J. Avian Biol. 41: 1�11, 2010

doi: 10.1111/j.1600-048X.2009.04794.x# 2010 The Authors. J. Compilation # 2010 J. Avian Biol.

Received 30 December 2008, accepted 2 June 2009

Absence of male reproductive skew, along with high frequency ofpolyandry and conspecific brood parasitism in the lekking Houbarabustard Chlamydotis undulata undulata

Loıc Lesobre, Frederic Lacroix, Eric Le Nuz, Yves Hingrat, Toni Chalah and Michel St. Jalme

Loıc Lesobre (correspondence), Frederic Lacroix, Eric Le Nuz, Yves Hingrat, Toni Chalah, Emirates Ctr. for Wildlife Propagation, Province deBoulemane PO Box 47, 33250 Missour, Morocco. Email: llesobre@ecwp.net.ma � Michel St. Jalme, Mus. Natl. d’Histoire Nat. (MNHN),UMR 7204 MNHN-CNRS-Paris VI ‘‘Conservation des especes, restauration et suivi des populations’’, Menagerie du Jardin des Plantes, 57 rueCuvier, 75005 Paris, France.

Behavioural studies have led to the perception that lekking species experience a high male reproductive skew as aconsequence of females’ selective mate choice. In addition, observations suggest that females copulate only once andtherefore polyandry seems unlikely as females are supposed to choose the best male available. In order to analyse themating strategy of the Houbara bustard, an endangered lekking species under reinforcement in eastern Morocco, we usedmicrosatellite data to perform paternity analyses. None of our observations followed common expectations under a lekmating system: we found no male reproductive skew suggesting no apparent selective female mate choice and no apparentmale benefit from lekking. In contrast, a high level of polyandry (60 % of the nests) was recorded suggesting that spermcompetition may operate. In addition, we present another case of conspecific brood parasitism in a lekking species andthis was an unexpected alternative strategy for a species presenting high parental cost and low fecundity. The increasingnumber of studies contradicting common assumptions on lekking species suggests that alternative breeding strategies suchas males pursuing an off-lek mating strategy, female polyandry and even conspecific brood parasitism might be morewidespread in lekking species than previously thought.

Behavioural studies in lekking species have led to theperception that female choice is often relatively constrainedand that such species experience a high male reproductiveskew as a consequence of females’ selective mate choice(Bradbury and Gibson 1983, Lewin 1988). In addition,previous observations of copulations on avian leks suggestedthat females copulate only once in most species (Birkheadet al. 1987, Wiley 1991, Alatalo et al. 1996). However, thisperception may not reflect the situation in all lekking species(Lanctot et al. 1997). Females’ alternative breeding strategies,such as multiple mating, may be important. More copulationevents increase the possibility that a female can mate withhigh ranking males especially if such males not available atthe time of a given copulation event (Møller 1992, Petrieet al. 1992). In lekking species, polyandry primarily seemsunlikely because females are suppose to choose the best maleavailable (Hoglund and Alatalo 1995), but studies indicatethat females could mate with several males in species such aspeafowl Pavo cristatus (Petrie et al. 1992), buff breastedsandpiper Tryngites subruficollis (Lanctot et al. 1997) andruffs Philomachus pugnax (Lank et al. 2002). This createsthe potential for sperm competition.

In order to answer many of the basic questions on theevolution of lekking and the mechanisms of sexual selec-

tion, knowledge of true paternity among offspring isessential and molecular paternity analysis may often berequired for its confirmation (Alatalo et al. 1996). This isparticularly true in species where observations of copula-tions are rare and in leks with large inter-male distances(referred as exploded leks, Hoglund and Alatalo 1995). Inthose situations, long distances between males do notpermit simultaneous observations of all displaying males.

Answering such questions is not only important in thecontext of behavioural ecology and in the understanding ofthe evolution of mating systems, but it has implications inconservation biology, specifically for the management ofdeclining populations. Unequal distribution of matingsuccess, whenever present, will affect the breeders’ sex-ratioand contribute to a decrease of the effective population size(Crow and Kimura 1970, Blumstein 1998, Quader 2005,Stiver et al. 2008). This will negatively affect the main-tenance of both heterozygosity and allelic diversity and thatin turn will jeopardize the viability of declining populations(Lacy 1987, Ballou and Lacy 1995, Blumstein 1998,Frankham 2005, Quader 2005, Robert et al. 2007).

The evolution model through which lekking evolved in aspecies may impact the frequency of polyandry and malereproductive success (Bradbury and Gibson 1983). In the

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hotshot model, the dominance of some males creates ahighly skewed mating success between males. In thealternative hotspot model, displaying males cluster in areaswith high female density to increase their mate encounterrate (Bradbury and Gibson 1983, Bradbury et al. 1986). Inthe latter model, male reproductive skew may be less severebecause inter-male competition is reduced. Further, inter-male dominance processes, which restrict female matechoice, are also reduced (Bradbury and Gibson 1983,Foster 1983, Beehler and Foster 1988, Alatalo et al.1996). The idea of a severely unequal distribution comesmainly from studies of classical lek species, which displayshort inter-male distances (Kokko et al. 1998, Jiguet et al.2000). However, in exploded leks, inter-male distance ishigher and this may reduce inter-male competition poten-tially leading to a less severe male reproductive skew.

The Houbara bustard Chlamydotis undulata undulata isan endangered lekking species that experienced a severedecline during the past 20 years (Saint Jalme and VanHeezik 1996, Schultz and Seddon 1996, Goriup 1997,Tourenq et al. 2005). This led to the creation, in 1996, of areinforcement program in Morocco with the objective ofsupplementing wild populations with captive bred indivi-duals (Lacroix 2003). For the reasons mentioned above,every reinforcement program requires a good understandingof the species’ biology (IUCN 1998), especially its matingsystem (Blumstein 1998). Therefore, behavioural aspects ofthe Houbara bustard’s mating system have been intensivelyaddressed. Recent studies in Morocco showed that themating system conforms to the definition of an explodedlek (Hingrat et al. 2004, Hingrat and Saint Jalme 2005),which seems to be the most common mating strategyamongst bustards (Jiguet et al. 2000, Jiguet et al. 2002). Inthe Houbara bustard, lekking has evolved through thehotspot model. Females’ home ranges have been shown toencompass several displaying males from different leks(Hingrat et al. 2004, Hingrat et al. 2008), thus providingthe potential for polyandry, sperm competition and crypticfemale choice.

Analyses of spatial relationships between displayingmales and nesting females in Houbara Bustards revealedthat nests had an aggregative distribution and werepreferentially located at the periphery of larger leks (Hingratet al. 2008). Because females exhibit annual fidelity tonesting areas and tend to aggregate in the best nestinghabitats, some males could then experience the highestfemale density and attempt to locate their displaying siteas close as possible to these areas of high female density(Hingrat et al. 2008). Since females are not nesting withinleks (Fig. 1), peripheral males will then experience thehighest female density.

A settlement strategy leading males to establish at thelek’s periphery might then be successful, especially for malesaiming at fathering replacement clutches. Moreover, a largenumber (close to 30 %) of displaying males are not locatedwithin leks (Hingrat et al. 2008), and this might be theconsequence of an off-lek mating strategy (Oring 1982,Rubenstein 1982, Lank and Smith 1987, Laska et al. 1992,Lanctot et al. 1997, Semple et al. 2001). We thereforetested whether or not there were alternative strategies in theHoubara bustard mating system. We used moleculartechniques to study both maternity and paternity, to

evaluate the occurrence of multiple paternities, and toassess the extent of male reproductive skew. We alsoinvestigated where the majority of copulations took place(i.e. within or outside leks, at the periphery of leks, etc.) andwhether genetic factors such as male heterozygosity orrelatedness levels influenced females’ mate choice.

Materials and methods

Samples

The focus of our study was a reproductive populationlocated in a protected area of 700 km2 in the south-easternpart of Morocco (Fig. 1). As part of a captive propagationprogram, eggs were collected under official agreementbetween 2001 and 2006 and hatched in captivity toincrease the number of founders. During the same period,brooding females and displaying males were sampled forblood in order to conduct paternity analyses. Birds werecaught using nylon snares placed around either the nest orwild chicks (to catch females) or around a bait female on thedisplaying site of males (Seddon et al. 1999, Hingrat et al.2004, Hingrat and Saint Jalme 2005).

Nesting females were located as described by Hingratet al. (2004) using the help of local shepherds while fieldworkers were looking for females exhibiting characteristicnesting behaviours (Gaucher 1995, Combreau et al. 2002).Once a nest was identified, and if no nest of the broodingfemale had already been collected during the same year, thewhole clutch was brought back to ECWP captive facilities.Whenever possible, females were caught for blood samplingand equipped with a unique metal band (or subcutaneousmicrochips) as well as a radio-transmitter. If a previous nestof the brooding female had already been sampled during thesame year, the new nest was checked regularly until eggshatched and the chicks were captured at the age of 15 d forblood sampling and equipped with a unique temporaryplastic band. If the nest was abandoned before hatching, anautopsy was done to sample the embryos for later geneticanalysis. Following this protocol, a total of 120 chicks weresampled out from 65 nests (Table 1, Fig. 1) and a total of47 brooding females were identified of which ten were notsampled (10 nests, 14 chicks).

Since 2000, displaying males were identified via aprotocol of systematic counting (Hingrat et al. 2004). Fiftythree displaying males were spotted in 2000 and 98 in 2006(Table 1). The apparently reduced number of displayingmales in 2002 and 2003 was due to lower sampling effortduring these years. The displaying males identified in 2006were grouped into leks according to the method describedby Hingrat and Saint Jalme (2005). Eleven leks werethen delineated (Fig. 1), encompassing 64 out of the 98displaying males. Between 2002 and 2006, 70 males,representing 71.4 % of all displaying males, were caughtand blood- sampled: 47 were lekking males (67.1 %) and23 were solitary males (32.9 %). Special attention was paidto sampling males with the highest displaying rates. Becausemales exhibit fidelity to their displaying site through theyear (Hingrat et al. 2004), we assumed that a sampled malewas present the previous years on the same display site.Therefore, all males sampled at the end of the study period

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were included in the pool of candidate fathers for chickssampled in all years. Taking into account the increasingnumber of displaying males on the area, this probably led to

an over-estimation of the number of candidates for somechicks; but this was the most conservative way to listcandidate fathers for every chick.

Table 1. Samples collected, between 2002 and 2006, for a paternity analysis in a Houbara Bustard population in the south eastern part ofMorocco. For each year is indicated the number of sampled and genotyped chicks, the total number of nests, the number of identified andsampled females, as well as the number of displaying and sampled males. The total number of males and females is lower than the sum of allsamples through the years because some females had their nest collected for several years and males exhibit high fidelity to their displayingsites.

Sampled chicks Genotyped chicks Total nests Identified females Sampled females Displaying males Sampled males

2001 2 2 1 1 1 53 92002 32 29 13 11 9 32 92003 17 17 9 9 8 27 112004 17 17 9 8 7 46 202005 15 13 10 10 8 63 382006 37 31 23 22 18 98 70

Total 120 109 65 47 37 70

Figure 1. Location of the study area in eastern Morocco. Spatial distribution, within this area, of Houbara bustard samples used for thepaternity analysis. Data were collected between 2002 and 2006. 120 chicks from 65 nests and 70 out of 98 displaying males weresampled. The position of these 70 males (for the year 2006) and 65 nests (for all years) are shown on this map.

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Microsatellite genotyping

All DNA extractions were made using NucleoSpin-tissuekits from Macherey-Nagel (Duren, Germany) and geno-typing was carried out by GENOSCREEN (Lille, France).We genotyped 14 loci designed for the Houbara bustardChlamydotis undulata undulata (Chbel et al. 2002), andthree designed for the great bustard Otis tarda (Pitra et al.2004): A10, A113a, A120, A18, A2, A205, A208, A21,A210, A29, D110, D117, D118, D119, O26, O27 andO38. The locus A113a was amplified with redesignedprimers because of null alleles.

Amplifications were performed in a final volume of20 ml, with 0.2 ml of Taq Polymerase (5 U/ml), 1.5 ml ofdNTPs (0.24 mM), 1.5 ml of MgCl2 (25 mM) and 2 mlof DNA. The primer concentration for each locus rangedfrom 0.125 mM to 1 mM. PCR conditions were: hot start at948 C for 10 min, followed by 40 cycles of 948 C for 30 s,558 C for 1 min and 728 C for 30 s, with a final extensionstep of 728 C for 10 min. In total, genotypes were obtainedfor 109 chicks, 37 females and 73 males (Table 1).

Data analysis

Linkage disequilibrium and departure from Hardy-Wein-berg equilibrium were tested with GENEPOP 4.0 (Rousset2008), and the frequency of null alleles for each locus wasestimated with CERVUS 3.0 (Marshall et al. 1998,Kalinowski et al. 2007).

Whenever the genotype of the brooding female wasavailable, our first step was to confirm the mother withCERVUS 3.0. We tested the brooding female of 95 chicks(53 nests) and the final decision was based on LOD scoresof putative females (negative LOD score implied that thecandidate was less likely the parent than any other candidaterandomly chosen in the population). Chicks whose motherwas not confirmed were considered of unknown mother forsubsequent paternity analyses.

Next, we determined the confidence level of paternitiesusing CERVUS 3.0 (95 % strict and 80 % relaxed), usingthe following parameters for all simulations: 10 000 cycles,98 candidate fathers, 74.1 % of candidate fathers sampled,91 % of loci typed and 1.49 % of genotyping error rate.The proportion of loci genotyped was directly estimatedfrom the mean proportion of individuals genotypedwhereas the genotyping error rate was deducted fromgenotype mismatches between chicks and their confirmedmother.

A father was assigned to a chick whenever the paternitywas assigned with a minimum confidence level of 80 %, orwhen no genotype mismatches were found in the trio‘‘female/chick/candidate male’’ (Lebigre et al. 2007). Theobserved mating success distribution for all sampled maleswas compared to a Poisson distribution under the assump-tion of random distribution of mating success (Focardi andTinelli 1996).

The polyandry rate was computed as the percentage ofnests with more than one male fathering chicks. For this, weonly considered nests where a father was assigned to allconfirmed sibs.

To investigate whether males gain more reproductivesuccess to aggregate, we compared with a x2 test theproportions of chicks fathered by lekking males vs. chicksfathered by solitary males. Next, we compared the propor-tion of lekking males confirmed as fathers vs. the totalproportion of lekking males in the population with aStudent t-test. We used a Pearson correlation to correlatethe number of chicks fathered by one male with the numberof males displaying in the same lek. For this test, the lek sizeof a solitary male was considered to be one.

The potential difference of breeding success betweencentral and peripheral males within leks was tested bycomparing the average distance to the centre of the lek ofboth confirmed and candidate fathers with a Mann -Whitney U-test. For this, we computed the centre of eachlek as the arithmetic mean of all displaying sites within thelek. We also used a Mann - Whitney U-test to test whetherfathers were significantly closer from nests than candidateswith.

We estimated the female density experienced by eachmale as the number of nests identified within 10 km aroundeach displaying site and correlated this estimation with themale breeding success, again using a Pearson correlation.Individual heterozygosity was computed with GENE-POP 4.0 and average heterozygosity levels were comparedbetween confirmed fathers and candidates with a Mann -Whitney U-test. Inbreeding avoidance was investigated bycomparing average relatedness between two groups ofdyads: the first group involving layers and candidate fatherswhile the second involved layers and confirmed fathers.Many relatedness estimators are available in the literatureand their efficiency varies greatly across datasets and needsto be tested to select the most accurate for each specificdataset (Blouin et al. 1996, Van de Casteele et al. 2001,Butler et al. 2004, Russello and Amato 2004). Oursimulations (see appendix) showed that the most efficientestimator within our dataset was the likelihood basedestimator of (Goodnight and Queller 1999). We thereforeused this estimator which was computed with KINGROUP(Konovalov et al. 2004).

Results

The exact test for genotypic disequilibrium was significantbetween loci D110 and D118. Null allele frequencieshigher than 0.05 were recorded in A210, D117, D119 andO26 (respectively 0.071; 0.131; 0.056 and 0.166). A210,D110, D117, D119 and O26 were therefore removed fromfurther analyses.

With the 12 remaining loci, we identified up to 20alleles per locus and the mean allele number per locus was7.3. The observed heterozygosity (Ho) ranged from 0.33 to0.84 with a mean Ho of 0.56. No deviation from Hardy-Weinberg equilibrium was found in the whole sample.

One female was not compatible with the seven chicks ofher nests at three different seasons. This was probably theconsequence of human error (i.e. labelling of tubes). Thesechicks were not considered for the maternity analysis. On atotal number of 120 sampled chicks, 11 were not success-fully genotyped and the female was not sampled for 21 ofthem. The maternity analysis was therefore conducted with

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88 chicks. For 29 eggs, the brooding female was notconfirmed by genetic analysis indicating that some femaleslaid eggs in other females’ nests. In order to evaluateprecisely the rate of conspecific brood parasitism (CBP), weonly took into account the nests with a minimum of twochicks that were genotyped, and for which the mother wasnot rejected for all chicks. Eggs were laid by two females inseven out of 26 nests (26.9 %), with a higher proportion ofparasitized nests in three than in two eggs clutches(Table 2). Most of the parasitized nests were found in theyears 2004 (four out seven nests) and 2006 (two out ofseven nests).

A father was assigned to 68 chicks (49 nests) out of 109analysed (Table 3). These 68 chicks were sired by 39 males,which is therefore the minimum number of males thatreproduced in the area. The distribution of the number ofchicks sired by males fitted a Poisson distribution (Fig. 2,l�0.971; x2�4.108; df�2; P�0.128). This meant thatthe paternity did not differ from a random distribution andthat we did not observe a skewed breeding success amongstmales. Fathers sired between one and five chicks with anaverage of 1.7 chicks per father.

To evaluate the occurrence of possible multiple pater-nity, we considered the nests where the female wasconfirmed and for which a father was attributed to aminimum of two chicks (n�5). Three of them (60 %)were sired by two different males. The proportion of fatherswas not significantly higher (x2�1.20; df�1; P�0.273)in lekking males (44 %; 28 out of 64) than in solitary males(32 %; 11 out of 34). While lekking males represented65 % of all displaying males (64 out of 98); theyrepresented 72 % of confirmed fathers (28 out of 39),and fathered 74 % of the chicks (50 out of 68). However,these last two proportions (lekking males amongst con-firmed fathers, and chicks fathered by lekking males)were not significantly different from the proportion oflekking males in the populations (Student t-test, P�0.216and P�0.221 respectively) indicating that lekking malesdid not obtain higher breeding success than solitary ones. Inaddition, the number of chicks fathered by a male was notcorrelated to the size of the lek it was displaying in (n�70,R�0.204, P�0.09), confirming that males in bigger leksdid not have a higher breeding success. Within leks,the distance to the centre of the lek was not significantlydifferent between confirmed fathers and candidates(dist fathers�945 m, n�28; dist candidates�952 m, n�19;

Mann - Whitney U-test, U�260, P�0.905): central orperipheral males did not acquire more breeding success.

The average distance between displaying males and nestswas not significantly different for confirmed fathers thancandidates (dist fathers�12.5 km, n�65; dist candidates�13.2 km, n�4238; Mann - Whitney U-test, U�127499.5, P�0.303).

Finally, average heterozygosity in confirmed fathers (n�39, Ho�0.541) and in candidates (n�31, Ho�0.529)was not significantly different (Mann - Whitney U-test,U�604, P�0.995). And the average relatedness betweenfemales and confirmed fathers (r�0.044, n�35) orcandidates (r�0.029, n�2300) were not significantlydifferent (Mann - Whitney U-test, U�39434.5, P�0.838).

Discussion

Our results on Houbara bustard are not consistent withearlier findings of severe male reproductive skew in lekkingspecies (Bradbury and Gibson 1983, Lewin 1988, Alataloet al. 1992, Hoglund et al. 1993, Kokko et al. 1998). Werecorded that mating success did not vary from a randomdistribution amongst males. Moreover, no relationship wasfound between males’ reproductive success and theirgeographic location or their position within or outsideleks. Heterozygosity and relatedness with the females werealso not related to males’ breeding success; although thesefactors were proven to positively affect female mate choicein some other lekking species (Fiske et al. 1998, Brownet al. 2000, Rintamaki et al. 2001, Hoglund et al. 2002,Shorey 2002, Morales et al. 2003, Loyau et al. 2005).Similar outcomes have been found in other avian species,such as low variance in male reproductive success in thebuff-breasted sandpiper (Lanctot et al. 1997), the absence ofcorrelation between reproductive success and either maledisplay characteristics in the white-bearded manakin Man-acus manacus (Shorey 2002) or heterozygosity in the blue-crowned manakin Lepidothrix coronata (Duraes et al. 2009).

In Houbara bustards, the low male reproductive variancewas associated with a successful off-lek male mating strategyand no apparent reproductive benefit of lekking. Suchstrategy has been previously suggested in some lekkingspecies like the Pacific harbour seal Phoca vitulina richardii(Hayes et al. 2004), the fallow deer Dama dama (Apollonioet al. 1992) and several avian species: the buff-breastedsandpiper (Lanctot et al. 1997), the lesser bird of paradiseParadisaea minor (Laska et al. 1992), the ruff (Lank andSmith 1987), and the sage grouse Centrocercus urophasianus(Semple et al. 2001). The benefit of such a strategy is toallow more males (young or low ranking males) access toreproduction by reducing inter-male sexual competition.

Our results suggested that females adopted two alter-native mating strategies: polyandry and conspecific broodparasitism. Both were primarily supposed to be at reducedrates in birds, but the improvement of molecular techniqueshas led to numerous studies rejecting this assumption(Arnold and Owens 2002). Thus, multiple copulationswere identified in avian lekking species such as peafowl(Petrie et al. 1992), buff-breasted sandpiper (Lanctot et al.1997), sage grouse (Semple et al. 2001), and at a very high

Table 2. Yearly distribution, between 2002 and 2006, of conspecificbrood parasitism in a Houbara bustard population among clutcheswith different sizes (parasitized nests/total). Only clutches with aconfirmed mother and a minimum of two genotyped chicks wereconsidered.

Clutch size Total

2 eggs 3 eggs

2002 0/4 1/3 1/72003 0/3 0/2 0/52004 1/2 3/3 4/52005 0/2 0/0 0/22006 1/6 1/1 2/7

Total 2/17 5/9 7/26

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rate in the ruff where a minimum of 59 % of nests wereconcerned (Lank et al. 2002). Amongst lekking species,Houbara bustards therefore exhibit one of the highest levelsof polyandry with 60 % of multiple paternities.

In the Houbara bustard, males contribute nothing butgenes to the reproduction. A polyandrous strategy musttherefore have evolved through either fertility insuranceprocesses (Krokene et al. 1998) and/or genetic benefits(Bradbury and Gibson 1983). The fertility insurancehypothesis may lead to both positive and negatives correla-tion between the rate of polyandry and the rate of infertileeggs. Indeed, if females are aware of the fertility status oftheir mate, we should expect only some females to engage inpolyandry and that most nests will present no multiplepaternity. On the contrary, if females are not aware of thefertility status of their mate, most nests will present multiplepaternities (Kempenaers and Dhondt 1993). Since noinformation about male infertility in the wild was available,this hypothesis can not be ruled out in the Houbara bustardwithout further investigation.

Genetic benefits involve intrinsic effects of paternalgenes in offspring (good genes model) and/or a defencemechanism against genetic incompatibility (genetic com-patibility model, Jennions and Petrie 2000, Tregenza andWedell 2000, Zeh and Zeh 2001, Charmantier et al. 2004,Simmons 2005). Good genes and genetic compatibilitymodels have different outcomes in terms of males’reproductive success. The good genes model produceshighly skewed reproductive success in males as a conse-quence of a strong female selection for the best male.

Genetic compatibility leads to a greater variability in femalemate choice with more males gaining access to reproduction(Neff and Pitcher 2005). According to these predictions,our results seemed in favour of the genetic compatibilitymodel operating in the study population despite noapparent effect on female choice from both male hetero-zygosity and relatedness of the potential pair. Nevertheless,it is not possible to extend this assumption to the wholespecies because both models could coexist in a populationor a species, at different rates in a dynamic equilibrium(Petrie and Lipsitch 1994, Petrie and Kempenaers 1998,Colegrave et al. 2002, Neff and Pitcher 2005, Oh andBadyaev 2006). The Houbara bustard might then be arelevant model to integrate both good genes and geneticcompatibility models in the study of the evolution of matechoice and to test the hypothesis of a dynamic equilibriumbetween both models.

Polyandry supposes successive copulations with differentmales and entails sperm competition that is associated withcryptic female choice (Andersson and Simmons 2006). Insuch cases, although some males may gain more copula-tions, incompatibility patterns may produce reduced breed-ing success from these males and induce relaxed sexualselection on males’ reproductive traits (Colegrave et al.2002, Andersson and Simmons 2006). The increasingnumber of studies highlighting polyandry in lekking speciessuggests that sperm competition may be more widespreadthan previously stated (Birkhead and Møller 1992). InHoubara bustards, the potential for sperm competitionthrough high sperm storage capacities was revealed byearlier studies on artificial insemination (Saint Jalme et al.1994). This potential was confirmed by paternity analysesin a captive population, where 80 % of the chicks werefathered by the last male used for insemination (Lesobre,unpubl. data) as predicted by the last male precedencemodel (Birkhead 1998). Potential benefits of cryptic femalechoice would be to enhance genetic compatibility throughheterozygote advantage (inbreeding avoidance), selectionfor coadapted gene complexes (outbreeding avoidance) and/or avoidance of selfish genetic elements (such as segregationdistorters, maternal lethal effect or cytoplasmic parasites/symbionts; Tregenza and Wedell 2000).

The second alternative strategy adopted by females wasconspecific brood parasitism (CBP) and was observed in27 % of the nests. So far, CBP has been recorded in fewlekking species although it has already been described inabout 240 avian species in which it may affect up to 50 %of broods (Lyon 1993, Yom Tov 2001, Arnold and Owens2002). After a review of 95 avian species, Arnold and

Table 3. Yearly distribution, between 2002 and 2006, of paternity results in a Houbara Bustard population in the south-eastern part ofMorocco. ‘‘Assigned’’: number of chicks for which a father was assigned; ‘‘Non assigned’’: chicks for which no father was assigned withenough confidence level to be confirmed; ‘‘Not sampled’’: chicks for which the father was not present in our sample; ‘‘Non analysed’’: chicksfor which the number of genotyped loci was too low for analysis.

Assigned father Non assigned father Non sampled father Non analysed chicks Total

2001 2 22002 13 6 9 322003 8 2 7 4 172004 12 2 3 172005 11 1 1 152006 22 2 5 2 37

Total 68 13 25 8 120

Figure 2. Observed and predicted distributions of male reproduc-tive success amongst 70 displaying males of a population ofHoubara bustard in the south-eastern part of Morocco. Thesedistributions were computed using a paternity analysis conductedbetween 2002 and 2006. The observed distribution fitted aPoisson distribution with l�0.971.

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Owens (2002) predicted that CBP should occur at lowerrates in species with high parental care and low levels offecundity, such as the Houbara Bustard. In this species,inter-seasonal variations of reproductive investment (clutchsize) have been recorded, in the wild and in captivity. Theseare related to climatic conditions and food availability(van Heezik et al. 2002). In good years in terms of foodavailability, costs related to maternal investment arereduced and CBP could be an alternative strategy. Indeed,CBP was mainly recorded in 2004 and 2006, years withunusually high rainfall levels in Morocco during spring(ECWP unpubl. data). We then suggest that Houbarabustard females exhibit opportunistic behaviour of engagingin CBP only under favourable environmental conditions.Nevertheless, and because of our low sample size for eachyear, further study is required.

To summarise, our results, which showed no malereproductive skew, no relationship between male reproduc-tive success and males’ geographic location and nodifference of reproductive success between lekking malesor solitary ones, did not follow those expected under a lekmating system. These expectations were mostly based onstudies of classical lekking species. However, the matingsystem in Houbara bustards still fits the definition of a lekthat is an aggregation of displaying males visited by thefemales for the sole purpose of fertilization (Hoglund andAlatalo 1995). In the Houbara bustard, the selection of thedisplaying site is driven by female density, and the origin ofleks matches the hotspot model (Hingrat et al. 2008). Insuch cases, inter-male competition is reduced and inter-male dominance processes, which restrict female matechoice, are also reduced (Bradbury and Gibson 1983,Foster 1983, Beehler and Foster 1988, Alatalo et al.1996). The relative importance of female mate choicemay then increase. There may be more room for diversity inthis choice, which could then be driven by both good geneand genetic compatibility models (Neff and Pitcher 2005).This would lead to a more relaxed reproductive skew thanin classical leks. Furthermore, in a previous study (Lesobre2008), we showed that kin selection between lekking malesis unlikely to occur in exploded leks compared to classicalleks. It therefore seems that constraints are divergentenough between both types of leks that we can avoidextrapolating conclusions from one to the other forprocesses leading to their establishment, maintenance orrespective outcomes in terms of males’ and females’benefits.

The absence of highly skewed male mating success alongwith alternative mating strategies allowing more individualsto breed in the population has significant implications for theconservation of Houbara bustards. These phenomena indir-ectly contribute to the maintenance of the population’seffective size and its genetic diversity. The Houbara bustard’smating system is therefore not a ‘‘threat’’ to the viability ofdeclining populations (Blumstein 1998, Frankham 2005,Quader 2005). It should therefore not be considered anegative concern for the reinforcement program of theHoubara bustard in North Africa.

Acknowledgements � The authors would like to thank His HighnessSheikh Mohamed Bin Zayed Al Nahyan, Founder and Sponsor.We greatly thank His Excellency Mohammed Al Bowardi, General

Manager of the Office of Mohammed Bin Zayed Al Nahyan andJacques Renaud, Manager of the Emirates Centre for WildlifePropagation, for their support. We are most grateful to allpeople involved in data collection and Mrs Duthoy Stephanie(GENOSCREEN) for the lab work. Many thanks to reviewerswho provided their valuable comments on the manuscript andspecial thanks to those who helped with correcting the English.

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Appendix 1: Choice of relatedness estimator

We used relatedness methods, instead of dyad classificationmethods, because they make no a priori assumption onkinship levels, and give a continuous value of r, whereasclassification methods provide the probability of belongingto one relationship category in comparison with anotherone (Ritland 2000, Blouin 2003, Butler et al. 2004).

The use of a relatedness estimator has a direct impact onthe reliability of results and on the increase in number ofmarkers, and is the only way to reduce the samplingvariance during relatedness estimation (Van de Casteeleet al. 2001). The performance of such estimators is testedthrough the evaluation of their efficiency in classifyingdyads, through their behaviour towards various populationcompositions, and through their misclassification error ratewhen assigning relatedness categories (Blouin et al. 1996,Van de Casteele et al. 2001, Butler et al. 2004, Russello andAmato 2004).

We tested seven estimators: rHK (Konovalov and Heg2008), rLi (Li et al. 1993), rLR (Lynch and Ritland 1999),rML (Goodnight and Queller 1999), rMLG (Milligan 2003),rQG (Queller and Goodnight 1989) and rWang (Wang2002). SpageDi (Hardy and Vekemans 2002) was used tocompute rLi and rWang, whereas Kingroup (Konovalov et al.2004) was used to compute rHK, rQG, rLR, rML and rMLG.

Allelic frequencies of the reference sample were used togenerate genotypes for dyads of each of the followingrelatedness categories: non-related (NR), half-sib (HS), full-sib (FS) and parent/offspring (PO). These dyads were usedto simulate 15 samples of 1 000 dyads each: four werecomposed on the basis of a single relatedness category andeleven were composed on the basis of various proportions ofeach relatedness category. The first four samples were usedto obtain, for each estimator in each relatedness category,the distribution of r, the mean bias, the standard deviation

(as an estimation of the sampling variance) and themisclassification rate, which was computed empirically asdescribed by Blouin et al. (1996). The second set of sampleswas used to evaluate the fraction of the total variance thatcould be explained by true relatedness, within a samplecomposed of various relatedness categories (Van de Casteeleet al. 2001, Russello and Amato 2004). This fraction wasexpressed as the ratio between the variance due torelatedness categories and the total variance.

No significant deviation from normality was found forany relatedness distribution. The estimator rLi presented thelowest bias for non-related dyads (Appendix 1) whereas thelowest biases for related dyads were given by rML (HS), rHK

(FS) and rLi (PO). For each relatedness category the loweststandard deviation was obtained for rMLG, which in eachcase presented a significant bias (Appendix 1). The lowestmisclassification error rates were obtained with rML andrMLG (Appendix 2). The estimator rML outperformed otherrelated category estimators, whereas rLR had the bestclassification rate for non-related dyads. Finally, the fractionof total variance expressed by true relatedness was max-imized for both rML and rMLG (Appendix 3). Performanceswere globally poor when the sample was dominated by full-sibs (i.e. population I), with the fraction of total varianceexplained by classical relatedness ranging from 41.5 % (rLR)to 54.4 % (rMLG).

The best performance was obtained with both rML andrMLG. However, rML allows negative values, whereas rMLG

ranges between 0 and 1. Negatives values of r indicate thatindividuals are less related than two randomly generated,unrelated individuals, which could occur in the case ofrecent immigration (Queller and Goodnight 1989, Kono-valov and Heg 2008). Because of the potentially highdispersion in Houbara bustards, as measured with bothgenetic (Lesobre 2008), and telemetric data (ECWP,unpubl. data), we finally chose to use rML.

Appendix 1. Average relatedness, standard deviation (in brackets) and bias computed for 1 000 dyads in each relatedness category (NR: non-related; HS: half sib; FS: full sib and PO: parent/offspring). Significant bias values, as shown by the Student t-test, are indicated with a *(P50.05), **(P50.01) or ***(P50.001).

NR HS FS PO

rHK r �0.009 (0.223) 0.233 (0.196) 0.500 (0.170) 0.493 (0.105)bias 0.009 0.017** 0.000 0.007*

rLi r �0.004 (0.192) 0.239 (0.185) 0.494 (0.170) 0.499 (0.109)bias 0.004 0.011 0.006 0.001

rLR r �0.005 (0.122) 0.241 (0.202) 0.497 (0.207) 0.498 (0.180)bias 0.005 0.009 0.003 0.002

rML r 0.010 (0.135) 0.247 (0.168) 0.509 (0.154) 0.493 (0.089)bias 0.010* 0.003 0.009 0.007**

rMLG r 0.060 (0.103) 0.265 (0.165) 0.513 (0.150) 0.528 (0.065)bias 0.060*** 0.015** 0.013** 0.028***

rQG r �0.009 (0.190) 0.230 (0.182) 0.493 (0.171) 0.492 (0.122)bias 0.009 0.020*** 0.007 0.008*

rWang r �0.005 (0.198) 0.238 (0.190) 0.494 (0.172) 0.498 (0.095)bias 0.005 0.012 0.006 0.002

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Appendix 3. Fraction of total variance explained by true relatedness, computed as the ratio between the factorial variance (i.e. relatednesscategory) and the total variance. Results are presented for eleven samples, for various combinations of relatedness categories. The theestimators with the best performance for each case are indicated with*.

I II III IV V VI VII VIII IX X XI

NR 0.1 0.1 0.1 0.25 0.25 0.25 0.4 0.5 0.6 0.6 0.8HS 0.1 0.1 0.1 0.25 0.25 0.25 0.2 0.2 0.2 0.2 0FS 0.8 0 0.4 0 0.5 0.25 0.2 0.2 0.2 0 0.1PO 0 0.8 0.4 0.5 0 0.25 0.2 0.1 0 0.2 0.1

rHK 0.468 0.620 0.524 0.641 0.538 0.568 0.603 0.532 0.477 0.502 0.491rLi 0.479 0.646 0.532 0.670 0.557 0.599 0.625 0.571 0.524 0.563 0.542rLR 0.415 0.438 0.406 0.603 0.54 0.571 0.637 0.619 0.608 0.607 0.658rML 0.544* 0.699 0.578 0.749 0.637* 0.682 0.731 0.691 0.642 0.666 0.690rMLG 0.514 0.779* 0.603* 0.799* 0.629 0.705* 0.765* 0.725* 0.668* 0.742* 0.782*rWang 0.480 0.596 0.516 0.662 0.574 0.605 0.657 0.594 0.552 0.579 0.564rQG 0.466 0.667 0.527 0.689 0.549 0.594 0.627 0.556 0.516 0.547 0.535

Appendix 2. Misclassification error rates of seven relatedness estimators, tested for four relatedness categories (NR: non-related; HS: half sib;FS: full sib and PO: parent/offspring). Simulations were run with 1 000 dyads created from the allelic frequencies of a Houbara bustardreference sample.

Simulated category rHK rLi rLR rML rMLG rQG rWang

NR 0.317 0.264 0.141 0.189 0.152 0.276 0.277HS 0.528 0.5 0.576 0.489 0.532 0.494 0.518FS 0.202 0.234 0.295 0.195 0.207 0.227 0.223PO 0.087 0.105 0.268 0.01 0.021 0.146 0.071Average error rate 0.284 0.276 0.320 0.221 0.228 0.286 0.272

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