Fowl Play:
Molecular evidence of interbreeding
between wild and feral domestic Helmeted Guineafowl
Numida meleagris
A.L. Walker
Percy FitzPatrick Institute of African Ornithology
University of Cape Town
Rondebosch, 7701
Cape Town, South Africa.
Supervisor: Prof T.M. Crowe
Thesis submitted in partial fulfilment of the requirements for the degree of Master of
Science in Conservation Biology, University of Cape Town.
June 2000
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The copyright of this thesis rests with the University of Cape Town. No
quotation from it or information derived from it is to be published
without full acknowledgement of the source. The thesis is to be used
for private study or non-commercial research purposes only.
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1. Title: Fowl Play: Molecular evidence of interbreeding between wild and feral domestic
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Helmeted Guineafowl Numida meleagris
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2. Authors: A.L. Walker, C.S. Ratcliffe, R.C.K. Bowie and T.M. Crowe
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3. Full Postal Address: Percy FitzPatrick Institute, University of Cape Town,
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Rondebosch, 7701 South Africa
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4. Date Received: XX XXXX 2000
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5. Keywords: conservation, Numida meleagris, Helmeted Guineafowl, hybridisation,
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mitochondrial DNA.
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6. Corresponding Author: Assoc. Prof. Timothy M. Crowe; Percy FitzPatrick Institute,
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University of Cape Town, Private Bag, Rondebosch 7701, South Africa; Tel. +27-21-
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650-3292/1; Fax: +27-21-650-3295; E-mail:
[email protected];.Q9-:.!1~L~g_~~~
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7. Running Title: INTRASPECIFIC HYBRIDISATION IN GUINEAFOWL
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28 pages -- Total
= 4206 words -- Abstract 179 words
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Abstract
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Since the late 1980s, many populations of the Helmeted Guineafowl Numida meleagris in
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the Midlands of KwaZulu-Natal, South Africa have collapsed or gone locally extinct.
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They have also become considerably more fragmented .thanthey were in the 1970s, when
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seasonal hunting bags were massive. This study investigates the possibility that
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hybridisation between introduced domestic guineafowl, originating from West Africa's
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N. m. galeata, and southern African guineafowl, N. m. coronata, might have contributed
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to these population collapses. There is morphological evidence of such hybridisation in
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wild populations and it is known that domestic guineafowl do not survive well in the
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wild. Molecular analysis of the control region ofmtDNA confirmed the occurrence of the
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domestic guineafowl haplotype in individuals present in wild populations of KwaZulu-
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Natal. The development of a simple diagnostic test based on restriction fragment-length
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polymorphisms allowed for a rapid and inexpensive assessment of hybridisation in wild
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guineafowl populations. The low level at which such hybridisation appears to occur at
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present suggests that genetic 'pollution' due to introgression from domestic guineafowl
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may not be a major factor negatively influencing wild guineafowl populations.
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Introduction
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One of Africa's most widespread and abundant gamebirds, the Helmeted Guineafowl
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Numida meleagris (hereafter called guineafowl), can be found in a broad range of sub-
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Saharan, open-country vegetation types (Crowe et al. 1986). Of the nine subspecies of
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Helmeted Guineafowl, only N. m. coronata, occurs within South Africa (Crowe 1978).
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Over the last century this gamebird has undergone a dramatic expansion, both in range
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and population size, due to deliberate translocations by humans and the ability of
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guineafowl to exploit low-level, agriculture conducted in a mosaic of natural vegetation
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types and urban landscapes (Little 1997). This guineafowl has also provided
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wingshooters (gamebird hunters) with superb quarry, making it the most popular upland
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gamebird in South Africa (Crowe 2000a). However, since the late 1980s, guineafowl
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populations in the Midlands of KwaZulu-Natal province, which once supported some of
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the best guineafowl wingshooting in Africa, have collapsed, in some cases -to local
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extinction (Johnson 1984; Pero & Crowe 1996). A range of agents, including the
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widespread use ofagrochemicals (especially pesticides), disease, illegal hunting and loss
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of habitat due to increases in the proportion of land under intensive, mono-culture
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cultivation have been proposed as important factors responsible for the decline and
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extinction of guineafowl populations (Pero & Crowe 1996; Malan & Benn 1999; Crowe
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2000b). This decline in the guineafowl populations highlights the detrimental impact
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modern agricultural practices may have had on many species of wildlife, causing changes
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in, orlosses of, biological diversity (Potts 1980; O'Connor & Shrubb 1986; Rand et al.
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1988).
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The plight of guineafowl populations in KwaZulu-Natal is of great economic and
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ecological concern because of its value as a gamebird and as an indicator species of a
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healthy African landscape rich in birdlife (Little & Crowe 1994; Pero & Crowe 1996;
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Crowe 2000b). An opportunistic omnivore, this guineafowl also eats large. quantities of
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destructive insects and weeds, contributing to the production of healthy crops.(Little et al.
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1995; Witt et al. 1995) and indirectly increasing agricultural revenues. From a
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commercial wingshooting perspective, guineafowlprovide a tangible financial return
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(approximately US$20 per bird) which can supplement incomes in rural communities
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(Crowe & Little 1993), thereby fostering the development of a conservation ethic based
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on sustainable use (Pero & Crowe, 1996).
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Wolff and Milstein (1987) suggested that another potential threat to guineafowl
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populations is the introduction of domesticated guineafowl into wild populations. This is
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because interbreeding between domestic and wild birds might undermine the ability of
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their offspring to survive in the wild (Hastings Belshaw 1985; Crowe 2000b). The
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guineafowl has been domesticated since the times of ancient Greece and Rome, with the
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most recently domesticated stock originating from West Africa (N. m. galeata) (Ghigi
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1936) and introduced repeatedly world-wide (Long 1981; Hastings Belshaw 1985;
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Donkin 1991). Overthe centuries, selective breeding has produced domestic guineafowl
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with anatomy, plumage and behaviour quite distinct from that of wild guineafowl. The
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distinguishing characteristics of domestic guineafowl include: rapid growth to greater
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body mass; shorter yellow legs (black in wild type); white claws (black in wild type);
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reduced inclination to care for downy offspring; increased egg production and differing
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plumage, often with large amounts of white feathers (Hastings Belshaw 1985; Wolff &
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Milstein 1987). In addition to these novel features, domestic guineafowl possess
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attributes which characterise its wild ancestors (N. m. galeata), e.g. white faces (bluish in
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N. m. coronata) and rounded gape wattles (pennant-shaped inN. m. coronata) (Fig. 1).
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The guineafowl of KwaZulu-Natal currently occur as small populations persisting in
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fragmented habitats; and are often in close contact with introduced domestic guineafowl
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(Crowe 2000b; Ratcliffe & Crowe in review). These are conditions typical of other
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hybridised populations (Lehman et al. 1991; Ward et al. 1999). Hybrids and hybrid
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zones, particularly between subspecies are a natural occurrence (O'Brian & Mayr 1991).
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However, the interbreeding between native and introduced taxa, even on a small-scale,
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has the potential to cause widespread genetic introgression, jeopardising a species'
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genetic integrity. Indeed, the characteristics produced by artificial selection that make the
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domesticated guineafowl commercially valuable could undermine the viability of wild
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populations if interbreeding did take place(Crowe 2000b).
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Reports of individuals that are morphologically intermediate between wild and
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domestic guineafowl within wild guineafowl populations from Kwazulu-Natal suggest
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that such hybridisation is occurring (Ratcliffe & Crowe 1999). Furthermore, across
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Africa, individuals within the Helmeted Guineafowl species-complex at hybrid zones
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(intergrades) do show characters intermediate to the often strikingly distinct parental
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subspecies (Ghigi 1936; Crowe 1978). Similarly, hybrid offspring of guineafowl (as with
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other gaUifermse;g;Junglefowl) appear to be morphological intergrades of both the
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parental varieties (Ghigi 1936; Hastings Belshaw, 1985).
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Rossouw (1996) provided direct molecular evidence for the introgression of domestic
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guineafowl DNA into wild populations in South Africa when, as part of a study of the
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molecular phylogeography of Helmeted Guineafowl, she discovered diagnostic sequences
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(from D-loop - control region - of mtDNA) for domestic guineafowlin captive birds
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which appeared morphologically intermediate between wild and domestic birds. Much
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more disturbingly, she also found the domestic haplotype in one bird in the wild, which
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morphologically appeared to be a pure wild type guineafowl. Thus, there appeared to be
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morphologically undetectable genetic introgression from domestic guineafowl into wild
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populations.
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Based on molecular data, there are many cases reporting introgression after contact
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with introduced species (Largiader & Scholl 1996; Thulin et al. 1997; Ward et al. 1999).
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The non-recombinant inheritance and rapid substitution rate of mitochondrial DNA
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(mtDNA) provides an effective marker to screen forhybridisation at the individual and
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population level (Carr et al. 1986; Lehman et al. 1991; Thulin et al. 1997; Wardet al.
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1999). In this study, the occurrence and extent of genetic introgression in wild Helmeted
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Guineafowl from the provinces of KwaZulu-Natal and the Free State provinces of South
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Africa is assessed using a molecular analysis of sequence data. Sampling of areas was
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done in a more intensive manner than Rossouw's (1996) more broadscale study which
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examined relatively fewer samples, but from more localities, on an Africa-wide scale. In
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addition to sequencing analysis, a simple, relatively inexpensive, diagnostic test using
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restriction fragment-length polymorphisms (RFLPs) was sought to help identify domestic
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guineafowl mtDNA in wild guineafowl. .This method of genetic testing could allow a
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rapid and relatively inexpensive assessment of hybridisation in wild guineafowl
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populations, and prove to be invaluable In facilitating informed management and
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conservation.decisions.
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It is essential that issues pertaining to the damaging effects of such hybridisation be
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addressed for the imp~ementation of effective conservation •. and management strategies.
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Therefore, the identification of hybridisation in natural populations is an increasingly
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important issue in conservation biology. Moreover, it is fundamental for the sustainable
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utilisation of economically important species, such as the guineafowl, that the genetic
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integrity of wild populations is maintained (Largiader & Scholl 1996).
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Materials and methods
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Sample collection
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Guineafowl (n
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and were characterised morphologically as domestic, intermediate or wild, based on
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coloration of plumage, face and legs, and the shape of the wattles (Fig. 1). A further 20
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samples were obtained from a farm in the vicinity of Petrus Steyn in the Free State (270
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39' I" S; 28 0 7' 40" E). These individuals were described as either wild or possibly
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introgressed in appearance. There were very few specimens from the Free State described
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as putative hybrids «20% of a total bird bag>1000), whose identification was based on
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the presence of white claws, some yellow scales on the legs, or very pale grey
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backgroundplumage:
=
36) were collected from populations in the Midlands of KwaZulu-Natal
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Laboratoryprocedures
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DNA was obtained from liver and heart tissue preserved in saturated sodium chloride
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(NaCl) with 20 % dimethylsulphoxide (DMSO) (Amos & Hoelzel 1991). Total genomic
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DNA was extracted from 56 samples using a standard proteinase K digestion followed by
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phenol chloroform extraction (Sambrook et al. 1989).
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A ~550 bp fragment was amplified from total genomic DNA using primers that targeted
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the 5' end of the control region (D-loop) modified according to published chicken
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sequences (Desjardins & Marais 1990): L16746 5'-ACC CCA AGG ACT ACG GCT
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TGA A-' (Wenink, et al. 1994) and H522 5'-GeC TGA CCG AGG AAC CAG AG-3'
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(Quinn & Wilson 1993).
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Amplification reactions were performed using 0.2 U of Taq (Bioline), Ix Taqbuffer
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(lOx Stock), 2.5 roM MgCh, 3 mM dNTPs, 0.5 pmols of each primer and 1 ~L of DNA,
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in a total volume of 50 ~L. The thermal profile was 27 cycles at (94°C for 45 s; 68°C for
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45 s; 71°C for 60 s). The PCR products (10 ~L) were run on 1% agarose gels (Techcomp
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LTD) for 1 h at 80 V. Gels were stained with ethidium bromide and visualised using UV
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light. The PCR products were purified using a Concert" rapid PCR purification system
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(Life Technologies), a cycle sequenced using a dye terminator cycle sequencing kit and
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an ABI 373 STRETCH automated DNA sequencer. . For verification both strands were
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.sequenced separately. The sequences were aligned using DAPSA (Harley 1999).
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Identification ofRFLPs
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A restriction enzyme analysis (DNAMAN 4.13, Lynnon BioSoft) using sequence data
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identified a potential diagnostic RFLP site between domestic and wild N. meleagris
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within the mtDNAPCR product. Restriction enzyme digestion was, therefore, performed
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on amplified·DNA with MspI in an attempt to identify wild and domestichaplotypes by
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RFLP analysis. The fragments were separated on a 2% agarose gel for f:-3 h, stained
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with ethidium bromide and photographed under UV light.
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Results
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Morphology
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Each of the guineafowl collected was characterised as wild, domestic or intermediate
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(Fig. 1; Table 1).
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MIDNA Sequence data
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Sequence data identified six diagnostic sites in the control region, which distinguish wild
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from domestic haplotypes (Table 2). Pure domestic guineafowl occurred only in the
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populations from KwaZulu-Natal, and had a haplotype distinct from that of wild birds
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from KwaZulu-Natal and the Free State. In KwaZulu-Natal, three of the putative hybrids
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and one individual described as wild were identified as possessing the domestic
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haplotype (Table 1). In the Free State samples, all individuals described as wild and
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putatively hybridised possessed the wild bird haplotype (Table 1).
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RFLPs
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The MspI restriction enzyme digestion of the 550 bp mtDNA D-Ioop region produced
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distinct patterns for domestic and wild individuals, depending upon the presence or
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absence of a single restriction site (Table 3). This single restriction site corresponded to
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the third diagnostic site at the 190 bp position based on sequence data (Table 2). This
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MspI restriction site is absent in wild guineafowl. The validity of the diagnostic fragment
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pattern was confirmed with individuals whose haplotype had been identified by sequence
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data.
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Discussion
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Genetic evidence ofhybridisation
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The findings of this investigation confirm that there is intraspecific hybridisation between
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wild and domestic guineafowl populations in KwaZulu-Natal, and the identification of a
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diagnostic restriction fragment pattern provides a use:fulmethod for rapidly determining
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the mtDNA haplotype of any given individual. The presence of hybrids confirms the
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suspicion that morphologically intermediate individuals may indeed be hybrids. The lack
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of a 1 : 1 correspondence between morphological and molecular evidence of
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hybridisation may be a result of low levels of intraspecific mating or problems associated
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with using a maternally inherited molecular marker such as mtDNA. To assess the :full
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extent to which hybridisation has taken place between domestic and wild guineafowl
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populations in KwaZulu-Natal, a biparentally inherited nuclear marker needs to be used
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(Compton 1990).
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It is also possible that the decline of wild guineafowl populations, as a result of habitat
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fragmentation and modern agricultural practices (Ratcliffe & Crowe in review),
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facilitated hybridisation with introduced domestic guineafowl which were released in the
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hope of resuscitating declining populations (Crowe 2000b). There are several other well-
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documented examples where introduced domestic taxa have hybridised with native
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species under similar conditions (Hubbard et al. 1992; Butler 1994; Ward et al. 1999).
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The identification of only one morphologically 'wild' individual from KwaZulu-Natal
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with the domestic haplotype also confirms that birds with a wild-type morphology may
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have a hybrid ancestry. Thus, it is not always possible to identify hybrids
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morphologically, and both morphological and molecular analyses are necessary to
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confirm the occurrence of hybridisation conclusively (Rhymer & Simberloff 1996;
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Wilson & Bernatchez 1998). In addition, this result reiterates that, based upon this study,
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morphology may only be suggestive of hybridisation or the lack thereof, and not
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indicative.
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The apparent absence of introgressive hybridisation in populations from the Free State
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is, perhaps, not unexpected.
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defined individuals from this area as putative hybrids were much more subtle (i. e. traces
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of yellow on legs and one or more white claws on toes) than those apparent in
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intermediate birds from KwaZulu-Natal. In addition, no individuals described as pure
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domestic in morphology were identified in the populations from the Free State. The
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occurrence of discoloration on the feet may, therefore, be explained by natural local
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variation (e.g. private alleles, local inbreeding effects, etc.), and were not phenotypic
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expressions of introgression, rather a population-specific variation.
This is because the morphological characteristics that
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Conservation implications
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Although detailed study of habitat use by guineafowl (Ratcliffe & Crowe in review)
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suggests thatthe primary, underlying cause of populations collapses in the Midlands of
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KwaZulu-Natal is a breakdown in meta-population structure due to habitat destruction
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and fragmentation, hybridisation in wild guineafowl populations should be taken into
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account in the implementation of effective management strategies. The identification of
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populations with hybrids is essential if conservation efforts are to be targeted correctly,
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since the efficiency of reproduction in, and overall fitness of introgressed populations
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may be relatively low. The diagnostic restriction fragment pattern shown in this study is
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an easy and rapid genetic test in this identification process. The potential implications of
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hybridisation between wild and domestic for the conservation of guineafowl are
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multifaceted:
The mixing of gene pools and the loss of genotypically distinct populations,may
jeopardise the genetic integrity of the species (Rhymer & Simberloff 1996). An
important role of biological conservation is to safeguard against the loss of
genetic purity and locally adapted genetic variation, regardless of the taxonomic
status, to maintain levels of biological diversity.
Domesticated animals have been selected for survival and reproduction in
captivity; and not wild conditions. These individuals may have characteristics that
are maladaptive in the wild and, in turn, may reduce the viability of populations in
natural systems (Wolff & Milstein 1987; Rhymer &Simberloff 1996).
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Furthermore, reduced viability of hybrid individuals might exacerbate a decline in
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wild populations, or perhaps retard recovery.
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iii)
The maintenance of genetically unique populations is important for the
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sustainable management of this economically important species (Largiader &
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Scholl 1996). The implications of translocating or re-stocking populations with
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individuals with a hybrid ancestry would be counter-productive to the long-term
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success of populations. Indeed, the objective of the study by Rossouw (1996) was
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to determine the genetically most suitable donor populations for a conservation
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programme to restore guineafowl populations in depleted areas within KwaZulu-
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Natal.
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For the Helmeted Guineafowl, its economic value as a gamebird underpinning a local
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hunting industry might become undermined by the potentially detrimental impacts of
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hybridisation. However, the fact that only one 'wild-type' individual tested positive for
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DNA from domestic guineafowl suggests that the effects of morphologically undetectable
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introgression from domestic birds into wild populations may be relatively minor. Further
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research into this problem should focus on the breeding success of feral, genetically
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introgressed individuals that resemble wild birds to assess the viability of hybrids which
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have back-crossed with wild birds. In the meantime, mitigation measures which involve
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the removal ofmorphologicaHyintermediate birds from wild populations seem to be
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justified.
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Acknowledgements
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We are grateful to Dr. Colleen O'Ryan for the use of laboratory facilities and Geeta Eick
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and Carol Logan . for technical advice, and Krystal Tolley for comments on the
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manuscript. We thank Jacqueline Bishop for the diagrams used.in.Figure 1, and for her
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invaluable intellectual input, encouragement and support. We thank the National
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Research Foundation and the South African Department of Trade and Industry (THRIP
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Initiative) for financial support, and Leon Kruger, Mark Haldane and Peter Wales for
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help with collecting specimens.
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References
311
312
313
Amos B, Hoelzel AR (1991) Long-term preservation of whale skin for DNA analysis.
314
Report for the International WhalingC0111111ission,SpeciaFReport Issue 13, 99-
315
104.
316
317
Butler D (1994) Bid to protect wolves from genetic pollution. Nature, 370, 497.
318
319
Carr SM, Ballinger SW, Derr IN, Blankenship LI-I, Bickham JW (1986) Mitochondrial
320
analysis of hybridization between sympatric white-tailed deer and musk deer in
321
west Texas. Proceedings of the National Academy of Science USA, 83, 9576-
322
9580.
323
324
Compton DE (1990)· Application of biochemical and molecular markers to analysis of
325
hybridisation. In: Electrophoretic and isoelectric focusing techniques in fisheries
326
management (ed. Whitmore DD), pp. 241-264. Boca Raton.
327
328
Crowe TM (1978) The evolution of guineafowl (Galliformes, Phasianidae, Numidinae):
329
taxonomy, phylogeny, speciation and biogeography. Annals of the South African
330
Museum, 78, 43-136.
331
332
333
Crowe TM (2000a) Introduction. In: Gamebirds of Southern Africa. (eds. Little RM &
Crowe TM), pp.12-18. Stroik Publishers (Pty) Ltd, Cape Town.
17
334
335
336
Crowe TM (2000b) Helmeted Guineafowl. In: Gamebirds ofSouthern Africa. (eds. Little
RM & Crowe TM), pp.90-94. Stroik Publishers (Pty), Ltd, Cape Town.
337
338
Crowe TM, Little RM (1993) In the bag. Our Living World September 12-13
339
340
341
Crowe TM, Keith GS, Brown LH (1986) Galliformes. In: The Birds ofAfrica. Vol. 2 (eds.
Urban EK, Fry CH,Keith GC), pp. 1-75. Academic Press, London.
342
343
Desjardins P, Marais R (1990) Sequence and gene organisation of the chicken
344
mitochondrial genome: A novel gene order in higher vertebrates. Journal
345
Molecular Biology, 212, 599-634
346
347
348
Donkin RA (1991) Meleagrides: An Historical and Ethnogeographical Study of the
Guinea Fowl. Ethnographica, London.
349
350
Ghigi A (1936) Galline di Faraone e Tacchini. V.Hoepli, Milano.
351
352
353
Hastings Belshaw RH (1985) Guinea Fowl of the World. Nimrod Book Services.
Hampshire, England.
354
355
356
HarleY§II(1999)PAfSA. A computer program for aligning DNA and protein sequence
data. Version 4.0.
357
18
358
Hubbard AL, McOrist S, Jones TW, Biod R, Scott R, Easterbee N (1992) Is survival of
359
European wildcats Felis silvestris in Britain threatened by interbreeding with
360
domestic cats? Biological Conservation, 61, 203-208.
361
362
Johnson, DN (1984) Management of guineafowl on farmland. Wildlife Management:
363
Technical guides for farmers. Number 1. Second Edition. Natal Parks Board,
364
Pietermaritzburg.
365
366
Largiader CR, Scholl A (1996) Genetic introgression between native and introduced
367
brown trout Salmo trutta L. populations in the Rhone River Basin. Molecular
368
Ecology, 5, 417-426.
369
370
Lehman N, Eisenhawer A, Hansen K, Mech DL, Peterson RO, Wayne RK (1991)
371
Introgression of coyote mitochondrial DNA into sympatric North American gray
372
wolf populations. Evolution, 45,104-119.
373
374
Little RM (1997) Helmeted Guineafowl. In: The Atlas of Southern African Birds Volume
375
1: Non-Passerines (eds. Harrison JA, Allan DG, Underhill LG, Herremans M,
376
Tree AJ, Parker V, Brown CJ), pp. 308-309. BirdLife South Africa,
377
Johannesburg.
378
379
Little, R.M.,Crow€\T;M. (1994) Conservation implications of deciduous fruit farming
380
on birds in the Elgin district, Western Cape Province, South Africa. Trans. Roy.
381
Soc. S. Afr. 49, 185-197.
19
382
383
Little RM, Perrings JSA, Crowe TM (1995) Notes on diet of Helmeted Guineafowl
384
Numida meleagris on deciduous fruit farms in the Western Cape Province, South
385
Africa. South African Journal of Wildlife Research, 25, 144-146.
386
387
388
Long JL (1981) Introduced Birds of the World: The worldwide history,distributionand
influence of birds introduced to new environments. David & Charles, London.
389
390
Malan G, Benn GA (1999) Agricultural land-use patterns and thedecline of the Helmeted
391
Guineafowl Numida meleagris (Linnaeus 1766) in KwaZulu-Natal, South Africa.
392
Agriculture, Ecosystems and Environments, 73, 29-40.
393
394
395
O'Brian SJ, Mayr E (1991) Bureaucratic mischief: recognising endangered species and
subspecies. Science, 251, 1187-1188.
396
397
398
O'Connor RJ, Shrubb M (1986) Farming and Birds. Cambridge University Press,
Cambridge.
399
400
Pero LV, Crowe TM (1996) Helmeted Guineafowl Numida meleagris in KwaZulu-Natal:
401
a case for non-sustainability. South African Journal of Wildlife Research, 26, 123-
402
130.
403
20
21
428
429
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual,
2nd edn. Cold Spring Harbor Laboratory Press, New York.
430
431
Thulin CG, Jaarola M, Tegelstrom H (1997) The occurrence of mountain hare
432
mitochondrial DNA in wild brown hares. Molecular Ecology, 6, 463-467.
433
434
Ward TJ, Bielawski JP, Davis SK, Templeton, JW, Derr IN (1999) Identification of
435
domestic cattle hybrids in wild cattle and bison species: a general approach using
436
mtDNA markers and the parametric bootstrap. Animal Conservation, 2, 51-57.
437
438
Wenink PW, Baker AJ, Tilanus MGJ (1994) Mitochondrial control-region sequences in
439
two shorebird species, the Turnstone and the Dunlin, and their utility In
440
population genetic studies. Molecular Biology and Evolution, 11, 22-31.
441
442
Wilson CC, Bernatchez L (1998) The ghost of hybrids past: fixation of the arctic charr
443
(Salvelinus alpinus) mitochondrial DNA in an introgressed population of lake
444
trout (S.namaycush). Molecular Ecology, 7, 127-132.
445
446
Witt ABR, Little RM, Crowe TM (1995) The effectiveness of Helmeted Guineafowl
447
Numida meleagris (Linnaeus 1766) in controlling the banded fruit weevil
448
Phylctinus callosus (Schonherr 1826), and their impact on other invertebrates in
449
apple orchards in the Western Cape Province, South Africa. Agriculture,
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Ecosystems and Environment, 55, 169-179.
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452
453
Wolff SW, Milstein P le S (1987) Limiting factors for gamebirds in southern Africa.
South African Journal of Wildlife Research, Supplement 1, 51-53.
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Author information box:
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The present study was part of Andrew L. Walker's MSc project in Conservation Biology.
457
The work is part of a wider study by Charles Ratc1iffewhich identified causes of and
458
remedies to, the crash in Helmeted Guineafowlpopulations in the Midlands ofKwazulu-
459
Natal, looking at issues of land-use, disease, pesticides, genetic 'pollution' and utilisation
460
by humans. Associate Professor Tim Crowe co-ordinates the Gamebird Research
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Programme at the Percy FitzPatrick Institute, whose primary research areas are
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systematics, biogeography and conservation biology (based on the premise of sustainable
463
utilisation of natural resources).
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Figure caption
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466
Figure 1. Diagnostic morphological features of wild, South African N. m. coronata (a),
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wild, an intermediate, putative hybrid (b) and West AfricanN. m. galeata(c).
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469
25
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470
471
472
473
474
475
Table 1 Phenotypes and RFLP haplotypes (in parentheses domestic vs wild) of mtDNA
from Helmeted Guineafowl.
Wild
Phenotype
Intermediate
Domestic
8 (0 8)
12 (0 12)
o
14 (113)
14(311)
8(80)
22
26
8
Locality
Free State
KwaZulu-Natal
Number of Individuals
476
26
477
478
479
480
481
482
483
Table 2 Diagnostic sites for mtDNA haplotypes (i.e. wild and domestic) of guineafowl
from KwaZulu-Natal and the Free State, South Africa. The base-pair (bp) position
indicates the locus of each site on the amplified mtDNA D-Ioop (control) region. A dash
(-) indicates an indel site.
Haplotype
Wild
Domestic
1 (152 bp)
C
T
Diagnostic site ( base-pair position)
2 (173 bp) 3 (190 bp) 4 (225 bp) 5 (236 bp)
A
A
A
T
G
G
484
27
6 (279 bp)
~
A
485
486
487
Table 3 Restriction fragment patterns (and approximate sizes in kb) generated from MspI
digestion of550 bp peR-amplified D-Ioop (control) region ofmtDNA for guineafowl.
Fragment sizes (in bp)
Haplotype
Wild
Domestic
197
197
336
257
488
28
159