Molecular Cytogenetic Analysis of Karyotype and Y Chromosome Conservation in Species of the Genus Talpa (Insectivora).

The Talpidae family has a highly stable karyotype. Most of the chromosome studies in this mammal group, however, employed classical cytogenetic techniques. Molecular cytogenetic analyses are still scarce and, for example, no repeated DNA sequences have been described to date. In this work, we used sequence analysis, chromosomal mapping of a LINE1 retroelement sequence, as well as chromosome painting with a whole Y chromosome probe of T. occidentalis to compare the karyotypes of 3 species of the genus Talpa (T. occidentalis, T. romana, and T. aquitania). Our results demonstrate that in Talpa genomes LINE1 sequences are widely distributed on all chromosomes but are enriched in pericentromeric C-band-positive regions. In addition, these LINE1 accumulate on the Y chromosomes of the 3 Talpa species regardless of their euchromatic or heterochromatic condition. Chromosome painting shows that the Y chromosomes in these 3 species are highly conserved. Interestingly, they share sequences with heterochromatic blocks on chromosome pairs 14 and 16 and, to a lesser degree, with the pericentromeric regions of other autosomes. Together, our analyses demonstrate that the repetitive DNA content of chromosomes from Talpa species is highly conserved.

The complete mitochondrial genome of Talpa aquitania (Talpidae; Insectivora), a mole species endemic to northern Spain and southern France.

The complete mitogenome sequence of Talpa aquitania, a recently described Talpa species, was assembled using whole-genome sequencing data. It varies in length from 16,776 to 16,846 bp, contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L-strand replication, and a control region. In the control region, which varied from 1320 to 1390 bp, we identified the extended termination-associated sequence (ETAS-1 and ETAS-2) and the conserved sequence blocks (CSB-1, 2, 3, B, C, D, E, F). In addition, this region includes a 10 bp tandem repeat DNA sequence, with a variable number of repeats that suggest the existence of heteroplasmy. Phylogeny reconstructions based on Maximum Likelihood, Neighbor-joining and Bayesian inference analyses yielded phylogenies with similar topologies demonstrating that T. aquitania and T. occidentalis are sister species.

Karyotype Analysis of the New Talpa Species Talpa aquitania (Talpidae; Insectivora) from Northern Spain.

Karyotypes of 3 male Talpa specimens from northern Spain were analyzed. The mesostyles of upper molars and cytochrome b sequence analysis identified these specimens as belonging to Talpa aquitania, a new Talpa species recently described from northern Spain and southern France. We describe here for the first time the karyotype of Talpa aquitania. Its diploid number is 2n = 34 and NFa = 64, and all chromosomes including the sex chromosomes are biarmed, either metacentric or submetacentric. G-banding demonstrated that the karyotypes of T. aquitania and T. occidentalis (the most closely related species) are almost identical. However, the karyotype of T. aquitania differs from the karyotypes of both T. europaea and T. occidentalis in that it has a medium-sized biarmed Y chromosome rather than a dot-like chromosome and that chromosome 16 is submetacentric in T. aquitania but has a small p-arm in both T. europaea and T. occidentalis. Pericentromeric C-bands were scarce and only clearly visible in a few chromosomal pairs. In addition, C-banding demonstrated that half of the 14p, the 16p, and the Y chromosome are all heterochromatic. rDNA genes were located at the secondary constriction in autosomal pair 3, a common feature in the karyotypes of all Talpa species. Hybridization signals for telomeric repeats were found on the telomeres and the pericentric regions of some chromosomes and co-localized in the secondary constriction of pair 3 with the rDNA genes. In conclusion, the karyotype of T. aquitania from northern Spain is very similar to the karyotype of other species belonging to the genus Talpa.

Determining diet from faeces: Selection of metabarcoding primers for the insectivore Pyrenean desman (Galemys pyrenaicus).

Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements.

Mitogenomic sequences support a north-south subspecies subdivision within Solenodon paradoxus.

Solenodons are insectivores found only in Hispaniola and Cuba, with a Mesozoic divergence date versus extant mainland mammals. Solenodons are the oldest lineage of living eutherian mammal for which a mitogenome sequence has not been reported. We determined complete mitogenome sequences for six Hispaniolan solenodons (Solenodon paradoxus) using next-generation sequencing. The solenodon mitogenomes were 16,454-16,457 bp long and carried the expected repertoire of genes. A mitogenomic phylogeny confirmed the basal position of solenodons relative to shrews and moles, with solenodon mitogenomes estimated to have diverged from those of other mammals ca. 78 Mya. Control region sequences of solenodons from the northern (n = 3) and southern (n = 5) Dominican Republic grouped separately in a network, with FST = 0.72 (p = 0.036) between north and south. This regional genetic divergence supports previous morphological and genetic reports recognizing northern (S. p. paradoxus) and southern (S. p. woodi) subspecies in need of separate conservation plans.

Evolutionary History of the Nesophontidae, the Last Unplaced Recent Mammal Family.

The mammalian evolutionary tree has lost several major clades through recent human-caused extinctions. This process of historical biodiversity loss has particularly affected tropical island regions such as the Caribbean, an area of great evolutionary diversification but poor molecular preservation. The most enigmatic of the recently extinct endemic Caribbean mammals are the Nesophontidae, a family of morphologically plesiomorphic lipotyphlan insectivores with no consensus on their evolutionary affinities, and which constitute the only major recent mammal clade to lack any molecular information on their phylogenetic placement. Here, we use a palaeogenomic approach to place Nesophontidae within the phylogeny of recent Lipotyphla. We recovered the near-complete mitochondrial genome and sequences for 17 nuclear genes from a ∼750-year-old Hispaniolan Nesophontes specimen, and identify a divergence from their closest living relatives, the Solenodontidae, more than 40 million years ago. Nesophontidae is thus an older distinct lineage than many extant mammalian orders, highlighting not only the role of island systems as "museums" of diversity that preserve ancient lineages, but also the major human-caused loss of evolutionary history.

Molecular phylogenetics and phylogeographic structure of Sorex bedfordiae based on mitochondrial and nuclear DNA sequences.

The southeastern margin of the Tibetan Plateau is characterized by complex topography and a discontinuous landscape, creating a sky island situation. However, the way topography shapes genetic structures and demographic histories of endemic species has not been well studied. We examined the phylogeographic pattern and demographic histories of Sorex bedfordiae, a dispersal-limited small mammal, using three nuclear genes [1977bp] and two mitochondrial genes [1794bp] with comprehensive molecular approaches. We recovered five well-supported clades whose distributions are along mountain ridges and roughly subdivided by large rivers. Demographic expansions in the middle Pleistocene were strongly supported by both nuclear and mitochondrial genes. Our results support the hypothesis that sky island topography and river systems strongly affect the genetic structure of non-aquatic terrestrial species. We further clarify that S. bedfordiae and S. cylindricauda are valid sibling species, whereas S. excelsus is most likely a geographic subspecies of S. bedfordiae.

A new hantavirus from the stripe-backed shrew (Sorex cylindricauda) in the People's Republic of China.

Inspired by the recent discovery of genetically distinct hantaviruses from insectivore species worldwide, we performed a small-scale search for insectivore-borne hantaviruses. In this paper, we report the discovery of a new hantavirus, which was designated the Qian Hu Shan virus (QHSV). This virus was detected in the lung tissues of three stripe-backed shrews (Sorex cylindricauda), which were captured in the Yunnan Province, China. The full-length S genomic segment of the representative QHSV strain YN05-284 was 1661 nucleotides and is predicted to encode a nucleocapsid protein of 429 amino acids that starts at nucleotide position 48. It exhibited the highest similarity with other Sorex-related hantaviruses, with 68.1%-72.8% nucleotide and 71.9%-84.4% amino acid sequence identities. An analysis of a 1430-nucleotide region of the partial M segment exhibited approximately 54.4%-79.5% nucleotide and 43.2%-90.8% amino acid sequence identities to other hantaviruses. A comparison of a 432-nucleotide region of the L segment also showed similar degrees of identity, with 68.9%-78.4% nucleotide and 71.1%-93.8% amino acid sequence identities to other hantaviruses. Phylogenetic analyses using Bayesian methods indicated that QHSV shared the most recent common ancestor with other Sorex-related hantaviruses. The host was identified using a morphological assessment and verified using mitochondrial cytochrome b (mt-Cyt b) gene sequencing. A pair-wise comparison of the 1140-nucleotide mt-Cyt b gene sequence from the host demonstrated that the host was close to S. cylindricauda from Nepal with 94.3% identity. The virus-host association tanglegram, which was constructed using the Dendroscope software, indicated that the QHSV phylogeny and the host phylogeny were approximately matched, which suggests no evidence of host switching for QHSV. Our results contribute to a wider viewpoint regarding the heterogeneity of viruses that infect shrews.

Complete mitochondrial genome sequence of Nectogale elegans.

The elegant water shrew (Nectogale elegans) belongs to the family Soricidae, and distributes in northern South Asia, central and southern China and northern Southeast Asia. In this study, the complete mitochondrial genome of N. elegans was sequenced. It was determined to be 17,460 bases, and included 13 protein-coding genes (PCGs), 22 tRNA genes, 2 ribosomal RNA genes and one non-coding region, which is similar to other mammalian mitochondrial genomes. Bayesian inference and maximum likelihood methods were used to construct phylogenetic trees based on 12 heavy-strand concatenated PCGs. Phylogenetic analyses further confirmed that Crocidurinae diverged prior to Soricinae, and Sorex unguiculatus differentiated earlier than N. elegans.

Phylogeography and postglacial expansion of the endangered semi-aquatic mammal Galemys pyrenaicus.

BACKGROUND: Species with strict ecological requirements may provide new insights into the forces that shaped the geographic variation of genetic diversity. The Pyrenean desman, Galemys pyrenaicus, is a small semi-aquatic mammal that inhabits clean streams of the northern half of the Iberian Peninsula and is endangered in most of its geographic range, but its genetic structure is currently unknown. While the stringent ecological demands derived from its aquatic habitat might have caused a partition of the genetic diversity among river basins, Pleistocene glaciations would have generated a genetic pattern related to glacial refugia. RESULTS: To study the relative importance of historical and ecological factors in the genetic structure of G. pyrenaicus, we used mitochondrial and intronic sequences of specimens covering most of the species range. We show, first, that the Pyrenean desman has very low levels of genetic diversity compared to other mammals. In addition, phylogenetic and dating analyses of the mitochondrial sequences reveal a strong phylogeographic structure of a Middle Pleistocene origin, suggesting that the main lineages arose during periods of glacial isolation. Furthermore, both the spatial distribution of nuclear and mitochondrial diversity and the results of species distribution modeling suggest the existence of a major glacial refugium in the northwestern part of the Iberian Peninsula. Finally, the main mitochondrial lineages show a striking parapatric distribution without any apparent exchange of mitochondrial haplotypes between the lineages that came into secondary contact (although with certain permeability to nuclear genes), indicating incomplete mixing after the post-glacial recolonization. On the other hand, when we analyzed the partition of the genetic diversity among river basins, the Pyrenean desman showed a lower than expected genetic differentiation among main rivers. CONCLUSIONS: The analysis of mitochondrial and intronic markers in G. pyrenaicus showed the predominant effects of Pleistocene glaciations on the genetic structure of this species, while the distribution of the genetic diversity was not greatly influenced by the main river systems. These results and, particularly, the discovery of a marked phylogeographic structure, may have important implications for the conservation of the Pyrenean desman.

Chromosome evolution in Eulipotyphla.

We integrated chromosome painting information on 5 core-insectivora species available in the literature with new Zoo-FISH data for Iberian shrew (Sorex granarius) and Altai mole (Talpa altaica). Our analysis of these 7 species allowed us to determine the chromosomal features of Eulipotyphla genomes and to update the previously proposed ancestral karyotype for 2 main groups of the Sorex genus. The chromosome painting evidence with human painting probes (HSA) reveals the presence of the 2 unique associations HSA4/5 and 1/10p/12/22b, which support Eulipotyphla. There are a series of synapomorphies both for Erinaceidae (HSA3/1/5, 3/17, 11/15 and 10/20) and for Soricinae (HSA5/9, 6/7/16, 8/3/21 and 11/12/22). We found associations that link Talpidae/Erinaceidae (HSA7/8, 1/5 and 1/19p), Talpidae/Soricidae (HSA1/8/4) and Erinaceidae/Soricidae (HSA4/20 and 2/13). Genome conservation in Eulipotyphla was estimated on the basis of the number of evolutionary breaks in the ancestral mammalian chromosomes. In total, 7 chromosomes of the boreo-eutherian ancestor (BEA8 or 10, 9, 17, 18, 20-22) were retained in all eulipotyphlans studied; among them moles show the highest level of chromosome conservation. The integration of sequence data into the chromosome painting information allowed us to further examine the chromosomal syntenies within a phylogenetic perspective. Based on our analysis we offer the most parsimonious reconstruction of phylogenetic relationships in Eulipotyphla. The cytogenetic reconstructions based on these data do not conflict with molecular phylogenies supporting basal position of Talpidae in the order.

Karyotypes of six soricomorph species from Emei Shan, Sichuan Province, China.

We report the karyotypes of six species of the order Soricomorpha (Mammalia), based on specimens collected from Emei Shan (Mount Emei) in central Sichuan Province, China: two talpid species, Uropsilus andersoni (diploid chromosome number [2n]=34, fundamental number of chromosome arms Including two X chromosomes [FN]=56) and Scaptonyx fusicaudus (2n=34, FN=64); and four soricid species, Episoriculus sacratus (2n=58, FN=108), Anourosorex squamipes (2n=48, FN=96), Sorex cylindricauda (2n=30, FN=50), and Sorex bedfordiae (2n=24/25, FN=46/48). This is the first report of the karyotypes of all species except Sc. fusicaudus and A. squamipes. Episoriculus sacratus is considered to be a valid species in Sichuan, separated from the Nepalese E. soluensis based on differences in the karyotype. The karyotype of S. bedfordiae showed polymorphism due to the occurrence of a B chromosome. Differences in the karyotypes of S. cylindricauda and S. bedfordiae could be explained mostly by seven Robertsonian translocations revealed by G-band karyotypes.

Hantavirus evolution in relation to its rodent and insectivore hosts: no evidence for codivergence.

Hantaviruses are considered one of the best examples of a long-term association between RNA viruses and their hosts. Based on the appearance of strong host specificity, it has been suggested that hantaviruses cospeciated with the rodents and insectivores they infect since these mammals last shared a common ancestor, approximately 100 million years ago. We tested this hypothesis of host-virus codivergence in two ways: 1) we used cophylogenetic reconciliation analysis to assess the fit of the virus tree onto that of the host and 2) we estimated the evolutionary rates and divergence times for the Hantavirus genus using a Bayesian Markov Chain Monte Carlo method and similarly compared these with those of their hosts. Our reconciliation analysis provided no evidence for a history of codivergence between hantaviruses and their hosts. Further, the divergence times for the Hantavirus genus were many orders of magnitude too recent to correspond with the timescale of their hosts' speciation. We therefore propose that apparent similarities between the phylogenies of hantaviruses and their mammalian hosts are the result of a more recent history of preferential host switching and local adaptation. Based on the presence of clade-defining amino acids in all genomic segments, we propose that the patterns of amino acid replacement in these viruses are also compatible with a history of host-specific adaptation.

[An attempt to determine intermediate hosts of the tapeworm Echinococcus multilocularis in Poland]. / Próba wykrycia zywicieli posrednich tasiemca Echinococcus multilocularis na terenie Polski.

Echinococcus multilocularis, a tiny tapeworm (2-3 mm in length) occurs in the small intestine of red foxes and raccoon dogs in Poland. Small rodents, intermediate hosts of the tapeworm can ingest parasite eggs excreted in faeces by final host. Humans can become infected by accidental ingestion of the eggs. The resulting disease, alveolar echinococcosis, potentially lethal zoonosis, typically presents as an infiltrative tumor-like growth in the liver, which may invade neighbouring organs. The objective of the study was to determine intermediate host species for E. multilocularis in Poland. During the years 2004-2006, a total of 1425 animals, i.e. 1170 rodents (14 species), 238 insectivores (4 species) and 17 Mustelids (3 species) were trapped and autopsied for the presence of E. multilocularis protoscolices (larvae). Animals were captured in northern, north-eastern, central and southern Poland, areas where the high prevalence of the tapeworm was recorded in foxes. In 44 (3.1%) of examined animals were found cysts in the liver, however no typical lesions were affirmed. Infection was not confirmed using PCR technique with primers specific for E. multilocularis (EmCA90, EmTriple83).

Molecular phylogeny and divergence times of Malagasy tenrecs: influence of data partitioning and taxon sampling on dating analyses.

BACKGROUND: Malagasy tenrecs belong to the Afrotherian clade of placental mammals and comprise three subfamilies divided in eight genera (Tenrecinae: Tenrec, Echinops, Setifer and Hemicentetes; Oryzorictinae: Oryzorictes, Limnogale and Microgale; Geogalinae:Geogale). The diversity of their morphology and incomplete taxon sampling made it difficult until now to resolve phylogenies based on either morphology or molecular data for this group. Therefore, in order to delineate the evolutionary history of this family, phylogenetic and dating analyses were performed on a four nuclear genes dataset (ADRA2B, AR, GHR and vWF) including all Malagasy tenrec genera. Moreover, the influence of both taxon sampling and data partitioning on the accuracy of the estimated ages were assessed. RESULTS: Within Afrotheria the vast majority of the nodes received a high support, including the grouping of hyrax with sea cow and the monophyly of both Afroinsectivora (Macroscelidea + Afrosoricida) and Afroinsectiphillia (Tubulidentata + Afroinsectivora). Strongly supported relationships were also recovered among all tenrec genera, allowing us to firmly establish the grouping of Geogale with Oryzorictinae, and to confirm the previously hypothesized nesting of Limnogale within the genus Microgale. The timeline of Malagasy tenrec diversification does not reflect a fast adaptive radiation after the arrival on Madagascar, indicating that morphological specializations have appeared over the whole evolutionary history of the family, and not just in a short period after colonization. In our analysis, age estimates at the root of a clade became older with increased taxon sampling of that clade. Moreover an augmentation of data partitions resulted in older age estimates as well, whereas standard deviations increased when more extreme partition schemes were used. CONCLUSION: Our results provide as yet the best resolved gene tree comprising all Malagasy tenrec genera, and may lead to a revision of tenrec taxonomy. A timeframe of tenrec evolution built on the basis of this solid phylogenetic framework showed that morphological specializations of the tenrecs may have been affected by environmental changes caused by climatic and/or subsequent colonization events. Analyses including various taxon sampling and data partitions allow us to point out some possible pitfalls that may lead to biased results in molecular dating; however, further analyses are needed to corroborate these observations.

Chromosomal evolution in tenrecs (Microgale and Oryzorictes, Tenrecidae) from the Central Highlands of Madagascar.

Tenrecs (Tenrecidae) are a widely diversified assemblage of small eutherian mammals that occur in Madagascar and Western and Central Africa. With the exception of a few early karyotypic descriptions based on conventional staining, nothing is known about the chromosomal evolution of this family. We present a detailed analysis of G-banded and molecularly defined chromosomes based on fluorescence in situ hybridization (FISH) that allows a comprehensive comparison between the karyotypes of 11 species of two closely related Malagasy genera, Microgale (10 species) and Oryzorictes (one species), of the subfamily Oryzorictinae. The karyotypes of Microgale taiva and M. parvula (2n = 32) were found to be identical to that of O. hova (2n = 32) most likely reflecting the ancestral karyotypes of both genera, as well as that of the Oryzorictinae. Parsimony analysis of chromosomal rearrangements that could have arisen following Whole Arm Reciprocal Translocations (WARTs) showed, however, that these are more likely to be the result of Robertsonian translocations. A single most parsimonious tree was obtained that provides strong support for three species associations within Microgale, all of which are consistent with previous molecular and morphological investigations. By expanding on a recently published molecular clock for the Tenrecidae we were able to place our findings in a temporal framework that shows strong chromosomal rate heterogeneity within the Oryzorictinae. We use these data to critically examine the possible role of chromosomal rearrangements in speciation within Microgale.

[Hematozoon cenoses of small rodents and insectivora in the Orenburg Region].

A total of 2942 specimens of 15 species of ground rodents and insectova in the Orenburg Region were caught and examined during long-term studies. The investigators detected 7 taxonomic groups of hematozoons: rickettsia (Anaplasma sp., Grahamella sp., Haemobartonella sp.), protozoa (Trypanosoma sp., Plasmodium sp., Piroplasma sp.), and nematodes (Filariidae spp., larval stages). The authors give information on the species composition and infection extensiveness of individual systematic groups of small mammals, the most important morphometric and biological signs of blood parasites, and the specificity of parasite-host relations. The Eversmann hamster was found to have parasitic protozoa of the genera Trypanosoma and Piroplasma, which had not been earlier described in the scientific literature.