Biometrical analysis reveals major differences between the two subspecies of the European rabbit

The climatic oscillations that have occurred in the last few million years have strongly affected species distribution ranges. Highly divergent genetic lineages arose, some of which correspond to recognized subspecies that currently occupy small geographical areas. Understanding the implications of the genetic differences between these subspecies is crucial for proper conservation of Evolutionarily Significant Units. We use the two European rabbit subspecies, Oryctolagus cuniculus cuniculus and O. c. algirus, in the Iberian Peninsula as a model to investigate the repercussions at the biometric level of their largely recognized genetic differentiation. To accomplish this we analysed the ear and hind foot length, and the body mass of 999 adult rabbits from 27 locations across the distribution range of both subspecies in their native range, the Iberian Peninsula. Our results show biometric differences between the two subspecies, also explained by geographical location and sex, O. c. algirus being lighter and having shorter ear and hind foot lengths. We examine these findings under an evolutionary framework, and discuss their implications for current conservation efforts. Future research should focus on the ecological implications of these biometric differences, namely potential different habitat use and anti‐predatory strategies in the species' native range.     

Social environment and weather during early life influence gastro-intestinal parasite loads in a group-living mammal

Conditions experienced during early life have been frequently shown to exert long-term consequences on an animal’s fitness. In mammals and birds, the time around and shortly after weaning is one of the crucial periods early in life. However, little is known about how social and abiotic environmental conditions experienced around this time affect fitness-related traits such as endoparasite loads. We studied consequences of social interactions and rainy weather conditions around and after weaning on gastro-intestinal nematode loads in juvenile European rabbits Oryctolagus cuniculus. Infestations with the gastric nematode Graphidium strigosum and with the intestinal nematode Passalurus ambiguus were higher in animals experiencing more rain during early life. This might have been due to the higher persistence of nematodes’ infective stages outside the host body together with the animals’ lower energy allocation for immune defence under more humid and thus energetically challenging conditions. In contrast, infestations with P. ambiguus were lower in animals with more positive social interactions with mother and litter siblings. We propose that social support provided by familiar group members buffered negative stress effects on immune function, lowering endoparasite infestations. This is supported by the negative correlation between positive social behaviour and serum corticosterone concentrations, indicating lower stress in juveniles which integrated more successfully into the social network of their group. In conclusion, the findings offer a pathway showing how differences in the abiotic environment and social life conditions experienced early in life could translate into long-term fitness consequences via the effects on endoparasite loads.     

European rabbit research in the Iberian Peninsula: state of the art and future perspectives

The European rabbit (Oryctolagus cuniculus) is a high-profile prey, native from the Iberian Peninsula, the only region in the world where the two rabbit subspecies (O. cuniculus algirus and O. cuniculus cuniculus) currently co-exist in natural conditions. In this area, this important prey represents a keystone species and ecosystem engineer of Mediterranean landscapes, being also the most harvested and one of the most managed small-game species. Additionally, the species can create damage to crops in some parts of the Iberian Peninsula where it is regarded as an agricultural pest. The scientific interest towards the species is becoming increasingly apparent most likely as a repercussion of declining population trends over the last decades. The latter has been the result of the impact of habitat deterioration, viral diseases, unsustainable hunting, and predation. In this paper, I present a review of the scientific literature currently available on the European rabbit in the Iberian Peninsula. I discuss knowledge gaps and highlight priority research guidelines to suppress them, in an attempt to provide a general perspective to target research efforts more effectively. This analysis is particularly relevant due to the current vulnerability of rabbit populations in Iberia and to the recent news of cuts in scientific funding in most Mediterranean countries.     

Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species

Whereas in its natural host (Sylvilagus sps.) the effects of myxoma virus infections are benign, in European rabbit (Oryctolagus cuniculus), it causes a highly infectious disease with very high mortality rate, known as myxomatosis. There is evidence that, as with HIV-1 virus in human, myxoma virus may use chemokine receptors such as CCR5 of the host target cell for entry and activation of pathways of immune avoidance. We have characterized and compared CCR5 genes of leporid species with different susceptibility levels to myxomatosis. The CCR5 protein of O. cuniculus differs markedly from all those known from other species. The most striking was the replacement of a specific peptide motif of the second extracellular loop (ECL2) by a motif, which in other species characterizes the CCR2 molecules. While absent in Sylvilagus and Lepus species, this CCR2 imposed CCR5–ECL2 alteration was observed in all genomes of 25 European rabbits, representing the subspecies O. cuniculus algirus and O. cuniculus cuniculus. Allelic variation at the rabbit CCR5 locus confirmed that the gene conversion predates the subspecies split (1–2 Ma).     

Predictive modelling to aid the regional-scale management of a vertebrate pest

Extensive resources are allocated to managing vertebrate pests, yet spatial understanding of pest threats, and how they respond to management, is limited at the regional scale where much decision-making is undertaken. We provide regional-scale spatial models and management guidance for European rabbits (Oryctolagus cuniculus) in a 260,791 km² region in Australia by determining habitat suitability, habitat susceptibility and the effects of the primary rabbit management options (barrier fence, shooting and baiting and warren ripping) or changing predation or disease control levels. A participatory modelling approach was used to develop a Bayesian network which captured the main drivers of suitability and spread, which in turn was linked spatially to develop high resolution risk maps. Policy-makers, rabbit managers and technical experts were responsible for defining the questions the model needed to address, and for subsequently developing and parameterising the model. Habitat suitability was determined by conditions required for warren-building and by above-ground requirements, such as food and harbour, and habitat susceptibility by the distance from current distributions, habitat suitability, and the costs of traversing habitats of different quality. At least one-third of the region had a high probability of being highly suitable (support high rabbit densities), with the model supported by validation. Habitat susceptibility was largely restricted by the current known rabbit distribution. Warren ripping was the most effective control option as warrens were considered essential for rabbit persistence. The anticipated increase in disease resistance was predicted to increase the probability of moderately suitable habitat becoming highly suitable, but not increase the at-risk area. We demonstrate that it is possible to build spatial models to guide regional-level management of vertebrate pests which use the best available knowledge and capture fine spatial-scale processes.     

Extensive Genetic Polymorphism of Peptidases A, B, C, and D, in Wild Rabbit (Oryctolagus cuniculus) Populations from the Iberian Peninsula

Genetic polymorphism of peptidases A, B, C, andD in the wild rabbit (Oryctolagus cuniculus)was assessed by horizontal starch gel electrophoresis,in a total of 1003 individuals comprising 16 Iberian and 2 French populations and 1 domestic stock.Twenty-five different alleles were detected: 6 in PEPA,4 in PEPB, 8 in PEPC, and 7 in PEPD. The highest valuesof observed heterozygosity averaged over the four loci were obtained for the southwestern Iberianpopulations and a clinal loss of variability in anortheastern direction was detected. A clear separationbetween the two putative subspecies O. c.cuniculus and O. c. algirus was notobtained.     

Révision d’Oryctolagus lacosti (Lagomorpha,Mammalia) du Pliocène supérieur de Perrier (Auvergne,France)

Fossil leporids from the Upper Pliocene of the locality of Perrier-Étouaires (Auvergne, France) are here revisited. They were initially attributed to Lepus lacosti by Pomel (1853), and later to the genus Oryctolagus. This material had been neither accurately described nor figured until now. Thus, a lectotype and two paralectotypes have been chosen among the material of the original collection. The diagnostic character of Oryctolagus lacosti is its big size, similar to that of modern hares (Lepus), together with other morphological characters that fit in the variability range of European rabbits (Oryctolagus cuniculus). Fossils of leporids similar to those of Perrier have also been found in several Plio-Pleistocene localities from western Europe.     

Sharing of Endogenous Lentiviral Gene Fragments among Leporid Lineages Separated for More than 12 Million Years

Lentiviruses are causal agents of severe pathologies of a variety of mammals, including cattle and humans (e.g., AIDS and different types of lymphoma). While endogenous forms of lentivirus do not occur in these species, A. Katzourakis and coworkers (A. Katzourakis, M. Tristem, O. G. Pybus, and R. J. Gifford, Proc. Natl. Acad. Sci. USA 104:6261-6265, 2007) recently reported the presence in the genome of the European rabbit (Oryctolagus cuniculus) of multiple sequences defining a lentiviral subgroup elegantly referred to as RELIK (rabbit endogenous lentivirus type K). Sequence comparisons indicated that the RELIK ancestor may have integrated into the rabbit lineage more than 7 million years ago. We have substantiated this by producing sequence data certifying the sharing of RELIK sequences among leporid lineages that diverged some 12 million years ago.Lentiviruses are remotely related to known endogenous retroviruses (e.g., of the alpha- and betaretrovirus groups). Five subgroups of lentivirus have been described, each being restricted to a single mammalian family (1, 5, 14). Most intensively studied are the more recently discovered human immunodeficiency viruses. The origin of this virus group, however, remains obscure (11, 12), mainly because of the absence of endogenous forms, i.e., of virus that has integrated into the host genome. Compared to their exogenous counterparts, such germ line-imbedded copies are known to maintain much better their original sequence patterns and therefore provide valuable markers in evolutionary studies (3, 4). The discovery by Katzourakis and coworkers (6) of what appears to be the first reported endogenous lentivirus in any species therefore constitutes a crucial step toward our understanding of the history of lentivirus and associated diseases.Interestingly, it was found in the European rabbit (Oryctolagus cuniculus), a species without known lentiviral affections. It was achieved by data mining of the WGS (Whole Genome Shotgun) archives released by the rabbit genome project (genome project 12819). By measuring the genetic distances between segmentally duplicated regions, assuming a divergence rate of 4 × 10⁻⁹ nucleotide substitutions per site per year for the rabbit genome (7, 8), Katzourakis and coworkers dated the oldest intragenomic segmental duplication at 7 million years (My) and estimated at 11 My the maximal time depth of the RELIK evolutionary tree. The authors suggested that a more exact estimate of RELIK persistence times might be obtained by searching for the occurrence of related lentiviral sequences in other lagomorph species.We have done this for several lagomorph genera by targeting the RELIK gag gene sequence, which is among the more conserved gene regions of lentivirus (6). The gag sequence of the RELIK consensus region was at first subjected to intensive BLAST research on WGS data banks, both in the trace file archives (http://www.ncbi.nlm.nih.gov/blast/mmtrace.shtml) and in the assembled WGS reads (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The most closely related nonrabbit sequences were parts of the gag genes of equine infective anemia virus and of puma feline lentivirus (d > 0.75). Except for Oryctolagus cuniculus, no vertebrate sequence even remotely similar to the query was found in any of the available databases, including that of Ochotona princeps (pikas, or whistling hares), the only other lagomorph species for which WGS sequences are available. The order Lagomorpha is indeed composed of two families, which diverged more than 35 My ago (7, 8): Ochotonidae, with the genus Ochotona (pikas), and Leporidae (rabbits and hares), with 11 extant genera, Brachylagus, Caprolagus, Nesolagus, Pentalagus, Pronolagus, Poelagus, Romerolagus, Sylvilagus, Oryctolagus, Bunolagus, and Lepus (2, 7).Genomic DNA from tissue samples of Orytcolagus cuniculus algirus (OCA), Bunolagus monticularis (BM), Lepus granatensis (LG), and Sylvilagus brasiliensis (SB) was prepared using a Qiagen extraction kit (Qiagen, Vienna, Austria). Taxonomic verification for DNA samples was carried out using mitochondrial and nuclear markers. (Genomic DNA of Ochotona princeps was kindly made available by Dennis Lanning [Loyola University Chicago]. The specimen of Oryctolagus was collected in the aboriginal range of the species [Mértola, SE Portugal] [7].) Amplification of a 0.7-kb fragment of the RELIK gag gene (below GagC) was performed using the primer pair Gag_1F (5′-GGACGTCCCAGTCAAAAGAA-3′) and Gag_1R (5′-AGGGTTCTGGCATCAGCAAA-3′), designed according to the RELIK consensus sequence described in reference 6. The PCR conditions and procedures were standard and are available upon request. PCR products with the expected fragment size were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Sequencing of cloned and uncloned products was performed on an ABI PRISM 310 genetic analyzer. Sequence data were analyzed using the software packages MEGA4 (13, 15) and DnaSP4 (10).PCR amplification proved to be successful with the four leporid species but not with pikas. The sequencing of the uncloned PCR products revealed sequence patterns undeniably characteristic of GagC although often obscured by multiple polymorphic positions and frameshift mutations due to insertions and deletions (data not shown). Sequencing of cloned products was limited to two clones per individual. For Sylvilagus (SB), only one sequence was obtained, while the two sequences of one Portuguese wild rabbit (OCA) turned out to be identical, although the electrochromatographs of the crude PCR product revealed the presence of a variety of different sequences.For each species, the cloned sequences showed more than 90% similarity with GagC. These sequences were grouped per species (OCA, BM, SB, and LG; see above) and compared to the 14 WGS assemblies of the archive of Oryctolagus c. cuniculus (Occ01 to Occ14, forming the species group OCC). These assemblies were selected for their complete coverage of the RELIK gag region under study. The distance tree in Fig. ​Fig.11 visualizes the common history of RELIK-like sequences among the Oryctolagus, Bunolagus, Lepus, and Sylvilagus species. We note that, independently of the tree-building method used (including maximum likelihood) and analysis of synonymous and nonsynonymous distance trees (data not shown), there were no indications of separate clustering according to species origin (i.e., although the two BM sequences form one cluster, they are embedded within the OCC clade). This confirms that the initial genome insertion of the RELIK precursor must have occurred in a common ancestor of these species. Because ratios of nonsynonymous to synonymous evolutionary changes were consistently and significantly smaller than 1 (0.55 to 0.36) (6), we have estimated both synonymous and nonsynonymous divergence times. The pairwise distances among the rabbit WGS sequences were found to differ by almost an order of magnitude, implying that different pairs do not have the same history. In Table ​Table1,1, we therefore show the range of distance values between the sequence pairs rather than their averages.Open in a separate windowFIG. 1.Evolutionary-distance tree of homologous gag gene fragments of various leporid species. The neighbor-joining tree was obtained using the p distance method provided by the MEGA4 program (13). p distances are obtained by dividing the number of nucleotide differences by the total number of nucleotides compared. All positions containing gaps and missing data were eliminated from the data set (by using the complete deletion option [592 bp]). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown above the branches for percentages above 60. The tree is drawn to scale, with branch lengths shown in the same units as those of the evolutionary distances used to infer the distance tree. Values larger than 0.003 are shown below the branches. Sequences were aligned on the PCR target GagC, ignoring primer-imposed parts and frame-disrupting inserts (658 bp). The sequences were named according to species origin, as follows: Occ, Oryctolagus cuniculus cuniculus; Oca, Oryctolagus c. algirus; Bm, Bunolagus monticularis; Lg, Lepus granatensis; and Sb, Sylvilagus brasiliensis. Occ_01 to Occ_14 are derived from assembled WGS contigs. Their GenBank accession numbers are AAGW01007437, AAGW01043000, AAGW01086182, AAGW01127774, AAGW01133803, AAGW01183526, AAGW01222583, AAGW01228233, AAGW01228525, AAGW01252278, AAGW01327267, AAGW01433545, AAGW01516016, and AAGW01717649, respectively. The remaining sequences are those of cloned PCR products. Their GenBank accession numbers are shown in the text.

TABLE 1.

Ranges of synonymous nucleotide distances between pairs of RELIK elements within and between species groups^a

Sequencing of modern Lepus VDJ genes shows that the usage of VHn genes has been retained in both Oryctolagus and Lepus that diverged 12 million years ago

Among mammals, the European rabbit (Oryctolagus cuniculus) has a unique mechanism of generating the primary antibody repertoire. Despite having over 200 VH genes, the VH1 gene, the most d-proximal VH gene, is used in 80–90 % of VDJ rearrangements, while the remaining 10–20 % is encoded by the VHn genes that map at least 100 Kb upstream of VH1. The maintenance of the VHn genes usage in low frequency in VDJ rearrangements has been suggested to represent a relic of an ancestral immunologic response to pathogens. To address this question, we sequenced VDJ genes for another leporid, genus Lepus, which separated from European rabbit 12 million years ago. Approximately 25 VDJ gene sequences were obtained for each one of three Lepus europaeus individuals. We found that Lepus also uses the VHn genes in 5–10 % of its VDJ rearrangements. Our results show that the VHn genes are a conserved ancestral polymorphism that has been maintained in the leporids genome and is being used for the generation of VDJ rearrangements by both modern Lepus and Oryctolagus.     

Proteomic investigation of the effects of weight loss in the gastrocnemius muscle of wild and NZW rabbits via 2D‐electrophoresis and MALDI‐TOF MS

The study of changes within the key agents regulating metabolism during genetic upgrading because of selection can contribute to an improved understanding of genomic and physiological relationships. This may lead to increased efficiencies in animal production. These changes, regarding energy and protein metabolic saving mechanisms, can be highlighted during food restriction periods. In this study, a 20% weight reduction was induced in two rabbit breeds: New Zealand white, a selected meat producer (Oryctolagus cuniculus cuniculus), and Iberian wild rabbit (Oryctolagus cuniculus algirus), with the aim of determining differential protein expression in the gastrocnemius muscle within control (ad libitum) and restricted diet experimental animal groups, using techniques of two‐dimensional gel electrophoresis and peptide mass fingerprinting. Results show that l ‐lactate dehydrogenase, adenylate kinase, β enolase and α enolase, fructose bisphosphate aldolase A and glyceraldehyde 3‐phosphate dehydrogenase, which are enzymes involved in energy metabolism, are differentially expressed in restricted diet experimental animal groups. These enzymes are available to be further tested as relevant biomarkers of weight loss and putative objects of manipulation as a selection tool towards increasing tolerance to weight loss. Similar reasoning could be applied to 2D gel electrophoresis spots corresponding to the important structural proteins tropomyosin β chain and troponin I. Finally, a spot identified as mitochondrial import stimulation factor seems of special interest as a marker of undernutrition, and it may be the object of further studies aiming to better understand its physiological role.     

Recombination and Speciation: Loci Near Centromeres Are More Differentiated Than Loci Near Telomeres Between Subspecies of the European Rabbit (Oryctolagus cuniculus)

Recent empirical and theoretical studies suggest that regions of restricted recombination play an important role in the formation of new species. To test this idea, we studied nucleotide variation in two parapatric subspecies of the European rabbit (Oryctolagus cuniculus). We surveyed five loci near centromeres, where recombination is expected to be suppressed, and five loci near telomeres, where recombination is expected to be higher. We analyzed this multilocus data set using a divergence-with-gene flow framework and we report three main findings. First, we estimated that these subspecies diverged ~1.8 MYA and maintained large effective population sizes (O. c. algirus N_e ≈ 1,600,000 and O. c. cuniculus N_e ≈ 780,000). Second, we rejected a strict allopatric model of divergence without gene flow; instead, high rates of gene flow were inferred in both directions. Third, we found different patterns between loci near centromeres and loci near telomeres. Loci near centromeres exhibited higher levels of linkage disequilibrium than loci near telomeres. In addition, while all loci near telomeres showed little differentiation between subspecies, three of five loci near centromeres showed strong differentiation. These results support a view of speciation in which regions of low recombination can facilitate species divergence in the presence of gene flow.     

Colonization history of Mallorca Island by the European rabbit,Oryctolagus cuniculus,and the Iberian hare,Lepus granatensis (Lagomorpha: Leporidae)

The Mediterranean islands have a long history of human‐mediated introductions resulting in frequent replacements of their fauna and flora. Although these histories are sometimes well documented or may be inferred from paleontological studies, the use of phylogenetic and population genetic reconstruction methods provides a complementary perspective for answering questions related to the history of insular species. In the present study, we infer the colonization history of Mallorca (Balearic Islands) by the European rabbit (Oryctolagus cuniculus) and the Iberian hare (Lepus granatensis) using sequence variation of the mitochondrial DNA control region from continental and insular specimens (total of 489 sequences). Additionally, the taxonomic identity of Mallorcan L. granatensis was confirmed using a diagnostic nuclear marker. For both Mallorcan rabbits and hares, genetic diversity was comparable to the continental populations, suggesting the introduction of multiple lineages. Two Mallorcan haplogroups were found in hares, which likely correspond to two introduction events. Rabbits from Mallorca were identified as belonging to the subspecies Oryctolagus cuniculus cuniculus, and may have been originated both from Iberian and French populations. The molecular estimates of the timing of the colonization events of the Mallorcan lagomorphs are consistent with human‐mediated introductions by early settlers on the islands. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 748–760.     

Analysis of Low Frequency Protein Truncating Stop-Codon Variants and Fasting Concentration of Growth Hormone

Background

The genetic background of Growth Hormone (GH) secretion is not well understood. Mutations giving rise to a stop codon have a high likelihood of affecting protein function.

Objectives

To analyze likely functional stop codon mutations that are associated with fasting plasma concentration of Growth Hormone.

Methods

We analyzed stop codon mutations in 5451 individuals in the Malmö Diet and Cancer study by genotyping the Illumina Exome Chip. To enrich for stop codon mutations with likely functional effects on protein function, we focused on those disrupting >80% of the predicted amino acid sequence, which were carried by ≥10 individuals. Such mutations were related to GH concentration, measured with a high sensitivity assay (hs-GH) and, if nominally significant, to GH related phenotypes, using linear regression analysis.

Results

Two stop codon mutations were associated with the fasting concentration of hs-GH. rs121909305 (NP_005370.1:p.R93*) [Minor Allele Frequency (MAF) = 0.8%] in the Myosin 1A gene (MYO1A) was associated with a 0.36 (95%CI, 0.04 to 0.54; p=0.02) increment of the standardized value of the natural logarithm of hs-GH per 1 minor allele and rs35699176 (NP_067040.1:p.Q100*) in the Zink Finger protein 77 gene (ZNF77) (MAF = 4.8%) was associated with a 0.12 (95%CI, 0.02 to 0.22; p = 0.02) increase of hs-GH. The mutated high hs-GH associated allele of MYO1A was related to lower BMI (β-coefficient, -0.22; p = 0.05), waist (β-coefficient, -0.22; p = 0.04), body fat percentage (β-coefficient, -0.23; p = 0.03) and with higher HDL (β-coefficient, 0.23; p = 0.04). The ZNF77 stop codon was associated with height (β-coefficient, 0.11; p = 0.02) but not with cardiometabolic risk factors.

Conclusion

We here suggest that a stop codon of MYO1A, disrupting 91% of the predicted amino acid sequence, is associated with higher hs-GH and GH-related traits suggesting that MYO1A is involved in GH metabolism and possibly body fat distribution. However, our results are preliminary and need replication in independent populations.     

Phylogeny and evolutionary origins of the Leporidae: a review of cytogenetics, molecular analyses and a supermatrix analysis

• 1 We review current knowledge of the evolutionary relationships among species of Leporidae drawing on molecular, cytogenetic and morphological data. We highlight problems associated with retrieving phylogenetic information under conditions of a rapid radiation and the lack of phylogenetically informative cytogenetic and mitochondrial DNA characters. Most morphological features underpinning generic distinctions are subtle and prone to reversal and convergence and as a consequence, they generally provide little basis for assessing phylogenetic affinity.
• 2 We report the results of a supermatrix analysis that combines published nucleotide sequence data, unique insertion/deletion events, morphological characters and presumed geographical centres of origin of each genus. This represents the most comprehensive intergeneric comparison of the Leporidae thus far undertaken.
• 3 The monophyly of the 11 leporid genera is unambiguously supported. There is support for an Afroasian assemblage that comprises Poelagus, Pronolagus and Nesolagus, a primitive Lepus, with the problematic Bunolagus, Oryctolagus, Caprolagus and Pentalagus as derived species in a clade that also includes the closely related Brachylagus and Sylvilagus as sister taxa.
• 4 There is no support for the Palaeolaginae, although Romerolagus is an ancient lineage within the extant Leporidae.
• 5 We hold that of the polytypic genera Lepus remains the most problematic, and provide a working hypothesis that will hopefully encourage future research on the various hare species.

     

Selected subspecies names from Richter,Plantae Europeae,Vol. 1, 1890

The bookK. Richter, Plantae europeae, Vol. 1, 1890, is analyzed from the viewpoint of nomenclature of subspecies names. 264 subspecies names interesting from the standpoint of contemporary taxonomy and nomenclature are selected from the total number of 840 subspecies names for a commented list. Seventeen important cases are dealt with separately in greater detail. Eleven new nomenclatural combinations (two for species, nine for subspecies) are proposed in the Appendix. Types for two generic names (Acorellus Palla andJuncellus C. B. Clarke) are proposed. A new term “parabasionym” is applied for names establishing the priority of infraspecific epithets in the rank in question. The need to respect the newly accepted principle of priority of autonymic subspecies names at proposing new nomenclatural combinations is stressed. Examples ofRichter’s subspecies names having priority, subspecies names with necessary changes in their authorship and names having a possible importance as parabasionyms are enumerated in separate lists.     

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

Background

Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.

Results

The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.

Conclusion

The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.     

Differentiation of tortoises of the genera Testudo and Agrionemys (Testudinidae) based on the polymorphism of nuclear and mitochondrial markers

Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes, including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statistically significantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.