The Sarolga: Conservation Implications of Genetic and Visual Evidence For

Home , Antigone (bird), Brolga, Sarus crane

The sarolga: conservation implications of genetic and visual evidence for hybridization between the brolga Antigone rubicunda and the Australian sarus crane Antigone antigone gillae

T IMOTHY D. NEVARD,MARTIN H AASE,GEORGE A RCHIBALD I AN L EIPER and S TEPHEN T. GARNETT

Abstract To investigate the extent of suspected hybridiza- Introduction tion between the brolga Antigone rubicunda and the ybridization between apparently well-differentiated Australian sarus crane Antigone antigone gillae, first noted in the s, we analysed the genetic diversity of  feath- Hspecies is a commonly observed phenomenon and is widely studied in the context of speciation and repro- ers collected from breeding and flocking areas in north  ductive isolation among species (Arnold, ;Pennisi, Queensland, Australia. We compared these with samples  from birds of known identity, or that were phenotypically ). Hybridization has a variety of evolutionary conse- typical. Bayesian clustering based on  microsatellite loci quences, depending on the fitness of the hybrids, which may identified nine admixed birds, confirming that Australian range from sterility and reduced fertility, to hybrid vigour or heterosis, whereby hybrid individuals perform better and cranes hybridize in the wild. Four of these were backcrosses,  also confirming that wild Australian crane hybrids are fer- out-compete parental species (Burke & Arnold, ;  Pennisi, ). As a consequence, if species are rare and locally tile. Genetic analyses identified times more hybrids than  our accompanying visual field observations. Our analyses confined (Rhymer & Simberloff, ), genetic mixing can lead to the extinction of one or both parental species also provide the first definitive evidence that both brolgas  and sarus cranes migrate between the Gulf Plains, the prin- (Todesco et al., ). Eventually, hybridization can lead to speciation, with two parental species giving rise to a cipal breeding area for sarus cranes, and major non-breed-   ing locations on the Atherton Tablelands. We suggest that new species (Dowling & Secor, ; Soltis & Soltis, ), genetic analysis of shed feathers could potentially offer a highlighting the relevance of studying hybridization from the perspective of conservation biology. Allendorf et al. cost-effective means to provide ongoing monitoring of  this migration. The first observations of hybrids coincided ( ) noted that taxa that have arisen through natural hy- with significantly increased opportunities for interaction bridization warrant protection. However, they also noted between the two species when foraging on agricultural that increased anthropogenically related hybridization is crops, which have developed significantly in the Atherton causing extinction of many taxa (species, subspecies and lo- Tablelands flocking area since the s. As the sarus cally adapted populations) by both replacement and genetic crane is declining in much of its Asian range, challenges mixing, and they argued that hybrid taxa resulting from anthropogenic causes should therefore be protected only to the genetic integrity of the Australian sarus crane populations have international conservation significance. in exceptional circumstances. We do not yet know if hybrids of the brolga Antigone rubicunda and the Australian sarus Keywords Antigone antigone gillae, Antigone rubicunda, crane Antigone antigone gillae have arisen as a result of Australian sarus crane, brolga, evolution, hybridization, anthropogenic habitat change, nor if agricultural and con- introgression, migration monitoring servation policy has the potential to curtail this, but it is a matter for careful consideration. The starting point for this is gaining an appreciation of the scale of hybridization. Depending on the interaction of parental alleles, and their

TIMOTHY D. NEVARD* (Corresponding author, orcid.org/0000-0002-1287- dominance patterns as well as epistatic effects, hybrid in- 4595)IAN LEIPER and STEPHEN T. GARNETT Research Institute for the dividuals need not be phenotypically intermediate between Environment and Livelihoods, Charles Darwin University, Darwin, Northern their parents (Rieseberg et al., ). Also, introgression of Territory 0909, Australia. E-mail [email protected] alleles from one species into the other need not be symmet- MARTIN HAASE AG Vogelwarte, Zoologisches Institut und Museum, Universität Greifswald, Greifswald, Germany rical, as demonstrated in many cases of mitochondrial introgression (Toews & Brelsford, ). On the nuclear level, GEORGE ARCHIBALD International Crane Foundation, Baraboo, Wisconsin, USA genomic data indicate that hybridization and introgression *Also at: Wildlife Conservancy of Tropical Queensland, Ravenshoe, Queensland, Australia interacting with recombination and selection result in much more complex scenarios of genetic diversity (Payseur & Received  October . Revision requested  February . Accepted  May . First published online  May . Rieseberg, ).

Oryx, 2020, 54(1), 40–51 © 2019 Fauna & Flora International doi:10.1017/S003060531800073X Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.126, on 01 Oct 2021 at 07:52:30, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S003060531800073X The sarolga 41

PLATE 1 Typical brolga Antigone rubicunda (small red skull-cap type comb, dark wattle and grey legs), with unfledged chick, in Gulf Plains, Australia (Fig. ). (Photo: T. Nevard)

PLATE 2 Typical Australian sarus cranes Antigone antigone gillae (pink legs, red comb extending down the neck, whitish crown and darker grey colour), with first-year juvenile, in Atherton Tablelands, Australia (Fig. ). (Photo: T. Nevard)

Secondary contact of taxa that are well differentiated in allopatry may result in hybridization (Grant & Grant, ; Vijay et al., ), even among non-sister species (Koch et al., ). Such is the situation for the two Australian cranes, the brolga (Plate ) and the Australian subspecies of the sarus crane (Plate ). The brolga, which is endemic to Australasia (Australia and New Guinea), probably split from a common ancestor with the sarus FIG. 1 (a) Global distribution of the brolga Antigone rubicunda crane .–. million years ago (Krajewski et al., ). and the sarus crane Antigone antigone, and data collection sites Sarus cranes, whose range includes South and South-east in (b) the Gulf Plains and (c) Atherton Tablelands, Australia. Asia (Fig. ), were first recorded in Australia in the s Distribution data derived from BirdLife International and  NatureServe Bird Species Distribution Maps of the World () (Gill, ) but may have been isolated from Asian popula-     and The Australian Bird Guide (Menkhorst et al., ). Gulf tions for up to , years (Wood & Krajewski, ). Plains Interim Biological Regionalization Area derived from Hybridization between the brolga and sarus crane was Department of Environment & Energy (), and state roads first noted in French aviculture in  (Gray, ), when from Department of Transport & Main Roads ().

fertile hybrids between the brolga and the South-east Asian near Normanton, brolgas favoured deeper, generally coastal subspecies of the sarus crane Antigone antigone sharpii were marshes (Walkinshaw, ). However, they also occur in produced. In , when observing the flocking behaviour open forest and woodland, grassland and cultivated land of brolgas and sarus cranes on the Atherton Tablelands in (Lavery & Blackman, ). Queensland, Australia, Archibald () identified at least two and possibly six potential hybrids (for which he coined the name sarolga), and speculated that if hybridization were Australian sarus crane to increase, introgression could be significant in the conservation of cranes in Australia. Similar concerns led Jones Although the sarus crane is primarily a South Asian species, et al. () to recommend monitoring of the genetic integ- it is extinct in the Philippines and declining in much of its rity of Australian sarus cranes. The present research is the Asian range, particularly in Burma, Thailand and Indochina first to confirm and quantify the extent of natural hybridiza- (Meine & Archibald, ; Archibald et al., ), and is tion between the brolga and the Australian sarus crane. categorized as Vulnerable on the IUCN Red List (BirdLife International, ). The Australian sarus crane population is therefore of international conservation significance and Brolga hybridization with the brolga could potentially be a threat to the genetic integrity of the Australian subspecies. In Australia the brolga occurs across the north of the country Sarus cranes in Australia are almost entirely confined to in Queensland, the Northern Territory and Western Australia, Queensland (Fig. a), with known breeding areas along the andasfarsouthasVictoria,whereitisthreatened(Marchant Gulf Plains and on the Normanby River floodplains in & Higgins, ; Fig. a). In Queensland the species has been eastern Cape York Peninsula (Marchant & Higgins, ; recorded throughout the state (Marchant & Higgins, )but Barrett et al., ; Franklin, ). Most breeding records is now widespread and abundant only in the north. Although are from the Gulf Plains and concentrated in the Gilbert breeding occurs throughout its Queensland range (Marchant and Norman River catchments, where cranes make use of &Higgins,;Barrettetal.,), it appears to be focused both natural and man-made wetlands on cattle properties on the grasslands and open savannah woodland of the Gulf (Barrett et al., ). The only known major dry-season Plains, south-east of the Gulf of Carpentaria and in Cape flocking area for sarus cranes is the Atherton Tablelands York Peninsula. Scattered breeding occurs elsewhere, with and its immediate vicinity. Counts of dry-season flocks on flocking (non-breeding) regions including the Atherton the Atherton Tablelands provide the only current estimates Tablelands and south of Townsville on the north-east coast of sarus crane recruitment rates (Marchant & Higgins, ; of Queensland (Lavery & Blackman, ). Grant, ). An unknown proportion of the sarus crane The brolga population in northern Australia is generally population remains on the Gulf Plains and the adjacent in- considered to be stable and was estimated to comprise land area in the dry season, and therefore utilizes different ,–, individuals (Meine & Archibald, ). landscapes and food types during this period, as there are A total of , brolgas were recorded by Kingsford et al. no significant cropped areas. Migration routes followed by (), and all but  of these were in northern Australia. sarus cranes to the Atherton Tablelands from their breeding The only longitudinal counts are from north-east Queens- areas, and their habitat usage along the way, are currently land (Atherton Tablelands and Townsville regions), with an unknown. The importance of migration routes is becoming annual mean of c.  individuals recorded by community- increasingly understood, as knowledge of breeding and based naturalists during –, with a peak of c. , non-breeding locations alone is insufficient to secure the in  (E. Scambler, pers. comm.). protection of migratory species (Runge et al., ). The dispersal patterns and movements of brolgas across Estimates of total numbers of Australian sarus cranes their range are poorly understood (Marchant & Higgins, have varied from , to , individuals (E. Scambler, ). Brolgas undertake seasonal movements between breed- pers. comm.), but these have low reliability (Garnett & ing and non-breeding (flocking) habitats in response to rain Crowley, ). Annual (unpublished) counts undertaken and flooding (Marchant & Higgins, ). Flocks are formed by local members of BirdLife Australia on the Atherton during the dry season and pairs disperse to breeding sites in Tablelands (E. Scambler, pers. comm.) indicate –, the wet season. Although there is an increasing body of work individuals flocking in dry seasons during – but it on the species in the southern part of its range, especially in is not known what proportion of the total Australian sarus Victoria (Harding, ; Herring, , ), knowledge crane population these birds represent. There are no current of the behaviour and ecology of tropical brolgas remains estimates of population trends in Australia (E. Scambler, largely confined to earlier studies, such as those of Hughes pers. comm.), but it is thought that numbers of sarus cranes &Blackman(). Brolgas can use brackish waters, having have increased from perhaps as few as  on the Atherton a unique salt-gland, and when observed with sarus cranes Tablelands in the late s (G. Archibald, pers. obs.).

Aims In May , freshly shed feathers were collected oppor- tunistically from Miranda Downs cattle station (°.′ S, Although the presence of putative hybrid cranes on the °.′ E, elevation  m) on the Gulf Plains, and in Atherton Tablelands has been noted by experienced obser- September  from Innot Hot Springs (°.′ S, vers for many years, we hypothesized that it is possible that °.′ E,  m) on the southern edge of the these were non-hybrid birds, exhibiting intrinsic phenotypic Atherton Tablelands (Fig. c). Only feathers with minimal variability. We were therefore interested in testing the contamination from soil and vegetation were selected and degree of introgression (if any) in the crane population, as were picked from dry ground, using forceps, placed immedi- well as providing a benchmark for its future measurement ately in sealed plastic bags and refrigerated prior to analysis. both visually and genetically. To do this we needed to understand whether birds with the visual characteristics Molecular analysis that had led observers to believe that they were hybrids (sarolgas) were, in fact, hybrids. A total of  feathers were collected and prepared for geno-  We aimed to ( ) establish the extent of hybridization be- typing:  from the Gulf Plains and  from the Atherton tween the Australian sarus crane and the brolga, using mo- Tablelands. Fifteen samples (blood, feathers) were taken  lecular methods; ( ) seek evidence from these genetic data from reference birds, which were either phenotypically typ- for the presumed connectivity of breeding and flocking ical or for which a potential history of contact with the other  areas in the Gulf Plains and Atherton Tablelands; ( ) de- species could be excluded or minimized ( A. antigone scribe the distribution of individuals with hybrid character- antigone,  A. antigone gillae,  A. rubicunda; Table ; note  istics on the Atherton Tablelands; and ( ) discuss the results that including the non-Australian A. antigone antigone in primarily with respect to their relevance to biological con- the reference collection does not affect the veracity of the com- servation of the two species in Australia. parison with brolga). DNA was extracted using the slightly modified sodium dodecyl sulphate/salting-out protocol of Miller et al. (). To increase the yield, dithiothreitol Methods and Roti-PinkDNA (Carl Roth,Karlsruhe,Germany)were added. Initially we tested the  microsatellite loci that Field observations and sample collection had been used successfully for other species of crane (Hasegawa et al., ; Meares et al., )aswellasthe We observed cranes during July –June  in  sarus crane (Jones et al., ) and the brolga (Miller, monthly circuits of . km that encompassed  foraging ). Of these,  could be consistently amplified and sites distributed across all known crane flocking areas of scored (Table ). Polymerase chain reactions (PCRs) were the Atherton Tablelands (Figs d & ), which were visible conducted in a volume of  μl and contained  μlDNA from the road. At each site we counted adult and first-year (– ng),  μlof ×NH-based reaction buffer, juvenile brolgas and sarus cranes using  ×  binoculars, .–. mM MgCl solution (Table ), . mM of each pri- and examined potential hybrids (see below and Plate ) mer, . mM of dNTP, . μl of BioTaq DNA polymerase using a  × spotting scope. Individuals could not be distin- ( U/μl), . μlof% bovine serum albumin and sterile guished and were probably counted on multiple occasions ddHO. For two loci, GR and GR, we used the MyTaq and at multiple sites. However, enough cranes were counted mix (all products from Bioline, London, UK). The PCR pro- that there is no reason to suspect systematic bias in the es- file comprised an initial denaturation at  °C,  cycles timate of the proportion of visually distinguishable hybrids including denaturation at  °C, primer specific annealing amongst the wider crane population. (Table ) and extension at  °C, each for  s, and a final The initial identification of hybrids was based on the elongation at  °C for  minutes. Microsatellite alleles presence of at least four readily observable characteristics were separated on a xl Genetic Analyzer, together including at least two of (), () and () in Archibald’s with the GeneScan  LIZ Size Standard . (Applied () typology of sarolgas: () larger and heavier than Biosystems, Waltham, USA). Fragment sizes were deter- either parent species; () an irregular border to the comb; mined manually to avoid the inconsistencies in automatic () notches in the cap; () intermediate or mixed-colour calling that result from the arbitrariness of bin width legs; () a scalloped mantle; () brighter yellow eyes than definitions using GeneMapper . (Applied Biosystems, typical sarus cranes (which are more orange); and ()a Waltham, USA). To control the accuracy of size determin- small wattle. Although Archibald’s() typology was ation we repeated PCRs of samples that initially gave weak helpful, in overall appearance the sarolgas he described signals or had rare variants. In other cases, problematic PCR were more like atypical sarus cranes than atypical brolgas, samples that were initially typed in different runs, and there- whereas we also observed putative hybrids that looked fore possibly difficult to calibrate, were loaded on the same more like atypical brolgas (Plate b). plate to improve comparability.

FIG. 2 Distribution and clustering of brolga, sarus crane and sarolga visually identified at  sites on the Atherton Tablelands (Fig. ) during –. On some occasions, as a result of poor light and weather conditions, cranes could not be identified.

TABLE 1 Notes on  reference individuals of the sarus cranes Antigone antigone antigone and Antigone antigone gillae and the brolga Antigone rubicunda from which samples were taken for molecular analysis. Samples were taken from living or recently deceased wild birds that were phenotypically typical, or from birds whose lineages of descent were known not to have had contact with the other species.

No. of Species specimens Locality Notes A. antigone antigone 3 Lemgo Avicultural Collection, Breeding records show no interbreeding with other sarus Germany subspecies since their ancestors were collected from the wild in the 1970s A. antigone gillae 5 Lemgo Avicultural Collection, Breeding records show no interbreeding since their ancestors Germany were collected from the wild in the 1970s A. antigone gillae 2 Kairi, Queensland, Australia Wild birds; phenotypically typical sarus cranes A. rubicunda 3 Cairns Tropical Zoo, Breeding records show no interbreeding since collected from Queensland, Australia a wild population with no history of contact with sarus cranes A. rubicunda 2 Herbert River, Atherton Wild birds; phenotypically typical brolgas Tablelands, Queensland, Australia

TABLE 2 PCR specifications, genetic diversity and allele richness of microsatellite loci of brolgas and sarus cranes from the Gulf Plains (Gulf) and Atherton Tablelands (Table), Australia (Fig. ), with locus/dye (fluorophore at ′-end of forward primer), MgCl concentration, annealing temperature (T), number of alleles, genetic diversity and allelic richness. The following loci could not be consistently amplified or scored: Gamμ, Gamμ, Gamμ, Gamμ, Gamμ, Gamμ, Gamμ, Gamμ, GjM, GjM, GjM,GR (Hasegawa et al., ; Meares et al., ; Miller, ).

Gene diversity Allelic richness No. of alleles Brolga Sarus Brolga Sarus

Locus/dye MgCl2 (mM) T (°C) Brolga Sarus Gulf Table Gulf Table Gulf Table Gulf Table Gamμ3/FAM 1.5 59 7 6 0.618 0.654 0.162 0.229 6.994 6.965 3.183 2.875 Gamμ18/HEX 1.5 53 31 1 0.224 0.363 0.000 0.000 2.943 3.000 1.000 1.000 Gamμ24/HEX 2.25 60 5 51 0.578 0.628 0.204 0.338 4.943 4.998 3.732 4.000 Gamμ101b/HEX 2 59 4 3 0.173 0.203 0.349 0.326 3.000 2.566 2.273 2.999 GjM8/FAM 2 59 3 3 0.159 0.265 0.157 0.125 2.996 2.910 2.254 2.000 GjM13/HEX 2 60 51 31 0.722 0.557 0.498 0.042 5.000 3.815 2.276 1.875 GjM15/FAM 2 59 9 5 0.794 0.757 0.552 0.539 8.994 8.251 4.422 4.000 GjM48b/HEX 2 56 81 61 0.559 0.571 0.160 0.362 6.999 7.853 3.039 3.862 GR222/HEX 60 111 5 0.625 0.566 0.448 0.434 7.937 8.430 2.575 2.875 GR252/Cyanine3 60 5 3 0.727 0.741 0.512 0.442 5.000 5.000 2.996 2.000

 Heterozygote deficiency.  Multiplexed.

TABLE 3 The number of brolgas, sarus cranes and sarolgas observed across  sites on the Atherton Tablelands in Queensland, Australia (Fig. ) during –.

Life history No. of brolgas No. of sarus cranes No. of sarolgas stage (%) (%) (%) Unidentified* Total no. of cranes (%) Adult 24,870 (55.8) 12,245 (27.5) 84 (0.2) 37,199 (83.5) First-year juvenile 2,491 (5.6) 1,136 (2.5) 0 (0) 3,627 (8.1) Total 27,361 (61.4) 13,381 (30) 84 (0.2) 3,728 (8.4) 44,554 (100)

*Because of poor weather/light conditions.

As there was the possibility that feathers of a particular including a burn-in of ,. Pilot runs with longer bird were collected more than once, identical genotypes Markov chains yielded identical results. We used Structure were identified using Cervus .. (Kalinowski et al., ). Harvester .. (Earl & von Holdt, ) to analyse the Consequently,  samples ( Gulf Plains,  Atherton data, following Evanno et al. (), and STRUCTURE Tablelands,  captive reference A. antigone gillae that was PLOT V. (Ramasamy et al., ) and Adobe Illustrator identical to its sibling) were excluded from the following CS (Adobe Systems Inc., San José, USA) to render the analyses. In  of these cases the same genotype was found graphical output. We defined individuals with admixture in both localities. In these cases the sample from the Qof.–. as hybrids, following Devitt et al. (). Atherton Tablelands was excluded arbitrarily. Missing These thresholds were considerably more conservative in data were not considered to be problematic, as only .% our analysis as we used  loci, compared to three used by of the birds had missing data and only two of the nine Devitt et al. (). However, this restriction was necessary potential hybrids lacked data (at one locus each). It was because the loci used by Devitt et al. () had species- not possible to distinguish siblings from non-siblings specific alleles whereas the two species of cranes we com- because the genetic variance would be too large given the pared shared some alleles. We did not attempt to identify moderate number of markers. different classes of hybrids (F,F and their backcrosses) To identify the number of genetic clusters and potential because this would have required genotyping at least  hybrid individuals we conducted Bayesian clustering using loci (Fitzpatrick, ) and we had only  available. The STRUCTURE .. (Pritchard et al., ; Falush et al., Bayesian clustering approach was validated by k-means ), with K (number of clusters) ranging from one to clustering, which, in contrast to STRUCTURE, is free of five and  replicates, assuming the admixture model and, assumptions about population genetics, using GenoDive in different runs, either uncorrelated or correlated allele fre- .b (Meirmans & van Tienderen, ). Individuals quencies. The Markov chains ran for , generations, were clustered based on their allele frequencies according

to the pseudo F statistic of Caliński & Harabasz (; see Population genetics also Meirmans, ). More detailed population genetic analyses were con- In all STRUCTURE analyses the highest ΔK resulted from ducted based on the clusters identified by Bayesian assuming two clusters, and this was confirmed by k-means clustering, including descriptive statistics and tests for clustering. These clusters corresponded well to the species Hardy–Weinberg equilibrium, linkage disequilibrium, null brolga and sarus crane, as indicated by the reference birds alleles, and population differentiation, using FSTAT ... (Fig. ). As allele frequencies apparently differed consider- (Goudet, ), GenePop . (Raymond & Rousset, ; ably between the species, we based the subsequent ana- Rousset, ) and Micro-Checker . (Van Oosterhout lyses on the STRUCTURE model assuming uncorrelated et al., ). We estimated gene flow using the formula frequencies. Although the majority of birds were either  −   Nm = [( /FST) ]/ (where Nm is the number of effective pure brolga or pure sarus crane, some were admixed migrants) as well as based on private alleles; i.e. alleles (Fig. ). occurring only in one subpopulation (Barton & Slatkin, According to our criteria,  samples belonged to ), the latter in GenePop. Probabilities that two indivi- brolgas ( Gulf Plains,  Atherton Tablelands) and  duals were identical were calculated using Cervus, assuming to sarus cranes ( Gulf Plains,  Atherton Tablelands). either that the two birds were unrelated or, more con- Nine individuals (.%;  Gulf Plains,  Atherton Table- servatively, that they were full siblings (Waits et al., ). lands) had Q scores of .–. (reference: brolga) and In multiple tests, α was Bonferroni corrected. were identified as hybrids (Fig. ), a proportion c.  times greater than in our field observations. Four hybrids had alleles occurring only in brolgas at one or two loci, indicat- Lincoln-Petersen index ing that hybrids are fertile and can interbreed at least with brolgas or other hybrids. We applied the Lincoln–Petersen index to our ‘genetically After exclusion of reference birds and hybrids, we ana- ’ re-trapped birds (nGulf ×nTablelands/nboth) based on meth- lysed the species separately, dividing both into two subpo- odology described by Southwood & Henderson (). pulations corresponding to the localities. Genetic diversity is summarized in Table . Genetic diversity and allelic richness were both considerably higher in brolgas. Brolgas also  – Results exhibited private alleles, per locus, whereas sarus cranes had only eight private alleles, and four loci did not Field observations have any. This translates into much lower probabilities that two individuals will carry the same genotype in brolgas Our observations of cranes on the Atherton Tablelands are than in sarus cranes. The probabilities of finding two unre- − summarized in Table  and Fig. .Of, cranes identi- lated individuals with identical genotypes were . ×  for fiable to species level (of a total of , observations), only brolgas and . for sarus cranes. Assuming the indivi-  were classified as morphologically intermediate sarolgas duals were full siblings, these probabilities increased to (.%). Variation among these intermediate birds meant . and ., respectively. In brolgas and sarus cranes, it was not possible to assess the extent of potential hybrid- four and three loci, respectively, deviated from the Hardy– ization (Plate ). Weinberg equilibrium and the same loci had indications of Our observations of identifiable hybrids on the Atherton null alleles. However, this was probably because there was a Tablelands were concentrated in a relatively small number relatively high number of rare alleles (frequency , %) in of areas (Fig. ), including Innot Hot Springs, Tumoulin/ both species, and therefore it was not considered to be prob- Kaban, Hastie’s Swamp and the Mareeba Wetlands. lematic for our analyses. In any case, the general results of Figure  also illustrates that although they occur in mixed the STRUCTURE analysis did not change after exclusion flocks, brolgas and sarus cranes are differentially distributed of these loci. Linkage disequilibrium did not play a role in across the Atherton Tablelands, with higher numbers of either species. In both species, subpopulations were hardly     –  sarus cranes concentrated on more fertile volcanic soils clo- differentiated, with FST = . ( %CI . . )in ser to Atherton itself, and both brolgas and putative hybrids brolgas and . (−.–.) in sarus cranes. The sig- occurring predominantly in areas of lower intrinsic fertility. nificant result for brolgas is probably attributable to sample At sites with higher counts of either brolgas or sarus size and is not biologically relevant. Estimates of migration cranes, the species with smaller numbers in the flock tended based on the private allele method were high in both species, to have proportionally more first-year juveniles (Table ; with . and . migrants per generation for brolgas and  χ tests: P , . in both cases), potentially increasing sarus cranes, respectively. Transforming FST into estimates  −   opportunities for interactions between species while juveniles of migration using the formula Nm = [( /FST) ]/ ,we are at a critical age for pair-bonding. inferred . and . migrants, respectively.

PLATE 3 Heads and necks of typical species phenotypes and presumed hybrids. (a) Typical brolga; (b) sarolga (this bird was congruent with a typical brolga, except that it had pink legs, slightly more red on the nape of the comb than is typical of brolgas, and a very small, almost non-existent wattle; this combination of characters had not hitherto been recorded); (c) typical sarolga, with much shorter neck comb and slight wattle; and (d) typical Australian sarus crane. (Photos a, c & d: T. Nevard; b: B. Johnson)

continued to note the presence of apparent hybrids amongst TABLE 4 Total number of cranes (and % juveniles) at sites domi- dry season, non-breeding crane flocks on the Atherton nated by either brolgas or sarus cranes. Tablelands (Matthiessen, ) and elsewhere. We have No. of brolgas No. of sarus cranes shown that natural hybridization between brolgas and (% juveniles) (% juveniles) sarus cranes is happening and occurs more widely than is Brolga-dominated sites 25,510 (8.8) 2,273 (14.5) visually detectable. As hybridization is still relatively rare Sarus-dominated sites 1,851 (13.3) 11,308 (8.9) and brolgas and sarus cranes are differentially distributed Total 27,361 (9.1) 13,581 (9.8) on the Atherton Tablelands (Fig. ), its evolutionary influ- ence could potentially be relatively limited. However, a closer look at the allele combinations in the hybrids suggests – Lincoln Petersen index that introgression could become more widespread. Four of the birds had alleles that occur only in brolgas at one or two In  cases the same genotypes were collected at both Miranda loci. Although one of these birds had a membership index of Downs in the Gulf Plains and at Innot Hot Springs in the ., suggestive of being an F-hybrid, these pure brolga com- southern Atherton Tablelands. Of these, nine were attribut- binations indicate that the birds are hybrids of a later gen- able to brolgas and two to sarus cranes. According to the eration with a hybrid parent that back-crossed with a brolga probabilities of encountering two individuals carrying or another hybrid. Sarolgas therefore appear to be fertile at identical genotypes by chance with our sample sizes, only several generational recombinations. the two sarus cranes could be cases of chance identity, Longitudinal research on Darwin’s finches Geospiza spp. provided the birds were siblings (see above). Applying the has shown that they evolved from a common ancestor in the Lincoln–Petersen index (n ×n /n ; Southwood Gulf Tablelands both Galápagos archipelago in the last  million years (Grant & &Henderson,) to these results implies that at least Grant, ), with both natural selection and introgressive , brolgas and  sarus cranes could have moved between hybridization being key processes in their evolution. At this the Gulf Plains and Atherton Tablelands. stage it is not possible to predict the trajectory of brolga/ sarus crane introgression but Lamichhaney et al. () Discussion demonstrated that introgression at only a single locus may have important evolutionary consequences. Interaction of Since Archibald’s first observation of brolga–sarus crane introgression and selection may be complex and locality hybrids in  (Archibald, ), local observers have dependent, as shown for the European crow Corvus corone

(Vijay et al., ), in which genetic and phenotypic differentiation varies significantly across its range, with some boundaries being firm and others mobile. Ongoing brolga/ sarus crane introgression may provide alleles to either species that could facilitate their adaptation to environmental changes brought about by such stresses as climate change or agricultural intensification; however, whether the larger body size of hybrids identified by Archibald () confers a selective advantage over the parental species, potentially leading to its displacement, cannot yet be determined. The current Australian population of sarus cranes appears to have increased relatively rapidly from what is thought to have been small numbers in northern Australia since its formal identification in the s (Gill, ). Archibald () estimated the sarus crane population was c.  on the Atherton Tablelands in , when he identified at least two and possibly six potential brolga–sarus crane hybrids (Archibald, ). Crops have been grown in northern Australia since the late th century and agriculture expanded substantially on the Atherton Tablelands in the s, soon after Gill’s first observation of sarus cranes. Templeton et al. () found that changes in landscapes can be associated with genetic consequences, including introgression, which threat- ens species integrity (e.g. kākāriki Cyanorhamphus spp., Triggs & Daugherty, , and black-eared miners Manorina melanotis, Clarke et al., ). Landscape features and het- erogeneity can also be reflected in population genetic structure within and across species (Miller & Haig, ; Safner et al., ), and often changes in landscape are associated brolgas and sarus cranes, ordered horizontally by decreasing Q, with brolga as reference. The rectangle with habitat loss, contributing to population decline and  possibly resulting in changes in the genetic diversity of re- maining populations (Templeton et al., ; Keyghobadi, ). That the species with fewer individuals in a mixed- species flock has proportionally more first-year juveniles means that young brolgas and sarus cranes are more ex- posed to one another other at a time when imprinting is important (Horwich, ). This may make pairing with the other species more likely. The enduring pair bond and lon- gevity in both species (Johnsgard, ) suggests that, once formed, hybrid pairs could contribute numerous hybrid off- . The bar underneath indicates the origin of birds and reference specimens. 

. spring to the population. Although we cannot yet predict showing admixture (Q) among a total of  –  the trajectory of crane introgression in northern Australia,  . = 

K there are clear conservation implications. The sarus crane is thought to be declining in much of its Asian range, particularly in Burma, Thailand and Indochina (Meine &

plot with Archibald, ; Archibald et al., ). The maintenance of Australian sarus crane populations and their genetic integrity may therefore provide security for the species should populations continue to decline in Asia. However, there

STRUCTURE seems to be no practical way of eliminating Australian 

.3 crane introgression in the wild, as ( ) hybrids are not neces-

IG  F includes nine hybrids, with Q = sarily visually distinguishable in the field, ( ) Australian

cranes occur in remote and inaccessible areas, and () the Although feathers have the advantage of being a non- underlying behavioural stimuli are not yet understood. If re- invasive means of gathering genetic data on individuals, as lated to anthropogenically driven land-use change, brolgas noted by Hou et al. (), the probability of detecting and sarus cranes may be brought into ever-closer and hybrids was .  times greater using genetic analysis rather more frequent foraging and flocking proximity, potentially than visual observation. However, the boundaries between leading to more opportunities for hybridization. Therefore, pure and hybridized birds are unclear and are probably in- should the maintenance of genetically pure populations of definable given that hybrids are fertile, meaning that intro- Australian sarus cranes and northern Australian brolgas gression may have reached the point where it has no clear be considered desirable for conservation purposes, one or thresholds. A subject of future research could therefore be more well-managed extralimital populations may need to the definition of the relationship between genetics and be established. As far as we know, the only captive flock of phenological expression, using the feathers of known birds Australian sarus cranes is in Lemgo, Germany, whose ances- with hybrid phenotypes, and using data from more than the try dates from a wild capture in the s. This could poten-  loci we had available (Fitzpatrick, ). In addition, tially form a nucleus for a captive metapopulation, spread genomic approaches targeting coding loci (Payseur & across several institutions. Establishing a flock managed to Rieseberg, ) would provide a deeper insight into the maintain its genetic variability at this stage could also serve evolutionary consequences of the hybridization, as the ex- as insurance against climate change impacts, with some ample of Darwin’s finches has shown (Lamichhaney et al., models predicting a potential % decline in climatic ). However, to obtain DNA of sufficient quality and suitability for the Australian sarus crane by  (Garnett in the necessary quantities would necessitate catching et al., ). many more cranes to obtain tissue and blood samples. As we have collected feathers from what appear to be the Whilst acknowledging that the proportion of apparent same birds in both the Gulf Plains and Atherton Tablelands, hybrids identifiable by observers is likely to be an order of considering the low probabilities of encountering genetical- magnitude lower than are actually present in the crane ly identical individuals, we have been able to confirm what population, both visual observation and analysis of shed has hitherto only been assumed by birdwatchers in North feathers could provide valuable collateral data to monitor Queensland, that cranes migrate between breeding areas the trajectory of Australian crane introgression. in the Gulf Plains and the Atherton Tablelands, a distance of c.  km. There appears to be no material genetic differ- Acknowledgements We acknowledge the assistance of many people who have made this work possible, including Elinor Scambler and John ence between Gulf Plains and Atherton Tablelands cranes, Grant, who shared their insights on cranes on the Atherton Tablelands indicated by estimates of population differentiation and mi- and Gulf Plains; Inka Veltheim and Adam Miller in relation to brolgas in gration after exclusion of the duplicate genotypes. The con- Victoria; Silke Fregin, who managed our large number of samples and servation implications of this are significant, as both areas laboratory work at the University of Greifswald; Annabelle Olsson, will need to be targeted with appropriate conservation mea- who facilitated veterinary health clearance for our samples to be sent to Germany; Harry, Ruairidh and Bronwen Nevard, and Dominic and sures to ensure the survival of north Queensland’s brolgas Vera von Schwertzell, who assisted with sample collection; Hans Rehme and sarus cranes. There are currently no laws protecting of Lemgo, who provided access to his collection of cranes; Tom and species that migrate within Australia (Runge et al., ). Tanya Arnold (Miranda Downs cattle station), the Waddell family The identification of identical birds in both the Gulf (Woodleigh cattle station), Terry Trantor, Nick Reynolds, the Godfrey Plains and Atherton Tablelands based on shed feathers family and Fay Moller-Nielsen, who all provided access to their farms; highlights the potential of genetic analyses to explore migra- and Angela Freeman of the North Queensland Wildlife Trust, who provided funding for laboratory costs. tion patterns and potential trends, not only in Australian cranes but also in other species. Although our collection Author contributions Conception of research, fieldwork, sample of feathers was opportunistic rather than deliberately ran- collection, data analysis, writing: TDN; sample collection, supervision dom and our estimates using the Lincoln–Petersen index of laboratory work, genetic analysis, writing: MH; first identified the are therefore necessarily indicative, they are of the same sarolga, facilitation of research, writing: GA; GIS-based mapping, data analysis, writing: IL; institutional supervision, facilitation of research, order as other estimates of crane populations on the data analysis, writing: STG. Atherton Tablelands (Brolga –, counted, , calculated; sarus crane –, counted,  calculated). Conflicts of interest None. Similarly to the use of non-invasive genetic monitoring techniques elsewhere, such as for large carnivores in Ethical standards The research was carried out in accordance with Europe (Kaczensky et al., ), feather genetics could Charles Darwin University Animal Ethics Committee’s approval contribute to the assessment of population trends and A13019 and Queensland Department of Environment and Heritage Protection Scientific Purposes Permit WISP13984714. Export of movements of cranes within the Atherton Tablelands and samples to Greifswald University was undertaken under Australian between the Atherton Tablelands and other locations Customs permit ACLTEM47 K and CITES permit PWS2014-AU- where either species occurs. 001240. The authors abided by the Oryx Code of Conduct for authors.

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