BOLUS LIBRAR Y C24 0009 2776 1111111111111 11111 Phylogenetic positions ofNahan's Francolin Francolinus nahani and the Stone Partridge Ptilopachus petrosus: enigmatic African gamebirds Julia Wakeling Department of Botany and the Percy FitzPatrick Institute University of Cape Town November 2004 Supervisors: Professor T.M. Crowe and Professor T.A Hedderson A project in part fulfillment of the requirements for a BSc (Hons) degree in Systematics Nahan's Francolin Francolinus nahani I University of Cape Town The Stone Partridge Ptilopachus petrosus The copyright of this thesis vests in the author. 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. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town ABSTRACT Nahan's Francolin Francolinus nahani has both been given its own subgenus and has been associated with and compared to Latham's Forest Francolin (Peliperdix lathami). Using sequences of two mitochondrial genes, cytochrome b and NADH dehydrogenase subunit 2 (ND2) as well as a nuclear gene, avian ovomucoid intron G (OVOG), we found that Francolinus nahani is sister to the Stone Partridge (Ptilopachus petrosus). This relationship is revealed using parsimony analysis, and is supported by high jackknife values. Peliperdix lathami is part of the "Quail-Francolins" however its relationship within this clade is unresolved due to insufficient sampling. A clade consisting of Gallus, Bambusicola and the "Quail-Francolins" is shown, and the use of Gallus as an outgroup for pheasant and partridge phylogenetics is questioned. INTRODUCTION Nahan's Francolin Francolinus nahani, a rare and endangered gamebird found in northeastern Congo, and in west and south central Uganda, was first described by Dubois in 1905 (Urban et al., 1986; Hall, 1963; Johnsguard, 1988, Madge & McGowan, 2002). It was placed by Hall (1963) within the Genus Francolinus, which has traditionally been recognized as comprising 36 African and 5 Asian species, prior to molecular studies. In a reworking of the francolins, Crowe et al. (1992) placed Nahan's Francolin in a monotypic subgenus, Acentrortyx, within Genus Pternistis with 27 other francolins. F. nahani is found only in dense primary forest up to 1400m, is very shy and usually seen in pairs (Urban et al., 1986). Hall (1963) hypothesized that F. nahani could be closely related to Latham's Forest Francolin Peliperdix lathami, but also stated that similarities such as small size, general dark colouration and spotting, may be the result of convergence due to habitat. These two species have overlapping distributions, but that of P. lathami is much larger and extends further west, with F. nahani being rarer and more localized (Urban et al., 1986). The species are also not ecologically segregated, which may suggest that they do not have a close relationship (Hall, 1963). Hall (1963) proposed that F. nahani was part of her "Scaly" group, consisting of F. ahantensis, F. squamatus and F. griseostriatus, forest edge and savanna species. This is because, although much smaller it does share some characteristics with this group. For example, in F. nahani and all the species of the "Scaly" group, the sexes are similar, legs are red and there is a small area of bare skin below and behind the eye (although this is not brightly coloured and often overlooked). F. nahani is also similar to F. squamatus in that they both have an unpattemed head and vermiculated back. F. nahani and F. ahantensis have white streaking, and F. nahani and F. griseostriatus both have a crimson base to the bill. In an analysis of its position usmg Euclidean distances, Crowe & Crowe (1985) confirmed that F. nahani fell near the "Scaly" group and was grouped closest to F. 2 ahantensis. Whilst a number of other studies have used molecular evidence to examine francolin phylogenetics (e.g. Crowe et al., 1992 and Bloomer & Crowe, 1998), the DNA of F. nahani has not been used for phylogenetic analysis, and its relation to other galliforms is not confirmed. The "Scaly" group is currently placed in the genus Pternistis (Crowe et al., 1992). More recently, unpublished observations on the basis of behaviour and calls have suggested a close link to the Stone Partridge (B. Finch, M. Mills & C. Cohen, pers. comm.). Aims and Hypotheses In this study I analyse variation at a number of DNA loci to establish the phylogenetic relationships between Francolinus nahani and Peliperdix lathami, the "Scaly" group (Pternistis) as well as with the Stone Partridge, Ptilopachus petrosus. METHODS Approach and Materials Mitochondrial DNA (mtDNA) markers are often used for resolving vertebrate phylogenies because they are haploid and maternally inherited. They evolve rapidly enough to resolve closely related taxa, and yet slowly enough at some sites to resolve deeper relationships (Moore, 1995). However, mitochondrial genes are inherited as a single linkage group, and thus phylogenetic estimates from different mitochondrial genes are not independent (Moore, 1995). Recently, a number of studies have used nuclear genes, which may occur on distinct chromosomes, thus providing independent estimates I of phylogeny (Moore, 1995; Johnson & Clayton, 2000; Armstrong et al. , 2001). In this study, two mitochondrial genes (Cytochrome b and NADH dehydrogenase subunit 2 (ND2)) and one nuclear gene (Ovomucoid Intron j--(~OG)), are used to hypothesize the phylogenetic position of Francolinus nahani (( & Py t'ytochrome b is a region frequently used for avian phylogenetics, and thus there are many sequences available for comparison. ND2 has been used for a number of galliform studies ( e.g. Dimcheff et al., 2002), and was easily amplified. It was thus also used so that a larger character set was available for mitochondrial analysis. OVOG has been shown to be useful for avian 3 I phylogenetics, especially in comparison with mitochondrial genes (Armstrong, et al., 2001). A broad selection of galliforms has also been sequenced for this region (Armstrong, et al., 2001), making it useful for this study. Species sequenced in this study, and information pertaining to them, are listed in Table 1. Sequences extracted from GenBank are listed in Table 2. Laboratory Techniques I Total genomic DNA was extracted from blood, heart and liver tissue using the DNeasy animal tissue protocol provided with the DNeasy® tissue kit (Qiagen). Primers used for amplification and sequencing and indicated in Table 3. The initial Cytochrome b primers amplified 1337 base pairs. Due to the length of this region, an internal primer was also used (Table 3). The initial cytochrome b primer pair did not amplify the Pternistis griseostriatus and Pternistis leucoscepus DNA extractions, so galliform specific primers were used. Double stranded DNA templates were amplified by polymerase chain reaction (PCR) using 0.75 units of BIOTAQ™ DNA polymerase (Bioline) in 30µ1 reactions. Reactions also contained 1 x NH4 buffer, 2.5mM MgC}z, each dNTP at O. lmM and each primer at 0.3µM. Three µI of un-quantified stock DNA was used as a template. The thermal profile used for all three DNA regions comprised an initial denaturation step at 94°C for two minutes, followed by 30 cycles of 94°C for one minute, 52°C for one minute and 72°C for two minutes, with a final poymerisation step of 72°C for seven minutes. The PCR's were performed by a GeneAmp® PCR System 2700 (Applied Biosystems) Version 2.07. Amplified products were cleaned from solution or gel using the GFX™ PCR DNA and gel band purification kit (Amersham Biosciences), prior to cycle sequencing with the ABI PRISM® Big Dye™ Terminator V3.l cycle sequencing Ready Reaction Kit (Applied Biosystems). Amplification primers were also used for sequencing, and the reaction conditions were as per the ABI protocol. The thermal profile used for all three DNA regions comprised 25 cycles of 96°C for 30 seconds, 50°C for 15 seconds and 60°C 4 for four minutes. Sequencing products were resolved on an ABI PRISM® 3100 Genetic Analyser. Sequences were assembled and checked for incorrect base calling and the presence of stop codons using SeqMan II (LaserGene systems software, DNAstar, inc.). Consensus sequences were aligned by Clustal and adjusted manually using MegAlign (LaserGene systems software, DNAstar, inc.). Phylogenetic Analyses Phylogenetic relationships among taxa were analysed with the parsimony optimality criterion (e.g. Swofford et al., 1996), using PAUP4.0b10 (Swofford, 2001) mounted on an Apple Macintosh G4. Parsimony analyses were conducted with only parsimony I informative characters included, and no substitution or codon position weighting was applied. 10 000 random stepwise taxon addition replicates were run using a TBR branch­ swapping heuristic search, where two trees of length greater than or equal to 10 were kept per replicate. The trees obtained were then used for further TBR branch-swapping search, where the number of trees per replicate was not limited. A strict consensus tree was then computed. Nodal support was assessed by Jackknife (Farris et al., 1996) using 1000 replicates and excluding 33.67% of characters at each replicate. Once again a simple stepwise taxon addition heuristic search using TBR branch-swapping was used, and if it was limited at all, 100 trees were kept per replicate. RESULTS Summaries of data and tree statistics are provided in Table 4. For cytochrome b, a total of 1337 base pairs were sequenced for Francolinus nahani, Scleroptila shelleyi, Peliperdix sephaena, Pternistis afer and Peliperdix lathami and 776 base pairs for Pternistis griseostriatus and Pternistis leucoscepus. These sequences exceeded those from GenBank, so only 660 base pairs could be used in the analysis of 75 sequences. The strict consensus of all most parsimonious trees was found for each analysis, and jackknife values were added to each topology.