Analysis of genetic diversity at the DQA loci in African cattle: evidence for a BoLA-DQA3 locus

 We describe the development of a polymerase chain reaction (PCR)-based approach for analysis of genetic diversity at the DQA loci in African Bos indicus and Bos taurus cattle. This approach, equally effective in European and Asian cattle breeds, detects the presence or absence of DQA1 and most duplicated DQA2 genes. Nucleotide and predicted amino acid sequence analysis of the highly polymorphic second exons, in addition to analysis of the locus-specific and relatively non-polymorphic transmembrane, cytoplasmic, and 3-prime untranslated regions, has provided evidence for considerable diversity between each of the duplicated DQA2 genes. Therefore, we propose the designation BoLA-DQA3 for the previously unpublished alleles at the second DQA2 locus. Fourteen distinct PCR restriction fragment length polymorphism (RFLP) patterns, each identifying families of alleles at three DQA loci, can be distinguished. Nucleotide sequence analysis of new PCR-RFLP patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N’Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families. Received: 19 February 1997 / Revised: 28 February 1997     

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.     

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine Λgt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class IIA sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.     

Sequence variation at the major histocompatibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros)

 The variation at loci with similarity to DRB class II major histocompatibility complex loci was assessed in 313 beluga collected from 13 sampling locations across North America, and 11 narwhal collected in the Canadian high Arctic. Variation was assessed by amplification of exon 2, which codes for the peptide binding region, via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Two DRB loci were identified in beluga: DRB1, a polymorphic locus, and, DRB2, a monomorphic locus. Eight alleles representing five distinct lineages (based on sequence similarity) were found at the beluga DRB1 locus. Although the relative number of alleles is low when compared with terrestrial mammals, the amino acid variation found among the lineages is moderate. At the DRB1 locus, the average number of nonsynonymous substitutions per site is greater than the average number of synonymous substitutions per site (0.0806 : 0.0207, respectively;P<0.01). Most of the 31 amino acid substitutions do not conserve the physiochemical properties of the residue, and 21 of these are located at positions implicated as forming pockets responsible for the selective binding of foreign peptide side chains. Only DRB1 variation was examined in 11 narwhal, revealing a low amount of variation. These data are consistent with an important role for the DRB1 locus in the cellular immune response of beluga. In addition, the ratio of nonsynonymous to synonymous substitutions is similar to that among primate alleles, arguing against a reduction in the balancing selection pressure in the marine environment. Two hypotheses may explain the modest amount of Mhc variation when compared with terrestrial mammals: small population sizes at speciation or a reduced neutral substitution rate in cetaceans. Received: 15 July 1997 / Revised: 24 March 1998     

Characterization of Mhc-DRB allelic diversity in white-tailed deer (Odocoileus virginianus) provides insight into Mhc-DRB allelic evolution within Cervidae

 Although white-tailed deer (Odocoileus virginianus) are one of North America's best studied mammals, no information is available concerning allelic diversity at any locus of the major histocompatibility complex in this taxon. Using the polymerase chain reaction, single-stranded conformation polymorphism analysis, and DNA sequencing techniques, 15 DRB exon 2 alleles were identified among 150 white-tailed deer from a single population in southeastern Oklahoma. These alleles represent a single locus and exhibit a high degree of nucleotide and amino acid polymorphism, with most amino acid variation occurring at positions forming the peptide binding sites. Furthermore, twenty-seven amino acid residues unique to white-tailed deer DRB alleles were detected, with 19 of these occurring at residues forming contact points of the peptide binding region. Significantly higher rates of nonsynonymous than synonymous substitutions were detected among these DRB alleles. In contrast to other studies of Artiodactyla DRB sequences, interallelic recombination does not appear to be playing a significant role in the generation of allelic diversity at this locus in white-tailed deer. To examine evolution of white-tailed deer (Odvi-DRB) alleles within Cervidae, we performed a phylogenetic analysis of all published red deer (Ceel-DRB), roe deer (Caca-DRB), and moose (Alal-DRB) DRB alleles. The phylogenetic tree clearly shows a trans-species persistence of DRB lineages among these taxa. Moreover, this phylogenetic tree provides insight into evolution of DRB allelic lineages within Cervidae and may aid in assignment of red deer DRB alleles to specific loci. Received: 25 June 1998 / Revised: 2 September 1998     

Polymorphism and selection in the major histocompatibility complex <Emphasis Type="Italic">DRA</Emphasis> and <Emphasis Type="Italic">DQA</Emphasis> genes in the family Equidae

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.     

Analysis of polymorphisms in the major expressed class I locus (B-FIV) of the chicken

 We analyzed the polymorphic nature of eleven alleles expressed by the major class I locus (B-FIV) in chickens. Similar to mammalian class I loci, the nucleotide substitutions with high variability occur in exons 2 and 3 encoding the α1 and α2 domains. However, the nonsynonymous to synonymous ratio of nucleotide substitutions in exon 3 encoding the α helix and β sheets is reversed compared with HLA. The region of exon 3 encoding the α2 helix demonstrates a much lower nonsynonymous to synonymous ratio, suggesting evolutionary selection of a more conserved α2 helix in B-FIV compared with HLA. Amino acid residues with high Wu-Kabat variability are typically located in positions predicted to impact antigen presentation. B-FIV amino acid residues predicted to interact with the CDR1α region of the T-cell receptor (Tcr) demonstrate less variability than in mouse and human class I alleles. The combination of a reduced nonsynonymous to synonymous ratio in exon 3 encoding the α2 helix and the limited variability in CDR1α contact residues is discussed with regard to concerted evolution between a minimal major histocompatibility complex and compaction of Tcr variable gene segments in the chicken. Received: 18 Juli 1997 /  Revised: 21 November 1997     

Genetic variation in the major histocompatibility complex of the European brown hare (Lepus europaeus) across distinct phylogeographic areas

The major histocompatibility complex is one of the best studied systems in vertebrates providing evidence for the long-term action of selection. Here, we examined the intra- and inter-population genetic diversity of the MHC class II DRB locus in European brown hare (Lepus europaeus) and correlated the results with genetic variability already estimated from the MHC DQA locus and from maternally (mitochondrial DNA (mtDNA)) and biparentally (allozymes, microsatellites) inherited loci. L. europaeus showed remarkable genetic polymorphism in both DQA and DRB1 loci. The Anatolian populations exhibited the highest genetic polymorphism for both loci. Balancing selection has established increased variability in the European populations despite the founder effects after the last glaciation. Different evolutionary rates were traced for DRB1 and DQA loci, as evidenced by the higher number of common DRB1 than DQA alleles and the greater differences between DRB1 alleles with common origin in comparison with DQA alleles. The high number of rare alleles with low frequencies detected implies that frequency-dependent selection drives MHC evolution in the brown hare through the advantage of rare alleles. Both loci were under the influence of positive selection within the peptide-binding region. The functional polymorphism, recorded as amino acid substitutions within the binding pockets, fell also within distinct geographic patterns, yet it was much narrower than the genetic polymorphism. We hypothesize that certain structural and functional characteristics of the binding pockets set limitations to the actual shape of genetic polymorphism in MHC.     

Allelic diversity at the Mhc-DQA locus of woodmouse populations (Apodemus sylvaticus) present in the islands and mainland of the northern Mediterranean

Aim Polymorphism at neutral markers and at MHC loci in rodent populations living on islands is generally low. The main genetic factors that may contribute to a reduced level of genetic variability are genetic drift, reduced gene flow and founder events. We investigated the pattern of polymorphism at the second exon of the Mhc‐DQA gene in island populations of Apodemus sylvaticus and in their mainland counterparts to investigate the pattern of MHC polymorphism in a phylogeographical context and to assess the impact of insularity on diversity at this locus. Location Eight north Mediterranean populations of Apodemus sylvaticus were studied, including five island populations (Majorca, Minorca, Porquerolles, Port‐Cros and Sicily) and three mainland populations. Methods cDNA sequencing and nucleotide sequences analyses. Synonymous and non‐synonymous substitutions were examined at the PBR and non‐PBR sites. The DQA allelic distribution in populations was compared with the woodmouse phylogeography. Results This study presents novel DQA alleles. High polymorphism of the DQA locus is recorded in natural populations of A. sylvaticus with 13 alleles being widely distributed irrespective of the geographical origin and palaeoclimatic history of populations. The DQA locus does not show the expected pattern for non‐synonymous substitutions at the PBR sites. However, island populations show a weak loss of polymorphism in comparison with their mainland counterparts. Main conclusions The DQA locus in the woodmouse seems to be subject to weak selection and does not allow resolution of phylogeographical relationships among European woodmouse populations. The presence of at least three alleles in island populations and the maintenance of five alleles between the two European lineages over 1.5 Myr suggest that balancing selection may act within populations, and more precisely within island populations, to maintain genetic variability. This study shows that phylogeographical studies are a prerequisite for any genetic investigation of selected genes in natural populations.     

Larval stages of the crab Mithrax tortugae (Brachyura: Mithracidae) with comparisons between all species of Mithrax

The postembryonic larval stages of Mithrax tortugae are described, illustrated and compared with the described zoeae of other Mithrax species. Mithrax tortugae showed morphological features in all the stages of larval development that differed from those observed in other species of Mithrax, especially M. hispidus. In the Zoea I stage, M. tortugae and M. pleuracanthus lacked the minute spine on the dorsal spine observed in M. hispidus; M. tortugae exhibited a terminal spine on the inner lobe of the coxal endite of the maxilla, which was not observed in M. hispidus or M. pleuracanthus. Also, M. tortugae exhibited furcae with spines that are not spinulated, whereas in M. hispidus and M. pleuracanthus these spines are spinulated. In the Zoea II stage, M. tortugae showed a terminal spine on the coxal endite of the maxillula, whereas in M. hispidus and M. pleuracanthus this spine is absent. In the Megalopa stage, we also observed differences in the sternal plate setation between M. tortugae and M. hispidus, where M. tortugae had eight simple setae and M. hispidus showed two simple and four plumodenticulate setae. Partial sequences of the 16S rRNA and COI genes of the parental female were analysed, providing additional evidence for species identification. Together, our analyses of larval morphology and the results of the molecular analyses reinforced recognition of the relationships among M. tortugae, M. hispidus and M. pleuracanthus.     

Structure and evolution of the promoter regions of the DQA genes

HLA-DQ antigens are unique among the class II antigens in that their chains are highly polymorphic. In the present study, we characterized the general structure of the promoter regions of the DQA genes derived from different DR haplotypes and defined their nucleotide sequence polymorphisms. The promoter of each DQA1 allele contains three sequence motifs which are not present in non-DQA related class II genes: one identical to a tumor necrosis factor (TNF) response element, one similar to an NFB binding element, and one similar to a W motif. All DQA alleles lack TATA and CCAAT boxes in the proximal promoter region but carry other sequence elements characteristic of MHC class II genes, including S, X, X2, and Y boxes, and a pyrimidine-rich tract upstream of the X box. Nucleotide sequence polymorphisms among the various DQA1 alleles were noted within the promoter region, with some of the differences mapping within, or close to, regulatory elements that are important for the expression of MHC class II genes. All DQA1 alleles carry an unrearranged, full length, Alu-Sx related repeat immediately upstream of the proximal promoter region. This repeat was not present in the DQA2 (DXA) genes analyzed, confirming that DQ locus duplication probably occurred before integration of the Alu repeat into the primordial DQA1 locus, some 31–43 million years (myr) ago. The DQA2 promoter region is highly conserved between DR4 and DR3 haplotypes, with the degree of conservation exceeding that expected from the neutral mutation rate.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accession numbers M97 454-M97 464. Correspondence to: E. Morzycka-Wroblewska.     

Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa

The human secretor type α(1,2)fucosyltrans-ferase gene (FUT2) polymorphism was investigated in Xhosa and Caucasian populations of South Africa by polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing. Six new base substitutions were found in the coding region of FUT2. A single base (C) deletion at nucleotide 778, which led to a frame shift and produced a stop codon at codon 275, was responsible for the enzyme inactivation. Three nonsynonymous base substitutions, A40G (Ile¹⁴Val), C379T (Arg¹²⁷Cys), and G481A (Asp¹⁶¹Asn), and two synonymous base substitutions, A375G (Glu¹²⁵) and C480T (His¹⁶⁰), were also identified in functional alleles. As a result, seven new alleles, Se ⁴⁰ , Se ⁴⁸¹ , Se ^40,481 , Se ^357,480 , Se ^357,379,480 , Se ³⁷⁵ , and se ^357,480,778 were identified. Population studies revealed that an allele containing a nonsense mutation G428A (Trp¹⁴³stop) (se ⁴²⁸ ) was the common null allele in both Xhosa and Caucasian populations, whereas an allele containing a missense A385T (Ile¹²⁹Phe) mutation (se ^357,385 ), which is the common null allele in Orientals, was found to be absent from both populations. The heterozygosity rates of FUT2 genotypes were as high as 0.75 in the Xhosa population and 0.65 in the Caucasian population. Therefore, the extensive polymorphism and race specificity of the FUT2 gene make it suitable for application as a new tool in genetic studies of modern human evolutionary history. Received: 23 March 1998 / Accepted: 9 May 1998     

Evolutionary history of an MHC gene in two leporid species: characterisation of <Emphasis Type="Italic">Mhc-DQA</Emphasis> in the European brown hare and comparison with the European rabbit

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloning–sequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers FJ225335–FJ225346.     

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole,Microtus pennsylvanicus

Summary The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNA^Lys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNA^Lys is concentrated in the TC loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (>92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.     

Crassostrea angulata Bindin Gene and the Divergence of Fucose-Binding Lectin Repeats Among Three Species of Crassostrea

Bindin is a major protein for species-specific recognition between sperm and congenetic egg in many free-spawning marine invertebrates. We cloned a novel bindin gene from the oyster Crassostrea angulata by 3′ and 5′ rapid amplification of cDNA ends. The full-length bindin cDNA was 1,049 bp with a 771-bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8,508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Both neighbor-joining and minimum-evolution phylogenetic trees show that haplotype an1 was close to Crassostrea gigas while an2 and an3 were close to Crassostrea sikamea. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types according to their size and the F-lectin repeat they were located in. Intron-4 may play an important role in recombination. We compared the number of nonsynonymous substitutions (Dn) and synonymous substitutions (Ds) per nucleotide site among 19 F-lectin haplotypes of the three species. Dn/Ds ratios suggested that positive selection occurred between C. gigas and C. sikamea and between C. gigas and C. angulata. Nine positive selected positions (p > 90%) are identified among 19 haplotypes of three species. They are located on the F-lectin binding face around the three recognition motif residues. We assume that these nine clustered amino acids are related with species-specific recognition.     

The transcription of MGAT4A glycosyl transferase is increased in white cells of peripheral blood of Type 2 Diabetes patients

Background

The giant panda (Ailuropoda melanoleuca) is one of the most endangered animals due to habitat fragmentation and loss. Although the captive breeding program for this species is now nearly two decades old, researches on the genetic background of such captive populations, especially on adaptive molecular polymorphism of major histocompatibility complex (MHC), are still limited. In this study, we characterized adaptive variation of the giant panda's MHC DQA gene by PCR amplification of its antigen-recognizing region (i.e. the exon 2) and subsequent single-strand conformational polymorphism (SSCP) and sequence analyses.

Results

The results revealed a low level of DQA exon 2 diversity in this rare animal, presenting 6 alleles from 61 giant panda individuals. The observed polymorphism was restricted to 9 amino acid substitutions, all of which occurred at and adjacent to positions forming the functionally important antigen-binding sites. All the samples were in Hardy-Weinberg proportions. A significantly higher rate of non-synonymous than synonymous substitutions at the antigen-binding sites indicated positive selection for diversity in the locus.

Conclusion

The DQA allelic diversity of giant pandas was low relative to other vertebrates. Nonetheless, the pandas exhibited more alleles in DQA than those in DRB, suggesting the alpha chain genes would play a leading role when coping with certain pathogens and thus should be included in conservation genetic investigation. The microsatellite and MHC loci might predict long-term persistence potential and short-term survival ability, respectively. Consequently, it is recommended to utilize multiple suites of microsatellite markers and multiple MHC loci to detect overall genetic variation in order to design unbiased conservation strategies.     

Speciation in sympatric species of land snails from the genus Trochulus (Gastropoda,Hygromiidae)

The identification and designation of land snail species in the genus Trochulus on the basis of shell characteristics are problematic because of their great phenotypic plasticity. Some genetic analyses have proved inconclusive, with much variation within populations and apparent gene flow among them. We examined this issue by morphometric and molecular approaches on the morphologically similar species T. coelomphala, T. hispidus and T. striolatus, co‐occurring in the Alpenvorland of Germany. While these species differed in shell and reproductive system morphology, there were forms that turned out intermediate in shell characters between T. coelomphala and T. hispidus but had genital morphology similar to T. coelomphala. Phylogenetic analysis, however, showed that these forms clustered neither with T. coelomphala nor T. hispidus but are sister to T. striolatus from the same region, which suggests that they evolved by way of sympatric speciation. Further, these analyses suggest that T. coelomphala diverged within T. hispidus; a crossing experiment indicated that they were interfertile. Expanding the study to include all available Trochulus sequences enabled us to infer evolutionary relationships between them and showed that T. hispidus is polyphyletic. Some Trochulus samples of one nominal species were grouped within others. The combination of phenotypic plasticity and possible mitochondrial DNA introgression illustrates the complex nature of evolutionary processes and the need for caution in the application of traditional taxonomic practice.     

Sequence variability analysis on major histocompatibility complex class II DRB alleles in three felines

The variation of the exon 2 of the major histo-compatibility complex (MHC) class II gene DRB locus in three feline species were examined on clouded leopard (Neofelis nebulosa), leopard (Panthera pardus) and Amur tiger (Panthera tigris altaica). A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2. Exon 2 encodes the β1 domain which is the most variable fragments of the MHC class II molecule. Single-strand conformational polymorphism (SSCP) analysis was applied to detect different MHC class II DRB haplotypes. Fifteen recombinant plasmids for each individual were screened out, isolated, purified and sequenced finally. Totally eight distinct haplotypes of exon 2 were obtained in four individuals. Within 237 bp nucleotide sequences from four samples, 30 variable positions were found, and 21 putative peptide-binding positions were disclosed in 79 amino acid residues. The ratio of nonsynonymous substitutions (d _N ) was much higher than that of synonymous substitutions (d _S ), which indicated that balancing selection probably maintain the variation of exon 2. MEGA neighbor joining (NJ) and PAUP maximum parsimony (MP) methods were used to reconstruct phylogenetic trees among species, respectively. Results displayed a more close relationship between leopard and tiger; however, clouded leopard has a comparatively distant relationship form the other two. __________ Translated from Zoological Research, 2006, 27(2): 181-C188 [译自:动物学研究]