The importance of genetic research in zoo breeding programmes for threatened species: the African...

The importance of genetic research in zoobreeding programmes for threatened species: theAfrican dwarf crocodiles (genus Osteolaemus) asa case studyF. SCHMIDT1*, F. A. FRANKE2,3, M. H. SHIRLEY4, K. A. VLIET5 & V. L. VILLANOVA6

1Zoo Leipzig, Pfaffendorfer Strasse 29, D-04105 Leipzig, Germany, 2Animal Evolution &Development, Institute of Biology, University Leipzig, Talstrasse 33, D-04103 Leipzig,Germany, 3Molecular Evolution and Animal Systematics, Institute of Biology, UniversityLeipzig, Talstrasse 33, D-04103 Leipzig, Germany, 4SFM Safari Gabon, B.P. 1107,Libreville, Gabon, 5Department of Biology, University of Florida, 876 Newell Drive,Gainesville, Florida, 32611, USA, and 6Department of Biology, University of CentralFlorida, 4000 Central Florida Boulevard, Orlando, Florida 32816, USAE-mail: [email protected]

The threatened African dwarf crocodiles (genusOsteolaemus) are distributed throughout West and CentralAfrica. Traditionally two subspecies were described(Osteolaemus tetraspis tetraspis and Osteolaemustetraspis osborni), although recent molecular studiesdemonstrate the presence of three allopatric lineages thatshould be recognized as full species.These highly divergenttaxa are distributed in the three major forested biogeo-graphic zones of western Africa: Congolian (Osteolaemusosborni), Lower Guinean (Osteolaemus tetraspis) andUpper Guinean (Osteolaemus sp. nov. cf. tetraspis).Largely because of their diminutive size, dwarf crocodilesare regularly kept in zoos and aquariums worldwide. InEurope, the collection is managed by a European studbookcoordinated by Leipzig Zoo, Germany, since 2006, whileAmerican zoological institutions do not yet manage thesespecies as part of a studbook programme. To facilitate exsitu conservation efforts, it is important to identify accu-rately each individual to the appropriate species followingthe latest systematic understanding of the genus. Populationaggregation analysis with mitochondrial and nuclear genesequences was used for both species identification anddetection of interspecific hybridization. The results of ourstudy show that only European collections house all threeOsteolaemus taxa, although only a single individualO. osborni was confirmed. The most prevelant speciespresent in both European and North American institutionswas O. tetraspis. Additionally, several O. sp. nov. cf.tetraspis were identified, likely originating from theSenegambia region, especially in the North American col-

lections.This will represent an important resource for futureconservation efforts as Osteolaemus are highly threatenedin this region of West Africa. Unfortunately, both zoopopulations showed relatively high frequencies (c.25–28%) of hybridization between O. tetraspis and O. sp.nov. cf. tetraspis bred in captivity. We highly recommendthat zoological institutions ensure they know the speciesidentity of the Osteolaemus they maintain and worktogether to transfer individuals into single-species coloniesto avoid further hybridization. In the USA, this may neces-sitate the creation of a studbook programme. It may alsoprove valuable to consider a cooperative programmebetween the EuropeanAssociation of Zoos andAquaria andthe Association of Zoos & Aquariums, wherein each Asso-ciation focuses its resources largely on a singleOsteolaemus species. This would, however, require trans-Atlantic transfer of individuals. The case study of dwarfcrocodiles in zoological institutions reinforces the impor-tance of genetic research in conservation-breeding pro-grammes, highlights the potential for collaboration betweenEuropean and American zoological institutions for the exsitu conservation of threatened wildlife, and foreshadowssome of the regulatory challenges in managing captivepopulations internationally.

Key-words: captive breeding; ex situ conservation;genetic barcoding; hybridization; osborni;Osteolaemus; population aggregation analysis;tetraspis.

*Corresponding author: Fabian Schmidt, Zoo Leipzig, Pfaffendorfer Str. 29, D-04105 Leipzig, Germany. E-mail:[email protected]

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GENETIC RESEARCH IN ZOO BREEDING PROGRAMMES: AFRICAN DWARF CROCODILES 1

Int. Zoo Yb. (2015) 49: ••–••DOI:10.1111/izy.12082

Int. Zoo Yb. (2015) 49: ••–•• © 2014 The Zoological Society of London

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INTRODUCTION

African dwarf crocodiles of the genusOsteolaemus have had a muddled taxonomichistory. The genus was originally describedwith a single content species, Osteolaemustetraspis, by Cope (1861) from a specimencollected in the ‘Ogobai’ (= Ogooué) RiverBasin of Gabon. Later, Murray (1862)described Crocodilus frontatus from a dwarfcrocodile collected in the Benué River ofNigeria, and Lilljeborg (1867) describedHalcrosia afzelii from a dwarf crocodile col-lected in Sierra Leone. Interestingly, neitherof these latter authors referenced Cope’swork on O. tetraspis, suggesting they simplywere not aware of it, and both taxa weresynonymized with the nominate species(Boulenger, 1889).

Schmidt, K. P. (1919) described a fourthdwarf crocodile species from the Ituriforest of the Congo Basin, even placingit in a monotypic genus (Osteoblepharon)to account for what he felt were conside-rable morphological differences from Osteo-laemus. In reality, it amounted to only fourcranial characters and the genus Osteo-blepharon was finally refuted by Wermuth(1953) who reduced the genus Osteolaemusto a single species with two allopatric sub-species: Osteolaemus tetraspis osborni fromthe Congo Basin and Osteolaemus tetraspistetraspis from everywhere else.

This taxonomy stood until 2009 when aseries of largely genetic studies were imple-mented to test the taxonomic hypothesis oftwo species within O. tetraspis. Eaton et al.(2009) sampled individuals wild caught inthe Congo Basin (Republic of Congo), theOgooué Basin (Gabon), and the UpperGuinea biome (Ghana and Cote d’Ivoire).Their results supported the previously recog-nized Congo Basin (O. osborni) and OgoouéBasin (O. tetraspis) forms, but also revealedthat West African individuals were a third,equally divergent form. Eaton et al. (2009)proposed that the three lineages shouldbe regarded as full species correspondingto the three major biogeographic zones ofwestern Africa: Congolian (the Congo Basin:

Osteolaemus osborni), Lower Guinean(Ogooué River Basin of Gabon, as wellas Equatorial Guinea, portions of Came-roon and coastal Congo: O. tetraspis), andUpper Guinean (from Ghana and Coted’Ivoire westwards: Osteolaemus sp. nov. cf.tetraspis). The authors proposed possible bio-geographic barriers, such as the DahomeyGap, the Cameroon Volcanic Line and theBatéké Plateau, keeping these species inallopatry (Fig. 1), but recommended thatfurther sampling be undertaken before theexact distributional limits would be known.All three taxa were depicted side by side forthe first time in Shirley & Eaton (2010),which clearly demonstrated their crypticmorphology, although also hinted at the pres-ence of distinguishing morphological charac-ters awaiting in-depth study (Plate 1).

Following Eaton et al.’s groundbreak-ing work, Shirley, Vliet et al. (2014) includeda more geographically comprehensive Osteo-laemus sample set in a species delimitationand phylogenetic analysis focused on the sys-tematics of the genus Mecistops (slender-snouted crocodiles). Not only did theysupport the results of Eaton et al. (2009)using robust Bayesian species delimitationmethods but also provided the first estimateof divergence timing for dwarf crocodilesusing fossil-calibrated phylogenetic recon-struction. Shirley, Vliet et al. (2014) showedthat the three Osteolaemus last shared acommon ancestor ± 11 Mya (9–14 Mya)when the two western species diverged fromO. osborni, and that O. tetraspis and O. sp.nov. cf. tetraspis have been isolated for ± 8Mya (5·5–10·5 Mya). Also, since the workcarried out by Eaton et al. (2009), bothFranke et al. (2013) and Smolensky et al.(2014) found significant evidence that theCameroon Volcanic Line is not the biogeo-graphic barrier between O. tetraspis andO. sp. nov. cf. tetraspis. Instead, they foundthat the former taxon might be distributed allthe way to Togo, indicating that the DahomeyGap, an extensive upland savannah extendingfrom western Ghana, through Benin andTogo (see Fig. 1 in Smolensky et al., 2014),is the next most likely biogeographic-barrier

2 REPTILE CONSERVATION


candidate. Additionally, Franke et al. (2013)and Shirley, Villanova et al. (2014) detectedsignificant subspecific phylogeographicstructure within O. sp. nov. cf. tetraspis iden-tifying two evolutionary significant units: onein the Upper Guinea forests of Ghana, Coted’Ivoire and Liberia (haplogroup I), and theother restricted to the Senegambian forestblock of Senegal, Gambia and Guinea(haplogroup II).

Morphological differentiation betweenany Osteolaemus taxa has only beendescribed for cranial characters, preventingreliable diagnosis of living specimens unlesstheir geographic origin is known. This hasconsiderably hindered efforts to managespecimens in European Association of Zoosand Aquaria (EAZA) and Association ofZoos & Aquariums (AZA) institutions, asonly about ten individuals have confidentcollection-locality data. Dwarf crocodilesare among the most represented crocodiliansin EAZA and AZA collections largelybecause of their diminutive size, tractabilityand relative ease with which they breed incaptivity. These zoo populations represent apotentially significant conservation resourceas wild Osteolaemus populations continueto decline. Osteolaemus tetraspis, the sole

dwarf crocodile recognized by the Interna-tional Union for Conservation of Nature(IUCN), has been listed as Vulnerable onthe IUCN Red List since 1996 (IUCN,2014). Across its distribution, the majorthreats to the species are habitat alterationand hunting for the bushmeat trade.However, the impacts of these threats varythroughout the ranges of dwarf crocodiles.For example, the soon to be recognizedWest African Osteolaemus species faces farmore critical threats to its conservation,such as the bushmeat trade, habitat destruc-tion by commercial logging and degradationof wetland habitats (Shirley et al., 2009;Eaton, 2010).

Conservation breeding in zoos often facesthe classical dilemma of whether to manageindividuals from naturally isolated popula-tions separately, or should they be combinedto reduce the risk of inbreeding depressionand ensure sufficient gene diversity and theadaptive potential of the captive population(Edmands, 2007; Senn et al., 2014). Whilethese two issues are often the focus ofconservation-breeding discussion, the lack oftaxonomic resolution leading to inadvertenthybridization and the loss of unique popula-tions should be of equal concern in the

Fig. 1. Updated distribution of the genus Osteolaemus following the results of the genetic analysis carried outwith wild-caught captive animals of known provenance for this study and the results of Smolensky et al. (2014),which provided evidence that O. tetraspis extends as far west as Togo.



modern era of taxonomic discovery(Kitchener, 2010; Shirley, Villanova et al.,2014). Aside from introgression and simplymuddling species boundaries, the risks ofmixing different species are outbreedingdepression and hybrid sterility (Edmands,2007; Senn et al., 2014). Ultimately, hybridi-zation may eliminate the conservation valueof such populations and, therefore, solutionsmust be considered carefully. Clarificationof taxonomic issues using molecular tech-niques has significant potential to inform

such conservation-breeding decision making(Kitchener, 2010).

In 2006, EAZA established a Europeanstudbook (ESB), maintained at Leipzig Zoo,Germany, in order to manage captive Osteo-laemus populations better. No studbook orcoordinated breeding programme has yetbeen established by the AZA. Underscoringthe link to in situ conservation efforts for thesecrocodiles, the ESB has been approachedby African reptile parks seeking founderstock for future breeding and reintroduction

Plate 1. The three different Osteolaemus taxa are very difficult to distinguish morphologically, makingOsteolaemus a cryptic species complex. Top image: Osteolaemus tetraspis (below) and Osteolaemus sp. nov. cf.tetraspis (above). Zoo Leipzig, Germany. Bottom image: Osteolaemus osborni. Zoologico Santillana del Mar, Spain.



purposes. To support this link to grass rootsconservation initiatives in Africa, we under-took to identify Osteolaemus maintained inEAZA and AZA institutions utilizing thelatest systematic and taxonomic researchdescribed here. Individuals were alsoscreened for evidence of hybridization toprevent the exportation of hybrid individualsfor in situ reintroduction efforts.

MATERIALS AND METHODS

Appropriate institutional veterinary or curato-rial staff were asked to take samplesand send blood cards from all knownOsteolaemus in the EAZA and AZA popula-tions. Samples for a total of 129 non-relatedindividual animals were received from zoos(91), private collections (17) and museums(21) in the EAZA region. Including thesiblings/offspring of these non-related indi-viduals (also maintained within the popula-tion), the total number of samples receivedwas 172, c. 92·5% of the EAZA captive popu-lation and c. 82·5% of all founders/potentialfounders in this population (Schmidt, F.,2013a). Thirty-two samples were receivedfrom nine AZA collections (accessionnumbers and analyses published in Shirley,Villanova et al., 2014). There is no studbookfor Osteolaemus in AZA institutions, so it isnot possible to know precisely the total size ofthe AZA Osteolaemus population or the pro-portion of the total population these 32samples represent. Based on recent Interna-tional Species Information System (ISIS)Zoological Information Management System(ZIMS) data (October 2013), combined withinformation from an older (2011) AZACrocodilian Advisory Group (CAG) spacesurvey for AZA institutions that do not reportto ISIS (Crocodilian Advisory Group, 2012),the AZA population is believed to be roughly71 crocodiles at 24 institutions. As such, the32 AZA samples received would represent c.45% of the AZA captive population. For theOsteolaemus in EAZA, 34 of the 129 sampleswere newly sequenced as part of this study and75 samples had been sequenced as part of theFranke et al. (2013) study. No new individuals

were sequenced from the AZA for the studyreported here (but see Shirley, Villanova et al.,2014).

A 565 bp fragment of the mitochondrialcytochrome c oxidase subunit I (COI) and a 642bp fragment of the nuclear lactate dehydro-genaseA (LDH-A) were sequenced. Extractionof genomic DNA, polymerase chain reactionand sequencing protocols, as well as sequencemanipulation and alignment, were describedin Franke et al. (2013) and Shirley, Villanovaet al. (2014). These genes were selected toenable comparison with the extensive publishedOsteolaemus data from Eaton et al. (2009).The GenBank accession numbers for the 27non-hybrid individuals, of the 34 newlysequenced EAZA individuals, are KM406124–KM406150 (COI) and KM406151–KM406177 (LDH-A) (Appendix S1).

Species identification was carried out usingboth genes in a population aggregation analysis(PAA) framework (Davis & Nixon, 1992).Unique haplotypes (PAA1) and fixednucleotide positions (PAA2) that segregated thethree Osteolaemus species within a referencedata set of wild-caught, known-locality individ-uals were searched for [96 reference sequencespublished by Eaton et al., (2009), Shirley,Villanova et al. (2014) and Shirley, Vliet et al.(2014): these studies found a total of 83 variablebases for COI segregating the three Osteo-laemus species, although the number of fixedsites segregating any two species ranged from44 to 57, and five variable LDH-A sites; threesites segregated O. osborni from both otherspecies and two sites segregated O. sp. nov. cf.tetraspis from both other species]. Zoo speci-mens were assigned to species by aligning theirsequences with the wild references and match-ing unique haplotypes and fixed nucleotidepositions in both COI and LDH-A. Hybridswere detected by examining the LDH-Alineage-specific diagnostic sites in the post-sequencing chromatograms for heterozy-gosities, which manifest as double peaks,whereas non-hybrid individuals will show onlya single peak indicating homozygosity. Whereavailable, both the COI and LDH-A parentalhaplotypes were used to confirm detectedhybrids.



RESULTS

All three Osteolaemus species were found incollections within EAZA and AZA, althoughthe taxa were disproportionately represented(Table 1). Only a single O. osborni has beenfound in captivity to date, at Zoo deSantillana del Mar, Cantabria, Spain (anEAZA facility). No O. osborni appear toexist in the AZA. Osteolaemus tetraspis wasthe most abundant dwarf crocodile speciesin both populations, representing 60·5%and 59% of the tested individuals in theEAZA and AZA populations, respectively.Osteolaemus sp. nov. cf. tetraspis repre-sented only 11% and 16% of EAZA andAZA populations, respectively. Both O. sp.nov. cf. tetraspis mitochondrial haplogroupsdescribed by Franke et al. (2013) and Shirley,Villanova et al. (2014) were present in theEAZA population, although Upper Guineaanimals were somewhat more numerous. Incontrast, all West African individuals ana-lysed in the AZA were from haplogroup II.Detailed data sets for the results of the PAAanalysis and GenBank accession data for allreference sequences have been previouslypublished in Eaton et al. (2009), Franke et al.(2013) and Shirley, Villanova et al. (2014).

Hybrid individuals were detected at anoverall rate of 25–28% in the two populations(Table 1). Discordance between the maternaland paternal haplotypes for both gene regionssupported the hybrid identifications in threeand six cases in the EAZA and AZA popula-tions, respectively. Only O. tetraspis × O. sp.nov. cf. tetraspis hybrids were detected, andhybridization occurred in both directions

(i.e. !! and "" of both species will repro-duce with the opposite sex of the opposingspecies). Comparison of our genetic resultswith genealogical accounts for the identifiedhybrid individuals (i.e. in the ESB or inZIMS) confirmed that hybrids were F1, F2 andbackcrosses in both EAZA and AZA collec-tions. No known wild-caught individuals (i.e.F0) were identified as hybrids.

An error was identified in the records forOsteolaemus in at least one case from theAZA population. Five offspring purportedlyfrom a pair of wild-caught Osteolaemus at theMemphis Zoo, TN, USA (0564 + 0565, nowdeceased and not sequenced as part of thisstudy) were sequenced. One of the recordedoffspring possessed O. tetraspis mtDNA andwas homozygous for O. tetraspis LDH-A,while the remaining four offspring possessedboth O. sp. nov. cf. tetraspis mtDNA andhomozygous LDH-A alleles. This individualwas unmarked and no records were found toindicate its origin. Following confiscationby police authorities, this individual wasthought to originate from Memphis Zoo andwas ‘returned’ there. However, our analysesclearly indicate that this individual was notfrom the Memphis breeding group.

While O. osborni is reasonably distinctphysically and can be identified by externalfeatures, O. tetraspis and O. sp. nov. cf.tetraspis truly represent cryptic species and,at the time of writing, it is still not possible todistinguish them by external morphology(Plate 1). There are anecdotal reports ofnoticeable differences among hatchlings andjuveniles of these taxa, including snoutshape, eye colour, and the pigmented bands

Table 1. Proportional occurrence of Osteolaemus taxa in the European Association of Zoos and Aquaria (EAZA)(n = 172 animals) and the Association of Zoos & Aquariums (AZA) (n = 32 animals) collections in 2012.

TAXON

EAZA AZA

NO. FREQUENCY NO. FREQUENCY

Osteolaemus tetraspis 104 60·5% 19 59%Osteolaemus osborni 1 0·5% 0 0%Osteolaemus sp. nov. cf. tetraspis 19 11% 5 16%Hybrids 48 28% 8 25%



on the torso and tail (Plate 2). Our resultstentatively suggest that these external fea-tures may be used to distinguish dissimilarlooking juveniles. For example, sequencedjuveniles that had light greenish–yellowisheyes all had O. sp. nov. cf. tetraspishaplotypes compared with those with darkbrown eyes, which all had O. tetraspishaplotypes. This result is very preliminaryowing to the small sample size, and furtherwork is needed to confirm these assumptions.

DISCUSSION

The 34 specimens analysed as part of thisstudy were all readily identifiable to one ofthe three species proposed by previous

authors (e.g. Eaton et al., 2009; Franke et al.,2013; Shirley, Villanova et al., 2014; Shirley,Vliet et al., 2014), which continues tosupport the hypothesis of Osteolaemus as acryptic species complex (Appendix S1).Additionally, our results, revealing both thetaxonomic identities of individuals and thepresence of a significant number of hybrids,require decisions to be made as to howEAZA and AZA institutions should managedwarf crocodiles in the future.

In some managed breeding programmes,the decision was made to continue breedinghybrids, even after genetic analysis. Forexample, ESB programmes for Beadedlizards Heloderma horridum and Galapagosgiant tortoises Geochelone nigra justified

Plate 2. Juveniles. Top image: Animals with copper, brown and golden bands, upturned snouts and dark eyes. Inthis case the animals originate from Ogooué Basin parents. Zoo Leipzig, Germany. Bottom image: Animal withblack and yellow bands, wide snouts and light greenish-yellowish eyes. In this case the animal originates fromWest African parents. Walter Zoo, Gossau, Switzerland.



continuation of subspecific hybridization forreasons of small captive population size, theimportance of keeping the species as a modelfor research or education purposes in EAZAcollections (Furrer et al., 2006) and to ensurea genetically healthy population (Draeby,2008). In the last-mentioned situation, EAZAbelieved that the removal of hybrids andseparation of subspecies into separate breed-ing populations would have left too few indi-viduals to retain adequate gene diversity.Similarly, a recent study on the CriticallyEndangered Dama gazelle Nanger dama pro-vided evidence that, despite phenotypic dif-ferences among populations, it should beregarded as single species without a sub-specific division. In future, captive breedingshould involve mixing the three currentlyseparated populations in zoos to maximizegene diversity and minimize inbreeding(Senn et al., 2014). Another justification formaintaining ongoing hybridization comesfrom the captive Amur leopard Pantherapardus orientalis population, which isstrongly influenced by a founder animal fromanother subspecies, whose genetic introgres-sion simply cannot be bred out. As a result,however, the captive population shows muchhigher gene diversity than the remnant wildpopulation, which suffers from a remarkablyreduced genetic diversity (Uphyrkina et al.,2002).

In other examples, where hybrids formed arelatively minor proportion of the populationand could be excluded from further breedingwithout significantly decreasing the genediversity in the studbook, the decision wastaken to form several taxonomically puresubpopulations in the breeding programme.EAZA established a European EndangeredSpecies Programme (EEP) for the WestAfrican chimpanzee Pan troglodytes verus(Carlsen & de Jongh, 2006). A strategy wasdeveloped to ensure that breeding onlyoccurred in genetically pure chimpanzees topreserve a healthy and self-sustaining popu-lation in EAZA, whereas hybrid animals arebeing phased out (Hvilsom et al., 2013).However, decisions may differ from region toregion; in contrast to EAZA, AZA decided to

manage their chimpanzees at the specieslevel (Carlsen & de Jongh, 2006).

All of the above discussion, however, dealswith the much more common issue of inter-subspecific hybridization in captivity. TheOsteolaemus issue could be argued to be bio-logically more significant at the species leveland, as such, the Osteolaemus ESB decidedto divide the population of dwarf crocodilesinto three separate species following ourgenetic results. The decision is justified bythe fact that 72% of tested individuals in theEAZA population were assignable to a puretaxonomic unit [11% to O. sp. nov. cf.tetraspis and 60·5% to O. tetraspis (and 0·5%for the single O. osborni)] and managing toprevent further hybridization only meant thatc. 28% of individuals would have to beexcluded because of their hybrid status(Table 1).

The impact on gene diversity of differentmanagement scenarios was modelled usingthe population management software PMx(Lacy et al., 2012), and we found that man-aging the species separately could still resultin the long-term maintenance of gene diver-sity for each species (> 90% for 100 years)(Schmidt, F., 2013a). Because crocodiles arelong-lived animals, the ESB still includesmany founder and potential founder (F0)specimens that contribute to maintaininghigh gene diversity within the subpo-pulations. For example, the O. tetraspis inEAZA collections descended from 14founder animals, of which 11 are still alive.Gene diversity is 93·6% and would beincreased to 98·5% by including 20 addi-tional founders (Schmidt, F., 2013a). Thereare at least 20 potential founders in theEAZA population – F0 specimens living inthe population that have not yet reproduced –making this a feasible management objective.The smaller O. sp. nov. cf. tetraspis popula-tion in EAZA descends from only fivefounder animals, of which four are stillliving. The resulting relatively low genediversity of 85·9% could still be increased to95·8% by including seven additional poten-tial founders (Schmidt, F., 2013a), which arealso available and living in the population.



The comparative effects of ‘removing’ (i.e.by managing species separately) threefounder and three potential founder individ-uals from the generalized Osteolaemus popu-lation do not result in significantly lower genediversity. Rather, gene diversity of each indi-vidual species increases by now striving tobreed the potential founder individuals thatwere previously not given breeding opportu-nities. In light of this, the ESB decided toexclude all hybrids from further breeding,separate several mixed founder pairs, recom-bine individuals into taxonomically purebreeding pairs, and continue the studbookwith two taxonomically pure breedingsubpopulations representing O. tetraspis andO. sp. nov. cf. tetraspis.

Additionally, specific recommendationswere made for the O. osborni, O. sp. nov. cf.tetraspis and hybrid populations (Schmidt, F.,2013a). It was recommended that O. osbornishould no longer be maintained in EAZAinstitutions. A population cannot comprise asingle individual and the threatened status ofthis species in the wild will likely preventfuture importation of founder stock. The ESBrecommended including O. sp. nov. cf.tetraspis haplogroup II individuals (all !!)originating from Senegambia in breedinggroups for the time being but pairing themonly with F1 "" (i.e. they were not to bepaired with founder or potential founder "")pending genetic results for the AZA popula-tion. If haplogroup II crocodiles were foundin abundance in the AZA, then the decisionwas to send the EAZA !! to AZA collec-tions. Similarly, the ESB recommendedphasing out hybrid individuals in the ESBpopulation by discontinuing any breedingand placing them outside the ESB at facilitiesthat would not breed them, in order to freespace for taxonomically pure individuals.The ESB further recommended that untestedanimals be treated as hybrids pending futureevaluation of their taxonomic status andinclusion in the ESB. These recommenda-tions led to 29 transfers of adult Osteolaemusbetween European zoological institutions(Schmidt, F., 2013a,b), resulting in 19 poten-tial breeding pairs for pure O. tetraspis and

six potential breeding pairs for pure O. sp.nov. cf. tetraspis. Through this specificallyfocused management action, the proportionof hybrids in the population was decreasedfrom 28% to 21%, while the proportion ofpure O. tetraspis increased from 60·5% to68% (Schmidt, F., 2014).

At the time of writing, the AZA CAGmaintains eight managed programmes for theCritically Endangered and Endangeredcrocodilian species: two yellow Species Sur-vival Plan (SSP) cooperative breeding pro-grammes and six red SSPs. As is the case inEAZA, AZA institutions have a long historyof keeping Osteolaemus. Since the mid-1970s, many institutions have maintainedand bred dwarf crocodiles in their collec-tions. Because of the abundance of speci-mens, and because the conservation status ofdwarf crocodiles was thought to be less direthan that of other crocodilians, the CAGnever sought to manage this species within anSSP coordinated programme or other stud-book. Our new understanding of taxonomicdiversity in Osteolaemus and the results ofthe present genetic analyses, however, clearlyrequire the CAG to contemplate how best toaddress this newly recognized situation.

Unfortunately, the eight AZA SSP pro-grammes already in place face significantspace shortages, making it difficult to bringthese populations up to target populationsizes, and space issues limit the future sus-tainability of some programmes. There isgrowing pressure within the AZA communityto reduce the number of managed pro-grammes to help alleviate space shortages.While no decisions have been made, itappears reasonably unlikely that the CAGwill choose to create new official studbookprogrammes for dwarf crocodiles and othermeans of managing the species may need tobe considered. One possible solution previ-ously discussed is for one or more zoo pro-fessionals to serve as ‘taxon champions’,familiarizing themselves with all dwarfcrocodiles within AZA institutions, makingunofficial recommendations regarding theirmanagement and, possibly, maintaining anunofficial studbook for this genus.



The CAG will publish its next RegionalCollection Plan in 2015. Decisions regar-ding the creation of a formal studbook orother population management plan forOsteolaemus will need to be made by thistime. Whether the CAG initiates a formalprogramme for dwarf crocodiles or not, thisdocument will include recommendations forAZA collections holding Osteolaemus.These recommendations will be quite similarto those detailed above for EAZA facilities.For example, all untested Osteolaemus mustnot be bred until they are tested. Pure popu-lations of O. tetraspis and O. sp. nov. cf.tetraspis must be maintained by separatingindividuals found in mixed groups. Hybridanimals should be removed from collec-tions, although these may prove useful aseducation-programme animals in some cases.Private, responsible facilities capable ofcaring for these crocodiles could serve thezoo community by taking these specimens. Inthis case, all hybrids should be permanentlymarked before leaving AZA facilities toavoid these animals re-entering the popula-tion at a later date.

One possible solution to managingOsteolaemus within the EAZA and AZApopulations is the wholesale exchange ofcrocodiles across the Atlantic. For instance,transferring all O. sp. nov. cf. tetraspis (bothhaplotype I and haplotype II) from EAZAinstitutions to the AZA and all O. tetraspisfrom AZA collections to EAZA collectionswould allow these two species to be managedcompletely separately without the possibilityof accidental hybridization and, furthermore,would strengthen the size and gene diversityof populations of both species. This wouldalso eliminate the issue of managing twospecies of crocodiles that, at least to the zoo-going public, look absolutely identical to oneanother, and remove the need to justify thisdecision to administrators and the public.However, an exchange such as this would bean enormously difficult undertaking andwould require significant regulatory over-sight because Osteolaemus crocodiles arelisted in Appendix I of the Convention onInternational Trade in Endangered Species of

Wild Fauna and Flora (CITES) and in theUSA Endangered Species Act. Permits wouldbe required for numerous animals from amultitude of institutions, and this would onlyaddress the regulatory hurdles. Institutionswould have to agree to the transfers, healthchecks would have to be performed, and thecosts of the transfers would have to be budg-eted and approved. Thus, despite its logic, itseems very unlikely that an exchange such asthis could ever be carried out successfully.Additionally, further investigation may revealthat it makes more sense to manage O. sp.nov. cf. tetraspis on a global level by main-taining the two different haplogroups sepa-rately across the Atlantic – a simpler taskowing to the smaller number of individuals.

Another notable finding of our study wasa significant discrepancy in institutionalresponse rates between EAZA and AZAinstitutions, likely a result of differences inadministrative mechanisms [i.e. through theESB in EAZA versus Taxon Advisory Group(TAG)-motivated research in AZA]. Zoosand aquariums were much more responsiveto the initiative under the ESB. This is addi-tionally noteworthy considering the level ofpermitting required to send specimens ofCITES-listed taxa across borders, in somecases between institutions within Europe (butnot necessarily in the European Union) andEuropean Union member states. In additionto the lack of an Osteolaemus studbook pro-gramme, low participation rates among AZAinstitutions might indicate a reluctance on thepart of veterinary staff to capture and restraincrocodiles solely for the purpose of drawingblood for a research project. It is possible thatadministrators in AZA considered this pro-ject as independent research conducted by agraduate student, rather than an offer to iden-tify their crocodile taxa, and did not feelcompelled to participate; even though theirparticipation had been officially requested bythe TAG. The results of this investigationclearly indicate the necessity of genetic sam-pling for the appropriate taxonomic and exsitu conservation management of thesespecies in captivity. As the results of ourstudy suggest the need for proper species



identification and pedigree analysis, westrongly recommend that institutions housingunsampled individuals should refrain frombreeding until the taxonomic identities ofthese specimens are determined. Ultimately,such institutions will be required to sponsortheir own genetic analyses in order to partici-pate in future studbooks or other coordinatedbreeding programmes, which heightens therisk that potentially important breedinganimals in the AZA population will beexcluded from the breeding programme.

ACKNOWLEDGEMENTSWe thank all the zoos and museums that providedsamples to be analysed. F.S. and F.A.F. thank S. Martinfor facilitating sampling of French individuals. F.S. andF.A.F. thank M. Schlegel for providing laboratory facil-ities and A.-M. Geißler for laboratory assistance.M.H.S., K.A.V. and V.L.V. thank the Riverbanks Zoo andGardens, Colombia, SC, USA, for funding this work andJ. Austin for mentoring the laboratory work.

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Manuscript submitted 19 May 2014; revised1 September 2014; accepted 28 October 2014

SUPPORTING INFORMATIONAdditional Supporting Information may befound in the online version of this article atthe publisher’s web-site:

Appendix S1. List of 34 African dwarfcrocodiles Osteolaemus spp from Europeanzoo and aquarium collections that werenewly sequenced for this study.A 565 bp fragment of the mitochondrialcytochrome c oxidase subunit I (COI) anda 642 bp fragment of the nuclear lactatedehydrogenase A (LDH-A) were sequenced.ESB, European studbook; species, Osteo-laemus sp. nov. cf. tetraspis, Osteolaemustetraspis, Osteolaemus tetraspis × Osteo-laemus sp. nov. cf. tetraspis (hybrids).



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