Molecular evidence for the pattern and timing of cladogenesis in dasyurid marsupials

Recent molecular studies have provided estimates of phylogeny for nearly all living and recently extinct species in the Order Dasyuromorphia, the dominant clade of insectivorous‐carnivorous marsupials in Australasia. We review these studies along with morphology‐based ones, and present an analysis of all cytochrome b, 12S rRNA, and protamine Pl gene sequences available. In light of these results, we provide a revised suprageneric classification and assess the implications of molecular and paleontological data for dasyurid cladogenesis. Molecular results divide extant dasyurids (Dasyuridae) into four major clades apart from the numbat (Myrmecobiidae) and thylacines (Thylacinidae). We recognize these clades as tribes Dasyurini (Dasyurus, Phascolosorex, and allied genera) and Phascogalini (Antechinus, Murexia, Phascogale) in the Subfamily Dasyurinae, and tribes Sminthopsini (Sminthopsis, Ningaui, Antechinomys) and Planigalini (Planigale) in the Subfamily Sminthopsinae. Each tribe shows a basal radiation of lineages corresponding to genera or species groups. Our results concur with the most recent previous synthesis of dasyurid phylogeny in many respects, but subsumption of New Guinean ‘phascolosoricines’ and ‘muricines’ within Dasyurini and Phascogalini, respectively, constitute significant differences. In particular, the sister‐pairing of ‘phascolosoricines’ with a Dasyurus‐Sarcophilus clade implied by molecular data is difficult to reconcile with anatomy. Divergence rates of mitochondrial sequences are calibrated approximately by comparing thylacine‐to‐dasyurid distances with the age of the oldest thylacinid (Badjcinus, latest Oligocene). Estimated cladogenic dates suggest that extant subfamilies shared a common ancestor around 24 Mya and that major radiations began late in the mid‐Miocene, consistent with the results of previous paleontological studies. The late‐middle and late Miocene corresponds to an episode of faunal turnover in Australian marsupials (including the decline of thylacinid and bandicoot genera, as well as the rise of dasyurids) and to a time when uplift of the New Guinean highlands accelerated the transition from rainforest to drier habitats. Our findings are consistent with the hypothesis that continent‐wide climate changes modulated macroevolution across these independent marsupial clades.     

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the α-globin gene family in mammals by isolating and characterizing α-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar α-globin family consists of at least four genes including a single adult-expressed gene (α), two embryonic/neonatally expressed genes (ζ and ζ′), and θ-globin, each orthologous to the respective α-, ζ-, and θ-globin genes of eutherian mammals. The results suggest that the θ-globin lineage arose by duplication of an ancestral adult α-globin gene and had already evolved an unusual promoter region, atypical of all known α-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that θ-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human θ-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three α-like and one β-like globin genes linked in the order 5′–ζ–α–θ–ω–3′ existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar ζ-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.Reviewing Editor: Dr. John Oakeshott     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Phylogenetic structure of the marsupial family dasyuridae based on cytochromeb DNA sequences

Archer provided the most recent and comprehensive suprageneric classification of dasyurid marsupials. Five extant subfamilies, two with constituent tribes, were recognized on the basis of morphological, serological, and allozyme data. Phylogenetic relationships among these groups, however, were totally unresolved. Subsequent molecular studies suggested that the endemic New Guinean subfamilies Muricinae and Phascolosoricinae are parts of larger Australian clades. Our objective in this study was to test the monophyly of Archer's seven groups and estimate relationships among them using DNA sequences from the mitochondrial cytochromeb (cyt-b) gene. We report 657 bp ofcyt-b from 32 dasyuroid species. Phylogenetic analysis of these data leads to the following conclusions: (1) muricines form a clade within Phascogalinae that includes endemic New GuineanAntechinus species; (2) the two genera of Phascolosoricinae are part of a more inclusive Dasyurinae; (3) Sminthopsinae is monophyletic, but the tribes Sminthopsini and Planigalini are not; and (4) the dasyurine tribes Dasyurini and Parantechini are probably not monophyletic. Relationships among Sminthopsinae, Dasyurinae (including phascolosoricines), and Phascogalinae (including muricines) remain unresolved.     

Systematic relationships within the dasyurid marsupial tribe Sminthopsini--a multigene approach.

We report analyses of complete DNA sequences of the mitochondrial cytochrome b (1146 bp), 12S rRNA (974 bp), partial control region (371 bp) loci, and the nuclear protamine P1 (616 bp) gene from all but one species (Sminthopsis butleri) of the dasyurid marsupial tribe Sminthopsini, as well as several outgroups. Parsimony analyses of combined nuclear and mitochondrial data suggest that Antechinomys is sister to a clade consisting of Sminthopsis and Ningaui. Parsimony, maximum-likelihood, and mixed-model distance analyses consistently resolve several species groups within Sminthopsis. The Macroura group includes S. macroura, S. virginiae, S. douglasi, and S. bindi; S. butleri is also included here on the basis of partial 12S rRNA sequences. S. crassicaudata is resolved as sister to the Macroura clade. The Murina group includes S. murina, S. leucopus, S. gilberti, S. dolichura, and S. archeri. S. griseoventer and S. aitkeni are resolved as a clade, and there is moderate support for a group consisting of the genetically divergent species S. psammophila, S. hirtipes, S. youngsoni, and S. ooldea (possibly along with S. longicaudata and S. granulipes). Compositions of species groups are partially congruent with clades previously proposed on the basis of morphological data. Radiations within Sminthopsini appear to be coincident with major environmental changes since the mid-Miocene.     

DNA phylogeny of the marsupial wolf resolved.

The phylogenetic position of the recently extinct marsupial ''wolf'', or thylacine (Thylacinus cynocephalus), has been a source of contention in mammalian systematics for nearly a century. Thylacines were endemic to Australasia, but possessed striking anatomical similarities to Oligo-Miocene borhyaenid marsupials of South America. At issue has been whether these features are indicative of common ancestry or convergent adaptation to carnivory. Recent morphological studies have supported both conclusions. Although current marsupial classifications group thylacines with Australian dasyuromorphians, this putative clade is characterized by mostly primitive morphological features. Attempts to determine thylacine affinities with ancient protein and DNA analyses have supported, but not resolved, a dasyuromorphian placement. We report 1546 bp of mitochondrial DNA sequence (from cytochrome b and 12S rRNA genes) and 841 bp of nuclear protamine gene sequence from the thylacine and representatives of all or most other marsupial orders. Phylogenetic analysis of these sequences shows unambiguously that thylacines are members of Dasyuromorphia, and suggests a late Oligocene or very early Miocene divergence of familial lineages.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Evolutionary change at the5S Dna loci of species in theTriticeae

Previous studies have shown that two separate loci for 5S DNA sequences may exist within a species. Two size classes have been tentatively assigned in the ranges 327–468 bp (chromosome 1), and 469–500 bp (chromosome 5) and the entire data-base was subjected to various numerical taxonomic analyses. The results confirm the existence of two lineages of 5S DNA sequences represented by the two size classes.Hordeum vulgare is separate from the species known also asCritesion and, together withDasypyrum, occupy an intermediate position between the two size classes. The 5S DNA units fromTriticum spp. that have also been namedAegilops spp. in other classifications appear as a distinct group withinTriticum spp. at both loci. The consensus 5S DNA sequence fromPsathyrostachys is remote fromHordeum s.l. in contrast to opinions based on morphology. Other aspects are detailed and discussed, including the inadequacy of the computing methods used as well as the need for more data in view of the large amount of homoplasy. The merit of using the spacer for phylogenetic inference is also discussed.     

The evolution of reproductive strategies in dasyurid marsupials: implications of molecular phylogeny

Dasyurid marsupials show a remarkable diversity of reproductive patterns ranging from aseasonal polyoestry to restricted annual breeding in which males synchronously die after a brief mating season. Previous studies have categorized dasyurid reproduction into six strategies, defined on the basis of five life-history characters. We provide an up-to-date summary of reproductive traits in dasyurid species and examine the evolution of these characters on a phylogeny for the family recently obtained from DNA sequence data. Our results suggest that reproductive evolution in modern dasyurids is characterized by a basal separation of subfamily lineages employing Strategy II (monoestrous females, restricted breeding season, 11 months to maturity; Dasyurinae) and Strategy V (polyoestrous females, extended breeding season, 8–11 months to maturity; Sminthopsinae). Strategies I (male die-off) and III (facultative polyoestry) appear to have arisen several times from Strategy II or V ancestors, and Strategy IV appears to have arisen within Sminthopsis from a Strategy V ancestor. Strategy VI (aseasonal breeding) has arisen independently in each of the four major dasyurid lineages (tribes), and is highly (but not perfectly) correlated with New Guinean endemism. This scenario is not strongly affected if reproductive characters are optimized on an alternative phylogeny more consistent with morphology-based opinions on species relationships. When evaluated in light of current habitat associations and geographic distributions, the reproductive data suggest that the Miocene diversification of modern dasyurids may have been correlated with the invasion of dry forest or woodland habitats.     

Complete Structural Organization of the Human α1(V) Collagen Gene (COL5A1): Divergence from the Conserved Organization of Other Characterized Fibrillar Collagen Genes

Genes that encode the vertebrate fibrillar collagen types I–III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization ofCOL5A1,the human gene that encodes the α1 chain of fibrillar collagen type V. The structure ofCOL5A1is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I–III.COL5A1has 66 exons, which is greater than the number of exons found in the genes for collagen types I–III. The increased number of exons is partly due to the increased size of the pro-α1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I–III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region ofCOL5A1compared to the organization of the triple-helix coding regions of the genes for collagen types I–III. Of particular interest is the increase of 54 bp exons in this region ofCOL5A1,strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure ofCOL5A1to the highly conserved structure of the genes of collagen types I–III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes.     

Phylogenetic relationships of dasyuromorphian marsupials revisited

We reassessed the phylogenetic relationships of dasyuromorphians using a large molecular database comprising previously published and new sequences for both nuclear (nDNA) and mitochondrial (mtDNA) genes from the numbat (Myrmecobius fasciatus), most living species of Dasyuridae, and the recently extinct marsupial wolf, Thylacinus cynocephalus. Our molecular tree suggests that Thylacinidae is sister to Myrmecobiidae + Dasyuridae. We show robust support for the dasyurid intrafamilial classification proposed by Krajewski & Westerman as well as for placement of most dasyurid genera, which suggests substantial homoplasy amongst craniodental characters presently used to generate morphology‐based taxonomies. Molecular dating with relaxed molecular clocks suggests that dasyuromorphian cladogenesis began in the Eocene, and that all three dasyuromorphian families originated prior to the end of this epoch. Radiation within Thylacinidae and Dasyuridae had occurred by the middle to late Oligocene, consistent with recognition of primitive thylacinids (e.g. Badjcinus turnbulli) in the later Oligocene and of putative dasyurids (e.g. Barinya wangala) by the early Miocene. We propose that all four extant dasyurid tribes were in existence by the early Miocene and that most modern dasyurid genera/species were established before the later Miocene. This is in marked contrast to the popularly accepted advocation of their origins in the latest Miocene–early Pliocene. © 2015 The Linnean Society of London     

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.     

Molecular phylogeny of the Herpestidae (Mammalia, Carnivora) with a special emphasis on the Asian Herpestes

Until now, phylogenetic studies of the mongooses (Carnivora, Herpestidae) have not included an exhaustive sampling of the Asian members of this family. In this study, we used mitochondrial (Cytochrome b and ND2), nuclear (β-fibrinogen intron 7 and Transthyretin intron 1) sequences from almost all of the recognized mongoose species to produce a well-resolved phylogeny of the Herpestidae. We also performed molecular dating analyses to infer divergence dates of the different lineages within the Herpestidae. Our results confirmed the paraphyly of the Herpestes genus and other phylogenetic relationships, which previously had only been moderately supported. The Asian herpestid species were found to form a monophyletic group within the Herpestidae. Within the Asian species, a cyto-nuclear conflict was discovered between the small Indian mongoose (Herpestes auropunctatus), the Indian gray mongoose (Herpestes edwardsii) and the Javan mongoose (Herpestes javanicus), which may have occurred through interspecific hybridization. This study inferred an Early Miocene origin for the Herpestidae and a Middle Miocene origin for the Asian mongooses.     

Molecular phylogeny of the lionfish genera Dendrochirus and Pterois (Scorpaenidae, Pteroinae) based on mitochondrial DNA sequences

This study investigates the molecular phylogeny of seven lionfishes of the genera Dendrochirus and Pterois. MP, ML, and NJ phylogenetic analysis based on 964 bp of partial mitochondrial DNA sequences (cytochrome b and 16S rDNA) revealed two main clades: (1) “Pterois” clade (Pterois miles and Pterois volitans), and (2) “Pteropterus–Dendrochirus” clade (remainder of the sampled species). The position of Dendrochirus brachypterus either basal to the main clades or in the “Pteropterus–Dendrochirus” clade cannot be resolved. However, the molecular phylogeny did not support the current separation of the genera Pterois and Dendrochirus. The siblings P. miles and P. volitans are clearly separated and our results support the proposed allopatric or parapatric distribution in the Indian and Pacific Ocean. However, the present analysis cannot reveal if P. miles and P. volitans are separate species or two populations of a single species, because the observed separation in different clades can be either explained by speciation or lineage sorting. Molecular clock estimates for the siblings P. miles and P. volitans suggest a divergence time of 2.4–8.3 mya, which coincide with geological events that created vicariance between populations of the Indian and Pacific Ocean.     

Revision and reclassification of three Plasmopara species based on morphological and molecular phylogenetic data

Based on the results of morphological and DNA sequence (partial D1–D3/D7–D8 nuLSU and partial nuSSU-ITS1-5.8S rDNA) data, three species of Plasmopara are revised and reclassified. A species of Plasmopara parasitic on Scorzonera, invalidly published several times, is assigned to a new genus and species under Novotelnova scorzonerae. Plasmopara euphrasiae sp. nov. is segregated from P. densa, and P. centaureae-mollis is revised and relegated to synonymy of Bremia centaureae. All taxa are described and illustrated.     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.     

Restriction fragment length polymorphism (RFLP) analysis of bovine nuclear protein genes

Summary We have recently cloned both the bovine protamine (Krawetz et al. 1987, DNA 6: 47–57) and high mobility group (HMG-1) cDNAs (Pentecost and Dixon 1984, Bioscience Reports 4: 49–57). They have been used as probes for Restriction Fragment Length Polymorphism analysis of male-female pairs of different species and breeds, within the genus Bos. Utilizing this approach we have studied inheritance, chromosomal location and gene copy number of the bovine protamine and HMG-1 genes. This revealed that these nuclear protein genes are highly conserved suggesting that selective pressure has maintained their gene structures during evolution. A polymorphic Taq 1 restriction fragment was identified that was shown to be a heritable marker. These genes are not sex-linked and are present in a single copy for protamine and at least two copies for the HMG-1.     

SURVEY AND SUMMARY: exon-intron organization of genes in the slime mold Physarum polycephalum

The slime mold Physarum polycephalum is a morphologically simple organism with a large and complex genome. The exon–intron organization of its genes exhibits features typical for protists and fungi as well as those characteristic for the evolutionarily more advanced species. This indicates that both the taxonomic position as well as the size of the genome shape the exon–intron organization of an organism. The average gene has 3.7 introns which are on average 138 bp, with a rather narrow size distribution. Introns are enriched in AT base pairs by 13% relative to exons. The consensus sequences at exon–intron boundaries resemble those found for other species, with minor differences between short and long introns. A unique feature of P.polycephalum introns is the strong preference for pyrimidines in the coding strand throughout their length, without a particular enrichment at the 3′-ends.     

A comparative analysis of complete plastid genomes from Prangos fedtschenkoi and Prangos lipskyi (Apiaceae)

Prangos fedtschenkoi (Regel & Schmalh.) Korovin and P. lipskyi Korovin (Apiaceae) are rare plant species endemic to mountainous regions of Middle Asia. Both are edificators of biotic communities and valuable resource plants. The results of recent phylogenetic analyses place them in Prangos subgen. Koelzella (M. Hiroe) Lyskov & Pimenov and suggest they may possibly represent sister species. To aid in development of molecular markers useful for intraspecific phylogeographic and population‐level genetic studies of these ecologically and economically important plants, we determined their complete plastid genome sequences and compared the results obtained to several previously published plastomes of Apiaceae. The plastomes of P. fedtschenkoi and P. lipskyi are typical of Apiaceae and most other higher plant plastid DNAs in their sizes (153,626 and 154,143 bp, respectively), structural organization, gene arrangement, and gene content (with 113 unique genes). A total of 49 and 48 short sequence repeat (SSR) loci of 10 bp or longer were detected in P. fedtschenkoi and P. lipskyi plastomes, respectively, representing 42–43 mononucleotides and 6 AT dinucleotides. Seven tandem repeats of 30 bp or longer with a sequence identity ≥90% were identified in each plastome. Further comparisons revealed 319 polymorphic sites between the plastomes (IR, 21; LSC, 234; SSC, 64), representing 43.8% transitions (Ts), 56.1% transversions (Tv), and a Ts/Tv ratio of 0.78. Within genic regions, two indel events were observed in rpoA (6 and 51 bp) and ycf1 (3 and 12 bp), and one in ndhF (6 bp). The most variable intergenic spacer region was that of accD/psaI, with 21.1% nucleotide divergence. Each Prangos species possessed one of two separate inversions (either 5 bp in ndhB intron or 9 bp in petB intron), and these were predicted to form hairpin structures with flanking repeat sequences of 18 and 19 bp, respectively. Both species have also incorporated novel DNA in the LSC region adjacent to the LSC/IRa junction, and BLAST searches revealed it had a 100 bp match (86% sequence identity) to noncoding mitochondrial DNA. Prangos‐specific primers were developed for the variable accD/psaI intergenic spacer and preliminary PCR‐surveys suggest that this region will be useful for future phylogeographic and population‐level studies.     

Restriction fragment length variants in the marsupialSminthopsis crassicaudata

A fully pedigreed colony of the dasyurid marsupialSminthopsis crassicaudata has provided material for establishing two panels of DNA samples: a broad-based test panel and a two-generation family panel. These have been used to search for genetic markers in the form of restriction fragment length variants. The molecular probes—pSG-2H, a region of theS. crassicaudata embryonic -globin gene; pB8.BS, a region of the human ubiquitin gene, and p3-21a1:1, a region of the processed pseudogene of phosphoglycerate kinase-1 of the macropodid marsupialMacropus robustus—were hybridized to Southern blots ofEcoR1-digested DNA from the panels. Analysis of these blots when probed with pSG-2H provided evidence of two alleles segregating at a singleEcoR1 site. Analysis of the same blots when probed with pB8.BS suggested allelic variation at two closely linkedEcoR1 sites. Probing the blots with p3-21a1:1 produced a complex pattern of bands resembling DNA fingerprints. The presence of a 12.3-kb band was found to conform to a simple autosomal dominant mode of inheritance. Analysis of the family data, for each probe, revealed no significant departure from Mendelian inheritance. This work has provided additional genetic markers that will enhance the use ofS. crassicaudata as a model marsupial species and has demonstrated that a high level of genetic variability has been maintained in the marsupial colony.This project was supported by grants from the Australian Research Council and the University of Adelaide.