95% CI: 3,948–125,498097, 95% CI: 1,587–19,354

Beast L. l. lagothricha 809, 95% CI: 1,439–18,776L. l. lugens 898, 95% CI: 801–17,187

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included in the study, which would be necessarywere contemporary hybridation to account forour key result of the lack of reciprocal monophylyof L. l. lugens and L. l. lagotricha. However, ourresults imply a significant level of gene flow betweenthe subspecies, and thus these individuals should infact be hybrids or descendants of those, thoughpresumably not sampled from the contact zone.Individual M2 is a female with a very clear L. l.lugens phenotype but L. l. lagotricha mtDNA,showing silver gray coat, darker blackish chest, anda dark blackish crown with a silver mid-sagittalstreak. Individual V3 is also a female, which showsa black chest and ventral side of the extremities, anda gray brownish grizzled coat color in the rest ofthe body. This coat coloration is reminiscent ofthat described by Fooden [1963] for the Caqueta-Magdalena divide, and we have classified it asL. l. lugens. It could be argued that this individualpresents an intermediate phenotype. SupplementaryFigure 1, accessible online, provides pictures of thisindividual for the interested reader.

Differences between estimated migration ratesare not likely owing to the inclusion of directionalityin the calculations, but to the different methods usedfor the calculations. If an average is made, account-ing for the estimated population size scalars obtainedin the same IM calculation, a value of 11 is obtained,which is still much higher than 0.68, as estimated byHudson et al.’s [1992] equations. The latter resultsare more sensitive to sample size than coalescentsimulations, thus directional estimates are likelymore accurate. Both results are consistent with thesubspecies scheme suggesting some population struc-ture with limited gene flow from L. l. lagothricha toL. l. lugens. It was our expectation to find some levelof structuring given the large area spanned by eachsubspecies distribution and the relatively smallcontact area in the Caqueta region [Hernandez-Camacho & Cooper, 1976], see Figure 1. A detailedanalysis which incorporates spatial genetics from thetwo subspecies will certainly provide evidence todetermine the level of structuring between the taxa.Nonetheless, the level of separation indicates thatthe taxa should be considered distinct evolutionaryunits for conservation purposes, because they couldrepresent a very early state of allopatric speciation.Efforts to protect L. l. lugens should arguably bedirected toward the Sierra de la Macarena popula-tion, where they show high population density[Stevenson, 2001] and have been studied for aprolonged period of time [Defler, 2004; Stevenson &Link, 2008].

It has been proposed that the four L. lagothrichasubspecies originated during the Pleistocene,following the Pleistocene forest refugia hypothesis[Hernandez-Camacho et al., 1992]. Our results forthe species TMRCA are in the lower end of this timeframe, extending into the beginning of the Holocene.

This result does not seem to support the Pleistocenerefugia hypothesis, because under this hypothesisthe splitting of the two taxa would have occurredearlier, between 100,000 and 600,000 years ago.The large confidence intervals obtained precludeexcluding this possibility, however.

The higher nucleotide diversity observed inL. l. lagothricha is likely owing to its broadergeographical distribution, which should allow for alarger population size. Our results place the TMRCAof L. lagothricha at some time during the latePleistocene, most likely diversifying independentlyof the Pleistocene refugia; the main separation factorbetween the two subspecies would then be limitedgene flow, possibly owing to a relatively small contactarea. Our data set does not allow determining whichof the two subspecies, if any, might be ancestralbecause the estimates of the TMRCA are verysimilar. The obtained tree topology would indicatethat L. l. lagothricha is a more derived taxon, butnode support is too low and it should not beinterpreted in that way. An analysis, including theother L. lagothricha subspecies, will certainly help toclarify the historical forces acting on its evolution.

Both methods for estimating TMRCA rely on acoalescent MCMC approach and as such should notbe affected by sample size, because L. lagothrichaestimation is based on 16 individuals and a samplesize of 10 is sufficient to estimate one parameterwithout any more changes in its variance [Harding,1996]. Estimations of the TMRCA varied betweenmethods, but the time frame where they place theTMRCA of the species is the same. The mutationrate we used comes from Hodgson et al.’s data, whospecifically calculated it for New World monkeymitochondrial coding regions [Hodgson et al., 2009]and of the relative value of the D-loop, which isknown to be an order of magnitude higher [Ruvoloet al., 1993]. Adding more samples could potentiallyincrease L. l. lagothricha TMRCA and consequentlythat of L. lagothricha. However, it is unlikely thatthis value will vary significantly because the resultis based on a coalescent simulation. The fact thatthe data does not behave in an optimal clock-wisemanner, and that the COII shows little variability,introduces additional noise to the estimation;however, given the very recent divergence times,there seems to be no alternative to this calculationbecause neutrally selected nuclear genes would showvirtually no variability.

Overall, our results suggest that separating ofthe two taxa into distinct species, as proposed byGroves [2001], is not justified. The original sub-species status is clearly a more appropriate way ofdescribing their relationship and is in agreementwith the existence of intermediate phenotypes[Defler, 2004]. Although sample size for this studywas small, the implications of the results are unlikelyto change by increasing sampling. The lack of

Am. J. Primatol.


reciprocal monophyly observed in the phylogeneticanalyses, for example, will not change by addingmore terminal taxa. It is also unlikely to change byadding more loci because, given its faster rate ofmutation, no difference is expected to be found innuclear genes if none is found in the mitochondria.Estimating migration rates under coalescent simula-tions would most likely have the same tendency,because coalescent simulations are very robust tosmall sample sizes [Harding, 1996]. Our results suggesta late Pleistocene diversification of L. lagothrichawith the subspecies starting to differentiate owingto unequal gene flow between them; however, theformer interpretation should be taken with cautionuntil the analyses can be replicated, including theother subspecies.

ACKNOWLEDGMENTS

The authors express their gratitude to threeanonymous reviewers and Anthony Di Fiore, thereview editor, for valuable comments on the article;to ACOPAZOA and its participating institutions fortheir collaboration with the research; to SantiagoHerrera for his critical comments on the study andarticle; and to Jason Garry for his comments on theform of the article. The research was carried outwith permission by the Colombian Ministerio deAmbiente, Vivienda y Desarrollo Territorial giventhrough Resolucion 521 del 1 de abril de 2008,Resolucion 751 del 13 de mayo de 2008, Resolucion803 del 16 de mayo de 2008, and Contrato de Accesoa Recursos Geneticos para Investigacion Cientıficasin Interes Comercial No 17.

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