Ancient host specificity within a single species of brood parasitic bird Claire N. Spottiswoodea,b,1, Katherine Faust Stryjewskic, Suhel Quadera,2, John F. R. Colebrook-Robjentd,3, and Michael D. Sorensonc aDepartment of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom; bPercy FitzPatrick Institute, Department of Science and Technology/ National Research Foundation Centre of Excellence, University of Cape Town, Rondebosch 7701, South Africa; cDepartment of Biology, Boston University, Boston, MA 02215; and dMusumanene Farm, Choma, Zambia Edited by John C. Avise, University of California, Irvine, CA, and approved August 30, 2011 (received for review June 14, 2011) Parasites that exploit multiple hosts often experience diversifying spite interbreeding among males and females reared by different selection for host-specific adaptations. This can result in multiple hosts. In both cuckoos and cowbirds, however, females with strains of host specialists coexisting within a single parasitic identical or closely related mtDNA haplotypes were associated species. A long-standing conundrum is how such sympatric host with different hosts, indicating frequent host shifts over evolu- races can be maintained within a single parasitic species in the face tionary time (13, 14). Parentage analyses based on both micro- of interbreeding among conspecifics specializing on different satellite loci (12, 17) and radio telemetry (18) indicate that hosts. Striking examples are seen in certain avian brood parasites female common cuckoos occasionally lay eggs in the nest of such as cuckoos, many of which show host-specific differentiation more than one host species. A recent study showing slight but in traits such as host egg mimicry. Exploiting a Zambian egg col- significant genetic structure among male common cuckoos as- lection amassed over several decades and supplemented by recent sociated with different hosts (17) suggests an alternative model fieldwork, we show that the brood parasitic Greater Honeyguide in which egg mimicry evolves through strong selection on auto- Indicator indicator exhibits host-specific differentiation in both egg somal genes in partially isolated local populations. Thus, the size and egg shape. Genetic analysis of honeyguide eggs and chicks stability and evolutionary longevity of cuckoo gentes and their show that two highly divergent mitochondrial DNA lineages are adaptation to specific hosts remains uncertain. associated with ground- and tree-nesting hosts, respectively, indi- Clearer patterns of genetic diversification in relation to host cating perfect fidelity to two mutually exclusive sets of host species use are perhaps most likely in tropical species, which may ex- for millions of years. Despite their age and apparent adaptive perience relatively greater climatic stability (19–21) and hence diversification, however, these ancient lineages are not cryptic longer periods of host–parasite coevolution (22); the vast ma- species; a complete lack of differentiation in nuclear genes shows jority of avian brood parasites are tropical in distribution (23). In that mating between individuals reared by different hosts is suffi- this study, we investigate both phenotypic and genotypic di- ciently frequent to prevent speciation. These results indicate that versification in relation to host use in the Greater Honeyguide host specificity is maternally inherited, that host-specific adapta- Indicator indicator. This Afrotropical species is perhaps best tion among conspecifics can be maintained without reproductive known for its remarkable mutualistic interactions with human isolation, and that host specificity can be remarkably ancient in honey gatherers (24), but is also an obligate brood parasite that evolutionary terms. exploits a range of host species at any given geographical loca- tion (25). Parasitized hosts suffer high fitness costs because the coevolution | gentes | phylogeography | W chromosome hypothesis young honeyguide stabs host young to death as soon as they hatch, using needle-sharp hooks at the tips of its bill (25, 26). pecies interactions can be powerful generators of evolution- Greater honeyguides parasitize hole-nesting host species that Sary diversity (1, 2). In host–parasite relationships, different produce eggs of varying color, size, and shape. Host species are hosts can exert divergent selection for parasitic specialization. drawn primarily from the Coraciiformes and Upupiformes, some Although this sometimes results in speciation in the parasitic of which have eggs that differ in coloration from the invariably lineage (3–5), in other cases multiple strains of host specialists white eggs of honeyguides (Fig. 1). Color differences, however, coexist within a single parasitic species. A textbook example is are likely to be imperceptible in the darkness of deep tree holes – seen in avian brood parasites such as cuckoos, in which the and terrestrial burrows. A more important aspect of host para- individuals of a single parasitic species specialize on different site matching may be tactile cues provided by egg size and shape (27–29), which we might thus predict to show host-specific spe- hosts and show phenotypic differentiation in traits such as host fi egg mimicry (6, 7). How phenotypically distinct host races [also cialization. In this study, we rst test for evidence of phenotypic differentiation in egg size and shape among honeyguides para- known as “gentes” in the context of avian brood parasitism (8)] sitizing different hosts. We then test for evidence of genetic can evolve and be maintained over evolutionary time in the face differentiation in relation to host use, with particular interest of interbreeding among conspecifics has long remained para- in comparing patterns of differentiation in maternally inherited doxical (9–12). Genetic data have provided important insights by allowing a test of whether lineages of parasitic females consistently spe- Author contributions: C.N.S. and M.D.S. designed research; C.N.S., K.F.S., J.F.R.C.-R., and cialize on particular host species. Studies of the common cuckoo M.D.S. performed research; C.N.S., K.F.S., S.Q., and M.D.S. analyzed data; and C.N.S., S.Q., Cuculus canorus (13) and the shiny cowbird Molothrus bonar- and M.D.S. wrote the paper. iensis (14), both of which show host-specific phenotypic differ- The authors declare no conflict of interest. entiation in egg traits (7, 15), have revealed subtle genetic This article is a PNAS Direct Submission. differentiation in mitochondrial DNA (mtDNA) haplotype fre- 1To whom correspondence should be addressed. E-mail: [email protected]. quencies in relation to host use. These results are potentially 2Present address: National Centre for Biological Sciences, GKVK Campus, Bellary Road, consistent with the long-standing hypothesis that egg mimicry Bangalore 560065, India. might be controlled by genes on the maternally inherited W 3Deceased November 17, 2008. ’ chromosome [Punnett s sex chromosome hypothesis (10, 16)], This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thereby allowing host-specific adaptation of female lineages de- 1073/pnas.1109630108/-/DCSupplemental. 17738–17742 | PNAS | October 25, 2011 | vol. 108 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1109630108 Downloaded by guest on September 25, 2021 Fig. 2. Size (A) and shape (B) of Greater Honeyguide eggs in relation to those of host eggs in the same nest. Data points are individual parasitized Fig. 1. Host and Greater Honeyguide eggs from representative parasitized clutches from the Choma District of Zambia, with different host species in- clutches for each host species in Zambia. Host eggs are in the left column and dicated (A, Inset). The regression lines (solid lines) result from generalized parasite eggs are in the right column. Subtle host-specific differentiation in estimating equations that take into account nonindependence of parasit- honeyguide egg size and shape (Fig. 2) is visible even to the human eye: note ized clutches within host species. The dotted lines show the expectation if EVOLUTION the small honeyguide egg from a little bee-eater nest and the rounder egg there were perfect correspondence between parasite and host egg traits, ’ from a striped kingfisher nest. Clutches were selected from near the middle emphasizing that honeyguides do not perfectly match their hosts eggs. of the honeyguide egg size and shape distributions. (Photos courtesy of Note that the axes are displayed on a log scale so the untransformed units M. D. Anderson, B. Danckwerts, and W. R. Tarboton.) are displayed. mtDNA vs. biparentally inherited nuclear DNA. Fidelity of Do Greater Honeyguides Show mtDNA Genetic Divergence in Relation female lineages to particular hosts combined with assortative to Host Use? To test for genetic divergence among honeyguide females parasitizing different host species, we sequenced mating of males and females raised by the same host species Methods would be reflected in both genomes and could lead to speciation. mtDNA ( ) from samples of known host origin: shell In contrast, if host-specific female lineages mate randomly with membranes from honeyguide eggs in parasitized clutches and respect to the host species of males, we would expect to find blood or tissue from parasitic nestlings (Table S2). We found that two highly divergent mtDNA lineages (genetic distance = host-related genetic structure in mtDNA only. 14.8% for the ND2 gene) were perfectly associated with two A Results groups of host species (Fig. 3 ): those