MOLECULAR INVESTIGATION OF PHYLOGENETIC RELATIONSHIPS AND PHYLOGEOGRAPHY OF POLISH PHEASANTS USING THE MITOCHONDRIAL CYTOCHROME B GENE SEQUENCE

Piotr Czyżowski, Mirosław Karpiński, Leszek Drozd
Department of Pet Breeding and Wildlife Management, University of Lifes Science in Lublin, Poland

ABSTRACT

Samples of soft tissue (muscle) from wild and pen-reared ring-neck pheasants (Phasianus colchicus) were collected from the central and western regions of Lublin Upland by hunting. This population has been regularly introduced into wild populations each year for the last thirty years. No sequence differences were identified within a 236 bp conserved part of the cytochrome b gene from pheasants sourced with wild and pen-reared populations. We also investigated differences in cytochrome b sequences from Polish pheasant populations and Asian pheasants (evaluated in other studies). We found that genetic distances between mtDNA haplotypes of pheasants show only minor variations, which suggest that the evaluated region of the cytochrome b gene is well conserved within the genus Phasianus.

Key words: cyt b, mtDNA cytochrome b, pheasant, polymorphism.

INTRODUCTION

Pheasants are some of the most widely kept game birds in captivity [6]. Wildlife management practices of ring-necked pheasants in Poland and in all of Europe are based on birds that are released from breeding farms to augment wild populations. The interventions are not always successful because pen-reared pheasants have low breading success [12,15]. Ring-necked pheasants released in hunting regions are thought to negatively influence the wild pheasant population [9,17]. The results of both morphological and ethological analyses of both wild and pen-reared pheasants in Lublin Upland, conflict with traditional results that suggest a poor survival and reproductive success between that wild and pen-reared populations [14].

Modern molecular biology techniques enable comparisons between nucleotide and amino acid sequences of different populations to evaluate genetic diversity and phylogeny. Many studies have demonstrated that polymorphic loci or gene sequences are often conserved between closely related taxa [4]. Mitochondrial DNA has many advantages as a genetic marker for phylogenetic analyses  [13]. In avian studies, the cytochrome b (cyt b) gene has been sequenced more often than all other mitochondrial genes. Cyt b sequences are approximately 61% of all avian mitochondrial gene sequences in GenBank [21].

Investigation of the genetic status of the pheasants is important for effective long-term management of natural and artificial populations. The aim of his paper is to compare the basic genetic parameters between two pheasant populations. The first is a wild population reared without human interference. The second is a farm-bred pheasant population. In this paper, we report on the genetic variability detected in both natural and artificial pheasant (Phasianus colchicus) populations using mitochondrial cyt b gene sequence analysis.

MATERIAL AND METHODS

The samples of soft tissue (muscle) from wild and pen-reared ring-neck pheasants (Phasianus colchicus) were collected from the central and western regions of Lublin Upland by hunting. Lublin Upland is located in southeastern Poland. It is mainly lowland with continental climate. Woods cover 24.6% of the territory and agriculture is not particularly developed. Vegetation season persists commonly for 208 to 218 days in the west, and for 220 days in the south-west. The mean annual temperature is 7–8°C. The temperature of vegetation season is 14°C to 15°C. The annual sum of atmospheric precipitation is 500–600 mm.

The wild pheasants (n=25) originated from eastern Lublin Upland. This wild pheasant population was reared without human interference. The second group (n=23) was the introduced (with human interference) population from central Lublin Upland. This population has been regularly introduced into wild populations each year for the last thirty years. The distance between both study areas is about 100 kilometers.

Genomic DNA was extracted from soft tissues using the QIAamp DNA Mini Kit following the manufacturers instructions. A part of the cyt b gene sequence (missing the tRNA-Glu fragment) was amplified using PCR. Primers Cyt b1 (5' -cca tcc aac atc tca gca tga tga aa- 3' and Cyt b 2 (5'-gcc cct cag aat gat att tgt cct ca-3') were used to amplify a 359 bp fragment (307 bp without primers). The PCR conditions were as follows: an initial denaturation at 94°C for 1 min followed by 35 cycles of 94°C for 5 s, 55°C for 30 s, annealing temperature at 58°C and the final elongation at 72°C was prolonged to 3 minutes [5]. PCR products were purified using the QIA-quick PCR purification kit according to the manual. The PCR products were purified and then sequenced using a DNA sequencer (ABI Prism 377, Perkin-Elmer). The sequences were analyzed using Chromas software and then subjected to a BLAST search [1].

DNA sequences from the EMBL database were used to compare cyt b gene sequences(236 bps of the total 1140 bp gene) from pheasants (Phasianus colchicus) used in this study to cyt b sequences of pheasants from other studies: P. colchicus voucher (GenBank accession no. DQ115315) [2], P. colchicus (GenBank accession no. AY368054) [23] and P. versicolor (GenBank accession no. AY368058) [23], P. colchicus (GenBank accession no. AY870274) [22], P. colchicus (GenBank accession no. AF028798) [10].

A method using single nucleotide polymorphism (SNP) is a rapid method was to distinguish differences in mtDNA lineages. SNP genotyping sequences were calculated using the DnaSP 4.0 freeware program [18]. We estimated nucleotide sequence diversity (Pi), the number of monomorphic (invariable) sites (Msites), the number of polymorphic (variable) SNPs (Psites), the number of haplotypes (Hn), the haplotype diversity (Hd), and the variance of haplotype diversity (VHd). Nucleotide diversity is defined as the average number of nucleotide differences per site between any two DNA sequences. From this distance data, we constructed an mtDNA phylogenetic tree using PHILIP software version 3.5 [8] with the Gene-Bee program. Partial nucleotide sequences from Gallus gallus (GenBank accession no. X52392) [7] from Tetrao urrogallus (GenBank accession no. AB120132) [18] were used as outgroups.

RESULTS AND DISCUSSION

Mitochondrial DNA (mtDNA) sequences are commonly used to evaluate vertebrate phylogenies. MtDNA markers evolve rapidly, making investigation among closely related species possible. However, mtDNA sequences also contain slowly evolving sites that facilitate the investigation of deeper relationships. Between closely related taxa, cyt b sequences diverge faster than do nuclear DNA sequences between more distant taxa. Sequence divergence continues to increase in nuclear DNA [3].

After alignment of these sequences and elimination of regions where homology of nucleotides was ambiguous, we obtained a conserved, 236 bp (Table 1) piece of the cyt b gene. The main differences in this region were reported previously [10,11,16]. Cyt b gene sequences from 47 cyt b sequences gave consensus sequences (P.Lub.all, Table 2). Only one sequence was different (P.Lub.15, Table 2). In our study, genetic distances between mtDNA haplotypes of pheasants showed only minor variations within the 236 bp region (Table 1). We observed only one difference between wild and pen-reared pheasants; however, this difference appeared in only one wild pheasant specimen. The mutation is a cytosine to thymine substitution at position 84. Sequences obtained in this study and previously published sequences showed differences in several positions: at position 27, a guanine to cytosine [2], at position 84, a cytosine to thymine [2,23], at position 228, a cytosine to thymine, and at position 234, a guanine to adenine [2,10,22,23].

A total number of 4 polymorphic mutations (Eta) at nucleotide sites were identified over the 236 bp (excluding all sites with alignment gaps). Single nucleotide polymorphism (SNP) analysis revealed 232 monomorphic sites (Msites) and four variable or polymorphic sites (Psites). We also evaluated haplotype and nucleotide diversity. The number of haplotypes (Hn) was 5 and the value of haplotype diversity (Hd) was 0.857.  The SNP variance of haplotype diversity (VHd) was 0.01880 (SD=0.137) and the nucleotide diversity (Pi) was 0.00767 (Table 3).

The genetic distances between pheasant populations from the Lublin region (including wild-P.Lub.15 and pen reared pheasants -P.Lub.all) and other populations (P. Zhan, P.Versic.,       P. Wen, P. An, P. Kim.; Figs. 1 and 2) were much smaller than expected based on the large geographical distances separating the populations. Most of the mtDNA cyt b sequences found in the Lublin region are genetically close.

The sequence distance within the Polish pheasant population was 0.004 (Fig. 2). The sequence distance within populations evaluated in Asia ranged from 0.007 to 0.022. It is interesting that the distance between the pheasant populations examined by An et al. [2] and Zhan and Zhang [23] (GenBank accession no. AY368054) was exactly the same (0.004) as the distance found in this study. A possible explanation for these results might be related to the fact that in avians, the average age of alleles may be about 1.5 million years [19], which is ample time for a mutation to mould a nucleotide diversity level.

Estimations of nucleotide divergence between sequences from Poland (this study) and sequences from Kimball et al. [10], Zhan and Zhang [23], Wen et al. [22], An et al. [2] are interspersed (Fig.1). Genetic distances between evaluated sequences are shown in Fig. 2. The last row gives information about nucleotide distances between the outgroup species, Gallus gallus and Tetrao urrogallus. This is an example of monophyly of the pheasant lineage, which is well supported by our cyt b analysis. Our study has shown that species of the genus Phasianus are extremely low most specific groupings. Both the distance SNP analyses and the phylogenetic tree analyses suggested that the pheasants we investigated are derived from one maternal source. The analyses showed a significant (or nearly significant) reduction in the number of haplotypes and in the haplotype diversity values.

CONCLUSIONS

1. Due to a strong conservatism of cytochrobe b gene at mitochondrial DNA, no differences in nucleotide sequence between wild and pen-reared ring-neck pheasants (Phasianus colchicus) from the central and western regions of Lublin Upland were found.

2. Two phylogenetic pheasant groups were distinguished od a base of studied fragment of cytochrome b. Studied pheasant population from Lublin region and birds from Asia belong to the same phylogenetic group.

3. The genetic distances between pheasant populations from the Lublin region and other populations from Asia were much smaller than expected based on the large geographical distances separating the populations. Most of the mtDNA cyt b sequences found in the Lublin region are genetically close.

REFERENCES

Accepted for print: 4.11.2008

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