DoesEulemur cinereiceps exist? Preliminary evidence from genetics and ground surveys in southeastern Madagascar

American Journal of Primatology 70:372–385 (2008)

RESEARCH ARTICLE

Does Eulemur cinereiceps Exist? Preliminary Evidence From Genetics andGround Surveys in Southeastern Madagascar

STEIG E. JOHNSON1�, RUNHUA LEI2, SARA K. MARTIN3, MITCHELL T. IRWIN4, AND EDWARD E. LOUIS2

1Department of Anthropology, University of Calgary, Calgary, Canada2Center for Conservation and Research, Henry Doorly Zoo, Omaha, Nebraska3Interdepartmental Doctoral Program in Anthropological Sciences, Stony Brook University, Stony Brook, New York4Department of Biology, McGill University, Montreal, Canada

Although appearing in the literature as early as 1890, the brown lemur form Eulemur cinereicepshas recently resurfaced as a potentially valid taxon, distinct from neighboring, presumably closelyrelated species such as white-collared lemurs (Eulemur albocollaris). We propose two scenarios for thepotential separation of E. cinereiceps and E. albocollaris: (1) coastal and interior populations representtwo distinct taxa and (2) the coastal population north of the Manampatrana River (the localityfor purported museum specimens of E. cinereiceps) represents a distinct species from E. albocollarisfound south of the river and in the interior escarpment forests. We tested these hypotheses usingdata from ground surveys and genetic sampling. Surveys were conducted in coastal forest fragmentsboth north and south of the Manampatrana River in July–August 2006. Genetic samples were collectedat two coastal sites and one interior forest. We used maximum parsimony, maximum likelihood,and neighbor-joining analyses on mitochondrial DNA regions to determine if populations from differentsites clustered into diagnosable clades. Results from field surveys confirmed the presence offorms commonly referred to as E. albocollaris at the two southern coastal forests; no consistentphenotypic differences across sites were observed. All genetic analyses yielded identical results: coastaland interior populations do not cluster into separate groups, thus rejecting the first hypothesis.Eulemur species and all other day-active lemurs have apparently been extirpated from coastal forestsnorth of the Manampatrana. Owing to the absence of lemurs from the northern coastal localities, wecould not conclusively support or reject the second scenario. However, based on examination of theoriginal plates and museum specimens, as well as the biogeographic patterns typical of this region, westrongly suspect that all populations from this area belong to a single species. We conclude withremarks regarding the apparent priority of E. cinereiceps for this taxon. Am. J. Primatol. 70:372–385,2008. r 2007 Wiley-Liss, Inc.

Key words: Eulemur cinereiceps; Eulemur albocollaris; white-collared lemur; mitochondrial DNA;biogeography; taxonomy; southeastern Madagascar

INTRODUCTION

The brown lemurs (Eulemur fulvus and relatedspecies) are among the most widespread primates inMadagascar [Johnson, 2006; Mittermeier et al.,2006b; Tattersall, 1982]. However, anthropogenicdisturbance in last two millennia has resulted in thepresently discontinuous range. The central plateau,which likely contained a mosaic of forest and moreopen habitats before humans [Godfrey et al., 1997],has been largely deforested; remaining brown lemurhabitats are confined to the periphery of Madagascar[Tattersall, 1982; Tattersall & Sussman, 1998], withsome taxa having discontinuous ranges incorporat-ing both eastern and western forests.

The taxonomy of this diverse group is conten-tious. Previously, brown lemurs were considered asingle polytypic species (E. fulvus), with six recog-

nized subspecies: E. fulvus fulvus (the commonbrown lemur), E. f. rufus (the red-fronted lemur),E. f. albocollaris (the white-collared lemur, WCL),E. f. collaris (the collared lemur), E. f. sanfordi

Published online 20 November 2007 in Wiley InterScience(www.interscience.wiley.com).

DOI 10.1002/ajp.20501

Received 4 May 2007; revised 30 August 2007; revision accepted16 October 2007

Contract grant sponsors: The University of Calgary; TheCommittee for Research and Exploration of the NationalGeographic Society; Contract grant number: 6613.99; Contractgrant sponsors: Margot Marsh Foundation Grant; PrimateAction Fund.

�Correspondence to: Steig E. Johnson, Department of Anthro-pology, University of Calgary, 2500 University Dr. NW, Calgary,AB T2N 1N4, Canada. E-mail: [email protected]

rr 2007 Wiley-Liss, Inc.

(Sanford’s lemur), and E. f. albifrons (the white-fronted lemur) [Mittermeier et al., 1994]. However,recent cytogenetic and molecular genetic evidencehas supported the elevation of E. albocollaris and E.collaris, the only two brown lemur taxa that cannotproduce fertile hybrids, to full species [Djlelati et al.,1997; Wyner et al., 1999a]. Groves [2001], notingdistinct phenotypic appearance and craniodentalfeatures [Tattersall & Schwartz, 1991] among brownlemur taxa, suggested further splitting, with species-level designation for all recognized subspecies.

There is some evidence that such extensivereclassification is not yet warranted. Tattersall[1993] noted that homoplasy plagues phylogeneticanalyses based on anatomical characters in thisgroup. Moreover, no genetic analyses have thus farbeen able to sort E. f. fulvus, E. f. rufus, E. f. albifrons,and E. f. sanfordi into diagnosable clades; Wyner et al.[1999a] found no markers to distinguish among thesesubspecies and Pastorini et al. [2000] identified cladesthat cross-cut the recognized subspecies. It should benoted that both of these studies found E. albocollarisand E. collaris were sister taxa, separated from otherbrown lemurs, but they disagreed over whether toelevate these taxa to species.

Recently, there has been discussion concerning apossible seventh brown lemur taxon [Groves, 2001;Mittermeier et al., 2006b]. E. cinereiceps wasdescribed (as Lemur (Prosimia) macaco cinereiceps)by Groves [1974] as a distinct ‘‘white-cheeked’’variety from the Farafangana region, separatedgeographically from neighboring E. collaris. Thename is based on plates from Milne-Edwards andGrandidier [1890], but unfortunately no text accom-panied these plates. Schwarz [1931] concluded thattwo mounted specimens from the Paris NationalMuseum were the individuals depicted by Milne-Edwards and Grandidier [1890]. The localities forthese specimens were Farafangana and Salohy[north of Farafangana; Schwarz, 1931]. Schwarz[1931] included E. cinereiceps among the synonymsfor E. collaris. Shortly after Groves’ [1974] publica-tion, Rumpler [1975] proposed E. albocollaris(as Lemur fulvus albocollaris) based on karyoptypicdivergence (2N 5 48) from E. collaris (2N 5 50, 51,52) individuals of known capture locations.

Despite the apparent priority of E. cinereicepsover E. albocollaris in nomenclature, a considerabledebate ensued [Groves, 1974, 2001; Tattersall, 1979,1982]. Tattersall [1979, 1982] rejected E. cinereicepsas the senior designation, suggesting the Milne-Edwards and Grandidier [1890] plates did notresemble the WCL of southeastern Madagascar. Inaddition, the individuals depicted in the plates andthe mounted specimens were females, which are notdiagnostic for southeastern brown lemurs [white-collared and collared lemur females are largelyindistinguishable; Tattersall, 1982]. Thus, Tattersall[1982] supported Rumpler’s [1975] description of

E. albocollaris as the brown lemur taxon in theregion in question. Groves [2001] disagreed andcontinued to support the seniority of E. cinereiceps,but also suggested that E. cinereiceps andE. albocollaris could represent two distinct taxa. Hedescribed E. cinereiceps separately and explicitlycontrasted it with E. albocollaris: ‘‘[c]ompared withfemales of E. collaris (and so presumably ofE. albocollaris) they are much lighter and redder;the cheeks are light gray, with no trace of orange orwhite whiskers; the muzzle is very light, not black[Groves, 2001:78].’’ Subsequently, Mittermeier et al.[2006b] reported a captive female observed in 2005 inFarafangana that closely resembled the Milne-Edwards and Grandidier [1890] plate and Parisspecimens. They reserved judgment as to the validityof a separate E. cinereiceps, but recommendedfurther surveys in the vicinity of Farafangana toevaluate its status.

Field surveys and genetic sampling over the past10 years have clarified the geographic distribution ofWCL [Irwin et al., 2005; Johnson & Overdorff, 1999;Johnson & Wyner, 2000; Wyner et al., 1999a, 2002].They are found in the eastern escarpment rain forestcorridor from the Mananara River in the south (theboundary with E. collaris) to the Andringitra Massifin the north, where they form a hybrid zone with themore northerly E. fulvus rufus [Irwin et al., 2005].North of Andringitra, populations with E. fulvusrufus phenotypes are found at least as far southas Ankopakopaka [Goodman et al., 2001; Fig. 1].Within the WCL range, the remaining forest ishighly fragmented, and coastal populations inthe fragments south of Farafangana are separatedfrom the interior corridor by approximately 40 km ofdeforested area (Fig. 1). The Manampatrana Riverdoes not serve as a barrier to WCL in the interior[Johnson & Wyner, 2000; Fig. 1]. There are no recentsurveys describing coastal Eulemur populationsimmediately north of Farafangana (i.e., north ofthe mouth of the Manampatrana River and thelocality for at least one of the Paris specimens).

The objective of this study is to investigate thepotential separation of WCL into E. cinereiceps andE. albocollaris. Specifically, we evaluate two possiblescenarios for their division:

(1) Coastal and interior populations represent twodistinct taxa. In this case, coastal populationsshould have claim to E. cinereiceps based on theParis specimens’ localities (Farafangana region).We suggest that distinct interior populationswould retain the binomial Eulemur albocollaris,as this term has prevailing usage in the literature[Johnson, 2006; Mittermeier et al., 2006b; Over-dorff & Johnson, 2003; Wyner et al., 2002].

(2) Coastal populations north of Farafangana(i.e., north of the Manampatrana River mouth)

Am. J. Primatol.

Does Eulemur cinereiceps Exist? / 373

represent a distinct taxon. In this case, popula-tions north of Farafangana would representE. cinereiceps and E. albocollaris would belocated south of Farafangana (e.g., Manombo)as well as in the interior corridor (e.g., Vevembe).This possibility is at least potentially supportedby the locality information for the Parisspecimens: one is found at Salohy, north ofFarafangana and the Manampatrana River[Schwarz, 1931].

To test these hypotheses, we present data fromrecent ground surveys and genetic sampling ofbrown lemurs in the region in question. If scenario1 holds, we anticipate phylogenetic analyses willindicate that individuals from interior and coastalpopulations can be sorted consistently into distinctclades based on mitochondrial DNA (mtDNA) se-

quences. If scenario 2 holds, we expect that coastalpopulations north and south of the ManampatranaRiver will cluster in separate clades, even if southerncoastal populations are not found to differ frominterior populations. If no consistent differencesamong populations are found, this would suggestthat all belong to the same species. As the distinc-tions between taxa are not presently well estab-lished, we will refer to brown lemur populations fromthe region in question collectively as WCL.

In examining the question of divergence amongWCL populations, we adopt the phylogenetic speciesconcept, in which species are defined as the smallestcluster of individuals that are diagnosably distinctfrom other taxa [i.e., share apomorphic characters;Cracraft, 1983]. The phylogenetic species concept isby no means conservative in assigning populations asdistinct taxa, particularly in comparison with other

Fig. 1. Study region. Ground survey sites include: Sakanany, Analalava, Manombo, and Mahabo. Genetic sampling sites for the presentanalysis include: Vevembe, Manombo, and Mahabo. Additional localities include: Evendra [white-collared lemurs; Johnson & Wyner,2000], Andringitra [white-collared � red-fronted lemur hybrids; Wyner et al., 2002], Ankopakopaka, Ranomafana, Kianjavato, andVatovavy [red-fronted lemurs; Goodman et al., 2001; Wyner et al., 1999a].

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374 / Johnson et al.

species definitions relying on reproductive isolation,such as the biological species concept [Mayr, 1942].In this first attempt to examine potential species-level distinctions in WCL, we present phylogramsbased on mtDNA sequence data of wild-caughtindividuals from target populations. However, wecaution that phylograms based on limited charactersets (e.g., mtDNA) may not be sufficient for describ-ing species diversity [Rubinoff, 2006]. If the studypopulations can be sorted into distinct clades by thepresent techniques [commonly employed in lemurphylogenetics; Pastorini et al., 2000; Wyner et al.,1999a], we recommend further analyses, includingsampling multiple genetic loci, quantitative analysisof morphological characters, and field studies ofcontact zones to further assess the validity ofassigning WCL to multiple taxa.

There are significant conservation implicationsfor determining the relationships among thesepopulations. WCL, with their limited rangeand ongoing threats from anthropogenic distur-bance, are presently listed among the most endan-gered primates in the world [Mittermeieret al., 2006a]. If this species should be divided intoseparate taxa, each form would certainly be criticallyendangered and would require distinct manage-ment plans.

METHODS

Ground Surveys

We conducted ground surveys in the Farafanga-na region in July–August 2006. Sites south ofFarafangana included: Mahabo (S 231 11.1750 E 47143.0950; altitude 5 18 m) and Manombo (S 231 01.6970

E 471 43.8380; altitude 5 36 m; Fig. 1). Sites north ofFarafangana included: Analalava (S 221 38.1830 E 47144.5260; altitude 5 88 m) and Sakanany (S 221 34.3410

E 471 51.7470; altitude 5 18 m; Fig. 1).Mahabo contains approximately 1,500 ha of

degraded littoral rain forest. Manombo SpecialReserve and adjacent classified forest contains15,730 ha of a mosaic of anthropogenic matrix,lowland rain forest, and littoral rain forest. Defor-estation and hunting continue at both sites. The twonorthern sites are the closest remaining forests toSalohy, the locality for one of the Paris specimens[Schwarz, 1931; Fig. 1]. Analalava, near the village ofAndramena, consists of approximately 70 ha ofhighly degraded lowland rain forest. Sakanany is athin strip of littoral rain forest (ca. 200 ha). Theforest is heavily and actively degraded, with virtuallyno large trees (410 cm diameter at breast height)remaining (i.e., there are few remaining potentialfood sources for frugivorous lemurs).

All sites were surveyed during daylight hours,using existing trails where present to minimizedisturbance. Survey effort varied depending on thesize of the forest fragment; however, most fragments

were small enough to investigate a majority of theforested area. The northern sites were investigatedfor one to three days by three to five observerssearching independently or in teams of two to threeindividuals. Survey effort was estimated in personhours per hectare of forest. We surveyed the south-ern sites of Manombo and Mahabo to confirm thepresence of WCL, examine phenotypic variationamong populations, and to collect DNA samples(see below). These populations are currently subjectsfor ongoing behavioral ecology research. Searches atthese sites were therefore nonrandom, as knownsocial groups were targeted. Pelage characters wereassessed visually and compared qualitatively withpublished descriptions of WCL phenotypes [Tatter-sall, 1982] and previous observations elsewhere[Johnson & Overdorff, 1999; Johnson & Wyner,2000]. Brown lemur taxa are generally easilydistinguished by facial pelage, particularly in males[Tattersall, 1982]. Phenotypic characters were notincluded in quantitative phylogenetic analyses.

Genetic Sampling and Analysis

Samples were collected at Manombo (N 5 10individuals) and Mahabo (N 5 6) in April 2006(Table I). This sample augmented previous collec-tions at Manombo in 2000 (N 5 2; Table I). Samples(N 5 11) were also collected at Vevembe (S 22147.065, E 471 11.1100) in 2000 (Fig. 1; Table I). Thisinterior corridor forest is 65 km west of Farafangana(Fig. 1). Outgroups include Eulemur and other lemurtaxa from across Madagascar (Table I). On the basisof the null hypothesis of no population differencesamong WCL, we refer to test individuals from allsites as E. albocollaris in all figures and tables,recognizing that different nomenclature may need tobe adopted for different populations depending onresults.

Study animals (all adults) were immobilizedwith a CO2 projection rifle with 10 mg/kg of Telazol(Fort Dodge, Overland Park, Kansas). DNA wasextracted from 2.0-mm biopsies using a phenol-chloroform extraction [Sambrook et al., 1989]. Weanalyzed the following regions of the mtDNA: thedisplacement loop or control region [D-loop; Bakeret al., 1993; Wyner et al., 1999b] and a fragment ofthe cytochrome oxidase subunit III gene, NADH-dehydrogenase subunits 3, 4L, and 4 (ND3, ND4L,and ND4) as well as the tRNAGly, tRNAArg, tRNAHis,tRNASer, and partial tRNALeu genes [subsequentlyreferred to as the PAST fragment; Pastorini et al.,2000]. Using 50 ng of genomic DNA, the D-loop(552–555 bp) and the PAST (2388 bp) fragmentswere amplified using the following conditions: 941Cfor 4 min, 941C for 30 sec, 471C for 45 sec, 721C for45 sec for 35 cycles, 721C for 10 min. The sampleswere electrophoresed on a 1.2% agarose gel to verify

Am. J. Primatol.


TABLE I. Samples (29 Eulemur albocollaris and 41 outgroups total) Used in the Present Genetic Analyses

ID# Site Taxon SexPAST GenBank

accession no.D-loop GenBank

accession no.

HABO6.1 Mahabo Eulemur albocollaris F EF552610 EF552658HABO6.2 Mahabo Eulemur albocollaris F EF552611 EF552659HABO6.3 Mahabo Eulemur albocollaris M EF552612 EF552660HABO6.4 Mahabo Eulemur albocollaris M EF552613 EF552661HABO6.9 Mahabo Eulemur albocollaris F EF552614 EF552662HABO6.10 Mahabo Eulemur albocollaris M EF552615 EF552663M151 Manombo Eulemur albocollaris F EF552616 EF552664M152 Manombo Eulemur albocollaris M EF552617 EF552665MBO6.2 Manombo Eulemur albocollaris M EF552618 EF552666MBO6.3 Manombo Eulemur albocollaris M EF552619 EF552667MBO6.4 Manombo Eulemur albocollaris F EF552620 EF552668MBO6.5 Manombo Eulemur albocollaris M EF552621 EF552669MBO6.6 Manombo Eulemur albocollaris F EF552622 EF552670MBO6.7 Manombo Eulemur albocollaris M EF552623 EF552671MBO6.8 Manombo Eulemur albocollaris M EF552624 EF552672MBO6.9 Manombo Eulemur albocollaris F EF552625 EF552673MBO6.10 Manombo Eulemur albocollaris M EF552626 EF552674MBO6.11 Manombo Eulemur albocollaris M EF552627 EF552675VVEV1 Vevembe Eulemur albocollaris M EF552628 EF552676VVEV2 Vevembe Eulemur albocollaris F EF552629 EF552677VVEV3 Vevembe Eulemur albocollaris M EF552630 EF552678VVEV4 Vevembe Eulemur albocollaris F EF552631 EF552679VVEV5 Vevembe Eulemur albocollaris M EF552632 EF552680VVEV6 Vevembe Eulemur albocollaris M EF552633 EF552681VVEV7 Vevembe Eulemur albocollaris M EF552634 EF552682VVEV8 Vevembe Eulemur albocollaris M EF552635 EF552683VVEV9 Vevembe Eulemur albocollaris M EF552636 EF552684VVEV10 Vevembe Eulemur albocollaris M EF552637 EF552685VVEV11 Vevembe Eulemur albocollaris F EF552638 EF552686RANO261 Ranomafana Avahi laniger M AY582559 AY584496RANO67 Ranomafana Avahi laniger M AY582558 AY584495ANK33 Ankarafantsika Avahi occidentalis F AY582560 AY584497RANO229 Ranomafana Cheirogaleus major F AY582563 AY254050GAR8 Manongarivo Cheirogaleus medius M AY582562 AY584498DOG8 Midongy du Sud Eulemur collaris F EF552591 EF552639AND25 Andohahela Eulemur collaris F EF552592 EF552640MER16 Analamera Eulemur coronatus F EF552608 EF552656ANKA3 Ankarana Eulemur coronatus F EF552609 EF552657BET31 Betampona Eulemur fulvus albifrons M EF552593 EF552641JAR11 Anjanaharibe-Sud Eulemur fulvus albifrons F EF552596 EF552644ZAH19 Zahamena Eulemur fulvus fulvus M EF552594 EF552642ANK3 Ankarafantsika Eulemur fulvus fulvus — EF552595 EF552643RANO45 Ranomafana Eulemur fulvus rufus M AY582561 AY585738ISA2.3 Isalo Eulemur fulvus rufus M EF552599 EF552647ANAL4 Analamera Eulemur fulvus sanfordi M EF552597 EF552645MER12 Analamera Eulemur fulvus sanfordi M EF552598 EF552646LOKO4.10 Lokobe Eulemur macaco macaco M EF552604 EF552652LOKO4.25 Lokobe Eulemur macaco macaco M EF552605 EF552653MIT40 Antrema Eulemur mongoz F EF552602 EF552650MIT39 Antrema Eulemur mongoz F EF552603 EF552651RANO25 Ranomafana Eulemur rubriventer F EF552600 EF552648MERY9 Marojejy Eulemur rubriventer F EF552601 EF552649LAZA5.02 Sahamalaza (Ankarafa) Euleumur macaco flavifrons F EF552606 EF552654LAZA5.08 Sahamalaza (Ankarafa) Euleumur macaco flavifrons F EF552607 EF552655RANO351 Ranomafana Hapalemur aureus M AY582549 AY584489RANO352 Ranomafana Hapalemur aureus M AY582550 AY254048RANO61 Ranomafana Hapalemur griseus griseus — AY582551 AY584490RANO62 Ranomafana Hapalemur griseus griseus F AY582552 AY584491ANAL2.23 Analamera Hapalemur griseus occidentalis M AY582554 AY584493

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the polymerase chain reaction (PCR) product andpurified using QIAquick PCR purification kit (QIA-GEN cat. no. 28106, Valencia, CA). The cleanedproducts were cycle sequenced using a big dye-terminator sequencing kit (Applied Biosystems,Foster City, CA). The sequences were analyzed bycapillary electrophoresis with an Applied BiosystemsPrizm 3100 genetic analyzer. A suite of internalsequencing primers from Pastorini et al. [2000, 2001]was used to generate the PAST fragment. Thesequence fragments were aligned to generate aconsensus sequence using Sequencher (Gene Corp,Ann Arbor, MI), and the consensus sequences werealigned using Clustal X [Thompson et al., 1997]. Allsequences have been deposited in GenBank and thesequence data and information are available fromthe referenced accession numbers (Table I).

Maximum parsimony (MP), maximum likeli-hood (ML), and neighbor-joining (NJ) analyses wereperformed for the phylogenetic study of thecombined D-loop and PAST fragments sequencedata with PAUP� Version 4.0b10 software [Swofford,2001]. The trees described in this study are allconsensus trees except for the bootstrap analysis(all trees are presented as phylograms for presenta-tion purposes only). Bootstrap analyses were accom-plished with 4,000 replicates, with ten randomadditional heuristic searches per replicate. Onlynodes with greater than 50% support were reported.The NJ tree was generated using the Tamura–Neimodel [Tamura & Nei, 1993]. A stepwise additionwas selected for MP and ML analyses, and correc-tions for nucleotide sequence data suggested byKimura [1980] were used with the NJ analyses.Gaps were considered as a fifth character in MPanalyses, whereas gaps were treated as missing datain the NJ analyses. The ML trees were estimated viathe heuristic search. For the substitution model, thetransition/transversion ratios were estimated inMacClade [Maddison & Maddison, 1992], and adiscrete approximation to g distribution was esti-

mated for among site rate variation. The defaultsettings were maintained for all other settings, thusyielding the equivalent of the HKY model [Hasegawaet al., 1985]. In addition to character-based phyloge-netic analysis of DNA sequences, PAUP software[Swofford, 2001] and MEGA Version 3.1 [Kumaret al., 2004] were used to calculate genetic distance.

All research procedures complied with protocolsapproved by Institutional Animal Care andUse Committee (US) and Animal Care Committee(Canada), and adhered to the legal requirements ofthe Government of Madagascar.

RESULTS

Surveys

WCL presence was confirmed at Manombo andMahabo forests (Fig. 1). Three social groups with sixto 11 individuals were identified at Mahabo. Twosocial groups of four to eight individuals wererecorded at Manombo. We detected no evidentdifferences in coat patterns or facial markingsbetween these populations, nor did either differ frominterior WCL populations [e.g., Vevembe; S.E.J.,personal observation]. Analalava was searched for3.25 person hours (ca. 0.05 hr/ha). Search time atSakanany forest was approximately 95 person hours(ca. 0.48 hr/ha). No Eulemur or other lemur taxonwas observed in either forest, nor was there any signof lemur activity (feeding remains, feces). Localinformants believed Eulemur was still present butcould not consistently identify lemur species inphotographs.

Genetic Analyses

mtDNA sequence data were completed forD-loop and PAST fragments for 29 WCL (Table I).Relationships among species or genera were consis-tent in all analyses (Figs. 2–4). Generally, there wasvery high support in both MP and NJ analyses with

TABLE I. Continued

ID# Site Taxon SexPAST GenBank

accession no.D-loop GenBank

accession no.

GAR9 Manongarivo Hapalemur griseus occidentalis M AY582553 AY584492JAR4 Anjanaharibe-Sud Indri indri F DQ855969 DQ856049MIZA5.3 Maromizaha Indri indri F DQ855967 DQ856050ANAL5 Analamera Lepilemur septentrionalis F AY582564 AY769363ANK7 Ankarafantsika Microcebus ravelobensis F AY582545 AY159695RANO250 Ranomafana Microcebus rufus M AY582546 AY159722KIAN124 Kianjavato Prolemur simus F AY582548 AY584488RANO338 Ranomafana Prolemur simus F AY582547 AY254049RANO332 Ranomafana Propithecus edwardsi M AY582556 AY585739MOR68 Beroboka Propithecus verreauxi verreauxi — AY582557 AF354712FAN21 Fandriana Varecia variegata variegata F AY582555 AY584494

Mitochondrial DNA sequence data for each sample are available from GenBank under the listed accession numbers.

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Fig. 2. Neighbor-joining phylogram derived from the D-loop and PAST fragment combined DNA sequence data from the 29 Eulemuralbocollaris individuals with 41 outgroup taxa. Values above branches indicate the number of changes between nodes. Values withincircles indicate support of bootstrap pseudoreplicates.

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respect to the branching order of genera and species(Figs. 2–4). On the basis of phylogenetic inferences ofthe NJ, MP, and ML analyses of the sequencealignments, WCL individuals from the three differ-ent sites were clustered together with 100% boot-

strap support. The absolute distance and the Kimuratwo-parameter distance measures are presented inTables II and III. The absolute distances among WCLindividuals are 2–9 bp for D-loop and 1–11 bp forPAST (Tables II and III).

Fig. 3. Maximum parsimony phylogram derived from the D-loop and PAST fragment-combined DNA sequence data from the 29Eulemur albocollaris individuals (one of six most parsimonious trees). Values above branches indicate number of changes betweennodes. Values within circles indicate support of bootstrap pseudoreplicates. Length 5 5,574; Consistency index (CI) 5 0.4760; Retentionindex (RI) 5 0.7875; Rescaled consistency index (RC) 5 0.3748; Homoplasy index (HI) 5 0.5240.

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Fig. 4. Maximum-likelihood phylogram derived from the D-loop and PAST fragment-combined DNA sequence data from the 29 Eulemuralbocollaris individuals. The phylogram presented with branch lengths proportional to the number of changes (values specified on thebranches). We obtained the Maximum-likelihood phylogram (-ln likelihood 5 26,784.84) from the PAST alignment from a transition/transversions ration of 4.54 (k5 9.72) and g shape parame 0.38.

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Am. J. Primatol.


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;2

6,

VV

EV

5;

27

,V

VE

V8

;2

8,

VV

EV

11

.

Am. J. Primatol.


DISCUSSION

Our ground surveys confirmed the presence ofWCL at the coastal forests south of the Manampa-trana River: Manombo and Mahabo. We found noconsistent pattern of phenotypic variation betweenthese populations, or between these groups andinterior populations, unlike the established brownlemur species and subspecies [Mittermeier et al.,2006b; Shedd & Macedonia, 1991]. These resultswere expected based on recent research at Manombo[Johnson & Overdorff, 1999; Ratsimbazafy, 2002]and other published observations from Mahabo[Mittermeier et al., 2006b].

Genetic analyses indicate the monotypy of WCLpopulations. Using multiple mtDNA regions, everyanalysis yielded identical results: all WCL from twocoastal (Manombo and Mahabo) and one interior site(Vevembe) clustered together as a single species,with high bootstrap support and very short geneticdistances relative to recognized lemur species andsubspecies (Figs. 2–4). Moreover, none of thesepopulations formed a distinct lineage within thisspecies. Specifically, Manombo and Mahabo indivi-duals formed mixed clades. Individuals from theinterior site Vevembe could be expected to clustermore exclusively, owing to the relative geographicisolation of this population. Nonetheless, two indivi-duals from this site (VVEV5 and VVEV8) consis-tently clustered with the coastal populations. Onepossibility is that these individuals represent recentmigrants from the coastal region. Both individualsare adult males (Table I), the sex that commonlydisperses in brown lemurs [Overdorff et al., 1999].However, this is unlikely given the substantialdistance between the coastal forests and Vevembe(450 km) and the lack of suitable habitat betweenthese sites (Fig. 1). Thus, we suggest that the lack ofdistinct geographic clustering among populationsfrom the coast south of the Manampatrana Riverand the interior forests indicates gene flow acrossthis region before isolation caused by anthropogenicdisturbance. Further research with larger samplesizes and additional loci may show additional sub-structure and biased directionality in gene flow(suggested as east to west here).

The two fragments we surveyed north of theManampatrana River, Analalava and Sakanany,appeared to be the most likely locations for lemursto persist in a region severely impacted by habitatloss. They are also very near Salohy, a localityreported for the mounted specimens of whatSchwarz [1931] referred to as E. cinereiceps (Fig. 1).However, we did not detect Eulemur or any otherday-active lemur species at these sites. Therefore, itseems that brown lemurs have been extirpated fromthe coastal plain between Manombo (south of theManampatrana) and the lowland forests of Vatovavyand Kianjavato, within the range of E. fulvus rufus

[Mittermeier et al., 2006b; Tattersall, 1982](although we note our surveys were brief). As aconsequence, we were unable to investigate directlywhether the Manampatrana River divides distinctforms of WCL on the coastal plain. However,previous studies of interior WCL found shareddiagnostic characters between populations found tothe south (Vevembe) and to the north (Evendra) ofthe Manampatrana River [Johnson & Wyner, 2000].Thus, this river is not a boundary for brown lemurtaxa in the eastern escarpment forests.

With the evidence gathered from ground surveysand genetic sampling, we may now evaluate the twoscenarios for the separation of the two possible formsof WCL: E. albocollaris and E. cinereiceps. The firsthypothesis suggests an east–west separation, withE. albocollaris found in the escarpment rain forestsand E. cinereiceps found in the littoral and lowlandrain forests of the east coast. On the basis of thelack of distinct clustering among individuals fromVevembe (interior) or Manombo/Mahabo (coast), wecan provisionally reject this hypothesis.

The second scenario suggests a north–southgeographic division at the Manampatrana River. Asnoted, we are unable to test directly this hypothesiswith the data presented here, as northern popula-tions may have been extirpated. We may, however,be able to draw inferences from other evidence. First,we have suggestions of what E. cinereiceps wouldhave looked like from the original plates and theParis specimens [Milne-Edwards & Grandidier,1890; Schwarz, 1931]. There is also the photographof the captive female of unknown provenance inFarafangana from 2005 [Mittermeier et al., 2006b].In describing the female mounted specimens, Groves[2001] noted that the body pelage and muzzle arerelatively light in color, and the cheeks are not white(a WCL trait) or red (a collared lemur trait).However, in examining photographs of these speci-mens [Mittermeier et al., 2006b], we cannot identifytraits that suggest important differences from WCLat known extant localities (e.g., Vevembe, Manom-bo). The body pelage of the museum specimensappears to have faded substantially postmortem, sowe do not feel coat color (hue) is a valid distinction.Moreover, the gray muzzle is consistent with extantWCL females. Finally, white or red cheeks are traitsfound only in male WCL or collared lemurs,respectively [Tattersall, 1982], so their absence isexpected for female WCL specimens. As for the plate[Milne-Edwards & Grandidier, 1890] and Farafan-gana captive female [Mittermeier et al., 2006b],again we find no clear differences between theindividuals depicted and WCL females observedduring our surveys and captures. In other words,all sources seem to depict the same species. However,females are not particularly useful in diagnosis ofsoutheastern brown lemurs as WCL and collared

Am. J. Primatol.


lemur females are largely indistinguishable [Mitter-meier et al., 2006b; Tattersall, 1982].

Patterns of lemur biogeography offer additionallines of evidence. In recent geological history, lemurpopulations in coastal areas have been subject tosevere climatic fluctuations and likely range contrac-tions [Goodman & Ganzhorn, 2004]. Lemur speciesmay have maintained high elevational ranges owingto the relative stability of vegetational zones in mid-high elevation forests [Goodman & Ganzhorn, 2004].Indeed, there is not a single species that is confinedto lowland habitats in eastern Madagascar; allspecies found in the coastal forests maintain broadranges that include higher elevation interior forests[Goodman & Ganzhorn, 2004]. Therefore, it seemsunlikely that the WCL that occupied the coastalforests north of Farafangana at least until the late19th century (i.e., the Paris specimens) would be adistinct taxon from WCL still currently found inadjacent forests such as Evendra [Johnson & Wyner,2000]—particularly in light of the absence of clearpelage differences.

From the available evidence, we may tentativelyconclude that E. cinereiceps does not exist as aseparate taxon from E. albocollaris. Moreover, ifa distinct brown lemur species once existed in anarrow coastal plain north of the ManampatranaRiver, this animal would now likely be extinct. Thisevidence suggests a response to the question posed inthe title. However, we believe that the issue ofpriority in nomenclature for WCL warrants revisit-ing and E. cinereiceps may be retained. As discussedabove, the cinereiceps designation first appeared asa plate [Milne-Edwards & Grandidier, 1890]. Accord-ing to the International Commission on ZoologicalNomenclature (ICZN), names accompanied only byillustrations may be valid for taxa proposed before1931 [Article 12.2.7; ICZN, 1999]. Such a precedentcan be reversed if the senior name has not been usedsince 1899 and the junior synonym [in this case,E. albocollaris; Rumpler, 1975] has been in commonuse in the previous 50 years [Article 23.9.1; ICZN,1999]. However, both Schwarz [1931] and Groves[1974] used cinereiceps to refer to what we can inferare WCL by their descriptions of localities andphenotypes, respectively. Accordingly, we concludethat, if indeed WCL represent a single species,priority should be given to the senior synonym,E. cinereiceps [Milne-Edwards & Grandidier, 1890].

The reemergence of E. cinereiceps as a validtaxon would perhaps be more dramatic if it repre-sented a truly ‘‘new’’ species. However, the availableevidence suggests that there is a single WCL species,recently connected by gene flow. Our findings alsounderscore the ‘‘critically endangered’’ conservationstatus of WCL, as they seem to have been extirpatedfrom large areas of their already limited range.Future research should examine more closely therelationships among the presently fragmented popu-

lations. In addition, management plans shouldconsider immediate steps to maintain the viabilityof all remaining individual populations as well asensuring the connectivity of these populations topreserve genetic diversity.

ACKNOWLEDGMENTS

We thank the Association Nationale pour laGestion des Aires Protegees (ANGAP), the Ministeredes Eaux et Forets (MEF), and the Universited’Antananarivo for permission to conduct researchin Madagascar. We gratefully acknowledge ourfunding sources: University of Calgary (S.E.J.), theCommittee for Research and Exploration of theNational Geographic Society (6613.99) (E.E.L.),Margot Marsh Foundation Grant (E.E.L.), PrimateAction Fund (S.K.M., E.E.L.). E.E.L. and R.L. alsoacknowledge the generosity of Bill and BernieceGrewcock for their long-term support and commit-ment, which gave the CCR its direction and identity;this research would not be possible without thesupport of the Ahmanson Foundation, the TheodoreF. and Claire M. Hubbard Family Foundation, andthe James Family. We are also grateful to theInstitute for the Conservation of Tropical Environ-ments (ICTE), Madagascar Institut Pour la Con-servation des Ecosystemes Tropicaux (MICET),Durrell Wildlife Conservation Trust (DWCT), theMissouri Botanical Garden (MBG), the CentreValBio (CVB), Benjamin Andriamihaja, PatriciaWright, Anna Feistner, Chris Birkinshaw, JonahRatsimbazafy, and Karen Samonds. For assistance inthe field, we thank Hubert Emilien Andriamaharoa,Fidimalala Bruno Ralainasolo, Olivia V. Randrianar-imalalasoa, and Justin Solo. Many thanks to ColinGroves for his insightful comments and pointing usto the appropriate sections of the Zoological Code. Wealso thank the three anonymous reviewers for theirhelpful comments. This research complied withprotocols approved by Institutional Animal Careand Use Committee (US), Animal Care Committee(Canada), and the Government of Madagascar(Tripartite Committee).

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