african elephant genetics: enigmas and anomalies

Journal of Genetics (2019) 98:83 © Indian Academy of Scienceshttps://doi.org/10.1007/s12041-019-1125-y

PERSPECTIVES

African elephant genetics: enigmas and anomalies†

ALFRED L. ROCA1,2∗

1Department of Animal Sciences, and 2Carl R. Woese Institute for Genomic Biology, University of Illinois atUrbana-Champaign, Urbana, IL 61801, USA*E-mail: [email protected].

Received 5 November 2018; revised 28 March 2019; accepted 27 May 2019

Keywords. elephants; mito-nuclear; subspecies; effective population size; glacial refugia.

During the last two decades, our understanding of thegenetics of African elephant populations has greatly increas-ed. Strong evidence, both morphological and genetic, sup-ports recognition of two African elephant species: thesavanna elephant (Loxodonta africana) and the forest ele-phant (L. cyclotis). Among elephantids, phylogeographicpatterns for mitochondrial DNA are highly incongruent withthose detected using nuclear DNA markers, and this incon-gruence is almost certainly due to strongly male-biased geneflow in elephants. As our understanding of elephant popula-tion genetics has grown, a number of observations may beconsidered enigmatic or anomalous. Here, several of theseare discussed. (i) There are a number of within-species mor-phological differences purported to exist among elephantsin different geographic regions, which would be difficult toreconcile with the low genetic differentiation among popula-tions. (ii) Forest elephants have a higher effective populationsize than savanna elephants, with nuclear genetic markersmuch more diverse in the forest elephants than savanna ele-phants, yet this finding would need to be reconciled with thelife history of the two species. (iii) The savanna and forestelephants hybridize and produce fertile offspring, yet full-genome analysis of individuals distant from the hybrid zonesuggests that gene flow has been effectively sterilized for atleast ∼500,000 years. (iv) There are unexplored potentialramifications of the unusual mito–nuclear patterns amongelephants. These questions are considered in light of highmale and low female dispersal in elephants, higher varianceof reproductive success among males than females, and ofhabitat changes driven by glacial cycles and human activity.

†This is one of the articles of collections on ’Conservation Genetics’.

Introduction

Morphological analyses of skull dimensions of African ele-phants from widespread locations in Africa have revealedcomplete separation morphologically between forest andsavanna elephants, with a few intermediaries primarilyin habitat transition zones (Groves and Grubb 2000a, b;Grubb et al. 2000). Various nuclear genetic studies havealso reported that forest and savanna elephants are genet-ically distinct and deeply divergent (Roca et al. 2001;Comstock et al. 2002; Rohland et al. 2010; Palkopoulouet al. 2018), with a limited degree of ongoing hybridizationbetween the two species (Roca et al. 2001; Comstock et al.2002; Mondol et al. 2015). Genetic polymorphisms includ-ing indels (insertion-deletion variants) that are commonin one species may be completely or almost completelyabsent from the other, further demonstrating the lack ofnuclear gene flow between forest and savanna elephants(figure 1) (Roca et al. 2001). Hybrid elephants near the for-est edge carry genetic markers typical of both species, yetthe genetic variants in ones species do not introgress intothe other species (figure 1) (Roca et al. 2001, 2005), empha-sizing the genetic isolation of savanna elephants fromforest elephants, and strongly supporting their status asdistinct species (Mayr 1942, 1963, 1969; Roca et al. 2001,2005, 2007). This separation is also supported by differ-ences in behaviour and life history traits (Grubb et al. 2000;Turkalo et al. 2017, 2018). Further, millions of years ofgenetic divergence separate forest and savanna elephants, aseparation almost as deep as that between Asian elephants(Elephas maximus) and woolly mammoths (Mammuthusprimigenius) (Rohland et al.2010; Palkopoulou et al.2018).

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83 of 12 Alfred L. Roca

Figure 1. Differences in the phylogeographic patterns of nuclear and mitochondrial markers among forest and savanna elephants. Setsof pie charts indicates for each locality the frequencies of species-typical genetic markers including: maternally-inherited mtDNA (left),paternally-inherited Y chromosome (right) or biparentally-inherited markers (centre). In each pie chart, the green colour indicatesmarkers within clades typical of forest elephants (including mitochondrial ‘F clade’), while the blue colour indicates markers withinclades typical of savanna elephants (including mitochondrial ‘S clade’). Total indicate the number of individuals (for mtDNA, Ychromosomes) or combined number of chromosome segments (biparentally inherited markers) examined. At locales with an asterisk,the number of forest-typical and savanna-typical markers was significantly different (P < 0.05) between mtDNA and biparentallyinherited markers. Inset beneath the map are the expected pattern for a recent hybrid (left), while the term ‘conplastic’ is moretypically used for laboratory mice in which mtDNA from one strain is bred into a second strain by backcrossing hybrid females tomales only from the second strain (Yu et al. 2009). This pattern is analogous to those found in localities in which savanna elephantscarry mtDNA derived from forest elephants, but show no evidence of forest elephant nuclear gene introgression, and implies a similarpattern of backcrossing. The biparentally inherited markers are three unlinked X-chromosome intronic segments; similar patterns arepresent in diploid autosomal markers (Roca et al. 2001; Ishida et al. 2011, 2013; Mondol et al. 2015). Forest localities: DS, DzangaSangha; LO, Lope; OD, Odzala. Savanna localities: AB, Aberdares; AM, Amboseli; BE, Benoue; CH, Chobe; HW, Hwange; KE,central Kenya; KR, Kruger; MA, Mashatu; MK, Mount Kenya; NA, Namibia; NG, Ngorongoro; SA, Savuti; SE, Serengeti; SW,Sengwa; TA, Tarangire; WA, Waza; ZZ, Zambezi. Garamba (GR) is in a transition zone of vegetation in Congo that includes bothhabitats and both elephant species (White 1983; Groves and Grubb 2000a, b). Dark green is tropical forest habitat; light green is theforest-savanna transition zone of vegetation (White 1983). Figure is from my own publication which permits reuse of figures withthis reference to the original publication (Roca et al. 2005).

Analyses of nuclear genomes indicate that the forestand savanna elephants have been genetically isolated forat least 500,000 years. By contrast, mtDNA patternsshow frequent introgression of forest elephant mtDNAin savanna elephant populations (figure 1) (Eggert et al.2002; Nyakaana et al. 2002; Debruyne 2005; Johnsonet al. 2007; Ishida et al. 2011, 2013). Male elephants dis-perse from their natal social group, while females do not(Douglas-Hamilton 1972; Moss 2001). Thus, unlike all theother genetic markers for which gene flow can be medi-ated by males, there is little gene flow for mtDNA dueto nondispersal of females that remain with core socialgroups (Ishida et al. 2011). The discordant mtDNA pat-terns detected in elephants are thus attributable to sexdifferences in dispersal (Roca et al. 2005, 2007; Roca and

O’Brien 2005). In many cases of interspecies hybridization,mitochondrial but not nuclear DNA crosses the speciesbarrier when females are the nondispersing sex (Petit andExcoffier 2009).

As the number of elephant population genetic studieshas increased, a number of findings have been reportedthat appear to be anomalous or enigmatic. Those thatmay be considered anomalous appear at first glance tobe inconsistent with what may be expected, especially inlight of other known aspects of elephant biology. Othersmay be considered enigmatic because they are puzzlingor difficult to explain, often because there may not beenough information to infer strong conclusions aboutthe causes or consequences of some facet of elephantgenetics. This article considers several different aspects of

African elephant genetics of 12 83

elephant genetics that may be considered to fall into thesecategories.

Regional morphological differences within Africanelephant species are purported to exist, althoughwithin-species genetic differentiation is low

There are a number of reputed geographic differences inthe morphology of forest and of savanna elephants withintheir respective species ranges. For example, the desertdwelling elephants of Namibia are said to be taller andleaner, with longer legs and larger feet than other popula-tions of savanna elephants, and they have been proposedas a distinct subspecies (Ishida et al. 2018). Several taxaof pygmy elephants have been said to inhabit the tropicalforests of Africa (Frade 1955; Groves and Grubb 2000a, b;Debruyne et al. 2003). Other regional differences have beenreported among both forest and savanna elephants, withover 20 subspecies proposed, largely based on purportedmorphological differences (Frade 1955).

Reports of geographic differences in the morphologyof different populations of African elephants are difficultto reconcile with genetic data. Among savanna elephants,male dispersal and gene flow are quite high, and this wouldtend to limit the degree of genetic differentiation amongpopulations of savanna elephants from various regionsof the continent (Sukumar 2003). FST is a commonlyused estimate of genetic substructure among populations,which ranges in value from zero (no substructure) toone. Low FST values have been estimated for savannaelephants across their range; for example, between ele-phants in eastern and southern Africa the value of FSTusing microsatellite markers has been reported as just 0.016(Comstock et al. 2002). Likewise, there is evidence fromthe distribution of indels (insertion-deletion variants) thatgene flow has been extensive among the forest elephantsacross central Africa (Roca et al. 2001, 2015). The esti-mated FST between forest elephant populations in theeastern Congolian forest block and those in the westernCongolian forest block is just 0.035 (Ishida et al. 2018),and populations across the central African rainforest donot display great genetic distinctiveness (figure 2) (Ishidaet al. 2018). These low estimates for limited geographic dif-ferentiation within each species are supported by variousstudies using different types of nuclear DNA data (Rocaet al. 2001; Ishida et al. 2011; Ahlering et al. 2012).

A number of hypotheses may be proposed to explainthe purported regional morphological differences amongelephants. The first would be that many of the reputedregional differences are anecdotal and may not stand upto scrutiny. Many subspecies of African elephants wereproposed based on the features of a single specimen, andmay represent individual variation that was taken to bemeaningful when typological considerations dominatedtaxonomy. For the desert dwelling elephants in Namibia,

Figure 2. Genetic differentiation is low within forest and withinsavanna elephants. Forest and savanna elephants are distinctspecies, isolated from nuclear gene flow for at least ∼500,000years (Palkopoulou et al. 2018). By contrast, within each species,high levels of gene flow limits genetic differentiation among pop-ulations. (a) Savanna elephant are reputed to show geographicvariation in morphology. For example, the Namibian desertelephant is said to be taller and leaner, with longer legs andlarger feet than other savanna elephants, and they have beenproposed as a distinct subspecies (Ishida et al. 2018). However,genetic analyses comparing desert elephants to other elephantswithin Namibia found no evidence for genetic differentiationusing either microsatellites (e.g. the principal co-ordinate analy-sis shown) or mitochondrial DNA sequences (not shown) (Ishidaet al. 2016). (b) For forest elephants, genetic differentiation is alsoquite limited across central Africa. Analysis of microsatellitesusing a Bayesian clustering approach almost completely parti-tioned forest from savanna elephants (not shown). When onlyforest elephants were analysed, an incomplete pattern of parti-tioning was present between eastern and western localities withinthe Congolian forest block (Ishida et al. 2018), consistent withlow levels of genetic structure and high levels of gene flow acrossthe tropical forests of central Africa (Roca et al. 2001). Bothimages are reproduced under the terms of the Creative Com-mons Attribution License; panel A is from (Ishida et al. 2016);panel B is from (Ishida et al. 2018).

which are said to differ in morphology from other savannaelephants, no genetic differentiation was found when theywere compared with other Namibian savanna elephants(figure 2) (Ishida et al. 2016). In a study that measuredthe shoulder heights of savanna elephants using a digitalphotogrammetric method (Shrader et al. 2006), savannaelephants were found to attain similar asymptotic shoulderheights across the 10 populations (Shrader et al. 2006). For


a population in eastern Africa and another in southernAfrica for which the age of individuals was known, ele-phants in both localities were found to grow at the samerate (Shrader et al. 2006).

Another possible explanation for reported difference inmorphology is that age structure among different pop-ulations may vary, especially in areas where elephantshave been heavily hunted for the ivory trade, and puta-tive morphological differences may reflect this (Joneset al. 2018). As an cautionary note, some specimensassigned as belonging to proposed taxa of pygmy ele-phants turned out to be young specimens of the forestelephant once their skulls were examined and comparedto those of other L. cyclotis and the age of the indi-viduals was estimated using their molar eruption stage(Groves and Grubb 2000a, b). Finally, if morphologicalfeatures can be shown to vary geographically within theforest elephant or within the savanna elephant species, itis possible that environmental factors such as nutrition orpathogens may play a role. These factors may differ region-ally and may have an influence on elephant growth rates orpatterns.

Are west and central African forest elephantsgenetically distinctive?

There may be one major exception to the lack of dif-ferentiation within African elephant species. The densetropical humid forest of west and central Africa, whichcomprise most of the home range of the forest elephant,is fragmented in two by the Benin (or Dahomey) Gap, aca. 200 km wide corridor dominated by open vegetation(Demenou et al. 2018). The Benin gap may have causedthe isolation of forest elephant populations west and eastof the Gap, perhaps sufficiently to allow them to differen-tiate genetically. Genetic differentiation between west andcentral African forest elephants would be of interest forconservation management, since in west Africa numbersof elephant are quite low with their range very fragmented,more so than in other African regions (Thouless et al.2016).

Although the possibility needs to be better established,some genetic analyses have suggested that forest elephantsin west Africa have a different evolutionary history thanthose of central Africa (Eggert et al. 2002; Ishida et al.2018; Palkopoulou et al. 2018). Any genetic differencesbetween forest elephants in the two regions may havebeen augmented by hybridization of west (but not cen-tral) African forest elephants withPaleoloxodon, an extinctAfrican elephantid, as suggested by genomic comparisons(Meyer et al. 2017; Palkopoulou et al. 2018). There havealso been assertions that forest elephants in the two for-est blocks may be somewhat different morphologically(Bosman and Hall-Martin 1989).

By contrast, genetic similarity between west and centralAfrican forest elephants has been emphasized by otherstudies (Debruyne et al. 2003; Capelli et al. 2006; Ishidaet al. 2011). The forest elephants of west and centralAfrica are genetically similar enough for genetic methodsthat estimate provenance to have cross-assigned elephantsbetween the two regions (Ishida et al. 2011). By con-trast, such misassignment never occurs between forestand savanna elephants when similar methods are used(Wasser et al. 2004). Thus, west African forest elephantsare unlikely to represent a distinct species from forestelephants in central Africa, even should forest elephantsbetween the two regions be genetically differentiated.Overall, the taxonomic status of forest elephants in westAfrica remains something of an enigma (Groves 2000;Roca et al. 2015; Ishida et al. 2018).

Other points are worth noting when discussing westAfrican elephants. First, there is no support for the claimthat forest and savanna habitats across west Africa containa single elephant morphological type that is intermediatebetween the morphology of the forest elephant and thatof the savanna elephant (Roca et al. 2015). This assertion(Johnson et al. 2007) appears to have been based on abook chapter by Fernando Frade (Frade 1955). Frade inhis chapter shows a map reporting the subspecies desig-nations of elephant museum specimens collected acrossAfrica (Frade 1955; Roca et al. 2015). The map listed westAfrican specimens as unknown, i.e. not assigned by anyprevious authors to a putative subspecies. This map mayhave been misunderstood to suggest that elephants in westAfrica had been judged to be of unknown or interme-diate morphology between forest and savanna elephants.This would be a misinterpretation of Frade’s work, whichunequivocally states that Africa’s elephants fall into onlytwo distinct species, with a range extending across westAfrica for each of the two species (Frade 1934, 1955; Rocaet al. 2015). More recently, analyses of the full genomesequences of forest and savanna elephants found no evi-dence of admixture between the two species in the genomeof a west African forest elephant (Palkopoulou et al. 2018).

A final point is that, as elephant populations are iso-lated, they would be increasingly subject to the effects ofgenetic drift and inbreeding. This could lead to geneticand morphological differences among the isolated pop-ulations, especially among forest elephants, as the highlevel of forest elephant genetic diversity would enablegreater differentiation among populations following iso-lation. The elephants of west Africa may be more greatlyaffected, because habitat fragmentation and decimationof elephant populations occurred earlier there and to agreater degree than in other regions (Roth and Douglas-Hamilton 1991; Barnes 1999). In west Africa, the range ofthe forest elephant currently extends into savanna habitats(Groves 2000; Mondol et al. 2015) (although it is not clearif this was true historically), and the degree of hybridiza-tion between forest and savanna elephant species appears


to be somewhat greater than in other regions (Roca et al.2005). Increased opportunities for interspecies hybridiza-tion could also potentially enable greater differentiationafter isolation.

The higher nuclear genetic polymorphism in forestthan in savanna elephants would need to be reconciledwith their life histories

Genetic diversity has been examined within the twoAfrican elephant species using nuclear sequences andmicrosatellite genotypes (Roca et al. 2001, 2005; Com-stock et al. 2002; Rohland et al. 2010; Ishida et al. 2011;Palkopoulou et al. 2018). Nuclear genetic diversity washigher among forest elephants than among savanna ele-phants in all of these studies, indicating that the two specieshave different patterns in the generation or loss of geneticdiversity (Roca et al. 2015). Further, the higher nucleargenetic diversity in forest elephants is evident even in stud-ies that included a larger number of savanna elephantsfrom a broader geographic range when compared with theforest elephants.

By contrast, a higher effective population size (Ne) hasbeen estimated for the forest elephant than for the savannaelephant (figure 3) (Rohland et al. 2010). The higher Neamong forest elephants would be need to be consideredin light of a recently conducted study of life history andreproductive patterns in the forest elephant (Turkalo et al.2017, 2018). Forest elephants were found to have a later ageof first calving, and a much longer inter-calving intervalthan had been reported for various savanna populations(Turkalo et al. 2017, 2018). When r/K selection theory isconsidered, K -strategists tend to have relatively reducedeffective population sizes relative to r-strategists (Romigu-ier et al. 2014). Although all elephant species would be con-sidered to be K -selected, an older age of first calving andgreater intercalving interval among forest elephants wouldbe need to be reconciled with the larger Ne and highernuclear genetic diversity among forest elephants thansavanna elephants (Roca et al. 2001, 2005; Comstock et al.2002; Rohland et al. 2010; Ishida et al. 2011; Palkopoulouet al. 2018). At least three hypotheses may be reasonable.

Forest elephant range likely retreated into tropical forestrefugia during glacial cycles (Ishida et al. 2018). Thus onepossibility may be that high diversity of forest elephantsmay in part be due to the long-term isolation of pop-ulations, which can preserve regional genetic differences(Ishida et al. 2018). The overall diversity of the speciescould then have increased once the populations becamecontiguous and panmictic, due to reversal of the Wahlundeffect (Ishida et al. 2018). Under this hypothesis, the forestelephant has a temporarily heightened genetic diversity,which would be gradually reduced by drift (Ishida et al.2018).

A second hypothesis is that the variance in reproductivesuccess in male forest elephants may be lower than thatof savanna elephants. Among savanna elephants there is ahigh degree of male–male competition, with older largermales having much greater reproductive success thanyounger smaller males (Slotow et al. 2000; Hollister-Smithet al. 2007; Rasmussen et al. 2008). Because many malesdo not reproduce, their genetic diversity is lost to the nextgeneration, reducing the effective population size relativeto census size. Under this alternative hypothesis, forestelephant males may have less variance in reproductivesuccess, with a larger proportion of males contributingto the next generation than is the case for savanna ele-phants. The genetic contribution to the next generationby a larger proportion of forest elephant males wouldtend to increase Ne relative to that of the savanna ele-phant for a given census size. If this hypothesis was true,it would be unclear whether innate social behaviours (e.g.reduced male–male competition, or differences in femalechoice) or environmental factors (e.g. different reproduc-tive patterns due to the dense forest habitats) would beresponsible.

A third hypothesis is based on the fossil record ofproboscideans. For most of the Pliocene and PleistoceneEpochs, taxa assigned to the genusPaleoloxodon (althoughsometimes assigned to another genus) predominated inthe savannas of Africa (Maglio 1973; Kingdon 1979;Sanders et al. 2010; Palkopoulou et al. 2018). The mostrecent of these taxa, Paleoloxodon (recki) iolensis, dis-appears from the fossil record towards the end of thePleistocene (Sanders et al. 2010), after which the extantsavanna elephant is believed to have expanded in range(Kingdon 1979). If the current population of Loxodontaafricana derives from a smaller founding population,then the lower Ne of this species may at least in partreflect a smaller ancestral population size before theextinction of Paleoloxodon (Kingdon 1979) (Roca et al.2001).

One aspect of the high diversity of nuclear geneticsequences among forest elephants is that when sequencesare aligned, a high number of indels have been detectedamong forest elephants, relative to the number of indels insavanna elephants (Roca et al. 2001, 2005). Among forestelephants known to be male that are Sanger sequenced,no polymorphisms are detected, since males would onlycarry one X chromosome. On the other hand, polymor-phisms are often detected among female forest elephants,including many indels, since females carry two X chromo-somes. Small indels of the type detected in forest elephantsequence alignments have been attributed to replicationslippage, recombination, unequal crossing over, or imper-fect repair of double strand breaks (Messer and Arndt2007). Several forest elephant indels are found across cen-tral African forest populations, and thus have been subjectto gene flow across the east–west expanse of the Con-golian forest block (Roca et al. 2001, 2005, 2015). The


Figure 3. Relationships, genetic diversity and gene flow among proboscidean species. The black horizontal arrows represent geneflow inferred from the genomes of living and extinct species, with arrow thickness corresponding to inferred gene flow (Palkopoulouet al. 2018). Note that although gene flow has been detected across elephant species and genera, there has been no detectable geneflow between the extant forest and savanna African elephant species for ∼500,000 years (Palkopoulou et al. 2018). The tree depictsmore ancient relationships that can be inferred despite subsequent gene flow (Meyer et al. 2017; Palkopoulou et al. 2018). Theta (θ ),a gauge of effective population size and genetic diversity, is indicated for the three living species (Rohland et al. 2010). Note thatforest elephant nuclear genetic diversity is more than three times higher than that of the savanna elephant. Branch lengths and splitsare not drawn to scale. The dotted line segments and shaded areas represent limited periods of gene flow between incipient species(Palkopoulou et al. 2018). The labels consisting of N and subscripts refer to common ancestors at internal nodes are not relevant tothis review. Values of θ are from Rohland et al. (2010), while the tree figure is from (Palkopoulou et al. 2018) and may be reproducedin a review article (http://www.pnas.org/page/about/rights-permissions) provided that this full journal reference is cited (Palkopoulouet al. 2018).

high number of indels is consistent with the higher Nedetected for forest elephants than for other elephantids(Rohland et al. 2010). The high number of indels maysuggest that the forest elephant species is a combina-tion of different ancient lineages that evolved separatelyfor long periods of time, before combining into a sin-gle population. If the indels had previously occurredin locally restricted forest refugia, sufficient time hasoccurred since the central African forests became con-tiguous for gene flow to carry the indels across thisregion.

One may also consider that the high current geneticdiversity of forest elephants, and the detection of highnumbers of indels would be more likely if ancient iso-lated populations had similar sizes at the point that theybecame contiguous due to expansion of forest habitats.Equally large contributions from different refugia wouldhave helped to equalize the frequencies of genetic vari-ants after the forests became contiguous, fostering thepersistence of nuclear genetic diversity and increasing theamount of time required for the loss of alleles to drift.Such a hypothesis would, of course, be quite specula-tive.

There is a dearth of nuclear gene flow even in thepresence of a hybrid zone between forest and savannaelephants

As noted above, elephant mtDNA phylogeographic pat-terns do not conform to those revealed using nuclearmarkers, due to highly sex-biased dispersal and gene flow.The mito–nuclear incongruence detected between Africanelephant species is consistent with patterns detected inother cases of interspecies hybridization involving taxain which females are the nondispersing sex (Petit andExcoffier 2009). The discordant mtDNA and nucleargenetic patterns have unfortunately been a source of con-fusion in genetic studies, some of which have wronglyconcluded that mtDNA results are novel or contradictoryto other studies that relied on nuclear DNA markers (Rocaet al. 2007; Ishida et al. 2011).

Phylogenies based on mtDNA have detected two lin-eages among African elephants, designated the ‘S’ and ‘F’clades (Debruyne 2005), with an estimated divergence of5.5 Mya (Brandt et al. 2012). The ‘S’ (or ‘savanna’) clademtDNA haplotypes are only carried by savanna elephants,whereas no forest elephant in the deep tropical forest


has been found to carry a haplotype within the S clade(figure 1) (Ishida et al. 2011). By contrast, F (or ‘forest-derived’) clade mtDNA haplotypes are carried by all forestelephants, and for that reason the clade is believed tooriginate in forest elephant populations (figure 1) (Ishidaet al. 2011). Yet F clade mtDNA has regularly introgressedinto savanna elephant populations. Because many savannaelephant populations across eastern and southern Africacarry F clade mtDNA but do not show evidence of nucleargenes originating in forest elephants (figure 1), a process oflargely unidirectional hybridization and backcrossing offorest and hybrid females to savanna males can be inferred(figure 4) (Roca et al. 2005, 2007; Ishida et al. 2011, 2013).

The genetic patterns present in African elephant species,in which mitochondrial but not nuclear introgressionbetween species can be detected (Cahill et al. 2013; Liet al. 2016), are somewhat analogous to those in ‘conplas-tic’ inbred mouse strains (Boyse 1977; Yu et al. 2009). A‘conplastic’ mouse is one in which the mtDNA is the onlylocus transferred from one strain to a second strain. A con-plastic mouse is generated after a female from one strain ishybridized to a male from a second strain. In subsequentgenerations, female offspring are always backcrossed tomales of the second strain. This gradually converts thenuclear genetic background of the mice to that of the sec-ond strain, but the mice retain the mtDNA present inthe original female of the first strain (Yu et al. 2009). Ananalogous process appears to have occurred in the case ofelephants and other species in which the mtDNA appearsto derive from one species or lineage, but the nuclear mark-ers no longer show evidence of hybridization (figure 1)(Roca et al. 2007; Cahill et al. 2013; Li et al. 2016).

In many savanna elephant populations, the numbers ofindividuals examined for mtDNA and nuclear sequencesis quite high (figure 1) (Roca et al. 2005). For example,across eastern and southern Africa, savanna elephantsat 15 localities have been sequenced for the presenceof F clade (forest-elephant-derived) mtDNA or S-clademtDNA (present only among savanna elephants), andwere also sequenced for three X-linked gene segments(BGN, PHKA2 and PLP) that distinguish between for-est and savanna elephants. Considering the nuclear genesegments, among eastern and southern African savannaelephant populations there was not a single nuclearsequence of forest elephant origin among 1623 nucleargene segments sequenced, even though 38 of 214 ele-phants carried F clade (forest-derived) mtDNA (figure 1)(Roca et al. 2005). For the populations that carried Fclade mtDNA, the difference in forest elephant-derivedvs savanna elephant-derived mtDNA and forest elephant-derived vs savanna elephant-derived nuclear sequences wasvery strongly supported statistically (figure 1), and the pat-tern is inconsistent with random mating (Roca et al. 2005).

Of course, there are differences to consider when com-paring the analogous mito–nuclear patterns detected insavanna elephants to those in conplastic mice. One is that

laboratory mouse strains undergo deliberate inbreeding,unlike the elephants. A second, and critical difference isthat conplastic mice are generated through human iso-lation of strains in the laboratory and manipulation ofmating patterns. In the case of elephants, the inferredpatterns of mating would require a natural explanation.The two species are parapatric, with ranges overlappingat the edges of the Congolian forest block where vegeta-tion zones change from tropical forest to savanna habitats(figure 1) (Roca et al. 2005). Thus geographic isolationcannot be used to explain why the two types of elephantsshould remain genetically distinctive (Grubb et al. 2000;Roca et al. 2015; Stoffel et al. 2015). Because the twoAfrican elephant types are parapatric and appear to readilyhybridize where their habitats overlap, the absence of geneflow between the forest and savanna elephants for at leastthe last ∼500,000 years (figure 3) (Palkopoulou et al. 2018)requires an explanation that would make sense in light ofthe widespread hybridization. A number of observationsmay be considered when framing potential explanations.

First, because mitochondrial gene flow from forestelephants into savanna elephant populations is readilydetected (figure 1) (Roca et al. 2005; Lei et al. 2008, 2009),female elephant hybrids are successfully passing on theirmtDNA, i.e. successfully reproducing. Thus a consider-able female–male difference in hybrid reproductive successwould be critical for understanding the discrepant mito–nuclear pattern in savanna elephants (figure 4) (Roca et al.2007). As in the case of conplastic mice, the mito–nuclearpattern detected in savanna elephants requires not onlyhybridization between forest and savanna elephants, butspecifically requires for female forest elephants to havehybridized with savanna males. That backcrossing had tooccur repeatedly between hybrid females and nonhybridsavanna males is supported by the absence of detectablelevels of forest elephant nuclear alleles in these savannaelephant populations (figure 1) (Roca et al. 2005; Leiet al. 2009). To have removed the forest elephant nuclearcontribution from populations with high frequencies offorest-elephant-derived F-clade mtDNA, generations ofrepeated backcrossing to savanna elephant males can beinferred, since this would be necessary to dilute out theforest elephant contribution from the nuclear gene pool(figure 4) (Roca et al. 2007).

Second, at least some male hybrids between forestand savanna elephants are not physiologically sterile. Atleast one individual hybrid has been shown to carry aY-chromosome sequence from a forest elephant, an indi-cation that the forest elephant contributed the paternal lin-eage for this individual (Roca et al.2005). Additionally, ele-phants in some parts of the hybrid zone have been reportedto show evidence of hybridization between the two speciesin both directions (Mondol et al. 2015). However, thiswould not detract from the very strong statistical supportshowing many populations carry forest elephant-derivedmtDNA but do not carry forest elephant-derived nuclear


Figure 4. Hybridization and backcrossing between the twospecies of African elephant. In this schema, the forest ele-phant component of the nuclear genome would be diluted andcompletely replaced in herds that retained residual maternallyinherited forest-typical mtDNA haplotypes (Roca et al. 2005;Roca and O’Brien 2005). This pattern of backcrossing can beinferred from mito-nuclear patterns in some savanna elephantpopulations that carry forest elephant-derived mtDNA but donot carry traces of forest elephant nuclear DNA (figure 1) (Rocaet al. 2005). This pattern is also supported when one consid-ers the distribution of morphological types and nuclear geneticpatterns among African elephants, and compares them to the pat-terns seen for mitochondrial DNA (figure 1) (Groves and Grubb2000a, b; Roca et al. 2001, 2005, 2007; Debruyne 2005; Ishidaet al. 2011, 2013). Where savanna and forest elephant rangesmeet, hybridization occurs (Groves and Grubb 2000a, b; Rocaet al. 2001; Mondol et al. 2015). Given that reproductive successamong male elephants depends largely on body size (Slotow et al.2000; Hollister-Smith et al. 2007; Rasmussen et al. 2008), and thatthe deleterious effects of hybridization may differentially harmmale hybrids (Haldane 1922), large savanna males are likely tohave greater reproductive success than smaller forest or hybridmales. However, other species isolation mechanisms (Coyne andOrr 2004) may be involved.

sequences (figure 1) (Roca et al. 2005). Thus, even in theabsence of physiological sterility, the reproductive successof male hybrids across many generations is sufficientlyreduced relative to nonhybrid males that they effectivelydo not contribute to the gene pool of savanna elephants inthe long term (Roca et al. 2007; Palkopoulou et al. 2018).

What causes a relative lack of reproductive successamong hybrid forest-savanna male elephants? One argu-ment has been that male–male competition may be in partresponsible (Roca et al. 2005, 2007). Reproductive successamong savanna elephant males is known to depend onbody size and age, mediated by periods of musth in whichtestosterone increases and males become more aggres-sive towards other males (Poole 1989; Slotow et al. 2000;Hollister-Smith et al. 2007; Rasmussen et al. 2008). Fully-grown forest elephant males are only half as massive asfully-grown savanna elephant males (Groves et al. 1993;

Groves and Grubb 2000a, b). Although, the role of bodysize in reproductive success in hybrid zones has not beenexamined, it is plausible that the difference in body sizebetween male savanna elephants and forest- or hybrid- ele-phant males may play a role in the backcrossing of hybridfemales to unhybridized savanna males (figure 4) (Rocaet al. 2005, 2007).

However, it would take many generations for the genepool of hybrid elephants to be replaced by backcrossingonly to savanna elephant males. In the production of con-plastic strains of mice, typically 10 generations have beenused to ensure conversion of the gene pool of hybrid miceto one that matches just with one of the parental strains(Markel et al.1997). Yet in elephants, before 10 generationsof backcrossing of hybrid females to savanna males haveoccurred, it would seem plausible that some hybrid maleswould begin to approach nonhybrid savanna elephantmales in body size, and thus, that some level of forestelephant nuclear contribution would be detectable in pop-ulations of savanna elephants that carry F-clade mtDNA.The absence of detectable levels of forest elephant contri-bution to these herds would suggest that perhaps otherspecies-isolation mechanisms (Coyne and Orr 2004) maybe involved in keeping the two elephant species distinct,i.e., in precluding reproductive success in hybrid males evenafter generations of backcrossing.

Finally, there has been no genetic introgression detectedat all from savanna elephants into forest elephants otherthan at the edge of their range. Even though many savannaelephant populations carry F-clade mtDNA derived fromforest elephants, no S-clade mtDNA has ever been detectedin populations deep within the tropical forest, even afterextensive sampling and mtDNA sequencing of forestelephants across central and west Africa (Ishida et al.2011, 2013). In a geographically more limited examina-tion of nuclear gene sequences among forest elephants,savanna elephant-typical nuclear gene sequences have notbeen detected among elephants in the deep tropical for-est (figure 1), although both forest and savanna elephantnuclear (and mtDNA) sequences are present in the hybridzone of Garamba (Roca et al. 2001, 2005), where bothtypes of elephants are found (Groves and Grubb 2000b).The lack of nuclear introgression from savanna elephantsto populations in the tropical forest for at least ∼500,000years (figure 3) (Palkopoulou et al. 2018) suggests that geneflow of any type from savanna elephants to forest elephantsis effectively sterilized.

Thus, additional species-isolation mechanisms would beneeded to explain why savanna elephant nuclear gene flowinto forest elephant populations, deep in the tropical foresthas never been detected, and a genomic level appears tobe have not occurred for at least ∼500,000 years (figure 3)(Palkopoulou et al. 2018). Since hybrid males would pre-sumably be larger than forest elephant males, factors otherthan body size must play a role in precluding introgressionof savanna elephant nuclear or mtDNA into the forest


elephant gene pool. Such factors would also help explainthe inferred lack of reproductive success of hybrid malesin the savannas. It would be speculative to discuss whichspecies isolation mechanisms (Coyne and Orr 2004) mayplay a role African elephants. There is no evidence for anyparticular mechanism in elephants. But some factor bey-ond body size may play a role in maintaining the speciesbarrier to nuclear gene flow from forest to savanna ele-phants, or to any gene flow in the reverse direction, evenin the presence of a hybrid zone.

Do the very different phylogeographic patterns fornuclear and mitochondrial DNA in elephantids havean effect on cellular mitochondrial function?

A final consideration regards the potential effect ofthe quite different phylogeographic patterns for mtDNAand nuclear markers among African elephants (figure 1)and elephantids in general (Ishida et al. 2011; Roca2015; Meyer et al. 2017). While in forest elephants,nuclear genetic diversity is quite high, this diversity is notgeographically structured, at least across central Africa(figure 2) (Ishida et al. 2018). Also, nuclear gene flow ishigh between the eastern and western ends of the Congo-lian forest block, given that populations carry the sameset of indels (Roca et al. 2001, 2005, 2015). By contrast,while all forest elephants carry mtDNA haplotypes that arepart of the ‘F clade,’ this clade is further subdivided intofive distinct ‘subclades,’ each with a geographically limiteddistribution within the range of the forest elephant (Ishidaet al. 2013). For example, the ‘western subclade’ is foundonly in west Africa, in the Guinean forest block, and hasnot been detected in central African populations (Eggertet al. 2002; Ishida et al. 2013). The west-central subcladeis found both in west Africa, and also in the western partof the Congolian forest block. The three other subclades(designated north-central, east-central and south-central)tend to be found in greater frequency within the Congolianforest block in the ordinal direction indicated by their des-ignation, with all three subclades completely absent fromwest Africa (Ishida et al. 2013). The pattern for mtDNAmay reflect the isolation of forest elephant populations inforest refugia during glacial cycles (Ishida et al. 2018). Thepattern would also reflect limited gene flow of females,which would allow the local formation and local persis-tence of mtDNA haplotypes while preventing the longdistance movement of mtDNA haplotypes across forestregions (Ishida et al. 2018). By contrast, gene flow dueto dispersal of males from natal social groups for genera-tion after generation would have mediated the transfer ofnuclear markers across the length of the central Africanforest (Ishida et al. 2018).

Savanna elephants also display differences betweenmitochondrial and nuclear phylogeographic patterns(figure 1) (Roca et al. 2005; Ishida et al. 2011; de Flamingh

et al. 2018). Nuclear haplotypes that are completely absentamong forest elephants predominate among savanna ele-phants south, east and north of the tropical forest (figure 1)(Roca et al. 2001, 2005). A high level of nuclear gene flowis evident from the low genetic differentiation detectedamong savanna elephant populations across these regionsof Africa (Comstock et al. 2002). By contrast, mtDNA dis-plays a very different phylogeographic pattern in savannaelephants. The S clade, which is found only amongsavanna elephants, is further subdividided into three well-supported subclades (Ishida et al. 2011, 2013). While the‘savanna-wide’clade is found among savanna elephantsacross most of their range, the northern-central savannasubclade is found across north-central African Sudaniansavanna belts into east Africa, while haplotypes within thesoutheast savanna clade are carried by elephants in south-ern and part of eastern Africa (Ishida et al. 2013). ThemtDNA phylogeography of savanna elephants is furthercomplicated by the introgression of forest elephant derivedF clade haplotypes into savanna populations. Savanna ele-phants carry haplotypes that resemble (or are identical to)the haplotypes carried by forest elephants to which theyare geographically proximate (Johnson et al. 2007; Ishidaet al. 2011, 2013); thus savanna elephants in northern Tan-zania (which otherwise show no evidence of hybrid origin)tend to carry haplotypes within the east-central subcladeof the F clade, which is common in forest elephants of theeastern Congolian forest (figure 1) (Ishida et al. 2013).

It may be possible for these quite distinctive nuclearand mitochondrial patterns to have consequences for thefitness of elephant populations. Proteins encoded by themitogenome form complexes that are involved in cellu-lar metabolism. The proteins encoded by the mitogenomeform these complexes with proteins encoded by thenuclear genome. One may consider whether high levelsof nuclear gene flow and lack of geographic structureamong nuclear genes intraspecies in elephants would affectmitochondrial function and organismal fitness, given thatnuclear-encoded proteins interact with proteins coded forby the mitochondrial genome. Variants of the mitogenomemay be geographically constrained due to limited femaledispersal, and thus may be quite distinctive locally orregionally. Three potential consequences may be worthconsidering.

First, there is likely to be a great persistence of mtDNAhaplotypes across generations, even if these haplotypesmay reduce the fitness of the organism by having a neg-ative effect on mitochondrial function. Whereas purifyingselection would tend to remove genetic variants that lowerthe fitness of organisms, such selection would have tobe mediated through a selective sweep in which the vari-ants that provide greater fitness replace those that lead tolower fitness. However, a selective sweep across the rangeof a species would require gene flow across that range(Petit and Excoffier 2009). Since mtDNA is only trans-mitted by females, and female elephants are philopatric, a


selective sweep across the range of the elephants could bethwarted by nondispersal of females. Thus it is possiblethat mitochondrial function may be negatively affected bythe mtDNA haplotypes carried by some elephants, evenif not sufficiently compromised for selection to readilyremove the haplotypes in the absence of female-mediatedmitochondrial gene flow (Petit and Excoffier 2009).

A somewhat speculative corollary may be that selec-tion could become more effective at removing haplo-types should elephants migrate across long distances.The mtDNA-encoded proteins form complexes that areinvolved in bioenergetics. The mtDNA variants thatreduce fitness may persist while females remain in a region.However, the migration of females across long distancesmay enable selection favouring females in which mito-nuclear protein complexes have not been compromised byslightly harmful mutations in the mtDNA. It is possiblethat long distance migration of females would enable selec-tion against mtDNA that carries mutations that negativelyimpact mitochondrial bioenergetics.

A final possibility is that there may come a point atwhich the level of fitness is compromised sufficiently forpurifying selection to remove the mitochondrial variantseven in a matrilocal species. For example, a mutation in anuclear gene that codes for a protein in a mitochondrialcomplex could conceivably increase fitness among males,potentially driving a selective sweep across the range of thespecies. Potentially, the novel variant could be compatiblewith proteins encoded by some haplotypes of mtDNA,but incompatible with proteins encoded by other haplo-types. If this were the case, then the selective sweep of anuclear-encoded variant could potentially lower the fit-ness of females with certain mitochondrial haplotypes tosuch a degree that their lineage may not survive. Thusin principle mutations in nuclear genes involved in mito-chondrial function could, during a selective sweep of anuclear-encoded variant, drive the disappearance of haplo-types or haplogroups of mtDNA that proved incompatiblewith the novel variant. The incompatibility could occurif their mtDNA encoded for proteins that failed to formeffective complexes with the novel nuclear-encoded pro-teins undergoing a selective sweep through male-mediateddispersal. It is notable that experiments involving the trans-fer of mitochondria from one species into cell lines ofa related species have found that the function of mito-nuclear protein complexes may be disrupted, most likelydue to the inability of nuclear and mtDNA-encoded pro-teins to properly interact (Barrientos et al. 1998; McKenzieet al. 2003).

Although, the disappearance of one of two major mito-chondrial clades in the woolly mammoth occurred longbefore the extinction of the species (Debruyne et al. 2008;Gilbert et al. 2008), there is no evidence to indicatethat mito-nuclear interactions played a role. Nonethe-less, consistent with the strongly male-biased disper-sal across elephantid taxa, discordance in mito-nuclear

phylogeographic patterns has been reported not justbetween the two species of African elephant, but alsowithin-species in savanna elephants, forest elephants andAsian elephants (Fernando et al. 2000; Fleischer et al.2001; Eggert et al. 2002; Nyakaana et al. 2002; Debruyne2005; Roca et al. 2005; Johnson et al. 2007; Lei et al. 2009;Ishida et al. 2013; de Flamingh et al. 2018); and, in analy-ses involving extinct taxa, between Columbian mammoth(Mammuthus columbi) and woolly mammoth species (Enket al. 2011; Palkopoulou et al. 2018), between Paleolox-odon and the extant forest elephant (Meyer et al. 2017;Palkopoulou et al. 2018), and among woolly mammothpopulations (Debruyne et al. 2008; Gilbert et al. 2008).Thus among elephantids, the typical phylogeographic pat-tern is for mtDNA to be discordant with morphologyor with nuclear genetic structure (Roca 2015; Roca et al.2015). Analyses of their mito-nuclear sequences and ofhow their genetic diversity may have affected mitochon-drial protein complexes may provide insights into theevolution of proboscidean lineages.

In conclusion, we have considered several enigmatic oranomalous findings detected by genetic studies of Africanelephants. These relate primarily to sex differences amongelephants, which are central to understanding elephantpopulation genetics. These sex differences distort geneticpatterns, and these genetic patterns in elephantids havebeen prone to repeated misinterpretation. One theme ofthis study is that, relative to females, male elephants havemuch greater variance in reproductive success, leading todistortions in effective population sizes among markers,away from ratios that would be expected if the sexes hadsimilar variance in reproductive success. One can speculatethat differences between the two extant African speciesin the degree of reproductive variance may potentiallyaccount for higher effective population size among for-est elephants. Sex differences in the dispersal of elephantshave strongly distorted mtDNA phylogeographic patternsrelative to the patterns inferred for any other geneticmarker (since even the X chromosome, though presentin a single copy in males, is transmitted by males acrossthe landscape, unlike mtDNA). Thus, mtDNA which isonly transmitted by females, has a phylogeographic pat-tern highly discrepant from those of other genetic markers,both within and across species. Since mtDNA is one ofthe most widely used genetic markers, the distortion ofits phylogeographic patterns relative to all other mark-ers has unfortunately led to misleading conclusions aboutpopulation genetic patterns. The discrepant mito–nuclearpatterns do allow inferences to be made regarding the roleof males and females in the genetic isolation of the twoAfrican elephant species (even in the face of hybridiza-tion), and may suggest potential ramifications for proteincomplexes involved in mitochondrial function. This studyhighlights the anomalous or enigmatic outcomes of geneticresearch, so that these may be further considered in futurestudies.


Acknowledgements

For enabling part of the primary research that forms the basisfor this study, we thank the US Fish and Wildlife Service AfricanElephant Conservation Fund, and specifically grants AFE1816-F18AP00819 and AFE1606-F16AP00909.

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